Stimulation of Cl- secretion by extracellular ATP does not depend on increased cytosolic Ca2+ in HT-29.cl16E

1995 ◽  
Vol 269 (6) ◽  
pp. C1457-C1463 ◽  
Author(s):  
X. Guo ◽  
D. Merlin ◽  
R. D. Harvey ◽  
C. Laboisse ◽  
U. Hopfer
Keyword(s):  
Protein Kinase ◽  
Purinergic Receptors ◽  
Extracellular Atp ◽  
Equilibrium Potential ◽  
Tetraacetic Acid ◽  
Whole Cell ◽  
Cl Secretion ◽  
Ht 29 ◽  
Cl Conductance ◽  
Stimulation Of

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.

CaMKII mediates stimulation of chloride conductance by calcium in T84 cells

1991 ◽  
Vol 260 (4) ◽  
pp. C877-C882 ◽  
Author(s):  
R. T. Worrell ◽  
R. A. Frizzell
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Peptide Inhibitor ◽  
Secretory Cells ◽  
T84 Cells ◽  
Whole Cell ◽  
Regulatory Pathways ◽  
Kinase C ◽  
Cl Conductance ◽  
Stimulation Of

We used the secretory colonic cell line T84 to study the regulatory pathways controlling the Ca-stimulated Cl conductance [GCl(Ca)]. Under whole cell patch clamp, basal (unstimulated) current levels averaged 73 +/- 9 pA/20 pF (n = 93) and increased to 600 +/- 100 pA/20 pF (n = 53; at +100 mV) on exposure to 1-2 microM ionomycin. Bath application of the calmodulin (CaM) antagonists trifluoperazine, calmidazolium, or sphingosine (50 microM) reversibly inhibited GCl(Ca), whereas the protein kinase C antagonists H7 and phloretin (50 microM) were without effect. This suggests that increases in intracellular Ca stimulate GCl(Ca) via a CaM-dependent process rather than activating Cl channels directly. To assess the involvement of protein kinases in the Ca-dependent stimulation of Cl conductance, we employed pseudosubstrate peptide inhibitors of protein kinase C (PKC) and the Ca/CaM-dependent protein kinase II (CaMKII). Cellular concentrations of inhibitors during whole cell recording were estimated to be 4-20 times the inhibitory constant values for kinase inhibition observed in vitro. Pipette solutions containing the PKC peptide inhibitor PKC-(19-36) (7.5 microM) had no effect on GCl(Ca). In contrast, stimulation of GCl(Ca) by ionomycin was abolished when pipette solutions contained 10 microM CaMKII peptide inhibitor CaMKII-(273-302). The truncated peptide CaMKII-(284-302) (20 microM) lacks the CaMKII inhibitory domain and did not affect GCl(Ca). These data suggest that CaM, acting through the multifunctional CaMKII, mediates the Ca-dependent stimulation of Cl conductance in colonic secretory cells.

Activation of CFTR Cl- conductance in polarized T84 cells by luminal extracellular ATP

1995 ◽  
Vol 268 (2) ◽  
pp. C425-C433 ◽  
Author(s):  
M. J. Stutts ◽  
E. R. Lazarowski ◽  
A. M. Paradiso ◽  
R. C. Boucher
Keyword(s):  
Adenosine Receptor ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Extracellular Atp ◽  
T84 Cells ◽  
Apical Cell Membrane ◽  
Cl Secretion ◽  
Cl Conductance

Luminal extracellular ATP evoked a bumetanide-sensitive short-circuit current in cultured T84 cell epithelia (90.2 +/- 18.2 microA/cm2 at 100 microM ATP, apparent 50% effective concentration, 11.5 microM). ATP appeared to increase the Cl- conductance of the apical membrane but not the driving force for Cl- secretion determined by basolateral membrane K+ conductance. Specifically, the magnitude of Cl- secretion stimulated by ATP was independent of basal current, and forskolin pretreatment abolished subsequent stimulation of Cl- secretion by ATP. Whereas ATP stimulated modest production of adenosine 3',5'-cyclic monophosphate (cAMP) by T84 cells, ATP caused smaller increases in intracellular Ca2+ and inositol phosphate activities than the Ca(2+)-signaling Cl- secretagogue carbachol. An inhibitor of 5'-nucleotidase, alpha,beta-methyleneadenosine 5'-diphosphate, blocked most of the response to luminal ATP. The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline blocked both the luminal ATP-dependent generation of cAMP and Cl- secretion when administered to the luminal but not submucosal bath. These results demonstrate that the Cl- secretion stimulated by luminal ATP is mediated by a A2-adenosine receptor located on the apical cell membrane. Thus metabolism of extracellular ATP to adenosine regulates the activity of cystic fibrosis transmembrane conductor regulator (CFTR) in the apical membrane of polarized T84 cells.

Conductive properties of a rabbit cortical collecting duct cell line: regulation by isoproterenol

1992 ◽  
Vol 262 (4) ◽  
pp. F578-F582 ◽  
Author(s):  
P. Dietl ◽  
N. Kizer ◽  
B. A. Stanton
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cell Line ◽  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Conductive Properties ◽  
Cl Conductance ◽  
Kinase A

The present study was carried out to characterize the membrane conductive properties of RCCT-28A cells, a continuous cell line derived from rabbit cortical collecting duct (CCD). RCCT-28A cells have many phenotypic properties of acid-secreting intercalated cells (A-IC). Using the whole cell patch-clamp technique, we found that the cells are conductive to Cl-, but not to Na+ or K+. The beta-adrenergic agonists isoproterenol (2 x 10(-6) M) and adenosine 3',5'-cyclic monophosphate (cAMP, 10(-4) M) increased the whole cell Cl- conductance. Protein kinase A (150 nM) in the patch pipette (i.e., intracellular solution) also increased whole cell Cl- conductance. Because isoproterenol increases cAMP levels in these cells, we conclude that isoproterenol stimulates the Cl- conductance by increasing cell cAMP, which in turn activates protein kinase A. In contrast, vasopressin does not increase cAMP in these cells and did not increase the Cl- conductance. In conclusion, these experiments show that RCCT-28A cells, like A-IC, are conductive only to Cl-. Thus RCCT-28A cells are a good model with which to study Cl- channels in the collecting duct.

Recruitment of purinergically stimulated Cl- channels from granule membrane to plasma membrane

1996 ◽  
Vol 271 (2) ◽  
pp. C612-C619 ◽  
Author(s):  
D. Merlin ◽  
X. Guo ◽  
K. Martin ◽  
C. Laboisse ◽  
D. Landis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Inhibitory Effects ◽  
Fungal Metabolite ◽  
Cl Secretion ◽  
Fusion Inhibitors ◽  
Granule Membrane ◽  
Cl Channels ◽  
Ht 29 ◽  
Cl Conductance

HT29-Cl.16E and HT29-Cl.19A are two different subcloned cell lines derived from the human adenocarcinoma cell line HT-29. They are similar in their ability to grow and differentiate to polarized epithelial cells but differ in that HT29-Cl.16E is goblet cell-like with many mucin granules, whereas HT29-Cl.19A lacks mucin granules. Extracellular ATP stimulates Cl- secretion in both cell lines through luminal purinergic P20 receptors and, in HT29-Cl.16E, also mucin secretion release. To evaluate whether fusion of mucin granules is associated with an increase in Cl- conductance of the plasma membrane, the effects of two fusion inhibitors on luminal Cl- conductance were measured. Blockage of actin depolymerization with phalloidin (1 microM) inhibited purinergically stimulated but not adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated luminal Cl- efflux by 50% in HT29-Cl.16E. The same treatment was without effect in HT29-Cl.19A. The fungal metabolite wortmannin, which is an inhibitor of regulated exocytosis in leukocytes, at 100 nM inhibited Cl- secretion by 70% in HT29-Cl.16E. This inhibition was not a direct effect on purinergically stimulated Cl- channels because wortmannin concentrations of up to 1 microM did not affect the secretory response in HT29-Cl.19A. The wortmannin inhibition of Cl- secretion is associated with an inhibition of granule fusion as judged by electron microscopy. The differential inhibition of Cl- secretion in the related HT-29 clones that differ with respect to the presence of mucin granules indicates that 1) the granule fusion inhibitors, phalloidin and wortmannin, have no direct inhibitory effects on purinergically and cAMP-activated Cl- channels, 2) a major portion of purinergically but not cAMP-activated Cl- channels is associated with granule fusion in HT29-Cl.16E, and 3) the signaling pathways for Cl- secretion and granule fusion are not completely identical.

Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl- secretion in epithelial cells

1994 ◽  
Vol 266 (1) ◽  
pp. C254-C268 ◽  
Author(s):  
A. P. Morris ◽  
S. A. Cunningham ◽  
A. Tousson ◽  
D. J. Benos ◽  
R. A. Frizzell
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Brefeldin A ◽  
Microtubule Organizing Center ◽  
Cl Secretion ◽  
Organizing Center ◽  
Reversible Time ◽  
Golgi Network ◽  
Ht 29 ◽  
Cl Conductance

The relationship between adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and the cellular location of the cystic fibrosis transmembrane conductance regulator (CFTR) was determined in both polarized (Cl.19A) and unpolarized (parental) HT-29 colonocytes expressing similar levels of CFTR mRNA and protein. CFTR immunolocalized to the apical membrane domain of polarized colonocytes exhibiting cAMP-responsive Cl- secretion. In contrast, CFTR staining was perinuclear in unpolarized colonocytes, which gave little or no cAMP-stimulated Cl- conductance responses. Thus cAMP-stimulated Cl- secretion coincided with an apical localization of CFTR. Brefeldin A (BFA) was used to perturb glycoprotein targeting in these cells. In polarized colonocytes, BFA caused a reversible, time-dependent decrease in the Cl-conductance response to cAMP but not Ca2+. Apical CFTR redistributed into large coalesced intracellular vesicles, located within the same plane as the microtubule organizing center, a marker for the trans-Golgi network (TGN). In preconfluent monolayers or unpolarized HT-29 cells, BFA had no effect on CFTR staining, which remained perinuclear. Mature, Golgi-processed CFTR protein was isolated from both polarized and unpolarized colonocytes. Thus the mechanism for polarization-dependent apical membrane CFTR targeting and the acquisition of cAMP-dependent Cl- secretion lies at or beyond the late Golgi-TGN in epithelial cells.

The role of an inwardly rectifying chloride conductance in postsynaptic inhibition

1994 ◽  
Vol 72 (1) ◽  
pp. 273-284 ◽  
Author(s):  
K. Staley
Keyword(s):  
Protein Kinase ◽  
Dentate Gyrus ◽  
Maximum Amplitude ◽  
Chloride Conductance ◽  
Equilibrium Potential ◽  
Inward Rectification ◽  
Tetraacetic Acid ◽  
Recording Time ◽  
Inwardly Rectifying

