valinomycin and calmidazolium

Apoptosis of murine thymocytes induced by either methylprednisolone or valinomycin was studied by flow cytometry. The apoptosis induced by methylprednisolone followed three stages: an initial decrease in cell volume, indicated by a fall in forward scatter accompanied by faint ethidium bromide staining, a second stage in which the cells became brightly stained by ethidium bromide, and a final stage when the cells were apparently less fluorescent as the nuclei disintegrated into apoptotic bodies. As the forward scatter of cells decreased there was a simultaneous depolarization of the cells and an elevation of intracellular calcium. These early changes preceded the fragmentation of the DNA which also preceded the intense staining of the cells by ethidium bromide. Methylprednisolone-induced apoptosis was inhibited by low concentrations (1 x 10(-7) M) of valinomycin and nonactin, neither of which could themselves induce apoptosis at these low concentrations. Cadmidazolium and cycloheximide arrested the program at an early stage. Okadaic acid allowed volume loss and ethidium bromide staining to proceed in the absence of DNA fragmentation. At high concentrations (1 x 10(-5) M) valinomycin induced a form of apoptosis, but nonactin only caused the cells to fragment. The valinomycin-induced apoptosis, although it involved the degradation of DNA and the disintegration of the nuclei into apoptotic bodies, differed from the methylprednisolone apoptosis as it did not involve a decrease of cell volume and was not inhibited by cycloheximide or affected by okadaic acid.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Cells, Cultured; Cycloheximide; DNA Damage; Ethers, Cyclic; Flow Cytometry; Imidazoles; In Vitro Techniques; Macrolides; Male; Methylprednisolone; Mice; Okadaic Acid; Thymus Gland; Valinomycin

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca2+ (K1/2 = 0.08 microM Ca2+) potassium (maximally at 50 mM K+), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC50 = 5 microM) and the calmodulin inhibitor R24571 (IC50 = 5 microM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K+, stimulated calcium uptake, suggesting that the calcium pump either countertransports K+, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na+, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca2+ in this condition.

Topics: 4-Chloromercuribenzenesulfonate; Adenosine Triphosphate; Animals; Calcimycin; Calcium; Calmodulin; Carrier Proteins; Cattle; Cell Membrane; Chloromercuribenzoates; Cyclic AMP; Cytosol; Imidazoles; Lens, Crystalline; Membrane Proteins; p-Chloromercuribenzoic Acid; Potassium; Protein Kinases; Sodium-Calcium Exchanger; Sodium-Potassium-Exchanging ATPase; Valinomycin; Vanadates

8-Bromo cyclic AMP (cAMP) (10(-4) M) inhibits Na absorption in isolated chicken enterocytes as has been reported previously. Direct measurements of intracellular pH (pHi) using 5,6-carboxyfluorescein diacetate showed that both 8-bromo cAMP and the diuretic amiloride (10(-3) M) stimulated a persistent decrease in pHi of approximately 0.1 pH units, effects that were Na dependent and were not additive when cells were stimulated with both agents. These results suggest inhibition of an amiloride-sensitive Na/H exchange by cAMP. Direct measurements of intracellular Ca [Ca]i were also made using quin 2. 8-Bromo cAMP (10(-4) M) stimulated an immediate and persistent (greater than 10 min) increase in [Ca]i of approximately 20 nM, an effect that was not dependent on extracellular Ca. Pretreatment of cells with the specific calmodulin inhibitor calmidazolium (10(-7) M) and the intracellular Ca-buffering agent MAPTAM blocked cAMP's effects on pH and Na uptake, but did not interfere with amiloride's effects. An increase in [Ca]i stimulated by the Ca ionophore A23187 (10(-6) M) was sufficient by itself to decrease pHi and inhibit amiloride-sensitive Na influx in isolated enterocytes. We conclude that cAMP stimulates the release of endogenous Ca in isolated enterocytes. This increase in [Ca]i appears to be essential for inhibition of amiloride-sensitive Na-H exchange by this cyclic nucleotide.

Topics: Alprostadil; Amiloride; Aminoquinolines; Animals; Buffers; Butyrates; Calcimycin; Calcium; Carrier Proteins; Chelating Agents; Chickens; Cyclic AMP; Glycine; Hydrogen-Ion Concentration; Imidazoles; In Vitro Techniques; Intestine, Small; Nigericin; Sodium-Hydrogen Exchangers; Valinomycin

Calcium ionophore (A23187)-stimulated prostaglandin (PG) E2 and I2 (measured as 6-keto PGF1 alpha) release from cultured rabbit coronary microvessel endothelial (RCME) cells in a time- and dose-dependent manner. A23187-stimulated PG release was reduced by the calcium channel blockers nifedipine, verapamil and diltiazem and by the intracellular calcium blocker, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate. A23187-stimulated PG release was significantly reduced by lowering the calcium concentration in the buffer to concentrations of 0.2 mM or less. A23187-stimulated 45Ca uptake was not inhibited by nifedipine (0.5 microM), diltiazem (10 micron) or verapamil (50 microM) although these same concentrations of calcium channel blockers significantly inhibited A23187-stimulated PG release. However, these concentrations of calcium blockers did inhibit K+ (10 mM)-valinomycin (5 microM)-stimulated 45Ca uptake, indicating that, although RCME cells probably have voltage-dependent calcium channels and although calcium influx via these channels is blocked by the calcium channel blockers, voltage-dependent calcium influx plays little or no role in A23187-stimulated 45Ca influx and PG release. KCl-valinomycin significantly stimulated PG release, but this increase was not significantly affected by either nifedipine (0.5 microM) or diltiazem (10 microM) despite complete inhibition of KCl-valinomycin-stimulated 45Ca influx. Verapamil (50 microM) exhibited a small but significant suppression of KCl-valinomycin-stimulated PG release. These observations most likely indicate that calcium influx by voltage-dependent calcium channels plays little or no role in the events leading to either A23187- or KCl-valinomycin-stimulated PG release. The calmodulin antagonists, trifluoperazine and calmidazolium, also reduced A23187-stimulated PG release. In vitro studies of porcine pancreatic phospholipase (PL) A2 activity suggested that the inhibitory actions of trifluoperazine, but not the calcium antagonists, may be mediated by direct inhibitory actions on PLA2. Studies with [3H]arachidonic acid (AA)- and [14C]stearic acid-prelabeled RCME cells suggested that A23187 stimulated both PLA2 and PLC activity, leading to the release of AA. Studies with exogenous AA indicated that reducing calcium availability by reducing buffer calcium concentrations resulted in an enhanced conversion of exogenous AA to PGs. RCME cells incubated in nominally calcium-free buffer exhibited a decreased rate of AA

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Calcium Channel Blockers; Calmodulin; Cells, Cultured; Coronary Vessels; Endothelium; Imidazoles; Kinetics; Microcirculation; Prostaglandins; Rabbits; Trifluoperazine; Valinomycin