Energetics of muscarinic stimulation of smooth muscle.
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Biomedical subjects
P A Longhurst
Publications and source records attributed to P A Longhurst.
The influence of streptozotocin-induced diabetes mellitus on the sensitivity of rat urinary bladder body and base strips to changes in extracellular calcium.
1. The influence of calcium on contractile responses of bladders from control and 2 month streptozotocin-diabetic rats was investigated. 2. Removal of calcium from the bathing medium caused rapid decreases in the contractile responses of bladder body and base strips to carbachol. The responses of strips from control rats were reduced more by calcium removal than were strips from diabetics. 3. Replacement of calcium caused dose-dependent increases in contraction to carbachol. The responses of bladder body strips from diabetic rats to carbachol were significantly greater at all calcium concentrations than were those of controls. There were no differences in the responsiveness of bladder base strips to carbachol. 4. In contrast, bladder body strips from diabetic rats were more sensitive to calcium than were strips from controls, with an IC50 value for calcium of 0.38 mM vs 0.72 mM for controls. 5. At the calcium concentration of Krebs buffer (2.5 mM), contractile responses were near maximal, and there were no differences in sensitivity. 6. The calcium antagonist nifedipine caused dose-dependent decreases in the contractile responses of bladder base and body strips to nerve stimulation. The responses to nerve stimulation were more sensitive to nifedipine than were those to carbachol. There were no differences between controls and diabetics in the sensitivity of bladder strips to nifedipine. 7. The findings suggest that although increases in sensitivity to calcium are observed in bladder body strips from streptozotocin-diabetic rats, they are unlikely to be responsible for the increases in maximal contractile response to nerve stimulation and contractile agents.
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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.
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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.
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Studies on experimental bladder outlet obstruction in the cat: long-term functional effects.
Experimental bladder outlet obstruction in cats induces a significant increase in the in vivo leakage pressure. The effects of obstruction on the detrusor function were analyzed, using control, 3, 6 and 12 month obstructed cats. The in vivo leakage pressures were substantially increased in all obstructed groups. From a previous study on the short-term effects of obstruction in the cat; although there was an immediate increase in pressure, there were no significant differences between control bladders and two week obstructed bladders in: bladder weight, peak pressure response to field stimulation and bethanechol, and emptying of the in vitro whole bladder model. In this current study, although there was no direct relationship between duration of obstruction and severity of the impaired pharmacological response of the isolated whole bladder, there was a direct relationship between the magnitude of the increase in bladder weight, leakage pressure, and impaired functional response of the in vitro whole bladder. In this regard the cats were separated into four groups: controls and shams (average bladder weight 2.95 gm.), obstructed group 1 with bladder weights less than 6.0 gm. obstructed group 2 with bladder weights between 6 and 10 gm., and obstructed group 3 with bladder weights greater than 10 gm. The results can be summarized as follows: A) In vivo leakage pressures were significantly increased in all obstructed groups, and progressively higher in proportion to the bladder weights. B) Bladder capacity significantly increased in all, being greatest in obstructed group 3. C) The magnitude of the response to field stimulation of obstructed groups 1 and 2 were significantly greater than the response of control bladders at virtually all frequencies. The frequency response curve of obstructed group 3 was similar to control. D) The ability of the bladder to empty in response to field stimulation was significantly reduced in all obstructed groups. The magnitude of the decrease was proportional to the increase in bladder weight. E) There were no differences between control and obstructed bladders in the pressure response to bethanechol or KCl; the ability of both bethanechol and KCl to empty the bladder was reduced in the obstructed groups. In general, the presence of bladder enlargement was accompanied by significant alterations in the ability of the in vitro bladder to empty in response to field stimulation, bethanechol, and KCl.(ABSTRACT TRUNCATED AT 400 WORDS)
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Urinary bladder function in the tight-skin mouse.
