Download Pertussis Toxin-Insensitive Phosphoinositide Hydrolysis, Membrane

436
Pertussis Toxin-Insensitive Phosphoinositide
Hydrolysis, Membrane Depolarization,
and Positive Inotropic Effect of
Carbachol in Chick Atria
Tsunemi Tajima, Yasuhiro Tsuji, Joan Heller Brown, and Achilles J. Pappano
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Muscarinic agonists can stimulate rather than inhibit cardiac muscle in some preparations. In left atria
from hatched chicks, treatment with pertussis toxin reversed the membrane action of carbachol from
hyperpolarization to depolarization and reversed the inotropic effect of carbachol from negative to
positive. Acetylcholine also depolarized the membrane and increased the force of contraction in atria
from pertussis-toxin-treated chicks although oxotremorine did not. These cholinergic responses were
blocked by atropine but not by adrenoceptor antagonists, suggesting that they are mediated via
muscarinic receptors and are not due to actions of endogenously released catecholamines. Muscarinic
receptor stimulation leads to two distinct biochemical responses in chick atria: inhibition of adenylate
cyclase and activation of phosphoinositide (PI) hydrolysis. The former is lost in atria from
pertussis-toxin-treated chicks, whereas the PI response persists. The pharmacologic characteristics
of the PI response resemble those of the depolarization and positive inotropic response. Both are
insensitive to blockade by pertussis toxin, require high concentrations of carbachol, and are elicited
by acetylcholine but not by oxotremorine. The present study suggests that muscarinic agonist-induced
PI turnover may be responsible for the membrane depolarization and positive inotropic effects of
carbachol and acetylcholine; that an increase in Na+ conductance underlies these responses; and that
it is stimulated either by an increase of intracellular calcium mobilized by inositol triphosphate and/or
by activation by protein kinase C. (Circulation Research 1987;61:436-445)
C
arbachol inhibits the calcium-dependent action
potential and contractions in the atrium by an
action on muscarinic receptors (mAChR).' The
mAChR are linked to the catalytic unit of adenylate
cyclase by Gi( a guanine nucleotide binding protein that
transduces the receptor signal into inhibition of enzymatic activity. The attendant reduction of cyclic
adenosine 3',5'monophosphate (cAMP) synthesis and
of the phosphorylation of membrane components
diminishes the entry of Ca2+ through voltagedependent channels.2"4 In frog ventricular muscle,
acetylcholine inhibits the opening of Ca2+ channels by
activating a cyclic guanosine monophosphate (cGMP)dependent phosphodiesterase that diminishes cAMP
content.5 Muscarinic receptors are also linked to
activation of atrial K + channels through a guanine
nucleotide binding protein, apparently G, and/or GO.6~9
Guanine nucleotide binding proteins obtained from
human erythrocytes10 and from bovine cerebral cortex"
have been shown to activate muscarinic K+ channels
directly in isolated atrial cell membrane patches.
From the Division of Pharmacology, Department of Medicine,
School of Medicine, University of California at San Diego, La Jolla,
Calif., and the Department of Pharmacology, University of Connecticut Health Center, Farmington, Conn.
Supported by USPHS grants HL-13339 (A.J.P.) and HL-17682
and HL-28143 (J.H.B.).
Address for reprints: A.J. Pappano, Department of Pharmacology, University of Connecticut Health Center, Farmington, CT
06032.
Received December 12, 1986; accepted April 3, 1987.
Although there is significant disagreement between
these reports concerning the suitability of Go (from
brain) and the role of a/3y vs. fiy subunits as
components of the reaction, the results substantiate the
previously advanced conclusion that no second messenger need be involved for muscarinic agonists to open
K+ channels.7"9 Muscarinic agonists thereby increase a
specific K + conductance, hyperpolarize the membrane,
and decrease action potential duration , 79 Acetylcholine
is proposed to act on specific K + channels that are
distinct from background K channels (iKI) in sinoatrial
node, atrial muscle, and Purkinje fibers.12"15 The
reduced influx of Ca2+ and the increased efflux of K+
both contribute to the negative inotropic action of
muscarinic agonists in atrial muscle.1
Treatment of chicks with pertussis toxin (islet
activating protein, IAP) under conditions in which 90
to 95% of G, and Go are ribosylated16 prevents the
inhibition of adenylate cyclase and Ca2+ entry, the
activation of K+ channels, and the negative inotropic
effect of carbachol.9 Also, under these conditions,
carbachol stimulates rather than inhibits atrial cells by
depolarizing the membrane and exerting a positive
inotropic action. Because these stimulant effects are
pertussis-toxin-insensitive, they apparently occur via
muscarinic receptors whose link to intracellular effectors is independent of G( and G,,.'6
Carbachol stimulates the hydrolysis of phosphoinositides (PI) in embryonic chick atria and chick heart
cells through activation of mAChR. l7 This effect is not
blocked by treatment of chick heart cells with pertussis
Tajima et al
PI Hydrolysis and Muscarinic Stimulation
toxin.18 These observations, together with those concerning depolarization and positive inotropic actions of
carbachol in the hatched chick atrium, prompted the
hypothesis that carbachol may be acting through the PI
cycle to regulate ion permeability and force development in a novel manner that results in stimulation.
The studies reported here describe the stimulant
effects of muscarinic agonists on membrane potentials
and contractions and test the hypothesis that these
responses are related to activation of PI hydrolysis.
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Materials and Methods
Pertussis Toxin Treatment
White Leghorn chicks (5 to 7 days after hatching)
were injected intravenously (jugular vein) either with
1.0 to 1.5 fxg of pertussis toxin (List Biochemicals,
Campbell, Calif.) or with an equal volume (50 fi\)
of 0.9% NaCl and housed in an incubator at 35° C.
After 2 to 3 days, the animals were decapitated, their
hearts were removed, and the atria were retained for
experiments.
Electrophysiology and Contraction Experiments
Left atrial muscle from pertussis toxin or vehicletreated chicks was used for electrophysiology and
contraction experiments. Atrial muscle strips were
superfused with aerated (95% 0 , - 5 % CO2) modified
Tyrode's solution (raM concentrations: Na+ 149.3, K+
5.4, Ca2+ 1.8, Mg2+ 1.0, Cl" 148, HC<V 11.9,
H2P<V 0.4, and glucose 5.5) at 37° C. Membrane
potentials were recorded with standard microelectrode
techniques. Cells were impaled with glass microelectrodes filled with 3 M KCI (tip resistance 15 to 30 Mfl).
The membrane potential was led through a high input
impedance preamplifier (WPI type 701, World Precision Instruments, New Haven, Conn.) and was displayed on an oscilloscope. A single sucrose gap method
was used to obtain an estimate of changes in membrane
resistance produced by carbachol and to adjust the
steady membrane potential to desired values by passing
constant current pulses through the tissue.
This experimental setup and its limitations have been
described in a report from this laboratory.l9 Assuming
that internal longitudinal resistance is constant, the
change in electronic potential produced by a drug in
response to a constant current pulse yields an estimate
of the ratio of membrane resistance (AR) by the relation
( R . W R ^ J - k t A V ^ A V ^ , , ) . The assumption of
constant internal longitudinal resistance has been
challenged20 because acetylcholine (1.1 xlO" 5 M)
increased the resistivity of the intercellular pathway
between atrial cells by 25%. Changes in polarization
resistance up to 29% at the end of 500-yum fiber bundles
could escape detection by our recording procedures, as
previously reported.19 For these reasons, it is possible
for muscarinic agonists to increase membrane conductance to Na+ and yet not be observed, particularly when
polarization resistance is low before drug addition.
When membrane resistance is increased by addition of
20 mM Cs + , the aforementioned limitations are partially offset, and a reduction in membrane resistance
437
consistent with the ability of carbachol to increase
membrane permeability to Na+ has been measured.2'
(It is of interest that Korth and Kuhlkamp observed no
depolarization when carbachol increased intracellular
Na + activity in the presence of normal Tyrode's solution yet were able to detect a 2.5-3 mV depolarization
after membrane resistance had been increased by
addition of Ba2+ or the omission of K+ from the
Tyrode's solution.) Data were taken only from cells in
which a stable impalement was maintained throughout
the experiment.
Muscle contractions were recorded with a force
displacement transducer (FT03C, Grass Instrument
Co., Quincy, Mass.) using a technique described
previously.22 Left atrial strips (approximately 1 X 4 mm)
were set at a rest length that provided maximum force
development. The muscles were paced by a punctate
silver electrode (100 yuM diameter) with rectangular
voltage pulses (1.5 x threshold voltage, 0.5 millisecond duration, 3 Hz).
Phosphoinositide Measurements
Right and left atria from untreated or pertussistoxin-treated chicks were removed and labelled with
20 /LtCi/ml [3H]inositol (New England Nuclear, Boston,
Mass.) for 90 minutes in a bicarbonate buffered Krebs
salt solution gassed with 95% O 2 -5% CO2. After
removing [3H]inositol and replacing with fresh medium, drug (in 10 mM LiCl) was added for 30 minutes.
To end the assay, atria were blotted dry and frozen in
Freon cooled with liquid nitrogen. Frozen atria were
homogenized in 10% TCA and the pellet removed by
centrifugation in a Beckman microfuge B (Somerset,
N.J.) for 60 seconds. The supernatant was washed 5
times with 4 volumes of ice-cold, water-saturated ether
to remove TCA. The sample was run on ion-exchange
columns and the inositol phosphate ([3H]InsP) collected, as described elsewhere.23 Data were normalized for
tissue protein content, and assayed by the method of
Bradford.24 Normalized data from right and left atria
were not different and were therefore combined.
Measurements are given as mean±SEM. The statistical significance between sample means was estimated with Student's t test.
Results
Carbachol Depolarization of Atrial Membranes From
Pertussis-Toxin-Treated Chicks: Concentration and
Agonist Dependence
Carbachol (10~8 to 10"6 M) hyperpolarized the
resting membrane in cells from saline-treated animals.8
As shown in Figure 1A, a high concentration of
carbachol (10" 4 M) evoked a relatively small hyperpolarization that tended to dissipate during exposure.
As we suggested previously,16 the reduced magnitude of
the carbachol-induced hyperpolarization at 10~4 M is
probably the result of the concomitant generation of a
depolarization. The occurrence of carbachol-induced
depolarization is seen when the drug-induced hyperpolarization is prevented by treatment with pertussis
toxin (Figure IB). Carbachol depolarized the mem-
438
Circulation Research
Carb IO"*M
Carb
IO"5M
I. A. P.
SALINE
Vol 61, No 3, September 1987
IO"4M
Carb
icr 4 M
B
I
I
Carb IO' 4 M
Carb
Atropine
Alropine
+
+
IO"4M
10 mV
IO" 3 M
i
I min
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FIGURE 1. Effects of carbachol (10 " M) on resting potential
in atrial cellsfrom saline (A, A') and lAP-treated (B,B') chicks.
Carbachol hyperpolarized membrane of atrial cell from salinetreated chick (A); this effect was blocked by3xlO~'M atropine
(A')- Carbachol depolarized membrane of atrial cell from
IAF'-treated chick (B); atropine also blocked this response (B ')•
Voltage and time calibrations apply to all records. Initial resting
potential was — 80 mV in A,A' and — 77 mV in B,B'.
brane by 5 mV, and the depolarization was sustained
throughout the 2 minutes of supervision with the drug.
