Ascorbate-stimulated active Na+ transport in rabbit ciliary epithelium

In a physiological medium (134 mM Na+ concentration), unidirectional blood-to-aqueous and aqueous-to-blood Na+ fluxes across the isolated rabbit ciliary epithelium are large, rendering the detection of a net transport difficult. At 134 mM an active component for Na+ may be obscured by diffusional fluxes and a bidirectional Na(+)-Cl- cotransport. Considering that the active transport saturates at about 30 mM, experiments were performed at this reduced Na+ concentration to minimize the influence of diffusional pathways. A net blood-to-aqueous Na+ flux that ranged from 0.25 to 0.81 mu eq/hr was obtained. Addition of ascorbic acid to the aqueous side under this condition increased the blood-to-aqueous flux with little effect on the flux in the opposite direction. Ouabain inhibited both the Na+ and ascorbate-stimulated Na+ transport. The increase in blood-to-aqueous Na+ flux by ascorbate was also observed in tissues bathed with [Na+] closer to physiological levels (100 mM). These results indicate that the rabbit ciliary epithelium transports Na+ into the posterior chamber. Since aqueous ascorbate stimulates Na+ transport, it may be implicated in both Na+ movement and aqueous humor secretion. However, the rate of Na+ transport can only account for a small fraction of total aqueous humor production.     

Active transport of ascorbate across the isolated rabbit ciliary epithelium

The transepithelial transport of ascorbate across the isolated rabbit ciliary epithelium (CE) was investigated. Unidirectional 14C-ascorbate fluxes were measured in the presence of equal concentrations of ascorbate on both sides of the tissue within the range of 0.025 to 1 mM. The blood to aqueous (Bl----Aq) flux increased from 6 to 95 nmoles/hr and showed nonlinearity and saturation. The aqueous to blood (Aq----Bl) flux increased, for the same range, from 0.5 to 23 nmoles/hr in a linear fashion. The permeability calculated from the Aq----Bl flux was similar to the CE permeability for mannitol suggesting that the Aq----Bl flux is mainly paracellular. The flux ratio Bl----Aq/Aq----Bl was between 4 to 12. Anoxia, ouabain and low Na+ in the media inhibited the Bl----Aq flux indicating that the transport system requires energy and a Na+ gradient. 3-O-methyl-D-glucose, D-isoascorbic acid and phlorizin also inhibited the Bl----Aq flux, suggesting that ascorbate and glucose may share a common carrier mechanism. Although the isolated CE preparation was clearly capable of flux separation and active transport, the rate of ascorbate transport measured in vitro is insufficient to maintain the aqueous ascorbate concentration observed in vivo.     

P2 purinergic receptor-coupled signaling in the rabbit ciliary body epithelium

PURPOSE: To identify and characterize P2 purinergic receptors and their signaling pathways in the epithelial cells of the rabbit ciliary body. METHODS: Real-time fluorescence ratio imaging of the intact fura-2-loaded nonpigmented ciliary body epithelial (NPE) cells of rabbit were used to record changes in the intracellular free calcium concentration ([Ca(2+)](i)), in response to a number of purinergic agonists and antagonists. The effects of some of these drugs on the inositol phosphate (IP) levels in ciliary processes were also examined. RESULTS: Adenosine diphosphate (ADP), adenosine triphosphate (ATP), and uridine triphosphate (UTP) dose dependently increased the [Ca(2+)](i) and IP levels. The [Ca(2+)](i) increases induced by ADP and UTP were distinguishable, both kinetically and pharmacologically. The effect of ADP on [Ca(2+)](i) was mimicked by a number of P2Y(1)-selective agonists, and was blocked by three P2Y(1)-receptor-specific antagonists. The [Ca(2+)](i) increases elicited by ADP (or its analogs) and UTP were additive. CONCLUSIONS: Rabbit ciliary body epithelium possesses both P2Y(1) and P2Y(2) metabotropic purinergic receptor subtypes, which differentially use the IP(3)/Ca(2+) second-messenger pathway.     

Characterization of human and rabbit pigmented and nonpigmented ciliary body epithelium

Nonpigmented epithelial (NPE) and pigmented epithelial (PE) cells were carefully dissected from both human and rabbit ciliary processes and have been maintained in vitro and partially characterized by morphology and immunocytochemical techniques using polyclonal and monoclonal antibodies against S-100 proteins, collagen type I and type III. The tissue distribution of these proteins was studied in formalin fixed deparaffinized tissue sections of human and rabbit eyes by immunoperoxidase staining techniques. Both NPE and PE cell lines from human and rabbit showed hexagonal morphology by light microscopy; distinct granules containing pigment could be visualized in the PE cell lines, but not in the NPE cells. Antibodies against S-100 proteins stained NPE layer intensely and PE layer slightly in the human tissue sections. The staining was less intense in rabbit tissues than human tissues. The ciliary body stroma was positive for collagen type III and negative for collagen type I or S-100.     

Nonpigmented cells of the rabbit ciliary body epithelium. Tissue culture and voltage-gated currents.

The aqueous humor of the eye is thought to be secreted by the epithelium of the ciliary body. This epithelium has been difficult to study, in part because of its complicated morphology. The authors attempted to circumvent this difficulty by growing the epithelial cells in tissue culture. A procedure is described for producing pure primary cultures of rabbit nonpigmented ciliary body epithelial cells. This procedure was used with whole-cell patch-clamp recording to characterize voltage-activated currents in the nonpigmented cells. These experiments show that most nonpigmented cells contain two kinds of currents: a rapidly activating and inactivating inward current, carried by Na+ and blocked by tetrodotoxin (TTX), and a more slowly activating and inactivating outward current, blocked by tetraethylammonium (TEA+), Ba2+, and 4-aminopyridine (4-AP) and presumably carried by K+. Both of these currents have been observed in freshly dissociated cells and in cultures up to 7 days old. The voltage-gated currents in ciliary body epithelial cells are remarkably similar to those of neurons and raise the possibility that these epithelial cells are capable of spike propagation.     

Stimulation of active sodium-potassium transport by hydrogen peroxide in cultured rabbit nonpigmented ciliary epithelium.

PURPOSE: Studies were conducted to examine the effect of hydrogen peroxide on active sodium-potassium transport in a cell line derived from nonpigmented ciliary epithelium of the rabbit eye. METHODS: Studies were carried out using a rabbit nonpigmented ciliary epithelium cell line. 86Rb uptake by intact cells was measured in the presence or absence of ouabain. The ouabain-sensitive potassium (86Rb) uptake rate was used as an index of the rate of active sodium-potassium transport. Cell sodium content was measured by atomic absorption spectrophotometry. Na,K-ATPase activity was determined by measuring ATP hydrolysis in the presence or absence of ouabain, using membrane material isolated by centrifugation of cell homogenates. RESULTS: Ouabain-sensitive potassium (86Rb) uptake rate measured in cells that had been preincubated with 200microM hydrogen peroxide for either 30 min or 60 min was increased to 196% and 181% of the control uptake rate, respectively. Lesser concentrations of hydrogen peroxide caused lesser degrees of stimulation. 200microM hydrogen peroxide caused an increase of cell sodium content. Such a change of cell sodium content is likely to be responsible, at least in part, for the observed stimulation of active sodium-potassium transport. However, the response may also be partly dependent on activation of a protein kinase since the serine/threonine protein kinase inhibitors staurosporine (1microM) and H-89 (20microM) were both found to prevent the stimulatory effect of 200microM hydrogen peroxide on ouabain-sensitive potassium (86Rb) uptake. Interestingly, neither H-89 nor staurosporine prevented the elevation of sodium content in cells that received 200microM hydrogen peroxide. CONCLUSIONS: Taken together, these findings suggest a low concentration of hydrogen peroxide causes increased sodium entry into the cell and also activates a protein kinase-dependent mechanism for sodium pump stimulation. The protein kinase-dependent mechanism does not appear to be triggered by an increased rate of sodium entry since staurosporine did not prevent the stimulation of ouabain-sensitive potassium (86Rb) uptake elicited by an increase in sodium permeability caused by amphotericin B.     

Spontaneous fluid transport across isolated rabbit and bovine ciliary body preparations

PURPOSE: To quantify spontaneous fluid transport across the isolated ciliary bodies of rabbit and bovine and to determine their osmotic permeabilities. METHODS: A complete annulus of ciliary body was mounted in a custom-designed chamber appropriate for detecting net fluid movement across the in vitro preparation. RESULTS: A net fluid flow in the blood-to-aqueous direction was measured. It was generally observed that tissue freshness is a critical parameter for detection of such flow. The spontaneous, baseline fluid transport rate lasted, on average, approximately 4 hours. This flow solely reflects the secretory activity of the isolated ciliary epithelium, since the in vitro arrangement precludes contributions from ultrafiltration. Both the isolated rabbit and bovine ciliary body epithelia transported fluid in the absence of an external osmotic or pressure gradient. After corrections for area and possible collapse of the processes, a total flux rate of approximately 23 microL/hour or 13% of the in vivo flow in rabbit was estimated. This value agrees with predictions of ionic fluxes and short-circuit current measurements, which are also obtained in vitro. The fluid flow is bicarbonate dependent in rabbit and chloride dependent in bovine, consistent with ionic transport mechanisms described in these species. Ouabain inhibited the fluid flow across both species, indicating dependence on active ionic transport. Irrespective of the spontaneous fluid transport, a flow elicited by an osmotic gradient allowed for a calculation of the osmotic permeability coefficient (P(f); approximately 10(-3) cm/s) in line with reports in other epithelia. In addition, mannitol permeability (5.6 x 10(-6) cm/sec) was similar to that measured in "tight" epithelia, as determined by measurements of radiolabeled fluxes of the sugar across rabbit ciliary bodies mounted in the chambers used for the present fluid transport study. CONCLUSIONS: This work demonstrates that isolated ciliary epithelial preparations transport fluid in the blood-to-aqueous direction. The present observations suggest that mounting arrangements for measuring volumetric fluid flow across the ciliary epithelium is suitable for future studies directed toward the pharmacological control of secretion.     

Adrenergic effects on the isolated rabbit ciliary epithelium.

Norepinephrine, epinephrine, phenylephrine and isoproterenol have no effect on fluid secretion across the isolated rabbit ciliary epithelium, but all four drugs increase the hydraulic conductivity, or passive water permeability. By using a series of adrenergic antagonists (two α- and two β-compounds) it was possible to examine the type of receptor present in the ciliary epithelium which was responsible for the increased leakiness of the membrane. The results indicate that both α- and β-receptors exist in the ciliary epithelium and influence membrane permeability. From a consideration of the various influencing factors on intraocular pressure, the action of the drugs on lowering intraocular pressure is strongly suggested be on true outflow facility and pseudofacility.     

Muscarinic cholinergic inhibition of adenylate cyclase in the rabbit iris-ciliary body and ciliary epithelium

The effects of cholinergic agents on hormone-stimulated cyclic AMP (cAMP) accumulation were investigated in iris-ciliary body segments, excised ciliary processes, and isolated ciliary epithelium from albino rabbit eyes. In all three tissue preparations, the cholinergic agonist carbamylcholine markedly inhibited the stimulation of cAMP biosynthesis by vasoactive intestinal peptide VIP--a potent activator of nonpigmented ciliary epithelial adenylate cyclase. Carbamylcholine also attenuated cAMP increases mediated by isoproterenol, prostaglandin E2, and forskolin. The effects of carbamylcholine on VIP-induced cAMP synthesis were concentration dependent (EC50 = 23 nM), mimicked by selective muscarinic cholinergic agonists (oxotremorine, pilocarpine), and antagonized by atropine. Carbamylcholine- and clonidine-mediated inhibition of VIP-stimulated cAMP accumulation in ciliary processes were nonadditive, indicating that inhibitory muscarinic and alpha 2-adrenergic receptors coexist on VIP-responsive target cells. These findings suggest that the cholinergic system may have a direct role in modulation of ciliary epithelial adenylate cyclase and aqueous humor secretion.     

An electrophysiologic study of rabbit ciliary epithelium

Microelectrode recordings from cells in rabbit ciliary epithelium have been made in vitro. Ionophoresis of Lucifer Yellow dye from microelectrodes during measurements of potential confirmed that the recordings were intracellular. Dye passed from the impaled cells into adjacent cells in both the nonpigmented and pigmented layers of the epithelium. Electrical coupling between epithelial cells also was observed. The mean (+/- SD) values of the potential measured across the basolateral membranes of the nonpigmented cells was -65 +/- -15 mV (n = 77); the mean value of the input resistance at this intracellular recording site was 37 +/- 28 M omega (n = 17). The membrane potential was reduced by raising the concentration of extracellular potassium but unaffected by changes in the concentrations of sodium, chloride, or bicarbonate ions. After a period of deprivation of extracellular potassium, the cells hyperpolarized without a measurable change in membrane resistance when potassium was restored to the bathing solution; this transient response to potassium was abolished by preincubation with ouabain or by bathing the epithelium in a solution lacking sodium. It was concluded that the ciliary epithelial cells are permeable to potassium but exhibit only a low permeability to sodium, chloride, or bicarbonate ions; that the cells possess an electrogenic Na/K pump; and finally, that all of the cells in the epithelium function as a syncytium.     

Metabolism of arachidonic acid by isolated rabbit ciliary epithelium.

We examined the ability of rabbit ciliary epithelium to metabolize arachidonic acid in vitro. The epithelium was homogenized and incubated with 14C-labeled arachidonic acid. 14C-labeled metabolites were extracted and then separated by thin layer chromatography. The range of arachidonic acid metabolites synthesized by ciliary epithelium was compared to the metabolites generated by rabbit iris-ciliary body. Ciliary epithelium produced substantial amounts of arachidonic acid metabolites that comigrated with 5-HETE and 12-HETE. Authenticity of the 12-HETE produced by ciliary epithelium was confirmed by gas chromatography/mass spectrometry. The ciliary epithelium generated only small amounts of the cyclooxygenase products, PGF2 alpha, PGE2, PGD2 and 6k-PGF1 alpha. In contrast, the iris-ciliary body produced large amounts of cyclooxygenase products such as PGF2 alpha and PGD2. The ability of the ciliary epithelium to generate 12-HETE is noteworthy since 12(R)-HETE is known to be capable of lowering intraocular pressure.     

丝裂霉素C对兔眼睫状体无色素上皮细胞的毒性作用

目的 观察兔眼巩膜咬切术中应用０．２ｍｇ／ｍｌ丝裂霉素Ｃ（ＭＭＣ）后,睫状体无色素上皮细胞的病理形态学的改变,探讨ＭＭＣ对睫状体的毒性作用。方法 在兔眼巩膜咬切术中用含０．２ｍｇ／ｍｌ的ＭＭＣ０．１ｍｌ的海绵块,置于Ｔｅｎｏｎ囊和巩膜之间共５分钟,并用２０ｍｌ生理盐水冲洗,术后第７天及２８天,采用光镜观察睫状体,透射电镜观察其无色素上皮细胞。结果 术后第７天睫状体无色素上皮细胞水肿,线粒体丰富、肿