The role of c-kit-positive interstitial cells in mediating phasic contractions of bladder strips from streptozotocin-induced diabetic rats
Vahabi, B., McKay, N., Lawson, K. and Sellers , D. (2010) The role of c-kit-positive interstitial cells in mediating phasic contractions of bladder strips from streptozotocin-induced diabetic rats. British Journal of Urology. ISSN 1464-4096 Available from: http://eprints.uwe.ac.uk/13765
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Publisher's URL: http://dx.doi.org/10.1111/j.1464-410X.2010.09507.x
OBJECTIVE To investigate the role of c-kit-positive interstitial cells (ICCs) in mediating muscarinic receptor-induced phasic contractions of isolated bladder strips from streptozotocin(STZ)-induced diabetic rats and to confirm the expression and location of ICCs in the rat bladder. MATERIALS AND METHODS Bladders were removed from STZ-induced diabetic rats at 1, 4 and 12 weeks after induction of diabetes and from age-matched controls. To investigate the functional role of ICCs in mediating phasic contractions, bladder strips were isolated from control and diabetic rats and mounted in tissue baths. Strips were stimulated with low concentrations of the muscarinic receptor agonist carbachol (CCH; 0.1 µm) to induce phasic contractions and the effect of increasing concentrations (1–50 µm) of imatinib (Glivec® or Gleevec®, formerly STI571), a c-kit tyrosine kinase inhibitor, was then investigated. For molecular studies, to detect expression of the c-kit tyrosine kinase receptor (c-kit), total cellular RNA was extracted from rat bladders and reverse-transcribed to obtain complementary DNA (cDNA). Reverse transcription-polymerase chain reaction (RT-PCR) was then performed using primers specific to the c-kit sequence and amplified products separated by agarose gel electrophoresis. Amplified PCR products were excised from the gel, sequenced and compared with the known c-kit sequence to confirm their identity. For immunohistochemical detection, whole mount preparations of control rat bladders were fixed in acetone and labelled using antibodies directed to the ICC marker c-kit. RESULTS In functional studies, CCH induced phasic contractions in bladder strips from control and diabetic rats. Bladder strips from 1-week diabetic rats showed CCH-induced phasic contractions, which were greater in amplitude, but had lower frequency, than the controls, whilst no such differences were apparent at later time points of diabetes. Imatinib decreased the amplitude and the frequency of the CCH-induced phasic contractions in both control and diabetic tissues in a concentration-dependent manner, although in diabetic tissues this effect was only seen at the higher concentrations of imatinib. RT-PCR of bladder cDNA yielded a single amplicon of 480 bp. The sequence of this amplicon showed a 98% homology with the published c-kit sequence, thus confirming c-kit mRNA expression in both control and 1-week diabetic rat bladder. Expression of c-kit protein was also detected in a network of cells on the edge of and between smooth muscle bundles of control rat bladders by positive immunoreactivity to c-kit specific antibodies. CONCLUSION These data show the presence of c-kit-positive ICCs in rat urinary bladder and their importance in mediating muscarinic receptor-induced phasic contractions of bladder strips from control and diabetic rats. The role of these ICCs does not seem to be significantly altered by the diabetic state.