Peptidergic modulation of in situ canine intrinsic cardiac neurons

Peptides.Vol. 14, pp. 191-202, 1993

0196-9781/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.

Printed in the USA.

Peptidergic Modulation of In Situ Canine Intrinsic Cardiac Neurons J. A. A R M O U R , L M. H. H U A N G

AND

F. M. S M I T H

Departments of Physiology and Biophysics, and Anatomy, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada R e c e i v e d 25 J u n e 1992 ARMOUR, J. A., M. H. HUANG AND F. M. SMITH. Peptidergicmodulation of in situ canine intrinsic cardiac neurons. PEPTIDES 14(2) 191-202, 1993.--In order to determine which peptides are involved in modulating intrinsic cardiac neurons, angiotensin II, atrial natriuretic peptide, bradykinin, calcitonin gene-related peptide, enkephalin, neuropeptide Y, oxytocin, substance P, and vasoactive intestinal peptide dissolved in saline were administered individually by microinjeetion adjacent to spontaneously active canine intrinsic cardiac neurons. No neuronal or cardiac responses were elicited when saline was administered into active loci or when peptides were administered into loci with no spontaneous activity. Each peptide elicited neuronal responses when administered into active loci in most animals, bradykinin eliciting neuronal responses in every active locus studied. Concomitant cardiovascular responses were elicited in many cases when every peptide except atriopeptin was studied. After cardiac decentralization, neuronal and cardiovascular responses to repeat doses of peptides occurred with less frequency than before decentralization, implying that connections with central and other intrathoracic neurons can influence the function of peptide-sensitive intrinsic cardiac neurons. After atropine and timolol administration, cardiovascular, but not neuronal, responses to peptides were eliminated, indicating that cardiovascular responses were dependent upon efferent parasympathetic and sympathetic neurons. It is concluded that a number of neuropeptides may be involved in regulation of cardiac function by intrinsic cardiac neurons. Angiotensin Oxytocin

Atriopeptin Substance P

Bradykinin Calcitonin gene-related peptide Vasoactive intestinal peptide

E N K E P H A L I N , neuropeptide Y (NPY), oxytocin, substance P, and vasoactive intestinal peptide (VIP) can directly or indirectly modify stellate and middle cervical ganglion neurons that are involved in regulating the m a m m a l i a n heart (5,6). Anatomical evidence has indicated that calcitonin gene-related peptide (CGRP) (36), N P Y (16,21), substance P (9,16,25,29,34), and VIP (21,41) are associated with neuronal somata in, or on, the m a m m a l i a n heart. Furthermore, primary cardiac afferent neurons have been proposed to contain C G R P and substance P (29). In accordance with these finding, recent physiological evidence has demonstrated that some peptides can affect canine intrinsic cardiac neurons that modulate cardiac rate and force of contraction (8). Thus, peptidergic neurons in both extrinsic and intrinsic cardiac ganglia are known to be involved in cardiac regulation. Bradykinin, oxytocin, and substance P can modify in situ m a m m a l i a n intrinsic cardiac neurons such that cardiodynamics are affected (8). However, it is not known how these peptides affect activity generated by these neurons. Furthermore, it is not known whether other peptides can modulate intrinsic cardiac neuronal activity. Thus, the present experiments were devised to determine whether nine different neuropeptides can affect the activity generated by in situ canine intrinsic cardiac neurons

Enkephalin

Neuropeptide Y

and, if so, in what manner. The peptides studied were angiotensin II, atriopeptin, bradykinin, CGRP, [D-Ala2,D-LeuS]enkephalin (DADLE), NPY, oxytocin, substance P, and VIP. Furthermore, these investigations were performed to determine whether peptide-induced neuronal responses can be associated with alterations in cardiodynamics. Studies were performed in neuraily intact as well as acutely decentralized preparations in order to determine if the connections of intrinsic cardiac neurons with extrinsic cardiac ones, including neurons in the central nervous system, are important for the initiation of responses by specific peptides. METHOD

Animal Preparation Twenty-five mongrel dogs of either sex, weighing 19-26 kg, were sedated with sodium thiopental (12-15 mg/kg IV) and anesthetized with alpha chloralose (100 mg/kg IV). Thereafter, alpha chloralose (25 mg/kg IV) was administered as a bolus every 1.5 h throughout the experiments or more frequently, as required. Following intubation, positive-pressure ventilation was initiated and maintained using a Bird Mark 7A ventilator. A bilateral thoracotomy was made in the fourth intercostal space.

t Requests for reprints should be addressed to J. A. Armour, Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada.

191

192 The ventral pericardium was incised and retracted laterally to expose the ventral right atrial deposit of fat, which contains the ventral component of the right atrial ganglionated plexus (18). A miniature solid-state pressure transducer (Konigsberg Instruments, model PI90; 5 mm diameter, 1.5 mm thick) was inserted into the midwall region of the left ventricular ventral wall to record regional intramyocardial pressure (7). This sensing device was employed since intraventricular pressure represents a less sensitive index for detecting ventricular force changes induced by efferent autonomic neurons (12). Since the left ventricular ventral wall is richly innervated (5,7,12), this region was chosen for the sensor placement. Left ventricular chamber pressure was measured using a Bentley Trantec model 800 transducer connected to a Cordis #7 catheter inserted into that chamber via a femoral artery. All data, including a lead II electrocardiogram, were recorded on an Astro-Med, Inc. model MT 9500 eight-channel rectilinear recorder. The output of the recorder was led to a computer utilizing a 12-bit analog-to-digital converter. Data were stored on 20-Mb Bernouille disks for later analysis.

Neuronal Recording Neurons in the right atrial ventral ganglionated plexus were studied. These neurons lie embedded in subepicardial fat on the ventral surface of the right atrium, as has been described elsewhere (18). To minimize epicardial motion during each cardiac beat, a circular ring of heavy-gange wire was gently placed around the epicardial fat of the ventral surface of the right atrium. A tungsten microelectrode-micropipette assembly, mounted on a micromanipulator, was placed over the epicardial fat so that the assembly could be slowly advanced into the fat to search for neuronal activity. The indifferent electrode was attached to structures adjacent to the heart. Signals were amplified by a Princeton Applied Research model 113 amplifier that had bandpass filters set at 300 Hz to 10 kHz and an amplification range of 100-500×. The output of this device was further amplified (50-200×) and filtered (bandwidth 100 Hz to 2 kHz) using an optically isolated amplifier (Applied Microelectronics Institute, Nova Scotia, Canada). The output of this signal conditioner was led to a Nicolet model 207 oscilloscope for monitoring neuronal activity. Signals from the conditioner unit were also led to the recording and storage devices described above. With the aid of a dissecting microscope, the fat on the ventral surface of the right atrium was explored with the microelectrode at depths ranging from the surface of the fat to regions adjacent to cardiac musculature. The latter was identified by an increase in the amplitude of the ECG artifact. One to three loci were identified in the right atrial ganglionated plexus of each animal from which action potentials were recorded, individual units being identified by the amplitude and shape of their action potentials. Periodic motion at the recording site occurred due to cardiac and respiratory dynamics, thereby inducing minor fluctuations in the amplitude of individual action potentials generated by a given unit over time. Such fluctuations in the amplitude of action potentials generated by one unit were found to vary by less than 10 uV over several minutes, action potentials retaining the same configurations over time. Thus, action potentials recorded in a given locus with the same configuration and amplitude (_+10 # V ) were considered to be generated by a single unit. Action potentials with signal-to-noise ratios greater than 3:1 were analyzed. The frequency of activity generated by a given unit was analyzed for 10-s periods before and after administration of each pelatide. A 25% alteration of baseline activity following peptide administration was required to classify a unit

ARMOUR. HUANG AND SMITH as generating increased or decreased activity. Action potential data were grouped according to whether activity increased, decreased, or remained unchanged. Heart rate, as well as left ventricular intramyocardial and chamber systolic pressures, was measured for l0 consecutive cycles before and after chemical administration. Spontaneous fluctuations of cardiodynamics were minimal over minute periods of time, presumably due to the effects of anesthesia. For example, heart rate variability was less than 5 beats/min and systolic pressure fluctuations were less than 5 mmHg during control conditions. Thus, thresholds for classifying induced changes were chosen to be above these ranges. In order to rule out the possibility that recorded action potentials were generated by intracardiac axons, the recording electrode was also inserted into an intrinsic cardiac nerve (the ventral lateral cardiac nerve) in four animals in an attempt to record spontaneous activity propagated by intrinsic cardiac axons.

Administration of Peptides Peptides were applied to the intrinsic cardiac nervous system via two different methods. 1. In 21 dogs, peptides were applied in 5-#1 quantities adjacent to epicardial ganglia from which spontaneous activity arose. 2. In four dogs, peptides were administered in 10-100-ul quantities into the regional arterial supply of the right atrial ganglionated plexus.

Local app#cation. The locus identified in the right atrial ventral ganglionated plexus of each of the 21 dogs that contained the greatest number (two to six) of spontaneously active units was chosen for investigation. A four-barrel glass micropipette (Activational Systems), with an attached tungsten recording microelectrode, was used for local chemical application. This pipette was pulled to a fine tip and broken back to an overall tip diameter of 15-20 #m. The electrode (Frederick Haer 25-10-3) was glued in parallel to the micropipette so that the tip of the electrode extended ~ 25 um beyond the pipette tip. The open ends of the four-barrel micropipette were connected via 30-era polyethylene tubes to four 25-tsl Hamilton syringes (model PB 702-1) held in 0.5-#1 repeating dispensers (Hamilton model PB 600-1). This permitted each of the four syringes to deliver microliter quantities of a chemical into the tissue adjacent to the tip of the recording electrode. Saline (10/A of 0.9% w/v NaCI solution) was administered into active loci via local microinjection in six dogs using one oftlie pipettes. Regional arterial administration. In four additional animals, peptides were administered into the regional artery that supplies the right atrial ventral ganglionated plexus in 10-100-t~l quantities. The arterial branch that supplies these neurons courses cranially over the ventral surface of the right atrium. A PE-50 catheter was inserted into the branch of the right coronary artery arising immediately proximal to the root oftbe artery that supplied the plexus. The catheter was secured in place by ligatures, thereby ensuring that infused chemicals were delivered to the artery perfusing the right atrial ganglionated plexus and other local tissues. Pep#des. Nine peptides obtained from Sigma Chemical Co. (St. Louis, MO) were administered in pharmacological doses. The peptides investigated were: 1. human synthetic angiotensin II acetate salt, concentration 0.37 raM, in 10 animals; 2. atriopeptin I, concentration 0.56 raM, in 10 animals; 3. bradykinin acetate salt, concentration 0.75 raM, in 15 animals;

PEPTIDERGIC MODULATION OF INTRINSIC CARDIAC NEURONS 4. human synthetic CGRP, concentration 0.21 mM, in eight animals; 5. DADLE, concentration 0.90 mM, in eight animals; 6. neuropeptide Y (NPY), concentration 0.23 mM, in eight animals; 7. oxytocin acetate salt, concentration 0.01 ICU/ul, in eight animals; 8. substance P, concentration 0.32 mM, in nine animals; and 9. VIP, concentration 0.45 mM, in nine animals. These agents were administered in random order. A maximum of four neurochemicals were studied in each animal when chemicals were applied locally. All peptides were dissolved in normal saline and were delivered in 5-ul doses. When smaller doses were administered, activity changes were induced, but with less consistency. When larger doses were administered, activity was altered in a similar fashion as occurred when 5-ul volumes were injected. As larger doses increased the likelihood that a peptide would leak out of the fat into the systemic circulation, 5 ul doses were studied.

