摘要:

Abstract:Neurogranin, a peptide capable of binding the calcium‐poor form of calmodulin, was tested in vitro for its ability to modulate a typical calmodulin target. The target employed was the calcium/calmodulin‐dependent form of nitric oxide synthase, which is produced by several different types of neurons. Neurogranin for the study was purified from perchloric acid‐soluble calf brain proteins by a combination of calmodulin‐Sepharose affinity chromatography and reverse‐phase HPLC. The protocol yielded highly purified neurogranin that was active in assays using purified nitric oxide synthase. The titration of the enzyme activity with neurogranin demonstrated a concentration‐dependent effect of the peptide on enzyme activation. Subsequent analysis of the ability of increased calcium concentrations to activate the enzyme was performed in the presence of different amounts of neurogranin. The effect of neurogranin on the calcium‐dependent activation of the enzyme was to depress enzyme activity in the range of 0.2 to ∼1 µMcalcium. Treatment of the neurogranin peptide with protein kinase C eliminated its inhibition on nitric oxide synthase activation. Treatment of the protein kinase C‐phosphorylated peptide with calcineurin did not restore the ability of neurogranin to inhibit enzyme activity, whereas treatment with alkaline phosphatase did restore this ability. These results suggest that neurogranin may serve as a member of a unique class of endogenous calmodulin inhibitor that functions to regulate the activation of calmodulin‐requirin

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010092.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Cultured granule cells grown in serum‐containing medium with a “low K+” concentration (10 mM) underwent apoptosis after maturation for 5 days in vitro (5 DIV), a time that coincides with the developmental decline in the activity of metabotropic glutamate receptors (mGluRs) coupled to polyphosphoinositide hydrolysis. The mGluR agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD) prevented the development of low K+‐induced apoptosis and the presence of the drug was critical at 6 and 7 DIV, i.e., after the drop of mGluR activity. The neuroprotective action of 1S,3R‐ACPD was prevented by the mGluR antagonist (RS)‐α‐methyl‐4‐carboxyphenylglycine (MCPG) and was mimicked byN‐methyl‐d‐aspartate or carbamylcholine but not by agonists of the mGluR subtypes negatively linked to adenylyl cyclase. In cultures treated either with Li+—which reduced polyphosphoinositide response to concentrations of glutamate (5 µM) that approximate those physiologically present in the incubation medium—or MCPG, the development of low K+‐induced apoptosis already occurred at 4 DIV. Thus, the activation of mGluRs coupled to polyphosphoinositide hydrolysis by endogenous glutamate could contribute to protect cultured granule cells against ap

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010101.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The β4and β10thymosins are G‐actin binding proteins that exhibit complex patterns of expression during rat cerebellar development. Their expression in vivo is initially high in immature granule cells and diminishes as they migrate and differentiate, ceasing altogether by postnatal day 21. Thymosin β4is present in a subset of glia throughout postnatal development, and its synthesis is also induced in maturing Bergmann glia. In contrast, thymosin β10is only present at very low levels in a very small subpopulation of glia in the adult cerebellum. To study the factors differentially regulating expression of the β‐thymosins, we characterized their patterns of expression in primary cultures of rat cerebellum. Both β‐thymosins were initially expressed in granule cells, although expression, especially of thymosin β4, was truncated compared with the in vivo time course. As in vivo, thymosin β4was synthesized at much higher levels in astrocytes and microglia in cultures from postnatal cerebellum than was thymosin β10. Unlike in vivo, the latter was expressed in glia cultured from fetal cerebellum. The similarities between the in vivo and in vitro expression of the β‐thymosins show that modulation of tissue culture conditions could be used to identify factors regulating β‐thymosin expression in vivo. The differences would identify regulatory mechanisms that are not evident from the i

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010109.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The autacoid platelet‐activating factor (PAF) takes part in a complex network of interactions regarding the cellular components of nervous tissues. Efforts aimed at characterizing the effects of PAF in the brain have been recently focalized on neurons because PAF exerts pleiotropic effects on these cells. Less attention has instead been paid to the glial component of the brain. We have used microglial cell lines immortalized from 13‐day‐old mouse embryo brains by amyc‐transducing retrovirus. When exposed to physiological doses of PAF, immortalized microglial cells showed increases in intracellular free calcium concentrations due to release of calcium from internal stores, as well as to extracellular calcium influxes. These profiles of reactivity were independent from the immortalizing process, being observable in primary microglial cultures and in immortalized clones showing different proliferative rates. PAF was also able to induce transient expression of the c‐fosprotooncogene in serum‐starved cultures and induced a strong chemotactic response in microglial cells. In contrast with control macrophage cultures, PAF did not promote prostaglandin or leukotriene synthesis in immortalized cells. This was most likely due to the low amount of total arachidonic acid found in immortal microglia, with respect to that observed in freshly isolated cells. Our data suggest that several of the effects observed after PAF stimulation might be independent from PAF‐induced arachidonic acid metabolism. The availability of an in vitro microglial model might now help in studying the proinflammatory effects of PAF, both direct or microglia mediated, in the neura

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010121.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The direct effect of melatonin and related agonists on Li+‐amplified phosphoinositide breakdown was studied in chick brain slices prelabeled withmyo‐[2‐3H]‐inositol. The melatonin receptor agonist 6‐chloromelatonin (10–100 µM) increased, in a concentration‐dependent manner, the accumulation of inositol phosphates (IP) in chick brain slices. This effect of 6‐chloromelatonin (10 µM) was rapid as transient increases in IP3/IP4(maximal increase, 29% at 20 s) and IP2levels (maximal increase, 36% at 1 min) were observed, followed by a slower but sustained increase in IP1level (30% at 5 min), when the amount of IP3/IP4and IP2had already been decreased to the control level. The phosphoinositide response elicited by 6‐chloromelatonin (10 µM) was dependent on the presence of extracellular calcium. Direct stimulation of membrane phospholipase C by 6‐chloromelatonin (10 µM) in isolatedmyo‐[2‐3H]inositol‐prelabeled optic tectum membranes was dependent on the presence of guanosine‐5′‐O‐(3‐thio)triphosphate (1 µM), thus suggesting that G protein(s) link melatonin receptor activation to phospholipase C stimulation. The competitive melatonin receptor antagonist luzindole (10–100 µM) inhibited in a concentration‐dependent manner the IP1accumulation stimulated by 6‐chloromelatonin (10–100 µM); however, it did not affect the accumulation stimulated by 5‐hydroxytryptamine (10 µM). By contrast, methysergide (10 µM) completely inhibited 5‐hydroxytryptamine (10 µM)‐, but not 6‐chloromelatonin (10 µM)‐, induced IP1accumulation. Melatonin receptor agonists increased IP1accumulation in a concentration‐dependent manner reaching different maximal responses.N‐Acetyl‐5‐hydroxytryptamine was more potent than melatonin in increasing IP1accumulation, suggesting activation of a melatonin receptor site other than the ML‐1 melatonin receptor (i.e.,N‐acetyl‐5‐hydroxytryptamine ≥ melatonin). In conclusion, these results demonstrate that activation of a melatonin receptor with pharmacological characteristics different from those of the ML‐1 sub

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010130.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:We have recently demonstrated that bovine adrenal medulla contains a soluble phospholipase A2(PLA2), which is localized in the cytosol. In the present study, this PLA2was purified 1,097‐fold using sequential concanavalin A, hydrophobic interaction, anion exchange, gel filtration, and an additional anion exchange chromatography. The enzyme is activated over the range of 20–1,000 µMCa2+and has a pH optimum near 8.0. On sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, the protein has a molecular mass of 26 kDa and an isoelectric point of 4.6 as revealed by isoelectric focusing. The cytosolic PLA2is not inhibited by NaCl, and the enzymatic activity is stimulated at low concentrations of Triton X‐100 (0.01%) and deoxycholate (1 mM) but inhibited at higher concentrations (0.1% and 3 mM, respectively) of these detergents. Furthermore, heat treatment (57°C, 5 min) reduced the enzymatic activity by 80%, whereas glycerol (30%) increased the activity.p‐Bromophenacylbromide, a frequently used irreversible inhibitor of type II PLA2, has little effect until 100 µM, and 2–10 mMdithiothreitol totally inactivated the enzyme. The purified PLA2displays a preference for phosphatidylcholine as a substrate but hydrolyzes phospholipid substrates with arachidonic acid or linoleic acid esterified at thesn‐2 position to the same extent. It is concluded that the chromaffin cell cytosolic PLA2, which was isolated and characterized in this study, represents a type of PLA2that has not been formerly reported in chromaffin cells. Additional research on the chromaffin cell cytosolic PLA2will help to reveal whether the enzyme is importan

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010139.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:This study attempts to determine if the medial (MSO) and lateral superior olive (LSO), medial nucleus of the trapezoid body (MNTB), ventral nucleus of the lateral lemniscus (VNLL), and central nucleus of the inferior colliculus (ICc) contain glutamatergic synaptic endings. Micropunch and microdissection procedures provided fresh samples of these auditory nuclei for the measurement of the high‐affinity uptake and electrically evoked release of exogenousd‐[3H]ASP. The study also determined if the LSO and MSO contain glycinergic synaptic endings by measuring uptake and release of [14C]‐Gly in these nuclei, and whether the MNTB, VNLL, and ICc contain GABAergic endings by assessing the uptake and release of [14C]GABA in these structures. Several strategies optimized the evoked Ca2+‐dependent release of the labeled amino acids. These included the enhancement of high‐affinity uptake during loading of the markers into the tissues, inhibition of uptake during the subsequent measurement of release, and use of an electrical stimulus current that evoked maximal Ca2+‐dependent release. Each of these nuclei manifested the high‐affinity uptake and the evoked Ca2+‐dependent release ofd‐[3H]Asp, suggesting the presence of synaptic endings that may use Glu or Asp as a transmitter. Similar findings suggest the presence of glycinergic synaptic endings in the LSO and MSO, and of GABAergic synaptic endings in the MN

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010147.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:This study attempts to determine if projections ascending from the guinea pig cochlear nucleus (CN) could be glutamatergic and/or aspartatergic. Multiple radio frequency lesions were made to ablate the right CN. The ablation was verified histologically. To identify the principal targets of CN efferents, silver impregnation methods were used to localize the preterminal degeneration of fibers in transverse sections of the brainstem 5 and 7 days after CN ablation. CN efferents projected heavily to the lateral superior olive (LSO) ipsilaterally, the medial superior olive (MSO) bilaterally, and contralaterally to the medial (MNTB) and ventral (VNTB) nuclei of the trapezoid body, the ventral (VNLL) and intermediate nuclei of the lateral lemniscus and the central nucleus of the inferior colliculus (ICc). There were smaller projections to the lateral nucleus of the trapezoid body ipsilaterally, the dorsal and dorsomedial periolivary nuclei bilaterally, and the dorsal nucleus of the lateral lemniscus contralaterally. There were sparse projections to the VNLL and ICc ipsilaterally and the CN contralaterally, and a very sparse projection to the contralateral LSO. To determine if CN efferents were glutamatergic and/or aspartatergic, the fresh brainstem was sectioned transversely and samples of the LSO, MSO, MNTB, VNLL, and ICc were taken to measure the electrically evoked release and the uptake ofd‐[3H]Asp and [14C]Gly or [14C]GABA 3–5 days after the CN ablation. The release studies suggest that only certain of the histologically identified projections ascending from the CN may be glutamatergic and/or aspartatergic. CN ablation depressedd‐[3H]Asp release in the MSO bilaterally and in the contralateral MNTB and VNLL, suggesting that the CN efferents to these nuclei may use glutamate or aspartate as a transmitter. It was unclear whether a marginal depression ofd‐[3H]Asp release in the ipsilateral LSO reflected the presence of glutamatergic CN projections to this nucleus.d‐[3H]Asp release in the ICc was unaffected, suggesting that CN efferents to this nucleus may not be glutamatergic. There were no deficits ind‐[3H]Asp uptake. [14C]Gly release from the LSO and MSO was unchanged. [14C]Gly uptake was unchanged in the MSO and depressed only in the contralateral LSO, possibly reflecting subnormal uptake activity in endings contributed by contralateral MNTB cells that had lost their CN efferents. [14C]GABA uptake in the MNTB, VNLL, and ICc was unchanged. [14C]GABA release was unchanged in the VNLL and ICc. [14C]GABA release was depressed only in the contralateral MNTB, possibly reflecting the loss of a small complement of GABAergic CN efferents and the reaction of GABAergic projections from the contralateral VNTB to their loss of C

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010161.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Sustained activation of protein kinase C significantly enhanced a secondary (slow) phase in the depolarization‐induced release of glutamate from isolated hippocampal nerve endings. The phorbol ester, 4β‐phorbol 12,13‐dibutyrate, was used to sustain the activation of presynaptic protein kinase C for a prolonged (10‐min) period, and then this relatively water‐soluble phorbol ester was removed by superfusion before a 2‐min stimulus of continuous membrane depolarization. These conditions were used to investigate the persistent effects of sustained protein kinase C activation on the magnitude of the slow phase of evoked glutamate release, in which the efficiency of synaptic vesicle mobilization and recycling may be primary determinants of response magnitude. It is reported here that sustained protein kinase C activation selectively increased the Ca2+‐dependent component of glutamate release during a prolonged phase of K+‐induced depolarization. The magnitude of this persistent effect on Ca2+‐dependent glutamate release was directly related to the dose of 4β‐phorbol 12,13‐dibutyrate and the duration of exposure that was used to prime the release apparatus, was observed using two alternative synaptosomal preparations, and was evident regardless of the depolarizing stimulus used (elevated [KCl] or 4‐aminopyridine). However, 4β‐phorbol 12,13‐dibutyrate did not alter the release induced by the Ca2+ionophore ionomycin. Thus, the persistent effects of protein kinase C activation on a prolonged phase of glutamate release were dependent on the route of Ca2+influx. The finding that voltage‐regulated Ca2+channel blockers were able to neutralize completely the 4β‐phorbol 12,13‐dibutyrate‐dependent facilitation of K+‐evoked glutamate release provided further support for this conclusion. Thus, 4β‐phorbol 12,13‐dibutyrate significantly potentiated the sustained release of glutamate without altering the strict requirement that is normally displayed by synaptosomes

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010172.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:The phorbol ester 4β‐phorbol 12,13‐dibutyrate increases the final extent of Ca2+‐dependent glutamate release during the continuous depolarization of the synaptosomal plasma membrane. Based on this finding, we suggested that the sustained activation of protein kinase C has a positive influence on the efficiency of synaptic vesicle recycling in the presence of saturating concentrations of Ca2+. Previous work from our laboratory demonstrated that this 4β‐phorbol 12,13‐dibutyrate‐dependent enhancement of synaptic vesicle recycling persists following the removal of 4β‐phorbol 12,13‐dibutyrate, requires localized Ca2+entry through voltage‐regulated channels, and is insensitive to the protein kinase inhibitor staurosporine. In the present study, we examined the possibility that the facilitation of glutamate release may be propagated through interactions between the protein kinase C‐ and multifunctional Ca2+/calmodulin‐dependent protein kinase pathways. However, our data argue strongly against the involvement of such a mechanism in the persistent enhancement of sustained glutamate release. We observed that 4β‐phorbol 12,13‐dibutyrate did not increase the availability of cytosolic free calmodulin or the level of autonomous Ca2+/calmodulin‐dependent protein kinase activity. In addition, we determined the effects of various serine/threonine kinase and phosphatase inhibitors on the phorbol ester‐dependent enhancement of sustained glutamate release and found that protein kinase C increased the extent, but not the duration, of Ca2+‐dependent glutamate release through a kinase‐independent mechanism. Given our finding that the actin‐depolymerizing agent cytochalasin D totally occluded the effect of 4β‐phorbol 12,13‐dibutyrate on release, we postulate that protein kinase C signals may be transduced through direct interactions between protein kinase C isoforms and cy

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64010181.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY