PHARMACOLOGICAL CHARACTERIZATION OF THE 3': 5' CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ACTIVITY AND MUSCARINIC ACETYLCHOLINE RECEPTORS OF BRAIN COATED VESICLES.
Item
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Title
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PHARMACOLOGICAL CHARACTERIZATION OF THE 3': 5' CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ACTIVITY AND MUSCARINIC ACETYLCHOLINE RECEPTORS OF BRAIN COATED VESICLES.
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Identifier
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AAI8611383
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identifier
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8611383
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Creator
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SILVA ORTIZ, WALTER I.
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Contributor
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Saul Puszkin
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Date
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1986
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Language
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English
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Publisher
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City University of New York.
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Subject
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Health Sciences, Pharmacology | Biology, Neuroscience
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Abstract
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Bovine brain coated vesicles (CV) can display a high degree of microheterogeneity. This degree would be dependent on the contribution of diverse subcellular, cellular and neuroanatomical regions to the CV population. Two cargo molecules are described in this thesis with their respective pharmacological properties. These cargo molecules are 3':5'-cyclic nucleotide phosphodiesterase (PDE) and the muscarinic acetylcholine receptor (mAChR). The PDE activity is insensitive to calcium and stimulated by cyclic GMP. The basal hydrolysis has a broad alkaline pH optimum in contrast to the cGMP-stimulated hydrolytic activity which has a neutral pH optima. Analysis of the dose response curves for the hydrolysis of both cyclic cAMP and cGMP reveal very similar Kd and Vmax values. The kinetic behavior of the enzyme suggests positive cooperativity. Nonetheless, an alternative equilibrium kinetics model is proposed which can explain the kinetic behavior of the CV type II PDE activity. The inhibition profile of the CV PDE by papaverine, IBMX and theophylline can support partially the kinetic model, in addition to certain experimental observations. The presence of a type II PDE in coated vesicles could suggest a differential subcellular partitioning of the known PDE isozymes.;The mAChR had a Kd of 25 pM and a Bmax of 341 fmol/mg protein. Competition experiments with both agonists and antagonists revealed heterogeneity of binding affinities. The binding site with high affinity for oxotremorine displayed poor sensitivity to GTP. The high and low affinity binding sites for carbachol could be interpreted in terms of distinct pre and postsynaptic receptors (Mash, et al., 1985). Nonetheless, the differences in the percentage of high affinity between both agonists, carbachol and oxotremorine leads to caution in the use of the latter approach in the interpretation of the data. Atropine did not display shallow inhibition or competition curves in contrast to the hydrophylic antagonist N-methyl-scopolamine. The heterogeneity of binding affinities for muscarinic drugs in CV as well as the PDE data is discussed in the text in the context of CV subpopulations (Pfeffer and Kelley, 1985 and Weidenmann et al., 1985).
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
