| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
2001
Ca^<2+> entry pathway activated by sphingosine-1-phosphate (S1P) was examined in human umbilical vein endothelial cells (HUVECs) by measuring intracellular Ca^<2+> concentration ([Ca^<2+>]_i), whole-cell membrane currents and single channel activity. Application of S1P to HUVECs induced a slowly developing increase in [Ca^<2+>_i. When Ca^<2+> was absent in the bathing solution, S1P did not affect [Ca^<2+>]_i Addition of Ca^<2+> to the bathing solution, however, produced a sustained increase in [Ca^<2+>_i in the presence of S1P, suggesting that influx of Ca^<2+> plays an obligatory role in elevation of [Ca^<2+>_i by S1P. Pretreatment of HUVECs with pertussis toxin (PTX) abolished S1P-induced elevation of [Ca^<2+>]_i. When whole-cell membrane currents were recorded under the monitoring of [Ca^<2+>]_i, the application of S1P induced a tiny inward current (I_<S1P>) which was followed by the elevation of [Ca^<2+>]_i. The reversal potential of I_<S1P> revealed that I_<S1P> is a ***-selec
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tive cation (NSC) current. When S1P was included in the pipette solution in the excised inside-out patch clamp configuration, ***gle channel with a conductance of 17 pS was activated, depending on the presence of intracellular GTP. These results suggest that S1P has a novel function to activate a NSC-channel in a GTP-dependent manner via a PTX-sensitive G-protein. The NSC-channel activated by S1P acts as a Ca^<2+> entry pathway in endothelium and may regulate the cell-proliferation. These results have been published in J. Physiol. (537, 431-441, 2001)
2002
Effects of palmitoylcarnitine (palcar), a lipid that is released from various types of cells under the ischemic conditions, on [Ca^<2+>]_i were examined in HUVECs and compared with those of S1P. Application of palcar elevated [Ca^<2+>]_i in HUVECs and its potency was about 30 times lower than that of S1P. Response to 3 μM palcar in each HUVEC clearly paralleled that to 0.3 μM S1P. In addition, HUVECs that were treated with PTX failed to respond to palcar as werll as to S1P. These results suggest that palcar has a novel action on huvecs as a potential agonist of receptors for S1P. These results have been published in J. Pharmacol. Sci. (in press, 2003) Less
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