Modulation of guanosine triphosphatase activity of G proteins by arachidonic acid in rat Leydig cell membranes

Previous results from our group have indicated that arachidonic acid decrea se cAMP production through a modification of heterotrimeric G proteins. In the present study, we have characterized the high affinity GTPase activity present in Leydig cell membranes and its regulation by fatty acids. The hig h-affinity GTPase activity, measured as [gamma(32)P] GTP hydrolysis rate, w as both time and protein concentration dependent. Arachidonic acid elicited a dose-dependent inhibition of enzyme activity with an IC50 = 26.7 +/- 1.1 mu M. The existence of only two double bonds in Linoleic acid is reflected by a decrease in its inhibitory activity(IC50 = 34 +/- 2.3 mu M) Saturated fatty acids showed no effect at this level. The kinetic analysis as interp reted by Lineweaver-Burk plots, indicated that 50 mu M arachidonic acid had no effect on the apparent affinity for GTP, but resulted in a 40% decrease s in the maximal velocity of the reaction. Arachidonic acid modulation of G TPase activity was not attenuated by blocking eicosanoid metabolism with in hibitors of B'-lipoxygenase, cyclooxygenase, or epoxygenase P-450. The addi tion of arachidonic acid to per tussis toxin-treated membranes had no effec t on the enzyme activity, indicating that arachidonic acid does not modify the GTPase activity present in G(alpha s) protein. However, ADP-ribosylatio n with cholera toxin followed by arachidonic acid treatment led to a furthe r 40% inhibition when compared with cholera toxin treatment alone. These re sults allowed us to postulate that arachidonic acid inhibits the GTPase act ivity of G(i) protein family. To further analyze the mechanism of arachidon ic acid inhibition of GTPase activity, the effect of arachidonic acid on th e [S-35]GTP gamma S binding was studied. No effect of this fatty acid on GT P binding was found. Combining our previous results with those found here, we can conclude that arachidonic acid maintains G(i) proteins in their acti ve state, which in turn inhibit adenylate cyclase and results in decrease c AMP levels.