Protein kinase C is involved in carbachol-induced calcium release in permeabilized rat detrusor having interstitial cystitis

M Denizalti, NT Durlu-Kandilci, TE Bozkurt, I Sahin-Erdemli. Hacettepe University, Faculty of Pharmacy, Department of Pharmacology, 06100, Ankara, Turkey

Interstitial cystitis is a syndrome characterized by chronic inflammation of the bladder in which detrusor smooth muscle contractions can vary. It has been known that carbachol-induced contractions in bladder of rats increase in cyclophosphamide-induced experimental cystitis1,2 . However the mechanism of this change is unclear. The aim of the present study is to investigate the role of protein kinase C and other intracellular calcium release pathways in carbachol-induced detrusor contractions in a rat model of interstitial cystitis. Cyclophosphamide (150 mg/kg, dissolved in saline) was injected to rats (Sprague-Dawley, female, 200-250 g) intraperitoneally once a day on days 1, 4 and 7 to induce interstitial cystitis. Control groups were injected with saline (%0.9 NaCl). 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 ANOVA/Bonferroni. P<0.05 was accepted as significant. Tissues were permeabilized with 40 µM β-escin for 30 min. Carbachol-induced contractions were significantly increased from 21.2±1.6% (control, n=6) to 44±4.4% in cystitis (n=8, P<0.05). Protein kinase C inhibitor GF-109203X (5 µM) inhibited this increased response in cystitis group approximately 73% (n=9, P<0.05) where in control group approximately 49% (n=7, P<0.05). Carbachol-induced contractions were significantly decreased (25.8±3.2%, n=7, P<0.05) in the presence of sarcoplasmic reticulum ryanodine channel blocker ryanodine (10 μM) in cystitis group but were not changed (16.2±1.7%, n=6, P>0.05) in control group. Similarly, carbachol-induced contractions were significantly decreased (17.2±2.2%, n=6, P<0.05) in the presence of sarcoplasmic reticulum IP3 receptor blocker heparin (1 mg/ml) in cystitis group but not in control group (13.4±3.3%, n=7, P>0.05). Carbachol-induced calcium sensitization responses 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 increased in cystitis (from 15.8±2.2%, n=8 to 24.7±2.8%, n=8, P<0.05). The calcium sensitization responses were both significantly inhibited in the presence of GF-109203X in control group approximately 46% (n=5) and in cystitis group approximately 82% (n=6). Interstitial cystitis enhances both carbachol-induced calcium release from intracellular stores and calcium sensitization3 in detrusor smooth muscle. Activation of protein kinase C pathway may be the robust mechanism in this increased carbachol contraction response in cystitis.

1 Toxicol. Appl. Pharmacol. 2005;208:163-169

2 J. Auton. Nerv. Syst. 2000;80(3):130-136

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