TY - JOUR
T1 - Vasoactive intestinal peptide-stimulated Cl- secretion
T2 - Activation of cAMP-dependent K+ channels
AU - Izu, Leighton T
AU - McCulle, S. L.
AU - Ferreri-Jacobia, M. T.
AU - Devor, D. C.
AU - Duffey, M. E.
PY - 2002/4/1
Y1 - 2002/4/1
N2 - 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 (ISC) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased ISC from 3 ± 0.4 μA/cm2 to 75 ± 11 μA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 μM), reduced ISC 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 (ECl- = -2 mV), and activated inward current (179 ± 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (EK+ = -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.
AB - 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 (ISC) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased ISC from 3 ± 0.4 μA/cm2 to 75 ± 11 μA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 μM), reduced ISC 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 (ECl- = -2 mV), and activated inward current (179 ± 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (EK+ = -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.
KW - cAMP-dependent protein kinase (PKA)
KW - Cl secretion
KW - Colon
KW - Intestine
KW - K conductance
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U2 - 10.1007/s00232-001-0145-7
DO - 10.1007/s00232-001-0145-7
M3 - Article
C2 - 12148841
AN - SCOPUS:0036537733
VL - 186
SP - 145
EP - 157
JO - Journal of Membrane Biology
JF - Journal of Membrane Biology
SN - 0022-2631
IS - 3
ER -