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• W2079239630 abstract "Circulating hormones produce rapid changes in the Cl− permeability of liver cells through activation of plasma membrane receptors coupled to heterotrimeric G-proteins. The resulting effects on intracellular pH, membrane potential, and Cl− content are important contributors to the overall metabolic response. Consequently, the purpose of these studies was to evaluate the mechanisms responsible for G-protein-mediated changes in membrane Cl− permeability using HTC hepatoma cells as a model. Using patch clamp techniques, intracellular dialysis with 0.3 mm guanosine 5′-O-(3-thiotriphosphate) (GTPγS) increased membrane conductance from 10 to 260 picosiemens/picofarads due to activation of Ca2+-dependent Cl− currents that were outwardly rectifying and exhibited slow activation at depolarizing potentials. These effects were mimicked by intracellular AlF4− (0.03 mm) and inhibited by pertussis toxin (PTX), consistent with current activation through Gαi. Studies using defined agonists and inhibitors indicate that Cl− channel activation by GTPγS occurs through an indomethacin-sensitive pathway involving sequential activation of phospholipase C, mobilization of Ca2+ from inositol 1,4,5-trisphosphate-sensitive stores, and stimulation of phospholipase A2 and cyclooxygenase (COX). Accordingly, the conductance responses to GTPγS or to intracellular Ca2+were inhibited by COX inhibitors. These results indicate that PTX-sensitive G-proteins regulate the Cl− permeability of HTC cells through Ca2+-dependent stimulation of COX activity. Thus, receptor-mediated activation of Gαi may be essential for hormonal regulation of liver transport and metabolism through COX-dependent opening of a distinct population of plasma membrane Cl− channels. Circulating hormones produce rapid changes in the Cl− permeability of liver cells through activation of plasma membrane receptors coupled to heterotrimeric G-proteins. The resulting effects on intracellular pH, membrane potential, and Cl− content are important contributors to the overall metabolic response. Consequently, the purpose of these studies was to evaluate the mechanisms responsible for G-protein-mediated changes in membrane Cl− permeability using HTC hepatoma cells as a model. Using patch clamp techniques, intracellular dialysis with 0.3 mm guanosine 5′-O-(3-thiotriphosphate) (GTPγS) increased membrane conductance from 10 to 260 picosiemens/picofarads due to activation of Ca2+-dependent Cl− currents that were outwardly rectifying and exhibited slow activation at depolarizing potentials. These effects were mimicked by intracellular AlF4− (0.03 mm) and inhibited by pertussis toxin (PTX), consistent with current activation through Gαi. Studies using defined agonists and inhibitors indicate that Cl− channel activation by GTPγS occurs through an indomethacin-sensitive pathway involving sequential activation of phospholipase C, mobilization of Ca2+ from inositol 1,4,5-trisphosphate-sensitive stores, and stimulation of phospholipase A2 and cyclooxygenase (COX). Accordingly, the conductance responses to GTPγS or to intracellular Ca2+were inhibited by COX inhibitors. These results indicate that PTX-sensitive G-proteins regulate the Cl− permeability of HTC cells through Ca2+-dependent stimulation of COX activity. Thus, receptor-mediated activation of Gαi may be essential for hormonal regulation of liver transport and metabolism through COX-dependent opening of a distinct population of plasma membrane Cl− channels. Liver cells undergo rapid changes in the rate of transport of ions, glucose, and bile acids in response to changing physiological demands (1.Graf J. Haussinger D. J. Hepatol. 1996; 24 Suppl. 