Long-term High Osmolality Activates Na⁺-H⁺Exchange and Protein Kinase C in Aortic Smooth Muscle Cells

Abstract

The effect of long-term exposure to hypertonic medium on Na(+)-H+ exchange activity was studied in cultured vascular smooth muscle (VSM) cells by using a combination of 22Na+ influx and pH measurement with the pH-sensitive dye BCECF. Incubation of VSM cells in high-osmolality medium (510 mOsm/L) for 48 hours significantly increased the acid-stimulated 22Na+ influx (control, 3.16 +/- 0.41 nmol/mg protein per minute; high osmolality, 6.40 +/- 0.66 nmol/mg protein per minute; P < .01) and Na(+)-dependent pHi recovery (control, 0.29 +/- 0.06 pH/min; high osmolality, 0.65 +/- 0.13 pH/min; P < .03). Activation of Na(+)-H+ exchange was osmolality dependent and reached maximal stimulation at approximately 700 mOsm/L. Na(+)-H+ exchanger stimulation was independent of serum in the culture media. Na(+)-H+ exchanger isoform (NHE-1) mRNA in VSM cells cultured in high-osmolality medium was unchanged from that in VSM cells cultured in control medium, indicating an absence of transcriptional regulation by high osmolality. Long-term high osmolality significantly increased protein kinase C (PKC) activity in cultured VSM cells, as assessed by phosphorylation of a PKC-specific substrate (control, 20.9 +/- 2.1 pmol phosphorylation/mg protein per minute; high osmolality, 33.6 +/- 2.9 pmol phosphorylation/mg protein per minute; P < .01). Downregulation of PKC by preincubation of VSM cells with 0.1 mumol/L phorbol 12-myristate 13-acetate (PMA) prevented osmolality-induced stimulation of the Na(+)-H+ exchanger (control plus PMA, 0.27 +/- 0.05 pH/min; high osmolality plus PMA, 0.33 +/- 0.08 pH/min; P > .05).(ABSTRACT TRUNCATED AT 250 WORDS)