Cells Of Urinary Bladder Research Articles

Peculiarities of Signal Transduction from Arginine-Vasopressin Receptors Located on the Apical Surface of Epithelial Cells of the Frog Urinary Bladder and on the Amoeba Outer Membrane

Mechanisms of action of arginine-vasopressin (AVP) on water transport across the wall of urinary bladder of the frog R. temporaria L. and the outer membrane of the amoeba A. proteus were considered. AVP and its functional analogs were added to the basolateral and apical surfaces of the frog urinary bladder membrane and to the amoeba outer surface. In amoeba the AVP effect was evaluated from action on frequency of contractions of contractile vacuole that represents its organ of water—electrolyte homeostasis. It was shown that the system of signal transduction from the apical AVP receptors differed considerably from that located on the basolateral membrane and had a marked similarity with the system of signal transduction from AVP-sensitive structures on the outer membrane of amoeba. The urinary bladder apical membrane as well as the amoeba outer membrane has a mixed type of sensitivity to AVP with unidirectional (not antagonistic like on the basolateral membrane) involvement of systems coupled to receptors of the V1 and V2 type. We suggest that the system of signal transduction from apical AVP receptors corresponds to the earlier stage of evolutionary development.

Immunocytochemical studies on translocation of phosphorylated aquaporin-h2 protein in granular cells of the frog urinary bladder before and after stimulation with vasotocin

We have generated a specific antibody against phosphorylated aquaporin-h2 (pAQP-h2) protein to investigate the role of phosphorylation in the translocation of AQP-h2 protein within the granule cells of the urinary bladder of the frog (Hyla japonica). The antibody was generated against a synthetic peptide (ST-160) corresponding to amino acids 255-268, with a phosphorylated Ser-262, a residue that is putatively phosphorylated by protein A kinase. Using this antibody, we found, by Western blot analysis, that phosphorylation of the AQP-h2 protein rapidly increased within 2 min after vasotocin (AVT) stimulation and remained at a higher than normal level for 15 min. Moreover, quantitative immunoelectron microscopy indicated that the location of the AQP-h2 protein dramatically changed after AVT stimulation. Before stimulation, pAQP-h2 protein was localized in only a small number of intracellular vesicles near the nucleus of the granular cells, whereas the labeling density of the intracellular vesicles and the apical membrane rapidly increased after stimulation. This finding was also confirmed by the results of an immunofluorescence study. Thus, phosphorylation of AQP-h2 protein seems to be essential for translocation of the protein from the cytoplasmic pool to the apical plasma membrane of the granular cells in frog urinary bladder.

Distribution and fate of cocaine‐ and amphetamine‐regulated transcript peptide (CARTp)‐expressing cells in rat urinary bladder: A developmental study

We examined the distribution and fate of cocaine- and amphetamine-regulated transcript peptide (CARTp)(55-102)-immunoreactive (IR) structures in the neonatal and adult rat urinary bladder. Double-labeling studies examining CARTp with tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), or choline acetyltransferase (ChAT) were performed in wholemounts of urothelium or detrusor or cryostat sections of the bladder. In younger animals (postnatal day [P]1, P3), CARTp-IR cell bodies in detrusor smooth muscle were observed in large clusters ( approximately 100 cells/cluster) at the ureteral insertion and along thick bundles of nerve fibers at the bladder base. The total number of CARTp-IR cells was significantly reduced (by five-fold) at P14, and this reduced number persisted into adulthood. The decrease in the number of CARTp-expressing cells was complemented with positive staining for cleaved caspase-3, suggesting that apoptosis contributed to this decrease. At birth (P1), all CARTp-IR cells expressed the neuronal marker Hu. After birth, CARTp was expressed by some neurons (CARTp-IR, Hu-IR) that represent intramural ganglion cells and by cells that lacked a neuronal phenotype (CARTp-IR, Hu-) but did express TH. Neither of these cell populations expressed ChAT immunoreactivity in adult bladder. These cells (CARTp-IR, Hu-, TH-IR) may represent paraganglion or small intensely fluorescent (SIF) cells. The percentage of colocalization of CARTp-IR and nNOS or TH was dependent on postnatal age and showed an inverse relationship. At P1, 67.1 % of CARTp-IR cells expressed nNOS immunoreactivity. Decreased colocalization was observed with increasing postnatal age. In contrast, 19.5% of CARTp-IR cells expressed TH at P1, but colocalization increased with postnatal age. The suburothelial plexus lacked CARTp-IR nerve fibers until P14, when nerve fibers with varicosities were observed in the urethra and bladder neck region. In summary, we demonstrate 1) a decrease in the number of CARTp-IR cells in rat detrusor in early postnatal development; 2) apoptotic events in the bladder during early postnatal development; 3) rostral migration of CARTp-IR cells from the ureteral insertion toward the bladder body during postnatal development; 4) the presence of different populations of CARTp-IR cells, some with and others without a neuronal phenotype; and (5) age-dependent changes in chemical coding of CARTp-IR cells with postnatal development. This study demonstrates that CARTp-IR intramural ganglia and CARTp-IR paraganglion or SIF cells exist in the postnatal and adult rat bladder, although the role of these cell types remains to be determined.

