Robert M. Levin

Anoxia and Corporal Smooth Muscle Dysfunction: A Model for Ischemic Priapism

The hemodynamics of penile flaccidity, erection and detumescence requires corporal smooth muscle to function across a wide variation in pO2. The present study describes the effect of anoxia on corporal smooth muscle response to field stimulation and pharmacologic agonists and antagonists of erection. The response of isolated strips of rabbit corpus cavernosal tissue to field stimulation, phenylephrine, bethanechol, ATP and KCL was determined under oxygenated and anoxic conditions. The results can be summarized as follows: 1) Anoxia eliminated spontaneous contractile activity and reduced basal tissue tension to a minimum. 2) Neither field stimulation nor pharmacological agents (ATP, bethanechol, isoproterenol) could relax basal tension below that induced by anoxia alone. 3) Under anoxic conditions alpha-adrenergic agonists produced poorly sustained phasic contractile responses; anoxia eliminated tonic contractile responses to phenylephrine. 4) In normoxic conditions field stimulation of smooth muscle precontracted with phenylephrine produced frequency-dependent graded relaxations; under anoxic conditions field stimulation yielded contractile responses at all frequencies. Our data suggest that corporal smooth muscle tone, spontaneous contractile activity, the contractile response to alpha-agonists and field stimulated relaxation depend on the state of corporal oxygenation. The inability of alpha-stimulation to induce a tonic contraction of corporal smooth muscle under anoxia in vitro parallels the failure of penile injection of alpha-adrenergic agonists to relax ischemic priapism.

The Influence of Ovariectomy and Estradiol Replacement on Urinary Bladder Function in Rats

Female Fischer 344 rats were ovariectomized or sham operated and treated with oil or estradiol cypionate (100 mg./100 gm./month) for two or four months. Rats were then placed in metabolism cages for measurement of micturition characteristics, and bladders were removed for bladder strip studies. Ovariectomy had no effects on micturition characteristics. However, estradiol treatment of ovariectomized rats caused significant increases in water consumption and urine excretion, and in mean and maximal micturition volumes compared to both ovariectomized and sham-operated rats. These effects were more pronounced at four months. Estradiol treatment also caused significant increases in bladder body mass, while ovariectomy was without effect. Two months after ovariectomy and/or estradiol treatment, there were no differences in contractile responses of bladder body or base strips to contractile agents when compared to shams. However, after four months, ovariectomy caused significant decreases in contractile responsiveness to nerve stimulation. ATP, carbachol, and KCl compared to sham-operated rats. Estradiol treatment caused increased responsiveness to nerve stimulation, ATP, carbachol, and KCl compared to ovariectomized rats, and to carbachol compared to sham operated rats. Possible causes for the effects of ovariectomy on bladder contractility include decreases in calcium influx. Although estradiol reversed the effects of ovariectomy on bladder function, in addition we observed some indirect effects which were probably the result of estradiol-induced polyuria and increases in bladder mass.

Length-Tension Relationship of Urinary Bladder Strips from Streptozotocin-Diabetic Rats

Streptozotocin-induced diabetes mellitus or the consumption of 5% sucrose in place of drinking water cause an increase in rat urinary bladder capacity and mass. Length-tension curves were generated using bladder body strips isolated from control, diabetic, or sucrose-drinking rats to determine whether the length-tension relationship was altered by the bladder hypertrophy associated with diabetic and nondiabetic diuresis. In addition, we compared the data using three different methods of expression: (1) absolute grams tension developed; (2) grams tension/100 mg tissue, and (3) grams tension/mm2. The cross-sectional area of strips from diabetic rats was increased compared to the other two groups. The length-passive tension curves for all three groups increased with increasing tissue length to a plateau. No optimal resting length for generation of active tension was found. Strips from diabetic and sucrose-drinking rats reached the plateau at a slightly larger passive tension than did strips from control rats. In addition, strips from diabetic and sucrose-drinking rats generally developed a greater active tension than did strips from control rats depending on the method of data presentation used. The data suggest that the complex nonlinear arrangement of smooth muscle fibers in the bladder wall results in the unusual length-tension curves generated by urinary bladder strips (as compared to skeletal or vascular smooth muscle). This relationship would be of benefit in the urinary bladders of early-stage diabetic patients before neuropathy development, where the stretching of the bladder wall would allow accommodation without any compromise of bladder function. The data indicate that comparisons of bladder strip function from control and diabetic rats should be done at passive tensions of greater than or equal to 2 g to ensure that maximal active tension is generated.

