Helen Turner - Academia.edu (original) (raw)

Papers by Helen Turner

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1996

Amiloride (0.5 mM) inhibited the rate of entry of Na + into corneal endothelial cells by more tha... more Amiloride (0.5 mM) inhibited the rate of entry of Na + into corneal endothelial cells by more than half ((0.76 + 0.10) to (0.21 + 0.10) /xEq cm-2 h-~). The same concentration of amiloride caused only minimal disturbance to corneal hydration control by the endothelium (range 0-12%). Amiloride (0.5 mM) and acetazolamide (1 mM) reversibly inhibited trans-endothelial short circuit current by about a half. Their combined effect was not additive. Acetazolamide (1 raM) reduced net HCO~-flux across the short-circuited endothelium by about the same amount ((0.50 + 0.11) /xEq cm-2 h-i) that amiloride (0.5 mM) reduced Na" entry into the cells ((0.55 + 0.14) /zEq cm-2 h-~). Low concentrations of amiloride (10 p.M) had little effect on the transport characteristics of the endothelium, indicating that Na + entry into the endothelial cells under physiological conditions is not primarily through Na" channels. The data are consistent with an Na+/H + exchanger acting in tandem with carbonic anhydrase through a pathway which could have a regulatory role on endothelial transport via its effect on Na ÷ re-entry. Residual trans-endotheliai HCO 3 transport, apparently unaffected by amiloride or acetazolamide inhibition, is calculated to be of sufficient magnitude to maintain corneal hydration.

American Journal of Physiology-Cell Physiology, 2001

The effects of serotonin [5-hydroxytryptamine (5-HT)] on the transepithelial electrical propertie... more The effects of serotonin [5-hydroxytryptamine (5-HT)] on the transepithelial electrical properties of the short-circuited rabbit conjunctiva were examined. With this epithelium, the short-circuit current ( I sc) measures Cl− secretion plus an amiloride-resistant Na+ absorptive process. Apical addition of 5-HT (10 μM) elicited a prompt I screduction from 14.2 ± 1.2 to 10.9 ± 1.2 μA/cm2 and increased transepithelial resistance from 0.89 ± 0.05 to 1.03 ± 0.06 kΩ · cm2(means ± SE, n = 21, P < 0.05). Similar changes were obtained with conjunctivae bathed without Na+ in the apical bath, as well as with conjunctivae preexposed to bumetanide with the Cl−-dependent I sc sustained by the parallel activities of basolateral Na+/H+ and Cl−/HCO[Formula: see text] exchangers. In contrast, the 5-HT-evoked effects were attenuated by the absence of Cl−(Δ I sc = −0.5 ± 0.2, n = 5), suggesting that reduced Cl−conductance(s) is an effect of 5-HT exposure. In amphotericin B-treated conjunctiva and in ...

Molecular vision, Jan 22, 2010

Dual specificity phosphatases (DUSPs) modulate the duration and magnitude of phospho-activation o... more Dual specificity phosphatases (DUSPs) modulate the duration and magnitude of phospho-activation of Erk1/2, p38 and JNK1/2, the terminal kinases (TKs) of the mitogen activated protein kinase (MAPK) cascades. Three DUSPs, DUSP1, DUSP5, and DUSP6, are overexpressed in ocular surface side population stem cells (SPSCs). Our objective was to identify the impact of these enzymes on TK phosphorylation and proliferation of corneal epithelial cells. SV40 immortalized (sv) and expanded fresh human corneal epithelial cells (efHCECs) were transduced with lentivectors to elicit expression of shRNAmir against DUSP1, DUSP5, and JNK1 to thereby create the DUSP1i, DUSP5i and JNKi cell sublines, or overexpress DUSP6 (henceforth DUSP6(+)), respectively. TK phosphorylation status and proliferation rates were determined by immunoblotting and (3)H thymidine uptake. In both ef and svHCECs, EGF supplementation after a 24 h serum starvation caused a rapid 5-15 min spike in the phosphorylation of all three TK...

Investigative Opthalmology & Visual Science, 2009

PURPOSE. To define the molecular signature of limbal SP cells and identify signaling pathways ass... more PURPOSE. To define the molecular signature of limbal SP cells and identify signaling pathways associated with the phenotype of these putative stem cells. METHODS. Primary cultures of pig limbal epithelial cells stained with Hoechst 33342 were sorted by flow cytometry into SP and non-SP cells, and purified RNA was processed for microarray analysis with an oligonucleotide spotted array. Expressed transcripts for which SP and non-SP expressions differed by more that 1.5-fold in each paired set and by twofold overall were considered to be differentially expressed. Differential expression was validated by quantitative PCR and immunostaining. Data-mining methods were used to identify cellular processes that are either salient or depressed in the SP cells. RESULTS. The microarray identified approximately 9000 distinct, expressed, and identifiable genes. Of those, 382 and 296 were either over-or underexpressed in the SP cells, respectively. Overrepresentation analysis indicated that SP cells are in a low metabolic and biosynthetic state. In addition, a pattern of elevated MXD1, MAXI2, DUSP5, p27/KIP1, and p57/KIP2 and decreased Cyclin D and CDK genes can be expected to slow intrinsic and mitogen-induced G 1-to-S cell cycle transition. SP cells were also rich in genes associated with stem cell phenotype and genes providing protection against oxidative and/or xenobiotic damage. CONCLUSIONS. Microarray analysis of pig limbal SP cells yielded a molecular signature underscoring a phenotype characterized by slow cycling and low metabolic activity. The results provide valuable insights for the preservation and/or replication of epithelial stem cells.

Investigative Opthalmology & Visual Science, 2009

PURPOSE. Side-population (SP) cells isolated from limbal and conjunctival epithelia derive from c... more PURPOSE. Side-population (SP) cells isolated from limbal and conjunctival epithelia derive from cells that are slow cycling in vivo, a known feature of tissue stem cells. The purpose of this study was to define the molecular signature of the conjunctival SP cells and identify markers and signaling pathways associated with the phenotype of these cells. METHODS. Overnight cultures of freshly isolated human conjunctival epithelial cells stained with Hoechst 33342 were sorted by flow cytometry into SP and non-SP cohorts. Isolated RNA was processed for microarray analysis using a commercial oligonucleotide spotted array. Results were validated at the gene and protein levels by quantitative PCR and immunologic methods. Data mining methods were used to identify cellular processes relevant for stem cell function. RESULTS. Comparative analyses of transcripts expression based on present and absent software calls across four replicate experiments identified 16,993 conjunctival epithelial transcripts including 10,266 unique known genes of ϳ24,000 represented in the array. Of those genes, 1254 and 363 were overexpressed (Ͼ2-fold) or underexpressed (Ͻ0.5-fold), respectively, in the SP. The overexpressed set included genes coding for proteins that have been associated with (1) embryonic development and/or stem cell self renewal (MSX, MEIS, ID, Hes1, and SIX homeodomain genes); (2) cell survival (e.g., CYP1A1 to degrade aromatic genotoxic compounds); (3) cycling rate (e.g., DUSPs and Pax6 to foster slow cycling); and (4) genes whose expression is not typical in epithelia (e.g., CD62E). CONCLUSIONS. The molecular signature of conjunctival SP cells is consistent with a stem cell phenotype. Their gene expression patterns underpin slow cycling and plasticity, features associated with tissue stem cells. The results provide valuable insights for the preservation and/or expansion of epithelial stem cells.

Experimental Eye Research, 2000

The regulation of Na + transport by cAMP in freshly isolated rabbit conjunctival epithelium, a ti... more The regulation of Na + transport by cAMP in freshly isolated rabbit conjunctival epithelium, a tissue exhibiting both Cl − secretion and Na + absorption, was examined. Bulbar-palpebral segments of conjunctiva were mounted between Ussing-type hemichambers under short-circuit conditions in Cl − free media. In this situation, the short-circuit current (I sc) measures an amiloride-resistant Na + absorptive process in the apical-to-basolateral direction. Apical additions (each at 10 µ) of cAMP-elevating compounds, forskolin, rolipram, IBMX and epinephrine all stimulated the Na +-dependent I sc by $ 3n5-4n5 µA cm −# (minimal 40 % increase) and reduced transepithelial resistance (R t) by at least 7 % (P 0n05). Pre-exposure (1 hr) to the protein kinase A (PKA) inhibitor H-89 (10 µ), which in itself inhibited the I sc by 0n5 µA cm −# , attenuated the I sc responses of the cAMP-elevating agents (P 0n05, unpaired data). In reverse, H-89 promptly decreased the I sc by 1n5-2n5 µA cm −# and increased R t by 5 % (P 0n05) in tissues pre-stimulated with either forskolin or an epinephrine plus IBMX combination. Additions of epinephrine or rolipram to apically permeabilized preparations using amphotericin B, increased the I sc by 12 and 22 % respectively over baseline and reduced R t by 6 % (P 0n05). Similarly, in the presence of a transepithelial K + gradient (apical to basolateral) and amphotericin B, cAMP elevation stimulated K diffusion across the preparation by at least 1n8 µA cm −# and decreased the R t by 4 % (P 0n05), changes that were reversed by subsequent H-89 addition. Under Cl − rich conditions, pretreatment with 5 m Ba# + reduced the basal I sc by 59 % and blocked the cAMP-induced I sc stimulations typically seen in the presence of the anion. The results provide evidence for a PKA-regulated, Ba# +-inhibitable (voltage insensitive) basolateral K + conductance in rabbit conjunctival epithelial cells. The action of Cl − secretogogues acting via cAMP on basolateral K + channel activity indicates that endogenous levels of cAMP may play a role in the regulation of Cl − secretion and Na + absorption in the conjunctiva.

Experimental Eye Research, 2001

Earlier work from this laboratory demonstrated a bumetanide-inhibitable K uptake activity in cult... more Earlier work from this laboratory demonstrated a bumetanide-inhibitable K uptake activity in cultured bovine lens epithelial cells, but not at the anterior surfaces of intact bovine lenses isolated in an Ussingtype chamber. Presently the distribution of the bumetanide-sensitive Na-K-2Cl À cotransporter within the lens was reexamined. To complement previous results, 86 Rb uptake experiments were done in a chamber design that limited exposure of the radiolabel to speci®c surfaces of rabbit lenses under shortcircuit conditions. In addition, the cotransporter protein (NKCC1, but not NKCC2) was immune-detected in Western blots. For the latter, membrane preparations were obtained from capsule-plus-epithelial specimens, and from three cortical fractions, i.e. the anterior, equatorial, and posterior regions, as well as a ®fth, nuclear fraction. K in¯uxes across the anterior-polar, equatorial, and posterior-polar surfaces were 0. 375, 0. 348 and 0. 056 mEq (hr cm 2) À1 respectively, rates that were not signi®cantly reduced by the presence of 0. 1 mM bumetanide (P 4 0. 15, as unpaired data). In contrast, bumetanide-sensitive K in¯ux rates were measured across the anterior and equatorial surfaces under hypertonic, but not under hypo-osmotic conditions. In culture, bumetanide and ouabain were equipotent in reducing by approximately half the K uptake of quiescent, rabbit lens epithelial cells under control, iso-osmotic conditions, indicating a cell-culture induced up-regulation of the cotransport activity by an undetermined mechanism. The immunoblotting of lens membrane proteins elicited approximately 170±180 kDa bands accordant with the identity of the NKCC1 isoform in the epithelial and cortical equatorial fractions. Thus, NKCC1 was readily demonstrated using membrane specimens taken from within the lens. Its activity in the intact organ may be activated by conditions fostering cell shrinkage, and perhaps, agents stimulating epithelial cell elongation, given its distribution within the lens.

Current Eye Research, 2003

To characterize the serotonin (5-HT) receptors linked to the modulation of adenylyl cyclase activ... more To characterize the serotonin (5-HT) receptors linked to the modulation of adenylyl cyclase activity in rabbit, porcine and human conjunctivae. Serotonin receptor-subtype expression was examined using reverse transcription-polymerase chain reaction (RT-PCR) and receptor subtype-specific polyclonal antibodies for the immunofluorescent labeling of conjunctival cryosections. In addition, measurements of the effects of serotonergics on the short-circuit current (I(sc)) across rabbit and porcine conjunctivae were contrasted. RT-PCR assays indicated the expression of 5-HT(1B ) and 5-HT(1D) receptors, subtypes negatively coupled to adenylyl cyclase, in the rabbit conjunctiva. This approach also suggested the co-expression of 5-HT(1B), 5-HT(1D), 5-HT(1F), 5-HT(4) and 5-HT(7) mRNA&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s in the porcine conjunctiva, and 5-HT( 1D), 5-HT(1F) and 5-HT(7) in the human conjunctiva. Since the 5-HT(4) and 5-HT(7) receptors are positively linked to adenylyl cyclase, these results implied that the porcine and human tissues exhibited subtypes both positively and negatively linked to the enzyme. However, immunohistochemical observations, using currently available antibodies solely localized the 5-HT(7) moiety in the porcine and human epithelia, suggested that the 1B/1D forms may be minor elements. Consistent with this prospect, 5-HT was a stimulant of the transepithelial I(sc) across the porcine conjunctiva, an opposite response from earlier findings that demonstrated inhibitory effects by 5-HT on the rabbit I(sc), which are now explained by the localization of the 1B/1D receptors in the rabbit stratified epithelium. The 5-HT receptors expressed by mammalian conjunctivae are not identical. In terms of 5-HT receptor expression, the porcine tissue may be a more appropriate model for human, than is the rabbit, in that 5-HT may serve as a secretagogue in the human epithelium.

Investigative Opthalmology & Visual Science, 2007

PURPOSE. To determine global mRNA expression levels in corneal and conjunctival epithelia and ide... more PURPOSE. To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. METHODS. cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression Ͼ1% of the ␤-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest. RESULTS. The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA 2-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb. CONCLUSIONS. Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that are potentially active in the function of corneal and conjunctival epithelia.

Current Eye Research, 2003

To characterize the effects of cAMP-elevating stimuli on the rabbit translens electrical paramete... more To characterize the effects of cAMP-elevating stimuli on the rabbit translens electrical parameters and examine the distribution of beta adrenoceptors about the epithelial surface. The electrophysiological experiments encompassed the isolation of lenses within a vertically arranged, Ussing-type chamber under short-circuit conditions, an approach that allowed for measurements of short-circuit current (I(sc)) across, in separate experiments, discrete surface regions. Epithelial beta receptors were localized by immunofluorescent labeling of lens cryosections primarily exposed to a polyclonal antibody against human beta( 2)-adrenoceptors. Reverse transcription - polymerase chain reaction (RT-PCR) was used to generate cDNA (using specific primers based upon the sequence of the previously cloned human beta(2) receptor) from rabbit lens RNA extracted from mechanically sequestered anterior and equatorial epithelial cells. Asymmetrical I(sc) reductions with increases in translens resistance were elicited with epinephrine, isoproterenol, terbutaline, forskolin, and a lipid-permeable cAMP analogue. Electrical changes were recorded across the anterior aspect and not observed when the above compounds were applied to solutions bathing the equatorial and posterior surfaces. Immunohistochemical observations indicated the expression of beta receptors from the anterior epithelium to the equatorial region. RT-PCR yielded cDNA of expected basepair length for the apparent fragment of the beta(2)-adrenoceptor, which exhibited a sequence homology 90% identical with its human equivalent in both the anterior and equatorial epithelia. The cAMP-sensitive conductance(s) appear limited to the anterior epithelium and undetectable equatorially. The asymmetrical I(sc) responses do not seem to arise from a spatial heterogeneity in epithelial receptor expression.

Current Eye Research, 2003

To characterize the effects of cAMP-elevating stimuli on the rabbit translens electrical paramete... more To characterize the effects of cAMP-elevating stimuli on the rabbit translens electrical parameters and examine the distribution of beta adrenoceptors about the epithelial surface. The electrophysiological experiments encompassed the isolation of lenses within a vertically arranged, Ussing-type chamber under short-circuit conditions, an approach that allowed for measurements of short-circuit current (I(sc)) across, in separate experiments, discrete surface regions. Epithelial beta receptors were localized by immunofluorescent labeling of lens cryosections primarily exposed to a polyclonal antibody against human beta( 2)-adrenoceptors. Reverse transcription - polymerase chain reaction (RT-PCR) was used to generate cDNA (using specific primers based upon the sequence of the previously cloned human beta(2) receptor) from rabbit lens RNA extracted from mechanically sequestered anterior and equatorial epithelial cells. Asymmetrical I(sc) reductions with increases in translens resistance were elicited with epinephrine, isoproterenol, terbutaline, forskolin, and a lipid-permeable cAMP analogue. Electrical changes were recorded across the anterior aspect and not observed when the above compounds were applied to solutions bathing the equatorial and posterior surfaces. Immunohistochemical observations indicated the expression of beta receptors from the anterior epithelium to the equatorial region. RT-PCR yielded cDNA of expected basepair length for the apparent fragment of the beta(2)-adrenoceptor, which exhibited a sequence homology 90% identical with its human equivalent in both the anterior and equatorial epithelia. The cAMP-sensitive conductance(s) appear limited to the anterior epithelium and undetectable equatorially. The asymmetrical I(sc) responses do not seem to arise from a spatial heterogeneity in epithelial receptor expression.

Current Eye Research, 2000

To investigate possible regional expression of transport systems in the conjunctival epithelium g... more To investigate possible regional expression of transport systems in the conjunctival epithelium given distinct differences in morphological appearance between the bulbar and palpebral epithelia as well as variations found in the proportions of Na absorptive versus Cl secretory activities in electrophysiological studies. Mouse monoclonal antibodies against the alpha1-subunit of Na-K-ATPase and Na-K-Cl cotransporter (NKCC1) and a rabbit polyclonal antibody against the Na-glucose cotransporter (SGLT1) were used in immunoblotting and immunofluorescent labeling of frozen fixed sections isolated from either the bulbar and palpebral regions of the conjunctiva. Western blot analysis clearly demonstrated the presence of Na-K-ATPase, NKCC1 and SGLT1 proteins in both bulbar and palpebral conjunctiva. Indirect immunofluorescence studies on bulbar and palpebral conjunctival portions revealed intense staining by the anti-NKCC1 and anti-alpha1-Na-K-ATPase antibodies exclusively at the basolateral surfaces, whereas the anti-SGLT1 antibody was used with porcine conjunctiva to elicit strong and unambiguous staining along the apical plasma membrane. Proteins that mediate the transport activities of the Na-K-ATPase and Na-K-Cl cotransporter are uniformly distributed throughout the palpebral and bulbar regions of the rabbit conjunctival epithelium. Although the Na-glucose cotransporter could be detected in immunoblots of the rabbit, this cotransporter appears to be uniformly distributed as well, based upon immunohistochemical sections of the pig conjunctiva. Thus, it appears likely that mechanisms for Cl secretion and Na absorption reside in both bulbar and palpebral segments of the conjunctival epithelium.

Pflügers Archiv European …, 2000

Corneal stromal hydration is maintained by an active HCO3 – transport mechanism located in the co... more Corneal stromal hydration is maintained by an active HCO3 – transport mechanism located in the corneal endothelium. Whilst modulation of transport activity by changes in intracellular cAMP concentration have been noted, the site of effect is undefined. To resolve this ...

Experimental eye …, 1999

Protein kinase C (PKC) activation elicits diverse cell-type specific effects on key epithelial tr... more Protein kinase C (PKC) activation elicits diverse cell-type specific effects on key epithelial transporters. The present work examined the influence of phorbol esters, which are known activators of PKC isoenzymes, on the short-circuit current (I sc), a direct measure of net transcellular electrolyte transport, of the rabbit conjunctiva. In this preparation, the I sc measures a Na +-dependent, bumetanide-inhibitable Cl − transport in the basolateral-to-apical direction plus an amiloride-resistant Na + absorptive process in the opposite direction. Additions of phorbol 12-myristate-13-acetate (PMA) to the basolateral bathing media did not affect the transepithelial electrical parameters ; but its introduction to the apical bath at 1 and 10 µ elicited a transient ($ 2 min duration) I sc spike followed by a sustained reduction relative to the control level. Such PMA-elicited I sc reductions were from 14n0p2n0 to 3n1p0n8 µA cm −# (p...'s, n l 3) at 1 µ and from 16n5p1n9 to 4n6p0n7 µA cm −# (n l 22) at 10 µ. The former concentration failed to produce extensive I sc reductions in 3 other experiments. Similar results were obtained with phorbol 12,13-dibutyrate (PDBu). Its apical administration at 0n1 µ reduced the I sc from 18n5p4n1 to 7n8p2n0 (n l 3), and from 16n5p2n9 to 6n9p1n2 (n l 7) when introduced at 1 µ. The phorbol-evoked I sc reductions occurred without a simultaneous change in transepithelial resistance (R t). However, after about 15-20 min, R t gradually declined by about 25 %. In contrast to these results, treatment with a phorbol ester known not to activate PKC (4-α-PMA) did not affect the electrical parameters when added at 10 µ. PMA-and PDBu-evoked I sc reductions could be obtained with conjunctiva that were (1) pretreated with bumetanide, (2) bathed in Cl −-free media, and (3) pretreated with amphotericin B, changes consistent with a likely inhibition of the basolateral Na + \K + pump. Such I sc inhibitions were attenuated with conjunctiva pre-exposed to 1 µ staurosporine, a nonselective kinase inhibitor known to suppress PKC activity. Staurosporine, in itself, produced a rapid 26 % I sc inhibition (n l 15) along with a 17% R t increase upon its apical introduction. These electrical responses were less extensive in Cl −-free media and absent in amphotericin B-treated conjunctiva, suggesting the presence of a kinase-mediated regulation of apical channels for both Na + and Cl −. Overall, these results imply that in addition to previously demonstrated epinephrine-elicited, up-regulation of Cl − secretion, mechanisms may also exist, via PKC activation, to suppress Na + \K + pumping and consequently reduce transepithelial transport rates.

Molecular vision, 2004

METHODS Tissue samples and RNA extraction: This work followed the Guiding Principles in the Care ... more METHODS Tissue samples and RNA extraction: This work followed the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80-23). Adult male New Zealand White rabbits (2.0-2.5 kg) were killed with an overdose of sodium pentobarbital. Non-ocular tissues (kidney, lung, muscle, and skin) were collected using a fresh sterile disposable scalpel for each, dissected into cubes (side length 3-5 mm), rinsed in sterile phosphate buffered saline, pH 7.4 (PBS), and snap frozen in

Experimental Eye Research, 2006

Current eye research, 2002

To determine the presence of the cystic fibrosis transmembrane conductance regulator (CFTR) in th... more To determine the presence of the cystic fibrosis transmembrane conductance regulator (CFTR) in the conjunctiva and examine the possibility of its regional expression in rabbit, rat and porcine conjunctival epithelia given distinct differences in morphological appearance between the bulbar and palpebral epithelia. Two specific anti-CFTR antibodies, against different epitopes in the R domain of the CFTR molecule were used in immunofluorescent labeling of frozen fixed sections isolated from the bulbar and palpebral regions of fresh rabbit, porcine and rat conjunctivae. CFTR expression was also determined in the rabbit conjunctival epithelium using RT-PCR methods. CFTR immunoreactivity in the conjunctival epithelium exhibits polarized expression and is associated with the apical domain of conjunctival epithelial cells. An identical pattern of staining obtained in porcine cryosections using either of the anti-human CFTR antibodies confirmed the specificity of the positive apical staining. RT-PCR analysis produced bands at the predicted size for CFTR mRNA transcripts in both bulbar and palpebral portions of the rabbit conjunctival epithelium. Apical localization of CFTR in the conjunctival epithelium is consistent with the function of this protein as a chloride channel or as a regulator of channel activity. The identification of CFTR in both bulbar and palpebral portions of the conjunctiva provides evidence that the mechanisms for Cl secretion reside throughout the conjunctiva. This finding suggests that manipulations of the CFTR Cl channel could affect transepithelial Cl transport rates and water movement into the tear film.

Experimental eye research, 2001

Acid-base transporters of rabbit and porcine conjunctival epithelia were identi®ed and localized ... more Acid-base transporters of rabbit and porcine conjunctival epithelia were identi®ed and localized with immunoblotting and immunohistochemical techniques using speci®c antibodies against carriers commonly found in epithelia, i.e. the Cl À /HCO À 3 exchanger (AE2), Na /H exchanger (NHE-1,-2,-3) and the electrogenic Na-(n)HCO À 3 cotransporter (NBC). Western blot analysis demonstrated that anti-AE2 reacted with an approximate 170 kDa protein in both rabbit and pig cell membranes prepared from separately isolated bulbar and palpebral conjunctivae. NHE1 was similarly identi®ed in these distinct conjunctival regions but results with anti-NBC were ambiguous. Histochemical examinations indicated that the AE2 and NHE1 proteins reside on the basolateral surfaces of the plasma membrane throughout the multilayered tissue. The immunostaining of porcine cryosections for AE2 and rabbit sections for NHE1 was speci®c, because of its abolishment following either pre-absorption with the corresponding peptide or omission of the primary antibody. Screening with anti-NBC produced weak staining of the sections that appeared to be non-speci®c. For con®rmation of these results, the acid-base transporters present in rabbit cell cultures of conjunctival epithelia were ascertained from the changes in intracellular pH (pH i) evoked upon sequential superfusion with media of altered composition. This approach readily obtained Na-and Cl À-dependent pH i effects consistent with the existence of Cl À /HCO À 3 and Na /H exchange activities. Evidence for the presence of NBC could not be acquired, thereby substantiating the observations from the immunodetection techniques. The identity and location of the antiporters that were found suggested that these elements could contribute to transcellular Cl À transport in the basolateral-to-apical direction. To test this possibility, the effects of AE and/or NHE inhibition were determined on the bumetanide-insensitive Cl À-dependent short-circuit current across rabbit conjunctivae freshly isolated in Ussing-type chambers. These experiments revealed that such current is indeed sustained by the antiporters. Results with acetazolamide further suggested that the contribution of the acid-base transporters towards transepithelial Cl À secretion is variable and dependent upon individual rates of metabolic CO 2 production. Overall, the present study provides an initial identi®cation of the acid-base transporters present in the conjunctiva. Besides their likely role in intracellular pH regulation, the parallel, basolateral expression of AE2 and NHE1 indicates that these elements do not directly contribute to the pH of the tear ®lm but may complement the Na-2Cl À-K cotransporter in effectuating Cl À secretion.

Experimental Eye Research, 2000

The regulation of Na + transport by cAMP in freshly isolated rabbit conjunctival epithelium, a ti... more The regulation of Na + transport by cAMP in freshly isolated rabbit conjunctival epithelium, a tissue exhibiting both Cl − secretion and Na + absorption, was examined. Bulbar-palpebral segments of conjunctiva were mounted between Ussing-type hemichambers under short-circuit conditions in Cl − free media. In this situation, the short-circuit current (I sc ) measures an amiloride-resistant Na + absorptive process in the apical-to-basolateral direction. Apical additions (each at 10 µ) of cAMP-elevating compounds, forskolin, rolipram, IBMX and epinephrine all stimulated the Na + -dependent I sc by $ 3n5-4n5 µA cm −# (minimal 40 % increase) and reduced transepithelial resistance (R t ) by at least 7 % (P 0n05). Pre-exposure (1 hr) to the protein kinase A (PKA) inhibitor H-89 (10 µ), which in itself inhibited the I sc by 0n5 µA cm −# , attenuated the I sc responses of the cAMP-elevating agents (P 0n05, unpaired data). In reverse, H-89 promptly decreased the I sc by 1n5-2n5 µA cm −# and increased R t by 5 % (P 0n05) in tissues pre-stimulated with either forskolin or an epinephrine plus IBMX combination. Additions of epinephrine or rolipram to apically permeabilized preparations using amphotericin B, increased the I sc by 12 and 22 % respectively over baseline and reduced R t by 6 % (P 0n05). Similarly, in the presence of a transepithelial K + gradient (apical to basolateral) and amphotericin B, cAMP elevation stimulated K diffusion across the preparation by at least 1n8 µA cm −# and decreased the R t by 4 % (P 0n05), changes that were reversed by subsequent H-89 addition. Under Cl − rich conditions, pretreatment with 5 m Ba# + reduced the basal I sc by 59 % and blocked the cAMP-induced I sc stimulations typically seen in the presence of the anion. The results provide evidence for a PKA-regulated, Ba# + -inhibitable (voltage insensitive) basolateral K + conductance in rabbit conjunctival epithelial cells. The action of Cl − secretogogues acting via cAMP on basolateral K + channel activity indicates that endogenous levels of cAMP may play a role in the regulation of Cl − secretion and Na + absorption in the conjunctiva.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1996

