An increase in [Ca2+]i activates basolateral chloride channels and inhibits apical sodium channels in frog skin epithelium.
The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 microM) increased [Ca2+]i from 66 +/- 9 to 137 +/- 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (Isc) to drop from 26.4 to 10.6 microA cm-2 (n = 19, P<0.001) concomitant with a depolarization of the cells from -79 +/- 1 to -31 +/- 2 mV (n = 18, P<0.001). Apical sodium permeability (PaNa) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. PaNa decreased from 8.01.10(-7 )to 3.74.10(-7) cm s-1 (n = 7, P = 0.04) following an increase in [Ca2+]i. A decrease in apical sodium permeability per se would tend to decrease Isc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca2+]i. This was confirmed in wash-out experiments with 36Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 +/- 5.01 to 62.63 +/- 13.3 nmol.min-1 cm-2 (n = 5, P = 0.04). We conclude that a small increase in [Ca2+]i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption.
Brodin
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Biochemical Department, August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100, Denmark.
Rytved
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KA
Nielsen
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D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004594:Electrophysiology; D004848:Epithelium / Q000378:metabolism; D005260:Female; D007425:Intracellular Membranes / Q000378:metabolism; D008297:Male; D009994:Osmolar Concentration; D010539:Permeability; D011188:Potassium / Q000378:metabolism; D011896:Rana temporaria; D012867:Skin / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D026941:Sodium Channel Blockers; D019284:Thapsigargin / Q000494:pharmacology
D018118:Chloride Channels; D002712:Chlorides; D026941:Sodium Channel Blockers; D019284:Thapsigargin; D012964:Sodium; D011188:Potassium; D002118:Calcium