High resolution 2-deoxyglucose localization in olfactory epithelium

The olfactory epithelium of the salamander and the mouse hasbeen analyzed for patterns of activity elicited by odor stimulation.A high-resolution adaptation of the 2-deoxyglucose (2DG) methodwas used, involving freeze-substitution in anhydrous acetonecombined with nuclear emulsion autoradiography, according toSejnowski et al. (1980). In animals exposed to the odor of amylacetate, the autoradiograms of 2 µg thick sections showedrestricted regions of high [¹⁴C]2DG and [³H]2DG uptake. Withinthese regions, there was a characteristic pattern of diffuselocalization in the superficial supranuclear zone; small clumpsor strands of grains in the receptor cell nuclei layer; andthick clumps of grains in the basal cell layer. The relationof these patterns of differential odorinduced activity in thecells of the epithelium is discussed. ^* Present address: Department of Membrane Research, The WeizmannInstitute of Science, Rehovot, Israel     

Internalization of odorant-binding proteins into the mouse olfactory epithelium

The detection of odorants in vertebrates is mediated by chemosensory neurons that reside in the olfactory epithelium of the nose. In land-living species, the hydrophobic odorous compounds inhaled by the airstream are dissolved in the nasal mucus by means of specialized globular proteins, the odorant-binding proteins (OBPs). To assure the responsiveness to odors of each inhalation, a rapid removal of odorants from the microenvironment of the receptor is essential. In order to follow the fate of OBP/odorant complexes, a recombinant OBP was fluorescently labeled, loaded with odorous compounds, and applied to the nose of a mouse. Very quickly, labeled OBP appeared inside the sustentacular cells of the epithelium. This uptake occurred only when the OBP was loaded with appropriate odorant compounds. A search for candidate transporters that could mediate such an uptake process led to the identification of the low density lipoprotein receptor Lrp2/Megalin. In the olfactory epithelium, megalin was found to be specifically expressed in sustentacular cells and the Megalin protein was located in their microvilli. In vitro studies using a cell line that expresses megalin revealed a rapid internalization of OBP/odorant complexes into lysosomes. The uptake was blocked by a Megalin inhibitor, as was the internalization of OBPs into the sustentacular cells of the olfactory epithelium. The results suggest that a Megalin-mediated internalization of OBP/odorant complexes into the sustentacular cells may represent an important mechanism for a rapid and local clearance of odorants.     

Inositol polyphosphate derivative inhibits Na+ transport and improves fluid dynamics in cystic fibrosis airway epithelia

Amiloride-sensitive, epithelial Na⁺ channel (ENaC)-mediated, active absorption of Na⁺ is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, D-myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na⁺ and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and ²²Na⁺ flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives. epithelial Na⁺ channels; fluid absorption     

Choline uptake across the ventricular membrane of neonate rat choroid plexus

The uptake of[³H]choline from thecerebrospinal fluid (CSF) side of the rat neonatal choroid plexus wascharacterized in primary cultures of the choroidal epithelium grown onsolid supports. Cell-to-medium concentration ratios were ~5 at 1 minand as high as 70 at 30 min. Apical choline uptake was facilitated; theK_m was ~50µM. Several organic cations (e.g., hemicholinium-3 and N¹-methylnicotinamide)inhibited uptake. The reduction or removal of externalNa⁺ or the addition of 5 mM LiClhad no effect on uptake. However, increasing externalK⁺ concentration from 3 to 30 mMdepolarized ventricular membrane potential (70 to 15 mV)and reduced uptake to 45% of that for the control. Treatment with 1 mMouabain or 2 mM BaCl₂ reduced uptake 45%, and intracellular acidification reduced uptake to ~90%of that for controls. These data indicate that the uptake of choline from CSF across the ventricular membrane of the neonatal choroidal epithelium is not directly coupled toNa⁺ influx but is sensitive toplasma membrane electrical potential.

     

Learning-induced change in neural activity during acquisition and consolidation of a passive avoidance response in the rat

Time-dependent alterations in neural activity have been established during the acquisition and consolidation of a stepdown passive avoidance paradigm. Change in neural activity was established by administering a glucose analogue, [³H]2-deoxyglucose, 50min prior to sacrifice and estimating perchloric acid soluble counts in nine hand dissected brain regions. Change in [³H]2-deoxyglucose uptake was closely paralleled in both trained and yoked animals for up to 40min following task acquisition however the striatum was the only area to exhibit a task-specific increase in [³H]2-deoxyglucose uptake at 20–30min after training. Longterm changes in neural activity were also apparent as the amygdala and brainstem showed increased [³H]2-deoxyglucose uptake at the 24h time point. No further paradigm-specific changes were apparent at 48 h. These findings are concluded to suggest that the striatum is involved in the early events of acquiring a passive avoidance response and the amygdala and brainstem during the later events.     

A monoclonal antibody directed against high Mr salivary mucins recognizes the SO3-3Gal{beta}1-3GlcNAc moiety of sulfo-Lewisa: a histochemical survey of human and rat tissue

Using a panel of synthetic oligosaccharides attached to a polyacrylamidecarrier, the epitope of monoclonal antibody F2, evoked to highM_r salivary mucins, was mapped to the SO₃-3Galß1-3GlcNAc-moiety of the sulfo-Le^a antigen. Using immunochemical techniques,the expression of the F2-epitope was investigated in a numberof different isolated human mucin species, as well as in humanand rat tissue specimens. The mAb F2 bound to high M_r salivarymucins, cervical mucins, colon mucins and gallbladder mucins,but not to low M_r salivary mucins nor to gastric mucins. Immunohistochemicalscreening of human tissues with mAb F2 revealed a positive reactionwith a number of epithelia, including the (sero)mucous salivaryglands, the goblet cells of the colon, submucosal glands ofthe lung, the lining epithelium of cervical and esophageal glands,the suprabasal skin keratinocytes, and Hassall's corpusclesof the thymus. No staining was found in normal breast, pancreas,small intestine, spleen, and lymph nodes. Normal gastric glandswere negative, but gastric intestinal metaplastic glands stronglystained with the antibody. In rat tissues, mAb F2 labeled epithelialcells of salivary glands, colon and stomach. In addition toepithelial cells, extracellular matrix components in rat thymusand skin were labeled by mAb F2. No labeling of erythrocytes,granulocytes, lymphocytes or bone marrow cells was found byFACScan analysis. The present data shows a tissue specific distributionof the F2-epitope in cells from the epithelial lineage in humanand rat. epithelial tissue sulfo-Lewis^a mucins mAbs immunohistochemistry     

The effects of α- and β-adrenergic agents,Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent V_max without altering the K_m. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl₂ also increased uptake. Lowering of the perfusate Ca²⁺ concentration from 1.27 to 0.1 mM Ca²⁺, addition of the Ca²⁺ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca²⁺ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca²⁺, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca²⁺ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca²⁺ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca²⁺ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca²⁺ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca²⁺-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca²⁺-dependent and a Ca²⁺-independent component. Reversal studies suggest an additional role for Ca²⁺ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Effects of air flow on rat electroolfactogram

The electroolfactogram (EOG) previously has been used to demonstrate the regional distribution of rat olfactory epithelial odorant responses. Here, we evaluated the effects of airflow parameters on EOGs in two preparations: one where odorants were directly applied to the epithelium (opened preparation) and one where odorants were drawn through the nasal passages by an artificial sniff (closed preparation). EOG rise times served as one measure of odorant access. For isoamyl acetate (but not for limonene), rise times were slower in the lateral recesses of the closed (but not the opened) preparation. Polar odorants (amyl acetate, carvone and benzaldehyde) evoked smaller responses in the closed preparation than in the opened preparation, and these responses were particularly depressed in the lateral regions of the closed preparation. Responses to nonpolar hydrocarbon odorants (limonene and benzene) were equal in the lateral regions of both preparations, but were somewhat depressed in the medial region of the closed preparation. The responses to some polar odorants in the closed preparation were sensitive to changes in airflow parameters. These data suggest that the sorptive properties of the nose contribute substantially to determining the response of the epithelium and act to increase differences produced by inherent receptor mechanisms.     

Purinergic agonists stimulate lens Na-K-ATPase-mediated transport via a Src tyrosine kinase-dependent pathway

The Na-K-ATPase is vital for maintenance of lens transparency. Past studies using intact lens suggested the involvement of tyrosine kinases in short-term regulation of Na-K-ATPase. Furthermore, in vitro phosphorylation of a lens epithelial membrane preparation by Src family kinases (SFKs), a family of nonreceptor tyrosine kinases, resulted in modification of Na-K-ATPase activity. Here, the effect of purinergic agonists, ATP and UTP, on Na-K-ATPase function and SFK activation was examined in the rabbit lens. Na-K-ATPase function was examined using two different approaches, measurement of ouabain-sensitive potassium (⁸⁶Rb) uptake by the intact lens, and Na-K-ATPase activity in lens epithelial homogenates. ATP and UTP caused a significant increase in ouabain-sensitive potassium (⁸⁶Rb) uptake. Na-K-ATPase activity was increased in the epithelium of lenses pretreated with ATP. Lenses treated with ATP or UTP displayed activation of SFKs as evidenced by increased Western blot band density of active SFK (phosphorylated at the active loop Y416) and decreased band density of inactive SFKs (phosphorylated at the COOH terminal). A single PY416-Src immunoreactive band at 60 kDa was observed, suggesting not all Src family members are activated. Immunoprecipitation studies showed that band density of active Src, and to a lesser extent active Fyn, was significantly increased, while active Yes did not change. Preincubation of the lenses with SFK inhibitor PP2 abolished the ATP-induced increase in ouabain-sensitive potassium (⁸⁶Rb) uptake. The results suggest selective activation of Src and/or Fyn is part of a signaling mechanism initiated by purinergic agonists that increases Na-K-ATPase-mediated transport in the organ-cultured lens. Src kinase; receptors     

Unexpected similarity of intestinal sugar absorption by SGLT1 and apical GLUT2 in an insect (Aphidius ervi, Hymenoptera) and mammals

Sugars are critical substrates for insect metabolism, but little is known about the transporters and epithelial routes that ensure their constant supply from dietary resources. We have characterized glucose and fructose uptakes across the apical and basolateral membranes of the isolated larval midgut of the aphid parasitoid Aphidius ervi. The uptake of radiolabeled glucose at the basal side of the epithelium was almost suppressed by 200 microM cytochalasin B, uninhibited by phlorizin, and showed the following decreasing rank of specificity for the tested substrates: glucose > glucosamine > fructose, with no recognition of galactose. These functional properties well agree with the expression of GLUT2-like transporters in this membrane. When the apical surface of the epithelium was also exposed to the labeled medium, a cation-dependent glucose uptake, inhibited by 10 microM phlorizin and by an excess of galactose, was detected suggesting the presence in the apical membrane of a cation-dependent cotransporter. Radiolabeled fructose uptakes were only partially inhibited by cytochalasin B. SGLT1-like and GLUT5-like transporters were detected in the apical membranes of the epithelial cell by immunocytochemical experiments. These results, along with the presence of GLUT2-like transporters both in the apical and basolateral cell membranes of the midgut, as we recently demonstrated, allow us to conclude that the model for sugar transepithelial transport in A. ervi midgut appears to be unexpectedly similar to that recently proposed for sugar intestinal absorption in mammals.     

Stereo architecture of the interface of the epithelial cell layer and connective tissue core of the foliate papilla in the rabbit tongue.

The three-dimensional relationship between the epithelial cell layer and the underlying connective tissue core (CTC) of the foliate papilla of the rabbit tongue was studied by scanning electron microscopy after removal of the epithelial cell layer. The foliate papillae were fixed in Karnovsky's fixative, and the epithelial cell layers were exposed to long-term hydrochloric acid treatment (3.5 N HCl for 2-3 weeks at room temperature). The foliate papillae consisted of ridges and grooves located on the posterolateral margin of the tongue. They appeared as linear projections or ridges of lingual mucosa roughly perpendicular to the longitudinal axis of the tongue. These projections or ridges were parallel to one another and separated by grooves. After removal of the epithelium, two kinds of CTC folds appeared: one was the septal fold of CTC which runs in the central portion under each linear projection or ridge, and the other consisted of two sheets of groove side folds of CTC which run along both sides of the former and face the groove side epithelium. It was revealed that there are two sheets of septal epithelial processes, and each of them fits in between each septal fold and groove side fold of CTC. Numerous taste buds were located in the groove side epithelia, and their pores faced the surface of the groove. On the hollow surfaces that appeared on the surface of the groove side fold of CTC after removal of the epithelial cells with taste buds, nerve-terminal-like structures were encountered. Some openings of the ducts of small lingual glands were arranged linearly on the underside of the basal portion of each groove side epithelium.     

Evidence for electrogenic active ion transport across the frog olfactory mucosa in vitro

We present the first electrophysiological evidence for electrogenicion transport across the frog olfactory mucosa in vitro. Whenthe isolated dorsal mucosa was placed in an Ussing chamber andbathed symmetrically in amphibian Ringer's, the ciliated sidebecame electronegative (V = –5.2 mV ± 0.7 mV).The resistance of the mucosal preparation was 148 ± 4 cm². The true short-circuit current was obtained as the intersectionof the I–V curve with the current axis after correctingfor the series solution resistance. The average value of theshort-circuit current was 35.9 µA/cm². The I–V relationwas linear over the applied potential range of ± 16mV.The magnitude of the specific resistance of the olfactory mucosais comparable to values reported for various actively transportingrespiratory and oral cavity epithelia. Because the geometricalarea of the aperture used to normalize both the short-circuitcurrent and the resistance undoubtedly underestimates the actualarea of the dorsal olfactory epithelium, the specific resistanceand the short-circuit current are probably underestimated andoverestimated, respectively. Therefore, the nominally low resistanceneed not imply a leaky epithelium. Substitution of NO₃^–for Cl^– caused the current to increase and the resistanceto decrease. These results suggest that cation absorption playsa role in the sign of the short-circuit current. The in vitropreparation responded to the odorant ethyl n-butyrate by givingan electro-olfactogram (EOG)-like voltage transient which wassuperimposed on the steady-state potential created by activeion transport. The significance of these results is discussedfrom the perspective of the peripheral events surrounding olfactorytransduction.     

Mesenchyme cells degrade epithelial basal lamina glycosaminoglycan

We investigated whether turnover of basal lamina glycosaminoglycan (GAG), an active process during epithelial morphogenesis, involves the mesenchyme. Fixed, prelabeled, isolated mouse embryo submandibular epithelia were prepared retaining radioactive surface components, as determined by autoradiographic and enzymatic studies, and a basal lamina, as assessed by electron microscopy. Recombination of mouse embryo submandibular mesenchyme with these epithelia stimulates the release of epithelial radioactivity when the labeled precursor is glucosamine or glucose but not when it is amino acid. The release is linear with time during 150 min incubation. Augmented release of epithelial label requires living mesenchyme which must be close proximity with the epithelia. Although heterologous mesenchymes, including lung, trachea, and jaw, stimulate the release of submandibular epithelial label, epithelial tissues do not. The label released by intact submandibular mesenchyme from prelabeled epithelia is in GAG and in two unique fractions: heterogeneous materials of tetrasaccharide or smaller size and N-acetylglucosamine. Enzymatic treatment of the heterogeneous materials revealed the presence of glycosaminoglycan-derived oligosaccharides. These unique products were not obtained by incubating prelabeled epithelia with a mesenchymal cell extract, suggesting that intact mesenchymal cells are required. N-Acetylglucosamine was also released when mesenchyme was recombined with living prelabeled epithelia which contained labeled basal laminar GAG. Our results establish that submandibular epithelial basal lamina GAGs are degraded by submandibular mesenchyme. We propose that one mechanism of epithelial-mesenchymal interaction is the degradation of epithelial basal laminar GAG by mesenchyme.     

THE SHELL-FORMING MANTLE EPITHELIUM OF BIOPHALARIA GLABRATA (PULMONATA): ULTRASTRUCTURE, PERMEABILITY AND CYTOCHEMISTRY

In the freshwater snail Biomphalaria glabrata, the outer mantleepithelium and the transitional epithelium from the outer mantleepithelium to the belt are characterized by apical vesiclesof different electron density and vacuoles including lipid dropletsand fibrillar structures. Wide intercellular spaces predominatein the transitional epithelium. In addition to belt desmosomes,freeze fracture studies detect septate junctions as apical intercellularjunctions. The permeability of the septate junctions is testedby injecting anaesthetized snails with solutions containingperoxidase and lanthanum-nitrate. The septate junctions appearto be impermeable for the protein and inhibit permeation ofthe ion. Alkaline phosphatase is detected at the light microscope levelin the transitional and outer mantle epithelium. At the electronmicroscope level localization of alkaline phosphatase is restrictedto the apical and basal cell membranes of the same epithelia.An ATPase with low affinity to Ca²⁺ and Mg²⁺ ions is observedin the lumen of vacuoles in the proximal belt, the transitionaland the outer mantle epithelium. The possible role of the vacuolesas a site of synthesis or modification of the intercrystallinematrix is discussed. A high affinity Ca²⁺/Mg²⁺ ATPase is detectedwith the help of Electron Spectroscopic Imaging in the cellmembranes and in membranes of the rER. A model for solute transport through the epithelia to the extrapallialspace is proposed. (Received 18 August 1992; accepted 4 January 1993)     

Epithelial permeability induced by neutrophil transmigration is potentiated by hypoxia: Role of intracellular cAMP

Mucosal tissues, such as the lung and intestine, are primary targets for ischemic damage. Under these conditions, neutrophil (polymorphonuclear leukocyte; PMN) infiltration into the protective epithelium has been implicated as a pathophysiologic mediator. Because PMN transepithelial migration results in increased paracellular permeability, and because our previous data revealed that epithelial hypoxia enhances PMN transmigration, we hypothesized that macromolecular permeability may be altered in epithelium exposed to hypoxia and reoxygenation (H/R) in the presence of PMNs. Human intestinal epithelia (T84) were grown on permeable supports, exposed to cellular hypoxia (pO₂ 20 torr) for 0–72 hr, and examined for increases in PMN-evoked permeability by using standard flux assays. Increasing epithelial hypoxia potentiated PMN-induced permeability of labeled paracellular tracers (size range 3–500 kD). Such increases were blocked by monoclonal antibody (mAb) to the PMN integrin CD11b (82 ± 1% decreased compared with control mAb) and were partially blocked by anti-CD47 mAb(51 ± 1%). Assessment of barrier recovery revealed that monolayers exposed to H/R were significantly diminished in their ability to reseal following PMN transmigration (recovery of 36 ± 6% in H/R vs. 94 ± 2% in normoxic controls). Because intracellular cyclic AMP (cAMP) has been demonstrated to regulate epithelial permeability, and because PMN-derived compound(s), (i.e., 5′-adenosine monophosphate; AMP) elevate epithelial cAMP, we examined the impact of hypoxia on epithelial cAMP responses. These experiments revealed that hypoxic epithelia were diminished in their ability to generate cAMP, and pharmacologic elevation (8-bromo-cAMP) of intracellular cAMP in hypoxic cells normalized both PMN-induced permeability changes and restoration of barrier function. These results support a role for PMN in increased intestinal permeability associated with reperfusion injury and imply a substantial role for cAMP signaling in maintenance of permeability during PMN transmigration. J. Cell. Physiol. 176:76–84, 1998. © 1998 Wiley-Liss, Inc.     

Distribution of parvalbumin,cytochrome oxidase activity and 14C-2-deoxyglucose uptake in the brain of the zebra finch

Summary The visual system of adult zebra finches was investigated 1) immunocytochemically for the distribution of the Ca²⁺-binding protein parvalbumin, 2) for the activity of the respiratory enzyme cytochrome oxidase, and 3) for the uptake of 2-deoxyglucose. In the visual system, only nuclei of the tecto-fugal pathway and related nuclei were labeled by the parvalbumin antiserum (ectostriatum, nucleus rotundus, tectum opticum, nucleus postero-ventralis, nucleus praetectalis, nucleus subpraetectalis, nucleus isthmipars parvocellularis and-magnocellularis, nucleus isthmoopticus). Additionally, parvalbumin-positive nuclei such as area entorhinalis, area a in the hyperstriatum accessorium, nucleus septalis medialis and nucleus habenularis are described. With few exceptions there was a striking correlation of parvalbumin-positive and cytochrome oxidase-positive nuclei of the visual system. Most of the areas with high levels of parvalbumin and cytochrome oxidase were labeled with 2-deoxyglucose as well. Nucleus posteroventralis showed labeling below background intensity. 2-Deoxyglucose uptake primarily reflects energy demands of actual electrical activity, i.e., of the Na⁺-K⁺ pump, while cytochrome oxidase supposedly indicates the long-term energy demands of various metabolic pathways. Consequently, high cytochrome oxidase activity together with large 2-deoxyglucose uptake in the tecto-fugal pathway might be due to the high spontaneous and evoked electrical activity. Parvalbumin concentrations in the same areas (and in auditory areas, see Braun et al. 1985I) suggest as one possibility that special Ca²⁺ mechanisms are present in neuronal systems that can reach high levels of electrical activity.     

RNA interference targeted to multiple P2X receptor subtypes attenuates zinc-induced calcium entry

A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca²⁺-dependent Cl^– channels via stimulation of Ca²⁺ entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca²⁺ in human airway epithelial cells that translates into stimulation of sustained secretory Cl^– transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. On the basis of these studies, the Ca²⁺ entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X₄, P2X₅, and P2X₆ subtypes in non-CF (16HBE14o^–) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca²⁺ entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X₄ and P2X₆ (but not P2X₅) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca²⁺ entry markedly in fura-2 Ca²⁺ measurements and "knocked down" protein by >65%. These data suggest that multiple P2X receptor Ca²⁺ entry channel subtypes are expressed in airway epithelia. P2X₄ and P2X₆ may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype. purinergic receptors; zinc receptors; airway epithelia; cystic fibrosis; therapy     

Uptake of horseradish peroxidase from CSF into the choroid plexus of the rat,with special reference to transepithelial transport

Summary Protein uptake from cerebral ventricles into the epithelium of the choroid plexus, and transport across the epithelium were studied ultrastructurally in rats. Horseradish peroxidase (HRP, MW 40,000) was used as protein tracer. Steady-state ventriculo-cisternal perfusion with subatmospheric pressure (-10cm of water) in the ventricular system was applied. HRP dissolved in artificial CSF was perfused from the lateral ventricles to cisterna magna for various times, and ventriculo-cisternal perfusion, vascular perfusion or immersion fixation with a formaldehyde-glutaraldehyde solution was performed.Coated micropinocytic vesicles containing HRP were seen both connected with the apical, lateral and basal epithelial surface and within the cells. Heavily HRP-labeled vesicles were often fused with the lining membrane of slightly labeled or unlabeled intercellular spaces. Since the apical tight junctions of the epithelium never appeared open or never contained HRP in the spaces between the fusion points, and since the intercellular spaces between adjacent epithelial cells below the junctions only infrequently contained tracer after 5 min, by increasing amounts after 15–60 min of HRP perfusion, a vesicular transport of HRP from the apical epithelial surface to the intercellular spaces, bypassing the tight junctions, is suggested.In addition to the transepithelial transport, micropinocytic vesicles also transported HRP to the lysosomal apparatus of the epithelial cells. With increasing length of exposure to HRP, a sequence of HRP-labeled structures could be evaluated, from slightly labeled apical vacuoles and multivesicular bodies to very heavily labeled dense bodies.     

Basonuclin in murine corneal and lens epithelia correlates with cellular maturation and proliferative ability

Basonuclin is a zinc finger protein with highly restricted tissue distribution. It has been found in abundance only in keratinocytes of stratified epithelia and the germ cells of the testis and ovary. We studied the expression pattern of basonuclin in relation to cellular proliferation and differentiation in murine corneal and lens epithelia, two self-renewing tissues in the eye which contain cells that proliferate throughout life. Mouse corneal and lens epithelial cells at various stages of development were labeled with BrdU for 90 min to detect cells in S phase and to establish proliferative rates. Whole eyes of mouse or rat were processed for frozen sections and cellular basonuclin was detected by either a rabbit antimouse- or a rabbit anti-human-basonuclin antibody. Basonuclin was expressed in virtually all cells in the basal layer of corneal epithelium and in the pre-equatorial lens epithelium, the respective proliferative compartments of adult corneal and lens epithelia. Basonuclin expression in corneal epithelium began at post-natal life day 4, first in a few cells and then spread to virtually all basal cells at day 20. Basonuclin was consistently absent in limbal epithelium. Lens basonuclin, which was detected earlier than that of the cornea, was confined to the pre-equatorial epithelium and was absent in equatorial cells that expressed p57KIP2, an early differentiation marker for these cells. An important distinction between corneal and lens basonuclin is that the former is predominantly nuclear whereas the latter cytoplasmic.     

Lanthanum is transported by the sodium/calcium exchanger and regulates its activity

La³⁺ uptake was measured in fura 2-loaded Chinese hamster ovary cells expressing the bovine cardiac Na⁺/Ca²⁺ exchanger (NCX1.1). La³⁺ was taken up by the cells after an initial lag phase of 50-60 s and achieved a steady state within 5-6 min. Neonatal cardiac myocytes accumulated La³⁺ in a similar manner. La³⁺ uptake was due to the activity of the exchanger, because no uptake was seen in nontransfected cells or in transfected cells that had been treated with gramicidin to remove cytosolic Na⁺. The low rate of La³⁺ uptake during the lag period resulted from insufficient cytosolic Ca²⁺ to activate the exchanger at its regulatory sites, as shown by the following observations. La³⁺ uptake occurred without a lag period in cells expressing a mutant of NCX1.1 that does not exhibit regulatory activation by cytosolic Ca²⁺. The rate of La³⁺ uptake by wild-type cells was increased, and the lag phase was reduced or eliminated, when the cytosolic Ca²⁺ concentration was increased before initiating La³⁺ uptake. La³⁺ could substitute for Ca²⁺ at very low concentrations to activate exchange activity. Thus preloading cells expressing NCX1.1 with a small quantity of La³⁺ increased the rate of exchange-mediated Ca²⁺ influx by 20-fold; in contrast, cytosolic La³⁺ partially inhibited Ca²⁺ uptake by the regulation-deficient mutant. With an estimated K_D of 30 pM for the binding of La³⁺ to fura 2, we conclude that cytosolic La³⁺ activates exchange activity at picomolar concentrations. We speculatively suggest that endogenous trace metals might activate exchange activity under physiological conditions. fura 2; NCX1.1; myocyte