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
The interaction of nitric oxide synthase (NOS) with a
variety of proteins plays an important role in the regu-
lation of NO production [1–4]. Of these interacting
proteins, heat shock protein 90 (HSP90) has been pro-
posed 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 Ca
2+
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 haem-
deficient 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 degrada-
tion of NOS and HSP90 [17–26].

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 stabil-
ization 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.
Abbreviations
[Ca
2+
]
i
, intracellular Ca
2+
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
We have recently demonstrated that the suscep-
tibility to calpain degradation of purified endothelial
NOS (eNOS) and neuronal NOS (nNOS) is signifi-
cantly 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 sub-
strates. Furthermore, when NOS isozymes are associ-
ated with this binary complex, they also become
resistant to proteolytic degradation. These observa-

HSD.
We report here that, in the brain and aorta of HSD-
treated rats, the extent and patterns of proteolytic deg-
radation of NOS isozymes and HSP90 are similar to
those previously detected in Jurkat and BAE-1 cells
loaded with Ca
2+
[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 degra-
dation.
Results
Levels of HSP90 and NOS isozymes in rat brain
and aorta
The level of HSP90 and the type of NOS isoform pres-
ent in rat brain and aorta were determined by immu-
noblotting (Fig. 1). In brain, nNOS was the most
preferentially expressed isoform, together with traces
of eNOS (Fig. 1A). In aorta, only eNOS isozyme was
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
A
B
C
Fig. 1. NOS isozymes and HSP90 expressed in brain and aorta of

]
i
, HMS rats were also
used, as a limited increase in [Ca
2+
]
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. Fur-
ther direct evidence in support of calpain activation in

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
A
B
Fig. 2. 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 proce-
dures. The values reported are the arithmetical means ± standard
deviation of five different experiments carried out on five different
animals of each strain.
M. Averna et al. In vivo degradation of NOS and HSP90 by calpain
FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS 2503
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

and aorta of NMS and HMS rats treated with HSD for 4 weeks.
The data reported are the arithmetical means ± standard deviation
of five different experiments carried out on five different animals of
each strain.
Animal Treatment
a
Total
calpastatin
activity (%)
b
15 kDa
fragment
activity (%)
c
Loss of total
calpastatin
activity (%)
d
Brain
NMS None 97 ± 5 3 ± 0.3 0
NMS HSD 40 ± 2 40 ± 3 20
HMS None 90 ± 6 10 ± 2 0
HMS HSD 5 ± 1 49 ± 2 46
Aorta
NMS None 99 ± 5 1 ± 1 0
NMS HSD 16 ± 2 25 ± 2 59
HMS None 64 ± 4 16 ± 2 20
HMS HSD 2 ± 0.5 14 ± 1 84
a
NMS and HMS rats were fed for 4 weeks with HSD as described

+ HSDControl
Fig. 3. 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 fol-
lowed 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 l
M C.I.1, were
submitted to 8% SDS-PAGE followed by immunoblotting. Desmin
and talin were detected with specific mAbs.
In vivo degradation of NOS and HSP90 by calpain M. Averna et al.
2504 FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS
of conditions favouring and stabilizing the heterocom-
plex 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 degrada-
tion 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 proteo-
lytic degradation of NOS in vivo still remain to be
defined. One of these molecular signals could be

to be addressed. The first question concerns the vul-
nerability of different NOS isoforms to proteolysis
in vivo under conditions of small changes to [Ca
2+
]
i
.
The second question concerns the capacity of HSP90
to protect NOS in vivo against proteolytic degrada-
A
B
CD
Fig. 4. 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 materi-
als 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.
M. Averna et al. In vivo degradation of NOS and HSP90 by calpain
FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS 2505
tion. Finally, a third question involves the possi-
ble 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 [Ca
2+
]

aorta. The reduced availability of HSP90 in aorta can
thus explain the increased vulnerability of eNOS rela-
tive to nNOS to proteolysis. On the basis of these find-
ings, we propose a novel mechanism in which HSP90
can provide functional stability of NOS isozymes
under conditions characterized by an alteration in
intracellular Ca
2+
homeostasis.
Experimental procedures
Materials
Leupeptin C.I.1, aprotinin, phosphatase inhibitor cocktail I
and II, NADPH, calmodulin, FAD, FMN, tetrahydrobiop-
terin, l-arginine and aldolase were purchased from Sigma
Aldrich, Milan, Italy. l-[
14
C]arginine (925 Bq; specific activ-
ity, 1Æ14 · 10
11
BqÆmol
)1
), Sephacryl S-300, Sephadex G-
200 resins, Superose
Ò
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

