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600
Cholinergic Sensitivity of the Denervated
Canine Heart
DONALD V. PRIOLA AND HAROLD A. SPURGEON
With the technical assistance of Constantine Anagnostelis
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SUMMARY We determined whether the extrinsically denervated heart becomes supersensitive to acetylcholine
(ACh) and nicotine (NIC). Fifteen mongrel dogs were subjected to total extrinsic cardiac denervation. The negative
inotropic responses of their hearts were compared with those of 15 control dogs with respect to ACh (0.005-1.0 pig)
and NIC (10-300 fig) administered intracoronary. All dogs were placed on cardiopulmonary bypass, and atrial and
ventricular contractility were evaluated using a four-chamber, isovolumic technique. The denervated hearts showed
no detectable supersensitivity to ACh. Because the effects of ACh at the doses used are almost completely on the
decentralized cardiac muscle cells, the results indicate that the cardiac muscle is an exception to the increased
sensitivity usually produced by decentralization. When the positive inotropic effects of NIC were blocked by practolol
in the control hearts, the negative inotropic effects of the intrinsic parasympathetic neurons (IPN) are seen clearly.
These intrinsic neural elements are specifically activated by NIC. The denervated atria and ventricles displayed NIC
dose-response curves that consistently were shifted to the left when compared to control animals after 0,-blockade.
These results indicate the IPN, which are denervated by the surgical procedure, do become more sensitive to NIC.
The increases in sensitivity to NIC were between 2- and 6-fold, comparable to the increased sensitivity to norepinephrine observed in denervated hearts. The postjunctional IPN supersensitivity probably occurs as a result of spreading of
nicotinic receptors on the ganglion cell membrane.
WALTER B. CANNON formulated a "law of denervation" based upon extensive experimental work with neuroeffector systems.1 Simply put, this "law" stated that
a denervated organ becomes supersensitive, after a time,
to its usual neurotransmitter substance. The most exquisite example of this denervation supersensitivity is seen in
skeletal muscle whose sensitivity to acetylcholine increases
as much as 1000-fold following denervation.2 Other structures show varying degrees of supersensitivity, depending
on many factors, among which is whether the denervation
is pre- or postganglionic.3
The heart, once thought to be an exception to this law,
demonstrates an increase in sensitivity to norepinephrine
(NE) of 1.5-10-fold following denervation,4"6 depending
on whether one is measuring atrial contractility, ventricular contractility, or heart rate .5 The question of whether or
not the heart becomes supersensitive to cholinergic agents
following denervation is, however, unsettled. The scanty
data which exist are often inconclusive and contradictory.7-8
The following experiments were performed to determine whether the extrinsically denervated heart becomes
supersensitive to cholinergic agents which may interact
From the Departments of Physiology and Pharmacology, The University of New Mexico, School of Medicine, Albuquerque, New Mexico.
Supported by Grant No. HL-10869 from the National Institutes of
Health and a Cooperative Grant from the American and New Mexico
Heart Associations.
Dr. Priola was the recipient of Research Career Development Award
KO 5 HL-15, 912 from the National Heart and Lung Institute.
Dr. Spurgeon's present address is: Gerontology Research Center, National Institutes of Health, Baltimore City Hospitals, Baltimore, Maryland.
Address for reprints: Donald V. Priola, Ph.D., Department of Physiology, The University of New Mexico, School of Medicine, Albuquerque,
New Mexico 87131.
Received November 8, 1976; accepted for publication April 11, 1977.
with myocardium and cardiac parasympathetic neural elements. Two types of agents were employed: one that
primarily interacts with the effector cells (acetylcholine)
and one that primarily interacts with the intrinsic parasympathetic ganglion cell (nicotine). In this manner, it was
hoped that denervation-induced sensitivity changes in
either the effector cell or the intrinsic innervation, or both,
could be determined.
Methods
CHRONIC EXPERIMENTS
Fifteen mongrel dogs were anesthetized with thiopental
sodium (20-25 mg/kg) and subjected to total extrinsic
denervation of the heart using the method of Geis et al.9
The procedure consists of two operations separated by an
interval of 1-2 weeks. In the first operation, the left
atrium is separated from its mediastinal attachments from
the superior to the inferior junctions with the interatrial
septum and reanastomosed. At the same time, the aorta
and pulmonary artery are circumferentially stripped of
their adventitial layers. In the second operation, the entire
wall of the right atrium, as well as the interatrial septum, is
incised and reanastomosed. The latter incision is done
under inflow occlusion. This technique produces a heart
that has been effectively separated from all mediastinal
connections which may act as pathways for both sympathetic and parasympathetic cardiac nerves. It produces a
heart that is postganglionically denervated with regard to
its sympathetic nerves, but preganglionically denervated
with regard to its parasympathetic nerves.
A period of 3-6 weeks was allowed for recovery before
these dogs were employed in the acute phase of the experiments. This is ample time for development of supersensi-
CHOLINERGIC SENSITIVITY OF THE HEART/Pno/a and Spurgeon
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tivity to norepinephrine which usually reaches its maximum in 1-2 weeks.5 Prior to their acute use, the adequacy
of denervation was established by supramaximal stimulation of the cervical vagi (30 Hz, 5 msec, 2-10 V) and the
stellate ganglia (10 Hz, 5 msec, 10 V). In addition, tyramine, 500 ptg, was injected into the coronary circulation.
A change of >10% from control values in atrial contractility, ventricular contractility, or heart rate in response to
any one of these procedures was considered sufficient to
eliminate the animal from inclusion in the experimental
data. No negative inotropic responses to vagal stimulation
were observed in the denervated dogs used in this study.
In addition, atrial and ventricular myocardial samples
were removed after the experiment and analyzed for catecholamine content (both epinephrine and NE). All the
denervated dogs used in this study showed NE levels of
<0.02 fig/g wet weight in all chambers sampled. Total
catecholamine levels (NE + epinephrine) averaged 0.05
± 0.01 fig/g wet weight in hearts from the denervated
dogs.
ACUTE EXPERIMENTS
In addition to the denervated animals, 15 control dogs
were used in these experiments. All dogs were anesthetized with thiopental sodium (25-30 mg/kg, supplemented
as necessary) and the chests were opened by transsternal
thoracotomy in the fourth or fifth interspace. The cervical
vagi and stellate ganglia were isolated and prepared for
stimulation and the left carotid artery was cannulated for
pressure recording and subsequent drug injections. The tip
of the carotid cannula was positioned just at the aortic side
of the aortic valve. Once cardiac bypass was established,
drugs injected into this cannula would then enter the
coronary circulation directly because of the retrograde
nature of arterial perfusion during bypass. Total cardiopulmonary bypass was accomplished in the usual manner.
Venous return from the venae cavae was drained into a
disc oxygenator, oxygenated in an atmosphere of 100%
O2, warmed, filtered, and returned to the dog via a femoral artery. Sumps in both ventricles were placed under airrelieved suction and the coronary and bronchial blood was
collected and returned to the oxygenator.
Arterial pH was measured at intervals throughout the
experiments with an Instrumentation Laboratories bloodgas analyzer and ranged between 7.35 and 7.40. Arterial
blood temperature was monitored with a thermistor probe
(Yellow Springs Instruments) placed directly in the arterial inflow line. The blood temperature was maintained
between 34 and 36°C by warming the oxygenator with
heat lamps. Arterial pressure was monitored by placing a
catheter in the ascending aorta with a pressure transducer
(Trantec). In these experiments, arterial pressure varied
between 75 and 125 mm Hg.
To record the contractile activity of the cardiac chambers, a four-chamber isovolumic technique was used. This
technique and the method of drug injection have been
described in earlier publications.10' " Saline-filled balloons
in each of the four chambers were used to record isovolumic pressures. Diastolic pressures were adjusted to 1-5
mm Hg in the atria and 3-8 mm Hg in the ventricles,
601
pressures equivalent to those recorded in the open-chest
dog preparation. The heart rate was held constant by
pacing from the right atrium. With heart rate, preload,
and afterload eliminated as variables, changes in isovolumic pressures very closely reflect changes in chamber
contractility.
