concanamycin-a and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

concanamycin-a has been researched along with 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein* in 3 studies

Other Studies

3 other study(ies) available for concanamycin-a and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

ArticleYear
Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.
    Analytical biochemistry, 2010, Mar-15, Volume: 398, Issue:2

    Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC(50)=26 microM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved.

    Topics: Drug Evaluation, Preclinical; Enzyme Inhibitors; Flow Cytometry; Fluoresceins; High-Throughput Screening Assays; Hydrogen-Ion Concentration; Macrolides; Saccharomyces cerevisiae; Spectrometry, Fluorescence; Vacuolar Proton-Translocating ATPases; Vacuoles; Yeasts

2010
Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase.
    The Journal of experimental biology, 2008, Volume: 211, Issue:Pt 5

    Blowfly salivary gland cells have a vacuolar-type H(+)-ATPase (V-ATPase) in their apical membrane that energizes secretion of a KCl-rich saliva upon stimulation with serotonin (5-hydroxytryptamine, 5-HT). We have used BCECF to study microfluometrically whether V-ATPase and carbonic anhydrase (CA) are involved in intracellular pH (pH(i)) regulation, and we have localized CA activity by histochemistry. We show: (1) mean pH(i) in salivary gland cells is 7.5+/-0.3 pH units (N=96), higher than that expected from passive H(+) distribution; (2) low 5-HT concentrations (0.3-3 nmol l(-1)) induce a dose-dependent acidification of up to 0.2 pH units, with 5-HT concentrations >10 nmol l(-1), causing monophasic or multiphasic pH changes; (3) the acidifying effect of 5-HT is mimicked by bath application of cAMP, forskolin or IBMX; (4) salivary gland cells exhibit CA activity; (5) CA inhibition with acetazolamide and V-ATPase inhibition with concanamycin A lead to a slow acidification of steady-state pH(i); (6) 5-HT stimuli in the presence of acetazolamide induce an alkalinization that can be decreased by simultaneous application of the V-ATPase inhibitor concanamycin A; (7) concanamycin A removes alkali-going components from multiphasic 5-HT-induced pH changes; (8) NHE activity and a Cl(-)-dependent process are involved in generating 5-HT-induced pH changes; (9) the salivary glands probably contain a Na(+)-driven amino acid transporter. We conclude that V-ATPase and CA contribute to steady-state pH(i) regulation and 5-HT-induced outward H(+) pumping does not cause an alkalinization of pH(i) because of cytosolic H(+) accumulation attributable to stimulated cellular respiration and AE activity, masking the alkalizing effect of V-ATPase-mediated acid extrusion.

    Topics: Animals; Carbonic Anhydrases; Cytophotometry; Diptera; Fluoresceins; Homeostasis; Hydrogen-Ion Concentration; Macrolides; Oxygen; Salivary Glands; Serotonin; Vacuolar Proton-Translocating ATPases

2008
Na+-independent proton secretion in MDCK-C11 cells.
    Pflugers Archiv : European journal of physiology, 2000, Volume: 441, Issue:2-3

    In this work we studied the proton secretion mechanisms in recently cloned MDCK-C11 cells. We measured intracellular pH (pHi) in monolayers grown on permeable filters, using the pH-sensitive probe BCECF and an inverted epifluorescence microscope. The cells have a basal pHi of 7.20+/-0.01 (n=136) and after an acid-releasing NH4Cl pulse pHi recovered at a rate (dpHi/dt) of 0.167+/-0.006 pH units/ per minute (n=20). This rate decreased significantly when Na+ was removed from both cell surfaces, and was further reduced when they were both perfused with a solution containing no Na+ and K+. pHi recovery fell again in the presence of concanamycin (at a concentration of 4.6x10(-8) M; a specific inhibitor of the vacuolar H+-ATPase). When Na+ was removed from the apical or the basolateral side, pHi recovery (in pH units per minute) was significantly reduced to 0.099+/-0.008 (n=11) and 0.086+/-0.01 (n=10), respectively. The Na+-independent mechanism of pHi recovery was significantly inhibited by the presence of 5 x 10(-5) M Schering 28080 (an inhibitor of the H+-K+-ATPase) at the apical side (0.065+/-0.01 versus 0.099+/-0.008 pH units per minute, P<0.05), but not at the basolateral side (0.072+/-0.01 versus 0.086+/-0.01 pH units per minute). On the other hand, concanamycin inhibited the Na+-independent pHi recovery when applied apically (0.0304+/-0.005 pH units per minute, n=7) and basolaterally (0.025+/-0.004 pH units per minute, n=7). From these results we conclude that monolayers of MDCK-C11 cells have a Na+/H+ exchanger and a concanamycin-sensitive H+-ATPase on their apical and basolateral membranes; and a K+-dependent, Schering 28080-sensitive H+-K+-ATPase on their apical side.

    Topics: Ammonium Chloride; Animals; Anti-Bacterial Agents; Cell Line; Dogs; Enzyme Inhibitors; Fluoresceins; Hydrogen-Ion Concentration; Imidazoles; Kidney; Macrolides; Microscopy, Fluorescence; Proton Pump Inhibitors; Proton-Translocating ATPases; Sodium; Solutions

2000