Beta adrenoceptors and cyclic amp production in muscle cells isolated from adult heart

Abstract

1. Adult rat heart muscle cells were isolated after retrograde perfusion of the heart with a collagenase-containing solution. About 80% of the isolated cells were elongated and excluded trypan blue. 2. Binding of (-) [3H] dihydroalprenolol ([3H]DHA) to isolated myocardial cells did not fulfil a major criterion for β-adrenoceptor binding, namely, stereoselectivity. This was not due to the absence of β-adrenoceptors in myocardial cells as [3H]DHA binding to particulate material prepared from these cells was stereoselective. Results of experiments carried out in an attempt to explain the lack of stereoselective binding of [3H] DHA to whole myocardial cells were explained. 3. Binding of (±)[125I] iodocyanopindolol ([125I]CYP) to isolated myocardial cells was saturable and stereoselective. The order of potency of β-agonists in competing for [125I] CYP binding sites was the same as their relative potency in stimulating cyclic AMP production. Dissociation constants for β-antagonists estimated from competition for [125I]CYP binding sites correlated with those estimated from inhibition of isoprenaline-stimulated cyclic AMP production. Scatchard analysis of [125I]CYP saturation isotherms indicated that binding was to a single class of sites and the KD was 37 pM. From the Bmax, the number of β-adrenoceptors per cell was calculated assuming that each receptor site binds to one molecule of radioligand. The number of β-adrenoceptors per cell increased in proportion to the cell size during growth of the heart within the range investigated (0.26 to 0.98 g wet weight). However, the number of β-adrenoceptors per μm2 of the cell surface ares was constant 25 β-adrenoceptors per μm2). The compound Ro 03-7894, reported to be an irreversible β-antagonist, could not be used to block varying proportions of myocardial cell β-adrenoceptors in order to determine cyclic AMP production coupled to the remaining receptors, since its binding to isolated myocardial cells dissociated after washing. Evidence for the presence of "spare" β-adrenoceptors in isolated myocardial cells was obtained by using non-radioactive cyanopindolol to block varying proportions of receptors and then determining cyclic AMP production coupled to the remaining receptors. Myocardial cells produced maximal amounts of isoprenaline-stimulated cyclic AMP despite blockade of about 50% of the β-adrenoceptors.