Oral Kohki Tea and its protective effect against in vitro ischemic damage to the bladder

AIMS: Results of several studies indicate that ischemia/reperfusion (I/R) is an etiological factor in the contractile dysfunctions induced by partial bladder outlet obstruction in animal models. In support of this hypothesis, pretreatment of rabbits with Kohki Tea (Engelhardtia chrysolepis), a Japanese herbal drink very high in antioxidant activity, significantly reduced the contractile dysfunctions induced by partial outlet obstruction. The current study was designed to determine if pretreating rabbits with Kohki Tea could protect the bladder against the contractile damage induced by in vitro ischemia followed by re-oxygenation. METHODS: Forty-eight New Zealand White rabbits were separated into two groups of 24; Group 1 was pretreated by oral gavage for 3 weeks with Kohki Tea and Group 2 received vehicle (water). Each rabbit was anesthetized with pentobarbital. The urinary bladder was rapidly removed and eight longitudinal muscle strips were cut from the bladder body. Each strip was mounted in a separate 15-ml bath containing Tyrode's solution with glucose (1 mg/ml) and maintained at 37 degrees C. All strips were equilibrated for 30 min with a gas mixture of 95% O2 and 5% CO2. At the end of this period of time, all strips were stimulated with field stimulation (FS) carbachol and KCl. After the last wash, the aeration was changed to hypoxic mixture (nitrogen-CO2) without glucose. At the end of 2 hr, the aeration was changed back to the normal 95% O2 and 5% CO2, and glucose was added to the buffer. After 1 hr of re-oxygenation, a second set of stimulations was performed. In order to represent hyperreflexia, the strips were stimulated at 32-Hz FS at 5-min intervals during the hypoxic period in half of the in vitro experiments. RESULTS: The results showed that Kohki Tea pretreatment protected the bladder's response to FS from the detrimental effects of repetitive stimulation and the detrimental effects of both in vitro ischemia and repetitive stimulation on the contractile responses to carbachol and KCl. CONCLUSIONS: These data are consistent with the concept that Kohki Tea acts by protecting the bladder from cellular damage caused by hypoxia and the generation of free radicals.     

Normal detrusor is more sensitive than hypertrophied detrusor to in vitro ischemia followed by re-oxygenation

Partial outlet obstruction results in marked metabolic as well as contractile alterations. Specifically, the ratio of anaerobic to oxidative metabolism is significantly greater in hypertrophied than normal bladder smooth muscle, lactate dehydrogenase (LDH) and lactic acid production are increased, and the contractile apparatus is altered to allow for metabolically more efficient tension generation. In addition, contractile responses of hypertrophied bladder are apparently more resistant than those of normal bladder to hypoxia. In the current experiment, we studied the effects of in vitro ischemia (hypoxia + substrate deprivation) followed by an in vitro model of reperfusion (re-oxygenation + substrate replacement) on contractile responses of normal and hypertrophied urinary bladder strips. We used repetitive field stimulation (FS) during the hypoxic period as a model for hyperreflexia. The purpose of the current study was to compare the responses of normal and hypertrophied bladder smooth muscle to repetitive stimulation in the presence of in vitro ischemia followed by re-oxygenation and substrate replacement. Thirty-two rats were separated into four groups of eight each. The rats in groups 1 and 3 were subjected to partial outlet obstruction. Two weeks later, all rats were anesthetized; their bladders were isolated and cut into four strips. Each strip was mounted in an isolated bath, and after 1-hour incubation in Tyrode's solution containing glucose (in the presence of O(2)), contractile responses to FS, carbachol, and KCl were determined. After this first set of stimulations, the strips were incubated without glucose and in the presence of N(2) for 30 minutes and 1 hour (groups 1 and 2); and for 2 and 4 hours (groups 3 and 4). For groups 1 and 2, the tissues were stimulated at 5-minute intervals with FS at 32 Hz, 1-millisecond duration, 3-second trains (in vitro model of hyperreflexia). For groups 3 and 4, no stimulations were performed during the ischemic period. At the end of the ischemic period, all strips were washed and incubated for 1 hour in the presence of O(2) and with glucose. At the end of this incubation, all strips received a second set of stimulations. a) Partial outlet obstruction resulted in a significant increase in bladder weight. b) Responses to in vitro ischemia: After in vitro ischemia, contractile responses of both normal and hypertrophied tissues to FS were reduced to a significantly greater degree than were responses to carbachol and KCl. The rate of development of contractile dysfunction was significantly greater in normal bladder tissue strips than in hypertrophied bladder strips. c) Responses to repetitive stimulation: The rate of development of contractile dysfunction was significantly greater in all strips subjected to repetitive stimulation than in those not repetitively stimulated; in addition, normal bladder strips were more sensitive than hypertrophied strips to hypoxia and substrate deprivation-induced contractile dysfunction. The rate of contractile failure induced by in vitro ischemia followed by re-oxygenation and substrate replacement was significantly greater for normal bladder strips than for hypertrophied bladder strips. These results indicate that, after partial outlet obstruction, the hypertrophied tissue is more resistant than normal tissue to hypoxia and substrate deprivation.     

Reperfusion injury of the rat bladder is worse than ischemia

PURPOSE: Previous studies have demonstrated that in vivo and in vitro ischemia of the bladder results in decreased contractile responses. However, to our knowledge the effect of reperfusion following ischemia of the bladder is not known. MATERIALS AND METHODS: Adult male rats were subjected to bilateral bladder ischemia and varying periods of reperfusion. In vivo ischemia was created for 4 hours by reversibly clamping the 2 vesical arteries for 4 hours. Reperfusion was produced by removing the clamps and allowing the animals to recover for 1 day, 1 week or 1 month after surgery. Following recovery bladders strips were studied using field stimulation (FS), carbachol and KCl. The maximal contractile response and rate of response generated were recorded digitally and analyzed. RESULTS: The maximal responses to FS, carbachol and adenosine triphosphate (ATP) were not decreased by 4-hour ischemia alone, whereas the response to KCl was decreased significantly. The contractile responses to FS and KCl were significantly decreased after 1 day and 1 week of reperfusion. Responses after 1 month of reperfusion were increased significantly compared with responses after 1 week of reperfusion. The responses to ATP were not affected by ischemia or reperfusion. The contractile response to KCl was significantly more sensitive to ischemia than the responses to carbachol, ATP or FS, whereas the contractile response to FS was significantly more sensitive to reperfusion than the other forms of stimulation. CONCLUSIONS: This study demonstrates clearly that injury by reperfusion following ischemia is more detrimental than the effects of ischemia alone and FS contraction is the most sensitive form of stimulation to reperfusion damage. This study also demonstrates the ability of the bladder to recover from ischemic and reperfusion injuries.     

Correlation between the structure and function of the rabbit urinary bladder following partial outlet obstruction

PURPOSE: To understand the relationship between contractile and structural changes in the obstructed bladder, rabbit bladder was partially obstructed for up to 70 days and alterations in tension response to field stimulation and carbachol were compared with alterations in ultrastructure and innervation of detrusor smooth muscle (SM). The effect of partial outlet obstruction on the physiological responses to field stimulation (FS) (nerve mediated contraction) and carbachol (receptor mediated contraction) were correlated with the structure and innervation of the detrusor smooth muscle (SM) of the same animal during a 70 day period. MATERIALS AND METHODS: 28 rabbits were subjected to 1 to 70 days of mild partial outlet obstruction. Sham operated rabbits were euthanized at 7, 14, 28, and 70 days post-obstruction. At each time period, isolated strips of bladder body were mounted in individual baths and the contractile response to FS and carbachol determined. Three additional strips from each bladder were fixed for electron microscopy. RESULTS: Bladder mass increased rapidly during the first 7 days after obstruction, was constant for the next 7 days, and then continued to increase gradually. Dysfunction of the contractile response to FS was noted as early as 3 days and progressively increased over the 70-day study period. The decrease in the response to FS increased at a significantly faster rate than the decrease in the contractile response to carbachol. In ultrastructure studies, at 3 and 7 days post-obstruction the majority of SM cells displayed the characteristics of hypertrophy. At 28 days some SM cells displayed loosely packed myofilaments and an irregular distribution of sarcoplasmic dense bodies. At 70 days swollen mitochondria were present in all cell types of the bladder wall. Evidence of axonal degeneration was first observed at 7 days post-obstruction and became more extensive thereafter. No evidence of mitotic figures, nerve growth cones or regenerating SM cells was observed. CONCLUSIONS: Prolonged partial bladder outflow obstruction is accompanied by a progressive decrease in contractility of SM. The present study describes the structural damage that occurs in the bladder wall in response to partial outlet obstruction and correlates these observations with the contractile dysfunction with which it is associated. Furthermore, mitochondrial damage in vessels and fibroblasts is suggestive of bladder wall ischemia.     