Capsaicin receptor TRPV1 in urothelium of neurogenic human bladders and effect of intravesical resiniferatoxin

To study TRPV1 immunoreactivity in the urothelium of patients with neurogenic detrusor overactivity (NDO) before and after treatment with resiniferatoxin (RTX) and controls. Functional capsaicin TRPV1 receptors have been demonstrated in urothelial cells of rodent urinary bladder, and TRPV1-knockout mice exhibit diminished nitric oxide and stretch-evoked adenosine triphosphate release from urothelial cells. In patients with NDO, TRPV1 suburothelial nerve density is increased, which is reversed by successful treatment with intravesical RTX. However, the role of urothelial TRPV1 in human bladder disorders is unknown. Flexible cystoscopic bladder biopsies were obtained from 14 patients with NDO before and after treatment with RTX and from 8 control patients. Using a specific antibody for immunostaining, TRPV1 immunoreactivity in the urothelium was quantified by image analysis. TRPV1 immunoreactivity was observed in basal and apical urothelial cells. Basal cell layer TRPV1 immunoreactivity was significantly increased in NDO compared with control bladders (P = 0.003). In 5 patients who responded clinically to RTX, basal cell layer and total urothelial TRPV1 immunoreactivity decreased significantly after treatment (P = 0.032 and P = 0.016, respectively). The decreases in the basal cell layer TRPV1 immunoreactivity after RTX were comparable to the decreases in suburothelial TRPV1 nerve fibers in the biopsies previously studied from the same patients. Increased urothelial TRPV1 in patients with NDO may play a role in the pathophysiology, in concert with increased TRPV1 nerve fibers. Although it is not known whether similar pathogenic mechanisms are involved in the increase of urothelial and neuronal TRPV1, both may be targeted by successful RTX therapy.

Organization of Ca 2+ Release Units in Excitable Smooth Muscle of the Guinea-Pig Urinary Bladder

Ca 2+ release from internal stores (sarcoplasmic reticulum or SR) in smooth muscles is initiated either via pharmaco-mechanical coupling due to the action of an agonist and involving IP3 receptors, or via excitation-contraction coupling, mostly involving L-type calcium channels in the plasmalemma (DHPRs), and ryanodine receptors (RyRs), or Ca 2+ release channels of the SR. This work focuses attention on the structural basis for the coupling between DHPRs and RyRs in phasic smooth muscle cells of the guinea-pig urinary bladder. Immunolabeling shows that two proteins of the SR: calsequestrin and the RyR, and one protein the plasmalemma, the L-type channel or DHPR, are colocalized with each other within numerous, peripherally located sites located within the caveolar domains. Electron microscopy images from thin sections and freeze-fracture replicas identify feet in small peripherally located SR vesicles containing calsequestrin and distinctive large particles clustered within small membrane areas. Both feet and particle clusters are located within caveolar domains. Correspondence between the location of feet and particle clusters and of RyR- and DHPR-positive foci allows the conclusion that calsequestrin, RyRs, and L-type Ca 2+ channels are associated with peripheral couplings, or Ca 2+ release units, constituting the key machinery involved in excitation-contraction coupling. Structural analogies between smooth and cardiac muscle excitation-contraction coupling complexes suggest a common basic mechanism of action.

