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Title: Guanyl nucleotide interactions with dopaminergic binding sites labeled by (/sup 3/H)spiroperidol in human caudate and putamen: guanyl nucleotides enhance ascorbate-induced lipid peroxidation and cause an apparent loss of high affinity binding sites

Abstract

The human caudate and putamen contain two high affinity binding sites for (/sup 3/H)spiroperidol. Both of these affinity states exhibit dopaminergic selectivity. Ascorbic acid, at 0.1 mM, induces a slow loss of the low affinity component of (/sup 3/H)spiroperidol binding in these tissues. The addition of guanyl nucleotides to the ascorbate produces a more rapid loss of (/sup 3/H)spiroperidol binding which includes a loss of the highest affinity state for (/sup 3/H)spiroperidol. Ascorbate induces lipid peroxidation in human caudate and putamen, an effect that is further enhanced by guanyl and inosine nucleotides. In the absence of ascorbate, guanyl nucleotides have no effect on (/sup 3/H)spiroperidol binding but do decrease the affinity of dopamine at each affinity state greater than 60-fold. In the absence of ascorbate, guanyl nucleotides apparently decrease agonist affinity at human brain dopamine2-binding sites without causing an interconversion of agonist affinity states.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Case Western Reserve Univ. School of Medicine, Cleveland, OH (USA)
OSTI Identifier:
5304056
Resource Type:
Journal Article
Resource Relation:
Journal Name: Mol. Pharmacol.; (United States); Journal Volume: 33:2
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; DOPAMINE; RECEPTORS; LIPIDS; METABOLISM; NUCLEOTIDES; BIOCHEMICAL REACTION KINETICS; SPIPERONE; AFFINITY; ASCORBIC ACID; IN VITRO; MAN; TRACER TECHNIQUES; TRITIUM COMPOUNDS; AMINES; ANIMALS; AROMATICS; AUTONOMIC NERVOUS SYSTEM AGENTS; CARDIOTONICS; CARDIOVASCULAR AGENTS; DRUGS; HYDROXY COMPOUNDS; ISOTOPE APPLICATIONS; KINETICS; LABELLED COMPOUNDS; MAMMALS; MEMBRANE PROTEINS; NEUROREGULATORS; ORGANIC COMPOUNDS; PHENOLS; POLYPHENOLS; PRIMATES; PROTEINS; REACTION KINETICS; SYMPATHOMIMETICS; VERTEBRATES; VITAMINS 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Andorn, A.C., Bacon, B.R., Nguyen-Hunh, A.T., Parlato, S.J., and Stitts, J.A.. Guanyl nucleotide interactions with dopaminergic binding sites labeled by (/sup 3/H)spiroperidol in human caudate and putamen: guanyl nucleotides enhance ascorbate-induced lipid peroxidation and cause an apparent loss of high affinity binding sites. United States: N. p., 1988. Web.
Andorn, A.C., Bacon, B.R., Nguyen-Hunh, A.T., Parlato, S.J., & Stitts, J.A.. Guanyl nucleotide interactions with dopaminergic binding sites labeled by (/sup 3/H)spiroperidol in human caudate and putamen: guanyl nucleotides enhance ascorbate-induced lipid peroxidation and cause an apparent loss of high affinity binding sites. United States.
Andorn, A.C., Bacon, B.R., Nguyen-Hunh, A.T., Parlato, S.J., and Stitts, J.A.. 1988. "Guanyl nucleotide interactions with dopaminergic binding sites labeled by (/sup 3/H)spiroperidol in human caudate and putamen: guanyl nucleotides enhance ascorbate-induced lipid peroxidation and cause an apparent loss of high affinity binding sites". United States. doi:.
@article{osti_5304056,
title = {Guanyl nucleotide interactions with dopaminergic binding sites labeled by (/sup 3/H)spiroperidol in human caudate and putamen: guanyl nucleotides enhance ascorbate-induced lipid peroxidation and cause an apparent loss of high affinity binding sites},
author = {Andorn, A.C. and Bacon, B.R. and Nguyen-Hunh, A.T. and Parlato, S.J. and Stitts, J.A.},
abstractNote = {The human caudate and putamen contain two high affinity binding sites for (/sup 3/H)spiroperidol. Both of these affinity states exhibit dopaminergic selectivity. Ascorbic acid, at 0.1 mM, induces a slow loss of the low affinity component of (/sup 3/H)spiroperidol binding in these tissues. The addition of guanyl nucleotides to the ascorbate produces a more rapid loss of (/sup 3/H)spiroperidol binding which includes a loss of the highest affinity state for (/sup 3/H)spiroperidol. Ascorbate induces lipid peroxidation in human caudate and putamen, an effect that is further enhanced by guanyl and inosine nucleotides. In the absence of ascorbate, guanyl nucleotides have no effect on (/sup 3/H)spiroperidol binding but do decrease the affinity of dopamine at each affinity state greater than 60-fold. In the absence of ascorbate, guanyl nucleotides apparently decrease agonist affinity at human brain dopamine2-binding sites without causing an interconversion of agonist affinity states.},
doi = {},
journal = {Mol. Pharmacol.; (United States)},
number = ,
volume = 33:2,
place = {United States},
year = 1988,
month = 2
}
  • The interactions of dopaminergic agonists and antagonists with /sup 3/H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of (/sup 3/H)dopamine and (/sup 3/H)apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/(/sup 3/H)dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific (/sup 3/H)dopamine binding are significantly correlated withmore » their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and (/sup 3/H)flupentixol-binding activities. The affinities of agonists to inhibit D3 specific (/sup 3/H)dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/(/sup 3/H)flupentixol competition curves. Both D3 specific (/sup 3/H) dopamine binding and the high affinity agonist-binding component of dopamine/(/sup 3/H)flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor.« less
  • In order to develop more selective methods for labeling brain dopamine receptors, this study describes in detail the properties of 2-amino-6,7,-(/sup 3/H)dihydroxy-1,2,3,4,-tetrahydronaphthalene ((/sup 3/H) ADTN) binding to dopaminergic sites in rat, calf, and human brain. (/sup 3/H)ADTN labeled two distinct types of dopaminergic binding sites in the brain striatum of the rat, calf, and human. Very low concentrations of dopamine and dopaminergic catecholamines (with IC50 values of 1 to 10 nM) inhibited the binding of (/sup 3/H)ADTN to both sites. Neuroleptics, however, inhibited the binding of (/sup 3/H)ADTN in two distinctly separate concentration ranges, with IC50 values of 0.15 tomore » 40 nM at one site and 100 and 50,000 nM at the other site. The site with high affinity for dopamine and low affinity for neuroleptics had binding properties that corresponded to those of the previously characterized D3 site). The (/sup 3/H)ADTN binding site with high affinity for neuroleptics demonstrated binding characteristics similar to a site labeled by /sup 3/H-Neuroleptics. (/sup 3/H)Apomorphine appeared to label the same two sites as (/sup 3/H)ADTN, while (/sub 3/H)dopamine labeled only the D3 site. Scatchard analysis of (/sup 3/H)ADTN or (/sub 3/H)apomorphine binding, under conditions for selective labeling of the low affinity neuroleptic site (D3) and the high affinity site for neuroleptics, detected a density of 70 fmol/mg of protein for each. The density of the D3 site in the calf striatum (170 fmol/mg of protein) was much greater than that of the high affinity neuroleptic site (50 fmol/mg). In the rat, the dissociation constant (KD) of (/sup 3/H)ADTN was 2 nM for both sites. (/sup 3/H)Apomorphine, however, had a higher affinity for the D3 site (KD.1.6 nM) than for the high affinity neuroleptic site (KD.4.2 nM).« less
  • (/sup 3/H)Dihydroergotamine (DE) labels a population of binding sites in rat brain membranes with an affinity of approximately 70 pM in both hippocampus (maximal binding at saturation (Bmax) = 340 fmol/mg of protein) and cerebral cortex (Bmax = 250 fmol/mg of protein). Specific binding typically comprises about 97% of total binding at the Kd of the radioligand when nonspecific binding is determined in the presence of 100 nM unlabeled DE. Association kinetics at 37 degrees C are consistent with a uniform association rate constant for all sites labeled. Specific binding is completely reversible with addition of excess unlabeled DE, butmore » dissociation does not proceed with simple first-order kinetics, suggesting the presence of more than one discrete binding site. Competition studies with selective drugs reveal alpha adrenergic, 5-HT1A and 5-HT1B components of (/sup 3/H)DE specific binding. When phentolamine (500 nM) is included to block alpha receptors and DPAT (100 nM) or spiroxatrine (500 nM) is included to block 5-HT1A receptors, specific binding is exclusively to sites with drug affinities characteristic of 5-HT1B receptors. Under these 5-HT1B-selective conditions, (/sup 3/H)DE binding is about 90% specific, with a Kd of about 50 to 60 pM and a Bmax of 96 fmol/mg of protein in hippocampus and 77 fmol/mg of protein in cortex. (/sup 3/H)DE binding to 5-HT1B sites is very slowly dissociable, with a T1/2 of greater than 2 h at 37 degrees C. 5-HT1B antagonists and DE itself yield competition curves at (/sup 3/H)DE-labeled 5-HT1B sites that are adequately fit assuming a single site in nonlinear regression analysis. Addition of 100 microM guanylyl 5'-imidodiphosphate appears to convert nearly all 5-HT1B sites to those having low affinity for agonists while having a much smaller effect on the binding of (/sup 3/H)DE.« less
  • Ascorbate acts as an antioxidant by protecting human serum from lipid peroxidation induced by azo dye-generated free radicals. On the other hand, ascorbate is readily oxidized in the presence of transition metal ions, (especially cupric ion) and accelerates lipid peroxidation in tissue homogenates by producing free radicals. Interestingly, the authors observed an antioxidant rather than an expected prooxidant role of ascorbate when human serum supplemented with 1.2 mmol/L ascorbate underwent lipid peroxidations initiated by 2mmol/L copper sulfate. The antioxidant role of ascorbate was confirmed by studying the conventional thiobarbituric acid reactive substances (TBARS) as well as by observing the protectivemore » effect of ascorbate on the copper-induced peroxidation of unsaturated and polyunsaturated fatty acids. The antioxidation protection provided by ascorbate was comparable to that of equimolar {alpha}-tocopherol when incubated for 24h. However, lipid peroxidation products were lower in serum supplemented with {alpha}-tocopherol after 48h of incubation. This effect may be attributed to the binding of copper by serum proteins, thus preventing direct interaction between cupric ions and ascorbate. This proposed mechanisms is based on the observation that the concentration of ascorbate decreased more slowly in serum than in phosphate buffer at physiological pH.« less
  • Putative parathyroid hormone (PTH) receptors in canine renal membranes were affinity labeled with /sup 125/I-bPTH(1-34) using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 4-azido-benzoate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a major 85,000 molecular weight (M/sub r/) PTH binding component, the labeling of which was inhibited by nanomolar concentrations of unlabeled PTH and by micromolar concentrations of 5'-guanylyl imidodiphosphate (Gpp-(NH)p). Labeling was not influenced by the unrelated peptides insulin and arginine vasopressin. Minor PTH binding components of M/sub r/ 55,000 and 130,000 were also seen, and labeling of these was likewise sensitive to unlabeled PTH and to Gpp(NH)p. Omissionmore » of protease inhibitors during the isolation of plasma membranes resulted in the loss of the M/sub r/ 85,000 PTH binding species and the appearance of an M/sub r/ 70,000 form. Several minor PTH binding components also were observed. Equilibrium binding studies showed that such membranes had an affinity for PTH indistinguishable from that in membranes isolated with protease inhibitors and displaying a major M/sub r/ 85,000 PTH binding species. The authors conclude that the major form of the adenylate cyclase coupled PTH receptor in canine renal membranes is an M/sub r/ 85,000 protein. An endogenous enzyme, probably a lysosomal cathepsin, can cleave this form to produce an M/sub r/ 70,000 receptor that retains full functional activity with respect to high-affinity, guanyl nucleotide sensitive PTH binding. The ability to covalently label the PTH receptor in high yield represents a major step toward the structural characterization of this important detector molecule.« less