Effects of dextromethorphan on in vitro contractile responses of mouse and rat urinary bladders

PURPOSE: Dextromethorphan (DXM) is a cough-suppressing ingredient in a variety of over-the-counter cough and cold medications. Dextromethorphan elevates the threshold for coughing primarily through a central mechanism. At doses recommended for treating coughs the drug is safe and effective. At much higher doses, DXM produces dissociative effects similar to those of phencyclidine and ketamine. Opioid analgesics structurally related to DXM also inhibit bladder contractions and produce urinary retention through a non-opioid mechanism. This study evaluated the direct effects of DXM on in vitro contractile responses of rat and mouse urinary bladders. METHODS: Male rats and mice were anaesthetized and their bladders removed. Bladder strips were suspended in 15 ml oxygenated Tyrode's solution containing glucose. Bladder strip contractions were evoked by field stimulation (FS), carbachol or elevated KCl concentrations and contractile responses recorded. The strips were then exposed to 3 microM (DXM) for 30 min and re-stimulated. This sequence was repeated at 10, 30, and 100 microM DXM. RESULTS: (a) The rat bladder generated significantly greater tension than the mouse bladder. (b) Dextromethorphan produced a dose-dependent inhibition of the response to FS that was approximately equal for rat and mouse bladders. FS at 8 or 32 Hz was significantly more sensitive to DXM inhibition than 2 Hz. (c) The response to carbachol was more sensitive to inhibition by DXM than the responses to FS or KCl. CONCLUSIONS: These results demonstrate that DXM inhibits bladder contractions in vitro and that mouse and rat bladders are affected to approximately the same extent.     

Effects of partial outlet obstruction on bladder-strip sensitivity to glucose deprivation: an in vitro study in the rat

Summary Partial outlet obstruction has been shown to result in contractile and metabolic dysfunctions. Specifically, there is a greater reduction in the response to field stimulation (FS) in comparison with the responses to bethanechol and KCl, a greater reduction in the tonic response to stimulation in comparison with the phasic response, and a reduction in oxidative metabolism of glucose accompanied by an increase in the glycolytic metabolism of glucose. The specific aim of the current study was to correlate the effects of partial outlet obstruction on the contractile responses of isolated strips of bladder smooth muscle to repetitive stimulation in the presence and absence of glucose. Adult male Sprague-Dawley rats were subjected to partial outlet obstruction by the surgical placement of silk ligatures around the urethra. After 2 weeks, each rate was anesthetized, the bladder was excised, and isolated strip studies were performed. These studies demonstrated that the maximal phasic response to FS was significantly decreased in the obstructed strips as compared with controls, with no difference being noted for responses to bethanechol or KCl; the tonic responses to all forms of stimulation were significantly decreased after obstruction, with the tonic response to FS being decreased to a greater degree than were the tonic responses to bethanechol and KCl; and in the absence of glucose, the tonic responses of control strips to all forms of stimulation were reduced to a greater degree than were the phasic responses. These studies demonstrate that the tonic response to FS is extremely sensitive to fatigue induced by repetitive stimulation.     

Effect of age on the response to in vitro ischemia and reperfusion of the rabbit bladder

BACKGROUND/AIMS: Urinary bladder dysfunction secondary to outlet obstruction is a common condition, seen in both children and adults. Using in vitro models of ischemia/reperfusion (I/R), we compared the contractile and biochemical responses of bladders isolated from young and old rabbits. MATERIALS AND METHODS: Fourteen male New Zealand White rabbits were separated into two groups of 7. Group 1 (young) included rabbits 6-7 weeks old and group 2 (old) consisted of rabbits 2 years old. Isolated bladder body strips were subjected to in vitro I/R, and the effects on contractility were determined. The strips were then frozen and stored for quantitative malondialdehyde (MDA) analysis. RESULTS: Contractile responses of young and old rabbit bladders to all forms of stimulation--field stimulation (FS), carbachol and potassium chloride (KCl)--were equal. The rate of tension to 32 Hz FS was significantly higher for group 1 in comparison with group 2. The old and young rabbits were equally sensitive to I/R in regard to FS, but the old rabbits were more sensitive to I/R in regard to carbachol and KCl stimulation. Basal MDA concentration of both young and aged bladder strips were similar. Ischemia mediated a significant increase in MDA in bladder strips from both young and old rabbits, but the MDA level was significantly greater for the young than for the old. CONCLUSION: Although the level of oxidative damage was greater in the young rabbit bladders, functionally, the old rabbit bladders were more sensitive to I/R damage.     

Protein kinase c regulates purinergic component of neurogenic contractions in mouse bladder

PURPOSE: We evaluate the role of protein kinase C in excitatory purinergic neurotransmission in the mouse bladder. MATERIALS AND METHODS: In isolated mouse detrusor strips contractile responses to electrical field stimulation were mostly mediated by neural released acetylcholine and adenosine triphosphate (ATP). The changes in neurotransmission were measured indirectly by recording the contraction of detrusor strips in response to repetitive electrical field stimulation by trains of electrical pulses at 8 Hz. 1 second in duration. RESULTS: A protein kinase C activator, 1 to 2.5 nM. (beta-phorbol-12,13-dibutyrate (beta-PDBu), but not the inactive form alpha-phorbol-12,13-dibutyrate, significantly enhanced neurogenic detrusor contractions. The purinergic component of the evoked detrusor contractions in the presence of atropine was specifically sensitive to this enhancing effect by beta-PDBu but the cholinergic component in the alpha,beta-methylene ATP treated detrusors remained unaffected. This enhancing effect of beta-PDBu was dependent on the extracellular calcium (Ca2+) concentration. A P and/or Q type Ca2+ channel blocker, 0.1 and 0.3 microM. omega-conotoxin-MVIIC, and protein kinase C inhibitors, 0.3 and 1 microM. staurosporine and 0.3 and 1 microM. bisindolylmaleimide I but not 0.1 and 0.3 microM. omega-conotoxin-GVIA, an N type Ca2+ channel blocker, abolished the effect of beta-PDBu. Moreover, beta-PDBu did not affect the muscle responses induced by the exogenous agonists carbachol or alpha,beta-methylene ATP and potassium chloride. CONCLUSIONS: These results suggest that the activation of Ca2+ channel, especially the P and/or Q type, may be involved in the enhancing effect of protein kinase C activator beta-PDBu on muscle contractions elicited by excitatory purinergic neurotransmission in the mouse detrusor strips.     

Effect of age on hydrogen peroxide mediated contraction damage in the male rat bladder

PURPOSE: Recent studies introduced the concept that reactive oxygen species (ROS) may be a major factor in the progressive deterioration of bladder function induced by benign prostatic hyperplasia in men and following partial outlet obstruction in animals. We determined whether bladder contraction sensitivity to ROS changes with aging. Using H2O2 to simulate ROS damage we compared the sensitivity of the contractile responses of bladder smooth muscle isolated from young and elderly rats to H2O2. MATERIALS AND METHODS: Male 3-month-old (young) and 12-month-old (elderly) Sprague-Dawley rats were used for this study with 24 per group. Each rat was anesthetized and the bladder was excised. Two longitudinal strips were cut from the bladder body. Each strip was placed in individual 15 ml baths containing oxygenated Tyrode's solution at 37C. Each strip was stimulated at 32 Hz for 20 seconds with pulses 1 millisecond in duration at 80 V. After electrical field stimulation (EFS) the response to 20 microM carbachol and 120 mM KCl was determined. The bath solution was then exchanged for H2O2 solutions of 6 different concentrations including 0%, 0.0625%, 0.125%, 0.25%, 0.5% and 1.0%. After 1 hour of exposure to H2O2 the tissues were washed free of H2O2, and the response to EFS, carbachol and KCl were measured again. At the end of the experiment the strips were weighed and frozen at -70C for malondialdehyde analysis. RESULTS: The magnitude of the contractile responses of the young and elderly rats to all forms of stimulation were equal. Hydrogen peroxide caused a dose dependent decrease in the contractile responses of bladder strips to all forms of stimulation. Contractile responses to carbachol and KCl were more sensitive to H2O2 than to EFS. Contractile responses of bladder strips isolated from elderly rats were significantly more sensitive to H2O2 damage than strips isolated from young rats. Malondialdehyde generation of bladder strips isolated from elderly rats was significantly greater than those from young rats. CONCLUSIONS: It is suggested that aging increases the sensitivity of detrusor contraction to oxidative damage.     

Inhibition of the contractile responses of isolated human and rat bladders by clenbuterol

PURPOSE: We investigated the inhibition of the contractile responses of human continent and unstable detrusor muscle by the beta2 agonist clenbuterol as well as the inhibition of electrical field stimulation evoked contractile responses of isolated rat bladder muscle strips by orally administered clenbuterol. MATERIALS AND METHODS: The contractile responses of human continent and unstable detrusor muscle strips to electrical field stimulation (0.05 milliseconds, 0.5 to 80 Hz.) were measured before and after adding 10(-9) to 10(-4) M. clenbuterol in vitro. In addition, 6 rats per group were dosed orally with 2 microg x kg(-1) clenbuterol daily acutely (1 dose) or chronically (1 dose daily for 8 days), or with distilled water to serve as controls. The contractile response to electrical field stimulation of strips of isolated detrusor muscle was then measured. Serum clenbuterol levels were analyzed in duplicate by enzyme-linked immunosorbent assay and high performance liquid chromatography. RESULTS: In vitro clenbuterol significantly inhibited the electrical field stimulation evoked contractile responses of detrusor muscle strips from unstable but not continent human bladders. A significant inhibitory effect of clenbuterol on the electrical field stimulation evoked contractile response of rat detrusor muscle was observed after chronic but not acute oral dosing (p <0.01). Serum clenbuterol levels measured by enzyme-linked immunosorbent assay and high performance liquid chromatography were not significantly different. CONCLUSIONS: Clenbuterol or related beta2-adrenoceptor agonists may represent a useful therapeutic strategy for detrusor muscle overactivity.     

Effect of partial bladder outlet obstruction on nitrotyrosine levels and their correlation with contractile function

AIMS: It has been demonstrated that partial bladder outlet obstruction (PBOO) causes free radical generation that, in turn, results in cellular and subcellular damage. We tested the hypothesis that nitration of proteins is associated with contractile dysfunctions in obstructive bladder disease. METHODS: Thirty rabbits were subjected to 1-28 days of partial outlet obstruction. Sham operated rabbits served as controls. Western blotting was used to determine the amount of nitrotyrosine level at the protein level. At each time point, isolated strips of bladder body were mounted in individual baths and the contractile response to field stimulation (FS), carbachol, and KCl determined. RESULTS: Bladder weight increased rapidly during the first 7 days and then increased slowly thereafter. There was a fourfold increase in the amount of nitrotyrosine in the 7 day obstructed groups when compared to sham controls and the levels remain elevated at 14 and 28 days of obstruction. Contractile dysfunction in response to FS (8 and 32 Hz) was noted as early as 1 day after obstruction and increased progressively over the study period. The decrease in response to carbachol and KCl was significant only after 3 days of obstruction and the progressive increase in dysfunction was slower than with FS. CONCLUSIONS: PBOO is accompanied by an increase in nitrotyrosine, a marker of free radical damage. Simultaneously there was a progressive decrease in contractility of detrusor smooth muscles (DSMs). Nitrotyrosine may be usable as a marker of free radical damage and reperfusion injury.     

Oxidative stress reduces the muscarinic receptor function in the urinary bladder

AIMS: Several pathophysiological conditions in the urinary bladder, for example, ischemia/reperfusion and inflammation are characterized by the formation of reactive oxygen species (ROS). The ROS are highly toxic because they can destroy proteins, DNA, and lipids. The aim of this study was to investigate the effect of oxidative stress on excitation-contraction coupling of detrusor smooth muscle. MATERIALS AND METHODS: Smooth muscle strips were dissected from pig urinary bladder and mounted in organ baths. Oxidative stress was mimicked by the addition of Cumene hydroperoxide (CHP), a lipophilic hydroperoxide, to the organ baths. Contractile responses to electrical field stimulation (EFS: 4-32 Hz), carbachol (10(-8)-3 x 10(-5) M), potassium (65.3 mM), and ATP (1 mM) were monitored before and after the addition of CHP. RESULTS: Responses of detrusor strips to EFS were for the greater part based on neurogenic stimulation and the release of acetylcholine. CHP diminished contractile responses to EFS and carbachol to the same extent. The pD(2) value of the carbachol concentration-response curve decreased significantly after exposure to 0.1 mM, 0.4 mM, 0.8 mM CHP. Furthermore the maximal effect obtained with carbachol was significantly reduced after 0.1 mM, 0.4 mM, and 0.8 mM CHP treatment. Contractions induced by potassium and ATP were significantly less affected by oxidative stress compared to EFS- and carbachol-induced responses of comparable amplitude. CONCLUSIONS: The results of our study demonstrate that oxidative stress induced by CHP affects pig bladder contractility. The muscarinic receptor signaling system is severely damaged. L-type calcium channels and the contractile system are less affected and cholinergic nerves remain largely unaffected.     

Comparative physiology and biochemistry of rat and rabbit urinary bladder

OBJECTIVE: To compare directly the biochemistry and contractile responses of rat and rabbit bladder to different stimuli. Materials and methods Sexually mature male New Zealand White rabbits and Sprague Dawley rats were compared. Each bladder was excised while the animal was anaesthetized; longitudinal bladder strips were cut and then mounted in an organ bath. Tension (2 g) was placed on all strips and each underwent field stimulation (FS) for a total of 20 s at 1-32 Hz, 1 ms and 80 V and was exposed to carbachol (100 micromol/L), ATP (2 mmol/L) and KCl (120 mmol/L). The tension was monitored continually using a polygraph and data stored digitally in a computer. The responses to each stimulus were determined as the maximum tension generated, maximum rate of tension generation and duration to a maximum response. The Ca2+- ATPase activity of the rat and rabbit bladder was determined. Bladder pressures were then predicted from the strip data using Laplace's law and compared with published values. RESULTS: Contractile responses (per unit tissue mass) of rat bladder strips were significantly greater than those of rabbit bladder strips at all frequencies of FS and to carbachol, KCl and ATP. The rate of contractile force generated by rat bladder strips in response to all stimuli were significantly greater than that generated by rabbit strips. Rabbit bladder strips took significantly longer to generate maximum tension than did rat bladder strips in response to pharmacological stimuli. In response to FS, rat strips took significantly longer than rabbit strips to generate maximum tension. Although the predicted rat bladder pressures were significantly greater than those for rabbit, the predicted pressures for both the rat and rabbit were significantly lower than the pressure responses of the isolated whole bladder model. The contractile data correlated well with the Ca2+-ATPase activity data; rat bladder had seven times the enzyme activity of rabbit bladder. CONCLUSION: Per unit mass, rat bladder is capable of generating more than five times the tension of rabbit bladder. Similarly, the rate of tension generation by rat bladder is three to five times greater than that by rabbit bladder. The duration to maximum tension generated in response to FS compared with pharmacological stimuli was affected by the inherent difference in the rate of contractile response to electrical activation compared with agents which diffuse through tissue, and by the difference in size between rat and rabbit bladder smooth muscle cells.     

Effects of cromakalim (BRL 34915) and pinacidil on normal and hypertrophied rat detrusor in vitro

Normal and hypertrophied rat detrusor were investigated in vitro with regard to effects of the K(+)-channel openers pinacidil and cromakalim. Both drugs abolished spontaneous contractile activity and induced a relaxation of normal and hypertrophied detrusor preparations. In both types of preparation, contractions elicited by K+, carbachol or electrical field stimulation were depressed in the presence of the K(+)-channel openers. Responses induced by K+ or electrical stimulation were more reduced in the hypertrophied than in the normal detrusor. Both K(+)-channel openers increased the efflux of 86Rb+ in a concentration-dependent way and this increase was similar in normal and hypertrophied detrusor. If applicable to man, this data suggest that K(+)-channel openers may be effective in the treatment of bladder instability secondary to outflow obstruction.     

Effect of bladder ischaemia/reperfusion on superoxide dismutase activity and contraction

OBJECTIVES: To correlate the effect of bilateral in-vivo bladder ischaemia/reperfusion on superoxide dismutase activity (SOD) and then to correlate this with contractile responses to various forms of stimulation. MATERIALS AND METHODS: Twenty mature male New Zealand White rabbits were divided into five equal groups: group 1 (controls); group 2, 2 h of in-vivo bilateral bladder ischaemia; and groups 3-5, 2 h of in-vivo ischaemia followed by 1, 7 or 14 days of reperfusion (recovery). At the end of the treatment period, bladder strips were incubated and placed in isolated baths for contractile studies. The contractile responses to field stimulation, carbachol (10 micromol/L), ATP and KCl were determined. The balance of the bladder body was separated into muscle and mucosa sections and analysed for SOD activity. RESULTS: There were few effects on contraction either directly after ischaemia or after 1 day of reperfusion. However, all contractile responses were significantly reduced at 7 and 14 days after ischaemia. SOD activity of the detrusor muscle was reduced significantly immediately after ischaemia and at 7 and 14 days of reperfusion. SOD activity of the mucosa was significantly greater than that of the muscle, and was significantly reduced by both ischaemia and all times of reperfusion. CONCLUSIONS: These studies show clearly that both ischaemia and reperfusion result in significantly lower activity of SOD, and in contractile dysfunctions, and that reperfusion results in greater decreases in both SOD activity and contractile responses than ischaemia alone.     

Alterations in purinergic and cholinergic components of contractile responses of isolated detrusor contraction in a rat model of partial bladder outlet obstruction

OBJECTIVE: To study the effect of 3 weeks of partial bladder outlet obstruction (BOO), compared to a sham operation, on the cholinergic and purinergic components of detrusor contractile responses to agonists and to electrical field stimulation (EFS); the expression of P2X receptor subtypes was also examined. MATERIALS AND METHODS: Partial BOO was induced in female Sprague-Dawley rats by surgically applying a jeweller's silver 'jump' ring around the urethra, such that the urethra was constricted but not closed. Sham-operated female rats underwent an identical procedure without placement of a ring. RESULTS: After 3 weeks of partial BOO the rat bladders became significantly hypertrophied, doubling in weight. Spontaneous activity was markedly increased, but the contractile response to a single bolus of KCl (120 mM) was unaltered. The neurogenic-induced contractile responses of strips of detrusor from obstructed bladders were significantly greater than those from sham-operated bladders, and the responses of strips of detrusor from obstructed bladders to EFS showed a significantly greater atropine-sensitive component than sham-operated detrusor. However, the response of detrusor strips to EFS that was susceptible to desensitization by alpha,beta-methylene ATP was not significantly changed in obstructed bladders. The sensitivity of the strips from obstructed bladders to carbachol, ATP and beta,gamma-methylene ATP was less than in sham-operated detrusor. Immunohistochemical studies showed no difference in the P2X receptor subtypes expressed on detrusor smooth muscle from obstructed and sham-operated rats. CONCLUSION: In the rat, after moderate bladder hypertrophy, the atropine-sensitive component was significantly up-regulated, but the ATP-sensitive component was marginally reduced, although not significantly. These results suggest that up-regulation of the P2X component of bladder contraction seen in humans with bladder instability, and in other species models of BOO, is not mirrored in the rat, or occurs later in the pathological process of bladder hypertrophy.     

α-Adrenoceptor function before and after chemical sympathectomy in human and feline detrusor muscles

Summary Isolated bladder segments from man and cat were treated with 6-hydroxydopamine (6-OHDA) in vitro. Chemical sympathectomy was evaluated with fluorescence microscopy and found to be very similar to the effect of 6-OHDA administered in vivo to cats. Isometric smooth muscle contractile responses were induced by field stimulation (FS). The amplitude of the responses increased after denervation. The effects of -adrenoceptor agonists and antagonists on the FS-induced contractile responses were compared before and after treatment with 6-OHDA. The reduction in the contractile responses after the addition of noradrenaline to the feline bladder strips was more pronounced after treatment. Phentolamine induced an increase in contractile responses before treatment, an effect not seen afterwards in human bladder strips but which persisted in feline bladder strips. Selective -adrenoceptor agonists did not alter the contractile responses in denervated strips. It is suggested that the function of the -adrenoceptors in the detrusor is to inhibit neuronally mediated contractile responses of smooth muscle.     

Agonist-induced contraction and accumulation of inositol phosphates in the guinea-pig detrusor: evidence that muscarinic and purinergic receptors raise intracellular calcium by different mechanisms

In vitro contractile responses to electrical stimulation, ATP, histamine, and carbachol were measured in strips of guinea pig detrusor. The contractile responses were redetermined at intervals after replacement of the Krebs bicarbonate buffer with a nominally calcium-free medium. Agonist induced accumulation of [3H]-inositol phosphates was measured in a suspension of detrusor slices. Electrical stimulation (five second train; frequency 20 Hz; pulse width 100 microseconds) produced a contraction that was abolished by tetrodotoxin (10(-6) M) and reduced by approximately 50% in the presence of atropine (10(-8) M). This atropine resistant component was abolished by desensitization of the purine receptors with alpha, beta-methylene ATP, confirming that the response was mediated by nerves that released ATP and acetylcholine. Carbachol, ATP, and histamine produced concentration dependent contractions in guinea-pig detrusor strips. The response to ATP was much more dependent on extracellular calcium than the response to carbachol. Muscarinic, but not purine-receptor stimulation induced the accumulation of [3H]-inositol phosphates. These data suggest that ATP stimulates a purine receptor which opens a membrane ion channel and allows an influx of calcium while muscarinic receptor stimulation can mobilize intracellular calcium via hydrolysis of inositol phospholipid and production of the second messenger inositol triphosphate.     

Aging effects on contractility of longitudinal and circular detrusor and trigone of rat bladder.

PURPOSE: Aging is associated with bladder dysfunction, including difficult voiding and urinary leakage. Voiding involves reduction in the bladder lumen in all dimensions brought about by contraction of the meshwork of longitudinal, circular and oblique layers of detrusor smooth muscles. Most in vitro physiological studies of the effects of aging on bladder function used the longitudinal detrusor. To understand the region specific effects of aging on bladder function the contractile responses of longitudinal and circular detrusor, and trigone segments of the bladder from young and old rats were monitored. MATERIALS AND METHODS: These studies were performed using male Fisher 344 rats 6 months (young) and 27 months (old) old obtained through the National Institute on Aging. Each rat was anesthetized and the bladder was isolated. From each bladder a strip of longitudinal detrusor, circular detrusor and trigone was isolated and mounted in an in vitro multi-muscle chamber containing normal physiological solution at 37C. Isometric contractions of the 3 bladder strips were monitored after electrical field stimulation, 120 mM. potassium and 1 to 1,000 microM. bethanechol using a digital oscilloscope. RESULTS: In longitudinal detrusor from old rats there was no significant difference in the contractions evoked by electrical stimulation or high potassium but there was a significant reduction in contractions evoked by bethanechol compared with the responses of longitudinal detrusor from young rats. In circular detrusor from old rats there was a significant increase in contractions evoked by electrical stimulation and a slight increase in contractions produced by high potassium but no significant change in contractions evoked by bethanechol compared with the responses of circular detrusor from young rats. In trigone from old rats there was a significant decrease in contractions evoked by electrical stimulation, high potassium and bethanechol compared with young trigone. CONCLUSIONS: The reduction in contractions evoked by bethanechol suggests an age related reduction in muscarinic receptors in the longitudinal detrusor of aged rats. An increase in contractions evoked by electrical stimulation without a change in contractions evoked by bethanechol suggests a decrease in compliance caused by an increase in collagen in the circular detrusor of aged rats. A general decline in all contractile responses, including those evoked by high potassium, suggests reduced membrane depolarization in the trigone of aged rats. The effect of aging is specific to different regions and functional components of the bladder, probably due to changes in muscarinic receptors, collagen and depolarization.     

Response of the fetal sheep bladder to urinary diversion

PURPOSE: In adults urinary diversion results in bladder atrophy and a rapid decrease in contractile function. Little is known about the effects of urinary diversion on bladder development. In this regard we characterized the responses of fetal sheep bladder strips obtained from animals that underwent urinary diversion. MATERIALS AND METHODS: Urinary diversion was performed on fetal sheep after 90 days of gestation (term 147 days) and bladder tissue was obtained 2 weeks later. Contractile and relaxant responses of full-thickness bladder strips were tested. RESULTS: Bladders from fetal sheep subjected to urinary diversion weighed significantly less than control fetal bladders. Histological studies demonstrated marked connective tissue infiltration and the reorganization of smooth muscle elements. Carbachol stimulated a tonic contraction, while field stimulation administered during the tonic contraction elicited a phasic relaxation or a biphasic response, consisting of an initial relaxation and then a phasic contraction in control and diverted bladders. Contractile responses of defunctionalized strips to carbachol were significantly less than those of control bladder strips. Contractile responses of defunctionalized bladder strips to field stimulation at 1 Hz. were significantly greater than those of control strips. Responses of the 2 sets of fetal bladder strips to higher frequencies were similar, as were the contractile responses to adenosine triphosphate and KCl. Field stimulated relaxations in the presence of carbachol stimulated contraction of defunctionalized bladder strips were significantly greater than those of control strips, while the relaxant responses of each set of fetal bladder strips to isoproterenol and nitroprusside were similar. CONCLUSIONS: Urinary diversion in normal fetal sheep resulted in marked structural changes, reduced carbachol stimulation and increased field stimulation relaxation.