Download Is acetylcholine an autocrine growth factor via the

Nuclear expression of -opioid receptors in a human mesothelial cell line
Amir Khorram-Manesha*, Cecilia Bengtssonb, Gunnar Nylunda, Svante
Nordgrenb, and Dick S. Delbrob,c
a
Department of Surgery, Kungälv Hospital, SE-442 83 Kungälv, Sweden
Department of Surgery, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden
c
School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar,
Sweden
b
*
Corresponding author.: Tel.: +46 303 98000.
E-mail address: [email protected]
Abstract
We have demonstrated by immunochemical means that -opioid receptors (MORs) are
expressed in a human mesothelial cell line with a predominantly nuclear localization. This
could suggest that such receptors are targets at intracrine cell signalling, but the biological
significance of this finding is obscure.
Introduction
We recently reported that the formation of experimentally induced postoperative abdominal
adhesions in rat can be markedly inhibited by the provision of morphine to the drinking water
of the animals for three weeks, postoperatively. The adhesions arise a consequence of an
inflammation-induced imbalance between fibrin deposition and fibrinolysis, a process being
initiated by the surgical trauma (Khorram-Manesh et al., 2006). The mechanism of action for
morphine to inhibit adhesion formation has not been elucidated, but it is conceivable that this
compound acts as an antiinflammatory agent. It is well-known that morphine down-regulates
the innate as well as adaptive immune systems directly via activation of -opioid receptors
(MORs) in the immune cells (Sharpe, 2006), and indirectly, via central activation of adrenal
glucocorticoid release (Sacerdote, 2006).
The mesothelial cells play a pivotal role for healing after peritoneal damage, but may also
participate in adhesion formation by way of secreting mediators that interfere negatively with
fibrinolysis (diZerega 1997; Yao et al., 2004; Boland and Weigel, 2006). We proposed as one
possible mechanism of action for adhesion prevention by morphine, the down-regulation of
mesothelial cells with regard to the release of pro-adhesion mediators. -opioid receptors have
been demonstrated on pleural mesothelial cells (see Khorram-Manesh et al., 2006), but, to the
best of our knowledge, no studies have been performed concerning the expression of opioid
receptors in peritoneal mesothelial cells.
The aim of the current study was to investigate in a human, mesothelial cell line whether these
cells express MORs, which, thus, may be a target for morphine in prevention of postoperative
adhesions.
Materials and methods
Cell culture
The human mesothelial cell line (ATCC CRL 9444; a kind gift from the laboratory of Dr. I.
Mattsby-Baltzer, Department of Clinical Bacteriology, Sahlgrenska University Hospital,
Göteborg, Sweden) was maintained in culture in McCoy’s 5a medium (Invitrogen,
Stockholm, Sweden) supplemented with 1%L-glutamine (Bio Whittaker Europe, Verviers,
Belgium) and 1% penicillin–streptomycin (Invitrogen) in the presence of 4% fetal calf serum
(FCS, Invitrogen). A split ratio of 1:8 once weekly was chosen, with a change of medium in
between (McCoy’s 5a plus 2% FCS). Cells were renewed after approximately 22 passages.
For immunocytochemistry, the cells were seeded in chamber slides (2 ml; 100,000 cells/ml).
On day 5 following seeding, the cells were prepared for either for immunocytochemistry or
for Western blotting. The experiments were repeated at least three times, unless specified
otherwise.
Immunocytochemistry
Before fixing the cells with phosphate-buffered formaldehyde, pH 7.4 (Substratavdelningen,
Sahlgrenska University Hospital, Göteborg, Sweden) for 25 min, the cells were washed twice
with PBS. Fixation was followed by a rinse (3-5 min) in TBS. Endogenous peroxidase was
blocked with 0.3% hydrogen peroxidase (VWR International, Stockholm, Sweden) in
methanol (Merck, Stockholm, Sweden) for 30 min followed by the blocking of unspecific
protein binding with 2% normal horse serum PK6200 (ImmunKemi, Järfälla, Sweden) for 1 h
in a moist chamber. The primary antibody: a polyclonal rabbit anti-MOR antibody (Sta Cruz,
USA, 1:50-1:200) diluted in horse serum was added and incubated in a moist chamber at 4C
over-night. The following day, the slides were washed in TBS (2 x 5 min), incubated with the
secondary antibody for 30 min (also in a moist chamber), followed by a second wash in TBS
(2 x 5 min). For staining, the slides were incubated with the ABC-reagents (Vectastain Elite
ABC; Vector Laboratories, Burlingame, CA) for 30 min in a moist chamber, again rinsed with
TBS (2 x 5 min). Positive immunoreactivity was visualised with 3-diaminobenzidine
tetrahydrochloride (DAB, DakoCytomation), resulting in brown staining. The color reaction
was stopped after about 8 min by a rinse in water for 10 min. The cells were then
counterstained with Mayer’s hematoxylin (Histolab, Göteborg, Sweden) for about 1–2 min,
rinsed in water for 5 min before being mounted with Faramount Aqueous Mounting Medium
(DakoCytomation); they were then photographed under a light microscope (Nikon Eclipse
E400 & Nikon Digital Camera DXM 1200; Upplands Väsby, Sweden). Negative controls
were performed by excluding the primary antibody and incubating the cells instead with horse
serum, resulting in no immunoreactivity.
Protein Extraction
The culture medium was removed and the cell culture was rinsed with PBS and scraped into
0.5 ml of ice-cold homogenization buffer (25 mM) Hepes, pH 7.4 containing EDTA (0.1
mmol), 0.25% deoxycholate, 2.5% Triton X-100, and 0.01mg/ml each of
phenylmethylsulphonyl fluoride, trypsin inhibitor, leupeptin, antipain, chymostatin, and
pepstatin. The cells were freeze-thawed and disrupted by passage through a 21-gauge needle.
After centrifugation at 10,000 x g for 45 min, the protein concentration was determined by the
BCA protein assay reagent.
Western Blot Analysis
The method has been described previously (Jacobsson et al. 2006). In brief, mesothelial cells
were kept on ice and were rinced twice in PBS and were then lysed in RIPA lysis buffer,
containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1
mM EDTA, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1% Triton X-100, 1% sodium
deoxycholate, and 0.1% SDS. The cells were freeze-thawed and disrupted by passage through
a 21-gauge needle. After centrifugation at 10,000 x g for 45 min, the protein concentration
was determined by the BCA protein assay reagent. A lysate volume corresponding to 20 g of
total protein was added to each well. The proteins were separated by SDS-PAGE for 50 min
at 200 V using NuPAGE 4-12% Bis-Tris gel (Invitrogen) and MOPS SDS running buffer
supplemented with NuPAGE antioxidant (Invitrogen). Western blot at 30 V for 60 min was
used to transfer buffer supplemented with NuPAGE antioxidant and 30% methanol. The
nitrocellulose membrane was stained with Ponceau S solution (Sigma-Aldrich) for 10 min,
and was rinsed with distilled water until sharp red bands appeared. The membrane was
blocked for 60 min with a blocking solution and was probed for 60 min with: rabbit
polyclonal antibody against MOR diluted 1:200. The membrane was rinsed with TBST
(containing 50 mM Tris-HCl, 150mM NaCl, and 0.5% Tween 20, pH 7.5). The secondary
antibody was goat anti-rabbit AP-conjugated antibody (Santa Cruz Biotechnology) at 1:5000.
The immunoreactivity was finally visualized with a chemiluminescent detection system that
utilizes enzyme-linked immunodetection (Western Star). The chemiluminiscence reaction was
detected using hyperfilm ECL (Amersham Pharmacia Biotech, Uppsala, Sweden).
Results
MORs are expressed in mesothelial cells predominantlyin the nuclei.
The antibody to MOR produced, in a concentration dependent fashion, immunoreactivity in
the cells, which was to a major extent observed in the nuclei. (Fig. 1).
Fig. 1. Human mesothelial cells display immunoreactivity
to the anti-MOR antibody (concentration: 1:100).
Bar is 25m.
Western blotting demonstrates MOR expression in the nuclear fraction of the mesothelial
cells.
When separate cytosol, and nuclear fractions were prepared of the mesothelial cells, it was
evident that a predominant part of the MOR expression was located in the nuclei, displaying a
clear-cut band at approximately 50 kDa, in all likelihood corresponding to the molecular
weight of the MOR (i.e. 51 kDa).
Discussion
In the current study, undertaken with a human mesothelial cell line, we addressed the
problem whether the cells express MORs, which would be the very first condition in order to
explain an adhesion-preventing action of of morphine (cf. Khorram-Manesh et al., 2006). We
could confirm such an expression by immunocytochemistry, which, interestingly, was
confined predominantly to the nuclear compartment. Such a localization could be verified by
fractionized Western blotting when a cytosolic and a nuclear fraction had been prepared.
We recently reported that MORs are expressed in human colon cancer as well as
macroscopically tumor free, adjacent colon tissue with a predominantly nuclear localization
(Nylund et al., 2007). MORs belong to the class of G-protein coupled receptors. It is
becoming increasingly established that such receptors may in addition to their plasma
membrane localization, also be expressed in the nuclear membrane, being targets for
intracrine signal transduction (Gobeil et al., 2006). Studies are in progress in our laboratory in
order to elucidate the biological significance of the currently reported nuclear MORs.
Acknowledgements
The present study was supported by the Anna-Lisa and Bror Björnsson Foundation (D.
Delbro), the Assar Gabrielsson Foundation (D. Delbro), the Göteborg Medical Society (A.
Khorram-Manesh, G. Nylund), the King Gustaf V Jubilee Clinic Foundation (D. Delbro), the
LUA-ALF agreement (S. Nordgren), the University of Kalmar (D. Delbro) and the Västra
Götalandregionen (G. Nylund).
References
Boland, G.M., Weigel, R.J. (2006) Formation and prevention of postoperative abdominal
adhesions, J Surg Res, 132:3-12
Gobeil, F., Fortier, A., Zhu, T., Bossolasco, M., Leduc, M., Grandbois, M., Heveker, N.,
Bkaily, G., Chemtob, S., Barbaz, D. (2006) G-protein-coupled receptors signalling at the cell
nucleus: an emerging paradigm, Can J Physiol Pharmacol, 84:287-297
Khorram-Manesh, A., Ardakani, J.V,, Behjati, H.R., Nylund, G., Delbro, D. (2006) The effect
of opioids on the development of postoperative intra-abdominal adhesions, Dig Dis Sci,
51:560-565
Nylund, G., Pettersson, A., Bengtsson, C., Khorram-Manesh, A., Nordgren, S., Delbro, D.S.
(2007) Functional Expression of mu-Opioid Receptors in the Human Colon Cancer Cell Line,
HT-29, and their Localization in Human Colon, Dig Dis Sci, Aug 7; [Epub ahead of print]
Sacerdote, P. (2006) Opioids and the immune system, Palliat Med, 20, suppl 1:9-15
Sharp, B.M. (2006) Multiple opioid receptors on immune cells modulate intracellular
signalling, Brain Behav Immun, 20:9-14
Yao, V., McCauley, R., Cooper. D., Platell, C., Hall, J.C. (2004) Peritoneal
mesothelial cells produce cytokines in a murine model of peritonitis,
Surg Infect, 5:229–236
diZerega, G.S. (1997) Biochemical events in peritoneal tissue repair,
Eur J Surg, Suppl 577:10–16