Histamine H3 receptors are involved in regulating the release of norepinephrine (NE), in both central and peripheral nervous systems. We investigated the effect of R-α-methylhistamine (R-HA), a selective H3 receptor agonist, and thioperamide (Thiop), a selective H3 receptor antagonist, on ischemia/reperfusion-induced changes in carrier-mediated NE release and cardiac function in isolated rat heart. Hearts were subjected to 40-minute ischemia followed by 30-minute reperfusion. Ischemia/reperfusion evoked massive NE release, which was markedly suppressed by the treatment with desipramine (DMI), a neuronal NE transporter blocker. Ischemia/reperfusion-induced cardiac dysfunction (decreases in left ventricular developed pressure, LVDP, and the first derivative of left ventricular pressure, dP/dt, and a rise in left ventricular end diastolic pressure, LVEDP) was also improved by the DMI treatment. The treatment with R-HA also significantly decreased the excessive NE release induced by the ischemia/reperfusion, improved the recovery of LVDP and dP/dt, and suppressed the rise in LVEDP. Thiop did not affect NE release and cardiac function after the reperfusion. When R-HA was administered concomitantly with Thiop, R-HA failed to attenuate ischemia/reperfusion-induced NE release and cardiac dysfunction. Thus, it seems likely that the ischemia/reperfusion-induced carrier-mediated NE release in rat hearts is negatively regulated by the activation of H3 receptors, probably located on cardiac noradrenergic nerve endings.
Histamine receptors with 7 transmembrane-spanning proteins have been classified into 3 subtypes; H1, H2, and H3. 1,2 It is known that histamine H3 receptors (H3Rs)-signaling pathway, which is different from those of H1 and H2 receptors, involves pertussis toxin-sensitive Gi/Go protein and a decrease in calcium influx through N-type calcium channels. 1,3 H3Rs are located on central histaminergic nerve endings and act as presynaptic autoreceptors, which modulate the biosynthesis and release of histamine. 4,5 Recently, it has been reported that H3Rs act as heteroreceptors present on noradrenergic, cholinergic, dopaminergic, and serotonergic nerve endings, and are involved in the regulation of neurotransmitter release. 1
Norepinephrine (NE) release from sympathetic nerve endings occurs mainly by 2 pathways: Ca2+-dependent exocytotic release and Ca2+-independent carrier-mediated release via the activation of NE transporter (NET) in the outward direction. 6,7 When an impulse reaches to sympathetic nerve endings, Ca2+ entry through Ca2+ channel is enhanced and subsequently NE release occurs by exocytosis depending on a rise in axoplasmic Ca2+ concentration. In the brain, 8,9 heart, 6,10 and mesenteric artery, 11 the stimulation of H3Rs is known to inhibit the Ca2+-dependent exocytotic NE release. We also found that the activation of H3Rs suppressed an electrical nerve stimulation-induced NE overflow, which is mediated by the exocytotic process, in the dog kidney. 12
In physiological conditions, NE released from sympathetic nerve endings is retrieved from extracellular space via NET utilizing Na+ gradient. In protracted myocardial ischemia, the intraneuronal Na+ is accumulated in anoxic conditions, and this triggers a massive NE efflux via a reverse mode of NET. 13 Excessively released NE is considered to be closely associated with the myocardial ischemia/reperfusion injury, including arrhythmia and myocardial damage. 14–17 It has been reported that activation of H3Rs inhibits the carrier-mediated NE release during myocardial ischemia in guinea pigs, 7 humans, 18 and mice. 19 However, studies in rats failed to demonstrate a significant role of H3Rs for NE release from sympathetic nerves under physiological and/or pathologic conditions. 20–22
To evaluate the role of H3Rs located on noradrenergic nerve endings during ischemia/reperfusion in isolated rat hearts, we investigated the effects of a selective H3R agonist and antagonist on the ischemia/reperfusioninduced changes in carrier-mediated NE release and cardiac function.
From the Department of Pharmacology, Osaka University of Pharmaceutical Sciences, Osaka, Japan.
Received for publication May 1, 2003; accepted October 30, 2003.
Supported in part by a Grant-in-Aid for High Technology Research and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan.
Reprints: Yasuo Matsumura, PhD, Department of Pharmacology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. E-mail: firstname.lastname@example.org
Male Sprague-Dawley rats (280–380 g; Japan SLC, Inc., Shizuoka) were used. The animals were housed in a light-controlled room with a 12-hour light:dark cycle and were allowed ad libitum access to food and water. Animals were maintained at the departmental animal care facility of the Osaka University of Pharmaceutical Sciences in accordance with the guidelines of the Recommendations from the Declaration of Helsinki. Experimental protocols and animal care methods in the experiments were approved by the Experimental Animal Research Committee at Osaka University of Pharmaceutical Sciences.
Isolated Rat Heart Preparation
Sprague-Dawley rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Hearts were rapidly excised, connected via the aorta to Langendorff apparatus (IPH-W2, Labo Support, Osaka, Japan), and perfused retrogradely at a constant pressure of 80 mm Hg with the perfusate (Krebs-Henseleit solution) consisting of the following composition (mM): NaCl 118.1, KCl 4.6, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 24.8, glucose 10. The perfusate was continuously bubbled with a gas mixture of 95% O2/5% CO2 (pH 7.4) and temperature was maintained at 37°C throughout the experiment. A latex balloon filled with water was inserted into the left ventricle through the left atrium and attached to a pressure transducer (DX-360, Nihon Kohden, Tokyo, Japan). Left ventricular developed pressure (LVDP) and left ventricular end diastolic pressure (LVEDP) were measured by amplifier for pressure measurement (AP601G, Nihon Kohden) and the maximum value of the first derivative of left ventricular pressure (dP/dtmax) was measured by derivative operation unit (EQ621G, Nihon Kohden) and these parameters were recorded using PowerLab/4sp (ADInstruments). The balloon volume was adjusted to provide an LVEDP of 10 mm Hg. After the stabilization of 20 to 30 minutes, the experiment was started.
After stabilization, hearts were subjected to global ischemia for 40 minutes by clamping the aortic cannula followed by reperfusion for 30 minutes. Drugs were perfused 30 minutes before ischemia and during reperfusion. Cardiac function (LVDP, dP/dtmax, LVEDP) measurements were performed just before drugs administration, during ischemia, and after the reperfusion.
NE concentration in coronary effluent was measured using high-performance liquid chromatography and an amperometric detector (ECD-100, Eicom, Kyoto, Japan), as reported. 23
R-HA and Thiop were purchased from Sigma Chemical (St. Louis, MO), and other chemicals were obtained from Nacalai Tesque (Kyoto, Japan) and Wako (Osaka, Japan). R-HA, Thiop, and DMI were dissolved in Krebs-Henseleit solution.
All values were expressed as mean ± SEM. For statistical analysis, we used the one-way analysis of variance combined with Dunnett multiple range test for multiple comparisons. Differences were considered significant when P values were less than 0.05.
Effects of DMI, a Neuronal Norepinephrine Transporter Blocker, on Ischemia/Reperfusion-Induced Norepinephrine Release
The cumulative NE overflow in coronary effluent during 5 minutes after the reperfusion was much higher than the pre-ischemic levels (pre-ischemic levels approximately 855 pg/5 minutes). The majority of NE was released 2 minutes after reperfusion; the NE level in coronary effluent 5 minutes after reperfusion was far less. The treatment with DMI significantly attenuated the ischemia/reperfusion-induced enhancement of NE release in coronary effluent in a dose-dependent manner (DMI 10 nM, 4840 ± 1595 pg/5 minutes; DMI 100 nM, 2487 ± 447 pg/5 minutes; DMI 1 μM, 1494 ± 334 pg/5 minutes vs control, 23718 ± 3699 pg/5 minutes) (Fig. 1).
Effects of DMI on Ischemia/Reperfusion-Induced Cardiac Dysfunction
Perfusion of DMI at concentrations of 10 nM, 100 nM, and 1 μM produced no significant changes in basal cardiac function. As shown in Figure 2A, the pre-ischemic level of LVDP was markedly reduced by 40-minute ischemia and reperfusion, but a dose-dependent improvement was observed in the presence of DMI. When 1 μM of DMI was perfused, the diminished LVDP restored to the pre-ischemic level at 30 minutes after the reperfusion. Qualitatively similar results were also seen in the case of dP/dtmax. As shown in Figure 2B, LVEDP was elevated gradually during 40-minute ischemia, and its level further increased after the reperfusion. DMI efficiently improved the changes of LVEDP after the reperfusion, in a dose-dependent manner, although this drug did not significantly affect the levels of LVEDP during ischemia.
Effects of R-HA, a Selective H3R Agonist, and Thiop, a Selective H3R Antagonist, on Ischemia/Reperfusion-Induced Norepinephrine Release
Administration of R-HA significantly and markedly suppressed the ischemia/reperfusion-induced enhancement of NE release in coronary effluent during 5 minutes of reperfusion, compared with control (R-HA 100 nM, 8551 ± 2328 pg/5 minutes; R-HA 1 μM, 2820 ± 671 pg/5 minutes). Thiop did not affect NE release (23000 ± 3147 pg/5 minutes); however, the R-HA-induced suppressive effect on NE release was abolished by a combination of R-HA and Thiop (Fig. 3).
Effects of R-HA and Thiop on Ischemia/Reperfusion-Induced Cardiac Dysfunction
No significant changes in cardiac function were observed by R-HA or Thiop perfusion before the ischemia. As shown in Figure 4A, the decrease in LVDP after 40-minute ischemia and reperfusion was significantly improved by the treatment with R-HA. However, the R-HA–induced improvement was abolished by superimposing Thiop perfusion. Figure 4B illustrates the changes in LVEDP levels during ischemia and after the reperfusion. The treatment with R-HA further increased the early phase of LVEDP during ischemia, but efficiently suppressed the elevation observed after the reperfusion. R-HA–induced alternations in LVEDP were completely abolished by a concomitant administration of Thiop.
It is well known that the decrease of oxygen supply by ischemia causes ATP depletion and intracellular acidosis due to lactate production. In sympathetic nerve endings under the ischemic condition, free axoplasmic NE massively accumulates due to incapability of the driving force for NE storage because vesicular storage of NE depends on H+ gradient and ATP in physiological conditions. An increase in axoplasmic H+ activates Na+/H+ exchanger (NHE), consequently leading to an influx of Na+ in exchange for H+. Furthermore, inhibition of Na+/K+ ATPase activity by ATP depletion results in accumulation of axoplasmic Na+. This Na+ accumulation triggers an excessive axoplasmic NE release via a reversal of NET from intracellular space to extracellular space. 13 It has been considered that, in protracted myocardial ischemia, this carrier-mediated NE release is the major mechanism for the NE overflow from the nerve endings. 24
In the present study, we observed marked protective effects of R-HA on ischemia/reperfusion-induced cardiac dysfunction and NE overflow in isolated rat hearts. The effects of R-HA were abolished by a concomitant treatment with Thiop, thereby suggesting that H3Rs play a role in regulating negatively the ischemia/reperfusion-induced NE release. This NE release seems to occur via the carrier-mediated NET system, since the treatment with DMI efficiently suppressed it. Carrier-mediated NE release has been described in ischemic human cardiac tissues. 25 In guinea pig 7 and human 18 hearts, the stimulation of H3Rs has been shown to attenuate the carrier-mediated NE release by inhibiting NHE-mediated intraneuronal accumulation of Na+. Our results using isolated ischemic rat hearts are in agreement with the above findings. On the other hand, Mazenot et al 22 showed that histamine H3 receptor stimulation failed to modulate NE release induced by ischemia in isolated rat hearts and suggested that unlike other species, the rat appears to be insensitive to H3-histaminergic receptor modulation of ischemia-induced NE release. The reason for discrepancy is unknown, but some differences in experimental conditions including the duration of ischemia may be partly related to the conflicting results.
In our study, Thiop itself did not influence the ischemia/reperfusion-induced NE release. This suggests that endogenous histamine dose not function as an inhibitory modulator of carrier-mediated NE release in the ischemic rat heart. Alternatively, there is a possibility that endogenous histamine is not released from the ischemic rat heart. On the other hand, an enhanced histamine release in post-ischemic heart has been observed in guinea pigs 7 and humans. 18 Findings that a selective H3R antagonist such as Thiop or clobenpropit can potentiate the NE release in the post-ischemic heart of the above species strongly suggests the functional role of endogenous histamine in sympathetic nerve endings of the ischemic heart. Thus, further studies are required to elucidate whether histamine is endogenously released and plays a role as an inhibitory modulator of carrier-mediated NE release in the post-ischemic heart of rats.
Increased intracellular Ca2+ concentration has been considered to be one of the important factors in ischemia/reperfusion-induced injury. In sympathetic nerve endings under ischemic conditions, accumulated axoplasmic Na+ is extruded not only via the reversal of NET but also via the reverse mode of Na+/Ca2+ exchanger (NCX). It has been demonstrated that NCX is largely responsible for excessive Ca2+ influx (Ca2+ overload) during the ischemia/reperfusion, which results from intracellular Na+ accumulation. 26–28 So far, several studies have demonstrated that NHE inhibitors decrease Na+ accumulation induced by ischemia/reperfusion and consequently suppress intracellular Ca2+ overload via the reverse mode of NCX. 28–30 In addition, since NHE inhibitors can attenuate the carrier-mediated NE release, 7,18 they are likely to attenuate the post-ischemic increase in intracellular Ca2+ concentration by reducing NE-induced Ca2+ entry through voltage-dependent Ca2+ channels or receptor-operated Ca2+ channels and IP3-induced Ca2+ mobilization from sarcoplasmic reticulum. 31 Since H3Rs stimulation is associated with a reduced NHE activity, 7,18 it is suggested that H3Rs can improve myocardial ischemia/reperfusion injury by attenuating carrier-mediated excessive NE release and/or by suppressing intracellular Ca2+ overload via the reverse mode of NCX, which occurs secondary to the inhibition of NHE.
R-HA–induced suppression of NE release immediately after the reperfusion resulted in an improvement of ischemia/reperfusion-induced cardiac dysfunction in rat hearts. Also, Imamura et al 7 demonstrated that there was a direct correlation between NE release and the severity of reperfusion arrhythmias and that activation of H3Rs reduced NE release and the incidence of ventricular fibrillation by 50% in guinea pig hearts. It is speculated that an overactivity of cardiac conduction pathway following massive NE release is largely involved in the myocardial ischemia-induced cardiac dysfunction and arrhythmia. 31,32 Indeed, severe ventricular arrhythmias are the main cause of sudden cardiac death in acute myocardial infarction and in postinfarct patients, 31 and elevation of plasma NE levels is a predictable factor in the development of heart failure, acute ischemic syndromes, and cardiovascular mortalities. 33 Recently, Koyama et al 19 demonstrated that hearts of animals lacking H3 receptors release much more NE when subjected to ischemia, than hearts with wild-type. This finding accentuates the relevance of H3R as a major cardioprotective mechanism in myocardial ischemia. Accordingly, it seems likely that negative regulation of NE release by H3R agonists may confer a therapeutic approach to heart failure, cardiac hypertrophy, and myocardial infarction associated with sympathetic overactivated states, in addition to myocardial ischemia/reperfusion injury.
In conclusion, we suggest that functional H3Rs in post-ischemic rat hearts play an important role as an inhibitory modulator of noradrenergic neurotransmission and that the inhibitory action probably leads to attenuating the ischemia/reperfusion-induced cardiac dysfunction.
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