Expression of stress fibers in bullfrog mesothelial cells in response to tension

The relationship between stress fibers and tension in mesothelial cells of the bullfrog small intestine was examined by fluorescence cytochemistry using en face mesothelial cell preparations. In nontreated controls, actin revealed by rhodamine-phalloidin staining was localized only along the margins of the mesothelial cells. On the other hand, many stress fibers were formed in the mesothelial cells within 5-7 min after stretching of the intestinal wall in a given direction. The orientation of stress fibers within the cells was coincident with the direction of the tension applied. These cytoplasmic fibers disappeared almost completely from the mesothelial cells within 30 min after the release of tension. According to a difference in the intensity of tension necessary for stress fiber expression, the intestinal mesothelial cells were classified into two groups. Furthermore, cells containing stress fibers in each group showed a rapid increase in number once a given value of tension was applied. The present results indicate that the mesothelial cells of bullfrog small intestine may develop stress fibers to counteract tension exerted on the intestinal wall. Such stress fibers may serve to maintain cellular integrity by strengthening the cellular attachment to subepithelial tissue.     

A morphologic and histochemical study of the mesentery in the guinea pig

The guinea pig mesentery is a uniform, continuous, thin (18 micron) sheet of connective tissue covered by a single layer of flattened mesothelial cells on both surfaces. Tight and gap junctions provide for cell-to-cell adhesion among mesothelial cells. These cells possess numerous micropinocytotic vesicles; a conspicuous basal lamina separates the mesothelium from the underlying connective tissue. Most of the material found between the two serous coverings consisted of a three-dimensional meshwork of abundant collagenous fibers intermingled with a sparse net of very thin (0.4 micron) elastic fibers. Two distinct populations of collagen fibrils are segregated into different compartments of the mesentery. One population is formed of thick (56 nm) fibrils which associate to form closely packed fibers. The second population, composed of loosely arranged thin (38 nm) fibrils which do not become assembled into fibers, is found underlying the basal lamina that separates the mesothelium from the connective tissue. These observations strongly suggest that the mesentery contains both collagens type I and type III. The guinea pig mesentery contains 6.8 mg of sulfated glycosaminoglycans/g dry weight. Most of these glycosaminoglycans (78%) were identified as dermatan sulfate, whilst the rest (22%) corresponded to heparan sulfate.     

Inhibitory effect of curcumin on the invasion of rat ascites hepatoma cells in vitro and ex vivo

Curcumin, a yellow pigment in turmeric, is a food factor withantioxidative activity. The effect of curcumin on the proliferation and invasion of the rat ascites hepatoma AH109Acells was studied in vitro and ex vivo assay systems. Especially, a co-culture system of the hepatoma cellswith mesothelial cells derived from rat mesentery was employed to investigate the invasive motility. Curcumin suppressed thehepatoma slipping motility in a dose-dependent manner up to 5 M and thereafter maintained the effect up to 20 M, whereas this substance exerted little influence on the proliferation of the hepatoma cells at the same concentrations. Sera obtained from rats orally given curcumin also inhibited the AH109A cellular invasive movement when added to the culturemedium. Hepatoma cells previously cultured with hypoxanthineand xanthine oxidase showed a highly invasive activity. Curcumin and curcumin-loaded rat sera suppressed this reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with hypoxanthine, xanthine oxidase and either of curcumin samples. These resultssuggest that the antioxidative property of curcumin may beinvolved in its anti-invasive action.     

Role of marginal stress fibers formed in the rat vascular endothelial cells

Fluorescence cytochemistry using en face preparations of rat vascular endothelial cells (ECs) revealed the localization of actin, fibronectin (FN) and fibronectin receptor (FNR) along not only central stress fibers (SFs) but also the cell margins. Electron microscopy showed very close proximity between the topographical distribution of intracellular microfilament bundles and that of subendothelial FN in the EC margins. Therefore, these basal and marginal actin cables may be comparable to the well-established central SFs present in ECs. Formation of the central SFs was induced in ECs or mesothelial cells in response to tension, by which their cellular integrity seems to be effectively maintained. However, even when central SF formation was inhibited by cytochalasin D, the ECs with marginal SFs showed high resistance to mechanical tension, whereas mesenteric mesothelial cells having no such fibers easily lost their integrity. Thus, together with central SFs, the marginal SFs characteristic of rat vascular ECs may play an essential role in strengthening cell-matrix adhesion.     

The fate and role of openings formed in the mesentery of bullfrog tadpoles,with reference to the contour of mesothelial cells

Using morphological techniques, histological changes of the mesentery were observed during the development of the bullfrog, Rana catesbeiana. The tadpoles of this species had many openings all over the mesentery from the duodenum through the large intestine. Most of the openings were elliptical and less than 3 × 2 mm in size. The openings became remarkably decreased in size and number with rapid narrowing of the mesentery occurring during the period of metamorphic climax, and had almost completely disappeared by the end of metamorphosis. Appearance and disappearance of the openings were closely correlated with the changes in the dimensions of the mesentery. Furthermore, in parallel with these changes in the openings, a noticeable alteration occurred in the shape of the mesothelial cells of the mesentery. In tadpoles having no mesenteric openings, the mesothelial cells had a polygonal contour, which became transformed once the openings were formed in the mesentery. The shapes of the transformed cells were classified into two types, one having many radiating cell processes and the other a very slender and spindle-shaped contour. Both types of cells eventually became transformed into a definitive type of cell exhibiting a roundish polygonal contour by the end of metamorphosis. From these findings it was concluded that the growing mesentery might, of necessity, give rise to the openings and transformation of the mesothelial cells to enable rapid lengthening and shortining of the intestinal tract to occur during the postembryonic development of anuran amphibians.     

Differential effects of tumor necrosis factor and asbestos fibers on manganese superoxide dismutase induction and oxidant-induced cytotoxicity in human mesothelial cells

We compared induction of manganese superoxide dismutase (MnSOD) by asbestos fibers and tumor necrosis factor (TNF) using cultures human mesothelial cells. Transformed pleural mesothelial cells (MET 5A) were exposed for 48 h to amosite asbestos fibers (2 g/cm²), to TNF (10 Ng/ml), and to the combination of these two. TNF and amosite+TNF caused significant MnSOD mRNA upregulation. Similarly MnSOD specific activity was increased by TNF (290% increase) and the amosite+TNF combination (313% increase) but not by amosite alone. In cell injury experiments, amosite and amosite+TNF exposures caused significant cell membrane injury when assessed by lactate dehydrogenase release, which was 31% and 57% higher than in the unexposed cells. However, only the amosite+TNF combination caused significant depletion of cellular high-energy nucleotide when expressed as percentage of [¹⁴C]denine labeling in cellular high-energy nucleotides. The nucleotide levels were 91.5 ± 2.0% in the unexposed cells, 89.9 ± 3.9% in amosite-exposed cells, 90.1 ± 2.2% in TNF-exposed cells, and 79.8 ± 9.4% in amosite+TNF-exposed Amosite+TNF-exposed cells were also most sensitive to menadione (20 mol/L, 2 h), a compound which generates superoxide radicals intracellularly. In conclusion, our data suggests that in human mesothelial cells inflammatory cytokines but not asbestos fibers alone can cause MnSOD induction. In this study, however amosite asbestos+TNF treatment rendered these cells more vulnerable to oxidant-induced cell damage despite elevated MnSOD activity.Abbreviations MnSOD manganese superoxide dismutase - TNF tumor necrosis factor - LDH lactate dehydrogenase     

