Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes.
A high affinity Ca2+/Mg2+ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca2+ (less than or equal to microM) believed to approximate the range of Ca2+ in the synaptosomal cytosol (0.1 to 5.0 microM). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca2+-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca2+-stimulated ATP hydrolysis was characterized by a reduction in Vmax for Ca2+. Levorphanol pretreatment reduced the Hill coefficient (HN) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca2+ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca2+/Mg2+ ATPase in synaptic membranes. Levorphanol (10 microM), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca2+-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca2+-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35-40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction in Vmax for both Ca2+ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca2+-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007981:Levorphanol / Q000494:pharmacology; D008297:Male; D009270:Naloxone / Q000494:pharmacology; D009294:Narcotics / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology; D013570:Synaptic Membranes / Q000201:enzymology* / Q000648:ultrastructure
D002147:Calmodulin; D009294:Narcotics; D007981:Levorphanol; D009270:Naloxone; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium