ATP enhances spontaneous calcium activity in cultured suburothelial myofibroblasts of the human bladder

Background

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves. They express purinergic receptors and show calcium transients in response to ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Since ATP concentration is likely to be very low during the initial filling phase, we hypothesized that sMF Ca²⁺ activity is affected even at very low ATP concentrations. We investigated ATP induced modulation of spontaneous activity, intracellular calcium response and purinergic signaling in cultured sMF.

Methodology/Principal Findings

Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10⁻¹⁶ to 10⁻⁴ mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18%±1.65 of the sMF (N = 48 experiments). ATP significantly increased calcium activity even at 10⁻¹⁶ mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1 µM; A-317491, 1 µM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence.

Conclusions/Significance

Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca²⁺ response in cultured sMF at very low concentrations, most likely involving P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.     

Ultrastructure and permeability of lymph node microvasculature in the mouse

Summary The microvasculature of lymph nodes and Peyer's patches consists of arterioles, capillaries and venules. The postcapillary segment comprises high-endothelial venules (HE venules) as well as ordinary venules. In order to study the ultrastructure of the microvasculature, particularly with respect to the nature of intercellular junctions, lanthanum and ruthenium red were used as tracers. Furthermore, to evaluate the permeability properties of the different segments of the microvasculature, intravenously injected horseradish peroxidase (HRP; MW: 40,000) was used.All segments of the microvasculature are permeable to HRP. However, the mechanism of transport across the vascular wall varies in the different segments, apparently correlated with a gradual decrease in number of transport vesicles and a gradual attenuation in the sealing of the endothelial cells. Tight junctions are present in arterioles, and it is assumed that HRP reach the basal lamina exclusively by vesicular transport. Incomplete or focal tight junctions are present in the capillaries, and both intercellular and vesicular pathways are observed. In the venules the intercellular pathway seems to be the dominant one, while vesicular transfer is negligible. However, some micropinocytic vesicles in the HE venule endothelial cells probably represent the initial stage of an intracellular digestion.     

成年牦牛心室壁微血管的形态特征

用ABS血管铸型、扫描电镜观察法和血管炭素墨水灌注、组织切片法研究了成年牦牛心脏微血管的构筑特征,首次对各级微血管的管径和毛细血管的密度进行了测量,并对成年牦牛心室壁的微血管进行了分类.结果显示:成年牦牛心脏微动脉、毛细血管前微动脉和毛细血管的管径平均值分别为为 78.50±10.23 μm ,16.24±2.27 μm ,6.57±2.28 μm.其管径范围分别为12.5～100 μm,12.50～19.99 μm,6.25～12.50 μm.成年牦牛心脏微动脉一般经3-4级分支才发出毛细血管,其第一、第二、第三和第四级分支的管径平均值分别为87.64±4.87 μm, 69.46±6.67 μm, 48.52±5.77 μm,30.45±5.44 μm.其范围分别为79.55～95 μm, 59.31～79.55 μm,37.50～59.31 μm,19.99～37.50 μm.成年牦牛心肌层毛细血管的密度为2 528±263根/mm2,靠近心外膜处毛细血管的密度为1 864±179根/mm2,心内膜毛细血管的密度为1 636±235根/ mm2.成年牦牛心脏微动脉铸型表面呈典型的"树皮样"结构,偶尔可见卵圆形的内皮细胞核压痕.成年牦牛心脏毛细血管前微动脉铸型形态呈锥状,铸型表面有环行缩窄.成年牦牛心脏的毛细血管铸型表面光滑,有环形缩窄,无内皮细胞核压痕.成年牦牛心肌层中毛细血管与心肌纤维平行,并形成"H"形或"Y"形的广泛吻合,而在靠近心内膜处毛细血管形态较为扭曲,毛细血管多形成平面或立体的吻合.成年牦牛心脏微静脉管径多在300 μm以下,管腔扁且不规则,微静脉铸型呈"树根"样结构.     

Morphological characteristics of the microvasculature in healing myocardial infarcts.

Myocardial infarction (MI) is associated with an angiogenic response, critical for healing and cardiac repair. Using a canine model of myocardial ischemia and reperfusion, we examined the structural characteristics of the evolving microvasculature in healing MI. After 7 days of reperfusion, the infarcted territory was rich in capillaries and contained enlarged, pericyte-poor "mother vessels" and endothelial bridges. During scar maturation arteriolar density in the infarct increased, and a higher percentage of microvessels acquired a pericyte coat (60.4 +/- 6.94% after 28 days of reperfusion vs 30.17 +/- 3.65% after 7 days of reperfusion; p<0.05). The microvascular endothelium in the early stages of healing showed intense CD31/PECAM-1 and CD146/Mel-CAM immunoreactivity but weak staining with the Griffonia simplicifolia lectin I (GS-I). In contrast, after 28 days of reperfusion, most infarct microvessels demonstrated significant lectin binding. Our findings suggest that the infarct microvasculature undergoes a transition from an early phase of intense angiogenic activity to a maturation stage associated with pericyte recruitment and formation of a muscular coat. In addition, in the endothelium of infarct microvessels CD31 and CD146 expression appears to precede that of the specific sugar groups that bind the GS-I lectin. Understanding of the mechanisms underlying the formation and remodeling of the microvasculature after MI may be important in designing therapeutic interventions to optimize cardiac repair.     

Effects of relaxin on the microvasculature of mouse mammary gland

The effects of RLX on the microvasculature of the mouse mammary gland are reported. RLX (pure porcine standard NIH-RXN-P1) at a dose of 3 GPU was administered subcutaneously to virgin adult mice ovariectomized 12 days before. The mammary glands were removed 18-20 h after RLX injection and their examination by light microscopy did not reveal any substantial growth-response to the hormone. Histology and morphometry indicated striking dilation of microvessels, especially capillaries, and electron microscopy revealed an increase in the micropinocytotic vesicles, thus suggesting enhanced transendothelial transport of substances. Such phenomena, which were independent of a release of granules by mast cells, may represent an important component of the mammotrophic action of RLX.     

Effects of airway distension on leukocyte recruitment in the mouse tracheal microvasculature.

We have shown previously that excessive distention of the rat trachea during mechanical ventilation results in enhanced leukocyte recruitment to the airway (Lim LH and Wagner EM. Am J Respir Crit Care Med 168:1068-1074, 2003). The objectives of this study were to develop a mouse model of positive end-expiratory pressure (PEEP)-induced leukocyte recruitment to the airway and begin to pursue molecular mechanisms that may contribute to the in vivo observation of increased leukocyte adhesion after PEEP exposure. We studied C57BL/6 wild-type mice and mice deficient in P-selectin or intercellular adhesion molecule-1 (ICAM-1) exposed to intermittent PEEP (8 cmH(2)O) applied five times for a 1-min duration, at 10-min intervals. After the imposed ventilatory stress, during normal ventilation (0.2 ml/breath, no PEEP), leukocyte adhesion in tracheal postcapillary venules was determined using intravital microscopy. PEEP induced a time-dependent increase in leukocyte adhesion that was significantly increased between 0 and 60 min (P < 0.01). Furthermore, PEEP-induced leukocyte adhesion at 60 min was ablated in P-selectin- and ICAM-1-deficient mice. These findings demonstrate the essential nature of both P-selectin and ICAM-1 within airway postcapillary venular endothelium for leukocyte recruitment after airway distension.     

Immunohistochemical localization of angiotensin II in the mouse pancreas

Previous studies have suggested the presence of a tissue renin--angiotensin II system in the pancreas. These studies were based on the observation of several key components of the renin--angiotensin II system using molecular biological, biochemical and pharmacological approaches. In the present study, angiotensin II was localized immunohistochemically in the mouse pancreas using an indirect immunoperoxidase-staining technique. The results showed that angiotensin II-like immunoreactivity was localized predominantly in the endothelial cells of pancreatic blood vessels and the epithelial cells of pancreatic ducts from a subgroup of the vessels and ducts. Compared with those found in the pancreatic blood vessels and ductal system, a less pronounced immunoreactivity for angiotensin II was also observed in the acinar cells and in the smooth muscle layers overlying the pancreatic ducts as well as the blood vessels. However, no angiotensin II-like immunoreactivity was detected in the islet cells. Taken together with previous findings, the present results suggest a local angiotensin II-forming system in the mouse pancreas, which may be a significant autocrine or paracrine modulator of diverse pancreatic functions, including regulation of pancreatic blood flow and pancreatic anion secretion     

Immunohistochemical localization of calmodulin in mouse brain

Calmodulin is a well-known calcium-binding protein which is ubiquitous in the plant and animal kingdoms and regulates many cellular processes. In this paper, the distribution of calmodulin in mouse brain was studied immunohistochemically using specific anti-calmodulin IgG which was raised in the rabbit by immunization with native calmodulin. Immunoreactive staining was observed in the cells in almost all areas of the brain, but its intensity varied. Some areas were stained heavily even in the presence of high concentration of NaCl. These differed from those stained immunohistochemically with antibody against other calcium-binding proteins, parvalbumin or vitamin D-dependent calcium-binding protein.     

Cytokine effects on gap junction communication and connexin expression in human bladder smooth muscle cells and suburothelial myofibroblasts

Background

The last decade identified cytokines as one group of major local cell signaling molecules related to bladder dysfunction like interstitial cystitis (IC) and overactive bladder syndrome (OAB). Gap junctional intercellular communication (GJIC) is essential for the coordination of normal bladder function and has been found to be altered in bladder dysfunction. Connexin (Cx) 43 and Cx45 are the most important gap junction proteins in bladder smooth muscle cells (hBSMC) and suburothelial myofibroblasts (hsMF). Modulation of connexin expression by cytokines has been demonstrated in various tissues. Therefore, we investigate the effect of interleukin (IL) 4, IL6, IL10, tumor necrosis factor-alpha (TNFα) and transforming growth factor-beta1 (TGFβ1) on GJIC, and Cx43 and Cx45 expression in cultured human bladder smooth muscle cells (hBSMC) and human suburothelial myofibroblasts (hsMF).

Methodology/Principal Findings

HBSMC and hsMF cultures were set up from bladder tissue of patients undergoing cystectomy. In cytokine stimulated cultured hBSMC and hsMF GJIC was analyzed via Fluorescence Recovery after Photo-bleaching (FRAP). Cx43 and Cx45 expression was assessed by quantitative PCR and confocal immunofluorescence. Membrane protein fraction of Cx43 and Cx45 was quantified by Dot Blot. Upregulation of cell-cell-communication was found after IL6 stimulation in both cell types. In hBSMC IL4 and TGFβ1 decreased both, GJIC and Cx43 protein expression, while TNFα did not alter communication in FRAP-experiments but increased Cx43 expression. GJ plaques size correlated with coupling efficacy measured, while Cx45 expression did not correlate with modulation of GJIC.

Conclusions/Significance

Our finding of specific cytokine effects on GJIC support the notion that cytokines play a pivotal role for pathophysiology of OAB and IC. Interestingly, the effects were independent from the classical definition of pro- and antiinflammatory cytokines. We conclude, that connexin regulation involves genomic and/or post-translational events, and that GJIC in hBSMC and hsMF depend of Cx43 rather than on Cx45.     

Immunohistochemical localization of taurine in various tissues of the mouse

Summary The localization of taurine was investigated in several tissues of the mouse. Immunohistochemical methods using a polyclonal antibody for taurine derived from rabbits was used in these studies. This method was used since it is a simple procedure and the results are clear and reliable. Tissues were fixed with paraformaldehyde, embedded in paraffin and treated in a microwave oven before using an avidin-biotin-complex method (ABC method). Control staining was accomplished by employing absorption staining using various amino acids: taurine, arginine, cysteine, hypotaurine and others. For purposes of comparison, radioautography (RAG) with³H-taurine was performed to confirm the reliability of the immunohistochemical staining compared with the localization of the³H-taurine incorporation in endothelial cells of the blood vessels of several tissues. In this investigation, immunoreactivity was broadly observed in many tissues: Purkinje cells of the cerebellum, glia cells of brain tissue, cardiac muscle cells, matrices of the bone, mucus granules of goblet cells of the intestines, and brown adipose cells of the fetus. Although the meaning of this widespread localization of taurine can not be explained completely, we surmise that taurine may have a different function in each of the tissues. In addition, taurine reactivity was observed in cell nuclei which was evidence of the presence of taurine in the nuclei.     

Immunohistochemical analysis of neuron types in the mouse small intestine

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/± TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.     

Immunohistochemical study of calmodulin in developing mouse testis

The purpose of this study was to determine the localization of calmodulin in the developing mouse testis by the indirect immunoperoxidase method. In addition, the amount of calmodulin in pachytene spermatocytes, spermatids, and residual bodies isolated from the mouse testis and epididymal spermatozoa was quantitated by the adenylate cyclase activation assay and by enzyme immunoassay. The relative levels of calmodulin in the developing mouse testis and in the isolated testicular germ cells were confirmed by western transfer staining. The level of immunoreactive calmodulin was very low in the testes from immature animals. In testes from the mature mouse, calmodulin was found to be localized in spermatocytes and spermatids, but was not found in spermatogonia, Sertoli cells, and interstitial cells. By contrast, immunochemical staining of tubulin was extremely intense in Sertoli cells. Biochemical determinations also showed that pachytene spermatocytes, round spermatids, spermatozoa, and residual bodies contained 14.9 micrograms, 15.8 micrograms, 2.3 micrograms and 5.2 micrograms of calmodulin per mg of protein, respectively. Both the immunochemical and the biochemical studies revealed that levels of calmodulin were high in the spermatocytes and in the round spermatids, as compared to the level in spermatozoa. This fact strongly suggests that the large amount of calmodulin in mammalian testes may be associated primarily with meiotic divisions and/or spermatogenesis.     

Immunohistochemical markers in the evaluation of tumors of the urinary bladder: a review

The clinical utility of immunohistochemistry in the diagnosis of tumors of the urinary bladder is well established. With recent advances in molecular biology and novel technologies, several biomarkers have emerged as important adjuncts in the diagnosis of lesions of the bladder. When used in conjunction with careful histologic examination, immunohistochemistry can be a valuable aid in classifying adenocarcinoma presenting in the bladder and mesenchymal lesions of the bladder and in establishing the urothelial origin of a metastatic tumor. In addition, a number of biomarkers may prove to be important indicators of prognosis or response to chemotherapy.     

Immunohistochemical localization of erythropoietin in the rat and mouse submandibular gland

Synopsis There is a great deal of evidence to indicate that organs other than the kidney are involved in erythropoietin production. In this paper, it is reported that erythropoietin has been localized with an immunocytochemical method in the granular ducts of the submandibular glands of the rat and mouse.     

Immunohistochemical distribution of epimorphin in human and mouse tissues

A novel protein epimorphin has been identified as a mesenchymal signal factor. We reported previously ubiquitous expression of epimorphin in normal skin and a significant increased expression in diseased human skin. The present immunofluorescence study was conducted to determine systematically the distribution of epimorphin in adult human organs with an anti-epimorphin monoclonal antibody. Epimorphin was found to be widely distributed in all human organs examined. It was present in the connective tissue adjacent to or around various epithelial tissues, muscles and vessels. In particular, strong staining was present on the endomysium of muscles, the adventitia of blood vessels, along the sinusoidal lining of hepatocytes and connective tissue around epithelial cells, exocrine and endocrine glands. The results suggest that epimorphin may play a key role in maintaining normal tissue structure and interaction between mesenchymal tissue and epithelial tissue in vivo. ©; 1998 Chapman & Hall     

Immunohistochemical characteristics of chronic erosion of the gastric mucosa

By methods of immunohistochemistry, light and electron microscopies gastrobiopsies from regions of acute and chronic erosions and border zones of gastric mucosa were studied in 36 patients with erosive gastritis. There was determined the decrease of amount of IgA-producing cells against a background of the increase of IgG-producing plasmocytes in the zone of mucosal defect. IgG-precipitations and fixation of heterogeneous complement in fibrinoid necrosis of erosive defect were revealed. Mucosal microcirculatory disturbances stipulated by immunocomplex injuries are found to be mosaic. There is given proof of the role of second local immunity disturbances in origin and chronization of erosive defects of gastric mucosa.