Effects of NaCl concentration on adenylate cyclase regulation by prostaglandins and guanine nucleotides.

Abstract

Na+ has been implicated as a requirement for the inhibition of adenylate cyclase by hormones and neurotransmitters. This study examines effects of salt concentration on neuroblastoma plasma membranes that occur in the absence of an inhibitory hormone. The adenylate cyclase response to stimulatory agonists (GTP plus PGE1 (3), PGI2 or PGE2) was influenced by NaCl. As the [NaCl] increased to 150 mM, an increase in maximal activity and a decrease in apparent affinity was observed. At concentrations above 150 mM, NaCl decreased prostaglandin affinity and progressively decreased maximal activation. The GTP requirement was not altered by 30 or 150 mM NaCl in the presence of PGE1 or PGI2. The rate of Gpp(NH)p stimulated activity increased as the [NaCl] was increased in the assay. This increased rate was conserved when membranes activated in the presence of Gpp(NH)p and NaCl were reassayed in the absence of guanine nucleotide or salt. The salt evoked rate increase was proportionally greater at submaximal MgCl2 concentrations. The concentration requirement for Mg2+ was reduced by salt for adenylate cyclase in the presence of GTP or Gpp(NH)p. However, the enzyme stimulated by hormone exhibited a Mg2+ requirement that was low in the absence of salt and could not be further reduced by increased [NaCl]. Alternative monovalent cations (150 mM Li+, K+, Cs+, but not choline or tetramethylammonium) and anions (SO4=) substituted for NaCl. The observed effects were reversible upon washing the membranes and neither ouabain nor tetrodotoxin altered the response. These effects may result from a conformational alteration of a protein particularly sensitive to neutral salts in the assay.