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Am J Physiol Gastrointest Liver Physiol 290: G535-G542, 2006. First published November 3, 2005; doi:10.1152/ajpgi.00297.2005
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LIVER AND BILIARY TRACT

Defects in cGMP-PKG pathway contribute to impaired NO-dependent responses in hepatic stellate cells upon activation

Roman E. Perri,1 Daniel A. Langer,1 Suvro Chatterjee,1 Simon J. Gibbons,2 Jay Gadgil,1 Sheng Cao,1 Gianrico Farrugia,2 and Vijay H. Shah1

1Gastroenterology Research Unit, Department of Physiology, and Tumor Biology Program, and 2Enteric Neuroscience Program, Mayo Clinic, Rochester, Minnesota

Submitted 1 July 2005 ; accepted in final form 20 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
NO antagonizes hepatic stellate cell (HSC) contraction, although activated HSC in cirrhosis demonstrate impaired responses to NO. Decreased NO responses in activated HSC and mechanisms by which NO affects activated HSC remain incompletely understood. In normal rat HSC, the NO donor diethylamine NONOate (DEAN) significantly increased cGMP production and reduced serum-induced contraction by 25%. The guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) abolished 50% of DEAN effects, whereas the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) reiterated half the observed DEAN response, suggesting both cGMP-dependent protein kinase G (PKG)-dependent and -independent mechanisms of NO-mediated antagonism of normal HSC contraction. However, NO donors did not increase cGMP production from in vivo activated HSC from bile duct-ligated rats and showed alterations in intracellular Ca2+ accumulation suggesting defective cGMP-dependent effector pathways. The LX-2 cell line also demonstrated lack of cGMP generation in response to NO and a lack of effect of ODQ and 8-BrcGMP in modulating the NO response. However, cGMP-independent effects in response to NO were maintained in LX-2 and were associated with S-nitrosylation of proteins, an effect reiterated in primary HSC. Adenovirus-based overexpression of PKG significantly attenuated contraction of LX-2 by 25% in response to 8-BrcGMP. In summary, these studies demonstrate that NO affects HSC through cGMP-dependent and -independent pathways. The HSC activation process is associated with maintenance of cGMP-independent actions of NO but defects in cGMP-PKG-dependent NO signaling that are improved by PKG gene delivery in LX-2 cells. Activating targets downstream from NO-cGMP in activated HSC may represent a novel therapeutic target for portal hypertension.

protein kinase G; guanylate cyclase

NO REGULATES HEPATIC STELLATE cell (HSC) function in important ways including inhibition of HSC proliferation, migration, and contraction, with the latter function being particularly relevant to sinusoidal blood flow regulation (5). In prototypical vascular beds, endothelial cells produce NO via the endothelial NO synthase (eNOS) isoform (30) with NO diffusing abluminally to adjacent contractile cells to promote vasodilation. This occurs in large part by NO binding of the heme moiety of soluble guanylate cyclase (sGC), thereby activating cGMP generation and inducing a downstream signaling cascade. This cascade includes activation of a kinase downstream from cGMP, protein kinase G (PKG), and subsequent regulation of intracellular Ca2+. Effects of intracellular Ca2+ accumulation are complex, as global increases contribute to cell contraction but focal increases can actually cause relaxation through hyperpolarization mediated by the large-conductance Ca2+-activated K+ (KCa) channel (19, 36). NO effects on contractile cells occur independent of cGMP as well, including through S-nitrosylation, a posttranslational regulatory modification of protein cysteine thiols by NO (32, 33). Prior studies demonstrated that NO antagonizes contraction in activated HSC, as in other contractile cells (15); however, the pathways by which NO exerts its dynamic effects on HSC are less well characterized compared with those of other contractile cells.

Portal hypertension is characterized by alterations in paracrine and endocrine vasodilatory and vasoconstricting factors that result in hemodynamic disturbances in various circulatory beds (23). Increases in intrahepatic vascular resistance contribute to portal hypertension, and in this regard, the process of HSC activation from a quiescent to a proliferative, contractile phenotype has been implicated in the pathway of increased hepatic resistance (26, 29). Owing to the well-documented deficiency of NO generation from liver endothelial cells that contributes to increased intrahepatic vascular resistance in cirrhosis and portal hypertension (11, 25, 31), organic nitrates constitute a rational therapy to antagonize the contractile HSC phenotype. However, in experimental models, it has been demonstrated that nitrate compounds that are typically potent vasorelaxing agents in normal liver possess an impaired vasorelaxant capacity in cirrhotic liver (6). For example, in the isolated, perfused rat liver system, livers from animals with cirrhosis evidenced diminished vasodilation in response to the organic nitrate nitroglycerin compared with control animals (6). Because of the clinical utility of NO donors as a treatment option in portal hypertension (3), the mechanisms responsible for impairment of NO responses after HSC activation are of both scientific and clinical significance.

