Subclassification of β-adrenergic receptors in cultured rat cardiac myoblasts and fibroblasts

Abstract

β-Adrenergic receptors in primary cultures of neonatal rat cardiac cells were identified with the radioligand [125I]iodohydroxybenzylpindolol ([125I]HYP). At the time of cell plating, a differential attachment procedure was employed to separate myocardial (M) cells from fibroblast-like (F) cells. After 3-4 days, the cultures enriched in M cells were still more than 80% pure and the cultures enriched in F cells were more than 95% pure. For binding studies, confluent cell layers were nonenzymatically detached as single cell suspensions. Both M and F cells contained a limited number of β-adrenergic receptors (M, 7600 ± 2100 sites/cell; F, 9000 ± 2400 sites/cell) which had very high affinity [M, K(d) = 88 ± 33 pM; F, K(d) = 71 ± 23 pM] for [125I]HYP. For each cell type, the binding sites were stereoselective for the l-isomers of agonists and antagonists. Further binding studies on the relative potency of β-agonists showed that the β-receptors in M cells could be subclassified as β1 (isoproterenol>epinephrine≃norepinephrine), whereas the β-receptors in F cells were more typical of β2 (isoproterenol>epinephrine>norepinephrine). The order of potency of these catecholamines in stimulating the adenylate cyclase activity in M and F cells was consistent with the order of potency observed in binding studies. Practolol, a β1 inhibitor, was 30 times more effective as a competitor of [125I]HYP binding in M cells than in F cells. It thus appears that, whereas M and F cells share a similar number of receptors per cell and similar affinities for [125I]HYP, the receptors in the two cell types nevertheless can be distinguished on the basis of their subclassification. These results also emphasize the importance of obtaining a homogeneous cell population for studies of the β-adrenergic receptor in cultured cardiac tissue.