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Quantification of beta-adrenergic receptors in canine cardiac myocytes using autoradiography and an internal standard
Abstract:
The type and amount of adrenergic receptors in different tissues are important determinants of their response to adrenergic stimulation in normal and pathologic states. Autoradiographic analysis of receptor binding has the advantage that receptor sites can be localized over different tissue compartments. However, quantitative assessment of receptor sites through autoradiography can only be made by using radioactive standards. The purpose of this study was to investigate the use of an internal standard method for quantitative autoradiographic receptor studies by analyzing the binding of the beta-receptor antagonist, [3H]dihydroalprenolol, to canine cardiac tissue. Sections from dog heart were incubated in [3H]dihydroalprenolol to label the beta-adrenergic receptors in the absence of (total binding) and in the presence of (nonspecific binding), 10(-5) M (+/-) propranolol. Specific binding was calculated as total minus nonspecific binding. Half of the sections were scraped off of the slides and assayed by scintillation spectrometry, and half of the sections were set up for light microscopic autoradiography. The autoradiograms were exposed, developed and stained, and grain density was quantified by using an automated image analyzer. By plotting grain density per tissue section against cpm per tissue section for total, nonspecific and specific binding, curves for efficiency of the autoradiographs were obtained. There were no significant differences among the slopes of the three lines. Also, there were no significant differences among the efficiencies of the total, nonspecific and specific binding at different concentrations of [3H]dihydroalprenolol using a two way analysis of variance. Thus, grain counts from the autoradiographs can be used to estimate the concentration of beta-adrenergic receptors in cardiac myocytes. By using these methods it was calculated that canine cardiac myocytes have 5.12 X 10(9) receptor sites/mm3.