Development of the innervation of fetal mesenteric microvasculature

Summary The innervation of the mesenteric microvasculature was studied in fetal and neonatal rabbits with the aid of methods demonstrating fluorescence of catecholamines and cholinesterase activity as well as a silver impregnation procedure. The results showed that: (1) adrenergic nerve fibers were present, coursing independently in the mesentery by day twenty-one of gestation, and were found routinely in the adventitia of arterioles and venules by day 25 of gestation; (2) cholinesterase positive cells and fibers of the myenteric plexus were present by day 18 of gestation but cholinergic fibers were not present in the mesentery until day 26; the latter not being associated with blood vessels; and (3) nerve fibers in the mesentery thought to be sensory stained positively with the Holmes silver method on day 18 of gestation.Supported by grants from the Akron Heart Association and the Heart Association of Southwestern Ohio.Recipient of N.I.H. Research Career Development Award AM-42, 370.     

Use of mesothelial cell cultures to assess the carcinogenic potency of mineral or man made fibers

Natural mineral fibers may produce pulmonary cancers and mesothelioma. In contrast with lung cancer, the incidence of fiber-induced mesothelioma is not enhanced in smokers compared to non smokers. It is therefore of special interest to use mesothelial cells to study the toxicity of natural or man made mineral fibers. Several years ago, we have developed a method to culture rat pleural mesothelial cells (RPMC). We have first studied the effects of asbestos fibers by the application of in vitro tests formerly developed to determinedthe genotoxicity and transforming potency of soluble xenobiotics. Moreover, we have determined whether RPMC expressed cytochromes P450 known to metabolize polycyclic aromatic hydrocarbons. This paper reviews the results obtained so far. It has been found that asbestos fibers produced a cell transformation and a gentoxicity characterized by the formation of aneuploid cells, abnormal anaphases, chromosomal aberrations and DNA repair (UDS). In addition, RPMC expressed different forms of cytochromes P450. It is nowadays suggested that the tumorigenic potency of asbestos fibers may be related to the fiber dimensions, to their surface properties and in vivo biopersistence; this term involves the fiber solubility in biological medium and the fiber epuration from the lung by clearance mechanisms. Experiments are now in progress to determine whether the in vitro effects are dependent on the fiber parameters suggested as playing a role in the carcinogenic potency.Abbreviations B[a]P benzo[a]pyrene - CAs chromosomal aberrations - FBS fetal bovine serum - MM malignant mesothelioma - MMF man made fibers - MMMF man made mineral fibers - RPMC rat pleural mesothelial cell - TEM transmission electron microscopy - UDS unscheduled DNA synthesis - UICC Union Internationale Contre le Cancer     

Galanin-, neuropeptide Y- and enkephalin-like immunoreactivities in catecholamine-storing paraganglia of the fetal guinea pig and newborn pig

Summary The occurrence and distribution of several neuropeptides and transmitter enzymes have been investigated by means of indirect immunofluorescence histochemistry in preaortal and carotid body-like paraganglia of the fetal guinea pig and the newborn pig. Preaortal paraganglia from the celiac and inferior mesenteric ganglion regions in fetal guinea pigs showed cell bodies immunoreactive (IR) for tyrosine hydroxylase (TH), dopamine -hydroxylase (DBH), neuropeptide Y (NPY), galanin (GAL) and metenkephalin (ENK). Almost all cells were IR for TH and DBH, whereas NPY-like immunoreactivity (-LI), GAL-LI and ENK-LI occurred less frequently. Direct double-labeling revealed the coexistence of NPY/GAL, NPY/ENK and GAL/ENK in paraganglion cells from the celiac and inferior mesenteric region. Nerve fibers and terminals were IR for ENK; fibers IR for calcitonin-gene-related peptide (CGRP) were present in the inferior mesenteric ganglion region. Preaortal paraganglia cells from the newborn pig showed TH-LI, DBH-LI, GAL-LI and ENK-LI, the distribution pattern being similar to that seen in the guinea pig; however, NPY-LI was absent. Carotid-body-like paraganglia from the newborn pig showed cell bodies IR to TH, GAL and ENK. Few cells were seen with DBH-LI. A rich supply of nerve fibers with CGRP-LI was present; some fibers exhibited ENK-LI and CCK-LI. In the adjacent superior cervical ganglion, ganglion cell bodies showed immunoreactivity to TH, DBH and NPY. A small number of cells were positive for GAL, CGRP and vasoactive intestinal polypeptide (VIP). Physiological activation of the paraganglia, leading to release or increase in catecholamines, may also change the content of the neuropeptides present in the paraganglia.     

