Barry B. Wolfe - US grants

The overall goal of this research program is to increase our understanding of the factors involved in the regulation of receptors for autonomic neurotransmitters and hormones. These studies are designed to investigate autoradiographically the localization of Alpha1-, Beta1-, and Beta2-adrenergic receptors in rat kidney and to characterize the biochemical and physiological response to stimulation of each of these receptors. The effects of altered levels of stimulation on the density and properties of these receptors will be determined. Additionally, the functional significance of such alterations will be studied by measuring the biochemical and physiological response to the stimulation of each of these receptors in kidney preparations from control and treated rats. In particular, isoproterenol-stimulated adenylate cyclase activity and norepinephrine-stimulated phosphatidylinositol breakdown will be characterized and utilized as measures of the biochemical response to activation of Beta- and Alpha1-adrenergic receptors, respectively. Similarly, isoproterenol-stimulated renin release or norepinephrine-stimulated, prazosin-inhibitable inhibition of renin release from slices of kidney or isolated glomeruli will be characterized and utilized as physiological correllates of Beta and Alpha1-adrenergic receptor stimulation, respectively. The degree of receptor stimulation will be altered utilizing the chronic administration of several commonly used therapeutic interventions. Thus, Beta-receptor stimulation will be decreased by the administration of propranolol or pindolol, the latter possessing a significant degree of intrinsic sympathomimetic activity, as well as the administration of a Beta1- or a Beta2-selective antagonist. These studies are designed to examine the possibility that the subtypes of renal Beta-adrenergic receptors can be independently regulated and to determine the functional consequences of such selective regulation. Stimulation of Alpha1-adrenergic receptors will be decreased by the administration of prazosin and the functional consequences of this will be determined. Receptor stimulation will be increased by the chronic administration of several types of antidepressants as well as the direct administration of catecholamines. The effects on receptor density will be determined autoradiographically at high anatomical resolution. Additionally, the effects on the functional correllates of these binding sites will be determined for each receptor type.

Subtypes of muscarinic cholinergic receptors have been described using both pharmacological and molecular biological tools. It appears that there are at least three receptors identifiable with drugs while the genes for five muscarinic receptors have been cloned. Thus, the pharmacological tools in current use appear inadequate to identify definitively a given subtype in a tissue expressing multiple subtypes. Thus, we have developed a set of antibodies selective for each of the molecularly defined subtypes of muscarinic receptor. These antibodies will be used to examine the regulation of muscarinic receptors in rats as well as in clonal cell lines maintained in culture. In addition to these tools to identify selectively a given subtype, we will utilize cells that normally do not express muscarinic receptors but have been transfected stabily with cDNA encoding a given genetically defined subtype of muscarinic receptor. The specific aims of the project are: 1. To characterize each of the known subtypes of muscarinic receptors utilizing cell lines expressing only a single genetically defined subtype. Several radioligands will be employed to determine if any can be utilized as a "selective" radioligand under precisely defined conditions. The second. messenger systems associated with each subtype will be examined. 2. To examine the regulation of each of the subtypes of muscarinic receptors In cell lines of "natural" origin. The phenomena of desensitization and down-regulation will be examined for each receptor subtype. The regulation by protein kinase C will be examined. Potential phosphorylation or palmitoylation of the receptor(s) will be studied. The identity of the G protein(s) to which the receptor(s) couple will be determined. The effects of various hormones on receptor expression will be studied. The possibility that mRNA encoding a given subtype is regulated will be examined. 3. To examine the regulation of subtypes of muscarinic receptors In rats. The effects of chronic administration of an antagonist (e.g. atropine), a cholinesterase inhibitor (e.g. tetrahydroaminoacridine), and a direct acting agonist (e.g. oxotremorine) on the expression of each of the subtypes will be examined using antibodies selective for each subtype. Since muscarinic receptors are involved in many aspects of normal and abnormal physiology and behavior, drugs affecting these receptors could be clinically useful. Up till now, however, side effects have limited the usefulness of such agents; but with a fuller understanding of the subtypes of muscarinic receptors it may be possible to target specific subtypes with drugs to ameliorate the symptoms of, for example, Alzheimers disease, Parkinson's disease, peptic ulcer, and hypertension.

The overall goal of the studies proposed here is to increase our knowledge of muscarinic cholinergic receptors at the molecular level. We now know from molecular biological experiments that multiple subtypes of these receptors exist. However, due to the fact that no drugs exist that are highly selective for a given subtype, we currently have no tools with which to study the individual subtypes in the presence of any of the other subtypes. Muscarinic receptors are involved in a variety of processes in the human body including memory and learning, thermal regulation, heart rate, Gl secretions etc. Drugs that can selectively affect these processes could be therapeutically useful in the management of Alzheimer's disease, peptic ulcer, hypertension etc. Development of such agents relies on understanding the exact localization and function of the various subtypes. The goals of the current application are: 1. To develop polyclonal and monoclonal antibodies specifically directed against each of the known subtypes of muscarinic cholinergic receptors. 2. To utilize these antibodies to develop assays to quantify subtypes of receptors in homogenates of tissues that contain multiple subtypes. With this assay, to measure the density of each subtype in a number of key organs and brain regions. 3. To utilize these antibodies to determine which parts of the molecule are involved in ligand binding, G-protein interactions, and production of second messengers. These studies will also include tests of the current model of secondary structure of the receptors (i.e. which portions are on the inside of the cell and which are on the outside.) 4. To utilize these antibodies to examine the distribution of receptor subtypes in thin sections of tissue both at the visible and light microscope level.

Deficits in cholinergic function have been implicated in the loss of ability to learn and remember which occurs in Alzheimer's Disease and to some extent in normal aging. Several reports have indicated that both muscarinic and nicotinic receptors are affected as a function of aging and in Alzheimer's Disease. Several subtypes of both muscarinic and neuronal nicotinic receptors have been cloned but drugs selective enough to distinguish between the subtypes do not currently exist. Thus, antibodies with selectivity to each of the subtypes of muscarinic nicotinic receptors have been generated utilizing fusion proteins corresponding to the unique portions of each molecule. We propose to utilize these antibodies: 1. To examine the hypothesis that certain subtypes of muscarinic (m1-m5) or neuronal nicotinic receptors are specifically affected in Alzheimer's Disease. 2. To determine the effects of normal aging on the density of each of the subtypes of muscarinic and nicotinic receptors in human brain. 3. To determine the effects of normal aging on the density of the subtypes of muscarinic and nicotinic receptors in rat brain. Additionally, experiments will be conducted using biochemical (cyclic AMP accumulation or phosphoinositide hydrolysis) and physiological assays (prolactin release) to examine the hypothesis that these responses are blunted in aging due to changes in specific receptors. 4. To examine the hypothesis that in aged rats the observed large differences from rat to rat in the ability to learn is mirrored by a loss of a specific subtype(s) of muscarinic or nicotinic cholinergic receptor in the hippocampus or cortex. Thus, as rats age some will perform well on a Morris swim maze and some will perform poorly. In collaboration with Dr. Michela Gallagher at the University of North Carolina, we will examine the density of each subtype of muscarinic and nicotinic receptor in the brains of aged (24-26 mo.) rats which have been tested behaviorally within one week of euthanasia. Preliminary data from Dr. Gallagher's laboratory demonstrate a change in muscarinic receptors from hippocampus of aged rats that show a learning deficit but not from aged rats that do not show a deficit. These experiments should provide important information about the precise aspects of cholinergic receptors affected in aging and in AD and may provide insight into potential therapeutic targets for e.g. Alzheimer's Disease as well as other cognitive disorders.

The major objective of this project is to understand the role of ionotropic glutamate receptors in the cognitive decrements often associated with aging. The first aim includes a determination of the relationship between phosphorylation of NMDA and AMPA receptor subunits and cognitive performance in young and aged rats. Additionally, the level of neurotransmitter release stimulated by NMDA receptors will be examined in young and aged, behaviorally-characterized rats. The second aim characterized the role of phosphorylation of NMDA and AMPA subunits in a specific learning paradigm by using a behavioral/electrophysiological procedure developed by Dr. Eichenbaum which produces learning-related alterations in synaptic efficacy in specific hippocampal pathways. This aim is based on the observation that protein phosphorylation plays an important in LTP and that phosphorylation of specific subunits of ionotropic glutamate receptors subunits appears to be a major mechanism for regulating receptor function. Thus, if phosphorylation is induced by the learning paradigm in young rats, this phenomenon will be examined in aged, behaviorally-characterized rats. The third aim of this project, a collaboration with Drs. Gallagher and McKinney, is to study glucocorticoid receptors and associated transcription factors and their relationship to hippocampal aging. A great deal of evidence implicates dysregulation of glucocorticoids in the negative consequences of aging, particularly in the hippocampus. Experiments are designed to determine if glucocorticoid receptors or APl, which can be associated, are altered in aging and if this alteration is related to cognitive decline. In these studies the parallel to the human condition is clear in that, like the rats used in the experiments, some humans age with little or no loss of cognitive abilities while others tend to have a serious decline in cognition associated with aging. A better understanding of the biochemical events responsible for this difference may lead to novel approaches to alter the age-related cognitive decline.

NMDA receptors are Ca++-gating inotropic receptors. They are multi- subunit hetero-oligomers assembled from two classes (NR1 and NR2) of subunits. The one NR1 gene is processed into at least seven distinct splice variants and the four NR2 genes produce proteins that combine, in unknown combinations and stoichiometry, with NR1 to form receptors of distinct physiological characteristics depending on subunit composition. We have developed a battery of antibodies that can selectively recognize each of the subunits on immunoblots and can selectively immunoprecipitate a specific subunit. By solubilizing receptors in a gentle detergent, the subunit interactions remain intact and co- immunoprecipitation of multiple subunits with a specific antibody yields information regarding subunit associations in situ. This project proposes: (1) to determine the subunit combinations that assemble into NMDA receptors in various regions of the rat brain, (2) to generate cell lines stably expressing NMDA receptors of defined subunit composition that represent the major species of NMDA receptors expressed, (3) to examine the functional properties of NMDA receptors of defined subunit composition, (4) to measure the stoichiometry of NMDA receptor subunits in assembled NMDA receptors, and (5) to examine the possibility that agrin mediates tyrosine phosphorylation and/or aggregation of NMDA receptors. Results from these studies will increase our knowledge of the structure, function, location, and pharmacology of these receptors. Because NMDA receptors are involved in many important functions such as synaptic plasticity, which is vital for normal CNS development and may underlie the processes of learning, memory, and excitotoxicity, which may be involved in ischemic brain damage as well as several neurological diseases, drugs that affect the function of these receptors could be useful in the treatment of stroke,neurodegenerative diseases, and control of chronic pain to name a few examples. However, until we understand the various types and properties of the receptors that are expressed, side-effects will dominate any drugs that act via these receptors.

NMDA receptors are Ca++-gating inotropic receptors. They are multi- subunit hetero-oligomers assembled from two classes (NR1 and NR2) of subunits. The one NR1 gene is processed into at least seven distinct splice variants and the four NR2 genes produce proteins that combine, in unknown combinations and stoichiometry, with NR1 to form receptors of distinct physiological characteristics depending on subunit composition. We have developed a battery of antibodies that can selectively recognize each of the subunits on immunoblots and can selectively immunoprecipitate a specific subunit. By solubilizing receptors in a gentle detergent, the subunit interactions remain intact and co- immunoprecipitation of multiple subunits with a specific antibody yields information regarding subunit associations in situ. This project proposes: (1) to determine the subunit combinations that assemble into NMDA receptors in various regions of the rat brain, (2) to generate cell lines stably expressing NMDA receptors of defined subunit composition that represent the major species of NMDA receptors expressed, (3) to examine the functional properties of NMDA receptors of defined subunit composition, (4) to measure the stoichiometry of NMDA receptor subunits in assembled NMDA receptors, and (5) to examine the possibility that agrin mediates tyrosine phosphorylation and/or aggregation of NMDA receptors. Results from these studies will increase our knowledge of the structure, function, location, and pharmacology of these receptors. Because NMDA receptors are involved in many important functions such as synaptic plasticity, which is vital for normal CNS development and may underlie the processes of learning, memory, and excitotoxicity, which may be involved in ischemic brain damage as well as several neurological diseases, drugs that affect the function of these receptors could be useful in the treatment of stroke,neurodegenerative diseases, and control of chronic pain to name a few examples. However, until we understand the various types and properties of the receptors that are expressed, side-effects will dominate any drugs that act via these receptors.