Pharmacology & Pharmacy, 2011, 2, 256-265
doi:10.4236/pp.2011.24033 Published Online October 2011 (http://www.SciRP.org/journal/pp)
Modulation of c-Jun NH2-Terminal (JNK) by
Cholinergic Autoantibodies from Patients with
Sjögren’s Syndrome
Enri Borda1,2, Daniela Passafaro1, Silvia Reina1,2, Leonor Sterin-Borda1,2
1Pharmacology Unit, School of Dentistry, Buenos Aires University, Buenos Aires, Argentina; 2National Research Council of Argen-
tina (CONICET), Buenos Aires, Argentina.
Received June 27th, 2011; revised July 20th, 2011; accepted September 14th, 2011.
ABSTRACT
Background: We wanted to determine (via an immunopharmacological approach) whether the c-Jun NH2 terminal
kinase (JNK) cascade is phosphorylated in the submandibular gland by carbachol and cholinergic autoantibodies (IgG)
present in the sera of patients with primary Sjögren’s syndrome (pSS) by interaction and activation of salivary gland
muscarinic acetylcholine receptors (mAChRs). Methods: The JNK, PGE2 and NOS assays were measured in rat sub-
mandibular gland with pSS IgG and carbachol alone or in the presence of different blocker agents. Results: pSS IgG-
activated M3 mAChRs stimulated JNK phosphorylation whereas the activation of M1 mAChRs by carbachol stimulated
JNK phosphorylation involving calcium-activated mechanism. The intracellular pathway leading to pSS IgG-induced
biological effects on JNK activity involved activation of protein kinase C (PKC), inducible nitric oxide synthase (iNOS)
and cyclooxygenase-2 (COX-2) enzymes. Also, activation of COX-2 and COX-1 by pSS IgG and carbachol-induced
PGE2 generation were involved. Conclusion: These results may contribute to better understanding the modulatory role
of JNK enzymes by cholinergic autoantibodies from pSS patients acting on mAChR in rat submandibular gland.
Keywords: JNK, pSS IgG, Autoantibodies, PGE2, NOS, Carbachol, Cholinergic Antagonists, Enzymatic Inhibitors
1. Introduction
Primary Sjögren’s syndrome (pSS) is a systemic auto-
immune disease of unknown etiology. Many autoanti-
bodies have been reported to be present in pSS [1]. In
particular; antibodies to the antigens SS-A/Ro and SS-B/
La are commonly used in the diagnosis of pSS [2]. The
presence of subtypes M1 [3], M3 [4,5] and M4 [6] specific
autoantibodies in 83% - 90% persons with pSS [7] is an
important advance towards understanding the pathogene-
sis of pSS not only in terms of impaired glandular func-
tion, but also because of peripheral parasympathetic
dysfunction associated with the disease [8,9].
IgG molecules from serum samples from patients with
pSS have been reported to bind to glandular cholinore-
ceptors and act as partial agonists. IgG molecules have
been reported to not only activate the receptor, but also to
impair the response to the authentic cholinergic agonist
[10], suggesting a defect in post-receptor signaling [11].
The most direct mechanism for preventing target organs
from carrying out their function is that of early agonist-
mediated activation of cholinoreceptors initiated by
autoantibodies [12] which bind to, and persistently acti-
vate, cholinoreceptors [13]. Subsequently, the agonistic
activity displayed by these autoantibodies may induce the
desensitization [14], internalization and/or intracellular
degradation of cholinoreceptors, leading to a progressive
decrease in the expression and activity of these receptors
[12].
Xerostomia and keratoconjunctivitis sicca result from
lymphocyte infiltration of the salivary gland [15] and
lachrymal gland [16]. The infiltrating cells interfere with
glandular function by cell-mediated glandular destruction
and production of autoantibodies that interfere with cho-
linoreceptors [17]. Dental caries resulting from the loss
of salivary flow may be associated with periodontal dis-
ease [18].
Prostaglandins (PGs) are among the most relevant lo-
cal mediators that participate in the modulation of acini
cell functions under basal conditions [19]. PGs are re-

Modulation of c-Jun NH-Terminal (JNK) by Cholinergic Autoantibodies from Patients with Sjögren’s Syndrome257
2
leased in large amounts during inflammation or in early
stages of autoimmune diseases. In particular, overpro-
duction of PGs has been shown to occur in neuroinflam-
matory diseases [20]. Also, nitric oxide (NO) plays a key
part in the pathophysiology of systemic and chronic in-
flammatory disease and in the neurodegenerative process
[21]. Recent evidence has indicated that there is constant
crosstalk between NO and PGs biosynthesis pathways
involved in the pathological mechanisms underlying cer-
tain inflammatory disorders [21,22].
In general, muscarinic acetylcholine receptor (mAChR)
subtypes are grouped according to their functional cou-
pling. This can be via mobilization of intracellular cal-
cium (M1, M3, M5) through the activation of phospholi-
pase C (PLC), which results in the release of the second
messenger inositol 1,4,5-triphosphate (IP3) or by inhibit-
tion of adenylate cyclase (M2, M4), which results in re-
duction of the intracellular levels of cyclic adenosine
monophosphate [23]. The same receptor may generate
more than one set of intracellular second messengers and
considerable crosstalk exists between signaling cascades
[24]. The ability of these receptors to stimulate or inhibit
cell growth has been attributed to differences in cell
models, but the mechanisms involved in these cell type-
dependent differences in growth response are unknown
[25].
Mitogen-activated protein kinases (MAPKs) are acti-
vated by a diverse array of extracellular stimuli and regu-
late various cellular responses [26,27]. MAPK family
members include c-Jun NH2-terminal kinase (JNK) [28].
JNK is activated by cellular stress (ultraviolet and
gamma radiation), osmotic and heat shock, inhibitors of
protein synthesis, and inflammatory cytokines (tumor
necrosis factor (TNF)-α, interleukin (IL)-1), but also
weakly by growth factors (epidermal growth factor, EGF)
[28,29]. JNK activation has been implicated in the im-
mune response, oncogenic transformation, apoptosis [28,
30] and activation of two major transcription factors:
activator protein 1 (AP-1) and nuclear factor-kappa B
(NF-kB) [31,32]. In turn, AP-1 and NF-kB induce the
transcription of several genes involved in acute and
chronic inflammation as well as diseases of connective
tissue [32]. The gene for the inducible isoform of nitric
oxide synthase (iNOS) is involved in inflammation [32].
MAPK regulation by G protein-coupled receptors
(GPCRs) appears to be a widespread phenomenon. It is
also likely to mediate the proliferative and hypertrophic
responses of cells to various hormones, neurotransmitters
and local mediators that act at this class of receptor [33].
JNK activation has also been demonstrated for several
GPCRs, including M1 and M2 mAChR [34-36], angio-
tensin [37], α1-adrenergic [38], thrombin [39] and endo-
thelin-1 [40]. Some studies support the notion of mobile-
zation of intracellular calcium and activation of protein
kinase C (PKC) [41] in cholinergic receptor-mediated
JNK activation [35,37].
2. Aim
The aim of the present work was to examine the effect of
cholinergic autoantibodies present in the sera of pSS pa-
tients and the authentic cholinergic agonist carbachol on
JNK phosphorylation. We found that M1 and M3 mAChRs
were coupled to JNK in the submandibular glands of rats.
However, carbachol preferentially stimulated M1 mAChRs
whereas pSS IgG stimulated M3 mAChRs. Both, the ac-
tivation of M3 and M1 mAChRs by pSS IgG and car-
bachol stimulated JNK phosphorylation. The pSS IgG
stimulation effect appeared to be mediated by activation
of PKC, iNOS and cyclo-oxygenase-2 (COX-2) whereas
the carbachol stimulatory effect on JNK was mainly as-
sociated with intracellular calcium-activated endothelial
nitric oxide synthase (eNOS) and neuronal nitric oxide
synthase (nNOS) with COX-1 stimulation. Also, the re-
sults indicated that in the JNK activation phenomenon,
by pSS IgG and carbachol on salivary gland participated
cholinergic M3 and M1 receptor, respectively. Our results
suggested that activation of JNK by pSS IgG indicate
that the enzyme may be involved in the pathological
process of chronic sialodenitis present in the course of
pSS.
3. Methodology
3.1. Drugs
Carbachol, pirenzepine, J104291, verapamil, calphostin
C and thapsigargin were obtained from Sigma-Aldrich;
FR-122047, DuP 697, methylisothiourea sulphate, L-
NIO and NZ were from Tocris Cookson (Ellisville, MO,
USA). Stock solutions were freshly prepared in the ap-
propriate buffers. The drugs were diluted in a water bath
to achieve the final concentrations stated in the text.
