Abstract
Mitogens and vasoconstrictors stimulate many of the same early intracellular signals (e.g. phospholipase C and protein kinase C activation) in vascular smooth muscle cells (VSMC). Despite these shared signals, angiotensin II is not mitogenic for cultured VSMC. The nonmitogenic effect of angiotensin II suggests that other intracellular signals associated with growth should differ between mitogens and vasoconstrictors. Because of the importance of intracellular pH (pHi) in growth, we compared the effects of 10% calf serum, 10 ng/ml platelet-derived growth factor, and 100 nM angiotensin II on pHi and Na+/H+ exchange. All agonists stimulated a rapid (less than 1 min) rise in pHi mediated by Na+/H+ exchange. However, exposure of growth-arrested VSMC to these agonists for 24 h caused significant differences in pHi: 7.18 (10% serum), 7.16 (platelet-derived growth factor), 6.99 (angiotensin II), and 7.08 (0.4% serum). Na+/H+ exchange activity was measured in acid-loaded cells by the ethyl isopropyl amiloride-sensitive influx of Na+ and efflux of H+. Both techniques showed that exposure to 10% serum caused approximately 45% decrease in Na+/H+ exchange activity without significant change in angiotensin II-treated cells. Thus, although the rapid changes in pHi and Na+/H+ exchange function are the same for angiotensin II and mitogens, the long term effects differ. The data suggest that differences in pHi regulatory mechanisms are important in determining whether an agonist causes VSMC hypertrophy or hyperplasia.
Highlights
Exposure of growth-arrested vascular smooth muscle cells (VSMC) to 10% serum and platelet-derived growth factor (PDGF) caused a decrease in Na+/H+ exchange activity and an increase in pHi whereas angiotensin II had no effect on activity and caused a decrease in pHi. These findings suggest that differences in pHi regulatory mechanisms are important in determining whether an agonist causes cell hypertrophy or hyperplasia
In the series of experiments described below we investigated the effects of these agonists on pHi and Na+/H’
The most important findings of this study are that pHi and Na+/H’ exchange activity are altered in hypertrophied VSMC as compared with hyperplastic VSMC
Summary
13160, HL-07042, and HL-36561 from the National Institutes of Health and by a grant from the National Science Foundation. Cell Culture-VSMC were isolated from rat thoracic aorta by enzymatic digestion as described by Travo et al (12). The vessels were incubated for 30 min at 37 “C in 1 ml/aorta Hanks’ solution containing 175 units/ml type I collagenase. 10% fetal calf serum in an air, 5% CO, incubator. Enzymatic digestion cell(s); BCECF, 2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; was completed by incubating the vessel for 90-120 min in 1 ml/aorta. EGF, epidermal growth factor; EIPA, ethyl isopropyl amiloride; Hanks’ solution containing 175 units/ml type I collagenase and 0.5. [Na’],, extracellular [Na+]; pH,, intracellular pH; PDBu, phorbol aorta 20% calf serum in DMEM, and the cells were pelleted by. VSMC were plated at 1 X lo[4] cells/cm* in DMEM kinase C; MOPS, 4-morpholinepropanesulfonic acid; DMEM, Dulcontaining 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, becco‘s modified Eagle’s medium.
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