Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium

Abstract

Background: The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis.

Method/principal findings: Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH.

Conclusions: The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

Figure 1. Establishment of primary culture of rat caput epididymal epithelium.

A: In the above is the fluorescence image of cultured rat caput epididymal epithelial cells showing FITC immunoreactivity for pan keratin, in the middle is bright field image of caput epididymal epithelial cells and in the below is overlapping image of the fluorescence and bright field images; B: In the above of native control group is the fluorescence image of cultured rat caput epididymal epithelial cells without the application of primary antibody, in the middle is bright field image of caput epididymal epithelial cells and in the below is overlapping image of the fluorescence and bright field images.

Figure 2. HCO₃ ⁻-induced intracellular acidification recovery in rat caput epididymal epithelium.

A: pHi calibration linear of rat caput epididymal epithelium; B: Representative record chart showing the function of HCO₃ ⁻ in NH₄Cl induced acidification recovery in rat caput epididymal epithelium. In the HEPES-KH solution, 40 mM NH₄Cl induced intracellular alkalization. Then, in the HCO₃ ⁻ free condition containing 1 mM amiloride induced transient intracellular acidification, and the recovery was inhibited. After that, the cells were perfused with HCO₃ ⁻-KH solution with 1 mM amiloride, and the intracellular acidification was recovered. NH₄Cl, 40 mM NH₄Cl; ami, 1 mM amiloride; Each point represents the mean ±S.E.M. n = 4.

Figure 3. Na⁺ dependent acidification recovery in rat caput epididymal epithelium.

A: Representative record chart showing involvement of Na⁺ in the HCO₃ ⁻ mediated recovery for NH₄Cl induced acidification in rat caput epididymal epithelium. The HEPES-KH solution containing 40 mM NH₄Cl caused intracellular alkalization. Washing the NH₄Cl with Naf-HEPES-KH solution would arouse intracellular acidification with no remarkable recovery. When the perfusion solution was changed into Naf-HCO₃ ⁻-KH solution, the recovery was still depressed. When the perfusion solution was changed into normal KH solution, the intracellular acidification recovered to basal level; B: The summary of the results showing the effect of Na⁺ on HCO₃ ⁻ mediated intracellular acidification recovery. HCO₃ ⁻ mediated intracellular acidification recovery in Fig. 2B is set as control group. Data are expressed as the change of the pH value per minute. NH₄Cl, 40 mM NH₄Cl; ami, 1 mM amiloride; Naf, Na+ free; Ctl, control; Columns and error bars are mean ± S.E.M. (n = 3; ** P<0.01 vs Ctl).

Figure 4. DIDS inhibits HCO₃ ⁻ induced intracellular acidification recovery in rat caput epididymal epithelium.

A: The record chart showing that DIDS inhibited the HCO₃ ⁻ mediated recovery for NH₄Cl induced acidification in the rat caput epididymal epithelium. The HEPES-KH solution containing 40 mM NH₄Cl caused intracellular alkalization. Wash the NH₄Cl with HEPES-KH solution with 1 mM amiloride would arouse intracellular acidification with no remarkable recovery. The recovery was still suppressed when the solution was changed into HCO₃ ⁻ -KH solution containing 1 mM DIDS and 1 mM amiloride; B: The summary of results showing the effects of DIDS on the HCO₃ ⁻ mediated intracellular acidification recovery. HCO₃ ⁻ mediated intracellular acidification recovery in Fig. 2B is set as control group. Data are expressed as the change of the pH value per minute. NH₄Cl, 40 mM NH₄Cl; ami, 1 mM amiloride; DIDS, 1 mM DIDS; Ctl, Control; Columns and error bars are mean ± S.E.M. (n = 5; **P<0.01 vs Ctl).

Figure 5. NBC on the apical membrane of rat epididymal epithelium regulates epididymal apical pH.

In the control group, the cultured rat caput epididymal epithelium was immersed in Cl⁻ free KH solution without any treatment of DIDS (n = 7). 1 mM DIDS was applied to the apical(n = 3) or basolateral(n = 3) side of the cultured epithelial monolayer and incubated for 1 hour. Ctl, Control; DIDS, 1 mM DIDS; ap, apical; bl, basolateral; N.S., no significant difference vs Ctl; All bars represent mean ± S.E.M. (*P<0.05 vs Ctl).