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ENDOCRINE AND DIABETES
Department of Pharmacology, School of Pharmaceutical Sciences (K.N., T.Y., T.H., K.H.), and Department of Pharmacology, School of Medicine (M.O., S.I., K.O.), Showa University, Tokyo, Japan; and Department of Pharmacology, School of Medicine, St. Mariannna University, Miyamae-ku Kawasaki, Kanagawa, Japan (T.K., S.K.)
Received September 6, 2007; accepted October 31, 2007.
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). Consequently, these severe diseases may be life-threatening. These serious dysfunctions were noted in lifestyle-related diseases; however, dysfunctions of the urinary organs, including dysuria and frequent urination, negatively impact social situations (Bremnor and Sadovsky, 2002; Kowalczyk, 2003). It is generally accepted that normalization of urinary dysfunction is beneficial to maintain patient quality of life.
Hypertension, a major lifestyle-related disease, has been investigated by many groups (Sonkusare et al., 2006; Mehta and Griendling, 2007). An association between autonomic nerve dysfunction (Velez-Roa et al., 2004; Mancia et al., 2007) or renal disease (Williams et al., 2007) and hypertension was suggested; additionally, alterations of urinary bladder function were also reported (Schneider et al., 2005). Bladder smooth muscle tissue isolated from spontaneously hypertensive rat (SHR) was indicative of overcontraction (Persson et al., 1998). These findings suggested that enhancement of bladder contraction causes various urinary dysfunctions. Overexpression or sensitization of muscarinic receptors was noted as the mechanism (Schneider et al., 2005). Moreover, intracellular calcium sensitization (Sherer et al., 2000; Rajasekaran et al., 2005) and correlation of autonomic nerve alteration (Steers et al., 1999) were also hypothesized.
In some patients of hypertension, hyperlipidemia, cardiovascular diseases, and diabetes indicate not a single disease, but multiple diseases. Recently, multiple types are increasing. This situation creates diversification and complicates classification of tissue dysfunctions. In particular, the possibility exists that hypertension may be accompanied by hyperlipidemia. This combined hypertension and hyperlipidemia is believed to lead to arteriosclerosis and/or other serious cardiovascular diseases. Not unexpectedly, bladder responses may be characteristically altered under conditions of hypertension and hyperlipidemia. However, alteration of the response has not been demonstrated.
In 2003, spontaneously hypertensive and hyperlipidemic rat (SHHR) was generated from SHR and hyperlipidemic rat (Kumai et al., 2003; Amagasa et al., 2005). SHHR is characterized by systolic blood pressure of approximately 150 mm Hg and plasma cholesterol concentration exceeding 150 mg/dl. Moreover, plasma catecholamine levels and low-density lipoprotein expression were higher in SHHRs than in Sprague-Dawley (SD) rats. In the aorta, both endothelial degeneration and lipid deposits were also reported (Kumai et al., 2004). Based on these findings, we concluded that SHHR is a novel beneficial tool for application as a multiple hypertensive and hyperlipidemic model. The objective of this study was to evaluate differences in isolated bladder smooth muscle contractility between SD rat and SHHR bladders and to examine the correlation between the intracellular signaling pathway and bladder smooth muscle dysfunction.
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Materials and Methods |
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Experimental Models. SHHR (male, 7–9 months of age) and age-matched control (SD) rats were used in this study. The animals were prepared as described previously (Kumai et al., 2003). Rats were housed at constant room temperature (23 ± 1°C) with a 12-h light/dark cycle. The rats were fed standard rat chow including 5% fat (CE-2; Clea, Tokyo, Japan). Food and water were available ad libitum, and the rats grew satisfactorily. All procedures were performed according to the guilting principles for the care and use of laboratory animals for the Japanese Pharmacological Society.
Blood Collection and Plasma Biochemical Assays. Blood specimens, which were obtained from the inferior vena cava under pentobarbital anesthesia, were mixed with a 3.2% sodium citrate solution at a ratio of 9:1. The citrated plasma supernatant was used for chemical assays. Total cholesterol (TC) and triacylglycerol (TG) levels were measured as described previously (Kumai et al., 2003).
