Pressure- and parathyroid-hormone-dependent Ca2+ transport in rabbit connecting tubule: role of the stretch-activated nonselective cation channel.
To characterize the Ca2+ transport process across the apical membrane of the rabbit connecting tubule (CNT), we examined the effects of luminal pressure on parathyroid hormone (PTH)-dependent apical Ca2+ transport in this segment perfused in vitro. An increase of perfusion pressure (0.2 to 1.2 KPa) caused cytoplasmic free Ca2+ concentration ([Ca2+]i) to increase by 42 +/- 11 nM in Fura-2 loaded perfused CNT. The response was accentuated when 10 nM PTH was added to the bath (101 +/- 30 nM, n = 6). Addition of 0.1 mM chlorphenylthio-cAMP (CPT-cAMP) to the bath also augmented the [Ca2+]i response to pressure from 36 +/- 16 to 84 +/- 26 nM (n = 3). Under steady perfusion pressure at 1.2 KPa, PTH (10 nM) increased [Ca2+]i by 31 +/- 7 nM (n = 5), whereas it did only slightly by 6 +/- 2 nM (n = 12) at 0.2 KPa. The pressure-dependent increase of [Ca2+]i was abolished by removing luminal Ca2+ (n = 3), and was not affected by 0.1 and 10 microM nicardipine (n = 4) in the presence of 10 nM PTH. Cell-attached patch clamp studies on the apical membrane of everted CNT with pipettes filled with either 200 mM CaCl2 or 140 mM NaCl revealed channel activities with conductances of 42 +/- 2 pS (n = 4) or 173 +/- 7 pS (n = 5), respectively. An application of negative pressure (-4.9 KPa) to the patch pipette augmented its mean number of open channels (NPo) from 0.005 +/- 0.001 to 0.022 +/- 0.005 in the Ca(2+)-filled pipette, and was further accelerated to 0.085 +/- 0.014 (n = 3) by 0.1 mM CPT-cAMP. In the Na(+)-filled pipette, similar results were obtained (n = 3), and CPT-cAMP did not activate the stretch-activated channel in the absence of negative pressure (n = 3). These results suggest that a stretch-activated nonselective cation channel exists in the apical membrane of the CNT and that it is activated by PTH in the presence of hydrostatic pressure, allowing entry of Ca2+ transport from the apical membrane.
Taniguchi
J
J
Department of Pharmacology, Jichi Medical School, Tochigi, Japan.
Takeda
M
M
Yoshitomi
K
K
Imai
M
M
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D017136:Ion Transport / Q000187:drug effects; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials; D009529:Nicardipine / Q000494:pharmacology; D010281:Parathyroid Hormone / Q000494:pharmacology; D010477:Perfusion; D011312:Pressure; D011817:Rabbits; D013873:Thionucleotides / Q000494:pharmacology
D010281:Parathyroid Hormone; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D009529:Nicardipine; D000242:Cyclic AMP; D002118:Calcium