1985 |
Yamamura, Henry I |
K02Activity Code Description: Undocumented code - click on the grant title for more information. |
Psychotropic Drugs and Central Cholinergic Receptors
Until a shorttime ago, studies of neurotransmitter receptors in the brain have relied primarily upon neurophysiological techniques. Recently, however biochemical methods for the direct measurement of receptors have become available. We have demonstrated the specific binding of muscarinic receptors using QNB and CD, nicotinic receptors using Naja naja, GABAergic receptors using muscimol, dopamine receptors using spiroperidol and benzodiazepine receptors using flunitrazepam. We have used these ligands as tools in solving some of the problems in neuropharmacology. We have found biochemical receptor alterations in discrete brain regions from postmortem brain samples of persons dying from Huntington's, Parkinson's, Alzeheimer's Disease and found Schizophrenic brain samples. I am interested in finding new ligands in the future so that novel neurotransmitter and drug receptors can be labeled. We are currently identifying the barbiturate and nonbenzodiazepine receptors in brain. An important area of the research involves the mechanism of coupling between the receptor and the effector (perhaps the guanine nucleotide binding site or adenylate cyclase). Other important areas involve receptor regulation and receptor heterogeneity. Neurotransmitter receptors appear to be regulated in an up or down direction depending upon neuronal activity. We are interested in determining the exact mechanism of this regulation. Neurotransmitter receptors for the opiate, alpha- and beta-adrenergic, dopamine, histamine and muscarinic cholinergic systems exist as subtypes. Receptor binding studies have complimented classical pharmacological studies in this respect. We are interested in determining the mechanism of receptor heterogeneity. We have clues which indicate that ions, and guanine nucleotides are important agents in illustrating receptor homogeneity.
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1990 — 1992 |
Yamamura, Henry I |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Psychotropic Drugs &Muscarinic Receptor Types
In our initial studies, we found that the human neuroblastoma (SH-SY5Y) cell line consistently expresses a high density of mAChRs (about 220 fmol/mg protein or 25,000 receptors/cell). Most of the mAChRs in the SH-SY5Y cells showed high affinity for [3H]PZ, suggesting an M1 nature of these muscarinic receptors. To our knowledge, the SH-SY5Y cell line is the only cell line with high affinity [3H]PZ binding to_the mAChRs on the intact cells. The SH-SY5Y cells have a functional PI system and an adenylate cyclase system. The existence of these effector systems offers a unique opportunity to study the effector coupling mechanisms of the M1 receptors in a homogeneous neuronal cell line. Our initial data suggest that these M1 receptors are coupled to the PI system. In this proposal, we will further examine the pharmacological properties of the mAChRs in SY-SY5Y cells using several new selective muscarinic ligands and compare these data with a transfected B82 system which is thought to contain only one type of mAChr. i.e., M1. Furthermore, we will also examine the second messenger system coupled to the receptors in order to obtain a better understanding of the molecular basis for the function of the mAChRs in both cell lines. Although our initial pharmacological studies indicate that the predominant mAChRs on the SH-SY5Y cells are of the M1 type, since there could be more than one mAChR showing high affinity for PZ (40), an unambiguous definition of this receptor requires a knowledge of its primary structure by gene cloning and sequencing. However, this will only be done if the mAChRs in the SH-SY5Y cell line is unique. This study will yield clone(s) which express a constant population of M1 receptors with pharmacological and biological properties of the native M1 receptors. This study will provide in depth information on the functional mechanism of neural mAChRs and their genetic regulation. This knowledge will have importance in the drug treatment of CNS disorders with cholinergic deficits such as senile dementia of the Alzheimer's type (SDAT), Huntington's disease (HD) and Parkinson's disease(PD). Furthermore, these cell lines will be used as models for the development of selective drugs which act at a single type of mAChR thereby improving therapeutic effects while reducing side effects.
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1991 — 1993 |
Yamamura, Henry I |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Gastrointestinal Control by Neuropeptides
NO PARENT ABSTRACT AVAILABLE
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1992 — 2001 |
Yamamura, Henry I |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Biochemical Characterization of Opioid Ligands and Receptors
The existence of opioid receptor subtypes would be consistent with the discoveries of multiple muscarinic, adrenergic, and serotonergic receptor subtypes and would provide new opportunities for opioid pharmacology. these opportunities would result from the presence of new receptor targets for opioid drug development. It may be possible to show that certain opioid receptor subtypes mediate desirable effects like analgesia without mediating dangerous effects like respiratory depression. The focus of this proposal concerns the identification and characterization of mu and delta opioid receptor heterogeneity. The specific aims of this proposal are: 1. To test our hypothesis that there are at least two subtypes of the delta opioid receptor by the examination of their ligand binding properties using our recently developed radiolabeled radioligands. 2. To use radioligand binding methods to test hypotheses related to the mu- delta opioid receptor complex. these hypotheses include: A. That DPDPE acts at a mu opioid receptor coupled delta opioid receptor through an allosteric mechanism that serves to potentiate mu opioid agonist-induced analgesia in the brain. B. That the enkephalin analogue DALCE selectively antagonizes the delta opioid receptor acted upon by DPDPE but not that of the highly delta selective agonist deltorphin II. C. That the isothiocynante derivative of naltrindole (NTII) selectively blocks the receptor responsible for deltorphin II induced analgesia but does not recognize the delta opioid site acts upon by DPDPE. D. That D-Tca-CTAP where D-Tca is an analogue of D-Trp and a novel bivalent enkephalin analogue biphalin act at coupled mu and delta opioid receptors through a self-potentiation mechanism.
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1994 — 1998 |
Yamamura, Henry I |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Biochemical Studies and Cloning of Delta Subtypes
This proposal is part of a program project that seeks to produce and characterize non-peptidic compounds acting at delta opioid receptors. A basic hypothesis of this program project is that drugs acting on delta opioid receptors will be potent analgesics, but will lack many of the undesirable side effects of the opiate drugs now in use. Furthermore, it is hypothesized that there are multiple delta opioid receptor subtypes and that an additional increase in therapeutic specificity can be achieved by drugs selective for particular delta receptor subtypes. Two recent developments, the discovery of the non-peptidic compound BW373U86 and the cloning of mouse delta opioid receptors, provide a foundation for the testing of these hypotheses and the work proposed here. BW373U86 is the only known selective delta receptor agonist that is not a peptide. The racemic mixture of BW373U86 was resolved into its two (+) and (-) isomers by Dr. Kenner Rice who proposes to have each form labeled with tritium for studies by radioligand binding. We propose to do this characterization by tissue homogenate binding and receptor autoradiography studies using mouse and rat neural tissue. Parallel studies with radiolabeled forms of the established ligands [4'-Cl- Phe4]DPDPE and naltrindole in addition to binding inhibition studies designed to characterize the site(s) labeled by BW373U86 will be used to define its properties at CNS receptors. This information will assist the design of new analogs of BW373U86 and help to explain some of the unusual pharmacological properties of this novel compound. Some of these properties, including high potency at delta receptors in the mouse vas deferens but low analgesic potency suggest that BW373U86 may be selective for delta receptor subtypes. We also intend to explore the hypothesis of delta opioid receptor subtypes by cloning mouse and human cDNAs encoding the proposed subtypes. Since at least one subtype of the mouse delta opioid receptor has been cloned, we will use polymerase chain reaction methods to product oligonucleotide probes selective for delta opioid receptors. These probes will be used to initially screen mouse and subsequently human brain cDNA libraries for the presence of multiple delta opioid receptors. The identification of cDNAs for delta receptor subtypes is important since selective radioligands are not available which impedes the development of selective drugs for these subtypes. Furthermore, the only way by which any human delta opioid receptors can be studied is through the use of recombinant cells expressing these receptors. cDNAs for delta receptors will be expressed in mammalian cells to produce cell lines having defined delta receptor subtypes. The cell lines expressing different human delta opioid receptor subtypes will be used for the development of specific radioligand binding and functional assays for the screening and characterization of the new compounds proposed elsewhere by this program project proposal.
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1997 — 2001 |
Yamamura, Henry I |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Bioanalytical Core
The Program Project that this Bioanalytical Core Proposal is part of intends to develop new opioid analgesic drugs that will replace existing narcotics. It is anticipated that these new drugs will be as effective as existing opiate drugs but will be safer and have less abuse potential. The Bioanalytical Core Proposal provides essential analytical services to other parts of the associated Program Project responsible for the development of new opioid peptide and peptide-like compounds. These services include measurements of binding afinity at the different opioid receptor types, measurements of potency using isolated tissue assays, determination stability against degradation by tissue, studies of the ability of these compounds to pass membrane barriers and analgestic activity after direct injection into the brain and after systemic injection. The basic information on opioid receptor binding affinity and potency will be obtained for all compounds under development. Compounds selected from these studies will be further tested using the other three approaches. Stability, membrane permeation and analgesic activity studies will identify new compounds most likely to have clinical utility as new opioid drugs.
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2003 — 2006 |
Yamamura, Henry I |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanism of Adenylyl Cyclase Superactivation
DESCRIPTION (provided by applicant):Analgesics that act through the delta opioid receptor have low addictive potential but similarly to classical opiates, display tolerance after long-term treatment. Chronic opioid receptor stimulation leads to a compensatory increase in adenylyl cyclase (AC) activity, called AC superactivation. We have demonstrated that an AC isoenzyme (AC VI) is phosphorylated upon chronic 5 opioid agonist (SNC 80) treatment in CHO cells transfected with the human delta opioid receptor (hDOR/CHO). We hypothesize that phosphorylation of AC VI isinvolved in adenylyl cyclase superactivation, and that superactivation is involved in tolerance to chronic delta opioid agonists. In preliminary experiments we found that alpha-transducin, a putative scavenger of G protein beta-gamma-subunits, attenuated both chronic SNC 80 mediated phosphorylation of AC VI and AC superactivation. In this proposal we will use other, independent methods, to study the involvement of G protein beta-gamma-subunitsin cellular responses to acute- and chronic SNC 80 treatment. Attenuation of AC superactivation and AC VI phosphorylation by these methods will confirm the role of G protein beta-gamma-subunits in chronic SNC 80-mediated respolises. Subsequently we will show that free beta-gamma-subunits, released upon chronic SNC 80 treatment, regulate the activity of second messenger regulated protein kinases and Raf-1 protein kinase in hDOR/CHO cells. Finally, we will demonstrate that depletion of the protein kinase responsible for phosphorylation of AC VI in hDOR/CHO cells also attenuates chronic delta opioid agonist mediated AC superactivation. Better understanding of the molecular mechanisms of drug tolerance at the human delta opioid receptor should aid in the development of longer acting analgesics with fewer side effects.
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2007 |
Yamamura, Henry I |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Bioanalytical Facility |
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2007 |
Yamamura, Henry I |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Functional Domains of the Delta Opioid Receptor |
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2009 — 2013 |
Vanderah, Todd W [⬀] Yamamura, Henry I |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Biochemical Core
by the Chemistry Core. These compounds will be designed as opioid agonists and bradykinin/dynorphin antagonists in order to produce potent and efficacious antinociception targeting the pathology of neuropathic pain while eliminating antinocicpetive tolerance. The Biochemical Core contains 7 aims that will test such novel compounds. The in vitro pharmacological data in particular will provide timely feedback to the Chemistry Core on the structure-activity relationship (SAR) to further inform chemistry design. The initial binding and functional characterization of all novel compounds (est. 20 to 50 compounds/year) is necessary and essential for target-based drug discovery. To identify lead compounds, we must evaluate their affinity at multiple opioid and bradykinin receptors, and their apparent biological activity at each of these receptors. Studies will include in vitro tissue assaysto determine agonist and antagonist activity as well as novel compound activity at calcium channles using calcium fluorimetric analysis in transfectd cells. We will use a number of in vivo animal models to identify whether such novel bi-functional compounds produce antinociception as well as antihyperalgesia in inflammatory and chronic pain states. Finally, in vivo studies will be performed to detemine whether such compounds will result in antinocicpetive tolerance. Overall, Studies will be performed to identify molecules with agonist activity at one receptor and concurrent antagonist actions at a second receptor. The biochemical core provides dedicated equipment, personnel and expertise in data analysis for the entire project. It serves to centralize the use and maintenance of shared equipment and the technical training of personnel to use these equipment, management and oversight of animal protocols required by IACUC and Radiation Control, as well as to ensure data and information sharing with the Chemistry Core and the other projects. The biochemical core will synergize with projects A, C and D by providing lead compounds to directly test their hypotheses. RELEVANCE (Seeinstructions): Inflammatory and chronic neuropathic pains are growing areas of unmet medical need. Clinically, chronic pain remains poorly controlled by available therapies and thus adversely impacts quality of life (Arner & Meyerson, 1988). One reason for this lack of effect is the absence of compounds that specifically target the pathology of neuropathic pain. The goal of the chemistry &biochemical core of this PPG are to synthesize and test compounds that result in potent and efficacious antinociception while eliminating tolerance. PROJECT/
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2009 — 2013 |
Varga, Eva V Yamamura, Henry I |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Functional Regulation of Opioid Signalling
Sustained morphine treatment was shown to increase the concentration of excitatory Gs protein-coupled neuromodulators (such as PGE2 and dynorphin) and augment pain neurotransmitter release in the spinal cord. In Project C we will investigate the role of cAMP-regulated signaling pathways in the regulation of pain neurotransmitter (CGRP) release from cultured neonatal rat primary sensory (DRG) neurons by PGE2 and a non-opioid fragment of spinal dynorphin, dyn2-13. In addition, since earlier we have shown that sustained morphine treatment leads to a Raf-1 -mediated sensitization of adenylyl cyclase(s) (AC superactivation) towards excitatory agents in recombinant cells, in Project C we also will investigate the physiological role of Raf-1-mediated AC superactivation in the sensitization of basal and/or capsaicin-evoked CGRP release from sensory neurons after sustained morphine-treatment. We hypothesize that Raf-1-mediated AC superactivation sensitizes primary sensory neurons to Gs protein-coupled neuromodulators leading to augmented basal and/or evoked CGRP release upon sustained morphine treatment. To evaluate this hypothesis we shall I. investigate the role of Raf-1 in the sensitization of cAMP formation in cultured neonatal rat DRG neurons toward the Gs protein-coupled excitatory neuromodulators, PGE2 and dyn2-13; II. test the role of cAMP, cAMP-dependent protein kinase (PKA) and Raf-1 in the regulation of basal and/or capsaicinevoked CGRP release by PGE2 and dyn2-13 in cultured neonatal rat DRG neurons before and after sustained morphine treatment; and III. study the effect of selected novel compounds - prepared in the Synthetic Core and Project A - on cAMP concentration and basal and capsaicin-evoked CGRP release in cultured neonatal rat DRG neurons, before and after sustained opioid agonist treatment.
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