sq-23377 and propionic-acid

The hypothesis that changes in cytosolic pH effect the release from intracellular compartments of stored calcium in Trypanosoma brucei was addressed by the use of procyclic and bloodstream trypomastigotes of T. brucei loaded with the fluorescent reagents 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) to measure intracellular pH (pHi), or fura 2 to measure intracellular free calcium ([Ca2+]i). Experiments were performed in EGTA-containing buffers, so increases in [Ca2+]i reflected release of stored calcium rather than Ca2+ entry. Nigericin reduced pHi and increased [Ca2+]i in loaded cells, whilst propionate reduced pHi, but did not affect [Ca2+]i, and NH4Cl increased both variables, so there appears to be no correlation between pHi and [Ca2+]i. Treatment of the cells with the calcium ionophore ionomycin under similar conditions (nominal absence of extracellular Ca2+) resulted in an increase of [Ca2+]i which was greatly increased by addition of either NH4Cl, nigericin or the vacuolar H(+)-ATPase inhibitor bafilomycin A1. Similar results were obtained when the order of additions was reversed or when digitonin-permeabilized cells were used with the Ca2+ indicator arsenazo III. The results suggest that more Ca2+ is stored in this acidic compartment in procyclic than in bloodstream forms. Taking into account the relative importance of the ionomycin-releasable and the ionomycin-plus-NH4Cl-releasable Ca2+ pools, it is apparent that a significant amount of the Ca2+ stored in T. brucei trypomastigotes is present in the acidic compartment thus identified.

Topics: Ammonium Chloride; Animals; Anti-Bacterial Agents; Calcium; Cytosol; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Ionomycin; Ionophores; Kinetics; Macrolides; Nigericin; Propionates; Proton-Translocating ATPases; Trypanosoma brucei brucei; Vacuoles

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.

Topics: Ammonium Chloride; Animals; Cells, Cultured; Cyclic AMP; Cyclic GMP; Hydrogen-Ion Concentration; Ionomycin; Isoproterenol; Male; Nifedipine; Phorbol Esters; Pineal Gland; Propionates; Protein Kinase C; Rats; Rats, Sprague-Dawley

Using a Coulter-based cell sizing method, we have previously demonstrated that, in response to cytoplasmic acidification by 140 mmol/l sodium propionate, both the mean initial rate of amiloride-sensitive platelet volume swelling and the net volume change achieved at steady-state are greater in essential hypertensives than in normotensives. In the present study, we extend this observation by showing that, in response to graded propionate exposure (56-140 mmol/l), steady-state amiloride-sensitive volume responsiveness (as percentage increase over baseline) increases linearly, and the mean slope of the line relating amiloride-sensitive volume change and propionate concentration is increased in hypertensives (0.40 +/- 0.02 versus 0.32 +/- 0.02% per mmol/l propionate, P less than 0.003). In 56 mmol/l propionate, average amiloride-sensitive platelet swelling is significantly less in hypertensives than in normotensives (7.6 +/- 0.8 versus 11.1 +/- 0.9%, P less than 0.05), but in 140 mmol/l propionate, swelling is significantly increased in hypertensives (40.8 +/- 1.7 versus 36.2 +/- 1.5%, P less than 0.05). Since platelet intracellular calcium concentration is elevated in some hypertensives and Ca2+ is known to stimulate Na(+)-H+ antiport, the transport system that is the primary determinant of amiloride-sensitive cell swelling during propionate incubation, we studied the effects of the Ca2+ ionophore, ionomycin, on volume regulation. In both normotensives and hypertensives, ionomycin (2 x 10(-10 to 2 x 10(-7) mol/l) causes dose-related increases in amiloride-sensitive platelet swelling during graded propionate exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Amiloride; Blood Platelets; Calcium; Carrier Proteins; Female; Humans; Hydrogen-Ion Concentration; Hypertension; Ionomycin; Male; Propionates; Sodium-Hydrogen Exchangers

