Role of G(i)alpha2-protein in opioid tolerance and mu-opioid receptor downregulation in vivo.

Although opioid receptors are G-protein coupled, the role that specific G-protein subunits play in the development of opioid tolerance and the regulation of opioid receptor number is not well understood. In the present study, we used a G((i)alpha2) antisense oligodeoxynucleotide (ODN) to examine the contribution of G((i)alpha2) proteins to mu-opioid tolerance and receptor downregulation in the mouse. Mice were injected intracerebroventricularly (ICV) and into the spinal intrathecal space (IT) for 4-5 consecutive days (30 microg/site/day), with an antisense ODN or a mismatch ODN directed at mRNA for the G((i)alpha2) subunit of G-proteins. Controls were treated with dH(2)O. On the second day of ODN treatment continuous subcutaneous (SC) infusion of etorphine (200 microg/kg/day) or morphine (40 mg/kg/day + 25 mg pellet) was begun. Control mice were implanted with inert placebo pellets. Three days later, pumps and pellets were removed and mice were tested for morphine analgesia or mu-opioid receptor density was determined in whole brain. Etorphine produced significant tolerance (ED(50) shift = approximately 11-fold) and downregulation of mu-opioid receptors (approximately 25%). Morphine treatment produced significant tolerance (ED(50) shift approximately 9-fold), but no mu-opioid receptor downregulation. Antisense treatment reduced G((i)alpha2) protein levels in striatum and spinal cord by approximately 25%. G((i)alpha2) antisense reduced the acute potency of morphine. G((i)alpha2) antisense blocked the development of tolerance to morphine treatment and reduced the development of tolerance to etorphine treatment. Antisense did not have any effect on etorphine-induced mu-opioid receptor downregulation. In another experiment, 7-day treatment with morphine or etorphine similarly increased G((i)alpha2) mRNA and protein abundance in spinal cord. Overall, these results support an important role for G((i)alpha2)-protein in the acute effects of opioids and opioid tolerance. However, G((i)alpha2) is not required for agonist-induced mu-opioid receptor density regulation in vivo.

mu-Opioid receptor downregulation contributes to opioid tolerance in vivo.

The present study examined the contribution of downregulation of mu-opioid receptors to opioid tolerance in an intact animal model. Mice were implanted subcutaneously with osmotic minipumps that infused etorphine (50-250 microg/kg/day) for 7 days. Other mice were implanted subcutaneously with a morphine pellet (25 mg) or a morphine pellet plus an osmotic minipump that infused morphine (5-40 mg/kg/day) for 7 days. Controls were implanted with an inert placebo pellet. At the end of treatment, pumps and pellets were removed, and saturation binding studies were conducted in whole brain ([3H]DAMGO) or morphine and etorphine analgesic ED(50)s were determined (tail-flick). Morphine tolerance increased linearly with the infusion dose of morphine (ED(50) shift at highest infusion dose, 4.76). No significant downregulation of mu-receptors in whole brain was observed at the highest morphine treatment dose. Etorphine produced dose-dependent downregulation of mu-opioid receptor density and tolerance (ED(50) shift at highest infusion dose, 6.97). Downregulation of mu-receptors only occurred at the higher etorphine infusion doses (> or =150 microg/kg/day). Unlike morphine tolerance, the magnitude of etorphine tolerance was a nonlinear function of the dose and increased markedly at infusion doses that produced downregulation. These results suggest that mu-opioid receptor downregulation contributes to opioid tolerance in vivo. Therefore, opioid tolerance appears to rely upon both "receptor density-dependent" and " receptor density-independent" mechanisms.

Role of cAMP-dependent protein kinase (PKA) in opioid agonist-induced mu-opioid receptor downregulation and tolerance in mice.

Studies suggest that acute and chronic opioids can regulate the cAMP-dependent protein kinase (PKA) signaling pathway and that changes in this pathway may be involved in opioid tolerance. In the present study, we examined the role of cAMP-PKA on mu-opioid receptor downregulation and tolerance in mice. Mice were injected intracerebroventricular (i.c.v.) and intrathecal (i.t.) once a day with an antisense oligodeoxynucleotide directed at the mRNA for the alpha catalytic subunit of mouse PKA. Controls were treated with saline or a mismatch oligodeoxynucleotide. On day 2 of treatment, mice were implanted s.c. with a 25-mg morphine pellet and an osmotic minipump infusing morphine (40 mg/kg/day) for 3 days. Other mice were implanted with an osmotic minipump infusing etorphine (125, 250 microg/kg/day) for 2 days. Control mice were implanted s.c. with inert placebo pellets. At the end of treatment, pumps and pellets were removed and mice tested for morphine or etorphine analgesia. Other mice were sacrificed and mu-opioid receptor binding assays conducted in whole brain. Both infusion doses of etorphine produced significant tolerance (ED(50) shift = 3.6 and 6.3-fold). The higher etorphine infusion produced downregulation of mu-receptor density ( approximately 30%) while the lower infusion dose of etorphine did not. Morphine treatment also produced significant tolerance in mice (ED(50) shift = 4.5-fold), but no receptor downregulation. Antisense to PKA partially blocked tolerance induced by the higher dose of etorphine, but had no effect on receptor downregulation. On the other hand, antisense to PKA completely blocked tolerance induced by morphine and the lower infusion dose of etorphine. The mismatch oligodeoxynucleotide had no effect on any measure. These results suggest that PKA has a limited role in opioid agonist-induced receptor downregulation. However, the partial block of tolerance for the high infusion dose of etorphine and the complete block of tolerance for morphine and the low infusion dose of etorphine suggests that PKA may play a critical role in tolerance that is "receptor-regulation-independent."

In vivo regulation of mu-opioid receptor density and gene expression in CXBK and outbred Swiss Webster mice.

Chronic in vivo treatment with the opioid agonist etorphine downregulates mu-opioid receptor density, produces tolerance, and regulates gene expression in the mouse. After cessation of treatment, there is an increase in mu-opioid receptor mRNA level associated with the recovery of mu-opioid receptors. However, the effect of etorphine on the regulation of mRNA during treatment is currently not known. In this study, etorphine-induced changes in mu-opioid receptor density, mRNA, and opioid analgesic potency were determined in two mouse strains that differ in basal mu-opioid receptor density in brain. CXBK mice (mu-opioid receptor deficient) and outbred Swiss Webster mice were implanted s.c. with placebo pellets (controls) or etorphine minipumps (250 microg/kg/day) for 1-7 days and mu-opioid receptor density or mRNA levels in whole brain were assessed or mice were tested for etorphine analgesia following 7 days of treatment. In control CXBK mice, mu-receptor density was approximately 40% less than that for the Swiss Webster, although mRNA abundance was similar in both strains. Etorphine's potency was 4-fold greater in control Swiss Webster compared to CXBK mice. Etorphine treatment decreased ( approximately 25-40%) mu-receptor density similarly in both strains throughout treatment. The magnitude of analgesic tolerance to etorphine was 8-fold in both mouse strains. Etorphine produced a biphasic effect on receptor mRNA in both strains with levels decreased (25%) by 3 days and increased (30-40%) at 7 days. mRNA levels remained elevated (55%) 16 h following the end of the 7 day etorphine treatment. Taken together, these data suggest that in vivo etorphine treatment that produces mu-opioid receptor downregulation and tolerance, can regulate mu-opioid receptor mRNA abundance. Receptor downregulation may initially induce decreases in mRNA levels since downregulation preceded a decrease in gene expression. Prolonged (>3 days) receptor downregulation may be responsible for increasing message levels and may be important in recovery of receptors following treatment. In addition, the magnitude of changes in receptor density, mRNA, and tolerance were similar in both CXBK and Swiss Webster mice, indicating that the mechanisms required for receptor regulation and its functional consequences are independent of basal mu-opioid receptor density.

The effect of nimodipine on opioid antagonist-induced upregulation and supersensitivity.

Regulation of calcium flux has been suggested to play a role in acute and chronic effects of opioids. Previous studies have shown calcium channel blockers can inhibit opioid agonist-induced downregulation of mu-opioid receptors and may reduce the magnitude of tolerance. In the present study, we determined if calcium channel blockade would affect increases in opioid receptor density and functional supersensitivity produced by chronic opioid antagonist treatment in the mouse. Mice were implanted subcutaneously with a 15-mg naltrexone (NTX) or placebo pellet. Mice also were implanted with an osmotic minipump that infused nimodipine (100 microg/kg/day) or a second placebo pellet. This protocol yielded four groups: nimodipine-NTX; nimodipine-placebo; placebo-NTX; placebo-placebo. On the seventh day, pumps and pellets were removed. Twenty-four hours later, a morphine dose-response study was conducted (tail flick); or mice were sacrificed and saturation binding studies ([3H]DAMGO) were performed in whole brain. NTX treatment significantly increased the analgesic potency of morphine by approximately 60%. Nimodipine increased the potency of morphine by approximately 50%. For mice treated with both nimodipine and NTX, there was an additive effect on morphine potency ( approximately 120% increase). In binding studies, NTX increased the density of mu-opioid receptors similarly ( approximately 60-70%) in the presence and absence of nimodipine treatment, with no change in affinity. No effect of chronic nimodipine alone on mu-opioid receptor binding was observed. These data indicate that NTX-induced upregulation and supersensitivity are independent of calcium channel blockade by nimodipine. These results contrast with those from tolerance and downregulation studies, and confirm suggestions that different substrates mediate chronic opioid agonist and antagonist-induced effects in vivo. Finally, in a separate study, morphine potency was unaffected by acute nimodiopine (100 microg/kg; SC), suggesting that prolonged exposure to this calcium channel blocker is required to increase morphine potency.

Opioid receptor upregulation in mu-opioid receptor deficient CXBK and outbred Swiss Webster mice.

Chronic in vivo treatment with opioid antagonists increases opioid receptor density and the potency of opioid agonists without altering receptor mRNA levels. To determine if basal receptor density affects opioid receptor upregulation, we examined the effect of chronic naltrexone treatment on mu-opioid receptor density and mRNA in two mice strains that differ in mu-opioid receptor density. CXBK mice (mu-opioid receptor deficient) and outbred Swiss Webster mice were implanted s.c. with a placebo or 15 mg naltrexone pellet for 8 days, the pellets removed and 24 hr later opioid receptor density (mu, delta) and receptor mRNA level (mu) determined in whole brain; or morphine dose-response studies conducted. In placebo-treated CXBK mice, mu-opioid receptor density was approximately 40% less than in Swiss Webster mice, although mu-opioid receptor mRNA abundance was similar in both strains. In placebo-treated CXBK mice, morphine potency was approximately 6-fold less than Swiss Webster mice. Naltrexone treatment increased morphine potency (1.7-fold) and mu- (approximately 90%) and delta- (approximately 20-40%) opioid receptor density in CXBK and Swiss Webster mouse brain similarly. Mu-opioid receptor mRNA was unchanged by naltrexone treatment in either strain. There was no difference in the basal or naltrexone-treated whole brain G(i alpha2) protein levels in CXBK or Swiss Webster mouse. These data indicate that a deficiency in mu-opioid receptors does not alter the regulation of opioid receptors by opioid antagonists in vivo, and suggest that adaptive responses to chronic opioid antagonist treatment are independent of opioid receptor density.

Preproenkephalin mRNA and enkephalin levels in the adult Syrian hamster: the influence from glucocorticoids.

Proenkephalin (Penk) gene structure in hamsters and humans are similar but they differ from rats. In this study hamster Penk gene expression was examined after hypophysectomy+/-glucocorticoid receptor blockade with RU 486 (mifepristone). In contrast to rats, basal Penk gene expression in hamster adrenals did not change after treatments that reduced both the influence from glucocorticoids and phenylethanolamine-N-methyltransferase mRNA levels. Meanwhile, striatal preproenkephalin mRNA levels increased under these conditions.

The effects of antisense to Gialpha2 on opioid agonist potency and Gialpha2 protein and mRNA abundance in the mouse.

In this study, mice received a single intracerebroventricular (i.c.v. ) injection of an antisense oligodeoxynucleotide (ODN) directed towards the mRNA of Gialpha2. Controls received a saline or a nonsense ODN injection. The subsequent effects on protein levels and mRNA of Gialpha2 were determined in mouse striatum, as well as, the effect on opioid ([d-Ala2, d-Leu5]-enkephalin; DADLE) inhibition of cyclic AMP (cAMP) formation in striatum and morphine analgesic potency. At 48 h after treatment, maximal inhibition (Emax) of cAMP formation was significantly reduced for the antisense group compared to controls. Antisense ODN treatment only changed the Emax and did not significantly alter the IC50s of the dose-effect curves for inhibition of cAMP formation. Antisense ODN, but not nonsense ODN, significantly reduced morphine's analgesic potency by >2-fold, 48 h following treatment. Using a quantitative immunoblotting procedure, antisense treatment was shown to decrease striatal Gialpha2 protein 48 h after antisense injection, while there were no changes in protein levels at 2, 12 and 24 h. In contrast, no changes in Gialpha2 mRNA in mouse striatum were noted at any time after antisense treatment. Taken together, these data suggest that Gialpha2 mediates opioid-induced analgesia and opioid inhibition of cAMP production in the mouse. These data also suggest that antisense reduces target protein by a mechanism independent of changes in mRNA abundance.

Acute ethanol exposure decreases the analgesic potency of morphine in mice.

Chronic (7 days), forced ethanol drinking can decrease the analgesic potency of opioid agonists in mice. In the present study, the effect of short-term ethanol treatment was examined using forced ethanol access and ethanol injection protocols. Mice were given forced access to 1, 3 or 7% (v/v) ethanol for 24 hr and then tested for s.c. morphine analgesia using the tailflick assay. Controls had access to water. Another group of mice was injected i.p. with 2.5 g/kg ethanol or water 4 times over a 21 hr period and tested 3 hr after the final injection for morphine analgesia. Other mice were injected once i.p. with 1, 2 or 3 g/kg ethanol or water and tested 24 hr later using the tailflick. In the forced access study, ethanol dose-dependently decreased morphine's analgesic potency with the highest dose (7%) producing a 1.6-fold shift in the ED50. This decrease in morphine potency was similar to that found in a related study using 7% ethanol for 7 days (1.8-fold shift). Repeated ethanol injections significantly reduced the analgesic potency of morphine (1.9-fold shift), whereas, a single injection of 1, 2 or 3 g/kg ethanol did not alter the potency of morphine. Control studies indicated that neither 24 hr water nor food deprivation affected morphine potency. Overall, these data show that sustained exposure to ethanol over a 24 hr period will dose-dependently decrease morphine's analgesic potency.

The effect of cumulative dosing on the analgesic potency of morphine in mice.

Opioid analgesic potency can be evaluated using cumulative dosing, in which subjects are repeatedly administered a drug and tested after each dose until a criterion effect is reached. Although many laboratories use cumulative dosing, the effects of varying the starting dose and the magnitude of the increment dose on morphine analgesia (tail flick) in mice have not been evaluated. In experiment 1. mice were injected with the same starting dose [0.5 mg/kg subcutaneously (SC)] and 30 min later were tested for analgesia. Mice that were not analgesic were administered an increment dose (0.5, 1.0, 2.0, 2.5, or 3.0 mg/kg) and retested. The process was continued until all mice were analgesic. There was a significant effect of increment dose on morphine potency, with the relative potency increasing as the increment dose was increased. In experiment 2, different starting doses (0.5, 1.0, 2.0, or 3.0 mg/kg) were used with a constant increment dose of 1.0 mg/kg. There was a significant effect of starting dose on the potency of morphine, with the relative potency increasing as the starting dose increased. To determine if increment and starting dose affect tolerance estimates, mice were implanted SC with a 25- or 75-mg morphine or placebo pellet for 7 days and then tested using cumulative dose-response. Changes in the increment dose significantly affected the degree of tolerance for mice implanted with a 25-mg morphine pellet but not for mice implanted with a 75-mg morphine pellet. Changes in the starting dose did not significantly alter estimates of tolerance. Overall, these data indicate that the starting dose and increment dose can impact on morphine's potency determined by cumulative dosing protocols. Furthermore, estimates of tolerance can be affected by dosing parameters in the cumulative dosing protocol. These results suggest that cumulative dosing procedures should be standardized across experiments.

Synthesis and antinociceptive activity of [D-Met2, Pro5] enkephalin [N1,5-beta-D-2,3,4,6-O-tetraacetylglycosyl]--amide and [D-Met2, Pro5] enkephalinamide.

Tetra-O-acetylgalactopyranosylamine and tetra-O-acetylglucopyranosylamine of D-Met2, Pro5 enkephalin were designed and synthesized to enhance their membrane penetration, biological activity and resistance to proteolytic hydrolysis. Three approaches to the synthesis were attempted, which lead to a new synthetic scheme with a higher yield and enhanced ease of purification. The improved procedure involved attaching the tetra-O-acetylglycopyranosylamine to a t-Boc-Gly-Phe-Pro-OH peptide, removing the t-Boc, and condensing it with t-Boc-Tyr-D-Met-OH. Biological evaluation in vivo showed that these acetylglycopyranosylamine derivatives bind to mu and delta opioid receptors in homogenate binding assays and possess analgesic activity. The analgesic potency was less than that of the parent compound D-Met2, Pro5 enkephalin. These acetylglycopyranosylamine derivatives showed enhanced lipophilicity compared to their parent compound by a partition coefficient study and they also showed greater membrane permeability, using the rabbit cornea as a model system. These derivatives also are resistant to hydrolytic enzymes as compared to the endogenous met-enkephalin when evaluated in homogenized iris-ciliary body and aqueous humor from rabbit eyes.

The effect of ethanol drinking on opioid analgesia and receptors in mice.

Recent studies suggest substantial interactions between opioids and ethanol (EtOH). Both in vivo and in vitro experiments indicate that EtOH can regulate opioid systems and that opioids can modify EtOH consumption. In the present studies, we examined if EtOH consumption altered opioid receptors and the potency of opioid analgesics. Mice were given unlimited access to 6-7% EtOH alone for 7 days or were allowed to drink increasing concentrations (3-6%) of EtOH over 13-14 days. Controls had access to water. The EtOH groups drank significantly less volume than controls, although there were no significant differences in body weight or baseline nociception. The analgesic (tail flick) potency of SC morphine was decreased by approximately 1.6-2.0-fold in EtOH-treated mice. A single acute dose of EtOH (1 g/kg) that produced blood alcohol levels in excess of that for 7 day exposure to EtOH, did not change morphine's analgesic ED50, suggesting that chronic exposure to EtOH was necessary for the reduction in potency. The change in morphine potency was not due to pharmacokinetic differences because EtOH consumption did not modify the concentration of morphine in brain and spinal cord. The analgesic potency of a delta-opioid receptor agonist (ICV DSLET) was also decreased by approximately 2-fold. Saturation binding studies indicated no changes in the density or affinity of brain and spinal cord delta-opioid ([3H]DPDPE, [3H]DSLET, [3H]DeltorphinII) and mu-opioid ([3H]DAMGO) receptors. Similarly, there was no significant effect of EtOH on delta-opioid receptor mRNA in either brain or spinal cord preparations. Taken together, these data suggest that EtOH consumption decreases the analgesic potency of opioids in mice through a mechanism that is unrelated to pharmacokinetics or opioid receptor changes in brain and cord.

Time-dependent effects of in vivo pertussis toxin on morphine analgesia and G-proteins in mice.

Previous studies have indicated a long-duration of effect of in vivo pertussis toxin (PTX) on morphine analgesia in the mouse. However, the time-course of potency changes in morphine analgesia as determined in dose-response studies and biochemical correlates of PTX treatment have not been reported to date. Therefore, in the present studies the effects of in vivo PTX on morphine analgesia ED50 and PTX-catalyzed incorporation of [32P]-ADP-ribose and synapsin content in mouse spinal cord were examined. Mice were injected IT & ICV with saline or PTX (total dose = 0.2 microg) and tested for systemic morphine analgesia (tail-flick) 1, 10, 16 & 40 days later. There was no significant decrease in morphine potency 1 day following PTX treatment, whereas PTX produced a significant decrease in morphine potency at 10, 16 & 40 days. Concurrent decreases in the incorporation of [32P]-ADP-ribose in spinal cord by PTX were observed on days 10, 16 & 40. No changes were observed in synapsin content which suggests that the effect was not nonspecific. This study indicates that in vivo PTX produces co-ordinate long-lasting effects in both functional (analgesia) and biochemical (Gi/o-proteins) assays.

