pH(i) responses to osmotic cell shrinkage in the presence of open-system buffers.

Changes in plasma volume in vivo cause rapid changes in extracellular pH by altering the plasma bicarbonate concentration at a constant Pco(2) (Garella S, Chang BS, and Kahn SI. Kidney Int 8: 279, 1975). Few studies have examined the possibility that changes in cell volume produce comparable changes in intracellular pH (pH(i)).

Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells.

Using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+ concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between approximately 6.

Inadequacy of high K+/nigericin for calibrating BCECF. II. Intracellular pH dependence of the correction.

In the accompanying study [G. Boyarsky, C. Hanssen, and L.

Inadequacy of high K+/nigericin for calibrating BCECF. I. Estimating steady-state intracellular pH.

Intracellular pH (pHi) was measured in single vascular smooth muscle (VSM) cells, cultured from rabbit abdominal aorta, using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) on a microscope-based fluorescence system. Three lines of evidence are presented that using nigericin along with high external K+ to calibrate intracellular BCECF produces systematic errors in pHi. 1) The intrinsic buffering power (beta int), measured using weak bases (e.

Superiority of in vitro over in vivo calibrations of BCECF in vascular smooth muscle cells.

We measured intracellular pH (pHi) in single vascular smooth muscle cells (VSM) cultured from rabbit abdominal aorta, using 2',7'-biscarboxyethyl-5(6)carboxyfluorescein (BCECF) on a microscope-based fluorimetric system. We previously found substantial errors introduced by using high K+/nigericin to calibrate intracellular BCECF (1). We also previously demonstrated that the necessary correction (pHcor) to the high K+/nigericin-calibrated pHi was linearly dependent on pHi, increasing with increasing pHi (2).

pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats.

1. We used the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to study the regulation of intracellular pH (pHi) in single pyramidal neurons freshly isolated from the hippocampal CA1 region of immature (2- to 10-day-old) and more mature (21- to 30-day-old) rats. 2.

Intracellular pH regulation in single cultured astrocytes from rat forebrain.

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.

Effect of diabetes on Na(+)-H+ exchange by single isolated hepatocytes.

We used the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to examine intracellular pH (pHi) regulation in single hepatocytes isolated from control rats and rats with either spontaneous or drug-induced diabetes mellitus (DM). In the absence of CO2-HCO3-, both control and DM cells recovered from cellular acid loads applied by the NH4+ prepulse technique. Because the pHi recovery was blocked by either Na+ withdrawal or ethylisopropylamiloride in both control and DM cells, it was presumably mediated by Na(+)-H+ exchange.

Intracellular-pH dependence of Na-H exchange and acid loading in quiescent and arginine vasopressin-activated mesangial cells.

We studied intracellular pH (pHi) regulation in the absence of HCO3- in single mesangial cells (MCs) with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(and -6)carboxyfluorescein. Our approach was to acid load the cells by an NH+4 prepulse and to monitor the subsequent pHi recovery. Previous work on MCs and other cells has shown that the recovery is prevented by adding ethylisopropyl amiloride (EIPA) or removing Na+ before the recovery begins, suggesting that at low pHi only Na-H exchange contributes to the recovery.

Arginine vasopressin enhances pHi regulation in the presence of HCO3- by stimulating three acid-base transport systems.

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.

pH regulation in single glomerular mesangial cells. II. Na+-dependent and -independent Cl(-)-HCO3- exchangers.

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.

pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3-.

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+.

Effects of angiotensin II and vasopressin on intracellular pH of glomerular mesangial cells.

We investigated changes in intracellular pH (pHi) of cultured rat glomerular mesangial cells (MCs) exposed to angiotensin II (ANG II) and arginine vasopressin (AVP). pHi of quiescent MCs, passage 2-5, and grown on glass cover slips, was assessed by spectrofluorometry using the pH-sensitive dye, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The steady-state pHi of MCs in a pH 7.

Calcium signals recorded from cut frog twitch fibers containing tetramethylmurexide.

The Ca indicator tetramethylmurexide was introduced into cut fibers, mounted in a double-Vaseline-gap chamber, by diffusion from the end-pool solutions. The indicator diffused rapidly to the central region of a fiber where optical recording was done and, if removed, diffused away equally fast. The time course of concentration suggests that, on average, a fraction 0.