Polarized distribution of actin isoforms in gastric parietal cells.

The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.     

Polarized secretion of urokinase-type plasminogen activator by epithelial cells.

Numerous epithelial cell types produce and secrete plasminogen activators (PAs) and/or PA inhibitors (PAIs). When epithelial cells were grown on polycarbonate filters and their apical and basolateral secretion products analyzed, PA activity accumulated in a highly polarized fashion; depending upon the cell line, the compartment of PA accumulation was either apical (MDCK I cells and HBL-100 cells) or basolateral (LLC-PK1, CaCo-2, and HeLa cells). By contrast, PAI-1 was recovered in roughly equal amounts in both compartments. Basolateral accumulation of urokinase-type plasminogen activator (uPA), but not its apical targeting, required an acidic compartment and the integrity of the cytoskeleton. Polarity of uPA accumulation did not result from removal of the free enzyme from the opposite compartment through its binding to the cell surface. Transfection with wild-type or mutated murine uPA demonstrated that neither the "growth factor" domain nor the kringle domain is required for the appropriate sorting of the protein. We propose that polarized secretion of PAs is one mechanism whereby cells spatially control extracellular proteolysis.     

Polarized expression of CD74 by gastric epithelial cells.

CD74 is known as the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) that regulates the cell biology and functions of MHC class II molecules. Class II MHC and Ii expression was believed to be restricted to classical antigen-presenting cells (APC); however, during inflammation, other cell types, including mucosal epithelial cells, have also been reported to express class II MHC molecules. Given the importance of Ii in the biology of class II MHC, we sought to examine the expression of Ii by gastric epithelial cells (GEC) to determine whether class II MHC molecules in these nonconventional APC cells were under the control of Ii and to further support the role that these cells may play in local immune and inflammatory responses during Helicobacter pylori infection. Thus we examined the expression of Ii on GEC from human biopsy samples and then confirmed this observation using independent methods on several GEC lines. The mRNA for Ii was detected by RT-PCR, and the various protein isoforms were also detected. Interestingly, these cells have a high level expression of surface Ii, which is polarized to the apical surface. These studies are the first to demonstrate the constitutive expression of Ii by human GEC.     

Lysolecithin actions on vascular smooth muscle cells.

Oxidation of low density lipoprotein increases its atherogenic potential. During oxidation there is an extensive conversion of lecithin to lysolecithin. In rat aortic smooth muscle cells, 2-25 micrograms/ml lysolecithin elevated cytosolic calcium concentration up to 560%. Lysolecithin (10-20 micrograms/ml) increased [3H]thymidine incorporation from 15 cpm/mg cell protein (controls) up to 189 cpm/mg cell protein. Lysolecithin (10 micrograms/ml) potentiated the PDGF-induced (50 ng/ml) [3H]thymidine incorporation up to 6.3 times. The results indicate that lysolecithin could induce mechanisms, by which oxidized low density lipoproteins could promote cell growth and thus contribute to atherosclerosis.     

Rapid actions of aldosterone: lymphocytes, vascular smooth muscle and endothelial cells.

The genomic theory of steroid action has been the unquestioned dogma for the explanation of steroid effects over the past four decades. Despite early observations on rapid steroid effects being clearly incompatible with this theory, only recently has nongenomic steroid action been recognized more widely and led to a critical reappraisal of unsolved questions about this dogma. Evidence for nongenomic steroid effects come from all fields of steroid research now, and mechanisms of agonist action are studied with regard to membrane receptors and second messengers involved. A prominent example of a receptor/effector-cascade for nongenomic steroid effects has been described for rapid aldosterone effects in various cell types, including lymphocytes, cultured vascular smooth muscle, and endothelial cells involving nonclassical membrane receptors with a high affinity for aldosterone, but not for cortisol, and phosphoinositide turnover. As another important second messenger, [Ca2+]i is consistently increased by aldosterone within 1-2 min. In vascular smooth muscle cells, calcium is released from perinuclear stores, while in endothelial cells a predominant increase of subplasmalemmal calcium is seen. Effects are half maximal at physiological concentrations of free aldosterone (0.1 nmol/L), while cortisol is inactive up to 0.1 micromol/L; the classical mineralocorticoid antagonist canrenone is ineffective in blocking the action of aldosterone. The data show that intracellular signaling for nongenomic aldosterone effects also involves calcium, but pathways of cell activation may vary between different cell types. Future research will have to target the cloning of the first membrane receptor for steroids, and the evaluation of the clinical relevance of these rapid steroid effects.     

Polarized sorting and trafficking in epithelial cells

The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain, which are separated by tight junctions. The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules. This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers. Here we review the recent advances in the field of polarized sorting in epithelial cells. We especially highlight the role of lipid rafts in apical sorting.     

UV irradiation of polar cells of Drosophila melanogaster eggs. II. Estimation of the number of polar cells at the origin of the germinal line

Polar cap cells of Drosophila eggs were irradiated with UV doses ranging from 750 to 1200 erg/mm². One of the important observations relates to the incidence of sterility among the surviving males and its dependence on the dose of UV irradiation. Histological examination of the sterile males showed that such males have agametic testes.     

Polarized functions and permeability properties of rat epididymal epithelial cells in vitro.

Cultured rat caput and cauda epididymidal epithelial cells are shown to exhibit polarized properties characteristic of functioning epithelia. When grown on plastic substrates coated with reconstituted basement membrane, confluent monolayers of cells from both regions formed domes characteristic of other transporting epithelia. Immunocytochemical localization of three proteins characteristically associated with epithelial junctional complexes revealed that uvomorulin, zonula occludens 1 and cingulin were present in cultured epididymal epithelial cells and that their distribution was similar to that in the epididymal epithelium in vivo. These three molecules were not found in epididymal stromal cells. Cells from both regions growing in two compartment chambers developed an electrical resistance across the monolayer with a magnitude characteristic of high resistance epithelia. The optimal plating density of cells was 0.75 x 10(6) cells cm-2. The presence of reconstituted basement membrane on the filters did not affect the resistance of the cells. Inulin passage from basal to apical chambers was less than 2% over 24 h. These results show that several polarized functions of epididymal epithelial cells can be maintained in culture and that this type of culture system is useful for studying the function of the epididymis in vitro.     

Polarized entry and release in epithelial cells of Black Creek Canal virus, a New World hantavirus.

Black Creek Canal (BCC) virus is a newly identified hantavirus from Florida which is carried by the cotton rat (Sigmodon hispidus) and is associated with hantavirus pulmonary syndrome (HPS). We have investigated the interaction of BCC virus with polarized epithelial cells to examine whether entry and release of this virus occur at specific plasma membrane domains. The polarized Vero C1008 monkey kidney cell line was grown on permeable filters and infected with BCC virus either through the apical or basolateral surface. As shown by indirect immunofluorescence and radioimmunoprecipitation analysis, cells infected through the apical surface demonstrated a high level of susceptibility to BCC virus infection. In contrast, Vero C1008 cells infected basolaterally exhibited a barely detectable level of BCC virus-synthesized proteins. Titration of virus from apical and basolateral media of infected cells has demonstrated that virus titers released from the apical surface are about 1,200-fold greater than the titer of virus released into the basolateral media. The site of BCC virus release from polarized cells is, therefore, different from that previously described for release of other members of the family Bunyaviridae and may reflect one of the determinants of hantavirus pathogenesis. In addition, we have shown that BCC viral glycoproteins are expressed at the plasma membrane on the apical surface of polarized cells. Electron microscopy studies of the infected cells revealed evidence of BCC virus budding at the plasma membrane. This strongly indicates that, in contrast to most other members of the Bunyaviridae, BCC virus is assembled at the plasma membrane. Since the same site of virus assembly was recently described for Sin Nombre virus, it is likely that all of the new American hantaviruses associated with HPS utilize this same type of virus maturation.     

Metabolism,cellular actions,and cytotoxicity of selenomethionine in cultured cells

Selenomethionine metabolism and the biochemical basis for its cytotoxicity were analyzed in cultured human and murine lymphoid cells. The metabolic pathways were also addressed, using purified mammalian enzymes and crude tissue extracts. Selenomethionine was found to be effectively metabolized to S-adenosylmethionine analog, and that analog was further metabolized in transmethylation reactions and in polyamine synthesis, similarly to the corresponding sulphur metabolites of methionine. Selenomethionine did not block these pathways, nor was there a specific block on the synthesis of DNA, RNA, or proteins when added to the culture medium. Selenomethionine showed cytotoxicity at above 40 microM levels. Yet, low selenomethionine levels (10 microM) could replace methionine and support cell growth in the absence of methionine. Selenomethionine toxicity took place concomitantly with changes in S-adenosylmethionine pools. D-form was less cytotoxic than L-form. Methionine concentration modified the cytotoxicity. Together, this indicates that selenomethionine uptake and enzymic metabolism are involved in the cytotoxicity in a yet unknown way.     

Polarized plasma membrane domains in cultured endothelial cells

To determine whether distinct plasma membrane domains exist in endothelial cells, we infected monolayer cultures of macro- and microvascular endothelial cells with enveloped RNA viruses known to bud selectively from either the apical or basal surface in polarized epithelial cells. We found that vesicular stomatitis (VSV) and Sendai virus emerge asymmetrically from cultured endothelial cells. This provides direct evidence for the existence of polarized plasma membrane domains in vascular endothelial cells.     

Polarized expression of Shaker channels in epithelial cells

The polarized distribution of ion channels into an apical or a basolateral domain is a fundamental feature of the transporting-epithelial phenotype. To study the molecular motifs of the channel that may serve as addressing signal(s), as well as the cellular mechanisms that interpret it and deliver the protein accordingly, we study the fate of transfected ShIR K+ channels (a non-inactivating Shaker channel) tagged with an HA epitope, as well as several other deletants and mutants. Surface expression is triggered by Ca2+-activated cell-cell contacts, through a cascade including a phospholipase C, a protein kinase C, and the cytoskeleton of actin and tubulin, and is partially impaired by suppressing N-glycosylation with tunicamycin. Using domain-specific biotinylation we show that the channel is delivered preferentially to the basolateral domain thanks to a segment between amino acids 571 and 613, and is retained on the membrane surface due to a region involving the last three amino acids (threonine, aspartic acid, valine, TDV) of the COOH terminal. Its association with the cytoskeleton seems to take the form of a scaffold comprising actin, a-actinin, b-tubulin, mLin7 and CASK. We also observe that membrane expression of ShIR channels depends entirely on its sequence of amino acids and the conformation that the molecule may adopt, but not on its ability to translocate K+ across the membrane.     

Cellular and molecular actions of adrenomedullin in glomerular mesangial cells.

Adrenomedullin (AM), a potent vasodilatory and hypotensive peptide produces several biological outcomes in glomerular mesangial cells. Mesangial cells are important in the pathogenesis of glomerulonephritis, and therefore the actions of AM on mesangial cells have important clinical and therapeutic implications. This minireview describes the various actions of AM on mesangial cell function and the signal transduction mechanisms involved. As in other systems, most actions of AM can be explained by increase in cAMP levels in the cell, although a few exceptions remain. The fact that most data obtained to date has been in culture, the physiological significance of the actions of AM in mesangial cells is discussed.     

Endocrine,paracrine and autocrine actions of prolactin on immune cells

The immune response is regulated by locally released factors, collectively referred to as cytokines. Data on the human immune system have convincingly demonstrated that the hormone prolactin (PRL), in addition to exerting its endocrine control on the immune system, acts as a cytokine in that it is released within the immune system and regulates the lymphocyte response by paracrine and autocrine mechanisms. Both lymphocyte and pituitary PRLs are under the control of immune factors. Synthesis of human PRL by lymphocytes is induced by T-cell stimuli, while increased release of PRL by the pituitary, observed in vivo after immune challenge, may be mediated by cytokines produced by monocyte-macrophages. Since hyperprolactinemia and hypoprolactinemia are both immunosuppressive, physiological levels of circulating PRL must be necessary to maintain basal immunocompetence. The effects of Cyclosporin (CsA) on IL-2 and PRL gene activation and the analysis of the intracellular signaling events downstream IL-2 and PRL receptors suggest coordinate actions of these two cytokines during T cell activation.     

On polar auxin transport in plant cells

We present here explicit mathematical formulas for calculating the concentration, mass, and velocity of movement of the center of mass of the plant growth regulator auxin during its polar movement through a linear file of cells. The results of numerical computations for two cases, (a) the conservative, in which the mass in the system remains constant and (b) the non-conservative, in which the system acquires mass at one end and loses it at the other, are graphically presented. Our approach differs from that of Mitchison's (Mitchison 1980) in considering both initial effects of loading and end effects of substance leaving the file of cells. We find the velocity varies greatly as mass is entering or leaving the file of cells but remains constant as long as most of the mass is within the cells. This is also the time for which Mitchison's formula for the velocity, which neglects end effects, reflects the true velocity of auxin movement. Finally, the predictions of the model are compared with two sets of experimental data. Movement of a pulse of auxin through corn coleoptiles is well described by the theory. Movement of auxin through zucchini shoots, however, shows the need to take into account immobilization of auxin by this tissue during the course of transport.     

Uptake,recycling, and antioxidant actions of alpha-lipoic acid in endothelial cells

Alpha-lipoic acid, which becomes a powerful antioxidant in its reduced form, has been suggested as a dietary supplement to treat diseases associated with excessive oxidant stress. Because the vascular endothelium is dysfunctional in many of these conditions, we studied the uptake, reduction, and antioxidant effects of alpha-lipoic acid in cultured human endothelial cells (EA.hy926). Using a new assay for dihydrolipoic acid, we found that EA.hy926 cells rapidly take up and reduce alpha-lipoic acid to dihydrolipoic acid, most of which is released into the incubation medium. Nonetheless, the cells maintain dihydrolipoic acid following overnight culture, probably by recycling it from alpha-lipoic acid. Acute reduction of alpha-lipoic acid activates the pentose phosphate cycle and consumes nicotinamide adenine dinucleotide phosphate (NADPH). Lysates of EA.hy926 cells reduce alpha-lipoic acid using both NADPH and nicotinamide adenine dinucleotide (NADH) as electron donors, although NADPH-dependent reduction is about twice that due to NADH. NADPH-dependent alpha-lipoic acid reduction is mostly due to thioredoxin reductase. Pre-incubation of cells with alpha-lipoic acid increases their capacity to reduce extracellular ferricyanide, to recycle intracellular dehydroascorbic acid to ascorbate, to decrease reactive oxygen species generated by redox cycling of menadione, and to generate nitric oxide. These results show that alpha-lipoic acid enhances both the antioxidant defenses and the function of endothelial cells.