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(Circulation Research. 1996;78:863-869.)
© 1996 American Heart Association, Inc.

Articles

Histamine H₃-Receptor–Mediated Inhibition of Calcitonin Gene-Related Peptide Release From Cardiac C Fibers

A Regulatory Negative-Feedback Loop

Michiaki Imamura, Neil C.E. Smith, Monique Garbarg, Roberto Levi

From the Department of Pharmacology (M.I., N.C.E.S., R.L.), Cornell University Medical College, New York, NY, and Unité de Neurobiologie et Pharmacologie (M.G.), INSERM U109, Paris, France.

Correspondence to Roberto Levi, MD, Department of Pharmacology, Cornell University Medical College, 1300 York Ave, New York, NY 10021. E-mail rlevi@med.cornell.edu.

	Abstract

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Abstract Antidromic stimulation of cardiac sensory C fibers releases calcitonin gene-related peptide (CGRP), which increases heart rate, contractility, and coronary flow. C-fiber endings are closely associated with mast cells, and CGRP may release mast-cell histamine. Because prejunctional histamine H₃-receptors inhibit transmitter release from autonomic nerves, we tested the hypothesis that H₃-receptors modulate CGRP release in the heart. CGRP released by bradykinin in the electrically paced guinea pig left atrium and by capsaicin in the spontaneously beating isolated heart caused marked positive inotropic and chronotropic effects, respectively. Capsaicin significantly enhanced the overflow of CGRP (fivefold) and histamine (twofold) into the coronary effluent. All of these effects were prevented by prior chemical destruction of C fibers in vivo. The H₃-receptor agonist imetit attenuated the inotropic response to bradykinin by 50%. Imetit also decreased the capsaicin-induced tachycardia and the increase in CGRP overflow by 50%. Imetit, however, did not modify the response to exogenous CGRP. The effects of imetit were blocked by the H₃-receptor antagonist thioperamide. Notably, thioperamide by itself potentiated the capsaicin-evoked increases in heart rate and CGRP overflow (by 25% and 50%, respectively). Thus, our findings identify a negative-feedback loop, whereby CGRP releases histamine from cardiac mast cells and histamine in turn inhibits CGRP release by activating H₃-receptors on C-fiber terminals. Because CGRP release is augmented in pathophysiological conditions, such as septic shock, heart failure, and acute myocardial infarction, modulation of CGRP release may be clinically relevant.

Key Words: histamine H₃-receptors • calcitonin gene-related peptide release • bradykinin • sensory C-fiber endings • mast cells

	Introduction

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The heart is richly innervated by peptidergic sensory nerves.^{1 2} Many agents, including capsaicin, the pungent ingredient in red peppers of the genus Capsicum,^{1 2 3} and endogenous mediators, such as bradykinin,⁴ stimulate sensory nerves, which send impulses both orthodromically and antidromically. Antidromic impulses elicit the release of several peptides from sensory nerve endings. Among these are calcitonin gene-related peptide (CGRP), substance P, and tachykinins.²

CGRP has both positive chronotropic and inotropic effects and is a potent vasodilator.^{1 2 5} Because marked changes in plasma CGRP concentration occur in congestive heart failure,⁶ septic shock,⁷ and acute myocardial infarction,⁸ CGRP may play a role in such states. Accordingly, an understanding of the mechanisms that modulate CGRP release could prove important.

Morphological and functional studies suggest an interaction between sensory fibers and mast cells.^{9 10 11 12} Indeed, neuropeptide expression has been demonstrated in nerve fibers closely associated with mast cells in various tissues.⁹ Notably, CGRP releases histamine from mast cells.¹⁰ Further, by activating inhibitory prejunctional H₃-receptors, histamine inhibits the release of various neurotransmitters from adrenergic^{13 14 15 16} and cholinergic¹⁷ nerve endings.