In the present studies, primary rat HSC isolates from normal and injured liver were utilized in conjunction with complementary studies in human HSC and an activated HSC cell line, LX-2, to 1) delineate the relative roles of cGMP-dependent and cGMP-independent pathways in the dynamic responses of NO in normal HSC and 2) better characterize mechanisms of deficient NO responses and signaling mechanisms in HSC upon activation such as that which occurs in liver disease. We have found that normal HSC exhibit significant attenuation of contraction in response to complementary NO donors through both cGMP-dependent and cGMP-independent mechanisms. However, in vivo activated HSC isolated from bile duct-ligated (BDL) liver demonstrate impaired cGMP generation in response to NO donors and alterations in intracellular Ca2+ accumulation. This defective phenotype is recapitulated in the highly activated LX-2 cell line, in which NO effects are largely through cGMP-independent pathways including protein S-nitrosylation. Additionally, the defective cGMP-dependent responses are reconstituted in LX-2 by adenoviral PKG overexpression. These studies support the concept that, in HSC, targets downstream of NO including sGC and PKG may be defective and may constitute new targets for future therapy of portal hypertension.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation and culture. Animals were maintained with standard chow and water diet and received care as per institutional guidelines. All animal studies were submitted, approved, and reviewed by the Mayo Clinic Institutional Animal Care and Use Committee. HSC were isolated from both sham-operated and 4-wk BDL male Sprague-Dawley rats as we described previously (12, 17). In brief, rat livers were perfusion digested with collagenase and pronase. Nonparenchymal and parenchymal cell fractions were separated by centrifugation, and HSC were further purified by density-gradient centrifugation with an Accudenz gradient containing a 15.6% layer and an 8.2% layer. The gradient was centrifuged at 20,000 rpm for 25 min at 20°C. The band located above the 8.2% Accudenz layer was retrieved, and the cells were resuspended in culture medium and plated on collagen-coated plasticware. Cell purity routinely approximated 95% at the time of study, as assessed by expression of smooth muscle actin by immunofluorescence, which was performed in representative preparations. Upon confluence, which occurred at ~7–10 days, cells were passed onto collagen lattices for contraction assays or, alternatively, passed into wells for cGMP assay. We were unable to demonstrate significant contractility of primary HSC isolated from normal rats under the influence of endothelin-1 within the first 5 days after isolation, as examined by analysis of cell area by individual cell microscopy (n = 15). However, HSC that had reached confluence at 7–10 days after isolation were readily contractile when passaged onto collagen lattices. LX-2 cells (37), kindly provided by Scott Friedman (Mount Sinai School of Medicine, New York, NY), were used as a model of activated HSC to complement in vivo activated BDL HSC for specific experimental protocols (i.e., transfection) in which experiments from primary HSC were not technically feasible. These spontaneously immortalized human HSC (HHSC) have been extensively characterized as a cell type sharing many phenotypic features with the highly activated form of HSC (17, 37). Nitrosylation experiments were also performed in HHSC isolated from human resection specimens and used at less than passage 5 (Sciencell, San Diego, CA).

Adenoviral transduction. Adenoviral transduction was performed, as we described previously (12), using vectors encoding PKG-1beta (AdPKG), eNOS (AdeNOS), or green fluorescent protein (GFP) control (AdGFP) (12). AdPKG was a kind gift from Ken Bloch (Massachusetts General Hospital, Boston, MA; Ref. 4). Vectors were transduced at a multiplicity of infection of 25, which achieved transduction approximating 90% with minimal toxicity in LX-2.

Collagen lattice assay. Contraction of HSC was performed as previously described with some minor modifications (26). Briefly, individual wells of a 24-well culture dish were incubated with PBS containing 1% BSA (500 µl/well) for 1 h at 37°C and then washed twice with PBS and allowed to air dry. Collagen gels were prepared by mixing 60% type I rat tail collagen (Upstate Laboratories), 10% 10x MEM (GIBCO), 10% 0.2 M HEPES, and 20% DMEM (GIBCO) at 4°C to make a final concentration of collagen of 2.4 mg/ml. The solution was added to the culture wells and incubated for 1 h at 37°C. Rat HSC or LX-2 were layered on top of the collagen lattice at a concentration of 2.5 x 105 cells/ml and serum starved for 24 h. Contraction was induced by incubation of duplicate wells with 10% FBS or, alternatively, endothelin-1 (ET-1, 10–7 M; Sigma) as indicated in individual experiments. To assess NO-dependent responses, the NO donor diethylamine NONOate (DEAN) was added at 45-min intervals, starting immediately after addition of the contractile agonist, to achieve a final concentration of 1 mM. Multiple preliminary experiments were performed to optimize the conditions of this assay in our hands. Cell concentrations between 1.0 x 105 and 5.0 x 105 cells/ml were studied before we settled on the final, optimal cell concentration. In addition, we omitted serum-starved conditions, as well as altering the time course of addition of contractile agonist and NO donors. For example, NO donors were added only once, at t = 0, at 30-min increments, and at 90-min increments before the optimal time frame of administration of NO donors was determined. In some experiments, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 50 µM) was added 30 min before DEAN, or, alternatively, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP; 50 µM) or sodium nitroprusside (SNP; 75 µM increments to a total dose of 300 µM) was used in place of DEAN. Concentrations of SNP, ODQ, and 8-BrcGMP were based on prior studies that had established effective doses of these compounds (17). Contraction and NO effects on contraction were assessed by the change in lattice area measured 3 h after addition of the contractile agonist.

Measurement of intracellular Ca2+ levels. Changes in intracellular Ca2+ concentration were measured by digital fluorescent microscopy using the Ca2+ indicator fura-2 (18). HSC isolated from sham-operated and BDL rats were plated on glass coverslips and incubated with 2.5 µM fura-2 AM dissolved in 0.02% Pluronic F-127–0.1% DMSO in a saline buffer (in mM: 137 NaCl, 5 KCl, 1.2 MgCl2, 2 CaCl2, 10 D-glucose, 10 HEPES, pH 7.4 with NaOH) for 30 min at 37°C. The saline buffer was then replaced with fresh buffer, and the cells were incubated for a further 30 min at 37°C to allow complete cleavage of the fura-2 precursor. Efficient loading and complete cleavage of the dye were assessed by observing the even distribution and stable ratios of the emitted fluorescence. Coverslips were mounted in a shallow chamber over an inverted-stage fluorescence microscope (Olympus IX71, Melville, NY) and continuously perfused with saline (2–3 ml/min). Compounds were applied in the perfusate as indicated in RESULTS. Cells were illuminated alternately with 340- and 380-nm light from a xenon source and a filter wheel (Sutter, Novato CA). Emitted light passed through a 480-nm dichroic mirror and was collected at 510 nm with a 12-bit charge-coupled device camera (Hamamatsu, Hamamatsu City, Japan). Data were collected and converted to ratios of the emitted signal for the two excitation wavelengths with a commercial acquisition system (MetaFluor, Universal Imaging, Downingtown, PA). Image pairs could be collected every 0.3 s with this system. Data were analyzed as percent change of emitted fluorescence, which reflects the increase in intracellular Ca2+ that was detected based on emission/excitation characterization of fura-2 as measured in individual cells. Ca2+-free solutions were made by omitting CaCl2 and adding 100 µM EGTA to give an estimated free Ca2+ concentration of 50 nM.

Assessment of thiol S-nitrosylation. Protein S-nitrosylation of cells was measured with the biotin switch protocol described by Jaffrey et al. (13). A commercial nitrosylation detection assay was also used in some experiments, with similar results (Nitro-Glo; PerkinElmer, Boston, MA), In brief, cells were incubated with vehicle or varying concentrations of SNP (0–10 mM, 20 min at 37°C) or, alternatively, transduced with AdGFP or AdeNOS. After preparation of cell lysates, free thiols were blocked with 20 mM methylmethanethiosulfonate. Nitrosothiol bonds were reduced with 2.5 mM ascorbate and modified with 4 mM N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide. Protein lysates were separated by SDS-PAGE and analyzed by Western blot using anti-biotin antibody. In separate experiments, thiol-bound NO was confirmed by the Saville reaction (34). In brief, cells were incubated with the NO donor S-nitrosoglutathione (GSNO) for 20 min. After treatment with the NO donor, cells were lysed and samples were incubated with vehicle or 0.2% HgCl2, to liberate NO from thiol groups (34). NO liberated from thiols was measured by Greiss assay by subtracting values of vehicle from that obtained with HgCl2.

Western blotting. Cells were homogenized in a lysis buffer [50 mM Tris·HCl, 0.1 mM EGTA, 0.1 mM EDTA, 100 mM leupeptin, 1 mM PMSF, 1% (vol/vol) NP-40, 0.1% deoxycholic acid, pH 7.5]. Protein quantification of samples was performed with the Lowry assay. Detergent-soluble protein lysates were separated by SDS-PAGE on a 12% acrylamide gel, and proteins were electroblotted onto nitrocellulose membranes (30). The membranes were washed in Tris-buffered saline with 0.1% Tween, blocked in 5% milk, and incubated with PKG MAb (Stressgene). Coomassie staining was performed on membranes to confirm equal protein loading and transfer between lanes, or, alternatively, membranes were reprobed for actin.

cGMP assay. cGMP was measured as we described previously (8), with some minor modifications. Cells were cultured in six-well plates in DMEM with 10% FBS. All measurements were performed in the presence of the phosphodiesterase inhibitor IBMX (0.1 mM). Cells were treated with DEAN (500 µM), SNP (300 µM), or vehicle for 20 min at 37°C. The medium was removed from the wells, and the cells were rinsed with PBS. cGMP was extracted with 65% ice-cold ethanol (300 µl) and quantified with an RIA kit from Amersham (TRK500). Radioactive counts were measured by scintillation counter. Amounts of cGMP in each sample were determined by plotting results against a standard curve obtained by creating samples of a known cGMP concentration. Absolute values of cGMP were measured per well as indicated.

Statistical analysis. Data are presented as means ± SE. Data were analyzed with paired and unpaired Student's t-tests as appropriate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
NO-dependent inhibition of normal rat HSC contraction occurs through cGMP-dependent and -independent pathways. Primary HSC were cultured to confluence and plated onto collagen lattices. HSC were incubated with 10% FBS to stimulate contraction and treated with DEAN, SNP, or vehicle. FBS induced contraction to 39 ± 6% of the original size of the collagen lattices (P < 0.05). HSC incubated with DEAN demonstrated significant attenuation of agonist-induced contraction compared with HSC incubated with vehicle (Fig. 1A; for ease of comparison, data are depicted as % inhibition of FBS-induced contraction). The representative collagen lattice wells and accompanying graph in Fig. 1A show quantitative degree of inhibition of contraction in response to DEAN from cumulative experiments (P < 0.05, n = 4). For pharmacological specificity, we also examined the NO donor SNP, which demonstrated inhibition of agonist-induced contraction very similar to that seen with DEAN (29 ± 6%). The role of cGMP-PKG in NO-mediated inhibition of HSC contraction was determined by pretreating FBS/DEAN-stimulated HSC with the sGC inhibitor ODQ or using the soluble cGMP analog 8-BrcGMP (50 µM) in place of DEAN and comparing antagonism of serum-induced contraction to that achieved by DEAN alone. Pretreatment of HSC with ODQ reduced DEAN effects by 50% compared with DEAN alone (DEAN effect is depicted as 100% to allow % comparison to other groups). 8-BrcGMP inhibited contraction of HSC in lieu of the NO donor DEAN, although this level of effect was only ~50% of that observed in response to DEAN. Only DEAN-treated cells showed a statistically significant inhibition of contraction compared with treatment with FBS alone (Fig. 1B; P < 0.05 vs. FBS alone, n = 4). We next directly measured cGMP production in response to exogenous NO donors. After treatment with DEAN (500 µM) for 20 min, HSC isolated from normal rats demonstrated greater than threefold increased accumulation of cGMP compared with HSC incubated with vehicle (Fig. 1C; P < 0.05, n = 4). For pharmacological specificity, we also measured cGMP generation in response to a complementary NO donor, SNP, which evidenced markedly enhanced cGMP accumulation as well (vehicle: 9 pmol cGMP/mg protein; SNP: 682 pmol cGMP/mg protein; n = 3 independent cell preps). These studies indicate that NO donors act on HSC through both cGMP-PKG-dependent and cGMP-PKG-independent pathways, with both pathways necessary for maximum NO-dependent inhibition of agonist-induced contraction in HSC.


Figure 1
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  		Fig. 1. Hepatic stellate cells (HSC) respond to the NO donor diethylamine NONOate (DEAN) via cGMP-dependent and cGMP-independent mechanisms. Rat HSC were isolated and cultured 7–10 days later on collagen lattices for measurement of contraction in response to combinations of serum, DEAN, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) or, alternatively, cultured in wells for measurement of cGMP accumulation in response to DEAN. A: HSC in collagen lattices were contracted with FBS and then treated with DEAN, which was added at 45-min intervals starting immediately after addition of FBS, to achieve a final concentration of 1 mM. Representative collagen lattices and graph demonstrate % inhibition of FBS-mediated lattice contraction in response to DEAN. Arrows indicate the border of lattice contraction. In response to FBS, lattices contracted to 39 ± 6% of the original collagen lattice size. Statistically significant inhibition of contraction was observed in response to DEAN after FBS stimulation (*P < 0.05, n = 4). B: effects of DEAN on modulation of HSC contraction after FBS administration were examined in absence and presence of ODQ and in response to 8-BrcGMP in lieu of DEAN administration. Although DEAN showed statistically significant inhibition of FBS-mediated contraction (depicted as 100% to allow % comparison to other groups), pretreatment of HSC with ODQ before DEAN treatment reduced DEAN-mediated responses by 50%. 8-BrcGMP promoted only 50% of the inhibitory effect on contraction of HSC compared with DEAN (*P < 0.05 compared with FBS alone, n = 4 independent experiments). C: incubation of HSC with DEAN significantly increased cGMP accumulation over 20 min compared with vehicle (*P < 0.05, n = 4).

 
Interrogation of NO-dependent cGMP generation, PKG expression, and calcium signaling from in vivo activated HSC. Primary HSC isolated after induction of liver injury have an activated phenotype characterized by enhanced contractility (27, 37). Prior studies in the isolated, perfused rat liver preparation demonstrated impaired NO-dependent hepatic vascular relaxation in rats after liver injury and HSC activation (6). To determine whether the highly contractile phenotype of activated HSC may be associated with impaired generation of the NO second messenger cGMP, HSC were isolated from BDL liver and assayed for cGMP generation in response to DEAN. HSC from BDL rats, in contrast to control HSC, did not demonstrate an increase in cGMP production after stimulation with DEAN [Fig. 2A; P = not significant (NS), n = 5]. This is in contradistinction to control cells, which demonstrated a robust threefold increase in cGMP accumulation in response to DEAN (Fig. 1C).


Figure 2
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  		Fig. 2. Impaired NO-dependent cGMP levels and Ca2+ signaling from in vivo activated HSC. HSC were isolated from bile duct-ligated (BDL) rat liver and plated in wells for DEAN-induced cGMP measurements, into dishes for preparation of SDS-PAGE/Western blot analysis, or onto coverslips to measure changes in intracellular Ca2+ in response to endothelin-1 (ET-1) and sodium nitroprusside (SNP) with fura-2. A: HSC isolated from BDL rat livers demonstrate impaired cGMP accumulation in response to DEAN [P = not significant (NS), n = 4]. For comparison, the amount of cGMP produced by HSC isolated from sham-operated rats when stimulated by DEAN is also presented (*P < 0.05, n = 4) B: HSC isolated from BDL rat liver evidence similar protein kinase G (PKG) protein levels compared with HSC isolated from sham-operated rat liver as assessed by Western blot analysis (blot depicts 2 independent sham preps and 2 independent BDL preps representative of 4 animals studied from each group). beta-Actin immunoblot is shown as loading control. C:. ET-1 increased intracellular Ca2+ in HSC from both sham-operated and BDL rats with more prominent increases detected in activated HSC from BDL animals compared with HSC from sham-operated animals (*P < 0.05, n = 25–35 cells studied per group from 2 independent cell preps). Data were analyzed as % change of emitted fluorescence, which reflects the increase in intracellular Ca2+ that was microfluoroscopically detected based on emission/excitation characterization of fura-2.

 
We next examined PKG protein levels from HSC isolated from sham-operated and BDL rat liver. Cultured HSC were lysed and prepared for SDS-PAGE and Western blot analysis with a PKG MAb. This analysis demonstrated that PKG protein levels were not diminished in HSC from BDL rats compared with sham-operated rats (Fig. 2B; n = 4 independent samples from each group).

To further examine the consequences of impaired NO-dependent cGMP generation on intracellular Ca2+ dynamics in activated HSC, we measured the effects of ET-1 and the NO donor SNP on changes in intracellular Ca2+, using fura-2 in HSC isolated from sham-operated animals and activated HSC from BDL animals. ET-1 (0.1 µM) markedly increased global intracellular Ca2+ in both sham-operated and BDL HSC. However, a significantly greater percent increase in intracellular Ca2+ accumulation was detected in activated HSC from BDL animals (Fig. 2C; P < 0.05, n = 30 sham-operated and 25 BDL cells from 2 independent cell preps). The response to ET-1 was due to release from intracellular Ca2+ stores as it was not blocked by incubating cells in Ca2+-free saline buffer (data not shown). Interestingly, the NO donor SNP (100 µM) slightly increased global intracellular Ca2+ in HSC from both sham-operated and BDL rats; however, these changes were much smaller than those observed with ET-1 (ET-1: sham operated 0.065 ± 0.076, BDL 0.106 ± 0.124; SNP: sham 5.3 ± 3.6 x 10–4, BDL 9.2 ± 8.1 x 10–4; n = 25–35 cells per group).

Mechanisms of NO-mediated inhibition of contraction in LX-2 model of activated HSC. We next examined cGMP generation in response to DEAN from the constitutively activated HHSC cell line LX-2, which recapitulates the phenotype of highly activated HSC (37). Similar to that observed in HSC isolated from BDL liver, LX-2 demonstrated impaired cGMP production after stimulation with DEAN (Fig. 3A; P = NS). Again, this result is in marked contrast with the significantly increased cGMP production seen in control HSC after stimulation with DEAN. In light of the defect in cGMP generation in LX-2, we next examined the cGMP-PKG-mediated effects of NO by assessing effects of the sGC inhibitor ODQ on DEAN-mediated inhibition of serum-induced contraction as well as the effect of the cGMP analog 8-BrcGMP (50 µM) on serum-induced contraction. Consistent with a deficit in the cGMP-PKG pathway, pretreatment with ODQ (50 µM) did not influence DEAN-mediated effects (Fig. 3B; P = NS, n = 4). Furthermore, minimal inhibition of serum-induced contraction in response to 8-BrcGMP was observed in LX-2 and was significantly less compared with DEAN (Fig. 3B; P < 0.05, n = 4; DEAN is depicted as 100% to allow easy % comparison to other groups). The lack of effect of ODQ and 8-BrcGMP in LX-2 is in marked contrast to HSC isolated from control animals, which demonstrated both a cGMP-PKG-dependent and a cGMP-PKG-independent component of NO-mediated antagonism of contraction (Fig. 1). These data provide evidence that the hyporesponsiveness of activated HSC to NO donors (6) may be due to loss of NO-dependent cGMP generation during the activation process, leaving only cGMP-independent pathways intact for NO-mediated responses. These studies also support the utility of the LX-2 cell line in mimicking some of the phenotypic abnormalities seen in activated HSC from BDL rats.


Figure 3
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  		Fig. 3. LX-2 cells recapitulate a model of activated HSC with impaired NO-dependent cGMP responses. LX-2 cells were plated in wells for DEAN-induced cGMP measurements or onto collagen lattices to assay contraction in response to combinations of FBS, DEAN, ODQ, and 8-BrcGMP. A: LX-2 did not evidence increased cGMP accumulation in response to DEAN (P = NS, n = 4). For comparison, the amount of cGMP produced by HSC isolated from sham-operated rats stimulated by DEAN is also presented, which was significantly greater than cGMP amounts seen in both (*P < 0.05). B: pretreatment of cells with ODQ did not impair DEAN-induced antagonism of LX2 contraction. 8-BrcGMP evidenced significantly less inhibition of FBS-mediated contraction compared with DEAN (*P < 0.05, 8-BrcGMP vs. DEAN, n = 5; DEAN effect is depicted as 100% to allow easy comparison to other groups).

 
Recent studies suggest that cGMP-independent effects of NO may occur through pathways mediated by S-nitrosylation of target cellular proteins (13, 32). To examine whether S-nitrosylation occurs in LX-2 cells after treatment with NO, we increased NO gradients in LX-2 with NO donors or, alternatively, eNOS gene transfer and proceeded with a biochemical assay to detect protein S-nitrosylation from LX-2 cell lysates, the biotin switch. Treatment of cells with the NO donor SNP or with adenoviral gene transfer of eNOS was associated with an increase in the level of S-nitrosylation of specific intracellular target proteins as indicated by Western blot using the biotin switch method (Fig. 4, A and B). In particular, a 75-kDa protein was prominently S-nitrosylated in response to either SNP or AdeNOS. S-nitrosylation of LX-2 cells was further confirmed by detecting NO release from GSNO-treated LX-2 cells after liberating NO groups with HgCl2, which showed a 90% increase in the HgCl2-driven NO release from GSNO-treated cells (data not shown). To confirm that nitrosylation occurs in response to NO donors in primary stellate cells, HHSC were incubated with GSNO and protein S-nitrosylation was detected by the biotin switch method. S-nitrosylation of several proteins was detected in response to GSNO, including a protein of 75 kDa, similar to that observed in LX2 (Fig. 4C).


Figure 4
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  		Fig. 4. NO donors increase S-nitrosylation of intracellular proteins in LX-2 and human HSC (HHSC). LX-2 were treated with SNP or alternatively transduced with adenoviral vector encoding endothelial nitric oxide synthase (AdeNOS), whereas HHSC were treated with S-nitrosoglutathione (GSNO). Control LX-2 were incubated with vehicle or transduced with adenoviral vector encoding green fluorescent protein (AdGFP), respectively. Cells were prepared for detection of protein thiol S-nitrosylation by the biotin switch method. A: LX-2 incubated with SNP evidenced a concentration-dependent increase in S-nitrosylation of a protein with a molecular mass of 75 kDa. Positive control was LX-2 cells incubated with the nitrosylating compound provided in the assay. B: cells transduced with AdeNOS evidenced an increase in the level of S-nitrosylation of a specific intracellular target protein of 75 kDa (arrow). C: HHSC were incubated with GSNO. S-nitrosylation was detected by biotin switch. S-nitrosylation of several proteins was detected in response to GSNO, including a protein of 75 kDa (arrow), similar to that observed in LX2. Equal protein loading was confirmed by Coomassie stain.

 
AdPKG delivery promotes cGMP-mediated inhibition of LX-2 contractile responses. Owing to the defect in NO-dependent signaling in LX-2, we next sought to determine whether we could reconstitute 8-BrcGMP-dependent downstream effects with PKG gene delivery using ET-1 as a contractile agonist. LX-2 were transduced with AdGFP or AdPKG and passed onto collagen lattices to assess modulation of contraction in response to vehicle or 8-BrcGMP (50 µM) after ET-1 (10–7 M)-induced contraction. ET-1 induced contraction to 43 ± 4.2% of the original collagen lattice size in LX-2 cells (P < 0.05 vs. no ET-1; ET-1 is depicted as 0% inhibition of contraction in Fig. 5). AdPKG-transduced cells treated with 8-BrcGMP demonstrated significantly less contraction in response to ET-1 (Fig. 5; P < 0.05, n = 7) compared with the level of contraction in cells treated with 8-BrcGMP after transduction with AdGFP or cells transduced with AdPKG, with vehicle in place of 8-BrcGMP. These data demonstrate that overexpression of PKG reconstituted cGMP-mediated downstream effects in the highly activated HSC cell line LX-2 with the complementary contractile agonist ET-1.


Figure 5
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  		Fig. 5. Transduction of LX-2 with adenoviral vector encoding PKG (AdPKG) promotes cGMP-dependent modulation of contraction. LX-2 were transduced with AdPKG or AdGFP and passaged onto collagen lattices. Transduction of LX-2 with AdPKG resulted in dramatically increased levels of PKG protein as assessed by Western blot analysis from cell lysates (data not shown). ET-1 contracted LX-2 collagen lattices to 43 ± 4.2% of the original collagen lattice size (P < 0.05; depicted as 0% inhibition of contraction). AdPKG- or AdGFP-transduced LX-2 in collagen lattices were contracted with ET-1 and then treated with 8-BrcGMP (50 µM) or vehicle. LX-2 transduced with AdPKG and treated with 8-BrcGMP demonstrated significantly greater inhibition of ET-1-mediated contraction compared with LX-2 transduced with AdGFP or LX-2 transduced with AdPKG in absence of 8-BrcGMP (*P < 0.05, n = 7).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The prototypical pathway by which NO mediates its vasorelaxant effects in contractile cells is via stimulation of sGC, production of cGMP, subsequent activation of PKG, and finally initiation of a phosphorylation cascade that culminates in cell relaxation (19). HSC isolated from normal rats utilize this prototypical pathway as evidenced by increased DEAN-induced cGMP production and inhibition of agonist-induced contraction in response to DEAN that is partially inhibited by ODQ and partially mimicked by 8-BrcGMP. In addition, the intermediate effects of ODQ and 8-BrcGMP in normal HSC suggest that NO-mediated effects also occur through cGMP-independent pathways in normal HSC. Prior studies demonstrated cGMP-independent NO-mediated relaxation of contractile cells such as smooth muscle, with a number of findings pointing toward a mechanism of NO-dependent S-nitrosylation of target proteins within contractile cells (32). Therefore, in vivo, the maximum observed responses to NO donors in normal HSC may require the combination of cGMP-dependent and cGMP-independent pathways. However, it should be recognized that although normal HSC are contractile in culture after 7–10 days, as depicted in our studies, under normal physiological circumstances the contractile phenotype of normal quiescent HSC remains controversial (14, 38).

Presently, treatment of portal hypertension has largely focused on targeting the vascular component of portal hypertension because of its dynamic and reversible nature (3). With regard to the dynamic component of increased intrahepatic resistance, prior studies in human cirrhosis and animal models of portal hypertension demonstrated that NO generation from liver endothelial cells is diminished (11, 25, 28). Because of the unique perisinusoidal location of HSC, it is anticipated that HSC serve as effector cells for NO signals. Diminished NO generation therefore contributes to diminished NO responses in HSC, resulting in increased intrahepatic vascular resistance (23, 24, 26). In addition to endothelial dysfunction, more recent studies have suggested that defects may exist within HSC that contribute to diminished responsiveness to NO (6, 7). For example, recent studies from Dudenhoefer et al. (6) demonstrate that in the isolated, perfused rat liver system livers from animals with cirrhosis evidence impaired vasodilation in response to NO donors, which may be due to increased expression of phosphodiesterase-5 in cirrhotic rat liver (20). The present studies identify alterations in NO-dependent signaling that occur in HSC upon activation, which may contribute to the deficient vasodilation in response to NO that occurs in cirrhosis. Specifically, we have identified a number of defects in the cGMP-dependent NO pathway that are evident in HSC upon activation. Both in vivo activated HSC from BDL rats and the highly activated LX-2 line failed to generate cGMP in response to NO donors. Furthermore, ODQ failed to attenuate the DEAN-mediated inhibition of contractile responses in activated HSC, and 8-BrcGMP evidenced minimal effect in modulating agonist-induced contraction, indicating defects in the sGC-PKG pathway in activated HSC. Thus the remaining NO-dependent downstream responses that occur in HSC upon activation require cGMP-PKG independent pathways. Using complementary approaches, we showed that NO-induced S-nitrosylation in activated HSC may account for a component of cGMP-independent NO-mediated inhibition of contraction. In particular, we identified a 75-kDa intracellular protein in LX-2 that was prominently nitrosylated in response to both an NO donor and NOS overexpression. However, more detailed mass spectrometric studies are required to definitively establish the nature of nitrosylated proteins in HSC and their potential role in HSC dynamic responses. A similar nitrosylation paradigm was proposed to contribute to NO-mediated relaxation in smooth muscle (32). Although a definitive target for S-nitrosylation-dependent smooth muscle cell relaxation has not been established, a number of putative targets have been identified, most notably specific channel proteins whose modification may allow for smooth muscle cell relaxation in the absence of cGMP/PKG activation (2, 10, 21, 32). Other recent studies suggest that cAMP and prostaglandin cross talk may provide a redundancy for cGMP signaling in HSC as well (7).

ET-1, serum, and other HSC agonists cause a rise in intracellular Ca2+ due to release from intracellular stores that culminates in cellular contractility (9, 15, 22). The present studies confirm previous studies that demonstrated that ET-1 markedly increases intracellular Ca2+ in normal HSC and that increases are more prominent in activated HSC from BDL rats (15, 27). Interestingly, in our studies NO donors did not act to reduce global intracellular Ca2+ levels and, in fact, tended to paradoxically increase intracellular Ca2+, albeit by a very subtle amount. The lack of reduction in intracellular Ca2+ in response to NO is consistent with a number of recent studies demonstrating that NO signals in contractile cells may promote smooth muscle relaxation by transiently increasing local Ca2+ gradients, with these "calcium sparks" activating KCa channels (35). Indeed, Gasull et al. (9) recently demonstrated that NO-mediated inhibition of contraction occurs in part through activation of the large-conductance KCa channel in HSC. However, the enhanced ET-1-dependent Ca2+ accumulation that occurred in HSC from BDL rats dwarfed the small effects of NO that we detected on Ca2+ dynamics, and thus enhanced constrictor effects may be a contributor to impaired responses to NO through the cGMP pathway that are detected in activated HSC from BDL rats. However, other as yet unidentified defects are likely to coexist as well.

Owing to the defects that we detected in cGMP signaling in activated HSC, we also tested whether modulation of targets downstream from sGC may be effective in improving cGMP-dependent modulation of contraction in activated HSC. As PKG is an established downstream effector of cGMP, and PKG-1beta is the most abundant isoform within contractile cells (19), we targeted this molecule to assess for improvement in the ability of cGMP to modulate HSC contraction. Consistent with this premise, PKG overexpression in LX-2 cells enhanced the effects of exogenous cGMP on antagonism of HSC contraction. However, exogenous cGMP was required to detect PKG induced responses, and because of the deficient cGMP generation in HSC from BDL rats, an in vivo benefit from PKG gene delivery would likely require use of a constitutive active construct that does not require cGMP substrate (16).

Thus, although nitrates represent a rational and clinically established therapy targeting the increased intrahepatic resistance of cirrhosis (3), the present studies elucidate a number of signaling defects in activated HSC that may explain, in part, their limited effectiveness in the treatment of portal hypertension (1). These studies also suggest that approaches designed to target signaling pathways in HSC downstream from defects in sGC, such as PKG, may provide a novel means of modulating vascular resistance in the liver in health and disease.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institutes of Health Grants DK-59615, R01-DK-59388, and P50-CA-102701 Pancreatic Cancer specialized programs of research excellence Development Grant (V. H. Shah).


   ACKNOWLEDGMENTS
 
The authors thank Scott Friedman for providing LX-2 cells, Ken Bloch for providing the PKG adenovirus, and Bill Sessa for providing the GFP and eNOS adenovirus.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. H. Shah, Gastroenterology Research Unit, Al 2-435, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (e-mail: shah.vijay{at}mayo.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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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