Bladder Smooth Muscle Preparation. The urinary bladder was isolated from pentobarbital-anesthetized (35 mg/kg) rats. Bladders were rinsed in physiological salt solution (PSS); subsequently, fat and connective tissue were removed from both sides. PSS, which was supplemented with 118 mM NaCl, 5.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 1.4 mM NaH2PO4, 21.4 mM NaHCO3, and 11.1 mM glucose, was aerated with 95% O2 and 5% CO2 at 37°C. Prior to measurements, the wet weight of each tissue was determined.
Isometric Force Measurement. Each tissue was positioned vertically in a temperature-controlled 5-ml organ bath. One end of the tissue was connected to a strain gauge transducer (Type T-7–8-240; Orienteck, Tokyo, Japan) to monitor contractile responses. Bladder contractions were normalized to cross-sectional area with the following equation: cross-sectional area (millimeters squared) = (2 x wet weight (milligrams))/(1.06 x circumference (millimeters)), where 1.06 is smooth muscle tissue density (milligrams per millimeter cubed). Resting tension was set at 10 mN, where the tissue length was established in the optimal range for force generation.
Measurement of Relative Rho Activity. Bladders were stimulated under various conditions similar to force measurement under resting tension (10 mN). Subsequently, tissues were powdered at liquid nitrogen temperature. The samples were resuspended in a homogenization buffer containing 20 mM MOPS, pH 7.2, 250 mM sucrose, 1 mM dithiothreitol, 1 mM EGTA, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 50 µg/ml soybean trypsin inhibitor (buffer A); homogenates were centrifuged (1000g for 5 min) to remove nuclei. The supernatant was decanted, after which pellets were washed once with sucrose-free buffer A (buffer B). Next, the combined supernatants were recentrifuged (20,000g for 30 min). The pellets were resuspended in buffer B. Finally, total membrane fractions were collected by centrifugation of the supernatant (100,000g for 60 min) and resuspended in buffer B. The supernatant served as the cytosolic fraction. Total Rho A activities were measured with a specific Rho A activation assay kit (G-LISA; Cytoskeleton Inc., Denver, CO). Membrane and cytosolic fractions (25 µg of protein) were assayed and results were detected as relative luminescence units (RLU) over background signal (background incubation with assay reagents alone instead of cellular fractions).
Measurement of Total Mass of Diacylglycerol. The total mass of diacylglycerol (DG) in each tissue was measured in a manner similar to that described in a previous report (Nobe et al., 1993). Isolated tissues were treated under various conditions in 200 µl of PSS. The reaction was terminated upon the addition of chloroform/methanol (1:2 by volume, 750 µl). Tissues were homogenized; subsequently, water and chloroform (200 µl of each) were added. The mixture was shaken, followed by centrifugation at 1000g. The lower phase was removed and dried under N2 gas. The residue was redissolved in chloroform (concentration, 2 µl/mg wet weight tissue). This sample was spotted on a thin layer chromatography plate (Merck, Whitehouse Station, NJ; Silica gel 60 with concentrating zone). DG separation was effected with diethyl ether/heptane/acetic acid (75: 25:1 by volume). The plates were dried and stained with 0.03% Coomassie Brilliant Blue solution containing 30% methanol and 100 mM NaCl for 30 min; plates were destained for 5 min in dye-free staining solution. Each thin-layer chromatography plate was scanned; moreover, the density of each band was calculated using NIH image software. Total mass of DG was determined from a dioreoyl-glycerol standard curve. Results are expressed as nanograms per milligram wet weight of tissue.
D-[14C]Glucose Incorporation into DG. Tissues were prelabeled with 33 mCi/ml D-[14C]glucose contained within normal PSS and high-glucose (HG)-PSS (twice the glucose concentration of normal PSS) at 37°C for 60 min (Inoguchi et al., 1994; Lee et al., 2004). CCh and/or additional reagents were introduced following this treatment. Following termination of these reactions, total lipids were extracted as described above. DG was separated on Silica Gel G thin-layer plates and developed in hexane/ether/acetic acid (60:40:1). Spots on the silica gel were removed by scraping; subsequently, radioactivity of [14C]DG was measured on a liquid scintillation counter.
Data Analysis. Values are presented as means ± S.E.M. obtained from at least four to six animals. Statistical analyses for simple comparisons were performed using Student's t test. Multiple comparisons were conducted with analysis of variance for repeated measures followed by the Student-Newman-Keuls test.
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Alteration of CCh-Induced Isometric Force Responses in SHHR and SD Rat Bladder. Resting levels of isometric force in SD rat and SHHR bladders averaged 0.96 ± 0.02 and 0.98 ± 0.04 mN/mm2 (n = 5), respectively. Cumulative addition of CCh induced significant increases in isometric force. Typical responses of individual SD rat and SHHR bladders are shown in Fig. 1A; the averaged concentration-response relationships from these experiments for both bladder types are summarized in Fig. 1B. Maximal increase in force response in SD rat bladder in the presence of 30 µM CCh was 13.14 ± 0.21 mN/mm2 (n = 5). At higher CCh levels, force development declined. Decreases at high concentrations of CCh (30 µM) have been reported (Lundbeck and Sjogren, 1992). The basis for this decline is not known with certainty; however, it may be attributable to G-proteinmediated inhibition associated with high levels of muscarinic receptor activation. In SHHR, the maximal increase in isometric force was depressed considerably (6.93 ± 0.30 mN/mm2; n = 5). Significant decreases were also evident between 1.0 and 100 µM CCh. EC50 values for SD rat and SHHR bladders were 2.5 and 7.5 µM, respectively. The dose-response curve for SHHR displayed a substantial rightward shift in comparison with that of SD rat. The responses to CCh were responsible for this shift following a rise in PSS for 15 min.
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Effects of CaCl2 Replacement on CCh-Induced Increase in Isometric Force in SD Rat and SHHR Bladders. The depressed responses of SHHR are consistent with a decrease in calcium channel activity. CaCl2-replaced PSS (nominally Ca2+-free PSS) was used with respect to this approach; summarized data are presented in Fig. 2. In SD rat bladder, pretreatment with Ca2+-free PSS for 5 min did not alter isometric force resting level. In contrast, the response was severely blunted by cumulative CCh stimulation; isometric force at 30 µM CCh was 45.0 ± 3.82% (n = 5) of that of the control (response in normal-PSS). In SHHR bladder, pretreatment with Ca2+-free PSS also markedly reduced the isometric force response to CCh without affecting the resting level. CCh (30 µM)-induced force development in Ca2+-free PSS was 3.56 ± 0.64 mN/mm2 (n = 5), and the inhibition ratio (45.7 ± 13.8%) was similar to that of SD rat. Significant rates of reduction relative to SD rat were detected at concentrations of 10 to 30 µM CCh.
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Alteration of Bladder Contractility under HG Conditions in SD Rat and SHHR Bladder. CCh-induced force development in HG-PSS was assessed employing a strategy similar to that of the rho A assay under HG conditions (Fig. 5). Pretreatment of SD rat bladder with HG-PSS for 30 min did not alter the nonstimulated resting level (1.05 ± 0.07 mN/mm2; n = 5). However, the CCh-induced dose-dependent increase in force development displayed significant enhancement. Apparent enhancement in comparison with the responses in normal-PSS was detected following treatment with 0.3 to 30 µM CCh. Submaximal response induced by 30 µM CCh was 13.8 ± 0.23 mN/mm2 (n = 5). The EC50 value in HG-PSS was 2.1 µM. In SHHR bladder, the CCh-induced increase in force development was not affected by extracellular glucose accumulation. Force developments induced by 30 µM CCh in normal- and HG-PSS were 7.09 ± 0.19 and 7.52 ± 0.54 mN/mm2 (n = 5), respectively.
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Alteration of Intracellular DG Formation under Normal and HG Conditions. The roles of DG in phosphatidylinositol turnover-mediated contraction have been identified; thus, to establish alteration of the intracellular signaling system in SD rat and SHHR, levels of DG, a major contractile factor, were measured (Fig. 6). In SD rat bladder, the endogenous DG level in the resting state was 138.5 ± 11.1 ng/mg wet weight tissue (n = 5) (Fig. 6A). This level rose markedly in the presence of 30 µM CCh to 246.3 ± 14.0 ng/mg wet weight tissue (n = 5). Under HG conditions, the resting level of DG increased significantly to 271.6 ± 12.7 ng/mg wet weight tissue (n = 5). DG increased to 338.4 ± 10.7 ng/mg wet weight tissue (n = 5) following stimulation with 30 µM CCh. In SHHR bladder, resting and CCh-treated values of total mass of DG in normal PSS were 134.1 ± 10.9 and 186.4 ± 7.36 ng/mg wet weight tissue (n = 5), respectively. A meaningful increase in terms of CCh stimulation was not evident. Moreover, enhancement in HG-PSS in SHHR was blunted; resting and CCh-treated values were 150.4 ± 15.2 and 194.9 ± 11.0 ng/mg wet weight tissue (n = 5), respectively. Significant differences between normal- and HG-PSS were not observed.
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Effects of Protein Kinase C Inhibitors on CCh-Induced Bladder Contractility under HG Conditions in SD Rat and SHHR. To identify a relationship between protein kinase C (PKC) and bladder contraction in SD rat and SHHR, three types of PKC inhibitors were introduced. Calphostin C is a general-type (nonisoform-specific type) PKC inhibitor. Gö6976 and rottlerin, calcium-dependent and -independent PKC inhibitors, respectively, were selected (Fig. 7, inset). To assess the specific inhibitory effects of the PKC inhibitors, time and dose dependence of the inhibitors were measured (data not shown). Based on these preliminary trials, each condition was adopted as the minimum concentration in the critical inhibitory range. Treatment with 30 µM CCh under HG conditions apparently increased SD rat bladder contraction in a manner similar to that of Fig. 1. Both Calphostin C and Gö6976 significantly reduced CCh-induced force development (57.8 ± 3.6 and 50.9 ± 2.7% of control response; n = 5). In contrast, rottlerin did not affect the response (92.6 ± 9.5% of control response; n = 5). Similar trials were performed under HG conditions. In the nonstimulated resting state in HG-PSS, force level was indistinct from the corresponding level in normal-PSS (1.1 ± 0.1 mN/mm2; n = 5); however, significant enhancement of force development was evident following treatment with 30 µM CCh (12.8 ± 0.3 mN/mm2; n = 5). A similar pattern in terms of isometric force changes in the presence of Calphostin C was detected under HG conditions in SD rat bladder. However, marked inhibition was observed in the presence of rottlerin (44.6 ± 3.4% of control response; n = 5); Gö6976 did not alter the force response.
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The inhibitory effects of rottlerin on the CCh-induced dose-dependent enhancement of force development were also measured (Fig. 7A). In a manner similar to that of Fig. 5B, CCh-induced dose-dependent increase in force development was confirmed under normal glucose conditions. This response exhibited meaningful enhancement during stimulation with 0.3 to 100 µM CCh under HG conditions. Pretreatment with 1 µM rottlerin significantly reduced the isometric force in the presence of 1 to 100 µM CCh, although resting level was unaffected. This inhibitory effect was apparent only in HG-PSS. Dose-response curves for CCh in the presence of rottlerin in normal- and HG-PSS were striking.
In SHHR bladder, enhancement of force development induced by 30 µM CCh under normal-glucose conditions was confirmed (resting and maximal levels were 1.0 ± 0.02 and 4.6 ± 0.2 mN/mm2, respectively; n = 5). Pretreatment of tissues with Calphostin C reduced CCh-induced responses (5.5% of control; n = 5); however, meaningful attenuation of force development was not evident in the presence of rottlerin (over 70% of control responses remained). Pretreatment of bladders with Gö6976 significantly reduced force development (1.2 ± 0.1 mN/mm2, respectively; n = 5). This inhibitory effect resembled that of Calphostin C. Under HG conditions, effects of CCh and PKC inhibitors were measured; moreover, patterns similar to the responses in normal-PSS emerged. Extracellular glucose-dependent alteration of force development was not detected. The dose-response curve for CCh treatment in SHHR bladder was not impacted by extracellular glucose levels as in Fig. 5B (Fig. 7B). Pretreatment of SHHR bladder with 1 µM rottlerin did not influence the CCh-induced dose-response curves.
To confirm the relationship between force development and PKC activity in the presence or absence of rottlerin, the effect of rottlerin on PKC activity in total membrane fractions was measured (Fig. 8). In SD rat, PKC activity in the membrane fraction of the nonstimulated resting state was 6.2 ± 0.6 pmol/min/mg protein (n = 5). This activity increased markedly upon treatment of tissue with 30 µM CCh (20.1 ± 1.4 pmol/min/mg protein; n = 5). The calcium-independent PKC inhibitor, rottlerin, slightly reduced CCh-induced PKC activation; however, this result was not significant. Similar trials were performed under HG conditions. In the nonstimulated resting state in HG-PSS, PKC activity in the membrane fraction (17.6 ± 1.8 pmol/min/mg protein; n = 5) was significantly greater in comparison with the corresponding value in normal PSS. This increase was maintained during CCh stimulation (20.2 ± 0.9 pmol/min/mg protein; n = 5). However, rottlerin (1 µM; 5 min) suppressed PKC activity in HG-PSS (8.9 ± 0.4 pmol/min/mg protein; n = 5).
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; Biourge et al., 1997); 9-month-old SHHR did not display this stage. Furthermore, sustained elevation of fasting blood glucose level was not evident in SHHR and SD rat. Meaningful elevation of glucose level was detectable via the oral glucose tolerance test at 7 months of age. These results indicated that the selected SHHR stage (9 months of age) critically demonstrated both hypertension and hyperlipidemia; however, obesity and diabetic hyperglycemia were absent. These data are indicative of the onset of these conditions in the early stage of the model. The significance of this investigation with respect to the early stages pertains to the provision of novel findings in terms of prevention and early care regarding multiple hypertension and hyperlipidemia mediated by an understanding of the initial dysfunction of the tissue.
Treatment of bladder smooth muscle tissue with CCh induced time- and dose-dependent contraction in both SD rat and SHHR (Fig. 1A). However, the response in SHHR was significantly lower than that in SD rat (Fig. 1B). Enhancements of contraction in diabetic models (Gupta et al., 1996; Waring and Wendt, 2000) and SHR (Rajasekaran et al., 2005) have been described. Consequently, reduction of contraction in SHHR is a characteristic of bladder dysfunction in combined hypertension and hyperlipidemia. Increased bladder output was also documented (Drake et al., 2006), and it may be correlated with dysuria and frequent urination in hypertensive patients. In terms of previous findings regarding enhanced bladder contractility in hypertension, why is bladder contraction reduced in SHHR?
Based on the results that SHHR bladder indicated inverted dysfunction from the SHR, we surmised that reduction of contractility might be governed by original mechanisms. We initially measured alteration of muscarinic receptor function (Fig. 1B) and intracellular calcium dependence (Fig. 2). In these experiments, pA2 values in SD rat and SHHR were similar (8.48 and 8.72, respectively). We concluded that alteration of muscarinic receptor function was not a major reason for reduced contraction.
CCh-induced contraction in SD rat and SHHR bladders was significantly attenuated (approximately 50% of each response in normal-PSS). However, differences between SD rat and SHHR remained. These results indicated that alterations of muscarinic receptor and extracellular calcium dependencies in SD rat and SHHR were not involved with the reduction of contraction. We therefore focused on rho and the rho kinase-mediated pathway in terms of a calcium-independent smooth muscle regulatory mechanism. This pathway plays an important role in bladder smooth muscle contraction as well as in other types of smooth muscle contraction (Nobe and Paul, 2001; Tsai and Jiang, 2006). A specific rho kinase inhibitor, Y27632, significantly inhibited CCh-induced SD rat bladder contraction (Fig. 3). The dose-response curve derived from Y27632 inhibition in SD rat was similar to the control dose-response curve in SHHR. However, addition of Y27632 did not alter contraction in SHHR. These data indicated that the rho-rho kinase pathway was involved in CCh-induced bladder smooth muscle contraction in SD rat; additionally, activity of the pathway was reduced in SHHR. This reduction may lead to bladder dysfunction in SHHR. To confirm inactivation of the rho-rho kinase pathway, cellular rho activities were measured (Fig. 4). From the CCh-induced increase in rho activation was suppressed only in SHHR, it was suggested that suppression of rho activation leads to reduced bladder smooth muscle responsiveness mediated by rho kinase inactivation.
Reduction of contractile responses in bladder smooth muscle isolated from hypertensive and hyperlipidemic rats was observed for the first time in the current study. This phenomenon does not involve alteration of muscarinic receptor or extracellular calcium dependence, e.g., SHR; however, participation of the rho-rho kinase pathway in this dysfunction was strongly suggested.
In SHHR, hypertension and hyperlipidemia are aggravated with age. After 7 months of age, marked increases in blood glucose level were also detected in oral glucose tolerance test. Although the selected stage of combined hypertension and hyperlipidemia in SHHR did not follow with respect to chronic hyperglycemia (Table 1), the relationship between extracellular glucose accumulation and CCh-induced contractile responses in bladder was assessed.
HG conditions were introduced in our previous study involving diabetic vascular smooth muscle measurement (Nobe et al., 2003, 2004). CCh-induced SD rat bladder contraction was significantly enhanced under HG conditions, but it was not evident in SHHR (Fig. 5). This finding indicated that SHHR bladder lacks glucose sensitivity. In diabetic bladder dysfunction under HG conditions, Stevens et al. (2006) noted that bladder overcontraction is involved in diabetic dysfunction. Reduced bladder contraction was also reported (Su et al., 2004). Our results in SHHR may support future possibilities. As an important point in the current investigation, it was indicated that reduction of bladder smooth muscle contraction in SHHR occurred prior to sustained elevation of plasma glucose level. It was thought that glucose sensitivity was suppressed prior to the rat's progression to the stage of chronic hyperglycemia. This desensitization, which was described initially in this study, might be critical in terms of a thorough understanding of bladder dysfunction in combined hypertension and hyperlipidemia.
Dysfunction of SHHR bladder contraction in normal PSS arose due to inactivation of the rho-rho kinase pathway; consequently, rho activity in the membrane fraction was measured under HG conditions (Fig. 4). These results suggested that suppression of glucose sensitivity in SHHR bladder is not correlated with activity of the rho-rho kinase pathway. We previously reported that enhancement of smooth muscle contraction under HG conditions involves the DG-PKC pathway in some vascular tissue as an additional regulatory factor of bladder contraction (Nobe et al., 2003, 2004). It is generally accepted that CCh-induced bladder smooth muscle contraction is mediated by phosphatidylinositolbisphosphate hydrolysis and DG formation in a manner similar to that of other types of excitable cells (Nord, 1996; Nofer et al., 2000). Individual PKC isoforms might undergo activation by a specialized DG species (Kanoh et al., 1990). Moreover, some activated-PKC isoform types accelerate phosphatidylinositol turnover via activation of DG kinase.
Accumulation of endogenous DG during CCh stimulation was observed in SD rat bladder (Fig. 6A). Under HG conditions, incorporated glucose is utilized by the glycolytic pathway as well as in DG formation via a de novo synthesis pathway (Mancia et al., 2007). HG treatment induced an elevation in resting DG level, which was maintained during CCh stimulation. DG formation derived from incorporated glucose also increased (Fig. 6B). These findings suggested that sustained basal DG level under HG conditions in SD rat bladder results from an increase in the formation of DG derived from glucose. This enhanced DG level might contribute to overactivation of PKC and DG kinase. PKC activity in the membrane fraction was significantly enhanced under HG conditions (Fig. 8). Therefore, these data revealed that HG enhances intracellular DG level via a de novo synthesis pathway in SD rat bladder.
Is PKC activation under HG conditions distinguishable from PKC activation in normal-PSS? De Witt et al. (2001) described rottlerin inhibition of calcium-independent isoforms of PKC (novel PKC-
,-
,-
,-
). Pretreatment with rottlerin significantly attenuated CCh-induced SD rat bladder contraction (Fig. 7A) in HG-PSS. Glucose-dependent activation of PKC in the membrane fraction was also inhibited (Fig. 8). The inhibitory effects of rottlerin were similar to the effect of general (isoform-nonselective) PKC inhibitor types, namely, Calphostin C (Fig. 7A, inset). Moreover, rottlerin did not alter contractile response or PKC activity under normal glucose conditions (Figs. 7A and 8). These results suggested that DG is unnaturally formed from incorporated glucose under HG conditions, which leads an activated calcium-independent isoform of PKC. This activation may induce glucose-dependent overcontraction in SD rat bladder. It is a normal physiologic response in bladder function under HG conditions; absence of this response may be associated with bladder dysfunction.
In SHHR bladder, enhancement of total DG level was not observed in HG-PSS; furthermore, DG formation from incorporated glucose was undetectable in HG-PSS (Fig. 6). A calcium-independent isoform of PKC activation was not detected due to the absence of glucose-dependent DG (Figs. 6B and 8). Consequently, contractile response in SHHR bladder under HG conditions was unaltered by pretreatment with rottlerin (Fig. 7B). These data suggested that suppression of glucose dependence in SHHR bladder is responsible for the reduction of DG formation from incorporated glucose, which may be attributable to inactivation of the glucose transporter in bladder smooth muscle.
We examined bladder responsiveness in a combined hypertensive and hyperlipidemic rat. Prior to any indication of hyperglycemia in this model, glucose utilization diminished, and reduction of bladder smooth muscle contraction occurred. Under this pathological condition, we hypothesized that improvement with respect to reduced formation of DG from glucose and/or reduced activity of calcium-independent PKC isoforms contribute to the normalization of bladder function.
Diminished bladder smooth muscle contraction was detected initially under combined hypertensive and hyperlipidemic conditions in this investigation. The reduction of bladder contractility in SHHR involved inactivation of the rho-rho kinase pathway. The absence of extracellular glucose dependence was observed prior to hyperglycemia indications in SHHR. The occurrence of alteration of contractile response prior to hyperglycemia onset was critical with respect to a thorough understanding of bladder dysfunction. Mechanistically, this phenomenon suggested that this alteration mediates a reduction in calcium-independent PKC activity followed by formation of DG from incorporated glucose. The current findings afforded novel information with respect to establishment of pharmacotherapy for bladder dysfunction.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: SHR, spontaneously hypertensive rat; SHHR, spontaneously hypertensive and hyperlipidemic rat; SD, Sprague-Dawley; CCh, carbachol; Atr, atropine sulfate; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo-(2,3-a)pyrrolo(3,4-c)-carbazole; Y27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; TC, total cholesterol; TG, triacylglycerol; PSS, physiological salt solution; RLU, relative luminescence unit(s); DG, diacylglycerol; HG, high-glucose; PKC, protein kinase C.
Address correspondence to: Dr. Koji Nobe, Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. E-mail: kojinobe{at}pharm.showa-u.ac.jp
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  K. Nobe, T. Yamazaki, N. Tsumita, T. Hashimoto, and K. Honda Glucose-Dependent Enhancement of Diabetic Bladder Contraction Is Associated with a Rho Kinase-Regulated Protein Kinase C Pathway J. Pharmacol. Exp. Ther., March 1, 2009; 328(3): 940 - 950. [Abstract] [Full Text] [PDF]
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, p < 0.01 versus nonstimulated resting level.