1: 53-77PubMed Google Scholar, 2.Boyer J.L. Graf J. Meier P.J. Annu. Rev. Physiol. 1992; 54: 415-438Crossref PubMed Scopus (137) Google Scholar). Recent studies have emphasized a key role for plasma membrane Cl− channels in this response (3.Roman R.M. Bodily K.O. Wang Y. Raymond J.R. Fitz J.G. Hepatology. 1998; 28: 1073-1080Crossref PubMed Scopus (48) Google Scholar, 4.Bodily K. Wang Y. Roman R. Sostman A. Fitz J.G. Hepatology. 1997; 25: 403-410Crossref PubMed Scopus (30) Google Scholar, 5.Roman R.M. Smith R.L. Feranchak A.P. Clayton G.H. Doctor R.B. Fitz J.G. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 280: G344-G353Crossref PubMed Google Scholar). Under basal conditions, membrane Cl− permeability is low. However, increases in cell volume or hormonal stimulation increase membrane Cl− permeability ∼20-fold through opening of Cl− channels in the plasma membrane. The resulting efflux of Cl− is not only essential for regulation of cell volume (5.Roman R.M. Smith R.L. Feranchak A.P. Clayton G.H. Doctor R.B. Fitz J.G. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 280: G344-G353Crossref PubMed Google Scholar, 6.Haddad P. Beck J.S. Boyer J.L. Graf J. Am. J. Physiol. 1991; 261: G340-G348PubMed Google Scholar) but also modulates a broad range of transport and metabolic processes through effects on intracellular pH (7.Gleeson D. Corasanti J.G. Boyer J.L. Am. J. Physiol. 1990; 258: G299-G307Crossref PubMed Google Scholar), membrane potential (8.Capiod T. Ogden D.C. Proc. R. Soc. Lond. B Biol. Sci. 1989; 236: 187-201Crossref PubMed Scopus (19) Google Scholar), and bile formation (6.Haddad P. Beck J.S. Boyer J.L. Graf J. Am. J. Physiol. 1991; 261: G340-G348PubMed Google Scholar). Whereas definition of cellular mechanisms involved in Cl− channel regulation represents an important focus for modulating liver cell and organ function, little is known about the specific channels and signaling pathways involved. In previous studies, the biophysical properties of a volume-sensitive Cl− current (ICl,swell) that functions to maintain liver cell volume within a narrow physiological range have been defined (4.Bodily K. Wang Y. Roman R. Sostman A. Fitz J.G. Hepatology. 1997; 25: 403-410Crossref PubMed Scopus (30) Google Scholar, 5.Roman R.M. Smith R.L. Feranchak A.P. Clayton G.H. Doctor R.B. Fitz J.G. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 280: G344-G353Crossref PubMed Google Scholar, 9.Fitz J.G. Sostman A.H. Am. J. Physiol. 1994; 266: G544-G553PubMed Google Scholar). Current activation depends on rapid translocation of protein kinase Cα to the plasma membrane, release of ATP into the extracellular space, and autocrine activation of P2 purinergic receptors (3.Roman R.M. Bodily K.O. Wang Y. Raymond J.R. Fitz J.G. Hepatology. 1998; 28: 1073-1080Crossref PubMed Scopus (48) Google Scholar, 10.Wang Y. Roman R. Lidofsky S.D. Fitz J.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12020-12025Crossref PubMed Scopus (299) Google Scholar). In addition, large increases in Cl− conductance are observed after exposure to angiotensin II and noradrenaline (11.Ogden D.C. Capiod T. Walker J.W. Trentham D.R. J. Physiol. (Lond.). 1990; 422: 585-602Crossref Scopus (57) Google Scholar), hormones that bind to receptors coupled to heterotrimeric G-proteins (8.Capiod T. Ogden D.C. Proc. R. Soc. Lond. B Biol. Sci. 1989; 236: 187-201Crossref PubMed Scopus (19) Google Scholar). However, it is not known whether these hormone-activated Cl− channels are the same as those involved in ICl,swell, and whether they are regulated by distinct signaling pathways. In liver cells and other epithelial cells, the mechanisms for activation of different Cl− channels including the ICl,swell are not known (12.Begenisich T. Melvin J.E. J. Membr. Biol. 1998; 163: 77-85Crossref PubMed Scopus (83) Google Scholar). It is well documented that 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) 1The abbreviations used are: NPPB5-nitro-2-(3-phenylpropylamino)-benzoic acidAAarachidonic acidPLCphospholipase CCOXcyclooxygenasePTXpertussis toxinGTPγSguanosine 5′-O-(3-thiotriphosphate)pSpicosiemenspFpicofaradPLA2phospholipase A2IP3inositol 1,4,5-trisphosphateERendoplasmic reticulum 1The abbreviations used are: NPPB5-nitro-2-(3-phenylpropylamino)-benzoic acidAAarachidonic acidPLCphospholipase CCOXcyclooxygenasePTXpertussis toxinGTPγSguanosine 5′-O-(3-thiotriphosphate)pSpicosiemenspFpicofaradPLA2phospholipase A2IP3inositol 1,4,5-trisphosphateERendoplasmic reticulum effectively inhibits opening of different Cl− channels (12.Begenisich T. Melvin J.E. J. Membr. Biol. 1998; 163: 77-85Crossref PubMed Scopus (83) Google Scholar, 13.Strange K. Emma F. Jackson P.S. Am. J. Physiol. 1996; 270: C711-C730Crossref PubMed Google Scholar). In addition to its function as a Cl− channel blocker, NPPB is also a potent inhibitor of the enzymatic activity of cyclooxygenase in a manner analogous to indomethacin and aspirin (14.Breuer W. Skorecki K.L. Biochem. Biophys. Res. Commun. 1989; 163: 398-405Crossref PubMed Scopus (20) Google Scholar). Cyclooxygenases are present in liver cells and utilize arachidonic acid as a substrate to generate prostaglandins that are known to modulate a broad range of diverse physiological processes (15.Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2212) Google Scholar). 5-nitro-2-(3-phenylpropylamino)-benzoic acid arachidonic acid phospholipase C cyclooxygenase pertussis toxin guanosine 5′-O-(3-thiotriphosphate) picosiemens picofarad phospholipase A2 inositol 1,4,5-trisphosphate endoplasmic reticulum 5-nitro-2-(3-phenylpropylamino)-benzoic acid arachidonic acid phospholipase C cyclooxygenase pertussis toxin guanosine 5′-O-(3-thiotriphosphate) picosiemens picofarad phospholipase A2 inositol 1,4,5-trisphosphate endoplasmic reticulum Based on these considerations, the purpose of these studies was to evaluate the potential role of heterotrimeric G-proteins on the Cl− conductance of a model liver cell line and the specific signaling pathways involved. Using patch clamp techniques, intracellular dialysis with GTPγS to activate heterotrimeric G-proteins increased membrane permeability to Cl− by ∼25-fold due to opening of plasma membrane Cl− channels. The current was biophysically distinct from ICl,swell and was mediated through a PTX-sensitive pathway that involves stimulation of phospholipase C, subsequent release of Ca2+ from endoplasmic reticulum, activation of phospholipase A2, and increases in cyclooxygenase activity. Thus, agonist binding to Gαi-coupled receptors is closely associated with activation of membrane Cl− channels through an indomethacin-sensitive pathway, and the signaling pathways and channels involved represent potential sites for pharmacological modulation of liver cell transport and metabolism. All experiments were performed in HTC cells derived from rat hepatoma using methods described previously (9.Fitz J.G. Sostman A.H. Am. J. Physiol. 1994; 266: G544-G553PubMed Google Scholar,16.Barnard G.F. Erickson S.K. Cooper A.D. J. Clin. Invest. 1984; 74: 173-184Crossref PubMed Scopus (28) Google Scholar). These cells have been used widely as a stable model of hepatocyte ion transport because they exhibit signaling pathways and ion channels analogous to those found in primary hepatocytes (4.Bodily K. Wang Y. Roman R. Sostman A. Fitz J.G. Hepatology. 1997; 25: 403-410Crossref PubMed Scopus (30) Google Scholar, 9.Fitz J.G. Sostman A.H. Am. J. Physiol. 1994; 266: G544-G553PubMed Google Scholar, 17.Heaton J.H. Krett N.L. Alvarez J.M. Gelehrter T.D. Romanus J.A. Rechler M.M. J. Biol. Chem. 1984; 259: 2396-2402Abstract Full Text PDF PubMed Google Scholar, 18.Sung C.K. Goldfine I.D. Biochem. Biophys. Res. Commun. 1992; 189: 1024-1030Crossref PubMed Scopus (26) Google Scholar). In addition, HTC cells express insulin and other receptors that are linked to heterotrimeric G-proteins (9.Fitz J.G. Sostman A.H. Am. J. Physiol. 1994; 266: G544-G553PubMed Google Scholar, 19.Sanchez-Margalet V. Diabetologia. 1999; 42: 317-325Crossref PubMed Scopus (20) Google Scholar). Cells were plated on coverslips and maintained at 37 °C in a 5% CO2 and 95% air atmosphere in culture media composed of minimal essential medium (Invitrogen) supplemented with 5% fetal calf serum, 2 mml-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Before study, coverslips containing cells were removed from culture media and placed in an extracellular solution that contained 142 mm NaCl, 4 mm KCl, 1 mm KH2PO4, 2 mm MgCl2, 2 mm CaCl2, 10 mm d-glucose, and 10 mm HEPES/NaOH. For patch clamp recordings, cells were dialyzed with different pipette solutions as indicated. The standard pipette solution contained 130 mm KCl, 10 mm NaCl, 1 mm EGTA, 0.5 mm CaCl2, 2 mm MgCl2, and 10 mm HEPES/NaOH (free [Ca2+] = ∼0.1 μm). In some experiments to move Cl−equilibrium potential to about −40 mV, intracellular [Cl−] was decreased by dialysis with a low Cl− solution that contained 130 mmK-glutamate, 20 mm NaCl, 1 mm EGTA, 0.5 mm CaCl2, 2 mm MgCl2, and 10 mm HEPES/NaOH. To buffer [Ca2+]i to lower levels, cells were dialyzed with a solution that contained 125 mm KCl, 10 mmNaCl, 5 mm EGTA, 2 mm MgCl2, and 10 mm HEPES/NaOH. The activity of endogenous G-proteins was increased by adding the nonhydrolyzable GTP analog GTPγS (0.3 mm) to the different pipette solutions. To stimulate heterotrimeric G-proteins more specifically, cells were dialyzed with an aluminum fluoride solution that contained 120 mm KCl, 10 mm NaCl, 10 mm NaF, 0.03 mmAlCl3, 1 mm EGTA, 0.5 mmCaCl2, 2 mm MgCl2, and 10 mm HEPES/NaOH. Under these conditions, the concentration of AlF4− was 30 μm (20.Hess S.D. Doroshenko P.A. Augustine G.J. Science. 1993; 259: 1169-1172Crossref PubMed Scopus (107) Google Scholar). To buffer [Ca2+]i to 1 μm, cells were dialyzed with a solution that contained 125 mmcesium-d-glutamate, 10 mm NaCl, 5 mm EGTA, 4.45 mm CaCl2, 2 mm MgCl2, and 10 mm HEPES/CsOH. This solution contained low [Cl−] and high [Cs+] to block Ca2+-activated K+channels in HTC cells. The free [Ca2+]i was determined as described previously (21.Kilic G. Angleson J.K. Cochilla A.J. Nussinovitch I. Betz W.J. J. Physiol. (Lond.). 2001; 532: 771-783Crossref Scopus (26) Google Scholar). The pH of all solutions was 7.25. Osmolarity of the extracellular solution was 300 mosmol/kg, and the osmolarity of the pipette solutions was 270–275 mosmol/kg. All compounds were purchased from Sigma. Using patch clamp techniques, plasma membrane conductance was measured in the whole-cell recording configuration. Cells were voltage-clamped at −40 mV, and membrane conductance was determined every 3 s by applying 4-ms voltage pulses (pulse amplitude, 40 mV). The current response was used to determine the conductance and the capacitance as described previously (22.Lindau M. Neher E. Pflugers Arch. 1988; 411: 137-146Crossref PubMed Scopus (500) Google Scholar). To compare GTPγS-activated conductance responses from different cells, the conductance was normalized to the initial capacitance (measure of cell surface area) and expressed in pS/pF. Whole-cell currents were measured in response to intracellular GTPγS, after membrane capacitance and access resistance were compensated. Whole-cell currents in response to voltage pulses were filtered with an 8-pole Bessel filter at a 1 kHz cut-off frequency and sampled every 0.5 ms. To determine reversal potential, a voltage ramp from −90 mV to 90 mV (duration, 0.2 s) was applied. Reversal potentials were determined taking into account the corrections for liquid junction potentials. With standard external solution and low Cl−pipette solutions, the liquid junction potential was measured to be 5 mV. The activity of phospholipase A2 was monitored using an engineered phospholipid molecule, PED6 (Molecular Probes), as described previously (23.Farber S.A. Olson E.S. Clark J.D. Halpern M.E. J. Biol. Chem. 1999; 274: 19338-19346Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). PED6 is a substrate for PLA2 and consists of a phospholipid labeled with a fluorescent molecule (BODIPY) and a quencher molecule (dinitrophenyl). PED6 is nonfluorescent in the solution but becomes fluorescent after removal of dinitrophenyl by activated PLA2. Consequently, the fluorescence intensity of cleaved PED6 is a direct measure of PLA2 activity (23.Farber S.A. Olson E.S. Clark J.D. Halpern M.E. J. Biol. Chem. 1999; 274: 19338-19346Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar,24.Farber S.A. Pack M. Ho S.Y. Johnson I.D. Wagner D.S. Dosch R. Mullins M.C. Hendrickson H.S. Hendrickson E.K. Halpern M.E. Science. 2001; 292: 1385-1388Crossref PubMed Scopus (270) Google Scholar). Before the experiments, cells were incubated with PED6 (0.3 μg/ml) for 2 h and then washed to remove PED6 from extracellular media. The intracellular PED6 fluorescence was monitored using an oil immersion Olympus objective ×60 (NA = 1.2) and a SensicamQE camera controlled by SlideBook 3.0 software (Intelligent Imaging Innovations). Images were acquired every 30 s from regions containing cells. Background fluorescence was determined from regions containing no cells and was subtracted from the cell fluorescence. All fluorescence data were analyzed using IgorPro 3.14 software (Wavemetrics). To evaluate the role of pertussis toxin-sensitive heterotrimeric G-proteins on the GTPγS-evoked conductance, cells were incubated overnight with pertusiss toxin (200 ng/ml) from Bordetella pertussis. The potential involvement of PLC was assessed by including neomycin sulfate or spermine (inhibitors of PLC) with GTPγS in the standard pipette solution. The involvement of arachidonic acid in GTPγS response was assessed in two manners: different concentrations of arachidonic acid were included in the standard pipette solution, or cells were exposed directly to arachidonic acid in the extracellular solution. The potential role of cyclooxygenase (COX) was assessed in similar manners. Cells were exposed to the COX inhibitor NPPB (20 μm), or indomethacin (10 μm) or aspirin (100 μm) was included in the standard pipette solution together with GTPγS or 1 μm [Ca2+]i. These reagents were purchased from Calbiochem. All experiments were performed at 24 °C. Data are expressed as the means ± S.E. Results were compared using Student's t test on paired or unpaired data. To evaluate whether GTP-binding proteins are capable of regulating the plasma membrane conductance of HTC cells, the cell interior was dialyzed with GTPγS (0.3 mm) through a patch pipette while measuring whole-cell conductance. GTPγS is a nonhydrolyzable GTP analog that increases the activity of a broad range of GTP-dependent proteins. Representative recordings of normalized conductance (pS/pF) from cells dialyzed with (top trace) and without (bottom trace) GTPγS are shown in Fig. 1A. Under control conditions, membrane conductance was small and remained stable during recordings. Intracellular GTPγS stimulated a large increase in conductance that reached maximal values within ∼2 min and then slowly decreased over time. In 12 of 18 cells dialyzed with GTPγS, multiple transient increases in conductance were observed (Fig. 1A). The peak conductance of the first transient was always the largest and was taken for analysis. In Fig. 1B, the peak conductance of cells dialyzed with or without GTPγS is shown. These findings indicate that intracellular GTPγS leads to a ∼ 25-fold increase in the membrane conductance of HTC cells. To characterize the properties of the GTPγS-activated conductance, current-voltage relations were measured in solutions of differing ionic composition. The dominant response, observed in 8 of 11 cells, is illustrated in Fig. 2A. With standard pipette solution, currents reversed near the Cl−equilibrium potential of 0 mV, were outwardly rectifying, and exhibited slow activation at depolarizing potentials above 10 mV. In the remaining 3 of 11 cells, currents also reversed near 0 mV and were outwardly rectifying but showed slow inactivation at depolarizing potentials, properties suggestive of the ICl,swell, the Cl− current activated by volume increase of HTC cells (4.Bodily K. Wang Y. Roman R. Sostman A. Fitz J.G. Hepatology. 1997; 25: 403-410Crossref PubMed Scopus (30) Google Scholar,10.Wang Y. Roman R. Lidofsky S.D. Fitz J.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12020-12025Crossref PubMed Scopus (299) Google Scholar). The reversal potential of all GTPγS-evoked currents was −3.0 ± 2.1 mV (Fig. 2B, 14 cells). Decreasing intracellular [Cl−] from 145 to 25 mmshifted the reversal potential of currents to −39.7 ± 5.4 mV (Fig. 2B, 10 cells), close to the new Cl−equilibrium potential. These findings indicate that the GTPγS-stimulated conductance is due to opening of Cl−channels, and the dominant response involves an outwardly rectifying conductance that shows slow activation at depolarizing potentials. In many cells, intracellular GTPγS has been shown to activate ICl,swell (25.Voets T. Manolopoulos V. Eggermont J. Ellory C. Droogmans G. Nilius B. J. Physiol. (Lond.). 1998; 506: 341-352Crossref Scopus (137) Google Scholar, 26.Doroshenko P. Neher E. J. Physiol. (Lond.). 1992; 449: 197-218Crossref Scopus (120) Google Scholar, 27.Doroshenko P. Penner R. Neher E. J. Physiol. (Lond.). 1991; 436: 711-724Crossref Scopus (54) Google Scholar, 28.Mitchell C.H. Zhang J.J. Wang L. Jacob T.J. Am. J. Physiol. 1997; 272: C212-C222Crossref PubMed Google Scholar). In HTC cells, activation of ICl,swell is mediated by an autocrine mechanism that is mediated by release of ATP into extracellular space and activation of purinergic receptors (10.Wang Y. Roman R. Lidofsky S.D. Fitz J.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12020-12025Crossref PubMed Scopus (299) Google Scholar). To assess whether GTPγS stimulates opening of Cl− channels through ATP release, cells were exposed to apyrase, an enzyme that rapidly hydrolyzes ATP and completely inhibits opening of ICl,swell in HTC cells (10.Wang Y. Roman R. Lidofsky S.D. Fitz J.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12020-12025Crossref PubMed Scopus (299) Google Scholar). A representative recording is shown in Fig. 3. The presence of apyrase to eliminate extracellular ATP had no effect on the response to GTPγS because the peak of GTPγS-activated conductance in the presence of apyrase (227 ± 37 pS/pF; 5 cells) was not significantly different from the values measured in the absence of apyrase (261 ± 38 pS/pF; 18 cells; p > 0.30). Thus, intracellular GTPγS activates Cl− channels in HTC cells through a mechanism different from ATP release. The conductance response to GTPγS could result from activation of heterotrimeric G-proteins (29.Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4700) Google Scholar) or inactivation of certain small monomeric GTP-binding proteins (30.Takel K. McPherson P.S. Schmid S.L. De Camilli P. Nature. 1995; 374: 186-190Crossref PubMed Scopus (650) Google Scholar, 31.Johannes L. Lledo P.M. Roa M. Vincent J.D. Henry J.P. Darchen F. EMBO J. 1994; 13: 2029-2037Crossref PubMed Scopus (186) Google Scholar). To distinguish between these possibilities, cells were dialyzed with a pipette solution that contained aluminum fluoride instead of GTPγS. AlF4− is thought to permanently activate heterotrimeric G-proteins (29.Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4700) Google Scholar) but to have no effect on monomeric GTP-binding proteins (32.Kahn R.A. J. Biol. Chem. 1991; 266: 15595-15597Abstract Full Text PDF PubMed Google Scholar). A representative recording is shown in Fig. 4A. Similar to GTPγS, AlF4− (30 μm) stimulated multiple transient increases in conductance that inactivated over several minutes. Moreover, the reversal potential of AlF4−-activated currents was close to Cl− equilibrium potential, and currents were outwardly rectifying (Fig. 4B). However, the peak of AlF4−-activated conductance was 455 ± 97 pS/pF (5 cells), significantly larger than the peak of GTPγS-activated response (p > 0.05). These observations demonstrate that selective activation of heterotrimeric G-proteins by AlF4− mimics the response to intracellular GTPγS in HTC cells. Heterotrimeric G-proteins that are sensitive to PTX are essential for the response of liver cells to many hormones (33.Sanchez-Margalet V. Gonzalez-Yanes C. Santos-Alvarez J. Najib S. Cell Mol. Life Sci. 1999; 55: 142-147Crossref PubMed Scopus (13) Google Scholar, 34.Berven L.A. Barritt G.J. FEBS Lett. 1994; 346: 235-240Crossref PubMed Scopus (21) Google Scholar, 35.Berven L.A. Hughes B.P. Barritt G.J. Biochem. J. 1994; 299: 399-407Crossref PubMed Scopus (33) Google Scholar). To test whether the heterotrimeric G-proteins activated by GTPγS are PTX-sensitive, cells were incubated overnight in the presence of PTX. Pretreatment with PTX substantially decreased the response to GTPγS (Fig. 4A), decreasing the magnitude of the GTPγS-activated conductance to 86 ± 11 pS/pF (4 cells; p < 0.05). Finally, whole-cell currents were measured after exposure to noradrenaline (1 μm), a physiological agonist that activates receptors coupled to Gαi. A representative recording is shown in Fig. 4C. Noradrenaline activated currents (4 cells) analogous to those stimulated by GTPγS, with properties including slow activation at depolarizing potentials, outward rectification, and reversal potential close to Cl−equilibrium potential (data not shown). Taken together, these findings suggest that the response to GTPγS is mediated in large part by activation of pertussis toxin-sensitive G-proteins of the Gαi family. In liver cells, stimulation of Gαi could open Cl− channels directly through G-protein-channel interactions or indirectly through activation of intermediary signaling pathways. One pathway that has been linked to PTX-sensitive G-proteins in liver involves stimulation of phospholipase C and subsequent mobilization of Ca2+ through inositol 1,4,5-trisphosphate (IP3)-sensitive channels in the endoplasmic reticulum (ER) (36.Yang L.J. Baffy G. Rhee S.G. Manning D. Hansen C.A. Williamson J.R. J. Biol. Chem. 1991; 266: 22451-22458Abstract Full Text PDF PubMed Google Scholar). To test this possibility, cells were treated with neomycin (1 mm) or spermine (1 mm) to inhibit PLC. Each inhibitor decreased the amplitude of the GTPγS-stimulated conductance response by ∼ 80% (Fig. 5). Similarly, intracellular dialysis with heparin, a blocker of IP3-sensitive channels (37.Bakowski D. Glitsch M.D. Parekh A.B. J. Physiol. (Lond.). 2001; 532: 55-71Crossref Scopus (136) Google Scholar), or buffering of [Ca2+]i to low levels by dialysis with a pipette solution containing 5 mm EGTA substantially inhibited the response to GTPγS (Fig. 5). These observations suggest that the response to GTPγS is mediated in large part by effects of Gαi on PLC, leading to release of Ca2+from intracellular stores. The release of Ca2+ from ER is essential for the response of liver cells to different hormones (8.Capiod T. Ogden D.C. Proc. R. Soc. Lond. B Biol. Sci. 1989; 236: 187-201Crossref PubMed Scopus (19) Google Scholar, 11.Ogden D.C. Capiod T. Walker J.W. Trentham D.R. J. Physiol. (Lond.). 1990; 422: 585-602Crossref Scopus (57) Google Scholar, 35.Berven L.A. Hughes B.P. Barritt G.J. Biochem. J. 1994; 299: 399-407Crossref PubMed Scopus (33) Google Scholar). To test whether intracellular Ca2+ is capable of activating membrane conductance without participation of heterotrimeric G-proteins, cells were treated with thapsigargin (20 μm), an inhibitor of intracellular Ca2+-ATPase that causes release of Ca2+ from the endoplasmic reticulum of hepatocytes (38.Berven L.A. Crouch M.F. Katsis F. Kemp B.E. Harland L.M. Barritt G.J. J. Biol. Chem. 1995; 270: 25893-25897Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). As shown in Fig. 6A, exposure to thapsigargin activated Cl−-selective currents with properties of those activated by GTPγS (5 cells). These data indicate that the release of Ca2+ from endoplasmic reticulum per se is capable of activating Cl− conductance in HTC cells. In liver, PTX-sensitive Gαi and increases in intracellular [Ca2+] lead to the activation of a Ca2+-dependent PLA2, resulting in generation of arachidonic acid (39.Tong L.J. Dong L.W. Liu M.S. Mol. Cell. Biochem. 1998; 189: 55-61Crossref PubMed Google Scholar). To evaluate whether this mechanism is operative in HTC cells, the activity of PLA2 was monitored by measuring PED6 fluorescence (24.Farber S.A. Pack M. Ho S.Y. Johnson I.D. Wagner D.S. Dosch R. Mullins M.C. Hendrickson H.S. Hendrickson E.K. Halpern M.E. Science. 2001; 292: 1385-1388Crossref PubMed Scopus (270) Google Scholar) as shown in Fig. 6, B and C. After loading with PED6, cells were placed in dye-free solution, and intracellular fluorescence was measured in the absence or presence of thapsigargin. In the absence of thapsigargin, cells had a low level of baseline fluorescence likely resulting from basal PLA2 activity. Fluorescence intensity gradually decreased over time as the PLA2-cleaved fluorescent dye slowly diffused out of the cell interior (11 cells). Interestingly, exposure to thapsigargin to increase [Ca2+]i markedly increased PED6 fluorescence (Fig. 6C, 6 cells). In 4 of 6 cells, the fluorescence pattern showed multiple transients that decayed slowly after reaching a peak, similar to the pattern observed with the conductance response to GTPγS. These results suggest that in HTC cells, mobilization of Ca2+ from the ER leads to rapid activation of Ca2+-dependent PLA2. Ca2+-dependent activation of PLA2would be anticipated to increase local concentrations of arachidonic acid (AA) (40.Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar), which serves as a substrate for generation of prostaglandins by cyclooxygenase (41.Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1837) Google Scholar). To test whether AA per se is capable of activating membrane conductance, cells were treated with increasing concentrations of AA (1–25 μm). Under basal conditions, with the standard pipette solu" @default.
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• W2079239630 date "2002-04-01" @default.
• W2079239630 modified "2023-10-14" @default.
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