Ligand binding profiles of U-69, 593-sensitive and-insensitive sites in human cerebral cortex membranes: evidence of kappa opioid receptors heterogeneity

Receptor binding studies were performed to characterize the properties of subtypes of kappa opioid receptors in membrane preparations of human cerebral cortex. [3H]U69,593 ([3H]U69), a selective kappa 1-agonist, and [3H]diprenorphine ([3H]DIP), a non-selective opioid antagonist, in the presence of 1 microM each of DAMGO, DPDPE and U-69 to block mu-, delta-, and kappa 1-sites, labeled single population of binding sites, respectively. [3H]U-69 binding sites (KD = 3.8 +/- 0.2 nM, Bmax = 6.3 +/- 0.2 fmol/mg protein) had a binding profile that correspond to kappa 1-receptor. That is, dynorphin A (1-13) (Dyn A), bremazocine (BZC), U50,488H (U50), (-)ethylketocyclazocine (EKC) and nor-binaltorphimine (nor-BNI) bound to this site with high affinities. [3H]DIP labeled binding sites (Kd = 7.3 +/- 0.2 nM, Bmax = 102 +/- 9 fmol/mg protein) that were not sensitive to U-50, but to BZC, EKC and nor-BNI. These results indicate that kappa 1 and Kappa 2 opioid receptors exist in human cerebral cortex with different ligand binding profiles.     

Logistic regression analysis of twin data: estimation of parameters of the multifactorial liability-threshold model

This study with the rat evaluated the contribution of omega-conotoxin GVIA-(omega-CgTx) and verapamil-sensitive Ca2+ channels in behavioural, antinociceptive and thermoregulatory responses to intracerebroventricular (i.c.v.) injection of [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO), [D-Pen2,D-Pen5]enkephalin (DPDPE) and dynorphin A-(1-17), which are selective agonists for putative mu, delta and kappa-opioid receptors, respectively. The rats treated with omega-CgTx (8-32 pmol i.c.v.) showed transient, dose-dependent shaking behaviour, hyperalgesia and hypothermia which gradually disappeared within 4 h. The behaviour of the rats was normal by 24 h. Histological examination of brain sections showed morphological alterations of neurons in the hippocampus, medial-basal hypothalamus and pyriform cortex. antinociception, catalepsy and thermoregulatory responses elicited by DAMGO (0.4 and 2.0 nmol) were significantly prolonged and potentiated by verapamil (20 pmol i.c.v. 15 min before) or omega-CgTx (8 pmol 24 h before). Antinociception and hypothermia induced by DPDPE were antagonized by verapamil and omega-CgTx, whereas only omega-CgTx prevented the behavioural arousal observed after DPDPE. Similarly, hypothermia induced by dynorphin A-(1-17) (5.0 nmol) and by the kappa-opioid receptor agonist U50,488H (215 nmol) was antagonized by the two Ca2+ channel blockers but only omega-CgTx prevented the barrel rolling and bizarre postures caused by the opioid peptide.     

Prostaglandin E2 suppression of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by interacting with prostanoid receptors of the EP3-subtype

1. We have demonstrated recently that exogenous prostaglandin E2 (PGE2) inhibits electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. In the present study, we have attempted to characterize the pre-junctional prostanoid receptor(s) responsible for the inhibitory action of PGE2 and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea-pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS-evoked [3H]-ACh release. 2. In epithelium-denuded tracheal strips pretreated with indomethacin (10 microM), PGE2 (0.1 nM-1 microM) inhibited EFS-evoked [3H]-ACh release in a concentration-dependent manner with an EC50 and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP-receptor agonist, PGF2alpha and the stable thromboxane mimetic, U46619 (each at 1 microM), also inhibited [3H]-ACh release by 48%, 41% and 35%, respectively. PGD2 (1 microM) had no significant effect on [3H]-ACh release. 3. The selective TP-receptor antagonist, ICI 192,605 (0.1 microM), completely reversed the inhibition of cholinergic neurotransmission induced by U-46619, but had no significant effect on similar responses effected by PGE2 and PGF2alpha. 4. A number of EP-receptor agonists mimicked the ability of PGE2 to inhibit [3H]-ACh release with a rank order of potency: GR63799X (EP3-selective) > PGE2 > M&B 28,767 (EP3 selective) > 17-phenyl-omega-trinor PGE2 (EP1-selective). The EP2-selective agonist, AH 13205 (1 microM), did not affect EFS-induced [3H]-ACh release. 5. AH6809 (10 microM), at a concentration 10 to 100 times greater than its pA2 at DP-, EP1- and EP2-receptors, failed to reverse the inhibitory effect of PGE2 or 17-phenyl-omega-trinor PGE2 on [3H]-ACh release. 6. These results suggest that PGE2 inhibits [3H]-ACh release from parasympathetic nerves supplying guinea-pig trachea via an interaction with prejunctional prostanoid receptors of the EP3-receptor subtype. Evidence for inhibitory prejunctional TP- and, possibly, IP-receptors was also obtained although these receptors may play only a minor role in suppressing [3H]-ACh release when compared to receptors of the EP3-subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea-pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.     

