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The human megakaryoblastic cell lines HEL, MEG-01, and DAMI express preproendothelin-1 mRNA. This investigation was designed to find out whether they could also express endothelin-converting enzyme (ECE) and release mature endothelin (ET). RT-PCR applied to RNA isolated from the cell lines amplified fragments of the expected size. The amplified cDNA of MEG-01 was submitted to restriction enzymes, which generated the expected subfragments. Membrane ECE activity was phosphoramidon-sensitive, in contrast to the cytosolic activity capable of producing ET-1 from big ET-1. The three cell lines produced ir-ET in a time-dependent manner. These results show that human megakaryoblastic cell lines express functional, phosphoramidon-sensitive and insensitive ECE activity and produce mature ET.

Because it has been shown that human platelet precursors from normal bone marrow express preproendothelin-1 (ET-1) mRNA, this investigation was designed to find out whether these cells could synthesize and release mature ET-1 and express ET receptors. Therefore, we examined the expression of endothelin-converting enzyme (ECE) mRNA and of mRNAs for ET receptors in cells purified from normal bone marrow and measured immunoreactive (ir)ET in their culture medium. RT-PCR applied to RNAs from platelet precursors yielded amplified fragments of the expected sizes of 567, 428, and 299 bp for ECE, ET B and ET A receptors, respectively. These cells released ir-ET into the culture medium in a time-dependent fashion. These results raise the possibility of autocrine actions of the intrinsic ET system in bone marrow platelet precursor cells.

The presence of endothelin (ET) receptors and the nature of the subtype and expression of ET were investigated in the human megakaryoblastic cell line MEG-01. By the RT-PCR procedure, we have shown that both ETA and ETB receptor subtype mRNAs are expressed in the cells. However, binding experiments have shown that the selective ETB receptor antagonist BQ788, but not the selective ETA receptor antagonist BQ123, competes with the specific binding of [125I]ET-1. Using immunocytochemistry, RIA, and RT-PCR Southern blot, we have shown that MEG-01 cells express ET-1. In addition, ET (1-21)-like immunoreactivity was released from the cells into the culture medium, and this release was modulated by thrombin. These data suggest that an ET-1-mediated autocrine loop could occur in the human megakaryoblastic MEG-01 cell line.

The effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) were investigated on carbachol-contracted guinea-pig trachea. ET-1 and ET-3 induced a dose-dependent relaxation of the precontracted preparations. ET-1-induced relaxation was attenuated by hemoglobin and methylene blue. By contrast, ET-3-induced relaxation was not affected by hemoglobin or methylene blue. The large conductance Ca(2+)-activated K(+)-channel blocker charybdotoxin antagonized ET-1- and ET-3-induced relaxations. These results show that charybdotoxin-sensitive K+ channels are involved in both ET-1 and ET-3 relaxant activities, whereas the nitric oxide pathway is involved only in ET-1 relaxant effects.