摘要:

In recent years, the enzyme Ca2+/calmodulin‐stimulated protein kinase II1(CaM‐PK II) as attracted a great deal of interest. CaM‐PK II is the most abundant calmodulin‐stimulated protein kinase in brain, where it is particularly enriched in neurons (Ouimet et al., 1984; Erondu and Kennedy, 1985; Lin et al., 1987; Scholz et al., 1988). Neuronal CaM‐PK II has been suggested to be involved in several phenomena associated with synaptic plasticity (Lisman and Goldring, 1988; Kelly, 1992), including long‐term potentiation (Malinow et al., 1988; Malenka et al.,1989), neurotransmission (Nichols et al., 1990; Siekevitz, 1991), and learning (for review, see Rostas, 1991). This enzyme has also been postulated to be selectively vulnerable in several pathological condition, including epilepsy/kindling (Bronstein et al.,1990; Wu et al., 1990), cerebral ischemia (Taft et al., 1988), and organophosphorus toxicity (Abou‐Donia and La

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08428.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:The selective agonists for the metabotropic glutamate receptor and the ionotropic non‐Ar‐methyl‐D‐aspar‐tate (NMDA) glutamate receptor, (±)‐l‐aminocyclopen‐tane‐/ra/w‐l,3‐dicarboxylic acid (ACPD) and (R,S)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA), respectively, increased the cyclic GMP (cGMP) content in cerebellar slices prepared from adult rats. The ACPD‐induced rise in cGMP level was blocked by compounds known to antagonize metabotropic glutamate receptors, such as DL‐2‐amino‐3‐phosphonopropionic acid and L‐2‐amino‐4‐phosphonobutyric acid, but not by ionotropic glutamate receptor antagonists, D‐2‐amino‐5‐phosphonovale‐ric acid and 6 ‐ cyano ‐ 7 ‐ nitroquinoxaline ‐2,3– dione (CNQX), whereas the AMPA‐induced rise in cGMP level was suppressed by CNQX. Both rises in cGMP level involved nitric oxide synthase (NOS), becauseNG‐methyl‐L‐arginine (NMLA), an inhibitor of NOS, blocked both cGMP level rises, and excess L‐arginine reversed the effect of NMLA. After lithium chloride treatment, which could exhaust phosphatidylinositol phosphates, ACPD no longer increased cGMP levels, whereas AMPA was still effective. In a calcium‐free medium, ACPD still induced a rise in cGMP level, whereas AMPA did not. When the molecular layer was isolated to determine the cGMP content separately from that in the rest of the cerebellar cortex, it was found that ACPD raised the cGMP level mainly in the molecular layer, whereas AMPA raised it in both sections. These results suggest that ACPD enhances the cGMP level through activation of NOS independently of extracellular calcium, most likely by calcium release from intracellular stores triggered by metabotropic glutamate receptors linked to phosphoinositide breakdown, whereas AMPA activates the enzyme by calciu

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08429.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:In this study, the importance of the Mg2+blockade of theN‐methyl‐D‐aspartate (NMDA) receptor during metabolic stress was examined in embryonic day 13 chick retina. Retina exposed to mild conditions of metabolic stress (i.e., blockade of glycolysis with 1 mMiodoacetate for 30 min) underwent acute histological somal and neuritic swelling and an increase in γ‐aminobutyric acid (GABA) release into the medium. These acute signs of metabolic stress were eliminated by NMDA antagonists present during pharmacological blockade of glycolysis, occurred in the absence of a net increase in extracellular glutamate or aspartate, and were not affected by the presence or absence of Ca2+in the incubation medium. One possible explanation for the activation of NMDA receptors in the absence of an increase in extracellular ligand is that NMDA sensitivity during metabolic stress may be governed at the receptor level. Depolarization of membrane potential during metabolic stress may result in the loss of the Mg2+blockade from the NMDA receptor channel, resulting in an increased potency for glutamate. To test this, the dose‐response characteristics for NMDA, glutamate, and kainate in the presence or absence of extracellular Mg2+and the effects of Mg2+on metabolic inhibition were examined. The potency for NMDA‐ or glutamate‐mediated acute toxicity was enhanced two‐ to fivefold in the absence of Mg2+. Omission of Mg2+greatly decreased the minimal concentration of agonist needed to produce acute excitotoxicity; 25 versus 5 μMfor NMDA and 300 versus 10 μMfor glutamate in 1.2 or zero Mg2+, respectively. Elevating external Mg2+to 20 mMcompletely protected against NMDA‐mediated acute toxic effects. In contrast, varying external Mg2+had no effect on kainate‐induced toxicity. Acute toxicity caused by inhibition of metabolism was not potentiated in the absence of Mg2+but was attenuated by elevating extracellular Mg2+. The protective effect of Mg2+during metabolic inhibition was not additive with NMDA antagonists, suggesting that the action of Mg2+was at the level of the NMDA receptor. These findings are consistent with the hypothesis that the Mg2+block is lifted during metabolic inhibition and may be the primary event resulting in NM

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08430.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:The activity of multifunctional calcium/calmodulin‐dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia‐induced inhibition of the enzyme, CaM kinase II was purified to>1,000‐fold from brains of control and ischemic gerbils. The characteristics of CaM kinase II from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase II was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase II activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase II was not different from control enzyme in affinity for calmodulin, Ca2+, Mg2+, or exogenously added substrate or rate of autophosphorylation. CaM kinase II isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3) directed toward the β subunit of the enzyme. In addition, ischemia caused a significant decrease in affinity of CaM kinase II for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase II, the covalent‐binding ATP analog 8‐azidoadenosine‐5′‐[α‐32P]triphosphate was used. Covalent binding of 25 μMazido‐ATP was decreased 40.4 ± 12.3% in ischemic CaM kinase II when compared with control enzyme (n = 5;p<0.01 by paired Student'sttest). Thus, CaM kinase II levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase II and that results in an apparent decreas

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08431.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:In synaptosomal membranes from rat and monkey brain cortex, the addition of petroselenic (18:1,cis‐Δ6) acid, oleic (18:1, m‐Δ9) acid, and vaccenic (18:1,cis‐Δ11) acid or their corresponding methyl esters at 0.5 μmol/mg of membrane protein caused a similar 7–10% decrease in the microviscosity of the membrane core, whereas at the membrane surface the microviscosity was reduced 5–7% by the fatty acids but only 1% by their methyl esters. Concomi‐tantly, the fatty acids, but not the methyl esters, inhibited the specific binding of the tritiated μ‐, δ‐, and K‐opioids Tyr‐D‐Ala‐Gly‐(Me)Phe‐Gly‐ol (DAMGO), [D‐Pen2,D‐Pen5]‐enkephalin (DPDPE), and U69,593, respectively. As shown with oleic acid, the sensitivity of opioid receptor binding toward inhibition by fatty acids was in the order δ>μκk, whereby the binding of [3H]DPDPE was abolished, but significant inhibition of [3H]U69,593 binding, determined in membranes from monkey brain, required membrane modification with a twofold higher fatty acid concentration. Except for the unchangedKDof [3H]U69,593, the inhibition by oleic acid involved both theBmaxand affinity of opioid binding. Cholesteryl hemisuccinate (0.5–3 μmol/mg of protein), added to membranes previously modified by fatty acids, reversed the fluidization caused by the latter compounds and restored inhibited μ‐, δ‐, andk‐opioid binding toward control values. In particular, theBmaxof [3H]‐DPDPE binding completely recovered after being undetectable. The results implicate membrane surface fluidity in the modulation of opioid receptor binding, reveal distinct sensitivity of δ, μ, andKreceptors toward that modulation, and identify unsaturated fatty acids and cholesterol as

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08432.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:Somatostatin (SRIF) is a neurotransmitter that produces its multiple effects in the CNS through interactions with membrane‐bound receptors. Subtypes of SRIF receptors are found in the CNS that are distinguished by their sensitivities to the cyclic hexapeptide MK‐678, such that SRIF, receptors are sensitive to MK‐678 and SRIF2receptors are insensitive to MK‐678. In the present study, we further examined the selectivities of a series of structurally diverse SRIF analogues for SRIF receptor subtypes. SRIF receptors were labeled by125I‐Tyr11SRIF, which has indistinguishable affinities for SRIF receptor subtypes. The inhibition by MK‐678 was incomplete, indicating this peptide is highly selective for a subtype of SRIF receptor that we have termed the SRIF, receptor. The binding of125I‐MK‐678 to SRIF, receptors was monophasically inhibited by SRIF, the octapeptides (such as SMS‐201–995), and the hexapeptides (such as MK‐678), consistent with the highly selective labeling of a subtype of SRIF receptor. In contrast, the smaller CGP‐23996‐like analogues did not inhibit125I‐MK‐678 binding to SRIF, receptors. The binding of125I‐CGP‐23996 to SRIF receptors was inhibited by SRIF and the octapeptides with Hill coefficients of<1, indicating that128I‐CGP‐23996 labels multiple SRIF receptor subtypes. The hexapeptides and CGP‐23996‐like compounds produced only partial inhibitions of125I‐CGP‐23996 binding, which were additive, indicating selective interactions of these compounds with the different receptor subpopulations labeled by125I‐CGP‐23996.125I‐Tyr11‐SRIF binding and125I‐CGP‐23996 binding to SRIF receptors were likewise only partially affected by 100 μMguanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS), a concentration that completely abolishes specific125I‐MK‐678 binding to SRIF, receptors. The component of125I‐CGP‐23996 labeling that was sensitive to GTPγS was also MK‐678 sensitive. Thus, two subpopulations of SRIF receptors exist in the CNS. The SRIF, receptor is sensitive to cyclic hexapeptides such as MK‐678 and to GTPγS but insensitive to smaller CGP‐23996‐like compounds. The SRIF2receptor is sensitive to the CGP‐23996‐like compounds and can be selectively labeled by125I‐CGP‐23996 in the presence of high concentrations of the hexapeptides or GTPγS because, unlike the SRIF, receptor, the SRIF2receptor is insensitive to these agents. The SRIF receptor subtype‐selective peptide analogues will be usef

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08433.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:Calcitonin gene‐related peptide‐like immunoreactivity (CGRP‐LI) was measured in selected regions of the cervical, thoracic, and lumbar spinal cord of untreated rabbits and, following intrathecal injection of the serotonergic neurotoxin 5,7‐dihydroxytryptamine (5,7‐DHT), in the thoracolumbar cord in rats using a sheep antiserum raised against tyrosine0calcitonin gene‐related peptide28‐37. In the cervical, thoracic, and lumbar segments of the rabbit spinal cord, CGRP‐LI levels were 15–50‐fold higher in the dorsal than in the ventral grey region in the same segment. The only segmental variation in CGRP‐LI levels was in the dorsal white region, where levels in the thoracic cord were lower than those in cervical or lumbar segments. Within individual spinal segments, the pattern of distribution of CGRP‐LI in the rabbit spinal cord was analogous to that in other species previously examined, including rat, human, and cat spinal cord. Intrathecal injection of 5,7‐DHT, which caused 85–91% depletion of 5‐hydroxytryptamine and 5‐hydroxyindoleacetic acid from the thoracolumbar ventral spinal cord, did not affect choline acetyltransferase activity, which is colocalized with CGRP in motoneurones in this spinal cord region. In contrast, intrathecal 5,7‐DHT produced a threefold increase in CGRP‐LI in the ventral thoracolumbar cord, suggesting that spinal motoneurones selectively increase production of CGRP 10 days after neurotoxin‐induced denervatio

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08434.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:It is suggested that norepinephrine (NE) plays a role during transient forebrain ischemia. NE may have a protective action against neuronal cell death in the hippocampus, or it may be one of the causes of injurious ischemic effects. We used the microdialysis technique to study extracellular NE levels in the rat hippocampus before, during, and after 30 min of transient incomplete forebrain ischemia (induced by four‐vessel occlusion) to describe the time course of NE in this condition. There was a maximal increase (fivefold) in extracellular NE after 10 min of reflow only when the electroencephalogram was isoelectric. NE levels returned to baseline 40 min after release of the carotid clamps and remained constant for the next 80 min. Thus there appears to be a transient NE overflow in the hippocampus during ischemia, closely related to the complete loss of brain electrical activit

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08435.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:When the supernatant fractions from extracts of control and nerve growth factor (NGF)‐ or dibutyryl cyclic AMP‐treated PC12D cells were applied to DEAE‐Sepharose columns and proteins were eluted with a gradient of NaCl, three separate peaks of kinase activity that phosphorylated microtubule‐associated proteins (MAPs) were recovered. Enhancement of the kinase activity in peak 1 was noted in the case of dibutyryl cyclic AMP‐treated cells. In contrast, the kinase activity in the third peak was markedly elevated, in terms of the ability to phosphorylate MAP1 and MAP2, in the case of the extract from NGF‐treated cells. This activity was designated previously as NGF‐dependent MAP kinase. The apparent molecular mass of the active kinase was 45–50 kDa. The apparentKmvalue was 35 μMfor ATP with either MAP1 or MAP2 as substrate. When the kinase activity in the fractions from the DEAE‐Sepharose column was assayed in the presence of Mn2+instead of Mg2+, another NGF‐stimulated kinase activity was detected in the fractions eluted by a lower concentration of NaCl than that which eluted the Mg2+‐activated kinase. Other growth factors, namely, epidermal growth factor and basic fibroblast growth factor, also stimulated the activity of NGF‐dependent MAP kinase. Possible involvement of the kinase in the outgrowth of neurites has been suggested. The NGF‐induced activation of NGF‐dependent MAP kinase was blocked by the presence of K‐252a. In contrast, the activation of NGF‐dependent MAP kinase by basic fibroblast growth factor and by epidermal growth factor was not blocked, but actually stimulated by K‐252a, a result that correlates well with the analogous actions of the drug on the outgrowth of neurites that is induced by these growth factors. The latter observation strengthens the possibility of a close relationship between the outgrowth of neurites and the acti

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08436.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract:The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15‐kDa proteolipid, presents a very high sequence homology with theN,N′‐dicyclohexylcarbodiimide (DCCD)‐binding proteolipid subunit of the vacuolar‐type H+‐ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium‐dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either fromTorpedosynaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15‐kDa proteolipid subunit of the mediatophore. These results suggest that although the 15‐kDa proteolipid subunits of the mediatophore and the vacuolar H+‐ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium‐dependent ACh release and that the two proteins have a different

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1992.tb08437.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY