John Williamson - US grants

The major goal of this project is to take advantage of the recent advances made in the field of 31p and 13C nuclear magnetic resonances (NMR) spectroscopy to study the energetics and metabolism of perfused rat hearts under different conditions of substrate supply and work. Emphasis will be given to the use of 13C-enriched substrates to study regulation of the citric acid cycle, its interactions with the malate-asparate cycle and mechanisms for increasing or decreasing net pool sizes of citric acid cycle intermediates. 13C NMR spectra will be obtained using both intact hearts and perchloric acid extracts. The latter will also be used for enzymatic assay of metabolites to aid identification and quantitation of resonances obtained from individual 13C-labeled carbon atoms from the 13C spectra. The information provided from 13C specific labeling patterns of intermediates, together with knowledge of their total pool sizes as determined by metabolic analyses, will be used to construct a flux model of the citric acid cycle and interrelated transamination steps. The model will be used to test various hypotheses concerning regulation of flux in the citric acid cycle under different conditions of substrate supply and work output of the heart during the steady state as well as transition states when cycle flux is nonuniform. The following major specific aims will be addressed. 1. Identification of rate-controlling steps in the citric acid cycle and assessment of enzyme control strengths by inhibitor titration studies. 2. Assessment of the existence and metabolic significance of metabolite compartmention and substrate channeling. 3. Effects of varying flux rates through the malate-asparate cycle on the regulation of flux through citrate syntase, Alpha-ketoglutarate dehydrogenase and asparate aminotransferase. 4. Investigation of the mechanisms responsible for causing net changes of the tissue contents of citric acid cycle intermediates. 5. Effects of acetoacetate in the absence and presence of propionate on flux through succinate thiokinase and regulation of the citric acid cycle from Alpha-keto-glutarate to malate. 6. Investigation of citric acid cycle function in reoxygenated post-ischemic hearts.

The long term objective of this project is to elucidate the molecular mechanisms and physiological significance of hormone activated signaling systems in hepatocytes. A common mechanism by which many hormones and growth factors exert their effects on cell function is by phospholipase C (PLC)-mediated activation of inositol lipid breakdown, changes of Ca2+ flux and covalent modification of proteins. Complex feedback mechanisms regulate receptor-mediated cell signaling processes and an imbalance between these factors results in abnormal metabolism or cell growth. An elucidation of these fundamental biochemical events is important for understanding the etiology of disease states such as diabetes and cancer. This proposal will address the following specific aims: 1) the mechanism of signal transduction of hepatocyte growth factor (HGF) in rat hepatocytes, 2) the mechanisms of Ca2+ influx into hepatocytes mediated by Ca2+ mobilizing hormones (co-mitogens) and growth factors, 3) the mechanisms of synergistic or inhibitory interactions between growth factors and co-mitogenic hormones and 4) receptor coupling to phospholipase C isoenzymes alpha and delta. It is hypothesized that the signal transduction pathway involves tyrosine phosphorylation of PLC-gamma and possibly a G-protein for enhanced production of inositol 1,4,5-P3 and diacylglycerol with associated intracellular Ca2+ mobilization and Ca2+ influx. This hypothesis will be tested using anti-phosphotyrosine and anti-PLC-gamma antibodies for immunoprecipitation and Western blotting, together with analytical procedures and Ca2+ measurements using hepatocytes loaded with fura-2. Ca2+ influx mechanisms mediated by phenylephrine and peptide hormones, which overproduce Ins 1,4,5-P3 will be compared with those mediated by HGF and other growth factors to determine whether separate processes are involved that may account for synergistic effects on cell growth by augmenting cytosolic free Ca2+ during the cell cycle. The techniques to be used for measurement of hormone-mediated Ca2+ influx include whole cell patch clamp, Mn2+ quench of the fura-2 fluorescence and Ca2+ readdition to Ca2+-depleted cells. Inhibitory interactions between transforming growth factor beta (TGFbeta) and mitogenic growth factors will be investigated by measuring effects on Ca2+ flux and the effects of okadaic acid to inhibit protein phosphatases. The hypothesis that receptors for co-mitogenic hormones are coupled to PLC isoenzymes other than PLC-gamma will be investigated by use of immunoprecipitating antibodies to PLC-alpha and PLC-delta with solubilized rat liver membranes to identify complexes between alpha1-adrenergic, vasopressin and angiotensin II receptors, G-proteins and particular PLC isoenzymes.

The objectives of this proposal are to use isolated myocytes prepared by collagenase digestion of perfused rat and guinea pig hearts to study various aspects of calcium homeostasis and its perturbation under conditions of hormonal stimulation and interactions leading to irreversible tissue injury. The use of Quin 2 and other intracellular Ca2+ indicators will be thoroughly investigated as tools to measure the intracellular free Ca2+ and Ca2+ transients induced by electrical stimulation of the myocytes. Contraction of single cells will be monitored by video-microscopy and compared with the fluorescence changes of intracellular probes using a computerized image analysis system. A dual channel fluorometer for simultaneous measurement of Quin 2-Ca fluorescence and pyridine nucleotide fluorescence will be used with bulk cell suspensions. In addition, permeant and non-penetrating membrane potential-sensitive and pH-sensitive fluorescent probes will be used to monitor continuously the electrical potential across the plasma and mitochondrial membranes and proton gradients. Results will be compared with measurements of adenine nucleotide and creatine phosphate contents assayed by classical sampling techniques to assess the energy state of the cells. The calcium distribution between mitochondria and other vesicular pools will be investigated using rapid cell disruption techniques and by sequential additions of uncoupling agent and the Ca2+ ionophore A23187, with arsenazo III as an extracellular Ca2+ indicator. Calcium transport across the plasma membrane will be monitored by means of a Ca2+-selective electrode and Ca2+ sensitive dyes. Using combinations of the above techniques, emphasis will be given to understanding the relationships between altered energy status of the cell, intracellular free Ca2+, the calcium distribution and plasma membrane integrity during oxygen and substrate deprivation and upon reinitiation of oxidative phosphorylation. Further studies will relate to the mechanism of sarcolemma damage and the relationship between the Ca2+ transient, the source of contractile calcium and the role of mitochondria in the regulation of cytosolic free Ca2+ and the activity of mitochondrial Ca2+-sensitive dehydrogenase. In addition, we will investigate whether inositol trisphosphate, the product of phosphatidylinositol 4,5-bisphosphate breakdown, acts as a Ca2+-mobilizing second messenger in cardiac tissue, and if so its intracellular site and mechanisms of action will be studied. Further studies will utilize 31P and 1H NMR techniques to investigate post-ischemic reperfusion of rat hearts.

A research program is described which will be directed towards the characterization of growth factor-medicated Ca2+ responses in human parenchymal liver cells using liver biopsy material. This study relates to an extension of the parent grant concerning epidermal growth factor (EGF) and hepatocyte growth factor (HGF) actions on the liver. The physician experience of the Hungarian collaborator and his experience with measurement of growth factor effects on Ca2+ homeostasis in single cells will be combined to apply advanced techniques to human liver cells. First, attention will be focussed on establishing a reliable technique for measurement of intracellular Ca2+ in fura-2-loaded single human hepatocytes to characterize "normal" Ca2+ homeostasis of these cells with an emphasis on the effects of the recently described HGF. The involvement of a receptor tyrosine kinase in HGF-induced Ca2+ mobilization will be evaluated by tyrosine kinase inhibitors. The amount of releasable Ca2+ from non-mitochondrial and mitochondrial pools of human hepatocytes will be assessed by microperfusion of the specific Ca2+-mobilizing agents such as thapsigargin and mitochondrial uncouplers. Furthermore, purified antibodies raised against different components of the signaling pathway (G-proteins, phospholipase C isozymes) will be microinjected into human hepatocytes. This powerful approach has the potential for giving very specific answers to questions related to hormone-induced signal transduction in human liver. Second, growth factor-induced signal transduction and Ca2+ homeostasis will be investigated throughout the pathological evolution of alcoholic liver disease (ALD). We hypothesize that cellular changes in the hepatocytes are accompanied by specific alterations in cellular Ca2+ homeostasis corresponding in severity to the stage of ALD. A higher basal Ca2+ and a reduced cellular responsiveness to the effect of Ca2+ mobilizing hormones and growth factors is expected in relation to a decrease in the capacity of hormone-sensitive pools to sequester Ca2+. Assessment of the size of non-mitochondrial and mitochondrial Ca2+ pools will allow an evaluation of abnormal redistribution in the intracellular Ca2+. The inhibitory properties of transforming growth factor beta (TGF- beta) as well as the effect of protein kinase C-stimulating phorbol esters on HGF-induced Ca2+ response will be also determined. Knowledge of Ca2+ homeostasis and HGF-related early biochemical events of human parenchymal liver cells obtained throughout this project is of direct relevance to the development of new strategies for the prevention, prognostication, and treatment of alcoholic liver disease.

This IGERT program addresses America's long-term need for top-notch scientists capable of translating research results into new technologies and new businesses. It combines excellent interdisciplinary science education with entrepreneurial and business training. Faculty and students from two Mississippi universities will form teams and develop joint projects exploring how polymer science and medicinal chemistry can synergistically extend the methods of making and evaluating new materials, new bioactive agents and new applications of biomaterials. Participants will be centered in the School of Pharmaceutical Science at the University of Mississippi and the School of Polymers and High Performance Materials at the University of Southern Mississippi. They will be tied together through research projects, broad education in business and entrepreneurialism, and interactions with industry in the US and abroad. Specific components of the intellectual merit include: Basic research projects coupling medicinal/pharmaceutical science and polymeric materials; Discipline-centered, interdisciplinary and entrepreneurial education involving students-teaching-students, workshops, seminars, short-courses, industrial internships and international experiences; and Continuous reinforcement of the discipline and entrepreneurial components by team-building activities, video conferencing plus joint proposals, presentations, publications and business plans.

Broader impact of this program is inherent in the materials and methods being developed, and in how the projects and business plans are implemented: Within the state, bringing together two excellent programs to teach and do research at a truly innovative and socially important science interface; Within the US, by developing a successful model for research and teaching in science that uniquely combines structured education, training and hands-on experience in business and entrepeneurialism; and World-wide, through learning from and teaching students and industrial practitioners in other countries and other disciplines.

Students experiencing this program will be able to start their own companies, perhaps even at or before graduation, and to be highly effective members of larger company research and development teams. The methods and education materials developed will also be made available to members of the local community, industry in the state and throughout the nation, and students and faculty at universities everywhere through workshops, seminars, books and training manuals, and distance-learning courses and degrees. It is expected that this IGERT program will serve as a model for integration of science and entrepeneurial education that have immediate and long-term impact on other programs in Mississippi, and will result in new businesses that will enhance economic development in the state and quality of life everywhere.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries. In this sixth year of the program, awards are being made to institutions for programs that collectively span the areas of science and engineering supported by NSF.

? DESCRIPTION (provided by applicant): Heart failure is a major public health problem contributing to various comorbid medical conditions, physical and cognitive disability. There is increasing evidence that heart failure adversely affects the brain. We have previously shown that heart failure is associated with cognitive impairments, particularly so-called executive functions. We have also shown that CRT improves cognitive performance in patients with heart failure. The proposed study will delineate mechanisms underlying the effects of heart failure on brain function and determine if cognitive benefits of cardio-resynchronization therapy (CRT) are due to enhanced cerebral metabolic and hemodynamic function. There are several potential interacting contributions to brain and cognitive changes in heart failure, including neuro-inflammation, reduced cerebral perfusion and changes in cerebral hemodynamics. Neuroimaging provides access to several potentially powerful biomarkers of alterations in brain structure, function (e.g., fMRI) and hemodynamics (ASL and transcranial Doppler), as well as pathophysiology (DTI and MRS). We know from work we have published in patients with cerebrovascular disease that regional structural damage to white matter and neuropsychological performance predict recovery over time. To date, no published studies have examined neuronal, metabolic and vascular brain changes after CRT, nor have there been studies examining cognitive, functional, and structural brain changes pre-CRT and their impact on recovery. Recent advances in neuroimaging technologies allow for the imaging of patients with CRT devices. We hypothesize that: 1) Patients with cognitive impairment, no dementia who undergo CRT will improve in behaviors dependent on fronto-subcortical systems including processing speed, memory encoding, working memory and task switching. 2) Patients who undergo CRT will exhibit improvements in fMRI indices including default network attributes and task dependent fMRI response. 3) Patients with baseline neuropsychological deficits in cognitive tests of intentional/executive behaviors will be more likely to improve after CRT in the absence of white matter disease in fronto-subcortical systems, implying that the mechanism of impairment is changes in cerebral hemodynamics. 4) Cerebral hemodynamics and metabolite profiles will normalize after CRT. The groups will have equal proportions of cognitive impairment and no cognitive impairment, enabling us to clarify the mechanisms of brain dysfunction in heart failure and their relation to cognition. This study will capitalize on a powerful quasi-experimenta manipulation that will provide a unique window into the effects of heart failure and CRT on the brain.

Patients with amnestic mild cognitive impairment (MCI) often have a compromised quality of life (QOL). Cognitive impairment is a major contributor to decrements in QOL and progression of MCI often leads to loss of independence and withdrawal from social participation. MCI, in many patients, is an early expression of neurodegenerative disease. Patients with MCI frequently convert to Alzheimer's disease (AD) (12-16 percent by some estimates per year). Treatments for MCI are of limited scope and availability and of limited effectiveness. Thus, there is great need for treatments that can improve cognition and extend QOL in patients with MCI. Early intervention (prior to the development of dementia) is more likely to successfully treat this population. We propose to investigate the effect of a non-invasive and safe intervention that should have direct influence on brain systems underlying AD, transcutaneous vagal nerve stimulation (tVNS). This promising approach has not yet been studied in patients with MCI. The hippocampus is a structure that deteriorates in AD. Further, studies have suggested that the locus coeruleus (LC), the brainstem nucleus that is the brain's sole source of norepinephrine (NE), may be one of the first structures that deteriorates in patients with AD. The release of NE in the hippocampus and frontal lobes has an important role in cognition and is critical in mediating memory and attention. The ascending portions of the vagus nerve form synapses within the nucleus of the solitary tract, which projects to the LC and to the hippocampus. The LC also projects directly to the hippocampus. Thus, vagal nerve stimulation (VNS) may ameliorate symptoms of MCI. We have demonstrated, in patients with epilepsy, that VNS improves memory; however, VNS has not been used to treat patients with MCI. VNS can now be performed without surgery by transcutaneous stimulation of the auricular branch with electrodes on the external ear. tVNS has the potential to improve cognition and may even alter the course of decline in patients with MCI. We will employ a multimodal MRI-based neuroimaging approach combined with comprehensive and targeted cognitive testing to assess changes with tVNS in cognition in patients with MCI. We will evaluate the effects of tVNS on 60 patients who have been diagnosed with MCI. To maximize statistical power, we will employ a cross-over design with tVNS and control stimulation conditions (stimulating an area on the external ear that does not have nerve endings that connect to the vagus). Very little in the way of mechanistic data or understanding of individual differences in response to tVNS in MCI/AD has been published. Thus, this is a necessary study to evaluate the potential utility of tVNS to enhance cognitive performance in patients with MCI. These data may serve as a platform for supporting the development of a clinical trial with this technology.