We have developed a technique to measure the fluorescence of a pH-sensitive dye (2,7-biscarboxyethyl-5(6)-carboxyfluorescein) in single glomerular mesangial cells in culture. The intracellular fluorescence excitation ratio of the dye was calibrated using the nigericin-high-K+ approach. In the absence of CO2-HCO3-, mesangial cells that are acid loaded by an NH+4 prepulse exhibit a spontaneous intracellular pH (pHi) recovery that is blocked either by ethylisopropylamiloride (EIPA) or removal of external Na+. This pHi recovery most probably reflects the activity of a Na+-H+ exchanger. When the cells are switched from a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution to one containing CO2-HCO3-, there is an abrupt acidification due to CO2 entry, which is followed by a spontaneous recovery of pHi to a steady-state value higher than that prevailing in HEPES. Both the rate of recovery and the higher steady-state pHi imply that the application of CO2-HCO3- introduces an increase in net acid extrusion from the cell. One third of total net acid extrusion in CO2-HCO3- is EIPA sensitive and most likely is mediated by the Na+-H+ exchanger. The remaining two thirds of acid extrusion could be caused by a decrease in the background acid-loading rate and/or the introduction of a new, HCO3- -dependent acid-extrusion mechanism. The HCO3- -induced alkalinization cannot be accounted for by a HCO3- -induced reduction in the acid-loading rate. The latter can be estimated by applying EIPA in the absence of HCO3- and observing the rate of pHi decline. We found that this acid-loading rate is only about one fifth as great as the total net acid extrusion rate in the presence of HCO3-. Indeed, two thirds of net acid extrusion in HCO3- is blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), an inhibitor of HCO3- -dependent transport. Furthermore, the effects of EIPA and SITS were additive. Thus, in the presence of CO2-HCO3-, a SITS-sensitive-HCO3- -dependent transporter is the dominant mechanism of acid extrusion. This mechanism also accounts for the increase in steady-state pHi on addition of CO2-HCO3-.

Growth factors raise intracellular pH (pHi) by stimulating Na+/H+ exchange in the absence of HCO3-. In mutant cells that lack the Na+/H+ exchange activity, this alkalinization does not occur, and the cells do not proliferate without artificial elevation of pHi. It has therefore been widely suggested that an early pHi increase is a necessary signal for mitogenesis. In the presence of HCO3- however, growth factors fail to raise pHi in A431 cells, renal mesangial cells and 3T3 fibroblasts. In mesangial cells, arginine vasopressin (AVP) raises pHi in the absence of HCO3-, but lowers it when HCO3- is present; growth is stimulated under both conditions. We report here that, in the presence of HCO3-, AVP stimulates two potent HCO3- transporters, as well as the Na+/H+ exchanger. These are the Na+-dependent and Na+-independent Cl-/HCO3- exchangers. Our results indicate that AVP causes acidification in the presence of HCO3- because, at the resting pHi, it stimulates Na+-independent Cl-/HCO3- exchange (which lowers pHi) more than it stimulates the sum of Na+/H+ exchange and Na+-dependent Cl-/HCO3- exchange (both of which raise pHi). The stimulation of three acid-base transporters by the growth factor AVP greatly enhances the ability of the cell to regulate pHi.

1. We used the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to study the regulation of intracellular pH (pH1) in single pyramidal neurons freshly isolated from the hippocampal CAI region of immature (2-to 10-day-old) and more mature (21-to 30-day-old) rats.2. Whether isolated from immature or mature rats, neurons had a broad range of initial pHi values (6 3-7 7) when the cells were examined in solutions buffered with Hepes and no CO2/HCO3-. The initial pH, distribution for neurons isolated from immature rats was best fitted with a Gaussian distribution with a mean of 6-95. In contrast, the initial pHi distribution for neurons isolated from mature rats was best fitted with the sum of two Gaussian distributions with means of 6-68 and 7-32.3. When neurons with a relatively low initial pHi in Hepes-buffered solutions were acid loaded, pH, recovered very slowly. Neurons with a relatively high initial pHi recovered rapidly. The rate constant for the exponential pHi recovery increased with initial pHi. All pHi recoveries required Nae. 4. Both for neurons with a relatively high (>7 705) and a relatively low (<7 05) initial pHi, net acid extrusion rates (Jttal = dpHi/dt x buffering power) decreased linearly with increasing pH,. Compared with the line for neurons with a relatively low initial pHi, that for neurons with a relatively high pH1 had a significantly greater slope and was alkaline shifted by 0-6-0-7 pH units.5. Removing external Nae in the absence of C02/HC03-caused pH1 to decrease by -0 3 in neurons with a relatively low initial pH,, and by -0 5 in neurons with a relatively high initial pHi. This initial acidification was followed by a slower, partial pH, recovery in -32% of neurons with a relatively low initial pH,, but only -14 % of neurons with a relatively high pHi.6. When exposed to C02/HC03-, all neurons initially acidified. Neurons with a relatively low initial pHi recovered to a pHi -0-2 pH units greater than the initial value. Among neurons with higher initial pHi values, some did not recover at all, whereas others recovered to a value similar to or above the initial pHi. On average, the final C02/HC03-pH1 for neurons with a relatively high initial pH1 was similar to the pHi in Hepes buffer. Neurons with a relatively high pHi in Hepes buffer continued to be more alkaline (by -0f2 pH units) in C02/HC03.

We used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to further characterize the mechanisms of intracellular pH (pHi) regulation in renal mesangial cells. In the accompanying paper [Am. J. Physiol. 255 (Cell Physiol. 24): C844-C856, 1988], we showed that acid extrusion from mesangial cells is mediated by both an ethylisopropylamiloride (EIPA)-sensitive Na+-H+ exchanger and a 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-sensitive-HCO3(-)-dependent mechanism. In this study, we examined the ionic dependencies of pHi-regulatory mechanisms in the presence of CO2-HCO3-. We found that in CO2-HCO3-, approximately 90% of the net acid extrusion occurring during recovery from an acid load is blocked by removing external Na+. Short-term (less than 15 min) removal of external Cl- has little effect on the rate of recovery in CO2-HCO3-. In contrast longer periods of external Cl- removal (1-2 h) blocks 40-60% of the rate of recovery, which is consistent with the hypothesis that a large fraction of the SITS-sensitive-HCO3(-)-dependent recovery mechanism described in the preceding paper is also Na+- and Cl(-)-dependent. Therefore, this Cl(-)-dependent component is probably mediated by a Na+-dependent Cl(-)-HCO3- exchanger. As much as 16% of total acid extrusion is insensitive to EIPA and long-term Cl- removal but is blocked by SITS. Thus either 1-2 h of Cl- removal is insufficient to wash out all internal Cl-, or a small component of acid extrusion is mediated by a Cl(-)-independent mechanism, such as the electrogenic Na+/HCO3- cotransporter. We also studied the effect on pHi of the removal and readdition of external Cl-, observing pHi changes consistent with the existence of a Na+-independent Cl(-)-HCO3- exchanger, which would presumably function as an acid loader. In contrast to the Na+-H+ exchanger and Na+-dependent Cl(-)-HCO3- exchanger, which are stimulated at low pHi, the Cl(-)-HCO3- exchanger is stimulated at high pHi. Thus the acid-extruding and acid-loading mechanisms have opposite pHi dependencies.

We used the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to monitor intracellular pH (pHi) in single astrocytes cultured from the forebrain of neonatal rats. When exposed to a nominally CO2/HCO3(-)-free medium buffered to pH 7.40 with HEPES at 37 degrees C, the cells had a mean pHi of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO2 and 25 mM HCO3(-) caused the steady-state pHi to increase by an average of 0.35, suggesting the presence of a HCO3(-) -dependent acid-extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HCO3(-)-free (HEPES-buffered) solutions, the application and withdrawal of 20 mM extracellular NH4+ caused pHi to fall to a value substantially below the initial one. pHi spontaneously recovered from this acid load, stabilizing at a value approximately 0.1 higher than the one prevailing before the application of NH4+. In other experiments conducted on cell bathed in HEPES-buffered solutions, removing extracellular Na+ caused pHi to decrease rapidly by 0.5. Returning the Na+ caused pHi to increase rapidly, indicating the presence of an Na(+)-dependent/HCO3(-)-dependent acid-extrusion mechanism; the final pHi after returning Na+ was approximately 0.08 higher than the initial value. This pHi recovery elicited by returning Na+ was not substantially affected by 50 microM ethylisopropylamiloride (EIPA), but was speeded up by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS).(ABSTRACT TRUNCATED AT 250 WORDS)