Activation of an apical Cl– conductance by extracellular ATP in Necturus gallbladder is mediated by cAMP and not by [Ca2+]i

1999 ◽  
Vol 438 (4) ◽  
pp. 486-496 ◽  
Author(s):  
C. Vank ◽  
E. Frömter ◽  
G. Kottra
Keyword(s):  
Extracellular Atp ◽  
Necturus Gallbladder ◽  
Cl Conductance

scholarly journals Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium.

1992 ◽  
Vol 99 (2) ◽  
pp. 241-262 ◽  
Author(s):  
G A Altenberg ◽  
J S Stoddard ◽  
L Reuss
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Resistance ◽  
Membrane Voltage ◽  
Gallbladder Epithelium ◽  
K Channels ◽  
Necturus Gallbladder ◽  
Electrophysiological Effects ◽  
Paracellular Diffusion ◽  
Cl Conductance

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.

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.

Potassium-induced cell swelling in Necturus gallbladder epithelium

1988 ◽  
Vol 254 (5) ◽  
pp. C643-C650 ◽  
Author(s):  
C. W. Davis ◽  
A. L. Finn
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Basolateral Membrane ◽  
Cell Volume Regulation ◽  
Cell Swelling ◽  
Gallbladder Epithelium ◽  
Bathing Medium ◽  
Necturus Gallbladder ◽  
Per Se ◽  
Cl Conductance

In Necturus gallbladder epithelium, elevation of mucosal K+ to 95 mM in the presence of 10 mM Na+ resulted in cell swelling at a rate of 3.2% original volume per minute, followed by volume-regulatory shrinking. When Na+ was completely removed from or when amiloride (10(-4) M) was added to the mucosal medium, K+-induced cell swelling was abolished. In the presence of 10 mM Na+, 1 mM Ba2+ abolished and substitution of mucosal Cl- by NO-3 had no effect on K+-induced swelling. Thus solute entry following elevation of mucosal K+ is effected by separate K+ and Cl- pathways. Furthermore, substitution of 95 mM K+ for Na+ in the mucosal bathing medium leads to the development of a Cl- conductance in the basolateral membrane as long as some Na+ remains in the medium. However, cell swelling induced by mucosal dilution does not lead to the appearance of a Cl- conductance. Thus the activation of this conductance requires both swelling and membrane depolarization. These results show that 1) high mucosal K+ leads to cell swelling due to the entry of Cl- along with K+ and the Cl- can enter across either membrane, 2) the Cl- pathways require the presence of mucosal Na+, and 3) cell volume regulation is activated by an increase in volume per se, i.e., a hyposmotic exposure is not required for volume regulation to occur.

Regulation of apical membrane ion transport in Necturus gallbladder

1992 ◽  
Vol 263 (1) ◽  
pp. C187-C193 ◽  
Author(s):  
J. L. Garvin ◽  
K. R. Spring
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Cell Swelling ◽  
Gallbladder Epithelium ◽  
Necturus Gallbladder ◽  
Camp Levels ◽  
Nacl Cotransport ◽  
Cl Conductance ◽  
Membrane Potential Difference ◽  
Membrane Ion Transport

Na and Cl movement through the apical membrane of Necturus gallbladder epithelium was investigated using electrophysiological and light microscopic measurements. Changes in membrane potential difference, fractional resistance of the apical membrane, and transepithelial resistance caused by changes in apical bath Cl concentration revealed the presence of a Cl conductance in the apical membrane of control tissues that was apparently not present in the preparations studied by other investigators. This Cl conductance was blocked by bumetanide (10(-5) M) or by the inhibitor of adenosine 3',5'-cyclic monophosphate (cAMP) action, the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS; 0.5 mM). Treatment of the tissues with Rp-cAMPS also eliminated bumetanide-sensitive cell swelling in the presence of ouabain and activated an amiloride-sensitive swelling, changes consistent with inhibition of NaCl cotransport and the activation of Na-H and Cl-HCO3 exchange. We conclude that the mode of NaCl entry into Necturus gallbladder epithelial cells is determined by the level of cAMP. When cAMP levels are high, entry occurs by NaCl cotransport; when cAMP levels are low, parallel exchange of Na-H and Cl-HCO3 predominates. These observations explain the previous disagreements about the mode of NaCl entry into Necturus gallbladder epithelial cells.

Chloride secretory response to extracellular ATP in human normal and cystic fibrosis nasal epithelia

1992 ◽  
Vol 263 (2) ◽  
pp. C348-C356 ◽  
Author(s):  
L. L. Clarke ◽  
R. C. Boucher
Keyword(s):  
Cystic Fibrosis ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Chloride Secretion ◽  
Extracellular Atp ◽  
Nasal Epithelium ◽  
Secretory Process ◽  
Cl Secretion ◽  
Epithelial Cultures ◽  
Cl Conductance

Chloride secretion across cystic fibrosis (CF) airway epithelia is effectively regulated by pathways associated with intracellular Ca2+ metabolism, but not by mechanisms dependent on protein kinase A or C. In a search for therapeutically useful agonists, we investigated the effects of extracellular ATP on the Cl- secretory process in human normal and CF nasal epithelial cultures with double-barreled Cl- selective microelectrodes. When applied to the basolateral membrane of normal, but not CF, nasal epithelium, extracellular ATP (10(-4) M) stimulated a small increase in Cl- secretion that was primarily associated with a hyperpolarizing conductance in the basolateral membrane. In contrast, ATP applied to the apical (luminal) membrane of either normal or CF nasal epithelium stimulated a greater increase in Cl- secretion that was associated with activation of an apical membrane Cl- conductance. The increases in Cl- current and apical conductance were greater in CF tissues and attained maximal values similar to normal nasal epithelium. We conclude 1) that basolateral application of ATP indirectly induces Cl- secretion by activating a basolateral (K+) conductance and is an effective secretagogue only in normal nasal epithelium and 2) that luminally applied ATP is an effective Cl- secretagogue that activates the apical membrane Cl- conductance of normal and CF nasal epithelia to an equivalent level.

Multiple modes of regulation of airway epithelial chloride secretion by extracellular ATP

1994 ◽  
Vol 267 (5) ◽  
pp. C1442-C1451 ◽  
Author(s):  
M. J. Stutts ◽  
J. G. Fitz ◽  
A. M. Paradiso ◽  
R. C. Boucher
Keyword(s):  
Chloride Secretion ◽  
Extracellular Atp ◽  
Disulfonic Acid ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Transmembrane Conductance ◽  
Cl Secretion ◽  
Tightly Coupled ◽  
Cf Transmembrane Conductance Regulator ◽  
Cl Conductance

Cultured normal and cystic fibrosis (CF) airway epithelia were exposed to 5'-(N-ethylcarboxamido)-adenosine (NECA), ATP, or ionomycin. NECA activated a sustained, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-insensitive Cl- secretory response in normal but not CF, consistent with stimulation of the CF transmembrane conductance regulator (CFTR). In normal and CF, ionomycin or ATP induced Cl- secretion with an initial peak that was inhibited > 50% by DIDS, but in normals there was a prolonged current that was not inhibited by DIDS. The ATP and ionomycin responses in CF were of greater magnitude, and the prolonged phase was inhibited by DIDS. Although we expected ATP to regulate Cl- conductance through intracellular Ca2+ activity, ATP further stimulated Cl- secretion in tissues pretreated to maximally elevate intracellular Ca2+ activity. ATP also activated whole cell Cl- currents in cells dialyzed with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Thus ATP and ionomycin regulate a Cl- conductance that is distinct from CFTR, but the regulation by ATP is not tightly coupled to intracellular Ca2+ activity. Alternatively, ATP regulates separate Ca(2+)-sensitive and Ca(2+)-insensitive Cl- conductances. Furthermore, extracellular ATP activates DIDS-resistant Cl- secretion in normal but not CF cultured epithelia, consistent with activation of CFTR by extracellular ATP.

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.

Extracellular ATP increases [Ca2+]i in distal tubule cells. II. Activation of a Ca2+-dependent Cl− conductance

2000 ◽  
Vol 279 (1) ◽  
pp. F102-F111 ◽  
Author(s):  
Isabelle Rubera ◽  
Michel Tauc ◽  
Michel Bidet ◽  
Catherine Verheecke-Mauze ◽  
Guy De Renzis ◽  
...  
Keyword(s):  
Cell Line ◽  
Time Course ◽  
Distal Tubule ◽  
Extracellular Atp ◽  
Acetoxymethyl Ester ◽  
P2y2 Receptors ◽  
Intracellular Ca ◽  
Immortalized Cell ◽  
Potency Order ◽  
Cl Conductance

We characterized Cl− conductance activated by extracellular ATP in an immortalized cell line derived from rabbit distal bright convoluted tubule (DC1). 125I− efflux experiments showed that ATP increased 125I− loss with an EC50 = 3 μM. Diphenylamine-2-carboxylate (10−3 M) and NPPB (10−4 M) abolished the125I− efflux. Preincubation with 10 μM 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-acetoxymethyl ester or 10−7 M thapsigargin inhibited the effect of ATP. Ionomycin (2 μM) increased125I− efflux with a time course similar to that of extracellular ATP, suggesting that the response is dependent on the intracellular Ca2+ concentration ([Ca2+]i). The ATP agonist potency order was ATP ≥ UTP > ATPγS. Suramin (500 μM) inhibited the ATP-induced 125I− efflux, consistent with P2 purinoceptors. 125I− effluxes from cells grown on permeable filters suggest that ATP induced an apical efflux that was mediated via apical P2 receptors. Whole cell experiments showed that ATP (100 μM) activated outwardly rectifying Cl− currents in the presence of 8-cyclopentyl-1,3-dipropylxanthine, excluding the involvement of P1 receptors. Ionomycin activated Cl−currents similar to those developed with ATP. These results demonstrate the presence of a purinergic regulatory mechanism involving ATP, apical P2Y2 receptors, and Ca2+ mobilization for apical Cl− conductance in a distal tubule cell line.

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