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.