Bertil Hille - US grants

DESCRIPTION (provided by applicant): This proposal concerns the regulation of ion-channel function by G-protein-coupled receptor (GPCR) signaling to membrane lipids. It focuses on the hypothesis that the function of many ion channels depends on the concentration of one rare phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane. The kinetics of signaling steps from M1 muscarinic receptors to phospholipase C will be measured to test the hypothesis that they are fast, perhaps reflecting a preformed signaling complex. The kinetics of the metabolic steps that deplete and replenish PIP2 will be measured to understand the cellular sources and dynamics of PIP2. All results will be fitted with a comprehensive kinetic model to provide additional information on the mechanisms of the signaling cascade. The ability of PIP2 concentration changes and muscarinic signaling to modulate function of KCNQ channels, several voltage-gated K+ channels (Kv channels), and voltage-gated Ca2+ channels (Cav channels) will be studied. The ability of arachidonic acid to modulate KCNQ and Cav channels will be analyzed. Physiological mechanisms for arachidonic acid production initiated by GPCR inputs will be defined. The methods will include patch-clamp electrophysiology, fluorescence resonance energy transfer, dynamic targeting of enzymes to cellular membranes, confocal microscopy, and chemical analysis. Most of the studies will be done on cell lines but a small number will be done in nerve cells of rodents to demonstrate the relevance to mammalian physiology. This work lays the basis for understanding hormonal control of mental state and the actions of many drugs of biological psychiatry. Many drugs of abuse and drugs of psychiatry act on the signaling systems studied here. The involuntary nervous system talks to its targets by the signaling mechanisms elucidated here.

A Symposium under the auspices of the Society of General Physiologists is planned for September 5-8, 1985, at the Marine Biological Laboratory in Woods Hole, Massachusetts. The subject is: Proteins of Excitable Membranes. The meeting is designed to stimulate an exchange of ideas between membrane physiologists and molecular biologists at a time when new molecular approaches are first being applied to membrane functions. Sessions will focus on three major classes of molecules: transmitter-activated channels, voltage-gated channels, and transport ATPases. These include the acetylcholine receptor, sodium channel, calcium channel, the sodium/potassium ATPase and calcium ATPase. Twenty speakers have been invited, including two from overseas. The symposium will encourage an interdisciplinary synthesis of the best structural and functional ideas for the most widely studied devices of excitable membranes. It will attract a large audience, many of whom will contribute posters, and the full proceedings will be published (as the 39th volume in the series of Society of General Physiologists Symposia) to illustrate the value of an interdisciplinary approach to membrane physiology. The program will consist of four invited lecture sessions, two contributed abstract sessions, and two keynote addresses.

DESCRIPTION (provided by applicant):Intracellular Ca 2+ signaling and the regulation of vesicular exocytosis are two fundamental physiological properties of all eukaryotic cells. They have been analyzed in detail in only a few exemplar cell types. We need precise descriptions in each cell type to understand the implications for disease and therapy. This project will study pancreatic beta-cells, pancreatic ductal epithelium, chromaffin cells, pituitary gonadotropes, and sympathetic neurons. It will use patch clamp biophysical methods and optical Ca 2+ reporters to quantitate sources and sinks of Ca 2+ in differentiated mammalian endocrine, nerve, and epithelial cells. One focus will be on Ca 2+ buffering and Ca 2+ clearance. In these cells, cytoplasmic buffering and four membrane clearance processes shape the Ca 2+ transient and thus regulate secretion of the endocrine hormones insulin, adrenaline, and gonadotropins and the secretion of mucus of the gastrointestinal tract. Defects in regulation of secretion underlie some forms of diabetes, infertility, cystic fibrosis, and digestive disorders. We need basic understanding to inspire new therapeutic approaches. This project will determine a kinetic model for the secretory vesicle pools of pancreatic ductal epithelium, including their regulation by Ca 2+, protein kinases, and other physiological variables. The Ca 2+ buffering and Ca 2+ clearance mechanisms of sympathetic neurons, pancreatic beta-cells, and pancreatic ductal epithelial cells will be dissected and described by a quantitative model. Our analysis of the Ca 2+ dynamics within the endoplasmic reticulum of gonadotropes and within the mitochondria of chromaffin cells will be deepened. What are the Ca 2+ buffering and flux properties of these organelles? Our work in these cells concerns processes whose failure leads to disease and whose modulation offers new therapies.

laboratory mouse; tissue /cell culture; voltage /patch clamp

DESCRIPTION (provided by applicant):Intracellular Ca 2+ signaling and the regulation of vesicular exocytosis are two fundamental physiological properties of all eukaryotic cells. They have been analyzed in detail in only a few exemplar cell types. We need precise descriptions in each cell type to understand the implications for disease and therapy. This project will study pancreatic beta-cells, pancreatic ductal epithelium, chromaffin cells, pituitary gonadotropes, and sympathetic neurons. It will use patch clamp biophysical methods and optical Ca 2+ reporters to quantitate sources and sinks of Ca 2+ in differentiated mammalian endocrine, nerve, and epithelial cells. One focus will be on Ca 2+ buffering and Ca 2+ clearance. In these cells, cytoplasmic buffering and four membrane clearance processes shape the Ca 2+ transient and thus regulate secretion of the endocrine hormones insulin, adrenaline, and gonadotropins and the secretion of mucus of the gastrointestinal tract. Defects in regulation of secretion underlie some forms of diabetes, infertility, cystic fibrosis, and digestive disorders. We need basic understanding to inspire new therapeutic approaches. This project will determine a kinetic model for the secretory vesicle pools of pancreatic ductal epithelium, including their regulation by Ca 2+, protein kinases, and other physiological variables. The Ca 2+ buffering and Ca 2+ clearance mechanisms of sympathetic neurons, pancreatic beta-cells, and pancreatic ductal epithelial cells will be dissected and described by a quantitative model. Our analysis of the Ca 2+ dynamics within the endoplasmic reticulum of gonadotropes and within the mitochondria of chromaffin cells will be deepened. What are the Ca 2+ buffering and flux properties of these organelles? Our work in these cells concerns processes whose failure leads to disease and whose modulation offers new therapies.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Calcium signaling and the regulation of exocytosis are central issues in the physiology of all animal cells. We seek quantitative understanding of such signaling through biophysical experiments in electrically excitable and non-excitable mammalian cell lines: PC12 pheochromocytoma cells, tsA epithelial cells, and pancreatic duct epithelial cells. Two long-term hypotheses guide this work: (a) that Ca2+ clearance and the regulation of exocytosis take different forms in different cells and are tuned to the physiological role of each cell;and (b) that several intracellular organelles make significant contributions to cellular Ca2+ dynamics. The aims in this grant period are: (1) To test the hypothesis that accumulation and release of Ca2+ by secretory granules can make significant contributions to cellular Ca2+ signaling during physiological responses. (2) To measure the amplitude of receptor-evoked inositol 1,4,5, trisphosphate (IP3) elevations and to test the hypothesis that Ca2+ signaling via IP3 is terminated by rapid metabolism of IP3 by IP3 5- phosphatase followed by rapid reuptake of Ca2+ into the endoplasmic reticulum Ca2+ stores. And (3) To test the hypothesis that cytoskeletal tracks and fast cytoskeletal remodeling participate in the mobilization of secretory granules from reserve pools into secretion-competent pools. The work requires a range of biophysical techniques including: patch clamp of ion currents;amperometric and capacitance measurements of exocytosis;transfection of genetically targeted probes, indicators, and cellular proteins;ratiometric photometry and fluorescence resonance energy transfer (FRET) of indicators;video fluorescence imaging;total internal reflection microscopy (TIRF);confocal microscopy;and quantitative kinetic modeling. Modeling by Virtual Cell will concern the compartmental dynamics of Ca2+ and production and breakdown of IP3.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project concerns the regulation of ion-channel function by G-protein-coupled receptor (GPCR) signaling to membrane lipids. It focuses on the hypothesis that the function of many ion channels depends on the concentration of one rare phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane. The kinetics of signaling steps from M1 muscarinic receptors to phospholipase C will be measured to test the hypothesis that they are fast, perhaps reflecting a preformed signaling complex. The kinetics of the metabolic steps that deplete and replenish PIP2 will be measured to understand the cellular sources and dynamics of PIP2. All results will be fitted with a comprehensive kinetic model to provide additional information on the mechanisms of the signaling cascade. The ability of PIP2 concentration changes and muscarinic signaling to modulate function of KCNQ channels and voltage-gated Ca2+ channels (CaV channels) will be studied. The methods will include patch-clamp electrophysiology, fluorescence resonance energy transfer, dynamic targeting of enzymes to cellular membranes, confocal microscopy, and chemical analysis. Analysis and interpretation of these experiments will be facilitated with the Virtual Cell modeling and simulation software. Most of the studies will be done on cell lines. This work lays the basis for understanding hormonal control of mental state and the actions of many drugs of biological psychiatry and of drugs of abuse.

DESCRIPTION (provided by applicant): The pineal gland is an endocrine organ in the brain that is primarily regulated by noradrenaline released by the sympathetic nervous system. It is the part of our circadian clock system that broadcasts the night hormone, melatonin, to all of the body. The night state of pinealocytes is accompanied by extensive changes in gene expression relative to the day state. Many expressed genes are those expressed by a sister lineage, photoreceptors, except that mammalian pinealocytes are not photosensitive. Our overall hypothesis is that pinealocytes switch between two very different electrophysiological excitability states that contribute to regulated secretion of melatonin and to entrainment and maintenance of circadian rhythms. Using biophysical techniques, the night and day states of cultured rat pinealocytes will be contrasted in terms of their ion channel complement, calcium signal dynamics, receptor activation, second messenger levels, and secretory mechanisms. The state changes will be achieved by preincubating cultured primary cells in appropriate neurotransmitters. Cells will be dissociated from rat pineal glands, placed in cell culture, treate with norepinephrine to mimic night, and studied under the microscope with techniques such as whole-cell gigaseal recording, photometry of calcium-sensitive dyes, amperometry and HPLC of neurotransmitters, and use of various live-cell indicators for second messengers. Night pinealocytes are hypothesized to have electrical excitability related to that of photoreceptors, and day pinealocytes are hypothesized to be relatively quiescent. The hypothesis that serotonin is secreted from pinealocytes by quantal exocytosis whereas melatonin and N-acetyl serotonin are secreted by hydrophobic diffusion will also be tested. Understanding the rhythmic secretory mechanisms of the pineal will make an important contribution towards treating sleep disorders, seasonal affective responses to short days, and loss of attention due to jet lag and shift work. PUBLIC HEALTH RELEVANCE: Cells of the pineal gland in the brain secrete melatonin, the hormone of night. We will study how their excitability differs in the secreting night state from th quiescent day state. Under- standing the rhythmic secretory mechanisms of the pineal will make an important contribution towards treating sleep disorders, seasonal affective responses to short days, and loss of attention due to jet lag and shift work.