KATP channels regulate mitogenically induced proliferation in primary rat hepatocytes and human liver cell lines. Implications for liver growth control and potential therapeutic targeting.

To determine whether K(ATP) channels control liver growth, we used primary rat hepatocytes and several human cancer cell lines for assays. K(ATP) channel openers (minoxidil, cromakalim, and pinacidil) increased cellular DNA synthesis, whereas K(ATP) channel blockers (quinidine and glibenclamide) attenuated DNA synthesis. The channel inhibitor glibenclamide decreased the clonogenicity of HepG2 cells without inducing cytotoxicity or apoptosis. To demonstrate the specificity of drugs for K(+) channels, whole-cell patch-clamp recordings were made. Hepatocytes revealed K(+) currents with K(ATP) channel properties. These K(+) currents were augmented by minoxidil and pinacidil and attenuated by glibenclamide as well as tetraethylammonium, in agreement with established responses of K(ATP) channels. Reverse transcription of total cellular RNA followed by polymerase chain reaction showed expression of K(ATP) channel-specific subunits in rat hepatocytes and human liver cell lines. Calcium fluxes were unperturbed in glibenclamide-treated HepG2 cells and primary rat hepatocytes following induction with ATP and hepatocyte growth factor, respectively, suggesting that the effect of K(ATP) channel activity upon hepatocyte proliferation was not simply due to indirect modulation of intracellular calcium. The regulation of mitogen-related hepatocyte proliferation by K(ATP) channels advances our insights into liver growth control. The findings have implications in mechanisms concerning liver development, regeneration, and oncogenesis.

Potassium channels are ubiquitous in eukaryotic cells and play roles in resting membrane potential, frequency of action potential, and membrane potential repolarization rates (1). Membrane currents through ATP-sensitive K ϩ channels, K ATP , have been recorded in diverse cell types, including pancreatic ␤ cells; skeletal, cardiac, and vascular myocytes; neurons; and renal epithelial cells (2)(3)(4)(5)(6)(7). The K ATP channel is a heteromultimer composed of at least two types of subunits, an inwardly rectifying K ϩ channel (Kir6.x) and a sulfonylurea receptor (SUR), that belongs to the ATP-binding cassette superfamily (8). Kir6.x subunits form the pore, and SUR subunits impart regulatory activity (9). The K ATP channels are inhibited by sulfonylureas, e.g. glibenclamide and tolbutamide, and activated by diverse substances designated as potassium channel openers (10,11), which include benzopyrans (cromakalim, levcromakalim, and pinacidil), cyanoguanidines, thiogormamides, pyrimidines (minoxidil), and benzothiadiazines (diazoxide). The activity of K ATP channels is dependent upon the energy state of the cell. Various subunits (Kir6.1, Kir6.2, SUR1, and SUR2) form channel subtypes with differences in expression among different organs (10). Kir6.2 and SUR1 form the characteristic K ϩ channel in pancreatic ␤ cells, where an increase in intracellular ATP levels, such as following increased glucose availability, causes channel closure, plasma membrane depolarization, and activation of voltage-sensitive calcium channels, which finally triggers insulin release (10,12).
It is noteworthy that potassium channels regulate the growth and proliferation of many cell types. For example, receptor-coupled, voltage-sensitive, and calcium-sensitive K ϩ channels regulate proliferation in lymphocytes, adipocytes, and epithelial cells derived from the skin, breast, bladder, and stomach (13)(14)(15). This regulation seems to occur at the level of cellular DNA synthesis because inhibition of K ϩ channel activity synchronizes cells in G 0 /G 1 (16). Also, in fibroblasts, the Ca 2ϩ -sensitive potassium channel has been found to regulate intracellular signal transduction events that concern mitogenic activity (17).
Information concerning the role of K ϩ channels in hepatocytes has been relatively limited. Early studies with primary rat hepatocytes by Sawanobori et al. (18) showed a wide range of resting membrane potentials, along with non-rectifying linear current-voltage relationships and no effect on these currents after norepinephrine or Ca 2ϩ administration. In guinea pig hepatocytes, Capiod and Ogden (19) have established the presence of Ca 2ϩ -activated, delayed-rectifier K ϩ currents. Similarly, Ca 2ϩ -sensitive and cAMP-dependent K ϩ channel currents were reported in HTC rat hepatoma cells by Lidofsky (20). Embryonic chick hepatocytes have been shown to possess large conductance voltage-gated and Ca 2ϩ -activated channels (21). Henderson et al. (22) found inwardly rectifying K ϩ channels in primary rat hepatocytes that were unperturbed by voltage or Ca 2ϩ stimulation. K ϩ channel activity was also found to regulate additional processes in hepatocytes, such as cell volume (23).
Our own interest in hepatic K ϩ channels concerned potential relationships between K ϩ channel activity and liver growth control, which involves growth factor-mediated activation of intracellular signaling cascades and other cellular events (24). The general hypothesis was that modulation of K ϩ channel activity would affect hepatic DNA synthesis. We studied primary rat hepatocytes as well as several established human cancer cell lines to analyze the effects of K ATP channel regulation upon cell proliferation. The results showed that K ATP channels play significant roles in regulating hepatocyte proliferation.
Cell Culture-Primary hepatocytes were isolated from F344 rats with a two-step perfusion using 0.03% collagenase as described previously (25). Primary hepatocytes were pelleted in 45% Percoll TM in Hanks' balanced salt solution (Amersham Pharmacia Biotech, Uppsala, Sweden) at 1000 ϫ g for 10 min at 4°C to obtain viable cells. Cell viability was determined by trypan blue dye exclusion. Cells were plated on borosilicate coverslips (Corning Inc., Horseheads, NY) and tissue culture dishes coated with rat tail collagen. Primary hepatocytes were cultured in RPMI 1640 medium containing 100 units/ml penicillin, 100 g/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Norcross, GA). HepG2, HuH-7, and HFL cells are cancerous liver epithelial cell lines derived from the human liver (26 -28). These cells were cultured in Dulbecco's minimal essential medium (Life Technologies, Inc.) with antibiotics and fetal bovine serum. Cell morphology was observed under phase-contrast microscopy. All experiments were in triplicate at least and repeated many times.
Cell Proliferation Assays-Changes in the number of cells were determined by plating cells at a density of 5 ϫ 10 4 /cm 2 in 24-well plates. The medium was changed every 48 h. Cells were trypsinized and counted in a modified Neubauer hemocytometer at 1, 3, and 5 days after culture.
To determine DNA synthesis rates, primary hepatocytes were plated at a density of 2 ϫ 10 4 /cm 2 , and HepG2, HuH-7, and HFL cells were plated at 1 ϫ 10 4 /cm 2 . Two hours later, drugs affecting potassium channel activity or vehicle alone was added to cultured cells. Primary rat hepatocytes were cultured with and without 10 ng/ml hHGF and incubated with drugs for 44 -46 h. HepG2, HuH-7, and HFL cells were cultured for 22-23 h. This was followed by the addition of 1 Ci of [ 3 H]thymidine (Sigma) for 1 h. Cells were washed twice with ice-cold phosphate-buffered saline, and trichloroacetic acid-precipitable DNA was extracted as described previously (29). [ 3 H]Thymidine incorporation into DNA was measured by liquid scintillation counting with normalization to DNA content. All experiments were in triplicate at least and performed on 3-10 occasions.
Colony-forming assays were undertaken by plating 1000 cells in 100-mm culture dishes. The drugs were added 72 h after plating the cells. The culture medium was changed every 48 h until colonies became visible at 18 days of cell culture. To count colonies, cells were fixed in 100% ethanol for 10 min, followed by staining with 4% crystal violet in 0.2 M citric acid. The number of colonies was counted with an AlphaImager 2000 instrument (Alpha Innotech Corp., San Leandro, CA).
Cell Viability Assays-To determine changes in cell viability, utilization of thiazolyl blue (MTT; Sigma) was measured. Cells were plated at a density of 1.3 ϫ 10 5 /cm 2 in 96-well plates. After drug treatments, cells were incubated with 1 mg/ml MTT in serum-free RPMI 1640 medium for 60 min at 37°C. The MTT-containing medium was removed; cells were lysed; and the intracellular formazan product was solubilized in isopropyl alcohol for quantitation at A 560. In aliquots, total protein was measured by the Bio-Rad assay using bovine ␥-globulin standards. To demonstrate apoptosis, cells were fixed in 75% methanol and 25% glacial acetic acid for 15 min at room temperature, washed twice with water, air-dried, and incubated for 15 min with 8 g/ml Hoechst 33258. The stained cells were examined by epifluorescence at 365-400 nm. The terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) technique was used to demonstrate apoptosis with a commercial kit (Roche Molecular Biochemicals). Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 30 min, followed by quenching of endogenous peroxidase activity with 0.3% H 2 O 2 in methanol for another 30 min at room temperature. Cells were then incubated with the TUNEL reaction mixture at 37°C for 30 min. The peroxidase system was used to visualize nuclei with evidence of apoptotic lesions.
Electrophysiological Analysis of K ϩ Channel Activity-The whole-cell configuration of the patch-clamp technique was used to record currents under voltage-clamp conditions (30). Glass pipettes with tip diameters of ϳ1-2 m were fabricated from borosilicate capillary tubing and fire-polished. Cells on coverslips were placed in a perfusion chamber on an inverted microscope equipped with patch-clamp micromanipulators. The chamber perfusion flow rate was 750 l/min, and the bath volume was 250 l. The external solution contained 170 mM Tris, pH 7.4, 1 mM MgCl 2 , 4 mM KCl, 15 mM NaCl, and 5 mM glucose. The internal pipette solution contained 140 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, and 5 mM UDP. The pH was adjusted to 7.2 with KOH. All recordings were obtained without ATP in solutions. All experiments were performed at room temperature. The holding potential was maintained at Ϫ20 or Ϫ33 mV, and voltage steps were applied from 80 to Ϫ160 mV in 20-mV increments. An Axopatch-1C patch-clamp amplifier (Axon Instruments, Inc., Foster City, CA) controlled by a PC computer was used for recordings. Data were analyzed with pClamp6 software (Axon Instruments, Inc.).
Analysis of Calcium Responses in Cells-Changes in intracellular calcium levels were demonstrated in HepG2 cells and primary rat hepatocytes plated on glass-bottomed microwells (MatTek Corp., Ashland, MA). Cells were loaded with a 15 M concentration of the intracellular calcium indicator Fura-2/AM (Molecular Probes, Inc., Eugene, OR) at 37°C for 45 min. Loaded cells were rinsed with Tyrode's solution (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 12 mM NaHCO 3 , 0.45 mM NaH 2 PO 4 , 5.5 mM glucose, and 5 mM HEPES, pH 7.0 -7.2) and examined under an epifluorescence microscope. The ratio of Fura-2 fluorescence emitted at 340-and 380-nm excitation wavelengths was obtained using a combined system of optic filter wheel (Sutter Instrument Co., Burlingame, CA) and shutter (Uniblitz, Rochester, NY) driven by an OEI computer (Universal Imaging Corp., West Chester, PA). The images were acquired with an intensified CCD camera and analyzed with Metafluor imaging system software (Universal Imaging Corp.). Fura-2 fluorescence ratio images were continuously acquired at a rate of 0.3 Hz before and after inducing Ca 2ϩ fluxes with ATP to HepG2 cells at an effective concentration of 50 M. To establish conditions for demonstrating Ca 2ϩ fluxes in primary rat hepatocytes, we stimulated cells with hHGF in concentrations ranging from 5 to 600 ng/ml within 1-3 h after plating cells as well as after 24 h of cell culture. Hepatocytes were pulsed with hHGF for 2 min and then switched to Tyrode's solution. After pilot studies to determine effective conditions, primary rat hepatocytes were pulsed with 12 ng/ml hHGF for 2 min, followed by incubation in Tyrode's solution and sequential recordings at 5-min intervals for 15-20 min. Intracellular calcium levels were obtained from regions of interest by measuring the ratio of Fura-2 intensity during excitation at 340 and 380 nm after applying the following calibration equation: (31), R is the ratio intensity, R min is the ratio of the intensity at zero calcium, R max is the ratio of the intensity at saturated calcium, F 380(min) is the fluorescence intensity at zero calcium at 380 nm (insensitive to calcium), and F 380(max) is the fluorescence intensity with saturated calcium at 380 nm.
Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis-Total RNA was extracted from cells with the guanidium thiocyanate method using Trizol reagent (Life Technologies, Inc.). A commercial system (Access RT-PCR, Promega, Madison, WI) was used for one-step reverse transcription-PCR for amplifying rat Kir6.1, Kir6.2, SUR1, and SUR2 as well as ␤-actin to serve as an internal control for semiquantitative comparisons with previously described primers (6). In addition, primers were synthesized in our DNA synthesis facility to amplify human Kir6.1, Kir6.2, SUR1, and SUR2. The effective primers for human sequences were as follows: Kir6.2, 5Ј-AGC CCA AGT TCA GCA TCT CTCC (forward) and 5Ј-CCA GAA ATA GCA TAG TGA CAA GTGCC (reverse); and SUR1, 5Ј-GAC CCG CAG GAA GGA GAT GAC (forward) and 5Ј-ACC ACA ACT GGA CAG CAG GAAC (reverse). Control reactions, where no reverse transcription was performed and where no template RNA was added, were included. cDNAs were amplified for 50 cycles by denaturing at 92°C for 2.5 min, annealing at 55°C for 1.5 min, extension at 72°C for 1 min, with final extension at 72°C for 10 min. The PCR products were resolved on 2% agarose gels.
