The role of extracellular association of HGF with satellite cells and stretch activation was examined by assaying satellite cell activation in response to stretch as a function of pH. The pH range used in these experiments encompassed the normal range of pH encountered physiologically. By adjusting the pH of medium (Figure ), 25% stretch was applied at 12-s intervals from 12 to 36 h postplating. As illustrated in Figure , activation in control unstretched cultures was pH dependent with a peak at pH 7.23. Activation in stretch-treated cultures exhibited the same pH optimum. Activation above control in stretch treatment only occurred between pH 7.1 and 7.5 with the maximum stretch effect at pH 7.23.
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 (more ...)
The effect of pH on stretch-induced activation could be due to the effect on HGF release from the extracellular matrix or to the effect of pH on HGF binding to its signaling receptor, c-met. To investigate whether the effect was due to changes in HGF activity as a function of pH, unstretched control cultures were subjected to exogenous HGF from 12 to 36 h postplating. Figure illustrates the effect of pH on activation of unstretched satellite cells by HGF. In contrast to results with stretched and unstretched cells in Figure , HGF stimulated activation at all pH treatments examined, between 6.9 and 7.8. The HGF and control curves peaked at pH 7.23 but were parallel over the entire range. This suggests that the pH effect on stretch activation is not due to changes in the ability of HGF to interact with c-met on the target cell to generate the appropriate intracellular signals.
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 (more ...)
The issue was further examined by assessing the effect of pH on HGF binding to a c-met-Fc chimera in an ELISA assay. Figure shows a standard curve for HGF binding in the assay (A) and the effect of pH on HGF binding to c-met (B). Over the pH range used in the previously described cell culture assays, there was no difference in HGF binding to c-met, which agrees with the biological effect of HGF on satellite cell activation in Figure .
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 (more ...)
The second potential explanation for the affect of pH on stretch activation of satellite cells was a pH-dependent alteration in release of HGF from the extracellular domain. Release of HGF into medium in stretched cultures as a function of pH was investigated by immunoblot analysis (Figure ). Very little HGF was present in stretch-conditioned medium at pH 7.0 or 7.1 (Figure , top). Maximum release occurred at pH 7.2, with diminishing amounts at pH 7.4 and 7.7. No HGF was detected in unstretched control cultures (Figure , bottom) at any pH. Maximum release occurred at the same pH as maximum stretch-induced satellite cell activation in Figure .
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 (more ...)
The release of biologically active HGF was further examined by stretching cells at pH 7.7 for variable periods of time from 2 to 20 h (Figure ). Conditioned medium generated from satellite cells that were stretched for 2, 10, or 20 h did not stimulate satellite activation above the levels produced in cultures that received conditioned medium from unstretched cultures. These results demonstrated that even very long periods of stretch could not generate an activation response if pH was unfavorable for stretch-induced release of HGF.
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 (more ...)
The second objective of this study was to investigate the potential involvement of NO in HGF release and satellite cell activation in response to mechanical stretch. In experiments described in Figure , satellite cells were cultured for 12 h and then fed serum-free DMEM containing 1.0 mg of NaHCO3
/ml in the presence or absence of stretch and with one of the following: l
-Arg, or SNP. Conditioned media were subsequently assayed for satellite cell activation as described previously. As seen in Figure A, stretch-conditioned medium (b) and stretch-conditioned medium plus control antibody (c) stimulated significant increases in BrdU incorporation (p < 0.01) relative to unstretched cell conditioned medium (a). Stretch-conditioned medium plus anti-HGF antibody (d) did not stimulate activation relative to the control, and the addition of HGF negated the neutralizing effect of anti-HGF (e). These results are consistent with results from Tatsumi et al. (2001)
and serve as important controls for the other treatments within this experiment.
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 (more ...)
Stretch-conditioned medium was also produced in the presence of d-NAME (f), l-Arg (g), or l-NAME (h) and subsequently tested for activation activity in cultures of unstretched cells. Conditioned medium from stretched cells treated with d-NAME (f) or l-Arg (g) stimulated activation, but conditioned medium from cells stretched in the presence of l-NAME (h), an inhibitor of NOS, did not generate satellite cell-activating activity. When HGF was added to stretch-conditioned medium that was generated in the presence of l-NAME (i) and subsequently assayed for activation activity in cultures of unstretched cells, activation activity was restored, indicating that l-NAME did not directly inhibit satellite cell activation or inhibit HGF action. The final treatment was the addition of an NO-generating compound, SNP, to unstretched cultures (j). Conditioned medium from these cultures stimulated activation comparable to stretch alone.
Figure B displays results of immunoblots analyzing the presence of HGF in medium from the experiment. These immunoblots indicate that the α subunit of HGF (bands in the 60-kDa range) was not found in medium from unstretched cultures (lane a), but was present in medium from stretched cultures (lane b), as seen previously (Tatsumi et al., 2001
). Cultures stretched in the presence of d
-NAME (lane f) or l
-Arg (lane g) also released HGF into medium, but cells stretched in the presence of l
-NAME (lane h) had diminished amounts of HGF in medium. The addition of SNP, a NO donor, to unstretched cultures stimulated the release of HGF into medium (lane k), similar to the effect of mechanical stretch.
The involvement of NO production was further examined by determining the presence of different NOS isoforms in satellite cells and by directly demonstrating that NOS activity is stimulated when satellite cells are subjected to mechanical stretch. Figure presents an immunoblot of 12-h cultured satellite cells and positive controls for eNOS and nNOS (provided with antibodies by Transduction Laboratories); three lanes containing crude lysates of 22,000 cells each were blotted for each form of NOS. Bands corresponding to eNOS and nNOS were present, and there were some additional bands of lower molecular weight immunoreactive proteins in lysates. A very faint band with an apparent molecular mass of 140 kDa that comigrated with the endothelial cell lysate positive control was seen in each of the three lysates blotted with anti-eNOS. Blots for nNOS in the same three lysates also contained bands that comigrated with the 155-kDa band of pituitary nNOS in the positive control. Immunostaining of cultures within the same experiment showed positive staining for nNOS in 96.3 ± 0.52% of the cells, compared with positive eNOS staining of 8.24 ± 0.73% of cells; these cultures were 96.7 ± 0.49% positive for c-met, 95.4 ± 1.0% positive for desmin, and 96.8 ± 0.63% positive for HGF. Although a significant amount of immunoreactive protein was present in blots for eNOS, only a minor percentage of cells were immunopositive for eNOS. Resolution of this apparent discrepancy awaits further experimentation. Nonetheless, these data suggest that both forms of NOS are present in cultures of satellite cells, but on a cell-by-cell basis, immunostaining indicated that nNOS expression was more prevalent in the cell population.
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 (more ...)
Figure describes experiments in which NOS activity was assayed in stretched and control cells as measured by the production of NOx. Figure A presents data showing the time course of NOS activity in stretched and control cells and demonstrates that a significant (p < 0.01) increase in NOx was detectable as early as 1 h after initiation of stretch. The difference between NOS activity in stretched and unstretched cells continued to increase for as long as 20 h after the start of stretch. In Figure B, the effects of l-NAME (b), d-NAME (c), l-Arg (d), and control medium (a) on NOx production by unstretched cells (open bars) and by stretched cells (solid bars) are compared. NOS activity was significantly increased by stretch in cultures fed control medium, medium containing d-NAME, or medium containing l-Arg. In stretched cultures treated with l-NAME, however, NOS activity was inhibited.
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 (more ...)