Determination of the roles of cADPR and NAADP as intracellular calcium mobilizing messengers in S1P-induced contractions in rat bladders having IC/PBS.

AIMS: Interstitial cystitis/painful bladder syndrome (IC/PBS) is characterized by lower abdominal pain and increased frequency and urgency of urine. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that plays role in calcium homeostasis in smooth muscle. The intracellular calcium mobilizing secondary messengers are also involved in smooth muscle contraction. The role of intracellular calcium storing depots in S1P-induced contraction was investigated in permeabilized detrusor smooth muscle having cystitis. MAIN METHODS: IC/PBS was induced by cyclophosphamide injection. The detrusor smooth muscle strips isolated from rats were permeabilized with ß-escin. KEY FINDINGS: S1P-induced contraction was increased in cystitis. S1P-induced enhanced contraction was inhibited by cyclopiazonic acid, ryanodine and heparin showing involvement of sarcoplasmic reticulum (SR) calcium stores. Inhibition of S1P-induced contraction by bafilomycin and NAADP suggested the participation of lysosome-related organelles. SIGNIFICANCE: IC/PBS triggers S1P-induced increase in intracellular calcium from SR and lysosome-related organelles in permeabilized detrusor smooth muscle.

Trypsin-induced elevated contractile responses in a rat model of interstitial cystitis/bladder pain syndrome: Involvement of PAR2 and intracellular Ca2+ release pathways.

AIMS: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory disease with unclear etiology. Different receptors play a role in the pathophysiology including protease activated receptors (PARs). The present study aimed to investigate the subtypes and the effects of PARs on contractility using permeabilized detrusor smooth muscle strips in IC/BPS. MAIN METHODS: IC/BPS was induced by cyclophosphamide injection. Histopathological analysis, PCR for detecting PAR proteins, western blotting for indicating PAR2 protein expression levels and myograph recording for measuring contractile force were used. KEY FINDINGS: The present study reveals that in rat bladder PAR1 and PAR2 but not PAR4 were found to be expressed. The first evidence was revealed where trypsin-induced contractions in rat permeabilized detrusor were potentiated in CYP-induced cystitis. Moreover, the functional inhibition of trypsin-induced contractions by selective PAR2 antagonist (ENMD-1068) and the supporting immunoblotting results emphasized that the main PAR subtype involved in IC/BPS model in rat bladder is PAR2. Our data emphasize the prominent role of IP3 in cystitis pathology besides ryanodine channels. Trypsin-induced Ca2+sensitization contractions were also higher in cystitis. Both Rho kinase and protein kinase C played a role in this increased Ca2+sensitization situation. SIGNIFICANCE: The present paper highlights the intracellular pathways that are involved in trypsin-induced contractions mainly via PAR2 in permeabilized bladder detrusor smooth muscle in a rat model of IC/BPS.

Rho Kinase and Protein Kinase C Pathways are Responsible for Enhanced Carbachol Contraction in Permeabilized Detrusor in a Rat Model of Cystitis.

Interstitial cystitis is a syndrome characterized by detrusor overactivity and chronic inflammation of the bladder. The mechanisms responsible for the altered smooth muscle contractility remain poorly understood. The aim of the study was to investigate the role of intracellular signalling pathways in carbachol-induced detrusor contraction in a rat model of interstitial cystitis. Cyclophosphamide (150 mg/kg, dissolved in saline) was injected to rats (Sprague-Dawley, female, 200-250 g) intraperitoneally once a day on days 1, 4 and 7 to induce interstitial cystitis. Control groups were injected with saline (0.9% NaCl). Detrusor smooth muscle strips were mounted in 1-ml organ baths containing HEPES-buffered modified Krebs' solution and permeabilized with 40 µM ß-escin for 30 min. Carbachol-induced contractions were significantly increased from 21.2 ± 1.6% (saline-treated) to 44 ± 4.4% in cyclophosphamide-treated group. The Rho kinase inhibitor Y-27632 (8.8 ± 2%) and the protein kinase C inhibitor GF-109203X (11.7 ± 2.8%) inhibited the increased contractile response (44 ± 4.4%) in rats with cystitis. The increased carbachol-induced contraction (44 ± 4.4%) was also significantly inhibited by the sarcoplasmic reticulum ryanodine channel blocker ryanodine (25.8 ± 3.2%) and the sarcoplasmic reticulum IP3 receptor blocker heparin (17.2 ± 2.2%) in cystitis. RhoA protein levels in the bladder of cyclophosphamide-treated rats were significantly increased while pan-protein kinase C (α, ß and γ isoforms) protein expression was unaltered between experimental groups. Carbachol-induced calcium sensitization at constant and clamped calcium (pCa 6) was also increased in cystitis (from 15.8 ± 2.2% to 24.7 ± 2.8%). This increased response (24.7 ± 2.8%) was significantly inhibited by both Y-27632 (7.9 ± 0.7%) and GF-109203X (4.4 ± 1.5%). We conclude that interstitial cystitis is characterized by an enhanced carbachol contractile response as well as by calcium sensitization of the detrusor smooth muscle. Activation of Rho kinase and protein kinase C pathways may be the molecular culprits responsible for the augmented muscarinic response observed in cystitis.

Enhancement of S1P-induced contractile response in detrusor smooth muscle of rats having cystitis.

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 ß-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 ß-escin. S1P (50µM)-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 Y-27632 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.

The effect of cannabinoids on dinitrofluorobenzene-induced experimental asthma in mice.

Cannabinoids have anti-inflammatory effects and can produce bronchodilation in the airways. We have investigated the effects of cannabinoids on tracheal hyperreactivity and airway inflammation in dinitrofluorobenzene (DNFB)-induced experimental non-atopic asthma in mice. 5-hydroxytryptamine (5-HT)-induced contraction response was enhanced while carbachol- and electrical field stimulation-induced contractions, and isoprenaline-induced relaxation responses were remained unchanged in DNFB group. The increased 5-HT-induced contractions were inhibited by incubation with either atropine or tetrodotoxin. DNFB application resulted in increased macrophage number in the bronchoalveolar lavage fluid (BALF). In vivo ACEA (CB1 agonist) treatment prevented the increase in 5-HT contractions, while JWH133 (CB2 agonist) had no effect. However, neither ACEA nor JWH133 prevented the increase in macrophage number in BALF. In vitro ACEA incubation also inhibited the increase in 5-HT contraction in DNFB group. These results show that cannabinoid CB1 receptor agonist can prevent tracheal hyperreactivity to 5-HT in DNFB-induced non-atopic asthma in mice.