Chronic infusion of morphine to guinea pigs produced selective changes in Im agonist binding properties in cerebrocortical membrane preparations. Employing the pselective opioid agonist MePhe4, in direct binding studies and in competition of labeled antagonist binding, we found that the major changes were a decrease in the number of sites with high affnity for agonist, a small reduction in total receptor number, and a loss in the ability of guanosine 5'-[y-thio]triphosphate to regulate binding. A fraction of high-affinity p receptors appeared to retain their high affinity for agonist and their sensitivity to guanine nucleotide analogue after the induction of morphine tolerance, possibly because the morphine concentrations achieved in brain were insufficient to uncouple all p receptors from associated guanine nucleotide-binding regulatory proteins. Some membrane preparations were treated with pertussis toxin, which has been shown to functionally uncouple IL opioid receptors from their effector systems. In these preparations, a single agonist-affinity state of the receptor was observed. The apparent dissociation constant for this affinity state in pertussis toxin-treated membranes was similar to the lower-affinity state observed in preparations from morphinetolerant animals. In contrast to the changes observed at , opioid binding sites, no significant changes in agonist affinity or binding density were observed for selective 8 or K agonists, consistent with the development of selective tolerance at Ip receptors.The mechanisms by which opiate drugs induce tolerance and dependence are not clearly understood. A number of observations suggest that changes occur in the function of the receptors through which the tolerance-inducing drug acts to evoke its primary pharmacologic effect. A significant fraction of the receptors must be activated for tolerance and dependence to develop (1,2). In tissues where more than one type of opioid receptor is present, the occurrence of receptorselective tolerance (3-5) also suggests that receptor-specific mechanisms are critical in the tolerance process. Studies of the interaction of agonists with 8 opioid receptors in NG108-15 neuroblastoma-glioma hybrid cells in culture after chronic receptor activation have demonstrated that sustained agonist exposure leads initially to a loss of the ability of 8 agonists to regulate their effector system (in this case, to inhibit adenylate cyclase), followed later by a reduction in the number of 8 receptors detected by radiolabeled ligand binding (6). Recently, similar results have been reported in a study of the chronic activation by morphine of 1 opioid receptors in 7315c pituitary tumor cells maintained in primary culture (7). Thus, in these cell systems in which opioid receptors negatively regulate adenylate cyclase, sustained exposure to morphine resulted in an initial desensitization followed by receptor down-regulation.Early studies examining the binding of opioid receptors in brain membranes from small mammals following treatment wi...

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Opioid agonists selective for mu-, delta-, and kappa-receptors are all capable of regulating the stimulated release of noradrenaline from three terminal fields (cortex, hippocampus, and cerebellum) of the noradrenergic projections from locus coeruleus in the guinea pig brain. Intracerebroventricular injections of pertussis toxin abolished the ability of a mu-selective agonist and of a delta-selective agonist to inhibit stimulated noradrenaline release, but left unaffected the concentration-related inhibition of NE release by a kappa agonist. Thus, mu- and delta-receptors have been shown to be coupled to their effector system in these noradrenergic neurons via guanyl nucleotide binding proteins (G proteins) which are sensitive to pertussis toxin, while kappa-receptors in the same neurons appear to be coupled through a different mechanism which is significantly less sensitive to pertussis toxin. In contrast to opioid receptor regulation of noradrenaline release in guinea pig hippocampus, mu-, but not delta- or kappa-agonists are capable of regulation of stimulated noradrenaline release from rat hippocampus and cortex, and kappa-, but not mu- or delta-agonists are capable of inhibiting the stimulated release of dopamine from rat striatum and cortex. Pertussis toxin injections significantly attenuated mu-agonist inhibition of noradrenaline release, but had no effect on the ability of a kappa-selective agonist to regulated dopamine release, confirming the insensitivity of the kappa-receptor-effector coupling system to pertussis toxin.

An isolation procedure was developed for the extraction of genomic DNA and Random Amplified Polymorphic DNA (RAPD) analysis using individual soybean embryos. This procedure can be used to quickly and efficiently isolate DNA from a large number of individuals. DNA isolations were analyzed for total yield, integrity, and usefulness as a template in RAPD analysis.

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