C5a에 의해 자극된 호중구에서 세포내 칼슘동원에 대한 Protein Tyrosine Kinase의 조절작용

Regulatory Action of Protein Tyrosine Kinase in Intracellular Calcium Mobilization in C5a-stimulated Neutrophils

본 연구는 C5a에 의해 자극된 호중구에서 세포내 칼슘유리와 세포외부로부터 칼슘유입에 있어 protein kinase C와 protein tyrosine kinase의 관여 여부를 조사하였다. Protein kinase C 억제제인 staurosporine과 H-7은 C5a에 의해 자극된 호중구에서 세포내 칼슘유리를 억제하였으나, 세포막을 교차한 칼슘유입이나 세포내 칼슘농도 증가에 영향을 나타내지 않았다. C5a에 의한 세포내 칼슘유리와 칼슘유입은 protein tyrosine kinase 억제제인 genistein과 methyl-2,5-dihydroxycinnamate에 의해서 억제 되었다. ADP에 의해 야기된 세포내 칼슘농도의 증가는 genistein과 methyl-2,5-dihydroxycinnamate에 의해서 억제되었으나 staurosporine과 H-7의 영향은 받지 않았다. Genistein과 methyl-2,5-dihydroxy-cinnamate는 thapsigargin을 처리한 호중구에서 칼슘유입을 감소시켰으나 이에 대한 staurosporine 과 H-7의 효과는 나타나지 않았다. 호중구를 phorbol 12-myristate 13-acetate로 전처치하였을때 세포내 칼슘증가에 미치는 C5a의 자극 효과는 감소하였다. 이상의 결과로 부터 protein tyrosine kinase는 C5a에 의해 활성화된 호중구에서 세포내 칼슘유리와 세포막을 교차한 칼슘유입의 조절에 관여할 것으로 추정된다.

The present study was done to examine the involvement of protein kinase C and protein tyrosine kinase in intracellular $Ca^{2+}$ mobilization in C5a-stimulated neutrophils. Although protein kinase C inhibitors, staurosporine and H-7 inhibited intracellular $Ca^{2+}$ release in C5a-stimulated neutrophils, they did not affect $Ca^{2+}$ influx across the plasma membrane and elevation of $[Ca^{2+}]_i$ C5a-induced intracellular $Ca^{2+}$ release and $Ca^{2+}$ influx were inhibited by protein tyrosine kinase inhibitors, genistein and methyl-2,5-dihydroxycinnamate. ADP-evoked elevation of $[Ca^{2+}]_i$ was inhibited by genistein and methyl-2,5-dihydroxycinnamate but was not affectd by staurosporine and H-7. Genistein and methyl-2,5-dihydroxycinnamate reduced the store-regulated $Ca^{2+}$ influx in thapsigargin-treated neutrophils, while the effect of staurosporine and H-7 was not detected. When neutrophils were preincubated wih phorbol 12-myristate 13-acetate, the stimulatory effect of C5a on the elevation of $[Ca^{2+}]_i$ was reduced. These results suggest that protein tyrosine kinase may be involved in control of intracellular $Ca^{2+}$ release and $Ca^{2+}$ influx across the plasma membrane in C5a-activated neutrophils.