Inhibition of protein phosphorylation by opioid-inhibited adenylyl cyclase in rat brain membranes.
Opioid inhibition of adenylyl cyclase is a major second messenger system associated with opioid receptors in brain. To identify membrane phosphoproteins whose phosphorylation state is modulated by opioid inhibition of adenylyl cyclase, rat striatal membranes were preincubated with opioid agonists in the presence of 500 microM 5'-adenylyl-imidodiphosphate (which acted as a substrate for adenylyl cyclase, but not for protein kinase) before addition of [gamma-32P]ATP. Under these conditions, adenylyl cyclase in the membranes formed cyclic AMP, which stimulated cyclic AMP-dependent protein kinase. This process was confirmed by observing forskolin-stimulated phosphorylation of two bands of MW 85 and 63 kDa, which were also stimulated directly by cyclic AMP. Forskolin-stimulated phosphorylation of these two bands was inhibited by 15 to 30% by opioid agonists such as D-Ala2-Met5-enkephalinamide. This inhibition of phosphorylation was mediated by opioid receptors, because it required both sodium and GTP, and was blocked by naloxone. These results suggest that these two proteins may be primary targets of opioid-inhibited adenylyl cyclase in striatal membranes.
Fleming
L M
LM
Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina.
Ponjee
G
G
Childers
S R
SR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000502:physiology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism*; D000242:Cyclic AMP / Q000096:biosynthesis; D004723:Endorphins / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008297:Male; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D051381:Rats
D000067956:Adenylyl Cyclase Inhibitors; D004723:Endorphins; D011506:Proteins; D000266:Adenylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases