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Differences in the mechanisms underlying tolerance and mu-opioid receptor desensitization resulting from exposure to opioid agonists of different efficacy have been suggested previously. The objective of this study was to determine the effects of protein kinase C (PKC) and G protein-coupled receptor kinase (GRK) inhibition on antinociceptive tolerance in vivo to opioid agonists of different efficacy. A rapid (8-h) tolerance-induction model was used where each opioid was repeatedly administered to naive mice. Animals were then challenged with the opioid after injection of a kinase inhibitor to determine its effects on the level of tolerance. Tolerance to meperidine, morphine, or fentanyl was fully reversed by the PKC inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)carbazole (Gö6976). However, in vivo tolerance to [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) was not reversed by PKC inhibition. The novel small-molecule GRK inhibitors beta-adrenergic receptor kinase 1 inhibitor and 2-(8-[(dimethylamino) methyl]-6,7,8,9-tetrahydropyridol[1,2-a]indol-3-yl)-3-(1-methylindol-3-yl)maleimide (Ro 32-0432) did not reverse the tolerance to meperidine, fentanyl, or morphine but did reverse the tolerance to DAMGO. To correlate GRK-dependent DAMGO-induced tolerance with mu-opioid receptor desensitization, we used in vitro whole-cell patch-clamp recording from mouse locus coeruleus neurons and observed that the GRK inhibitors reduced DAMGO-induced desensitization of mu-opioid receptors, whereas the PKC inhibitor had no effect. These results suggest that tolerance induced by low- and moderate-efficacy mu-opioid receptor agonists is dependent on PKC, whereas tolerance induced by the high-efficacy agonist DAMGO is dependent on GRK.

The present study characterizes the involvement of the N-methyl-D-aspartic acid receptors (NMDARs) in mediating thermal hyperalgesia induced by activation of group I metabotropic glutamate receptors (mGluRs). Intrathecal administration of the mGluR1/5 agonist (S)-3,5-DHPG [(S)-3,5-dihydroxyphenylglycine] to mice resulted in significant hyperalgesia as assessed by the tail immersion test. The pretreatment of mice i.t. with CGS 19755 (selective antagonist of the NMDAR), CGP 78608 [[(1S)-1-[[(7-bromo-1,2,3,4-tetrahydro-2,3-dioxo-5-quinoxalinyl)methyl]amino]ethyl]phosphonic acid] (selective antagonist at the glycine-binding site of the NMDAR), ifenprodil and Ro 25-6981 (selective antagonists of the NR2B subunit of the NMDAR), bisindolylmaleimide I and Go-7874 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] (inhibitors of protein kinase C), or PKI-(14-22)-amide [Myr-N-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-NH(2)] (inhibitor of protein kinase A) dose-dependently inhibited the hyperalgesia induced by i.t. administration of the mGluR1/5 receptor agonist (S)-3,5-DHPG. In contrast, i.t. pretreatment of mice with NVP-AAM077 [[(R)-[(S)-1-(4-bromophenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid] (selective antagonist of the NR2A subunit of the NMDAR) or DT-3 [H-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Leu-Arg-Lys-Lys-Lys-Lys-Lys-His-OH] (inhibitor of protein kinase G) had no effect on (S)-3,5-DHPG-mediated hyperalgesia. We also show for the first time that i.t. injection of pSM2 (pShag Magic version 2)-grin2b (coding for an short-hairpin RNA to the NR2B subunit of the NMDAR) resulted in a dose-dependent decrease in the NR2B protein and blockade of hyperalgesia induced by activation of the mGluR1/5 in (S)-3,5-DHPG-treated mice. Taken together, our results suggest the hypothesis that mGluRs are coupled to the NMDAR channels through the NR2B subunit in the spinal cord and that this coupling involves the activation of protein kinase C and protein kinase A.

Presently, there are numerous structural classes of cannabinoid receptor agonists, all of which require solubilization for experimental purposes. One strategy for solubilizing water-insoluble tetrahydrocannabinols is conversion of the phenolic hydroxyl to a morpholinobutyryloxy substituent. The hydrochloride salts of these analogs are water-soluble and active in vivo when administered in saline. The present investigation demonstrated that hydrochloride salts of numerous substituted butyryloxy esters are water-soluble and highly potent. The substitutions include piperidine, piperazine, and alkyl-substituted amino moieties. It was also discovered that incorporation of a nitrogenous moiety in the alkyl side chain increased the pharmacological potency of tetrahydrocannabinol. For example, an analog containing a pyrazole in the side chain (O-2545) was found to have high affinity and efficacy at cannabinoid 1 (CB(1)) and CB(2) receptors, and when dissolved in saline, it was highly efficacious when administered either intravenously or intracerebroventricularly to mice. A series of carboxamido and carboxylic acid amide analogs exhibited high pharmacological potency, but their hydrochloride salts were not water-soluble. On the other hand, incorporation of imidazoles into the terminus of the side chain led to water-soluble hydrochloride salts that were highly potent when administered in saline to laboratory animals. It is now possible to conduct cannabinoid research with agonists that are water-soluble and thus obviating the need of solubilizing agents.

It has been suggested that the cannabinoid receptor type 1 (CB1), a G protein-coupled receptor, is internalized after agonist binding and activation of the second messenger pathways. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor, thus contributing to tolerance. Alterations in phosphorylation of proteins in the signal transduction cascade after CB1receptor activation could also alter tolerance to cannabinoids. We addressed our hypothesis by evaluating the role of several kinases in antinociceptive tolerance to Delta(9)-tetrahydrocannabinol (THC). We evaluated cAMP-dependent protein kinase (PKA) using KT5720, a PKA inhibitor; protein kinase C (PKC) using bisindolylmaleimide I, HCl (bis), a PKC inhibitor; cGMP-dependent protein kinase (PKG) using KT5823, a PKG inhibitor; beta-adrenergic receptor kinase (beta-ARK) using low molecular weight heparin (LMWH), a beta-ARK inhibitor; and phosphatidylinositol-3 kinase (PI3-K) using 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a PI3-K inhibitor and PP1, a Src family tyrosine kinase inhibitor. The cAMP analog used was dibutyryl-cAMP and the cGMP analog used was dibutyryl-cGMP. Our data indicate that selective kinases may be involved in cannabinoid tolerance. Mice and rats were rendered tolerant to Delta(9)-THC. The PKG inhibitor KT5823, the beta-ARK inhibitor LMWH, the PI3-K inhibitor LY294002, and inhibition of PKC by bis had no effect on tolerance. At a higher dose, bis attenuated the antinociceptive effect of delta(9)-THC in nontolerant mice. PP1, the Src family tyrosine kinase inhibitor, and KT5720, the PKA inhibitor, reversed THC-induced tolerance. In addition, inhibition of PKA reversed a decrease in dynorphin release shown to accompany THC tolerance in rats. These data support a role for PKA and Src tyrosine kinase in phosphorylation events in delta(9)-THC-tolerant mice.

The antinociceptive effects of various mu opioids given p.o. alone and in combination with Delta-9-tetrahydrocannabinol (Delta9-THC) were evaluated using the tail-flick test. Morphine preceded by Delta9-THC treatment (20 mg/kg) was significantly more potent than morphine alone, with an ED50 shift from 28.8 to 13.1 mg/kg. Codeine showed the greatest shift in ED50 value when administered after Delta9-THC (139.9 to 5.9 mg/kg). The dose-response curves for oxymorphone and hydromorphone were shifted 5- and 12.6-fold, respectively. Methadone was enhanced 4-fold, whereas its derivative, l-alpha-acetylmethadol, was enhanced 3-fold. The potency ratios after pretreatment with Delta9-THC for heroin and meperidine indicated significant enhancement (4.1 and 8.9, respectively). Pentazocine did not show a parallel shift in its dose-response curve with Delta9-THC. Naloxone administration (1 mg/kg s.c.) completely blocked the antinociceptive effects of morphine p.o. and codeine p.o. The Delta9-THC-induced enhancement of morphine and codeine was also significantly decreased by naloxone administration. Naltrindole (2 mg/kg s.c.) did not affect morphine or codeine antinociception but did block the enhancement of these two opioids by Delta9-THC. No effect was seen when nor-binaltorphimine was administered 2 mg/kg s.c. before morphine or codeine. Furthermore, the enhancements of morphine and codeine were not blocked by nor-binaltorphimine. We find that many mu opioids are enhanced by an inactive dose of Delta9-THC p.o. The exact nature of this enhancement is unknown. We show evidence of involvement of mu and possibly delta opioid receptors as a portion of this signaling pathway that leads to a decrease in pain perception.

Fentanyl and morphine are administered to human neonates and infants to provide analgesia and sedation during painful and stressful procedures. These opioids have often been shown to produce tolerance and dependence during continuous intravenous infusion. In neonatal animals, morphine produces tolerance and dependence, yet little is known about fentanyl. This report describes the first model for studying opioid tolerance and dependence in neonatal animals with use of osmotic minipumps. Postnatal day 6 rat pups were anesthetized and then remained naive or were surgically implanted subcutaneously with Alzet osmotic minipumps containing either saline or fentanyl (100 microg/kg/hr). Tolerance and dependence were assessed 72 hr after implantation. The ED50 values for fentanyl antinociception in the tail-flick test were not different between naive and saline pump-implanted animals. However, the fentanyl pump-implanted animals were tolerant to fentanyl. The tolerance observed was not the result of gender, developmental changes, fentanyl distribution or changes in fentanyl metabolism. These results indicate that continuous administration of fentanyl via osmotic minipump can render normal neonatal rats tolerant and physically dependent on fentanyl in 72 hr. Withdrawal precipitated by naloxone (5 mg/kg s.c.) in the fentanyl pump-implanted animals was characterized by increased spontaneous activity, micturition/defecation, wall climbing, abdominal stretching, tremors, scream on touch and spontaneous vocalization. This new model may provide a tool for studying the long-term consequences of neonatal opioid exposure in juvenile and adult animals.

Calcium (Ca++) administered into the i.c.v. space of mice has been reported to block opioid-induced antinociception dose dependently. These studies were conducted to test the hypothesis that Ca++ i.c.v. blocks the antinociceptive effects of morphine i.c.v. as a consequence of transmembrane Ca++ influx and Ca++ release from intracellular pools. Mice were injected with voltage-sensitive Ca++ channel antagonists at a dose that did not affect morphine antinociception to determine whether this pretreatment would prevent the inhibitory effects of Ca++. Nimodipine (12 nmol i.c.v.) was ineffective in preventing the inhibitory effects of Ca++ (100 nmol i.c.v.), whereas omega-conotoxin GVIA (3.3 pmol i.c.v.) completely prevented the inhibition by Ca++ of morphine antinociception. Other experiments were conducted to determine whether blocking Ca++ release from Ca++/caffeine-sensitive microsomal pools with ryanodine would prevent the inhibitory effects of Ca++. Ryanodine (2 nmol i.c.v.) significantly attenuated the inhibition by Ca++ of morphine antinociception. Another hypothesis to be tested was that stimulation of Ca++ release from intracellular pools would, like Ca++, block morphine antinociception. Thapsigargin (0.002-30 nmol i.c.v.), which increases cytosolic Ca++ by depleting Ca++ from inositol 1,4,5-trisphosphate-sensitive microsomal pools, dose-dependently blocked the antinociceptive effects of morphine. The results of this study indicate that Ca++ blocked morphine antinociception by stimulating Ca++ influx through omega-conotoxin GVIA-sensitive channels and by stimulating Ca++ release from Ca++/caffeine-sensitive microsomal pools.

In studying the interactions between handling mice and their subsequent analgesic response to an intrathecally (i.t.) administered mu-opioid agonist, DAMGO, it was found that suspending ICR mice by the tail for 1, 5, or 20 sec, 10 min before the tail-flick test, enhanced DAMGO by 5.3-, 7.4- and 23.6-fold, respectively, compared with mice maintained in a level posture. This enhancement was not accompanied by a change in the rostral flow of [3H]-DAMGO (25 ng, i.t.) to the brain (3.7% in 10 min), in its distribution along the neuraxis or in its systemic absorption. However, i.c.v. administration of beta-endorphin (1-27), an antagonist of epsilon opioid receptors, abolished the enhancement of i.t. DAMGO without affecting its basal analgesic potency. Pretreatment with the delta-opioid antagonist naltrindole (5.6 nmol, i.t.,-30 min) also blocked the enhancement of DAMGO without significantly affecting its basal analgesic potency. Alternatively, this same dose of naltrindole injected i.c.v. failed to block the enhancement of DAMGO in suspended mice. A 20-sec suspension failed to enhance i.t. kappa and delta-agonists, but it did enhance i.t. morphine. In mouse strain comparisons, i.t. DAMGO was more potent in C57BL/6J and DBA/2J mice than in C3H/HeJ and ICR mice, but DAMGO was enhanced by a 20-sec suspension in all strains tested. Thus suspending mice by the tail evoked a reflex enhancement of spinal mu agonist-induced analgesia that probably involved both the supraspinal release of beta-endorphin (an endogenous epsilon agonist) and the subsequent spinal release of an endogenous delta-receptor agonist in the reflex pathway.

To the authors' knowledge, the effect of i.t. administered calcium on thermoregulation in mice has not been investigated. Calcium administration (i.t.) induced hypothermia in mice. It was found that calcitonin gene-related peptide (CGRP) (i.t.) also produced hypothermia. Because opioids have well documented thermoregulatory effects, the authors evaluated whether the hypothermia induced by calcium and CGRP was the result of the release of opioids. Calcium induced hypothermia at different ambient temperatures (4 degrees C, 22 degrees C and 30 degrees C) in intact mice. Similarly treated spinalized mice maintained body temperature. Using laser Doppler flowmetry, there was a significant increase in blood flow in the tails of calcium-injected mice vs. those of vehicle-injected mice. Both naloxone and naltrindole failed to block the hypothermic effects of calcium (i.t.). Nor-binaltorphimine (i.t.) significantly blocked calcium (i.t.)-induced changes in body temperature. CGRP (i.t.) produced hypothermia for 15 hr postinjection, with the maximum decrease at 3 hr. CGRP induced hypothermia in intact and sham-lesioned mice but not in spinalized mice. CGRP (i.c.v.) also produced hypothermia (onset, 15-min postinjection) followed by the peak effect at 1 hr with recovery to baseline temperature by 2 hr. Subthreshold doses of calcium and CGRP given in combination produced greater than additive hypothermia. The hypothermic effects of CGRP were reversed by naloxone, naltrindole and nor-binaltorphimine. CGRP produced significant hypothermia in both morphine-tolerant and nontolerant mice. Chronic administration of CGRP in nontolerant and morphine-tolerant mice did not alter hypothermia after pretreatment with CGRP (i.t.).(ABSTRACT TRUNCATED AT 250 WORDS)

Mice injected with calcium in the intrathecal (i.t.) space display dose-dependent antinociception in the tail-flick test. The aims of this study were to evaluate whether endogenous opioids mediate the antinociceptive effects of calcium (i.t.) and to determine if antinociception resulted from calcium acting directly in segmental spinal sites. Mice spinalized at T6 to T8 were more sensitive to the antinociceptive effects of calcium (150-600 nmol i.t.) than sham-lesioned mice. In intact mice, naloxone (138-275 pmol i.t.) and naltrindole (2.8-22 nmol i.t.) dose-dependently blocked the antinociceptive effects of calcium (600 nmol i.t.), with inhibitory dose-50 (ID50) values of 235 picomol and 11.4 nanomol, respectively. nor-Binaltorphimine (nor-BNI) (14-54 nmol i.t.) did not antagonize the antinociceptive effects of calcium (i.t.). Furthermore, the calcium (i.t.) dose-response curve was shifted right-ward by naloxone (206 pmol i.t.) and naltrindole (5.5 nmol i.t.). nor-BNI (54 nmol i.t.) was ineffective in shifting the dose-response curve. In spinalized mice, naloxone (206-687 pmol i.t.) and naltrindole (11-44 nmol i.t.) blocked the antinociceptive effects of calcium (i.t.), with ID50 values of 342 and 19.2 nmol, respectively. nor-BNI did not antagonize antinociception. In addition, the calcium (i.t.) dose-response curve was shifted right-ward by naloxone (275 pmol i.t.) and naltrindole (11 nmol i.t.). The dose-response curve was not shifted by nor-BNI (54 nmol i.t.). A 4-hr pretreatment with the irreversible mu receptor antagonist beta-funaltrexamine (0.01-0.4 nmol i.t.) blocked [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin but not [D-Pen2,5]enkephalin or calcium (i.t.)-mediated antinociception.(ABSTRACT TRUNCATED AT 250 WORDS)