Abstract
Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Cytoplasmic pH (pH i ) has been shown to be an important parameter in cell physiology, regulating namely cell metabolism and differentiation. However, membrane transport mechanisms involved in pH i regulation mechanisms of Sertoli cells have not yet been elucidated. In this work, pH i was determined using the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Addition of weak acids resulted in rapid acidification of the intracellular milieu. Sertoli cells then recovered pH i by a mechanism that was shown to be sensitive to external Na+. pH i recovery was also greatly reduced in the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and amiloride. These results point toward the action of an Na+-driven HCO −3 /Cl− exchanger and/or an Na+/HCO −3 cotransporter and the action of the Na+/H+ exchanger on pH i regulation in the experimental conditions used. pH i recovery was only slightly affected by ouabain, suggesting that the inhibition of Na+/K+-ATPase affects recovery indirectly, possibly via the shift on the Na+ gradient. On the other hand, recovery from the acid load was independent of the presence of concanamycin A, a specific inhibitor of the V-type ATPases, suggesting that these pumps do not have a relevant action on pH i regulation in bovine Sertoli cells.
Abbreviations
- BCECF:
-
2′,7′-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein
- DIDS:
-
4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid
- DMEM:
-
Dulbecco's Modified Eagle’s medium
- DMSO:
-
Dimethyl sulfoxide
- FSH:
-
Follicle Stimulating Hormone
- HBSSf :
-
Calcium- and magnesium-free Hank’s Balanced Salt Solution
- PBS:
-
Phosphate Buffered Solution
- pH e :
-
Extracellular pH
- pH i :
-
Intracellular pH
- SEM:
-
Standard error of the mean
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Acknowledgement
P. F. Oliveira was supported by Fundação para a Ciência e a Tecnologia (SFRH/BPD/20965/2004).
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Oliveira, P.F., Sousa, M., Barros, A. et al. Membrane Transporters and Cytoplasmatic pH Regulation on Bovine Sertoli Cells. J Membrane Biol 227, 49–55 (2009). https://doi.org/10.1007/s00232-008-9139-z
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DOI: https://doi.org/10.1007/s00232-008-9139-z