In a model of cerebral hypoxia-ischemia in the immature rat, widespread brain injury is produced in the ipsilateral hemisphere, whereas the contralateral hemisphere is left undamaged. Previously, we found that calpains were equally translocated to cellular membranes (a prerequisite for protease activation) in the ipsilateral and contralateral hemispheres. However, activation, as judged by degradation of fodrin, occurred only in the ipsilateral hemisphere. In this study we demonstrate that calpastatin, the specific, endogenous inhibitor protein to calpain, is up-regulated in response to hypoxia and may be responsible for the halted calpain activation in the contralateral hemisphere. Concomitantly, extensive degradation of calpastatin occurred in the ipsilateral hemisphere, as demonstrated by the appearance of a membrane-bound 50-kDa calpastatin breakdown product. The calpastatin breakdown product accumulated in the synaptosomal fraction, displaying a peak 24 h post-insult, but was not detectable in the cytosolic fraction. The degradation of calpastatin was blocked by administration of CX295, a calpain inhibitor, indicating that calpastatin acts as a suicide substrate to calpain during hypoxia-ischemia. In summary, calpastatin was up-regulated in areas that remain undamaged and degraded in areas where excessive activation of calpains and infarction occurs.Hypoxic-ischemic brain damage is an important contributor to long term neurological sequelae in term and pre-term infants (1-3). The intracellular calcium concentration increases during anoxia/ischemia (4) followed by a secondary phase of cellular calcium overload simultaneous with or slightly preceding development of hypoxic-ischemic neuronal damage (5, 6). Activation of calcium-dependent enzymes is considered to be an early feature in this process (7). Calpains (EC 3.4.22.17) are calciumactivated, nonlysosomal, neutral cysteine proteases proposed to participate in many important intracellular processes, such as turnover of cytoskeletal proteins and regulation of kinase activity and transcription factors (8, 9). The activity of calpains is strictly regulated by calcium concentrations and interaction with calpastatin (the endogenous inhibitor protein), membrane phospholipids, and a multitude of other factors. The ubiquitous distribution of calpains and the complex regulation of their activity indicate that these proteases play important roles under both physiological and pathological conditions. Calpain activation, as judged by the appearance of specific fodrin (10) (also called brain spectrin (11)) breakdown products (FBDP) 1 has previously been demonstrated in adult (12-16) and neonatal (17, 18) models of ischemia and hypoxia. Activation of calpains and selective degradation of preferred substrates precede neuronal degeneration, indicating that these proteases are activated during hypoxia-ischemia (HI) and in the early phase of reperfusion after HI, preceding neuronal death (12, 13, 16 -19). In vivo administration of inhibitors of calpain activity has been sho...