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The present study examines the stimulatory effect of opioids on adenosine 3':5'-cyclic monophosphate (cyclic AMP) production in the human neuroblastoma cell line SK-N-SH, and its dependence on calcium. We show that, in this culture, the mu-opioid selective agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin stimulates cyclic AMP production by 30% in a naloxone-reversible manner. This stimulation is completely dependent on calcium and involves the activation of calcium/calmodulin since it is abolished in the presence of EGTA, calcium channel blockers or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). The results suggest that the activation of calcium/calmodulin dependent adenylyl cyclases by opioids in SK-N-SH cells is secondary to the induction of calcium influx and the consequent elevation of intracellular calcium level.

The hypothesis that mu-opioid agonists having low antinociceptive efficacy might be more susceptible to interference with G-protein coupling than mu-opioid agonists having higher antinociceptive efficacy was tested. Supraspinal antinociceptive efficacy for the three mu-opioid agonists morphine, [D-Ala2, NMePhe4, Gly5-ol]-enkephalin (DAMGO) and sufentanil in the mouse 55 degrees C warm-water tail-flick test was evaluated 18-24 h after intracerebroventricular (i.c.v.) administration of beta-funaltrexamine (beta-FNA). The beta-FNA pretreatment (0.2-2.0 nmol) attenuated antinociception in the order morphine > DAMGO > sufentanil, consistent with previous reports of their relative antinociceptive efficacy. The association of efficacy with G-protein coupling was then assessed by determining sensitivity to i.c.v. (0.1-3.0 micrograms) pertussis toxin (PTX) or cholera toxin (CTX). The effect of PTX on equiantinociceptive doses was in the inverse order of agonist efficacy. CTX augmented sufentanil-induced antinociception. Morphine- and DAMGO-induced antinociception were unaffected by CTX. These data suggest that: (i) highly efficacious mu agonists (viz., sufentanil) couple more efficiently to PTX-sensitive inhibitory Gi-proteins than do agonists of lower efficacy (viz., morphine, DAMGO) and (ii) highly efficacious mu agonists have greater capacity to utilize CTX-sensitive stimulatory Gs-proteins than do mu-agonists with lower efficacy.

C57BL/6J (B6) mice self-administer substantial quantities of morphine compared to DBA/2J (D2) mice, and most of the genetic component of this strain difference has been attributed to a locus on chromosome 10 in the vicinity of the mu opioid receptor gene. To compare binding characteristics of mu opioid receptor populations between the two strains, mice were given single daily injections of a long-acting preparation of morphine sulfate (80 mg/kg, s.c.) or saline for a period of seven days, and euthanatized six hours after the last injection. Brains were removed and dissected into specific regions. Receptor binding studies were performed on frontal cortex and striatum. Data were analyzed using non-linear regression, and Kd and Bmax comparisons made between strains and treatments. Specific [3H]DAMGO binding in striatum indicates that the density of mu opioid receptors in saline-treated B6 mice and saline-treated D2 mice does not differ significantly. After repeated morphine injection, B6 mice exhibited a decrease in striatal [3H]DAMGO binding, indicating a downregulation of receptor density by approximately 45% (p=.0003 vs saline-treated B6), a phenomenon not observed in D2 mice. In frontal cortex, no differences in [3H]DAMGO binding were observed between strains or treatment groups. These results demonstrate a significant difference between mu opioid receptor regulation in B6 and D2 mice, and may underlie well documented strain differences in specific opioid-related behaviors.

Studies of human and animal central nervous system receptor degradation and measurement postmortem are important as human tissue can rarely be collected under ideal experimental conditions. Correlating the change in binding of opioid receptor subtypes over time will help define the conditions under which human studies may be valid. The present study was designed to investigate the rate at which opioid receptor subtypes degrade postmortem. Brains from rats or cerebelli from guinea pigs were kept at 22 degrees C or 4 degrees C at times from zero to 24 hours to simulate two common human collection techniques; room temperature and morgue refrigeration. Tissue homogenates from these brains were analysed for mu1, mu2, delta, kappa1 and kappa3 opioid receptor binding using standard radioligand binding techniques. At room temperature mu1, mu2 and delta opioid receptor binding was reduced between 6 and 12 hours, whereas kappa1 and kappa3 binding was reduced after 24 hours. At 4 degrees C mu1, mu2, delta, kappa1 and kappa3 binding remained constant over the 24 hour period.

Guinea pig ileum longitudinal muscle/myenteric plexus preparations contain functional mu- and kappa-opioid receptors. A preparation with blocked kappa receptors was obtained by pretreating guinea pigs with 0.1-10.0 mg/kg intraperitoneal doses of a new kappa-selective affinity label, DIPPA (2-(3,4-dichlorophenyl)-N-methyl-N-[1S-1-(-3-isothiocyanatophenyl)-2-(-1 -pyrrolidinyl) ethyl] acetamide). Determination of IC50 values for the mu-selective agonist DAMGO ([D-Ala2,MePhe4,Gly(ol)5]-enkephalin) and the kappa-selective agonist U 69,593 ([5alpha,7alpha,8beta]-(+)-N-methyl-N-[7-(1-pyrrolidin yl)-1-oxaspiro-(4,5)-dec-8-yl]) showed that 0.5 mg/kg DIPPA at 48 h produced reliable, near-complete blockade of kappa-opioid activity but a minimal shift for kappa-opioid agonism. This new assay should be useful for studying mixed agonists/antagonists that produce strong kappa-opioid receptor agonism, which prevents determination of mu-opioid receptor antagonism.

Enhanced sensitivity to some of the behavioral effects of the opioid antagonist naltrexone (NTX) develops following once-weekly injections of cumulative doses of the drug. Rats treated with this regimen of NTX injections show enhanced sensitivity to the operant response rate decreasing effects of NTX and NTX-induced salivation. The enhanced sensitivity is long-lasting and appears to be produced through conditioning processes. We have conducted saturation binding assays to assess possible changes in the number and affinity of mu and delta opioid receptors in cortical, midbrain and hindbrain membrane preparations from Long-Evans rats treated once weekly for 8 weeks with cumulative doses of the drug (1, 3, 10, 30 and 100 mg/kg). 3H-DAMGO (0.5-21 nM) and 3H-pCl-DPDPE (0.04-4 nM) were used to characterize mu and delta receptors, respectively. NTX treatment had no effect on 3H-DAMGO binding in cortex, but decreased binding in midbrain and increased binding in hindbrain relative to saline-treated controls. Saturation analyses revealed that these differences reflected changes in the number, but not the affinity of mu receptors. NTX treatment also increased the amount of 3H-pCl-DPDPE bound to delta receptors in midbrain and hindbrain, but not in cortex. Again, these changes were due to changes in the number of receptors. Thus, chronic NTX differentially affects the number of mu and delta opioid receptors in various brain regions.

The intracerebroventricular (i.c.v.) injection to mice of a polyclonal antibody raised against the peptide sequence 208-216 (TKYRQGSID) of cloned rat mu opioid receptor, reduced the analgesic potency of DAMGO, morphine and beta-endorphin-(1-31) when studied 48 h later in the tail-flick test. Antinociception elicited by delta agonists, DPDPE and [D-Ala2]-Deltorphin II, and by the kappa agonist U-50488H, was fully expressed in mice undergoing this treatment. The specific binding displayed by 0.6 nM [3H]-DAMGO was reduced in membranes preincubated with the antiserum, whereas no change could be detected for 3 nM [3H]-DPDPE or 2 nM [3H]-U-69593 labelling delta and kappa opioid receptors respectively. Naloxonazine, irreversible antagonist of the pharmacologically defined mu 1 opioid receptor, and beta-funaltrexamine (beta-FNA), that also displays irreversible antagonism at mu 1/2 receptors, when injected i.c.v. 24 h before the opioids significantly reduced the activity of DAMGO and morphine. In mice treated with naloxonazine, but not with beta-FNA, the antibody further reduced the remaining analgesic effect of DAMGO and morphine. Thus, both the antibody and beta-FNA blocked a wider population of mu opioid receptors than that tagged by naloxonazine.

The affinity of morphine, codeine, dihydrocodeine and their glucuronides for mu-, delta-, and kappa-opioid receptors was investigated. Binding was studied on guinea-pig brain homogenates with [3H]DAMGO, [3H]DPDPE, and [3H]U69593. The substitution of the free phenolic group of morphine caused a decrease in binding at opioid receptors without affecting the mu/delta-ratio nor that of mu/kappa. Glucuronidation of the 6-hydroxyl group of morphine, codeine or dihydrocodeine did not affect the affinity to mu-receptors, slightly increased the affinity for delta-receptors and reduced the affinity for kappa-receptors. The 6-glucuronides possess a decreased selectivity for mu-receptors over delta-receptors whereas that for mu- over kappa-receptors was increased. It is concluded that chemical variations at 3- and 6-position of morphine independently affect the affinity to opioid receptor subtypes.

Spinally administered mu opioid agonists produce potent antinociception and inhibition of gastrointestinal transit. Blockade of 5-hydroxytryptamine (5-HT) or norepinephrine (NE) uptake potentiates intrathecal (i.t.) DAMGO antinociception. To determine whether 5-HT and NE uptake blockade will also potentiate the gastrointestinal inhibition, mice were treated with zimelidine, desipramine or saline, followed by i.t. DAMGO and tested for tailflick antinociception or inhibition of gastrointestinal transit. DAMGO produced antinociception dose-dependently (ED50 = 4.6 ng). Zimelidine (10 mg/kg, s.c., 1 hr before DAMGO) produced a 6.2-fold leftward shift in the antinociceptive dose-response curve (ED50 = 0.73 ng). Desipramine produced a 5.3-fold shift (ED50 = 1.4 ng). DAMGO also produced a dose-dependent inhibition of gastrointestinal transit (ED50 = 117 ng). However, zimelidine or desipramine treatment did not affect DAMGO inhibition of gastrointestinal transit (ED50 = 80 ng.).

Characteristics of opioid receptors binding in the Mongolian gerbil cerebellum were determined by in vitro radioligand binding technique. Homogenate of cerebellar membranes possessed a binding capacity for 3H-DAMGO, a mu-agonist, and for 3H-diprenorphine, an antagonist of mu, delta and kappa-receptors. These bindings were saturable, and characterized by high affinity (Kd values: 1.55 +/- 0.43 nM for 3H-DAMGO and 0.56 +/- 0.20 nM for 3H-diprenorphine) and high density (Bmax: 127.8 +/- 23.8 fmoles/mg protein for 3H-DAMGO and 135.8 +/- 9.03 fmoles/mg protein for 3H-diprenorphine). Autoradiographically, the binding sites were predominantly located in the molecular layer of the cerebellum. 3H-DPDPE, a delta-agonist, showed only very small binding capacity to the cerebellar membranes. The kappa-agonist, 3H-U-69593, also showed very small binding capacity to the cerebellar membranes except in the early postnatal period. Thus, the gerbil cerebellum can be considered as a tissue containing a homogeneous population of mu-opioid receptors.

The relative binding to mu, delta, and kappa opiate receptors was characterized for the brain peptides Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2), and two fragments of Tyr-W-MIF-1 (Tyr-Pro-Trp and Tyr-Pro-Trp-Gly) previously shown to have antagonist as well as agonist activity in the guinea pig ileum. Tyr-MIF-1 had relatively low affinity (Ki = 1 microM at the mu site) but high selectivity (400- and 700-fold greater affinity for mu over delta and mu over kappa binding). Tyr-W-MIF-1 (Ki = 71 nM at the mu site) showed higher affinity binding to all three sites than Tyr-MIF-1 while retaining 200-fold selectivity for mu over delta and kappa receptors. The affinity of the fragments of Tyr-W-MIF-1 was lower for mu but higher for delta receptors. We also tested two cyclized analogs of Tyr-W-MIF-1 that were about 200-fold more active than the parent compound in producing analgesia. These analogs showed higher affinity binding to all three opiate receptors. One of the analogs showed binding affinity to mu sites (Ki = 1.3 nM) that was within 3-fold of that of the potent analog of enkephalin, DAMGO. Thus, brain peptides with an N-terminal Tyr-Pro, rather than the Tyr-Gly-Gly-Phe sequence typical of other endogenous opiates, can provide high selectivity for mu opiate receptors. Analogs based on one of them, Tyr-Pro-Trp-Gly-NH2, show high affinity as well as potent analgesic activity.

Intracerebroventricular (i.c.v.) administration to mice of IgGs raised against alpha subunits of Gi2 or Gx/z transducer proteins lessened the activation of low Km GTPase induced by morphine, DAMGO and DADLE in P2 membranes from mouse periaqueductal grey matter (PAG). In mice injected with anti Gi2 alpha, DADLE, DPDPE and [D-Ala2] Deltorphin II, but not beta-endorphin-(1-31), antagonized the analgesic activity of morphine. Conversely, following anti Gx/z alpha, morphine antagonized the antinociceptive potency of DADLE. It is concluded that opioids display diverse efficacy at mu-Gi2 and mu-Gx/z complexes to produce supraspinal analgesia in mice.

A polyclonal antiserum directed against the first 16 amino acids of the N-terminal sequence of the murine delta opioid receptor was raised in rabbits. The intracerebroventricular (i.c.v.) injection to mice of the anti delta receptor IgGs impaired the antinociception produced by DPDPE, [D-Ala2]-Deltorphin II, DADLE and beta-endorphin-(1-31) when studied 24 h later in the tail-flick test. Antinociception produced by morphine and DAMGO was fully expressed in mice undergoing this treatment. The selective delta antagonist ICI 174864 (0.8 nmols/mouse, i.c.v.) significantly reduced the antinociceptive activity of opioids to the extent observed after giving the antibodies. ICI 174864 did not decrease further the antinociception that remained after the anti delta receptor serum. The specific binding displayed by 3 nM [3H]-DPDPE was reduced in membranes pre-incubated with the antiserum, whereas no change could be detected for 0.6 nM [3H]-DAMGO labelling mu receptors. This experimental approach revealed the delta component of opioid-evoked supraspinal antinociception in mice.

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.

Human neuroblastoma cells were tested for the presence of opioid receptors. [3H]Diprenorphine binds to NMB cell membranes with a KD value of 0.46 +/- 0.13 nM and Bmax of 534 +/- 22 fmol/mg protein. The presence of mu, delta, and kappa opioid receptors was tested by displacing [3H]diprenorphine specific binding by the selective agonists DAMGO, DPDPE, and U50,488H, respectively. Using this procedure, the data suggest that the NMB neuroblastoma cells express the three opioid receptor types with the abundance of delta receptors (about 60%) and minor, yet substantial populations of mu and kappa receptors (about 20% each).

Intracerebroventricular (i.c.v.) administration of immune sera raised against Gi2 alpha subunits to mice, significantly reduced the supraspinal antinociceptive effect of opioids when evaluated 24 h later in the tail-flick test. Antisera directed against Gi1 alpha subunits did not modify this opioid activity. In mice injected with sera anti-Gx/z alpha, the mu-preferential agonists, DAMGO and morphine, and the endogenous mu/delta opioid peptide beta-endorphin-(1-31) displayed a reduced antinociceptive activity, whereas, the potency of the delta-selective agonists DPDPE and [D-Ala2]Deltorphin II, was not altered. This reduction was present for 3 to 7 days and returned to the control values after 10 days. Anti-Gi2 alpha and anti-Gx/z alpha, but not anti-Gi1 alpha, reduced the specific binding of [3H]DAMGO to the opioid receptor in PAG. These results suggest the ability of the mu receptor to interact in vivo with different classes of G transducer proteins (Gx/z/Gi2) to produce an effect. This work also indicates a functional role of the pertussis toxin insensitive Gx/z protein, on the mu-mediated (but not delta-mediated) supraspinal antinociception in mice.

We examined the effect of insulin on the analgesic potency of DAMGO, a selective agonist of mu-opioid receptors, in mice which had been rendered diabetic by an injection of streptozotocin (STZ; 200 mg/kg, i.v.) 1 and 2 weeks previously. The analgesic potency of DAMGO, based on the tail-flick test, was significantly decreased in both the 1 and 2-week diabetic mice as compared to its effect in age-matched controls. Sensitivity to the analgesic potency of DAMGO returned to normal when insulin was administered to STZ-induced 1-week diabetic mice. However, pretreatment with insulin had no significant effect on the sensitivity to the analgesic potency of DAMGO in 2-week diabetic mice. These results suggest that the serum glucose levels may possibly be responsible for the altered potency of mu-opioid agonists only during the incipient stages of diabetes.

The present voltage-clamp data from guinea pig colonic mucosa indicate that mu opioid receptors both enhance and inhibit net chloride secretion stimulated by PGE1 plus theophylline. Actually, enhancement by the mu opioid agonist DAGO (0.2 nmol/l) declines and finally turns into inhibition with increasing net chloride secretion observed in control preparations taken from the same animals. Moreover, inhibition by the mu opioid antagonist CTOP-NH2 (100 nmol/l) increases with increasing inhibition (or decreasing enhancement) by the mu opioid agonist DAGO. Thus, the mu opioid agonist DAGO and the mu opioid antagonist CTOP-NH2 display opposite effects on net chloride secretion only to a limited degree but otherwise show parallel, either stimulatory or inhibitory, effects. To explain this complex function of mu opioid receptors in the control of net chloride secretion, a working hypothesis is being proposed.

To elucidate the role of opioids in regulating hibernation, the modulatory effects of different opioids on 35 mM K(+)-stimulated [3H]-5-HT release from brain slices were examined in the Richardson's ground squirrels. DAGO ([D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin), a specific mu agonist, evoked a significant dose-dependent (10(-7)-10(-5) M) inhibition of K(+)-stimulated 5-HT release from hippocampal slices of the non-hibernating squirrels. The inhibitory effect of DAGO was attenuated by either the opioid antagonist naloxone (10(-6) M) or the voltage dependent sodium channel blocker tetrodotoxin (TTX, 10(-6) M). The inhibitory effect of DAGO persisted in the hibernating squirrels; however, a ten fold higher concentration of DAGO (10(-6)-10(-5) M) was required to elicit a significant inhibition. In contrast, kappa agonist U50488 (10(-5) M) exerted a significant enhancement of K(+)-stimulated 5-HT release from hippocampal slices of the non-hibernating squirrels. This enhancement was blocked by either the specific kappa antagonist nor-binaltorphimine (10(-6) M) or TTX (10(-6) M). However, in the hibernating squirrels, the stimulatory effect of U50488 (10(-5) M) on 5-HT release was absent. DAGO and U50488 had no modulatory effects on K(+)-stimulated 5-HT release from the hypothalamic slices of either the non-hibernating or hibernating squirrels. These results demonstrate that the modulatory effects of opioids on 5-HT release are receptor-specific and state-dependent, indicating the complex nature of the roles of different opioids in regulating hibernation.

The antinociception induced by beta-endorphin given intracerebroventricularly (i.c.v.) has been previously demonstrated to be mediated by the release of Met-enkephalin and subsequent stimulation of delta receptors in the spinal cord for antinociception. The present study was designed to determine what type of opioid receptor, delta 1 or delta 2, in the spinal cord is involved in i.c.v. beta-endorphin-induced antinociception. Antinociception was assessed by the tail-flick test in male ICR mice. NTB (0.2-20 nmol) and NTI (0.22-2.2 nmol), selective delta 2 receptor antagonists, given intrathecally (i.t.) dose-dependently attenuated i.c.v. beta-endorphin-induced inhibition of the tail-flick response. On the other hand, BNTX (0.02-2.2 nmol), a selective delta 1 receptor antagonist, given i.t., did not block i.c.v. beta-endorphin-induced antinociception. The tail-flick inhibition induced by DAMGO, a mu receptor agonist, or U50,488H, a kappa receptor agonist, was not blocked by i.t. BNTX, NTB or NTI. It is concluded that delta 2 but not delta 1 receptors in the spinal cord are involved in i.c.v. beta-endorphin-induced antinociception.

Recent reports provided evidence that at least two delta opioid receptors may mediate antinociception in mice. In this study, we studied further the involvement of delta opioid receptor subtypes in mediating antinociception at spinal sites in mice using subtype selective agonists and antagonists. The antinociceptive ED50 values (95% C.I.) of i.t. administered DPDPE [(D-Pen2, D-Pen5)enkephalin] (delta 1 receptor agonist) and DELT II [(D-Ala2)deltorphin II] (delta 2 receptor agonist) were 6.3 (5.2-7.6) and 6.4 (5.4-7.7) nmol/mouse, respectively. Administration of BNTX, s.c. increased the antinociceptive ED50 value of DPDPE 5.9-fold whereas that of DELT II was not changed significantly. On the other hand administration of naltriben (NTB, the benzofuran derivative of naltrindole), s.c. increased the antinociceptive ED50 value of DELT II 12.5-fold but did not alter that of DPDPE. Similarly administration of BNTX, i.t. increased the antinociceptive ED50 value of DPDPE 4-fold without altering significantly that of DELT II. NTB given i.t. enlarged the antinociceptive ED50 of DELT II 11-fold without affecting significantly that of DPDPE. BNTX, s.c. did not alter the antinociceptive ED50 values of the mu-agonists, DAMGO [(D-Ala2,MePhe4,Gly-ol5) enkephalin] and morphine or that of the kappa-agonist, U50,488H [trans(+/-)-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl-cyclohexyl]benzeneacetamide] These results demonstrate that BNTX is highly selective for delta 1 opioid receptors at spinal sites. Also, the present data provide for the involvement of both delta 1 and delta 2 opioid receptors in mediating antinociception at spinal sites in mice.

In order to investigate the role of mu-opioid receptor regulation in catecholaminergic neurotransmission during morphine tolerance/dependence and supersensitivity, we measured changes in number and functional properties of two distinct types of mu receptors in the brain of rats chronically treated with morphine and naltrexone. In membranes of striatum and cortex of morphine treated rats the binding of mu ligand [3H]DAMGO was unaltered, whereas an increase in mu binding sites was found in these brain regions of naltrexone treated rats. The ability of the mu agonist DAMGO to inhibit the dopamine D-1 receptor stimulated cAMP production in striatal slices and the electrically evoked release of [3H]noradrenaline from cortical slices was unaffected in both experimental groups. The major changes found were an increased D-1 receptor stimulated cAMP production and an enhanced release of noradrenaline in morphine treated rats and a decreased D-1 receptor stimulated cAMP production in naltrexone treated rats. These data support the hypothesis that tolerance and supersensitivity to morphine and other mu-opioids may be caused by up- and down-regulated neuronal second messenger systems linked to mu-opioid receptors, rather than by changes in the sensitivity of the mu-opioid receptor itself.

Selective fluorescence labeling of opioid receptor subclasses on SK-N-SH cultured cells has been accomplished using labeled polyclonal anti-idiotypic antibodies along with subclass-selective opioid agonists (DPDPE, delta-selective; DAMGO, mu-selective) as blocking reagents. Labeling of the cells was examined using conventional fluorescence microscopy. Co-localization of mu- and delta-opioid receptors on SK-N-SH cells has been studied by double labeling fluorescence experiments. In agreement with our own, and other workers', previous observations on NG108-15 cells, a subpopulation of viable cells in asynchronous cultures are labeled. Among those SK-N-SH cells that are labeled, both subclasses of receptors are seen. On the basis of sequential blocking experiments we interpret our combined results to be consistent with a model where mu- and delta- binding sites reside on different subunits of a multimeric complex.

The brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) was tested for its effects on electrically stimulated contractions in the guinea pig ileum assay. Tyr-MIF-1 acted as an opiate agonist in reducing these contractions. Its IC50 was about 9 microM, and its effects were reversed by naloxone and CTOP. The ability of Tyr-MIF-1 also to antagonize the inhibitory effects of opiates on electrically stimulated contractions was more evident in the ileum removed from a guinea pig tolerant to morphine or after partial inactivation of opiate receptors with beta-CNA. Similar results were observed with hemorphin. The endogenous peptide Tyr-MIF-1 and the blood-derived peptide hemorphin, therefore, can act as agonists as well as antagonists in the guinea pig ileum. The effects as antagonists are best observed in preparations of ileum with reduced receptor reserve (tolerant or beta-CNA treated) and are consistent with the idea that properties of endogenous peptides as opiate antagonists are enhanced in the tolerant state.

SH-SY5Y (human neuroblastoma) cultured cells, known to have mu-opioid receptors, have been used to assess and compare the ability of eight representative mu-selective compounds from diverse opioid families to recognize and activate these receptors. A wide range of receptor affinities spanning a factor of 10,000 was found between the highest affinity fentanyl analogs (Ki = 0.1nM) and the lowest affinity analog, meperidine (Ki = 1 microM). A similar range was found for inhibition of PGE1-stimulated cAMP accumulation with a rank order of activities that closely paralleled binding affinities. Maximum inhibition of cAMP accumulation by each compound was about 80%. Maximum stimulation of GTPase activity (approximately 50%) was also similar for all compounds except the lowest affinity meperidine. Both effects were naloxone reversible. These results provide further evidence that mu-receptors are coupled to inhibition of adenylate cyclase and that the SH-SY5Y cell line is a good system for assessment of mu-agonists functional responses.

The effect of glucose on the binding characteristics of opiate receptor subtypes was investigated in brain membranes from normoglycaemic lean Aston (C57BL/6J) mice using [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO), [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) and [3H]U69,593 as selective ligands for mu, delta and kappa opiate receptors respectively. The equilibrium dissociation constants (Kd) and maximal binding capacities (Bmax) of [3H]DAMGO and [3H]DPDPE were unaltered by 20mM glucose in vitro. Similarly, [3H]U69,593 binding was not modified by increasing the concentration of glucose from 0 to 20mM (P between 0.10 and 0.05), or by the presence of 20mM fructose and of 20mM 3-O-me-glucose, a non-metabolisable sugar, in the incubation medium. The nonselective opiate ligand, [3H]diprenorphine, bound with similar affinity and binding capacity to brain membranes prepared from control and streptozotocin-diabetic Swiss (CD1) mice. The addition of 20mM glucose or of 20mM fructose in vitro induced no changes in their binding parameters. The affinity and binding capacity of [3H]U69,593 to STZ-diabetic Swiss mouse brain membranes was not significantly different to that of normoglycaemic controls; 20mM glucose in vitro had no effect on ligand binding to kappa sites in STZ-diabetic mouse brain membranes. We conclude that glucose does not interact directly with the opiate receptor to modfy it in such as way as could explain the altered sensitivity to different opioid agonists seen in obese and hyperglycaemic animal models in vivo.

Picomol doses of the acetylated derivative of beta-endorphin-(1-31), injected intracerebroventricularly (icv) in mice, reduced the analgesic activity of morphine, etorphine and beta-endorphin-(1-31), while the efficiency of DAGO and DADLE in producing analgesia was enhanced. The effects of the delta agonists DPDPE and [D-Ala2]-Deltorphin II were not altered by this treatment. After alpha N-acetyl beta-endorphin-(1-31) injection, morphine antagonized the analgesia of DAGO. The regulatory effect of alpha N-acetyl beta-endorphin-(1-31) was exhibited when giving the peptide both before (up to 24 h) and after the opioids. Naloxone did not prevent or reverse that modulatory activity; moreover, pretreatment with the acetylated peptide did not change the pA2 value displayed by the antagonist at the mu receptor. The antinociceptive activity of the alpha 2-adrenoceptor agonist clonidine was also increased in mice treated with alpha N-acetyl beta-endorphin-(1-31). The reducing activity of alpha N-acetyl beta-endorphin-(1-31) upon morphine- and beta-endorphin-induced analgesia was not exhibited in mice undergoing treatment with pertussis toxin or N-ethylmaleimide, agents known to impair the function of Gi/Go transducer proteins. However, the enhancing activity displayed by this peptide upon DAGO- DADLE and clonidine-evoked antinociception was still manifested. These results confirm and strengthen the idea of alpha N-acetyl beta-endorphin-(1-31) acting as a non-competitive regulator of mu opioid- and alpha 2-adrenoceptor-mediated supraspinal antinociception. A neural substrate acted on by both receptors (likely Gi/Go transducer proteins) appears to be involved in the effects of that neuropeptide.

A radiolabelled form of deltorphin II was synthesized by catalytic tritiation using [p-IPhe3]-deltorphin II as a precursor. The ligand labels rat brain membranes with a Kd value of 1.9 nM, and the Bmax was found to be 92 fmol/mg protein. This new tritiated ligand exhibits high affinity for the delta opioid binding site, whereas its binding to the mu type is weak and extremely low for the kappa type. Mu/delta and kappa/delta selectivity ratios were about 900 and 10,000, respectively. The highly delta selective binding properties of this new radioligand suggest that it could serve as an excellent tool for investigating the delta opioid receptors in various species.

Changes in functional responsiveness of spinal opioid receptors in monoarthritic rats were investigated at the behavioral and the molecular level. After intrathecal administration of morphine, D-Ala2-D-Leu5-enkephalin (DADLE), D-Pen2-D-Pen5-enkephalin (DPDPE) and dynorphin monoarthritic rats showed an enhanced antinociceptive response as measured by a tail-flick latency. No such changes were observed following administration of the selective kappa agonists U50,488H and U69,593. The opioid mu and delta receptor agonists (0.1-1.0 microM) inhibited the basal, as well as the forskolin-stimulated cAMP formation in spinal cord slices obtained from monoarthritic rats, whereas no significant changes were found in control animals. Higher concentrations of the mu and delta opioid receptor agonists were required to attenuate the cAMP level in spinal cord of control animals. The selective kappa agonists U50,488H and U69,593 did not influence the cAMP formation in monoarthritic or control animals. Additionally, we found that the GppNHp-stimulated level of cAMP was higher in the spinal cord slices of monoarthritic rats, which points to an enhanced responsiveness of the adenylate cyclase effector system to the action of this GTP analog. Our data suggest that the enhanced antinociceptive response to intrathecally administered opioids in monoarthritic rats may be connected with the increased sensitivity of adenylate cyclase to the inhibitory effects of mu and delta agonists.

These studies examined the effect of cocaine on the analgesia produced by systemically and centrally administered opioid agonists. Cocaine (50 mg/kg, s.c.) increased the analgesic potency of systemic, ICV and IT morphine; and the ICV and IT analgesic effects of the delta selective peptide, [D-Pen2,D-Pen5]enkephalin (DPDPE). Cocaine also increased the analgesic potency of the mu selective ligand [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) administered ICV. However, cocaine did not alter the ED50 for IT DAGO. GC-MS studies indicated that brain cocaine concentration was approximately 3.0 micrograms/g wet weight 45 min following s.c. administration. These results suggest that cocaine-induced increases in opioid analgesic potency are mediated at brain mu and delta receptors and spinal mu receptors. Furthermore, there might be functional differences between spinal and supraspinal sites at which DAGO produces analgesia.

In this study we investigated the development of cross-tolerance among intrathecally (i.t.)- administered mu and delta opioid receptor selective peptides in beta-funaltrexamine (beta-FNA)-treated mice. Tolerance to the antinociceptive effect of i.t. administered DPDPE was accomplished by administration of 16 nmol/mouse of DPDPE, i.t. 3 hr before testing in beta-FNA-treated mice (10 mumol/kg, s.c., 24 hr before the experiment). Cross-tolerance developed to the antinociceptive effect of i.t. administered DADLE but not to those of DSLET or DAMGO. DSLET (0.1 nmol/mouse i.t.) administration in beta-FNA-treated mice resulted in tolerance development to its antinociceptive effect. The same pretreatment resulted in a marginally significant increase in the antinociceptive ED50 value of DPDPE. There was no cross-tolerance to the antinociceptive effect of i.t. administered DADLE or DAMGO. These results provide further evidence for the existence of delta opioid receptor subtypes where DADLE and DPDPE interact with one site and DSLET with a different one.

Delta opioid binding sites were assayed using [3H][D-ala2,D-leu5]enkephalin and rat brain membranes depleted of mu binding sites with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimid a zole-HCI. [D-Pen2,D-Pen5]enkephalin (DPDPE), [D-Pen2,L-Pen5]enkephalin, [D-Ala2]deltorphin-I and [D-Ala2]deltorphin-II inhibition curves were characterized by slope factors (Hill coefficients) less than 1. The low slope factor of DPDPE persisted in the presence of 50 microM 5'-guanylyimidodiphosphate in the assay Quantitative analysis of [D-ala2,D-leu5]enkephalin, DPDPE and [D-Ala2]deltorphin-I binding surfaces resolved two binding sites. Whereas [D-ala2,D-leu5]enkephalin had equal affinity for both sites, DPDPE and [D-Ala2]deltorphin-I had high affinity for the high capacity binding site, and low affinity for the low capacity binding site. These data support pharmacological studies demonstrating delta receptor subtypes which mediate antinociception.

Cholera toxin, an agent that impairs the function of Gs transducer proteins, was injected (0.5 microgram/mouse, icv) and the antinociceptive activity of opioids and clonidine was studied 24h later in the tail-flick test. In these animals, an enhancement of the analgesic potency of morphine, beta-endorphin and clonidine could be observed. Cholera toxin did not modify the antinociception evoked by the enkephalin derivatives DAGO and DADLE. Pertussis toxin that catalyses the ADP ribosylation of alpha subunits of Gi/Go regulatory proteins was given icv (0.5 microgram/mouse). This treatment reduced the analgesic effect of opioids and clonidine. However, while the analgesia elicited by DAGO, DADLE and clonidine was greatly decreased, the effect of morphine and beta-endorphin was reduced to a moderate extent. It is concluded that Gi/Go regulatory proteins functionally coupled to opioid and alpha 2 receptors are implicated in the efficacy displayed by opioids and clonidine to produce supraspinal analgesia. Moreover, these two receptors are susceptible to regulation by a process that might involve a Gs protein.

The binding affinity to the mu receptor of some opioids chemically related to morphine and some of their metabolites was examined in rat brain homogenates with 3H-DAMGO. The chemical group at position 6 of the molecule had little effect on binding (e.g. morphine-6-glucuronide Ki = 0.6 nM; morphine = 1.2 nM). Decreasing the length of the alkyl group at position 3 decreased the Ki values (morphine less than codeine less than ethylmorphine less than pholcodine). Analgesics with high clinical potency containing a methoxyl group at position 3 (e.g. hydrocodone, Ki = 19.8 nM) had relatively weak receptor binding, whilst their O-demethylated metabolites (e.g. hydromorphone, Ki = 0.6 nM) had much stronger binding. Many opioids may exert their pharmacological actions predominantly through metabolites.

alpha N-acetyl human beta-endorphin-(1-31) injected icv to mice antagonized the analgesic activity of beta-endorphin-(1-31) and morphine whereas the analgesia evoked by DADLE and DAGO was enhanced by this treatment. The modulatory activity of alpha N-acetyl beta-endorphin-(1-31) was exhibited at remarkable low doses (fmols) reaching a maximum that persisted even though the dose was increased 100,000 times. The regulatory effect of a single dose of the acetylated neuropeptide lasted for 24h. The activity of alpha N-acetyl human beta-endorphin-(1-31) was partially retained by the shorter peptide alpha N-acetyl human beta-endorphin-(1-27) and to a lesser extent by beta-endorphin-(1-27), beta-endorphin-(1-31) lacked this regulatory activity on opioid analgesia. Acetylated beta-endorphin-(1-31) displayed a biphasic curve when competing with 5 pM [125I]-Tyr27 human beta-endorphin-(1-31) specific binding, the first step (20 to 30% of the binding) was abolished with an apparent IC50 of 0.35 nM, and the rest with an IC50 of 200 nM. It is suggested that alpha N-acetyl beta-endorphin-(1-31) changed the efficiency of the opioid analgesics by acting upon a specific substrate that is functionally coupled to the opioid receptor, presumably the guanine nucleotide binding regulatory proteins Gi/Go.

beta-Funaltrexamine (beta-FNA) irreversibly blocks morphine analgesia, lethality and its inhibition of gastrointestinal transit, confirming that these actions involve mu receptors. In dose-response studies, beta-FNA antagonized all the actions with similar potencies (ID50 values of 12.1, 11.3 and 12.3 mg/kg, respectively). beta-FNA also reduced intra-cerebroventricular and intrathecal DAMGO analgesia equally well (ID50 values of 6.09 and 7.7 mg/kg, respectively). Naloxanazine blocked systemic morphine analgesia (ID50 value 9.5 mg/kg) and supraspinal DAMGO analgesia (ID50 value 6.1 mg/kg) as potently as beta-FNA. However, against spinal DAMGO analgesia, morphine's inhibition of gastro-intestinal transit or lethality, naloxonazine (ID50 values 38.8, 40.7 and 40.9 mg/kg, respectively) was significantly less active than beta-FNA (p less than 0.05). beta-FNA remains a valuable tool in the classification of mu opioid actions. Within the mu category, actions can be defined as either mu 1 (naloxonazine-sensitive) or mu 2 (naloxonazine-insensitive).

The objective of this study was to determine the binding affinities of (+/-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]- N-phenylpropanamide-HCI (RTI-4614-4), which is an analog of (+)-cis-3-methylfentanyl for opioid receptor subtypes. The Ki values (nM) of this agent for opioid receptor subtypes were as follows: mu (0.0055), delta (148), kappa 1 (84.8), kappa 2a (2275), and kappa 2b (22.3). The selectivity of this agent for the mu binding site was 27,000 vs. the delta binding site, 15,400 vs. the kappa 1 binding site, 413,700 vs the kappa 2a and 4,054 vs the kappa 2b binding site. In contrast, two other fentanyl analogs, N-(2-(4-methylpyridinyl))-N-(1-phenethyl-4-piperidinyl) 2-furamide and N-(2-pyrazinyl)-N-(1-phenethyl-4-piperdinyl)2-furamide had considerably higher Ki values at, and were less selective for, the mu binding site. Since RTI-4614-4 is composed of a mixture of four stereoisomers, the resolution of these isomers should permit identification of an extremely potent and selective agent for the opioid mu receptor.

We have measured mu and delta opioid receptor sites on intact SK-N-SH and NG108-15 neuroblastoma cells, respectively, in culture. Use of 125I-beta-endorphin (beta E) as a tracer, together with beta E(6-31) to block high-affinity non-opioid binding in both cell lines, permitted the measurement of cell surface mu and delta opioid receptor sites. Labeling was at delta sites in NG108-15 cells and predominantly at mu sites in SK-N-SH cells. Pretreatment with the mu and delta agonist, DADLE, caused a rapid loss of cell surface delta receptor sites in NG108-15 cells, but failed to reduce significantly mu receptor density in SK-N-SH cells.

Opiates such as morphine have profound antidiarrheal and constipating actions due in part to their ability to modify intestinal ion transport. This study was undertaken to examine the presence of opiate binding sites in the porcine distal jejunum, a gut segment analogous to the human ileum. Specific binding sites for the tritiated, mu-selective opioid agonist [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) were localized to the basal portion of villous and crypt cells of the intestinal epithelium by receptor autoradiography. These binding sites may represent enkephalin receptors capable of modulating active electrolyte transport.

The purpose of the present study was firstly to determine whether morphine and (D-Ala2, NMe-Phe4, Gly-ol)-enkephalin (DAGO), a highly selective mu-agonist, increased intracellular free calcium of rat myocytes and secondly to determine whether opioid receptors were involved. Two series of experiments were performed. In the first, the effect of morphine and DAGO on intracellular free calcium (Cai) of cultured isolated myocytes was studied with a spectrophotometric method using fura2-AM as the fluorescent Ca2+ indicator. In the second, the effect of morphine on Cai of isolated ventricular myocytes from rats which had received chronic daily injection of morphine for two weeks or myocytes which had been incubated in a solution with morphine for 12 hr was studied. It was found that both morphine at 100-250 microM and DAGO at 23-75 microM increased Cai dose-dependently and that the effect was significantly antagonized by naloxone at a concentration of 50 microM, which itself did not cause any significant alteration in Cai. Pretreatment with morphine also abolished the morphine-induced increase in Cai of isolated myocytes. The results suggest that morphine increases Cai by directly activating the cardiac receptors (most likely micro-receptors) on the membrane of ventricular myocytes.

The enantiomers in the alpha and beta series of cyclazocine were evaluated for their ability to bind to phencyclidine (PCP) and mu-opioid receptors in order to determine their receptor selectivity. The affinity of (-)-beta-cyclazocine for the PCP receptor was 1.5 greater than PCP itself. In contrast, (-)-alpha-cyclazocine, (+)-alpha-cyclazocine, and (+)-beta-cyclazocine were 3-, 5- and 138-fold less potent than PCP, respectively. Scatchard analysis of saturable binding of [3H]Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) also exhibited a homogeneous population of binding sites with an apparent KD of 1.9 nM and an estimated Bmax of 117 pM. [3H]Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) binding studies revealed that (-)-alpha-cyclazocine (KD = 0.48 nM) was 31-, 1020- and 12,600-fold more potent than (-)-beta-cyclazocine, (+)-alpha-cyclazocine and (+)-beta-cyclazocine, respectively, for binding to the mu-opioid receptor. These data show that, although (-)-beta-cyclazocine is a potent PCP receptor ligand consistent with its potent PCP-like discriminative stimulus effects, it shows little selectivity for PCP receptors since it also potently displaces mu-opioid binding. However, these cyclazocine isomers, due to their extraordinary degree of stereoselectivity, may be useful in characterizing the structural requirements for benzomorphans having activity at the PCP receptor.

Guinea pig brain membranes treated with cyanogen bromide (CNBr) demonstrate a loss in the number of mu opioid receptors and a lower binding affinity of delta opioid receptors. These receptor changes are irreversible. Results from ligand protection experiments support the hypothesis that the location of the methionine groups, the sites at which CNBr cleaves peptides, differs between these two types of opioid receptors. Kappa receptors are significantly less sensitive to the action of CNBr than mu or delta receptors.

[3H][D-Pen2, D-Pen5]enkephalin binding to intact NG 108-15 cells has been measured under physiological conditions of temperature and medium. The dissociation constant (7.78 nM), receptor density (385 fmol/mg protein), and Hill slope (1.06) values measured under these conditions are consistent with values obtained by others using membranes prepared from these cells. Kinetic analysis of the radioligand binding to these cells show biphasic association and monophasic dissociation processes suggesting the presence of different receptor affinity states for the agonist. The data show that the binding affinity of [3H][D-Pen2, D-Pen5]enkephalin under physiological conditions is not substantially different to that measured in 50 mM Tris buffer using cell membrane fractions. Unlike DPDPE, the mu opioid agonists morphine, normorphine, PL-17, and DAMGO, have much lower affinity for the delta receptor measured under these conditions than is observed by studies using 50 mM Tris buffer. The results described here suggest that this assay may serve as a useful model of delta opioid receptor binding in vivo.

Opiate receptors have been identified within the striatum and some have been localized presynaptically to nigrostriatal neurons. Using unilateral ablative lesions of the substantia nigra, we examined binding in the ipsilateral and contralateral striata. Lesions significantly lowered both 3H[D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) and 3H[D-Ala2,Leu5]enkephalin (DADL) binding. The inclusion of competitors in these assays revealed a decrease in both mu1 and mu2 receptors. Mu1 binding was slightly more sensitive to the lesioning than mu2 binding. Selective mu1 and mu2 binding assays supported these observations. No change in delta binding was observed in the lesioned striata. These studies raise the possibility that both mu1 and mu2, but not delta, receptors are localized presynaptically on nigrostriatal neurons.

It is known that under some conditions the administration of opioid agonists will stimulate food intake. However, the lack of receptor selectivity of some of the agonists which produce this effect leaves open the question of which receptor types are actually involved. In the experiments presented here, rats were given intracerebroventricular injections of Dynorphin 1-17 (DYN), [D-ala2MePhe4,-Gly-ol5]enkephalin (DAGO), and [D-ser2, leu5]enkephalin-thr6 (DSLET); these peptides are thought to be selective agonists at kappa, mu and delta opioid receptors, respectively. All three peptides stimulated food intake in non-deprived rats at doses in the 3-10 nmol range; water intake was also increased in some cases. Generally, DYN stimulated feeding at a lower dose than DAGO or DSLET and the magnitude of the effect tended to be greater. On the other hand, DAGO more consistently increased water intake. In some cases, DYN also caused episodes of "barrel-rolling" and postural abnormalities, whereas DAGO had sedative and/or cataleptic effects. These results are interpreted as an involvement of more than one opioid receptor types in the regulation of appetite, possibly with separate opioid systems contributing to food and water intake.

The D-optimal design, a minimal sample design that minimizes the volume of the joint confidence region for the parameters, was used to evaluate binding parameters in a saturation curve with a view to reducing the number of experimental points without loosing accuracy in binding parameter estimates. Binding saturation experiments were performed in rat brain crude membrane preparations with the opioid mu-selective ligand [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO), using a sequential procedure. The first experiment consisted of a wide-range saturation curve, which confirmed that [3H]-DAGO binds only one class of specific sites and non-specific sites, and gave information on the experimental range and a first estimate of binding affinity (Ka), capacity (Bmax) and non-specific constant (k). On this basis the D-optimal design was computed and sequential experiments were performed each covering a wide-range traditional saturation curve, the D-optimal design and a splitting of the D-optimal design with the addition of 2 points (+/- 15% of the central point). No appreciable differences were obtained with these designs in parameter estimates and their accuracy. Thus sequential experiments based on D-optimal design seem a valid method for accurate determination of binding parameters, using far fewer points with no loss in parameter estimation accuracy.

Fifteen generations of selective breeding were used to produce lines (strains) of mice which differ markedly from one another in levorphanol-induced antinociception on the hot plate assay. These are the high antinociceptive response (HAR) and low antinociceptive response (LAR) selection lines, which now differ by over 5-fold in the i.p. dose of levorphanol doubling control (no drug or saline) latency scores. We sought to determine if these large genetically-mediated differences in antinociceptive sensitivity bred into these selection lines with i.p. levorphanol would generalize equally to a series of enkephalin analogues known to differ in their selectivity for mu and delta opioid receptors. DAGO (D-ala2, MePhe4, Gly-ol5 enkephalin), a highly mu selective agent, produced a 67-fold difference between HAR and LAR mice in the slopes of the dose-response curves on the hot plate assay, while DSLET (D-ser2, leu enkephalin Thr6), a delta selective agent, only produced a 5.4-fold difference via the i.c.v. route. DADLE (D-ala, D-ser enkephalin) a slightly delta preferring ligand, was found to be intermediate (17.4-fold difference). These findings demonstrate that selective breeding has been quite successful in altering those genes which control analgesia due to mu selective agents, while relatively little change has occurred in those genes which control analgesia due to delta agonists. Thus, analgesia mediated by the former has been genetically dissociated from analgesia mediated by the latter, implying that DAGO has mechanisms of action largely dependent of DSLET on the hot plate assay. These findings are consistent with the contention that the mu receptor mediates analgesia produced by DAGO, while a different receptor (presumably delta) mediates much of the analgesic effects of DSLET.

The effect of cyclo(Leu-Gly) on U-50,488H- induced pharmacological actions was determined in male Sprague-Dawley rats. Intraperitoneal (i.p.) administration of U-50,488H to rats produced analgesia (tail-flick) and increased urinary output. Cyclo (Leu-Gly) (1-4 mg/kg, s.c.) antagonized the analgesic response to U-50,488H (25 mg/kg; i.p.). A dose of 10 mg/kg (i.p.) of U-50,488H increased the spontaneous urinary output which was antagonized by cyclo (Leu-Gly) (1-4 mg/kg; s.c.). To determine whether cyclo (Leu-Gly) was acting as a kappa-opioid receptor antagonist, the effect of cyclo (Leu-Gly) on the binding of [3H]ethylketocyclazocine (EKC) to membranes of rat cerebral cortex and spinal cord was determined. The IC50 values of cyclo(Leu-Gly) in displacing [3H]EKC from its binding sites in cortex and spinal cord were 1.44 and 0.40 mM, respectively. Chronic administration of U-50,488H (25 mg/kg; i.p., b.i.d.) for 4 days induced tolerance to its analgesic effect. The latter was not affected by cyclo(Leu-Gly) (2 to 8 mg/kg; s.c.) given once a day for 4 days. It is concluded that cyclo(Leu-Gly) antagonizes acute actions of U-50,488H and that such effects of cyclo(Leu-Gly) are not mediated via a direct action on kappa-opioid receptors.

The purpose of this study was to evaluate the effects of the millimicrons opioid agonist D-Ala-2-Me-Phe-4-Gly-ol enkephalin (DAGO) on catecholamine-induced arrhythmias. Arrhythmias were produced, in the rat, by continuous infusion of epinephrine until the development of fatal arrhythmias that were usually ventricular fibrillation. Intracerebroventricular (ICV) administration of DAGO, 3 nmol, significantly (p less than 0.05) shifted to the right the relationship between epinephrine and both the onset of ventricular arrhythmias and the development of fatal arrhythmias. Naloxone, 1 mg/kg i.v., prevented these effects of DAGO. Atropine, 1 mg/kg i.v. or 20 micrograms/kg ICV, prevented the shift in these dose response relationships. Antagonism of DAGO's effects on arrhythmias could not be explained by an alteration of the blood pressure response to epinephrine. However, DAGO significantly increased blood pressure and decreased heart rate in separate experiments in animals that did not receive epinephrine and atropine prevent the heart rate and blood pressure effects of DAGO. These data show that 1) the millimicrons opioid receptor agonist DAGO suppresses epinephrine-induced arrhythmias, 2) the site of action can be within the CNS, 3) there is a role for the central parasympathetic nervous system to mediate the effect of DAGO and 4) endogenous opioids could modulate catecholamine-induced cardiac arrhythmias.

It has been proposed that various opiate receptor subtypes mediate different cardiovascular responses to centrally administered opioids. We evaluated this hypothesis in chloralose-urethane anesthetized cats by monitoring the cardiovascular and respiratory responses to relative mu [morphine, morphiceptin, D-Ala2, MePhe4, Gly-ol5 enkephalin (DAGO)] and delta [D-Ala2, D-Leu5enkephalin (DADL)] agonists microinjected (0.5 ul/kg) into the caudal region of the Nucleus of Tractus Solitarius (NTS). Dynorphin (1-13), an endogenous opioid which exhibits selective affinity towards the kappa receptor, was also tested. Dynorphin at a dose of 50 nMol/kg did not alter cardiovascular or respiratory variables. Morphine (10-54 nMol/kg) and DAGO (50 nMol/kg) had no effect on blood pressure, heart rate or respiratory rate; morphiceptin (100-320 nMol/kg) caused tachycardia only at the highest dose. DADL (10-100 nMol/kg) elicited a dose-dependent depression of blood pressure. High doses of DADL depressed heart rate and respiratory rate. The depressor effects of DADL were reversed by low doses of naloxone (0.1 mg/kg). This dose of naloxone also elicited pressor responses in cats treated with the other opioids and reversed the morphiceptin-induced tachycardia. These data indicate that opioid agonists differ with regard to their cardiovascular and respiratory effects following microinjection into the NTS of anesthetized cats, with the delta agonist DADL showing greatest activity.