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

DE Selley, CS Breivogel and SR Childers

Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157.

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.

Volume 44, Issue 4, pp. 731-741, 10/01/1993
Copyright © 1993 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				I. Bileviciute-Ljungar, M. Spetea, Y. Guo, J. Schutz, P. Windisch, and H. Schmidhammer Peripherally Mediated Antinociception of the {micro}-Opioid Receptor Agonist 2-[(4,5{alpha}-Epoxy-3-hydroxy-14beta-methoxy-17-methylmorphinan-6beta-yl)amino]acetic Acid (HS-731) after Subcutaneous and Oral Administration in Rats with Carrageenan-Induced Hindpaw Inflammation J. Pharmacol. Exp. Ther., April 1, 2006; 317(1): 220 - 227. [Abstract] [Full Text] [PDF]

				G. T. Whiteside, J. M. Boulet, and K. Walker The Role of Central and Peripheral {micro} Opioid Receptors in Inflammatory Pain and Edema: A Study Using Morphine and DiPOA ([8-(3,3-Diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl]-acetic Acid) J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1234 - 1240. [Abstract] [Full Text] [PDF]

				K. J. Valenzano, W. Miller, Z. Chen, S. Shan, G. Crumley, S. F. Victory, E. Davies, J.-C. Huang, N. Allie, S. J. Nolan, et al. DiPOA ([8-(3,3-Diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]dec-3-yl]-acetic Acid), a Novel, Systemically Available, and Peripherally Restricted Mu Opioid Agonist with Antihyperalgesic Activity: I. In Vitro Pharmacological Characterization and Pharmacokinetic Properties J. Pharmacol. Exp. Ther., August 1, 2004; 310(2): 783 - 792. [Abstract] [Full Text] [PDF]

				G. T. Whiteside, J. E. Harrison, M. S. Pearson, Z. Chen, Y. Rotshteyn, P. I. Turchin, J. D. Pomonis, L. Mark, K. Walker, and K. C. Brogle DiPOA ([8-(3,3-Diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]dec-3-yl]-acetic Acid), a Novel, Systemically Available, and Peripherally Restricted Mu Opioid Agonist with Antihyperalgesic Activity: II. In Vivo Pharmacological Characterization in the Rat J. Pharmacol. Exp. Ther., August 1, 2004; 310(2): 793 - 799. [Abstract] [Full Text] [PDF]

				V. Zachariou, D. Georgescu, N. Sanchez, Z. Rahman, R. DiLeone, O. Berton, R. L. Neve, L. J. Sim-Selley, D. E. Selley, S. J. Gold, et al. Essential role for RGS9 in opiate action PNAS, November 11, 2003; 100(23): 13656 - 13661. [Abstract] [Full Text] [PDF]

				J. Sawynok Topical and Peripherally Acting Analgesics Pharmacol. Rev., March 1, 2003; 55(1): 1 - 20. [Abstract] [Full Text] [PDF]

				H. Machelska and C. Stein Immune Mechanisms in Pain Control Anesth. Analg., October 1, 2002; 95(4): 1002 - 1008. [Full Text] [PDF]

				L. Valle, O. Pol, and M. M. Puig Intestinal Inflammation Enhances the Inhibitory Effects of Opioids on Intestinal Permeability in Mice J. Pharmacol. Exp. Ther., April 13, 2001; 296(2): 378 - 387. [Abstract] [Full Text]

				M. M. Puig and O. Pol Peripheral Effects of Opioids in a Model of Chronic Intestinal Inflammation in Mice J. Pharmacol. Exp. Ther., December 1, 1998; 287(3): 1068 - 1075. [Abstract] [Full Text]

				L. Zhou, Q. Zhang, C. Stein, and M. Schäfer Contribution of Opioid Receptors on Primary Afferent Versus Sympathetic Neurons to Peripheral Opioid Analgesia J. Pharmacol. Exp. Ther., August 1, 1998; 286(2): 1000 - 1006. [Abstract] [Full Text]

				C. Stein The Control of Pain in Peripheral Tissue by Opioids N. Engl. J. Med., June 22, 1995; 332(25): 1685 - 1690. [Full Text] [PDF]