Analgesic, respiratory and heart rate effects of cannabinoid and opioid agonists in rhesus monkeys: antagonist effects of SR 141716A

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.     

Streptozotocin-induced diabetes selectively enhances antinociception mediated by delta 1- but not delta 2-opioid receptors

We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of delta-opioid receptor agonists [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective delta 1-opioid receptor antagonist, but not with naltriben (NTB), a selective delta 2-opioid receptor antagonist. There were no significant differences in the antinociceptive effect of [D-Ala2]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala2]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal delta 1-opioid receptor-mediated antinociception, but are normally responsive to activation of delta 2-opioid receptors.     

Enhanced kappa-opioid receptor-mediated analgesia by antisense targeting the sigma1 receptor

In the current study, we used an antisense oligodeoxynucleotide targeting the recently cloned sigma1 receptor to assess its functions within the nervous system. Sigma1 antagonists potentiate the analgesic actions of opioids. Similarly, the antisense probe targeting the sigma1 receptor enhanced the analgesic activity of the kappa1-opioid receptor agonist U50,488H (trans-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeacetamidel++ +) and the kappa3-opioid receptor agonist naloxone benzoylhydrazone. A mismatch control was inactive. These results confirm the role of sigma1 receptors in an anti-opioid analgesic system and illustrate the utility of antisense approaches towards the elucidation of sigma receptor functions.     

Carrier-dependent and Ca(2+)-dependent 5-HT and dopamine release induced by (+)-amphetamine, 3,4-methylendioxymethamphetamine, p-chloroamphetamine and (+)-fenfluramine

1. The mechanism underlying 5-hydroxytryptamine (5-HT) and/or dopamine release induced by (+)-amphetamine ((+)-Amph), 3,4-methylendioxymethamphetamine (MDMA), p-chloroamphetamine (pCA) and (+)-fenfluramine ((+)-Fen) was investigated in rat brain superfused synaptosomes preloaded with the 3H neurotransmitters. 2. Their rank order of potency for [3H]-5-HT-releasing activity was the same as for inhibition of 5-HT uptake (pCA > or = MDMA > or = (+)-Fen > > (+)-Amph). Similarly, their rank order as [3H]-dopamine releasers and dopamine uptake inhibitors was the same ((+)-Amph > > pCA = MDMA > > (+)-Fen). We also confirmed that the release induced by these compounds was prevented by selective transporter inhibitors (indalpine or nomifensine). 3. [3H]-5HT and/or [3H]-dopamine release induced by all these compounds was partially (31-80%), but significantly Ca(2+)-dependent. Lack of extracellular Ca2+ did not alter uptake mechanisms nor did it modify the carrier-dependent dopamine-induced [3H]-dopamine release. (+)-Amph-induced [3H]-dopamine release and pCA- and MDMA-induced [3H]-5-HT release were significantly inhibited by omega-agatoxin-IVA, a specific blocker of P-type voltage-operated Ca(2+)-channels, similar to the previous results on (+)-Fen-induced [3H]-5-HT release. 4. Methiothepin inhibited the Ca(2+)-dependent component of (+)-Amph-induced [3H]-dopamine release with high potency (70 nM), as previously found with (+)-Fen-induced [3H]-5-HT release. The inhibitory effect of methiothepin was not due to its effects as a transporter inhibitor or Ca(2+)-channel blocker and is unlikely to be due to its antagonist properties on 5-HT1/2, dopamine or any other extracellular receptor. 5. These results indicate that the release induced by these compounds is both 'carrier-mediated' and Ca(2+)-dependent (possibly exocytotic-like), with the specific carrier allowing the amphetamines to enter the synaptosome. The Ca(2+)-dependent release is mediated by Ca(2+)-influx (mainly through P-type Ca(2+)-channels), possibly triggered by the drug interacting with an unknown intracellular target, affected by methiothepin, common to both 5-HT and dopamine synaptosomes.     

Selective inhibition of [D-Ala2, Glu4]deltorphin antinociception by supraspinal, but not spinal, administration of an antisense oligodeoxynucleotide to an opioid delta receptor

Evidence in vivo has suggested the existence of subtypes of the delta opioid receptor (DOR), which have been termed delta 1 and delta 2. These proposed DOR subtypes are thought to be activated by [D-Pen2, D-Pen5]enkephalin (DPDPE, delta 1) and [D-Ala2, Glu4]deltorphin (delta 2). Recent work in which an antisense oligodeoxynucleotide (oligo) to a cloned DOR was administered by the intrathecal (i.th.) route has demonstrated a reduction in the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin, but not of [D-Ala2, NMPhe4, Gly-ol]enkephalin (DAMGO, mu agonist) in mice. The present investigation has extended these observations by administering the same DOR antisense oligo sequence by the intracerebroventricular (i.c.v.) route and evaluating the antinociceptive actions of i.c.v. agonists selective for delta, mu and kappa receptors. I.th. treatment with DOR antisense oligo, but not mismatch oligo, significantly inhibited the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin but not of i.th. DAMGO or U69,593 (kappa agonist), confirming previous data. In contrast, i.c.v. DOR antisense oligo, but not mismatch oligo, selectively inhibited the antinociceptive response to i.c.v. [D-Ala2, Glu4]deltorphin without altering the antinociceptive actions of i.c.v. DPDPE, DAMGO or U69,593. The data suggest that the cloned DOR corresponds to that pharmacologically classified as delta 2 and further, suggest that this delta receptor subtype may play a major role in eliciting spinal delta-mediated antinociception.     

Differential agonist regulation of the human kappa-opioid receptor

Opiates are potent analgesics used clinically in the treatment of pain. A significant drawback to the chronic use and clinical effectiveness of opiates is the development of tolerance. To investigate the cellular mechanisms of tolerance, the cloned human kappa-opioid receptor was stably expressed in human embryonic kidney (HEK 293) cells, and the effects of opioid agonist treatment were examined. The receptor-expressing cells showed specific high-affinity membrane binding for a kappa-selective opioid, 3H-labeled (+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidiny l)-1-oxaspiro [4,5] dec-8-yl] benzeneacetamide ([3H]U69,593), and a nonselective opioid antagonist, [3H]diprenorphine. Pretreatment with pertussis toxin or guanosine 5'-O-(3-thiotriphosphate) reduced [3H]69,593 binding, indicating that the human K receptor coupled to G proteins of the Gi or Go families in HEK 293 cells. The receptor-mediated inhibition of adenylyl cyclase was abolished by pertussis toxin pretreatment and was blocked by a kappa-selective antagonist, norbinaltorphimine. A 3-h pretreatment with a kappa-selective agonist, (+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488), caused receptor down-regulation, whereas no receptor down-regulation was found after levorphanol pretreatment. U50,488 or dynorphin A(1-17) pretreatments (3 h) desensitized the ability of U50,488 or dynorphin A(1-17) to inhibit cyclic AMP accumulation, as evidenced by a decrease in functional potency. Also, U50,488 pretreatment desensitized the ability of levorphanol to inhibit forskolin-stimulated cyclic AMP accumulation. In contrast, pretreatment of cells with either levorphanol or a potent nonselective opioid, etorphine, resulted in no apparent receptor desensitization. Taken together, these results demonstrate that the human kappa receptor is differentially regulated by selective and nonselective opioid agonists, with selective agonists able to desensitize the receptor.     

Regulation of hsp expression during rodent spermatogenesis

As part of an effort to develop peptides with selective kappa-opioid antagonist activity, a series of N-alkylated [D-Pro10]dynorphin A-(1-11) derivatives were made through solid-phase peptide synthesis: R-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-D-Pro-LysOH, where R = N-benzyl, N-cyclopropylmethyl, N,N-dicyclopropylmethyl, or N,N-diallyl. These derivatives and dynorphin A-(1-13)NH2 were evaluated for kappa-opioid receptor binding affinity and potency as inhibitors of adenylyl cyclase. Equilibrium competition binding experiments using [3H]diprenorphine (approximately 600 pM) were performed on membranes prepared from cultured Chinese hamster ovary (CHO) cells stably expressing the rat kappa-opioid receptor. Tissue prepared from this cell line was used to evaluate opioid peptide inhibition of forskolin-stimulated (50 microM) adenylyl cyclase activity. Displacement of [3H]diprenorphine specific binding by these peptides was observed with a rank order of affinity (Ki, nM) = [D-Pro10]dynorphin A-(1-11) (0.13) > dynorphin A-(1-13)NH2 (0.34) > N-cyclopropylmethyl- (1.4) > N,N-dicyclopropylmethyl- (12.6) approximately N-benzyl- (18.3) approximately N,N-diallyl-[D-Pro10]dynorphin A-(1-11) (26.0). A similar rank order was observed for potency of adenylyl cyclase inhibition (IC50, nM): [D-Pro10]dynorphin A-(1-11) (0.12) approximately dynorphin A-(1-13)NH2 (0.19) > N-cyclopropylmethyl- (2.7) > N,N-dicyclopropylmethyl- (13.2) approximately N,N-diallyl- (18.0) approximately N-benzyl-[D-Pro10]dynorphin A-(1-11) (36.4). The peptides differed in their percent maximal inhibition of adenylyl cyclase activity: dynorphin A-(1-13)NH2 (100%) approximately N-cyclopropylmethyl- (94.3%) approximately [D-Pro10]dynorphin A-(1-11) (87.9%) > N-benzyl- (71.4%) > N,N-dicyclopropylmethyl- (23.6%) approximately N,N-diallyl-[D-Pro10]dynorphin A-(1-11)(18.9%). As the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) derivatives were found to have only weak partial agonist activity with respect to adenylyl cyclase inhibition, they were evaluated for their ability to reverse dynorphin A-(1-13)NH2 (10 nM) inhibition of adenylyl cyclase activity. N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) reversed dynorphin A-(1-13)NH2 inhibition to levels equal to the maximal inhibition produced by N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) alone. This weak partial agonism combined with nanomolar potency render the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) compounds promising leads for further attempts to synthesize peptide kappa-opioid receptor antagonists.     

Characterization, expression, and immunohistochemical localization of 5 alpha-reductase in human skin

The effect of the mu opioid agonist DAGO, delta opioid agonist DPDPE and kappa opioid agonist U50,488H on 3H-dopamine (3H-DA) uptake was studied in synaptosomes prepared from rat striatum and nucleus accumbens. Over the range of concentrations tested (1 nM-10 microM) DAGO and DPDPE were devoid of effects on 3H-DA uptake in the striatum and the nucleus acumbens. In contrast, U50,488H significantly decreased 3H-DA uptake in both structures. The inhibition of uptake induced by the kappa agonist was not reversed in the presence of the opiate antagonists naloxone (10 microM) or nor-binaltorphimine (0.1 microM). Dynorphin A (1-13) also induced a significant reduction in 3H-DA uptake in both structures at the concentrations of 10 and 30 microM. This inhibitory effect was not reversed by naloxone (10 microM). These data suggest that kappa opioid agonists modulate dopamine uptake in the striatum and the nucleus accumbens and their effects may not be due to an activation of opioid receptors.     

Activation of the cloned human kappa opioid receptor by agonists enhances [35S]GTPgammaS binding to membranes: determination of potencies and efficacies of ligands

Activation of kappa receptors inhibits adenylate cyclase, enhances K+ conductance and reduces Ca++ conductance via pertussis toxin-sensitive G proteins. We recently cloned a human kappa opioid receptor and stably expressed it in Chinese hamster ovary (CHO) cells. In this study, the effects of activation of the human kappa receptor by agonists on [35S]GTPgammaS binding to CHO cell membranes were examined. The presence of GDP and Mg++ was essential for the kappa agonist (-)-U50,488H-induced increase in [35S]GTPgammaS binding to be observed and the optimal concentration was 3 microM and 5 mM, respectively. The presence of 100 mM Na+ was necessary to produce the maximal signal-to-background ratio. (-)U50,488H-induced increase in [35S]GTPgammaS binding was time- and tissue concentration-dependent. (-)U50,488H increased [35S]GTPgammaS binding in a dose-dependent manner with an EC50 of 3.1 nM. (+)-U50,488H had no effect, which indicates that this effect is stereospecific. Naloxone (1 microM) or norbinaltorphimine (10 nM) shifted the dose-response curve of (-)-U50,488H to the right by 100-fold. These results indicate that enhancement of [35S]GTPgammaS binding by (-)-U50,488H is a kappa receptor-mediated event. Pretreatment of the cells with pertussis toxin, but not cholera toxin, abolished the (-)-U50,488H-induced increase in [35S]GTPgammaS binding, which indicates the involvement of Gi and/or Go proteins. [35S]GTPgammaS binding induced by (-)-U50,488H had a Kd value of 0.34 +/- 0.08 nM and a Bmax value of 431 +/- 29 fmol/mg protein. The rank order of potencies of opioid ligands tested in stimulating [35S]GTPgammaS binding was dynorphin A 1-17 > (+/-)-ethylketocyclazocine > beta-funaltrexamine, (-)-U50,488H, tifluadom > nalorphine > pentazocine, nalbuphine > buprenorphine. Dynorphin A 1-17, (+/-)-ethylketocyclazocine, (-)-U50,488H, tifluadom and beta-funaltrexamine were full agonists, but nalorphine and pentazocine were partial agonists producing maximal responses of 68% and 23% of those of full agonists, respectively. Nalbuphine and buprenorphine had low levels of agonist activities. Norbinaltorphimine and naloxone were antagonists devoid of activities. Enhancement of [35S]GTPgammaS binding by kappa agonists provides a simple functional measure for receptor activation and can be used for determination of potencies and efficacies of opioid ligands at the kappa receptor.     

The delta2-opioid receptor subtype stimulates phosphoinositide metabolism in mouse periaqueductal gray matter

The delta(delta)-opioid agonists [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II increased the formation of inositol phosphates (IPs) in mice periaqueductal gray matter (PAG) slices pre-labeled with myo-[3H]inositol. Both delta-agonists caused an increase in IP accumulation in a dose-dependent manner (1-100 microM) and which was pertussis toxin (0.5 microg/mouse, i.c.v.) sensitive. This effect was blocked by the delta-antagonist ICI-174.864 (10 microM). The presence of subtypes of the delta-opioid receptor (delta1 and delta2) in PAG has been suggested by pharmacological studies. In this brain structure, naltrindrole 5'-isothiocyanate (5'-NTII), but not 7-benzylidenenaltrexone (BNTX), antagonized the effects of DPDPE and [D-Ala2]deltorphin II, suggesting the involvement of a population of delta receptors sensitive to the delta2-antagonist NT II on this effect. To further investigate the participation of delta-receptor subtypes in the stimulation of IPs formation, mice were injected with antisense oligodeoxynucleotides (ODNs) directed to nucleotides 7-26 or 2946 of the cloned delta-receptor mRNA, and PAG slices from these animals were used in in vitro assays. The results demonstrate that the reported increase of phosphoinositide (PI) hydrolysis depends on the agonist activation of the delta2-opioid receptor subtype in the PAG.     

Effects of kappa opioid receptor-selective agonists on responses of pelvic nerve afferents to noxious colorectal distension

The aim of this study was to examine the effects of kappa-opioid receptor selective agonists on responses of mechanosensitive afferent fibers in the pelvic nerve. Single-fiber recordings were made from pelvic nerve afferents in the decentralized S1 dorsal root of the rat. A total of 572 afferent fibers in the S1 dorsal root were identified by electrical stimulation of the pelvic nerve; 252 (44%) responded to noxious colorectal distension (CRD; 80 mmHg). Of these 252 fibers that responded to CRD, 100 were studied further. All 100 fibers gave monotonic increases in firing to increasing pressures of CRD. Eighty-eight fibers had low thresholds for response (mean: 3 mmHg) and 12 fibers had high-thresholds for response (mean: 28 mmHg). Responses of 17 fibers also were tested after instillation of 5% mustard oil (MO) into the colon. The resting activity of 16/17 fibers significantly increased after MO instillation; 13 (77%) also exhibited sensitization of responses to graded CRD when tested 30 min after intracolonic MO instillation. The effects of kappa1-opioid receptor preferring agonists (U50,488H, U69,593 and U62,066), the kappa2-opioid receptor preferring agonist bremazocine, and the kappa3-opioid receptor preferring agonist naloxone benzoylhydrazone (nalBzoH) were tested on responses of 64 mechanosensitive afferent fibers to noxious CRD. All five agonists dose-dependently inhibited afferent fiber responses to noxious CRD. Doses producing inhibition to 50% of the control response to CRD did not differ among the five agonists, ranging from approximately 4 to 15 mg/kg. The effects of kappa1, kappa2, and kappa3 receptor agonists were attenuated by naloxone; two kappa-opioid receptor-selective antagonists were ineffective. There were no differences in the dose-response relationships of these drugs for fibers recorded from untreated and irritant-treated colons. Conduction velocities of the fibers remained unaffected after high doses of all tested agonists. In an in vitro study, U50,488 (10(-4) M) did not produce any significant change in the tension of colonic smooth muscle. These results document that responses of mechanosensitive pelvic nerve afferent fibers innervating the colon are inhibited by kappa-opioid receptor agonists having varying affinities for putative kappa-opioid receptor subtypes. The inhibitory effects of these drugs likely are mediated by an action at receptors associated with the afferent fibers. The receptor at which these effects are produced is kappa-opioid-like but clearly different from the kappa-opioid receptor characterized in the CNS and is perhaps an orphan receptor.     

Selective in vivo binding of [3H]naltriben to delta-opioid receptors in mouse brain

Naltriben (NTB) is a selective antagonist for the putative delta2-opioid receptor. We have determined the regional kinetics and pharmacological profile of [3H]naltriben in vivo in mouse brain. After i.v. administration to CD1 mice, [3H]naltriben uptake and retention were high in striatum, cortical regions and olfactory tubercles, and low in superior colliculi and cerebellum. Robust rank order correlation was found between [3H]naltriben uptake in discrete brain regions and prior delta-opioid receptor binding determinations in vitro and in vivo. [3H]Naltriben binding in vivo was saturable, and was blocked by the delta-opioid receptor antagonist naltrindole, but not by the mu-opioid receptor antagonist cyprodime or the K-opioid receptor agonist (trans)-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]ben zeneacetamide mesylate (U50,488H). (E)-7-Benzylidenenaltrexone (BNTX), a selective antagonist for the putative delta1-opioid receptor, was 9.6- to 12.9-fold less potent than naltriben as an inhibitor of [3H]naltriben binding. Thus, the sites labeled by [3H]naltriben in vivo may correspond to the delta2-opioid receptor subtype. Such assignment is not definitive, particularly considering the 4-fold higher brain uptake of naltriben as compared to (E)-7-benzylidenenaltrexone. Moreover, the regional distribution of [3H]naltriben in brains from CXB-7/BY (CXBK) mice, a strain that shows supraspinal delta1- but not delta2-opioid receptor agonist effects, was quite similar to that found for CD1 mice.     

delta Opioid receptor subtypes activate inositol-signaling pathways in the production of antinociception

To analyze the selectivity of delta receptor subtypes to regulate different classes of G proteins, the expression of the alpha-subunits of Gi2, Gi3, Go1, Go2, Gq and G11 transducer proteins was reduced by administration of oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. Mice receiving antisense ODNs to Gi2 alpha, Gi3 alpha, Go2 alpha and G11 alpha subunits showed an impaired antinociceptive response to all the delta agonists evaluated. An ODN to Go1 alpha specifically blocked the antinociceptive effect of the agonist of delta-1 receptors, [D-Pen2,5]enkephalin (DPDPE), without altering the activity of [D-Ala2]deltorphin II or [D-Ser2]-Leu-enkephalin-Thr (DSLET). In mice treated with an ODN to Gq alpha, the effects of the agonists of delta-2-opioid receptors were reduced, but not those of DPDPE. Thus, Go1 proteins are selectively linked to delta-1-mediated analgesia, and Gq proteins are related to delta-2-evoked antinociception. After impairing the synthesis of Go1 alpha subunits, DPDPE exhibited an antagonistic activity on the antinociception produced by [D-Ala2]deltorphin II. After treatment with ODNs complementary to sequences in Gq alpha or PLC-beta 1 mRNAs, the analgesic capacity of [D-Ala2]deltorphin II was diminished. However, the delta-2-agonist did not alter the antinociceptive activity of DPDPE. An ODN complementary to nucleotides 7 to 26 of the murine delta receptor reduced the analgesic potency of [D-Ala2]deltorphin II, but not that observed for DPDPE. In these mice, [D-Ala2]deltorphin II did not antagonize the effect of DPDPE. These results suggest the existence of different molecular forms of the delta opioid receptor, and the involvement of inositol-signaling pathways in the supraspinal antinociceptive effects of delta agonists.     

Kappa receptor activation attenuates L-trans-pyrrolidine-2,4-dicarboxylic acid-evoked glutamate levels in the striatum

The effects of local kappa receptor activation and blockade on extracellular striatal glutamate levels evoked by reverse microdialysis of L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) were investigated. L-trans-PDC elevates extracellular glutamate levels in vivo by acting as a competitive substrate for plasma membrane excitatory amino acid transporters. The selective kappa-opioid receptor agonist U-69593 (1-100 nM) significantly attenuated L-trans-PDC-stimulated glutamate levels in a concentration-dependent manner. The selective kappa receptor antagonist nor-binaltorphimine (1-100 nM) reversed the U-69593-induced decrease in L-trans-PDC-evoked glutamate levels also in a concentration-dependent manner, indicating that the U-69593-induced reduction was mediated by kappa receptor activation. In addition, nor-binaltorphimine significantly elevated basal extracellular glutamate levels, implying that kappa receptors tonically regulate glutamate efflux in the striatum. Previous data from this laboratory have shown that L-trans-PDC-evoked extracellular glutamate levels are partially calcium-sensitive. The present study demonstrated that the inhibition of L-trans-PDC-evoked glutamate levels by reduced calcium perfusion was not altered by U-69593. Therefore, kappa receptors regulate the calcium-dependent component of L-trans-PDC-evoked extracellular glutamate levels in the striatum.     

Anti asialoglycoprotein receptor antibodies and soluble interleukin-2 receptor levels as marker for inflammation in autoimmune hepatitis

Previous results using an amphibian model showed that systemic and spinal administration of opioids selective for mu, delta and kappa-opioid receptors produce analgesia. It is not known whether non-mammalian vertebrates also contain supraspinal sites mediating opioid analgesia. Thus, opioid agonists selective for mu (morphine; fentanyl), delta (DADLE, [D-Ala2, D-Leu5]-enkephalin; DPDPE, [D-Pen2, D-Pen5]-enkephalin) and kappa (U50488, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methanesulfonate; CI977, (5R)-(544alpha,744alpha,845beta)-N-methyl-N-[7-(1-p yrr olidinyl)-1-oxaspiro[4,5]dec-8yl]-4-benzofuranaceta mide++ + monohydrochloride) opioid receptors were tested for analgesia following i.c.v. administration in the Northern grass frog, Rana pipiens. Morphine, administered at 0.3, 1, 3 and 10 nmol/frog, produced a dose-dependent and long-lasting analgesic effect. Concurrent naltrexone (10 nmol) significantly blocked analgesia produced by i.c.v. morphine (10 nmol). ED50 values for the six opioids ranged from 2.0 for morphine to 63.9 nmol for U50488. The rank order of analgesic potency was morphine > DADLE > DPDPE > CI977 > fentanyl > U50488. These results show that supraspinal sites mediate opioid analgesia in amphibians and suggest that mechanisms of supraspinal opioid analgesia may be common to all vertebrates.     

The effect of dopamine on hepatic blood flow in patients undergoing epidural anesthesia

1. The effect of two D3/2 dopamine receptor agonists, LY-171555 (quinpirole) and 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) on spontaneous [3H]-acetylcholine ([3H]-ACh) release were investigated in rat striatal synaptosomes. 2. Quinpirole and 7-OH-DPAT inhibited in a concentration-dependent manner the basal efflux of [3H]-ACh with similar Emax (maximal inhibitory effect) values (29.95 +/- 2.91% and 33.19 +/- 1.21%, respectively). Significant differences were obtained between the pEC50 (-log of molar concentration) of quinpirole (7.87 +/- 0.12) and 7-OH-DPAT (7.21 +/- 0.17; P < 0.01). 3. Different concentrations (0.3-10 nM) of haloperidol (D2/3 dopamine receptor antagonist) shifted to the right the concentration-response curves elicited by quinpirole and 7-OH-DPAT, without modifications in the Emax. 4. Slopes of a Schild plot obtained with haloperidol in the presence of quinpirole and 7-OH-DPAT were not significantly different from unity (0.85 +/- 0.05 and 1.17 +/- 0.11, respectively) and consequently haloperidol interacted with a homogeneous receptor population. The pKB values of haloperidol obtained from Schild regression were 9.96 +/- 0.15 (in presence of quinpirole) and 9.90 +/- 0.09 (in presence of 7-OH-DPAT). 5. Specific binding of [3H]-YM-09151-2 to membranes of striatal synaptosomes and cells expressing D2 and D3 dopamine receptors was inhibited by haloperidol. Analysis of competition curves revealed the existence of a single population of receptors. There were no differences between the estimated pKi (-log of molar concentration) values for synaptosomes (8.96 +/- 0.02) and cells expressing D2 receptors (8.81 +/- 0.05), but the pKi value from cells expressing D3 dopamine receptors differed significantly (8.48 +/- 0.06; P < 0.01). 6. In conclusion, the data obtained in the present study indicate that quinpirole and 7-OH-DPAT, two D3/2 dopamine receptor agonists, inhibit the spontaneous [3H]-ACh efflux and this effect is competitively antagonized by haloperidol and probably mediated through dopamine D2 receptors.     

Involvement of potentially distinct neurotensin receptors in neurotensin-induced stimulation of striatal [3H]dopamine release evoked by KCl versus electrical depolarization

We intended to determine whether the effect of neurotensin (NT) on K+ and electrically evoked [3H]dopamine (DA) release from rat and guinea-pig striatal slices involved different mechanisms and/or receptors. In the two species, NT and three NT agonists were found to exhibit different relative potencies to enhance K+- and electrically-evoked [3H]DA release. NT(1-13) increased [3H]DA release with EC50 values in the nanomolar range and Emax values in the range of 100% of control. NT(8-13) and Eisai hexapeptide were both as active as NT(1-13) under K+ depolarization, but did not exceed 40% of the NT(1-13) effect under electrical depolarization. In rats, when [3H]DA release was stimulated with two successive K+ depolarizations, in the presence of NT(1-13), the NT effect during the second exposure to K+ was drastically decreased, suggesting that the NT receptor was desensitized. The desensitization process was essentially observed on Emax values, EC50 values being weakly affected. Similar results were obtained in guinea pig. In contrast, with two electrical depolarizations or with two different depolarizations (K+ followed by electrical), the NT effect during the second depolarization was not significantly affected. Concerning NT antagonists, SR 48692 antagonized with IC50 values in the nanomolar range the NT(1-13) stimulated K+-evoked [3H]DA release but did not affect, up to 10(-6) M, the NT(1-13) enhancement of electrically stimulated [3H]DA release. On the contrary, SR 142948A antagonized the NT(1-13) effect on K+- and electrically-evoked [3H]DA release. In conclusion, these results suggest the possible existence of potentially distinct neurotensin receptors differentially involved in the control exerted by NT on DA release under KCl vs electrical depolarization.