56.3) mAbs were produced as indicated in [35] and [36],
respectively.
Animals
NMS and HMS rats [37] were housed in controlled condi-
tions (22 ± 1 °C; humidity, 50 ± 5%; lighting, 8–20 h).
Systolic blood pressure was measured by tail-cuff plethys-
mography [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 ± 5 and 145 ± 10 mmHg, respec-
tively.
Experimental hypertension
Experimental hypertension was induced in 60-day-old rats
by feeding ad libitum with a standard rat chow and provid-
ing NaCl dissolved in tap water at a concentration of
10 gÆL
)1
for a period of time ranging from 15 to 30 days.
Each animal received approximately 0.7 gÆday
)1
of NaCl.
Where indicated, 25 lm C.I.1 was dissolved in tap water in
the presence of 10 gÆL
)1
NaCl, and administered to NMS
and HMS rats for 4 weeks [28]. Each rat received 0.5–
0.7 mgÆday
)1
of C.I.1. Experiments were carried out follow-

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 arith-
metical means ± standard deviation of five different experiments carried out on five different animals of each strain.
M. Averna et al. In vivo degradation of NOS and HSP90 by calpain
FEBS Journal 275 (2008) 2501–2511 ª 2008 The Authors Journal compilation ª 2008 FEBS 2507
were then blotted onto a nitrocellulose membrane and
saturated with a NaCi/P
i
solution, pH 7.5, containing 5%
powered milk. The blots were probed with specific antibod-
ies, followed by a peroxidase-conjugated secondary anti-
body 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 (Bio-
Rad 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
EGTA, 0.14 m NaCl, pH 7.4 (immunoprecipitation buffer),
containing 1% Triton X-100, 10 lgÆ mL
)1
aprotinin,
20 lgÆmL
)1
leupeptin, 10 lgÆmL

)1
and the eluted proteins were collected in
500 lL fractions. The molecular weights of the eluted pro-
teins were calculated from the elution volumes of ferritin
(M
r
= 450 kDa) and aldolase (M
r
= 160 kDa), utilized as
standard proteins.
A
B
C
Fig. 7. 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 4 °C with IgG1-HSP90 antibody (see Experimental
procedures), as reported also in [7,44,45]. The mixtures were then
incubated for 1 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 5 min at 90 °C and submit-
ted 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 dif-
ferent 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 chromatog-
raphy (see Experimental procedures). Aliquots (30 lL) of each eluted

)at37°C. After
30 min, 2 mL of ice-cold stop buffer (50 mm Hepes,
pH 5.5, containing 5 mm EDTA) was added. These incuba-
tions were then submitted to anion exchange chromatogra-
phy using 2 mL of packed Dowex 50W8 Na
+
form resin
pre-equilibrated with stop buffer. l-Citrulline was eluted by
washing the resin with 3 mL of stop buffer, and the radio-
activity present was counted in a liquid scintillation coun-
ter. One unit of NOS activity was defined as the amount of
enzyme producing 1 pmol citrullineÆmin
)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 pro-
tein each), prepared as described above from untreated or
treated NMS and HMS rat brain and thoracic aorta homo-
genates, were submitted to 12% SDS-PAGE [28]. Calpasta-
tin 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 Investimenti della Ricerca di Base and Progetti di

McCabe TJ, Zoellner S, Garcia-Cardena G, Zhou Z,
Gratton J & Sessa WC (2004) Vanadate is a potent acti-
vator of endothelial nitric-oxide synthase: evidence for
the role of the serine ⁄ threonine kinase akt and the
90 kDa heat shock protein. Mol Pharmacol 65, 407–
415.
8 Song Y, Zweier JL & Xia Y (2001) Heat-shock protein
augments neuronal nitric oxide synthase activity by
enhancing Ca
2+
⁄ calmodulin binding. Biochem J 355,
357–360.
9 Minami Y, Kimura Y, Kawasaki H, Suzuki K & Yaha-
ra I (1994) The carboxy-terminal region of mammalian
HSP90 is required for its dimerization and function
in vivo. Mol Cell Biol 14, 1459–1464.
10 Billecke SS, Bender AT, Kanelakis KC, Murphy PJM,
Lowe ER, Kamada Y, Pratt WB & Osawa Y (2002)
HSP90 is required for heme binding and activation of
apo-neuronal nitric-oxide synthase. J Biol Chem 277,
20504–20509.
11 Dunbar AY, Kamada Y, Jenkins GJ, Lowe ER, Bille-
cke SS & Osawa Y (2004) Ubiquitination and degrada-
tion of neuronal nitric-oxide synthase in vitro: dimer
stabilization protects the enzyme from proteolysis. Mol
Pharmacol 66, 964–969.
12 Osawa Y, Lowe ER, Everett AC, Dunbar AY & Bille-
cke SS (2003) Proteolytic degradation of nitric oxide
synthase: effect of inhibitors and role of HSP90-based
chaperones. J Pharmacol Exp Ther 304, 493–497.

alho AP & Carvalho CM (2003) Neuronal nitric oxide
synthase proteolysis limits the involvement of nitric
oxide in kainate-induced neurotoxicity in hippocampal
neurons. J Neurochem 85, 791–800.
22 Walker G, Pfeilschifter J, Otten U & Kunz D (2001)
Proteolytic cleavage of inducible nitric oxide synthase
(iNOS) by calpain I. Biochim Biophys Acta 1568, 216–
224.
23 Su Y & Block ER (2000) Role of calpain in hypoxic
inhibition of nitric oxide synthase activity in pulmonary
endothelial cells. Am J Physiol Lung Cell Mol Physiol
278, 1204–1212.
24 Bellocq A, Doublier S, Suberville S, Perez J, Escoubet
B, Fouqueray B, Puyol DR & Baud L (1999) Somato-
statin increases glucocorticoid binding and signalling in
macrophages by blocking the calpain-specific cleavage
of HSP90. J Biol Chem 274, 36891–36896.
25 Laine
´
R & Ortiz de Montellano PR (1998) Neuronal
nitric oxide synthase isoforms a and l are closely
related calpain sensitive proteins. Mol Pharmacol 54,
305–312.
26 Hajimohammadreza I, Raser KJ, Nath R, Nadimpalli
R, Scott M & Wang KKW (1997) Neuronal nitric oxide
synthase and calmodulin-dependent protein kinase IIa
undergo neurotoxin-induced proteolysis. J Neurochem
69, 1006–1013.
27 Averna M, Stifanese R, DeTullio R, Salamino F, Bertuc-
cio M, Pontremoli S & Melloni E (2007) Proteolytic deg-

tory effect of di- and tripeptidyl aldehydes on calpains
and cathepsins. J Enzym Inhib 3, 195–201.
34 Lu Q & Mellgren RL (1996) Calpain inhibitors and ser-
ine protease inhibitors can produce apoptosis in HL-60
cells. Arch Biochem Biophys 334, 175–181.
35 Melloni E, De Tullio R, Averna M, Tedesco I, Salami-
no F, Sparatore B & Pontremoli S (1998) Properties of
calpastatin forms in rat brain. FEBS Lett 431, 55–58.
36 Pontremoli S, Melloni E, Damiani G, Salamino F,
Sparatore B, Michetti M & Horecker BL (1988) Effects
of a monoclonal anti-calpain antibody on responses of
stimulated human neutrophils. Evidence for a role for
proteolytically modified protein kinase C. J Biol Chem
263, 1915–1919.
37 Bianchi G, Ferrari P & Berber BR (1984) The Milan
hypertensive strain. In: Handbook of Hypertension,
Vol. 4 (de Jong W, ed.), pp. 328–349. Elsevier Science
Publisher.
38 Byrom FB & Wilson CA (1938) A plethysmographic
method for measuring systolic blood pressure in the
intact rat. J Physiol 93, 301–304.
39 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of proteins
utilizing the principle of protein–dye binding. Anal
Biochem 72, 248–254.
40 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
41 Palejwala S & Goldsmith LT (1992) Ovarian expression
of cellular Ki-ras p21 varies with physiological status.