Antagonistic property of buprenorphine for putative epsilon-opioid receptor-mediated G-protein activation by beta-endorphin in pons/medulla of the mu-opioid receptor knockout mouse.

beta-Endorphin is a non-selective opioid peptide which binds mu-, delta- and putative epsilon (beta-endorphin-sensitive non-mu-, non-delta- and non-kappa(1)-)-opioid receptors. We have previously reported that beta-endorphin-produced G-protein activation is mediated by the stimulation of both mu- and putative epsilon-opioid receptors. The present study was designed to further characterize this putative epsilon-opioid receptor-mediated G-protein activation in the pons/medulla membrane obtained from mice lacking mu-opioid receptor, using a guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS)-binding assay. beta-Endorphin and the mu-opioid receptor agonist [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) increased the [(35)S]GTPgammaS binding in a concentration-dependent manner (0.001-10 microM), and at 10 microM beta-endorphin and DAMGO produced approximately 250 and 120% increases of [(35)S]GTPgammaS binding in the pons/medulla membrane obtained from wild-type mice, respectively. In the pons/medulla membrane obtained from mu-opioid receptor knockout mice, beta-endorphin-stimulated [(35)S]GTPgammaS binding was only partially attenuated and a more than 100% increase by 10 microM beta-endorphin still remained, while DAMGO failed to produce any increase in [(35)S]GTPgammaS binding. The residual increase in [(35)S]GTPgammaS binding by 10 microM beta-endorphin in mu-opioid receptor knockout mice was partially but significantly attenuated by the putative epsilon-opioid receptor partial agonist beta-endorphin (1-27), but not by the delta-opioid receptor antagonist naltrindole or the kappa(1)-receptor antagonist norbinaltorphimine. Furthermore, buprenorphine significantly attenuated the residual increase in [(35)S]GTPgammaS binding by 10 microM beta-endorphin in mu-opioid receptor knockout mice. The present results indicate that beta-endorphin activates G-protein by stimulation of putative epsilon-opioid receptors in the condition lacking the mu-opioid receptor, and buprenorphine acts as an antagonist for putative epsilon-opioid receptors in this condition.

Evidence for epsilon-opioid receptor-mediated beta-endorphin-induced analgesia.

Among the opioid receptors, which have been pharmacologically classified as mu, delta, kappa and epsilon, the existence of the epsilon receptor has been controversial, and this receptor is generally not recognized as a member of the opioid peptide receptor family because it has not been precisely characterized. However, results from pharmacological, physiological and opioid receptor binding studies clearly indicate the presence of epsilon-opioid receptors, which are distinct from mu-, delta- or kappa-opioid receptors. This putative epsilon-opioid receptor is stimulated supraspinally by the endogenous opioid peptide beta-endorphin, which induces the release of Met-enkephalin, which, in turn, acts on spinal delta2-opioid receptors to produce antinociception. In this article, this beta-endorphin-sensitive epsilon-opioid receptor-mediated descending pain control system, which is distinct from that activated by the mu-opioid receptor agonist morphine, is described and the physiological role of the beta-endorphin-mediated system in pain control activated by cold-water swimming and intraplantar injection of formalin is discussed.

Acute antinociceptive tolerance and asymmetric cross-tolerance between endomorphin-1 and endomorphin-2 given intracerebroventricularly in the mouse.

Development of tolerance in mice pretreated intracerebroventricularly with mu-opioid receptor agonist endomorphin-1, endomorphin-2, or [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO) was compared between endomorphin-1- and endomorphin-2-induced antinociception with the tail-flick test. A 2-h pretreatment with endomorphin-1 (30 nmol) produced a 3-fold shift to the right in the dose-response curve for endomorphin-1. Similarly, a 1-h pretreatment with endomorphin-2 (70 nmol) caused a 3.9-fold shift to the right for endomorphin-2. In cross-tolerance experiments, pretreatment with endomorphin-2 (70 nmol) caused a 2.3-fold shift of the dose-response curve for endomorphin-1, whereas pretreatment with endomorphin-1 (30 nmol) caused no change of the endomorphin-2 dose-response curve. Thus, mice acutely tolerant to endomorphin-1 were not cross-tolerant to endomorphin-2, although mice made tolerant to endomorphin-2 were partially cross-tolerant to endomorphin-1; an asymmetric cross-tolerance occurred. Pretreatment with DAMGO 3 h before intracerebroventricular injection of endomorphin-1, endomorphin-2, or DAMGO attenuated markedly the antinociception induced by endomorphin-1 and DAMGO but not endomorphin-2. It is proposed that two separate subtypes of mu-opioid receptors are involved in antinociceptive effects induced by endomorphin-1 and endomorphin-2. One subtype of opioid mu-receptors is stimulated by DAMGO, endomorphin-1, and endomorphin-2, and another subtype of mu-opioid receptors is stimulated solely by endomorphin-2.

Reduced hyperalgesia induced by nerve injury, but not by inflammation in mice lacking protein kinase C gamma isoform.

Protein kinase C is one of protein kinases which might be involved in the nerve injury- or inflammation-induced hyperalgesia. The present study was designed to investigate the hyperalgesia with thermal paw-withdrawal test induced by sciatic nerve ligation or by intraplantar injection of a complete Freund's adjuvant solution in protein kinase C gamma knockout and its wild-type mice. Either sciatic nerve ligation or intraplantar injection of a complete Freund's adjuvant caused a marked decrease of the paw-withdrawal latency only on the ipsilateral, but not on the contralateral side of the paw in wild-type mice. This ipsilateral hyperalgesia induced by sciatic nerve ligation was significantly attenuated in protein kinase C gamma knockout mice. On the other hand, the ipsilateral hyperalgesia induced by complete Freund's adjuvant remained about the same in protein kinase C gamma knockout mice as in wild-type mice. The results indicate that protein kinase C gamma is involved in the development of the thermal hyperalgesia induced by nerve ligation, but not by complete Freund's adjuvant-induced inflammation.

Release of [Met5]enkephalin from the spinal cord by intraventricularly administered endomorphin-2, but not endomorphin-1 in the anesthetized rat.

Effects of intraventricular injection of endomorphin-1, endomorphin-2 and beta-endorphin on the release of immunoreactive [Met(5)]enkephalin from the spinal cord were studied in pentobarbital anesthetized rats. Intraventricular injection of endomorphin-2, but not endomorphin-1, caused an increased release of immunoreactive [Met(5)]enkephalin in the spinal perfusates. Beta-endorphin given intraventricularly also increased the release of immunoreactive [Met(5)]enkephalin in an amount 15-fold higher than that produced by endomorphin-2. The increase of the release of immunoreactive [Met(5)]enkephalin induced by endomorphin-2 was blocked by mu-opioid receptor antagonist CTOP. Our result suggests that endomorphin-2 stimulates another subtype of mu-opioid receptor different from that acted by endomorphin-1 at the supraspinal site and subsequently increases the release of [Met(5)]enkephalin from the spinal cord.

Differential antinociceptive effects induced by intrathecally administered endomorphin-1 and endomorphin-2 in the mouse.

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for mu-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the mu (1)-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the delta(2)-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the delta(1)-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met(5)]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of mu (1)-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met(5)]enkephalin which subsequently act on kappa-opioid receptors and delta(2)-opioid receptors to produce antinociception.

Up-regulation of spinal mu-opioid receptor function to activate G-protein by chronic naloxone treatment.

The effects of repeated s.c. administrations of an mu-opioid receptor antagonist naloxone on the G-protein activation induced by mu-opioid receptor agonists [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO), endomorphin-1 and endomorphin-2 in the mouse spinal cord was studied, monitoring guanosine-5'-o-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding. All mu-opioid receptor agonists concentration-dependently increased the [35S]GTPgammaS binding. The increases of [35S]GTPgammaS binding induced by agonists were significantly enhanced in mice pretreated with naloxone. Under the present condition, chronic treatment with naloxone significantly increased the density of [3H]DAMGO binding sites with an increase in K(d) values in spinal cord membranes, indicating an increase in mu-opioid receptors on the membrane surface. These findings suggest that chronic treatment with an mu-opioid receptor antagonist naloxone leads to the supersensitivity to activate G-protein by mu-opioid receptor agonists with an increase in mu-opioid receptors in membranes of the mouse spinal cord.

Different motivational effects induced by the endogenous mu-opioid receptor ligands endomorphin-1 and -2 in the mouse.

The present study was designed to investigate the motivational effects of the newly discovered endogenous mu-opioid receptor ligands, endomorphin-1 and endomorphin-2, using the conditioned place preference paradigm in mice. The binding properties of these peptides were first examined using an opioid binding assay. In membranes obtained from the mouse whole brain, the binding of [3H][D-Ala2, NMePhe4, Gly(ol)5]enkephalin (DAMGO; mu), but not of [3H][D-Phe2, D-Phe5]enkephalin (DPDPE; delta) or [3H]U69593 (kappa) selectively and concentration-dependently competed with that of endomorphin-1 and endomorphin-2, indicating that both endomorphin-1 and endomorphin-2 are specific ligands for mu-opioid receptors in the brain. Endomorphin-1 (1-30 nmol/mouse) given i.c.v. produced a dose-related place preference. This effect was abolished by pre-treatment with the mu-opioid receptor antagonist beta-funaltrexamine but not the delta-opioid receptor antagonist naltrindole or the kappa-opioid receptor antagonist nor-binaltorphimine. In contrast, endomorphin-2 (5.6 nmol/mouse) produced place aversion. This aversive effect was inhibited by nor-binaltorphimine as well as beta-funaltrexamine, but not by naltrindole. The place aversion produced by endomorphin-2 was also attenuated by pre-treatment with antiserum against the endogenous kappa-opioid receptor ligand dynorphin A (1-17). These findings indicate that endomorphin-1 may produce its rewarding effect via mu-opioid receptors. On the other hand, the aversive effect induced by endomorphin-2 may be associated with the stimulation of endomorphin-1-insensitive mu-opioid receptors and the activation of dynorphinergic systems in the mouse brain.

Differential antinociception induced by spinally administered endomorphin-1 and endomorphin-2 in the mouse.

We have previously demonstrated that the antinociception induced by either endomorphin-1 or endomorphin-2 given supraspinally is mediated by the stimulation of mu-opioid receptors. However, the antinociception induced by endomorphin-2 given supraspinally contains additional components, which are mediated by the spinal release of dynorphin A (1-17) acting on kappa-opioid receptors and the spinal release of [Met5]enkephalin acting on delta2-opioid receptors in the spinal cord. The present studies were performed to determine whether there are any differential effects on the tail-flick inhibition induced by endomorphin-1 and endomorphin-2 given intrathecally (i.t.) in mice. Endomorphin-1 or endomorphin-2 given i.t. inhibited the tail-flick response in a dose-dependent manner. The tail-flick inhibition induced by endomorphin-1 was blocked by i.t. pretreatment with mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Try-Orn-Thr-Pen-Thr-NH2 (CTOP), but not kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI), delta1-opioid receptor antagonist 7-benzylidene naltrexamine (BNTX), or delta2-opioid receptor antagonist naltriben (NTB). In contrast, the tail-flick inhibition induced by endomorphin-2 given i.t. was blocked by i.t. pretreatment with CTOP or nor-BNI, but not BNTX or NTB. Intrathecal pretreatment with antiserum against dynorphin A (1-17), but not antiserum against [Met5]enkephalin, [Leu5]enkephalin, or beta-endorphin, blocked the tail-flick inhibition induced by i.t.-administered endomorphin-2. None of these antisera attenuated the i.t.-administered endomorphin-1-induced tail-flick inhibition. It is concluded that the tail-flick inhibition induced by endomorphin-1 and endomorphin-2 given spinally is mediated by the stimulation of mu-opioid receptors. However, the tail-flick inhibition induced by spinally injected endomorphin-2 contains an additional component, which is mediated by the spinal release of dynorphin A (1-17) acting on kappa-opioid receptors in the spinal cord. We propose that there are at least two different subtypes of micro-opioid receptors for endomorphin-1 and endomorphin-2 to produce antinociception in the spinal cord.

Lack of the involvement of mu1-opioid receptor subtype on motivational effects induced by the endogenous mu-opioid receptor ligands endomorphin-1 and -2 in the mouse.

The present study was designed to investigate the role of mu-opioid receptor subtypes in the motivational effect of endogenous mu-opioid receptor ligands, endomorphin-1 and -2. In C57BL/6J mice, endomorphin-1 produced a significant place preference, whereas endomorphin-2 exhibited a significant place aversion. These effects were abolished by a mu1/mu2-opioid receptor antagonist beta-funaltrexamine. Under these conditions, both endomorphin-1 and -2 produced their motivational effects in mu1-opioid receptor-deficient CXBK mice, indicating the mu2-opioid receptor involvement. Furthermore, in the lower midbrain including ventral tegmental area, both endomorphin-1 and -2 equally produced dose-related increases in guanosine-5'-o-(3-[35S] thio) triphosphate bindings in C57BL/6J and CXBK mice. These findings indicate that endomorphin-1 and -2 may produce distinct motivational effects via respective mu2-opioid receptor isoforms in the mouse. Furthermore, endomorphin-1 and -2 produced the mu1-resistant G-protein activation in the mouse lower midbtrain.

Antisera against endogenous opioids increase the nocifensive response to formalin: demonstration of inhibitory beta-endorphinergic control.

The roles of endogenous opioid peptides in the brain in the modulation of nocifensive responses to formalin in ICR mice were studied. Mice were pretreated intracerebroventricularly (i.c.v.) with rabbit antiserum against beta-endorphin, [Leu5]enkephalin, [Met5]enkephalin or dynorphin A-(1-17) 1 h prior to intraplantar injection of formalin (0.5%, 25 microl) and the nocifensive licking responses were then observed. Pretreatment of mice with antiserum against beta-endorphin enhanced the second phase, but not the first phase of the nocifensive responses to formalin. Pretreatment with antiserum against [Leu5]enkephalin also caused a small but statistically significant enhancement of the second phase, but not the first phase of nocifensive responses to formalin. On the other hand, pretreatment with antiserum against [Met5]enkephalin or dynorphin A-(1-17) did not affect the nocifensive response to formalin. Our results indicate that beta-endorphinergic, and to a lesser extent, [Leu5]enkephalinergic systems are activated at the supraspinal sites to attenuate the nocifensive responses to formalin stimulation.

Involvement of spinal protein kinase Cgamma in the attenuation of opioid mu-receptor-mediated G-protein activation after chronic intrathecal administration of [D-Ala2,N-MePhe4,Gly-Ol(5)]enkephalin.

The present study was designed to investigate the role of a protein kinase C (PKC) isoform in the uncoupling of the mu-opioid receptor from G-proteins after repeated intrathecal injection of a selective mu-receptor agonist, [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO), in the spinal cord of mice. The activation of G-proteins by opioids was measured by monitoring the guanosine-5'-o-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding. Mice were injected intrathecally with saline or DAMGO once a day for 1-7 d. At 24 hr after every injection the spinal cord membranes were prepared for the assay. The enhanced [(35)S]GTPgammaS binding by mu-agonists DAMGO, endomorphin-1, or endomorphin-2 was attenuated clearly in spinal cord membranes obtained from mice that were treated intrathecally with DAMGO for 5 and 7 d, but not for 1 or 3 d. By contrast, no change in levels of [(35)S]GTPgammaS binding induced by the delta-receptor agonist SNC-80 or kappa-receptor agonist U-50,488H was noted in membranes obtained from mice that were treated with DAMGO. Concomitant intrathecal administration of a specific PKC inhibitor Ro-32-0432 with DAMGO blocked the attenuation of DAMGO-induced G-protein activation that was caused by chronic DAMGO treatment. Western blotting analysis showed that chronic DAMGO treatment increased the levels of PKCgamma, but not PKCalpha, PKCbetaI, and PKCbetaII isoforms, in spinal cord membranes. Furthermore, mice lacking PKCgamma failed to exhibit the desensitization of the DAMGO-stimulated [(35)S]GTPgammaS binding after repeated DAMGO injection. These findings indicate that repeated intrathecal administration of DAMGO may activate the PKCgamma isoform and in turn cause a desensitization of mu-receptor-mediated G-protein activation in the mouse spinal cord.

TRK-820, a selective kappa-opioid agonist, produces potent antinociception in cynomolgus monkeys.

TRK-820 ((-)-17-cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6b-[N-methyl-trans-3-(3-furyl)acrylamide]morphinan hydrochloride) has been shown to be a potent opioid kappa-receptor agonist with pharmacological properties different from those produced by kappa1-opioid receptor agonists in rodents. To ascertain whether or not these properties of TRK-820 would be extended to primates, the antinociceptive effect of TRK-820 was evaluated in cynomolgus monkeys by the hot-water tail-withdrawal procedure. TRK-820 given intramuscularly (i.m.) produced a potent antinociceptive effect that was 295- and 495-fold more potent than morphine with the 50 degrees C and 55 degrees C hot-water tests, respectively, and 40-fold more potent than U-50,488H and 1,000-fold more potent than pentazocine in the 50 degrees C hot-water test. The duration of antinociceptive effects of TRK-820 treatment (0.01 and 0.03 mg/kg, i.m.) lasted more than 6 h, which was much longer than those of U-50,488H. The antinociception produced by the higher dose (0.03 mg/kg, i.m.) of TRK-820 was not inhibited by nor-binaltorphimine (3.2 and 10 mg/kg, s.c.) or by naloxone (0.1 mg/kg, s.c.), although the antinociception induced by a lower dose of TRK-820 (0.01 mg/kg, i.m.) was inhibited by nor-binaltorphimine (10 mg/kg, s.c.). The same doses of nor-binaltorphimine and naloxone effectively inhibited the antinociception induced by the higher doses of U-50,488H (1.0 mg/kg, i.m.) and morphine (10 mg/kg, i.m.), respectively. These results indicate that the antinociception induced by TRK-820 is less sensitive to nor-binaltorphimine and suggest that it is mediated by the stimulation of a subtype of kappa-opioid receptor different from the kappa-opioid receptor in cynomolgus monkeys.

Inverse agonist action of Leu-enkephalin at delta(2)-opioid receptors mediates spinal antianalgesia.

Dynorphin A(1-17) given intrathecally releases spinal cholecystokinin to produce an antianalgesic action against spinal morphine in the tail-flick test in CD-1 mice. The present study showed that following the cholecystokinin step, a delta(2)-opioid inverse agonist action of Leu-enkephalin (LE), was involved. Pretreatment with intrathecal LE antiserum eliminated dynorphin and cholecystokinin-8s antianalgesia. A small dose of LE intrathecally produced antianalgesia that like that from dynorphin A(1-17) and cholecystokinin was eliminated by naltriben but not 7-benzylidenenaltrexone (delta(2)- and delta(1)-opioid receptor antagonist, respectively). This LE step was followed by N-methyl-D-aspartate (NMDA) receptor activation. MK801, an NMDA receptor antagonist, eliminated the antianalgesia from dynorphin A(1-17), cholecystokinin-8s, and LE. Furthermore, none of the three were effective against morphine analgesia in 129S6/SvEv mice possibly because of their deficiency in NMDA receptor response. In 129S6/SvEv mice, [D-Ser(2)]-Leu-enkephalin-Thr analgesia was not attenuated by LE; thus, this delta(2)-analgesic agonist and LE inverse agonist action did not occur through competition at the same delta(2)-receptor in CD-1 mice. In CD-1 mice, a linear sequence of dynorphin A(1-17) --> cholecystokinin --> LE --> NMDA receptors was indicated: cholecystokinin antiserum inhibited cholecystokinin but not LE; naltriben inhibited LE but not NMDA. The uniqueness of LE in linking dynorphin A(1-17), cholecystokinin, delta(2)-opioid, and NMDA receptor activation may unify the separate known mechanisms involved in the antiopioid actions of these components against morphine.

Enhanced mu-opioid responses in the spinal cord of mice lacking protein kinase Cgamma isoform.

The protein kinase C (PKC)gamma isoform is a major pool of the PKC family in the mammalian spinal cord. PKCgamma is distributed strategically in the superficial layers of the dorsal horn and, thus, may serve as an important biochemical substrate in sensory signal processing including pain. Here we report that mu-opioid receptor-mediated analgesia/antinociception and activation of G-proteins in the spinal cord are enhanced in PKCgamma knockout mice. In contrast, delta- and kappa-opioidergic and ORL-1 receptor-mediated activation of G-proteins in PKCgamma knockout mice was not altered significantly relative to the wild-type mice. Deletion of PKCgamma had no significant effect on the mRNA product of spinal mu-opioid receptors but caused an increase of maximal binding of the mu-opioid receptor agonist [3H][d-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin in spinal cord membranes obtained from PKCgamma knockout mice. These findings suggest that deletion of PKCgamma genes protects the functional mu-opioid receptors from degradation by phosphorylation. More importantly the present data provide direct evidence that PKCgamma constitutes an essential pathway through which phosphorylation of mu-opioid receptors occurs.