1. The relationship of the activation of a voltage-sensitive chloride conductance [GCl(V)] to the chloride transmembrane equilibrium potential (ECl) and the consequent role of this conductance in determining the effect of the gamma-aminobutyric acid-A (GABAA) receptor-mediated transmembrane chloride (Cl-) flux were investigated with the use of whole-cell recordings in the CA1 and dentate gyrus regions of adult rat hippocampal slice preparations. 2. GCl(V) was inwardly rectifying, with significant conductance only at membrane potentials more negative than ECl. For all tested neuronal Cl- concentrations, the activation of GCl(V) could be described by a Boltzman equation with an average half-activation voltage 15 mV negative to ECl, a slope factor of 14 mV, and a maximum conductance of 5 microS. There was no time-dependent inactivation of GCl(V). 3. GCl(V) was modulated by intracellular divalent cations. When magnesium was omitted from the electrode solution, the inward rectification of GCl(V) was unchanged, but the maximum amplitude of GCl(V) increased by a factor of 1.7. GCl(V) was blocked by bath application of 100 microM zinc (Zn2+), but not when 1–6 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) were present in the electrode solution. 4. GCl(V) was increased by 10 microM norepinephrine, and by activation of protein kinase A (PKA) with 1 mM 8-bromoadenosine cyclic monophosphate (8-Br cAMP). GCl(V) was blocked by activation of protein kinase C (PKC) with 10 microM phorbol 12,13-dibutyrate (PdBu) or 1-oleoyl-2-acetyl-sn-glycerol (OAG). 5. GCl(V) was present in all tested CA1 pyramidal neurons but no dentate gyrus neurons. In standard extracellular solution, the amplitude of GCl(V) was initially negligible but increased with recording time, suggesting that under normal conditions GCl(V) is blocked by an endogenous divalent cation or downregulated by PKC. 6. In current-clamp recordings, the steady-state resting membrane potential (RMP) diminished with Cl- loading, from -73 mV (4 mM electrode Cl-) to -27 mV (131 mM electrode Cl-). When GCl(V) was blocked with PdBu, there was no change in the RMP with Cl- loading. When electroneutral Cl- transport was blocked, voltage-clamp experiments using electrode Cl- concentrations of 4–131 mM demonstrated that ECl changed in parallel with the holding potential, but not when GCl(V) was blocked by PdBu.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart.

1993 ◽  
Vol 101 (5) ◽  
pp. 629-650 ◽  
Author(s):  
T C Hwang ◽  
M Horie ◽  
D C Gadsby
Keyword(s):  
Steady State ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Ventricular Myocytes ◽  
Protein Phosphatases ◽  
Pipette Solution ◽  
Whole Cell ◽  
Endogenous Protein ◽  
High Level ◽  
Cl Conductance

The regulation of cardiac Cl- conductance by cAMP-dependent protein kinase (PKA) and cellular phosphatases was studied in isolated guinea pig ventricular myocytes by using wide-tipped, perfused pipettes to record whole-cell currents. Exposure to forskolin (Fsk) or isoproterenol (Iso) elicits a Cl- conductance that results exclusively from PKA-dependent phosphorylation because it can be completely abolished, or its activation fully prevented, by switching to pipette solution containing PKI, a synthetic peptide inhibitor of PKA. The Cl- conductance activated by micromolar concentrations of either agonist reached its steady-state amplitude in 1-2 min and was deactivated promptly and entirely, usually within 2 min, upon washing out the agonist, implying a continuous high level of activity of endogenous protein phosphatases. Accordingly, intracellular application of okadaic acid or microcystin, both potent inhibitors of protein phosphatases 1 and 2A, during exposure to Fsk enhanced the steady-state Cl- conductance and slowed its deactivation after washing out the Fsk. Maximal potentiation of the conductance, by approximately 60%, was obtained with pipette concentrations of approximately 10 microM okadaic acid (or approximately 5 microM microcystin) and did not result from an increase in the apparent affinity for Fsk. In the presence of maximally effective concentrations of okadaic acid and/or microcystin, deactivation of the enhanced Cl- conductance upon washout of agonist was incomplete, with about half of the conductance persisting indefinitely. That residual conductance did not reflect continued action of PKA because it was insensitive to PKI, but was identified as a fraction of the activated Cl- conductance by its biophysical characteristics. The results suggest that complete deactivation of the PKA-regulated cardiac Cl- conductance requires dephosphorylation by a type 1 and/or 2A phosphatase, but that partial deactivation can be accomplished by activity of some other phosphatase(s). These findings are consistent with sequential phosphorylation of a protein, probably the Cl- channel itself, at two different kinds of sites. The resulting phosphoproteins can be distinguished on the basis of their different contributions to whole-cell Cl- conductance.

Carbachol induces oscillations in membrane potential and intracellular calcium in a colonic tumor cell line, HT-29

1997 ◽  
Vol 273 (4) ◽  
pp. C1186-C1193 ◽  
Author(s):  
P. Sand ◽  
T. Svenberg ◽  
B. Rydqvist
Keyword(s):  
Membrane Potential ◽  
Reversal Potential ◽  
Tumor Cell Line ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Control Solution ◽  
Whole Cell ◽  
Calcium Dependent ◽  
High K ◽  
Ht 29

The patch-clamp technique was used to study the effects of carbachol (CCh) on HT-29 cells. During CCh exposure, the cells ( n = 23) depolarized close to the equilibrium potential for Cl−([Formula: see text]; −48 mV) and the membrane potential then started to oscillate (16/23 cells). In voltage-clamp experiments, similar oscillations in whole cell currents could be demonstrated. The whole cell conductance increased from 225 ± 25 pS in control solution to 6,728 ± 1,165 pS (means ± SE, n = 17). In substitution experiments (22 mM Cl− in bath solution,[Formula: see text]= 0 mV), the reversal potential changed from −41.6 ± 2.2 mV (means ± SE, n = 9) to −3.2 ± 2.0 mV (means ± SE, n = 7). When the cells were loaded with the calcium-sensitive fluorescent dye, fluo 3, and simultaneously patch clamped, CCh caused a synchronous oscillating pattern of fluorescence and membrane potential. In cell-attached patches, the CCh-activated currents reversed at a relative membrane potential of 1.9 ± 3.7 mV (means ± SE, n = 11) with control solution in the pipette and at 46.2 ± 5.3 mV (means ± SE, n = 10) with a 15 mM Cl− solution in the pipette. High K+ (144 mM) did not change the reversal potential significantly ( P ≤ 0.05, n = 8). In inside-out patches, calcium-dependent Cl−channels could be demonstrated with a conductance of 19 pS ( n = 7). It is concluded that CCh causes oscillations in membrane potential that involve calcium-dependent Cl−channels and a K+ permeability.

Purinergic agonists, but not cAMP, stimulate coupled granule fusion and Cl- conductance in HT29-Cl.16E

1997 ◽  
Vol 273 (3) ◽  
pp. C804-C809 ◽  
Author(s):  
X. W. Guo ◽  
D. Merlin ◽  
C. Laboisse ◽  
U. Hopfer
Keyword(s):  
Adenylyl Cyclase ◽  
Cell Line ◽  
Purinergic Receptors ◽  
Secreting Cell ◽  
Supramaximal Stimulation ◽  
Cell Capacitance ◽  
Purinergic Stimulation ◽  
Cl Conductance ◽  
Similar Peak ◽  
Stimulation Of

Cl- conductance and capacitance were simultaneously measured in the mucin-secreting cell line, HT29-Cl.16E (Cl.16E), and its sister cell line, HT29-Cl.19A (Cl.19A), which lacks mucin granules. Purinergic stimulation by extracellular ATP transiently increased Cl- conductance in both cell lines with similar peak increases of 0.92 and 1.00 nS/pF in Cl.16E and Cl.19A cells, respectively (baseline of 0.08 nS/pF). Cell capacitance increased only in Cl.16E cells (17% above baseline of 22 pF in Cl.16E and 1% above baseline of 18 pF in Cl.19A cells). Wortmannin inhibited the purinergically activated Cl- conductance and capacitance increases in Cl.16E by 50 and 80%, respectively, but had no effects in Cl.19A cells. In Cl.16E cells, adenosine 3',5'-cyclic monophosphate (cAMP) signaling increased Cl- conductance from 0.08 to 0.52 nS/pF without changing capacitance. Cl- secretion in Cl.16E monolayers was additive in response to supramaximal stimulation of purinergic receptors and adenylyl cyclase, even though granule fusion is nine times greater with purinergic than adenylyl cyclase stimulation. In conclusion, 1) wortmannin does not directly inhibit activation of Cl- conductance, 2) at least 50% of purinergically activated Cl- conductance in Cl.16E is associated with granule fusion, and 3) cAMP-activated Cl- conductance is not associated with granules.

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