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...

Summary:The role of GABA in regulating cerebral mi crovessels was examined in the parenchyma of the hippocam pus and the surface of the neocortex. Microvessels were moni tored in in vitro slices using computer-assisted videomicros copy, and synaptically evoked field responses were simultaneously recorded. ,/-Aminobutyric acid (GAB A) and the GABAA receptor agonist, muscimol, elicited vasodilation in hippocampal microvessels, whereas the GABAB receptor agonist, badofen, elicited constriction. The muscimol-induced dilation persisted in the presence of the nitric oxide synthase inhibitor, N-nitro-L-arginine, indicating that this response is not mediated by nitric oxide. Inhibition of neuronal discharge ac tivity with tetrodotoxin did not alter this dilation, but it fully blocked the constrictor response to baclofen. These data sug gest that GABAB-mediated, but not GABAA-mediated, re sponses are dependent on action potential generation. The GABAA receptor antagonists, bicuculline and picrotoxin, elicConverging lines of evidence suggest that local neu rons are major participants in the regulation of regional cerebral blood flow (CBF). At rest, regional CBF is di rectly correlated with the level of local neuronal activity (Lou et aI., 1987). Furthermore, CBF increases in a spa tially and temporally precise manner during enhanced neuronal activity (Fox and Raichle, 1986). This vascular response frequently is dissociated from changes in re gional cerebral metabolism, suggesting that these blood flow changes are not necessarily a consequence of meta bolic alterations (Lenniger-Follert and Lubbers, 1976; Nakai et a!., 1983; Fox and Raichle, 1986; Lou et aI., 992ited constriction, suggesting a tonic dilatory influence by en dogenous GABA. Bicuculline-induced constriction was not at tenuated by tetrodotoxin. In contrast, these vessels were unre sponsive to the GABAB receptor antagonist, 2-hydroxysac lofen. Hippocampal microvessels dilated in response to mod erate hypoxia, and this response persisted in the presence of bicuculline, indicating that the hypoxia-induced dilation is not mediated by an action at GABAA receptors. In arterioles lo cated on the surface of the neocortex (i.e., not embedded in the parenchyma of the brain), muscimol elicited vasodilation, whereas bicuculline was ineffective. These results suggest that although these vessels are responsive to GABA, the local con centration of endogenous GABA is insufficient to elicit a tonic effect at rest. These findings raise the possibility that GABA plays a role in local neurovascular signaling in the parenchyma of the brain.

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