Biomedical subjects

M Coca-Prados

Publications and source records attributed to M Coca-Prados.

At least 55 records · Page 3Linked to original sources

PubMed1995

Immortalization of cat iris sphincter smooth muscle cells by SV40 virus: growth, morphological, biochemical and pharmacological characteristics.

The purpose of this study was to establish immortalized cell cultures of cat iris sphincter smooth muscle cells for a model investigating ocular receptors and their signal transduction pathways. Cultured cat iris sphincter muscle cells were immortalized by viral transformation with SV40 virus and the morphological and immunocytochemical properties of the normal and immortalized cells were investigated. The transformed cell clone, SV-CISM-2, was further characterized biochemically and pharmacologically. The normal muscle cells showed characteristics of smooth muscle cells, as judged by their growth and the presence of smooth muscle alpha-actin and desmin. After seven passages the normal cells ceased to proliferate. In contrast, the immortalized cells retained their proliferative ability for more than 220 population doublings over 55 passages. The transformation phenotype in these cells was confirmed by their expression of the large T-antigen, the incorporation of viral DNA into cellular DNA, growth in agarose and in low-serum medium, and complete loss of contact inhibition. The immortalized cells expressed smooth muscle alpha-actin, desmin and MLC protein. Biochemical and pharmacological studies on the SV-CISM cells revealed the presence of several functional receptors including muscarinic cholinergic, beta-adrenergic, peptidergic (substance P and endothelin). Platelet-activating factor, and prostaglandin (PG). Muscarinic stimulation of these cells resulted in: (a) a dose-dependent increase in the release of arachidonic acid (AA) and (PGs) and enhancement in the production of inositol trisphosphate (IP3); and (b) a substantial increase in MLC phosphorylation (118%), an indicator of smooth muscle contractility. The stimulatory effects of carbachol on these responses were completely blocked by atropine, a muscarinic receptor antagonist. This study constitutes the first successful immortalization of iris sphincter smooth muscle cells. The SV-CISM-2 cells can serve as an important model system for investigations on the biochemical and pharmacological properties of receptors and their signal transduction pathways in smooth muscle. The advantage of these cells over normal iris sphincter cells is that they can be propagated over many generations without alterations in their morphological, biochemical and physiological characteristics.

Animals↗

PubMed1995

Isolation and characterization of cell-specific cDNA clones from a subtractive library of the ocular ciliary body of a single normal human donor: transcription and synthesis of plasma proteins.

A subtractive cDNA library was developed for the purpose of identifying cell-specific genes expressed within the human ocular ciliary body, a tissue responsible for regulating aqueous humor secretion and intraocular pressure. Partial DNA sequence of a large number of cDNA clones and homology searches of nucleic acid and protein databases revealed significant homologies to at least 90 independently known genes. A group of biologically significant genes that were previously not known to have transcriptional expression in the ciliary body, complement component C4; alpha 2 macroglobulin; selenoprotein-P; and apolipoprotein D, were further demonstrated by Northern hybridization. Antibodies to these and other proteins (i.e., tyrosinase-related protein and pigment epithelium-derived factor) confirmed their cell-restricted expression in ciliary epithelial cells (pigmented, nonpigmented), or vascular endothelial cells. We provide evidence that two human plasma proteins, complement component C4 and alpha 2-macroglobulin are metabolically labeled with [35S]methionine in ciliary processes explants, suggesting that the ciliary body is an organ of synthesis and secretion of plasma proteins present in aqueous humor. These results challenge the notion that plasma proteins in aqueous humor are imported from outside of the eye. The subtractive cDNA library reported in this work should be very useful for identifying potential candidate genes in ocular abnormalities affecting the ciliary body, or involved in the regulation of intraocular pressure.

Adult↗

PubMed1995

PKC-sensitive Cl- channels associated with ciliary epithelial homologue of pICln.

Swelling activates and protein kinase C (PKC) downregulates Cl- channels in cultured nonpigmented ciliary epithelial (NPE) cells. We now report that the PKC inhibitor staurosporine upregulates whole cell Cl- currents isosmotically. The kinetics and current-voltage relationship are similar to those of volume-activated Cl- channels of these cells. These properties are inconsistent with cloned ClC-0, ClC-1, ClC-2, and MDR1 channels but could reflect the cystic fibrosis transmembrane conductance regulator (CFTR) channel or the Cl- channel regulator pICln. CFTR mRNA was undetectable by Northern analysis of cultured NPE cells or ciliary body tissue. In contrast, a human pICln probe obtained by polymerase chain reaction cloning and showing 90% identity with the rat cDNA clone detected high levels of transcripts in NPE cells. The level was low in tissue, where the NPE message was diluted by RNA from other cells. We conclude that NPE cells display staurosporine-activated Cl- channels [gSt(Cl)] likely identical with the volume-activated channels. The same cells expressing gSt(Cl) transcribe mRNA for a novel homologue (pHCBICln) of pICln that may regulate Cl- transport into the aqueous humor.

Alkaloids↗

PubMed1995

Cell-specific expression of the human Na+,K(+)-ATPase beta 2 subunit isoform in the nonpigmented ciliary epithelium.

PURPOSE: To evaluate the patterns of expression of beta subunit isoforms of the Na+,K(+)-ATPase and H+,K(+)-ATPase in the human eye and to determine the cell-specific distribution of the beta 2 subunit in the human ciliary epithelium. METHODS: Total RNA extracted from human ocular tissues was screened by Northern blot analysis with cDNA probes for the human Na+,K(+)-ATPase subunit isoforms (beta 1 and beta 2) or the H+,K(+)-ATPase (alpha and beta) subunits. Antibodies were raised to the amino and carboxyl terminal regions of the human beta 2 isoform. Polymerase chain reaction was used to verify the expression of beta 2 subunit in nonpigmented ciliary epithelial cells (NPE). RESULTS: Transcripts for the Na+,K(+)-ATPase beta 1 and beta 2 subunit isoforms were present at different levels in all the ocular tissues except the lens, which expressed only beta 1. No transcripts for the alpha or beta subunits of the H+,K(+)-ATPase were detected in the eye. Isoform beta 2 specific anti-peptide antibodies V15E (N-terminus) and A18R (C-terminus) recognized a 55- to 60-kDa protein in the ciliary epithelium and the core protein of 32 kDa after N-glycanase treatment. Immunocytochemical localization within the ciliary epithelium indicates that the Na+,K(+)-ATPase beta 2 isoform is expressed preferentially in the NPE cells. The expression of Na+,K(+)-ATPase beta 2 isoform in the human NPE cell line, ODM-2, was confirmed by polymerase chain reaction amplification and Southern blot analysis. CONCLUSIONS: The Na+,K(+)-ATPase beta 2 subunit isoform, but not H+,K(+)-ATPase, was expressed widely in ocular tissues of the human eye. The restricted cellular distribution of beta 2 isoform within the NPE cells represents an important differential gene marker associated with the multiple alpha subunit isoforms of Na+,K(+)-ATPase.

Aged↗

PubMed1994

Cloning of a novel cDNA homologous to CHIP28 water channel from ocular ciliary epithelium.

Water channel protein (CHIP28) has been initially identified from human erythrocyte and subsequently localized in human kidney, rat kidney and bovine corneal endothelium. We report here cloning of homologous cDNA (CHIP29) from an expression cDNA library constructed from bovine ciliary epithelium poly A+ that codes a 271 amino acid, 28.8 kDa protein. At the amino acid level, bovine CHIP29 is 91% identical to the CHIP28 protein. Analysis of the deduced amino acid sequence indicated a hydrophobic protein with six membrane spanning domains, one N-linked glycosylation site, two conserved NPA boxes common to MIP26 family proteins, and conserved amino acid residue cysteine 189 common to water channels. Northern blotting revealed a major mRNA of 2.8 Kb size expressed in bovine ciliary epithelium. The results presented here confirm the presence of water channel gene in the bovine ciliary epithelium, a primary site for the aqueous humor production.

Amino Acid Sequence↗

PubMed1994

Nucleotide sequence of a cDNA for the beta 2 subunit isoform of Na+,K(+)-ATPase from human retina.

Using as probe the entire human liver cDNA clone coding for the beta 2 subunit isoform of the Na+,K(+)-ATPase, which lacks the initiation codon ATG, and the entire 5'-untranslated region (Martin-Vasallo, P., Dackowski, W., Emanuel, J.R. and Levenson, R. (1989) J. Biol. Chem. 264, 4613-4618), we isolated a larger clone from a directional human adult retina cDNA library (Swaroop, A. and Xu, J. (1993) Cytogenet. Cell Genet. 64, 292-294). This clone, pNH beta 2, shows 100% homology with the nucleotide sequence of the human liver cDNA clone and also contains additional 407 nucleotides in the 5'-untranslated region, the initiation codon and a poly(A) tail. Northern blot hybridization analysis reveals that the human mRNA (3.6 kb) is approx. 300 nucleotides larger than the major transcript size expressed in rat (3.3 kb). The larger human size mRNA for the human beta 2 Na+,K(+)-ATPase indicates species differences in gene processing.

Amino Acid Sequence↗

PubMed1994

cDNA from human ocular ciliary epithelium homologous to beta ig-h3 is preferentially expressed as an extracellular protein in the corneal epithelium.

The non-pigmented ciliary epithelium is largely responsible for the formation of aqueous humor in the mammalian eye. To provide a basis for studies at the molecular level, a directional expression cDNA library was constructed in Uni-ZAP XR vector from poly A+ RNA of the human non-pigmented ciliary epithelial derived ODM-2 cell line. Fifty-three cDNA clones were isolated from the library and characterized by partial sequence analysis. Approximately 49% of the clones exhibited homology with known genes in the GenBank/EMBL databases. The putative identification of these clones may reflect the transcriptional activity of the ODM-2 cells in culture. One of the identified clones, ODM-42-I, was found to be specific and highly expressed in the corneal epithelium. This clone had an exact match with a recently discovered human gene, beta ig-h3 (Skonier et al., 1992, DNA Cell Biol., 11:511-522), which codes a surface recognition protein, inducible by transforming growth factor beta (TGF-beta), and containing a putative binding site (RDG) for integrins. The ODM-42-I cDNA clone displays a distinctive pattern of expression found in the human eye, expressed almost exclusively in the cornea. Further studies, using sera from a synthetic peptide to the carboxy-terminal region of ODM-42-I, reveal that the protein is heterogeneous in charge and is preferentially expressed on the extracellular surface of corneal epithelial cells, and might share immunologic properties with integrins beta 1.

Animals↗

PubMed1994

EP2-receptor stimulated cyclic AMP synthesis in cultured human non-pigmented ciliary epithelium.

Prostaglandins are locally produced mediators of physiologic function which act through specific receptors to influence cellular response pathways. We have conducted a series of experiments designed to obtain a preliminary pharmacologic characterization of the prostaglandin E2 receptor subtype mediating increased synthesis of adenosine 3',5'-cyclic monophosphate (cyclic AMP) in an SV40 transformed line of human non-pigmented ciliary epithelium (ODMC2). Human non-pigmented epithelial (NPE) cells were grown to confluency, preincubated with indomethacin (to prevent endogenous PGE2 synthesis) and with isobutyl-methyl-xanthine (to prevent breakdown of cAMP), challenged with PGE2 or receptor-selective prostanoids and extracted. Prostanoid-stimulated cAMP synthesis was determined using a protein binding assay and normalized to cellular protein. Prostanoid agonists known to be relatively selective for the EP2 receptor subtype stimulated cAMP synthesis by human NPE cells. EP1 and EP3, IP, FP and DP selective prostanoids had negligible effects on stimulation of cAMP synthesis. The results of these studies lead us to conclude that human NPE cells express receptors of the EP2 subtype which mediate PGE2 stimulated synthesis of cyclic AMP.

Cell Line, Transformed↗

PubMed1994

Inhibition of Na(+)-K(+)-ATPase activates Na(+)-K(+)-2Cl- cotransporter activity in cultured ciliary epithelium.

Inhibition of Na(+)-K(+)-ATPase activates Na(+)-K(+)-2Cl- cotransporter activity in cultured ciliary epithelium. Am. J. Physiol. 266 (Cell Physiol. 35): C198-C205, 1994.--86Rb uptake experiments were conducted to measure Na(+)-K(+)-ATPase activity and Na(+)-K(+)-2Cl- cotransporter activity in a cell line derived from rabbit nonpigmented ciliary epithelium. The presence of a Na(+)-K(+)-2Cl- cotransporter was supported by the observation of a bumetanide-sensitive 86Rb uptake component that was dependent on the extracellular concentration of both sodium and chloride. Potassium influx mediated by the Na(+)-K(+)-2Cl- cotransporter and Na(+)-K(+)-ATPase accounted for approximately 46 and 33% of total potassium uptake, respectively, whereas both ouabain- and bumetanide-resistant uptake accounted for 9%. Inhibition of the Na(+)-K(+)-ATPase had a stimulatory effect on Na(+)-K(+)-2Cl- cotransporter activity, which was dependent on the extent and duration of Na(+)-K(+)-ATPase inhibition. Ouabain treatment stimulated the potassium (86Rb) efflux rate and reduced intracellular potassium ([K]i). Potassium channel blockers suppressed the ouabain-activated potassium efflux and inhibited the ouabain-induced activation of the Na(+)-K(+)-2Cl- cotransporter. We conclude that Na(+)-K(+)-ATPase inhibition leads to the opening of potassium channels, which exacerbates the depletion of cellular potassium; Na(+)-K(+)-2Cl- cotransporter stimulation caused by the fall of [K]i overrides the tendency of increased cellular sodium to inhibit the cotransporter.

Animals↗

PubMed1994

Isolation of cDNA clones encoding the 80-kd subunit protein of the human autoantigen Ku (p70/p80) by antisera raised against ciliary processes of human eye donors.

PURPOSE: To isolate and characterize human nonpigmented ocular ciliary epithelial cDNA clones by screening a cDNA library constructed from the established human nonpigmented ciliary epithelial cell line (ODM-2) with sera raised against ciliary processes of human eye donors (cadavers). METHODS: An ODM-2 cDNA expression library in lambda Uni-ZAP-XR vector was screened with sera of mouse anti-human ciliary processes. The cDNA inserts from plaque-purified clones were sequenced by the dideoxy-chain termination method with T3 and T7 polymerase. Sera were assayed by Western blot analysis and indirect immunofluorescence and were then compared with sera, enriched in anti-Ku antibodies, collected from patients with scleroderma polymyositis overlap syndrome. RESULTS: Immunoscreening of 2.5 x 10(5) independent clones with sera induced experimentally in total tissue homogenates of human ciliary processes led to the isolation of four cDNA-positive clones. Three clones, designated A1, D1, and D3 and containing inserts measuring 1.4 kb to 1.8 kb, were found to be identical in DNA sequence analysis to that of the 80-kd subunit protein of the human autoantigen Ku (p70/p80). The fourth clone, designated D2, failed to show any significantly similarity with previously known genes. Cell extracts from human ciliary processes and ODM-2 cells were analyzed by immunoblotting with anti-human ciliary process sera. The pattern of proteins immunodetected was compared with the profile of proteins immunodetected with human anti-Ku sera from a patient with scleroderma polymyositis overlap syndrome. These results demonstrated that sera from anti-human ciliary processes recognize a variety of antigens in ODM-2 cells and are strikingly rich in antibodies to the 80-kd subunit protein of Ku (p70/p80). Furthermore, indirect immunofluorescence analysis supported the observation that anti-human ciliary processes sera labeled nuclear antigens in a human ciliary epithelium cell line and cross species in bovine ciliary epithelial cell, thus demonstrating the nuclear localization of Ku antigen. The mRNA in the human eye was found to be widely expressed in all tested ocular tissues and in ODM-2 cells. CONCLUSIONS: The ocular ciliary epithelium induces a species-specific immune response in xenogenic animals. In particular, the human ciliary epithelium elicits a strong immune recognition of the 80-kd subunit protein of the human autoantigen Ku (p70/p80) in ODM-2 cells.

Animals↗

PubMed1994

Pathways signaling the regulatory volume decrease of cultured nonpigmented ciliary epithelial cells.

PURPOSE: The authors identify the signaling pathways for the regulatory volume decrease (RVD) of nonpigmented ciliary epithelial (NPE) cells. The RVD is a regulatory response triggered by swelling and reflecting KCl release by NPE cells. METHODS: The cell volumes of human nonpigmented ciliary epithelial cells were measured in suspension by electronic cell sorting. Measurements were conducted in test solutions of constant ionic strength, but osmolality was varied by sucrose. RESULTS: Cyclic AMP (cAMP), forskolin, PGE2, the PKC-inhibitor staurosporine, and increasing cytoplasmic Ca2+ activity with thapsigargin all enhanced the RVD. Leukotrienes A4, D4, E4, and the protein phosphatase inhibitor okadaic acid had no detectable effect under the current experimental conditions. The cyclooxygenase inhibitor indomethacin, the epoxygenase inhibitors ketoconazole and SKF 525A, and the PKC activator DiC8 all downregulated the RVD. The addition of the cation ionophore, gramicidin, increased the RVD. In the presence of gramicidin, cAMP, PGE2, and indomethacin did not affect the RVD, but ketoconazole, DiC8, and the calcium-calmodulin blocker trifluoroperazine still inhibited--and staurosporine still enhanced--the RVD. Many of these observations are strikingly different from results reported with other cells. Anisosmotic swelling did not increase intracellular cAMP concentration. CONCLUSIONS: The pathways signaling the regulatory responses to swelling are unique for each cell type. The authors propose that hypotonic swelling of NPE cells stimulates arachidonic acid turnover, triggering PGE2-mediated upregulation of K+ channels and epoxide-mediated upregulation of Cl- channels. Swelling may also reduce endogenous PKC activity, further upregulating Cl- channels. Calcium-calmodulin plays a permissive role in upregulating the Cl- channels.

Arachidonic Acid↗

PubMed1993

Characterization of soluble guanylyl cyclase in transformed human non-pigmented epithelial cells.

Topical application of nitro vasodilators, such as sodium nitroprusside, reduces intraocular pressure. In brain and blood vessels, nitro vasodilators activate soluble guanylyl cyclases, producing cGMP. The location and molecular identity of ocular guanylyl cyclases are unknown. We studied transformed human non-pigmented ciliary epithelial cells, whose parental cells are responsible for the production of aqueous humor. Sodium nitroprusside increased cGMP 40 to 60 fold in a time and concentration dependent manner (EC50 40 to 100 microM). Methylene blue inhibited this effect (IC50 0.6 microM in the presence of 100 microM sodium nitroprusside). We also detected mRNA for the alpha 1 and beta 1, but not the beta 2, subunit isoforms of soluble guanylyl cyclase in these cells by Northern blotting. We conclude that transformed non-pigmented epithelial cells contain an active heterodimeric soluble guanylyl cyclase composed of at least alpha 1 and beta 1 subunits.

Blotting, Northern↗

PubMed1993

Studies on regulation of the ascorbic acid transporter in a cell line derived from rabbit non-pigmented ciliary epithelium.

A cell line was derived from rabbit non-pigmented ciliary epithelium. The non-pigmented ciliary epithelium is one of the two cell layers which secrete aqueous humor into the eye and concentrate ascorbic acid in the newly-formed fluid. The cultured non-pigmented epithelial cells accumulated ascorbic acid at a rate of 3-5 pmol/micrograms protein per h. As in freshly-isolated native tissue, the ascorbate uptake mechanism was sodium-dependent and could be inhibited by phloretin (apparent Ki = 2-10(-5) M). Phorbol 12,13-dibutyrate (PDBu), a protein kinase C activator, reduced the ascorbate uptake rate. The PDBu effect was concentration-dependent; at a concentration of 10(-6) M, PDBu reduced the ascorbate uptake rate to 65% of the control value. PDBu reduced the maximal rate of ascorbate uptake (determined at 200-500 microM external ascorbate) but caused no detectable change in the Km for ascorbic acid (approx. 80 microM). The PDBu-induced inhibition of ascorbate uptake persisted in the presence of ouabain and in low sodium (25 mM Na) medium, suggesting that the effect is not secondary to a change in the sodium gradient. Furthermore, no detectable elevation of cell sodium content was seen in cells equilibrated with 22Na prior to PDBu treatment. The PDBu-induced inhibition of ascorbate uptake was apparently mediated by protein kinase C because the effect was not observed in the presence of staurosporine (10(-6) M), a protein kinase C inhibitor, or in cells in which protein kinase C was downregulated. These observations suggest that activation of protein kinase C causes inhibition of the ascorbate transporter in this cultured cell line.

Alkaloids↗

PubMed1993

Differential expression of the cellular retinaldehyde-binding protein in bovine ciliary epithelium.

Differential expression of the cellular retinaldehyde-binding protein (CRALBP), a likely component of the visual cycle, has been observed in the ocular ciliary epithelium, a bilayer of neurepithelial cells involved in the active transport of aqueous humor. Antibody probes raised to bovine ciliary epithelium were used to isolate a cDNA encoding CRALBP from a bovine ciliary epithelium cDNA expression library. Northern analysis of poly A+RNA isolated from three different regions of the neonate ciliary epithelium revealed a three-fold higher level of CRALBP expression in the most anterior portion of the pars plicata region relative to the posterior pars plana region. Western blot analysis using CRALBP-specific antibodies also demonstrated that the level of CRALBP expression was higher in the most anterior portion of the ciliary epithelium and gradually decreases toward the posterior region in both neonate and adult bovine eyes. Using indirect immunofluorescence and frozen-sectioned bovine ciliary epithelium, CRALBP was localized to the pigmented ciliary epithelial cell layer along the entire ciliary epithelium. In the nonpigmented ciliary epithelial cell layer, CRALBP was only detected in the pars plana region. Pigmented ciliary epithelial cells have also been cultured and CRALBP expression demonstrated for several continuous passages by Northern and Western analysis. These results raise questions regarding the physiological role of CRALBP in the ciliary epithelium and the involvement of the protein in the defining regional and functional boundaries with this tissue.

Animals↗

PubMed1993

Cloning of the bovine plasma selenium-dependent glutathione peroxidase (GP) cDNA from the ocular ciliary epithelium: expression of the plasma and cellular forms within the mammalian eye.

In the anterior segment of the mammalian eye, the ocular ciliary epithelium produces the aqueous humor, a fluid that nourishes and protects the avascular tissues from oxidative stress. This report details the results of a study of molecular cloning, sequencing, and expression of plasma glutathione peroxidase (GPx-P) from the bovine ocular ciliary epithelium. The bovine GPx-P cDNA contains an open reading frame of 226 amino acids with a calculated molecular weight of 24,860. The corresponding amino acid sequence showed an overall identity of 88% with the human GPx-P, 88.5% with the rat GPx-P, and 46.4% with the cellular bovine glutathione peroxidase (GPx-1). The levels of GPx-P and GPx-1 transcripts in ocular tissues were analyzed and the ciliary epithelium was found to express the highest levels of GPx-P transcripts in human and bovine eyes, whereas the cornea of calf eyes expressed the highest levels of GPx-1 transcripts. Surprisingly, the lens, on which oxidants have profound effects leading to cataract formation, expressed the lowest levels of GPx-P and GPx-1 transcripts in human donor eyes. These results provide new evidence of differential gene expression of the GPx-P and GPx-1 forms in the mammalian eye and stresses the functional role of the ocular ciliary epithelium in protecting the anterior segment of the eye from oxidative damage.

Amino Acid Sequence↗

PubMed1993

Calcium-dependent regulation of cation transport in cultured human nonpigmented ciliary epithelial cells.

We performed 86Rb flux studies to examine Na-K-adenosinetriphosphatase (ATPase), Na-K-2Cl cotransporter, and potassium channel activity in an established cell line derived from human nonpigmented ciliary epithelium (ODM2). The elevation of intracellular calcium by A23187 (3 microM) or thapsigargin (200 nM) increased both ouabain-sensitive potassium (86Rb) uptake (Na-K-ATPase mediated) and ouabain-insensitive potassium (86Rb) uptake. The ouabain-insensitive component could be inhibited substantially by bumetanide (0.1 mM), suggesting the involvement of a Na-K-2Cl cotransporter. The increase of potassium (86Rb) uptake caused by thapsigargin could be prevented by the intracellular calcium buffer 1,2-bis(2-amino-phenoxy)ethane N,N,N',N'-tetraacetic acetoxymethyl ester (BAPTA/AM); in BAPTA/AM-treated cells, the thapsigargin stimulation of the bumetanide-sensitive portion of 86Rb uptake was abolished. After A23187 (5 microM), the 86Rb efflux rate was significantly increased; the increase could be blocked partially by quinidine (0.1 mM) and partially by bumetanide, suggesting that potassium channels and the Na-K-2Cl cotransporter contribute to the effect. We propose that the cell potassium loss after activation of quinidine-sensitive potassium channels is involved in the calcium-induced activation of Na-K-ATPase because 0.1 mM quinidine and 100 mM external potassium both markedly inhibited the A23187-induced increases of the ouabain-sensitive component of potassium (86Rb) uptake. Calcium-induced stimulation of the Na-K-2Cl cotransporter may not be linked to channel activation.

Biological Transport↗

PubMed1992

Isolation of a cDNA encoding a glutathione S-transferase (GST) class-pi from the bovine ocular ciliary epithelium.

We have used a polyclonal antiserum to bovine ciliary epithelium, a secretory tissue involved in the formation of aqueous humor, to immunoscreen a directional lambda gt11 Sfi-Not cDNA expression library prepared from bovine ciliary epithelium poly(A)+ RNA. After immunoscreening 6 x 10(5) independent clones, 41 cDNA clones positive for ciliary epithelium were isolated and characterized. About one-third of the positive cDNA clones were found to be identical and to encode a glutathione S-transferase (GST) class-pi. The largest bovine GST cDNA clone isolated, pCN11, contains an open reading frame of 630 bases, encoding a protein of 210 amino acids with a calculated molecular weight of 23,335 Da. The corresponding amino acid sequence showed an overall identity of 85.6% with the human, and 85.2% with the rat and mouse GST class-pi. Northern analysis of bovine ocular tissues revealed that the GST class-pi gene encodes a 0.8-kilobase mRNA which is expressed most abundantly in cornea, ciliary epithelium and retina, and in lower levels in iris and lens. Cell lines derived from non-pigmented or pigmented bovine ciliary epithelium also showed high levels of GST-pi mRNA expression. These results provide additional evidence for differential gene expression of GST class-pi mRNA in various areas of the bovine eye.

Animals↗

PubMed1992

Regulatory volume decrease by cultured non-pigmented ciliary epithelial cells.

Cells (ODM C1-2/SV40) derived from human non-pigmented ciliary epithelial cells were studied by electronic cell sizing. The time course of the cell volume (vc) was monitored after suspending cells in paired experimental and control, isosmotic and hyposmotic solutions of identical ionic composition. Following anisosmotic cell swelling, the cells displayed the regulatory volume decrease (RVD) previously described. The RVD primarily reflects loss of cell KCl since: (1) the K(+)-channel blockers quinidine and Ba2+ both inhibit the RVD; and (2) replacement of external Cl- with gluconate or addition of the Cl- channel blocker NPPB also inhibits the RVD. Bicarbonate has previously been reported to speed the RVD. This action likely reflects pH dependence of the channels since: (1) increasing the external pH speeds the RVD, whether or not HCO3- is present; and (2) DIDS (a blocker of Cl- channels and of Cl-/HCO3- exchange) is an effective inhibitor of the RVD, even after blocking Cl-/HCO3- exchange by removing external HCO3-. The RVD could also be inhibited by reducing the availability of Ca2+, either by omitting Ca2+ from the external medium or by blocking mobilization of intracellular Ca2+ with TMB-8. Furthermore, the RVD was slowed and incomplete in the presence of the calcium/calmodulin blocker trifluoperazine. We conclude that anisosmotic swelling triggers a series of events, mediated at least in part by calcium/calmodulin, leading to the extrusion of KCl through parallel K+ and Cl- channels.

Bicarbonates↗