digitonin and fura-2-am

A fourth intracellular Ca2+ pool in Leishmania donovani was identified by permeabilizing plasma membrane with digitonin. In Fura 2 loaded cells Ca2+ was released synergistically when mitochondrial function was blocked by antimycin and oligomycin. Vanadate did not have any effect if applied before incorporation of these mitochondrial poisons. However, the same inhibitor which inhibits Ca2+-ATPase activity of endoplasmic reticulum was able to release Ca2+ at a slow rate when added after antimycin and oligomycin. Alkalization of cytoplasmic pH allowed further release of Ca2+ essentially from the acidocalcisome. Purified glycosomes could mediate Ca2+ uptake mechanism in presence of vanadate whereas bafilomycin, a specific and potent inhibitor of vacuolar proton pump did not have any effect. Glycosomal Ca2+-ATPase activity was optimum at pH 7.5. The apparent Km for calciumin presence of vanadate was 12 nM. Taken together, it may be suggested that a vanadate-insensitive Ca2+-ATPase is present in the membrane of this microbody. Presence of glycosomal Ca2+ was further confirmed by imaging of Ca2+ activity in the Fura 2 loaded purified organelle using confocal laser. Results reveal that newly localized glycosomal calcium may essentially be an effective candidate to play a significant role in cellular function.

Topics: Animals; Antimycin A; Calcimycin; Calcium; Calcium-Transporting ATPases; Digitonin; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Ionophores; Leishmania donovani; Macrolides; Microbodies; Microscopy, Confocal; Oligomycins; Spectrometry, Fluorescence; Uncoupling Agents; Vanadates

Intracellular movement of vesiculated pigment granules in angelfish melanophores is regulated by a signalling pathway that triggers kinesin and dyneinlike microtubule motor proteins. We have tested the relative importance of intracellular Ca2+ ([Ca2+]i) vs cAMP ([cAMP]i) in the control of such motility by adrenergic agonists, using fluorescence ratio imaging and many ways to artificially stimulate or suppress signals in these pathways. Fura-2 imaging reported a [Ca2+]i elevation accompanying pigment aggregation, but this increase was not essential since movement was not induced with the calcium ionophore, ionomycin, nor was movement blocked when the increases were suppressed by withdrawal of extracellular Ca2+ or loading of intracellular BAPTA. The phosphatase inhibitor, okadaic acid, blocked aggregation and induced dispersion at concentrations that suggested that the protein phosphatase PP-1 or PP-2A was continuously turning phosphate over during intracellular motility. cAMP was monitored dynamically in single living cells by microinjecting cAMP-dependent kinase in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine respectively (Adams et al., 1991. Nature (Lond.). 349:694-697). Ratio imaging of F1CRhR showed that the alpha 2-adrenergic receptor-mediated aggregation was accompanied by a dose-dependent decrease in [cAMP]i. The decrease in [cAMP]i was both necessary and sufficient for aggregation, since cAMP analogs or microinjected free catalytic subunit of A kinase-blocked aggregation or caused dispersal, whereas the cAMP antagonist RpcAMPs or the microinjection of the specific kinase inhibitor PKI5-24 amide induced aggregation. Our conclusion that cAMP, not calcium, controls bidirectional microtubule dependent motility in melanophores might be relevant to other instances of non-muscle cell motility.

Topics: Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Animals; Calcium; Carbachol; Cell Aggregation; Cyclic AMP; Digitonin; Egtazic Acid; Epinephrine; Ethers, Cyclic; Fishes; Fluorescent Dyes; Fura-2; Ionomycin; Kinetics; Melanophores; Microscopy, Fluorescence; Okadaic Acid; Organelles; Phosphoprotein Phosphatases; Protein Kinases