The endothelium is a critical component of the vasculature in serving as a barrier between the blood and surounding tissues and as a source of many vasoactive mediators such as nitric oxide (NO) and endothelin-1 (ET-1) [l]. As part of the inflammatory response, particularly in the lung, the permeability of the endothelium is increased allowing humoural and cellular elements of the blood access to underlying tissue. Control of endothelial barrier function in general is mediated by a variety of factors including thrombin, oxidants, and inflammatory cytokines such as interleukin-1 (IL-1) and tumour necrosis factor-a (TNF-a) [2]. It is also possible that endothelium-derived mediators such as NO and ET-1 could participate in regulation of endothelial permeability. The aim of this study was to investigate production of ET-1 by bovine pulmonary artery endothelial cells (BPAEC) and the potential role of ET-1 in modulating endothelial barrier function in vitro.Transcription of the prepro-ET-1 gene in BPAEC was first demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers (see Figla). Secretion of ET-1 by BPAEC was then investigated by collecting medium conditioned by confluent cells for 4, 12 or 24 h and subjecting it to radioimmunoassay (RIA) using an antiserum to the peptide's Cterminus. Indirect evidence for the presence of endothelin converting enzyme (ECE), which converts pro-ET-1 to ET-1, was obtained using the ECE inhibitor, phosphoramidon (PA). As shown in Fig. lb, BPAEC secreted ET-1 in a time-dependent manner, reaching 1980 fmoV106 cells at 24 h. Secretion was significantly inhibited at all time points by lO'M phosphoramidon, implying the presence of ECE inlon BPAEC.To investigate potential regulation of ET-1 secretion by TNF-a, confluent BPAEC monolayers were exposed to medium containing a range of TNF-a concentrations for 4, 8 or 24 h. Conditioned medium was collected, extracted by Sep-Pak C18 chromatography and analysed for ET-1 by RIA as above. As shown in Table 1, TNF-a elicited a dose-depndent increase in ET-1 secretion from BPAEC at all times examined, culminating in a greater than 2-fold increase at 24 h.To study the potential role of ET-1 in modulating endothelial barrier function either directly (by itself) or indirectly (as part of TNF-a response) BPAEC grown on Transwell assemblies as previously described [3] were used. In brief, BPAEC grown to confluency on porous Transwell inserts were exposed to either TNF-a or ET-1 for 1 or 20 h. Following washing, trypan blue labelled bovine serum albumin (TB-BSA) was added to the upper chamber of the insert while buffer was added to the lower chamber. After a 60 min incubation at 3 7 T , buffer from the lower chamber was sampled, TB-BSA transfer measured spectrophotometrically at 590 nm and expressed as a percentage of that initially added to the upper chamber of the Transwell insert. 1 2 3 4 5 6 m m I 3 3 g 1000 li d Figure 1 : (A) RT-PCR of prepro-endothelin-1. Lane 1 = 100 bp ladder. Lane 2 = no template control. La...
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