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ABOLITION BY ESTROGEN OF NEURONAL NITRIC OXIDE SYNTHASE SMOOTH MUSCLE RELAXATION IN THE FEMALE MICE URETHRA
73
74
SECOND MESSENGER PATHWAYS FOLLOWING DETRUSOR M3 RECEPTOR STIMULATION
ABOLITION BY ESTROGEN OF NEURONAL NITRIC OXIDE SYNTHASE SMOOTH MUSCLE RELAXATION IN THE FEMALE MICE URETHRA
Daly D.1, Chess-Williams R.1, Chapple C.2 1 University of Sheffield, Biomedical Science, Sheffield, United Kingdom, 2Royal Hallamshire Hospital, Urology, Sheffield, United Kingdom INTRODUCTION & OBJECTIVES: Muscarinic receptors are the main targets for anticholinergic drugs used to treat detrusor overactivity; however little is known about the 2nd messenger pathways involved. Understanding these pathways may reveal novel targets for pharmological intervention. M3 receptor stimulation results in the production IP3 and DAG, which ultimately results in intracellular calcium release and an influx of extracellular calcium respectively. M3 receptors can also increase DAG via phospholipase D. We previously reported that the α1-adrenoreceptor exhibits agonist specificity. The aim of this study was to identify the signal transduction mechanism involved in the carbachol or acetylcholine (Ach) induced contraction of the porcine detrusor smooth muscle. MATERIAL & METHODS: Strips of female pig detrusor muscle were set up under 1g tension in gassed Krebs-bicarbonate solution at 37°C. Concentration-response curves were obtained to carbachol or Ach in the absence and presence of drugs that interfere with the second messenger pathways or calcium movements. Results are expressed as the mean ± SEM for pEC50 values and maximum responses for carbachol. Paired Student’s t-test was used for statistical comparisons. RESULTS: Carbachol produced concentration–dependant contractions of the detrusor smooth muscle with a mean pEC50 of 5.51±0.11 and a maximum contraction of 9.86g±2.09g (n=13). The presence of ryanodine (30mM), which inhibits intracellular calcium release, had no significant effect on carbachol-induced maximal contractions or pEC50.The IP3 receptor antagonist 2-aminoethoxydiphenylborane (2APB, 30μM) also had no significant effect on maximal contractions or pEC50 . In contrast, the L- type calcium channel blocker nifedipine (1μM), significantly reduced responses to carbachol by 85.9±4.5% (P=0.003, n=11). Incubation with low calcium Krebs(0.95mM n=8) or calcium-free Krebs (nominal n=8) caused significant falls in maximum contraction (39.7±9.6%, P=0.006 and 98.1±0.8%, P=0.0001 respectively). The phospholipase C inhibitor U71322 (30uM), had no significant effect on carbachol induced contractions, but the phospholipase D inhibitor butan-1-ol (n= 8) significantly reduced maximal responses to carbachol by 73.7±4.6% (p=0.001). When using Ach (in the presence of physostigmine, 100nM), nifedipine and 2ABP had similar effects as with carbachol, nifedipine significantly inhibiting responses by 83.0%±2.8 (control pEC50 = 5.0±0.1; nifedipine pEC50=4.3±0.2) and 2ABP having no effect. CONCLUSIONS: The results suggest that pig detrusor contraction to carbachol is mediated with little contribution from intracelluar calcium stores. A pathway may be involved whereby M3 stimulation activates PLD resulting in DAG formation and contraction via extracellular calcium influx. No differences were observed between carbachol and Ach in these experiments.
Gamé X.1, Praddaude F.2, Arnal J.L.3, Escourrou G.4, Tack I.2, Allard J.2, Rischmann P.1, Ader J.L.2, Sarramon J.P.1, Malavaud B.1 1 Chu Rangueil, Service d’Urologie, Toulouse Cedex 4, France, 2Chu Rangueil, Laboratoire de Physiologie Rénale, Ufr Médecine, Toulouse, France, 3Chu Rangueil, Inserm U397 et Laboratoire de Physiologie, Toulouse, France, 4Chu Rangueil, Laboratoire d’Anatomie Pathologique, Toulouse, France
INTRODUCTION & OBJECTIVES: To assess the implication of estrogen and neuronal Nitric Oxide synthase (nNOS) in urethral relaxation. MATERIAL & METHODS: 4 weeks-old female mice C57/BL6, were split into 3 groups : ovariectomized (group I), Sham-operated (group II), ovariectomized receiving 17b-estradiol (E2) pellet releasing 80 μg/kg/day of E2 for 60 days (group III). Six weeks later, the animals were treated either by a competitive inhibitor of nNOS : 7-Nitroindazole (7NI, 50 mg/kg IP) or by the vehicle. Under anesthesia (ketamine + xylazine), a 22-gauge angiocatheter was inserted percutaneous into the bladder. The bladder was distended via the suprapubic catheter by saline. Pressures were measured (Gould TA400 Pression Transducer) and the leak point pressure defined by oozing of fluid at the meatus was determined. Results were compared using a Mann-Whitney’s test. RESULTS: Although leak point pressure was similar in the group I and II (4.25 ±0.66 cmH2O vs. 4.90 ±0.89 cmH2O; n=5 and 5 ; P = 0.31), leak point pressure was significantly higher in the group III compared to group I (8.75 ±2.29 cmH2O ; n=6 ; P = 0.004) and group II (P = 0.004). Administration of 7-NI resulted in an increase of leak point pressure in group I (9.90 ±5.13 cmH2O vs. 4.25 ±0.66 cmH2O; n=5 ; P = 0.0317) and in group II (7.60 ±1.55 cmH2O vs. 4.90 ±0.89 cmH2O; n=5; P = 0.0317). On the other hand, leak point pressure was not statistically different in group III (10.67 ±6.12 cmH2O vs. 8.75 ±2.29 cmH2O; n = 6; P = 0.70). CONCLUSIONS: nNOS mediated urethral smooth muscle relaxation is abolished in presence of high and prolonged doses of estradiol. The tonic action of nNOS on urethral smooth muscle relaxation is observed even in the absence of estrogen.
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GLYCOSAMINOGLYCANS’ BEHAVIOUR FOLLOWING PROTAMINE INDUCED CYSTITIS IN RATS
EXCRETION OF URINARY GLYCOSAMINOGLYCANS DURING NORMAL PREGNANCY AND PUERPERIUM IN YOUNG WOMEN
Soler R.1, Bruschini H.1, Truzzi J.C.1, Alves M.T.2, Leite K.R.2, Camara N.O.3, Mendes A.4, Martins J.R.4, Pimentel L.G.1, Nader H.4, Srougi M.1, Ortiz V.1
Maroclo M., Cabral C., Pereira S., Sampaio F., Cardoso L.
1
Federal University of Sao Paulo, Urology, Sao Paulo, Brazil, 2Federal University of Sao Paulo, Pathology, Sao Paulo, Brazil, 3Federal University of Sao Paulo, Nephrology, Sao Paulo, Brazil, 4Federal University of Sao Paulo, Molecular Biology, Sao Paulo, Brazil
State University of Rio de Janeiro, Department of Anatomy, Rio de Janeiro, Brazil
INTRODUCTION & OBJECTIVES: Impaired barrier function of bladder epithelium and subsequent infiltration of urine contents are the supposed initial events in the pathophysiology of interstitial cystitis (IC). Altered urinary GAG levels have been suggested as markers for IC, such as hyaluronic acid (HA) and sulfated GAG (S-GAG) (related to severe disease) and GAG replacement therapy is one of the main therapeutic approaches for IC. We evaluated urinary GAG behaviour and bladder inflammation after intravesical protamine sulfate (PS) injury, simulating IC.
INTRODUCTION & OBJECTIVES: Urinary glycosaminoglycans (GAG) are known to protect the urothelium against infections and stone formation. In addition, we have shown recently that urinary GAG excretion may be modulated by sexual hormones in non-pregnant young women (Maroclo MVO, Pereira SD, Sampaio FJB, Cardoso LEM. J Urol 2005;173:1789-1792). Here we investigated whether normal pregnancy affects urinary GAG excretion.
MATERIAL & METHODS: Cystitis was induced in adult female Wistar rats (n=7 per day) by bladder instillation of 200 μL of PS (10 mg) for 30 minutes. Controls (n=5 per day) were instilled with 200 μL of saline. Urine was collected during 24 hours in a metabolic cage. The rats (n=108) were sacrificed at days 1 to 7, 10 and 14 after the PS instillation and their bladders were removed for morphometric analysis of inflammatory cells. S-GAG and HA were measured in all groups and in non-manipulated rats (day 0) by a noncompetitive fluorescence-based assay and by a micro electrophoresis technique, respectively, and normalized to creatinine. Additionally, [35S]-inorganic sulfate was intraperitoneally injected in rats (cystitis and control) on day 0, 1, 5 and 7 to assess S-GAG turn over. RESULTS: Compared to controls, neutrophils were increased on the first 2 days and lymphocytes on the 5th to 7th days (p<0.05). Both urinary HA and S-GAG had increased levels on the first days and after the 5th day, with a maximum level on 7th day (graph 1). Urine analysis after [35S] instillation revealed a higher concentration of sulfate labelled compounds on 5th and especially on 7th day, compared to day 0 and their respective controls, suggesting an increased GAG turn over at this period (fig. 1).
