chlornaltrexamine and Morphine-Dependence

The mu opioid receptor, MOR, displays spontaneous agonist-independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPgammaS binding to cell membranes and cAMP levels in intact cells. beta-CNA, C-CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6alpha- and 6beta-naltrexol and -naloxol, and 6beta-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6beta-naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone-induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine-dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.

Topics: Analgesics, Opioid; Animals; Benzylidene Compounds; Cell Line; Cell Membrane; Cyclic AMP; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Kidney; Magnesium; Male; Mice; Mice, Inbred ICR; Morphine; Morphine Dependence; Naloxone; Naltrexone; Narcotic Antagonists; Nociceptors; Receptors, Opioid, mu; Signal Transduction; Substance Withdrawal Syndrome; Sulfur Radioisotopes; Transfection; Tritium

We have measured the opioid receptor reserve in the guinea pig ileum myenteric plexus by means of the site-directed alkylating agent, beta-chlornaltrexamine. Treatment of the tissue with low (less than 10 nM) concentrations of beta-chlornaltrexamine caused a parallel shift of the log concentration-response curves for both normorphine and dynorphin A-(1-13). Analysis of the resulting curves indicated that the Kd values were 1.5 +/- 0.5 X 10(-6) and 10 +/- 4 X 10(-9), respectively. Using the naloxone Ke to distinguish between the mu and kappa receptors in this tissue, we found that the receptor selectivities of normorphine and dynorphin A-(1-13) were unchanged after a maximum parallel shift, thus demonstrating that there are both spare mu and spare kappa receptors present. The spare-receptor fraction for both receptor types was about 90%. In morphine-tolerant preparations (chronic pellet implantation), there was an apparent reduction in the fraction of spare mu receptors without any change in the apparent affinity of normorphine. Reduction in the spare receptor fraction does not necessarily imply reduction in the number of binding sites. We suggest that this reduction in receptor reserve is the basis of opioid tolerance, since the agonist concentration needed to produce a given effect is expected to increase as the receptor reserve decreases.

Topics: Animals; Guinea Pigs; Humans; Male; Morphine Dependence; Muscle Contraction; Muscle, Smooth; Myenteric Plexus; Naltrexone; Receptors, Opioid

Topics: Animals; Female; Guinea Pigs; Humans; Ileum; In Vitro Techniques; Male; Morphine Dependence; Morphine Derivatives; Muscle Contraction; Muscle, Smooth; Naloxone; Naltrexone; Narcotic Antagonists

We observed that the spinal cord of rats was involved in development of tolerance and dependence to morphine that was administered systemically by s.c. morphine-pellet implantation. Rats, surgically fitted with intrathecal catheters, were injected intrathecally (i.t.) with saline or 2.4 nmol beta-chlornaltrexamine (beta-CNA), an irreversible opiate antagonist. Twenty-four h later, animals were implanted s.c. with either placebo or morphine pellets. Seventy-two h after implanting pellets, development of tolerance or dependence was assessed. Control animals implanted with morphine pellets became tolerant to analgesia induced by i.p. injections of morphine as determined by the use of tail flick and hot plate analgesic assays. beta-CNA pretreatment antagonized the effects of i.p. injections of morphine and blocked development of tolerance in morphine-implanted animals. Dependence was assessed by observing several characteristic signs of precipitated withdrawal. Treatment with beta-CNA before morphine treatment antagonized naloxone-induced expression of withdrawal for all signs observed, except weight loss. We conclude that the spinal cord plays a significant role in development of tolerance and dependence induced by systemically administered opiates.

Topics: Animals; Drug Tolerance; Humans; Male; Morphine; Morphine Dependence; Naloxone; Naltrexone; Rats; Rats, Inbred Strains; Reaction Time; Spinal Cord

Chlornaltrexamine (CNA) produces ultralong-lasting (3--6 days) narcotic antagonism in mice and persistent stereospecific binding to rat-brain homogenate. Protection studies in mice suggest that CNA mediates its narcotic antagonist effects by interacting with the same receptors that are occupied by naloxone. A single icv dose of CNA also has been found to inhibit the development of physical dependence in mice for at least 3 days. These studies suggest that CNA exerts its sustained effects by selective covalent association with opioid receptors.

Topics: Alkylating Agents; Analgesics; Animals; Humans; In Vitro Techniques; Male; Mice; Molecular Conformation; Morphine; Morphine Dependence; Naloxone; Naltrexone; Narcotic Antagonists; Nitrogen Mustard Compounds; Rats; Receptors, Opioid; Time Factors