Phasic Contractions in Urinary Bladder from Juvenile versus Adult Pigs

Aims

Alterations in properties of the bladder with maturation are relevant physiologically and pathophysiologically. The aim of this study was to investigate alterations in bladder properties with maturation in juvenile vs. adult pig, focussing on differences between layers of the bladder wall (mucosa vs. detrusor) and the presence and functional contribution of interstitial cells (ICs).

Methods

Basal and cholinergic-induced phasic contractions (PCs) in mucosal and denuded-detrusor strips from juvenile and adult pigs were assessed. Expression of c-kit, a marker of ICs, was investigated in the mucosa and the detrusor layers of the pig bladder. The functional role of ICs in mediating PCs was examined using imatinib.

Results

Mucosal strips from juvenile and adult pig bladders demonstrated basal PCs whilst denuded-detrusor strips did not. PCs of mucosal strips from juvenile pigs were significantly greater than those from adult bladders. Immunoreactivity for c-kit was detected in mucosa and detrusor layers of pig bladder. Histological studies demonstrated a distinct layer of smooth muscle between the urothelium and bladder detrusor, termed the muscularis mucosa. Imatinib was only effective in inhibiting PCs in mucosal strips from juvenile pigs. Imatinib inhibited the carbachol-induced PCs of both juvenile and adult denuded-detrusor strips, although strips from juvenile bladders demonstrated a trend towards being more sensitive to this inhibition.

Conclusions

We confirm the presence of c-kit positive ICs in pig urinary bladder. The enhanced PCs of mucosal strips from juvenile animals could be due to altered properties of ICs or the muscularis mucosa in the bladders of these animals.     

Adrenergic innervation of the lower urinary tract in cattle

Studies were carried out on 8 sexually immature male calves. Sections of the ureters, urinary bladder, and urethra were cut with a freezing microtome and the method of Ky?s?la et al. (1980) was used to visualize the adrenergic nerve fibres. It was found that bovine ureters possessed weak innervation; most of the nerves was located in the muscular membrane, and only in the paravesical part, sparse nerve fibres were found in the submucosa of this one. Apex of the urinary bladder was more weakly supplied with the adrenergic nerves than the corpus, whereas bladder's trigonum and cervix possessed numerous nerve fibres in both muscular and submucosal membranes. The distribution pattern of adrenergic nerves in the urethra was similar to that of urinary bladder's cervix. The presence of adregeneric nerve fibres was found in submucosal layer of both the urinary bladder and the urethra. Part of the nerves was connected with blood vessels of the organs under study.     

Adrenergic innervation of the ureters, urinary bladder, and urethra in pigs

Studies were conducted on 4 sexually mature and 4 immature pigs. Scraps of the ureters, urinary bladder, and urethra were cut with a freezing microtome. Fluorescence method of Torre and Surgeon (1976) was used to reveal the adrenergic innervation. It was found that the ureters were weakly supplied with the adrenergic nerves; most of the nerves were located in the muscular and submucosal membranes. Apex of the urinary bladder possessed the weakest innervation. More nerves were found in particular layers of the bladder corpus whereas bladder trigonum and cervix possessed numerous nerves. Adrenergic innervation of the urethra was similar to that of the urinary bladder's cervix. Adrenergic nerves were present in the serous and muscular membranes of both the urinary bladder and the urethra. Part of the nerve fibres was connected with blood vessels of the organs under study.     

The role of alpha 1L-adrenoceptor in rat urinary bladder: comparison between young adult and aged rats

