The degradation of troponin (Tn) subunits by calpain was studied by incubating either isolated cardiac Tns or myocardial cryosections with two different calpain isoenzymes isolated from rat skeletal muscle. Western-blot analysis with monoclonal antibodies against TnI and TnT showed that mu-calpain was at least ten times more active than m-calpain in degrading TnI and TnT both in vitro and in situ. TnC was completely resistant to both proteinase forms. Phosphorylation by cyclic AMP-dependent protein kinase (PKA) isolated from rat skeletal muscle reduced the sensitivity of TnI to degradation. This effect in combination with an increased efficiency of the endogenous inhibitor [Salamino, De Tullio, Michetti, Mengotti, Melloni and Pontremoli (1994) Biochem. Biophys. Res. Commun. 199, 1326-1332] probably reduces the proteolytic activity of calpain in cells on PKA stimulation. Conversely, phosphorylation by protein kinase C (PKC) resulted in a twofold increase in the degradation of TnI. Degradation by m-calpain was not modified by Tn phosphorylation. The different sensitivity to mu-calpain might be related to changes in TnI oligomeric structure. Indeed, on PKC phosphorylation, the apparent molecular mass of TnI calculated from the distribution coefficient of Tn complex in Sephadex G-100 matrix was reduced from 90 to 30 kDa suggesting dissociation of the Tn complex.

Calpains, the thiol proteinases of the calciumdependent proteolytic system, are regulated by a natural inhibitor, calpastatin, which is present in brain tissue in two forms. Although both calpastatins are highly active on human erythrocyte calpain, only one form shows a high inhibitory efficiency with both rat brain calpain isozymes. The second calpastatin form is almost completely inactive against homologous proteinases and can be converted into an active one by exposure to a phosphoprotein phosphatase, also isolated from rat brain. Phosphorylation of the active calpastatin by protein kinase C and protein kinase A promotes a decrease in its inhibitory efficiency. The interconversion between the two inhibitor forms seems involved in the adjustment of the level of intracellular calpastatin activity on specific cell requirements.

Cachexia is a syndrome characterized by profound tissue wasting that frequently complicates malignancies. In a cancer cachexia model we have shown that protein depletion in the skeletal muscle, which is a prominent feature of the syndrome, is mostly due to enhanced proteolysis. There is consensus on the views that the ubiquitin/proteasome pathway plays an important role in such metabolic response and that cytotoxic cytokines such as TNFα are involved in its triggering (Costelli and Baccino, 2000), yet the mechanisms by which the relevant extracellular signals are transduced into protein hypercatabolism are largely unknown. Moreover, little information is presently available as to the possible involvement in muscle protein waste of the Ca 2+ -dependent proteolysis, which may provide a rapidly activated system in response to the extracellular signals. In the present work we have evaluated the status of the Ca 2+ -dependent proteolytic system in the gastrocnemius muscle of AH-130 tumour-bearing rats by assaying the activity of calpain as well as the levels of calpastatin, the natural calpain inhibitor, and of the 130 kDa Ca 2+ -ATPase, both of which are known calpain substrates. After tumour transplantation, total calpastatin activity progressively declined, while total calpain activity remained unchanged, resulting in a progressively increasing unbalance in the calpain/calpastatin ratio. A decrease was also observed for the 130 kDa plasma membrane form of Ca 2+ -ATPase, while there was no change in the level of the 90 kDa sarcoplasmic Ca 2+ -ATPase, which is resistant to the action of calpain. Decreased levels of both calpastatin and 130 kDa Ca 2+ -ATPase have been also detected in the heart of the tumour-bearers. These observations strongly suggest that Ca 2+ -dependent proteolysis was activated in the skeletal muscle and heart of tumour-bearing animals and raise the possibility that such activation may play a role in sparking off the muscle protein hypercatabolic response that characterizes cancer cachexia. © 2001 Cancer Research Campaign http://www.bjcancer.com

Localization of the two main components of the Ca2+-dependent proteolytic system has been investigated in human neuroblastoma LAN-5 cells. Using a monoclonal antibody which recognizes the N-terminal calpastatin domain, it has been shown that this inhibitory protein is almost completely confined in two granule-like structures not surrounded by membranes. Similar calpastatin distribution has been found in other human and in murine cell types, indicating that aggregation of calpastatin is a general property and not an exclusive characteristic of neuronal-like cells. The existence of such calpastatin aggregates is confirmed by the kinetics of calpastatin-activity release during rat liver homogenization, which does not correspond to the rate of appearance of cytosolic proteins or to the disruption of membrane-surrounded organelles. Calpastatin distribution is affected by the intracellular increase in free Ca2+, which results in calpastatin progressively becoming a soluble protein. However, calpain is distributed in the soluble cell fraction and, in activating conditions, partially accumulates on the plasma membrane. Similar behaviour has been observed in calpastatin localization in LAN-5 cells induced with retinoic acid, suggesting that the proteolytic system is activated during the differentiation process of these cells. The involvement of calpastatin in controlling calpain activity, rather than its activation process, and the utilization of changes in calpastatin localization as a marker of activation of the system is discussed.

We have previously reported that, in neuroblastoma LAN-5 cells, calpastatin is in an aggregated state, close to the cell nucleus [de Tullio, Passalacqua, Averna, Salamino, Melloni and Pontremoli (1999) Biochem. J. 343, 467-472]. In the present paper, we demonstrate that aggregated calpastatin is predominantly in a phosphorylated state. An increase in intracellular free [Ca2+] induces both dephosphorylation of calpastatin, through the action of a phosphoprotein phosphatase, and its redistribution as a soluble inhibitor species. cAMP, but not PMA-induced phosphorylation, reverses calpastatin distribution favouring its aggregation. This intracellular reversible mechanism, regulating the level of cytosolic calpastatin, could be considered a strategy through which calpain can escape calpastatin inhibition, especially during earlier steps of its activation process.

Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90–NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced.

Hypertensive rats from the Milan strain show a significant decrease in calpastatin activity as compared with normotensive control animals. Calpastatin deficiency is age-related and highly relevant in kidney, heart, and erythrocytes and of minor entity in brain tissue. In normotensives the changes during aging in the levels of calpastatin activity and mRNA are consistent with an increase of calpastatin protein. In hypertensive rats such a relationship during aging is not observed, because a progressive accumulation of mRNA is accompanied by a lower amount of calpastatin protein as compared with control rats. Together with the low level of calpastatin in kidney of hypertensive rats, a progressive accumulation of an active 15-kDa calpastatin fragment, previously shown to represent a typical product of calpain-mediated calpastatin degradation, is also observed. Evidence for such intracellular proteolysis by Ca 2؉ -activated calpain is provided by the normalization of the calpastatin level, up to that of control animals, in hypertensive rats treated with drugs known to reduce both blood pressure and intracellular Ca 2؉ influx. Further evidence is provided by the disappearance, in these conditions, of the 15-kDa calpastatin fragment. These data allow the conclusion that calpastatin degradation is a relevant part of the overall mechanism for regulating calpain activity.

The regulatory mechanism of calpain activity is primarily based on the physiological control of intracellular Ca 2ϩ homeostasis, predominantly exerted by the plasma membrane Ca 2ϩ -ATPase and, more specifically, by the expression in all mammalian cells of a natural protein inhibitor named calpastatin (1-7).Calpastatin is a protein endowed with peculiar molecular properties characterized by the presence of four identical inhibitory domains, each one possessing, in its free form, an inhibitory capacity almost equivalent to that expressed by the native calpastatin molecule (2-4, 8 -11). Moreover, calpastatin also behaves as a substrate of calpain, which is degraded to single inhibitory domains as well as to inactive products as a result of more extensive digestion (8,(12)(13)(14)(15)(16). Conservative fragmentation has been attributed to -calpain activity, and degradation to inactive peptides attributed to digestion by m-calpain (8, 16).Despite this information and the numerous reports on the structural properties of calpain (17-22), including the presence of different isoforms in various tissues (23-27), the biological function of this protease and the efficiency of its "in vivo" intracellular regulation remain uncertain.It is often suggested (28 -34) that in brain, calpain is directly implicated in neurodegeneration and neuronal cell death as a consequence of a massive Ca 2ϩ influx occurring under pathological conditions, such as hypoxia and ischemia. This statement has been largely based on the protective effects exerted by synthetic protease inhibitors and by overexpression of calpastatin (35-40). However, in these reports, the fact that a large excess of calpastatin is normally present in brain is not considered, and no explanation has been given on the possible ineffectiveness of the inhibitor under the experimental conditions used (31). It must also be considered that the severe and irreversible cell damage observed under these pathological conditions occurs in the presence of a massive Ca 2ϩ influx, associated with the collapse of the membrane potential. On the basis of these considerations, it seems therefore reasonable to assume that in brain cells, as well as in other cell types, a moderate alteration in Ca 2ϩ homeostasis, even prolonged for a long period of time, could evoke adaptive compensatory defense mechanisms capable of preserving cell integrity.To explore in vivo the existence in the brain of such mechanisms and to characterize their molecular aspects, we have treated normal normotensive Milan strain rats (NMS) 2 with a high salt (sodium) diet (HSD) known to promote elevation in blood pressure in response to a mild increase in intracellular free [Ca 2ϩ ] in vascular smooth muscle, via an alteration in the Na ϩ /Ca 2ϩ exchanger (41)(42)(43). A role of the exchanger in the alteration of calcium homeostasis in brain has been previously suggested (44,45). To amplify the effects of this treatment we have exposed hypertensive rats (HMS) of the same Milan strain (46) to the same diet, because these an...