HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 33, 30871-30877, August 17, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/33/30871    most recent M102362200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Roe, M. W. Articles by Lidofsky, S. D. Search for Related Content PubMed PubMed Citation Articles by Roe, M. W. Articles by Lidofsky, S. D. Social Bookmarking                      What's this?

Purinergic-independent Calcium Signaling Mediates Recovery from Hepatocellular Swelling

IMPLICATIONS FOR VOLUME REGULATION^*

Michael W. Roe, Ann L. Moore, and Steven D. Lidofsky^§¶

From the Departments of  Medicine and ^§ Pharmacology, University of Vermont College of Medicine, Burlington, Vermont 05401

Received for publication, March 16, 2001, and in revised form, June 1, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Swelling of hepatocytes and other epithelia activates volume-sensitive ion channels that facilitate fluid and electrolyte efflux to restore cell volume, but the responsible signaling pathways are incompletely defined. Previous work in model HTC rat hepatoma cells has indicated that swelling elicits ATP release, which stimulates P2 receptors and activates Cl channels, and that this mechanism is essential for hepatocellular volume recovery. Since P2 receptors are generally coupled to Ca²⁺ signaling pathways, we determined whether hepatocellular swelling affected cytosolic [Ca²⁺], and if this involved a purinergic mechanism. Exposure of HTC cells to hypotonic media evoked an increase in cytosolic [Ca²⁺], which was followed by activation of K⁺ and Cl currents. Maneuvers that interfered with swelling-induced increases in cytosolic [Ca²⁺], including extracellular Ca²⁺ removal and intracellular Ca²⁺ store depletion with thapsigargin, inhibited activation of membrane currents and volume recovery. However, the swelling-induced increases in cytosolic [Ca²⁺] were unaffected by either extracellular ATP depletion with apyrase or blockade of P2 receptors with suramin. These findings indicate that swelling elicits an increase in hepatocellular Ca²⁺, which is essential for ion channel activation and volume recovery, but that this increase does not stem from activation of volume-sensitive P2 receptors. Collectively, these observations imply that regulatory responses to hepatocellular swelling involve a dual requirement for a purinergic-independent Ca²⁺ signaling cascade and a Ca²⁺-independent purinergic signaling pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Epithelia face substantial osmotic stresses from the vectorial transport of solutes that cause cell swelling and challenge cellular integrity. An adaptive response to cell swelling, termed regulatory volume decrease (RVD),¹ provides a dynamic safeguard against tissue injury produced by such stresses. RVD is mediated, in part, by the opening of swelling-activated K⁺ and Cl channels in the plasma membrane, which leads to fluid and electrolyte efflux and consequent restoration of cell volume (1). Although it has been well appreciated that activation of both K⁺ and Cl channels is critical for RVD, the mechanisms that couple cell swelling to ion channel activation and RVD exhibit tissue diversity.

Among epithelia, hepatocytes are particularly susceptible to dynamic perturbations in cell volume, given the central role of the liver in nutrient uptake and metabolism (2). Whereas emerging evidence suggests that hepatocellular volume per se is an important determinant of several critical organ level functions, including glucose metabolism and bile formation (2), the mechanisms that govern hepatocellular volume regulation remain to be defined. We and others (3, 4) have provided evidence for the involvement of purinergic signaling in this process. In both HTC rat hepatoma cells and human hepatocytes, hypotonic swelling elicits ATP release, which stimulates P2 purinoreceptors, the activation of which leads to the opening of volume-sensitive Cl channels (3, 4). This purinergic mechanism is essential for hepatocellular RVD. Although it is known that swelling-induced ATP release requires activation of phosphatidylinositol 3-kinase (5) and involves a putative member of the ATP-binding cassette family (6), the downstream effectors that couple osmosensitive P2 receptors to ion channel opening and RVD are unknown.

In many cell types, including hepatocytes, activation of P2 receptors elicits increases in cytosolic [Ca²⁺] ([Ca²⁺]_i) through stimulation of phospholipase C, intracellular inositol trisphosphate (IP₃) formation, and activation of IP₃ receptors (7, 8). However, P2 receptors have also been reported to be coupled to Ca²⁺-independent cellular effectors (9-12). This leaves unresolved whether Ca²⁺ is involved in osmosensitive purinergic signaling in hepatocytes.

The role of Ca²⁺ in hepatocellular volume regulation is also uncertain. Hepatocellular swelling has been reported to elicit the opening of Ca²⁺-permeable cation channels and transiently increase [Ca²⁺]_i (13). However, other investigations (14) have suggested that swelling does not affect hepatocyte [Ca²⁺]_i. The reasons for these discrepancies are not apparent. Whether Ca²⁺ mediates hepatocellular RVD is controversial as well. Removal of extracellular Ca²⁺ inhibited RVD in one study (13) but was without effect in another (15). On the other hand, swelling-induced liver cell membrane hyperpolarization, indicative of K⁺ channel activation, and activation of volume-sensitive hepatocellular Cl channels have each been shown to be Ca²⁺-dependent (16, 17). These observations support a role for Ca²⁺ in hepatocellular volume regulation, but it is unclear how swelling affects [Ca²⁺]_i and if swelling-mediated changes in [Ca²⁺]_i involve volume-sensitive purinergic signaling.

Here we report that swelling elicits increases in [Ca²⁺]_i in HTC rat hepatoma cells through intracellular Ca²⁺ mobilization and Ca²⁺ influx. Our findings demonstrate further that intracellular Ca²⁺ mobilization is necessary for activation of Ca²⁺ influx, volume-sensitive K⁺ and Cl channels, and RVD but that swelling-evoked increases in [Ca²⁺]_i are independent of purinergic signaling pathways. In light of our previous studies (3, 6) of volume-sensitive purinergic signaling in HTC cells, these findings support a model in which adaptive responses to hepatocellular swelling require the dual actions of a purinergic independent Ca²⁺ signaling pathway and a Ca²⁺-independent purinergic pathway.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- HTC rat hepatoma cells, a model liver cell line (3, 6, 17-19), were grown at 37 °C in a humidified 5% CO₂ atmosphere in minimal essential medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin as described previously (19). One day prior to all experiments, cells were seeded onto glass coverslips.

Measurement of [Ca²⁺]_i-- [Ca²⁺]_i was measured by dual-wavelength emission ratiometric laser scanning confocal microspectrofluorimetry, using the Ca²⁺-sensitive fluorescent dyes fluo-3 and fura-red (20, 21). This method takes advantage of the fact that fluo-3 and fura-red fluorescence increase and decrease, respectively, as a function of [Ca²⁺]. In this way, the ratio (R) of fluo-3 to fura-red fluorescence is a function of [Ca²⁺] and is unaffected by changes in dye concentration expected to occur during changes in cell volume.

HTC cells were loaded with fluo-3 and fura-red by incubation for 20-45 min at room temperature in the presence of 5 µM fluo-3 acetoxymethyl ester (fluo-3-AM) and 15 µM fura-red-AM (each from Molecular Probes) dissolved in a physiological buffer (standard extracellular solution (SES)). SES consisted of (in mM) 140 NaCl, 4 KCl, 1 CaCl₂, 2 MgCl₂, 1 KH₂PO₄, 10 glucose, and 10 HEPES (pH 7.4). Each coverslip was placed into a perfusion chamber (Warner Instruments R-26G) mounted on an Olympus BX-50 upright fluorescence microscope equipped with a Bio-Rad MRC 1024 laser scanning confocal system. Cells were continuously perfused at 3-4 ml/min with SES. Fluorescence was excited with the 488 nm line of an argon-krypton laser, and emission was detected simultaneously at 522 (fluo-3) and 680 nm (fura-red). Cells were visualized with a 60× water immersion quartz objective (Olympus LUM Plan F1) and outlined for study using computer-controlled data acquisition software (Bio-Rad Time Course). Following a 10-15-min rinse with SES, changes in [Ca²⁺]_i were measured in a field-of-view consisting of 10-30 cells. Measurements of fluo-3 and fura-red fluorescence emission intensity were acquired every 5-30 s. In most studies, changes in [Ca²⁺]_i were inferred from changes in the relative fluorescence ratio, calculated by dividing R at each time point by R₀, the fluorescence ratio measured at the first time point (t = 0) of exposure to hypotonic solution. In studies involving long term [Ca²⁺]_i measurements (>5 min), R₀, was taken to be the average fluorescence ratio 1-2 min prior to exposure to hypotonic solution.

Hypotonic swelling was produced by perfusion with solutions identical to SES except that the NaCl concentration was 84 mM. In selected experiments, CaCl₂ was not added to hypotonic solutions in order to achieve nominally Ca²⁺-free conditions. All experiments were performed at room temperature. Nitrendipine was from Calbiochem, and unless otherwise indicated, all reagents were from Sigma.

Measurement of Cell Volume-- Cell volume was determined by three-dimensional reconstructions of optical sections using laser scanning confocal microscopy (22). Cells were loaded with calcein, a fluorescent dye that exhibits cytosolic distribution, by incubation for 30 min at room temperature with SES containing 5 µM calcein-AM (Molecular Probes). Calcein fluorescence excitation and emission wavelengths were at 488 and 530 nm, respectively. For each experiment, images consisting of 8-12 optical sections, beginning at a region adjacent to the cover glass and progressing by serial (x, y) scans to the top of the cells, were acquired at 1-min intervals. The cross-sectional area of each cell in an optical section was determined by computer image processing (Scion Image for Windows, version Beta 4.02). The areas for all sections through a cell were summed and converted to volume (V) by multiplying the total cross-sectional area by the z axis spacing (2 µm) between the optical sections (23). Relative cell volume was calculated by dividing V by the mean cell volume, V₀, measured during perfusion at room temperature with SES 5 min prior to application of hypotonic solutions. The extent of cell volume recovery after swelling (%RVD) was calculated from Equation 1,

(Eq. 1)

where V_max is the relative maximum volume after swelling, and V₁₅ is the relative cell volume 15 min after exposure to hypotonic solutions.

Measurement of Membrane Currents-- Whole-cell currents were measured using patch clamp recording techniques as described previously (3, 19). Cells on coverslips were placed in a perfusion chamber (Warner Instruments R-26G) mounted on the stage of an inverted microscope equipped with Hoffman modulation contrast optics. Hypotonic swelling was produced by changing the perfusion solution from SES to a solution identical in composition except that the NaCl concentration was 98 mM. The pipette solution contained (in mM) 10 NaCl, 130 KCl, 0.5 CaCl₂, 2 MgCl₂, 1 EGTA, and 10 HEPES (pH 7.30). With these solutions, the reversal potential for K⁺ is approximately 80 mV under both basal and hypotonic conditions, and the reversal potential for Cl is close to 0 mV (1 mV under basal conditions and +7 mV under hypotonic conditions). This approach has been successfully used to detect outward swelling-activated K⁺ currents at a potential of 0 mV, and inward swelling-activated Cl currents at a potential of 80 mV (18). In selected experiments, [Ca²⁺] in the patch pipette was lowered by increasing the concentration of EGTA to 5 mM and withholding CaCl₂. All experiments were performed at room temperature.

Statistics-- All results are presented as means ± S.E., where n represents the number of cells studied. Statistical comparisons were made with the use of Student's unpaired t test, and p < 0.05 was considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hypotonic Stress Increases [Ca²⁺]_i-- Dual-wavelength emission ratiometric imaging and laser scanning confocal microscopy were used to determine the effect of hypotonic challenge on [Ca²⁺]_i and volume in HTC cells. Exposure to hypotonic solution (40% reduction in NaCl concentration) resulted in a rapid increase in the relative fluorescence ratio of fluo-3 to fura-red, a measure of [Ca²⁺]_i (Fig. 1A). [Ca²⁺]_i remained elevated for ~5 min and then gradually decreased to basal levels within 10 min. In parallel experiments, under basal conditions, cell volume was calculated to be 3.8 ± 0.1 pl (n = 55), which is in the range of values reported previously for hepatocytes and HTC cells (3, 19, 22, 24). Exposure to hypotonic solution (40% reduction in NaCl concentration) produced a rapid increase in cell volume that was followed by a decline toward basal values (Fig. 1B). The onset of the increase in cell volume and in [Ca²⁺]_i occurred ~1 min after hypotonic exposure. This suggested that the changes in [Ca²⁺]_i seen were the result of cell swelling and supports the concept that Ca²⁺ serves as a sensor for changes in liver cell volume.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Swelling increases [Ca2+]i in HTC cells. A, effect of hypotonic challenge on [Ca2+]i. Cells were loaded with the Ca2+-sensitive dyes fluo-3 and fura-red and subsequently exposed to hypotonic solution (40% reduction in NaCl concentration), as indicated by the gray bar. [Ca2+]i was measured via ratiometric laser scanning confocal microspectrofluorimetry. Changes in [Ca2+]i were inferred from the ratio of fluo-3 to fura-red fluorescence, normalized to mean basal values, and measured 2 min prior to hypotonic exposure (see "Experimental Procedures"). Data represent means ± S.E. of 76 cells. B, effect of hypotonic challenge on cell volume. Cells were loaded with the fluorescent dye calcein and then exposed to hypotonic solution (as above). Cell volume was determined via laser scanning confocal microscopy and normalized to basal values, as described under "Experimental Procedures." Data represent means ± S.E. of 26 cells.

To examine the mechanisms responsible for swelling-evoked increases in [Ca²⁺]_i, we performed a series of experiments in which contributions of Ca²⁺ influx or intracellular store release were inhibited. In the nominal absence of extracellular Ca²⁺ (which would disable Ca²⁺ influx), hypotonic exposure elicited only a transient increase in [Ca²⁺]_i (Fig. 2). Under these conditions, the maximum relative fluorescence ratio (analyzed on a cell-by-cell basis) was 1.47 ± 0.03 (n = 106), versus 1.59 ± 0.03 (n = 116) in the presence of extracellular Ca²⁺. These results suggested that the initial increase in [Ca²⁺]_i that was elicited by swelling arose primarily from intracellular Ca²⁺ mobilization, whereas the prolonged [Ca²⁺]_i increase stemmed from Ca²⁺ influx. The swelling-activated increases in [Ca²⁺]_i were unaffected by 1 µM nitrendipine, a selective inhibitor of L-type voltage-dependent Ca²⁺ channels (data not shown). This suggests that the volume-sensitive Ca²⁺ influx pathways did not involve L-type Ca²⁺ channels.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Swelling-evoked increases in [Ca2+]i result from intracellular Ca2+ mobilization and Ca2+ influx. Cells were exposed to hypotonic solutions (40% reduction in NaCl concentration) as indicated by the gray bar. Swelling was evoked by exposure to control hypotonic solution (top), to hypotonic solution in the nominal absence of extracellular Ca2+ (middle), and to thapsigargin-containing hypotonic solution following a 10-min preincubation with 1 µM thapsigargin in the presence of extracellular Ca2+ (bottom). Changes in [Ca2+]i were inferred from the relative fluo-3/fura-red fluorescence ratio (see "Experimental Procedures"). The controls used for these studies were different than the cells depicted in Fig. 1, for which a substantially longer time course was followed. Data represent the means ± S.E. of 51 to 116 cells for each condition.

To determine the nature of swelling-evoked intracellular Ca²⁺ mobilization, we exposed cells to thapsigargin, an agent that specifically inhibits sarcoendoplasmic reticulum Ca²⁺- ATPases in hepatocytes and many other cell types and depletes endoplasmic reticulum Ca²⁺ stores (25). Application of 1 µM thapsigargin alone produced a transient increase in [Ca²⁺]_i that fell to basal levels within 10 min (data not shown). However, following a 10-min incubation with 1 µM thapsigargin in the presence of extracellular Ca²⁺ (Fig. 2), both the initial and prolonged increases in [Ca²⁺]_i elicited by hypotonic exposure were nearly abolished (maximal fluorescence ratio 1.11 ± 0.02, n = 51; p < 0.05, compared with control conditions). This suggests not only that Ca²⁺ mobilization from the endoplasmic reticulum contributes to increases in [Ca²⁺]_i elicited by hepatocellular swelling but that Ca²⁺ influx is dependent on intracellular Ca²⁺ mobilization.

Swelling-activated Membrane Currents and Ca²⁺-- The [Ca²⁺]_i measurements described above support the hypothesis that intracellular Ca²⁺ store release and Ca²⁺ influx serve as volume regulatory signals evoked by hepatocellular swelling, and by extension, signals that would trigger RVD. Since K⁺ and Cl channels serve as downstream mediators of RVD, we tested whether swelling-evoked K⁺ and Cl currents were Ca²⁺-dependent. In agreement with previous reports (3, 17, 18), exposure of HTC cells to hypotonic solution (30% reduction in NaCl concentration) elicited increases (within 2-3 min after exposure) in outwardly rectifying membrane currents (Fig. 3, A and B). The outward currents at 0 mV and inward currents at 80 mV (see "Experimental Procedures") correspond to swelling-activated K⁺ and Cl currents, respectively (18). To determine the dependence of these currents on Ca²⁺, we exposed HTC cells to hypotonic solutions under conditions in which [Ca²⁺]_i was buffered to <10 nM by including in the pipette (intracellular) solution 5 mM EGTA and withholding CaCl₂. Under these conditions, both swelling-activated K⁺ currents and Cl currents were inhibited by greater than 75% compared with control conditions (p < 0.05, Fig. 3C). This suggested that activation of K⁺ and Cl channels by swelling required increases in [Ca²⁺]_i.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Dependence of swelling-activated K+ and Cl currents on intracellular Ca2+ mobilization and Ca2+ influx. A, using patch clamp recording techniques (see "Experimental Procedures"), current transients in this representative cell were elicited by step changes in membrane voltage from a holding potential of 40 mV over a range from 100 to +100 mV under basal conditions and 5 min following exposure to hypotonic solution (30% reduction in NaCl). B, relation between membrane current (normalized to cell capacitance, pA/pF) and membrane voltage under basal conditions and 5 min following exposure to hypotonic solution. Data represent means ± S.E. of 13 cells. C, effects of altering intracellular and extracellular Ca2+ on K+ currents and Cl currents. K+ currents (top, recorded at 0 mV) and Cl currents (bottom, recorded at 80 mV) were measured (see "Experimental Procedures") under basal conditions and 5 min after exposure to hypotonic solution under control conditions, with intracellular Ca2+ chelation with 5 mM EGTA, in the nominal absence of extracellular Ca2+ (0 Ca) and following a 10-min preincubation with 1 µM thapsigargin in the presence of extracellular Ca2+ (Thaps). Currents have been normalized to cell capacitance, and data represent means ± S.E. of 8-13 cells for each condition (*, p < 0.05, compared with hypotonic control; NS, not significantly different from hypotonic control).

We next determined whether channel activation required swelling-evoked Ca²⁺ influx or intracellular Ca²⁺ store release. Exposure of cells to nominally Ca²⁺-free hypotonic solutions was associated with a 60% reduction in the amplitude of swelling-activated K⁺ currents (p < 0.05). By contrast, the amplitude of swelling-activated Cl currents was not significantly affected under these conditions (Fig. 3C). This indicated that activation of K⁺ currents by swelling was, in part, dependent on Ca²⁺ influx but that activation of Cl currents was not. The dependence of swelling-activated currents on intracellular Ca²⁺ mobilization was examined by depleting endoplasmic reticulum Ca²⁺ stores with a 10-min exposure to 1 µM thapsigargin prior to swelling. Thapsigargin inhibited swelling-activated Cl currents by more than 85% (p < 0.05) and abolished the swelling-activated K⁺ currents (Fig. 3C). These results suggest that activation of both K⁺ and Cl channels by hepatocellular swelling requires intracellular Ca²⁺ mobilization.

RVD and Ca²⁺-- The preceding results are consistent with the hypothesis that adaptive responses to hepatocellular swelling are regulated by Ca²⁺ influx and intracellular store release. We further assessed the roles of these pathways in RVD, using laser scanning confocal microscopy, to determine the Ca²⁺ dependence of RVD (Fig. 4). When Ca²⁺ influx was disabled by exposure to nominally Ca²⁺-free hypotonic solution (40% reduction in NaCl), %RVD was partially but significantly reduced (60.1 ± 5.6, n = 3 groups of 9-10 cells, compared with 118.2 ± 19.6, n = 3 groups of 7-11 cells, under control conditions, p < 0.05). By contrast, when Ca²⁺ release from the endoplasmic reticulum was prevented by a 10-min preincubation with 1 µM thapsigargin, %RVD was nearly abolished (11.1 ± 5.7, n = 3 groups of 12-16 cells). These findings suggest that RVD is dependent on and differentially regulated by Ca²⁺ influx and intracellular store release.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Dependence of cell volume recovery following swelling on intracellular Ca2+ mobilization and Ca2+ influx. Cell swelling was elicited by exposure to hypotonic solution (40% reduction in NaCl concentration), as indicated by the gray bar. Relative cell volume was determined (see "Experimental Procedures") under control conditions (top), in the nominal absence of extracellular Ca2+ (middle), and following a 10-min preincubation with 1 µM thapsigargin in the presence of extracellular Ca2+ (bottom). Data represent means ± S.E. of 26 to 41 cells for each condition.

Volume-sensitive Purinergic Signaling and [Ca²⁺]_i-- Our results thus far indicate that HTC cell swelling increases [Ca²⁺]_i and that these increases are necessary for volume regulatory responses. Previous work (3) has shown that autocrine signaling via ATP is essential for regulation of HTC cell volume and that ATP evokes increases in [Ca²⁺]_i in these cells via stimulation of purinoreceptors (26). We therefore tested whether swelling-induced increases in [Ca²⁺]_i involved a purinergic pathway. To address this question, the ATP hydrolase apyrase and the broad spectrum P2 receptor antagonist suramin were employed at concentrations that we have shown previously to block swelling-activated Cl currents and RVD in HTC cells (3). Neither apyrase (grade VI, 3 units/ml) nor suramin (100 µM) prevented swelling-mediated increases in [Ca²⁺]_i (Fig. 5). In parallel controls, however, apyrase and suramin (at identical concentrations used in studies above) significantly inhibited the increase in [Ca²⁺]_i elicited by exogenous application of 10 µM ATP. In the presence of apyrase, the peak relative fluorescence ratio induced by ATP was 1.7 ± 0.1% that induced by ATP alone (n = 37 cells), and in the presence of suramin, it was 22 ± 1% that induced by ATP alone (n = 40 cells). Collectively, these findings indicate that swelling-induced increases in [Ca²⁺]_i are independent of volume-sensitive purinergic signaling pathways.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Swelling-evoked increases in [Ca2+]i are independent of P2 purinoreceptor-mediated signaling pathways. Cells were exposed to hypotonic solutions (40% reduction in NaCl concentration) as indicated by the gray bar. Swelling was evoked by exposure to control hypotonic solution (top), to hypotonic solution in the presence of apyrase (3 units/ml, middle), and to hypotonic solution in the presence of suramin (100 µM, bottom). Exposure to inhibitors is indicated by the open bar. Changes in [Ca2+]i were expressed as the relative fluo-3/fura-red fluorescence ratio (see "Experimental Procedures"). Data represent the means ± S.E. of 80-137 cells for each condition.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we have provided evidence that strongly supports an important role for Ca²⁺ in hepatocellular volume regulation. Hypotonic swelling of HTC cells elicited a transient increase in [Ca²⁺]_i via intracellular Ca²⁺ store release and Ca²⁺ influx, and each of these pathways was coupled distinctly to the ion channels that mediate volume recovery. Ca²⁺ store discharge provided a signal that activated volume-sensitive Cl channels, whereas activation of volume-sensitive K⁺ channels required both intracellular Ca²⁺ store release and extracellular Ca²⁺ influx. We have also demonstrated that the Ca²⁺ signals generated by swelling, while necessary for RVD, did not involve activation of P2 purinergic pathways that have been implicated as essential for volume recovery after hepatocellular swelling (3, 4). Taken together, these observations support the hypothesis that Ca²⁺ and purinergic signaling pathways independently govern hepatocellular volume regulation.

While many (but not all) cell types utilize Ca²⁺ as an intracellular messenger in volume regulation (1, 27), information concerning the effects of hepatocellular swelling on [Ca²⁺]_i has been limited and conflicting. One study suggested that hepatocellular swelling activates Ca²⁺ influx (13). This interpretation was based on the observation that hypotonic challenge elicited increases in [Ca²⁺]_i that were prevented by chelation of extracellular Ca²⁺. However, the chelation conditions employed decreased resting [Ca²⁺]_i (raising the possibility of intracellular store depletion), and this left the underlying mechanisms responsible for swelling-mediated increases in [Ca²⁺]_i unclear. Another study showed that hypotonic challenge did not affect [Ca²⁺]_i in hepatocytes that were transiently pretreated with the Ca²⁺-mobilizing agonists ATP and vasopressin (14). Although it was suggested that cell swelling does not influence [Ca²⁺]_i, it is possible that the failure to observe swelling-evoked [Ca²⁺]_i increases under these conditions resulted from depletion of volume-sensitive intracellular Ca²⁺ stores by agonist-mediated Ca²⁺ mobilization. Our findings reconcile the apparently disparate conclusions of these previous studies in that we have shown that hepatocellular swelling elicits both intracellular Ca²⁺ store release and Ca²⁺ influx.

Although we have not yet definitively determined the underlying mechanisms that trigger the activation of these Ca²⁺ signaling pathways, our results suggest that swelling-evoked intracellular Ca²⁺ store release and Ca²⁺ influx are interdependent. In particular, Ca²⁺ store depletion with thapsigargin abolished the swelling-evoked increases in [Ca²⁺]_i in the presence of extracellular Ca²⁺. This supports the concept that the Ca²⁺ influx is mediated by channels that open in response to discharge of intracellular Ca²⁺ stores. However, whether swelling-mediated Ca²⁺ influx occurs through such store-operated Ca²⁺ channels or previously described mechanosensitive cation channels (13) requires further study.

Although the intracellular sites responsible for swelling-evoked Ca²⁺ mobilization have not been fully defined in this study, abolition of [Ca²⁺]_i increases by thapsigargin strongly implicates a contribution by the endoplasmic reticulum. This is consistent with observations in other cell types in which swelling has been shown to elicit release of endoplasmic reticulum Ca²⁺ stores via IP₃ receptors (28), ryanodine receptors (29), or by undefined pathways independent of either receptor type (30, 31). Although the mechanisms that underlie swelling-mediated hepatocellular Ca²⁺ store release are uncertain, the observation that prolonged hypotonic exposure increases intracellular levels of IP₃ in hepatocytes (32) raises the intriguing possibility that swelling elicits mobilization of intracellular Ca²⁺ consequent to activation of phospholipase C and IP₃ receptors. Indeed, liberation of diacylglycerol by osmosensitive stimulation of phospholipase C would account for previous findings concerning swelling-mediated activation of protein kinase C in HTC cells (17).

Our findings implicate an essential role for Ca²⁺ in the regulation of ion channels that are involved in hepatocellular volume recovery and suggest that the source of cellular Ca²⁺ is a key determinant in channel regulation. We found that inhibiting Ca²⁺ influx reduced the amplitude of swelling-activated K⁺ currents, whereas either chelation of intracellular Ca²⁺ with EGTA or intracellular Ca²⁺ store depletion with thapsigargin ablated such currents. These results indicate that Ca²⁺ influx and intracellular store release each contribute to activation of volume-sensitive K⁺ channels. This could occur in two ways. First, Ca²⁺ influx and Ca²⁺ mobilization could each contribute to increases in local [Ca²⁺]_i that activate a common population of K⁺ channels. Alternatively, there could be two populations of volume-sensitive K⁺ channels, one of which is activated in response to Ca²⁺ influx and one in response to intracellular store release. Consistent with this possibility, two distinct types of swelling-activated K⁺ channels have been reported in hepatocytes. A large conductance Ca²⁺-regulated K⁺ channel has been observed to open in response to cell swelling evoked by alanine uptake (33). A small conductance K⁺ channel has been identified on the basis of fluctuation analysis of whole-cell currents evoked by hypotonic challenge (34). The Ca²⁺ dependence of this channel has not been studied, however, and it remains to be determined whether either the large or small conductance K⁺ channels could be selectively activated by Ca²⁺ influx or Ca²⁺ store release in hepatocytes.

In contrast to K⁺ channels, our observations suggest that volume-sensitive Cl channels are distinctly regulated by the source of hepatocellular Ca²⁺. We found that intracellular dialysis with EGTA or exposure to thapsigargin inhibited swelling-activated Cl currents but that inhibition of Ca²⁺ influx had no effect. Our findings thus extend previous work (17) on the Ca²⁺ dependence of hepatocellular volume-sensitive Cl channels by demonstrating that channel activation requires release of intracellular Ca²⁺ stores but not Ca²⁺ influx. It is probable that the Cl channels are not directly Ca²⁺-activated, since hepatocellular swelling-activated Cl channels exhibit voltage dependence that is different from that of Ca²⁺-activated Cl channels (35). Rather, the Ca²⁺ dependence of swelling-activated Cl channels likely reflects a requirement for activation of the Ca²⁺-dependent protein kinase C (PKC) isoform PKC (17). If this is the case, our data would suggest that under conditions of swelling, the site of interaction between Ca²⁺ and PKC lies close to the site of Ca²⁺ release from the endoplasmic reticulum.

Consistent with a Ca²⁺ dependence for the activation of volume-sensitive K⁺ and Cl channels, the results reported here indicate a Ca²⁺ dependence for hepatocellular RVD as well. In light of previous observations (3, 6) that release of ATP and autocrine activation of P2 receptors are also required for volume recovery after HTC cell swelling, our current findings have several additional implications. First, in contrast to many purinergic signaling pathways, the volume-sensitive purinergic pathway does not employ Ca²⁺ as an intracellular mediator. If this were not the case, then apyrase and suramin (which block hepatocellular RVD) would have inhibited the observed swelling-elicited increases in [Ca²⁺]_i. Second, the site of swelling-elicited ATP release is likely to be in close proximity to the volume-sensitive P2 receptor. If not, then it is likely that activation of a more distant P2 receptor coupled to increases in [Ca²⁺]_i would have been observed. Taken together, our observations support a model in which two distinct signaling pathways are each required for volume recovery after hepatocellular swelling, a purinergic-independent Ca²⁺ signaling pathway and a Ca²⁺-independent purinergic pathway (Fig. 6).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Proposed model for hepatocellular volume regulation involving dual purinergic-independent Ca2+ signaling cascades and Ca2+-independent P2Y purinoreceptor signaling pathways. In this model, increases in [Ca2+]i are triggered by intracellular Ca2+ release from the endoplasmic reticulum (ER), which leads to extracellular Ca2+ influx and activation of K+ and Cl channels. Swelling also elicits ATP release, which stimulates P2Y receptors that are coupled to Cl channel activation via a Ca2+-independent mechanism. Both the Ca2+ and purinergic signaling pathways are required for volume recovery.

It is tempting to speculate how Ca²⁺-independent purinergic signaling occurs in the context of hepatocellular volume regulation. Our data suggest that it occurs via activation of P2Y receptors, as opposed to P2X receptors, which form Ca²⁺-permeable cation channels, the activation which would be expected to increase [Ca²⁺]_i. At least four P2Y receptors (P2Y1, P2Y2, P2Y4, and P2Y6) have been identified in hepatocytes (36, 37), and each of these can couple to trimeric G proteins that are linked to Ca²⁺-independent signaling pathways. For example, each of these purinoreceptor isoforms can activate in a Ca²⁺-independent manner pathways mediated by the GTPase Rho (11), which has been suggested to regulate volume-sensitive Cl channels in intestinal epithelial cells and vascular endothelium (38, 39). Moreover, other intracellular signaling effectors that regulate volume-sensitive ion channels, such as cyclic AMP and mitogen-activated protein kinases (40, 41), have been shown in some cases (e.g. in leukocytes, astrocytes, and pancreatic duct epithelial cells) to be coupled to P2Y receptor activation via Ca²⁺-independent pathways (9, 10, 12). It remains to be determined which P2Y receptor isoforms are activated in response to hepatocellular swelling and which downstream purinergic effectors participate in RVD.

In summary, our findings implicate an important role for Ca²⁺ in recovery from hepatocellular swelling and suggest that the endoplasmic reticulum participates in osmosensing and osmoregulation. Moreover, the observations reported here raise the possibility of a novel volume regulatory mechanism involving coincidence detection of independent signals arising from increases in cell Ca²⁺ and from stimulation of volume-sensitive P2Y receptors. Such a mechanism would provide a powerful means of tight control of liver cell volume and, by extension, maintenance of critical organ level functions.

	ACKNOWLEDGEMENTS

We thank Drs. Elisabeth Barfod and Joseph Brayden for helpful discussions and Drs. Gary Mawe and Mark Nelson for critical comments on the manuscript.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant DK47849 and the American Diabetes Association (both to S. D. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Burgess 414 MFU, University of Vermont, Burlington, VT 05401. Tel.: 802-847-5990; Fax: 802-847-4928; E-mail: steven.lidofsky@uvm.edu.

Published, JBC Papers in Press, June 18, 2001, DOI 10.1074/jbc.M102362200

	ABBREVIATIONS

The abbreviations used are: RVD, regulatory volume decrease; [Ca²⁺]_i, cytosolic Ca²⁺ concentration; IP₃, inositol trisphosphate; AM, acetoxymethyl ester; SES, standard extracellular solution; PKC, protein kinase C.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				G. Li and J. E. Olson Purinergic activation of anion conductance and osmolyte efflux in cultured rat hippocampal neurons Am J Physiol Cell Physiol, December 1, 2008; 295(6): C1550 - C1560. [Abstract] [Full Text] [PDF]

				P. Olivero, E. Leiva-Salcedo, L. Devoto, and A. Stutzin Activation of Cl- Channels by Human Chorionic Gonadotropin in Luteinized Granulosa Cells of the Human Ovary Modulates Progesterone Biosynthesis Endocrinology, September 1, 2008; 149(9): 4680 - 4687. [Abstract] [Full Text] [PDF]

				E. T. Barfod, A. L. Moore, M. W. Roe, and S. D. Lidofsky Ca2+-activated IK1 Channels Associate with Lipid Rafts upon Cell Swelling and Mediate Volume Recovery J. Biol. Chem., March 23, 2007; 282(12): 8984 - 8993. [Abstract] [Full Text] [PDF]

				E. T. Barfod, A. L. Moore, R. F. Melnick, and S. D. Lidofsky Src Regulates Distinct Pathways for Cell Volume Control through Vav and Phospholipase C{gamma} J. Biol. Chem., July 8, 2005; 280(27): 25548 - 25557. [Abstract] [Full Text] [PDF]

				A. A. Mongin and H. K. Kimelberg ATP regulates anion channel-mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms Am J Physiol Cell Physiol, January 1, 2005; 288(1): C204 - C213. [Abstract] [Full Text] [PDF]

				M. V. Espelt, P. N. Mut, G. Amodeo, G. Krumschnabel, and P. J. Schwarzbaum Volumetric and ionic responses of goldfish hepatocytes to anisotonic exposure and energetic limitation J. Exp. Biol., February 1, 2003; 206(3): 513 - 522. [Abstract] [Full Text] [PDF]

				P. R. Junankar, A. Karjalainen, and K. Kirk The Role of P2Y1 Purinergic Receptors and Cytosolic Ca2+ in Hypotonically Activated Osmolyte Efflux from a Rat Hepatoma Cell Line J. Biol. Chem., October 18, 2002; 277(43): 40324 - 40334. [Abstract] [Full Text] [PDF]

				A. L. Moore, M. W. Roe, R. F. Melnick, and S. D. Lidofsky Calcium Mobilization Evoked by Hepatocellular Swelling Is Linked to Activation of Phospholipase Cgamma J. Biol. Chem., September 6, 2002; 277(37): 34030 - 34035. [Abstract] [Full Text] [PDF]

				A. A. Mongin and H. K. Kimelberg ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes Am J Physiol Cell Physiol, August 1, 2002; 283(2): C569 - C578. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/33/30871    most recent M102362200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Roe, M. W. Articles by Lidofsky, S. D. Search for Related Content PubMed PubMed Citation Articles by Roe, M. W. Articles by Lidofsky, S. D. Social Bookmarking                      What's this?