Kappa-opioid Agonist Research Articles

Discrimination of a Single Dose of Morphine Followed by Naltrexone: Substitution of Other Agonists for Morphine and Other Antagonists for Naltrexone in a Rat Model of Acute Dependence

Rats were trained to discriminate 4-h pretreatment with 10 mg/kg morphine and 15-min pretreatment with 0.3 mg/kg naltrexone (morphine-->naltrexone) from pretreatment with saline and 0.3 mg/kg naltrexone (saline-->naltrexone). The discrimination seems to derive from interoceptive stimuli from antagonist-precipitated withdrawal from acute morphine dependence. The purpose of this study was to extend pharmacological characterization of the discrimination by testing opioid agonists other than morphine and antagonists other than naltrexone. Of seven mu-opioid agonists tested in place of morphine, only two (heroin and levorphanol) substituted completely for it; trials completed on the morphine-->naltrexone-appropriate lever increased as a function of agonist and naltrexone dose. Agonists with intrinsic efficacy higher (etorphine, fentanyl, and methadone) or lower (buprenorphine and meperidine) than that of morphine substituted only partially. However, when naltrexone was administered during continuous infusion of fentanyl or methadone via s.c. osmotic pump, rats responded as if they had received morphine-->naltrexone; discriminative responding correlated with global withdrawal scores. Rats responded primarily on the saline-->naltrexone-appropriate lever when naltrexone was administered after pretreatment with dextrorphan, the dextrorotatory isomer of levorphanol, or kappa-opioid agonists (5-alpha,7-alpha,8-beta)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-benzeneacetamide (U69,593) and spiradoline. Antagonists with no intrinsic efficacy at mu-opioid receptors (naloxone and diprenorphine) substituted completely for naltrexone, whereas those with some efficacy (nalorphine and levallorphan) substituted partially. Thus, morphine-->naltrexone-like stimulus control of behavior by drugs administered acutely requires pretreatment with certain mu-opioid agonists and a pure antagonist, is independent of agonist efficacy, and is stereoselective. Interoceptive stimuli from naltrexone-precipitated opioid withdrawal are more similar across morphine-like agonists during chronic dependence than they are during acute dependence.

Effects of mixed-action kappa/mu opioids on cocaine self-administration and cocaine discrimination by rhesus monkeys.

kappa-Opioid agonists may functionally antagonize some behavioral effects of cocaine, but the role of mixed kappa/mu receptor activity is unclear. The effects of three mixed kappa/mu agonists (MCL-101, (-)cyclorphan, and Mr2034) and one kappa-selective agonist (enadoline) on cocaine self-administration and cocaine discrimination were compared in rhesus monkeys. Acute treatment with all kappa agonists dose dependently reduced cocaine-maintained responding and produced a downward shift in the cocaine self-administration dose-effect curve (0.001-0.32 mg/kg/inj, i.v.). During 7 days of chronic treatment, (-)cyclorphan (0.0032-0.032 mg/kg/h) and MCL-101 (0.0032-0.032 mg/kg/h) each dose dependently reduced cocaine self-administration maintained by a dose near the peak of the cocaine self-administration dose-effect curve. MCL-101 (0.032 mg/kg/h) produced selective and sustained decreases in cocaine self-administration, whereas (-)cyclorphan (0.032 mg/kg/h) had selective but transient effects. In addition, these mixed kappa/mu agonists produced fewer side effects (some salivation) than the kappa-selective agonist (sedation, salivation, emesis). However, none of these kappa agonists substituted for or antagonized cocaine's discriminative stimulus effects in monkeys trained to discriminate cocaine (0.4 mg/kg, i.m.) from saline. Thus, kappa and mixed kappa/mu-opioid agonists may reduce cocaine self-administration without altering cocaine's discriminative stimulus effects. Mixed kappa/mu agonists appear to offer some advantages over selective kappa agonists as potential treatments for cocaine abuse.

Dextromethorphan and ketamine potentiate the antinociceptive effects of mu- but not delta- or kappa-opioid agonists in a mouse model of acute pain.

Animal and clinical studies have reported potentiation of opioid antinociception by NMDA receptor antagonists such as ketamine and dextromethorphan. The aim of this study was to compare these clinically available NMDA antagonists in combination with classical morphine, mu-selective fentanyl-like opioids, the delta-opioid agonist SNC80 and the kappa-opioid agonist U50,488H. Using a mouse hot-plate test, dose-response relationships were first determined for all compounds individually and then for opioids co-administered with fixed doses of ketamine or dextromethorphan. All compounds were administered intraperitoneally ED(50) values were calculated from the proportion of animals failing to exhibit any response within a fixed cut-off criterion of 30 s. To varying degrees, all compounds produced increases in response latencies over time. Dextromethorphan produced lower ED(50) values for morphine, fentanyl and sufentanil but exerted no effect on the potency of SNC80 or U50,488H. Similarly, ketamine potentiated the antinociceptive potency of morphine, fentanyl and sufentanil but not SNC80 or U50,488H. In summary, these results support the use of mu-opioid agonists in combination with NMDA antagonists, but suggest that there may be no advantage in combining dextromethorphan or ketamine with delta- or kappa-opioids in the management of acute pain.

Effect of Bremazocine, a Kappa-Opioid Receptor Agonist, on Inositol Phosphate Formation in Isolated Iris-Ciliary Bodies

The goal of the current study was to examine the effect of the kappa-opioid agonist, bremazocine (BRE), on inositol phosphate (IP) formation in the rabbit iris-ciliary body (ICB). Concentrations of BRE (10^–7 to 10^–5M) augmented levels of IP. Incubation of ICBs with BRE (10^–6M) produced a time-dependent increase in IP levels that peaked at 60 s and declined to basal levels by 5 min. The increase in IP levels produced by BRE (10^–6M) was inhibited by the kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI, 10^–7 to 10^–5M) and by activation of PKC with PDBu (10^–7M). These results demonstrate that kappa-opioid receptor activation by BRE in the rabbit ICB is linked to IP production. Thus, opioid agonist-induced increases in IP activity could play a role in BRE-induced increases in atrial natriuretic peptide release and alterations in aqueous humor dynamics.

Chronic cocaine increases kappa-opioid receptor density: lack of effect by selective dopamine uptake inhibitors.

Continuous infusion of cocaine or the selective dopamine uptake inhibitors GBR 12909 or RTI-117 increases locomotor stimulation, to which partial tolerance occurs. In addition, all three drugs produce significant decreases in tyrosine hydroxylase immunoreactivity in caudate putamen and nucleus accumbens core, suggesting a decreased dopaminergic tone. An interaction between cocaine and opioids has long been documented. Chronic cocaine significantly increases mu and kappa-opioid receptors and treatment with a kappa-opioid agonist markedly reduces the behavioral effects of cocaine. In addition, chronic cocaine, but not GBR 12909, increases prodynorphin gene expression in caudate putamen. To further understand the interaction between cocaine and the kappa-opioid system, the effects of a chronic continuous infusion for 14 days of cocaine or one of the selective dopamine uptake inhibitors GBR 12909 or RTI-117 via osmotic minipump were examined on kappa-opioid receptors using the selective kappa-opioid ligand [3H] U-69593. [3H] U-69593 binding density was significantly increased in caudate putamen, nucleus accumbens shell, claustrum, and endopiriform nucleus after cocaine, while neither GBR 12909 nor RTI-117 had any effect. The increased kappa-opioid receptor densities observed following cocaine are likely not related to dopamine uptake inhibition, since they were not produced by selective dopamine uptake inhibitors. These findings suggest that regulation of kappa-opioid receptors by cocaine may be via inhibition of serotonin or norepinephrine uptake, by a combination of effects on two or three monoamine transporters, or by a mechanism unrelated to transporter inhibition.

Effects of Bremazocine on Self-Administration of Smoked Cocaine Base and Orally Delivered Ethanol, Phencyclidine, Saccharin, and Food in Rhesus Monkeys: A Behavioral Economic Analysis

There is increasing evidence that kappa-opioid receptor agonists modulate cocaine-maintained behavior, and limited findings implicate the involvement of kappa-opioid receptors in ethanol-maintained behaviors. The purpose of the present study was to investigate the effects of bremazocine, a kappa-opioid agonist, on the self-administration of smoked cocaine base and oral ethanol in rhesus monkeys (Macaca mulatta). To determine the selectivity of bremazocine, the effects of bremazocine pretreatment on the oral self-administration of phencyclidine (PCP), saccharin, and food were also examined. Adult male rhesus monkeys were trained to self-administer oral ethanol, PCP, saccharin (n = 8), food (n = 6), or smoked cocaine base (n = 6) and water during daily sessions. Bremazocine (0.00032-, 0.001-, and 0.0025-mg/kg i.m.) injections were given 15 min before session. The 4 days of stable behavior before pretreatment served as baseline. Demand curves (consumption x fixed ratio; FR) were obtained for smoked cocaine base, ethanol, and PCP by varying the cost (FR) of drug deliveries and measuring consumption (deliveries). Bremazocine (0.001 mg/kg) was administered at each FR value in nonsystematic order. Results indicate that bremazocine dose dependently reduced cocaine, ethanol, PCP, and saccharin intake. Food intake was affected less by bremazocine than the other substances in five of the six monkeys. Generally, bremazocine treatment reduced the demand for cocaine, ethanol, and PCP as well as other measures of response strength. These results extend the findings that kappa-agonists reduce the self-administration of drug and nondrug reinforcers to smoked cocaine base and oral ethanol, PCP, and saccharin in rhesus monkeys.

Inhibition of cAMP accumulation by kappa-receptor activation in isolated iris-ciliary bodies: role of phosphodiesterase and protein kinase C.

The present study was designed to examine the roles of protein kinase C (PKC) and phosphodiesterase (PDE) in modulating the action of kappa receptor stimulation on cAMP accumulation in isolated iris-ciliary bodies (ICBs) of New Zealand White rabbits. The kappa receptor agonist, (+/-)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl)methylpiperidine (BRL-52537) (BRL), and the PKC activator, phorbol 12,13-dibutyrate (PDBu), both caused a concentration-dependent inhibition of forskolin-stimulated cAMP production. The inhibitory effect of BRL on cAMP levels was significantly reduced in the presence of the selective kappa receptor antagonist, norbinaltorphimine (10(-6) M), but the effect of PDBu was not, thus supporting the involvement of kappa-opioid receptors in the response to BRL. In the presence of 3-isobutyl-1-methylxanthine or rolipram (10(-5) M), the inhibitory effect of BRL or PDBu (10(-6) M) on cyclic AMP accumulation was abolished. In the presence of the selective PKC antagonist, chelerythrine (10(-6) M), the inhibitory effect of PDBu or BRL (10(-6) M) was significantly reduced. Direct measurement of PDE activity demonstrated the ability of BRL and PDBu (10(-6) M) to augment the activity of these enzymes. Preincubation of ICBs with rolipram (10(-5) M) or chelerythrine (10(-6) M) caused significant reversal of both BRL- and PDBu-induced increases in PDE activity. These results indicate that stimulation of PKC and PDE4 activity is part of the complex mechanism whereby kappa-opioid receptor agonists reduce levels of cAMP in the rabbit ICB. This mechanism of action could contribute to the ability of kappa-opioid agonists to suppress aqueous flow rate and to lower intraocular pressure.

Characterization of mu, kappa, and delta opioid binding in amphibian whole brain tissue homogenates.

Opioid agonists produce analgesia in mammals through the activation of mu, kappa, or delta opioid receptors. Previous behavioral and binding studies from our laboratory using an amphibian model suggested that mu, kappa, or delta opioid agonists may activate a single type of opioid receptor in the grass frog, Rana pipiens. In the present study, kinetic, saturation, and competitive binding profiles for three opioid radioligands, [(3)H]DAMGO ([D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin) (mu-selective), [(3)H]U65953 [(5 alpha, 7 alpha,8 beta)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide] (kappa-selective), and [(3)H]DPDPE ([D-Pen(2),D-Pen(5)]-enkephalin) (delta-selective) were determined using frog whole brain homogenates. Kinetic analyses and experimentally derived values from saturation experiments gave affinity constants (K(D)) in the low nanomolar range. The density of opioid binding sites (B(max)) was 224.4, 118.6, and 268.9 fmol/mg for mu, kappa, and delta opioid radioligands, respectively. The affinity values did not significantly differ among the three opioid radioligands, but the kappa radioligand bound to significantly fewer sites than did the mu or delta radioligands. K(i) values for unlabeled mu, kappa, and delta competitors, including highly selective opioid antagonists, were consistent with each radioligand selectivity profile. The present data suggest that mu, kappa, and delta opioid radioligands bind to distinct opioid receptors in amphibians that are surprisingly similar to those found in mammalian brain.

Effect of kappa opioid agonists on visceral nociception induced by uterine cervical distension in rats

Although uterine distension in rats results in an escape reflex, there exists no model of uterine cervical distension (UCD), the pain stimulus during the first stage of labor. The aims of this study were to develop such a model in virgin rats and to test whether peripherally restricted kappa opioid receptor (KOR) agonists (ADL 10-0101, ADL 10-0102, ADL 10-0116) inhibit responses to UCD. Under intravenous (i.v.) pentobarbital and alpha-chloralose anesthesia, fine metal rods were inserted in both uterine cervical osses through a small midline laparotomy. UCD was performed by manual separation of the rods (25–100 g). Single-unit afferent responses in hypogastric nerve or reflex rectus abdominis electromyographic (EMG) activity were determined before and after i.v. KOR agonists. UCD resulted in a stimulus-dependent increase in single-unit afferent activity. Units could be characterized as low threshold (mean threshold 6.6±2.7 g), or high threshold (mean threshold 55±8.8 g); all were C fibers, all responded to topical bradykinin. ADL 10-0116 (10 mg/kg) reduced the afferent response to UCD. Reflex EMG response occurred over a distension force range of 25–100 g, unaffected by i.v. saline. All three KOR agonists produced a dose-dependent, naloxone-reversible inhibition of the EMG response with a potency relationship of ADL 10-0102 (ED 50 0.04 mg/kg)>ADL 10-0101 (ED 50 0.65 mg/kg)=ADL 10-0116 (ED 50 0.60 mg/kg). These data support the use of acute UCD as a noxious stimulus, inducing afferent and reflex activity. Like other visceral stimuli, UCD is sensitive to inhibition by KOR agonists.

Estrogen reduces efficacy of mu- but Not kappa-opioid agonist inhibition in response to uterine cervical distension.

Although the uterine cervix is a common source of acute and chronic visceral pain in women, there is practically no neurobiological investigation of nociception from this visceral organ. With use of a novel model of uterine cervical distension nociception in rats, the estrogen dependency of opioid agonist-induced inhibition was investigated. Sprague Dawley rats were anesthetized with halothane and bilateral ovariectomy was performed, after which placebo or estrogen treatment was administered for 1 week. Animals were reanesthetized and fine metal rods were inserted into the uterine cervix for manual distension. Reflex contraction of the rectus abdominis in response to distension was recorded before and after cumulative dosing with the mu-opioid agonist morphine and the kappa-opioid agonist (-)U50488. Uterine cervical distension increased reflex abdominal muscle contraction with a threshold of 75 g, regardless of estrogen treatment. Morphine and (-)U50488 reduced the reflex response to cervical distension in a dose-dependent manner. Estrogen reduced the inhibitory effect of morphine but not that of (-)U50488. It has been suggested that mu-opioid agonists are less potent in females than males, whereas kappa-opioid agonists are more potent in females than males. These data suggest that estrogen may influence the action of opioids, at least against visceral pain, which may explain this sex difference. In addition, these data suggest that kappa-opioid agonists may be effective in the treatment of pain originating from the uterine cervix, regardless of estrogen status.

Orphanin FQ inhibits capsaicin-induced thermal nociception in monkeys by activation of peripheral ORL1 receptors.

1. Orphanin FQ (OFQ), an endogenous peptide for ORL1 receptors, has been identified. Although the actions of OFQ have much in common with those of opioid peptides at the cellular level, behavioral studies in rodents seem conflicting. 2. The aim of this study was to investigate the potential pronociceptive or antinociceptive function of peripheral ORL1 receptors in primates. Experiments were conducted to verify whether local administration of OFQ can attenuate capsaicin-induced nociception and whether peripheral ORL1 receptors selectively mediate the local action of OFQ in monkeys. 3. Capsaicin (100 microg) was administered subcutaneously in the tail to locally evoke a nociceptive response (thermal allodynia/hyperalgesia), which was manifested as a reduced tail-withdrawal latency in normally innocuous 46 degreeC warm water. 4. Co-administration of OFQ (1--30 microg) with capsaicin in the tail dose-dependently inhibited thermal nociception. However, a locally effective dose of OFQ (30 microg), when applied in the back, did not inhibit capsaicin-induced nociception. 5. OFQ-induced local antinociception was antagonized by a small dose (10 microg) of J-113397, a selective ORL1 receptor antagonist, in the tail. Similarly, s.c. administration of 10 microg of J-113397 in the back did not antagonize local antinociception of OFQ. 6. In addition, s.c. administration of either OFQ or J-113397 in the tail alone did not change its thermal nociceptive threshold. Local administration of opioid receptor antagonists selective for mu, kappa, and delta opioid receptors did not antagonize OFQ-induced local antinociception. Local administration of J-113397 also did not interfere with the local actions of mu, kappa, and delta opioid agonists in the tail. 7. These results provide the first functional evidence that activation of peripheral ORL1 receptors produces thermal antinociception in primates and this action is independent of antinociception produced at classical opioid receptors.

Regulation of dynorphin gene expression by kappa-opioid agonist treatment.

The effects of K-opioid agonist treatment on prodynorphin mRNA expression in the rat brain were studied. Rats were treated with the selective kappa-opioid agonist U-69593 or vehicle for 5 days and prodynorphin mRNA was measured on day 8 (3 days after the last injection) or 22 (17 days after the last injection). On day 8 prodynorphin mRNA was increased in the hypothalamus and decreased in the striatum, frontal cortex, and hippocampus of rats treated with U-69593. On day 22, prodynorphin mRNA was increased in the hypothalamus, frontal cortex and striatum of U-69593 treated rats. These findings suggests that kappa-opioid receptor agonist treatment has long-term, continually changing effects on prodynorphin mRNA expression.

Effects of gonadal steroid hormone treatments on opioid antinociception in ovariectomized rhesus monkeys.

Gonadal steroid hormones altered opioid antinociception under some conditions in rodents, and we reported previously that chronic estradiol enhanced kappa but not mu opioid antinociception in ovariectomized rhesus monkeys. Sex differences have also been observed in the antinociceptive effects of opioid agonists. These findings suggest that gonadal hormones may modulate opioid antinociception. To extend our previous studies of estradiol by examining the effects of progesterone alone, estradiol in combination with progesterone, and testosterone alone on opioid antinociception in ovariectomized rhesus monkeys. Opioid effects were studied during chronic treatment with vehicle (sesame oil) or with progesterone alone (P; 0.32 mg/kg per day), a combination of progesterone+estradiol (P+E; 0.32 mg/kg per day P + 0.002 mg/kg per day E), or testosterone alone (T; 0.32 mg/kg per day). Opioid antinociception in a warm-water tail-withdrawal procedure was examined with the selective kappa opioid agonist U50,488, the selective mu agonist morphine, and the two mixed-action opioids butorphanol and nalbuphine. The steroid treatment regimens produced physiological levels of progesterone and estradiol similar to peak levels observed during the luteal phase of the menstrual cycle and physiological levels of testosterone similar to those observed in intact males. Treatment with P, P+E, or T did not alter baseline thermal nociception. P+E significantly increased the potency of U50,488 at 50 degrees C but not at 54 degrees C. Gonadal hormone treatments had little or no effect on antinociception produced by morphine, butorphanol, or nalbuphine at either temperature. These findings further suggest that chronic treatment with physiological levels of gonadal hormones may modulate the antinociceptive effects of U50,488 in ovariectomized rhesus monkeys.

Cholera toxin-B subunit blocks excitatory opioid receptor-mediated hyperalgesic effects in mice, thereby unmasking potent opioid analgesia and attenuating opioid tolerance/dependence

In a previous study we demonstrated that injection (i.p.) of low doses of GM1 ganglioside in mice rapidly attenuates morphine’s analgesic effects. This result is consonant with our electrophysiologic studies in nociceptive types of dorsal root ganglion (DRG) neurons in culture, which showed that exogenous GM1 rapidly increased the efficacy of excitatory (Gs-coupled) opioid receptor functions. By contrast, treatment of DRG neurons with the non-toxic B-subunit of cholera toxin (CTX-B) which binds selectively to GM1, blocked the excitatory, but not inhibitory, effects of morphine and other bimodally-acting opioid agonists, thereby resulting in a net increase in inhibitory opioid potency. The present study provides more direct evidence that endogenous GM1 plays a physiologic role in regulating excitatory opioid receptor functions in vivo by demonstrating that cotreatment with remarkably low doses of CTX-B (10 ng/kg, s.c.) selectively blocks hyperalgesic effects elicited by morphine or by a kappa opioid agonist, thereby unmasking potent opioid analgesia. These results are comparable to the effects of cotreatment of mice with morphine plus an ultra-low dose of the opioid antagonist, naltrexone (NTX) which blocks opioid-induced hyperalgesic effects, unmasking potent opioid analgesia. Low-dose NTX selectively blocks excitatory opioid receptors at their recognition site, whereas CTX-B binds to, and interferes with, a putative allosteric GM1 regulatory site on excitatory opioid receptors. Furthermore, chronic cotreatment of mice with morphine plus CTX-B attenuates development of opioid tolerance and physical dependence, as previously shown to occur during cotreatment with low-dose NTX.

Resensitization of blood pressure response to mu-opioid peptide agonists after acute desensitization.

IV administration of mu-opioid peptide agonists (DAMGO, DALDA, and [Dmt(1)]DALDA) results in a transient, naloxone-sensitive, increase in blood pressure in awake sheep. Despite significant differences in pharmacokinetics, these blood pressure responses all last < 15 min. The lack of correlation between half-life and duration of action suggested rapid desensitization. When a second dose of the same agonist was repeated 30 min later, the response was completely abolished. An increase in blood pressure and rapid desensitization was also observed with the kappa-opioid agonist (U50488H), whereas delta-agonists (DPDPE and DELT) had no effect on blood pressure. The response to DAMGO was abolished after prior exposure to DAMGO or DALDA, but there was no evidence of cross-desensitization between mu and delta, or mu and kappa, opioid agonists. Full resensitization of the blood pressure response occurred by 4 h for DAMGO (t(1/2) = 15 min) and by 48 h for [Dmt(1)]DALDA (t(1/2) = 1.8 h). These data support our hypothesis that the transient nature of the blood pressure response to mu-opioid agonists is caused by rapid desensitization and suggest that the rate of resensitization is dependent on the pharmacokinetics of the agonist.

The kappa-opioid agonist, asimadoline, alters cytokine gene expression in adjuvant arthritis.

We have previously found that the kappa-opioid agonist, asimadoline, attenuates adjuvant arthritis in a dose-dependent, antagonist-reversible manner. To elucidate possible mechanisms, we investigated the effects of asimadoline (5 mg/kg/day i.p.) or vehicle on in vivo cytokine expression and T-cell recruitment in adjuvant arthritis. Arthritis severity was assessed every 3-4 days for 21 days. Rats were killed on days 0, 13 and 21 post-induction and synovial membrane and inguinal lymph nodes were removed for mRNA extraction. Changes in cytokine mRNA expression were measured using reverse transcription-polymerase chain reaction (RT-PCR) and densitometry. T cells in joints were quantified by immunohistochemistry. Asimadoline significantly decreased arthritis severity at day 13, with a concomitant decrease in synovial membrane expression of cytokines interleukin-17 and transforming growth factor-beta (TGF-beta) mRNA at day 13, and no change in T cell numbers in the joints of arthritic rats. By contrast, in the inguinal lymph nodes, expression of tumour necrosis factor was increased at day 13 and TGF-beta mRNA was increased throughout. An altered balance, therefore, in the pro- and anti-inflammatory effects of TGF-beta by asimadoline might explain its striking anti-arthritic actions.

The Fas/FasL apoptotic pathway is involved in kappa-opioid-induced apoptosis of human endometrial stromal cells.

Human endometrium expresses specific kappa-opioid binding sites and their endogenous ligands, the dynorphins. In neural crest-derived tissues, kappa-opioids affect apoptosis, a phenomenon of major significance in endometrial stroma physiology. Our hypothesis was that endometrial kappa-opioids may play a role in endometrial stromal cell apoptosis. Thus, we examined the effect of the synthetic kappa-opioid agonist, U69593, on the apoptotic rate of human endometrial stromal cells in primary culture. Apoptosis of endometrial stromal cells was elevated after 3 h exposure to 100 nmol/l U69593, and remained elevated for up to 3 days. This effect was dose-dependent and was reversed by the general opioid antagonist, naloxone, suggesting that it is mediated via opioid receptors. In parallel, semi-quantitative Western blot and flow cytometry analysis showed that U69593 caused a rapid but transient up-regulation of Fas protein, suggesting that its effect on apoptosis is mediated by activation of the Fas/FasL apoptotic pathway. Additionally, U69593 increased the content of the anti-apoptotic members of the Bcl-2 family of proteins, the Bcl-2 and Bcl-x(L), whereas it had no significant effect on the apoptosis-promoting homologues Bax, Bcl-x(S) and Bak. This implies that a transient survival mechanism is activated in stromal cells as a parallel rescue response to the apoptosis-inducing factor. In conclusion, our data suggest that endometrial opioid dynorphins may participate in the apoptotic processes related to endometrial tissue remodelling during early pregnancy or menstruation.

Kappa Opioid Agonist-Induced Changes in IOP: Correlation with3H-NE Release and cAMP Accumulation

Opioid receptors have been demonstrated to modulate various functions in the eye. This research project was designed to determine and compare the effects of kappa opioid agonists on selected parameters that influence ocular hydrodynamics. Experiments determined the effects of two relatively selective kappa opioid receptor agonists, ICI 204 448 (ICI), which has limited ability to penetrate the blood–brain barrier, and spiradoline mesylate on: (1) in vivo parameters, intraocular pressure (IOP) and pupil diameter (PD); and (2) in vitro parameters, neurotransmitter release and cAMP accumulation, in the ciliary body. Dark-adapted, reverse light cycle New Zealand white (NZW) male rabbits were used in all experiments. In in vivo experiments, intraocular pressures and pupil diameters were measured by a pneumatonometer and an optistick, respectively, before and after drug administration. Baseline readings were taken at 0.5 and 0hr prior to agonist administration. Postdrug IOP and PD measurements were made at 0.5, 1, 2, 3, 4 and 5hr after agonist application. In some experiments, the relatively selective kappa antagonist, norbinaltorphimine was applied 30min prior to agonist application. In in vitro experiments, the release of tritiated norepinephrine (3H-NE) was measured from perfused electrically stimulated iris ciliary bodies and expressed as the percent change of the control. Basal and isoproterenol-stimulated cyclic AMP concentrations in iris ciliary bodies were quantified by radioimmunoassay techniques in the presence and absence of ICI and spiradoline. ICI and spiradoline decreased IOP in a dose-dependent manner in normal rabbits, but only spiradoline produced significant changes in PD. The kappa opioid receptor antagonist, norbinaltorphimine, antagonized the hypotensive effects of spiradoline and ICI in IOP experiments. Both kappa agonists inhibited the release of norepinephrine from perfused iris ciliary bodies. Isoproterenol- stimulated cAMP levels in iris ciliary bodies were suppressed by both kappa receptor agonists. The antagonism by norbinaltorphimine suggests that ICI and spiradoline lower IOP by activating kappa opioid receptors in the eye. The bilateral effects of unilaterally applied spiradoline on PD indicate that this kappa agonist activates receptors in the iris and/or the brain. The inhibition of norepinephrine release and cAMP accumulation in the iris ciliary body by ICI and spiradoline suggests that there are both pre- and postjunctional sites of action for kappa agonists.

The effects of benzodiazepines on human opioid receptor binding and function.

We performed in vitro studies to investigate the potential interaction of benzodiazepines with cloned human opioid receptor subtypes. Midazolam, chlordiazepoxide, and diazepam directly displaced [(3)H]-diprenorphine binding from kappa and delta receptors, but not mu receptors, whereas flumazenil was inactive. These benzodiazepines also stimulated (35)S-GTPgammaS binding in membranes containing human kappa receptors, and the effect of midazolam was prevented by a selective kappa antagonist. Midazolam was also weakly active at delta-receptor activation, whereas all three were inactive at mu receptors. The results suggest that the analgesic efficacy reported for intrathecal benzodiazepines may be attributed, in part, to direct interaction with kappa-opioid receptors. Several human and animal studies have shown analgesic effects of benzodiazepines after spinal injection. Our results show that large concentrations of midazolam, chlordiazepoxide, and diazepam displace the binding of [(3)H]-diprenorphine-an opiate radioligand from kappa receptors. In an in vitro functional assay, midazolam is a weak agonist at the delta-opioid receptor, whereas all three benzodiazepines are kappa-opioid agonists. These findings may partially explain the mechanism of benzodiazepine-induced spinal analgesia.

The kappa-opioid antagonist GNTI reduces U50,488-, DAMGO-, and deprivation-induced feeding, but not butorphanol- and neuropeptide Y-induced feeding in rats

Antagonists selective for either kappa- [e.g. nor-binaltorphimine (nor-BNI)] and mu- (e.g. beta-funaltrexamine) opioid receptors have previously been shown to reduce both kappa- and mu-opioid-induced feeding. In the present studies, the anorectic effects of GNTI, a newly synthesized antagonist selective for kappa-opioid receptors, were studied in rats. GNTI (0.032–0.32 nmol; i.c.v.), administered 15 min prior to food access, reduced feeding induced by the kappa-opioid agonist U50,488 (producing a 70% maximal decrease), the mu-opioid agonist DAMGO (90% maximal decrease), and 24 h acute food deprivation (60% maximal decrease). GNTI did not reduce the orexigenic effects of butorphanol, an agonist that binds to both kappa- and mu-opioid receptors, and neuropeptide Y (NPY). Taken together, these results suggest that GNTI is a potent anorectic agent and opioid antagonist in rats. Like nor-BNI, GNTI reduced feeding induced by both kappa- and mu-opioid agonists. However, unlike nor-BNI, GNTI did not alter the orexigenic effects of butorphanol or NPY. Given the selectivity of GNTI and its effectiveness in several of the present experiments, its potency, and its short duration of action compared to nor-BNI, GNTI may serve to be a useful tool to study behavioral effects mediated by kappa-opioid receptors.