Abstract
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+].) 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+]; 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+]; 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+]; was abolished by removing luminal Ca2+ (n = 3), and was not affected by 0.1 and 10 μm 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 (NP 0 ) from 0.005 ± 0.001 to 0.022 ± 0.005 in the Ca2+-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.
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We appreciate Ms. Hisayo Hosaka and Ms. Yuki Oyama for their technical assistance and Ms. Keiko Sakai for her secretarial work. This research was supported by grants from the Ministry of Education and Culture of Japan (No. 05670054) and from Yamanouchi Foundation for Research on Metabolic Disorders (1992–1993).
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Taniguchi, J., Takeda, M., Yoshitomi, K. et al. Pressure- and parathyroid-hormone-dependent Ca2+ transport in rabbit connecting tubule: Role of the stretch-activated nonselective cation channel. J. Membarin Biol. 140, 123–132 (1994). https://doi.org/10.1007/BF00232900
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DOI: https://doi.org/10.1007/BF00232900