Download the relative importance of nervous, humoral and intrinsic

J. exp. Biol. (i977). 7«>, 77-92
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77
THE RELATIVE IMPORTANCE OF
NERVOUS, HUMORAL AND INTRINSIC MECHANISMS IN
THE REGULATION OF HEART RATE AND STROKE VOLUME
IN THE DOGFISH SCYLIORHINUS CANICULA
BY S. SHORT, P. J. BUTLER AND E. W. TAYLOR
Department of Zoology and Comparative Physiology
University of Birmingham, Birmingham B15 2TT, U.K.
(Received 24 February 1977)
SUMMARY
Experiments involving supra-maximal electrical stimulation of the vagus
have indicated that the stimulation of the peripheral cut ends of the branchial
cardiac branches produces a more intense cardio-inhibition than the stimulation of visceral cardiac branches. It is suggested that the visceral cardiac
branches may have a mainly sensory function. In no case could cardioacceleration be obtained during vagal stimulation either before or after
injection of atropine, and any increases in stroke volume that occurred
accompanied reductions in heart rate. This relationship was considered to be
a manifestation of Starling's Law of the heart and it has been concluded that
there is no augmentary sympathetic innervation to the dogfish heart.
Evidence also indicates that the Starling relationship is responsible for the
increase in stroke volume which accompanies the bradycardia during
hypoxia. Circulating catecholamines do not appear to be of importance in
this response although they are concerned in cardio-vascular regulation
during normoxia.
INTRODUCTION
Elasmobranchs are of interest in that they possess two distinct pairs of cardiac
branches of the vagus. One arises from the visceral branch (Marshall & Hurst, 1905)
and the other from the post-branchial branch of the fourth branchial division of the
vagus (Norris & Hughes, 1920). The cardio-inhibitory action of these nerves and their
roles in the response to hypoxia have been described previously (Lutz, 1930; Taylor,
Short & Butler, 1977).
It has recently been shown that there is an extensive cardio-regulatory sympathetic
innervation of the heart in some teleosts (Govyrin & Leonteva, 1965; Otsuka &
Tomisawa, 1969; Gannon & Burnstock, 1969), the adrenergic fibres presumably
entering with the vagus (Gannon, 1971) as they do in amphibians (Gaskell, 1886;
Langley & Orbeli, 1911). In contrast, Gannon, Campbell & Satchell (1972) illustrated
a very sparse adrenergic innervation in elasmobranchs, which was confined to the
us venosus, and they considered that this may account for the slight reduction in
o-atrial conduction time on stimulation of the ducti Cuvieri, which was reported by
K
78
S. SHORT, P. J. BUTLER AND E. W. TAYLOR
Izquiredo (1930). The balance of evidence would suggest that the sparse adrenergiq
innervation of the elasmobranch heart is not involved in the control of heart rate
(Bottazzi, 1902; Lutz, 1930). It therefore appears that the nervous innervation of the
elasmobranch heart is entirely inhibitory and that any increase in heart rate or stroke
volume will be dependent upon the release of vagal tone, or upon intrinsic or humoral
regulatory mechanisms. At present, there is no clear indication of the relative roles of
these regulatory mechanisms in the intact fish.
Recent evidence has suggested that the majority of the vagal activity to the dogfish
heart during normoxia is exerted via the branchial cardiac branches, the visceral
cardiac branches being of secondary importance in this respect (Taylor et al. 1977).
In view of the importance of the two pairs of cardiac vagi for cardiac control in
elasmobranchs, the present study was undertaken to determine their function using
electrical stimulation techniques. We wished to see if heart rate or stroke volume
could be augmented at any intensity or frequency of stimulation, either before or
after injection of atropine. The relative importance of circulating catecholamines and
Starling's Law of the heart in determining heart rate and blood flow through the
ventral aorta during normoxia and during the response to hypoxia has also been
investigated, by using adrenergic receptor blocking agents.
METHODS
The general anatomical arrangement of the innervation to the heart and gills in the
dogfish (Scyliorhinus canicula L.) was described by Taylor et al. (1977). The cardiac
vagi of four freshly killed fish were exposed and separated from surrounding connective tissue. They were fixed in situ using Bouin's fluid and then removed and embedded in paraffin wax. Five /im sections were stained using the Masson's Trichrome
technique (Masson, 1929; Gurr, 1962). Sections were viewed and photographed
using a photomicroscope (Leitz, Ortholux II). The number of fibres present in each
cardiac nerve was counted from suitable photographs.
Eighty-four dogfish of either sex whose mass ranged between 0-52 and 1-15 kg
were obtained from the aquaria of the Marine Biological Association, Plymouth, and
were transferred to holding tanks in Birmingham which contained aerated, recirculated and filtered sea water at 13-5 ± 1 °C, where they were allowed to acclimate for at
least two weeks prior to experimentation.
The fish were anaesthetized by placing them in aerated sea water containing
approximately 0-04 g I"1 of MS 222 (Sandoz Ltd). The actual concentration used
varied between animals. The anaesthetized fish were then placed on an operating
table in a constant-temperature room held at the acclimation temperature, and
artificially irrigated with sea water containing MS 222. Polythene cannulae (Portex
Ltd) containing heparinized dogfish saline were inserted into the first right afferent
branchial artery, the caudal artery to a level just posterior to the iliac arteries in the
dorsal aorta, and into the caudal vein at a level just posterior to the renal portal veins
(for injection of drugs). In those fish (41) in which blood flow was measured, a
cannulating electro-magnetic flow transducer (Biotronex Ltd.), 1 cm in length and
with a bore of approximately 1 mm, was inserted into the ventral aorta between
2nd and 3rd branchial arteries. This allowed the measurement of blood flow to
Cardio-regulation in dogfish
79
first two pairs of afferent branchial arteries (cf. Butler & Taylor, 1975). The pulsatile
flow measured by this technique has been termed ' stroke flow' and it is taken as an
indication of stroke volume. A previous investigation has shown that these values
represent approximately 37% °f t n e actual cardiac output and stroke volume respectively, and that the same proportion of blood flows through the flow probe during
the bradycardia induced by hypoxia (Taylor et al. 1977). The probe included a
catheter for the measurement of the ventral aortic blood pressure, and thus an
afferent branchial artery was not cannulated during this procedure.
The vagus nerves of those fish used for the electrical stimulation experiments
were exposed on both sides by cutting along the lateral line from a point dorsal to the
spiracle to the pectoral girdle. The muscle was incised until the anterior cardinal
sinus was exposed. By paring away the connective tissue it was possible to gain access
to the posterior branches of the vagus without damaging the sinus (cf. Taylor et al.
1977). The blood loss during this procedure was therefore minimal. The wounds were
temporarily sutured with linen thread. In those experiments involving the cardiac
chronotropic responses to electrical stimulation of the vagi, the forebrain was separated
from the mid-brain and destroyed by extirpation, and the spinal cord was destroyed
at a level just posterior to the vagal outflow by pithing from the posterior. This
procedure immobilized the animal without affecting any of the cardiovascular
responses to vagal stimulation. In six fish the anterior cardinal sinus was exposed and a
polythene cannula was pushed through the connective tissue on its dorsal surface
and pushed posteriorly along the sinus and down the ductus Cuvieri until its tip
lay within the sinus venosus. The cannula was then secured to the muscle, and the
muscle and skin were sutured. This procedure was executed with care so that the
connective tissue of the anterior cardinal sinus formed a tight fit around the cannula
which did not permit haemorrhage or the entry of air into the circulatory system. By
this means measurement of central venous pressure could be made via a pressure
transducer (S.E. Labs: S.E.M. 4-86).
The fish were then clamped into a Perspex experimental holding tank which
contained approximately 8 1 of aerated, recirculated and filtered sea water maintained
at 13-5 + 0-5 °C. They were left undisturbed for 3 h prior to experimentation to allow
recovery from the effects of the operation and anaesthetic (Butler & Taylor, 1971;
Taylor et al. 1977). The stitches securing the wounds were removed and the muscles
retracted to display the vagus, the seawater level being controlled so that it covered
the spiracles but left the wounds clear. The animal was found to irrigate normally
under these conditions, whilst access to the vagi was obtained.
The visceral cardiac (the cardiac branch arising from the visceral branch of the
vagus) or the branchial cardiac (the cardiac branch arising from the post-branchial
branch of the branchial division to the last gill arch) branches of the vagus were
cleared of the surrounding connective tissue and raised onto silver hook electrodes.
It was possible to pick up the visceral cardiac on to the electrodes free of any other
nerves. However, the branchial cardiac was picked up at a level central to the division
of the pre and post-trematic branches, since access to the branchial cardiac branch
itself necessitated the undesirable procedure of opening the anterior cardinal sinus.
her the branchial branch of the vagus to the fourth gill arch (the third vagal
ision) or that to the third gill arch were used in those experiments which involved
t
80
S. SHORT, P. J. BUTLER AND E. W. TAYLOR
the central stimulation of the branchial vagi. The positions adopted for the stimulatioif
of the vagus are illustrated diagrammatically in Fig. i. The nerves were stimulated
at intensities ranging from o-i to 60 V, frequencies between 1 and 500 Hz and a
pulse width of 1 ms. In some experiments the effect of vagal stimulation after injection
of 0-2 mg kg"1 of atropine sulphate (Sigma) was tested. The abilities of each of the
cardiac vagi to elicite a bradycardia was investigated at stimulus intensities which
produced a maximal effect. It was not practical to use such a procedure during the
experiments involving the simultaneous stimulation of two cardiac vagi, or those
concerned with the response to changes in stimulus frequency, as a less intense
bradycardia was required in order to study these responses. The central cut ends of the
vagal branches were also stimulated at voltages that would produce a submaximal
effect since higher stimulus voltages were found to disturb the animal. An external
switch was set to trigger square wave pulses of predetermined intensity, duration
and frequency from a physiological stimulator (Farnell instruments) and also to
trigger an event marker on a 4-channel rectilinear pen recorder (Devices: M4).
Dorsal and ventral aortic blood pressures were displayed via two pressure transducers
(S.E. Labs: S.E.M. 4-86), and blood flow was measured by a Biotronex BL-610
pulsed logic electromagnetic flow meter set to an upper frequency response of 50 Hz.
This equipment was calibrated at the end of an experiment with the dogfish's blood
held at the experimental temperature and stroke volume calculated by integrating
the flow waveform (Butler & Taylor, 1975). In those experiments where the effects of
adrenergic beta receptor blocking agents were tested, the peripheral cut ends of the
cardiac vagi were electrically stimulated at a frequency of 50 Hz, a pulse width of 1 ms
and at various intensities (up to 1 V) so that a reduction in heart rate of approximately
50% was obtained. This reduction in heart rate was comparable to that induced
during hypoxia when the Px Of was reduced to approximately 30 mmHg.
It was found that 0-4 mg kg"1 of DL-propranolol HC1 (Sigma) was sufficient to
abolish the cardio-vascular effects of o-1 mg kg" 1 of the pure adrenergic beta-receptor
stimulating agent DL-isoproterenol sulphate (Sigma). Injection of o-i mg kg" 1 of
arterenol tartrate (Sigma) or epinephrine tartrate (Sigma) in animals in which 0-4
mg kg"1 of Propranolol had previously been injected caused variable circulatory
changes which could be abolished by 0-4 mg kg"1 of the alpha receptor blocking
agents phentolamine mesylate BP (Rogitine, Ciba) or dihydroergotamine tartrate
(Sigma). These doses of alpha and beta adrenergic receptor blocking agents were,
therefore, applied as standard and their potency was tested at the end of an experiment by injecting the relevant agonists.
Blood flow along the ventral aorta was monitored during the following experimental
situations:
(1) During the response to electrical stimulation of the peripheral cut end of the
branchial cardiac branch of the vagus, during normoxia, both before and after
injection of Propranolol (ten fish).
(2) During the response to hypoxia both before and after injection of Propranolol
(eight fish). In these experiments hypoxia was rapidly induced using a technique
that has been described elsewhere (Butler & Taylor, 1971). The P 7 Oi was reduced to
approximately 30 mmHg within 1 min and then maintained at this level for 10 m f l
(3) During the responses to electrical stimulation of the peripheral cut end of the
Cardio-regulation in dogfish
81
IX
Branchial branches of X
Branchial cardiac vagus
Visceral cardiac vagus
Fig i. A diagrammatic illustration of the cranial nerves IX and X of the dogfish, Scyliorhinus canicula (left side, dorsal view). The positions adopted for stimulation are indicated
by arrows.
branchial cardiac branch of the vagus during hypoxia, in cardiac vagotomized fish,
both before and after injection of Propranolol (four fish).
(4) During the response to a rapidly induced hypoxia lasting appioximately
10 min after injection of both Propranolol and Rogitine (three fish).
in the present report all mean values are expressed plus or minus the standard
ror of the mean and the number of observations put in parentheses. Differences
t
82
S. SHORT, P. J. BUTLER AND E. W. TAYLOR
between means were determined by Student's t test or, where applicable, by
paired comparisons technique (Bailey, 1959). The term 'significant' refers to the
95 % l eve l °f confidence (P < 0-05).
RESULTS
Histology
The branchial cardiac vagi contain approximately 420 myelinated fibres, whereas
the visceral cardiac vagi contain approximately 300 myelinated fibres (Fig. 2). There
was no appreciable variation of the number of fibres in the cardiac branches of
different animals. No non-myelinated fibres could be observed and no differences
between the left and right sides of the fish were apparent.
The cardio-inhibitory response to electrically stimulating the peripheral cut end of the
vagus
This series of experiments was performed on 45 dogfish of mass 0-74 ± 0-02 kg.
During normoxia (P7to, = 147 ± 3 (5)) the mean Pa> 0 | was 88 ± 2 (5) mmHg, mean
heart rate wa9 29 + 2 (45) beats min"1, and mean ventral and dorsal aortic pressures
were 31 ±1 (38) mmHg and 25 ±1 (35) mmHg respectively. Cardio-inhibitory
responses to vagal stimulation could be obtained from fish for up to 3 days without
any appreciable change in the levels of the measured variables.
The peripheral stimulation of the visceral cardiac branches of the vagus produced
inhibition in only 42 % of experiments (whatever the intensity of stimulation) although
ineffective nerves were often capable of producing cardio-inhibition if they were
stimulated centrally, when one or both of the branchial cardiac vagi were still intact.
This would, therefore, indicate that the absence of response was due to the nerve
being ineffective centrifugally rather than there being a deterioration in its physiological condition. Ineffective visceral cardiac vagi were found on either the left or
right side of the animal, or occasionally on both sides. The peripheral stimulation
of the branchial cardiac branch of the vagus always produced a profound cardioinhibition and no differences between the left or right sides of the fish were apparent.
Electrically stimulating the cardiac vagi, at voltages that were above threshold for all
fibres, revealed that the branchial cardiac branches were able to cause almost complete
cardiac inhibition (92 ±2 (13)% reduction in heart rate), whereas the visceral cardiac
branches were only able to cause a 50 ± 19 (10) % reduction in rate (Fig. 3).
In experiments that were undertaken on 20 animals, the cardiac nerves were
stimulated at frequencies ranging from 1 to 500 Hz and it was found that the degree
of cardio-inhibition was dependent upon the stimulus frequency. The degree of
inhibition was optimal when the cardiac nerves were stimulated at frequencies
between 25 and 50 Hz (Fig. 4). There appeared to be no appreciable differences
between the spectrum of frequency responses of the branchial cardiac (n = 18)
and visceral (n = 6) vagi, although the mean response at 10 Hz was significantly
higher for the branchial cardiac vagi (P < 0-05). In all other experiments 50 Hz was
Fig. 3. Transverse sections of a left branchial cardiac branch of the vagus (top), and a right
visceral cardiac branch (bottom). Note the much larger number of fibres in the branchial
cardiac vagus.
Journal of Experimental Biology, Vol. 70
Fig. 2
0-1 mm
S. SHORT P. J. BUTLER AND E. W. TAYLOR
(Facing p. 82)
Cardio-regulation in dogfish
83
Adopted as the routine frequency of stimulation, except when looking for augmentary
Effects at low stimulation frequencies.
Simultaneous stimulation of the peripheral cut ends of the cardiac vagi
In 24 experiments, pairs of cardiac nerves were individually stimulated at submaximal voltages both before and after simultaneous stimulation so that it was
possible to compensate for any change in the intensity of cardio-inhibition that
occurred whilst the cardiac vagi were raised on to the electrodes. Fig. 5 shows a
result obtained from an individual fish and suggests that the effect of stimulating
the two nerves together was greater than simple summation of their individual
effects. In order to obtain mean values for the effect of stimulating various paired
combinations of the cardiac nerves, the effect upon heart rate of stimulating any one
nerve was obtained by averaging the effects both before and after combined stimulation and compared with the effect of stimulating the two nerves together. On
average, the bradycardia produced by simultaneously stimulating any two cardiac
vagi was little more than a summation of their individual effects (Fig. 6).
The cardiac effects of centrally stimulating the vagal branches
The electrical stimulation of the central cut end of the branchial cardiac, visceral
cardiac or branchial branches of the vagus (89 experiments on 26 animals) produced
a cardio-inhibition when all other cardiac branches were intact. No differences were
apparent in the capabilities of the branchial, branchial cardiac or visceral cardiac
branches of the vagus, with respect to their ability to elicit a 'reflex' bradycardia
by the electrical stimulation of cut central ends.
Sequential sectioning of the cardiac efferent branches during central stimulation
revealed that the cardio-inhibitory response was both ipsilateral and contralateral,
and that the visceral cardiac was less capable of relaying this ' reflex' motor activity
to the heart than the branchial cardiac branches. Not only was the intensity of cardioinhibition reduced when the 'reflex' information was conveyed to the heart via the
visceral cardiac, as opposed to the branchial cardiac vagi, but the former were often
found to be ineffective in producing any cardio-inhibition at all. Cardiac nerves, which
did not convey efferent activity to the heart in response to central stimulation of
other nerve tracts were tested to see if they were effective in producing cardioinhibition by peripheral stimulation. It has been mentioned that only 42% of the
visceral cardiac vagi were effective in producing cardio-inhibition when stimulated
peripherally, and in these experiments it was found that only a third of these effective
visceral cardiac branches were capable of inhibiting the heart in response to central
stimulation of other nerve tracts, as opposed to 30 of the 31 branchial cardiac branches
that were tested.
The absence of cardio-acceleratory responses to vagal stimulation
Neither the peripheral stimulation of the cardiac branches of the vagus nor the
central stimulation of the branchial or cardiac branches resulted in cardio-acceleration
| t any of the frequencies (1-500 Hz) or intensities (o-i-6o V) of stimulation investiPited (105 experiments on 69 animals). All the cardio-inhibitory responses to
S. SHORT, P. J. BUTLER AND E. W. TAYLOR
Time(min)
jG 5 0
o.
* * *
[
0-35 V 0-40 V
0-45 V
0-50 V
0-55 V
0-60 V
0-65 V
iI
0-50 V
0-60 V
0-70 V
0-80 V
0-90 V
100 V
Fig. 3. (A) Cardio-inhibition produced by electrically stimulating the peripheral cut ends
of the right branchial cardiac branch of the vagus (50 Hz, 1 ms) of a 063 kg female dogfish.
Note that an almost complete cardio-inhibition occurs at high stimulus voltages (as indicated under each trace). The time marker indicates minute intervals and applies to all traces.
(B) Obtained from a 055 kg male dogfish during periods of electrically stimulating the
peripheral cut end of the left viscera] cardiac branch of the vagus (50 Hz, 1 ms). Note that
during supramaximal stimulation the intensity of the resultant cardio-inhibition is much
less than that in trace (A).
80
70
60
2 50
c
40
I
I 30
I
20
10
0
I
0 10
25
50
100
250
Frequency of stimulation (Hz)
Fig. 4. The mean effect of increasing the frequency of stimulation of the cardiac branches
of the vagus on the resulting percentage reduction in heart rate. There was no significant
difference between the inhibitory effects of the branchial cardiac (full line) and visceral cardiac
(dotted line) branches of the vagus at all frequencies of stimulation with the exception of 10 Hz
where the branchial cardiac nerves (18 experiments) elicited a significantly more intense
cardio-inhibition than the visceral cardiac vagi (6 experiments).
stimulation of these nerve tracts were abolished by injection of cc2 mg kg"1 of atropine
sulphate into the caudal vein.
A bradycardia elicited by either vagal stimulation or acetylcholine injection was
not followed by a post-inhibitory tachycardia in animals with normal blood pressur
and heart rates, although it was often observed in animals that had deteriorated a:
1
Cardio-regulation in dogfish
40
00 o
3
CA
£
0
40
u
0
u
a.
1s
I
LBCp
RVCp
RVCp
LBCp
Fig. 5. The effect of electrically stimulating the peripheral cut ends of the left branchial cardiac
(LBCp) and right visceral cardiac (RVCp) branches of the vagus, both individually and
simultaneously (S), on the heart rate and blood pressures of a o#5» kg female dogfish. The
time marker indicates minute intervals.
30
20
10
Fig. 6. Histograms illustrating the mean effects of stimulating the peripheral cut ends of two
cardiac vagi both individually and simultaneously, on the heart rate of the dogfish (n = 24).
(a) Initial heart rate; (6) inhibition initiated by left branchial cardiac branch; (c) inhibition
initiated by left visceral cardiac branch; (d) when (6) and (c) simultaneously stimulated; («)
repeat (6); (/), repeat (c).
displayed extremely low heart rates (9 beats min"1) and mean blood pressures
(17 mmHg in ventral aorta).
Changes in cardiac stroke flow occurring during peripheral stimulation of the cardiac vagi
In nine animals the stroke flow to the first two pairs of afferent branchial arteries
was measured during the electrical stimulation of the peripheral cut ends of the
cardiac branches of the vagus. It may be seen from Fig. 7 that the reduction in heart
rate is associated with an increased stroke flow. This increase was not further potentiated by stimulation at different frequencies or intensities. The increases in stroke
flow that were observed were purely related to a reduction in heart rate, and there
were no differences between the visceral cardiac and branchial cardiac vagi in this
k c t . Injection of 02 mg kg- 1 of atropine sulphate abolished the bradycardia
icited by vagal stimulation, and the associated increase in stroke volume. No further
86
S. SHORT, P. J. BUTLER AND E. W. TAYLOR
Heart rate = 35 beatsmin" 1
Stroke flow=015 ml
Heart rate = 18 beats min ~'
Stroke flow=0-26 ml
Venous pressure
(mmHg)
Stroke flow 30
(ml)
0
Ventral aortic 50 |~
blood pressure g \_
(mmHg)
-1
Fig. 7. The effect of stimulating the peripheral cut end of the left visceral cardiac branch of
the vagus (06 V, 50 Hz and 1 ms pulses) on the heart rate, stroke flow, central venous pressure
and ventral aortic blood pressure. The values of heart rate and stroke flow are indicated at
two points on the trace. The trace was obtained from a male dogfish (mass O'8o kg). The
period of stimulation is indicated on the event marker trace, and the time marker indicates
1 s intervals.
effects could be produced at any of the intensities (o-i-6o V) or frequencies (1-500
Hz) of stimulation investigated (five animals).