Tight-skin mice develop hypertrophy of connective tissue and tendons, associated with increases in collagen concentration in skin, heart, lungs, and tail. The bladders from these mice have not previously been examined. Because of the purported importance of collagen in bladder wall structure and compliance, we examined collagen content, micturition characteristics, and length-tension relationships in bladders from tight-skin mice. Bladder collagen content and concentration were approximately 70% greater in 5-6 month tight-skin mice than age-matched controls, but bladder mass, protein content, and protein concentration were similar. Tight-skin mice urinated larger volumes more frequently during the light cycle, and the functional bladder capacity appeared to be greater than that of controls. There was a small shift to the right of the passive length-tension curves of bladder strips from tight-skin mice, but the shift was not statistically significant. The magnitude of active tension development was the same. The data suggest that bladder collagen concentration does not necessarily determine bladder capacity or compliance. It is suggested that other factors, such as the ratio of collagen subtypes or the collagen:elastin ratio may have more importance for the maintenance of bladder distension.
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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.
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Effects of sensitization on the permeability of urothelium in guinea pig urinary bladder.
Permeability of the guinea pig urinary bladder was investigated in a model of experimental cystitis induced by intravesical antigen challenge following sensitization. Guinea pigs were sensitized by intraperitoneal injections of ovalbumin (10 mg./kg.) given on days 1, 3, and 5. The studies described were done four weeks after the last injection. Controls (injected with saline) were used at the same time as sensitized animals. Each group (control and sensitized), was divided into two subgroups; guinea pigs challenged with intravesical ovalbumin (10 mg./ml., one ml.) and those receiving one ml. saline intravesically. Immediately following the antigen (or saline) challenge, one ml. of 14C-urea urea was placed into the bladder for two hours. We examined the peripheral blood concentrations of 14C-urea at periods of time up to 120 minutes. There was a progressive increase in the blood level of 14C-urea with time only in the sensitized group challenged with antigen (ovalbumin). There was no 14C-urea present in the blood of the sensitized group without antigen challenge, or in either unsensitized group. We also measured isotope concentration in the bladders and found a significantly higher concentration of isotope in the bladders from ovalbumin-treated sensitized guinea pigs. We believe that this animal model of cystitis is an improvement over previous models because of its physiological relevance. In this model, cystitis is produced without mechanical or chemical damage to the bladder mucosa. A discussion of the model in relation to interstitial cystitis is presented.
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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.
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Effects of sensitization on female guinea pig urinary bladder function: in vivo and in vitro studies.
Although bladder inflammation is known clinically to produce a variety of symptoms including urgency, frequency, and pain, there are only a few experimental studies that directly relate bladder inflammation with urodynamic and functional alterations. We have used the sensitized guinea pig model to study the effects of inflammation on micturition parameters, cystometry, and in vitro bladder contractility. This model depends on the allergic response of the bladder mucosa to ovalbumin, an otherwise non-irritative agent, as an antigen. In vivo exposure of the bladder to ovalbumin via urethral catheterization induced a prompt and marked increase in the number of micturitions in antigen-sensitized guinea pigs. Ovalbumin had no effects on the micturition parameters in the control group. Using in vivo cystometry, intravesical exposure to ovalbumin induced a significant decrease in both the pressure at which micturition was induced, and the volume at which micturition was induced. Ovalbumin had no effect on cystometric parameters of the control animals. In vitro exposure of whole-bladder preparations to ovalbumin induced a significant contractile response only in the bladders isolated from the sensitized guinea pigs. The responses of the isolated bladders to field stimulation and bethanechol were identical for bladders from both sensitized and control animals. In conclusion, exposure of the bladder to ovalbumin in the sensitized animal induced an increase in the frequency of micturitions and a decrease in the pressure and volume at which micturition was induced. Thus, intravesical exposure of the bladder mucosa to a substance that the bladder has been sensitized to can induce alterations in micturition that are consistent with the clinical symptoms of "urgency and frequency".
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Changes in bladder function in the one year spontaneously diabetic BB rat.
Micturition characteristics and in vitro urinary bladder function were investigated in insulin-treated spontaneously diabetic BB rats and age-matched non-diabetic controls one year after the onset of diabetes. BB rats weighed less than controls and were hyperglycemic. Diabetic rats consumed larger volumes of water and excreted larger volumes of urine than controls. The frequency of micturitions and the mean volumes of urine excreted per micturition were significantly increased in BB rats compared to age-matched controls. Associated with the micturition changes in the BB rats were significant increases in bladder body mass. Contractile responses of strips from bladder bodies and bases were measured in response to nerve stimulation, carbachol, phenylephrine, ATP, and KCl. No significant differences between controls and diabetics were found in the absolute contractile responses of bladder body strips to nerve stimulation, carbachol, ATP, or KCl. However, if the data were transformed to correct for the increases in tissue mass in the diabetics, there were significant decreases in the responses of bladder body strips from BB rats to carbachol, ATP, and KCl, but not to nerve stimulation. Even after transformation, there were no differences in the responses of bladder base strips to carbachol, phenylephrine, or KCl. The data indicate that significant changes in micturition characteristics are evident one year after the onset of diabetes in the spontaneously diabetic BB rat. These changes are slow in development, since they are absent six months after the onset of diabetes. The changes in micturition and bladder strip contractility are qualitatively similar to, but quantitatively modest in comparison with those caused by streptozotocin-induced diabetes mellitus. The quantitative differences are probably attributable to an ameliorative effect of the insulin received by the BB rat.
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Urinary bladder function 6 months after the onset of diabetes in the spontaneously diabetic BB rat.
Urinary bladder function was examined in the spontaneously diabetic BB rat six months after the onset of diabetes. Diabetes caused significant decreases in rat weight and increases in bladder body weights and in vivo bladder capacities compared to age-matched controls, but no changes in the weights of bladder bases. The absolute contractile responses of urinary bladder body and base strips to nerve stimulation, carbachol, 5-hydroxytryptamine, ATP, phenylephrine, and KCl were unaltered by diabetes. However, when the data were corrected for tissue mass, there were slight but not significant decreases in contractile responses of strips from diabetic rats. There were increases in total muscarinic receptor numbers and calcium channel binding sites in bladder bodies from BB rats as a result of the increases in tissue mass. The data indicate that the six month-diabetic BB rat differs from the streptozotocin-diabetic rat in the sensitivity of the urinary bladder to the complications of diabetes, probably as a result of the insulin treatment required to keep BB rats alive.
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In vitro contractile responses of vasa deferentia from spontaneously diabetic BB rats.
1. The effects of diabetes of 6 months' duration on autonomic function of the vas deferens were examined in spontaneously diabetic BB rats and diabetes-resistant BB rats. 2. Diabetes caused significant decreases in rat and testes weights, but had no effects on the weights of vasa deferentia compared to diabetes-resistant controls. 3. The contractile responses of vasa deferentia to nerve stimulation and to carbachol were increased compared to controls. However, the contractile responses of vasa deferentia from diabetic BB rats to ATP, phenylephrine, and 120 mM KCl were unaltered. 4. The increased responses to carbachol were associated with increases in the muscarinic receptor density, but no changes were detected in the number of dihydropyridine binding sites. 5. The data indicate that, in contrast to vasa deferentia from streptozotocin-diabetic rats which exhibit decreases in responsiveness to nerve stimulation and a non-specific supersensitivity to contractile agents, vasa deferentia from diabetic BB rats have no changes in sensitivity, and increased contractile responses to only nerve stimulation and carbachol. These differences in urogenital sensitivity to diabetes may be related to the insulin treatment which diabetic BB rats require to keep them alive.
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Comparison of the contractile and metabolic effects of muscarinic stimulation with those of KCl.
Urinary bladder emptying is mediated primarily by a coordinated contraction of the bladder body in response to parasympathetic stimulation and muscarinic receptor activation. In previous studies we presented evidence that the contractile response to bethanechol stimulation could be dissociated from the metabolic response through the use of diltiazem (calcium channel blockade). The conclusion from these studies was that muscarinic stimulation resulted in a significant increase in metabolic activity which was not directly associated with contraction. KCl stimulates contraction in isolated strips by directly depolarizing the membrane rather than from binding to specific membrane receptors. The current study directly compares the metabolic and contractile activity of bethanechol (muscarinic stimulation) with KCl (direct membrane depolarization). Isolated strips of rabbit 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 of isolated bladder smooth muscle strips 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 a isometric force transducer connected to a Grass model D polygraph. The results can be summarized as follows. (1) Both bethanechol and KCl stimulate a sharp decrease in the NADH/NAD ratio, a rapid increase in intracellular-free calcium, and a slower increase in contractile force.(ABSTRACT TRUNCATED AT 250 WORDS)
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Creatine kinase activity of urinary bladder and skeletal muscle from control and streptozotocin-diabetic rats.
