Bladder Interstitial Cells Of Cajal Research Articles

Expression and activity of pyruvate kinase in bladder interstitial cells of cajal in rats with diabetic cystopathy

Objective To investigate the expression and activity of pyruvate kinase in interstitial cells of Cajal (ICCs) in the bladder of rats with diabetic cystopathy (DCP). Methods Forty 8-week-old female rats were divided into two groups randomly, 31 as the experiment group and the rest 9 as the control group. The experiment group was intraperitoneally injected with streptozotocin (STZ) to establish diabetic models and 18 rats were succeeded to be induced. Nine diabetic rats were further raised for 12 weeks getting DCP rats. ICCs from bladder muscle were cultured. The expression of pyruvate kinase protein and mRNA was detected by Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR) respectively, and pyruvate kinase activity was measured. Results The expression levels of pyruvate kinase protein and mRNA and activity in DCP rats were reduced significantly as compared with those of the diabetic rats and control rats [(0.537±0.015), (0.624±0.026), (0.098±0.010) U/g·prot respectively, vs. (0.722±0.026), (0.739±0.045), (0.178±0.017) U/g·prot, vs. (0.923±0.031), (1.000±0.022), (0.277±0.030) U/g·prot; P<0.01]. Conclusion The reduction of pyruvate kinase activity in ICCs may play an important role in the development of DCP. Key words: Diabetic cystopathy; Pyruvate kinase; Interstitial cells of cajal

The function of hyperpolarization-activated cyclic nucleotide-gated channel in diabetic cystopathy.

We aimed at investigating changes in the expression and physiological function of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in interstitial cells of Cajal (ICC) in diabetic state. Twenty adult female Sprague-Dawley (SD) rats were randomly assigned to control and Zucker diabetic fatty (ZDF) group. The protein and mRNA expression of HCN isoforms and C-kit in the rat bladders were detected using Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The bladder contraction was evaluated using a bladder smooth muscle strip test. Whole cell patch-clamp techniques were used to detect the activity of HCN channels. Immunofluorescent staining was used to the positive expression of HCN and C-kit in ICC. cAMP, as HCN channel-specific stimulant, could increase the frequency and amplitude of spontaneous contractions in both group, while cAMP inducing contraction of ZDF rats, was still significantly lower compared with the control group. Acute bladder ICCs were isolated by collagenase digestion. Classic Ih current pattern was recorded on ICCs while Ih current amplitude of ICCs from ZDF diabetic rats was significantly lower than the control group. The expression and mRNA of HCN1-4 isoforms in ZDF diabetic rats were both significantly lower compared with the control group. Meanwhile, the number of c-kit positive cells in ZDF diabetic rats showed no significant differences compared with controls. The morphological structure of ICC in the bladder of ZDF rats was relatively loose and the number of their cell process was apparently decreased. The structure of ICCs in ZDF rats was relatively loose, their connection to each other was also diminished. The expression of HCN was down-regulated and its response to cAMP was also decreased. HCN channels in bladder ICCs might regulate detrusor contraction. Changes in HCN expression and activity in bladder ICCs might be one of the most important mechanisms of diabetic cystopathy.

Relationship between the change of bladder interstitial cells of Cajal and bladder compliance post-spinal cord transection at different time points in rats

Objective To observe the change of bladder compliance and bladder interstitial cells of Cajal (ICCs) post-suprasacral spinal cord transection at different time points in rats and the effect of Imatinib mesylate on the complicance of neurogenic bladder caused by long-term suprasacral spinal cord transaction in rats. Methods One hundred and two female Wistar rats were randomly divided into 6 groups: group A (sham group of 6 weeks); group B (suprasacral spinal cord transaction group of 6 weeks); group C and group E (sham group of 12 weeks); group D and group F (suprasacral spinal cord transaction group of 12 weeks). The rats in groups A to D were used to study the change of bladder compliance and bladder ICCs post-suprasacral spinal cord transection at different time points. The rats in group E and group F were used to study the effect of Imatinib mesylate on the complicance of neurogenic bladder caused by long-term suprasacral spinal cord transaction. Spinal cord transection at T10 level was built in spinal cord transaction groups, while group A, group C and group F underwent sham operaion. Urodynamics was detected at 6th or 12th week after surgery. We observed the changes of ICCs in the bladder wall by immunohistochemistry. Urodynamics was also detected in group E and group F post-surgery 12 weeks before and after intravenous administration of Imatinib mesylate (5 μmol and 20 μmol respectively). Results The bladder compliance was significantly increased [(0.146±0.107) ml/cmH2O vs. (0.066±0.019) ml/cmH2O, P=0.017] and the number of ICCs was significantly decreased [(3.180±1.976) vs. (4.920±1.564), P=0.014] in group B as compared with group A. The bladder compliance was significantly lower [(0.046±0.023) ml/cmH2O vs. (0.063±0.013) ml/cmH2O] and the number of ICCs was significantly higher [(7.710±2.024) vs. (4.750±1.138), P=0.000] in group D than in group C. The bladder compliance in group E was increased by intravenous administration of 20 μmol Imatinib mesylate [from (0.070±0.029) ml/cmH2O to (0.096±0.036) ml/cmH2O]. It increased the bladder compliance of group F in a dose-dependent manner. The bladder compliance in group F was increased by 5 μmol and 20 μmol Imatinib mesylate from (0.047±0.021) ml/cmH2O to (0.065±0.019) ml/cmH2O and (0.077±0.019) ml/cmH2O respectively, P=0.000. Conclusion The bladder compliance was increased first, then decreased and the number of ICCs changed adversely in the T10 spinal cord transection rats. The bladder compliance of the rats at 12th week post-suprasacral spinal cord transection could be improved by inhibiting the ICCs function. Not only the fibrosis, but also the number of ICCs of the bladder wall increased might be the pathological basis of the reduced bladder compliance in suprasacral spinal cord transaction rats. Key words: Spinal cord injury; Neurogenic bladder; Bladder compliance; Interstitial cells of Cajal; Imatinib mesylate

EP3 activation facilitates bladder excitability via HCN channels on ICCs

EP3 is a receptor for prostaglandin E2 (PGE2), and although its effect on bladder excitability has attracted considerable attention, the underlying mechanism remains unclear. To investigate whether the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the interstitial cells of Cajal (ICCs) of the bladder are involved in the effect of EP3 activation on bladder excitability, wild-type mice, HCN1 knockout (HCN1−/−) mice and rats were used in our study. Double immunofluorescence staining and immunoprecipitation assays demonstrated the interaction between EP3 and the HCN channels. Sulprostone is a selective agonist of EP3. The current density of HCN channels was enhanced by sulprostone or PGE2 using whole-cell patch clamping. Western blot analyses showed that the expression levels of HCN1 and HCN4 were higher in bladders that had undergone intravesical instillation with sulprostone than in bladders treated with normal saline (NS). Both PGE2 and sulprostone increased the calcium concentration of the ICCs, and their effects were inhibited by ZD7288 (antagonist of HCN channels) treatment. In bladder detrusor strip testing, both PGE2 and sulprostone enhanced the amplitude of the bladder detrusor in HCN1−/− mice; however, these effects were less than those in the wild-type mice. Furthermore, the effects of PGE2 and sulprostone were inhibited by ZD7288. Taken together, our results indicate that EP3 is expressed in bladder ICCs and facilitates bladder excitability via HCN channels. This study provides more comprehensive insights into the mechanism between inflammation and bladder excitability and highlights methods that can resolve bladder hyperactivity.

Distribution of interstitial cells of Cajal in the bladders of fetal rats with retinoic acid induced myelomeningocele.

Myelomeningocele (MMC) is one of the most common reason of neurogenic bladder dysfunction in children. Although neurogenic bladder dysfunction occurrence is related with bladder innervation, also there are some changes seen in the smooth muscle and neural cells of the bladder. Interstitial cells of Cajal (ICC) are the pacemaker cells found in organs with peristaltic activity. Although it has been shown that ICC are diminished in the rat urinary bladder with traumatic spinal cord injury, there is no data about ICC in fetal rat bladders with MMC. This study has been conducted to investigate the ICC in the bladders of fetal rats with retinoic acid induced MMC. Time dated pregnant Wistar albino rats were divided into 3 groups. In MMC group, dams were fed with gavage solution containing 60 mg/kg all-trans retinoic acid dissolved in olive oil on 10. embryologic day. Sham group animals were fed only olive oil. Control group dams were fed with standard rat chow. Fetuses were delivered by cesarean section and harvested on 22. embryologic day. MMC was identified by observing MMC sacs at the back of the fetuses. Distribution of ICCs were evaluated using immunohistochemical staining. ICCs were found in all groups, which have the same morphological features that had been described earlier in the gastrointestinal tract and the bladder. The density of the ICC in the MMC group was found to be significantly decreased when compared with the control and the sham groups (p<0.05). The density of the ICC in the urinary bladder decreased in the neurogenic bladder developed in MMC.

