摘要:

Abstract:High‐affinity choline transport (HAChT), the rate‐limiting and regulatory step in acetylcholine (ACh) synthesis, is selectively localized to cholinergic neurons. Hemicholinium‐3 (HC3), a potent and selective inhibitor of HAChT, has been used as a specific radioligand to quantify HAChT sites in membrane binding and autoradiographic studies. Because both HAChT velocity and [3H]HC3 binding change as in vivo activity of cholinergic neurons is altered, these markers are also useful measures of cholinergic neuronal activity. Evidence that [3H]HC3 is a specific ligand for HAChT sites on cholinergic terminals is reviewed. The ion requirements of HAChT and [3H]HC3 binding indicate that sodium and chloride are required for recognition of both choline and [3H]HC3. A common recognition site is also indicated by the close correspondence of the potency of HC3 and choline analogues for inhibiting both HAChT and [3H]HC3 binding. The parallel regional distributions of both markers in adult brain, during development and after specific lesions, all indicate specific cholinergic localization. The close association of HAChT and [3H]HC3 binding sites is also supported by parallel regulatory changes occurring after in vivo drug treatments and in vitro depolarization. Overall, the data indicate a close association between HAChT and [3H]HC3 binding and are consistent with the sites being identical. Methodologic considerations in using [3H]HC3as a ligand and considerations in interpretation of results are also disc

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb03277.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:A large body of evidence suggests that disturbances of Ca2+homeostasis may be a causative factor in the neurotoxicity induced by excitatory amino acids (EAAs). The route or routes by which an increase in intracellular calcium concentration ([Ca2+]i) is mediated in vivo are presently not clarified. This may partly reflect the complexity of intact nervous tissue in combination with the relative unspecific action of the available “calcium antagonists,” e.g., blockers of voltage‐sensitive calcium channels. By using primary cultures of cortical neurons as a model system, it has been found that all EAAs stimulate increases in [Ca2+]ibut via different mechanisms. By using the drug dantrolene, it has been shown that 2‐amino‐3‐(3‐hydroxy‐5‐methylisoxazol‐4‐yl)propionate (AMPA) apparently exclusively stimulates Ca2+influx through agonist‐operated calcium channels and voltage‐operated calcium channels. Increased [Ca2+]idue to exposure to kainate (KA) is for the major part caused by influx, as in the case of AMPA, but a small part of the increase in [Ca2+]imay be attributed to a release of Ca2+from intracellular stores. Quisqualate (QA) stimulates Ca2+release from an intracellular store that is independent of Ca2+influx; presumably this store is activated by inositol phosphates. The increase in [Ca2+]idue to exposure to glutamate orN‐methyl‐d‐aspartate (NMDA) may be compartmentalized into three components, one of which is related to influx and the other two to Ca2+release from internal stores. Only one of the latter stores is dependent on Ca2+influx with regard to release of Ca2+, whereas the other is activated by some other second messengers or, alternatively, directly coupled to the receptor. In muscles dantrolene is known to inhibit Ca2+release from the sarcoplasmic reticulum, and also in neurons dantrolene inhibits an equivalent release from one or more hitherto unidentified internal Ca2+pool(s). By using this drug it has been possible to show to what extent these Ca2+stores are involved in the toxicity observed subsequent to exposure to the EAAs. It turned out that dantrolene, even under conditions allowing Ca2+influx, inhibited toxicity induced by QA, NMDA, and glutamate, whereas that induced by AMPA or KA was unaffected. In combination with the findings that dantrolene inhibited release from the intracellular stores activated by QA, NMDA, and glutamate, it may be concluded that Ca2+influx per se is not the primary event causing toxicity following exposure to these EAAs in these neurons. However, it may certainly be involved in the cases of toxicity induced by AMPA and KA. Finally, it should be pointed out that this model only serves as a much simplified working hypothesis and that the situati

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb03278.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Two forms of rat brain cytosolic diacylglycerol kinase (EC 2.7.1.107) were separated by heparin‐agarose column chromatography. These forms, designated DGK‐I and DGK‐II, were not interconvertible as determined by rechromatography. DGK‐I and DGK‐II had respective molecular masses of 88 and 180 kDa, as measured by Sepharose 6B chromatography. Both forms preferred diacylglycerol over monoacylglycerol and were insensitive to R59022. DGK‐II, but not DGK‐I, was activated by an activator substance prepared from chicken egg yolk. DGK‐II was activated by a rat brain cytosolic activator and was exclusively sensitive to 5′‐AMP‐mediated inactivation. Further studies revealed that these two forms had the following distinct characteristics: (a) substrate specificity, (b) inhibition by heparin, (c) sensitivity to lysine‐containing polyamino acids, and (d) responses to different phospholipids. In general, DGK‐II was more responsive to various inhibitors and activators, making it a prime candidate