The urinary bladder depends on intracellular ATP for the support of a number of essential intracellular processes including contraction. The concentration of ATP is maintained constant primarily via the rapid transfer of a phosphate from creatine phosphate (CP) to ADP catalyzed by the enzyme creatine kinase (CK). Since muscular pathologies associated with diabetes are in part related to intracellular alterations in metabolism, we have characterized the CK activity in both skeletal muscle and urinary bladder from control and streptozotocin-diabetic rats. The following is a summary of the results: 1) Bladder tissue from control rats showed linear kinetics with a Vmax = 390 nmoles/mg protein/min, and a Km = 275 microM. 2) Urinary bladder tissue isolated from diabetic rats displayed biphasic kinetics with Vmax = 65 and 324 nmoles/mg protein/min, and Km's = 10 microM and 190 microM respectively. 3) Skeletal muscle isolated from control rats showed linear kinetics with an approximate Vmax of 800 nmoles/mg protein/min and a Km of 280 microM CP. 4) Homogenates of skeletal muscle from diabetic rats showed complex kinetics not separable into distinct component forms. 5) The Km for ADP for both skeletal muscle and bladder was approximately 10 microM. These studies demonstrate that whereas bladders isolated from both control and diabetic rats possess a low-affinity isomer(s) of CK with similar maximum enzymatic activity, there is a high affinity isomer present within the urinary bladder muscle of diabetic rats that is not present in bladder tissue isolated from control rats. Skeletal muscle isolated from both diabetic and control rats exhibited a maximal activity 2 to 3 times higher than that of the bladder.
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Effect of bladder outlet obstruction on the morphology, physiology, and pharmacology of the bladder.
Bladder outlet obstruction secondary to benign prostatic hyperplasia induces numerous changes in bladder morphology, physiology, and pharmacology. These changes have been studied experimentally in various animal models, and while each species has advantages and disadvantages, it is unclear which is most like man. It has been shown that tissue hypertrophy leading to an increase in tissue mass develops rapidly after bladder outlet obstruction. Ischemia induced by the obstruction results in acute muscle dysfunction. The degree of functional impairment is directly related to the degree of tissue hypertrophy. However, the bladder contractile apparatus appears to have a surprising regenerative ability, such that recovery of bladder function becomes obvious 14 days after obstruction. Urodynamic changes include an increase in urinary frequency and voiding pressure and a decrease in voided volume. Clinically, involuntary bladder contractions are often present. Determination of which of these specific aspects of outlet obstruction the investigator is interested in studying will dictate the selection of the most appropriate animal model.
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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.
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The effects of testosterone or insulin treatment on contractile responses of the rat vas deferens following castration or streptozotocin-induced diabetes mellitus.
1. Castration and streptozotocin-induced diabetes produce significant decreases in serum testosterone levels accompanied by decreased vas deferens weights, a decreased responsiveness to nerve stimulation, and altered contractile responses to carbachol and phenylephrine. 2. Treatment of castrated rats with testosterone for 8 weeks prevented the decreased vas deferens weights and contractile changes associated with castration. 3. Treatment of diabetic rats with testosterone for 8 weeks prevented the decreased vas deferens weights and the supersensitivity to contractile agonists associated with diabetes. Testosterone treatment only partially prevented the decreased response to nerve stimulation. 4. Treatment of diabetic rats with testosterone plus insulin for 8 weeks prevented the decreased vas deferens weights and decreased the sensitivity to carbachol and phenylephrine compared to controls. Testosterone plus insulin treatment prevented the decreased response to nerve stimulation. 5. There were no differences in the IC50 values for nitrendipine among any of the groups studied, suggesting that the contractile changes observed in vasa deferentia following castration or diabetes are not the result of changes in calcium movements. 6. The results suggest that decreased testosterone levels are at least partially responsible for the changes in contractility of the vas deferens of streptozotocin-diabetic rats.
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