Involvement of the G_i/o/cGMP/PKG pathway in the AT₂-mediated inhibition of outer cortex proximal tubule Na⁺-ATPase by Ang-(1–7)

The molecular mechanisms involved in the Ang-(1–7) [angiotensin-(1–7)] effect on sodium renal excretion remain to be determined. In a previous study, we showed that Ang-(1–7) has a biphasic effect on the proximal tubule Na⁺-ATPase activity, with the stimulatory effect mediated by the AT₁ receptor. In the present study, we investigated the molecular mechanisms involved in the inhibition of the Na⁺-ATPase by Ang-(1–7). All experiments were carried out in the presence of 0.1 nM losartan to block the AT₁ receptor-mediated stimulation. In this condition, Ang-(1–7) at 0.1 nM inhibited the Na⁺-ATPase activity of the proximal tubule by 54%. This effect was reversed by 10 nM PD123319, a specific antagonist of the AT₂ receptor, and by 1 μM GDP[β-S] (guanosine 5′-[β-thio]diphosphate), an inhibitor of G protein. Ang-(1–7) at 0.1 M induced [³⁵S]GTP[S] (guanosine 5′-[γ-[³⁵S]thio]triphosphate) binding and 1 μg/ml pertussis toxin, an inhibitor of G_i/o protein, reversed the Ang-(1–7) effect. Furthermore, it was observed that the inhibitory effect of Ang-(1–7) on the Na⁺-ATPase activity was completely reversed by 0.1 μM LY83583, an inhibitor of guanylate cyclase, and by 2 μM KT5823, a PKG (protein kinase G) inhibitor, and was mimicked by 10 nM d-cGMP (dibutyryl cGMP). Ang-(1–7) increased the PKG activity by 152% and this effect was abolished by 10 nM PD123319 and 0.1 μM LY83583. Taken together, these data indicate that Ang-(1–7) inhibits the proximal tubule Na⁺-ATPase by interaction with the AT₂ receptor that subsequently activates the G_i/o protein/cGMP/PKG pathway.