Attenuation of enkephalin activity in neuroblastoma X glioma NG108-15 hybrid cells by phospholipases.
The role of membrane phospholipids in enkephalin receptor-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma X glioma NG108-15 hybrids was studied by selective hydrolysis of lipids with phospholipases. When NG108-15 cells were treated with phospholipase C from Clostridium welchii at 37 degrees C, an enzyme concentration--dependent decrease in adenylate cyclase activity was observed. The basal and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF-5'-guanylylimidodiphosphate (Gpp(NH)p)-sensitive adenylate cyclase activities. Further, Leu5-enkephalin inhibition of basal or PGE1-stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level. [3H]D-Ala2-Met5-enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment. Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p. Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N-component. Further, treatment of NG108-15 hybrid cell membrane with phospholipase C at 24 degrees C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding. The reduction in enkephalin potency could be reversed by treating NG108-15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate. The enkephalin activity in NG108-15 cells was not altered by treating the cells with phospholipase A2 o phospholipase C from Bacillus cereus. Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000700:Analgesics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives* / Q000378:metabolism; D005910:Glioma / Q000378:metabolism*; D006822:Hybrid Cells / Q000378:metabolism*; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D010740:Phospholipases / Q000494:pharmacology*; D010741:Phospholipases A / Q000494:pharmacology*; D054467:Phospholipases A2; D051381:Rats; D011957:Receptors, Opioid / Q000187:drug effects / Q000378:metabolism*; D010738:Type C Phospholipases / Q000494:pharmacology*
D000700:Analgesics; D008563:Membrane Lipids; D011957:Receptors, Opioid; D004744:Enkephalin, Methionine; C013801:enkephalin-Met, Ala(2)-; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D000262:Adenylyl Cyclases