Purinergic-induced ion current in monolayers of C7-MDCK cells: role of basolateral and apical ion transporters.
This study examines purinergic modulation of short-circuit current (I(SC)) in monolayers of C7- and C11-MDCK cells resembling principal and intercalated cells from collecting ducts. In C7 monolayers, basolateral and apical application of ATP led to similar elevation of I(SC), consisting of a transient phase with maximal I(SC) increment of approximately 10 microA/cm2 terminating in 2-3 min, and a sustained phase with maximal I(SC) less than 2 microA/cm2 and terminating in 10 min. ATP-induced I(SC) was insensitive to the presence of Na+, Cl- and inhibitors of K+ (Ba2+, charibdotoxin (ChTX), clotrimazole (CLT), apamin) and Na + (amiloride) channels in the mucosal solution. Inhibitors of Cl- channels, DIDS and NPPB, added to apical membranes at a concentration of 100 microM, did not affect ATP-induced I(SC), whereas at 500 microM, NPPB inhibited it by 70-80%. Substitution of Cl- with NO3- in serosal medium decreased ATP-induced I(SC) by 2-3-fold and elevation of [K+]o from 6 to 60 mM changed its direction. Basolateral NPPB inhibited I(SC) by 10-fold with ED50 of approximately 30 microM, whereas ChTX (50 nM) and CLT (2 microM) diminished this parameter by 30-50%. Suppression of Na+, K+, Cl- cotransport with bumetanide did not affect the transient phase of ATP-induced I(SC) and slightly diminished its sustained phase. ATP increased ouabainand bumetanide-resistant K+ (86Rb) influx across the basolateral membrane by 7-8-fold, which was partially inhibited with ChTX and CLT. ATP-treated C11 cells exhibited negligible I(SC), and their presence did not affect I(SC) triggered by ATP in C7 cells. Thus, our results show that transcellular ion current in ATP-treated C7 cells is mainly caused by the coupled function of apical and basolateral anion transporters providing transient Cl- secretion. These transporters possess different sensitivities to anion channel blocker NPBB and are under the control of basolateral K+ channels(s) inhibited by ChTX and CLT.
Bourcier
N
N
Centre de recherche, Centre hospitalier de l'Université de Montréal-Hôtel-Dieu, Quebec, Canada.
Grygorczyk
R
R
Gekle
M
M
Berthiaume
Y
Y
Orlov
S N
SN
D000255:Adenosine Triphosphate / Q000008:administration & dosage; D000818:Animals; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D004285:Dogs; D004594:Electrophysiology; D004837:Epinephrine / Q000008:administration & dosage; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques / Q000379:methods; D015221:Potassium Channels / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015222:Sodium Channels / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism
D018118:Chloride Channels; D015221:Potassium Channels; D018048:Receptors, Purinergic P2; D015222:Sodium Channels; D028021:Sodium-Potassium-Chloride Symporters; D000255:Adenosine Triphosphate; D004837:Epinephrine