Localization of α7 nicotinic acetylcholine receptor mRNA and protein within the cholinergic anti-inflammatory pathway.

Electrical stimulation of the vagus nerve attenuates tumor necrosis factor (TNF) synthesis by macrophages and reduces the systemic inflammatory response. Current evidence suggests that the α7 nicotinic acetylcholine receptor present in the celiac/superior mesenteric ganglia is a key component in vagus nerve signaling to the spleen; however, there is currently no direct anatomical evidence that the α7 receptor is present in the murine celiac/superior mesenteric ganglia. Our study addresses this deficiency by providing anatomical evidence that the α7 receptor is expressed within the celiac/superior mesenteric ganglia and splenic nerve fibers using immunohistochemistry and quantitative polymerase chain reaction (qPCR). α7 receptor mRNA is highly expressed in the celiac/superior mesenteric ganglia and at low levels in the spleen compared to the brain. Double-labeling for α7 and tyrosine hydroxylase shows that α7 receptor protein is present on noradrenergic neurons within the ganglia and prejunctionally on noradrenergic nerve fibers within the spleen. The α7 receptor in the ganglia provides a possible location for the action of α7-selective agonists, while prejunctional α7 receptor expressed on splenic nerves may induce an increase in norepinephrine release in a positive feedback system enhanced by lymphocyte-derived acetylcholine.

Development of cardiac parasympathetic neurons, glial cells, and regional cholinergic innervation of the mouse heart.

Very little is known about the development of cardiac parasympathetic ganglia and cholinergic innervation of the mouse heart. Accordingly, we evaluated the growth of cholinergic neurons and nerve fibers in mouse hearts from embryonic day 18.5 (E18.5) through postnatal day 21(P21). Cholinergic perikarya and varicose nerve fibers were identified in paraffin sections immunostained for the vesicular acetylcholine transporter (VAChT). Satellite cells and Schwann cells in adjacent sections were identified by immunostaining for S100ß calcium binding protein (S100) and brain-fatty acid binding protein (B-FABP). We found that cardiac ganglia had formed in close association to the atria and cholinergic innervation of the atrioventricular junction had already begun by E18.5. However, most cholinergic innervation of the heart, including the sinoatrial node, developed postnatally (P0.5-P21) along with a doubling of the cross-sectional area of cholinergic perikarya. Satellite cells were present throughout neonatal cardiac ganglia and expressed primarily B-FABP. As they became more mature at P21, satellite cells stained strongly for both B-FABP and S100. Satellite cells appeared to surround most cardiac parasympathetic neurons, even in neonatal hearts. Mature Schwann cells, identified by morphology and strong staining for S100, were already present at E18.5 in atrial regions that receive cholinergic innervation at later developmental times. The abundance and distribution of S100-positive Schwann cells increased postnatally along with nerve density. While S100 staining of cardiac Schwann cells was maintained in P21 and older mice, Schwann cells did not show B-FABP staining at these times. Parallel development of satellite cells and cholinergic perikarya in the cardiac ganglia and the increase in abundance of Schwann cells and varicose cholinergic nerve fibers in the atria suggest that neuronal-glial interactions could be important for development of the parasympathetic nervous system in the heart.

Localization of multiple neurotransmitters in surgically derived specimens of human atrial ganglia.

