Andrew P. Thomas - US grants

The objective of this study is to characterize the second messenger system which mediates the actions of calcium- dependent hormones in liver cells. These hormones, which include epinephrine and vasopressin, stimulate the breakdown of inositol lipids in the plasma membrane with a resultant release of two second messengers; diacylglycerol which activates protein kinase C and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) which mobilizes intracellular calcium. The mechanism by which Ins(1,4,5)P3 releases calcium will be investigated and special emphasis will be placed on the regulation of this system by other cellular constituents and hormones. Of particular interest in this respect is the apparent potentiation by GTP of Ins(1,4,5)P3-induced calcium release, which we and others have described. The calcium release system will be studied in preparations of permeabilized hepatocytes and isolated sub-cellular fractions. The hypothesis will be investigated that the calcium mobilization system consists of an Ins(1,4,5)P3-activated calcium channel in the endoplasmic reticulum, with associated regulatory proteins whose activity can be modulated by guanine nucleotides. This proposal will be tested by measurements of the kinetics of calcium release and reuptake using fluorescent calcium indicators and by examining the communication between different intracellular pools of calcium. In addition to macroscopic calcium transport measurements, microscopic and single channel calcium currents will be measured using microsomes reconstituted into planar lipid bilayers. These studies will give insight into the physiological responses to the wide range of calcium-dependent hormones which have important effects in almost all mammalian cell types. Attempts will be made to identify the protein components of the calcium release system by using photoaffinity labels, with a view to longer term plans to reconstitute the Ins(1,4,5)P3-sensitive calcium channel. A further focus of this investigation will be the mechanisms by which the calcium- mobilizing hormones interact with other classes of hormones such as insulin.

biomedical equipment purchase;

The heart is a major locus for the toxic and lethal effects of cocaine. An important action of cocaine in the heart is to inhibit presynaptic catecholamine reuptake, leading to an elevated sympathomimetic state. While this undoubtedly plays a role in the harmful effects of cocaine, it is probable that other factors also contribute to cocaine cardiotoxicity since most sympathomimetic interventions do not have the deleterious effects associated with cocaine use. In preliminary experiments using isolated ventricular heart muscle cells, we have observed a direct action of cocaine to interfere with the cytosolic Ca2+ transients responsible for excitation-contraction coupling. It is proposed that the direct actions of cocaine on cardiomyocytes may contribute to the cardiotoxic effects of cocaine. In particular, the combination of elevated catecholamine levels (which activate specific Ca2+ flux components of the Ca2+ transient), together with the inhibitory actions of cocaine, could lead to abnormalities of excitationcontraction coupling. The objective of the present proposal is to characterize the direct effects of cocaine on the Ca2+ transient and contractility of isolated cardiac myocytes and to elucidate the mechanisms responsible for these effects. In addition, the interactions between cocaine and catecholamines at the level of excitation-contraction coupling will be investigated in the-isolated cardiomyocyte system. The primary experimental approaches will involve the use of fluorescent Ca2+ indicators and a high-speed digital imaging system to follow Ca2+ fluxes in electrically stimulated myocytes at the single cell level. This technique allows us to measure the kinetic parameters and subcellular distribution of these Ca2+ fluxes with millisecond time resolution, while simultaneously monitoring cell contraction. We will also use the whole-cell configuration of patch-clamp to study the effects of cocaine on individual sarcolemmal ion conductances under voltage-clamp. The voltage-clamp and Ca2+ imaging methods will be combined to investigate how the effects of cocaine on ion channels might play a role in the inhibitory effect of cocaine on the Ca2+ transient. The long term objective of the proposed study is to understand the basic mechanisms which underlie the cardiotoxic actions of cocaine and to determine how each of these mechanisms contributes to the harmful effects of cocaine in vivo.

The heart is a major locus for the toxic and lethal effects of cocaine. An important action of cocaine in the heart is to inhibit presynaptic catecholamine reuptake, leading to an elevated sympathomimetic state. While this undoubtedly plays a role in the harmful effects of cocaine, it is probable that other factors also contribute to cocaine cardiotoxicity since most sympathomimetic interventions do not have the deleterious effects associated with cocaine use. In preliminary experiments using isolated ventricular heart muscle cells, we have observed a direct action of cocaine to interfere with the cytosolic Ca2+ transients responsible for excitation-contraction coupling. It is proposed that the direct actions of cocaine on cardiomyocytes may contribute to the cardiotoxic effects of cocaine. In particular, the combination of elevated catecholamine levels (which activate specific Ca2+ flux components of the Ca2+ transient), together with the inhibitory actions of cocaine, could lead to abnormalities of excitationcontraction coupling. The objective of the present proposal is to characterize the direct effects of cocaine on the Ca2+ transient and contractility of isolated cardiac myocytes and to elucidate the mechanisms responsible for these effects. In addition, the interactions between cocaine and catecholamines at the level of excitation-contraction coupling will be investigated in the-isolated cardiomyocyte system. The primary experimental approaches will involve the use of fluorescent Ca2+ indicators and a high-speed digital imaging system to follow Ca2+ fluxes in electrically stimulated myocytes at the single cell level. This technique allows us to measure the kinetic parameters and subcellular distribution of these Ca2+ fluxes with millisecond time resolution, while simultaneously monitoring cell contraction. We will also use the whole-cell configuration of patch-clamp to study the effects of cocaine on individual sarcolemmal ion conductances under voltage-clamp. The voltage-clamp and Ca2+ imaging methods will be combined to investigate how the effects of cocaine on ion channels might play a role in the inhibitory effect of cocaine on the Ca2+ transient. The long term objective of the proposed study is to understand the basic mechanisms which underlie the cardiotoxic actions of cocaine and to determine how each of these mechanisms contributes to the harmful effects of cocaine in vivo.

The effects of hormones such as catecholamine, vasopressin and angiotensin II on hepatic metabolism are mediated by alterations in the concentration of cytosolic free Ca2+ ([Ca2+]i). These [Ca2+]i changes are brought about, at least in part, by the action of the second messenger inositol 1,4,5-triphosphate (IP3). Biochemical studies in populations of hepatocytes have demonstrated a relatively simple relationship between IP3 and [Ca2+]i changes. However, studies carried out at the single cell level have revealed that the [Ca2+]i responses to these hormones are organized in the form of [Ca2+]i hormone concentration. These frequency-modulated [Ca2+]i oscillations may serve a number of signaling functions that cannot readily be achieved in other ways. There is a further level of organization of these [Ca2+]i signals at the subcellular level, since [Ca2+]i oscillations initiate from a discrete locus within each cell and then spread through the cell as waves of Ca2+ that propagate with constant velocity and amplitude. We suggest that Ca2+ waves play an important role in distributing [Ca2+]i signals, signal transduction from the plasma membrane may be essential for full hormonal responsiveness in large cytoplasm by binding to a limited sinusoidal membrane domain. Patterns of [Ca2+]i oscillation can be modified by a number of other agents, including insulin and glucagon, and as such the [Ca2+]i oscillation systems may be important loci for the interactions between various classes of hormones. The present proposal is directed towards elucidating the mechanisms that are responsible for generating [Ca2+]i oscillations and waves within hepatocytes. In addition, the subcellular distribution of major components of the Ca2+ signaling system will be examined to determine how these components give rise to specific polarized initiation site for Ca2+ waves, and how the intercellular organization contributes to the propagation of the Ca2+ waves. Experiments will be carried out using permeabilized cells to examine the structure and functional regulation of the intracellular Ca2+ storage pools, and intact cells to elucidate how these properties are integrated in the generation of oscillatory Ca2+ waves. A major components of these studies will rely on imaging approaches to the measurement of dynamic Ca2+ movements within both intact and permeabilized cells, including some novel techniques that permit spatially-resolved studies of Ca2+ stores and IP3 action at the subcellular level.

The effects of hormones such as catecholamines and vasopressin on hepatic metabolism are mediated by alterations in the concentration of cytosolic free Ca2+ ([Ca2+]c), largely as a result of Ca2+ mobilization from intracellular stores by the second messenger inositol 1,4,5- trisophosphate (IP3). The [Ca2+]c responses to these hormones are organized in the form of [Ca2+]c oscillations and waves whose frequency is controlled by agonist dose. Frequency-modulated [Ca2+]c oscillations may serve a number of signaling functions, including improved fidelity, sensitivity and targeted regulation of specific processes. [Ca2+}c waves are also likely to play a role in propagating [Ca2+]c signals to distal parts of the cell, in signaling between cells and in establishing polarized functional responses. It is clear that the principal element of the [Ca2+]c oscillation mechanism is the IP3 receptor Ca2+ channel (IP3R), which is sensitive to positive and negative feedback regulation by Ca2+ and IP3. However, the evidence also indicates that other Ca2+ signaling components may be involved in establishing the spatial and temporal organization of [Ca2+]c signaling, either by modulating the Ca2+ feedback regulation of the IP3R or by superimposing additional Ca2+ - feedback loops on the essential IP3R oscillator. We propose to investigate how these additional inputs interact with IP3-dependent [Ca2+]c oscillations to enrich the frequency range and spatial pattern of oscillatory [CA2+]c waves. Our studies are designed to address the following questions: 1. Does Ca2+ - feedback on IP3 metabolism (phospholipase C or IP3 3-kinase) play a role in generating [Ca2+]c oscillations and waves? 2. What is the role of the ryanodine receptor in Ca2+ release and regenerative Ca2+ wave propagation? 3. What are the consequences of the close coupling between IP3-dependent Ca2+ release and mitochondrial Ca2+ uptake for the temporal and spatial organization of [Ca2+]c signals? 4. What defines the polarized initiation site for [Ca2+]c waves in hepatocytes; subcellular heterogeneity in IP3R function or spatially localized interactions of the IP3R with other Ca2+ signaling components? These studies will make use of biochemical, molecular and imaging techniques for studies of Ca2+ signaling in intact hepatocytes, together with a recently developed permeabilized cell system in which we can reconstitute IP3-dependent Ca2+ oscillations and waves.

alcoholism /alcohol abuse; bioimaging /biomedical imaging; fluorimetry; imaging /visualization /scanning

Chronic alcohol consumption leads to alterations in the contractile function of the heart and alcoholism is a leading cause of cardiomyopathy. Alcohol also depresses heart function through a direct interaction with the cardiac muscle cells during acute exposure and it is likely that adaptive responses to these acute effects contribute to the etiology of alcoholic heart disease. A number of targets for the acute effects of alcohol have been identified and several of these impact on the pathway of excitation-contraction coupling (E-C coupling). However, the mechanisms underlying the chronic effects of alcohol on the heart remain obscure. In this study we will investigate the mechanisms responsible for the acute and chronic actions of alcohol in isolated ventricular muscle cells obtained from the hearts of rats maintained on an alcohol feeding protocol for prolonged periods, and in control animals. We will determine how the acute effects of alcohol on individual elements of the E-C coupling cascade are integrated to give rise to the depression of contractility. In the context of the chronic effects of alcohol, we have recently identified a potentially important lesion in the regulation of cardiac E-C coupling. Specifically, the activation of the rate of rise and amplitude of the [Ca2+]i transients by beta-adrenergic agonists is greatly decreased in alcohol-fed rats. By contrast, the beta-adrenergic stimulation of the relaxation phase is unaffected. This defect appears to be distal to beta- adrenergic receptor activation and cAMP formation. However, the L-type voltage-dependent Ca2+ channels of these "alcoholic" cardiomyocytes give reduced currents compared to their paired controls, and are largely resistant to activation by isoproterenol. This loss of Ca2+ channel activity is associated with an increased density of Ca2+ channel alpha1 subunit measured as dihydropyridine binding. We will investigate the mechanism of this novel effect and examine the role of the altered Ca2+ channel properties in the contractile dysfunction of these cells. We hypothesize that chronic alcohol consumption modifies the expression level and subunit composition of the Ca2+ channels. Such a defect could contribute to the depression of contractility and may be a precipitating factor in the development of other aberrant adaptive processes m the heart that eventually lead to cardiac failure. These studies will be carried out using molecular and cellular physiology approaches to study the mechanisms and components of cardiac muscle E-C coupling in cardiomyocytes from control and alcohol-fed rats.

Funds are sought to cover partial support of the Gordon Research Conference on Calcium Signaling, to be held at New England College, Henniker, NH, August 8-13, 1999. The meeting provides a unique multidisciplinary forum for interchange of ideas and information in the field of calcium signaling. Since calcium signaling is a fundamental means of cellular control common to all eukaryotic organisms, from yeast to mammalian cells, the meeting derives particular strength by stimulating the interchange of basic information on cell regulation from workers using a rich variety of different organisms and cellular systems. The primary objective is to facilitate successful interactions between new and established investigators. Highest priority will be given to the selection and provision of funds to support junior scientists (student and postdoctoral trainees), women scientists, and under-represented minority scientists. The conference will include 8 sessions with research presentations and extensive discussion, each lead by a prominent member of the field with experience in maximizing interactive exchange. There will also be a keynote address given by Dr. James W. Putney, Jr. of MEHS, a pioneer in the calcium signaling field. In addition, there will be two poster sessions and opportunities for junior scientists selected from the poster presenters to make oral presentations of their work within the main sessions. Each of the planned sessions will focus on timely and central aspects of calcium signaling, including the molecular nature and regulation of intracellular IP3 receptors, the spatial and temporal control of calcium signaling events, the role of mitochondria in calcium signaling and as mediators of cell injury and apoptosis, targets of calcium signaling in the brain and in peripheral tissues, and recent advances in the methodology for studying calcium and other intracellular ions. Participants include a combination of established and successful younger investigators representing an array of cellular systems and different techniques ranging from molecular biological analysis and manipulation of the calcium signaling machinery to sophisticated and pioneering micro- imaging technology to observe the generation and propagation of calcium signals. Basic disciplines of participants include biochemistry, physiology, cell biology, molecular biology, biophysics, pathology and pharmacology; however, all have a primary interest in understanding the mechanisms and significance of calcium signaling events in cells. The topics to be addressed at this meeting are important to our understanding of a number of health problems, including issues related to environmental health, cancer biology, cystic fibrosis, diabetes, neurodegenerative diseases and aging, to name a few.

[unreadable] DESCRIPTION (provided by applicant): Chronic alcoholism is a major cause of cardiomyopathy in humans and leads to a variety of changes that interfere with contractile function in the hearts of ethanol-fed animal models. Features of alcoholic cardiomyopathy include decreased cardiac output, impaired myocardial contractility and atrial dysrhythmias. There are also direct cardiodepressant effects of alcohol during acute administration. Some of the features of alcoholic heart disease may reflect adaptive changes induced by these acute ethanol actions. Our studies during the previous granting period have identified a number of targets of acute ethanol action within the excitation-contraction (E-C) coupling cascade, including sarcolemmal Na+ and Ca2+ channels, which lead to depression of the cytosolicCa2+ ([Ca2+]i) transients that drive contraction. We have also identified specific changes in E C coupling in the hearts of ethanol-fed rats, which suggest a selective lesion in the L-type Ca2+ channel and its regulation by cAMP-dependent protein kinase. In the work proposed here, we will further investigate the elements of E C coupling that are affected by acute ethanol, with a view to elucidating which of these is likely to contribute to the deficient [Ca2+]i transients. However, the principal aims of the proposed studies will be to characterize the changes in E C coupling following chronic ethanol consumption, and to determine the mechanism of these changes and how they lead to deficiencies in cardiac function. Specifically, we will investigate the following linked hypotheses: Alterations in the channel subunit isoform composition and/or expression leads to a channel that is defective, either in (1) basal channel properties, (2) coupling to intracellular Ca2+ release pathways, (3) regulation by the normal cAMP-dependent signaling pathway, and/or (4) localization within the cardiomyocyte. These experiments will utilize a combination of [Ca2+]i imaging, electrophysiology and molecular biology approaches. The proposed work will yield new insights into the changes in E C coupling that underlie cardiac dysfunction induced by chronic ethanol consumption. These findings may also have broader implications in elucidating the causal factors associated with the development of other aberrant adaptive processes in the heart, which eventually lead to cardiac failure [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The effects of hormones such as catecholamines and vasopressin on hepatic metabolism and secretion are mediated by alterations in the concentration of cytosolic free Ca2+ ([Ca2+]c), largely as a result of Ca2+ mobilization from intracellular stores by the second messenger inositol 1,4,5-trisphosphate (IP3). The [Ca2+]c signals elicited by these hormones are organized in the form of [Ca2+]c oscillations and waves, whose frequency is controlled by hormone dose. Frequency-modulated [Ca2+]c oscillations may serve a number of regulatory functions, including improved fidelity, sensitivity and targeted regulation of specific processes. While the role of IP3-receptors in this pathway is well established, it has recently become evident that other Ca2+ release and Ca2+-feedback components contribute to extend the temporal and spatial range of [Ca2+]c oscillations. One way in which this might be achieved is through interplay between IP3R and the other major class of intracellular Ca2+ release channels, the ryanodine receptors (RyRs). RyRs are typically activated by Ca2+ through a Ca2+-induced Ca2+ release (CICR) mechanism, but they can also be activated by another second messenger, cyclic-ADPribose (cADPR). Although the RyRs have been well-characterized in excitable tissues (primarily muscle and brain), relatively little is known about the identity and properties of RyRs in nonexcitable cells. We have recently identified and cloned a unique RyR isoform from rat hepatocytes (RyR1b), which is derived from the RyR1 gene, but with an alternative start site that gives rise to a protein of only 40% the size of full length muscle RyRs. We hypothesize that RyR1b has distinct functional properties that underlie its contribution to [Ca2+]c signaling in hepatocytes and other nonexcitable secretory cells, where it may be the principal RyR isoform. Specifically, we propose that RyR1b plays a key role in enhancing and sustaining ER Ca2+ release initiated by IP3R activation. We will examine the potential role of RyR1b in two specific aims: In Aim 1 we will carry out heterologous expression studies to investigate the function and regulatory properties of RyR1b, including regulation by [Ca2+]c, cADPR and its interactions with IP3 and the IP3R. In Aim 2 we will investigate the subcellular distribution and regulation of RyR1b in primary hepatocytes, and determine how it contributes to the temporal and spatial pattern of [Ca2+]c signaling during hormonal stimulation of these cells. RyR1b appears to be a major new addition to the superfamily of intracellular Ca2+ release channels, with distinct properties. It may have specific functions that are tuned to the signaling requirements of nonexcitable secretory epithelia and endocrine cells. Thus, the proposed work is innovative and has the potential for high impact, with significant biomedical health relevance. PUBLIC HEALTH RELEVANCE: The proposed work will investigate the role and regulation of a newly discovered calcium signaling protein (RyR1b) that appears to play a role in mediating the effects of hormones on liver function. It is also postulated to participate in signaling in other tissues, including the pancreas and cells of the digestive tract. Since derangements of signaling in these tissues are frequently associated with disease states (eg. hepatitis, cholestasis, pancreatitis), characterization of the properties of RyR1b has significant potential public health impact. Its unique molecular structure may yield a novel therapeutic target.

DESCRIPTION (provided by applicant): Malaria is a leading cause of morbidity and mortality in the Third World and resistance of Plasmodium parasites to antimalarial treatments is a significant and growing problem. Novel drug targets and treatment approaches are urgently required, but this is an under-resourced problem in the pharmaceutical industry. The purpose of this proposal is to validate a malarial signaling pathway as a novel drug target, using a subset of compounds with antimalarial activity recently released by a major pharmaceutical company, together with a related group of compounds already validated for other clinical uses. The proposed target is a malarial melatonin receptor (pMTR) that is postulated to subvert the host hormone melatonin to regulate parasite proliferation and entrain the Plasmodium cell cycle to be synchronized with the host circadian rhythm. Validation of pMTR as a potential target for small molecule drug therapy would represent a paradigm shift, both in terms of the novel pathway identified and the proposed mechanism of therapeutic action. The present work is focused on the red blood cell (RBC) stage of P. falciparum infection, which is responsible for the predominant disease symptoms, including anemia, cerebral malaria, multi-organ failure and, significantly, periodic fevers that occur on a 48 h cycle. Our previous studies have provided evidence that melatonin can initiate an intracellular calcium signaling cascade involving the second messenger IP3, leading to enhanced parasitemia and stimulating progression through the cell cycle. We hypothesize that the Plasmodia melatonin receptor (pMTR) represents a novel antimalarial drug target to decrease proliferation and synchronization of the RBC cycle. The specific aims of the proposed project are: 1. To validate pMTR as a potential small molecule chemical target and assess the effects of putative pMTR modulators on P. falciparum proliferation and synchronization. These studies will determine an initial activity profile and elucidate the mechanism of drug action, while at the same time providing additional information on the pMTR signaling pathway. 2. To develop chemical probe-derived affinity reagents to identify the pMTR protein, with the eventual goal to annotate and clone the gene. 3. To determine the efficacy of pMTR modulators in combination with established antimalarial drugs that are not thought to act through the pMTR calcium signaling pathway. 4. To examine the effect of the small molecule inhibitors of pMTR identified in Aim 1 in vivo using rodent models, in order to further assess the viability of pMTR as a potential small molecule drug target. The work will be carried out by a multidisciplinary team, with expertise in calcium and G-protein signaling, malaria biology, parasitology, chemical biology and medicinal chemistry. Techniques include live-cell fluorescence imaging, imaging flow cytometry, in vivo malaria models, and synthetic chemistry.