Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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Effect of hemolysate on tyrosine phosphorylation

Fig. 1: The level of tyrosine phosphorylation of cellular proteins by Western blotting.

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Fig. 2: Hemolysate-induced [Ca²⁺]_i elevation in rat basilar smooth muscle cells.

The effect of hemolysate on [Ca²⁺]_i was concentration-dependent (Fig. 3).

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Fig. 3: The effect of hemolysate on [Ca²⁺]_i.

In the absence of extracellular Ca²⁺ (buffer also contained 1.0 mM EGTA), the addition of 10% hemolysate raised [Ca²⁺]_i to a peak level and a sustained plateau, significantly less than the peak and plateau [Ca²⁺]_i responses of the cells to hemolysate in the presence of extracellular Ca²⁺ (Fig. 4).

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Fig. 4: Hemolsaye-induced [Ca²⁺]_i elevation in the absence of external Ca²⁺.

These data indicate that [Ca²⁺]_i entry may be an important component in both peak and plateau [Ca²⁺]_i responses to hemolysate.

Effects of tyrosine kinase inhibitors on hemolysate-induced [Ca²⁺]_i

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Fig. 5: Hemolysate-induced [Ca²⁺]_i elevation in pre-incubated cell with genistein.

Increasing the genistein concentration to 100 M further reduced the response to hemolysate (Fig. 6).

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Fig. 6: Inhibitory effect of genistein on [Ca²⁺]_i by hemolysate.

Tyrphostin A51, a synthetic tyrosine kinase inhibitor that is structurally different from genistein, produced a similar inhibitory effect to the [Ca²⁺]_i response to hemolysate (Fig. 7, Fig. 8).

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Fig. 7: Hemolysate-induced [Ca²⁺]_i elevation in pre-incubated cell with tyrphostin A51.

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Fig. 8: Inhibitory effect of tyrphostin A51 on [Ca²⁺]_i by hemolysate.

To test the specificity of those tyrosine kinase inhibitors, tyrphostin A1, an inactive analogue of tyrphostins, was used in a similar fashion. Tyrphostin A1 (30 M) did not alter the [Ca²⁺]_i response evoked by hemolysate.

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Fig. 9: Inhibitory effect of staurosporine on [Ca²⁺]_i by hemolysate.

Effects of tyrosine kinase inhibitors on fractionated hemolysate-induced [Ca²⁺]_i

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Fig. 10: Small molecular weight hemolysate-induced [Ca²⁺]_i elevation in rat basilar smooth muscle cells.

The effect of the small molecular weight fraction of hemolysate on [Ca²⁺]_i was also dose dependent (Fig. 11).

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Fig. 11: The effect of small molecular weight hemolysate on [Ca²⁺]_i.

In contrast, the large molecular weight fraction of hemolysate (>10 kD) did not evoke a marked response in [Ca²⁺]_i (Fig. 12).

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Fig. 12: The effect of large molecular weight hemolysate on [Ca²⁺]_i.

Genistein (30 M) pretreatment for 5 min inhibited both the initial peak and the sustained phase of [Ca²⁺]_i evoked by the small molecular weight hemolysate fraction (Fig. 13).

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Fig. 13: Small molecular weight hemolysate-induced [Ca²⁺]_i elevation in pre-incubated cell with genistein.

Increasing the genistein concentration to 100 M further reduced the [Ca²⁺]_i response to the small molecular weight fraction (Fig. 14).

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Fig. 14: Inhibitory effect of genistein on [Ca²⁺]_i by small molecular weight hemolysate.

A similar inhibitory response was obtained by tyrphostin A51, significantly reducing both the initial peak and the plateau [Ca²⁺]_i responses to small molecular weight hemolysate fraction (Fig. 15, Fig. 16).

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Fig. 15: Small molecular weight hemolysate-induced [Ca²⁺]_i elevation in pre-incubated cell with tyrphostin A51.

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Fig. 16: Inhibitory effect of tyrphostin A51 on [Ca²⁺]_i by small molecular weight hemolysate.

The [Ca²⁺]_i peak response to a 10% concentration of the large molecule fraction was nearly completely inhibited by genistein or tyrophostin A51.

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