Pharmacological block of Ca(2+)-activated Cl- current in rat vascular smooth muscle cells in short-term primary culture.
Ca(2+)-activated Cl- currents were studied in isolated cells from rat portal vein smooth muscle in short-term primary culture using the whole-cell patch-clamp technique. Cl- currents can be activated separately by Ca2+ release from intracellular stores (in response to external applications of caffeine or noradrenaline) and by Ca2+ influx through voltage-dependent Ca2+ channels. The effects of several Cl- channel blockers and of spironolactone (a substance known to reduce internal Ca2+ loading) on both Cl- and Ca2+ currents were examined. Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and diphenylamine-2,2'-dicarboxylic acid (DPC) inhibited the Ca(2+)-activated Cl- current (IC50 values between 16.5 and 306 microM) with no effects on the inward Ca2+ current and on internal Ca2+ loading (testing by measuring the Ca(2+)-activated K+ current). These results indicate that the inhibition of Cl- current by these compounds is due to a direct interaction with the Cl- channel. In contrast, spironolactone inhibited both K+ and Cl- currents (IC50 = 7.6 microM) by reducing the amount of Ca2+ located in the internal stores, whereas the Cl- current activated by Ca2+ current through T-type Ca2+ channels was unchanged. This preparation and the protocols developed in this study appears to be appropriate for analysis of substances interfering with Cl- channels or intracellular Ca2+ stores.
Baron
A
A
Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, INSERM CJF 88-13, Université de Bordeaux II, France.
Pacaud
P
P
Loirand
G
G
Mironneau
C
C
Mironneau
J
J
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D013148:Spironolactone / Q000494:pharmacology
D000873:Anthracenes; D018118:Chloride Channels; D002712:Chlorides; D008565:Membrane Proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D013148:Spironolactone; D002110:Caffeine; C010030:9-anthroic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium