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Keywords:

  • acute urinary retention;
  • KATP channel;
  • nicorandil;
  • cromakalim;
  • glibenclamide;
  • apoptosis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERNCES

What's known on the subject? and What does the study add?

Acute urinary retention (AUR) and catheterization for AUR (AURC) or drainage of the urine is a well established cause of bladder dysfunction. Previously, we reported that the induction of AURC significantly reduced contractile responses to both carbachol and KCl compared with a control group, and that this reduction was prevented by nicorandil and cromakalim in a dose-dependent manner; however, although we reported a possible beneficial effect of nicorandil and cromakalim on bladder dysfunction caused by AURC, its molecular mechanism is still unknown.

Our study establishes that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via up-regulation of both KIR6.1 and KIR6.2 with a subsequent decrease in oxidative stress and decreased induction of apoptosis in the bladder.

OBJECTIVE

  • • 
    To investigate whether ATP-sensitive potassium (KATP) channel openers prevent bladder injury after acute urinary retention (AUR) and subsequent catheterization for AUR (AURC) in the rat.

MATERIALS AND METHODS

  • • 
    Eight-week-old male Sprague–Dawley rats were divided into five groups: a sham-operated control group, an AUR group, and three AUR groups treated with: nicorandil (10 mg/kg); cromakalim (300 µg/kg); or glibenclamide (5 mg/kg).
  • • 
    AUR was induced by intravesical infusion of 3.0 mL of saline via cystostomy with simultaneous clamping of the penile urethra and, after 30 min of AUR, the bladder was allowed to drain for 60 min.
  • • 
    After the experimental period, bladder function was assessed using organ bath techniques (carbachol and KCl), and by measuring tissue levels of 8-isoprostane, a marker of oxidative stress. The participation levels of KATP channel pores were investigated using ELISA and real-time PCR methods, respectively.
  • • 
    The degree of apoptosis was estimated using the TUNEL method in the bladder smooth muscle and epithelium.

RESULTS

  • • 
    The AURC group showed significantly decreased contractile responses to carbachol and KCl, and significant increases in tissue 8-isoprostane levels and apoptosis index in the epithelium compared with the control group.
  • • 
    Nicorandil and cromakalim, but not glibenclamide, significantly prevented these AURC-induced alterations.
  • • 
    The expressions of KIR6.1 and KIR6.2 mRNAs were significantly up-regulated by the induction of AURC.
  • • 
    Nicorandil and cromakalim, but not glibenclamide, significantly up-regulated expressions of KIR6.1 and KIR6.2 mRNAs in the bladder compared with the AUR group.

CONCLUSION

  • • 
    Our data indicate that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via activation of KATP channels, with a subsequent decrease in oxidative stress and decreased induction of apoptosis.

Abbreviations
AUR

acute urinary retention

AURC

catheterization for AUR

IR

ischaemia-reperfusion

IPreC

ischaemic preconditioning

KATP

ATP-sensitive potassium

sarcKATP

sarcolemmal KATP

mitoKATP

mitochondrial KATP

nucKATP

nuclear KATP

SUR

sulfonylurea receptor

NO

nitric oxide

Nic 10

nicorandil 10 mg/kg

Crom 300

cromakalim 300 µg/kg

Glib 5

glibenclamide 5 mg/kg

BBF

bladder blood flow

EC50

the concentration of agonist that produces half-maximal contractile response

Emax

maximal contractile response

ROS

reactive oxygen species.

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERNCES

Acute urinary retention (AUR) and subsequent catheterization for AUR (AURC) or drainage of the urine is a well established cause of bladder dysfunction. Indeed, several reports have indicated that the biochemical and metabolic status of the bladder is altered after prolonged overdistension [1,2]. Moreover, there is increasing evidence that ischaemia-reperfusion (IR) injury might be at least partly responsible for the prolonged bladder dysfunction occurring after AUR [3,4]. AUR induces a reduction in blood flow to bladder tissue resulting in ischaemia and after subsequent catheterization, the blood supply recovers, allowing reperfusion [4–6]. We have reported that AURC and/or IR lead to oxidative stress, inflammation and neutrophil infiltration, which might contribute to the delayed recovery of the bladder function [4–8].

Ischaemic preconditioning (IPreC), first described by Murry et al. [9], is a protective manoeuvere that includes brief periods of ischaemia that offer protection against subsequent longer ischaemic periods. Many studies in cardiac protection with IPreC suggest that the mechanism of cardiac IPreC involves the activation and opening of ATP-sensitive potassium (KATP) channels. There are three types of KATP channels: the sarcolemmal (sarcKATP), the mitochondrial (mitoKATP), and the nuclear (nucKATP) channel [10]. Although the mitoKATP channel plays a critical role in IPreC and apoptosis, its molecular identity remains unclear [10]. The sarcKATP channel is thought to be composed of a tetramer of inward rectifier K+ channels (KIR6.x) surrounded by four subunits of sulfonylurea receptors (SURs). Based on similarities between mitoKATP and sarcKATP channels, it has been proposed that the mitoKATP channel may also consist of KIR6.x and SUR subunits [10]. KATP channel openers may have a direct impact on the underlying pathophysiological mechanism of tissue injury after an IR insult, which is called pharmacological IPreC. In the genitourinary system, we have reported that IPreC significantly prevents IR injury in the bladder [11], and that the KATP channel openers nicorandil and cromakalim induce pharmacological IPreC to prevent additional damage caused by IR in the testis and kidney [12,13].

Nicorandil (2-nicotiamidoethyl-nitrate ester), a KATP channel opener and nitric oxide (NO) donor, is used in the treatment of angina and acute heart failure [14]. Cromakalim is a potassium channel opening vasodilator, which acts on KATP channels causing membrane hyperpolarization of smooth muscle cell membranes by pulling their membrane potential away from the threshold. Nicorandil is reported to be a selective mitoKATP channel opener, and cromakalim is reported to be a non-selective KATP channel opener, whereas glibenclamide is reported to be a non-selective KATP channel inhibitor [15].

Previously, we reported that the induction of AURC significantly reduced the contractile responses to both carbachol and KCl compared with the control group, and that this reduction was prevented by nicorandil and cromakalim in a dose-dependent manner [6]; however, although we reported a possible beneficial effect of the pharmacological IPreC on bladder dysfunction caused by AURC, its molecular mechanism is still unknown. The aim of the present study was to investigate the molecular mechanism of KATP channel openers, nicorandil and cromakalim, and KATP channel inhibitor, glibenclamide, in the bladder dysfunction induced by AURC.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERNCES

ANIMALS AND EXPERIMENTAL DESIGN

All rat experiments were conducted in accordance with the guidelines set by the Tottori University Committee for Animal Experimentation. The rat experimental model was created as described in previous reports from this laboratory [4–6]. Eight-week-old male Sprague–Dawley rats (220–260 g; SLC, Shizuoka, Japan) were randomly divided into five groups: a sham-operated control (Cont group), an AUR group (30 min of AUR and allowed 60 min to drain), and three AUR groups treated with: nicorandil 10 mg/kg (Nic 10 group), cromakalim 300 µg/kg (Crom 300 group), or glibenclamide 5 mg/kg (Glib 5 group [n= 6 in each group]). The above doses were chosen after performance of preliminary studies, which indicated that these doses of drugs significantly prevented bladder dysfunction after AURC. Each drug was administered i.p. 30 min before the induction of AUR. Each rat was anaesthetized with sodium pentobarbital (50 mg/kg, i.p.). The rat penile urethra was then clamped with a small clip (Sugita standard aneurysm clip, holding force 145 g; Mizuho Ikakogyo, Tokyo, Japan); subsequently a cystostomy was made and 3.0 mL (0.6 mL/min) of saline was infused with an infusion pump (TOP-5200; TOP, Tokyo, Japan) to induce AUR. Thirty minutes after inducing AUR, the cystostomy tube was opened and the bladder was allowed to empty for another 60 min. The rats were killed with an overdose of pentobarbital (60 mg/kg, i.p.). The isolated bladders were used in organ bath experiments or frozen at –80 °C for measurements of tissue 8-isoprostane levels, KIR6.1 mRNA and KIR6.2 mRNA.

SIMULTANEOUS MEASUREMENTS OF INTRAVESICAL PRESSURE AND BLOOD FLOW IN THE BLADDER

A cystometry catheter was connected to an external pressure transducer (P2310; Gould, Eastlake, OH, USA) for the measurement of intravesical pressure. Simultaneously, the rat bladder blood flow (BBF) was measured in vivo with a laser Doppler flowmeter (BRL-100; Bioresearch Co., Nagoya, Japan) according to the method we described in previous reports [4–6].

TISSUE PREPARATION AND MEASUREMENT OF CONTRACTILE FORCE OF THE BLADDER

Functional studies were conducted according to methods described in previous reports [4–6]. Briefly, longitudinal strips of the posterior wall of the bladder dome with mucosa were mounted in organ baths (25 mL) containing Krebs-Henseleit solution, and bubbled with 5% CO2 and 95% O2 (37 °C). One hook was suspended from a transducer (type 45196 A; San-ei Instruments, Tokyo, Japan), and the lower hook was fixed to a plastic support leg attached to a micrometer (Mitutoyo, Tokyo, Japan). Each strip was equilibrated unstretched for 30 min. A load of 1.0 g was applied to each strip by micrometer adjustment, and the load was readjusted to this level 30 min later. The changes in the tone of the strips were measured isometrically using force transducers, and the data were recorded on a personal computer using the Chart v3.6.9 software and the PowerLab/16sp data acquisition system (AD Instruments, Castle Hill, Australia). The cumulative concentration response curves to carbachol were constructed, and the contractile forces to 100 mM of KCl were also measured.

MEASUREMENT OF 8-ISOPROSTANE IN THE BLADDER

The 8-isoprostane concentrations in the bladder were measured using ELISA according to the manufacturer's instructions (STAT-8-Isoprostane EIA kit, Cayman Chemical Company, Ann Arbor, MI, USA). Tissue 8-isoprostane levels were normalized by protein contents. Protein was determined using a commercial kit (Protein Assay Rapid Kitwako, Wako Pure Chemical Industries, Osaka, Japan).

REAL-TIME PCR OF KIR6.1 AND KIR6.2 MESSENGER RNAS

Real-time PCR was performed according to our previous reports [16]. The mRNAs were purified using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The reverse transcriptase (RT) mixture (20 µL) containing 1 µg of total RNA was prepared and incubated at 37 °C for 60 min. Fifteen micro liters of the mixture were used for real-time PCR, which was carried out using a Light Cycler system with a LightCycler-FastStart Hybridization Probe kit (Roche Diagnostics, Tokyo, Japan). The primers and probe sequences specific to the genes of KIR6.1 (GeneBank Accession: NM_017099), KIR6.2 (GeneBank Accession: NM_031358) and β-actin (GeneBank Accession: NM_031144) were used according to our previous reports [16]. The primer and probe of the β-actin used were from the LightCycler-Primer/Probe Set (rat), and was used as the internal standard. A total of 5 µL of cDNA solution was used for the sample.

PROTEIN EXTRACTION FOR WESTERN BLOT ANALYSIS

Briefly, bladder samples (30 mg) were homogenized in 150 µL lysis buffer (1% NP-40, 0.05% sodium deoxycholate, 0.1% SDS, 100 mM NaCl, 50 mM Tris/HCl, pH 7.5, 4.0 mM EDTA and Complete Protease Inhibitor Cocktail tablet (Roche Diagnosis, Indianapolis, IN, USA). The homogenate was subjected to centrifugation at 10 000 g for 20 min. The supernatant was collected and used for colometric detection and quantitation of total protein (Pierce® BCA Protein Assay Kit, Thermo Scientific, Loughborough, UK).

DETERMINATION OF CASPASE-3, BCL-2 AND BAX BY WESTERN BLOT ANALYSIS

The protein samples (50 µg) were subjected to SDS-polyacrylamide gel electrophoresis (15% gradient gels). Proteins were electrophoretically transferred to nitrocellulose membranes, blocked with TBS, 0.1% Tween 20 (PBS-T) containing 5% non-fat dried milk, washed with TBS-T, and incubated overnight at 4 °C on a shaker with antibodies for cleaved caspase-3 (1:1000), Bcl-2 (1:1000), Bax (1:1000) and anti-β-actin (1:500) in TBS-T containing 5% non-fat dried milk. The blots were washed with TBS-T (6 × 10 min) and incubated for 1 h at 4 °C on a shaker with secondary antibody conjugated with horseradish peroxidase (1:3000) in TBS-T containing 5% non-fat dried milk. After thorough washing with PBS-T (6 × 10 times) the detection was performed using enhanced chemiluminescence reagent.

The antibodies used were as follows: rabbit monoclonal antibody, which detects the endogenous levels of the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175 (Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal antibody for Bcl-2 (Cell Signaling Technology, Inc, Danvers, MA, USA), mouse monoclonal antibody for Bax (BD Pharmingen, Franklin Lakes, NJ, USA), and rabbit polyclonal antibody for anti-β-actin (AnaSpec, Inc., San Jose, CA, USA). Anti-β-actin was used as a loading control for normalization.

TUNEL ASSAY IN THE BLADDER SMOOTH MUSCLE AND EPITHELIUM

Testicular DNA fragmentation was evaluated with TUNEL assay as described previously [17] using a commercial kit (Apop Tag Plus Peroxidase In Situ Apoptosis Detection Kit, Chemicon Laboratories, Temecula, CA, USA). TUNEL-positive cells displayed brown staining within the nucleus of apoptotic cells. TUNEL-positive cells were quantified under high-power magnification (×400) by an investigator (H.O.) who was blinded to the studies, and were expressed as numbers per 100 nuclei in smooth muscle and epithelium separately. Additionally, the TUNEL-positive cell number per 100 nuclei was randomly examined under a light microscope in each slide.

DATA ANALYSIS

The concentration of agonist that produces half-maximal contractile response (EC50) and maximal contractile response (Emax) values were obtained by a Macintosh computer (G3) located with Chart version 3.6.9 software and a PowerLab/16sp data acquisition system. The contractile data were calculated as grams of active force per cross-sectional area in mm2. The cross-sectional area was calculated by using the following equation: cross-sectional area = weight/(length × 1.05), where 1.05 is the assumed density of the smooth muscle [4–6]. The EC50 values were calculated as geometric means, and Emax values were calculated as arithmetic means. The expressions of KIR6.1 and KIR6.2 mRNAs were quantified according to the expressions of β-actin mRNAs in the experimental rat bladder domes. Data are shown as means (sem) of six separate determinations in each group. The density of the protein bands for capase-3, Bcl-2 and Bax obtained from western blot were analysed using Image J software (1.44m, National Institutes of Health) and normalized by anti β-actin. A statistical comparison of differences between groups was performed using anova and Fisher's multiple comparison tests. A P value of <0.05 was considered to indicate statistical significance.

DRUGS AND CHEMICALS

Nicorandil was kindly supplied by Chugai Pharmaceutical Co. Ltd (Tokyo, Japan). Cromakalim and glibenclamide were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the other chemicals are available commercially.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERNCES

MEASUREMENT OF INTRAVESICAL PRESSURE AND BBF DURING URINARY RETENTION AND SUBSEQUENT CATHETERIZATION

The results of the simultaneous measurement of intravesical pressure and rat BBF during AUR and subsequent AURC are shown in Fig. 1. In all the experimental groups, AUR increased intravesical pressure and decreased BBF. AURC decreased the intravesical pressure and increased BBF. Although treatment with nicorandil or cromakalim did not alter BBF during the catheterization phase, treatment with glibenclamide decreased BBF during the catheterization phase compared with the other groups. BBF in the Glib 5 group was significantly smaller than that in the Crom 300 group at the 4-min timepoint, and also BBF in the Glib 5 group was significantly smaller than that in the AUR group at 6, 7 and 8 min. All of these timepoints were during the overdistention phase. There were no significant differences in BBF among the groups at any point during the drainage phase. By contrast, there were no significant differences in the intravesical pressure of the bladder among any of the other groups during the experimental period. Our data suggest that treatment with KATP channel openers or a KATP channel inhibitor did not alter the intravesical pressure during AUR and subsequent AURC. Furthermore, treatment with KATP channel inhibitor significantly decreased BBF during the catheterization phase.

image

Figure 1. Intravesical pressure and BBF, measured simultaneously for the duration of the experiment. Cont, control rats; AUR, AURC rats; Nico 10, AURC rats treated with nicorandil at 10 mg/kg body weight; Crom 300, AURC rats treated with cromakalim at 300 µg/kg body weight; Glib 5, AURC rats treated with glibenclamide at 5 mg/kg body weight.

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MEASUREMENT OF CONTRACTILE RESPONSE TO CARBACHOL AND 100 MM KCL

The Emax and EC50 values for the contractile response of bladder strips to carbachol and the response to KCl (100 mM) were determined, and the results are shown in Table 1. The Emax values of carbachol as well as the contractile responses induced by 100 mM KCl in the AUR group were significantly lower than those in the Cont group. Treatment with cromakalim significantly prevented this injury, but still the Emax values of carbachol and contractile responses induced by 100 mM KCl in the Crom300 group were significantly lower than those in the Cont group. Treatment with nicorandil significantly prevented this injury and returned the values to the control levels. Treatment with glibenclamide failed to prevent bladder smooth muscle dysfunction. The EC50 values of carbachol were similar in all groups (Table 1).

Table 1.  Functional studies in the experimental rats
GroupEmax,g/mm2EC50, 10–6 MKCl, g/mm2
  • Data are means (sem) of six separate determinations in each group. Emax and EC50 values are for carbachol effect in experimental rat bladder strips. KCl means contractile force to 100 mM KCl.

  • *

    Significantly different from control group (P < 0.05).

  • Significantly different from AUR group (P < 0.05).

  • Significantly different from Nic 10 group (P < 0.05).

  • §

    Significantly different from Crpm 300 group (P < 0.05).

Cont4.53 (0.15)1.03 (0.11)3.19 (0.14)
AUR2.65 (0.29)*‡§1.13 (0.19)1.62 (0.20)*
Nico 104.05 (0.33)1.23 (0.20)2.52 (0.23)*†
Crom 3003.36 (0.17)*1.57 (0.27)2.25 (0.10)*†
Glib 52.68 (0.24)*‡§0.89 (0.13)§1.71 (0.16)*‡§

MEASUREMENT OF 8-ISOPROSTANE IN THE BLADDER

The levels of 8-isoprostane in the experimental rat bladder are shown in Fig. 2. The tissue level of 8-isoprostane in the AUR group was significantly higher than that in the Cont group. Treatment with nicorandil as well as treatment with cromakalim significantly inhibited this up-regulation which returned to the control levels. Treatment with glibenclamide failed to inhibit the up-regulation of 8-isoprostane levels in the bladder.

image

Figure 2. 8-isoprostane concentrations in the bladder. Cont, control rats; AUR, AURC rats; Nico 10, AURC rats treated with nicorandil at 10 mg/kg body weight; Crom 300, AURC rats treated with cromakalim at 300 µg/kg body weight; Glib 5, AURC rats treated with glibenclamide at 5 mg/kg body weight. *Significantly different from the control group. Significantly different from the AUR group. #Significantly different from the Glib 5 group.

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MEASUREMENT OF KIR6.1 AND KIR6.2 MRNAS IN THE BLADDER

The expression levels of KIR6.1 mRNAs in the bladder were >100 times greater than those of KIR6.2 mRNAs in the Cont group. The expression levels of KIR6.1 mRNAs as well as the expression levels of KIR6.2 mRNAs in the AUR group were significantly up-regulated compared with the Cont group. Treatment with nicorandil and cromakalim significantly increased the expressions of both KIR6.1 and KIR6.2 mRNAs (Fig. 3). By contrast to nicorandil and cromakalim, treatment with glibenclamide did not significantly increase in the expression levels of KIR6.1 mRNAs and KIR6.2 mRNAs compared with those in the AUR group.

image

Figure 3. Expressions of KIR6.1 and KIR6.2 messenger RNAs in the bladder. Cont, control rats; AUR, AURC rats; Nico 10, AURC rats treated with nicorandil at 10 mg/kg body weight; Crom 300, AURC rats treated with cromakalim at 300 µg/kg body weight; Glib 5, AURC rats treated with glibenclamide at 5 mg/kg body weight. Data are means (sem) of six separate determinations in each group. The expressions of KIR6.1 and KIR6.2 mRNAs were quantified according to the expressions of β-actin mRNAs in the experimental rat bladder domes. *Significantly different from the Cont group. Significantly different from the AUR group.

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EXPRESSIONS OF CASPASE-3, BCL-2 AND BAX IN THE RAT BLADDER

Figure 4 shows western blot analysis in the experimental rat bladder. Although levels of proteins caspase-3, Bcl-2 and Bax were detected, there were no significant differences in expressions of caspase-3 and Bax or Bcl-2 among each group examined.

image

Figure 4. Western blot analysis for cleaved caspase-3, Bcl-2 and Bax in the rat bladder. Cont,control rats; AUR, AURC rats; Nico 10, AURC rats treated with nicorandil at 10 mg/kg body weight; Crom 300, AURC rats treated with cromakalim at 300 µg/kg body weight; Glib 5, AURC rats treated with glibenclamide at 5 mg/kg body weight. Data are means (sem) of five separate determinations in each group.

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DETECTION OF APOPTOSIS IN THE BLADDER SMOOTH MUSCLE AND EPITHELIUM

Apoptosis in the experimental bladder was characterized using the TUNEL technique. Data are summarized in Table 2 and typical TUNEL-positive cells are shown in Fig. 5. A large number of TUNEL-positive cells were observed in the epithelium in the AUR group. Treatment with nicorandil, as well as treatment with cromakalim significantly diminished the AURC-induced apoptosis index in the epithelium, which was reduced to the control level. Treatment with glibenclamide, however, failed to inhibit the increased apoptosis index. Interestingly, there were no significant differences in the apoptosis index of the detrusor smooth muscle among any of the groups examined.

Table 2.  TUNEL index in the experimental rat bladder
GroupEpithelium, %Detrusor smooth muscle, %
  • Data are means (sem) of six separate determinations in each group.

  • *

    Significantly different from control group Cont (P < 0.05).

  • Significantly different from AUR group (P < 0.05).

  • Significantly different from Nic 10 group (P < 0.05).

  • §

    Significantly different from Crpm 300 group (P < 0.05).

Cont4.22 (0.44)1.39 (0.18)
AUR9.26 (1.05)*1.30 (0.15)
Nico 105.23 (0.51)1.16 (0.18)
Crom 3004.84 (0.60)1.51 (0.19)
Glib 57.30 (0.61)*‡§1.15 (0.18)
image

Figure 5. TUNEL staining in the rat bladder. A large number of TUNEL-positive cells were observed in the epithelium in the AUR group. By contrast, there were few TUNEL-positive cells in the detrusor smooth muscle in all groups examined. Original magnification: ×100.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERNCES

In the present study, we clearly showed that nicorandil and cromakalim prevented AURC-induced bladder dysfunction via activation of KATP channels with a subsequent inhibition of oxidative stress and a decreased apoptosis index. In addition, the present data confirmed that AURC induced contractile dysfunction in the bladder detrusor muscle. Furthermore, the preventive effect of KATP channel openers on AURC-induced bladder dysfunction was shown by organ bath techniques. These data corroborate and support previous findings from this laboratory [6]. It is well known that one of the main causes of IR injury is oxidative stress in the tissue. Reperfusion and re-oxygenation of ischaemic bladder tissue increase the generation of reactive oxygen species (ROS) metabolites, which cause oxidative damage and cell apoptosis. Isoprostanes, prostaglandin-like end products of arachidonic acid peroxidation, are produced as the end products of free-radical injury to arachidonoyl-containing lipids and are known as unique markers of oxidative stress. It has been suggested that 8-isoprostane (8-iso prostaglandin F2α) is an oxidative stress marker and may play an important role in oxidative stress-related smooth muscle dysfunction [18]. In the present study, we showed that AURC induced oxidative stress in the bladder and that KATP channel openers, but not the KATP channel closer, significantly prevented the production of 8-isoprostane.

The mechanism of the preventive effect IPreC on IR injury is suggested in some tissues. Preconditioning represents the condition where transient exposure of cells to an initiating event leads to protection against subsequent, potentially lethal stimuli. Several different potassium channels have been identified in the inner mitochondrial membrane and their activation may initiate preconditioning in the tissue. Activation of these channels allows potassium ions to flow into mitochondria, which results in depolarization. The open probability of the KATP channel is directly regulated by the intracellular level of ATP or the ratio of ATP to ADP, allowing the channel to couple the metabolic state of the cell to membrane potential [19]. KATP channels contribute in the maintenance of the resting membrane potential and regulating action potential duration. KATP channel inhibitors are the most potent inducer of preconditioning owing to their combined effects on mitochondrial membrane depolarization and ROS production. There are three possible mechanisms by which KATP channel opening protects against IR injury. First, opening of the mitoKATP channel followed by mitochondrial swelling could improve mitochondrial ATP production and/or handling [20]. Second, the protective effect of mitoKATP channel activation could be mediated by decreasing Ca2+ overloading of mitochondria [21]. Third, it has been shown that opening of the mitoKATP channel may increase ROS generation by mitochondria [22]. This increase could lead to protein kinase C activation, which is known to be important during cell protection. In addition, nicorandil is known to be an NO donor. It is possible that in the experimental periods, NO from nicorandil dilates vessels to increase the BBF. Furthermore, nitrate produced from NO, has a protective effect against cardiac IR injury [23].

In the present study, we detected apoptosis by using the TUNEL method. It has been shown that AURC significantly increased the apoptosis index in the epithelium but not in the smooth muscle. Levin et al. [24] reported that the rate of high energy phosphate degradation of the mucosa is significantly greater than the rate of high energy phosphate degradation of the smooth muscle in the rabbit anoxia bladder. Moreover the same authors suggested that the mucosa is more sensitive to the ischaemic damage than smooth muscles in the rabbit bladder [24], which supports our findings. Nicorandil and cromakalim, but not glibenclamide, administration significantly inhibited the induction of apoptosis, which was reduced to the control levels. To investigate the possible pathway of apoptosis, we performed western blot analyses for Bax, Bcl-2 and activated/cleaved caspase-3 expression levels in the experimental rat bladder; however, we did not detect any significant alterations in these protein expressions (Fig. 4). Recently, Li and Oh [25] reported that activation of PARP might be involved in apoptosis in the rat bladder after AUR and subsequent emptying via energy depletion and suppression of Akt activity. They also reported that induction of AUR significantly increased the malondialdehyde levels and apoptosis, decreased the NAD+ levels and Bcl-2:Bax ratio in the rat bladder [25]. Their protocol was 60 min of over-distension, followed by 2 h of drainage, while ours was 30 min of over-distension, followed by 1 h of drainage. It is possible that AURC-induced injury in the present study was milder than that in the study by Li and Oh, which could be why we could not detect changes in expressions of apoptosis-related proteins in the whole bladder.

KATP channels in smooth muscle cells consist of K+ channel pore KIR6.1 or KIR 6.2 coupled with SUR1, SUR2A, SUR2B, forming channels. Kajioka et al. [26] reported that KATP channels in pig detrusor mainly consist of KIR6.1 and SUR2A, instead of SUR2B, while immunohistochemistry showed coexpression of SUR1 and SUR2B in human detrusor, although SUR2B plays the major role [26–28]. Previously, we reported that the predominant KATP channel pore in the rat bladder was KIR6.1 [16]. In the present study, the expressions of KIR6.1 and KIR6.2 were up-regulated by AURC, while nicorandil and cromakalim, but not glibenclamide, treatment increased the additional up-regulation of KIR6.1 and KIR6.2. It is possible that the role and distributions of KIR6.1 and KIR6.2 are in smooth muscle and in urothelium but, in the present study, we did not separate the bladder tissue into smooth muscle and urothelium. Further experiments are required to clarify these issues. Wang et al. reported that pretreatment with diazoxide, a mitoKATP channel opener, increased the expression of KIR6.1 mRNA in cultured hippocampal neurons and gerbil brain, and also that diazoxide played a major protective role on cerebral IR injury through increasing the expression of KIR6.1 mRNA [29]. In contrast to this report, the KIR6.2 subunit has been shown to provide protection during hypoxia-induced generalized seizure using mutant mice lacking the KIR6.2 subunit of KATP channels [30]. In addition, Héron-Milhavet et al. [31] also reported a protective effect of KATP channels and the KIR6.2 subunit brain against hypoxia-ischaemia injury. In their reports, the transgenic overexpression of KIR6.2 in the forebrain significantly protects mice from the hypoxic–ischaemic injury and neuronal damage seen in stroke [31], but it is still unclear which channel pore type plays the major role in the detrusor smooth muscle in this condition.

In conclusion, nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via up-regulation of both KIR6.1 and KIR6.2 with a subsequent decrease in oxidative stress and decreased induction of apoptosis in the bladder.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERNCES

This study was supported by a grant in aid from the Ministry of Education, Science, and Culture of Japan (#20591880).

REFERNCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERNCES