Mu-opioid receptors selectively regulate basal inhibitory transmission in the central amygdala: lack of ethanol interactions

Endogenous opioid systems are implicated in the actions of ethanol. For example, mu-opioid receptor (MOR) knockout (KO) mice self-administer less alcohol than the genetically intact counterpart wild-type (WT) mice (Roberts et al., 2000). MOR KO mice also exhibit less anxiety-like behavior than WT mice (Filliol et al., 2000). To investigate the neurobiological mechanisms underlying these behaviors, we examined the effect of ethanol in brain slices from MOR KO and WT mice using sharp-electrode and whole-cell patch recording techniques. We focused our study in the central nucleus of the amygdala (CeA) because it is implicated in alcohol drinking behavior and stress behavior. We found that the amplitudes of evoked inhibitory postsynaptic currents (IPSCs) or inhibitory postsynaptic potentials (IPSPs) were significantly greater in MOR KO mice than WT mice. In addition, the baseline frequencies of spontaneous and miniature GABA(A) receptor-mediated inhibitory postsynaptic currents were significantly greater in CeA neurons from MOR KO than WT mice. However, ethanol enhancements of evoked IPSP and IPSC amplitudes and the frequency of miniature IPSCs were comparable between WT and MOR KO mice. Baseline spontaneous and miniature excitatory postsynaptic currents (EPSCs) and ethanol effects on EPSCs were not significantly different between MOR KO and WT mice. Based on knowledge of CeA circuitry and projections, we hypothesize that the role of MOR- and GABA receptor-mediated mechanisms in CeA underlying reinforcing effects of ethanol operate independently, possibly through pathway-specific responses within CeA.     

Interaction between opioid antagonists and amphetamine: evidence for mediation by central delta opioid receptors.

Naloxone, the opioid receptor antagonist, attenuates the effects of amphetamine in a wide range of behavioral paradigms. To determine the role of the opioid receptor subtypes in this phenomenon, subtype-selective opioid receptor antagonists were administered intracisternally to rats either as a 15-min [naloxone methiodide (NX.M) and naltrindole (NTI)] or a 24-hr [beta-funaltrexamine (beta-FNA) and norbinaltorphimine (nBNI)] pretreatment. Cumulative dose-response curves to amphetamine were constructed (saline, 0.1, 0.4, 1.6 and 6.4 mg/kg s.c.) with dosing every 30 min. Motor activity (gross and fine movements) was recorded for 20 min, commencing 10 min postinjection. Amphetamine dose-dependently increased both fine and gross movements. NX.M (30 micrograms) and NTI (10 and 30 micrograms) attenuated the gross activity response to amphetamine but did not alter the increase in fine movements. Lower doses of NX.M (2.0 and 10 micrograms) potentiated the fine activity response to amphetamine without any effect on the gross movements. Pretreatment with beta-FNA (1.25-20 micrograms), nBNI (10 and 30 micrograms) or NX.M (5.0 mg/kg s.c.) did not influence the response to amphetamine. However, beta-FNA and nBNI blocked the antinociceptive effects of morphine and spiradoline, respectively, indicating that these antagonists were tested under appropriate conditions for opioid receptor blockade. These data indicate a central site of action for the opioid antagonist-amphetamine interaction. The ability of NX.M (i.c.) and NTI, but not beta-FNA or nBNI, to influence the motor activity response to amphetamine implicates delta receptors in the opioid-mediated modulation of the behavioral stimulant effects of amphetamine.     

Anti-allodynic effects of peripheral delta opioid receptors in neuropathic pain

The analgesic effects of local administration of opioid agonists into peripheral tissues in alleviating pain have been well documented in both clinical and preclinical studies, although few studies have examined their effects in neuropathic pain. In this study, we investigated the anti-allodynic effects of peripherally acting delta opioid receptor (DOR) agonists in a rat model of neuropathic pain. Peripheral nerve injury (PNI) produced a time-dependent decrease in mechanical withdrawal thresholds that was attenuated by local administration into the hind paw of either morphine or the DOR agonist deltorphin II. Using Western blotting techniques, no change in DOR protein expression was detected in DRG ipsilateral to the site of injury compared to contralateral. However, an up-regulation of DOR protein was found in neuropathic DRG compared to sham, suggesting that there may be a bilateral increase in the expression of DOR following PNI. Results obtained from immunohistochemical studies confirmed up-regulation in small and large DRG neurons in neuropathic compared to sham animals. Additionally, there was an increase in DOR protein within the ipsilateral sciatic nerve of neuropathic animals compared to sham and contralateral neuropathic conditions indicating the occurrence of receptor trafficking to the site of injury. Taken together, our findings suggest that functional peripheral DORs are present in sensory neurons following PNI and validate the development of selective DOR agonists for alleviating neuropathic pain.     

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.     

Endogenous opioid peptide inhibition of the central actions of angiotensin

Effects of endogenous opioid peptides (leucine5-enkephalin; methionine5-enkephalin; and beta-endorphin) and morphine on isoleucine5-angiotensin II (All)-stimulated increase in plasma vasopressin concentration, blood pressure and drinking behavior were characterized in conscious rats. Plasma vasopressin concentration, drinking frequency and latency were measured 90 sec after intracerebroventricular (i.v.t.) All in animals pretreated with enkephalins, beta-endorphin or morphine, i.v.t. Vasopressin was measured by radioimmunoassay. A consistent dose-related, naloxone-sensitive inhibition of the All-stimulated increase in plasma vasopressin concentration and drinking behavior (frequency) occurred after enkephalins, beta-endorphin or morphine. beta-Endorphin and morphine were longer acting and more potent than enkephalins. In other experiments, All (i.v.t.) pressor activity, drinking volume and latency were measured at hourly intervals after opiates, i.v.t. The All-stimulated increase in mean blood pressure and drinking volume were inhibited by endogenous opioid peptides and morphine, i.v.t. Naloxone prevented opiate inhibition of the All pressor response. Endogenously synthesized opiates may modulate All activity as naloxone potentiated the pressor response and decreased water consumption after All, i.v.t. Continuous i.v. infusion of leucine-enkephalin did not affect All drinking or pressor activity, indicating a central nervous system site of inhibition. Opiate inhibition of the central actions of All appeared independent of sedation; head shakes or wet dog shakes occurred at effective doses of enkephalins. Naloxone did not affect basal blood pressure or plasma vasopressin concentration. Endogenous opioid peptides may modulate the central actions of angiotensin II affecting blood pressure and hydration.     

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)     

Continuous intrathecal opioid analgesia: tolerance and cross-tolerance of mu and delta spinal opioid receptors

The tolerance effects of continuous intrathecal infusions of opioids at mu and delta receptors were studied in rats. These effects were compared to those of chronic systemic morphine. A chronic intrathecal infusion of the relatively selective delta agonist, [D-Ala2, D-Leu5]enkephalin (DADLE), produced a larger degree of tolerance to DADLE than to the highly specific mu-activating morphiceptin analog [N-methyl-Phe3, D-Pro4]morphiceptin (PL017). The slope of the analgesic dose-response curve for the highly specific delta agonist, cyclic [D-Penicillamine2, D-Penicillamine5]enkephalin (DPDPE), was significantly different (flatter) from those of mu agonists or DADLE. High-dose infusion of PL017 induced a 61-fold parallel shift of the dose-response curve for PL017. This same treatment also induced a corresponding flattened, nonparallel change of the dose-response curve for DADLE. This altered curve for DADLE was very similar in slope to that of DPDPE. Pretreatment with the irreversible mu antagonist, beta-funaltrexamine, caused a parallel rightward shift of the dose-response curve for PL017 but did not affect DPDPE activity. beta-Funaltrexamine treatment induced a nonparallel rightward shift of the dose-response curve for DADLE with a change of slope similar to that of DPDPE. These findings demonstrate a mixed mu-delta analgesic activity for the compound DADLE, which is often referred to as a prototypic delta agonist. These interactions differ from prior reports of none or minimal mu-ligand interactions with DADLE. Despite the cross-reactivity of DADLE to mu receptors, DADLE remains a more effective analgesic than do mu agonists in states of mu receptor tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic ablation of delta opioid receptors in nociceptive sensory neurons increases chronic pain and abolishes opioid analgesia

Opioid receptors are major actors in pain control and are broadly distributed throughout the nervous system. A major challenge in pain research is the identification of key opioid receptor populations within nociceptive pathways, which control physiological and pathological pain. In particular, the respective contribution of peripheral vs. central receptors remains unclear, and it has not been addressed by genetic approaches. To investigate the contribution of peripheral delta opioid receptors in pain control, we created conditional knockout mice where delta receptors are deleted specifically in peripheral Na_V1.8-positive primary nociceptive neurons. Mutant mice showed normal pain responses to acute heat and to mechanical and formalin stimuli. In contrast, mutant animals showed a remarkable increase of mechanical allodynia under both inflammatory pain induced by complete Freund adjuvant and neuropathic pain induced by partial sciatic nerve ligation. In these 2 models, heat hyperalgesia was virtually unchanged. SNC80, a delta agonist administered either systemically (complete Freund adjuvant and sciatic nerve ligation) or into a paw (sciatic nerve ligation), reduced thermal hyperalgesia and mechanical allodynia in control mice. However, these analgesic effects were absent in conditional mutant mice. In conclusion, this study reveals the existence of delta opioid receptor-mediated mechanisms, which operate at the level of Na_V1.8-positive nociceptive neurons. Delta receptors in these neurons tonically inhibit mechanical hypersensitivity in both inflammatory and neuropathic pain, and they are essential to mediate delta opioid analgesia under conditions of persistent pain. This delta receptor population represents a feasible therapeutic target to alleviate chronic pain while avoiding adverse central effects.     

Contribution of mu and delta opioid receptors to the pharmacological profile of kappa opioid receptor subtypes

Molecular cloning has identified three opioid receptors: mu (MOR), delta (DOR) and kappa (KOR). Yet, cloning of these receptor types has offered little clarification to the diverse pharmacological profiles seen within the growing number of novel opioid ligands, which has led to the proposal of multiple subtypes. In the present study, utilizing in vitro and in vivo methods including the use of opioid receptor knockout mice, we find that certain antinociceptive effects of the KOR‐1 and KOR‐2 subtype‐selective ligands (+)‐(5α,7α,8β)‐N‐Methyl‐N‐[7‐(1‐pyrrolidinyl)‐1‐oxaspiro[4.5]dec‐8‐yl]‐benzene‐acetamide (U69, 593) and 4‐[(3,4‐Dichlorophenyl)acetyl]‐3‐(1‐pyrrolidinylmethyl)‐1‐piperazine‐carboxylic acid methyl ester fumarate (GR89, 696), respectively, are potentiated by antagonism of MOR and DOR receptors. We believe that our findings can be best explained by the existence of KOR‐DOR and KOR‐MOR heteromers. We only find evidence for the existence of these heteromers in neurons mediating mechanical nociception, but not thermal nociception. These findings have important clinical ramifications as they reveal new drug targets that may provide avenues for more effective pain therapies.     

Neuroanatomical sites mediating the central actions of beta-endorphin as mapped by changes in glucose utilization: involvement of mu opioid receptors.

Local cerebral glucose utilization, which is a correlate of neuronal activity, was measured to obtain information on the neuroanatomical sites mediating the different behaviors elicited by i.c.v. administration of the opioid peptide beta-endorphin (beta-END). The selective mu and delta opioid receptor antagonists d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH), respectively, were used to characterize the opioid receptor type involved in the actions of beta-END. beta-END was found to produce profound increases in glucose utilization in limbic regions such as the lateral septal nucleus, the amygdalo-hippocampal transition area, the nucleus accumbens and the hippocampal formation. The ventral hippocampus proved the most sensitive structure, displaying increases in glucose utilization of up to 200%; changes in the dorsal part amounted up to 100%. Only moderate effects were induced by beta-END in motor areas, such as the substantia nigra, pars reticulata and the nucleus ruber. This regional pattern of changes is assumed to underlie the epileptogenic-, motivational-, mood- and possibly memory-modulating actions of beta-END. The effects of beta-END on local cerebral glucose utilization were blocked by pretreatment with the mu antagonist, CTOP, whereas the selective delta opioid antagonist ICI 174,864 was less effective. An involvement of predominantly mu opioid receptors in the central actions of beta-END is, therefore, suggested.     

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)     

The role of central mu opioid receptors in opioid-induced itch in primates

Pruritus (itch sensation) is a significant clinical problem. The aim of this study was to elucidate the roles of opioid receptor types and the site of action in opioid-induced itch in monkeys. Observers who were blinded to the conditions counted scratching after administration of various drugs. Intravenous (i.v.) administration of mu opioid receptor (MOR) agonists (fentanyl, alfentanil, remifentanil, and morphine) evoked scratching in a dose- and time-dependent manner. However, the kappa opioid agonist U-50488H [trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)-benzeneacetamide] and delta opioid agonist SNC80 [(+)-4-[(alphaR)-alpha-[2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-methoxybenzyl]-N,N-diethylbenzamide] did not increase scratching. Intrathecal (i.t.) administration of peptidic MOR agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO, 0.00032-0.01 mg) evoked scratching, but i.v. DAMGO (0.01-1 mg/kg) did not increase scratching. A similar difference between i.t. and i.v. effectiveness was seen with morphine. Antagonist studies revealed that i.v. administration of an opioid receptor antagonist (naltrexone, 0.0032-0.1 mg/kg) dose dependently attenuated scratching induced by i.v. fentanyl (0.018 mg/kg) or morphine (1 mg/kg). However, a peripherally selective opioid antagonist (quaternary naltrexone, 0.0032-0.32 mg/kg) did not block i.v. fentanyl- or morphine-induced scratching. Moreover, a histamine antagonist (diphenhydramine, 0.1-10 mg/kg), failed to attenuate scratching induced by i.t. morphine (0.032 mg) or i.v. morphine (1 mg/kg). Pretreatment with a selective MOR antagonist (clocinnamox, 0.1 mg/kg), but not kappa or delta opioid antagonists (nor-binaltorphimine or naltrindole), blocked i.t. morphine-induced scratching. Together, these data suggest that MOR, not other opioid receptor types or histamine, mediates scratching evoked by opioid analgesics. More important, this study provides in vivo pharmacological evidence that activation of central MOR plays an important role in opioid-induced itch in primates.     

Interaction between medullary and spinal delta1 and delta2 opioid receptors in the production of antinociception in the rat

Previous work supports the existence of two types of delta opioid receptor (delta1 and delta2) and a role of both subtypes in the spinal cord and the ventromedial medulla (VMM) in the production of antinociception. Although it is well established that spinal and supraspinal mu opioid receptors interact in a synergistic manner to produce antinociception, little is known about the interaction of delta opioid receptors. This study used isobolographic analysis to determine how delta1 and delta2 opioid receptors in the VMM interact with their respective receptors in the spinal cord to produce antinociception. Concurrent administration of the delta1 opioid receptor agonist [D-Pen2,D-Pen5]enkephalin at spinal and supraspinal sites in a fixed-dose ratio produced antinociception in an additive manner in the tail-flick test. In contrast, concurrent administration of very low doses of the delta2 opioid receptor agonist [D-Ala2,Glu4]deltorphin at spinal and medullary sites produced antinociception in a synergistic manner. However, as the total dose of [D-Ala2,Glu4]deltorphin increased, this interaction converted to additivity. These observations suggest that different mechanisms mediate the antinociceptive effects of different doses of delta2 opioid receptor agonists. The difference in the nature of the interaction produced by delta1 and delta2 opioid receptor agonists provides additional evidence for the existence of different subtypes of the delta opioid receptor. These results also suggest that delta2 opioid receptor agonists capable of crossing the blood-brain barrier will be more potent or efficacious analgesics than delta1 opioid receptor agonists after systemic administration.     

Peripheral opioid receptors mediating antinociception in inflammation. Evidence for involvement of mu, delta and kappa receptors

This study examined a possible peripheral site of action of opioids in the modulation of the response to noxious pressure on inflamed tissue. Rats developed a unilateral localized inflammation upon injection of Freund's complete adjuvant into one hindpaw. 4-6 days after inoculation, intraplantar administration of mu, delta and kappa selective agonists [D-Ala2,N-methyl-Phe4,Gly-ol5]-en-kephalin (1 micrograms), [D-Pen2,5]-enkephalin (40 micrograms) and U-50, 488H (50 micrograms) produced marked antinociceptive effects in inflamed but not noninflamed paws. Equivalent doses applied systemically (s.c. and i.v.) were without effect. Dose dependency and stereospecificity of these effects were demonstrated using (-)- and (+)-morphine and (-)- and (+)-tifluadom. Furthermore, by use of (-)- and (+)-naloxone, dose-dependent and stereospecific antagonism was shown. Lastly, reversal of effects of [D-Ala2,N-methyl-Phe4,Gly-Ol5]-enkephalin, [D-Pen2,5]-enkephalin and U-50,488H by mu, delta and kappa selective antagonists D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, ICI 174,864 and nor-BNI, respectively, indicated that these agents interact with discriminable populations of receptors. These observations suggest that several selective opioid agonists can modulate responses to noxious pressure through a peripheral opioid receptor-specific site of action in inflammation and that these receptors possess distinguishable pharmacological characteristics resembling those of mu, delta and kappa receptors.     

Presynaptic opioid receptor subtypes in the rabbit ear artery

In segments of rabbit ear arteries preincubated with [3H]noradrenaline, Leu-enkephalin, D-Ala2-D-Leu-enkephalin and ethylketocyclazocine concentration dependently reduced the overflow of tritium and the vasoconstriction elicited by field stimulation (120 pulses every 14 min, 1 Hz, 0.3 msec pulse duration). The effects of Leu-enkephalin and ethylketocyclazocine were antagonized by naloxone which, given alone, increased the evoked overflow of tritium at the high concentration of 10 microM. Morphine failed to produce inhibition, and at 100 microM actually increased evoked 3H-overflow. Continued exposure to Leu-enkephalin desensitized the tissue to this opioid; there was no cross-desensitization to ethylketocyclazocine. In arteries not preincubated with [3H]noradrenaline, normorphine, fentanyl and morphiceptin did not change the vasoconstrictor response (5 pulses every min, 5 Hz, 0.3 msec pulse duration). Among various peptide agonists, Leu-enkephalin, D-Ala2-D-Leu-enkephalin and Met-enkephalin were the most potent inhibitors. In a series of peptides with C-terminal extensions of the Met-enkephalin chain, the potency decreased in the order Met-enkephalin greater than Met-enkephalin-Arg-Gly-Leu greater than Met-enkephalin-Arg-Phe greater than BAM-12P greater than beta-endorphin. In a series of peptides with C-terminal extensions of the Leu-enkephalin chain, the potency decreased in the order Leu-enkephalin greater than dynorphin1-13 greater than dynorphin1-9 greater than alpha-neo-endorphin greater than dynorphin1-8 greater than dynorphin1-6 greater than dynorphin1-17. The delta-selective antagonist ICI 154129 counteracted the effect of Met-enkephalin but not that of dynorphin1-13, whereas naloxone counteracted the effect of either agonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic alpha1 adrenergic receptors differentially regulate synaptic glutamate and GABA release to hypothalamic presympathetic neurons

The hypothalamic paraventricular nucleus (PVN) neurons that project to the spinal intermediolateral cell column and brainstem are important for the control of sympathetic outflow. Stimulation of alpha(1) adrenergic receptors in the PVN increases sympathetic outflow, but the cellular mechanisms remain unclear. In this study, we determined the role of alpha(1) adrenergic receptors in the regulation of glutamatergic and GABAergic synaptic inputs to spinally projecting PVN neurons. Whole-cell and cell-attached patch-clamp recordings were performed on retrogradely labeled PVN-spinal neurons in rat brain slices. Bath application of 10 to 100 microM phenylephrine, an alpha(1) adrenergic receptor agonist, significantly increased the frequency of spontaneous excitatory postsynaptic currents in a concentration-dependent manner. This effect was blocked by the alpha (1)adrenergic receptor antagonists prazosin or corynanthine. Phenylephrine also significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) but not the amplitude and decay constant of mEPSCs. Furthermore, activation of alpha(1) adrenergic receptors with phenylephrine or cirazoline significantly decreased the frequency of spontaneous inhibitory postsynaptic currents and miniature inhibitory postsynaptic currents, and this effect also was blocked by corynanthine. In addition, 50 microM phenylephrine significantly increased the firing rate of 13 labeled PVN neurons from 3.16 +/- 0.42 to 5.83 +/- 0.65 Hz. However, phenylephrine failed to increase the firing of most labeled PVN neurons in the presence of GABA(A) and ionotropic glutamate receptor antagonists. Thus, these data suggest that activation of alpha (1)adrenergic receptors increases the excitability of PVN presympathetic neurons primarily through augmentation of glutamatergic tone and attenuation of GABAergic inputs.     

Inverse agonism and neutral antagonism at wild-type and constitutively active mutant delta opioid receptors

The delta opioid receptor modulates nociceptive and emotional behaviors. This receptor has been shown to exhibit measurable spontaneous activity. Progress in understanding the biological relevance of this activity has been slow, partly due to limited characterization of compounds with intrinsic negative activity. Here, we have used constitutively active mutant (CAM) delta receptors in two different functional assays, guanosine 5'-O-(3-thio)triphosphate binding and a reporter gene assay, to test potential inverse agonism of 15 delta opioid compounds, originally described as antagonists. These include the classical antagonists naloxone, naltrindole, 7-benzylidene-naltrexone, and naltriben, a new set of naltrindole derivatives, H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-TicPsi[CH2N]Cha-Phe-OH [TICP(Psi)], as well as three 2',6'-dimethyltyrosine-1,2,3,4-tetrahydroquinoline-3-carboxylate (Dmt-Tic) peptides. A reference agonist, SNC 80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide], and inverse agonist, ICI 174864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu), were also included. In a screen using wild-type and CAM M262T delta receptors, naltrindole (NTI) and close derivatives were mostly inactive, and TIPP behaved as an agonist, whereas Dmt-Tic-OH and N,N(CH3)2-Dmt-Tic-NH2 showed inverse agonism. The two latter compounds showed negative activity across 27 CAM receptors, suggesting that this activity was independent from the activation mechanism. These two compounds also exhibited nanomolar potencies in dose-response experiments performed on wild-type, M262T, Y308H, and C328R CAM receptors. TICP(Psi) exhibited strong inverse agonism at the Y308H receptor. We conclude that the stable N,N(CH3)2-Dmt-Tic-NH2 compound represents a useful tool to explore the spontaneous activity of delta receptors, and NTI and novel derivatives behave as neutral antagonists.     

Antagonist-induced up-regulation of the putative epsilon opioid receptor in rat brain: comparison with kappa, mu and delta opioid receptors

We reported previously that in the presence of appropriate blocking agents [3H]ethylketocyclazocine selectively labels a non-mu, non-delta, non-kappa binding site with opioid receptor characteristics. We now report that chronic treatment with the opiate antagonist naltrexone dramatically increases the number of these binding sites in rat brain, as well as the number of mu, delta and kappa receptors. This finding further supports the concept that the [3H]ethylketocyclazocine site is an opioid receptor and previously reported evidence indicating that it might be the beta-endorphin-specific epsilon receptor that has been hypothesized to exist for some time. The affinity of naltrexone for the putative epsilon site in vivo appears to be similar to its affinity for mu and delta receptors, since a single 30-mg naltrexone pellet was sufficient to induce a maximal rate of up-regulation for all three types of sites. In contrast, a maximal rate of kappa receptor up-regulation requires at least four naltrexone pellets and, therefore, the affinity of naltrexone for this site in vivo appears to be considerably lower than for the other sites. Unexpectedly, we found that the naltrexone-induced increase in binding to all four types of receptors continued to increase through 60 days of naltrexone exposure, the longest treatment period examined, and that the increases in receptor binding were linear with exposure time. This finding is contradictory to the generally held view that antagonist-induced opioid receptor up-regulation in brain increases asymptotically, leveling off after a relatively brief treatment period.     

Presynaptic dopamine receptor stimulation in the cardiovascular actions of lergotrile

The involvement of dopamine receptors present at different sites in the hypotensive and bradycardic actions of lergotrile was investigated. Intravenous administration of lergotrile to pentobarbital-anesthetized dogs decreased blood pressure, heart rate and renal vascular resistance and increased renal blood flow. These effects were prevented by prior treatment with sulpiride or with hexamethonium plus atropine. When administered to phentolamine-treated animals, lergotrile failed to alter either blood pressure or renal blood flow, but exerted and an enhanced bradycardic effect. Lergotrile significantly inhibited the positive chronotropic and renal vasoconstrictor effects elicited by cardiac and renal sympathetic nerve stimulation, which could be prevented by prior treatment with sulpiride, suggesting that stimulation of presynaptic dopamine receptors was responsible for the inhibition of sympathetic nerve function caused by lergotrile. Administration of lergotrile into the vertebral artery did not produce any cardiovascular changes, whereas i.c.v. lergotrile caused a transient tachycardia followed by a bradycardia of greater magnitude and duration than that seen after i.v. administration. These studies suggest that lergotrile exerts its hypotensive and bradycardiac effects via stimulation of presynaptic dopamine receptors resulting in subsequent inhibition of sympathetic nerve function. Dopamine receptors present in the brain may contribute to the bradycardia, but vascular dopamine receptors do not appear to be involved in the cardiovascular actions of lergotrile.