Inverse expression of uroplakins and inducible nitric oxide synthase in the urothelium of patients with bladder outlet obstruction.

OBJECTIVE: To assess the expression and distribution of uroplakins, protein subunits of the asymmetric unit membrane (AUM), and inducible nitric-oxide synthase (iNOS) in the urinary bladder urothelium of patients with bladder outlet obstruction (BOO) caused by benign prostatic hyperplasia (BPH). PATIENTS AND METHODS: Urinary bladder urothelium samples from 15 men (mean age 69 years) with BOO secondary to BPH were processed for light and electron immunocytochemistry. Uroplakins and iNOS were detected, and areas of apical surface covered with AUM were compared with those of iNOS-positive urothelial cells. RESULTS: Areas of superficial urothelial cells with no AUM were found in all obstructed bladder samples. The immuno-electron microscopy showed that the uroplakin-positive cells had the characteristic appearance of terminally differentiated umbrella cells, whereas cells from the uroplakin-negative regions were undifferentiated, typically showing microvilli on their apical surface. iNOS was not detected in areas with continuous AUM staining, but was readily detected in the uroplakin-negative areas. There was an inverse correlation between the intensity of uroplakin and iNOS staining. CONCLUSIONS: In patients with BOO associated with BPH, some superficial urothelial cells lacked the AUM, suggesting focal compromise of the blood-urine permeability barrier. In such relatively undifferentiated urothelial zones there was an accompanying increase in the expression of iNOS, which marks perturbed urothelial differentiation and may modulate bladder response to the outlet obstruction.

An EPR study of the secretion of G-CSF heterologous protein from Pichia pastoris.

The biologically active protein known as human granulocyte colony stimulating factor (G-CSF) can be efficiently secreted from the transformed GS115 Pichia pastoris (GS115/pPIC9/G-CSF), which contains an alpha-mating-factor prepro signal sequence and an alcohol oxidase I promoter, both introduced using the pPIC9 plasmid. To study the P. pastoris G-CSF protein-secretion process, changes to the plasma membrane's lateral domain structure were monitored using electron paramagnetic resonance (EPR). The GS115 and its transformed analogue show that the plasma membrane can be described by fluid-disordered and fluid-ordered lateral domains. The relative proportion of these domains is a sensitive parameter that is able to describe the membrane's involvement in the protein-excretion process. Here, P. pastoris GS115 served as a control for us to compare with the GS115/pPIC9/G-CSF heterologous protein-secreting cells. Electron paramagnetic resonance studies using the spin-probe 5-doxyl methyl ester of palmitic acid [MeFASL (10,3)] showed that during cultivation in a glycerol medium all types of cells had a relatively high proportion of cell-membrane fluid-disordered domains, while during the production phase the G-CSF protein-secreting cells showed a decrease in the proportion of fluid-disordered domains. We ascribe this effect to the vesicular lipid exchange caused by the fusion of secretary vesicles with the plasma membrane during exocytosis. Using electron microscopy and immunocytochemistry intracellular vesicles containing the G-CSF protein were detected. Our studies support the exocytotic mechanism of the heterologous protein secretion.

Restoration of the rat urothelium after cyclophosphamide treatment.

Processes leading to the recovery of a normal three-layered urothelium from a hyperplastic urothelium induced by cyclophosphamide (CP) treatment in rats have been investigated. A single intraperitoneal (ip) dose of CP caused extensive loss of cells from urothelium, but the remaining cells started to express epidermal growth factor receptor (EGFR) in their plasma membranes. On day 2 after CP injection, proliferating cell nuclear antigen (PCNA) immunohistochemistry showed a rapid increase in positively stained nuclei, from which a hyperplastic urothelium developed, composed of undifferentiated cells expressing EGFR over the entire plasma membrane. Subsequently, EGFR gradually disappeared from the apical plasma membrane but remained in the basolateral membranes. After day 6, PCNA-positive nuclei in all cell layers decreased, except in basal cells. Apoptotic cells were detectable by the TUNEL assay at day 2, and increased in number in all layers of the hyperplastic urothelium until day 10, returning to the control levels by day 14. Electron microscopic evidence showed that apoptotic cells were either pinched off into the bladder lumen or phagocytosed by the neighbouring urothelial cells. Thus, the urothelium responds to the damage by intense proliferation for a week, resulting in an undifferentiated hyperplastic state. Differentiation of superficial cells then begins and damaged cells are gradually removed by apoptosis until the three-layered urothelium is fully restored by two weeks following CP treatment.

Postnatal restoration of the mouse urinary bladder urothelium.

Mouse urothelium is disrupted just before birth, followed by a postnatal restoration process which includes cell proliferation, death and differentiation. We assessed urothelial proliferation by the expression of proliferating cell nuclear antigen (PCNA), desquamation by electron microscopy, and apoptosis by TUNEL staining and urothelial differentiation by the expression of uroplakins and cytokeratin 20 (CK20) as well as the apical plasma membrane maturation. Our results indicated that urothelial proliferation was high from birth until about the 14th postnatal day. A majority of basal cells and even occasional superficial cells were PCNA positive during the first 5 postnatal days. Cell death occurred during the first 9 postnatal days. Between birth and day 5, single cells underwent apoptosis, whereas between days 6 and 9 cells mainly desquamated. CK20 and uroplakins were expressed in all superficial cells in postnatal urothelium. Their subcellular distribution characteristically changed in accordance with the progressive differentiation of superficial cells. During the urothelial postnatal development, proliferation activity slowly decreases to the proliferatively quiescent urothelium of the adult animal. Apoptosis is present in the first 9 postnatal days and within a few days of this period it appears simultaneously with desquamation. Superficial urothelial cells gradually differentiate, which is reflected in the changeable morphology of the apical plasma membrane.

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein.

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca(2+)-dependent activator protein for secretion), a protein required for Ca(2+)-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca(2+) elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca(2+) dependencies. A threshold of approximately 10 microM Ca(2+) was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca(2+) activity < 10 microM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca(2+) and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.

Urothelial cell detachment and differentiation in urinary bladder.

In developing and in repairing bladder, proliferation of the transitional urothelium is followed by cell detachment--desquamation or apoptosis. Proliferation results in formation of terminally differentiated superficial cells and this process may be followed by checking the cells on the presence of differentiation markers. The formation of an asymmetric unit membrane (AUM) structure (plaque) on the cell surface is in correlation with urothelial differentiation. Thus, the microstructure of the luminal surface of the urinary bladder provides a very convenient differentiation biomarker. The surface of immature cells showed a pattern of microvilli. The progress of differentiation was associated with microvili arranged in rows finally forming the characteristic pattern of ridges in terminally differentiated cells. These results demonstrate that the characteristic surface pattern and the AUM plaque formation in the apical plasma membrane of superficial urothelial cells are associated with specific morphology, and patterns and thus help detect differentiation level of cell.

Actin filaments during terminal differentiation of urothelial cells in the rat urinary bladder.

The localisation of actin filaments was studied in rat urothelial cells during differentiation which accompanied regeneration after cell damage induced by cyclophosphamide (CP). By immunofluorescence it was established that actin filaments equally stained along the cell circumference in basal and intermediate cells, while basolateral cell membrane expression was found in terminally differentiated superficial cells. During regeneration, after CP treatment, simple urothelial hyperplasia developed with smaller cuboidal superficial cells, in which actin filaments were equally distributed under the apical and basolateral plasma membranes. As demonstrated by immunoelectron microscopy, the apical surface of these superficial cells was covered with microvilli containing bundles of actin filaments. Within 1 week, the urothelium reverted to its normal three-layer thickness. Superficial cells became larger and flattened and the unthickened apical plasma membrane matured into a thick asymmetric unit membrane. Concomitantly actin filaments disappeared from apical areas of superficial cells while remaining abundant at basolateral areas. Our results indicate that in the urothelium subcellular distribution of actin filaments can be considered as a marker of cell differentiation.

Uroplakins and cytokeratins in the regenerating rat urothelium after sodium saccharin treatment.

A sodium saccharin (NaSac) diet was used to induce cell damage and regeneration in the urothelium of the male rat urinary bladder. Foci of terminally differentiated superficial cell exfoliation were detected after 5 weeks and their number increased after 10 and 15 weeks of the diet. At the sites of superficial cell loss, regenerative simple hyperplasia developed. Within 5 weeks of NaSac removal, regeneration re-established normal differentiated urothelium. In order to follow urothelial differentiation during regeneration we studied the expression of uroplakins and cytokeratins by means of immunocytochemistry and immunohistochemistry, respectively. Normal urothelium was characterised by terminally differentiated superficial cells which expressed uroplakins in their luminal plasma membrane and cytokeratin 20 (CK20) in the cytoplasm. Basal and intermediate cells were CK20 negative and cytokeratin 17 (CK17) positive. In hyperplastic urothelium all cells synthesised CK17, but not CK20. Differentiation of the superficial layer was reflected in three successive cell types: cells with microvilli, cells with rounded microridges and those with a rigid-looking plasma membrane on the luminal surface. The cells with microvilli did not stain with anti-uroplakin antibody. When the synthesis of uroplakins was detected rounded microridges were formed. With the elevated expression of uroplakins the luminal plasma membrane becomes rigid-looking which is characteristic of asymmetric unit membrane of terminally differentiated cells. During differentiation, synthesis of CK17 ceased in superficial cells while the synthesis of CK20 started. These results indicate that during urothelial regeneration after NaSac treatment, specific superficial cell types develop in which the switch to uroplakin synthesis and transition from CK17 to CK20 synthesis are crucial events for terminal differentiation.

Reorganisation of the urothelial luminal plasma membrane in the cyclophosphamide treated rats.

The luminal surface of differentiated urothelium is characterised by the presence of asymmetric unit membrane (AUM) which contains four major integral proteins, uroplakins. Cyclophosphamide (CP) causes extensive denudation of the urothelium that is followed by regeneration. In this study the differentiation of the urothelial luminal plasma membrane was examined in CP treated rats by electron microscopy. Single dose of cyclophosphamide was injected and rats were killed after 1, 3, 7, 14 or 28 days. One day after the treatment only few cells remained in the urothelium. They had a flat surface and did not stain with anti-AUM antibody. During regeneration cells with short or pleomorphic microvilli and latter cells with ropy and leafy microridges appeared. Cells with either type of microvilli were not labelled whereas cells with microridges were labelled with anti-AUM. At day 28 the luminal surface was normal and labelled. These results show a good correlation of morphogenesis and the expression of uroplakins.

Endocytosis during postnatal differentiation in superficial cells of the mouse urinary bladder epithelium.

This electron microscopical study was performed in order to follow the endocytic pathway of horseradish peroxidase and colloidal gold tracers and to determine the involvement of endocytosis in postnatal differentiation in superficial cells of the mouse urinary bladder epithelium. Morphometric analyses of late endosomes/multivesicular bodies from day of birth to day 25 were performed. The internalisation and intracellular transport of luminal plasmalemma to multivesicular bodies via endocytic vesicles, early endosomes and pleomorphic compartments was established. Dynamic changes in endocytic activity took place within the first few days of postnatal differentiation. During this period the number of multivesicular bodies changed in an inverse ratio to their size. After the third day endocytic activity gradually approached the low rate of adult urothelium.