The heart rate, stroke flow and total flow changes that occur during reductions in
heart rate of approximately 30% and 50%, elicited by electrical stimulation of the
vagus, are presented in Table 1. A reduction in heart rate of 28^8 ± 4*2 (9) % resulted in
a 33'° ± io-6 (9)% increase in strokeflowand as a consequence no significant reduction
in total flow occurred. However, a 52-2 + 5*3 (9)% reduction in heart rate resulted in a
38-6 ± 8-8 (9)% increase in stroke flow. This change in stroke flow did not completely
compensate for the induced bradycardia so that a significant reduction in the total
flow occurred. In six animals the central venous pressure was measured and was
found to be —3-4 mmHg at rest and became more positive ( — 2-1 mmHg) during a
50 % reduction in heart rate induced by either electrical stimulation of the vagus or
by hypoxia (Fig. 7).
The effects of Propranolol on cardiac stroke flow
These experiments were performed on dogfish of mass 075 + 0-04 (25) kg and the
mass of the dogfish was not significantly different between experimental categories.
In control fish (those used for the hypoxia experiments) the mean normoxic values of
ventral and dorsal aortic blood pressure were 29 ± 2 (8) and 24 ± 1 (7) mmHg respectively. The corresponding mean values for other experimental categories did not
differ from these values by more than 8 mmHg (cf. Short, 1976). However, significant
differences occurred in the levels of the measured variables between experimental
categories, either as a result of vagotomy or injection of adrenergic receptor blocking
agents (cf. Fig. 8). In general, the vagotomized animals had an increased heart rate
and blood pressures and a reduced stroke flow (cf. Short, 1976). The results obtained
from 17 animals in this series of experiments have indicated that there is no significant
change in heart rate after injection of Propranolol. However, it is evident that this
adrenergic y?-receptor blocking agent severely effects the circulatory system, since
significant reductions occur in stroke flow (18 + 4%), total flow (2215%), mean
ventral aortic pressure (12 ±3%) and mean dorsal aortic blood pressure (19 + 4%).
There was an increase in stroke flow during the bradycardia induced by electri^
stimulation of the peripheral cut end of the vagus during normoxia (Fig. 7), and thrc
Cardio-regulation in dogfish
87
Table 1. The mean ( ± s.E. of mean) values of the measured variables in unanaesihetized
dogfish at rest during normoxia and during a reduction in heart rate of approximately
3 0 % (^4) and 5 0 % (B), elicited by the electrical stimulation of the peripheral cut ends
of the vagus
Resting
(A) Heart rate (beats min-1)
Stroke flow (ml)
Total flow (ml kg' 1 min"1)
40-2 ±»-8
o-i7±o-oi
(B) Heart rate (beats min"1)
Stroke flow (ml)
Total flow (ml kg"1 min"1)
O-I8±O-OI
9-6±II
99 ±07
During
cardioinhibition
(« = 9)
28-5 ±2-2*
o-aa±o-oi #
8-8±o-9
o-as±o-oi#
6o±i-3»
• Signifies significant differences (P < 002) using the paired comparisons technique.
0-45
r
Stim/
Norm
Stim/Norm/
Prop
Hyp
Hyp/
Prop
Stim/
Hyp
Stim/Hyp/
Prop
040
_
0-35
1
•5 0-30
0-25
o
w5
-h
0-20
015
0-10
Initial heart rate 40±3(9) 36 ±2 (10) 31 ± 2 (8) 29±3(8) 48±1(4) 35±4(4)
(beats min"1)
Reduced heart rate 19±3 (9) 15±2(10) 18±2(8) 17±2(8) 18±1(4)
(beats min"')
16±2(4)
Fig. 8. The mean ( ± 8.E. of mean) values of stroke flow that occur in resting dogfish (plain
histograms) and during a reduction in heart rate of approximately 50% (dotted histograms)
initiated by: Stim/Norm, electrical stimulation of the peripheral cut end of the cardiac vagi
during normoxia (9 animals); Stim/Norm/Prop, Stim/Norm after injection of Propranolol
(10 animals); Hyp, hypoxia (8 animals); Hyp/Prop, hypoxia after injection of Propranolol
(8 animals); Stim/Hyp, electrical stimulation of the vagus during hypoxia in vagotomized fish
(4 animals), and Stim/Hyp/Prop, Stim/Hyp after injection of Propranolol (4 animals). The
mean ( ± s.B. of mean) initial and reduced heart rates are entered below each set of histograms.
Significant changes in stroke flow during cardio-inhibition are indicated by • . Arrows between histograms indicate when injection of Propranolol had a significant effect on the
response.
88
S. SHORT, P. J. BUTLER AND E. W. TAYLOR
stroke flow response was not affected by injection of Propranolol (Fig. 8). However,
injection of Propranolol abolished the increased stroke flow that occurred in association with the bradycardia induced by hypoxia (Fig. 8), and this response was not
alleviated by injection of an a-receptor blocking agent. Propranolol also had similar
action on the response to electrically stimulating the peripheral cut end of the cardiac
vagus during hypoxia (in vagotonmed animate).
During electrical stimulation of the peripheral cut end of the vagus, total blood
flow to the first two pairs of gills was approximately 6-o ml min -1 kg" 1 during both
normoxia and hypoxia, whereas the mean drop in blood pressure across the gills was,
on average, 7-4 mmHg during normoxia and 3-2 mmHg during hypoxia. This indicates
that the branchial blood vessels dilated during hypoxia and, using the conventional
calculation for vascular resistance (see Butler & Taylor 1975), that resistance in the
branchial vessels approximately halved. However, following the injection of Propranolol, hypoxia appeared to cause constriction in the branchial blood vessels, for
although total flow dropped to 2 ml min -1 kg"1, the pressure difference across the
gills was 7-4 mmHg giving a calculated resistance in the branchial vasculature approximately 10 x greater than during hypoxia before injection of Propranolol.
DISCUSSION
The vagal control of heart rate
Selective cardiac nerve transection techniques have previously been employed
to demonstrate that the branchial cardiac vagi convey the majority of the tonic
inhibitory activity to the heart during normoxia and are responsible for the greater
part of the reflex bradycardia initiated by hypoxia. The visceral cardiac nerves
appeared to be of secondary importance in both these respects (Taylor et al. 1977).
These observations have been extended by this investigation in that the branchial
cardiac vagi have been shown to have a greater capacity to inhibit the heart, when
stimulated electrically, than the visceral cardiac vagi, and furthermore, the latter are
often ineffective in producing a cardio-inhibition. However, no difference in the
abilities of the cardiac nerves to convey afferent activity could be detected. Evidence
therefore suggests that although the branchial cardiac vagi have both sensory and
motor capabilities the visceral cardiac vagi may have a mainly sensory function. It
is possible that a proportion of the motor fibres contained within the visceral cardiac
vagi may innervate regions other than the cardiac pacemaker and this could, to some
extent, explain the different cardiac chronotropic abilities of the cardiac vagi. In this
connexion it is also interesting that the visceral cardiac vagi contain substantially
fewer fibres than the branchial cardiac vagi. The results presented here indicate that
the effects of the cardiac branches of the vagus interact at the myocardium. It is
therefore possible that the visceral cardiac vagi may modulate the effects of the
branchial cardiac vagi, but, since the former are often ineffective in producing cardioinhibition, their motor effects are probably only of secondary importance.
Recent investigations have shown that the afferent activity in cranial nerves IX, X,
V and VII is of importance in generating the inhibitory vagal tone upon the heart
(Butler, Taylor & Short 1977). In the present investigation it was evident that t l 4
electrical stimulation of the central cut ends of the branchial branches of the vagus
Cardio-regulation in dogfish
89
initiated cardio-inhibition, and that this response was both ipsilateral and contralateral.
In addition, it was apparent that the branchial cardiac vagi were more effective in
conveying inhibitory activity to the heart when stimulated at the central cut end of
the cardiac or branchial branches of the vagus.
The central stimulation of the branchial branches of the vagus always produced
cardio-inhibition, whatever the stimulus intensity or frequency. These results are in
direct contrast with those obtained in a number of teleosts (Kulaev, 1957, 1958) and
elasmobranchs (Rodionov, 1959), where although strong stimuli caused cardioinhibition weak stimuli were reported to cause cardio-acceleration. Similar investigations on fish (Cobb & Santer, 1973) and other vertebrates (Bulbring & Burns,
1949; Marshal & Vaughan Williams, 1956; Burn & Rand, 1957; Jensen, 1958;
Misu & Kirpekar, 1968) lead to the suggestion that reliable cardio-acceleratory
responses to vagal stimulation or acetylcholine application can be obtained in preparations which had been allowed to deteriorate. In general, the heart was exposed and
beating at a low rate or had become quiescent. This investigation has indicated that
these responses do not occur in dogfish when the heart is not exposed and the heart
rate and blood pressures are normal. The present investigation on intact dogfish was
unable to demonstrate a tachycardia on recovery from vagal stimulation or administration of acetylcholine unless the animal was allowed to deteriorate until the heart
rate was low. Therefore this phenomena is probably also dependent upon experimental conditions, and this may perhaps explain why some authors have reported a
recovery tachycardia (Fange & Ostlund, 1954; Cobb & Santer, 1973) and others have
noted its absence (McWilliam, 1885; McKay, 1931; Randal, 1966).
Control of cardiac stroke flow
The cardiac adrenergic innervation of teleosts is thought to reach the heart via the
vagus (see introduction for references). In support of previous investigations (Young,
1931, 1933) it is thought unlikely that adrenergic fibres pass within the cardiac vagus
of elasmobranchs since no non-myelinated fibres could be observed in sections of the
cardiac nerves. There is physiological evidence that adrenergic nerves are absent in
the vagus, since no heart rate increases can be initiated by electrical stimulation of
this nerve (Bottazzi, 1902; Lutz, 1930) and such a conclusion has been borne out by
the present investigation. In addition, however, it has been shown that increases in
stroke flow cannot be initiated by vagal stimulation at any intensity or frequency of
stimulation after administration of atropine. The only increases in stroke flow that
occurred in normal circumstances were directly related to reductions in heart rate
and were, therefore, considered to be a manifestation of Starling's Law of the heart.
The results indicate that moderate reductions in heart rate do not cause reductions
in cardiac output. In fact, three fish displayed a mean increase in total flow of 23 %
during a reduction in heart rate of 25 % and thus, relatively small reductions in heart
rate may result in an increase in cardiac output. It is possible that this mechanism
may explain the increase in cardiac output that was associated with small (but nonsignificant) reductions in heart rate during the responses of S. canicula to hypoxia at
7 °C (Butler & Taylor, 1975).
The effects of Propranolol on the circulatory system may be due to the removal
t>f the effects of catecholamines or to local anaesthetic properties of the drug (Eliash &
go
S. SHORT, P. J. BUTLER AND E. W. TAYLOR
Weinstock, 1971; Lee et al. 1975). However, moderately high concentrations of
circulating catecholamines have been demonstrated in S. canicula (Mazeaud, 1969;
Butler, Taylor, Capra & Davidson, 1978) and it seems likely that they will have important
effects on the circulatory system. Recent investigations on Squalus acanthias have shown
that Propranolol blocks the dilator tone of catecholamines on the gill vasculature and
potentiates a vasco-constrictor response (Capra & Satchell, 1974; Capra, 1975). In
addition, it has been shown that Propranolol blocks the positive inotropic responses to
catecholamines in the isolated heart (Capra, 1975). The present results, obtained by
injection of Propranolol, indicate that blood catecholamines are exerting a dilator
tone on the branchial vasculature and are augmenting stroke volume during normoxia.
The former of these is not thought to be due to side-effects of Propranolol, since
large concentrations of this drug result in a decreased peripheral resistance in mammals (Lee et al. 1975). Our results also suggest that circulating catecholamines have
an increased dilator effect on the branchial blood vessels during hypoxia which
masks a potential vasoconstriction (cf. Satchell 1962).
An increased stroke flow is associated with the cardio-inhibition induced by
electrical stimulation of the peripheral cut end of the vagus during normoxia, and
it has been shown that this relationship is unaffected by Propranolol (cf. Butler &
Taylor, 1975; Taylor et al. 1977). The increase in stroke volume that is associated
with the bradycardia induced by hypoxia is abolished by Propranolol, thus suggesting
that circulating catecholamines may be of importance (cf. Butler & Taylor, 1975).
In contradiction, cardiac vagotomy abolishes both the bradycardia and the rise in
stroke volume during hypoxia (Taylor et al. 1977) and no adrenergic fibres can be
demonstrated in these cardiac branches. These results may seem paradoxical. However, it has been shown that hypoxia alone was not responsible for the discrepancy in
the results, as stroke flow increases during the bradycardia induced by electrically
stimulating the cardiac vagi during hypoxia, in vagotomized fish. This response could,
however, be abolished by Propranolol and it would seem that the lack of a rise in
stroke volume during hypoxia results from the combined depressant effects of
hypoxia and Propranolol on myocardial contractility. Indeed, Propranolol has been
demonstrated to cause direct myocardial depression in mammals (Lee et al. 1975) and
may reduce myocardial blood flow (Drake, 1976). It is certainly possible that the
latter may have more severe consequences during hypoxia. In addition, no significant
increases in stroke volume occurred during hypoxia after both alpha and beta adren ergic receptor blockade. It seems therefore that the lack of an increased stroke
flow during hypoxia after injection of Propranolol was not due to a potentiation of
alpha adrenergic receptor stimulation.
The evidence indicates that although circulating catecholamines may be of importance in maintaining cardiac activity, vascular resistance and blood pressures during
normoxia, they appear to be of little importance in increasing the cardiac stroke
volume during the response to hypoxia. The increase in cardiac stroke flow that
occurs is a manifestation of Starling's Law of the heart and this relationship is such
that no significant reduction in cardiac output occurs during moderate reductions
in heart rate.
The authors wish to thank the Science Research Council for financial support.
Cardio-regulation in dogfish
91
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