Regulation of Intracellular pH is Altered in Cardiac Myocytes of Ovariectomized Rats

Figures

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  Figure 1. NHE function in OVX myocytes is increased in comparison to sham myocytes. A, Representative traces of intracellular pH (pHi) during the application of an ammonium pulse (20 mmol/L NH4Cl) in sham and OVX ventricular myocytes perfused with HEPES buffer. B, Average proton efflux (JH) carried by NHE calculated at different pHi values during the recovery from acidosis in sham and OVX myocytes. The values were fitted with the following linear regression (sham: JH= −6.00pHi + 42.07; R2, 0.97; and OVX: JH= −7.85pHi + 55.53; R2, 0.99). P‐value vs sham is depicted above each average point. N indicates number or rats for each group; NHE, sodium‐proton exchanger; R2, R‐Squared.

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  Figure 2. NHE protein expression in OVX hearts is greater than in sham hearts. Representative immunoblots showing the increased expression of NHE in OVX heart homogenates in comparison to sham (SH). Average data are depicted in the bar graphs as percentage of NHE/GAPDH respect to Sham. *P<0.05 vs Sham. N indicates number of hearts for each group; NHE, sodium/proton exchanger; OVX, ovariectomized rats.

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  Figure 3. NBC isoform activity. A, Superimposed representative traces of intracellular pH (pHi) during the application of an ammonium pulse in the absence (nonimmune serum as control) or presence of the serum containing the functional inhibitory antibody against NBCe1 (a‐L3; 1:500). The experiments were performed in the continuous presence of the NHE inhibitor HOE 642 (10 μmol/L). B, Average bicarbonate influx (JHCO3) calculated at pHi 6.85 in the absence (control) or presence of a‐L3. NBCn1 activity was estimated as the pHi recovery in the presence of a‐L3, whereas NBCe1 activity was calculated as the control pHi recovery (total NBC) minus the NBCn1 activity. N indicates number of rats for each group; NBCe1, electrogenic sodium‐bicarbonate cotransporter; NBCn1, neutral sodium‐bicarbonate cotransporter; OVX, ovariectomized rats.

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  Figure 4. NBCe1 activity is impaired in OVX myocytes. A, Representative traces of intracellular pH (pHi) recorded in sham and OVX ventricular myocytes during the application of a potassium pulse (45 mmol/L K+). B, Average data of pHi alkalinization induced by the hyperkalemic solution in sham and in OVX myocytes. Data are expressed as increase of pHi units after 15 minutes in comparison to the 0 time point in high‐K+ solution. **P<0.01 vs sham. N indicates number of rats for each group; NBCe1, electrogenic sodium‐bicarbonate cotransporter; OVX, ovariectomized rats.

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  Figure 5. NBC protein expression did not change between sham and OVX hearts. A, Representative immunoblots showing the expression of NBCn1 and NBCe1 in sham and OVX heart homogenates. B, Average data are depicted in the bar graphs as percentage of NBCn1/GAPDH or NBCe1/GAPDH relative to sham. N indicates number of hearts for each group; NBCe1, electrogenic sodium‐bicarbonate cotransporter; NBCn1, neutral sodium‐bicarbonate cotransporter; OVX, ovariectomized rats; Sh, sham.

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  Figure 6. Relative contribution of the transporters to the pHi recovery in bicarbonate solution. A, Representative traces of intracellular pH (pHi) during the application of an ammonium pulse (20 mmol/L NH4Cl) in sham and OVX ventricular myocytes perfused with bicarbonate buffer. B, Average bicarbonate influx (JHCO3‐) at pHi 6.85 in sham and OVX myocytes, evidencing the relative contributions of NHE, NBCn1, and NBCe1 to the total recovery. *P<0.05 vs sham. N indicates number of rats for each group; NHE, sodium/proton exchanger; NBCe1, electrogenic sodium‐bicarbonate cotransporter; NBCn1, neutral sodium‐bicarbonate cotransporter; OVX, ovariectomized rats.

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  Figure 7. The modifications of the alkalinizing transporters lead to an increase in the pHi during the steady‐state only in HEPES buffer. A, Average final pHi after the ammonium pulse in HEPES buffer in sham and OVX ventricular myocytes. B, Average final pHi after the ammonium pulse in bicarbonate buffer in sham and OVX ventricular myocytes. C, Maximal acidification during the ammonium pulse in HEPES buffer in sham and OVX ventricular myocytes. D, Maximal acidification during the ammonium pulse in bicarbonate buffer in sham and OVX ventricular myocytes. *P<0.05 vs sham. N indicates number of rats for each group; OVX, ovariectomized rats.

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  Figure 8. p‐Akt expression: representative immunoblots showing the expression of p‐Akt in sham and OVX heart homogenates. Average data are depicted in the bar graphs as percentage of p‐Akt/Akt relative to sham. To normalize the amount of the phosphorylated proteins, the membranes were stripped and probed with an antibody that specifically detects total Akt. *P<0.05 vs sham. N indicates number of hearts for each group; OVX, ovariectomized rats; SH, sham.

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  Figure 9. Platelets NHE activity. A, Representative traces of pHi recorded in platelets during the recovery of an ammonium pulse in platelets perfused with HEPES buffer. B, Average rate of recovery from acidosis, depicted as change in pH units per minute at pHi 6.8. *P<0.05 vs sham. N indicates number of rats for each group; NHE, sodium/proton exchanger; OVX, ovariectomized rats.