Evidence for mu opioid receptor mediation of enkephalin-induced electroencephalographic seizures

The opioid receptor types involved in the mediation of enkephalin-induced electroencephalographic (EEG) seizures were studied in unanesthetized, freely moving rats. Four receptor-selective peptide ligands were evaluated for effectiveness in producing nonconvulsive EEG seizures after i.c.v. administration; these included the mu agonist, [D-Ala2-N-methyl-Phe4-Gly5-ol]enkephalin (DAGO), the mixed mu-delta agonist, [D-Ala2-D-Leu5]enkephalin (DADLE), and the selective delta agonists, [D-Pen2-D-Pen5]enkephalin and [D-Pen2-L-Pen5]enkephalin. Only DAGO and DADLE were found to produce EEG seizures, with DAGO being 9 times more potent than DADLE. DAGO produced a greater number of seizure episodes with a greater overall incidence compared with DADLE, reflecting its potent effect to elicit EEG seizure activity in these rats. Injections of [D-Pen2-D-Pen5]enkephalin or [D-Pen2-L-Pen5]enkephalin, even at the highest doses tested, failed to produce seizure activity. Behaviorally, the DAGO and DADLE EEG seizures were nonconvulsive but were temporally associated with episodic bursts of wet-dog shakes. The enkephalin-induced responses were extremely sensitive to antagonism by naloxone and completely blocked by pretreatment with the irreversible mu antagonist beta-funaltrexamine. The selective delta opioid receptor antagonist ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH) was ineffective. The use of the most selective agonists and antagonists for mu and delta opioid receptors suggests that, in rats, enkephalin-induced EEG seizures are mediated exclusively by mu opioid receptors and not by delta opioid systems.     

Direct dependence studies in rats with agents selective for different types of opioid receptor

The objective of this study was to describe, quantitate and compare naloxone-induced abstinence syndromes in rats infused centrally (Sylvian aqueduct) with agonists that are currently the most selective for mu [( D-Ala2, MePhe4, Gly-ol5]enkephalin), delta [( D-Pen2, D-Pen5]enkephalin) and kappa (3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]benzeneacetamide) (U-50,488H) opioid receptors, respectively. Morphine, ethylketazocine and dynorphin A served as reference compounds. After 70 hr of infusion from s.c. implanted osmotic minipumps, three levels of abstinence were associated with the injection of naloxone (3 mg/kg s.c.): 1) negligible syndromes (scores of less than 21) were obtained in rats on water or the kappa-directed ligands, U-50,488H and dynorphin A; 2) a low-to-moderate abstinence score (37-38) was recorded with rats receiving [D-Pen2, D-Pen5]enkephalin and ethylketazocine; and 3) a high abstinence score (64-73) was obtained with rats on morphine and DAGO. These results reinforce the concept of developing selective, nonbenzomorphan kappa agonists as clinically useful analgesics and emphasize that, when evaluating new analgesics, high selectivity for delta receptors does not, in itself, guarantee freedom from physical dependence.     

Kappa receptor regulation of dopamine release from striatum and cortex of rats and guinea pigs

The effects of opioid agonists with selectivity for kappa, mu and delta types of opioid receptors on the K+-stimulated release of [3H]dopamine (DA) from striatum and cortex of rat and guinea pig loaded previously with the monoamine have been studied. The kappa agonist U50488H did not affect base-line release of [3H]DA measured in 5 mM K+, but produced a dose-dependent inhibition of the release of [3H]DA stimulated by 20 mM K+ from slices of striatum in rat and guinea pig, with an IC50 of about 0.5 nM in each case. In contrast, the mu-selective agonist, Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, and the delta-selective agonist, [D-Pen2-D-Pen5]enkephalin, did not inhibit stimulated release from the slice preparations at concentrations up to 1 microM. The inhibitory effects of U50488H were antagonized by naloxone, and the potent and selective kappa antagonist, nor-binaltorphimine (nor-BNI). Similar results were obtained when release of [3H]DA from rat and guinea pig cortex slices was examined. In guinea pig cortex, the maximum inhibition of DA release induced by U50488H was 80% of control-stimulated fractional release. In rat cortex and in striatum of both species the maximum release was about 40% of control fractional release. Thus, in the guinea pig, the mesocortical dopaminergic pathway appears more sensitive to the inhibitory effects of U50488H than the nigrostriatal system. The effects of the opioids on the K+ (12.5 mM)-stimulated release of [3H]DA from guinea pig striatal synaptosomes also were determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mu and delta opioid receptors inhibit serotonin release in rat hippocampus

We studied the influence of opioid agonists on the release of serotonin (5-HT) elicited by K+ (20 mM) in superfused slices of rat hippocampus. K+-evoked outflow of serotonin was inhibited significantly up to 50% in the presence of the mu-selective agonist [D-Ala2,N-methyl-Phe4,Gly5-ol]enkephalin (DAGO) and of the delta-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE). U50,488H a selective kappa agonist, at concentrations between 0.1 to 1 microM, produced an inhibition of 5-HT-release lower than that observed in the presence of mu and delta agonists. The delta antagonist ICI 174,864 (N,N-diallyl-Tyr1,Aib2,Aib3)Leu-enkephalin potently inhibited the effect of DPDPE but did not affect the inhibition produced by DAGO. In contrast, the mu-selective antagonist D-Phe-Cys-Tyr-D-Trp-Nle-Thr-Pen-Thr-NH2 at 1 microM significantly reversed the inhibitory effect produced by a maximal dose of DAGO (0.1 microM) but not the corresponding effect produced by a maximal dose of DPDPE (1 microM). Naloxone was a competitive antagonist of DAGO but noncompetitive antagonist of DPDPE. Treatment of hippocampal slices with pertussis toxin did not alter the K+-evoked release of 5-HT but abolished the inhibitory effect of both DAGO and DPDPE.     

Role of mu and kappa opioid receptors in conditional fear-induced analgesia: the antagonistic actions of nor-binaltorphimine and the cyclic somatostatin octapeptide, Cys2Tyr3Orn5Pen7-amide

When rats are returned to an environment in which they previously received electric shock they show a reduction in sensitivity to painful stimuli. This conditional fear-induced analgesia was measured using the formalin test. The octapeptide Cys2Tyr3Orn5Pen7-amide (CTOP; 0, 10, 40 and 160 ng/rat) dose-dependently reversed conditional analgesia when administered i.c.v. The 40-ng dose partially attenuated fear-induced analgesia, whereas the 160-ng dose reversed it completely. Using similar procedures, CTOP was tested for its ability to reverse the analgesia produced by i.c.v. administered [D-Ala2,-NMPhe4, Glyol5]-enkephalin, [D-Pen2,D-Pen5]-enkephalin and U50488H, which are highly selective opioid agonists at mu, delta and kappa receptors, respectively. At 40 ng/rat, CTOP reversed the analgesia produced by the mu selective ligand but not that produced by the delta ligand or the kappa ligand. At 80 ng CTOP antagonized the analgesia produced both by both enkephalin analogs but not U50488H. Nor-binaltorphimine (0, 1, 3, 10 and 30 micrograms/rat) had no effect on conditional analgesia. However, the 10- and 30-micrograms doses could reverse completely the analgesia produced by U50488H. Therefore, it appears that mu and delta, but not kappa receptors, are involved in this opioid form of stress-induced analgesia.     

Supraspinal and spinal potency of selective opioid agonists in the mouse writhing test

Three agonists with the highest degree of selectivity available for mu ([D-Ala2,NMePhe4,Gly-ol]enkephalin, DAGO), delta ([ D-Pen2,D-Pen5]enkephalin, DPDPE) and kappa (U-50,488H, U50) opioid receptors were compared for their activity in inhibiting acetic acid-induced writhing in mice. Additionally, three reference agonists for mu (morphine), delta ([ D-Ala2,D-Leu5]enkephalin, DADLE) and kappa (ketocyclazocine, KC) receptors were also studied in this test. The agonists were given directly into the lateral cerebral ventricle (i.c.v.) or into the lumbar spinal subarachnoid space (intrathecal), and the potency of each compound was compared across injection sites and with data previously obtained in a thermal analgesic test (mouse hot-plate test). The rank order of potency for inhibition of writhing after i.c.v. administration was DAGO greater than DADLE greater than morphine greater than DPDPE; KC and U50 showed no significant activity by this route. After intrathecal administration, the compounds inhibited writhing with a rank potency order of DAGO greater than KC greater than morphine = DADLE greater than DPDPE greater than U50. All compounds were more potent in inhibiting writhing at spinal sites than at supraspinal sites; DPDPE and DAGO were 15 and 24 times more potent after intrathecal than after i.c.v. administration, respectively. The proposed delta agonists DPDPE and DADLE inhibited writhing at both spinal and supraspinal sites. Further, although the proposed kappa-acting compounds KC and U50 were effective at relatively low doses at spinal levels, these compounds lacked activity at supraspinal sites at doses not causing sedation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Roles of central and peripheral mu, delta and kappa opioid receptors in the mediation of gastric acid secretory effects in the rat

The opioid receptors involved in the mediation of gastric acid secretory effects were studied in the pylorus-ligated rat. The effects of i.c.v. and i.v. administration of morphine and mu ([D-Ala2, NMePhe4, Gly5-ol]enkephalin and Tyr-Pro-NMePheD-Pro-NH2)-, delta ([D-Pen2,D-Pen5]enkephalin)- and kappa-selective [trans-3,4-dichloro-N-methyl-N-[2-91-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate (U-50,488H), dynorphin-(1-9), dynorphin-(1-17), nalorphine, alpha-neoendorphin and ethyl-ketocyclazocine) opioid receptor agonists on gastric volume and acid output were examined. Morphine, [D-Ala2, NMePhe4, Gly5-ol]enkephalin and Tyr-Pro-NMePhe-D-Pro-NH2 decreased gastric acid secretion more potently after i.c.v. than after i.v. administration. The inhibitory effect of i.v. administered morphine on gastric acid secretion was not blocked by the quaternary opioid antagonist naltrexone methylbromide when given s.c. However, when naltrexone methylbromide was administered i.c.v., it blocked completely the effects of i.c.v. morphine and partially antagonized the effects of i.v. morphine, indicating a central site of action for morphine. The delta-selective agonist [D-Pen2,D-Pen5]enkephalin did not alter gastric acid secretion after i.c.v. or i.v. administration. The kappa-selective opioid agonist U-50,488H produced a dose-dependent increase in gastric acid secretion after i.v. but not i.c.v. administration. The other kappa-selective agonists tested did not produce a significant increase in gastric acid secretion after i.c.v. or i.v. administration. The increase in gastric acid secretion produced by U-40,488H was blocked by pretreatment with the opioid receptor antagonist naloxone, the nonselective muscarinic receptor antagonist atropine and the M1 selective muscarinic receptor antagonist pirenzepine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opioid modulation of basal intestinal fluid transport in the mouse: actions at central, but not intestinal, sites

Central and peripheral sites of opioid action on net basal fluid transport were studied in loops of jejunum in urethane-anesthetized mice. Intracerebroventricular administration of morphine, [D-Ala2, N-MePhe4, Gly-ol]enkephalin (DAMGO), D-Pen2, D-Pen5] enkephalin (DPDPE) or U50,488H produced dose-related increases in net basal intestinal absorption. Intracerebroventricular DAMGO was approximately 2.6, 1278 and 2674 times more potent than morphine, DPDPE and U50,488H, respectively. The increase in net basal fluid absorption mediated by i.c.v. administration of all these compounds, except DPDPE, was antagonized in a dose-related manner by coadministration of i.c.v. naloxone, an opioid antagonist which did not produce any effects when given alone. Neither the increase in net basal fluid absorption produced by DPDPE nor the fluid transport effects produced by the other agonists tested was antagonized by the delta antagonist, N,N-diallyl-Tyr-alpha-aminoisobutyric acid [( Aib]-Aib-Phe-Leu-OH) and no effects were observed with this delta antagonist alone. Intracerebroventricular administration of beta-funaltrexamine (18.8 nmol, 4 hr before testing) blocked the i.c.v. effects of DAMGO, but not those of U50,488H. In contrast to the effects seen following i.c.v. administration of these agonists, no changes in net basal fluid transport were obtained by the i.p. route for DAMGO, DPDPE, [D-Ala2,D-Leu5]enkephalin, [D-Ala2, Met5]enkephalinamide or U50,488H; of the compounds tested, only morphine produced an increase in net basal fluid absorption after i.p. administration. The effects of i.c.v. or i.p. morphine were blocked by i.c.v. SR 58002C, a quaternary opioid antagonist which had no effects alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of delta opioid binding sites in the bovine pineal.

