Atef ME, Anand-Srivastava MB. Enhanced expression of Gq␣ and PLC-␤1 proteins contributes to vascular smooth muscle cell hypertrophy in SHR: role of endogenous angiotensin II and endothelin-1. Am J Physiol Cell Physiol 307: C97-C106, 2014. First published April 24, 2014 doi:10.1152 doi:10. /ajpcell.00337.2013 Gq␣ signaling has been shown to contribute to cardiac hypertrophy. In addition, angiotensin II (ANG II) was shown to induce vascular smooth muscle cell (VSMC) hypertrophy through Gq␣ signaling; however, the studies on the role of Gq␣ and PLC-␤1 proteins in VSMC hypertrophy in animal model are lacking. The present study was therefore undertaken to examine the role of Gq␣/PLC-␤1 proteins and the signaling pathways in VSMC hypertrophy using spontaneously hypertensive rats (SHR). VSMC from 16-wk-old SHR and not from 12-wk-old SHR exhibited enhanced levels of Gq␣/PLC-␤1 proteins compared with age-matched Wistar-Kyoto (WKY) rats as determined by Western blotting. However, protein synthesis as determined by [ 3 H]leucine incorporation was significantly enhanced in VSMC from both 12-and 16-wk-old SHR compared with VSMC from age-matched WKY rats. Furthermore, the knockdown of Gq␣/ PLC-␤1 in VSMC from 16-wk-old SHR by antisense and small interfering RNA resulted in attenuation of protein synthesis. In addition, the enhanced expression of Gq␣/PLC-␤1 proteins, enhanced phosphorylation of ERK1/2, and enhanced protein synthesis in VSMC from SHR were attenuated by the ANG II AT1 and endothelin-1 (ET-1) ETA receptor antagonists losartan and BQ123, respectively, but not by the ETB receptor antagonist BQ788. In addition, PD98059 decreased the enhanced expression of Gq␣/PLC-␤1 and protein synthesis in VSMC from SHR. These results suggest that the enhanced levels of endogenous ANG II and ET-1 through the activation of AT1 and ETA receptors, respectively, and MAP kinase signaling, enhanced the expression of Gq␣/PLC-␤1 proteins in VSMC from 16-wk-old SHR and result in VSMC hypertrophy.Gq␣ protein; PLC-␤1 protein; SHR; VSMC; hypertrophy THE HETEROTRIMERIC GUANINE nucleotide regulatory protein (G protein), composed of three subunits (␣, ␤, and ␥), plays a crucial role in the regulation of cardiovascular functions through the activation of several signal transduction systems including adenylyl cyclase and phosphatidyl inositide system (39). Based on ␣-subunit sequence similarity, G␣ proteins are divided into four families G s , G i/o , G q/11 , and G 12/13 . G s and G i proteins regulate the activity of adenylyl cyclase whereas the activation of G q ␣ by a G protein-coupled receptor (GPCR) stimulates phospholipase C-␤ (PLC-␤), which hydrolyzes inositol biphosphate (PIP2) and produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] (IP 3 ) and diacylglycerol (DAG) (23) and activates protein kinase C (PKC) (7,43).Alterations in the levels of G q ␣ protein and associated signaling pathways appear to contribute to the impaired cellular functions in several pathological states including diabetes, hyperglycemia, and cardiac hypertrophy (1, 12...

We showed previously that vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) exhibit overexpression of Gqα/PLCβ1 proteins, which contribute to increased protein synthesis through the activation of MAP kinase signaling. Because oxidative stress has been shown to be increased in hypertension, the present study was undertaken to examine the role of oxidative stress and underlying mechanisms in enhanced expression of Gqα/PLCβ1 proteins and VSMC hypertrophy. Protein expression was determined by Western blotting, whereas protein synthesis and cell volume, markers for VSMC hypertrophy, were determined by [(3)H]-leucine incorporation and three-dimensional confocal imaging, respectively. The increased expression of Gqα/PLCβ1 proteins, increased protein synthesis, and augmented cell volume exhibited by VSMCs from SHRs were significantly attenuated by antioxidants N-acetyl-cysteine (NAC), a scavenger of superoxide anion, DPI, an inhibitor of NAD(P)H oxidase. In addition, PP2, AG1024, AG1478, and AG1295, inhibitors of c-Src, insulin-like growth factor receptor (IGFR), epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor (PDGFR), respectively, also attenuated the enhanced expression of Gqα/PLCβ1 proteins and enhanced protein synthesis in VSMCs from SHRs toward control levels. Furthermore, the levels of IGF-1R and EGFR proteins and not of PDGFR were also enhanced in VSMCs from SHRs, which were attenuated significantly by NAC, DPI, and PP2. In addition, NAC, DPI, and PP2 also attenuated the enhanced phosphorylation of IGF-1R, PDGFR, EGFR, c-Src, and EKR1/2 in VSMCs from SHRs. These data suggest that enhanced oxidative stress in VSMCs from SHRs activates c-Src, which through the transactivation of growth factor receptors and MAPK signaling contributes to enhanced expression of Gqα/PLCβ1 proteins and resultant VSMC hypertrophy.

The discovery of endothelin (ET) in 1988 has led to considerable effort to unravel its implication in health and disease and the mechanisms evoked by ET. ET-1 and related signaling aberrancies are believed to be implicated in the pathogenesis of diverse cardiovascular diseases, such as hypertension, atherosclerosis, hypertrophy and diabetes. The endothelin system consists of three potent vasoconstrictive isopeptides, ET-1, ET-2 and ET-3, signaling through two G protein coupled receptors, ETA and ETB, which are linked to multiple signaling pathways. Activated signaling transduction pathways include the modulation of the adenylyl cyclase/cAMP pathway through stimulatory (Gs) and inhibitory (Gi) G proteins, activation of the phosphoinositide pathway through the activation of proteins Gq/11, generation of oxidative stress, growth factor receptor-related mitogenic events, such as the activation of phosphatidylinositol-3 kinase pathway, phosphoinositide pathway and activation of the mitogen-activated protein (MAP) kinase cascade. The levels of ETA and ETB receptors as well as the signaling pathways activated by these receptors are altered in several cardiovascular diseases including hypertension, hypertrophy, atherosclerosis, diabetes, etc. In this review, we provide an overview of the signaling events modulated by ET-1 in vascular smooth muscle cells in both physiological and pathological conditions.

Gqα signaling has been implicated in cardiac hypertrophy. In addition, angiotensin II (Ang II) was also shown to induce its hypertrophic effect through Gqα and PKCδ activation. We recently showed the role of enhanced expression of Gqα/PLCβ1 proteins in vascular smooth muscle cell (VSMC) hypertrophy, however, the role of PKCδ in VSMC hypertrophy in animal model is still lacking. The present study was therefore undertaken to examine the role of PKCδ and the associated signaling mechanisms in VSMC hypertrophy using 16-week-old spontaneously hypertensive rats (SHR). VSMC from 16-week-old SHR exhibited enhanced phosphorylation of PKCδ-Tyr311 and increased protein synthesis, marker of hypertrophy, as compared to WKY rats which was attenuated by rottlerin, an inhibitor of PKCδ. In addition, knocking down of PKCδ by PKCδ-siRNA also attenuated enhanced protein synthesis in VSMC from SHR. Furthermore, rottlerin attenuated the increased production of superoxide anion, NAD(P)H oxidase activity, increased expression of Gqα, phospholipase C (PLC)β1, insulin like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) proteins in VSMC from SHR. In addition, the enhanced phosphorylation of c-Src, PKCδ-Tyr311, IGF-1R, EGFR and ERK1/2 exhibited by VSMC from SHR was also attenuated by rottlerin. These results suggest that VSMC from SHR exhibit enhanced activity of PKCδ and that PKCδ is the upstream molecule of reactive oxygen species (ROS) and contributes to the enhanced expression of Gqα and PLCβ1 proteins and resultant VSMC hypertrophy involving c-Src, growth factor receptor transactivation and MAP kinase signaling.