AN ABSTRACT OF THE THESIS OF
MASTER OF SCIENCE
ROBERT EDWARD STUART for the degree of
December 11, 1981
in FISHERIES presented on
Title:
PHYSIOLOGICAL AND PHARMACOLOGICAL INVESTIGATION OF THE
VASCULARIZED AND NONVASCULARIZED TELEOST HEART WITH ADRENERGIC,
CHOLINERGIC AND CORONARY DATING AGENTS
Abstract approved:
Redacted for Privacy
Lavern J. Weber
Teleost hearts can be classified into two types, vascularized
(coronaries) and nonvascularized, based on the arrangement of the
ventricular myocardium.
Anatomical investigation of the buffalo sculpin
(Enophrys bison) heart has revealed that it lacks coronary circulation.
To see if the nonvascularized heart obeys Starling's law of the heart
and to characterize it as to the type of adrenergic receptors present,
isolated sculpin hearts were attached by the sinus venosus to a modified
Langendorff perfusion apparatus.
A pressure transducer was placed in
series between the bulbus arteriosus and a vertical 15 cm length of
glass tubing so as the heart pumped perfusate a resting pressure would
be established for:the heart to work against.
Heart rate, systolic
pressure, and cardiac output were monitored.
As atrial perfusion pressure was increased, the sculpin heart
responded with a positive inotropy and an increase in cardiac output.
Heart rate did not change.
Epinephrine (0.64 pg /ml) accentuated the
pulse pressure respone, causing an overall increase in pressure and
heart rate.
Acetylcholine (1.0 pg/ml) resulted in a negative chronotropy
and a positive inotropy.
The data indicate that the contractility
increases with elevated filling pressure, demonstrating Starling's law.
Isoproterenol results in positive chronotropy which could be
blocked by propranolol.
Methoxamine was without effect.
The sculpin
heart then appears to be under the control of beta adrenergic receptors.
A further study was carried out to determine what effect vasoactive
drugs would have on the fish heart.
Chum salmon (Oncorhynchus keta)
were used as a source of vascularized hearts.
Hearts were mounted on
the Langendorff perfusion apparatus as described previously with the
exception that'the coronary artery was perfused in the salmon hearts
via a cannula attached to a perfusion pump.
The buffalo sculpin heart showed no responses when exposed to
sodium nitrite (1 pg/ml), nitroglycerin (100 pg /mi), or perhexiline
(1 pg/ml) while prenylamine (10 pg/ml) caused negative chronotropy and
increased stroke volume.
Nitroglycerin (100 pg/ml) and sodium nitrite
(1 pg/ml) appeared to have no direct effect on the myocardial tissue of
the chum salmon.
Sodium nitrite, when administered through the coronary
artery, produced a prolonged and gradual increase in coronary pressure
which continued after their removal.
results.
Nitroglycerin produced no conclusive
Acetylcholine (0.5 pg/ml) decreased heart rate, increased
systolic pressure, stroke volume and vascular resistance by both atrial
and coronary administration but these changes were greater when acetylcholine was administered through the atrium.
Epinephrine (0.5 pg/ml) administered through the coronary artery
had no significant effect on any of the parameter's measured.
When per-
fused through the chambers, stroke volume declined but none of the other
parameters were changed.
Physiological and Pharmacological Investigation of the
Vascularized and Nonvascularized Teleost Heart with Adrenergic,
Cholinergic and Coronary Dilating Agents
by
Robert Edward Stuart
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed December 11, 1981
Commencement June 1982
APPROVED:
Redacted for Privacy
Professor of Fisheries in charge of major
Redacted for Privacy
Head of Department of Fisheries and Wildlife
Redacted for Privacy
Dean of Graduatk school
Date thesis is presented
U
December 11, 1981
Typed by Anita deMercado Stuart for
Robert Edward Stuart
ACKNOWLEDGMENTS
The completion of this thesis would not have been possible without
the encouragement, guidance, and love of many people.
those is my major professor, Dr. Lavern J. Weber.
Foremost among
I wish to thank him
for always being available whenever I had questions or problems.
The
Thursday night groups, the Sunday dinners and the time spent spear
fishing all contributed in some way to my education and will long be
remembered.
I would also like to thank the members of my committee, Dr. James
L. Hedtke, Dr. John R. Smith, Dr. Robert E. Olson and Dr. Paul Kifer.
Additionally, special thanks go to friend and fellow student Daniel B.
Gant for helping me relieve my frustrations through the strangulationrelaxation technique.
By rights 50% of this thesis belongs to my wife, Anita.
She's the
one who listened to my rantings and ravings when everything seemed to
go wrong.
She had faith in me when I had lost faith in myself and was
always a source of understanding and love.
Without her this thesis
would never have been put on paper, especially since she typed it.
To
her go my love and thanks.
I'm greatly indebted to my parents, Buren B. Stuart and Gertrude
M. Stuart.
Their love and encouragement was always given generously
and was unrelenting.
This work was supported by NIEHS ESO 1926.
TABLE OF CONTENTS
Page
CHAPTER I.
INTRODUCTION
1
MATERIALS AND METHODS
7
RESULTS
12
DISCUSSION
25
CHAPTER II.
29
INTRODUCTION
29
MATERIALS AND METHODS
RESULTS
Sculpin
Salmon
31
34
34
45
DISCUSSION
61
BIBLIOGRAPHY
68
APPENDIX
75
LIST OF FIGURES
Page
Fig.
1
Fig. 2
Fig. 3
Schematic showing attachment of heart to perfusion
manifold and pressure transducer
8
Electronmicrograph and light photomicrographs of the
ventricular myocardium of the buffalo sculpin and
chum salmon
13
Strip chart recordings of in vivo and in vitro heart
rate and pulse pressure from the buffalo sculpin
exposed to adrenergic and cholinergic agonists and
antagonists
15
Fig. 4
Changes in heart rate, pulse pressure and stroke volume
17
over a two hour period for the control hearts
Fig. 5
Effects on heart rate, stroke volume, pulse pressure
and cardiac output of hearts exposed to varying atrial
perfusion pressures in the absence and presence of
epinephrine (0.64 pg/ml)
21
Effects on heart rate, stroke volume, pulse pressure
and cardiac output of hearts exposed to varying atrial
perfusion pressures in the absence and presence of
acetylcholine (1.0 pg/ml)
23
Fig. 6
Fig. 7
Changes in heart rate, pulse pressure and stroke volume
35
over a 50 minute interval for the control hearts
Fig. 8
Heart rate, pulse pressure and stroke volume changes in
the isolated buffalo sculpin heart exposed to the
vasodilators sodium nitrite (1.0 pg/m1) and nitro37
glycerin (100 pg/ml)
Fig. 9
Strip chart recordings of heart rate and pulse pressure
from the isolated buffalo sculpin heart exposed to the
vasodilators prenylamine, perhexiline, nitroglycerin
and sodium nitrite
Fig. 10
Changes in heart rate, pulse pressure and stroke volume
in the isolated buffalo sculpin heart administered the
vasodilators perhexiline (1 pg/ml) and prenylamine
(10 pg/m1)
Fig. 11
39
41
Strip chart recordings of heart rate, pulse pressure
and coronary pressure from isolated salmon hearts
exposed to acetylcholine (0.5 pg/m1) and epinephrine
(0.5 p9 /ml)
46
LIST OF FIGURES, Contd.
Page
Fig.
Fig.
Fig.
Fig.
Fig.
12
13
14
15
16
Graph of heart rate, pulse pressure, stroke volume
and coronary pressure in the isolated salmon heart
during administration of acetylcholine (1.0 pg/m1)
first through the coronary artery and then through the
sinus venosus
48
Effects of epinephrine (0.5 pg/ml) on heart rate, pulse
pressure, stroke volume and coronary pressure in the
Drug was added first through
isolated salmon heart.
the coronary and then through the sinus venosus
51
Strip chart recordings of heart rate, pulse pressure
and coronary pressure from isolated salmon hearts
exposed to the vasodilators sodium nitrite (1 pg/m1)
and nitroglycerin (100 lig/m1)
53
Changes in heart rate, pulse pressure, stroke volume
and coronary pressure in the isolated salmon heart
exposed to sodium nitrite (1 pg/ml) first through the
coronary and then through the sinus venosus
55
Changes in heart rate, pulse pressure, stroke volume
and coronary pressure in the isolated salmon heart
exposed to nitroglycerin (100 pg/m1) first through the
coronary and then through the sinus venosus
57
LIST OF TABLES
Page
TABLE I.
Summary of the Effects of Acetylcholine, Epinephrine
and Vasodilators on the Isolated Buffalo Sculpin Heart
TABLE II. Summary of the Effects of Acetylcholine, Epinephrine
and Vasodilators on the Isolated Salmon Heart
44
60
PHYSIOLOGICAL AND PHARMACOLOGICAL INVESTIGATION OF THE
VASCULARIZED AND NONVASCULARIZED TELEOST HEART WITH ADRENERGIC,
CHOLINERGIC AND CORONARY DILATING AGENTS
CHAPTER I
INTRODUCTION
Since Motti's review of the fish cardiovascular system in 1957,
increasingly more work has been done on fish physiology and pharmacology.but there is still a relative paucity of information in this
field.
With over 20,000 species of fish, 96% of these represented
by the infraclass Teleostei (Nelson, 1976), extending over a wide
range of habitats, only a few species have been studied extensively.
With such a diverse group it is difficult to make, generalizations
based on the knowledge of only a few species.
Anatomically, the cardiovascular system of the fish shows some
conformity among the many species of fish, with the exception of
the cyclostomes and lungfishes.
The heart lies within a thin,
compliant pericardium located just beneath and slightly posterior
to the gills.
Venous blood is pumped by the heart through the
gills and systemic circulation and then returns to the heart.
The
teleost heart is a myogenic, branchial heart formed of typical
vertebrate cardiac muscle fibers (Randall, 1970; Satchell, 1971).
It consists of four chambers in series:
the sinus venosus, atrium,
ventricle and noncontractile but very elastic bulbus arteriosus,
thus forming a single circuit pump (Hildebrand, 1974).
2
To meet the circulatory demands of the tissues the heart
requires a regulatory mechanism.
The two factors determining
cardiac output are stroke volume and heart rate (Rushmer, 1961;
Satchell, 1971).
As in mammals, the teleost heart is controlled
aneurally and neurally.
A basic aneural control mechanism is
demonstrated by Starling'd law of the heart which states that the
strength of contraction is a function of the initial length of the
muscle fiber (Rushmer, 1961; Randall, 1970).
Bennion (1968) and
Butler (1976), using the isolated hearts from trout and shark
respectively, have confirmed that Starling's law is applicable to
the hearts of these two species.
It is believed that when the
pacemaker tissue is extended in the heart it is more sensitive to
stretch and depolarization (Jensen, 1961).
Neural mechanisms for controlling the heart in fish share some
similarities with those in mammals.
In the mammal, neural control
is mediated through the parasympathetic and sympathetic nervous
systems.
Hagfishes, Myxine and Eptatretus (Cyclostomata), appear to
have aneural hearts receiving no vagal supply (Jensen, 1961; Falck,
et al, 1961).
Neural control of the other fish heart, however, is
quite controversial.
It was observed that the isolated trout heart
could respond to catecholamines (Fange and Ostlund, 1954; Bennion,
1968).
With fluorescence histochemistry, catecholamine-containing
nerve fibers were found in the hearts of the cod, Gadus morhua
(Holmgren, 1977), Salmo trutta and S. irideus (Gannon and Burnstock,
1969), Carassius auratus, and several other species (Cameron, 1979).
Although it is commonly believed that acetylcholine is the major
neurotransmitter in the fish vagus nerve, Euler and Fange (1961)
also found norepinephrine in the vagus nerves of the cod (Gadus
morhua).
Randall (1970) reports that all fish hearts, with the
exception of the hagfish, are innervated by a branch of the vagus.
Stimulation of the vagus results in bradycardia (Randall, 1970)
which can be blocked by atropine (Mott, 1957; Chan and Chow,- 1976;
Gannon and Burnstock, 1969, Randall, 1966; Seibert, 1979).
Many
fish exhibit a reflex bradycardia (Cameron, 1979; Peyraud-Waitzenegger,
et al., 1980) to visual, auditory and mechanical stimuli which
suggests the slowing of the heart is due to an increasing level of
vagal control (Randall, 1970).
Saito (1973) observed both inhibitory
and excitatory responses in the carp heart during or after vagal
stimulation, which were abolished by atropine.
It has been found by Stevens and Randall
(1967a) and by Cameron
(1978) that a resting vagal tone is present in many fish.
Trout,
(Stevens and Randall, 1967; Wood and Shelton, 1980) Asiatic eel
(Anguilla japonica (Chan and Chow, 1976) and cod (Helgason, 1973)
are reported to have little cardiac vagal tone while the eel Anguilla
anguilla (Seibert, 1979) and the sea robin (Roberts, 1968) demonstrated
considerable vagal tone to the heart.
In Anguilla anguilla Seibert
(1979) proposed that the degree of vagal tonus may depend upon
temperature.
No sympathetic fibers could be found in the heart of plaice,
Pleuronectes platessa (Falck, et al., 1966) or in the eel, Anguilla
japonica (Chow and Chan, 1975).
Burnstock (1969) has reported
sympathetic fibers running in the vagus nerve of fish.
Sympathetic
control of the fish heart may also be mediated through circulating
4
catecholamines released by chromaffin tissue (Nakano and Tomlinson,
1967; Holmgren, 1977; Wahlquist and Nilsson, 1977).
It appears
that there may be interspecific variation in the sympathetic
innervation of the fish heart and that sympathetic control is
exerted by one or both of these mechanisms.
Adrenoreceptors have
been demonstrated pharmacologically in the isolated and intact
fish hearts.
Falck, et al
(1966) using the isolated hearts of
Lampetra fluviatilis and Pleuronectes platessa, found adrenoreceptors
of the beta type.
Cardiac beta receptors were also demonstrated
in the intact hearts of Salmo gairdneri (Wood and Shelton, 1980)
and the isolated (Holmgren, 1977) and intact (Wahlquist and Nilsson,
1977) hearts of the cod, Gadus morhua.
al
Peyraud-Waitzenegger, et
(1980), using Anguilla anguilla, demonstrated a seasonal variation
in the type of cardiac adrenoreceptors.
In summer, the fish
exhibited beta adrenoreceptor mediated activity whereas in winter,
alpha activity predominated.
Anguilla anguilla is reported to
have both alpha and beta adrenoreceptors (Forster, 1975) while
Anguilla japonica demonstrated only alpha activity (Chan and Chow,
1976).
In the isolated heart of the elasmobranch Squalus acanthias
a negative chronotropic response was shown to be mediated by alpha
receptors (Capra and Satchell, 1976a).
Fish appear then to exhibit
an interspecific, and perhaps, a seasonal variation in the type of
cardiac adrenoreceptors.
The myocardial catecholamines epinephrine and norepinephrine
in fish are reported to be lower than in mammals with the predominant
catecholamine being epinephrine (Euler and Frige, 1961; Burnstock,
1969; Lee, et al., 1970; Randall, 1970).
In the isolated heart of
Squalus acanthias epinephrine and norepinephrine produced positive
inotropy but epinephrine resulted in positive chronotropy while
norepinephrine exerted a negative chronotropic effect.
The potency
ratio was reported to be epinephrine (10):norepinephrine (1000)
(Capra and Satchell, 1976a).
Catecholamines produced no chronotropic
effect in the eel Anguilla japonica (Chan and Chow, 1976), although
a positive chronotropy in the cod, Gadus morhua was produced
(Holmgren, 1977; Wahlquist and Nilsson, 1977).
Flige and Ostlund
(1954) demonstrated positive chronotropy and inotropy in the
isolated fish heart exposed to epinephrine and norepinephrine
while Helgason (1973) found a decrease in heart rate in the intact
cod with these catecholamines.
The isolated heart of the rainbow
trout responded to epinephrine with positive inotropy and a decrease
in heart rate (Bennion, 1968).
The catecholamines affect the rest
of the circulatory system as well, dilating the gill vasculature
and constricting the general systemic vasculature (Ostlund and
Fange, 1962).
The general consensus until recently was that all teleostean
hearts were vascularized.
According to Randall (1970) the heart
in all fishes, with the exception of the hagfish, had a coronary
supply.
However, Santer and Walker (1980) found that of 93 species
of teleosts examined, only 19 possessed a coronary vasculature and
in these 19 species plus the anguillids the ventricular myocardium
was composed of an outer compact layer of muscle and an inner
spongy layer.
Of 14 elasmobranch species examined, all possessed
6
vascularized hearts.
Physiological and pharmacological studies of
the isolated fish heart have not taken this into account in the
past.
Dissection studies have revealed that the buffalo,sculpin
(Enophrys bison), a marine teleost, contains no coronary vasculature.
Light and electron micrographs (Bacon, unpublished data) revealed
a ventricle composed of only a spongy, trabeculated myocardium,
consistent with the nonvascularized hearts described by Santer and
Walker (1980).
A physiological and pharmacological study was
carried out on both the intact and isolated heart of the-buffalo
sculpin.
1.
The object of this experiment was:
To determine if the nonvascularized heart obeyed Starling's
law.
2.
To determine its response to acetylcholine and epinephrine.
3.
To determine the type of adrenoreceptors present.
MATERIALS AND METHODS
Buffalo sculpin (Enophrys bison) were collected from Yaquina
Bay, Newport, Oregon by otter trawl and maintained in aerated,
running sea water at 12°C.
weekly.
A diet of chopped herring was fed once
Animals weighed from 300 to 1100 gms.
The animals were pithed and a midventral incision made to
expose the heart.
The heart was excised so as to include the sinus
venosus and the bulbus arteriosus.
Isolated hearts were immediately
placed in cold marine teleost saline (hereafter referred to as
saline) (NaCl: 7.25g/1; KC1: 0.38 g/1; CaC12.2H20:
0.23 g/1; NaHCO3:
1.0 g/1; MgSO47H20: 0.23 g/1; glucose: 1.00 g/1; Trizma Base: 0.80
g/1; Trizma HC1: 6.85 g/1), at pH 7.8 and at an osmolality of 350
mOs/kg, flushed with saline to remove residual blood or clots, and
weighed.
Hearts were then mounted on a modified Langendorff coronary
perfusion apparatus by the sinus venosus and the bulbus arteriosus
was cannulated with PE 90 tubing attached to a pressure transducer
(Statham P23De).
A 15cm length of glass tubing was mounted vertically
to the exhaust port of the pressure transducer to establish a resting
pressure (Fig. 1).
The heart was perfused through the chambers with
saline maintained at 12°C, (Neslab RTE-8 refrigerated circulating
bath) pH 7.8 and also superfused with saline.
All recordings were
made with a Clevite Brush Mark 220 recorder.
The modified Langendorff apparatus allowed the perfusion
pressure to be varied.
Hearts were allowed to equilibrate at the
lowest perfusion pressure (0.8 kPa) for 30 min. before introducing
drugs or altering perfusion pressures.
After equilibration, the
8
Figure 1. Schematic showing attachment of heart to perfusion manifold
and pressure transducer. The exhaust ports on the manifold
allow atrial perfusion pressure to be varied. Small arrows
indicate path of perfusate through heart, transducer and
vertical length of glass tubing.
Manifold
6
venosus
4Atrium
Transducer
<--Ventricle
4,
Bulbus
arteriosus
Figure
1
10
perfusion pressure was raised to 1.1 kPa, 1.3 kPa, and 1.6 kPa at 5
min. intervals and finally returned to 0.8 kPa for an additional 5
min. to determine if the heart would recover its initial rate and
pulse pressure.
Saline with either L-epinephrine bitartrate (Sigma)
or acetylcholine chloride (Sigma) was then perfUsed through the
heart repeating the above sequence.
Four hearts were used with each
drug and each heart served as its own control.
To characterize the autonomic receptors in the heart the
following drugs were used:
DL-isoproterenol HC1 (Sigma), DL-
propranolol, methoxamine HC1 (Burroughs Wellcome Co.), acetylcholine
Cl and atropine sulfate (J. L. Baker Chemical Co.).
mounted and allowed to equilibrate as described.
Hearts were
During this time,
heart rate, pulse pressure and cardiac output were recorded.
