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.beta-gal). Ad.CMV.secECE (1-10 x 10(9) 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.beta-gal groups (0.3-0.5 pM). When Ad.CMV.secECE was co-injected with Ad.CMV.ET-1 (2.5 x 10(9) pfu/ml each), plasma ET-1 levels were significantly elevated compared to the control group co-injected with Ad.CMV.secECE and Ad.CMV.beta-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 ETA 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.
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Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas, Southwestern Medical Center, Dallas 75235-9050, USA.
Emoto
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deWit
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Yanagisawa
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D000257:Adenoviridae Infections / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D001381:Azepines / Q000494:pharmacology; D001794:Blood Pressure / Q000187:drug effects; D002417:Cattle; D018076:DNA, Complementary / Q000096:biosynthesis; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1 / Q000096:biosynthesis* / Q000097:blood; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000096:biosynthesis / Q000097:blood; D006801:Humans; D006973:Hypertension / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D063065:Organophosphonates / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000096:biosynthesis / Q000097:blood; D051381:Rats; D017208:Rats, Wistar; D044022:Receptor, Endothelin A; D013777:Tetrazoles / Q000494:pharmacology
D001381:Azepines; D018076:DNA, Complementary; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; D016232:Endothelins; D007211:Indoles; D063065:Organophosphonates; D011480:Protease Inhibitors; D011498:Protein Precursors; D044022:Receptor, Endothelin A; D013777:Tetrazoles; C079574:FR 139317; C089987:CGS 26303; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes