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Beschreibung

Millipore's Ready-to-Assay GPCR frozen cells are designed for simple, rapid calcium assays with no requirement for intensive cell culturing. Millipore has optimized the freezing conditions to provide cells with high viability and functionality post-thaw. The user simply thaws the cells and resuspends them in media, dispenses cell suspension into assay plates and, following over night recovery, assays for calcium response. The endogenous catecholamines epinephrine and norepinephrine have profound effects on smooth muscle activity, cardiac function, carbohydrate and fat metabolism, hormone secretion, neurotransmitter release, and central nervous system actions. These activities are mediated by GPCRs belonging to two subfamilies, the alpha- and beta-adrenoceptors (Bylund et al., 1994). The three members of the alpha1 subclass of adrenoceptors, alpha1A, alpha1B and alpha1D, couple to Gq, and promote contraction of vascular and urinary tract smooth muscle, relaxation of intestinal smooth muscle, increased contractile force in the heart, and glycogenolysis and gluconeogenesis in the liver. The different subtypes have overlapping distributions and variably contribute to these effects depending on species and tissue. The alpha1D adrenergic receptor mediates smooth muscle contraction in several tissues. In the vasculature, activation of alpha1D increases blood pressure (Tanoue et al., 2002, Hosoda et al., 2005). In the urinary tract, alpha1D promotes bladder contraction. Antagonists of alpha1 receptors are used to treat bladder outlet obstruction, and this effect is thought to be mediated by alpha1D (Chen et al., 2005). The alpha1D adrenergic receptor has a relatively long N-terminal extracellular domain, and truncation of this domain has been shown to increase expression of the receptor at the cell surface (Pupo et al., 2003). Millipore's cloned human alpha1D -expressing cell line contains a version of alpha1D lacking residues 2-79. The cell line is made in the Chem-1 host, which supports high levels of recombinant alpha1D expression on the cell surface and contains high levels of the promiscuous G protein to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for agonists and antagonists at alpha1D.