Heterotrimeric G-proteins Activate Cl− Channels through Stimulation of a Cyclooxygenase-dependent Pathway in a Model Liver Cell Line*

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 AlF 4 − (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 (1,2). Recent studies have emphasized a key role for plasma membrane Cl Ϫ channels in this response (3)(4)(5). Under basal conditions, membrane Cl Ϫ permeability is low. However, increases in cell volume or hormonal stimulation increase membrane Cl Ϫ permeability ϳ20-fold through opening of Cl Ϫ channels in the plasma membrane. The resulting efflux of Cl Ϫ is not only essential for regulation of cell volume (5,6) but also modulates a broad range of transport and metabolic processes through effects on intracellular pH (7), membrane potential (8), and bile formation (6). Whereas definition of cellular mechanisms involved in Cl Ϫ 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 volumesensitive Cl Ϫ current (I Cl,swell ) that functions to maintain liver cell volume within a narrow physiological range have been defined (4,5,9). Current activation depends on rapid translocation of protein kinase C␣ to the plasma membrane, release of ATP into the extracellular space, and autocrine activation of P2 purinergic receptors (3,10). In addition, large increases in Cl Ϫ conductance are observed after exposure to angiotensin II and noradrenaline (11), hormones that bind to receptors coupled to heterotrimeric G-proteins (8). However, it is not known whether these hormone-activated Cl Ϫ channels are the same as those involved in I Cl,swell , and whether they are regulated by distinct signaling pathways.
In liver cells and other epithelial cells, the mechanisms for activation of different Cl Ϫ channels including the I Cl,swell are not known (12). It is well documented that 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) 1 effectively inhibits opening of different Cl Ϫ channels (12,13). In addition to its function as a Cl Ϫ channel blocker, NPPB is also a potent inhibitor of the enzymatic activity of cyclooxygenase in a manner analogous to indomethacin and aspirin (14). 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).
Based on these considerations, the purpose of these studies was to evaluate the potential role of heterotrimeric G-proteins on the Cl Ϫ conductance of a model liver cell line and the specific signaling pathways involved. Using patch clamp techniques, intracellular dialysis with GTP␥S to activate heterotrimeric G-proteins increased membrane permeability to Cl Ϫ by ϳ25fold due to opening of plasma membrane Cl Ϫ channels. The current was biophysically distinct from I Cl,swell and was mediated through a PTX-sensitive pathway that involves stimulation of phospholipase C, subsequent release of Ca 2ϩ from endoplasmic reticulum, activation of phospholipase A 2 , and increases in cyclooxygenase activity. Thus, agonist binding to G␣ i -coupled receptors is closely associated with activation of membrane Cl Ϫ 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.

EXPERIMENTAL PROCEDURES
Cell Preparation-All experiments were performed in HTC cells derived from rat hepatoma using methods described previously (9,16). 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 (4,9,17,18). In addition, HTC cells express insulin and other receptors that are linked to heterotrimeric G-proteins (9,19). Cells were plated on coverslips and maintained at 37°C in a 5% CO 2 and 95% air atmosphere in culture media composed of minimal essential medium (Invitrogen) supplemented with 5% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 g/ml streptomycin.
Solutions for Electrophysiological Studies-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 KH 2 PO 4 , 2 mM MgCl 2 , 2 mM CaCl 2 , 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 CaCl 2 , 2 mM MgCl 2 , and 10 mM HEPES/NaOH (free [Ca 2ϩ ] ϭ ϳ0.1 M). In some experiments to move Cl Ϫ equilibrium potential to about Ϫ40 mV, intracellular [Cl Ϫ ] was decreased by dialysis with a low Cl Ϫ solution that contained 130 mM K-glutamate, 20 mM NaCl, 1 mM EGTA, 0.5 mM CaCl 2 , 2 mM MgCl 2 , and 10 mM HEPES/NaOH. To buffer [Ca 2ϩ ] i to lower levels, cells were dialyzed with a solution that contained 125 mM KCl, 10 mM NaCl, 5 mM EGTA, 2 mM MgCl 2 , and 10 mM HEPES/NaOH. The activity of endogenous G-proteins was increased by adding the nonhydrolyzable GTP analog GTP␥S (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 mM AlCl 3 , 1 mM EGTA, 0.5 mM CaCl 2 , 2 mM MgCl 2 , and 10 mM HEPES/NaOH. Under these conditions, the concentration of AlF 4 Ϫ was 30 M (20). To buffer [Ca 2ϩ ] i to 1 M, cells were dialyzed with a solution that contained 125 mM cesium-D-glutamate, 10 mM NaCl, 5 mM EGTA, 4.45 mM CaCl 2 , 2 mM MgCl 2 , and 10 mM HEPES/CsOH. This solution contained low [Cl Ϫ ] and high [Cs ϩ ] to block Ca 2ϩ -activated K ϩ channels in HTC cells. The free [Ca 2ϩ ] i was determined as described previously (21). 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.
Conductance and Current Measurements-Using patch clamp techniques, plasma membrane conductance was measured in the whole-cell recording configuration. Cells were voltage-clamped at Ϫ40 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). To compare GTP␥S-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 GTP␥S, 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 Ϫ90 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 Ϫ pipette solutions, the liquid junction potential was measured to be 5 mV.
Detection of PLA 2 Activity-The activity of phospholipase A 2 was monitored using an engineered phospholipid molecule, PED6 (Molecular Probes), as described previously (23). PED6 is a substrate for PLA 2 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 PLA 2 . Consequently, the fluorescence intensity of cleaved PED6 is a direct measure of PLA 2 activity (23,24).
Before the experiments, cells were incubated with PED6 (0.3 g/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 ϫ60 (NA ϭ 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).
Cell Treatments-To evaluate the role of pertussis toxin-sensitive heterotrimeric G-proteins on the GTP␥S-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 GTP␥S in the standard pipette solution. The involvement of arachidonic acid in GTP␥S 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 M), or indomethacin (10 M) or aspirin (100 M) was included in the standard pipette solution together with GTP␥S or 1 M [Ca 2ϩ ] i . These reagents were purchased from Calbiochem.
All experiments were performed at 24°C. Data are expressed as the means Ϯ S.E. Results were compared using Student's t test on paired or unpaired data.

RESULTS
To evaluate whether GTP-binding proteins are capable of regulating the plasma membrane conductance of HTC cells, the cell interior was dialyzed with GTP␥S (0.3 mM) through a patch pipette while measuring whole-cell conductance. GTP␥S 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) GTP␥S are shown in Fig. 1A. Under control conditions, membrane conductance was small and remained stable during recordings. Intracellular GTP␥S stimulated a large increase in conductance that reached maximal values within ϳ2 min and then slowly decreased over time. In 12 of 18 cells dialyzed with GTP␥S, 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 GTP␥S is shown. These findings indicate that intracellular GTP␥S leads to a ϳ 25-fold increase in the membrane conductance of HTC cells.
To characterize the properties of the GTP␥S-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 Ϫ 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 I Cl,swell , the Cl Ϫ current activated by volume increase of HTC cells (4,10). The reversal potential of all GTP␥S-evoked currents was Ϫ3.0 Ϯ 2.1 mV (Fig. 2B, 14 cells). Decreasing intracellular [Cl Ϫ ] from 145 to 25 mM shifted the reversal potential of currents to Ϫ39.7 Ϯ 5.4 mV (Fig. 2B, 10 cells), close to the new Cl Ϫ equilibrium potential. These findings indicate that the GTP␥S-stimulated conductance is due to opening of Cl Ϫ channels, and the domi-nant response involves an outwardly rectifying conductance that shows slow activation at depolarizing potentials.
In many cells, intracellular GTP␥S has been shown to activate I Cl,swell (25)(26)(27)(28). In HTC cells, activation of I Cl,swell is mediated by an autocrine mechanism that is mediated by release of ATP into extracellular space and activation of purinergic receptors (10). To assess whether GTP␥S stimulates opening of Cl Ϫ channels through ATP release, cells were exposed to apyrase, an enzyme that rapidly hydrolyzes ATP and completely inhibits opening of I Cl,swell in HTC cells (10). A representative recording is shown in Fig. 3. The presence of apyrase to eliminate extracellular ATP had no effect on the response to GTP␥S because the peak of GTP␥S-activated conductance in the presence of apyrase (227 Ϯ 37 pS/pF; 5 cells) was not significantly different from the values measured in the absence of apyrase (261 Ϯ 38 pS/pF; 18 cells; p Ͼ 0.30). Thus, intracellular GTP␥S activates Cl Ϫ channels in HTC cells through a mechanism different from ATP release.
The conductance response to GTP␥S could result from activation of heterotrimeric G-proteins (29) or inactivation of certain small monomeric GTP-binding proteins (30,31). To distinguish between these possibilities, cells were dialyzed with a pipette solution that contained aluminum fluoride instead of GTP␥S. AlF 4 Ϫ is thought to permanently activate heterotrimeric G-proteins (29) but to have no effect on monomeric GTPbinding proteins (32). A representative recording is shown in Fig. 4A. Similar to GTP␥S, AlF 4 Ϫ (30 M) stimulated multiple transient increases in conductance that inactivated over several minutes. Moreover, the reversal potential of AlF 4 Ϫ -activated currents was close to Cl Ϫ equilibrium potential, and currents were outwardly rectifying (Fig. 4B). However, the peak of AlF 4 Ϫ -activated conductance was 455 Ϯ 97 pS/pF (5 cells), significantly larger than the peak of GTP␥S-activated response (p Ͼ 0.05). These observations demonstrate that selective activation of heterotrimeric G-proteins by AlF 4 Ϫ mimics the response to intracellular GTP␥S in HTC cells.
Heterotrimeric G-proteins that are sensitive to PTX are essential for the response of liver cells to many hormones (33)(34)(35). To test whether the heterotrimeric G-proteins activated by GTP␥S are PTX-sensitive, cells were incubated overnight in the presence of PTX. Pretreatment with PTX substantially decreased the response to GTP␥S (Fig. 4A), decreasing the magnitude of the GTP␥S-activated conductance to 86 Ϯ 11 pS/pF (4 cells; p Ͻ 0.05). Finally, whole-cell currents were measured after exposure to noradrenaline (1 M), a physiological agonist that activates receptors coupled to G␣ i . A representative recording is shown in Fig. 4C. Noradrenaline activated currents (4 cells) analogous to those stimulated by GTP␥S, with properties including slow activation at depolarizing potentials, outward rectification, and reversal potential close to Cl Ϫ equilibrium potential (data not shown). Taken together, these findings suggest that the response to GTP␥S is mediated in large part by activation of pertussis toxin-sensitive G-proteins of the G␣ i family.
In liver cells, stimulation of G␣ i could open Cl Ϫ 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 Ca 2ϩ through inositol 1,4,5-trisphosphate (IP 3 )sensitive channels in the endoplasmic reticulum (ER) (36). 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 GTP␥S-stimulated conductance response by ϳ 80% (Fig. 5). Similarly, intracellular dialysis with heparin, a blocker of IP 3 -sensitive channels (37) low levels by dialysis with a pipette solution containing 5 mM EGTA substantially inhibited the response to GTP␥S (Fig. 5). These observations suggest that the response to GTP␥S is mediated in large part by effects of G␣ i on PLC, leading to release of Ca 2ϩ from intracellular stores.
The release of Ca 2ϩ from ER is essential for the response of liver cells to different hormones (8,11,35). To test whether intracellular Ca 2ϩ is capable of activating membrane conduct-ance without participation of heterotrimeric G-proteins, cells were treated with thapsigargin (20 M), an inhibitor of intracellular Ca 2ϩ -ATPase that causes release of Ca 2ϩ from the endoplasmic reticulum of hepatocytes (38). As shown in Fig.  6A, exposure to thapsigargin activated Cl Ϫ -selective currents with properties of those activated by GTP␥S (5 cells). These data indicate that the release of Ca 2ϩ from endoplasmic reticulum per se is capable of activating Cl Ϫ conductance in HTC cells.
In liver, PTX-sensitive G␣ i and increases in intracellular [Ca 2ϩ ] lead to the activation of a Ca 2ϩ -dependent PLA 2 , resulting in generation of arachidonic acid (39). To evaluate whether this mechanism is operative in HTC cells, the activity of PLA 2 was monitored by measuring PED6 fluorescence (24) 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 PLA 2 activity. Fluorescence intensity gradually decreased over time as the PLA 2 -cleaved fluorescent dye slowly diffused out of the cell interior (11 cells). Interestingly, exposure to thapsigargin to increase [Ca 2ϩ ] 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 GTP␥S. These results suggest that in HTC cells, mobilization of Ca 2ϩ from the ER leads to rapid activation of Ca 2ϩ -dependent PLA 2 .
Ca 2ϩ -dependent activation of PLA 2 would be anticipated to increase local concentrations of arachidonic acid (AA) (40), which serves as a substrate for generation of prostaglandins by   FIG. 3.

Hydrolysis of extracellular ATP does not inhibit GTP␥S-activated conductance in HTC cells. Normalized conductance was measured in cells dialyzed with GTP␥S (0.3 mM) in the absence (left) or presence (right) of extracellular apyrase (2 units/ml). Apyrase hydrolyzes ATP, prevents activation of purinergic receptors, and inhibits I Cl,swell in HTC cells.
Dashed lines indicate the initial conductance level after the break-in. Note that apyrase did not prevent activation of membrane conductance by GTP␥S.

FIG. 4. AlF 4
؊ and noradrenaline activate whereas PTX inhibits Cl ؊ conductance in HTC cells. A, representative normalized conductances from cells dialyzed with a fluoride solution (30 M AlF 4 Ϫ , left trace) or GTP␥S (0.3 mM, right traces). AlF 4 Ϫ activated currents analogous to those activated by GTP␥S. The response to GTP␥S was inhibited by overnight incubation with PTX (200 ng/ml). Dashed lines indicate initial conductance level after the break-in. B, current response of AlF 4 Ϫ -evoked conductance to a voltage ramp (from Ϫ90 mV to 90 mV, 0.2 s in duration). Note that the current is outwardly rectifying with reversal potential close to 0 mV. C, exposure to noradrenaline (1 M; NA) to stimulate receptordependent activation of G␣ i activated whole-cell currents with analogous properties. Voltage protocol was as defined in Fig. 2A.

FIG. 5. Conductance response to GTP␥S requires activation of PLC and release of Ca 2؉ from IP 3 -sensitive intracellular stores.
The normalized peak conductance was measured in whole-cell configuration from HTC cells dialyzed with a standard pipette solution that contained 0.3 mM GTP␥S (18 cells). Inclusion of a PLC inhibitor (neomycin (4 cells) or spermine (5 cells); 1 mM for both treatments) in the intracellular solution markedly decreased the peak conductance (p Ͻ 0.02). Similarly, the presence of heparin to block IP 3 -sensitive channels (1 mg/ml; 4 cells; p Ͻ 0.04) or strong Ca 2ϩ buffering with 5 mM EGTA (4 cells; p Ͻ 0.02) in the standard pipette solution prevented activation of the conductance by GTP␥S.
cyclooxygenase (41). To test whether AA per se is capable of activating membrane conductance, cells were treated with increasing concentrations of AA (1-25 M). Under basal conditions, with the standard pipette solution containing 0.1 M [Ca 2ϩ ] i , arachidonic acid had minimal effects on membrane conductance (Fig. 7). To assess the alternative possibility that the GTP␥S-activated conductance requires stimulation of COX activity and generation of prostaglandins from AA, the effects of putative inhibitors of COX on the response to GTP␥S were evaluated. NPPB is an effective blocker of Cl Ϫ channels in HTC cells (4), and it may function either by direct effects on the open channel configuration or indirectly through inhibition of COX activity (14). Exposure of cells to NPPB when GTP␥S-evoked currents were already maximal had no immediate effect on the conductance (3 cells; data not shown), indicating that NPPB is not likely to act directly on the open channel configuration. However, pretreatment of liver cells with NPPB for several minutes before GTP␥S dialysis inhibited the conductance by ϳ90% (Fig. 7). Similarly, the GTP␥S-stimulated Cl Ϫ conductance was also blocked by indomethacin or aspirin, potent inhibitors of COX activity (Fig. 7).
These findings suggest that GTP␥S, by increasing [Ca 2ϩ ] i , may regulate Cl Ϫ permeability indirectly through a cyclooxygenase-dependent pathway. To test this possibility more directly, additional studies were performed in cells dialyzed with a pipette solution that contained 1 M [Ca 2ϩ ] i in the absence or presence of the COX inhibitor indomethacin. A representative recording is shown in Fig. 8A. Increases in [Ca 2ϩ ] i activated currents with biophysical properties analogous to those activated by GTP␥S or thapsigargin, including slow activation and outward rectification at depolarizing potentials (Fig. 8, A and B, 7 cells). Unlike GTP␥S-activated currents, Ca 2ϩ -activated currents did not inactivate during several minutes of recording (Fig. 8C). With low [Cl Ϫ ] in the intracellular solution (25 mM), the reversal potential of these currents was Ϫ42.8 Ϯ 1.8 mV (Fig. 8B, 5 cells), close to a Cl Ϫ equilibrium potential. Notably, treatment with indomethacin prevented activation of currents by Ca 2ϩ (Fig. 8, C and D, 6  cells). These results suggest that intracellular Ca 2ϩ is not likely to regulate Cl Ϫ channels directly. It is more likely that GTP␥S stimulates a signaling pathway that involves activation of G␣ i , subsequent mobilization of Ca 2ϩ from the ER, activation of Ca 2ϩ -dependent PLA 2 , and stimulation of COX activity (Fig.  9). Thus, the prostaglandin products of COX are likely to represent signaling molecules that mediate changes in the Cl Ϫ permeability of HTC cells due to the activation of hormone receptors coupled to heterotrimeric G-proteins. DISCUSSION Liver cell transport and metabolism are closely regulated by circulating hormones in response to changing physiological demands. The principal findings of these studies of a model liver cell line suggest that receptors coupled to PTX-sensitive G-proteins (G␣ i ) modulate membrane Cl Ϫ permeability through a sequence of events involving activation of phospholipase C, mobilization of Ca 2ϩ from IP 3 -sensitive stores, activation of phospholipase A 2 , and generation of cyclooxygenase FIG. 6. Release of Ca 2؉ from the ER stimulates Cl ؊ currents and increases the activity of PLA 2 . A, whole-cell currents were measured after exposure to thapsigargin (20 M; TG) using the voltage protocol defined in Fig. 2. Note that currents demonstrated outward rectification and slow activation at depolarizing potentials (5 cells). B, intracellular fluorescence was measured in cells loaded with PED6, a reporter molecule sensitive to changes in PLA 2 activity. Exposure to thapsigargin (20 M; arrow) was followed by multiple transient increases in cellular fluorescence. C, fluorescence images of the cell shown in B obtained before (1) and after (2) exposure to thapsigargin. Fluorescence of PED6 appears to be distributed throughout the cytosol and not in nucleus, with several localized regions of increased PLA 2 activity. Scale bar, 5 m. products that mediates changes in channel open probability. Accordingly, intracellular Ca 2ϩ does not appear to regulate these channels directly but activates an indomethacin-sensitive conductance that is biophysically distinct from that activated by increases in cell volume. Consequently, this represents a candidate for the Cl Ϫ conductance activated by multiple G␣ i -coupled receptors in hepatocytes and a potential target for modulating liver metabolic functions through effects on membrane Cl Ϫ permeability.
Intracellular application of GTP␥S has been utilized as a nonspecific probe to assess the effects of G-proteins on membrane permeability. In endothelial cells (25), pigmented ciliary epithelial cells (28), and adrenal chromaffin cells (26,27), GTP␥S activates Cl Ϫ currents that closely resemble the volume-activated conductance I Cl,swell . Because HTC cells express the I Cl,swell at high density (4, 10), activation of I Cl,swell by GTP␥S was anticipated. Instead, the dominant conductance exhibited properties more compatible with a Ca 2ϩ -activated Cl Ϫ conductance (I Cl(Ca) ), including outward rectification and slow activation at depolarizing potentials. The I Cl(Ca) was also observed after increasing [Ca 2ϩ ] i in secretory epithelia (42)(43)(44)(45), smooth muscle cells (46), and adipocytes (47). Whereas in these cells the I Cl(Ca) has roles in fluid secretion and response to neurotransmitters, the physiological roles of this current in liver cells are not readily apparent. However, even small changes in membrane Cl Ϫ permeability influence the membrane potential of hepatocytes, which plays an essential role in regulation of gluconeogenesis (48) as well as uptake of bile acids, amino acids, and HCO 3 Ϫ (49,50). In that light, it is intriguing that receptors for insulin (33), vasopressin (35), epidermal growth factor (36), and possibly other agonists signal in part via PTX-sensitive G-proteins. Moreover, analogous currents are detected in HTC cells after exposure to both insulin (51) and noradrenaline (Fig. 4). Consequently, it is attractive to speculate that opening of I Cl(Ca) represents an important regulatory event for these agonists.
In liver (36) and other cell types (52)(53)(54), PTX-sensitive Gproteins activate PLC and lead to increases in [Ca 2ϩ ] i due to Ca 2ϩ mobilization from IP 3 -sensitive stores (8,11). These general conclusions regarding initiation of signaling via G␣ i and Ca 2ϩ mobilization are supported by three primary observations. First, intracellular AlF 4 Ϫ , a more specific activator of heterotrimeric G-proteins, stimulated currents indistinguishable from those activated by GTP␥S (Fig. 4A). Second, pretreatment with PTX to down-regulate G␣ i markedly inhibited the current response (Fig. 4B). Finally, inhibition of PLC or blockade of IP 3 -sensitive Ca 2ϩ release prevented current activation by GTP␥S. Notably, increases in [Ca 2ϩ ] i stimulated by thapsigargin or dialysis with Ca 2ϩ -containing pipette solutions activated the same current in the nominal absence of changes in G␣ i activity. Because the responses to increased [Ca 2ϩ ] i or GTP␥S were inhibited by indomethacin and other inhibitors of cyclooxygenase, these findings suggest the presence of important functional interactions between PLC-and COX-dependent signaling pathways in liver cells.
These interactions appear to be mediated in part by Ca 2ϩdependent stimulation of PLA 2 as detected by the effects of thapsigargin on the fluorescence of cells loaded with the PLA 2 substrate PED6. The resulting increase in arachidonic acid production does not appear to regulate Cl Ϫ channels directly because cell treatments with arachidonic acid under basal [Ca 2ϩ ] i (ϳ0.1 M) failed to activate currents. This differs from some cell types in which exposure to arachidonic acid results in increased biological effects of prostaglandins, indicating that basal COX activity is capable of synthesizing prostaglandins (55,56). However, arachidonic acid represents the only known substrate for COX (40), implying that Ca 2ϩ mobilization either directly (Fig. 9) or through other signaling events increases COX activity in HTC cells, leading to generation of prostaglandins. This putative sequence is further supported by the observation that I Cl(Ca) activated by intracellular dialysis with 1 M [Ca 2ϩ ] is largely prevented by indomethacin. Moreover, these findings are consistent with reports in other cell types that PLA 2 activity and concomitant release of arachidonic acid are closely regulated by [Ca 2ϩ ] i (57). Furthermore, because COX proteins are predominantly localized in the ER (58), and the production of prostaglandins depends on [Ca 2ϩ ] i (59), it is likely that high [Ca 2ϩ ] produced at the outer leaflet of the ER after activation of IP 3 -sensitive channels may influence the generation of prostaglandins.
Assuming that these findings are relevant to liver cells in vivo, three additional points merit further clarification. First, the potential relationship between I Cl(Ca) and I Cl,swell is not Pulse potentials are the same as those indicated in Fig. 2. B, response of Ca 2ϩ -activated currents to voltage ramp (from Ϫ90 mV to 90 mV, 0.2 s in duration). With the low Cl Ϫ pipette solution, Ca 2ϩ -activated currents reversed near the Cl Ϫ equilibrium potential and demonstrated slow activation at depolarizing potentials. C, representative conductance recordings are shown from cells dialyzed with 1 M [Ca 2ϩ ] containing solution in the absence (top trace) or presence (bottom trace) of indomethacin (10 M). D, normalized peak conductance response of Ca 2ϩ -activated channels in the absence (5 cells) or presence of indomethacin (6 cells; p Ͻ 0.04).
FIG. 9. Proposed model for GTP␥S-dependent activation of Cl ؊ currents. These findings suggest the presence of a signaling cascade regulated by PTX-sensitive G␣ i that involves sequential activation of PLC, mobilization of Ca 2ϩ through IP 3 -sensitive channels in the ER, stimulation of PLA 2 to cleave AA from membrane phospholipids, and generation of COX products (prostaglandins (PG)) that mediate changes in Cl Ϫ channel open probability. fully defined. Whereas the most direct interpretation of these findings is that different channels are involved, the molecular identity of these Cl Ϫ channels is not known, and GTP␥S also activated currents with properties of I Cl,swell in a minority (ϳ25%) of cells. Consequently, it is possible that GTP␥S stimulates more than one signaling pathway and that a single channel protein could exhibit different properties according to the dominant signaling pathway involved. In HTC cells, CLC-2 chloride channel proteins appear to contribute to cell volume regulation, but mRNA for CLC-3, CLC-6, and CLC-7 is also expressed (5). Ultimately, it will be important to link these molecular candidates with specific physiological functions in liver cells. Second, whereas it is clear that COX products of arachidonic acid are necessary for channel activation, it is not clear which prostaglandins are involved. Moreover, the mechanistic links between Ca 2ϩ mobilization and COX activation are not defined. Also, it is not apparent which COX isoforms are involved in channel activation. Third, whereas hepatocytes produce small amounts of prostaglandins under basal conditions (60), a dramatic increase in production is observed in regenerating hepatocytes after partial liver removal (61). Consequently, it is attractive to speculate that prostaglandin-dependent regulation of membrane Cl Ϫ permeability could have broader roles in other liver functions as well. Whereas prostaglandins have been implicated in the regulation of hepatocyte glycogenolysis (62) and oxygen consumption (63), it is not clear whether the actions of prostaglandins are mediated by direct effects on channel proteins or activation of specific receptors.
In summary, these findings suggest the presence of a signaling cascade regulated by PTX-sensitive G␣ i that involves sequential activation of phospholipase C, Ca 2ϩ mobilization from IP 3 -sensitive stores, activation of phospholipase A 2 , and generation of cyclooxygenase products that mediate changes in channel open probability. Because the Cl Ϫ channels involved are biophysically distinct from those activated by cell volume, they represent a candidate for the Cl Ϫ conductance activated by multiple G-protein-coupled receptors in hepatocytes and a potential target for modulating liver metabolic functions through effects on membrane Cl Ϫ permeability.