New evidence of a dihydropyridine-activated cationic channel in the MDCK cell line.

Newborn rat distal cells express an apical Ca2+ channel activated by dihydropyridine drugs. Similarly, in Madin-Darby canine kidney (MDCK) cells, nifedipine increased Ca2+i in a concentration-dependent manner (IC50=4 µM) in fura-2-loaded cells. Response to nifedipine was abolished by EGTA, suggesting that it depends on extracellular calcium. Ca2+ channel antagonist isradipine and agonist BayK8644 increased Ca2+i indicating that this effect is related to the dihydropyridine group. Diltiazem (20 µM) and gadolinium (200 µM) decreased the nifedipine effect (62 and 43%, respectively). Lanthanum (100 µM) did not change the response. Valinomycin clamping of the membrane potential did not modify nifedipine-induced increment, indicating that it was unrelated to potassium fluxes. We performed whole cell clamp experiments in MDCK cells maintained at -50 mV with perfusion solution containing 10 mM CaCl2. Nifedipine (20 µM) induced an increase in current (1.2±0.3 nA), which was partially inhibited by Gd3+. No significant current was induced by nifedipine in the presence of 0.5 mM EGTA. To determine the effects of nifedipine on the membrane potential, we performed oxonol fluorescence experiments. The addition of nifedipine or Bay K8644 induced depolarization, highly dependent on external sodium. Nifedipine (20 µM) induced depolarization of 6.9±0.8 mV (n=21). EC50 to nifedipine was in the 10 µM range. We conclude that MDCK cells exhibit a dihydropyridine-activated cationic channel.

Nifedipine-activated Ca(2+) permeability in newborn rat cortical collecting duct cells in primary culture.

To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).