title: Stimulation of Cl- secretion by extracellular ATP does not depend on increased cytosolic Ca2+ in HT-29.cl16E.
abstract: Extracellular ATP and elevated cytosolic Ca2+ concentration ([Ca2+]i) are major secretagogues for Cl- in the goblet cell-like clone cl.16E derived from colonic HT-29 cells. The involvement of [Ca2+]i as a messenger for the purinergically stimulated Cl- secretion was investigated using whole cell patch-clamp and Ussing chamber techniques, as well as [Ca2+]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 microM extracellular ATP increased the current at +50 mV to 41 +/- 10 pA/pF, with a half-maximal effective dose (ED50) of approximately 3 microM. The current was transient, usually lasting 1-2 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 [Ca2+]i, with an ED50 of approximately 1 microM and maximal changes (at 200 microM) from baseline (71 +/- 3 nM) to 459 +/- 50 nM. The ATP-dependent Cl- conductance increase was not diminished when [Ca2+]i was clamped at 100 nM using a Ca(2+)-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering system. However, the ATP effect did require some basal level of Ca2+ because clamping [Ca2+]i at < 10 nM abolished activation of the Cl- conductance. The presence of the protein kinase A inhibitor H-89 or the protein kinase C 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 [Ca2+]i, suggesting the involvement of either another signaling pathway or direct activation of Cl- channels by purinergic receptors.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
authors.forename: X
authors.initials: X
authors.lastname: Guo
authors.forename: D
authors.initials: D
authors.lastname: Merlin
authors.forename: R D
authors.initials: RD
authors.lastname: Harvey
authors.forename: C
authors.initials: C
authors.lastname: Laboisse
authors.forename: U
authors.initials: U
authors.lastname: Hopfer
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology*; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002712:Chlorides / Q000378:metabolism* / Q000502:physiology; D003600:Cytosol / Q000378:metabolism*; D004553:Electric Conductivity; D005110:Extracellular Space / Q000378:metabolism*; D006801:Humans; D018408:Patch-Clamp Techniques; D015290:Second Messenger Systems; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
chemical_list: D002712:Chlorides; D000255:Adenosine Triphosphate; D002118:Calcium