FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2005313237> ?p ?o ?g. }

• W2005313237 endingPage "2665" @default.
• W2005313237 startingPage "2656" @default.
• W2005313237 abstract "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. 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. The regulatory mechanism of calpain activity is primarily based on the physiological control of intracellular Ca2+ homeostasis, predominantly exerted by the plasma membrane Ca2+-ATPase and, more specifically, by the expression in all mammalian cells of a natural protein inhibitor named calpastatin (1Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (779) Google Scholar, 2Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2329) Google Scholar, 3Murachi T. Biochem. Int. 1989; 18: 263-294PubMed Google Scholar, 4Saido T.C. Sorimachi A. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (616) Google Scholar, 5Huang Y. Wang K.K.W. Trends Mol. Med. 2001; 7: 355-362Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 6Goll D.E. Thompson V.F. Taylor R.G. Zalewska T. Bioessays. 1992; 14: 549-556Crossref PubMed Scopus (185) Google Scholar, 7Barnoy S. Kosower N.S. Int. J. Biochem. Cell Biol. 2006; 39: 253-261Crossref PubMed Scopus (10) Google Scholar).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 (2Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2329) Google Scholar, 3Murachi T. Biochem. Int. 1989; 18: 263-294PubMed Google Scholar, 4Saido T.C. Sorimachi A. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (616) Google Scholar, 8De Tullio R. Averna M. Salamino F. Pontremoli S. Melloni E. FEBS Lett. 2000; 475: 17-21Crossref PubMed Scopus (31) Google Scholar, 9De Tullio R. Sparatore B. Salamino F. Melloni E. Pontremoli S. FEBS Lett. 1998; 422: 113-117Crossref PubMed Scopus (41) Google Scholar, 10Betts R. Weinsheimer S. Blouse G.E. Anagli J. J. Biol. Chem. 2003; 278: 7800-7809Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 11Betts R. Anagli J. Biochemistry. 2004; 43: 2596-2604Crossref PubMed Scopus (20) Google Scholar). 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 (8De Tullio R. Averna M. Salamino F. Pontremoli S. Melloni E. FEBS Lett. 2000; 475: 17-21Crossref PubMed Scopus (31) Google Scholar, 12Tompa P. Mucsi Z. Orosz G. Friedrich P. J. Biol. Chem. 2002; 277: 9022-9026Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 13Averna M. De Tullio R. Salamino F. Minafra R. Pontremoli S. Melloni E. J. Biol. Chem. 2001; 276: 38426-38432Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 14Sorimachi Y. Harada K. Saido T.C. Ono T. Kawashima S. Yoshida K. J. Biochem. 1997; 122: 743-748Crossref PubMed Scopus (64) Google Scholar, 15Blomgren K. Hallin U. Andersson A.L. Puka-Sundvall M. Bahr B.A. McRae A. Saido T.C. Kawashima S. Hagberg H. J. Biol. Chem. 1999; 274: 14046-14052Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 16Nakamura M. Inomata M. Imajoh S. Suzuki K. Kawashima S. Biochemistry. 1989; 28: 449-455Crossref PubMed Scopus (70) Google Scholar). Conservative fragmentation has been attributed to μ-calpain activity, and degradation to inactive peptides attributed to digestion by m-calpain (8De Tullio R. Averna M. Salamino F. Pontremoli S. Melloni E. FEBS Lett. 2000; 475: 17-21Crossref PubMed Scopus (31) Google Scholar, 16Nakamura M. Inomata M. Imajoh S. Suzuki K. Kawashima S. Biochemistry. 1989; 28: 449-455Crossref PubMed Scopus (70) Google Scholar).Despite this information and the numerous reports on the structural properties of calpain (17Strobl S. Fernandez-Catalan C. Brown M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 588-592Crossref PubMed Scopus (314) Google Scholar, 18Brandenburg K. Harris F. Dennison S. Seydel U. Phoenix D. Eur. J. Biochem. 2002; 269: 5414-5422Crossref PubMed Scopus (35) Google Scholar, 19Dainese E. Minafra R. Sabatucci A. Vachette P. Melloni E. Cozzani I. J. Biol. Chem. 2002; 277: 40296-40301Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 20Dutt P. Spriggs C.N. Davies P.L. Jia Z. Elce J.S. Biochem. J. 2002; 367: 263-269Crossref PubMed Scopus (30) Google Scholar, 21Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 22Moldoveanu T. Campbell R.L. Cuerrier O. Davies P.L. J. Mol. Biol. 2004; 343: 1313-1326Crossref PubMed Scopus (76) Google Scholar), including the presence of different isoforms in various tissues (23Sorimachi H. Imajoh-Ohmi S. Emori Y. Kawasaki H. Ohno S. Minami Y. Suzuki K. J. Biol. Chem. 1989; 264: 20106-20111Abstract Full Text PDF PubMed Google Scholar, 24Sorimachi H. Ishiura S. Suzuki K. J. Biol. Chem. 1993; 268: 19476-19482Abstract Full Text PDF PubMed Google Scholar, 25Kinbara K. Sorimachi H. Ishiura S. Suzuki R. Biochem. Pharmacol. 1998; 56: 415-420Crossref PubMed Scopus (41) Google Scholar, 26Nakajima T. Fukiage C. Azuma M. Ma H. Shearer T.R. Biochim. Biophys. Acta. 2001; 1519: 55-64Crossref PubMed Scopus (33) Google Scholar, 27Duguez S. Bartoli M. Richard I. FEBS J. 2006; 273: 3427-3436Crossref PubMed Scopus (99) Google Scholar), the biological function of this protease and the efficiency of its “in vivo” intracellular regulation remain uncertain.It is often suggested (28Nixon R.A. Saito K.I. Grynspan F. Griffin W.R. Katayama S. Honda T. Mohan P.S. Shea T.B. Beermann M. Ann. N. Y. Acad. Sci. 1994; 747: 77-91Crossref PubMed Scopus (239) Google Scholar, 29Banik N.L. Matzelle D.C. Gantt-Wilford G. Osborne A. Hogan E.L. Brain Res. 1997; 752: 301-306Crossref PubMed Scopus (106) Google Scholar, 30Tsuji T. Shimohama S. Kimura J. Shimizu K. Neurosci Lett. 1998; 248: 109-112Crossref PubMed Scopus (81) Google Scholar, 31Shields D.C. Schaecher K.E. Saido T.C. Banik N.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11486-11491Crossref PubMed Scopus (147) Google Scholar, 32Rami A. Neurobiol Dis. 2003; 13: 75-88Crossref PubMed Scopus (148) Google Scholar, 33Yamashima T. Cell Calcium. 2004; 36: 285-293Crossref PubMed Scopus (192) Google Scholar, 34Higuchi M. Tomioka M. Takano J. Shirotani K. Iwata N. Masumoto H. Maki M. Itohara S. Saido T.C. J. Biol. Chem. 2005; 280: 15229-15237Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) that in brain, calpain is directly implicated in neurodegeneration and neuronal cell death as a consequence of a massive Ca2+ 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 (35Ray S.K. Hogan E.L. Banik N.L. Brain Res. Brain Res. Rev. 2003; 42: 169-185Crossref PubMed Scopus (168) Google Scholar, 36Kawamura M. Nakajima W. Ishida A. Ohmura A. Miura S. Takada G. Brain Res. 2005; 1037: 59-69Crossref PubMed Scopus (64) Google Scholar, 37Yoshikawa Y. Hagihara H. Ohga Y. Nakajima-Takenaka C. Murata K.Y. Taniguchi S. Takaki M. Am. J. Physiol. Heart Circ. Physiol. 2005; 288: 1690-1698Crossref PubMed Scopus (43) Google Scholar, 38Wang K.K.W. Nath R. Posner A. Raser K.J. Buroker-Kilgore M. Hajimohammadreza I. Probert Jr., A.W. Marcoux F. W Ye Q. Takano E. Hatanaka M. Maki M. Caner H. Collins J.L. Fergus A. Lee K.S. Lunney E.A. Hays S.J. Yuen P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6687-6692Crossref PubMed Scopus (255) Google Scholar, 39Squier M.K. Sehnert A.J. Sellins K.S. Malkinson A.M. Takano E. Cohen J.J. J. Cell. Physiol. 1999; 178: 311-319Crossref PubMed Scopus (116) Google Scholar, 40Limaye P.B. Bhave V.S. Palkar P.S. Apte U.M. Sawant S.P. Yu S. Latendresse J.R. Reddy J.K. Mehendale H.M. Hepatology. 2006; 44: 379-388Crossref PubMed Scopus (46) Google Scholar). 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 (31Shields D.C. Schaecher K.E. Saido T.C. Banik N.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11486-11491Crossref PubMed Scopus (147) Google Scholar). It must also be considered that the severe and irreversible cell damage observed under these pathological conditions occurs in the presence of a massive Ca2+ 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 Ca2+ 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) 2The abbreviations used are: NMS, normotensive Milan strain rats; HMS, hypertensive Milan strain rats; CI-1, calpain inhibitor 1; PM, plasma membrane; HSD, high salt (sodium) diet; mAb, monoclonal antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 2The abbreviations used are: NMS, normotensive Milan strain rats; HMS, hypertensive Milan strain rats; CI-1, calpain inhibitor 1; PM, plasma membrane; HSD, high salt (sodium) diet; mAb, monoclonal antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. with a high salt (sodium) diet (HSD) known to promote elevation in blood pressure in response to a mild increase in intracellular free [Ca2+] in vascular smooth muscle, via an alteration in the Na+/Ca2+ exchanger (41Aviv A. Am. J. Hypertens. 1996; 9: 703-707Crossref PubMed Scopus (27) Google Scholar, 42Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Crossref PubMed Scopus (1443) Google Scholar, 43Iwamoto T. Kita S. Zhang J. Blaustein M. Arai Y. Yoshida S. Wakimoto K. Komuro I. Katsuragi T. Nat. Med. 2004; 10: 1193-1199Crossref PubMed Scopus (231) Google Scholar). A role of the exchanger in the alteration of calcium homeostasis in brain has been previously suggested (44Zeitz O. Maass A.E. Van Nguyen P. Hensmann G. Kogler H. Moller K. Hasenfuss G. Janssen P.M.L. Circ. Res. 2002; 90: 988-995Crossref PubMed Scopus (84) Google Scholar, 45Li S. Jiang Q. Stys P.K. J. Neurophysiol. 2000; 84: 1116-1119Crossref PubMed Scopus (111) Google Scholar). To amplify the effects of this treatment we have exposed hypertensive rats (HMS) of the same Milan strain (46Bianchi G. Ferrari P. Berber B.R. de Jong W Handbook of Hypertension. 4. Elsevier, 1984: 328-349Google Scholar) to the same diet, because these animals are characterized by an unbalanced calpain-calpastatin system in heart, kidney, and erythrocytes (13Averna M. De Tullio R. Salamino F. Minafra R. Pontremoli S. Melloni E. J. Biol. Chem. 2001; 276: 38426-38432Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), probably due to a genetically determined deregulation of intracellular Ca2+ homeostasis.In the present study, we report that after 2 weeks of HSD treatment, as expected, [Ca2+]i increases in the aorta and also in brain cells of NMS rats. These modifications induced upon HSD treatment are more pronounced in the brain of HMS rats, probably because of a constitutively slightly elevated basal level of [Ca2+]i.Thus, on the basis of these differences, both animals represent appropriate models to study in vivo the effects of the increase in [Ca2+]i on the calpain-calpastatin system. This relationship has been explored on the basis of: (i) changes in the level of calpain activity during HSD treatment; (ii) appearance of the activated calpain form; (iii) degradation of calpain protein substrate such as plasma membrane Ca2+-ATPase and fodrin; and (iv) levels and molecular properties of calpastatin. The results obtained provide a new understanding of how the activity of the calpain-calpastatin system is regulated in vivo to prevent irreversible cell damage.EXPERIMENTAL PROCEDURESMaterials—Leupeptin, aprotinin, calpain inhibitor 1 (CI-1), monoclonal anti-fodrin antibody, and monoclonal anti-m-calpain (Domain III/IV) were purchased from Sigma. Calpain inhibitor PD151746 and 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF) were obtained from Calbiochem. [γ-32P]ATP was purchased from Amersham Biosciences.Human erythrocyte calpain was purified and assayed as reported previously (47Michetti M. Salamino F. Tedesco I. Averna M. Minafra R. Melloni E. Pontremoli S. FEBS Lett. 1996; 392: 11-15Crossref PubMed Scopus (63) Google Scholar). One unit of calpain activity is defined as the amount causing the production of 1 nmol of acid-soluble NH2 groups per hour under the conditions reported previously (48Pontremoli S. Melloni E. Viotti P.L. Michetti M. Salamino F. Horecker B.L. Arch. Biochem. Biophys. 1991; 288: 646-652Crossref PubMed Scopus (51) Google Scholar). Calcium Green-1/AM was purchased from Molecular Probes.Animals—To induce experimental hypertension, adult male normotensive (NMS) and hypertensive (HMS) Milan strain rats (46Bianchi G. Ferrari P. Berber B.R. de Jong W Handbook of Hypertension. 4. Elsevier, 1984: 328-349Google Scholar) of about 60 days old were housed under controlled conditions (22 °C ± 1; humidity 50% ± 5%; lighting 8–20 h), were fed on a standard rat chow, and drank ad libitum NaCl dissolved in tap water at a concentration of 10 g/liter for 15, 30, or 90 days. Each rat received ∼0.7 g of NaCl every day. Alternatively, 25 μm CI-1 was dissolved in tap water in the presence or in the absence of 10 g/liter NaCl and administrated to NMS and HMS rats at the beginning of the experiment for 2 or 4 weeks. Each rat received ∼0.7 mg of CI-1 every day. Experiments were conducted in accordance with the institutional ethical guidelines. During the course of the experiments, no appreciable changes were observed either in food consumption or body weight.Systolic blood pressure was measured by tail-cuff plethysmography (W + W Electronic, BP recorder 8005) on preheated rats (37 °C) following the procedure originally described by Byrom and Wilson (49Byrom F.B. Wilson C.A. J. Physiol. (Lond.). 1938; 93: 301-304Crossref Scopus (102) Google Scholar). Experiments were performed using five animals for each treatment.Ca2+ Imaging—NMS and HMS rats untreated and treated with HSD (high salt diet) for 2 weeks, were sacrificed by decapitation. Brains were immediately collected, and the tissue was dispersed by gently squeezing in 10 mm oxygenated HEPES containing 0.14 m NaCl, 5 mm KCl, 5 mm glucose, 1 mm MgCl2, 1 mm CaCl2 and 1% bovine serum albumin, pH 7.4 (buffer A). The suspensions were then filtered trough a gauze, and the tissue fragments were incubated at 37 °C for 30 min with 15 μm calcium green 1-AM (50Martínez-Zaguilán R. Parnami G. Lynch R.M. Cell Calcium. 1996; 19: 337-339Crossref PubMed Scopus (45) Google Scholar). Cells were then washed with buffer, A to remove the flourophore excess, and fluorescence was detected with a Bio-Rad MRC 1024 confocal microscope, connected to a Nikon Diaphot 200 microscope equipped with an N.A. 0.75 objective (×40).Thoracic aorta were rapidly excised from the same animals, placed in oxygenated buffer A and stripped of connective tissue. Arterial segments (0.2 × 0.5 cm) were incubated in buffer A with 15 μm calcium green 1-AM. After 30 min at 37 °C, the dye excess was removed by several washes in Buffer A and dye-loaded arteries, prepared as described in Refs. 43Iwamoto T. Kita S. Zhang J. Blaustein M. Arai Y. Yoshida S. Wakimoto K. Komuro I. Katsuragi T. Nat. Med. 2004; 10: 1193-1199Crossref PubMed Scopus (231) Google Scholar and 51Zhang J. Wier W.G. Blaustein M.P. Am. J. Physiol. 2002; 283: H2692-H2705Crossref Scopus (38) Google Scholar, were imaged by means of a Bio-Rad MRC 1024 confocal microscope connected to a Nikon Diaphot 200 microscope equipped with N.A. 0.75 objective (×40).The fluorescence intensity in each collected image was quantified using LaserPix software (Bio-Rad). Each image was divided in ten sections, and the fluorescence value of each part was determined and the arithmetical mean was evaluated. The mean intensity value for each animal tissue utilized for these were calculated from the analysis of ten different images. Variation of the values was taken as an indication of corresponding changes in [Ca2+]i.Determination of Ca2+-ATPase Activity in Rat Brain—NMS and HMS rats were sacrificed by decapitation, the brains were rapidly removed, quickly frozen in liquid nitrogen, and stored at -80 °C. To detect both plasma membrane and endoplasmic reticulum Ca2+-ATPase activities, aliquots (0.3 g) were minced and re-suspended in 10 volumes of ice-cold 50 mm Tris-HCl buffer, pH 7.4 containing 0.5 mm 2-mercaptoethanol, 0.1 mg/ml leupeptin, 10 μg/ml aprotinin, and 2 mm AEBSF. The samples were homogenized in a Potter-Elvehjem homogenizer and lysed by sonication, and the particulate material was collected by centrifugation (100,000 × g for 10 min). Membranes were washed, and Ca2+-ATPase activity was measured as described (52Wang K.K.W. Villalobo A. Roufogalis B.D. Arch. Biochem. Biophys. 1988; 260: 696-704Crossref PubMed Scopus (72) Google Scholar).Separation of Calpastatin Forms by Ion-exchange Chromatography—Freshly collected brains (2 g) from NMS and HMS rats were minced, homogenized in a Potter-Elvehjem homogenizer, and lysed by sonication in 3 volumes of chilled 50 mm sodium borate buffer, pH 7.5, containing 1 mm EDTA, 0.5 mm 2-mercaptoethanol, 0.1 mg/ml leupeptin, 10 μg/ml aprotinin, and 2 mm AEBSF (buffer B). The particulate material was discarded by centrifugation (100,000 × g for 10 min), the clear supernatant (crude extract) was heated at 100 °C for 3 min, and centrifuged at 100,000 × g for 10 min. The soluble material (heated extract) was collected and loaded onto a Source 15 Q (Amersham Biosciences) column (1.5 × 3 cm) equilibrated in 50 mm sodium borate buffer, pH 7.5, containing 0.1 mm EDTA and 0.5 mm 2-mercaptoethanol. The adsorbed proteins were eluted with a linear gradient (40 ml) 0–0.35 m NaCl and collected in 1-ml fractions. Calpastatin activity was measured on aliquots of each fraction, as previously described (48Pontremoli S. Melloni E. Viotti P.L. Michetti M. Salamino F. Horecker B.L. Arch. Biochem. Biophys. 1991; 288: 646-652Crossref PubMed Scopus (51) Google Scholar). One unit of calpastatin activity is defined as the amount of inhibitor required to inhibit one unit of human erythrocyte calpain.Separation of Calpain Isoforms by Ion-exchange Chromatography—Freshly collected brains (2 g) from NMS and HMS rats were minced, homogenized in a Potter-Elvehjem homogenizer and lysed by sonication in 3 volumes of chilled buffer B without proteases inhibitors. Crude extracts prepared as described above were collected and loaded onto a Source 15 Q under the same conditions described for calpastatin ion-exchange chromatography. Calpain activity was assayed as reported (47Michetti M. Salamino F. Tedesco I. Averna M. Minafra R. Melloni E. Pontremoli S. FEBS Lett. 1996; 392: 11-15Crossref PubMed Scopus (63) Google Scholar).Identification of Rat Brain Calpain and Calpastatin Species by Immunoblot Analysis—Crude extracts from brains (50 μg) of NMS and HMS rats prepared as described above, underwent SDS-polyacrylamide gel electrophoresis (12%). Proteins were then transferred to a nitrocellulose membrane (Bio-Rad) by electroblotting. Membranes were probed with anti-μ-calpain monoclonal antibody 56.3 (53Pontremoli S. Melloni E. Damiani G. Salamino F. Sparatore B. Michetti M. Horecker B.L. J. Biol. Chem. 1988; 263: 1915-1919Abstract Full Text PDF PubMed Google Scholar) or anti-m-calpain (Domain III/IV) monoclonal antibody (Sigma) or with anti-calpastatin monoclonal antibody 35.23 (54Melloni E. De Tullio R. Averna M. Tedesco I. Salamino F. Sparatore B. Pontremoli S. FEBS Lett. 1998; 431: 55-58Crossref PubMed Scopus (34) Google Scholar), followed by a peroxidase-conjugated secondary antibody as described (55Palejwala S. Goldsmith L.T. Proc. Natl. Acad. Sci. 1992; 89: 4202-4206Crossref PubMed Scopus (42) Google Scholar) and then developed with an ECL detection system (Amersham Biosciences).Separation and Quantification of Calpastatin Species in Rat Brain, following SDS-Polyacrylamide Gel Electrophoresis—Aliquots (1.5 mg of protein) of brain extract obtained from NMS and HMS were submitted to SDS-polyacrylamide gel electrophoresis (12%) divided in ten lanes. Calpastatin species were identified following protein extraction from the gel, as previously described (13Averna M. De Tullio R. Salamino F. Minafra R. Pontremoli S. Melloni E. J. Biol. Chem. 2001; 276: 38426-38432Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Calpastatin activity was measured as described in Ref. 48Pontremoli S. Melloni E. Viotti P.L. Michetti M. Salamino F. Horecker B.L. Arch. Biochem. Biophys. 1991; 288: 646-652Crossref PubMed Scopus (51) Google Scholar.Determination of Total Calpain Activity in Brain Crude Extracts from NMS and HMS Rats—Crude extracts from brains of NMS and HMS rats (25 mg) untreated and treated with HSD for 4 weeks were prepared, as described above, without protease inhibitors. Calpain activity was measured directly on samples of each crude extract in the usual assay mixture containing 1 mm CaCl2 (56Melloni E. Sparatore B. Salamino F. Michetti M. Pontremoli S. Biochem. Biophys. Res. Commun. 1982; 106: 731-740Crossref PubMed Scopus (92) Google Scholar).Alternatively, to determine total calpain activity, 25 mg of the same samples were immediately loaded onto a Phenyl-Sepharose column (Amersham Biosciences). This chromatographic step separates calpastatin from calpain (57De Tullio R. Stifanese R. Salamino F. Pontremoli S. Melloni E. Biochem. J. 2003; 375: 689-696Crossref PubMed Scopus (41) Google Scholar).Levels of Calpastatin mRNA in the Brain of NMS and HMS Rats—Total RNA was isolated from brain of both NMS and HMS rats by extraction with guanidium thiocyanate (58Sambrook J. Russel D.W. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1989Google Scholar), quantified, and immediately reverse-transcribed. First strand cDNA synthesis was performed by means of the Superscript RNase H- Reverse Transcriptase kit (Invitrogen) using random examer primers.Equal amounts (1 μl) of each sample were co-amplified in the presence of primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calpastatin. The primers and the amplification conditions were the same as reported in Refs. 9De Tullio R. Sparatore B. Salamino F. Melloni E. Pontremoli S. FEBS Lett. 1998; 422: 113-117Crossref PubMed Scopus (41) Google Scholar and 13Averna M. De Tullio R. Salamino F. Minafra R. Pontremoli S. Melloni E. J. Biol. Chem. 2001; 276: 38426-38432Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, and mRNA levels were quantitatively determined with the method described by Ref. 59Chelly J. Kaplan J.C. Mire S.G. Kahn A. Nature. 