IN-VITRO ENZYMATIC PROCESSING OF RADIOLABELED BIG ET-1 IN HUMAN KIDNEY

We have investigated enzymatic processing of big ET-1 in sections of h uman renal cortex by examining selected binding characteristics of the radiolabelled precursor and cleaved peptide. Sections of histological ly normal human kidney obtained from patients undergoing nephrectomy f or hypemephroma (50-74 years, N = 10, male or female) were incubated w ith 0.1 nM [I-125]-ET-1, [I-125]-Tyr(13) big ET-1 or [I-125]-Tyr(31) b ig ET-1 in culture media at 37 degrees to facilitate enzymatic activit y. Specific binding measured from sections incubated with [I-125]-Try( 13) big ET-1 (which would yield [I-125]-ET-1 on enzymatic cleavage) wa s 39.7 +/- 2.5%. This was significantly reduced to 19.0 +/- 2.0% follo wing co-incubation with 10 mu M thiorphan, an inhibitor of neutral end opeptidase (NEP) but not the putative endothelin converting enzymes (E CE). No further reduction in specific binding was obtained with 100 mu M thiorphan, indicating that this is a maximal effect. However phosph oramidon (100 mu M), an inhibitor of ECE and NEP, almost abolished spe cific binding, indicating that both NEP and ECE cleave big ET-1 in the kidney. No specific binding was detected when sections were labelled with [I-125]-Tyr(31) big ET-1 (which would be expected to yield [I-125 ] labelled C-terminal fragment). Binding of the product of processed [ I-125]-Tyr(13) big ET-1 was inhibited mainly by the ETB selective anta gonist (BQ788 = 75.1 +/- 2.1% inhibition; FR139317 = 9.7 +/- 7.3% inhi bition), consistent with the predominance of this subtype in human kid ney. We conclude that big ET-1 is processed by NEP and ECE in human ki dney and that the cleaved product binds predominantly to the ETB recep tor subtype. ECE may be a therapeutic target in the attenuation of ren al diseases in which ET-1 has been implicated. (C) 1998 Elsevier Scien ce Inc.