Catalytic Activation of Guanylate Cyclase Atrial/Natriuretic Factor Receptor by Combined Effects of ANF and GTPγS in Plasma Membranes of Leydig Tumor Cells: Involvement of G-Proteins

Guanosine 5′-(γ-thio) triphosphate (GTPγS) 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 GTPγS 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′(γ-thio)triphosphate, adenosine 5′-(β,γ-imino)triphosphate, GDP, and guanosine 5′-(2-O-thiodiphosphate) (100 μM 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, AlF−4 , and benzalkonium chloride. The saturation binding assay of [35S]GTPγS showed the dissociation constant (Kd) of 2.3 × 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]GTPγS-binding capacity, however, without affecting its affinity constant. Pretreatment of plasma membranes with antibodies against Gsα subunit attenuated the GTPγS-stimulated GC activity, whereas antibodies against Giα subunit enhanced the stimulatory effect of GTPγS on GC catalytic activity of GC-A/ANF-R. However, the antibodies against Goα 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.