Previous studies from this laboratory have demonstrated that angiotensin II (Ang II) and arginine vasopressin (AVP) are potent hypertrophic agents in cultured rat aortic smooth muscle cells. The present study identified major proteins that accumulate in Ang H-induced and AVP-induced hypertrophic cells and initiated studies of the mechanisms that contribute to their accumulation. Smooth muscle cell hypertrophy induced by Ang H and/or AVP (1 ,uM each) was associated with widespread increases in the content of many cellular proteins that were resolved by one-and two-dimensional gel electrophoresis. However, increases were also selective in nature, with increases in certain individual proteins, including actin (twofold to threefold), vimentin (2.5-fold to sevenfold), tropomyosin (threefold to sixfold), and myosin heavy chain, far exceeding overall increases in cellular protein content (20-40%o). Increases in actin content were due largely to increased expression of smooth muscle ar-actin (3.6-to 7.5-fold), as opposed to nonmuscle f-actin (1.7-to 2.5-fold). Increases in smooth muscle a-actin were accompanied by a fivefold to eightfold increase in smooth muscle a-actin mRNA, indicating that these changes were not due exclusively to translational controls. Results demonstrate that contractile agonist-induced hypertrophy in cultured smooth muscle cells is due, in part, to increased expression of smooth muscle contractile proteins. Furthermore, the fact that Ang II and AVP induced selective increases in smooth muscle a-actin suggests that these agonists may not only regulate growth of vascular smooth muscle but may also promote expression of smooth muscle-specific contractile proteins during differentiation of vascular smooth muscle. (Circulation Research 1991;68:288-299 Results showed that, for a given level of blood pressure lowering, the converting enzyme inhibitor, captopril, was more effective in preventing hypertrophy than was hydralazine, a direct SM relaxant.Results of these studies suggested that hypertrophy was not simply a response to elevated blood pressure and implicated a possible role for angiotensin II (Ang II) in mediation of vascular SMC growth. Furthermore, observations that captopril also reduced aortic SM content in normotensive Wistar-Kyoto rats suggested that Ang II might also play a role in maintenance of vascular mass under normal conditions. Results of recent studies in cultured SMCs provide further support for the hypothesis that contractile agonists may mediate SM hypertrophy. We"1,12 and others13,14 have shown that the contractile agonists Ang 11 and arginine vasopressin (AVP) stimulated hypertrophy but not hyperplasia of rat aortic SMCs in culture. Contractile agonist-induced increases in protein content of hypertrophied SMCs were concentration dependent, were inhibited by specific receptor antagonists, and were due to an increase in protein synthesis, rather than to a change in the overall rate of protein degradation.1112 However, the cellular mechanisms whereby these contractile agonists i...

Recent studies suggest that phospholipid-sensitive, Ca 2+ -dependent protein kinase C participates in contractile responses of vascular smooth muscle. This study characterizes vascular reactivity to protein kinase C activators in stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). Helical strips of mesenteric arteries were mounted in organ chambers for measurement of isometric contractions (responses were normalized as a percentage of maximal force in response to 100 mM KC1; in SHRSP, 350 ± 16 mg; in WKY, 335 ± 21 ing). Arteries from SHRSP contracted faster and developed greater force than arteries from WKY (168 ± 9% vs 143 ± 3%) in response to the phorbol ester, 12-Otetradecanoylphorbol-13-acetate. Arteries from SHRSP (0.6 x 10-8 M) were more sensitive to the phorbol ester than those from WKY (2.2 x 1(H M), as indicated by the dose of the phorbol ester required to produce 50% of the maximal response to KC1. Additionally, SHRSP arteries were more sensitive to the contractile effects of mezerein, a non-phorbol ester activator of protein kinase C. Ca 2+ -free solution (1.0 mM EGTA) and verapamil (10-7 M) caused relaxation (approximately -60%) of contractions in response to the phorbol ester (10-* M). Addition of 10-6 M of the phorbol ester to arteries that were preincubated in Ca 2+ -free solution (1.0 mM EGTA for 30 minutes) elicited submaximal contractions (in SHRSP, 26 ± 4%; in WKY, 38 ± 7%). Upon addition of 1.6 mM Ca 2+ , arteries from SHRSP contracted faster (t^, =2.7 ± 0.6 minutes) than those from WKY (8.2 ± 0.5 minutes). These data indicate that increased vascular responsiveness in SHRSP may result from enhanced activity of the protein kinase C branch of the Ca 2+ messenger system. (Hypertension 9 [Suppl III]: IIM50-III-154, 1987) KEY WORDS • stroke-prone spontaneously hypertensive rats • mesenteric artery • protein kinase C • phorbol ester K CENT evidence suggests that in vascular smooth muscle, phosphatidylinositol 4,5-bisphosphate of the plasma membrane is hydrolyzed by phospholipase C in response to certain hormones and neurotransmitters.'-5 A product of this hydrolysis, inositol 1,4,5-trisphosphate, has been proposed to be a messenger for Ca 2+ release from intracellular stores. 6 -7 Furthermore, Nishizuka 8 has shown that another product of phosphatidylinositol turnover, diacylglycerol, can activate protein kinase C (C-kinase). Diacylglycerol, along with phosphatidylserine, greatly increases the affinity of C-kinase for Ca 2+ to the submicromolar range, thereby rendering this enzyme fully From the Department of Physiology, The University of Michigan, Ann Arbor, Michigan.

Recent studies suggest that serotonergic receptor activation is coupled to phospholipase C-mediated phosphoinositide hydrolysis, which results in the release of intracellular second messengers. The purpose of this study was to determine whether altered phosphoinositide metabolism is the basis for augmented vascular responsiveness to serotonin in genetic hypertension. Thoracic aortic segments isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY) were labeled with myo-[3H]inositol and stimulated with serotonin in the presence of LiCl. Accumulation of [3H]inositol phosphates was then quantitated by column chromatography. Basal inositol phosphate accumulation and basal incorporation of myo-[3H]inositol into aortic cell membranes from SHRSP was not significantly different from WKY values. At 2.6 x 10(-7) to 2.6 x 10(-4) M serotonin, phosphoinositide metabolism was significantly augmented in aortae from SHRSP compared with WKY. Depolarization (100 mM KCl) did not increase phosphoinositide hydrolysis above basal levels in SHRSP or WKY. 2-Nitro-4-carboxyphenyl-N,N-diphenyl carbamate (NCDC), an inhibitor of phospholipase C, prevented the serotonin-induced phosphoinositide metabolism. NCDC also partially inhibited phasic contractions (responses in calcium-free solution) to serotonin in aortas from SHRSP and WKY. In conclusion, abnormal phosphoinositide metabolism may be one mechanism responsible for the characteristic increase in vascular reactivity to serotonin in hypertension.

Previous studies have demonstrated that angiotensin II (ANG II) and arginine vasopressin (AVP) stimulate increased protein synthesis and cellular hypertrophy in cultured rat aortic smooth muscle cells (SMC). The aim of this study was to explore the hypothesis that ANG II- and/or AVP-induced increases in protein synthesis are mediated by autocrine secretion of platelet-derived growth factor (PDGF)-AA. Results demonstrated that ANG II or AVP increased expression of PDGF-A, but not -B, chain mRNA. Additionally, conditioned media from ANG II- and AVP-treated SMC had increased mitogenic activity for Swiss 3T3 cells, which could be inhibited with a neutralizing antibody to PDGF-AA. However, PDGF-AA-neutralizing antibodies did not inhibit ANG II- or AVP-induced increases in protein synthesis, and exogenous PDGF-AA did not stimulate increased protein synthesis. Furthermore, no PDGF-alpha receptors were evident based on 125I-labeled PDGF-AA binding studies. In summary, results indicate that ANG II- or AVP-induced increases in protein synthesis were not dependent on autocrine secretion of PDGF-AA.

This study examined the calcium dependency of contractions in arteries from rats made hypertensive by aortic coarctation and in rats with genetic hypertensive (stroke-prone spontaneously hypertensive rats). Mesenteric artery and aortic strips were suspended in tissue baths for isometric force recording and contractions to two drugs were characterized: 1) a phorbol ester, TPA (12-O-tetrade-canoylphorbol-13-acetate), and 2) the calcium channel agonist, Bay K 8644. Thoracic aortae and mesenteric arteries from hypertensive rats were more sensitive to the contractile properties of the protein kinase C activator TPA than comparable arteries from normotensive rats. In thoracic aortae from coarcted rats, the contractile activity of Bay K 8644 was potentiated compared to normotensive values. In the presence of 19.2 mmol/L KCl, responses to Bay K 8644 in thoracic aortae from normotensive rats were potentiated and did not differ from coarcted values. In contrast, contractions to Bay K 8644 and TPA in abdominal aortae obtained below the coarctation were not different from normotensive values. Upon exposure to 26.2 mmol/L KCl, contractions to Bay K 8644 in abdominal aortae were potentiated and those in aortae from coarcted rats did not differ from sham values. Contractile responses to both drugs were blocked by nifedipine and verapamil and responses were attenuated in calcium-free solution. We conclude that calcium channel function and its regulation by protein kinase C contribute to altered vascular reactivity in hypertension. Further, these abnormalities have a pressure dependency, because they did not occur in abdominal aortae from coarcted rats.