European Journal of Pharmacology xxx (xxxx) xxx–xxx
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European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Full length article
Enhancement of S1P-induced contractile response in detrusor smooth
muscle of rats having cystitis
⁎
Irfan Anjuma, Merve Denizaltia, Hilmi Burak Kandilcib, Nezahat Tugba Durlu-Kandilcia, ,
Inci Sahin-Erdemlia
a
b
Hacettepe University, Faculty of Pharmacy, Department of Pharmacology, 06100 Sıhhiye-Ankara, Turkey
Ankara University, Faculty of Medicine, Department of Biophysics, 06100 Sıhhiye-Ankara, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
Sphingosine 1-phosphate
Interstitial cystitis
Rat
Permeabilization
Detrusor smooth muscle
Interstitial cystitis is a chronic disease characterized by lower abdominal pain and some nonspecific symptoms
including an increase in urinary frequency and urgency. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that controls smooth muscle tone via G-protein coupled receptors (S1P1–3 receptors). S1P production is
known to take place both in physiological states and some pathological situations, such as in overactive bladder
syndrome. The intracellular mechanism of S1P-induced contractile response was investigated in β-escin permeabilized detrusor smooth muscle of rats having cyclophosphamide-induced cystitis. The bladder was isolated
from rats and detrusor smooth muscle strips were permeabilized with β-escin. S1P (50 µM)-induced contraction
and calcium sensitization response were significantly increased in cystitis. S1P-induced augmented contractile
response was inhibited by S1P2 receptor antagonist JTE-013 and S1P3 receptor antagonist suramin. S1P2 receptor protein expressions were increased in cystitis, where no change was observed in S1P3 expressions between control and cystitis groups. S1P-induced contraction was reduced by Rho kinase (ROCK) inhibitor Y27632 and protein kinase C (PKC) inhibitor GF-109203X in both control and cystitis group. S1P-induced increased calcium sensitization response was decreased by ROCK inhibitor and PKC inhibitor in cystitis. Our
findings provide the first evidence that interstitial cystitis triggers S1P-induced increase in intracellular calcium
in permeabilized detrusor smooth muscle of female rats. Both S1P2 and S1P3 receptors are involved in S1P
mediated enhanced contractile response. The augmentation in S1P-induced contraction in interstitial cystitis
involves both PKC and ROCK pathways of calcium sensitization.
1. Introduction
Interstitial cystitis, also known as painful bladder syndrome, is a
urogenital tract disease that is defined by lower abdominal pain and
some non specific symptoms such as increased frequency and urgency
of urine, i.e. overactive detrusor (Moutzouris and Falagas, 2009). It may
be characterized pathologically by infection, urothelial dysfunction,
autoimmunity, chronic inflammation and mast cell activation in
bladder (Dasgupta and Tincello, 2009). Interstitial cystitis can alter
detrusor smooth muscle contractile activity (Nazif et al., 2007) and may
enhance the normal contractile activity of the bladder, leading to urination during urine filling phase (Masago et al., 2009).
Sphingosine 1-phosphate (S1P), a bioactive lipid, supports cell
growth and discourages programmed cell death (Spiegel and Milstien,
2003). Calcium mobilization, cell proliferation and cell survival is induced by S1P intracellularly while extracellular functions involve cell
⁎
migration, development and maturation of blood vessels (Van Brocklyn
et al., 1998). S1P stimulates specific five subtypes (S1P1–5 receptors) of
G-protein coupled receptors (GPCRs) which belong to endothelial differentiation (EDG) gene receptors family.
S1P induces detrusor smooth muscle contraction by fast release of
calcium from internal stores by stimulating S1P2 receptors. S1P1, S1P2
and S1P3 receptors have been found through out the lower urinary tract
of female rat but the ratio of the expression of these receptors varies
(Sandhu et al., 2009). S1P2 receptors activate protein kinase C (PKC)
and Rho-kinase (ROCK) to increase calcium sensitivity by phosphorylating CPI-17 and inhibiting myosin light chain phosphatase (MLCP) in
male rabbits (Kendig et al., 2013). S1P-induced detrusor contraction is
mediated by both S1P2 and S1P3 receptors involving phospholipase C
(PLC) and ROCK pathways in Sprague Dawley female rats (Sandhu
et al., 2009). Besides these physiological processes, S1P has also a role
in pathophysiology. S1P signalling pathway is significantly up
Corresponding author.
