Uneven cellular expression of recombinant alpha2A-adrenoceptors in transfected CHO cells results in loss of response in adenylyl cyclase inhibition.
Two populations of Chinese hamster ovary (CHO) cells expressing similar numbers of recombinant human alpha2A-adrenergic receptors (alpha2A-AR) showed different capacity to inhibit adenylyl cyclase (AC) activity. Cells transfected with an integrating vector exhibited agonist-dependent inhibition of forskolin-stimulated AC, whereas cells transfected with a non-integrating episomal vector showed no inhibition. Fluorescent microscopy and flow cytometry revealed a very uneven receptor distribution in the episomally transfected cell population. Monoclonal cell populations were expanded from this parent population. Most clones lacked significant amounts of receptors, while a few expressed receptors at high density; these exhibited efficient agonist-dependent inhibition of forskolin-stimulated AC activity. Thus, dense receptor expression in only a few cells is not sufficient to evoke a significant inhibitory response in a functional assay where AC is stimulated in all cells. Consequently, a false negative result was produced. Furthermore, the cell population transfected with an integrating vector showed loss of homogeneity with increasing passage number.
Björk
Susann
S
Department of Pharmacology and Clinical Pharmacology, University of Turku, Itäinen Pitkäkatu 4, FI-20520 Turku, Finland. susann.bjork@utu.fi
Vainio
Minna
M
Scheinin
Mika
M
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D016466:CHO Cells; D005576:Colforsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors; D006801:Humans; D018341:Receptors, Adrenergic, alpha-2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
C547768:ADRA2A protein, human; D000067956:Adenylyl Cyclase Inhibitors; D018341:Receptors, Adrenergic, alpha-2; D011994:Recombinant Proteins; D005576:Colforsin