Lack of effect of the alpha2C-adrenoceptor Del322-325 polymorphism on inhibition of cyclic AMP production in HEK293 cells.
OBJECTIVE
The alpha(2C)-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human alpha(2C) polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells.


METHODS
Human alpha(2C) wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200-2320 fmol.mg(-1) protein) was measured following agonist treatment.


RESULTS
Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325.


CONCLUSIONS
The alpha(2C) WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the alpha(2C)-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.
Montgomery
M D
MD
Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska 69198-5800, USA.
Bylund
D B
DB
D000262:Adenylyl Cyclases / Q000378:metabolism*; D058669:Adrenergic alpha-2 Receptor Antagonists; D000316:Adrenergic alpha-Agonists / Q000378:metabolism / Q000494:pharmacology*; D001667:Binding, Competitive; D000068438:Brimonidine Tartrate; D002460:Cell Line; D003000:Clonidine; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D006801:Humans; D016014:Linear Models; D009638:Norepinephrine; D011110:Polymorphism, Genetic; D011810:Quinoxalines; D011869:Radioligand Assay; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection
C547774:ADRA2C protein, human; D058669:Adrenergic alpha-2 Receptor Antagonists; D000316:Adrenergic alpha-Agonists; D020536:Enzyme Activators; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D005576:Colforsin; D000068438:Brimonidine Tartrate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D003000:Clonidine; D009638:Norepinephrine