Interventions Neuronal activity and cardiac variables were recorded continuously on paper during control states and after chemical administration. Recordings were obtained immediately before and following peptide administrations, as well as later if activity changes persisted. Five to 10 min were allowed to elapse between each peptide administration. Following completion of the series of peptide and saline injections into an active site, acute decentralization was performed by sectioning the right and left thoracic vagosympathetic trunks and associated cardiopulmonary nerves immediately caudal to the middle cervical ganglia while the electrode-pipette assembly remained in place. This procedure disconnected intrinsic cardiac neural elements from other intrathoracic and central neurons. Peptide administrations were then repeated. If cardiovascular responses were elicited by repeat doses of peptides, these agents were then reinjected following administration of atropine sulphate (l mg/kg, IV) and again after administration of timolol maleate (1 mg/kg, IV). In order to determine whether neuronal or cardiodynamic responses induced by a neuropeptide were due to the effects exerted by the agent on intrinsic cardiac neurons or on axons of passage, peptides were administered adjacent to small branches of the ventral lateral cardiac nerve in the left atrial ventral fat. Peptides were also administered into loci in the right atrial ganglionated plexus where spontaneous activity was not recorded. The effects of such administrations on cardiac variables were also recorded. Finally, peptides were administered into the superior vena cava in the same doses as those given locally in order to determine whether they could exert detectable cardiovascular effects if they entered the systemic circulation.

Data Analysis When different peptides were administered into a site, some failed to elicit any response in a given animal. When no response was elicited by a peptide from a particular site, data so derived were not analyzed. Heart rate, peak systolic intramyocardial pressure, and peak systolic left ventricular chamber pressure were measured for five consecutive beats and their mean +_ SEM calculated. Individual action potentials were identified, as described above, and counted for 10-s periods. This was done immediately prior to and during maximal responses elicited following chemical application. Responses elicited by each neuropeptide were

193

evaluated in two ways. The first was to compare data obtained immediately before each administration with data obtained at the point of maximum change after administration o f a peptide, using the two-tailed Student's t-test for paired data. The second was to group data according to whether neural activity was increased or decreased following administration of each peptide. Activity changes were ascribed when neuronal activity changed by more than 20% from baseline values. RESULTS

Identification of Active Sites and Injection of Vehicle When the majority of sites in the collection of fat on the ventral surface of the right atrium was explored with a microelectrode, no spontaneous activity was identified. Two to four loci from which spontaneous activity could be recorded were identified in the right atrial ganglionated plexus of each animal. Peptides were administered into those sites that displayed the greatest number of spontaneously active units in the right atrial ganglionated plexus of each animal. Some spontaneously active units exhibited activity that was phase-related to the cardiac cycle during control conditions. Three to eight (average 5.2 _+ 1.9) spontaneously active units, as determined by amplitudes and shapes of individual action potentials, were identified at each site following application of a neuropeptide. When saline was injected into active loci in six dogs, neuronal activity or monitored cardiac variables were unaffected. When the microelectrode tip was placed in fat adjacent to intrinsic cardiac nerves in right atrial ganglionated plexi or directly into the ventrolateral cardiac nerve on the left atrium, either no action potentials were detected or small-amplitude action potentials, which had signal-to-noise ratios less than 2" l, were recorded. When peptides were administered adjacent to such nerves, no changes were detected in recorded activity or cardiovascular variables.

Intra-Arterial Administration of Peptides When saline was administered into the coronary artery that supplied blood to the right atrial ganglionated plexus in four animals, neuronal activity was unaffected. When 10-100 ~1 of substance P was injected into that artery, activity changed. In some animals the doses required to initiate neuronal responses were greater than that required for local administration. Systemic vascular pressure also became reduced within ~ 15 s of administration of larger doses in two of four animals. This systemic vascular hypotension presumably could have been due to substance P rapidly reaching the systemic circulation in sufficient quantities to directly alter total peripheral vascular resistance. When angiotensin was administered, systemic vascular pressure was increased in two of four animals, presumably because sufficient quantities of angiotensin entered the systemic circulation to alter total peripheral vascular resistance directly. As we could not be certain whether a peptide exerted local or distant effects when administered into the regional blood supply of a ganglionated plexus, this study was not continued.

Effects of Local Administration of Peptides in Intact Preparations When a peptide was injected into loci in epicardial fat from which no spontaneous activity was recorded, no cardiovascular effects were elicited. These data indicate that the doses ofpeptides employed were low enough that peptides did not leach away from the injection site and enter the circulation in sufficient quantities to directly modify vascular or other neural tissues. In

194

ARMOUR, HUANG AND SMITH

addition, w h e n peptides were administered adjacent to a m a j o r left atrial c a r d i o p u l m o n a r y nerve or into right atrial fat loci t h a t subsequently proved to contain nerves b u t not ganglia, no changes in neural or cardiovascular variables were detected. Thus, peptides did not appear to affect axons of passage when administered in the c o n c e n t r a t i o n s used in this study. Each peptide studied altered the s p o n t a n e o u s activity generated by adjacent intrinsic cardiac neurons, some inducing changes in n e u r o n a l activity with greater frequency t h a n others (Table 1). If activity increased following peptide administration, activity generated by more t h a n o n e unit usually was e n h a n c e d in any given locus. F u r t h e r m o r e , r e c r u i t m e n t of previously inactive units frequently occurred (Fig. 1). In contrast, at other loci activity was reduced following a d m i n i s t r a t i o n of a peptide. T h e e n h a n c e m e n t or suppression o f ongoing activity was dep e n d e n t o n the locus investigated a n d the peptide studied (Table 2). Heart rate, left ventricular intramyocardial systolic pressure, a n d left ventricular c h a m b e r systolic pressure were also modified following local a d m i n i s t r a t i o n of peptides, but less often t h a n n e u r o n a l activity (Table 2). Angiotensin II modified n e u r o n a l activity in eight o f 10 investigated sites (Table 1), e n h a n c i n g it in seven sites a n d dim i n i s h i n g it in one (Table 2). W h e n activity was e n h a n c e d , left ventricular intramyocardial systolic pressure increased in three a n i m a l s (Fig. 1). H e a r t rate a n d left ventricular c h a m b e r systolic

pressure were reduced in the a n i m a l in which activity was reduced. Atriopeptin modified recorded activity in seven of l0 animals (Table 1). Activity was increased in six a n i m a l s a n d decreased in one animal. N o n e of the m o n i t o r e d cardiovascular variables were altered following local application o f a t r i o p e p t i n . Bradykinin elicited responses with the greatest frequency of all peptides tested, neuronal activity being affected in every, animal tested. F u r t h e r m o r e , n e u r o n a l activity was only e n h a n c e d by this peptide (Table l ). Heart rate a n d intramyocardial systolic pressure were increased in three animals c o n c o m i t a n t with increases in activity, as was intramyocardial systolic pressure in a n o t h e r a n i m a l (Table 2). In one case, left ventricular intramyocardial a n d c h a m b e r pressures subsequently r e m a i n e d elevated even though neuronal activity had decreased below baseline values (Fig. 2). Local a d m i n i s t r a t i o n of C G R P resulted in e n h a n c e m e n t , not depression, of activity in seven of eight a n i m a l s (Table I). Even though this peptide failed to elicit cardiovascular responses when administered into the systemic circulation, it produced positive c h r o n o t r o p i c a n d inotropic responses when applied locally in four of seven instances in which neuronal responses were elicited (Table 2). W h e n D A D L E was investigated, activity was e n h a n c e d in six o f eight dogs. No site was identified in which activity was

TABLE 1 HEART RATE (HRL LEFT VENTRICULAR INTRAMYOCARDIAL SYSTOLIC PRESSURE (IMP), LEFT VENTRICULAR CHAMBER SYSTOLIC PRESSURE (LVP), AND NEURONAL ACTIVITY (IMPULSES PER SECOND) ELICITED BEFORE AND FOLLOWING ADMINISTRATION OF NINE DIFFERENT PEPTIDES Neurochemical Control Angiotensin p value Control Atriopeptin p value Control Bradykinin p value Control CGRP p value Control DADLE p value Control NPY p value Control Oxytocin Control Substance P p value Control VIP

n (dogs)

HR (beats/min)

IMP (mmHg)

IVP (mmHg)

Activity tiPS)

8/10

139 -+ 8 138 _+ 7

154 _+ 7 189 _+ 18

136 _+ 15 171 _+ 24

7/10

137 _ 8 137 _+ 8

134 -+ 5 134-+ 5

125 -+ 9 135 +_ 9

15/15

114 + 8 121 _+ 9

127 + 7 135 _+ 10

130 + ~ 129 _+ 5

7/8

147 + 9 152+ 4

131 + 6 133 + 7

134_+ 7 147_~ 12

6/8

146 _+ 8 162 -+ 12

155 + 10 151 +_ 10

131 _+ 8 126 _+ 11

7/8

141+ 7 144 -+ 4

121-+ 9 122 -+ 10

134-+ 9 138 -+ 12

8.4 + 2.3 15.6 + 3.0 0.02 5.8 _+ 0.8 16.6 _+ 3.9 0.03 4.5 -+ 0.6 14.3 _+ 2.6 0.00 I 8.3 z 1.2 30.0±6.1 0.007 9.3 + 3.1 29.6 _+ 8.2 0.01 11.1-+2.2 17.l _+ 5.1

6/8

117 -" 10 114 -+ 10 119 ___ 8 129 +_ 11

120-+ 15 120 _+ 15 133 ___ 14 164 ___20 0.03 137 + 17 124-+ 9

117 -+ 118 -+ 126 -+ 127 -+

10.1 8.8 9.8 9.3

8/9

8/9

128 -+ 10 143 -+ 15

8 8 5 7

129 +_ 8 128 -+ 8

_ 3.0 -+ 2.9 -+ 2.3 -+ 3.2

10.6 -+_2.6 13.3 +_ 6.5

The number of animals that elicited neuronal responses following administration of each peptide are tabulated. Averagecardiovascularand neuronal values obtained when responsive sites were studied with each peptide are provided.

PEPTIDERGIC MODULATION

OF INTRINSIC CARDIAC NEURONS

195

A EKG

Lvp,O

(mmHg}

0

500 msec

% ~,~-.~-4--~~ .

.

~

~

.,_

0

500 m s e c

L

I/

FIG. 1. Administration of angiotensin (between panels) into a locus in the right atrial ganglionated plexus before (A) and after (B) acute decentralization. Previously inactive units were activated by angiotensin, peak activity being greater in the intact state. A concomitant increase in left ventricular chamber systolic pressure (LVP) was elicited. The vertical bar beside neuronal activity (bottom trace) represents l0 #V, which is similar for all other figures. The same abbreviations are also used in other figures.

196

ARMOUR, HUANG AND SMITH

TABLE 2 CHANGES INDUCED IN NEURONAL ACTIVITY GROUPED ACCORDING TO WHETHER ACTIVITY INCREASED OR DECREASED FOLLOWING ADMINISTRATION OF EACH PEPTIDE.

Chemical

Effect

n

Angiotensin

Increase Decrease Increase Decrease Increase Decrease Increase Decrease Increase Decrease Increase Decrease Increase Decrease Increase Decrease Increase Decrease

0 1 0 0 3 0 2 0 4 0 1 0 1 1 6 0 2 1

Atriopeptin Bradykinin CGRP DADLE NPY Ox~aocin Substance P VIP

HR (beats/rain)

-153-144 --120 +_ 24-156 _ 36 144 ± 2-163 -150 _ 8-170 -96-124 -112-120 96-72 120 _ 7-150 -120 ± 12-213 96-85

+ 3 _+ 10"

+ 10t _ 31

n

IMP (mmHgl

3 0 0 0 4 1 1 0 2 2 1 0 0 0 6 0 1 0

140 _+ 4-233 ---156 ± 13-201 106-98 122-148 -147 +_ 13-170 165 + 22-132 150-165 ---145 ± 13-216 -140-170 --

± 33

_+ 15"

+ 15 ± 7

+ 23t

n

LVP (mmHg)

2 l 0 0 0 0 3 0 0 2 1 0 0 0 3 0 1 1

148 _+ 4-273 140-115 ----135 _ 6-166 --139 + 23-100 140-205 ---133 ± 16-148 -150-165 156-127

± 17

± 14

± 5

_+ 14

n

Activity (IPS)

7 1 6 1 15 0 7 0 6 0 4 3 2 4 4 4 5 3

6.4 _+ 1.4-15.9 ± 3.5t 22.0-15.6 4.9 +_ 0.4-21.1 ± 4.4110.0-7.8 4.5 ± 0.6-14.3 ± 2.6t -8.3 ± 1.2-30.0 ± 6.1# -9.3 +_ 3.1-29.6 ± 8.2t -7.7 + 1.4-23.9 ± 8.2 10.7 _+ 3-5.3 + 2.1 5.1 ± 2.5-17.0 ± 2.0 9.6 ± 2.8-3.6 + 1.7 6.7 + 2.8-14.8 ± 6.2 8.9 +_ 3.2-4.2 ± 1.8 9.2 ± 1.8-19.6 ± 9.1 12.8 ± 4.0-2.9 ± 1.0

Changes in heart rate (HR), left ventricular intramyocardial systolic pressure (IMP), and left ventricular chamber systolic pressure (LVP) that accompanied neuronal responses are tabulated according to whether activity was enhanced or diminished. With regard to substance P, positive chronotropic and inotropic responses were elicited when sites were studied in which activity was decreased as well as increased. n is the number of dogs in which each type of response was elicited. * p <0.01. f p < 0.001.

reduced subsequent to local application o f D A D L E . Cardiovascular variables were either increased or decreased as a consequence o f D A D L E administration. It should be n o t e d that suppression o f left ventricular systolic pressure was elicited in two a n i m a l s in which n e u r o n a l activity was e n h a n c e d (Table 2). Systemic vascular a d m i n i s t r a t i o n o f D A D L E in the doses employed for local injections did n o t elicit cardiovascular changes. Local a d m i n i s t r a t i o n of N P Y resulted in e n h a n c e d activity in four a n i m a l s a n d d i m i n i s h e d activity in three others, with one a n i m a l showing n o response. W h e n activity was enhanced, previously silent units were recruited (Fig. 3). In one o f the animals in which activity was increased, c o n c o m i t a n t augmentation of heart rate a n d force occurred (Table 2). W h e n oxytocin was a d m i n i s t e r e d locally, activity increased in two animals a n d decreased in four others. W h e n oxytocin increased n e u r o n a l activity, previously inactive units were also activated. Cardiovascular variables were unaffected by this peptide with the exception o f heart rate, which was modified in two animals (Table 2). Substance P elicited n e u r o n a l responses in eight o f nine animals tested. These responses were a c c o m p a n i e d by cardiovascular responses in the highest p r o p o r t i o n o f a n i m a l s with respect to all peptides tested. H e a r t rate a n d left ventricular i n t r a m y o cardial systolic pressure were a u g m e n t e d in six a n i m a l s (Table 2). Local application o f substance P elicited either e n h a n c e m e n t or reduction o f activity, d e p e n d i n g o n the locus tested (Table 2). For instance, in one a n i m a l substance P induced a reduction in n e u r o n a l activity that was a c c o m p a n i e d by increases in b e a n