Magnitude of tolerance to fentanyl is independent of mu-opioid receptor density.

The effect of a mu-opioid receptor irreversible antagonist on the development of tolerance to fentanyl was determined in mice. Mice were injected with saline or clocinnamox (3.2 mg/kg, i.p.) and 4 h later mice implanted s.c. with a placebo pellet or an osmotic minipump that infused fentanyl (0.165 mg/kg per day) for 3 days. Fentanyl pumps and placebo pellets were removed on the third day following implantation and 4 h later mu-opioid receptor saturation binding studies in whole brain ([3H][D-Ala2,MePhe4,Gly-ol5]enkephalin: DAMGO) or fentanyl analgesic dose-response studies (tailflick assay) were conducted. Fentanyl infusions and clocinnamox both significantly reduced the potency of fentanyl by 2.8- and 2.4-fold, respectively. When fentanyl and clocinnamox were administered together, a significant 5.0-fold reduction in fentanyl potency relative to the saline-placebo group was observed, which represents an additive effect of clocinnamox and fentanyl. The ED50 of fentanyl in clocinnamox-treated mice was shifted 2.1-fold by fentanyl infusion relative to the clocinnamox-placebo group. This is comparable to the 2.8-fold shift in the ED50 produced by fentanyl infusion in saline-treated mice. In binding studies, fentanyl produced a small (-9%) reduction in Bmax, while clocinnamox significantly reduced (-41%) mu-opioid receptor density without altering affinity (Kd). In the clocinnamox-fentanyl group, there was a 50% reduction in Bmax, which is similar to the additive effect observed in analgesia studies. These data indicate that changes in mu-opioid receptor density prior to the development of tolerance to fentanyl do not impact on the magnitude of tolerance.

The effect of mu-opioid receptor antisense on morphine potency and antagonist-induced supersensitivity and receptor upregulation.

The present study examined the effect of in vivo antisense oligodeoxynucleotide treatment on naltrexone (NTX)-induced functional supersensitivity and mu-opioid receptor up-regulation in mice. On day 1 mice were implanted S.C. with a NTX or placebo pellet and injected I.T. and I.C.V. with dH2O or oligodeoxynucleotides. The oligodeoxynucleotides were designed so that they were either perfectly complementary to the first 18 bases of the coding region of mouse mu-opioid receptor mRNA, or had one (Mismatch-1) or four (Mismatch-4) mismatches. On days 3, 5, 7, and 9, mice were again injected I.T. and I.C.V. with dH2O or one of the oligodeoxynucleotides. After the final injections on day 9, placebo and NTX pellets were removed, and 24 h later mice were tested for morphine analgesia or sacrificed for saturation binding studies ([3H]DAMGO). Naltrexone increased the analgesic potency of morphine in dH2O treated mice by approximately 70%. In binding studies, NTX significantly increased density of brain (approximately 60%) and spinal cord (approximately 140%) mu-opioid receptors without affecting affinity. The mu-opioid antisense and the oligodeoxynucleotide with one mismatch (Mismatch-1) significantly reduced the potency of morphine by approximately twofold in placebo-treated mice. The oligodeoxynucleotide with four mismatches (Mismatch-4) did not significantly alter morphine potency. When placebo-treated mice were treated with either the antisense to the mouse mu-opioid receptor, Mismatch-4 or Mismatch-1 there were no significant changes in the density of mu-opioid receptors. Thus, mu-opioid antisense significantly reduced morphine potency without changing mu-opioid receptor density. When NTX and oligodeoxynucleotide treatments were combined, there was no change in NTX-induced supersensitivity and mu-opioid receptor upregulation. These data suggest that opioid antagonist-induced supersensitivity and upregulation of mu-opioid receptors does not involve changes in gene expression.

In vivo homologous regulation of mu-opioid receptor gene expression in the mouse.

Regulation of the mu-opioid receptor gene by opioid analgesic drugs has not been observed in rats and mice following in vivo treatments that produce tolerance. Although in vivo heterologous regulation of mu-opioid receptor mRNA by non-opioid compounds has been reported, the failure to observe changes in mu-opioid receptor mRNA levels in vivo after treatment with opioid agonists raised the possibility that in vivo homologous regulation by agonists may not occur. Therefore, in the present study, the effect of a high intrinsic efficacy opioid receptor agonist on opioid receptor density, gene expression and tolerance was determined. Mice were infused with etorphine for 7 days using an osmotic minipump, then the pump was removed and studies conducted 16-168 h later. Etorphine (50-250 microg/kg/day) infusion produced significant dose-dependent tolerance to the analgesic (tailflick) effects of etorphine, as well as dose-dependent mu-opioid receptor downregulation in brain at 16 h following the end of the infusion. Mu-opioid receptor density returned to control levels over a 168 h period following the end of etorphine (250 microg/kg/day) infusion. Similarly, the magnitude of tolerance decreased over the same period. Evaluation of changes in brain mu-opioid receptor mRNA 16 h following etorphine infusion indicated that there was dose-dependent increase in steady-state levels, with no significant change in GAPDH mRNA. The increase in mu-opioid receptor mRNA was approximately 55-65% over control at the highest etorphine infusion dose. Mu-opioid receptor mRNA returned to control levels over a 168 h period following the end of etorphine (250 microg/kg/day) infusion. These data suggest that the increase in mu-opioid receptor mRNA following the termination of etorphine treatment may drive the recovery of mu-opioid receptors. These data are the first demonstration of in vivo homologous regulation of mu-opioid receptor gene expression in the mouse by an opioid receptor agonist that produces tolerance and receptor downregulation.

The effect of in vivo ethanol consumption on cyclic AMP and delta-opioid receptors in mouse striatum.

In this study the effect of in vivo ethanol consumption on cyclic AMP (cAMP) and [D-Ala2,D-Leu5]enkephalin (DADLE) inhibition of forskolin-stimulated cAMP production was examined in mouse striatum. Effects of ethanol on striatal delta-opioid receptor (DOR) density and mRNA were also examined. Mice had unlimited access to 7% (v/v) ethanol alone or water for 1 or 7 days and were then sacrificed and striatum removed for analysis. There was no difference in basal cAMP formation between water and ethanol-treated mouse striatum following 7 day treatment, and a small, but statistically significant increase in basal cAMP in the ethanol group following 1 day treatment. Both 1 day and 7 day ethanol treatment did not significantly alter the percentage increase in cAMP following treatment with 10 microM forskolin. There was a significant effect of ethanol treatment on the maximum inhibitory effect of DADLE on forskolin-stimulated cAMP formation following both 1 and 7 day ethanol treatment. The DADLE IC50 was unaffected by ethanol treatment. Saturation binding studies ([3H]Deltorphin II) indicated no effect of ethanol on Bmax or Kd in striatum. Similarly, no difference between water and ethanol-treated was observed for DOR mRNA in striatum. These data indicate that ethanol consumption can alter opioid regulation of cAMP formation. However, this effect is not related to changes in any delta-opioid receptor parameters that were examined.

The effect of the irreversible mu-opioid receptor antagonist clocinnamox on morphine potency, receptor binding and receptor mRNA.

In these experiments, the effect of the irreversible mu-opioid receptor antagonist clocinnamox on the potency of morphine, opioid receptor binding and mu-opioid receptor mRNA was examined. Mice were injected with clocinnamox (0.32-12.8 mg/kg) and the analgesic potency of morphine was examined 24 h later. Clocinnamox produced a dose-dependent decrease in the potency of morphine; and at the higher dose of clocinnamox the maximal analgesic effect was not observed following doses of morphine in excess of 500 mg/kg s.c. In saturation binding studies in brain, clocinnamox (0.32-25.6 mg/kg) dose-dependently decreased mu-opioid ([3H][D-Ala2,MePhe4,Gly-ol5]enkephalin; DAMGO) receptor Bmax with relatively minimal effects on Kd. Binding to delta-opioid receptor ([3H][D-Pen2,D-Pen5]enkephalin; DPDPE) and kappa-opioid receptor ([3H](5,7,8)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec -8-yl) benzeneacetamide; U69,593) was not affected by clocinnamox. The effect of clocinnamox was time-dependent in that the greatest changes in morphine potency and mu-opioid receptor density were observed within 24 h of administration and decreased with time (336 h). Although mu-opioid receptor density was decreased to less than 30% of control 24 h following clocinnamox (12.8 mg/kg) and had increased to 80% by 5 days, a solution hybridization assay for mu-opioid receptor mRNA transcript revealed no changes in the steady-state levels of this mRNA. These studies indicate that clocinnamox is an irreversible antagonist at the mu-opioid receptor since it appears to selectively affect receptor density with minimal effects on affinity. Furthermore, clocinnamox produces time- and dose-dependent changes in Bmax and these changes appear to be unrelated to changes in mu-opioid receptor mRNA. It is possible that the repopulation of brain by mu-opioid receptors following clocinnamox is mediated by an existing pool of receptors that are activated following treatment.

