calcimycin and 3-3--dipropyl-2-2--thiadicarbocyanine

The cationic fluorescent probe, DiSC3(5) was used to measure the membrane potential in human platelets. Hyperpolarization was induced by the addition of Ca2+ to the medium and also by the addition of the Ca2+ ionophore, A23187. In the absence of extracellular Ca2+ ([Ca2+]o) there was no response to A23187. The threshold concentration for [Ca2+]o was 20 microM and for A23187 was 12 nM. The increase polarity induced by [Ca2+]o was not affected by various K+ channel blockers. However, the effect of A23187 was inhibited by quinine and charybdotoxin, while apamin, tetraethylammonium, and the calmodulin inhibitors trifluoperazine and compound R24571 were ineffective. The resting membrane potential was -66 +/- 0.9 mV and was decreased by quinine. There are three conclusions from this study: (i) Ca2+-activated K+ channels exist in human platelets; (ii) they are the type that are apamin insensitive, charybdotoxin sensitive; and (iii) they may contribute to the resting membrane potential.

Topics: Adult; Apamin; Benzothiazoles; Blood Platelets; Calcimycin; Calcium; Carbocyanines; Female; Fluorescent Dyes; Humans; Kinetics; Male; Membrane Potentials; Middle Aged; Potassium Channels; Quinine; Tetraethylammonium; Tetraethylammonium Compounds

The applicability of the potential-sensitive dye diS-C3-(5) for the study of A23187 + Ca2+ induced plasma membrane hyperpolarization was tested in rat brain synaptosomes. An appropriate dye synaptosome ratio was chosen for the fluorescence titration dye in Ca-free Krebs-Ringer solution. The fluorescence intensity of the probe was increased upon the addition of Ca2+ (1 microM) to the synaptosomes in the presence of A23187 (1 microM). The effect of Ca2+ + A23187 persisted in a Na+-free medium or when Na+ channels were inhibited by tetrodotoxin as well as in high K+-depolarized synaptosomes (75 microM KCl). In the presence of oligomycin or a protonophore (1 microM) the effect of Ca2+ + A23187 was suppressed. This suggests that the A23187-induced fluorescence increase is due to a depolarization of intrasynaptosomal mitochondria. Therefore, the use of the dye diS-C3-(5) for the study of Ca-induced hyperpolarization does not seem to be feasible unless a quantitative model of changes in fluorescence related to the plasma and mitochondrial membrane potentials is elaborated.

Topics: Animals; Benzothiazoles; Brain; Calcimycin; Calcium; Calcium Channels; Carbocyanines; Cell Membrane; Dose-Response Relationship, Drug; Fluorescent Dyes; In Vitro Techniques; Intracellular Membranes; Membrane Potentials; Mitochondria; Rats; Spectrometry, Fluorescence; Synaptosomes

Quinine inhibits mitogenesis at the same concentration (10(-4) M) as that which blocks Ca++-dependent potassium transport in lymphocytes. Lower quinine concentrations (10(-8)-10(-6) M) induce a comitogenic effect which is most pronounced when Ca++-ionophore A23187 is used as a mitogen. Thus, activation of Ca++-dependent K+-channels is not necessary to trigger mitogenesis, but is important for further stages of the process.

Topics: Benzothiazoles; Calcimycin; Calcium; Carbocyanines; Cell Membrane Permeability; Cells, Cultured; Concanavalin A; DNA; Fluorescent Dyes; Humans; Ion Channels; Lymphocyte Activation; Membrane Potentials; Mitogens; Phytohemagglutinins; Potassium; Quinine

Polymorphonuclear leukocytes undergo a series of morphological and biochemical changes in response to various chemical stimuli. Transmembrane potential change is an early event that follows stimulation and membrane depolarization may act as a trigger for superoxide generation. To determine if there is a correlation between membrane depolarization and superoxide generation, we investigated the effects of different membrane modulators on stimulus-dependent depolarization. The membrane modulators mepacrine, chlorpromazine and cepharanthine inhibited the superoxide generation produced by chemotactic peptide, FMLP, and/or digitonin in neutrophils. Inhibitory profiles of the activation parameters, however, demonstrate that membrane depolarization is not associated with superoxide generation: FMLP-induced depolarization was inhibited by the modulators tested and was accompanied by the suppression of superoxide generation, but the depolarization produced by digitonin was stimulated somewhat by these drugs. Our results indicate that receptor-mediated membrane depolarization is not a necessary event for the activation of superoxide generation by digitonin.

Topics: Animals; Benzothiazoles; Calcimycin; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Digitonin; Fluorescent Dyes; Guinea Pigs; Kinetics; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxygen Consumption; Potassium; Spectrometry, Fluorescence; Superoxides

We have studied Microciona prolifera cells as a model for inflammation and secretion. Dissociated in Ca-, Mg-free seawater with 2.5 mM EDTA, the cells aggregate when exposed to Ca (greater than 5 mM) and Ca ionophores. Extracellular Ca is not required over the course of aggregation; brief pulses of Ca suffice. Aggregation was induced by A23187 in excess EDTA after cells were prepared by pulse Ca. It appeared that Ca ionophore stimulated the secretion of Microciona aggregation factor (MAF) to a locus or in a form inaccessible to external EDTA. Pulse-induced aggregation depended on MAF because it was inhibited by MAF fragments, which are ligands for MAF-binding sites. Sponge cells were preloaded with three fluorescent dyes that monitor aspects of stimulus-secretion coupling: 1) 3,3'-dipropylthiadicarbocyanine iodide (dis-C3-(5)), a carbocyanine dye presumed to report changes in membrane potential; 2) 9-aminoacridine (9AA), which presumably reports secretion from acid vesicles; and 3) chlortetracycline (CTC), presumed to report mobilization of membrane-associated Ca. Exposure of cells either to constant Ca or to pulse Ca stimuli caused prompt decreases in the fluorescence of cells with diS-C3-(5) and increases in fluorescence of cells with 9AA. In contrast, although constant Ca provoked decreases in fluorescence of cells with CTC, a pulse Ca was without effect. Moreover, inhibitors of stimulus-response coupling (e.g., aspirin, sodium salicylate, 5 mM; diclofenac, 100 microM) inhibited sponge aggregation induced by either constant or pulse stimuli. In contrast, like the endogenous mediator of inflammation, leukotriene B4, trienoic alkyl catechols (urushiol) from poison ivy provoked aggregation. These studies suggest the utility of this marine model for analysis of stimulus-response coupling in cells of higher species that also respond to secretagogues in the absence of external Ca.

Topics: Aminacrine; Animals; Anti-Inflammatory Agents; Benzothiazoles; Calcimycin; Calcium; Carbocyanines; Catechols; Cell Adhesion Molecules; Cell Aggregation; Chlortetracycline; Fluorescence; Fluorescent Dyes; Inflammation; Porifera; Potassium; Proteins