Stem cells and differentiation stages in the limbo-corneal epithelium.
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Animals↗
Biomedical subjects
J M Wolosin
Publications and source records attributed to J M Wolosin.
Connexin50, a gap junction protein of macrogliaP6n the mammalian retina and visual pathway.
Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunocytochemistry were used to study the expression of gap junction proteins (connexins; Cx) in the rat and rabbit retina. RT-PCR of rabbit total retinal RNA using primers selected for the human Cx50 (alpha 8 Cx) DNA template yielded cDNA fragments of the predicted base pair size. Western blots of rat and rabbit retinal membrane preparations probed with a monoclonal antibody which recognizes Cx50 in the lens of several mammalian species revealed a single band (MW 50 kD), identical to that recognized in lens membrane extracts. In frozen retinal sections of both species, the same monoclonal antibody as well as two polyclonal antisera raised against a synthetic peptide from the C-terminal region of the human Cx50 polypeptide labeled Müller cells and astrocytes. In Müller cells, labeling was strongest in the endfeet and in the filamentous processes ensheathing the photoreceptors. Extending from the neural retina, Cx50-like immuno-reactivity was detected in astrocytes of the optic nerve and along retinal projections within the CNS. Our data indicate that Müller cells and astrocytes of mammalian retinas and throughout the visual pathway are coupled through gap junctions composed of connexin50.
Animals↗
Differentiation-dependent expression of alpha-2,3-sialyltransferase in rabbit corneal epithelium.
PURPOSE: Lectin studies have shown that in the rabbit corneal epithelium, alpha-2,3 sialylation of O-linked glycans differentiates limbal and corneal epithelial cell phenotypes. Because sialic acid can be regulated at the level of the expression of sialyltransferases (STs), the purpose of the present study was to analyze the expression of alpha-2,3STs in this tissue. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to generate ST cDNA from total rabbit corneal epithelium RNA using primers selected from the sequences of three previously cloned STs capable of catalyzing the transfer of sialic acid to O-linked oligosaccharides, human placental Galbeta-1,3GalNAc-Galbeta-1,4GluNAcalpha-2,3ST (STZ), and mouse brain Galbeta-1,3GalNAcalpha-2,3ST types I and II (ST3Gal I and ST3Gal II). Tissue distribution of mRNA was assayed by fluorescent in situ hybridization. A synthetic peptide whose sequence was deduced from a cloned cDNA fragment was synthesized and used to prepare an anti-ST goat antiserum. The molecular weights of immunodetectable polypeptides and their distribution in cryostat sections of the limbocorneal area were investigated by western blot analysis and indirect immunofluorescence, respectively. RESULTS: RT-PCR yielded cDNA of expected basepair length for STZ and ST3(Gal II. The rabbit STZ cDNA was 86% identical with its human equivalent. Its mRNA was confined to the cornea, mainly in basal epithelial cells, and was not expressed in the limbus. Western blot analysis identified a band at 37 kDa whose binding was abolished by preincubation of the antiserum with the immunization peptide. Immunohistologic analysis revealed the presence of immunoreactive epitopes in all basal cells of the cornea but not in the limbus. CONCLUSIONS: STZ mRNA and the enzyme itself are expressed in the basal layer of the corneal epithelium but are absent in the limbus. This enzyme's de novo expression seems thus responsible for the differential expression of alpha-2,3 sialylation along the limbocorneal differentiation axes. At least one more alpha-2,3ST is also present in the epithelium.
Animals↗
Effect of heptanol on the short circuit currents of cornea and ciliary body demonstrates rate limiting role of heterocellular gap junctions in active ciliary body transport.
Rabbit ciliary body and cornea were mounted in Ussing-type chambers in Tyrode's under voltage clamp and the effects of heptanol, a gap junction inhibitor, on the short circuit current generated by each of the respective epithelia were determined. Studies were carried out either in control conditions or following amphotericin B permeabilization of either the basolateral membrane of the nonpigmented epithelium of the ciliary body or the apical membrane of the corneal epithelium, respectively. Previous studies have shown that, following these permeabilizations, short circuit currents are established, reflecting aqueous (or tear)-to-serosa Na+ fluxes, and that Na+ translocation through gap junctions connecting the individual layers of these tissues constitutes the major rate limiting step. Heptanol inhibited most of the short circuit current of the amphotericin B-modified ciliary body and cornea and of the unmodified ciliary body epithelium (control). In all these cases, the apparent IC50 was about 0.8 M. In the unmodified corneal epithelium, where ion translocation across the apical membrane constitutes the main rate limiting step for active secretion, 0.4 or 0.8 mM heptanol induced short circuit current increases; partial inhibition was observed only at high concentrations known to cause maximal inhibition of junctional permeability. Heptanol also enhanced the volume regulatory decrease of cultured human NPE cells, a process dependent on cell swelling-induced stimulation of Cl- and K+ permeabilities. Combined with our previous results demonstrating the lack of heptanol effects on other epithelial functions, these data suggest that the effect of heptanol on the active ciliary body transepithelial transport is primarily due to inhibition of the nonpigmented-pigmented junctional path and that this path is a potential site of rate limitation for the secretory process.
Alcohols↗
Stem cells of the corneal epithelium lack connexins and metabolite transfer capacity.
The stem cells of the corneal epithelial lineage are confined to the basal cell layer of the limbus, a vascularized outer corneal rim. These slow cycling cells of great proliferative potential maintain the corneal epithelial mass. Since cell-cell communication plays an important role in development and differentiation, we conducted a comparative examination of the expression of two corneal connexins, C x 43 and C x 50, and the tracer transfer capacity of the limbal and corneal epithelia using the scrape loading technique. C x 43 is abundantly expressed in the basal cell layer of the epithelium covering the cornea, but is essentially absent from the mouse, human, neonatal rabbit, and chicken limbal epithelium. In the adult rabbit the limbal epithelium displays an overall weak C x 43 immunoreactivity, but C x 43-free isolated basal cells can be distinguished. C x 50 is expressed throughout the corneal epithelium of the three mammalian corneas, but is not detectable in the limbus. Scrape loading experiments in the rabbit yielded results which were consistent with the immunohistological findings; limbal epithelium lacked tracer (lucifer yellow) transfer capacity, strongly suggesting the absence of functional gap junctions. Altogether, our results demonstrate the incompetence of stem cells for gap junction-mediated cell-to-cell communication. This property may reflect the need of these unique cells to maintain a distinct intracellular environment.
Aged↗
Connexin distribution in the rabbit and rat ciliary body. A case for heterotypic epithelial gap junctions.
PURPOSE: To evaluate the distribution of different alpha- and beta-type connexins (Cx) present in the dual layered ciliary body epithelia (CBE) of both rabbit and rat. METHODS: Immunocytochemical detection of Cx26, Cx32, Cx43, and Cx50 was performed on frozen sections of rabbit and rat ciliary body using indirect immunofluorescent methods. The identity of the antigens recognized by the monoclonal primary antibodies was further confirmed by Western immunoblots. Double labeling experiments based on either conventional or confocal microscopy were carried out to establish the exact spatial relationship between different connexins. RESULTS: Connexin 50 was found only in the nonpigmented epithelium (NPE) at apical and basolateral membranes, whereas Cx43 was observed exclusively and at a very high concentration in the pigmented epithelium (PE), primarily in the apical cell membrane, with minimal extension to the proximal lateral zone. The correct antigenicity of the antibodies was confirmed by Western blots of rabbit ciliary body membranes. In rabbit, the Cx26 antibody detected an antigen that was abundant in the NPE and was weakly expressed in the PE. In rat, however, the Cx26 staining was confined to capillary wall endothelia. Western blots of ciliary body and liver membranes and liver immunohistology indicated that the Cx26 antibody used does not recognize rabbit Cx26. Cx32 did not yield any substantial epithelial labeling in either species. CONCLUSIONS: The distribution of Cx50 around the entire NPE cell perimeter suggests its involvement in NPE-NPE cell homotypic gap junctions. The concentration of Cx43 and Cx50 at the apical membranes of the PE and NPE cells, respectively, and their complete absence from the opposite cell suggest that these connexins may participate in the formation of heterotypic gap junctions, either with each other or with other yet unidentified connexins.
Animals↗
Alterations in connexin expression and cell communication in healing corneal epithelium.
PURPOSE: The aim of this study was to examine cell-to-cell metabolite transfer and connexin distribution in the rabbit corneal epithelium, in the stationary state, and during wound healing. METHODS: Rabbit corneas were wounded with a surgical tool, producing a 3-mm-wide elongated debridement. Corneas were allowed to heal in vivo for up to 45 hours. Monoclonal antibodies against connexins Cx 26, Cx 32, Cx 43, and Cx 50 were used to stain cryostat sections. Cell-to-cell metabolite transfer capacity was assessed by a modification of the scrape-loading technique using lucifer yellow as the organic ion tracer. RESULTS: The rabbit corneal epithelium contains Cx 43 and Cx 50, localized in the cell's plasma membrane, as shown previously for other species. Cx 26 and Cx 32 are not detectable. Tracer transfer occurred in both basal and suprabasal cell layers. After wounding, the migrating epithelial monolayer lacked Cx 43 and Cx 50. This change was apparent 6 hours after injury and persisted until complete wound closure (approximately 24 hours). The Cx 50 membrane stain was increased elsewhere, in particular in the transition zone between monolayered and multilayered epithelium. Consistent with the expression changes, migrating cells displayed no or minimal cell-to-cell tracer transfer, whereas in the periphery of the wound, tracer transfer was enhanced in comparison to the control specimen. CONCLUSIONS: Corneal epithelial healing involves biphasic changes in the expression of connexins and cell-to-cell communications. These alterations may be critical for the optimization of the healing response.
Animals↗
Ca2+ mobilization and interlayer signal transfer in the heterocellular bilayered epithelium of the rabbit ciliary body.
1. 'Ratiometric' fura-2 methodology in slice preparations and 'intensitometric' fluo-3 measurements of confocal images were used to simultaneously monitor Ca2+ mobilization in the two distinct, apically joined cell layers which constitute the ciliary body epithelium (CBE): the non-pigmented (NPE) and pigmented (PE) epithelia. 2. Both methods yielded comparable results regarding Ca2+ responses in the syncytium upon stimulation with adrenergic and cholinergic agonists. 3. The alpha 1-adrenoceptor agonist phenylephrine elicited a moderate [Ca2+]i increase in the PE, whereas NPE [Ca2+]i remained unchanged or exhibited a slight diminution. 4. In combination with carbachol, the alpha 2-adrenoceptor agonist brimonidine elicited large Ca2+ increases (> 10-fold) in both the NPE and PE cell layers, even though previous studies indicated the absence of an alpha 2-adrenergic effect on [Ca2+]i in the PE. The onset, as well as the peak of the Ca2+ responses in PE cells frequently exhibited a small delay with respect to adjacent NPE cells. No such time difference was observed between adjacent NPE cells. 5. Pre-incubation of the ciliary body in Ca(2+)-free solution under conditions known to elicit overt NPE-PE separation abolished the alpha 2-adrenocholinergic response in the PE. 6. Addition of heptanol to the perfusate, to block gap-junctional communication, caused a small [Ca2+]i decrease in the NPE and a slight increase in PE[Ca2+]i. Subsequently, the Ca2+ mobilization in the Pe in response to the brimonidine and carbachol combination was either blocked or showed a substantial delay. The Ca2+ mobilization in the NPE, in contrast, remained unchanged. 7. We conclude that the heterocellular syncytium exhibits rectificatory behaviour with respect to Ca2+ mobilization; responses originating within the NPE are easily transferred to the PE, while the reverse does not occur.
Adrenergic alpha-Agonists↗
Comparative adrenocholinergic control of intracellular Ca2+ in the layers of the ciliary body epithelium.
PURPOSE: Both nonpigmented epithelia (NPE) and pigmented epithelia (PE) of the ciliary body are thought to participate in the formation of aqueous humor and its pharmacologic regulation. The aim of this study was to identify the similarities and differences in intracellular Ca2+ ([Ca2+]i) changes in each of the two layers in response to adrenergic and cholinergic inputs. METHODS: Incubation of the Dutch belted rabbit ciliary body (CB) in low-[Ca2+] solution was used to induce the functional dissociation of the NPE and PE layers from each other. These layers or the intact undissociated CB were loaded with the fluorescent Ca2+ indicator fura-2 and mounted in superfusion chambers for fluorometric measurement of [Ca2+]i. RESULTS: In the NPE of the intact CB epithelium or in the isolated NPE, 10 microM acetylcholine (ACh), 1 microM brimonidine (UK 14304), or 1 microM epinephrine each elicited minimal rises in [Ca2+]i. On the other hand, the combination of either adrenergic drug with ACh resulted in large mobilizations of this cation. The alpha 1-adrenergic agonist phenylephrine was unable to induce Ca2+ mobilization in the isolated NPE and failed to do so in 10 of 12 intact CB specimens. In the isolated PE, both epinephrine and phenylephrine elicited substantial similar [Ca2+]i increases, ACh induced a smaller and slower rise, and the response to its combination with either adrenergic drug was essentially additive. UK 14304 (+/- ACh) had no measurable effect in these cells. CONCLUSIONS: Each layer of the CBE exhibits distinct alpha-adrenergic control mechanisms. The NPE contains an alpha 2-adrenergic mechanism highly dependent on the cholinergic tone. The PE, in contrast, contains an alpha 1-adrenergic pathway that operates independently of cholinergic input. This segregation of mechanisms provides a basis for highly complex regulatory responses in the intact, syncytially organized ciliary body epithelium.
Acetylcholine↗
Adreno-cholinergic modulation of junctional communications between the pigmented and nonpigmented layers of the ciliary body epithelium.
PURPOSE: Cell-to-cell communications between the epithelial layers of the ciliary body may be critical for aqueous humor production. The aim of this study was to identify pharmacologic agents that affect this path. METHODS: Whole New Zealand rabbit ciliary bodies were mounted in Ussing-type chambers with Ca2+(-)free and Ca2+(-)rich Tyrode's in the nonpigmented (NPE; aqueous) and pigmented (PE; serosa) epithelial side hemichambers, respectively. The NPE of the PE were then permeabilized, either selectively to monovalent ions with amphotericin B or nonselectively to small solutes with digitonin. Resultant active transport activities were tracked as short circuit currents (ISCS). RESULTS: Permeabilization of the NPE with either 10 microM amphotericin B or 10 micro M digitonin led to an aqueous-to-serosa-positive ISC. This ISC was inhibited by serosal-side ouabain and heptanol, indicating movement of Na+ from permeabilized NPE to the PE by the interlayer junctional path, followed by PE-to-serosa active Na+ transport. Permeabilization of the PE with amphotericin B elicited an ISC in the opposite direction, This ISC was abolished by aqueous-side ouabain and by heptanol, consistent with sequential PE to NPE Na+ translocation, followed by active, NPE-to-aqueous transport. Acetylcholine, epinephrine, norepinephrine, and the alpha 1-adrenergic agonist phenylephrine, but not brominidine, an alpha 2-adrenergic agonist, each caused an approximately 50% reduction of these currents. The inhibitions were fully dependent on serosal-side Ca2+ and were blocked by one calmodulin inhibitor, trifluoperazine, but not by another, calmidazolium. CONCLUSIONS: The above observations provide evidence that cholinergic or alpha 1-adrenergic activation of the PE causes Ca2+(-)dependent inhibition of the NPE-PE junctional path. A triflouperazine-sensitive entity, which may be distinct from calmodulin, is involved in the inhibition.
Adrenergic Agonists↗
Alpha-2,3 sialylation differentiate the limbal and corneal epithelial cell phenotypes.
PURPOSE: The initial differentiation event for the corneal epithelial cell lineage occurs as the limbally localized stem cells yield, through mitosis, the highly proliferative, transiently amplifying corneal peripheral cells. This differentiation is characterized by the expression of tissue-specific cytokeratins, as well as the loss of alpha-enolase and pigmentation. All these are intracellular events. The aim of this study was to identify and characterize, through lectin analysis, changes in cell surface properties associated with differentiation. METHODS: Cryostat sections of the limbo-corneal area from freshly dissected pigmented rabbit corneas were stained with fluorescent lectins. RESULTS: Peanut lectin (PNA; binds to Ser/Threo-GalNAc-beta-1,3-Gal, if the Gal residue is not sialylated) stained the plasma membrane of all layers of the conjunctiva and limbus but was excluded from corneal cell membranes. Maakia amurensis agglutinin (MAA; binds to sialic acid attached to galactose through alpha-2,3 bonds in either N-glycans or O-glycans) stained exclusively corneal cell plasma membrane. After complete tissue desialylation, all corneal plasma membranes became PNA positive with equal stain intensity across both sides of the limbo-corneal margin. The binding of the agglutinins from Limax flavus (binds unselectively to sialic acid) and Sambucus nigra (binds to sialic acid attached through alpha-2,6 bonds) to the basement membrane displayed a large increase at the corneal side of limbo-corneal demarcation. CONCLUSIONS: Limbal (stem) cells express on the cell surface unsialylated galactose residues that are recognized by PNA and that lack any sialic acid bound through alpha-2,3 bonds. The initial differentiation involves sialylation of these residues and the concurrent appearance of alpha-2,3 sialic acid residues, suggesting expression or activation of alpha-2-3 sialytransferase. Changes in basement membrane composition, charge, or both may underpin this expression.
Animals↗
Synthesis of rabbit corneal epithelial glycocalyx in vitro.
A method to study the synthesis and cellular processing of epithelial apical membrane glycoproteins in the rabbit cornea was developed. Fluorescent derivatives of wheat germ agglutinin (WGA; alpha-N-acetylglucosamine and sialic acid hapten affinities), succinylated WGA (alpha-N-acetylglucosamine hapten affinity) and concavalin A (Con A; D-mannose and D-glucose hapten affinity) were reacted with the corneal surface and the extent of binding attained was measured by en face, microscope-aided fluorophotometry. Minimal binding of succinylated WGA and a large reduction in WGA binding following neuraminidase treatment demonstrated that the attachment of WGA to the corneal surface occurred via sialic acid residues, i.e. via structures associated with terminal glycosylation. Corneas were treated with digitonin to induce the exfoliation of the outer squamous-like cell layers. The time-dependent changes in lectin binding density at the apical surface of the newly exposed intrastratal cells were then determined. Binding densities for WGA and Con A at the time of exfoliation of the digitonin-devitalized squamous cell layers (< 2 hr post-devitalization) were similar to the densities measured at the surface of untreated corneas. Over the subsequent 18-20 hr, the WGA and Con A binding increased by 2.63 +/- 0.24 and 3.0 +/- 0.68 (+/- S.D., n = 4) fold, respectively. The effect of inhibitors of transcription (actinomycin D, alpha-amanitin), translation (cycloheximide), core glycosylation of polypeptides (tunicamycin), endoplasmic reticulum glucosidases (deoxinojirimycin) and Golgi mannosidase (swainsonine) indicated that the increases were underpinned by new glycoprotein synthesis driven by a stable, pre-existing mRNA pool. Retinoic acid (2 microM) inhibited the increase in WGA binding by 55 +/- 6% (n = 4) but did not affect the Con A density increase suggesting that this agent either, modifies the terminal glycosylation pattern of apical membrane proteins and/or inhibits the synthesis of proteins bearing sialic acid. Actinomycin D or alpha-aminitin reverted the retinoic acid action, indicating that the retinoid effect is mediated by induced gene expression.
Animals↗
Na(+-) Cl(-)- and HCO3(-)-dependent base uptake in the ciliary body pigment pigment epithelium.
Segments of whole ciliary body dissected from Dutch belted rabbits were incubated for 60 min at 36 degrees C in a 30 microM Ca2+ Ringer's. The inner limiting membrane with its adherent non-pigmented epithelium then was mechanically removed from the surface. The newly-exposed viable layer of pigmented cells was then loaded with the fluorescent probe 2'-7'-bis (carboxymethyl)-5(6) carboxyfluorescein by incubating the segments for 45 min at RT with the cell permeable acetoxymethoxy form of the dye. These loaded tissues were perfused in a flow-through chamber, mounted on the heated stage of a microscope equipped for quantitative epifluorescence, and the intracellular pH (pHi) of small groups of cells was derived from the ratio of emission intensities generated by excitations at 490 and 440 nm, respectively. In N[2-hydroxyethyl] piperazine-N"-[2 ethane sulfonic acid](Hepes)-buffered Ringer's the intracellular pH was 7.23 +/- 0.21 (+/- S.D., n = 20). Replacement of 28 mM Hepes by 28 mM HCO3-/5% CO2 led to a 0.14 +/- 0.04 increase in pHi. This increase required the presence of Na+ and Cl- and was inhibited by 0.2 mM diisothiocyanatostilbene-2-2'-disulfonic acid. These observations as well as characteristic pHi, responses to the removal or introduction of Na+ or Cl- indicated the presence in the pigmented cells of a Na(+)- and Cl(-)-dependent HCO3- transporter responsible for base uptake.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Separation of the rabbit ciliary body epithelial layers in viable form: identification of differences in bicarbonate transport.
Sections of whole ciliary (CB) dissected from Dutch belted rabbits were incubated for 2 hr at 36 degrees C in a 30 microM Ca2+ Ringer's. This incubation resulted in the spontaneous dissociation of the two cell layers comprising this epithelium, each remaining firmly cohesive with its own basement membrane. The inner limiting membrane with its adherent non-pigmented epithelium (NPE) was then mechanically removed from the surface exposing the apical surface of the pigmented epithelium (PE). Ultrastructural examination revealed no noxious effects in most cells although gross morphological changes in the NPE cells were noted. The newly separated layers were loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF. Most cells of both layers acquired stable BCECF fluorescence indicating viability. To achieve a preliminary evaluation of differences in PE and NPE bicarbonate transport, dye-loaded tissues were perfused in a flow-through chamber which was mounted on a microscope equipped for quantitative epifluorescence. The intracellular pH (pHi) of groups of cells (5-10) was derived from the ratio of emission intensities generated by excitations at 490 and 440 nm. In Hepes-buffered Ringer's the pHis for the PE and NPE were 7.20 +/- 0.10 and 7.33 +/- 0.14 (+/- S.D., n = 6), respectively. Replacement of 28 mM Hepes by 28 mM HCO3-/5% CO2 led to a 0.13 pHi increase in the PE and a decrease of 0.27 U in the NPE. The pHi responses of the two cell layers to removal and/or reintroduction of Na+ and/or Cl- were also highly dissimilar.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
ZO-1 in corneal epithelium; stratal distribution and synthesis induction by outer cell removal.
The tight junctions (TJs) present exclusively in between the superficial cells (SC) are an important component for the barrier and ion secretory functions of the corneal epithelium. Electrophysiological studies have demonstrated that digitonin-induced devitalization of overlying cells induces the de novo generation of a paracellular barrier between the intrastratal cells becoming exposed to the outer surface. It was also shown that the closer the exposed cells to the SC position the higher their competency for this activity. We have now examined the spatial and quantitative distribution of ZO-1, a protein closely associated with the cytosolic face of TJs in the rabbit. Immunohistology showed: (a) no detectable ZO-1 in basal cells; (b) incipient punctuate accumulations in the wing cells; (c) numerous foci and a diffuse cytosolic staining in the squamous (SQ) cells; and (d) strong staining at the apical TJ locations in the superficial SQ layer. Immunoblot analysis of separated layers showed a SQ:basal cell ZO-1 concentration ratio in excess of 100. Devitalization of the SQ layers induced strong ZO-1 synthesis in the newly exposed wing cells where electron microscopy showed time-dependent development of TJs. A similar ZO-1 increase was observed in the basal cells after removal of all suprabasal cells by a low [Ca2+] incubation method. These results provide a biochemical correlate to the previous electrophysiological findings and show that expression of ZO-1 in the corneal epithelium is intimately related to the development of the superficial cell phenotype.
Animals↗
Ontogeny of corneal epithelial tight junctions: stratal locale of biosynthetic activities.
PURPOSE: On reaching the surface position, corneal epithelial cells assemble a tight paracellular barrier rapidly. The purpose of this study was to identify at what maturation stage the cells acquire biochemical elements underpinning this ability. METHODS: Rabbit corneas were subjected to a digitonin-exposure protocol that results in the devitalization and desquamation of the three outermost cell layers of the 5-6-layer thick epithelium and thus, exposes winglike cells to the tear surface. Corneas were then mounted in Ussing-type chambers to measure transepithelial resistance and solute fluxes. RESULTS: After the devitalization treatment transepithelial resistance remained near baseline for 2 hours and then rose to 8.24 +/- 3.12 K omega.cm2 (+/- SD, n = 15) during the next 8 hours. This increase was matched by increases in the resistance to the paracellular flow of mannitol. Transmission electron microscopy confirmed de novo formation of tight junctions over this time span in the new surface. The addition of transcription (actinomycin D; alpha-amanitin), translation (cycloheximide), core glycosylation (tunicamycin), and endoplasmic reticulum-to-Golgi traffic (brefeldin A) inhibitors immediately after the devitalization protocol prevented the transepithelial resistance raise. Introducing time delays between the cell devitalization and the addition of these biosynthesis inhibitors reduced their effect; a time delay of 90 minutes abolished the inhibition by alpha-amanitin. CONCLUSION: Devitalization of overlying cells induces the assembly of a tight paracellular barrier in the wing cells. The process requires and includes the synthesis of essential transcripts and proteins that were initially absent in these cells. In vivo, the synthesis of these elements must occur as cells reach the squamous stage of maturation.
Amanitins↗
Evidence for parallel Na(+)-H+ and Na(+)-dependent Cl(-)-HCO3- exchangers in cultured bovine lens cells.
BCECF, a cell-entrapable dye with a pH-sensitive fluorescence spectrum, was used to identify transport mechanisms contributing to pH homeostasis of cultured bovine lens epithelial cells. Cells from a spontaneously established lineage were grown on glass coverslips that fit diagonally in a standard curvette and intracellular pH (pHi) was measured. Under perfusion with a CO2-HCO3(-)-free medium (pH 7.45), pHi was 7.19 +/- 0.21 (mean +/- S.D., n = 94 cell preparations). Cell acidifications (pHi to 6.65, n = 8) induced by the 'NH(4+)-loading' method were rapidly followed by a Na(+)-dependent, amiloride-inhibitable pHi recovery. Introduction of a CO2-HCO3(-)-rich medium (pH 7.45) resulted in a small acidification (0.18 +/- 0.04 U, n = 16; P < 0.002) due to rapid CO2 entry and an ensuing slow alkalinization to a pHi near the control CO2-HCO3(-)-free value. Subsequent removal of Cl- resulted in a further alkalinization of 0.18 +/- 0.02 U (n = 13; P < 0.001). This Cl- effect was completely inhibited by the absence of Na+, but was insensitive to amiloride, suggesting the presence of a Na(+)-dependent Cl(-)-HCO3- exchanger. Consistent with this posit, the reintroduction of Na+ to cells perfused in the absence of the cation with a HCO3(-)-containing, amiloride-complemented solution resulted in a gradual recovery from the acidic pHi induced by the baseline conditions (n = 6). The amiloride-insensitive, Na(+)- and HCO3(-)-dependent recovery was completely inhibited in cells pre-incubated with DIDS.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Bicarbonate transport mechanisms in rabbit ciliary body epithelium.
Sections of whole ciliary body dissected from Dutch belted rabbits were incubated with the cell entrappable pH probe BCECEF-AM. This led to a highly specific localization of epifluorescence emission at the exposed, non-pigmented cell layer (npe) of the dual layered epithelium that covers this organ. The BCECF-loaded tissue sections were superfused in a flow-through chamber and the intracellular pH (pHi) of small groups (10-20) of cells was derived from the ratio of the emission intensities derived from excitations at 490 and 440 nm. In CO2/HCO3- Ringer's, npe pHi = 7.09 +/- 0.11. Replacement of CO2/HCO3- by Hepes increased pHi by 0.22 +/- 0.02, indicating alkali secretory activity under the bicarbonate-rich conditions. Replacement of Cl- by gluconate elicited a rapid, 0.6-U increase in pHi. This effect exhibited little dependence on Na+ and was inhibited by 0.5 mM dihydro-4,4'-diisothiocyanatostilbene -2,2'-disulfonate (H2DIDS). These results indicate the presence of an electroneutral Cl-/base exchange activity. Elevation of [K-] (by partial replacement of Na+) also elicited increases in pHi. In Cl(-)-free media pHi reached 7.8-8.0, a condition under which intracellular [HCO3-] is at least twice as high as its extracellular value. This effect did not occur in the absence of Na+. The Na(+)-dependent high [K+]-induced pHi increase was inhibited by H2DIDS. The effects of Ba2+ on pHi, alone and in combination with high [K+], as well as that of full K+ removal, suggested that the link between high [K+] and pHi increase was mainly due to the effect of cell depolarization on an electronegative Na+ dependent HCO3- transporter. Under normal physiological conditions, the two acid/base transport systems are the main determinants of npe pHi.
Animals↗