Secretion of endothelin-1 and endothelin-3 by human cultured vascular smooth muscle cells.
1. It is generally accepted that endothelial cells secrete endothelin (ET) to the underlying media which mediates the contractile effects of ET. However, there is some evidence that animal vascular smooth muscle cells (VSMCs) also secrete ET. We cultured VSMCs from human vessels representative of a number of different vascular beds to determine whether human VSMCs endogenously secrete ET. 2. VSMCs explanted from adult arterial vessels secrete picomolar quantities of immunoreactive mature ET: coronary artery 226.6 +/- 58.8 pM/10(6) cells (n = 7), thoracic aorta 169.5 +/- 105.4 pM/10(6) cells (n = 3), left internal mammary artery 102.4 +/- 23.1 pM/10(6) cells (n = 3) and saphenous vein 69.4 +/- 19.9 pM/10(6) cells (n = 3), as well as from umbilical vein (HUVSMCs) 38.3 +/- 4.3 pM/10(6) cells (n = 3). Secretion of immunoreactive big ET-1 was also detected: coronary artery 249.1 +/- 59.4 pM/10(6) cells (n = 7), thoracic aorta 120.0 +/- 13.4 pM/10(6) cells (n = 3), left internal mammary artery 170.0 +/- 68.2 pM/10(6) cells (n = 3), saphenous vein 105.1 +/- 30.7 pM/10(6) cells (n = 3) and from umbilical vein 146.3 +/- 7.4 pM/10(6) cells (n = 3). Comparable, intracellular levels of immunoreactive big ET-1 and mature ET were also detected in cultured VSMCs. 3. Since enzyme-dispersed VSMCs are thought to be more differentiated and more closely resemble their in vivo counterparts, and these enzyme-dispersed VSMCs from human umbilical vein (HUVSMCs) also secreted the greatest levels of immunoreactive peptides, they were characterized further. Reverse transcription-polymerase chain reaction assay demonstrated that HUVSMCs express ET-1 mRNA. High performance liquid chromatography coupled to radioimmunoassay revealed that HUVSMCs secrete ET-1 and ET-3, in addition to big ET-1. However, levels of ET are not altered by 100 AM phosphoramidon,an inhibitor of metalloproteases or by 100 microM pepstatin A, an aspartyl protease inhibitor.4. In concordance, KD and Bmax values for [125I]-ET-l saturation binding are not altered in HUVSMC cultures incubated for 24 h with 100 microM phosphoramidon (431 +/- 218 PM and 31.1 +/- 12.7 fmol mg-1;mean =/- s.e.mean, n = 3) or 100 microM pepstatin A (381 +/- 169PM and 19.9 +/- 7.8 fmol mg-1, n = 3) as compared to controls (355 +/- 99 pM and 33.3 +/- 9.3 fmol mg-1; n = 3). This observation indicates the absence of an autocrine 'unmasking' effect for ET receptors.5. HUVSMCs synthesize and secrete immunoreactive ET-1, ET-3 and big ET-1, and possess intracellular levels of immunoreactive mature ET and big ET-1. There is some evidence of common cellular mechanisms between growth factors and vasoconstrictor peptides, suggesting a close relationship between contraction and proliferation. Since the development of various vascular pathologies such as atherosclerosis, hypertension and after vessel injury has been attributed to alterations in the normal growth pattern of VSMCs, the role of ET in these diseases may be of significance.
Yu
J C
JC
Clinical Pharmacology Unit, University of Cambridge, Addenbrooke's Hospital.
Davenport
A P
AP
D000962:Antimalarials / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D019332:Endothelin-1; D016232:Endothelins / Q000096:biosynthesis / Q000378:metabolism*; D005260:Female; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D011863:Radioimmunoassay; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
D000962:Antimalarials; D019332:Endothelin-1; D016232:Endothelins; D006020:Glycopeptides; D010436:Pepstatins; D011480:Protease Inhibitors; D011498:Protein Precursors; D011506:Proteins; D012333:RNA, Messenger; C014195:Streptomyces pepsin inhibitor; C008890:phosphoramidon; C031375:pepstatin