1988; 333: 858-860Crossref PubMed Scopus (652) Google Scholar, following the modifications reported in Ref. 13Averna M. De Tullio R. Salamino F. Minafra R. Pontremoli S. Melloni E. J. Biol. Chem. 2001; 276: 38426-38432Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar. To minimize tube-to-tube variation, each experiment was carried out in triplicate.Levels of Plasma Membrane Ca2+-ATPase mRNA in the Brain of NMS and HMS Rats—Equal amounts (1 μl) of the same cDNA samples prepared for the quantification of calpastatin mRNA levels were co-amplified in the presence of primers specific for GAPDH and plasma membrane Ca2+-ATPase. Ca2+-ATPase primers were chosen to detect all the forms of plasma membrane calcium pumps present in the brain tissue (60Strehler E.E. Zacharias D.A. Physiol. Rev. 2001; 81: 21-50Crossref PubMed Scopus (481) Google Scholar). The oligonucleotide used were: sense primer 5′-AAGAAAATGATGAAGGACAACAAC-3′ and antisense primer 5′-CCTTCCAGGCACAGGAA-3′. The PCR conditions were: denaturation (94 °C for 40 s), annealing (52 °C for 30 s) and extension (70 °C for 60 s). Amplification was carried out as described in Ref. 13Averna M. De Tullio R. Salamino F. Minafra R. Pontremoli S. Melloni E. J. Biol. Chem. 2001; 276: 38426-38432Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar and Ca2+-ATPase mRNA levels were quantitatively determined with the method described by Ref. 59Chelly J. Kaplan J.C. Mire S.G. Kahn A. Nature. 1988; 333: 858-860Crossref PubMed Scopus (652) Google Scholar, following the modifications reported in Ref. 13Averna M. De Tullio R. Salamino F. Minafra R. Pontremoli S. Melloni E. J. Biol. Chem. 2001; 276: 38426-38432Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar. To minimize tube-to-tube variation, each experiment was carried out in triplicate.RESULTSEffect of HSD Treatment on Ca2+ Homeostasis in Aorta and in the Brain of NMS and HMS Rats—The effect of an increased [Ca2+]i in normal rat brain (normotensive Milan strain, NMS), was studied using animals treated for 2 weeks with a high salt (NaCl) diet (HSD), known to promote aorta contraction accompanied by an elevation of its [Ca2+]i (41Aviv A. Am. J. Hypertens. 1996; 9: 703-707Crossref PubMed Scopus (27) Google Scholar, 42Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Crossref PubMed Scopus (1443) Google Scholar, 43Iwamoto T. Kita S. Zhang J. Blaustein M. Arai Y. Yoshida S. Wakimoto K. Komuro I. Katsuragi T. Nat. Med. 2004; 10: 1193-1199Crossref PubMed Scopus (231) Google Scholar). In parallel we have submitted the same treatment to genetically determined hypertensive rats of the same strain (HMS).According to previous reports (43Iwamoto T. Kita S. Zhang J. Blaustein M. Arai Y. Yoshida S. Wakimoto K. Komuro I. Katsuragi T. Nat. Med. 2004; 10: 1193-1199Crossref PubMed Scopus (231) Google Scholar), we have observed that in NMS rats, HSD treatment produces elevation in blood pressure (Fig. 1A) from 105 to 145–150 mm Hg, accompanied by a 1.4–1.5 increase in fluorescence recorded following exposure of aorta slices to calcium green fluorescence (Fig. 1B). These data indicate that HSD treatment promotes a contraction of aorta smooth muscle mediated by an alteration in Ca2+ homeostasis. In HMS rats the elevated blood pressure (Fig. 1A) is associated to a constitutively increased [Ca2+] ranging from 1.8–1.9-fold (Fig. 1B). This calcium concentration is further increased (2.3-fold) by 2-week treatment with HSD (Fig. 1B).Because the calcium green loading of aorta slice" @default.
• W2005313237 created "2016-06-24" @default.
• W2005313237 creator A5004437812 @default.
• W2005313237 creator A5014147321 @default.
• W2005313237 creator A5030915444 @default.
• W2005313237 creator A5055337483 @default.
• W2005313237 creator A5071383401 @default.
• W2005313237 creator A5072213514 @default.
• W2005313237 creator A5085308198 @default.
• W2005313237 creator A5091744245 @default.
• W2005313237 date "2007-01-01" @default.
• W2005313237 modified "2023-10-17" @default.
• W2005313237 title "Regulation of Calpain Activity in Rat Brain with Altered Ca2+ Homeostasis" @default.
• W2005313237 cites W1542485314 @default.
• W2005313237 cites W1565542770 @default.
• W2005313237 cites W1574047547 @default.
• W2005313237 cites W1599440851 @default.
• W2005313237 cites W1603617892 @default.
• W2005313237 cites W1639940444 @default.
• W2005313237 cites W179348653 @default.
• W2005313237 cites W1963650112 @default.
• W2005313237 cites W1967791344 @default.
• W2005313237 cites W1968645356 @default.
• W2005313237 cites W1970374401 @default.
• W2005313237 cites W1973963193 @default.
• W2005313237 cites W1974089466 @default.
• W2005313237 cites W1979515209 @default.
• W2005313237 cites W1982748988 @default.
• W2005313237 cites W1983477773 @default.
• W2005313237 cites W1984196084 @default.
• W2005313237 cites W1987703691 @default.
• W2005313237 cites W1987933233 @default.
• W2005313237 cites W1991764697 @default.
• W2005313237 cites W1992136431 @default.
• W2005313237 cites W1993528483 @default.
• W2005313237 cites W1994577092 @default.
• W2005313237 cites W2003424773 @default.
• W2005313237 cites W2003597344 @default.
• W2005313237 cites W2016459361 @default.
• W2005313237 cites W2019802143 @default.
• W2005313237 cites W2029955024 @default.
• W2005313237 cites W2030054492 @default.
• W2005313237 cites W2031721553 @default.
• W2005313237 cites W2034546847 @default.
• W2005313237 cites W2036532215 @default.
• W2005313237 cites W2038517223 @default.
• W2005313237 cites W2038721029 @default.
• W2005313237 cites W2042867596 @default.
• W2005313237 cites W2044802305 @default.
• W2005313237 cites W2045267492 @default.
• W2005313237 cites W2047669710 @default.
• W2005313237 cites W2055782143 @default.
• W2005313237 cites W2057995162 @default.
• W2005313237 cites W2068301543 @default.
• W2005313237 cites W2069465345 @default.
• W2005313237 cites W2069509382 @default.
• W2005313237 cites W2074562451 @default.
• W2005313237 cites W2078205307 @default.
• W2005313237 cites W2083308741 @default.
• W2005313237 cites W2088868590 @default.
• W2005313237 cites W2089625101 @default.
• W2005313237 cites W2091797889 @default.
• W2005313237 cites W2091949456 @default.
• W2005313237 cites W2094587177 @default.
• W2005313237 cites W2104645386 @default.
• W2005313237 cites W2107070633 @default.
• W2005313237 cites W2111853402 @default.
• W2005313237 cites W2120531949 @default.
• W2005313237 cites W2123665894 @default.
• W2005313237 cites W2144139389 @default.
• W2005313237 cites W2144970165 @default.
• W2005313237 cites W2148220372 @default.
• W2005313237 cites W2169436026 @default.
• W2005313237 cites W2169550834 @default.
• W2005313237 cites W2172682370 @default.
• W2005313237 cites W2206400534 @default.
• W2005313237 cites W2235497272 @default.
• W2005313237 cites W2267906346 @default.
• W2005313237 cites W2335815721 @default.
• W2005313237 cites W2404157374 @default.
• W2005313237 doi "https://doi.org/10.1074/jbc.m606919200" @default.
• W2005313237 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17135258" @default.
• W2005313237 hasPublicationYear "2007" @default.
• W2005313237 type Work @default.
• W2005313237 sameAs 2005313237 @default.
• W2005313237 citedByCount "24" @default.
• W2005313237 countsByYear W20053132372012 @default.
• W2005313237 countsByYear W20053132372013 @default.
• W2005313237 countsByYear W20053132372014 @default.
• W2005313237 countsByYear W20053132372015 @default.
• W2005313237 countsByYear W20053132372016 @default.
• W2005313237 countsByYear W20053132372017 @default.
• W2005313237 countsByYear W20053132372018 @default.
• W2005313237 countsByYear W20053132372019 @default.
• W2005313237 crossrefType "journal-article" @default.
• W2005313237 hasAuthorship W2005313237A5004437812 @default.
• W2005313237 hasAuthorship W2005313237A5014147321 @default.
• W2005313237 hasAuthorship W2005313237A5030915444 @default.

• next