Trypsin-induced contractile responses are enhanced in permeabilized detrusor smooth muscle in a rat model of cystitis

I. Anjum¹, M. Denizalti¹, G. Simsek²,N. T. Durlu-Kandilci¹. ¹Department of Pharmacology, Hacettepe University, Ankara, Turkey, ²Department of Biophysics, Ankara University, Ankara, Turkey.

Introduction: Protease-activated receptors (PAR_1,2,3,4) belong to G-protein coupled receptor family. Trypsin causes smooth muscle contraction via PAR₂ receptors, both in physiological and pathological situations.^1,2,3 It has been shown that activation of PAR₂ stimulates release of prostaglandins from mucosal layer and thereby contracts bladder smooth muscle². Furthermore, trypsin-induced contractions were increased in cystitis without any change in PAR₂receptor expression, but in detrusors with urothelium³. In the present study, The intracellular mechanisms of trypsin-induced contractile response were investigated in permeabilized detrusor smooth muscle of rats without urothelium, in a model of cystitis.

Method: The study protocol was approved by the University Animal Ethics Committee (2016/07-06). 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 (urothelium denuded) were mounted in 1 ml organ baths containing modified Krebs\' solution. Tissues were permeabilized with 40 μM β-escin for 30 min. Isometric contractions were expressed as % of 80 mM K⁺. PAR₂ receptor protein levels were determined by Western blotting. Data were given as mean±S.E.M. P<0.05 was accepted as significant.

Results: Trypsin (1 mM)-induced contractile responses were increased from 10±1% (control,n=6) to 14±1% in cystitis (P<0.05,n=7). These contractions were inhibited 60% by PAR₂receptor antagonist ENMD-1068 (5mM) in control. PAR₂ receptor protein expression was also increased 30% in cystitis (compared to control,P<0.05,n=3-4). Trypsin-induced contractions were inhibited 40% by sarcoplasmic reticulum IP₃ receptor blocker heparin (1mg/ml) in control where 64% in cystitis (P<0.05,n=6). Moreover, contractions were inhibited 70% by sarcoplasmic reticulum ryanodine channel blocker ryanodine (10mM) in control but 35% in cystitis (n=4). Contractions were decreased to 7±1% in control and to 9±1% in cystitis by Rho kinase inhibitor Y-27632 (1μM) (P<0.05,n=5-7). These responses were also attenuated by protein kinase C inhibitor GF-109203X (5μM) in both groups, 5±1% in control and 8±1% in cystitis (P<0.05,n=6).

Conclusion: Our findings provide the first evidence that cystitis increases trypsin-induced contractions in permeabilized detrusor smooth muscle of rats. The augmentation in trypsin-induced contractions via PAR₂ receptors in cystitis involves an increase in intracellular calcium release from sarcoplasmic reticulum and moreover an activation in both Rho kinase and protein kinase C pathways.

References:

1. Kubota et al. (2003). Naunyn-Schmiedeberg’s Arch Pharmacol 367: 588-591.

2. Nakahara et al. (2003). Naunyn-Schmiedeberg’s Arch Pharmacol 367: 211-213.

3. Nakahara et al. (2004). Naunyn-Schmiedeberg’s Arch Pharmacol 369: 212-219.