Brain Research, 493 (1989) 23-32 23 Elsevier BRE 14656 Guanine nucleotide regulation of [125I]fl-endorphin binding to NG108-15 and SK-N-SH cell membranes: specific cation requirements Dana E. Selley and Jean M. Bidlack Department of Pharmacology, The University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642 (U.S.A.) (Accepted 27 December 1988) Key words: Guanosine triphosphate; fl-Endorphin; Neuroblastoma cell; Opioid receptor, p-; Opioid receptor, 6-; Opioid peptide Regulation of [~:5I]flh-endorphin binding by guanine nucleotides was investigated in membrane preparations from two opioid receptor-containing cell lines: NG108-15, which contains only b opioid receptors, and SK-N-SH, which contains predominantly # opioid receptors. In contrast to the binding of the b-selective agonist [3H][D-penicillamine2,D-penicillamineS]enkephalin to NG108-15 cell membranes, and of the p-selective agonist [3H][D-Ala2,MePhe4,Gly-olS]enkephalin to SK-N-SH cell membranes, [125I]fl h- endorphin binding to NG108-15 and SK-N-SH cell membranes was not altered by guanosine triphosphate (GTP) or guanylyl-5"-imidodiphosphate (Gpp(NH)p) in the absence of cations. However, in the presence of NaCI, [125I]flh-endorphinbinding to both cell lines was inhibited by GTP and Gpp(NH)p in a concentration-dependent manner. In SK-N-SH cell membranes, the ability of sodium to promote regulation of [125I]flh-endorphinbinding by GTP was mimicked by the monovalent cations lithium and potassium, but not by the divalent cations magnesium, calcium, or mangenese. In NG108-15 cell membranes, only sodium was effective in promoting inhibition of [125I]flh-endorphinbinding by GTP. The effect of GTP or Gpp(NH)p in the presence of sodium was also observed with guanosine diphosphate, but not guanosine monophosphate or any of the non-guanine nucleotides tested. These results indicate that the presence of monovalent cations is required for regulation of [~25I]flh-endorphinbinding by guanine nucleotides, and that the specificity of this cation requirement differs between the p and b receptor-containing cell lines. INTRODUCTION Current evidence suggests the involvement of guanosine triphosphate (GTP)-binding regulatory proteins, termed N or G proteins, in the coupling of p and 6 opioid receptors to a variety of second messenger/effector systems, including inhibition of adenylate cyclase activity ~'23"32,stimulation of guanyl- ate cyclase activity 24, inhibition of calcium conduc- tance ~6, and stimulation of potassium conductance 28. The inhibitory effect of guanine nucleotides upon opioid agonist b i n d i n g 4"5'9-12'3°'35"36 is thought to be a result of this interaction of opioid receptors with the Ni protein ~s'32'33. Although the endogenous opioid peptide fl-endorphin has been shown to bind to B and 6 opioid receptors 22 and to inhibit adenylate cyclase activity 3A9'25'26, we have shown that human [125I-Tyr27]fl-endorphin ([125I]flh-endorphin) binding to rat brain membranes was not inhibited by guanine nucleotides in the absence of cations 31. However, in the presence of monovalent cations at concentra- tions sufficient to inhibit [12sI]flh-endorphin binding by themselves, [125I]flh-endorphin binding was sup- pressed by GTP and guanylyl-5"-imidodiphosphate (Gpp(NH)p) in a concentration-depenent manner. The order of potency of the monovalent cations in promoting inhibition of [125I]flh-endorphin binding by GTP was: sodium > lithium > potassium. The divalent cations, magnesium, calcium, and mange- nese were not effective in promoting inhibition of [125I]flh-endorphin binding by GTP, even though the divalent cations alone were potent inhibitors of binding. In this report, we investigated the regulation by Correspondence: J.M. Bidlack, Department of Pharmacology, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642, U.S.A. 0006-8993/89/$03.50 ~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)