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Am J Physiol Regul Integr Comp Physiol 275: R1683-R1689, 1998;
0363-6119/98 $5.00
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Vol. 275, Issue 5, R1683-R1689, November 1998

Canine intrinsic cardiac neurons involved in cardiac regulation possess NK1, NK2, and NK3 receptors

G. W. Thompson1, D. B. Hoover2, J. L. Ardell3, and J. A. Armour1

1 Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7; 2 Department of Pharmacology, College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614-0577; and 3 Department of Physiology, University of South Alabama, Mobile, Alabama 36688-0002

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

To determine whether intrinsic cardiac neurons involved in cardiac regulation possess neurokinin (NK) receptor subtypes, we administered selective NK receptor agonists individually (100 µM; 0.1 ml) into the coronary arterial blood supply of right atrial intrinsic cardiac neurons of 18 anesthetized dogs. The selective NK1 receptor agonist [Sar9,Met(O2)11]-substance P depressed the spontaneous activity of right atrial neurons (26.7 ± 6.7 to 13.0 ± 4.0 impulses/min; P < 0.05) in 11 dogs and augmented such activity in the other 5 dogs (8.0 ± 3.1 to 27.8 ± 8.7 impulses/min; P < 0.05). Local administration of the selective NK2 receptor agonist [beta -Ala8]-NKA-(4---10) depressed right atrial neuronal activity (27.3 ± 6.4 to 14.7 ± 3.8 impulses/min; P < 0.05), whereas the selective NK3 receptor agonist senktide augmented such activity (18.9 ± 6.4 to 53.1 ± 12.0 impulses/min; P < 0.05). Left ventricular chamber pressure fell when selective NK1 and NK2 receptor agonists were administered. Increases in heart rate and right ventricular intramyocardial systolic pressure occurred when the selective NK3 receptor agonist was studied. Administration of a selective NK1 or NK2 receptor antagonist altered neuronal activity, with no subsequent change in activity occurring after administration of its respective receptor agonist. Receptor autoradiography demonstrated tachykinin receptors associated with ventral right atrial intrinsic cardiac neurons. It is concluded that intrinsic cardiac neurons involved in cardiac regulation possess NK1, NK2, and NK3 receptors and that some intrinsic cardiac neurons receive tonic input via endogenously released NKs.

neurokinin

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

CARDIOVASCULAR AFFERENT neurons possess the neurokinins (NKs) substance P (SP) and NKA (16, 18, 25). Anatomic evidence indicates that the somata of some mammalian intrinsic cardiac neurons possess SP receptors (1, 14). Peptides such as SP modify the activity generated by some intrinsic cardiac neurons (4) such that concomitant changes in cardiodynamics occur in situ (6) and in vitro (22). It remains to be determined which NK receptor subtypes are associated with canine intrinsic cardiac neurons.

The present experiments evaluated whether NK1, NK2, and NK3 receptors are associated with canine intrinsic cardiac neurons. Neurons in the right atrial ganglionated plexus were investigated because this population of neurons is involved in cardiac regulation (28). To characterize tachykinin-sensitive intrinsic cardiac neurons, we administered the selective NK1 receptor agonist [Sar9,Met(O2)11]-SP (26), the selective NK2 receptor agonist [beta -Ala8]-NKA-(4---10) (19), and the selective NK3 receptor agonist senktide (19) individually to intrinsic right atrial neurons via their local arterial blood supply. The selective NK1 receptor antagonist Win-51708 (2) and the selective NK2 receptor antagonist L-659877 (19) were then administered sequentially to determine whether selective tachykinin receptor blockade influences intrinsic cardiac neuronal activity in situ. Agonists were administered in the presence of these NK receptor antagonists to determine whether the NK1- or NK2-selective NK receptor antagonists modify the effects induced by receptor-selective agonists. Finally, receptor autoradiography was used to determine the distribution of neurons possessing NK receptors throughout the right atrial ganglionated plexus. In this manner, we sought to determine whether mammalian intrinsic cardiac neurons involved in cardiac regulation possess NK1, NK2, and NK3 receptors and, if so, the capacity of neurons associated with such receptors to influence cardiodynamics.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Adult mongrel dogs (n = 24) of either sex weighing between 16 and 24 kg were used in this study. Eighteen dogs were used for the in situ experiments, and six dogs were used for the anatomic studies. All experiments were performed in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals." These experiments were approved by the institutional animal care and use committees of Dalhousie University and the University of South Alabama.

General methods. Animals were anesthetized with a loading dose of Pentothal sodium (15-20 mg/kg iv), followed with maintenance doses (5 mg/kg iv to effect every 5-10 min) for the duration of the surgical procedures. Noxious stimuli were applied periodically to a paw throughout the experiments to ascertain the adequacy of the anesthesia. After anesthesia induction, animals were intubated and positive-pressure ventilation was maintained with a Bird Mark 7A ventilator using a gas mixture of 95% O2 and 5% CO2. After the initial surgical procedures were completed (see below), Pentothal sodium (a short-acting anesthetic agent) was replaced with alpha -chloralose (a long-lasting anesthetic) administered as a bolus (50 mg/kg iv), with repeat doses (25 mg/kg iv) administered every hour or less throughout the experiments as required. When neuronal activity was recorded, spontaneous activity was suppressed for 5-10 min after bolus injections of alpha -chloralose due to the neuronal depressor effects of this agent. Therefore, at least 10 min were allowed to elapse after such injections before recordings proceeded.

A bilateral thoracotomy was made in the fifth intercostal space. The ventral pericardium was incised and retracted laterally to expose the ventral right atrial deposit of fat that contains the ventral component of the right atrial ganglionated plexus (27). One miniature solid-state pressure transducer (model P190; 5 mm in diameter and 1.5 mm thick; Konigsberg Instruments, Pasadena, CA) was inserted into the midwall region of the right ventricular conus, and another was inserted into the midwall region of the left ventricular ventral wall to record regional intramyocardial pressures. These sensing devices were used because intraventricular pressure and regional ventricular length represent less-sensitive indexes for detecting ventricular force changes induced by efferent autonomic neurons (5, 8). Because these ventricular regions are richly innervated (4), they were chosen for sensor placement. Pressures in the left atrial chamber, left ventricular chamber, and aorta were measured using Bentley Trantec model 800 transducers connected to a PE-50 catheter placed in the left atrial cavity, a Cordis no. 7 pigtail catheter inserted into the left ventricular chamber via a femoral artery, and a Cordis no. 6 catheter inserted into the descending aorta via the other femoral artery, respectively. All data, including a lead II electrocardiogram, were recorded on an Astro-Med model MT 9500 eight-channel rectilinear recorder. Data were stored for later analysis on VHS tapes (T120 Scotch; 3M Canada, London, Ontario) using a videocassette recorder (model 820; A. R. Vetter, Rebersburg, PA).

Neuronal recording. The activity generated by neurons that lie embedded in subepicardial fat on the ventral surface of the right atrium was recorded, as described by us elsewhere (9). To minimize epicardial motion during each cardiac beat, a circular ring of heavy-gauge wire was gently placed around the epicardial fat of the ventral surface of the right atrium. The recording microelectrode had a 10-µm diameter, an exposed tip of 50 µm, and an impedance of 9-11 MOmega at 1,000 Hz. The fat was explored with the tungsten microelectrode mounted on a micromanipulator at depths ranging from the surface of the fat to regions adjacent to cardiac musculature. Proximity to cardiac musculature was indicated by increases in the amplitude of the ECG artifact. The indifferent electrode was attached to the pericardium.

Signals from intrinsic cardiac neurons were differentially amplified by a Princeton Applied Research model 113 amplifier, which had band pass filters set at 300 Hz to 10 kHz and amplification ranges of ×100-500. The output of this device, further amplified (×50-200) and filtered (band width 100 Hz to 2 kHz) by means of an optically isolated amplifier (Applied Microelectronics Institute, Halifax, Nova Scotia, Canada), was led to a Nicolet model 207 oscilloscope and to a Grass AM8 audio monitor. Loci in epicardial fat were identified, from which action potentials with signal-to-noise ratios >3:1 were recorded, individual units being identified by the amplitude of their action potentials. Using these techniques and criteria, the microelectrode does not record action potentials generated by axons of passage but rather records action potentials generated by cell bodies and/or dendrites (4, 9). 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 amplitudes of recorded action potentials varied by <10 µV over several minutes, action potentials retaining the same configurations over time. Thus action potentials recorded in a given locus with the same amplitude (±10 µV) were considered to be generated by a single unit. Action potentials with signal-to-noise ratios >3:1 were analyzed. The frequency of activity generated by the somata and/or dendrites of neurons in each investigated locus was analyzed for 30-s periods before and after administration of each chemical. A 25% alteration of baseline spontaneous activity (i.e., change in firing frequency) after peptide administration was required to classify a unit as generating increased or decreased activity. Action potential data were grouped according to whether activity increased, decreased, or remained unchanged.

Chemical administration. Chemicals were administered as a bolus in 0.1-ml volumes into the regional arterial supply of the right atrial neurons studied. As previously described (4), a side branch of the right coronary artery that arises immediately proximal to the root of the sinus nodal artery supplying blood to neurons in the right atrial ganglionated plexus was cannulated with a PE-50 catheter; this catheter was secured in place by ligatures. Chemicals were delivered into the regional arterial blood perfusing right atrial neurons and other distal tissues via this catheter. To control for systemic effects of injected chemicals, we administered each agonist into the bloodstream of the descending aorta in the same doses as used for local coronary artery administration. Each agonist was administered into the local coronary artery and systemic blood at least two times due to the tachyphylaxis that they may exhibit.

The agonists, obtained from Research Biochemicals International (Natick, MA), were administered in pharmacological doses and in random order. The chemicals investigated were 1) the selective NK1 receptor agonist [Sar9,Met(O2)11]-SP (0.1 ml of a 100 µM solution), 2) the selective NK2 receptor agonists [beta -Ala8]-NKA-(4---10) (0.1 ml of a 100 µM solution) and GR-64349 (0.1 ml of a 100 µM solution), and 3) the selective NK3 receptor agonist senktide (0.1 ml of a 100 µM solution).

Because smaller doses of each chemical induced neuronal responses with less consistency and because larger doses increased the likelihood that each chemical would enter the systemic circulation in sufficient doses to affect distant tissues, 0.1 ml of 100 µM doses of these selective NK receptor agonists were used. Each receptor agonist was then studied after individual administration of either the NK1 receptor antagonist Win-51708 (0.1 ml of a 100 µM solution) or the NK2 antagonist L-659877 (0.1 ml of a 100 µM solution) into the regional arterial blood supply of the right atrial ganglionated plexus. The effects of these selective antagonists were investigated in random order. Systemic administration of each agonist was also tested after local arterial administration of an NK1 or NK2 receptor antagonist.

Data analysis. Heart rate, peak systolic left atrial pressure, peak systolic intramyocardial pressure (right and left), and peak systolic left ventricular chamber pressure were measured for 30-s periods before and after chemical administration. Their means ± SE were calculated. Individual action potentials were identified as described in Neuronal recording and counted for 30-s periods. This was done immediately before (baseline control) and during maximal responses elicited after each chemical application. Data obtained at the point of maximum change after administration of a chemical were compared with baseline control data using the two-tailed Student's t-test for paired data.

Histological studies. After anesthesia and surgical preparation as described in General methods, right atrial tissue including the right atrial ganglionated plexus and underlying myocardium was removed from six animals not previously exposed to NK receptor agonists or antagonists. These tissues were placed on specimen plates using OCT compound (Ted Pella, Redding, CA), frozen rapidly with powdered dry ice, placed immediately in 50-ml plastic tubes, and stored at -80°C. Subsequently, these tissues were removed and 20-µm serial sections were cut from them using a microtome cryostat held at -20°C. Groups of three adjacent sections obtained from serial tissue sections were thaw mounted onto separate glass slides that had been coated two times with chrome alum-gelatin. Two to four adjacent sections were placed on each glass slide. After the sections had dried, the slides containing adjacent sections from each set of tissue were stored at -20°C, awaiting subsequent staining with hematoxylin and eosin. These stained sections were used to determine which samples contained intrinsic cardiac ganglia to know which adjacent sections could be used for the autoradiographic studies. The remaining sections were stored at -80°C for subsequent autoradiographic analysis.

Receptor autoradiography. NK receptors associated with right atrial tissues were identified using the following compounds: 125I-labeled NKA, 125I-labeled eledoisin (20, 23), and 125I-labeled [MePhe7]-NKB (2,200 Ci/mmol; NEN, Boston, MA). Although none of these radioligands is specific for a single receptor subtype, 125I-labeled NKA has been shown to be more selective for NK1 and NK2 receptors, whereas 125I-labeled eledoisin is highly selective for the NK3 receptor (21, 23). 125I-labeled [MePhe7]-NKB has also been shown to be selective for NK3 receptors (23). However, very high amounts of nonspecific binding of 125I-labeled [MePhe7]-NKB to tissues were observed, and thus we did not study the binding characteristics of this radioligand further. Slide-mounted sections were preincubated in 50 mM Tris · HCl (pH 7.4) for 10 min at room temperature before being incubated in buffer and radioligand. To determine total binding of the radioligand, we incubated one slide in each set for 2 h at room temperature in 50 mM Tris · HCl buffer (pH 8.0) containing 3 mM MnCl2, 0.02% BSA, 40 mg/l bacitracin, 2 mg/l chymostatin, 4 mg/l leupeptin, and 0.1 nM 125I-labeled NKA or 125I-labeled eledoisin. Slides with adjacent sections were also incubated in the same buffer with the addition of 1 µM unlabeled NKA or [MePhe7]-NKB (Peninsula, Belmont, CA) so that nonspecific binding of 125I-labeled NKA and 125I-labeled eledoisin, respectively, to tissues could be analyzed. After incubation with the radioligand, slides were rinsed four times for 5 min each in 50 mM Tris · HCl (pH 7.4) at 4°C and then dipped briefly into cold deionized water. Excess water remaining on the slides was carefully removed with gauze. These sections were dried at room temperature using an electric fan. Subsequently, these slides were placed in X-ray cassettes with 125I microscales and Hyperfilm-3H (Amersham, Arlington Heights, IL). Films were processed by standard methods after exposure for 4 wk (125I-labeled NKA) or 8 wk (125I-labeled eledoisin) at 4°C. These slides were stained with hematoxylin and eosin to identify the ganglia in each tissue studied. A microcomputer-assisted imaging device (Imaging Research) was used to quantify the signals obtained from each film autoradiogram. Stained sections were evaluated to determine whether specific binding sites were localized to ganglia, blood vessels, atrial myocardium, or adipose tissue.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Identification of active sites. Spontaneous activity generated by intrinsic cardiac neurons was recorded from one locus in each right atrial ganglionated plexus studied. Three to five spontaneously active units, as determined by the amplitudes of individual action potentials, were identified at each locus. The activity generated by a single unit as determined by amplitude was used for later analysis. When saline was administered into the coronary artery, which supplied blood to the ventral right atrial ganglionated plexus, neuronal activity and cardiac indexes were unaffected. Systemic administration of the selective NK1 receptor agonist [Sar9,Met(O2)11]-SP or the selective NK2 receptor agonist [beta -Ala8]-NKA induced minor systemic vascular hypotension after the first, but not the second, administration. Thus neuronal activity, cardiac indexes, and aortic pressure were not changed overall when each selective NK agonist was administered individually into the systemic circulation in the doses studied.

Neuronal responses to agonists. One or more of the selective NK receptor agonists modified the spontaneous activity generated by neurons within the ventral right atrial ganglionated plexus of every animal studied (Table 1). In some animals, previously quiescent neurons were recruited as well (Fig. 1). Neuronal responses elicited during the second administration of each agonist were similar to those previously induced.

                              
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  		Table 1.   Neuronal activity responses


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  		Fig. 1.   Regional arterial administration of the selective neurokinin 3 receptor (NK3) agonist senktide (0.1 ml; 100 µM at arrow) activated previously quiescent right atrial neurons (bottom trace). Minor increases in left atrial chamber (LAP) and left ventricular intramyocardial (LV IMP) systolic pressures occurred concomitantly. ECG, lead II electrocardiogram.

[Sar9,Met(O2)11]-SP attenuated the activity generated by right atrial neurons by 51% in 11 of 16 dogs tested while enhancing the activity generated by such neurons in the other 5 dogs (Table 1). Minor reduction in left ventricular chamber systolic pressure occurred (139 ± 10 to 98 ± 10 mmHg; P < 0.05) concurrently with induced neuronal activity changes. Heart rate and left atrial systolic pressure as well as right and left ventricular intramyocardial systolic pressures remained unchanged.

When [beta -Ala8]-NKA was administered to the right atrial ganglionated plexus, the activity generated by neurons therein decreased by 46% in 10 of 15 animals, increasing in the other 5 dogs studied (Table 1). As with the NK1 receptor agonist, a minor reduction in left ventricular chamber systolic pressure occurred (137 ± 9 to 113 ± 10 mmHg; P < 0.05) concurrently with induced neuronal activity changes. Remaining recorded cardiac variables did not change significantly. The NK2 receptor agonist GR-64349 did not affect the spontaneous activity generated by intrinsic cardiac neurons overall when administered to six animals.

Senktide enhanced the activity generated by intrinsic cardiac neurons by 181% in 14 dogs (Fig. 1 and Table 1) while decreasing the activity generated by such neurons in the remaining 4 dogs. Local coronary artery administration of senktide increased right ventricular intramyocardial systolic pressure (29 ± 2 to 35 ± 3 mmHg; P < 0.01) without affecting other recorded cardiovascular variables, including heart rate.

Antagonist administration. When the selective NK1 receptor antagonist Win-51708 was administered into the regional arterial blood supply of right atrial neurons (Table 1; 5 animals), the activity generated by them increased (18.4 ± 6.4 to 34.6 ± 10.5 impulses/min; P < 0.05). Cardiac indexes remained unaffected. In contrast, neuronal activity decreased (24.3 ± 5.5 to 6 ± 2.1 impulses/min; P < 0.05) in seven animals when the selective NK2 receptor antagonist L-659877 was administered into their local coronary arterial blood supply (Table 1 and Fig. 2). The change in neuronal activity induced by L-659877 was accompanied by a minor reduction in left ventricular chamber systolic pressure (118 ± 5.5 to 101 ± 5.5 mmHg; P < 0.05). Other recorded cardiac variables remained unchanged.


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  		Fig. 2.   Regional arterial administration of the selective NK2 receptor antagonist L-659877 (0.1 ml of a 100 µM solution at arrow) suppressed spontaneous activity generated by right atrial neurons. Heart rate, LAP, and left ventricular chamber pressure (LVP) remained unchanged.

All recorded cardiac indexes, including heart rate, peak systolic left atrial pressure, peak systolic intramyocardial pressure (right and left), and peak systolic left ventricular chamber pressure, as well as neuronal activity, were not altered when [Sar9,Met(O2)11]-SP was administered to right atrial neurons after local arterial administration of the selective NK1 receptor antagonist. Likewise, the NK2 receptor agonist failed to induce responses in the presence of the NK2 receptor antagonist L-659877. In those animals in which minor reductions in left ventricular chamber and aortic systolic pressures had been induced after systemic administration of the NK1 receptor agonist, systemic vascular hypotension continued to be induced after regional arterial administration of the antagonist to that receptor subtype, indicating the regional nature of the receptor blockade.

Receptor autoradiography. Right atrial ganglionated plexuses and adjacent atrial tissues obtained from three dogs were analyzed by autoradiography for specific 125I-labeled NKA binding sites. 125I-labeled NKA was found to be associated with the relatively large local coronary arteries identified in right atrial fat and adjacent atrial tissue. No association of this radioligand with intrinsic cardiac ganglia, adipose tissue, or atrial myocytes was detected (Fig. 3, A-D, and Table 2). Specific binding of 125I-labeled NKA to canine tracheal tissue (not shown) was identified. Sections of tissue obtained from three different dogs were evaluated for 125I-labeled eledoisin binding. Specific sites for this radioligand were associated with right atrial ganglia and local blood vessels (Fig. 3, E-K, and Table 2). Although most ganglia contained specific binding sites for 125I-labeled eledoisin, some ganglia in each fat pad studied remained unlabeled. Labeling within individual intrinsic cardiac ganglia also was not uniform, being found in one or more regions of some intrinsic cardiac ganglia. The density of 125I-labeled eledoisin binding sites was less in canine intrinsic cardiac ganglia than in adjacent blood vessels. The specific binding of 125I-labeled NKA to coronary arteries was greater than that of 125I-labeled eledoisin (Table 2).


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  		Fig. 3.   Photographs demonstrating localization of 125I-labeled NKA and 125I-labeled eledoisin binding sites in right atrial tissues obtained from 1 dog. Autoradiograms (B, C, F, G, J, and K) were used as negatives to produce reverse-image photographs in which autoradiographic grains appear white on a black background. Sets composed of 3 horizontal plates (A-C, E-G, and I-K) show hematoxylin-and-eosin-stained sections and matched autoradiograms for total and nonspecific binding, respectively. Scale bars in C, G, and K are equal to 1 mm and apply to A-C, E-G, and I-K. Scale bar in H is equal to 100 µm and applies also to D. Arrows in A and B identify coronary arteries that were labeled by 125I-labeled NKA. Ganglion (g), right atrial myocardial tissue (a), and fat (f) present in these sections were not labeled by 125I-labeled NKA. D and H: enlarged views of ganglion g in A. Arrowheads in E and F identify ganglia that were labeled with 125I-labeled eledoisin. Arrows in I and J identify coronary arteries in these tissues. Specific binding sites for 125I-labeled eledoisin were not detected in adjacent fat or atrial myocardium (E-G and I-K). Nonspecific binding to right atrial tissues is shown in higher-magnification photomicrographs of G and K.

