Premium
Endothelin ET A receptor expression in human cerebrovascular smooth muscle cells
Author(s) -
Yu Julie C.M.,
Pickard John D.,
Davenport Anthony P.
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb15093.x
Subject(s) - endothelin receptor , receptor , microbiology and biotechnology , biology , endothelin 1 , vascular smooth muscle , binding site , endothelins , cell culture , complementary dna , medicine , endocrinology , biochemistry , smooth muscle , gene , genetics
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 subtypes 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 (RT‐PCR) assays and subsequent product separation by agarose gel electrophoresis revealed single bands corresponding to the expected product sizes encoding cDNA for ET A (299 base pairs) and ET B (428 base pairs) (n = 3 different cultures). 4 Autoradiography demonstrated the presence of specific binding sites for [ 125 I]‐ET‐1 which labels all ET receptors, and [ 125 I]‐PD151242, an ET A subtype‐selective antagonist which exclusively labels ET A receptors, but no specific binding was detected using ET B subtype‐selective [ 125 I]‐BQ3020 ( n = 3 different cultures, in duplicate). 5 In saturation binding assays, [ 125 I]‐ET‐1 bound with high affinity: K D = 0.8 ± 0.1 nm and B max = 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 ). [ 125 I]‐PD151242 also bound with similar affinity: K D = 0.4 ± 0.1 nm and B max = 388 ± 68 fmol mg −l (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 [ 125 I]‐ET‐I 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 ET A receptors. 6 Although messenger RNA encoding both ET A and ET B receptors was detected, autoradiographical analysis, as well as binding studies indicate that human cultured brain smooth muscle cells express only ET A receptor protein. Antagonism of this subtype may be necessary to block the actions of ET‐1 in the human cerebral resistance vessels in the vasospasm observed subsequent to subarachnoid haemorrhage.