RACK1 regulates angiotensin II-induced contractions of SHR preglomerular vascular smooth muscle cells.

The preglomerular microcirculation of spontaneously hypertensive rats (SHR) is hypersensitive to angiotensin (ANG) II, and studies have shown that this is likely due to enhanced coincident signaling between G protein subunits α_q (Gα_q; released by ANG II) and βγ (Gβγ; released by G_i-coupled receptors) to active phospholipase C (PLC). Here we investigated the molecular basis for the enhanced coincident signaling between Gβγ and Gα_q in SHR preglomerular vascular smooth muscle cells (PGVSMCs). Because receptor for activated C kinase 1 (RACK1; a scaffolding protein) organizes interactions between Gβγ, Gα_q, and PLC, we included RACK1 in this investigation. Cell fractionation studies demonstrated increased levels of membrane (but not cytosolic) Gβ, Gα_q, PLCβ₃, and RACK1 in SHR PGVSMCs compared with Wistar-Kyoto rat PGVSMCs. In SHR PGVSMCs, coimmunoprecipitation demonstrated RACK1 binding to Gβ and PLCβ₃, but only at cell membranes. Pertussis toxin (which blocks Gβγ) and U73122 (which blocks PLC) reduced membrane RACK1; however, RACK1 knockdown (shRNA) did not affect membrane levels of Gβ, Gα_q, or PLCβ₃ In a novel gel contraction assay, RACK1 knockdown in SHR PGVSMCs attenuated contractions to ANG II and abrogated the ability of neuropeptide Y (which signals via Gβγ) to enhance ANG II-induced contractions. We conclude that in SHR PGVSMCs the enlarged pool of Gβγ and PLCβ₃ recruits RACK1 to membranes and RACK1 then organizes signaling. Consequently, knockdown of RACK1 prevents coincident signaling between ANG II and the G_i pathway. This is the first study to implicate RACK1 in vascular smooth muscle cell contraction and suggests that RACK1 inhibitors could be effective cardiovascular drugs.

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