The myocardial response to catecholamines is significantly diminished in many types of shock or heart failure. The guinea pig heart is an ideal model for the study of shock, as it is relatively inexpensive, and the cardiovascular system of the guinea pig most closely resembles that of the human. Using this model, we have developed techniques to characterize and quantitate changes in β-adrenergic receptors (βAR) in the guinea pig heart after burn injury. Preliminary experiments were performed to determine the optimum binding conditions, e.g., incubation time and conditions, protein concentrations, rinsing, etc. Additional experintents were conducted using agonists and antagonists to characterize the rank order of potency and stereospecificity of the βAR. Crude membrane preparations (50 μg/250μl) from sham-burned and burned hearts were incubated with 8-10 concentrations of 125 I-cyanopindolol (10-450 pM) at 37°C for 1 hr. Under these conditions, binding assays were linear with respect to protein concentration and time. Alprenolol (10 μM) was used to determine nonspecific binding. The membrane preparations used in this study bound both agonists and antagonists with a rank order of potency and stereospecificity characteristic of a β-adrenergic receptor. Finally, agonist competition curves were performed with isoproterenol in the presence and absence of Gpp(NH)p to determine receptor regulation by the Gs protein. Analysis using computer-assisted techniques suggests that the fraction of high-affinity β-receptors is significantly reduced after burn injury (41.2 ± 4.7%) compared to sham-burned controls (54 ± 2%, P ≤ 0.023). The methods described here can be easily adapted to correlate cardiac dysfunction with changes in β-adrenergic receptors during many types of shock or trauma.
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