Functional Identification of Histamine H3-Receptors in the Human Heart
Abstract Norepinephrine release contributes to ischemic cardiac dysfunction and arrhythmias. Because activation of histamine H3-receptors inhibits norepinephrine release, we searched for the presence of H3-receptors directly in sympathetic nerve endings (cardiac synaptosomes) isolated from surgical specimens of human atria. Norepinephrine was released by depolarization with K+. The presence of H3-receptors was ascertained because the selective H3-receptor agonists (R)α-methylhistamine and imetit reduced norepinephrine release, and the specific H3-receptor antagonist thioperamide blocked this effect. Norepinephrine release was exocytotic, since it was inhibited by the N-type Ca2+-channel blocker ω-conotoxin and the protein kinase C inhibitor Ro31-8220. Functional relevance of these H3-receptors was obtained by showing that transmural electrical stimulation of sympathetic nerve endings in human atrial tissue increased contractility, an effect blocked by propranolol and attenuated in a concentration-dependent manner by (R)α-methylhistamine. Also, thioperamide antagonized the effect of (R)α-methylhistamine. Our findings are the first demonstration that H3-receptors are present in sympathetic nerve endings in the human heart, where they modulate adrenergic responses by inhibiting norepinephrine release. Since myocardial ischemia causes intracardiac histamine release, H3-receptor–induced attenuation of sympathetic neurotransmission may be clinically relevant.
- cardiac synaptosomes
- histamine H3-receptors
- norepinephrine release
- sympathetic nerve endings
- myocardial ischemia
Sympathetic overactivity with excessive norepinephrine release is a recognized cause of cardiac dysfunction and arrhythmias in myocardial ischemia1 and hypertension.2 Accordingly, it is important to identify mechanisms to attenuate norepinephrine release. Among these, inhibitory presynaptic receptors have received much attention.3 We and others have recently reported that histamine H3-receptors negatively modulate cardiac adrenergic function in the guinea pig both in vitro4 and in vivo.5 Because selective H3-receptor agonists attenuated the release but not the effects of norepinephrine, we interpreted these findings as an indication that H3-receptors are located prejunctionally. We further found that these receptors are quiescent in normal conditions but become activated in the setting of myocardial ischemia, when histamine is copiously released.6 If histamine H3-receptors were present in sympathetic nerve endings in the human heart, their activation could represent a novel mechanism to attenuate the deleterious effects of sympathetic overactivity. Therefore, the aim of the present study was to directly demonstrate the presence of H3-receptors in sympathetic nerve endings isolated from the human heart and to determine whether these receptors modulate adrenergic responses by decreasing norepinephrine release.
Materials and Methods
Specimens of right atrial tissue were obtained from 36 patients undergoing cardiopulmonary bypass for cardiac surgery (coronary artery bypass grafting, 23 patients; valve replacement, 13 patients) by following a protocol approved by our Institutional Review Board. All subjects gave informed consent. No patient was in congestive heart failure at the time of surgery. Nine of the 23 coronary artery bypass patients were chronically treated with β-adrenoceptor blocking agents. Preoperative treatment with β-blockers affected neither the response to field stimulation nor the release of norepinephrine from K+-depolarized synaptosomes. At the time of surgery, a piece of atrial appendage measuring ≈1 cm2 was removed from the atriotomy site. After excision, the specimen was placed in cold oxygenated Krebs-Henseleit solution of the following composition (mmol/L): NaCl 119.0, CaCl2 1.5, KCl 4.6, MgSO4 1.1, KH2PO4 1.2, NaHCO3 24.9, and glucose 10.0.
Norepinephrine Release From Cardiac Synaptosomes
Specimens of human right atrial tissue were freed from fat and connective tissues and minced in ice-cold 0.32 mol/L sucrose containing 1 mmol/L EGTA, pH 7.4. Synaptosomes were isolated as previously described,7 8 with the following modifications. Minced tissue was digested with 150 mg collagenase (type II, Worthington Biochemicals) per 10 mL HEPES-buffered saline solution (HBSS) per gram wet heart weight for 3 hours at 37°C. HBSS contained (mmol/L) HEPES 50 (pH 7.4), NaCl 144, KCl 5, CaCl2 1.2, MgCl2 1.2, glucose 10, and pargyline 1, to prevent enzymatic destruction of synaptosomal norepinephrine. After low-speed centrifugation (5 minutes at 1000g), the resulting pellet was suspended in 10 vol of 0.32 mol/L sucrose and homogenized with a polytetrafluoroethylene (Teflon)/glass homogenizer. The homogenate was spun at 650g for 10 minutes, and the pellet was rehomogenized and respun. The pellet containing cellular debris was discarded, and the supernatants from the last two spins were recentrifuged for 20 minutes at 20 000g at 4°C. This pellet, which contained cardiac synaptosomes, was resuspended in HBSS (500 μL), incubated with KCl (30 mmol/L) in the presence or absence of pharmacological agents for a total of 20 minutes in a water bath at 37°C, and recentrifuged for 20 minutes (20 000g at 4°C). The supernatant (final synaptosomal fraction) was assayed for norepinephrine content by high-performance liquid chromatography with electrochemical detection, as previously described.6 Protein content was measured by a modified Lowry procedure (Markwell et al9 ). The pellet was discarded. When high concentrations of KCl were used to depolarize synaptosomes, osmolarity was maintained constant by adjusting the NaCl concentration.
Pectinate muscles10 were carefully dissected free from the atrial wall and mounted vertically in a 10-mL double-walled Pyrex organ bath filled with Krebs-Henseleit solution (gassed with 95% O2/5% CO2 at 30°C, pH 7.4) and prepared for isometric tension measurement. The upper end of the pectinate muscle was connected by a silk thread to a force transducer (model 7D, 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 platinum electrodes. Pectinate muscles were continuously paced at 1 Hz with rectangular pulses (duration, 5 milliseconds; 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 30 to 60 minutes. Then, transmural sympathetic nerve stimulation was periodically superimposed on the continuous pacing.4 For this, intense field stimuli were delivered at a frequency of 1 Hz by a pair of spiral platinum electrodes and consisted of square-wave pulses 10 milliseconds in duration generated by the same stimulator (model S88, Grass Instruments) and synchronized with the pacing stimuli. The intensity of the current applied was 12 to 15 mA. Concentration-response relations were determined by adding the selective histamine H3-receptor agonist (R)α-methylhistamine11 to the bath in a cumulative fashion, allowing the contractile force to stabilize at each concentration (ie, every 2 minutes). Atropine (1 μmol/L) was added to the incubation medium to prevent the activation of muscarinic receptors by acetylcholine released from parasympathetic nerve terminals.
Values are expressed as mean±SEM. Analysis was by ANOVA followed by post hoc testing (Dunnett’s test). Student’s t test was performed for paired observations. A value of P<.05 was considered statistically significant.
Norepinephrine Exocytosis From Isolated Sympathetic Nerve Endings and Its Modulation by Histamine H3-Receptors
We tested the hypothesis that H3-receptors are located prejunctionally by determining the action of the selective H3-receptor agonist (R)α-methylhistamine directly on sympathetic nerve endings (cardiac synaptosomes) isolated from surgical specimens of human right atria. As shown in Figs 1⇓ and 2⇓, depolarization of synaptosomes with 30 mmol/L K+ elicited a 25% to 60% increase in endogenous norepinephrine release above basal levels. The N-type Ca2+ channel blocker ω-conotoxin (10 nmol/L) prevented this release (Fig 1A⇓). Norepinephrine release was also inhibited by 70% by the protein kinase C (PKC) inhibitor Ro31-8220 at 1 μmol/L (Fig 1B⇓). Neither ω-conotoxin nor Ro31-8220 by themselves affected basal norepinephrine release from unstimulated synaptosomes (norepinephrine release was 76±22 and 71±8.4 pg/mg of protein in the absence and presence, respectively, of ω-conotoxin [n=8] and 62±10 and 60±6.5 pg/mg of protein in the absence and presence, respectively, of Ro31-8220 [n=6]). As shown in Fig 2A⇓, (R)α-methylhistamine (0.3 μmol/L; pD2, 8.07±0.04 for inhibition of electrically evoked contraction of guinea pig ileum)12 reduced the K+-evoked norepinephrine exocytosis by 65%. The specific H3-receptor antagonist thioperamide (0.3 μmol/L; pA2, 8.5±0.09 in the guinea-pig ileum)12 13 blocked this effect. Another selective H3-receptor agonist, imetit (0.1 μmol/L), known to be three times as potent as (R)α-methylhistamine on the basis of inhibition of the binding of [3H](R)α-methylhistamine to rat brain membranes,14 also reduced by 65% the K+-evoked norepinephrine exocytosis (Fig 2B⇓). Again, the H3-receptor antagonist thioperamide (0.3 μmol/L) blocked this effect (Fig 2B⇓); thioperamide alone had no effect on K+-induced norepinephrine release (n=4; data not shown). Notably, the EC50 values for the effects of (R)α-methylhistamine and imetit for H3-receptors are in the nanomolar range and for H2- and H1-receptors are in the high micromolar range. Similarly, the Ki for thioperamide is in the nanomolar range for H3-receptors and in the high micromolar range for H2- and H1-receptors.13
Positive Inotropic Response of Human Right Atrium to Transmural Sympathetic Nerve Stimulation or to Norepinephrine Administration
To strengthen the functional relevance of these H3- receptors, we next subjected atrial tissue to transmural electrical stimulation of sympathetic nerve endings so as to elicit an adrenergically evoked inotropic response on which to study the effects of H3-receptor agonists. Pectinate muscles, isolated from human right atrial surgical specimens, were paced at 1 Hz. In the presence of atropine (1 μmol/L), transmural stimulation of sympathetic nerve endings elicited a greater than 10-fold increase in contractile force (from 0.08±0.02 to 1.37±0.16 g, n=11, P<.05). Exogenous norepinephrine (1 μmol/L) elicited a 15-fold increase in contractile force (from 0.027±0.003 to 0.410±0.123 g, n=11, P<.05). The β-adrenoceptor antagonist propranolol (1 μmol/L) inhibited these responses by 36.9±4.3% (n=7, P<.05) and 38.2±16.2% (n=5, P<.05), respectively. In contrast, the selective α2-adrenoceptor agonist UK 14,304 (0.1 μmol/L) inhibited only the increase in contractile force elicited by transmural stimulation by 33.6±8.7% (n=5), but not the response to exogenous norepinephrine (data not shown). This suggested that the contractile response to transmural stimulation was due to norepinephrine released from sympathetic nerve endings.
Effect of (R)α-Methylhistamine on the Positive Inotropic Responses of Human Right Atrium to Transmural Sympathetic Nerve Stimulation or to Norepinephrine Administration
As shown in Fig 3⇓, (R)α-methylhistamine at 0.03 to 10 μmol/L caused a concentration-dependent inhibition of the positive inotropic response of human right atrial tissue to transmural stimulation of sympathetic nerve endings. In contrast, (R)α-methylhistamine at 0.3 μmol/L failed to inhibit the positive inotropic effect of exogenous norepinephrine, suggestive of a prejunctional effect of (R)α-methylhistamine [norepinephrine at 1 μmol/L caused a 2.8±2.9% greater increase in contractility in the presence of (R)α-methylhistamine than in its absence; n=6]. In the presence of the histamine H3-receptor antagonist thioperamide (0.3 μmol/L), the concentration-response curve for the inhibitory effect of (R)α-methylhistamine was shifted markedly to the right (see Fig 3⇓). Furthermore, 1 μmol/L imetit inhibited the positive inotropic response of human right atrial tissue to transmural stimulation of sympathetic nerve endings by 21±5%; thioperamide (0.3 μmol/L) reduced this effect to 2.8±2.0% (n=5, P<.05 by paired t test).
These findings directly demonstrate that histamine H3-receptors are present on sympathetic nerve endings in the human heart, where they function to modulate adrenergic responses by decreasing norepinephrine exocytosis. We show in the present study that the selective H3-receptor agonists (R)α-methylhistamine11 and imetit14 inhibit the depolarization-induced release of norepinephrine from cardiac synaptosomes isolated from human myocardium and that these responses are antagonized by the specific H3-receptor antagonist thioperamide.13 The inference that the K+-induced depolarization of the synaptosomal preparation elicits an exocytotic release of norepinephrine is supported by our finding that the specific N-type Ca2+-channel antagonist ω-conotoxin15 inhibited this release. Also, the PKC inhibitor Ro31-822016 antagonized the K+-induced synaptosomal norepinephrine release. Since PKC participates in the exocytotic cascade,17 antagonism of norepinephrine release by PKC inhibition further supports the exocytotic nature of the norepinephrine release.
(R)α-Methylhistamine and imetit also attenuated the positive inotropic response of human myocardium to transmural stimulation of sympathetic nerve endings, and thioperamide antagonized the effect of (R)α-methylhistamine. The β-adrenoceptor antagonist propranolol inhibited the positive inotropic response to sympathetic nerve stimulation as well as the response to exogenous norepinephrine, whereas compound UK 14,304, a selective agonist at prejunctional inhibitory α2-adrenoceptors, attenuated only the response to sympathetic nerve stimulation. Thus, the inotropic effect of transmural stimulation was exclusively due to norepinephrine release from sympathetic nerve endings. Like the α2-agonist, (R)α-methylhistamine inhibited the effect of adrenergic nerve stimulation but not the response to administered norepinephrine. This confirms that histamine H3-receptors responsible for the action of (R)α-methylhistamine operate from a prejunctional site and demonstrates the functional relevance of these receptors.
The inhibitory effect of (R)α-methylhistamine on the inotropic response to adrenergic nerve stimulation reached a maximum of ≈20%. This is comparable to what we had previously observed in the guinea pig atrium.4 This seemingly modest inhibition does not detract from the physiological importance of H3-receptors. In fact, we had found that the H3-receptor–mediated inhibition of the positive chronotropic effect to cardiac sympathetic stimulation amounts to 30%4 6 and that the inhibition of norepinephrine release in ischemia/reperfusion models exceeds 50%.6 18
Although H3-receptor activation inhibited both the inotropic response to adrenergic nerve stimulation and the K+-induced synaptosomal release of norepinephrine, greater inhibitory effects were achieved in synaptosomes than in pectinate muscles (65% versus 20%, respectively). This discrepancy most likely reflects the difference between the two preparations. Whereas in synaptosomes we directly measured norepinephrine release in response to K+-induced depolarization, in atrial tissue we measured the increase in contractility elicited by field stimulation. In any event, the fact that (R)α-methylhistamine and imetit significantly inhibited both responses, whereas thioperamide antagonized them, strongly indicates that both responses are mediated by H3-receptors.
We had previously investigated H3-receptor signaling in the guinea pig heart and demonstrated the involvement of a pertussis toxin–sensitive Gi/Go protein and a decrease in Ca2+ influx through N-type channels.4 Given the similarity of H3-receptor–mediated effects in human and guinea pig hearts, we suspect that coupling of H3-receptors to inhibitory G proteins in the human heart may ultimately result in an inhibition of Ca2+ entry via N-type channels. Because inhibition of PKC with compound Ro31-8220 decreased K+-induced norepinephrine release from synaptosomes, it is possible that PKC inhibition plays a role in the modulation of norepinephrine release associated with H3-receptor activation. Indeed, H3-receptors have been found to be coupled to a pertussis toxin–sensitive G protein and to decrease phosphoinositide turnover in a human gastric carcinoma cell line.19 Histamine H3-receptor–mediated decrease in phosphoinositide turnover and diacylglycerol formation could decrease PKC activity and, therefore, norepinephrine release from sympathetic nerve endings.
Although we had access only to human atrial specimens, in view of our findings in the guinea pig heart6 18 and canine ventricular myocardium (unpublished data, 1995), we believe that histamine H3-receptors are also present on sympathetic nerve endings in human ventricle. Therefore, our discovery that sympathetic nerve endings in the human heart harbor histamine H3-receptors that can reduce exocytotic norepinephrine release may be clinically relevant. Indeed, myocardial infarction is often accompanied by arrhythmias that can be fatal.1 Sympathetic overactivity and excessive norepinephrine release may increase metabolic demand, thus aggravating the primary ischemia and initiating a vicious cycle leading to further myocardial damage.20 Therefore, negative modulation of norepinephrine release from cardiac sympathetic nerves is a crucial protective mechanism. In the ischemic guinea pig heart, sufficient amounts of histamine are released to activate H3-receptors and attenuate norepinephrine exocytosis.6 Accordingly, our findings imply that H3-receptors may mitigate the consequences of the sympathetic overactivity that accompanies myocardial ischemia in humans and suggest new therapeutic strategies to alleviate dysfunctions associated with this condition.
This study was supported by National Institutes of Health grants HL-34215 and HL-46403. We gratefully acknowledge the help of Dr O. Wayne Isom, Chairman of the Department of Cardiothoracic Surgery of Cornell University Medical College, in providing us with surgical specimens of human right atria. Compound Ro31-8220 was a gift of Dr T.J. Hallam, Roche Research Centre, Welwyn Garden City, Herts, UK. Imetit was graciously provided by Prof C.R. Ganellin, Department of Chemistry, University College, London, UK.
Preliminary version presented at the meeting of Experimental Biology ’95, Atlanta, Ga, April 9-13, 1995, and published in abstract form (FASEB J. 1995;9:A933).
- Received March 20, 1995.
- Accepted April 21, 1995.
- © 1995 American Heart Association, Inc.
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