摘要:

Abstract:Many processes in the CNS depend on calcium. The calcium signal is transduced into an intracellular response via Ca2+‐binding proteins, including calbindin D‐28K. In many laboratories, polyclonal antibodies against chicken in testinal calbindin D‐28K have been used to study its localization in the brain (normal and degenerated) of various species, including humans, but some of these antisera cross‐reacted with other proteins, including calretinin. We purified recombinant rat brain calbindin D‐28K to raise antisera in rabbits and purified a recombinant rat–chicken calbindin D28K hybrid protein to immunize mice for the generation of monoclonal antibodies. These antisera were highly specific for calbindin D‐28K, as demonstrated by two‐dimensional Western blotting analysis. Immunohistochemical analyses combined with in situ hybridization studies demonstrated that calbindin D‐28K in the Purkinje cells of the cerebellum is independent of vitamin D. The antibodies described here will be important tools for studying the regulation of expression of calbindin D‐28K and its biological function in the b

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04879.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The in vivo mechanisms underlying the dopamine (DA)‐releasing actions of veratrine and ouabain in the striatum of halothane‐anaesthetised rats have been investigated using brain microdialysis. Relevant catecholamines and indoleamines were separated and quantified using HPLC combined with an electrochemical detection system. Veratrine (10 μg/ml‐1 mg/ml) and ouabain (10 μM‐1 mM) were added to the medium perfusing the dialysis probes. Both compounds increased dialysate DA content in a dose‐related manner. Dialysate levels of the DA metabolites 3,4‐dihydroxyphenylacetic acid and homovanillic acid and the serotonin metabolite 5‐hydroxyindoleacetic acid were reduced by both veratrine and ouabain. Veratrine‐induced DA efflux was maximal in the first 20‐min sample collected after drug infusion began, whereas the maximal effect of ouabain was not observed until 20–40 min after administration began. Veratrine‐induced DA efflux was unaffected by systemic injection of the DA uptake inhibitor nomifensine but was inhibited by either coperfusion of tetrodotoxin (TTX) or removal of calcium from the perfusing buffer. These data suggest that veratrine induces release of DA via a carrier‐independent mechanism, perhaps involving an exocytotic release process. In contrast, ouabain‐induced DA release was reduced by nomifensine but was inhibited to a lesser degree by calcium depletion and TTX. Detailed analyses of these data suggest that although ouabain initially induces release of DA via a carrier‐dependent mechanism, an exocytotic process may also be involved. The finding that ouabain‐induced DA efflux exhibits a degree of TTX and calcium sensitivity suggests that membrane depolarisation caused by Na+,K+‐ATPase blockade opens voltage‐gated sodium channels and initiates an exocytotic release of DA. The intracellular pools of DA involved in the release of DA induced by veratrine and ouabain were also examined. Depletion of vesicular pools of DA by pretreatment with reserpine reduced the amount of DA release induced by both agents, although this effect was only significant in the case of veratrine. However, in reserpinised animals the residual amount of DA release induced by veratrine was inhibited by nomifensine, a result suggesting that DA may be released via a carrier‐dependent process in the absence of vesicular DA. Newly synthesised pools of DA were also depleted by pretreatment with the DA synthesis inhibitor α‐methyl‐p‐tyrosine. Under these conditions, both veratrine‐ and ouabain‐induced DA efflux was reduced. In the case of veratrine, newly synthesised DA is presumably used to replenish the vesicular store released by depolarisation. A similar process may also operate with ouabain, although the newly synthesised pool of DA may also be released di

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04880.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The brain microdialysis technique has been used to examine the in vivo effects of potassium and tyramine on dopamine (DA) release and metabolism in the striatum of halothane‐anaesthetised rats. Increasing the concentration of potassium perfusing the dialysis probe (30–120 mM) induced a dose‐related efflux of DA. A dose‐related release of DA was also observed following addition of tyramine (1–100 μM) to the perfusing buffer. High concentrations of potassium were found to reduce the dialysate content of the DA metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid and the serotonin metabolite 5‐hydroxyindoleacetic acid. No such effect was observed even when using the highest concentration of tyramine tested. Potassium‐evoked DA release was facilitated by pretreatment with the DA uptake inhibitor nomifensine, was inhibited by depletion of extracellular calcium, and was not significantly affected by tetrodotoxin (TTX). The effect of tyramine on DA efflux was inhibited by nomifensine and was insensitive to both TTX and calcium depletion. These data suggest that potassium and tyramine induce release of DA via different mechanisms. Potassium‐induced DA release involves a carrier‐independent process and may utilise an exocytotic release mechanism. On the other hand, tyramine‐induced DA release would appear to involve a carrier‐dependent process. Depletion of vesicular stores of DA by pretreatment with reserpine did not significantly affect potassium‐induced DA release, whereas a marked inhibition of the effects of tyramine was noted. However, in reserpinised animals the potassium‐induced release of DA was inhibited by nomifensine, a result suggesting that a carrier‐dependent release mechanism operates in the absence of vesicular DA. Inhibition of DA synthesis by pretreatment with α‐methyl‐p‐tyrosine (α‐MPT) reduced potassium‐evoked DA release. However, this treatment only impaired the effect of tyramine when α‐MPT was given 120 min before tyramine, when vesicular DA stores are also depleted. These data suggest that tyramine induces release of DA from vesicular stores, whereas the effect of potassium involves newly synthesised DA. Because DA metabolite levels are reduced by potassium but not by tyramine, these data suggest that extracellular DOPAC is d

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04881.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The role of glucocorticoids in the modulation of central α2‐receptor mechanisms was investigated by in vitro receptor binding studies. [3H]Clonidine and [3H]idazoxan were used as radioligands. The α2‐receptor subtypes and guanine nucleotide sensitivity were studied in homologue and heterologue displacement experiments following hydrocortisone treatment (25 mg/kg s.c.) for 10 days. High and low agonist affinity states of the α2‐receptor could be identified in3H‐antagonist‐agonist and3H‐agonist‐antagonist displacement experiments, which may correspond to different regulatory protein‐nucleotide associated forms of the receptor. In the presence of 10 μMGTP, the high‐affinity binding was depressed. Following hydrocortisone treatment, there was no detectable change either in the affinity or the binding site concentration of clonidine in homologue displacement (“cold saturation”) experiments. The affinity of idazoxan, however, was depressed. The effect of GTP was similar to the controls in this experimental arrangement. In contrast, in heterologue binding studies the high‐affinity binding site was not demonstrable and the amount of low‐affinity binding increased following the hydrocortisone treatment. The high‐affinity site reappeared in the presence of GTP. The change in GTP sensitivity suggests that the nucleotide regulatory system may be involved in the action of adrenal steroids on c

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04882.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Creatine has been used previously to alter the energy balance of neurons in brain slices. In the present experiments, it was found to reduce the accumulation of γ‐[3H]aminobutyric acid [3H]GABA) as synthesized from [3H]glutamic or [3H]glutamic acid in slices of rat neostriatum. The lowest effective concentration was 5 mM. Creatine (25 mM) was also effective when the degrading enzyme of GABA, i.e., GABA‐α‐oxoglutarate transaminase, was blocked by gabaculine. Creatine (25 mM) did not inhibit the uptake and subsequent accumulation of [3H]GABA. Thus, indirect evidence was obtained that creatine decreased the activity of the synthesizing enzyme of GABA, i.e., glutamate decarboxylase. When the direct effect of creatine (25 mM) on glutamate decarboxylase was studied in vitro, the agent indeed decreased the activity of the enzyme. Creatine (25 mM) also diminished the release of [3H]GABA (expressed as dpm/mg wet weight) from rat neostriatal slices, probably by reducing its synthesis and thus its readily releasable pool. These data are of importance for studies with creatine in complex neuronal systems, because they show that the agent changes not only neuronal energy balance, but also synthesis and release of the ubiquitous transmitt

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04883.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The contribution of dopamine (DA) afferents to the regulation of β‐adrenergic receptor sensitivity (isoproterenol‐stimulated adenylate cyclase activity) in the rat prefrontal cortex was investigated by comparing the effects of lesions affecting either both DA and noradrenaline (NA) or NA fibers alone. Bilateral 6‐hydroxydopamine (6‐OHDA) lesions made in the ventral tegmental area destroyed ascending DA and to a variable extent ascending NA fibers innervating the prefrontal cortex. Two opposite effects were observed depending on the extent of cortical NA denervation: (a) When NA denervation was complete (<4% of controls), a marked increase in the isoproterenol‐sensitive adenylate cyclase activity (+78%) was found. The amplitude of this denervation supersensitivity was similar to that occurring following complete and selective destruction of NA innervation induced by bilateral 6‐OHDA injections made into the pedunculus cerebellaris superior. (b) When 6‐OHDA injections into the ventral tegmental area led to a partial destruction of cortical NA afferents (10–40% of control values), a hyposensitivity of the isoproterenol‐induced adenylate cyclase activity (–30%) was observed. This effect contrasted with the moderate supersensitivity seen in rats with partial, but selective, destruction of NA innervation (pedunculus cerebellaris superior lesions). The hyposensitivity of β‐adrenergic receptors obtained in rats with partial lesions of cortical NA fibers, but devoid of cortical DA innervation, suggests that DA neurons may regulate, under certain conditions, the denervation supersensitivity of

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04884.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM‐binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM‐binding and glycosylation sites was ∼ 16 kDa. Cerebral receptors were32P‐phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C‐terminal segment including a part of the third intracellular loop, because a32P‐labeled peptide of 12–14 kDa reacted with anti‐(m 1 C‐terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of ∼ 17 kDa containing the [3H]PrBCM‐binding site, but not the glycosylation sites, was partly converted to a peptide of ∼ 12 kDa on treatme

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04885.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Recent studies indicate that circulating peptides or plasma proteins, such as insulin or transferrin, or modified proteins, such as cationized albumin, undergo receptor‐mediated or absorptive‐mediated transport through the brain capillary wall, i.e., the blood‐brain barrier (BBB). Although morphologic studies such as autoradiography or immunoperoxidase labeling can demonstrate transport of blood‐borne protein into brain, there is a need for a rapid, sensitive, and quantifiable physiology‐based technique for comparing the relative rates of transport of several different blood‐borne peptides or proteins into brain. Therefore, the present investigations describe a carotid arterial infusion technique coupled with a capillary depletion method for quantifying transport of blood‐borne cationized albumin, cationized IgG, and acetylated low‐density lipoprotein (LDL). Because differentiation of true transcytosis into the postcapillary compartment of brain parenchyma from binding and/or endocytosis to the brain microvasculature is important, the present studies use a dextran density centrifugation step to deplete brain homogenate of the vasculature. In addition,3H‐labeled native albumin is used as a vascular space marker to account for release of capillary contents into the postcapillary supernatant following homogenization of brain. This study demonstrates rapid transport of cationized IgG or cationized albumin into brain, as these compounds achieve a volume of distribution of 20–30 μl/g within 10 min of arterial perfusion. Conversely, acetylated LDL, although rapidly bound by cerebral microvasculature, is shown not to undergo transport into the postcapillary compartment of brain parenchyma. These studies provide the basis for a sensitive, quantifiable technique for studying transport of radiolabeled blood‐borne peptides and proteins across the BBB o

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04886.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:We measured proenkephalin (PEK) mRNA levels in the anterior and medial aspects of the caudate‐putamen (CPU) and in the nucleus accumbens (NAc) of the rat by in situ hybridization histochemistry after chronic treatment for 21 days with typical (haloperidol and prolixin) and atypical (molindone, thioridazine, and clozapine) neuroleptics. Chronic administration with these drugs resulted in PEK mRNA levels that were 60–80% higher than controls in the anterior and medial aspects of the CPU but only 25–30% over controls in the NAc. All three atypical neuroleptics studied increased PEK mRNA in the following order: anterior‐CPU, thioridazine>clozapine and molindone; medial‐CPU, thioridazine and molindone>clozapine; and NAc, thioridazine>>molindone and clozapine. Chronic treatment with the specific dopamine D2 antagonist sulpiride also caused elevation in PEK mRNA levels in all three brain regions studied whereas the specific serotonin S2 receptor blocker, cinanserin, had no significant effects on PEK mRNA levels. These results are consistent with the hypothesis that elevated levels of the enkephalins in the mesolimbic system may be necessary for antipsychotic activity. They also support the idea that the undesirable motoric signs and symptoms observed after chronic treatment with typical neuroleptics may not be the result of increased levels of enkephalins in the basal ganglia because atypical neuroleptics which are almost totally devoid of these side effects caused similar increases in PEK mRNA i

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04887.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Schwann cells, on receiving the correct signal, will encircle an axon and wrap it with a myelin sheath. To begin examining some of the mechanisms underlying the process of myelination in vitro, we isolated Schwann cells from the sciatic nerves of neonatal rats and generated large cell populations with cholera toxin. The immunological and biochemical properties of these secondary Schwann cells were characterized after five to seven passages in the absence of axonal contact. These cells continued to express antigens found in both myelinating (P0and 2′,3′‐cyclic nucleotide phosphohydrolase) and nonmyelinating cells in vivo (A5E3 and glial fibrillary acidic protein) in addition to the markers common to both types of cells (Ran‐1, 217c, S‐100, and laminin). Biochemical analyses showed that these cells synthesize the very‐long‐chain fatty acids (22–26 carbon atoms) found in myelin membranes. Moreover, the enzymes required for the synthesis of myelin glycolipids (including sphingosine acyltransferase, UDP‐galactose:ceramide galactosyltransferase, and cerebroside sulfotransferase) were still active, and metabolic labeling studies showed that galactocerebroside and sulfatide were synthesized even though the galactocerebroside pool was insufficient to be detected by immunostaining. Secondary Schwann cells also synthesized four species of myelin basic protein and the major structural glycoprotein in myelin, P0. The pathway necessary for glycosylation of P0protein remained active, and an analysis of the oligosaccharide chain revealed that 70% was processed to a complex form. In summary, we found that secondary Schwann cells still express most of the immunological markers of differentiated cells and continue to synthesize low levels of myelin components. Therefore, Schwann cells do not dedifferentiate in culture, as pr

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb04888.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY