Nonpigmented Research Articles

Electron Probe X-Ray Microanalysis of Rabbit Ciliary Epithelium

Rabbit iris–ciliary bodies were preincubated in control and experimental Ringer's solutions before quick freezing, cryosectioning, dehydration and electron probe X-ray microanalysis. After preincubation in a baseline bicarbonate-free Cl -Ringer's solution, the ciliary epithelial intracellular Na +, K +and Cl -concentrations were estimated to be 15±3, 162±14 and 46±5mmolkg -1intracellular water, respectively. The water and elemental Na, K, Cl and P contents were similar in the non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells. As expected, inhibition of the Na,K-exchange pump by preincubation with ouabain markedly increased the intracellular Na content, and markedly reduced the intracellular K content, verifying the validity of the experimental analysis. The Cl -channels of the NPE cells likely play a critical role in determining the rate of aqueous humor formation. Therefore, we have examined the effects of altering Cl -transport on the intracellular composition in this initial microprobe study of the ciliary epithelium. As expected, exposure to bicarbonate increased the intracellular Cl and water contents. Replacement of external Cl -by NO 3 -was twice as effective as replacement by gluconate in leaching Cl -out of the intracellular compartment. An unexpected finding was that NO 3 -replacement of internal Cl -substantially increased the intracellular Na and decreased the intracellular K content, possibly by stabilizing the Na,K-pump in the E 1P form and inhibiting enzyme activity.

Ion transport asymmetry and functional coupling in bovine pigmented and nonpigmented ciliary epithelial cells

The solute and water transport properties of the bovine ciliary epithelium were studied using isolated pigmented (PE) and nonpigmented (NPE) cells. It was shown that these cells were functionally coupled by demonstrating dye diffusion between paired PE and NPE cells after microinjection of lucifer yellow. Electronic cell sizing was used to measure cell volume changes of isolated PE and NPE cells in suspension after anisosmotic perturbations and after transport inhibition under isosmotic conditions. The PE cells showed the presence of a regulatory volume increase when subjected to osmotic shrinkage with NaCl, whereas the NPE cells did not demonstrate a regulatory volume increase under these conditions. In contrast, the NPE cells exhibited a regulatory volume decrease when subjected to osmotic swelling, whereas the PE cells did not recover from swelling. The regulatory volume decrease in NPE cells was inhibited by increased bath K or pretreatment with quinine (1 mM). The presence of a bumetanide-sensitive mechanism capable of moving measurable amounts of solute and water, probably Na-K-2Cl cotransport, was demonstrated in the PE cells but absent in the NPE cells. Bumetanide produced a dose-dependent shrinkage of PE cells at concentrations as low as 1 microM. Isosmotically reducing bath Cl, Na, or K concentration caused a rapid shrinkage of PE cells that was bumetanide inhibitable. The asymmetry of transport properties in PE and NPE cells supports a functional syncytium model of aqueous humor formation (39) across the two layers of the ciliary epithelium wherein ion uptake from the blood is carried out by the PE cells and ion extrusion by the NPE cells. Gap-junction coupling between the cells allows the ions taken up by the PE cells to move into the NPE cells. Extrusion of Na by the Na-K pump across the aqueous facing (basolateral) membranes of the NPE cells, most likely accompanied by Cl, determines the formation of the aqueous humor.

Immunocytochemical similarities between retinal photoreceptors and pinealocytes of various vertebrates including man

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-α), phosphodiesterase (PDE-α), and cGMP-gated channel α-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.

Melatonin and the immune system

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-α), phosphodiesterase (PDE-α), and cGMP-gated channel α-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.

Fluorophotometry of bicarbonate and ion transport in the isolated, native pigmented (PE) and non pigmented (NPE) layers of the rabbit ciliary body eptthelium

Fluorophotometry of bicarbonate and ion transport in the isolated, native pigmented (PE) and non pigmented (NPE) layers of the rabbit ciliary body eptthelium

Human ciliary body in organ culture.

Ciliary body explants from 30 human eyes were maintained in organ culture up to 14 days. The age of the donors ranged from 45 to 85 years, the post mortem time from 4 to 22 hours. The ciliary epithelium as well as the underlying stroma were studied light- and electronmicroscopically before incubation and after 1, 3, 5, 7 and 14 days of culture. At the same time intervals, the localization of Na/K-ATPase and carbonic anhydrase (CA) were examined histochemically. If the cells were already damaged before incubation in medium (9 cases), they did not recover in culture. Best results were obtained after 3 to 5 days of culture with a survival rate of more than 90% after 3 days and more than 70% after 5 days, respectively. Both the nonpigmented (NPE) and the pigmented epithelium (PE) of the pars plicata in culture retained the morphological characteristics of epithelia involved in active secretion, namely elaborate infoldings of the cell membranes, numerous mitochondria in the cytoplasm and high activity of Na/K-ATPase and CA. In addition the adjacent capillaries were still fenestrated. After longer incubation times (7-14 days) the NPE and PE cells were filled with increasing amounts of lipid droplets and glycogen granules, indicating changes in metabolism.

Cellular distribution and differential gene expression of the three alpha subunit isoforms of the Na,K-ATPase in the ocular ciliary epithelium.

We have investigated the localization and pattern of expression of the three alpha subunit isoforms of Na,K-ATPase in the transporting ciliary epithelium of the bovine eye. Using specific cDNA probes and antisera to the alpha 1, alpha 2, and alpha 3 isoforms of Na,K-ATPase, we demonstrated that mRNAs and polypeptides for the three distinct forms of the Na,K-ATPase alpha subunit (alpha 1, alpha 2, and alpha 3) were expressed in the ciliary epithelium in vivo. Immunochemical localization of the three alpha isoforms of Na,K-ATPase in two ultrastructurally different regions of the ciliary epithelium (namely, the pars plicata and pars plana) revealed that the three alpha isoforms of Na,K-ATPase were distributed in a distinct fashion in the basolateral plasma membrane domains of nonpigmented (NPE) and pigmented (PE) cells. The NPE cells in the pars plicata showed an immunoreactive signal to all the three alpha isoforms; in the pars plana, they showed immunoreactive signals only for the alpha 1 and alpha 2 isoforms but not for alpha 3. The PE cells, in both the pars plana and pars plicata regions, showed an immunoreactive signal only for the alpha 1 isoform; immunoreactive signals were not detected for alpha 2 and alpha 3. To verify the differential immunostaining patterns of NPE and PE cells, specific antibodies for each of the three alpha subunit isoforms of Na,K-ATPase were applied to immunoblots containing microsomal fractions from flow cytometric-sorted cells (NPE and PE). Our results indicate that alpha 1, alpha 2, and alpha 3 polypeptides were present in microsomal fractions of NPE cells of the pars plicata and pars plana and that the alpha 1 polypeptide was the only polypeptide present in the PE cells from both regions of the ciliary epithelium. These results also revealed that the alpha 3 isoform epitope recognized by the monoclonal antibody McB-X3.1 in the pars plicata is not readily accessible in the pars plana. A cell line was established from the ciliary epithelium of a bovine eye by viral transformation with simian virus 40. In culture, this cell line expressed all three alpha isoforms at the mRNA and polypeptide levels, suggesting that the line may have derived from the NPE layer.

Mechanically stripped pigmented and non-pigmented epithelium of the shark ciliary body: Morphology and transepithelial electrical properties

Sections of intact ciliary epithelium and mechanically stripped non-pigmented (NPE) and pigmented (PE) cell layers of adult sharks (Squalus acanthias) were mounted in Ussing-type chambers (area 0.1 cm2). Addition of 10(-5) M forskolin to the aqueous side of intact epithelium significantly increased short-circuit current (Isc) within 15 min and a maximum of approx. 30 microA cm-2 was reached after 45-60 min. Transepithelial potential difference (V) increased from -0.8 mV (aqueous side negative as compared with blood/stromal side) to -1.5 mV, whereas resistance (R) was unchanged (50 omega cm2). Forskolin was without effect when applied to the blood side. In stripped PE preparations (R 15 omega cm2), 10(-5) M forskolin applied to the apical side induced a qualitatively similar change of Isc and V compared with the intact tissue. The forskolin-induced effects were fully reversed by 10(-4) M bumetanide and were not dependent on pretreatment of the tissue with 10(-3) M BaCl2. In stripped NPE preparations resistance was usually less than 10 omega cm2 and was not stable. This is consistent with the morphologic observation that although tight junctions were still demonstrable in stripped NPE cells, the apical membranes were damaged. In preparations taken for light and electron microscopy the stripped PE layer revealed intact epithelial cells. In particular, the basal thirds of the stripped PE cells were in very close contact with each other. These attachment zones may have the appearance of tight junctions. Thus the PE cells of the shark ciliary epithelium can be successfully isolated for transepithelial transport studies. The adenylate cyclase system is present in PE cells, and transepithelial transport of chloride may be regulated by intracellular cAMP.

Expression of Na,K‐ATPase alpha subunit isoforms in the human ciliary body and cultured ciliary epithelial cells

We have analyzed the expression of Na,K-ATPase alpha subunit isoforms in the transporting ciliary processes of the human eye and in cultured cells derived from non-pigmented (NPE) and pigmented (PE) ciliary epithelium. Northern hybridization analysis shows that the mRNAs encoding all the three distinct forms of Na,K-ATPase alpha subunit [alpha 1, alpha 2, and alpha 3] are expressed in the human ciliary processes in vivo. Immunohistochemical analysis using antibodies specific for each of the three alpha subunit isoforms confirms that these polypeptides are present in the microsomal fraction from the human ciliary processes. The monoclonal antibody McB2, which is specific to the Na,K-ATPase alpha 2 subunit isoform, has been found to decorate specifically the basolateral membrane domains of NPE cells but not of the PE cells, suggesting its expression in vivo only in the ocular NPE ciliary epithelium. However, cultured cells derived from the NPE and PE layers exhibit a different pattern of expression of mRNA and protein for the Na,K-ATPase alpha subunit isoforms when compared to the tissue. Both the NPE and PE cells express alpha 1 and alpha 3 mRNA and polypeptide, whereas alpha 2 mRNA and polypeptide are undetectable in these cells. The established cell lines derived from the NPE layer express comparable levels of the alpha 1 and alpha 3 isoforms of Na,K-ATPase as detected in the primary culture. However, the established NPE cell lines are also distinguishable from the normal PE cells when analyzed by Western blot analysis with A x 2 antibodies. The results presented here clearly show that the NPE and PE cells in the ciliary body have a distinct expression of Na,K-ATPase alpha subunit isoforms as compared to cultured cells.

Evidence that the α and α(+) isoforms of the catalytic subunit of (Na +,K +)-ATPase reside in distinct ciliary epithelial cells of the mammalian eye

Polyclonal antibodies against the canine kidney (Na +,K +)-ATPase were used to examine the localization and distribution of this protein in intact ciliary processes (CP) from bovine eyes by indirect immunofluorescence. The basolateral surface of non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells was found to be stained specifically for the (Na +,K +)-ATPase. Immunoblot analysis of intact CP, separated PE and NPE cells by density gradients and cultured ciliary epithelial cells, revealed two forms of the catalytic subunit of the (Na +,K +)-ATPase: the α and α(+). The α(+) form was enriched in NPE cells while α was in PE cells.

Enzymes of Mercapturate Pathway in Cultured Bovine Ciliary Epithelial Cells

Relative enzyme activities of the mercapturate pathway were determined in bovine ciliary pigmented (PE) and nonpigmented (NPE) epithelial cells in culture. Glutathione S-transferase activity was greater in PE cells than in NPE cells. Gamma-glutamyl transpeptidase activity appeared to be exclusively associated with NPE cells; the enzyme activity was virtually absent in PE cells. The level of cystine aminopeptidase activity was about the same in NPE and PE cells. N-acetyl transferase was predominantly found in NPE cells. If a similar enzyme distribution should exist in vivo in the bicellular layer of ciliary epithelium, the present result suggests that both NPE and PE cells are involved in the detoxification of xenobiotics via the mercapturate pathway, glutathione conjugation occurring primarily in the PE cells and the reactions catalyzed by the membrane-bound enzymes gamma-glutamyl transpeptidase and N-acetyl transferase taking place mainly in the NPE cells.

Visual cliff behavior of mice as a function of genetic differences in eye characteristics

The effects of type of breeding on visual cliff avoidance is evaluated using three groups of mice with different genetically determined eye characteristics—pigmented (P), non-pigmented (NP) and rod-deficient (RD)-tested on 2 occasions. The results show significant group differences on latency and descents. P and NP groups show high degree of cliff avoidance and NP significantly less descents than P. Strain differences were evident on latency and total descents for RD and on bolus count for NP. Latency decreased significantly from first to second test. Inter- and intraspecies differences are emphasized and genetic analysis of determinants of cliff avoidance suggested.