RESUMEN

We characterized the functional and molecular properties of a volume-regulated anion channel (VRAC) in SV40-immortalized rabbit corneal epithelial cells (tRCE), since they mediate a robust regulatory volume decrease (RVD) response during exposure to a hypotonic challenge. Whole-cell patch clamp-monitored chloride currents and light-scattering measurements evaluated temporal cell-volume responsiveness to hypoosmotic challenges. Exposure to 200 mOsm medium elicited an outwardly-rectifying current (VACC), which was reversible upon reperfusion with isotonic (300 mOsm) medium. VACC and RVD were chloride-dependent because either chloride removal or application of NPPB (100 microM) suppressed these responses. VACC behavior exhibited voltage-dependent inhibition in the presence of DIDS (500 microM), whereas inhibition by both NPPB (100 microM) and niflumic acid (500 microM) was voltage-independent. VACC was insensitive to glibenclamide (250 microM), verapamil (500 microM) or removal of extracellular calcium. Phorbol dibutyrate, PDBu, (100 nM) had no effect on activated VACC. However, preincubation with PDBu prior to hypotonic challenge prevented VACC and RVD responses as well as prolonged characteristic time. An inactive phorbol ester analogue had no effect on RVD behavior. Moreover, Northern blot analysis verified expression of ClC-3 gene transcripts. The presence of ClC-3 transcripts along with the correspondence between the effects of known ClC-3 inhibitors on VACC and RVD suggest that ClC-3 activation underlies these responses to hypotonic-induced cell swelling.

Asunto(s)

Canales de Cloruro/metabolismo , Cloruros/metabolismo , Epitelio Corneal/metabolismo , Animales , Northern Blotting , Línea Celular Transformada , Tamaño de la Célula , Canales de Cloruro/genética , Impedancia Eléctrica , Soluciones Hipotónicas , Técnicas de Placa-Clamp , ARN Mensajero/genética , Conejos

RESUMEN

BACKGROUND: Intraocular pressure (IOP) is maintained by a balance between aqueous humour (AH) production (dependent on sodium transport across a ciliary epithelial bi-layer) and drainage (predominantly through the trabecular meshwork). In peripheral epithelial tissues, sodium and water transport is regulated by corticosteroids and the 11beta-hydroxysteroid dehydrogenase (11beta-HSD) isozymes (11beta-HSD1 activating cortisol from cortisone, 11beta-HSD2 inactivating cortisol to cortisone). AIM: To analyse expression of 11beta-HSD in the human eye and investigate its putative role in AH formation. DESIGN: Multipart prospective study, including a randomized controlled clinical trial. METHODS: The expression of 11beta-HSD1 in normal human anterior segments was evaluated by in situ hybridization (ISH). RT-PCR for 11beta-HSDs, glucocorticoid and mineralocorticoid receptors (GR, MR) was performed on human ciliary body tissue. AH cortisol and cortisone concentrations were measured by radioimmunoassay on specimens taken from patients with primary open-angle glaucoma (POAG) and age-matched controls. Randomized, placebo-controlled studies of healthy volunteers and patients with ocular hypertension (OHT, raised IOP but no optic neuropathy) assessed the effect of oral carbenoxolone (CBX, an inhibitor of 11beta-HSD) on IOP. RESULTS: ISH defined expression of 11beta-HSD1 in the ciliary epithelium, while RT-PCR analysis of ciliary body tissue confirmed expression of 11beta-HSD1, with additional GR and MR, but not 11beta-HSD2 expression. In both POAG patients and controls, AH concentrations of cortisol exceeded those of cortisone. The CBX-treated healthy volunteers who demonstrated the largest change in urinary cortisol metabolites, indicative of 11beta-HSD1 inhibition, had the greatest fall in IOP. Patients with OHT showed an overall reduction of IOP by 10% following CBX administration, compared to baseline (p<0.0001). DISCUSSION: CBX lowers IOP in patients with ocular hypertension. Our data suggest that this is mediated through inhibition of 11beta-HSD1 in the ciliary epithelium. Selective and topical inhibitors of 11beta-HSD1 could provide a novel treatment for patients with glaucoma.

Asunto(s)

Carbenoxolona/farmacología , Inhibidores Enzimáticos/uso terapéutico , Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Presión Intraocular/efectos de los fármacos , Hipertensión Ocular/tratamiento farmacológico , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2 , Anciano , Humor Acuoso/química , Humor Acuoso/enzimología , Cortisona/análisis , Método Doble Ciego , Femenino , Humanos , Hidrocortisona/análisis , Masculino , Antagonistas de Receptores de Mineralocorticoides , Hipertensión Ocular/fisiopatología , Estudios Prospectivos , Receptores de Glucocorticoides/antagonistas & inhibidores

RESUMEN

PURPOSE: Carbonic anhydrase enzymes (CAs) are universally involved in many fundamental physiological processes, including acid base regulation and fluid formation and movement. In glaucoma patients, CA inhibitors are very effective in lowering intraocular pressure by reducing the rate of aqueous humour secretion mediated by the CAs in the ciliary epithelium. In this work, we investigated the expression and tissue distribution of two recently discovered CA genes CA9 (CAIX) and CA12 (CAXII) in fetal, neonatal, and adult human eyes with and without glaucoma. METHODS: CAIX and CAXII expression in 16 normal and 10 glaucomatous eyes, and in cultured non-pigmented ciliary epithelial cells (NPE) from normal and glaucoma eye donors was assessed by immunostaining. In addition, northern blot hybridisation was performed to assess expression of CA4, CA9, and CA12 mRNA in cultured NPE cells from normal and glaucoma donors. RESULTS: CAXII was localised primarily to the NPE with its expression prominent during embryonic eye development but which decreased significantly in adults. CAIX expression in the NPE was very low. The epithelium of cornea and lens occasionally expressed both enzymes at low levels during development and in adult eye, and no expression was detected in the retina. The NPE from glaucoma eyes expressed higher levels of CAXII, but not CAIX, in comparison with normal eyes. This expression pattern was retained in cultured NPE cell lines. NPE cells from a glaucoma patient showed a five-fold increase in the CA12 mRNA level with no detectable expression of CA9 mRNA. Also, no expression of the CA4 gene encoding a GPI anchored plasma membrane protein was detected on these northern blots. CONCLUSIONS: Transmembrane CAIX and CAXII enzymes are expressed in the ciliary cells and, thus, may be involved in aqueous humour production. CA12 may be a targeted gene in glaucoma.

Asunto(s)

Anhidrasas Carbónicas/genética , Membrana Celular/enzimología , Glaucoma/genética , Northern Blotting , Anhidrasas Carbónicas/metabolismo , Células Cultivadas , Cuerpo Ciliar/citología , Cuerpo Ciliar/enzimología , Células Epiteliales/enzimología , Regulación Enzimológica de la Expresión Génica , Glaucoma/enzimología , Glaucoma/patología , Humanos , Inmunohistoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

BACKGROUND: Renin-angiotensin system (RAS) components have been identified in human ciliary body and aqueous humour, pointing to a role for the RAS in the regulation of aqueous humour dynamics. Here, the authors examine the functional expression of a RAS and the effects of angiotensin II (AII) on a signal transduction pathway and ion secretion mechanism in cultured human ciliary body non-pigmented epithelium (HNPE). METHODS: RAS expression was examined in cultured HNPE cells using polymerase chain reaction (PCR) analysis. Secretory function was determined using spectrofluorescence imaging microscopy to measure cell calcium (Ca(2+)(I)) and volume responses. Single channel patch clamp techniques were employed to investigate ion channel activity. RESULTS: PCR analysis demonstrated the expression of angiotensinogen and the AT(1b) receptor in HNPE cells. A large conductance potassium (BK) channel (mean 190 (SEM 5.6) pS, n = 22 cells), was observed in plasma membrane patches. This channel was calcium sensitive with channel open probability (Po) increasing with increasing Ca(2+)(I) (K(0.5) 10.79 (0.44) microM Ca(2+), Hill coefficient of 1.04 (0.04)). AII (100 nM) increased the number (N) of active BK channels in HNPE cells and also the probability of channel opening (Po). N.P(o) increased from 0.008 (0.002) to 1.38 (0.4) following the addition of AII (p=0.0064). AII also induced a rapid rise in Ca(2+)(I) from resting values of 112 (14) nM to a peak of 992 (106) nM (p<10(-4)). A simultaneous cell volume reduction of 24.70% (3.34%) (p<10(-4)) occurred during this calcium signal. Losartan (1 microM) significantly blocked the AII induced BK channel activation (p=0.0131), the Ca(2+)(I) response (p<10(-4)), and the AII induced volume effect (p=0.0046). CONCLUSION: It was demonstrated that AII activates a Ca(2+)(I) signalling system which subsequently increases potassium ion channel activity. These effects are accompanied simultaneously by cell volume loss, indicating that AII acts as receptor operated secretagogue in HNPE cells. The ability of an AT(1) receptor antagonist to inhibit these processes may thus offer a new family of pharmaceutical agents to the current armamentarium in the treatment of glaucoma.

Asunto(s)

Cuerpo Ciliar/citología , Epitelio Pigmentado Ocular/citología , Sistema Renina-Angiotensina/fisiología , Angiotensina II/farmacología , Calcio/fisiología , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Cuerpo Ciliar/metabolismo , Electrofisiología , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio , Técnicas de Placa-Clamp , Epitelio Pigmentado Ocular/metabolismo , Canales de Potasio Calcio-Activados/efectos de los fármacos , Canales de Potasio Calcio-Activados/fisiología , Receptores de Angiotensina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología

RESUMEN

Purines regulate intraocular pressure. Adenosine activates Cl(-) channels of nonpigmented ciliary epithelial cells facing the aqueous humor, enhancing secretion. Tamoxifen and ATP synergistically activate Cl(-) channels of pigmented ciliary epithelial (PE) cells facing the stroma, potentially reducing net secretion. The actions of nucleotides alone on Cl(-) channel activity of bovine PE cells were studied by electronic cell sorting, patch clamping, and luciferin/luciferase ATP assay. Cl(-) channels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 microM. UTP triggered ATP release. The second messengers Ca(2+), prostaglandin (PG)E(2), and cAMP activated Cl(-) channels without enhancing effects of 100 microM ATP. Buffering intracellular Ca(2+) activity with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid or blocking PGE(2) formation with indomethacin inhibited ATP-triggered channel activation. The Rp stereoisomer of 8-bromoadenosine 3',5'-cyclic monophosphothioate inhibited protein kinase A activity but mimicked 8-bromoadenosine 3',5'-cyclic monophosphate. We conclude that nucleotides can act at >1 P2Y receptor to trigger a sequential cascade involving Ca(2+), PGE(2), and cAMP. cAMP acts directly on Cl(-) channels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure.

Asunto(s)

Adenosina Trifosfato/fisiología , Calcio/fisiología , Canales de Cloruro/fisiología , Cuerpo Ciliar/fisiología , AMP Cíclico/fisiología , Dinoprostona/fisiología , Animales , Bovinos , Tamaño de la Célula/efectos de los fármacos , Cuerpo Ciliar/citología , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Luciferasas/química , Nucleótidos/farmacología , Técnicas de Placa-Clamp , Pigmentación/fisiología , Transducción de Señal/efectos de los fármacos , Uridina Trifosfato/farmacología

RESUMEN

Here we report the expression, in the human ocular ciliary epithelium and in a human nonpigmented (NPE) ciliary epithelial cell line, of genes usually restricted to cone and rod photoreceptor cells of the retina. By RT-PCR and DNA sequencing we identified the expression of rhodopsin and components linked to its deactivation, including rhodopsin kinase, recoverin, and visual arrestin. We also detected the expression of transducin (T-alpha), phosphodiesterase (PDE-alpha), and cGMP-gated channel alpha-subunits. Cultured NPE cells responded to treatment with phorbol ester by enhancing the expression of rhodopsin mRNA three- to fourfold. Indirect immunofluorescence of the intact ciliary epithelium with monoclonal antibodies (MAbs) against rhodopsin, rhodopsin kinase, and visual arrestin revealed labeling preferentially restricted to the NPE cells. Furthermore, Western blot analysis of whole lysates from the pars plicata region of the human ciliary epithelium with MAbs demonstrated immunochemical cross-reactivity with proteins of molecular mass similar to rhodopsin (36 kDa), rhodopsin kinase (64 to 66 kDa), and arrestin (48-52 kDa) from the human retina. These results provide the first molecular evidence that components of a non-visual phototransduction pathway are expressed in the human ocular NPE ciliary epithelium, which may be linked to circadian entrainment tasks.

Asunto(s)

Cuerpo Ciliar/metabolismo , Células Epiteliales/metabolismo , Proteínas del Ojo , Fototransducción/fisiología , Lipoproteínas , Proteínas del Tejido Nervioso , Animales , Anticuerpos Monoclonales , Arrestina/biosíntesis , Arrestina/genética , Western Blotting , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Bovinos , Línea Celular , Cuerpo Ciliar/citología , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Células Epiteliales/citología , Técnica del Anticuerpo Fluorescente Indirecta , Quinasa 1 del Receptor Acoplado a Proteína-G , Hipocalcina , Humanos , Immunoblotting , Inmunohistoquímica , Canales Iónicos/biosíntesis , Canales Iónicos/genética , Hidrolasas Diéster Fosfóricas/biosíntesis , Hidrolasas Diéster Fosfóricas/genética , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genética , Subunidades de Proteína , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Recoverina , Retina/citología , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rodopsina/biosíntesis , Rodopsina/genética , Análisis de Secuencia de ADN , Transducina/biosíntesis , Transducina/genética

RESUMEN

PURPOSE: To examine the effects of extracellular adenosine 5-triphosphate (ATP) on intracellular pH ([pH](i)) in cultured human non-pigmented ciliary body epithelium (HNPE). METHODS: Intracellular pH was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF. RESULTS: In 5%CO(2)/HCO(3)(-) buffered Ringer's the resting [pH](i) was 7.25 +/- 0.006 (mean +/- SEM). Application of 10 microM ATP significantly decreased [pH](i) to 7.00 +/- 0.007 (P < 10(-5), n = 14). In the presence of 1 mM suramin, a P(2) receptor inhibitor, this process was significantly blocked. This [pH](i) effect required the presence of Cl(-) and was significantly inhibited by 0.1 mM diisothiocyanatostilbene-2-2'-disulfonic acid or acetazolamide (500 microM), indicating the involvement of a Cl(-)/HCO(3)( +) exchange mechanism. This response exhibited little dependence on external Na(+) and remained unaffected by the addition of the Na(+)/H( +) exchanger inhibitor amiloride (1 mM). Clamping intracellular calcium levels by incubation in the cell permeable calcium chelator, the acetoxymethyl ester form of BAPTA (100 microM) in low extracellular calcium solution (pCa9) did not affect the ATP-induced [pH](i) signal. In addition, the vacuolar H(+)-ATPase (V-ATPase) inhibitor, bafilomycin A(1) (1 microM), failed to alter the [pH](i) transient. CONCLUSION: We have demonstrated that extracellular ATP leads to a sustained increase in [H(+)](i) in HNPE cells via a purinergic receptor activated pathway which is independent of the intracellular calcium signaling system. This study demonstrates that the ATP induced [pH]( i) transient is mediated through an upregulation in Cl(-)/HCO( 3)(-) exchange across the plasmamembrane in HNPE cells.

Asunto(s)

Adenosina Trifosfato/farmacología , Antiportadores de Cloruro-Bicarbonato/metabolismo , Cloruros/metabolismo , Cuerpo Ciliar/citología , Macrólidos , Epitelio Pigmentado Ocular/efectos de los fármacos , Antibacterianos/farmacología , Calcio/metabolismo , Células Cultivadas , Fluoresceínas , Colorantes Fluorescentes , Humanos , Concentración de Iones de Hidrógeno , Microscopía Fluorescente , Epitelio Pigmentado Ocular/metabolismo , ATPasas de Translocación de Protón/antagonistas & inhibidores , Receptores Purinérgicos/metabolismo , Espectrometría de Fluorescencia , Suramina/farmacología , Regulación hacia Arriba

RESUMEN

PURPOSE: To determine the effects of extracellular ATP on calcium signaling in cultured human non-pigmented ciliary body epithelium (HNPE). METHODS: Intracellular calcium (Ca(2+)(i)) was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the fluorescent dye Fura-2. RESULTS: Nucleotides caused a transient oscillatory increase in Ca(2+)(i) with a potency order of ATP = UTP > ADP > AMP> alpha,beta-methylene-ATP. Treatment with thapsigargin (100 nM), an inhibitor of endoplasmic Ca(2+)-ATPase pumps, produced a sustained increase in Ca(2+)(i). Subsequent exposure to ATP caused a rapid reduction in Ca(2+)(i) and this effect was reduced by pre-exposure to vanadate and to a lesser extent in sodium free solution. Prolonged exposure to ATP in the presence of thapsigargin caused a transient spike increase in Ca(2+)(i) which was prevented by exposure to low extracellular Ca(2+) (1 nmol/l), verapamil, nifedipine or the microfilament disrupting agent, cytochalasin B. CONCLUSIONS: These results provide evidence for ATP mobilisation of Ca(2+) from intracellular stores via P2Y2 receptor activation in HNPE cells. ATP also primarily activates a vanadate-sensitive Ca(2+ )-ATPase pump, in addition to having a smaller effect on the Na( +)/ Ca(2+) exchanger in terminating the calcium signal. Capacitative calcium entry, possibly via an L-type Ca(2+) channel, is implicated in generating a calcium signal following emptying of intracellular stores and is sensitive to cytoskeleton disruption. ATP can thus regulate a potent intracellular signal for secretion, suggest-ing that purinergic receptors may provide a therapeutic target in glaucoma.

Asunto(s)

Adenosina Trifosfato/farmacología , ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Cuerpo Ciliar/citología , Epitelio Pigmentado Ocular/efectos de los fármacos , Transducción de Señal , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Células Cultivadas , Colorantes Fluorescentes/metabolismo , Fura-2/metabolismo , Humanos , Microscopía Fluorescente , Epitelio Pigmentado Ocular/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2 , Intercambiador de Sodio-Calcio/metabolismo , Espectrometría de Fluorescencia , Tapsigargina/farmacología

RESUMEN

Mutations in the the glaucoma gene GCL1A, also known as trabecular meshwork glucocorticoid response (TIGR) or myocilin (Myoc), have been shown to be associated with juvenile-onset primary open-angle glaucoma. Very little is known about the pattern of expression of the TIGR gene in human ocular tissues. In-situ hybridization experiments demonstrated the localization of TIGR mRNA in cells throughout the iris, ciliary muscle, and the filtering portion of the trabecular meshwork of normal eye donors. The expression of TIGR protein was investigated by Western blot using an epitope-directed antibody to the carboxy terminus region of TIGR. This antibody was able to distinguish a recombinant TIGR fusion protein from a truncated TIGR form containing the naturally occurring Gln(368)-->stop mutation. In tissue extracts from the iris, ciliary body, and trabecular meshwork, the antibody recognized a major protein band of 57-kDa molecular mass. Deglycosylation treatment with PNGase F, NANase II, and O-glycosidase indicated that the 57-kDa protein in these tissues was unglycosylated. In agreement with this observation, in coupled in-vitro transcription/translation systems, the 57-kDa TIGR protein was unaffected by the presence of the processing and glycosylation activities of canine pancreatic microsomal membranes. These findings support the view that the expression of TIGR mRNA in cells of the iris, ciliary body, and trabecular meshwork correlates with that of TIGR protein, and that the 57-kDa TIGR protein was unglycosylated. These results, which are in contrast with earlier reports, raise the possibility that the TIGR protein might be processed into distinct forms in a tissue-specific manner.

Asunto(s)

Cuerpo Ciliar/metabolismo , Proteínas del Ojo/genética , Glicoproteínas/genética , Iris/metabolismo , ARN Mensajero/biosíntesis , Malla Trabecular/metabolismo , Adulto , Anciano , Western Blotting , Proteínas del Citoesqueleto , Cartilla de ADN/química , Electroforesis en Gel de Poliacrilamida , Proteínas del Ojo/biosíntesis , Expresión Génica , Glicoproteínas/biosíntesis , Humanos , Hibridación in Situ , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

Medical therapy of glaucoma commonly aims at slowing aqueous humor formation by the ocular ciliary epithelial bilayer, but underlying mechanisms are poorly understood. The first step in secretion is NaCl uptake from the stroma into the pigmented ciliary epithelial (PE) cell layer by electroneutral transporters. After crossing gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, solute is released into the aqueous humor. Published data have indicated that both paired Na+/H+ and Cl-/HCO3- antiporters and the Na+-K+-2Cl- symporter are involved in net uptake. The molecular identities of the paired antiporters have not been elucidated. We have studied continuously cultured bovine PE cells. Acid-activated 22Na+ uptake was inhibited by cariporide, EIPA (ethyl-isopropyl-amiloride) and amiloride, at concentrations characteristic of the NHE-1 isoform. Videomicroscopy of BCECF-loaded PE cells verified the presence of an EIPA-inhibitable Na+/H+ antiporter. Removing external Cl- also triggered an alkalinization, which was Na+-independent and could be inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Application of hypotonicity followed by return to isotonicity triggered a regulatory volume increase, which was pharmacologically similar to the uptake mechanisms described for intact rabbit ciliary epithelium. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of RNA from the human ciliary body detected expression of the AE2 Cl-/HCO3- exchanger, but not of AE1, cAE3 or bAE3. Immunostaining of bovine PE cells also revealed the presence of AE2 epitope. We conclude that paired NHE-1 Na+/H+ and AE2 Cl-/HCO3- antiporters are important components in the initial step in aqueous humor formation.

Asunto(s)

Proteínas de Transporte de Anión , Antiportadores/metabolismo , Cuerpo Ciliar/fisiología , Pigmentación , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Antiportadores/genética , Bovinos , Línea Celular Transformada , Antiportadores de Cloruro-Bicarbonato , Cuerpo Ciliar/citología , Cuerpo Ciliar/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Microscopía por Video , Modelos Biológicos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas SLC4A , Sodio/metabolismo

RESUMEN

Chloride release from nonpigmented ciliary epithelial (NPE) cells is a final step in forming aqueous humor, and adenosine stimulates Cl(-) transport by these cells. Whole cell patch clamping of cultured human NPE cells indicated that the A(3)-selective agonist 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-be ta-D-ribofuranuronamide (IB-MECA) stimulated currents (I(IB-MECA)) by approximately 90% at +80 mV. Partial replacement of external Cl(-) with aspartate reduced outward currents and shifted the reversal potential (V(rev)) from -23 +/- 2 mV to -0.0 +/- 0.7 mV. Nitrate substitution had little effect. Perfusion with the Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid inhibited the currents. Partial Cl(-) replacement with aspartate and NO(3)(-), and perfusion with NPPB, had similar effects on the swelling-activated whole cell currents (I(Swell)). Partial cyclamate substitution for external Cl(-) inhibited inward and outward currents of both I(IB-MECA) and I(Swell). Both sets of currents also showed outward rectification and inactivation at large depolarizing potentials. The results are consistent with the concept that A(3)-subtype adenosine agonists and swelling activate a common population of Cl(-) channels.

Asunto(s)

Canales de Cloruro/fisiología , Cilios/fisiología , Células Epiteliales/fisiología , Receptores Purinérgicos P1/fisiología , Adenosina/análogos & derivados , Adenosina/farmacología , Células Cultivadas , Canales de Cloruro/efectos de los fármacos , Cilios/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Humanos , Agonistas del Receptor Purinérgico P1 , Receptor de Adenosina A3

RESUMEN

Purines alter aqueous humour secretion by the bilayered ciliary epithelium. Adenosine but not ATP shrinks non-pigmented ciliary epithelial (NPE) cells by activating Cl- channels. We now report effects of ATP on pigmented ciliary epithelial (PE) cells. Cultured bovine PE cells were studied volumetrically by electronic cell sorting. ATP and tamoxifen acted synergistically to shrink PE cells. Neither ATP nor tamoxifen alone had a consistent effect on cell volume. The tamoxifen, ATP-activated shrinkage required Cl- release since the response was blocked by removing Cl- and was inhibited by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate and 4,4'-diisothiocyano-2,2'-disulfonic acid. The modulating effect of tamoxifen could have reflected many actions of tamoxifen. Our data do not support the suggestion that tamoxifen inhibits protein kinase C (PKC) or calcium-calmodulin, or that it acts on histamine or carbachol receptors. The shrinkage produced by ATP and tamoxifen was blocked by 17beta-oestradiol, but not 17alpha-oestradiol. The cooperative interaction between tamoxifen and ATP was not mediated by an enhanced rise in [Ca2+]i. The results indicate that tamoxifen interacts synergistically with ATP to activate Cl- release by the PE cells.

Asunto(s)

Adenosina Trifosfato/farmacología , Canales de Cloruro/metabolismo , Cuerpo Ciliar/efectos de los fármacos , Tamoxifeno/farmacología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/análogos & derivados , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Adenosina/farmacología , Animales , Calcio/metabolismo , Bovinos , Línea Celular , Tamaño de la Célula/efectos de los fármacos , Canales de Cloruro/antagonistas & inhibidores , Cloruros/metabolismo , Cuerpo Ciliar/metabolismo , Estradiol/farmacología , Cinética , Nitrobenzoatos/farmacología , Proteína Quinasa C/metabolismo , Receptores de Estrógenos/metabolismo , Receptores Histamínicos/metabolismo , Receptores Muscarínicos/metabolismo

RESUMEN

1. We used whole-cell patch-clamp recording techniques and noise analysis of whole-cell current to investigate the properties of hyposmotic shock (HOS)-activated Cl- channels in SV40-transformed rabbit non-pigmented ciliary epithelial (NPCE) cells. 2. Under conditions designed to isolate Cl- currents, exposure of cells to hyposmotic external solution reversibly increased the whole-cell conductance. 3. The whole-cell current activated with a slow time course (> 15 min), exhibited outward rectification and was Cl- selective. 4. The disulphonic stilbene derivatives 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS, 0. 5 mM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS, 0.5 mM) produced a voltage-sensitive block of HOS-activated Cl- current at depolarized potentials, whereas niflumic acid produced a voltage-independent block of the current. 5. Under Ca2+-free conditions, HOS stimulation still reversibly activated the Cl- current, but the amplitude of current was reduced and the time course of current activation was slower compared with control (P < 0. 05). 6. The non-specific kinase inhibitor H-7 (100 microM), upregulated HOS-activated Cl- current amplitude in all cells tested (P < 0.05). 7. Noise analysis of whole-cell Cl- current indicated that cell swelling activated a high density of small conductance Cl- channels (< 1 pS). 8. We conclude that HOS primarily activates a high density of volume-sensitive small conductance Cl- channels in rabbit NPCE cells, and that Ca2+ and phosphorylation are involved in channel regulation.

Asunto(s)

Canales de Cloruro/metabolismo , Cuerpo Ciliar/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Calcio/metabolismo , Línea Celular , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/efectos de los fármacos , Cuerpo Ciliar/citología , Cuerpo Ciliar/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Ácido Niflúmico/farmacología , Presión Osmótica , Técnicas de Placa-Clamp , Fosforilación , Inhibidores de Proteínas Quinasas , Conejos , Sistemas de Mensajero Secundario , Estilbenos/farmacología

RESUMEN

PURPOSE: It has been proposed that pI(Cln), a highly acidic protein, is a candidate gene product related to the swelling-activated chloride (Cl-) channel Icl.swell in mammalian cells. However, no consensus has been reached as to whether this relationship is direct or indirect. Recently the cDNA for pI(Cln) was isolated from human ciliary epithelial cells. To learn more about the structure-function of pI(Cln) we attempted to: i) overexpress pI(Cln) as a fusion protein in bacteria; ii) carry out its purification; iii) generate polyclonal antibodies to study its expression and cellular localization in the ciliary epithelial cells; and iv) determine whether cell-swelling affects pI(Cln) expression in ciliary epithelial cells. METHODS: The open reading frame (ORF) of human pI(Cln) was subcloned in the pET-20b(+) plasmid and established as a recombinant vector in E. coli BL21(DE3)pLysS cells. Upon induction with iso-propyl-beta-thio-galactopyranoside (IPTG), pI(Cln) was isolated as a His-Tag fusion protein and purified to homogeneity. Polyclonal antibodies were raised in rabbits after immunization with pI(Cln) purified protein, and its expression and cellular distribution in ciliary epithelial cells determined by Western blot, immunoprecipitation and indirect immunofluorescence respectively. Cell-swelling effect on ciliary epithelial cells was carried out upon treatment of cultured cells with hypotonic solution up to 60 min and pI(Cln) expression measured by Northern and Western blot analysis. RESULTS: By Western blot analysis or immunoprecipitation, pI(Cln) antisera recognized a main band of 37-kDa in total cell extracts from ciliary body or metabolically labeled ciliary epithelial cells. By indirect immunofluorescence, pI(Cln) antibodies stained the cytoplasm of NPE in the intact tissue, and the perinuclear region of cultured ciliary epithelial cells. When subjected to hypotonic treatment, NPE cells did not induce translocation of pI(Cln) protein from the cytoplasm into the plasma membrane, nor changes in pI(Cln) expression at the protein level, but did down regulate up to 30% the level of pI(Cln) mRNA in continued hypotonic treatment. CONCLUSIONS: These observations indicate that, contrary to previous suggestions, the pI(Cln) protein is not likely to be in contact with the plasma membrane of ciliary epithelial cells, and its influence on Cl- -channel activity is more likely to be expressed indirectly, (i.e. through cytoskeletal restructuring).

Asunto(s)

Canales de Cloruro/metabolismo , Cuerpo Ciliar/efectos de los fármacos , Cuerpo Ciliar/metabolismo , Soluciones Hipotónicas/farmacología , Canales Iónicos , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Células Cultivadas , Canales de Cloruro/genética , Canales de Cloruro/inmunología , Cuerpo Ciliar/citología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Sueros Inmunes/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/metabolismo , Distribución Tisular/fisiología

RESUMEN

Here we report the isolation, characterization and chromosome localization of a subtracted cDNA (CBS-1) isolated from the human ocular ciliary body which encodes a novel protein. As is deduced from the nucleotide sequence of the cDNA, CBS-1 contains an open reading frame consisting of 182 amino acids, with a molecular weight of 19.5kDa. CBS-1 shares significant nucleotide and amino acid sequence identities (residues 51 to 182) with a hypothetical 15.5kDa protein in the ANSA-GAP intergenic region (yeaA) of Escherichia coli, and the carboxyl terminal region of pilB, a transcription factor involved in the regulation of expression of pili, from Neisseria gonorrhoeae. Interestingly, CBS-1 also shares significant identity with the carboxyl terminus of the peptide-methionine sulfoxide reductase (MsrA), a repair enzyme, from Helicobacter pylori and Streptococcus pneumoniae. However, the amino terminal of CBS-1 (residues 23 to 43), which lacks homology to the amino terminal region of gonococcal pilB or pneumococcal MsrA, exhibits significant identity in a stretch of 20 amino acids, with glycine-rich proteins. By Northern blot, CBS-1, hybridized to a 0.6 to 0.7kb transcript in size, is expressed ubiquitously in many tissues, but most abundantly in the retina and ocular ciliary body, skeletal muscle and heart. An epitope-directed antibody to an amino acid sequence at the carboxyl terminus of CBS-1 recognized a main protein of 19.5kDa in ocular ciliary body extracts, and a 23kDa protein in total extracts from E. coli MC1061 cells, which expresses high levels of MsrA. The CBS-1 gene was mapped to human chromosome 10p12 between markers WI-8535 and WI-4724, and is tightly linked to the two STRP markers of D10S1789 and D10S550. We suggest that the CBS-1 gene encodes a mammalian transcription factor related to the bacterial pilB and certain bacterial MsrA homologues.

Asunto(s)

Proteínas Bacterianas/genética , Cromosomas Humanos Par 10 , Oxidorreductasas/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cuerpo Ciliar/enzimología , ADN Complementario , Técnica del Anticuerpo Fluorescente Indirecta , Helicobacter pylori/enzimología , Humanos , Metionina Sulfóxido Reductasas , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Streptococcus pneumoniae/enzimología

RESUMEN

The expression of the natriuretic peptide system in the human ocular ciliary epithelium (CE) and in cultured nonpigmented (NPE) ciliary epithelial cells was examined. By RT-PCR and DNA sequencing, we demonstrated that the CE and NPE cells express mRNA for (i) ANP; (ii) BNP; (iii) NPR-A, NPR-B, and NPR-C receptors; and (iv) the neutral endopeptidase 24.11. Radioimmunoassay results indicate that BNP is secreted by cultured NPE cells at much higher levels than ANP. NPR-A and NPR-B receptors elicited a cGMP response to ANP, BNP, and CNP, in a rank order of potency (CNP >> ANP >/= BNP), indicative that the NPR-B receptor is predominant in NPE cells. A71915, an inhibitor of NPR-A activity, attenuated (65-75%) cGMP response to ANP and BNP, but not to CNP. C-ANP4-23 elicited an inhibitory effect (30-37%) on basal levels of cAMP in NPE cells and on forskolin NPE-treated cells, indicative that the NPR-C receptor is functional in these cells. PMA induced, in NPE cells, a long-term downregulation (75-85%) of NPR-C receptor mRNA, but not of NPR-A or NPR-B receptor mRNA, suggesting a differential regulation of NPR-C receptor mRNA via activation of PKC. Collectively, our data provide molecular evidence that all the components of the natriuretic peptide system with the exception of CNP are coexpressed in the ocular NPE ciliary epithelial cells, where they may function as local autocrine/paracrine modulators to influence eye pressure.

Asunto(s)

Factor Natriurético Atrial/metabolismo , Cilios/metabolismo , Ojo/metabolismo , Péptido Natriurético Encefálico/metabolismo , Factor Natriurético Atrial/genética , Secuencia de Bases , Línea Celular , Preescolar , Medios de Cultivo Condicionados , Cartilla de ADN , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/metabolismo , Ojo/citología , Humanos , Péptido Natriurético Encefálico/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

The generation of expression and subtractive libraries from the ocular ciliary body and cultured ciliary epithelial cells has been instrumental in the cloning, identification and characterization of many genes which, overall reflect a representative profile of transcripts expressed in ciliary nonpigmented, ciliary pigmented and ciliary muscle cells. The cell-specific expression of some of these genes (i.e. a neurotrophic factor, a gene associated with juvenile open glaucoma, and a visual component) reveal a degree of cell differentiation with a diversity of functions and properties higher than previously thought. The protection from light-induced oxidative reactions, free radicals and detoxification, may be partially attributed to the high level of expression in the ciliary epithelium of antioxidative enzymes (i.e., glutathione S-transferase, glutathione peroxidases, selenoprotein-P). The expression of genes encoding plasma proteins (i.e., complement component C4, alpha2-macroglobulin, apolipoprotein D) is in contrast with the view that plasma proteins in aqueous humor are synthesized outside the eye (i.e., liver). The identification of neuropeptide-processing enzymes (i.e., prohormone convertases, carboxypeptidase E, peptidyl-glycine-alpha-amidating monoxigenase), neuropeptides (i.e., secretogranin II, neurotensin) and regulatory peptides (i.e., atrial natriuretic peptide and angiotensinogen) with hypertensive and hypotensive activities provide the molecular basis to support the view that the ciliary epithelium is a neuroepithelium with neuroendocrine functions. We propose a working model to demonstrate that aqueous humor and intraocular pressure are under neuroendocrine control through regulatory peptides synthesized and released by the ciliary epithelium and targeting the peptide producing cells at the inflow system by an autocrine mechanism and/or cells at the outflow system (i.e., trabecular meshwork cells) by a paracrine mechanism. Finally, we hypothesize that these mechanisms could be entrained in the light-dark cycle following the circadian rhythm of aqueous humor and intraocular pressure.

Asunto(s)

Cuerpo Ciliar/fisiología , Expresión Génica/fisiología , Cuerpo Ciliar/metabolismo , ADN Complementario/genética , Epitelio/fisiología , Biblioteca de Genes , Humanos , Sistemas Neurosecretores/fisiología

RESUMEN

Adenosine stimulates Cl- channels of the nonpigmented (NPE) cells of the ciliary epithelium. We sought to identify the specific adenosine receptors mediating this action. Cl- channel activity in immortalized human (HCE) NPE cells was determined by monitoring cell volume in isotonic suspensions with the cationic ionophore gramicidin present. The A3-selective agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) triggered shrinkage (apparent Kd = 55 +/- 10 nM). A3-selective antagonists blocked IB-MECA-triggered shrinkage, and A3-antagonists (MRS-1097, MRS-1191, and MRS-1523) also abolished shrinkage produced by 10 microM adenosine when all four known receptor subtypes are occupied. The A1-selective agonist N6-cyclopentyladenosine exerted a small effect at 100 nM but not at higher or lower concentrations. The A2A agonist CGS-21680 triggered shrinkage only at high concentration (3 microM), an effect blocked by MRS-1191. IB-MECA increased intracellular Ca2+ in HCE cells and also stimulated short-circuit current across rabbit ciliary epithelium. A3 message was detected in both HCE cells and rabbit ciliary processes using RT-PCR. We conclude that human HCE cells and rabbit ciliary processes possess A3 receptors and that adenosine can activate Cl- channels in NPE cells by stimulating these A3 receptors.

Asunto(s)

Canales de Cloruro/metabolismo , Cuerpo Ciliar/metabolismo , Células Epiteliales/metabolismo , Receptores Purinérgicos P1/fisiología , Adenosina/análogos & derivados , Adenosina/farmacología , Adulto , Animales , Cuerpo Ciliar/citología , Cuerpo Ciliar/efectos de los fármacos , Cuerpo Ciliar/fisiología , Conductividad Eléctrica , Humanos , Iris/efectos de los fármacos , Iris/fisiología , Masculino , Agonistas del Receptor Purinérgico P1 , ARN Mensajero/metabolismo , Conejos , Receptores Purinérgicos P1/metabolismo

RESUMEN

Here we report the coexpression of the neuropeptide galanin and GalR-1 galanin receptors in the human ciliary epithelium, a bilayer of neuroepithelial cells [nonpigmented (NPE) and pigmented (PE)] with neuroendocrine functions, and in a cell line (ODM-2) derived from the NPE cells. Stimulation of ODM-2 cells with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or forskolin resulted in an up-regulation (two- to threefold) of galanin mRNA expression. Procaterol, a selective beta2-adrenergic agonist, and the catecholamine isoproterenol exerted a long-term down-regulation on galanin mRNA expression when added alone or in combination with PMA or forskolin. These actions exerted by procaterol or isoproterenol were abolished in the presence of ICI 118,551, a selective beta2-adrenergic antagonist. A radioimmunoassay for galanin peptide indicated that galanin or a galanin-like product is present in the human aqueous humor fluid and is accumulated with time in the culture medium of ODM-2 cells. It is interesting that norepinephrine, which exhibited no effect on galanin mRNA expression, induced a down-regulation in the level of galanin or galanin-like product accumulated in the medium of cultured ODM-2 cells to levels even lower than those induced by beta2-adrenergic agonists. This effect is best explained by the concomitant up-regulation (four- to fivefold) of GalR-1 galanin receptor transcripts induced through the activation of alpha2-adrenergic receptors. These findings support the view that pathways elicited by the activation of alpha2- and beta2-adrenergic receptors influence the expression of galanin and GalR-1 galanin receptors in ciliary epithelial cells.

Asunto(s)

Cuerpo Ciliar/metabolismo , Galanina/metabolismo , Receptores de Neuropéptido/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Humor Acuoso/metabolismo , Línea Celular , Cuerpo Ciliar/citología , Cuerpo Ciliar/efectos de los fármacos , Clonidina/análogos & derivados , Clonidina/farmacología , Colforsina/farmacología , Medio de Cultivo Libre de Suero/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Galanina/genética , Humanos , Norepinefrina/farmacología , Precursores de Proteínas/genética , ARN Mensajero/metabolismo , Receptores Adrenérgicos alfa/fisiología , Receptores Adrenérgicos beta/fisiología , Receptores de Galanina , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

PURPOSE: The authors recently established a link between swelling-activated myo-inositol efflux and chloride movement via anion channels in cultured bovine lens epithelial cells (BLECs). To further define this pathway, the relationship between cell volume, myo-inositol movement and mRNA expression of pI(Cln), a proposed chloride channel regulatory protein was investigated. METHODS: To demonstrate the effect of cell volume changes on pIcln transcription, BLECs were exposed to either hypertonic or hypotonic medium conditions. For rapid cellular shrinkage, BLECs were maintained at confluence in physiologic medium (257+/-2 mosm) then transferred to sodium hypertonic medium (473+/-6 mosm) or raffinose hypertonic medium (452+/-2 mosm). For rapid cellular swelling, cells were switched from sodium hypertonic medium to physiologic medium+/-tamoxifen. The expression of pI(Cln) mRNA was determined by Northern blot analysis. RESULTS: Upon cell volume reduction (increasing intracellular osmolality), BLECs upregulate the expression of pI(Cln) mRNA. Contrastly, when cell volume rapidly increases (decreasing intracellular osmolality), BLECs moderately downregulate pIcln mRNA, with expression levels reaching near physiologic control by 24 h. Blockage of swelling-activated chloride movement and osmolyte efflux with either tamoxifen or niflumic acid enhances the downregulation of pIcln mRNA expression. CONCLUSIONS: In cultured BLECs, pI(Cln) transcriptional regulation appears to be responsive to cell volume fluctuations. These data suggest a converse relationship exists between pIcln mRNA expression and changes in cell volume.

Asunto(s)

Tamaño de la Célula/fisiología , Canales de Cloruro/metabolismo , Células Epiteliales/metabolismo , Canales Iónicos , Cristalino/metabolismo , ARN Mensajero/metabolismo , Animales , Northern Blotting , Bovinos , Células Cultivadas , Canales de Cloruro/genética , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Soluciones Hipertónicas , Inositol/metabolismo , Cristalino/citología , Ácido Niflúmico/farmacología , Tamoxifeno/farmacología