Differential coupling of opioid binding sites to guanosine triphosphate binding regulatory proteins in subcellular fractions of rat brain.
In this study, we present evidence for the occurrence of mu, delta, and kappa opioid binding sites in synaptic plasma membranes (SPM) and microsomes of rat brain. Binding to all three opioid classes was inhibited by 5'-guanylylimidodiphosphate (Gpp[NH]p) in SPM, while microsomal sites proved to be insensitive to this GTP analog. Sensitivity was restored upon solubilization of microsomes with digitonin, suggesting that opioid receptors are physically separated from G proteins in this fraction. Modulation of microsomal binding by Na+ and Mn++ was greater than that of SPM. Pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation revealed the presence of G proteins with alpha-subunit molecular weights of 40 kDa in both subcellular fractions. Basal low Km GTPase activity in SPM was greater than in microsomes. Etorphine elicited a concentration-dependent stimulation of guanosine triphosphatase (GTPase) activity in SPMs but not in microsomes, indicating functional coupling of opioid receptors to G protein in the former and an uncoupling in the latter. Microsomes from 3-day-old rat brain contained more mu opioid sites and they were more sensitive to Gpp(NH)p inhibition than those in adults. These results are consistent with the hypothesis that opioid binding sites in adult microsomes are internalized and G protein uncoupled, while those in neonates are newly synthesized, coupled receptors.
Szücs
M
M
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged.
Coscia
C J
CJ
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001923:Brain Chemistry / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D037342:Pertussis Toxin; D011485:Protein Binding; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism*; D015398:Signal Transduction; D012964:Sodium / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism*; D013570:Synaptic Membranes / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D011957:Receptors, Opioid; D010566:Virulence Factors, Bordetella; D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D012964:Sodium; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins