Antibodies targeting human endothelin-1 receptors reveal different conformational states in cancer cells.

The endothelin axis (endothelins and their receptors) is strongly involved in physiological and pathological processes. ET-1 plays a crucial role in particular in tumor diseases. Endothelin-1 receptors (ET(A) and ET(B)) are deregulated and overexpressed in several tumors such as melanoma and glioma. We studied the binding of 24 monoclonal antibodies directed against human ET(B) receptors (hET(B)) to different melanoma cell lines. Few of these mAbs bound to all the melanoma cell lines. One of them, rendomab B49, bound to ET(B) receptors expressed at the surface of human glioma stem cells. More recently, we produced new antibodies directed against human ET(A) receptor (hET(A)). Several antibodies have been isolated and have been screened on different tumoral cells lines. As for the mAbs directed against the hET(B) receptor only some of new antibodies directed against ET(A) receptor are capable to bind the human tumoral cell lines. Rendomab A63 directed against hET(A) is one of them. We report the specificity and binding properties of these mAbs and consider their potential use in diagnosis by an in vivo imaging approach.

Pharmacological in vitro evaluation of new substance P-cyclodextrin derivatives designed to drug targeting towards NK1-receptor bearing cells.

Some biological properties of new bifunctional conjugates designed for drug targeting were evaluated through in vitro experiments. Eight peptidylcyclodextrin compounds were used, which correspond to modified beta- or gamma-cyclodextrin (CD) grafted on neuropeptide substance P (SP) or a shorter derivative (SP(4-11)). Using anti-SP and anti-CD antibodies as molecular probes, we showed that the main structural features of the two moieties of these adducts were preserved. Binding experiments, using CHO cells expressing the human SP-specific NK1 receptor, demonstrated the functionality of all peptidylcyclodextrin derivatives, which exhibited IC50 values in a 10(-9)-10(-7) M range. All compounds were able to induce a pharmacological response, triggering phosphatidylinositol turnover with EC50 values in the same range as the natural ligand. Moreover, autoradiography analysis of rat spinal corn sections proved that [125I]SP binding was dose-dependently displaced by one selected compound (a gamma-CD-SP), showing a similar affinity of this adduct for the rat neurokinin 1 receptor. Our observations demonstrate that these peptidylcyclodextrins efficiently target NK1 receptor-expressing cells.

A monoclonal antibody directed against the neurokinin-1 receptor contains a peptide sequence with similar hydropathy and functional properties to substance P, the natural ligand for the receptor.

Monoclonal antibody (mAb) PS12, obtained using the complementary peptide methodology, mimics the neuropeptide substance P (SP) in recognizing the SP-binding domain of the neurokinin-1 receptor (NK1R) and eliciting production of polyclonal antibodies cross-reacting with SP with a high affinity (Déry et al., 1997. J. Neuroimmunol. 76, 1-9). The aim of the present study was to investigate which structural features of mAb PS12 might account for this molecular mimicry. Cloning and sequencing of variable regions of both light (VL) and heavy (VH) chains of this 'SP-like' antibody did not indicate any primary sequence homology between SP and any antibody region. Instead, they revealed a striking similarity between the hydropathic profile of SP and that of an 11-amino-acid region in the light chain encompassing the second complementarity determining region (CDR2). When applied to CHO cells expressing the human NK1R, a synthetic extended 17-amino-acid peptide (denoted CDR2L) corresponding to this VL region inhibited the high-affinity binding of radiolabeled SP and antagonized the SP-induced inositol phosphate production. Moreover, a re-examination of the sequences of several antibodies that previously served in the design of CDR-derived bioactive peptides indicated that these antibodies also carried the hydropathic image of the respective ligands that they mimic. In agreement with previous observations on artificial synthetic peptides, our data thus suggest that the molecular mimicry between natural proteins (i.e. antibody and hormone, for example) could be understood on a structural level directly related, at least in part, to hydropathic homology. These results could then guide the search for bioactive paratope-derived peptides of potential pharmacological interest. We also observed inverse hydropathy between multiple CDRs of mAb PS12 (including CDR3H and CDR3L) and the peptide epitope, confirming the importance of hydropathic complementarity in antigen-antibody interactions.

Muscarinic M2-receptors in rat thoracic dorsal root ganglia.

The occurrence and distribution of the muscarinic M2-receptor subtype (M2R) was investigated in rat thoracic dorsal root ganglia (DRG). Messenger RNA for M2R was demonstrated by RT-PCR in total RNA from DRG. Immunoreactivity to M2R-protein was localized to 26% of sensory neurons, the majority of them (85%) belonging to the size class of 25-40 microm in diameter. Double-labeling (immuno)histochemistry revealed that all M2R-immunoreactive neurons bind the lectin, I-B4, whereas they are generally devoid of substance P-immunoreactivity. These data show the presence of M2R on a subpopulation of presumably nociceptive primary afferent neurons, thereby extending previous pharmacological and electrophysiological studies that indicated a role of M2R and/or M4R in inhibition of calcium channel currents in rat sensory neurons (Wanke, E., Bianchi, L., Mantegazza, M., Guatteo, E., Macinelli, E. and Ferroni, A., Muscarinic regulation of Ca2+ currents in rat sensory neurons: channel and receptor types, dose-response relationships and cross-talk pathways. Eur. J. Neurosci., 6 (1994) 381-391).

Identification in the NK1 tachykinin receptor of a domain involved in recognition of neurokinin A and septide but not of substance P.

The three mammalian tachykinins, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), exert their physiological effects through specific receptors, NK1, NK2 and NK3, respectively. However, homologous binding studies have recently demonstrated that, contrary to the generally accepted belief, NKA could bind NK1 receptor with high affinity (Hastrup and Schwartz, 1996). Using COS-7 cells expressing the human NK1 receptor, we show that two simultaneous point mutations (E193L and V195R) in a restricted five amino acid sequence (the (193-197) region), selected because of its hydropathic complementarity with the common C-terminal extremity of tachykinins, abolish both the high-affinity binding and highly potent biological activity of NKA, without affecting those of SP. In addition, the same mutations also suppressed the high functional activity of septide, a synthetic SP atypical agonist ([pGlu6-Pro9] SP 6-11). These results suggest that the (193-197) region, located at the end of the second extracellular loop of the receptor, could be part of a common high-affinity binding domain for both NKA and septide, distinct from the SP binding site.

Localization of the neurokinin 1 (NK-1) receptor in the human antrum and duodenum.

A newly available antibody to the neurokinin-1 receptor (NK-1r) has made it possible to determine the distribution of the NK-1r receptor in human tissue. In the present study the distribution of the NK-1r and substance P have been determined in the human antrum and duodenum by immunocytochemistry. The NK-1r was present on myenteric and submucosal neurons and nerve fibers of the gastro-enteric nervous system. In addition, the receptor was present on spindle-shaped cells in the circular muscle layer, endothelial cells and a population of mucosal cells. In the submucosal plexus NK-1r immunoreactive neurons were surrounded by substance P containing fibers. These results indicate an extensive cellular expression of the NK-1r in the human antrum and duodenum.

Substance P receptor immunodetection in the spinal cord: comparative use of direct anti-receptor antibody and anti-complementary peptide antibody.

The immunolocalization of substance P (SP) receptors was compared in the rat spinal cord using either a direct anti-substance P NK1-receptor antibody (anti-SPR) or an anti-complementary peptide antibody (anti-CP). The first antibody recognizes an intracellular epitope, the C-terminal tail of the NK1-receptor. The second antibody recognizes an extracellular epitope located at or near the ligand-binding domain because anti-CP antibody and SP were previously shown to compete for binding to the receptor. At the light microscope level, it was observed that anti-CP antibody labels both laminae I and II of the dorsal horn, while anti-SPR antibody labels exclusively lamina I, except at the lumbar level. This could suggest that spinal NK1 receptors are heterogeneous. Anti-SPR antibodies may recognize an NK1 receptor subclass confined to lamina I. Conversely, anti-CP antibody may recognize either another receptor subclass or two different subclasses present in laminae I and II. At the electron microscope level, labeling was localized either on the intracellular or the extracellular face of the plasma membrane depending on the location of the epitope recognized by both antibodies on the transmembrane receptor. However, using either antibody, the ultrastructural labeling was found at non-junctional sites, suggesting that SP may act in a non-synaptic manner on all putative receptor subclasses.

Importance of hydropathic complementarity for the binding of the neuropeptide substance P to a monoclonal antibody: equilibrium and kinetic studies.

In a previous study (Boquet et al., 1995, Molec. Immunol. 32, 303-308) we observed remarkable inversions of hydropathic profiles between complementarity determining regions (CDRs) of an anti-substance P monoclonal antibody (SP31) and the corresponding 5 amino acid C-terminal peptide epitope. Here we demonstrate the importance this hydropathic complementarity by measuring the immunoreactivity of SP-related peptides which have been modified in their C-terminal parts so that hydropathic profile has been conserved (by substituting amino acids in the epitope) or modified (by mixing the sequence of amino acids in the epitope). Experiments performed in equilibrium conditions using a competitive enzyme immunoassay showed that most of the peptides conserving the hydropathic profile of SP epitope were recognized by mAb SP31 (even if marked variations in affinity were observed), while those for which the hydropathic profile was modified exhibited very low or undetectable affinity. The kinetic parameters (ka and kd) of peptide-antibody interactions were determined using Surface Plasmon Resonance technology (BIACORE 2000). These measurements showed that all peptides recognized by mAb SP31 had similar association rate constants (close to 2 x 10[5] M[-1] s[-1]), differences in binding affinities essentially resulting from differences in dissociation rate constants (ranging from 1.61 x 10[-4] to 1.15 x 10[-1] s[-1]). From these results, it was concluded that hydropathic complementarity between the epitope and the paratope could be a necessary but not sufficient condition for establishing high-affinity binding. We hypothesize that hydropathic interactions may play a critical role during the first contacts between antibody CDRs and the peptide, possibly by favouring reorganization of water molecules at the antibody-peptide interface.

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P.

Monoclonal antibodies to the binding site of the NK1 receptor for the neuropeptide substance P were produced in mice using the complementary or antisense peptide methodology. Among several anti-peptide monoclonal antibodies, we selected the mAb12 antibody which specifically crossreacted, through its paratope, with a binding site present on membranes from rat parotid gland cells, with an affinity close to 2 x 10(-7) M and with membranes from CHO cells expressing human brain NK1 receptors. Immunocytochemical investigations using mAb12 revealed immunostaining whose distribution in the dorsal horns of rat spinal cord fits well with the known location of NK1 receptors. In both biochemical and immunocytochemical experiments, the competition occurring between the antibody and substance P, or a substance P-protein conjugate, indicates that mAb12 recognizes a membrane epitope located at or near the substance P binding domain on the NK1 receptor. Immunization of mice with mAb12 led to the production of specific anti-substance P antibodies, again suggesting that mAb12 shares common structural features with the neuropeptide. This monoclonal antibody can now be used in further biochemical or cytochemical characterizations of NK1 receptors. Owing to its fine specificity, mAb12 could also serve as a molecular model for designing peptides, possibly displaying pharmacological properties in the various processes in which substance P is involved, e.g. immunomodulation, inflammation or chronic pain.

Systemic capsaicin pretreatment fails to block the decrease in food-motivated behavior induced by lipopolysaccharide and interleukin-1beta.

The physiological and behavioral disturbances observed during an infection can be reproduced by systemic administration of proinflammatory cytokines (e.g., interleukin (IL)-1, IL-6, tumor necrosis factor-alpha) or lipopolysaccharide (LPS), a potent inducer of these cytokines. It is now well established that these molecules induce their effects by acting centrally, however, the mechanisms by which they reach central structures are not clear. We have earlier proposed that the humoral immune message is converted to a central neural activation by the action of cytokines on peripheral terminations of afferent neurons. Subdiaphragmatic vagotomy abolishes several effects of peripherally injected IL-1beta and LPS (e.g., decreased food-motivated behavior and social exploration, central expression of cytokines). To further define the nature of the peripheral fibers implicated in this phenomenon, we used a potent sensory neurotoxin, capsaicin, to selectively destroy C-fiber afferents. Adult rats were injected I.P. with a total dose of 25 mg/kg capsaicin in a series of 10 injections over a 48-h period. Adult mice were injected I.P. with a total dose of 75 mg/kg in a series of seven injections over a 7-day period. Although capsaicin treatment altered visceral chemosensory function, corneal and pain sensitivity, vagal-mediated anorexic effects of cholecystokinin, and depleted levels of substance P in the thoracic spinal cord, it was completely ineffective in blocking the decrease in food-motivated behavior induced by IL-1beta (4 microg/rat I.P. in rats) and LPS (250 microg/kg I.P. in rats and 400 microg/kg I.P. in mice). Thus, other afferents besides capsaicin-sensitive C-fibers appear to be involved in the transduction of cytokine effects during inflammatory and infectious events.

Use of conformationally constrained peptides for a topographical analysis of the combing site of a monoclonal anti-substance P antibody.

The topography of the binding site of a monoclonal anti-substance P antibody directed toward the C-terminal pentapeptide of substance P, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, was analyzed further using a wide range of constrained analogues of substance P. Results obtained in the present study show the following: (a) The binding subsites of Phe7 and Phe8 are large and deep, accommodating various side chains, including nonaromatic amino acids. (b) In contrast, the binding pockets for Gly-Leu-Met-NH2 appear more restrictive. Consequently, five residues in the peptide are necessary for the high binding affinity to the antibody, the C-terminal tripeptide determining the binding specificity. These data, which appear to contradict those previously published, illustrate the limits of conclusions drawn from studies generally carried out using exclusively Ala-substituted peptides. In addition, the present results indicate that the topography of the binding site of this monoclonal antibody differs from that of the specific substance P neurokinin-1 receptor, in agreement with the differences observed in the fine specificities of these two substance P binding macromolecules.

Immunoreactive substance P and calcitonin-gene-related peptide (CGRP) in rat milk and in human milk and infant formulas.

This study examined the presence of substance P and calcitonin-gene-related peptide (CGRP) immunoreactivities in various milks and infant formulas. Rat milk was obtained from lactating dams between parturition and weaning (0, 2, 5, 10, 15, and 20 d postpartum). Samples of human milk were obtained from seven multiparous, nonsmoking white women, and newborn infant formulas were purchased from local stores. Substance P and CGRP were measured by competitive enzyme immunoassay using acetylcholinesterase-peptide conjugates as tracers. In rats, substance P and CGRP were below detectable concentrations in amniotic fluid from the last day of gestation. In contrast, in milk the concentrations of substance P and CGRP-like immunoreactivities were high on the first day of lactation (3.1 +/- 0.2 and 23.1 +/- 1.5 micrograms/L, respectively), then dropped after day 2 (1.6 +/- 0.7 and 7.5 +/- 0.4 microgram/L, respectively) and remained fairly constant until weaning. Significant concentrations of substance P and CGRP were found in human milk (129.2 +/- 27 ng/L and 4.5 +/- 0.7 microgram/L, respectively, at 15 wk), but substance P or CGRP could not be detected in any of the formulas tested. These data show that milk contains high concentrations of immunoreactive substance P and CGRP. In rats the absence of peptides in amniotic fluid suggests that there is a flood of peptides into the gastrointestinal tract of neonates when suckling is initiated. Significant concentrations of substance P and CGRP in human milk but not in infant formulas may therefore have physiologic implications for neonatal nutrition.

Ultrastructural study of substance P receptors in the dorsal horn of the rat spinal cord using monoclonal anti-complementary peptide antibody.

A monoclonal antibody directed against a peptide (PS5) specified by RNA complementary to the mRNA coding for substance P (SP), was used to label SP receptors in the rat spinal cord as demonstrated by light and electron microscopy. An immunocytochemical method (avidin-biotin-peroxidase) was used on vibratome sections from rats perfused with paraformaldehyde. Immunoreactivity was observed principally in the two superficial layers of the dorsal horn, in lamina X and the region of motoneurons. The labeling was absent when the antibody was preincubated with the complementary peptide (PS5) used as immunogen. Competition between the anti-complementary peptide antibody and different ligands was tested by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors before addition of the antibody. A specific agonist (SP) or antagonist (spantide, RP 67580) at 10(-6)M led to total absence of labeling. These results indicate that under our experimental conditions, the anti-complementary peptide antibody recognizes a SP binding site in the rat spinal cord. Electron microscopic study of the two superficial laminae of the dorsal horn showed that immunolabeling was mainly localized extracellularly at apposing neuronal plasma membranes. It was mostly associated with axodendritic or axosomatic appositions. Occasionally labeling was observed between two axon terminals. In all cases, these appositions were non-junctional. Generally, neuronal processes involved in these appositions did not contain large granular vesicles. These observations suggest that SP may act in a diffuse, nonsynaptic manner probably on targets distant from SP release sites.

Prevaccination with diluted Freund adjuvant prevents the development of chronic pain and transient release of cerebrospinal fluid substance P in adjuvant-induced arthritis in rats.

Parallel time courses of preclinical and behavioural pain-related parameters and levels of substance P-like immunoreactivity in plasma (plasma-SPLI) and cerebrospinal fluid (CSF-SPLI) were studied in 2 groups of rats injected with an arthritogenic solution (concentrated Freund adjuvant) over a 9-week post-infection (PI) period; 1 group was pretreated with saline (control) and 1 pretreated with diluted Freund adjuvant (immunized). In control rats all symptoms of adjuvant-induced arthritis (AIA) developed while in immunized rats AIA symptoms were significantly reduced or did not appear. A significant increase in plasma-SPLI was obvious as early as the 2nd week PI and remained at this level in both groups of animals until the end of the 9-week PI observation period, but with a significantly higher increase in control versus immunized group at all stages. In contrast, CSF-SPLI transiently peaked only in the control group at 3 weeks PI whereas CSF-SPLI values did not differ from one week to another in both groups of rats. These results suggest that successive injections of diluted Freund adjuvant impairs the development of chronic inflammation and pain in AIA in rats, as well as the transient increase in SP release in CSF at 3 weeks PI, but not the long-lasting increased SP release in plasma. Since there is a clear dissociation between our biochemical and preclinical and behavioral data, this study does not provide evidence for the role of substance P as a possible biologic marker of chronic pain either in plasma or in CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

Antipeptide polyclonal antibodies that recognize a substance P-binding site in mammalian tissues: a biochemical and immunocytochemical study.

We used complementary peptide methodology to obtain antibodies against the receptor for the neuropeptide substance P, specifically directed at the ligand-binding domain. Rabbits were immunized with two distinct peptides derived from the sequence of the RNA complementary to the mRNA for substance P. Binding experiments revealed that antipeptide polyclonal antibodies were able to recognize, through their paratope, a specific binding site on the rat parotid cell membranes. Substance P and antibodies competed for this binding site, because preincubation of membranes in the presence of substance P significantly reduced antibody binding, and conversely, preincubation of membranes in the presence of antibodies partly inhibited the binding of radioiodinated substance P. Immunocytochemical experiments performed on the rat cervical spinal cord show that the distribution of labeling by antibodies is similar to that observed by conventional autoradiography using 125I-substance P. Here again, control experiments demonstrated that antibodies and substance P were competing for the same binding site on the spinal cord. These biochemical and immunocytochemical data indicate that antipeptide antibodies recognize a substance P membrane binding site in nervous and nonnervous mammalian tissues. This site is likely to correspond to the NK1 specific receptor for substance P.

A behaviorally active dose of lipopolysaccharide increases sensory neuropeptides levels in mouse spinal cord.

To assess whether peripheral immune stimuli activate sensory afferents at behaviorally active doses, we measured the effects of lipopolysaccharide (LPS) on the levels of sensory neuropeptides in the spinal cord. LPS (10 micrograms/mouse i.p.) increased the levels of substance P, neurokinin A, and calcitonin gene-related peptide in the spinal cord, the maximum being observed 1 hr post-injection. Pretreatment with indomethacin at a dose (5 mg/kg i.p.) which completely blocked the decrease in food-motivated behavior induced by LPS abrogated this effect.

Time course of variations in rabbit cerebrospinal fluid levels of calcitonin gene-related peptide- and substance P-like immunoreactivity in experimental subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Cerebral vasospasm after subarachnoid hemorrhage may result partially from the imbalance between vasodilator and vasoconstrictor factors. The vasodilator peptides substance P and calcitonin gene-related peptide contained in the trigeminovascular system are involved in the vasomotor phenomenon occurring after subarachnoid hemorrhage. The delayed arterial narrowing may reflect the time course of the release of these peptides. Therefore, we followed the time course of the changes in cerebrospinal fluid immunoreactivity of substance P and calcitonin gene-related peptide in a model of experimental subarachnoid hemorrhage. METHODS: Cerebrospinal fluid samples were taken in the basal state and at 30 minutes, 24 hours, and 3 days after a single injection of 1 mL autologous arterial blood into the cisterna magna of rabbits using a percutaneous suboccipital route. Substance P-like and calcitonin gene-related peptide-like immunoreactivities were determined in centrifuged cerebrospinal fluid and plasma by use of enzyme immunoassay. RESULTS: Early (30 minutes) after induced subarachnoid hemorrhage, there was a large increase in cerebrospinal fluid substance P-like immunoreactivity (P < .01) and calcitonin gene-related peptide-like immunoreactivity (P < .01). Arterial and hemorrhagic cerebrospinal fluid levels of substance P-like immunoreactivity were different (P < .03), indicating that the increased cerebrospinal fluid level did not result only from the blood contamination. Twenty-four hours after induced subarachnoid hemorrhage, the immunoreactivities of substance P and calcitonin gene-related peptide remained significantly higher than the basal level (P < .01). At day 3, both immunoreactivities had decreased to a level nonsignificantly different from the basal level. CONCLUSIONS: The early high values of the cerebrospinal fluid immunoreactivities for substance P and calcitonin gene-related peptide, apart from the contamination by arterial blood, probably resulted from the depletion of neurotransmitter peptides from the trigeminovascular fibers.

Neurochemistry, connectivity and plasticity of small intensely fluorescent (SIF) cells in the rat superior cervical ganglion.

Applying double-labelling immunofluorescence, the peptide content of solitary and clustered small intensely fluorescent (SIF) cells, identified by an antiserum to a selective membrane glycoprotein marker, synaptophysin, was correlated with the presence of catecholamines in the rat superior cervical ganglion. Most of synaptophysin-immunoreactive solitary and clustered SIF cells apparently contained dopamine (indicated by tyrosine hydroxylase-TH) but not noradrenaline (indicated by dopamine-beta-hydroxylase-DBH). Frequently, immunoreactivities for substance P or rarely, neuropeptide Y were colocalized in TH-immunolabeled cells of both types. Immunostaining for vasoactive intestinal polypeptide was found only in solitary SIF cells and was visible in TH-immunoreactive, as well as in TH-nonreactive cells. Very few solitary SIF cells were TH- and DBH-immunoreactive. Solitary and clustered SIF cells, as a rule, were encircled by leu-enkephalin-positive fibres which were also met-enkephalin-arg6-phe7-immunoreactive, indicating proenkephalin as precursor. SIF cells were additionally approached by varicose fibres which contained immunoreactivity for calcitonin gene-related peptide (CGRP) but not for enkephalins. As observed by immuno-electronmicroscopy, fibres that were immunostained for leu-enkephalin or CGRP, deeply invaginated into SIF cell somata. In addition to close membrane appositions, CGRP-immunolabeled fibres exhibited efferent synaptic contacts wih elements of SIF cell clusters. SIF cells were non-reactive to enkephalin-antisera in control ganglia and after transection of the postganglionic nerves (axotomy); but both types exhibited leu-enkephalin in preganglionically transected ganglia (decentralization) in which enkephalin-immunoreactive fibre baskets were absent. Synthesis of enkephalin in SIF cells after decentralization was confirmed by in situ hybridization demonstrating intracytoplasmic proenkephalin messenger-RNA. The findings are indicative for a differential neurochemical equipment of SIF cells in the rat superior cervical ganglion, which mainly is independent to a topographical classification. Moreover, they demonstrate the involvement of two neuropeptides in preganglionic SIF cell innervation. Finally, the observations indicate the capacity of SIF cells for proenkephalin-expression in response to preganglionic denervation.

Nitric oxide synthase in cardiac nerve fibers and neurons of rat and guinea pig heart.

Participation of nitric oxide (NO) in the autonomic innervation of rat and guinea pig hearts was investigated by applying the NADPH diaphorase technique and immunohistochemistry with NO synthase antiserum. We present evidence that NO synthase is localized in cardiac ganglion cells and nerve fibers innervating the sinuatrial and atrioventricular nodes, the myocardium, local neurons, coronary arteries, and pulmonary vessels, suggesting an involvement of NO in neurogenic heart rate regulation, myocardial cell function, neuronal transmission in cardiac ganglia, and coronary as well as pulmonary vasodilation.