Hydrogen peroxide inhibits Ca2+ efflux through plasma membrane Ca2+-ATPase in mouse parotid acinar cells.

Intracellular Ca2+ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide (H2O2) on cytosolic Ca2+ accumulation in mouse parotid acinar cells. Intracellular Ca2+ levels were slowly elevated when 1 mM H2O2 was perfused in the presence of normal extracellular Ca2+. In a Ca2+-free medium, 1 mM H2O2 still enhanced the intracellular Ca2+ level. Ca2+ entry tested using manganese quenching technique was not affected by perfusion of 1 mM H2O2. On the other hand, 10 mM H2O2 induced more rapid Ca2+ accumulation and facilitated Ca2+ entry from extracellular fluid. Ca2+ refill into intracellular Ca2+ store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca2+ release from Ca2+ store was not affected by 1 mM H2O2 in permeabilized cells. Ca2+ efflux through plasma membrane Ca2+-ATPase (PMCA) was markedly blocked by 1 mM H2O2 in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected H2O2-induced Ca2+ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of H2O2 under pathological conditions may lead to cytosolic Ca2+ accumulation and that the primary mechanism of H2O2-induced Ca2+ accumulation is likely to inhibit Ca2+ efflux through PMCA rather than mobilize Ca2+ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.

Ethanol suppresses carbamylcholine-induced intracellular calcium oscillation in mouse pancreatic acinar cells.

Oscillation of intracellular calcium levels is closely linked to initiating secretion of digestive enzymes from pancreatic acinar cells. Excessive alcohol consumption is known to relate to a variety of disorders in the digestive system, including the exocrine pancreas. In this study, we have investigated the role and mechanism of ethanol on carbamylcholine (CCh)-induced intracellular calcium oscillation in murine pancreatic acinar cells. Ethanol at concentrations of 30 and 100 mM reversibly suppressed CCh-induced Ca2+ oscillation in a dose-dependent manner. Pretreatment of ethanol has no effect on the store-operated calcium entry induced by 10 µM of CCh. Ethanol significantly reduced the initial calcium peak induced by low concentrations of CCh and therefore, the CCh-induced dose-response curve of the initial calcium peak was shifted to the right by ethanol pretreatment. Furthermore, ethanol significantly dose-dependently reduced inositol 1,4,5-trisphosphate-induced calcium release from the internal stores in permeabilized acinar cells. These results provide evidence that excessive alcohol intake could impair cytosolic calcium oscillation through inhibiting calcium release from intracellular stores in mouse pancreatic acinar cells.

Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells.

Intracellular calcium (Ca2+) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H2O2) on intracellular Ca2+ accumulation in mouse pancreatic acinar cells. Perfusion of H2O2 at 300 µM resulted in additional elevation of intracellular Ca2+ levels and termination of oscillatory Ca2+ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca2+. Antioxidants, catalase or DTT, completely prevented H2O2-induced additional Ca2+ increase and termination of Ca2+ oscillation. In Ca2+-free medium, H2O2 still enhanced CCh-induced intracellular Ca2+ levels and thapsigargin (TG) mimicked H2O2-induced cytosolic Ca2+ increase. Furthermore, H2O2-induced elevation of intracellular Ca2+ levels was abolished under sarco/endoplasmic reticulum Ca2+ ATPase-inactivated condition by TG pretreatment with CCh. H2O2 at 300 µM failed to affect store-operated Ca2+ entry or Ca2+ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca2+ uniporter blocker, failed to attenuate H2O2-induced intracellular Ca2+ elevation. These results provide evidence that excessive generation of H2O2 in pathological conditions could accumulate intracellular Ca2+ by attenuating refilling of internal Ca2+ stores rather than by inhibiting Ca2+ extrusion to extracellular fluid or enhancing Ca2+ mobilization from extracellular medium in mouse pancreatic acinar cells.