In vivo degradation of nitric oxide synthase (NOS) and heat shock protein 90 (HSP90) by calpain is modulated by the formation of a NOS–HSP90 heterocomplex Monica Averna, Roberto Stifanese, Roberta De Tullio, Franca Salamino, Sandro Pontremoli and Edon Melloni Department of Experimental Medicine (DIMES)-Biochemistry Section, and Centre of Excellence for Biomedical Research (CEBR), University of Genoa, Italy Keywords Ca2+ homeostasis; calpain; calpastatin; heat shock protein 90; nitric oxide synthase Correspondence S Pontremoli, Department of Experimental Medicine (DIMES)-Biochemistry Section, University of Genoa, Viale Benedetto XV, 1-16132 Genoa, Italy Fax: +39 010 518343 Tel: +39 010 3538128 E-mail: pontremoli@unige.it (Received October 2007, revised 19 February 2008, accepted 11 March 2008) doi:10.1111/j.1742-4658.2008.06394.x We have shown previously that isolated heat shock protein 90 (HSP90) and nitric oxide synthase (NOS), once associated in a heterocomplex, become completely resistant to calpain digestion In this study, it is shown that, in vivo, under conditions of calpain activation, the protection of NOS degradation occurs In addition, the extent of NOS degradation is a function of the level of HSP90 expression Thus, in rat brain, which contains a large excess of HSP90, almost all neuronal NOS is associated with the chaperone protein In this condition, neuronal NOS retains its full catalytic activity, although limited proteolytic conversion to still active low-molecular-mass (130 kDa) products takes place In contrast, in aorta, which contains much smaller amounts of HSP90, endothelial NOS is not completely associated with the chaperone, and undergoes extensive degradation with a loss of protein and catalytic activity On the basis of these findings, we propose a novel role of the HSP90–NOS heterocomplex in protecting in vivo NOS from proteolytic degradation by calpain The efficiency of this effect is directly related to the level of intracellular HSP90 expression, generating a high HSP90 to NOS ratio, which favours both the formation and stabilization of the HSP90–NOS heterocomplex This condition seems to occur in rat brain, but not in aorta, thus explaining the higher vulnerability to proteolytic degradation of endothelial NOS relative to neuronal NOS The interaction of nitric oxide synthase (NOS) with a variety of proteins plays an important role in the regulation of NO production [1–4] Of these interacting proteins, heat shock protein 90 (HSP90) has been proposed to exert a relevant role for both NOS function and stability [1,5–7] Thus, HSP90 may serve as an allosteric positive modulator of NOS isozymes by inducing the acquisition of the active conformation or by enhancing the affinity of NOS for the Ca2+ sensor calmodulin [8] It has also been proposed that the association of NOS with HSP90 favours the correct insertion of the haem group into apo-NOS and the formation of stable NOS dimers [9,10] As the haemdeficient monomeric NOS form following treatment with HSP90 inhibitors is rapidly polyubiquitinated and degraded by the proteasome pathway, HSP90 has been considered to be indirectly involved in the selective proteolytic degradation of NOS [11–16] In addition to proteasome degradation, several reports have indicated that, in extreme cytotoxic conditions, calpain becomes uncontrollably activated, producing extensive degradation of NOS and HSP90 [17–26] Abbreviations [Ca2+]i, intracellular Ca2+ concentration; C.I.1, synthetic calpain inhibitor-1; eNOS, endothelial nitric oxide synthase; HMS, hypertensive Milan strain; HSD, high-sodium diet; HSP90, heat shock protein 90; iNOS, inducible NOS; NMS, normotensive Milan strain; nNOS, neuronal nitric oxide synthase; NOS, nitric oxide synthase FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS 2501 In vivo degradation of NOS and HSP90 by calpain M Averna et al We have recently demonstrated that the susceptibility to calpain degradation of purified endothelial NOS (eNOS) and neuronal NOS (nNOS) is significantly reduced in the presence of equimolar amounts of HSP90 [27] Using immunoprecipitation studies, it has also been established that the protective effect is caused by HSP90-specific recruitment by active calpain molecules In this associated form, HSP90 becomes resistant to digestion, although the protease still retains 50% of its proteolytic activity against external substrates Furthermore, when NOS isozymes are associated with this binary complex, they also become resistant to proteolytic degradation These observations imply a correlation between the vulnerability of NOS isozymes and the availability of HSP90 to generate stable ternary complexes This relationship is strongly supported by the different digestibility of NOS in Jurkat and BAE-1 cells, expressing high and low levels of HSP90, respectively To verify the occurrence of such a protective effect in vivo, we used normotensive Milan strain (NMS) rats as a model Thus, we induced a mild elevation of intracellular Ca2+ concentration ([Ca2+]i) by the administration of a high-sodium diet (HSD) [28], and studied calpain degradation of NOS and HSP90 in brain and aorta To amplify the range of fluctuations in [Ca2+]i, hypertensive Milan strain (HMS) rats were also used, as they are characterized by a constitutive elevation in [Ca2+]i and a higher responsiveness to HSD We report here that, in the brain and aorta of HSDtreated rats, the extent and patterns of proteolytic degradation of NOS isozymes and HSP90 are similar to those previously detected in Jurkat and BAE-1 cells loaded with Ca2+ [27] As the differences in expression of HSP90 in the two rat tissues are similar to those present in these cell models [27], we propose that the occurrence of conditions which favour the formation and stabilization of proteolytically resistant complexes of NOS with HSP90 are crucial in determining the in vivo resistance of NOS and HSP90 to calpain degradation Results Levels of HSP90 and NOS isozymes in rat brain and aorta The level of HSP90 and the type of NOS isoform present in rat brain and aorta were determined by immunoblotting (Fig 1) In brain, nNOS was the most preferentially expressed isoform, together with traces of eNOS (Fig 1A) In aorta, only eNOS isozyme was 2502 A B C Fig NOS isozymes and HSP90 expressed in brain and aorta of NMS rats (A) Aliquots (50 lg protein) of NMS rat brain soluble material, obtained as described in Experimental procedures, were submitted to 6% SDS-PAGE and blotted as described previously NOS isozymes were detected with the specific mAbs (B) Aliquots (50 lg protein) of NMS rat thoracic aorta total lysate, obtained as described in Experimental procedures, were submitted to 6% SDSPAGE and blotted as described previously NOS isozymes were detected with specific mAbs (C) HSP90 levels were detected from the same samples as reported in (A) and (B) using the specific mAb The immunoreactive bands detected in (A–C) were quantified (see bars) as described in Experimental procedures The values reported are the arithmetical means ± standard deviation of five different experiments carried out on five different animals of each strain detectable (Fig 1B) In both tissues, no expression of inducible NOS (iNOS) was found (Fig 1A,B) HSP90 was present in rat brain in amounts six- to sevenfold FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS M Averna et al In vivo degradation of NOS and HSP90 by calpain higher than in aorta, resulting in a much higher HSP90 to NOS ratio in brain (Fig 1C) A Calpain activation in rat brain and aorta following HSD treatment To promote in vivo calpain activation, NMS rats were treated with HSD, which has been established previously to induce a mild elevation in [Ca2+]i, slightly higher in aorta than in brain [28] To amplify the range of elevation in [Ca2+]i, HMS rats were also used, as a limited increase in [Ca2+]i in both aorta and brain has been found to be constitutively present in these animals To assess the in vivo activation of calpain, we relied on the following well-established methods: (a) the occurrence of calpain consumption [26,29–31]; (b) a specific pattern of calpastatin digestion, resulting in an imbalance within the proteolytic system [32]; and (c) the degradation of calpain target proteins [26,30] As shown in Fig 2A, following HSD treatment, the levels of both l- and milli-calpain isoforms were reduced to a limited extent in brain, whereas, in aorta (Fig 2B), the decrease in the two protease isoforms was more pronounced Moreover, in brain, the natural inhibitor of calpain, calpastatin, was preferentially converted into still active 15 kDa fragments (Table 1), whereas, in aorta, the inhibitor was predominantly inactivated As both the inactivation and fragmentation of calpastatin are known to be produced by active calpain [32], these observations further indicate that calpain is activated in both tissues, although at a higher rate in aorta Further direct evidence in support of calpain activation in aorta was provided by the degradation of talin and desmin in HSD-treated rats (Fig 3A) Indeed, this process was completely prevented (Fig 3B) by the administration to the animals of synthetic calpain inhibitor-1 (C.I.1) [33,34] Digestion of HSP90 and NOS in brain and aorta of normotensive and hypertensive rats treated with HSD Following HSD treatment, no appreciable changes in NOS activity occurred in the brain of NMS rats, although a small fraction of the native 160 kDa synthase was converted into the still active 130 kDa form (Fig 4A) The level of HSP90 remained unchanged during the period of treatment (Fig 4A) By contrast, in aorta, more than 50% of native eNOS progressively disappeared (Fig 4B,D), together with a significant degradation of HSP90, which was only partially B Fig Levels of calpain isoforms and calpain substrates in the aorta of NMS and HMS rats treated with HSD Aliquots (100 lg protein) of brain soluble material (A) and aorta total lysate (B), prepared as described in Experimental procedures, from untreated or 4-week HSD-treated NMS and HMS rats, were submitted to 8% SDS-PAGE, followed by immunoblotting revealed with serum-l-calpain mAb 56.3 [36] and monoclonal IgG milli-calpain The immunoreactive material was detected and quantified as described in Experimental procedures The values reported are the arithmetical means ± standard deviation of five different experiments carried out on five different animals of each strain replaced by an 84 kDa form (Fig 4B,D) The involvement of calpain in these digestion processes was demonstrated by the protective effect on both HSP90 and NOS degradation of the administration to the HSD-treated NMS animals of C.I.1 (Fig 5) In the brain of hypertensive rats, in spite of a preexisting condition of altered Ca2+ homeostasis, no appreciable changes in total nNOS activity or the level of HSP90 were observed (Fig 6A,C) By contrast with NMS rats, a small fraction of a still active 130 kDa form was already present in the brain of untreated HMS rats and increased following HSD treatment (Fig 6A) However, in aorta, the digestion of eNOS and HSP90 appeared to be more extensive (Fig 6B) Indeed, approximately 80–90% of eNOS protein and FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS 2503 In vivo degradation of NOS and HSP90 by calpain M Averna et al Table Levels of native and 15 kDa calpastatin species in brain and aorta of NMS and HMS rats treated with HSD for weeks The data reported are the arithmetical means ± standard deviation of five different experiments carried out on five different animals of each strain Animal Brain NMS NMS HMS HMS Aorta NMS NMS HMS HMS Treatmenta Total calpastatin activity (%)b 15 kDa fragment activity (%)c 97 40 90 ± ± ± ± 40 10 49 ± ± ± ± 0.3 2 20 46 None HSD None HSD 99 16 64 ± ± ± ± 0.5 25 16 14 ± ± ± ± 2 Desmin Talin Loss of total calpastatin activity (%)d None HSD None HSD A 59 20 84 NMS B activity, together with 60–70% of HSP90, were lost (Fig 6B,D) The degradation pattern of nNOS in the brain of HSD-treated rats, resulting in the accumulation of the still active 130 kDa form, can be reproduced in in vitro conditions if nNOS digestion by calpain is carried out in the presence of HSP90 [27] This finding can also explain the large extent of digestion of eNOS in aorta, in which, in association with a higher degree of calpain activation, a lower level of HSP90 is also present Identification of HSP90–NOS heterocomplexes in aorta and brain lysates In order to explore the relationship between the HSP90 to NOS ratio and the formation of calpainresistant heterocomplexes, we first studied, by immunoprecipitation analysis, the association of the two proteins in brain and aorta As shown in Fig 7A, following the addition of IgG1-HSP90 antibody to crude extracts of brain or aorta, NOS was immunoprecipitated, indicating a specific association of the two proteins We then determined the amount of each enzyme 2504 NMS +HSD HMS +HSD Desmin Talin a NMS and HMS rats were fed for weeks with HSD as described in Experimental procedures b Total calpastatin activity was measured as described in Experimental procedures and [28,43] c Aliquots (1.5 mg protein) of the soluble material, obtained as described previously from untreated or 4-week HSD-treated NMS and HMS rat brain and thoracic aorta homogenates, were submitted to 12% SDS-PAGE divided into 10 lanes [28] The 15 kDa calpastatin species were identified on the basis of their electrophoretic mobility, and quantified following extraction from the gel by measuring their inhibitory activity as described previously [28,42] d The loss of calpastatin activity was calculated by subtracting the sum of the percentage of the active calpastatin species from 100 HMS Control + HSD + HSD + C.I Fig Levels of calpain substrates in aorta of NMS and HMS rats treated with HSD and C.I.1 Aliquots (50 lg protein) of aorta total lysate (A), prepared as described in Experimental procedures, from untreated and HMS rats, were submitted to 8% SDS-PAGE followed by immunoblotting Samples (50 lg protein) of aorta total lysate (B) from untreated or 4-week HSD-treated NMS rats, in the absence (+HSD) or presence (+HSD+C.I.1) of 25 lM C.I.1, were submitted to 8% SDS-PAGE followed by immunoblotting Desmin and talin were detected with specific mAbs involved in such complexes by submitting samples of crude extracts of rat brain and aorta to gel filtration chromatography As shown in Fig 7B, in brain, nNOS eluted entirely as a single peak at a volume corresponding to a molecular mass higher than that of the free native enzyme HSP90 was eluted in two peaks: the first coincident with that of nNOS, and the second containing more than 60% of total chaperone protein, with an elution volume identical to that of free HSP90 Thus, all nNOS appeared to be engaged in a complex with HSP90, whereas the major fraction of the chaperone was present in the free form In aorta (Fig 7C), approximately 85–90% of eNOS was recovered in association with HSP90 and the remaining 10–15% was found in the free form; however, the amount of HSP90 recovered as free protein was much lower than that engaged in the complex The large difference in the amount of free chaperone observed in the two tissues is indicative of the existence FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS M Averna et al In vivo degradation of NOS and HSP90 by calpain B A C D Fig In vivo digestion of NOS and HSP90 in NMS rats during HSD treatment Aliquots (20 lg protein) of rat brain soluble material (A) and aliquots (50 lg protein) of rat aorta total lysate (B), obtained as described in Experimental procedures, from untreated or HSD-treated NMS rats, were submitted to 6% SDS-PAGE and blotted as described previously nNOS, eNOS and HSP90 were detected with specific mAbs (C) nNOS (open circles) and HSP90 (filled circles) immunoreactive materials detected in (A) and the corresponding nNOS activity (open squares) were quantified as described in Experimental procedures (D) eNOS (open circles) and HSP90 (filled circles) immunoreactive materials detected in (B) and the corresponding eNOS activity (open squares) were quantified as described in Experimental procedures The values reported are the arithmetical means ± standard deviation of five different experiments carried out on five different animals of each strain of conditions favouring and stabilizing the heterocomplex much more efficiently in brain than in aorta This could explain the higher susceptibility of eNOS to calpain digestion Discussion Although several reports [11–26] have indicated that calpain and the proteasome pathway are the two major systems responsible for the proteolytic degradation of NOS, some pertinent questions still remain unsolved Indeed, although it has been established, especially by the use of NOS and HSP90 inhibitors, that proteasome-promoted degradation selectively removes inactive structurally damaged NOS forms, or monomeric haem-deficient isozyme species [11–16], the precise molecular events that trigger the proteolytic degradation of NOS in vivo still remain to be defined One of these molecular signals could be altered or decreased HSP90 function, favouring the accumulation of abnormal or monomeric NOS molecules and their degradation by the proteasome system [1,12] Furthermore, proteolytic degradation of NOS by calpain has been described in conditions of extreme cytotoxicity [17,19,21,23,26] In these experiments, as a result of high Ca2+ overload, several calpain targets, including NOS, can undergo proteolytic digestion For this reason, the degradation of NOS can be attributed to an overactivation of calpain rather than to a selective regulated proteolytic mechanism In previous studies, we have observed that HSP90 is five- to tenfold less susceptible than nNOS and eNOS to calpain degradation [27] as a result of the formation of a calpain–HSP90 complex in which the protease can no longer degrade the bound chaperone NOS isozymes, once recruited into the HSP90–calpain binary complex, also become resistant to calpain digestion This protective effect may be of physiological relevance, as conditions promoting NO production also induce calpain activation Thus, the formation of NOS–HSP90 complexes may provide a new insight into the understanding of the mechanisms involved in modulating NO production In such a case, the availability of adequate amounts of HSP90 becomes the limiting factor Our study poses new important questions that need to be addressed The first question concerns the vulnerability of different NOS isoforms to proteolysis in vivo under conditions of small changes to [Ca2+]i The second question concerns the capacity of HSP90 to protect NOS in vivo against proteolytic degrada- FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS 2505 In vivo degradation of NOS and HSP90 by calpain M Averna et al a loss of catalytic activity In contrast, in aorta, both eNOS and HSP90 were highly degraded The different vulnerability of the two NOS isoforms to proteolytic degradation is strictly related to the availability of HSP90, which is expressed in higher concentrations in the brain than in the aorta Furthermore, the patterns of digestion of eNOS and nNOS observed in HSD-treated animals are identical to those previously obtained in reconstructed systems containing the synthases together with different levels of HSP90 Our data suggest that a large reservoir of HSP90 maintains all NOS engaged in a calpain-resistant heterocomplex, which is protected from proteolysis, even under conditions of prolonged protease activation This conclusion is further supported by the finding reported here that, in brain, the nNOS–HSP90 complex is in equilibrium with a large amount of stabilizing free chaperone, a condition that does not occur in aorta The reduced availability of HSP90 in aorta can thus explain the increased vulnerability of eNOS relative to nNOS to proteolysis On the basis of these findings, we propose a novel mechanism in which HSP90 can provide functional stability of NOS isozymes under conditions characterized by an alteration in intracellular Ca2+ homeostasis A B Fig Levels of NOS isozymes and HSP90 in brain and aorta of NMS rats treated with HSD and C.I.1 Aliquots of brain soluble material (20 lg protein) and of aorta total lysate (50 lg protein), obtained as described in Experimental procedures, from untreated or 4-week HSD-treated NMS rats, in the absence (+HSD) or presence (+HSD+C.I.1) of 25 lM C.I.1, were submitted to 6% SDSPAGE followed by immunoblotting, revealed with IgG1-eNOS or IgG1-nNOS mAbs (A) or IgG1-HSP90 mAb (B) The immunoreactive material of eNOS, nNOS and HSP90 was detected and quantified as described in Experimental procedures The values reported are the arithmetical means ± standard deviation of five different experiments carried out on five different animals of each strain tion Finally, a third question involves the possible relationship between such protection and the well-known different expression of HSP90 in various tissues To answer these questions, we have used animals treated with HSD, which has been shown previously to induce an increase in the level of [Ca2+]i and a correlated calpain activation [28] This increase in [Ca2+]i is more intense in aorta than in brain Under these conditions, in brain, no change in the level of HSP90 was observed, although a limited and conservative degradation of nNOS occurred without 2506 Experimental procedures Materials Leupeptin C.I.1, aprotinin, phosphatase inhibitor cocktail I and II, NADPH, calmodulin, FAD, FMN, tetrahydrobiopterin, l-arginine and aldolase were purchased from Sigma Aldrich, Milan, Italy l-[14C]arginine (925 Bq; specific activity, 1Ỉ14 · 1011 BqỈmol)1), Sephacryl S-300, Sephadex G200 resins, SuperoseÒ 12 10 ⁄ 300 GL column and protein G-Sepharose were obtained from GE Healthcare, Milan, Italy Ferritin was purchased from Boehringer Mannheim, Mannheim, Germany Dowex 50W8 resin (Na+ form) was obtained from Bio-Rad Laboratories, Milan, Italy 4-(2Aminoethyl)benzenesulfonylfluoride (AEBSF) was obtained from Calbiochem (Missiagua, Canada) The ECLÒ Detection System was obtained from GE Healthcare Monoclonal antibodies (mAbs) nNOS, eNOS, iNOS and HSP90 antibodies were purchased from BD Transduction Laboratories, Milan, Italy b-Actin and milli-calpain antibodies were obtained from Sigma Aldrich, Milan, Italy Desmin and talin antibodies were purchased from Novus Biologicals, Littleton, CO, USA IgG1-calpastatin (mAb 35.23) and serum l-calpain (mAb FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS M Averna et al In vivo degradation of NOS and HSP90 by calpain A B C D Fig In vivo digestion of NOS and HSP90 in HMS rats during HSD treatment Aliquots (20 lg protein) of rat brain soluble material (A) and aliquots (50 lg protein) of rat aorta total lysate (B), obtained as described in Experimental procedures, from untreated or HSD-treated HMS rats, were submitted to 6% SDS-PAGE and blotted as described previously nNOS, eNOS and HSP90 were detected with