摘要:

Abstract:Although [3H]imipramine is a selective radioli‐gand for the 5‐hydroxytryptamine (5‐HT) transporter in human platelets, its affinity for binding to the 5‐HT transporter complex at 0°C (0.6 nM) is significantly higher than its potency for inhibition of [3H]5‐HT uptake at the physiological temperature of 37°C (Ki= 29 nM). As this apparent discrepancy could be related to the assay temperature, we studied the thermodynamics of drug interaction with the 5‐HT transporter at assay temperatures between 0°C and 37°C, using as radioligands [3H]imipramine (0°C and 20°C) and [3H]paroxetine (20°C and 37°C), a newly available probe for the 5‐HT transporter. At 20°C,Kivalues of 14 tricyclic and nontricyclic drugs for inhibition of [3H]imipramine and [3H]paroxetine binding to human platelet membranes were highly significantly correlated (r = 0.98, p<0.001), validating the use of these two radioligands to study the 5‐HT transporter over a temperature range larger than was previously possible with [3H]imipramine alone. The affinity of imipramine for the 5‐HT transporter is progressively enhanced with decreasing incubation temperature, thus favoring the selectivity of [3H]imipramine for the 5‐HT transporter at 0°C. At 37°C, theKiof imipramine for inhibition of [3H]paroxetine binding is 32 nM, and equals itsKivalue for inhibition of 5‐HT uptake into human platelets. With the exception of chlorimipramine, other tricyclic 5‐HT uptake inhibitors showed a temperature sensitivity in their interaction with the 5‐HT transporter similar to that of imipramine. On the other hand, the temperature sensitivity of nontricyclic 5‐HT uptake inhibitors was much less pronounced. Moreover,Kivalues for drug inhibition of [3H]5‐HT uptake into human platelets were significantly (p<0.05) better correlated with the correspondingKivalues for inhibition of [3H]paroxetine binding at 37°C (r = 0.97) than with theirKivalues for inhibition of [3H]imipramine binding at 0°C (r =0.87). Therefore, drug inhibition of [3H]paroxetine binding to the 5‐HT transporter at 37°C may be more representative of 5‐HT uptake inhibitory activity than its inhibition of [3H]imipramine binding to

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb04098.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:Two distinct forms of cysteine sulfinate decarboxylase (CSD), respectively, CSDI and CSDII, have already been separated in rat brain. One of them, CSDII, appeared to be closely associated wtih glutamate decarboxylase (GAD). We have investigated whether the taurine concentration in brain was dependent on CSDII activity in vivo. CSDI and CSDII activities were specifically measured in crude brain extracts after selective immunotrapping. After 4 days of chronic treatment of mice with γ‐acetylenic γ‐aminobutyric acid, a drastic and identical decrease in CSDII and GAD activities was observed in the brain. Taurine concentration and CSDI activities were not significantly altered. Following striato‐nigral pathway lesioning in the rat brain, GAD and CSDII show an identical 80% decrease in the substantia nigra. In contrast, CSDI activity and taurine concentration in the substantia nigra were similarly but only slightly affected with an about 30% decrease. Our results provide further evidence that GAD and CSDII are indeed the same enzyme. They show that CSDII does not play any role in the biosynthesis of taurine in vivo. Our findings suggest that CSDI might be the biosynthetic enzyme for taurine in vivo and that there might be some endings projecting into the substantia nigra that contain CSDI and

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb04099.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:Cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme for taurine, has been shown to exist in two forms in rat brain, respectively CSDI and CSDII, one of which (CSDII) is considered to be in fact glutamate decarboxylase (GAD). CSDI assay after immunotrapping was made possible by using an anti‐CSD antiserum raised in sheep immunized with a partially purified CSD fraction from liver. This antiserum immunoprecipitated both liver CSD and brain CSDI activities with the same affinity but did not inhibit their enzymatic activities. The immunotrapping of CSDI was selective without any contamination by GAD/CSDII activity. The immunotrapped CSD activity, which corresponded exactly to the amount of CSD not precipitated by a GAD/CSDII antiserum, was not inhibited by a specific irreversible GAD inhibitor. A quantitative, selective and sensitive assay was thus developed by measuring CSD activity on the solid phase after immunotrapping. Kinetic parameters of the immunotrapped enzyme remained unchanged. CSDI activity represented only a fraction, around 20% with saturating concentration of substrate, of the total CSD activity in rat brain homogenate. This indicates that most studies on total CSD activity dealt essentially with CSDII activity that is indeed GAD. Regional and subcellular distributions of CSDI have been determined. CSDI activity was about threefold higher in the richest (cerebellum) compared to the poorest (striatum) region without any correlation with GAD/CSDII distribution. Subcellular distribution showed a fourfold enrichment of CSDI activity in the synaptosomal fraction. The precise role of CSDI and CSDII in the biosynthesis of taurine in vivo remains to be elucidate

