Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. I. Biochemical studies

Seubert, Peter; Ivy, Gwen O.; Larson, John; Lee, Jenny; Shahi, Kavian; Baudry, Michel; Lynch, Gary

doi:10.1016/0006-8993(88)90638-5

Cited by 72 publications

(22 citation statements)

References 33 publications

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“…Breakdown of cytoskeletal proteins such as MAP-2 has been described as one of the earliest stages of postischemic neurodegeneration (Matesic and Lin, 1994). One of the main enzymes involved in this process is calpain 1 whose activation depends, in turn, on stimulation of NMDA receptors and the concomitant elevation of cytosolic Ca 2+ (Regehr and Tank, 1990;Seubert et al, 1988;Siman and Noszek, 1988). Since excitability of NMDA receptors has been shown to be closely modulated by adenosine A 1 receptors (Schubert and Kreutzberg, 1990;Schubert et al, 1995), it is therefore quite likely that stimulation of the latter by ADAC may result in a significant impairment of NMDA receptor-mediated Ca 2+ influx with consequential depression of calpain 1 activity.…”

Section: Discussionmentioning

confidence: 99%

Postischemic administration of adenosine amine congener (ADAC): analysis of recovery in gerbils

Lubitz

Lin²,

Paul

et al. 1996

European Journal of Pharmacology

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Although adenosine receptor-based treatment of cerebral ischemia and other neurodegenerative disorders has been frequently advocated, cardiovascular side effects and an uncertain therapeutic time window of such treatment have constituted major obstacles to clinical implementation. Therefore, we have investigated the neuroprotective effects of the adenosine A 1 receptor agonist adenosine amine congener (ADAC) injected after either 5 or 10 min ischemia at 100 μg/kg. When the drug was administered at either 6 or 12 h following 5 min forebrain ischemia, all animals were still alive on the 14th day after the occlusion. In both ADAC treated groups neuronal survival was approximately 85% vs. 50% in controls. Administration of a single dose of ADAC at times 15 min to 12 h after 10 min ischemia resulted in a significant improvement of survival in animals injected either at 15 or 30 min, or at 1, 2, or 3 h after the insult. In all 10 min ischemia groups, administration of ADAC resulted in a significant protection of neuronal morphology and preservation of microtubule associated protein 2 (MAP-2). However, postischemic Morris' water maze tests revealed full preservation of spatial memory and learning ability in animals injected at 6 h. On the other hand, the performance of gerbils treated at 12 h postischemia was indistinguishable from that of the controls. Administration of ADAC at 100 μg/kg in non-ischemic animals did not result in bradycardia, hypotension, or hypothermia. The data indicate that when ADAC is used postischemically, the most optimal level of protection is obtained when drugs are given at 30 min to 6 h after the insult. Although the mechanisms involved in neuroprotective effects of adenosine A 1 receptor agonists require further studies, the present results demonstrate the feasibility of their clinical applications.

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Section: Discussionmentioning

confidence: 99%

Postischemic administration of adenosine amine congener (ADAC): analysis of recovery in gerbils

Lubitz

Lin²,

Paul

et al. 1996

European Journal of Pharmacology

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“…It has been reported that ,u-calpain is autolyzed in human leukemic Molt-4 cells with treatment of calcium ionophore A23187 (24). It is also well established that that alpha-spectrin, which is one of its preferred cytoskeletal substrates, was degraded into two proteolytic fragments (150 kDa and 145 kDa) (5,24,35,36). This distinct pattern of fragmentation of a-spectrin has been used as an assay for calpain activity in intact cells (28,37,38).…”

Section: Methodsmentioning

confidence: 99%

An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective.

Wang¹,

Nath

Posner

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

256

171

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Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) K; values for ,u-and m-calpains of 0.21 ,uM and 0.37 ,IM, respectively, (ii) high specificity for calpains relative to other proteases, (iii) uncompetitive inhibition with respect to substrate, and (iv) it does not shield calpain against inactivation by the active-site inhibitor trans-(epoxysuccinyl)-L-leucyl-amido-3-methylbutane, suggesting a nonactive site action for PD150606. The recombinant calcium-binding domain from each of the large or small subunits of ,u-calpain was found to interact with PD150606. In low micromolar range, PD150606 inhibited calpain activity in two intact cell systems. The neuroprotective effects of this class of compound were also demonstrated by the ability of PD150606 to attenuate hypoxic/hypoglycemic injury to cerebrocortical neurons in culture and excitotoxic injury to Purkinje cells in cerebellar slices.Calpain (EC 3.4.22.17) is a class of cytosolic cysteine protease that is activated by elevated intracellular calcium (1-3). Interest in this class of enzymes has grown substantially in recent years because it has been implicated in the pathophysiology of several degenerative conditions; including cerebral ischemia, myocardial ischemia, and cataract (4-8). The unifying features of these pathological conditions are that calcium serves as a trigger for cellular injury and that calpain may represent a crucial mediator of the degenerative response. Uncontrolled activation of calpain leads to cytoskeletal protein (e.g., spectrin) breakdown, degradation of many receptor proteins (e.g., epidermal growth factor receptor), and enzyme systems (e.g., protein kinase C and calmodulin-dependent kinases) and consequently cell death (1,8). Several studies have shown that peptidic inhibitors of calpain protect cells in models of ischemic, hypoxic and/or excitotoxic neuronal injury (6, 9-13). However, a clear interpretation of the role of calpain in these studies has been hampered by the lack of selectivity of these peptidic inhibitors (8).The predominant forms of calpain in mammalian tissues are ,u-calpain and m-calpain, requiring low and high micromolar calcium, respectively, for in vitro activation. Both of these isoforms are heterodimers in which the large subunit (80 kDa) contains a distinct cysteine protease domain and a calcium-binding domain with four helix-loop-helix (EF-hand) structures (14 MATERIALS AND METHODSProtease Assays and Kinetic Studies. The calpain microplate assay was carried out as described (15). Briefly, a mixture containing 0.5 mg/ml casein, 20 mM DTT, 50 mM Tris-HCl (pH 7.4), 0.01 unit purified ,u-calpain (human erythrocytes) or m-calpain (r...

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“…21 A silver staining method was chosen since proteolysis of fibrillic macromolecules that are stained by silver starts a few minutes after onset of ischemia. 22,23 For SIS, the slides were submerged for 2 minutes into a silver impregnation solution (see below), which was shaken vigorously. Then the slides were washed in distilled water 6 times for 1 minute before they were transferred to a vigorously shaken developer solution for 3 minutes (see below).…”

Section: Silver Infarct Stainingmentioning

confidence: 99%

Early Delineation of Ischemic Tissue in Rat Brain Cryosections by High-Contrast Staining

Vogel

Möbius

Kuschinsky

1999

Stroke

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Background and Purpose-After short periods of ischemia, commonly used staining methods yield only moderate differences in optical contrast between normal and damaged brain tissue when gray-scale images are used for computer-assisted image analysis. We describe a high-contrast silver infarct staining (SIS) method that allows an early delineation of ischemic tissue as soon as 2 hours after middle cerebral artery occlusion (MCAO) in rat brain cryosections. Methods-Rats were subjected to permanent MCAO for 2, 4, 6, and 48 hours. The optical densities were quantified in nonischemic white and gray matter and in damaged tissue from gray-scale images of serial sections with the use of a video camera-based image analyzing system. SIS, hematoxylin-eosin, Nissl, and nitroblue tetrazolium stainings were performed in cryosections, and 2,3,5-triphenyltetrazolium hydrochloride (TTC) staining was performed in unfrozen vibratome sections. In addition, the range of reduced cerebral blood flow (CBF) in areas demarcated by SIS was determined in iodo[ 14 C]antipyrine autoradiograms of adjacent cryosections. Results-At all times after MCAO, only SIS showed significantly (PϽ0.01) lower optical densities in damaged than in normal brain tissue for both white and gray matter. TTC staining was as effective as SIS 6 and 48 hours after MCAO. The tightest correlation between areas of reduced SIS and of reduced CBF was found at a mean ischemic CBF of 22.3 mL/100 g per minute. This corresponds to a CBF range of 0 to 44 mL/100 g per minute in areas of reduced SIS. Conclusions-In contrast to other staining methods, SIS allows a reliable delineation of ischemic brain tissue (core plus penumbra) from nonischemic white and gray matter of rat brain cryosections as soon as 2 hours after MCAO. (Stroke. 1999;30:1134-1141.)

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Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. I. Biochemical studies

Cited by 72 publications

References 33 publications

Postischemic administration of adenosine amine congener (ADAC): analysis of recovery in gerbils

Postischemic administration of adenosine amine congener (ADAC): analysis of recovery in gerbils

An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective.

Early Delineation of Ischemic Tissue in Rat Brain Cryosections by High-Contrast Staining

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