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Am J Physiol Gastrointest Liver Physiol 290: G7-G13, 2006. First published August 25, 2005; doi:10.1152/ajpgi.00337.2005
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LIVER AND BILIARY TRACT

[Ca2+]i-independent contractile force generation by rat hepatic stellate cells in response to endothelin-1

Andrew C. Melton,1,2 Anuj Datta,1 and Hal F. Yee, Jr.1,2

1Liver Center and Department of Medicine, University of California San Francisco, San Francisco; and 2Department of Physiology, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, California

Submitted 20 July 2005 ; accepted in final form 22 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The contractile force generated by hepatic stellate cells in response to endothelin-1 contributes to sinusoidal blood flow regulation and hepatic fibrosis. This study's aim was to directly test the widely held view that changes in cytosolic Ca2+ concentration ([Ca2+]i) mediate stellate cell force generation. Contractile force generation by primary cultures of rat hepatic stellate cells grown in three-dimensional collagen gels was directly and quantitatively measured using a force transducer. Stellate cell [Ca2+]i, myosin activation, and migration were quantified using standard techniques. [Ca2+]i was modulated using ionomycin, BAPTA, KCl, and removal of extracellular Ca2+. Removal of extracellular Ca2+ did not alter endothelin-1-stimulated force development or [Ca2+]i. Ionomycin, a Ca2+ ionophore, triggered an increase in [Ca2+]i that was three times greater than that stimulated by endothelin-1, but only induced 16% of the force and 38% of the myosin regulatory light chain (MLC) phosphorylation induced by endothelin-1. Physiological increases in [Ca2+]i induced by hyperkalemia had no effect on contractile force. Loading BAPTA, a Ca2+ chelator, in stellate cells completely blocked endothelin-1-induced increases in [Ca2+]i but had no effect on endothelin-1-stimulated force generation or MLC phosphorylation. In contrast, Y-27632, a selective rho-associated kinase inhibitor, inhibited endothelin-1-stimulated force generation by at least 70% and MLC phosphorylation by at least 80%. Taken together, these observations indicate that changes in [Ca2+]i are neither necessary nor sufficient for contractile force generation by rat stellate cells. Our results challenge the current model of contractile regulation in hepatic stellate cells and have important implications for our understanding of hepatic pathophysiology.

myosin light chain phosphorylation; myosin light chain kinase; rho-associated kinase; cytosolic calcium concentration

MOUNTING EVIDENCE INDICATES that generation of contractile force by hepatic stellate cells in response to endothelin-1 contributes to the regulation of sinusoidal blood flow and the development of fibrosis (1, 7, 9, 17, 23, 29, 37). Thus understanding the signal transduction pathways that control stellate cell force generation has considerable importance to defining the pathogenesis of and developing treatments for diverse disorders of the liver. At present, it is generally recognized that changes in cytosolic Ca2+ concentration ([Ca2+]i) mediate force generation by stellate cells (2, 3, 8, 15, 20, 26–29, 31), but this notion has heretofore not been directly tested.

Several lines of evidence support a critical role for Ca2+ in the regulation of stellate cell force generation. First, agonists such as thrombin, ANG II, and endothelin-1 that activate Ca2+-signaling pathways in stellate cells (3, 15, 26, 27) also induce stellate cell contraction (3, 15, 27, 30, 32, 38). Second, in stellate cells, expression of plasma membrane Ca2+ channels, magnitude of Ca2+ influx through these channels, and [Ca2+]i each correlate with the proportion of stellate cells that exhibits reductions in cell surface area (2, 8). Third, contractile force generated by stellate cells is mediated by myosin II (14, 32, 43), a protein known to be activated by Ca2+-dependent phosphorylation of the myosin regulatory light chain (MLC) in other cell types (12, 21, 34). Taken together, these studies have provided persuasive, but circumstantial, evidence to support the prevailing belief that changes in [Ca2+]i govern contractile force generation in stellate cells.

The aim of the present study was to directly test the hypothesis that the generation of contractile force by stellate cells in response to endothelin-1 is mediated by alterations in [Ca2+]i. Our results suggest that changes in [Ca2+]i are neither necessary for endothelin-1-stimulated contractile force generation nor sufficient to induce a characteristic contractile response. These findings challenge a widely held model of stellate cell contractile regulation and have important implications for understanding the pathophysiological mechanisms underlying cirrhosis.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Materials. Dulbecco's modification of Eagle's medium (DMEM), FBS, L-glutamine, penicillin/streptomycin, nonessential amino acids, sodium pyruvate, and trypsin-EDTA were purchased from Irvine Scientific (Irvine, CA). Fura 2-AM and BAPTA-AM were obtained from Molecular Probes (Portland, OR). Type I collagen (rat tail) was obtained from Upstate (Waltham, MA), endothelin-1 and Y-27632 were from Calbiochem (La Jolla, CA), and ionomycin was from Sigma (St. Louis, MO). All other reagents were obtained from Fisher (Hampton, NH).

Cell isolation and culture. Rat hepatic stellate cells were isolated and grown in primary culture as described (6, 39). After isolation by density gradient centrifugation, cells were grown on plastic dishes in DMEM supplemented with 10% FBS, 200 mM L-glutamine, 50,000 units penicillin G, 50,000 units streptomycin, 1% nonessential amino acids, and 0.5 mM sodium pyruvate at 37°C in 5% CO2. Media-containing serum was changed every 3 days.

Measurement of [Ca2+]i. After 7 days in culture, stellate cells were washed with serum-free DMEM and removed from the dishes by adding 1 ml of five times trypsin-EDTA for 1 min at 37°C. The cells were then placed on 9 x 22 mm cover glasses at a density of 6,000 cells/glass. Cells were next washed two times with Ca2+-containing buffer (in mM: 135 NaCl, 5 KCl, 0.8 MgCl, 1.2 CaCl2, 0.8 NaH2PO4, 10 HEPES, and 5 glucose, pH 7.4) or Ca2+-free buffer (in mM: 135 NaCl, 5 KCl, 3 MgCl, 0.8 NaH2PO4, 10 HEPES, 5 glucose, and 2 EGTA, pH 7.4) and incubated with 5 µM of the Ca2+-sensitive dye fura 2-AM (diluted in Ca2+-containing or Ca2+-free buffer) for 30 min at 25°C. Extracellular fura 2-AM was then removed by washing two times with either Ca2+-containing or Ca2+-free buffer. Cells were then placed in a fluorospectrophotometer (F-2000; Hitachi) with continuous stirring of either Ca2+-containing or Ca2+-free buffer at 37°C. Agonists and antagonists were added directly to the buffer in the fluorospectrophotometer. Fluorescence emission at 510 nm was recorded every 0.5 s after excitation at 340- and 380-nm wavelengths. {Delta}F340/F380 is the change in the fluorescence emission ratio (340 nm/380 nm) relative to the average baseline fluorescence emission ratio before agonist treatment. Results presented include data collected from at least three independent stellate cell isolations. Statistical significance was determined using Student's t-test.

Quantitation of contractile force generation. The force generated by stellate cells within an elastic three-dimensional collagen gel was determined as described (38). Stellate cells were removed from the dishes (as described above) after 7–10 days in culture and suspended in type I collagen at a density of 5 x 105 cells/gel. After 3–4 days, collagen gels were attached to an isometric force transducer (Harvard Apparatus, Holliston, MA), stretched to their original length, and placed in a 37°C organ bath filled with either Ca2+-containing or Ca2+-free buffer. Agonists and antagonists were added directly to the bath. Changes in isometric tension were recorded through an analog-to-digital converter (DAQ-500; National Instruments, Austin, TX) attached to a PC-type computer (Dell, Round Rock, TX) running data acquisition software (Virtual Bench; National Instruments). Results presented include data collected from at least three independent stellate cell isolations. Statistical significance was determined using Student's t-test.

Measurement of wound-induced migration. Wound-induced migration was determined as previously described (36). Stellate cells were isolated, plated on glass cover slips in supplemented DMEM, and grown to confluency. Cell-free wounds (width = 367 ± 20 µm; n = 18 wounds) were created by dragging the tip of a sterile 20-µl plastic pipette tip across the cover slip. After wounding, media was changed to 0.5% FBS-supplemented DMEM containing carrier, endothelin-1 with carrier, or endothelin-1 with BAPTA for 24 h. Digital images of the wounds were acquired immediately after wounding and 24 h later. Wound-induced migration was determined by measuring the percent change in the surface area of the cell-free wounds.

Determination of MLC phosphorylation. MLC phosphorylation was measured as described (41, 44). After 7 days in primary culture, stellate cells were removed from the dishes (as described above) and placed on plastic dishes. Stellate cells were equilibrated in either Ca2+-containing or Ca2+-free buffer, and agonists or antagonists were added directly to the dish. Protein samples were suspended in 9 M urea buffer, loaded on a 10% acrylamide-glycerol gel, and then transferred to nitrocellulose. Immunoblot was performed with an antibody directed against MLC (no. 9828; see Ref. 36). Phosphorylated and unphosphorylated forms of MLC were detected using the enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ). This immunoblot method permitted determination of MLC phosphorylation as the magnitude of the phosphorylated MLC signal as a percentage of the magnitude of the unphosphorylated and phosphorylated MLC signal from a given sample lane [i.e., phosphorylated MLC signal/(unphosphorylated MLC signal + phosphorylated MLC signal)]. Results presented include data collected from at least three independent stellate cell isolations. Statistical significance was determined using Student's t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Extracellular Ca2+ is not necessary for endothelin-1-stimulated contractile force generation. Previous work has suggested that influx of extracellular Ca2+ triggers contractile force generation by stellate cells (2, 8, 13, 20). If extracellular Ca2+ induces contractile force generation, then we would expect removal of extracellular Ca2+ to abolish or reduce the force generated in response to endothelin-1 (2 nM). Removal of extracellular Ca2+ did not alter endothelin-1-stimulated contractile force development. As shown in Fig. 1A, stellate cells treated with endothelin-1 in Ca2+-free buffer generated the same amount of force as cells treated with endothelin-1 in Ca2+-containing media. These observations suggest that extracellular Ca2+ is not necessary for endothelin-1-stimulated contractile force generation by stellate cells.



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  		Fig. 1. Extracellular Ca2+ is not necessary for endothelin-1 (ET-1)-induced contractile force generation and Ca2+ signaling in stellate cells. A: contractile force generated by stellate cells in response to endothelin-1 (2 nM) in Ca2+-containing buffer (left) or Ca2+-free buffer (right). Histogram results represent the endothelin-1-induced force generated as a percentage of force generated by endothelin-1 in Ca2+-containing buffer within the same stellate cell isolation. Each bar represents a mean ± SE (n = 5 experiments). Representative contractile force tracings for each respective experimental condition are depicted above each bar. Arrowheads indicate the time when endothelin-1 was added to the media. B: change in cytosolic Ca2+ concentration ([Ca2+]i) in response to endothelin-1 (2 nM) in Ca2+-containing buffer (n = 8) and Ca2+-free buffer (n = 5) presented as the change in the fluorescence emission ratio (340 nm/380 nm) relative to the average baseline fluorescence emission ratio before agonist treatment ({Delta}F340/F380). Each point represents the mean ± SE. Arrowheads indicate the time when endothelin-1 was added.

 
To further investigate this unexpected result, we determined whether extracellular Ca2+ was necessary for endothelin-1-mediated [Ca2+]i mobilization in stellate cells. We measured [Ca2+]i in the presence and absence of extracellular Ca2+. Endothelin-1 stimulated a 0.19 ± 0.05 {Delta}F340/F380 in Ca2+-containing media, and a 0.20 ± 0.06 {Delta}F340/F380 in Ca2+-free buffer (Fig. 1B). These data, that endothelin-1 triggers normal elevations in [Ca2+]i in the absence of extracellular Ca2+, add additional support to the conclusion that extracellular Ca2+ is not required for endothelin-1-stimulated force generation.

Extracellular influx of Ca2+ is not sufficient to induce generation of contractile force. We next tested whether influx of extracellular Ca2+ is sufficient to induce generation of contractile force by stellate cells using the Ca2+-selective ionophore ionomycin. Ionomycin (1 µM) induced a robust increase in [Ca2+]i (0.62 ± 0.06 {Delta}F340/F380) within 45 s of addition (Fig. 2A) that was more than three times larger than that stimulated by endothelin-1 (2 nM). Despite causing a markedly greater rise in [Ca2+]i, ionomycin (1 µM) only induced 38 ± 12 dyn of force (Fig. 2B). In contrast, endothelin-1 (2 nM) produced 232 ± 28 dyn of force (Fig. 2B). These results suggest that even a superphysiological increase in [Ca2+]i is insufficient to stimulate a magnitude of contractile force similar to that observed in response to endothelin-1.



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  		Fig. 2. Ca2+ influx is not sufficient for contractile force generation. A: representative [Ca2+]i tracings in stellate cells after treatment with either endothelin-1 (2 nM; {circ}; n = 8) or ionomycin (Iono; 1 µM; {bullet}; n = 8). Arrowhead indicates the time when endothelin-1 or ionomycin was added. B: representative contractile force tracings after treatment with either endothelin-1 (2 nM; {circ}; n = 10) or ionomycin (1 µM; {bullet}; n = 3). Arrowhead indicates the time when endothelin-1 or ionomycin was added. C: representative contractile force tracing of stellate cells treated with hyperkalemic buffer (100 mM KCl; n = 7). The recording gap just before addition of hyperkalemic buffer is an artifact resulting from washout of Ca2+-containing buffer and replacement with hyperkalemic Ca2+-containing media. Arrowhead indicates the time when hyperkalemic buffer was added.

 
Stellate cell exposure to 50 and 100 mM KCl has been shown to increase [Ca2+]i by opening voltage-operated Ca2+ channels (VOCC; see Ref. 2). To investigate whether elevations in [Ca2+]i through VOCC are sufficient to induce contractile force generation, we transiently depolarized the stellate cell plasma membrane with hyperkalemic media. We observed no change in the force generated by stellate cells after exposure to buffer containing 100 mM KCl (Fig. 2C). These findings indicate that extracellular influx of Ca2+ is not capable of inducing contractile force generation by stellate cells.

Increases in [Ca2+]i are not necessary for endothelin-1-induced stellate cell contractile force generation. To test whether elevations in [Ca2+]i mediate contractile force generation in response to endothelin-1, stellate cells were loaded with the Ca2+-selective chelator BAPTA. Endothelin-1 (2 nM)-stimulated elevations in [Ca2+]i were completely abrogated in BAPTA-loaded cells (Fig. 3A). Stellate cells loaded with BAPTA generated the same magnitude of force in response to endothelin-1 (2 nM) as cells not loaded with BAPTA (Fig. 3B). These observations suggest that increases in [Ca2+]i are not necessary for endothelin-1-induced stellate cell contractile force generation.



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  		Fig. 3. Ca2+ chelation by BAPTA has no effect on endothelin-1-induced stellate cell contractile force generation. A: change in [Ca2+]i in response to endothelin-1 (2 nM) in stellate cells not loaded (left; n = 8) and loaded with 10 µM BAPTA (right; n = 4) presented as {Delta}F340/F380. Stellate cells were loaded with BAPTA for 30 min before endothelin-1 treatment. Each point represents the mean ± SE. Arrowheads indicate the time when endothelin-1 was added. B: contractile force generated by stellate cells in response to endothelin-1 (2 nM) in stellate cells not loaded (left) and loaded with 10 µM BAPTA for 30 min (right). Histogram results represent the endothelin-1-induced force generated as a percentage of force generated by endothelin-1 in cells not loaded with BAPTA within the same stellate cell isolation. Each bar represents a mean ± SE (n = 3). Representative contractile force tracings for each respective experimental condition are depicted above each bar. Arrowheads indicate the time when endothelin-1 was added to the media.

 
As a positive control to verify a functional effect of BAPTA, we studied the effects of BAPTA loading on endothelin-1-stimulated wound-induced migration of stellate cells. First, we verified our prior observation (36) that endothelin-1 enhances the rate of wound-induced migration (Fig. 4). Next, we found that loading stellate cells with BAPTA completely abolished endothelin-1-stimulated wound-induced migration (Fig. 4). The finding that loading stellate cells with BAPTA blocks endothelin-1-induced increases in [Ca2+]i and migration, but does not alter endothelin-1-stimulated force generation, further supports the conclusion that increases in [Ca2+]i are not necessary for contractile force generation.



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  		Fig. 4. Loading stellate cells with BAPTA abolishes endothelin-1-stimulated wound-induced migration. Wound-induced migration of stellate cells in the absence of treatment (left), in response to endothelin-1 (2 nM; middle), and in response to endothelin-1 (2 nM) after loading with 10 µM BAPTA (right). Endothelin-1 and BAPTA were present in the buffer for the duration of the experiment. Histogram results represent the percentage reduction in wound area after 24 h. Note that the apparent increase in wound area demonstrated with ET-1 + BAPTA treatment results from retraction of the wound edge that occurs early after wounding even under control conditions, as we reported (36). Each bar represents a mean ± SE (n = 6).

 
Elevations in [Ca2+]i are not required for myosin activation. The force of cellular contraction is powered by myosin II, which is activated by phosphorylation of the MLC (12, 21, 34). Therefore, to confirm that alterations in [Ca2+]i do not mediate generation of contractile force in response to endothelin-1, we also quantified MLC phosphorylation in response to endothelin-1. Endothelin-1 (2 nM) stimulated a 37 ± 2% increase in MLC phosphorylation that was sustained for at least 10 min (Fig. 5A). In comparison, strongly increasing [Ca2+]i with ionomycin (1 µM) caused only a 14 ± 4% increase in MLC phosphorylation that rapidly declined below baseline (Fig. 5A). MLC phosphorylation in response to endothelin-1 was not altered significantly by blocking changes in [Ca2+]i with BAPTA (Fig. 5B). These results provide additional evidence to support the concept that changes in stellate cell [Ca2+]i play little role, if any, in the regulation of myosin-dependent force generation in response to endothelin-1.



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  		Fig. 5. Changes in [Ca2+]i are neither sufficient nor necessary for characteristic myosin regulatory light chain (MLC) phosphorylation. A: time course of MLC phosphorylation after treatment with endothelin-1 (2 nM; {bullet}; n = 3) or ionomycin (1 µM; {blacklozenge}; n = 5). Data are presented as the change in the fraction of myosin that is phosphorylated divided by the basal fraction of myosin that is phosphorylated, multiplied by 100. Inset: representative immunoblot demonstrating unphosphorylated (No P), monophosphorylated (Mono), and diphosphorylated (Di) forms of MLC before and 0.5, 2, and 10 min after exposure to 2 nM endothelin-1. B: MLC phosphorylation in stellate cells in the absence of treatment (basal; left), in response to endothelin-1 (2 nM; middle), and in response to endothelin-1 (2 nM) after loading with 10 µM BAPTA for 30 min (right). Histogram results represent the change in the fraction of myosin that is phosphorylated divided by the basal fraction of myosin that is phosphorylated, multiplied by 100, after 2 min of endothelin-1 (2 nM) treatment. Each bar represents a mean ± SE (n = 3). *P < 0.05 relative to basal levels (carrier-only treated cells) of MLC phosphorylation.

 
A rho-associated kinase signaling pathway mediates contractile force generation and MLC phosphorylation. If elevations in [Ca2+]i mediate endothelin-1-stimulated contractile force generation, then inhibition of alternative signaling pathways should have little or no effect on force. To test this, we employed Y-27632, a selective inhibitor of rho-associated kinase (5, 40). Y-27632 dramatically decreased endothelin-1-stimulated contractile force generation by 34% at 50 µM and 70% at 100 µM (Fig. 6A). Y-27632 also blocked endothelin-1-stimulated MLC phosphorylation by 69% at 50 µM and 81% at 100 µM (Fig. 6B). These data suggest that the Ca2+-independent rho-associated kinase signaling pathway contributes substantially to regulation of stellate cell myosin activation and force generation. These results suggest a rho-associated kinase signaling pathway as the mechanism through which endothelin-1 controls stellate cell force generation independent of [Ca2+]i.



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  		Fig. 6. Rho-associated kinase inhibition blocks endothelin-1-induced contractile force generation and MLC phosphorylation. A: effect of 30 min of exposure to different concentrations of Y-27632 (selective rho-associated kinase inhibitor) on contractile force generation in response to 2 nM endothelin-1. Histogram results represent maximal contractile force generation after Y-27632 treatment as a percentage of maximal force generation in response to endothelin-1 alone. Each bar represents a mean ± SE (0 µM Y-27632, n = 8; 10 µM Y-27632, n = 3; 50 µM Y-27632, n = 3; 100 µM Y-27632, n = 3). *P < 0.05 relative to carrier + 2 nM endothelin-1. B: effect of 30 min of exposure to different concentrations of Y-27632 on MLC phosphorylation in response to 2 nM endothelin-1 stimulation for 2 min. Histogram results represent the percentage of MLC phosphorylation after Y-27632 treatment compared with endothelin-1-stimulated MLC phosphorylation alone. Each bar represents a mean ± SE (0 µM Y-27632, n = 3; 10 µM Y-27632, n = 3; 50 µM Y-27632, n = 3; 100 µM Y-27632, n = 2). *P < 0.05 relative to carrier + 2 nM endothelin-1.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The principal aim of this study was to directly test the widely held belief that changes in [Ca2+]i mediate contractile force generation by stellate cells in response to endothelin-1 (15, 26, 28, 29, 45). The key findings of this study were that 1) increases in [Ca2+]i were not necessary for endothelin-1-stimulated force generation, 2) physiological increases in [Ca2+]i were not sufficient to initiate contractile force generation, and 3) inhibition of Ca2+-independent rho-associated kinase signaling pathways blocked most endothelin-1-stimulated force generation. These results, which strongly suggest that contractile force generation by hepatic stellate cells in response to endothelin-1 occurs independent of changes in [Ca2+]i, provide compelling evidence that directly refutes current understanding of contractile regulation in hepatic stellate cells.

The hypothesis that alterations in [Ca2+]i mediate contractile force generation by stellate cells was first postulated based on observations from several laboratories that substances that activate Ca2+-signaling pathways in stellate cells, such as endothelin-1, thrombin, and ANG II (3, 15, 26, 27), also provoke stellate cell contraction (3, 15, 27, 30, 32). However, none of these prior studies directly investigated the contribution of [Ca2+]i to stellate cell contractile force generation by modulating [Ca2+]i. In this study, we found that increasing [Ca2+]i by depolarizing the plasma membrane did not induce stellate cell force generation. Moreover, we observed that even superphysiological increases in [Ca2+]i triggered by ionomycin were capable of stimulating only a small increase in contractile force. We also demonstrated that abolishing endothelin-1-induced elevations in [Ca2+]i with BAPTA had no effect on stellate cell force generation in response to endothelin-1. In addition to quantifying the effects of BAPTA on [Ca2+]i using a Ca2+-sensing dye, we verified a functional effect of loading stellate cells with BAPTA by showing that this Ca2+ chelator completely abrogated wound-induced migration. By modulating [Ca2+]i, we have demonstrated that changes in [Ca2+]i are neither necessary for, nor sufficient to, induce contractile force generation by stellate cells.

It has been reported that L-type VOCC mediate rat hepatic stellate cell Ca2+ influx (2, 22) and contraction (2). In the current study, we found that removal of extracellular Ca2+ had no effect on contractile force generation in response to endothelin-1. In fact, extracellular Ca2+ was not even necessary for endothelin-1-stimulated increases in [Ca2+]i. This observation is consistent with prior studies of endothelial, epithelial, and smooth muscle cells, in which activation of G protein-coupled receptors results in Ca2+ release from intracellular stores (4, 24, 33). Moreover, as discussed above, we observed that triggering influxes of Ca2+ with membrane depolarization or ionomycin had no and little effect on contractile force, respectively. Discrepancies between the current findings and prior studies may be explained by substantial differences in the methods used to assay contractile force. We quantified contraction by directly measuring the force generated by stellate cells grown in a three-dimensional collagen gel with a sensitive force transducer. In the previous studies, investigators assayed contraction by determining the fraction of stellate cells grown on cover slips that demonstrated a reduction in cell area of >8%. Although changes in stellate cell surface area may correlate with contractile force generation, such changes could instead reflect alterations in cellular adhesion or volume, three-dimensional remodeling of the cytoskeleton, or focusing artifacts. Moreover, the physiological relevance of an 8% diminution in stellate cell area is unclear. Based on the current and published data, we conclude that, although Ca2+ influx through Ca2+ channels may mediate stellate cell morphology, Ca2+ influx does not appear to play a role in contractile force generation in stellate cells.

We previously demonstrated that myosin II couples endothelin-1 to contractile force generation in stellate cells (32). In cell types, such as smooth muscle cells, in which contractile regulation has been thoroughly characterized, it has been shown that Ca2+-activated myosin light chain kinase activates myosin through the phosphorylation of the MLC (12, 21, 34). Thus it has previously been presumed that endothelin-1-induced increases in [Ca2+]i should activate myosin II and subsequently stimulate contractile force generation by stellate cells (29). However, several laboratories have shown over the past decade that contractile force generation by certain nonmuscle cell types, including fibroblasts and endothelial cells, is controlled predominantly by rho-associated kinase signaling pathways, with Ca2+ signaling pathways playing a subordinate role (16, 25, 34, 46). Furthermore, recent data indicate that pathways that signal through rho-associated kinase contribute to the regulation of stellate cell morphology (42–44), migration (14, 36, 43), and apoptosis (10), and also to the development of hepatic fibrosis (14, 19, 35). New studies also suggest that, in smooth muscle, Ca2+ entry specifically through VOCC stimulates contraction in part through rho-associated kinase signaling (11, 18). However, our data indicate that this signaling event is unlikely to occur in stellate cells based on the finding that hyperkalemia-induced membrane depolarization did not induce changes in contractile force generation.

In the current study, we observed that the selective rho-associated kinase inhibitor Y-27632 is a powerful inhibitor of endothelin-1-stimulated MLC phosphorylation and force generation. The effects of Y-27632 were observed in the low micromolar range (50–100 µM), which is similar to the concentrations of this agent that have been shown to block collagen lattice shrinkage by rat hepatic stellate cells (43) and contraction of chicken fibroblasts (46). It is interesting that the effective inhibitory constant (Ki) of Y-27632 in intact cells is >100 times greater than the reported Ki (140 nM) determined in vitro (40). The reason for this reproducible finding is unknown. Therefore, our results suggest that, in contrast to certain cell types, such as smooth muscle cells, where Ca2+ mediates force generation, stellate cell contraction is mediated primarily by rho-associated kinase signaling pathways.

Based on these findings, we propose a new scheme for the regulation of contractile force generation by rat hepatic stellate cells. In this model, endothelin-1 stimulates contractile force generation by stellate cells via a pathway signaling through rho-associated kinase rather than Ca2+. This revision in our knowledge has major implications for our understanding of sinusoidal blood flow and hepatic fibrosis, since myosin-dependent contraction is thought to play key roles in stellate cell regulation of sinusoidal resistance (29, 32, 38), chemotaxis (14, 36, 43), cell survival (10), and extracellular matrix contraction (7). Finally, our data, which suggest that stellate cell contraction and smooth muscle cell contraction are predominantly regulated by different signaling pathways, offer the potential for the development of novel therapeutic agents that selectively target the generation of contractile force by stellate cells.


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 ABSTRACT
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H. F. Yee, Jr. was supported by NIH Grant R01 DK-61532 and A. C. Melton by the NIH-funded Training Program in Alcoholic Liver and Pancreatic Diseases Grant T32 AA-07578. A. C. Melton and A. Datta were supported by Hefni Fellowships from the Technical Training Foundation.


   ACKNOWLEDGMENTS
 
We thank the National Institutes of Health (NIH)-supported Cell and Tissue Biology Core Facility of the University of California San Francisco Liver Center (DK-26743) and the NIH-supported Nonparenchymal Liver Cell Core of the Research Center for Alcoholic Liver and Pancreatic Diseases at USC (R24AA-12885) for providing hepatic stellate cells in primary culture. We thank Jiu Han for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. F. Yee, UCSF Liver Center Lab, San Francisco General Hospital, Bldg. 40, Rm. 4102, 1001 Potrero Ave., San Francisco, CA 94110 (e-mail: hyee{at}medsfgh.ucsf.edu)

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.


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 REFERENCES
 

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