Dysfunction of the intrinsic cardiac nervous system is implicated in the genesis of atrial and ventricular arrhythmias. While this system has been studied extensively in animal models, far less is known about the intrinsic cardiac nervous system of humans. This study was initiated to anatomically identify neurotransmitters associated with the right atrial ganglionated plexus (RAGP) of the human heart. Biopsies of epicardial fat containing a portion of the RAGP were collected from eight patients during cardiothoracic surgery and processed for immunofluorescent detection of specific neuronal markers. Colocalization of markers was evaluated by confocal microscopy. Most intrinsic cardiac neuronal somata displayed immunoreactivity for the cholinergic marker choline acetyltransferase and the nitrergic marker neuronal nitric oxide synthase. A subpopulation of intrinsic cardiac neurons also stained for noradrenergic markers. While most intrinsic cardiac neurons received cholinergic innervation evident as punctate immunostaining for the high affinity choline transporter, some lacked cholinergic inputs. Moreover, peptidergic, nitrergic, and noradrenergic nerves provided substantial innervation of intrinsic cardiac ganglia. These findings demonstrate that the human RAGP has a complex neurochemical anatomy, which includes the presence of a dual cholinergic/nitrergic phenotype for most of its neurons, the presence of noradrenergic markers in a subpopulation of neurons, and innervation by a host of neurochemically distinct nerves. The putative role of multiple neurotransmitters in controlling intrinsic cardiac neurons and mediating efferent signaling to the heart indicates the possibility of novel therapeutic targets for arrhythmia prevention.

Cholinergic neurons of mouse intrinsic cardiac ganglia contain noradrenergic enzymes, norepinephrine transporters, and the neurotrophin receptors tropomyosin-related kinase A and p75.

Half of the cholinergic neurons of human and primate intrinsic cardiac ganglia (ICG) have a dual cholinergic/noradrenergic phenotype. Likewise, a large subpopulation of cholinergic neurons of the mouse heart expresses enzymes needed for synthesis of norepinephrine (NE), but they lack the vesicular monoamine transporter type 2 (VMAT2) required for catecholamine storage. In the present study, we determined the full scope of noradrenergic properties (i.e. synthetic enzymes and transporters) expressed by cholinergic neurons of mouse ICG, estimated the relative abundance of neurons expressing different elements of the noradrenergic phenotype, and evaluated the colocalization of cholinergic and noradrenergic markers in atrial nerve fibers. Stellate ganglia were used as a positive control for noradrenergic markers. Using fluorescence immunohistochemistry and confocal microscopy, we found that about 30% of cholinergic cell bodies contained tyrosine hydroxylase (TH), including the activated form that is phosphorylated at Ser-40 (pSer40 TH). Dopamine beta-hydroxylase (DBH) and norepinephrine transporter (NET) were present in all cholinergic somata, indicating a wider capability for dopamine metabolism and catecholamine uptake. Yet, cholinergic somata lacked VMAT2, precluding the potential for NE storage and vesicular release. In contrast to cholinergic somata, cardiac nerve fibers rarely showed colocalization of cholinergic and noradrenergic markers. Instead, these labels were closely apposed but clearly distinct from each other. Since cholinergic somata expressed several noradrenergic proteins, we questioned whether these neurons might also contain trophic factor receptors typical of noradrenergic neurons. Indeed, we found that all cholinergic cell bodies of mouse ICG, like noradrenergic cell bodies of the stellate ganglia, contained both tropomyosin-related kinase A (TrkA) and p75 neurotrophin receptors. Collectively, these findings demonstrate that mouse intrinsic cardiac neurons (ICNs), like those of humans, have a complex neurochemical phenotype that goes beyond the classical view of cardiac parasympathetic neurons. They also suggest that neurotrophins and local NE synthesis might have important effects on neurons of the mouse ICG.

Substance P modulates nicotinic responses of intracardiac neurons to acetylcholine in the guinea pig.

Application of substance P (SP) to intracardiac neurons of the guinea pig causes slow depolarization and increases neuronal excitability. The present study was done to determine the influence of SP on fast excitatory responses of intracardiac neurons to ACh. Intracellular recording methods were used to measure responses of intracardiac neurons in whole mount preparations of atrial ganglionated nerve plexus from guinea pig hearts. Local pressure ejection of 100 microM SP (1 s) from a glass micropipette caused slow depolarization of all neurons (n = 38) and triggered action potential generation in 47% of the cells tested. Bath application of SP (0.5-100 microM) caused a dose-dependent depolarization of intracardiac neurons but rarely evoked action potentials, even at the highest concentration. However, such treatment with SP enhanced nicotinic responses evoked by local pressure ejections of ACh (10 mM, 10- to 100-ms duration) in 77% of intracardiac neurons studied (n = 52). A significant increase in amplitude of ACh-evoked fast depolarization occurred during treatment with 0.5 microM SP (13.0 +/- 1.8 mV for control vs. 17.7 +/- 1.9 mV with SP present, n = 7, P = 0.019). At higher concentrations of SP, enhancement of the response to ACh resulted mainly in action potential generation. However, responses to ACh were attenuated by SP in 15% of the intracardiac neurons studied. This attenuation occurred primarily during exposure to 10 and 100 microM SP and was manifest as a reduction in amplitude of nicotinic fast depolarization or inhibition of ACh-evoked action potentials. These findings support the conclusion that SP could function as a neuromodulator and neurotransmitter in intracardiac ganglia of the guinea pig.

Distribution of cocaine- and amphetamine-regulated transcript peptide in the guinea pig intrinsic cardiac nervous system and colocalization with neuropeptides or transmitter synthetic enzymes.

This study was conducted to establish the presence of cocaine- and amphetamine-regulated transcript peptide (CARTp) immunoreactivity in neurons and fibers within guinea pig atrial whole-mount preparations containing the intrinsic cardiac ganglia. Many cardiac ganglia, but not all, in a given whole-mount preparation, were innervated by CARTp-immunoreactive (IR) fibers. Following explant culture of whole mounts for 72 hours, the CARTp-IR fiber networks were absent, but the number of CARTp-IR neurons was increased markedly. These observations suggested that the majority of the CARTp-IR fibers in the intracardiac ganglia were derived from sources extrinsic to the heart. In control whole-mount preparations, very few CARTp-positive neurons were present. The few intrinsic CARTp-IR neurons also exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they make up a small subpopulation of cholinergic postganglionic neurons. Some CARTp-IR neurons also exhibited nitric oxide synthase (NOS) immunoreactivity, indicating that they were nitrergic as well. We compared the immunohistochemical staining patterns of CARTp-IR fibers with the staining patterns of a number of other neurotransmitters or neurotransmitter synthetic enzymes that mark specific extrinsic inputs. The CARTp-IR fibers were not immunoreactive for ChAT, tyrosine hydroxylase, calcitonin gene-related peptide, or substance P. However, virtually all CARTp-IR fibers exhibited immunoreactivity to neuronal NOS (a marker for nitric oxide-producing neurons). CARTp-IR cells and NOS-IR cells were present in the nodose ganglia. In addition, CARTp-IR neurons in the nodose also were stained positively for NADPH-diaphorase. Thus, we propose that most CARTp-IR fibers within the guinea pig intrinsic cardiac ganglia are vagal afferent fibers that also contain NOS.

Chronic decentralization of the heart differentially remodels canine intrinsic cardiac neuron muscarinic receptors.

The objective of the study was to determine if chronic interruption of all extrinsic nerve inputs to the heart alters cholinergic-mediated responses within the intrinsic cardiac nervous system (ICN). Extracardiac nerve inputs to the ICN were surgically interrupted (ICN decentralized). Three weeks later, the intrinsic cardiac right atrial ganglionated plexus (RAGP) was removed and intrinsic cardiac neuronal responses were evaluated electrophysiologically. Cholinergic receptor abundance was evaluated using autoradiography. In sham controls and chronic decentralized ICN ganglia, neuronal postsynaptic responses were mediated by acetylcholine, acting at nicotinic and muscarinic receptors. Muscarine- but not nicotine-mediated synaptic responses that were enhanced after chronic ICN decentralization. After chronic decentralization, muscarine facilitation of orthodromic neuronal activation increased. Receptor autoradiography demonstrated that nicotinic and muscarinic receptor density associated with the RAGP was unaffected by decentralization and that muscarinic receptors were tenfold more abundant than nicotinic receptors in the right atrial ganglia in each group. After chronic decentralization of the ICN, intrinsic cardiac neurons remain viable and responsive to cholinergic synaptic inputs. Enhanced muscarinic responsiveness of intrinsic cardiac neurons occurs without changes in receptor abundance.

Regional localization and abundance of calcitonin gene-related peptide receptors in guinea pig heart.

Calcitonin gene-related peptide (CGRP) is a neurotransmitter that is released within the heart during myocardial ischemia. The present study was done to determine the regional localization and abundance of CGRP receptors in the guinea pig heart. CGRP binding sites in 20 microm frozen sections of heart were labeled using [125I]CGRP. Non-specific binding was determined in the presence of 1 microm unlabeled CGRP or CGRP(8-37). Significant amounts of specific CGRP binding were identified in atrial and ventricular myocardium, all portions of the conducting system, coronary arteries, the aorta and pulmonary trunk and intracardiac ganglia. Specific binding of CGRP to the left atrium was two-fold higher than binding to the right atrium (0.667+/-0.052 v 0.340+/-0.029 fmol/mg tissue, n=5, CGRP(8-37)group). In contrast to the atria, a lower and uniform density of CGRP receptors occurred in contractile tissue of the ventricular myocardium (e.g. 0.239+/-0.013 fmol/mg left ventricle, n=5). The highest concentration of CGRP receptors in guinea pig cardiac tissue occurred at the bundle of His and the bundle branches (0.752+/-0.087 and 0.713+/-0.138 fmol/mg tissue, respectively, n=5). CGRP receptors were localized to coronary vessels throughout the heart and to the ascending aorta and pulmonary trunk. Lastly, intracardiac ganglia exhibited moderate levels of specific [125I]CGRP binding (0.475+/-0.043 fmol/mg, n=5). These findings support the concept that CGRP can have direct effects on atrial and ventricular function as well as coronary flow. The high density of CGRP receptors in the distal conducting system and the presence of CGRP receptors in intracardiac ganglia further suggest that CGRP could have important effects on cardiac conduction velocity and parasympathetic regulation of the heart.

NK(3) receptors in the feline nucleus tractus solitarius are not involved with the muscle pressor response.

Isometric muscle contractions cause an increase in mean arterial pressure and heart rate. Previously, we showed that substance P (SP) is released from sites in the feline medial nucleus tractus solitarius (mNTS) in response to isometric muscle contractions, and that it most likely interacted with NK(1) tachykinin receptors at these sites. This study was undertaken to determine whether other tachykinin receptors in this area of the brainstem are involved with the muscle pressor response. Receptor autoradiography, using [(125)I]Bolton-Hunter SP and [(125)I] [MePhe(7)] neurokinin B to label NK(1) and NK(3) receptors, respectively, indicated that NK(3) tachykinin receptors are as abundant as NK(1) and NK(3) receptors, respectively, indicated that NK(3) tachykinin receptors are as abundant as NK(1) receptors in this region of the feline brainstem Injections of the specific NK(3) receptor antagonist, SR 142801 (0.1 to 10 microM) into the mNTS did not modify the pressor response or the heart rate response to isometric muscle contractions. Injection of SR142801 into the NTS prior to the injection of the NK(1) antagonist, GR82334 did not affect the action of GR82334 to attenuate the muscle pressor reflex. We conclude that NK(3) receptors in the NTS are not involved with the regulation of cardiovascular function during activation of the muscle pressor response.

Increased ganglionic responses to substance P in hypertensive rats due to upregulation of NK(1) receptors.

Intravenous injection of substance P (SP) increases renal nerve firing and heart rate in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) by stimulating sympathetic ganglia. Blood pressure is increased in SHRs but lowered in WKYs. This study assesses the role of neurokinin-1 (NK(1)) receptors in mediating the ganglion actions of SP. Rats for functional studies were anesthetized and then treated with chlorisondamine. Renal nerve, blood pressure, and heart rate responses to intravenous injection of the NK(1) receptor agonist GR-73632 were similar but less than those to equimolar doses of SP in SHRs. GR-73632 only slightly increased renal nerve firing and heart rate and lowered blood pressure in WKYs. The NK(1) receptor antagonist GR-82334 (200 nmol/kg iv) blocked the ganglionic actions of GR-73632 and the pressor response to SP in SHRs. It reduced the renal nerve and heart rate responses by 52 and 35%. This suggests that the pressor response to SP is mediated by ganglionic NK(1) receptors and that NK(1) receptors also have a prominent role in mediating the renal nerve and heart rate responses to SP. Quantitative autoradiography showed that NK(1) receptors are more abundant in the superior cervical ganglia of SHRs. RT-PCR showed increased abundance of NK(1) receptor mRNA in SHRs as well. These observations suggest that the greater ganglionic stimulation caused by SP in SHRs is due to upregulation of NK(1) receptors.

Tachykinin receptor subtypes in the isolated guinea pig heart and their role in mediating responses to neurokinin A.

Selective tachykinin agonists were used to identify cardiac and coronary responses mediated by specific tachykinin receptor subtypes in isolated, perfused guinea pig hearts. Receptor desensitization with selective agonists and blockade with selective antagonists were used to determine the role of specific subtypes in generating responses to neurokinin A (NKA). Dose-dependent cardiac and coronary effects were evoked by bolus injections of ¿Sar(9), Met(O(2))(11)substance P (¿Sar(9),Met(O(2))(11)SP), GR64349, and ¿MePhe(7)neurokinin B (¿MePhe(7)NKB) (selective agonists for NK(1), NK(2), and NK(3) receptors, respectively). Each agonist caused bradycardia, but GR64349 was most effective (34 +/- 4% decrease in heart rate with 32 nmol, n = 8). Prominent increases in ventricular contractility and perfusion pressure also occurred with 32 nmol of GR64349 (25 +/- 6 and 33 +/- 4%, respectively). ¿Sar(9), Met(O(2))(11)SP was unique in having a high potency for decreasing ventricular contractility and perfusion pressure. Bolus injections of 25 nmol of NKA decreased rate (48 +/- 2%, n = 51), increased contractility (26 +/- 2%), and had biphasic effects on perfusion pressure (24 +/- 1% decrease followed by 9.2 +/- 1.4% increase). Desensitization with GR64349 or treatment with the NK(2) antagonist SR48968 reduced the bradycardic response to NKA by greater than 75% and eliminated the positive inotropic response. The remaining bradycardia occurred through NK(3) receptors. Desensitization with ¿Sar(9),Met(O(2))(11)SP or NK(1) blockade with FK888 eliminated the coronary relaxant action of NKA and enhanced the pressor response. It is concluded that three tachykinin receptor subtypes are present in the guinea pig heart and that each contributes to the overall response evoked by NKA.

Endogenous tachykinins cause bradycardia by stimulating cholinergic neurons in the isolated guinea pig heart.

The purpose of this study was to determine if endogenous tachykinins can cause bradycardia in the isolated perfused guinea pig heart through stimulation of cholinergic neurons. Capsaicin was used to stimulate release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac afferents. A bolus injection of 100 nmol capsaicin increased heart rate by 26 +/- 7% from a baseline of 257 +/- 14 beats/min (n = 6, P < 0.01). This positive chronotropic response was converted to a minor bradycardic effect in hearts with 1 microM CGRP-(8-37) present to block CGRP receptors. The negative chronotropic response to capsaicin was markedly potentiated in another group of hearts with the further addition of 0.5 microM neostigmine to inhibit cholinesterases. In this group, capsaicin decreased heart rate by 30 +/- 10% from a baseline of 214 +/- 6 beats/min (n = 8, P < 0.05). This large bradycardic response to capsaicin was inhibited by 1) infusion of neurokinin A to desensitize tachykinin receptors or 2) treatment with 1 microM atropine to block muscarinic receptors. The latter observations implicate tachykinins and acetylcholine, respectively, as mediators of the bradycardia. These findings support the hypothesis that endogenous tachykinins could mediate axon reflexes to stimulate cholinergic neurons of the intrinsic cardiac ganglia.