THE ABILITY OF INSULIN TREATMENT TO REVERSE OR PREVENT THE CHANGES IN URINARY BLADDER FUNCTION CAUSED BY STREPTOZOTOCIN-INDUCED DIABETES MELLITUS

1. The effects of insulin treatment on in vivo and in vitro urinary bladder function in streptozotocin-diabetic rats were investigated. 2. Diabetes of 2 months duration resulted in decreases in body weight and increases in fluid consumption, urine volume, frequency of micturition, and average volume per micturition; effects which were prevented by insulin treatment. 3. Insulin treatment also prevented the increases in contractile responses of bladder body strips from diabetic rats to nerve stimulation, ATP, and bethanechol. 4. Diabetes of 4 months duration also resulted in decreases in body weight, and increases in fluid consumption, urine volume, frequency of micturition, and average volume per micturition, effects which were reversed by insulin treatment for the final 2 months of the study. 5. Insulin treatment reversed the increases in contractile responses of bladder body strips from diabetic rats to nerve stimulation, ATP, and bethanechol. 6. The data indicate that the effects of streptozotocin-induced diabetes on urinary bladder function are both prevented and reversed by insulin treatment.

Update on bladder smooth-muscle physiology

SummaryThe urinary bladder responds to distension induced by a number of different stresses with rapid and substantial increases in bladder mass and concomitant alterations in the contractile responses to neuronal stimulation, pharmacological simulation by autonomic agonists, and membrane depolarization. Furosemide, sucrose, or diabetes-induced diuresis, as well as outlet obstruction and overdistension all produce similar effects on the bladder. Accompanying the increases in bladder mass and contractile changes are increases in DNA synthesis and [3H]-thymidine uptake. Autoradiographic studies have localized the increased DNA synthesis following bladder distension initially to the urothelium, followed by slower increases in labelling of the lamina propria and extramural connective tissue. The net result of these compartmental differences in DNA synthesis is a reorganization of the structural relationships between smooth-muscle cells, the connective-tissue matrix, and the extrinsic connective-tissue lamina. This may contribute to the functional changes which occur after severe overdistension. Increases in the expression of heat-shock protein-70, basic fibroblast growth factor, N-ras, and c-myc, and decreases in transforming growth factor-beta occurred acutely after obstruction, suggesting that these changes may play a role in obstruction-induced bladder hypertrophy. Removal of the obstruction induces apoptosis of urothelial and connective tissue elements in the bladder, accompanied by increases in transforming growth factor-beta and decreases in basic fibroblast growth factor genes, and a reversal of the bladder dysfunction. Therefore the bladder hyperplasia after outlet obstruction and the regression following removal of the obstruction seem to be directly opposing processes governed by gene expression.

Factors underlying the increased sensitivity to field stimulation of urinary bladder strips from streptozotocin-induced diabetic rats.

1 The responses of bladder strips from control, streptozotocin‐diabetic, and sucrose‐drinking rats to electrical field stimulation were investigated. Sucrose‐drinking rats were included as additional controls because they have enlarged bladders as a result of non‐diabetic diuresis. 2 Bladder strips from diabetic rats developed more spontaneous activity than those from the two control groups. Indomethacin reduced the amplitude and frequency of spontaneous contractions suggesting that they resulted from endogenous prostaglandin formation. Tetrodotoxin (TTX) had little effect, while α,β‐methylene ATP caused increases in spontaneous activity. 3 Bladder strips from diabetic rats responded to field stimulation with greater contractions than controls in the absence of antagonists as well as in the presence of atropine and α,β‐methylene ATP. Increasing TTX concentrations caused a step‐wise depression of the contractile response to electrical stimulation which was not affected by preincubation with either atropine or α,β‐methylene ATP. 4 Atropine and indomethacin had no effect on stength‐duration curves constructed to measure threshold contractile responses to five pulses stimulation. The curves were shifted to the right by both TTX and α,β‐methylene ATP, indicating that the responses were neurogenic in nature and at least partially, the result of stimulation of P2‐purinoceptors. In the absence of drugs, bladder strips from diabetics responded at lower voltages and pulse widths than those of control and sucrose‐drinking rats, suggesting that they were more excitable. 5 The response curve of bladder strips from diabetics to field stimulation at increasing voltage was shifted upwards and to the left compared to strips from control or sucrose‐drinking rats. 6 Bladder strips from diabetics responded to stimulation at increasing pulse width with greater responses than those from control or sucrose‐drinking rats. At 1.0 ms pulse width, the TTX‐resistant response of strips from diabetic rats was still greater than that of the other groups, indicating that a myogenic component was also involved. 7 The data suggest that bladder strips from diabetic rats are more excitable than those of control or sucrose‐drinking rats. This may result from diabetes‐induced decreases in bladder lipid or other membrane changes, and/or be a result of partial depolarization, perhaps related to diabetic neuropathy.

Effects of Pregnancy on Muscarinic Receptor Density and Function in the Rabbit Urinary Bladder

The contractile response of the rabbit urinary bladder to field stimulation consists of both cholinergic and purinergic components. In general, approximately 60% of the contractile response to field stimulation is cholinergic and 40% is purinergic. Although the purinergic response represents a significant proportion of the initial (phasic) pressure response to field stimulation of the isolated whole bladder, it contributes only 10-15% of the ability of field stimulation to empty the bladder. The current study investigates the effects of pregnancy on the contractile responses of the isolated urinary bladder to cholinergic and purinergic stimulation. The results of these studies indicate that pregnancy induces substantial changes in the physiology and pharmacology of the urinary bladder. The following data are consistent with the theory that pregnancy substantially increases the relative purinergic component of the response to field stimulation (and presumably neuronal stimulation): (1) there was a significantly greater response of the bladders isolated from pregnant rabbits to low-frequency field stimulation; (2) atropine was more effective at inhibiting the pressure generation of bladders isolated from virgin female rabbits; (3) field stimulation was more effective at emptying bladders isolated from virgin female rabbits; (4) the response of the bladders from pregnant rabbits to bethanechol was significantly reduced, whereas the response to ATP was significantly increased. In addition to these effects of pregnancy on bladder physiology, pregnancy induced a 50% decrease in the muscarinic receptor density of the urinary bladder body, which correlated very well with the 50% decrease in the contractile response to bethanechol.