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb04100.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:A method has been developed for the simultaneous in vivo measurement of local rates for methionine incorporation into cerebral protein in the rat. It is based on the use of l‐[35S]methionine as a tracer for reflecting the bidirectional exchange of methionine between plasma and brain and its incorporation into cerebral protein, using a dynamic three‐compartment model. An operational equation based on this model has been derived in terms of deter‐minable variables. The method has been applied to the normal freely moving rat and to the rat under chloral hydrate anesthesia. In the freely moving rat, the values of methionine incorporation into cerebral protein in the gray matter vary widely from structure to structure (50–300 nmo1/100 g/min), with the highest values in structures related to neurosecretory functions, e.g., supraoptic and para ventricular nuclei. The values for white matter are more uniform (24–28 nmol/100 g/min) at levels approximately six‐ to sevenfold lower than for gray matter. Chloral hydrate anesthesia depresses the rate of methionine incorporation in all the structures examined. Anesthesia did not reduce the heterogeneity normally present within

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb04101.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:Specific binding of [3H]N‐propylnorapomorphine ([3H]NPA) to 3,4‐dihydroxyphenylethylamine (dopamine) D‐2 receptors was investigated in rat striatum in vitro. For various dopamine receptor substances, the rank order of potency to inhibit [3H]NPA binding was spiroperidol ≧NPA ≥ LY 171555 ≥ SCH 23390 ≥ SKF 38393. A single high‐affinity binding site was found in membranes prepared in either Tris‐citrate buffer or imidazole buffer; the affinity constants were 0.11 and 0.76 nM, respectively. The number of receptors (33 pmol/g wet weight) was independent of whether the membranes were prepared in Tris‐citrate buffer or imidazole buffer and was similar to the number of receptors estimated by [3H]spiroperidol binding to dopamine receptors. Irradiation inactivation of frozen whole rat striata showed a monoexponential loss of [3H]NPA binding sites without a change in the binding affinity. The target size of the [3H]NPA binding site was 81,000 daltons, which shows that the functional molecular entity to bind the dopamine D‐2 agonist was smaller than the molecular entity to bind the dopamine D‐2 antagonist [3H]spiroperidol (target

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb04102.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:[3H]SKF 38393 (2,3,4,5,‐tetrahydro‐7,8‐dihy‐droxy‐l‐phenyl‐lH‐3‐benzazepine) binds with high affinity to 3,4‐dihydroxyphenylethylamine (dopamine) D‐l receptors in rat striatum in vitro (KD= 7 and 14 nMin nonfrozen and frozen striatum, respectively). The number of binding sites (5max) was ∼ 80.0 pmol/g of original tissue, a value similar to the Bmaxfor the dopamine D‐l antagonist SCH 23390. Nondisplaceable [3H]SKF 38393 binding was ∼45% of total binding. Irradiation (0–4 Mrad) of frozen whole striata decreased the number of [3H]SKF 38393 binding sites monoexponentially without changing the binding affinity. The functional molecular mass for the agonist dopamine D‐1 binding site was 132,800 daltons, which is higher than the functional molecular mass of the antagonist dopamine D

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb04103.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:Lead buffers (citrate and Tiron) were used to investigate the effects of low concentrations (0.1–6 μM) of Pb2+on stimulus‐secretion coupling in isolated bovine chromaffin cells. Nicotinic agonists and high K elicit secretion by enhancing Ca2+influx into chromaffin cells. Pb2+inhibited the catecholamine secretion in response to 500 μMcarbachol and 77 mMK+depolarization but was without significant effect on basal secretion. Pb2+also inhibited the influx of45Ca occurring in response to these agents. TheK0.5values for inhibition suggest that the carbachol‐evoked flux is more sensitive to Pb2+than influx in response to a direct depolarization. When extracellular calcium was lowered in the absence of Pb2+, both secretion and45Ca entry were reduced. The effects of Pb2+were comparable to those of lowered Ca2+.22Na influx through nicotinic receptor‐mediated channels, measured in the presence of tetrodotoxin (2 μM) and ouabain (50 μM), was inhibited by Pb2+. The results suggest that Pb2+inhibits exocytotic catecholamine secretion by inhibiting Ca2+influx. The differential sensitivity to Pb2+of K‐ and carbachol‐evoked45Ca flux, coupled with the22Na measurements, indicates that Pb2+inhibits the movement of ions through acetylcholine‐induced channels as well as through voltage‐sensiti

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb04104.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:Agents that stimulate secretion also accelerate the rate of Pb uptake into adrenal medullary cells. For example, when cells are suspended in a medium containing 5 μMPb2+, depolarization by 77 mMK increases the rate of Pb uptake from 12 ± 1 to 47 ± 5 μmol/(L cells × min). K‐induced Pb uptake has an apparentKmfor Pb2+of 2.6 μM, and is antagonized by Ca2+with aK0.5of 1.4 mM. The Ca channel blocker D‐600 inhibits Pb entry with aK0.5of 0.4 μM. Pb uptake is also stimulated by the Ca channel agonist BAY K 8644. These observations suggest that Pb passes through Ca channels. The permeability of the channels to Pb appears to be at least 10 times the permeabi

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb04105.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:Alterations in neostriatal dopamine metabolism, release, and biosynthesis were determined 3, 5, or 18 days following partial, unilateral destruction of the rat nigrostriatal dopamine projection. Concentrations of dopamine and each of its metabolites, 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3‐methoxytyra‐mine (3‐MT) were markedly decreased in the lesioned stri‐ata at 3, 5, or 18 days postoperation. The decline in striatal high‐affinity [3H]dopamine uptake closely matched the depletion of dopamine at 3 and 18 days postoperation. However, neither DOPAC, HVA, nor 3‐MT concentrations were decreased to as great an extent as dopamine at any time following lesions that depleted the dopamine innervation of the striatum by>80%. In these more severely lesioned animals, dopamine metabolism, estimated from the ratio of DOPAC or HVA to dopamine, was increased two‐ to fourfold in the injured hemisphere compared with the intact hemisphere. Dopamine release, estimated by the ratio of 3‐MT to dopamine, was more increased, by five‐ to sixfold. Importantly, the HVA/dopamine, DOPAC/dopamine, and 3‐MT/dopamine ratios did not differ between 3 and 18 days postlesioning. The rate of in vivo dopamine biosynthesis, as estimated by striatal DOPA accumulation following 3,4‐dihydroxyphenylalanine (DOPA) decarboxylase inhibition with NSD 1015, was increased by 2.6‐ to 2.7‐fold in the surviving dopamine terminals but again equally at 3 and 18 days postoperation. Thus, maximal increases in dopamine metabolism, release, and biosynthesis occur rapidly within neostriatal terminals that survive a lesion. This mobilization of dopaminergic function could contribute to the recovery from the behavioral deficits of partial denervation by increasing the availability of dopamine to neostriatal dopamine receptors. However, these presynaptic compensations are not sufficient to account for the protracted (at least 3‐week) time course of sensorimotor recovery that has been observed following

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb04106.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY

摘要:

Abstract:The unidirectional influx of biotin across cerebral capillaries, the anatomical locus of the blood‐brain barrier, was measured with an in situ rat brain perfusion technique employing [3H]biotin. Biotin was transported across the blood‐brain barrier by a saturable system with a one‐half saturation concentration of ∼ 100 μM. The permeability‐surface area products were 10−4s−1with a biotin concentration of 0.02 μMin the perfusate. Probenecid, pantothenic acid, and nonanoic acid but not biocytin or biotin methyles‐ter (all 250μM) inhibited biotin transfer through the blood‐brain barrier. The isolated rabbit choroid plexus was unable to concentrate [3H]biotin from medium containing 1 nM[3H]biotin. These observations provide evidence that: (1) biotin is transported through the blood‐brain barrier by a saturable transport system that depends on a free carboxylic acid group, and (2) the choroid plexus is probably not involved in the transfer of biotin between blood an

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1987.tb04107.x

出版商:Blackwell Publishing Ltd    

年代:1987

数据来源: WILEY