Amiloride (0.5 mM) inhibited the rate of entry of Na + into corneal endothelial cells by more tha... more Amiloride (0.5 mM) inhibited the rate of entry of Na + into corneal endothelial cells by more than half ((0.76 + 0.10) to (0.21 + 0.10) /xEq cm-2 h-~). The same concentration of amiloride caused only minimal disturbance to corneal hydration control by the endothelium (range 0-12%). Amiloride (0.5 mM) and acetazolamide (1 mM) reversibly inhibited trans-endothelial short circuit current by about a half. Their combined effect was not additive. Acetazolamide (1 raM) reduced net HCO~-flux across the short-circuited endothelium by about the same amount ((0.50 + 0.11) /xEq cm-2 h-i) that amiloride (0.5 mM) reduced Na" entry into the cells ((0.55 + 0.14) /zEq cm-2 h-~). Low concentrations of amiloride (10 p.M) had little effect on the transport characteristics of the endothelium, indicating that Na + entry into the endothelial cells under physiological conditions is not primarily through Na" channels. The data are consistent with an Na+/H + exchanger acting in tandem with carbonic anhydrase through a pathway which could have a regulatory role on endothelial transport via its effect on Na ÷ re-entry. Residual trans-endotheliai HCO 3 transport, apparently unaffected by amiloride or acetazolamide inhibition, is calculated to be of sufficient magnitude to maintain corneal hydration.

American Journal of Physiology-Cell Physiology, 2001

The effects of serotonin [5-hydroxytryptamine (5-HT)] on the transepithelial electrical propertie... more The effects of serotonin [5-hydroxytryptamine (5-HT)] on the transepithelial electrical properties of the short-circuited rabbit conjunctiva were examined. With this epithelium, the short-circuit current ( I sc) measures Cl− secretion plus an amiloride-resistant Na+ absorptive process. Apical addition of 5-HT (10 μM) elicited a prompt I screduction from 14.2 ± 1.2 to 10.9 ± 1.2 μA/cm2 and increased transepithelial resistance from 0.89 ± 0.05 to 1.03 ± 0.06 kΩ · cm2(means ± SE, n = 21, P < 0.05). Similar changes were obtained with conjunctivae bathed without Na+ in the apical bath, as well as with conjunctivae preexposed to bumetanide with the Cl−-dependent I sc sustained by the parallel activities of basolateral Na+/H+ and Cl−/HCO[Formula: see text] exchangers. In contrast, the 5-HT-evoked effects were attenuated by the absence of Cl−(Δ I sc = −0.5 ± 0.2, n = 5), suggesting that reduced Cl−conductance(s) is an effect of 5-HT exposure. In amphotericin B-treated conjunctiva and in ...

Molecular vision, Jan 22, 2010

Dual specificity phosphatases (DUSPs) modulate the duration and magnitude of phospho-activation o... more Dual specificity phosphatases (DUSPs) modulate the duration and magnitude of phospho-activation of Erk1/2, p38 and JNK1/2, the terminal kinases (TKs) of the mitogen activated protein kinase (MAPK) cascades. Three DUSPs, DUSP1, DUSP5, and DUSP6, are overexpressed in ocular surface side population stem cells (SPSCs). Our objective was to identify the impact of these enzymes on TK phosphorylation and proliferation of corneal epithelial cells. SV40 immortalized (sv) and expanded fresh human corneal epithelial cells (efHCECs) were transduced with lentivectors to elicit expression of shRNAmir against DUSP1, DUSP5, and JNK1 to thereby create the DUSP1i, DUSP5i and JNKi cell sublines, or overexpress DUSP6 (henceforth DUSP6(+)), respectively. TK phosphorylation status and proliferation rates were determined by immunoblotting and (3)H thymidine uptake. In both ef and svHCECs, EGF supplementation after a 24 h serum starvation caused a rapid 5-15 min spike in the phosphorylation of all three TK...

Investigative Opthalmology & Visual Science, 2009

PURPOSE. To define the molecular signature of limbal SP cells and identify signaling pathways ass... more PURPOSE. To define the molecular signature of limbal SP cells and identify signaling pathways associated with the phenotype of these putative stem cells. METHODS. Primary cultures of pig limbal epithelial cells stained with Hoechst 33342 were sorted by flow cytometry into SP and non-SP cells, and purified RNA was processed for microarray analysis with an oligonucleotide spotted array. Expressed transcripts for which SP and non-SP expressions differed by more that 1.5-fold in each paired set and by twofold overall were considered to be differentially expressed. Differential expression was validated by quantitative PCR and immunostaining. Data-mining methods were used to identify cellular processes that are either salient or depressed in the SP cells. RESULTS. The microarray identified approximately 9000 distinct, expressed, and identifiable genes. Of those, 382 and 296 were either over-or underexpressed in the SP cells, respectively. Overrepresentation analysis indicated that SP cells are in a low metabolic and biosynthetic state. In addition, a pattern of elevated MXD1, MAXI2, DUSP5, p27/KIP1, and p57/KIP2 and decreased Cyclin D and CDK genes can be expected to slow intrinsic and mitogen-induced G 1-to-S cell cycle transition. SP cells were also rich in genes associated with stem cell phenotype and genes providing protection against oxidative and/or xenobiotic damage. CONCLUSIONS. Microarray analysis of pig limbal SP cells yielded a molecular signature underscoring a phenotype characterized by slow cycling and low metabolic activity. The results provide valuable insights for the preservation and/or replication of epithelial stem cells.

Investigative Opthalmology & Visual Science, 2009

PURPOSE. Side-population (SP) cells isolated from limbal and conjunctival epithelia derive from c... more PURPOSE. Side-population (SP) cells isolated from limbal and conjunctival epithelia derive from cells that are slow cycling in vivo, a known feature of tissue stem cells. The purpose of this study was to define the molecular signature of the conjunctival SP cells and identify markers and signaling pathways associated with the phenotype of these cells. METHODS. Overnight cultures of freshly isolated human conjunctival epithelial cells stained with Hoechst 33342 were sorted by flow cytometry into SP and non-SP cohorts. Isolated RNA was processed for microarray analysis using a commercial oligonucleotide spotted array. Results were validated at the gene and protein levels by quantitative PCR and immunologic methods. Data mining methods were used to identify cellular processes relevant for stem cell function. RESULTS. Comparative analyses of transcripts expression based on present and absent software calls across four replicate experiments identified 16,993 conjunctival epithelial transcripts including 10,266 unique known genes of ϳ24,000 represented in the array. Of those genes, 1254 and 363 were overexpressed (Ͼ2-fold) or underexpressed (Ͻ0.5-fold), respectively, in the SP. The overexpressed set included genes coding for proteins that have been associated with (1) embryonic development and/or stem cell self renewal (MSX, MEIS, ID, Hes1, and SIX homeodomain genes); (2) cell survival (e.g., CYP1A1 to degrade aromatic genotoxic compounds); (3) cycling rate (e.g., DUSPs and Pax6 to foster slow cycling); and (4) genes whose expression is not typical in epithelia (e.g., CD62E). CONCLUSIONS. The molecular signature of conjunctival SP cells is consistent with a stem cell phenotype. Their gene expression patterns underpin slow cycling and plasticity, features associated with tissue stem cells. The results provide valuable insights for the preservation and/or expansion of epithelial stem cells.

Experimental Eye Research, 2000

The regulation of Na + transport by cAMP in freshly isolated rabbit conjunctival epithelium, a ti... more The regulation of Na + transport by cAMP in freshly isolated rabbit conjunctival epithelium, a tissue exhibiting both Cl − secretion and Na + absorption, was examined. Bulbar-palpebral segments of conjunctiva were mounted between Ussing-type hemichambers under short-circuit conditions in Cl − free media. In this situation, the short-circuit current (I sc) measures an amiloride-resistant Na + absorptive process in the apical-to-basolateral direction. Apical additions (each at 10 µ) of cAMP-elevating compounds, forskolin, rolipram, IBMX and epinephrine all stimulated the Na +-dependent I sc by $ 3n5-4n5 µA cm −# (minimal 40 % increase) and reduced transepithelial resistance (R t) by at least 7 % (P 0n05). Pre-exposure (1 hr) to the protein kinase A (PKA) inhibitor H-89 (10 µ), which in itself inhibited the I sc by 0n5 µA cm −# , attenuated the I sc responses of the cAMP-elevating agents (P 0n05, unpaired data). In reverse, H-89 promptly decreased the I sc by 1n5-2n5 µA cm −# and increased R t by 5 % (P 0n05) in tissues pre-stimulated with either forskolin or an epinephrine plus IBMX combination. Additions of epinephrine or rolipram to apically permeabilized preparations using amphotericin B, increased the I sc by 12 and 22 % respectively over baseline and reduced R t by 6 % (P 0n05). Similarly, in the presence of a transepithelial K + gradient (apical to basolateral) and amphotericin B, cAMP elevation stimulated K diffusion across the preparation by at least 1n8 µA cm −# and decreased the R t by 4 % (P 0n05), changes that were reversed by subsequent H-89 addition. Under Cl − rich conditions, pretreatment with 5 m Ba# + reduced the basal I sc by 59 % and blocked the cAMP-induced I sc stimulations typically seen in the presence of the anion. The results provide evidence for a PKA-regulated, Ba# +-inhibitable (voltage insensitive) basolateral K + conductance in rabbit conjunctival epithelial cells. The action of Cl − secretogogues acting via cAMP on basolateral K + channel activity indicates that endogenous levels of cAMP may play a role in the regulation of Cl − secretion and Na + absorption in the conjunctiva.

Experimental Eye Research, 2001

Earlier work from this laboratory demonstrated a bumetanide-inhibitable K uptake activity in cult... more Earlier work from this laboratory demonstrated a bumetanide-inhibitable K uptake activity in cultured bovine lens epithelial cells, but not at the anterior surfaces of intact bovine lenses isolated in an Ussingtype chamber. Presently the distribution of the bumetanide-sensitive Na-K-2Cl À cotransporter within the lens was reexamined. To complement previous results, 86 Rb uptake experiments were done in a chamber design that limited exposure of the radiolabel to speci®c surfaces of rabbit lenses under shortcircuit conditions. In addition, the cotransporter protein (NKCC1, but not NKCC2) was immune-detected in Western blots. For the latter, membrane preparations were obtained from capsule-plus-epithelial specimens, and from three cortical fractions, i.e. the anterior, equatorial, and posterior regions, as well as a ®fth, nuclear fraction. K in¯uxes across the anterior-polar, equatorial, and posterior-polar surfaces were 0. 375, 0. 348 and 0. 056 mEq (hr cm 2) À1 respectively, rates that were not signi®cantly reduced by the presence of 0. 1 mM bumetanide (P 4 0. 15, as unpaired data). In contrast, bumetanide-sensitive K in¯ux rates were measured across the anterior and equatorial surfaces under hypertonic, but not under hypo-osmotic conditions. In culture, bumetanide and ouabain were equipotent in reducing by approximately half the K uptake of quiescent, rabbit lens epithelial cells under control, iso-osmotic conditions, indicating a cell-culture induced up-regulation of the cotransport activity by an undetermined mechanism. The immunoblotting of lens membrane proteins elicited approximately 170±180 kDa bands accordant with the identity of the NKCC1 isoform in the epithelial and cortical equatorial fractions. Thus, NKCC1 was readily demonstrated using membrane specimens taken from within the lens. Its activity in the intact organ may be activated by conditions fostering cell shrinkage, and perhaps, agents stimulating epithelial cell elongation, given its distribution within the lens.

Current Eye Research, 2003

To characterize the serotonin (5-HT) receptors linked to the modulation of adenylyl cyclase activ... more To characterize the serotonin (5-HT) receptors linked to the modulation of adenylyl cyclase activity in rabbit, porcine and human conjunctivae. Serotonin receptor-subtype expression was examined using reverse transcription-polymerase chain reaction (RT-PCR) and receptor subtype-specific polyclonal antibodies for the immunofluorescent labeling of conjunctival cryosections. In addition, measurements of the effects of serotonergics on the short-circuit current (I(sc)) across rabbit and porcine conjunctivae were contrasted. RT-PCR assays indicated the expression of 5-HT(1B ) and 5-HT(1D) receptors, subtypes negatively coupled to adenylyl cyclase, in the rabbit conjunctiva. This approach also suggested the co-expression of 5-HT(1B), 5-HT(1D), 5-HT(1F), 5-HT(4) and 5-HT(7) mRNA&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s in the porcine conjunctiva, and 5-HT( 1D), 5-HT(1F) and 5-HT(7) in the human conjunctiva. Since the 5-HT(4) and 5-HT(7) receptors are positively linked to adenylyl cyclase, these results implied that the porcine and human tissues exhibited subtypes both positively and negatively linked to the enzyme. However, immunohistochemical observations, using currently available antibodies solely localized the 5-HT(7) moiety in the porcine and human epithelia, suggested that the 1B/1D forms may be minor elements. Consistent with this prospect, 5-HT was a stimulant of the transepithelial I(sc) across the porcine conjunctiva, an opposite response from earlier findings that demonstrated inhibitory effects by 5-HT on the rabbit I(sc), which are now explained by the localization of the 1B/1D receptors in the rabbit stratified epithelium. The 5-HT receptors expressed by mammalian conjunctivae are not identical. In terms of 5-HT receptor expression, the porcine tissue may be a more appropriate model for human, than is the rabbit, in that 5-HT may serve as a secretagogue in the human epithelium.

Investigative Opthalmology & Visual Science, 2007

PURPOSE. To determine global mRNA expression levels in corneal and conjunctival epithelia and ide... more PURPOSE. To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. METHODS. cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression Ͼ1% of the ␤-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest. RESULTS. The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA 2-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb. CONCLUSIONS. Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that are potentially active in the function of corneal and conjunctival epithelia.

Current Eye Research, 2003

To characterize the effects of cAMP-elevating stimuli on the rabbit translens electrical paramete... more To characterize the effects of cAMP-elevating stimuli on the rabbit translens electrical parameters and examine the distribution of beta adrenoceptors about the epithelial surface. The electrophysiological experiments encompassed the isolation of lenses within a vertically arranged, Ussing-type chamber under short-circuit conditions, an approach that allowed for measurements of short-circuit current (I(sc)) across, in separate experiments, discrete surface regions. Epithelial beta receptors were localized by immunofluorescent labeling of lens cryosections primarily exposed to a polyclonal antibody against human beta( 2)-adrenoceptors. Reverse transcription - polymerase chain reaction (RT-PCR) was used to generate cDNA (using specific primers based upon the sequence of the previously cloned human beta(2) receptor) from rabbit lens RNA extracted from mechanically sequestered anterior and equatorial epithelial cells. Asymmetrical I(sc) reductions with increases in translens resistance were elicited with epinephrine, isoproterenol, terbutaline, forskolin, and a lipid-permeable cAMP analogue. Electrical changes were recorded across the anterior aspect and not observed when the above compounds were applied to solutions bathing the equatorial and posterior surfaces. Immunohistochemical observations indicated the expression of beta receptors from the anterior epithelium to the equatorial region. RT-PCR yielded cDNA of expected basepair length for the apparent fragment of the beta(2)-adrenoceptor, which exhibited a sequence homology 90% identical with its human equivalent in both the anterior and equatorial epithelia. The cAMP-sensitive conductance(s) appear limited to the anterior epithelium and undetectable equatorially. The asymmetrical I(sc) responses do not seem to arise from a spatial heterogeneity in epithelial receptor expression.

Current Eye Research, 2003

To characterize the effects of cAMP-elevating stimuli on the rabbit translens electrical paramete... more To characterize the effects of cAMP-elevating stimuli on the rabbit translens electrical parameters and examine the distribution of beta adrenoceptors about the epithelial surface. The electrophysiological experiments encompassed the isolation of lenses within a vertically arranged, Ussing-type chamber under short-circuit conditions, an approach that allowed for measurements of short-circuit current (I(sc)) across, in separate experiments, discrete surface regions. Epithelial beta receptors were localized by immunofluorescent labeling of lens cryosections primarily exposed to a polyclonal antibody against human beta( 2)-adrenoceptors. Reverse transcription - polymerase chain reaction (RT-PCR) was used to generate cDNA (using specific primers based upon the sequence of the previously cloned human beta(2) receptor) from rabbit lens RNA extracted from mechanically sequestered anterior and equatorial epithelial cells. Asymmetrical I(sc) reductions with increases in translens resistance were elicited with epinephrine, isoproterenol, terbutaline, forskolin, and a lipid-permeable cAMP analogue. Electrical changes were recorded across the anterior aspect and not observed when the above compounds were applied to solutions bathing the equatorial and posterior surfaces. Immunohistochemical observations indicated the expression of beta receptors from the anterior epithelium to the equatorial region. RT-PCR yielded cDNA of expected basepair length for the apparent fragment of the beta(2)-adrenoceptor, which exhibited a sequence homology 90% identical with its human equivalent in both the anterior and equatorial epithelia. The cAMP-sensitive conductance(s) appear limited to the anterior epithelium and undetectable equatorially. The asymmetrical I(sc) responses do not seem to arise from a spatial heterogeneity in epithelial receptor expression.

Current Eye Research, 2000

To investigate possible regional expression of transport systems in the conjunctival epithelium g... more To investigate possible regional expression of transport systems in the conjunctival epithelium given distinct differences in morphological appearance between the bulbar and palpebral epithelia as well as variations found in the proportions of Na absorptive versus Cl secretory activities in electrophysiological studies. Mouse monoclonal antibodies against the alpha1-subunit of Na-K-ATPase and Na-K-Cl cotransporter (NKCC1) and a rabbit polyclonal antibody against the Na-glucose cotransporter (SGLT1) were used in immunoblotting and immunofluorescent labeling of frozen fixed sections isolated from either the bulbar and palpebral regions of the conjunctiva. Western blot analysis clearly demonstrated the presence of Na-K-ATPase, NKCC1 and SGLT1 proteins in both bulbar and palpebral conjunctiva. Indirect immunofluorescence studies on bulbar and palpebral conjunctival portions revealed intense staining by the anti-NKCC1 and anti-alpha1-Na-K-ATPase antibodies exclusively at the basolateral surfaces, whereas the anti-SGLT1 antibody was used with porcine conjunctiva to elicit strong and unambiguous staining along the apical plasma membrane. Proteins that mediate the transport activities of the Na-K-ATPase and Na-K-Cl cotransporter are uniformly distributed throughout the palpebral and bulbar regions of the rabbit conjunctival epithelium. Although the Na-glucose cotransporter could be detected in immunoblots of the rabbit, this cotransporter appears to be uniformly distributed as well, based upon immunohistochemical sections of the pig conjunctiva. Thus, it appears likely that mechanisms for Cl secretion and Na absorption reside in both bulbar and palpebral segments of the conjunctival epithelium.

Pflügers Archiv European …, 2000

Corneal stromal hydration is maintained by an active HCO3 – transport mechanism located in the co... more Corneal stromal hydration is maintained by an active HCO3 – transport mechanism located in the corneal endothelium. Whilst modulation of transport activity by changes in intracellular cAMP concentration have been noted, the site of effect is undefined. To resolve this ...

Experimental eye …, 1999

Protein kinase C (PKC) activation elicits diverse cell-type specific effects on key epithelial tr... more Protein kinase C (PKC) activation elicits diverse cell-type specific effects on key epithelial transporters. The present work examined the influence of phorbol esters, which are known activators of PKC isoenzymes, on the short-circuit current (I sc), a direct measure of net transcellular electrolyte transport, of the rabbit conjunctiva. In this preparation, the I sc measures a Na +-dependent, bumetanide-inhibitable Cl − transport in the basolateral-to-apical direction plus an amiloride-resistant Na + absorptive process in the opposite direction. Additions of phorbol 12-myristate-13-acetate (PMA) to the basolateral bathing media did not affect the transepithelial electrical parameters ; but its introduction to the apical bath at 1 and 10 µ elicited a transient ($ 2 min duration) I sc spike followed by a sustained reduction relative to the control level. Such PMA-elicited I sc reductions were from 14n0p2n0 to 3n1p0n8 µA cm −# (p...'s, n l 3) at 1 µ and from 16n5p1n9 to 4n6p0n7 µA cm −# (n l 22) at 10 µ. The former concentration failed to produce extensive I sc reductions in 3 other experiments. Similar results were obtained with phorbol 12,13-dibutyrate (PDBu). Its apical administration at 0n1 µ reduced the I sc from 18n5p4n1 to 7n8p2n0 (n l 3), and from 16n5p2n9 to 6n9p1n2 (n l 7) when introduced at 1 µ. The phorbol-evoked I sc reductions occurred without a simultaneous change in transepithelial resistance (R t). However, after about 15-20 min, R t gradually declined by about 25 %. In contrast to these results, treatment with a phorbol ester known not to activate PKC (4-α-PMA) did not affect the electrical parameters when added at 10 µ. PMA-and PDBu-evoked I sc reductions could be obtained with conjunctiva that were (1) pretreated with bumetanide, (2) bathed in Cl −-free media, and (3) pretreated with amphotericin B, changes consistent with a likely inhibition of the basolateral Na + \K + pump. Such I sc inhibitions were attenuated with conjunctiva pre-exposed to 1 µ staurosporine, a nonselective kinase inhibitor known to suppress PKC activity. Staurosporine, in itself, produced a rapid 26 % I sc inhibition (n l 15) along with a 17% R t increase upon its apical introduction. These electrical responses were less extensive in Cl −-free media and absent in amphotericin B-treated conjunctiva, suggesting the presence of a kinase-mediated regulation of apical channels for both Na + and Cl −. Overall, these results imply that in addition to previously demonstrated epinephrine-elicited, up-regulation of Cl − secretion, mechanisms may also exist, via PKC activation, to suppress Na + \K + pumping and consequently reduce transepithelial transport rates.

Molecular vision, 2004

METHODS Tissue samples and RNA extraction: This work followed the Guiding Principles in the Care ... more METHODS Tissue samples and RNA extraction: This work followed the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80-23). Adult male New Zealand White rabbits (2.0-2.5 kg) were killed with an overdose of sodium pentobarbital. Non-ocular tissues (kidney, lung, muscle, and skin) were collected using a fresh sterile disposable scalpel for each, dissected into cubes (side length 3-5 mm), rinsed in sterile phosphate buffered saline, pH 7.4 (PBS), and snap frozen in

Experimental Eye Research, 2006

Current eye research, 2002

To determine the presence of the cystic fibrosis transmembrane conductance regulator (CFTR) in th... more To determine the presence of the cystic fibrosis transmembrane conductance regulator (CFTR) in the conjunctiva and examine the possibility of its regional expression in rabbit, rat and porcine conjunctival epithelia given distinct differences in morphological appearance between the bulbar and palpebral epithelia. Two specific anti-CFTR antibodies, against different epitopes in the R domain of the CFTR molecule were used in immunofluorescent labeling of frozen fixed sections isolated from the bulbar and palpebral regions of fresh rabbit, porcine and rat conjunctivae. CFTR expression was also determined in the rabbit conjunctival epithelium using RT-PCR methods. CFTR immunoreactivity in the conjunctival epithelium exhibits polarized expression and is associated with the apical domain of conjunctival epithelial cells. An identical pattern of staining obtained in porcine cryosections using either of the anti-human CFTR antibodies confirmed the specificity of the positive apical staining. RT-PCR analysis produced bands at the predicted size for CFTR mRNA transcripts in both bulbar and palpebral portions of the rabbit conjunctival epithelium. Apical localization of CFTR in the conjunctival epithelium is consistent with the function of this protein as a chloride channel or as a regulator of channel activity. The identification of CFTR in both bulbar and palpebral portions of the conjunctiva provides evidence that the mechanisms for Cl secretion reside throughout the conjunctiva. This finding suggests that manipulations of the CFTR Cl channel could affect transepithelial Cl transport rates and water movement into the tear film.

Experimental eye research, 2001

Acid-base transporters of rabbit and porcine conjunctival epithelia were identi®ed and localized ... more Acid-base transporters of rabbit and porcine conjunctival epithelia were identi®ed and localized with immunoblotting and immunohistochemical techniques using speci®c antibodies against carriers commonly found in epithelia, i.e. the Cl À /HCO À 3 exchanger (AE2), Na /H exchanger (NHE-1,-2,-3) and the electrogenic Na-(n)HCO À 3 cotransporter (NBC). Western blot analysis demonstrated that anti-AE2 reacted with an approximate 170 kDa protein in both rabbit and pig cell membranes prepared from separately isolated bulbar and palpebral conjunctivae. NHE1 was similarly identi®ed in these distinct conjunctival regions but results with anti-NBC were ambiguous. Histochemical examinations indicated that the AE2 and NHE1 proteins reside on the basolateral surfaces of the plasma membrane throughout the multilayered tissue. The immunostaining of porcine cryosections for AE2 and rabbit sections for NHE1 was speci®c, because of its abolishment following either pre-absorption with the corresponding peptide or omission of the primary antibody. Screening with anti-NBC produced weak staining of the sections that appeared to be non-speci®c. For con®rmation of these results, the acid-base transporters present in rabbit cell cultures of conjunctival epithelia were ascertained from the changes in intracellular pH (pH i) evoked upon sequential superfusion with media of altered composition. This approach readily obtained Na-and Cl À-dependent pH i effects consistent with the existence of Cl À /HCO À 3 and Na /H exchange activities. Evidence for the presence of NBC could not be acquired, thereby substantiating the observations from the immunodetection techniques. The identity and location of the antiporters that were found suggested that these elements could contribute to transcellular Cl À transport in the basolateral-to-apical direction. To test this possibility, the effects of AE and/or NHE inhibition were determined on the bumetanide-insensitive Cl À-dependent short-circuit current across rabbit conjunctivae freshly isolated in Ussing-type chambers. These experiments revealed that such current is indeed sustained by the antiporters. Results with acetazolamide further suggested that the contribution of the acid-base transporters towards transepithelial Cl À secretion is variable and dependent upon individual rates of metabolic CO 2 production. Overall, the present study provides an initial identi®cation of the acid-base transporters present in the conjunctiva. Besides their likely role in intracellular pH regulation, the parallel, basolateral expression of AE2 and NHE1 indicates that these elements do not directly contribute to the pH of the tear ®lm but may complement the Na-2Cl À-K cotransporter in effectuating Cl À secretion.

Experimental Eye Research, 2000

The regulation of Na + transport by cAMP in freshly isolated rabbit conjunctival epithelium, a ti... more The regulation of Na + transport by cAMP in freshly isolated rabbit conjunctival epithelium, a tissue exhibiting both Cl − secretion and Na + absorption, was examined. Bulbar-palpebral segments of conjunctiva were mounted between Ussing-type hemichambers under short-circuit conditions in Cl − free media. In this situation, the short-circuit current (I sc ) measures an amiloride-resistant Na + absorptive process in the apical-to-basolateral direction. Apical additions (each at 10 µ) of cAMP-elevating compounds, forskolin, rolipram, IBMX and epinephrine all stimulated the Na + -dependent I sc by $ 3n5-4n5 µA cm −# (minimal 40 % increase) and reduced transepithelial resistance (R t ) by at least 7 % (P 0n05). Pre-exposure (1 hr) to the protein kinase A (PKA) inhibitor H-89 (10 µ), which in itself inhibited the I sc by 0n5 µA cm −# , attenuated the I sc responses of the cAMP-elevating agents (P 0n05, unpaired data). In reverse, H-89 promptly decreased the I sc by 1n5-2n5 µA cm −# and increased R t by 5 % (P 0n05) in tissues pre-stimulated with either forskolin or an epinephrine plus IBMX combination. Additions of epinephrine or rolipram to apically permeabilized preparations using amphotericin B, increased the I sc by 12 and 22 % respectively over baseline and reduced R t by 6 % (P 0n05). Similarly, in the presence of a transepithelial K + gradient (apical to basolateral) and amphotericin B, cAMP elevation stimulated K diffusion across the preparation by at least 1n8 µA cm −# and decreased the R t by 4 % (P 0n05), changes that were reversed by subsequent H-89 addition. Under Cl − rich conditions, pretreatment with 5 m Ba# + reduced the basal I sc by 59 % and blocked the cAMP-induced I sc stimulations typically seen in the presence of the anion. The results provide evidence for a PKA-regulated, Ba# + -inhibitable (voltage insensitive) basolateral K + conductance in rabbit conjunctival epithelial cells. The action of Cl − secretogogues acting via cAMP on basolateral K + channel activity indicates that endogenous levels of cAMP may play a role in the regulation of Cl − secretion and Na + absorption in the conjunctiva.