Stimulation of cl- secretion by extracellular atp does not depend
on increased cytosolic ca2+ in the mucin-secreting, epithelial cell
line ht29-cl.16e.
Guo, Xiaowei, Didier Merlin, Robert D. Harvey, Christian Laboisse, and
Ulrich Hopfer.
Department of Physiology and Biophysics, School of Medicine, Case
Western Reserve University, Cleveland, OH, 44106-4970 and
Universit[acute]e de Nantes, Facult[acute]e, de M[acute]edecine,
Groupe de Recherche: Fonctions S[acute]ecr[acute]etoires des
Epitheliums Digestifs, F-44035 Nantes (France)
APStracts 2:0225C, 1995.
Extracellular ATP and elevated cytosolic Ca2+ ([Ca]i) are major
secretagogues for Cl- in the goblet cell-like clone Cl.16E derived
from colonic HT29 cells. The involvement of [Ca]i as a messenger for
the purinergically stimulated Cl- secretion was investigated using
whole-cell patch-clamp and Ussing chamber techniques, as well as
[Ca]i measurements using fura-2 loaded cells. Under voltage clamp
conditions, the whole cell current at +50 mV was 3+/-1 pA/pF under
unstimulated conditions. Stimulation of purinergic receptors with 200
[mu]M extracellular ATP increased the current at +50 mV to 41+/-10
pA/pF, with an ED50 of about 3 [mu]M. The current was transient,
usually lasting 1 to 2 min, and the current-voltage relationship was
approximately linear between -70 and +50 mV. Evidence that the ATP
-stimulated current was carried by Cl- included: 1) The reversal
potential of the current closely followed the Cl- equilibrium
potential, and 2) the stimulated current was absent when Cl- was
removed from both bath and pipette solutions. Exposure to ATP also
increased [Ca]i with an ED50 of about 1 [mu]M and maximal changes (at
200 [mu]M) from baseline (71+/-3 nM) to 459+/-50 nM. The ATP
-dependent Cl- conductance increase was not diminished when [Ca]i was
clamped at 100 nM using a Ca2+/BAPTA or Ca2+/EGTA buffering system.
However, the ATP effect did require some basal level of Ca2+, as
clamping [Ca]i at <10 nM abolished activation of the Cl-
conductance. The presence of the PKA inhibitor H89 or the PKC
inhibitor staurosprine did not change the ATP-activated Cl-
conductance. These data demonstrate that the ATP-stimulated increase
in Cl- current does not require an increase in [Ca]i, suggesting the
involvement of either another signaling pathway or direct activation
of Cl- channels by purinergic receptors.
Received 28 March 1995; accepted in final form 25 May 1995.
APS Manuscript Number C174-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 8 June 1995.