Recently, one of us suggested the presence of inhibitory H₃-receptors on sensory nerve endings in the rat lung and spleen,¹¹ whereas others have found that H₃-receptors negatively modulate the release of substance P from sensory nerve endings in rat skin.¹⁸ Thus, we questioned whether mast cells in the heart functionally interact with sensory peptidergic fibers and whether CGRP may modulate its own release via a histamine-mediated negative-feedback loop. We report that in the guinea pig heart, CGRP released from C fibers stimulates mast cells to release histamine. Histamine then inhibits the release of CGRP via inhibitory H₃-receptors on C-fiber endings.

	Materials and Methods

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Inotropic Response of the Left Atrium
Male Hartley guinea pigs (Hilltop) weighing 250 to 300 g were killed by cervical dislocation under light anesthesia with CO₂ vapor. The rib cage was rapidly dissected away, exposing the heart. The heart was excised and placed in a dissection dish filled with oxygenated Krebs-Henseleit solution of the following composition (mmol/L): NaCl 118.12, KCl 4.83, CaCl₂ 2.5, MgSO₄ 2.37, KH₂PO₄ 1.0, NaHCO₃ 24.99, and glucose 11.1. The left atrium was carefully dissected and mounted vertically in a 10-mL double-walled Pyrex organ bath filled with Krebs-Henseleit solution, gassed continuously with 95% O₂/5% CO₂ (pH 7.4), and maintained at 30°C. The upper end of the left atrium was connected by a silk thread to a force transducer (model FT03, Grass Instruments). A resting tension of 1 g was applied. The lower end of the preparation was clamped between two small plastic plates containing a pair of stimulating platinum electrodes. The left atrium was continuously paced at 1 Hz with rectangular pulses, 1 millisecond in duration and intensity 20% above threshold voltage, generated by a stimulator (model S88, Grass Instruments) coupled to an isolation unit (model SIU5, Grass Instruments). The preparations were allowed to stabilize for at least 60 minutes. Atropine (1 µmol/L) was added to the incubation medium to prevent activation of muscarinic receptors by acetylcholine released from parasympathetic nerve terminals. Enalaprilat (10 µmol/L) was also added to prevent bradykinin metabolism by kininase II. Various receptor agonists and antagonists were added to the organ bath 20 minutes before bradykinin or CGRP administration.

Isolated Heart Perfusion
Guinea pigs were killed by cervical dislocation under light anesthesia. The rib cage was rapidly dissected away, exposing the heart. A cannula was inserted into the aorta, and the heart was perfused through this cannula at constant pressure (40 cm H₂O) with Krebs-Henseleit solution equilibrated with 95% O₂/5% CO₂ and maintained at 37°C (pH 7.4). The heart was transferred to a Langendorff apparatus, where it was suspended via the aortic cannula and allowed to beat spontaneously. The sinus rate was determined from a surface electrogram recorded from the right atrium and the left ventricle, recorded at a paper speed of 50 mm/s on a Grass model 7WC8PA oscillograph. Hearts were perfused for 30 minutes before experimentation to stabilize the rate of beating. Coronary effluent was collected every 5 minutes over a 20-minute period: one collection before and three collections during perfusion with capsaicin. The collected effluents were weighed and subsequently analyzed for histamine and CGRP.

Capsaicin Pretreatment In Vivo
Guinea pigs were anesthetized with pentobarbital (25 mg/kg IP) and artificially ventilated with a rodent respirator (Harvard Apparatus). Theophylline (100 mg/kg IP) was given to counteract respiratory impairment. Capsaicin (total dose, 50 mg/kg SC) was administered 6 hours before in vitro experimentation; this has been shown to cause a total loss of CGRP-containing nerves within the heart.^{1 3}

Preparation/Concentration of Samples
Acetic acid was added to 6 mL of each sample of coronary effluent to yield a final concentration of 0.2 mol/L. Acidified samples (5 mL) were desalted using SEP-PAK C-18 cartridges (Waters), lyophilized, and resuspended into 250 µL of CGRP assay matrix buffer. Assays for histamine and/or CGRP were performed on the concentrated samples.

Histamine and CGRP Assays
The concentrated samples were stored at -20°C for a short period of time (ie, <2 weeks). Samples were then thawed and assayed for histamine and/or CGRP content, with the use of two commercial enzyme immunoassay (EIA) kits (histamine EIA kit from Immunotech International Co, Inc, and human CGRP EIA kit from Peninsula Laboratories, Inc). The detection limits of the kits were 0.02 pmol and 2 fmol for histamine and CGRP, respectively.

Statistics
Values are given as mean±SEM. Analysis by Student's t test was performed for paired or unpaired observations. Comparison of more than two groups was performed by ANOVA followed by Bonferroni's t test. A value of P<.05 was considered statistically significant.

Drugs
The following drugs were used: atropine sulfate, bradykinin, capsaicin, pyrilamine maleate, and theophylline (Sigma Chemical Co); CGRP and CGRP_8-37 (Peninsula Laboratories); capsazepine (Cookson Chemicals); enalaprilat (a gift of Merck Sharp & Dohme Research Laboratories); HOE 140 (a gift of Hoechst AG); imetit (a gift of Dr C.R. Ganellin, University College London); pentobarbital sodium solution (Fort Dodge Laboratories); L-propranolol hydrochloride (a gift of Wyeth-Ayerst Laboratories); thioperamide maleate (Research Biochemicals International); and tiotidine (a gift of Stuart Pharmaceuticals). L-Propranolol, thioperamide, and tiotidine were dissolved in dimethyl sulfoxide; capsaicin was dissolved in 5% ethanol. Further dilutions were made with distilled water; at the concentrations used, dimethyl sulfoxide and ethanol affected neither cardiac function nor mediator release.

	Results

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Inotropic Response of the Guinea Pig Left Atrium to Bradykinin: Mediation by CGRP and Modulation by Histamine H₃-Receptors
In the presence of bradykinin (1 µmol/L), the positive inotropic response of the guinea pig left atrium to direct electrical stimulation (square pulses; voltage, 20% above threshold; duration, 1 millisecond; frequency, 1 Hz) slowly increased to a 3.3±0.4-fold maximum in 2 to 3 minutes (mean±SEM, n=11). Propranolol (1 µmol/L) did not affect the bradykinin-induced inotropic effect (ie, contractility increased to a 3.0±0.4-fold maximum; n=3). In contrast, the selective bradykinin B₂-receptor antagonist HOE 140 (30 nmol/L) completely blocked the response to bradykinin (Fig 1A), indicating that B₂-receptors mediate this effect. Furthermore, when left atria were isolated from guinea pigs pretreated in vivo with capsaicin (50 mg/kg, 6 hours earlier), a procedure known to destroy afferent sensory C fibers,¹ bradykinin's inotropic effect was almost completely abolished (Fig 1B). Since this suggested an obligatory role of C fibers in the inotropic effect of bradykinin, we next investigated whether bradykinin's effect is mediated by CGRP released from C fibers. As shown in Fig 1C, the CGRP receptor antagonist CGRP_8-37 (1 µmol/L) markedly antagonized the inotropic effect of bradykinin (1 µmol/L) as well as that of CGRP (30 nmol/L) (Fig 1D). This suggested that CGRP released from C fibers mediates the inotropic effect of bradykinin.