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb03279.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of protein phosphorylation in rat striatal synaptosomes. Two major synaptosomal phosphoproteins of ∼80 and ∼60 kDa, possibly synapsin I and tyrosine hydroxylase, were especially sensitive to neurocatin. Immunoprecipitation experiments confirmed that the 60‐kDa protein is the enzyme tyrosine hydroxylase. At low concentrations of neurocatin (to ∼7.5 ng/100 μl of suspension), incorporation of32P orthophosphate into these proteins increased with increasing neurocatin concentration. At 7.5 ng of neurocatin, incorporation of the label into the two proteins increased by 22 and 26%, respectively. Concentrations of neurocatin>7.5 ng/100 μl caused progressive decrease in incorporation of32P into many synaptosomal proteins; by a concentration of neurocatin of ∼45 ng/100 μ/l, the level of32P incorporation into many proteins was ≤70% of control. The effects of neurocatin on synaptosomal protein phosphorylation were also dependent on the time of incubation. At a constant concentration of ∼7.5 ng/100 μl of neurocatin, increased incorporation of 32P into many proteins was measurable within 0.5 min and was maximal by 1 min. Incubation times>2.0 min, showed progressive decrease in32P incorporation. Removing extrasynaptosomal Ca2+with EGTA attenuated the increased32P incorporation induced by low neurocatin concentrations, suggesting that calcium plays a role in neurocatin‐induced phosphorylation of rat striatal synaptosomal proteins. The reduced incorporation of label induced by high neurocatin concentrations, however, was not calcium dependent. The effects of neurocatin on the level of32P incorporation into proteins were observed only in intact synaptosomes, consistent with this compound acting through receptors on

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb03280.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The biosynthesis of phosphatidylserine in mammalian tissues is catalyzed by the serine base exchange enzyme. The activity of this membrane‐bound enzyme can be manipulated by amphiphiles. Amphiphilic cations, such as oleylamine, W‐7, chlorpromazine, and didodecyldimethylamine, stimulate the serine base exchange activity. Amphiphilic anions, such as bis(2‐ethylhexyl) hydrogen phosphate and cholesterol sulfate, inhibit the serine base exchange activity. These effects are more pronounced at pH 7.0 than at the pH optimum of 8.5 for this enzyme. Both the stimulators and the inhibitors alter theVmaxvalues without changing theKmvalue for serine, suggesting that their mechanism of action is related to interactions of the membrane‐bound cosubstrate, phosphatidylethanolamine, with the membrane‐bound enzyme. The optimal concentration of stimulator varies with the amount of membrane protein present; however, supraoptimal concentrations cause inhibitions. It is proposed that the amphiphilic cations enhance the interaction of the phosphorylethanolamine moiety of the membrane‐bound cosubstrate with the enzyme and the amphiphilic anions interfere with such an interaction. Some of the pharmacological properties of these amphiphilic cations, employed clinically as antidepressants, may be mediated by modulation of the serine base exchange enzy

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb03281.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Using fura‐2 microfluorometry, I investigated the mechanism by which non‐N‐methyl‐d‐aspartate (NMDA) receptor agonists increase the cytosolic free calcium concentration ([Ca]in) in single cerebellar Purkinje cells isolated from 3–10‐day‐old rats. Kainate and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate dose‐dependently increased the cytosolic free Na+concentration, which was measured using sodium‐binding benzofuran isophthalate microfluorometry, confirming the Na+influx through ion channels linked to non‐NMDA receptors. The [Ca2+] increases induced by relatively lower concentrations of agonists were entirely dependent on external Ca2+and were reduced by removal of external Na+or by addition of a Ca2+channel blocker, D600. The results indicate that the non‐NMDA agonist–induced [Ca]inincrease was due mainly to Ca2+influx through voltage‐dependent Ca2+channels, which were activated by a massive Na+influx. On the other hand, higher concentrations of agonists dose‐dependently increased [Ca]inunder conditions in which activation of voltage‐dependent Ca2+channels were blocked by a combination of Na+removal with D600. These [Ca]inincreases were Ca2+dependent and little affected by adding a competitive NMDA antagonist. Non‐NMDA agonists also stimulated influxes of Mn2+and Co2+, both of which can be monitored by quenching fura‐2 fluorescence under the same conditions. These results suggest that ion channels linked to non‐NMDA receptors on immature

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb03282.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:We studied the CSF amino acid levels of 42 patients with newly diagnosed epilepsy before treatment with antiepileptic medication and during monotherapy with either vigabatrin or carabamzepine. The present study shows that patients with newly diagnosed epilepsy have elevated levels of the excitatory amino acid glutamate in CSF. Vigabatrin monotherapy effectively prevents the appearance of seizures in patients with high baseline CSF glutamate levels. In these patients, vigabatrin not only elevates the levels of γ‐aminobutyric acid, but also decreases the elevated levels of glutamate in CSF, which may also be important to the antiepileptic efficacy of vigabatrin. Patients with low CSF glutamate levels did not benefit from vigabatrin‐induced changes in amino acid levels and successful monotherapy with carbamazepine did not affect CSF amino acid levels. The elevation of γ‐aminobutyric acid is thus not the only way to achieve seizure control and there are several factors underlying the generation and control of seizures. Follow‐up of the patients with high baseline glutamate CSF levels will show if the observed abnormalities are related to the severity of epilepsy in individual patients and if early treatment with vigabatrin of these patients could prevent the development of intractable

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb03283.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:Noradrenaline release from sympathetic nerve terminals was evoked by electrical nerve stimulation of an isolated segment of rat tail artery. This release was recorded by a carbon fiber electrode combined with differential pulse amperometry. The active part of the electrode (one carbon fiber 8 μm in diameter and 50 μm in length) was placed in close contact with the arterial surface. The oxidation current appearing at +120 mV and corresponding to the local noradrenaline concentration at the electrode surface was recorded every 0.5 s. No oxidation current was detected under resting conditions, but electrical stimulation evoked an immediate increase in this current. This response was suppressed when tetrodotoxin was added to the perfusion medium and was enhanced when noradrenaline reuptake was inhibited by cocaine. The amplitude of the response was increased with increasing stimulation frequencies (2–25 Hz) and train lengths (1–16 pulses). Finally, the time resolution of the method (0.5 s) was good enough to show that noradrenaline release precedes the postsynaptic response, i.e., the electrically evoked contraction of the a

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb03284.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The effects of mild stress on nonoxidative glucose metabolism were studied in the brain of the freely moving rat. Extracellular lactate levels in the hippocampus and striatum were monitored at 2.5‐min intervals with microdialysis coupled with an enzyme‐based flow injection analysis system. Ten minutes of restraint stress led to a 235% increase in extracellular lactate levels in the striatum. A 5‐min tail pinch caused an increase of 193% in the striatum and 170% in the hippocampus. Local application of tetrodotoxin in the striatum blocked the rise in lactate following tail pinch and inhibited the subsequent clearance of lactate from the extracellular fluid. Local application of the noncompetitiveN‐methyl‐d‐aspartate receptor antagonist MK‐801 had no effect on the tail pinch‐stimulated increase in lactate in the striatum. These results show that mild physiological stimulation can lead to a rapid increase in nonoxidative glucose metaboli

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb03285.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract:The involvement of B‐50, protein kinase C (PKC), and PKC‐mediated B‐50 phosphorylation in the mechanism of Ca2+‐induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin‐O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP‐dependent manner as a result of an elevation of the free Ca2+concentration from 10−8to 10−5MCa2+The Ca2+sensitivity in the micromolar range is identical for [3H]NA and endogenous NA release, indicating that Ca2+‐induced [3H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca2+‐induced NA release was inhibited by either N‐ or C‐terminal‐directed anti‐B‐50 antibodies, confirming a role of B‐50 in the process of exocytosis. In addition, both anti‐B‐50 antibodies inhibited PKC‐mediated B‐50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC‐mediated B‐50 phosphorylation in Ca2+‐induced NA release. PKC pseudosubstrate PKC19‐36, which inhibited B‐50 phosphorylation (IC50value, 10−5M), failed to inhibit Ca2+‐induced NA release, even when added before the Ca2+trigger. Similar results were obtained with PKC inhibitor H‐7, whereas polymyxin B inhibited B‐50 phosphorylation as well as Ca2+‐induced NA release. Concerning the Ca2+sensitivity, we demonstrate that PKC‐mediated B‐50 phosphorylation is initiated at a slightly higher Ca2+concentration than NA release. Moreover, phorbol ester‐induced PKC down‐regulation was not paralleled by a decrease in Ca2+‐induced NA release from streptolysin‐O‐permeated synaptosomes. Finally, the Ca2+‐ and phorbol ester‐induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B‐50 is involved in Ca2+‐induced NA release from streptolysin‐O‐permeated synaptosomes. Evidence is presented challenging a role of PKC‐mediated B‐50 phosphorylation in the mechanism of NA exocytosis after Ca2+influx. An involvement of PKC or PKC‐mediated B‐50 phosphorylation before the Ca2+trigger is not ruled out. We suggest that the degree of B‐50 phosphorylation, rather than its phosphorylation after PKC activation i

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb03286.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY