Sodium nitroprusside augments human lung fibroblast collagen gel contraction independently of NO-cGMP pathway

X. D. Liu, C. M. Skold, T. Umino, J. R. Spurzem, D. J. Romberger, and S. I. Rennard

Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198-5300


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO) relaxes vascular smooth muscle in part through an accumulation of cGMP in the target cells. We hypothesized that a similar effect may also exist on collagen gel contraction mediated by human fetal lung (HFL1) fibroblasts, a model of wound contraction. To evaluate this, HFL1 cells were cultured in three-dimensional type I collagen gels and floated in serum-free DMEM with and without various NO donors. Gel size was measured with an image analyzer. Sodium nitroprusside (SNP, 100 µM) significantly augmented collagen gel contraction by HFL1 cells (78.5 ± 0.8 vs. 58.3 ± 2.1, P < 0.01), whereas S-nitroso-N-acetylpenicillamine, 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride, NONOate, and NG-monomethyl-L-arginine did not affect the contraction. Sodium ferricyanide, sodium nitrate, or sodium nitrite was not active. The augmentory effect of SNP could not be blocked by 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, whereas it was partially reversed by 8-(4-chlorophenylthio) (CPT)-cGMP. To further explore the mechanisms by which SNP acted, fibronectin and PGE2 production were measured by immunoassay after 2 days of gel contraction. SNP inhibited PGE2 production and increased fibronectin production by HFL1 cells in a concentration-dependent manner. CPT-cGMP had opposite effects on fibronectin and PGE2 production. Addition of exogenous PGE2 blocked SNP-augmented contraction and fibronectin production by HFL1 cells. Therefore, SNP was able to augment human lung fibroblast-mediated collagen gel contraction, an effect that appears to be independent of NO production and not mediated through cGMP. Decreased PGE2 production and augmented fibronectin production may have a role in this effect. These data suggest that human lung fibroblasts in three-dimensional type I collagen gels respond distinctly to SNP by mechanisms unrelated to the NO-cGMP pathway.

nitric oxide; guanosine 3',5'-cyclic monophosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SODIUM NITROPRUSSIDE (SNP), a nitric oxide (NO) donor, is a well-known vascular smooth muscle relaxant and is frequently used as a vasodilator in clinical practice. A principal mechanism of action of SNP is to generate NO, which subsequently activates the soluble form of guanylate cyclase (sGC) after binding to its heme moiety, thus causing cGMP accumulation in target cells (17, 28). The increase in cGMP through this pathway is believed to be the major mechanism underlying many of the cardiovascular and neural actions of NO and NO donors (2, 5, 15, 26).

Fibroblast-mediated type I collagen gel contraction is a well-established in vitro model of tissue contraction, which characterizes wound healing and tissue fibrosis (6). Many substances such as growth factors and cytokines are able to augment or inhibit the process of collagen gel contraction (3, 9, 19, 23, 25). Based on the recognized effects of the NO-cGMP pathway, we hypothesized that SNP and other NO donors may also have an inhibitory effect on human lung fibroblast-mediated type I collagen gel contraction. Accordingly, the purpose of this study was to determine whether human fetal lung (HFL1) fibroblast-mediated contraction of collagen gels is inhibited by NO donors. However, our results indicate that SNP augments the type I collagen gel contraction mediated by HFL1 cells, an effect not shared by other NO donors. SNP appears to mediate this effect by increasing fibroblast fibronectin production while decreasing PGE2 production rather than an effect mediated via the NO-cGMP pathway.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Type I collagen (rat tail tendon collagen) was extracted from rat tail tendons by a previously published method (16). Briefly, tendons were excised from rat tails. After repeated washing with Tris-buffered saline and dehydrating by 50, 75, 95, and 100% ethanol, type I collagen was extracted in 6 mM acetic acid. Protein concentration was determined by weighing a lyophilized aliquot from each lot of collagen solution.

The agents used were purchased from the company indicated: SNP (Sigma); S-nitroso-N-acetylpenicillamine (SNAP, Calbiochem); 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1; Cayman Chemical); diethylenetetraamine (DETA) NONOate (Cayman Chemical); NG-monomethyl-L-arginine (L-NMMA, Cayman Chemical); 8-(4-chlorophenylthio) (CTP)-cGMP sodium salt (Sigma); sodium ferricyanide (Fluka); PGE2 (Sigma); 1H-[1,2,4]oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ, Sigma); and indomethacin (Sigma).

Cell culture. HFL1 cells (lung, diploid, human) were purchased from the American Type Culture Collection (Manassas, VA). The cells were cultured in DMEM (GIBCO) supplemented with 10% FCS, 100 µg/ml penicillin, 250 µg/ml streptomycin, 2.5 µg/ml Fungizone, and 2 mM L-glutamine. Cells were refed three times weekly in 100-mm tissue culture dishes (Becton Dickinson Labware, Lincoln Park, NJ), and confluent cells were passaged at a ratio of 1:4. Cells were used between the 15th and 22nd passages.

Collagen gel preparation and contraction assay. Gels were prepared using a previously described method (18). Briefly, collagen gels were prepared by mixing rat tail tendon collagen, distilled water, and four times concentrated DMEM so that the final mixture resulted in 0.75 mg/ml of collagen, a physiological ionic strength, 1× DMEM, and 3 × 105 cells/ml. After this, 0.5-ml aliquots of the mixture were cast into each well of 24-well tissue culture plates (Falcon). After gelation was completed, generally within 20 min at room temperature, the gels were gently released from the 24-well tissue culture plates and transferred into 60-mm tissue culture dishes (Falcon), which contained 5 ml of prewarmed serum-free DMEM with the indicated concentration of varying agents. The gels were then incubated at 37°C in a 5% CO2 atmosphere. The area of each gel was measured with an image analyzer (Optomax V, Burlington, MA) every day. Data are expressed as the percentage of area compared with the original gel size.

Extraction of cells and DNA assay. To estimate cell number in three-dimensional collagen gels, DNA was assayed fluorometrically with Hoechst dye 33258 (Sigma) by a modification of a previously published method (14). Collagen gels were dissolved with collagenase (0.25 mg/ml in serum-free DMEM) for 2 h at 37°C. Cell pellets were collected by centrifugation at 500 g for 10 min and frozen at -80°C. The supernatant solution following digestion was saved at -80°C and subsequently used for fibronectin and PGE2 assay (see Measurement of fibronectin by ELISA and Measurement of PGE2 by RIA). One day after being frozen, cell pellets were thawed and resuspended in 1 ml of distilled water. After this, cells were briefly sonicated and the suspensions were mixed with 2 ml of TNE buffer (3 M NaCl, 10 mM Tris, and 1.5 mM EDTA, pH 7.4) containing 2 µg/ml of Hoechst 33258. Fluorescence intensities were measured with a fluorescence spectrophotometer (LS-5, Perkin-Elmer) with excitation at 356 nm and emission at 458 nm. Cell number was then calculated by comparison to a standard curve relating cell number to fluorescence.

Measurement of fibronectin by ELISA. For quantification of fibronectin production, a previously reported method was used (1). Briefly, the medium in which the gels were floated and the supernatant solutions after gel digestion (see Extraction of cells and DNA assay) were harvested and stored at -80°C. Fibronectin concentration was determined by an ELISA, which is specific for human fibronectin and does not detect bovine fibronectin (22).

Measurement of PGE2 by RIA. The same samples used for fibronectin quantification were used for determination of PGE2 concentration using RIA as described by the manufacturer (PGE2 RIA kit, Perseptive Diagnostics). Briefly, 100 µl of samples or standards were mixed with 100 µl of [3H]PGE2 tracer and 100 µl of PGE2 antibody in a siliconized glass tube and incubated at 4°C for 16-24 h. After this, 750 µl of magnetic dextran-coated charcoal were added to each tube, vortexed for 3-5 s, and centrifuged at 4°C (1,000 g for 15-20 min). Supernatants were then decanted into scintillation vials, and a volume of scintillation fluid was added to each vial and counted in a liquid scintillation counter. Data were calculated and are expressed as picograms per milliliter.

Statistical analysis. Each condition in every experiment included three replicate gels, and the data presented are the means ± SE of these triplicates. Each experiment was repeated on multiple occasions, and each figure is a representative experiment. The number of replicates in experiments is provided in each figure legend. Data were compared by an unpaired two-tailed Student's t-test or one-way ANOVA. Values of P < 0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fibroblast-mediated collagen gel contraction is augmented by SNP but not by other NO donors. To observe the effect of NO on collagen gel contraction mediated by fibroblasts, several NO donors were added to the medium in which fibroblasts containing collagen gels were floated. Among the NO donors tested, SNP significantly augmented collagen gel contraction mediated by fibroblasts (Table 1). This significant augmentation was observed in all eight replicate experiments. In contrast, SNAP, SIN-1 chloride, DETA NONOate, and L-arginine caused a minimal inhibition of contraction, which was not statistically significant (Table 1). The effect of SNP was observed within 24 h after release, and the difference between SNP-treated and control cultures persisted for the 5 days of incubation (Fig. 1). By comparison, serum and transforming growth factor-beta 1 (TGF-beta 1) were able to augment the collagen gel contraction mediated by HFL1 fibroblasts as well (Table 1). L-NMMA, a NO synthase inhibitor, did not alter collagen gel contraction mediated by fibroblasts either when added alone or in the presence of SNP, other NO donors, serum, or TGF-beta 1 (Table 1). The ability of SNP to augment contraction was concentration dependent over a range from 0.01 to 100 µM, a range used in previous studies with SNP (15, 26) (Fig. 2). SNP did not affect cell number as assessed by DNA content over the 2 days of exposure (Fig. 2).

                              
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Table 1.   Effect of NO donors on collagen gel contraction mediated by human fetal lung fibroblasts



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Fig. 1.   Time course of collagen gel contraction by human fetal lung (HFL1)-1 fibroblasts in presence and absence of sodium nitroprusside (SNP, 100 µM). HFL1 fibroblasts were cast into type I collagen gels as described in MATERIALS AND METHODS. After polymerization, gels were released into serum-free DMEM with and without 100 µM SNP. Gel size was measured daily for 5 days. Data are presented as means ± SE of samples in triplicate. Error bars not visible are contained within lines on graph. This figure is representative of 8 different experiments.



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Fig. 2.   Effect of SNP on collagen gel contraction mediated by HFL1 cells: concentration dependence. HFL1 fibroblasts were cast into type I collagen gels as described in MATERIALS AND METHODS. After polymerization, gels were released into serum-free DMEM with and without varying concentrations of SNP. Gel size was measured after 2 days at which time cell number was assessed by DNA content. This figure is representative of 3 different experiments.

Effect of SNP metabolites on collagen gel contraction. To determine whether the effect of SNP on fibroblast-mediated collagen gel contraction was due to SNP per se or might be mediated by its potential metabolites, sodium ferricyanide, sodium nitrate, and sodium nitrite were tested. None had any effect on collagen gel contraction mediated by HFL1 cells (Fig. 3).


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Fig. 3.   Effect of sodium ferricyanide, sodium nitrate, and sodium nitrite on collagen gel contraction mediated by HFL1 cells. Collagen gels containing HFL1 cells were prepared as described in MATERIALS AND METHODS. After polymerization, gels were released into serum-free DMEM with and without freshly prepared SNP, sodium ferricyanide (Na-FeCN), sodium nitrate, or sodium nitrite. Gel size was measured after 2 days. Data presented are means ± SE of samples in triplicate. Error bars not visible are contained within lines on graph. Agents added to medium are 100 µM in final concentration. This figure is representative of 3 different experiments.

Role of cGMP and sGC. To determine whether SNP augmentation of fibroblast-mediated collagen gel contraction might be mediated through stimulation of sGC and increased levels of cGMP, ODQ (a specific sGC inhibitor) was tested. ODQ did not abolish the ability of SNP to augment fibroblast-mediated collagen gel contraction. In contrast, at some concentrations, there was a small, but significant, further potentiation of gel contraction (Fig. 4). Moreover, CPT-cGMP, an analog of cGMP, not only inhibited HFL1 cell-mediated collagen gel contraction in a concentration-dependent manner (Fig. 5) but also partially blocked the augmentative effect of SNP on the collagen gel contraction mediated by HFL1 cells (Fig. 6). Inhibition of gel contraction was not due to cytotoxicity of CPT-cGMP because low concentrations slightly, but significantly, increased cell number and higher concentrations were without effect.


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Fig. 4.   Effect of guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) on SNP-augmented collagen gel contraction. Collagen gels containing HFL1 cells were prepared as described in MATERIALS AND METHODS. After polymerization, gels were released into serum-free DMEM with and without 100 µM SNP and varying concentrations of ODQ. Gel size was measured after 2 days. This figure is representative of 2 different experiments.



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Fig. 5.   Concentration-dependent effect of 8-(4-chlorophenylthio) (CPT)-cGMP on collagen gel contraction mediated by HFL1 cells. HFL1 fibroblasts were cast into type I collagen gels as described in MATERIALS AND METHODS. After polymerization, gels were released into serum-free DMEM with and without varying concentrations of CPT-cGMP. Gel size was measured after 2 days. This figure is representative of 2 different experiments.



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Fig. 6.   Effect of SNP and/or CPT-cGMP on collagen gel contraction mediated by HFL1 cells. HFL1 fibroblasts were cast into collagen gels as described in MATERIALS AND METHODS. After polymerization, gels were released into serum-free DMEM with and without SNP and/or CPT-cGMP. Gel size was measured daily after release. This figure is representative of 3 different experiments.

Role of fibronectin and PGE2 in SNP augmentation of collagen gel contraction by HFL1 cells. Fibronectin and PGE2 modulate fibroblast-mediated collagen gel contraction in response to some stimuli. Therefore, production of fibronectin and PGE2 by HFL1 cells in the three-dimensional collagen gel culture system were measured in response to SNP and other NO donors. SNP increased fibronectin production by HFL1 cells and decreased PGE2 production in a concentration-dependent manner (Fig. 7). SIN-1 increased PGE2 production but had no effect on fibronectin, whereas NONOate decreased fibronectin production and had no effect on PGE2 (Table 2). Interestingly, as with contraction, CPT-cGMP had the opposite effect from SNP, decreasing fibronectin and increasing PGE2 production by HFL1 cells (Fig. 8), consistent with a role for fibronectin and PGE2 as autocrine or paracrine mediators of this process. Indomethacin was also able to augment gel contraction, further supporting a role for cyclooxygenase production as autocrine or paracrine regulators of gel contraction (Fig. 9). Because SNP further augmented the increased contraction induced by indomethacin alone, PGE2 inhibition does not appear to be the only mechanism for the SNP effect. Similarly, the slight but significant augmentation observed when indomethacin was added to SNP is consistent with the direct measurements, which demonstrate that SNP does not completely inhibit PGE2 production.


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Fig. 7.   Effect of SNP on fibronectin (Fn) and PGE2 production by HFL1 cells in 3-dimensional collagen gel culture: concentration dependence. Collagen gels were prepared and floated in medium containing varying concentrations of SNP as described in MATERIALS AND METHODS. After 48 h, floating media were harvested and gels were dissolved with collagenase. Fibronectin was measured by ELISA and PGE2 by RIA in both floating medium and solubilized gel supernate. DNA content in cell pellets was quantified and used to determine cell number. Data presented represent 1 of 2 independent experiments.


                              
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Table 2.   Effect of NO donors on fibronectin and PGE2 production by human fetal lung fibroblasts



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Fig. 8.   Effect of CPT-cGMP with and without SNP on fibronectin (Fn) and PGE2 production by HFL1 cells in 3-dimensional collagen gels. Collagen gels containing HFL1 cells were prepared and floated in serum-free (SF) DMEM with and without 100 µM SNP and/or 100 µM CPT-cGMP as described in MATERIALS AND METHODS. After 48 h, floating media were harvested and gels were dissolved, and PGE2 and fibronectin concentrations were measured in both. DNA content in cell pellets was quantified and used to determine cell number. A: fibronectin production by HFL1 cells in 3-day collagen gels. B: PGE2 production by HFL1 cells in 3-day collagen gels. Data presented represent 1 of 2 independent experiments.



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Fig. 9.   Effect of SNP and indomethacin (Indo) on HFL1 cell-mediated collagen gel contraction. Collagen gels containing HFL1 cells were prepared as described in MATERIALS AND METHODS. Gels were released into serum-free DMEM with and without 2 µM indomethacin and with and without 100 µM SNP. Gel size was measured on day 2. Data presented are means ± SE of triplicate samples. Error bars not visible are contained within lines on graph. This figure is representative of 3 different experiments.

Exogenous PGE2 inhibits collagen gel contraction as well as decreases fibronectin production by HFL1 cells stimulated by SNP. To both confirm the role of PGE2 as a mechanism of SNP-augmented collagen gel contraction and to determine whether the effect of SNP was reversible, three-dimensional collagen gels were incubated in the presence and absence of SNP with and without exogenous PGE2. Exogenous PGE2 inhibited collagen gel contraction in a concentration-dependent manner in both the presence and absence of SNP (Fig. 10). In parallel, exogenous PGE2 inhibited fibronectin production by HFL1 cells in both the presence and absence of SNP in a concentration-dependent manner (Fig. 11).


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Fig. 10.   Exogenous PGE2 attenuates SNP augmentation of HFL1 cell-mediated collagen gel contraction. Collagen gels containing HFL1 cells were prepared as described in MATERIALS AND METHODS. After polymerization, gels were released into serum-free DMEM with and without SNP in presence of varying concentrations of PGE2. After 48 h, gel size was measured. This figure is representative of 3 different experiments.



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Fig. 11.   Exogenous PGE2 attenuates SNP augmentation of HFL1 cell fibronectin (Fn) production. Collagen gels containing HFL1 cells were prepared as described in MATERIALS AND METHODS. After polymerization, gels were released into serum-free DMEM with and without SNP in presence of varying concentrations of PGE2. After 48 h, gels were then dissolved with collagenase and fibronectin concentration was measured by ELISA in floating medium and in solubilized gel supernate. DNA content in cell pellets was quantified and used to determine cell number. This figure is representative of 3 different experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The current study demonstrates that SNP augments fibroblast-mediated contraction of type I collagen gels in a concentration-dependent manner. This contrasts to other exogenous NO donors that have no effect. The effect of SNP is not blocked by ODQ, an inhibitor of sGC. The cGMP analog CPT-cGMP inhibits collagen gel contraction. This suggests that the SNP effect is not mediated through cGMP. SNP was associated with a decrease in PGE2 and an increase in fibronectin production. These findings suggest that SNP may differ from other NO donors and may be able to affect fibroblast-mediated collagen gel contraction by an action independent of the NO-cGMP pathway.

The current study was originally conceived based on the well-known properties that NO relaxes endothelial cell and smooth muscle cell contraction through stimulating sGC and thus leading to an increase in cGMP (13, 17). In contrast to the expected result, however, NO donors had no effect on fibroblast-mediated collagen gel contraction with the exception of SNP. Similarly, L-NMMA, an inhibitor of NO synthase, also had no effect on collagen gel contraction. These results suggest that NO does not have a role in mediating collagen gel contraction under the culture conditions used. Interestingly, the fibroblasts were capable of responding to the cGMP analog CPT-cGMP. This agent caused a concentration-dependent inhibition of collagen gel contraction. This result suggests therefore that the HFL1 fibroblasts, under the conditions used, may not be able to activate sGC in response to NO.

SNP differed from the other NO donors and clearly affected fibroblast-mediated collagen gel contraction. Its effect, however, was opposite to that observed with CPT-cGMP. Specifically, SNP augmented collagen gel contraction. This effect appears to be independent of the NO-cGMP pathway. Differing effects of SNP from other NO donors have been observed in other systems. In contrast to the relaxant effect of SNP on vascular smooth muscle cells, results of studies of the effect of SNP on airway smooth muscle (lung strips obtained from trachealis from the hilar bronchus) are varied (7, 10, 31). SNP failed to relax lung strips precontracted with KCl, histamine, phorbol myristate acetate, or U-46619. U-46619-precontracted bronchial rings, however, were effectively relaxed by either SNP or NaNO2 (4, 8, 29, 30).

How SNP activates fibroblast-mediated gel contraction is unclear. Photolytical inactivation of the SNP was without effect (data not shown). Similarly, neither sodium nitrite nor sodium nitrate had any effect. How SNP interacts with cellular targets therefore remains to be determined. SNP was, however, associated with an increase in fibronectin production and a decrease in PGE2 production.

Previous reports have demonstrated an increase in PGE production with NO donors (11, 12, 24), an effect reproduced in the current study with SIN-1. However, our result with SNP, an inhibition of PGE production, differs from previous reports (11, 20). Disparate effects of NO donors and NO-mediated pathways on fibronectin production have been observed previously by investigators using different cell culture systems. Studer and co-workers (27) found that NO donor SNAP inhibited basal and thromboxane-stimulated fibronectin synthesis by mesangial cells. In contrast, Pellegatta et al. (21) found that fibronectin synthesis and secretion increased both in the EA.hy 926 cell line and human umbilical vein endothelial cells after prolonged exposure to tumor necrosis factor-alpha or interferon-gamma due to induction of NO synthesis. The differences in PGE response may also reflect differences in species, target cells, and in vitro culture conditions.

Other agents that modulate fibroblast-mediated collagen gel contraction also appear to act, at least in part, through PGE2 and fibronectin. In this regard, PGE2 is a potent inhibitor of fibroblast-mediated collagen gel contraction. Glucocorticoids augment fibroblast-mediated collagen gel contraction through their ability to inhibit PGE2 release (25). Fibronectin can augment fibroblast-mediated collagen gel contraction. Cigarette smoke can attenuate fibroblast-mediated gel contraction, and inhibition of fibronectin production accounts for part of this activity (3). Thus the ability of SNP to inhibit PGE2 and to stimulate fibronectin production is consistent with its action in increasing fibroblast-mediated collagen gel contraction. Interestingly, CPT-cGMP inhibits collagen gel contraction while stimulating PGE2 release and inhibiting fibronectin release.

The effect of SNP on fibroblast-mediated collagen gel contraction appears to be readily reversible. The addition of exogenous PGE2, for example, can block the SNP effect. Similarly, the addition of exogenous CPT-cGMP can also antagonize the effect of SNP.

Although no effect of NO donors on gel contraction was observed in the current study, this does not exclude a role for NO donors in regulating fibroblast-mediated collagen gel contraction. It may be possible, for example, for fibroblasts to induce mechanisms by which NO could lead to increased accumulation of cGMP. Results from the current study suggest that this would result in an inhibition of collagen gel contraction. It is possible therefore that such a pathway could represent an inducible mechanism for regulation of collagen gel contraction.

Taken together, the results of the current study demonstrate that SNP is capable of augmenting collagen gel contraction. The mechanism for this augmentation appears to be independent of the ability of SNP to serve as an NO donor.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. I. Rennard, Univ. of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, Nebraska 68198-5125 (E-mail: srennard{at}unmc.edu).

Received 19 July 1999; accepted in final form 15 December 1999.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 278(5):L1032-L1038
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