No abstract

The a-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, a-gustducin, is described here to be also expressed in the stomach and intestine. The a-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.It is generally believed that the epithelium lining the inner surface of the gut can sense chemical components of the lumenal contents. This chemosensory information appears to be important for the regulation of various aspects of gastrointestinal secretion, resorption, and motility (1, 2). Classical examples of intestinal chemosensitivity are the dependence of gastric emptying on the chemical nature of the nutrients present in the small intestine and the involvement of chemical preabsorption information in short-term regulation of food intake (2). The cellular and molecular basis for chemoreception in the gut is hitherto unknown. In this study we addressed the question ofwhether the epithelium of the gut might express a-gustducin, the GTP-binding a-subunit of a trimeric Gprotein complex that is specific for taste receptor cells of the tongue (3). Here we show that a-gustducin is also expressed in the epithelium of the gut where it is associated with a specialized cell type long known under the names brush cell, tufted cell, or caveolated cell (4-6). The function of this cell type, which is present in humans, rats, and probably all other mammals, had been enigmatic until now. MATERIALS AND METHODSAntibodies and Immunostaining. A polyclonal antibody specific for a-gustducin was raised in a rabbit immunized with a synthetic peptide comprising amino acid residues 92-113 of the rat a-gustducin sequence (3). This sequence stretch is unique for a-gustducin and is not present in the sequences of any other known G-protein. Antibodies were affinity-purified to the peptide adsorbed to nitrocellulose (7,8). Polyclonal rabbit antibodies specific for chromogranin A and serotonin (9) and mouse monoclonal antibodies to villin (Dianova, Hamburg, Germany) and cytokeratin 18 (Progen, Heidelberg) were also used in this study. Indirect immunofluorescence was applied to 1-,um thick tissue sections of quick-frozen and Epon-embedded tissues as described (8). For doubleimmunofluorescence sections were incubated with a mixture of the rabbit antibody against a-gustducin and mouse monoclonal antibodies either specific for villin or cytokeratin 18. Primary antibodies were diluted with PBS: anti-gustducin (1:200), anti-chromogranin (1:4,000), anti-serotonin (1:10,000), anti-villin (0.1 /Lg/ml-1), anti-cytokeratin 18 (0.5 ,ug/ml-1). As secondary antibodies fluorescein isothiocyanate-labeled goat anti-mouse IgG and tetramethylrhodamine isothiocyanate-labeled goat anti-rabbit IgG (Dianova) were used at concentrations of 0.1 Ag/ml-1.Immunoblotting. Various tissue...

Abstract. Microvascular pericytes are believed to be involved in various functions such as regulation of capillary blood flow and endothelial proliferation. Since pericytes represent a morphologically heterogeneous cell population ranging from circular smooth musclelike to elongated fibroblast-like morphology it is possible that regulation of blood flow (via contractility) and control of endothelial proliferation (as well as other metabolic functions) may be accomplished by different subsets of pericytes. In the present study we provide evidence for heterogeneity of pericytes at the molecular level by using two novel technical approaches. These are (a) immunostaining of whole mounts of the microvascular beds of the rat mesentery and bovine retina and (b) immunoblotting studies of microdissected retinal microvessels. We show that pericytes of true capillaries (midcapiUaries) apparently lack the smooth muscle isoform of tx-actin whereas transitional pericytes of pre-and postcapillary microvascular segments do express this isoform. Thus, regulation of capillary blood flow may be accomplished by the smooth muscle-related pre-and postcapillary pericytes whereas the nonmuscle pericytes of true capillaries may play a role in other functions.

Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function. It is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and backwards. The integrity of the BBB is compromised in many disorders of the human CNS; therapeutical strategies for several of these diseases include treatment with glucocorticoids, but the molecular basis of how glucocorticoids regulate BBB permeability is not understood. Here, we report the generation and characterization of a murine immortalized brain (cerebral) capillary endothelial (cEND) cell line which expresses the BBB marker occludin at intercellular tight junctions (TJ). Hydrocortisone at physiological concentrations induced upregulation of occludin, accompanied by a threefold enhancement of transendothelial electrical resistance to values up to 1000 Ωcm 2 . Insulin enhanced the glucocorticoid response. At the molecular level, hydrocortisone induces increase of occludin at protein and mRNA levels by activation of the glucocorticoid receptor (GR) and its binding to putative glucocorticoid responsive elements in the occludin promoter. At the same time, insulin potentiated the ligand-dependent GR transactivation via induction of the GR in this in vitro system. This study thus provides insights into the molecular processes of barrier genesis, and may help to elucidate mechanisms of brain pathology at the microvascular level.

Homeostasis of the central nervous system (CNS) microenvironment is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and back. Many disorders change the functionality and integrity of the BBB. Glucocorticoids are being used sucessfully in the treatment of some disorders while their effects on others are questionable. In addition, conflicting results between clinical and experimental experience using animal models has arisen, so that the results of molecular studies in animal models need to be revisited in an appropriate in vitro model of the human BBB for more effective treatment strategies. Using the human brain microvascular endothelial cell line hCMEC/D3, the influence of glucocorticoids on the expression of barrier constituting adherens junction and tight junction transmembrane proteins (VE-cadherin, occludin, claudins) was investigated and compared to other established BBB models. In hCMEC/D3 cells the administration of glucocorticoids induced expression of the targets occludin 2.75 ± 0.04-fold and claudin-5 up to 2.32 ± 0.11-fold, which is likely to contribute to the more than threefold enhancement of transendothelial electrical resistance reflecting barrier tightness. Our analyses further provide direct evidence that the GC hydrocortisone prevents endothelial barrier breakdown in response to pro-inflammatory stimuli (TNFα administration), which could be demonstrated to be partly based on maintenance of occludin levels. Our studies strongly suggest stabilization of BBB function as a mode of GC action on a molecular level in the human brain vasculature.

To investigate the participation of microvascular pericytes in the process of capillary sprouting, we examined whole-mount preparations of the rat mesentery by use of a double immunofluorescence approach. Angiogenesis was induced by intraperitoneal injections of either the mast cell-degranulating substance compound 48/80 or tumor cell-conditioned medium. Capillary sprouts were visualized by staining with rhodamine-conjugated phalloidin and pericytes were simultaneously stained by an antibody to the intermediate filament protein desmin. Developing pericytes were negative for the smooth-muscle isoform of alpha-actin, but were clearly reactive for desmin. Pericytes appear to be involved in the earliest stages of capillary sprouting. Pericytes were regularly found lying at and in front of the advancing tips of endothelial sprouts. At many sites pericytes were seen to bridge the gap between the leading edges of opposing endothelial sprouts, which were apparently preparing to merge, suggesting that pericytic processes may serve as guiding structures aiding outgrowth of endothelial cells.

As hemoglobin begins to denature, it forms hemichromes that cross-link the major erythrocyte membrane-spanning protein, band 3, into clusters. These clusters provide the recognition site for antibodies directed against senescent cells. These antibodies bind to the aged red cell and trigger its removal from circulation.

Plectin, a major linker and scaffolding protein of the cytoskeleton, has been shown to be essential for the mechanical integrity of skin, skeletal muscle, and heart. Studying fibroblast and astroglial cell cultures derived from plectin (-/-) mice, we found that their actin cytoskeleton, including focal adhesion contacts, was developed more extensively than in wild-type cells. Also it failed to show characteristic short-term rearrangments in response to extracellular stimuli activating the Rho/Rac/Cdc42 signaling cascades. As a consequence, cell motility, adherence, and shear stress resistance were altered, and morphogenic processes were delayed. Furthermore, we show that plectin interacts with G-actin in vitro in a phosphatidylinositol-4,5-biphosphate-dependent manner and associates with actin stress fibers in living cells. The actin stress fiber phenotype of plectin-deficient fibroblasts could be reversed to a large degree by transient transfection of full-length plectin or plectin fragments containing the amino-terminal actin-binding domain (ABD). These results reveal a novel role of plectin as regulator of cellular processes involving actin filament dynamics that goes beyond its proposed role in scaffolding and mechanical stabilization of cells.