Merle S. Olson - US grants

The research described in this application is designed to characterize the regulatory properties of the pyruvate dehydrogenase multienzyme complex in the rat brain. Metabolic flux through the pyruvate dehydrogenase reaction, the activation state of the enzyme complex and the level of phosphorylation of the enzyme will be determined in mitochondrial and synaptosomal preparations derived from several individual regions of the brain taken from adult rats and from rats during their early developmental stages. The regulation of pyruvate dehydrogenase or pyruvate metabolism in general will be investigated in perfused brains derived from adult rats and developing rats. The effects of alterations in the hormal and/or the nutritional state of the animal on the regulatory parameters for the pyruvate dehydrogenase complex will be investigated in this metabolic preparation. The long range goal of this research project is to document the crucial relationship between the ability ofthe brain to regulate its important energy generating pathways and the maintenance of neuro-logical competence in this tissue.

The major objective of the proposed research program is to elucidate regulatory mechanisms for two multienzyme complexes involved in mirochondrial energy production in cardiac tissue. The regulation of the pyruvate and the branched chain Alpha-keto acid dehydrogenase enzyme complexes by two forms of covalent modification (e.g., phosphorylation and acylation) will be investigated using the purified enzyme complexes from bovine heart, isolated rat heart mitochondria and in the isolated perfused rat heart. Experimental support will be sought for our contention that these two enzyme complexes are activated by their respective natural substrates but inactivated by the substrates for the other complex, e.g., pyruvate activates pyruvate dehydrogenase but inactivate the branched chain complex while the branched chain Alpha-keto acids activate the branched chain complex but inactivate the pyruvate complex. Of particular interest will be our suggestion that the pyruvate dehydrogenase kinase may be regulated by acylation/acetylation by various Alpha-keto acid substrates or Coenzyme A derivatives. Finally, the effects of various metabolic and hormonal states on the regulation of the pyruvate and branched chain dehydrogenase complexes will be investigated in perfused heart preparations. Experiments are proposed to test a suggestion that insulin exerts its stimulatory effect on the pyruvate dehydrogenase complex through an effect of a (e.g., disulfide containing) mediator substance on a reactive thiol moiety on the pyruvate dehydrogenase kinase. It is anticipated that the proposed studies will provide a necessary and unique perception of how the regulation of these two very important enzyme complexes is crucial for the maintenance of an appropriate energy generating capability in my myocardium.

This proposal outlines our experimental approach to three important regulatory problems involved in liver metabolism. The first of these problems concerns the characterization of a regulatory relationship between the ketogenic and gluconeogenic pathways mediated by the mitochondrial monocarboxylate translocator. We have proposed that the exchange of mitochondrial acetoacetate for cytosolic pyruvate via the monocarboxylate translocator is a primary regulator of the supply of precursors for the gluconeogenic pathway. Several experiments are proposed to test and to elaborate upon this hypothesis in various liver-derived metabolic systems. Considerable effort will be given to the development of specific inhibitors of the monocarboxylate translocator with an ultimate goal of isolating and characterizing the molecular properties of this important mitochondrial substrate transport system. Second, we have proposed studies to define certain aspects of the regulatory effects of Alpha-adrenergic agonists such as epinephrine in the liver. Experiments have been suggested a) to define the source of the calcium ions which are released into the liver cytosol upon Alpha-stimulation and b) to characterize the effects of Alpha-adrenergic stimulation on various mitochondrial processes. Finally, the regulatory properties of the hepatic glycine cleavage system will be investigated in perfused rat livers and in isolated liver mitochondria. The possible regulatory effects of various alternative substrates (e.g., fatty acids, amino acids and ketone bodies) and nucleotide species (e.g., adenine and pyridine nucleotides) will be assessed. Possible inhibitory effects of several compounds on mitochondrial glycine transport will be evaluated. Changes in the glycine cleavage system will be monitored in livers derived from animals in several nutritional/hormonal state (e.g., fed, fasted diabetic, etc.). Ultimately we would like to define the relationship between the activity of the glycine synthase multienzyme complex in the liver and various clinically defined hyperglycinemic states.

DESCRIPTION (Adapted from applicant's abstract): Endothelin is a 21 amino acid peptide synthesized as three isopeptides by vascular endothelial cells exposed to various types of stress or injury. All major organs including lung, kidney heart, brain and liver have been shown to both produce and respond to the agonist effects of endothelin in autocrine and paracrine signaling mechanisms. The liver is one of the most responsive tissues exhibiting both hemodynamic and glycogenolytic responses at low nM concentrations and specific signal transduction responses in cultured hepatic-derived cells at pM concentrations. The objective of the proposed research program is to characterize regulatory mechanisms in which endothelin participates in the mammalian liver in physiological and pathophysiological situations. Specific experimental goals include: a) characterization of endothelin-mediated signaling mechanisms or responses in hepatic-derived cultured cells; specifically, endothelin receptors on sinusoidal endothelial cells will be identified and characterized and endothelin-mediated synthesis of lipid and peptide secondary messengers will be characterized; b) investigation of the capability of hepatic sinusoidal endothelial cells to synthesize endothelin and the elucidation of factors and conditions which regulate endothelin synthesis; c) characterization of endothelin-mediated signaling mechanism operative in various pathophysiological models of systemic and hepatic injury such as endotoxic shock, hepatic ischemia/reperfusion injury, and obstructive jaundice. Successful outcomes from the proposed research will provide mechanistic insight into the role and the importance of probably the most potent vasoactive mediator yet discovered in the hepatic responses to inflammation/injury. Understanding the biochemical or molecular details of the endothelin receptor-mediated signaling mechanisms and the metabolic functions which are responsive to endothelin in pathophysiological episodes may facilitate the development of appropriate therapeutic interventions designed to minimize or ameliorate impairment of hepatic function following exposure to systemic or specific hepatic trauma.

biomedical equipment resource; biomedical equipment purchase;

During the past decade the accomplishments of our research effort have established the liver as a novel and important model in which to characterize the autocrine and paracrine mediator response of platelet- activating factor. Our experiments have provided insights into the nature and regulatory characteristics of the PAF receptor and have provided at least a glimpse of the importance of PAF as a mediator of hepatic responses to systemic and localized hepatic pathophysiology. PAF is synthesized in Acting through specific receptors and well defined signal transduction mechanisms, activation of glycogenolysis and glucose output. Temporally later in a trauma-response view, represents a key factor in regulating the entry of inflammatory cells from the circulation into the compromised liver. In the next grant period we will pursue four major experimental issues designed to characterize: 1) the regulation of two key of PAF receptor mRNA synthesis, and 4) the role of PAF as an inflammatory mediator in three relevant models of hepatic injury, e.g., ischemia/reperfusion, bile duct ligation-induced jaundice and sepsis/endotoxemia. In each of these hepatic injury models we have measured an increase in the hepatic PAF content and we need to understand the consequences of this finding which impinge upon the hemodynamic and metabolic functions of the liver. Our efforts will be aided greatly by several novel reagents developed during the past grant period, the most important of which are specific anti-PAF receptor antibodies to be employed in studies of the regulatory mechanisms of the proposed study will be the solubilization, stabilization and purification of the acety1CoA::lysoPAF acetyltransferase which most probably is the key regulatory step in PAF synthesis in macrophage-type cells. Successful outcomes from our proposed experiments will make a significant contribution to our knowledge-based concerning the inter- and intracellular signaling mechanisms operative in the mammalian liver as its responds to pathophysiological episodes.