E-mail address: ndurlu@hacettepe.edu.tr (N.T. Durlu-Kandilci).
http://dx.doi.org/10.1016/j.ejphar.2017.08.043
Received 3 March 2017; Received in revised form 25 August 2017; Accepted 25 August 2017
0014-2999/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Anjum, I., European Journal of Pharmacology (2017), http://dx.doi.org/10.1016/j.ejphar.2017.08.043
European Journal of Pharmacology xxx (xxxx) xxx–xxx
I. Anjum et al.
changes were made by moving the Perspex block. The contractile force
was measured by a sensitive force transducer (Swema, Stocholm,
Sweden) connected to a computer using Biopac Student Lab Pro 3.7.3
(Commat Ltd, Turkey) software.
regulated in partial urethral obstruction in male rats with the activation
of the RhoA/ROK pathway. It has also been reported that S1P2, S1P3
receptors and RhoA, ROKα expressions have been increased. Moreover,
S1P-induced contraction is increased in bladder strips from rats having
partial urethral obstruction (Memduh Aydin et al., 2009). The level of
sphingosine kinase-1 expression has been increased on urothelium in
bladder species from patients with neurogenic detrusor overactivity
(Quentin Ballouhey et al., 2015).
Detrusor smooth muscle has a spontaneous rhythmic activity (tone)
that is independent of neurogenic control, which is enhanced in overactive bladder syndrome and interstitial cystitis (Sjogren et al., 1982).
Since S1P is known to have a role in calcium homeostasis of smooth
muscle cells (Watterson et al., 2005), it is of importance to evaluate
whether S1P disturbs contractile activity of detrusor smooth muscle in
interstitial cystitis animal model. The objective of the present study was
to evaluate the intracellular mechanisms of S1P-induced contractile
response in permeabilized detrusor smooth muscle of rats having interstitial cystitis. The receptor subtypes, the role of intracellular calcium stores and the mechanism of calcium sensitization involved S1Pinduced contractile response was investigated. We have now uncovered
a prominent role for the bioactive sphingolipid metabolite, sphingosine1-phosphate (S1P), in regulating detrusor smooth muscle tone in rat
interstitial cystitis model.
2.5. Experimental Protocols
Stable contractile responses were achieved to 80 mM K+ (see
below) and 50 μM carbachol in intact strips. Strips were then moved
into relaxing solution containing 4 mM EGTA with 1 min interval for 2
times. Then the strips were permeabilized with 40 μM β-escin in relaxing solution for 30 min at pH 6.8. After that, the strips were washed
in relaxing solution containing 4 mM EGTA for 1 min and in relaxing
solution containing 0.05 mM EGTA solution for 3 times with 1 min
interval. Strips were accepted as permeabilized if the maximum tension
obtained by 100 μM calcium after permeabilization was found to be
greater than the tension produced by 80 mM K+ in the same strip before permeabilization (Endo et al., 1977).
After this permeabilization procedure, intracellular calcium stores
were loaded by activating solution including 1 μM calcium (prepared in
0.05 mM EGTA) and calmodulin (1 μM) for 10 min. Then, a contractile
response was elicited by S1P (50 μM) in the presence of GTP (100 μM).
In some experiments carried out to evaluate the effect of different inhibitors on S1P-induced contractile response, S1P responses were obtained in the presence of S1P2 receptor inhibitor JTE-013 (10 μM), S1P3
receptor inhibitor suramin (50 μM), Rho kinase inhibitor Y-27632
(1 μM) or protein kinase C inhibitor GF-109203X (5 μM).
The same protocol was repeated in the presence of sarcoplasmic
reticulum Ca2+-ATPase pump inhibitor cyclopiazonic acid (CPA;1 μM)
and mitochondrial proton pump inhibitor carbonyl cyanide p-trifluromethoxyphenylhydrazone (FCCP; 1 μM) to induce calcium sensitization. Calcium sensitization responses were then obtained in the presence of Rho kinase inhibitor Y-27632 (1 μM) and protein kinase C
inhibitor GF-109203 X (5 μM).
2. Materials and methods
2.1. Animal welfare and ethical statement
The study protocol was approved by the Hacettepe University
Animal Ethics Committee (2014/34-6). Female Sprague Dawley rats
(200–250 g), kept under 12-h light/dark period with food and water ad
libitum were used. A total of 84 animals were used in our studies.
2.2. Experimental model
Cyclophosphamide (CYP, 150 mg/kg, dissolved in saline) was injected into rats intraperitoneally once a day on days 1, 4 and 7 to induce
interstitial cystitis. Control groups were injected with saline (0.9%
NaCl), in respectively. On the 8th day, rats were used in studies.
2.6. Western Blotting
Freshly isolated rat bladders were immersed in liquid nitrogen to
snap freeze. ∼ 50 mg solid tissue was pulverized under liquid nitrogen
and the powder homogenized with an electric homogenizer in 15 ml of
ice cold lysis buffer (150 mM sodium chloride, 1% NP-40, 50 mM Tris
pH 8.0). Samples were centrifuged for 20 min at 13,572 g at 4 °C in a
micro centrifuge. Total amount of proteins (~20 µl) were resolved by
SDS-PAGE and transferred to a PVDF membrane and detected using
rabbit polyclonal and monoclonal antibodies raised against EDG-5
(Santa Cruz/sc-25491, 1/200) and EDG-3 (Abcam/ab108370, 1/
10000), in respectively. Immunoblots were stripped off (1 ml 20% SDS
+ 9 ml TBST + 70 µl β-mercaptoethanol) for 30 min at 50 °C and then
reprobed to visualize β-actin using mouse monoclonal antibody (Santa
Cruz/sc-47778, 1/1000). ECL was used to analyse protein expression
levels.
2.3. Histopathological analysis
Rats were euthanized by carbon dioxide inhalation and were killed
by carotid artery bleeding. The urinary bladders were carefully removed on 8th day after CYP injection to assess bladder histopathology.
Tissue samples were fixed in 10% formaldehyde for at least 24 h at
room temperature. Following fixation, tissue samples were embedded
in paraffin, sectioned (5 µm slices) and stained with haematoxylin and
eosin. 5 tissue sections per bladder were obtained. Histopathological
damage was evaluated (in a blinded fashion) based on epithelial damage, haemorrhage, inflammatory cell infiltration and edema.
2.4. Tissue preparation
2.7. Solutions and drugs used in experiments
Rats were euthanized by carbon dioxide inhalation and were killed
by carotid artery bleeding. The urinary bladders were isolated and
placed in Hepes buffered modified Krebs’ solution. The mucosa and
connective tissues were removed from the bladder under a dissecting
microscope. Small strips (150–250 µm in diameter, 3–4 mm in length)
of smooth muscle were dissected from the urinary bladder. A small
hook was tied to one end of a strip to attach it to the transducer, and a
snare of 5/0 surgical silk captured the other end and was used to mount
the strip in a fixed position in 1 ml chamber in one of a series of small
chambers in a Perspex block. The chamber was filled with Hepes buffered modified Krebs’ solution at room temperature and the strips were
equilibrated for 30 min under a resting tension of 100 mg. Solution
All solutions and drugs were prepared by using 18.2 MΩ cm deionized water except FCCP, S1P, CPA and GF-109203X. FCCP was dissolved in ethanol, S1P was dissolved in methanol and CPA and GF109203X were dissolved in DMSO but none of the vehicle affected the
contractile response when tested alone.
2.7.1. Solution used for intact tissues
Hepes buffered modified Krebs' solution contained (mM) NaCl 126;
KCl 6; CaCl2 2; MgCl2 1.2; glucose 14 and HEPES 10.5. The pH was
adjusted to 7.2 with NaOH. 80 mM K+ Krebs’ solution was prepared by
replacing NaCl with an equivalent amount of KCl.
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3.3.2. The role of S1P receptor subtypes in S1P-induced detrusor smooth
muscle contraction
S1P produces its effects by activating its GPCRs. In order to investigate the receptor subtypes involved in S1P-induced detrusor
muscle contraction, S1P2 selective receptor antagonist JTE-013 and
S1P3 selective receptor antagonist suramin were used. After permeabilization, detrusor smooth muscle strips were incubated with JTE-013
(10 µM) and suramin (50 µM) for 15 min and then strips were subjected
to S1P (50 µM). S1P- induced detrusor contraction was significantly
reduced in control (13.01 ± 1.39, n = 7) and CYP-treated group of rats
(17.38 ± 4.92 n = 6) by S1P2 selective receptor antagonist JTE-013.
S1P-induced detrusor contraction was also reduced in both control
(12.10 ± 2.43, n = 6) and cystitis group of rats (13.47 ± 3.65, n = 6)
by S1P3 selective receptor antagonist suramin as shown in Figs. 4A and
B.
2.7.2. Solutions used for permeabilized tissues
Relaxing solution contained (mM) K propionate 130; MgCl2 4;
Na2ATP 4; tris-maleate 20; creatine phosphate 10; EGTA 4 and creatine
phosphokinase 3.3 units/ml; protease inhibitor leupeptin (1 μM). The
pH of this solution was adjusted to 6.8 with KOH. Activating solutions
were the same as relaxing solution except that EGTA was lowered to
0.05 mM, free Ca2+ concentration was adjusted to the desired value
and calmodulin (1 μM) added as specified. GTP (100 μM) was also
added when S1P was used to activate the S1P receptors. The free Ca2+
concentration was calculated using a computer programme ("Bound
and Determined", Brooks and Storey 1992) and expressed as the negative logarithm (pCa). When drugs were added to an organ chamber,
they were made up in relaxing solution containing 0.05 mM EGTA, and
the concentration given was the estimated final concentration.
2.7.3. Drugs used
Drugs were β-escin, carbamylcholine chloride (carbachol), sphingosine 1-phosphate (S1P), JTE-013, suramin, creatine phosphokinase,
leupeptin, ethylene glycol-bis(β-aminoethylether)-N,N,N ′,N′-tetraacetic acid (EGTA), adenosine 5′triphosphate (Na2ATP), GTP, CPA,
calmodulin, FCCP, dimethylsulphoxide (DMSO) from Sigma (St.
Louis,Missouri) and creatine phosphate disodium salt, (R)-(+)-trans-N(4-pyridyl)−4-(1-aminoethyl)-cyclohexanecarboxyamide 2HCl (Y27632) and bisindolylmaleimide 1 HCl (GF-109203X) from Calbiochem
(Nottingham,UK) and Endoxan (Cyclophosphamide, 1 g) from Pfizer.
3.3.3. Expressions of S1P2 and S1P3 receptor proteins
In whole bladders, densitometric quantification of western blots
indicated that S1P2 (EDG-5) protein expression were significantly increased in CYP-treated group of rat bladders when compared to controls
but for S1P3 (EDG-3) there is no change in the expressions between
experimental groups (Figs. 5A and B).
3.3.4. The effect of Y-27632 or GF-109203X on S1P-induced contraction
S1P-induced detrusor smooth muscle contraction was also obtained
in the presence of ROCK inhibitor Y-27632 (1 µM) and PKC inhibitor
GF-109203X (5 µM). S1P-induced contraction was significantly decreased in control (13.44 ± 2.39, n = 6) and CYP-treated group of rats
(12.51 ± 1.39, n = 6) by ROCK inhibitor. S1P-induced contractile response was also inhibited in both control (12.18 ± 1.32, n = 6) and
cystitis group of rats (8.82 ± 1.66, n = 6) by PKC inhibitor as shown in
Figs. 6A and B.
2.8. Data Analysis
Contractions were expressed as % of the response to 80 mM K+
elicited in intact tissues before permeabilization. Data were given as
mean ± S.E.M. of n experiments. Statistical analyses were carried out
by using unpaired Student's t-test for comparing two groups. P < 0.05
was accepted as statistically significant.
3.3.5. S1P-induced calcium sensitization
S1P-induced calcium sensitization response elicited in the presence
of sarcoplasmic reticulum calcium-ATPase pump inhibitor CPA (1 µM)
and mitochondrial blocker FCCP (1 µM) had been increased
(16.52 ± 2.64, n = 6) in permeabilized detrusor smooth muscle isolated from CYP-treated group of rats compared to control group
(8.37 ± 1.01, n = 6) as shown in Fig. 7.
3. Results
3.1. Histopathological changes in rat bladder having cyclophosphamide
induced cystitis
Haemorrhage in mucosa and muscle (muscularis propria) layer, and
inflammatory cell infiltration in subepithelial layer were observed in
bladder isolated from rats in cyclophosphamide-induced cystitis group
as compared to control group (Fig. 1).
3.3.6. The effect of Y-27632 or GF-109203X on S1P-induced calcium
sensitization
The calcium sensitization response in control group increased with
ROCK inhibitor Y-27632 (14.62 ± 2.063, n = 6) and PKC inhibitor GF109203X (13.54 ± 1.245, n = 6) but were inhibited in CYP-treated
group of rats (11.53 ± 1.702, n = 6), (8.23 ± 1.29, n = 6) in respectively as shown in Figs. 8A and B reaching back to control group response.
3.2. Intact detrusor smooth muscle
80 mM K+-induced contraction response was obtained from intact
detrusor smooth muscle strips. There was not any significant difference
in control and cystitis group responses (Fig. 2, control group
230.65 mg ± 11.85, cystitis group 230.26 mg ± 13.94; n = 35)
(P > 0.05).
4. Discussion
In the present study, the intracellular signalling pathways and the
receptor subtypes involved in S1P-induced contractile response due to
interstitial cystitis were evaluated in detrusor smooth muscle of rats, by
using chemically permeabilized strips. The cause of interstitial cystitis
is still unknown, but our findings may help understanding how different
intracellular pathways may play a role in overactivity of bladder in
cystitis. In throughout the study, whole bladders were used for Western
Blotting while functional studies had been performed on urothelium
denuded detrusor strips.
The contractile activity of detrusor smooth muscle is predominantly
regulated by acetylcholine but nonadrenergic, noncholinergic (NANC)
mechanisms also have small contributions (Smith and Chapple, 1994).
Recently, it has been shown that a new mediator of NANC pathway, S1P
is also involved in detrusor contractions (Watterson et al., 2007). S1P-
3.3. Permeabilized detrusor smooth muscle
3.3.1. S1P-induced contraction
Rat bladder smooth muscle strips were mounted for isometeric force
recording under the basal tension of 100 mg and were subjected to S1P
(50 µM) or solvent alone (methanol) after permeabilization. S1P produced initial fast phasic contractions followed by very slow but sustained contractions yielding tonic response in rat detrusor smooth
muscles (Fig. 3A). S1P-induced contraction was increased significantly
(42.90 ± 5.98, n = 8) in bladder smooth muscle strips isolated from
CYP-treated group of rats as compared to control group (20.07 ± 1.51,
n = 11) as shown in Fig. 3B.
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Fig. 1. Histopathological images of bladder isolated from rats of control (A) and cystitis (B) group after hematoxylin and eosin staining (n = 4–5).
characterized by inflammation, urgency, frequency, painful urination
and fullness (Nazif et al., 2007). It may be caused by bacterial infection
or by noninfectious conditions, mostly of unknown etiology (Lee et al.,
2014). Systemic injection of cyclophosphamide in rats and mice has
been widely used to induce interstitial cystitis (Bjorling et al., 2011).
Acrolein is the metabolic end product of cyclophosphamide. It enters
into epithelial cells of bladder and activates the production of reactive
oxygen species and nitric oxide species. Nitric oxide species lead to
peroxynitrite production. Increased level of peroxynitrite damages
DNA, lipids and proteins causing necrotic cell death (Korkmaz et al.,
2007). Thus, acrolein produces edema, haemorrhage and ulceration of
urinary bladder wall (Batista et al., 2006; Cox, 1979). In this study,
epithelial damage, edema, mucosal and submucosal haemorrhage and
subepithelial inflammatory cells infiltration after haematoxylin and
eosin staining were observed in isolated urinary bladder of rats treated
with cyclophosphamide (shown in Fig. 1), in parallel with previous
findings (Golubeva et al., 2014; Joshi et al., 2008; Korkmaz et al.,
2007).
It is widely known that interstitial cystitis changes detrusor smooth
muscle contractile activity. In our experiments, contractions aroused
from depolarization by high potassium was not changed in intact detrusor strips (Fig. 2). S1P produced an initial phasic contraction followed by a consistent tonic contraction of permeabilized detrusor, as in
parallel with literature (Watterson et al., 2007). S1P-induced contraction was increased more than two folds in cystitis compared to control
(Fig. 3). The increased contraction elicited by S1P in cystitis showed
that detrusor was more responsive to S1P, in accordance with partial
urethral obstructive rat model (Memduh Aydin et al., 2009). Both interstitial cystitis and partial urethral obstruction are manifested by
overactive bladder tone. It has been demonstrated that detrusor contraction to carbachol was increased in Escherichia coli-induced cystitis
Fig. 2. The contractile response elicited with 80 mM K+ in intact detrusor smooth muscle
isolated from control and CYP-treated group of rats (P > 0.05 compared to control group;
n = 35).
induced contractions were also observed in male Sprague Dawley rats
(Memduh Aydin et al., 2009) although some researchers could not get
this response in other species of rat (Kendig et al., 2013). Our finding
that S1P-induced contractile force in female rats was lower than that of
carbachol (data not shown) is also consistent with those previous studies in rabbits (Kendig et al., 2013; Watterson et al., 2007).
The role of calcium sensitization pathways, contractile proteins and
intracellular stores in action of agonists can be evaluated by using
permeabilization technique. Chemically permeabilized muscle enables
to control intracellular calcium (Nasu, 1989). Among the permeabilization agents, β-escin is a saponin ester that opens pores on the cell
membrane allowing passage of molecules up to 150 kDa such as IP3 and
heparin, and it retains receptor–effector coupling (Iizuka et al., 1994;
Sei Kobayashi et al., 1989; Toshio Kitazawal et al., 1989). Some small
endogenous substances, i.e. GTP and calmodulin, may escape out of the
cell during permeabilization so these should be replaced exogenously.
Interstitial cystitis is a syndrome of bladder hypersensitivity
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Fig. 3. Contraction of permeabilized detrusor smooth
muscle by S1P (50 µM) (A). The contractile response
elicited with S1P (50 µM) in permeabilized detrusor
smooth muscle isolated from control and CYP- treated
group of rats (B) (*P < 0.05 compared to control
group; n = 8–11).
Fig. 4. The contractile response elicited with S1P (50 µM) in the
absence and presence of JTE-013 (10 µM) (A) and suramine
(50 µM) (B) in permeabilized detrusor smooth muscle isolated
from control and CYP-treated group of rats (*P < 0.05 compared
to control response, **P < 0.05 compared to CYP group; n =
6–7).
urinary bladder and brain tissues (Sandhu et al., 2009; Zhang et al.,
2010). S1P1 receptors are coupled to Gi protein and activation leads to
an increase in intracellular calcium in endothelial cells (Zhang et al.,
2010) but not in other tissues (Hu et al., 2006; Rapizzi et al., 2007;
Siehler and Guerini, 2006). S1P2 and S1P3 receptors are coupled to Gq
(Weng et al., 2005). Moreover, an increase in urination reflex was
shown in male rat models of interstitial cystitis induced by cyclophosphamide (Aronsson et al., 2015).
There are 5 subtypes in S1P receptor family where S1P1, S1P2 and
S1P3 receptors have been widely distributed in liver, lung, kidney,
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Fig. 5. Western blots for S1P2 (EDG-5) (A) and S1P3 (EDG-3) (B) in control and CYP-treated group rat bladders detected using rabbit polyclonal antibodies. EDG-5 and EDG-3 protein
levels were normalized to β-actin protein levels detected by the mouse monoclonal antibody (*P < 0.05 compared to control group, n = 3–4).
Sandhu et al., 2009; Watterson et al., 2007).
In order to evaluate the mechanism of enhanced S1P2 and S1P3
receptor induced contractile responses, protein expressions were studied in whole bladders. Our results propose that, in a cystitis bladder rat
model, enhanced S1P2 (EDG-5) receptor signalling pathway as a result
of an increase in EDG-5 expression may be associated with augmented
S1P-induced detrusor smooth muscle contraction. Since we demonstrated the increased S1P2 (EDG-5) expression in whole bladder, we
cannot argue that the increased function in denuded detrusor strips is
specific to the enhanced EDG-5 expression in detrusor smooth muscle.
Although the majority of the bladder tissue consists of muscle layer, a
potential contribution of EDG-5 receptors in urothelium cannot be
protein and activation leads to an increase in intracellular calcium via a
release from intracellular calcium stores (Hopson et al., 2011; Hu et al.,
2006; Murakami et al., 2010; Rapizzi et al., 2007). S1P-induced contractions were reduced by both S1P2 receptor antagonist, JTE-013 and
S1P3 receptor antagonist, suramin (35% and 40%, in respectively)
showing the role of these two receptor subtypes in detrusor. Besides, the
inhibition of S1P-induced contraction by these antagonists was more
pronounced in cystitis compared to that of control; i.e. S1P2 receptor
was blocked by 60% and S1P3 receptor was inhibited by 69%. Our
functional results in urothelium denuded permeabilized bladders are in
parallel with other studies showing both S1P2 and S1P3 receptors are
responsible for bladder contraction induced by S1P (Kendig et al., 2013;
Fig. 6. The contractile response elicited with S1P (50 µM) in the
absence and presence of Y-27632 (1 µM) (A) and (B) GF-109203X
(5 µM) in permeabilized detrusor smooth muscle isolated from
control and CYP-treated group of rats (*P < 0.05 compared to
control response, **P < 0.05 compare to CYP group; n = 6–8).
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These inhibitors also blocked the increased S1P-induced contraction in
cystitis by 71% and 79%, in respectively, showing the involvement of
both pathways (Fig. 6). In one study, the researchers investigated the
role of PKC pathway in rats having lipopolysaccharides-induced cystitis
showing that detrusor contraction is dependent on PKC (Weng et al.,
2005). In another study evaluating the importance of ROCK, it has been
concluded that this pathway is involved in dysfunction in rats with
hydrochloric acid-induced cystitis (Shimizu et al., 2013). S1P receptors
are coupled to Gi/o, Gq, G12 and G13 proteins but PLC-PKC pathway is
activated by S1P3 receptors and ROCK pathway is activated by mainly
S1P2 receptors (Igarashi et al., 2001; Nofer et al., 2004). Both PKC and
ROCK were involved in S1P-induced detrusor contractions in rabbits,
mainly depending on ROCK (Kendig et al., 2013). Inhibition by both
PKC and ROCK may suggest an interaction between these two enzymes,
as a cross talk (Niiro et al., 2003; Patil and Bitar, 2006; Wang et al.,
2009, 2012).
Intracellular calcium level and smooth muscle contraction doesn’t
coordinate in parallel always (Somlyo and Himpens, 1989). The contraction of smooth muscle is also related to calcium sensitization which
doesn’t depend on intracellular calcium level (A. Horowitz, 1996; Gong
et al., 1992). The physiological phenomenon that is responsible for the
increase in smooth muscle contractile force by inhibition of MLCP at a
constant calcium and MLCK level is calcium sensitization (Somlyo,
2002). Inhibition of MLCP is carried out either by ROCK or CPI-17.
ROCK phosphorylates the MYPT1 component while CPI-17, activated
by PKC, phosphorylates the PPIc component of MLCP to inhibit MLCP
activity (Somlyo and Somlyo, 1998). In the presence of sarcoplasmic
reticulum calcium-ATPase pump inhibitor CPA and mitochondrial
blocker FCCP at constant calcium level, S1P-induced calcium sensitization response was increased significantly in the cystitis group (97%,
Fig. 7). The inhibition of this increased calcium sensitization response
in cystitis by ROCK and PKC inhibitors showed the involvement of both
pathways. However, S1P-induced calcium sensitization in control group
was increased significantly by ROCK and PKC inhibitors. This increase
Fig. 7. S1P (50 µM)-induced calcium sensitization response in permeabilized detrusor
muscle isolated from control and CYP-treated group of rats (*P < 0.05 compared to
control group; n = 6).
excluded. For example, endothelial cells have the potential to synthase
and secrete S1P via its receptors located in their cell membranes
(Berdyshev et al., 2011) where it can act on muscular cells in autocrine
or paracrine manner. Furthermore, S1P might induce the tonic contraction of lymphatic muscles by activating EDG-5 receptors (Kimizuka
et al., 2013).
On the other hand, for S1P3 (EDG-3) there is no change in the expressions between control and cystitis groups. Therefore, under our
experimental conditions, in a cystitis bladder rat model, EDG-3 contribution in augmented S1P-induced detrusor smooth muscle contractions occur in a total EDG-3 receptor expression independent manner.
Hence, increased S1P contractions through S1P3 (EDG-3) receptors in
muscle cells may be related with an increased affinity or efficacy of the
receptor, in cystitis-induced overactive bladders. However, probable
increased expression of EDG-3 in detrusor smooth muscle masked by a
decrease in urothelium tissue should also be considered.
ROCK inhibitor Y-27632 inhibited S1P contraction by 33% where
PKC inhibitor GF-109203X inhibited this response by 39% in control.
Fig. 8. The calcium sensitization response elicited with S1P
(50 µM) in the presence of Y-27632 (1 µM) (A) or GF-109203X
(5 µM) (B) in permeabilized detrusor smooth muscle isolated
from control and CYP-treated group of rats (*P < 0.05 compared
to control response, **P < 0.05 compare to CYP group; n = 6).
7
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5. Conclusion
The present study was designed to determine whether or not S1P
induced intracellular signalling is involved in augmented detrusor
smooth muscle activity in interstitial cystitis. Under our experimental
conditions, S1P enhanced isometric contractile force in permeabilized
rat detrusor having interstitial cystitis. Both S1P2 and S1P3 receptor
subtypes are involved in this increased S1P-induced contractile force.
According to our results, in a cystitis bladder rat model, increased S1P2
receptor protein expression in cystitis indicated up regulation of S1P
signalling pathway while S1P3 contribution in augmented S1P-induced
detrusor smooth muscle contractions occur in a total S1P3 receptor
expression independent manner. An increased calcium sensitization
response in interstitial cystitis may also be responsible for S1P-induced
augmented contractile response. Both ROCK and PKC signalling pathways participate in S1P-induced contractile force and calcium sensitization response in permeabilized detrusors from rats having cystitis.
In conclusion, dysregulation of S1P signalling pathway may contribute in interstitial cystitis-induced overactive bladder pathology. The
findings of this study may help enlighten the molecular changes of
bladder physiology in interstitial cystitis of different causes i.e. cyclophosphamide treatment in oncology. Therefore, the investigation of
intracellular mechanisms of mediators which has a role in detrusor
innervation under pathological conditions may be of importance in
developing new drugs for the treatment of bladder dysfunction.
Acknowledgement
This study was supported by Hacettepe University Scientific
Research Projects Coordination Unit (Project number: 014D08301002).
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