calcimycin and triphenylmethylphosphonium

We investigated the Na-dependent Ca transport in purified bovine luminal and basolateral renal tubular membranes. Na-dependent Ca uptake was observed in basolateral but not in luminal kidney tubular membranes. 45Ca uptake in basolateral membrane vesicles loaded with Na and suspended in K buffer was significantly greater than that observed in vesicles loaded with Na and suspended in Na buffer. The Na ionophore ETH-227 inhibited Na-dependent Ca uptake indicating that the Ca uptake was dependent on Na gradient. The Ca taken up in the presence of Na gradient could be released by the Ca ionophore A-23187 suggesting that Ca was accumulated inside the vesicles. In vesicles loaded with 45Ca, addition of Na to the media promoted Ca efflux. Methyl triphenylphosponium uptake and Ca uptake were significantly higher in the presence of a Na gradient as compared to those observed in the presence of other monovalent cation gradients, indicating the specificity for Na gradient and arguing against a calcium-activated sodium conductance pathway. The Na-dependent Ca uptake varied with intravesicular Na concentration with an apparent Km of 20-40 mM. The Km for Ca of the Na-dependent Ca uptake was 50 microM and the Vmax was 0.2 nmol/mg protein/min. The Na-dependent Ca uptake was inhibited by LaCl3, tetracaine and verapamil, unaffected by quinidine and amiloride, and slightly stimulated by chymotrypsin. These data demonstrate that the Na-dependent Ca transport by renal basolateral membranes is mediated by a Na-Ca exchange system and not by a calcium-activated sodium conductance pathway.

Topics: Acetamides; Amiloride; Animals; Biological Transport, Active; Calcimycin; Calcium; Calcium Channel Blockers; Cattle; Cell Membrane; Ionophores; Kidney Cortex; Kidney Tubules; Kinetics; Onium Compounds; Sodium; Trityl Compounds

The membrane potential of L1210 murine leukemia cells was assessed by use of the tritiated lipophilic cation probe triphenylmethylphosphonium bromide. The potassium equilibrium potential of the cells was found to be -71 +/- 7 mV. The resting membrane potential was partly dissipated by the protonophore m-chlorocarbonylcyanidephenylhydrazone (10 microM), but was unaffected by ouabain (1 mM) and apparently by the calcium ionophore A23187 (2.5 microM). Monensin (20 microM) caused a hyperpolarization which, since it was blocked by ouabain, was presumed to be brought about by activation of the Na+K+-ATPase via an elevated cytoplasmic Na+ concentration. Adriamycin at concentrations as high as 5 X 10(-4) M brought about no change in the resting potential of the cells. Also, cytotoxic concentrations of adriamycin, unlike ouabain, had no effect on rubidium-86 transport into L1210 cells, nor upon a monensin-induced increased in rubidium-86 uptake. The results suggest that although adriamycin is capable of interaction with the plasma membrane, and may exert its cytotoxicity at this locus, changes in ion flux mediated by Na+K+-ATPase or those capable of changing the membrane potential do not appear to be implicated in its mechanism of action.

Topics: Animals; Calcimycin; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Cell Membrane; Doxorubicin; Leukemia L1210; Membrane Potentials; Mice; Monensin; Onium Compounds; Ouabain; Sodium-Potassium-Exchanging ATPase; Trityl Compounds

Rat heart mitochondria respiring on succinate in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) released Ca2+ on the calcium/sodium antiporter until a steady state was reached. Addition of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) did not perturb this steady state. Thermodynamic analysis showed that if a Ca2+/nNa+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would cause an obvious change in extramitochondrial free [Ca2+]. Therefore the endogenous calcium/sodium antiporter of mitochondria catalyses electroneutral Ca2+/2Na+ exchange.

Topics: Animals; Calcimycin; Calcium; Carrier Proteins; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Proteins; Methylamines; Mitochondria, Heart; Onium Compounds; Rats; Sodium; Sodium-Calcium Exchanger; Thermodynamics; Trityl Compounds

1. We have monitored the plasma-membrane potential of lymphocytes by measuring the accumulation of the lipophilic cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). 2. The mitogen concanavalin A causes a decrease in TPMP+ accumulation by pig lymphocytes corresponding to a 3 mV depolarization with 2 1/2 min. Concanavalin A does not alter 86Rb+ uptake in the first 30 min. 3. In contrast concanavalin A increased TPMP+ accumulation and the rate of Rb+ uptake in mouse thymocytes. This is consistent with a previous proposal that the mitogen induces a hyperpolarization of mouse thymocytes as a result of stimulation of a Ca2+-dependent K+ channel. 4. Studies with the calcium ionophore A23187 and quinine (an inhibitor of the Ca2+-dependent K+ channel) suggest that the channel is partially closed in mouse resting thymocytes but is almost fully active in pig resting cells. Thus concanavalin A hyperpolarizes mouse thymocytes by activating the Ca2+-dependent K+ channel but cannot do so in pig lymphocytes because the channel is already maximally activated. 5. The 3mV depolarization of pig cells cannot be explained by a decrease in electrogenic K+ permeability.

Topics: Animals; Calcimycin; Concanavalin A; In Vitro Techniques; Lymphocyte Activation; Membrane Potentials; Mice; Mice, Inbred Strains; Onium Compounds; Quinine; Rubidium; Swine; Thermodynamics; Thymus Gland; Trityl Compounds

Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O-2) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H. M., and G. Weissmann. 1978. Proc, Natl, Acad, Sci. U. S. A. 75: 3818--3822). To further define the inter-relationship of these functions, studies were done with two indirect probes of membrane potential, 3-3'-dipentyloxacarbocyanine and triphenylmethylphosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O-2 production (chronic granulomatous disease [CGD]), and from patients with defective degranulation and/or chemotaxis (Cheddiak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore A23187 and phorbol myristate acetate (PMA). The results obtained with 3-3'-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with CGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells of f-Met-Leu-Phe and PMA could not be attributed to the absence of O-2, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.

Topics: Adolescent; Adult; Calcimycin; Carbocyanines; Chediak-Higashi Syndrome; Chemotactic Factors; Chemotaxis, Leukocyte; Child; Child, Preschool; Female; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Male; Membrane Potentials; Neutrophils; Onium Compounds; Superoxides; Tetradecanoylphorbol Acetate; Tetraphenylborate; Trityl Compounds; Valinomycin

The accumulation of the lipophilic cation, triphenylmethylphosphonium, has been employed to determine the resting membrane potential in human erythrocytes, turkey erythrocytes, and rat white adipocytes. The triphenylmethylphosphonium cation equilibrates rapidly in human erythrocytes in the presence of low concentrations of the hydrophobic anion, tetraphenylborate. Tetraphenylborate does not accelerate the uptake of triphenylmethylphosphonium ion by adipocytes. The cell associated vs. extracellular distribution of the triphenylmethylphosphonium ion is proportional to changes in membrane potential. The distribution of this ion reflects the membrane potential determining concentration of the ion with dominant permeability in a "Nernst" fashion. The resting membrane potentials for the human erythrocyte, turkey erythrocyte, and rat white adipocyte were found to be -8.4 +/- 1.3, -16.8 +/- 1.1, and -58.3 +/- 5.0 mV, respectively, values which compare favorably with values obtained by other methods. In addition, changes in membrane potential can be assessed by following triphenylmethylphosphonium uptake without determining the intracellular water space. The method has been successfully applied to a study of hormonally induced changes in membrane potential of rat white adipocytes.

Topics: Adipose Tissue; Animals; Calcimycin; Cations, Monovalent; Cell Membrane; Cell Membrane Permeability; Erythrocytes; Humans; Kinetics; Male; Mathematics; Membrane Potentials; Onium Compounds; Rats; Tetraphenylborate; Trityl Compounds; Turkeys; Valinomycin

Chemotaxis is migration of organisms to higher concentrations of attractant or lower concentrations of repellent. Understanding the switch than controls whether the flagella rotate counterclockwise for swimming or clockwise for tumbling (thrashing about without making much forward progress) is central to understanding chemotaxis of peritrichous bacteria, since chemotaxis results from selective suppression of tumbles. Depletion of divalent cation by chelating agents in the presence of A23187, an ionophore that conveys divalent cation across membrane, causes incessant tumbling in Bacillus subtilis. Small additions of MgCl2 prevent this tumbling. In this tumbling condition, the bacteria which normally swim extensively when given attractant, do not respond even to 10 mM alanine, a strong attractant. MnCl2, by contrast to others potentiated by the ionophore. Permanent cations, including tetraphenylarsonium ion and triphenylmethylphosphonium ion, cause permanent swimming, even in the presence of A23187 and chelating agents. We propose that divalent cation, probably Mg2+ ion, binds to the switch to cause swimming and that, in the absence of divalent cation at the switch, the bacterium tumbles.

Topics: Alanine; Arsenicals; Bacillus subtilis; Calcimycin; Calcium; Cell Membrane; Chelating Agents; Chemotaxis; Edetic Acid; Egtazic Acid; Magnesium; Manganese; Onium Compounds; Organophosphorus Compounds; Trityl Compounds; Uncoupling Agents