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Different susceptibility of red cell membrane proteins to calpain degradation
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 298, No. 1, October, pp. 287-292, 1992
Different Susceptibility of Red Cell Membrane to Calpain Degradation Franca Salamino,’ Roberta De Tullio, Edon Melloni, and Sandro Pontremoli Institute
of Biochemistry,
University
of Genoa,
Paola Mengotti,
Proteins
Pier Luigi Viotti,
Genoa, Italy
Received October 3, 1991, and in revised form June 4, 1992
The presence of low levels of calpastatin activity in erythrocytes of hypertensive rats affects regulation of calpain activity so it is highly susceptible to activation within physiological fluctuations in [Ca2+]. Under identical conditions, in red cells of normotensive rats, calpain activation is efficiently controlled by the high levels of calpastatin activity, and a progressive increase in proteinase activity can only be observed in parallel with a decrease in the level of calpastatin. In intact erythrocytes from hypertensive rats exposed to small variations in [Ca2’], degradation of anion transport protein (band 3) and Ca2+-ATPase appears as a primary event indicating that these two transmembrane proteins are probably early recognized as targets of intracellular calpain activity. Furthermore, band 3 protein seems to be structurally modified in erythrocytes from hypertensive rats, as indicated by its increased susceptibility to degradation in the presence of lo-50 I.LM Ca’+. In addition, when exposed to progressive and limited increases in [Ca2+], erythrocytes from hypertensive rats, but not those from normotensive rats, show a high degree of fragility that can be restored to normal values by inhibition of calpain. These results indicate that, within fluctuations in [Ca2’] close to physiological values, regulation of calpain activity is efficiently accomplished in normal erythrocytes but is completely lost in cells from hypertensive animals. Regulation is of critical importance in maintaining normal structural and functional properties of selective red cell membrane and cytoskeletal proteins, among which band 3 and Ca2+-ATPase appear to be the substrates with highest susceptibility to digestion by calpain. o 1992 Academic
Press,
Inc.
i Author to whom correspondence should be addressed. * Abbreviations used: MHS, Milan hypertensive strain; MNS, Milan normotensive strain; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. 0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Red cells from human subjects affected with essential hypertension and from rats of the Milan hypertensive strain (MHS)2 are characterized by a variety of molecular lesions mostly concerning membrane and cytoskeletal proteins (l-8). These alterations have been attributed to a defective control of calpain activity due to the presence of low levels of calpastatin, the natural proteinase inhibitor (9-12). However, no precise demonstration has yet been provided indicating an enhanced activation of calpain in intact red cells of MHS rats, promoted by variations in [Ca2+] too small to induce activation of the proteinase in control cells. The occurrence of these events, as well as their implication in structural modifications of membrane and cytoskeletal proteins, has been investigated under conditions in which variations in the intracellular concentration of Ca2+ have been induced in intact erythrocytes from rats of Milan normotensive (MNS) and hypertensive strains. In the present paper, we report comparative data showing that increased cell fragility, as well as alterations in the molecular structure of band 3 and of Ca2+-ATPase, can be related to an uncontrolled activation of calpain occurring in red cells of hypertensive rats. MATERIALS
AND
METHODS
Adult males from MNS and MHS rats with masses of about 220-250 g were obtained as described (13). Calcium ionophore A23187 and leupeptin were purchased from Sigma Chemical Co. [y-32P]ATP (3000 Ci/ mmol) was obtained from Amersham International. All other chemicals were reagent grade. Acid-denatured globin was prepared as described by Winterhalter et al. (14). In the extracellular medium, due to the absence of chelating agents, the Ca*+ added was considered total Ca2+ concentration. Packed erythrocytes obtained from Loading of erythrocytes with Ca”. MNS and MHS rats (2 ml) and deprived of leukocytes and platelets (16) were suspended in 10 vol of 10 mM Hepes, pH 7.4, containing 5 mM KCl, 140 mM NaCl, and 5 mM glucose. Elevation of intracellular Ca*+ levels was obtained by exposure of the red cells to the ionophore A23187 in the presence of a specified concentration of Ca*‘. To avoid interference by chelating agents, the extracellular medium was prepared without EDTA and the Ca*’ concentration was considered the total 287
Inc. reserved.
