Effects of nociceptin on cardiac norepinephrine and acetylcholine release evoked by ouabain

Brain Research 904 (2001) 153–156 www.elsevier.com / locate / bres

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Effects of nociceptin on cardiac norepinephrine and acetylcholine release evoked by ouabain Toji Yamazaki*, Tsuyoshi Akiyama, Hidezo Mori Department of Cardiac Physiology, National Cardiovascular Center Research Institute, 5 -7 -1 Fujishirodai, Suita, Osaka 565 -8565, Japan Accepted 27 March 2001

Abstract We investigated whether the novel peptide, nociceptin, modulates neuronal transmission at autonomic nerve endings. Using a cardiac dialysis technique, the effects of locally applied nociceptin on cardiac acetylcholine (ACh) and norepinephrine (NE) release were examined in anesthetized cats. Dialysis probes were implanted in the left ventricular wall, with the concentration of dialysate NE or ACh serving as an indicator of NE or ACh output at cardiac sympathetic or parasympathetic nerve endings. Locally applied ouabain evoked increases in NE and ACh output. Nociceptin suppressed the ouabain induced ACh increment. The ouabain induced NE release was not altered by nociceptin. However, in the presence of desipramine (a NE uptake inhibitor), nociceptin suppressed the ouabain-induced NE release. Inhibition by nociceptin of ouabain-induced release of NE or ACh was blocked by pretreatment with nocistatin (a nociceptin action blocking peptide). Nociceptin-induced inhibition of ACh or NE release is attributable to pre-synaptic modulation rather than a reversal of the ouabain effect. These findings demonstrate that nociceptin inhibits cardiac autonomic neurotransmission via a presynaptic opioid receptor-like1(ORL1) receptor.  2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Opioid receptors Keywords: ORL1 receptor; Autonomic nerve; Heart; Pre-synaptic; Nocistatin

It is well known that the opioid system plays a critical role in the regulation of cardiovascular function [14]. Recently, the novel neuropeptide, nociceptin, has been suggested to be the endogenous ligand for the opioid receptor-like1 (ORL1) receptor which is expressed in the central [3] and peripheral nervous system [15]. Further, a nociceptin binding site has been shown in the rat heart [4]. Nociceptin dose dependently produced transient hypotension and bradycardia [6]. Nociceptin-induced hemodynamic changes were affected by vagotomy or guanethidine treatment. These data suggested that nociceptin exerts modulatory actions on the cardiac autonomic pathway. Recently, we developed a new method of monitoring myocardial interstitial norepinephrine (NE) and acetylcholine (ACh) levels to serve as indices of endogenous NE [18] and ACh release [1] from post-ganglionic sympathetic *Corresponding author. Tel.: 181-6-6833-5012; fax: 181-6-68728092. E-mail address: [email protected] (T. Yamazaki).

and parasympathetic nerve endings. In the present study, we investigated the possibility that nociceptin induces neurotransmitter release at the cardiac sympathetic and parasympathetic nerve endings. Furthermore, we examined whether nociceptin modulates the release of the neurotransmitter evoked by ouabain. To avoid the influence of ouabain and nociceptin on systemic hemodynamic changes, we administered these agents locally through the dialysis probe. Adult cats were anesthetized with pentobarbital sodium. The electrocardiogram, heart rate and mean arterial blood pressure were simultaneously monitored. With a fine guiding needle, one or two dialysis probes for dialysate sampling were implanted in the midwall of the anterolateral region of the left ventricle. We designed a transverse dialysis probe (13 mm length, 0.31 mm O.D., and 0.2 mm I.D.; PAN-1200, 50 000 molecular weight cutoff, Asahi Chemical, Japan) [18]. The dialysis probe was perfused at a rate of 2 ml / min with Ringer’s solution containing the cholinesterase inhibitor, eserine (100 mM). Sampling

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02456-8

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periods were 15 min in duration (one sampling volume530 ml). Two-thirds of the dialysate sample was used for the ACh measurement, and the remaining one-third for the NE measurement. We measured NE and ACh concentrations using separate types of high-performance liquid chromatography with electrochemical detection. Detailed settings for the liquid chromatography have been described elsewhere [1,18]. There was no statistically significant difference in basal dialysate NE levels between vehicle and nociceptin pretreatment (0.2260.04, 0.2860.04, 0.2960.03 nM, vehicle, nociceptin (1 mM), nociceptin (10 mM), respectively) (mean6S.E., n56). The left panel in Fig. 1 shows the dialysate NE response to locally administered ouabain at 100 mM. Ouabain induced increases in dialysate NE levels. Pretreatment with nociceptin at 1 and 10 mM did not suppress the ouabain induced dialysate NE responses. There was no statistically significant difference in basal dialysate ACh levels between vehicle and nociceptin pretreatment (0.6460.32, 0.6560.13, 0.7660.38 nM, vehicle, nociceptin (1 mM), nociceptin (10 mM), respectively) (mean6S.E., n56). The right panel in Fig. 1 shows the dialysate ACh response to locally applied ouabain. Ouabain caused increases in dialysate ACh levels. Pretreatment with nociceptin (1 and 10 mM) suppressed ouabain induced dialysate ACh responses. To eliminate the carrier-mediated outward NE transport evoked by ouabain, a membrane NE transport inhibitor was locally co-administered and the effect of nociceptin on the ouabain-induced NE response was examined. Pretreatment with the neuronal NE uptake inhibitor, desipramine (100 mM), suppressed the ouabain induced NE release. Pretreatment with nociceptin1desipramine further suppressed it (Fig. 2). Inhibition by nociceptin of ouabaininduced release of NE or ACh was blocked by pretreat-

Fig. 2. The effects of nociceptin (10 mM) on dialysate norepinephrine (NE) responses to locally applied ouabain (100 mM) in the pretreatment with desipramine (DMI, 100 mM). Data represent mean6S.E.M. of five separate samples. * Indicates significantly different from desipramine alone (P,0.05, assessed by Student’s non-paired t-test).

ment with nocistatin (nociceptin action blocking peptide, 10 mM) (Fig. 3). In the present study, ouabain alone evoked the release of endogenous neurotransmitter at the cardiac autonomic nerve endings. In the implanted region of the dialysis probe, adjacent tissue contains few pre-ganglionic sympathetic and parasympathetic nerve endings [1,18]. Local administration of ouabain and / or nociceptin did not affect hemodynamic parameters. Therefore, these data suggest that ouabain induced NE and ACh release from surrounding post-ganglionic sympathetic and parasympathetic nerve endings, respectively. Nociceptin suppressed the ouabain-induced ACh release. Furthermore, this suppression was reversed by addition of

Fig. 1. Left panel: the effects of nociceptin on dialysate norepinephrine (NE) responses to locally applied ouabain (100 mM). Each column is a 15-min collection period and numbers in the axis of abscissas represent the time course of locally applied ouabain. Data represent mean6S.E.M. of six separate samples. Right panel: the effects of nociceptin on dialysate acetylcholine (ACh) responses to locally applied ouabain (100 mM). Each column is a 15-min collection period and numbers in the axis of abscissas represent the time course of locally applied ouabain. Data represent mean6S.E.M. of six separate samples. * Indicates significantly different from vehicle group (P,0.05, assessed by analysis of variance, Neuman–Keuls’ test).

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Fig. 3. The effects of nocistatin (a nociceptin action blocking peptide, 10 mM) on dialysate norepinephrine (NE) and acetylcholine (ACh) responses to locally applied ouabain (100 mM) in the pretreatment with nociceptin (10 mM). Data represent mean6S.E.M. of five separate samples. * Indicates significantly different from nociceptin alone (P,0.05, assessed by Student’s non-paired t-test). DMI; desipramine (100 mM).

nocistatin [11]. These data suggest that nociceptin interacts with pre-synaptic orphan ORL1 receptors, which modulate the ACh release evoked by ouabain. This conclusion is consistent with other studies in which nociceptin inhibited the ACh release evoked by electrical field stimulation [12] or light [10]. Alternatively, nociceptin may interact with ouabain-binding sites at the parasympathetic nerve endings [4]. If this is the case, nociceptin alone would augment ACh release. However, without ouabain, nociceptin alone did not augment ACh release. The ORL1 receptor is mainly detected in the brain [3], but recently a nociceptin binding site has been shown in membrane preparation of rat heart [4]. Furthermore, using reverse transcription and the polymerase chain reaction, the ORL1 receptor mRNA was detected in rat heart. An increase in the mRNA for ORL1 receptor was correlated with a change in nociceptin binding activity. These data suggest the presence of ORL1 receptors in heart. Actually nociceptin modulated cardiac inotropic and chronotropic action [5,6]. Nociceptin had no effect on the amount of NE release evoked by ouabain. At the cardiac sympathetic nerve endings, two distinct mechanisms (exocytosis and carriermediated outward transport) contribute to the amount of NE release evoked by ouabain [18]. In vitro studies have revealed that pre-synaptic modulation of neurotransmitter release is involved in intracellular Ca 21 homeostasis [8,17]. If ouabain-induced NE release is mainly derived from Ca 21 -independent carrier mediated NE outward transport, suppression of NE release by nociceptin might be masked. We examined this hypothesis. When Ca 21 independent carrier-mediated outward NE transport was blocked by desipramine, nociceptin suppressed the NE release evoked by ouabain. These data suggest that desipramine-insensitive NE release was suppressed by nociceptin. Furthermore, nociceptin induced suppression of NE release was reversed by addition of nocistatin.

Nociceptin induced suppression of NE release also is mediated via orphan ORL1 receptors. An alternative explanation is that nociceptin itself may act as a NE uptake blocker. Dumon et al. [4] suggested that nociceptin as well as Dynorphin A has inhibitory effect on membrane NE uptake. If nociceptin acts as an inhibition of the NE transporter, nociceptin inhibits bidirectional NE transport. In the present study, nociceptin alone failed to alter basal NE level, suggesting it did not impair NE uptake. Furthermore, in the absence of desipramine, ouabain-induced NE release was not suppressed by nociceptin. These data suggest that the interaction of nociceptin with the membrane NE transporter may contribute very little to nociceptin-induced inhibition of NE release. Or rather, nociceptin-induced inhibition of NE release seems to be attributable to pre-synaptic modulation. This conclusion is in agreement with other studies in which nociceptin modulated noradrenergic neurotransmission by acting on pre-synaptic orphan ORL1 receptors at the nerve endings [2,16]. Our data suggest that nociceptin exerted an inhibitory effect on the release of neurotransmitter from cardiac sympathetic and parasympathetic nerve endings. Several in vitro studies suggest nociceptin-induced inhibitory mechanisms of neurotransmitter release. An in vitro study demonstrated that nociceptin inhibits voltage-dependent Ca 21 channel currents, including N- and T types [8]. A recent study indicated that the ORL1 receptor is functionally coupled with a G-protein-activated K 1 (GIRK) channel, and the ORL1 receptor mRNA and GIRK mRNA coexist in hippocampus neurons [7]. These neurophysiological interactions of nociceptin with Ca 21 and K 1 channel may lead to the modulation of neurotransmitter release. Furthermore, in the supraoptic nucleus, the orphanin-FQ / nociceptin receptor activates a hyperpolarizing current via an inward-rectifier K 1 channel, which may lead to the inhibition of neurotransmitter release [13]. In thyroid

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follicular cells, there is co-localization of a rectifying K 1 channel and Na 1 -K 1 ATPase [9]. These studies implied a close functional coupling between the rectifying K 1 channel and Na 1 -K 1 ATPase. Taken together, these data suggest that nociceptin is functionally coupled with Na 1 K 1 ATPase via a rectifying K 1 channel. Nociceptin may modulate ouabain-induced release of neurotransmitter through the above mentioned mechanisms. In conclusion, nociceptin suppresses ACh release but has no effect on the NE release evoked by ouabain. However, in the presence of desipramine, nociceptin suppresses the NE release evoked by ouabain. These data suggest that nociceptin-induced inhibition of ACh or NE release is attributable to pre-synaptic modulation rather than a reversal of the ouabain effect.

Acknowledgements This study was supported by grants-in-aid for scientific research form the Ministry of Education, Science, and Culture and by a grant from the Technology Agency, Encourage system of COE.

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