The role of c-kit-positive interstitial cells in mediating phasic contractions of bladder strips from streptozotocin-induced diabetic rats

Bahareh Vahabi, Neil G. McKay, Kim Lawson, Donna J. Sellers

Research output: Contribution to journalArticleResearchpeer-review

27 Citations (Scopus)

Abstract

What's known on the subject? and What does the study add? Although the functional significance of ICCs in the bladder is still not clear, they have been linked with detrusor overactivity. In this study we demonstrate the presence of c-kit positive interstitial cells in rat urinary bladder for the first time and their importance in mediating muscarinic receptor-induced phasic contractions of bladder strips from control and diabetic rats. The role of these cells does not seem to be significantly altered by the diabetic state. 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.

Original languageEnglish
Pages (from-to)1480-1487
Number of pages8
JournalBJU International
Volume107
Issue number9
DOIs
Publication statusPublished - May 2011

Fingerprint

Streptozocin
Urinary Bladder
Muscarinic Receptors
Polymerase Chain Reaction
Reverse Transcription
Complementary DNA
Proto-Oncogene Proteins c-kit
Muscarinic Agonists
Agar Gel Electrophoresis
Antibodies
Receptor Protein-Tyrosine Kinases
Carbachol
Acetone
Baths
Protein-Tyrosine Kinases
Smooth Muscle
Imatinib Mesylate

Cite this

@article{e9c3d58fbf3a400bb07f25ca3bcc1ca8,
title = "The role of c-kit-positive interstitial cells in mediating phasic contractions of bladder strips from streptozotocin-induced diabetic rats",
abstract = "What's known on the subject? and What does the study add? Although the functional significance of ICCs in the bladder is still not clear, they have been linked with detrusor overactivity. In this study we demonstrate the presence of c-kit positive interstitial cells in rat urinary bladder for the first time and their importance in mediating muscarinic receptor-induced phasic contractions of bladder strips from control and diabetic rats. The role of these cells does not seem to be significantly altered by the diabetic state. 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{\circledR} or Gleevec{\circledR}, 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.",
author = "Bahareh Vahabi and McKay, {Neil G.} and Kim Lawson and Sellers, {Donna J.}",
year = "2011",
month = "5",
doi = "10.1111/j.1464-410X.2010.09507.x",
language = "English",
volume = "107",
pages = "1480--1487",
journal = "British Journal of Urology",
issn = "1464-410X",
publisher = "Wiley-Blackwell",
number = "9",

}

The role of c-kit-positive interstitial cells in mediating phasic contractions of bladder strips from streptozotocin-induced diabetic rats. / Vahabi, Bahareh; McKay, Neil G.; Lawson, Kim; Sellers, Donna J.

In: BJU International, Vol. 107, No. 9, 05.2011, p. 1480-1487.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - The role of c-kit-positive interstitial cells in mediating phasic contractions of bladder strips from streptozotocin-induced diabetic rats

AU - Vahabi, Bahareh

AU - McKay, Neil G.

AU - Lawson, Kim

AU - Sellers, Donna J.

PY - 2011/5

Y1 - 2011/5

N2 - What's known on the subject? and What does the study add? Although the functional significance of ICCs in the bladder is still not clear, they have been linked with detrusor overactivity. In this study we demonstrate the presence of c-kit positive interstitial cells in rat urinary bladder for the first time and their importance in mediating muscarinic receptor-induced phasic contractions of bladder strips from control and diabetic rats. The role of these cells does not seem to be significantly altered by the diabetic state. 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.

AB - What's known on the subject? and What does the study add? Although the functional significance of ICCs in the bladder is still not clear, they have been linked with detrusor overactivity. In this study we demonstrate the presence of c-kit positive interstitial cells in rat urinary bladder for the first time and their importance in mediating muscarinic receptor-induced phasic contractions of bladder strips from control and diabetic rats. The role of these cells does not seem to be significantly altered by the diabetic state. 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.

UR - http://www.scopus.com/inward/record.url?scp=79955385779&partnerID=8YFLogxK

U2 - 10.1111/j.1464-410X.2010.09507.x

DO - 10.1111/j.1464-410X.2010.09507.x

M3 - Article

VL - 107

SP - 1480

EP - 1487

JO - British Journal of Urology

JF - British Journal of Urology

SN - 1464-410X

IS - 9

ER -