Urol Sci 2010;21(4):175−179
O R I G I N A L A RT I C L E
The Effect of Calcium on the Response of Rabbit Urinary Bladder Muscle and Mucosa to Hydrogen Peroxide Kiroloss Hanna1, Mira Ibrahim1, Robert E. Leggett1, Robert M. Levin1,2,3* 1
Albany College of Pharmacy and Health Sciences, Albany, NY, USA Stratton VA Medical Center, Albany, NY, USA 3 Albany Medical College, Albany, NY, USA 2
Objective: The specific aim of this investigation was to determine the effect of hydrogen peroxide on calcium ATPase of the rabbit urinary bladder smooth muscle and mucosa in the presence of varying concentrations of calcium. Hydrogen peroxide can inhibit enzyme activity via oxidation of the proteins and is often used as a model of free radical damage. The activity of calcium ATPase is increased by increasing the calcium concentration in the buffer. In addition, calcium can also increase the activation of hydrolytic enzymes such as calpain and phospholipase A2. Materials and Methods: The effect of 0%, 0.045%, 0.9% and 0.18% hydrogen peroxide on calcium ATPase activity was quantitated in rabbit urinary bladder smooth muscle and mucosa in the presence of 3 mM, 10 mM or 30 mM of calcium chloride. Results: The results were as follows: (1) Calcium ATPase activity increased with increasing calcium. The muscle and mucosa had similar activities at 3 mM and 10 mM calcium, but the mucosal activity was significantly higher than the muscle at 30 mM. (2) Hydrogen peroxide inhibited enzyme activity of the smooth muscle and contractile activity in a dose-dependent manner to approximately the same extent. (3) Hydrogen peroxide inhibited enzyme activity for both muscle and mucosa. (4) The muscle was significantly more sensitive to hydrogen peroxide than was the mucosa. (5) The mucosa had a significantly higher total antioxidant activity than the muscle. Conclusion: The increased sensitivity of the muscle to hydrogen peroxide may be due in part to its lower total antioxidant activity.
Received: May 10, 2010 Revised: June 14, 2010 Accepted: July 17, 2010
KEY WORDS: calcium; calcium ATPase; hydrogen peroxide; oxidative damage; oxidative stress; SERCA
*Corresponding author. Albany College of Pharmacy and Health Sciences, 106 New Scotland Avenue, Albany, NY 12208, USA. E-mail:
[email protected]
1. Introduction Reactive oxygen species (ROS) and oxidative membrane damage have been implicated as contributing factors in tissue injury associated with aging and numerous diseases.1–5 One especially important ROS is hydrogen peroxide (H2O2), which is formed in vivo by the dismutation of superoxide radicals.6,7 There is increasing evidence suggesting that H2O2 induces cell damage depending on the concentration of the oxidant generated in various cell types.8,9 Normally, antioxidant mechanisms are able to ©2010 Taiwan Urological Association. Published by Elsevier Taiwan LLC.
limit or prevent the adverse effects of H2O2. However, as one ages, these antioxidant mechanisms decrease, which makes oxidative damage more prevalent with aging.10 Urinary bladder obstructive dysfunction secondary to benign prostatic hyperplasia is common in the elderly.11 Recent studies have clearly demonstrated that increased ROS in the bladder is a major etiology of obstructive bladder dysfunction and the subsequent decreased contractile function.12–14 Calcium ATPase (CaATPase) is very important for bladder smooth muscle and mucosal function.15 CaATPase exists in several forms in the bladder as 175
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2. Methods These studies were approved by the Institutional Animal Care and Use Committee of the Stratton VAMC, Albany, NY. Each of five rabbits was anesthetized with pentobarbital (25 mg/kg) and the bladder rapidly excised. The bladder body was separated from the base at the level of the ureteral orifices. Three longitudinal strips (1 × 0.5 cm) were then obtained from the ventral side of the bladder body. Each strip was mounted in a separate 15-mL bath containing Tyrode’s solution maintained at 37°C and equilibrated with a mixture of 95% oxygen and 5% carbon dioxide. An initial resting tension of 2 g was applied for 30 minutes, and the responses were recorded isometrically using a force displacement transducer connected to a Grass Polygraph Model D (Grass Technologies, West Warwick, RI, USA). Electrical field stimulation at 32 Hz was applied and the contraction recorded. H2O2 was added and the strips were incubated for 15 minutes and then stimulated again. This was repeated at three H2O2 concentrations including a control (0%): 0.045%, 0.09% and 0.18%. The smooth muscle and mucosa of the remaining bladder body were separated by blunt dissection, frozen under liquid nitrogen and stored at –80°C until analyzed. Tissue samples of both muscle and mucosa were removed from the freezer and weighed. The samples were homogenized at 100 mg/mL in 50 mM Tris buffer (pH 7.4). The homogenized samples were then centrifuged at 2500 rpm for 10 minutes and the pellet discarded. The supernatant was saved for the biochemical analysis. While on ice, 375 μL of each supernatant (muscle or mucosa) was added to a 10 mL test tube; 50 μL of calcium chloride (pH 7.4) was added so that the final concentration was 3 mM, 10 mM or 30 mM. Then, 300 μL of H2O2 was added so that the final concentration was 0%, 0.045%, 0.9% or 0.18%. The reaction was started by adding 25 μL of 20 mM ATP (pH 7.4). All tubes were then placed in a 37°C water bath for 40 minutes. The test tubes were then removed from the water bath and 500 μL of 12.5% trichloroacetic acid was 176
added to all tubes including the standard curve tubes. This was followed by the addition of 500 μL of the ferrous sulfate-ammonium molybdate solution to each test tube. All samples were then transferred to microcentrifuge tubes and spun for a period of 2 minutes at a speed of 9000 rpm. The absorbance of each supernatant at 650 nm was quantitated using a Hitachi spectrophotometer (model U-2001; Hitachi Ltd., Tokyo, Japan). The standard curve used potassium phosphate (KH2PO4) at a range of 0–2 mM. For the antioxidant (CUPRAC) assay,19 tissues were homogenized at 50 mg/mL in 0.05 M Tris buffer and spun at 2500 rpm for 10 minutes. The supernatant was extracted and used to make sample aliquots at 50 mg/mL, 25 mg/mL and 12.5 mg/mL in 0.05 M Tris buffer. The ascorbic acid standard was diluted with water to concentrations of 1 mM and 500, 250, 125, 63 and 31 μM. A 0 μM control (water) was also used. Then, 0.5 mL of sample or standard aliquots were added in duplicate to glass test tubes followed by 0.5 mL of 10 mM copper (II) chloride, 0.5 mL of 7.5 mM neocuproine and 0.5 mL of 1 M ammonium acetate at a pH of 7.00. Test tubes were then capped and left to incubate for 30 minutes at room temperature. After incubating, absorbance readings were taken for all samples and standards at 450 nm in a Hitachi U-2001 spectrophotometer (Hitachi Ltd.). Antioxidant quantitative data were calculated as the concentration of ascorbic acid required to have the same antioxidant activity as the test sample per mg protein. Statistical analyses utilized analysis of variance followed by the Bonferroni test for individual differences among groups.
3. Results Figure 1 presents the effect of increasing Ca++ concentrations on CaATPase activity. At 3 mM and 10 mM Ca++ concentrations, muscle and mucosa CaATPase activity was 6
Muscle Mucosa
∗
5 Calcium ATPase acvity (μmol Pi/mg protein)
it does elsewhere. Thapsigargin-sensitive CaATPase is the form that regulates calcium (Ca++) entering the sarcoplasmic reticulum. It is generally referred to as SERCA. (The thapsigargin-insensitive form is the enzyme that regulates the movement of Ca++ out of the cell.) In general, SERCA is significantly less abundant than the thapsigargininsensitive form. Together, they regulate the entrance and exit of Ca++ from the cell and control contraction in the smooth muscle and secretion in the mucosa. Partial outlet obstruction results in a significant decrease in both forms of CaATPase activity in both rabbits (muscle and mucosa) and obstructive bladder dysfunction in man.16–18 In this study, we determined the sensitivity of rabbit bladder contraction, smooth muscle CaATPase, and mucosal CaATPase using H2O2 to simulate ROS damage.
4 3 2 1 0 3 mM
10 mM Calcium concentraon
30 mM
Figure 1 Effect of calcium chloride concentration on calcium ATPase activity of the rabbit urinary bladder smooth muscle and mucosa. Each bar is the mean ± SEM of 5 individual rabbits. *Significantly different from muscle (p < 0.05).
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H2O2 and bladder calcium ATPase approximately the same. However, at 30 mM Ca++, the enzyme activity of the mucosa was significantly higher than that of the muscle. Figure 2 shows the dose response curves of CaATPase (at 30 mM Ca++) and contraction (to 32 Hz field stimulation) to H2O2. Both contraction and CaATPase activity responded in a dose-dependent manner to H2O2, showing a similar sensitivity.
CaATPase Contracon
100 ∗
16
∗
60 ∗
40
∗ ∗
20 0 0.000
0.045 0.090 H2O2 concentraon (%)
0.180
Figure 2 Effect of increasing concentrations of hydrogen peroxide on contraction of isolated strips of bladder base to field stimulation (32 Hz) and smooth muscle calcium ATPase. Each bar is the mean ± SEM of 5 individual rabbits. *Significantly different from 0% hydrogen peroxide (p < 0.05).
Calcium ATPase acvity (% of 0 H2O2)
A
Muscle Mucosa
120 100 † ∗
60 40
Calcium ATPase acvity (% of 0 H2O2)
∗†
∗ ∗
20 0 0.000
C
†
0.045 0.090 H2O2 concentraon (%)
0.180
12
8
4
0 Muscle
Mucosa
Figure 4 Total antioxidant activity of the muscle and mucosal homogenate. *Significantly different from muscle (p < 0.05). B
140
80
∗
∗ Total anoxidants (μm AA equivalents)
80
Calcium ATPase acvity (% of 0 H2O2)
Contracle response to FS (32 Hz) calcium ATPase acvity (% of 0 H2O2)
120
Figure 3 presents the effects of H2O2 at 3 mM, 10 mM and 30 mM Ca++, respectively. H2O2 caused a dosedependent decrease in the activities of both muscle and mucosa. The muscle was significantly more sensitive to H2O2 than was the mucosa. The sensitivity of the muscle and mucosa to H2O2 was similar at all Ca++ concentrations. Figure 4 presents the total antioxidant levels in muscle and mucosa. These data demonstrate that the mucosa has a significantly higher overall antioxidant level than the muscle tissue.
140
Muscle Mucosa
120 100 80
∗
∗†
∗
60
∗†
∗
40 20
∗
0 0.000
0.045 0.090 H2O2 concentraon (%)
0.180
120 Muscle Mucosa
†
100
†
∗
80 60
∗† ∗
40
∗
20 0 0.000
0.045 0.090 H2O2 concentraon (%)
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0.180
Figure 3 Effect of increasing concentrations of hydrogen peroxide (H2O2) on smooth muscle and mucosal calcium ATPase at: (A) 3 mM calcium; (B) 10 mM calcium; (C) 30 mM calcium. Each bar is the mean ± SEM of 5 individual rabbits. *Significantly different from 0% H2O2 (p < 0.05); †significantly different from muscle (p < 0.05).
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4. Discussion The incidence of benign prostatic hyperplasia increases with age.11,20 Partial outlet obstruction in rabbits is utilized to represent the effect of the enlarged prostate on bladder function.21,22 Recent studies have demonstrated clearly that bladder outlet obstruction induces cyclical ischemia/reperfusion that leads to the generation of ROS, and that ROS participates in the progressive deterioration of bladder function that is observed during bladder decompensation.23,24 Biochemical studies have demonstrated that CaATPase activity decreases significantly following partial outlet obstruction, and the decrease is proportional to the decreased contractile function of the bladder.16 Intracellular free Ca++ can vary considerably in smooth muscle cells between the stimulated state and relaxed state.25–28 Alternately, Ca++ concentration in the mucosa would remain relatively stable in comparison to the smooth muscle. Although the Ca++ concentrations utilized in these experiments are not physiological, we believe that the differences seen between muscle and mucosa do have physiological relevance. The results of this investigation revealed several differences between the CaATPase activity of the bladder muscle and mucosa. First, the mucosal CaATPase was significantly more sensitive to Ca++ than that of the smooth muscle at high Ca++ concentrations. Second, contraction and smooth muscle CaATPase have approximately the same sensitivity to H2O2. Third, the smooth muscle CaATPase was significantly more sensitive to H2O2 than was the mucosal CaATPase. Lastly was the demonstration that the mucosa had a significantly higher total antioxidant activity than the smooth muscle, which may account for the greater resistance of the mucosa to oxidative stress. Published studies have clearly demonstrated that antioxidants such as resveratrol can protect CaATPase activity impaired by both disease and reaction to free radical damage.29–32 Our own studies have also clearly demonstrated the protective effect of both specific antioxidants such as alpha lipoic acid and coenzyme Q10, and natural products high in antioxidant activity such as grapes and Kohki tea on obstructive and ischemic bladder dysfunction.33–38 The similarity between the effects of H2O2 on contraction and smooth muscle CaATPase activity and the previously demonstrated similarity between the effects of partial outlet obstruction on both contraction and CaATPase activity lend support to the importance of oxidative stress as a major etiological factor in obstructive and ischemic bladder dysfunction.39–43
and in part by the Capital Region Medical Research Foundation.
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Acknowledgments This material is based upon work supported in part by the Office of Research and Department of Veterans Affairs 178
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