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Modulation of Guanosine Triphosphatase Activity of G Proteins by Arachidonic Acid in Rat Leydig Cell Membranes¹

Unidad de Neuroendocrinología Molecular, Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, E-28871 Alcalá de Henares-Madrid, Spain

Address all correspondence and requests for reprints to: M. Pilar López-Ruiz, Unidad de Neuroendocrinología Molecular, Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, E-28871 Alcalá de Henares-Madrid, Spain. E-mail: bqplr{at}bioqui.alcala.es

Previous results from our group have indicated that arachidonic acid decrease 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 high-affinity GTPase activity, measured as [³²P] GTP hydrolysis rate, was both time and protein concentration dependent. Arachidonic acid elicited a dose-dependent inhibition of enzyme activity with an IC₅₀ = 26.7 ± 1.1 µM. The existence of only two double bonds in linoleic acid is reflected by a decrease in its inhibitory activity (IC₅₀ = 34 ± 2.3 µM). Saturated fatty acids showed no effect at this level. The kinetic analysis as interpreted by Lineweaver-Burk plots, indicated that 50 µM arachidonic acid had no effect on the apparent affinity for GTP, but resulted in a 40% decreases in the maximal velocity of the reaction. Arachidonic acid modulation of GTPase activity was not attenuated by blocking eicosanoid metabolism with inhibitors of 5'-lipoxygenase, cyclooxygenase, or epoxygenase P-450. The addition of arachidonic acid to pertussis toxin-treated membranes had no effect on the enzyme activity, indicating that arachidonic acid does not modify the GTPase activity present in G_s protein. However, ADP-ribosylation with cholera toxin followed by arachidonic acid treatment led to a further 40% inhibition when compared with cholera toxin treatment alone. These results allowed us to postulate that arachidonic acid inhibits the GTPase activity of G_i protein family. To further analyze the mechanism of arachidonic acid inhibition of GTPase activity, the effect of arachidonic acid on the [³⁵S]GTPS binding was studied. No effect of this fatty acid on GTP binding was found. Combining our previous results with those found here, we can conclude that arachidonic acid maintains G_i proteins in their active state, which in turn inhibit adenylate cyclase and results in decrease cAMP levels.