All drugs were administered intracoronary via the carotid cannula in volumes of 0.5-1.0 ml followed by a 1- to
2-ml wash with normal saline. Acetylcholine (ACh), in
doses of 0.005-1.0 fig, was used to produce the expected
negative inotropic responses of the cardiac muscle. Nicotine HO (NIC) was used to activate the intrinsic parasympathetic neural elements (IPN). The doses employed were
10-300 /ig. These doses have been shown to produce a
highly selective activation of IPN which results in negative
inotropic responses of both atria and ventricles." These
responses to NIC can be blocked totally by neural or
ganglionic blocking agents, while the responses to ACh
are completely unaffected." Thus, the two agents can be
employed to study the effects of denervation on both the
decentralized effector cell and the denervated IPN. In
dogs with intact cardiac nerves, NIC also induces the
release of NE from postganglionic sympathetic neurons.
This results in an indirect, positive inotropic drug effect. In
seven experiments on control dogs, these effects were
abolished with 2-4 mg of intracoronary practolol, a /3,adrenergic blocking agent. The responses of these control
animals after practolol were used as the control group to
detect changes in sensitivity of the intrinsic innervation to
NIC in the denervated dogs.
Changes in isovolumic pulse pressures in response to the
injections were measured and expressed as percent change
from control ± SEM. Significance of differences between
the means of responses in the two groups of dogs was
analyzed by Student's Mest. A level of P < 0.05 was
considered to indicate a significant difference.
Results
RESPONSES TO ACh
Typical responses of a normal and a denervated dog to
ACh, 0.5 jxg, are shown in Figure 1. Both hearts were
CONTROL
DENERVATED
RA 1
RV
so-
.5mcg ACH
FIGURE 1 Recordings which illustrate typical responses of an
intact heart (left panel) and a denervated heart (right panel) lo 0.5
fj.g acetylcholine (ACh) (arrows). Isovolumic pressures from the
right atrium (RA), right ventricle (RV), left atrium (LA), and left
ventricle (LV) are shown. Calibrations (center) are in mm Hg.
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CIRCULATION RESEARCH
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paced at a constant rate from the right atrium (RA). In the
normal heart, both RA and left atrial (LA) isovolumic
pressures decrease within a few beats following injection
and show significant recovery even before the maximum
ventricular depressions have been reached. Both the right
(RV) and left (LV) ventricles display negative inotropic
responses to ACh, but they develop more gradually (1520 sec) and recover more slowly. These differential atrial
and ventricular responses to injected ACh have been reported earlier" and also are typical of atrial and ventricular responses to vagus nerve stimulation.10 The responses
of the denervated heart to the same dose of ACh (right
panel) are not remarkably different either quantitatively
or qualitatively from those of the control animal. In this
particular dog, the LA was not responsive to ACh at this
dose level. It is important to note that in no case is there a
significant positive component to the cardiac responses to
ACh. These recordings are quite representative of what
was observed overall; i.e., injection of ACh in doses of up
to 1.0 /xg, intracoronary, rarely produced any significant
positive inotropic response. These observations are in
sharp contrast to those for NIC which are described below.
The apparent lack of supersensitivity to ACh observed
in the denervated dogs is substantiated by the overall
comparative data shown in Figure 2. In only one chamber
and at only one dose level (RA, 0.01 /xg) is the response
of the denervated heart significantly greater than that of
the normal heart. In fact, the opposite appears to be true,
i.e., in most instances, the denervated heart exhibits less
depression in response to ACh than does the intact heart.
In four cases, these lesser responses are significantly different at the P — 0.05 level. As previously reported for
denervated animals," the most responsive chambers to
ACh in the normal dogs were the RA and LA. The
RA
VOL. 41, No. 5, NOVEMBER
1977
ventricles generally displayed about 50% of the negative
inotropic responses of the atria. Except for the RA, these
differences are not nearly as clear in the denervated dogs.
In both groups of dogs, inotropic responses do not show a
clear relationship to ACh dose. Based on the data shown
in Figure 2, it does not appear that the denervated heart
shows any supersensitivity to the negative inotropic effects
of ACh.
RESPONSES TO NIC
Intracoronary injection of NIC has been shown to activate the intrinsic parasympathetic ganglion cells (IPN) in
the heart." Although the cardiac muscle cells undergo
parasympathetic decentralization following chronic extrinsic cardiac denervation, these intrinsic neural elements are
denervated by the procedure.12 The comparative responses to NIC of the two groups should, therefore, reflect
any supersensitivity of the IPN.
Typical responses of control hearts (before and after j3,blockade), as well as denervated hearts, to 50 /ig of NIC
are shown in Figure 3. Prior to /3,-blockade (left panel),
the negative inotropic responses produced by nicotinic
activation of the IPN are masked by concurrent release of
NE from postganglionic sympathetic nerves. This results
from stimulation of nicotinic receptors on the adrenergic
nerve fiber.13' H This indirect NIC action is apparent in the
responses of the RV in Figure 3. After elimination of this
indirect action by /3,-blockade (central panel) or denervation (right panel), only negative inotropic responses are
produced. These negative inotropic effects of NIC are
qualitatively similar to those produced by ACh (Fig. 1), in
that the atrial depression has a short latency and a fast
recovery (RA), while the ventricular responses are slower
in both developing and recovering (RV and LV, right
LA
RV
LV
j I control
• denervated
I
<
T
a:
i-
z
o
o
I
T
a.
o
.01
.05
.5
s
.01
I
I
.05
.1
-S
81
.85
.1
.01
.05
ACH DOSE,meg INTRACORONARY
FIGURE 2 Summary of responses of intact (open bars) and denervated hearts (closed bars) to acetylcholine (ACh). Each bar is the mean of
15 experimental values. SEM is shown by the brackets. Chambers are as in Figure I. Significant differences (P < 0.05) of denervated from
control responses at a given dose are indicated by • .
CHOLINERGIC SENSITIVITY OF THE HEART/Priola and Spurgeon
CONTROL
no blockade
CONTROL
DENERVATED
after A blockade
RA1
RV
LA
Slmcg NICOTINE
51 m e g NICOTINE
51 meg NICOTINE
FIGURE 3 Recordings illustrating typical responses of intact
hearts before (left panel) and after (center panel) f},-blockade with
practolol and denervated hearts (right panel) to the same dose of
nicotine (arrows). Left and center panel recordings are from the
same dog. Calibrations are in mm Hg. Chambers are as in Figure
1.
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panel). The responses of the denervated dog shown in
Figure 3 suggest an increase in sensitivity to NIC as a
result of denervation of the IPN. After /3,-blockade, the
control heart shows little negative inotropic response to 50
/ng NIC, whereas the denervated heart exhibits significant
RA, RV, and LV negative inotropy in response to the
same dose.
When the overall responses of the control (without /3,blockade) and denervated hearts are compared, some
clear differences emerge (Fig. 4). At the lower doses of
NIC (25 and 50 fig), the control animals show responses
that are entirely negative. At the higher doses (100 and
200 /tig), most of the responses are positive. Obviously,
the net response of a chamber to NIC is the resultant of
the indirectly mediated positive response and the indirectly mediated IPN negative response. In the case of the
denervated dogs, the degeneration of the postganglionic
sympathetic nerves eliminates the source of NE for the
indirect positive response. Therefore, all NIC responses
observed in the denervated dogs were negative. On this
basis, many of these responses were significantly different
from those of the normal dogs. Positive responses to NIC
in the 15 denervated animals were very rare; i.e., only
nine of 168 observations (5%) were positive. In the 15
intact dogs, 56 of 131 responses to NIC were positive
(43%).
To provide suitable control responses with which the
responses of the denervated animals might be compared,
seven intact dogs were given NIC following /3,-blockade
with practolol. Figure 5A-D compares the NIC doseresponse curves for the four heart chambers in these two
groups. After/3,-blockade, only 10 of 153 responses of the
intact dogs to NIC were positive (6.5%). Overall, the
denervated dogs displayed an increased sensitivity to NIC.
Although the differences between the mean values of the
two groups were not all statistically significant, the doseresponse curves for the denervated group were consistently shifted to the left, compared with intact dogs, after
/3,-blockade. In Figure 5A, the RA curve for the denervated dogs is significantly shifted to the left, indicating an
increase in sensitivity of 3- to 5-fold, depending upon
603
which portion of the curves one selects for comparison.
Responses of the denervated animals at 25 and 50 /xg NIC
are significantly greater than control. There is no significant difference between maximum responses in the two
groups except that they occur at different dose levels. The
sensitivity changes in the LA (Fig. 5B) are not as clear.
Although the dose-response curve for the denervated dogs
is shifted to the left, only the 25 /Ag point is clearly
statistically different from control. The magnitude of the
shift is approximately 2-fold. There is also evidence in the
RV responses that denervation increases sensitivity to NIC
(Fig. 5C). Although responses to the two lower doses of
NIC do not appear to be different between the two groups,
the denervated dogs show a significantly higher response
at 100 /ng. The RV is the only chamber in which denervation did not produce an apparent decrease in the threshold
dose of NIC. This latter change is clear in the doseresponse curves of Figure 5D. The LV of the denervated
dogs exhibits an increased sensitivity to NIC throughout
the dose range studied. The difference at only one dose
level (100 /Ltg) is statistically significant. Depending on the
method of comparison used, the LV of the denervated
dogs appears to be 2-4 times more sensitive to NIC than
that of the control animals. If one uses the horizontal
displacement of the dose-response curve, the increase
could be as much as 6-fold.
As reported earlier," these responses to NIC could
subsequently be blocked by 10-30 /xg of tetrodotoxin,
intracoronary, which had no effect on responses to ACh.
Discussion
On the basis of the data presented, it is clear that the
extrinsically denervated heart does not become supersensitive to ACh (Fig. 2). The lack of supersensitivity is
undoubtedly related to the fact that the surgical denervation produces a decentralization rather than a denervation
of the atrial and ventricular effector cells. This contrasts
with data on other organ systems3'I5> 16 which indicate a
modest (2- to 4-fold) increase in sensitivity following dedenervated
control
RA
25 5* 1M 211
25 56 IN 2M
25 51 IN 2M 25 SO IN 2M
nicotine dose, meg intracoronary
FIGURE 4 Summary of isovolumic inotropic responses to nicotine
before /J,-blockade of intact (open bars) and denervated hearts
(closed bars). Ordinate indicates percent change of isovolumic
pressure from control, and dose of nicotine is shown on abscissa.
Each value represents the mean of 15 experiments and the bracket
indicates the SEM. Chambers are as in Figure I; • = P < 0.05.
CIRCULATION RESEARCH
604
IS
RIGHT ATRIUM
—Control After
Ri Blockade
, Cardiac Denervated
3VOLUAHIC PRESSURE,
FROM (CONTROL
y
/
5
SO
/
J
a;"-
I
1977
LEFT ATRIUM
• Contol After
Bi Blockade
Cardiac Denervated
UJ
V
VOL. 4 1 , No. 5, NOVEMBER
'10-
1
31
40
20
NICOTINE, meg
30
40
SO 60 70 <(
s'o
9'oibo
200
300
NICOTINE,meg
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111
K
3
ffig
KrO.Z
UO20-
RIGHT VENTRICLE
•-Control After
81 Blockade
-Cardiac Denervated
25-
L..
lll_
LEFT VENTRICLE
—Control After
Bi Blockade
° -Cardiac Denervated
20-
Q.Z
**"i
|8.sH
_iO 1 5
OK
-1-'"
>"•
O
8? H
40
SO 60 70 80 90100
300
NICOTINE, meg
~30^
40
50
60 70
I 90100
—I—
200
301
NICOTINE.mcg
FIGURE 5 A -D, Dose-response curves for nicotine as measured in the right atrium (A), left atrium (B), right ventricle (C), and left ventricle
(D) of normal dogs after (3,-blockade (solid lines) and of denervated dogs (broken lines). The average percent decrease of isovolumic
pressure from control is shown on the ordinate, and the dose of nicotine is shown on the abscissa on a log scale. Each point represents the
average of 15 experiments, and the bracket indicates the SEM. Curvesfittedby eye. Values for denervated dogs that significantly differ from
control (P < 0.05) are indicated by • .
centralization. As early as 1936, Cannon and Rosenblueth'6A showed that the denervated superior cervical
ganglion displayed a response threshold to ACh that was
only one-fourth that of the innervated ganglion. The decentralized nictitating membrane was used as the test system. The phenomenon of supersensitivity following decentralization has also been demonstrated in vas deferens
and other effector cells.16 Very little information has been
available regarding cholinergic sensitivity of the denervated heart. Recently, Jacobs and his co-workers7 have
reported that extrinsic cardiac denervation does not appear to alter the bradycardia in response to ACh. However, these workers did show an increased tendency for
atrioventricular (AV) block in response to ACh, suggesting a possible increase in AV nodal sensitivity. More
recently, Hageman et al.8 have shown that the perfused
sinoatrial (SA) node of selectively parasympathectomized
animals does not show an increased sensitivity to ACh
injected directly into the isolated SA nodal artery. It
appears that the inotropic sensitivity of the denervated
heart to ACh also is an exception to the general observations following decentralization. If atrial and ventricular
muscle do show any moderately increased cholinergic sensitivity following extrinsic denervation, perhaps the techniques used in these experiments were not sensitive
enough to demonstrate it. There is considerable response
variability in the methods used because of the stress placed
upon the animal, variation in the pattern of drug delivery
through the coronary circulation, and the nature of the
isovolumic pressure-recording technique. It may be that
this variability would be enough to obscure small changes
in sensitivity. However, the data of Figure 2 are quite clear
and appear to rule out such marginally detectable changes.
It is more likely that the heart is indeed an exception to the
usual enhancement produced by chronic decentralization.
There is no technique presently available for selectively
eliminating the IPN in order to obtain truly cholinergically
denervated cardiac muscle cells. It is conceivable that
chronic ganglionic blockade as used in other organs15
might accomplish this objective. Until elimination of the
IPN can be accomplished, the question of whether denervated cardiac muscle becomes supersensitive to ACh will
remain unanswered.
As we have shown previously, the negative inotropic
CHOLINERGIC SENSITIVITY OF THE HEART/Priola and Spurgeon
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effects of NIC at these doses are primarily the result of
activating the IPN." Denervation of the 1PN (which would
be expected from the surgical technique used) does result
in an increased sensitivity to nicotinic stimulation (Fig.
5A-D). Although these increases are not statistically significant in all chambers at all dose levels, the NIC doseresponse curves in the denervated dogs are consistently
shifted to the left of the control curves. There are enough
statistically significant differences to conclude that denervation supersensitivity of the IPN does occur. This supersensitivity is only apparent, however, when the indirect
positive inotropic effects of NIC in the control dogs are
blocked by practolol. In the absence of practolol (Fig. 4),
the effects of NIC on the IPN are obscured by release of
catecholamine from sympathetic postganglionic nerves.
This phenomenon has been described previously by a
number of workers.14' l7~20 That the sources of this positive
inotropic effect of NIC are the sympathetic cardiac neural
elements is verified by the complete absence of positive
inotropic effects in the denervated animals (Fig. 4).
The increased sensitivity of the IPN is probably the
result of a postsynaptic change which involves a "spreading" or increased number of drug-sensitive receptors on
the membrane of the ganglion cell similar to the well
known effect of denervation on the cell membranes of
skeletal muscle.2'21 Kuffler and his co-workers22 have
shown that the area of chemosensitivity of IPN cells in the
interatrial septum of the frog heart increases greatly after
section of the vagosympathetic trunks. The time course of
development of this IPN supersensitivity (4-8 days) correlates well with the usual time course for supersensitivity
development.16 Therefore, the increased responsiveness to
NIC seen in the cardiac-denervated animals probably represents a true postjunctional or type II16 supersensitivity of
the intrinsic nerves of the heart.
No such supersensitivity was observed with ACh (Fig.
2). Neither was ACh effective in producing indirect positive inotropic responses similar to those of NIC. There are
at least two reasons for these findings. First of all, nicotinic
receptors of the cardiac nerves are probably much less
sensitive to ACh than are muscarinic receptors on the
cardiac muscle cells. Blumenthal et al.23 have shown that
intracoronary doses of ACh from 0.01 to 1.0 fxg produce
only negative inotropic effects on the canine LV that can
be blocked by atropine. In order to produce positive
inotropic effects, the dose of ACh had to be raised to 20300 j/.g, and prior administration of neostigmine and atropine was necessary. These positive responses could then
be blocked completely by intracoronary administration of
tetraethylammonium chloride.23 These observations indicate a vastly greater ACh sensitivity of cardiac muscarinic
receptors, compared with nicotinic receptors. This would
explain why ACh did not produce any positive inotropic
responses at the doses employed in this study (0.01-0.5
/xg). Even with an increased nicotinic sensitivity of the
IPN following denervation, the doses of ACh used probably were still insufficient to produce IPN activation. These
speculations assume, of course, that the nicotinic receptors
on the IPN and those on the sympathetic nerve endings
have approximately the same characteristics. Second, the
605
inotropic responses to both direct and indirect actions of
ACh (muscarinic and nicotinic) are qualitatively identical.
Therefore, any effects of IPN stimulation which would be
produced at the doses used would probably be much less
than the direct effects of ACh on the effector cells. Thus,
IPN stimulation by ACh would not be detectable under
the present experimental circumstances even if it should
occur at the doses used.
The adrenergic supersensitivity produced by cardiac denervation4"6 would be considered a type I or "deviation"
supersensitivity according to Fleming's classification;16
i.e., a change in the percent of an administered amount of
a drug that is available in the biophase to interact with its
receptors. This results from the loss of the catecholamine
reuptake mechanism that had existed in the degenerated
sympathetic nerve endings. In contrast, the decentralization supersensitivity observed in the present experiments
would fit Fleming's classification as type II or "nondeviation" supersensitivity; i .e., enhanced responsiveness of the
target cells (IPN). It is interesting to note that these two
diverse mechanisms produce the same order of magnitude
of supersensitivity, 2- to 10-fold.
The chamber that illustrates the enhanced sensitivity to
NIC most clearly is the RA (Fig. 5A). This correlates well
with anatomical evidence that indicates the largest concentration of parasympathetic ganglion cells is in the R A .24' 25
References
1. Cannon WB: A law of denervation. Am J Physiol 198: 737-750, 1939
2. Miledi R: The acetylcholine sensitivity of frog muscle fibers after
complete or partial denervation. J Physiol (Lond) 151: 1-23, 1960
3. Fleming WW, McPhillips JJ, Westfall, DP: Postjunctional supersensitivity and subsensitivity of excitable tissues to drugs. Ergeb Physiol 68:
55-119,1973
4. Dempsey PJ, Cooper T: Supersensitivity of the chronically denervated
feline heart. Am J Physiol 215: 1245-1249, 1968
5. Priola, DV: Individual chamber sensitivity to norepinephrine after
unilateral cardiac denervation. Am J Physiol 216: 604-614, 1969
6. Spann JF, Sonnenblick EH, Cooper T, Chidsey CA, Willman VL,
Braunwald E: Cardiac norepinephrine stores and the contractile state
of heart muscle. Circ. Res. 19: 317-325, 1966
7. Jacobs HK, Randall WC, Kaye MP: Responses of the parasympathectomized canine heart to neurohumora! stimulation (abstr). Physiologist 17: 254, 1974
8. Hageman GR, Urthaler F, James TN: Absence of supersensitivity to
acetyl choline in the extrinsically denervated canine sinus node (abstr).
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Heterogeneity of the Hypoxic State in Perfused Rat
Heart
CHARLES STEENBERGEN, GILBERT DELEEUW, CLYDE BARLOW, BRITTON CHANCE, AND
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JOHN R.
WILLIAMSON
SUMMARY Tissue oxygen gradients were examined in the saline-perfused rat heart by NADH fluorescence
photography. In high flow hypoxia, where the coronary flow was maintained and the arterial oxygen tension was
gradually reduced, oxygen extraction was virtually complete before oxygen consumption was significantly diminished.
Inadequate oxygen delivery resulted in a well defined pattern of anoxic zones. The anoxic zones were several hundred
microns in width, an order of magnitude greater than intercapillary distances. In low flow hypoxia (ischemia), where
the arterial oxygen tension remained at its control value and the coronary flow was diminished, anoxic zones also
developed, following the same pattern as in high flow hypoxia. However, in ischemia, the anoxic areas developed
while the effluent oxygen tension was significantly greater than zero. Whereas respiratory acidosis between pH 7.3
and 6.9 resulted in vasodilation, below pH 6.8 there was a marked increase in vascular resistance. Anoxic zones
appeared despite only a slight change in effluent oxygen tension from the control. In high flow hypoxia, ischemia, and
acidosis-induced ischemia, the anoxic zones disappeared when control perfusion conditions were restored. The data
demonstrate that tissue oxygen gradients are very steep in the hypoxic state, so that ischemia and hypoxia result in
discrete heterogeneous areas of anoxic tissue bounded by sharp areas where the oxygen supply is sufficient to maintain
normal mitochondrial oxidative function. In these states in which oxygen delivery is less than oxygen demand,
coronary perfusion appears to be regulated at the level of the arterioles rather than the capillaries.
MYOCARDIAL ENERGY production is predominantly
determined by aerobic metabolism. ATP synthesis from
glycolysis during anoxia is insufficient to maintain cardiac
work at its normoxic level, even though glycolytic flux is
increased significantly.1"3 Therefore, the delivery of oxygen to the myocardial cells is crucial for normal function.
The heart has a rich network of capillaries which provide
adequate blood supply under the normal range of work
loads so that oxygen delivery does not limit oxygen consumption.4 However, when either the arterial Po2 or the
blood flow is lowered below critical levels, oxygen delivery
can diminish until it limits aerobic ATP synthesis. The
nature of the oxygen gradients in the hypoxic myocardium
under these conditions is not well understood.
Several theoretical treatments of this issue have been
previously presented.5 Initially, oxygen was assumed to be
From the Department of Biochemistry and Biophysics. University of
Pennsylvania, Philadelphia. Pennsylvania.
Supported by National Institutes of Health Grants HL-14461. HL18708, and GM-07170.
Address for reprints: Dr. John R. Williamson, University of Pennsylvania. Department of Biochemistry and Biophysics, School of Medicine-G3,
Philadelphia. Pennsylvania 19104.
Received December 23. 1976; accepted for publication May 5. 1977.
supplied to the tissue based on diffusion according to
Fick's law. More recently, the importance of myoglobinfacilitated oxygen transport has been recognized." The two
principal theoretical models of oxygen delivery are
Krogh's cylinder model, which assumes that the venous
Po2 limits oxygen diffusion from the capillary, and Diemer's cone model, which recognizes that capillary Po2
varies from the arterial to the venous end but requires that
blood flow in adjacent capillaries be in opposite directions.
Both models predict that, in the hypoxic myocardium, the
anoxic zones should be confined to the tissue at the greatest distance from a capillary. More complex mathematical
models based on a single capillary and its surrounding
tissue have been devised, but they also predict that oxygen
supply will first become limiting in the '"lethal" corners."
According to these models, the size of the anoxic areas
may increase as the arterial P02 is decreased, but the tissue
immediately adjacent to a capillary should remain well
oxygenated and the tissue Po2 should decline as the distance from a capillary increases. Grunewald8 has attempted to determine the effect of inhomogeneity in the
coronary circulation, but. in the absence of experimental
data, he could onlv conclude that the tissue Po2 distribu-
Cholinergic sensitivity of the denervated cannine heart.
D V Priola and H A Spurgeon
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Circ Res. 1977;41:600-606
doi: 10.1161/01.RES.41.5.600
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Copyright © 1977 American Heart Association, Inc. All rights reserved.
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