Invited Symposium: Stroke/Cerebral Vasospasm



Materials & Methods


Discussion & Conclusion



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Tyrosine Kinase In Cerebral Vasospasm

Contact Person: S. Iwabuchi, MD, PhD (iwabuchi@med.toho-u.ac.jp)

Discussion and Conclusion

The results with hemolysate and smooth muscle cells were very different from those we obtained previously with endothelial cells. We had found that the addition of a red blood cell lysate to cultured vascular endothelial cells induced a much greater transient increase in the level of the tyrosine phosphorylation of two proteins, approximately 60 and 110 kDa.16) The level of phosphorylation of both was maximal between 1 and 2 min and returned to basal within 10 min. The initial [Ca2+]i peak in endothelial cells was unchanged in Ca2+-free solutions indicating the peak was solely due to store release whereas there is both an initial Ca2+ influx and store release in the muscle cells.

Tyrosine kinases do not appear to have the same roles in controlling [Ca2+]i in the two cell types. While genistein reduced the [Ca2+]i peak in the muscle cells, the endothelial peak was unchanged indicating no role of tyrosine kinases for signalling Ca 2+ release in those cells. Genistein nearly completely eliminated the plateau phase of the hemolysate response in endothelial cells but it only caused a partial inhibition of the muscle [Ca2+]i plateau phase. Based upon the results with staurosporine, PKC has a role in opening Ca2+ channels for the secondary influx in muscle while it had no apparent role in controlling [Ca2+]i responses to hemolysate in endothelial cells.16) Although the [Ca2+]i responses to hemolysate appear to be identical in endothelial and smooth muscle cells, the controlling mechanisms are distinct.

A careful review of the [Ca2+]i responses of the smooth muscle cells to hemolysate indicates that they are the result of a multi-component system. There may be multiple active molecules in hemolysate and there may be multiple receptors and/or signaling pathways. The possibility of multiple active components in hemolysate can not be ruled out. The large molecule fraction obviously elicits a response in the cells but the effect on [Ca2+]i is small. Some components in hemolysate could have indirect effects upon [Ca2+]i through crosstalk of signaling pathways. Their activity could modify responses to other molecules.

[Ca2+]i changes in the smooth muscle cells are modified but not solely controlled by tyrosine kinases. In terms of the [Ca2+]i peak response to hemolysate, tyrosine kinases could have roles in signaling receptor binding at the membrane, triggering Ca2+ store release, or opening the channels for the initial Ca2+ influx. Hemolysate-evoked tyrosine kinase phosphorylation may be involved in the regulations of both Ca2+ release and Ca2+ entry in cerebral smooth muscle cells.

The concentrations of genistein and tyrphostin A51 (30-100 M) used in this study are in the same range as reported by others and are comparable to that required for inhibition of tyrosine kinase.15) At such concentrations, these agents selectively inhibit tyrosine kinases but are without effect against other kinases, such as cyclic adenosine monophosphate dependent protein kinase (PKA) or protein kinase C (PKC).15) In this study, genistein and tyrphostin A51, two structurally different inhibitors, significantly attenuated both the transient peak and the sustained plateau phase in [Ca2+]i, while tyrphostin A1 failed to exert an effect in cerebral smooth muscle cells. Genistein has some effect on other protein kinases and signal transduction systems besides tyrosine kinases, whereas tyrphostin A51 selectively inhibits tyrosine kinases.20)21) Therefore, two structurally different tyrosine kinase inhibitors, genistein and tyrphostin A51, showed similar inhibition of hemolysate-induced [Ca2+]i response, further supporting the view that in cerebral arteries, tyrosine kinases are involved in the regulation of [Ca2+]i elevated by hemolysate.

Tyrosine kinases are key elements in cellular signal transduction pathways and play important roles in the regulation of smooth muscle tone. Tyrosine kinases consist of three general subclasses: (1) membrane receptor tyrosine kinases, including the insulin receptor and receptors for epidermal growth factor and platelet-derived growth factor; (2) cytosolic non-receptor protein tyrosine kinases such as the protooncogene products Abl and Fes; and (3) membrane-associated non-receptor tyrosine kinases related closely to pp60v-src.14) A large number of potential substrates for these tyrosine kinases have been identified including those, such as inositol 1,4,5-triphosphate (IP3) receptors, phospholipase C_ and MAP kinase, that are believed to be directly involved in cell signaling.22)23) G-protein, as a substrate for tyrosine phosphorylation has also been documented recently.24) In human platelets and fibroblasts, the tyrosine kinases have been found to be primarily involved in the regulation of Ca2+ entry pathway,25)26) whereas, in smooth muscle cells receptor activation, such as by phenylephrine, 5-HT, vasopressin or endothelin, increases protein tyrosine phosphorylation which regulates both Ca2+ release from internal Ca2+ stores and Ca2+ entry.15)

We conclude that hemolysate activates tyrosine phosphorylation in rat basilar smooth muscle cells. Elevation of tyrosine kinase activity may play a role in Ca2+ release and entry. Tyrosine kinase inhibitors reduce [Ca2+]i responses to hemolysate and its small and large molecule fractions. Tyrosine kinase inhibitors may be useful in the management of cerebral vasospasm following aneurysmal subarachnoid hemorrhage.

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Iwabuchi, S MD; Zhang, J MD PhD.; Aoki, K MD.; Marton, L PhD.; Kimura, H MD.; Samejima, H MD; (1998). Tyrosine Kinase In Cerebral Vasospasm. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/zhang/iwabuchi0452/index.html
© 1998 Author(s) Hold Copyright