specific mAbs (C) nNOS (open circles) and HSP90 (filled circles) protein detected in (A) and the corresponding nNOS activity (open squares) were quantified as described in Experimental procedures (D) eNOS (open circles) and HSP90 (filled circles) immunoreactive materials detected in (B) and the corresponding eNOS activity (open squares) were quantified as described in Experimental procedures The values reported are the arithmetical means ± standard deviation of five different experiments carried out on five different animals of each strain 56.3) mAbs were produced as indicated in [35] and [36], respectively ing the institution’s ethical guidelines During the course of the experiments, no appreciable changes were observed in food consumption and body weight Animals NMS and HMS rats [37] were housed in controlled conditions (22 ± °C; humidity, 50 ± 5%; lighting, 8–20 h) Systolic blood pressure was measured by tail-cuff plethysmography [W&W Electronic, BP recorder 8005 (Huntsinlle, AL, USA)] on prewarmed (37 °C) rats, following the procedure originally described by Byrom and Wilson [38] Normotensive and hypertensive rats showed mean arterial blood pressures of 100 ± and 145 ± 10 mmHg, respectively Experimental hypertension Experimental hypertension was induced in 60-day-old rats by feeding ad libitum with a standard rat chow and providing NaCl dissolved in tap water at a concentration of 10 gỈL)1 for a period of time ranging from 15 to 30 days Each animal received approximately 0.7 gỈday)1 of NaCl Where indicated, 25 lm C.I.1 was dissolved in tap water in the presence of 10 gỈL)1 NaCl, and administered to NMS and HMS rats for weeks [28] Each rat received 0.5– 0.7 mgỈday)1 of C.I.1 Experiments were carried out follow- Preparation of tissue homogenates NMS and HMS rats were sacrificed by decapitation; the brain was immediately removed, minced, homogenized in a Potter–Elvehjem homogenizer and sonicated in three volumes of 50 mm sodium borate buffer, pH 7.5, containing mm EDTA, 0.5 mm 2-mercaptoethanol, 0.1 mgỈmL)1 leupeptin and mm AEBSF (buffer A) The particulate material was discarded by centrifugation (100 000 g for 10 min) Thoracic aorta was rapidly excised from the same animals After the removal of the adhering connective tissue, the tissue was cut into several segments (approximately mm each), homogenized in a Potter–Elvehjem homogenizer and lysed by sonication in three volumes of buffer A The protein concentration was determined following the procedure of Bradford [39] Immunoblot Rat brain and aorta lysates (20–50 lg) were diluted in a final volume of 100 lL of the SDS-PAGE loading buffer and submitted to 6% SDS-PAGE [40] The protein bands FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS 2507 In vivo degradation of NOS and HSP90 by calpain M Averna et al Fig Identification of NOS–HSP90 association in rat brain and aorta (A) Aliquots (500 lg protein) of brain and aorta crude extract, prepared as described in Experimental procedures, were incubated overnight at °C with IgG1-HSP90 antibody (see Experimental procedures), as reported also in [7,44,45] The mixtures were then incubated for h at room temperature with 50 lL of protein G-Sepharose The particles were collected and washed three times with immunoprecipitation buffer The particles were then suspended in SDS-PAGE loading solution, heated for at 90 °C and submitted to 6% SDS-PAGE NOS isozymes and HSP90 were identified with specific mAbs (see Experimental procedures) The values reported are the arithmetical means ± standard deviation of five different experiments carried out on five different animals of each strain (B, C) Aliquots (500 lg protein) of the soluble material of brain homogenate and thoracic aorta total lysate, obtained from NMS rats as described previously, were submitted to gel filtration chromatography (see Experimental procedures) Aliquots (30 lL) of each eluted fraction were suspended in SDS-PAGE loading solution [40] and submitted to 6% SDS-PAGE, followed by immunoblotting HSP90 (filled circles) and NOS isoforms (open circles) were probed with the appropriate antibody The immunoreactive material was quantified as described in Experimental procedures A B C were then blotted onto a nitrocellulose membrane and saturated with a NaCi/Pi solution, pH 7.5, containing 5% powered milk The blots were probed with specific antibodies, followed by a peroxidase-conjugated secondary antibody as described previously, and then developed with the ECL Detection System [41] The immunoreactive material was detected with a Bio-Rad Chemi Doc XRS apparatus and quantified using quantity one 4.6.1 software (BioRad Laboratories) The procedure was made quantitative by the use of known amounts of proteins submitted to SDS-PAGE and staining with the appropriate antibody The bands were then scanned, and the areas of the peaks obtained were used to create a calibration curve Immunoprecipitation Brain and thoracic aorta, excised from NMS rats, were lysed in ice-cold 20 mm Tris ⁄ HCl, 2.5 mm EDTA, 2.5 mm 2508 EGTA, 0.14 m NaCl, pH 7.4 (immunoprecipitation buffer), containing 1% Triton X-100, 10 lgỈmL)1 aprotinin, 20 lgỈmL)1 leupeptin, 10 lgỈmL)1 AEBSF and phosphatase inhibitor cocktail I and II (10 lgỈmL)1), followed by brief sonication Cell lysates were centrifuged at 12 000 g for 15 at °C, and protein quantification of the supernatants was performed using the Lowry assay For the immunoprecipitations,  500 lg of detergent-soluble protein (crude extract) was previously precleared with protein G-Sepharose, and then incubated in the presence of lg of IgG1-HSP90 mAb at °C overnight Protein G-Sepharose was then added and incubated for an additional hour The immunocomplexes were washed three times with immunoprecipitation buffer, heated in SDS-PAGE loading buffer for [40] and submitted to 6% SDS-PAGE Proteins were then transferred by electroblotting onto a nitrocellulose membrane, and immunoblotting analysis was performed as described above Identification of NOS–HSP90 association by gel filtration Aliquots (0.5 mg protein) of the soluble material of brain homogenate and thoracic aorta total lysate, obtained from NMS rats as described previously, were submitted to gel filtration chromatography on a SuperoseÒ 12 10 ⁄ 300 GL column (total volume, 24 mL) equilibrated in buffer A containing 50 mm NaCl using an FPLC system The flow rate was 100 lLỈmin)1 and the eluted proteins were collected in 500 lL fractions The molecular weights of the eluted proteins were calculated from the elution volumes of ferritin (Mr = 450 kDa) and aldolase (Mr = 160 kDa), utilized as standard proteins FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS M Averna et al Aliquots (30 lL) of each eluted fraction were suspended in SDS-PAGE loading buffer [40] and submitted to 6% SDS-PAGE Proteins were then transferred to a nitrocellulose membrane by electroblotting, and immunoblotting analysis was performed as described above The immunoreactive material was detected and quantified as described above Assay of NOS activity NOS activity was assayed by detecting the production of citrulline from l-[14C]arginine, as reported previously [23] with the following modifications Aliquots (100 lg protein) of the crude homogenate were incubated in a total volume of 250 lL in buffer A containing mm NADPH, 200 mm calmodulin, 20 lm tetrahydrobiopterin, lm FAD, lm FMN, lm l-arginine and 925 Bq of l-[14C]arginine (specific radioactivity, 1Ỉ14 · 1011 BqỈmol)1) at 37 °C After 30 min, mL of ice-cold stop buffer (50 mm Hepes, pH 5.5, containing mm EDTA) was added These incubations were then submitted to anion exchange chromatography using mL of packed Dowex 50W8 Na+ form resin pre-equilibrated with stop buffer l-Citrulline was eluted by washing the resin with mL of stop buffer, and the radioactivity present was counted in a liquid scintillation counter One unit of NOS activity was defined as the amount of enzyme producing pmol citrullinmin)1 in the specified conditions Separation and quantification of calpastatin species in rat brain and aorta Aliquots of the soluble material (10 lanes with 100 lg protein each), prepared as described above from untreated or treated NMS and HMS rat brain and thoracic aorta homogenates, were submitted to 12% SDS-PAGE [28] Calpastatin species were identified following protein extraction from the gel, as described previously [42] Calpastatin activity was measured as described in [43] Acknowledgements This work was supported in part by grants from Min` istero Haliano per I’Universita e la Ricerca, Fondo per gli 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In vivo degradation of NOS and HSP90 by calpain M Averna et al Table Levels of native and 15 kDa calpastatin species in brain and aorta of NMS and HMS rats treated with HSD for weeks The data... compilation ª 2008 FEBS M Averna et al In vivo degradation of NOS and HSP90 by calpain higher than in aorta, resulting in a much higher HSP90 to NOS ratio in brain (Fig 1C) A Calpain activation in. .. inactivated As both the inactivation and fragmentation of calpastatin are known to be produced by active calpain [32], these observations further indicate that calpain is activated in both tissues, although