Heterologous in vivo processing of human preproendothelin 1 into bioactive peptides.
Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET-1 serum. ET-1-like immunoreactivity was detected in oocytes injected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured. These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis.
Fabbrini
M S
MS
Istituto Biosintesi Vegetali, Consiglio Nazionale delle Ricerche, Milan, Italy.
Vitale
A
A
Patrono
C
C
Zamai
M
M
Vaghi
F
F
Caiolfa
V
V
Monaco
L
L
Benatti
L
L
D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D019332:Endothelin-1; D016232:Endothelins / Q000032:analysis* / Q000235:genetics* / Q000494:pharmacology; D005260:Female; D015723:Gene Library; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology; D009841:Oligonucleotides; D009865:Oocytes / Q000502:physiology; D010449:Peptide Mapping; D010920:Placenta / Q000502:physiology; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000235:genetics; D011817:Rabbits; D011863:Radioimmunoassay; D014684:Venae Cavae / Q000187:drug effects / Q000502:physiology; D014981:Xenopus
D019332:Endothelin-1; D016232:Endothelins; D009841:Oligonucleotides; D011498:Protein Precursors; D012313:RNA