Differences in the mechanisms underlying tolerance and μ-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-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) was not reversed by PKC inhibition. The novel small-molecule GRK inhibitors β-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 μ-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 μ-opioid receptors, whereas the PKC inhibitor had no effect. These results suggest that tolerance induced by low- and moderate-efficacy μ-opioid receptor agonists is dependent on PKC, whereas tolerance induced by the high-efficacy agonist DAMGO is dependent on GRK.

Background and purpose: The ability of an agonist to induce desensitization of the µ‐opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist‐specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high‐efficacy agonist). Experimental approach: MOR function was measured in locus coeruleus neurons, by using whole‐cell patch‐clamp electrophysiology, in rat and mouse brain slices (both wild‐type and protein kinase C (PKC)α knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C‐kinase (RACK), and in vivo viral‐mediated gene‐transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G‐protein‐coupled receptor kinases (GRKs). Key results: Morphine‐induced desensitization was attenuated by using RACK inhibitors that inhibit PKCα, but not by other isoform‐specific inhibitors. Further, the PKC component of morphine‐induced desensitization was absent in locus coeruleus neurons from PKCα knockout mice. The PKC‐enhanced morphine‐induced desensitization was not affected by over‐expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO‐induced MOR desensitization was independent of PKC activity but was reduced by over‐expression of the GRK2 DNM but not by that of a GRK6 DNM. Conclusions and implications: In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCα‐mediated, heterologous mechanism and DAMGO by a GRK‐mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.

In morphine tolerance a key question that remains to be answered is whether mu-opioid receptor (MOPr) desensitization contributes to morphine tolerance, and if so by what cellular mechanisms. Here we demonstrate that MOPr desensitization can be observed in single rat brainstem locus coeruleus (LC) neurons following either prolonged (> 4 h) exposure to morphine in vitro or following treatment of animals with morphine in vivo for 3 days. Analysis of receptor function by an operational model indicated that with either treatment morphine could induce a profound degree (70-80%) of loss of receptor function. Ongoing PKC activity in the MOPr-expressing neurons themselves, primarily by PKC alpha, was required to maintain morphine-induced MOPr desensitization, because exposure to PKC inhibitors for only the last 30-50 min of exposure to morphine reduced the MOPr desensitization that was induced both in vitro and in vivo. The presence of morphine was also required for maintenance of desensitization, as washout of morphine for > 2 h reversed MOPr desensitization. MOPr desensitization was homologous, as there was no change in alpha(2)-adrenoceptor or ORL1 receptor function. These results demonstrate that prolonged morphine treatment induces extensive homologous desensitization of MOPrs in mature neurons, that this desensitization has a significant PKC-dependent component and that this desensitization underlies the maintenance of morphine tolerance.

CD38 is a 45 kDa monomer enzyme complex consisting of both ADP-ribosylcyclase as well as cADP hydrolase. C38 has been discovered in both neurons and glia of the brain, however, its role in signal transduction is not fully characterized. Experiments were conducted to determine its role in morphine-induced analgesia in the periaqueductal gray (PAG) of male Swiss-Webster mice. PAG was removed 30-min following acute administration of 8 mg/kg morphine s.c. Gene expression of CD38 was significantly increased in the morphine-treated mice compared to naive mice. Western Immunoblotting demonstrated an increase in the expression of the more active 110 kDa homodimer form of CD38. In addition, the ADP-ribosylcyclase conversion of β-NGD+ to cGDPR was significantly increased. All of these effects were completely blocked in mice co-treated with 1 mg/kg naloxone s.c. Other experiments determined the specific activity of CD38 in morphine-induced analgesia. Nicotinamide is a negative feedback inhibitor of CD38 ADP-ribosylcyclase. Nicotinamide injected i.p. dose-dependently antagonized the antinociceptive effects of 8 mg/kg morphine in the 56 °C tail-withdrawal test. Furthermore, a 500 mg/kg nicotinamide dose decreased the potency of morphine by 9.7-fold. These results are supported by data from male CD38−/− knockout mice, which exhibited a decreased analgesic response to morphine in comparison to male C57BL/6J wild-type mice. Funded by NIH grants: R01-DA-01647, T32-DA-07027, K05-DA-00480, HL-57244, HL-75316.

This study tested the hypothesis that NAD(P)H oxidase plays a role in morphine antinociceptive tolerance formation. Antinociceptive tolerance was induced in male Swiss Webster mice following morphine treatment for 24 hours. The effects of chronic morphine treatment on NAD(P)H oxidase mRNA levels, protein levels, and enzyme activity were determined in the periaqueductal gray (PAG) and cerebral cortex from these mice. NAD(P)H oxidase activity, as measured by the conversion rate of NADH to NAD+ using HPLC, significantly increased by 47.6% in homogenates from the PAG, while NAD(P)H oxidase activity was not significantly altered in the cerebral cortex. The NAD(P)H oxidase inhibitor, diphenylene iodonium, completely blocked basal and morphine-increased NAD(P)H oxidase activity in the PAG. Western blot analysis revealed that protein levels of the NADPH oxidase subunits gp91phox, p47phox, and p67phox significantly increased in the PAG of morphine-tolerant mice, but not in the cerebral cortex. Real-time PCR (RT-PCR) analysis revealed that gp91phox mRNA levels were significantly increased in the PAG of morphine-tolerant mice. These results indicate that NAD(P)H oxidase is present in PAG neurons and that chronic morphine treatment increases the expression of various subunits of this enzyme, leading to enhanced superoxide production in this brain area. Future studies will continue to elucidate the functional significance of enhanced NAD(P)H oxidase activity in morphine antinociceptive tolerance expression. (Supported by NIH Grants HL57244, HL75316 and DA01647).

The process of translocation of PKC from the cytosol to membranes is tightly regulated by RACK1 anchoring proteins, defined as Receptors for Activated C Kinase (RACK). The amino acid sequence allows for only a specific PKC isoform to undergo active translocation to that site, allowing for the phosphorylation of specific target proteins with the addition of appropriate co-factors such as diacylglycerol, phosphatidylserine, and calcium (with conventional PKC isoforms). To date the RACKs for PKC betaI, betaII, gamma, delta, theta, epsilon and eta have been developed for research purposes. Swiss-Webster mice implanted with placebo or 75 mg morphine pellets developed significant tolerance 3-days later in the 56 °C tail-withdrawal and hot-plate tests, and tolerance to morphine-induced hypothermia. Pretreatment i.c.v. with RACK1 inhibitor to PKC betaI, betaII, delta, theta and eta all failed to significantly reverse morphine tolerance in any of these tests. However, the RACK1 inhibitors to PKC gamma and epsilon significantly reversed tolerance in each of the tests. PKC epsilon has been located predominantly on primary afferent neurons on pre-synaptic terminals in the spinal cord, while PKC gamma is located on postsynaptic sites associated with glutamate release and the activation of NMDA receptors. PKC alpha translocates to F-actin, and therefore does not have a specific RACK1 site. However, PKC alpha has also been implicated in morphine tolerance. These data demonstrate that PKC gamma and epsilon must undergo active translocation in order to mediate morphine tolerance. Funded by NIDA grants: R01-DA-01647, T32-DA-0027, K05-DA-00480.

Relatively few studies have compared the effects of tetrahydrocannabinols and anandamide-like cannabinoids following repeated dosing. Whereas pronounced tolerance develops to many of the in vivo pharmacological effects of Δ9-tetrahydrocannabinol with repeated dosing, tolerance to anandamide-induced effects is typically less noted. In the present study, we examined cross-tolerance between Δ9-tetrahydrocannabinol and anandamide-like compounds (anandamide, 2-methylanandamide, and O-1812) in a tetrad of in vivo tests sensitive to cannabinoid action, including spontaneous activity, tail flick, rectal temperature, and a ring immobility test of catalepsy. Six intraperitoneal injections of Δ9-tetrahydrocannabinol 10 mg/kg over a period of 4 days resulted in the development of pronounced tolerance to all of its in vivo effects. In contrast, task specificity was observed in cross-tolerance to anandamide and its analogs: antinociception (all three compounds), suppression of spontaneous activity (2-methylanandamide and O-1812), catalepsy (O-1812), and hypothermia (none of the compounds). Furthermore, when it occurred, the magnitude of cross-tolerance was notably smaller. These results suggest that anandamide-like cannabinoids may have a unique pharmacology that only partially overlaps with that of Δ9-tetrahydrocannabinol and other traditional cannabinoids. Although the basis for this unique pharmacology has not as yet been determined, it is possible that regional specificity of cannabinoid CB1 receptor downregulation and endocannabinoid release induced by repeated dosing with Δ9-tetrahydrocannabinol may play a role.

The present study investigated the effect of morphine antinociceptive tolerance on Protein Kinase A (PKA) activity in mouse brain (periaqueductal gray (PAG), thalamus, medulla) and lumbar spinal cord (LSC). A model was developed in which mice expressed a 21-fold level of morphine antinociceptive tolerance following implantation of a 75-mg morphine pellet for 15 days. Cytosolic and particulate PKA activity was measured directly in homogenates from the PAG, thalamus, medulla and LSC which studies have shown play a role in morphine-induced analgesia. In addition, a kinetic analysis of cytosolic and particulate PKA activity in homogenates from these regions was conducted and PKA V(max) and K(m) values were determined. Results demonstrated that chronic morphine treatment did not alter PKA activity or PKA kinetics in mouse brain. Moreover, particulate PKA activity/kinetics were not altered in LSC. However, cytosolic PKA activity was significantly increased in LSC following morphine treatment for 15 days. Furthermore, an increase in cytosolic PKA V(max) was observed in LSC. These results suggest that spinal and supraspinal PKA activity are differentially altered during morphine tolerance in mice. Thus, neurons in mouse brain and LSC that comprise the pain pathway descending from the brainstem and ending in the spinal cord respond differently to chronic morphine treatment.

The present study investigated the effect of different levels of Delta-9-tetrahydrocannabinol (Delta(9)-THC) antinociceptive tolerance on Protein Kinase A (PKA) activity in mouse brain and spinal cord. To strengthen this investigation, a positive control was developed to demonstrate the assay utilized in this study was sensitive enough to detect an increase in PKA activity in the anatomical regions utilized in this study. The membrane-permeant and phosphodiesterase-resistant cAMP analog 8-Bromoadenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-8-Br-cAMPS) was utilized for the development of this positive control and this compound produced an increase in PKA activity in several mouse brain regions (i.c.v.) and lumbar spinal cord (i.t.) following its administration. Models were then developed in which mice expressed either a 13-fold or 49-fold level of Delta(9)-THC antinociceptive tolerance following chronic treatment with 10mg/kg Delta(9)-THC or 80mg/kg Delta(9)-THC for 6.5 days. Basal and total cytosolic and particulate PKA activities were measured directly in homogenates from the striatum, hippocampus, cerebellum, cortex and lumbar spinal cord. Results from this study indicate that chronic exposure to Delta(9)-THC does not produce an increase in PKA activity in these mouse brain regions or spinal cord. Future work is needed to determine the role of PKA in cannabinoid tolerance in mice.

Two peptide fragments of native Protein Kinase A inhibitor (PKI), PKI-(6-22)-amide and PKI-(Myr-14-22)-amide, significantly reversed low-level morphine antinociceptive tolerance in mice. The inhibition of Protein Kinase A (PKA) activity by both peptide fragments was then measured in specific brain regions (thalamus, periaqueductal gray (PAG), and medulla) and in lumbar spinal cord (LSC), which in previous studies have been shown to play a role in morphine-induced analgesia. In drug naive animals, cytosolic PKA activity was greater than particulate PKA activity in each region, while cytosolic and particulate PKA activities were greater in thalamus and PAG compared to medulla and LSC. The addition of both peptides to homogenates from each region completely abolished cytosolic and particulate PKA activities in vitro. Following injection into the lateral ventricle of the brain of drug naive mice and morphine-tolerant mice, both peptides inhibited PKA activity in the cytosolic, but not the particulate fraction of LSC. In addition, cytosolic and particulate PKA activities were inhibited by both peptides in thalamus. These results demonstrate that the inhibition of PKA reverses morphine tolerance. Moreover, the inhibition of PKA activity in specific brain regions and LSC from morphine-tolerant mice by PKI analogs administered i.c.v. is evidence that PKA plays a role in morphine tolerance.

Summary Background : Infants placed on extracorporeal membrane oxygenation (ECMO) or mechanical ventilation often need continuous morphine infusions for pain relief and sedation. The resulting physical dependence requires an additional 2–3‐week hospital stay to taper the morphine to avoid withdrawal. Buprenorphine effectively blocks abstinence in dependent adults, and in infants it could accelerate or eliminate the tapering schedule, thereby enabling earlier hospital dismissals. Methods : Morphine‐dependent infant rats were used in this study to determine the effectiveness of buprenorphine in blocking abstinence. Postnatal day‐14 (P14) rats were implanted with osmotic minipumps that delivered saline (1 μ l·h −1 ) or morphine (2 mg·kg −1 h −1 ) for 72 h. The minipumps were then removed to allow the rats to undergo spontaneous morphine withdrawal. Results : The withdrawal period lasted approximately 72 h out of a 96‐h observation period. The following signs were significant during these hours: wet‐dog shakes, 1–72 h; abdominal stretches, 1–72 h; forepaw tremors, 1–24 h; splayed hind‐limbs, 1–72 h; ptosis, 4–72 h; and evoked vocalization, 4 and 8 h. A single 1 mg·kg −1 buprenorphine dose significantly decreased wet‐dog shakes from 1 to 72 h, abdominal stretches from 1 to 48 h, forepaw tremors and splayed hind‐limbs 1–8 h, and ptosis and evoked vocalization at 4 and 8 h. Repeated administration of 1 mg·kg −1 buprenorphine before pump removal and at 24, 48 and 72 h resulted in a greater magnitude of blockade of abstinence throughout the 96‐h observation period. Conclusions : Buprenorphine may prove to be a suitable drug for treating opioid withdrawal in human infants.

Morphine antinociceptive tolerance in the tail-flick test is completely reversed by inhibitors of protein kinase C (PKC) or cAMP-dependent protein kinase (PKA). The effects of these inhibitors on tolerance to supraspinally mediated antinociception, such as the hot-plate test was unknown, as well as their effects in tests of mechanical nociception. The PKC inhibitors bisinolylmaleimide I ((2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) and Gö-7874 {2[1[(3-Dimethylaminopropyl)-5-methozyindol-3-yl]-3-(1H-indol-3-yl) hydrochloride} completely reversed the tolerance to morphine in both the hot-plate and tail-pinch tests. Similarly, the PKA inhibitor KT-5720 (8R, 9S, 11S)-(−)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one also reversed tolerance in both tests. The role of PKC and PKA in mediating tolerance to morphine-induced hypothermia was also investigated. Bisinolylmaleimide I, Gö-7874 and KT-5720 only partly reversed the 32-fold level of tolerance induced by the morphine pellets. However, co-administration of bisinolylmaleimide I with KT-5720 or Gö-7874 with KT-5720 completely reversed the tolerance. This demonstrates that tolerance in a non-behavioral system involves the actions of PKC and PKA.

Intracerebroventricular (i.c.v.) injection of phospholipase C inhibitors and structurally dissimilar PKC inhibitors were shown to completely reverse morphine antinociceptive tolerance in mice. Since Group I metabotropic glutamate receptors (mGlu1 and mGlu5) activate phospholipase C through Gαq Gα11 proteins, we hypothesized that morphine tolerance could occur through an increase in mGlu1 and mGlu5 receptor stimulation. Seventy-two hours after implantation of placebo or 75 mg morphine pellets, mice were tested in the 56 °C warm-water tail-withdrawal test following i.c.v. injection of vehicle or test drug. The mGlu1 receptor antagonist CPCCOEt (7-(Hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) partly but significantly reversed morphine tolerance. The mGlu5 receptor antagonist MPEP (2-Methyl-6-(phenylethynyl)pyridine hydrochloride) also partly reversed the antinociceptive tolerance. Co-administering CPCCOEt with MPEP completely reversed the tolerance. Furthermore, the mixed mGlu1/mGlu5 antagonist AIDA ((RS)-1-Aminoindan-1,5-dicarboxylic acid) also completely reversed the tolerance. Thus, greater mGlu1 and mGlu5 receptor stimulation during morphine tolerance may lead to persistent activation of the phosphatidylinositol cascade.

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 endoplasmic reticulum inside neurons can provide enormous amounts of releasable Ca2+ to increase cytosolic Ca2+ levels through the activation of endoplasmic membrane ion channels. Ryanodine (RyR) channels release Ca2+ into the cytosol when activated by Ca2+ influx through voltage-gated channels, or by cyclicADP ribose. Inositol tris-phosphate (IP3) channels are stimulated by phospolipid metabolism and the release of IP3. The hypothesis was tested that drugs that bind RyR or IP3 channels would affect the anesthetic potency of bupivacaine. The radiant heat tail-flick test was used to assess for anesthesia following subcutaneous infiltration of bupivacaine and Ca2+ modulating drugs in the tails of mice. No musculature is contained in the tail that could result in motor block. The RyR channel agonists 4-chloro-m-cresol and poly-L-lysine significantly reduced the anesthetic potency of bupivacaine. The plant alkaloid ryanodine elicited a bi-phasic effect, with low concentrations blocking bupivacaine anesthesia, and a high concentration enhancing anesthesia. Alternatively, the RyR channel antagonist dantrolene sodium dose-dependently increased bupivacaine's potency. However, the IP3 channel drugs were inactive. The IP3 agonist adenophostin A failed to affect bupivacaine anesthesia. Furthermore, bupivacaine was unaffected by the IP3 channel antagonists xestospongin C or low molecular weight heparin. Our results indicate that only the RyR channel drugs modulated the anesthetic effects of bupivacaine. Electrophysiological and molecular studies of sensory dorsal root ganglia neurons, the source of Adelta and C-fiber nociceptors, have demonstrated the presence of RyR3 Ca2+ release channels. This provides the first evidence that RyR channels might affect bupivacaine anesthesia in some fashion.

Sodium channel drugs were used to modulate the anesthetic effects of bupivacaine in mice. Anesthesia was measured following perisciatic injection of bupivacaine with vehicle or neurotoxin in the popliteal region. The site 1 Na+ channel blocker tetrodotoxin alone was inactive, but increased the anesthetic effects of bupivacaine. We hypothesized that the site 2 and site 3 Na+ channel openers veratridine and anemone toxin II (ATXII), respectively, would antagonize bupivacaine. Paradoxically, both drugs enhanced bupivacaine. In bupivacaine-treated mice, a significant correlation was observed between limb weakness scores and paw withdrawal latencies. The correlation coefficients were higher when tetrodotoxin, veratridine, or ATXII was coadministered with bupivacaine. In conclusion, veratridine and ATXII may have increased the stimulus-dependent binding of bupivacaine to Na+ channels, thereby increasing the anesthetic effects of bupivacaine.

We have previously reported that intracerebroventricular (i.c.v.) injection of either a PKC or PKA inhibitor completely reversed the expression of 5- to 8-fold morphine antinociceptive tolerance. We developed a model of 45-fold morphine tolerance that included a 75-mg morphine pellet and twice daily morphine injections. PKC inhibitor doses of bisindolylmaleimide I and Gö-7874 that completely reversed 8-fold tolerance only partly reversed the 45-fold level of antinociceptive tolerance. A component of tolerance was resistant to PKC inhibition, since even higher inhibitor doses failed to further reverse the high level of morphine tolerance. In addition, the 45-fold tolerance was only partly reversed by the PKA inhibitor KT-5720 at a dose previously cited by others to reverse 5-fold tolerance. Another PKA inhibitor 4-cyano-3-methylisoquinoline only partly reversed the morphine tolerance as well. In other experiments PKC and PKA inhibitors were co-administered together to determine their effectiveness for completely reversing the 45-fold level of morphine tolerance. Co-administering either bisindolylmaleimide I with KT-5720, or Gö-7874 with KT-5720, completely reversed the high level of tolerance. The high level of morphine tolerance was also completely reversed by co-administering Gö-7874 with 4-cyano-3-methylisoquinoline. Thus, high levels of morphine tolerance may reflect increases in protein phosphorylation by the terminal kinases of both the adenylyl cyclase and phosphatidylinositol cascades in brain and spinal cord areas critical to the expression of antinociception.