We examined the role of the alpha1L-adrenoceptors in the urinary bladder of young adult and aged rats in vitro. In the isolated body of the urinary bladder (corpus vesicae), phenylephrine-induced contractions were significantly facilitated in aged rats. Either prazosin, a non-selective alpha1-adrenoceptor antagonist, or JTH-601, an alpha1L-adrenoceptor antagonist, competitively inhibited the phenylephrine-induced contraction of isolated body of the urinary bladder. The antagonistic effect of JTH-601 was almost equipotent between young adult and aged rats (pA2 values were 9.61+/-0.12 and 9.79+/-0.07, respectively), although a statistically significant difference was noted for that of prazosin (pA2 values were 9.49+/-0.09 and 9.19+/-0.06, respectively). In macroscopic autoradiographic studies, specific binding of [3H]JTH-601 (5nM) was seen widely in the muscle layer of urinary bladder, but no differences were noted between young adult and aged rats. In the present study, there was no evidence to suggest a role of the alpha1L-adrenoceptors in the body of rat urinary bladder. On the other hand, alpha1A-adrenoceptors may play an important role in an age-related increase of alpha1-adrenoceptors response in this tissue. These results suggest that a facilitation of contractile response mediated by alpha1A-adrenoceptors may be a cause of unstable bladder in aged persons.     

Increased expression of non-muscle myosin heavy chain-B in connective tissue cells of hypertrophic rat urinary bladder

Expression of the non-muscle myosin heavy chain-B (NM-MHC-B, also denoted as the embryonic smooth muscle myosin heavy chain, SMemb) was examined in rat urinary bladder during growth in response to a partial urinary outflow obstruction. Following obstruction, the weight of the urinary bladder increased more than five-fold within 10 days. Immunohistochemistry with a polyclonal antiserum against the C-terminal sequence of NM-MHC-B revealed very few NM-MHC-B immunoreactive cells in the control urinary bladders. In hypertrophic bladders, the number of NM-MHC-B immunoreactive cells markedly increased. The majority of such cells were found in the interstitium surrounding smooth muscle bundles and also in the subserosal and submucosal layers. Western blot analysis showed that the NM-MHC-B expression was transient; the content of NM-MHC-B immunoreactive material had doubled 10 days after obstruction and then declined towards the control level after 6 weeks. Immunohistochemistry revealed co-localization of NM-MHC-B and vimentin within the same cells. NM-MHC-B did not co-localize with smooth muscle actin, suggesting that the source of NM-MHC-B is not a de-differentiated smooth muscle cell or myofibroblast but a non-muscle cell possibly reacting to tissue distension or stress. The NM-MHC-B-positive cells could have a role in the production of extracellular matrix and growth factors or be involved in modulation of spontaneous contractile activity.     

Butylated hydroxyanisole inhibits the growth of HeLa cervical cancer cells via caspase-dependent apoptosis and GSH depletion

To evaluate the effects of in vitro ischemia/reperfusion on contractile response to field stimulation (FS), free fatty acid (FFA) content, phospholipid (PL) content, and malondialdehyde (MDA) levels of the rabbit urinary bladder. There is significant evidence that ischemia/reperfusion injury is linked to obstructive bladder dysfunction secondary to men with benign prostatic hyperplasia (BPH). Twelve New Zealand White male rabbits were separated into two groups of six rabbits each. Each rabbit was euthanized, and the bladder was surgically removed intact for whole bladder incubation. The bladders in Group 1 received a 3-h incubation under normal oxygenated physiological conditions. These bladders received electrical field stimulation (32?Hz) after 1 and 3?h. The bladders associated with Group 2 received a 1-h incubation under normal oxygenated physiological conditions. At the end of this 1-h period, the bladders were subjected to FS. After a maximal pressure response was recorded, the stimulation was turned off and the bath medium was changed to one equilibrated with 95% nitrogen, 5% oxygen without glucose (ischemic medium) and incubated for 1?h with field stimulations (32?Hz) occurring at 5-min intervals to represent overactive bladder dysfunction. At the end of this hour of ischemia with repetitive stimulation, the bath was changed to an oxygenated medium with glucose for a 1-h period after which the stimulation was repeated. At the end of the experimental period, each bladder was opened longitudinally and the muscle and mucosa separated by blunt dissection, frozen under liquid nitrogen, and stored at -80°C for biochemical analyses. Each tissue was fractionated by differential centrifugation into nuclear, mitochondrial, synaptosomal, and supernatant (cytosol) components. PL, FFA, and MDA content were analyzed for each fraction using standard biochemical techniques. The bladder contractile responses decreased during the period of in vitro ischemia and returned to only 30% of control after reperfusion. In vitro ischemia/reperfusion showed the following: (1) There was a modest but significant decrease in the FFA content of the microsomes of the muscle and significant increases in the FFA content of the nuclei and mitochondria of the mucosa. (2) There were decreases in the PL content of the homogenate and microsomes of the muscle and decreases in the PL content of the homogenate, microsomes, and supernatant of the mucosa. (3) Significant increases were observed in the MDA levels of the homogenate, mitochondria, and microsomes of both the muscle and mucosa. The significant increases in the lipid peroxidation of the bladder smooth muscle are consistent with the marked decrease in the contractile ability of the bladder following ischemia/reperfusion. The specific increased lipid peroxidation of the mitochondrial and microsomal components is consistent with the specific dysfunctions of the mitochondria and innervations observed following I/R in earlier published studies. The marked increases in lipid peroxidation in the mucosa associated with the loss of PL and FFA from this component are consistent with the significant dysfunction in both the antiadherence and antipermeability properties of the mucosa and may play a major role in the symptomatic nature of I/R-linked diseases of the bladder.     

Nonmuscle Myosin motor of smooth muscle

Nonmuscle myosin can generate force and shortening in smooth muscle, as revealed by studies of the urinary bladder from mice lacking smooth muscle myosin heavy chain (SM-MHC) but expressing the nonmuscle myosin heavy chains A and B (NM-MHC A and B; Morano, I., G.X. Chai, L.G. Baltas, V. Lamounier-Zepter, G. Lutsch, M. Kott, H. Haase, and M. Bader. 2000. Nat. Cell Biol. 2:371-375). Intracellular calcium was measured in urinary bladders from SM-MHC-deficient and SM-MHC-expressing mice in relaxed and contracted states. Similar intracellular [Ca2+] transients were observed in the two types of preparations, although the contraction of SM-MHC-deficient bladders was slow and lacked an initial peak in force. The difference in contraction kinetics thus do not reflect differences in calcium handling. Thick filaments were identified with electron microscopy in smooth muscle cells of SM-MHC-deficient bladders, showing that NM-MHC can form filaments in smooth muscle cells. Maximal shortening velocity of maximally activated, skinned smooth muscle preparations from SM-MHC-deficient mice was significantly lower and more sensitive to increased MgADP compared with velocity of SM-MHC-expressing preparations. Active force was significantly lower and less inhibited by increased inorganic phosphate. In conclusion, large differences in nucleotide and phosphate binding exist between smooth and nonmuscle myosins. High ADP binding and low phosphate dependence of nonmuscle myosin would influence both velocity of actin translocation and force generation to promote slow motility and economical force maintenance of the cell.     

Bovine papillomavirus type 2 detection in the urinary bladder of cattle with chronic enzootic haematuria

The bovine papillomavirus type 2 (BPV-2) involvement in the aetiology of chronic enzootic haematuria associated to bracken fern ingestion has been suggested for a long time. However, a few reports have shown the presence of the BPV-2 in urinary bladder tumors of cattle. The aim of this study was to investigate the presence of the BPV-2 infection in the urinary bladder of cattle with chronic enzootic haematuria in Brazilian cattle herds. Sixty-two urinary bladders were collected from adult cattle in beef herds from the north region of the state of Paraná, Brazil. According to clinical and pathological finds the specimens were distributed in three groups: the group A was constituted by 22 urinary bladders with macroscopic lesions collected at necropsy of cattle with clinical signs of chronic enzootic haematuria; the group B by 30 urinary bladders with macroscopic lesions collected in a slaughterhouse of cows coming from bracken fern-endemic geographical region; and the group C (control) by 10 urinary bladders without macroscopic lesions collected from asymptomatic cattle in a bracken fern-free geographical region. By a semi-nested polymerase chain reaction (PCR) assay, with an internal control, a fragment of the BPV-2 L1 gene with 386 bp length was amplified in 36 (58%) urinary bladder. The rate of BPV-2 positive urinary bladders was 50% (11/22) for group A, 80% (24/30) for group B, and 10% (1/10) for group C (control). The rate of the positive results found in groups A and B that included urinary bladder samples with macroscopic lesions was 67% (35/52) and the detection of the BPV-2 in both groups was significantly higher (P < 0.05) than in the control group. RFLP with Rsa I and Hae III enzymes evaluated the specificity of the BPV-2 amplicons. The PCR internal control that amplified a 626 bp fragment of the ND5 gene of the bovine mitochondrial genome was amplified in all analyzed samples and excluded false-negatives or invalid results in the semi-nested PCR. These results suggest the BPV-2 involvement in the chronic enzootic haematuria aetiology and open the perspective of the development of new strategies for the control of this disease that is the major cause of economical losses in beef herds from many Brazilian geographical regions.     

Developmental changes in spontaneous smooth muscle activity in the neonatal rat urinary bladder

Changes in spontaneous activity of the urinary bladder during postnatal development were examined in muscle strips from the base and dome of bladders from 1- to 5-wk-old rats. Activity was analyzed using fast Fourier transformation (FFT), nonlinear cross prediction, and the Shannon entropy test. Spontaneous activity was not detected in strips from 1- to 5-day-old rats but was observed in 50% of strips from 6- to 7-day-old rats and was prominent in strips from 2-wk-old animals. FFT analysis revealed one peak in activity, which was significantly faster in the bladder base (0.21 +/- 0.03 Hz) than in the dome (0.08 +/- 0.01 Hz). A second peak at approximately 0.5 Hz was detected at 3-5 wk of age. Atropine but not tetrodotoxin decreased the amplitude of spontaneous contractions, whereas carbachol, a muscarinic agonist, unmasked or stimulated spontaneous activity. These data suggest that slow rhythmic activity observed previously in neonatal whole bladders is generated by pacemaker cells in the bladder base or dome. The emergence of faster activity in bladders from older animals may reflect the development of multiple pacemaker sites, which would reduce coordination within the bladder wall and improve storage function in the mature bladder.     

The effect of partial outlet obstruction on prostaglandin generation in the rabbit urinary bladder

Partial outlet obstruction of the urinary bladder has been demonstrated to induce specific dysfunctions in cellular and sub-cellular membrane structures within the bladder's smooth muscle and mucosal compartments. Recent studies have linked these membrane dysfunctions to alterations in phospholipid metabolism leading to mobilization of free arachidonic acid, the precursor for synthesis of prostaglandins (PG). The purpose of this study was to determine if partial outlet obstruction of the urinary bladder induces changes in the capacity of bladder smooth muscle and mucosa to generate PG. PG were isolated from control and partially obstructed urinary bladder smooth muscle and mucosa of male New Zealand White (NZW) rabbits. PG concentrations (PGE2, PGF2alpha and PGI2, as its stable metabolite 6-keto-PGF1alpha) were determined after 30 minute incubations using enzyme-linked immunoassay (ELISA) kits. In both control and obstructed rabbit urinary bladders, PG generation was significantly higher in isolated mucosa than muscle tissues. A significantly higher concentration of PGF2alpha, and 6-keto-PGF1alpha was measured in obstructed muscle tissue relative to controls. The concentration of 6-keto-PGF1alpha was also significantly higher than the concentrations measured for PGE2 and PGF2alpha in both control and obstructed smooth muscle samples. The generation of PGE2 was significantly higher in rabbit urinary bladder mucosa than either PGF2alpha or 6-keto-PGF1alpha in both control and obstructed samples. The capacity of obstructed mucosal tissue to generate 6-keto-PGF1alpha was significantly higher than control tissue, while no significant differences in PGE or PGF2alpha generation were noted. These data suggest obstruction of the urinary bladder induce specific elevations in PG in both smooth muscle and mucosal tissues.     

Prostaglandins and cyclic nucleotides in the urinary bladder of a rabbit model of partial bladder outlet obstruction

Bladder outlet obstruction (BOO) is a common disorder that is associated with altered bladder structure and function. For example, it is well established that BOO results in hypertrophy and hyperplasia of the bladder smooth muscle as well as detrusor instability. Since prostaglandins (PGs) and cyclic nucleotides (cyclic AMP [cAMP] and cyclic GMP [cGMP]) mediate both smooth muscle tone and proliferation, it is reasonable to suggest that changes in their levels may be involved in the pathophysiology of BOO-associated bladder disorders. Hence, the objective of this study was to investigate cyclic AMP, cyclic GMP and prostaglandins in the bladder of a rabbit model of BOO. BOO was induced in adult male New Zealand White rabbits. After 3 weeks, urinary bladders were excised, weighed and cut into segments. They were then incubated with stimulators of PGs, cAMP and cGMP and the formation of PGs, cAMP and cGMP were measured using radioimmunoassays. There was a significant increase in the obstructed bladder weights (P=0.002). The formation of PGE2, PGI2, cAMP and cGMP was significantly diminished in the detrusor (P<0.05) and bladder neck (P<0.05) in the BOO bladders compared to age-matched controls. Since PGE2, PGI2, cAMP and cGMP are known to inhibit the proliferation of smooth muscle cells (SMCs), the decreased synthesis of these factors, in BOO, may play a role in bladder SMC hypertrophy/hyperplasia. Our study points to the possible use of drugs that modulate the NO-cGMP and/or PG-cAMP axes in BOO-associated bladder pathology.     

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.     

Alteration in expression of myosin isoforms in detrusor smooth muscle following bladder outlet obstruction

Partial urinary bladder outlet obstruction (PBOO) in men, secondary to benign prostatic hyperplasia, induces detrusor smooth muscle (DSM) hypertrophy. However, despite DSM hypertrophy, some bladders become severely dysfunctional (decompensated). Using a rabbit model of PBOO, we found that although DSM from sham-operated bladders expressed nearly 100% of both the smooth muscle myosin heavy chain isoform SM-B and essential light chain isoform LC_17a, DSM from severely dysfunctional bladders expressed as much as 75% SM-A and 40% LC_17b (both associated with decreased maximum velocity of shortening). DSM from dysfunctional bladder also exhibited tonic-type contractions, characterized by slow force generation and high force maintenance. Immunofluorescence microscopy showed that decreased SM-B expression in dysfunctional bladders was not due to generation of a new cell population lacking SM-B. Metabolic cage monitoring revealed decreased void volume and increased voiding frequency correlated with overexpression of SM-A and LC_17b. Myosin isoform expression and bladder function returned toward normal upon removal of the obstruction, indicating that the levels of expression of these isoforms are markers of the PBOO-induced dysfunctional bladders. bladder remodeling; bladder dysfunction; SM-A; LC_17a; benign prostatic hyperplasia     

Differential expression of caveolin-3 in mouse smooth muscle cells in vivo

Expression of caveolin-1 and -3 in mouse smooth muscle cells in vivo was examined by immunohistochemistry. Caveolin-1 was detected in almost all smooth muscles examined, except for the pupillary dilator muscle, whereas caveolin-3 was present only in smooth muscles of some specific tissues. In the eye, the pupillary sphincter muscle was intensely positive for caveolin-3, whereas the ciliary muscle and pupillary dilator muscle were negative. In the gastrointestinal tract, caveolin-3 was detected in the inner circular layer, but not in the outer longitudinal layer. Vascular smooth muscle cells of the resistance-sized artery in the uterus and corpus cavernosum were intensely positive for caveolin-3, whereas those of the aorta were only weakly positive and those of the vena cava were negative. Caveolin-3 was also detected in smooth muscle cells of the urinary bladder, ureter, prostatic vas deferens, and seminal vesicle. The different levels of caveolin-3 expression among various smooth muscle tissues were confirmed by Western blot analysis. Even within the same muscle, the relative expression levels of caveolin-1 and -3 were variable among neighboring cells, suggesting distinct fine regulation of expression of these two caveolins. Moreover, even in the same cell, caveolin-1 and -3 showed different distributions. These results indicate that the two caveolins form distinct caveolae in smooth muscles, and that caveolin-1 and -3 serve different functions. Their differential expression may therefore be related to the functional diversity of smooth muscles. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of the Japanese Government.     

