Vascular response of the rabbit bladder to short term partial outlet obstruction.

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

Intermittent catheterization limits rabbit bladder dysfunction in response to partial outlet obstruction.

INTRODUCTION: The initial response of the urinary bladder to outflow partial obstruction consists of distension, followed by a rapid increase in mass and concomitant functional adaptations. Subsequently, an indeterminate period of stabilized function and little increase in mass occurs (compensation). Finally, bladder mass again progressively increases, accompanied by deleterious changes in bladder morphology, biochemistry and pharmacology, and progressive loss of function (decompensation). The reported study was designed to determine whether limiting the level of bladder distension, using intermittent catheterization (IntCath), could protect the bladder from decompensation. MATERIALS AND METHODS: Sixteen male New Zealand White rabbits were separated into 4 groups of 4 rabbits each: control, control with IntCath, obstructed, and obstructed with IntCath. IntCath was performed with the animals under inhalation anesthesia. An 8 Fr. catheter was inserted through the urethra into the bladder every 8 hours and the urine drained. After 14 days of obstruction, bladders were removed from all rabbits; longitudinal strips were cut from the bladder body and suspended in individual organ baths. Contractile responses to field stimulation (FS), KCl, ATP, and carbachol were measured. RESULTS: There were no significant differences between control rabbits and controls with IntCath in bladder weights, compliance, and contractile responses to all stimuli. Bladder weights of both obstructed groups increased significantly compared to those of both control groups. Bladder weights of obstructed rabbits increased to a significantly greater extent than did those of obstructed rabbits with IntCath. Bladder compliance in the obstructed group was significantly lower than compliance in both control groups and in obstructed rabbits with IntCath. Both obstructed groups exhibited significantly decreased contractile responses to FS, ATP and KCl, compared to control groups. The response of the obstructed group with IntCath to FS and carbachol was significantly greater than the responses of the obstructed group without IntCath. CONCLUSIONS: These findings show that limiting distension with IntCath reduces the magnitudes of the increased bladder mass, the loss of bladder wall elasticity (compliance), and the impaired contractile responses which occur secondary to outflow obstruction.

Rabbit versus rat urinary bladder: effects of in vitro hypoxia.

PURPOSE: Studies indicate that bladder hypoxia may be an etiological factor for lower urinary tract dysfunction. Rat and rabbit are two species of experimental animals used frequently to study lower urinary tract function and dysfunction. The objective of this study was to compare directly effects of in vitro hypoxia on contractile responses of rat and rabbit urinary bladder to different forms of stimulation. METHODS: Sexually mature male New Zealand White rabbits and Sprague-Dawley rats were compared. Each bladder was excised while the animal was anesthetized, and longitudinal bladder strips were cut, then mounted in organ baths. A tension of 2 g was placed on all strips. Effects of 1, 2, 3 and 4 h hypoxia followed by 1 h of reoxygenation on contractile responses of bladder strips to field stimulation (FS), carbachol (100 micromol/l), ATP (1 mmol/l) and KCl (120 mmol/l) were determined. RESULTS: Contractility, per unit tissue mass, of rat bladder strips was significantly greater than that of rabbit bladder strips in response to FS (all frequencies), carbachol, KCl and ATP. Hypoxia (followed by reoxygenation) resulted in time-dependent progressive reduction in contractile responses of bladder strips to all stimuli. Rat bladder was significantly more sensitive to hypoxia than rabbit bladder in response to FS and carbachol. Hypoxia induced similar effects on rat and rabbit bladder responses to ATP and KCl. CONCLUSION: Rat bladder neurogenic and cholinergic responses are significantly more sensitive to hypoxia than are those of rabbit bladder, which may be due to the rat bladder's greater contractile force generation and previously reported higher Ca2+-ATPase activity.

Sucrose diuresis protects rat bladder from outlet partial obstruction-induced contractile dysfunction.

OBJECTIVES: Evidence is accumulating that bladder dysfunction caused by experimental partial obstruction of the bladder outlet can be reduced or reversed by treatment that results in upregulation of bladder function, even in the presence of obstruction. Inducing diuresis in rats or rabbits results in a significant increase in bladder mass and increased contractility in response to stimulation. The objective of the present study was to determine whether diuresis-induced amplification of bladder function in the rat could protect the bladder from contractile dysfunctions caused by partial outlet obstruction. METHODS: Thirty-two rats were separated into four groups of 8 rats each. Groups 2 and 4 were fed 5% sucrose instead of water; groups 1 and 3 were fed only water. Three weeks later, partial outlet obstructions were created in groups 3 and 4. After 4 weeks of obstruction, all bladders were rapidly excised and cut into longitudinal strips; each strip was mounted in an isolated muscle bath for contractile studies. RESULTS: Sucrose-induced diuresis caused a moderate but significant increase in bladder mass. Partial outlet obstruction stimulated significant increases in bladder mass in both water-drinking and sucrose-drinking groups; the bladder mass of sucrose-drinking rats, however, increased less than that of water-drinking rats. In water-drinking rats, partial outlet obstruction resulted in significantly decreased bladder strip contractility in vitro in response to field stimulation (1 to 32 Hz), carbachol (0.1 to 22 microM), and KCl (120 mM). After 3 weeks of sucrose-induced diuresis, partial obstruction of the rat bladder outlet did not result in decreased in vitro contractile responses to any form of stimulation applied. CONCLUSIONS: Sucrose-induced diuresis caused an increase in bladder mass and an increase in contractile strength, consequently protecting the rat bladder from the contractile dysfunctions that usually follow partial outlet obstruction.

Correlation of ischemia/reperfusion or partial outlet obstruction-induced spectrin proteolysis by calpain with contractile dysfunction in rabbit bladder.

OBJECTIVES: In the rabbit, both experimental ischemia and partial outlet obstruction of the urinary bladder induce similar dysfunctions with regard to the contractile responses to both field (neuronal) stimulation and postsynaptic receptor stimulation. Circumstantial evidence indicates that the pathologic response to both conditions is related to two connected processes-tissue ischemia and reperfusion injury-that result in a marked increase in intracellular calcium ([Ca2+]i), followed by the activation of the Ca(2+)-dependent neutral protease calpain. Calpain activation results in the proteolysis of specific membrane proteins, including those of neuronal membranes (resulting in progressive denervation of the detrusor) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), resulting in the previously reported decrease in SERCA. The current study is designed to generate direct support for the theory that both ischemia and partial outlet obstruction result in the activation of calpain. METHODS: Separate sets of rabbits were subjected to 1 or 2 hours of ischemia, followed by reperfusion for different lengths of time, or partial outlet obstruction for different lengths of time. We determined the state of calpain activation by quantitating tissue proteolysis of alpha-spectrin by Western blot analysis. Correlative organ bath studies were conducted to observe the contractile responses of bladder strips to field stimulation and bethanechol administration. RESULTS: (1) Sixty minutes of ischemia followed by 30 minutes of reperfusion resulted in (a) a reduction in the contractile responses to field stimulation and bethanechol (89% and 57%, respectively), and (b) a 72% decrease in native alpha-spectrin, with a concomitant 300% increase in its breakdown products (BDPs). Neither alpha-spectrin nor its BDPs had returned to control levels after 72 hours of reperfusion. (2) Twenty-four hours after the creation of a partial obstruction, alpha-spectrin BDP levels were increased 330%, then gradually fell to 130% of control levels by 14 days after obstruction. Concomitantly, the native alpha-spectrin level was decreased 74% 24 hours after obstruction and remained low through 7 days after obstruction. At 14 days after obstruction, the alpha-spectrin levels had recovered to 75% of control levels. CONCLUSIONS: These findings suggest that Ca(2+)-dependent proteolysis of the preferred calpain substrate alpha-spectrin in urinary bladder tissues is increased significantly by both ischemia/reperfusion and partial outlet obstruction. Temporally, proteolysis precedes the reduced muscle function resulting from these pathologic conditions.

Cellular and molecular aspects of bladder hypertrophy.

Bladder dysfunction secondary to benign prostatic hyperplasia (BPH) is a major affliction associated with ageing. As the disease slowly progresses, the bladder changes from a state of compensation to decompensation, in which there are severe, irreversible alterations in bladder function. Using a rabbit model of partial outlet obstruction we have identified three major cellular changes in the bladder which result from such obstruction. These include progressive denervation, mitochondrial dysfunction and disturbances of calcium storage and release from the sarcoplasmic reticulum. Our hypothesis is that outlet obstruction results in bladder hypertrophy which induces ischaemia. This leads to a release of intracellular calcium, leading to activation of specific enzymes and generation of free radicals. These then attack the membranes of nerves, sarcoplasmic reticulum and mitochondria. We have demonstrated that pretreatment of rabbits with Pygeum africanum extract (Tadenan) significantly reduced the severity of both the contractile and metabolic dysfunctions induced by partial outlet obstruction. Our current hypothesis is that Tadenan may either prevent the activation of degradative enzymes (or generation of free radicals), or protect the intracellular membranes against the destructive effects of free radicals or degredative enzymes. In conclusion, identifying cellular mechanisms responsible for bladder dysfunction induced by partial outlet obstruction provides new possibilities for non-surgical treatment of BPH. Our studies on Tadenan support this concept that the bladder provides a novel target for therapeutic intervention.

Bladder function in experimental outlet obstruction: pharmacologic responses to alterations in innervation, energetics, calcium mobilization, and genetics.

The two functions of the urinary bladder is to store urine at low intravesical pressures, and to periodically expel the urine through a coordinated contraction of the bladder and relaxation of the urethra. To a large extent, urinary bladder function depends upon the underlying structure of the organ as a whole, particularly on the inter-relationships among the smooth muscle, connective tissue, and neuronal elements. An alteration in the ratio of connective tissue to smooth muscle, for example, can significantly alter compliance and functional capacity, structurally impairing the bladder's ability to empty efficiently and fully. Thus, a change in structural compartmentation can affect bladder function independent of autonomic receptor density, response to receptor stimulation, and the contractile capabilities of the smooth muscle elements. Similarly, a selective alteration in either the afferent or efferent innervation of the bladder or urethra can induce significant alterations in the structural interrelationships between smooth muscle and connective elements. In addition, the bladder responds rapidly to alterations in urine volume and urethral resistance with marked changes in bladder and urethral structure and function, and these changes are under the controls of specific genes that are known to control cellular growth, hypertrophy, and hyperplasia. A knowledge of the mechanisms that control the response to specific forms of stress may lead to novel therapies for specific disease states.

Partial outlet obstruction of the rabbit bladder results in changes in the mitochondrial genetic system.

In the rabbit, partial outlet obstruction of the urinary bladder results in significant changes in the physiology, cellular structure, and cellular metabolism of that organ. One of the most striking changes observed is a 50% decrease in oxidative metabolism. Here we investigate whether the function of the mitochondrial (mt) genetic system is altered in rabbit bladder tissue following partial outlet obstruction. Southern analyses of total DNA prepared from bladder tissue excised as a function of time after initiation of partial outlet obstruction showed that the relative number of copies of the mt genome decreases as much as 10-fold during the first 7 d after obstruction, and that this attenuated mt genome copy number is maintained until at least 14 d post-obstruction. Northern analyses, in contrast, showed that mt COII and cytochrome b transcript levels initially decrease but recover to control levels by about 5 d after obstruction; that level is maintained through 14 d post-obstruction. Enzymatic analysis of cytochrome oxidase and NADH cytochrome c reductase activities in obstructed bladder tissue gave results which paralleled the pattern in the mt RNA analyses. Surprisingly, transcript levels for the mt-related nuclear COIV gene rapidly decreased to about 50% of control levels following obstruction and remained there until 14 d post-obstruction. These results indicate that partial outlet obstruction of the rabbit bladder leads to significant changes in the status and expression of the mt genetic system in bladder tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of partial outlet obstruction of the rabbit urinary bladder on ryanodine binding to microsomal membranes.

1. Partial outlet of obstruction of the rabbit urinary bladder results in a significant increase in the ability of ryanodine to inhibit field stimulated contraction. The current study determined the effect of outlet obstruction on ryanodine binding to microsomal membranes. 2. The results demonstrate: (i) Ryanodine binding is linear showing one set of ryanodine binding sites (Bmax = 28 +/- 3.5 fmol/mg protein; Kd = 4.2 nM). (ii) Total ryanodine binding was slightly increased at 1 and 3 day following obstruction. (iii) Binding increased approx 4-fold at 5 and 7 days post obstruction. (iv) No change in the Kds were noted at any time period. 3. The results of these studies indicate that smooth muscle hypertrophy secondary to partial outlet obstruction induces a marked increase in the role of intracellular calcium in the mediation of the contractile response to field stimulation.

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

The urinary bladder depends on intracellular ATP to support a number of essential intracellular processes including contraction. The concentration of ATP is maintained by mitochondrial oxidative phosphorylation, cytosolic glycolysis and the cytosolic activity of creatine kinase, the enzyme that catalysis the rapid transfer of a phosphate from creatine phosphate (CP) to ADP resulting in the formation of ATP. Prior studies in this lab and others have demonstrated that mitochondrial respiration is significantly lower in hypertrophied bladder tissue (induced by partial outlet obstruction of the white New Zealand Rabbit). In addition to decreased mitochondrial respiration, there are significant increases in glycolysis and lactic acid formation in the hypertrophied tissue. In view of the increased glycolysis and decreased mitochondrial function in the hypertrophied tissue, and the importance in creatine kinase in maintaining cytosolic levels of ATP, the current study was designed to determine if outlet obstruction induces any changes in the activity of creatine kinase. The following is a summary of the results: 1) The bladder mass increased from 2.2 +/- 0.2 gm to 11.5 +/- 1.6 gm at 7 days following outlet obstruction. 2) The intracellular concentrations of both ATP and CP were significantly reduced in the bladder tissue following 7 days of obstruction. 3) The percent of protein (per tissue mass) was significantly lower in the obstructed bladders, although the percent of soluble protein was similar. 4) Creatine kinase activity of control bladders showed linear kinetics with a Vmax = 1120 nmoles/mg protein/4 min and Km = 147 microM CP.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Alteration by perfluorodecalin of hepatic metabolism and excretion of phenobarbital.

The bile duct was cannulated in rats that had been infused intravenously with an emulsion of perfluorodecalin at intervals from 2 to 34 weeks earlier. After injection of [14C]phenobarbital, urine and bile were collected during the next 24 hr and were analyzed for phenobarbital and its metabolites. There was a decrease in the biliary excretion of phenobarbital and its metabolites for several weeks after infusion of perfluorodecalin, but conjugation of the metabolites was not decreased. The reduced excretion returned to normal after about 20 weeks.

Biophysical and catalytic properties of the phenobarbital p-hydroxylase--a cytochrome P-450 dependent mixed function oxidase.

1. A sensitive method to detect and quantify the products of phenobarbital (PB) hydroxylation by model chemical systems and by biological systems has been developed. 2. Chemical model systems hydroxylate PB in the p-position of the phenyl ring and form one or two additional oxidation products, while in vitro and in vivo (bile fistula rats) biological systems hydroxylate PB only in the p-position. 3. Phenobarbital hydroxylation rates in vitro are of the order of 0.007 nmol/nmol cytochrome P-450 per min. These values are decreased by pretreatment of the rats with inducing doses of phenobarbital. 4. Enzymes catalysing the p-hydroxylation reaction of phenobarbital are localized in the microsomes and have the biophysical and chemical properties that are usually associated with cytochrome P-450-dependent mixed function oxidases.

The metabolism and excretion of enzyme-inducing doses of phenobarbital by rats with bile fistulas.

The metabolism of enzyme-inducing doses of 14C-phenobarbital injected i. p. into bile duct-cannulated rats has been studied using improved chromatographic separation and quantification techniques. In animals with bile fistulas most of the 14C-phenobarbital was excreted in bile as p-hydroxyphenobarbital conjugated with glucuronic acid. In urine the main substance found was phenobarbital, with significant amounts of p-hydroxyphenobarbital and varying amounts of its glucuronide conjugate. Animals without bile fistulas excreted 80% dose of phenobarbital in the urine; metabolites were free phenobarbital, p-hydroxyphenobarbital and conjugated material. Approx 90% of the conjugated material was the glucuronide. Only free phenobarbital and p-hydroxyphenobarbital were found in the faeces. Animals drinking plain water excreted 50-65% dose of phenobarbital (80 mg/kg) in bile and the remainder mainly in the urine, whereas superhydrated animals (drinking 5% glucose and 0.9% NaCl) excreted 90% of the dose as free phenobarbital in the urine. Phenobarbital is the only labelled material detectable in hepatic tissue and portal, vena caval or aortic blood, which indicates that phenobarbital is the enzyme-inducing substance and that liver and kidney rapidly eliminate all metabolites. Metabolism of phenobarbital in vivo is a complex process involving interaction of hepatic and intestinal metabolism, partial readsorption from the intestinal tract and renal elimination.

Evidence for diplasmalogen as the major component of rabbit sperm phosphatidylethanolamine.

The question of whether diplasmalogens [1,2-di(O-1'-alkenyl) phosphatidyl derivatives] make up part of the plasmalogen component of cell phospholipids was examined using rabbit epididymal spermatozoa. These cells are readily obtained as a highly homogeneous suspension and long have been known to have high plasmalogen content. Phospholipids were determined by thin layer chromatography (TLC) with CuSO4 staining. Plasmalogens were determined by hydrolysis of the phospholipids with TCA/HCl, followed by TLC and CuSO4 staining. Ethanolamine derivatives were determined by ninhydrin. The phosphatidylethanolamine (PE) content of these cells was 29 +/- 2 micrograms/10(8) cells, 90% of which was assayed as diplasmalogen and 10% as diacyl PE. No monoplasmalogen could be detected. The presence of diplasmalogen as the major component of PE was given further support from infrared and proton nuclear magnetic resonance (1H-NMR) spectroscopy, which showed the presence of O-1'-alkenyl substituents but near absence of O-acyl substituents. The phosphatidylcholine (PC) content of the cells was 104 +/- 5 mu/10(8) cells, of which 50% was monoplasmalogen with the 1'-alkenyl group on the 2 position of the glycerol moiety. No diplasmalogen was found in PC. The other phospholipids in rabbit sperm were phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SP) and lysophosphatidylcholine (LPC). Phosphatidylserine (PS) and phosphatidylinositol (PI) were present at the limits of detectability of the TLC method. None of these phospholipids contained plasmalogen. The PE component of rabbit sperm phospholipids appears to differ from that of the other cells in having the previously unreported diplasmalogen as its major constituent.

(3-sn-Phosphatidyl)cholines (lecithins) in amniotic fluid.

Many currently used thin-layer chromatographic methods for phospholipid assay rely on charring the developed plate in the presence of cupric acetate. Saturated acyl phospholipids do not react. We find that substitution of cupric sulfate results in detection of both saturated and unsaturated phospholipids. By exploiting the difference with the two reagents, one can separately estimate the amounts of saturated phospholipid. The method described here is reproducible, and we illustrate its use in determinations of (3-sn-phosphatidyl)cholines (lecithins) in amniotic fluid from problem pregnancies.