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

2017 ◽  
Vol 312 (4) ◽  
pp. F565-F576 ◽  
Author(s):  
Xiao Zhu ◽  
Edwin K. Jackson
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Scaffolding Protein ◽  
Cell Fractionation ◽  
Ang Ii ◽  
Spontaneously Hypertensive ◽  
Wistar Kyoto

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 αq (Gαq; released by ANG II) and βγ (Gβγ; released by Gi-coupled receptors) to active phospholipase C (PLC). Here we investigated the molecular basis for the enhanced coincident signaling between Gβγ and Gαq in SHR preglomerular vascular smooth muscle cells (PGVSMCs). Because receptor for activated C kinase 1 (RACK1; a scaffolding protein) organizes interactions between Gβγ, Gαq, and PLC, we included RACK1 in this investigation. Cell fractionation studies demonstrated increased levels of membrane (but not cytosolic) Gβ, Gαq, 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, Gαq, 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 Gi 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.

SAM68: a downstream target of angiotensin II signaling in vascular smooth muscle cells in genetic hypertension

2004 ◽  
Vol 286 (5) ◽  
pp. H1954-H1962 ◽  
Author(s):  
Mohammed El Mabrouk ◽  
Quy N. Diep ◽  
Karim Benkirane ◽  
Rhian M. Touyz ◽  
Ernesto L. Schiffrin
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Kinase Activity ◽  
Muscle Cells ◽  
Regulatory Subunit ◽  
Ang Ii ◽  
Wistar Kyoto

We investigated whether phosphatidylinositol 3-kinase (PI3K) and 68-kDa Src associated during mitosis (SAM68) are involved in angiotensin II (ANG II) growth signaling in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). PI3K activity was assessed by measuring the phosphorylation of the regulatory subunit p85α and kinase activity of the catalytic 110-kDa subunit of PI3K. The PI3K-SAM68 interaction was assessed by coimmunoprecipitation, and SAM68 activity was evaluated by poly(U) binding. SAM68 expression was manipulated by SAM68 antisense oligonucleotide transfection. VSMC growth was evaluated by measuring [3H]leucine and [3H]thymidine incorporation as indexes of protein and DNA synthesis, respectively. ANG II increased the phosphorylation of p85α and kinase activity of the 110-kDa PI3K subunit in VSMCs from SHR and transiently increased p85α-SAM68 association. In Wistar-Kyoto (WKY) rat cells, ANG II increased SAM68 phosphorylation without influencing poly(U) binding. In SHR, ANG II did not influence SAM68 phosphorylation but increased SAM68 binding to poly(U). ANG II stimulated phosphoinositol phosphate synthesis by PI3K in SAM68 immunoprecipitates in both groups, with significantly enhanced effects in SHR. Inhibition of PI3K, using the selective inhibitor LY-294002, and downregulation of SAM68, by antisense oligonucleotides, significantly decreased ANG II-stimulated incorporation of [3H]leucine and [3H]thymidine in VSMCs, showing the functional significance of PI3K and SAM68. Our data demonstrate that PI3K and SAM68 are involved in ANG II signaling and that SAM68 is differentially regulated in VSMCs from SHR. These processes may contribute to the enhanced ANG II signaling and altered VSMC growth in SHR.

Role of Na(+)-Ca2+ exchange in agonist-induced changes in cytosolic Ca2+ in vascular smooth muscle cells

1994 ◽  
Vol 266 (3) ◽  
pp. C794-C799 ◽  
Author(s):  
Z. Zhu ◽  
M. Tepel ◽  
M. Neusser ◽  
W. Zidek
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Free Calcium ◽  
Ang Ii ◽  
Protein Kinase C Activity ◽  
Wistar Kyoto ◽  
Induced Changes

Changes in cytosolic free calcium concentration ([Ca2+]i) induced by angiotensin II (ANG II), arginine vasopressin (AVP), angiotensin III (ANG III), norepinephrine (NE), or thapsigargin were investigated after inhibition of the Na(+)-Ca2+ exchange in vascular smooth muscle cells (VSMC) from Wistar-Kyoto rats by use of the fluorescent dye technique. The ANG II-induced peak [Ca2+]i increase was significantly enhanced after inhibition of Na(+)-Ca2+ exchange by NiCl2 or 1,3-dimethyl-2-thiourea (DMTU): control, 99 +/- 9 (SE) nM (n = 64); NiCl2, 181 +/- 23 nM (n = 23; P < 0.01); DMTU, 182 +/- 35 nM (n = 10; P < 0.05). In the absence of external calcium, the inhibition of the Na(+)-Ca2+ exchange by NiCl2 also enhanced the ANG II-induced [Ca2+]i increase. Inhibition of Na(+)-Ca2+ exchange by removal of external sodium, which was replaced by choline, augmented the ANG II-induced [Ca2+]i increase to 174 +/- 26 nM (n = 11; P < 0.05 compared with control). The inhibition of the protein kinase C activity by isoquinoline-sulfonyl-O-2-methylpiperazine blocked the enhancing effect of NiCl2 on ANG II-induced [Ca2+]i increase. The inhibition of the Na(+)-Ca2+ exchange did not enhance the increase in [Ca2+]i induced by ANG III, NE, or thapsigargin. The AVP-induced changes in [Ca2+]i were not significantly different in the presence or absence of NiCl2. It is concluded that the recovery of resting [Ca2+]i after stimulation by ANG II is mediated by calcium efflux via the Na(+)-Ca2+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Blunted cGMP response to ANF in vascular smooth muscle cells of SHR

1988 ◽  
Vol 255 (5) ◽  
pp. C573-C580 ◽  
Author(s):  
M. Nakamura ◽  
A. Nakamura ◽  
B. Fine ◽  
A. Aviv
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Spontaneously Hypertensive Rat ◽  
Specific Binding ◽  
Poor Response ◽  
Muscle Cells ◽  
Spontaneously Hypertensive ◽  
Wistar Kyoto

Abnormalities in the coupling of atrial natriuretic factor (ANF) receptors with the guanosine 5'-cyclic monophosphate (cGMP) system in vascular smooth muscle cells (VSMCs) may play a role in the pathophysiology of hypertension in the spontaneously hypertensive rat (SHR). This concept was examined in cultured, aortic VSMCs (passages 6-10) from SHR, Wistar-Kyoto (WKY), and American Wistar (Wis) rats. Quiescent VSMCs of the SHR (serum deprived for 24 h) had higher ANF receptor density (Bmax) and lower affinity [i.e., increased equilibrium dissociation constant (Kd)] than cells from normotensive controls. Maximal binding (Bmax) (specific binding sites/cell) values for these cells were SHR 112,855 +/- 6,951, WKY 48,650 +/- 3,607, and Wis 36,122 +/- 2,607 (means +/- SE; P less than 0.001 for SHR vs. both WKY and Wis). The Kd values were (in nM) SHR 1.20 +/- 0.098, WKY 0.657 +/- 0.065, and Wis 0.37 +/- 0.037 (P less than 0.001 for SHR vs. both WKY and Wis). Despite their higher Bmax, VSMCs of the SHR showed a substantially lower maximal stimulation of cGMP accumulation in response to ANF: 987 +/- 29.3, 1,992 +/- 574.2, and 2,019 +/- 273.8 fmol.4 min-1.10(6) cells-1 for SHR, WKY, and Wis, respectively (P less than 0.01 for SHR vs. Wis and P less than 0.02 for SHR vs. WKY). Further experiments demonstrated that the poor response of SHR VSMCs to the ANF was not due to a population of receptors that did not couple to the particulate guanylate cyclase. Such findings demonstrate a dissociation of the cGMP response to ANF from the binding of the hormone to its receptors in VSMCs of the SHR compared with controls. This appears to represent a primary and innate defect in these cells that may contribute to the hypertensive process in the SHR.

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