Vitamin A Deficiency in Rats Induces Langerhans Cells in the Iliac Lymph Nodes Draining to the Squamous Metaplastic Urinary Bladder

We previously observed Langerhans cells (LC) in the squamous metaplastic urinary bladder in rats fed a vitamin A-deficient diet. Here we report the finding of LC in the iliac lymph nodes, one of the abdominal lymph nodes draining to the urinary bladder. LC had previously only been observed in the superficial and hilar lymph nodes, but never in the abdominal lymph nodes. This is also the first observation of Birbeck granule (BG)-positive LC in the iliac lymph nodes. LC were not found in the transitional mucosa of the urinary bladder. In the present experiments, the LC were observed in both the squamous metaplastic mucosa and lamina propria of the urinary bladder, and also in the iliac lymph nodes, which drain into the urinary bladder. BG-positive LC may mature not only in the squamous epithelia, but also in squamous metaplastic mucosa. LC in the lamina propria of the urinary bladder might migrate to the iliac lymph nodes, which are regional lymph nodes, where they could function in antigen presentation. LC in the skin play a role in antigen-antibody interactions, and the squamous metaplastic mucosa of the urinary bladder may be a similar environment, in which LC in the mucosa can migrate to the regional lymph nodes.

Effect of nefiracetam, a neurotransmission enhancer, on primary uroepithelial cells of the canine urinary bladder.

Repeated oral treatment of dogs with a high dose of nefiracetam is reported to induce hemorrhagic lesions in the urinary bladder. To delineate its pathogenesis, we established the primary culture of uroepithelial cells of the canine urinary bladder, and then explored the effect of nefiracetam on the cultured cells. Uroepithelial cells scraped from the connective tissues of the urinary bladder of naive dogs were suspended in the minimum essential medium containing dispase, and then resuspended in the keratinocyte medium to be 6.0-7.0 x 10(5) cells/ml. Afterward, they were added to an apical chamber with a 12-mm transwell filter, cultured for three days, and recultured in the keratinocyte medium containing 1 mM CaCl(2) for 20 days. Microscopically, these cultured cells consisted of three cell layers with high transepithelial electric resistance (TER; > 10,000 ohm-cm(2)). Immunofluorescence observations revealed ZO-1 and E-cadherin bands, and electron microscopic examinations displayed the superficial cells with the assembly of tight junctions. When the effect of nefiracetam and its five main metabolites (M-3, M-10, M-11, M-18, and M-20) on TER and the ZO-1 band was assessed using cultured cells, only M-18 significantly reduced TER in the coculture for 48 h or more. Both M-10 and M-18 exhibited a deformation of uroepithelial cells and a slight reduction of the ZO-1 band from 120 h later. In conclusion, this culture system possesses both functional and morphological features of the uroepithelium reflected in vivo, and M-18 may play a pivotal role in the impairment of uroepithelial cells, leading to the onset of the urinary bladder lesion in dogs due to nefiracetam.

Immunohistochemical Detection of Uroplakin III, Cytokeratin 7, and Cytokeratin 20 in Canine Urothelial Tumors

Immunohistochemistry for uroplakin III (UP III), cytokeratin 7 (CK 7), and cytokeratin 20 (CK 20) using commercially available antibodies was done in normal canine urinary bladder and 72 canine urinary bladder tumors that had been fixed in formalin and embedded in paraffin. Prolonged fixation (3–28 days) did not significantly alter the immunostaining for UP III. There was moderate reduction in the intensity for CK 7 and CK 20 after 1 week of fixation. UP III was detected in superficial (umbrella) cells and some intermediate cells of the normal urinary bladder, 7 of 7 transitional cell papillomas (TCPs), 50 of 55 transitional cell carcinomas (TCCs), and 4 of 5 metastatic TCCs. Staining was typically outlined in the plasma membrane, but diffuse or focal cytoplasmic staining was also observed. Intracytoplasmic lumina were usually positive for UP III. One squamous cell carcinoma of the bladder, 4 nonepithelial bladder tumors, and 285 nonurothelial tumors from different nonurinary locations were negative for UP III. CK 7 was detected in 7 of 7 TCPs, 53 of 54 TCCs, and 5 of 5 metastatic TCCs. The staining for CK 7 was diffuse cytoplasmic. CK 20 was detected in 1 of 7 TCPs, 37 of 54 TCCs, and 1 of 5 metastatic TCCs. The staining with CK 20 was cytoplasmic and weaker than with antibodies to UP III or CK 7. There was concurrent expression of UP III, CK 7, and CK 20 in 36 of 54 TCCs. UP III is a specific and sensitive marker for canine transitional epithelial (urothelial) neoplasms, detecting 91% of TCCs. Negative results may be observed with anaplastic tumors.

Immunohistochemical detection of uroplakin III, cytokeratin 7, and cytokeratin 20 in canine urothelial tumors.

Immunohistochemistry for uroplakin III (UP III), cytokeratin 7 (CK 7), and cytokeratin 20 (CK 20) using commercially available antibodies was done in normal canine urinary bladder and 72 canine urinary bladder tumors that had been fixed in formalin and embedded in paraffin. Prolonged fixation (3-28 days) did not significantly alter the immunostaining for UP III. There was moderate reduction in the intensity for CK 7 and CK 20 after 1 week of fixation. UP III was detected in superficial (umbrella) cells and some intermediate cells of the normal urinary bladder, 7 of 7 transitional cell papillomas (TCPs), 50 of 55 transitional cell carcinomas (TCCs), and 4 of 5 metastatic TCCs. Staining was typically outlined in the plasma membrane, but diffuse or focal cytoplasmic staining was also observed. Intracytoplasmic lumina were usually positive for UP III. One squamous cell carcinoma of the bladder, 4 nonepithelial bladder tumors, and 285 nonurothelial tumors from different nonurinary locations were negative for UP III. CK 7 was detected in 7 of 7 TCPs, 53 of 54 TCCs, and 5 of 5 metastatic TCCs. The staining for CK 7 was diffuse cytoplasmic. CK 20 was detected in 1 of 7 TCPs, 37 of 54 TCCs, and 1 of 5 metastatic TCCs. The staining with CK 20 was cytoplasmic and weaker than with antibodies to UP III or CK 7. There was concurrent expression of UP III, CK 7, and CK 20 in 36 of 54 TCCs. UP III is a specific and sensitive marker for canine transitional epithelial (urothelial) neoplasms, detecting 91% of TCCs. Negative results may be observed with anaplastic tumors.

Characterization of a new muscarinic receptor antagonist PNU-171990 in guinea pig, cat and human smooth muscle

The present study was done to characterize a new compound, PNU-171990, 2-diisopropyl aminoethyl 1-phenylcyclopentane carboxylate hydrochloride, with functional smooth muscle selectivity at least as high as tolterodine. In vitro homogenates of guinea pig cerebral cortex, parotid gland, heart, urinary bladder, and Chinese hamster ovary (CHO) cells expressing human muscarinic m 1–m 5 receptors PNU-171990 did not show selectivity for any subtype (p K i, 7.72–8.64). PNU-171990 caused a parallel shift in the concentration–response curve for carbachol-induced contraction of smooth muscle from guinea pig bladder (p K B, 7.65), guinea pig ileum (p K B, 8.48), and human ileum (p K B, 7.10). In vivo PNU-171990 inhibited urinary bladder contraction with a significantly lower ID 50 than on the salivary secretion (206 and 706 nmol/kg, respectively, P<0.05). In conclusion, PNU-171990 is a competitive and potent muscarinic receptor antagonist in vitro with a numerically better selectivity ratio for the bladder contraction over salivation in vivo than tolterodine.

Correlation between induction of DNA fragmentation in urinary bladder cells from rats and humans and tissue-specific carcinogenic activity

Seven chemicals, six of which are known to induce epithelial neoplasms of the urinary bladder in rats, were assayed for their ability to induce DNA damage in primary cultures of rat and human cells from urinary bladder mucosa, and in urinary bladder, liver and kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, were obtained in cells from both rats and humans with the following concentrations of five test compounds: 2-naphthylamine and N-nitrosodi- n-butylamine 0.5 and 1 mM, phenacetin 2 and 4 mM, cyclophosphamide from 2 to 8 mM, and o-toluidine 16 and 32 mM. Nitrilotriacetic acid (1–4 mM), a rat bladder carcinogen, and 4-aminobiphenyl (0.125–0.5 mM), a bladder carcinogen in humans but not in rats, gave a weak positive response in rats cells and a more marked response in humans cells. In terms of DNA-damaging potency, 4-aminobiphenyl, cyclophosphamide, phenacetin and 4 nitrilotriacetic acid were more active in human than in rat cells, whereas the converse occurred with 2-naphthylamine. Consistently with the results observed in vitro statistically significant dose-dependent increases in the average frequency of DNA breaks were detected in the urinary bladder mucosa of rats given p.o. single doses corresponding to 1 4 and 1 2 LD50 of six of the seven test compounds; the only one which gave a substantially negative response was 4-aminobiphenyl. With the exception of N-nitrosodi- n-butylamine which caused DNA damage in liver and of phenacetin and nitrilotriacetic acid which caused damage in kidney in agreement with their tumorigenic activity, any substantial evidence of DNA lesions in these two organs was absent in rats treated with 1 2 LD50 of the other 4 test compounds. These findings give evidence that urinary bladder genotoxic carcinogens may be identified by the DNA damage/Comet assay using as targets cells of urinary bladder mucosa, and show that the effect may be quantitatively different in cells from rats and from human donors.

Smooth muscle cells from human urinary bladder express connexin 43 in vivo and in vitro.

Smooth muscle cells (SMC) of the human urinary bladder are believed to be electrically coupled. However, it has not been successfully demonstrated the nature of the underlying cell-cell communication. Here, we used Western blot technique to demonstrate the gap junction protein connexin 43 (Cx 43) in human bladder musculature and in smooth muscle cell cultures. We found expression of Cx 43 in all samples of the detrusor (Mdv, n=6) and in five of six samples of the internal sphincter (Mv). Cx 43 expression was less in cell cultures, 60% showing Cx 43 expression. Iontophoretic application of the gap junction permeable fluorescent dye lucifer yellow revealed a mean of 2.6+/-2.2 (mean+/-SD, n=8) coupled cells in cultured smooth muscle cells. Our findings support the notion that Cx 43 is constitutively expressed in human bladder SMC in vivo. Thus, the function of the bladder is likely to be influenced by the organisation of smooth muscle cells into functional syncytia.

DNA Damage Induced by 4,4′-Methylenedianiline in Primary Cultures of Hepatocytes and Thyreocytes from Rats and Humans

4,4′-Methylenedianiline (MDA), an aromatic amine used in various industrial processes and previously found to induce tumor development in liver and thyroid of mice and rats, was evaluated for its DNA-damaging activity in primary cultures of hepatocytes and thyreocytes from rat and human donors. After exposure for 4 and 20 h to MDA concentrations ranging from 10 to 180 μM, a statistically significant increase in the frequency of DNA lesions was revealed by the Comet assay in primary hepatocytes and thyreocytes from donors of both species, the response being dose dependent up to 56–100 μM MDA. DNA fragmentation was more marked after 4 than after 20 h exposure in all four cell types. DNA was damaged to a lesser extent in human hepatocytes and thyreocytes than in corresponding rat cells and in both species in hepatocytes than in thyreocytes. In both rat and human hepatocytes a 20-h exposure to the same MDA concentrations elicited a modest amount of DNA repair synthesis, as evaluated by autoradiography. Evidence of a partial reduction of DNA damage, and therefore of only partial DNA repair, was observed in rat hepatocytes and in rat and human thyreocytes incubated for 16 h in MDA-free medium after a 4-h MDA treatment. A 4-h exposure to 56, 100, and 180 μM MDA did not induce DNA lesions in primary cultures of cells from three rat organs, kidney, urinary bladder mucosa, and brain, which are resistant to MDA carcinogenic activity. Under the same experimental conditions any evidence of DNA damage was absent in primary kidney and urinary bladder cells from human donors. Taken as a whole the results of this work indicate that MDA is specifically activated to DNA-damaging reactive species by hepatocytes and thyreocytes in both rats and humans and thus suggest that liver and thyroid might be the targets of the carcinogenic activity of MDA also in humans.

Identification by Flagellum Display of an Epithelial Cell- and Fibronectin-Binding Function in the SlpA Surface Protein of Lactobacillus brevis

Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of Lactobacillus brevis ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin. For functional analysis of the SlpA surface protein, different regions of the slpA gene were expressed as internal in-frame fusions in the variable region of the fliC(H7) gene of Escherichia coli. The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size. The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence. Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella. The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein. The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin. The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells. In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells. The results identify the S-layer of L. brevis ATCC 8287 as an adhesin with affinity for human epithelial cells and fibronectin and locate the receptor-binding region within a fragment of 81 amino acids in the N-terminal part of the molecule, which in native S-layer seems inaccessible to antibodies.

Localization of glucocorticoid receptor interacting protein 1 in murine tissues using two novel polyclonal antibodies

Glucocorticoid receptor interacting protein 1 (GRIP1) is a coactivator that binds to the nuclear hormone receptors in a ligand-dependent manner and mediates transcriptional activation of the target genes. The aim of this study was to investigate GRIP1 expression in various murine tissues and whether the protein is nuclear, cytoplasmic, or both. Two novel polyclonal antibodies against amino acids 34-47 and 468-481 of GRIP1 were raised and characterized in order to study the GRIP1 expression with immunohistochemistry. Transient transfection studies with COS cells showed a clearly nuclear staining pattern and also immunohistochemical localization of GRIP1 was mainly nuclear, but cytoplasmic expression was seen as well. GRIP1 was expressed in epithelial cells of the submandibular gland, gastrointestinal tract, pancreas, kidney, uterus, mammary gland, testis, prostate, trachea, lungs and adrenal gland. GRIP1 was also detected in stromal cells of colon, rectum, urinary bladder, vagina, uterus, mammary gland and trachea, and to a lesser extent in esophagus, ureter, urethra, thymus and spleen. Smooth muscle cells of the gastrointestinal and urinary tract, uterus, epididymis, prostrate and bronchioles expressed GRIP1. Blood vessels of many organs, capsule of the kidney and prostate, mesovarium, adipocytes of the mammary gland, pericardium and cartilage of the trachea were also GRIP1-positive. Liver, thyroid gland and striated muscle did not express GRIP1. GRIP1 was expressed in a wide variety of murine organs, and expression varied between cell types and organs. In addition to mainly nuclear localization of endogenous GRIP1, cytoplasmic expression was seen as well.

Local Ca(2+) transients and distribution of BK channels and ryanodine receptors in smooth muscle cells of guinea-pig vas deferens and urinary bladder.

1. The relationship between Ca(2+) sparks spontaneously occurring at rest and local Ca(2+) transients elicited by depolarization was analysed using two-dimensional confocal Ca(2+) images of single smooth muscle cells isolated from guinea-pig vas deferens and urinary bladder. The current activation by these Ca(2+) events was also recorded simultaneously under whole-cell voltage clamp. 2. Spontaneous transient outward currents (STOCs) and Ca(2+) sparks were simultaneously detected at -40 mV in approximately 50 % of myocytes of either type. Ca(2+) sparks and corresponding STOCs occurred repetitively in several discrete sites in the subplasmalemmal area. Large conductance Ca(2+)-dependent K(+) (BK) channel density in the plasmalemma near the Ca(2+) spark sites generating STOCs was calculated to be 21 channels microm(-2). 3. When myocytes were depolarized from -60 to 0 mV, several local Ca(2+) transients were elicited within 20 ms in exactly the same peripheral sites where sparks occurred at rest. The local Ca(2+) transients often lasted over 300 ms and spread into other areas. The appearance of local Ca(2+) transients occurred synchronously with the activation of Ca(2+)-dependent K(+) current (I(K,Ca)). 4. Immunofluorescence staining of the BK channel alpha-subunit (BKalpha) revealed a spot-like pattern on the plasmalemma, in contrast to the uniform staining of voltage-dependent Ca(2+) channel alpha1C subunits along the plasmalemma. Ryanodine receptor (RyR) immunostaining also suggested punctate localization predominantly in the periphery. Double staining of BKalpha and RyRs revealed spot-like co-localization on/beneath the plasmalemma. 5. Using pipettes of relatively low resistance, inside-out patches that included both clustered BK channels at a density of over 20 channels microm(-2) and functional Ca(2+) storage sites were obtained at a low probability of approximately 5%. The averaged BK channel density was 3-4 channels microm(-2) in both types of myocyte. 6. These results support the idea that a limited number of discrete sarcoplasmic reticulum (SR) fragments in the subplasmalemmal area play key roles in the control of BK channel activity in two ways: (i) by generating Ca(2+) sparks at rest to activate STOCs and (ii) by generating Ca(2+) transients presumably triggered by sparks during an action potential to activate a large I(K,Ca) and also induce a contraction. BK channels and RyRs may co-localize densely at the junctional areas of plasmalemma and SR fragments, where Ca(2+) sparks occur to elicit STOCs.

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.

Determination and Bioimaging Method for Nitric Oxide in Biological Specimens by Diaminofluorescein Fluorometry

A simple and sensitive assay and a cellular bioimaging method for nitric oxide (NO) were developed using a novel diaminofluorescein DAF-FM and its diacetate. DAF-FM is converted via an NO-specific mechanism to an intensely fluorescent triazole derivative. For the measurement of NO, the triazole derivative of DAF-FM was determined by reversed-phase high-performance liquid chromatography with fluorescence detection. In the presence of 1 μM DAF-FM, the concentrations of NOR-1, an NO donor, in the range of 2–200 nM were linearly related to the fluorescence intensity. This sensitive NO assay enabled us to detect the spontaneous and substance P-induced NO release from isolated porcine coronary arteries, both of which were dependent entirely on the NO synthase activity in vascular endothelial cells. We also obtained fluorescence images of cultured smooth muscle cells of the rat urinary bladder after loading with DAF-FM diacetate. In the cells pretreated with cytokines, the fluorescence intensity increased with time after DAF-FM loading. This increase in the fluorescence intensity was blocked by prior treatment of the muscle cells with an NO synthase inhibitor, NG-nitro-l-arginine methyl ester. Therefore, the present novel diaminofluorescein fluorometry should be useful not only for sensitive NO assay, but also for NO imaging in a variety of biological specimens.

Expression of the ETA and ETB Endothelin Receptor Subtype mRNA in Human Detrusor Cultured Smooth Muscle Cells

The aim of this study was to determine the expression of the endothelin receptor subtype mRNAs in human detrusor cultured smooth muscle cells using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). First strand cDNA was made from human detrusor cultured smooth muscle cells total RNA and used for PCR with primers designed to amplify fragments of the ET_A and ET_B endothelin receptor subtype cDNA sequences. Subcloned fragments of the ET_A and ET_B endothelin receptor cDNAs were used to synthesize digoxigenin-labeled cRNA probes by in vitro transcription. COS-7 cells transfected with the ET_A and ET_B receptor cDNAs were used as positive control and to confirm the absence of cross-hybridization due to sequence homology. Both ET_A and ET_B receptor mRNAs were detected by RT-PCR analysis. By ISH, both ET_A and ET_B receptor subtype mRNAs were detected. However, ET_A signal was much more intense than ET_B signal. These results indicate that mRNAs for both ET_A and ET_B receptors are expressed in detrusor smooth muscle cells of human urinary bladder. The ET_A receptor is the predominant detrusor ET receptor.

DNA damage induced by 3,3'-dimethoxybenzidine in liver and urinary bladder cells of rats and humans.

3,3'-Dimethoxybenzidine (DMB), a congener of benzidine used in the dye industry and previously found to be carcinogenic in rats, was evaluated for its genotoxic activity in primary cultures of rat and human hepatocytes and of cells from human urinary bladder mucosa, as well as in liver and bladder mucosa of intact rats. A similar modest dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic DMB concentrations ranging from 56 to 180 microM. Replicating rat hepatocytes displayed a modest increase in the frequency of micronucleated cells after a 48-h exposure to 100 and 180 microM concentrations. In primary cultures of human urinary bladder mucosa cells exposed for 20 h to 100 and 180 microM DMB, the Comet assay revealed a clear-cut increase of DNA fragmentation. In rats given one-half LD50 of DMB as a single oral dose, the GSH level was reduced in both the liver and urinary bladder mucosa, whereas DNA fragmentation was detected only in the bladder mucosa. Taken as a whole, these results suggest that DMB should be considered a potentially genotoxic chemical in both rats and humans; the selective effect on the rat urinary bladder might be the consequence of pharmacokinetic behavior.