Continuous monitoring of nitric oxide release from human umbilical vein endothelial cells

Direct measurement of nitric oxide (NO) release is pivotal for understanding its role in the regulation of vascular tone. However, data on the direct measurement of NO have been scarce. Recent description of NO-selective electrode has prompted us to examine NO release from endothelial cells using this approach. In the present study, we continuously monitored [NO] in the incubation medium conditioned by cultured human umbilical vein endothelial cells (HUVEC) with an amperometric NO-sensor. The HUVEC released NO on stimulation with several agonists such as alpha-thrombin, bradykinin, L-arginine and ionomycin; the responses were characterized by an initial rise and a subsequent sustained increase. Activation of Ca/calmodulin system resulted in a robust elevation in [NO], occasionally displaying an oscillatory component. Calmidazolium pretreatment attenuated the ionomycin-induced response. Pretreatment with phorbol ester suppressed the ionomycin-induced NO release from HUVEC. Forskolin pretreatment did not modify NO release elicited by ionomycin. These findings indicate that the synthesis/release of NO in endothelial cells is a Ca/calmodulin dependent step. Activation of protein kinase C interferes with the Ca/calmodulin-induced activation of NOS in endothelial cells. Thus, the present study shows that NO synthase is a substrate for phosphorylation by different kinases which modulate the activity of the enzyme as determined by continuous monitoring of NO release from endothelial cells using a specific NO-sensor.