Membrane Labeling Research Articles

Expression and localization of MT1-MMP and furin in the glomerular wall of short- and long-term diabetic rats

Diabetic glomerulopathy has been linked to shifts in balance between the synthetic and degradative pathways of the glomerular basement membrane (GBM), a key player in the permselectivity properties of the glomerular wall. The goal of this study was to trace the expression and localization of membrane type-1 metalloprotease (MT1-MMP) and its activating enzyme furin, key proteins involved in basement membrane turnover, in short- and long-term diabetic rat renal tissues. Quantitative immunogold was carried out for MT1-MMP and furin and their expression was evaluated in renal tissues of young and old, control and diabetic rats. To corroborate immunocytochemical findings, Western blots were performed on glomerular lysates. Electron microscopy revealed that the overall expression of MT1-MMP and furin is reduced in plasma membranes of all glomerular cell types of old normoglycemic animals, a phenomenon that is exacerbated in long-term diabetic animals. This observation supports the prevailing theory that diabetes fosters acceleration in the aging process. Interestingly, while biochemical results confirmed a decrease in MT1-MMP expression, an increase in furin was observed. Immunocytochemical studies resolved this discrepancy by tracing the increased furin expression in endoplasmic reticulum and Golgi membranes of podocytes, indicating that furin is retained in the secretory pathway in a diabetic environment. Disturbances at the molecular level of the otherwise tightly regulated MT1-MMP/furin interactions found at the cell surface must account for a lack in extracellular matrix remodeling, increased deposition of GBM material, and loss of glomerular filtration integrity.

Spectrin interacts with EVL (Enabled/vasodilator‐stimulated phosphoprotein‐like protein), a protein involved in actin polymerization

The alpha- and beta-spectrin chains constitute the filaments of the spectrin-based skeleton, which was first identified in erythrocytes. The discovery of analogous structures at plasma membranes of eukaryotic cells has led to investigations of the role of this spectrin skeleton in many cellular processes. The alphaII-spectrin chain expressed in nucleated cells harbours in its central region several functional motifs, including an SH3 (Src homology 3) domain. Using yeast two-hybrid screening, we have identified EVL [Enabled/VASP (vasodilator-stimulated phosphoprotein)-like protein] as a new potential partner of the alphaII-spectrin SH3 domain. In the present study, we investigated the interaction of the alphaII-spectrin SH3 domain with EVL and compared this with other proteins related to EVL [Mena (mammalian Enabled) and VASP]. We confirmed the in vitro interaction between EVL and the alphaII-spectrin SH3 domain by GST (glutathione S-transferase) pull-down assays, and showed that the co-expression of EVL with the alphaII-spectrin SH3 domain in COS-7 cells resulted in the partial delocalization of the SH3 domain from cytoplasm to filopodia and lamellipodia, where it was co-localized with EVL. In kidney epithelial and COS-7 cells, we demonstrated the co-immunoprecipitation of the alphaII-spectrin chain with over-expressed EVL. Immunofluorescence studies showed that the over-expression of EVL in COS-7 cells promoted the formation of filopodia and lamellipodia, and the expressed EVL was detected in filopodial tips and the leading edge of lamellipodia. In these cells over-expressing EVL, the alphaII-spectrin membrane labelling lagged behind EVL staining in lamellipodia and filopodia, with co-localization of these two stains in the contact area. In kidney epithelial cell lines, focused co-localization of spectrin with expressed EVL was observed in the membrane of the lateral domain, where the cell-cell contacts are reinforced. The possible link between the spectrin-based skeleton and actin via the EVL protein suggests a new way of integrating the spectrin-based skeleton in areas of dynamic actin reorganization.

A New Photostable Terrylene Diimide Dye for Applications in Single Molecule Studies and Membrane Labeling

A new terrylene diimide-based dye (WS-TDI) that is soluble in water has been synthesized, and its photophysical properties are characterized. WS-TDI forms nonfluorescing H-aggregates in water that show absorption bands being blue-shifted with respect to those of the fluorescing monomeric form. The ratio of monomeric WS-TDI to aggregated WS-TDI was determined to be 1 in 14 400 from fluorescence correlation spectroscopy (FCS) measurements, suggesting the presence of a large amount of soluble, nonfluorescent aggregates in water. The presence of a surfactant such as Pluronic P123 or CTAB leads to the disruption of the aggregates due to the formation of monomers in micelles. This is accompanied by a strong increase in fluorescence. A single molecule study of WS-TDI in polymeric films of PVA and PMMA reveals excellent photostability with respect to photobleaching, far above the photostability of other common water-soluble dyes, such as oxazine-1, sulforhodamine-B, and a water-soluble perylenediimide derivative. Furthermore, labeling of a single protein such as avidin is demonstrated by FCS and single molecule photostability measurements. The high tendency of WS-TDI to form nonfluorescent aggregates in water in connection with its high affinity to lipophilic environments is used for the fluorescence labeling of lipid membranes and membrane containing compartments such as artificial liposomes or endosomes in living HeLa cells. The superior fluorescence imaging quality of WS-TDI in such applications is demonstrated in comparison to other well-known membrane staining dyes such as Alexa647 conjugated with dextran and FM 4-64 lipophilic styryl dye.

Erythrocyte exit: Out, damned merozoite! Out I say!

A new study has combined video microscopy with fluorescent labeling of host and parasite membranes to follow Plasmodium falciparum merozoites as they exit their host erythrocyte. The result has yielded some arresting images, which make compelling viewing irrespective of whether or not you have an interest in cell motility in general or P. falciparum erythrocyte exit in particular. Moreover, this work injects important new insights into the long-running debate about the biological mechanisms that underpin merozoite release.

Regulation of aquaporin 1 in epithelia of vocal fold and trachea

We sought to identify and regulate a pathway for transcellular water flux in native epithelia of the ovine vocal fold and trachea. Immunogold localization visualized by transmission electron microscopy was used to identify presence of AQP1 and AQP2 in ovine vocal fold (N=2) and tracheal (N=2) epithelia. Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to examine regulation of AQP1 and AQP2 mRNA in ovine vocal fold (N=34) and tracheal (N=24) epithelia by vasopressin (4 hours, luminal exposure). In the vocal fold, plasma membrane labeling density of AQP1 and AQP2 was greater for squamous cells at the lumen than for deeper cells (AQP1 p< 0.02, AQP2 p<0.0001). Labeling of trachea was associated with the luminal plasma membrane of pseudostratified ciliated epithelial cells. In vocal fold epithelia only, vasopressin (but not sham control) significantly reduced CT values for AQP1 (p<0.01). No difference was seen in CT values of AQP2 in vocal fold or trachea following vasopressin exposure. Vasopressin-dependent increase of AQP1 mRNA in stratified squamous epithelium of the vocal fold suggests a hormonal mechanism for upregulation of water permeability in luminal vocal fold cells, possibly distinct from other regions of the airway. Supported by NIDCD #F31DC5733 (DL) and #K23DC00168 (KF).

E-cadherin Expression in Canine Malignant Mammary Tumours: Relationship to Other Clinico-Pathological Variables

The relationship between E-cadherin epithelial expression, as detected by immunohistochemical methods, and other clinico-pathological characteristics of canine malignant mammary tumours was studied in 77 tumours surgically removed from 45 female dogs. The immunohistochemical assessment was based on the estimated percentage of epithelial cells with membranous labelling. Reduction of E-cadherin expression was significantly related to size and ulceration of tumours but not to fixation to skin or underlying tissue; it was also related to lymph node metastasis, necrosis and infiltrative growth. Histological type (but not histological grade) was related to E-cadherin expression, with solid tumours more frequently lacking expression and tubulopapillary tumours showing increased expression as compared with the other types. The significant relationship between E-cadherin and other known factors of poor prognosis suggests that the loss of E-cadherin expression may have prognostic value in canine malignant mammary tumours.

Multigene family encoding 3',5'-cyclic-GMP-dependent protein kinases in Paramecium tetraurelia cells.

In the ciliate Paramecium tetraurelia, 3',5'-cyclic GMP (cGMP) is one of the second messengers involved in several signal transduction pathways. The enzymes for its production and degradation are well established for these cells, whereas less is known about the potential effector proteins. On the basis of a current Paramecium genome project, we have identified a multigene family with at least 35 members, all of which encode cGMP-dependent protein kinases (PKGs). They can be classified into 16 subfamilies with several members each. Two of the genes, PKG1-1 and PKG2-1, were analyzed in more detail after molecular cloning. They encode monomeric enzymes of 770 and 819 amino acids, respectively, whose overall domain organization resembles that in higher eukaryotes. The enzymes contain a regulatory domain of two tandem cyclic nucleotide-binding sites flanked by an amino-terminal region for intracellular localization and a catalytic domain with highly conserved regions for ATP binding and catalysis. However, some Paramecium PKGs show a different structure. In Western blots, PKGs are detected both as cytosolic and as structure-bound forms. Immunofluorescence labeling shows enrichment in the cell cortex, notably around the dense-core secretory vesicles (trichocysts), as well as in cilia. Immunogold electron microscopy analysis reveals consistent labeling of ciliary membranes, of the membrane complex composed of cell membrane and cortical Ca2+ stores, and of regions adjacent to ciliary basal bodies, trichocysts, and trafficking vesicles. Since PKGs (re)phosphorylate the exocytosis-sensitive phosphoprotein pp63/pf upon stimulation, the role of PKGs during stimulated exocytosis is discussed, in addition to a role in ciliary beat regulation.

Joint Meeting of the Society for Cutaneous Ultrastructure Research (32nd Annual Meeting of SCUR) with the International Society of Skin Pharmacology and Physiology (ISP) and the European Epidermal Barrier Research Group (Network) 1st–4th June 2005 HAMBURG, Germany

Kindler syndrome (KS; OMIM173650) is an unusual, autosomal recessive skin disorder associated with trauma-induced blisters in early life followed by photosensitivity, poikiloderma, and an increased risk of malignancy. Defects in the actin/focal adhesion associated protein kindlin-1 (also known as kindlerin) encoded by the gene KIND1 have been shown to cause this disease. In human epidermis, kindlin-1 is expressed in epidermal keratinocytes, particularly within basal keratinocytes with an increase in staining at the dermal-epidermal junction. We have undertaken a detailed ultrastructural and immunohistochemical study in KS (n¼4) and control skin (n¼3) to examine morphology and the labeling of basement membrane, actin cytoskeletal, and focal contact-associated proteins. Transmission electron microscopy of KS skin showed disruption and reduplication of the lamina densa, together with sub-lamina separation. The number and structure of hemi-desmosomes and anchoring filaments appeared normal, although there was focal disruption in desmosome- and hemidesmosome-keratin filament attachment. This disruption in normal keratin filament assembly was most obvious at dermal splits and was associated with disorganized substratum bundles of actin filaments. Fluorescence microscopy showed increased epidermal expression of actin, alpha actinin, talin, vinculin, tenascin C, and RACK-1 in KS skin but no change in labeling with antibodies to filamin, tensin, focal adhesion kinase, paxillin, or tropomyosin. Immunostaining for protein kinase C was markedly reduced in basal keratinocytes in KS skin compared to controls. Immunogold electron microscopy using kindlin-1 antibody in control skin cytoplasm showed labeling over ends of microfilament-like structures. Taken together, our findings reveal a close spatial and functional relationship between kindlin-1, actin, some focal contact proteins and regulatory molecules that link the actin skeleton to the integrin extracellular matrix receptors. We hypothesize that the function of kindlin-1 might be to bind to the ends of actin microfilaments, linking focal adhesion proteins to integrin receptors at hemidesmosomes, thereby limiting the elongation of actin microfilaments. Conversely, a lack of kindlin-1 disrupts focal adhesion linkage and allows the unregulated proliferation of actin microfilaments, causing perturbations in the other cytoskeletal networks.

Fluorescent, Acyl Chain-labeled Phosphatidylcholine Analogs Reveal Novel Transport Pathways across the Plasma Membrane of Yeast

Acyl chain-labeled NBD-phosphatidylcholine (NBD-PC) has been used to identify three gene products (Lem3p, Dnf1p, and Dnf2p) that are required for normal levels of inward-directed phospholipid transport (flip) across the plasma membrane of yeast. Although the head group structure of acyl chain-labeled NBD phospholipids has been shown to influence the mechanism of flip across the plasma membrane, the extent to which the acyl chain region and the associated fluorophore affect flip has not been assessed. Given the identification of these proteins required for NBD-PC flip, it is now possible to determine whether the fluorophore attached to a phospholipid acyl chain influences the mechanism of flip. Thus, flip of phosphatidylcholine molecules with three different Bodipy fluorophores (Bodipy FL, Bodipy 530, and Bodipy 581) was tested and compared with that of NBD-PC in strains carrying deletions in LEM3, DNF1, and DNF2. Deletion of these genes significantly reduced the flip of NBD-PC and Bodipy FL-PC but had no effect on that of Bodipy 581-PC and Bodipy 530-PC. These data, in combination with comparisons of the effect of ATP depletion, collapse of the proton electrochemical gradient across the plasma membrane, and culture density led to the conclusion that at least three different flip pathways exist in yeast that are selective for the structure of the fluorophore attached to the acyl chain of phosphatidylcholine molecules.

Interaction with the Na,K-ATPase and Tissue Distribution of FXYD5 (Related to Ion Channel)

FXYD5 (related to ion channel, dysadherin) is a member of the FXYD family of single span type I membrane proteins. Five members of this group have been shown to interact with the Na,K-ATPase and to modulate its properties. However, FXYD5 is structurally different from other family members and has been suggested to play a role in regulating E-cadherin and promoting metastasis (Ino, Y., Gotoh, M., Sakamoto, M., Tsukagoshi, K., and Hirohashi, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 365-370). The goal of this study was to determine whether FXYD5 can modulate the Na,K-ATPase activity, establish its cellular and tissue distribution, and characterize its biochemical properties. Anti-FXYD5 antibodies detected a 24-kDa polypeptide that was preferentially expressed in kidney, intestine, spleen, and lung. In kidney, FXYD5 resides in the basolateral membrane of the connecting tubule, the collecting tubule, and the intercalated cells of the collecting duct. However, there is also labeling of the apical membrane in long thin limb of Henle's loop. FXYD5 was effectively immunoprecipitated by antibodies to the alpha subunit of Na,K-ATPase and the anti-FXYD5 antibody immunoprecipitates alpha. Co-expressing FXYD5 with the alpha1 and beta1 subunits of the Na,K-ATPase in Xenopus oocytes elicited a more than 2-fold increase in pump activity, measured either as ouabain-blockable outward current or as ouabain-sensitive (86)Rb(+) uptake. Thus, as found with other FXYD proteins, FXYD5 interacts with the Na,K-ATPase and modulates its properties.

Proteolytic Processing of the Laminin α3 G Domain Mediates Assembly of Hemidesmosomes but Has No Role on Keratinocyte Migration

Laminin-5 (Lm5), the major adhesion ligand of basal epithelial cells, undergoes complex extracellular proteolytic processing that influences cell adhesion and migration. In tumor cell lines, the proteolytic truncation of the C-terminal G domain of the Lm alpha3 chain induces assembly of hemidesmosomes and downregulates cell migration. To define the biological functions of the alpha3 G domain processing in physiological conditions, we have expressed a series of mutant alpha3 complementary DNA in human primary alpha3-null keratinocytes immortalized by human papillomavirus E6E7 (HKalpha3 cells). Using monolayer and organotypic cell cultures we show that: (1) the hinge region between subdomains G3 and G4 carries the proteolytic cleavage sites; (2) nucleation of the hemidesmosomal proteins is independent of the proteolytic maturation of the alpha3 G domain, whereas formation of mature hemidesmosomes relies on proteolytic cleavage of alpha3; and (3) the proteolytic processing plays no role in cell migration, which suggests that nucleation of hemidesmosomal structures in culture does not reflect the migratory potential of the epithelial cells. Our results also demonstrate that HKalpha3 cells are a unique model system, which will be useful to dissect the functions and molecular interactions of Lm5.

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

It has become evident that the process of protein synthesis is performed by many cellular polypeptides acting in concert within the structural confines of protein complexes. In multicellular eukaryotes, one of these assemblies is a multienzyme complex composed of eight proteins that have aminoacyl-tRNA synthetase activities as well as three non-synthetase proteins (p43, p38, and p18) with diverse functions. This study uses electron microscopy and three-dimensional reconstruction to explore the arrangement of proteins and tRNA substrates within this "core" multisynthetase complex. Binding of unfractionated tRNA establishes that these molecules are widely distributed on the exterior of the structure. Binding of gold-labeled tRNA(Leu) places leucyl-tRNA synthetase and the bifunctional glutamyl-/prolyl-tRNA synthetase at the base of this asymmetric "V"-shaped particle. A stable cell line has been produced that incorporates hexahistidine-labeled p43 into the multisynthetase complex. Using a gold-labeled nickel-nitrilotriacetic acid probe, the polypeptides of the p43 dimer have been located along one face of the particle. The results of this and previous studies are combined into an initial three-dimensional working model of the multisynthetase complex. This is the first conceptualization of how the protein constituents and tRNA substrates are arrayed within the structural confines of this multiprotein assembly.

External amebocytes guard the pharynx entry in a tunicate (Ascidiacea)

In the present report, we describe the identification of unusual free amebocytes, completely exposed to seawater, which inhabit the inner surface of the oral and atrial siphons of the compound ascidian Botryllus schlosseri (Urochordata). The origin and biological role of these cells were studied by cytochemical and ultrastructural analysis. These amebocytes are mononucleate cells, with numerous round granules, varying in content, and long filopodia, which contact the cuticle protrusions of the tunic in the siphon. Histochemical, histoenzymatic and immunohistochemical assays were carried out under light microscopy on sections and on living and fixed cultured hemocytes. Results showed that the phagocytic blood cells and the free amebocytes of the siphons shared: (i) affinity for the α-mannose specific agglutinin of Narcissus pseudonarcissus (NPA), (ii) occurrence of hydrolytic activities of acid phosphatase and non-specific esterases inside lysosomal vesicles and large vacuoles, (iii) membrane labeling with the lipophilic dye PKH26 specific for phagocytic cells, (iv) anti-CD39 immunocytochemical labeling specific for lysosomes of mammalian macrophages. All histochemical data support the hypothesis that these cells are ‘sentinel cells’ belonging to the hyaline amebocyte population of the phagocytic differentiation line of the immunocytes, since they can also recognize and phagocytize carmine experimentally administered as target particles.

Localization of connexin 30 in the luminal membrane of cells in the distal nephron

Several isoforms of the gap junction protein connexin (Cx) have been identified in a variety of tissues that communicate intercellular signals between adjacent cells. In the kidney, Cx37, Cx40, and Cx43 are localized in the vasculature, glomerulus, and tubular segments in a punctuate pattern, typical of classic gap junction channels. We performed immunohistochemistry in the mouse, rat, and rabbit kidney to study the localization of Cx30 protein, a new member of the Cx family. The vasculature, glomerulus, and proximal nephron segments were devoid of staining in all three species. Unexpectedly, Cx30 was found throughout the luminal membrane of select cells in the distal nephron. Expression of Cx30 was highest in the rat, which also showed some diffuse cytosolic labeling, continuous from the medullary thick ascending limb to the collecting duct system, and with the highest level in the distal convoluted tubule. Labeling in the mouse and rabbit was much less, limited to intercalated cells in the connecting segment and cortical collecting duct, where the apical signal was particularly strong. A high-salt-containing diet and culture medium upregulated Cx30 expression in the rat inner medulla and in M1 cells, respectively. The distinct, continuous labeling of the luminal plasma membrane and upregulation by high salt suggest that Cx30 may function as a hemichannel involved in the regulation of salt reabsorption in the distal nephron.

In vivo allograft rejection in a bony fish Dicentrarchus labrax (L.): characterisation of effector lymphocytes

Lymphoid cell subpopulations involved in allograft rejection in the teleost Dicentrarchus labrax were characterised at the ultrastructural level and quantified by using monoclonal antibodies against T- and B-lymphocytes. T-cells positive for T-cell receptor beta-chain (TcR beta) were detected by reverse transcription/polymerase chain reaction (RT-PCR) and in situ hybridisation by using RNA probes for TcR beta. Flow cytometry detected a similar percentage of T- and B-lymphocytes (around 17%) in the leucocyte-enriched fraction from allografts. Two different types of T-lymphocytes (DLT 15-immunoreactive) infiltrating the allografts were identified by cytomorphology: small cells with high nuclear/cytoplasmic ratio and cells with a higher cytoplasmic content. RT-PCR revealed a single band (513 bp) corresponding to the TcR beta. In situ hybridisation showed that TcR beta-positive cells in the grafted muscle fibres were less numerous compared with DLT 15-positive cells, as evidenced in parallel sections, suggesting that cytotoxic cells might express different TcR phenotypes. DLIg 3-immunoreactive Ig-producing lymphocytes had: 1) a high nuclear/cytoplasmic ratio or 2) a larger size similar to that of pre-plasma cells (plasma cells lacked any membrane labelling).

Increased Toll-Like Receptor 4 Expression in Thymus of Myasthenic Patients with Thymitis and Thymic Involution

Thymic abnormalities are present in approximately 80% of myasthenia gravis (MG) patients, and the thymus seems to be the main site of autosensitization to the acetylcholine receptor. In view of findings that the innate immune system can generate an autoimmune response, we studied the expression of Toll-like receptors (TLRs) 2 to 5, key components of innate immunity signaling pathways, in 37 thymuses from patients with autoimmune MG. TLR4 mRNA levels were significantly greater in thymitis (hyperplasia with diffuse B-cell infiltration) and involuted thymus than in germinal center hyperplasia and thymoma. By immunohistochemistry and confocal microscopy, cells positive for TLR4 protein were rarely detected in thymoma. However, in thymitis TLR4 protein was mostly found on epitheliomorphic (cytokeratin-positive) cells located in close association with clusters of acetylcholine receptor-positive myoid cells in thymic medulla and also at the borders between cortical and medullary areas. B cells were never TLR4-positive. TLR4 protein was also present in remnant tissue of involuted thymus. This is the first finding of a possible link between innate immunity and MG. We speculate that in a subgroup of MG patients, an exogenous or endogenous danger signal may activate the innate immune system and give rise to TLR4-mediated mechanisms contributing to autoimmunity.

P systems with active membranes and without polarizations

P systems with active membranes but without using electrical charges (polarizations) are shown to be complete for generating recursively enumerable string languages when working on string objects and using only rules with membrane transitions as well as rules with membrane dissolving and elementary membrane division, but also when using various other kinds of rules, even including a new type of rules allowing for membrane generation. In particular, allowing for changing membrane labels turns out to be a very powerful control feature.

Tissue distribution of the “N-end rule” ubiquitin-conjugating enzyme, HR6, in the rat

The conjugation of multiple ubiquitin molecules is required for recognition and degradation of a protein by the proteasome. The ubiquitination pathway responsible for the bulk of constitutive protein degradation targets proteins carrying basic or large hydrophobic amino acids at the N-terminus. In mammalian cells, this "N-end rule" pathway requires the ubiquitin-conjugating enzyme HR6. Until now, it has not been known which mammalian tissues and cell types predominantly utilize this pathway for degradation. Therefore, the distribution and intracellular localization of HR6 was determined by indirect immunofluorescence techniques and protein blotting of adult rat tissues. Intense immunoreactivity against HR6 was detected in various epithelia, muscle, testis, peripheral neurons, chromaffin cells and macrophages, whereas lower HR6 protein levels were found in the gut or in the kidney. Autonomic and sensory neurons, glandular cells and spermatocytes revealed prominent nuclear HR6 immunoreactivity. Plasma membrane labeling was observed in peripheral neurons, spermatocytes and skeletal muscle cells. Smooth muscle cells, macrophages, endothelial and epithelial cells exhibited primarily cytoplasmic staining. The clear differences in the regional and intracellular distribution of HR6 are suggestive for the involvement of N-end rule protein degradation in various physiological processes dependent on cell type and subcellular structure.

