摘要:

Abstract:To investigate aspects of the biochemical nature of membrane‐bound dopamine D1 receptors, rat striatal homogenates were pretreated with heavy metal cations and some other chemical agents, and their effects on D1 receptors were subsequently determined using a standard [3H]CR)‐(+)‐8‐chloro‐2,3,4,5‐tetrahydro‐3‐methyl‐5‐phenyl‐1‐N‐3‐benzazepine ([3H]SCH 23390) binding assay. Incubation of striatal membranes with as little as 1 μMHg2+, 10 μMCu2+, and 10 μMCd2+completely prevented specific [3H]SCH 23390 binding. The effect of Cu2+, 1.5 μM was noncompetitive in nature, whereas 3‐5 μMCu2+afforded mixed‐type inhibition. The inhibitory effect of Cu2+was fully reversed by dithiothreitol (0.1‐1 mM). Cu2+(2 μM) did not affect the affinity of m‐flupenthixol or clo‐zapine for remaining [3H]SCH 23390 sites. A second series of cations, Co2+(30 μM), Ni2+(30 μM), Mn2+(1 mM), Ca2+(25 mM), and Ba2+(20 mM), inhibited specific [3H]SCH 23390 binding by 50% at the concentrations indicated. The thiol alkylating reagentN‐ethylmaleimide (NEM) (0.2 mM) reduced specific binding by 70%. The effect of NEM was completely prevented by coincubation with a D1 receptor saturating concentration of SCH 23390 (20 nM) or dopamine (10 μM). The results indicated that the dopamine D1 receptor is a thiol protein and that a

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb05721.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:The sensitivity of choline transport to inhibition by ethylcholine mustard aziridinium (ECMA) was studied in several tissues. Choline transport was found to be inhibited irreversibly by ECMA in guinea pig and rat synapto‐somes but not inhibited in erythrocytes or kidney slices. If this finding can be extended to other tissues ECMA sensitivity may provide a simple criterion for identifying the choline carrier associated with cholinergic tissu

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb05722.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

AbstractA synthetic derivative of γ‐aminobutyric acid (GABA), SR 95531 [2‐(3′‐carboxy‐2′‐propyl)‐3‐amino‐6‐p‐methoxyphenylpyridazinium bromide], has recently been reported, on the basis of biochemical and in vivo microion‐tophoretic studies, to be a potent, selective, competitive, and reversible GABAAantagonist. In the present study, the binding of [3H]SR 95531 to washed, frozen, and thawed rat brain membranes was characterized. Specific binding was linear with tissue concentrations, had a pH optimum at neutrality, and was maximal at 4°C after 30 min of incubation. Pretreatment of the membranes with Triton X‐100 resulted in a 50% decrease of specific binding. Addition of iodide, thiocyanate, or nitrate to the incubation mixture decreased the affinity of [3H]SR 95531 for its binding site; Na+had no effect. Subcellular fractionation showed that 74% of the P2binding was in synaptosomes; 31% of the total homogenate binding was in P2and 50% in P3. The binding of [3H]SR 95531 was saturable; Scatchard analysis of the saturation isotherm revealed two apparent populations of binding sites (KDof 6.34 nMandBmaxof 0.19 pmol/mg of protein;KDof 32 nMandBmaxof 0.81 pmol/mg of protein). The binding of [3H]SR 95531 was reversible, and association and dissociation kinetics confirmed the existence of two binding sites. Only GABAAligands were effective displacers of [3H]SR 95531. GABAAantagonists were relatively more potent in displacing [3H]SR 95531 than [3H]GABA; the inverse was true for GABAAagonists. There were marked regional differences in the distribution of binding sites: hippocampus = cerebral cortex>thalamus = olfactory bulb = hypothala‐mus = amygdala = striatum>pons‐medulla and cerebellum. The surprisingly low density of binding sites in the cerebellum was owing to a marked reduction ofBmaxvalues at both the high‐ and the low‐affinity binding sites. In conclusion, the present results demonstrate specific, high‐affinity, saturable, and reversible binding of [3H]SR 95531 to rat brain membranes and strongly suggest that this radioligand labels the GABAArecep