Bovine pineal membranes were shown to possess a single class of high-affinity binding sites for the opioid peptide, [125I]iodiotyrosyl27-beta-endorphin (beta E) (Kd = 47 pM, Bmax = 2.4 fmol/mg of tissue). The rank order of potency at this beta E site was deltorphin greater than [D-Ser2]-Leu-enkephalin-Thr greater than [D-Pen2,D-Pen5]enkephalin much greater than dermorphin greater than [D-Ala2,MePhe4,Gly5-ol]enkephalin much greater than (5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1- oxaspiro(4,5)dec-8-yl]] benzeneacetamide (U69593) greater than [des-Tyr1]beta E greater than beta E(6-31). These results suggest that beta E binds to delta opioid sites and excludes the possibility of significant binding to mu, kappa and epsilon sites. The presence of delta binding sites was confirmed by use of the delta selective ligand [3H][D-Pen2,D-Pen5]enkephalin (Kd = 1.5 nM). The Bmax observed using [D-Pen2,D-Pen5]enkephalin is similar to that obtained with [125I]beta E, confirming that essentially all pineal opioid sites are of the delta type. The virtual absence of mu opioid sites was confirmed using the mu-selective opioid ligand [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin. These results suggest that endogenous or circulating opioid peptides may modulate pineal function by interaction with delta opioid sites.     

Alteration of thermoregulatory set point with opioid agonists

This paper focuses on the behavioral thermoregulatory effects of i.c.v. administration of [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO), cyclic [D-Pen2,D-Pen5]enkephalin (DPDPE) and U-50,488H, selective agonists for the mu, delta and kappa opioid receptors, respectively. Rats were tested in a thermally graded tunnel (thermocline) which allowed for simultaneous measurement of body temperature, ambient temperature selection and general ambulatory activity levels. DAMGO (0.3 micrograms) caused an increase in body temperature which was facilitated by the selection of a warm ambient temperature. Both DPDPE (30 micrograms) and U-50,488H (100 micrograms) caused decreases in body temperature which were accompanied by a selection of a cool ambient temperature. In each case there was evidence for a regulated change in body temperature, with DAMGO increasing thermoregulatory set point and DPDPE and U-50,488H decreasing set point. DAMGO and U-50,488H produced a depression of activity levels for the first 15 min after injection. DAMGO and DPDPE produced increases in activity levels which peaked after body temperature had returned toward base-line levels. These data characterize further the differentiable profiles of physiological effects produced by these three compounds. The central modulation of the control of body temperature by these opioid receptor agonists may reflect a role of endogenous opioids in thermoregulatory control.     

Central and peripheral opioid modulation of gastric relaxation induced by feeding in dogs

The influence of i.v. vs. i.c.v. administration of [( D-Ala2, MetPhe4, Gly-ol5]enkephalin (DAMGO) and morphine), kappa U 50488 and ethylketocyclazocine and delta ([D-Pen2, D-Pen5]enkephalin (DPDPE)] opioid agonists on gastric relaxation induced by a standard meal was evaluated in conscious dogs with strain-gauge transducers implanted on the gastric fundus and antrum. Under control conditions, the amplitude of the gastric relaxation in response to feeding was 2.46 +/- 0.23 g. Given i.v. 10 min before feeding, both U 50488 (10 micrograms/kg) and ethylketocyclazocine (10 micrograms/kg) significantly (P less than .01) reduced the amplitude of the gastric relaxation by 57 and 68%, respectively, whereas DAMGO and morphine (10 micrograms/kg i.v.) increased markedly the response to feeding by 67 and 51%, respectively. In contrast, DPDPE had no effect on the gastric relaxation induced by feeding. Previous administration of naloxone (0.3 mg/kg i.v.) or MR 2266 (0.3 mg/kg i.v.) blocked the effect of U 50488 on gastric relaxation with no effect per se on the amplitude of response; naloxone also blocked the increase in gastric relaxation induced by DAMGO. When administered i.c.v. (0.1 microgram/kg) DAMGO induced a significant (P less than .05) increase in the amplitude of gastric relaxation whereas U 50488 and DPDPE (0.1 and 1 microgram/kg i.c.v.) had no effect. The effect of i.c.v. DAMGO on gastric relaxation was unaffected by a previous i.v. administration of SR 58002C (1 mg/kg). Truncal vagotomy blocked the increase in gastric relaxation induced by DAMGO (10 micrograms/kg i.v.), but did not change the effect of U 50488 (10 micrograms/kg i.v.) on gastric relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action at the mu receptor is sufficient to explain the supraspinal analgesic effect of opiates

Mu, delta and kappa opiate receptors have been implicated in the production of analgesia. In order to study the relative contributions of these receptor types to supraspinally mediated analgesia, apparent pA2 values and rank order potencies were determined for i.c.v. injected highly selective opioid agonists in the mouse using a thermal nociceptive test. Drugs used included the prototypical mu agonist morphine, putative mu agonists [D-Ala2, N-methyl-Phe4, Gly5-ol] enkephalin and BAM 22P, putative delta agonists [D-Pen2, D-Pen5] enkephalin, [D-Thr2, Thr6, Leu5] enkephalin and [D-Ser2, Leu5, Thr6] enkephalin and the putative kappa agonists [trans-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methane sulfonate hydrate] and Dynorphin A (1-13). We were unable to demonstrate significant analgesic potencies for i.c.v. injected Dynorphin A (1-13) or BAM 22P in the absence of marked behavioral abnormalities. The rank order potency of the remaining compounds studied was found to be: [D-Ala2, N-methyl-Phe4, Gly5-ol] enkephalin greater than [D-Thr2, Thr6, Leu5] enkephalin greater than [D-Ser2, Leu5, Thr6] enkephalin greater than Morphine greater than [D-Pen2, D-Pen5] enkephalin greater than [trans-3, 4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methane sulfonate hydrate]. Apparent pA2 values of morphine, [D-Ala2, N-methyl-Phe4, Gly5-ol] enkephalin and [D-Pen2, D-Pen5] enkephalin in naloxone antagonism trials did not differ significantly. These results indicate that although both mu- and delta-selective ligands produce potent analgesia, a single receptor (mu) is sufficient to explain the supraspinal effects of opiate drugs.     

Kappa opiate agonists modulate the hypothalamic-pituitary-adrenocortical axis in the rat

Systemic injections of opiate agonists were made in male rats to elucidate the involvement of multiple opioid receptors in the stress response. As an index of activity in the hypothalamic-pituitary-adrenocortical axis, plasma corticosterone was measured by radioimmunoassay. Rats were injected with ethylketocyclazocine (EKC), U50488H, MR2034, bremazocine or tifluadom and sacrificed 1 hr later. These kappa agonists produced potent, dose-dependent, stereospecific increases in plasma corticosterone levels at doses far below those needed to elicit analgesia. These effects were reversed by opiate antagonists, naloxone or Win 44441-3, which by themselves caused dose-dependent decreases in plasma corticosterone. Animals made tolerant to the prototype kappa agonist, U50488H, showed an attenuated response to an acute injection of the drug. However, when animals made tolerant to morphine were injected acutely with U50488H, the drug caused a dramatic increase in corticosterone levels. In hypophysectomized rats, U50488H and L-EKC did not increase plasma corticosterone. The agonist/antagonists, butorphanol and cyclazocine, when injected, behaved like kappa agonists and increased plasma corticosterone levels potently. The mu opiates, morphine and etorphine, also had similar effects but were less potent and efficacious than the kappa agonists. The delta agonist D-Ala-D-Leu enkephalin showed similar results, confirming a mu and delta opioid input into the hypothalamic-pituitary-adrenocortical axis. There were concomitant increases in plasma adrenocorticotropin in morphine-, D-Ala-D-Leu enkephalin-, L-EKC- and U50488H-treated rats which were also seen in adrenalectomized rats. D-EKC and D-cyclazocine, which bind to sigma sites, had no effect on corticosterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extremely long-lasting antagonistic actions of nor-binaltorphimine (nor-BNI) in the mouse tail-flick test.

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

Pharmacologic evaluation of a cyclic somatostatin analog with antagonist activity at mu opioid receptors in vitro

The cyclic somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2(CTP) was evaluated for agonist and opioid antagonist actions and receptor selectivity in two bioassays: electrically stimulated guinea pig isolated ileum (GPI) and mouse isolated vas deferens (MVD). CTP (100, 300, 1000 nM) produced concentration-dependent antagonism of the mu agonist [Me-Phe3,D-Pro4]morphiceptin (PL017) in both the GPI and MVD. Schild analysis of the interactions between CTP and PL017 indicated competitive antagonism between these peptides (Schild slope GPI -0.97 +/- 0.16, Schild slope MVD -1.4 +/- 0.4), and also suggested that the mu receptors in the two tissues are not different (pA2 GPI 7.1 +/- 0.17, pA2 MVD 6.9 +/- 0.16). The effects of the delta selective agonist [D-Pen2,D-Pen5]enkephalin in the MVD were not antagonized by CTP. Likewise, in the GPI, CTP did not antagonize the kappa agonist (trans-3-4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl)benzenea cetamine (U50, 488H). In comparison, naloxone antagonized both PL017 and U50,488H in the GPI, as well as [D-Pen2,D-Pen5]enkephalin and PL017 in the MVD. In the MVD, CTP also exerted weak somatostatin-like actions (35% maximal inhibition) that could not be demonstrated in somatostatin-tolerant tissues. It also showed inhibitory actions at very high concentrations (3000 and 10,000 nM) that were blocked by both naloxone and the delta antagonist N,N-diallyl-Tyr-AIB-AIB-Phe-Leu-OH (ICI 174,864). ICI 174,864 antagonized [D-Pen2,D-Pen5]enkephalin in the MVD, but did not affect PL017. These results indicate that CTP is a selective mu receptor antagonist in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Indirect involvement of delta opioid receptors in cholecystokinin octapeptide-induced analgesia in mice

Sulfated cholecystokinin octapeptide (CCK-8; Asp-Tyr-SO3H-Met-Gly-Trp-Met-Asp-Phe-NH2) produced analgesia in mice when administered i.c.v. and tested in the acetic acid-induced writhing assay. The ED50 was found to be 0.03 nmol/mouse which was about 3, 24 and 714 times more potent than morphine. [D-Pen2,D-Pen5]enkephalin and U50,488H [trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)cyclohexyl] benzeneacetamidel], respectively. When administered i.t., CCK-8 produced partial analgesia of up to 22 to 23% at low doses ranging from 15 to 60 ng/mouse and hyperalgesia at doses over 120 ng/mouse. Naloxone, an opioid antagonist, inhibited the analgesia induced by CCK-8 (i.c.v. and i.t.) but potentiated hyperalgesia induced by CCK-8 (i.t.). Apparent pA2 value for CCK-8 (i.c.v.) against naloxone (s.c.) was 5.88 which was significantly different from those for morphine-naloxone and U50,488H-naloxone but was not significantly different from that for [D-Pen2,D-Pen5]enkephalin-naloxone. Studies using highly selective opioid antagonists showed that CCK-8-induced analgesia was significantly antagonized by the delta receptor antagonist, ICI154,129 [(Allyl)2-Tyr-gly-gly-psi-(CH2S)-Phe-Leu] but not by beta-funaltrexamine, a highly selective mu receptor antagonist or nor-binaltorphimine, a highly selective kappa receptor antagonist. Opioid receptor binding study using [3H]-[D-Ala2,D-Leu5]enkephalin (+unlabeled [D-Ala2,MePhe4,Gly-ol5]enkephalin) in mouse brain membrane preparations revealed that there were no changes in the maximum binding or Kd of delta opioid binding sites in the presence of CCK-8 (1 microM) in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiologic studies on the spinal antinociceptive action of kappa opioid agonists in the adult and 21-day-old rat

The spinal antinociceptive actions of the selective kappa opioid receptor agonists U50488H and U69593 were investigated in anesthetized adult rats and 21-day-old rat pups. Single unit extracellular recordings were made of dorsal horn neurons responding to both innocuous and noxious peripheral stimuli. Mixed effects on neuronal responses were seen after intrathecal administration of lower doses of the kappa agonists but with higher doses selective inhibitions of C fiber-evoked responses were produced by either U50488H or U69593 in both the adult (ED50 420 and 250 micrograms, respectively) and pup (ED50 63 and 13 micrograms, respectively). In the adult intrathecal U50488H similarly inhibited the more prolonged nociceptive response evoked by s.c. formalin. Intravenous U50488H (4 mg/kg) also produced a rapid and selective inhibition of nociceptive responses in the adult rat. Intrathecal administration of the nonselective opiate antagonist naloxone reduced the inhibitions mediated by U50488H and U69593 in the pup and the adult. However, intrathecal norbinaltorphimine, a selective kappa antagonist, only prevented the action of the kappa agonists in the pup and not in the adult. Kappa receptors are reported to be sparse in the adult rat spinal cord so developmental changes in this receptor may be responsible for the differential action of norbinaltorphimine in the rat pup and adult.     

A comparative analysis of monoaminergic involvement in the spinal antinociceptive action of DAMPGO and DPDPE

The antinociceptive properties of intrathecally (i.t.) administered [D-Ala2, N-methyl-Phe4, Gly5-ol]enkephalin (DAMPGO) and [D-Pen2, D-Pen5]enkephalin (DPDPE), selective opioid agonists for mu (mu) and delta (delta) sites, respectively, were compared in rats. DAMPGO and DPDPE elevated tail-flick latency (TFL) in a dose-dependent manner, and the spinal antinociceptive actions of both drugs were reversed by the opiate antagonist naloxone. These findings suggest that both DAMPGO and DPDPE interact with spinal opiate receptors to elevate TFL. Another set of experiments was done to determine the involvement of local spinal serotonin (5-HT) or norepinephrine (NE) in DAMPGO and DPDPE-induced spinal analgesia. Both the alpha 1 noradrenergic receptor antagonist WB-4101 and the alpha 2 blocker yohimbine failed to alter the antinociceptive actions of DAMPGO and DPDPE. Similarly, the 5-HT receptor antagonists pindolol, ritanserin and ICS 205-930 (selective for 5-HT1, 5-HT2 and 5-HT3 sites, respectively) failed to inhibit opioid-induced spinal analgesia. Thus, while DAMPGO and DPDPE produce antinociception via an interaction with spinal opioid receptors, apparently neither drug activates endogenous monoaminergic systems.     

Nor-binaltorphimine, a highly selective kappa-opioid antagonist in analgesic and receptor binding assays

Previously, we reported on an opioid antagonist, nor-binaltorphimine (nor-BNI), that had high selectivity for kappa opioid receptors in smooth muscle preparations. In this study, nor-BNI administered either s.c. or i.c.v. was shown to antagonize significantly the antinociceptive effects of the kappa opioid agonists, ethylketazocine and U-50,488H at doses that had no effect on the antinociceptive effect of mu agonists, morphine and [D-Ala3, MePhe4, Gly-ol5]enkephalin and the delta agonist, [D-Pen3, D-Pen5]enkephalin. Nor-BNI and U-50,488H were used to demonstrate that kappa opioid receptors in the spinal cord were more important than those located supraspinally for kappa-mediated analgesia. Nor-BNI also possessed high affinity and high selectivity for kappa opioid receptors in the receptor binding assay. However, the comparatively low selectivity of BNI in receptor binding studies did not correlate with the high pharmacologic selectivity for kappa receptors.     

Cardiovascular and respiratory effects of mu-, delta- and kappa-opiate agonists microinjected into the anterior hypothalamic brain area of awake rats

Relatively selective mu-, delta- and kappa-opiate agonists were microinjected into anterior hypothalamic and septal brain regions of the unanesthetized rat in order to investigate the potential role of specific opiate receptors in central cardiovascular regulation. Low doses (0.2-3 nmol) of both [D-Ala2,MePhe4,Gly-(ol)5] enkephalin (DAGO, mu-agonist) and [D-Ala2,D-Leu5]enkephalin (DADL, delta-agonist) caused dose-dependent increases in blood pressure and heart rate which were naloxone reversible. Higher doses (7.5-30 nmol) of DAGO and DADL produced pressor responses but had little effect on heart rate. The kappa-agonist MR 2034 had no effect on cardiovascular parameters at these doses. DAGO but not MR 2034 also depressed respiration at the higher doses resulting in hypoxia, hypercapnia and acidosis while DADL only slightly depressed respiration. DAGO was approximately 10-fold more potent than DADL in eliciting cardiovascular and respiratory responses. These findings implicate mu-receptors in mediating the cardiovascular and respiratory effects of opiates at anterior hypothalamic sites.