Modulation of gastrointestinal function by MuDelta, a mixed µ opioid receptor agonist/ µ opioid receptor antagonist.

BACKGROUND & PURPOSE: Loperamide is a selective µ opioid receptor agonist acting locally in the gastrointestinal (GI) tract as an effective anti-diarrhoeal but can cause constipation. We tested whether modulating µ opioid receptor agonism with δ opioid receptor antagonism, by combining reference compounds or using a novel compound ('MuDelta'), could normalize GI motility without constipation. EXPERIMENTAL APPROACH: MuDelta was characterized in vitro as a potent µ opioid receptor agonist and high-affinity δ opioid receptor antagonist. Reference compounds, MuDelta and loperamide were assessed in the following ex vivo and in vivo experiments: guinea pig intestinal smooth muscle contractility, mouse intestinal epithelial ion transport and upper GI tract transit, entire GI transit or faecal output in novel environment stressed mice, or four weeks after intracolonic mustard oil (post-inflammatory). Colonic δ opioid receptor immunoreactivity was quantified. KEY RESULTS: δ Opioid receptor antagonism opposed µ opioid receptor agonist inhibition of intestinal contractility and motility. MuDelta reduced intestinal contractility and inhibited neurogenically-mediated secretion. Very low plasma levels of MuDelta were detected after oral administration. Stress up-regulated δ opioid receptor expression in colonic epithelial cells. In stressed mice, MuDelta normalized GI transit and faecal output to control levels over a wide dose range, whereas loperamide had a narrow dose range. MuDelta and loperamide reduced upper GI transit in the post-inflammatory model. CONCLUSIONS AND IMPLICATIONS: MuDelta normalizes, but does not prevent, perturbed GI transit over a wide dose-range in mice. These data support the subsequent assessment of MuDelta in a clinical phase II trial in patients with diarrhoea-predominant irritable bowel syndrome.

Cardiovascular and renal responses produced by central orphanin FQ/nociceptin occur independent of renal nerves.

The present study investigated the role of the renal nerves in mediating the cardiovascular and renal responses produced by the central administration of the opioid-like peptide orphanin FQ/nociceptin (OFQ/N) in conscious Sprague-Dawley rats. In conscious rats, OFQ/N (10 microgram icv) produced a transient bradycardia and hypotension (nadir 20 min). Although renal sympathetic nerve activity (RSNA) initially remained unchanged, a delayed renal sympathoinhibitory response occurred after recovery (30 min) of blood pressure. By 30 and 70 min postinjection, RSNA decreased to 75 and 66% of control, respectively. Coinciding with the decrease in RSNA, central OFQ/N elicited a diuresis and antinatriuresis that occurred independent of changes in renal hemodynamics. In other studies, intracerebroventricular OFQ/N produced similar cardiovascular and renal excretory responses in bilaterally renal-denervated rats. Finally, in conscious sinoaortic deafferentiated rats, intracerebroventricular OFQ/N produced a rapid decrease in RSNA (55% of control, 10 min; 38% of control, 20 min) that paralleled the onset of the hypotension and bradycardia. These studies demonstrate that in conscious rats, intracerebroventricular OFQ/N produces a centrally mediated inhibition of RSNA which, due to activation of baroreflex mechanisms, is temporally dissociated from the hypotensive and bradycardia responses. As revealed in renal-denervated rats, the cardiovascular and renal excretory responses produced by central OFQ/N occur by a pathway that is independent of intact renal nerves or changes in renal hemodynamics.

Central administration of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)-NH2 and orphanin FQ/nociceptin (OFQ/N) produce similar cardiovascular and renal responses in conscious rats.

In vitro studies have shown that [Phe1Psi(CH2-NH)Gly2]OFQ/N(1-13)-NH2 (referred to as [FG]OFQ/N(1-13)-NH2) is the first selective antagonist to prevent the binding of the endogenous ligand orphanin FQ/Nociceptin (OFQ/N) at the orphan opioid-like receptor. In the present study, we examined the potential changes in cardiovascular and renal function produced by the i.c.v. injection of [FG]OFQ/N(1-13)-NH2 in conscious Sprague-Dawley rats. In conscious rats, i.c.v. injection of [FG]OFQ/N(1-13)-NH2 produced a marked and sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate (i.e., a water diuresis). The cardiovascular and renal excretory responses produced by i.c.v. [FG]OFQ/N(1-13)-NH2 were dose dependent and were similar in pattern but of longer duration than responses evoked by i.c.v. OFQ/N. In other animals, the i.c.v. injection of OFQ/N(1-13)-NH2, a potential metabolite of [FG]OFQ/N(1-13)-NH2, produced changes in cardiovascular and renal function that were comparable to those evoked by i.c.v. [FG]OFQ/N(1-13)-NH2. In contrast, OFQ/N(2-17), a fragment of OFQ/N [OFQ/N(1-17)], was inactive when administered centrally. Finally, studies were performed to determine whether [FG]OFQ/N(1-13)-NH2 may be an antagonist at the orphan opioid-like receptor receptor when administered centrally at a dose that alone was inactive. In these studies, i.c.v. pretreatment of animals with low-dose [FG]OFQ/N(1-13)-NH2 failed to prevent the cardiovascular and renal excretory response to i.c.v. OFQ/N. Although [FG]OFQ/N(1-13)-NH2 is reported to be an antagonist of the OFQ/N receptor in vitro, these findings indicate that this compound has agonist activity similar to that of the endogenous ligand OFQ/N when administered centrally in vivo.

Central alpha2-receptor mechanisms contribute to enhanced renal responses during ketamine-xylazine anesthesia.

We have recently developed an experimental approach to study central opioid control of renal function in anesthetized rats. This model system uses the intravenous infusion of the alpha2-agonist xylazine to enhance basal levels of urine flow rate and urinary sodium excretion in ketamine-anesthetized rats. This study examined the contribution of central and peripheral alpha2-adrenergic receptor mechanisms in mediating the enhanced renal excretory responses produced by xylazine. In ketamine-anesthetized rats, the enhanced levels of urine flow rate and urinary sodium excretion produced by the intravenous infusion of xylazine were reversed by the intravenous bolus injection of the alpha2-adrenoceptor antagonist yohimbine but not by the alpha1-adrenoceptor antagonist terazosin. In separate animals the intracerebroventricular administration of yohimbine only reduced urine flow rate by approximately 50% but did not alter urinary sodium excretion. The decrease in urine flow rate produced by intracerebroventricular yohimbine was reversed by the intravenous injection of a selective V2-vasopressin receptor antagonist. In a separate group of ketamine- and xylazine-anesthetized rats, the bilateral microinjection of yohimbine into the hypothalamic paraventricular nucleus (PVN) also significantly decreased urine flow rate by 54% without altering urinary sodium excretion. The microinjection of the beta-adrenoceptor antagonist propranolol into the PVN did not alter either renal excretory parameter. These results suggest that during intravenous infusion, xylazine increases urine flow rate by activating alpha2-adrenergic receptors in the PVN, which in turn decrease vasopressin release. The ability of alpha-adrenergic mechanisms in the PVN to selectively influence the renal handling of water, but not sodium, may contribute to the reported dissociation of the natriuretic and diuretic responses of alpha2-adrenoceptor agonists.

Renal excretory responses produced by the delta opioid agonist, BW373U86, in conscious rats.

Studies were performed in conscious Sprague-Dawley rats to characterize the changes in renal excretory function produced by activation of delta opioid systems. The intravenous infusion of 50 microgram/kg/min, BW373U86 (BW), a nonpeptide delta opioid receptor agonist, produced a significant increase in urine flow rate and urinary sodium excretion. The infusion of BW at a dose of 30 microgram/kg/min produced diuresis without affecting urinary sodium excretion. In contrast, BW did not alter either renal excretory parameter at a dose of 10 microgram/kg/min. The renal responses produced by BW occurred without changes in heart rate or mean arterial blood pressure. The diuretic and natriuretic responses produced by the i.v. infusion of BW (50 microgram/kg/min) were prevented by pretreatment of animals with the selective delta opioid receptor antagonist, naltrindole (1 mg/kg, i.v.). When administered alone, naltrindole (1 mg/kg, i.v.) failed to change any systemic cardiovascular or renal excretory parameter. In other groups of animals, the peripheral administration of the delta opioid receptor agonist, SNC80, also evoked a profound diuretic and natriuretic response (naltrindole sensitive) similar to that produced by BW. In contrast to these findings, the diuretic and natriuretic response produced by BW infusion (30 or 50 microgram/kg/min, i.v.) was abolished in rats having undergone chronic bilateral renal denervation. Together, these results demonstrate that the peripheral administration of BW373U86 or SNC80 produce marked diuretic and natriuretic responses in conscious Sprague-Dawley rats via a delta opioid receptor pathway and that intact renal nerves are required for mediating these responses. Although endogenous delta opioid systems do not appear to exert a tonic influence under basal conditions, these findings suggest that delta opioid pathways may evoke significant changes in renal excretory function under conditions in which these systems are activated.

Diuretic and antinatriuretic responses produced by the endogenous opioid-like peptide, nociceptin (orphanin FQ).

Nociceptin (orphanin FQ) is a novel peptide isolated from brain tissue that has an amino acid sequence most similar to that of the endogenous opioid peptide dynorphin A. Aside from this similarity, the association of nociceptin to the endogenous opioid peptide systems and the functional importance of this new peptide in vivo are not completely known. Here we report that nociceptin is physiologically active in vivo and produces marked changes in the renal excretion of water and sodium. In conscious Sprague-Dawley rats, intravenous infusion of nociceptin produced a profound increase in urine flow rate and decrease in urinary sodium excretion. In further studies, intracerebroventricular (i.c.v.) injection of nociceptin into conscious rats produced a concurrent diuresis (dose-dependent) and antinatriuresis. The magnitude and pattern of the central nociceptin-induced water diuresis was similar to that produced by i.c.v. dynorphin A. Whereas i.c.v. pretreatment with the selective kappa-opioid receptor antagonist, nor-binaltorphimine, completely prevented the renal responses produced by dynorphin A, this antagonist did not alter the diuresis or antinatriuresis produced by central nociceptin. Thus, these results indicate that in conscious rats, nociceptin produces a selective water diuresis via a central nervous system mechanism independent of kappa-opioid receptors. Together, these observations suggest that endogenous nociceptin may be a novel peptide involved in the central control of water balance and ultimately in the regulation of arterial blood pressure. In the future, analogues of nociceptin may prove to be the first clinically useful water diuretics for patients with water-retaining diseases.

Chronic treatment with morphine and ethanol, but not cocaine, attenuates IL-1 beta activation of FOS expression in the rat hypothalamic paraventricular nucleus.

Interleukin-1 beta (IL-1 beta) is a key mediator of immunological and pathological responses to stress, injury and disease and it has been suggested to have profound effects on neuroendocrine-immune functions. We have shown that central treatment with IL-I beta induces the expression of FOS proto-oncogene protein immunoreactivity (FOS-IR) in several hypothalamic nuclei including the paraventricular nucleus (PVN). Since FOS expression has been used as an anatomical marker of neuronal function, these results suggested that the involvement of IL-1 beta in the neuro-endocrine-immune axis may be mediated through the PVN. Treatment with several substances of abuse has been shown to modify immune function in vivo and in vitro. In this study, we compared the effects of morphine, ethanol and cocaine on IL-1 beta induction of FOS-IR in the rat hypothalamus. Acute treatment with morphine or ethanol induced FOS-IR in several nuclei including the PVN. Cocaine, which induced FOS-IR in the Caudate-Putamen (CPU), nucleus Accumbens (nAcc) and Locus Coeruleus (LC), however, did not induce FOS-IR in the PVN. Chronic treatment with morphine desensitized FOS responsiveness to morphine and IL-1 beta in the PVN since FOS-IR was no longer induced by IL-1 beta or morphine in the PVN after this treatment. Similarly, chronic ethanol treatment desensitized FOS responsiveness to ethanol and to IL-1 beta in the PVN. By contrast, chronic cocaine did not affect FOS responsiveness to IL-1 beta in the PVN even though the treatment was able to desensitize the FOS responsiveness to acute cocaine in the CPU, nAcc, and LC. These results suggest that the PVN may be a site where actions of IL-1 beta converge with those of morphine and ethanol, but not cocaine, to modulate neuro-endocrine-immune functions.

Picrotoxin-induced seizures modified by morphine and opiate antagonists.

The effects of naloxone, Tyr-MIF-1, and MIF-1 on morphine-mediated changes in susceptibility to picrotoxin-induced seizures were studied. Rats were pretreated with naloxone, MIF-1, Tyr-MIF-1, or saline. At 15-min intervals, they received a second pretreatment of morphine or saline and then were tested for seizures following a convulsant dose of picrotoxin. Several parameters of specific categories of seizures were scored. Morphine increased the number of focal seizure episodes, duration of postseizure akinesis, and incidence of generalized clonic seizures. Naloxone tended to block the morphine-mediated changes in susceptibility. Tyr-MIF-1 had effects similar to naloxone on duration of postseizure immobility but tended to potentiate the effects of morphine on focal seizure episodes. The effects of morphine and the opiate antagonists on focal seizure episodes and postseizure duration suggest the general involvement of several types of opiate receptors in these picrotoxin-induced behaviors. However, the observation of antagonistic effects for Tyr-MIF-1 on immobility but agonistic effects for focal seizures suggests that the type of effect exerted by opiate agents may depend upon other neuronal variables.

Prolonged analgesia after intracerebroventricular Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2).

A peptide recently isolated from human and bovine brain, Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2), was tested for its effects on nociception in the tail-flick test after intracerebroventricular injection in the rat. Tail-flick latencies were significantly increased with a rapid onset and remained significantly elevated for at least 50 min. Naloxone reversed the effect of the peptide, indicating opiate receptor involvement in the response. Met-enkephalin at the same dose produced only slight antinociception. Some animals showed 'barrel-rolling' behavior in addition to the analgesia; this behavior was unusually short-lived, not a prerequisite for the analgesia, and had no apparent persistent effects. The results show that, in addition to previously described opiate-like actions (binding to the mu-receptor and inhibition of electrically induced contractions of the guinea pig ileum), Tyr-W-MIF-1 is capable of inducing significant analgesia.