Mechanisms and time course of beta1 adrenoceptor desensitisation in mammalian cardiac myocytes

To study the mechanisms and time course of beta1 adrenoceptor desensitisation in mammalian heart tissue neonatal rat cardiac myocytes (>90% pure) were cultured in serum free medium. Cells were exposed to 1 μmol·litre−1 (–)-isoprenaline for 30 min, 4 h, and 16 h. In myocyte membranes mean(SEM)) 125I-iodocyanopindolol binding was 167(46) pmol·litre−1 (n=5) and did not differ at 30 min, 4 h, or 16 h in control compared with (–)-isoprenaline treated cells. The maximum number of binding sites was 84(32) fmol·mg protein−1 and was unchanged at 30 min, but (–)-isoprenaline stimulated adenylate cyclase activity significantly decreased from 221(62) to 103(37) pmol·mg protein−1 30 min−1. (–)-Isoprenaline competition curves at 30 min showed a significant increase in the proportion of low affinity binding sites from 46% to 62% (n=5). By 4 h the maximum number of binding sites was significantly decreased by 54%, adenylate cyclase activity remained depressed, and agonist affinity decreased threefold in the (–)-isoprenaline treated cells. At 16 h (–)-isoprenaline treated cells showed alterations similar to the 4 h values in the maximum number of binding sites, adenylate cyclase activity, and affinity for (–)-isoprenaline. (–)-Isoprenaline stimulated adenylate cyclase activity took 72 h to recover after desensitisation. Overnight ultracentrifugation of the cytosol showed a significant 40% increase in beta adrenoceptor density in cells exposed to (–)-isoprenaline for 4 h (n=5), suggesting receptor internalisation. Thus a dissociation between beta adrenoceptor density and loss of adenylate cyclase activity occurs within 30 min of (–)-isoprenaline exposure and is accompanied by a relative shift to a low affinity state for the agonist. Subseqent loss of beta receptors is probably due to translocation to the cytosol. These data suggest that alterations in agonist affinity for the beta adrenoceptor may be more important than receptor number in determining functional response in mammalian cardiac myocytes during early desensitisation, whereas subsequent desensitisation is associated with both persistent changes in agonist affinity and redistribution of beta adrenoceptors into the cytosol.