MATERIAL & METHODS: Urine specimens were obtained during the first (up to 20 weeks gestation) and second (> 20 weeks) halves of normal pregnancy from 9 women aged 17 to 30 years (mean ± SD, 23.2 ± 4.5 years). One additional specimen was collected from each women in the puerperium (45 days postpartum). Total urinary GAG was purified by precipitations with cetylpyridinium chloride and ethanol and its concentration was expressed as μg hexuronic acid per mg urinary creatinine. The relative concentration of GAG species was determined by agarose gel electrophoresis. Statistical comparisons were done using paired t-test. RESULTS: Urinary GAG excretion was 33% higher in the second half of gestation as compared to the first one (1.83±0.65 vs. 1.39±0.3, p<0.01). Linear regression between values of the two halves for all patients showed a significant correlation (r=0.851, p<0.004), which implies that figures were consistently higher in the second half independent of individual variations in baseline GAG excretion. In the puerperium excretion values decreased markedly (0.62±0.10, p<0.001) in relation to the second half and were similar to those of non-pregnant young women.
CONCLUSIONS: There were 2 peaks of urinary HA and S-GAG excretion after intravesical PS instillation. The first peak probably correlates to inactivation of native bladder GAG, increased epithelial permeability and desquamation. Based on the higher concentration of [35S] during the second raise, we may assume there was a GAG synthesis de novo, concomitant to the occurrence of lymphocyte inflammatory infiltrate. This phenomenon could be a natural response to either the inflicted damage or the resultant inflammation in order to regenerate the bladder epithelial barrier. Increased levels of S-GAG in the urine of IC patients might represent this response. However, somehow, this regeneration process seems to be impaired in patients with IC.
CONCLUSIONS: Although pregnancy is accompanied by enhanced predisposing factors for stone formation, such as urinary stasis, dehydration, hypercalciuria, and hyperuricuria, the incidence of urolithiasis in pregnant women is similar to that of the non-pregnant. This fact may be explained by our results, since higher urinary GAG content is known to have an inhibitory effect on stone formation. Eur Urol Suppl 2006;5(2):41
74
SECOND MESSENGER PATHWAYS FOLLOWING DETRUSOR M3 RECEPTOR STIMULATION
ABOLITION BY ESTROGEN OF NEURONAL NITRIC OXIDE SYNTHASE SMOOTH MUSCLE RELAXATION IN THE FEMALE MICE URETHRA
Daly D.1, Chess-Williams R.1, Chapple C.2 1 University of Sheffield, Biomedical Science, Sheffield, United Kingdom, 2Royal Hallamshire Hospital, Urology, Sheffield, United Kingdom INTRODUCTION & OBJECTIVES: Muscarinic receptors are the main targets for anticholinergic drugs used to treat detrusor overactivity; however little is known about the 2nd messenger pathways involved. Understanding these pathways may reveal novel targets for pharmological intervention. M3 receptor stimulation results in the production IP3 and DAG, which ultimately results in intracellular calcium release and an influx of extracellular calcium respectively. M3 receptors can also increase DAG via phospholipase D. We previously reported that the α1-adrenoreceptor exhibits agonist specificity. The aim of this study was to identify the signal transduction mechanism involved in the carbachol or acetylcholine (Ach) induced contraction of the porcine detrusor smooth muscle. MATERIAL & METHODS: Strips of female pig detrusor muscle were set up under 1g tension in gassed Krebs-bicarbonate solution at 37°C. Concentration-response curves were obtained to carbachol or Ach in the absence and presence of drugs that interfere with the second messenger pathways or calcium movements. Results are expressed as the mean ± SEM for pEC50 values and maximum responses for carbachol. Paired Student’s t-test was used for statistical comparisons. RESULTS: Carbachol produced concentration–dependant contractions of the detrusor smooth muscle with a mean pEC50 of 5.51±0.11 and a maximum contraction of 9.86g±2.09g (n=13). The presence of ryanodine (30mM), which inhibits intracellular calcium release, had no significant effect on carbachol-induced maximal contractions or pEC50.The IP3 receptor antagonist 2-aminoethoxydiphenylborane (2APB, 30μM) also had no significant effect on maximal contractions or pEC50 . In contrast, the L- type calcium channel blocker nifedipine (1μM), significantly reduced responses to carbachol by 85.9±4.5% (P=0.003, n=11). Incubation with low calcium Krebs(0.95mM n=8) or calcium-free Krebs (nominal n=8) caused significant falls in maximum contraction (39.7±9.6%, P=0.006 and 98.1±0.8%, P=0.0001 respectively). The phospholipase C inhibitor U71322 (30uM), had no significant effect on carbachol induced contractions, but the phospholipase D inhibitor butan-1-ol (n= 8) significantly reduced maximal responses to carbachol by 73.7±4.6% (p=0.001). When using Ach (in the presence of physostigmine, 100nM), nifedipine and 2ABP had similar effects as with carbachol, nifedipine significantly inhibiting responses by 83.0%±2.8 (control pEC50 = 5.0±0.1; nifedipine pEC50=4.3±0.2) and 2ABP having no effect. CONCLUSIONS: The results suggest that pig detrusor contraction to carbachol is mediated with little contribution from intracelluar calcium stores. A pathway may be involved whereby M3 stimulation activates PLD resulting in DAG formation and contraction via extracellular calcium influx. No differences were observed between carbachol and Ach in these experiments.
Gamé X.1, Praddaude F.2, Arnal J.L.3, Escourrou G.4, Tack I.2, Allard J.2, Rischmann P.1, Ader J.L.2, Sarramon J.P.1, Malavaud B.1 1 Chu Rangueil, Service d’Urologie, Toulouse Cedex 4, France, 2Chu Rangueil, Laboratoire de Physiologie Rénale, Ufr Médecine, Toulouse, France, 3Chu Rangueil, Inserm U397 et Laboratoire de Physiologie, Toulouse, France, 4Chu Rangueil, Laboratoire d’Anatomie Pathologique, Toulouse, France
INTRODUCTION & OBJECTIVES: To assess the implication of estrogen and neuronal Nitric Oxide synthase (nNOS) in urethral relaxation. MATERIAL & METHODS: 4 weeks-old female mice C57/BL6, were split into 3 groups : ovariectomized (group I), Sham-operated (group II), ovariectomized receiving 17b-estradiol (E2) pellet releasing 80 μg/kg/day of E2 for 60 days (group III). Six weeks later, the animals were treated either by a competitive inhibitor of nNOS : 7-Nitroindazole (7NI, 50 mg/kg IP) or by the vehicle. Under anesthesia (ketamine + xylazine), a 22-gauge angiocatheter was inserted percutaneous into the bladder. The bladder was distended via the suprapubic catheter by saline. Pressures were measured (Gould TA400 Pression Transducer) and the leak point pressure defined by oozing of fluid at the meatus was determined. Results were compared using a Mann-Whitney’s test. RESULTS: Although leak point pressure was similar in the group I and II (4.25 ±0.66 cmH2O vs. 4.90 ±0.89 cmH2O; n=5 and 5 ; P = 0.31), leak point pressure was significantly higher in the group III compared to group I (8.75 ±2.29 cmH2O ; n=6 ; P = 0.004) and group II (P = 0.004). Administration of 7-NI resulted in an increase of leak point pressure in group I (9.90 ±5.13 cmH2O vs. 4.25 ±0.66 cmH2O; n=5 ; P = 0.0317) and in group II (7.60 ±1.55 cmH2O vs. 4.90 ±0.89 cmH2O; n=5; P = 0.0317). On the other hand, leak point pressure was not statistically different in group III (10.67 ±6.12 cmH2O vs. 8.75 ±2.29 cmH2O; n = 6; P = 0.70). CONCLUSIONS: nNOS mediated urethral smooth muscle relaxation is abolished in presence of high and prolonged doses of estradiol. The tonic action of nNOS on urethral smooth muscle relaxation is observed even in the absence of estrogen.
75
76
GLYCOSAMINOGLYCANS’ BEHAVIOUR FOLLOWING PROTAMINE INDUCED CYSTITIS IN RATS
EXCRETION OF URINARY GLYCOSAMINOGLYCANS DURING NORMAL PREGNANCY AND PUERPERIUM IN YOUNG WOMEN
Soler R.1, Bruschini H.1, Truzzi J.C.1, Alves M.T.2, Leite K.R.2, Camara N.O.3, Mendes A.4, Martins J.R.4, Pimentel L.G.1, Nader H.4, Srougi M.1, Ortiz V.1
Maroclo M., Cabral C., Pereira S., Sampaio F., Cardoso L.
1
Federal University of Sao Paulo, Urology, Sao Paulo, Brazil, 2Federal University of Sao Paulo, Pathology, Sao Paulo, Brazil, 3Federal University of Sao Paulo, Nephrology, Sao Paulo, Brazil, 4Federal University of Sao Paulo, Molecular Biology, Sao Paulo, Brazil
State University of Rio de Janeiro, Department of Anatomy, Rio de Janeiro, Brazil
INTRODUCTION & OBJECTIVES: Impaired barrier function of bladder epithelium and subsequent infiltration of urine contents are the supposed initial events in the pathophysiology of interstitial cystitis (IC). Altered urinary GAG levels have been suggested as markers for IC, such as hyaluronic acid (HA) and sulfated GAG (S-GAG) (related to severe disease) and GAG replacement therapy is one of the main therapeutic approaches for IC. We evaluated urinary GAG behaviour and bladder inflammation after intravesical protamine sulfate (PS) injury, simulating IC.
INTRODUCTION & OBJECTIVES: Urinary glycosaminoglycans (GAG) are known to protect the urothelium against infections and stone formation. In addition, we have shown recently that urinary GAG excretion may be modulated by sexual hormones in non-pregnant young women (Maroclo MVO, Pereira SD, Sampaio FJB, Cardoso LEM. J Urol 2005;173:1789-1792). Here we investigated whether normal pregnancy affects urinary GAG excretion.
MATERIAL & METHODS: Cystitis was induced in adult female Wistar rats (n=7 per day) by bladder instillation of 200 μL of PS (10 mg) for 30 minutes. Controls (n=5 per day) were instilled with 200 μL of saline. Urine was collected during 24 hours in a metabolic cage. The rats (n=108) were sacrificed at days 1 to 7, 10 and 14 after the PS instillation and their bladders were removed for morphometric analysis of inflammatory cells. S-GAG and HA were measured in all groups and in non-manipulated rats (day 0) by a noncompetitive fluorescence-based assay and by a micro electrophoresis technique, respectively, and normalized to creatinine. Additionally, [35S]-inorganic sulfate was intraperitoneally injected in rats (cystitis and control) on day 0, 1, 5 and 7 to assess S-GAG turn over. RESULTS: Compared to controls, neutrophils were increased on the first 2 days and lymphocytes on the 5th to 7th days (p<0.05). Both urinary HA and S-GAG had increased levels on the first days and after the 5th day, with a maximum level on 7th day (graph 1). Urine analysis after [35S] instillation revealed a higher concentration of sulfate labelled compounds on 5th and especially on 7th day, compared to day 0 and their respective controls, suggesting an increased GAG turn over at this period (fig. 1).
MATERIAL & METHODS: Urine specimens were obtained during the first (up to 20 weeks gestation) and second (> 20 weeks) halves of normal pregnancy from 9 women aged 17 to 30 years (mean ± SD, 23.2 ± 4.5 years). One additional specimen was collected from each women in the puerperium (45 days postpartum). Total urinary GAG was purified by precipitations with cetylpyridinium chloride and ethanol and its concentration was expressed as μg hexuronic acid per mg urinary creatinine. The relative concentration of GAG species was determined by agarose gel electrophoresis. Statistical comparisons were done using paired t-test. RESULTS: Urinary GAG excretion was 33% higher in the second half of gestation as compared to the first one (1.83±0.65 vs. 1.39±0.3, p<0.01). Linear regression between values of the two halves for all patients showed a significant correlation (r=0.851, p<0.004), which implies that figures were consistently higher in the second half independent of individual variations in baseline GAG excretion. In the puerperium excretion values decreased markedly (0.62±0.10, p<0.001) in relation to the second half and were similar to those of non-pregnant young women.
CONCLUSIONS: There were 2 peaks of urinary HA and S-GAG excretion after intravesical PS instillation. The first peak probably correlates to inactivation of native bladder GAG, increased epithelial permeability and desquamation. Based on the higher concentration of [35S] during the second raise, we may assume there was a GAG synthesis de novo, concomitant to the occurrence of lymphocyte inflammatory infiltrate. This phenomenon could be a natural response to either the inflicted damage or the resultant inflammation in order to regenerate the bladder epithelial barrier. Increased levels of S-GAG in the urine of IC patients might represent this response. However, somehow, this regeneration process seems to be impaired in patients with IC.
CONCLUSIONS: Although pregnancy is accompanied by enhanced predisposing factors for stone formation, such as urinary stasis, dehydration, hypercalciuria, and hyperuricuria, the incidence of urolithiasis in pregnant women is similar to that of the non-pregnant. This fact may be explained by our results, since higher urinary GAG content is known to have an inhibitory effect on stone formation. Eur Urol Suppl 2006;5(2):41