Expression of c-fos-like immunoreactivity in the feline brainstem in response to isometric muscle contraction and baroreceptor reflex changes in arterial pressure.

This study compared whether activation of muscle ergoreceptor afferents caused by isometric muscle contraction, activation of baroreceptor afferents induced by i.v. infusion of phenylephrine, or baroreceptor afferent inactivation, caused by carotid artery occlusion, elicit similar patterns of c-Fos induction in brainstem areas. Adult cats were anesthetized with alpha-chloralose, and in each case, the experimental intervention caused an increase in the arterial blood pressure. There were two sets of control experiments: in both, animals underwent the same surgical procedures but then either remained at rest for the entire study, or the tibial nerve was stimulated, as in the contraction group, following muscle paralysis with tubocurarine. Following the procedures, animals rested for 90 min to allow neuronal expression of c-Fos. Control cats showed very little c-Fos immunoreactivity (c-Fos-ir) in the brainstem. Muscle contraction induced c-Fos-ir expression mainly in the nucleus tractus solitarius, lateral reticular nucleus, lateral tegmental field, vestibular nucleus, subretrofacial nucleus, spinal trigeminal tract and in a lateral region of the periaqueductal grey (P 0.5-1.0). The majority of the c-Fos-ir was found in brainstem areas contralateral to the contracted muscle. In addition, muscle contraction induced c-Fos-ir in the dorsal horns of spinal segments L6-S1 on the ipsilateral side of the spinal cord. Phenylephrine infusion caused c-Fos-ir expression in the nucleus tractus solitarius, spinal trigeminal tract, solitary tract, and dorsal motor nucleus of the vagus. No c-Fos-ir was apparent in the periaqueductal grey. Carotid occlusions induced c-Fos-ir expression in the area postrema, nucleus tractus solitarius, solitary tract, and spinal trigeminal tract. Expression was bilateral. Areas that exhibited c-Fos-ir correspond to sites previously reported to release various neuropeptides in response to muscle contraction or carotid occlusions. These results indicate that the exercise pressor reflex and baroreflex activate similar, but not completely identical, sites in the brainstem.

Signaling mechanisms for muscarinic receptor-mediated coronary vasoconstriction in isolated rat hearts.

Signaling mechanisms for muscarinic receptor-mediated vasoconstriction in coronary resistance arteries were studied in potassium-arrested isolated rat hearts perfused at a constant flow rate. The cholinergic agonist bethanechol was given by bolus injection or constant infusion. Perfusion pressure was monitored as an indicator of coronary vascular resistance. Bolus injection of bethanechol evoked a phasic vasoconstriction in a dose-dependent manner, whereas infusion of bethanechol evoked a tonic vasoconstriction without producing tachyphylaxis. Bethanechol-induced phasic vasoconstriction was eliminated by perfusion with a Ca(2+)-free buffer. The L-type voltage-operated Ca(2+) channel blocker nifedipine decreased the maximal constrictor response to bethanechol by 59 +/- 2% (n = 4, P <.001), whereas the putative receptor-operated Ca(2+) channel blocker SK&F 96365 converted this vasoconstriction into vasodilation that was not mediated by nitric oxide. The protein kinase C inhibitor chelerythrine reduced the maximal phasic vasoconstrictor response to bethanechol by 78 +/- 2% (n = 6, P <.001) Bethanechol-induced tonic vasoconstriction was rapidly converted to a sustained vasodilation during infusion of SK&F 96365 or nifedipine, whereas infusion of chelerythrine gradually attenuated the tonic response to bethanechol. Results from other experiments do not support a role for phospholipase A(2)-dependent mediators in generating coronary vasoconstrictor responses to bethanechol. It is concluded that voltage-independent receptor-operated Ca(2+) channels, voltage-operated Ca(2+) channels, and protein kinase C are major signaling components for muscarinic receptor-mediated contraction of rat coronary resistance arteries.

Actions of tachykinins within the heart and their relevance to cardiovascular disease.

Substance P and neurokinin A are tachykinins that are co-localized with calcitonin gene-related peptide (CGRP) in a unique subpopulation of cardiac afferent nerve fibers. These neurons are activated by nociceptive stimuli and exhibit both sensory and motor functions that are mediated by the tachykinins and/or CGRP. Sensory signals (e.g., cardiac pain) are transmitted by peptides released at central processes of these neurons, whereas motor functions are produced by the same peptides released from peripheral nerve processes. This review summarizes our current understanding of intracardiac actions of the tachykinins. The major targets for the tachykinins within the heart are the intrinsic cardiac ganglia and coronary arteries. Intrinsic cardiac ganglia contain cholinergic neurons that innervate the heart and coronary vasculature. Tachykinins can stimulate NK3 receptors on these neurons to increase their excitability and evoke spontaneous firing of action potentials. This action provides a mechanism whereby tachykinins can indirectly influence cardiac function and coronary tone. Tachykinins also have direct effects on coronary arteries to decrease or increase tone. Stimulation of NK1 receptors on the endothelium causes vasodilation mediated by nitric oxide. This effect is normally dominant, but NK2 receptor-mediated vasoconstriction can also occur and is augmented when NK1 receptors are blocked. It is proposed that these ganglion stimulant and vascular actions are manifest by endogenous tachykinins during myocardial ischemia.

Substance P evokes bradycardia by stimulation of postganglionic cholinergic neurons.

Substance P (SP) evokes bradycardia that is mediated by cholinergic neurons in experiments with isolated guinea pig hearts. This project investigates the negative chronotropic action of SP in vivo. Guinea pigs were anesthetized with urethane, vagotomized and artificially respired. Using this model, IV injection of SP (32 nmol/kg/50 microl saline) caused a brief decrease in heart rate (-30+/-3 beats/min from a baseline of 256+/-4 beats/min, n = 27) and a long-lasting decrease in blood pressure (-28+/-2 mmHg from baseline of 51+/-5 mmHg, n = 27). The negative chronotropic response to SP was attenuated by muscarinic receptor blockade with atropine (-29 +/- 9 beats/min before vs -8 +/- 2 beats/min after treatment, P = 0.0204, n = 5) and augmented by inhibition of cholinesterases with physostigmine (-23 +/- 6 beats/min before versus -74 +/- 20 beats/min after treatment, P = 0.0250, n = 5). Ganglion blockade with chlorisondamine did not diminish the negative chronotropic response to SP. In another series of experiments, animals were anesthetized with sodium pentobarbital or urethane and studied with or without vagotomy. Neither anesthetic nor vagotomy had a significant effect on the negative chronotropic response to SP (F3,24 = 1.97, P = 0.2198). Comparison of responses to 640 nmol/kg nitroprusside and 32 nmol/kg SP demonstrated that the bradycardic effect of SP occurs independent of vasodilation. These results suggest that SP can evoke bradycardia in vivo through stimulation of postganglionic cholinergic neurons.

Characterization of responses to neurokinin A in the isolated perfused guinea pig heart.

Goals of this study were to identify and characterize effects of neurokinin A (NKA) in isolated guinea pig hearts. Bradycardia, augmentation of ventricular contractions, and reduction of perfusion pressure were prominent responses to bolus injections of NKA (0. 25-25 nmol). NKA was more potent than substance P (SP) in causing bradycardia but did not differ in potency for lowering perfusion pressure. Doses of SP of 25 nmol or less decreased ventricular force, whereas 100 nmol caused a biphasic response. The percent decrease in heart rate produced by 25 nmol NKA was reduced from 58.0 +/- 4.8 to 39.6 +/- 3.5% in the presence of 1 microM atropine (n = 5). The positive inotropic response to 25 nmol of NKA in spontaneously beating hearts was replaced by a negative inotropic response during pacing (22.5 +/- 3.3% increase vs. 11.7 +/- 1.7% decrease, n = 5). Reserpine pretreatment did not affect the positive inotropic response to NKA. Specific binding sites for 125I-labeled NKA were localized to intracardiac ganglia and coronary arteries but not to myocardium. It was concluded that 1) negative chronotropic responses to NKA involve cholinergic and noncholinergic mechanisms, and 2) the positive inotropic response is an indirect action.

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

To determine whether intrinsic cardiac neurons involved in cardiac regulation possess neurokinin (NK) receptor subtypes, we administered selective NK receptor agonists individually (100 microM; 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.

Differentiation of muscarinic receptors mediating negative chronotropic and vasoconstrictor responses to acetylcholine in isolated rat hearts.

The primary goal of this study was to determine the extent that selective muscarinic receptor antagonists could discriminate between the chronotropic and coronary vasoconstrictor responses to acetylcholine in isolated rat hearts perfused at constant flow rate. Bolus injections of acetylcholine caused dose-dependent decreases in heart rate and increases in perfusion pressure. The ED50 (95% confidence) of acetylcholine for decreasing rate was 0.463 (0.336-0.640) nmol and the dose that increased perfusion pressure by 30 mm Hg (ED30 mmHg) was 3.19 (2.00-5.08) nmol. The M2 selective antagonist methoctramine (3.16 microM) produced a 307-fold increase in the ED50 for bradycardia but had no significant effect on the pressor response to acetylcholine. In marked contrast, the M3 antagonist hexahydrosiladifenidol displayed a distinct preference for inhibiting coronary vasoconstrictor responses to acetylcholine. When present at 316 nM, this drug produced a 66-fold increase in the ED30 mmHg but only a 6-fold increase in the ED50 for bradycardia. The M1 selective antagonist pirenzepine (316 nM) produced a 5- to 7-fold increase in both parameters. Pretreatment with pertussis toxin (25 microg/kg, i.p.) essentially eliminated acetylcholine-evoked bradycardia although pressor responses persisted with some reduction. These observations demonstrate that cardiac and coronary vascular effects of acetylcholine can be clearly discriminated with specific muscarinic antagonists. Furthermore, they provide evidence that the M3 receptor subtype mediates the vasoconstrictor effect of acetylcholine on resistance vessels in rat heart.

Preconditioning of isolated rabbit cardiomyocytes: no evident separation of induction, memory and protection.

Cardiomyocytes isolated from rabbit hearts were preconditioned in vitro by 10 min of ischemia or treatment with 100 microM adenosine. Protection was assessed as average integrated mortality following osmotic swelling and determination of viability by trypan blue exclusion over 60-180 min ischemia. Repetitive sub-maximal stimulations with 1 microM adenosine amplified the protective response. Treatment with adenosine only at the onset of prolonged ischemia afforded a dose-dependent protection. The PKC inhibitor calphostin C (500 nm) blocked preconditioning and, when added during ischemic incubation of non-preconditioned cells, significantly increased injury. The memory of adenosine-induced preconditioning decayed over a 60 min post-incubation period. Light activation of calphostin C initially added to preconditioned ischemic cells in the dark indicated that a 10 min period of PKC activity at the onset of ischemia affords full protection. The reversible PKC inhibitors chelerythrine (5 microM) or staurosporine (100 nM) added only to bracket induction of ischemia, reduced but did not abolish protection. Protection was abolished when either drug was present during induction and a subsequent 30 min post-incubation period. Staurosporine included during initiation and post-incubation but washed out in the final 5 min of post-incubation allowed significant protection to occur. It is concluded that a single adenosine receptor-stimulation induces protection as it preconditions, and PKC activity appears to be required for both induction and protection. Memory may reside in post-receptor amplification of an initial protective response.