                              
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  		Table 2.   Quantification of 125I-labeled NKA and 125I-labeled eledoisin binding

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The functional data obtained from the investigation reported herein support the concept that canine intrinsic cardiac neurons possess NK receptors, such neurons being sensitive to exogenously applied selective NK1, NK2, or NK3 receptor agonists (Table 1 and Fig. 1). That neuronal effects induced by NK1 or NK2 receptor agonists no longer occurred in the presence of NK1 or NK2 receptor antagonists, respectively, supports the contention that NK receptor subtypes are associated with intrinsic cardiac neurons. Because some intrinsic cardiac neurons were modified by more than one NK agonist, it appears that some intrinsic cardiac neurons may possess more than one NK receptor subtype.

The selective NK1 receptor agonist [Sar9,Met(O2)11]-SP decreased right atrial neuronal activity in more animals than the number of animals in which it activated neurons (Table 1). The selective NK2 receptor agonist [beta -Ala8]-NKA induced similar responses. On the other hand, senktide enhanced the activity generated by intrinsic cardiac neurons in most animals studied (n = 14 dogs), depressing neuronal activity in only four dogs. Thus, although there were relatively equal numbers of intrinsic cardiac neurons whose activities were influenced by the NK1, NK2, or NK3 receptor agonists (Table 1), the response characteristics of neurons possessing NK1 and NK2 receptors differed from those expressing NK3 receptors.

Local arterial administration of an NK1 or NK2 receptor antagonist modified the spontaneous activity generated by many investigated right atrial neurons (Table 1 and Fig. 2). These data indicate that some intrinsic cardiac neurons receive tonic input via endogenously liberated NKs. NK1 or NK2 receptor agonists no longer affected the activity generated by ventral right atrial neurons in the presence of their selective antagonists. These data further support the contention that intrinsic cardiac neurons possess NK receptor subtypes.

Recent physiological evidence indicates that SP as well as other tachykinin receptor-selective agonists are capable of modifying cardiac neurons in intrathoracic extrinsic (3) and intrinsic (6, 12) cardiac ganglia that are involved in cardiac regulation. Anatomic and functional data indicate that mammalian intrinsic cardiac neurons (6, 7) and primary cardiac afferent neurons (4, 6) possess NK receptors. For example, Hardwick and colleagues (12) demonstrated that SP, NKA, and senktide depolarized guinea pig intracardiac neurons in vitro via a nonspecific cationic inward current (12). Furthermore, using specific tachykinin receptor blockers, they found that these tachykinins activated such neurons via NK3 receptors. Although our data demonstrating neuronal depressing effects induced by the more specific NK1 and NK2 receptor agonists differ from that found in their study, our results on the effects observed in situ of senktide activation via NK3 receptors support their findings observed in vitro. Thus NKs influence intrinsic cardiac neurons in vitro (11, 13, 17) as well as in situ (4).

As has been shown with respect to SP-sensitive intrinsic cardiac neurons studied in vitro (22) or in situ (4), cardiovascular variables were modified in some instances when NK-sensitive right atrial neurons were affected, particularly when senktide was tested. When a sufficient population of intrinsic cardiac neurons is modified by SP, enhancement of cardiac variables occurs via activation of cardiac sympathetic efferent neurons (4). That enhancement of right ventricular intramyocardial systolic pressure occurred when senktide was tested presumably was due to the fact that some sympathetic efferent cardiac neurons that innervate the right ventricle were directly or indirectly activated (4). The effects induced by senktide presumably were not the result of direct modification of the regional arterial blood supply because the NK receptors identified on these vessels by autoradiography were presumed to be the NK1 receptor subtype (10, 15). On the other hand, the effects that [Sar9,Met(O2)11]-SP induced may have been due in part to alterations in the local arterial blood supply to intrinsic cardiac neurons mediated via the NK1 receptors that are associated with such arteries. That neuronal activity and, for that matter, cardiovascular variables were not affected significantly when each agonist was administered individually in the same doses into the systemic circulation indicates that the neuronal responses induced by local coronary arterial administration were not secondary to the chemical entering the systemic circulation in sufficient quantities to affect distant tissues such as resistance arteries. Rather, neuronal responses induced when chemicals were administered to right atrial neurons via their regional coronary arterial blood supply appeared to be due to chemical modification of local somata and/or dendrites as opposed to distant tissues, including ventricular myocytes.

