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(Circulation Research. 1995;76:530-535.)
© 1995 American Heart Association, Inc.

Articles

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

Manoocher Soleimani, Gurinder Singh, Jesus H. Dominguez, Randy L. Howard

From the Department of Medicine, Indiana University School of Medicine, and Veterans Affairs Medical Center, Indianapolis, Ind.

Correspondence to Manoocher Soleimani, MD, Nephrology Section, Department of Medicine, Fesler Hall 108, 1120 South Dr, Indiana University School of Medicine, Indianapolis, IN 46202-5116.

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 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 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 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 gene 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.

Key Words: Na⁺-H⁺ exchanger • high osmolality • vascular smooth muscle cells

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