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb05723.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:The total levels of butyrylcholinesterase (BChE) activity and, more specifically, the distribution of BChE molecular forms were measured in the human neocortex during fetal development. Both the amount of total activity and the abundance of the different molecular forms (G1and G4) remained relatively constant between gestational ages of 8‐22 weeks and were similar to those observed in samples of cortex from aged brain. In addition, in both Alzheimer‐type and parkinsonian dementia, the levels of total BChE activity as well as the relative abundance of the G1and G4molecular forms were similar to those observed in control tissue. Hence, both the levels of total activity and the distribution of molecular forms did not change significantly either during fetal development or in the neurodegenerative disorders of Alzheimer‐type and parkinsonian dementias. Because these situations are accompanied by changes in the cortical cholinergic system (including an increase and decrease in levels of the G4form of acetylcholinesterase, respectively), it is concluded that, at least in the human neocortex, BChE is unrelated to cholinergic neurotransmission associated with subcortical cholinergic projection f

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb05724.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:Treatment of chick embryos in ovo with hydro‐cortisone‐21‐phosphate (a single dose of 150 μg) caused a marked reduction of retinal thymidine kinase activity 24 h later. The inhibitory effect was highest (65–70%) in 8–10‐day‐old embryos and declined with age, disappearing after day 15. It was accompanied by a reduction in thickness of the retinal layers. Adrenocorticotropic hormone (ACTH) treatment (10 μg daily for 2 days) also produced an age‐dependent inhibitory effect on retinal thymidine kinase, whereas treatment with a single dose of 200 μg of metopir‐one, a compound that prevents the 11β‐hydroxylation of steroid molecules in the adrenal glands, impeded the decrease in thymidine kinase activity that normally occurs in chick embryo retina after day 9 of development. In addition, metopirone prevented the inhibition exerted by ACTH on thymidine kinase activity but had no effect on the a

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb05725.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:Conditions have been developed for an L‐[3H]glu‐tamate binding assay in which 85‐95% of the specific binding is to a site that corresponds to theN‐methyl‐D‐aspartate subclass of acidic amino acid receptors. Incubation of synaptic plasma membranes with L‐[3H]glutamate in 50 mMTris/acetate, pH 7.4, for 2‐20 min at 2°C results in binding with pharmacological characteristics of the electrophysio‐logically definedN‐methyl‐D‐aspartate receptor. The fraction of glutamate binding to this subclass of receptors, relative to the total, decreases with both increased time and temperature. This binding is reversible, is concentrated in the synaptic plasma membrane fraction, has a pH optimum of 7.0‐7.4, and is linear with respect to tissue protein concentration. The binding is unaffected by 1 mMconcentrations of the anions sulfate, chloride, bromide, thiocyanate, phosphate, acetate, nitrate, or carbonate and the monovalent cations potassium or ammonium. However sodium and the divalent cations copper, cobalt, zinc, cadmium, and manganese decrease binding to thi

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb05726.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:Membrane fractions prepared from astrocytes grown in culture exhibit a specific binding site for L‐[3H]glu‐tamate that is Cl−‐dependent and Na+‐independent. The binding site is a single saturable site with aKDof about 0.5 μM, is inhibited by L‐aspartate, L‐cysteate, and quisqualate, and is insensitive to kainate,N‐methyl‐D‐aspartate, α‐ami‐no‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate, and 2‐ami‐no‐4‐phosphonobutyrate. The pharmacological characteristics of the binding site indicate that it is distinct from any site previously described in synaptic membrane preparations. Comparisons of ionic requirements, ligand specificity, and inhibitor sensitivities, however, suggest the described binding is the first step in a Cl−‐dependent high‐affinity glutamate uptake system. Such binding studies provide a useful model system in which to investigate the close association between excitatory amino acids, astrocytes, the termination of glutamate's excitatory action by high‐affinity uptake, and the excitotoxic action of

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb05727.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:Independent protein kinases in the synaptic junction (SJ) isolated from rat cerebrum were characterized. SJ showed a protein kinase activity, phosphorylating intrinsic proteins, even in the absence of cyclic AMP or Ca2+plus calmodulin (CaM) exogenously added. The activity was affected neither by Ca2+concentrations in the physiological fluctuation range nor by the addition of specific ligands such as glutamate, aspartate, acetylcholine, and concanavalin A. The activity was not due to cyclic AMP‐dependent protein kinase in SJ, since the activity was not inhibited by an inhibitor protein for cyclic AMP‐dependent protein kinase, and since synapsin I was not specifically phosphorylated whereas cyclic AMP‐dependent kinase appeared to phos‐phorylate selectively the protein in SJ. Phosphorylation of SJ proteins by the independent kinases was about one‐third of that of the Ca2+/CaM‐dependent protein kinase intrinsic to SJ. The apparentKmfor ATP was estimated to be 700 μM. Proteins of 16K Mrand 117K Mrwere specifically phosphorylated under the basic condition (in the absence of the substances known to activate specifically protein kinases), as well as six other proteins both under the basic conditions and in the presence of Ca2+and CaM. The phosphorylation of 150K Mr, 60K Mr, 51K Mr, and 16K MrSJ proteins was enhanced after prephosphorylation of SJ proteins by intrinsic kinase in the presence of Ca2+and CaM. This fact suggested that a part of the independent kinase activity was attributable to the autonomous form of Ca2+/ CaM‐dependent kinase which was derived from its auto‐phosphorylation, and that the Ca2+/CaM‐dependent auto‐phosphorylation affected the microtubules in the postsyn‐aptic region inasmuch as the 16K Mrprotein occurred in both brain cytosol and microtubule protein fraction p

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb05728.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:The concentrations of the acidic dopamine (DA) catabolites homovanillic acid (HVA) and 3,4‐dihydroxy‐phenylacetic acid (DOPAC) measured in human CSF are supposed to reflect the “turnover” of DA in the brain. The notion of “turnover” is, however, not synonymous with impulse nerve activity in the dopaminergic systems. Significant amounts of DOPAC and HVA could, indeed, be demonstrated in brain structures wherein dopaminergic inner‐vation has not been documented. It must also be noted that DA is not only a neurotransmitter itself, but also a precursor of norepinephrine and epinephrine. Furthermore, in lumbar CSF, levels of biogenic amine catabolites partially reflect metabolism in the spinal cord and may have limited relevance to neurotransmission in the brain. To elucidate these points further, we determined the concentrations of DOPAC and HVA in 22 areas of six human brains and eight levels of six human spinal cords. The data were correlated with the concentration of DA. Quantitative determinations were done using HPLC with electrochemical detection, after solvent and ion‐pair extraction. In this study, significant amounts of both DOPAC and HVA were demonstrated in brain structures not previously associated with dopaminergic innervation. The relatively lower DA concentration in these structures suggests that in these regions, the DOPAC and HVA concentrations are unrelated to dopaminergic neurotransmission. The possible role of capillary walls and glial cells in the catabolism of DA must be further evaluated. The demonstration of DOPAC and HVA in the spinal cord is another argument against the hypothesis that CSF levels of HVA and DOPAC reflect closely the activity of the dopaminergic syste

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb05729.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:It had previously been thought that muscarinic cholinergic receptors utilize an influx of extracellular calcium for activation of adrenomedullary catecholamine secretion. However, it has recently been demonstrated that muscarinic receptors on isolated adrenal chromaffin cells can elevate cytosolic free calcium levels in a manner independent of extracellular calcium, presumably by mobilizing intracellular calcium stores. We now demonstrate that muscarinic receptor‐mediated catecholamine secretion from perfused rat adrenal glands can occur under conditions of extracellular calcium deprivation that are sufficient to block both nicotine‐ and electrically stimulated release. Three independent conditions of extracellular calcium deprivation were used: (a) nominally calcium‐free perfusion solution (no calcium added), (b) EGTA‐containing calcium‐free perfusion solution, and (c) perfusion solution containing the calcium channel blocker verapamil. Secretion was evoked from the perfused glands by either transmural electrical stimulation or injection of nicotine or muscarine into the perfusion stream. Each condition of calcium deprivation was able to block nicotine‐ and electrically stimulated catecholamine release in an interval that left muscarine‐evoked release largely unaffected. The above results demonstrate that muscarine‐evoked catecholamine secretion from perfused rat adrenal glands can occur in the absence of extracellular calcium, presumably by mobilization of intracellular calcium. The latter may be due to muscarinic receptor‐mediated generation of inosi

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb05730.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY