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Single isolated rat hepatocytes were used to investigate the influence of intracellular pH (pHi) on hormone-induced cytosolic Ca2+ oscillations, using videofluorescence microscopy. Although pHi did not vary after alpha-adrenergic stimulation, manipulations of pHi induced pronounced alterations in the frequency of oscillations. Increasing the resting pHi with ammonium chloride (5-20 mM), trimethylammonium (2-10 mM), or triethylammonium (1.2-8 mM) reduced the frequency of oscillations. A change in pHi of > 0.25 was sufficient to reversibly inhibit oscillations. This effect could be overcome by increasing the agonist concentration or by adding 8-bromoadenosine 3',5'-cyclic monophosphate, an agent known to potentiate the alpha-adrenergic response. Cellular acidification, obtained by the ammonium prepulse method as well as by application of acetate or the ionophore nigericin, in the continuous presence of agonist was accompanied by a modest frequency increase of the oscillations, leading in some cases to an overstimulated state. This study indicates that pHi, within a range of values expected to occur in vivo (0.1-0.2 pH units), exerts a chronotropic effect on phenylephrine-induced Ca2+ oscillations. In contrast, oscillations induced by ADP or vasopressin were pHi invariant.

The lateral intercellular spaces (LIS) of reabsorptive epithelia are the site of the proposed local osmotic gradient responsible for transepithelial transport. We developed techniques for loading the LIS of living cultured renal cells (MDCK and LLC-PK1) with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), visualizing LIS geometry, measuring pH, and determining the BCECF diffusion coefficient within the LIS. The LIS pH was remarkably constant and differed substantially from that of the superfusate in both the presence and absence of HCO3 or CO2. The LIS of MDCK cells had a pH of 7.66 +/- 0.04 in bicarbonate-free solutions of pH 7.0, 7.4, or 7.8. In bicarbonate-containing solutions, MDCK LIS pH was acidic to the superfusate by 0.3-0.4 units. In the absence of bicarbonate, the LIS of LLC-PK1 cells was markedly acidic (6.83 +/- 0.05), becoming alkaline by approximately 0.25 units in the presence of bicarbonate. Gradients in pH or dye concentration were not detected within the LIS. The diffusion coefficient of BCECF within the LIS was approximately equal to that seen in free solution.

To assess the role of nonionic diffusion of salicylic acid (pKa = 3) in the terminal nephron, we measured the passive permeability of [14C]salicylic acid in rabbit cortical collecting ducts isolated and perfused in vitro. This segment can produce and maintain a maximal pH gradient between blood and tubular fluid. When peritubular pH was kept constant at pH 7.4 the apparent permeability of salicylic acid (P', 10(-6) cm/s) was 6.2 +/- 1.1 at a luminal pH of 6.0, 17.2 +/- 5.3 at a luminal pH of 5.5, and 39.0 +/- 4.7 at a luminal pH of 5.0. These permeabilities were in close correlation with the percentage of nonionized salicylic acid present at each pH, indicating that only the nonionized molecule can diffuse across the collecting duct epithelium. By recalculating the permeability, taking into account only the concentration of the nonionized salicylic acid molecules, we obtained the apparent permeability of nonionized salicylic acid, which was no longer pH dependent and averaged 4,345 +/- 460 x 10(-6) cm/s. The apparent activation energy of this diffusion process was 9.3 +/- 1.2 kcal/mol as calculated from an Arrhenius plot.