Atropine (3 x 10~7 M) prevented carbachol from evoking hyperpolarization (Figure 1A') and depolarization
(Figure IB') of atrial cells from saline- and pertussistoxin—treated animals, respectively.
Pertussis toxin treatment blocks the decrease of
polarization resistance (Rp,,,), that is, the increase of K+
conductance elicited by 10~6 M carbachol.9 Similar
results were obtained when 10"4 M carbachol was
tested in atria from pertussis-toxin-treated animals.
Membrane polarization resistance (R^,) averaged
7.0 ± 1.8 Kft {n = 7) in unstimulated atrial cells before
addition of carbachol and 7.2 ± 1 . 2 Kft in the presence
of 10~4 M carbachol (p>0.1). (The failure to detect an
increased membrane conductance to Na + under these
conditions can be accounted for by the experimental
limitations described in "Materials and Methods.")
The concentration dependence for the depolarization
evoked by carbachol is illustrated in Figure 2. This
experiment was done with agonist application from a
pipette (50 /urn tip diameter) containing the desired
drug concentration. The magnitude of the carbacholinduced depolarization under steady-state conditions
was the same if the drug was applied by superfusion.
As shown in Figure 2, depolarization amounted to 1
mV, 5 mV, and 6.3 mV for 10"5, 10"4, and 10~3 M
carbachol, respectively.
Acetylcholine also depolarized atrial membranes
from pertussis-toxin-treated chicks, but oxotremorine
did not. A representative experiment is shown in Figure
3. Superfusion with carbachol (10~4 M) elicited a 5.5
mV depolarization while an equimolar concentration of
acetylcholine depolarized the membrane by 1.4 mV
(Figure 3). Oxotremorine (10" 4 M) had no effect on the
resting potential of atrial cells. The results of all
experiments in which these muscarinic agonists were
tested are shown in Figure 4. Clearly, carbachol had the
lowest threshold (10~5 M) and evoked the largest
I m——i
i n i oc mv
«w
FIGURE 2. Concentration dependence of carbachol-induced
depolarization in atrial cell from lAP-treated chick. A 50-fxm
diameter pipette containing given carbachol concentration in
Tyrode's solution was placed in bath within 1 mm of tissue,
where it remained for 25 seconds. Depolarization produced by
carbachol increased in amplitude and rate of initiation as
carbachol concentration increased from I0'! to IO'J M. Initial
resting potential was — 72 mVfor each record. Voltage and time
calibrations apply to all records.
depolarization (6.9±0.5 mV at 10"3 M). Depolarization by acetylcholine was not significant below a
concentration of 10"4 M. At 10"3 M, the acetylcholineinduced depolarization (1.9±0.2 mV) was approximately 25% of that evoked by carbachol. Oxotremorine
was remarkable insofar as it failed to depolarize atrial
membranes from pertussis-toxin-treated chicks at any
concentration from 10~6 M to 10~3 M.
Voltage Dependence
Depolarization by carbachol was also influenced by
membrane voltage. The single sucrose gap method was
used to set the membrane potential at a desired value,
and the amplitude of the depolarization by carbachol
was measured. The results of all experiments (8
different tissues) are shown in Figure 5. The depolarization induced by 10~4 M carbachol was maximal
Carbachol IO"4M
Acetylcholine 10" M
Oxotremorine
IO'4M
I min
I5mv
FIGURE 3. Agonist dependence for muscarinic-induced depolarization. Records are from 3 atrial cells from as many
lAP-treated chicks. At arrows, superfusion was changed to one
containing carbachol, acetylcholine, or oxotremorine, all at
10'' M. Voltage and time calibrations apply to all records.
Initial resting potential before drug addition was —72 mV
(carbachol), -78 mV (acetylcholine), and —79 mV (oxotremorine).
Tajima et al
PI Hydrolysis and Muscarinic Stimulation
>
CAHB4CH0L ( n . 6 )
ACETYLCHOLINE
O—D OXOTREMORINE <
o
a.
AGONIST (M)
FIGURE 4. Summary of concentration dependence and agonist
dependence for depolarization of atrial cells from IAP-treated
chicks. Ordinate: depolarization amplitude in mV; abscissa:
agonist concentration. Number of cells given in parentheses.
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(~4.7 mV) between - 7 0 and - 8 0 mV; the average
resting potential in these preparations was —76 ± 2
mV before application of current to change membrane
voltage. At membrane potentials more negative than
— 90 mV and more positive than —50 mV, the
amplitude of the carbachol-induced depolarization
decreased. Because the multicellular preparations used
are subject to ion depletion and accumulation at
hyperpolarized and depolarized potentials, respectively, a more precise determination of the voltage dependence for carbachol-induced depolarization will have
to await voltage clamp experiments on isolated atrial
myocytes. However, our results for the voltage dependence of carbachol-induced depolarization are similar
to those of others25-26 who have studied the voltage
dependence of digitalis-induced transient inward currents in multicellular cardiac Purkinje fibers.
In atrial cells from saline-treated animals, carbachol
(10~4 M) hyperpolarized the resting membrane by
almost 4 mV (Table 1). The reversal potential (Erev) for
carbachol was —84 ± 2 . 3 mV (Table 1). Assuming
1
kj
o
6
5
CL
s
M
a.
<
o
439
that the intracellular K+ concentration in atria from
hatched chicks is the same (120 to 130 mM) as in atria
from 18-day embryos,27 the estimated K+ equilibrium
potential ranges from —82.2 to —84.3 mV. Therefore, the results of these experiments in saline-treated
chicks are consistent with the well-known ability of
muscarinic agonists to increase a specific K+ conductance in atrial cells.' 21213 By contrast, carbachol (10~4
M) depolarized the atrial membrane of pertussistoxin-treated chicks by 5 ± 0.7 mV. The magnitude of
the depolarization was reduced at voltages between
— 50 mV and — 10 mV. The possibility that there is a
reversal potential for the carbachol-induced depolarization at voltages more positive than — 10 mV could
not be tested because the control of membrane potential
at voltages less negative than — 10 mV is uncertain.
Lack of Effect of Tetrodotoxin and Verapamil
The depolarization induced by carbachol was not
significantly antagonized by either tetrodotoxin (TTX,
3 x 10"7M) or verapamil (10" 6 M). In 9 individual cells
from different preparations, carbachol (10~4 M) depolarized the resting membrane by 4 . 2 ± 0 . 2 mV in the
absence of TTX and by 4.5 ± 0 . 3 mV in the presence
of TTX. Carbachol depolarized by 5.2 ± 0 . 6 mV in the
absence and by 5.2±0.5 mV in the presence of
verapamil (n = 8). The tissues were exposed to either
TTX or verapamil for at least 30 minutes before
addition of carbachol, a time sufficient to produce
steady-state changes in the action potential rate of rise
and duration, respectively, by the channel-blocking
compounds.
Effect of Changes in Extracellular Ionic Constituents
In initial experiments, 20 mM Cs + was added to the
Tyrode's solution to block resting K+ conductance
(gK1). As described by others,28 the current-voltage
relation became linear (from — 100 to — 40 mV) in the
presence of Cs + (data not shown), and R^, increased to
13.1 ± 2.8 KCl (n = 7) from an initial value of 7.0 ± 1.8
Kft (/7<0.01). The membrane depolarized to
- 57 ± 4.0 mV because of the Cs + -induced block of K+
current (Table 2). At this initial value of the membrane
potential, the depolarization by carbachol (4.1 ± 0 . 5
mV) was not significantly different (p>0.1) from that
observed before Cs + (3.8 ± 0 . 3 mV). When the initial
resting Vm in Cs + was adjusted (compensated) to the
value observed before Cs + ( — 79 mV), the depolarization evoked by carbachol was larger (Table 2). These
results confirm the conclusion suggested above that
UJ
-110
-90
-TO
-50
-30
-10
MEMBRANE POTENTIAL (mV)
FIGURE 5. Voltage-dependence of depolarization induced by
carbachol (10'' M) in atrial cells from IAP-treated chicks.
Ordinate: depolarization amplitude in mV: abscissa: membrane
potential in mV. Number of cells is shown in parentheses.
Membrane potential was set by constant current pulses applied
through tissue in single sucrose gap chamber. Carbachol was
added to superfusion fluid, and drug-induced depolarization
was measured at steady-state (2 minutes) in each instance.
Table 1. Resting Potential Changes by Carbachol (10~4 M)
in Pertussis-Toxin-Treated and Control Atrial Cells
Resting potential (mV)
A*
Initial
Treatment
+ Carbachol
Ercv (mV)
Saline (n = 5) - 7 7 ± 2 . i i - 8 1 :t 1.9 - 3 .9+1.1 - 8 4 ± 2 . 3
Pertussis toxin
9
- 7 6 ± l . i i - 7 1 :11.7 + 5 ,0±0.7
*Membrane hyperpolarization indicated by minus sign, membrane depolarization by plus sign.
Circulation Research
440
Table 2. Effect of Cs + (20 mM) on Carbachol (10~4 M)Induced Depolarization*
Depolarization
Initial resting
amplitude (mV)
potential (mV)
Before Cs +
In Cs +
Uncompensated
- 7 4 + 0.9
3.8±0.3
-57±4.0
4.1 ±0.5
- 7 4 + 0.9
8.9±0.8t
Compensatedt
*n = 1 cells examined in each condition; all cells are from pertussis-toxin-treated animals. tRefers to condition where initial resting
potential was adjusted to value observed in the absence of Cs +
before supervision with carbachol. tp<0.005 when compared
with depolarization in Cs + (uncompensated).
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carbachol-induced depolarization did not arise from a
reduction of K+ conductance. A similar result was
obtained in the presence of 0.2 mM Ba2+ where carbachol retained its ability to depolarize the membrane.
In the presence of 20 mM Cs + , resting K+ channels
(iKi) activated by membrane voltage and K+ channels
activated by muscarinic agonists are blocked.15-28 Additionally, the presence of pertussis toxin prevents the
opening of K+ channels by muscarinic agonist.7"9 In the
presence of 20 mM Cs + and treatment with pertussis
toxin, carbachol (10~4 M) decreased R^, from an initial
value of 13.1 ± 2 . 8 to 8.2±1.7 Kfl (n = 7, p<0.0\)
when it depolarized atrial cells. If carbachol were
somehow able to increase K+ conductance despite the
presence of Cs + and pertussis toxin treatment, membrane hyperpolarization rather than depolarization
should have occurred.
The observation that carbachol depolarized the
membrane under these conditions indicated that the
agonist might increase resting Na + conductance. The
supposition was tested in a series of experiments in
which the extracellular Na + concentration ([Na + ]J was
reduced by replacement with isotonic sucrose. Carbachol (10~4 M) depolarized atrial membranes 3.7 ± 0 . 3
mV (n = 24) in 150 mM [Na + ] 0 . The amplitude of the
depolarization averaged 87 ± 9% of initial value in 75
mM [Na + ] 0 (n = 8); 61 ± 7% of initial value in 37.5 mM
[Na + ] o (n= 10);and53±8%ofinitialvaluein 13 mM
[Na + ] 0 (/i =11). The dependence on extracellular
sodium concentration is consistent with the hypothesis
that Na + conductance increased when carbachol depolarized. In the experiments with sucrose, [C\']o was
reduced along with [Na + ] o . From measurements of Cl"
distribution in frog ventricle, it was not possible to rule
out active C\~ transport although there is no evidence
for such transport.29 If the [Cl"], were greater than that
expected from passive distribution in avian atrial
muscle, the possibility could not be excluded that
carbachol depolarized by increasing Cl" efflux. Moreover, the ionic strength of the bathing solution decreased when sucrose replaced NaCl, thereby modifying the activities of other ions and making it more
difficult to assess the eventual contributions of ions
such as K+ and Ca2+ to the depolarization. In order to
avoid these problems, an attempt was made to duplicate
these experiments with choline chloride as the replace-
Vol 61, No 3, September 1987
ment for NaCl. However, when 75 mM choline chloride
replaced 75 mM NaCl, the carbachol-induced depolarization was eliminated. The most likely explanation
is that choline acts as an antagonist of carbachol
at muscarinic receptors. The IC50 (concentration to
displace 50% of bound ligand) of choline needed
to displace the muscarinic antagonist ligand, 3H-quinuclidinyl benzilate, from binding sites in brain was
1.8±0.6 mM.30 Our experiments were done at more
than 30 times this concentration.
Atrial Contractions
In atria from saline-treated animals, carbachol
evoked a characteristic negative inotropic effect with a
threshold concentration near 10~8 M and maximum
inhibition at about 10~6 M.9 By contrast, carbachol
evoked a positive inotropic effect in atria from
pertussis-toxin-treated chicks. The threshold concentration was >10" 6 M for the positive inotropic effect.
As shown in Figure 6, carbachol (10~5 to 10~3 M)
evoked a graded increase in the force of contractions
in atria from pertussis-toxin-treated chicks. The results of all experiments with carbachol are shown in
Figure 7. The positive inotropic effect of 10"4 M
carbachol, which increased force more than twofold,
was unchanged in the presence of propranolol (3 X 10"7
M). However, atropine (3 x 10"7 M) prevented carbachol from increasing the force of contraction (Figure
7). Acetylcholine also evoked a positive inotropic
effect in atria from pertussis-toxin-treated animals.
The threshold concentration was >10" 4 M, and force
averaged only 113±7% (n = 5) of initial at 10"3 M
(data not shown). However, physostigmine (10~6 M)
permitted the detection of a positive inotropic effect of
acetylcholine at lower concentrations (>10 =<f 'M), and
the maximum effect at 10"3 M was 172 ± 15% of the
initial value (n = 3).
The negative inotropic effect of oxotremorine seen
in saline-treated animals (n = 3) was largely but not
completely overcome by pertussis toxin treatment
(Figure 8). However, unlike carbachol and acetylchoTension
I 300mg
CONTROL
IO~5M
CARBACHOL
IO" 4 M
IO~3M
FIGURE 6. Positive inotropic effect of carbachol in atrial
muscle from IAP (pertussis toxin)-treated chick. Upper trace is
time marker; largest signals are minute marks. Lower trace is
developed force (calibration, 300 mg) in atrial muscle strip
pacedat3 Hz. Propranolol (3 X 10'7 M) was present in Tyrode's
solution throughout experiment. Introduction of indicated
concentrations of carbachol is shown by downward deflections
of time trace.
Tajima et al
441
PI Hydrolysis and Muscarinic Stimulation
250
• IAP
fc I A P+ Propfonolol
l I A P + Propronolol -f Atropine
(n = 3)
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0L
C
10"'
IO"
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10"
10"
CARBACHOL(M)
FIGURE 7. Summary of contraction experiments with carbachol in atria from lAP-treated chicks. Ordinate: force of
contraction as % initial (= 100%); abscissa: molar carbachol
concentration. Positive inotropic effect of carbachol was same
in IAP alone (circles) and in IAP plus 3 X 10'7 M propranolol
(triangles). However, addition of 3 X 10~7 M atropine to latter
solution (squares) was associated with complete blockade of
positive inotropic effect of carbachol.
line, oxotremorine was not able to increase the force of
contraction in atria from pertussis-toxin-treated animals, even at 10~4 M.
Phosphoinositide Hydrolysis
The ability of muscarinic agonists to promote
[3H]InsP formation was examined in chick atria. As
shown in Figure 9, carbachol stimulated phosphoino-
100
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o
50
h-
u
<x
<r
touz
I0"9
IO~8
I0"7
OXOTREMORINE
I0"6
10"
lagonist]
FIGURE 9. Agonist-induced stimulation of 'H-inositol phosphate (['HJInsP) production in chick atria. Ordinate: ['HJInsP
in cpmlmg protein; abscissa: carbachol or oxotremorine was
added to labelled chick atria in presence of 10 mM LiCI at
concentrations indicated. Unstimulated atria (no agonist)
received only 10 mM LiCI. Values shown are means±SEM
(n = 3) from representative experiment.
sitide hydrolysis in a concentration-dependent fashion.
The threshold concentration was 10~6 M, and [3H]InsP
formation was maximal at > 3 x 10~4 M. There was
little or no effect of oxotremorine on phosphoinositide
hydrolysis, even at concentrations as great as 3 x 10~4
M (Figure 9). Acetylcholine at 10~3 M gave approximately half the response elicited by a maximal
concentration of carbachol, and the response to carbachol was inhibited by 10"6 M atropine (Table 3) but
not by curare (data not shown). Verapamil did not
significantly decrease carbachol-stimulated [3H]InsP
formation (not shown), a finding consistent with the
lack of effect of this Ca2+ channel blocker on the
carbachol-induced depolarization.
Carbachol-induced stimulation of [3H]InsP formation was unchanged in atria from pertussis-toxin—
Table 3. Effects of Cholinergic Agonists and Antagonists on
Phosphoinositide Hydrolysis
[3H]InsP
(cpm/mg protein)
(M)
FIGURE 8. Blockade of negative inotropic effect of oxotremorine by IAP treatment. Ordinate: force of contraction as %
initial ( = 100%); abscissa: molar concentration of oxotremorine. In saline-treated animals (open circles), oxotremorine
exerted significant negative inotropic effect at 10'" M; contractions could not be evoked at >/0"° M. In lAP-treated animals
(solid circles), negative inotropic effect was largely ( — 75%) but
not completely prevented. All tissues were paced at 3 Hz.
Acetylcholine (10~ 3 M)
110±12
324±17
230 + 25
Carbachol (10" 3 M)
+ atropine (IO~"6 M)
132 it 12
Control
Carbachol (lO" 3 M)
Data are means ± SEM (n = 6). Acetylcholine was tested in the
presence of 10~ 6 M physostigmine.
Circulation Research
442
treated chicks (Figure 10). This finding is consistent
with observations made on embryonic chick heart
cells.18 In atria from saline-treated chicks (n = 8),
carbachol increased [3H]InsP by ~450 cpm/mg protein above the basal [3H]IP level (~fourfold). In atria
from pertussis-toxin-treated chicks (n = 9), the basal
[3H]InsP was somewhat higher, but the increase elicited
by carbachol (~475 cpm) was nearly the same as that
in untreated tissue (Figure 10). The maximal formation
of [3H]InsP by carbachol was not significantly different
(p = 0.4) in the absence and presence of pertussis toxin
treatment.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Discussion
In atria from pertussis-toxin-treated chicks, carbachol increased the hydrolysis of phosphoinositides,
depolarized the membrane, and exerted a positive
inotropic effect. These phenomena shared several
characteristic pharmacologic features that included 1)
concentration dependence, 2) agonist selectivity, 3)
antagonist sensitivity, and 4) resistance to blockade by
pertussis toxin.
The concentrations of carbachol required to hydrolyze phosphoinositides, depolarize the membrane, and
increase the force of contraction in atria ranged from
S:10~6 M to 10"3 M. This concentration range is
different from that (10~8 to 10"6 M) required to increase
K+ conductance,9 to decrease Ca2+ conductance,9 and
to inhibit isoproterenol-stimulated cAMP accumulation17-31 in chick heart. Acetylcholine was an agonist for
all three responses, although it was less efficacious than
carbachol. Oxotremorine did not promote phosphoinositide hydrolysis, nor did it depolarize the membrane or increase the force of contraction. Atropine
blocked the ability of carbachol to stimulate phosphoinositide hydrolysis, depolarize the membrane, or
increase the force of contraction. Propranolol had no
effect on the depolarization and positive inotropic
action of carbachol, and neither propranolol nor
_
a
e
T
800
I
I
at
600
E
-H
400
5
200
1
1
W///
re
n
Cont
W/A
Carb
- Pertussis toxin
Cont
Carb
+ Pertussis toxin
FIGURE 10. Pertussis toxin does not block carbachol-induced
stimulation oflnsP production. Atria from chicks treated or not
treated with 1.5 fig pertussis toxin were labelled and then
incubated with vehicle (10 mM LiCl control) or carbachol (1
mM) for 30 minutes. Ordinate: [3H]InsP in cpm/mg protein.
Values shown are mean±SEM (n = 4-5).
Vol 61, No 3, September 1987
prazosin antagonized the increase in InsP accumulation
produced by carbachol. These results indicate that
depolarization, the positive inotropic response, and PI
hydrolysis are all mediated via muscarinic receptors
and make it unlikely that release of endogenous
norepinephrine by carbachol is involved. The same
pharmacologic profile is seen in cat papillary muscle.32
In this preparation, carbachol also increased phosphoinositide hydrolysis and the force of contraction,
whereas oxotremorine was without effect on either
phenomenon. Moreover, atropine but not phenoxybenzamine, propranolol, or hexamethonium opposed
the stimulation of phosphoinositide hydrolysis and the
positive inotropic effect of carbachol in this preparation.32 The positive inotropic effects of ACh in
cardiac Purkinje fibers33 and of carbachol on guinea pig
papillary muscle21 were also blocked by atropine but
not by propranolol, phentolamine, hexamethonium, or
verapamil.
Pertussis toxin treatment does not block the increased phosphoinositide hydrolysis, membrane depolarization, or the positive inotropic effect of carbachol
in chick atrial tissue. 91618 Under these conditions of
pertussis toxin treatment, > 9 0 % of the labelling of
guanine nucleotide binding proteins Gi and Go is
eliminated, indicating that more than 90% of these G
proteins are ribosylated.16 Thus, if either of these
proteins is involved as a transducer for the stimulant
effects of carbachol in atria, it must be that 5-10% of
G^GJ is adequate to transduce these effects. However,
under these conditions of pertussis toxin treatment,9 it
has been reported that the affinity of muscarinic
receptors (mAChR) for carbachol is decreased, muscarinic inhibition of adenylate cyclase is virtually
abolished, and the hyperpolarization resulting from
increased K + conductance is inhibited. These data
argue that the link between mAChR and cellular
effectors responsible for the stimulant actions of
carbachol is not a pertussis-toxin-sensitive protein (G,
or Go) and that neither inhibition of adenylate cyclase
nor activation of K + conductance is involved.
It is possible that an as yet unidentified G protein
serves as a transducer for the stimulant actions of
carbachol. Muscarinic receptor mediated activation of
phospholipase C in mouse exocrine pancreas cells and
the stimulation of IP3 formation caused by carbachol
depend on guanine nucleotides.34 The "G" protein
transducer of this muscarinic effect is not inhibited by
pretreatment with either pertussis toxin or cholera
toxin. In embryonic chick heart cells permeabilized
with saponin, the formation of [3H]inositol phosphates
is also stimulated by guanine nucleotides (manuscript
in preparation).35 Since the PI response in the chick
heart cells is not blocked by either pertussis toxin or
cholera toxin,'8-35 it appears that a novel G protein may
regulate PI hydrolysis in the myocardium, as suggested
for other cells.18-34
The present study has obtained pharmacologic evidence consistent with the concept that the increased
hydrolysis of phosphoinositides, membrane depolarization, and positive inotropic effect are mediated
Tajima et al
PI Hydrolysis and Muscarinic Stimulation
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through a common pathway and may be casually
related to each other. One hypothesis for a causal
relation among these responses is that certain muscarinic agonists increase inositol trisphosphate (IP3)
concentration within the cell. As a second messenger,
IP3 would release Ca2+ from sarcoplasmic reticulum
(SR) stores. Indeed, in chick heart cells, IP3 is
increased within 10 seconds after the addition of
carbachol.35 Furthermore, IP3 (^30 /xM) has been
shown to induce the release of Ca2+ from the SR of
mechanically skinned rat ventricular cells.36 Even at
200 /xM, IP3 had no effect either on mitochondria or on
myofilaments in this preparation. Although Ca2+ release from SR by IP3 was judged incompatible with a
role in excitation-contraction coupling in heart
muscle,36 the ability of IP3 to increase force development by releasing Ca2+ is consistent with our hypothesis that it could serve as a second messenger in the
positive inotropic effect of carbachol.
An alternative hypothesis, still involving the PI
response as a primary mediator, would be that protein
kinase C is activated because of the formation of
diacylglycerol. Hypothetically, protein kinase C could
phosphorylate and thereby activate plasma membrane
ion channels, or it could phosphorylate and alter the
calcium sensitivity of the myofilaments. Among the
variety of cardiac membrane and myofibrillar proteins
shown to be substrates for protein kinase C35 is a 15 kDa
plasma membrane protein that may be involved in
voltage-sensitive Ca2+ channels.3738
The possibility that the positive inotropic effect of
muscarinic agonists arises from increased entry of Ca2+
through voltage-sensitive channels seems remote. In
canine cardiac Purkinje fibers, acetylcholine evoked an
atropine-sensitive positive inotropic effect at concentrations from 10~8 to 10"4 M." Acetylcholine also
increased action potential duration at 50% (5.4 mM
K + -Tyrode's solution) and restored Ca2+-dependent
action potentials to fibers depolarized by 22 mM [K + ] o
but only at concentrations ^ 1 0 " 5 M. However, two
observations mil itated against the view that the positive
inotropic effect of acetylcholine arose from increased
Ca2+ entry through voltage-sensitive channels.33 First,
the positive inotropic effect could be detected at
acetylcholine concentrations that had no effect on
action potential duration at 50% and did not restore
Ca2+-dependent action potentials. Second, the positive
inotropic effect was not antagonized by verapamil.
The positive inotropic effect was independent of catecholamine release. Indeed, it was suggested that the
positive inotropic effect of acetylcholine in Purkinje
fibers was similar under certain conditions to that seen
in working myocardium."
In sheep cardiac Purkinje fibers, acetylcholine increased action potential duration at concentrations as
low as 10"7 M.39-40 This effect, which was attributed to
a reduction of inward background currents,41 was
prevented by atropine and was independent of endogenous catecholamines.39-40 Because this action of
acetylcholine seemed specific for sheep cardiac Purkinje fibers,3940 its applicability to the positive inotropic
443
effect of muscarinic agonists in other cardiac tissues is
unknown.
Our experimental results16 in avian atrium and those
of others21 in mammalian ventricle indicate that carbachol and acetylcholine do not increase voltagedependent Ca2+ entry since neither the plateau amplitude nor the action potential duration was significantly
increased when the positive inotropic effect occurred.
That the membrane depolarization could arise from
an effect of increased intracellular Ca2+ (rather than
from Ca2+ entry through voltage-sensitive channels) is
suggested by experiments in sheep cardiac Purkinje
fibers. Strophanthidin induces transient depolarizations that are generated by a transient inward current
carried primarily by Na+.25 It was proposed that this
current develops because strophanthidin inhibits the
Na+ pump and elevates intracellular Ca2+, which then
activates a passive plasma membrane channel that
admits Na+ and K+.25 In our hypothesis, elevated
intracel lular Ca2 + released by IP3 action on the SR could
serve a similar function. The elevated intracellular Ca2+
would activate a plasma membrane channel that admits
Na+ and thereby depolarizes the membrane. Neither
the carbachol-induced depolarization of atrial fibers
(present report) nor the strophanthidin-induced transient depolarization25 is directly sensitive to TTX or
verapamil, blockers of voltage-dependent Na + and
Ca2+ channels, respectively. In Purkinje fibers that are
either electrically stimulated or voltage-clamped so as
to increase Na + and Ca + entry through voltagedependent channels, TTX and Ca2+ channel blockers
can diminish transient inward currents indirectly by
virtue of having diminished Na + and Ca2+ currents,
respectively.2542
In guinea pig papillary muscle, a different hypothesis
has been advanced by Korth and Kiihlkamp2' whose
findings are very similar to ours. They also demonstrated an atropine-sensitive positive inotropic effect of
carbachol. The response was associated with an increased intracellular activity of Na + that was not
prevented by TTX and was attributed to muscarinic
agonist increasing Na+ permeability of the plasma
membrane.21 More recently, these investigators have
reported that carbachol was the most potent and
oxotremorine was the least potent muscarinic agonist
for evoking a positive inotropic effect in guinea pig
ventricle.43 Acetylcholine, which increased intracellular Na + activity by half as much as carbachol, was also
half as effective as carbachol in producing a positive
inotropic effect. In contrast to the results with all other
muscarinic agonists tested (carbachol, methacholine,
acetylcholine, and bethanecol), atropine did not prevent the positive inotropic effect of oxotremorine.
Moreover, stimulation of PI hydrolysis with eventual
modulation of cation fluxes was suggested as a possible
mechanism for the positive inotropic effect of choline
esters.43 The mechanism by which Na+ permeability
increased is not clear but again could involve a
protein-kinase-C-mediated phosphorylation. Where
their hypothesis differs from ours is Korth and Kiihlkamp's proposal that the entry of Na + was the initiating
444
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event that then brought about a positive inotropic effect
by stimulation of Na-Ca exchange. (Indeed, a similar
mechanism had originally been considered as a possible
explanation for the initiation of a transient inward
current by digitalis glycosides,25 but these authors
found this mechanism less satisfactory than did
others.26) The carbachol-induced increase of intracellular Na + was greatly diminished when [Ca 2+ ] o was 1.6
mM. 2 ' This finding is puzzling, for the initial entry of
Na+ should not be changed when [Ca2+]0 is reduced
unless there is some essential but as yet unknown effect
of [Ca 2+ ] o on the reaction mechanism.21 Resolution of
whether Na + permeability increases secondary to or
leads to increases in Ca2+ will require experiments with
isolated myocytes under space clamp conditions more
optimal than those available in multicellular preparations. In any event, the hypothesis that carbachol
affects Na + rather than K+ permeability is well
supported by two findings. First, muscarinic agonist
increased membrane conductance in atrial preparations
treated with pertussis toxin and Cs + sufficient to block
drug-induced changes of K + conductance. Second, the
carbachol-induced depolarization was directly proportional to extracellular Na + .
The stimulant effects of carbachol in chick atria cells
(depolarization and positive inotropy) are the opposite
of those (hyperpolarization and negative inotropy)
mediated by inhibitory guanine nucleotide binding
proteins Gi and Go. There is reason to think that such
stimulant actions are present, but masked, when G; and
Go develop and become fully functional. In very young
(4th incubation day) embryonic hearts,27 acetylcholine
and carbachol depolarized the membranes of sinoatrial
cells. The depolarization, like that described in the
present report, was diminished in the absence of
extracellular Na+27 and resistant to blockade by TTX.44
The ability of acetylcholine and carbachol to depolarize
the sinoatrial membrane in 4-day embryonic hearts was
related not only to the increased Na + entry but also to
the fact that the increase of K+ conductance by these
muscarinic agonists was not well developed. (An
increase of G^ and Go occurred between the 4th and 8th
incubation days in embryonic chick atria,45 and this
phenomenon was associated with an increased sensitivity of the tissue to the negative chronotropic effect
of carbachol,45 which had been attributed to an increased activation of K+ channels by muscarinic
agonist.27) The present experiments carried out with
atria from newborn chicks bear a strong similarity
insofar as the depolarization produced by carbachol and
acetylcholine becomes evident when treatment with
pertussis toxin prevents the increased K+ conductance.
The previous electrophysiologic27-44 and biochemical31
findings in the embryonic heart provide additional
evidence for the phosphoinositide hypothesis advanced
in this report. Carbachol (10~5 to 10~3 M) increased
[3H]InsP formation in embryonic chick hearts between
the 4th and 13th incubation days. Interestingly, the
greatest stimulation (eightfold increase) was detected
on the 4th incubation day; the stimulant effect declined
monotonically to a twofold increase on the 13th
Circulation Research
Vol 61, No 3, September 1987
incubation day.31
In summary, stimulant effects of muscarinic agonists
have been detected in chick atrial cells that appear to
be independent of the guanine nucleotide binding
proteins, G, and Go, and to be mediated through
mechanisms other than inhibition of adenylate cyclase
or activation of K+ conductance. Our findings have
been incorporated into a hypothesis in which the
stimulatory effect of muscarinic agonists on the myocardium is secondary to increased PI turnover. One
possible mechanism for this effect would be that IP3 is
the intracellular messenger for the eventual membrane
depolarization and positive inotropic effects observed.
Alternatively, activation of protein kinase C might
affect sarcolemmal ion channels. The precise ionic
mechanism for the depolarization and positive inotropic effect cannot be decided on the basis of present
findings. Either an activated intracellular calciumdependent passive ion transport reaction or an altered
sodium-calcium exchange would provide a novel
mechanism for the stimulant effects of parasympathetic
transmitter.
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membrane
WORDS • muscarinic
agonist • atrial
pertussis toxin •
depolarization • positive inotropic effect
phospholipase C • inositol trisphosphate • protein kinase C
KEY
Pertussis toxin-insensitive phosphoinositide hydrolysis, membrane depolarization, and
positive inotropic effect of carbachol in chick atria.
T Tajima, Y Tsuji, J H Brown and A J Pappano
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Circ Res. 1987;61:436-445
doi: 10.1161/01.RES.61.3.436
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Copyright © 1987 American Heart Association, Inc. All rights reserved.
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