4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and propionic-acid

Short-chain fatty acids (SCFAs) are the major solutes and the major anions in the colonic lumen. We studied the response of suspended HT29-18-C1 cells (an epithelial cell line derived from a human colon carcinoma) to SCFA exposure. Cellular response was evaluated by measurement of cell volume (Coulter counter), intracellular pH [pHi; measured fluorometrically with 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF)], and intracellular Na+, K+, and Cl- content (flame photometry and chloride titrator). Exposure to 130 mM propionate in isosmotic medium causes a rapid decrease in pHi and activates pHi recovery via amiloride-sensitive Na-H exchange. In the presence of propionate, Na-H exchange also causes cell swelling to a peak volume 11% above control cells and causes a 2.8-fold increase in intracellular Na+ content. After peak swelling, a regulatory-volume decrease (RVD) significantly reduced volume and intracellular Na+ returned to baseline. Other SCFAs (acetate, butyrate, and valerate) also elicit swelling and RVD. Activation of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) is required to return Na+ to normal levels and to indirectly provide ion gradients required for propionate-induced RVD, but Na(+)-K(+)-ATPase activity does not directly mediate RVD. When 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) is added in the presence of propionate, RVD was inhibited and cell Na+ content increased. Cl- depletion inhibited propionate-induced RVD and diminished the effect of SITS.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Biological Transport; Chlorides; Colon; Humans; Hypotonic Solutions; Ions; Propionates; Sodium; Tumor Cells, Cultured

36Cl- efflux was studied in the isolated rat lens under two conditions that are known to decrease internal pH. The first follows exposure to a pulse of ammonium chloride (50 mM) and the second accompanies exposure to an acidified propionate (20 mM) solution. Under acidifying conditions, a stimulation in 36Cl- efflux was observed, that was abolished on removing external Na+ and also on removing external Cl- and HCO3-. In the absence of external Cl-, the presence of HCO3- (16 mM) resulted in an increase in 36Cl- efflux during internal acidification. In the absence of internal acidification, the addition of 0.1 mM dibutyrylcAMP or 0.5 mM IBMX to the external medium produced a rapid increase in 36Cl- efflux. This stimulation was reduced by 0.2 mM SITS. Neither cAMP or IBMX had any significant effect on the electrical resistance of the lens membranes. It is suggested that a coupled SITS-sensitive, Na(+)-Cl(-)-H(+)-HCO3- exchange mechanism is activated when the lens internal pH falls and further that cAMP may play a role in regulating this mechanism.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Ammonium Chloride; Animals; Carrier Proteins; Chloride-Bicarbonate Antiporters; Chlorides; Hydrogen-Ion Concentration; Lens, Crystalline; Membrane Potentials; Membrane Proteins; Microelectrodes; Propionates; Rats; Rats, Inbred Strains; Sodium

Double-barrelled pH-sensitive microelectrodes were used to measure the intracellular pH (pHi) of neuropil glial cells and the pH of extracellular spaces (pH(e)) within isolated, intact ganglia of the leech Hirudo medicinalis. By application of a weak acid (propionate, 40 mM) or a weak base (ammonium, 20 mM) the total buffer capacity was estimated by changes in glial pHi and in pH(e). The buffering power of glial cells and in the extracellular spaces was increased by up to threefold in the presence of CO2/bicarbonate. The anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3-0.5 mM) reversed this increase in buffering power both in the glial cells and in the extracellular spaces. Inhibitors of the carbonic anhydrases reduced the CO2/bicarbonate-dependent increase in extracellular buffering power. The results suggest that extracellular H+ buffering dependent upon the availability of bicarbonate is linked to DIDS-sensitive bicarbonate transport across the glial membrane.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Bicarbonates; Buffers; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Extracellular Space; Hydrogen-Ion Concentration; Intracellular Fluid; Leeches; Microelectrodes; Neuroglia; Propionates

1. We measured intracellular pH (pHi) in snail neurones using pH-sensitive glass microelectrodes. We then calculated the intracellular buffering power (beta i) from the pHi changes associated with the influx or efflux of a variety of weak acids or bases. 2. The weak acid anions butyrate and propionate (20 mM) gave similar values for beta i but those measured using 20 mM-acetate were on average twice as great. 3. Although solutions were nominally CO2-free, blockage of pHi regulation with SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) increased the sizes of the pHi changes upon weak acid addition and removal. The corresponding measured values of beta i were on average 26% lower with SITS than without. 4. With pHi regulation blocked, the use of 2.7% CO2 to measure beta i gave beta i values similar to those measured with butyrate or propionate. These values were about 50% less than those previously measured in snail neurones using CO2. 5. beta i values calculated from the pHi changes due to the removal of 5 mM of the weak bases trimethylamine, procaine and NH4Cl were all similar and comparable to those measured using butyrate or propionate. Removing the influence of pHi regulation on the undershoots after NH4Cl removal was found to decrease the apparent measured values of beta i by 10%. 6. Combining all the data (except the values obtained using CO2 and acetate), and adjusting for the errors due to pHi regulation reducing the sizes of the pHi changes, we found that the mean value for beta i was 10.4 +/- 0.6 mM (+/- S.E.M.) at a mean pHi of 7.36 +/- 0.05. 7. We also investigated the relationship between beta i and pHi using ionophoretic acid injection. By means of step-wise injections, with pHi regulation blocked, we found that at normal pHi levels beta i remained relatively constant. However, at a pHi of less than about 6.8 beta i increased with decreasing pHi.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetates; Acetic Acid; Ammonium Chloride; Animals; Buffers; Butyrates; Butyric Acid; Ganglia; Helix, Snails; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Potentials; Methylamines; Neurons; Procaine; Propionates; Time Factors