Endothelin ETA receptor expression in human cerebrovascular smooth muscle cells.
1. Endothelin (ET) has been implicated in cerebrovasospasm for example, following subarachnoid haemorrhage, and blocking the interaction of ET with its receptors on cerebral vessels, may be of therapeutic benefit. The aim of our study was to characterize endothelin receptor sub-types on medial smooth muscle cells of human cerebral vessels. Cultures of vascular smooth muscle cells were explanted from human cerebral resistance vessels and characterized as human brain smooth muscle cells (HBSMCs). 2. Over a 48 h incubation period, HBSMC cultures secreted comparable levels of immunoreactive (IR) big endothelin-1 (big ET-1) and IR endothelin (ET): 12.7 +/- 10.3 and 8.3 +/- 5.6 pmol/10(6) cells, respectively (mean +/- s.e. mean from three different individuals), into the culture medium. 3. Total RNA was extracted from cultures of human brain smooth muscle cells. Reverse-transcriptase polymerase chain reaction (RI-PCR) assays and subsequent product separation by agarose gel electrophoresis revealed single bands corresponding to the expected product sizes encoding cDNA for ETA (299 base pairs) and ETB (428 base pairs) (n = 3 different cultures). 4. Autoradiography demonstrated the presence of specific binding sites for [125I]-ET-1 which labels all ET receptors, and [125I]-PD151242, an ETA subtype-selective antagonist which exclusively labels ETA receptors, but no specific-binding was detected using ETB subtype-selective [125I]-BQ3020 (n = 3 different cultures, in duplicate). 5. In saturation binding assays, [123I]-ET-1 bound with high affinity: KD = 0.8 +/- 0.1 nM and Bmax = 690 +/- 108 fmol mg-1. A one-site fit was preferred and Hill slopes were close to unity over the concentration range (10(-12) to 10(-8) M). [125I]-PD151242 also bound with similar affinity: KD = 0.4 +/- 0.1 nM and Bmax = 388 +/- 68 fmol mg-1 (mean +/- s.e. mean, n = 3 different cultures). Again, a one-site fit was preferred and Hill slopes were close to unity over the concentration range. Unlabelled PD151242 competed for the binding of [125I]-ET-1 monophasically and analysis of the competition curves indicated that a one-site fit was preferred over a two-site model, implying that the cultures express mainly ETA receptors. 6. Although messenger RNA encoding both ETA and ETB receptors was detected, autoradiographical analysis, as well as binding studies indicate that human cultured brain smooth muscle cells express only ETA receptor protein. Antagonism of this sub-type may be necessary to block the actions of ET-1 in the human cerebral resistance vessels in the vasospasm observed subsequent to subarachnoid haemorrhage.
Yu
J C
JC
Clinical Pharmacology Unit, University of Cambridge, Addenbrooke's Hospital.
Pickard
J D
JD
Davenport
A P
AP
D000368:Aged; D001345:Autoradiography; D001381:Azepines / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D001667:Binding, Competitive / Q000187:drug effects; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation / Q000187:drug effects*; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; D016232:Endothelins / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D006801:Humans; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D011863:Radioimmunoassay; D017466:Receptors, Endothelin / Q000096:biosynthesis*
D001381:Azepines; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; D016232:Endothelins; D009842:Oligopeptides; D010446:Peptide Fragments; D011498:Protein Precursors; D012333:RNA, Messenger; D017466:Receptors, Endothelin; C075771:BQ 3020; C087850:PD 151242