title: Bradykinin-stimulated Cl- secretion in T84 cells. Role of Ca2+-activated hSK4-like K+ channels.
abstract: Bradykinin (BK)-stimulated colonic Cl- secretion was studied in T84 colonic adenocarcinoma cells by measuring BK (50 nM)-evoked changes in cytosolic free [Ca2+] ([Ca2+]i), membrane conductance and transepithelial ion transport. In T84 cells grown on impermeable supports, BK stimulated a transient increase in [Ca2+]i as assessed by fura-2 ratio imaging. In cell-attached, patch-clamp recordings, BK transiently activated low-conductance K channels. These channels were activated/inactivated reversibly in inside-out patches by switching [Ca2+]i in the bath between 30 nM and 100 nM. Excised channels recorded with 160 mM [K+] in bath and pipette exhibited an inwardly rectifying current/voltage-relation, conductances of 10+/-1 pS and 34+/-4 pS (n=10) at positive and negative voltages, respectively, and a 15-fold lower permeability for Na+ than for K+. The mean open probability of these channels did not depend on voltage but increased with increasing [Ca2+]i with an apparent concentration for a half-maximal response (EC50) of 110 nM, resembling that of hSK4 K+ channels. Application of the reverse transcriptase-polymerase chain reaction technique showed hSK4 messenger ribonucleic acid (mRNA) to be expressed in T84 cells. Macroscopic currents in T84 cells showed a similar dependence on [Ca2+]i. Whole cell conductance (in nS/10pF) increased from 0.5+/-0. 1 (n=6) at 10 nM [Ca2+]i in the pipette solution to 1.5+/-0.2 (n=7) at 100 nM, and to 2.0+/-0.5 (n=7) at 1 microM due to activation of a K+ conductance. In Ussing-chambered T84 monolayers grown on filters, BK did not evoke a short-circuit current (Isc). When, however, the monolayers were pre-stimulated by forskolin (1 microM), BK further enhanced Cl-secretion (DeltaIsc=21+/-5 microA/cm2, n=10) transiently and biphasically. In conclusion, BK enhances cyclic adenosine monophosphate-stimulated Cl- secretion in T84 cells, probably via basolateral, Ca2+-liganded activation of low-conductance hSK4-type K+ channels.
authors.affiliation: Physiologisches Institut der Ludwig-Maximilians-Universität, Pettenkoferstrasse 12, D-80336 Munich, Germany.
authors.forename: S M
authors.initials: SM
authors.lastname: Huber
authors.forename: J
authors.initials: J
authors.lastname: Tschöp
authors.forename: G S
authors.initials: GS
authors.lastname: Braun
authors.forename: W
authors.initials: W
authors.lastname: Nagel
authors.forename: M F
authors.initials: MF
authors.lastname: Horster
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism*; D001920:Bradykinin / Q000494:pharmacology*; D020013:Calcium Signaling / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000378:metabolism*; D016257:Fura-2; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D024681:Potassium Channels, Calcium-Activated; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D002712:Chlorides; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D001920:Bradykinin; D016257:Fura-2