|Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
We studied dynamic behavior of cell-substrate contacts, especially focal contacts (FCs), of vascular endothelial cells (ECs) cultured on a glass surface using an interference reflection microscope. ECs were cultured in our flow culture chamber and by completely stopping culture medium flow, we created an in vitro ischemic condition. The FC was a highly stable structure, but, when ECs in a confluent monolayr were kept under the in vitro ischemic condition for 2 hr and then exposed to flow, they transiently lost a significant number of their FCs. ECs detached from the substrate if the flow was fast enough to generate fluid shear stress of - 10 dyn/cm^2. To identify the stress (es) inducing the disruption of FCs, we exposed static EC cultures to conditions which were expected to occur in our in vitro ischemia-reperfusion experiment. ATP depletion, ionophore-induced Ca^<2+> influx, or oxidative stress (H_2O_2, t-butyl hydroperoxide, 15-HPETE) did not cause the loss of FCs. When ECs were pretreated for 40 min with a low pH medium (pH 6.3) containing 20 mM lactate and recovered in a normal pH medium without lactate, they transiently lost their FCs in the similar manner observed in the ischemia-reperfusion experiment. Concomitant with the FC loss, tyrosine phosphorylation of proteins around 120 kDa was augmented. We identified two of these proteins : one is pp125^<FAK> and the other is PECAM-1. Auti-phosphotyrosine staining intensity of the FC increased at the time of the FC loss. Treatment of ECs with tyrosine kinase inhibitors protected ECs from losing their FCs. These results suggest a regulatory role for protein tyrosine phosphorylation in the stability of FCs.