Collagen and Bladder Function in Streptozotocin-Diabetic Rats: Effects of Insulin and Aminoguanidine

The effects of insulin (5 U/day subcutaneously for 60 days) and aminoguanidine (25 mg./kg./day via gavage for 60 days) on collagen concentration, resistance to enzymatic digestion with Pronase E, and the accumulation of advanced glycosylation end products in bladder tissue were studied in male streptozotocin-diabetic rats. The characteristic autofluorescence of glycosylated connective tissue was used to quantitate advanced glycosylation end products. Fluorescence was measured in digests of bladder tissue and expressed as fluorescence/micrograms. of hydroxyproline. Correlation to alterations in bladder function was made by studying in-vivo bladder micturition and in-vitro length-tension relations of bladder strips. Five groups of age-matched rats were studied: 1) controls, 2) controls treated with aminoguanidine, 3) diabetics, 4) diabetics treated with aminoguanidine, and 5) diabetics treated with insulin. The collagen concentration and the amount of collagen released by enzymatic digestion decreased while the connective tissue autofluorescence increased in bladders from diabetic rats. Insulin was able to prevent all of the observed changes while aminoguanidine protected against changes in accumulation of advanced glycosylation end products and resistance to enzymatic digestion but not against changes in collagen concentration. Stretchability of the bladder as measured by length-tension relations of bladder strips was inversely proportional to the amount of collagen, and therefore increased in diabetic rats. Diabetes of two months duration resulted in altered micturition pattern (increased fluid consumption, diuresis, micturition frequency, and average volume per micturition). Alterations in in-vivo and in-vitro bladder function were prevented by insulin treatment but not by aminoguanidine treatment. We have shown that the collagen component of the bladder wall changes in amount as well as in quality in the diabetic rat. Our data suggest that the amount, rather than the properties of collagen, is important for bladder function.

Ontogeny of Bladder Function in the Rabbit

Although numerous experimental studies have addressed urinary bladder innervation, physiology and pharmacology, little information is available concerning the ontogeny of bladder function. The present study describes the developmental aspects of bladder mass, bladder capacity, pressure development and emptying in white New Zealand rabbits one day of age through maturity (11 to 15 weeks of age). The following studies were performed: Cystometry, pressure generation, rate of pressure generation, and emptying responses to field stimulation, cholinergic and purinergic stimulation using the in vitro whole bladder model. The results of these studies can be summarized as follows: 1) body weight and bladder weight increased in proportion to each other, bladder capacity increase proportionally with development until eight weeks of age, then increased substantially greater than body and bladder weight between eight and 11 to 15 weeks of age; 2) the ability to empty is similar for all ages; 3) pressure responsiveness to field stimulation, bethanechol, and ATP is greater at one day? of age, intermediate at one week of age, and similar for the other age groups; 4) The response to ATP (purinergic transmitter) is of an equal magnitude to the cholinergic response at one day, and reduces rapidly to approximately 45% of the cholinergic response by four weeks. Desensitization of the bladders to ATP reduced the response to field stimulation in the one day bladders to a significantly greater degree than the other age groups. These functional results indicate a marked alteration in cholinergic and purinergic response between the one day and the four week old rabbit bladders, with the response of the one week old bladders in between. The responses of the four, eight, and 11 to 15 week bladders was similar for equal volumes even though the bladder mass increased over threefold. This indicates that the ability of the bladder to generate pressure (during development) is not directly related to bladder mass.

Comparative length-tension relationship of urinary bladder strips from hamsters, rats, guinea-pigs, rabbits and cats

1. Comparative passive tension-active tension curves were constructed for urinary bladder body strips from hamster, rat, guinea-pig, rabbit and cat. 2. Equally sized strips from rabbit and cat bladders had a significantly greater mass and cross-sectional area than strips from other species. 3. There was a greater change in cross-sectional area of strips from rabbit and cat bladders with increasing length than in strips from other species. 4. Cat bladder strips developed a significantly greater absolute active tension than did strips from all other species at passive tensions greater than 5 g. 5. The length-tension relationships of the urinary bladder differs from skeletal or vascular smooth muscle in that there is no significant decrease in active tension at strip lengths greater than L0. 6. The ability of the urinary bladder to generate active tension at tissue lengths considerably greater than L0 is a prime importance in the physiological role of the urinary bladder to accommodate and store urine.