In vivo role of endothelin-converting enzyme-1 as examined by adenovirus-mediated overexpression in rats

We previously reported that adenovirus-mediated overexpression of endothelin-1 (ET-1) elevates systemic blood pressure in rats. In this model, plasma big ET-1:ET-1 ratios were almost 30, whereas they were only 5 in the control group, suggesting that endothelin-converting enzyme (ECE) may be a rate-limiting step in the production of ET-1 under these conditions. To further investigate the role of ECE in vivo, we prepared recombinant adenovirus strains carrying a soluble, secretory form of bovine ECE-1 cDNA (Ad.CMV.secECE), human ET-1 cDNA (Ad.CMV.ET-1), and, as a control, E. coli lacZ (Ad.CMV.β-gal). Ad.CMV.secECE (1-10 × 10⁹ pfu/ml) was injected into the caudal vein of male Wistar rats and the animals were studied 96 h later. Immunoblot analysis of circulating plasma confirmed the expression of the soluble ECE-1. The plasma levels of big ET-1 and mature ET-1 were similar in Ad.CMV.secECE and Ad.CMV.β-gal groups (0.3-0.5 pM). When Ad.CMV.secECE was co-injected with Ad.CMV.ET-1 (2.5 × 10⁹ pfu/ml each), plasma ET-1 levels were significantly elevated compared to the control group co-injected with Ad.CMV.secECE and Ad.CMV.β-gal (10.2 ± 2.4 vs. 1.1 ± 0.2 pM). Big ET-1 levels were threefold higher (3.7 ± 1.1 vs. 1.2 ± 0.4 pM), and systemic blood pressure was significantly elevated (132 ± 3 vs. 90 ± 3 mm Hg) in the Ad.CMV.secECE + Ad.CMV.ET-1 group. Administration of an ECE inhibitor (CGS 26303, 30 mg/kg) significantly reduced the blood pressure in the Ad.CMV.secECE + Ad.CMV.ET-1 group (from 125 ± 5 to 74 ± 6 mm Hg) but not in the control group (from 85 ± 2 to 75 ± 3 mm Hg). Infusion of an ET_A antagonist (FR 139317; 0.2 mg/kg/min for 30 min) also significantly reduced the blood pressure only in the Ad.CMV.secECE + Ad.CMV.ET-1 group, without any significant effect in the control group. This study demonstrates that even though overexpression of ECE-1 in itself does not lead to systemic hypertension, the enzyme can be a crucial rate-limiting factor in the production of mature ET-1 in vivo. Furthermore, this model may prove to be useful for in vivo screening of ECE inhibitors.