Vasoactive intestinal peptide-stimulated Cl- secretion: activation of cAMP-dependent K+ channels.
Vasoactive intestinal peptide (VIP) stimulates active Cl- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K+ conductance. To demonstrate the role of this K- conductance, we measured short-circuit current (I(SC)) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I(SC) from 3 +/- 0.4 microA/cm2 to 75 +/- 11 microA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 microM), reduced I(SC) by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K+ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K+ current (131 +/- 26 pA, n = 15) when membrane potential was held at the Cl- equilibrium potential (E(Cl-) = -2 mV), and activated inward current (179 +/- 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (E(K+) = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nM), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba2+ , 5 mM), but not 293B, blocked this K+ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K + current flow to demonstrate the channels that underlie this K+ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca2+ concentration, [Ca2 +]i. Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K+ current (185 +/- 56 pA, n = 8) without altering [Ca2+]i. These results demonstrate that VIP activates a basolateral membrane K+ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.
Izu
L T
LT
Department of Physiology and Biophysics, State University of New York, University at Buffalo 14214, USA.
McCulle
S L
SL
Ferreri-Jacobia
M T
MT
Devor
D C
DC
Duffey
M E
ME
D002110:Caffeine / Q000008:administration & dosage; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000008:administration & dosage; D004553:Electric Conductivity; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D017136:Ion Transport; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013696:Temperature; D014660:Vasoactive Intestinal Peptide / Q000008:administration & dosage
D002712:Chlorides; D015221:Potassium Channels; D014660:Vasoactive Intestinal Peptide; D002110:Caffeine; D017868:Cyclic AMP-Dependent Protein Kinases