NKCC1 and NHE1 are abundantly expressed in the basolateral plasma membrane of secretory coil cells in rat, mouse, and human sweat glands

In isolated sweat glands, bumetanide inhibits sweat secretion. The mRNA encoding bumetanide-sensitive Na(+)-K(+)-Cl(-) cotransporter (NKCC) isoform 1 (NKCC1) has been detected in sweat glands; however, the cellular and subcellular protein localization is unknown. Na(+)/H(+) exchanger (NHE) isoform 1 (NHE1) protein has been localized to both the duct and secretory coil of human sweat duct; however, the NHE1 abundance in the duct was not compared with that in the secretory coil. The aim of this study was to test whether mRNA encoding NKCC1, NKCC2, and Na(+)-coupled acid-base transporters and the corresponding proteins are expressed in rodent sweat glands and, if expressed, to determine the cellular and subcellular localization in rat, mouse, and human eccrine sweat glands. NKCC1 mRNA was demonstrated in rat palmar tissue, including sweat glands, using RT-PCR, whereas NKCC2 mRNA was absent. Also, NHE1 mRNA was demonstrated in rat palmar tissue, whereas NHE2, NHE3, NHE4, electrogenic Na(+)-HCO(3)(-) cotransporter 1 NBCe1, NBCe2, electroneutral Na(+)-HCO(3)(-) cotransporter NBCn1, and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger NCBE mRNA were not detected. The expression of NKCC1 and NHE1 proteins was confirmed in rat palmar skin by immunoblotting, whereas NKCC2, NHE2, and NHE3 proteins were not detected. Immunohistochemistry was performed using sections from rat, mouse, and human palmar tissue. Immunoperoxidase labeling revealed abundant expression of NKCC1 and NHE1 in the basolateral domain of secretory coils of rat, mouse, and human sweat glands and low expression was found in the coiled part of the ducts. In contrast, NKCC1 and NHE1 labeling was absent from rat, mouse, and human epidermis. Immunoelectron microscopy demonstrated abundant NKCC1 and NHE1 labeling of the basolateral plasma membrane of mouse sweat glands, with no labeling of the apical plasma membranes or intracellular structures. The basolateral NKCC1 of the secretory coils of sweat glands would most likely account for the observed bumetanide-sensitive NaCl secretion in the secretory coils, and the basolateral NHE1 is likely to be involved in Na(+)-coupled acid-base transport.

Rapid Access of Antibodies to α5β1 Integrin Overexpressed on the Luminal Surface of Tumor Blood Vessels

Integrin alpha(5)beta(1) is overexpressed on endothelial cells of tumor vessels and is uniformly and rapidly accessible to antibodies in the bloodstream. Here, we determined whether antibodies rapidly gain access to integrin overexpressed on the abluminal (basolateral) surface of endothelial cells through vascular leakiness or whether the rapid accessibility results instead because the integrin is overexpressed on the luminal (apical) surface of endothelial cells due to loss of cell polarity. Using tumors in RIP-Tag2 transgenic mice as a model, we first compared the binding pattern of intravascular anti-alpha(5)beta(1) integrin antibody with the leakage pattern of nonspecific IgG. The distributions did not match: anti-alpha(5)beta(1) integrin antibody uniformly labeled the tumor vasculature, but IgG was located in patchy sites of leakage. We next injected an antibody to fibrinogen/fibrin, which resulted in patchy labeling of tumors that matched the leakage of IgG and the overall distribution of fibrin in tumors. Similarly, injected antibodies to the basement membrane protein fibronectin, a ligand of alpha(5)beta(1) integrin, or type IV collagen produced patchy sites of leakage instead of uniform labeling of vascular basement membrane. Differences in the kinetics of labeling, which for alpha(5)beta(1) integrin antibody was near maximal by 10 minutes but for the other antibodies gradually increased over 6 hours, indicated differences in accessibility of their respective targets. Isosurface rendering of confocal microscopic images was consistent with antibody binding to alpha(5)beta(1) integrin on the luminal surface of endothelial cells. Together, these findings indicate that the rapid accessibility of alpha(5)beta(1) integrin in RIP-Tag2 tumors results from overexpression of the integrin on the luminal surface of tumor vessels.