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FMRFamide-ACTIVATED SIGNAL TRANSDUCTION PATHWAYS IN THE
CROP-GIZZARD OF THE EARTHWORM, LUMBRICUS TERRESTRIS
HUMBOLDT STATE UNIVERSITY
By
Jamey Krauss
A Thesis
Presented to
The Faculty of Humboldt State University
In Partial Fulfillment
Of the Requirements for the Degree
Master of Arts
In Biology
May, 2007
FMRFamide-ACTIVATED SIGNAL TRANSDUCTION PATHWAYS IN THE
CROP-GIZZARD OF THE EARTHWORM, LUMBRICUS TERRESTRIS
HUMBOLDT STATE UNIVERSITY
By
Jamey Krauss
Approved by the Master’s Thesis Committee:
Bruce O’Gara, Major Professor
Date
Jacob Varkey, Committee Member
Date
Casey Lu, Committee Member
Date
Joe Szewczak, Committee Member
Date
Mike Mesler, Graduate Coordinator
Date
Chris A. Hopper, Interim Dean,
Research, Graduate Studies & International Programs
Date
ABSTRACT
FMRFamide-ACTIVATED SIGNAL TRANSDUCTION PATHWAYS IN THE
CROP-GIZZARD OF THE EARTHWORM, LUMBRICUS TERRESTRIS
Jamey Krauss
In this study, I examined the effects of FMRFamide on the isolated crop-gizzard
of the earthworm, Lumbricus terrestris. The peptide induced contractions of both the
longitudinal and circular muscles of the crop-gizzard at concentrations examined (10-9 to
10-5 M). The responses were quantified by measuring increases in basal tonus, peak
tension, integrated area, mean contraction amplitude, and contraction rate. FMRFamide
application induced concentration-dependent decreases in basal tonus increase, peak
tension, integrated area, and mean contraction amplitude of the longitudinal muscles.
However, FMRFamide application induced a biphasic response in contraction rate where
at low concentrations (10-9 to 10-7 M) there was an increase in contraction rate, but at
high concentrations (10-6 – 10-5 M) the rate decreased and approached control values.
FMRFamide application induced a complex multiphasic effect in basal tonus increase,
peak tension, and integrated area of the circular muscles. At low concentrations (10-9 –
10-8 M) there was a decrease in each FMRFamide-induced response, whereas at higher
concentrations (10-7 – 10-6 M) the FMRFamide-induced responses increased before
falling at the highest exposure (10-5 M). Additionally, FMRFamide induced a
concentration-dependent biphasic effect on mean contraction amplitude, whereas the
contraction rate revealed an excitatory trend as FMRFamide concentrations increased.
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The main aim of this study was to determine which signal transduction pathways were
activated by FMRFamide in the crop-gizzard. It was discovered that the crop-gizzard
lacks amiloride-sensitive sodium channels gated by FMRFamide. Second messenger
pathway manipulation experiments suggested that the phosphatidylinositol and
arachidonic acid pathways are involved in the FMRFamide-induced responses.
FMRFamide-induced responses were reduced by the protein kinase C inhibitors, H-7 (5
×10-5 M) and BIM I (10-5 M), calcium-calmodulin kinase II inhibitor, KN-62 (10-5 M),
phospholipase A2 inhibitor, 4-BPB (10-6 M), and the phospholipase A2 and phospholipase
C inhibitor, U-73122 (18 × 10-6 M). However, there was no evidence to suggest that the
cAMP or NO-induced cGMP second messenger pathways were involved in the
FMRFamide induced responses. FMRFamide-induced responses were unaffected by the
protein kinase A inhibitor, H-89 (10-6 M), adenylyl cyclase inhibitor, MDL-12,330A (10-5
M), and guanylyl cyclase inhibitor, ODQ (10-6 M). Additionally, application of the
cAMP analog, 8-Br-cAMP (10-5 M), NO donor, SNAP (10-5 M), and cGMP analog, 8-BrcGMP (10-5 M) produced contractile responses that did not resemble those induced by
FMRFamide. Certain drug treatments alone induced distinct contractile responses of the
crop-gizzard, indicating the role of specific transduction mechanisms in mediating cropgizzard spontaneous activity. Normal crop-gizzard spontaneous activity was altered by
the calmodulin inhibitor, W-7 (10-4 M), and the tyrosine kinase inhibitor, genistein (5 ×
10-5 M).
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ACKNOWLEDGEMENTS
Several individuals have been instrumental in assisting me throughout my thesis
project. First and foremost, I must extend my greatest thanks to Dr. Bruce O’Gara who
was an integral part in my research and development as a graduate student. Bruce
provided a wealth of knowledge, guidance, and support during my years at Humboldt
State University. My committee members, Dr. Jacob Varkey, Dr. Casey Lu, and Dr. Joe
Szewczak, also contributed valuable insight, expertise, and support during the course of
this project. Additionally, I must acknowledge Bruce and Jacob for their contributions to
my professional school aspirations. I look forward to staying connected to the O’Gara
and Varkey labs as I head to UCSF School of Pharmacy.
Finally, I want to thank my parents for their constant love and support over the
years. I feel truly blessed to belong to a family that is as special as ours. Last, but surely
not least, I thank Miranda Haggarty for her unwavering commitment to me during my
stay at Humboldt State.
v
TABLE OF CONTENTS
ABSTRACT .................................................................................................................. iii
ACKNOWLEDGEMENTS ............................................................................................ v
TABLE OF CONTENTS............................................................................................... vi
LIST OF TABLES....................................................................................................... viii
LIST OF FIGURES ....................................................................................................... ix
INTRODUCTION .......................................................................................................... 1
MATERIALS AND METHODS .................................................................................. 10
Drugs and Saline ............................................................................................... 10
Isolated Crop-Gizzard Preparation..................................................................... 11
FMRFamide Response Determination.................................................... 15
Attempt to Examine FMRFamide-Gated Sodium Channels.................... 17
Protocol to Examine the Effects of Pharmacological
Manipulation upon Second Messenger Pathways....................... 18
Statistics............................................................................................................ 19
RESULTS .................................................................................................................... 20
Quantification of Crop-Gizzard Responses to FMRFamide ............................... 20
FMRFamide-Induced Longitudinal Contractions ................................... 22
FMRFamide-Induced Circular Contractions........................................... 26
The Absence of Amiloride Sensitive FMRFamide-Gated Sodium Channels ...... 30
Effects of Manipulating Second Messenger Pathways on
FMRFamide-Induced Responses............................................................ 31
Effects of Manipulating the Phosphatidylinositol
Second Messenger Pathway ....................................................... 34
vi
Effects of Manipulating the Arachidonic Acid
Second Messenger Pathway ....................................................... 39
Effects of Manipulating the cAMP Second Messenger Pathway............. 40
Effects of Manipulating the NO-Induced cGMP
Second Messenger Pathway ....................................................... 43
Manipulations of Second Messenger Pathways
Alter Spontaneous Activity of Crop-Gizzard .............................. 45
DISCUSSION............................................................................................................... 51
Crop-gizzard Responses to FMRFamide............................................................ 51
FMRFamide-Induced Longitudinal Contractions ................................... 52
FMRFamide-Induced Circular Contractions........................................... 53
The Absence of Amiloride Sensitive FMRFamide-gated Sodium Channels ....... 54
Effects of Manipulating Second Messenger Pathways on
FMRFamide-induced Responses............................................................ 55
Effects of Manipulating the Phosphatidylinositol
Second Messenger Pathway ....................................................... 56
Effects of Manipulating the Arachidonic Acid
Second Messenger Pathway ....................................................... 59
Effects of Manipulating the cAMP Second Messenger Pathway............. 60
Effects of Manipulating the NO-induced cGMP
Second Messenger Pathway ....................................................... 61
Manipulation of Second Messenger Pathways Alters
Spontaneous Activity of the Crop-Gizzard ................................. 62
LITERATURE CITED ................................................................................................. 68
vii
LIST OF TABLES
Table
Page
1
Effects of phosphatdylinositol and arachidonic acid pathway manipulations
on FMRFamide-induced contractions of the crop-gizzard. A Dunnett’s
multiple comparison test was utilized to compare the drug
(FMRFamide + drug) and recovery treatments (FMRFamide) with the
control treatment (FMRFamide). Statistically significant treatments are
represented in bold print. The concentrations of FMRFamide (10-7M)
was kept constant through all pathway manipulation experiments...................... 36
2
Effects of cAMP and nitric oxide-induced cGMP pathway manipulations on
FMRFamide-induced contractions of the crop-gizzard. A Dunnett’s multiple
comparison test was utilized to compare the drug (FMRFamide + drug) and
recovery treatments (FMRFamide) with the control treatment (FMRFamide).
Donor and analog experiments required a paired t-test to compare mean
value responses between control (FMRFamide) and drug (analog or donor)
treatments. Recovery treatments were not applied in donor and analog
experiments (gray-shaded boxes). Statistically significant treatments are
represented in bold print. The concentrations of FMRFamide (10-7 M)
was kept constant through all pathway manipulation experiments...................... 41
3
Direct effects of drugs on the phosphatidylinositol, arachidonic acid, and
mitogen-activated protein kinase second messenger pathways in the
crop-gizzard. A Dunnett’s or Student-Newman-Keuls multiple comparison
test was utilized to compare the drug and combined FMRFamide + drug
treatments to the control FMRFamide treatment. Statistically significant
groups are represented in bold print. Integrated area analysis was limited to
only the FMRFamide and FMRFamide + drug treatments and required a
paired t-test to compare the mean value responses. The concentration of
FMRFamide (10-7 M) was kept constant through all pathway manipulation
experiments....................................................................................................... 47
viii
LIST OF FIGURES
Figure
Page
1
Chemical structure of FMRFamide (Sigma-Aldrich Inc., Saint Louis, MO).
Amino acids phenylalanine (F), methionine (M), arginine (R), and
phenylalanine (F) joined by peptide linkages. FMRFamide is the prototypical
member of the RFamide family and all related peptides retain the amino acids
arginine and amidated phenylalanine on the c-terminus as highlighted above. ..... 5
2
The isolated Lumbricus terrestris crop-gizzard experimental setup. The
crop-gizzard was suspended in a saline-filled organ chamber where a constant
circulation was maintained via a saline inflow and a suction outflow. An
isometric force transducer, placed above the crop-gizzard, monitored
contractions of the organ. The signals from the transducer were displayed and
recorded using a computer-based data acquisition system. (A) The crop-gizzard
positioned along a longitudinal axis in preparation to record longitudinal
muscle contractions. Microsurgical sutures were used to secure the
crop-gizzard in the organ chamber, with one suture affixed to the force
transducer and the other affixed through the organ chamber saline inflow
intersection. (B) The crop-gizzard positioned along a circular axis in
preparation to record circular muscle contractions. A polyester thread was
passed through the lumen of the crop-gizzard and subsequently tied to the
force transducer. A lone microsurgical suture was attached to the medial
lateral juncture of the crop-gizzard and then affixed through the organ
chamber saline inflow intersection..................................................................... 13
3
Parameters measured to quantify the response of the crop-gizzard to
FMRFamide. (A) Peak tension is the greatest tension produced in response
to the peptide. The maximal increase in basal tonus is measured as the
highest valley between phasic contractions. (B) Integrated area is measured
as the area under the contraction curve when FMRFamide was applied until
the basal tonus has returned to the baseline value. ............................................. 16
4
Contractile response of the crop-gizzard induced by 10-9 M FMRFamide. An
approximately 4-min application of FMRFamide caused a series of phasic
contractions superimposed upon an increase in basal tonus. Responses of the
crop-gizzard to other FMRFamide concentrations were similar in form,
although they differ in magnitude. ..................................................................... 21
ix
5
Longitudinal muscle contractile recordings showing the effects of increasing
FMRFamide concentrations on a single isolated crop-gizzard. An arrowed
line indicates the duration of FMRFamide application. A saline wash
immediately followed the peptide application. The molar concentration of
the added peptide is stated on the left of each recording..................................... 23
6
Concentration-response curves of the effects of FMRFamide on basal
tonus, peak tension, and integrated area from longitudinal muscles of the
crop-gizzard. (A) The effects of FMRFamide on maximal increase in
basal tonus. (B) The effects of FMRFamide on peak tension. (C) The effects
of FMRFamide on integrated area. Unless otherwise noted, in this and
subsequent figures each point represents the mean of ten different
crop-gizzard preparations and the vertical bars represent standard errors. .......... 24
7
Concentration-response curves of the effects of FMRFamide on percent
changes in mean contraction amplitude and contraction rate from longitudinal
muscles of the crop-gizzard. (A) The effects of FMRFamide on the percent
change in mean contraction amplitude. (B) The effects of FMRFamide on the
percent change in contraction rate. The dashed line in each plot indicates the
control values for each measurement. ................................................................ 25
8
Circular muscle contractile recordings of the effects of increasing FMRFamide
concentrations on a single isolated crop-gizzard. An arrowed line indicates the
duration of FMRFamide application. A saline wash immediately followed the
peptide application. The molar concentration of the added peptide is stated
on the left of each recording. ............................................................................. 27
9
Concentration-response curves of the effects of FMRFamide on basal tonus,
peak tension, and integrated area from circular muscles of the crop-gizzard.
(A) The effects of FMRFamide on maximal increase in basal tonus. (B) The
effects of FMRFamide on peak tension. (C) The effects of FMRFamide on
integrated area. Unless otherwise noted, in this and subsequent figures each
point represents the mean of eleven different crop-gizzard preparations and
the vertical bars represent standard errors. ......................................................... 28
10
Concentration-response curves of the effects of FMRFamide on percent
change in mean contraction amplitude and percent change in contraction rate
from circular muscles of the crop-gizzard. (A) The effects of FMRFamide on
the percent change in mean contraction amplitude. (B) The effects of
FMRFamide on the percent change in contraction rate. The dashed line
in each plot indicates the control values for each measurement.......................... 29
x
11
The effects of 10-4 M amiloride on the 10-7 M FMRFamide-induced contractile
activity of a single isolated crop-gizzard. The figure displays the contractile
responses of a single crop-gizzard to the three treatments. An arrowed line
indicates when a specific treatment was added to the organ chamber. A
saline wash immediately followed each treatment. Parallel line breaks in
this and following figures represent segments of deleted data corresponding
to extended saline wash periods. All contractile responses were produced
from a crop-gizzard orientated to record longitudinal muscle contractions
by the force transducer. ..................................................................................... 32
12
Quantification of the effects of 10-4 M amiloride on the 10-7 M FMRFamideinduced response. (A) Pretreatment of the crop-gizzard with 10-4 M amiloride
(N = 11) had no significant effect on maximal increase in basal tonus
( ! r2 = 1.19; p = 0.552; df = 2 [Friedman ANOVA on ranks]). Basal tonus
data were not normally distributed and consequently the data was displayed
using a box plot. The line in the center of the box represents the median, the
lower and upper limits of the box represent the 25th and 75th percentile
respectively, and the whisker bars represent the 10th and 90th percentiles.
(B) Pretreatment of the crop-gizzard with 10-4 M amiloride (N = 11) had no
significant effect on peak tension ( ! r2 = 3.82; p = 0.1482; df = 2 [Friedman
ANOVA on ranks]). (C) Pretreatment of the crop-gizzard with 10-4 M
amiloride (N = 11) had no significant effect on integrated area (F = 2.68; p =
0.093; df = 2, 20 [One way repeated-measures ANOVA]). A vertical bar
chart was used to display the normally distributed data, with the column bars
representing the treatment means and the whisker bars representing
respective standard errors. ................................................................................. 33
13
The inhibitory effects of 5 × 10-5 M H-7 on the 10-7 M FMRFamide-induced
contractile activity of a single isolated crop-gizzard. The figure displays the
contractile responses of a single crop-gizzard to the three treatments (dashed
lines). An arrowed line indicates when a specific treatment was added to the
organ chamber. A saline wash immediately followed each treatment................ 35
14
The inhibitory effects of 10-5 M BIM I on the 10-7 M FMRFamide-induced
contractile activity of a single isolated crop-gizzard. The figure displays the
contractile responses of a single crop-gizzard to the three treatments (dashed
lines). An arrowed line indicates when a specific treatment was added to the
organ chamber. A saline wash immediately followed each treatment................ 35
xi
15
The inhibitory effects of 10-5 M KN-62 on the 10-7 M FMRFamide-induced
contractile activity of a single isolated crop-gizzard. The figure displays the
contractile responses of a single crop-gizzard to the three treatments (dashed
lines). An arrowed line indicates when a specific treatment was added to the
organ chamber. A saline wash immediately followed each treatment................ 38
16
The inhibitory effects of 10-6 M 4-BPB on the 10-7 M FMRFamide-induced
contractile activity of a single isolated crop-gizzard. The figure displays the
contractile responses of a single crop-gizzard to the three treatments (dashed
lines). An arrowed line indicates when a specific treatment was added to the
organ chamber. A saline wash immediately followed each treatment................ 38
17
The different effects of 10-5 M 8-Br-cAMP and 10-7 M FMRFamide on a
single isolated crop-gizzard. The figure displays the contractile responses
of a single crop-gizzard to the two treatments (dashed lines). Treatment
applications were varied in order throughout the sample set. ............................. 42
18
The different effects of 10-5 M SNAP and 10-7 M FMRFamide on a single
isolated crop-gizzard. The figure displays the contractile responses of a
single crop-gizzard to the two treatments (dashed lines). Treatment
applications were varied in order throughout the sample set. ............................. 42
19
The direct effect of 10-4 M W-7 on a single isolated crop-gizzard. The figure
displays the contractile responses of a single crop-gizzard to three distinct
treatments (dashed lines). In this and following figures, contractions were
continuous between the drug and FMRFamide-drug treatments......................... 46
20
The direct effect of 18 x 10-6 M U-7122 on a single isolated crop-gizzard.
The figure displays the contractile responses of a single crop-gizzard to
three distinct treatments (dashed lines). ............................................................. 46
21
The direct effect of 5 x 10-5 M genistein on a single isolated crop-gizzard.
The figure displays the contractile responses of a single crop-gizzard to
three distinct treatments (dashed lines). ............................................................. 49
xii
INTRODUCTION
Two significant regions of the Lumbricus terrestris gut are the thin-walled crop,
wherein food is stored, and the thick cuticle-lined gizzard that extends from the crop and
mechanically grinds ingested material (Brusca 1990). Food is passed through the entire
alimentary canal via body movements associated with locomotion, as well as muscular
activity generated by the gut musculature (Brusca 1990; Edwards and Bohen 1996). The
musculature of the crop-gizzard is organized into inner circular and outer longitudinal
muscles that produce wave-like contractions to propel the ingesta into the intestine. The
crop apparently initiates and drives the muscular activity with frequent and vigorous
contractions, whereas the gizzard contracts secondarily with weak and irregular
contractions (Wu 1939a; Mill 1978; Krajniak and Khlor 1999). The organization of
individual muscle fibers is similar to that seen in the earthworm Eisenia foetida and
garden snail Helix aspersa. Specifically, Royuela et al. (1995, 2000) described the
muscle ultrastructure of the earthworm and snail intestines as a variant of obliquely
striated muscle with a high thick-thin filament volume in no true sarcomeric organization,
numerous mitochondria, and a well developed sarcoplasmic reticulum, all contributing to
the generation of a slow vigorous force with a high resistance to fatigue.
Neural control of the crop-gizzard is essential to gut motility and the overall
efficacy of digestion. Both the crop and the gizzard are under extrinsic neural control
with innervations arising from the circumpharyngeal connectives, via a subepithelial
nerve plexus, and from the ventral nerve cord, via septal nerves (Wu 1939a). In addition,
1
2
intrinsic neural control is demonstrated in isolated crop-gizzard experiments where the
isolated organ spontaneously contracts up to ten hours in a saline-filled chamber (Wu
1939a; Vassileva et al. 1982; Krajniak and Khlor 1999). The subepithelial nerve plexus
runs the entire length of the alimentary canal and consists of a network of nerves from the
stomatogastric system, a peripheral nervous branch originating beneath the cerebral
ganglia and extending from the circumpharyngeal connectives (Mill 1978). Wu (1939a)
demonstrated that direct electrical stimulation of the L. terrestris stomatogastric system
produced increases in tonus and contractile rate, whereas direct electrical stimulation of
the ventral nerve cord inhibited the contractile rhythm. This dual innervation reveals the
presence of two distinct neural pathways, one excitatory and one inhibitory that control
the crop-gizzard. In addition, Millot (1943a) revealed that segmental nerves entering the
surrounding body wall of the crop-gizzard form synapses in the peritoneum from which
nerves pass to the gut via unique septal pathways and elicit antagonistic responses in the
tone of the gut muscles. Specifically, direct electrical stimulation of the anterior
segmental nerve produces a distinct fall in tone, whereas the direct electrical stimulation
of the medial and/or posterior segmental nerves produces a rise in tone (Millot 1943a).
Wu (1939a) and Millot (1943b) concluded that excitatory and inhibitory activity is
produced by cholinergic and adrenergic nerves respectively, eliciting the stimulatory and
inhibitory effects upon the tone of the crop-gizzard. Contractile experiments involving
intact and isolated crop-gizzard preparations show acetylcholine to induce an excitatory
effect, whereas epinephrine induces a dose-dependent inhibitory effect (Wu 1939a;
Millot 1943a, 1943b; Vassileva et al. 1982; Krajniak and Khlor 1999).
3
In addition to acetylcholine and epinephrine, there is evidence that several other
neurotransmitters are involved in regulating annelid gut activity. The following
neurotransmitters either alter gut contractile activity or have been localized to gut tissue
via immunocytochemistry: norepinephrine, GABA, serotonin, dopamine, octopamine,
proctolin, annetocin, Eisenia tetradecapeptides (ETP), and Eisenia inhibitory
pentapeptides (EIPP) (Anctil et al. 1984, 1990; Telkes et al. 1996; Ukena et al. 1996a;
Reglödi et al. 1997; Krajniak and Khlor 1999; Barna et al. 2001; Oumi et al. 1994; Ukena
et al. 1995; Ukena et al. 1996b). In the polychaete, Chaetopterus variopedatus, Anctil et
al. (1984, 1990) described the presence of norepinephrine-immunoreactive neural
processes associated with the intestine and also demonstrated norepinephrine-induced
augmentation of intestinal tone. Telkes et al. (1996) detected GABA-immunoreactive
neurons in the cerebral ganglia, stomatogastric ganglia, enteric plexus, and individual
cells in the gut epithelium of L. terrestris, while Ukena et al. (1995a) demonstrated a
GABA-induced excitatory response on the E. foetida gut. Serotonin appears to perform a
significant role in the peripheral nervous system of oligochaetes since neural processes in
the body wall and the enteric nervous system reveal strong serotonin immunoreactivity
(Reglödi et al. 1997). Additionally, Krajniak and Khlor (1999) demonstrated that
serotonin induced a concentration-dependent inhibitory response on the L. terrestris cropgizzard. Immunoreactive evidence revealed the distribution of dopamine, octopamine,
and proctolin in the stomatogastric ganglia and enteric plexus of E. foetida (Barna et al.
2001). Also, Barna et al. (2001) demonstrated that dopamine and octopamine induced a
concentration-dependent excitatory effect on the E. foetida foregut, whereas proctolin did
4
not induce any significant contractile effect. Oumi et al. (1994) isolated annetocin, an
oxytocin-related peptide, from E. foetida, and Ukena et al. (1995a) demonstrated that this
neurotransmitter stimulated contractions in the earthworm crop-gizzard. Finaly, Ukena et
al. (1995b; 1996) isolated both ETP and EIPP from the gut of E. foetida, and documented
their respective excitatory and inhibitory actions on the spontaneous activity of the cropgizzard.
Investigations of additional neurotransmitters that induce activity on the gut of
annelids have been an area of emergent research in recent years. In particular, attention
has been directed on the RFamide neuropeptide family, where the actions of FMRFamide
have generated great interest. FMRFamide, a neuropeptide composed of four linked
amino acids (Figure 1), was first discovered in the cerebral ganglia of the mollusc,
Macrocallista nimbosa (Price 1977). After the initial discovery, FMRFamide and related
peptides were isolated from a large number of invertebrates, including several species
from the phylum Annelida. To date seven RFamide neuropeptides have been isolated
from Polychaeta and Hirudinea species and include FMRFamide, FTRFamide,
FLRFamide, YLRFamide, YMRFamide, GGKYMRFamide, and GDPFLRFamide
(Krajniak 2005). FMRFamide induces a range of responses from annelid muscles,
including excitatory effects on the esophagus, intestine, and body wall of the earthworm
E. foetida, and the heart, pharynx, and body wall of Hirudo medicinalis (Ukena et al.
1996; Csokyna et al. 2005; Thompson et al. 1992; O’Gara et al. 1999a; Norris et al.
1990). FMRFamide-induced relaxation on annelid muscles occurs in the esophagus of
Nereis virens, the crop-gizzard of E. foetida, and the body wall of Sabellastarte
5
Figure 1.
Chemical structure of FMRFamide (Sigma-Aldrich Inc., Saint Louis, MO). Amino acids
phenylalanine (F), methionine (M), arginine (R), and phenylalanine (F) joined by peptide linkages.
FMRFamide is the prototypical member of the RFamide family and all related peptides retain the
amino acids arginine and amidated phenylalanine on the c-terminus as highlighted above.
6
magnifica (Barratte et al. 1990; Ukena et al. 1996; Diaz-Miranda et al. 1992).
Additionally, Krajniak and Khlor (1999) revealed that FMRFamide induces a complex
concentration-dependent contractile response on the crop-gizzard of L. terrestris,
consisting of a biphasic change in rate and a decrease in contraction amplitude. The
distinct effects of FMRFamide on a variety of annelid muscle tissues suggest the
existence of different FMRFamide-activated signaling pathways in the different muscle
types.
To date no neuropeptides in the RFamide family have been isolated from
oligochaetes. However, in a recent unpublished report, FMRFamide sequence similarity
(EMBL: CF416445) was revealed in the humus earthworm Lumbricus rubellus (Jones et
al. unpublished). In addition, FMRFamide-like immunoreactivity was shown in the
nervous system of oligochaetes (Fujii et al. 1989). In particular, Fujii et al. (1989)
described FMRFamide-like immunoreactive neural processes associated with the E.
foetida gut. A recent immunocytochemical study of the L. terrestris nervous system
revealed the presence of FMRFamide-like immunoreactive cells in the cerebral ganglia,
ventral nerve cord, the stomatogastric ganglia and nerves, as well as the wall of the
foregut (Reglödi et al. 1997). Reglödi et al. (1997) reported staining of FMRFamide-like
immunoreactive fibers between the inner circular and outer longitudinal muscle bands of
the foregut. Reglödi et al. (1997) speculated that these fibers have their origin in
stomatogastric ganglia or ventral nerve cord; thus illustrating evidence for the possibility
of FMRFamide control on the Lumbricus gut. Krajniak and Khlor (1999) proposed that
7
the complex FMRFamide-induced response they observed from the crop-gizzard was
attributed to the activation of two different RFamide receptor subtypes, an excitatory one
with high affinity for FMRFamide and an inhibitory one with low affinity for the peptide.
