• Mechanisms of ET-1-mediated 02~ production in the rat aorta

      Loomis, E. D.; Department of Medicine (2004-06)
      The objectives of this project were to test the hj^othesis that in the rat aorta endothelin-I (ET-I) binds to the ETa receptor stimulating superoxide (O2’ ) production. Furthermore, we wanted to identify the mechanism through which ET-1-mediates O2' production. Chemiluminescent detection of O2” production using probes such as lucigenin has been widely used with enzyme systems, leukocytes, and vascular tissues. Our first goal was to develop a microplate high-throughput protocol for lucigeninamplified chemiluminesence detection of 0 2 'L We have developed a novel adaptation to lucigenin-based assays that allows up to 36 samples to be counted at virtually the same time. Recent studies have shown that NOS 3 can become uncoupled and produce O2* when deprived of its cofactor BH4 . In addition several authors have shown that ONOOoxidizes BH4 in vitro. Using the high-throughput lucigenin assay and dihydroethidine (DHE) staining we have shown that (1) ET-1 is able to stimulate 0 2 "^ production in both endothelium-intact and -denuded vessels through the ETA-receptor, (2) ET-1 stimulates O2’ production through both NAD(P)H oxidase and an endothelial source of NOS, and (3) addition of exogenous tetrahydrobiopterin (BH4 ) and inhibition of peroxynitrite (ONOO-) inhibit ET-I-mediated 0 2 *^ production. Therefore our data have led us to hypothesize that ET-I stimulates O2* production by activating NAD(P)H oxidase through the ETa receptor. O2' production by NAD(P)H oxidase leads to the formation of ONOO- and the degradation of BH4. The loss of BH4 leads to uncoupled NOS which then contributes to ET-1-mediated production. In addition, we have found that (1) ET-1 increases the production of interleukin- 6 (IL-6 ) and (2) ET-1-mediated O2' production adversely affects vascular contractility. Although these consequences do not appear to be due to NOS uncoupling, they help support the role of ET-1 in vascular dysfunction.