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.     

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.     

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.     

Possible mechanisms of salt-induced hypertension in Dahl salt-sensitive rats

The effects of acute and chronic administration of cocaine on the antinociception and tolerance to the antinociceptive actions of mu-(morphine), kappa-(U-50,488H), and delta-([D-Pen2,D-Pen5]enkephalin; DPDPE), opioid receptor agonists were determined in male Swiss-Webster mice. Intraperitoneal injection of 40 mg/kg of cocaine by itself produced weak antinociceptive response as measured by the tail-fick test but the lower doses were ineffective. Administration of morphine (10 mg/kg, SC), U-50,488H (25 mg/kg, IP) or DPDPE (10 microg/mouse, ICV) produced antinociception in mice. Cocaine (20 mg/kg) potentiated the antinociceptive action of morphine and DPDPE but had no effect on U-50,488H-induced antinociception. Administration of morphine (20 mg/kg, SC), U-50,488H (25 mg/kg, IP) or DPDPE (20 microg/mouse, ICV) twice a day for 4 days resulted in the development of tolerance to their antinociceptive actions. Tolerance to the antinociceptive actions of morphine and U-50,488H was inhibited by concurrent treatment with 20 or 40 mg/kg doses of cocaine; however, tolerance to the antinociceptive action of DPDPE was not modified by cocaine. It is concluded that cocaine selectively potentiates the antinociceptive action of mu- and delta- but not of the kappa-opioid receptor agonist. On the other hand, cocaine inhibits the development of tolerance to the antinociceptive actions of mu- and kappa- but not of delta-opioid receptor agonists in mice.     

Effect of 7-nitroindazole on tolerance to morphine, U-50,488H and [D-Pen2, D-Pen5] enkephalin in mice

The effects of 7-nitroindazole (7-NI), an inhibitor of the neuronal nitric oxide synthase (nNOS) which does not increase blood pressure, on tolerance to the antinociceptive activity of mu-(morphine), kappa-(U-50,488H) and delta-([D-Pen2, D-Pen5]enkephalin, DPDPE) opioid receptor agonists were determined in mice. Male Swiss-Webster mice were made tolerant by twice daily injections of morphine (20 mg/kg, s.c.), U-50,488H (25 mg/kg, i.p.) or DPDPE (20 micrograms/mouse, i.c.v.) for 4 days. When tested on day 5, tolerance to their antinociceptive activity was evidenced by decreased response in chronic drug treated mice in comparison to vehicle-injected mice. Concurrent administration of 7-NI (20, 40 or 80 mg/kg, i.p.) with DPDPE did not modify the development of tolerance to the antinociceptive action of DPDPE. However, 7-NI (40 or 80 mg/kg, i.p.) inhibited the development of tolerance to the antinociceptive activity of morphine and U-50,488H but the lower dose of 7-NI (20 mg/kg, i.p.) was not effective. Chronic administration of 7-NI by itself did not modify the acute response to morphine, U-50,488H or DPDPE. It is concluded that a specific inhibitor of nNOS can inhibit tolerance to the antinociceptive activity of mu- and kappa- but not of delta-opioid receptor agonists in mice.     

Treatment with antisense oligodeoxynucleotide to the opioid delta receptor selectively inhibits delta 2-agonist antinociception

Using approaches emphasizing differential antagonism of receptor selective agonists and cross-tolerance paradigms, evidence in vivo has suggested the existence of subtypes of opioid delta receptors, which have been termed delta 1 and delta 2. Recent work has elucidated the structure of an opioid delta receptor. The present investigation attempted to continue to test the hypothesis of subtypes of delta receptors and to correlate the cloned delta receptor with the existing pharmacological classification. Synthetic oligodeoxynucleotides (oligos) complementary to the 5' end of the cloned delta receptor coding region (antisense) or its corresponding sequence (sense) were given by intracerebroventricular (i.c.v.) administration to mice, twice-daily for 3 days and antinociceptive responses to selective agonists at putative delta 1 and delta 2 receptors were subsequently determined. Treatment with antisense, but not sense, oligo significantly inhibited the response to [D-Ala2,Glu4]deltorphin (delta 2 agonist), but not to [D-Pen2,D-Pen5]enkephalin (DPDPE, delta 1 agonist). Further, subsequent administration of DPDPE elicited a full antinociceptive response in the same antisense oligo treated mice which did not show a significant response to [D-Ala2,Glu4]deltorphin while antisense oligo treated mice which responded to DPDPE did not show antinociception when tested subsequently with [D-Ala2,Glu4]deltorphin. The data suggest that the cloned delta receptor corresponds to that pharmacologically classified as delta 2 and continue to support the concept of subtypes of opioid delta receptors.     

Involvement of dynorphin B in the antinociceptive effects of the cannabinoid CP55,940 in the spinal cord

Intrathecal administration of delta 9-tetrahydrocannabinol (delta 9-THC) but not the cannabinoid agonist CP55,940 enhances the antinociception produced by morphine. In addition, CP55,940- and delta 9-THC-induced antinociception is blocked by the kappa opioid antagonist norbinaltorphimine, and both cannabinoids are cross-tolerant to kappa agonists but do not act directly at the kappa receptor. Previous work in our laboratory has implicated dynorphins in the antinociceptive effects of delta 9-THC and its enhancement of morphine-induced antinociception. The goal of the present study was to evaluate the role of dynorphins in the antinociceptive effects of CP55,940 at the spinal level. Pretreatment of mice with antisera to dynorphin A(1-17), dynorphin A(1-8) or alpha-neoendorphin, all of which have been shown to retain specificity for blockade of their respective peptide in vivo, blocked the antinociceptive effects of delta 9-THC but not CP55,940. Dynorphin B produced antinociceptive effects on intrathecal administration to mice. Like CP55,940, dynorphin B failed to enhance the antinociceptive effects of morphine, whereas dynorphin A(1-17) and alpha-neoendorphin enhanced the antinociceptive effects of morphine. Using spinal catheterization of the rat, CP55,940 administration was shown to produce a significant release of dynorphin B concurrent with the production of antinociception. Our data suggest that CP55,940 induces a release of spinal dynorphin B that contributes at least in part to its antinociceptive effects in the spinal cord.     

Analysis of the role of delta opioid receptors in gastroprotection in the rat

The effect of delta opioid agonists - [D-Ala2, D-Leu5]-enkephalin (DADLE), [D-Pen2, D-Pen5]-enkephalin (DPDPE) and deltorphin II - on acidified ethanol induced gastric mucosal lesions was studied in the rat compared with that of morphine. It was found that DADLE, DPDPE, deltorphin II and morphine exerted a dose-dependent inhibition on the mucosal lesions injected subcutaneously, their ID50 values were 0.037, 1.8, 3.5 and 0.35 micromoles/kg, respectively. Naltrindole (10 mg/kg sc.), the selective delta opioid receptor antagonist, inhibited the gastroprotective effect of DADLE, DPDPE and deltorphin II, but it failed to antagonise the effect of morphine. Our results suggest that 1. delta receptors are involved in opioid-mediated gastroprotection, 2. ethanol-induced gastric mucosal damage in the rat may be a quick, simple in vivo model for screening opioid delta receptor agonists and antagonists in the periphery.     

Effects of mu and delta opioid agonists and antagonists on affective vocal and reflexive pain responses during social stress in rats

The present experiments evaluated the influence of intraventricular mu and delta opioid receptors on affective vocal and reflexive responses to aversive stimuli in socially inexperienced, as well as defensive and submissive responses in defeated, adult male Long-Evans rats. Defeat stress consisted of: (1) an aggressive confrontation in which the experimental intruder rat exhibited escape, defensive and submissive behaviors [i.e., upright, supine postures and ultrasonic vocalizations (USV)], and subsequently, (2) protection from the resident stimulus rat with a wire mesh screen for 10-20 min. Defeat stress was immediately followed by an experimental session with tactile startle (20 psi). The mu opioid receptor agonists morphine (0.1-0.6 microg i.c.v.) and [D-Ala2-N-Me-Phe4-Gly5-ol]-enkephalin (DAMGO; 0.01-0.3 microg i.c.v.), and the delta opioid receptor agonist [D-Pen2,5]-enkephalin (DPDPE; 10-100 microg i.c.v.) dose-dependently decreased startle-induced USV and increased tail-flick latencies in socially inexperienced and defeated rats. Of greater interest, morphine, DAMGO and DPDPE increased the occurrence of the submissive crouch posture, and defeated rats were more sensitive than socially inexperienced rats to the startle-induced USV-suppressive and antinociceptive effects of morphine and DPDPE. The antinociceptive effects of DAMGO were likewise obtained at lower doses in defeated rats. Finally, the USV-suppressive effects of morphine and DAMGO were reversed with the mu receptor antagonist naltrexone (0.1 mg/kg i.p.), but the USV-suppressive effects produced by DPDPE were not reversed with the delta receptor antagonist naltrindole (1 mg/kg i.p.). These results confirm mu, but not delta opioid receptor activation as significant in affective vocal, passive-submissive behavior, as well as reflexive antinociception. Furthermore, similar to previous studies with restraint and electric shock stress, the facilitation of mu opioid effects on vocal responses and antinociception is consistent with the proposal that defeat stress activated endogenous opioid mechanisms.     

Antisense oligodeoxynucleotides to the kappa-1 receptor block the antinociceptive effects of delta 9-THC in the spinal cord

Intrathecal pretreatment of mice with an antisense oligodeoxynucleotide directed against the kappa-1 receptor significantly reduced the antinociceptive effects of the kappa receptor agonist U50,488 as well as delta 9-THC, the major psychoactive ingredient found in cannabis. A mismatched oligodeoxynucleotide which contained four switched bases did not block the antinociception produced by U50,488 or delta 9-THC. Furthermore, kappa-1 antisense did not alter the antinociceptive effects of either the mu receptor-selective opioid DAMGO, or the delta receptor-selective opioid DPDPE. By using kappa-1 antisense, we were able to demonstrate that an interaction occurs between the cannabinoids and opioids in the spinal cord.     

Enkephalin analogs containing 4,4-difluoro-2-aminobutyric acid: synthesis and fluorine effect on the biological activity

Analogs of Met-enkephalin and [D-Pen2, D-Pen5]enkephalin (DPDPE) containing the partially fluorinated amino acid 4,4-difluoro-2-aminobutyric acid (DFAB) in the 2- or 3-position of the peptide sequence were synthesized and their opioid activities and receptor selectivities were determined in vitro. The linear fluorinated [D-DFAB2, Met5-NH2]enkephalin showed mu and delta agonist potencies comparable to those of natural [Leu5]enkephalin. The partially fluorinated DPDPE analogs behaved differently as compared with their non-fluorinated correlates. While L-amino acid substitution in position 3 of DPDPE usually resulted in higher delta agonist potency than D-amino acid substitution. [D-DFAB3]DPDPE turned out to be a more potent delta agonist than [L-DFAB3]DPDPE. Furthermore, [D-DFAB3]DPDPE showed over 100-fold higher delta agonist potency than [D-Abu3]DPDPE (Abu = 2-aminobutyric acid), indicating that the fluorine substituents interact favorably with a delta opioid receptor subsite.     

