Inhibition of ATP levels by quinidine in a human colonic epithelial cell line

1986 ◽  
Vol 250 (6) ◽  
pp. G806-G813
Author(s):  
K. Dharmsathaphorn ◽  
P. Huott ◽  
C. A. Cartwright ◽  
J. A. McRoberts ◽  
K. G. Mandel ◽  
...  
Keyword(s):  
Inhibitory Action ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Human Colon ◽  
Inhibitory Effect ◽  
K Channel ◽  
Epithelial Cell Line ◽  
Cl Secretion ◽  
Atp Levels ◽  
Similar Inhibitory Effect

The influence of quinidine, a putative K+ channel blocker, on Cl- secretion induced by vasoactive intestinal polypeptide (VIP) was investigated. Quinidine inhibited Cl- secretion induced by VIP in T84 cell monolayers. A similar inhibitory effect of quinidine on Cl- secretion was observed in an isolated human colon. However, in the isolated human colon, which absorbs Na+ avidly, inhibition of Na+ absorption predominated. In the T84 cell, the half-maximal inhibition by quinidine occurred at 60 microM, while 300 microM almost completely inhibited the VIP-induced Cl— secretion. Mucosal addition of quinidine was at least equally effective compared with serosal addition, suggesting that quinidine acts on the apical membrane or intracellularly. Quinidine had little or no effect on VIP-stimulated Cl- efflux in the first 15 min after its addition, suggesting that blockage of the Cl- exit pathway on the apical membrane is an unlikely mechanism. Similarly, quinidine did not inhibit the K+-recycling mechanism on the basolateral membrane in the first 15 min after its addition. The initial inhibitory action of quinidine corresponded better with its ability to decrease cellular ATP levels. Our study suggests that the depletion of cellular ATP levels may explain the initial inhibitory action of quinidine on electrolyte transport in the intestine, while the late effect is multifactorial.

Staphylococcus aureus alpha-toxin permeabilizes the basolateral membrane of a Cl(-)-secreting epithelium

1992 ◽  
Vol 263 (1) ◽  
pp. L104-L112 ◽  
Author(s):  
L. S. Ostedgaard ◽  
D. M. Shasby ◽  
M. J. Welsh
Keyword(s):  
Staphylococcus Aureus ◽  
Ion Channels ◽  
Prolonged Exposure ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Transport Processes ◽  
Epithelial Cell Line ◽  
Alpha Toxin ◽  
Cl Secretion ◽  
Cl Channels

Apical membrane ion channels control the rate of transepithelial electrolyte transport in many epithelia. One way to study such channels in their native location, the apical membrane, is to eliminate the resistance of the basolateral membrane to ion flow. Then the opening and closing of apical channels can be measured as a transepithelial current, free from the influence of basolateral membrane transport processes. To develop a method that would permeabilize an epithelial basolateral membrane to ions and nucleotides, we examined the effect of Staphylococcus aureus alpha-toxin on the Cl(-)-secreting T84 epithelial cell line. alpha-Toxin permeabilized the basolateral, but not the apical membrane to Cl-, adenosine 3',5'-cyclic monophosphate (cAMP), and GTP. However, the integrity of signal-transduction pathways, the regulation of apical membrane Cl- channels, and the transepithelial resistance remained intact. In the course of examining the effect of ATP, we found that the basolateral membrane contained purinergic receptors that both stimulated Cl- secretion on their own and, at high concentrations, inhibited cAMP-induced Cl- secretion. These effects of extracellular ATP were eliminated after prolonged exposure to ATP, suggesting receptor downregulation. In addition, depletion of intracellular ATP following permeabilization prevented cAMP-dependent regulation of apical Cl- channels. We conclude that alpha-toxin may prove to be a useful tool for studying the regulation and properties of apical membrane ion channels.

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.

scholarly journals Zinc inhibits cAMP-stimulated Cl secretion via basolateral K-channel blockade in rat ileum

2005 ◽  
Vol 288 (5) ◽  
pp. G956-G963 ◽  
Author(s):  
Kazi Mirajul Hoque ◽  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
K Channel ◽  
Isolated Cells ◽  
Rat Ileum ◽  
Anion Secretion ◽  
Cl Secretion ◽  
Ion Absorption ◽  
Isotonic Buffer

Zn, an essential micronutrient and second most abundant trace element in cell and tissues, reduces stool output when administered to children with acute diarrhea. The mechanism by which Zn improves diarrhea is not known but could result from stimulating Na absorption and/or inhibiting anion secretion. The aim of this study was to investigate the direct effect of Zn on intestinal epithelial ion absorption and secretion. Rat ileum was partially stripped of serosal and muscle layers, and the mucosa was mounted in lucite chambers. Potential difference and short-circuit current were measured by conventional current-voltage clamp method.86Rb efflux and uptake were assessed for serosal K channel and Na-K-2Cl cotransport activity, respectively. Efflux experiments were performed in isolated cells preloaded with86Rb in the presence of ouabain and bumetanide, whereas uptake experiments were performed in low-Cl isotonic buffer containing Ba and ouabain. Neither mucosal nor serosal Zn affected glucose-stimulated Na absorption. In contrast, forskolin-induced Cl secretion was markedly reduced by serosal but not mucosal addition of Zn. Zn also substantially reversed the increase in Cl secretion induced by 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) with half-maximal inhibitory concentration of 0.43 mM. In contrast, serosal Zn did not alter Cl secretion stimulated by carbachol, a Ca-dependent agonist. Zn inhibited 8-BrcAMP-stimulated86Rb efflux but not carbachol-stimulated86Rb efflux. Zn had no effect on bumetanide-sensitive86Rb uptake, Na-K-ATPase, or CFTR. We conclude from these studies that Zn inhibits cAMP-induced Cl secretion by blocking basolateral membrane K channels.

Two types of K+ channel in thick ascending limb of rat kidney

1994 ◽  
Vol 267 (4) ◽  
pp. F599-F605 ◽  
Author(s):  
W. H. Wang
Keyword(s):  
Apical Membrane ◽  
Rat Kidney ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Inside Out

We have used the patch-clamp technique to study the apical K+ channels in the thick ascending limb (TAL) of the rat kidney. Two types of K+ channels, a low-conductance and an intermediate-conductance K+ channel, were identified in both cell-attached and inside-out patches. We confirmed the previously reported intermediate-conductance K+ channel (72 pS), which is inhibited by millimolar cell ATP, acidic pH, Ba2+, and quinidine (4). We now report a second K+ channel in apical membrane of the TAL. The slope conductance of this low-conductance K+ channel is 30 pS, and its open probability is 0.80 in cell-attached patches. This channel is not voltage dependent, and application of 2 mM ATP in the bath inhibits channel activity in inside-out patches. In addition, 250 microM glyburide, an ATP-sensitive K+ channel inhibitor, blocks channel activity, whereas the same concentration of glyburide has no inhibitory effect on the 72-pS K+ channel. Channel activity of the 30-pS K+ channel decreases rapidly upon excision of patches (channel run down). Application of 0.1 mM ATP and the catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) restores channel activity. Furthermore, addition of 0.1 mM 8-(4-chlorophenylthio)-cAMP or 50-100 pM vasopressin in the cell-attached patches increases channel activity. In conclusion, two types of K+ channels are present in the apical membrane of TAL of rat kidney, and PKA plays an important role in modulation of the low-conductance K+ channel activity.

Arachidonic acid inhibits K channels in basolateral membrane of the thick ascending limb

2002 ◽  
Vol 283 (3) ◽  
pp. F407-F414 ◽  
Author(s):  
Rui-Min Gu ◽  
Wen-Hui Wang
Keyword(s):  
Arachidonic Acid ◽  
Basolateral Membrane ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Cytochrome P 450

We have used the patch-clamp technique to study the effect of arachidonic acid (AA) on the basolateral K channels in the medullary thick ascending limb (mTAL) of rat kidney. An inwardly rectifying 50-pS K channel was identified in cell-attached and inside-out patches in the basolateral membrane of the mTAL. The channel open probability ( P o) was 0.51 at the spontaneous cell membrane potential and decreased to 0.25 by 30 mV hyperpolarization. The addition of 5 μM AA decreased channel activity, identified as NP o, from 0.58 to 0.08 in cell-attached patches. The effect of AA on the 50-pS K channel was specific because 10 μM cis-11,14,17-eicosatrienoic acid had no significant effect on channel activity. To determine whether the effect of AA was mediated by AA per se or by its metabolites, we examined the effect of AA on channel activity in the presence of indomethacin, an inhibitor of cyclooxygenase, or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), an inhibitor of cytochrome P-450 monooxygenase. Inhibition of cyclooxygenase increased channel activity from 0.54 to 0.9. However, indomethacin did not abolish the inhibitory effect of AA on the 50-pS K channel. In contrast, inhibition of cytochrome P-450 metabolism not only increased channel activity from 0.49 to 0.83 but also completely abolished the effect of AA. Moreover, addition of DDMS can reverse the inhibitory effect of AA on channel activity. The notion that the effect of AA was mediated by cytochrome P-450-dependent metabolites of AA is also supported by the observation that addition of 100 nM of 20-hydroxyeicosatetraenoic acid, a main metabolite of AA in the mTAL, can mimic the effect of AA. We conclude that AA inhibits the 50-pS K channel in the basolateral membrane of the mTAL and that the effect of AA is mainly mediated by cytochrome P-450-dependent metabolites of AA.

scholarly journals Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder.

1987 ◽  
Vol 89 (4) ◽  
pp. 563-580 ◽  
Author(s):  
J R Demarest ◽  
A L Finn
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
K Channel ◽  
Channel Blockers ◽  
Na Transport ◽  
Transepithelial Na Transport ◽  
Pump Activity ◽  
Relationship Of

Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.

Modulation of Cl- secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK

1996 ◽  
Vol 271 (5) ◽  
pp. L785-L795 ◽  
Author(s):  
D. C. Devor ◽  
A. K. Singh ◽  
R. J. Bridges ◽  
R. A. Frizzell
Keyword(s):  
Cystic Fibrosis ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Secretory Response ◽  
K Channel ◽  
T84 Cells ◽  
Coordinate Regulation ◽  
Cl Secretion ◽  
Cl Channels ◽  
Subsequent Addition

We previously demonstrated that the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), stimulates a sustained Cl- secretory response across T84 monolayers by opening a Ca(2+)-dependent basolateral K+ channel. In the present work, we evaluated the effects on Cl-secretion of other benzimidazolones, NS-004 and NS-1619, which have been shown to open cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast to 1-EBIO, neither NS-004 nor NS-1619 stimulated a significant Cl- secretory current (Isc). Neither NS-004 nor NS-1619 increased Isc subsequent to forskolin stimulation. However, when added after 1-EBIO, NS-004 and NS-1619 stimulated large sustained increases in Isc. In addition, NS-004 and NS-1619 potentiated the effects of carbachol. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of NS-004 and 1-EBIO on the basolateral K+ (IK) and apical Cl- (ICl) currents. Both NS-004 and 1-EBIO increased ICl, and the stimulated currents were inhibited by glibenclamide. In contrast, NS-004 failed to significantly affect IK, but subsequent addition of 1-EBIO induced a large increase in IK. The effects of 1-EBIO, NS-004, and NS-1619 on the Ca(2+)-dependent K+ channel (KCa) in T84 cells was determined in excised inside-out patches. Neither NS-004 nor NS-1619 affected K+ channel activity, whereas the subsequent addition of 1-EBIO produced a marked channel activation. Results similar to those observed in T84 monolayers were obtained from murine airway cell primary cultures: NS-004 or NS-1619 had no effect on Isc, whereas 1-EBIO stimulated a sustained Cl- secretory response. The results demonstrate that activation of CFTR alone is insufficient to evoke transepithelial Cl- secretion. Activation of the basolateral membrane K+ channel is a necessary component of the secretory response. Thus the basolateral membrane KCa may be a novel pharmacological target in cystic fibrosis therapy.

Basolateral K+ channels in airway epithelia. II. Role in Cl- secretion and evidence for two types of K+ channel

1990 ◽  
Vol 258 (6) ◽  
pp. L343-L348 ◽  
Author(s):  
J. D. McCann ◽  
M. J. Welsh
Keyword(s):  
Epithelial Cells ◽  
Time Course ◽  
Basolateral Membrane ◽  
Airway Epithelial Cells ◽  
K Channel ◽  
Airway Epithelial ◽  
K Channels ◽  
Cl Secretion ◽  
Transient Component ◽  
Permeable Supports

We previously described a Ca2(+)-activated K+ channel (KCLIC) in airway epithelial cells [J. D. McCann, J. Matsuda, M. Garcia, G. Kaczorowski, and M. J. Welsh. Am. J. Physiol 258 (Lung Cell. Mol. Physiol. 2): L334-L342, 1990]. To determine whether the KCLIC channel is a basolateral membrane channel and to understand its role in Cl- secretion, we studied airway epithelial cells grown on permeable supports. When cells were stimulated with A23187, charybdotoxin (ChTX) inhibited Cl- secretion and 86Rb efflux at the same concentrations, indicating that the KCLIC channel is required for Ca2(+)-stimulated Cl- secretion. We also investigated the function of K+ channels in adenosine 3',5'-cyclic monophosphate-stimulated secretion. Addition of isoproterenol caused a biphasic increase in Cl- secretion; the time course of the transient component correlated with the time course of the isoproterenol-induced increase in Ca2+ concentration [( Ca2+]c). ChTX inhibited the transient component, but not the prolonged component of secretion; Ba2+ inhibited the sustained component. These results suggest that when cells are grown on permeable supports isoproterenol-induced secretion depends on activation of two types of K+ channel: the KCLIC channel that is stimulated initially and a ChTX-insensitive K+ channel that is stimulated during sustained secretion. This conclusion was supported by measurement of 86Rb efflux from cell monolayers

Glycosylation status of endogenous CFTR does not affect cAMP-stimulated Cl- secretion in epithelial cells

1993 ◽  
Vol 265 (3) ◽  
pp. C688-C694 ◽  
Author(s):  
A. P. Morris ◽  
S. A. Cunningham ◽  
D. J. Benos ◽  
R. A. Frizzell
Keyword(s):  
Cystic Fibrosis ◽  
Apical Membrane ◽  
Human Colon ◽  
Transmembrane Conductance Regulator ◽  
Membrane Domain ◽  
Glycosylation Pattern ◽  
Cl Secretion ◽  
Cell Extracts ◽  
Oligosaccharide Processing ◽  
Ht 29

Cystic fibrosis (CF) impairs Cl- secretion across epithelial tissues and is caused by mutations in an N-linked glycoprotein, the cystic fibrosis transmembrane conductance regulator (CFTR). We modified the glycosylation pattern of CFTR using inhibitors of oligosaccharide processing and determined their effects on both agonist-induced Cl- secretion and CFTR location in human colon (HT-29) cell lines. In both polarized and unpolarized HT-29 cells, immunoprecipitation of cell extracts using a monoclonal antibody against CFTR gave a single band at 170 kDa. Inhibitors of N-linked glycosylation reduced the molecular mass of this band: swainsonine by 10 kDa, deoxymannojirimycin by 30 kDa, and deoxynojirimycin by 10-20 kDa. However, the transepithelial Cl- current and conductance stimulated by adenosine 3',5'-cyclic monophosphate (cAMP)- or Ca(2+)-dependent secretagogues was not affected. In the polarized cells, CFTR was localized in the apical membrane domain. Treatment of the monolayers with glycoprocessing inhibitors did not affect CFTR's location. Thus, in human colonocytes that endogenously express CFTR, the extent of CFTR glycosylation does not influence the targeting of CFTR to the apical membrane domain or its function as an agonist-stimulated Cl- channel.

scholarly journals Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli.

1992 ◽  
Vol 99 (4) ◽  
pp. 591-613 ◽  
Author(s):  
T A Cummings ◽  
S C Kinnamon
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Taste Receptor ◽  
K Channel ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
K Channels ◽  
Voltage Dependent ◽  
Whole Cell Recordings ◽  
Inside Out

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.

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