Changes in hyperpolarization-activated cyclic nucleotide-gated channel expression and activity in bladder interstitial cells of Cajal from rats with detrusor overactivity.

To investigate changes in the expression and function of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, interstitial cells of Cajal (ICCs) were isolated from detrusor overactivity (DO) rats induced with partial bladder outlet obstruction (PBOO). We hypothesized that increased expression of HCN channels in ICCs would enhance the excitability of ICCs and bladder in DO rats. Forty adult female Sprague-Dawley rats were randomly assigned to control and DO groups. The expression of HCN isoforms in the rat bladders was detected using reverse transcription-polymerase chain reaction (RT-PCR). Whole-cell patch-clamp techniques and laser confocal microscopy were used to explore the effects of the HCN channel blocker ZD7288 on Ih current and intracellular calcium levels ([Ca(2+)]i) in freshly isolated ICCs. The effect of ZD7288 on bladder contraction was evaluated using a bladder smooth muscle strip test. The results of RT-PCR showed that HCN1-4 isoforms increased expression in the DO group compared with the control group. The current density of Ih in the bladder ICCs was increased. A moderate concentration of ZD7288 (50 μmol/L) significantly decreased the [Ca(2+)]i levels in freshly isolated ICCs from sham and DO bladders. The smooth strip tests indicated that ZD7288 (50 μmol/L) suppressed the amplitude of smooth muscle strip contractions in the sham and DO bladders. All HCN channel isoforms were highly expressed in bladders from the DO group, which was correlated with increased Ih currents in DO ICCs, suggesting that the HCN channels might play an important role in the pathological processes of DO.

Expression and function of muscarinic subtype receptors in bladder interstitial cells of cajal in rats.

To locate the muscarinic (M) M2 and M3 receptors in bladder interstitial cells of Cajal (ICCs) and to determine the effects of M2 and M3 agonists on bladder ICCs. A total of 30 adult male Sprague-Dawley rats weighing 225-250 g were used in this study. Double-labeled fluorescence of muscarinic receptors and c-kit was performed for co-localization. To evaluate the effect of muscarinic agents on the excitation of bladder ICCs, we analyzed the inward current of bladder ICCs using the whole-cell patch clamp. The effect of muscarinic agents on the carbachol-induced inward currents was evaluated with the whole-cell patch clamp. M2 and M3 receptors were confirmed in the stroma ICCs in rats' bladders with double-labeled immunofluorescence. Spontaneous action potential was observed in freshly isolated bladder ICCs. The carbachol-induced inward Ca2+ current in ICCs can be blocked by atropine. The M2 receptor antagonist methoctramine (1 μM) showed a weak inhibitory capability on the inward Ca2+ current [from 74.8 ± 9.6 to 63.3 ± 13.8 Pascal (pA), n = 12, P = .03]. While the M3 receptor antagonist 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) (1 μM) significantly inhibited the inward Ca2+ current (from 78.4 ± 11.2 to 17.3 ± 7.9 pA, n = 12, P < .001). Bladder ICCs express M2 and M3 cholinergic receptors. Most muscarinic cholinergic receptor antagonists, especially the M3 antagonists, can effectively inhibit the carbamylcholine- induced inward current of bladder ICCs.

The Effects of Acupuncture on Bladder Interstitial Cells of Cajal Excitability in Rats with Overactive Bladder

It is well known that acupuncture treatment has an effect on patients with an overactive bladder, but the mechanism of its action remains to be clarified. This study was aimed to investigate the effects of acupuncture on bladder overactivity, and the excitability of interstitial cells of Cajal of the bladder in a rat model of partial bladder outlet obstruction. Electroacupuncture (continuous wave, 30 Hz, 1 mA) was applied to stimulate the Ciliao point (BL32) and the Huiyang point (BL35) of rats for 20 min, 3 days. Results showed that acupuncture suppressed detrusor unstable contraction frequency and decreased detrusor maximum pressure in the bladder filling period. Compared with the normal control rats, HCN2 mRNA and protein expression within the bladder were upregulated and were reversed by electroacupuncture in overactive bladder rats as determined by RT-PCR, western blotting and immunohistochemistry. Moreover, in-vitro cell-cultured OAB rats bladder interstitial cells of Cajal intracellular Ca2+ concentration were higher than normal control rats, which were lowered after acupuncture treatment. These findings suggest that acupuncture stimulation can suppress bladder overactivity, and regulate the excitability of bladder interstitial cells of Cajal in treatment of overactive bladder myogenic mechanism.

Identification of T-type Calcium Channels in the Interstitial Cells of Cajal in Rat Bladder

To investigate the expression and function of T-type calcium channels in the interstitial cells of Cajal in rat bladders. Bladders were harvested from Sprague-Dawley rats. The expression of T-type calcium channels subtypes (α1G, α1H, and α1I) in interstitial cells of Cajal were identified by double-labeled immunofluorescence analysis and reverse transcription-polymerase chain reaction analysis in whole mount preparations of rat bladders. The function of T-type calcium channels in freshly isolated interstitial cells of Cajal was assessed by detecting the changes of intracellular calcium ([Ca(2+)](i)) with preloading fluo-3 AM, and by evaluating the changes of the phasic contractions of rat bladder strips after treating with mibefradil and glivec. Three T-type calcium channels subtypes, α1G, α1H, and α1I, colocalized with c-kit in bladder interstitial cells of Cajal by double-labeled immunofluorescence analysis, and this was confirmed using reverse transcription-polymerase chain reaction. The T-type calcium channels selective blocker, mibefradil (1 μM), significantly decreased the intracellular calcium concentration ([Ca(2+)](i)) in isolated interstitial cells of Cajal (P < .01) and inhibited the spontaneous phasic contraction of bladder strips (P < .01). Moreover, the c-kit receptor blocker, glivec, significantly decreased the [Ca(2+)](i) of interstitial cells of Cajal further (P < .01) and the spontaneous phasic contraction of bladder strips. T-type calcium channel subtypes were confirmed to colocalize in interstitial cells of Cajal in rats bladders, which might participate in the spontaneous activity of interstitial cells of Cajal and phasic contractions of bladder strips by modulating [Ca(2+)](i) in interstitial cells of Cajal.

Identification of a Hyperpolarization-activated Cyclic Nucleotide-gated Channel and Its Subtypes in the Urinary Bladder of the Rat

To investigate the distribution and effects of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel and its isoforms in bladder, especially in bladder interstitial cells of Cajal (ICC). Four HCN isoforms were detected in bladder tissue from rats using reverse transcription-polymerase chain reaction and Western blotting. The HCN1 subtype was observed in bladder ICCs by double-labeled fluorescence. The effect of the HCN blocker, ZD7288, was investigated using the bladder smooth muscle strip test. HCN1-4 isoforms were all identified in bladder ICCs using reverse transcription-polymerase chain reaction and Western blotting. Based on our semiquantitative analysis, HCN1 was found to be the most prominent isoform. The expression of HCN1 was confirmed in bladder ICCs by double-labeled fluorescence through colabeling of HCN1 and kit (CD117). ZD7288 significantly decreased the bladder excitation. All 4 HCN channel isoforms exist in the bladder, and they affect the bladder excitation, presumably via bladder ICCs.

Interstitial cells in the urinary bladder—localization and function

This review summarizes the currently available literature on the localization and proposed functions of a novel group of cells in the urinary bladder known as interstitial cells or interstitial cells of Cajal (ICC). On-line searches of "Pubmed" for bladder, c-Kit, ICC, interstitial cell and myofibroblast were performed to identify relevant studies for the review. The literature contains substantial data that several sub-populations of ICC are present in the wall of the mammalian urinary bladder. These are located in the lamina propria and within the detrusor with distinctive cell shapes and morphological arrangements. Bladder ICC are identified with transmission electron microscopy or by immunohistochemical labeling using antibodies to the Kit receptor which is an established ICC marker. Lamina propria-ICC form a loose network connected via Cx43 gap junctions and are associated with mucosal nerves. Detrusor ICC track the smooth muscle bundles and make frequent contacts with intramural nerves. Both groups of ICC exhibit spontaneous electrical and Ca2+-signalling and also respond to application of neurotransmitter substances including ATP and carbachol. There is emerging evidence that the expression of ICC is upregulated in pathophysiological conditions including the overactive bladder. There is now a convincing body of evidence that specialized ICC are present in the urinary bladder making important associations with other cells that make up the bladder wall and possessing physiological properties consistent with a role of bladder activity modulation.

Potential for control of detrusor smooth muscle spontaneous rhythmic contraction by cyclooxygenase products released by interstitial cells of Cajal

Interstitial cells of Cajal (ICCs) have been identified as pacemaker cells in the upper urinary tract and urethra, but the role of ICCs in the bladder remains to be determined. We tested the hypotheses that ICCs express cyclooxygenase (COX), and that COX products (prostaglandins), are the cause of spontaneous rhythmic contraction (SRC) of isolated strips of rabbit bladder free of urothelium. SRC was abolished by 10 μM indomethacin and ibuprofen (non-selective COX inhibitors). SRC was concentration-dependently inhibited by selective COX-1 (SC-560 and FR-122047) and COX-2 inhibitors (NS-398 and LM-1685), and by SC-51089, a selective antagonist for the PGE-2 receptor (EP) and ICI-192,605 and SQ-29,548, selective antagonists for thromboxane receptors (TP). The partial agonist/antagonist of the PGF-2α receptor (FP), AL-8810, inhibited SRC by ∼50%. Maximum inhibition was ∼90% by SC-51089, ∼80–85% by the COX inhibitors and ∼70% by TP receptor antagonists. In the presence of ibuprofen to abolish SRC, PGE-2, sulprostone, misoprostol, PGF-2α and U-46619 (thromboxane mimetic) caused rhythmic contractions that mimicked SRC. Fluorescence immunohistochemistry coupled with confocal laser scanning microscopy revealed that c-Kit and vimentin co-localized to interstitial cells surrounding detrusor smooth muscle bundles, indicating the presence of extensive ICCs in rabbit bladder. Co-localization of COX-1 and vimentin, and COX-2 and vimentin by ICCs supports the hypothesis that ICCs were the predominant cell type in rabbit bladder expressing both COX isoforms. These data together suggest that ICCs appear to be an important source of prostaglandins that likely play a role in regulation of SRC. Additional studies on prostaglandin-dependent SRC may generate opportunities for the application of novel treatments for disorders leading to overactive bladder.

Comparison of mechanical and electrical activity and interstitial cells of Cajal in urinary bladders from wild‐type and W/Wv mice

W/W(v) and wild-type murine bladders were studied to determine whether the W/W(v) phenotype, which causes a reduction in, but not abolition of, tyrosine kinase activity, is a useful tool to study the function of bladder interstitial cells of Cajal (ICC). Immunohistochemistry, tension recordings and microelectrode recordings of membrane potential were performed on wild-type and mutant bladders. Wild-type and W/W(v) detrusors contained c-Kit- and vimentin-immunopositive cells in comparable quantities, distribution and morphology. Electrical field stimulation evoked tetrodotoxin-sensitive contractions in wild-type and W/W(v) detrusor strips. Atropine reduced wild-type responses by 50% whereas a 25% reduction occurred in W/W(v) strips. The atropine-insensitive component was blocked by pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid in both tissue types. Wild-type and W/W(v) detrusors had similar resting membrane potentials of -48 mV. Spontaneous electrical activity in both tissue types comprised action potentials and unitary potentials. Action potentials were nifedipine-sensitive whereas unitary potentials were not. Excitatory junction potentials were evoked by single pulses in both tissues. These were reduced by atropine in wild-type tissues but not in W/W(v) preparations. The atropine-insensitive component was abolished by pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid in both preparations. Bladders from W/W(v) mice contain c-Kit- and vimentin-immunopositive ICC. There are similarities in the electrical and contractile properties of W/W(v) and wild-type detrusors. However, significant differences were found in the pharmacology of the responses to neurogenic stimulation with an apparent up-regulation of the purinergic component. These findings indicate that the W/W(v) strain may not be the best model to study ICC function in the bladder.

Two kinds of synchronized rhythmic activity of immature rat spinal motoneurons

To investigate the distribution and effects of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel and its isoforms in bladder, especially in bladder interstitial cells of Cajal (ICC).Four HCN isoforms were detected in bladder tissue from rats using reverse transcription–polymerase chain reaction and Western blotting. The HCN1 subtype was observed in bladder ICCs by double-labeled fluorescence. The effect of the HCN blocker, ZD7288, was investigated using the bladder smooth muscle strip test.HCN1-4 isoforms were all identified in bladder ICCs using reverse transcription–polymerase chain reaction and Western blotting. Based on our semiquantitative analysis, HCN1 was found to be the most prominent isoform. The expression of HCN1 was confirmed in bladder ICCs by double-labeled fluorescence through colabeling of HCN1 and kit (CD117). ZD7288 significantly decreased the bladder excitation.All 4 HCN channel isoforms exist in the bladder, and they affect the bladder excitation, presumably via bladder ICCs.