Intracellular pH measurement in primary monolayer cultures of rat epididymal cells.

Intracellular pH (pHi) of primary monolayer cultures of the rat epididymal cells has been measured using fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). When incubated in normal Krebs-Henseleit (K-H) solution containing 25 mM HCO3, cell monolayers exhibited a basal pHi of 7.17 +/- 0.08. Amiloride (0.5 mM) added to the cell monolayers caused an immediate and sustained fall in pHi by 0.43 +/- 0.05 pH unit. Similar effects were produced when extracellular Na ions were substituted by N-methyl-D-glucamine. Addition of SITS (0.5 mM) or DPC (0.6 mM) resulted in a transient intracellular alkalosis. Adrenaline (0.23 microM) caused a fall in pHi by 0.33 +/- 0.05 pH unit. The fall was slow and was achieved after 30 min (half time 9.9 min). The effect of adrenaline was reversible upon washing and was blocked by propranolol (2 microM). The effect of adrenaline was mimicked by forskolin (10 microM) and Br-cAMP (0.5 mM) which caused a fall in pHi by 0.35 +/- 0.03 and 0.35 +/- 0.02 pH unit respectively. Addition of diphenylamine-2-carboxylate (DPC, 0.6 mM) to the cuvette completely blocked the intracellular acidification produced by adrenaline or forskolin. However, addition of SITS (0.5 mM) did not prevent the intracellular acidosis induced by these agonists. If monolayers were first treated with forskolin (10 microM), the intracellular pH fell, when stabilized, subsequent addition of amiloride caused a further fall in pHi. When incubated in a Cl-free solution (Cl substituted by gluconate), cell monolayers exhibited a pHi of 7.23 +/- 0.07 (values not significantly different from monolayers incubated in HCO3 solution).(ABSTRACT TRUNCATED AT 250 WORDS)