rate, left ventricular intramyocardial systolic pressure, a n d left ventricular c h a m b e r systolic pressure (Fig. 4A). W h e n VIP was administered into active loci, unit activity was modified in eight o f nine dogs. Activity was e n h a n c e d in five dogs a n d depressed in three others. Cardiovascular variables were altered in three o f these animals, heart rate being augmented in the two a n i m a l s in which n e u r o n a l activity was enhanced, systolic pressure increasing in one o f these cases. In one a n i m a l there was a reduction in activity t h a t was associated with a conc o m i t a n t reduction in heart rate a n d systemic pressure. W h e n peptidergic effects were elicited, activity o f individual units was modified by m o r e t h a n one peptide in 17 animals. T h e effects o f various c o m b i n a t i o n s o f the nine peptides o n similar populations of n e u r o n s were not studied in e n o u g h animals to perform statistical analysis o n the cross-reactivity o f individual n e u r o n s to various peptides. Despite that, some n e u r o n s were identified that produced activity which was modified by two to three peptides. F o r instance, the activity generated by three neurons identified in two different loci was modified by atriooeptin, angiotensin, a n d bradykinin. Peptides (5qd volumes) were injected into the superior vena cava in order to d e t e r m i n e the systemic circulatory effects o f each peptide. W h e n angiotensin II was administered into the superior vena cava, left ventricle systolic pressure rose slightly in two o f 10 a n i m a l s studied. W h e n N P Y was a d m i n i s t e r e d into the superior vena cava, left ventricular systolicpressure was reduced by less t h a n 10 m m H g in o n e o f eight animals. W h e n substance P was administered into the superior v e n a cava, sys-

PEPTIDERGIC MODULATION OF INTRINSIC CARDIAC NEURONS

A

B

197

C

EKG

soo msec

Fr, r,, r,

J.l,i,

1'11'I

FIG. 2. Following acute decentralization, administration ofbradykinin into an active locus (between panels A and B) resulted in increased neuronal activity, tachycardia, and slight reduction in left ventricular intramyocardial systolic pressure (LV.IMP). (C) Within 15 s of administration activity was less than before, even though systolic pressure increased above the control value.

temic vascular pressure was reduced minimally in three of nine animals. When the other peptides were administered into the superior vena cava, monitored neuronal or cardiac variables did not change.

Effects of Peptides in Acutely Decentralized Preparations and Following Pharmacological Blockade Following disconnection of intracardiac neurons from the rest of the nervous system, including from other intrathoracic neurons, repeat administration of peptides into active loci resulted in altered neuronal activity in 62% of the previously responsive sites overall. In the majority of instances when a specific peptide was injected into a site that had induced cardiovascular responses before decentralization, either a blunted response was elicited (Fig. 4) or none occurred. Following decentralization, cardiovascular responses were elicited from 21% of the sites that previously had elicited such responses. Following decentralization, angiotensin II elicited neuronal and cardiovascular responses when administered to 50% of previously responsive sites. Atriopeptin elicited neuronal responses in one of six previously active sites after decentralization, no cardiovascular responses being elicited. Bradykinin produced neuronal responses from only five of 15 previously active sites; one of the four sites that previously had elicited cardiovascular responses still initiated cardiovascular responses. Calcitonin generelated peptide elicited neuronal responses when administered into four of seven previously active sites, heart rate and left ventrieular pressure being augmented in one instance. [D-Ala2,D-

LeuS]Enkephalin elicited neuronal responses from three of six previously active sites, heart rate and left ventricular pressure being augmented in one of these animals. Local administration of NPY modified neuronal activity in three of seven previously responsive sites following acute decentralization, heart rate and left ventricular pressure still being augmented in the one animal in which a similar response had been elicited before. Oxytocin elicited neuronal responses in only one of six previously active sites. Augmentation of heart rate accompanied this neural response. Substance P elicited neuronal response in only one animal following acute decentralization, neuronal responses being similar before and after decentralization in that case (Fig. 4). In this case cardiodepressor responses were elicited, in contrast to the augmentation that had occurred before decentralization. Vasoactive intestinal peptide initiated neuronal effects in five of eight previously active sites following acute decentralization, but no cardiovascular effects were observed. Thereafter, following atropine and timolol administration, neuronal responses were still elicited by local application of each peptide except CGRP and DADLE. Following pharmacological blockade, cardiodynamic variables were not affected by peptide administrations (Fig. 5). DISCUSSION Using immunohistochemical techniques, CGRP (36), NPY (22,30), substance P (9,16,25,29,34), and VIP (21,41 ) have been associated with somata in the mammalian intrinsic cardiac nervous system. Primary afferent neurons arising from cardiac re-

198

ARMOUR, HUANG AND SMITH

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FIG. 3. Administration of NPY into an active locus (between panels) resulted m increased spontaneous activity, without heart rate or pressure alterations. One unit, the one that gmerated action potentials with the largest signal-to-noise ratio, was recruited.

ceptors have been proposed to contain CGRP and substance P (29), and a variety of peptides have been associated with intracardiac axons (9,16,30,44). Recent physiological evidence has indicated that enkephalin, NPY, oxytocin, substance P, and VIP are capable of modifying intrathoracic extrinsic cardiac neurons involved in cardiac regulation (5,6). Furthermore, bradykinin, oxytocin, and substance P are capable of modifying intrinsic cardiac neurons such that concomitant cardiodynamic changes are induced (8). The results of the present experiments demonstrate that these and other peptides can affect the activity of in situ mammalian intrinsic cardiac neurons, some neurons being affected by more than one peptide. Substance P, or a closely related peptide, has been proposed to act as a neurotransmitter (24) or neuromodulator (23) mediating synaptic input from primary afferent neurons to central nervous system neurons. Substance P, when administered adjacent to spontaneously active intrinsic cardiac neurons, presumably modifies somata and dendrites rather than axons of passage, since activity propagated by axons of passage was unaffected by substance P. Activity propagated by axons of passage was unaffected by the other peptides as well. Substance P does not elicit cardiovascular responses when administered to intrathoracic autonomic axons of passage (5). In accord with this, cardiodynamics were unaffected when substance P was injected into an intrinsic cardiac nerve or into epicardial fat loci containing nerves but no ganglia. Thus, when this or other peptides

were administered into an active locus of a canine epicardial ganglionated plexus, the neuronal and cardiovascular changes thereby induced presumably were due to modification of somata and/or dendrites rather than axons of passage. Substance P reduces systemic vascular resistance when administered systemically (15). When substance P was administered into the regional arterial supply of the fight atrial ganglionated plexus in the present experiments, systemic vascular hypotension was induced in 50% of the eases. This hypotension presumably was due to alterations of systemic vascular resistance after this peptide reached the systemic vascular walls. Thus, substance P administration into the local arterial tree was considered to be an unacceptable method for studying the effects of this peptide on the intrinsic cardiac nervous system. This also applies to other peptides used in this study that can modify the peripheral vaseulature. Since hypotension never was induced when substance P was administered into epicardial fat loci, the cardiac augmentation produced by local application of substance P presumably was due to peptidergic effects exerted directly on intrinsic cardiac neurons rather than systemic vascular effects. This contention is supported by the finding that substance P usually failed to induce cardiovascular responses in decentralized preparations. Substance P also failed to induce cardiovascular responses when injected into sites in intact Im~ptwations from which no spontaneous activity was recorded. Presumably this was due to the fact that either no neurons were present in the vicinity of

PEPTIDERGIC MODULATION OF INTRINSIC CARDIAC NEURONS

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FIG. 4. Administration of substance P into an active locus before and after decentralization. (A) Substance P administration (administration occurred between left and middle panels) resulted in diminution of activity and augmentation in heart rate as well as left ventricular intramyocardial (IMP) and chamber (LVP) systolic pressures. Pressure returned to the control level within 45 s (fight panel). (B) Following acute decentralization, repeat administration of substance P into the same locus resulted in reduction in activity without accompanying augmentor responses. In contrast, left ventricular wall and chamber systolic pressures were minimally reduced.

the injection site or that, if neurons were present, they failed to be activated. The fact that some intrinsic cardiac neurons are not modified by substance P is indicated by the finding that this peptide failed to alter activity when applied to one of nine active loci.

Activity generated by intrinsic cardiac neurons was either increased or suppressed by local administration of substance P, depending on the locus investigated (Table 2). In keeping with previous findings (8), associated augmentation of cardiac chronotropism and inotropism occurred in six of the animals. Con-

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FIG. 5. Effects of pharmacological blockade on substance P-induced responses. (A) Following atropine administration, repeat doses of substance P into the same locus as illustrated in Fig. 4 resulted in a reduction in neuronal activity that was accompanied by decreased left ventricular chamber systolic pressure. (B) Following timolol administration, substance P initiated similar neuronal responses without changes in ventricular pressure.

200 siderable augmentation of left ventricular inotropism was elicited in these six animals when substance P was studied, implying that the number of efferent sympathetic cardiac neurons involved might have been considerable despite the fact that substance P presumably reached a limited population of intrinsic cardiac neurons. That no cardiovascular responses were elicited in two of the eight animals in which neuronal responses were elicited may have been due to the fact that the number of efferent cardiac neurons directly or indirectly affected by substance P was not sufficient to alter cardiodynamics. Neurons that were activated may also have controlled cardiac indices that were not monitored (i.e., atrial force). Furthermore, these neurons might have influenced noncardiovascular tissues, thereby leading to changes that would not have been detected. Acute decentralization blunted or eliminated responses to repeat doses of substance P (Fig. 4), as has been shown previously (8). If the cardiac effects of substance P were mediated solely by intrinsic cardiac neurons in the intact state, then similar cardiac responses should have been elicited by substance P before and after acute decentralization, particularly since similar neuronal responses usually were elicited in both states. On the other hand, when cardiovascular responses were elicited by substance P following acute decentralization, presumably these were due to modulation of the intrinsic cardiac neurons alone, as the connections with other intrathoracic and central neurons could not have been involved (3). These data suggest that substance P can affect different populations of intrinsic cardiac neurons, one exhibiting the capacity to involve central reflexes. Bradykinin increased neuronal activity in every site tested; no inhibition of activity was observed (Fig. 2; Tables 1 and 2). These data suggest that the population of intrinsic cardiac neurons with bradykinin receptors may be greater than those with receptors sensitive to the other peptides studied. Despite that, cardiodynamic responses were elicited with less frequency than occurred with substance P (Table 2). Cardiac variables were increased or decreased, depending on the site tested. Since cardiovascular augmentation sometimes occurred after neuronal activity had returned to or was below control values (Fig. 2), it appears that concomitant cardiac augmentation might have been due to activation of neurons distant from a recording site. Bradykinin is a peptide that exhibits potent systemic vasodilator properties, being rapidly degraded upon entering the blood stream (11). Systemic administration ofbradykinin in the doses employed for local injection did not affect systemic vascular pressure significantly. Furthermore, cardiovascular effects were not observed in the majority of instances following local injection of bradykinin after acute decentralization. Thus, it appears that bradykinin did not leak out of the fat and enter the circulation in sufficient quantities to modify peripheral vessels directly. When heart rate and left ventricular intramyocardial systolic pressure were augmented following administration ofbradykinin, activation of efferent sympathetic neurons presumably was involved, since following timolol administration these responses were not elicited. Bradykinin increased neuronal activity in a third of previously active sites after decentralization, hemodynamic effects being totally eliminated. Thus, as with substance P. central connectivity was found to be important in producing cardiovascular and neuronal responses. A number of other peptides also induced neuronal responses similar to those elicited by substance P. When angiotensin II, atriopeptin, NPY, oxytocin, and VIP were locally administered, neuronal activity was altered in the majority of sites tested; for each peptide tested, activity either increased or decreased, depending on the locus investigated (Table 2). Angiotensin modified neuronal activity in eight of 10 animals, associated positive

ARMOUR, HUANG AND SMITH inotropic effects being eliciting in three animals. This peptide exerts direct augmentor effects on cardiac myocytes (14,28). Since, when angiotensin was injected into inactive loci or into four of seven active loci, no cardiodynamies changes were produced, it is unlikely that this agent leaked from the injection site in sufficient quantities to directly affect cardiac myocy'tes. Efferent sympathetic cardiac neurons can be activated by angiotensin II (28). Such neurons probably were directly or indirectly activated by angiotensin II in the present experiments, since positive inotropic responses were not elicited following fladrenergic blockade. Cardiovascular responses elicited by angiotensin II in intact states were blunted or eliminated following acute decentralization (Fig. l). These data indicate that cardiac responses elicited by angiotensin-sensitive, intrinsic cardiac neurons involve central as well as local neurons. Atriopeptin, when administered systemically, exerted no hemodynamic responses, as has been reported previously (3 l). Atriopeptin can either excite or depress central neurons (42), acting as a neuromodulator in rat sympathetic ganglion (l 3). These results are in accord with the present findings in that intracardiac neuronal activity was either suppressed or enhanced following local application of atriopeptin (Table 2). Since atriopeptin modified the activity of intrinsic cardiac neurons without producing associated cardiac changes, this peptide may affect neurons that influence other tissues (i.e., coronary arteries) or neurons that do not exert short-term cardiodynamic effects. Calcitonin gene-related peptide, another peptide known to be involved in cardiovascular regulation (40), induced neuronal and cardiovascular responses when applied to active sites in the canine intrinsic cardiac nervous system. Like bradykinin and DADLE, local application of CGRP resulted only in increased activity. However, since a limited number of sites were investigated, these data do not assure that CGRP cannot exert direct or indirect inhibitory effects on intrinsic cardiac neurons. Neuropeptide Y has been associated with intrinsic cardiac axons (38) and has been shown to exert direct depressor effects on cardiac myocytes (1). Furthermore, NPY can act both preand postsynaptically in the central nervous system (39). It has been proposed that NPY and its related peptides can be released from efferent sympathetic nerve terminals and thereby inhibit cardiodepressor effects induced by efferent postganglionic parasympathetic neurons (26,32). In addition, NPY can modulate intrathoracic efferent sympathetic neurons involved in cardiac regulation (4,17). In the present study NPY modified intrinsic cardiac neuronal activity, activity being either increased or decreased depending on the locus examined (Table 2). Concomitant cardiovascular responses were elicited in only one animal, indicating that NPY may primarily exert modulator, rather that direct excitatory, effects on efferent cardiac neurons. Because considerable positive chronotropic and inotropic responses were induced in that case, it appears that efferent sympathetic neurons were involved. Opioids are also involved in cardiovascular regulation (20,35,37 ), including at the level of sympathetic ganglion synaptic transmission (5,10,2%33). In the present experiments, local administration of DADLE resulted in increased activity, not depression of activity. In association with these increases, heart rate increased in four of six instances. Pressures were affected in four animals. These data indicate that opioidergic synapses in the mammalian intrinsic cardiac nervous system may be involved in cardiodynamic regulation. Oxytocin and VIP modify intrathoracic extrinsic (5) and intrinsic (8) cardiac neurons to produce cardiodynamic changes. Furthermore, oxytocin has been shown to inhibit preganglionic sympathetic neurons (19). The present experiments demonstrate

PEPTIDERGIC MODULATION OF INTRINSIC CARDIAC NEURONS

that oxytocin and VIP can modify activity generated by intrinsic cardiac neurons, effects that can be associated with concomitant cardiovascular responses. Thus, oxytocin and VIP apparently can modify intrinsic cardiac neurons in a manner similar to bradykinin and substance P. As occurred when studying bradykinin and substance P, the other peptides elicited neuronal and cardiovascular responses less frequently following acute decentralization. However, since responses were still elicited following decentralization, it appears that a number of peptides are capable of modulating intrinsic cardiac neurons in the absence of central neuronal input. Cardiovascular responses were no longer elicited following/~-adrenergic and muscarinic blockade, even though neuronal responses were elicited. These data imply that when cardiac responses were elicited, these depended upon the direct or indirect activation of efferent autonomic neurons. Intrinsic cardiac neurons that contain muscarinic receptors (2) evidently were not involved in the majority of instances, since, with the exception of C G R P and DADLE, the capacity of intrinsic cardiac neurons to be modified by peptides was not detectably altered following atropine administration.

201

It is concluded that angiotensin, atriopeptin, bradykinin, CGRP, DADLE, NPY, oxytocin, substance P, and VIP can modify activity generated by in situ intrinsic cardiac neurons. Bradykinin, CGRP, and D A D L E enhanced neural activity whereas other peptides induced both increases and decreases in activity, depending on the neurons studied. Furthermore, some intrinsic cardiac neurons can be modified by more than one neuropeptide. Associated cardiovascular responses were elicited when neuronal activity was modified by the majority of these peptides. Cardiovascular responses depended upon intrinsic cardiac neurons and their connectivity with centrally located neurons. These data indicate that the function of intrinsic cardiac neurons can be modified by a number of neuropeptides. ACKNOWLEDGEMENTS This work was supported by a Medical Research Council of Canada grant (MT-10122) and the Nova Scotia Heart and Stroke Foundation. The authors gratefully acknowledge the technical assistance of Richard Livingston. M. H. Huang is supported, in part, by Totts Gap Medical Research Laboratories, Inc., Bangor, PA.

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