View larger version (22K): [in this window] [in a new window]   Figure 1. Bar graphs showing that the positive inotropic effect of bradykinin (BK) on the electrically paced (1 Hz) guinea pig left atrium is mediated by the release of calcitonin gene-related peptide (CGRP) from capsaicin-sensitive sensory C fibers. A, The specific BK B2-receptor antagonist HOE 140 (30 nmol/L) blocks the positive inotropic effect of BK (1 µmol/L). Basal contractile force in the absence and presence of HOE 140 was 0.46±0.17 (n=11) and 0.44±0.08 g (n=4), respectively. B, The inotropic effect of BK (1 µmol/L) is inhibited by in vivo pretreatment with capsaicin (50 mg/kg, 6 hours). Basal contractile force in control left atria was 0.46±0.17 g (n=11) and 0.79±0.10 g (n=7) after capsaicin administration in vivo. C, The CGRP receptor antagonist CGRP8-37 (1 µmol/L) inhibits the inotropic effect of BK (1 µmol/L). Basal contractile force in the absence and presence of CGRP8-7 was 0.46±0.17 (n=11) and 0.58±0.10 g (n=6), respectively. D, The CGRP receptor antagonist CGRP8-37 (1 µmol/L) inhibits the inotropic effect of exogenous CGRP (30 nmol/L). Basal contractile force in the absence and in the presence of CGRP8-37 was 0.45±0.07 and 0.48±0.07 g, respectively (n=6). Each bar represents the maximum increase in contractile force (mean±SEM) elicited by BK or CGRP. *P<.05 and **P<.01 vs control value by paired or unpaired Student's t test.

The specific histamine receptor antagonists pyrilamine (anti-H₁; 300 nmol/L; pA₂, 1 nmol/L¹⁹ ), tiotidine (anti-H₂; 3 µmol/L; K_b, 0.03 µmol/L²⁰ ), and thioperamide (anti-H₃; 300 nmol/L; K_i, 4.3 nmol/L²¹ ) all failed to modify the inotropic effect of bradykinin (Fig 2A), thus excluding a histamine mediation of this effect. In contrast, the selective histamine H₃-receptor agonist imetit (100 nmol/L) inhibited the inotropic effect of bradykinin by 50% (Fig 2B). The specific histamine H₃-receptor antagonist thioperamide (300 nmol/L) prevented the inhibitory effect of imetit. Notably, imetit did not affect the inotropic effect of exogenous CGRP: the CGRP-induced increase in left atrial contractile force was 390±29% and 380±71% in the absence (n=10) and presence of imetit (n=6), respectively.

View larger version (32K): [in this window] [in a new window]   Figure 2. Bar graphs showing that the positive inotropic effect of bradykinin (BK) on the electrically paced (1 Hz) guinea pig left atrium is not mediated by histamine but is attenuated when histamine H3-receptors are activated with imetit. A, The positive inotropic effect of BK (1 µmol/L) is unaffected by the histamine H1-receptor antagonist pyrilamine (Pyril, 300 nmol/L), the H2-receptor antagonist tiotidine (Tiot, 3 µmol/L), and the H3-receptor antagonist thioperamide (Thiop, 300 nmol/L). B, The H3-receptor agonist imetit (100 nmol/L) attenuates the inotropic effect of BK (1 µmol/L), and the H3-receptor antagonist Thiop (300 nmol/L) prevents imetit's effect. Each bar represents the maximum increase in contractile force (mean±SEM) induced by BK. Basal contractile forces were as follows: BK, 0.46±0.17 g (n=11); Pyril, 0.33±0.07 g (n=4); Tiot, 0.36±0.11 g (n=5); Thiop, 0.39±0.10 g (n=5); imetit, 0.62±0.07 g (n=8); and imetit+Thiop, 0.37±0.08 g (n=7). *P<.05 and P<.05 vs control and imetit, respectively, by ANOVA.

Chronotropic Response of Isolated Guinea Pig Heart to C-Fiber Stimulation: Mediation by CGRP and Modulation by Histamine H₃-Receptors
The findings in the guinea pig left atrium implied that histamine H₃-receptors may modulate the bradykinin-induced release of CGRP from sensory C fibers. To define the function of these modulatory H₃-receptors, we next investigated C-fiber activation in the entire guinea pig heart and assessed both CGRP and histamine release and associated functional responses.

As shown in Fig 3, continuous perfusion of the spontaneously beating isolated guinea pig heart with capsaicin at 30 and 100 nmol/L elicited a marked concentration-dependent tachycardia (ie, heart rate increased 25% and 40% after 10 and 5 minutes of capsaicin perfusion at 30 and 100 nmol/L, respectively). Neither the ß-adrenergic receptor antagonist propranolol (1 µmol/L) nor the muscarinic receptor antagonist atropine (1 µmol/L) affected the capsaicin-induced tachycardia (ie, the maximum increases in heart rate were 51±19 and 51±23 bpm [n=5 or 6] in the presence of atropine and propranolol, respectively). In contrast, when hearts were isolated from guinea pigs pretreated in vivo with capsaicin (50 mg/kg, 6 hours earlier) in order to destroy sensory C fibers, subsequent perfusion with capsaicin in vitro elicited no tachycardia (Fig 3A and 3B). Furthermore, in the presence of the capsaicin receptor antagonist capsazepine (10 µmol/L),²² the positive chronotropic effect of capsaicin was almost completely inhibited (Fig 3C). Notably, neither capsazepine nor pretreatment with capsaicin in vivo affected the basal rate of the isolated heart (see legend to Fig 3). These findings suggested that a neurotransmitter released from sensory C fibers, possibly CGRP, may mediate the chronotropic effect of capsaicin.

View larger version (20K): [in this window] [in a new window]   Figure 3. Graphs showing that the positive chronotropic effect elicited in isolated guinea pig hearts by continuous perfusion of capsaicin (30 and 100 nmol/L [control]) is inhibited by the capsaicin antagonist capsazepine (10 µmol/L) or prevented by prior destruction of sensory C fibers by the in vivo administration of capsaicin (50 mg/kg, 6 hours earlier; pretreated). Points represent the increase in heart rate at the corresponding time (mean±SEM). A, Basal heart rates were as follows: control, 179±12 bpm (n=4); capsaicin pretreatment, 189±8 bpm (n=8). B, Basal heart rates were as follows: control, 191±9 bpm (n=5); capsaicin pretreatment, 200±10 bpm (n=4). In panels A and B, control guinea pigs were anesthetized and ventilated as the treated guinea pigs; they also received theophylline and the capsaicin solvent (see "Materials and Methods" for details). C, Basal heart rates were as follows: control, 194±3 bpm (n=5); capsazepine, 180±3 bpm (n=4).

Because CGRP is known to release histamine from mast cells¹⁰ and because histamine has positive chronotropic effects mediated by H₂-receptors,²³ we next assessed the possible contribution of endogenous histamine to capsaicin-induced tachycardia. As shown in Fig 4, perfusion of isolated guinea pig hearts with capsaicin (30 nmol/L) elicited an increase in the overflow of both CGRP and histamine into the coronary effluent. CGRP overflow increased approximately twofold, threefold, and fivefold after 5, 10, and 15 minutes of capsaicin perfusion, respectively. Histamine overflow increased by 30% after 5 minutes of perfusion with capsaicin, reached an 80% maximum in the next 5 minutes, and remained 50% higher than basal level after 15 minutes of capsaicin perfusion. Notably, when hearts were isolated from guinea pigs pretreated in vivo with capsaicin (50 mg/kg, 6 hours earlier) in order to destroy sensory C fibers, subsequent perfusion with capsaicin in vitro failed to elicit any increase in CGRP and histamine overflow (Fig 4).

View larger version (20K): [in this window] [in a new window]   Figure 4. Bar graphs showing the increases in CGRP (A) and histamine overflow (B) into the coronary effluents of isolated guinea pig hearts elicited by the continuous perfusion of capsaicin (30 nmol/L). Hearts were isolated from control guinea pigs or from guinea pigs subjected to the in vivo administration of capsaicin (50 mg/kg SC, 6 hours earlier; pretreated). Note that the increases in CGRP and histamine overflows are prevented by prior destruction of C fibers with capsaicin in vivo. Bars (mean±SEM, n=7 for each experimental group) represent the percent changes in CGRP and histamine overflow for each 5-minute interval, before and during capsaicin perfusion. Basal overflow levels are reported in the legends to Figs 5 and 6. **P<.01 vs control by unpaired t test.

Shown in Fig 5 are the time courses of the tachycardia and increased histamine overflow elicited by capsaicin perfusion. The histamine H₂-receptor antagonist tiotidine (3 µmol/L) did not affect the basal heart rate but partially inhibited the capsaicin-induced tachycardia by 30% (Fig 5A). Thus, activation of H₂-receptors by released histamine appeared to contribute only in minimal part to the chronotropic effects of capsaicin.

View larger version (19K): [in this window] [in a new window]   Figure 5. Graphs showing that the tachycardia elicited by perfusion of isolated guinea pig hearts with capsaicin (30 nmol/L), which is associated with an increase in histamine overflow into the coronary effluent, is only partially inhibited by the histamine H2-receptor antagonist tiotidine (3 µmol/L). A, Points represent increases in heart rate (mean±SEM) in the absence (control, n=10) and in the presence of tiotidine (n=4). Basal heart rates were as follows: control, 194±5 bpm; tiotidine, 187±5 bpm. *P<.05 and **P<.01 vs corresponding control value by unpaired Student's t test. B, Bars represent the amount of histamine detected in each 5-minute period, before and during perfusion with capsaicin (mean±SEM, n=7). *P<.05 and **P<.01 vs precapsaicin value by paired Student's t test.

We next investigated whether histamine H₃-receptors modulate CGRP release from sensory C fibers in the isolated guinea pig heart. As shown in Fig 6A, the histamine H₃-receptor agonist imetit (100 nmol/L) inhibited the capsaicin-induced tachycardia by 50%. Imetit also inhibited the capsaicin-induced increase in CGRP overflow into the coronary effluent by 50% (Fig 6B). Both of these inhibitory effects were blocked by the H₃-receptor antagonist thioperamide (300 nmol/L). Notably, thioperamide by itself potentiated the capsaicin-induced tachycardia as well as the increase in CGRP overflow, by 25% and 50%, respectively (Fig 6A and 6B). Neither imetit nor thioperamide affected basal heart rate (see legend to Fig 6) and basal CGRP overflow (Fig 6B).

View larger version (33K): [in this window] [in a new window]   Figure 6. Graphs showing that histamine H3-receptor activation negatively modulates the tachycardia and enhanced CGRP overflow elicited by capsaicin perfusion (30 nmol/L) in isolated guinea pig hearts. The concentration of the H3-receptor agonist imetit was 100 nmol/L and that of the H3-receptor antagonist thioperamide was 300 nmol/L. A, Points represent the increases in heart rate (mean±SEM) in control conditions (n=10) and in the presence of imetit alone (n=11), imetit and thioperamide together (n=11), and thioperamide alone (n=8). Basal heart rates were as follows: control, 197±5 bpm; imetit, 208±5 bpm; imetit and thioperamide together, 196±4 bpm; and thioperamide alone, 204±6 bpm. *P<.05 vs corresponding control value by ANOVA. B, Each bar represents the amount of CGRP detected in each 5-minute period, before and during perfusion with capsaicin (mean±SEM) in control conditions (n=7) and in the presence of imetit alone (n=5), imetit and thioperamide together (n=5), and thioperamide alone (n=4). *P<.05 vs corresponding control value by ANOVA. P<.05 and P<.001 vs precapsaicin value by paired t test.

	Discussion

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Our results identify a negative-feedback loop, which functions to modulate CGRP release from cardiac sensory C fibers. CGRP released by C-fiber activation in turn releases mast-cell histamine, which then attenuates CGRP release by acting at inhibitory H₃-receptors on C-fiber terminals (see Fig 7). This conclusion is based on our findings that (1) chemical stimulation of cardiac C fibers elicits the release of CGRP and histamine, both of which are prevented by prior C-fiber destruction; (2) activation of H₃-receptors inhibits the release and the effects of endogenous CGRP but not the effects of exogenous CGRP; and (3) H₃-receptor blockade potentiates both the release and the chronotropic effect of endogenous CGRP.

View larger version (20K): [in this window] [in a new window]   Figure 7. The proposed negative-feedback loop linking sensory C-fiber terminals to mast cells in the heart: (1) Antidromic stimulation of afferent C fibers by bradykinin or capsaicin causes the release of CGRP in an efferent direction. (2) Released CGRP stimulates local mast cell to release histamine (HA). (3) Released histamine stimulates prejunctional inhibitory H3-receptors (H3R) on C fibers. (4) H3-Receptors negatively modulate CGRP release. Released histamine contributes in part to the chronotropic effects of CGRP.

We found that bradykinin, a well-known releaser of CGRP from C fibers,⁴ increased the force of contraction of the atropinized, electrically paced, isolated guinea pig left atrium. The receptor involved in this action is most likely the bradykinin B₂-receptor, since compound HOE 140, a specific B₂-receptor antagonist,²⁴ completely blocked it. The fact that CGRP has marked positive inotropic effects,^{1 2 5} that the CGRP receptor antagonist CGRP_8-37²⁵ blocked the inotropic effect of bradykinin, and that destruction of C fibers by in vivo pretreatment with capsaicin¹ prevented it indicates that the positive inotropic effect of bradykinin is CGRP mediated.

Although cardiac C-fiber activation may release substance P and neurokinin A together with CGRP,^{1 2} only CGRP has strong positive inotropic and chronotropic effects.² Furthermore, although CGRP is a potent histamine-releasing agent¹⁰ and histamine has positive inotropic effects,²³ H₁- and H₂-receptor antagonists did not affect the bradykinin-induced increase in contractility, excluding the possibility that it might be histamine-mediated.

Most important, the selective H₃-receptor agonist imetit²⁶ markedly reduced the inotropic effect of bradykinin, and imetit's effect was blocked by the H₃-receptor antagonist thioperamide.²¹ Because imetit did not affect the positive inotropic effect of exogenous CGRP, our findings suggest that H₃-receptors on C-fiber terminals modulate the release of CGRP. We further substantiated this notion in the spontaneously beating guinea pig heart, a suitable model for the assay of CGRP and histamine release. In the isolated heart, chemical stimulation of C fibers with capsaicin^{1 2 3} elicited a conspicuous release of CGRP and a marked tachycardia, consistent with the well-known chronotropic effect of CGRP.^{1 27} Prior destruction of C fibers by in vivo pretreatment with capsaicin prevented CGRP release and associated tachycardia. Notably, C-fiber stimulation elicited significant histamine release, which was also prevented by in vivo capsaicin administration. This indicates that in the heart histamine release is tied to CGRP release, consistent with the notions that CGRP stimulates mast cells to release histamine¹⁰ and that cardiac histamine is localized almost exclusively in mast cells.^{23 28 29}

Cardiac C-fiber activation may release substance P together with CGRP.^{1 2} However, it is unlikely that substance P plays a role in this autoregulatory mechanism in the guinea pig heart. In fact, although substance P releases histamine from rat mast cells, it does not do so in guinea pig tissues,¹⁰ unless very high nonphysiological concentrations of substance P are used.^{30 31}

We had previously reported that ischemia/reperfusion augments histamine overflow into the coronary effluent.¹⁵ Low pH and high K⁺, both associated with myocardial ischemia, contribute to CGRP release.^{1 2 8} Indeed, in the guinea pig heart, the release of CGRP is potentiated during ischemia.³² Hence, activation of sensory C fibers and release of CGRP could contribute to enhance histamine overflow during ischemia.

Given the strong positive chronotropic effects of histamine,²³ it was important to determine how much of the capsaicin-induced tachycardia was due to CGRP and how much was due to histamine. Our finding with the H₂-receptor antagonist tiotidine indicates that the release of endogenous histamine accounts for 30% of the tachycardia elicited by C-fiber stimulation. In contrast, blockade of H₃-receptors augmented the tachycardia elicited by C-fiber stimulation by 30%, an effect that was associated with an 50% increase in CGRP release. This suggests that histamine released by CGRP in turn attenuates CGRP release and associated tachycardia. Indeed, our findings with the selective H₃-receptor agonist imetit, which decreased the release of CGRP from C fibers and the associated tachycardia, support the concept that histamine released from mast cells is the mediator of this negative-feedback loop by which CGRP turns off its own release and that prejunctional H₃-receptors are involved in this action.

This interpretation concurs with the functional relationship between mast cells and capsaicin-sensitive C fibers previously described in the spleen and lungs.^{11 33} Identified by their specific proteases, mast cells were found in close apposition to CGRP-immunoreactive fibers, whose activity was modulated by prejunctional autoinhibitory H₃-receptors.^{11 33} Mast cells and capsaicin-sensitive C fibers have also been found to interact in other organs in rodents. In spite of the heterogeneity of mast cell sensitivity to neuropeptides among species and tissues, a functional link between nerves and mast cells has been suggested in humans as in rodents.^{9 12 34} Accordingly, mast cells were observed in close apposition to nerves in human skin and airways as well as in the intestine.^{9 12} In the intestine, mast cells proximal to nerves showed signs of activation in ulcerative colitis and inflammatory bowel disease.⁹ Also, consistent with mast cell–nerve cross talk in humans, sensory neuropeptides have been shown to induce histamine release from human nasal mucosa in vitro³⁵ and from mast cells obtained from human skin¹⁰ and bronchoalveolar lavage.³¹ However, since mast cells may have a different activity depending on species and organs, the role of mast cells in the control of neural function in the human heart remains to be determined.

We had previously investigated H₃-receptor signaling in the guinea pig heart and demonstrated the involvement of a pertussis toxin–sensitive G_i/G_o protein and a decrease in Ca²⁺ influx through N-type channels.¹⁴ It is plausible that similar mechanisms apply to the negative modulation of CGRP release from sensory C fibers. Accordingly, coupling of H₃-receptors to inhibitory G proteins in C-fiber endings may result in an inhibition of Ca²⁺ entry via N-type channels, and this could ultimately attenuate CGRP release.

In conclusion, we propose a novel regulatory circuit whereby activation of afferent sensory C fibers in the heart leads sequentially to CGRP release from C-fiber endings, CGRP-induced release of histamine from local mast cells, activation of H₃-receptors on C-fiber endings, and eventual inhibition of CGRP release. Notably, histamine is present in the human heart in significant concentrations, localized almost exclusively in mast cells,^{23 28 29} and histamine H₃-receptors inhibit transmitter release in the human heart.¹⁶ Furthermore, CGRP is present in sensory fibers in the human heart,^{1 5 36} where, as in other tissues, mast cells and C-sensory nerves appear to closely interact.³⁷ Thus, there are strong indications that the inhibitory loop that we have identified in the guinea pig heart may also operate in the human heart. Inasmuch as CGRP release is augmented in a variety of human disease states, such as septic shock,⁷ heart failure,⁶ and acute myocardial infarction,⁸ our findings may offer new insights into pathophysiological mechanisms involved in these ailments.

	Acknowledgments

 
This study is dedicated to Prof Walter F. Riker on the occasion of his 80th birthday. This work was supported by National Institutes of Health grants HL-32415 and HL-46403. Dr Michiaki Imamura was a Fellow of the American Heart Association, New York City Affiliate, Inc. We wish to thank Dr Eiichiro Hatta for helping with some of the assays. We gratefully acknowledge the input of Dr Jean-Charles Schwartz in relation to the scheme depicted in Fig 7.

	Footnotes

 
Preliminary data were presented at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form (Circulation. 1995;92[suppl I]:I-568).

Received October 30, 1995; accepted January 18, 1996.

	References

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