Our anatomic data support the hypothesis that some intrinsic cardiac neurons possess NK receptors (Fig. 3, E and F). Definitive statements concerning the subtypes of NK receptors associated with canine intrinsic cardiac ganglia cannot be made based on our autoradiographic findings because 125I-labeled NKA and 125I-labeled eledoisin label more than one receptor subtype (21, 23, 24). Data obtained using 125I-labeled eledoisin indicate that NK receptors are associated with canine intrinsic cardiac ganglia (Fig. 3). It is known that 125I-labeled eledoisin binds with highest affinity to NK3 receptors (24). That senktide was most effective in activating canine intrinsic cardiac neurons (Table 1) supports our data that 125I-labeled eledoisin NK3 receptors are associated with a significant population of intrinsic cardiac neurons. Although the functional data indicate that NK2 receptors are associated with canine intrinsic cardiac neurons, we were unable to detect such receptors using 125I-labeled NKA binding. Because this radioligand has a significant affinity for NK1 and NK2 receptor subtypes, these NK receptors may be expressed on intrinsic cardiac neurons at levels below our limit of detection. On the other hand, a significant amount of 125I-labeled NKA binding was associated with regional coronary blood vessels. These sites presumably were NK1 receptors that have been implicated in mediating coronary vasodilator responses to tachykinins (10, 14).

Perspectives

Tachykinins are a family of neuropeptides widely distributed in the mammalian central and peripheral nervous systems. Their release from sensory nerves is responsible for many physiological activities, including neurogenic inflammation, pain transmission, central cardiovascular regulation, and other autonomic reflexes. The results of the present investigation indicate that populations of intrinsic cardiac neurons also possess NK1, NK2, and/or NK3 receptors. Furthermore, data obtained using selective NK receptor antagonists indicate that some intrinsic cardiac neurons receive tonic input via NKs in situ. That tachykinin-sensitive intrinsic cardiac neurons can activate cardiac sympathetic efferent neurons should be taken into account when considering modifying cardiac augmentor responses elicited during periods of stress such as that which occurs during myocardial ischemia. Furthermore, because such neurons also have the capacity to generate tachydysrhythmias, tachykinin receptor blockade may prove to be of therapeutic benefit in modifying cardiac dysrhythmias.

    ACKNOWLEDGEMENTS

The authors gratefully acknowledge the technical assistance of Richard Livingston.

    FOOTNOTES

This work was supported by Medical Research Council of Canada Grant MT-10122, the National Heart, Lung, and Blood Institute (Grants HL-54633 and HL-58140), and a grant-in-aid from the American Heart Association.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: J. A. Armour, Dept. of Physiology and Biophysics, Faculty of Medicine, Dalhousie Univ., Halifax, Nova Scotia, Canada B3H 4H7.

Received 8 April 1998; accepted in final form 6 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Allen, T. G. J., C. J. S. Hassal, and G. Burnstock. Mammalian intrinsic cardiac neurons in cell culture. In: Neurocardiology, edited by J. Andrew Armour, and Jeffrey L. Ardell. New York: Oxford Univ. Press, 1994, p. 115-138.

2.   Appell, K. C., B. J. Fragale, J. Loscig, S. Singh, and B. E. Tomczuk. Antagonists that demonstrate species differences in neurokinin-1 receptors. Mol. Pharmacol. 41: 772-778, 1992[Abstract].

3.   Armour, J. A. Peptidergic modulation of efferent sympathetic neurons in intrathoracic ganglia regulating the canine heart. Proc. Soc. Exp. Biol. Med. 191: 60-68, 1989[Abstract].

4.   Armour, J. A., M. H. Huang, and F. M. Smith. Peptidergic modulation of in situ canine intrinsic cardiac neurons. Peptides 14: 191-202, 1993[Medline].

5.   Armour, J. A., and W. C. Randall. Canine left ventricular intramyocardial pressure. Am. J. Physiol. 220: 1833-1839, 1971.

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Am J Physiol Regul Integr Compar Physiol 275(5):R1683-R1689
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society


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