pH-dependent regulation of the α-subunit of H+-K+-ATPase (HKα2)

Abstract

The H(+)-K(+)-ATPase α-subunit (HKα(2)) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα(2) plasma membrane expression is regulated by PKA (Codina J, Liu J, Bleyer AJ, Penn RB, DuBose TD Jr. J Am Soc Nephrol 17: 1833-1840, 2006) and in a separate study demonstrated that genetic ablation of the proton-sensing G(s)-coupled receptor GPR4 results in spontaneous metabolic acidosis (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). In the present study, we investigated the ability of chronic acidosis and GPR4 to regulate HKα(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 HKα(2) protein were monitored by immunoblot analysis, and HKα(2) mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα(2) mRNA; however, PKA activity and HKα(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 acid-stimulated HKα(2) expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα(2) protein by increasing PKA activity without altering HKα(2) mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα(2) and acid-base balance.

Fig. 1.

Chronic acidosis induced by reduction in media bicarbonate concentration increases the H⁺-K⁺-ATPase α-subunit (HKα₂) protein, but not time-dependent mRNA abundance. HEK-293 cells stably expressing HKα₂/NKβ₁ as described previously, were exposed for up to 24 h to reduced extracellular pH by reducing media NaHCO₃ concentration. Cell lysates were subsequently harvested and subjected to immunoblot analysis of HKα₂ and β-actin (used to verify similar loading) as described in methods. Bands from immunoblots were detected and quantified using the Odyssey Infrared Imaging System (Li-Cor). A: representative blot in which media pH was progressively reduced by reducing media HCO₃⁻ concentration at the beginning of the experiment; lysates were harvested after 24 h. B: fold-change in HKα₂ abundance relative to the value determined at pH 7.4 were calculated, and data were plotted as a function of media pH (media pH was measured at time of lysate harvest, means ± SE values were calculated, and 6 points, with X and Y error bars, were plotted); n = 23. C: immunoblots for HKα₂ and β-actin of lysates harvested from cells maintained in media pH 6.8 for different durations. D: values are means ± SE of HKα₂ from 3 different experiments.

Fig. 2.

Acidosis-induced increase in HKα₂ protein is reversible. A: cells were maintained in media pH ∼7.4 or ∼6.8 as indicated for 24 h, and then media was removed and replaced by media pH ∼7.4 or ∼6.8. Indicated pH values represent those measured for media after either the first (0–24 h) or second (24–48 h) 24-h treatment period. Cell lysates were harvested after each treatment period and subjected to immunoblot analysis of HKα₂ and β-actin as described in methods. B: values are means ± SE from 3 independent experiments. **P < 0.01.

Fig. 3.

Different methods of media pH reduction promote increased HKα₂ protein abundance. A: media pH was adjusted as indicated by addition of HCl. B: experiment was repeated 3 times, and fold-increase in HKα₂ vs. extracellular pH is shown. C: cells were cultured in incubators set for 5 or 10% CO₂ to achieve a media pH of 7.4 or 7.0, respectively. D: values are means ± SE for fold-increase in HKα₂ at 10 vs. 5% CO₂ from 3 independent experiments. ***P < 0.001.

Fig. 4.

Inhibition of carbonic anhydrase does not affect upregulation of HKα₂ during increase in CO₂. Cells were subjected to 24-h treatment in either 5 or 10% CO₂ in the presence of vehicle or 100 μM acetazolamide. A: representative immunoblot of HKα₂ and β-actin expression. B: values are means ± SE from 4 independent experiments. **P < 0.01.

Fig. 5.

Stimulators of protein kinase A (PKA) upregulate HKα₂ protein; decreasing extracellular pH increases PKA activity. A: 24-h treatment of cells with PKA-activating agents forskolin (FSK) or dibutyryl-cAMP (db-cAMP) increases HKα₂ protein expression in stably transfected cells. B: values are means ± SE derived from 4 independent experiments. C: stimulation of cells for 5 min with media of progressively lower pH causes a rapid increase in intracellular PKA activity, indicated by the increased expression of the 50-kDa band of the PKA substrate VASP. D: values are means ± SE for fold-increase in phospho-VASP levels from 3 independent experiments.

Fig. 6.

G protein proton-sensing receptor GPR4 expression increases basal and acid-induced PKA activity, HKα₂ expression. A: transient expression of GPR4 in HEK-293 cells increases both basal and acid-stimulated PKA activity. B: values are means ± SE depicting effect of GPR4 expression and reduced media pH on fold-expression of phospho-VASP levels (normalized to corresponding β-actin values); n = 4. C: progressive reduction in media pH to 6.9 further augments HKα₂ relative to that observed in vector-transfected control cells. D: values are means ± SE from 3 independent experiments. *P < 0.05. **P < 0.01.

Fig. 7.

Regulation of endogenous HKα₂ expression in m-IMCD-3 cells by extracellular pH. m-IMCD-3 cells were grown to near confluency, arrested 24 h in serum-free media, then subject to reduced extracellular pH by reduction of media bicarbonate concentration for another 24 h. A: representative immunoblot analysis of pH effect on HKα₂ expression. B: values are means ± SE of data derived from 3 independent experiments.