Curcumin counteracts loss of force and atrophy of hindlimb unloaded rat soleus by hampering neuronal nitric oxide synthase untethering from sarcolemma.

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J Physiol 592.12 (2014) pp 2637–2652

Curcumin counteracts loss of force and atrophy of hindlimb unloaded rat soleus by hampering neuronal nitric oxide synthase untethering from sarcolemma Maurizio Vitadello1 , Elena Germinario2,3 , Barbara Ravara2 , Luciano Dalla Libera1 , Daniela Danieli-Betto2,3 and Luisa Gorza2 1

CNR-Institute for Neuroscience, Padova section, Padova, Italy Department of Biomedical Sciences, University of Padova, Padova, Italy 3 Interuniversity Institute of Myology, Italy 2

The Journal of Physiology

Key points

r Attenuation of disuse muscle atrophy by means of a pharmacological approach represents the wanted solution for patients who cannot exercise.

r Nutraceutics, e.g. the vegetal polyphenol curcumin, are relatively safe substances. r By lowering oxidative stress, curcumin counteracted the loss of muscle mass and force of soleus muscles, reproduced in the laboratory rat by hindlimb unloading

r Curcumin effects are mediated by the chaperone protein Grp94, which maintains active neuronal nitric oxide synthase molecules at their physiological site in the skeletal myofibre.

r The systemic administration of very low doses of curcumin appears promising for counteracting muscle atrophy in bedridden patients.

Abstract Antioxidant administration aimed to antagonize the development and progression of disuse muscle atrophy provided controversial results. Here we investigated the effects of curcumin, a vegetal polyphenol with pleiotropic biological activity, because of its ability to upregulate glucose-regulated protein 94 kDa (Grp94) expression in myogenic cells. Grp94 is a sarco-endoplasmic reticulum chaperone, the levels of which decrease significantly in unloaded muscle. Rats were injected intraperitoneally with curcumin and soleus muscle was analysed after 7 days of hindlimb unloading or standard caging. Curcumin administration increased Grp94 protein levels about twofold in muscles of ambulatory rats (P < 0.05) and antagonized its decrease in unloaded ones. Treatment countered loss of soleus mass and myofibre cross-sectional area by approximately 30% (P ࣘ 0.02) and maintained a force–frequency relationship closer to ambulatory levels. Indexes of muscle protein and lipid oxidation, such as protein carbonylation, revealed by Oxyblot, and malondialdehyde, measured with HPLC, were significantly blunted in unloaded treated rats compared to untreated ones (P = 0.01). Mechanistic involvement of Grp94 was suggested by the disruption of curcumin-induced attenuation of myofibre atrophy after transfection with antisense grp94 cDNA and by the drug-positive effect on the maintenance of the subsarcolemmal localization of active neuronal nitric oxide synthase molecules, which were displaced to the sarcoplasm by unloading. The absence of additive effects after combined administration of a neuronal nitric oxide synthase inhibitor further supported curcumin interference with this pro-atrophic pathway. In conclusion, curcumin represents an effective and safe tool to upregulate Grp94 muscle levels and to maintain muscle function during unweighting.

C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society

DOI: 10.1113/jphysiol.2013.268672

Downloaded from J Physiol (jp.physoc.org) at NERL Consortia on June 15, 2014

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M. Vitadello and others

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(Received 21 November 2013; accepted after revision 31 March 2014; first published online 7 April 2014) Corresponding author Prof. Luisa Gorza, Department of Biomedical Sciences, Viale G. Colombo 3, 35121 Padova, Italy. Email: [email protected] Abbreviations AS, antisense; β-gal, β-galactosidase; BW, body weight; CSA, cross-sectional area; CSC, crosssectional circumference; DNPH, 2,4-dinitrophenylhydrazine; ER, endoplasmic reticulum; EV, empty vector; Grp94, glucose-regulated protein 94 kDa; HO-1, haem oxygenase 1; Hsp70, inducible heat shock protein 70; MDA, malondialdehyde; MHC, myosin heavy chain; MW, muscle weight; NADPH-d, NADPH diaphorase; 7-NI, 7-nitroindazole; RP, red Ponceau; RT, room temperature.

Introduction Disuse, as the consequence of different levels of unloading and inactivity, occurs frequently in clinical settings (i.e. limb immobilization or bed rest) and leads to rapid skeletal muscle atrophy and significant loss of force production and functional capacity (Powers et al. 2011a; Bodine, 2013). Hindlimb unloading (tail suspension), a model for muscle disuse in the laboratory rat, provided important information on the molecular mechanisms responsible for disuse muscle loss. Oxidative stress has been recently pinpointed as a major event that marks development of muscle atrophy and contributes to its progression (Powers et al. 2011a). The involvement of oxidative stress in muscle wastage represents the rationale for the administration of antioxidants to counteract loss of muscle mass (Bonetto et al. 2009). Although several scavengers of reactive oxygen species failed to attenuate muscle atrophy (Koesterer et al. 2002; Matuszczak et al. 2004; Farid et al. 2005; Desaphy et al. 2010), resveratrol and curcumin, among non-enzymatic antioxidants, apparently offered the best performance, probably because of their pleiotropic effects (Wyke et al. 2004; Jin & Li, 2007; Alamdari et al. 2009; Siddiqui et al. 2009; Momken et al. 2011; Smuder et al. 2012). Curcumin is a low molecular weight polyphenol, derived from the dietary spice turmeric, Curcuma longa. It acts as a scavenger of superoxide anion and hydroxyl radicals and as a pro-oxidant (Sandor et al. 2007; Singh, 2007; Hatcher et al. 2008), and interferes with several different signal transduction pathways (Calabrese et al. 2008; Gupta et al. 2013; Shehzad & Lee, 2013). In vitro and in vivo acute administration of curcumin hampers NF-κB activation (Singh & Aggarwal, 1995; Smuder et al. 2012), which induces the expression of ‘atrogenes’ in muscle (reviewed by Mourkioti & Rosenthal, 2008). Nevertheless, controversial results concerning NF-κB regulation and maintenance of muscle mass were reported after curcumin treatment of disuse muscle atrophy (Farid et al. 2005; Vazeille et al. 2012; Smuder et al. 2012). This could be explained both by the low bioavailability of orally administered curcumin (Gupta et al. 2013) and the major involvement of the non-classical NF-kB pathway in atrophy development (Li et al. 2008). In contrast, curcumin counteracted sepsis-induced muscle

atrophy efficaciously, without apparently affecting the expression of the NF-kB-dependent ubiquitin ligase MuRF (Jin & Li, 2007; Alamdari et al. 2009). This body of evidence suggests that this drug operates through different mechanisms. Previous studies showed that curcumin upregulates the expression of the transcription factor Nrf2, a transactivator of several antioxidant genes, when administered in vitro at a concentration higher than 5 μM (Calabrese et al. 2008; Shehzad & Lee, 2013). Among Nrf2 target genes is haem oxygenase 1 (HO-1) a redox-sensitive inducible protein (Motterlini et al. 2000), which is upregulated in hindlimb muscles of tail-suspended rats (Hunter et al. 2001) and during human experimental bed-rest (Dalla Libera et al. 2009). Curcumin also increased the in vitro expression of other stress proteins, such as the heat shock protein 70 kDa (Hsp70; Khan & Heikkila, 2011), whose upregulation in unloaded muscles was shown to mitigate atrophy (Senf et al. 2008). Moreover, as an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) calcium pump, curcumin can induce the ER stress response (Bakhshi et al. 2008). We demonstrated that, in the myogenic C2C12 cell line, a brief exposure to low concentrations of curcumin selectively upregulated the ER chaperone/stress protein glucose-regulated protein 94 kDa (Grp94), which mediated a delayed antioxidant cytoprotective response (Pizzo et al. 2010). Interestingly, Grp94 overexpression, induced in the unloaded soleus muscle by cDNA electroporation, attenuated myofibre atrophy significantly (Vitadello et al. 2013). To assess whether curcumin upregulates in vivo the expression of one or more of these stress proteins and to investigate its efficacy in preventing muscle loss due to disuse, we administered curcumin to ambulatory and tail-suspended rats. The expression levels of Grp94, Hsp70 and HO-1, degree of protein and lipid carbonylation and changes in mass and tension development of soleus muscle were investigated. Our results show that curcumin administration counteracted the loss of mass and force of 7-day unloaded soleus muscle, by involving the Grp94 chaperone as the prominent player and mechanistically preserving the sarcolemmal localization of neuronal nitric oxide synthase (nNOS).



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Curcumin counters muscle wastage

Methods

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Muscles that contained less than 20 transfected, non-regenerating, myofibres were excluded from the study.

Animals

Six-week-old female Wistar rats (n = 130), weighing between 140 and 160 g, were used. Animals were killed after inducing anaesthesia with isofluorane, except when muscles were used for physiological measurements (see later). Soleus muscles were excised, weighed and frozen in liquid nitrogen. Curcumin treatment. Curcumin (Sigma, Milan, Italy) was

dissolved in dimethyl sulphoxide at a 50 mM concentration and further diluted with sterile saline, to decrease solvent concentration below 0.1%, and administered by intraperitoneal injection at different dosages and frequencies as described in the Results section. Controls were injected with vehicle only. Body weight (BW) was checked daily. Forty animals were used in this part of the study. Hindlimb unloading. Hindlimb unloading was performed using tail suspension as previously described (Dalla Libera et al. 2009), following the recommendations provided by the European Convention for the protection of Vertebrate Animals used for Experimental and Scientific purposes (Council of Europe number 123, Strasbourg, 1985) and after receiving authorization by the Animal Ethics Committee of the University of Padova and the Italian Health Ministry (127/2011B). Animals were weighed, caged individually and randomly assigned to four experimental subgroups: (i) 10-day vehicle-treated freely ambulating controls; (ii) 10-day curcumin-treated freely ambulating controls; (iii) 10-day vehicle-treated and 7-day hindlimb unloaded animals; (iv) 10-day curcumin-treated and 7-day hindlimb-unloaded animals. Forty animals were used for unloading. Muscle gene transfer. A bicistronic construct containing β−galactosidase (β-gal) cDNA and a portion of rabbit grp94 cDNA in antisense (AS) orientation, was used for in vivo transfer into soleus muscles of curcumin and vehicle-treated rats. Empty vector (EV) contained β-gal cDNA only. As previously described, soleus muscle was injected with 50 μg of purified plasmid and DNA electroporated with a train of six 20 ms electrical impulses at 209 V cm−1 with 200 ms intervals (Vitadello et al. 2013). The day after muscle transfection, animals were weighed and either tail-suspended or let free to ambulate, to form four groups of five rats each: (i) freely ambulating and vehicle-treated AS-transfected animals; (ii) hindlimb unloaded and vehicle-treated AS-transfected animals; (iii) hindlimb unloaded and curcumin-treated AS-transfected animals; and (iv) hindlimb unloaded and curcumin-treated EV-transfected animals. After 7 days, animals were weighed and killed.

Curcumin and 7-nitroindazole treatments. The nNOS-

specific inhibitor 7-nitroindazole (7-NI) was administered daily by intraperitoneal injection at 50 mg kg−1 as previously described (Suzuki et al. 2007; Vitadello et al. 2013). Additional rats were injected either with curcumin or with vehicle, or with both 7-NI and curcumin, to form four different groups of five rats each: (i) 7-NI-treated rats; (ii) vehicle-treated rats; (iii) 7-NI- and curcumin-treated rats; and (iv) curcumin-treated rats. On the third day after drug or vehicle administration, animals were tail suspended. After 7 days of hindlimb unloading, animals were weighed and killed. Western blot and Oxyblot

For Western blot analysis, muscle cryosections were homogenized in the presence of SDS and in the absence of bromophenol blue and β-mercaptoethanol and used for protein determination, as previously described (Tarricone et al. 2008). Fifty micrograms of lysate were separated on 10% linear SDS-PAGE, transferred to nitrocellulose and stained with red Ponceau (RP). Parallel nitrocellulose strips were incubated with mouse monoclonal antibodies specific for HO-1 (ABI-OSA-111; Enzo Life Sciences, Farmingdale, NY, USA) and Hsp70 (ABI-SPA-810; Enzo Life Sciences), and with rabbit polyclonal antibodies specific for Grp94 (ABI-SPA-850; Enzo Life Sciences). After incubation with the appropriate secondary antibody conjugated with peroxidase (Santa Cruz Biotech., Heidelberg, Germany and Dakocytomation, Milan, Italy), blots were revealed using chemiluminescence (Bio-Rad Laboratories S.r.l., Rome, Italy). Oxyblot assay was performed as previously described (Dalla Libera et al. 2009; Vitadello et al. 2013), using 12 μg of muscle homogenates for reaction with 2,4-dinitrophenylhydrazine (DNPH; Oxyblot Oxidized Protein Detection Kit; Millipore, Vimodrone, Italy). After SDS-PAGE, proteins transferred to nitrocellulose membranes were first stained with RP and then incubated with anti-DNPH antibody, following the manufacturer’s instructions. Blots were developed using a chemiluminescence detection system. To identify the nature of carbonylated polypeptides, blots were stripped and incubated with rabbit polyclonal anti-actin antibodies (20–33; Sigma). Densitometry of Oxyblots and Western blots was performed on scanned autoradiographic films using the National Institutes of Health Image J software. Values were normalized using the corresponding densitometric value of rat serum albumin visualized by RP staining (Renaud et al. 2013; Vitadello et al. 2013), except for actin



