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The mechanisms by which Pi depletion rapidly regulates gene expression and cellular function have not been clarified. Here, we found a rapid increase in intracellular ionized calcium [Ca(2+)](i) by phosphate depletion in LLC-PK(1) cells using confocal microscopy with the green-fluorescence protein based calcium indicator "yellow cameleon 2.1." The increase of [Ca(2+)](i) was observed in the presence or absence of extracellular Ca(2+). At the same time, an approximately twofold increase in intracellular inositol 1,4,5-triphosphate (IP(3)) occurred in response to the acute Pi depletion in the medium. Furthermore, 2-aminoethoxydiphenyl borate completely blocked the [Ca(2+)](i) increase induced by Pi depletion. These results suggest that Pi depletion causes IP(3)-mediated release of Ca(2+) from intracellular Ca(2+) pools and rapidly increases [Ca(2+)](i) in LLC-PK(1) cells.

The awareness of phosphorus intake is important because hyperphosphatemia and hypophosphatemia both impair bone metabolism. Phosphorus consumption from food was obtained from values in the Food Balance Sheet (PBS) of Japan from 1960 to 1995. The amounts of phosphorus calculated from the FBS increased gradually from 1,243 mg/d in 1960 to 1,332 mg/d in 1975 and to 1,421 mg/d in 1995. This is explained by the increased consumption of cow's milk and milk products, meat, and chicken eggs. The main foods supplying phosphorus in 1995 were cereals, milk and milk products, fishes and shellfishes, and vegetables; their contributions were 24.4, 15.8, 14.2, and 10.9%, respectively. The phosphorus-to-calcium ratio calculated from the FBS was 3.51 in 1960, which decreased to 2.89 in 1975 and 2.44 in 1995. Therefore total phosphorus consumption in 1995 was presumably more than 1,500 mg/d when imported food containing phosphorus and the consumption of phosphorus-containing food additives in Japan are also considered. These findings suggest that the phosphorus consumption estimated from the FBS is increasing and that more attention should be paid to the maintenance of healthy bones in Japan, where the average amount of calcium intake is less than 600 mg/d.

Reactive oxygen species, generated by reduction-oxidation (redox) reactions, have been recognized as one of the major mediators of ischemia and reperfusion injury in the brain. Reactive oxygen species-induced cerebral events are attributable, in part, to the change in intracellular signaling molecules including mitogen-activated protein (MAP) kinases. Big MAP kinase 1 (BMK1), also known as ERK5, is a newly identified member of the MAP kinase family and has been reported to be sensitive to oxidative stress. In the present study, we examined the effect of H(2)O(2) on BMK1 activity in PC12 cells, and we investigated the pathophysiological implication of BMK1. Findings showed that BMK1 was rapidly and significantly activated by H(2)O(2) in a concentration-dependent manner in PC12 cells. BMK1 activation by H(2)O(2) was inhibited by both PD98059 and U0126, which were reported to inhibit MEK5 as well as MEK1/2. c-Src was suggested to be involved in BMK1 activation from the experiments with herbimycin A and PP2, specific inhibitors of Src family kinases. Transfection of kinase-inactive Src also inhibited H(2)O(2)-induced BMK1 activation. In addition, H(2)O(2) treatment of cells induced an enhancement of DNA binding activity of MEF2C, a downstream transcription factor of BMK1 in PC12 cells. Finally, pretreatment of cells with PD98059 and U0126 resulted in an increase in cell death including apoptosis by H(2)O(2) in ERK1/2 down-regulated cells as well as in intact PC12 cells. These findings suggest that c-Src mediated BMK1 activation by H(2)O(2) may counteract ischemic cellular damage probably through the activation of MEF2C transcription factor.