Mercury-arc photolysis: A method for examing second messenger regulation of endothelial cell monolayer integrity

Abstract

Cell-cell apposition in bovine pulmonary endothelial cell monolayers was modulated by inducing transient increases in intracellular adenosine 3′:5′-cyclic monophosphate (cAMP) and 1,4,5-inositol triphosphate (IP 3). This was accomplished by mercury-arc flash photolysis of o-nitrobenzyl derivatives of the second messengers (caged compounds). Second messenger release by the mercury-arc lamp was determined by radioimmunoassay of cAMP to have a t 1 2 of approximately 8 min. Each second messenger induced the phosphorylation of a distinct subset of cytoskeletal proteins; however, both IP 3 and cAMP increased vimentin phosphorylation. Actin isoform patterns were not altered by the second messengers. Intracellular pulses of IP 3 in pulmonary endothelial cells caused disruption of endothelial monolayer integrity as determined by phase-contrast microscopy and by visualization of actin stress fibers with rhodamine-phalloidin. Intracellular pulses of cAMP increased cell-cell contact, cell surface area, and apposition. IP 3 appeared to have its greatest effect on the actin peripheral band. In silicone rubber contractility assays this agent caused contraction of pulmonary microvascular endothelial cells as visualized by an increase in wrinkles beneath the cells. On the other hand, cAMP appeared to effect both the peripheral band and centralized actin domains. Caged cAMP caused relaxation of endothelial cells as visualized by a disappearance of wrinkles beneath the cells.