Nonpigmented Epithelial Cells Research Articles

Regulation of Na,K-ATPase expression by endothelin-1 in transformed human ciliary non-pigmented epithelial (HNPE) cells.

Endothelin-1 (ET-1) (1-100 nM) decreases the activity of Na,K-ATPase, a key enzyme responsible for aqueous humor formation, in transformed human non-pigmented ciliary epithelial (HNPE) cells. The present study sought to determine if ET-1 alters the expression of the catalytically active alpha subunit of Na,K-ATPase in HNPE cells and identify mechanisms underlying these effects. We report that acute (15 and 30 min) treatment with ET-1 results in an increase in mRNA expression of the alpha 1 subunit of Na,K-ATPase. Similar to ET-1's effects, ouabain (100 microM), a selective inhibitor of Na,K-ATPase, and monensin (10 microM), a sodium ionophore, also increased Na,K-ATPase expression in HNPE cells. The increase in Na,K-ATPase expression by short-term treatment with ouabain and monensin was dependent on their ability to elevate intracellular sodium concentrations. However, acute ET-1 treatment mediated increase in Na,K-ATPase expression was independent of changes in intracellular sodium. A prolonged (24 hr) ET-1 treatment results in an increase in both mRNA and protein levels of the alpha 1 subunit of Na,K-ATPase. These observations suggest that ET-1 could play a homeostatic role in modulating aqueous humor formation by having differential effects on the activity and expression of Na,K-ATPase by the ciliary epithelium in the eye.

Ectonucleotidases of the rabbit ciliary body nonpigmented epithelium.

To investigate the expression of both the message and function of ENPP1 (a member of the ectonucleotide pyrophosphatase/phosphodiesterase family, also known as PC-1), NTPD1 (a member of the ectonucleoside 5'-triphosphate diphosphohydrolase family, CD39), and ecto-5'-nucleotidase (CD73) in rabbit ciliary body nonpigmented epithelial (NPE) cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to reveal the presence of mRNAs of ectonucleotidases in NPE cells. Real-time fluorescence ratio imaging of the intact fura-2-loaded NPE cells was used to record changes in the intracellular calcium concentration. RT-PCR analysis revealed the expression of mRNAs for ENPP1, NTPD1, and ecto-5'-nucleotidase, but not NTPD2 (ecto-ATPase, or CD39L1), in the rabbit NPE cells. The ENPP1 inhibitor pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), and to a lesser degree the nonspecific ectonucleotidase antagonist 6-N,N-diethyl-beta-gamma-dibromomethylene-D-adenosine 5-triphosphate (ARL 67156), reduced the [Ca(2+)](i) increase elicited by the combination of acetylcholine (ACh) and cAMP. However, both inhibitors significantly enhanced the [Ca(2+)](i) increase generated by uridine triphosphate (UTP). The ecto-5'-nucleotidase inhibitor alphabeta-meADP significantly diminished the [Ca(2+)](i) increase evoked by ACh+cAMP, but not that generated by UTP. The A(1)-specific adenosinergic receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) significantly blocked the response to ACh+cAMP. These observations suggest that rabbit NPE cells possess at least three distinct ectonucleotidases capable of catalyzing the stepwise hydrolysis of adenine and pyrimidine nucleotides, as well as cAMP, thus shaping the purinergic-receptor-coupled signaling in these cells.

Oxysterol 7α-Hydroxylase (CYP39A1) in the Ciliary Nonpigmented Epithelium of Bovine Eye

The CYP39A1 oxysterol 7α-hydroxylase preferentially catalyzes the 7α-hydroxylation of 24-hydroxycholesterol and has been suggested to play a role in the alternative bile acid synthesis pathway in the liver. The presence of CYP39A1 oxysterol 7α-hydroxylase has been reported only in the liver. To investigate the physiologic characteristics of the ciliary processes in bovine ocular tissues, we raised a mAb, 42C, against nonpigmented epithelial (NPE) cells, which have tight junctions that act as a blood-aqueous barrier and are involved in producing aqueous humor and maintaining ocular homeostasis. Immunohistochemical analysis showed that 42C antibody reacted intensely with an antigen in the NPE cells of the ciliary processes but not with other ocular tissues. The SDS-PAGE profile of immunoaffinity-purified antigens from bovine ciliary processes showed a predominant protein of molecular mass of 44.0 kDa. The amino acid sequence of this antigenic protein was identical to human CYP39A1 oxysterol 7α-hydroxylase. Immunoreactivity with 42C antibody was found only in hepatocytes and ocular tissues. These data suggest a new physiologic function for the CYP39A1 oxysterol 7α-hydroxylase in addition to the production of bile acids and provide new insight into the physiologic role of the ciliary NPE cells concerning the metabolism of sterols in the eye.

Cannabinoid CB 1 receptor expression, activation and detection of endogenous ligand in trabecular meshwork and ciliary process tissues

Elevated intraocular pressure is the primary risk factor for glaucoma. Cannabinoids interact with molecular targets in the eye and lower intraocular pressure by an unknown mechanism. The purpose of the present study was to examine eye tissues for functional cannabinoid receptors of the neuronal, CB 1 class, and an endogenous ligand, anandamide. The trabecular meshwork and ciliary processes are the primary structures of the eye that contribute to intraocular pressure and thus were our focus. Total RNA, frozen sections, cellular membranes and primary cultures of cells were prepared from both bovine and cadaveric human tissues. Using cannabinoid CB 1 receptor-specific oligodeoxynucleotide primers, cannabinoid CB 1 receptor antiserum, and cannabinoid-specific compounds (CP-55,940, WIN55,212-2 and SR-141716A), the presence of cannabinoid CB 1 receptors in ciliary processes and trabecular meshwork was determined. Using reverse transcription-polymerase chain reaction, we identified mRNA encoding cannabinoid CB 1 receptor protein in ciliary process and trabecular meshwork cells. Specific binding of anti-CB 1 immunoglobulin-G in tissue sections localized cannabinoid CB 1 receptor protein to the non-pigmented epithelial cells of the ciliary process and cells of the trabecular meshwork. While CP-55,940 and WIN55,212-2 failed to stimulate [ 35S]GTPγS binding in membrane preparations from trabecular meshwork and ciliary process, CP-55,940 significantly stimulated whole cell [ 35S]GTPγS binding by 51% over basal in ciliary process epithelial cells and 69% over basal in trabecular meshwork cells permeabilized with 5 μM digitonin ( p<0.001). Specificity of agonist stimulation was verified by complete blockade with the specific cannabinoid CB 1 receptor antagonist, SR-141716A. Moreover, activation of cannabinoid CB 1 receptors by CP-55,940 resulted in a 2.3±0.3 and 1.7±0.3-fold stimulation of cAMP accumulation in trabecular meshwork and ciliary process cells, respectively ( p<0.01). Lastly, anandamide was detected in human trabecular meshwork (3.08 pmol/g), ciliary process (49.42 pmol/g) and neurosensory retinal (4.48 pmol/g) tissues. These data, for the first time, demonstrate in a single study the presence of both CB 1 mRNA and protein in trabecular meshwork and ciliary processes from two different species. Activation of heterotrimeric G-proteins and stimulation of cAMP accumulation by cannabinoids in vitro suggest that their intraocular pressure-lowering effects in vivo result from activation of cannabinoid CB 1 receptors in the trabecular meshwork and increase aqueous outflow.

Melatonin Receptor mRNA and Protein Expression in Xenopus laevis Nonpigmented Ciliary Epithelial Cells

Melatonin is an output signal of the circadian clock, and may regulate diurnal rhythms in ocular tissues. A role for melatonin has been suggested in the circadian changes in intraocular pressure (IOP). Changes in IOP may be due partially to changes in the rate of aqueous humor secretion, which is produced by the nonpigmented epithelium of the ciliary body. To examine the mechanism by which melatonin may influence ciliary epithelium function and perhaps the IOP diurnal rhythm, immunocytochemistry with an antibody directed against the Mel1c melatonin receptor subtype was performed on sections of Xenopus eyes. Melatonin receptor immunoreactivity was observed in the basolateral regions of the nonpigmented epithelial cells of the ciliary body. Receptor immunoreactivity was also observed in cells of the retina, as has been previously reported. Specific immunoreactivity was not observed in the epithelium of the iris or pigmented ciliary epithelium. In situ hybridization of the Xenopus eye revealed expression of Mel1c but not Mel1b receptor mRNA in the nonpigmented ciliary epithelium. These results provide evidence that the nonpigmented epithelia of the ciliary body are direct targets for melatonin, and supports previous work that melatonin may influence the rate of aqueous humor secretion by ciliary epithelium, and perhaps the circadian rhythm of IOP.

Constitutive Expression and Localization of COX-1 and COX-2 in Rabbit Iris and Ciliary Body

Prostaglandins are involved in the regulation of intraocular pressure and the blood-aqueous barrier of the eye, and are used for the treatment of glaucoma. The decrease of the constitutively expressed PG-synthesizing enzyme cyclooxygenase-2 (COX-2) has been demonstrated in the ciliary non-pigmented epithelial layer of patients with primary open angle glaucoma. Little is known about the distribution of COX-1 and COX-2 in animals. We investigated this in the iris and ciliary body of the normal rabbit eye. The presence of COX-1 and COX-2 in freshly excised iris and ciliary body tissue from adult New Zealand White albino rabbits was demonstrated by real-time RT-PCR, and Western blot analysis. The localization of both isoforms and of the neuron-specific protein gene product 9.5 was determined by indirect immunofluorescence. Both enzymes are expressed in the iris and the ciliary body. Immunofluorescence studies including double staining techniques localized COX-1 and COX-2 to about 50% of cells in the stromal tissue of iris and ciliary body, mainly on the corneal side. They were co-localized in about 75% of these cells. Whereas all stained cells were positive for COX-1, COX-2 showed a gradient-like distribution in the stroma, with some restriction of expression near the epithelial layers, which we clearly showed to be completely negative for both COX-1 and COX-2. Also, neuronal elements did not show COX-1 or COX-2 immunoreactivity. These results establish the presence of COX-1 and COX-2 on the RNA and protein levels in normal, unstimulated rabbit iris and ciliary body. The pattern of distribution suggests a role for both enzymes in maintaining the physiology of the eye. In contrast to our results in man, non-pigmented epithelial cells of the ciliary body did not express immunoreactivity. This could account for differences in the regulation of intraocular pressure and/or blood-aqueous barrier between human and rabbit eyes.

Establishment of Three Cell Lines Derived from Frog Melanophores

Abstract Three cell lines have been established from melanotic and amelanotic melanophores of wild and albino Rana nigromaculata and albino Rana porosa brevipoda after experiencing three culture crises. The population doubling rate increased with culture time. Cells from both wild and albino R. nigromaculata changed shape twice in ten years. They began as large pigmented or non-pigmented epithelial-like cells at the beginning of the cultures. The melanophores of albino R. nigromaculata unexpectedly became pigmented shortly after the beginning of the culture. Over time, they transiently aquired a small, non-pigmented, bipolar morphology and finally became large non-pigmented epithelial-like cells after about 8 to 10 years. On the contrary, cells from albino R. porosa brevipoda remained large, non-pigmented, epithelial-like cells over the entire course of the culture. Pigmented cells contained many mature melanosomes, while non-pigmented epithelial like cells contained premelanosomes with characteristic int...

Phenylarsine oxide inhibits phosphate uptake in human ciliary non-pigmented epithelial cells.

Phenylarsine oxide (PAO), a sulfhydryl modifying reagent and a widely used inhibitor for tyrosine phosphatases and endocytosis, was tested on the level of phosphorylation in human nonpigmented ciliary epithelial ocular (HNPE) cells. Pretreatment with (PAO, 10 microM) for 30 min followed by incubation with 32Pi to stimulate endogenous phosphorylation surprisingly resulted in a total reduction in 32Pi labeled proteins. PAO (10-50 microM) dose-dependently inhibited both sodium-dependent and -independent phosphate uptake in cells. p-Hydroxymercuribenzoate (pHMB, 10 microM), another sulfhydryl modifying reagent failed to mimic PAO effects. However, metabolic inhibitors (iodoacetamide (0.1 mM) and 2,4-dinitrophenol (DNP, 0.5 mM) also mimicked PAO effects, suggesting that the inhibition of ATP production may be responsible for attenuation of both phosphate uptake mechanisms. However, sodium-dependent phosphate uptake in isolated plasma membrane vesicles pretreated with PAO was also significantly lower than control vesicles treated with dimethlysulfoxide (DMSO), suggesting that PAO may be directly targeting a component of the sodium-dependent cotransporter. It is suggested that PAO is a novel inhibitor of phosphate uptake in HNPE cells that acts indirectly by inhibiting ATP production and directly by inhibiting the Na-dependent cotransporter.

Localization of Secreted Protein Acidic and Rich in Cysteine (SPARC) Expression in the Rat Eye

Secreted protein acidic and rich in cysteine (SPARC) is a secreted glycoprotein protein which modulates cell shape and cell-matrix interactions and has been implicated in the regulation of angiogenesis, vascular permeability and cataract formation. In situ hybridization and immunohistochemical studies for SPARC were performed to determine the cell and tissue distribution of SPARC in rat eye. Studies demonstrated SPARC mRNA and protein co-localization at all sites. In the retina SPARC mRNA and protein were localized predominantly to the Müller and ganglion cells. Within the choroid, SPARC was found in vascular endothelial cells and fibroblasts; in the sclera SPARC was present in blood vessels and fibroblasts. SPARC was also present in the non-pigmented epithelial cells of the ciliary body, and in the epithelium of the lens capsule and cornea. The demonstrated anatomical distribution of SPARC in the rat eye is consistent with several of the biological functions ascribed to this matricellular protein and provides a rational basis for its examination in pathological conditions.

Colocalization of tight junction proteins, occludin and ZO-1, and glucose transporter GLUT1 in cells of the blood-ocular barrier in the mouse eye.

The facilitative glucose transporter GLUT1 is abundant in cells of the blood-ocular barrier and serves as a glucose transport mechanism in the barrier. To see the relationship between the glucose transfer function and junctional proteins in the barrier, we examined the localization of GLUT1 and the tight junction proteins, occludin and ZO-1, in the mouse eye. Their localization in the retina, ciliary body, and iris was visualized by double-immunofluorescence microscopy and immunogold electron microscopy. Occludin and ZO-1 were colocalized at tight junctions of the cells of the barrier: retinal pigment epithelial cells, non-pigmented epithelial cells of the ciliary body, and endothelial cells of GLUT1-positive blood vessels. Occludin was restricted to these cells of the barrier. ZO-1 was found, in addition, in sites not functioning as a barrier: the outer limiting membrane in the retina, in the cell border between pigmented and non-pigmented epithelial cells in the ciliary body, and GLUT1-negative blood vessels. These observations show that localization of occludin is restricted to tight junctions of cells of the barrier, whereas ZO-1 is more widely distributed.

PIClnCan Regulate Swelling-Induced Cl−Currents in Either Layer of Rabbit Ciliary Epithelium

Swelling-induced Cl−currents were investigated in freshly prepared non-pigmented epithelial (NPE) and pigmented epithelial (PE) cells of the rabbit ciliary body using the whole-cell patch clamp technique. Exposure of both NPE and PE cells to hypotonic stress induced Cl−currents that exhibited outward rectification and were insensitive to Ca+2. We found that swelling-induced Cl−currents in PE cell are observed shortly after isolation. The swelling-induced Cl−current showed little or no inactivation at positive membrane voltages and was sensitive to 100 μM NPPB and 100 μM DIDS. Injection of cRNA encoded rabbit pIClninto Xenopus oocytes produced an outwardly rectifying Cl−current displaying features consistent with the swelling-induced Cl−current in epithelium. pIClnis ubiquitous in the ciliary epithelium. It participates in the equilibration of short term tonicity alterations, a phenomenon underlying mechanisms with larger and slower amplitudes for aqueous secretion by these cells.

Regulation of Endothelin-1 in Human Non-Pigmented Ciliary Epithelial Cells by Tumor Necrosis Factor-α

Endothelins (ET) are potent vasoactive peptides present in many ocular structures and are formed from precursor Big endothelins (Big ET-1) by the action of an endothelin-converting enzyme (ECE). ET-1 is thought to decrease intraocular pressure by contracting the ciliary muscle thus enhancing the outflow of aqueous humor through the Canal of Schlemm and trabecular meshwork. However, the mechanisms involved in the regulation of endothelin-1 (ET-1) synthesis and release in ocular tissues have not been fully characterized. In this study we examined the effect of tumor necrosis factor-α(TNF-α; 10 nm), a proinflammatory cytokine, on the cellular mechanisms leading to ET-1 synthesis and release in SV-40 transformed human ciliary non-pigmented epithelial cells (HNPE). ET-1 and Big endothelin-1 (Big ET-1) immunoreactivity was time-dependently increased following TNF-αtreatment. Phorbol esters (PMA), activators of PKC, also raised the immunoreactive levels of ET-1 and Big ET-1 while, staurosporine, a PKC inhibitor (20 nm), decreased ET-1 levels in TNF-α-stimulated cells. Pre-treatment with phosphoramidon (1 μm) an ECE-inhibitor, followed by TNF-αstimulation, decreased ir-ET-1 levels. Cycloheximide (9 μm), a protein synthesis inhibitor, decreased TNF-α-stimulated levels for ir-ET-1 and ir-Big ET-1, suggesting that TNF-αmay be directly regulating ET-1 expression at the ET-1 gene. Our data indicates that TNF-α regulates ET-1 levels in HNPE cells possibly by activating PKC either to stimulate protein synthesis and/or to enhance ET-1 secretion. These results suggest that ET-1 released from the ciliary body may play an important role in aqueous humor dynamics following cytokine activation.

Histochemical mapping of NADPH-diaphorase in monkey and human eyes

Purpose. To localize nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in normal human and monkey eyes.Methods. NADPH-d activity in ocular tissue sections was revealed by histochemical staining.Results. NADPH-d staining was found in the anterior and posterior ocular segment of both species. In the anterior segment, reaction product was present in the corneal epithelium and endothelium, iris dilator and sphincter, lens capsule, ciliary muscle, and non-pigmented epithelial cells and stroma of the ciliary processes. Staining density in the longitudinal ciliary muscle was higher than in the other ciliary muscle regions in the human; such regionalization was less apparent in the monkey. The trabecular meshwork stained minimally if at all in both species. In the posterior segment, staining was seen around choroidal blood vessels and choroidal nerves, and in the retinal pigment epithelial, photoreceptor, bipolar, inner plexiform and ganglion cell layers.Conclusions. NADPH-d is widespread in human and monkey eyes, indicating significant and presumably functionally relevant NO production in the various ocular structures. Curr. Eye Res. 17: 370–379, 1998.

Endothelin receptor A is expressed and mediates the [Ca2+]i mobilization of cells in human ciliary smooth muscle, ciliary nonpigmented epithelium, and trabecular meshwork

PURPOSE. To identify which endothelin receptor subtype is expressed and is functional in the human ciliary body and trabecular meshwork, tissues that regulate aqueous humor dynamics. METHODS. Immunocytochemistry was used to characterize the primary culture cells of normal human ocular cells. Endothelin receptor gene expression was probed with reverse transcription of polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca 2+ ] i) mobilization was measured with video image microscopy using Fura-2AM as a fluorescent probe. RESULTS. Identities of primary cultures, human ciliary smooth muscle (HCSM), ciliary nonpigmented epithelial (HCE), and trabecular meshwork (HTM) cells were confirmed by immuno-cytochemistry, using cell-specific markers and observing typical cell morphologies. The presence of endothelin receptor A (ET A) was detected with RT-PCR in all three types of cells. The mRNA phenotype was verified with restriction enzyme BamHI digestion. No ET B receptor subtype expression was detected with RT-PCR under the cell culture conditions used. The [Ca 2+ ] i of HCSM cells was increased from 57 ± 7 nM to 328 ± 108 nM (n = 23; mean ± SE; P < 0.05) by 1 nM endothelin-1 (ET-1). In HCE cells, [Ca 2+ ] i increased from 40 ± 3 nM to 90 ± 10 nM (n = 55) (P < 0.001) with the same concentration of ET-1. Similarly, ET-1 (1 nM) increased the [Ca 2+ ] i from 51 ± 6 nM to 185 ± 47 nM (n = 19) (P < 0.001) in the HTM cells. The agonist for ET B, S6c, had no effect on [Ca 2+ ] i transients in all three cell types. No ET B receptor expression was detected in these cell types under the experimental and culture conditions. CONCLUSION. ET A receptor is expressed and is possibly responsible for mediating the signal for [Ca 2+ ] i mobilization by ET-1 in human ciliary smooth muscle, ciliary nonpigmented epithelial cells, and trabecular meshwork cells.

Functional and morphological differentiation of nonpigmented ciliary body epithelial cells grown on collagen rafts

We have examined the effect of alteration in cell shape on promoting differentiated morphology and physiology in cultured nonpigmented epithelial cells from the ciliary body. We have grown pure populations of nonpigmented cells on collagen gels released from the culture dish to create collagen rafts. Shortly after the gels were detached, the cells shrank in diameter and increased in height while they contracted the gel. Concurrently, the actin cytoskeleton reorganized to the cell cortex as found in vivo. After this differentiated morphology developed, large changes in intracellular Ca2+ could be elicited by simultaneous activation of acetylcholine and epinephrine or acetylcholine and somatostatin receptors as seen in intact tissue. Explant cultures of isolated nonpigmented cell layers maintained their actin distribution and also showed synergistic Ca2+ increases. Spread cells, grown on rigid substrates, had a disorganized cytoskeleton and rarely showed synergism. These data suggest that the mechanism underlying synergistic Ca2+ responses in the ciliary body is functional in nonpigmented cells grown on collagen rafts. In addition, this pathway appears to be sensitive to the disposition of the cell's cytoarchitecture.

An Animal Model for Cidofovir (HPMPC) Toxicity: Intraocular Pressure and Histopathologic Effects

Intravitreal cidofovir has been shown to be a long acting and highly efficacious treatment for CMV retinitis; however decrease in IOP is an adverse effect. We wanted to determine the effect of cidofovir on intraocular pressure (IOP) in the guinea pig, and rabbit eye to develop an animal model of cidofovir induced ocular hypotony and to study the histopathology of this toxicity. Twenty-eight guinea pig eyes were injected with cidofovir yielding final intravitreal concentrations of 25, 200, 625 and 2000 μg ml−1. Eighteen eyes of pigmented rabbits were injected with cidofovir yielding final intravitreal concentrations of 625 and 2000 μg ml−1. A carefully calibrated low volume displacement manometer system using a micro-transducer was used to determine the IOP measurements in the guinea pig and rabbit eyes. Histology was evaluated using light and electron microscopy. Injection of 6.25 μg of cidofovir intravitreally (vitreous concentration of 25 μg ml−1) is the highest non-toxic dose in the guinea pig; the IOP was unchanged at two and four weeks after injection with this dose; histologically the eyes were normal. A single injection of 50 μg of cidofovir intravitreally (vitreous concentration of 200 μg ml−1) caused a long lasting (9.3 mmHg) decrease in IOP (approximately 50% of baseline). At this dose there were only mild and variable histologic changes in the ciliary body and the retina. Higher doses of 156.25 μg and 500 μg of cidofovir (vitreous concentrations of 625, and 2000 μg ml−1, respectively) caused moderate to severe ciliary body and retinal changes. In rabbit eyes there was a mild but statistically insignificant pressure drop with doses of 875 μg cidofovir intravitreally (vitreous concentration of 625 μg ml−1); retina was within normal limits after injection with this dose, there were mild changes in the ciliary body. There was a total destruction of ciliary body and loss of nonpigmented epithelial cells with injections of 2800 μg of cidofovir intravitreally (vitreous concentration of 2000 μg ml−1); retina was relatively well preserved. The guinea pig eye shows similar reduction in IOP and ciliary body changes as are seen in the human eye after intravitreal cidofovir and also appears to have a similar dose-response curve. However, the reduction of IOP caused by cidofovir occurs in the guinea pig eye at a concentration 40 times higher than was observed in the human eye.

Synergistic Rise in Ca2+Produced by Somatostatin and Acetylcholine in Ciliary Body Epithelial Cells

The purpose of these experiments was to demonstrate the presence of somatostatin receptors on the nonpigmented epithelial cells of the rabbit ciliary body and their link with intracellular Ca2+homeostasis. Freshly excised rabbit ciliary processes and nonpigmented cell layer explants were loaded with the fluorescent dye fura-2, and free-Ca2+concentration ([Ca2+]i) in the nonpigmented cells was measured with fluorescence ratio imaging. The cells were continuously perfused, and drugs were added to the perfusate. Somatostatin-14 (SS14, 0.1–1.0 μm) or acetylcholine (ACh, 10 μm) applied alone produced small increases in [Ca2+]i. However, SS14 (0.1 μm) in combination with ACh (10 μm) induced a massive increase in [Ca2+]i(25.7±3.3 times the baseline level,n=28). The dose-response curve for SS14 (in the presence of 10 μmACh) was sigmoidal with an EC50of 3.9 nmand Hill coefficient of 2.5, indicating the requirement for multiple SS receptor activation. Somatostatin-28 could mimic the effect of SS14, although a much higher concentration was required. Shifting the SS14 dose–response curve to the right by about two-orders of magnitude resulted in a fit to the SS28 data. The response to ACh+SS14 could not be blocked by the α2-adrenergic blocker yohimbine (Yoh, 10 μm) or the A1-specific adenosinergic antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 μm). Incubation of the tissue with pertussis toxin (PTx, 1 μg ml−1) did not alter the response to ACh alone but eliminated the synergistic effect of somatostatin. We conclude that nonpigmented epithelial cells of the rabbit ciliary body possess a novel somatostatin receptor whose activation can synergistically potentiate the rise in [Ca2+]iproduced by ACh. This potentiation appears to occur via a pertussis-toxin-sensitive pathway, perhaps through Gi.

Synergistic Increase in Ca2+Produced by A1Adenosine and Muscarinic Receptor Activation via a Pertussis-Toxin-Sensitive Pathway in Epithelial Cells of the Rabbit Ciliary Body

The combined effects of adenosine and acetylcholine on the intracellular free-Ca2+concentration in nonpigmented epithelial cells of the rabbit ciliary body were investigated using fura-2 fluorescence-ratio imaging. Acetylcholine (10 μM) by itself produced a modest increase in [Ca2+]i. Acetylcholine in combination with adenosine, or with the A1-specific agonists N6-cyclohexyl-adenosine, N6-cyclopentyl-adenosine and (R)-N6-(2-phenyl-1-methylethyl)-adenosine (0.1–1 μM), induced a massive increase in [Ca2+]i, which could be blocked by the A1-specific antagonist 8-cyclopentyl-1,3-dipropylxanthine. However, the A2-specific agonist 2-[(p-2-carboxyethyl)-phenethylamino]-5′-N-ethylcarboxamide-adenosine and the antagonist 3,7-dimethyl-1-(2-propynyl)xanthine were without effect. Incubation of the tissue with pertussis toxin did not alter the response to ACh alone but eliminated the synergistic effect of adenosine (or of epinephrine). It was concluded that in the epithelial cells of the rabbit ciliary body, adenosine and epinephrine synergistically potentiate the rise in [Ca2+]iproduced by ACh. This potentiation appears to occur via a pertussis-toxin-sensitive pathway, perhaps through Gi.

Relationship between caffeine-induced ocular hypertension and ultrastructure changes of non-pigmented ciliary epithelial cells in rats.

The purpose of this study was to morphologically assess a possible mechanism for caffeine-induced ocular hypertension. Taking into consideration the relationship between the secretion of aqueous humor and the ultrastructure of the ciliary body, the time course of the morphological features in the ciliary epithelium when caffeine was administered intravenously to male Wistar rats was investigated by electron-microscopy. These morphological findings were also compared with the changes in the intraocular pressure (IOP). A significant increase in IOP was noted 15 min and 1 hr after a single dosing of caffeine alone. This change disappeared in all animals within 2 hr after dosing. The IOP in the animals receiving caffeine and the beta-blocker befunolol, which lowers the IOP by inhibiting aqueous humor secretion, decreased significantly from 15 min after dosing, and this change persisted 2 hr after dosing. In electron-microscopy 15 min and/or 1 hr after dosing with caffeine, a slight dilatation in the lateral intercellular spaces near the basement membrane of the non-pigmented ciliary epithelium was observed and the interdigitations between the non-pigmented epithelial cells were intact. Reversal of these changes was observed 2 hr after dosing. On the other hand, the lateral intercellular spaces between the non-pigmented epithelial cells were markedly dilated and the interdigitations were disorganized following dosing with caffeine alone and in combination with befunolol. These results described here indicate that the intravenous administration of caffeine causes ocular hypertension and also changes in the non-pigmented ciliary epithelium, suggesting an enhancement of aqueous humor transportation. This paradigm in the rat is considered to be useful to further assess caffeine-induced ocular hypertension and for use as an animal model in glaucoma research associated with an aqueous humor secretion.

Detection and immunolocalization of macrophage migration inhibitory factor in rat iris and ciliary epithelium

To elucidate the role of macrophage migration inhibitory factor (MIF) in ocular inflammation, we examined the localization of MIF in the normal anterior uveal tract of rats. Immunohistochemistry using an anti-MIF antibody revealed that MIF was present in non-pigment epithelial cells of the ciliary body and the epithelial cells of the iris. Western blot analysis of these tissues showed a single band specific for MIF protein. The expression of MIF mRNA in these tissues was further confirmed by reverse transcription-polymerase chain reaction. Since MIF is known to be a potent proinflammatory cytokine, identification of the protein in iris and ciliary epithelial cells suggests the possibility that it may play an important role in ocular inflammation.