| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1993: ¥800,000 (Direct Cost: ¥800,000)
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| Research Abstract |
To characterize parathyroid hormone (PTH) -dependent luminal Ca^<2+> transport in rabbit connecting tubule (CNT), I examined the effects of luminal pressure on this transport. Cytoplasmic Ca^<2+> concentraion ([Ca^<2+>]_i) in fura 2-loaded CNT perfused in vitro was elevated by 42<plus-minus>11nM by increasing luminal pressure (0.2 to 1.2KPa).Basolateral 10nM PTH accelerated the response to 101<plus-minus>30nM.The response was augmented from 36<plus-minus>16nM to 84<plus-minus>26nM by 0.1mM chlorphenyl-thio-cAMP (CPT-cAMP). Under steady perfusion pressure at 1.2KPa, 10nM PTH increased [Ca^<2+>]i by 31<plus-minus>7nM,whereas it did by 6([SY.+-.[)2nM at 0.2KPa. The pressure dependent increase of [Ca^<2+>]_i was abolished by removing luminal Ca^<2+> in the presence of 10nM PTH,and was not affected by luminal 0.1 or 10 mu M nicardipine. Both 100 mu M nifedipine and benijipine suppressed by 24<plus-minus>6% and by 30<plus-minus>11% of [Ca^<2+>]i increased by 50nM PTH,respectively, but 10 mu M Gd^<3+> did by 90<plus-minus>19%. 10nM PTH hyperpolarized transmural voltage, followed by depolarization. Luminal Gd^<3+> (10 mu M) reduced only depolarization by 60<plus-minus>19%. Cell-attached patch clamp studies on the apical membrane of everted CNT using pipette filled with either 200mM CaCl_2 or 140mM NaCl revealed channel activities (42<plus-minus>2pS or 173<plus-minus>7pS,respectively). Negative pressure (-4.9KPa) applied to patch pipette augmented its mean number of open channels from 0.005<plus-minus>0.001 to 0.022<plus-minus>0.005 in the Ca^<2+>-filled pipette, and was further accelerated to 0.085<plus-minus>0.014 by 0.1mM CPT-cAMP.In the Na^+-filled pipette, similar results were obtained, and CPT-cAMP did not activate unstretch channels. These results revealed that stretch-activated nonselective cation channel is a dominant route of Ca^<2+> transport via luminal membrane of rabbit CNT and that PTH stimulates it in the presence of luminal pressure or fluid flow.
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