Statistical Analysis-Data are expressed as mean Ϯ S.D. The data were analyzed with SigmaStat 2.0 software (Jandel Scientific, San Rafael, CA). The significance of differences was determined by Student's t tests, 2 test, and analysis of variance, as appropriate.

RESULTS
The general experimental design concerned analysis of proliferation in cultured hepatocytes after the addition of drugs known either to open K ATP channels (minoxidil, cromakalim, and pinacidil) or to inhibit K ATP channel opening (quinidine and glibenclamide). Since primary rat hepatocytes exhibit limited DNA synthesis in culture, we induced DNA synthesis in these cells by exposure to 10 ng/ml hHGF for 48 h.
K ATP Channel Activity and Proliferation of Cultured Hepatocytes-Initial experiments were undertaken to analyze the effects of DNA synthesis in primary rat hepatocytes. Minoxidil, cromakalim, and pinacidil increased DNA synthesis in hHGFstimulated rat hepatocytes by severalfold (up to ϳ20-fold; p Ͻ 0.05) (Fig. 1A). Stimulation of rat hepatocytes with hHGF itself increased DNA synthesis by 4 -10-fold compared with untreated control cells (p Ͻ 0.05). Interestingly, DNA synthesis was not increased in rat hepatocytes treated with K ϩ channel openers in the absence of hHGF. The optimal stimulatory drug concentrations were as follows: minoxidil, 500 nM (range tested, 100 -1000 nM); cromakalim, 5 M (range tested, 5-25 M); and pinacidil, 100 M (range tested, 5-200 M). In contrast, culture of rat hepatocytes with tetraethylammonium, quinidine, or glibenclamide significantly inhibited hHGF-induced DNA synthesis. More extensive analysis with quinidine (12.5-250 M) and glibenclamide (50 -200 M) showed that hHGF-induced DNA synthesis was significantly inhibited. Use of these drugs suppressed DNA synthesis induced by the combination of hHGF and K ϩ channel openers. In a typical experiment, 10 ng/ml hHGF increased DNA synthesis by 15 Ϯ 3-fold and hHGF plus 500 nM minoxidil by 32 Ϯ 6-fold above untreated control cells, whereas in response to 12.5 or 25 M quinidine, DNA synthesis induced by hHGF and minoxidil decreased to 17 Ϯ 1-fold and decreased further to 7 Ϯ 1-fold above untreated control cells with 50 M quinidine (p Ͻ 0.05, analysis of variance). Treatment of rat hepatocytes with glibenclamide showed efficient suppression of hHGF-mediated DNA synthesis (Fig.  1B).
To exclude whether inhibition of DNA synthesis could have been due to drug toxicity, utilization of MTT by cells was analyzed. It was noteworthy that neither quinidine nor glibenclamide was cytotoxic to our primary hepatocytes or HepG2, HuH-7, and HFL cell lines across the range of drug concentrations tested. However, changes in the morphology of primary hepatocytes were observed following treatment with glibenclamide (Fig. 2). The most characteristic feature was that cells appeared more rounded in culture dishes. When DNA fragmentation was analyzed by staining HepG2 cells or primary rat hepatocytes with the DNA-binding Hoechst 33258 dye, there was no evidence of increased apoptosis (Fig. 2, E and F). TUNEL staining showed the presence of some apoptosis in untreated control primary rat hepatocytes at 48 h of cell culture (ϳ10%), which was in agreement with the generally limited survival of primary hepatocytes under these conditions. In contrast, only occasional apoptotic nuclei were detected in hepatocytes following culture with hHGF. TUNEL positivity in glibenclamide-treated hepatocytes did not increase, and apop-tosis rates remained similar to those in untreated control cells or hHGF-treated cells (Fig. 2, G and H). In control liver tissue from rats subjected to ischemia-reperfusion injury, TUNEL staining showed extensive apoptosis.
Further studies were undertaken in cultured HepG2, HuH-7, and HFL cells. These cells did not show significant changes in DNA synthesis after culture with 10 ng/ml hHGF. Therefore, studies were conducted by culturing these cells for 24 h with either 25 M quinidine or 100 M glibenclamide in the absence of hHGF. The studies showed that both K ϩ channel inhibitors suppressed DNA synthesis significantly by 2-5-fold in HepG2, HuH-7, and HFL cells (p Ͻ 0.05, t tests). In contrast, within 24 h of culture, exposure to 500 nM minoxidil increased DNA synthesis in HepG2 cells by 1.5-fold (p Ͻ 0.05, t test). Although a trend for greater DNA synthesis was observed in HuH-7 and HFL cells cultured with 100 M pinacidil, this did not reach statistical significance, indicating a difference in the responsiveness of various cell lines.
To determine whether inhibition of DNA synthesis was associated with sustained impairment in cell replication, we cultured HepG2 cells with 100 M glibenclamide and measured cell numbers in culture dishes for up to 5 days. The number of untreated control cells increased by 468 Ϯ 94% between 1 and FIG. 1. Effects of K ؉ channel activity on DNA synthesis in primary rat hepatocytes. A, effects of pinacidil, minoxidil, and cromakalim, which are K ϩ channel openers, on DNA synthesis in cells treated with 10 ng/ml hHGF (HGF). In response to hHGF stimulation alone, DNA synthesis increased by ϳ10-fold (p Ͻ 0.05). When hHGFtreated cells were exposed to K ϩ channel openers, DNA synthesis increased further (p 5 days of culture. In contrast, the number of glibenclamidetreated cells increased by 350 Ϯ 44% during this period (p Ͻ 0.05, analysis of variance). Additional studies were undertaken to demonstrate whether clonogenic capacity of HepG2 cells was altered by glibenclamide treatment. In our colony-forming assay, at 18 days of culture, 1735 Ϯ 61 colonies formed in dishes containing control cells, which were treated by vehicle alone, whereas 934 Ϯ 51 colonies (56 Ϯ 9%) formed in dishes containing 100 M glibenclamide (p ϭ 0.001, t test). This suggested that inhibition of K ϩ channel activity affected DNA synthesis and cell proliferation, including perturbation of clonogenic capacity.
Molecular Expression of K ATP Channels in Hepatocytes-The intact rat liver contained Kir6.1 and SUR1 mRNAs as well as SUR2 mRNA, although the abundances of Kir6.1 and SUR2 were greater in the rat heart compared with the rat liver (Fig.   3A). The expression of Kir6.2 mRNA in the intact rat liver was uncertain in our hands; and therefore, analysis of Kir6.2 expression in rat hepatocytes was not pursued further. When primary rat hepatocytes were cultured for 48 h, it was still possible to detect Kir6.1, SUR1, and SUR2 mRNAs, although SUR2 mRNA was detected only at low levels (Fig. 3B). Neither hHGF nor glibenclamide altered the overall abundance of Kir6.1 or SUR2 mRNA in primary rat hepatocytes. In contrast, SUR1 mRNA expression was perturbed. In rat hepatocytes treated with glibenclamide alone compared with no treatment, the relative SUR1 mRNA expression, after normalization for ␤-actin expression, was consistently decreased (Fig. 3B). Densitometric scanning showed that this decrease ranged from 0.25 to 0.5 in three independent experiments performed. In two

FIG. 3. RT-PCR analysis of K ATP channels in hepatocytes.
A, analysis of mRNAs for the presence of Kir6.1, SUR1, and SUR2 in F344 rat liver and heart. RT-PCR was with 2 g of total RNA. All samples were treated with RNase-free DNase before analysis to eliminate contamination with genomic DNA. The same RNA sample was analyzed with (ϩ) and without (Ϫ) reverse transcriptase (RT). Lane 1 contained DNA size markers with sizes of the bands indicated on the left in base pairs. Lanes 2 and 3 were probed for Kir6.1 mRNA; lanes 4 and 5 were probed for SUR1 mRNA; and lanes 6 and 7 were probed for SUR2 mRNA. The expected sizes of PCR products in base pairs (bp) were as follows: Kir6.1, 247 bp; SUR1, 181 bp; SUR2, 144 bp; and ␤-actin, 209 bp. The channels were expressed in both the heart and liver, although Kir6.1 and SUR2 mRNAs seemed to be more readily amplified in the heart. B, effect of glibenclamide treatment on K ATP expression in rat hepatocytes. Cells were cultured for 48 h before RT-PCR of total cellular RNA. Lane 1, DNA size markers; lane 2, cells treated with vehicle (Me 2 SO) alone; lane 3, glibenclamide-treated cells; lane 4, total rat liver RNA; lane 5, PCR mixture alone. Densitometric scanning of the bands was used for quantitation, and data were normalized to ␤-actin expression. There was no significant change in Kir6.1 expression. However, glibenclamide treatment down-regulated SUR1 mRNA expression (to 0.25 of the control in the experiment here). This finding was reproduced in all three independent experiments performed. of these three experiments, the addition of hHGF prevented glibenclamide-induced decreases in SUR1 mRNA expression. These data suggested that glibenclamide inhibited SUR1 expression, whereas hHGF could potentially prevent glibenclamide-induced inhibition of SUR1 expression. Our findings concerning Kir6.1 and SUR expression in primary rat hepatocytes were in agreement with the observations of Kir6.1 protein expression by Suzuki et al. (32), who studied plasma and mitochondrial membranes from primary rat hepatocytes with immunofluorescence. Also, Inoue et al. (33) showed binding of radiolabeled glibenclamide to hepatocyte membranes, which is in agreement with the presence of SUR in hepatocytes. Further verification of our findings concerning modulation of SUR expression will be facilitated by the availability of reliable antibodies, e.g. Western blot analysis, but this is lacking at present.
In HepG2, HuH-7, and HFL cells, we were able to detect Kir6.2 as well as SUR2 mRNAs by RT-PCR. We were unable to obtain PCR products for Kir6.1 and SUR1 in our cell lines.
There was no change in Kir6.2 and SUR2 mRNA expression after treatment with glibenclamide alone, which was similar to primary rat hepatocytes.
Electrophysiological Studies of K ϩ Channel Activity-To determine the functional expression of K ATP channels in hepatocytes, we performed whole-cell current recordings with the patch-clamp technique. ATP, an inhibitor of K ATP channel activity, was excluded from the internal pipette solution, and 5 mM UDP, a channel activator, was included.
In primary hepatocytes cultured for 24 h, the reversal potential shifted toward the equilibrium potential for K ϩ upon the addition of minoxidil (n ϭ 6) and pinacidil (n ϭ 3) (Fig. 4A and Table I). The current-voltage plot in Fig. 4A shows a minoxidilactivated current with some inward rectification in isolated hepatocytes. This current was inhibited by glibenclamide (Table I), which was in agreement with K ATP channel activity. The subtraction of current recorded in the presence of glibenclamide from K ϩ channel openers yielded an inward current of In HepG2 cells, we recorded a pinacidil-activated, outwardly rectifying current that reversed at Ϫ55 Ϯ 16 mV (n ϭ 8) (Fig.  4B). The reversal potential in these cells shifted toward the equilibrium potential for K ϩ from Ϫ48 Ϯ 26 to Ϫ55 Ϯ 16 mV upon the addition of pinacidil (n ϭ 3). This current was partially inhibited by both glibenclamide and tetraethylammonium. K ϩ channel openers and antagonists affected these currents at drug concentrations that also altered cellular DNA synthesis.
Analysis of Intracellular Calcium following Stimulation of Glibenclamide-treated HepG2 Cells with ATP-These studies showed that mean basal intracellular calcium levels in HepG2 cells were 141 Ϯ 2.4 nM (n ϭ 35). Upon stimulation of cells with 50 M ATP, which was applied to the cells externally, intracellular calcium levels increased significantly (by 4-fold) (Fig. 5). The intracellular calcium levels increased rapidly, followed by a sustained plateau during observations lasting ϳ90 s. When cells were treated with 100 M glibenclamide (n ϭ 50), mean resting calcium levels were 155 Ϯ 2 nM (p ϭ 0.98), similar to untreated control cells (Table II). Cells treated with glibenclamide immediately before analysis responded to ATP stimulation with a 3 Ϯ 1-fold increase in intracellular calcium. Similarly, HepG2 cells cultured with 100 M glibenclamide for 24 h prior to analysis showed basal intracellular calcium levels as well as responses to ATP stimulation that were not significantly different from those of untreated control cells ( Fig. 5 and Table II). There was not a delay in the response of cells to ATP stimulation, nor was a difference elicited in the magnitude of intracellular calcium fluxes.
In primary hepatocytes cultured for Ͻ3 h, hHGF stimulation was effective in activating Ca 2ϩ fluxes. On the other hand, when primary hepatocytes had been in culture for 24 h, hHGF stimulation (up to 600 ng/ml) was completely ineffective. Therefore, we restricted our analysis to primary hepatocytes that were cultured for Ͻ3 h, which was adequate for cell attachment to dishes. In untreated primary hepatocytes, mean basal intracellular calcium levels were 292 Ϯ 24 nM (n ϭ 20). When hHGF was added to the hepatocytes, most cells (60 -80%), but not all, responded with increased Ca 2ϩ fluxes. It was noteworthy that intracellular Ca 2ϩ levels did not change during the hHGF pulse itself. However, 5 min after removing hHGF from the solution, Ca 2ϩ levels increased by 1.6 Ϯ 0.1-fold (p Ͻ 0.001, t test), which was sustained for 15-20 min, although intracellular Ca 2ϩ levels oscillated in some hepatocytes. After incubation with 100 M glibenclamide for 5 min, intracellular Ca 2ϩ levels declined slightly in primary hepatocytes (n ϭ 36) from 344 Ϯ 19 to 242 Ϯ 16 nM (p Ͻ 0.05, t test). However, when glibenclamide-treated cells were pulsed with 12 ng/ml hHGF, intracellular Ca 2ϩ levels increased by 1.55 Ϯ 0.1-fold at 5 min (p Ͻ 0.001, t test). This induction of intracellular Ca 2ϩ flux was identical to control primary hepatocytes that had not been treated with glibenclamide (p ϭ not significant).

DISCUSSION
These studies provide new knowledge concerning K ATP channels in hepatocytes and their biological significance with respect to liver growth control. Our work showed functional K ATP channels in freshly isolated primary hepatocytes as well as in hepatocyte-derived cell lines, which was previously unknown. The currents recorded from primary rat hepatocytes and HepG2 cells were consistent with K ATP channels because these currents were observed in the presence of UDP in internal solution, without ATP, and with K ϩ as the major permeant cation. Moreover, the addition of K ϩ channel openers to the external solution shifted the reversal potential toward the equilibrium potential for K ϩ .
Previously recorded hepatic K ϩ currents have been either linear or Ca 2ϩ -dependent inward rectifiers (18,19). Our electrophysiological data are in agreement with additional demonstrations of K ϩ channel mRNA expression in liver cells. Kir6.1 and SUR1 mRNAs were well expressed in the intact rat liver as well as in isolated primary rat hepatocytes, whereas SUR2 was expressed weakly. In our cell lines, we detected Kir6.2 and a Data demonstrate pinacidil-activated currents, which were obtained by subtraction analysis of currents under untreated basal conditions and after pinacidil treatment of cells.
b Data show the K ATP component of the total currents and were obtained by subtraction analysis of currents in glibenclamide-and pinacidiltreated cells. SUR2 mRNAs. These findings concerning regulation of SUR1 in rat hepatocytes are in agreement with previous studies in chick cardiomyocytes showing that K ϩ channel openers and glibenclamide regulated K ATP activity (34). The K ϩ currents in primary rat hepatocytes and HepG2 cells were different, as indicated above. The difference in the properties of currents in primary rat hepatocytes and HepG2 cells might be due to the expression of different K ATP channel subunits.
K ϩ channels are known to regulate proliferation in many cell types, although the type of K ϩ channel involved in this varies, as indicated by the identification of small conductance Ca 2ϩactivated K ϩ channels in fibroblasts (17), voltage-activated and calcium-gated K ϩ channels in lymphocytes (16), and K ATP channels in bladder cells (14). Interestingly, activation of K ϩ channels occurs early during mitogenic stimulation, occurring prior to DNA synthesis in hematopoietic cells as well as in neural cells (16,35,36). In fibroblasts, small conductance Ca 2ϩ -activated K ϩ channels are involved in activating intracellular signaling, such as Ras signaling (17). Similarly, overexpression of ras or raf proto-oncogenes, which are downstream effectors of the intracellular signaling pathways, increases K ϩ channel current density in cultured fibroblasts (37,38).
Our work shows that in primary rat hepatocytes as well as in human liver cell lines, quinidine and glibenclamide dose-dependently inhibit cell proliferation. Although quinidine is a relatively nonspecific potassium channel blocker, glibenclamide has specificity for K ATP channels. In cultured primary hepatocytes, it was necessary to add a growth factor (hHGF) to elicit DNA synthesis because these cells show very limited proliferative activity. Various cancer cell lines were presumably capable of producing autocrine factors to sustain higher levels of proliferation. The efficacy of glibenclamide in suppressing cell proliferation in primary hepatocytes without the addition of hHGF (Fig. 1B) may suggest interference with growth factor-independent pathways, although suppression of growth factor-dependent increases in DNA synthesis was more obvious. Whether other activities of glibenclamide in cells, such as regulation of the cystic fibrosis transmembrane conductance regulator chloride channel, GLUT2 or GLUT4 glucose transporters, and glucose synthesis or storage (39), could contribute to regulating cell proliferation will require further analysis. It is noteworthy that although the pancreatic K ATP channel responds to nanomolar concentrations of glibenclamide, such responses can vary widely in cell-and tissue-specific fashions, ranging from 10 Ϫ7 to 10 Ϫ5 M (40,41). Also, glibenclamide is largely bound to serum proteins, and the total effective concentrations in cultured cells may not be reflective of free glibenclamide concentrations achieved in vivo (42).
K ATP channels play significant roles in liver growth control as indicated by stimulation of DNA synthesis as well as by elicitation of characteristic currents following exposure to K ϩ channel openers specific for the K ATP channel. On the other hand, we found that quinidine and glibenclamide, which block the K ATP channel, inhibited DNA synthesis both with and without growth factor stimulation in hepatocytes. These findings indicate that K ATP channels play critical roles in hepatocytes. Also, K ATP channel activity was preserved despite oncogenic transformation in cancer cell lines. Persistent inhibition of proliferation in glibenclamide-treated HepG2 cells was in agreement with the lack of redundancy in regulation of cell proliferation by K ATP channels. Previous studies with bladder cancer cells showed that K ϩ channel blockers inhibited cell proliferation without cytotoxicity (14), similar to our findings in hepatocytes.
Hepatocyte growth factor acts on hepatocytes via the c-Met receptor, which is a receptor tyrosine kinase, with activation of intracellular signaling pathways, including calcium-mediated signals. Also, hepatocyte growth factor exhibits morphogenic and motogenic activities in cells (24). It was noteworthy that our glibenclamide-treated cells underwent morphological alterations in culture. Several mechanisms could have been responsible for this change. Some K ϩ channels and other ion channels regulate cell volume, which may have secondary effects upon mitogenesis as well (23). Also, membrane depolarization associated with inhibition of K ϩ channels could modulate calcium channels with perturbation of signal transduction mechanisms. Fluxes in intracellular calcium ions as well as in other inorganic ions participate in mediating mitogenic stimuli, including hHGF (24,43,44). Hepatocyte and epidermal growth factors have previously been shown to elicit Ca 2ϩ fluxes in freshly isolated primary rat hepatocytes (45). It is noteworthy that glibenclamide did not alter hHGF-stimulated increases in intracellular Ca 2ϩ fluxes. Furthermore, stimulation of glibenclamide-treated HepG2 cells with ATP elicited intracellular calcium fluxes, which was similar to previous studies of Ca 2ϩ fluxes in HepG2 cells (46). These findings indicate that K ATP channels regulated cell proliferation independently of intracellular Ca 2ϩ response.
Further analysis of hepatic K ϩ channel activity will offer insights into liver growth control. An understanding of the hepatic activity of K ϩ channel openers and blockers may offer novel drug targets for regulating cell proliferation events. A working model would suggest that modulation of K ATP channels on the cell membrane alters intracellular processes involved in DNA synthesis. Potential interactions among intracellular regulators in the context of K ATP channel activity can be examined in our systems described here. For instance, we are currently defining alterations in cellular gene expression following inhibition of K ATP channel activity at a genome-wide level. Initial findings indicate perturbations in specific genes involved in growth factor activity and cell cycle regulation. Inhibitors of cell signaling mechanisms have recently been found to have potent anticancer effects in intact animals (47), which suggests that insights into K ATP channel activity in liver cancer could potentially offer novel therapeutic targets.
Insights into the regulation of hepatic K ATP channel activity  between footnotes a, b, and c, as well as between footnotes d, e, and f, were not statistically significant.
will help define potential hepatotoxic manifestations of sulfonylureas and related drugs (48), which are poorly understood at present, but could involve perturbation of cell cycling or cell survival. Finally, amplification of hepatic growth factor responses with effective drugs capable of modulating K ATP activity might also find applications. For instance, this might facilitate hepatic gene transfer for somatic gene therapy with specific vectors requiring DNA synthesis (49) as well as liver repopulation with transplanted hepatocytes for cell therapy.