Acute hyperglycaemia and mast-cell-dependent mitogenesis in rat

Hyperglycaemia is considered to be of possible aetiological significance for proliferative lesions in diabetes. We recently found that mast-cell-mediated proliferation in mesentery and skin is augmented in hyperglycaemic rats with longstanding diabetes. We now report a study to examine whether acute hyperglycaemia affects the proliferative response of fibroblasts and mesothelial cells that follows local mast-cell secretion in healthy rats. Hyperglycaemia was achieved by continuous intravenous infusion of glucose over a period of 2.5 h preceding and 9.5 h following administration of the mast-cell secretagogue compound 48/80. The 48/80 was given intraperitoneally which causes rapid mast-cell secretion and prompt subsequent mesenteric mitogenic stimulation. Hyperglycaemia thus persisted throughout the prereplicative period, during which stimulated cells are recruited into the cell cycle from a quiescent state. Because the ensuing mesenteric mitogenic response, estimated by specific DNA activity and mitosis counting, was unaffected by glucose infusion, acute hyperglycaemia itself appears to be of no aetiological significance for the accelerated mast-cell-dependent mesenteric mitogenesis in the diabetic rat.     

Deterministic cellular descendance and its relationship to the branching of plant organ axes

Summary. A double-wall map L-system, designated as S_5-5, was developed to simulate the cellular pattern found at the summit of shoot apices of Psilotum nudum. Commencing from a 3-sided autoreproductive founder cell, fives steps of simulation established a basic set of ten different cell types. Continuing the simulation beyond the fifth step revealed that, in addition to the regular production of new 3-sided cells, a group of autoreproductive 5-sided cells came into being. A close correspondence exists between the cells of the two-dimensional simulation and the two-dimensional cellular patterns found on the epidermis of the apices of Psilotum species. The 3-sided cells produced during the simulation correspond to the potentially organogenetic 3-sided cells that can be seen upon the apical surfaces. Successive generations of these 3-sided apical cells (which are actually 4-sided tetrahedral cells when viewed in three dimensions) and their immediate descendants are thought to be selected to organise the successive pairs of apices that bring about the repeated bifurcation of the Psilotum shoots. The 5-sided cells contribute to the cellular pavements which separate these pairs of organogenetic centres, each with their 3-sided apical cells. The cellular patterns simulated by the S_5-5 system may also correspond to the cellular patterns found on the surfaces of some other pteridophyte apices, including that of the rhizophores of Selaginella species. Tritiated-thymidine labelling of rhizophore apices revealed a group of nonproliferating cells that was associated with rhizophore bifurcation and which may correspond to a group of pavement cells. Nonproliferating cells, by regulating the siting of new organogenetic centres, may have evolved as an accompaniment to branching events such as the bifurcation of root and organ axes.Correspondence and reprints: School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, United Kingdom.     

Immunocytochemical demonstration of peptidergic cells in the pond snail Lymnaea stagnalis with an antiserum to the molluscan cardioactive tetrapeptide FMRF-amide

Summary With an antiserum to the molluscan cardioactive tetrapeptide FMRF-amide immunoreactive perikarya and nerve fibers were identified in the central and peripheral nervous system of the pond snail Lymnaea stagnalis. Their localization is described. The same antiserum yielded reactive product in particular cells of the epithelium of the alimentary tract. The use of two different fixatives, glutaraldehyde, and a mixture of glutaraldehyde, picric acid, and acetic acid (GPA) showed that certain nerve cells can be identified only in material fixed with either the one or the other of these two fixatives, a result which indicates that in Lymnaea more than one FMRF-amide-like substance may occur.Positive axon endings were found in the periphery of various nerves, i.e., in places where neurohormones are released into the blood. Other fibers were found to end, probably synaptically, on other neurons, on epithelial cells in the stomach, and between muscle cells in various parts of the body, e.g., in the heart. In these cases the FMRF-amide-like substance may function as a neurotransmitter or a neuromodulator.     

Role of actin filaments in shape formation of mesenteric mesothelial cells of the bullfrog

The role of actin filaments in the development of cellular shape in the mesenteric mesothelium of the bullfrog was studied by using a simple, new technique for making en face preparations of mesothelial sheets. By using these mesothelial cell preparations, the distribution of actin was determined by means of fluorescence microscopy with 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin and that of myosin by means of immunofluorescence microscopy. Although fluorescence produced by both NBD-phallacidin and antimyosin staining was found exclusively along the margins of the cells, its intensity was altered in correspondence with changes in cell shape. For instance, tadpole-type mesothelial cells with either an irregular or very slender cell shape showed very weak fluorescence. On the other hand, frog-type mesothelial cells with a polygonal shape showed intense fluorescence at their margins and had circumferential bundles of actin filaments at their apices. Furthermore, intercellular junctions between the mesothelial cells developed as the cell shape became polygonal during metamorphosis. The present study showed that development of circumferential bundles of actin filaments and intercellular junctions may serve to establish and maintain the definitive polygonal cellular pattern in the mesenteric mesothelium of the bullfrog.     

The fine structure of the conducting system of the monkey heart (Macaca mulatta)

Summary In the sino-atrial (S-A) node of the monkey heart two types of muscle cells occur: 1. typical nodal cells which are the predominant cells and form the nodal fibers. 2. Intercalated clear cells with various diameters (4 to 12 m) and containing poorly developed myofibrils, rich in glycogen and demonstrating poor staining properties. These latter cells are dispersed, few in number, and never form discrete fibers of themselves, but are intercalated between the cell rows of the typical nodal fibers. Such intercalated clear cells become more numerous at the periphery of the node. Interconnection between the S-A node and the conventional atrial muscle is established by a progressive transformation of nodal fibers into atrial fibers producing an intermediate (or junctional) type of fiber at the nodal periphery. However, in addition, few nodal fibers make direct contact with the atrial cardiocytes. Our light and EM studies have failed to prove the existence of truly specialized internodal pathways. Nevertheless intercalated clear cells, nodal-like cells, junctional or intermediate type of cells are relatively frequent in valvular regions (Thebesian, Eustachian, A-V, fossa ovalis) and less frequent in other regions of the atrial wall.This study was conducted in part in the Department of Histology and Embryology of the Medical University in Budapest.     

Vasoactive intestinal peptide-immunoreactive cerebrospinal fluid-contacting neurons in the reptilian lateral septum nucleus accumbens

By means of immunocytochemical demonstration of vasoactive intestinal peptide (VIP) an accumulation of cerebrospinal fluid (CSF)-contacting neurons was found in a circumscribed region of the nucleus accumbens/lateral septum of eleven reptilian (chelonian, lacertilian, ophidian, crocodilian) species. Basal processes of these cells contribute to a subependymal plexus whose density displays considerable interspecific variation. VIP-immunoreactive nerve fibers occur also in the lateral septum and the nucleus accumbens where they encompass immunonegative cells in a basket-like pattern. The CSF-contacting neurons are surrounded by columnar ependymocytes frequently arranged in a pseudostratified manner. These specialized arrays of ependymal cells, however, occupy a more extended area than the VIP-immunoreactive CSF-contacting neurons and can be traced from the rostro-ventral pole of the lateral ventricle to the interventricular foramen. These observations suggest the existence of a telencephalic site of CSF-contacting neurons which may be more widespread than hitherto thought and which may participate in a circumventricular system of the lateral ventricle. Previous studies mainly performed with birds indicate that the VIP-immunoreactive CSF-contacting neurons of the nucleus accumbens might form a part of the encephalic (extraretinal and extrapineal) photoreceptor. However, further experiments are required to test this supposition since the VIP-immunoreactive neurons of the nucleus accumbens remained unlabeled by antibodies against bovine rodopsin and chicken cone-opsin in all eleven species analysed in this investigation.     

The Lectin ArtinM Induces Recruitment of Rat Mast Cells from the Bone Marrow to the Peritoneal Cavity

Background

The D-mannose binding lectin ArtinM is known to recruit neutrophils, to degranulate mast cells and may have potential therapeutic applications. However, the effect of ArtinM on mast cell recruitment has not been investigated.

Methodology

Male Wistar rats were injected i.p. with ArtinM or ConA (control). The ability of the lectin to degranulate peritoneal and mesenteric mast cells was examined. Recruitment of mast cells to the peritoneal cavity and mesentery after ArtinM injection was examined with or without depletion of peritoneal mast cells by distilled water.

Results

ArtinM degranulated both peritoneal and mesentery mast cells in vitro. Three days after i.p. injection of the lectin there were reduced numbers of mast cells in the peritoneal lavage, while at 7 days post injection of ArtinM, the number of peritoneal mast cells was close to control values. Since immature mast cells are recruited from the bone marrow, the effect of the lectin on bone marrow mast cells was examined. Injection of ArtinM resulted in an increased number of mast cells in the bone marrow. To determine if degranulation of mast cells in the peritoneal cavity was required for the increase in bone marrow mast cells, the peritoneal cavity was depleted of mast cells with ultrapure water. Exposure to ArtinM increased the number of mast cells in the bone marrow of rats depleted of peritoneal mast cells.

Conclusions

The ArtinM induced recruitment of mast cells from the bone marrow to the peritoneal cavity may partially explain the therapeutic actions of ArtinM.     

On the impulse conducting system of the monkey heart (Macaca mulatta)

Summary The atrio-ventricular (A-V) node of the monkey heart is located in the focus of converging atrial muscle. Three main atrial muscle strands, coming from the atrio-ventricular ring, the dorsal wall of the atria, and the ventral part of the atrial septum, converge in the nodal region where they overlap and are interconnected. The junctional type of fibers establishing interconnection between the atrial muscle and the nodal tissue are not strictly localized at the periphery of the node, but may be traced further, along the A-V ring and coronary sinus. The A-V node consists of a loose peripheral and a compact distal part. In the former, typical nodal fibers were found, while the compact part shows an important individual variation in structure and cell-types. In some monkey hearts, the nodal fibers gradually become broader bundle fibers, while in other specimens the junctional fibers surround the compact part and than penetrate the nodal-His (N-H) region. These junctional fibers become nodal fibers or are in terminal contact with large clear cells up to 50 in diameter. Clear cells of various diameters are often intercalated between the cell rows of the nodal and His-bundle fibers and may form a distinct cellular gate between the node and the His-bundle.This study was conducted in part in the Department of Histology and Embryology of the Medical University in Budapest.     

Normal mitotic reactivity of fibroblasts and mesothelial cells in thrombocytopenic rat

Mitotic reactivity following 48/80-induced mast-cell secretion was studied in the mesentery of rats made thrombocytopenic, 7 days following a single injection of melphalan. In spite of a low platelet count (7% of normal), the mitogenic reaction of the mesenteric fibroblasts and mesothelial cells was normal as judged by DNA-synthesis and mitotic index. The findings suggest that platelets and platelet-growth factors are not essential for the mast-cell-mediated mitogenic reaction of these two types of connective-tissue cells studied in vivo.