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Angiotensin II Regulation of the Na⁺ Pump Involves the Phosphatidylinositol-3 Kinase and p42/44 Mitogen-Activated Protein Kinase Signaling Pathways in Vascular Smooth Muscle Cells

Department of Internal Medicine (J.R.S.), University of Missouri-Columbia and the H. S. Truman Veterans Affairs Medical Center, Columbia, Missouri 65212; Departments of Cell Biology, Biochemistry, and Medicine (E.R.I., D.B.J., M.H.K., Q.S., N.M., G.G.), State University of New York-Health Science Center, Brooklyn, New York 11201; and Department of Biology (K.K.), Jichi Medical School, Tochigi, Japan

Address all correspondence and requests for reprints to: James R. Sowers, M.D., F.A.C.P., Professor of Medicine, Physiology, and Pharmacology, Thomas W. and Joan F. Burns Missouri Chair in Diabetology, Associate Dean for Clinical Research, Director of the M.U. Diabetes and Cardiovascular Center, Department of Internal Medicine, MA 410, Health Sciences Center, One Hospital Drive, DCO43.00, Columbia, Missouri 65212. E-mail: sowersj{at}health.missouri.edu.

This investigation used primary cultured rat vascular smooth muscle cells to examine angiotensin II (Ang II) regulation of Na⁺, K⁺-ATPase (Na⁺ pump) activity, and Na⁺ pump ₁- and ß₁-subunit gene transcription. This regulation was mediated through both phosphatidylinositol-3 kinase (PI3K) and p42/44 mitogen-activated protein kinase (p42/44^MAPK) signaling pathways. Both acute (10 min) and prolonged (24 h) treatment with Ang II stimulated Na⁺ pump activity. Also, prolonged exposure to Ang II (24 h) increased promoter transcription of the Na⁺ pump ₁- and ß₁-subunits. Furthermore, PI3K activities because well because p42/44^MAPK phosphorylation were increased within 10 min after Ang II treatment. To determine whether these stimulatory activities of Ang II are acting through Ang II receptors 1 and/or 2 (AT₁, AT₂), cells were pretreated with either AT₁ receptor blocker losartan or the AT₂ receptor blocker PD 123,319. Indeed, these treatments prevented the stimulatory effect of Ang II on Na⁺ pump activity at both acute and 24-h time points. Furthermore, the Ang II-stimulated ₁-subunit promoter transcription was inhibited by losartan but not by the AT₂ receptor blocker. These results indicate that Ang II acts through both the AT₁ and AT₂ receptor to up-regulate Na⁺ pump activity; however, Ang II regulates ₁-gene transcription through AT₁ but not AT₂ receptors. It was also observed that the Ang II-stimulated ß₁-subunit gene transcription is not mediated through either AT₁ or AT₂ receptors. To examine whether the Na⁺/H⁺ exchanger is involved in Ang II-stimulated Na⁺ pump activity, cells were pretreated with amiloride, a specific inhibitor of the Na⁺/H⁺ exchanger. This pretreatment prevented 24 h, but not acute, Ang II-stimulated Na⁺ pump activity. The 24-h Ang II-stimulated ₁-subunit promoter transcription was also inhibited by amiloride. This suggests that the prolonged effect of Ang II on Na⁺ pump activity is dependent on increased Na⁺/H⁺ exchange. Because Ang II treatment for 10 min increased PI3K activity because well because p42/44^MAPK phosphorylation, studies were performed to determine the involvement of PI3K and p42/44^MAPK signaling pathways in both Ang II-stimulated Na⁺ pump activity and ₁- and ß₁-gene transcription. Cells were pretreated with either the PI3K inhibitor wortmannin or the p42/44^MAPK inhibitor PD 98059. Ang II-stimulated PI3K or p42/44^MAPK activity was inhibited by these pretreatments. Furthermore, pretreatment of cells with the PI3K inhibitors wortmannin and LY29404 or the MAPK inhibitors U0126 and PD 98059 were all observed to inhibit Ang II-stimulated Na⁺ pump activity. To more specifically determine the role of PI3K in Ang II-regulation of ₁-and ß₁-gene transcription, cells were cotransfected with a dominant-negative p85 construct. Cotransfection with dominant-negative p85 reduced Ang II-stimulated ₁-but not ß₁-gene transcription in vascular smooth muscle cells. These results indicate that Ang II acts through PI3K/p42/44^MAPK signaling pathways to up-regulate Na⁺ pump activity and ₁-gene transcription and that Ang II-regulated ß₁-gene transcription is not mediated through either PI3K or p42/44 ^MAPK signaling pathways.

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