To investigate the role of vasoconstrictor hormones in vascular smooth muscle cell growth we have studied the effects of the potent vasoconstrictor angiotensin II on cell growth in a cultured rat aortic cell model. Angiotensin II was not mitogenic for these cells, as assessed by determining cell number, nor was it synergistic in this regard with 10% calf serum. However, 24-hour exposure to 100 nM angiotensin II caused an 80% increase in protein synthesis (compared with 0.4% increase with serum control) as measured by tritiated leucine incorporation. This was a "hypertrophic" response as indicated by a 30% increase in protein content and a 45% increase in cell volume. Angiotensin O-induced smooth muscle cell hypertrophy was maximal at 100 nM, had In hypertensive models such as aortic coarctation and experimental injury models of atherosclerosis, VSMC migration into and proliferation (hyperplasia) in the intima is the most dramatic pathological feature. Received March 28, 1988; accepted November 18, 1988. 107-111-day-old SHR that VSMC proliferation rather than hypertrophy or hyperploidy appeared to account for the increase in VSMC mass. Several factors have been implicated in growth of VSMC in these models. In experimental injury models, endothelial dysfunction or denudation may result in abnormal interactions between elements in the blood (platelets, polymorphonuclear leukocytes, and monocytes) and the vessel wall that lead to sustained release of a variety of growth factors including platelet-derived growth factor. These growth factors are both chemotactic and mitogenic for VSMC 67 and thus may contribute to VSMC migration and proliferation in this model. In certain hypertensive models the increased blood pressure may result in altered mechanical stress that stimulates VSMC growth in a manner analogous to the effects of stretch on skeletal muscle protein synthesis.8 This explanation is supported by data that demonstrate a significant correlation between blood pressure and aortic VSMC hypertrophy and polyploidy. 6 -7 Furthermore, reduction of blood pressure in the SHR model can decrease VSMC polyploidy and hypertrophy.

The cellular mechanisms responsible for abnormalities in spontaneously hypertensive rat (SHR) vascular smooth muscle cell (VSMC) growth and vasoreactivity are not defined. Because Na+/H' exchange, which we have previously demonstrated in cultured VSMC, plays an essential role in mediating growth factor responses, we hypothesized that abnormalities in SHR growth regulation might be reflected in the activity of this transporter. To test this hypothesis, we studied DNA synthesis and Na+/H' exchange (measured as the rate of amiloride-sensitive intracellular alkalinization or Na' influx) in early subcultures (< 6) of aortic VSMC from 12-wk-old SHR and Wistar Kyoto (WKY) animals. Serum-deprived SHR VSMC grew more rapidly in response to 10% serum with an increase in [3Hjthymidine incorporation of 439% compared with 191% in WKY controls. Basal intracellular pH (pHj) values determined by fluorescent pH measurements were 7.37±0.04 and 7.27±0.03 (P < 0.05) in early passage SHR and WKY, respectively. Acid recovery (initial pH1 = 6.8) by SHR VSMC was faster than by WKY VSMC as measured by alkalinization (1.8±0.6 vs. 0.8±0.2 mmol Hf/liter * min, P < 0.05) or by amiloride-sensitive 22Na' influx (14.5±1.2 vs. 4.0±0.5 nmol Na+/mg protein * min, P < 0.05). In comparison to WKY cells early passage SHR VSMC exhibited 2.5-fold greater alkalinization and amiloride-sensitive 22Na' influx in response to 100 nM angiotensin II. During serial passage, WKY cells acquired enhanced Na+/H' exchange and growth rates so that by passage 6, these differences were no longer present. These findings in early cultures of SHR VSMC, removed from the in vivo neurohumoral milieu, suggest that increased Na+/H' exchange in SHR may reflect alterations in Na' homeostasis that might contribute to altered SHR VSMC function such as enhanced growth and vasoreactivity.

Myotubes of the C2 mouse muscle cell line form clusters of ACh receptors (AChRs) at apparently random sites along their length when cultured alone, and near sites of nerve-muscle contact when cocultured with neurons. We find in aneural cultures that myotubes of a C2 variant, S27, which is defective in glycosaminoglycan synthesis, express the AChR on their surface, but do not form clusters. S27 cells in aneural cultures also express the 43 kDa protein but do not cluster it. The altered distribution of laminin and collagen IV on the surface of S27 myotubes suggests that the basal lamina is abnormal. Neither the addition of exogenous proteoglycans or conditioned medium from wild-type C2 cells, nor the growth of S27 cells on substrates rich in basal lamina elements caused clusters to appear on S27 myotubes in aneural cultures. When cultured with primary neurons, however, S27 myotubes formed large clusters of the AChR near sites of neurite contact. The clusters were coincident with patches of the 43 kDa protein. Prelabeling experiments indicate that at least some AChRs in the clusters arise through aggregation. Although Torpedo agrin induces AChR clusters on C2 myotubes, it does not do so on S27 cells. Our experiments suggest that the spontaneous formation of clusters of AChRs and the 43 kDa protein in aneural cultures of myotubes depends upon the normal synthesis of muscle proteoglycans, and that nerve-induced clustering does not. Thus, there appear to be multiple mechanisms for the formation of AChR clusters.

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