Activation of μ‐opioid receptors (MORs) on enteric neurons during opioid treatment inhibits gastrointestinal peristalsis and leads to constipation. This is the most significant side affect associated with opioid treatment. Peripherally active MOR antagonists can minimize constipation without affecting analgesia. Previously, we described the 6β‐Naltrexamine derivative NAP as a novel peripherally selective MOR antagonist. NAP was 300x more potent than methylnaltrexone at improving intestinal motility in morphine tolerant mice. However, NAP partially antagonized morphine antinociception in the mouse tail‐flick assay. Efforts to reduce CNS activity led to the development of BNAP. Radioligand binding studies show BNAP maintained high MOR affinity (K­i = 0.76 ± 0.09 nM). In the mouse tail flick assay BNAP (10mg/kg) did not antagonize morphine‐induced antinociception. BNAP also competitively inhibited morphine‐induced contractions in circular muscle preparations from the mouse distal and proximal colon. Both NAP and BNAP antagonized morphine‐induced contractions with equal efficacy at the doses tested (1, 10, and 100 nM). Interestingly, they were 10x more potent in the proximal colon. This suggests differences in MOR receptor density or splice variants between proximal and distal colon. In conclusion, BNAP is a more peripherally selective MOR antagonist than NAP and has equal potency in isolated tissue preparations. Thus, BNAP is a suitable lead for further study and development as a novel peripherally selective MOR antagonist. Supported by DA024009, DA024002, and DA007027.

Background and purpose: Chronic morphine administration produces tolerance in vivo and attenuation of μ opioid receptor (MOR)‐mediated G‐protein activation measured in vitro , but the relationship between these adaptations is not clear. The present study examined MOR‐mediated G‐protein activation in the CNS of mice with different levels of morphine tolerance. Experimental approach: Mice were implanted with morphine pellets, with or without supplemental morphine injections, to induce differing levels of tolerance as determined by a range of MOR‐mediated behaviours. MOR function was measured using agonist‐stimulated [ 35 S]guanylyl‐5′‐ O ‐(γ‐thio)‐triphosphate ([ 35 S]GTPγS) and receptor binding throughout the CNS. Key results: Morphine pellet implantation produced 6‐12‐fold tolerance in antinociceptive assays, hypothermia and Straub tail, as measured by the ratio of morphine ED 50 values between morphine‐treated and control groups. Pellet implantation plus supplemental injections produced 25‐50‐fold tolerance in these tests. In morphine pellet‐implanted mice, MOR‐stimulated [ 35 S]GTPγS binding was significantly reduced only in the nucleus tractus solitarius (NTS) and spinal cord dorsal horn in tissue sections from morphine pellet‐implanted mice. In contrast, MOR‐stimulated [ 35 S]GTPγS binding was significantly decreased in most regions examined in morphine pellet+morphine injected mice, including nucleus accumbens, caudate‐putamen, periaqueductal gray, parabrachial nucleus, NTS and spinal cord. Conclusions and implications: Tolerance and the regional pattern of apparent MOR desensitization were influenced positively by the level of morphine exposure. These results indicate that desensitization of MOR‐mediated G‐protein activity is more regionally widespread upon induction of high levels of tolerance, suggesting that this response contributes more to high than low levels of tolerance to CNS‐mediated effects of morphine. British Journal of Pharmacology (2007) 151 , 1324–1333; doi: 10.1038/sj.bjp.0707328

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.

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.

Our laboratory demonstrated that morphine exhibits a modulatory control over the glyburide-binding site (sulfonylurea receptor) of the ATP-gated K(+) channel. This study evaluated the effect of chronic morphine administration on the sulfonylurea receptor during tolerance and physical dependence. ICR and Swiss-Webster mice were rendered tolerant to morphine by pellet implantation and were withdrawn by pellet removal. Alterations in the B(max) and K(D) were evaluated in mouse spinal cord using the radiolabeled ATP-gated K(+) channel blocker glyburide. The ED(50) for Swiss-Webster mice shifted from 13 to 451 mg/kg and thus they were more tolerant to morphine than ICR mice (ED(50) shift from 12 to 120 mg/kg). Swiss-Webster mice were also dependent to morphine only when the morphine pellet was in place, unlike ICR mice, which were dependent for 48 h after morphine pellet removal. Glyburide binding increased during chronic morphine treatment in Swiss-Webster mice by over 2-fold (from 294 to 635 fmol/mg of protein). This was not observed in ICR mice. In Swiss-Webster mice, chronic morphine treatment also significantly increased the K(D) by 3-fold (from 0.38 to 1.1 nM), whereas there was no change in affinity for ICR mice. Both strains of mice remained tolerant for 2 days after spontaneous withdrawal from morphine. However, the only increases in the B(max) and K(D) of glyburide were observed in Swiss-Webster mice that were highly tolerant to morphine. These results indicate that a high degree of tolerance is needed to alter ATP-gated potassium channels.

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.

The hypoglycemic effect of morphine (40 micrograms) injected intrathecally (i.t.) was studied with regard to disposition of i.v. [14C]glucose and [3H]2-deoxyglucose and was compared with the effects of two other hypoglycemic agents, insulin (1 IU/kg, s.c.) and xanthan gum (50 mg/kg, i.p.). Mice given i.t. morphine or s.c. insulin exhaled a greater amount of 14CO2 from i.v. [14C]glucose than did control mice given i.t. saline, whereas there was less 14CO2 expiration in xanthan-treated mice. In morphine-treated mice there was less 14C in liver, brain and blood, and more 3H in kidney and hindleg muscle than in control mice. Insulin-treated mice had more 14C in muscle, less 14C in liver, brain, kidney and blood, and less 3H in liver and blood. In xanthan-treated mice, levels of both radiolabels were higher in liver, brain and kidney. Much lower glycogen content in muscle and depletion of liver glycogen occurred in morphine-treated mice, compared with control mice. Spinal transection completely inhibited the hypoglycemic effect of morphine, whereas adrenalectomy caused no inhibition. Morphine, insulin and xanthan appear to be acting by different mechanisms, although the hypoglycemic effects of both morphine and insulin appear to be due largely to an increased glucose uptake by muscle.