FLOW-RELATED RESPONSES OF INTRACELLULAR INOSITOL PHOSPHATE LEVELS IN CULTURED AORTIC ENDOTHELIAL-CELLS

In vitro and in vivo evidence indicates that hemodynamic wall shear stress evokes a diversity of biological responses in vascular endothelial cells, ranging from cell shape changes to alterations in low density lipoprotein receptor expression. The signal transduction mechanisms by which the level of fluid mechanical shear stress is recognized by the endothelial cell and translated into these diverse biological responses remain to be elucidated. The present study focuses on the association between the onset of elevated shear stress and activation of the phosphoinositide signal transduction pathway, as measured by the intracellular release of inositol phosphates, in cultured bovine aortic endothelial cells (BAECs). BAECs were seeded, grown to confluence on large polyester sheets, and preincubated with 0.3 muCi/ml [H-3]inositol for 24 hours before insertion in parallel-plate flow chambers for exposure to high shear stress (HS) at 30 dynes/cm2 or low shear stress (LS) at <0.5 dyne/cm2 for periods ranging from 15 seconds to 24 hours. The induction of HS was associated with an early, transient but significant increase (142%, HS/LSx100%) in inositol trisphosphate (IP3) measured at 15 seconds of shear stress exposure followed by a major peak in IP3 (189%) observed at 5 minutes after HS onset. After these initial increases, IP3 levels returned to near resting levels within 30 minutes of continued HS exposure and then continued to decline to significantly lower (75%) levels relative to IS-treated cells within 4 hours and remained lower throughout the remainder of the 24-hour HS exposure. IS-treated cells exhibited no significant changes in inositol phosphate levels throughout the 24-hour exposure periods. Exposure of BAECs to shear stress of 60 dynes/cm2 resulted in an approximately fourfold increase in IP3 levels (396%) measured at 5 minutes, almost double the levels measured in cells exposed to 30 dynes/cm2 for 5 minutes. Pretreatment of BAECs for 30 minutes with 5 mM neomycin, an inhibitor of phosphoinositide metabolism, before HS exposure inhibited both the early increases in inositol phosphates and subsequent cell elongation and alignment observed in untreated BAECs simultaneously exposed to HS without inhibiting protein synthesis. These results indicate that the exposure of cultured BAECs to elevated wall shear stress is associated with an early biphasic IP3 increase followed by a resetting of intracellular inositol phosphate concentrations to levels below that observed in static cultured BAECs. Furthermore, neomycin inhibition of this IP3 response to shear stress is associated with an inhibition of one of the major endothelial biological responses to shear stress, i.e., cell shape change and orientation. These results provide evidence that the phosphoinositide signal transduction pathway may be an important component in transduction of the mechanical shear stress signal into at least some of the vascular endothelial responses to flow.