Therapeutic potential of PKC inhibitors in painful diabetic neuropathy.

Diabetic neuropathy accompanied by anomalies in pain perception is one of the most frequent complications in insulin-dependent diabetes in humans. Many clinical and experimental studies have suggested that diabetes or hyperglycaemia alters pain sensitivity. In humans, diabetic neuropathy can be associated with burning, tactile hypersensitivity. Behavioural reactions of hyperalgesia in animal models of diabetes have been described. However, the aetiology of these disturbances is still unknown, although metabolic factors such as hyperglycaemia or neurotransmitter alteration may be involved. Activation of protein kinase C (PKC) has been implicated in changes in pain perception. Phorbol esters, which activate PKC, enhance the thermal hyperalgesia in diabetic mice and enhance nociceptive responses after tissue injury induced by formalin. Electrophysiological experiments have shown that activation of PKC leads to long-lasting enhancement of excitatory amino acid-mediated currents in dorsal horn neurones and trigeminal neurones. Thus, activation of PKC may underlie the neuronal sensitisation that produces hyperalgesia in diabetic neuropathy.

Activation of G-proteins in the mouse pons/medulla by beta-endorphin is mediated by the stimulation of mu- and putative epsilon-receptors.

The activation of mu-, delta- and kappa1-opioid receptors by their respective agonists increases the binding of the non-hydrolyzable GTP analog guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS) to G proteins. Beta-endorphin is an endogenous opioid peptide which binds nonselectively to mu-, delta- and putative epsilon-opioid receptors. The present experiment was designed to determine which opioid receptors are involved in the stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the mouse pons/medulla. The mouse pons/medulla membranes were incubated in an assay buffer containing 50 pM [35S]GTPgammaS, 30 microM GDP and various concentrations of beta-endorphin. Beta-endorphin (0.1 nM-10 microM) increased [35S]GTPgammaS binding in a concentration-dependent manner, and 10 microM beta-endorphin produced a maximal stimulation of approximately 260% over baseline. This stimulation of [35S]GTPgammaS binding by beta-endorphin was partially attenuated by the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA), but not by the delta-opioid receptor antagonist naltrindole (NTI) or the kappa1-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Beta-endorphin stimulated [35S]GTPgammaS binding by about 80% in the presence of 10 microM beta-FNA, 30 nM NTI and 100 nM nor-BNI. The same concentrations of these antagonists completely blocked the stimulation of [35S]GTPgammaS binding induced by 10 microM [D-Ala2,NHPhe4,Gly-ol]enkephalin, [D-Pen(2,5)]enkephalin and U50,488H, respectively. Moreover, the residual stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the presence of the three opioid receptor antagonists was significantly attenuated by 100 nM of the putative epsilon-opioid receptor partial agonist beta-endorphin (1-27). These results indicate that the stimulation of [35S]GTPgammaS binding induced by beta-endorphin is mediated by the stimulation of both mu- and putative epsilon-opioid receptors in the mouse pons/medulla.

Implications of Ca(2+)-activated Cl(-) channels in the delta-opioid receptor-mediated antinociception in the mouse spinal cord.

The present study was designed to investigate the role of Ca(2+)-activated Cl(-) channels in the spinal opioid receptor-mediated antinociception using the mouse tail-flick assay. The antinociception induced by intrathecal (i.t.) administration of a selective delta-opioid receptor agonist [D-Ala(2)]deltorphin II (10 microgram, i.t.) was significantly attenuated by i.t.-pretreatment with selective Ca(2+)-activated Cl(-) channel blockers, IAA-94, flufenamic acid and niflumic acid. By contrast, IAA-94 had no effect on the antinociception induced by i.t.-treated with either the selective mu-opioid receptor agonist [D-Ala(2),N-MePhe(4), Gly-ol(5)]enkephalin or the kappa-opioid receptor agonist U-50,488H. The present results provide evidence for the first time that the Ca(2+)-activated Cl(-) channel is, at least in part, implicated in the delta-, but not the mu- and kappa-opioid receptor-mediated antinociception in the mouse spinal cord.

Differential involvement of mu(1)-opioid receptors in endomorphin- and beta-endorphin-induced G-protein activation in the mouse pons/medulla.

Several genetic mouse models of differential sensitivity to opioids have been used to investigate the mechanisms underlying individual variation in responses to opioids. The CXBK mice are inbred recombinant mice which have a lower level of mu(1)-opioid receptors than their parental strain. Endomorphin-1 and endomorphin-2 are endogenous opioid peptides that are highly selective for mu-opioid receptors, while beta-endorphin, which is also an endogenous opioid peptide, is non-selective for mu-, delta- and putative epsilon-opioid receptors. The present study was designed to investigate the effects of these endogenous opioid peptides on G-protein activation by monitoring guanosine-5'-o-(3-[35S]thio)triphosphate binding to pons/medulla membranes of CXBK mice and their parental strain C57BL/6 ByJ mice. Endomorphin-1 (0.1-10 microM), endomorphin-2 (0.1-10 microM) and beta-endorphin (0.1-10 microM) increased guanosine-5'-o-(3-[35S]thio)triphosphate binding to the pons/medulla membranes from C57BL/6 ByJ and CXBK mice in a concentration-dependent manner. However, the increases of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by either endomorphin-1 or endomorphin-2 in CXBK mice were significantly much lower than those in C57BL/6ByJ mice. However, no significant difference was found in the increases of the guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by beta-endorphin in C57BL/6 ByJ and CXBK mice. Moreover, whereas the increase of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by 10 microM endomorphin-1 or endomorphin-2 were almost completely blocked by a mu-opioid receptor antagonist beta-funaltrexamine (10 microM) in both strains, the increase of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by 10 microM beta-endorphin was attenuated to approximately 70% of stimulation by co-incubation with 10 microM beta-funaltrexamine in both strains. The residual stimulation of [35S]guanosine-5'-o-(3-thio)triphosphate binding by 10 microM beta-endorphin in the presence of 10 microM beta-funaltrexamine was further attenuated by the addition of putative epsilon-opioid receptor partial agonist beta-endorphin (1-27) (1 microM) in both strains. Like the endomorphins, the synthetic mu-opioid receptor agonist [D-Ala(2),N-MePhe(4), Gly-ol(5)]enkephalin at 10 microM showed lower increases of guanosine-5'-o-(3-[35S]thio)triphosphate binding in CXBK mice than those in C57BL/6ByJ mice. However, there was no strain difference in the stimulation of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by 10 microM of the selective delta(1)-opioid receptor agonist [D-Pen(2,5)]enkephalin, delta(2)-opioid receptor agonist [D-Ala(2)]deltorphin II or kappa-opioid receptor agonist U50,488H. The results indicate that the G-protein activation by endomorphin-1 and endomorphin-2 in the mouse pons/medulla is mediated by both mu(1)- and mu(2)-opioid receptors. Moreover, beta-endorphin-induced G-protein activation in the mouse pons/medulla is, in part, mediated by mu(2)- and putative epsilon-, but not by mu(1)-opioid receptors.

Involvement of spinal protein kinase C in thermal hyperalgesia evoked by partial sciatic nerve ligation, but not by inflammation in the mouse.

Activation of several protein kinases contributes to the development of hyperalgesia evoked by injuries. The present study was designed to investigate the role of protein kinase C in the spinal cord in thermal hyperalgesia evoked by sciatic nerve ligation or by intraplantar injection of complete Freund's adjuvant. The paw withdrawal latency on the ipsilateral side, but not on the contralateral side, was markedly decreased after sciatic nerve ligation. Intraplantar injection of complete Freund's adjuvant also caused markedly decreases of the paw withdrawal latency. Intrathecal pretreatment with protein kinase C inhibitor calphostin C (100 and 250 ng) attenuated the decrease of the paw withdrawal latency evoked by sciatic nerve ligation. In contrast, the decrease of the paw withdrawal latency evoked by inflammation was only slightly attenuated by intrathecal pretreatment with calphostin C. The results indicate that protein kinase C in the spinal cord is involved in the development of the thermal hyperalgesia evoked by nerve ligation and is much less involved in the thermal hyperalgesia by complete Freund's adjuvant's-induced inflammation.