Role of c-Jun N-Terminal Kinase (JNK) Activation in Micturition Reflexes in Cyclophosphamide (CYP)-Induced Cystitis in Female Rats

Abstract

c-Jun N-terminal Kinase (JNK) is member of the Mitogen-Activated Protein Kinase (MAPK) family, activated through phosphorylation following cytokine exposure and stress. In this study, phosphorylation of JNK was examined in the urinary bladder with CYP-induced cystitis and the effects of SP600125, a selective inhibitor of phosphorylation of JNK, on urinary bladder function were assessed using conscious, open outlet, cystometry with continuous instillation of intravesical saline. We induced bladder inflammation in adult female Wistar rats by injecting CYP intraperitoneally to produce acute (150 mg/kg; 4 hr), intermediate (150 mg/kg; 48 hr) and chronic (75 mg/kg; every third day for 10 days) treatments. Western blotting of urinary bladder demonstrated a significant (p ≤ 0.01) increase (i.e., phosphorylation) in JNK activation with 4 hr and 48 hr CYP-induced cystitis. Immunohistochemistry and image analyses demonstrated a significant (p ≤ 0.01) increase in JNK activation in the urothelium with 4 hr and 48 hr CYP-induced cystitis. Blockade of JNK phosphorylation significantly (p ≤ 0.01) increased bladder capacity and intercontraction void intervals in CYP-treated rats (4 hr and 48 hr). Furthermore, blockade of JNK phosphorylation reduced (p ≤ 0.01) neuropeptide (substance P, calcitonin gene-related peptide) expression in the urinary bladder with CYP-induced cystitis (4 hr and 48 hr). In contrast, blockade of JNK phosphorylation was without effect on bladder function or neuropeptide expression in urinary bladder in control (no inflammation) rats. Blockade of JNK phosphorylation may represent a novel target for improving urinary bladder function with CYP-induced cystitis.

The Effects of Tempol on Cyclophosphamide‐Induced Oxidative Stress in Rat Micturition Reflexes

“…Tissue processing and ELISAs were performed as described previously [ 18 , 30 ]. Briefly, rats from control ( n = 6 each) and all experimental groups ( n = 6 each) were deeply anesthetized (4% isoflurane), and a thoracotomy was performed.…”

“…Individual rat bladders were dissected, weighed, and placed in Tissue Protein Extraction Reagent (1 g tissue/20 mL; Pierce Biotechnology, Woburn, MA) with complete protease inhibitor cocktail tablets (Roche Applied Science, Mannheim, Germany) and stored at −80°C. On the day of assay, individual bladders were disrupted with a Polytron homogenizer until being homogeneous and centrifuged (10,000 rpm for 10 min) [ 18 , 30 ], and the supernatant was used for total protein estimation and CGRP (Phoenix Pharmaceuticals, Inc., Burlingame, CA), Sub P (Phoenix Pharmaceuticals), and 3-NT (Millipore Corporation, Bellerica, MA) quantification. Total protein was determined by the Coomassie Plus Protein Assay Reagent Kit (Pierce) [ 18 , 30 ] and CGRP, Sub P, and 3-NT were quantified using standard 96-well ELISA plates (Phoenix Pharmaceuticals; Millipore Corporation) according to the manufacturer's recommendations.…”

“…On the day of assay, individual bladders were disrupted with a Polytron homogenizer until being homogeneous and centrifuged (10,000 rpm for 10 min) [ 18 , 30 ], and the supernatant was used for total protein estimation and CGRP (Phoenix Pharmaceuticals, Inc., Burlingame, CA), Sub P (Phoenix Pharmaceuticals), and 3-NT (Millipore Corporation, Bellerica, MA) quantification. Total protein was determined by the Coomassie Plus Protein Assay Reagent Kit (Pierce) [ 18 , 30 ] and CGRP, Sub P, and 3-NT were quantified using standard 96-well ELISA plates (Phoenix Pharmaceuticals; Millipore Corporation) according to the manufacturer's recommendations. For determination of Sub P and CGRP content in voided cystometric fluid, void volumes were collected following each micturition event in control and CYP-treated groups with and without Tempol (only vehicle) administration during conscious cystometry (see below).…”

“…Tetramethylbenzidine or LumiGLO was the substrate, and the enzyme activity or luminescence was measured. The CRGP standard provided with this protocol generated a standard curve from 0 to 100 ng/mL ( R 2 = 0.998, P ≤ 0.0001) for bladder samples [ 30 ]. The Sub P standard provided with this protocol generated a standard curve from 0 to 25 ng/mL ( R 2 = 0.998, P ≤ 0.0001) for bladder samples [ 30 ].…”

“…The CRGP standard provided with this protocol generated a standard curve from 0 to 100 ng/mL ( R 2 = 0.998, P ≤ 0.0001) for bladder samples [ 30 ]. The Sub P standard provided with this protocol generated a standard curve from 0 to 25 ng/mL ( R 2 = 0.998, P ≤ 0.0001) for bladder samples [ 30 ]. The nitrated-BSA standard provided with this protocol generated a standard curve from 1.5 to 100 μ g/mL ( R 2 = 0.997, P ≤ 0.0001) for bladder samples.…”