Catalytic activation of guanylate cyclase/atrial natriuretic factor receptor by combined effects of ANF and GTP gamma S in plasma membranes of Leydig tumor cells: involvement of G-proteins

Abstract

Guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) exhibited a modulatory role in the catalytic activation of guanylate cyclase-A/atrial natriuretic factor receptor (GC-A/ANF-R) in the plasma membrane preparations of murine Leydig tumor (MA-10) cells. Both atrial natriuretic factor (ANF) and GTP gamma S synergistically stimulated the guanylate cyclase (GC) activity of GC-A/ANF-R in a dose- and time-related manner. Other nucleotides and their analogs such as ATP, adenosine 5'-(gamma-thio)triphosphate, adenosine 5'-(beta,gamma-imino)triphosphate, GDP, and guanosine 5'-(2-O-thiodiphosphate) (100 microM each) did not show any discernible effect on GC catalytic activity of GC-A/ANF-R. A significant stimulation of GC activity was observed in the presence of mastoparan, AlF4-, and benzalkonium chloride. The saturation binding assay of [35S]GTP gamma S showed the dissociation constant (Kd) of 2.3 x 10(-9) M and the binding capacity (Bmax) of 76 pmol/mg protein in the plasma membrane preparations of MA-10 cells. ANF increased the [35S]GTP gamma S-binding capacity, however, without affecting its affinity constant. Pretreatment of plasma membranes with antibodies against Gs alpha subunit attenuates the GTP gamma S-stimulated GC activity, whereas antibodies against Gi alpha subunit enhanced the stimulatory effect of GTP gamma S on GC catalytic activity of GC-A/ANF-R. However, the antibodies against Go alpha subunit did not show any effect on GC activity. These results provide the evidence that both Gs and Gi subunits of G-proteins seem to be involved in the regulation of GC catalytic activity of GC-A/ANF-R in the plasma membranes of MA-10 cells.