At 30
min. a drug solution was perfused through the heart for 5 min. at
the lowest perfusion pressure.
After this 5 min. drug treatment,
drug-free saline was reintroduced for 15 min. or until hearts regained
pre-drug rates.
Only one drug was used per heart.
Histological examination of the hearts was performed by Dr.
Robert Bacon at the Oregon Health Sciences University, Portland,
Oregon (Department of Anatomy) using light microscopy, transmission
electronmicroscopy and scanning electronmicroscopy.
In vivo studies of the buffalo sculpin were performed by
initially anesthetizing fish by alternate exposure of gills to
oxygenated sea water with and without benzocaine (70 mg/1).
A 24
inch length of PE 50 tubing filled with 500 I.U. heparinized saline
was inserted ventrally into the branchial artery in the second or
11
third gill arch.
The cannula was sutured into place and connected
to a Statham P23De pressure transducer.
Clevite Brush Mark 220 recorder.
Recordings were made with a
Animals were held in plexiglass
restraining boxes placed in aerated running sea water aquaria.
Recordings of heart rate and blood pressure were begun as soon as
the animals were placed in water.
A recovery period of two hours
was allowed before any tests were performed.
All drugs were prepared from concentrated stock solutions made
with glass distilled, deionized water adjusted to a pH of 4.0 with
HC1 and refrigerated.
Drug solutions to be administered in vivo or
in vitro were made just prior to administration with saline at the
appropriate osmolality.
Statistical analyses were by two way analysis of variance with
a multiple range test.
12
RESULTS
Figure 2A shows myocardial cells in the ventricle of the
buffalo sculpin.
myocardium.
They appear to resemble the typical vertebrate
Figure 2B pictures the ventricular wall of the sculpin
demonstrating the arrangement of the myocardial fibers.
No outer
compact layer of muscle is present and vascularization is completely
lacking in comparison to that of a salmonid (Fig. 2C).
No vasculari-
zation could be detected in this inner spongy layer of muscle of
either sculpin or salmon.
In vivo experiments with the buffalo sculpin demonstrated
that a reflex bradycardia could be elicited with visual stimuli
(Fig. 3A).
Accompanying this reflex bradycardia was a sharp
decrease in blood pressure.
Administration of 0.25 mg/kg atropine
abolished or reduced the bradycardia but resulted in a slight
increase in pressure when the animal was exposed to visual stimuli
(Fig. 3B).
In Vitro Experiments
Before the pharmacological tests were begun, hearts were
mounted on the perfusion apparatus at the lowest pressure for two
hrs to test their viability (Fig. 4).
Heart rate decreased over
the entire period, dropping 46% after two hrs.
Sixty-five percent
of this decrease occurred during the first 30 minutes.
Pulse
pressure and stroke volume increased over the two hr interval.
Pulse pressure increased by 33% at the end of two hrs and had
increased by 14% at 30 min.
Stroke volume increased by 63% over
the two hr test period and had increased by 36% at 30 min.
Cardiac
13
Figure 2
Electronmicrograph (mag. 3400X) of the ventricular
myocardium of the buffalo sculpin showing arrangement of
muscle fibers.
A.
Photomicrograph of the ventricular wall of the sculpin
heart (40X) showing trabecular arrangement of myocardial
Arrow indicates luminal
tissue and lack of vascularization.
side of wall.
B..
Photomicrograph of the ventricular wall of the salmon
heart (40X) showing compact myocardial tissue with coronary
Arrows indicate coronary vessels.
vessels.
C.
14
15
Figure 3. Strip,chart recordings of heart rate and pulse pressure
from the buffalo sculpin. A and B are in vivo recordings;
C through F are in the isolated heart. Horizontal and
vertical arrows indicate direction of recording and point
at which stimulus was administered, respectively. Pressure
in kPa.
In vivo recording demonstrating reflex bradycardia
induced by visual stimulus.
A.
In vivo recording in same fish as ,A showing the inhibition of reflex bradycardia following atropine (0.25 mg/kg)
(Atropine given at first arrow).
administration.
B.
C.
Acetylcholine (1.0 pg/ml) administration in the isolated
heart.
Acetylcholine. (1.0 pg /ml) following atropine (1.0 pg/ml)
administration.
D.
E.
Isoproterenol (1.0 pg/ml).
F.
Isoproterehol (1.0 pg /ml) following propranOlol
(1.0 pg/ml)
G.
Epinephrine (0.64 pg/ml).
16
6.0
s
4.0
1
Min.
8.0
4.0
1
I
!'ins.
4
10 sec.
2.0
1.0
2.0
E
1.0
10 sec.
1.5
1.0
10 sec.
2.0
C
0.5
25 sec.
ricure 3
17
Figure 4. Changes in heart rate, pulse pressure and stroke volume over
a two hour period for the control hearts (n = 4, x
S.E.).
O
O
STROKE VOLUME
0
PULSE PRESSURE(kPa)
HEART RATE
19
output remained relatively stable over the entire test period with
no significant changes occurring.
The greatest rate of change for
all parameters occurred within the first 30 mina
Since the least
amount of variation occurred between 30 and 90 min, all subsequent
studies were confined to this interval, the initial 30 min being
used as an equilibration period.
Acetylcholine (1.0 pg/m1) resulted in negative chronotropy
and positive inotropy (Figure 3C).
At a dose of 1.0 pg/m1 the
heart would be arrested for the entire duration of exposure or
would stop for short periods and then continue beating again,
resembling vagal escape.
These effects of acetylcholine could be
eliminated by washing the heart out with saline.
Administration
of atropine (1.0 pg/m1) for 5 min. followed by acetylcholine for 5
min. diminished or abolished the effects of acetylcholine
(Fig.
3D).
Epinephrine produced positive chronotropy and inotropy (Fig.
3G).
The chronotropic effect of epinephrine persisted long after
wash out with saline.
Exposure to a high concentration of epineph-
rine initially would mask the effects of a lower dose given later,
therefore each heart was used only once for each dose.
Isoproterenol increased heart rate, lowered pulse pressure
and decreased stroke volume (Fig. 3E).
As with epinephrine, the
chronotropic effect of isoproterenol persisted after reintroduction
of drug-free saline.
nol (Fig. 3F).
Propranolol abolished the effects of isoprotere-
20
Methoxamine was without effect.
Starling's Law
Increasing atrial perfusion pressure had no significant
effect on heart rate but stroke volume and pulse pressure both
increased.
Cardiac output also increased with increasing atrial
perfusion pressure (Fig. 5).
Increasing atrial perfusion pressure
in the presence of 0.64 pg/m1 epinephrine caused no change in
heart rate between pressures.
Stroke volume increased with atrial
perfusion pressure when epinephrine was present but was less than
in drug-free hearts.
Pulse pressure was greater with epinephrine
and continued to increase with rising atrial perfusion pressure.
Cardiac output increased with increasing atrial perfusion pressure
but was almost identical to that in the drug-free,hearts.
The same tests repeated with acetylcholine (1 pg/m1) resulted
in a negative chronotropic effect.
However, there appeared to be
little change in heart rate from one perfusion pressure to the
next in the presence of acetylcholine.
pressure were greater at all pressures.
Stroke volume and pulse
There was no significant
difference in cardiac output between non-treated hearts and those
administered acetylcholine (Fig. 6).
21
Figure 5. Effects on heart rate, stroke volume, pulse pressure and
cardiac output of hearts (n = 4) exposed to varying atrial
perfusion pressures in the absence (solid-bar) and presence
(crosshatched bar) of epinephrine (0.64 pg/m1).
S.E.).
22
bt)
40
20
0
MTS
0.16
MTS 8i Epi.
0.12
008
004
6
4
2
30 5
PI
45 70
P2
P3
Figure 5
P4
5075 MINS
pi
PRESSURE
23
Figure 6. Effects on heart rate, stroke volume, pulse pressure, and
cardiac output of hearts (n = 4) exposed to varying atrial
perfusion pressures in the absence (solid bar) and presence
(crosshatched bar) of acetylcholine (1.0 pg/m1) [Perfusion
P1 = 0.8 kPa; P2 = 1.1 kPa; P3 = 1.3 kPa; P4 =
pressures:
30-50 minutes were in absence of drug and 55-75
1.6 kPa.
(x ± S.E.)].
minutes were in presence of drug.
m
z
C
tn
to z
rn
n
\\\1\1..
HEART RATE
ROM
t \Mb, 111.
Ilk,\11
0
a)
STROKE VOLUME
111111L%\.\\B
0
0
1\121.\
PULSE PRESSURE(kPa)
1\11116,
CARDIAC OUTPUT
25
DISCUSSION
The ventricle of the sculpin heart is comprised entirely of
trabeculated muscle surrounded by an epicardium and endoCardium.
There is no outer compact layer of ventricular myocardium and
coronary vasculature is completely lacking.
The morphology of.the
buffalo sculpin ventricle is similar to that described by Santer and
Walker (1980) for nonvascularized teleost hearts which receive their
oxygen supply from venous blood in the ventricular lumen.
Santer
and Walker (1980) hypothesized that those teleost species expending
large amounts of metabolic energy throughout their lives would
require a vascularized myocardium, whereas less active fish would
not.
The buffalo sculpin is a relatively inactive bottom fish
remaining motionless most of the time except when feeding or disturbed.
Based on its level of activity, one would expect that the buffalo
sculpin heart would lack vascularization.
The reflex bradycardia demonstrated by the buffalo sculpin in
vivo and its reduction or abolishment by atropine is in agreement
with the literature (Stevens, et al., 1972; Chan and Chow, 1976;
Cameron, 1978).
Randall (1970) suggests that this bradycardia is
due to an increasing level of vagal tonus.
There appears to be
interspecific variation in the degree of vagal tonus to the teleost
heart (Cameron, 1978).
Wood and Shelton (1980) were unable to
demonstrate any cholinergic vagal tone in intact rainbow trout while
Priede (1974) was able to show an inhibitory vagal tonus in the same
species.
Seibert (1979) suggested the degree of vagal tonus depends
on temperature.
Roberts (1968) hypothesized that parasympathetic
26
activity was reduced in active species of teleosts but elevated in
less active species.
In Vitro Experiments
The relatively rapid heart rate at the beginning of perfusion
may have been due to catecholamines or other hormones released
during dissection.
As the effects of the catecholamines diminished
during the first 30 min. of perfusion the heart rate declined and
remained relatively stable thereafter.
The rate decrease was accompan-
ied by increased stroke volume and pulse pressure.
explained by Starling's law.
This may be
As rate declines, more time is available
for complete filling of the ventricle, which in turn stretches the
myocardial muscle fibers more.
The combination of increased filling
and increased stretch results in greater stroke volume and pulse
pressure.
In the isolated sculpin heart acetylcholine consistently
resulted in a positive inotropic and negative chronotropic response.
Capra and Satchell (1977a) fpund a negative chronotropic and inotropic
response in the isolated heart of Squalus acanthias, as one might
expect to see in an isolated mammalian heart.
In the intact animal
a negative inotropic response might be expected with administration
of acetylcholine due to vasodilation, but in the isolated heart one
might expect to see just the opposite.
As rate falls there will be
more time for relaxation resulting in increased stretching of the
myocardium.
The greater stretching would then be expected to result
in increased force of contraction.
In the isolated heart of the
buffalo sculpin, acetylcholine does not produce the expected negative
27
inotropy.
Atropine produces an inhibition of the acetylcholine
response as expected.
The increase in rate and pressure associated with epinephrine
administration is in agreement with the literature (Ffige and
Ostlund, 1954; Bennion, 1968).
Epinephrine administration caused a.
persistent rate increase whereas the positive inotropy was transient.
Administration of epinephrine to a failing or an arrhythmic heart
improved cardiac function and restored rhythmicity.
This phenomenon
was also reported by Bennion (1968) in the isolated heart-of the
rainbow trout.
The persistent chronotropic response elicited by
epinephrine may be due to its promotion of glycogenolysis.
The presence of beta receptors within the buffalo sculpin heart
is indicated by the positive chronotropy and negative inotropy
produced by isoproterenol and the blockade of these effects by
propranolol.
Furthermore, the absence of any effect with methoxamine
seems to indicate a lack of alpha receptors.
The predominance of
cardiac beta receptors concurs with evidence from other teleosts
(Falck, et al, 1966; Bennion, 1968).
However, Forster (1976b) found
both alpha and beta adrenergic receptors in the heart of Anguilla
anguilla.
Peyraud-Waitzenegger, et al (1980), also working with
Anguilla anguilla, reported seasonal variation in the predominant
type of adrenergic receptor, alpha receptors predominating in winter
and beta receptors in the summer.
In this study, seasonal variation
was not taken into account and whether such variation exists in the
bUffalo sculpin is not known.
28
The positive inotropic effect produced by increasing atrial
filling pressure demonstrates that this heart obeys Starling's law.
Bennion (1968) reported similar results for the isolated heart of
the trout.
As the input pressure increased so did the end diastolic
fiber length, and as a result the fibers contracted with greater
force.
Stroke volume and cardiac output increased as well.
In
Bennion's study, heart rate increased with increasing atrial perfusion
pressure.
However, in the sculpin heart, rising atrial perfusion
pressure resulted in a slight decrease or no change at all in heart
rate.
Bennion postulated that the rate increase produced by increasing
pressure was probably due to a direct effect of stretch on the
pacemaker fibers causing a change in ionic conductance.
Whether the
differences in heart rate response between these two species are
physiological in nature or due to differences in the mechanics of
the preparation is not known.
The two types of hearts may be different
in more ways than just vascularization.
29
CHAPTER II
INTRODUCTION
The mammalian coronary vascular system has attracted much
attention within the past three decades as a result of the high
incidence of coronary artery disease in man.
Much, if not the
majority, of the information on the physiology and pharmacology of
the coronary vasculature is clinical in nature.
Very little
literature is available describing the comparative aspects of the
coronary system.
Most research in this area has been conducted on
mammals, ignoring the other vertebrate groups.
By examining the
coronary vasculature system of lower vertebrates, some insight may
be gained in understanding the physiology and pathology of mammalian
coronary systems.
Until recently it was assumed that all vertebrate hearts were
vascularized.
However, Santer and Walker (1980), examining the
ventricles of teleosts were able to describe two basically different
arrangements of ventricular myocardium in fish.
The first type of
ventricular myocardium in fish is composed entirely of trabecular
muscle enclosed by the endocardium and epicardium, deriving its
oxygen supply from the venous blood in the lumen.
The second
arrangement of fish ventricular myocardium is made up of an outer
compact myocardium surrounding the trabecular muscle.
This cortical
layer is supplied with a coronary vasculature system derived from
epibranchial arteries (Watson and Cobb, 1979).
Belaud and Peyraud (1971), working with the isolated heart of
the Conger eel (Conger conger), cannulated and perfused what they
30
described as the coronary vasculature.
They recorded a coronary
blood flow between 0.1 and 0.3 ml/min at a pressure of 15 cm H20.
They found that electrically induced tachycardia diminished coronary
flow and that acetylcholine produced a coronary vasoconstriction
which could be blocked by atropine.
Physiological and pharmacological
investigatiOns of the isolated fish heart have been done with
little or no regard for perfusion Of the coronary vasculature in
those species with vascularized hearts.
Though the hearts of
several species of fish have been described physiologically and
pharmacologically the coronary vasculature has been neglected.
Unlike the mammalian heart the fish heart consists of a
single chambered atrium and a single chambered ventricle, which
receives its oxygen supply either from luminal venous blood or
from coronary vasculature derived from epibranchial arteries.
It
is possible to isolate the fish heart and perfuse the chambers and
the coronary vasculature independently of one another.
The present
study compares the effects of several coronary vasodilators and
neurotransmitters on the vascularized and non - vascularized fish
heart.
Additionally, it attempts to examine what differences, if
any, exist when administering the vasodilators first through the
coronary vasculature and then through the chambers of the heart.
31
MATERIALS AND METHODS
Buffalo sculpin (Enophrys bison) were collected by otter trawl
from Yaquina Bay, Newport, Oregon and held in running seawater aquaria
at 12°C.
Fish were fed a diet of chopped herring once weekly.
Experimental animals were killed by a blow to the head, then
weighed and bled by removing the tail.
The heart was excised through
a midventral incision so as to include the bulbus arteriosus and the
sinus venosus, and immediately placed in cold saline.
A cannula of
PE90 tubing flared at the tip was inserted into the sinus venosus and
tied in place.
Cold saline was introduced to clear the lumen of re-
maining blood and clots.
Finally, a second cannula of PE 90 tubing
was secured in the bulbus arteriosus.
The isolated heart was then connected to a modified Langendorff
coronary perfusion apparatus by the sinus venosus cannula.
One port
of a Statham P23De pressure transducer was attached to the bulbus
arteriosus cannula while.the other port was connected to a 15 cm length
of glass tubing so as to create a resting pressure of 1.5 kPa for the
heart to work against.
Returning adult male chum salmon (Oncorhynchus keta) were obtained
from Oregon State University's salmon hatchery on Whiskey Creek,
Netarts, Oregon.
The salmon were held in aerated, freshwater tanks at
12°C without food and were kept no longer than a week before being used.
The hearts of chum salmon were excised and prepared as described
above with the exception that the coronary artery was cannulated.
A
cannula made of a short length of polyethylene tubing and a blunted
hypodermiC needle was inserted into the coronary artery and sutured in
32
Cold saline was then injected to clear the coronary vasculature
place.
of blood and any possible clots.
The coronary artery cannula was connected to an infusion pump
(Harvard Apparatus Co., Inc.) via PE 90 tubing with an in-line
Statham P23ID pressure transducer.
The infusion pump was equipped
with two 60 ml Monoject syringes, each with a Luer lock 3-way stopcock,
so that it was possible to switch from one syringe to another.
After completion of the cannulations the heart was placed in a
temperature controlled (12°C) oxygenated organ bath.
The heart was
perfused with oxygenated teleost saline at 12°C and pH 7.8 at 1.1
kPa pressure.
Coronaries were perfused at a rate of 0.4 ml/min.
Coronary perfusion pressure varied between hearts.
Isolated hearts were perfused for 30 minutewith drug-free
teleost saline to allow equilibration.
After equilibration, drugs
were administered for a five minute interval followed by drug-free
saline.
Drugs were administered to the salmon hearts first through
the coronary vasculature followed by a drug-free wash of 15 minutes
and then the same drug was administered through the heart chambers.
Only one drug was used per heart.
Heart rate, pulse pressure and
coronary pressure were recorded with a Clevite Brush Mark 220
recorder.
Cardiac output was measured by collecting and weighing
perfusate exiting the bulbus arteriosus cannula.
Hearts of both species were sent to Oregon Health Sciences
University for histological examination.
Electron micrographs were
done by Dr. Robert Bacon, Dept. of Anatomy.
33
Drugs used were nitroglycerine (10% in lactose, Key Pharmaceuticals, Inc.), perhexiline maleate (Merrell National Laboratories),
prenylamine lactate (Hoechst-Roussell Pharmaceuticals, Inc.), Lepinephrine bitartrate (Sigma) and acetylcholine chloride (Sigma).
All drugs were prepared in deionized double-distilled water at a
concentration of 1 mg/ml.
Drug solutions were made fresh for each
experiment and kept refrigerated until used.
A microliter pipet
(Finnpipet) was used to withdraw the appropriate amount of concentrated
stock solution (1 mg/ml) and add it to 500 ml of saline.
The
drugged saline solution was prepared 5 to 10 minutes prior to its
use.
A flow rate of 0.4 ml/min in the coronary vasculature was found
to be the most optimum in terms of getting consistent recordings.
This rate resulted in perfusion pressures in the range of those
found in the dorsal aorta of salmon.
34
RESULTS
Sculpin
Controls
To determine the viability of the isolated heart on the
perfusion apparatus four hearts were used as controls.
During a
45 min. interval the mean heart rate decreased 41.3% from the
initial time.
The greatest drop in rate occurred during the first
15 min., 26.4%.
Pulse pressure and stroke volume did not change
significantly during the 45 min. test interval (Fig. 7).
Based on
this information, all subsequent hearts were allowed to equilibrate
for 15 min. before drugs were administered.
Sodium Nitrite
Sodium nitrite (1 pg/ml) was without significant effect on
heart rate, pulse pressure or stroke volume when administered to
the healthy heart (Fig. 8A and 9E).
However, when administered to
the arrhythmic failing heart sodium nitrite would occasionally
restore rhythmicity (Fig. 9F).
This was an inconsistent finding.
Nitroglycerin
Nitroglycerin (100 pg/ml) was without effect on the sculpin
heart.
No change could be seen in any of the parameters measured
(Fig. 8B and 9D).
Perhexiline
Perhexiline (1 pg/ml ) was without effect (Fig. 9C and 10A).
Prenylamine
Prenylamine (10 pg/ml) resulted in a significant fall in
heart rate accompanied by an insignificant rise in pulse pressure
35
Figure 7. Changes in heart rate, pulse pressure and stroke volume over
S.E.).
a 50 minute interval for the control hearts (n = 4, x
-1
O
0'
0
O
0
9
I
0
I
(MLS)
STROKE VOLUME
1
(kPa)
O
PULSE PRESSURE
HEART RATE
37
Figure 8. Heart rate, pulse pressure and stroke volume changes in the
isolated buffalo sculpin heart exposed to the vasodilators
sodium nitrite (1.0 pg/m1 - A) and nitroglycerin (100 pg /ml
Drugs were introduced at 15 minutes (arrow) and perfused
B).
(n = 4; x ± S.E.).
for 5 min.
38
RATE
20
PULSE
PRESSURE
( k Pa)
0.4
STROKE
VOLUME a-
50
HEART
RATE
B
40
T
4.0
PULSE
PRESS RE
(kPa)
STROKE
0.4
VOLUME
0.3
I0
20
MINS.
Figure 8
3
40
50
39
Figure 9. Strip chart recordings of heart rate and pulse pressure from
Vertical arrows indicate
the isolated buffalo sculpin heart.
point of drug administration and horizontal arrows indicate
direction of recording. Pressure in kPa.
B.
Prenylamine (10 pg/m1)
Prenylamine (10 pg/m1)
C.
Perhexiline (1 pg /ml)
D.
Nitroglycerin (100 pg/ml)
Sodium nitrite (1 pg/ml) in healthy heart
Sodium nitrite (1 pg/ml) in arrhythmic heart
A.
E.
F.
40
12 sec.
12 sc,c.
1.5
1-4
12 sec.
12 sec.
E
2.
[WiNlita
HANNON
;774sec.
2.0
1.0
10 sec.
Ficlure 9
41
Figure 10.
Changes in heart rate, pulse pressure and stroke volume
in the isolated buffalo sculpin heart administered the
A) and prenylamine
vasodilators perhexiline (1 pglml
Drugs were introduced at 15 minutes
(10 pg/ml - 13).
(n = 4,
(arrow) and perfused for 5 minutes.
42
5 0
A
HEART
RATE
30
PULSE
PRESSURE
(k Pa)
40
3.0
STROKE"
VOLUME
Q2
1
1
10
30
20
40
50
MINS.
B
40
HEART
RATE
20
PULSE
4
PRESSURE
(kPa)
3
05
STROKE
VOLUME
0.3
1
10
40
20
M INS.
Figure 10
50
43
and stroke volume (Fig. 10B).
perfusion.
Heart rate fell 32% during prenylamine
This negative chronotropy persisted even after prenylamine
was discontinued.
The transient increase in pulse pressure gave
way to gradually decreasing pressure and a continuing decrease in
heart rate 15 min. after discontinuation of prenylamine.
dramatic effect however was on heart rate.
effect of prenylamine on the sculpin heart.
The most
Fig. 9A shows the
In this recording
there was a noticeable increase in pulse pressure, however this
was not a consistent finding as figure 9B shows.
Stroke volume
increased slightly during the 5 min. drug exposure but after
removal of the prenylamine, stroke volume gradually decreased.
When the hearts were perfused beyond the normal 45 min. test
interval arrhythmias developed leading to failure,of the heart.
After administration of prenylamine the hearts never returned to
Pre-drug capacity.
The data are summarized in Table I.
44
TABLE I.
Summary of the Effects of Acetylcholine, Epinephrine
and Vasodilators on the Isolated Buffalo Sculpin Heart
Heart rate
Systolic Pressure
Acetylcholine
(0.5 (pg/m1)
+4,
fi
Epinpehrine
(0.5 ug/m1)
tt
Stroke Volume
4,
no change
no change
no change
Nitroglycerin
(100 pg/m1)
no change
no change
no change
Perhexiline
(10 ug/m1)
no change
no change
no change
Sodium Nitrite
(1 pg/m1)
Prenylamine
(10 ug/m1
t P < 0.05
4-4- = P < 0.01
no change
45
Salmon
Figure 11A shows the simultaneous recording of heart rate and
pulse presSure (top) and coronary pressure (bottom) in the isolated
salmon heart perfused with saline solution.
In a typical recording
such as this, pulse pressure preceded peak coronary pressure
indicating that ventricular contraction is probably producing the
oscillations in coronary pressure.
Acetylcholine
Acetylcholine (0.5 pg /ml) administered through the coronary
artery produced a slight, but insignificant, negative chronotropic
and positive inotropic effect.
significantly (22.6%).
increase.
Peak coronary pressure increased
Stroke volume showed a slight but insignificant
When the same dose was given through the sinus venosus
there was a highly significant drop in mean heart rate (88%) (P <
0.01) and a highly significant increase (61%) in pulse pressure.
Stroke volume and coronary pressure responded with significant
increases as well.
When drug-free saline was reintroduced the
heart returned to the pre-drug conditions (Fig. 12).
In Fig. 11B,
the lower recording shows the increase in coronary pressure when
0.5 pg /ml of acetylcholine is perfused through the artery.
There
is only a minor effect on pulse pressure and virtually no change
in rate.
But the same dose administered through the chambers of
the heart elicits a marked bradycardia (Fig. 11C).
Epinephrine
Epinephrine (0.5 pg/m1) administered through the coronary
artery had no significant effect on any of the parameters measured
46
Figure 11.
Strip chart recordings of heart rate, pulse pressure
and coronary pressure from isolated salmon hearts.
Upper recording is of heart rate and pulse pressure while
Vertical
the lower tracing is of coronary pressure.
arrows indicate point of drug administration and
horizontal arrows indicate direction of recording.
Pressure in kPa.
A.
Control salmon heart
Administration of 0.5 pg/ml acetylcholine through
Spike is an injection artifact.
the coronary artery.
B.
Same heart as in B but 15 minutes later but this
time acetylcholine (0.5 11g/m1) was given through the
sinus venosus.
C.
Epinephrine (0.5 pg /ml) administered through the
coronary artery.
D.
Epinephrine (0.5 pg/ml) administered through the
sinus venosus (same heart as D but 15 min. later).
E.
47
3.0
A
2.5
0.5
3.5
1.5
12 sec.
12 sec.
C
3.5
0_
2.5
12 sec.
E
2.5
0.5
12 sec.
Figure 11
48
Figure 12.
Graph of heart rate, pulse pressure, stroke volume and
coronary pressure in the isolated salmon heart during
administration of acetylcholine (1.0 pg /ml) first
through the coronary artery (15-20 min) and then through
the sinus venosus (35-40 min). The period of drug
exposure-occurs between the dashed and solid lines.
(n = 4, x ± S.E.).
C
n
0
0
to
0
0
0
1
V.
(kPa)
CORONARY PRESS.
9
cr,
(MLS)
STROKE VOL.
'
(kPa)
PULSE PRESSURE
HEART RATE
50
during the 5 min. exposure time.
But there was a gradual, but
significant, increase in heart rate after epinephrine was replaced
with drug-free saline.
There was a slight, but insignificant rise
in heart rate when epinephrine was perfused through the heart chambers.
Pulse pressure and coronary pressure appeared to be unaffected by
epinephrine through either route of administration.
Stroke volume
was not significantly altered by coronary administration of epinephrine
but showed a significant decline (16.2%) when epinephrine was
passed through the chambers of the heart (Fig. 11D, 11E and Fig.
13).
Sodium Nitrite
Sodium Nitrite (1 pg/m1) given through the coronary artery had
no effect on heart rate, pulse pressure or stroke volume during the
five minute perfusion time.
Coronary arterial pressure showed a
significant increase during this time and continued to increase
after sodium nitrite was replaced with drug-free saline.
in coronary pressure stabilized after 35 min.
This rise
Administration of
sodium nitrite through the heart chambers showed no effect on any
of the parameters measured (Fig. 1A, B and Fig. 15).
Nitroglycerin
The results of nitroglycerin (100 lig/m1) administration were
inconsistent whether given through the coronary artery or the sinus
venosus.
During the 5 min. interval when nitroglycerin was perfused
through the coronary vasculature no significant changes could be
detected in any parameters (Fig. 14C).
However, when nitroglycerin
was discontinued, coronary pressure began to rise and pulse pressure
showed a sharp decrease (Fig. 16).
51
Figure 13.
Effects of epinephrine (0.5 lig/m1) on heart rate, pulse
pressure, stroke volume and coronary pressure in the
Drug was added first through the
isolated salmon heart.
coronary artery (15-20 min) and then through the sinus
The period of drug exposure
venosus (35-40 min).
occurs between the dashed and solid lines (n = 4,
+
x - S.E.).
65
HEART
RATE
45
PULSE
4
PRESS URE
( k Pa)
3
1
STROKE 0.4
VOLUME
0.3
CORONARY
PRE SSURE
(kPa)
2
H
C
10
Figure 13
20
30
M INS.
I
40
50
60
53
Figure 14.
Strip chart recordings of heart rate, pulse pressure and
coronary pressure from isolated salmon hearts. Upper
recording is of heart rate and pulse pressure while the
lower tracing is of coronary pressure. Vertical arrows
indicate point of drug administration and horizontal
Pressure in kPa.
arrows indicate direction of recording.
Sodium nitrite (1 pg/ml) administered through the
coronary artery.
A.
Sodium nitrite (1 pg/ml) administered through the
sinus venosus (same heart as in A,' 15 min later).
B.
Nitroglycerin (100 pg/ml) administered through the
coronary artery.
C.
Nitroglycerin (100 pg/m1) administered through the
sinus venosus (same heart as in C, 15 min later).
D.
54
A
2.5
['i.,1\ki'i'd'u'\,\,,I\Jklk,i'i\l"\i'dkillgjl\i'd\,1\ittill'i'ill'il,ildiliiilt;Vd
0.5
5.0
2.5
r-4
At`
0--
12. sec.
B
C
2.5r
2.5
liklak*
1.0
3.0
4.0--
IteggiltIMAINNikl
1-1
1.57
12 sec.
0
12 sec.
D
3.0
0.
5-
2.0
[e.*".~V,...JAAMMVAMMoMyanyowwwWhy,wo004",
1-4
12 sec.
0
Figure 14
55
Figure 15.
Changes in heart rate, pulse pressure, stroke volume
and coronary pressure in the isolated salmon heart
The drug was
exposed to sodium nitrite (1 pg/ml).
administered first through the coronary artery (15-20
min) and then through the sinus venosus (35-40 min).
The period of drug exposure occurs between the dashed
and solid lines (n = 4, x ± S.E.).
O
O
CORONARY PRESS.
(kPa)
(AILS)
r)
O
(kPa)
STROKE VOL. PULSE PRESSURE
CT
CT
4=.
HEART RATE
c^1
cn
57
Figure 16.
Changes in heart rate, pulse pressure, stroke volume and
coronary pressure in the isolated salmon heart exposed
to nitroglycerin (100 pg /ml). The drug was administered
first through the coronary artery (15-20 min) and then
The period of
through the sinus venosus (35-40 min).
drug exposure occurs between the dashed and solid lines
±
S.E.).
(n = 4, x
CORONARY PRESSURE(kPa)
STROKE VOLUME
PULSE PRESSURE(kPa)
HEART RATE
59
Administration through the heart chambers in some cases resulted
in a negative chronotropic and positive inotropic response (Fig.
14D) while increasing stroke volume and decreasing coronary pressure,
(Fig. 16).
However, in other cases, nitroglycerin was entirely
without effect.
The data for nitroglycerin are inconclusive and
more work is needed.
See Table II for a summary of the results.
60
Summary of the Effects of Acetylcholine, Epinephrine
TABLE II.
and Vasodilators on the Isolated Salmon Heart
Systolic
Pressure
Stroke
Volume
H*
C
H
C
H
C
H
44
T
++
fi
++
+
t
--
+
--
Heart
Rate
Acetylcholine
(0.5 pg/m1)
Epinephrine
(0.5 pg/ml)
Sodium Nitrite
Coronary
Pressure
--
--
--
--
--
--
--
--
f
--
(1 pg/ml)
Nitroglycerin
(100 pg/ml
* C = Coronary; H = Heart chambers
**
= no change
+ = P < 0.05
44 = P < 0.01
61
DISCUSSION
The interpretation of the data obtained in this study will
rely heavily on comparisons to mammalian studies since very little
information exists on the pharmacology and physiology of the fish
coronary system.
Sculpin
The negative results obtained with perhexiline, sodium nitrite,
and nitroglycerin indicate that in the fish heart these drugs have
no direct effect on the myocardial tissue itself.
Winsor (1970),
using dogs, demonstrated that the major pharmacological properties
of perhexiline was dilation of the coronary and systemic vascular
beds resulting in increased coronary flow and decreased heart
rate.
During exercise-induced tachycardia, Winsor also described
a propranolol-like effect of perhexiline in lowering maximum heart
rate during exercise.
However, perhexiline did not lower the
resting rate and it is not considered a beta blocker.
Winsor
describes a reflex mechanism for the action of perhexiline on the
heart.
As coronary flow is increased and systemic vascular resistence
decreased the heart rate slows in response to a lower work load.
Though Hudak, et al., (1970) concurs with Winsor on the dominant
action of perhexiline on the systemic and coronary vascular beds,
they suggest that perhexiline-induced bradycardia in dogs is due
to a direct effect on cardiac membrane.
If there is a direct
effect of perhexiline on the cardiac membrane it is not demonstrated
in the present study.
This conflict with Hudak's findings could
well be due to species differences.
62
The vasodilatory effect of the nitrates and nitrites in mammals
is well documented in the literature.
Their principal pharmacological
action is dilation of vascular smooth muscle.
Cardiac output,
coronary blood flow, ventricular work and myocardial efficiency have
been reported to be increased, decreased or unchanged by nitroglycerin.
Using a modification of the Warburg.techniques, Honig, et al.,
(1960) found that the oxygen uptake of cardiac muscle was unaffected
by direct exposure to nitroglycerin.
They also found that cardiac
efficiency was unchanged by nitroglycerin in the isolated, but
metabolically supported, heart functioning at constant rate and
volume.
It seems unlikely that the nitrates and nitrites act by
altering the oxygen consumption of resting heart muscle.
Nitroglycerin
administered in vivo and in vitro in the rat heart was reported by
Ogawa, et al., (1967) to significantly inhibit the monoamine oxidase
activity of the rat heart mitochondria and thereby increasing the
norepinephrine content of the heart.
If nitroglycerin has any MAO
inhibiting activity in the fish heart it was not detectable in any
of the physiological parameters measured in this study.
The bradycardia elicited by prenylamine in the sculpin heart
concurs with the results reported for mammalian hearts.
Obianwu
(1967) found that in various mammalian species prenylamine lowered
mean arterial blood pressure and heart rate and potentiated the
responses to norepinephrine and epinephrine in vivo.
Using eats,
Nielsen and Owman (1967) reported that prenylamine resulted in
reduction in the catecholamine content of both the central nervous
system and the sympatho-adrenal system thus preventing spontaneous
63
development of ventricular fibrillation during hypothermia by interfering with peripheral adrenergic mechanisms.
In rats, 100 mg/kg of
prenylamine administered subcutaneously reduced the serotonin level
of the brain and the myocardium by 30% and the norepinephrine concentration by 50-60% (SchOne and. Lindner, 1960).
The catecholamine-
depleting action of prenylamine could possibly account for the
bradycardia found in the buffalo sculpin.
Salmon
The vasodilatory action of acetylcholine in the mammalian
cardiovascular system is well documented.
In this study acetylcholine
caused constriction of the coronary artery of the salmon heart.
When considering the origin of the coronary vasculature of the fish
heart this acetylcholine-induced vasoconstriction, might be expected.
The coronary artery in fish originates from an epibranchial artery
(Watson and Cobb, 1979) and acetylcholine produces vasoconstriction
of the branchial vasculature (Randall, 1970).
Belaud and Peyraud
(1971), working with the isolated heart of the Conger eel (Conger
conger L.), reported an acetylcholine-induced vasoconstriction in
the cannulated coronary artery.
blocked by atropine.
This vasoconstriction could be
However, Santer and Walker (1980), studying
the morphology of the ventricles of teleost and elasmobranch hearts,
were unable to detect a coronary vasculature supply in either Conger
conger or Anguilla anguilla.
What Belaud and Peyraud refer to as a
coronary artery may have been an epicardial vascular system.
Santer
(1976), working with older (9-16 years) plaice (Pleuronectes platessa),
found in the hearts of older fish only an extensive epicardial
64
vascH1,,,.r system, as revealed by arterial and venous profiles.
This
vascular system was confined to the epicardium and did not penetrate
the myocardium to form a vascular bed, nor was it derived from
extracardiac vessels.
When administered through the coronary artery, acetylcholine
resulted in a slight, but statistically insignificant, bradycardia.
In the isolated mammalian heart mounted on a Langendorff perfusion
apparatus administration of acetylcholine results in marked bradycardia (Broadley, 1979).
The bradycardia was accompanied by slight
but insignificant increases in pulse pressure and stroke volume.
The acetylcholine-induced bradycardia resulting from administration
through the sinus venosus of the salmon heart was significantly,
greater, indicating that route of exposure is quite important.
A
possible explanation for this difference in response may involve the
concentration of acetylcholine coming in contact with the sinoatrial (SA) node.
Coronary blood flow in the fish empties into the
coronary sinus at the base of the atrium after passing through the
coronary vasculature of the sinus, atrium and ventricle.
In this
study coronary perfusion rate was 0.4 ml/min in contrast to an
atrial perfusion rate of 8 ml/min so that the relative amount of
perfusate entering the atrium through the coronary route was minimal.
Therefore the SA node will be exposed to a greater quantity of drug
per unit of time when the drug is perfused through the atrium than
when the same concentration of drug is administered through the
coronary vasculature.
Furthermore, in this preparation, any drug
entering the atrium through the coronary sinus was being diluted by
65
perfusate entering from the sinus venosus, thus further diminishing
the quantity of drug reaching the SA node.
In the classical Langendorff
heart preparation, using the isolated guinea pig heart, 0.5 pg of
acetylcholine administered through the coronary artery resulted in a
significant bradycardia shortly after administration of the drug
In the mammalian heart there appears to be better
(Broadley, 1979).
communication between the coronary vasculature and myocardial tissue,
allowing neurohumoral agents access to receptors within the myocardium.
Acetylcholine has no chronotropic or inotropic effects within the
salmon heart until it reaches the atrium indicating that acetylcholine
is not eliciting any effect on the myocardial tissue while it is
within the coronary artery.
This may be.due to impermeability of
the coronary wall or to a lack of cholinergic receptors in the
ventricular myocardium.
Accompanying the bradycardia resulting from atrial perfusion of
acetylcholine were increases in pulse pressure and stroke volume.
As the heart slowed it had more time to relax, thus increasing the
tension on the muscle fibers resulting in greater force of contraction
and stroke volume.
An increase in coronary pressure resulted from
atrial perfusion of acetylcholine.
This may have been an indirect
effect due to extravascular compresSion from the increased force of
contraction and greater filling of the heart.
The initial reaction
to the acetylcholine was transient cardiac arrest which caused the
atrium to fill maximally, thus creating a back pressure at the
coronary sinus.
This could have also contributed to the increased
coronary pressure.
66
Epinephrine (0.5 pg/ml) administered through the coronary
artery had no significant effect on any of the parameters measured.
When given through the chambers of the heart, rate was increased and
stroke volume fell.
In the fish cardiovascular system, epinephrine
is reported to produce positive chronotropic and positive inotropic
effects (Capra and Satchel], 1977).
Broadly (1979) reported a
biphasic response in the coronary artery of the isolated guinea pig
to epinephrine.
An initial short vasoconstriction was followed by
a more prolonged and prominent fall in pressure.
Epinephrine is
reported to produce vasodilation in fish branchial vasculature.
(Ostlund and Fnge, 1962) thus the coronary vasculature might be
expected to respond in a similar manner.
The absence of effects may
be dose related, perhaps requiring a higher dose.
Tachycardia
produced by atrially perfused epinephrine appeared to have no
indirect effects on coronary vasculature through extravascular
compression.
The rise in coronary pressure produced by NaNO2, indicative of
vasoconstriction, is counter to the expected vasodilation normally
seen with the nitrates and nitrites in mammals.
continued even after discontinuing NaNO2.
Vasoconstriction
Accompanying this vasocon-
striction was an insignificant rise. in rate and fall in pressure.
Rubin, et al., (1963) reported a transient increase in heart rate
and lowered systemic arterial pressure when NaNO2 was administered
to the dog.
The transient tachycardia in the dog may have been a
feedback response to the lowered arterial pressure.
It is difficult
to speculate on the probable cause of this vasoconstriction.
More
67
information is needed on the physiology and pharmacology of the fish
coronary arterial system.
Sodium nitrite administered through the
atrium had no significant effects on any of the parameters being
measured although there was a noticeable fall in heart rate.
As in
the sculpin it appears that the nitrites and nitrates do not exert
any statistically significant direct effect on the myocardial tissue
of the chum salmon.
Their predominent effect appears to be on the
vasculature with secondary effects on the heart itself.
Nitroglycerin, as with NaNO2, appears to have no direct effect
on the myocardial tissue of the chum salmon.
After administration
through the coronary there was a prolonged and gradual increase in
coronary pressure which continued after removal of the nitroglycerin.
Again this is inconsistent with the reported action of the
organic nitrates.
As with NaNO2, there was an insignificant drop in
rate when nitroglycerin is given through the atrium.
Statistically
this drop in rate is insignificant but there appears to be some
action of the nitrates and nitrites on the fish heart.
This negative
chronotropic effect is also accompanied by a positive inotropic
effect and an increase in stroke volume.
The data on the nitrites
and nitrates is not conclusive and more data is needed.
68
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APPENDIX
75
To understand the anatomy, physiology and pharmacology of the
teleost heart it is necessary to first look at the mammalian heart
since most of our information has been gained through mammalian
studies.
The mammalian heart can be described as a double circuit
pump arranged in series, composed of two atria and two ventricles.
One side of the heart propels venous blood through the pulmonary
vasculature for oxygenation.
Oxygenated blood returns to the left
side of the heart from where it is pumped through the systemic
vasculature.
A typical teleost heart performs the same basic function
as the mammalian heart with the exception that it consists of one
atrium and one ventricle pumping venous blood to the gills for
oxygenation and from here the blood passes to the systemic circulation.
To perform its task effectively the myocardial tissue itself
must receive an oxygenated blood supply.
In the mammalian heart,
coronary arteries, which arise at the root of the aorta, supply the
heart muscle with oxygenated blood.
The right coronary artery
supplies principally the right ventricle and atrium while the left
coronary supplies the left ventricle and atrium.
In the dog the
left coronary artery supplies about 85% of the myocardium, whereas
in humans the relative flow in the right and left coronary arteries
is more variable, with the greatest percentage of individuals showing
dominance of the right artery.
The venous drainage of the coronary system can be divided into
(Berne and Levy, 1977) a superficial drainage system consisting of
the anterior cardiac veins and (Berne, 1974) the great cardiac
veins, all of which drain into the coronary sinus (Berne, 1974).
76
Most of the coronary venous blood, after passing thorugh the
myocardial capillary beds, returns to the right atrium through the
coronary sinus while some reaches the right atrium via the anterior
coronary veins.
A second deep system of veins, the arteriosinusoidal,
the arterioluminal and the thebesian vessels provide vascular communication between the myocardial vessels and the chambers of the heart.
These deeper veins represent only a minor contribution to the total
The arteriosinusoidal channels are small arteries which lose
flow.
their arterial structure as they penetrate the chamber walls and
divide into irregular, endothelium-lined sinuses.
These sinuses
connect with other sinuses and with capillaries and communicate with
the cardiac chambers.
The arterioluminal vessels are small arteries
or arterioles that open directly into the atria and ventricles.
The
thebesian vessels are small veins that connect capillary beds directly
with the cardiac chambers and also communicate with cardiac veins
and other thebesian veins.
It appears that the minute vessels of
the myocardium form an intercommunicating plexus of subendocardial
vessels.
It is postulated that myocardial tissue can derive nutrition
from the cardiac chambers through these channels.
In the mammal it
has been found that blood in the cardiac chambers penetrates a short
distance into the endocardium, but does not constitute a significant
source of oxygen and nutrients to the myocardium (Berne and Levy,
1977).
Factors Influencing Coronary Blood Flow
Perfusion of the coronary vasculature is determined by the
aortic pressure which is generated by the heart itself.
Alterations
77
in aortic pressure influence the coronary blood flow, but other
factors operate to maintain an adequate flow of blood to the myocardium.
The ability to maintain a relatively stable coronary flow rate
during changes in perfusion pressure is termed autoregulation of
blood flOw and three mechanisms have been proposed to explain this;
these are the tissue pressure, metabolic and myogenic hypotheses
(Abel and McCutcheon, 1979).
The tissue pressure hypothesis proposes
that as perfusion pressure increases so does capillary hydrostatic
pressure and this results in greater movement of fluid from the
intravascular compartment to the interstitial space.
interstitial pressure serves to compress
increase vascular resistance.
This elevated
the thin-walled vessels and
A reduction in perfusion pressure
would therefore lead to reduced resistance.
According to the metabolic
hypothesis vascular resistance is closely linked to myocardial
metabolic activity and that a chemical mediator produced in the
tissue acts to change vascular resistance in accordance with the
metabolic activity of the heart.
(Increased myocardial metabolic
activity results in lowered coronary resistance whereas reduced
cardiac metabolism increases coronary resistance).
The myogenic
hypothesis maintains that stretch or increased tension of the vascular
smooth muscle, produced by increased perfusion pressure, elicits an
enhanced contractile response of the smooth muscle and thereby
returns blood flow to the level existing before elevation.
Extravascular compression produced by the contracting myocardium
influences coronary blood flow by its squeezing effect on the coronary
blood vessels.
Depending on the diastolic pressure gradient and the
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diastolic time, 70 to 90 percent of the coronary flow normally
occurs during diastole.
Flow in the coronary sinus tends to be
opposite in phase to the arterial inflow pattern, venous flow is
increased during systole because contraction forces blood out of
capillary and venous beds (Berne and Levy, 1977).
During tachycardia,
when diastolic time is shortened, the heart spends more time in the
contracted state, thus increasing extravascular resistance and
restricting coronary blood flow.
Because of increased myocardial
metabolic activity during tachycardia the net effect on coronary
resistance is a decrease due to the reduction in intravascular
resistance by dilation of the arterioles.
During bradycardia mechani-
cal restriction of coronary blood flow is less as are the metabolic
demands of the myocardium.
Metabolic factors, which reduce intra-
vascular resistance, predominate over mechanical factors, which
increase extravascular resistance, in conditions such as tachycardia.
Neural Regulation of the Coronary Vasculature
The coronary vasculature receives both sympathetic and parasympathetic innervation (Berne, 1974; Berne and Levy, 1977; Abel and
McCutcheon, 1979).
Although circulating acetylcholine acts as a
vasodilator in mammalian coronary arteries, the role of coronary
cholinergic fibers is not clear.
Vagus nerve stimulation has little
direct effect on coronary arterial resistance.
In the fibrillating
heart with the coronary arteries perfused at a constant pressure, or
in the beating, paced heart with flow measured in late diastole when
there is essentially no extravascular compression, small increments
in coronary blood flow can be observed with stimulation of the
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peripheral ends of the vagus (Berne and Levy, 1977).
It is likely
that the vagi exert less effect on coronary resistance in the normal
animal, because of the overriding influence of metabolic mechanisms
(Berne and Levy, 1977).
Acetylcholine-induced coronary artery
vasodilation can be blocked by atropine.
Coronary dilation can
occur reflexly via the vagi by chemoreceptor activation.
In order to evaluate the direct effects of sympathetic nerve
stimulation on the coronary vasculature it has been necessary to
eliminate the accompanying inotropic, chronotropic and metabolic
factors which mask the effects on the coronary vessels.
Both alpha
and beta receptors have been demonstrated in the coronary vasculature.
The larger surface vessels contain primarily alpha receptors while
the deeper nutrient vessels are largely beta controlled.
Additionally,
betal receptors predominate in the larger vessels and beta2 receptors
predominate in the smaller one (Berne, 1974).
Using hearts in
ventricular fibrillation it has been found that NE causes an initial
coronary constriction followed quickly by vasodilation.
The physio-
logical importance of the coronary beta receptors has been questioned
due to the interference of cardiac inotropic and chronotropic responses
of sympathetic nerve stimulation.
By using practolol, myocardial,
but not vascular beta receptors can be blocked.
Stimulation of the
cardiac sympathetic nerves after practolol administration results in
an increase in coronary resistance and this increased coronary
resistance is attenuated if the alpha receptors are blocked with
phentolamine (Abel and McCutcheon, 1979).
80
Coronary resistance can also be altered reflexly by the baroreceptors.
In addition there seems to be some tonic activity in the
sympathetic fibers that innervate the coronary resistance vessels,
since acute cardiac denervation results in some decrease in coronary
resistance (Abel and McCutcheon, 1979).
Metabolic Factors Regulating Coronary Blood Flow
The levels of myocardial metabolic activity and the magnitude
of coronary blood flow are closely correlated.
A direct relationship
exists between the oxygen requirements of the myocardium and the
degree of coronary blood flow.
This relationship is seen in the
perfused isolated heart as well as the intact heart.
With the onset
of ventricular fibrillation, there occurs an increase in coronary
blood flow due to reduced myocardial metabolic demands and reduced
extravascular compression.
prefibrillation level.
