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    Characterization of the Hypersensitive Response of Glycogen Phosphorylase to Catecholamine Stimulation in Primary Culture Diabetic Cardiomyocytes: A Thesis

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    Authors
    Buczek-Thomas, Jo Ann
    Faculty Advisor
    Thomas B. Miller, Jr.
    Academic Program
    Biochemistry and Molecular Pharmacology
    UMass Chan Affiliations
    Department of Biochemistry
    Document Type
    Doctoral Dissertation
    Publication Date
    1992-08-01
    Keywords
    Glycogen Phosphorylase
    Receptors
    Catecholamine
    Diabetes Mellitus
    Experimental
    Enzyme Activation
    Heart
    Myocytes
    Cardiac
    Epinephrine
    Rats
    Sprague-Dawley
    Animal Experimentation and Research
    Carbohydrates
    Cardiovascular System
    Cells
    Endocrine System Diseases
    Macromolecular Substances
    Nutritional and Metabolic Diseases
    Organic Chemicals
    Tissues
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    Abstract
    The primary goal of my thesis research was to characterize the basis for the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in primary culture diabetic cardiomyocytes. Toward this goal, I have investigated several key regulatory sites in this signaling pathway which could promote the hypersensitive activation of phosphorylase. Specifically, I investigated (1) which adrenergic receptors are involved in mediating the hypersensitive response of glycogen phosphorylase to epinephrine stimulation; (2) whether the presence of fatty acid metabolites affects phosphorylase activation; (3) whether the hypersensitive response of phosphorylase results from altered signal transduction through the β-adrenergic receptor system or from a post-receptor defect; and (4) the potential role for phosphorylase kinase in mediating the hypersensitive response of phosphorylase to catecholamine stimulation. The basis for adrenergic receptor mediation of the catecholamine-induced activation of glycogen phosphorylase was investigated in adult rat cardiomyocytes isolated from normal and alloxan-diabetic animals. Cells derived from diabetic animals exhibited a hypersensitive response to epinephrine stimulation which was apparent 3 hours after cell isolation and was further enhanced upon maintenance of the myocytes in culture for 24 hours. Normal cells initially lacked the hypersensitive response to epinephrine stimulation although upon maintenance of these cells in culture for 24 hours, the hypersensitive response was acquired in vitro. To assess alpha- and beta- adrenergic mediation of the response, normal and diabetic cardiomyocytes were incubated with propranolol, a β-receptor antagonist, prior to direct α1receptor stimulation with phenylephrine. Both normal and diabetic myocytes failed to undergo activation of phosphorylase in 3 or 24 hour cell cultures. In addition, the effects of epinephrine on phosphorylase activation were completely inhibited by propranolol whereas prazosin, an α-receptor antagonist, was unsuccessful. This data suggests that the hypersensitive response of glycogen phosphorylase in normal and diabetic cardiomyocytes is solely mediated through β-adrenergic receptor activation. Since the accumulation of various fatty acid metabolites can affect certain enzymes and signal transduction pathways within the cell, the potential effect of various fatty acid metabolites on phosphorylase activation was investigated. To determine the potential effects of fatty acid metabolites on phosphorylase activation in cultured cardiomyocytes, normal and alloxan-diabetic cells were incubated with either carnitine or palmitoylcarnitine prior to stimulation with epinephrine. Pretreatment of cardiomyocytes with or without carnitine or palmitoylcarnitine for 3 or 24 hours before epinephrine stimulation failed to alter phosphorylase activation. The addition of exogenous carnitine in the absence and presence of insulin was also unsuccessful in attenuating the hypersensitive phosphorylase activation response in 3 and 24 hour, normal and alloxan-diabetic derived cardiomyocytes. To determine if carnitine palmitoyltransferase 1 (CPT-1) activity was responsible for the hypersensitive response of phosphorylase in the diabetic myocytes, both normal and diabetic myocytes were maintained for 3 and 24 hours in the absence and presence of etomoxir, a CPT-1 inhibitor. Subsequent activation of phosphorylase by epinephrine in normal and diabetic myocytes was unaltered in the presence of etomoxir. Collectively, these data fail to support a critical role for fatty acid metabolite involvement in the hypersensitive activation of glycogen phosphorylase in acute, alloxan-diabetic cardiomyocytes. To assess potential G-protein involvement in the response, normal and diabetic-derived myocytes were incubated with either cholera or pertussis toxin prior to hormonal stimulation. Pretreatment of cardiomyocytes with cholera toxin resulted in a potentiated response to epinephrine stimulation whereas pertussis toxin did not affect the activation of this signaling pathway. To determine if the enhanced response of phosphorylase activation resulted from an alteration in adenylyl cyclase activation, the cells were challenged with forskolin. After 3 hours in primary culture, diabetic cardiomyocytes exhibited a hypersensitive response to forskolin stimulation relative to normal cells. However, after 24 hours in culture, both normal and diabetic myocytes responded identically to forskolin challenge. The present data suggest that a cholera toxin sensitive G-protein mediates the hypersensitive response of glycogen phosphorylase to catecholamine stimulation in diabetic cardiomyocytes. This response, which is present in alloxan-diabetic cells, and is induced in vitroin normal cardiomyocytes, is primarily due to a defect at a post-receptor site. To assess the role of phosphorylase kinase in the hypersensitive activation of glycogen phosphorylase in the diabetic heart, phosphorylase kinase activity was measured initially in perfused hearts (to optimize the assay parameters) and subsequently in primary culture cardiomyocytes. Results from these experiments demonstrate that the present method for measuring phosphorylase kinase activity is a reliable indicator of the enzyme's activity in the heart, although the assay conditions must be further optimized before this system can be applied to the measurement of phosphorylase kinase activity in primary cultured cardiomyocytes.
    DOI
    10.13028/d744-zw50
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/32312
    Rights
    Copyright is held by the author, with all rights reserved.
    ae974a485f413a2113503eed53cd6c53
    10.13028/d744-zw50
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