valinomycin and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

Effect of potassium channel openers on membrane potential of rat liver mitochondria was studied. It has been found that potassium channel opener RP 66471 induces depolarization of the mitochondrial membrane. Since neither the inhibition of mitochondrial respiration nor the uncoupling of mitochondria was observed concomitantly, the specific effect on the mitochondrial potential is postulated. Most likely the effect is caused by the increase of permeability of the inner mitochondrial membrane to potassium ions. Interestingly, however, it was found that no other potassium channel openers tested but RP 66471 was able to induce depolarization of mitochondrial membrane.

Topics: Animals; Antihypertensive Agents; Benzoates; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Intracellular Membranes; Ion Channel Gating; Kinetics; Light; Lipid Bilayers; Membrane Potentials; Mitochondria, Liver; Niacinamide; Nicorandil; Potassium; Potassium Channels; Pyridines; Rats; Scattering, Radiation; Valinomycin

Phospholipid vesicles loaded with Quin-2 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) have been used to investigate the effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin. At an external pH of 7.0, a delta pH (inside basic) of 0.4-0.6 U decreases the rate of Ca2+ transport into the vesicles by severalfold under some conditions. The apparent extent of transport is also decreased. In contrast, raising the pH by 0.4-0.6 U in the absence of a delta pH increases both of these parameters, although by smaller factors. The relatively large effects of a delta pH on the transport properties of Ca2+ ionophores seem to reflect a partial equilibration of the transmembrane ionophore distribution with the H+ concentration gradient across the vesicle membrane. This unequal distribution of ionophore can cause a very slow or incomplete ionophore-dependent equilibration of delta pCa with delta pH. A second factor of less certain origin retards full equilibration of delta pCa when delta pH = 0. These findings call into question several ionophore-based methods that are used to investigate the regulatory activities of Ca2+ and other divalent cations in biological systems. Notable among these are the null-point titration method for determining the concentration of free cations within cells and the use of ionophores plus external cation buffers to calibrate intracellular cation indicators. The present findings also indicate that the transport mode of Ca2+ ionophores is more strictly electroneutral than was thought, based upon previous studies.

Topics: Aminoquinolines; Biological Transport; Calcimycin; Calcium; Fluoresceins; Ionomycin; Ionophores; Kinetics; Liposomes; Membrane Potentials; Models, Biological; Nigericin; Osmolar Concentration; Phosphatidylcholines; Structure-Activity Relationship; Valinomycin

Intracellular pH (pHi) regulation has been investigated in cells isolated from the proximal ceca of the chicken. pHi was measured with the pH-sensitive dye, 2',7'-bis(carboxyethyl)-5 (6)-carboxyfluorescein in nominally HCO(3-)-free solutions. Under resting conditions the pHi was 7.08. Removal of extracellular Na+ decreased pHi by approx. 0.24 pH units and the subsequent addition of Na+ increased pHi towards the control value. This Na(+)-dependent pHi recovery was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Following an intracellular acidification, by abrupt withdrawal of NH4Cl, pHi alkalinized in the nominally absence of Na+. Rotenone, N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, iodoacetic acid and SCH 28080 inhibited the Na(+)-independent pHi recovery rate by 82, 82, 67, 74, 77 and 50% respectively. Bafilomycin A1 was without effect. Na(+)-independent cell alkalization was stimulated by external K+. In the presence of N-ethylmaleimide addition of Na+ induced a rapid pHi recovery. The initial rate of this recovery exhibited first-order dependence on Na+ concentration and it was inhibited by EIPA. The initial rate of Na(+)-dependent cell alkalization increased with a Hill coefficient greater than one when pHi was reduced from 7.2 to 6.2. The 'set point' for the exchanger is approx. 7.5. These studies demonstrate that in cecal epithelial cells exist at least two mechanisms for proton secretion: a Na(+)-H+ exchanger and a Na(+)-independent proton transport system.

Topics: 4-Chloro-7-nitrobenzofurazan; Animals; Anti-Ulcer Agents; Cecum; Cells, Cultured; Chickens; Dicyclohexylcarbodiimide; Digitonin; Epithelium; Ethylmaleimide; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Imidazoles; Iodoacetates; Iodoacetic Acid; Kinetics; Nigericin; Rotenone; Sodium; Spectrometry, Fluorescence; Time Factors; Valinomycin

Activated neutrophils undergo a large burst of metabolic acid generation, yet maintain their cytosolic pH (pHi) within physiological limits. To analyze the underlying regulatory mechanisms, pHi was measured fluorimetrically in suspensions of human neutrophils. In acid loaded but otherwise unstimulated cells, pHi recovered rapidly via Na+/H+ exchange. Upon Na+ removal, recovery from an imposed acid load was negligible. Phorbol ester activation of acidified cells induced a rapid recovery of pHi partly due to a Zn(2+)-sensitive H(+)-conductive pathway. A third component of the regulatory response was apparent in Na(+)-free media containing Zn2+. Acid extrusion through this alternate pathway was voltage sensitive and capable of translocating H+ equivalents against their electrochemical gradient. This active H+ transport was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by nanomolar doses of bafilomycins A1 or B1, suggesting the involvement of vacuolar (V)-type H+ pumps. Cytosolic alkalinization was accompanied by extracellular acidification, indicative of translocation of H+ equivalents across the surface membrane and consistent with the sensitivity of the alkalinization to changes in plasma membrane potential. The activity of the V-type H+ pumps was virtually undetectable in resting cells, becoming apparent only after treatment with phorbol esters or other, chemically unrelated agonists of protein kinase C. These H+ pumps are likely to play a role in pHi homeostasis during the metabolic burst that accompanies neutrophil activation during infection and inflammation.

Topics: Adult; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carrier Proteins; Cell Membrane; Cytosol; Diglycerides; Fluoresceins; Homeostasis; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Neutrophils; Onium Compounds; Protein Kinase C; Proton-Translocating ATPases; Sodium-Hydrogen Exchangers; Spectrometry, Fluorescence; Tetradecanoylphorbol Acetate; Vacuoles; Valinomycin

To study the control of renal ammoniagenesis, a technique was developed to estimate simultaneously intracellular (pHi) and intramitochondrial (pHm) pH in suspensions of rat renal cortical tubules. pHi was estimated with the fluorescent probe 2',7'biscarboxyethyl-5(6)-carboxy-fluorescein (BCECF). The intracellular distribution of the weak acid 5,5-dimethyloxazolidine-2,4-dione (DMO) allowed calculation of pHm with the use of values of pHi obtained with BCECF and tubule mitochondrial content. At medium pH (pHe) 7.4, pHi was 7.08 +/- 0.02. Over the pHe range 7.0-7.7, pHi was linearly related to pHe, but the pH gradient across the cell membrane decreased as pHe was lowered. No difference in the relationship between pHe and pHi was obtained when tubules were incubated in the presence of a nonbicarbonate or bicarbonate-buffered medium. Changes in pHe with bicarbonate-buffered media resulted in identical pHi values, whether the changes were induced by altered bicarbonate or CO2 content. At pHe 7.4, pHm was 7.78 +/- 0.6 in bicarbonate-buffered medium but was higher (0.2-0.3 pH units) when tubules were bathed in nonbicarbonate-buffered medium. pHm was linearly related to pHi in either buffer. The pH gradient across the inner mitochondrial membrane was also positively correlated with pHe. The present studies indicate the suitability of the techniques for estimating pHi and pHm simultaneously in suspensions of rat renal cortical tubules. Parallel changes occur in both intracellular compartments when pHe is altered. pHm, which is approximately 0.7 pH units greater than pHi, decreases in acute acidosis. This decrease may be important in stimulating renal ammoniagenesis, possibly by activation of alpha-ketoglutarate dehydrogenase.

Topics: Animals; Citrate (si)-Synthase; Cytosol; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Imidazoles; Kidney Cortex; Kidney Tubules; Male; Mitochondria; Protons; Rats; Rats, Inbred Strains; Spectrometry, Fluorescence; Valinomycin

Cultured rat hepatocytes were treated with potassium cyanide, an inhibitor of cytochrome oxidase; valinomycin, a K+ ionophore; carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophore; and the ATP synthetase inhibitor oligomycin. The effect of these agents on the viability of the cells was related to changes in ATP content and the deenergization of the mitochondria. The ATP content was reduced by over 90% by each inhibitor. All of the agents except oligomycin killed the cells within 4 h. With the exception of oligomycin, the mitochondrial membrane potential as measured by the distribution of [3H]triphenylmethylphosphonium collapsed with each of the agents. Monensin, a H+/Na+ ionophore, potentiated the toxicity of cyanide and CCCP, whereas the toxicity of valinomycin was reduced. The effect of cyanide and monesin on the cytoplasmic pH of cultured hepatocytes was measured with the fluorescent probe, 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Cyanide promptly acidified the cytosol, and the addition of 10 microM monensin caused a rapid alkalinization of the cytosol. A reduction of pH of the culture medium from 7.4 to 6.6 and 6.0 prevented the cell killing both by cyanide alone and by cyanide in the presence of monensin. However, neither monensin nor extracellular acidosis had any effect on the loss of mitochondrial energization in the presence of cyanide. It is concluded that ATP depletion per se is insufficient to explain the cell killing with cyanide, CCCP, and valinomycin. Rather, cell killing is better correlated with a loss of mitochondrial energization. With cyanide an intracellular acidosis interferes with the mechanism that couples collapse of the mitochondrial membrane potential to lethal cell injury.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Survival; Cells, Cultured; Cytoplasm; Drug Interactions; Energy Metabolism; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Liver; Male; Membrane Potentials; Mitochondria, Liver; Monensin; Oligomycins; Onium Compounds; Potassium Cyanide; Rats; Rats, Inbred Strains; Trityl Compounds; Valinomycin

The transmembrane pH gradient maintained by nonrespiring, uncoupled heart mitochondria has been estimated using the distribution of methylamine and of 5,5-dimethyl-2,4-oxazolidinedione (DMO) and compared with the delta pH reported by the fluorescent probe 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under these conditions the protonmotive force approaches zero and the membrane potential (delta psi) should equal 59 delta pH (P. Mitchell and J. Moyle (1969) Eur. J. Biochem. 7, 471-484). The delta pH reported by DMO corresponds closely to that estimated by BCECF and is consistent with a Donnan potential of no greater than about -30 mV (interior negative) for nonenergized mitochondria in a sucrose medium. This potential appears to result from the presence of immobile negative charges in the matrix and is eliminated by addition of 10 to 25 mM KCl. Measurements of delta pH using the methylamine and of delta tsi using the distribution of 42K+ in the presence of valinomycin result in an apparent overestimation of these parameters due to binding of these components to negative sites on the membrane. Increasing ionic strength decreases this contribution of surface potential, but significant binding can still be detected in 100 mM KCl. These studies suggest that 42K+ (or 86Rb+) is far from an ideal probe for measuring delta tsi in respiring mitochondria and may significantly overestimate this parameter, especially in sucrose media.

Topics: Animals; Cattle; Diffusion; Dimethadione; Fluoresceins; Hydrogen-Ion Concentration; Membrane Potentials; Methylamines; Mitochondria, Heart; Osmolar Concentration; Oxazoles; Oxygen Consumption; Potassium; Rubidium; Valinomycin

The pH-sensitive, fluorescent, cytoplasmic-trapped dye 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to measure intracellular (pHi) and pH electrode to measure extracellular pH (pHo) in suspensions of gastric glands isolated from rabbit stomachs. The fluorescence of BCECF-loaded glands was calibrated in terms of pHi by equilibrating pHo and pHi using ionophores or digitonin and titrating pHo to different values. An APPENDIX is included that covers details of dye calibration and interpretation of fluorescence signals. Glands incubated in NaCl Ringer solution had pHi 7.11. Na+-free Ringer solution caused pHi to decrease reversibly to 6.80. Na+-dependent alkalinization of pHi followed a similar time course to the acidification of pHo. These changes were blocked by 1 mM amiloride. When gland cells were acidified (using two different techniques) realkalinization was completely Na+ dependent but was independent of the presence of Cl-; also, neither high extracellular K+ concentration ([K+]o) nor high [K+]o plus 10(-5) M valinomycin affected the rates of Na+-dependent alkalinization. A neutral Na+-H+ exchanger was implicated. Glands also exhibited Cl(-)-dependent changes of pHi that were blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (2 X 10(-4) M). A Cl(-)-OH-(HCO3-) exchanger was indicated. Other studies showed that intracellular buffering capacity was approximately 45 mM (pH-1) and that the apparent proton conductance of gland cell membranes was small.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Ammonium Chloride; Animals; Carrier Proteins; Chloride-Bicarbonate Antiporters; Chlorides; Digitonin; Fluoresceins; Gastric Mucosa; Hydrogen-Ion Concentration; Male; Monensin; Nigericin; Ouabain; Potassium; Rabbits; Rotenone; Sodium; Sodium-Hydrogen Exchangers; Trialkyltin Compounds; Valinomycin