1985 |
Deth, Richard C |
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. |
A-Adrenergic Coupling Events in Arterial Smooth Muscle @ Northeastern University
The series of cellular events which are initiated by agonist occupation of Alpha[unreadable]1[unreadable] adrenergic receptors are only partially understood. While it is recognized that an increase in cytosolic Ca[unreadable]2+[unreadable] mediates such Alpha[unreadable]1[unreadable] receptor responses as contraction of arteries and stimulation of hepatic glycogenolysis, the reactions leading to Ca[unreadable]2+[unreadable] mobilization are unclear. Since activation of Alpha[unreadable]1[unreadable] receptors is associated with a Ca[unreadable]2+[unreadable] independent increase in the generation of the membrane phospolipids phosphatidic acid (PA) and phosphatidyl inositol (PI), it has been suggested that these changes lead to observed increases in Ca[unreadable]2+[unreadable] influx and intracellular Ca[unreadable]2+[unreadable] release. To test the validity of this suggestion we will quantitate the relationship between Alpha[unreadable]1[unreadable] receptor occupany and coupling events in the rabbit aorta. In addition to measurements with the full agonists norepinephrine and phenylephrine we will compare the effects of a series of imidazoline derivatives which are graded partial agonists so as to draw inferences on the basis of their submaximal effectiveness. Occupancy will initially be determined for both full and partial agonists using contractile response as an endpoint. Radioligand binding studies on isolated membranes using the receptor subtype-specific radioligands [unreadable]3[unreadable]H-prazosin and [unreadable]3[unreadable]H-yohimbine will also be used to characterize the interaction of agonists with Alpha[unreadable]1[unreadable] and Alpha[unreadable]2[unreadable] receptors respectively. Subsequent studies will measure the simulation of [unreadable]45[unreadable]Ca release by increasing concentrations of agonists. [unreadable]32[unreadable]P-labelling of PA and PI will also be assayed at the same agonist concentrations. The results of contraction, receptor binding, Ca[unreadable]2+[unreadable] flux and phospholipid turnover measurements will be compared so as to address the following questions: 1) Is the relationship between receptor occupany and response linear when computed using each of the above receptor-initiated parameters? 2) Are non-linear relationships similar for different steps in the coupling process? 3) Is the pattern of PA (or PI) formation compatible with its role as a receptor-operated Ca[unreadable]2+[unreadable] gating mechanism? 4) Do phospholipid and Ca[unreadable]2+[unreadable] flux events indicate the presence of a receptor reserve? 5) Is the pattern of Ca[unreadable]2+[unreadable] handling by arterial smooth muscle cells similar at different levels of receptor-initiated activation.
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0.958 |
1986 — 1988 |
Deth, Richard C |
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. |
Pi Cycle Related A1 Receptor Coupling Events in Arteries @ Northeastern University
Ca2+ mobilizing hormones such as norepinephrine acting on Alpha-1 adrenergic receptors are thought to initiate receptor coupling events by their ability to activate a plasma membrane phospholipase C which selectively hydrolyzes plasma membrane polyphosphoinositides. In arterial smooth muscle both intracellular Ca2+ release and Ca2+ influx result from Alpha-1 receptor activation, however, the sequences of events causing these Ca2+ movements has not been established. Our studies during years one and two of this project show that PI cycle phospholipids are selectively hydrolyzed, indicating that the products of phosphatidyl inositol 4.5 bisphosphate hydrolysis (DAG and IP3) may be responsible for Ca2+ influx and Ca2+ release respectively. In the current proposal this hypothesis is to be tested and the mechanism by which these two agents cause Ca2+ movements in arterial smooth muscle is to be investigated. Conditions which modify Alpha-1 receptor coupling events will also be studied. Three areas can be identified: 1. Verification of IP3 and DAG formation and delineation of their relationship to Ca2+ release and Ca2+ influx. This includes a study of the action of phorbol esters which activate protein kinase C in a manner similar to DAG and a study of the possible role of Na+/H+ exchange in Ca2+ influx. 2. Dynamic aspects of Alpha-1 receptor function. Consequences of pre-activation of the PI-cycle pathway by either Alpha-1 agonists or angiotensin II will be determined along with the influence of c-AMP- or c-GMP- dependent protein kinase systems on Alpha--1-receptor coupling events. 3. Impact of Alpha-1 receptor-induced non-PI cycle phospholipid changes. Experiments will detail receptor-induced changes in phosphatidylcholine synthesis to determine how they may modulate Alpha-1 receptor coupling events. Particular emphasis will be placed on observed species differences between rat and rabbit arteries. This project should not only provide specific information on the mechanism of Alpha-1-adrenergic coupling events in arteries, but will help to identify conditions which may modulate performance of this receptor system under physiologic conditions. Our results will have a direct bearing on our knowledge of cardiovascular physiology and pathophysiology and may provide insight into better management of hypertension and related disorders.
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0.958 |
1989 — 1993 |
Deth, Richard C |
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. |
Protein Kinase Control of Arterial A1 &A2 Receptors @ Northeastern University
Alpha-1 adrenergic receptors cause increased vascular tone via a G-protein mediated hydrolysis of phosphatidylinositoldiphosphate (PIP2), IP3-induced Ca2+ release and diacylglycerol (DAG) activation of protein kinase C (PKC). The coupling pathway for alpha-2 receptors is not as well established although work form other tissues has focused interest on a receptor associated increase of Na+/H+ exchange. Recent evidence indicated that PKC may act as a modulator of alpha-1 receptor coupling via phosphorylation of the receptor itself or at subsequent steps. Receptor-activated hydrolysis of phospholipids other than PIP2 (e.g. phosphatidylinositol or phosphatidylcholine) may be a function of the level of kinase activity. This proposal is directed toward elucidating the importance of PKC and cGMP-dependent protein kinase regulation at arterial alpha-1 receptors. In addition, a study of alpha-2 receptor coupling pathways and its regulation by these kinases will be undertaken to permit a more comprehensive view of the function of these closely related receptor systems in vascular tissues. The approach taken will be to measure the comparative hydrolysis of different phospholipids in isolated tissues upon receptor activation under conditions where protein kinase C or protein kinase G have been elevated (by phorbol ester or nitroprusside respectively) or reduced (by staurosporine or LY 83583). The specific influence of these kinase on receptor binding will be assayed in radioligand binding studies using plasma membrane fractions isolated from bovine aorta after prior treatment to provide kinase activation or inhibition conditions. In these membrane preparations we will also measure agonist-induced phospholipid hydrolysis in order to correlate binding of agonists with functional responses. These studies will include examination of the GTP and Ca2+ requirements, as well as the influence of pH on phospholipase activity. Studies on alpha-2 receptors will focus on the possible role of changes in pHi mediated by increased Na+/H+ exchange and protein kinase C activation in contractile responses of the isolated saphenous vein. Binding studies will be utilized to demonstrate or exclude involvement of a G-protein in receptor coupling and the influence of kinase activation on agonist binding. Results from these experiments will provide further insight into the role of protein kinase and Na+/H+ exchange activities in regulating agonist-induced arterial contraction.
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0.958 |
1994 — 1997 |
Deth, Richard C |
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. |
Alpha-2 Adrenergic Receptor Coupling Pathways @ Northeastern University
Alpha-2 adrenergic receptors (alpha2R) play a critical role in mediating the influence of the sympathetic nervous on vascular smooth muscle, especially in the microvasculature. This role demands that the alpha2R coupling mechanism be able to integrate extrinsic neuronal activity with local needs in order to achieve optimal vascular tone and blood flow. This project will investigate the nature of the alpha2R coupling pathway, such as G proteins and effectors which are involved, and will examine how the efficacy and character of coupling is modulated. Coupling will be studied using both contraction and biochemical endpoints in isolated vascular tissue (rabbit saphenous vein) and GTP binding and biochemical endpoints in cultured cells (primarily PC12 cells) expressing a cloned alpha2R subtype. Previous work has established the ability of unoccupied alpha2R to cause a significant level of G protein activation, termed precoupling,and the regulation of precoupling is an important focus of the project. Since preliminary data indicated that precoupling can involve both vasoconstrictictor and vasodilator pathways, depending upon protein kinase C (PKC) activation, we will further investigate the mechanism of its control. In follow-up to earlier radioligand binding and GTP binding studies in isolated membranes, the influence of cytoplasmic Na+ levels on agonist efficacy will be examined, as well as its interplay with H+ levels. While the alpha2R coupling pathway is recognized to be highly dependent upon extracellular Ca2+, this dependence may reflect its essential role in providing for activation of PKC and for facilitating the activity of phospholipases D and A . The ability of alpha2R to stimulate these phospholipases under varied levels of Ca2+ will also be investigated. Preliminary data has indicated an essential role of tyrosine kinase activation in alpha2R vascular responses, but not in alpha1R responses. Since receptor-induced PLD activation has been shown in other tissues to require tyrosine kinase activity, we will determine whether this is the basis for the kinase in vascular smooth muscle as well. An integrated working hypothesis is presented which has the potential to provide a mechanistic basis for the observed adaptability of alpha2R responsiveness. Since micro-vascular dysfunction is thought to play an important role in several forms of hypertension, as well as in other disorders, the improved understanding provided by these studies will have both physiologic and pathophysiologic significance.
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0.958 |
2011 — 2012 |
Deth, Richard C |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Effect of Drugs of Abuse On Neuronal Redox and Methylation Status @ Northeastern University
DESCRIPTION (provided by applicant): Commonly abused drugs are diverse in their chemical and pharmacological profiles, but they act on neural systems which support attention and consciousness, and their repetitive use leads to a state of dependence, characterized by withdrawal syndromes. While the neurochemical events leading to dependence remain only partially understood, there is increasing evidence for the involvement of epigenetic mechanisms such as DNA methylation. Studies our lab have identified a critical role for the cysteine/glutamate uptake transporter EAAT3 (excitatory amino acid transporter 3) in controlling redox and methylation status in human neuronal cells. EAAT3-mediated cysteine transport is stimulated by dopamine, involving the D4 dopamine receptor, which has been linked to the frequency of drug abuse. Preliminary studies show that EAAT3- mediated cysteine uptake is also sensitive to opiates and ethanol, as well as nicotine and cannabinoids, suggesting that it may serve as a nexus for the epigenetic effects of abused drugs. To investigate this possibility, we will carry out a detailed investigation of the influence of selected drugs of abuse on redox and methylation status of cultured human neuroblastoma cells and rat-derived primary cultured neurons. We will measure the dose and time-dependent effects of morphine, amphetamine and alcohol on the cellular uptake of [35S]-cysteine, which primarily reflects EAAT3 activity. Under the same conditions, we will measure drug-induced changes of intracellular thiols (reduced and oxidized forms of glutathione (GSH and GSSG), cysteine and homocysteine) as well as methylation-related metabolites methionine, S- adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and cystathione. The ratio of GSH to GSSG is an indication of cellular redox status, while SAM to SAH is a reflection of methylation capacity. Changes in global DNA methylation and CpG island methylation status will be measured to assess the epigenetic effects of each drug.. Based upon preliminary data, we hypothesize that these parameters will be responsive to the selected drugs of abuse, and that a new steady-state redox and methylation status will be attained in their sustained presence, representing adaptive neuronal responses to their presence. We will monitor time-dependent changes in cysteine uptake and cellular redox/methylation status upon drug removal, representing a reversal of the initial adaptive responses, analogous to withdrawal and evaluate the influence of a previous exposure on subsequent redox and methylation responses to the same or different drug (i.e. sensitization or tolerance and cross-reactivity). Finally, we will use qRT-PCR to evaluate changes in transcription of selected redox and methylation-related genes, which may mediate adaptive responses to drugs of abuse during their presence and/or contribute to the withdrawal state in their absence. Experimental results for each drug will be incorporated into a computational model of the relevant metabolic pathways. This exploratory project will therefore examine a novel hypothesis related to the mechanisms of drug addiction. PUBLIC HEALTH RELEVANCE: The proposed project will carry out a detailed study of how morphine, methamphetamine, alcohol each affect sulfur metabolism and antioxidant status in cultured human neuroblastoma cells and primary cultured neurons. The results will provide an entirely new way of thinking about how drugs of abuse affect the brain and could lead to improved ways to treatment to treat drug addiction.
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0.958 |