Opioid-inhibited adenylyl cyclase in rat brain membranes: lack of correlation with high-affinity opioid receptor binding sites.
Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.
Nijssen
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Department of Physiology and Pharmacology, Bowman Grey School of Medicine, Wake Forest University, Winston-Salem, NC 27103.
Sexton
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Childers
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D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D005576:Colforsin / Q000494:pharmacology; D004723:Endorphins / Q000378:metabolism / Q000494:pharmacology*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D004745:Enkephalins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008297:Male; D009270:Naloxone / Q000031:analogs & derivatives / Q000494:pharmacology; D010741:Phospholipases A / Q000494:pharmacology; D054467:Phospholipases A2; D051381:Rats; D017207:Rats, Sprague-Dawley; D011957:Receptors, Opioid / Q000032:analysis / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology
D000067956:Adenylyl Cyclase Inhibitors; D004723:Endorphins; D004745:Enkephalins; D011957:Receptors, Opioid; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D005576:Colforsin; D009270:Naloxone; D012965:Sodium Chloride; D004743:Enkephalin, Leucine; C034318:enkephalin, Ser(2), Leu(5), Thr(6)-; C034643:naloxonazine; D006160:Guanosine Triphosphate; D012969:Sodium Fluoride; D012964:Sodium; D010741:Phospholipases A; D054467:Phospholipases A2; D000262:Adenylyl Cyclases