Penelope A. Longhurst

The Functional Effects of Long-Term Outlet Obstruction on the Rabbit Urinary Bladder

The current study investigated the relationship between duration of outlet obstruction, magnitude of bladder mass, and functional dysfunction on the rabbit urinary bladder. Following the production of obstruction with the cuff model, bladder wet weight increased to twice control weight within one week, and then slowly to four times control weight by one month, and remained at this level for the six month period. Bladder capacity decreased significantly by one week but returned to control volumes by one month. The in vitro ability of the bladder to empty in response to field stimulation and bethanechol decreased significantly in the one and two week obstructed bladders and remained decreased for six months. One of the major observations of this study was the relatively large variation of bladder weight and histology observed for the one to six month obstructed rabbits. Although the bladders with mild mass increase (less than 3 gm./kg. body weight) had normal distribution of urothelium and muscular elements, the bladders with moderate mass increase (3 to 6 gm./kg.) had thick extrinsic connective tissue deposits and the bladders with severe mass increase (greater than 6 gm./kg.) had thick extrinsic and intrinsic connective tissue deposits and muscular degeneration. The percentage occurrence of mild, moderate and severe mass increase was approximately the same (58%, 30% and 12%, respectively) for the one, three, and six month groups. The bladders with mild mass increase had normal bladder capacities and increased pressure responses to field stimulation and bethanechol. The bladders with moderate-to-severe mass increase showed enlarged bladder capacities and had progressively smaller pressure responses. As the magnitude of bladder mass increased, the ability of the bladder to empty in response to field stimulation and bethanechol decreased proportionally. We conclude that the functional impairment of the bladder is related to the amount of extrinsic and intrinsic connective tissue and the degree of muscle degeneration.

Comparison of Urinary Bladder Function in Rats with Hereditary Diabetes Insipidus, Streptozotocin-Induced Diabetes Mellitus, and Nondiabetic Osmotic Diuresis

In vivo and in vitro bladder function were studied in three different models of increased diuresis: 1) Brattleboro rats with hereditary diabetes insipidus (di/di), 2) Sprague-Dawley rats with streptozotocin-induced diabetes mellitus (STZ), and 3) Sprague-Dawley rats with increased diuresis due to 5% sucrose added to the drinking water. When compared with controls, all three models showed bladder mass, increased water consumption and urine output, higher mean and maximal increased micturition volumes, and greater bladder capacity and compliance by in vitro cystometry. The changes were more extensive in di/di rats than in the STZ and sucrose-drinking rats. The concentration of bladder collagen decreased in all three rat models when compared with controls. However, the collagen concentration of STZ bladders was significantly lower than the collagen concentration of di/di and sucrose bladders, suggesting that the decrease in bladder collagen concentration associated with experimental diabetes mellitus is only partly related to the increased diuresis. Contractile function was studied using a whole bladder model. Responses of whole bladders from control and diabetic rats to electrical field stimulation, carbachol and KCl were identical. Volume-pressure relations of the isolated whole bladder showed that the magnitude of the contractile response to KCl is constant at intravesical volumes ranging from about 10 to 95% of cytometrical bladder capacity. Bladders from Brattleboro di/di rats and STZ rats showed a rightward shift of volume-passive pressure curves when compared with appropriate controls. Bladders from sucrose-drinking rats had volume-passive pressure curves similar to the bladders from controls. This study suggests that while contractile function remains intact with increased diuresis, the passive function changes, with the bladder becoming more distensible.

Effects of Partial Outlet Obstruction of the Rat Urinary Bladder on Micturition Characteristics, DNA Synthesis and the Contractile Response to Field Stimulation and Pharmacological Agents

Partial outlet obstruction is one of the major urological complications induced by benign prostatic hypertrophy (BPH). The current study describes the time course of the effect of mild partial outlet obstruction (in rats) on in vivo micturition parameters, DNA synthesis, and on the in vitro response of the bladder to field stimulation, bethanechol, methoxamine, ATP, and KCl. Mild partial outflow obstruction was created by placing a catheter (outside diameter: 1.70 mm.) transabdominally in front of the urethra, tying a ligature (2-zero silk) around both the urethra and catheter, and then removing the catheter. The micturition pattern was monitored for 2 days prior to surgery, and then continuously for 14 days following the surgery. The changes in bladder weight and the in vitro detrusor function of control (sham operated) and obstructed bladders (1, 3, 5, 7, 14 and 28 days after surgery) were examined. Micturition frequency in the dark cycle decreased immediately after the operation, and then increased linearly reaching a maximum at the 5th day, and stabilized at this increased level for the duration of the micturition study. The frequency of the dark cycle was also decreased immediately after the sham surgery and then gradually increased over the period of observation. Bladder weight increased by day 1 following surgery, and remained high throughout the 28 day study. The contractile response of the obstructed bladder base to field stimulation was reduced at days 1 and 3. The response then increased above control for day 5, reached a maximum response at day 7 and remained at this level for days 14 and 28. A similar pattern was observed for the contractile response of the bladder body to bethanechol and KCl, and for the bladder base to methoxamine and KCl. Both obstructed and sham surgeries increased bladder DNA content and 3H-thymidine incorporation, which reached maximal values on days 5 and 3, respectively. DNA content and 3H-thymidine incorporation of obstructed bladders were greater than those of sham operated bladders. In conclusion, partial outlet obstruction in the rat resulted in a progressive increase in bladder mass, an increase in micturition frequency, increases in the in vitro contractile response to field stimulation, bethanechol, methoxamine, and KCl, and increases in bladder DNA content and 3H-thymidine incorporation.

Comparison of Urinary Bladder Function in 6 and 24 Month Male and Female Rats

Micturition characteristics, collagen composition, and in vitro urinary bladder strip contractility were examined in young adult (six month) and old (24 month) male and female Fischer 344 rats. Although young female rats consumed significantly less water than young males, there were no differences in volumes of urine excreted. Old females excreted significantly more urine than old males, but there were no differences in volumes of water consumed. Old male rats had similar micturition frequencies during the light and dark cycles, in contrast to females and young males, where the number of micturitions during the dark cycle was significantly greater than those during the light cycle. The mean and maximal micturition volumes were significantly greater in old males compared to young males and old females during both the light and dark cycles. Bladders from female rats weighed significantly less than bladders from males of the same age, and the bladders from young rats weighed less than those of old rats. The protein and collagen concentrations were significantly less in bladder bodies from young females than old females. The amount of collagen resistant to digestion by Pronase, and thus thought to be cross-linked, was significantly greater in bladders from old rats compared to young. No differences between groups were found in the contractile responses of bladder base strips. There were trends for the absolute contractile responses of bladder body strips from old males to field stimulation, carbachol, ATP, and KCl to be larger than the other groups, and for strips from the young females to be smaller. The responses of strips from young females to field stimulation and KCl were significantly less than those of young males or old females, and responses to 10(-3) M ATP were less than those of old females. Responses of strips from old males to 60 mM KCl were significantly greater than those of young males. The differences in contractility could be attributed to the differences in strip mass. It appears, therefore, that urinary bladder function in male and female rats is unaffected by increasing age between 6 and 24 months.

Effects of Outlet Obstruction on Glucose Metabolism of the Rabbit Urinary Bladder

Bladder outlet obstruction has been shown to cause detrusor contractile dysfunction. To determine if alterations in bladder metabolism may in part underlie these functional defects, we investigated the effects of mild outlet obstruction on the glucose metabolism of the rabbit urinary bladder. Mild outlet obstruction was created in mature male rabbits by the surgical placement of a silicon sleeve around the bladder neck. Two weeks after surgery, the in vitro ability of the obstructed bladder tissues to metabolize glucose was compared to that of the controls. The results can be summarized as follows: 1) The bladder wet weight increased 2.3-fold following two weeks of obstruction. 2) Obstructed bladder tissues had a reduced glucose consumption as compared to the controls. 3) CO2 generation was significantly reduced by 31% in obstructed bladder tissues whereas lactate formation increased significantly by 22%. 4) Tissue concentrations of ATP, creatine phosphate, and glycogen before incubation showed no significant differences between control and obstructed bladder tissues. In summary, bladder tissues following two weeks obstruction showed a decrease in aerobic metabolism and an increase in anaerobic metabolism. Previous studies have indicated that the ability of the bladder to maintain a contraction and empty may be directly related to aerobic metabolism. Therefore, the decrease in aerobic metabolism (even in the presence of increased anaerobic metabolism) may in part explain the decreased ability of the obstructed bladder to empty.

Temporal Changes in Micturition and Bladder Contractility after Sucrose Diuresis and Streptozotocin-induced Diabetes Mellitus in Rats

Studies were done to compare the acute effects of streptozotocin-induced diabetes and sucrose consumption on micturition, bladder mass and contractile responses of bladder strips to field stimulation and contractile agonists. Micturition changes occurred gradually in diabetic rats, reached maximal values within 7 to 14 days, and were accompanied by significant increases in bladder mass after 7 days. Bladder strips from diabetics responded to field stimulation, carbachol and KCl with significantly greater contractions than did those from controls within 7 days. Sucrose-drinking rats had maximal increases in fluid consumption and micturition frequency on the first night after starting treatment. Increases in micturition volumes were slower to develop than in diabetics. Bladder mass was significantly increased 30 and 60 days after starting sucrose treatment. Bladder strips from sucrose-drinking rats responded to field stimulation and carbachol with significantly greater contractions than did those from controls only after 60 days. Monitoring of drinking and micturition patterns established that diabetic rats drink and urinate during both the dark and light cycles. In contrast, control and sucrose-drinking rats drink and urinate principally at night. The results demonstrate that differences in bladder function between diabetic and sucrose drinking rats are apparent during the first month after treatment begins. The data suggest that the effects of diabetes and sucrose consumption on contractile bladder function are related to the diuresis-induced increases in bladder mass.

Metabolic studies on rabbit bladder smooth muscle and mucosa

Recent studies indicate that the mucosa of the urinary bladder may play a major role in the maintenance of normal bladder function. The mucosal surface of the urinary bladder serves as a protective layer against the irritative solutes found in the urine. The integrity of this barrier can be broken by overdistension, anoxia, detergents, alcohols, bacterial infection and by contact with agents to which the mucosa has been sensitized.In view that both anoxia and ischemia can mediate a breakdown in the role of the mucosal layer as a permeability barrier, it is reasonable to assume that this function is dependent on cellular metabolism. As an initial investigation we have compared a variety of biochemical and metabolic parameters between the mucosal layer (consisting of the lamina propria, urothelium, and any connective tissue and vascular tissue within this layer); and the muscularis layer.The results of these studies demonstrated that the rate of glucose metabolism to lactic acid (LA) of the mucosa was more than three-fold greater than that of the smooth muscle. The rate of CO2 production of the mucosa was 60% greater than that of the unstimulated smooth muscle. The maximal activity of the mitochondrial enzyme citrate synthase was significantly greater in the mucosa than in the smooth muscle, however, the activity of malate dehydrogenase was similar for both tissues. The maximal activity of the cytosolic enzyme creatine kinase was more than two-fold greater in the bladder smooth muscle than in the mucosa; although the affinities of the creatine kinase isoforms of the mucosa were sigificantly greater than those of the muscle.Although the concentrations of ATP and ADP were similar in both muscle and mucosa, the level of creatine phosphate (CP) was over four-fold greater in the bladder muscle while the level of AMP in the muscle was only 58% of that in the mucosal epithelium.In summary, the rate of glucose metabolism was greater in the mucosa than in the smooth muscle although the concentrations of high energy phosphates (ATP+CP) was significantly greater in the smooth muscle. Future studies will be directed at identifying the specific cellular processes within the mucosal layer that relate to the function of the urothelium as a permeability barrier.

The role of cyclic nucleotides in guinea-pig bladder contractility

1 The effects of phosphodiesterase (PDE) inhibition and forskolin pretreatment on the contractile responses of guinea‐pig urinary bladder strips to electrical field stimulation, carbachol, ATP and KCl were studied. 2 Inhibition of cyclic AMP‐specific PDE4 isozymes by rolipram significantly reduced the contractile response of bladder strips to field stimulation. Rolipram also suppressed the contractile response to low concentrations of carbachol, but potentiated the response to high concentrations. The contractile response to ATP was significantly reduced by rolipram treatment, but that to KCl was unaltered. 3 Inhibition of cyclic GMP‐specific PDE5 isozymes by zaprinast had no effects on the contractile response of bladder strips to field stimulation, ATP or KCl. Zaprinast suppressed the contractile responses to 1u2003μM carbachol and potentiated the response to high concentrations. 4 Contractile responses to field stimulation and to carbachol after pretreatment with the adenylyl cyclase activator, forskolin, were qualitatively similar to those caused by rolipram treatment. β‐Adrenoceptor blockade with propranolol partially reversed the inhibitory effects of rolipram on the response to field stimulation. 5 Rolipram significantly reduced the contractile response of bladder strips from sensitized guinea‐pigs to ovalbumin challenge, but zaprinast was ineffective. PDE inhibition had similar effects on the responsiveness of control and of sensitized guinea‐pig bladder strips to field stimulation, carbachol, ATP and KCl. 6 The data suggest that the contractile response of guinea‐pig bladder strips can be modified by increases in cyclic AMP levels.

The Influence of Acute Overdistension on Rat Bladder Function and DNA Synthesis

Prolonged micturition problems are often encountered after long-term bladder overdistension caused by urinary retention. In animal studies, damage to the bladder wall innervation has been found following overdistension. Experimentally, acute overdistension has also been implicated in the pathogenesis of the response to partial outlet obstruction. In the present study we investigated the influence of overdistension on micturition volume and frequency, on in vitro bladder function using the whole bladder model and on 3H-thymidine uptake, localization and DNA synthesis. Overdistension was induced for 3 hours by forced diuresis and balloon obstruction. Another group of rats was catheterized for 3 hours but received no diuretic, nor was the balloon inflated. An additional group of controls was neither anesthetized nor catheterized. Overdistension caused a gradual increase in bladder mass which was maximal at 7 days. During the first 24 hours following overdistension, the frequency of micturition decreased, but normalized thereafter. A progressive decrease in the response to field stimulation was noted between 16 hours and 7 days following overdistension and remained at this level until 21 days. There were, however, no significant differences in the responses to carbachol, ATP and KCl. There was a 30% reduction in the ability of field stimulation to empty the bladder 16 hours after overdistension, but no impairment of the emptying ability of carbachol. Overdistension was followed by a significant increase in 3H-thymidine uptake, which was maximal at 2 days. 3H-thymidine labelling increased rapidly after overdistension and was maximal within 16 hours in the urothelium. In smooth muscle, connective tissue and lamina propria, maximal labelling occurred at 2 days. Catheterization alone caused a mild distension which was associated with a small, but statistically significant, increase in 3H-thymidine incorporation into DNA within 16 hours. The labelling was located primarily in the urothelium. Overdistension causes a proliferative reaction within the bladder wall. Its initial effects occur within the urothelium, and the later involvement of the subendothelial smooth muscle and connective tissue is directly proportional to the degree of bladder distension. Three weeks following overdistension, the bladders functional state was not completely recovered, although the urinary bladder was found to have a good capacity to adapt and compensate for the stress-induced changes caused by overdistension. It is, therefore, clear that overdistension may have long-lasting effects on the bladder.

Effects of Muscarinic Stimulation on Intracellular Calcium in the Rabbit Bladder: Comparison with Metabolic Response

Urinary bladder emptying is mediated primarily by a co-ordinated contraction of the bladder body in response to parasympathetic stimulation and muscarinic receptor activation. In a previous study using surface spectrophotometry to monitor the nicotinamide adenine dinucleotide (oxidized form)/nicotinamide adenine dinucleotide (reduced form) (NADH/NAD) ratio, we demonstrated that muscarinic stimulation results in a rapid decrease in this ratio which precedes the contractile response, and that the ED50 for the NADH response is significantly lower than the ED50 for contraction. The current study was designed to correlate changes in intracellular free calcium using FURA-2 fluorescence with both the contractile and metabolic response to muscarinic stimulation. Isolated strips of urinary bladder body were monitored in vitro for changes in intracellular free calcium, NADH/NAD ratio, and contraction. Intracellular free calcium was monitored by preincubation with FURA-2 AM and continuously measuring the fluorescence with an MB2 surface spectrofluorometer using excitation wavelengths of 340 and 380 nm, and an emission wavelength of 510 nm. The NADH/NAD ratio was monitored with the MB2 surface spectrophotometer using an excitation wavelength of 366 nm and an emission wavelength of 450 nm. Contraction was monitored using an isometric force transducer connected to a Grass model D polygraph. The results can be summarized as follows: (1) Bethanechol stimulates a sharp decrease in the NADH/NAD ratio, a rapid increase in intracellular free calcium, and a slower increase in contractile force. (2) The ED50 for NADH fluorescence was significantly less than the ED50 for either contraction or calcium fluorescence which were equal to each other.(ABSTRACT TRUNCATED AT 250 WORDS)