3.2. Animals
Male Wistar rats weighing 250 - 300 g from the Pharma-
cologic Bioterium (School of Dentistry, University of
Buenos Aires) were used throughout. The animals housed
in standard environmental conditions were fed with a
commercial pellet diet and water ad libitum. For surgical
removal of submandibular glands, the animals were sac-
rificed using ether. The experimental protocol followed
the Guide to The Care and Use of Experimental Animals
(DHEW Publication, NIH 80-23).
3.3. Subjects and Serological Tests
Females (age, 35 - 55 years) who had been diagnosed 7 -
15 y previously and who had been free of treatment for 8

Modulation of c-Jun NH-Terminal (JNK) by Cholinergic Autoantibodies from Patients with Sjögren’s Syndrome 258 2
months were selected from the metropolitan area of
Buenos Aires. The study population was 25 women with
pSS who presented with dry mouth, and 18 healthy
women (mean age, 45 ± 10 years) without systemic
disease (control group). The diagnosis of SS was based
on four or more of the criteria published elsewhere [42].
Biopsy results, degree of xerostomia and keratocon-
junctivitis sicca, and the results of serological tests in the
different groups were the same as previously reported
[10].
3.4. Peptides
A 25-mer peptide (K-R-T-V-P-D-N-Q-C-F-I-Q-F-L-S-
N-P-A-V-T-F-G-T-A-I) and a 24-mer peptide (E-R-T-
M-L-A-G-Q-C-Y-I-Q-F-L-S-Q-P-I-T-F-G-T-A-M) corre-
sponding to the amino-acid sequence of the second ex-
tracellular loop of the human M3 mAChRs and M1
mAChRs, respectively, were synthesized from F-moc-
amino acids activated using the 1-hydroxy benzo tria-
zole/dicyclo hexyl carbodimide (HOBt/DCC) strategy
and an automatic peptide synthesizer (Model 431A, Ap-
plied Biosystems, Foster City, CA, USA). The peptides
were desalted, purified by high-performance liquid
chromatography (HPLC), and subjected to amino-ter-
minal sequence analysis using automatic Edman degra-
dation and an Applied Biosystems 470A Sequencer. An
unrelated 25-mer peptide (S-G-S-G-S-G-S-G-S-G-S-G-
S-G-S-G-S-G-S-G-S-G-S-G-S) was synthesized as a ne-
gative control.
3.5. Purification of Human IgG
The serum IgG fraction from patients with pSS and from
normal individuals (control) was isolated using protein G
affinity chromatography as described elsewhere [9].
Briefly, sera were loaded onto the protein G affinity
column (Sigma-Aldrich, St Louis, MO, USA) equili-
brated with 1 M Tris-HCl (pH 8.0) and the columns were
washed with 10 volumes of the same buffer. The IgG
fraction was eluted with 100 mM glycine-HCl, pH 3.0,
and immediately neutralized. The concentration and pu-
rification of IgG were determined using a radial immu-
nodiffusion assay.
3.6. JNK Assay
Slices of submandibular glands of rats (20 mg) were in-
cubated for 30 min in 500 μL of Krebs-Ringer bicarbon-
ate (KRB) buffer and gassed with 5% CO2 in O2 at 37˚C.
pSS IgG or carbachol were added 15 min before the end
of the incubation period, and blockers added 15 min be-
fore the addition of different concentrations of pSS IgG
or carbachol. The submandibular gland was then ho-
mogenized in 1.0 mL of cell lysis buffer (product 9803).
We then followed the manufacturer instructions for the
PathScan Total and Phospho-SAPK/JNK kit (Sandwich
ELISA Kit; Cell Signalling Technology Incorporated,
Beverly, MA, USA). JNK results were expressed as the
optical density at 450 nm (OD 450 nm).
3.7. PGE2 Assay
Rat submandibular gland slices (20 mg) were incubated
for 60 min in 500 μL of KRB and gassed with 5% CO2 in
O2 at 37˚C. pSS IgG or carbachol were added 30 min
before the end of the incubation period and blockers
added 30 min before the addition of different concentra-
tions of pSS IgG or carbachol. The submandibular gland
was then homogenized in a 1.5-mL polypropylene mi-
crocentrifuge tube. We then followed the manufacturer
instructions for the PGE2 Biotrak Enzyme Immune Assay
System (ELISA; Amersham Biosciences, Piscataway, NJ,
USA). PGE2 results were expressed as picograms per
milligram of tissue wet weight (pg/mg tissue wet wt).
3.8. Nitric Oxide Synthase (NOS) Assay
NOS activity was measured in rat submandibular gland
tissue by production of [U-14C]-citrulline from [U-14C]-
arginine according to the procedure described for brain
slices [43]. Briefly, after 20-min preincubation in KRB
solution, tissues were transferred to 500 mL of pre-
warmed KRB equilibrated with 5% CO2 in O2 in the
presence of [U-14C]-arginine (0.5 mCi). Drugs were
added and the mixture incubated for 20 min under 5%
CO2 in O2 at 37˚C. Tissues were then homogenized with
an Ultraturrax homogenizer in 1 mL of medium contain-
ing 20 mM HEPES (pH 7.4), 0.5 mM ethyleneglycol
tetra-acetic acid (EGTA), 0.5 mM ethylenediamine tetra-
acetic acid (EDTA), 1 mM dithiothreitol, 1 mM leu-
peptin and 0.2 mM phenylmethylsulphonyl fluoride
(PMSF) at 4˚C. After centrifugation at 20,000 × g for 10
min at 4˚C, supernatants were applied to 2-mL columns
of Dowex AG 50 WX-8 (sodium form). [14C]-citrulline
was eluted with 3 mL of water and quantified by liquid
scintillation counting. The results were expressed as pi-
comol per gram tissue wet weight (pmol/g/tissue wet wt).
3.9. Statistical Analices
The unpaired Student’s t-test was used to determine sta-
tistical significance. Analysis of variance (ANOVA) and
a post-hoc test (Dunnett’s method or the Student-New-
man-Keuls test) were employed if a pairwise multiple
comparison procedure was necessary. p < 0.05 was con-
sidered significant.
3.10. Ethical Approval of the Study Protocol
The study protocol was approved by the Ethics Commit-
tee of the School of Dentistry at Buenos Aires University
(Buenos Aires, Argentina). The studies were conducted

Modulation of c-Jun NH-Terminal (JNK) by Cholinergic Autoantibodies from Patients with Sjögren’s Syndrome259
2
according to the tenets of the Declaration of Helsinki. All
participants provided written informed consent to par-
ticipate in the study.
4. Results
We initially determined the effects of different concen-
trations of carbachol and pSS IgG on JNK phosphoryla-
tion. Figure 1 shows the potential of serum IgG from
patients with pSS to stimulate JNK phosphorylation in a
concentration-dependent manner. The authentic cho-
linergic agonist carbachol increased JNK activity (Fig-
ure 1(a)). The maximal effect of carbachol and/or pSS
IgG on JNK activation was obtained at 1 × 10–6 M in
both cases. The level of total JNK protein was not modi-
fied by pSS IgG, carbachol or normal IgG concentrations
(Figure 1(b)). The data obtained with pSS IgG or car-
bachol therefore referred to their capacity to alter the
level of JNK phosphorylation. The maximal capacity to
stimulate the activity of the JNK enzyme in the presence
of 1 × 10–6 M pSS IgG was impaired in the presence of
4.5 × 10–9 M of the specific M3 mAChR antagonist
J104192, but a lack of action was seen when the specific
M1 mAChR antagonist pirenzepine (1 × 10–6 M) was used
(Figure 2(a)). Moreover, M3 mAChR synthetic peptide
(5 × 10–5 M) not M1 synthetic peptide (5 × 10–5 M) blunted
pSS IgG-inhibited JNK phosphorylation (Figure 2(a)).
Carbachol (1 × 10–6 M)-stimulated JNK activity was
blocked by the M1 cholinergic antagonist pirenzepine
(Figure 2(b)). To define the participation of NO, we
studied which isoforms of NOS might be implicated in
the action of pSS IgG on JNK enzyme activity. To
achieve this, rat submandibular gland tissue was incu-
bated with specific inhibitors of NOS isoforms. Inhibi-
tion of iNOS activity by methylisothiourea sulfate (1 ×
10–7 M) prevented the stimulatory action of pSS IgG on
JNK activation (Figure 3(a)). Conversely, inhibition of
the activity of eNOS by L-NIO (5 × 10–6 M) and nNOS
by NZ (5 × 10–5 M) was not observed. L-NMMA (1 ×
10–5 M) prevented pSS IgG-mediated stimulation on JNK
activity and a natural substrate of NOS, L-arginine (5 ×
10–5 M), reversed the effect of L-NMMA (data no
shown). pSS IgG induced an increase in the activity of
NOS in submandibular gland tissue (Figure 3(b)). The
stimulatory action of pSS IgG on NOS activity was ab-
rogated by the inhibition of iNOS activity by methyli-
sothiourea sulfate without modification by L-NIO and
NZ. J104129 and M3 synthetic peptide (but not piren-
zepine and M1 synthetic peptide) impaired the stimula-
tory action of pSS IgG. This indicated the participation
of the M3 mAChR in the action of pSS IgG upon NOS
activity.
Figure 4(a) shows the participation of COX-2 (but not
COX-1) in the stimulatory action of pSS IgG on JNK
(a) (b)
Figure 1. (a) Effect of pSS IgG (●), carbachol (○) and nor-
mal IgG (▲) on JNK activity in rat submandibular glands;
(b) total JNK protein (black column) and JNK phosphory-
lated (striped line). Each point represents the mean ± SEM
of six independent experiments done in duplicate.
(a) (b)
Figure 2. (a) Values of optical density of 1 × 10–6 M pSS IgG
alone or in the presence of M3 (J102941) and M1 (piren-
zepine) mAChR antagonists and M3 and M1 mAChR syn-
thetic peptide; (b) optical density of 1 × 10–6 M carbachol
alone or in the presence of M3 (J102941) and M1 (piren-
zepine) mAChR antagonists. Results are mean ± SEM of 10
independent patients in each group done in duplicate. *p <
0.001 vs basal; **p < 0.001 vs pSS IgG or carbachol.
activity. A reduction in pSS IgG-mediated activation was
observed in the presence of DuP 697 (5 × 10–8 M) but not
by FR-122047 (5 × 10–6 M), which are COX-2 and COX-1
enzymatic inhibitors, respectively. pSS IgG (1 × 10–6 M)
could trigger an increase in PGE2 generation in rat sub-
mandibular glands, and COX 2 inhibition (but not COX-1 -

Modulation of c-Jun NH2-Terminal (JNK) by Cholinergic Autoantibodies from Patients with Sjögren’s Syndrome 260
0
(a) (b)
Figure 3. (a) Concentration–response curves of pSS IgG alone (●) or in the presence of 1 × 10–4 M L-NMMA (♦), 1 × 10–7 M
methylisothiourea sulfate (▼), 5 × 10–5 M NZ (○) and 5 × 10–6 M L-NIO (▲) on JNK phosphorylation; (b) stimulation of NOS
activity by 1 × 10–6 M pSS IgG alone or in the presence of enzymatic inhibitors of NOS isoforms, M3 and M1 mAChR syn-
thetic peptides, and M3 and M1 mAChR antagonists. Basal values A and B are also shown. Values represent the mean ± SEM
of seven experiments in each group done in duplicate. *p < 0.001 vs basal; **p < 0.0001 vs pSS IgG.
0
(a) (b)
Figure 4. (a) Concentration-response curves of pSS IgG alone (●) or in the presence of 5 × 10–8 M DuP 697 (▲) and 5 × 10–6
M FR-122047 (○) on JNK phosphorylation. (b) Stimulation of PGE2 production by 1 × 10–6 M pSS IgG alone or in the pres-
ence of enzymatic inhibitors of COX-s isoforms, M3 and M1 mAChR synthetic peptides and M3 and M1 mAChR antagonists.
Basal values ((a) and (b)) were also shown. Values represent the mean ± SEM of eight experiments in each group done in
duplicate. *p < 0.001 vs basal; **p < 0.0001 vs pSS IgG.
Figure 5 shows a positive correlation between the in-
crement of NOS activity Figure 5 (a) and PGE2 produc-
tion Figure 5(b) in the function of JNK activation.
inhibition) blunted this action of pSS IgG on PGE2 pro-
duction (Figure 4(b)). Also, the M3 antagonist J104129
and M3 synthetic peptide (but not the M1 antagonist
pirenzepine and M1 synthetic peptide) diminished pSS
IgG-stimulated PGE2 production.
Table 1 shows the enzymatic pathways involved in the
pSS IgG-mediated stimulation of JNK and carbachol-

Modulation of c-Jun NH-Terminal (JNK) by Cholinergic Autoantibodies from Patients with Sjögren’s Syndrome261
2
.
.
.
.
.
(a) (b)
Figure 5. Correlation in the modulator effect of pSS IgG on JNK phosphorylation. Production of NOS and PGE2 was plotted
as a function of JNK activation. Values are the means of seven experiments in each group.
mediated stimulation of JNK. Inhibition of COX-1, NZ
or L-NIO inhibited the stimulatory action of carbachol on
JNK phosphorylation. M3 and M1 mAChR are calcium-
mobilizing receptors coupled to PLC [44], so we deter-
mined the contribution of calcium to JNK phosphoryla-
tion by pSS IgG and carbachol in rat submandibular
glands. Verapamil (1 × 10–5 M), an inhibitor of calcium
influx and thapsigargin (1 × 10–8 M), an inhibitor of en-
doplasmic reticulum Ca2+-ATPase modified pSS IgG-
mediated and carbachol-induced stimulation of JNK ac-
tivity (Table 2). On the other hand, calphostin C (5 ×
10–9 M), an inhibitor of PKC, only modify pSS IgG-me-
diated activation of JNK (Table 2).
5. Discussion
Primary SS IgG with cholinergic agonist activity has
been found in the sera of persons with Sjögren’s syn-
drome (SS), and the presence of these antibodies corre-
lated with inflammation of the salivary gland [6]. Here
we demonstrated the possible role of pSS IgG to induce
glandular inflammation through its capacity to trigger the
production of the proinflammatory substances NO and
PGE2. Moreover, the increased production of these pro-
inflammatory substances correlated with JNK phos-
phorylation.
Carbachol increased JNK activity mainly through the
M1 mAChR, but pSS IgG stimulated JNK activity pre-
dominantly through the M3 mAChR. Inhibition of the M3
mAChR by J104129 and M1 mAChR by pirenzepine
impaired pSS IgG and carbachol-induced increase in
JNK phosphorylation.
Table 1. Enzymatic pathways coupled to the effect of pSS
IgG and carbachol upon JNK phosphorylation.
JNK (% change)
Additions pSS IgG
(1 × 10–6 M)
Carbachol
(1 × 10–6 M)
Alone +163 ± 16 +189 ± 18
Methylisothiourea sulfate
(1 × 10–7 M) +70 ± 6.5* +195 ± 17
Nz (5 × 10–5 M) +161 ± 15 +140 ± 12*
L-NIO (5 × 10–6 M) +167 ± 16 +130 ± 13*
DuP 697 (5 × 10–8 M) +74 ± 6.8* +182 ± 17
FR-122047 (5 × 10–6 M) +159 ± 16 +136 ± 12*
Values are mean ± SEM of five experiments in each group carried out in
duplicate. *P < 0.001 vs pSS IgG or carbachol alone.
Table 2. Contribution of calcium on the action of pSS IgG
and carbachol upon JNK phosphorylation.
Additions pSS IgG
(1 × 10–6 M)
Carbachol
(1 × 10–6 M)
Alone +151 ± 15 186 ± 17
Verapamil (1 × 10–5 M) +117 ± 12* +124 ± 11*
Thapsigargin (1 × 10–6 M) +132 ± 12 +122 ± 11**
Calphostin C (5 × 10–9 M) +71 ± 8** +182 ± 19
Values are mean ± SEM of six experiments in each group carried out in
duplicate. *P < 0.001 vs alone. **P < 0.0001 vs alone.

Modulation of c-Jun NH-Terminal (JNK) by Cholinergic Autoantibodies from Patients with Sjögren’s Syndrome 262 2
The M3 and M1 mAChR subtypes are calcium-mobi-
lizing receptors coupled to PLC activation [45]. They
provide the second messenger inositol triphosphate (IP3)
and diacylglycerol (DAG) which mobilize intracellular
calcium and activate PKC [41]. We observed that car-
bachol-phosphorylated JNK was diminished by calcium
ATPase from a sarcoplasmic reticulum blocker whereas
the pSS IgG-mediated effect was prevented by a PKC
inhibitor. This indicated that JNK activation by M1
mAChRs appeared to be dependent upon calcium/cal-
modulin activity, whereas pSS IgG-activated JNK ap-
peared to be involve M3 mAChRs were associated with
PKC activation. In support of our observations, it was
shown that JNK activation in NIH3T3 cells by M1
mAChRs did not require PKC [34]. However, PKC inhi-
bition results in enhancement of JNK activity in CHO-
M3 cells [46,47].
In the present study, we showed that the activation of
M3 mAChRs in rat submandibular glands by pSS IgG
increased generation of PGE2. This was preceded by
iNOS activation, which in turn catalyzed COX-2 activity.
Our data indicated that iNOS dependent pathway was the
key factor for pSS IgG-induced PGE2 generation. More-
over, we demonstrated a positive correlation between
NOS activity or PGE2 production and the activation of
JNK phosphorylation triggered by pSS IgG. The fact that
inhibition of the activities of iNOS and COX-2 prevented
the pSS IgG-mediated stimulation of JNK phosphoryla-
tion, parallel with an increase in the production of NO
and PGE2, confirmed this issue. Conversely, carbachol-
stimulated JNK phosphorylation was inhibited by the
activities of eNOS, nNOS and COX-1, indicating that the
constitutive enzymes were involved.
eNOS and nNOS are constitutively expressed whereas
the expression of iNOS requires protein synthesis. Fur-
thermore, the reason why pSS IgG activated iNOS inde-
pendent of an increase in intracellular calcium concentra-
tion was because this isoform could produce large
amounts of NO for extended periods of time, far exceed-
ing the levels generated by the constitutive isoforms in
the submandibular gland [48]. Also, it has been docu-
mented that JNK regulates iNOS expression [49], it is
likely that JNK mediates IL-1β-induced expression of
iNOS in the lachrymal gland, which leads to inhibition of
neural-as well as agonist-induced protein secretion [50].
The JNK cascade is triggered through pSS IgG and
carbachol activating M3 and M1 mAChRs in rat subman-
dibular glands. However, these stimuli, which trigger the
production of NO and PGE2, could activate the JNK
cascade in the infiltrating T-cells in the salivary glands of
pSS patients. The JNK cascade could play an important
part in the pathogenesis of SS, and could be a potential
therapeutic target [51,52].
6. Conclusions
We conclude that, in pSS, the early agonist-mediated
activation of M3 mAChRs initiated by autoantibodies
binding to, and persistently activating, cholinoreceptors,
resulted in JNK stimulation and an increase in the pro-
duction of PGE2 and NO through iNOS activation. This
contributed to the inflammation of the submandibular
gland, eliciting a loss of secretory response of glandular
acini cells (dry mouth) and the lachrymal gland (dry eye)
in patients with pSS. An illustration of bringing together
the various systems studied and proposing a mechanism
by which pSS IgG and carbachol might induce JNK ac-
tivation, thereby triggering the production of proinflam-
matory mediators, is shown in Figure 6.
Figure 6. Proposed model to explain the mechanism whereby
pSS IgG and carbachol up-regulates NOS isoform and
PGE2 generation to provoke JNK activation in subman-
dibular gland. pSS IgG acting on M3 mAChR and car-
bachol acting on M1 mAChR activates PLC mediating pro-
duction of 1,2-diacylglycerol (DAG) and inositol triphos-
phate (IP3). IP3 triggering intracellular release of calcium
stores (Ca2+). Free calcium binds to calcium/calmodulin
complex (CaM) and sensitizes PKC activation via DAG.
Subsequent PKC translocation to the membrane and CaM
complex increase NOS activity through different isoform
that in turn increases NO production. The over production
of NO also triggers COX-1 and COX-2 activation. Alterna-
tively, the rise in cytosolic calcium activates phospholipase
A2 (PLA2) with activation of COX-1 (carbachol) and COX-2
(pSS IgG) which induces generation of PGE2, NO and PGE2
in the last instance, evoked JNK activation. Inhibitory
agents are indicated in italics.

Modulation of c-Jun NH-Terminal (JNK) by Cholinergic Autoantibodies from Patients with Sjögren’s Syndrome263
2
7. Acknowledgements
This work was supported by grants from Buenos Aires
University (grant number UBACyT O 003) and the Na-
tional Research & Technology Agency (PICT’s 02120
and 01647). The authors thank Mrs. Elvita Vannucchi
and Mr. Alejandro Thorton for their expert technical as-
sistance. There is no conflict of interest.
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