Currently, no reports exist attempting to explore the signal transduction processes
involved in the FMRFamide-induced contractile responses of the Lumbricus cropgizzard.
Signal transduction pathways are known to detect, amplify, and integrate diverse
external signals to generate physiological responses such as changes in enzyme activity,
gene expression, or ion channel activity. In particular, members of the RFamide
neuropeptide family appear to activate the phosphatidylinositol second messenger
pathway to mediate synaptic transmission, ion channel regulation, or contractile
responses. In the crayfish, Procambarus clarkia, Friedrich et al. (1998) revealed that
DRNFLRFamide (DF2) induced a long-lasting synaptic response in abdominal extensor
muscle cells through protein kinase C, an enzyme activated by the second messenger
diacylglycerol (DAG) via the phosphatidylinositol signal pathway. O’Gara et al. (1999a)
demonstrated that FMRFamide-induced contractile activity on the pharynx of the leech,
H. medicinalis, is at least partially mediated via protein kinase C. The additional
phosphatidylinositol pathway second messenger, inositol triphosphate (IP3), has been
reported to mediate FMRFamide-induced contractile responses in retractor muscles of the
snail, H. aspersa, and in the heart of the pond snail, Lymnaea stagnalis (Falconer et al.
1993; Willoughby et al. 1999). Furthermore, DF2 was shown to activate calmodulin
8
dependent protein kinase, an enzyme modulated by the phosphatidylinositol pathway, in
contractile responses of the crayfish abdominal extensor muscle (Noronha et al. 1995).
RFamide neuropeptides activate additional signal transduction processes
including cyclic adenosine monophosphate (cAMP), nitric oxide-induced cyclic
guanosine monophosphate (NO-induced cGMP), arachidonic acid second messenger
pathways, independent G protein-coupled receptors, and directly opening an amiloridesensitive sodium ion channel to elicit responses in invertebrate tissue. The existence of
cAMP second messenger pathways in RFamide-induced cellular responses is seen in the
heart of the bivalve, Mercenaria mercenaria, and the body wall of nematode, Ascaris
suum, where each tissue exhibits cAMP-dependent contractile responses after the
application of FMRFamide and other RFamide neuropeptides (Higgins et al. 1978;
Reinitz et al. 2000). The NO-induced cGMP second messenger pathway appears to
mediate the FMRFamide-induced slow activation of a sodium current in neuron R14 of
the sea slug, Aplysia californica (Ichinose and McAdoo 1989). Arachidonic acid appears
to serve as a second messenger in mediating the FMRFamide-induced slow activation of
potassium currents in the neuron L7 of A. californica (Piomelli et al. 1987). Cottrell
(1993) revealed that different RFamides activate an independent G protein-coupled
receptor that in turn slowly activates potassium currents in H. aspersa neurons.
Additionally, Cottrel (1997) discovered that FMRFamide directly gates an amiloridesensitive sodium channel in neurons of H. aspersa, the only report of a peptide directly
activating an ion channel. From these studies, it appears FMRFamide and related
RFamide peptides induce a variety of cellular responses via a number of signal
9
transduction processes. However, the role and importance of these signal processes in
the FMRFamide-induced activity of the annelid gut are largely unexplored.
In this report, I confirmed the results generated by Krajniak and Khlor (1999) in
demonstrating the sensitivity of the L. terrestris crop-gizzard to the neuropeptide
FMRFamide. Moreover, I recorded additional response parameters that more completely
describe the response of the crop-gizzard to FMRFamide. To generate a more
comprehensive view of the contractile activity induced by the peptide, I examined and
compared the effects of FMRFamide applications on the longitudinal and circular
muscles of the crop-gizzard. Finally, I investigated a number of signal transduction
pathways for their potential involvement in mediating the FMRFamide-induced effects or
spontaneous activity of the crop-gizzard.
MATERIALS AND METHODS
Earthworms, Lumbricus terrestris, were obtained from a local bait shop
(Bucksport Sporting Goods, Eureka, CA) and maintained individually in 300-ml paper
cups containing Magic Worm Bedding (Magic Products, Inc., Amherst Junction, WI).
The cups were covered with perforated plastic lids and stored in an incubator at 14 °C in
constant darkness.
Drugs and Saline
During the dissection and experiment, the crop-gizzard was superfused with a
physiological saline (normal earthworm saline) containing 26 mM Na2SO4, 25 mM NaCl,
4 mM KCl, 1 mM MgCl2, 6 mM CaCl2, 2 mM TRIS base, 55 mM sucrose and adjusted
to pH 7.40 with HCl (Drewes and Pax 1975). All experiments were conducted at room
temperature (21 – 25 °C). Peptides or drugs were purchased from the following
suppliers: FMRFamide, amiloride (N-amido-3,5-diamino-6-chloropyrazinecarboxamide
hydrochloride), 4-bromophenacyl bromide (4-BPB), W-7 (N-[6-aminohexyl]-5-chloro-1napthalenesulfonamide hydrochloride), KN-62 (1-[N,O-bis-(5-isoquinolinesulfonyl)-Nmethyl-L-tyrosyl]-4-phenylpiperazine, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1one), MDL-12,330A (cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine
hydrochloride), U-73122 (1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17yl)amino]hexyl]-1H-pyrrole-2,5-dione), 8-Br-cAMP (8-bromoadenosine 3’,5’-cyclic
10
11
monophosphate sodium salt), 8-Br-cGMP (8-bromoguanosine-3’,5’-cyclomonophosphate
sodium salt), and SNAP (S-nitroso-N-acetylpenicillamine) (Sigma-Aldrich, Inc., Saint
Louis, MO); H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide
dihydrochloride) and genistein (4’,5,7-trihydroxyisoflavone) (Axxora, LLC, San Diego,
CA); OBAA (3-[4-octadecyl]-benzoylacrylic acid) (Biomol Inc., Plymouth Meeting,
PA); BIM I (2-[1-(3-dimethylaminopropyl)-1H-indol-3-3-yl]-3-(1H-indol-3yl)maleimide hydrochloride) (EMD Biosciences, Inc., La Jolla, CA); ML-7 (1-[5iodonaphthalene-1-sulfonyl]-1H-hexahydro-1,4-diazepine hydrochloride) (Seikagaku
Corporation, Tokyo, Japan). FMRFamide was dissolved in NANOpure distilled water
(Barnstead International, Dubuque, IA) and frozen (-20 °C) in small aliquots. 4-BPB, H89, W-7, KN-62, ML-7, ODQ, MDL-12,330A, genistein, and SNAP were prepared as
concentrated stock solutions (10-1 – 10-2 M) dissolved in DMSO (Sigma-Aldrich, Inc.,
Saint Louis, MO). OBAA and U-73122 were prepared as concentrated stock solutions
(10-2 M; 1.8 × 10-3 M) dissolved in ethanol. All drugs were diluted to their final
concentration in normal earthworm saline just prior to the experiment.
Isolated Crop-Gizzard Preparation
Approximately 24 hrs prior to an experiment, earthworms were transferred from
their home cups into individual Petri dishes containing a 8 x 8 cm paper towel section
dampened with distilled water and returned to the incubator until dissection. This
procedure allowed the gut to clear of ingesta. Prior to dissection, earthworms were
immobilized by placing them in an ice bath for approximately 10 min. Following
12
immobilization, the worms were pinned dorsal side up in a frozen wax-bottomed
dissection tray and covered with ice-cold normal earthworm saline. A dorsal midline
incision was made from just anterior of the clitellum to the anterior end of the worm. The
crop-gizzard was freed from the body wall by severing the connective septa on the lateral
and ventral sides. The crop-gizzard was removed from the worm by severing the gut
anterior to the crop and posterior to the gizzard. The isolated crop-gizzard was removed
from the dissection tray and temporarily placed into a small saline-filled Petri dish. To
record contractions of the longitudinal muscles (Figure 2A), separate microsurgical
needles, bent into hook shapes and attached to 8-0 monofilament nylon suture material
(Ethicon 2808G, Sommerville, NJ), were inserted through the ends of the crop and the
gizzard. The suture that was attached to the gizzard was anchored to the bottom of a
small perfusion organ chamber (volume 0.8-ml; constructed from a 3-ml syringe), while
the suture attached to the crop was affixed to an overhead isometric force transducer
(FORT-10, WPI, Sarasota, FL). The crop-gizzard was placed under approximately 15 –
20 mN of tension and allowed to relax for approximately 1 hr under saline perfusion.
Most crop-gizzards arranged in this orientation immediately produced spontaneous
contractile activity. To record contractions of the circular muscles (Figure 2B), a 100%
polyester Talon® sewing thread was passed through the lumen of the crop-gizzard and a
lone microsurgical bent needle with its adjoined nylon suture was inserted through the
medial lateral juncture of crop-gizzard. The nylon suture was anchored to the bottom of a
new small perfusion organ chamber (volume 0.8-ml; constructed from a 5-ml syringe),
13
A
Force
Transducer
Suction Outflow
Crop-gizzard
Organ
Chamber
Display
Saline Inflow
B
Force
Transducer
Suction Outflow
Crop-gizzard
Organ
Chamber
Display
Saline Inflow
Figure 2.
The isolated Lumbricus terrestris crop-gizzard experimental setup. The crop-gizzard was
suspended in a saline-filled organ chamber where a constant circulation was maintained via a
saline inflow and a suction outflow. An isometric force transducer, placed above the crop-gizzard,
monitored contractions of the organ. The signals from the transducer were displayed and recorded
using a computer-based data acquisition system. (A) The crop-gizzard positioned along a
longitudinal axis in preparation to record longitudinal muscle contractions. Microsurgical sutures
were used to secure the crop-gizzard in the organ chamber, with one suture affixed to the force
transducer and the other affixed through the organ chamber saline inflow intersection. (B) The
crop-gizzard positioned along a circular axis in preparation to record circular muscle contractions.
A polyester thread was passed through the lumen of the crop-gizzard and subsequently tied to the
force transducer. A lone microsurgical suture was attached to the medial lateral juncture of the
crop-gizzard and then affixed through the organ chamber saline inflow intersection.
14
while the polyester thread was tied to the overhead isometric force transducer. The cropgizzard was placed under approximately 10 – 15 mN of tension and allowed to relax for
approximately 1 hr under saline perfusion. All crop-gizzards arranged in this orientation
revealed weaker contractile activity compared to the set-up for recording longitudinal
muscle contractions. At the conclusion of the 1 hr relaxation period, spontaneous
longitudinal contractions were approximately 4 mN in strength whereas spontaneous
circular contractions were approximately 1 mN in strength.
The output of the force transducer was fed into a transducer interface (ETH-200,
CB Sciences, Dover, NH), whose subsequent amplified output was fed into a computerbased data acquisition system (WINDAQ 200, Dataq Instruments, Akron, OH). The
signal from the force transducer was digitized at 50 samples/sec and recorded to disk.
Data analysis of recorded signals was performed using playback software of the data
acquisition system and Advanced CODAS software (Dataq Instruments, Akron, OH).
The organ chamber was continuously perfused with saline through an inlet at the
bottom of the chamber at an approximate rate of 1 ml/min; saline was removed from the
top of the chamber by suction via a 26-gauge syringe needle. The application of control
or experimental salines was controlled by a valve attached to reservoirs (5-ml and 60-ml)
containing each of the salines. Prior to experimental manipulations, the experimental
reservoir held an appropriate measured volume of drug-containing saline. The switching
of a valve connected to the experimental and control saline reservoirs initiated the
application of drug-containing saline. An air bubble introduced into the perfusion line
15
indicated the beginning and end of each treatment. Each application of FMRFamidecontaining saline was 4-ml in volume (5 times the volume of the organ chamber) and
approximately 4 min in duration and followed by a sufficient saline wash to remove the
FMRFamide-containing saline from the organ chamber and return the crop-gizzard to a
predetermined control tension (as FMRFamide-induced effects wore off). Event markers
were inserted within the data acquisition file to accurately note the beginning and end of
each treatment (i.e., an air bubble entering the organ chamber).
FMRFamide Response Determination
When determining concentration-response relationships, the lowest FMRFamide
concentration was applied first (10-9 M) and higher concentrations (10-8 to 10-5 M) were
added in sequential order. FMRFamide-induced responses were quantified by measuring
the maximal increase in basal tonus (greatest sustained increase in muscle tonus; Figure
3A), peak tension (greatest tension produced in response to the peptide; Figure 3A),
integrated area (area under the contraction curve during the response of the peptide;
Figure 3B), contraction rate (average number of contractions during the response period),
and mean contraction amplitude (average contraction height during response period)
during the observed contraction period. Contraction rate and mean contraction amplitude
recordings were adjusted to percent change values compared to control contractions prior
to FMRFamide application (control period was approximately 10 min.). FMRFamideinduced longitudinal contractions were recorded from 10 crop-gizzards, while
FMRFamide-induced circular contractions were recorded from 11 crop-gizzards. Data
16
A
Peak Tension
Maximum Increase
of Basal Tonus
B
Integrated Area
FMRFamide
Figure 3.
Parameters measured to quantify the response of the crop-gizzard to FMRFamide. (A) Peak
tension is the greatest tension produced in response to the peptide. The maximal increase in basal
tonus is measured as the highest valley between phasic contractions. (B) Integrated area is
measured as the area under the contraction curve when FMRFamide was applied until the basal
tonus has returned to the baseline value.
17
collected from FMRFamide response periods were plotted to generate concentrationresponse curves where response thresholds and relationships were determined.
