Review article

B. MALINOWSKA, G. GODLEWSKI, *E. SCHLICKER


FUNCTION OF NOCICEPTIN AND OPIOID OP4 RECEPTORS IN THE REGULATION OF THE CARDIOVASCULAR SYSTEM


Department of Experimental Physiology, Medical Academy, Bialystok, Poland.
*Department of Pharmacology and Toxicology, University of Bonn, Bonn, Germany.


  Nociceptin is the endogenous ligand of the opioid OP4 or ORL1 (opioid receptor-like1) receptor. It decreases blood pressure and heart rate in anesthetized and conscious rats and mice after its intravenous and intracerebroventricular injection in a manner sensitive to OP4 but not to OP1-3 (or , and µ opioid) receptor antagonists. OP4 receptors involved in the cardiovascular effects of nociceptin were identified on sensory afferent fibres, in brain areas including the nucleus tractus solitarii and the rostral ventrolateral medulla, on preganglionic and/or postganglionic sympathetic and parasympathetic nerve fibres innervating blood vessels and heart or directly on these target organs. These receptors do not seem to be tonically activated but may play a role in the pathophysiology of inflammation, arterial hypertension and cardiac or brain circulatory ischemia.

Key words:    nociceptin, ORL1/OP4 receptors, [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2, naloxone benzoylhydrazone, blood pressure, heart rate, cardiovascular system.



INTRODUCTION

In 1994, several groups described a cDNA clone from three species (human, rat and mouse), which has a homology of about 50 % to the cDNA of the three classical opioid receptors (1). One year later, the endogenous ligand at the receptor encoded by this cDNA clone was independently described by two groups (2, 3); the heptadecapeptide was termed orphanin FQ (2; the F and Q refer to the first and last amino acid residue of the heptadecapeptide, i.e. phenylalanine and glutamine) or nociceptin (3; the name which will be used in the present review). As shown in Fig. 1, the amino acid sequence of nociceptin shows similarities to that of endogenous opioids activating the three classical opioid receptors, (OP1), (OP2) and µ (OP3). The receptor activated by nociceptin was given several names including ORL1 or OP4 (the latter abbreviation will be used here; for more details of the nomenclature, see ref. 1, 4) and resembles the three classical opioid receptors with respect to the transduction mechanisms. Thus, the OP4 receptor is coupled to a Gi/o protein and as a consequence inhibits adenylyl cyclase (5) and Ca2+ influx via voltage-sensitive Ca2+ channels (6) and activates K+ efflux via voltage-sensitive K+ channels (6). Another property shared by the OP4 and the classical opioid receptors is that they occur presynaptically on a variety of neurones where they cause inhibition of the release of the respective neurotransmitter (6, 7).

Fig. 1. Structures of nociceptin and of some of the closely related endogenous peptides acting on classical opioid receptors.

In recent years, pharmacological tools necessary for the characterization of this receptor have been described. Thus, radioligands like [3H]-nociceptin (8) and [125I]-[Tyr14]nociceptin (2) became available. Autoradiographic studies using the latter two radioligands or based on agonist-stimulated binding of [35S]-guanylyl-5´-O-(-thio)-triphosphate ([35S]-GTPS) and immunohistological studies using an antibody against the N-terminus of the OP4 receptor revealed that this receptor has a wide-spread distribution in the CNS, which, however, markedly differs from that of the three classical opioid receptors (9). The distribution of the OP4 receptor mRNA, determined by in situ hybridization studies and Northern blot and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) analyses, is similar to that of the OP4 receptor protein (9). Furthermore, many efforts were directed towards the synthesis of selective antagonists at the OP4 receptor (which is resistant to blockade by naloxone, an antagonist at the three classical opioid receptors) and eventually led to a peptide, [Nphe1]nociceptin(1-13)NH2 (10) and to a small organic molecule, the Banyu compound (11). Moreover, antisense oligodeoxynucleotides directed towards the OP4 receptor mRNA have been described (3, 12, 13) and an OP4 receptor-deficient mouse has been generated (14). Another two knockout mice lacking the precursor protein from which nociceptin is generated (3) are available as well (15, 16). From this precursor protein not only nociceptin itself but, in addition, another peptide, termed nocistatin, is produced, which counteracts some of the effects of nociceptin without directly binding to OP4 receptors (17).

Since the classical opioids play an important role in the modulation of nociception, it was an interesting question whether this function would be affected by nociceptin as well. In the two early studies, in which nociceptin was identified as the endogenous ligand of the OP4 receptor, intracerebroventricular administration of the peptide elicited hyperalgesia in mice (2, 3). This surprising finding led the authors of one of the studies to choose the name „nociceptin“ for the newly identified transmitter (3). The pronociceptive effect of nociceptin at supraspinal sites was later confirmed by other investigators (1). When, however, administered at the level of the spinal cord, nociceptin usually elicits analgesia (1). Apart from the nociceptive system, nociceptin also affects learning and memory (18), feeding (19), anxiety (20) and numerous vegetative functions (1). In our own studies, we mainly focused on the effects of nociceptin on the cardiovascular system and, in the present review, a short synopsis of our own results and of the data reported by other authors to this topic will be given.


CARDIOVASCULAR EFFECTS OF NOCICEPTIN AND ITS ANALOGUES

Cardiovascular effects of nociceptin
The following facts suggest that nociceptin may modulate the function of the cardiovascular system: (i) the endogenous opiate system plays a role in mediating cardiovascular responses (for review, see 21); (ii) both nociceptin and OP4 receptors are present in neuronal tissues involved in the regulation of these functions (for details see below; see also ref. 9). Indeed, nociceptin exerts potent effects in the cardiovascular system in various species. In urethane or pentobarbitone anesthetized rats, its intravenous (i.v.) injection produces a transient, dose-dependent (0.1–100 nmol/kg) fall in systemic blood pressure, accompanied by a strong reduction in heart rate (22-30). The depressant effects of nociceptin in the rat cardiovascular system are shown in Fig. 2. For the highest dose of the peptide the hypotension (maximally 40–60 % of basal diastolic blood pressure) develops within 30–90 s and precedes bradycardia (maximally 20% of the basal heart rate) by about 15 s. Both effects are maintained for 10 min. We have also found that nociceptin 100 nmol/kg impairs respiration and this phenomenon may also contribute to the marked cardiovascular depression produced by the peptide (30). In addition, lower doses of nociceptin (10 and 30 nmol/kg) were shown to reduce cardiac output and total peripheral resistance in rats by about 15-20% (24, 31).

An influence of anaesthesia on the cardiovascular effects of nociceptin can be excluded since similar changes in blood pressure and in heart rate were observed in conscious rats (32). Intracerebroventricular (i.c.v.) injection of nociceptin also triggers hypotension and bradycardia (32-35). Both effects were fast in onset (1–3 min) and persisted for 30-40 min (32, 33). However, according to Shirasaka et al. (35), bradycardia appeared immediately after the injection and disappeared within 15 min, whereas hypotension developed more gradually and declined within 25 min.

Fig. 2. Influence of bilateral vagotomy alone or combined with pithing on the changes in diastolic blood pressure (DBP) and heart rate (HR) induced by i.v. injection of nociceptin to urethane anaesthetized rats. In pithed animals, vasopressin (0.04 – 0.4 IU/kg/ min) was routinely infused into the right femoral vein to raise diastolic blood pressure to about 80 mmHg (i.e. to the level determined in the other two groups). Means±S.E.M. of 3-21 rats. For comparison of the mean values the one-way analysis of variance (ANOVA) was used followed by the Dunnett test. *P<0.05; ***P<0.001 compared to the control rats.

The cardiodepressant action of nociceptin does not seem to undergo tachyphylaxis. A comparable degree of response was observed in two studies in which various doses of this peptide were examined either in the same (Fig. 2; from ref. 30) or in separate animals (27). Furthermore, nociceptin (100 nmol/kg) induced similar changes in both cardiovascular parameters regardless of whether it was given alone or after three lower doses (30).

The nociceptin-induced hypotension and bradycardia were also reported in anaesthetized and conscious mice (36-38) and in anaesthetized rabbits (39). Moreover, in anaesthetized mice nociceptin was found to decrease the contractility of the left ventricle and to decrease the aortic blood flow (37, 38).

By contrast, in conscious sheep, the i.v. injection of nociceptin resulted in an increase in blood pressure and in heart rate (40). These actions were blocked by a- and b-adrenoceptor antagonists, indicating that nociceptin enhances the sympathetic activity in sheep. Several reasons have been proposed to be responsible for this discrepancy (according to the reviews 38, 41). First, in contrast to other species, the nociceptin-evoked changes in cardiovascular parameters in sheep were partly diminished by naloxone, thus suggesting the participation of opioid receptors other than OP4 receptors in these effects. Interestingly, in sheep nociceptin stimulates the release of endogenous opioid peptides, which cause sympathetic activation. Such a sympathetic stimulation leading to a vasopressor response was noticed in sheep also after administration of the OP3 receptor agonists Tyr-D-Ala-Gly-Phe(NMe)-glycinol (DAMGO) and Tyr-D-Arg-Phe-Lys-NH2 (DALDA). Second, in rats and mice the basal heart rate and blood pressure are mainly dependent on the sympathetic tone, whereas in sheep they depend on the parasympathetic tone. Thus, the differences in nociceptin-induced effects are due to an stronger inhibition of the dominant autonomic drive, which leads to hypotension and bradycardia in rats and mice and to hypertension and tachycardia in sheep.

Are the cardiovascular effects of nociceptin mediated via OP4 receptors?
Before selective pharmacological tools, especially antagonists, for OP4 receptors have been developed, the nociceptin-evoked changes could only be described as naloxone-insensitive. Indeed, the cardiovascular effects of nociceptin could not be antagonized even by very high doses of the classical opioid receptor antagonist naloxone (30, 42, 43) or other OP1-3 receptor antagonists. Bigoni et al. (22) first reported that in anaesthetized rats the OP4 receptor antagonist [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 diminished the hypotension and bradycardia elicited by nociceptin (30 nmol/kg) and failed to influence similar effects induced by carbachol. The above results were later confirmed by Lin et al. (29). In conscious mice, the nociceptin-elicited hypotension and bradycardia were counteracted by [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 and by [Nphe1]nociceptin(1-13)NH2, another OP4 receptor antagonist (37, 38). In our own studies, [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 exerted marked agonistic activity in urethane-anaesthetized rats and therefore could not be used to determine the class of opioid receptor mediating the action of nociceptin (30; see below). The nociceptin-mediated decreases in blood pressure and heart rate were however attenuated by the less specific opioid OP4 antagonist naloxone benzoylhydrazone (30; see also Fig. 3). Generally, the above results demonstrate that nociceptin elicits its effects via the activation of opioid OP4 receptors in the cardiovascular system. These effects are not sensitive to the opioid OP1-3 receptor antagonist naloxone, but they are attenuated by OP4 receptor antagonists.

Cardiovascular effects of nociceptin analogues
Several structural modifications of nociceptin have been made to yield compounds that mimic the action of the parent peptide in the cardiovascular system. These compounds were examined in various preparations. Thus, i.v. administration of [Tyr1]nociceptin had a vasodepressor effect in anaesthetized rats (43) and rabbits (39). Nociceptin(1-13)NH2 appears to be the smallest peptide in which the activity of the natural peptide is preserved both after intravenous (22, 37) and intracerebroventricular (34) injection. In urethane anaesthetized rats (22) and in conscious mice (37) hypotension and bradycardia were reported after i.v. administration of nociceptin(1-17)NH2 and nociceptin(1-13)NH2 but not after administration of nociceptin(1-9)NH2. By contrast, the fall in blood pressure and in heart rate evoked by non-amidated nociceptin(1-13)OH appears only in the presence of thiorphan, an inhibitor of neutral endopeptidase (NEP 24.11), due to the high susceptibility of nociceptin(1-13)OH to enzymatic degradation (37).

Fig. 3. Influence of naloxone benzoylhydrazone (NBH) on the changes in diastolic blood pressure (DBP) and heart rate (HR) induced by i.v. injection of nociceptin, [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 (Phe) and Acetyl-RYYRIK-NH2 (Ac-RYYRIK-NH2) to urethane anaesthetized rats. Means ± S.E.M. of 3-21 rats. For comparison of the mean values the t-test for unpaired data was used. *P<0.05; **P<0.01 compared to the group, which did not receive NBH.

Cardiovascular effects of nociceptin antagonists
Are the opioid OP4 receptors tonically activated by endogenous nociceptin? This question might be solved by the use of OP4 receptor antagonists. If such a tonical activation occurred, one might expect that OP4 receptor antagonists, by interrupting this tonical action, would show effects opposite to those of endogenous nociceptin, i.e. would increase basal blood pressure or heart rate in anaesthetized or conscious rats, mice or rabbits. However, such a phenomenon has so far not been described.

Some opioid OP4 receptor antagonists have been shown to exhibit a partial agonistic property (for review, see 44). In our laboratory we examined the effects of two compounds, initially described as OP4 receptor antagonists, namely [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 and acetyl-RYYRIK-NH2, on the cardiovascular system of urethane-anaesthetized rats (30). Similar to nociceptin, i.v. injection of both compounds exerted marked reduction in basal blood pressure and heart rate. The rank order of potencies was: nociceptin acetyl-RYYRIK-NH2 >> [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2. In comparison to nociceptin, acetyl-RYYRIK-NH2 offers some advantages that could be useful in the model of the anesthetized rat. Thus, acetyl-RYYRIK-NH2, which is as potent and almost as efficient as nociceptin, does not impair respiration at higher doses. Furthermore, a complete dose-response curve could be constructed for this peptide and the cardiovascular response has a relatively long duration (30). By contrast, the potency of [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 appeared to be lower than that of nociceptin by about 2.3 log units (30). The maximum effect of [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 was not reached up to a dose of 1000 nmol/kg. The action of all substances was insensitive to the OP1-3 receptor antagonist naloxone but sensitive to naloxone benzoylhydrazone. The results clearly demonstrate that opioid OP4 receptors are involved in the cardiovascular responses evoked by acetyl-RYYRIK-NH2 and [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 (Fig. 3; see also 30).

An agonistic activity of [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 was also determined in anaesthetized and in conscious rats after its i.v. or i.c.v. injection (22, 34). In both cases, a reduction in blood pressure and heart rate was reported. Surprisingly, Lin et al. (29) failed to detect any changes in blood pressure after i.v. injection of a very high dose of this antagonist into anaesthetized rats.


MECHANISM OF CARDIOVASCULAR EFFECTS OF NOCICEPTIN

One of the important questions regarding the cardiovascular effects of nociceptin is where the opioid OP4 receptors, which mediate the effects of the peptide, are located. The following possibilities should be taken into consideration, namely the afferent sensory fibres, various cardiovascular centres in the CNS, pre- and postganglionic autonomic nerves supplying the resistance vessels and the heart, and direct vascular and cardiac effects. For the possible site of action of other opioid peptides, see review 21. Nociceptin seems to act via different central and peripheral sites of action. The response to i.v. administration of nociceptin seems to involve peripherally located OP4 receptors, as suggested by the fact that the peptide hardly crosses the blood-brain barrier; furthermore, doses which reduce blood pressure and heart rate after i.c.v. injection fail to alter the cardiovascular parameters after i.v. injection (32, 35). On the other hand, the CNS may be involved indirectly, i.e. via reflex loops. In this context, one should consider that nociceptin may affect cardiovascular function also by modulating the release of hormones or by changing kidney function. With respect to the latter action concerning the marked diuretic and antinatriuretic effects elicited by nociceptin, the reader is referred to the excellent review by Kapusta (41) where this topic is thoroughly discussed.

The possible mechanism of the cardiovascular actions of i.v. nociceptin was first proposed by Giuliani et al. (27). They demonstrated that the nociceptin-induced reduction in blood pressure was attenuated by pretreating anaesthetized rats with guanethidine but was unaffected by bilateral vagotomy. On the other hand, bradycardia was reduced after bilateral vagotomy or by guanethidine and was abolished by the combination of both treatments. The authors concluded that the pronounced depression of cardiovascular function by nociceptin is due to the inhibition of sympathetic tone with concomitant activation of the parasympathetic outflow.

In our studies, we examined the influence of bilateral vagotomy and pithing on the changes in blood pressure and heart rate following i.v. administration of nociceptin to rats. Since pithing results in destruction of both medulla oblongata and spinal cord, this preparation offers the opportunity to study only peripheral cardiovascular effects of compounds without any interference from reflex loops and from the brain regulatory centres. Thus, we could monitor solely the peripheral effects of nociceptin and compare them with responses observed in anaesthetized and conscious animals. Since blood pressure is markedly reduced by pithing, all pithed animals received an infusion of vasopressin, which restored diastolic blood pressure to about 85 mmHg (the level observed in anaesthetized animals). Animals which were vagotomized (but not pithed) were injected with propranolol in order to counteract the vagotomy-induced tachycardia.

As shown in Fig. 2, bilateral vagotomy in anaesthetized rats abolished the cardiac response to all doses of nociceptin but diminished the vasodepression only of the highest dose of this peptide (100 nmol/kg). Thus, we can assume that the reduction in blood pressure and in heart rate are mediated by different mechanisms which are independent and dependent from the activity of vagal fibres, respectively. The hypotensive response to the highest dose of this peptide may be partly associated with marked bradycardia or with the impairment of respiration observed after administration of this peptide (see discussion above). As nociceptin failed to change cardiovascular parameters in pithed and vagotomized rats (Fig. 2) our results clearly demonstrate that the inhibition of sympathetic activity accounts for the vasodepression induced by this peptide. Our study confirms and extends the earlier observations of Giuliani et al. (27) who examined a high dose of nociceptin (100 nmol/kg) only. Moreover, they applied guanethidine, a blocker of sympathetic activity, which decreased basal blood pressure by itself by more than 30%. This alteration of the baseline condition might per se significantly impair the vasodepressor action of nociceptin (27).

Effects of nociceptin on sensory transmission
Does nociceptin affect sensory transmission? This question was examined in various vascular and cardiac preparations containing sensory neurons. The antidromic vasodilation induced in small-diameter afferent fibres in hairless skin of the anesthetized rat hindlimb is predominantely mediated by calcitonin gene-releated peptide (CGRP; 45). Nociceptin given i.v. was shown to reduce the electrically evoked antidromic vasodilation in this experimental model (28). The maximum extent of inhibition was approximately 100% for the highest dose of nociceptin (100 nmol/kg). This effect was not blocked by nocistatin (28); this finding does, however, by no means exclude that OP4 receptors are involved since nocistatin, although interfering with some effects of nociceptin, is devoid of affinity for OP4 receptors (17).

CGRP is also the main non-adrenergic non-cholinergic (NANC) neurotransmitter released from the sensory neurons that produces a delayed positive inotropic effect in the heart. Nociceptin has been demonstrated to markedly inhibit the electrically stimulated release of CGRP from capsaicin-sensitive sensory nerve terminals in the guinea-pig left atrium in a concentration-dependent manner (46). The maximum inhibition (about 90 %) was reached for this peptide at the concentration of 1 mM. This action involves presynaptic opioid OP4 receptors since nociceptin failed to modify the positive inotropic effect of exogenous CGRP and the action of the peptide was not inhibited by a mixture of naltrindole, nor-binaltorphimine and naloxone to block NH2, OP2 and OP3 receptors, respectively.

As suggested by Kapusta (41) in his review, the stimulation of presynaptic receptors on capsaicin-sensitive sensory nerve terminals in guinea-pig left atrium may reduce central sympathetic outflow by activating cardiopulmonary vagal afferent fibres. Such an activation, observed for example during the Bezold-Jarisch reflex and vaso-vagal syncope, might augment central parasympathetic outflow via a reflex loop and inhibit sympathetic activity, leading to a reduction in blood pressure and heart rate. In line with this suggestion are the following findings: (i) the pattern of cardiovascular changes elicited by nociceptin (e.g. marked vasodilation and bradycardia) is similar to that observed in vaso-vagal syncope or the Bezold-Jarisch reflex; (ii) the cardiodepressant action of nociceptin is sensitive to bilateral vagotomy, guanethidine and pithing (27; see also Fig. 2). However direct experiments in vivo are required to confirm the suggestion of Kapusta (41).

Central mechanisms of cardiovascular effects of nociceptin
Morphological studies have revealed high to moderate density of both opioid OP4 receptors and endogenous nociceptin in brain regions involved in the central regulation of cardiovascular function, such as nucleus tractus solitarii (NTS), rostral ventrolateral medulla (RVLM), nucleus ambiguus (NAmb), various hypothalamic nuclei and hypothalamus-related areas (for review, see ref. 9, 47, 48). Only recently, functional studies have confirmed the possible participation of this nociceptinergic system in the regulation of the cardiovascular function.

The inhibition of the sympathetic nerve activity after central administration of nociceptin was demonstrated directly by Kapusta and Kenigs (33) and by Shirasaka et al. (35) in conscious rats. In both studies, the i.c.v. injection of this peptide produced bradycardia and hypotension associated with a decrease in the renal sympathetic nerve activity (RSNA). This sympathoinhibitory response occurred, however, either after the recovery of blood pressure to the preinjection value (33) or along with the changes in cardiovascular parameters (35). The nociceptin-evoked bradycardia was diminished by propranolol but not by atropine, indicating that inhibition of the cardiac sympathetic outflow is responsible for this effect (35). Interestingly, in conscious rats, in which the arterial baroreceptors were removed (sinoaortic-denervated rats), cardiovascular and RSNA responses to nociceptin were increased when compared to intact animals (35). The papers suggest that nociceptin (i) may modulate the sympathetic nerve activity via central mechanism(s) and (ii) that this action is, at least partially, dependent on the afferent inputs from the baroreceptors.

The nucleus tractus solitarii (NTS) is a medullary area where various cardiac sensory afferents terminate, including those of the baroreceptor, chemoreceptor, Bezold-Jarisch and Breuer-Hering reflexes (for review, see 49). This region proved to be one of the possible sites of the central action of nociceptin. The unilateral injection of nociceptin into the NTS elevated blood pressure and heart rate both in conscious and in pentobarbitone anesthetized rats (50). The nociceptin-induced changes in the cardiovascular system were blocked by nocistatin but appeared insensitive to naloxone, suggesting that opioid OP4 receptors may mediate the action of this peptide in the NTS. An activation of these receptors by nociceptin also modulates the baroreflex, as was shown in another study by Mao and Wang (51). They found that injection of nociceptin into the NTS attenuated the baroreceptor reflex bradycardia elicited by i.v. injection of phenylephrine. Again, this action of nociceptin was diminished by nocistatin but not by naloxone, suggesting the participation of OP4 receptors.

The rostral ventrolateral medulla (RVLM) is a key centre in the CNS responsible for the tonical pressor activity of the sympathetic nerve fibres. Chu et al. (52, 53) reported that the bilateral injection of nociceptin (10 nmol) into the RVLM in a-chloralose/urethane anesthetized rats reduced blood pressure and heart rate by 32% and 15%, respectively. The same dose of the peptide had no effect on cardiovascular activity after i.v. injection. Chu et al. (52, 54) also showed that nociceptin (0.3-10 nmol) diminished the spontaneous discharges of RVLM neurons in rat brain slices in a concentration-dependent fashion. The effects of nociceptin observed both under in vivo and in vitro conditions were counteracted by the opioid OP4 receptor antagonist [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2, but not by the OP1-3 receptor receptor antagonist naloxone (53, 54).

However, opioid OP4 receptors do not seem to alter the physiological activity of the RVLM since [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 did not change the basal cardiovascular parameters when given alone (53, 54). A similar conclusion may be reached with respect to the NTS in which nocistatin by itself was inactive (50, 51).

A functionally important pathway leads from the NTS to the nucleus ambiguus (NAmb), an area within the medulla oblongata containing preganglionic parasympathetic cardiac neurons. In a recent in vitro study, using whole-cell patch-clamp recordings, nociceptin has been shown to inhibit GABAergic inputs to the rat cardiac parasympathetic neurons in this brain region (55). In other words, nociceptin interrupts the tonical inhibition of the preganglionic parasympathetic neurons, thereby increasing the parasympathetic outflow. This mechanism might, at least in part, explain the nociceptin-induced bradycardia. In detail, Venkatesan et al. (55) found that nociceptin (100 µM) decreased the frequency of the spontaneous inhibitory postsynaptic currents (IPSCs) by 36%, suggesting that the site of action of nociceptin is presynaptic at the GABAergic neurons synapsing on the preganglionic parasympathetic neurons. In addition, nociceptin (100 µM) also inhibited the amplitude of the IPSCs (by 50%), leaving open the possibility that it has a postsynaptic site of action as well. This postsynaptic site was proven in additional experiments in which nociceptin (100 µM) decreased the postsynaptic current elicited by GABA by 37%.

The hypothalamic paraventricular nucleus (PVN) is one of the nuclei involved in the control of the function of the autonomic nervous system. This nucleus has been postulated as another possible site of action of nociceptin since in experiments with the whole cell patch-clamp recording technique, nociceptin and [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 have been found to hyperpolarize the membrane potential of rat PVN brain slices (56).

Peripheral effects of nociceptin on the activity of sympathetic fibres innervating blood vessels and heart
The model of the pithed rat was used in our laboratory to examine the influence of nociceptin on the neurogenic vasopressor and tachycardic responses. Electrical stimulation (1 Hz) of the preganglionic sympathetic nerve fibres produced an increase in blood pressure and heart rate by about 30 mmHg and 70 beats/min, respectively. Both neurogenic cardiovascular responses (which are predominantly related to the release of catecholamines) were inhibited by nociceptin (1 – 1000 nmol/kg) in a dose-dependent manner (maximally by about 60% at the highest dose), as shown in Fig. 4. The inhibitory effects of nociceptin were not modified by the opioid OP1-3 receptor antagonist naloxone (57, 58), but were diminished by the OP4 receptor antagonists naloxone benzoylhydrazone and [Phe1Y(CH2-NH)Gly2]-nociceptin(1-13)NH2 (Fig. 4). This clearly demonstrates that these effects are mediated through the activation of opioid OP4 receptors.

Three additional series of experiments were carried out to determine the exact location of the inhibitory opioid OP4 receptors. First, the possibility that these receptors are located postsynaptically on the end-organ could be excluded, since nociceptin failed to modify the noradrenaline and isoprenaline-induced increases in blood pressure (57) and in heart rate (58), respectively. Second, the nicotine-induced vasopressor and tachycardic responses were reduced by nociceptin in a manner sensitive to naloxone benzoylhydrazone but not to naloxone (57, 58; also Fig. 5). Since the degree of inhibition produced by the peptide was similar in animals stimulated electrically or by nicotine we concluded that the inhibitory OP4 receptors are located presynaptically on the postganglionic sympathetic nerve endings innervating the rat resistance vessels and the heart. Third, our in vitro experiments on isolated pieces of atrium have shown that nociceptin (studied in the presence of naloxone) reduces the electrically (1 Hz) evoked release of [3H]noradrenaline from sympathetic terminals in a manner sensitive to naloxone benzoylhydrazone. The maximum inhibitory effect of nociceptin (100 nM) amounted to about 60% (Fig. 5). Both our in vivo and in vitro results do, however, not exclude the possibility that OP4 receptors might also be present on the preganglionic nerve terminals.

Fig. 4. Influence of nociceptin (alone) or in the combination with naloxone benzoylhydrazone (NBH) or with [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 (Phe) on the increase in diastolic blood pressure (DBP) or heart rate (HR) induced by electrical stimulation of the preganglionic sympathetic nerves in pithed and vagotomized rats. Means±SEM of 4-9 rats. For comparison of the mean values the one-way analysis of variance (ANOVA) was used followed by the Dunnett test. *P<0.05, **P<0.01 compared to the corresponding group which did not receive NBH or Phe.

Fig. 5. Influence of nociceptin alone or in the combination with naloxone benzoylhydrazone (NBH) on the increase in heart rate (HR) induced by injection of nicotine 2 mmol/kg in pithed and vagotomized rats and on the electrically-evoked [3H]noradrenaline (NA) release from isolated pieces of rat atrium. Means±SEM of 4-9 rats. For comparison of the mean values the t-test for unpaired data was used. *P<0.05 compared to the corresponding group which did not receive NBH.

The presynaptic inhibitory OP4 receptors on the postganglionic sympathetic nerve fibres supplying the resistance vessels, but not the heart, of the rat appear to be tonically activated by endogenous nociceptin. Thus, an increase of the neurogenic vasopressor response by about 15% was obtained with the OP4 receptor antagonists naloxone benzoylhydrazone and [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 (but not with the OP1-3 antagonist naloxone; 57, 58).

Our observations concerning the occurrence of presynaptic inhibitory OP4 receptors on the postganglionic sympathetic nerve fibres supplying the heart and arteries are further supported by the reports by Bucher (59) and Trendelenburg et al. (60) who showed that nociceptin reduced the electrically-evoked release of [3H]noradrenaline from the isolated rat tail artery and from mouse atrium pieces, respectively. In both cases, again the effects proved to be insensitive to naloxone, but were blocked by naloxone benzoylhydrazone or [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2. In addition, nociceptin diminished the sympathetic inotropic response evoked by electrical field stimulation of the isolated guinea-pig atrium (46). The fact that OP4 receptor mRNA was found in the sympathetic ganglia of the guinea-pig (61) and the rat (62) fits well to the studies by Bucher (59), Trendelenburg et al. (60), Giuliani and Maggi (46) and our own investigations (57, 58).

OP4 receptor-mediated inhibition of noradrenaline release was also observed in two studies in which the release of the catecholamine was evoked by chemical rather than electrical stimulation. Using a cardiac dialysis technique to monitor the myocardial interstitial level of noradrenaline in anaesthetized cats treated with desipramine (a noradrenaline uptake inhibitor), nociceptin was shown to inhibit the oubain-elicited release of noradrenaline in a manner sensitive to nocistatin (63). Moreover, nociceptin inhibited [3H]noradrenaline release evoked by inhibition of glycolysis and oxidative phosphorylation (by iodoacetate and sodium cyanide, respectively, to provoke ischemia) from rat cardiac synaptosomal fractions (64).

In another paper, Dumont and Lemaire (65) showed that nociceptin decreases [3H]noradrenaline uptake in rat cardiac membrane preparations (although the involvement of OP4 receptors in this effect and also in the nociceptin-induced inhibition of ischemia-related inhibition of [3H]noradrenaline release remains to be proven). Interestingly, in spontaneously hypertensive rats (SHR), nociceptin turned out to be 1.6-fold more potent in inhibiting the uptake of [3H]noradrenaline than in Wistar Kyoto control animals (WKY). This effect in SHR was accompanied by a pronounced increase in the level of cardiac OP4 mRNA and in high affinity binding sites for nociceptin. By contrast, under physiological conditions the OP4 receptor mRNA appears to be very low (66) and the nociceptin precursor mRNA is undetectable (67) in hearts of adult rats. Based on the above findings an intriguing theory was proposed by Salis et al. (38) in their review, namely that (i) opioid OP4 receptors expressed in neonatal cardiomyocytes are down-regulated during ontogenesis, and that (ii) expression of OP4 receptors may become reactivated in adults under pathological states occurring in the cardiovascular system (e.g. ischemia, hypertension).

Some pieces of evidence suggest that nociceptin may also modify the function of the preganglionic sympathetic nerve fibres. In urethane-anaesthetized rats, intrathecal injection of nociceptin dose-dependently reduced blood pressure and heart rate (68). The maximum inhibitory effect of nociceptin occurred at 30 nmol/kg and amounted to 35% and 20% for the respective basal vascular and cardiac values. The depression of cardiovascular responses lasted for about 30 min and was insensitive to naloxone. Furthermore, using whole cell patch clamp recordings on spinal cord slices of the rat, Lai et al. (68) found that nociceptin suppressed the excitatory postsynaptic potentials (EPSPs) of sympathetic preganglionic neurons evoked by focal stimulation and hyperpolarized these neurons, both effects being insensitive to naloxone. The above results suggest that nociceptin may act both presynaptically (by inhibiting glutamate release from the neurons synapsing on the preganglionic sympathetic neurons) and postsynaptically (by reducing the membrane excitability of the preganglionic sympathetic neurons). The presynaptic site of action may be associated with the modulation of N-type Ca2+ channels; indeed, Larsson et al. (69) have shown that nociceptin acts as a potent inhibitor of these channels. Moreover, the modulatory role of nociceptin on the function and activity of the preganglionic sympathetic nerve fibres is also suggested by the occurrence of nociceptin-like immunoreactivity in the rat intermediolateral cell column (70).

Peripheral effects of nociceptin on the activity of the parasympathetic fibres innervating the heart

Our laboratory studied the influence of nociceptin on bradycardia induced by electrical stimulation (5 Hz) of the cardiac end of the transected right vagus nerve in pithed and vagotomized rats. We found that nociceptin diminished the neurogenic bradycardia in a dose-dependent manner (maximally by about 40% at 1000 nmol/kg; Fig. 6). The inhibitory effect of nociceptin was sensitive to naloxone benzoylhydrazone but resistant to naloxone. Additional experiments with methacholine were carried out in order to distinguish between a pre- and postjunctional site of action. We found that nociceptin failed to modify the methacholine-related decrease in heart rate. In summary, the electrically induced bradycardia in pithed rats is inhibited by OP4 receptors located presynaptically on the post- and/or preganglionic cardiac vagal nerve fibres. The OP4 receptors do not appear to be tonically activated since naloxone benzoylhydrazone did not change the vagally-induced bradycardia by itself (58).

Fig. 6. Influence of nociceptin alone or in the combination with naloxone benzoylhydrazone (NBH) on the decrease in heart rate (HR) induced by electrical stimulation of the transected right vagus nerve. Means±SEM of 5-9 rats. For comparison of the mean values the t-test for unpaired data was used. *P<0.05 compared to the corresponding group which did not receive NBH.

There are another two papers in which an inhibitory effect of nociceptin on acetylcholine release was described. In the study by Giuliani and Maggi (46), nociceptin inhibited the negative inotropic response elicited by acetylcholine (released in response to electrical stimulation) in the isolated guinea-pig left atria. Yamazaki et al. (63) found that nociceptin inhibits the oubain-induced release of acetylcholine from the left ventricular wall in anaesthetized cats, in a manner sensitive to nocistatin.

Direct effects of nociceptin on blood vessels
Direct vasorelaxation induced by nociceptin may also contribute to the hypotensive effect of the peptide. Such a response was first reported in anaesthetized rats in the model of the perfused hindquarters. Intraarterial injection of nociceptin was shown to reduce the perfusion pressure in this preparation (23, 42, 71, 72). Since adrenergic motor neurons projecting to this vascular bed had been sectioned during preparation, the possibility of modulation of the sympathetic drive by nociceptin was rather unlikely. Therefore, direct effects of nociceptin on blood vessels should be taken into consideration. The decrease in perfusion pressure was dose-dependent (1-30 nM, maximal fall by 35%) and naloxone-insensitive. However, as has been demonstrated very recently, it was antagonized by the OP4 receptor antagonist [NPhe1]nociceptin(1-13)-NH2, confirming the postsynaptic localization of OP4 receptors in vascular bed (72). The vasodilatation developed rapidly and lasted up to 6 min (for nociceptin at a dose of 3 nM). Similar to the nociceptin-evoked changes in the systemic circulation, the perfusion pressure in rat hindlimbs was diminished by [Tyr1]-nociceptin but not by nociceptin(2-17), nociceptin(1-11) and nociceptin(1-7) (42). The same group also demonstrated that nociceptin exhibits a direct vasodilator activity in the rat isolated pulmonary vasculature, which was again insensitive to naloxone (31). However, this peptide appeared much less potent in decreasing the vascular resistance in the pulmonary vessels than in the hindquarters of the rat since the highest concentration of nociceptin (30 nM) decreased the pulmonary resistance by about 4% as opposed to 35% in the hindquarters. A non-neurogenic vasorelaxation was also observed in the sympathetically denervated cutaneous vascular bed of the anaesthetized rat after intravenous injection of nociceptin (28). A dose of 100 nmol/kg of nociceptin decreased the vascular resistance by about 40% of the basal value; this effect lasted up to 4 min.

The direct vasorelaxant properties of nociceptin were also confirmed in vitro on isolated feline renal, carotid and femoral rings (73), in the rat aorta (74) and in the resistance arteries of the rat mesenteric vascular bed (75), precontracted with phenylephrine or the thromboxane analogue U46619.

In chloralose anaesthetized pigs, topical administration of nociceptin elicited pial artery dilatation of the pig in a manner sensitive to [Phe1(CH2-NH)Gly2]-nociceptin(1-13)NH2 but resistant to naloxone (76). Effects of this peptide were more pronounced in newborn than in juvenile animals (77). Interestingly, some pathological conditions e.g. brain injury (77, 78) or hypoxia/ischemia (79) are associated with a marked release of nociceptin into the cerebrospinal fluid. Both the release of nociceptin and the altered vessel responsiveness to the peptide may contribute to the impairment of cerebrovasodilatation induced by hypercapnia or N-methyl-D-aspartate following brain injury or hypoxic/ischemic incidences (78, 79, 80, 81, 82).

Mechanisms of vasodilator effects of nociceptin
Evidence has been provided that the direct vasodilation produced by nociceptin is endothelium-independent and does not involve the release of nitric oxide. Indeed, removal of the endothelium failed to alter the response of the isolated rat mesenteric artery bed to the peptide (75). Moreover, the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine (L-NAME) had no effect on the response to nociceptin in the latter model (75) nor did it influence the effect of nociceptin on perfusion pressure of the rat hindquarters (26). In line with the latter data are the findings by Armstead (76), who showed that piglet pial artery dilatation in response to nociceptin was not altered by another NO-synthase inhibitor, L-NNA (N- nitro-L-arginine), or by the protein kinase G inhibitor, Rp 8-Br cGMPs, and was not accompanied by an increase in the cGMP level in the cerebrospinal fluid. All the above observations clearly indicate that the vasorelaxant action of nociceptin does not involve an NO-cGMP-dependent pathway.

The possibility that prostaglandins are involved in the vasodilator effects of nociceptin had to be considered as well. The data in the literature are controversial. Thus, the nociceptin-elicited decrease in the perfusion pressure of the rat hindquarters was not modified by the cyclooxygenase inhibitor sodium meclofenamate (26). By contrast, pretreatment with indomethacin, another cyclooxygenase inhibitor (83, 84), partially restored the pial artery dilatation in the pig elicited by nociceptin (85). In addition, nociceptin led to an increase in 6-ketoPGF1alpha, the breakdown product of PGI2 (the receptor of which is positively coupled to adenylyl cyclase) in the cerebrospinal fluid of the pig. In addition, the level of cAMP itself was increased in the cerebrospinal fluid. Moreover, the nociceptin-evoked cerebrovasodilation was attenuated by glibencamide (KATP channel antagonist; 86) and by iberiotoxin (KCa channel blocker; 76). These data indicate that the dilation of the pig pial artery elicited by nociceptin results from the release of cAMP; the latter might activate calcium-sensitive (KCa) and ATP-dependent K+ (KATP) channels. On the other hand, KATP channels do not appear to be involved in the nociceptin-induced vasodilatation in the rat hindquarters since this vasodilatation was not affected by U-37883A, another KATP channel antagonist (26).

Other possible mechanisms of the direct vasodilation evoked by nociceptin have also been examined. The involvement of muscarinic, alpha-adrenergic or CGRP receptors in the vasorelaxation in the rat mesenteric artery vascular bed could be excluded because the response to the peptide was not modified by atropine, phentolamine or CGRP-(8-37) (75). An interesting finding was provided by Kimura et al. (87) who found that nociceptin stimulates the release of histamine from rat peritoneal mast cells and that intradermal application of the peptide increases the vascular permeability. This response was insensitive to naloxone but it was reduced by the histamine H1 receptor antagonist pyrilamine. The authors’ conclusion was that nociceptin induces an increase in vascular permeability through the release of histamine from mast cells.

Direct effects of nociceptin on heart
Little is known with respect to a direct influence of nociceptin on the heart. Quite surprisingly, nociceptin increased the rate of spontaneously beating cardiomyocytes obtained from cultured rat neonatal hearts in a concentration-dependent (0.1–10 mmol/l) manner (88). The maximum effect reached 65% of the response to the b-adrenergic agonist isoprenaline. The chronotropic action of nociceptin was blocked by the OP4 receptor partial agonist/antagonist acetyl-RYYRIK-NH2, which by itself did not exhibit any activity in this experimental model. Future studies have to show whether a positive chronotropic effect of nociceptin can also be elicited in the isolated heart or in vivo.


CONCLUDING REMARKS

Nociceptin, the endogenous ligand of the opioid OP4 receptors, has marked effects on the cardiovascular system. As shown in Fig. 7 it has different central and peripheral sites of action. Thus, the nociceptin-induced hypotension and bradycardia in the rat, mouse and rabbit may be directly due to its effect on the inputs from the sensory afferent neurones in blood vessels, heart or baroreceptors. In addition, nociceptin reduces the activity of the sympathetic nervous system acting on the rostral ventrolateral medulla and on pre- or postganglionic nerve fibres innervating the heart and the resistance vessels. Furthermore, the bradycardia elicited by nociceptin may involve the inhibition of the GABAergic inputs to the cardiac parasympathetic neurons in the nucleus ambiguous. The direct vasodilatory action of nociceptin should also be taken into consideration.

Fig. 7. Distribution of opioid OP4 receptors involved in the regulation of cardiovascular function. Various locations of OP4 receptors as suggested by functional studies are shown. Excitatory neurons are represented by solid lines, inhibitory neurons are represented by dotted lines. Abbreviations: CVLM - caudal ventrolateral medulla; NAmb - nucleus ambiguus; NTS - nucleus tractus solitarii; PVN - paraventricular nucleus; RVLM - rostral ventrolateral medulla.

One should, however, not overlook that there were some inconsistencies with respect to the cardiovascular effects of nociceptin in the rat. To give just two examples. (i) Topical administration of nociceptin to the nucleus tractus solitarii elicits an increase, rather than a decrease, in blood pressure and heart rate. (ii) In cultured myocytes, nociceptin increases, rather than decreases, heart rate. These effects may dampen the overall depressant influence of nociceptin on cardiovascular parameters. The most striking discrepancy, however, refers to the sheep in which i.v. injection of nociceptin, unlike in the rat, mouse or rabbit, elicits an increase in blood pressure and heart rate.

What is the role of nociceptin and its receptor in the regulation of the cardiovascular system? The opioid OP4 receptors involved in this function do not seem to be tonically activated under physiological conditions. In humans, the concentration of endogenous nociceptin detected in plasma is as low as 10 pg/ml 5.5 pM (89). However, it is upregulated in the dorsal root ganglia (of the rat) during peripheral inflammation (90). Under such conditions the role of the opioid OP4 receptors in the regulation of the cardiovascular system may increase. So far, a role of nociceptin was suggested in the pathophysiology of inflammation, cardiac or brain circulatory ischemia and arterial hypertension and OP4 receptor agonists might become drugs for antihypertensive treatment. OP4 receptor agonists may also represent a new class of anti-anxiety drugs (20; 91) and in this context the elucidation of cardiovascular effects of nociceptin may be of practical relevance in order to assess potential side effects. Further studies are needed to get a better understanding of the role of nociceptin and its receptor in the physiology and especially in the pathophysiology of the cardiovascular system.

Acknowledgements: This work was supported by Medical Academy of Bialystok grant No 3-13844.


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R e c e i v e d:  February 21, 2002
A c c e p t e d: July 11, 2002

Author’s address: Prof. Barbara Malinowska, Department of Experimental Physiology Medical Academy, 15-230 Białystok, Mickiewicza Str. 2A, Poland. Tel./fax (48-85) 7420148.