Modification of G protein-coupled functions by low-pH pretreatment of membranes from NG108-15 cells: increase in opioid agonist efficacy by decreased inactivation of G proteins.

Low-pH pretreatment increases opioid agonist efficacy in inhibiting adenylyl cyclase in brain membranes. The mechanism of this effect was examined in membranes from cultured NG108-15 cells. Pretreatment of NG108-15 membranes at pH 4.5 before assay at pH 7.4 produced the following modifications in G protein-mediated signal transduction: 1) decreased activation of adenylyl cyclase by Gs, 2) increased maximal inhibition of opioid agonist binding by sodium and by guanine nucleotides in the presence of sodium, and 3) increased maximal inhibition of adenylyl cyclase by agonists for G(i)-coupled receptors. These results are similar to those previously observed in rat brain membranes. The mechanism by which low-pH pretreatment increased receptor-mediated inhibition of adenylyl cyclase was investigated further by examining low-Km GTPase activity in low-pH-pretreated NG108-15 cell membranes. Low-pH pretreatment decreased basal and agonist-stimulated low-Km GTPase activity maximally in the absence of sodium and minimally in the presence of 120 mM NaCl. This change was due to a decrease in the Vmax of the enzyme, with no change in the Km for GTP, indicating that GTP hydrolysis was decreased without any decrease in the affinity of the G protein for GTP. Scatchard analysis revealed no decrease in the Bmax for high affinity opioid agonist binding, and Western blot analysis with a G(i)-specific antibody revealed no loss of G(i) protein, in low-pH-pretreated membranes. Moreover, concentration-effect curves for GTP in supporting opioid inhibition of adenylyl cyclase showed that low-pH pretreatment increased inhibition by the agonist only at GTP concentrations equal to or greater than the Km for GTP hydrolysis by the low-Km GTPase. Taken together, these results indicate that the efficacy of receptor-mediated inhibition of adenylyl cyclase can be increased by decreasing the maximal inactivation rate of G(i) subsequent to its activation by the receptor.