Supersensitivity to opioid analgesics following chronic opioid antagonist treatment: relationship to receptor selectivity.

The effect of chronic opioid antagonist treatment on the analgesic potency of six opioid agonists was compared to changes in opioid receptor density and the selectivity of each agonist for mu (DAMGO), delta (DPDPE) and kappa (U69,593) opioid receptors. Mice were implanted SC with a 15-mg naltrexone or placebo pellet for 8 days. The pellets were removed and 24 h later, mice were sacrificed and binding studies were conducted, or mice were tested in analgesia (tail-flick) dose-response studies. All six analgesics acted as full agonists for both placebo and naltrexone-treated mice. Naltrexone increased the analgesic potency of methadone, etorphine, fentanyl, meperidine, and oxycodone by 1.9-3.2-fold. The analgesic potency of propoxyphene was not increased significantly (1.3-fold). In saturation binding studies in brain homogenate, naltrexone increased the Bmax of mu, delta, and kappa opioid receptors by 86, 43, and 33%, respectively, without altering Kd. Competition binding studies for each receptor type were conducted in brains from untreated mice, and KIs were determined for each agonist. All agonists had greatest selectivity toward mu compared with delta and kappa receptors. There did not appear to be an obvious relationship between receptor selectivity and the magnitude of supersensitivity. These studies indicate that supersensitivity occurs for a broad range of opioid analgesics following chronic opioid antagonist treatment in the mouse. However, the selectivity of these agonists for mu, delta, and kappa receptors does not appear to correlate with differences in supersensitivity.

The effect of intrinsic efficacy on opioid tolerance.

BACKGROUND: The intrinsic efficacy of opioid analgesics has been suggested to play a role in the development of tolerance to these agents. However, the effect of differences in dosing protocol on tolerance to opioid analgesics of high or low efficacy has not been addressed. Therefore, the effect of opioid intrinsic efficacy on tolerance in mice was determined in protocols of continuous and intermittent administration of equieffective doses of opioid agonists. METHODS: Initial antinociceptive median effective doses (ED50s) for five opioid agonists that vary in intrinsic efficacy were estimated in untreated mice. Groups of mice received continuous infusions of morphine, fentanyl, or etorphine for 72 h or 7 days from osmotic minipumps implanted subcutaneously. The infusion doses were calculated as multiples of the initial antinociceptive ED50. An inert placebo was implanted subcutaneously in controls. At the end of treatment, the pumps and placebos were removed, and 4-24 h later, mice were tested in dose-response studies (tail flick) using the same drug that had been chronically administered. In another study using intermittent dosing, mice received subcutaneous injections every 24 h for 3 days of saline or morphine, etorphine, fentanyl, oxycodone, or meperidine, or received subcutaneous injections every 24 h for 7 days of saline or morphine, etorphine, or fentanyl. Daily doses were calculated as multiples of the initial antinociceptive ED50. Twenty-four hours after the last injection, mice were tested in dose-response studies. RESULTS: High-intrinsic-efficacy compounds (e.g., etorphine and fentanyl) produced less tolerance than a lower-intrinsic-efficacy drug (morphine) in 72-h and 7-day infusion studies. Tolerance for all compounds after intermittent treatment with equieffective doses was similar, and intrinsic efficacy had no effect on the magnitude of tolerance after intermittent dosing. CONCLUSIONS: These results indicate that the intrinsic efficacy of opioid analgesics is inversely related to the degree of tolerance after continuous infusion, but that intrinsic efficacy does not significantly affect tolerance after once-daily intermittent administration of these agents. These findings may be of clinical utility in understanding the development of tolerance to the antinociceptive effects of opioids.