Flow then gradually returns toward the
This increase in coronary resistance, which
occurs despite the elimination of the extravascular compression, is
a manifestation of the heart's ability to adjust its blood flow to
meet its energy needs (Berne and Levy, 1977).
The metabolic factors that control coronary blood flow appear
to be significant (Berne, 1974).
Numerous metabolic agents have
been suggested as the mediator of coronary blood flow.
Some of the
substances implicated are CO2, 02, lactic acid, hydrogen ions,
histamine, potassium ions, increased osmolarity, polypeptides, and
adenine nucleotides.
None of these agents has satisfied all the
criteria for the physiological mediators, although adenosine seems
to be the most acceptable (Berne and Levy, 1977).
81
Adenosine and its phosphorylated derivatives are all powerful
vasodilators.
When the myocardial metabolic rate increases, resulting
in increased oxygen consumption, there is a decrease in myocardial
partial pressure of oxygen.
This leads to a decrease in ATP and an
increase in inorganic phosphate, thereby increasing the amount of
adenosine.
As the extracellular concentration of adenosine increases,
it produces a vasodilation of the coronaries and an increase in
coronary blood flow to restore myocardial tissue oxygen to normal.
The increase in blood flow washes out the additional adenosine, thus
operating as a feedback mechanism to decrease adenosine and the
resultant coronary vasodilation (Berne, 1974).
Coronary Vasodilators
The drugs employed as coronary vasodilators,,although used for
conditions involving regional tissue ischemia, do not act specifically
on the coronary vasculature but on the general systemic vasculature.
A host of chemically dissimilar agents fall under the category of
vasodilators.
The earliest group of chemicals to be used as vasodi-
lators were the nitrites and nitrates.
Organic nitrates have been
used for the relief of anginal pain since the 1800's.
Despite all
the research done on nitroglycerin and its widespread use, its
mechanism of action remains controversial.
The basic pharmacological action of the nitrates and nitrites
is to relax smooth muscle, particularly vascular smooth muscle.
The
organic nitrates act as rapidly as the nitrites and do not liberate
NO2 in the blood (Rath and Krantz, 1942; Krantz, et al., 1962;
Nickerson, 1975), thus it appears that the nitrates have a direct
82
action on smooth muscle.
The relief of angina pectoris by nitro-
glycerin was attributed to vasodilation of occluded coronary vessels
and thereby restores bloow flow to the ischemic myocardial tissue.
However, it has been pointed out by Winbury (1969), that in an
ischemic area tissue hypoxia probably already causes nearly maximal
'arteriolar dilation because of autoregulation (Winbury, 1969).
The
nitrates and nitrites produce both a local and systemic redistribution
of blood flow (Mason and Braunwald, 1965; Fam and McGregor, 1968;
Winbury, 1969; Winbury, et al., 1971; Becker, 1971; Horowitz, et
al., 1971; Cohen, et al., 1973).
Mason and Braunwald (1965) found
that nitroglycerin reduced systemic arterial Pressure, elevated
forearm blood flow, lowered forearm vascular resistance and decreased
venous tone.
By reducing the tone of the capacitance vessels,
venous return to the heart is decreased thus diminishing the metabolic
activity of the heart (Sharpey-Schafer, 1962; Ablad and Mellander,
1963; Robin, et al., 1967).
At the local level the organic nitrates
cause a redistribution of myocardial blood flow to the ischemic
subendocardium by reducing extravascular compression on the deep
lying capillaries and collaterals (Winbury, et al., 1971).
By
dilating the larger coronary conductance vessels blood is shunted to
the passive collateral vessels leading to the ischemic area (Winbury,
1969; Winbury, et al, 1971; Fam and McGregor, 1968).
Fam and McGregor
(1964) suggest that nitroglycerin exerts its action only in the
presence of well developed collateral circulation.
Directly related to the reduction of coronary vascular resistance
is oxygen delivery to the myocardial tissue.
It is believed the
83
relief of ischemia results from enhancing oxygen delivery rather
than by altering load (Honig, et al, 1960).
However, they were able
to find no effect of nitroglycerin on oxygen consumption in the
resting heart muscle.
Brachfeld, et al., (1959) found that following
nitroglycerin administration, myocardial oxygen consumption increased,
as did coronary flow, mediated by a lowered coronary vascular resistance.
A reduction in 02 requirements, reported by Robin, et al.,
(1967) was attributed to decreased ventricular wall tension and
velocity of myocardial fiber shortening after nitroglycerin..
Nitro-
glycerin injected directly into the coronary artery selectively
increased subendocardial oxygen tension due to a redistribution of
coronary blood flow toward the subendocardium and a small direct
reduction in cardiac metabolism (Weiss and Winbury, 1972).
Another feasible therapeutic mechanism of the nitroglycerin
type drugs is their action as functional antagonists of norepinephrine.
Gillis and Melville (1970) found that nitroglycerin produced .only a
transient increase in coronary flow and exerted significant blockade
of the contractile force response induced by norepinephrine without
affecting the contractile force response induced by Ca2+.
No blockade
of the heart rate response_ induced by norepinephrine was observed.
Nitroglycerin also exerted significant blockade against the pressor
effects of norepinephrine.
Increasing doses of nitroglycerin produced
a tolerance to both the coronary flow changes and the cardiac adrenergic blockade.
effects.
Very little tolerance developed to the hypotensive
In addition to its coronary vasodilator action, nitroglycerin
appears to exert a significant degree of cardiac blockade.
The
84
.anti- epinephrine sympathetic effects of nitroglycerin regarding
myocardial function suggest that the therapeutic action of this drug
is not due to coronary dilation alone but also to a specific counteraction against the myocardial metabolic effects of an excess influx
of adreno-sympathetic epinephrine in the heart (Raab and Humphreys,
1947; Darby and Aldinger, 1960).
Systemic administration of nitroglyc-
erin was found to cause a fall in systemic blood pressure, eliminated
systolic bulging during coronary occlusion, and suppressed excitation
of sympathetic afferent cardiac fibers (Bosnjak, et al., 1981).
This nitroglycerin induced inhibition of cardiac afferents may have
been due to reduction of substances which are released during myocardial ischemia, redistribution of epicardial to endocardial blood
flow ratios, and elimination of systolic bulge.
Related to its role
as a functional antagonist of norepinephrine is its possible function
as an MAO inhibitor.
Ogawa, et al., (1967) demonstrated an increase
in myocardial norepinephrine content after administration of nitroglycerin and found that rat heart mitochondrial MAO was inhibited.
The increase in norepinephrine content after nitroglycerin administration may be explained by a reflex release of catecholamines (O'Rourke,
et al., 1971).
The nitrates, in particular the organic nitrates, have demonstrated
strong inhibition of ATPase activity (Krantz, et al., 1951; Honig,
et al., 1960).
In summary the major therapeutic mechanisms of nitroglycerin
are:
85
1.
A reduction in capacitance vessel resistance leading to
diminished venous return.
This has a beneficial effect on
myocardial oxygen requirements and coronary endocardial
perfusion by reducing mechanical compression.
2.
Dilation of large coronary vessels resulting in increased
collateral and redistributed flow.
3.
Central antagonism of the afferent limb of reflex vasoconstrictor innervation of coronary vasculature.
Perhexiline
Perhexiline maleate [2-(2,2-dicyclohexylethyl) piperidine]
increases myocardial blood flow and decreases oxygen consumption in
experimental animals and man.
The dominant action of perhexiline is
vasodilation of both coronary and systemic vascular beds (Hudak,
et al., 1970; Winsor, 1970).
Perhexiline has been reported to have
a propranolol-like effect (lowering maximum heart rate during exercise);
however, it does not lower the resting rate and is not considered a
beta-blocker (Winsor, 1970).
Perhexiline differs from nitroglycerin
in that coronary flow is maintained above control values with perhexiline despite a 40% fall in mean arterial
blood pressure and it acts
preferentially on the arterioles rather than on postarteriolar
vessels (Winsor, 1970; Hudak, 1970).
To summarize the effects of
perhexiline found by Hudak (1970) and Winsor (1970):
1.
Increases coronary flow
2.
Reduces heart rate (especially exercise-induced tachycardia)
3.
Nonspecific inhibition of cardiac responsiveness to sympathetic stimulation which may involve inhibition of ionic
transport
86
4.
A diuretic effect, possibly leading to ventricular volume
and tension reduction
5.
Quinidine-like effect on cardiac cells which provides
anti-dysrhythmia activity.
Prenylamine
Prenylamine IN(3'-phenyl-propyl-(2')-1,1-diphenyl-propyl-(3)amine; Segontin] is another agent employed in coronary insufficiency.
Its principle action appears to be moderate depletion of peripheral
and central catecholamines (Bohm, et al., 1960; Kochsiek, et al.,
1960; Juorio and Vogt, 1965; Nielson and Oman, 1967).
Obianwu
(1967) found that in various animal species prenylamine lowered the
mean arterial blood pressure and the heart rate, antagonized responses
to tyramine and isoprenyline and potentiated those to norepinephrine
and epinephrine.
The potentiation of the actions of norepinephrine
and epinephrine after prenylamine is due to inhibition of the uptake
process of these amines at specific binding sites.
Prenylamine
exhibits alpha and beta blocking action in vitro but only moderate
beta blockade in vivo (Obianwu, 1967).
Prenylamine's beta blocking
action may account for its beneficial effects in angina] patients.
Juorio and Vogt (1965) have likened the action of prenylamine to the
weaker analogs of reserpine.
Granular amine storage mechanisms in
adrenergic nerves are blocked by prenylamine (Nielsen, 1967).
In
vitro and in vivo tests with prenylamine have demonstrated good
vasodilator effects on the coronary vasculature without having
significant hemodynamic side effects such as decreased blood pressure
or increased heart rate (Kochsiek, et al., 1960; Lindner, 1960).
87
Prenylamine appears also to have very little, if any, peripheral
dilatory effect.
In summary of prenylamine:
1.
Increases coronary flow
2.
Depletes cardiac catecholamines and inhibits calcium
influx into cardiac cells thereby reducing basal tone and
responsiveness to sympathetic discharges and consequent
energy requirements.
3
Causes a slightly sustained decrease in blood pressure
which lowers aortic resistance and ventricular compression,
favoring reduced cardiac work and better coronary perfusion,
respectively.