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 pet minute; P<.01) and Na+-dependent pH(i) 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 ≃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 un changed 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 μmol/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). These results indicate that long-term exposure to hypertonic medium stimulates Na+-H+ exchange activity in cultured VSM cells and that this effect is independent of antiporter gent expression regulation. The results further demonstrate that the stimulatory effect of osmolality on Na+-H+ exchanger is mediated via posttranslational modification of the Na+-H+ exchanger by chronic PKC activation. The Na+-H+ exchanger may be involved in VSM cell volume regulation in long-term high osmolality.
- high osmolality
- Na-H exchanger
- vascular smooth muscle cells
ASJC Scopus subject areas
- Cardiology and Cardiovascular Medicine