sq-23377 and antimycin

Ca2+-induced exocytosis in neuronal and neuroendocrine cells involves ATP-dependent steps believed to 'prime' vesicles for exocytosis. Primed, docked vesicles are released in response to Ca2+ influx through voltage-gated Ca2+ channels. Neutrophils, however, do not possess voltage-gated Ca2+ channels and appear to have no docked vesicles. Furthermore, neutrophils have several types of granules with markedly different Ca2+ requirements for exocytosis. These differential Ca2+ dependencies were used as a tool to investigate the ATP dependence of different granule populations. Here we demonstrate distinct ATP requirements for release of neutrophil granule populations, with respect to rate as well as amplitude. Intracellular ATP was depleted to various levels, and exocytosis was stimulated with different Ca2+ concentrations and measured with the patch-clamp capacitance technique or by detecting enzyme release. Primary granule exocytosis displayed a distinct ATP dependence with an apparent K(m) of approximately 80 microM ATP and no ATP-independent exocytosis. Release of secondary and tertiary granules displayed a more shallow ATP dependence (K(m) approximately 330 microM), and more than 50% of secondary and tertiary granules appeared not to need ATP at all for their release. Individual granules in human neutrophils have distinct ATP requirements for exocytosis, suggesting that the ATP-sensitive elements are localised to the granules. Primary granule exocytosis has a very low affinity for ATP. Furthermore, substantial ATP-independent exocytosis of secondary and tertiary granule occurs despite the absence of docked granules. These characteristics should help neutrophils to fulfil their bactericidal functions at poorly irrigated sites of infection with low glucose supply.

Topics: Adenosine Triphosphate; Antimycin A; Buffers; Calcium; Calcium Signaling; Cell Membrane; Deoxyglucose; Dose-Response Relationship, Drug; Energy Metabolism; Exocytosis; Humans; Intracellular Fluid; Ionomycin; Neutrophils; Secretory Vesicles

The actin cytoskeleton of rabbit proximal tubules was assessed by deoxyribonuclease (DNase) binding, sedimentability of detergent-insoluble actin, laser-scanning confocal microscopy, and ultrastructure during exposure to hypoxia, antimycin, or antimycin plus ionomycin. One-third of total actin was DNase reactive in control cells prior to deliberate depolymerization, and a similar proportion was unsedimentable from detergent lysates during 2.5 h at 100,000 g. Tubules injured by hypoxia or antimycin alone, without glycine, showed Ca(2+)-dependent pathology of the cytoskeleton, consisting of increases in DNase-reactive actin, redistribution of pelletable actin, and loss of microvilli concurrent with lethal membrane damage. In contrast, tubules similarly depleted of ATP and incubated with glycine showed no significant changes of DNase-reactive actin or actin sedimentability for up to 60 min, but, nevertheless, developed substantial loss of basal membrane-associated actin within 15 min and disruption of actin cores and clubbing of microvilli at durations > 30 min. These structural changes that occurred in the presence of glycine were not prevented by limiting Ca2+ availability or pH 6.9. Very rapid and extensive cytoskeletal disruption followed antimycin-plus-ionomycin treatment. In this setting, glycine and pH 6.9 decreased lethal membrane damage but did not ameliorate pathology in the cytoskeleton or microvilli; limiting Ca2+ availability partially protected the cytoskeleton but did not prevent lethal membrane damage. The data suggest that both ATP depletion-dependent but Ca(2+)-independent, as well as Ca(2+)-mediated, processes can disrupt the actin cytoskeleton during acute proximal tubule cell injury; that both types of change occur, despite protection afforded by glycine and reduced pH against lethal membrane damage; and that Ca(2+)-independent processes primarily account for prelethal actin cytoskeletal alterations during simple ATP depletion of proximal tubule cells.

Topics: Actins; Adenosine Triphosphate; Animals; Antimycin A; Calcium; Cytoskeleton; DNA; Female; Fluorescent Dyes; Hypoxia; Ionomycin; Kidney Cortex; Kidney Tubules, Proximal; Phalloidine; Rabbits; Rhodamines; Time Factors

To better define the role of Ca2+ in pathophysiological alterations of the proximal tubule microvillus actin cytoskeleton, we studied freshly isolated tubules in which intracellular free Ca2+ was equilibrated with highly buffered, precisely defined medium Ca2+ levels using a combination of the metabolic inhibitor, antimycin, and the ionophore, ionomycin, in the presence of glycine, to prevent lethal membrane damage and resulting nonspecific changes. Increases of Ca2+ to > or = 10 microM were sufficient to initiate concurrent actin depolymerization, fragmentation of F-actin into forms requiring high-speed centrifugation for recovery, redistribution of villin to sedimentable fractions, and structural microvillar damage consisting of severe swelling and fragmentation of actin cores. These observations implicate Ca(2+)-dependent, villin-mediated actin cytoskeletal disruption in tubule cell microvillar damage under conditions conceivably present during pathophysiological states. However, despite prior evidence for cytosolic free Ca2+ increases of the same order of magnitude and similar structural microvillar alterations, Ca(2+)- and villin-mediated events did not appear to account for the initial microvillar damage that occurs during ATP depletion induced by antimycin alone or hypoxia.

Topics: Actins; Animals; Antimycin A; Calcium; Cytoskeletal Proteins; Cytoskeleton; Deoxyribonucleases; Drug Combinations; Female; Glycine; Ionomycin; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Microvilli; Phalloidine; Rabbits; Rhodamines; Staining and Labeling

We have examined the dependence of unesterified fatty acid accumulation by intact, freshly isolated proximal tubules on Ca2+, pH, and the cytoprotective amino acid, glycine, during injury induced by hypoxia, antimycin, or antimycin plus ionomycin. In the absence of glycine, similarly high levels of fatty acid accumulation were seen during all three injury conditions irrespective of whether tubules were incubated in normal 1.25 mM Ca2+ medium or in medium where Ca2+ was buffered to 0.1 microM, a maneuver which prevented injury-associated increase of cytosolic-free Ca2+ as measured with fura 2. In the presence of glycine, which strongly suppressed development of lethal membrane damage for at least 60 min and did not have any apparent direct effects on fatty acid accumulation, both Ca(2+)-independent and Ca(2+)-dependent components of fatty acid accumulation were discernible. The Ca(2+)-independent component accounted for approximately 2/3 of fatty acid accumulation and did not vary as Ca2+ ranged from 10 nM to 1 microM. Unequivocal Ca(2+)-dependent accumulation occurred when Ca2+ exceeded 10 microM. Lowering pH to 6.9 had a moderate, generalized suppressive effect on fatty acid accumulation, including the major Ca(2+)-independent component, irrespective of the presence of glycine. These data emphasize the role of Ca(2+)-independent fatty acid accumulation during proximal tubule cell injury, clarify the modulatory actions of the potent, intrinsic cytoprotective factors, glycine and reduced pH, and provide insight into the relationship between fatty acid accumulation and lethal membrane damage.

Topics: Acidosis; Animals; Antimycin A; Biological Transport; Calcium; Cell Hypoxia; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Fatty Acids, Nonesterified; Glycine; Hydrogen-Ion Concentration; In Vitro Techniques; Ionomycin; Kidney Cortex; Kidney Tubules, Proximal; Kinetics; L-Lactate Dehydrogenase; Male; Phospholipids; Rabbits; Time Factors

Lowering extracellular pH to less than 7.0 strongly protects isolated proximal tubules against ATP depletion and Ca(2+)-induced injury, but there is little information about alterations of intracellular pH (pHi) in renal tubules during either injury or its modification by decreasing medium pHi or other potent protective factors such as glycine. pHi was assessed with 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein during proximal tubule injury produced by simple ATP depletion with the electron transport inhibitor antimycin or by large increases of cytosolic free Ca2+ induced by treatment with the calcium ionophore ionomycin, alone and in combination with antimycin. Freshly isolated rabbit proximal tubules studied under superfusion conditions in the presence of probenecid were suitable for monitoring pHi during relatively prolonged and severe injury states. Probenecid, used to promote the retention of intracellular fluorophores, only minimally modified the injury response by transiently delaying lactate dehydrogenase release during antimycin treatment. The tubules did not exhibit spontaneous decreases of pHi during simple ATP depletion, but pHi fully equilibrated with cytoprotective decreases of medium pH. Irrespective of the presence of antimycin, ionomycin induced intracellular alkalinization in Ca(2+)-replete medium, which may have further enhanced the severity of injury. When medium Ca2+ was buffered to 100 nM, ionomycin induced intracellular acidification, which likely resulted from a combination of Ca2+/H+ exchange activity of the ionophore and H+ uptake during Ca(2+)-ATPase-mediated extrusion of Ca2+ released by ionomycin from intracellular pools. Alterations of pHi did not contribute to glycine cytoprotection because glycine did not affect the behavior of pHi during treatment with antimycin, ionomycin, or both agents in combination.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Animals; Antimycin A; Calcium; Culture Media; Fluoresceins; Fura-2; Glycine; Hydrogen-Ion Concentration; Ionomycin; Kidney Tubules, Proximal; Male; Rabbits