Involvement of Bradykinin in Trypsin-Induced Urinary Bladder Contraction in Cyclophosphamide-Treated Rats

Abstract

We have previously demonstrated that both trypsin and the PAR-2 activating peptide (SLIGRL-NH 2 ) contract isolated rat urinary bladders by the activation of urothelium PARs and the subsequent release of prostaglandins.3) Furthermore, we found that contractions induced by PAR-2 agonists could be partly mediated through stimulation of capsaicin-sensitive sensory nerves.4) The PAR-2-mediated responses were enhanced after the induction of cystitis with cyclophosphamide (CYP).5) These findings suggested that PAR-2 plays a role in regulating bladder contractility under physiological and pathological conditions. Bradykinin, a metabolite of the kallikrein-kinin system, has multiple actions in urinary bladders, including the ability to contract the detrusor smooth muscle, 6) stimulate sensory nerves 6,7) and evoke the release of cyclooxygenase (COX) products.8) Also, bradykinin might play a key role in the pathogenesis of experimental cystitis 9) and activation of the kallikrein-kinin system in urinary bladders of patients with interstitial cystitis has been reported. 10,11) Thus, the actions of bradykinin in urinary bladders resemble those of PAR-2 agonists.In the present study, we examined the role of bradykinin in contractions induced by trypsin and the PAR-2 agonist 2-furoyl-LIGRL-NH 2 12) in the urinary bladders from normal and CYP-induced cystitis rats. MATERIALS AND METHODS AnimalsThe protocol for the use of animals was approved by the Committee on the Care and Use of Laboratory Animals of Kitasato University.Male Wistar rats weighing 250-280 g were maintained on standard rat chow and tap water ad libitum with a 12 h light/ dark cycle in a quiet environment. Rats were treated with vehicle (saline) or CYP (150 mg/kg, intraperitoneally (i.p.)). Experiments were performed 48 h after the treatment. Preparation of Rat Urinary Bladder StripsThe rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and sacrificed. The urinary bladder was removed and placed in ice-cold Krebs-Ringer bicarbonate buffer (KRB, composition in mmol/l: NaCl, 119; KCl, 4.8; MgSO 4 , 1.2; KH 2 PO 4 , 1.2; CaCl 2 , 2.5; NaHCO 3 , 25 and glucose, 10.0) (pHϭ7.4). Four longitudinal strips (approximately 1 mmϫ2 mmϫ10 mm) were isolated from the bladder body.Measurement of Mechanical Activity One end of each strip was attached to an isometric force displacement transducer (model TB-611T, Nihon Kohden, Tokyo) by a cotton thread, and the other end was tied to a stainless-steel holder. Tension was digitized at a sampling rate of 2 Hz (model 15BXW-H4, Dacs Giken, Okayama) and stored on the hard disk of a personal computer. Strips were mounted in 10-ml jacketed organ baths filled with KRB gassed with 95% O 2 -5% CO 2 at 37°C. The preparations were placed under initial load (9.8 mN) and the resting tension was adjusted every 15 min. The tissues were allowed to equilibrate for 60 min and the bath fluid was changed every 15 min with fresh KRB. After the equilibration period, all preparations were contracted with acetylcholine (ACh) (100 mmol/l) to c...