The new fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) was used for determination of the cytosolic free Na+ concentration, [Na+]i, in human platelets. The dye could be loaded into platelets in the form of its acetoxymethyl ester (SBFI-AM). Calibration of the fluorescence in terms of [Na+]i was done by measuring the 345/385 nm excitation ratio (emission 490 nm) at various extracellular Na+ concentrations, [Na+]o, in the presence of gramicidin D. The 345/385 intensity ratio increased almost linearly when [Na+]i was stepwise raised from 20 to 60 mM. The basal value for [Na+]i was found to be 26.0 +/- 4.5 mM (n = 15). Incubation of platelets in Na(+)-free buffer decreased [Na+]i, whereas inhibition of the (Na+ + K+)-ATPase by 0.5 mM ouabain increased [Na+]i to 56 +/- 4 mM (n = 4) within 60 min. Activation of Na+/H+ exchange by exposing platelets to propionic acid also raised [Na+]i, and a comparable effect was produced by the Na+/H+ ionophore monensin. Activation of platelets with thrombin (0.1-0.5 unit/ml) also increased the 345/385 nm intensity ratio, an effect that was not seen in Na(+)-free buffer or after raising intracellular cAMP by treatment of platelets with prostaglandin E1. On the average, [Na+]i was raised to 59.5 +/- 5.3 mM (n = 15) at 10 min after addition of thrombin without a significant decrease for further 10 min. An increase in [Na+]i was also seen when platelets were challenged with the Ca2+ ionophore ionomycin, an effect that did not occur in the absence of Na+o. Our findings confirm earlier reports which demonstrated a rise in [Na+]i in stimulated platelets and show that SBFI is a useful tool for determination of [Na+]i in resting and stimulated platelets.

Topics: Alprostadil; Benzofurans; Blood Platelets; Carrier Proteins; Cyclic AMP; Cytosol; Ethers, Cyclic; Fluorescent Dyes; Humans; Ionomycin; Monensin; Ouabain; Platelet Activation; Propionates; Sodium; Sodium-Hydrogen Exchangers; Sodium-Potassium-Exchanging ATPase; Spectrometry, Fluorescence; Thrombin

In granulocytes harvested from human blood, an elevation of the cytosolic concentration of Ca2+ ions is by itself insufficient to activate the cell's respiratory burst. We report herein that, when granulocytes are "primed" by a 90-min preincubation with the recombinant human hemopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSFrh), elevation of the concentration of cytosolic Ca2+ ions ([Ca2+]i) becomes a more effective transduction signal capable of triggering the generation of substantial quantities of superoxide (O2-) anions by the cell. In these studies, we used four separate and independent maneuvers to induce elevation of [Ca2+]i: 1) depolarization of the cell's electrical potential through obliteration of the transmembrane Na+ and K+ gradients; 2) acidification of the cytoplasm using propionic acid; 3) addition of the calcium ionophore ionomycin; and 4) treatment of the cells with the monoclonal antibody to the C3bi receptor, PMN7C3. In all cases, elevation of [Ca2+]i through these manipulations resulted in the release of substantially greater quantities of O2- by GM-CSFrh-primed granulocytes than by unprimed, control cells. The generation of O2- was in all cases markedly reduced by chelation of either intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or extracellular Ca2+ with [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid. We conclude that during the process of GM-CSFrh priming, the metabolic assembly responsible for O2- anion production in the granulocyte becomes altered in such a way that a subsequent elevation in [Ca2+]i provides a potent signal for its activation.

Topics: Antibodies, Monoclonal; Calcium; Colony-Stimulating Factors; Ethers; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocytes; Growth Substances; Humans; Hydrogen-Ion Concentration; Ionomycin; Membrane Potentials; Oxidation-Reduction; Potassium; Propionates; Receptors, Complement; Recombinant Proteins; Superoxides; Valinomycin