calcimycin and tetraphenylphosphonium

Changes in the charge of sarcoplasmic reticulum (SR) vesicles are studied using lipophilic ions, which are adsorbed by the membrane phase. Upon addition of MgATP, phenyldicarbaundecaborane (PCB-) and tetraphenylboron (TPB-) are taken up by the SR vesicles, while tetraphenylphosphonium (TPP+) is released into the water phase. The PCB- uptake occurs as well under conditions when SR membrane is shunted by high Cl- concentration. MgATP induces minor additional binding of PCB- in the presence of oxalate and it is followed by release of the lipophilic anion from the vesicles. EGTA partly reverses the ATP effect, and calcium ionophore A23187 plus EGTA reverses it completely. Vesicles that were preliminarily loaded by Ca2+ demonstrated higher passive and lower ATP-dependent PCB- binding. Activation of isolated Ca2+-ATPase in the presence of 0.1 mM EGTA results in PCB- release into the medium and additional TPP+ binding to the enzyme. We suggest that the redistribution of the lipophilic ions between the water phase and SR membrane reflects charge changes in Ca2+-binding sites inside both SR vesicles and Ca2+-ATPase molecules in the course of Ca2+ translocation.

Topics: Adenosine Triphosphate; Adsorption; Animals; Boron Compounds; Calcimycin; Calcium; Calcium-Transporting ATPases; Egtazic Acid; Membranes, Artificial; Muscle Proteins; Onium Compounds; Organophosphorus Compounds; Oxalates; Protein Binding; Rabbits; Sarcoplasmic Reticulum; Tetraphenylborate

Tetraphenylphosphonium (TPP+) is a permeant lipophilic cation that accumulates in cultured cells and tissues as a function of the electrical membrane potential across the plasma membrane. This study was undertaken to determine whether TPP+ can be used for assessing membrane potential in intact lenses in organ culture.. Rat lenses were cultured in media containing 10 microM TPP+ and a tracer level of 3H-TPP+ for various times. 3H-TPP+ levels in whole lenses or dissected portions of lenses were determined by liquid scintillation counting. Ionophores, transport inhibitors, and neurotransmitters were also added to investigate their effects on TPP+ uptake. RESULTS. Incubation of lenses in low-K+ balanced salt solution and TC-199 medium, containing physiological concentrations of Na+ and K+, led to a biphasic accumulation of TPP+ in the lens that approached equilibrium by 12 to 16 hours of culture. The TPP+ equilibrated within 1 hour in the epithelium but penetrated more slowly into the fiber mass. The steady state level of TPP+ accumulation in the lens was depressed by 90% when the lenses were cultured in a medium containing high K+. The calculated membrane potential for the normal rat lens in TC-199 was -75 +/- 3 mV. Monensin (1 microM) and nigericin (1 microM), Na+H+ and K+H+ exchangers respectively, as well as the protonophore carbonylcyanide-m-chlorophenylhydrazone (CCCP, 10 microM) and the calcium ionophore A23187 (10 microM), abolished TPP+ accumulation and caused cloudiness of the lenses. The neurotransmitter acetylcholine at 50 microM decreased TPP+ accumulation in the lens, but this effect could be prevented by simultaneous application of 1 mM atropine.. TPP+ accumulation can be used as an indicator of changes in membrane potential in intact lenses, but because of the long time required to reach steady state, its utility is limited. The slow accumulation of TPP+ and its slow efflux from the lens under conditions known to depolarize membranes are consistent with a diffusion barrier in the deep cortex and nucleus of the lens.

Topics: Animals; Calcimycin; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Culture Media; Female; Indicators and Reagents; Lens, Crystalline; Male; Membrane Potentials; Monensin; Monitoring, Physiologic; Nigericin; Onium Compounds; Organ Culture Techniques; Organophosphorus Compounds; Rats; Rats, Sprague-Dawley

The mitochondrial membrane permeability transition induced by Ca2+ is inhibited by quinine in a dose-dependent fashion. Competition experiments strongly suggest that quinine displaces Ca2+ bound to the inner membrane. This is supported by experiments showing that quinine inhibits Ca2+-dependent but not Ca2+-independent mitochondrial swelling induced by phenylarsine oxide. As with Ca2+ chelators, quinine induces permeability transition pore closure preventing the contraction induced by poly(ethylene glycol) 2000 in mitochondria preswollen by incubation in KSCN medium containing Ca2+ and inorganic phosphate. These results suggest that quinine dislodges Ca2+ bound to the protein site, which triggers pore opening.

Topics: Animals; Antimycin A; Calcimycin; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Intracellular Membranes; Kinetics; Membrane Potentials; Mitochondria; Mitochondria, Liver; Mitochondrial Swelling; Onium Compounds; Organophosphorus Compounds; Permeability; Quinine; Rats; Thiocyanates

Glycine and beta-alanine actively loaded into brain synaptic plasma membrane vesicles were released into the external medium by using the classical depolarization agents high K+ and veratridine. This release occurs via a Ca2+-independent process. Measurements of membrane depolarization using tetraphenylphosphonium uptake show a close correlation between changes in the membrane potential and stimulation of the efflux process. Results shown herein and previously reported by our group (Aragón, M.C. and Giménez, C. (1986) Biochim. Biophys. Acta 855, 257-264; Agulló, L., Jiménez, B., Aragón, M.C. and Giménez, C. (1986) Eur. J. Biochem. 159, 611-617), suggest that the glycine and beta-alanine transport systems in synaptic plasma membranes are susceptible of modulation by changes in ionic fluxes and hence in the membrane potential, similar to those occurring during depolarization and repolarization.

Topics: Action Potentials; Alanine; Animals; beta-Alanine; Brain; Calcimycin; Calcium; Glycine; Kinetics; Male; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Potassium; Rats; Rats, Inbred Strains; Synaptic Membranes; Synaptosomes; Veratridine

GH3 rat anterior pituitary cells possess a Na+/Ca2+ exchange transport mechanism which is present in purified plasma membrane vesicles prepared from these cells. Imposition of an outwardly directed Na+ gradient in vesicles results in a marked concentrative uptake of Ca2+ which is abolished by the Ca2+ ionophore A23187. Transport activity depends on a sustained Na+ gradient. Dissipation of the driving force by treatment with Na+ ionophores or by passive gradient collapse abolished transport activity. The exchange reaction is completely reversible since addition of extravesicular Na+ enhances Ca2+ efflux from Ca2+ loaded vesicles. A kinetic analysis of Na+/Ca2+ exchange indicates saturation kinetics for both substrates with apparent values of Km for Na+ and Ca2+ of 17 mM and 5 microM, respectively, and a Vmax of about 8 nmol/min/mg of protein for Ca2+ uptake at 25 degrees C. In addition to Na+/Ca2+ exchange, the transporter functions in a Ca2+/Ca2+ exchange mode with an apparent Km of 20 microM and Vmax of 16 nmol/min/mg of protein for Ca2+ influx. Na+/Ca2+ exchange is not inhibited by protonophores indicating that Ca2+ flux does not occur via coupled Na+/H+, Ca2+/H+ exchange. Transport is inhibited by derivatives of the pyrazine diuretic amiloride. The pH dependency of Ca2+ uptake displays a sigmoidal relationship with stimulation of activity at alkaline pH and inhibition at acid pH. Furthermore, the reaction is electrogenic (i.e. more than 2 Na+ transported per Ca2+) as demonstrated by stimulated uptake of lipophilic cations during exchange and by effects of artificially imposed membrane potentials on the rate of Ca2+ transport. Plasma membrane vesicles prepared from bovine anterior pituitary glands also display Na+/Ca2+ exchange with many of the same characteristics. These results support the notion that Na+/Ca2+ exchange functions in Ca2+ homeostasis in pituitary cells.

Topics: Amiloride; Animals; Biological Transport, Active; Calcimycin; Calcium; Calcium Channel Blockers; Cattle; Cell Membrane; Cells, Cultured; Kinetics; Onium Compounds; Organophosphorus Compounds; Pituitary Gland, Anterior; Rats; Sodium