Role for pAKT in rat urinary bladder with cyclophosphamide (CYP)-induced cystitis

AKT phosphorylation following peripheral nerve injury or inflammation may play a role in somatic pain processes and visceral inflammation. To examine such a role in micturition reflexes with bladder inflammation, we induced bladder inflammation in adult female Wistar rats (200-300 g) by injecting cyclophosphamide (CYP) intraperitoneally at acute (150 mg/kg; 4 h), intermediate (150 mg/kg; 48 h), and chronic (75 mg/kg; every third day for 10 days) time points. Western blot analyses of whole urinary bladders showed significant increases (P ≤ 0.01) in phosphorylated (p) AKT at all time points; however, the magnitude of AKT phosphorylation varied with duration of CYP treatment. Immunohistochemical analyses of pAKT immunoreactivity (pAKT-IR) in cryostat bladder sections demonstrated duration-dependent, significant (P ≤ 0.01) increases in pAKT-IR in both the urothelium and detrusor smooth muscle of CYP-inflamed bladders. Additionally, a suburothelial population of pAKT-IR macrophages (CD68-, MAC2-, and F4/80-positive) was present in chronic CYP-treated bladders. The functional role of pAKT in micturition was evaluated using open, conscious cystometry with continuous instillation of saline in conjunction with administration of an inhibitor of AKT phosphorylation, deguelin (1.0 μg/10 μl), or vehicle (1% DMSO in saline) in control (no inflammation) and CYP (48 h)-treated rats. Bladder capacity, void volume, and intercontraction void interval increased significantly (P ≤ 0.05) following intravesical instillation of deguelin in CYP (48 h)-treated rats. These results demonstrate increased AKT phosphorylation in the urinary bladder with urinary bladder inflammation and that blockade of AKT phosphorylation in the urothelium improves overall bladder function.     

Insulin cells are found in the main and accessory urinary bladders of the painted turtle, Chrysemys picta

Insulin has been localized immunocytochemically to cells in the main and accessory urinary bladders of the painted turtle, Chrysemys picta, and represents an unusual addition to the specturm of regulatory peptides associated with the urinary bladder. These stellate to fibroblastoid cells often possess neural-like processes and are similar in morphology to neurotensin cells found in Chrysemys and Pseudemys urinary bladders. Radioimmunoassay of 2M acetic acid extracts of bladder tissue indicate that the insulin concentration of accessory bladder is several-fold greater than main bladder but considerably lower than the insulin content of pancreas. Pieces of accessory bladder incubated in vitro exhibit a stable insulin release into the medium over 1 hour, but release is unaltered by known insulin secretagogues. It is tempting to postulate an endocrine or paracrine regulatory function for these cells, but at present their role in Chrysemys bladder function remains unknown.     

Ex vivo deformations of the urinary bladder wall during whole bladder filling: Contributions of extracellular matrix and smooth muscle

As the complete understanding of urinary bladder function requires knowledge of organ level deformations, we conducted ex vivo studies of surface strains of whole bladders during controlled filling. The surface strains derived from displacements of surface markers applied to the posterior surface of excised rat bladders were tracked under slow filling with pressure and volume simultaneously recorded in the passive and completely inactivated states (i.e. with and without smooth muscle tone, respectively). Bladders evaluated in the passive state exhibited spontaneous contractions and larger average peak pressures (16.7 mmHg compared to 6.4 mmHg in the inactive state). Overall, the bladders exhibited anisotropic deformations and were stiffer in the circumferential direction, with average peak stretch values of ～2.3 and ～1.9 in the longitudinal and circumferential directions, respectively, for both states. Although bladders in the passive state were stiffer, they had similar average peak areal stretches of 4.3 in both states. However, differences early in the filling process as a result of a loss in smooth muscle tone in the inactive state resulted in longitudinal lengthening of 36%. Idealizing the bladder as a prolate spheroid, we estimated the wall stress–strain relation during filling and demonstrated that the intact bladder exhibited the classic stress–stretch relation, with a significantly protracted low stress region and peak stresses of 36 and 51 kPa in the longitudinal and circumferential directions, respectively. The present study fills a major gap in the urinary bladder biomechanics literature, wherein knowledge of the pressure–volume–wall stress–wall strain relation was explored for the first time in a functioning organ ex vivo.