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

Intracellular pH can have profound effects on tissue function, but little is known about how pH is regulated, buffered or affects the function of gastric smooth muscle. As the pH of gastric myocytes may alter with pathophysiological disturbance of the gastric lining, or reduction in blood flow to the stomach, these parameters were investigated. Intracellular pH was measured in strips of corpus from rats and guinea-pigs and pH perturbed by the addition of Na butyrate. pH regulation was investigated using pharmacological inhibitors and ionic substitutions. Resting pH was found to be around 7.0, and buffering power relatively high, compared to other muscles in both species. In the guinea-pig amiloride, EIPA and HOE694 prevented pH regulation from an acid load, but amiloride- and EIPA-insensitive pH-regulating mechanisms were found in the rat. The pH-regulatory mechanism present in the rat was also insensitive to DIDS, SITS and removal of external Cl-, but inhibited by Na+ substitution and HOE694. Acidification reduced gastric tone in both species. We conclude that pH alteration will significantly affect gastric contractility, despite a high capacity of the tissue to buffer and regulate pH change. The sensitivity to NHE inhibitors differs between rat and guinea-pig, suggesting that Na+/H+ exchanger isoform expression differs between gastric tissue.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acid-Base Equilibrium; Amiloride; Animals; Anti-Arrhythmia Agents; Buffers; Butyrates; Diuretics; Gastric Emptying; Gastric Mucosa; Guanidines; Guinea Pigs; Hydrogen-Ion Concentration; Methylamines; Muscle, Smooth; Rats; Sodium; Sulfones

Experiments were performed in isolated cat papillary muscles loaded with the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in the esterified form to study the effect of endothelin-1 (ET-1) on the activity of the Na(+)-independent Cl(-)-HCO(3)(-) exchanger. Exposure to ET-1 (10 nmol/L) raised pH(i) by 0.13+/-0.03 U (P<0.05) in papillary muscles superfused with nominally HCO(3)(-)-free solution, whereas no significant change was detected under CO(2)/HCO(3)(-)-buffered medium. However, if ET-1 was applied to muscles pretreated with the anion exchanger inhibitor 4-acetamido-4'-isothiocyanato-stilbene-2, 2'-disulfonic acid, pH(i) increased by 0.09+/-0.02 U (P<0.05) in the presence of CO(2)/HCO(3)(-) buffer. The rate of pH(i) recovery from trimethylamine hydrochloride-induced intracellular alkaline load was enhanced so that net HCO(3) efflux increased about three times in the presence of ET-1 (2.74+/-0.25 versus 9.66+/-1.29 mmol. L(-1). min(-1) at pH(i) 7.55, P<0.05). This effect was canceled by previous exposure to either 50 nmol/L PD 142,893 (nonselective endothelin receptor blocker) or 300 nmol/L BQ 123 (selective blocker of ET(A) receptors). BQ 123 also abolished angiotensin II-induced activation of the Na(+) independent Cl(-)-HCO(3)(-) exchanger. These results show that ET-1 increases the activity of the Na(+)-independent Cl(-)-HCO(3)(-) exchanger in cardiac tissue through the ET(A) receptors. Furthermore, our data suggest that the previously described angiotensin II-induced stimulation of the anion exchanger activity is mediated by endogenous ET-1.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Alkalies; Angiotensin II; Animals; Antiporters; Bicarbonates; Buffers; Carbon Dioxide; Cats; Chloride-Bicarbonate Antiporters; Endothelin Receptor Antagonists; Endothelins; Hydrogen-Ion Concentration; Methylamines; Myocardium; Oligopeptides; Peptides, Cyclic; Rats; Receptor, Endothelin A; Sodium

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