Endothelin-2 synthesis is stimulated by the type-1 tumour necrosis factor receptor and cAMP: comparison with endothelin-converting enzyme-1 expression

The synthesis of the vasoconstrictor peptide endothelin-2 (ET-2) is depende nt on hydrolysis of the biologically inactive intermediate big ET-2 by an e ndothelin-converting enzyme (ECE). Here, mechanisms inducing ET-2 synthesis have been investigated using the human renal adenocarcinoma cell line (ACH N). Synthesis of ET-2 by ACHN cells was inhibited by phosphoramidon (IC50 c ongruent to 11 mu M). To determine whether ET-2 synthesis occurs in paralle l with the metallopeptidase ECE-1, a putative processing peptidase for big ET-2, changes in the levels of their mRNAs were compared by semi-quantitati ve RT-PCR under conditions causing the upregulation of ET-2 synthesis. Tumo ur necrosis factor-alpha (TNF alpha), forskolin and a cell-permeable cAMP a nalogue (dibutyryl cAMP) caused concentration-dependent increases in ET-2 s ynthesis. Combination of forskolin or dibutyryl cAMP with TNF alpha produce d a significantly greater increase in ET-2 production than these agents alo ne, indicating that adenylate cyclase and TNF alpha induce ET-2 synthesis b y separate signalling pathwayls. Studies using receptor selective TNF alpha mutants, I-125-TNF alpha binding and TNF receptor mRNA showed that type-1 TNF receptors mediate the ET-2 response to TNF alpha. PreproET-2 mRNA level s were increased by TNF alpha at 1 h and 2 h, but returned to control level s at 4 h. Treatment with forskolin significantly increased preproET-2 mRNA levels after 1 h and 4 h. ACHN cells expressed ECE-1b and ECE-1c, but not t he ECE-1a isoform of this peptidase. RT-PCR for the combined isoforms ECE-1 b/c/d showed TNF alpha to increase mRNA levels at 2 h and 4 h. Forskolin ha d no effect on ECE-1b/c/d mRNA levels. Thus, expression of ET-2 and ECE-1b/ c/d mRNAs in ACHN cells do not display the co-ordinated regulation observed with typical peptide prohormone processing enzymes and their substrates.