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SALAMINO
amount of Ca**. Under these conditions, following the report of Kirkpatrick (16), the increase in intracellular free Ca*+ corresponds to approximately 80% of that originally present in the extracellular medium. Aliquots (2 ml) of the erythrocyte suspensions were incubated at 37°C for 30 min with 5 pM A23187 (final concentration) in the absence or in the presence of increasing Ca*+ concentrations and then the cells were collected by centrifugation at SOOgfor 10 min. When indicated, the cells were preloaded with 2 mM leupeptin or anti-calpain monoclonal antibody following the hypotonic dialysis encapsulation procedure described by Ropars et al. (18, 19). Briefly, erythrocytes (2 ml), at 80% hematocrit, were treated with 20 ~1 of a stock solution of 200 mM leupeptin of 20 mg/ml anti-calpain antibody, transferred into a dialysis bag, and dialyzed against 20 ml of 10 mM NaH,PO,-NaHCOs buffer, pH 7.5, containing 20 mM glucose. After 20 min at 4°C the suspension was recovered and resealed by adding 0.1 vol of a stock solution containing 100 mM NaH,P04, 5 mM adenine, 100 mM inosine, 100 mM pyruvate, 100 mM glucose, and 12% NaCl. The suspension was incubated for 30 min at 39°C and then the cells were collected and extensively washed with isotonic saline buffer. With this procedure, using labeled tracers, such as iodinated antibodies or r4C-labeled amino acids, we have calculated that approximately 30-40% of the materials remain entrapped in red cells. As control, erythrocytes were submitted to the same treatment with the omission of the proteinase inhibitor or anti-calpain monoclonal antibody. Phosphor-y&ion of erythrocytes membranes. Erythrocytes, loaded with A23187 in the presence of increasing Ca2+ concentrations, were lysed in 10 vol of 5 mM sodium phosphate buffer, pH 8.0, containing 1 mM EDTA. Erythrocyte membranes were washed extensively with the same buffer without EDTA until the hemoglobin was completely removed. The phosphorylation of erythrocyte membranes was carried out on aliquots (100 gg of proteins) of each sample as described by Pontremoli et al. (3). Assay of the erythrocyte membrane Ca2+-ATPase. Erythrocytes, loaded with A23187 in the presence or absence of Cazf, were lysed in 10 ml of 10 mM Tris/Cl, pH 7.4, containing 1 mM EDTA, and the membranes were prepared as described in (20). The radiolabeled Ca2+ pump was visualized through its phosphorilated intermediate in the presence of 1 mM Ca and 0.1 mM lanthanum nitrate, which stabilizes the aspartyl phosphate intermediate as described in (20). Samples, containing 80 pg of proteins, were submitted to electrophoresis on a polyacrylamide gradient (from 5 to 15%) in the presence of sodium dodecyl sulfate (20). C&pain and calpostatin assay. Erythrocytes, loaded with 5 pM A23187 as described above in the presence of different Ca2+ concentrations, were lysed in 5 vol of a solution of 1 mM EDTA at pH 7.0 and centrifuged at 10,OOOgfor 15 min. Aliquots of the cytosolic fraction (50 ~1) were used for calpain assay as previously described (21). For the assay of calpastatin activity, an aliquot (20 al) of the cytosolic fraction was diluted to 100 ~1, with distilled water, and heated at 90°C for 3 min. The precipitated material was discarded by centrifugation at 80,OOOgfor 3 min and calpastatin activity assayed under standard conditions (21) using 40 (~1of the clear supernatant. Preparation of anticalpain monoclonal antibody. Anticalpain monoclonal antibody (mAb 56.3) was prepared as previously described (22). RESULTS
Effect of Loading with Ca2+ on Degradation of Membrane Proteins in Red Cells from Normotensive and Hypertensive Rats Erythrocytes from MHS rats loaded with ionophore A23187 in the presence of increasing concentrations of Ca2+ show a significantly higher fragility as compared to cells obtained from MNS rats (Fig. 1). This fragility, revealed by the release of hemoglobin into the extracellular
ET AL.
50
100
[ 1
Cap+ pM
FIG. 1. Effect of loading with Ca2+ on the lysis of erythrocytes from normotensive and hypertensive rats. Erythrocytes from MNS or MHS rats were preloaded with or without leupeptin, then incubated with the indicated Ca2+ concentrations, in the presence of A23187, as described under Materials and Methods. The cells were then centrifuged at 8OOg for 10 min and the hemoglobin contents in the clear supernatant were determined by the absorbance at 540 nm. MNS (O), MNS loaded with leupeptin (0). MHS (A), MHS loaded with leupeptin (A).
medium, is profoundly reduced and becomes almost identical to that observed in control cells, when erythrocytes had been previously loaded with leupeptin, a competitive inhibitor of calpain. The Ca2+ dependency and the protective effect of leupeptin suggests that calpain is involved in the membrane damage responsible for the release of hemoglobin. The occurrence of structural modifications of membrane proteins was then comparatively investigated in red cells of both animal strains. As shown in Fig. 2, the electrophoretic pattern of the membrane-associated proteins from MNS rat erythrocytes, loaded with increasing [Ca2’] from 0.5 to 50 PM, reveals no appreciable structural modification. On the contrary, under identical conditions, membrane proteins of cells from MHS rats undergo a significant proteolytic degradation. At concentrations of Ca2+ between 1 and 5 PM, the intensity of the band corresponding to the anion transport protein progressively decreases and completely disappears at higher concentration of this metal ion. Also spectrin and 4.1 protein are progressively degraded, but the disappearance of the corresponding bands is much slower and can be observed only at the highest concentrations of Ca2+ used. The appearance in the electrophoretic pattern of new protein bands with low M, (Fig. Z), is probably due to the formation under these conditions of degradation products. If red cells from MHS rats had been preloaded with leupeptin or with the anticalpain monoclonal antibody 56.3, shown to prevent expression of the proteinase activity (22), and then exposed to concentrations of Ca2+ from 0.5 to 50 PM, the electrophoretic pattern of mem-
DIGESTION
OF ERYTHROCYTE
MNS
MEMBRANE
MHS
;. 3
0
0.5
1
5
10
20
50
0
0.5
1
5
10
20
50
[Ca*+] yM FIG. 2. Effect of loading with CaZf on the SDS-PAGE protein pattern of erythrocytes membranes from normotensive and hypertensive rats. Erythrocytes from MNS (A) or MHS (B) rats, loaded with Ca*+ and A23187 as reported under Materials and Methods, were lysed in 5 mM sodium phosphate buffer, pH 8.0 (5P8), containing 1 mM EDTA and centrifuged at 10,OOOgfor 10 min; the membranes were collected and washed in 5P8 containing 1 mM EDTA until the hemoglobin was completely removed. Samples (50 pg) of each membrane preparation were submitted to SDS-PAGE according to Fairbanks (37). The gels were stained with Coomassie blue, destained by free diffusion in 7% acetic acid, and dried under vacuum. The calcium concentrations used during the loading with 5 pM A23187 were indicated at the bottom of each lane. The numbers indicate the positions of the following proteins: 1 and 2, spectrin subunits; 3, band 3; 4, band 4.1.
brane proteins becomes similar to that observed with control cells (data not shown). These data demonstrate a different susceptibility to calpain digestion of membrane proteins, one of which, the anion transport protein, seems the primary target substrate selectively degraded at very low [Ca”]. This conclusion is strongly supported by the existence of a structural modification of band 3 undetectable in SDS gel electrophoresis and probably representing an early modification of the protein produced by calpain. In fact, in cells of MHS rats, the basal level of phosphorylation of band 3 is approximately two times higher than that in control cells (Fig. 3), in which a similar level of phosphorylation can be induced following loading with increasing Ca2+ concentrations. Maximum increase in 32P incorporation is observed at approximately 20 PM Ca2+ (Fig. 3), a concentration insufficient to promote appreciable degradation of this protein by calpain (see Fig. 2) but sufficient to induce a limited structural modification probably resulting in the unmasking of additional phosphorylation sites. Thus, loading with Ca2+ reproduces in control cells a situation that spontaneously occurs in erythrocytes of MHS rats. A more massive activation of calpain, as it occurs at relatively low concentrations of Ca2+ in cells from hypertensives or at higher concentrations in cells from nor-
PROTEINS
BY CALPAIN
289
motensive rats, causes a decrease in the phosphorylation of band 3 (Fig. 3) due to a progressive and extensive degradation of the protein. Also under these conditions if control cells are preloaded with leupeptin or with anticalpain monoclonal antibody 56.3, the increase in the extent of band 3 phosphorylation is prevented (data not shown). It is interesting to note that the phosphorylation of spectrin is not affected in cells from MNS rats following changes in the intracellular level of Ca2+ but tends to decrease in cells from MHS rats in correlation with the disappearance of the protein band (see Fig. 3). Similar modification in the phosphorylation of band 3 has been observed in erythrocytes from patients carrying essential hypertension (3, 23), suggesting that the alterations in band 3 are common to cells from hypertensives and may represent an early sign of the disease. Degradation of Ca2+-ATPase in Red Cells Enriched with Ca2’ The transmembrane protein Ca2+-ATPase, previously identified as a substrate of calpain (18), has also been investigated for its susceptibility to calpain digestion. Ca2+-ATPase, visualized through its 32P-phosphorylated intermediate stabilized by the presence of La3+ ions, undergoes a slow degradation process when erythrocytes of MNS rats are loaded with increasing concentrations of Ca2+ (Fig. 4). Disappearance of the original Ca2+-ATPase band becomes appreciable only around 30 PM and progressively increases as [Ca2’] reaches values of 100 PM (see also Fig. 5A, open bars). When cells from MHS rats
FIG. 3. Effect of loading with Ca2+ on the protein phosphorylation pattern of erythrocyte membranes from normotensive and hypertensive rats. Erythrocytes from MNS (A) or MHS (B) rats were treated as in Fig. 2 and then the membrane fractions were collected, incubated with labeled [y-32P]ATP, as described under Materials and Methods and submitted to SDS-PAGE. The gels were stained with Coomassie blue, dried, and exposed to autoradiography on a TRIMAX 3M film. The szPlabeled proteins present in each lane were analyzed with a Shimatzu CS-9000 densitometer and the area of each band was calculated. Band 3 (O), spectrin band (0).
290
SALAMINO
A
As shown in Fig. 6A, in cells from MNS rats, the level of calpastatin activity progressively decreases with the increase in the intracellular [Ca2+], whereas the amount of detectable calpain activity progressively increases with a kinetics inversely related to the disappearance of calpastatin. In these pretreated cells, Ca2+-ATPase becomes susceptible to degradation at [Ca”‘] comparable to that required in cells from MHS rats (Fig. 6B). These results further demonstrate that the extent at which intracellular calpain undergoes activation is directly related to the level of calpastatin, thus confirming that this unbalanced calpain/calpastatin ratio (7,9-11) is responsible for the increased protein degradation by calpain in cells from MHS rats at low Ca2+ concentration.
MNS illw 0
1
5
10
50
loo
IICg+ ,pM B
MHS %‘, 0
1
5
10
30
50
loo
[Ca2+1,$A FIG. 4. Effect of an increased intracellular [Ca’+] on the plasma membrane Ca*+-ATPase. Erythrocytes from MNS (A) and MHS (B) rats were loaded with increasing [Ca’+] as described under Materials and Methods. The cells were then lysed, the plasma membranes were collected, and the Ca’+-ATPase was visualized following exposure to [y-32P]ATP in t,he presence of the inhibitor La3+, which stabilizes the aspartyl-phosphate-intermediate (see Materials and Methods). The solubilized membranes were then submitted to PAGE in the presence of sodium dodecyl sulfate. The dried gels were then exposed to autoradiography for 36-48 h.
are loaded with Ca2+ (Fig. 4B), a significant decrease in the Ca2+-ATPase band occurs at 5-10 PM Ca2+, whereas the complete disappearance of the enzyme occurs at higher concentrations of the metal ion. Degradation of Ca2+-ATPase is profoundly reduced in cells preloaded with leupeptin (Fig. 5), suggesting calpain activation as the primary mechanism involved. Further support to this evidence was obtained by loading the cells with an antibody against calpain, shown to prevent expression of the proteinase activity (22). Under these conditions, degradation of Ca2+-ATPase is almost completely abolished in red cells both from MNS and MHS rats. Degradation of the Ca2’-ATPase as a Function Intracellular Level of Calpastatin
ET AL.
of the
The results presented so far (see Fig. 2 and Fig. 4) indicate that the Ca2+ concentrations required to trigger calpain digestion of specific target proteins in normotensive rats are approximately 5-10 times higher than those necessary in cells from hypertensive rats. This discrepancy could be attributed to the virtual absence of calpastatin in erythrocytes from MHS rats (9) and to a proteolytic degradation of calpastatin by activated calpain (24). In order to provide a direct demonstration of the correlation between calpastatin level and calcium concentration necessary to trigger intracellular calpain activity, red cells from MNS rats were incubated for 2 h with a concentration of Ca2+ (5 PM) insufficient to promote any appreciable degradation of membrane proteins (see Fig. 2).
DISCUSSION Human and rat erythrocytes contain an intracellular Ca2+-dependent proteolytic system (25-31) composed of a single type of calpain, namely calpain I (or p), and a large excess of the M, 70 calpastatin form (2), sufficient to maintain the proteinase in an inactive state. Regulation of this proteolytic system can thus be potentially altered under all pathological conditions involving an unbalanced
10
50
100
FIG. 5. Ca’+-ATPase acyl phosphate intermediate assay on erythrocyte membranes from MNS or MHS rats in the presence or in the absence of leupeptin or mAb 56.3. Erythrocytes (70% of hematocrit) from MNS (A) or MHS (B) rats were preloaded with 2 mM leupeptin or with 100 pg/ml of the anticalpain antibody mAb 56.3, obtained as described under Materials and Methods, following the procedure reported by Ropars et al. (18, 19). The cells were then collected, washed with isotonic saline solution, and then loaded with the indicated Ca2+ concentrations in the presence of A23187 as described under Materials and Methods. As control, erythrocytes were submitted to the same treatment with the omission of the proteinase inhibitor or mAb 56.3. The membranes, obtained after lysis of cells, were collected and Ca’+-ATPase was visualized as aspartyl-phosphate-intermediate. The gels, stained with Coomassie blue and dried under vacuum, were exposed to autoradiography. The autoradiographic films were scanned with a Shimatzu CS-9000 and the areas of the bands were calculated. Each bar represents the arithmetical mean of five different experiments. (0) Control, erythrocytes not preloaded with calpain inhibitors; (0) erythrocytes preloaded with leupeptin; @) erythrocytes preloaded with anticalpain monoclonal antibody.
DIGESTION
d
c3
25
5
b2+l,JJM
OF ERYTHROCYTE
0
5
MEMBRANE
10
20
ICa’l,JJM
FIG. 6. Correlation between level of calpastatin and the degradation of Ca*+-ATPase. (A) Erythrocytes from MNS rats were loaded in the presence of A23187 with the indicated [Ca”], as described under Materials and Methods, and then incubated for 2 h at 37°C. The cells were collected by centrifugation at SOOgfor 5 min and lysed in 5 vol of 1 mM EDTA, pH 7.0. Membranes were discarded and aliquots of the soluble fractions were used for the determination of calpain activity (0). Calpastatin activity (u) was assayed on aliquots of these soluble fractions, following heating at 90°C for 3 min. (B) MNS rat erythrocytes were loaded as in A with 5 pM Ca ‘+, in the presence of A23187, and incubated for 2 h at 37°C. The sample was then divided into three aliquots. The first was maintained under the same conditions for others 30 min; the other two aliquots were also incubated for 30 min at 37”C, but their Ca2+ concentrations were raised to 10 and 20 pM Ca*‘, respectively. The membrane fractions were then collected and analyzed for their content in Ca2+-ATPase (dotted bars), as described in the legend to Fig. 4. The Ca*+-ATPase activity was calculated from the areas of the radioactive bands following scanning of the autoradiography. The values are the mean of three different experiments. The amount of Ca*+-ATPase present in plasma membranes before the second treatment (the bar at 5 pM Ca”) was taken as 100%. The undotted bars refer to the Cazf-ATPase levels in cells directly loaded with the indicated [Ca”], without the first loading at 5 pM Cazt for 2 h.
presence of calpain versus calpastatin or vice versa or an abnormal increase in [Ca2’] (32-34). The former condition has been shown to occur in erythrocytes of humans affected with essential hypertension (11, 12), and is even more pronounced in erythrocytes from MHS rats (8, 10). In fact, in red cells from hypertensives, calpain is present in normal amounts, whereas the level of calpastatin is significantly lower than in control cells. Such an unbalanced proteolytic system has been considered to be responsible for the structural and functional alterations described in red cells from hypertensive subjects and from hypertensive animals (l-6). In the present paper we demonstrate that in red cells from MHS rats, calpain is present in a form highly susceptible to activation by Ca2+. Taking advantage of this naturally occurring abnormal biochemical condition, it has been possible to establish the primary target of the intracellular calpain activity in red cells. The transmembrane proteins band 3 and Ca2+-ATPase have been found to undergo structural modifications at [ Ca2+] close to the
PROTEINS
BY CALPAIN
291
physiological range and much lower than that required for degradation of spectrin and band 4.1 (see Figs. 2 and 4). The different Ca2+ requirements observed under in rho conditions for the degradation of various membrane protein substrates could be due to the specificity of the proteinase or to the localization of these proteins that favors accessibility to the proteinase, following the increase in the intracellular [Ca2’]. The different [Ca2+] requirements for degradation of the various protein substrates are in agreement with previous observations by Barrett et al. (35) and Mellgren (35), indicating that degradation of nuclear proteins by calpain II requires 10 times lower [Ca2’], as compared to other substrates (36). Furthermore, we have shown that the intracellular [Ca2+] necessary to express calpain activity in intact red cells correlates with the level of calpastatin. In fact, exposure of cells from normotensive rats to low [Ca”‘] results in the decrease of calpastatin activity and in the increase in the amount of calpain activity directly detectable in crude hemolysates. In red cell hemolysates of MHS rats almost all calpain activity is constitutively detectable due to the absence of calpastatin (9). Thus the altered level of calpastatin could be considered as the biochemical condition that produces an increased rate of Ca2+-ATPase degradation. Taken together these results suggest that alterations in the function of these proteins may represent an early event in red cells from hypertensives and an early marker for the diagnosis and the follow-up of the progression of the disease. Whether similar alterations in protein structure are present in other cell types of the hypertensive animal is still to be established, and will be explored in the future; this however can be an attractive hypothesis due to previous observations (15) indicating the presence in kidney cells of MHS rats of an imbalance in the calpain system similar to that present in red cells. ACKNOWLEDGMENTS This work was supported in part by grants from National Research Council (C.N.R.), Target Project “Prevention and Control Disease Factors,” SP8. Control of cardiovascular diseases (Grant 91.000237, PF 41), Target Project “Biotechnology and Bioinstrumentation,” Sottoprogetto Ingegneria Molecolare e Cellulare, and Target Project “Ingegneria Genetica,” SP4 Malattie Ereditarie.
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OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 298, No. 1, October, pp. 287-292, 1992
Different Susceptibility of Red Cell Membrane to Calpain Degradation Franca Salamino,’ Roberta De Tullio, Edon Melloni, and Sandro Pontremoli Institute
of Biochemistry,
University
of Genoa,
Paola Mengotti,
Proteins
Pier Luigi Viotti,
Genoa, Italy
Received October 3, 1991, and in revised form June 4, 1992
The presence of low levels of calpastatin activity in erythrocytes of hypertensive rats affects regulation of calpain activity so it is highly susceptible to activation within physiological fluctuations in [Ca2+]. Under identical conditions, in red cells of normotensive rats, calpain activation is efficiently controlled by the high levels of calpastatin activity, and a progressive increase in proteinase activity can only be observed in parallel with a decrease in the level of calpastatin. In intact erythrocytes from hypertensive rats exposed to small variations in [Ca2’], degradation of anion transport protein (band 3) and Ca2+-ATPase appears as a primary event indicating that these two transmembrane proteins are probably early recognized as targets of intracellular calpain activity. Furthermore, band 3 protein seems to be structurally modified in erythrocytes from hypertensive rats, as indicated by its increased susceptibility to degradation in the presence of lo-50 I.LM Ca’+. In addition, when exposed to progressive and limited increases in [Ca2+], erythrocytes from hypertensive rats, but not those from normotensive rats, show a high degree of fragility that can be restored to normal values by inhibition of calpain. These results indicate that, within fluctuations in [Ca2’] close to physiological values, regulation of calpain activity is efficiently accomplished in normal erythrocytes but is completely lost in cells from hypertensive animals. Regulation is of critical importance in maintaining normal structural and functional properties of selective red cell membrane and cytoskeletal proteins, among which band 3 and Ca2+-ATPase appear to be the substrates with highest susceptibility to digestion by calpain. o 1992 Academic
Press,
Inc.
i Author to whom correspondence should be addressed. * Abbreviations used: MHS, Milan hypertensive strain; MNS, Milan normotensive strain; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. 0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Red cells from human subjects affected with essential hypertension and from rats of the Milan hypertensive strain (MHS)2 are characterized by a variety of molecular lesions mostly concerning membrane and cytoskeletal proteins (l-8). These alterations have been attributed to a defective control of calpain activity due to the presence of low levels of calpastatin, the natural proteinase inhibitor (9-12). However, no precise demonstration has yet been provided indicating an enhanced activation of calpain in intact red cells of MHS rats, promoted by variations in [Ca2+] too small to induce activation of the proteinase in control cells. The occurrence of these events, as well as their implication in structural modifications of membrane and cytoskeletal proteins, has been investigated under conditions in which variations in the intracellular concentration of Ca2+ have been induced in intact erythrocytes from rats of Milan normotensive (MNS) and hypertensive strains. In the present paper, we report comparative data showing that increased cell fragility, as well as alterations in the molecular structure of band 3 and of Ca2+-ATPase, can be related to an uncontrolled activation of calpain occurring in red cells of hypertensive rats. MATERIALS
AND
METHODS
Adult males from MNS and MHS rats with masses of about 220-250 g were obtained as described (13). Calcium ionophore A23187 and leupeptin were purchased from Sigma Chemical Co. [y-32P]ATP (3000 Ci/ mmol) was obtained from Amersham International. All other chemicals were reagent grade. Acid-denatured globin was prepared as described by Winterhalter et al. (14). In the extracellular medium, due to the absence of chelating agents, the Ca*+ added was considered total Ca2+ concentration. Packed erythrocytes obtained from Loading of erythrocytes with Ca”. MNS and MHS rats (2 ml) and deprived of leukocytes and platelets (16) were suspended in 10 vol of 10 mM Hepes, pH 7.4, containing 5 mM KCl, 140 mM NaCl, and 5 mM glucose. Elevation of intracellular Ca*+ levels was obtained by exposure of the red cells to the ionophore A23187 in the presence of a specified concentration of Ca*‘. To avoid interference by chelating agents, the extracellular medium was prepared without EDTA and the Ca*’ concentration was considered the total 287
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amount of Ca**. Under these conditions, following the report of Kirkpatrick (16), the increase in intracellular free Ca*+ corresponds to approximately 80% of that originally present in the extracellular medium. Aliquots (2 ml) of the erythrocyte suspensions were incubated at 37°C for 30 min with 5 pM A23187 (final concentration) in the absence or in the presence of increasing Ca*+ concentrations and then the cells were collected by centrifugation at SOOgfor 10 min. When indicated, the cells were preloaded with 2 mM leupeptin or anti-calpain monoclonal antibody following the hypotonic dialysis encapsulation procedure described by Ropars et al. (18, 19). Briefly, erythrocytes (2 ml), at 80% hematocrit, were treated with 20 ~1 of a stock solution of 200 mM leupeptin of 20 mg/ml anti-calpain antibody, transferred into a dialysis bag, and dialyzed against 20 ml of 10 mM NaH,PO,-NaHCOs buffer, pH 7.5, containing 20 mM glucose. After 20 min at 4°C the suspension was recovered and resealed by adding 0.1 vol of a stock solution containing 100 mM NaH,P04, 5 mM adenine, 100 mM inosine, 100 mM pyruvate, 100 mM glucose, and 12% NaCl. The suspension was incubated for 30 min at 39°C and then the cells were collected and extensively washed with isotonic saline buffer. With this procedure, using labeled tracers, such as iodinated antibodies or r4C-labeled amino acids, we have calculated that approximately 30-40% of the materials remain entrapped in red cells. As control, erythrocytes were submitted to the same treatment with the omission of the proteinase inhibitor or anti-calpain monoclonal antibody. Phosphor-y&ion of erythrocytes membranes. Erythrocytes, loaded with A23187 in the presence of increasing Ca2+ concentrations, were lysed in 10 vol of 5 mM sodium phosphate buffer, pH 8.0, containing 1 mM EDTA. Erythrocyte membranes were washed extensively with the same buffer without EDTA until the hemoglobin was completely removed. The phosphorylation of erythrocyte membranes was carried out on aliquots (100 gg of proteins) of each sample as described by Pontremoli et al. (3). Assay of the erythrocyte membrane Ca2+-ATPase. Erythrocytes, loaded with A23187 in the presence or absence of Cazf, were lysed in 10 ml of 10 mM Tris/Cl, pH 7.4, containing 1 mM EDTA, and the membranes were prepared as described in (20). The radiolabeled Ca2+ pump was visualized through its phosphorilated intermediate in the presence of 1 mM Ca and 0.1 mM lanthanum nitrate, which stabilizes the aspartyl phosphate intermediate as described in (20). Samples, containing 80 pg of proteins, were submitted to electrophoresis on a polyacrylamide gradient (from 5 to 15%) in the presence of sodium dodecyl sulfate (20). C&pain and calpostatin assay. Erythrocytes, loaded with 5 pM A23187 as described above in the presence of different Ca2+ concentrations, were lysed in 5 vol of a solution of 1 mM EDTA at pH 7.0 and centrifuged at 10,OOOgfor 15 min. Aliquots of the cytosolic fraction (50 ~1) were used for calpain assay as previously described (21). For the assay of calpastatin activity, an aliquot (20 al) of the cytosolic fraction was diluted to 100 ~1, with distilled water, and heated at 90°C for 3 min. The precipitated material was discarded by centrifugation at 80,OOOgfor 3 min and calpastatin activity assayed under standard conditions (21) using 40 (~1of the clear supernatant. Preparation of anticalpain monoclonal antibody. Anticalpain monoclonal antibody (mAb 56.3) was prepared as previously described (22). RESULTS
Effect of Loading with Ca2+ on Degradation of Membrane Proteins in Red Cells from Normotensive and Hypertensive Rats Erythrocytes from MHS rats loaded with ionophore A23187 in the presence of increasing concentrations of Ca2+ show a significantly higher fragility as compared to cells obtained from MNS rats (Fig. 1). This fragility, revealed by the release of hemoglobin into the extracellular
ET AL.
50
100
[ 1
Cap+ pM
FIG. 1. Effect of loading with Ca2+ on the lysis of erythrocytes from normotensive and hypertensive rats. Erythrocytes from MNS or MHS rats were preloaded with or without leupeptin, then incubated with the indicated Ca2+ concentrations, in the presence of A23187, as described under Materials and Methods. The cells were then centrifuged at 8OOg for 10 min and the hemoglobin contents in the clear supernatant were determined by the absorbance at 540 nm. MNS (O), MNS loaded with leupeptin (0). MHS (A), MHS loaded with leupeptin (A).
medium, is profoundly reduced and becomes almost identical to that observed in control cells, when erythrocytes had been previously loaded with leupeptin, a competitive inhibitor of calpain. The Ca2+ dependency and the protective effect of leupeptin suggests that calpain is involved in the membrane damage responsible for the release of hemoglobin. The occurrence of structural modifications of membrane proteins was then comparatively investigated in red cells of both animal strains. As shown in Fig. 2, the electrophoretic pattern of the membrane-associated proteins from MNS rat erythrocytes, loaded with increasing [Ca2’] from 0.5 to 50 PM, reveals no appreciable structural modification. On the contrary, under identical conditions, membrane proteins of cells from MHS rats undergo a significant proteolytic degradation. At concentrations of Ca2+ between 1 and 5 PM, the intensity of the band corresponding to the anion transport protein progressively decreases and completely disappears at higher concentration of this metal ion. Also spectrin and 4.1 protein are progressively degraded, but the disappearance of the corresponding bands is much slower and can be observed only at the highest concentrations of Ca2+ used. The appearance in the electrophoretic pattern of new protein bands with low M, (Fig. Z), is probably due to the formation under these conditions of degradation products. If red cells from MHS rats had been preloaded with leupeptin or with the anticalpain monoclonal antibody 56.3, shown to prevent expression of the proteinase activity (22), and then exposed to concentrations of Ca2+ from 0.5 to 50 PM, the electrophoretic pattern of mem-
DIGESTION
OF ERYTHROCYTE
MNS
MEMBRANE
MHS
;. 3
0
0.5
1
5
10
20
50
0
0.5
1
5
10
20
50
[Ca*+] yM FIG. 2. Effect of loading with CaZf on the SDS-PAGE protein pattern of erythrocytes membranes from normotensive and hypertensive rats. Erythrocytes from MNS (A) or MHS (B) rats, loaded with Ca*+ and A23187 as reported under Materials and Methods, were lysed in 5 mM sodium phosphate buffer, pH 8.0 (5P8), containing 1 mM EDTA and centrifuged at 10,OOOgfor 10 min; the membranes were collected and washed in 5P8 containing 1 mM EDTA until the hemoglobin was completely removed. Samples (50 pg) of each membrane preparation were submitted to SDS-PAGE according to Fairbanks (37). The gels were stained with Coomassie blue, destained by free diffusion in 7% acetic acid, and dried under vacuum. The calcium concentrations used during the loading with 5 pM A23187 were indicated at the bottom of each lane. The numbers indicate the positions of the following proteins: 1 and 2, spectrin subunits; 3, band 3; 4, band 4.1.
