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Effects of glucocorticoids on Na + /H + exchange and growth in cultured vascular smooth muscle cells
Author(s) -
Berk Bradford C.,
Vallega Gino,
Griendling Kathy K.,
Gordon John B.,
Cragoe Edward J.,
Canessa Mitzy,
Alexander R. Wayne
Publication year - 1988
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041370302
Subject(s) - cycloheximide , vascular smooth muscle , medicine , endocrinology , intracellular , hydrocortisone , intracellular ph , chemistry , angiotensin ii , biology , protein biosynthesis , biochemistry , smooth muscle , blood pressure
We have examined the effects of hydrocortisone on growth and Na + /H + exchange in cultured rat aortic vascular smooth muscle cells (VSMC). Hydrocortisone (2 μM) treatment of growth‐arrested VSMC significantly decreased VSMC growth in response to 10% calf serum assayed by 3 H‐thymidine incorporation and cell number at confluence. This effect was associated with the appearance of an altered cell phenotype characterized by large, flat VSMC that did not form typical “hillocks.” Na + /H + exchange was also altered in hydrocortisone‐treated cells assayed by dimethylamiloride‐sensitive 22 Na + influx into acid‐loaded cells or by intracellular pH (pH i ) change using the fluorescent dye BCECF. Resting pH i was 7.25 ± 0.04 and 7.15 ± 0.05 in control and hydrocortisone‐treated cells, respectively (0.1 < P < 0.05). Following intracellular acidification in the absence of external Na + , pH i recovery upon addition of Na + was increased 89% in hydrocortisone‐treated cells relative to control. This was due to an increase in the V max for the Na + /H + exchanger from 17.5 ± 2.4 to 25.9 ± 2.0 nmol Na + /mg protein × min ( P < 0.01) without a significant change in K m . Treatment of VSMC with actinomycin D (1 μg/ml) or cycloheximide (10 μM) completely inhibited the hydrocortisone‐mediated increase in Na + /H + exchange, indicating a requirement for both RNA and protein synthesis. Because hydrocortisone altered the V max for Na + /H + exchange, in contrast to agonists such as serum or angiotensin II which alter the K m for intracellular H + or extracellular Na + , respectively, we studied the effect of hydrocortisone on activation of Na + /H + exchange by these agonists. In cells maintained at physiological pH i (7.2), the initial rate (2 min) of angiotensin II‐stimulated alkalinization was increased 66 ± 39% in hydrocortisone‐treated compared with control cells. Hydrocortisone caused no change in angiotensin II‐stimulated phospholipase C activity assayed by measurement of changes in intracellular Ca 2+ or diacylglycerol formation. However, angiotensin II and serum stimulated only small increases in Na + /H + exchange in acid‐loaded (pH i = 6.8) hydrocortisone‐treated cells. These findings suggest that hydrocortisone‐mediated increases in VSMC Na + /H + exchange occur in association with a nonproliferating phenotype that has altered regulation of Na + /H + exchange activation. We propose that hydrocortisone‐mediated growth inhibition may be a useful model for studying the role of Na + /H + exchange in cell growth responsiveness.