Regulation of ANP-stimulated guanylate cyclase in the presence of Mn2+ in rat lung membranes

The catalytic activity of guanylate cyclase (GCase) coupled to atrial natri uretic peptide (ANP) receptor depends on the metal co-factor, Mn2+ or Mg2+. ATP synergistically stimulates the ANP-stimulated GCase in the presence of Mg-2+. We have now shown the ATP regulation of the ANP-stimulated GCase in the presence of Mn2+ in rat lung membranes. ANP stimulated the GCase 2.1-f old compared to the control. ATP enhanced both the basal (basal-GCase) and the ANP-stimulated GCase maximally 1.7- and 2.3- fold compared to the contr ol, respectively, at a concentration of 0.1 mM. The stimulation by ATP was smaller in the presence of Mn2+ than in the presence of Mg2+. The addition of inorganic phosphate to the reaction mixture altered the GCase activities in the presence of Mn2+ with or without ANP and/or ATP. In the presence of 10 mM phosphate, ATP dose-dependently stimulated the basal GCase 5-fold co mpared to the control at a concentration of 1 mM and augmented the ANP-stim ulated GCase, which was 4.2-fold compared to the basal-GCase, 5.5-fold comp ared to the control at a concentration of 0.5 mM. Protein phosphatase inhib itors, okadaic acid (100 nM), H8 (1 mu M) and staurosporin (1 mu M), did no t alter the activity. Orthovanadate (1 mM), an inorganic phosphate analogue , significantly stimulated both the basal-GCase and the ANP-stimulated GCas e, which were inhibited by ATP. It was assumed that phosphate and orthovana date might interact with the GCase to regulate the activity in the opposite manner. This was the first report that inorganic phosphate and orthovanada te affected the ATP-regulation of the ANP-stimulated GCase in the presence of Mn2+.