brane proteins becomes similar to that observed with control cells (data not shown). These data demonstrate a different susceptibility to calpain digestion of membrane proteins, one of which, the anion transport protein, seems the primary target substrate selectively degraded at very low [Ca”]. This conclusion is strongly supported by the existence of a structural modification of band 3 undetectable in SDS gel electrophoresis and probably representing an early modification of the protein produced by calpain. In fact, in cells of MHS rats, the basal level of phosphorylation of band 3 is approximately two times higher than that in control cells (Fig. 3), in which a similar level of phosphorylation can be induced following loading with increasing Ca2+ concentrations. Maximum increase in 32P incorporation is observed at approximately 20 PM Ca2+ (Fig. 3), a concentration insufficient to promote appreciable degradation of this protein by calpain (see Fig. 2) but sufficient to induce a limited structural modification probably resulting in the unmasking of additional phosphorylation sites. Thus, loading with Ca2+ reproduces in control cells a situation that spontaneously occurs in erythrocytes of MHS rats. A more massive activation of calpain, as it occurs at relatively low concentrations of Ca2+ in cells from hypertensives or at higher concentrations in cells from nor-
PROTEINS
BY CALPAIN
289
motensive rats, causes a decrease in the phosphorylation of band 3 (Fig. 3) due to a progressive and extensive degradation of the protein. Also under these conditions if control cells are preloaded with leupeptin or with anticalpain monoclonal antibody 56.3, the increase in the extent of band 3 phosphorylation is prevented (data not shown). It is interesting to note that the phosphorylation of spectrin is not affected in cells from MNS rats following changes in the intracellular level of Ca2+ but tends to decrease in cells from MHS rats in correlation with the disappearance of the protein band (see Fig. 3). Similar modification in the phosphorylation of band 3 has been observed in erythrocytes from patients carrying essential hypertension (3, 23), suggesting that the alterations in band 3 are common to cells from hypertensives and may represent an early sign of the disease. Degradation of Ca2+-ATPase in Red Cells Enriched with Ca2’ The transmembrane protein Ca2+-ATPase, previously identified as a substrate of calpain (18), has also been investigated for its susceptibility to calpain digestion. Ca2+-ATPase, visualized through its 32P-phosphorylated intermediate stabilized by the presence of La3+ ions, undergoes a slow degradation process when erythrocytes of MNS rats are loaded with increasing concentrations of Ca2+ (Fig. 4). Disappearance of the original Ca2+-ATPase band becomes appreciable only around 30 PM and progressively increases as [Ca2’] reaches values of 100 PM (see also Fig. 5A, open bars). When cells from MHS rats
FIG. 3. Effect of loading with Ca2+ on the protein phosphorylation pattern of erythrocyte membranes from normotensive and hypertensive rats. Erythrocytes from MNS (A) or MHS (B) rats were treated as in Fig. 2 and then the membrane fractions were collected, incubated with labeled [y-32P]ATP, as described under Materials and Methods and submitted to SDS-PAGE. The gels were stained with Coomassie blue, dried, and exposed to autoradiography on a TRIMAX 3M film. The szPlabeled proteins present in each lane were analyzed with a Shimatzu CS-9000 densitometer and the area of each band was calculated. Band 3 (O), spectrin band (0).
290
SALAMINO
A
As shown in Fig. 6A, in cells from MNS rats, the level of calpastatin activity progressively decreases with the increase in the intracellular [Ca2+], whereas the amount of detectable calpain activity progressively increases with a kinetics inversely related to the disappearance of calpastatin. In these pretreated cells, Ca2+-ATPase becomes susceptible to degradation at [Ca”‘] comparable to that required in cells from MHS rats (Fig. 6B). These results further demonstrate that the extent at which intracellular calpain undergoes activation is directly related to the level of calpastatin, thus confirming that this unbalanced calpain/calpastatin ratio (7,9-11) is responsible for the increased protein degradation by calpain in cells from MHS rats at low Ca2+ concentration.
MNS illw 0
1
5
10
50
loo
IICg+ ,pM B
MHS %‘, 0
1
5
10
30
50
loo
[Ca2+1,$A FIG. 4. Effect of an increased intracellular [Ca’+] on the plasma membrane Ca*+-ATPase. Erythrocytes from MNS (A) and MHS (B) rats were loaded with increasing [Ca’+] as described under Materials and Methods. The cells were then lysed, the plasma membranes were collected, and the Ca’+-ATPase was visualized following exposure to [y-32P]ATP in t,he presence of the inhibitor La3+, which stabilizes the aspartyl-phosphate-intermediate (see Materials and Methods). The solubilized membranes were then submitted to PAGE in the presence of sodium dodecyl sulfate. The dried gels were then exposed to autoradiography for 36-48 h.
are loaded with Ca2+ (Fig. 4B), a significant decrease in the Ca2+-ATPase band occurs at 5-10 PM Ca2+, whereas the complete disappearance of the enzyme occurs at higher concentrations of the metal ion. Degradation of Ca2+-ATPase is profoundly reduced in cells preloaded with leupeptin (Fig. 5), suggesting calpain activation as the primary mechanism involved. Further support to this evidence was obtained by loading the cells with an antibody against calpain, shown to prevent expression of the proteinase activity (22). Under these conditions, degradation of Ca2+-ATPase is almost completely abolished in red cells both from MNS and MHS rats. Degradation of the Ca2’-ATPase as a Function Intracellular Level of Calpastatin
ET AL.
of the
The results presented so far (see Fig. 2 and Fig. 4) indicate that the Ca2+ concentrations required to trigger calpain digestion of specific target proteins in normotensive rats are approximately 5-10 times higher than those necessary in cells from hypertensive rats. This discrepancy could be attributed to the virtual absence of calpastatin in erythrocytes from MHS rats (9) and to a proteolytic degradation of calpastatin by activated calpain (24). In order to provide a direct demonstration of the correlation between calpastatin level and calcium concentration necessary to trigger intracellular calpain activity, red cells from MNS rats were incubated for 2 h with a concentration of Ca2+ (5 PM) insufficient to promote any appreciable degradation of membrane proteins (see Fig. 2).
DISCUSSION Human and rat erythrocytes contain an intracellular Ca2+-dependent proteolytic system (25-31) composed of a single type of calpain, namely calpain I (or p), and a large excess of the M, 70 calpastatin form (2), sufficient to maintain the proteinase in an inactive state. Regulation of this proteolytic system can thus be potentially altered under all pathological conditions involving an unbalanced
10
50
100
FIG. 5. Ca’+-ATPase acyl phosphate intermediate assay on erythrocyte membranes from MNS or MHS rats in the presence or in the absence of leupeptin or mAb 56.3. Erythrocytes (70% of hematocrit) from MNS (A) or MHS (B) rats were preloaded with 2 mM leupeptin or with 100 pg/ml of the anticalpain antibody mAb 56.3, obtained as described under Materials and Methods, following the procedure reported by Ropars et al. (18, 19). The cells were then collected, washed with isotonic saline solution, and then loaded with the indicated Ca2+ concentrations in the presence of A23187 as described under Materials and Methods. As control, erythrocytes were submitted to the same treatment with the omission of the proteinase inhibitor or mAb 56.3. The membranes, obtained after lysis of cells, were collected and Ca’+-ATPase was visualized as aspartyl-phosphate-intermediate. The gels, stained with Coomassie blue and dried under vacuum, were exposed to autoradiography. The autoradiographic films were scanned with a Shimatzu CS-9000 and the areas of the bands were calculated. Each bar represents the arithmetical mean of five different experiments. (0) Control, erythrocytes not preloaded with calpain inhibitors; (0) erythrocytes preloaded with leupeptin; @) erythrocytes preloaded with anticalpain monoclonal antibody.
DIGESTION
d
c3
25
5
b2+l,JJM
OF ERYTHROCYTE
0
5
MEMBRANE
10
20
ICa’l,JJM
FIG. 6. Correlation between level of calpastatin and the degradation of Ca*+-ATPase. (A) Erythrocytes from MNS rats were loaded in the presence of A23187 with the indicated [Ca”], as described under Materials and Methods, and then incubated for 2 h at 37°C. The cells were collected by centrifugation at SOOgfor 5 min and lysed in 5 vol of 1 mM EDTA, pH 7.0. Membranes were discarded and aliquots of the soluble fractions were used for the determination of calpain activity (0). Calpastatin activity (u) was assayed on aliquots of these soluble fractions, following heating at 90°C for 3 min. (B) MNS rat erythrocytes were loaded as in A with 5 pM Ca ‘+, in the presence of A23187, and incubated for 2 h at 37°C. The sample was then divided into three aliquots. The first was maintained under the same conditions for others 30 min; the other two aliquots were also incubated for 30 min at 37”C, but their Ca2+ concentrations were raised to 10 and 20 pM Ca*‘, respectively. The membrane fractions were then collected and analyzed for their content in Ca2+-ATPase (dotted bars), as described in the legend to Fig. 4. The Ca*+-ATPase activity was calculated from the areas of the radioactive bands following scanning of the autoradiography. The values are the mean of three different experiments. The amount of Ca*+-ATPase present in plasma membranes before the second treatment (the bar at 5 pM Ca”) was taken as 100%. The undotted bars refer to the Cazf-ATPase levels in cells directly loaded with the indicated [Ca”], without the first loading at 5 pM Cazt for 2 h.
presence of calpain versus calpastatin or vice versa or an abnormal increase in [Ca2’] (32-34). The former condition has been shown to occur in erythrocytes of humans affected with essential hypertension (11, 12), and is even more pronounced in erythrocytes from MHS rats (8, 10). In fact, in red cells from hypertensives, calpain is present in normal amounts, whereas the level of calpastatin is significantly lower than in control cells. Such an unbalanced proteolytic system has been considered to be responsible for the structural and functional alterations described in red cells from hypertensive subjects and from hypertensive animals (l-6). In the present paper we demonstrate that in red cells from MHS rats, calpain is present in a form highly susceptible to activation by Ca2+. Taking advantage of this naturally occurring abnormal biochemical condition, it has been possible to establish the primary target of the intracellular calpain activity in red cells. The transmembrane proteins band 3 and Ca2+-ATPase have been found to undergo structural modifications at [ Ca2+] close to the
PROTEINS
BY CALPAIN
291
physiological range and much lower than that required for degradation of spectrin and band 4.1 (see Figs. 2 and 4). The different Ca2+ requirements observed under in rho conditions for the degradation of various membrane protein substrates could be due to the specificity of the proteinase or to the localization of these proteins that favors accessibility to the proteinase, following the increase in the intracellular [Ca2’]. The different [Ca2+] requirements for degradation of the various protein substrates are in agreement with previous observations by Barrett et al. (35) and Mellgren (35), indicating that degradation of nuclear proteins by calpain II requires 10 times lower [Ca2’], as compared to other substrates (36). Furthermore, we have shown that the intracellular [Ca2+] necessary to express calpain activity in intact red cells correlates with the level of calpastatin. In fact, exposure of cells from normotensive rats to low [Ca”‘] results in the decrease of calpastatin activity and in the increase in the amount of calpain activity directly detectable in crude hemolysates. In red cell hemolysates of MHS rats almost all calpain activity is constitutively detectable due to the absence of calpastatin (9). Thus the altered level of calpastatin could be considered as the biochemical condition that produces an increased rate of Ca2+-ATPase degradation. Taken together these results suggest that alterations in the function of these proteins may represent an early event in red cells from hypertensives and an early marker for the diagnosis and the follow-up of the progression of the disease. Whether similar alterations in protein structure are present in other cell types of the hypertensive animal is still to be established, and will be explored in the future; this however can be an attractive hypothesis due to previous observations (15) indicating the presence in kidney cells of MHS rats of an imbalance in the calpain system similar to that present in red cells. ACKNOWLEDGMENTS This work was supported in part by grants from National Research Council (C.N.R.), Target Project “Prevention and Control Disease Factors,” SP8. Control of cardiovascular diseases (Grant 91.000237, PF 41), Target Project “Biotechnology and Bioinstrumentation,” Sottoprogetto Ingegneria Molecolare e Cellulare, and Target Project “Ingegneria Genetica,” SP4 Malattie Ereditarie.
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