Sphingosine-1-phosphate (S1P) induced contractile responses in detrusor smooth muscle of rats having cyclophosphamide induced cystitis

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that plays a role in smooth muscle contraction via G-protein coupled receptors (S1P1-5 receptors). S1P2and S1P3 receptors are known to activate smooth muscle contraction viaRhoA/ROK pathways. Besides some physiological events, S1P production also takes place in some chronic pathological situations as in overactive bladder syndrome1. Moreover, it was reported that S1P and Rho kinase pathways were upregulatedin partial urethral obstruction wherein accordingly, S1P2 and S1P3 receptors together with Rho A and ROKα expressions were elevated resulting in an increased contraction by S1P2. Interstitial cystitis is a syndrome characterized with chronical inflammation which causes overactive bladder. The physiological functions of the bladder may change in cystitis. In the present study, the intracellular mechanisms of changes in S1P-induced contractile responses were investigated following the permeabilization with β-escin in detrusor smooth muscle of rats having cyclophosphamide induced cystitis. The study protocol was approved by the University Animal Ethics Committee (2014/34-6). Cyclophosphamide (150 mg/kg, dissolved in saline)was injected into rats (Sprague-Dawley, female, 200-250 g) intraperitoneally once a day on days 1,4 and7 to inducecystitis. Control groups were injected with saline (%0.9 NaCl). The bladder from rats were isolated and then the mucosa and the connective tissues were removed under a dissecting microscope. Detrusor smooth muscle strips (approximately 150-250 µm in diameter and 3-4 mm in length) were mounted in 1 ml organ baths containing Hepes buffered modified Krebs' solution (NaCl 126; KCl 6; CaCl2 2; MgCl2 1.2; glucose 14 and HEPES 10.5 in mM) under a resting tension of 100 mg. Isometric contractions were recorded and expressed as % of 80 mM K+. Data are expressed as mean±S.E.M. Statistics was done by Student’s t test. P<0.05 was accepted as significant. Tissues were permeabilized with 40 µM β-escin for 30 min. S1P (50 µM)-induced contractions were significantly increased from 20.1±1.5% (control, n=11) to 42.9±6% in cystitis (n=8, P<0.05). This elevation in contractile response in cystitis group is significantly inhibited by S1P2 receptor selective antagonist JTE-013(10 µM) that is 22.9±5.4 %(n=4, P<0.05). S1P-induced calcium sensitization contractions elicited in the presence of sarcoplasmic reticulum Ca2+-ATPase pump inhibitor cyclopiazonic acid (CPA; 1 µM) and mitochondrial blocker carbonyl cyanide p-trifluromethoxyphenylhydrazone (FCCP; 1 µM) were also significantly increased in cystitis (from 8.4±1%, n=6 to 16.5±2.7%, n=6, P<0.05). In conclusion, interstitialcystitis enhances both S1P-induced calcium release from intracellular stores and calcium sensitization3in detrusorsmooth muscle. These functional preliminary data suggested the contractile response induced by S1P in permeabilized bladder smooth muscle in Sprague-Dawley ratswas coupled to S1P2 receptors. Moreover, the increase in S1P induced calcium sensitization contractions in cystitis group may also suggest the activation of Rho kinase and protein kinase C pathways.

1 FASEB J. 2007;21: 2818-2828

2 BJU Int. 2010;106: 562-571

3 Physiol. Rev. 2003;83: 1325-1358