Influence of temperature on activity of the isolated whole bladder preparation of neonatal and adult rats

The temperature sensitivity of in vitro whole bladder preparations from neonatal and adult rats with or without chronic partial urethral obstruction was investigated. After the bladder was filled to a volume eliciting isovolumetric contractions, temperature was changed between 19 and 38 degrees C. In all preparations, higher temperatures were associated with higher frequencies of spontaneous intravesical pressure waves (IVPW). In 1- to 2-wk-old neonates, IVPW amplitude increased as the temperature increased; however, in older neonates and normal adults, the opposite occurred. The transition period was at 3 wk of age when bladder volume also markedly increased. At this age as well as in adult rats with outlet obstruction, changing temperature had little influence on the amplitude of IVPW. Thus obstructed outlet bladders and 3-wk-old bladders had similar properties. It is concluded that the properties of bladder muscle are changed during postnatal maturation and that in 3-wk-old rats, when brain control of voiding is emerging, micturition is abnormal, leading to obstructive changes in bladder muscle.     

The effect of partial outlet obstruction on prostaglandin generation in the rabbit urinary bladder

Partial outlet obstruction of the urinary bladder has been demonstrated to induce specific dysfunctions in cellular and sub-cellular membrane structures within the bladder's smooth muscle and mucosal compartments. Recent studies have linked these membrane dysfunctions to alterations in phospholipid metabolism leading to mobilization of free arachidonic acid, the precursor for synthesis of prostaglandins (PG). The purpose of this study was to determine if partial outlet obstruction of the urinary bladder induces changes in the capacity of bladder smooth muscle and mucosa to generate PG. PG were isolated from control and partially obstructed urinary bladder smooth muscle and mucosa of male New Zealand White (NZW) rabbits. PG concentrations (PGE2, PGF2alpha and PGI2, as its stable metabolite 6-keto-PGF1alpha) were determined after 30 minute incubations using enzyme-linked immunoassay (ELISA) kits. In both control and obstructed rabbit urinary bladders, PG generation was significantly higher in isolated mucosa than muscle tissues. A significantly higher concentration of PGF2alpha, and 6-keto-PGF1alpha was measured in obstructed muscle tissue relative to controls. The concentration of 6-keto-PGF1alpha was also significantly higher than the concentrations measured for PGE2 and PGF2alpha in both control and obstructed smooth muscle samples. The generation of PGE2 was significantly higher in rabbit urinary bladder mucosa than either PGF2alpha or 6-keto-PGF1alpha in both control and obstructed samples. The capacity of obstructed mucosal tissue to generate 6-keto-PGF1alpha was significantly higher than control tissue, while no significant differences in PGE or PGF2alpha generation were noted. These data suggest obstruction of the urinary bladder induce specific elevations in PG in both smooth muscle and mucosal tissues.     

Effect of partial and complete blockade of vanilloid (TRPV1-6) and ankyrin (TRPA1) transient receptor potential ion channels on urinary bladder motor activity in an experimental hyperosmolar overactive bladder rat model

The study investigated the mechanisms through which the hyperosmolarity might induce detrusor overactivity (DO). We compared the bladder activity in response to partial and complete blockade of TRPV1-6 and TRPA1 receptors. Experiments were performed on 42 rats. DO was induced by using hyperosmolar saline. All animals were randomly divided into six groups. The measurements represent the average of five bladder micturition cycles. Hyperosmolar saline induced DO. The complete blockade of TRPV1-6 and TRPA1 prevented DO. The partial blockade of TRPV1 didn't prevented DO. In the voiding phase periodical bladder contractions complexes occurred leading to slow urine flow due to bladder distension. Ruthenium red and capsaicin resulted in complete disorganisation of detrusor muscle contractility impairing urine voiding and leading to constantly lasting urine retention in healthy rats. Conclusions: hyperosmolar-induced DO is mediated by TRPV and TRPA1 channels; the hyperosmolar stimuli of urinary bladder might be transmitted mostly via ruthenium red sensitivity pathway.     

Partial urethral obstruction enhances NADPH-diaphorase activity in the monkey (Macaca fascicularis) bladder: light and electron microscopic studies

The effect of partially obstructing the urethra on the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in neurons of the intramural ganglia of the monkey (Macaca fascicularis) bladder was examined by light and electron microscopy. Partial urethral ligation was done in adult male monkeys. The animals were sacrificed 2, 4 weeks after partial urethral obstruction. This was compared to controls (normal and sham operated). Urethral obstructed animals were observed to have increased urinary frequency and decreased urinary flow rate. Two weeks after urethral obstruction, the overall NADPH-d activity in the intramural ganglia of the bladder base was enhanced compared to control animals. The frequency of intensely stained NADPH-d positive neurons was increased compared to the control animals. About one-third of intensely stained NADPH-d positive neurons appeared to undergo degenerative changes. At 4 weeks after urethral obstruction, a wide occurrence of NADPH-d positive neurons in advanced stages of degeneration in the bladder base was observed. Cellular debris was strewn among normal looking ganglion cells and along the nerve processes. The proportion of intensely stained NADPH-d positive neurons was relatively lower than the controls. The total number of NADPH-d positive neurons and the nerve fibres in the entire bladder was significantly reduced when compared to control animals. Electron microscopy showed some NADPH-d activity in intramural ganglion cells in 2 weeks after partial urethral obstruction. NADPH-d reaction product (formazan) was deposited on the membranes of the rough endoplasmic reticulum, and the outer membranes of some mitochondria in the intramural neuron. At 4 weeks after urethral obstruction, NADPH-d was present in the membrane of the mitochondria and some mitochondria appeared swollen with disrupted cristae. Present results show that NADPH-d activity in neurons of the intramural ganglia of the monkey (Macaca fascicularis) urinary bladder was increased after two weeks and reduced after 4 weeks of partial urethral obstruction. It is speculated that the increased NADPH-d activity associated with partial urethral obstruction would lead to neuronal damage and death, which may contribute to detrusor overactivity. However, it warrants further investigation to understand the mechanism of neuronal cell death after partial urethral obstruction.     

Altered neurogenic and mechanical responses to acetylcholine, ATP and substance P in detrusor from rat with outlet obstruction

The well-known side effects of anticholinergic compounds used to treat urinary incontinence caused by detrusor overactivity have addressed the interest on other pharmacological intervention. The purpose of the present work was to investigate the possible changes in purinergic and cholinergic components of parasympathetic neurotransmission in obstructed rat bladders with detrusor overactivity, and to examine the effect of the association of suramin, atropine and indomethacin on nerve-mediated responses to electrical field stimulation (EFS). Mechanical responses to exogenous acetylcholine, ATP and substance P were also evaluated. Altered sensitivities to acetylcholine and to the sensory neurotransmitter substance P, but unchanged sensitivity to the stable ATP analogue alpha,beta-methyleneATP were observed in bladders from obstructed rats. Suramin and atropine inhibited purinergic and cholinergic components of the neurogenic responses evoked by EFS in detrusor strips from control and obstructed rats. Interestingly, suramin enhanced the antagonistic effect of atropine on neurogenic responses of detrusor strips at all frequencies of stimulation tested. Our results suggest that the association between an antimuscarinic drug and an antagonist of P2X purinoceptors such as suramin might be helpful to reduce the therapeutic dosage of the antimuscarinic drug, along with its side effects. This approach may be of interest in the therapy of patients with bladder incontinence caused by detrusor overactivity, which do not even respond to a maximal dosage of antimuscarinic drug.     

Vascular response of the rabbit bladder to chronic partial outlet obstruction

Partial outlet obstruction of the rabbit urinary bladder results, initially, in a rapid increase in bladder mass and remodeling of the bladder wall. Previously, it was shown that this response was characterized by serosal growth (thickening) which was apparent after 1 day of obstruction, before any visible vascularization was observed. After 1 week of obstruction, significant microvessel formation was seen in the transition region between the detrusor smooth muscle and the thickening serosa; after 2 weeks the entire serosa was vascularized.In this study we investigated the effect of chronic (4 week) partial outlet obstruction on microvessel density and distribution in the bladder wall immunohistochemically using CD31 as a marker for vascular endothelium. Transverse sections of bladder wall were examined after 4 weeks of no surgery, sham surgery or partial obstruction.The microvessel density of the obstructed rabbit bladder mucosa and detrusor smooth muscle increased relative to augmentation of these compartments while new vessels appeared in the thickening serosa. Although vessel density did not change with obstruction a significant shift in mean vessel circumference to the left occurred indicating a significant increase in the number of microvessels and small vessels consistent with angiogenesis.     

Functional and morphological modifications of the urinary bladder in aging female rats

In female Wistar/Rij rats, 10 and 30 mo old, the micturition profiles in conscious animals, the contractile responses of the isolated urinary bladder, and the histology of the vesical tissue have been investigated. During cystomanometry, 60% of conscious senescent rats, but only 25% of young adult rats, showed spontaneous contractions during the bladder-filling phase. In aging rats, micturition pressure and duration of micturition were significantly higher by approximately 40-50%. In contrast, bladder capacity, bladder compliance, micturition volume, and residual volume were not modified with age. In vitro, the contractile responses of the bladder body to KCl, carbachol, arecoline, and alpha,beta-MeATP were similar in tissues from young adult and senescent rats. In contrast, maximum responses to noradrenaline, but not phenylephrine, were two times greater in the older rats. Isoprenaline exhibited the same potency in relaxing KCl-precontracted bladder body of 10- and 30-mo-old animals. Morphometric analysis showed a significant increase in the mean thickness of the muscularis layer with age, whereas the collagen density significantly decreased in the muscularis and in the lamina propria layers. The fact that the majority of senescent rats displayed bladder instability and increased response to alpha-adrenergic agonists suggests that this strain of rat seems a good model for the aged human. However, other characteristics of the aging human urinary tract (urinary frequency, decreased cystometric capacity, and decreased detrusor contractility associated with fibrosis) are not present.     

Unmodified calcium concentrations in tumour necrosis factor receptor subtype-mediated apoptotic cell death

Partial bladder outlet obstruction of the rabbit bladder results in a rapid increase in mass characterized by remodeling of the bladder wall.In this study we investigated the effect of partial outlet obstruction on microvessel density and distribution in the bladder wall immunohistochemically using CD31 as a marker for vascular endothelium, and on blood flow using a fluorescent microsphere technique. Transverse sections of bladder wall were examined after 0 (unobstructed), 1, 3, 5, 7, and 14 days of obstruction. The microvasculature of obstructed rabbit bladder mucosa and detrusor smooth muscle apparently increased relative to augmentation of these compartments, while new vessels appeared in the thickening serosa. These vascular changes correlated with results showing that, at 1 week after obstruction, blood flow (ml/min/g tissue) to the mucosa and detrusor was unchanged.Thickening of the serosa, apparent after 1 day of obstruction, began before its vascularization. Then, 1 week post-obstruction, there was significant microvessel formation in the transition region between the detrusor smooth muscle and the increasing serosa; after 2 weeks, the entire serosa was vascularized. The vascularization of the muscle-serosal transition region and then the remaining serosa apparently precedes fibroblast differentiation, providing blood supply and thus metabolic support for this process.All obstructed rabbit bladders in this study were in a state of compensated function based on their weights. Our working hypothesis is that blood flow per unit tissue mass is normal in compensated obstructed bladders, thus allowing for normal contractile function and cellular metabolism. The results of this study indicate the presence of an augmented microvasculature in compensated obstructed rabbit bladders that provides adequate blood perfusion for normal function.     

Age-related changes in urethrovesical coordination in male rats: relationship with bladder instability?

The micturition profile in conscious animals and the urethrovesical coordination in anesthetized conditions were investigated in 6- and 24-mo-old male Sprague-Dawley rats. The in vitro pharmacological responses to KCl, electrical field stimulation (EFS), carbachol, phenylephrine, and isoprenaline were determined in the isolated bladder body, the bladder neck, and urethra. A morphometric and immunohistological study has been included. During conscious cystomanometry, 63% of the aging rats but only 25% of the adult rats showed spontaneous contractions during the bladder-filling phase. In conscious aging rats, basal pressure, threshold pressure, and micturition pressure were also significantly increased. In anesthetized aging rats, a decrease in resting urethral pressure at micturition threshold and the occurrence of a significant delay in urethral relaxation during micturition were associated with an increased residual volume. In all isolated tissues, contractile response to KCl was not modified with aging, whereas age-related decreases in maximal responses to carbachol in the bladder body and to phenylephrine and carbachol in the urethra were observed. In the bladder neck only, we found a significant decrease in the amplitude of neurogenic contractions associated with fibrosis but without decrease in nerve density. These experiments show significant modifications in the voiding pattern of aging rats associated with urethral dysfunction and with regionally specific pharmacological and structural changes of the urinary tract. We propose that aging in rats is characterized by an impairment of the urethrovesical coordination, leading to bladder dysfunctions similar to those induced by bladder outlet obstruction.     

Effect of outlet obstruction on pyruvate metabolism of the rabbit urinary bladder

Bladder function is dependent upon cellular metabolism of substrates and the adequate generation of high-energy phosphate compounds. Partial outlet obstruction induces a marked decrease in bladder function which is associated with a significant decrease in the oxidative metabolism of glucose.The current investigation was designed to determine whether the time course of the decrease in mitochondrial oxidation in the hypertrophied urinary bladder is similar to the time course of the contractile dysfunction observed. In these studies we determined: 1) the rate of ¹⁴C-pyruvate metabolism to ¹⁴CO₂ in control and obstructed tissue (1, 3, 5 and 7 days), and 2) the mitochondrial enzymatic activities of malate dehydrogenase and citrate synthase.The results can be summarized as follows: 1) The rate of pyruvate metabolism decreases by over 50% within one day following partial outlet obstruction, and remains at this level for the seven day period of study. 2) Kinetic analysis demonstrates that the change in enzymatic activity is related to a decrease in Vmax; the K_d for pyruvate is similar for control and after all time periods of obstruction. 3) The enzymatic activity of malate dehydrogenase and citrate synthase is reduced by over 50% within one day following partial obstruction, and remains at this level throughout the 7 day study period. These metabolic results correlate in time and duration with the decreased ability of the bladder to empty following partial outlet obstruction.     

Expression of stress proteins (HSP-70 and HSP-90) in the rabbit urinary bladder subjected to partial outlet obstruction

Partial obstruction of the rabbit bladder outlet induces a rapid hypertrophy characterized by increased bladder mass, increased smooth muscle content, and increased collagen deposition. In addition, partial outlet obstruction induces decreased contractile responses to both field stimulation and postsynaptic receptor stimulation. Although the morphological and contractile responses to partial outlet obstruction have been well characterized, there is little information on the cellular and molecular mechanisms of these changes. In a previous study, we demonstrated that one of the earliest genes to be expressed following partial outlet obstruction in rabbits was the gene expressing stress protein-70 (HSP-70). In order to further define the genetic and molecular basis of these responses, the expression of stress gene products HSP-70 and HSP-90 in rabbit urinary bladder subjected to partial outlet obstruction has been quantitatively evaluated by Western blot coupled with laser densitometry using anti-HSP-70 and-90 monoclonal antibodies. The data show that stress gene products HSP-70 and HSP-90 are constitutively expressed in control rabbit bladder tissue and transiently increased following partial outlet obstruction. Increased content of HSP-70 was detected at 6 hr after obstruction and reached a maximum (2.7-fold over the control level) at 24 hr. Increased HSP-90 was also detected at 6 hr but reached a maximum (4.5-fold over the control level) at 12 hr. By 7 day post-obstruction, the content of these two proteins returned to the control levels. This study suggests that alterations of stress gene expression resulting in increased HSP-70 and 90 may play an important role in the response of the bladder to partial outlet obstruction.     

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder

Purpose: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). Materials and methods: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. Results: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. Conclusions: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction. (Mol Cell Biochem 276: 143–148, 2005)     

Comparative evaluation of antioxidant reactivity within obstructed and control rabbit urinary bladder tissue using FRAP and CUPRAC assays

Partial urinary bladder outlet obstruction mediates cyclic ischemia and reperfusion resulting in the generation of both reactive oxygen species and reactive nitrogen species. It is theorized that with an increase in the level of free radicals, the level of protective antioxidants should decrease. To test this hypothesis, two electron transfer assays, the FRAP method and the CUPRAC method, were used to determine the level of antioxidant reactivity of obstructed and control bladder tissue. The results showed that the CUPRAC assay detected a significant decrease in the reactivity of antioxidants found within the obstructed bladder tissue as compared to the control bladder tissue in both the muscle and mucosa. The FRAP assay did not detect any difference between the muscle and mucosa of the obstructed and control bladder tissue.     

K-ATP opener-mediated attenuation of spontaneous bladder contractions in ligature-intact,partial bladder outlet obstructed rats

Symptoms of urinary frequency and urgency secondary to benign prostatic obstruction are common in elderly men. In many patients, these symptoms correspond to the urodynamic finding of involuntary detrusor contractions during filling cystometry (i.e., detrusor instability). Spontaneous non-voiding contractions during filling can be modeled in animals by subchronic, partial urethral obstruction. However, many investigators remove the obstructive ligature a few days prior to cystometrical evaluation (which may not be an ideal representation of the clinical situation where obstruction is still present), and all perform cystometry within 3 days post-bladder catheterization surgery (i.e., while considerable wound healing is present). In the current study, we evaluated the effects, after oral dosing, of three structurally diverse ATP-sensitive potassium channel openers (KCOs) on spontaneous contractions secondary to obstruction in rats with an intact obstructive ligature at the time of testing and 2 weeks post-bladder catheterization. ZD6169, WAY-133537 and a novel dihydropyridine KCO, A-278637, all significantly decreased spontaneous bladder contractions at 30 min post-dosing (p.o.). However, only ZD6169 (10 micromol/kg) and A-278637 (3 micromol/kg) attenuated such bladder contractions at doses that did not concurrently, significantly affect mean arterial blood pressure and heart rate. These data confirm the efficacy of KCOs to inhibit unstable contractions in obstructed rats, and they further demonstrate the positive effect of a novel, bladder-selective KCO, A-278637, in an animal model with potentially less artifact than in previous such models.     

Persistent detrusor overactivity in rats after relief of partial urethral obstruction

Detrusor overactivity (DO) persists after prostatectomy in 20% to 25% of patients with benign disease. Assuming that nonvoiding contractions (NVCs) can be used as a surrogate for DO in humans, the rat model of obstruction/deobstruction may allow us to study the pathophysiology of persistent DO after deobstruction. We investigated bladder function, with a special focus on NVCs, in rats by use of a new, modified method of obstruction and deobstruction and compared these results with those obtained by use of the conventional method. Seventy female Sprague-Dawley rats underwent 1) sham operation (n = 10), 2) obstruction by a modified method (Modif-Obs; n = 12), 3) obstruction/deobstruction by the conventional method (Conv-Obs/Deobs; n = 13), or 4) obstruction/deobstruction by the modified method (Modif-Obs/Deobs; n = 35). The Modif-Obs/Deobs animals were divided into subgroups with (DO+) and without (DO-) NVCs. Two weeks after partial urethral obstruction, the animals were deobstructed, and 1 wk later cystometry was performed with recording of intravesical and intra-abdominal pressures. NVCs were shown in all groups: Modif-Obs (80%), Conv-Obs/Deobs (100%), and Modif-Obs/Deobs (40%). In the Modif-Obs/Deobs group, bladder weight and the muscle-to-collagen ratio were higher in DO+ than in DO- rats. The Modif-Obs/Deobs group showed no mortality compared with 25% mortality in the Conv-Obs/Deobs group. The modified method may be more adequate for studying persistent DO after deobstruction, because it resulted in pressure/volume- and DO-related parameters similar to those found in the clinical situation. The persistence of DO after deobstruction may partly be due to irreversible changes in the bladder caused during the period of obstruction.     

Effect of aging on the response of biochemical markers in the rabbit subjected to short-term partial bladder obstruction

Purpose Partial bladder outlet obstruction (PBOO) results in marked biochemical alterations in the bladder. In this study, we focused on comparison of thapsigargin sensitive sarco/endoplasmic reticulum Ca²⁺ ATPase activity (SERCA) and Citrate Synthase after short term PBOO in young versus old rabbits. Materials and methods A total of 20 young and 20 mature male rabbits were divided into 4 sub-groups of 5 rabbits each (4 obstructed and 1 sham-control rabbit). The rabbits in the groups were evaluated after 1, 3, 7, and 14 days of obstruction, respectively. The activities of SERCA and citrate synthase were examined as markers for sarcoplasmic reticular calcium storage and release and mitochondrial function, respectively. Results The SERCA activity of bladder body smooth muscle in the young animals increased at 7 and 14 days. For the old rabbits, the SERCA activity decreased significantly by 1 day and remained this level throughout the course of obstruction, and was significantly lower than young at all time periods. The citrate synthase activity in the young animals decreased over the 1–7 days, and then returned toward control level by 14 days following obstruction. In the old animals, citrate synthase activity of bladder body smooth muscle progressively decreased over the course of the study, and was significantly lower in the old than the young animals after 14 days obstructed. Conclusion The urinary bladders of the young rabbits have a considerable greater ability to adapt to PBOO than do those of the old rabbits. The deterioration of mitochondrial and SR function may be important mechanisms underlying geriatric voiding dysfunction.     

Creatine kinase activity in normal and hypertrophied rabbit urinary bladder tissue (following partial outlet obstruction)

The urinary bladder depends on intracellular ATP to support a number of essential intracellular processes including contraction. The concentration of ATP is maintained by mitochondrial oxidative phosphorylation, cytosolic glycolysis and the cytosolic activity of creatine kinase, the enzyme that catalysis the rapid transfer of a phosphate from creatine phosphate (CP) to ADP resulting in the formation of ATP.Prior studies in this lab and others have demonstrated that mitochondrial respiration is significantly lower in hypertrophied bladder tissue (induced by partial outlet obstruction of the white New Zealand Rabbit). In addition to decreased mitochondrial respiration, there are significant increases in glycolysis and lactic acid formation in the hypertrophied tissue.In view of the increased glycolysis and decreased mitochondrial function in the hypertrophied tissue, and the importance in creatine kinase in maintaining cytosolic levels of ATP, the current study was designed to determine if outlet obstruction induces any changes in the activity of creatine kinase.The following is a summary of the results: 1) The bladder mass increased from 2.2 ± 0.2 gm to 11.5 ±1.6 gm at 7 days following outlet obstruction. 2) The intracellular concentrations of both ATP and CP were significantly reduced in the bladder tissue following 7 days of obstruction. 3) The percent of protein (per tissue mass) was significantly lower in the obstructed bladders, although the percent of soluble protein was similar. 4) Creatine kinase activity of control bladders showed linear kinetics with a V_max = 1120 nmoles/mg protein/4 min and K_m = 147 µM CP. 2) The creatine kinase activity of obstructed bladders also displayed linear kinetics with a V_max = 1125 nmoles/mg protein/4 min tissue, and K_m = 276 µM CP.These studies demonstrate that whereas both control and obstructed bladders have virtually identical maximum creatine kinase activities, the K_m for the obstructed tissue is significantly higher than the K_m for the control tissue. This may indicate that under cellular conditions (at sub-maximum substrate concentrations), the creatine kinase activity of the obstructed bladders may be significantly lower than the activity of the control bladders. In addition, the reduced tissue concentrations of ATP and CP would certainly be consistent with the reduced functional response to bethanechol and field stimulation.