Treatment of chronic allodynia in spinally injured rats: effects of intrathecal selective opioid receptor agonists

We examined the effects of intrathecal (i.t.) selective opioid receptor agonists in alleviating mechanical and cold allodynia in spinally injured rats. Both DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a mu-opioid receptor agonist) and DPDPE ([D-Phe2,D-Phe5]-enkephalin, a delta-opioid receptor agonist) dose-dependently relieved the chronic allodynia-like behavior at doses selective for their respective receptors. The anti-allodynic effect of DAMGO and DPDPE was reversed by the selective mu- and delta-opioid receptor antagonists CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) and naltrindole, respectively. In contrast, the selective kappa-opioid receptor agonist U50488H did not alleviate the allodynia-like behavior, but rather enhanced it. The anti-nociceptive and anti-allodynic effect of i.t. DAMGO was blocked by U50488H. Thus, activation of spinal mu- and delta-, but not kappa-opioid receptors produced anti-allodynic effect in this model of central pain. Drugs which act selectively on opioid receptor subtypes may be useful in managing chronic central pain of spinal cord origin.     

Segmental effects on motor function following different intrathecal receptor agonists and antagonists in rabbits

BACKGROUND: The occurrence of motor impairment after intrathecal drug administration is infrequently reported in the literature and the methods of determining motor function vary. METHODS: Motor function was examined in rabbits after a wide dose range of a variety of intrathecally administered opioid agonists, alpha-adrenergic agonists, non-competitive NMDA antagonists, a benzodiazepine agonist, a sigma agonist, paracetamol, isotonic and acidified saline. The opioids, sigma agonist and NMDA antagonists were additionally examined following pretreatment with naloxone. The opioid antagonists naltrindole and MR2266 (delta- and kappa-opioid receptor antagonists, respectively) were administered before the delta agonist and the kappa agonist. The alpha 2-adrenergic antagonist yohimbine was given before administration of dexmedetomidine and xylazine. Motor function was evaluated by a five-point scale of motor impairment ranging from normal function to total paralysis of the hindlegs. RESULTS: DPDPE (delta agonist), paracetamol, naloxone, naltrindole, yohimbine, isotonic and acidified saline did not affect motor function. MR2266 produced minor motor impairment. The alpha-adrenergic agonist dexmedetomidine reduced motor function slightly and dose independently. The remaining compounds affected motor function in a dose-dependent fashion. High doses of morphine produced hypersensitivity and myoclonus. An irreversible paralysis of the hindlegs was observed following intrathecal administration of the sigma agonist SKF10047 in high doses. Naloxone and MR2266 attenuated the effects of U50488H (kappa agonist). CONCLUSION: The present results reveal a dose-dependent reduction in motor function after intrathecal administration of some of the investigated compounds. The mechanisms behind these effects appear to be multifactorial.     

Opioid antagonist profile of SC nor-binaltorphimine in the formalin paw assay

The antinociceptive effects of mu and kappa agonists were examined after the systemic administration of the opioid antagonists nor-binaltorphimine (nor-BNI) and naloxone in the late response or tonic nociceptive phase of the mouse formalin assay. Initially, SC morphine (ED50, 0.97 mg/kg), racemic U-50488H (ED50, 0.79 mg/kg), (-)U-50488 (ED50, 0.41 mg/kg), and another agonist PD 117,302 (ED50, 0.28 mg/kg) were found to produce graded increases in the level of antinociception as measured by this procedure; naloxone, administered immediately before morphine and U-50488H, antagonized their antinociceptive actions. The effects of morphine and U-50488H then were evaluated 10 min to 96 h after the administration of nor-BNI. Subcutaneous nor-BNI at 30.0 mg/kg, but not at 3.0 or 10.0 mg/kg, attenuated the antinociceptive effects of morphine and U-50488H when the interval separating nor-BNI and the agonists was kept constant at 1 h. Time-course analysis of the effects of combinations of nor-BNI with morphine led to irregular findings: 10.0 mg/kg of nor-BNI lessened the effects of morphine (2.0 mg/kg) if the dosing interval was 10 min, whereas 30.0 mg/kg of nor-BNI attenuated the effects of morphine (2.0 mg/kg) if the dosing interval was 1 or 4 h; 10.0 mg/kg of nor-BNI also diminished the antinociceptive effects of U-50488H (1.7 mg/kg) only if the interval spacing the two drugs was 24 h. In comparison, a threefold higher dose of nor-BNI (30.0 mg/kg) reduced the effects of U-50488H (1.7 mg/kg) if the interval was 1 h or more. In these latter experiments, the antagonist effects of SC nor-BNI (30.0 mg/kg) were evident up to 96 h posttreatment. These results show that the mu opioid antagonist activity of nor-BNI is variable and that the kappa opioid antagonist selectivity of nor-BNI is a function of dose and treatment interval and is long-lasting even after systemic administration.     

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.     

Differential antagonism by MK-801 against antinociception induced by opioid receptor agonists administered supraspinally in mice

Various doses of MK-801 ((+/-)-5-methyl-10,11-dihydro-5H-dibenzo(a,d) cyclohepten-5, 10-imine maleate), a non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist (0.001-1 microgram) injected intracerebroventricularly (i.c.v.) alone did not show any antinociceptive effect. MK-801 (0.001-1 microgram i.c.v.) dose dependently attenuated the inhibition of the tail-flick and hot plate responses induced by i.c.v. administered morphine (1 microgram), [D-Pen2, D-Pen5]enkephalin (DPDPE; 10 micrograms), and U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeoce tamide ) 60 micrograms). However, the inhibition of the tail-flick and hot plate responses induced by i.c.v. administered beta-endorphin (1 microgram) was not changed by i.c.v. administered MK-801. Our results indicate that, at the supraspinal level, NMDA receptors are involved in the production of antinociception induced by supraspinally administered morphine, DPDPE, and U50,488H but not beta-endorphin.     

Kappa1- and kappa2-opioid receptors mediating presynaptic inhibition of dopamine and acetylcholine release in rat neostriatum

1. The effects of selective opioid receptor agonists and antagonists on N-methyl-D-aspartate (NMDA, 10 microM)-induced release of [3H]-dopamine and [14C]-acetylcholine (ACh) from superfused neostriatal slices were studied to investigate the possible occurrence of functional kappa-opioid receptor subtypes in rat brain. 2. The kappa receptor agonists (-)-ethylketocyclazocine ((-)-EKC), U69593 and the endogenous opioid peptide dynorphin A1-13 caused a naloxone-reversible inhibition of NMDA-induced [3H]-dopamine release, with pD2 values of about 9, 8.5 and 8.2, respectively, whereas both the mu agonist Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) and the delta agonist D-Pen2-D-Pen5-enkephalin (DPDPE) were ineffective in this respect. The inhibitory effect of submaximally effective concentrations of dynorphin A1-13, U69593 and (-)-EKC on NMDA-induced [3H]-dopamine release were not changed by the delta1/delta2-opioid receptor antagonist naltrindole (up to a concentration of 1 microM, but reversed by the kappa receptor antagonist nor-binaltorphimine (nor-BNI), with an IC50) as low as 0.02 nM, indicating the involvement of U69593-sensitive kappa1-opioid receptors. 3. NMDA-induced [14C]-ACh release was reduced in a naloxone-reversible manner by DPDPE (pD2 about 7.2), dynorphin A1-13 (pD2 6.7) and EKC (pD2 6.2), but not by U69593 and DAMGO. The inhibitory effect of a submaximally effective concentration of DPDPE, unlike those of dynorphin A1-13 and (-)-EKC, on NMDA-induced [14C]-ACh release was antagonized by naltrindole with an IC50 of 1 nM, indicating the involvement of delta-opioid receptors in the inhibitory effect of DPDPE. On the other hand, the inhibitory effects of dynorphin A1-13 and (-)-EKC on [14C]-ACh release were readily antagonized by nor-BNI with an IC50 of about 3 nM. A 100 fold higher concentration of nor-BNI also antagonized the inhibitory effect of DPDPE, indicating the involvement of U69593-insensitive kappa2-opioid receptors in the inhibitory effects of dynorphin A1-13 and (-)-EKC. 4. Although naloxone benzoylhydrazone (NalBzoH), displaying high affinity towards the putative kappa3-opioid receptor, antagonized the inhibitory effects of dynorphin A1-13 and (-)-EKC on [3H]-dopamine and [14C]-ACh release as well as that of U69593 on [3H]-dopamine release, it displayed a low apparent affinity (IC50 about 100 nM) in each case. 5. In conclusion, whereas activation of kappa1-opioid receptors causes presynaptic inhibition of NMDA-induced dopamine release, kappa2 receptor activation results in inhibition of ACh release in rat neostriatum. As such, this study is the first to provide unequivocal in vitro evidence for the existence of functionally distinct kappa-opioid receptor subtypes in the brain.     

Differences in the influence of AIN-purified and non-purified diets on the development of hypertension in SHR and DOCA-salt hypertensive rats

The effect of nicotine administered supraspinally on antinociception induced by supraspinally administered opioids was examined in ICR mice. The intracerebroventricular (i.c.v.) injection of nicotine alone at doses from 1 to 12 micrograms produced only a minimal inhibition of the tail-flick response. Morphine (0.2 micrograms), beta-endorphin (0.1 microgram), D-Pen2.5-enkephalin (DPDPE; 0.5 microgram), trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeocetamide (U50, 488H; 6 micrograms) caused only slight inhibition of the tail-flick response. Nicotine dose dependently enhanced inhibition of the tail-flick response induced by i.c.v. administered morphine (0.2 microgram) or beta-endorphin (0.1 microgram). The degree of enhancing effect of nicotine toward beta-endorphin-induced inhibition of the tail-flick response was greater than toward morphine-induced inhibition of the tail-flick response. However, i.c.v. administered nicotine at the same doses was not effective in enhancing the inhibition of the tail-flick response induced by DPDPE (0.5 microgram) or U50, 488H (6 micrograms) administered i.c.v. Our results suggest that stimulation of supraspinal nicotinic receptors may enhance antinociception induced by morphine (a mu-opioid receptor agonist) and beta-endorphin (an epsilon-opioid receptor agonist) administered supraspinally. However, the activation of nicotinic receptors at supraspinal sites may not be involved in enhancing the antinociception induced by DPDPE (a delta-opioid receptor agonist) or U50, 488H (a kappa-opioid receptor agonist) administered supraspinally.     

In vivo injection of antisense oligodeoxynucleotides to G alpha subunits and supraspinal analgesia evoked by mu and delta opioid agonists

For 5 consecutive days repeated intracerebroventricular (i.c.v.) administration of antisense oligodeoxynucleotides (ODNs) to G alpha subunit mRNAs was used to impair the function of mouse Gi1, Gi2, Gi3 and Gx/z regulatory proteins. Decreases of 20 to 60% on the G alpha-like immunoreactivity could be observed in neural structures of mouse brain, an effect that was not produced by a random-sequence ODN used as a control. The ODN to Gi1 alpha subunits lacked effect on opioid-evoked analgesia. In mice injected with the ODN to Gi2 alpha subunits the antinociceptive activity of all the opioids studied appeared greatly impaired. The ODN to Gi3 alpha subunits reduced the effects of the selective agonists of delta opioid receptors, [D-Pen2,5]-enkephalin and [D-Ala2]deltorphin II. Conversely, the analgesia evoked by opioids binding mu opioid receptors, [D-Ala2, N-MePhe4,Gly-ol5]enkephalin and morphine, appeared consistently and significantly attenuated in mice injected with the ODN to Gx/z alpha. The effect of the neuropeptide beta-endorphine-(1-31) agonist at mu and delta receptors was also reduced by ODNs to Gi3 alpha or Gx/z alpha subunits. l.c.v. injection of antibodies directed to these G alpha subunits antagonized opioid-induced analgesia with a pattern similar to that observed for the ODNs. Thus, the mu and delta opiod receptors regulate different classes of G transducer proteins to mediate the analgesic effect of agonists. The in vivo antisense strategy and the use of specific antibodies to G alpha subunits gave comparable results, indicating that in the neural tissue the mRNAs and the G alpha subunits can be accessed by the corresponding ODNs and IgGs.     

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.