Luminal Membrane Domains Research Articles

Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain.

The choroid plexus epithelial cells (CPECs) belong to a small group of polarized cells, where the Na+-K+-ATPase is expressed in the luminal membrane. The basic polarity of the cells is, therefore, still debated. We investigated the subcellular distribution of an array of proteins known to play fundamental roles either in establishing and maintaining basic cell polarity or in the polarized delivery and recycling of plasma membrane proteins. Immunofluorescence histochemical analysis was applied to determine the subcellular localization of apical and basolateral membrane determinants. Mass spectrometry analysis of CPECs isolated by fluorescence-activated cell sorting was applied to determine the expression of specific forms of the proteins. CPECs mainly express the cell-adhesive P-cadherin, which is localized to the lateral membranes. Proteins belonging to the Crumbs and partitioning defective (Par) protein complexes were all localized to the luminal membrane domain. Par-1 and the Scribble complex were localized to the basolateral membrane domain. Lethal(2) giant larvae homolog 2 (Lgl2) labeling was preferentially observed in the luminal membrane domain. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) was immunolocalized to the basolateral membrane domain, while phosphatidylinositol 4,5-bisphosphate (PIP2) staining was most prominent in the luminal membrane domain along with the PIP3 phosphatase, Pten. The apical target-SNARE syntaxin-3 and the basolateral target-SNARE syntaxin-4 were both localized to the apical membrane domain in CPECs, which lack cellular expression of the clathrin adaptor protein AP-1B for basolateral protein recycling. In conclusion, the CPECs are conventionally polarized, but express P-cadherin at cell-cell contacts, and Lgl2 and syntaxin-4 in the luminal plasma membrane domain.

The choroid plexus epithelium displays conventional cell polarization

The choroid plexus epithelium (CPE) has been studied extensively as a model cell in e.g. high rate fluid secretion because of the direct access to the luminal surface. The high‐rate secretion is orchestrated by an unusual plasma membrane distribution of key transport proteins in the CPE cells. Par example, the ubiquitously expressed Na, K‐ATPase accumulates in the luminal membrane domain of CPE cells. The same has been observed for other normally basolateral proteins such as NKCC1 and NHE1, but not for all basolateral proteins (e.g. AE2). The observation led to the hypotheses that the CPE cells exhibit an inverse cell polarity, despite a conventional localization of structural polarity elements in epithelial cells: basal membrane facing the interstitium, tight junctions close to the luminal surface and luminal specializations such as microvilli and cilia. In this study, we investigated the expression and subcellular distribution of basic cell polarity markers in the CPE cells. Immunohistochemical analysis revealed a basolateral domain accumulation of the membrane lipid PtdIns(3,4,5)P3, Scrib, E‐cadherin, and partitioning‐defective protein (Par) 1. Luminal membrane domain accumulation was observed for the atypical protein kinase aPKC, the plasma membrane target snare syntaxin‐3, whereas Par3 was localized at the tight junction area. These markers represent the molecular complexes known to determine luminal and basolateral identity and were, thus, all located as expected for mature and normally polarized epithelial cells. We conclude that these central polarity proteins are distributed normally in the choroid plexus cells, and that cell polarity as such does not seem to be involved in the unusual localizations of key transport proteins in these cells.Support or Funding InformationThe work was supported by the Danish Medical Research Council and Aarhus University Ideas programme.

Polarization of membrane associated proteins in the choroid plexus epithelium from normal and slc4a10 knockout mice

The choroid plexus epithelium (CPE) has served as a model-epithelium for cell polarization and transport studies and plays a crucial role for cerebrospinal fluid (CSF) production. The normal luminal membrane expression of Na+,K+-ATPase, aquaporin-1 and Na+/H+ exchanger 1 in the choroid plexus is severely affected by deletion of the slc4a10 gene that encodes the bicarbonate transporting protein Ncbe/NBCn2. The causes for these deviations from normal epithelial polarization and redistribution following specific gene knockout are unknown, but may be significant for basic epithelial cell biology. Therefore, a more comprehensive analysis of cell polarization in the choroid plexus is warranted. We find that the cytoskeleton in the choroid plexus contains αI-, αII-, βI-, and βII-spectrin isoforms along with the anchoring protein ankyrin-3, most of which are mainly localized in the luminal membrane domain. Furthermore, we find α-adducin localized near the plasma membranes globally, but with only faint expression in the luminal membrane domain. In slc4a10 knockout mice, the abundance of β1 Na+,K+-ATPase subunits in the luminal membrane is markedly reduced. Anion exchanger 2 abundance is increased in slc4a10 knockout and its anchor protein, α-adducin is almost exclusively found near the basolateral domain. The αI- and βI-spectrin abundances are also decreased in the slc4a10 knockout, where the basolateral domain expression of αI-spectrin is exchanged for a strictly luminal domain localization. E-cadherin expression is unchanged in the slc4a10 knockout, while small decreases in abundance are observed for its probable adaptor proteins, the catenins. Interestingly, the abundance of the tight junction protein claudin-2 is significantly reduced in the slc4a10 knockouts, which may critically affect paracellular transport in this epithelium. The observations allow the generation of new hypotheses on basic cell biological paradigms that can be tested experimentally in future studies.

P‐glycoprotein trafficking at the blood–brain barrier altered by peripheral inflammatory hyperalgesia

P-glycoprotein (ABCB1/MDR1, EC 3.6.3.44), the major efflux transporter at the blood-brain barrier (BBB), is a formidable obstacle to CNS pharmacotherapy. Understanding the mechanism(s) for increased P-glycoprotein activity at the BBB during peripheral inflammatory pain is critical in the development of novel strategies to overcome the significant decreases in CNS analgesic drug delivery. In this study, we employed the λ-carrageenan pain model (using female Sprague-Dawley rats), combined with confocal microscopy and subcellular fractionation of cerebral microvessels, to determine if increased P-glycoprotein function, following the onset of peripheral inflammatory pain, is associated with a change in P-glycoprotein trafficking which leads to pain-induced effects on analgesic drug delivery. Injection of λ-carrageenan into the rat hind paw induced a localized, inflammatory pain (hyperalgesia) and simultaneously, at the BBB, a rapid change in colocalization of P-glycoprotein with caveolin-1, a key scaffolding/trafficking protein. Subcellular fractionation of isolated cerebral microvessels revealed that the bulk of P-glycoprotein constitutively traffics to membrane domains containing high molecular weight, disulfide-bonded P-glycoprotein-containing structures that cofractionate with membrane domains enriched with monomeric and high molecular weight, disulfide-bonded, caveolin-1-containing structures. Peripheral inflammatory pain promoted a dynamic redistribution between membrane domains of P-glycoprotein and caveolin-1. Disassembly of high molecular weight P-glycoprotein-containing structures within microvascular endothelial luminal membrane domains was accompanied by an increase in ATPase activity, suggesting a potential for functionally active P-glycoprotein. These results are the first observation that peripheral inflammatory pain leads to specific structural changes in P-glycoprotein responsible for controlling analgesic drug delivery to the CNS.

Nhe1 is a luminal Na+/H+ exchanger in mouse choroid plexus and is targeted to the basolateral membrane in Ncbe/Nbcn2-null mice

The choroid plexus epithelium (CPE) secretes the major fraction of the cerebrospinal fluid (CSF). The Na(+)-HCO(3)(-) transporter Ncbe/Nbcn2 in the basolateral membrane of CPE cells is important for Na(+)-dependent pH(i) increases and probably for CSF secretion. In the current study, the anion transport inhibitor DIDS had no effect on the residual pH(i) recovery in acidified CPE from Ncbe/Nbcn2 knockout mouse by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-fluorescence microscopy in the presence of CO(2)/HCO(3)(-) (Ncbe/Nbcn2-ko+DIDS 109% of control, P = 0.76, n = 5). Thus Ncbe/Nbcn2 mediates the DIDS-sensitive Na(+)-dependent pH(i) recovery in the CPE. The Na(+)/H(+) exchanger-1 Nhe1 is proposed to mediate similar functions as Ncbe/Nbcn2 in CPE. Here, we immunolocalize the Nhe1 protein to the luminal membrane domain in mouse and human CPE. The Na(+)-dependent pH(i) recovery of Nhe1 wild-type (Nhe1-wt) mice in the absence of CO(2)/HCO(3)(-) was abolished in the Nhe1 knockout CPE (Nhe1-ko 0.37% of Nhe1-wt, P = 0.0007, n = 5). In Ncbe/Nbcn2-ko mice, Nhe1 was targeted to the basolateral membrane. Nevertheless, the luminal Na(+)-dependent pH(i) recovery was increased in Ncbe/Nbcn2-ko compared with wild-type littermates (Nhe1-ko 146% of Nhe1-wt, P = 0.007, n = 5). Whereas the luminal Nhe activity was inhibited by the Nhe blocker EIPA (10 microM) in the Ncbe/Nbcn2-wt, it was insensitive to the inhibitor in Ncbe/Nbcn2-ko (Ncbe/Nbcn2-ko+EIPA 100% of control, P = 0.98, n = 5). This indicates that a luminal EIPA-insensitive Nhe was induced in Ncbe/Nbcn2-ko CPE and that EIPA-sensitive Nhe activity was basolateral. The Nhe1 translocation in Ncbe/Nbcn2-ko CPE may reflect a compensatory response, which provides the cells with better means of regulating pH(i) or transporting Na(+) after Ncbe/Nbcn2 disruption.

Nhe1 is a luminal protein in the mouse choroid plexus epithelium that translocates to the basolateral surface in Ncbe‐null mice

The choroid plexus epithelium, CPE, secretes a major fraction of the cerebrospinal fluid, CSF. The Na+;HCO3− transporter, Ncbe/Nbcn2, in the basolateral membrane of CPE cells is important for Na+ dependent pHi increases and probably for CSF secretion. The Na+/H+ exchanger‐1, Nhe1, have been proposed to mediate similar functions in CPE. Nhe1 protein was immunolocalized to the luminal membrane domain in CPE and was absent in CPE from Nhe1 knockout, ko, littermates. The Na+‐dependent pHi recovery in the absence of CO2/HCO3− was dramatically decreased in the Nhe1‐ko mice compared to Nhe1‐wild type, wt, (ko 1.55% of wt, n=6, p=0.02) by fluorescence microscopy. In Ncbe‐ko mice, immunoreactive Nhe1 was targeted to the basolateral membrane, but luminal Na+ dependent pHi recovery was in fact increased in Ncbe‐ko compared to wt littermates (ko 146.1% of wt, n=5, p=0.007). In Ncbe‐wt mice, the luminal Nhe activity was expectedly inhibited by the Nhe blocker, 5′‐(N‐ethyl‐N‐isopropyl)‐amiloride (EIPA, 10µM), while in Ncbe‐ko, Nhe activity was insensitive to the inhibitor (ko 939.0% of wt, n=5, p=0.036). This indicates that a luminal, EIPA‐insensitive Nhe was induced and that EIPA‐sensitive Nhe activity was basolateral in Ncbe‐ko CPE. We speculate that the Nhe1 translocation in Ncbe‐ko CPE reflects a compensatory response, which provides the cells with better means of regulating pHi in the absence of Ncbe.

Characterization of growth and differentiation of normal human submandibular gland epithelial cells in a serum-free medium

Abstract Parenchymal tissue of human submandibular glands was cultured in a serum-free medium consisting of a 1:9 mixture of Dulbecco's modified Eagle's medium and MCDB 153 supplemented with 10 ng/ml epidermal growth factor, 10 μ M dexamethasone and 1 μg/ml insulin. Cultivation of the tissue in this medium resulted in propagation of loosely arranged epithelioid cells on plastic, without the necessity of a matrix. Epidermal growth factor significantly enhanced mitogenesis of cultured cells, which expressed specific high- and low-affinity receptors for epidermal growth factor. The epithelioid cells were found to represent the undifferentiated ultra-structure of ductal cells. Immunocytochemically, cultured epithelioid cells expressed antigens specific to basal cells of the intra- and interlobular ducts in situ, including cytokeratins 3 and 6 and cytokeratins 13 and 16, vimentin, and α–smooth muscle actin. Moreover, cytoplasm of the cells was immunostained using antibody against the basement membrane component, type IV collagen. These results suggested that cultured epithelioid cells are undifferentiated ductal cells, which have the characteristics of basal cells of the intra- and/or interlobular ducts. Cultured epithelioid cells maintained the characteristics for serial passage until the time that the cultures were confluent. On the other hand, several stratified foci developed on the confluent monolayer. The stratified cells were strongly positive for cytokeratins 3 and 6, but negative for vimentin, α-smooth muscle actin and type IV collagen. Moreover, the stratified cells were strongly stained with the antibody against epithelial membrane antigen. This antibody stained the luminal membrane domain of salivary epithelial cells. Electron micrograph of the vertical section through the foci revealed stratified cell layers with a gradual transition from basal cells to squamous epidermoid cells. This result suggests that cultured epithelioid cells, which have the characteristics of basal cells of the intra– and/or interlobular duct, have the potential to differentiate into luminal duct cells.

Characterization of growth and differentiation of normal human submandibular gland epithelial cells in a serum-free medium

Abstract Parenchymal tissue of human submandibular glands was cultured in a serum-free medium consisting of a 1:9 mixture of Dulbecco's modified Eagle's medium and MCDB 153 supplemented with 10 ng/ml epidermal growth factor, 10 μ M dexamethasone and 1 μg/ml insulin. Cultivation of the tissue in this medium resulted in propagation of loosely arranged epithelioid cells on plastic, without the necessity of a matrix. Epidermal growth factor significantly enhanced mitogenesis of cultured cells, which expressed specific high- and low-affinity receptors for epidermal growth factor. The epithelioid cells were found to represent the undifferentiated ultra-structure of ductal cells. Immunocytochemically, cultured epithelioid cells expressed antigens specific to basal cells of the intra- and interlobular ducts in situ, including cytokeratins 3 and 6 and cytokeratins 13 and 16, vimentin, and α-smooth muscle actin. Moreover, cytoplasm of the cells was immunostained using antibody against the basement membrane component, type IV collagen. These results suggested that cultured epithelioid cells are undifferentiated ductal cells, which have the characteristics of basal cells of the intra- and/or interlobular ducts. Cultured epithelioid cells maintained the characteristics for serial passage until the time that the cultures were confluent. On the other hand, several stratified foci developed on the confluent monolayer. The stratified cells were strongly positive for cytokeratins 3 and 6, but negative for vimentin, α-smooth muscle actin and type IV collagen. Moreover, the stratified cells were strongly stained with the antibody against epithelial membrane antigen. This antibody stained the luminal membrane domain of salivary epithelial cells. Electron micrograph of the vertical section through the foci revealed stratified cell layers with a gradual transition from basal cells to squamous epidermoid cells. This result suggests that cultured epithelioid cells, which have the characteristics of basal cells of the intra- and/or interlobular duct, have the potential to differentiate into luminal duct cells.

Separation of luminal and abluminal membrane enriched domains from cultured bovine aortic endothelial cells: monoclonal antibodies specific for endothelial cell plasma membranes

Two kinds of membrane (luminal and abluminal membrane domains) fractions have been isolated from bovine aortic endothelial cells by fractionation of whole cell homogenate on discontinuous sucrose density gradients. The luminal membrane domain was enriched 12–16-fold for angiotensin-converting enzyme activity and 8–10-fold in alkaline phosphatase activity. The abluminal membrane domain displayed an enrichment of 8-fold in (Na + + K +)-ATPase activity. Both of the membrane domains were minimally contaminated with mitochondria, microsomes and Golgi bodies, as assessed by their corresponding marker enzyme activities. 125I-labeling of endothelial cell monolayers by the Enzymo-Bead lactoperoxidase-catalyzed iodination procedure, followed by isolation of membranes, revealed that the radioactivity was predominantly associated with membranes enriched in angiotensin-converting enzyme activity, corresponding to the luminal membrane domain. However, when cells were radioiodinated in suspension culture, radioactivity was found equally associated in both the luminal and abluminal membrane fractions. Electron microscopy of freeze-fractured and sectioned material showed both luminal and abluminal membrane domains to be in the form of vesicles varying in size from 100 to 400 nm in diameter. To characterize the separation of endothelial cell membrane domains, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained, producing antibodies which bound to endothelial cells of arterial, venous and capillary origin. Two antibodies of these clones, XIVC 6 and XVD 2, were studied in more detail. In the ELISA assay, these antibodies reacted with bovine vascular endothelial cells, but not with human umbilical cord endothelial cells, nor with bovine corneal endothelial cells, smooth muscle cells or fibroblasts. Both of these antibodies are directed against an antigen of approximately 130 kDa, under reducing and non-reducing conditions, as assayed by the immunoprecipitation method. Western blot analysis of luminal and abluminal membrane fractions revealed that only MAb XVD 2 reacted with an antigen, indicating that the antibody XIVC 6 is directed against an epitope which is denatured by SDS. Moreover, MAb XVD 2 preferentially reacted with the luminal membrane compared to the abluminal membrane domain of the endothelial cell. These monoclonal antibodies do not react with platelet membrane proteins, indicating that this 130 kDa membrane antigen is not common to both endothelial cells and platelets. These monoclonal antibodies will be useful for monitoring the separation of luminal and abluminal membrane domains and in the immunoaffinity purification of these domains.

Endothelial cell membranes contain podocalyxin--the major sialoprotein of visceral glomerular epithelial cells.

Podocalyxin is the major sialoprotein in the glycocalyx of glomerular podocytes. Here we report on its extraglomerular localization, using a monospecific antibody which was obtained by affinity purification of IgG on nitrocellulose transfers of glomerular podocalyxin. By indirect immunofluorescence, podocalyxin was found in the blood vessels of several organs (lung, heart, kidney, small intestine, brain, pancreas, aorta, the periportal blood vessels in liver, and the central arteries of follicles of the spleen, but not in the endothelia that line the sinusoids of the latter organs). By immunoelectron microscopy--using immunogold conjugates in diffusion ("pre-embedding") and surface ("postembedding") procedures--podocalyxin was localized on the luminal membrane domain of endothelial cells, in a patchy distribution. The presence of podocalyxin was confirmed in SDS extracts of lung tissue by immunoblotting. We conclude that (a) podocalyxin is a widespread component of endothelial plasma membranes, (b) it is restricted to the luminal membrane domain, and (c) it is distributed unevenly on the endothelial cell surface.