Age-dependent degradation of calpastatin in kidney of hypertensive rats

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.     

Regulation of calpain activity in rat brain with altered Ca2+ homeostasis

Activation of calpain occurs as an early event in correlation with an increase in [Ca2+]i induced in rat brain upon treatment with a high salt diet for a prolonged period of time. The resulting sequential events have been monitored in the brain of normal and hypertensive rats of the Milan strain, diverging for a constitutive alteration in the level of [Ca2+]i found to be present in nerve cells of hypertensive animals. After 2 weeks of treatment, the levels of the plasma membrane Ca2+-ATPase and of native calpastatin are profoundly decreased. These degradative processes, more pronounced in the brain of hypertensive rats, are progressively and efficiently compensated in the brain of both rat strains by different incoming mechanisms. Along with calpastatin degradation, 15-kDa still-active inhibitory fragments are accumulated, capable of efficiently replacing the loss of native inhibitor molecules. A partial return to a more efficient control of Ca2+ homeostasis occurs in parallel, assured by an early increase in the expression of Ca2+-ATPase and of calpastatin, both producing, after 12 weeks of a high salt (sodium) diet, the restoration of almost original levels of the Ca2+ pump and of significant amounts of native inhibitor molecules. Thus, conservative calpastatin fragmentation, associated with an increased expression of Ca2+-ATPase and of the calpain natural inhibitor, has been demonstrated to occur in vivo in rat brain. This represents a sequential adaptive response capable of overcoming the effects of calpain activation induced by a moderate long term elevation of [Ca2+]i.     

Increased phosphorylation in red cell membranes of subjects affected by essential hypertension

In hemolysates of red cells from hypertensive patients the proteolytic activity of calpain is expressed at a rate approximately three fold higher than in red cells of normotensive subjects. Susceptibility to lysis upon exposure to ionophore A23187 and calcium, conditions that increase intracellular calpain activity, is also significantly enhanced in erythrocytes of hypertensive patients. In inside-out vesicles prepared from erythrocytes of these patients band 3 region undergoes a high extent of phosphorylation which is 1.5 fold higher than that occurring in control red cells from normotensive subjects. This increased phosphorylation can be reproduced in inside-out vesicles from erythrocytes of normal subjects following pretreatment with calpain. Taken together, these results suggest that the presence in erythrocytes of hypertensive subjects of an unregulated calpain dependent proteolytic activity may affect the structure of plasma membranes and determine an increased phosphorylation of intrinsic membrane proteins.     

Calpastatin level in spontaneously hypertensive rats

We studied calpastatin activity in erythrocytes of Milan hypertensive and prehypertensive rats, in their normotensive controls, in F1 and F2 hybrids, and in two inbred strains derived from F2, one hypertensive and the other normotensive. Our results show that the decrease in calpastatin activity observed in Milan hypertensive rats was not caused by hypertension, it was transmitted in a recessive way in heterozygous, and it was not correlated to hypertension.     

Is calpain activity regulated by membranes and autolysis or by calcium and calpastatin?

Although the Ca(2+)-dependent proteinase (calpain) system has been found in every vertebrate cell that has been examined for its presence and has been detected in Drosophila and parasites, the physiological function(s) of this system remains unclear. Calpain activity has been associated with cleavages that alter regulation of various enzyme activities, with remodeling or disassembly of the cell cytoskeleton, and with cleavages of hormone receptors. The mechanism regulating activity of the calpain system in vivo also is unknown. It has been proposed that binding of the calpains to phospholipid in a cell membrane lowers the Ca2+ concentration, [Ca2+], required for the calpains to autolyze, and that autolysis converts an inactive proenzyme into an active protease. Recent studies, however, show that the calpains bind to specific proteins and not to phospholipids, and that binding to cell membranes does not affect the [Ca2+] required for autolysis. It seems likely that calpain activity is regulated by binding of Ca2+ to specific sites on the calpain molecule, with binding to each site eliciting a response (proteolytic activity, calpastatin binding, etc.) specific for that site. Regulation must also involve an, as yet, undiscovered mechanism that increases the affinity of the Ca(2+)-binding sites for Ca2+.     

The role of calpain in the selective increased phosphorylation of the anion-transport protein in red cell of hypertensive subjects

The phosphorylation of the anion-transport protein (band 3) is selectively increased in human red cell membrane, following exposure of intact cells to ionophore and micromolar calcium. The phosphorylation is catalyzed by a membrane associated protein kinase distinct from either protein kinase C or Ca2+/calmodulin dependent protein kinase. We show that the increase in phosphorylation of band 3 is abolished if red cells had been pre-loaded with an inhibitor of calpain or with an anticalpain monoclonal antibody. Our findings suggest that calpain activity may control, both at a functional and at a structural level, the activity of this important transmembrane protein through the modulation of its susceptibility as a substrate of membrane bound protein kinase(s). Based on previous observations indicating the presence in erythrocytes from hypertensive patients of an uncontrolled intracellular calpain-mediated proteolytic system accompanied by an increased phosphorylation of band 3 protein(s), we suggest that our results may shed light on the type of molecular alteration which is associated with the hypertensive state.     

Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.     

Association of calpastatin with inactive calpain: a novel mechanism to control the activation of the protease?

It is generally accepted that the Ca(2+)-dependent interaction of calpain with calpastatin is the most relevant mechanism involved in the regulation of Ca(2+)-induced proteolysis. We now report that a calpain-calpastatin association can occur also in the absence of Ca(2+) or at very low Ca(2+) concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4-7. This calpastatin region recognizes a calpain sequence located near the end of the DII-domain. Interaction of calpain with calpastatins lacking these sequences becomes strictly Ca(2+)-dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca(2+)-free calpain and various recombinant calpastatin forms has been demonstrated by the following experimental results. Addition of calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when calpastatin was immunoprecipitated. The calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-calpastatin interaction at a physiological concentration of Ca(2+) represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca(2+) influx.     

Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration.     

Decreased level of calpain inhibitor activity in red blood cells from Milan hypertensive rats

In mature red cells of rats from Milan Normal (MNS) and Hypertensive Strains (MHS), the soluble Ca2+-dependent neutral proteinase (calpain) is present in similar amounts as the form requiring 0.1-0.2 mM Ca2+ for maximum catalytic activity. The amount of the endogenous calpain inhibitor, however, differs greatly in the red cells of the two strains. In red cells from hypertensive rats the activity of the inhibitor is 10 times less with a ratio of inhibitor to calpain activity (unit/unit) of 0.2; compared to red cells from normal rats, in which this ratio is approximately 2. This is the first demonstration of the existence, in a mammalian cell, of such a low ratio of calpain to inhibitor and implies the occurrence of a potentially "unregulated" intracellular soluble proteinase. This abnormal condition may be responsible for some of the structural and metabolic changes reported in rats of the genetically determined MHS strain.     

Detection and some properties of calpain II (high-Ca2+-requiring form of calpain) in carp (Cyprinus carpio) erythrocytes

In order to examine the existence of calpain I, a low (micromolar)-Ca2+-requiring form of calpain, in fish tissues, carp erythrocytes were chosen as the experimental material, since only calpain I is known to exist in mammalian erythrocytes. By DEAE-cellulose chromatography, calpain and calpastatin (specific inhibitor for calpain) were separated from carp erythrocyte hemolysate. Carp erythrocyte calpain is classified as calpain II, a high (millimolar)-Ca2+-requiring form of calpain, from the result of Ca2+-requirement for the activity.     

Interaction between catalytically inactive calpain and calpastatin. Evidence for its occurrence in stimulated cells

Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca(2+), substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca(2+)] or extensive diffusion of calpastatin into the cytosol without affecting Ca(2+) homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain-calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action.     

Regulatory differences between Ca(2+)-ATPase in plasma membranes from chicken (nucleated) and pig (anucleated) erythrocytes

Kinetic and regulatory properties of the plasma membrane Ca(2+)-ATPase activity from chicken (nucleated) erythrocytes were studied and compared to those from pig (anucleated) erythrocytes. In the absence of known activators: (1) Ca(2+) affinity for the Ca(2+)-ATPase activity from nucleated erythrocytes was 12-fold higher than that from pig erythrocytes, and thus the enzyme is sensitive to physiological Ca(2+) concentrations; (2) the enzyme from chicken erythrocytes showed two apparent Km values for ATP, as compared to one apparent Km value displayed by pig erythrocytes; (3) Ca(2+)-ATPase inserted in chicken erythrocyte membranes showed a low sensitivity to activation by phosphatidylinositol-4-phosphate; (4) when p-NPP was used as substrate, the activity of chicken erythrocytes was high, similar to that attained by pig erythrocytes, but barely sensitive to activation by dimethylsulfoxide and calmodulin. ATP hydrolysis was 10-fold lower than that displayed by pig erythrocytes and the maximal velocity was activated three-fold by calmodulin. The enzyme was insensitive to alkaline phosphatase treatment and showed a single phosphorylation band in electrophoresis, ruling out the possibility of previous modulation by endogenous kinases and/or by partial proteolysis. The differences may be attributed to some endogenous modulator, to distinct isoforms, or to a difference in the E(1)/E(2) states of the enzyme.     

Protein kinase C phosphorylates the carboxyl terminus of the plasma membrane Ca(2+)-ATPase from human erythrocytes.

Purified Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase) from human erythrocytes was phosphorylated with a stoichiometry of about 1 mol of phosphate/mol of ATPase at both threonine and serine residues by purified rat brain type III protein kinase C. In the presence of calmodulin, the phosphorylation was markedly reduced. Labeled phosphate from [gamma-32P]ATP was retained on an 86-kDa calmodulin-binding tryptic fragment of Ca(2+)-ATPase but not on 82- and 77-kDa non-calmodulin-binding fragments. Similarly, fragmentation of the phosphorylated Ca(2+)-ATPase by calpain I revealed that calmodulin-binding fragments (127 and 125 kDa) retained phosphate label whereas a non-calmodulin-binding fragment (124 kDa) did not. The calmodulin-binding domain, located about 12 kDa from the carboxyl terminus of the Ca(2+)-ATPase, was thus located as a site of protein kinase C phosphorylation. A synthetic peptide corresponding to a segment of the calmodulin-binding domain (H2 N-R-G-L-N-R-I-Q-T-Q-I-K-V-V-N-COOH) was indeed phosphorylated at the single threonine residue within this sequence. The additional serine phosphorylation site was carboxyl terminal to the calmodulin domain. Phosphorylation by purified type III protein kinase C (canine heart) antagonized the calmodulin activation of the Ca(2+)-ATPase, particularly at lower Ca2+ concentrations (0.2-1.0 microM). By contrast, a purified but unresolved protein kinase C isoenzyme mixture from rat brain stimulated the activity of Ca(2+)-ATPase prepared in asolectin, but not glycerol, by more than 2-fold in the presence of the ionophore A23187, without increasing its Ca2+ sensitivity. The results clearly indicate that human erythrocyte Ca(2+)-ATPase is a substrate of protein kinase C, but the effect of phosphorylation on the activity of the enzyme depends on the isoenzyme form of protein kinase C used and on the lipid associated with the Ca(2+)-ATPase.     

Association of calpain (Ca(2+)-dependent thiol protease) with its endogenous inhibitor calpastatin in myoblasts.

Calpain isozymes (intracellular, Ca(2+)-dependent thiol proteases) are present in the cytoplasm of many cells, along with their endogenous specific inhibitor, calpastatin. Previously, we found that the levels of mu-calpain and m-calpain (activated by microM and mM Ca(2+), respectively) remain about the same during myoblast differentiation and fusion. By contrast, the calpastatin level, which is high during the initial stages of differentiation, diminishes markedly before myoblast fusion, allowing the proteolysis that is required for myotube formation. In the present study, we used immunoprecipitation to investigate the molecular association between calpain and calpastatin in dividing myoblasts and in the initial stages of myoblast differentiation. Immunoprecipitation (IP) was performed in two ways: (1) IP of calpain, using an anti-calpain antibody that recognized both isozymes; and (2) IP of calpastatin (using anti-calpastatin). Calpastatin was co-precipitated when calpain was immunoprecipitated; calpain was co-precipitated when calpastatin was immunoprecipitated. The results indicate that calpastatin is associated with calpain in dividing myoblasts and in myoblasts during the initial stages of differentiation, thereby preventing calpain activation at this stage. Prior studies carried out in vitro have shown a Ca(2+)-dependent interaction of calpain with calpastatin. The results described here suggest that an association between calpain and calpastatin could occur within cells in the presence of physiological Ca(2+)levels. It is proposed that the status of cellular calpain-calpastatin association is modulated by cell constituents, for which some possibilities are suggested.     

Decreased level of calpain inhibitor activity in kidney from Milan hypertensive rats

Rat kidney contains two different calpain isozymes distinguishable on the basis of their Ca2+ requirement and of their activation mechanisms. The two calpain isozymes are present in comparable amounts in kidney of normotensive and hypertensive rats of the Milan strain. Conversely, the level of the natural inhibitor of calpain is significantly decreased in kidney of hypertensive rats as compared to control normotensive rats. This deficiency is more pronounced in the cortical region than in other kidney fractions. These results taken together with previous observations indicating the existence of an identical defect in red cells from the same hypertensive rat strain, (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Viotti, P., Michetti, M., Duzzi, L., and Bianchi, G. (1986) Biochem. Biophys. Res. Commun. 138, 1370-1375) emphasize the possible role of an unbalanced intracellular proteolytic system in the development of genetically determined hypertension.     

Characterization of the calpastatin defect in erythrocytes from patients with essential hypertension

In erythrocytes of patients with essential hypertension the level of calpastatin activity was found to be significantly lower than in red cells of normotensive subjects (1). We now demonstrate, by Western blot analysis, that the decreased inhibitory activity is due to a corresponding decrease in the amount of the inhibitor protein. This is also supported by the observation that calpastatins isolated and purified from erythrocytes of normotensive and hypertensive patients, have identical specific activity. Data are presented indicating that the decreased level of calpastatin cannot be ascribed to an accelerated decay of the inhibitor during the erythrocyte life span. Taken together the previous and present results further emphasize that an umbalanced proteolytic system may represent one of the molecular mechanisms responsible for those membrane abnormalities underlying the development of essential hypertension and its clinical complications.     

Elevated intracellular Ca2+ affects Lii-Nao countertransport in human red blood cells

Changes in cytoplasmic Ca2+ concentration and in Lii-Nao countertransport activity have been shown to be associated with essential hypertension. Elevated intracellular free [Ca2+], as well as abnormalities of Ca2+ binding and transport have been reported in cells from different tissues of hypertensive laboratory animals and essential hypertensive patients. Similarly, enhanced rates of Lii-Nao countertransport and the modified pattern of the temperature dependence of this activity in red blood cells from essential hypertensive patients have been previously demonstrated. The aim of the present study was to investigate possible interaction between changes in intracellular free [Ca2+] and the Lii-Nao exchange in human red blood cells. The ionophore ionomycin was used to allow Ca2+ incorporation into the cells in a dose-dependent manner. The elevation of intracellular [Ca2+], in turn, resulted in enhanced Li+ efflux from the cells. At 3 microM, ionomycin selectively and significantly enhanced the Lii-Nao countertransport but not Li+ leakage from the cells. EGTA totally abolished the effect of ionomycin, indicating that the effect is directly related to Ca2+. As low as 0.4 microM Ca2+ caused a statistically significant effect. The maximal effect of Ca2+ on the Lii-Nao countertransport was achieved around the external pH range of 6.8-7.5. In contrast, the leakage of Li+ was significantly enhanced by Ca2+ at a pH of 7.4 and above. Ca2+ did not affect the Km of the Lii-Nao countertransport for Li+. Amiloride, which inhibits Na+/H+ exchange, inhibited only 10% of the Ca2+-enhanced countertransport. It is concluded that Ca2+ may play a role in the regulation of Lii-Nao countertransport in erythrocytes.     

Quantitation of tissue calpain activity after isolation by hydrophobic chromatography

A rapid and reliable method for quantitating tissue calpains (Ca2+-activated, neutral, thiol proteases) was developed using hydrophobic chromatography with phenyl-Sepharose. Calpains I and II isolated by this method are free of endogenous inhibitor(s) (calpastatin), activator(s), and nonspecific proteases. These calpains expose hydrophobic regions in the presence of Ca2+ and bind tightly to phenyl-Sepharose. Inactivation of bound calpain is prevented by the addition of leupeptin (20 microM). Calpains I and II bound initially by phenyl-Sepharose in a Ca2+-dependent manner are then eluted successively on the basis of their Ca2+-independent binding to phenyl-Sepharose. Because calpastatin may prevent binding of calpain to phenyl-Sepharose by forming a protease-inhibitor complex in the presence of Ca2+, preadsorbing the protease to a suspension of phenyl-Sepharose beads initially in the absence of Ca2+ separates most of the calpain present in tissue extracts from calpastatin. The isolated calpains obtained are assayed by casein digestion. This quantitation procedure is suitable for measuring calpain activity in various tissues and cells including erythrocytes.     

The cytosol of human erythrocytes contains a highly Ca2+-sensitive thiol protease (calpain I) and its specific inhibitor protein (calpastatin)

The cytosol of human erythrocytes was found to contain a Ca2+-dependent thiol protease (calpain) and its specific inhibitor (calpastatin) by DEAE-cellulose chromatography at pH 8.0, although no proteolytic activity toward casein was detected in the unfractionated hemolysate. The protease required only 40 microM Ca2+ for 50% activation, indicating that it belongs to the highly Ca2+-sensitive type of calpain, namely, calpain I. It was not inactivated by heating at 58 degrees C for 10 min at pH 7.2, the optimal pH for its action on casein. The inhibitor comprised major and minor components, calpastatin H (Mr = 280,000) and caplastatin L (Mr = 48,000). Both were heat-stable proteins which were readily inactivated by tryptic digestion. The inhibition of erythrocyte calpain by erythrocyte calpastatin H or L was not due to sequestering of Ca2+ from the reaction medium by the inhibitor protein. The calpain preparation preferentially digests bands III and IVa of human erythrocyte membrane proteins, with little or no cleavage of the bands corresponding to spectrin.