: 1745-1755, 2010). In the present study, we investigated the ability of chronic acidosis and GPR4 to regulate HK␣2 expression in HEK-293 cells. Chronic acidosis was modeled in vitro by using multiple methods: reducing media pH by adjusting bicarbonate concentration, adding HCl, or by increasing the ambient concentration of CO 2. PKA activity and HK␣2 protein were monitored by immunoblot analysis, and HK␣ 2 mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HK␣ 2 mRNA; however, PKA activity and HK␣ 2 protein abundance increased when media pH decreased from 7.4 to 6.8. Furthermore, this increase was independent of the method used to create chronic acidosis. Heterologous expression of GPR4 was sufficient to increase both basal and acid-stimulated PKA activity and similarly increase basal and acidstimulated HK␣ 2 expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HK␣ 2 protein by increasing PKA activity without altering HK␣ 2 mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HK␣ 2 and acid-base balance.
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