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The results of a previous study in rats indicated that spiradoline has pharmacologically selective discriminative effects that are mediated by kappa-opioid receptors. However, the training dose, 3.0 mg/kg, increased response latencies, suggesting that it was relatively high. The current study was performed to characterize further the discriminative effects of spiradoline by using a lower training dose, 1.0 mg/kg, and testing a larger number of drugs for generalization with spiradoline. Rats were trained in a discrete-trial avoidance/escape procedure to discriminate 1.0 mg/kg spiradoline, SC, from saline in an average of 19.7 sessions; response latencies after saline and spiradoline were not different from each other. The rats generalized dose dependently and completely to other kappa-opioid agonists that have relatively high efficacy: ethylketocyclazocine, U69,593, and U50,488. They generalized partially to ketocyclazocine, (-)-N-allylnormetazocine, and DuP 747, and not at all to cyclazocine, butorphanol, nalorphine, and pentazocine, discriminable opioids that have relatively low efficacy at kappa-opioid receptors, or to morphine and dextromethorphan, discriminable drugs that do not act at kappa-opioid receptors. The discriminative effects of spiradoline were unaffected by the mu-opioid antagonist beta-funaltrexamine, but were blocked completely for at least 4 weeks by the kappa-opioid antagonist nor-binaltorphimine. Thus, spiradoline-like stimulus control of behavior remains kappa-opioid selective, and continues to have a high efficacy requirement even at a training dose that does not impair performance.

We examined the diversity of single receptor function by measuring receptor-G protein coupling in the baculovirus-Sf21 expression system. In comparative studies using Sf21 cell membranes expressing kappa-opioid receptor (KOR) plus Galpha(i1)beta(1)gamma(2) or KOR plus Galpha(oA)beta(1)gamma(2), there was no significant difference between both preparations in the K(i) values of various kappa-opioid ligands for the displacement of [(3)H]U69593 binding. However, a marked difference in the rank order of agonists to stimulate [(35)S]GTPgammaS binding was observed between both preparations. These findings suggest that agonist efficacy is dependent on the population of different G proteins expressed in various tissues.

There is strong evidence supporting the existence of multiple kappa receptors. Previous studies proposed that U69,593 and (+)-tifluadom act on different kappa receptor subtypes, kappa(1) (kappa(1)) and kappa(2) (kappa(2)), respectively. In this study, we investigated the effects of the kappa selective antagonist nor-binaltorphimine (Nor-BNI) on U69,593- and (+)-tifluadom-induced receptor-mediated stimulation of [(35)S]-GTP-gamma-S binding in the guinea pig caudate. The IC(50) value of Nor-BNI in the presence of a stimulating concentration of U69,593 (1 microM) was 0.19+/-0.02; while the IC(50) for Nor-BNI in the presence of (+)-tifluadom (1 microM) was 13.9+/- 1.62 nM. The mu-opioid receptor antagonist CTAP (10,000 nM) significantly reduced (+)-tifluadom-stimulated [(35)S]-GTP-gamma-S binding in rat brain sections and guinea pig brain membranes, indicating that (+)-tifluadom has mu agonist activity. Under conditions in which the mu agonist activity of (+)-tifluadom was blocked by 1000 nM CTAP the Ki value for Nor-BNI for inhibition of U69,593-stimulated [(35)S]-GTP-gamma-S binding was 0.036+/-.004 nM, whereas, its Ki value for the (+)-tifluadom-stimulated [(35)S]-GTP-gamma-S binding was 0.27+/-.015 nM. These results suggest that (+)-tifluadom and U69,593 activate pharmacologically different receptors. This study provides functional evidence in support of kappa receptor heterogeneity.

This study characterized the antinociceptive, respiratory and heart rate effects of the cannabinoid receptor agonists Delta-9-tetrahydrocannabinol (Delta-9-THC) and WIN 55212 ((R)-(+)-2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol-[1,2,3-de]-1, 4-benzoxazin-6-yl)(1-naphtalenyl)methanone monomethanesulfonate), N-arachidonyl ethanolamide (anandamide) and the mu and kappa opioid receptor agonists heroin and U69593, alone and in conjunction with a cannabinoid receptor antagonist, SR 141716A [N-(piperidin-1-1-yl)-5-(4-chlorophenyl)-1(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] and an opioid receptor antagonist, quadazocine, in rhesus monkeys (Macaca mulatta). Using 12 adult rhesus monkeys, latencies to remove the tail from a 50 degrees C water bath, respiration in 5% CO2 and heart rate were measured. When administered alone, SR 141716A (1.8, 5.6 mg/kg i.m.) did not alter nociception, respiration or heart rate. Delta-9-THC (0.1-10 mg/kg i.m.) and WIN 55212 (0.1-10 mg/kg i.m.) dose-dependently increased antinociception and dose-dependently decreased respiratory minute and tidal volumes and heart rate. These antinociceptive, respiratory and heart rate effects were reversed by SR 141716A but not by the opioid antagonist quadazocine (1 mg/kg i.m.). Anandamide (10 mg/kg i.m.) also produced antinociception. Heroin (0.01-10 mg/kg i.m.) and U69593 (0.01-3.2 mg/kg i.m.) also dose-dependently increased antinociception and decreased respiratory and heart rate measures; these effects were antagonized by quadazocine but not by SR 141716A. These results demonstrate selective and reversible antagonism of cannabinoid behavioral effects by SR 141716A in rhesus monkeys.

1. The calcium channel blockers (CCBs), nifedipine, nicardipine, diltiazem and verapamil, were used to displace the binding of [3H]-U69593 ((5a, 7a,8b)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5] dec-8-yl)-benzeneacetamide), a specific kappa-opioid agonist, in the rat cardiac sarcolemma. The CCBs competed with the binding of [3H]-U69593 (4 nM) in a dose-dependent manner. The displacing potency of verapamil was 55 times greater than that of nifedipine. 2. The effects of two CCBs, verapamil and nifedipine, on the arrhythmogenic action of kappa-receptor stimulation by a specific kappa-receptor agonist, U50,488H (trans-(+/-(-3),4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeacetamide methanesulphonate), were also studied in the rat isolated perfused heart. U50,488H 80-800 nmol dose-dependently induced arrhythmias, which were completely abolished by a selective kappa-receptor antagonist, nor-BNI (nor-binaltorphimine, 17,17'-(dicyclopropylmethyl)-6,6',7,7'-6,6'-imino-7,7'-binorphinan -3,4',14, 14'-tetrol), at 100 nmol. The arrhythmogenic effect was also attenuated by both verapamil and nifedipine in a dose-dependent manner. The ED50 values for verapamil and nifedipine were 2.75 and 63.7 nmol, respectively. The antiarrhythmic potencies of these two CCBs were correlated to their displacing potencies and inversely related to their well known potencies in inhibiting transmembrane Ca2+ influx in the cardiac muscle. 3. Measurement of [Ca2+]i in the absence of free extracellular Ca2+ by a spectrofluorometric method, with fura-2 as Ca2+ indicator, showed that U50,488H 5 x 10(-5) M slowly increased [Ca2+]i in single ventricular myocytes and this effect was abolished by pretreatment with nor-BNI (5 microM), or ryanodine (5 microM). Verapamil 1 and 10 microM abolished the effect of U50,488H in 37.5% (3 out of 8) and 100% (12 out of 12) of the cells studied, respectively. On the other hand, nifedipine 10 and 100 microM had no effect at all. Neither verapamil nor nifedipine exerted any significant effect on the caffeine-induced Ca2+ transient. 4. The observations suggest that CCBs may inhibit the actions of kappa-receptor stimulation at the level of the kappa-receptor.

Local anaesthetics and opioid drugs function synergistically to provide analgesia. In the present study, the nature of this synergy has been investigated using in vitro radioligand binding to determine whether the local anaesthetics bupivacaine and tetracaine modulate the binding of two kappa-opioid receptor ligands, [3H]U-69593 (5-alpha,7-alpha,8-beta-(-)-N-methyl-N[7-(1-pyrrolodinyl)-1-oxa spiro(4,5)dec-8-yl]-benzene acetamide) and [3H](-)-EKC (ethylketocyclazocine). [3H]U-69593 bound with a KD of 0.88 nM and a Bmax of 2.39 +/- 0.22 fmol/mg wet weight in guinea pig cerebellar membranes. The binding was inhibited by bremazocine and morphine with Hill slopes near unity and pI50 values of 9.96 and 6.84-6.86, respectively. [3H]U-69593 binding was inhibited by Gpp(NH)p (5'-guanylyl imidodiphosphate) and NaCl, consistent with an agonist action of the compound. The binding characteristics of the ligand were not changed by bupivacaine or tetracaine. [3H](-)-EKC bound with KD values of 0.55 and 0.97 nM and Bmax values of 4.22 and 0.99 fmol/mg wet weight in guinea pig cerebellar membranes and rat spinal cords, respectively. In the rat spinal cord, [3H](-)-EKC appeared to act as an agonist/antagonist, since the presence of Gpp(NH)p and NaCl only produced a small (21%) reduction in binding, but reduced the pI50 for the residual binding to inhibition by morphine from 6.33-6.39 to 5.95. As with [3H]U-69593, the binding characteristics of [3H](-)-EKC were not affected by bupivacaine or tetracaine. These studies demonstrate that effects of kappa-opioid receptor recognition site conformation are unlikely to explain the clinically observed synergy between local anaesthetics and opioids.

The present study examined the effects of the competitive NMDA antagonist, NPC 12626, on the analgesic effects of the specific kappa opiate receptor agonist, U69,593, in male deer mice. Intraperitoneal (i.p.) administration of NPC 12626 had no effect on the basal nociceptive sensitivity of reproductive male deer mice, as measured by latency of response to a thermal (50 degrees C) surface. NPC 12626 dose-dependently (0.05-1.0 mg/kg) reduced U69,593-induced analgesia. NPC 12626 at 1.0 mg/kg attenuated U69,593-induced analgesia in a manner comparable to that produced by the specific kappa opiate antagonist, nor-binaltorphimine. In contrast, this dose of NPC 12626 potentiated the analgesia produced by the predominantly mu agonist morphine (1.0 mg/kg). The non-competitive NMDA antagonist, MK-801, which has been previously indicated to affect kappa opiate analgesia, significantly reduced at 1.0 mg/kg, but did not block, the analgesia produced by U69,593 and in contrast to NPC 12626, slightly reduced morphine-induced analgesia. These findings suggest that the NMDA antagonist, NPC 12626, may, either directly or indirectly, have effects on kappa opiate receptor mediated mechanisms.

1. The evidence for kappa-receptor heterogeneity is equivocal. We have now investigated this question by comparing the effects of five putatively selective kappa-agonists. The parameters examined were: the relative potencies in depressing hindlimb flexor muscle reflexes to noxious pinch stimuli in both spinalized and sham-spinalized rats; the reversibility of these effects by naloxone; and the effects on blood pressure. 2. Two types of drug effect was discriminated. One drug group, represented by U-50,488, U-69,593 and PD-117,302, had a potency ratio between sham and spinalized rats approximately 10 fold lower than the other group, which comprised GR103545 and CI-977. 3. Under sham-spinalized conditions, CI-977 and GR103545 at high doses caused only sub-maximal reductions of spinal reflexes. U-50,488 was still active when superimposed on these high doses of GR103545. 4. Naloxone reversed all effects, but different doses were required between compounds, with GR103545 taking some 20 times higher doses of naloxone to cause reversal than did U-50,488. 5. The effects on mean arterial pressure were opposite between groups. 6. The results imply that more than one type of naloxone-sensitive non-mu opioid receptor must be involved in mediating these complex actions of ligands that have been claimed to be selective for kappa-receptors.

Previously, we showed that kappa-selective ligands inhibit adenylyl cyclase in guinea pig cerebellar membranes. The present studies explore the relationship between kappa 1 binding sites (as determined with [3H]U-69,593 binding) and kappa 1-inhibition of adenylyl cyclase (using U-50,488H) in guinea pig brain membranes. Various kappa opioids displaced [3H]U-69,593 binding at a single site with subnanomolar affinities. These agonists were several hundred-fold weaker in inhibiting adenylyl cyclase, but for most agonists the rank order of adenylyl cyclase inhibition paralleled the displacement of kappa 1 binding. The correlation of IC50 values for both adenylyl cyclase and binding was significant except for alpha-neo endorphin, which was relatively weak at inhibiting adenylyl cyclase despite a Ki value of 0.08 nM versus kappa 1 binding. Comparison between kappa 1 binding and kappa 1-inhibited adenylyl cyclase across eleven guinea pig brain regions revealed that kappa 1-inhibited adenylyl cyclase was highest in the cerebellum, absent in thalamus and superior colliculus, and moderate in other regions. In most regions, kappa 1 binding correlated with the efficacy of kappa 1-inhibited adenylyl cyclase. However, the hippocampus had high levels of kappa 1-inhibited adenylyl cyclase despite low levels of kappa 1 binding, while cortex exhibited a high density of kappa 1 sites but a relatively low level of kappa 1-inhibited adenylyl cyclase. Reaction of cerebellar kappa receptors with beta-chlornaltrexamine (beta-CNA) blocked both kappa 1 binding and kappa 1-inhibited adenylyl cyclase. The effect of beta-CNA on kappa 1-inhibited adenylyl cyclase was to inhibit efficacy with little decrease in agonist potency, thus suggesting no significant level of kappa receptor reserve for this effector system.

The duration of antagonistic action of nor-binaltorphimine (nor-BNI), a kappa antagonist, of antinociception resulting from selective opioid agonists, was examined using the mouse tail-flick assay as the endpoint. Nor-BNI (1 nmol, i.c.v. at -20 min) antagonized equiantinociceptive doses of the opioid kappa agonists (5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro (4,5)dec-8-yl) benzeneacetamide (U69,593) (70 nmol i.c.v.) or bremazocine (25 nmol i.c.v.), but did not antagonize antinociception produced by the mu opioid-selective [D-Ala2, NMePhe4, Gly-ol]enkephalin or the delta opioid-selective [D-Pen2, D-Pen5]enkephalin. Pretreatment with nor-BNI (1 nmol i.c.v.) antagonized the antinociceptive effects of U69,593 and bremazocine for up to 28 days. At all pretreatment times, the antinociceptive dose-response lines for these kappa agonists were displaced to the right to various degrees in a parallel fashion; an increasing rightward displacement of the U69,593 and bremazocine antinociceptive dose-response lines was observed at 1 and 3 days after a single nor-BNI pretreatment, with a gradual return toward the control level at later times after pretreatment. Increasing the dose of nor-BNI to 10 nmol produced only a transient blockade of equiantinociceptive doses of the mu selective agonist [D-Ala2, NMePhe4, Gly-ol]enkephalin and the delta selective agonist [D-Pen2, D-Pen5]enkephalin (at 20-30 min post-nor-BNI pretreatment).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of pretreatment with the kappa receptor nonequilibrium antagonist, (-)-UPHIT (1S,2S-trans-2-isothiocyanato-4,5-dichloro-N-methyl-N-[2-(1-pyrrol idinyl) cyclohexyl]benzeneacetamide), on U69,593 [(5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5) dec-8-yl)benzeneacetamide]- and bremazocine-induced antinociception was examined in mice. Both U69,593 and bremazocine produced antinociception in the warm water tail-flick test after i.c.v. administration. Pretreatment with the kappa antagonist, nor-binaltorphimine, at doses shown not to affect [D-Ala2, NMePhe4, Gly-ol]enkephalin- (mu-agonist) or [D-Pen2, D-Pen5]enkephalin (delta-agonist)-induced antinociception, significantly attenuated the effects of U69,593 and bremazocine, suggesting actions of these agonists at kappa receptors. Furthermore, beta-funaltrxamine (mu antagonist) and ICI 174,864 [N,N,-diallyl-Tyr-(alpha-aminoisobutyric acid)2-Phe-Leu-OH] (delta antagonist), had no effect on U69,593 or bremazocine in this test providing further evidence of kappa receptor-mediated activity. Pretreatment with (-)-UPHIT produced no effect alone and a long-lasting (up to 48 hr) antagonism of U69,593, but not bremazocine, antinociception. The antagonist actions of (-)-UPHIT did not alter the antinociceptive effects of [D-Ala2, NMePhe4, Gly-ol]enkephalin or [D-Pen2, D-Pen5]enkephalin. These data suggest that (-)-UPHIT is a selective, long-lasting kappa antagonist which can differentially antagonize the antinociception produced by these two kappa agonists. These data provide evidence in vivo supportive of kappa receptor subtypes in the mouse, and suggest that (-)-UPHIT may be a useful probe for the exploration of kappa receptor heterogeneity.

The kappa-opioid compound U-69,593 was studied in rats in vitro in binding assays to assess its selectivity at the single types of opioid sites and in vivo to assess its analgesic activity and effect on intestinal propulsion. In vitro the U-69,593 inhibition curve of [3H]-(-)-bremazocine binding suppressed at mu- and alpha-sites was biphasic and the inhibition constant (Kl) at the high-affinity site (10-18 nM) was two orders of magnitude smaller than the Kl at the low-affinity site. The Kl at mu- and alpha-sites were respectively 3.3 and 8.5 microM. Thus [3H]-(-)-bremazocine, suppressed at mu- and alpha-sites, may still bind more than one site, which U-69,593 might distinguish. In vivo U-69,593 i.p. prolonged the reaction time of rats on a 55 degrees C hot-plate and the dose of naloxone required to antagonize this effect was 40 times the dose that antagonized morphine-induced antinociception, suggesting the involvement of the kappa-receptor. In the intestinal transit test U-69,593 at doses between 0.5 and 15 mg/kg i.p. only slightly slowed intestinal transit of a charcoal meal in rats with no dose-relation; it partly but significantly antagonized morphine-induced constipation. These results suggest that the kappa-type of opioid receptor, with which U-69,593 interacts may induce analgesia, but has no appreciable role in the mechanisms of opioid-induced inhibition of intestinal transit in rats.