three cells To investigate the effect of ET 1 on COX two PGE2 sys tem, bEnd. three cells had been incubated with many concen trations of ET 1 for the indicated time intervals. The data showed that ET 1 induced COX 2 expression in a time and concentration dependent manner. There was a significant raise inside two four h, reached a maximal response inside six h, and declined within 24 h. ET 1 also time dependently induced COX 2 mRNA ex pression in bEnd. 3 cells, determined by RT PCR. There was a significant boost in COX two mRNA inside 30 min, and reached a maximal response within 2 h. Moreover, to confirm regardless of whether ET 1 induces COX 2 expression via the transcription activity of COX 2 promoter, cells had been transiently transfected with COX two promoter luciferase reporter construct and then sti mulated with ET 1 for the indicated time intervals.
As shown in Figure 1C, ET 1 time dependently induced COX two promoter luciferase activity in bEnd. three cells. A maximal response was obtained within four h. Our earlier studies have shown that selleck chemical COX two expression induced by BK or sphingosine 1 phosphate is mainly accountable for prostanoid release in many cell varieties. As a result, to figure out whether ET 1 could induce PGE2 biosynthesis, we collected the conditioned media and determined PGE2 levels by using an EIA kit. The results showed that ET 1 time dependently stimulated PGE2 re lease and also a significant PGE2 production was observed inside four h, reached a maximal response within six h and slightly declined within 24 h. These outcomes sug gested that ET 1 induces COX two PGE2 method through up regulating COX 2 gene expression in bEnd.
3 cells. ET 1 upregulates COX two expression through an ETB receptor ET 1 exerts its biological effects by way of ET receptors, which includes ETA and ETB, that are members of GPCR superfamily. Initially, we determined which subtypes of ET receptors are expressed selleckchem Omecamtiv mecarbil on bEnd. three cells by RT PCR. The data showed that ETB but not ETA receptors are expressed on bEnd. 3 cells. Next, to determine the subtypes of ET receptors involved in ET 1 induced COX two expression, pretreatment with BQ 788, but not BQ 123, attenuated the ET 1 induced COX 2 protein and mRNA expression, suggesting that ETB receptor is predominantly involved in these responses. To further confirm this note, transfection of cells with ETB siRNA considerably down regulated ETB protein expression and inhibited ET 1 induecd COX two expression.
These data suggested that ET 1 induced COX 2 expression is mediated via an ETB receptor dependent manner in these cells. Involvement of a Gi and Gq protein coupled ETB receptor in ET 1 induecd COX two expression ET receptor has been shown to be a pleiotropic GPCR for ET 1 which can be coupled to G proteins including Gi and Gq. To additional ascertain which of G proteins was involved in ET 1 induced COX 2 expression, pretreatment with either Gi protein antagonist GP antagonist 2 or Gq protein antagonist GP antagonist 2A con centration dependently attenuated ET 1 induced COX two protein and mRNA expression.