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. 2002 Aug;13(8):2909-18.
doi: 10.1091/mbc.e02-01-0062.

Release of hepatocyte growth factor from mechanically stretched skeletal muscle satellite cells and role of pH and nitric oxide

Ryuichi Tatsumi  1 , Akihito Hattori, Yoshihide Ikeuchi, Judy E Anderson, Ronald E Allen
Affiliations

Release of hepatocyte growth factor from mechanically stretched skeletal muscle satellite cells and role of pH and nitric oxide

Ryuichi Tatsumi et al. Mol Biol Cell. 2002 Aug.

Abstract

Application of mechanical stretch to cultured adult rat muscle satellite cells results in release of hepatocyte growth factor (HGF) and accelerated entry into the cell cycle. Stretch activation of cultured rat muscle satellite cells was observed only when medium pH was between 7.1 and 7.5, even though activation of satellite cells was accelerated by exogenous HGF over a pH range from 6.9 to 7.8. Furthermore, HGF was only released in stretched cultures when the pH of the medium was between 7.1 and 7.4. Conditioned medium from stretched satellite cell cultures stimulated activation of unstretched satellite cells, and the addition of anti-HGF neutralizing antibodies to stretch-conditioned medium inhibited the stretch activation response. Conditioned medium from satellite cells that were stretched in the presence of nitric-oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester hydrochloride did not accelerate activation of unstretched control satellite cells, and HGF was not released into the medium. Conditioned medium from unstretched cells that were treated with a nitric oxide donor, sodium nitroprusside dihydrate, was able to accelerate the activation of satellite cells in vitro, and HGF was found in the conditioned medium. Immunoblot analysis indicated that both neuronal and endothelial NOS isoforms were present in satellite cell cultures. Furthermore, assays of NOS activity in stretched satellite cell cultures demonstrated that NOS is stimulated when satellite cells are stretched in vitro. These experiments indicate that stretch triggers an intracellular cascade of events, including nitric oxide synthesis, which results in HGF release and satellite cell activation.

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Figures

Figure 1
Figure 1
Adjustment of culture medium pH. DMEM-10% HS was adjusted by varying the NaHCO3 concentration. (A) Equilibration rate with addition of various concentrations of NaHCO3 in a humidified atmosphere of 5% CO2 at 37°C. (B) Standard curve for pH and NaHCO3.
Figure 2
Figure 2
Effect of medium pH on stretch activation of satellite cells. (A) Stretch pattern applied to culture. (B) Effect of pH on BrdU incorporation in stretched (●) and control (○) satellite cell cultures at 36 h postplating. Points represent means and SE for four cultures per treatment.
Figure 3
Figure 3
Effect of pH on activation of unstretched satellite cells by HGF. (A) Dose-response curve for HGF and satellite cell activation, as assessed by BrdU labeling index at 36 h postplating. (B) pH dependence of HGF action on satellite cell activation at 36 h postplating: 2.5 ng HGF/ml (●) and control (○). Points represent means and SE for four cultures per treatment.
Figure 4
Figure 4
Effect of pH on HGF binding to c-met. An ELISA binding assay was conducted that used a c-met-Fc chimera bound to plate and soluble HGF ligand; bound HGF was detected by anti-HGF antibody conjugated to horseradish peroxidase. (A) Standard curve for HGF binding. (B) Effect of pH on HGF Binding. Points represent means and SE for four wells per treatment.
Figure 5
Figure 5
Effect of pH on release of HGF from stretched satellite cells. Medium was analyzed from stretched and unstretched control satellite cell cultures after 2-h treatment in DMEM beginning at 12 h postplating. Each lane represents proteins from a constant number of cultured satellite cells (1500 cells). Top, immunoblots show the effect of pH on HGF release into medium in stretched cultures. Bottom, immunoblots from unstretched control culture conditioned medium. The first lane in the top panel shows molecular weight standards, and the first lane in the bottom panel shows a control with pH 7.2 stretched medium and no first antibody.
Figure 6
Figure 6
Biological activity of stretch-conditioned medium generated from cultures maintained at pH 7.7. Conditioned medium was prepared by subjecting cultures to various periods of stretch in pH 7.7 medium. Conditioned medium was fed to cultures of unstretched satellite cells at 12 h postplating, and BrdU incorporation from 34 to 36 h postplating was assessed at 36 h postplating. Treatment conditioned media were prepared from 2-h unstretched cultures (a), 2-h stretched cultures (b), 10-h stretched cultures (c), and 20-h stretched cultures (d). Bars show means and SEs for four cultures per treatment.
Figure 7
Figure 7
Stretch activation is affected by altering HGF availability and NO metabolism. Serum-free DMEM, pH 7.2, was used to prepare conditioned medium from satellite cell cultures that were stretched or unstretched for 2 h beginning at 12 h postplating. Medium was analyzed in satellite cell activation assays at 36 h postplating or by immunoblots. (A) Effects on satellite cell activation by the following conditioned medium treatments: control medium from unstretched cultures (a), medium from stretched cells (b), stretch medium plus 2 μg/ml control antibody (c), stretch medium plus 2 μg/ml anti-HGF neutralizing antibody (d), stretch medium plus 2 μg/ml anti-HGF neutralizing antibody plus 20 ng/ml HGF (e), stretch in the presence of 10 μM d-NAME (f), stretch in the presence of 2.4 mM l-Arg (g), stretch in the presence of 10 μM l-NAME (h), stretch in the presence of 10 μM l-NAME followed by addition of 2.5 ng/ml HGF (i), and no stretch in the presence of 30 μM SNP (j). Bars represent the means and SEs from three cultures per treatment, and significant differences from control unstretched means are indicated (∗∗, p < 0.01). (B) Corresponding immunoblots of conditioned medium from the treatments in A: no stretch (a), stretch conditioned medium (b), stretch in the presence of d-NAME (f), stretch in the presence of l-Arg (g), stretch in the presence of l-NAME (h), no stretch in the presence of SNP (k), and control blot of stretch medium without primary antibody (l). Each lane represents proteins from a constant number of cultured satellite cells at 12 h postplating (2700 cells). STD, biotinylated molecular weight standards.
Figure 8
Figure 8
Immunoblot detection of eNOS and nNOS in 12 h satellite cell cultures. Molecular weight standards are shown in the first lane. Bands corresponding to eNOS (*) and nNOS (**) are indicated next to lanes of respective positive controls (PC1, human endothelial cell lysate; PC2 rat pituitary lysate). Three separate 12 h satellite cell culture lysates were analyzed for each form of NOS, cell lysates 1, 2, and 3 are designated under each lane. CNT represents blot of lysate 1 without primary antibody.
Figure 9
Figure 9
NOS activity in stretched and control cells. (A) Time course of NOS activity in stretched (●) and control (○) cells and in control medium without cells (⋄). (B) NOS activity in cultures subjected to the following treatments: control medium (a), 10 μM l-NAME (b), 10 μM d-NAME (c), 2.4 mM l-Arg (d), and control medium with no cells (e). Each treatment was applied to unstretched (open bars) and stretched (solid bars) cultures for 20 h from 12 to 32 h postplating. Each point or bar represents the mean and SE for three cultures per treatment, and significant differences from control unstretched means are indicated (∗∗, p < 0.01).
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