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.     

Electrically silent Na+ and Cl- fluxes across the rabbit ciliary epithelium

Unidirectional Na+, Cl-, and mannitol fluxes were measured across the isolated and short-circuited rabbit iris-ciliary body as functions of the Cl- concentration in the bathing solution (0, 26 and 80 mM). At constant Na+ concentration, Na+ fluxes increased on the average from 8.0 to 11.9 mu eq/hr as the Cl- concentration was raised and vice versa. Cl- fluxes also increased more than expected from simple diffusion; from 2.0 (26 mM) to 8.1 mu eq/hr (80 mM). Mannitol permeability (9.0 X 10(-7) cm/sec) was independent of the Cl- concentration and similar to that measured in "tight" epithelia. In Cl(-)-free solutions, there was good agreement between the measured electrical resistance and that calculated with the partial conductance equation. In Cl(-)-rich solutions, the calculated resistance was smaller than the measured resistance, suggesting electrically silent fluxes of Na+ and Cl-. These silent fluxes were of similar magnitude and possibly coupled with each other. This mechanism may provide an additional pathway for electrolyte movement across the ciliary epithelium.     

Dopamine stimulation of passive permeability and secretion in the isolated rabbit ciliary epithelium.

Dopamine, at concentrations between 1·25 × 10^?7m and 1·25 × 10^?3m, increases both fluid secretion and passive fluid permeability across the isolated rabbit ciliary epithelium. These effects can be blocked by α- and β-adrenergic antagonists but not by specific dopaminergic antagonists. The present data suggest that dopaminergic receptors are not present in the ciliary epithelium.     

The influence of cyclic AMP upon Na,K-ATPase activity in rabbit ciliary epithelium.

ATPase activity was measured in samples of freshly dissected rabbit ciliary epithelium. The epithelium was ruptured in distilled water, frozen briefly, and incubated at 37 degrees C in a buffer containing 100 mM NaCl and 32P-labeled adenosine triphosphate (ATP). The rate of ATP hydrolysis by the epithelium was linear for as long as 45 min. Ouabain (1 mM) reduced the ATP hydrolysis rate by approximately 50%. When the epithelium was preincubated for 10 min. in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (cAMP), the ouabain-sensitive (Na,K-ATPase) activity was diminished; ouabain-insensitive ATPase activity was not reduced. Preincubation of the epithelium with forskolin with isobutylmethylxanthine also reduced ouabain-sensitive ATPase activity. These observations suggest that the ciliary epithelium may have a mechanism for short-term modulation of Na,K-ATPase activity by cAMP. Such a mechanism could be linked to the ability of cAMP-dependent protein kinase to reduce Na,K-ATPase activity in the tissue.     

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.     

The hydraulic conductivity of rabbit ciliary epithelium in vitro

The rate of fluid movement across rabbit ciliary body in vitro has been measured as a function of applied hydrostatic pressure. Previous studies from other laboratories carried out with the ciliary body clamped between two half chambers have yielded a value of 4600 ± 600 μm/sec. We have presently reproduced those results by using a similar mounting procedure. On the other hand, however, we report the development of a technique for edge-damage-free mounting of the ciliary body with which the tissue is not clamped and tissue glue is employed in order to achieve a proper seal. With this last procedure, the values thus obtained for the hydraulic conductivity were much lower than the previous ones, namely, 210 μm/sec for pressure applied on the stromal side of the ciliary epithelium (pressure range: 0–10 cm H₂O, n = 13; T = 37°C), or 109 μm/sec (pressure difference range: 0–31 cm H₂O) for pressure applied from the aqueous side. We conclude that the notion of aqueous production by ultrafiltration cannot be supported by the present results.     

Quantitation of Na/K ATPase pump sites in the rabbit corneal endothelium

In these experiments, the binding of 3H . ouabain, a specific inhibitor of Na/K ATPase, was used to quantitate the density of Na/K ATPase pump sites in the rabbit corneal endothelium. The uptake of ouabain by the corneal endothelium shows two components: one that saturates at a ouabain concentration near 2 X 10(-7) M (specific binding), and one component that increases linearly with increasing glycoside concentration (nonspecific uptake). The nonspecific uptake can be accounted for by that ouabain equilibrating with the extracellular space, which, estimated by inulin space, amounts to 13.0 nl/mm2 of endothelium. The saturable component of endothelial ouabain uptake is displaced by K+ ions, which is consistent with this fraction being bound to Na/K ATPase. Maximal endothelial ouabain binding was measured as 20.7 fmoles/mm2 of endothelium, which corresponds to 3.0 X 10(6) pump sites per cell. The density of Na/K ATPase pump sites in the rabbit corneal endothelium is comparable to densities reported for several transporting epithelia. These data are consistent with the known function of the endothelium in corneal deturgescense and corroborate the importance of Na/K ATPase in endothelial fluid transport.     

Cyclic AMP-dependent stimulation of basolateral K(+)conductance in the rabbit conjunctival epithelium

The regulation of Na(+)transport by cAMP in freshly isolated rabbit conjunctival epithelium, a tissue exhibiting both Cl(-)secretion and Na(+)absorption, was examined. Bulbar-palpebral segments of conjunctiva were mounted between Ussing-type hemichambers under short-circuit conditions in Cl(-)free media. In this situation, the short-circuit current (I(sc)) measures an amiloride-resistant Na(+)absorptive process in the apical-to-basolateral direction. Apical additions (each at 10 microm) of cAMP-elevating compounds, forskolin, rolipram, IBMX and epinephrine all stimulated the Na(+)-dependent I(sc)by approximately 3.5-4.5 microA cm(-2)(minimal 40% increase) and reduced transepithelial resistance (R(t)) by at least 7% (P<0.05). Pre-exposure (1 hr) to the protein kinase A (PKA) inhibitor H-89 (10 microm), which in itself inhibited the I(sc)by 0. 5 microA cm(-2), attenuated the I(sc)responses of the cAMP-elevating agents (P< 0.05, unpaired data). In reverse, H-89 promptly decreased the I(sc)by 1.5-2.5 microA cm(-2)and increased R(t)by 5% (P<0.05) in tissues pre-stimulated with either forskolin or an epinephrine plus IBMX combination. Additions of epinephrine or rolipram to apically permeabilized preparations using amphotericin B, increased the I(sc)by 12 and 22% respectively over baseline and reduced R(t)by 6% (P<0.05). Similarly, in the presence of a transepithelial K(+)gradient (apical to basolateral) and amphotericin B, cAMP elevation stimulated K diffusion across the preparation by at least 1.8 microA cm(-2)and decreased the R(t)by 4% (P<0.05), changes that were reversed by subsequent H-89 addition. Under Cl(-)rich conditions, pretreatment with 5 m m Ba(2+)reduced the basal I(sc)by 59% and blocked the cAMP-induced I(sc)stimulations typically seen in the presence of the anion. The results provide evidence for a PKA-regulated, Ba(2+)-inhibitable (voltage insensitive) basolateral K(+)conductance in rabbit conjunctival epithelial cells. The action of Cl(-)secretogogues acting via cAMP on basolateral K(+)channel activity indicates that endogenous levels of cAMP may play a role in the regulation of Cl(-)secretion and Na(+)absorption in the conjunctiva.     

Bicarbonate transport mechanisms in rabbit ciliary body epithelium.

Sections of whole ciliary body dissected from Dutch belted rabbits were incubated with the cell entrappable pH probe BCECEF-AM. This led to a highly specific localization of epifluorescence emission at the exposed, non-pigmented cell layer (npe) of the dual layered epithelium that covers this organ. The BCECF-loaded tissue sections were superfused in a flow-through chamber and the intracellular pH (pHi) of small groups (10-20) of cells was derived from the ratio of the emission intensities derived from excitations at 490 and 440 nm. In CO2/HCO3- Ringer's, npe pHi = 7.09 +/- 0.11. Replacement of CO2/HCO3- by Hepes increased pHi by 0.22 +/- 0.02, indicating alkali secretory activity under the bicarbonate-rich conditions. Replacement of Cl- by gluconate elicited a rapid, 0.6-U increase in pHi. This effect exhibited little dependence on Na+ and was inhibited by 0.5 mM dihydro-4,4'-diisothiocyanatostilbene -2,2'-disulfonate (H2DIDS). These results indicate the presence of an electroneutral Cl-/base exchange activity. Elevation of [K-] (by partial replacement of Na+) also elicited increases in pHi. In Cl(-)-free media pHi reached 7.8-8.0, a condition under which intracellular [HCO3-] is at least twice as high as its extracellular value. This effect did not occur in the absence of Na+. The Na(+)-dependent high [K+]-induced pHi increase was inhibited by H2DIDS. The effects of Ba2+ on pHi, alone and in combination with high [K+], as well as that of full K+ removal, suggested that the link between high [K+] and pHi increase was mainly due to the effect of cell depolarization on an electronegative Na+ dependent HCO3- transporter. Under normal physiological conditions, the two acid/base transport systems are the main determinants of npe pHi.     

H2O2-modification of Na,K-ATPase. Alterations in external Na+ and K+ stimulation of K+ influx

Studies, at steady state, of the Na,K-ATPase dependent influx of K+ into bovine lenses in organ culture are used to characterize further the H2O2-modification of the Na+ pump. Control lenses display constants for interaction with external Na+ and K+ similar to those obtained for the erythrocyte. H2O2 treatment of the bovine lens leads to total loss of external Na+ stimulation and alteration of external K+ stimulation.     

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.     

Adrenergic stimulation of ciliary process epithelium causes surface membrane internalization

An ultrastructural change induced in the nonpigmented epithelium (NPE) of the ciliary processes by adrenergic stimulation in the albino rabbit was studied. Thirty min after topical treatment with 2% isoproterenol, an extensive intracellular membranous network, previously reported to be smooth endoplasmic reticulum, was revealed by electron microscopy. It was postulated that this network originated from the plasma membrane. Using cationized ferritin (CF) as an ultrastructural tracer, freshly isolated anterior segments were incubated in buffer containing 10(-5) M isoproterenol and 0.2% CF. As early as 10 min, and for at least 30 min, the isoproterenol-treated NPE cells contained a membranous network that was morphologically similar to that which occurs in vivo. CF particles were present within the network, indicating that the membranous network had originated at the cell surface. This labeling was prevented by pretreatment with the beta-adrenergic antagonist timolol maleate. In both treated and control ciliary processes, CF was present in the ciliary canals between the NPE and the underlying pigmented epithelium after 10 min incubation. This suggests that the NPE is able to transport CF from its basilar to apical surface. These experiments imply that the NPE is able to internalize rapidly large amounts of plasma membrane in response to adrenergic stimulation. This response may be part of the mechanism of adrenergic receptor desensitization, alteration of aqueous humor production, or another adrenergic response.     

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.