Uneven cellular expression of recombinant alpha2A-adrenoceptors in transfected CHO cells results in loss of response in adenylyl cyclase inhibition |
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Authors: | Björk Susann Vainio Minna Scheinin Mika |
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Affiliation: | Department of Pharmacology and Clinical Pharmacology, University of Turku, It?inen Pitk?katu 4, FI-20520 Turku, Finland. susann.bjork@utu.fi |
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Abstract: | 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. |
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