Ciliary Body Epithelial Cells Research Articles

Dihydropyridine-sensitive Ca 2+ Spikes and Ca 2+ Currents in Rabbit Ciliary Body Epithelial Cells

Intracellular microelectrode and whole-cell patch-clamp recordings were used to investigate a Ba 2+-induced regenerative depolarization and its underlying Ba 2+ current in the ciliary body epithelial cells of the rabbit eye. Exposure of these epithelial cells to 4-10 mmol l -1 Ba 2+ depolarized the membrane potential and caused the generation of one or more spikes, before the membrane potential reached a steady-state level. The spikes, but not the slow phase of depolarization, could be blocked with Co 2+ (2 mmol l -1), Gd 3+ (25 μmoll -1), La 3+ (20 μmol l -1 ), Cd 2+ (10 μmol l -1), verapamil (30 μmol l -) and nifedipine (1 μmol l -1). Tetrodotoxin at 100 nmol l -1 had no effect. In the absence of Na +, but in the presence of external Ba 2+, step depolarization of the membrane potential activated an inward current that could be blocked with Co 2+ (2 mmol l -1), Cd 2+ (10 μmol l -1) and nifedipine (1 mol l -1), but not with Ni 2+ (50 μmol l -1) or ω-conotoxin (1-10 μmol). This inward current could be enhanced with the dihydropyridine agonist (±)BAY K 8644 (1 μmol l -1). The inactivation characteristics of the inward current (v 1/2 = - 38·7 mV, k = 12·6 mV) is most like that seen in neurons. These findings indicate that the epithelial cells of the ciliary body possess dihydropyridine-sensitive, voltage-activated Ca 2+ channels.

Tissue Culture of Rabbit Ciliary Body Epithelial Cells on Permeable Supports

The aqueous humor is produced by the epithelium of the ciliary body, a complex structure encircling the anterior segment of the eye. Aqueous humor production occurs by active transport, but the mechanism of this process is not understood. To produce a preparation in which active transport can be investigated, we have attempted to prepare cultures suitable for measurements of ion and water flux. We have grown rabbit ciliary body epithelial cells on permeable supports, coated with several extracellular matrix proteins. We then examined the ability of these proteins to promote the growth of a differentiated layer of epithelial cells. Non-pigmented and pigmented cells formed sheets of contiguous cells when grown on a variety of support media. The most successful substrate was a permeable support produced by Falcon/Becton Dickinson coated with a mixture of collagen IV, laminin and heparan sulfate. Under these conditions, cultures could be maintained for several months, but pigmented cell cultures did not develop a measurable transepithelial resistance (TER), and the TER of non-pigmented cell cultures was typically only 20-30 Ω cm 2. Much higher TERs exceeding 200 Ω cm 2 could be measured from non-pigmented cell cultures 3-5 days after plating, but these high values were unstable. Examination of the cultures with electron microscopy revealed that the cells were partially differentiated with the formation of a basal lamina and intercellular junctions. Labelling with a specific monoclonal antibody marker for tight junction protein (ZO-1) suggested that non-pigmented cell cultures showed extensive tight junction formation. The low TER of the non-pigmented cell cultures appears therefore not to be due to the lack of tight junctions but rather to the presence of spaces between cells.

Modulation of volume and intracellular pH in the ciliary body epithelial cells

This paper investigates the accuracy of a matrix method proposed by other researchers to calculate time-varying flux control coefficients (dynamic FCCs) from elasticity coefficients by means of summation and connectivity theorems in the framework of metabolic control analysis. A mathematical model for the fed-batch penicillin V fermentation process is used as a case example for discussion. Calculated results reveal that this method produces significant calculation errors because the theorems are essentially valid only in steady state, although it may provide rough time-transient behaviors of FCCs. Strictly, therefore, dynamic FCCs should be directly calculated from the differential equations for metabolite concentrations and sensitivities.

Ca2+ spikes in ciliary body epithelial cells

Ca2+ spikes in ciliary body epithelial cells

Regulation of ICAM-1 expression on human non-pigmented ciliary body epithelial (NPE) and trabecular meshwork (HTM) cells

Regulation of ICAM-1 expression on human non-pigmented ciliary body epithelial (NPE) and trabecular meshwork (HTM) cells

Separation, identification, tissue culture, and physiological recording of ciliary body epithelial cells

The mechanism of NH4+ transport in inner medulla is not known. The purpose of these experiments was to study the process that is involved in ammonium (NH4+) transport in cultured inner medullary collecting duct (mIMCD-3) cells. Cells grown on coverslips were exposed to NH4+ and monitored for pHi changes by the use of the pH-sensitive dye BCECF. The rate of cell acidification following the initial cell alkalinization was measured as an index of NH4+ transport. The rate of NH4+ transport was the same in the presence or absence of sodium in the media (0.052±0.003 vs 0.048±0.004 pH/min, P>0.05), indicating that NH4+ entry into the cells was independent of sodium. The presence of ouabain, bumetanide, amiloride, barium, or 4,4′-di-isothiocyanostilbene-2-2′-disulfonic acid (DIDS) did not block the NH4+-induced cell acidification, indicating lack of involvement of Na+:K+-ATPase, Na+:K+:2 Cl− transport, Na+:H+ exchange, K+ channel, or Cl−/base exchange, respectively, in NH4+ transport. The NH4+-induced cell acidification was significantly inhibited in the presence of high external [K+] as compared to low external [K+] (0.018±0.001 vs. 0.049±0.003 pH/min for 140 mM K+ vs. 1.8 mM K+ in the media, respectively, P<0.001). Inducing K+ efflux by imposing an outward K+ gradient caused intracellular acidification by ∼0.3 pH unit in the presence but not the absence of NH4+. This K+ efflux-induced NH4+ entry increased by extracellular NH4+ in a saturable manner with a Km of ∼5 mM, blocked by increasing extracellular K+ and was not inhibited by barium. The K+ efflux-coupled NH4+ entry was electroneutral as monitored by the use of cell membrane potential probe 3,3′-dipropylthiadicarbocyanine. These results are consistent with the exchange of internal K+ with external NH4+ in a 1:1 ratio. The K+-NH4+ antiporter was inhibited by verapamil and Schering 28080 in a dose-dependent manner, was able to work in reverse mode, and did not show any affinity for H+ as a substrate, indicating that it is distinct from other NH4+-carrying transporters.We conclude that a unique transporter, a potassium-ammonium (K+/NH4+) antiport, is responsible for NH4+ transport in renal inner medullary collecting duct cells. This antiporter is sensitive to verapamil and Schering 28080, is electroneutral, and is selective for NH4+ and K+ as substrates. The K+/NH4+ antiporter may play a significant role in acid-base regulation by excretion of ammonium and elimination of acid.

Cytochrome P450-mediated prostaglandin [formula omitted] hydroxylase activities in porcine ciliary body epithelial cells

The ocular hypotensive effect of topically applied prostaglandins (PGs) is well documented. Although PGs introduced in the posterior chamber accumulate in the anterior tissues (e.g. iris/ciliary complex), little is known about the metabolism of PGs by these tissues. We have recently found that non-pigmented epithelial (NPE) cells and pigmented epithelial (PE) cells are readily separated from porcine ciliary body and cytochrome P450-dependent xenobiotic metabolism is considerably higher in NPE cells than in PE cells. We have therefore investigated in this study the cytochrome P450-mediated PG ω ω-1 hydroxylase activities of porcine ciliary epithelial cells. The NPE cells show about three times higher activities than do PE cells; the NPE cells, in fact, demonstrate the highest PG ω ω-1 hydroxylase activities among different ocular tissues. Both ω and ω-1 hydroxylases show broad substrate specificities and hydroxylate PGA1, A2, E1, E2, and lauric acid. The ω ω-1 hydroxylase activities of NPE and PE, as determined with PGA2 and lauric acid as substrates, are enhanced or induced by treatment of primary cultures of the individual epithelial cells with clofibrate, both activities reaching maximum levels within 48 hr of induction. The induced activities are inhibited almost completely by cycloheximide and actinomycin D. The ω ω-1 hydroxylase activities of both NPE and PE cells require NADPH and molecular oxygen, are associated with the microsomal fraction, respond to inducers such as clofibrate, and are inhibited by metyrapone and SKF525 A (inhibitors of P450 enzymes). These results support the suggestion that PG ω ω-1 hydroxylations by NPE and PE are cytochrome P450-mediated reactions. The primary association of P450-mediated ω ω-1 hydroxylase activities with NPE cells supports the contention that ciliary NPE cells play an important role in the metabolism of xenobiotics as well as endogenous substances (autacoids) in the anterior part of the eye.

The inhibition of Na, K-ATPase, and Mg-ATPase by timolol maleate in cultured non-pigmented epithelial cells of the ciliary body.

Bovine, non-pigmented, ciliary body epithelial cells were isolated and grown in culture to determine whether timolol maleate might affect the activity of their plasma membrane ATPases. The possible effects were tested in drug concentrations in a range of 5 x 10(-19) to 5 x 10(-5) M over an incubation period of 30 min at 37 degrees. Assays of specific activity showed that the drug significantly (p less than .001 for most concentrations) inhibited both Na,K-ATPase and Mg-ATPase. However, the inhibition was partially reversed in concentrations greater than 10(-6) M for Na,K-ATPase and 10(-5) M for Mg-ATPase. The latter enzyme also indicated a second partial reversal in activity at concentrations between 10(-12) and 10(-9) M. These reversals in activity suggest that more than one binding site is involved in the inhibition of both enzymes. Since Na,K-ATPase in non-pigmented, ciliary body cells is responsible for the generation of aqueous fluid and the intraocular pressure (IOP), this inhibition demonstrates a possible mechanism for the pharmacological action of timolol maleate in lowering IOP.

Sodium channels in ocular epithelia.

Voltage-gated, tetrodotoxin(TTX)-blockable sodium channels are found in most excitable cells and are the primary contributors to action potentials generated by many of these cells. To date, there has only been one report of a non-cultured vertebrate epithelial cell type containing TTX-blockable Na+ channels: rabbit non-pigmented ciliary body epithelial cells [Cilluffo MC et al. (1991) Invest Opthalmol Vis Sci 32: 1619-1629], and three reports of cultured epithelial cells containing TTX-blockable Na+ channels: rabbit non-pigmented and pigmented ciliary body epithelium [Ciluffo MC et al. (1991) Invest Opthalmol Vis Sci 32: 1619-1629; Fain GL, Farahbakhsh (1989) J Physiol (Lond) 417: 83-103] and human lens epithelium [Cooper K et al. (1990) J Membr Biol 117: 285-298]. We report here the presence of sodium currents in two different non-cultured, freshly dissociated transporting epithelial cell types: the rabbit corneal endothelium and the frog lens epithelium. We also report the occurrence of sodium currents in six additional cultured ocular epithelial cell types from three different species. These currents have a current/voltage (I/V) relationship consistent with traditional voltage-gated Na+ currents, are quinidine- and TTX-blockable (of the low-affinity TTX-sensitive type), and disappear following bath substitution of Na+ with Cs+ or K+.

Interactions of cultured rat synovial and ocular ciliary body cells with two strains of mycoplasma arthritidis

Strains of Mycoplasma arthritidis differ in their ability to cause joint and ocular inflammations. Although the reasons for this difference are not fully understood, pathogenic mycoplasmas commonly require close associations with the cells they damage. Using 3H-uridine labeled mycoplasma, we compared cellular interactions of in vitro cultivated rat synovial and ocular ciliary body epithelial cells with two American Type Culture Collection strains of M. arthriditis shown to differ in their virulence. Radiolabeling assays gave evidence of a stronger retention capability on cultured cells by the more pathogenic strain, 14152. Scanning electron microscopy demonstrated cellular associations with the two strains of mycoplasma, with more of the 14152 adhering to both cell types. Examination by transmission electron microscopy showed evidence of contact between the more virulent 14152 strain and both cell types, but no similar evidence with the comparatively less virulent strain, 19611. The pathogenicity of different strains of M. arthritidis may vary according to their ability to closely associate with specific target cells involved in the disease process.

Voltage-activated currents recorded from rabbit pigmented ciliary body epithelial cells in culture.

1. The whole-cell recording mode of the patch-clamp technique was used to investigate the presence of voltage-activated currents in the isolated pigmented cells from the rabbit ciliary body epithelium grown in culture. 2. In Ringer solution with composition similar to that of the rabbit aqueous humour, depolarizing voltage steps activated a transient inward current and a delayed outward current, while hyperpolarization elicited an inwardly rectified current. 3. The depolarization-activated inward current was mainly carried by Na+ and was blocked by submicromolar concentrations of tetrodotoxin. This current in many cells was sufficiently large to produce a regenerative Na+ spike. 4. The depolarization-activated outward current was carried by K+ and blocked by external TEA and Ba2+. Its activation appeared to be Ca2(+)-independent. 5. The hyperpolarization-activated inward current was almost exclusively carried by K+ and was blocked by Ba2+ and Cs+. For large hyperpolarizations below -120 mV, this current exhibited a biphasic activation with a fast transient peak followed by a slower sag, that appeared to be due to K+ depletion. 6. The voltage-dependent K+ conductances probably act to stabilize the cell membrane resting potential and may also play a role in ion transport. The function of the Na(+)-dependent inward current is unclear, but it may permit the electrically coupled epithelial cells of the ciliary body to conduct propagated action potentials.