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carbonylation, whose densitometric value was normalized to the corresponding value of RP staining. Immunohistochemistry, 2,4-dinitrophenylhydrazine immunohistochemistry, NADPH-diaphorase histochemistry and morphometric analyses. Identification of type 1 and

type 2A myofibres on serial consecutive soleus muscle cryosections was achieved using antimyosin heavy chain (MHC) mouse monoclonal antibodies, specific for type 1 MHC (clone BA-D5) and for type 2A MHC (clone SC-71) in indirect peroxidase immunohistochemistry, following previously described protocols (Tarricone et al. 2008; Vitadello et al. 2013). For control staining, sections were incubated with non-immune mouse immunoglobulin (1 μg ml−1 ; Sigma) as the primary antibody. The presence of transfected myofibres was demonstrated on cryosections by means of peroxidase immunohistochemistry with the rabbit polyclonal anti-β–gal antibody (5’Prime-3’Prime, Inc., Boulder, CO, USA). Before antibody incubations, sections were fixed 10 min at room temperature (RT) with 4% buffered paraformaldehyde, and endogenous peroxidase activity was inhibited by incubation in methanol added with 0.3% H2 O2 for 30 min at RT. To discriminate between atrophic transfected myofibres and regenerating ones, which could increase in frequency after transfection, serial cryosections were stained with a mouse monoclonal antibody specific for the embryonic MHC isoform (clone BF-G6), as described in Vitadello et al. (2013). Carbonylated myofibres were demonstrated by incubating cryosections with DNPH and, subsequently, with indirect immunoperoxidase to visualize adduct distribution, as described in Vitadello et al. (2013). Enzyme histochemistry for NADPH-diaphorase (NADPH-d) was used to demonstrate the presence of subsarcolemmal localization of active nNOS, and was performed on cryosections as previously described (Vitadello et al. 2013). Morphometric analyses were performed using the Zeiss Microscope Axioplan (Zeiss, Milan, Italy). Images were acquired using a Leica digital DFC 300FX camera and IM50 software (Leica Microsystems SRL, Milan, Italy). Myofibre cross-sectional area (CSA) was evaluated using Image J software. CSA value for each myofibre population (slow and fast fibres) corresponded to the mean of the CSA values measured from 20 to 50 fibres of the same muscle and was expressed in μm2 (Tarricone et al. 2008). The extent of positive sarcolemma for NADPH-d histochemistry was expressed as the percentage of the myofibre cross-sectional circumference (CSC) (Vitadello et al. 2013). Whole muscle contractile properties measurements

The experiments were performed in vitro as described previously (Germinario et al. 2008), using a vertical muscle

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apparatus (300B; Aurora Scientific Inc., Aurora, ON, Canada) containing a Ringer solution of the following composition: 120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2 , 3.15 mM MgCl2 , 1.3 mM NaH2 PO4 , 25 mM NaHCO3 , 11 mM glucose, 30 μM D-tubocurarine, pH 7.2–7.4, 30°C, bubbled with 95% O2 –5% CO2 . Muscles were stretched to the optimal length (i.e. the length that allowed maximal tension development in response to a single pulse) and electrically stimulated, by two parallel electrodes, with supramaximal pulses (0.5 ms duration) delivered by a Grass S44 electronic stimulator through a stimulus isolation unit (Grass SIU5, Warwick, RI, USA). Muscle response was recorded through an isometric force transducer (Harvard, Apparatus, Holliston, MA, USA) connected to an AT-MIO 16AD acquisition card (National Instruments, Rockville, MD, USA) and data were analysed by a specific module of the National Instruments Labview software. Tetanic stimulation was obtained by applying trains of supramaximal stimuli at 80 Hz frequency. Twitch and tetanic tensions were normalized to the muscle wet weight (specific tension, Ng−1 ). Myosin heavy chain electrophoresis

MHC were separated as described by Dalla Libera et al. (2010). Cryosections of soleus muscles were solubilized in SDS-PAGE buffer and loaded on to 7% gels containing 37.5% vol/vol glycerol. Gels were stained with Coomassie blue and the percentage distribution of type 1 and type 2A MHCs was determined by densitometric scan using Image J software. Determination of malondialdehyde

Malondialdehyde (MDA) concentration was determined in muscle samples using HPLC, as described by Mateos et al. (2005) with modifications. In brief, 10 cryosections of 12 μm were collected from each muscle biopsy and solubilized in 250 μl of a buffer containing 250 mM Tris pH 7.4, 0.2 M sucrose, 5 mM DTT. Samples were vortexed and sonicated for 5 min at RT. After addition of 50 μl of 6N NaOH and incubation for 30 min at 60°C, samples were added with 125 μl of 35% perchloric acid and centrifuged at 2800 g for 10 min at RT. From each supernatant, 250 μl were transferred to new tubes, added with 25 μl of a 5 mM DNPH solution in 2 M HCl and incubated for 30 min in the dark at RT. An aliquot of 50 μl of the reaction mixture was injected on to HPLC equipment (Bio-Rad Laboratories S.r.l.) with a Nucleosil 100 RP18 column (4 × 250 mm, 5-μm particle size, 300 A pore size; Bio-Rad Laboratories S.r.l.) and isocratically eluted with a mixture of 0.2% (v/v) acetic acid in water, and acetonitrile (62:38, v/v) at a flow rate of 0.6 ml min−1 at RT. Chromatograms were acquired at 310 nm. A calibration curve was prepared loading MDA dilutions in 0.1 M HCl in phosphate-buffered saline as experimental C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society


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samples. Chromatographic parameters (linearity, limits of detection and quantification, reproducibility and recovery) were calculated for the calibration curve. MDA concentration was expressed as nmol mg−1 of protein. Protein content was estimated as previously described (Dalla Libera et al. 2009) in parallel homogenates. Statistical analyses

All data were expressed as mean ± S.E.M. One-way ANOVA and Newman–Keuls or Bonferroni test for post hoc analysis were used for multiple comparisons. Within-subject ANOVA (randomized complete block design with participants matched on BW) and Bonferroni test for post hoc analysis were used to compare average CSA and CSC of transfected and untransfected myofibres of muscles obtained from ambulatory or unloaded rats of the same BW (matched participants). P value = 0.05 was set as the limit for significance. Statistical analyses were performed using SigmaStat software, version 2.0 (Jandel Europe, Munich, Germany) or Statistica software, version 5.1 (Statsoft Inc., Tulsa, OK, USA). Results Curcumin administration increases muscle Grp94 protein levels in the ambulatory rat

Doses of 20–40 μg kg−1 of curcumin were administered daily, for 3 days, intraperitoneally to two groups of standardly caged 6-week-old female rats and a third group was injected with vehicle. To adapt the treatment to the tail-suspended rat model, drug or vehicle was administered to subgroups of rats for further 7 days, either daily or on alternate days, monitoring animal BW (Fig. 1A). No difference in BW was detected after 3 days of treatment of either 20 μg kg−1 (not shown) or 40 μg kg−1 (Fig. 1A), whereas daily treatment for 10 days with 40 μg kg−1 of curcumin not only counteracted significantly the physiological increase in BW, but also decreased it (P < 0.001; Fig. 1A). However, when this latter dosage was administered daily for 3 days, and, subsequently, every other day, BW did not differ from vehicle-treated rats (Fig. 1A). Muscles obtained from rats treated for 3 days or for 10 days with this last protocol were then investigated for protein levels of Grp94 and other stress protein/chaperones, such as HO-1 and Hsp70 (Fig. 1B), whose expression is known to be affected by curcumin administration (Motterlini et al. 2000; Khan and Heikkila, 2011) and/or by muscle unloading (Hunter et al. 2001; Senf et al. 2008; Dalla Libera et al. 2009; Renaud et al. 2013, Vitadello et al. 2013). Whereas Grp94 protein levels increased significantly in soleus muscle of

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curcumin-treated rats (P = 0.04; Fig. 1C), the increase in Hsp70 and HO-1 protein levels did not appear to be statistically significant (Fig. 1D and E, P = 0.19 and 0.13, respectively). Curcumin administration counteracts atrophy and loss of force of unloaded soleus

Two rat groups were daily injected for 3 days with 40 μg kg−1 curcumin or vehicle and then randomly divided in two further groups, which were either hindlimb unloaded or let free to ambulate, and injected every other day. Seven-day unloading decreased soleus muscle weight (MW) and MW/BW ratio, compared to ambulatory rats (P < 0.001; Fig. 2A; Dalla Libera et al. 2009; Vitadello et al. 2013). Curcumin treatment during unloading attenuated the decrease of both soleus MW and MW/BW, which appeared about 30% higher than that of unloaded soleus muscle of vehicle-injected rats (P = 0.02; Fig. 2A). Similar results were obtained after measuring fibre CSA (Fig. 2B and C). Soleus fast and slow fibres of tail-suspended and curcumin-treated rats showed an equivalent decrease in atrophy degree, which corresponded to about 27% and 33%, respectively, compared to the 38% and 46% decrease observed in unloaded soleus of vehicle-treated rats (P < 0.02). Figure 3 illustrates the effects of curcumin treatment on twitch and tetanic tensions of soleus muscle of ambulatory and unloaded rats. Whereas curcumin administration did not change the absolute tension developed after a single twitch by ambulatory or unloaded soleus muscle (Fig. 3A), it increased the absolute tetanic tension of unloaded soleus muscle, compared to that of the vehicle-treated one (P < 0.05; Fig. 3B). Interestingly, the significant increase in maximum developed tension was still evident after normalization to MW (P < 0.005; Fig. 3C), suggesting that curcumin exerted additional effects on unloaded muscle contractility. Moreover, curcumin treatment maintained a force–frequency relationship closer to ambulatory levels (Fig. 3E), which is at variance with soleus muscle from vehicle-treated and tail-suspended rats. Fibre type and MHC compositions were therefore investigated. Seven days of hindlimb unloading did not significantly change the relative percentage of type 1 and 2A fibre populations, identified by immunohistochemistry using anti-MHC specific antibodies (mean and S.E.M. percentage of type 2A myofibres of ambulatory and unloaded soleus muscle 12.73% ± 2.66 and 15.75% ± 3.37, respectively, n of muscles = 7; approximately 1000 fibres were evaluated for each muscle). Comparably, the relative percentage of slow or fast 2A MHCs, determined after electrophoretic separation, was not changed (mean and S.E.M. percentage of 2A MHC of ambulatory and unloaded soleus muscles 20.00% ± 3.71 and 20.25% ± 2.05, respectively, n of muscles = 4).



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Curcumin administration did not affect the relative proportion of slow and 2A MHCs, neither in ambulatory soleus muscle nor in the unloaded one (mean and S.E.M. percentage of 2A MHCs of curcumin-treated ambulatory and unloaded soleus muscle 17.50% ± 3.75 and 22.50% ± 1.19, respectively, n of rats = 4). Curcumin administration counteracts oxidation of unloaded soleus muscle

Further analyses investigated whether the antioxidant property of curcumin counteracted the oxidative stress accompanying muscle unloading. Muscle protein carbonylation, evaluated by means of the Oxyblot assay, increased significantly after unloading (Dalla Libera et al. 2009; Renaud et al. 2013; Vitadello

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et al. 2013). Curcumin treatment antagonized the increase in protein carbonylation (P = 0.01, Fig. 4A and B). Densitometry of single carbonylated polypeptides revealed actin as one major protein involved (Fig. 4A and C). Actin carbonylation level normalized to the RP staining of the protein, whose identity was confirmed by immunostaining (not shown), increased after unloading (P = 0.01), whereas, in the presence of curcumin administration, it appeared significantly decreased (P = 0.05) (Fig. 4C). MDA represents an index of lipid oxidation and its muscle levels were determined using the highly specific HPLC technique (Fig. 4D). MDA concentration increased significantly in unloaded soleus muscle (P < 0.001), whereas it remained at ambulatory levels after curcumin treatment.

Figure 1. Effects of Cu treatment on BW and stress protein/chaperone levels of the soleus muscle A, bar graph show the percentage increment in BW after daily intraperitoneal administration of vehicle (white bars) or 40 μg kg−1 Cu (grey bars). Black bar indicates the relative increment of BW after administration of 40 μg kg−1 Cu on alternate days from day 3. Single asterisks indicate a significant difference vs. day 3 values; double asterisks indicate a significant difference vs. all the groups. ∗ P ࣘ 0.02; ∗∗ P = 0.001 (ANOVA). B, representative Western blot of soleus muscles of ambulatory rats in the absence and in the presence of a daily administration of 40 μg kg−1 Cu for 3 days and on alternate days from day 3 to day 10, labelled for Grp94, Hsp70 and HO-1. Staining of SA is shown as a reference for sample loading. C–E, bar graph show the normalized amount of Grp94 (C), Hsp70 (D) and HO-1 (E) protein levels in soleus muscles of vehicle-injected rats (C labeled bar graph) and after administration of Cu, as described in (B). Asterisks indicate a significant difference vs. C : ∗ P = 0.04 (ANOVA). n indicates the number of rats used in each experimental group. Values are means ± S.E.M. BW, body weight; Cu, curcumin; Grp94, glucose-regulated protein 94 kDa; HO-1, haem oxygenase 1; Hsp70, heatshock protein 70; SA, serum albumin. C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society


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Curcumin administration attenuates unloading atrophy by increasing Grp94 protein levels

We then investigated the effect of curcumin administration on the relative amount of Grp94, Hsp70 and HO-1 stress-proteins/chaperones, which are known to be variably affected by muscle unloading (Fig. 5; Naito et al. 2000; Hunter et al. 2001; Oishi et al. 2001, 2008; Lawler et al. 2006; Senf et al. 2008; Renaud et al. 2013; Vitadello et al. 2013). Western blot analysis confirmed the significant decrease in Grp94 relative protein levels (Renaud et al. 2013; Vitadello et al. 2013) and the four-fold increase in HO-1 levels (Hunter et al. 2001) in unloaded soleus muscle. Conversely, almost no change was detected in Hsp70 amount after 7 days unloading, consistently with Oishi et al. (2001). Curcumin treatment abolished

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the unloading-induced decrease of Grp94 protein levels (P < 0.01; Fig. 5A and B) and increased, without statistical significance, Hsp70 relative levels (P = 0.09; Fig. 5A and C). Furthermore, it antagonized the unloaded-induced increase in the HO-1 relative amount, which remained at ambulatory levels (P < 0.001; Fig. 5D). We then investigated whether curcumin counteracted muscle atrophy by acting upon Grp94 expression. Curcumin administration was combined with the in vivo transfection in soleus muscle of AS cDNA for Grp94 or of EV and the subsequent exposure to hindlimb unloading for 7 days. As both constructs contained β-gal cDNA, immunohistochemistry for β-gal was used to identify transfected myofibres (Fig. 6A). Unloaded soleus muscle of curcumin-treated rats showed lower CSA of AS-transfected myofibres, compared to

Figure 2. Effect of Cu treatment on soleus muscle and fibre atrophy of tail-suspended rats A, bar graph showing BW, soleus MW and MW/BW ratio of ambulatory and 7-day tail-suspended (U) rats, exposed before unloading to daily injection for 3 days with 40 μg kg−1 Cu or vehicle and then injected every other day. B, representative cryosections of soleus muscle of unloaded rats treated or not with Cu. Dark staining corresponds to immunoperoxidase reactivity with anti-slow myosin antibody. Bar: 100 μm. C, bar graph show CSA values of slow and fast fibres of ambulatory and U soleus muscles, in the presence or in the absence of Cu treatment. At least 50 fibres were examined for group in each muscle. Asterisks indicate statistically significant difference of U soleus values vs. ambulatory values and lines emphasize the difference between Cu-treated and vehicle-treated U muscles: P < 0.02 (ANOVA). n indicates the number of rats/muscles used in each experimental group. Values are means ± S.E.M. A, ambulatory; BW, body weight; CSA, cross-sectional area; Cu, curcumin; MW, muscle weight; U, unloaded. C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society


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untransfected ones, which, being affected by the curcumin treatment, displayed a significantly higher CSA value than that of myofibres of unloaded soleus muscle of vehicle-treated rats (Fig. 6B, within-subject ANOVA). Conversely, AS transfection did not apparently affect myofibre CSA of vehicle-treated unloaded muscles, nor it affected vehicle-treated ambulatory ones (Fig. 6B), whose transfected and untransfected fibre CSA was 1732.92 ± 164.37 μm2 and 1802.10 ± 227.51 μm2 , respectively. The efficiency of the AS construct in counteracting curcumin treatment was further proven by the persistence of increased CSA value detected after EV transfection and unloading of curcumin-treated soleus muscle (Fig. 6A and B; within-subject ANOVA P < 0.01). To investigate whether inhibition of curcumin-induced Grp94 expression failed to counteract oxidative stress,

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adjacent cryosections were incubated with DNPH and reacted with specific antibodies (Fig. 6C). The percentage of transfected carbonylated myofibres was about three-fold greater after transfection with AS compared to EV (Fig. 6D; Student’s t test, P = 0.01). Curcumin administration prevents unloading-induced loss of subsarcolemmal NADPH diaphorase activity

Delocalization of nNOS from sarcolemma represents one major initiating event leading to disuse muscle atrophy (Suzuki et al. 2007) and we demonstrated that it can be prevented by the expression of recombinant Grp94 (Vitadello et al. 2013). As curcumin administration maintained Grp94 protein amounts of the unloaded soleus muscles at levels comparable to those of the ambulatory ones, we investigated whether the treatment affected also

Figure 3. In vitro tension measurements on ambulant and unloaded soleus muscle A–C, effect of Cu treatment on ambulatory and U soleus muscle. A, isometric absolute twitch tension; B, isometric absolute tetanic tension; C, isometric specific tetanic tension (isometric absolute tension normalized to muscle wet weight). Asterisks indicate a statistically significant difference of U soleus values vs. ambulatory values and lines emphasize the difference between Cu-treated and vehicle-treated U muscles: ∗ P < 0.05 and ∗∗ P < 0.005. Values are means ± S.E.M. of at least six muscles. D and E, effect of Cu treatment on tension–frequency curve. Soleus muscle was stimulated at various frequencies under isometric conditions. Tension is expressed as relative to the maximum tetanic force (D) and as specific tension (E). Symbols indicate a significant difference: ∗ P < 0.05 and ∗∗ P < 0.005 between U and all groups; # P < 0.05 between U + Cu and ambulatory and between U + Cu and ambulatory + Cu. Values are means ± S.E.M. of at least six muscles. A, ambulatory; Cu, curcumin; U, unloaded; P, tension; Pt, twitch tension; P0 , tetanic tension. C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society



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subsarcolemmal nNOS distribution, by demonstrating the presence of the active enzyme at the sarcolemma by means of NADPH-d histochemistry. As previously shown by our and other laboratories (Rothe et al. 2005; Vitadello et al. 2013), NADPH-d histochemistry decorated discrete regions of myofibre sarcolemma in rat ambulatory soleus muscle (Fig. 7A). Unloaded soleus muscle showed a dramatic decrease in the percentage of reactive CSC for NADPH-d (P = 0.0002; Fig. 7A and B). At variance, in the presence of curcumin treatment, the positive CSC percentage for NADPH-d in unloaded myofibres did not significantly differ from that one observed in ambulatory muscle (Fig. 7A and B). The role of Grp94 in mediating the curcumin effect on active nNOS subcellular localization was further clarified by analysing the CSC percentage of NADPH-d–positive sarcolemma of AS Grp94cDNA-transfected myofibres of hindlimb unloaded and curcumin-treated rats (Fig. 7C),

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and by comparing it with those of both untransfected and EV-transfected myofibres (Fig. 7D). Results show that AS transfected myofibres displayed a significant lower CSC percentage of NADPH-d-positive sarcolemma (P < 0.01, within-subject ANOVA), despite curcumin treatment, providing further evidence for the requirement of Grp94. As nNOS untethering from the sarcolemma was hypothesized to contribute to muscle disuse atrophy by increasing sarcoplasmic nitrosative stress (Suzuki et al. 2007; Powers et al. 2011b), the specific inhibitor 7-NI was administered together with curcumin. Treatment with 7-NI did not affect Grp94 expression, which decreased significantly after unloading and was maintained at ambulatory levels when 7-NI treatment was combined with curcumin (Fig. 7E; P = 0.02, ANOVA). As shown by Suzuki et al. (2007) and recently confirmed by us (Vitadello et al. 2013), treatment with 7-NI attenuated atrophy of unloaded soleus. Curcumin administration

Figure 4. Effect of Cu treatment on protein carbonylation and MDA levels of ambulatory and unloaded soleus muscles Representative Oxy (left side) and corresponding RP staining of the same gel (right side) of muscle lysates, in the presence and in the absence of U and/or Cu administration. B and C, bar graph show the normalized levels of protein carbonylation (B) and those of actin carbonylation (C) in soleus muscles of ambulatory and U rats exposed or not to Cu. D, bar graph show MDA concentration of soleus muscles of ambulatory and U rats exposed or not to Cu. Single asterisk indicates significant difference (P ࣘ 0.05) vs. all groups; double asterisk indicates significant difference (P = 0.01) vs. values of ambulatory vehicle-treated rats (ANOVA). n indicates the number of rats used in each experimental group. Values are means ± S.E.M. Cu, curcumin; MDA, malondialdehyde; Oxy, Oxyblot; RP, Red Ponceau; SA, serum albumin; U, unloading. C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society


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combined to 7-NI treatment did not increase further CSA of unloaded myofibres (Fig. 7F), ruling out its influence on other signalling pathways involved in atrophy progression.

Discussion This study demonstrates that curcumin administration counteracts unloading-induced muscle protein carbonylation and atrophy. These effects can be mechanistically explained by the maintenance of Grp94 expression at physiological levels. In fact, Grp94 knock-down impaired both the attenuation of myofibre atrophy and oxidation and the sarcolemmal localization of active nNOS molecules despite curcumin treatment. Other mechanisms were excluded by the absence of additional anti-atrophic effects when curcumin was administered together with a specific nNOS inhibitor.

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Curcumin counteracts loss of mass and force of unloaded soleus muscle

Prevention of muscle disuse atrophy has been attempted in experimental animal models by administering non-enzymatic antioxidants, among which curcumin, as diet supplements, with contrasting results (Koesterer et al. 2002; Matuszczak et al. 2004; Farid et al. 2005; Arbogast et al. 2007; Servais et al. 2007; Momken et al. 2011). Here we show that the intraperitoneal injection of curcumin succeeded in attenuating soleus muscle atrophy of tail-suspended rats, similar to that observed in other models after acute administration (Jin & Li, 2007; Smuder et al. 2012). Our protocol of chronic curcumin administration was designed to avoid BW loss (Hatcher et al. 2008) and increase, at the same time, muscle protein levels of Grp94. Curcumin dosage appeared crucial in determining the effects on disuse muscle atrophy. No effect was reported by Vazeille et al. (2012) by daily intraperitoneal injection of 1 mg kg−1 curcumin, whereas in

Figure 5. Effect of Cu treatment on stress-protein/chaperone levels of the unloaded soleus muscle A, representative Western blot analysis of soleus muscles lysates of rats in the presence and in the absence of U and/or Cu administration, labelled with antibodies for Grp94, Hsp70 and HO-1. SA is shown as a reference for sample loading. B–D, bar graph show means ± S.E.M. of normalized levels of Grp94 (B), Hsp70 (C) and HO-1 (D) in soleus muscles of ambulatory and U rats exposed or not to Cu treatment. Asterisks indicate a significant difference: ∗ P = 0.05 vs. vehicle-treated ambulatory group; ∗∗ P ࣘ 0.01 vs. all groups (ANOVA), n indicates the number of muscles analysed in each experimental group. A, ambulatory; Cu, curcumin; Grp94, glucose-regulated protein 94 kDa; HO-1, haem oxygenase 1; Hsp70, heatshock protein 70; SA, serum albumin; U, unloading. C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society


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the present study a 25–50-fold lower dosage attenuated soleus muscle atrophy. The anti-atrophic effect of curcumin is consistent with similar findings obtained after diet supplementation with vitamin E (Servais et al. 2007), or administration of a soy protein derivative (Arbogast et al. 2007) or resveratrol (Momken et al. 2011). Although each of these compounds displayed direct antioxidant capacity, only the soy protein derivative (Arbogast et al. 2007), vitamin E (Servais et al. 2007) and curcumin (this study) hampered the increase

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in lipid and/or protein carbonylation secondary to muscle unloading. In addition, we demonstrated that curcumin treatment increases muscle-specific force development, in contrast to the soy protein derivative and resveratrol (Arbogast et al. 2007; Momken et al. 2011), but similar to allopurinol (Matuszczak et al. 2004). The loss of force of the unloaded soleus not only results from muscle atrophy, but also because fibres were dysfunctional in their ability to generate tension. As reported by Dalla Libera et al. (2005),

Figure 6. Effect of AS Grp94 transfection in Cu- and saline-treated soleus muscles of tail-suspended rats A, representative cryosections of Cu-treated soleus muscles after 7-day U and transfection with AS Grp94 cDNA (left panel) or the EV (right panel). Dark staining corresponds to immunoperoxidase reactivity for β-gal, which served as transfection marker (arrows). B, bar graph of means ± S.E.M. values of CSA of unloaded soleus muscle fibres transfected (β-gal+) and untransfected (–) with AS or EV in Cu-treated and untreated rats. At least 15 fibres were examined for group in each muscle. Asterisks indicate a significant difference P < 0.01 (within-subject ANOVA), n indicates the number of muscles analysed in each experimental group. Arrow indicates mean fibre CSA value of ambulatory AS transfected soleus muscles. C, adjacent cryosections to those depicted in (A) were reacted with DNPH and stained with indirect immunoperoxidase. Arrows indicate representative transfected myofibres positive for carbonylation. Bar: 100 μm. D, bar graph of the means ± S.E.M. values of the percentage of DNPH-positive myofibres detected among AS- or EV-transfected myofibres in soleus muscles of Cu-treated unloaded rats. At least 40 fibres were examined for group in each muscle. Asterisk indicates a significant difference P = 0.01 (Student’s t test), n indicates the number of muscles analysed in each experimental group. AS, antisense; β-gal, β-galactosidase; CSA, cross-sectional area; Cu, curcumin; DNPH, 2,4-dinitrophenylhydrazine; EV, empty vector. C 2014 The Authors. The Journal of Physiology C 2014 The Physiological Society


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Figure 7. Effect of Cu treatment on subsarcolemmal neuronal nitric oxide synthase localization of tail-suspended rats A, representative cryosections of soleus muscles of ambulatory rats, after 7-day U and after both U + Cu treatment, processed for NADPH-d histochemistry. Sarcolemmal dark staining corresponding to active neuronal nitric oxide synthase remains detectable in unloaded myofibres of Cu-treated rats. Bar: 80 μm. B, bar graph representing the percentage of fibre CSC positive for NADPH-d. At least 15 fibres with detectable sarcolemmal NAPDH-d reactivity were examined for group in each muscle. ∗ P < 0.001 vs. ambulatory and U + Cu (ANOVA). C, consecutive cryosections of a representative U + Cu soleus muscle after transfection with AS Grp94 cDNA, stained for NADPH-d histochemistry (left panel) or β-galactosidase immunoperoxidase (right panel). Arrows indicate poor subsarcolemmal NADPH-d staining in representative AS-transfected myofibres. Bar: 80 μm. D, bar graph representing the percentage of positive CSC for NADPH-d in U + Cu soleus muscles after transfection with AS or the EV. At least 15 fibres were examined for group in each muscle. ∗ P < 0.01 vs. all the other groups (within-subject ANOVA). E, left, representative Grp94 Western blot of ambulatory and U soleus muscle homogenates from 7-NI rats in the absence and in the presence of Cu treatment. Staining of SA is shown as a reference for sample loading. Right, bar graph show normalized levels of Grp94. ∗ P = 0.02 vs. ambulatory + 7-NI and U + 7-NI + Cu (ANOVA). F, bar graph of CSA values of U soleus muscle fibres from rats exposed or not to 7-NI combined with Cu. At least 30 fibres were examined per group in each muscle. ∗ P ࣘ 0.01 (ANOVA). Arrows indicate mean CSA values of slow (left arrow) and fast (right arrow) ambulatory muscle fibres. n indicates the number of rats used in each experimental group. Values are means ± S.E.M. A, ambulatory; AS, antisense; β-gal, β-galactosidase; CSA, cross-sectional area; CSC, cross-sectional circumference; Cu, curcumin; EV, empty vector; Grp94, glucose-regulated protein 94 kDa; NADPH-d, NADPH-diaphorase; 7-NI, 7-nitroindazole; SA, serum albumin; U, unloading.



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the oxidized state of myosin, actin and tropomyosin negatively affect the capacity of force development. The increased specific force displayed by unloaded soleus muscle of curcumin-treated rats compared to untreated ones can be interpreted as an ameliorated redox state. Here we show that curcumin treatment decreased actin carbonylation of unloaded soleus muscle. Grp94 mediates the curcumin effect against skeletal muscle atrophy

The attenuation of muscle mass and force loss obtained after curcumin administration implies the involvement of drug-specific mechanisms. Several lines of evidence indicate that curcumin may interact directly with different molecules and act on signal transduction pathways, in addition to performing as a radical scavenger (Singh, 2007; Hatcher et al. 2008; Gupta et al. 2013; Shehzad & Lee, 2013). In particular, curcumin was shown to hamper activation of the NF-κB, a major regulator of muscle ‘atrogenes’ (Mourkioti & Rosenthal, 2008). Even though recent investigations confirmed involvement of the classical NF-κB pathway in the development of disuse atrophy (van Gammeren et al. 2009), the presence of nuclear p65 NF-κB subunit was detected only occasionally in myofibre nuclei of 7-day unloaded soleus muscle (M. Vitadello and L. Gorza, unpublished observation), making the involvement of such a pathway in curcumin-treated muscles unlikely. On the other hand, the present protocol of curcumin administration unveiled a selective expression of Grp94 protein at levels similar to those detected in myogenic C2C12 cells in vitro (Pizzo et al. 2010). Curcumin did not change muscle levels of Hsp70, another chaperone/stress-protein involved in hampering disuse muscle atrophy (Naito et al. 2000; Senf et al. 2008). Strikingly, the treatment variably affected muscle expression of HO-1, a recognized curcumin target gene (Motterlini et al. 2000). The dosage employed did not increase the protein level in the ambulatory soleus, thus suggesting that the actual drug concentration was not sufficient to activate the Nrf2 pathway (Shehzad & Lee, 2013). Unexpectedly, curcumin counteracted the increase of HO-1 expression in unloaded muscles (Hunter et al. 2001). Such an effect might be accounted for oxidative stress blunting, a strong stimulus for HO-1 expression (Ryter et al. 2006), as shown by the lack of increase in protein carbonylation and MDA accumulation. Grp94 is highly expressed by mammalian skeletal muscle cells during differentiation and maturation (Gorza & Vitadello, 2000; Vitadello et al. 2010), when it plays a crucial role as the exclusive chaperone of insulin-like growth factors (IGFs; Wanderling et al. 2007). Conversely, Grp94 is scantily expressed in adult skeletal myofibres (Vitadello et al. 1998, 2010), and its level decreases in

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an apparently selective way during muscle unloading, compared to other ER resident chaperones (Vitadello et al. 2013). Changes in Grp94 protein level in ambulatory muscles, either by curcumin or AS transfection, did not affect myofibre size. In contrast, in unloaded soleus muscle, the maintenance of adequate amounts of Grp94 by cDNA transfection (Vitadello et al. 2013) and/or by curcumin (this study) is required to counteract fibre oxidative stress and atrophy. The cytoprotective effect of chaperones is usually explained by their ability to prevent protein aggregation and assist the refolding of denatured or partially folded proteins (Ma & Hendershot, 2004). Protein levels of the exclusive Grp94 clients’ IGFs are apparently not affected by muscle unloading (Vitadello et al. 2013). Although curcumin was demonstrated to improve insulin resistance (Na et al. 2011), which characterizes unloaded muscles (Nakao et al. 2009), it was also reported to hamper IGF-1 maturation by interfering with pre-proconvertases (Zhu et al. 2013). In addition to being an exclusive chaperone, Grp94 acts as a regulator of both store-releasable calcium (Vitadello et al. 2003; Bando et al. 2004; Pizzo et al. 2010) and nNOS subcellular distribution (Vitadello et al. 2013). Immunoprecipitation, membrane fractionation experiments and confocal microscopy showed that a number of Grp94 molecules interact with nNOS. Upregulation of Grp94 is required to counteract nNOS untethering from the sarcolemma of unloaded myofibres (Vitadello et al. 2013). Delocalization of nNOS active molecules from sarcolemma to sarcoplasm has been recognized as a major event favouring increased NO availability at this site and improving activation by nitrosylation of atrophy regulators, such as FoxO3a (Suzuki et al. 2007; Vitadello et al. 2013), and of molecules involved in calcium homeostasis, such as the ryanodine receptor (Salanova et al. 2008; Gentil et al. 2012). Here we show that curcumin administration during hindlimb unloading favoured the maintenance of active nNOS at the sarcolemma and that the drug-induced atrophy attenuation was blunted by AS grp94 cDNA. The absence of additional, curcumin-induced effects after nNOS inhibition by 7-NI administration provides a further piece of evidence enucleating this molecular pathway in curcumin anti-atrophic protection of unloaded soleus muscle. Although blunting of nNOS activity did not fully antagonize soleus muscle atrophy due to unloading (Suzuki et al. 2007; Vitadello et al. 2013), it significantly abolished the increase in muscle protein carbonylation (Vitadello et al. 2013). Such an effect also characterized curcumin administration. In particular, curcumin counteracted actin carbonylation, but not that of MHCs (our unpublished observations). Furthermore, absence of active nNOS antagonized muscle weakness



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caused by unloading (Suzuki et al. 2007). Similarly, curcumin administration prevented the force–frequency shift occurring after unloading. In conclusion, the findings presented here indicate curcumin as a safe pharmacological tool to attenuate unloading muscle atrophy and counteract force loss. Our study identifies the ER chaperone/stress protein Grp94 as the major transducer of these effects by preserving the physiological localization of active nNOS molecules at the sarcolemma of unloaded myofibres.

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Additional information Competing interests The authors have no competing financial interests.

Author contributions The experimental work was performed in the research laboratory of L.G., with the assistance of M.V. and B.R. MDA determination was performed by L.D-L. Physiological studies were performed by E.G. and D.D-B. L.G. initiated/designed the experiments and wrote the manuscript with assistance from M.V. and D.D-B. All authors approved the final version of the manuscript.

Funding The financial support of Agenzia Spaziale Italiana (grant OSMA-WP1B51–2 to L.G.) and of Miur-Prin 2009 (to D.D-B.) is gratefully acknowledged.



PGC1-α over-expression prevents metabolic alterations and soleus muscle atrophy in hindlimb unloaded mice.

Atrophy of the soleus muscle by hindlimb unweighting.

Purification of insoluble nitric oxide synthase from rat cerebellum.

Kinetic characteristics of nitric oxide synthase from rat brain.

Kinetic characteristics of nitric oxide synthase from rat brain.

A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage.

Colocalization of cannabinoid receptor 1 with somatostatin and neuronal nitric oxide synthase in rat brain hypothalamus.

Functional regulation of neuronal nitric oxide synthase expression and activity in the rat retina.

Simulated weightlessness affects the expression and activity of neuronal nitric oxide synthase in the rat brain.

Neuronal Nitric Oxide Synthase in Vascular Physiology and Diseases.

Nitric oxide signalling and neuronal nitric oxide synthase in the heart under stress.

Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from L-arginine.

Phosphorylated neuronal nitric oxide synthase in neuropathic pain in rats.

Selective Irreversible Inhibition of Neuronal and Inducible Nitric-oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide.

Intracallosal neuronal nitric oxide synthase neurons colocalize with neurokinin 1 substance P receptor in the rat.

Histochemical mapping of nitric oxide synthase in the rat brain.

Distinct conformational behaviors of four mammalian dual-flavin reductases (cytochrome P450 reductase, methionine synthase reductase, neuronal nitric oxide synthase, endothelial nitric oxide synthase) determine their unique catalytic profiles.

Glucose uptake during contraction in isolated skeletal muscles from neuronal nitric oxide synthase μ knockout mice.

Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum.

Pterins inhibit nitric oxide synthase activity in rat alveolar macrophages.

Induction of nitric oxide synthase in rat peritoneal neutrophils and its inhibition by dexamethasone.

Differential induction of brain, lung and liver nitric oxide synthase by endotoxin in the rat.

Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase: Acceleration of fracture healing via inducible nitric oxide synthase.

Calmodulin is a subunit of nitric oxide synthase from macrophages.