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

I. Anjum1, M. Denizalti1, H. B. Kandilci2, N. T. Durlu-Kandilci1, I. Sahin-Erdemli1. 1Department of Pharmacology, Hacettepe University, Ankara, TURKEY, 2Department of Biophysics, Ankara University, Ankara, TURKEY.

Introduction: Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that causes smooth muscle contraction via S1P1-3 receptors, both in physiological and pathological situations1,2,3. G-protein coupled receptors (GPCRs) have been known to activate smooth muscle contraction mainly via inositol 1,4,5-trisphosphate (IP3). It has been later shown that cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), two other major intracellular calcium mobilizing agents, may also participate in agonist-induced contraction4,5. The intracellular mechanism of S1P-induced contractile response and receptor subtypes were investigated in permeabilized detrusor smooth muscle of rats having cyclophosphamide induced cystitis.

Method: The study protocol was approved by the University Animal Ethics Committee (2014/34-6). Cyclophosphamide (150 mg/kg) was injected to rats (Sprague-Dawley,female,200-250g) intraperitoneally once a day on days 1, 4 and 7 to induce cystitis. Control group was injected with saline (NaCl 0.9 %) using the same protocol. Detrusor smooth muscle strips were mounted in 1 ml organ baths containing Hepes buffered modified Krebs' solution. Tissues were permeabilized with 40 µM β-escin for 30 min. Isometric contractions were expressed as % of 80 mM K+. S1P2 receptor protein levels were determined by Western blotting. Data were given as mean±S.E.M. P<0.05 was accepted as significant.

Results: S1P (50 µM)-induced contraction was significantly increased from 20±2% (control,n=11) to 43±6% in cystitis (n=8). This contraction was significantly inhibited by S1P2 receptor specific antagonist JTE-013 (10µM) in control (13±1%,n=7) and cystitis (17±5%,n=6) groups. S1P2 receptor protein expression was increased in cystitis group (59±16%,n=3). S1P-induced contraction was also significantly inhibited by S1P3 receptor antagonist suramin (50µM) both in control (12±2%,n=6) and cystitis rats (14±4%,n=6). S1P-induced contraction was reduced by IP3 receptor blocker, heparin (1mg/ml), in control (11±1%,n=6) and CYP-treated groups (12±3%,n=6). Moreover, bafilomycin (100nM), a vacuolar H+-ATPase inhibitor, blocked S1P-induced contraction in both control (9±2%,n=6) and CYP-treated groups (12± 4%,n=6).

Conclusion: According to the present data, in interstitial cystitis, it seems that both S1P2 and S1P3 receptors are involved in S1P-induced augmented contractile response in permeabilized detrusor smooth muscle. Increased expression of S1P2 receptor protein indicates the up regulation of S1P pathway in cystitis. Furthermore, our results suggest the involvement of both IP3 sensitive sarcoplasmic reticulum stores and acidic lysosomal intracellular calcium stores in S1P-induced augmented contractile response in cystitis.

References: 1Watterson et al. (2007) FASEB J.2,2818-2828. 2Aydin et al. (2010) BJU Int.106,562-572. 3Q.Ballouhey et al. (2015) IBJU.41,1141-1147. 4Boittin et al. (2003) J. Biol. Chem. 278,9602-9608. 5Churchill et al. (2003) Cell. 111,703-708.