thapsigargin and barium-chloride

CCAAT/enhancer binding protein beta (C/EBPβ), a transcription factor expressed in muscle satellite cells (SCs), inhibits the myogenic program and is downregulated early in differentiation. In a conditional null model in which C/EBPβ expression is knocked down in paired box protein 7+ (Pax7+) SCs, cardiotoxin (CTX) injury is poorly repaired, although muscle regeneration is efficient in control littermates. While myoblasts lacking C/EBPβ can differentiate efficiently in culture, after CTX injury poor regeneration was attributed to a smaller than normal Pax7+ population, which was not due to a failure of SCs to proliferate. Rather, the percentage of apoptotic SCs was increased in muscle lacking C/EBPβ. Given that an injury induced by BaCl2 is repaired with greater efficiency than controls in the absence of C/EBPβ, we investigated the inflammatory response following BaCl2 and CTX injury and found that the levels of interleukin-1β (IL-1β), a proinflammatory cytokine, were robustly elevated following CTX injury and could induce C/EBPβ expression in myoblasts. High levels of C/EBPβ expression in myoblasts correlated with resistance to apoptotic stimuli, while its loss increased sensitivity to thapsigargin-induced cell death. Using cancer cachexia as a model for chronic inflammation, we found that C/EBPβ expression was increased in SCs and myoblasts of tumor-bearing cachectic animals. Further, in cachectic conditional knockout animals lacking C/EBPβ in Pax7+ cells, the SC compartment was reduced because of increased apoptosis, and regeneration was impaired. Our findings indicate that the stimulation of C/EBPβ expression by IL-1β following muscle injury and in cancer cachexia acts to promote SC survival, and is therefore a protective mechanism for SCs and myoblasts in the face of inflammation.

Topics: Animals; Apoptosis; Barium Compounds; Cardiotoxins; CCAAT-Enhancer-Binding Protein-beta; Cell Line; Chlorides; Immunohistochemistry; Interleukin-1beta; Mice; Mice, Knockout; Mice, Transgenic; Myoblasts; Neoplasms; PAX7 Transcription Factor; RNA, Messenger; Thapsigargin; Up-Regulation

We studied intracellular calcium ([Ca(2+)](i)) in acid-secreting bone-attached osteoclasts, which produce a high-calcium acidic extracellular compartment. Acid secretion and [Ca(2+)](i) were followed using H(+)-restricted dyes and fura-2 or fluo-3. Whole cell calcium of acid-secreting osteoclasts was approximately 100 nM, similar to cells on inert substrate that do not secrete acid. However, measurements in restricted areas of the cell showed [Ca(2+)](i) transients to 500-1000 nM consistent with calcium puffs, transient (millisecond) localized calcium elevations reported in other cells. Spot measurements at 50-ms intervals indicated that puffs were typically less than 400 ms. Transients did not propagate in waves across the cell in scanning confocal measurements. Calcium puffs occurred mainly over regions of acid secretion as determined using lysotracker red DND99 and occurred at irregular periods averaging 5-15 s in acid secreting cells, but were rare in lysotracker-negative nonsecretory cells. The calmodulin antagonist trifluoperazine, cell-surface calcium transport inhibitors lanthanum or barium, and the endoplasmic reticulum ATPase inhibitor thapsigargin had variable acute effects on the mean [Ca(2+)](i) and puff frequency. However, none of these agents prevented calcium puff activity, suggesting that the mechanism producing the puffs is independent of these processes. We conclude that [Ca(2+)](i) transients in osteoclasts are increased in acid-secreting osteoclasts, and that the puffs occur mainly near the acid-transporting membrane. Cell membrane acid transport requires calcium, suggesting that calcium puffs function to maintain acid secretion. However, membrane H(+)-ATPase activity was insensitive to calcium in the 100 nM-1 microM range. Thus, any effects of calcium puffs on osteoclastic acid transport must be indirect.

Topics: Adenosine Triphosphate; Animals; Barium Compounds; Biological Transport; Calcium; Calcium-Transporting ATPases; Calmodulin; Cell Membrane; Cells, Cultured; Chickens; Chlorides; Dopamine Antagonists; Enzyme Inhibitors; Glass; Hydrochloric Acid; Lanthanum; Osteoclasts; Proton-Translocating ATPases; Thapsigargin; Trifluoperazine

Depletion of intracellular calcium stores induces transmembrane Ca2+ influx. We studied Ca(2+)- and Ba(2+)-permeable ion channels in A431 cells after store depletion by dialysis of the cytosol with 10 mM BAPTA solution. Cell-attached patches of cells held at low (0.5 microM) external Ca2+ exhibited transient channel activity, lasting for 1-2 min. The channel had a slope conductance of 2 pS with 200 mM CaCl2 and 16 pS with 160 mM BaCl2 in the pipette. Channel activity quickly ran down in excised inside-out patches and was not restored by InsP3 and/or InsP4. Thapsigargin induced activation in cells kept in 1 mM external Ca2+ after BAPTA dialysis. These channels represent one Ca2+ entry pathway activated by depletion of internal calcium stores and are clearly distinct from previously identified calcium repletion currents.

Topics: Barium Compounds; Benzoquinones; Calcium; Calcium Channels; Calcium Chloride; Calcium-Transporting ATPases; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line; Chlorides; Egtazic Acid; Electric Conductivity; Humans; Inositol Phosphates; Ion Channel Gating; Kinetics; Magnesium Chloride; Membrane Potentials; Terpenes; Thapsigargin; Time Factors; Tumor Cells, Cultured