We have explored the hypothesis that contractile agonists are important regulators of smooth muscle cell growth by examining the effects of one potent contractile agonist, angiotensin II (AII), on both cell proliferation and cellular hypertrophy. AII neither stimulated proliferation of cells made quiescent in a defined serum-free media nor augmented cell proliferation induced by serum or platelet-derived growth factor. However, AII did induce cellular hypertrophy of postconfluent quiescent cultures following 4 days of treatment, increasing smooth muscle cell protein content by 20% as compared with vehicle-treated controls. AII-induced hypertrophy was maximal at 1 microM, had an ED50 of 5 nM, and was blocked by the specific AII receptor antagonist Sar1,Ile8 AII. The cellular hypertrophy was due to an increase in protein synthesis, which was elevated within 6-9 hours following AII treatment, while no changes in protein degradation were apparent. AII was even more effective in inducing hypertrophy of subconfluent cultures, causing a 38% increase in protein content after 4 days of treatment (1 microM) and showing a maximal response at concentrations as low as 0.1 nM. Interestingly, in subconfluent cultures, AII treatment (1 microM, 4 days) was associated with a 50% increase in the fraction of cells with 4C DNA content with the virtual absence of cells in S-phase of the cell cycle, consistent with either arrest of cells in the G2 phase of the cell cycle or development of tetraploidy.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract. We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-[I (TGF-13), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-13 inhibited seruminduced proliferation of rat aortic smooth muscle cells (EDs0 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:7155-7160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-13. TGF-I~ concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47 % increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-I~-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in Gm were hypertrophied with respect to the corresponding ceils in vehicle-treated controls. Chronic treatment with TGF-I~ (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-I~ from these cultures was associated with the appearance of a significant fraction of cycling cells with >4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-I~ in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-I~.C ELLULAR enlargement or hypertrophy plays a prominent role in the postnatal growth of many tissues, as well as in physiological and pathological hypertrophy of a variety of tissues (4). However, relatively little is known regarding the mechanisms that control cell size. Even less is known concerning the control mechanisms for the DNA endoreduplication and polyploidy that often accompanies cellular hypertrophy, although its occurrence is widespread in eukaryotic cells in vivo, occurring in terminally differentiated cardiac myocytes (14,15,29), and neurons (22), as well as in nonterminally differentiated hepatocytes (8), smooth muscle cells (10,23,30), and other cell types (see review by Brodsky and Uryvaeva [8]).

hypertension-induced hypertrophy are unknown, but there is suggestive evidence that contractile agonists may play an etiologic role. Khokhar and Slater 8 found that the urinary excretion of arginine vasopressin was elevated in some patients with essential hypertension as compared with normotensive control subjects. Also, Crofton et al 9 reported that, in the deoxycorticosterone-salt rat model of hypertension, arginine vasopressin appears to play a major role in both the development and maintenance of the hypertension. Further evidence that contractile agonists may play a role in some of the structural changes observed in hypertension is provided by the observation that converting enzyme inhibitors are more effective at preventing or reversing both smooth muscle cell 10 and myocardial cell 11 hypertrophy in the spontaneously hypertensive rat than direct vasodilators and that the effects of converting enzyme inhibitors on hypertrophy are greater than predicted based on the magnitude of decreases in blood pressure. The hypothesis that contractile agonists might play some direct role in regulation of smooth mus- cle cell growth is intriguing as it may explain how a smooth muscle cell might alter its mass in response to an induced change in its work load. Because growth factors and contractile agonists share many of the same intracellular signaling mechanisms, it is not surprising that contractile agonists, such as arginine vasopressin and angiotensin II, have been reported to induce proliferation of a number of cell lines.12 -15 However, studies by Campbell-Boswell and Robertson, 16 in which cultured human aortic smooth muscle cells were used, found that neither angiotensin II nor arginine vasopressin was mitogenic for smooth muscle cells in the absence of 10% fetal bovine serum (FBS). Interestingly, although angiotensin II enhanced the proliferation of the human smooth muscle cells in serum, arginine vasopressin inhibited smooth muscle cell proliferation in response to 10% FBS. Recently, we reported that angiotensin II induced hypertrophy of quiescent cultured rat aortic smooth muscle cells but did not stimulate cellular proliferation either alone or in combination with either 10% FBS or plateletderived growth factor (2.5 ng/ml). 17 In the present study, we have addressed whether contractile agonists other than angiotensin II can modulate smooth muscle cell growth by studying the effect of arginine vasopressin on both proliferation and hypertrophy of cultured rat aortic smooth muscle cells. Materials and Methods Cell CultureRat thoracic aortic smooth muscle cells were isolated and cultured as previously described.17 Cells were harvested for passaging just before confluency (approximately 4-day intervals) with a trypsin-EDTA (0.05% trypsin, 0.02% EDTA, GIBCO Labs., Grand Island, New York) solution and plated at 3-5 xlO 3 cells/cm 2 . Passaged cells were grown in a one-to-one mixture of Dulbecco's Modified Eagle Medium (DMEM) (GIBCO Labs.) and Ham's F12 medium (F12, GIBCO Labs.), containing either 10% FBS (Hyclone...