During the last three years of this grant we discovered that the inotropic action of Beta-adrenergic agonists consists of separate and dissociable functional alterations of sarcolemmal calcium flux and sarcoplasmic reticulum calcium sequestration. Zero sodium solutions eliminate the positive inotropic action of isoproterenol, the stimulation of the slow inward calcium current and markedly reduce the rise in intracellular cyclic AMP without altering the relaxant effect of isoproterenol which decreases the time for peak tension development. Our recent success in the development of low background, high affinity reversible and irreversible probes for the Beta-adrenergic receptor and projected additional high molecular weight, electron dense and fluorescent probes should facilitate the functional and anatomical localization of Beta-adrenergic receptors and sites of cyclic AMP generation in the myocardium. The original observation by this laboratory that RNA and protein synthesis inhibitors prevent catecholamine refractoriness will be a focus for studies of the mechanism of catecholamine refractoriness. There is widespread confirmation that RNA and protein synthesis are involved in refractoriness in a wide variety of cell types and hormones. While cyclase can be isolated which shows some refractoriness, the quantitative aspects reflect only a fraction of the dramatic rise in isoproterenol stimulated cyclic AMP accumulation and subsequent refractoriness observed in whole cells. Because of this, acute cell fusion experiments have been initiated and have already demonstrated the ability to acutely transfer altered responsiveness from one cell to the next. This technique will be used to identify and isolate the factor(s) which impart refractoriness. Additional experiments are proposed in this supplemental application to further evaluate our recent finding that long term treatment of cultured cells with isoproterenol or cholera toxin results in a cyclic AMP mediated loss of Beta-adrenergic receptor binding sites. The mechanism of cyclic AMP mediated receptor loss will be investigated to determine if cyclic AMP reduces receptor synthesis or modifies receptor binding by some covalent modification of the receptor.
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