Attempt to Examine FMRFamide-Gated Sodium Channels
An amiloride-sensitive sodium channel that is directly gated by FMRFamide
occurs in the snail H. aspersa (Cottrel 1987). To examine the possibility of FMRFamide
acting upon this sodium channel in the earthworm crop-gizzard the following protocol
was used (n = 11). An isolated crop-gizzard was positioned in the organ chamber to
record muscle contractions from the longitudinal axis since early experiments revealed
FMRFamide elicited the larger responses from the longitudinal muscles. After the
relaxation period, the crop-gizzard was exposed to a control period consisting of a 4-ml
volume of 10-7 M FMRFamide and the response was recorded. This FMRFamide
concentration produced a clearly observable contractile response (Figure 5). Following
FMRFamide application, the crop-gizzard was washed in normal saline for 35 – 40 min
and afterward exposed to 9-ml (approximately 15 min of exposure time) of the sodium
channel blocker amiloride (10-4 M). Pretreatment with 10-4 M amiloride was followed by
the experimental period consisting of a 4-ml volume of 10-7 M FMRFamide and 10-4 M
amiloride. After an additional 35 – 45 min saline wash, a second application of 4-ml of
10-7 M FMRFamide was performed to measure the recovery of the crop-gizzard from the
amiloride treatment.
18
Protocol to Examine the Effects of
Pharmacological Manipulation upon
Second Messenger Pathways
Experiments conducted to examine the possible role of specific second messenger
pathways in mediating the FMRFamide-induced responses used the following protocol.
An isolated crop-gizzard was positioned in the organ chamber to record muscle
contractions from the longitudinal axis. After the initial relaxation period, the cropgizzard was exposed to a control application consisting of a 4-ml volume of 10-7 M
FMRFamide and the response was recorded. Following FMRFamide application, the
crop-gizzard was continuously washed in normal saline for 35 – 45 min. Drugs that
inhibit signal transduction pathways were then applied to the tissue for approximately 30
min prior to the experimental period consisting of a 4-ml volume of 10-7 M FMRFamide
and the drug. Following the FMRFamide and drug application, the tissue was washed in
normal saline for 35 – 45 min and a second control application was performed consisting
of a 4-ml volume of 10-7 M FMRFamide to measure recovery of the crop-gizzard from
the drug treatment. When drugs were dissolved in DMSO or ethanol, the solvent
concentration (0.01% – 1.0% of final solution) was added to each treatment applied to the
crop-gizzard.
The use of second messenger analogs or nitric oxide donors required slight
experimental adjustments. Following the relaxation period, the crop gizzard was exposed
to a control application consisting of a 4-ml volume of 10-7 M FMRFamide, followed by
a 40 – 60 min saline wash, and then the experimental period consisting of a 4-ml volume
of the specific drug. To ensure an unbiased response, the procedure was reversed (drug
19
applied first, followed by FMRFamide) for an equal number of crop-gizzards in each
experiment. Because SNAP was dissolved in DMSO, during the control period the cropgizzard was exposed to an identical concentration of the solvent (0.1%).
Statistics
Data collected from the drug investigations were subjected to statistical analysis
to determine if drug treatments induced significant effects. Statistics were performed
using Sigma Stat 1.0 (SigmaStat Inc., San Rafael, Ca) or NCSS 2004 (Number Cruncher
Statistical Systems, Kaysville, UT). Normality was determined with preliminary
descriptive statistics using the Kolmogorov-Smirnov test with Lilliefors’ correction.
When the crop-gizzard was exposed to three or more treatments, normally distributed
data were subjected to a repeated measures analysis of variance test (ANOVA) and the
values are presented as mean ± standard error. Data that were not normally distributed
were subjected to a Friedman repeated measures ANOVA on ranks and the values are
presented as medians (25th percentile, 75th percentile). If either ANOVA result was
significant (pvalue< 0.05), multiple comparison tests (Dunnett’s or Student Newman-Keuls
test) were utilized to identify differences between treatments. When the crop-gizzard was
exposed to only two treatments, paired t-tests (pvalue= 0.05) were used to compare each
treatment if the data were normally distributed. However, when data were not normally
distributed, a Wilcoxon signed rank test was utilized to compare each treatment.
RESULTS
Quantification of Crop-Gizzard Responses to FMRFamide
Contractions of the isolated crop-gizzard and its responses to FMRFamide
application were monitored with a force transducer (Figure 2). At the end of a
preliminary relaxation period of about 1 hr, the crop-gizzard exhibited a constant basal
tonus with spontaneous contractions ranging from approximately 2 contractions per
minute for longitudinal muscle recordings to approximately 1 contraction per minute for
circular muscle recordings. After the relaxation period, a 4-min application of
FMRFamide to the crop-gizzard produced a contractile response (Figure 4). During the
FMRFamide-induced response, the phasic contractions often exhibited an increase in
peak tension and contraction rate. The FMRFamide-induced responses were quantified
by measuring the maximal increase in basal tonus, the peak tension, the integrated area
under the contraction curve (Figure 3), the percent change in contraction amplitude, and
the percent change in contraction rate. Changes in basal tonus and peak tension are
proportional to the amount of work produced by the crop-gizzard; and for at least some
neurotransmitters each of these measured variables is pharmacologically separable
(O’Gara et al. 1999b). Integrated area allows the entire contraction curve to be analyzed
and is influenced by both response duration and contraction rate.
20
21
10-9 M FMRFamide
5 mN
5 min
FMRFamide
Figure 4.
Contractile response of the crop-gizzard induced by 10-9 M FMRFamide. An approximately
4-min application of FMRFamide caused a series of phasic contractions superimposed upon an
increase in basal tonus. Responses of the crop-gizzard to other FMRFamide concentrations were
similar in form, although they differ in magnitude.
22
FMRFamide-Induced Longitudinal Contractions
Preferential recording of longitudinal muscle contractions was achieved by
positioning a force transducer above an organ chamber where a crop-gizzard was
orientated along a longitudinal axis (Figure 2A). Contractile recordings depicting each
FMRFamide application (10-9 – 10-5 M) upon a single crop-gizzard are displayed in
Figure 5. Spontaneous or induced-contractions were more frequent during the initial
stages of an experiment and as the isolation time increased, the contractile activity
lessened. Each FMRFamide-induced response revealed an excitatory effect upon the
crop-gizzard along with a concentration-dependent decrease in response. The excitatory
effect and concentration dependence of the decrease in response amplitude are shown in
Figure 6, which plots the effects of FMRFamide upon basal tonus, peak tension, and
integrated area. The amplitude of each response variable decreased with higher
concentrations of FMRFamide up to at least 10-5 M (FMRFamide concentrations higher
than 10-5 M were not tested). Positive basal tonus measurements at each concentration
reveal the excitatory effect induced by FMRFamide (Figure 6A); FMRFamide did not
induce relaxation at any concentration tested. In addition, the shape of each
concentration-response curve shows the decreasing amplitude of each of these parameters
as peptide concentration increased (Figure 6).
Concentration-response relationships for percent change in mean contraction
amplitude and contraction rate are plotted in Figure 7. The application of FMRFamide at
23
5 mN
5 min
10-9
FMRFamide
10-8
10-7
10-6
10-5
Figure 5.
Longitudinal muscle contractile recordings showing the effects of increasing FMRFamide
concentrations on a single isolated crop-gizzard. An arrowed line indicates the duration of
FMRFamide application. A saline wash immediately followed the peptide application. The molar
concentration of the added peptide is stated on the left of each recording.
24
Basal Tonus Increase (mN)
3.0
A
2.5
2.0
1.5
1.0
0.5
0.0
10
8
-9
10
-8
10
-7
10
-6
10
-5
B
Peak Tension (mN)
7
6
5
4
3
2
1
0
10
Integrated Area (mN · s)
1200
-9
10
-8
10
-7
10
-6
10
-5
C
1000
800
600
400
200
0
10
-9
10
-8
10
-7
10
-6
10
-5
FMRFamide Concentration (M)
Figure 6.
Concentration-response curves of the effects of FMRFamide on basal tonus, peak tension,
and integrated area from longitudinal muscles of the crop-gizzard. (A) The effects of FMRFamide
on maximal increase in basal tonus. (B) The effects of FMRFamide on peak tension. (C) The
effects of FMRFamide on integrated area. Unless otherwise noted, in this and subsequent figures
each point represents the mean of ten different crop-gizzard preparations and the vertical bars
represent standard errors.
25
60
A
Percent Change in
Mean Contraction Amplitude (%)
50
40
30
20
10
0
-10
-20
-30
-40
10
480
-9
10
-8
10
-7
10
-6
10
-5
B
Percent Change in
Contraction Rate (%)
420
360
300
240
180
120
60
0
-60
10
-9
10
-8
10
-7
10
-6
10
-5
FMRFamide Concentration (M)
Figure 7.
Concentration-response curves of the effects of FMRFamide on percent changes in mean
contraction amplitude and contraction rate from longitudinal muscles of the crop-gizzard. (A) The
effects of FMRFamide on the percent change in mean contraction amplitude. (B) The effects of
FMRFamide on the percent change in contraction rate. The dashed line in each plot indicates the
control values for each measurement.
26
increasing concentrations revealed an inhibitory effect upon contraction amplitude. The
threshold concentration for the inhibition of contraction amplitude was between 10-8 and
10-7 M. However, FMRFamide had a concentration-dependent biphasic effect upon
contraction rate. At low concentrations (10-9 – 10-7 M) there was an increase in
contraction rate with a peak contraction rate attained at 10-7 M, whereas at high
concentrations (10-6 – 10-5 M) the rate decreased and approached control values.
FMRFamide-Induced Circular Contractions
Preferential recording of circular muscle contractions was achieved by positioning
a force transducer above an organ chamber where the longitudinal axis of the cropgizzard was positioned perpendicular to the thread transmitting contractile force to the
transducer (Figure 2B). Contractile recordings depicting each FMRFamide application
(10-9 – 10-5 M) upon a single crop-gizzard are displayed in Figure 8. FMRFamide
application produced concentration-dependent changes in contractions produced by the
circular muscles of the crop-gizzard (Figure 9). At low concentrations (10-9 – 10-8 M)
there was a decrease in each FMRFamide-induced response, whereas at higher
concentrations (10-7 – 10-6 M) the FMRFamide-induced responses increased before
falling at the highest exposure (10-5 M). This multiphasic effect of FMRFamide was seen
throughout each response parameter and emphasized by similar shaped response curves
(Figure 9). Additionally, positive basal tonus measurements at each concentration reveal
the excitatory effect induced by FMRFamide (Figure 9A).
27
2 mN
5 min
10-9
FMRFamide
10-8
10-7
10-6
10-5
Figure 8.
Circular muscle contractile recordings of the effects of increasing FMRFamide
concentrations on a single isolated crop-gizzard. An arrowed line indicates the duration of
FMRFamide application. A saline wash immediately followed the peptide application. The molar
concentration of the added peptide is stated on the left of each recording.
Basal Tonus Increase (mN)
1.2
A
28
1.0
0.8
0.6
0.4
0.2
0.0
3.0
B
10-9
10-8
10-7
10-6
10-5
10-9
10-8
10-7
10-6
10-5
10-9
10-8
10-7
10-6
10-5
Peak Tension (mN)
2.5
2.0
1.5
1.0
0.5
0.0
Integrated Area (mN · s)
350
C
300
250
200
150
100
50
0
FMRFamide Concentration (M)
Figure 9.
Concentration-response curves of the effects of FMRFamide on basal tonus, peak tension,
and integrated area from circular muscles of the crop-gizzard. (A) The effects of FMRFamide on
maximal increase in basal tonus. (B) The effects of FMRFamide on peak tension. (C) The effects
of FMRFamide on integrated area. Unless otherwise noted, in this and subsequent figures each
point represents the mean of eleven different crop-gizzard preparations and the vertical bars
represent standard errors.
29
Percent Change in
Mean Contraction Amplitude (%)
150
A
125
100
75
50
25
0
-25
-50
450
10-9
10-8
10-7
10-6
10-5
10-9
10-8
10-7
10-6
10-5
B
400
Percent Change in
Contraction Rate (%)
350
300
250
200
150
100
50
0
-50
FMRFamide Concentration (M)
Figure 10.
Concentration-response curves of the effects of FMRFamide on percent change in mean
contraction amplitude and percent change in contraction rate from circular muscles of the cropgizzard. (A) The effects of FMRFamide on the percent change in mean contraction amplitude.
(B) The effects of FMRFamide on the percent change in contraction rate. The dashed line in each
plot indicates the control values for each measurement.
30
The FMRFamide-induced responses were also quantified via mean contraction
amplitude and contraction rate. Contractile recordings revealed a concentrationdependent biphasic effect on mean contraction amplitude, whereas the contraction rate
revealed an excitatory trend as FMRFamide concentrations increased (Figures 8, 10).
Concentration-response relationships for percent change in mean contraction amplitude
and contraction rate are plotted in Figure 10. At low concentrations (10-9 – 10-8 M) there
was a decrease in mean contraction amplitude to the control value, whereas at high
concentrations (10-7 – 10-5 M) the mean contraction amplitude increased. However, the
application of FMRFamide at increasing concentrations revealed an excitatory effect
upon contraction rate with a sharp increase between 10-9 – 10-7 M and subsequently
reaching a plateau between 10-7 – 10-5 M.
The Absence of Amiloride Sensitive FMRFamide-Gated Sodium Channels
The main aim of this investigation was to attempt to explain the signal
transduction processes that mediate the FMRFamide-induced responses on the cropgizzard. My early results revealed that FMRFamide elicited the greatest response on the
longitudinal muscles of the crop-gizzard. Additionally, an application of 10-7 M
FMRFamide proved to elicit an average characteristic response (versus other tested
FMRFamide concentrations) in the longitudinal preparations. Consequently, 10-7 M
FMRFamide was selected as my control application with the crop-gizzard orientated to
record longitudinal muscle contractions in the amiloride sensitive sodium channel and
signal transduction experiments.
31
It is known that in the snail H. aspersa, FMRFamide can directly gate a Na+
channel that is blocked by amiloride (Cottrell 1997). Presence of FMRFamide-gated
sodium channels on the surface of the crop-gizzard could serve as a potent source for
muscle cell depolarization and ultimately be responsible for the observed FMRFamideinduced contractile activity. A decrease in the FMRFamide-induced response due to an
amiloride application would suggest the presence of amiloride-sensitive sodium channels.
An application of 10-4 M amiloride did not alter the response induced by an application of
10-7 M FMRFamide (Figure 11). Statistical analysis revealed that no significant
differences existed between contractile response measurements (basal tonus, peak
tension, or integrated area) between treatments (Figure 12). These results suggest that the
crop-gizzard lacks amiloride-sensitive sodium channels gated by FMRFamide.
Effects of Manipulating Second Messenger Pathways
on FMRFamide-Induced Responses
A collection of second messenger pathways was examined to determine their
possible role in the FMRFamide-induced responses of the crop-gizzard. Given the
apparent absence of FMRFamide-gated sodium channels, FMRFamide-induced effects
on the crop-gizzard are likely to be mediated by second messenger transduction systems.
Fifteen different drugs were used to manipulate specific steps within a number of second
messenger pathways. The ability of specific drugs to alter FMRFamide-induced
responses would indicate the role of specific transduction mechanisms in mediating cropgizzard responses.
32
2 mN
5 min
10-7 M FMRFamide
10-7 M FMRFamide
+
10-4 M Amiloride
10-7 M FMRFamide
Figure 11.
The effects of 10-4 M amiloride on the 10-7 M FMRFamide-induced contractile activity of a
single isolated crop-gizzard. The figure displays the contractile responses of a single crop-gizzard
to the three treatments. An arrowed line indicates when a specific treatment was added to the
organ chamber. A saline wash immediately followed each treatment. Parallel line breaks in this
and following figures represent segments of deleted data corresponding to extended saline wash
periods. All contractile responses were produced from a crop-gizzard orientated to record
longitudinal muscle contractions by the force transducer.
33
A
600
Integrated Area (mN · s)
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
12
Peak Tension (mN)
Peak Tension (mN)
Basal Tonus Increase (mN)
Basal Tonus Increase (mN)
2.00
C
500
400
300
200
100
0
FMRFamide
FMRFamide + Amiloride
FMRFamide
FMRFamide
FMRFamide + Amiloride
FMRFamide
FMRFamide
FMRFamide + Amiloride
FMRFamide
B
10
8
6
4
2
0
Figure 12.
Quantification of the effects of 10-4 M amiloride on the 10-7 M FMRFamide-induced
response. (A) Pretreatment of the crop-gizzard with 10-4 M amiloride (N = 11) had no significant
effect on maximal increase in basal tonus ( ! r2 = 1.19; p = 0.552; df = 2 [Friedman ANOVA on
ranks]). Basal tonus data were not normally distributed and consequently the data was displayed
using a box plot. The line in the center of the box represents the median, the lower and upper
limits of the box represent the 25th and 75th percentile respectively, and the whisker bars
represent the 10th and 90th percentiles. (B) Pretreatment of the crop-gizzard with 10-4 M
amiloride (N = 11) had no significant effect on peak tension ( ! r2 = 3.82; p = 0.1482; df = 2
[Friedman ANOVA on ranks]). (C) Pretreatment of the crop-gizzard with 10-4 M amiloride (N =
11) had no significant effect on integrated area (F = 2.68; p = 0.093; df = 2, 20 [One way repeatedmeasures ANOVA]). A vertical bar chart was used to display the normally distributed data, with
the column bars representing the treatment means and the whisker bars representing respective
standard errors.
34
Prior to manipulation experiments, the consistency of FMRFamide-induced
responses was determined in a set of eight control experiments. A crop-gizzard
preparation was exposed to three 10-7 M FMRFamide applications in intervals identical to
the second messenger pathway manipulation protocol. Recordings of basal tonus
increase (F = 2.34; p = 0.132; df = 2, 14 [One way repeated-measures ANOVA]), peak
tension (F = 2.53; p = 0.115; df = 2, 14 [One way repeated-measures ANOVA], and
integrated area (F = 2.34; p = 0.132; df = 2, 14 [One way repeated-measures ANOVA])
proved statistically indistinguishable between the three FMRFamide treatments.
Effects of Manipulating the Phosphatidylinositol Second Messenger Pathway
RFamide-activated signal pathways that utilize protein kinase C (PKC) have been
described to affect the duration and magnitude of musculature responses (Noronha et al.
1995; Friedrich et al. 1998; O’Gara et al. 1999a). H-7 is a nonselective protein kinase
inhibitor, but it has been reported to exhibit the greatest selectivity and potency toward
protein kinase C (Kawamoto and Hidaka 1984; Hidaka et al. 1984). Application of 5 ×
10-5 M H-7 reduced the response induced by the application of 10-7 M FMRFamide
(Figure 13). Statistical analysis revealed significant reductions in contractile response
measurements (basal tonus, peak tension, or integrated area) between treatments (Table
1). The effects of H-7 upon FMRFamide-induced responses were not reversible.
However, evidence of a possible crop-gizzard recovery following washout of H-7 is seen
35
2 mN
5 min
10-7 M FMRFamide
10-7 M FMRFamide
+
5 × 10-5 M H-7
10-7 M FMRFamide
Figure 13.
The inhibitory effects of 5 × 10-5 M H-7 on the 10-7 M FMRFamide-induced contractile
activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single
crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific
treatment was added to the organ chamber. A saline wash immediately followed each treatment.
4 mN
5 min
10-7 M FMRFamide
10-7 M FMRFamide
+
10-5 M BIM I
10-7 M FMRFamide
Figure 14.
The inhibitory effects of 10-5 M BIM I on the 10-7 M FMRFamide-induced contractile
activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single
crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific
treatment was added to the organ chamber. A saline wash immediately followed each treatment.
Table 1.
Target
Effects of phosphatdylinositol and arachidonic acid pathway manipulations on FMRFamide-induced contractions of the crop-gizzard. A
Dunnett’s multiple comparison test was utilized to compare the drug (FMRFamide + drug) and recovery treatments (FMRFamide) with the control
treatment (FMRFamide). Statistically significant treatments are represented in bold print. The concentrations of FMRFamide (10-7M) was kept
constant through all pathway manipulation experiments.
Drug
(M)
[n]
Phosphatidyl
inositol
pathway
PKC
H-7
PKA
(5×10-5 M)
[n = 8]
PKC
MLCK
Ca2+CaMK II
1.19 ± 0.26
Value of
statistic
0.50 ± 0.34 0.56 ± 0.32 F = 6.59
p = 0.01*
Peak tension (mN)
Control
Drug
3.56 ± 0.77
BIM I
(10-5 M)
[n = 10]
0.75
0.38
0.48
(0.71, 1.03) (0.24, 0.51) (0.26, 0.52)
ML-7
(10-5 M)
[n = 10]
0.97 ± 0.19
KN-62
(10-5 M)
[n = 7]
0.85 ± 0.11
0.67 ± 0.15 0.71 ± 0.18 F = 0.919
p = 0.425
4.76 ± 1.30
1.13 ± 0.15
0.66 ± 0.18 0.41 ± 0.09 F = 8.75
p = 0.03*
3.81 ± 0.72
Arachidonic
acid
pathway
PLPA 2
4-BPB
(10-6 M)
[n = 8]
PLPA 2
Increase in basal tonus (mN)
Control
Drug
Recovery
OBAA
(10-7 M)
[n = 7]
! r2
=
14.0
Recovery
1.90 ± 0.29 1.91 ± 0.28 F = 4.95
p = 0.024*
3.59
1.50
1.44
(2.16, 4.55) (0.89, 2.06) (0.77, 2.40)
p = 0.001*
0.92 ± 0.19 0.70 ± 0.15 F = 1.87
p = 0.183
Value of
statistic
! r2
=
! r2
=
12.8
Integrated area (mN·s)
Control
Drug
Recovery
Value of
statistic
400.0 ± 93.2
195.5 ± 39.0
F = 3.78
p = 0.048*
221.3
(134.6, 338.3)
! r2
220.0 ± 60.0
280.6
160.4
(210.2, 683.1) (108.0, 248.4)
p = 0.002*
2.58
2.07
1.23
(1.44, 3.83) (1.43, 4.06) (1.00, 1.67)
=
10.4
p = 0.006*
425.6 ± 105.8 387.9 ± 103.9
267.4 ± 103.9
F = 1.76
p = 0.20
1.84 ± 0.32 2.54 ± 0.64 F = 4.01
p = 0.046*
517.1 ± 98.0
329.8 ± 59.2
F = 7.81
p = 0.0067*
1.70 ± 0.47 0.81 ± 0.12 F = 19.4
p = 0.0001*
291.2
293.6
(231.8, 473.7) (132.1, 347.7)
133.0
(70.2, 279.1)
! r2
8.17
p = 0.017*
284.3 ± 68.8
=
7.75
p = 0.02*
1.29
0.77
0.48
(1.02, 2.24) (0.47, 1.80) (0.32, 0.52)
! r2
=
4.08
p = 0.13
4.21± 0.91
2.33 ± 0.61 1.88 ± 0.50 F = 4.78
p = 0.03*
504.6 ± 100.0 333.8 ± 82.7
309.5 ± 96.6
F = 3.26
p = 0.074
* Statistically significant (p < 0.05)
36
37
with the slight recovery in the amplitude of the increase in basal tonus measurement
(Figure 13, Table 1).
BIM I, an additional potent and selective PKC inhibitor (Toullec et al. 1991), was
applied to the crop-gizzard to determine the importance of the phosphatidylinositol
pathway in the FMRFamide-induced response. Application of 10-5 M BIM I reduced the
response induced by the application of 10-7 M FMRFamide (Figure 14). Statistical
analysis revealed significant reductions in contractile response measurements (basal
tonus, peak tension, or integrated area) between treatments (Table 1). The crop-gizzard
demonstrated a more pronounced recovery from the BIM I application (versus H-7) as
evident with the increase in FMRFamide-induced basal tonus and integrated area
following drug washout.
Myosin light chain kinase (MLCK), a calcium-calmodulin-dependent enzyme that
is activated via the phosphatidylinositol signal pathway, is known to regulate contractile
activity in smooth muscle (Rasmussen et al. 1987). ML-7, a selective inhibitor of myosin
light chain kinase, has been reported to reduce smooth muscle phasic contractions (Saitoh
et al. 1987; Morano 2003). However, the application of 10-5 M ML-7 failed to produce a
significant change in the FMRFamide-induced responses of the crop-gizzard (Table 1).
Despite the significant decrease reported in peak tension for the recovery treatment, a
multiple comparison test revealed that the FMRFamide-induced response during the drug
treatment was statistically similar to the control FMRFamide treatment.
An additional calcium-calmodulin kinase, termed calcium-calmodulin kinase II
(Ca2+-CaMK II), is known to interact with myosin light chain kinase and the SK ion
38
5 mN
5 min
10-7 M FMRFamide
10-7 M FMRFamide
+
10-5 M KN-62
10-7 M FMRFamide
Figure 15.
The inhibitory effects of 10-5 M KN-62 on the 10-7 M FMRFamide-induced contractile
activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single
crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific
treatment was added to the organ chamber. A saline wash immediately followed each treatment.
2 mN
5 min
10-7 M FMRFamide
10-7 M FMRFamide
+
10-6 M 4-BPB
10-7 M FMRFamide
Figure 16.
The inhibitory effects of 10-6 M 4-BPB on the 10-7 M FMRFamide-induced contractile
activity of a single isolated crop-gizzard. The figure displays the contractile responses of a single
crop-gizzard to the three treatments (dashed lines). An arrowed line indicates when a specific
treatment was added to the organ chamber. A saline wash immediately followed each treatment.
39
channel, events that can initiate muscle cell contraction (Ikebe and Reardon 1990; Tansey
et al. 1992; Kong et al. 2000). KN-62 is a selective calcium-calmodulin kinase II
inhibitor that has been suggested to influence smooth muscle contractions (Tokumitsu et
al. 1990). Application of 10-5 M KN-62 reduced the response induced by the application
of 10-7 M FMRFamide (Figure 15). Statistical analysis revealed significant reductions in
contractile response measurements (peak tension and integrated area) between treatments
(Table 1). However, there was no significant change in the increase in basal tonus
response. The effects of KN-62 on peak tension were reversible.
Effects of Manipulating the Arachidonic Acid Second Messenger Pathway
Arachidonic acid production has been linked to a variety of cellular events that
are involved in muscle cell contraction (Gong et al. 1992; Alcorn et al. 2002; Kiss 2005).
Phospholipase A2 (PLPA2), a necessary enzyme involved in the arachidonic acid pathway
is potently inhibited by 4-BPB (Synder et al. 1992). Inhibition of PLPA2 prevents
production of arachidonic acid within a cell. Application of 10-6 M 4-BPB reduced the
response induced by the application of 10-7 M FMRFamide (Figure 16). Statistical
analysis revealed significant reductions in all contractile response measurements (basal
tonus, peak tension, and integrated area) between treatments (Table 1). The inhibitory
effects of 4-BPB upon FMRFamide-induced responses were not reversible.
Another drug shown to inhibit PLPA2 is OBAA (Kohler 1991). However, OBAA
only partially altered the FMRFamide-induced responses in the crop-gizzard.
Application of 10-7 M OBAA produced a significant reduction in the 10-7 M
40
FMRFamide-induced peak tension response, but failed to alter the FMRF-induced
increase in basal tonus and integrated area (Table 1).
Effects of Manipulating the cAMP Second Messenger Pathway
A variety of steps in the cAMP signal transduction pathway were manipulated to
determine if this signaling system was involved in the FMRFamide responses of the cropgizzard. H-89 is a selective and potent inhibitor of cAMP-dependent protein kinase A, an
enzyme that has been reported to be involved in smooth muscle contractile responses
(Geilen 1992; Makhlouf and Murthy 1997). However, application of 10-6 M H-89 failed
to alter the FMRFamide-induced increase in basal tonus, peak tension, and integrated
area (Table 2).
MDL-12,330A is an adenylyl cyclase inhibitor and prevents the production of the
second messenger cAMP (Lippe and Ardizzone 1991). However, application of 10-5 M
MDL-12,330A did not produce significant changes in the FMRFamide-induced responses
(Table 2).
In a final investigation into the cAMP signal transduction pathway, a membrane
permeable cAMP analog, 8-Br-cAMP, was applied to the crop-gizzard and the recorded
responses were compared to the prior or subsequent responses induced by FMRFamide.
If FMRFamide-induced responses are dependent upon this system, then both 8-Br-cAMP
and FMRFamide application should produce some similarity in their respective induced
responses. Application of 10-5 M 8-Br-cAMP failed to produce a distinct contractile
response on the crop-gizzard despite a small increase in basal tonus (Figure 17; Table 2).
Table 2.
Target
Effects of cAMP and nitric oxide-induced cGMP pathway manipulations on FMRFamide-induced contractions of the crop-gizzard. A
Dunnett’s multiple comparison test was utilized to compare the drug (FMRFamide + drug) and recovery treatments (FMRFamide) with the control
treatment (FMRFamide). Donor and analog experiments required a paired t-test to compare mean value responses between control (FMRFamide)
and drug (analog or donor) treatments. Recovery treatments were not applied in donor and analog experiments (gray-shaded boxes). Statistically
significant treatments are represented in bold print. The concentrations of FMRFamide (10-7 M) was kept constant through all pathway
manipulation experiments.
Drug
(M)
[n]
Increase in basal tonus (mN)
Control
Drug
Recovery
H-89
(10-6 M)
[n = 8]
0.90 ± 0.20
adenylyl
cyclase
MDL12,330A
(10-5 M)
[n = 8]
1.27
1.36
1.11
(0.62, 1.94) (0.86, 2.72) (0.84, 1.42)
cAMP
analog
8-Br-cAMP
(10-5 M)
[n = 10]
1.05 ± 0.27
0.65 ± 0.17
ODQ
(10-6 M)
[n = 8]
0.89 ± 0.16
0.67 ± 0.11 0.55 ± 0.13 F = 2.72
p = 0.10
NO donor
SNAP
(10-5 M)
[n = 7]
1.12 ± 0.12
cGMP
analog
8-Br-cGMP
(10-5 M)
[n = 6]
0.87
0.75
(0.77, 1.55) (0.24, 1.81)
cAMP
pathway
PKA
NO-cGMP
pathway
guanylyl
cyclase
Value of
statistic
0.71 ± 0.16 0.63 ± 0.13 F = 2.13
p = 0.16
Peak tension (mN)
Control
Drug
Recovery
2.04
1.89
1.04
(1.61, 4.52) (0.95, 4.52) (0.60, 3.62)
Value of
statistic
! r2
=
! r2
=
3.68
Integrated area (mN·s)
Control
Drug
Recovery
Value of
statistic
364.1 ± 83.1
254.1 ± 58.7
F = 2.79
p = 0.10
521.5
(316.2, 1089)
! r2
318.6 ± 60.3
p = 0.16
! r2
=
3.47
p = 0.18
t = 2.96
p = 0.016*
3.10
3.11
3.85
(2.46, 7.81) (2.07, 7.14) (1.87, 7.54)
5.25
p = 0.072
428.4
692.2
(326.5, 856.5) (382.4, 1321)
2.20
2.33
(0.96, 6.97) (1.19, 3.11)
W = -15.0
p = 0.49
501.2 ± 142.7 355.2 ± 94.2
2.27
1.31
1.53
(1.29, 4.33) (0.91, 2.39) (0.65, 3.11)
! r2
464.4
278.8
(197.1, 498.0) (169.6, 336.0)
=
3.38
p = 0.18
0.48 ± 0.13
=
4.75
p = 0.09
t = 2.41
p = 0.039*
196.0
(61.5, 334.7)
! r2
=
5.25
p = 0.072
t = 3.32
p = 0.016*
5.72 ± 1.77
3.89 ± 1.24
t = 1.47
p = 0.19
817.1 ± 279.7 551.4 ± 304.4
t = 2.03
p = 0.039*
W = 1.0
p = 1.0
4.60 ± 1.30
5.04 ± 1.86
t = -0.245
p = 0.82
487.6 ± 128.9 829.3 ± 350.0
t = -0.81
p = 0.46
*Statistically significant (p < 0.05)
41
42
4 mN
5 min
10-5 M 8-Br-cAMP
10-7 M FMRFamide
Figure 17.
The different effects of 10-5 M 8-Br-cAMP and 10-7 M FMRFamide on a single isolated
crop-gizzard. The figure displays the contractile responses of a single crop-gizzard to the two
treatments (dashed lines). Treatment applications were varied in order throughout the sample set.
2 mN
5 min
10-7 M FMRFamide
10-5 M SNAP
Figure 18.
The different effects of 10-5 M SNAP and 10-7 M FMRFamide on a single isolated cropgizzard. The figure displays the contractile responses of a single crop-gizzard to the two
treatments (dashed lines). Treatment applications were varied in order throughout the sample set.
43
There were no observable differences in contractile responses (mean contraction
amplitude, peak tension, and contraction rate) between the 8-Br-cAMP treatment and the
normal spontaneous activity just prior to drug application. However, statistical revealed
significant differences in the increase in basal tonus and integrated area between the
FMRFamide and 8-Br-cAMP applications (Table 2). The peak tension responses were
similar between the FMRFamide and 8-Br-cAMP treatment.
Effects of Manipulating the NO-Induced cGMP Second Messenger Pathway
The nitric oxide-induced cGMP signal transduction pathway was explored to
determine if the FMRFamide-induced responses were mediated through this system.
This specific pathway is known to mediate relaxation events in smooth muscle (Makhlouf
and Murthy 1997). Manipulation of cGMP levels prior, during, or subsequent to
FMRFamide applications would reveal the role of the cGMP in the FMRFamide-induced
responses of the crop-gizzard. ODQ is a selective inhibitor of nitric oxide-sensitive
guanylyl cyclase, an enzyme necessary to produce cGMP (Garthwaitel et al. 1995).
Application of 10-6 M ODQ failed to produce significant changes in FMRFamideinduced increase in basal tonus, peak tension, or integrated area (Table 2).
If the crop-gizzard possesses a NO- induced cGMP transduction system coupled
to the muscle contraction mechanism, then application of the nitric oxide donor SNAP
would be expected to alter contractile activity of the organ. If FMRFamide-induced
responses are dependent upon this system, then both SNAP and FMRFamide application
should produce some similarity in their respective induced responses. Application of 10-5
44
M SNAP failed to produce a distinct contractile response on the crop-gizzard despite a
small increase in basal tonus (Figure 18; Table 2). There were no observable differences
in contractile responses (mean contraction amplitude, peak tension, and contraction rate)
between the SNAP treatment and the normal spontaneous activity just prior to drug
application. However, statistical analysis revealed significant differences in contractile
response parameters (basal tonus and integrated area) between the FMRFamide and
SNAP applications (Table 2). The peak tension responses were similar between the
FMRFamide and SNAP treatments.
In a final investigation into the nitric oxide-induced cGMP signal transduction
pathway, a cGMP analog, 8-Br-cGMP, was applied to the crop-gizzard in attempt to
elevate cGMP levels inside the muscle cells. If FMRFamide-induced responses are
dependent upon this system, then both 8-Br-cGMP and FMRFamide application should
produce some similarity in their respective induced responses. Application of 10-5 M
8-Br-cGMP failed to induce a distinct contractile response upon the crop-gizzard despite
a small increase in basal tonus (Table 2). There were no observable differences in
contractile responses (mean contraction amplitude, peak tension, and contraction rate)
between the 8-Br-cGMP treatment and the normal spontaneous activity just prior to drug
application. Additionally, application of 8-Br-cGMP produced statistically similar
changes in the increase in basal tonus, peak tension, and integrated area when compared
to the FMRFamide application (Table 2).
45
Manipulations of Second Messenger Pathways
Alter Spontaneous Activity of Crop-Gizzard
Certain drug applications alone produced distinct responses on the crop-gizzard.
Pretreatment of the crop-gizzard with particular drugs prior to FMRFamide-drug
treatments produced marked changes in basal tonus and peak tension. The direct effects
of these specific drugs demonstrate that the second messenger pathways targeted by each
drug are involved in regulating the spontaneous contractile activity of the crop-gizzard.
Additionally, in some experiments the combined FMRFamide-drug application produced
distinct responses on the crop-gizzard that differed from normal spontaneous activity or
responses induced by peptide and drug applications alone. However, these discoveries
provide little evidence to which transduction pathways are used during FMRFamideinduced responses.
W-7 is a compound that is known to act as a calmodulin antagonist, calciumcalmodulin phosphodiesterase inhibitor (Ca2+-CaMP), and myosin light chain kinase
inhibitor (Asano 1989). Application of 10-4 M W-7 demonstrated a marked increase in
basal tonus and peak tension when compared to normal crop-gizzard spontaneous activity
(Figure 19). Statistical analysis revealed that the increase in basal tonus in the W-7
treatment was significantly larger than the FMRFamide-induced increase in basal tonus
(Table 3). However, the combined FMRFamide-drug treatment failed to generate a
marked increase in basal tonus (Figure 19, Table 3). The peak tension responses in the
FMRFamide and W-7 treatments were statistically indistinguishable, but in the combined
FMRFamide-W-7 treatment the peak tension was significantly reduced. Additionally, the
46
3 mN
5 min
10-7 M FMRFamide
10-4 M W-7
10-7 M FMRFamide
+
10-4 M W-7
Figure 19.
The direct effect of 10-4 M W-7 on a single isolated crop-gizzard. The figure displays the
contractile responses of a single crop-gizzard to three distinct treatments (dashed lines). In this
and following figures, contractions were continuous between the drug and FMRFamide-drug
treatments.
5 mN
5 min
10-7 M FMRFamide
18 x 10-6 M U-73122
10-7 M FMRFamide
+
18 x 10-6 M U-73122
Figure 20.
The direct effect of 18 x 10-6 M U-7122 on a single isolated crop-gizzard. The figure
displays the contractile responses of a single crop-gizzard to three distinct treatments (dashed
lines).
Table 3.
Target
Direct effects of drugs on the phosphatidylinositol, arachidonic acid, and mitogen-activated protein kinase second messenger pathways in the
crop-gizzard. A Dunnett’s or Student-Newman-Keuls multiple comparison test was utilized to compare the drug and combined FMRFamide + drug
treatments to the control FMRFamide treatment. Statistically significant groups are represented in bold print. Integrated area analysis was limited
to only the FMRFamide and FMRFamide + drug treatments and required a paired t-test to compare the mean value responses. The concentration of
FMRFamide (10-7 M) was kept constant through all pathway manipulation experiments.
Drug
(M)
[n]
Phosphatidyl
inositol
pathway
CaM
W-7
Ca2+-CaMP (10-4 M)
MLCK
[n = 12]
Phosphatidyl
inositol &
Arachidonic
acid
pathways
PLPC
U-73122
PLPA 2
(18 x 10-6 M)
[n = 8]
MAP kinase
pathway
tyrosine
kinase
Genistein
(5 x 10-5 M)
[n = 12]
Increase in basal tonus (mN)
FMRFamide Drug
FMRFamide + Value of
Drug
statistic
Peak tension (mN)
FMRFamide
Drug
0.75
(0.37, 0.96)
1.24
0.26
(0.75, 1.80) (0.24, 0.62)
3.12
(2.24, 4.00)
2.74
1.38
(2.61, 4.10) (0.72, 2.97)
1.86 ± 0.24
2.09 ± 0.19
0.72 ± 0.13
F = 22.0
p = 0.0001*
7.03 ± 1.34
7.36 ± 1.34
1.35 ± 0.25
2.01 ± 0.42
1.39 ± 0.29
F = 3.57
p = 0.045*
3.97
(2.99, 9.00)
10.59
9.10
(4.97, 16.2) (5.71, 16.1)
! r2
=
10.7
p = 0.005*
Integrated area (mN·s)
FMRFamide
FMRFamide +
Drug
Value of
statistic
362.8
(218.0, 402.7)
173.1
(135.0, 308.4)
W = -28.0
p = 0.301
F = 1.88
p = 0.189
1192.7 ± 201.9
760.7 ± 129.9
t = 1.92
p = 0.097
! r2
824.7 ± 199.7
1370.0 ± 286.2
t = -1.90
p = 0.084
FMRFamide + Value of
Drug
statistic
5.31 ± 1.26
! r2
=
10.2
p = 0.006*
=
7.61
p = 0.022*
* Statistically significant (p < 0.05)
47
48
integrated area responses in the FMRFamide and FMRFamide-W-7 treatments were
statistically indistinguishable.
U-73122 is a potent phospholipase C inhibitor and selective PLPA2 inhibitor
(Yule and Williams 1992; Bleasdale et al. 1990). Application of 18 x 10-6 M U73122produced a complex response on the crop-gizzard that demonstrated similarities
and differences to the FMRFamide-induced response (Figure 20). The U-73122
application produced a marked increase in basal tonus and peak tension when compared
to normal crop-gizzard spontaneous activity. Statistical analysis revealed that the
increase in basal tonus between FMRFamide and U-73122 treatments were similar, but in
the combined FMRFamide-U-73122 the increase in basal tonus was significantly reduced
(Table 3). Additionally, peak tension and integrated area responses were statistically
indistinguishable between all measured treatments.
Genistein is a specific inhibitor of tyrosine kinase, an enzyme involved in the
mitogen-activated protein kinase pathway, and has been shown to inhibit the contraction
of several smooth muscle types (Akiyam et al. 1987; Wigetunge et al. 1992; Chopra et al.
1997; Palmier et al. 1999). Application of 5 x 10-5 M genistein alone or combined with
10-7 M FMRFamide induced distinct contractile responses upon the crop-gizzard (Figure
21). The genistein application produced a marked increase in basal tonus and peak
tension when compared to normal crop-gizzard spontaneous activity. Statistical analysis
revealed that the genistein-induced increase in basal tonus and peak tension were
significantly larger than the FMRFamide-induced responses (Table 3). Additionally, the
combined FMRFamide-genistein treatment demonstrated a similar increase in peak
49
10 mN
5 min
10-7 M FMRFamide
5 x 10-5 M Genistein
10-7 M FMRFamide
+
5 x 10-5 M Genistein
Figure 21.
The direct effect of 5 x 10-5 M genistein on a single isolated crop-gizzard. The figure
displays the contractile responses of a single crop-gizzard to three distinct treatments (dashed
lines).
50
tension to that seen in genistein application alone. However, the increase in basal tonus
and integrated area responses in the FMRFamide and FMRFamide-genistein treatments
were statistically indistinguishable.
DISCUSSION
The results of the experiments presented in this thesis confirm the work of
Krajniak and Khlor (1999) and indicate that the motility of the Lumbricus terristris cropgizzard is, in part, regulated by FMRFamide or other members of the RFamide family.
FMRFamide application induced contractions of the outer longitudinal and inner circular
muscles of the crop-gizzard. In addition, pharmacological manipulation of signal
transduction pathways demonstrated that the FMRFamide-induced and spontaneous
longitudinal contractions of the crop-gizzard appear to be mediated through calmodulin
and the phosphatidylinositol, arachidonic acid, and mitogen-activated second messenger
pathways.
Crop-gizzard Responses to FMRFamide
The results in this investigation create a more complete understanding of the role
of FMRFamide in crop-gizzard motility. In addition to replicating previous findings on
the FMRFamide-induced responses on the longitudinal muscles (Krajniak and Khlor
1999), my experiments are the first to document the FMRFamide-induced responses of
the circular muscles of an isolated crop-gizzard. Moreover, I measured five distinct
contractile parameters from each peptide-induced response to produce the most
comprehensive report of FMRFamide-induced activity on the L. terrestris gut to date.
51
52
FMRFamide-Induced Longitudinal Contractions
Previous experiments have effectively reported that FMRFamide induces a dosedependent reduction in contractile responses of the longitudinal muscles of the
earthworm crop-gizzard (Ukena et al. 1995a; Krajniak and Khlor 1999). In the present
study, all FMRFamide treatments elicited an excitatory effect on the crop-gizzard
(demonstrated by an increase in basal tonus after each peptide application) with the
amplitude of most contractile responses decreasing as FMRFamide concentrations
increased. My experiments demonstrated that FMRFamide application induced a
concentration-dependent decrease in contraction amplitude and a biphasic effect on
contraction rate, results that were also reported by Krajniak and Klor (1999). However,
small discrepancies existed between my findings and those by Krajniak and Khlor. The
threshold for the decrease in contraction amplitude appeared between 10-8 – 10-7 M
FMRFamide in my experiments versus the reported threshold of 10-9 – 10-8 M
FMRFamide by Krajniak and Khlor. Additionally, my experiments reported an increase
in contraction rate from 10-9 – 10-7 M FMRFamide followed by a decrease in contraction
rate from 10-7 – 10-5 M FMRFamide, whereas the work by Krajniak and Khlor revealed
an increase in contraction rate from 10-9 – 10-6 M FMRFamide and a decrease in
contraction rate from 10-6 – 10-5 M FMRFamide. These differences in threshold
concentrations between the different studies could be attributed to the number of
experiments performed (my investigation utilized 10 crop-gizzard preparations versus 5
53
used by Krajniak and Klor), procedural techniques, or equipment; however, the trends
reported in the two studies are qualitatively similar.
The concentration-dependent FMRFamide-induced decrease in contractile
responses is further demonstrated in the three new response parameters used in this
investigation. The maximal increase in basal tonus, peak tension, and integrated area all
demonstrated similar concentration-response relationships as each measurement
decreased with increasing FMRFamide concentrations. The addition of these three newly
reported parameters adds greater support to the conclusion drawn by Krajniak and Khlor
(1999) that FMRFamide largely induces concentration-dependent reduction in contractile
activity in the longitudinal muscles of the L. terrestris crop-gizzard.
FMRFamide-Induced Circular Contractions
Manipulation of the crop-gizzard orientation in relation to the force transducer
enabled spontaneous circular muscle contractions to be recorded over an extended period
of time. Direct recordings of FMRFamide-induced circular muscle contractions from an
isolated L. terrestris crop-gizzard have not been recorded until this investigation. The
application of increasing FMRFamide concentrations on this preparation produced a
different concentration-response relationship versus the longitudinal preparation. The
magnitude of the circular muscle responses was far smaller than the longitudinal muscle
responses, suggesting that longitudinal muscle activity dominates the FMRFamideinduced crop-gizzard response. In addition, high FMRFamide concentrations (10-7 – 10-6
M) elicited a concentration-dependent increase in each of the measured parameters,
54
whereas the FMRFamide-induced longitudinal responses decreased with increasing
concentrations. However, low FMRFamide concentrations (10-9 – 10-8 M) appeared to
parallel the concentration-dependent decrease demonstrated in the longitudinal muscle
recordings.
The concentration-dependent biphasic effect on the circular muscles may suggest
the presence of multiple FMRFamide receptor subtypes on the crop-gizzard. Specific
receptor subtypes may show greater affinity to different FMRFamide concentrations and
ultimately induce different contractile responses. FMRFamide and FLRFamide were
reported to elicit biphasic responses in F2 neurones of Helix aspersa at high and low
concentrations (Chen et al. 1995). Additional RFamide related neuropeptides have been
reported to act on multiple receptor subtypes in H. aspersa central neurons and elicit
distinct responses (Cottrel and Davies 1987; Chen et al. 1995). Several experiments have
isolated and sequenced annelid RFamide related peptides including FLRFamide,
FTRFamide, YMRFamide, YLRFamide, GGKYMRFamide, and GDPFLRFamide
(Krajniak and Price 1990; Barratte et al. 1990; Evans 1991; Salzet 1994). Thus, it
appears likely that the L. terrestris possesses several RFamide neuropeptides and that
multiple RFamide receptor subtypes may exist that have the potential to bind
preferentially to FMRFamide or other members of the RFamide family.
The Absence of Amiloride Sensitive FMRFamide-gated Sodium Channels
One hypothesis was that FMRFamide directly gates an amiloride-sensitive
sodium ion channel on the longitudinal muscle cell membrane inducing a fast influx of
55
sodium ions that ultimately triggers cell contraction. This specific sodium channel is the
only documented peptide-gated ion channel (Cottrel 1997). In a recent investigation of
the leech pharynx, amiloride failed to block the FMRFamide-induced contractile
responses (O’Gara et al. 1999a). Similarly, my results demonstrated that amiloridesensitive sodium channels were absent in the L. terrestris crop-gizzard and with no
reports of new peptide-gated ion channels it appears unlikely that ligand-gated ion
channels mediate the FMRFamide-induced contractile responses in the earthworm gut. It
is worth noting that a low sequence similarity was reported between the amiloride
sensitive FMRFamide-gated sodium channel and epithelial sodium channels and
degenerins, suggesting that peptides may indeed activate other ion channels in other
systems (Lingueglia et al. 1995).
Effects of Manipulating Second Messenger Pathways
on FMRFamide-induced Responses
The bulk of this study concentrated on an investigation into the possible role of
specific second messenger pathways in mediating the FMRFamide-induced responses of
the earthworm crop-gizzard. Fifteen drugs were applied to the crop-gizzard in attempt to
manipulate known signal transduction components that might mediate the FMRFamideinduced responses. To my knowledge, this is the first investigation into the FMRFamideinduced activation of second messenger pathways in oligochaetes. The results of this
study suggest that the actions of FMRFamide on the crop-gizzard are mediated through
the activation of protein kinase C, calcium-calmodulin kinase II, and arachidonic acid.
56
Furthermore the normal crop-gizzard spontaneous activity appears to be regulated by the
activation of tyrosine kinase, calmodulin protein, and myosin light chain kinase.
Effects of Manipulating the Phosphatidylinositol Second Messenger Pathway
My investigation revealed that the phosphatidylinositol second messenger
pathway appeared to be a likely mediator of the FMRFamide-induced contractile
responses in the crop-gizzard. The ligand-activated phosphatidylinositol pathway
proceeds via stimulation of phospholipase C by a specific G protein, allowing the
activated enzyme to hydrolyze phosphatidylinositol into the second messengers,
diacylglycerol (DAG) and inositol triphosphate (IP3). DAG is the major physiological
activator of protein kinase C whereas IP3 mobilizes intracellular calcium that in turn can
activate calmodulin-dependent kinases. The activation of protein kinase C or calciumcalmodulin kinases can lead to several cellular events including muscle cell contraction.
H-7 and BIM I, two different protein kinase C inhibitors, were applied to the
crop-gizzard to determine if protein kinase C is involved in the FMRFamide-induced
responses. Both drugs irreversibly inhibited the FMRFamide-induced responses by a
similar magnitude suggesting that protein kinase C is a likely mediator in the responses.
Active protein kinase C is known to induce tonic contraction of smooth muscle through
myosin light chain phosphorylation and inhibition of myosin light chain phosphatase
(Vorotnikov et al. 2002; Rasmussen el al. 1987; Masuo et al. 1994). In experiments
involving H-7 and BIM I, O’Gara et al. (1999a) concluded that the actions of
FMRFamide on the contractile activity of leech pharynx were at least partially mediated
57
via protein kinase C. In a signal transduction investigation involving spontaneously
active muscle strips from liver flukes, Graham et al. (2000) reported that a FMRFamiderelated neuropeptide stimulated mechanical activity through a protein kinase C-dependent
pathway. Thus, it appears likely that FMRFamide induces contractile activity upon the
earthworm crop-gizzard via the activation of protein kinase C.
Additionally, I examined the role of calcium-calmodulin kinases in mediating the
FMRFamide-induced contractile responses. It is well known that the activation of a
dedicated calcium-calmodulin-dependent kinase, termed myosin light chain kinase,
phosphorylates myosin light chain and activates the contractile myosin adenosine
triphosphatase (ATPase), events necessary for gastrointestinal smooth muscle contraction
in vertebrate systems (Makhlouf and Murthy 1997; Vorotniknov et al. 2002). Smooth
muscle myosin light chain kinases are also found in invertebrates and have been reported
to be activated by similar calcium-dependent events (Gallagher et al. 1997). However, in
my experiments the application of ML-7, a drug designed to inhibit myosin light chain
kinase, failed to alter the FMRFamide-induced contractile responses on the crop-gizzard.
Although it seems unlikely that myosin light chain kinase is absent in mediating
contractile events in the crop-gizzard, ML-7 proved ineffective in reducing the
FMRFamide-induced contractile response parameters. Perhaps ML-7 failed to
effectively select the myosin light chain kinase in the crop-gizzard, suggesting that ML-9
or other novel myosin light chain kinase inhibitors might demonstrate better selection and
effectiveness. Saitoh et al. (1987) reported that ML-9 and synthesized ML-9 derivatives
inhibited myosin light chain kinase in smooth muscle preparations.
58
KN-62 is a drug designed to inhibit the multifunctional calcium-calmodulindependent kinase II, an enzyme known to phosphorylate and regulate multiple cellular
targets including myosin light chain kinase in smooth muscle cells (Ikebe and Reardon
1990; Tansey et al. 1992). This study revealed that application of KN-62 significantly
decreased the FMRFamide-induced contractile responses of the crop-gizzard, suggesting
that this specific calcium-calmodulin kinase is involved in the FMRFamide-induced
contractile responses of the crop-gizzard and might serve to activate the myosin light
chain kinase. Despite the reported link of calcium-calmodulin-dependent kinase II to
myosin light chain kinase, the calcium-calmodulin enzyme has also been shown to affect
gastrointestinal cell excitability via the SK channel, a calcium-activated potassium
channel (Xia et al. 1998; Kong et al. 2000). The opening of the SK channel limits the
possibility of repetitive cell depolarizations, an event that is necessary in regulating
action potential frequency. Kong et al. (2000) demonstrated that KN-93, an additional
calcium-calmodulin-dependent kinase II inhibitor, decreased the open probability of the
SK channels in colonic myocytes. If SK channels are present in the crop-gizzard,
channel inhibition would increase the excitability of muscle cells and possibly induce
contractile activity. However, application of KN-62 significantly reduced all
FMRFamide-induced contractile parameters in the crop-gizzard, suggesting that SK
channels were not involved in the contractile responses. Thus, it appears that calciumcalmodulin kinase II activation of myosin light chain kinase is involved in the
FMRFamide-induced contractile responses of the crop-gizzard.
59
Effects of Manipulating the Arachidonic Acid Second Messenger Pathway
The ligand-activated arachidonic acid pathway proceeds via stimulation of
phospholipase A2 by a specific G protein, allowing the activated enzyme to hydrolyze
phospholipids into arachidonic acid and additional lipid products. Although the
arachidonic acid second messenger pathway is best known to produce prostaglandin
molecules inside cells, recent evidence reports that arachidonic acid can activate several
cellular targets including protein kinase C (Khan et al. 1995). In investigating the
arachidonic acid second messenger pathway, I found that inhibiting phospholipase A2
with 4-BPB significantly attenuated the FMRFamide-induced responses of the cropgizzard. Additionally, the crop-gizzard failed to recovery from the 4-BPB dose,
supporting the potent irreversibility of the drug (Kits et al. 1997; Kiss 2005). My
evidence suggests that arachidonic acid plays a role in mediating the FMRFamideinduced responses, possibly through the stimulation of protein kinase C, cell
depolarization, or generation of arachidonic acid metabolites that promote contraction. In
a vertebrate smooth muscle preparation, arachidonic acid was reported to increase basal
tonus levels and myosin light chain phosphorylation, and inhibit the dephosphorylation of
myosin light chain (Gong et al. 1992). Additionally, an arachidonic acid metabolite was
shown to activate a G protein-gated potassium channel in cardiac myocytes, an event that
contributes to contraction deceleration in the heart (Kurachi et al. 1989). Lipoxygenase
metabolites of arachidonic acid were also shown to mediate FMRFamide-induced SK
channel activity in Aplysia sensory neurons (Buttner et al. 1989). However, as previously
mentioned in this report, SK channels are unlikely to exist on the crop-gizzard. In an
60
experiment involving vertebrate smooth muscle strips and cholecystokinin (CCK), an
evolutionarily related peptide to FMRFamide, the contractile effects induced by the
peptide were inhibited by 4-BPB, suggesting that arachidonic acid is a mediator in the
peptide-induced responses (Alcon et al. 2002). Furthermore, Kiss (2005) recently
revealed that the activation of neuronal potassium channels by Mytilus inhibitory peptide,
a neuropeptide found in molluscs, was irreversibly inhibited by 4-BPB, demonstrating
that arachidonic acid is involved in mediating cellular hyperpolarization. The role of
arachidonic acid as an intracellular signal is linked to a variety of cellular responses, and
with this report, arachidonic acid appears to be involved in the FMRFamide-induced
contractile responses of the crop-gizzard.
Effects of Manipulating the cAMP Second Messenger Pathway
FMRFamide or related RFamide neuropeptides have been shown to induce
cAMP-dependent responses in a variety of invertebrate tissues (Higgins et al. 1978; Trim
et al. 1998; Reinitz et al. 2000). However, there was little evidence suggesting that the
cAMP second messenger pathway was involved in the FMRFamide-induced responses in
the crop-gizzard. H-89 and MDL-12,330A, drugs designed to inhibit protein kinase A
and adenylyl cyclase respectively, failed to significantly alter any of the FMRFamideinduced responses. A similar conclusion was reported in a cAMP second messenger
pathway investigation of the FMRFamide-induced contractile responses on the leech
pharynx (O’Gara et al. 1999a). Additionally, application of a membrane-permeable
cAMP analog, 8-Br-cAMP, on the crop-gizzard produced responses that were distinctly
61
different in appearance compared to FMRFamide-induced responses. However, similar
peak tension responses were observed in the 8-Br-cAMP and FMRFamide applications.
Thus, although elevated levels of cAMP inside crop-gizzard muscle cells induce
contractile activity, it is unlikely to be responsible for the FMRFamide-induced activity.
Similarly, spontaneous contractions of flatworm muscle are increased by 8-Br-cAMP, but
contractions induced by another RFamide peptide could not be linked to the cAMP
system (Graham et al. 2000). Additionally, Occor et al. (1985) revealed that FMRFamide
did not affect cAMP levels in snail sensory neurons and Flamm et al. (1987) reported the
same conclusion in lobster pyloric cells. Despite evidence that RFamides work through
the cAMP second messenger pathway in some preparations, the FMRFamide-induced
response of the earthworm crop-gizzard is unlikely to be mediated through the cAMP
second messenger pathway.
Effects of Manipulating the NO-induced cGMP Second Messenger Pathway
FMRFamide did not appear to activate the nitric oxide-induced cGMP second
messenger pathway or elevate cGMP levels in the crop-gizzard responses. The
application of ODQ, SNAP, and 8-Br-cGMP, drugs designed to inhibit guanylyl cyclase,
release nitric oxide, and increase cGMP levels respectively, failed to alter or mimic
FMRFamide-induced responses. Despite the similar contractile responses elicited in the
experiment involving FMRFamide and 8-Br-cGMP treatments, mere resemblance of
contractile responses is insufficient to infer a causal relationship. However, the two
remaining drugs, ODQ and SNAP, produced convincing results that FMRFamide was not
62
activating the nitric oxide-induced cGMP pathway or increasing cGMP levels. Worden
et al. (1994) reached a similar conclusion when they demonstrated that a FMRFamiderelated neuropeptide enhanced contractility in the lobster dactyl opener muscle, but the
peptide failed to elevate cGMP levels inside the muscle cells. Many studies have shown
that the nitric oxide-induced cGMP signaling pathway is involved in the relaxation of
smooth muscle preparations from a variety of organs (Ahn et al. 1997; Olsson and
Holmgren 1997; Choi and Farley 1998; Elphick 1998); however, my results along with
the work of Krajniak and Khor (1999) have not found any evidence of a FMRFamideinduced relaxation on the crop-gizzard. Activation of the nitric oxide-induced cGMP
signaling system by neuropeptides remains unexamined in annelids, but the results from
this study suggest that this second messenger pathway is absent in the FMRFamideinduced responses on the earthworm crop-gizzard.
Manipulation of Second Messenger Pathways Alters
Spontaneous Activity of the Crop-Gizzard
In a few experiments, second messenger pathway manipulations provided
evidence that normal crop-gizzard contractile activity is mediated by specific signal
pathways. For instance, the drugs W-7 and genistein produced distinct contractile
responses when applied to the crop-gizzard prior to FMRFamide application. It was
apparent that these drug-induced contractile responses provided evidence that calmodulin
and tyrosine kinase are involved in mediating crop-gizzard spontaneous activity.
Additionally, treatment of W-7 and U-73122 with the FMRFamide application produced
63
distinct contractile responses on the crop-gizzard that generated causal conclusions
regarding the involvement of calmodulin and the phosphatidylinositol signal pathway in
the FMRFamide-induced responses.
U-73122, a potent phospholipase C inhibitor and selective phospholipase A2
inhibitor, produced contractile responses that were similar to the FMRFamide-induced
increase in basal tonus and peak tension. From these results, it appears that the
phosphatidylinositol pathway and/or arachidonic acid pathway may be involved in the
normal spontaneous activity of the crop-gizzard. It has been reported that arachidonic
acid and lipoxygenase metabolites can induce smooth muscle relaxation, most notably
during vasodilation events (Pfister and Campbell 1992; Zhang et al. 2005). However,
recent investigations into vertebrate smooth muscle preparations involving U-73122 and
other phospholipase C inhibitors have reached opposing conclusions regarding the role of
the phosphatidylinositol pathway in the mediation of spontaneous contractile activity
(Balemba et al. 2005; Tanaka et al. 2003). Perhaps the normal spontaneous activity of
the crop-gizzard is partially mediated by the inhibitory products of the arachidonic acid
pathway; thus supporting the increased contractile responses generated by the
phospholipase A2 inhibitor, U-73122. I have already discussed that the FMRFamideinduced responses appear to be mediated by the phosphatidylinositol pathway based upon
the effects of the H-7, BIM I, and KN-62 treatments. When FMRFamide was combined
with U-73122, the contractile responses diminished with significant reductions found in
the basal tonus levels. U-73122 is known to inhibit invertebrate neuropeptide-induced
responses through the inactivation of the phosphatidylinositol pathway (Satake et al.
64
2003). Therefore it seems likely that the decreased contractile responses generated from
the combined FMRFamide-U-73122 treatment support the conclusion that FMRFamide
activates the phosphatidylinositol pathway in the crop-gizzard responses.
The application of genistein, a tyrosine kinase inhibitor, elicited a significant
elevation in basal tonus and peak tension when compared to the FMRFamide-induced
responses. Additionally, in the combined FMRFamide-genistein treatment, the induced
contractile responses maintained the elevated levels seen in the individual genistein
treatment, suggesting the absence of tyrosine kinase in the FMRFamide-activated signal
transduction. However, the actions of genistein alone indicate that tyrosine kinase is
involved in the regulation of the crop-gizzard spontaneous activity. Protein tyrosine
kinases occupy a large class of enzymes found in vertebrates and invertebrates that are
categorized into two groups, receptor or cellular tyrosine kinases. Each type of tyrosine
kinase has been shown to play a vital role in signaling pathways (mitogen-activated
protein kinase pathway) that initiate cellular processes such as proliferation,
differentiation, and development (Blenis 1993; Seger and Krebs 1995). In addition,
tyrosine kinase has been shown to increase contractile responses in a variety of vertebrate
smooth muscle preparations primarily through voltage-gated calcium influx (Chopra et
al. 1997; Palmier et al. 1999; Tolloczko et al. 2000). Genistein has been demonstrated to
block the tyrosine kinase-activated calcium influx and induce muscle relaxation
(Wigetunge et al. 1992; Chopra et al. 1997; Palmier et al. 1999). Despite the evidence of
genistein-induced relaxation in vertebrate smooth muscle, there exists no reported
tyrosine kinase signal pathway investigation in invertebrate smooth muscle to date. In
65
my investigation, it appears that genistein is inhibiting a tyrosine kinase signal pathway
that regulates crop-gizzard spontaneous activity via a unique signaling process that
relaxes basal tonus and reduces contractile strength.
W-7 is a drug known to have wide ranging actions including inhibition of
calmodulin, calcium-calmodulin phosphodiesterase, and myosin light chain kinase. W-7
elicited a response that was significantly greater than the FMRFamide-induced response.
The W-7-induced contractile response on the crop-gizzard was similar to contractile
recordings from a bovine smooth muscle experiment, where application of W-7 alone
induced contractions, possibly through a histamine-releasing mechanism (Asano 1990).
Wu (1939a) demonstrated that histamine induced an elevation of basal tonus on the
Lumbricus crop-gizzard. It has also been reported that W-7 might enhance intracellular
calcium motility in cardiac muscle cells through the modulation of the calcium inducedcalcium release mechanism during excitation-contraction coupling (Suziki et al. 1999).
My results demonstrate that the normal spontaneous activity of the crop-gizzard appears
to be mediated by a calmodulin-dependent event due to the excitatory effects induced by
W-7. However, when the FMRFamide application was combined with W-7, the
contractile responses were diminished with significant reductions found in both basal
tonus increase and peak tension. Thus, it appears W-7 does inhibit some contractile
FMRFamide-activated signal transduction pathways. W-7 has been shown to block
Substance P-induced contractions in smooth muscle through the inhibition of calmodulin
and internal calcium motility, or myosin light chain phosphorylation (Bitar et al. 1990;
Washabau 2002). Additionally, Suenaga et al. (2001) demonstrated that protein kinase C
66
activation induced a contractile response in a smooth muscle preparation, and the
response was delayed and weakened by W-7. From my earlier assertion that the
FMRFamide-induced contractile response of the crop-gizzard is mediated by the
phosphatidylinositol pathway and together with the FMRFamide-W-7-induced effect, I
believe that W-7 is inhibiting portions of the phosphatidyl inositol pathway that are
necessary for the FMRFamide-induced response. Thus, experiments involving W-7
exposed a new excitatory response on the L. terrestris crop-gizzard and strengthened the
link between FMRFamide and the phosphatidylinositol pathway.
In this thesis, I have presented the most comprehensive examination of
FMRFamide-induced activity on the oligochaete foregut to date. An early success of my
project was the validation of the original FMRFamide-induced dose-dependent response
curves reported by Krajniak and Khlor (1999). My attempt to reveal a more complete
contractile profile of FMRFamide-induced activity on the crop-gizzard was achieved by
constructing concentration-response curves for five different parameters on both the
longitudinal and circular muscles. The main aim of the investigation was to determine
FMRFamide-activated signal transduction pathways that mediate the contractile
responses of the crop-gizzard longitudinal muscles. I found no evidence that
FMRFamide activated an amiloride-sensitve sodium channel, but my results
demonstrated that the phosphatidylinositol, arachidonic acid, and mitogen-activated
protein kinase second messenger pathways are involved in mediating the peptide-induced
responses. Additionally, I found that the calmodulin protein and the phosphatidylinositol
second messenger pathway appear to be involved in the regulating normal crop-gizzard
67
spontaneous activity. Thus, this investigation is the first to identify specific signaling
pathways that are involved in both the FMRFamide-induced responses and normal
spontaneous activity of the L. terrestris crop-gizzard.
The role of FMRFamide or other RFamides in oligochaetes is still largely
unknown. However, with this investigation and the past works by Krajniak and Khlor
(1999), Reglodi et al. (1997), Ukena et al. (1995a), and Fugii et al. (1989), FMRFamide
appears to adopt a convincing role in regulating crop-gizzard activity. The significance
of FMRFamide alongside the established gut neuromodulators acetylcholine and
epinephrine is a question that remains unanswered. The identification of specific
signaling pathways involved in the FMRFamide-induced response helps increase the
understanding of the actual response mechanism and can prove valuable in future
cellular, molecular, or pharmacology investigations in the crop-gizzard or other systems.
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