Nonpigmented Research Articles

Unveiling the distribution of free and bound phenolic acids, flavonoids, anthocyanins, and proanthocyanidins in pigmented and non-pigmented rice genotypes.

The total phenolic content, phenolic acid profile, anthocyanins, proanthocyanidins, flavonoids, and antioxidant capacity of the whole-grain and bran portion of sixteen distinct rice genotypes that correspond to three distinct pericarp bran colors-black, red, and non-pigmented (NP)-were examined. Ten free and bound phenolic acids, as well as two flavonoids, were analyzed using HPLC-PDA. The flavonoids included kaempferol and catechin hydrate, and the free phenolic acids included gallic acid, 2,5-dihydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, chlorogenic acid, trans-cinnamic acid, trans-ferulic acid, p-coumaric acid, and sinapic acid. Trans-ferulic acid (207.39 mg/kg), p-hydroxybenzoic acid (94.36 mg/kg), and p-coumaric acid (59.75 mg/kg) were the principal bound phenolic acids in pigmented rice genotypes, whereas in NP genotypes they were trans-ferulic acid (95.61 mg/kg) and p-hydroxybenzoic acid (58.32 mg/kg). The main free phenolic acid was syringic acid (120.43 mg/kg) in all genotypes. 2,5-dihydroxybenzoic acid was also detected in NP genotypes, mainly in the bound form (4.88 mg/kg). NP genotypes Basmati 386 and Punjab Basmati 7 also displayed high content of bran flavonoids (1001 and 1028 mg CE/100g). The bound form of phenolics had significant DPPH and ABTS + activity. This study found wide diversity in the phenolic acid profile, total phenolic constituents, and antioxidant activity in the bran and whole grain of pigmented and NP rice. The individual phenolic acids in free and bound forms in different fractions of the grain were found to exert their antioxidant activity differently. The results obtained will provide new opportunities to improve the nutritional quality of rice with enhanced levels of phytochemicals in the ongoing breeding programs. Black rice bran contains a high level of phytochemicals and thus has a potent pharmaceutical role. This information would enhance the use of whole-grain and bran of pigmented rice in food product development by food technologists. Further studies may be focused on clinical trials with respect to cancer and diabetes.

Characterization and Regulation of Gap Junctions in Porcine Ciliary Epithelium.

Gap junctions provide a conduit between the intracellular fluids of the pigmented (PE) and non-pigmented (NPE) ciliary epithelial cells, and are therefore critical in the secretion of the aqueous humor (AH). However, opinions differ concerning the connexin (Cx) composition of the gap junctions. Therefore, we aimed to characterize the expression of Cx in the porcine ciliary epithelium (CE), a favorable model for humans; and determine the contribution of the highest expressed Cx to AH secretion. Freshly-harvested porcine CE cells were used. The mRNA and protein expressions of gap junctions were assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. The relative gene expressions of various Cx were determined by quantitative PCR. The gap junction permeability of isolated PE-NPE cell couplets was evaluated by Lucifer Yellow dye transfer. Using RT-PCR and WB, Cx43, Cx45, Cx47, Cx50, and Cx60 were present in porcine CE, with Cx43 being the most abundant isoform, having over 200-fold higher expression than other Cx. Cx43 was primarily localized in the PE-NPE interface and the basolateral membranes of PE cells. Knockdown of Cx43 by siRNA significantly reduced gene and protein expressions, resulting in reduction of transcellular fluid flow by 90%. Cx43 was found to be the major component of gap junctions in porcine CE. Consistent with results from a bovine model, our results support the important role of Cx43 in mediating AH secretion. This finding may shed light on the development of a novel ocular hypotensive agent.

Abstract A21: Functional differences among melanoma cells separated according to pigment content

Abstract Improved understanding of cancer progression is essential for the development of rational treatment strategies. In tumors that follow a cancer stem cell (CSC) model, targeting only small populations of tumorigenic cells should be sufficient to control disease. In contrast, targeting only some cancer cells will be unsuccessful if every cell in a tumor can convert plastically into another or can acquire genetic changes that enhance tumorigenesis. In these tumors, every cell needs to be eliminated. In melanoma, development of patient-derived xenografting (PDX) of melanomas in immunocompromised mice has permitted testing of the CSC model, yielding conflicting results. Surprisingly, none of these studies has comprehensively evaluated markers of melanocytic differentiation. This is mainly due to the paucity of markers of melanocytic differentiation that enable separation of more and less differentiated cells for side-by-side studies of in vitro clonogenesis and in vivo tumorigenesis. We observe within overtly pigmented melanomas significant heterogeneity in pigmentation of constituent cells; although some cells contain melanin pigment, the canonical marker of melanocytic differentiation, many cells usually a majority are non-pigmented. We have thus devised a novel flow cytometry method to separate melanoma cells according to their content of melanin. This method, which relies on the near-infrared emission properties of eumelanin, allows separation of pigmented (P) and non-pigmented (NP) melanoma cells from the same tumor (or cell culture) for side-by-side clonogenesis assays. Using this method, we surprisingly found that in B16-F10 mouse melanoma cells, NP cells are far more clonogenic than P cells from the same culture. Further, only NP cells recapitulated the heterogeneous phenotypes of the parental culture. In pigmented PDX melanomas, NP cells exhibited enhanced clonogenicity compared to their P cell counterparts in the same tumor, although variability among PDX melanomas was notable. This suggests that NP and P cells may be hierarchically related in pigmented melanoma cell lines and in some pigmented PDX melanomas, consistent with a CSC model, with NP cells primarily driving cell line or tumor propagation. Our data suggest that the CSC model may in fact apply to melanoma, at least in some contexts, despite the contradicting data published so far in studies of markers largely unrelated specifically to melanocytic differentiation. However, the abundance of NP cells in most evaluated pigmented cell lines and pigmented PDX tumors indicates that the hierarchical cellular structure of these entities is flat, such that, at least in most cases, a high proportion of melanoma cells would need to be eliminated to eradicate the disease. Citation Format: Clare G. Fedele, Rose Hyslop, Zoe Grant, Ralph Rossi, Mark J. Shackleton. Functional differences among melanoma cells separated according to pigment content. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A21.

In silico analysis of the molecular machinery underlying aqueous humor production: potential implications for glaucoma

BackgroundThe ciliary body epithelia (CBE) of the eye produce the aqueous humor (AH). The equilibrium between the AH production by the CBE and the outflow through the trabecular meshwork ultimately determines the intraocular pressure (IOP). An increased IOP is a major risk factor for primary open angle glaucoma (POAG). This study aims to elucidate the molecular machinery of the most important function of the CBE: the AH production and composition, and aims to find possible new molecular clues for POAG and AH production-lowering drugs.MethodsWe performed a gene expression analysis of the non-pigmented (NPE) and pigmented epithelia (PE) of the human CBE of post mortem eyes. We used 44 k Agilent microarrays against a common reference design. Functional annotations were performed with the Ingenuity knowledge database.ResultsWe built a molecular model of AH production by combining previously published physiological data with our current genomic expression data. Next, we investigated molecular CBE transport features which might influence AH composition. These features included caveolin- and clathrin vesicle-mediated transport of large biomolecules, as well as a range of substrate specific transporters. The presence of these transporters implies that, for example, immunoglobins, thyroid hormone, prostaglandins, cholesterol and vitamins can be secreted by the CBE along with the AH. In silico, we predicted some of the molecular apical interactions between the NPE and PE, the side where the two folded epithelia face each other. Finally, we found high expression of seven POAG disease genes in the plasma membrane of extracellular space of the CBE, namely APOE, CAV1, COL8A2, EDNRA, FBN1, RFTN1 and TLR4 and we found possible new targets for AH lowering drugs in the AH.ConclusionsThe CBE expresses many transporters, which account for AH production and/or composition. Some of these entries have also been associated with POAG. We hypothesize that the CBE may play a more prominent role than currently thought in the pathogenesis of POAG, for example by changing the composition of AH.

Gene Expression and Functional Annotation of the Human Ciliary Body Epithelia

PurposeThe ciliary body (CB) of the human eye consists of the non-pigmented (NPE) and pigmented (PE) neuro-epithelia. We investigated the gene expression of NPE and PE, to shed light on the molecular mechanisms underlying the most important functions of the CB. We also developed molecular signatures for the NPE and PE and studied possible new clues for glaucoma.MethodsWe isolated NPE and PE cells from seven healthy human donor eyes using laser dissection microscopy. Next, we performed RNA isolation, amplification, labeling and hybridization against 44×k Agilent microarrays. For microarray conformations, we used a literature study, RT-PCRs, and immunohistochemical stainings. We analyzed the gene expression data with R and with the knowledge database Ingenuity.ResultsThe gene expression profiles and functional annotations of the NPE and PE were highly similar. We found that the most important functionalities of the NPE and PE were related to developmental processes, neural nature of the tissue, endocrine and metabolic signaling, and immunological functions. In total 1576 genes differed statistically significantly between NPE and PE. From these genes, at least 3 were cell-specific for the NPE and 143 for the PE. Finally, we observed high expression in the (N)PE of 35 genes previously implicated in molecular mechanisms related to glaucoma.ConclusionOur gene expression analysis suggested that the NPE and PE of the CB were quite similar. Nonetheless, cell-type specific differences were found. The molecular machineries of the human NPE and PE are involved in a range of neuro-endocrinological, developmental and immunological functions, and perhaps glaucoma.

Cellular Localization of Glutamate and Glutamine Metabolism and Transport Pathways in the Rat Ciliary Epithelium

To investigate how glutamate and glutamine levels are established in the aqueous humor by identifying the transporters and metabolism pathways that contribute to the differential accumulation of glutamate and glutamine between the distinct epithelial cell layers that constitute the ciliary body. Postembedding immunohistochemistry and silver intensification were used to quantify the relative distributions of glutamate, glutamine, and related amino acids (aspartate, alanine, GABA, and glycine) in the pigmented (PE) and nonpigmented (NPE) epithelial cells of the ciliary body. Fluorescent immunocytochemistry was used to localize Na(+)-dependent glutamate transporters (EAAT1-5), glutamine transporters (LAT1, LAT2, and b(0,+)AT), and the enzyme glutamine synthetase (GS) in the ciliary epithelium. Intravitreal injection of the GS inhibitor methionine sulfoximine (MSO) or the EAAT functional probe D-aspartate was used to modulate GS activity and indirectly monitor glutamate uptake from the aqueous, respectively. Although glutamate, glutamine, and alanine were preferentially accumulated in NPE relative to PE cells, no such differential distribution of aspartate, GABA, or glycine was observed. This differential distribution of amino acids was abolished by a single injection of MSO that caused a decrease in glutamine and an increase in glutamate levels in NPE compared with PE cells. This amino acid distribution plus an observed strong labeling of EAAT3 in the interface between the PE and the NPE cell layers indicate that EAAT3 mediates the uptake of glutamate from the blood. Weaker EAAT3 labeling of the basolateral membranes of NPE cells, coupled with the accumulation of injected D-aspartate by the ciliary epithelium, indicates that NPE cells also mediate glutamate uptake directly from the aqueous. In contrast, the basolateral localization of LAT1 and b(0,+)AT in NPE cells suggest that these transporters may mediate glutamine efflux from the NPE cells into the aqueous. The basolateral membrane localization of EAAT3 and LAT1/b(0,+)AT in NPE cells indicates that the low glutamate and high glutamine levels observed in the aqueous are determined by glutamate uptake and glutamine efflux, respectively. Furthermore, the concentration gradient for glutamine efflux appears to be generated by the active accumulation of glutamate by EAAT3, located in the apical membrane of NPE cells and the subsequent conversion of the accumulated glutamate to glutamine by GS in NPE cells. This suggests that in contrast to fluid transport, which uses both the PE and the NPE cell layers, the transepithelial transport of glutamine occurs primarily in the NPE cell layer.

Pathways for ATP release by bovine ciliary epithelial cells, the initial step in purinergic regulation of aqueous humor inflow

ATP release by nonpigmented (NPE) and pigmented (PE) ciliary epithelial cells is the enabling step in purinergic regulation of aqueous humor formation, but the release pathways are unknown. We measured ATP release from primary cultures of bovine mixed NPE and PE (bCE) cells and transformed bovine NPE and PE cells, using the luciferin-luciferase reaction. Hypotonicity-triggered bCE ATP release was inhibited by the relatively selective blocker of pannexin-1 (PX1) hemichannels (probenecid, 1 mM, 47 ± 2%), by a connexin inhibitor (heptanol, 1 mM, 49 ± 4%), and by an inhibitor of vesicular release (bafilomycin A1, 25 ± 2%), but not by the P2X(7) receptor (P2RX(7)) antagonist KN-62. Bafilomycin A1 acts by reducing the driving force for uptake of ATP from the cytosol into vesicles. The reducing agent dithiothreitol reduced probenecid-blockable ATP release. Similar results were obtained with NPE and PE cell lines. Pannexins PX1-3, connexins Cx43 and Cx40, and P2RX(7) were identified in native cells and cell lines by RT-PCR. PX1 mRNA expression was confirmed by Northern blots; its quantitative expression was comparable to that of Cx43 by real-time PCR. Heterologous expression of bovine PX1 in HEK293T cells enhanced swelling-activated ATP release, inhibitable by probenecid. We conclude that P2RX(7)-independent PX1 hemichannels, Cx hemichannels, and vesicular release contribute comparably to swelling-triggered ATP release. The relatively large response to dithiothreitol raises the possibility that the oxidation-reduction state is a substantial regulator of PX1-mediated ATP release from bovine ciliary epithelial cells.

Electron microprobe analysis of rabbit ciliary epithelium indicates enhanced secretion posteriorly and enhanced absorption anteriorly

The rate of aqueous humor formation sequentially across the pigmented (PE) and nonpigmented (NPE) ciliary epithelial cell layers may not be uniform over the epithelial surface. Because of the tissue's small size and complex geometry, this possibility cannot be readily tested by conventional techniques. Rabbit iris-ciliary bodies were divided, incubated, quick-frozen, cryosectioned, and freeze-dried for electron probe X-ray microanalysis of the elemental contents of the PE and NPE cells. We confirmed that preincubation with ouabain to block Na(+),K(+)-ATPase increases Na(+) and decreases K(+) contents far more anteriorly than posteriorly. The anterior and posterior regions were the iridial portion of the primary ciliary processes and the pars plicata, respectively. Following interruption of gap junctions with heptanol, ouabain produced smaller changes in anterior PE cells, possibly reflecting higher Na(+) or K(+) permeability of anterior NPE cells. Inhibiting Na(+) entry selectively with amiloride, benzamil, or dimethylamiloride reduced anterior effects of ouabain by approximately 50%. Regional dependence of net secretion was also assessed with hypotonic stress, which stimulates ciliary epithelial cell regulatory volume decrease (RVD) and net Cl(-) secretion. In contrast to ouabain's actions, the RVD was far more marked posteriorly than anteriorly. These results suggest that 1) enhanced Na(+) reabsorption anteriorly, likely through Na(+) channels and Na(+)/H(+) exchange, mediates the regional dependence of ouabain's actions; and 2) secretion may proceed primarily posteriorly, with secondary processing and reabsorption anteriorly. Stimulation of anterior reabsorption might provide a novel strategy for reducing net secretion.

CAMP-activated maxi-Cl(-) channels in native bovine pigmented ciliary epithelial cells.

The eye's aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl(-) enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl(-) channels are expressed in PE cells, but their physiological significance is unclear. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl(-) channels with a unitary conductance of 272 +/- 2 pS (n = 80). Voltage steps from 0 to +/-80 mV, but not to +/-40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl(-) channels. cAMP also activated the maxi-Cl(-) channels in outside-out patches. In both cases, maxi-Cl(-) channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl(-) concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl(-) concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at +/-80 but not at +/-40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl(-) channels of native PE cells that contribute to Cl(-) release particularly from Cl(-)-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl(-) reabsorption into the ciliary stroma.

Inhibition of NHE-1 Na+/H+ exchanger by natriuretic peptides in ocular nonpigmented ciliary epithelium.

The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) display hypotensive effects in the mammalian eye by lowering the intraocular pressure (IOP), a function that is mediated by the bilayer ocular ciliary epithelium (CE), in conjunction with the trabecular meshwork. ANP regulates Na(+)/H(+) exchanger (NHE) activity, and inhibitors of NHE have been shown to lower IOP. We examined whether NPs influence the NHE activity of the CE, which is comprised of pigmented (PE) and nonpigmented (NPE) epithelial cells, by directly recording the rate of intracellular pH (pH(i)) recovery from its inner NPE cell layer. NPs inhibited, in a dose-dependent manner (1-100 nM), the rate of pH(i) recovery with the order of potency CNP > ANP > BNP, indicative that this inhibition is mediated by the presence of NPR type B receptors. 8-Bromo-cGMP (8-BrcGMP), a nonhydrolyzable analog of cGMP, mimicked NPs in inhibiting the rate of Na(+)-dependent pH(i) recovery. In contrast, ethylisopropyl amiloride (EIPA, 100 nM) or amiloride (10 microM) completely abolished the pH(i) recovery by NHE. 18alpha-Glycyrrhetinic acid (18alpha-GA), a gap junction blocker, attenuated the inhibitory effect of CNP on the rate of pH(i) recovery, suggesting that NHE activity in both cell layers of the CE is coregulated. This interpretation was supported, in part, by the coexpression of NHE-1 isoform mRNA in both NPE and PE cells. The mechanism by which the inhibitory effect of NPs on NHE-1 activity might influence the net solute movement or fluid transport by the bilayer CE remains to be determined.

Molecular Profiling and Cellular Localization of Connexin Isoforms in the Rat Ciliary Epithelium

The functionally distinct epithelial layers of the ciliary body act as a syncitium to produce the aqueous humour. Ultrastructural studies have shown that the pigmented (PE) and non-pigmented (NPE) cell layers of the ciliary epithelium are connected by gap junctions. However the molecular composition of gap junctions both between and within the two cell layers has not been comprehensively studied. To address this issue the authors have performed an extensive molecular screening of connexin (Cx) expression patterns in ciliary epithelium of the rat. Initially, mRNA was extracted from rat ciliary bodies, reverse-transcribed, and subjected to two rounds of PCR using primer sets designed against each of the 14 Cx isoforms known to be expressed in the rat. This initial screening protocol amplified eight candidate Cx isoforms (Cxs 26, 31, 33, 37, 40, 43, 45 and 46). The Cx isoforms identified in this initial screen were then first assigned to the ciliary epithelium itself (Cxs 26, 31, 40 and 43) or structures outside the epithelium (Cxs 37, 40, and 45) using immunohistochemistry performed on ciliary body whole mounts. No convincing evidence for either Cx 33 or 46 labelling was found in the ciliary body. Then the four Cx isoforms localized to the epithelium were further localized to specific membrane domains within the epithelial cell layers by performing high resolution imaging of the antibody labeling patterns obtained in cryosections. This enabled Cx26 and 31 to be specifically localized to spatially different gap junctions between NPE cells. Cx31 labeled gap junctions associated with an extensive network of membrane interdigitations found between NPE cells at their basal surfaces. In contrast Cx26 labeling in NPE cells was restricted to the basolateral membranes of adjacent NPE cells. Cx40 and Cx43 were both localized to the PE–NPE interface where they formed discrete homomeric/homotypic gap junction plaques. No convincing evidence was found for antibody labeling between PE cells. Thus it appears that intercellular communication, both within the NPE layer and between the PE and NPE cell layers, is mediated by gap junction channels that have distinctive permeability properties. In particular the results raise the possibility that the permeability of PE–NPE gap junctions can be modulated by changing the Cx43 : Cx40 expression ratio. Whether such a change in Cx expression ratios occurs and what effect it has on aqueous humour production and composition remains to be determined.

Molecular Evidence That Human Ocular Ciliary Epithelium Expresses Components Involved in Phototransduction

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.

Catecholaminergic Regulation of Na-K-Cl Cotransport in Pigmented Ciliary Epithelium: Differences Between PE and NPE

Pigmented (PE) and nonpigmented (NPE) ciliary epithelial cells comprise the ciliary epithelium, the site of aqueous humor formation in the eye. In man, catecholamines increase the rate of aqueous humor formation, but the mechanism underlying these effects is not understood. Recent evidence suggests that Na-K-Cl cotransport plays a central role in blood-to-aqueous chloride transport across ciliary epithelium in cow and rabbit. We therefore investigated whether catecholamines stimulate Na-K-Cl cotransport in human PE cells. Na-K-Cl cotransporter protein was detected as a 170kDa protein band on immunoblots. Immunofluorescence microscopy detected cotransporter on the basolateral membranes of the PE layer of ciliary epithelium from a human donor. Cotransporter immunofluorescence was also detected in cultured PE cells. Na-K-Cl cotransport activity measured as ouabain-insensitive bumetanide-sensitive86Rb uptake was stimulated by isoproterenol 1.6-fold, with an EC50=28n M and maximal stimulation at 1μM. Other transport mechanisms involved in86Rb uptake were not affected. Stimulation by 1 μM isoproterenol was blocked by 10n M ICI 118,551, a β2-specific receptor antagonist, whereas the receptor subtype-specific antagonists yohimbine (α2), prazosin (α1) and atenolol (β1) were ineffective. Norepinephrine stimulation (EC50=280n M) was also blocked by ICI 118,551. Dopamine stimulated Na-K-Cl cotransport 1.6-fold with an EC50=14μM. The dopamine effect could not be blocked by 10μM SCH 23390, a D1-antagonist, but was abolished by ICI 118,551. Forskolin and CPT-cAMP stimulated Na-K-Cl cotransport 1.79- and 1.71-fold, respectively, whereas the inactive forskolin analogue 1,9-dideoxyforskolin had no effect. However, high concentrations of the PKA inhibitors PKI amide 14–22 and KT 5720 were needed to inhibit both PKA activity in cell lysates and isoproterenol stimulation of cotransport. This finding may indicate the presence of a novel PKA isoform in PE cells. Inhibitors of other protein kinases, including myosin light chain kinase, protein kinase G, calmodulin-dependent kinase and tyrosine kinase, were without effect on stimulated Na-K-Cl cotransport. When EC50s for catecholaminergic stimulations of Na-K-Cl cotransport in PE were compared to those in NPE, values within five-fold of one another were seen for isoproterenol and norepinephrine. In contrast, dopamine was 28-fold more potent in NPE than in PE. The data suggest that both PE and NPE possess β2adrenergic receptors, but only NPE cells possess dopamine D1 receptors linked to Na-K-Cl cotransport.

Functional Expression of Components of the Natriuretic Peptide System in Human Ocular Nonpigmented Ciliary Epithelial Cells

The expression of the natriuretic peptide system in the human ocular ciliary epithelium (CE) and in cultured nonpigmented (NPE) ciliary epithelial cells was examined. By RT-PCR and DNA sequencing, we demonstrated that the CE and NPE cells express mRNA for (i) ANP; (ii) BNP; (iii) NPR-A, NPR-B, and NPR-C receptors; and (iv) the neutral endopeptidase 24.11. Radioimmunoassay results indicate that BNP is secreted by cultured NPE cells at much higher levels than ANP. NPR-A and NPR-B receptors elicited a cGMP response to ANP, BNP, and CNP, in a rank order of potency (CNP ⪢ ANP ≥ BNP), indicative that the NPR-B receptor is predominant in NPE cells. A71915, an inhibitor of NPR-A activity, attenuated (65–75%) cGMP response to ANP and BNP, but not to CNP. C-ANP4–23elicited an inhibitory effect (30–37%) on basal levels of cAMP in NPE cells and on forskolin NPE-treated cells, indicative that the NPR-C receptor is functional in these cells. PMA induced, in NPE cells, a long-term downregulation (75–85%) of NPR-C receptor mRNA, but not of NPR-A or NPR-B receptor mRNA, suggesting a differential regulation of NPR-C receptor mRNA via activation of PKC. Collectively, our data provide molecular evidence that all the components of the natriuretic peptide system with the exception of CNP are coexpressed in the ocular NPE ciliary epithelial cells, where they may function as local autocrine/paracrine modulators to influence eye pressure.

A3 adenosine receptors regulate Cl- channels of nonpigmented ciliary epithelial cells.

Adenosine stimulates Cl- channels of the nonpigmented (NPE) cells of the ciliary epithelium. We sought to identify the specific adenosine receptors mediating this action. Cl- channel activity in immortalized human (HCE) NPE cells was determined by monitoring cell volume in isotonic suspensions with the cationic ionophore gramicidin present. The A3-selective agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) triggered shrinkage (apparent Kd = 55 +/- 10 nM). A3-selective antagonists blocked IB-MECA-triggered shrinkage, and A3-antagonists (MRS-1097, MRS-1191, and MRS-1523) also abolished shrinkage produced by 10 microM adenosine when all four known receptor subtypes are occupied. The A1-selective agonist N6-cyclopentyladenosine exerted a small effect at 100 nM but not at higher or lower concentrations. The A2A agonist CGS-21680 triggered shrinkage only at high concentration (3 microM), an effect blocked by MRS-1191. IB-MECA increased intracellular Ca2+ in HCE cells and also stimulated short-circuit current across rabbit ciliary epithelium. A3 message was detected in both HCE cells and rabbit ciliary processes using RT-PCR. We conclude that human HCE cells and rabbit ciliary processes possess A3 receptors and that adenosine can activate Cl- channels in NPE cells by stimulating these A3 receptors.

Molecular identification and coexpression of galanin and GalR-1 galanin receptor in the human ocular ciliary epithelium: differential modulation of their expression by the activation of alpha2- and beta2-adrenergic receptors in cultured ciliary epithelial cells.

Here we report the coexpression of the neuropeptide galanin and GalR-1 galanin receptors in the human ciliary epithelium, a bilayer of neuroepithelial cells [nonpigmented (NPE) and pigmented (PE)] with neuroendocrine functions, and in a cell line (ODM-2) derived from the NPE cells. Stimulation of ODM-2 cells with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or forskolin resulted in an up-regulation (two- to threefold) of galanin mRNA expression. Procaterol, a selective beta2-adrenergic agonist, and the catecholamine isoproterenol exerted a long-term down-regulation on galanin mRNA expression when added alone or in combination with PMA or forskolin. These actions exerted by procaterol or isoproterenol were abolished in the presence of ICI 118,551, a selective beta2-adrenergic antagonist. A radioimmunoassay for galanin peptide indicated that galanin or a galanin-like product is present in the human aqueous humor fluid and is accumulated with time in the culture medium of ODM-2 cells. It is interesting that norepinephrine, which exhibited no effect on galanin mRNA expression, induced a down-regulation in the level of galanin or galanin-like product accumulated in the medium of cultured ODM-2 cells to levels even lower than those induced by beta2-adrenergic agonists. This effect is best explained by the concomitant up-regulation (four- to fivefold) of GalR-1 galanin receptor transcripts induced through the activation of alpha2-adrenergic receptors. These findings support the view that pathways elicited by the activation of alpha2- and beta2-adrenergic receptors influence the expression of galanin and GalR-1 galanin receptors in ciliary epithelial cells.

Organisation of microbenthic communities in intertidal Estuarine flats, a case study from the molenplaat (Westerschelde estuary, The Netherlands)

Summary The microbenthic communities of a tidal flat in the Westerschelde estuary were studied at 4 stations in late spring and early autumn 1996. Additional information on the diatom component of these communities was obtained from a one-year survey of these organisms. Total biomass of pigmented (PIG) protists greatly exceeded that of non-pigmented (NPIG) protists in late spring, especially at the more silty stations. However, in autumn, the ratio of PIG/NPIG protists was much lower and is In general, silty sediments appear to be characterized by considerable temporal changes in microbenthic biomass and composition, related to predictable seasonal changes in environmental conditions as well as episodic and stochastic events resulting in severe disturbance and resuspension. Sandy sediments may have more complex and resilient microbenthic communities, adapted to a continuous regime of disturbance in the top layers of sediments and with a less pronounced seasonality.

Potential contribution of epithelial Na+ channel to net secretion of aqueous humor.

The aqueous humor of the eye is secreted by the bilayered ciliary epithelium, consisting of the pigmented (PE) cell layer facing the stroma and the nonpigmented (NPE) cell layer facing the aqueous humor. Cells within each layer and between the two layers are linked by gap junctions, forming a ciliary epithelial syncytium. Unidirectional secretion from the stroma to the aqueous proceeds both through the cells (the transcellular pathway) and between the cells (the paracellular pathway). Net formation of aqueous humor must, however, be the algebraic sum of unidirectional secretion and unidirectional reabsorption from the aqueous humor back into the stoma. The mechanisms potentially underlying reabsorption of aqueous humor by the NPE cells have recently been addressed by studying the regulatory response (RVI) of anisosmotically shrunken NPE cells. The results indicated that epithelial Na+ channels with a high affinity to amiloride likely contribute to reabsorption of solute from the aqueous humor. We have substantiated this possibility by using Northern analysis to identify in human ciliary body RNA a 3.7-kb transcript corresponding to the alpha-subunit of the amiloride-sensitive, alpha beta gamma-ENaC epithelial sodium channel. We have also found that the Na(+)-channel inhibitor benzamil inhibits the RVI without affecting the cell volume of isotonic cell suspensions. This observation supports the hypothesis that the low conductance, highly selective epithelial Na+ channel is activated by shrinkage and contributes to unidirectional reabsorption as aqueous humor. Examples are provided of how the integrative regulation of aqueous humor formation can involve conjugate actions on both unidirectional secretion and reabsorption.

Ca2+ mobilization and interlayer signal transfer in the heterocellular bilayered epithelium of the rabbit ciliary body.

1. 'Ratiometric' fura-2 methodology in slice preparations and 'intensitometric' fluo-3 measurements of confocal images were used to simultaneously monitor Ca2+ mobilization in the two distinct, apically joined cell layers which constitute the ciliary body epithelium (CBE): the non-pigmented (NPE) and pigmented (PE) epithelia. 2. Both methods yielded comparable results regarding Ca2+ responses in the syncytium upon stimulation with adrenergic and cholinergic agonists. 3. The alpha 1-adrenoceptor agonist phenylephrine elicited a moderate [Ca2+]i increase in the PE, whereas NPE [Ca2+]i remained unchanged or exhibited a slight diminution. 4. In combination with carbachol, the alpha 2-adrenoceptor agonist brimonidine elicited large Ca2+ increases (> 10-fold) in both the NPE and PE cell layers, even though previous studies indicated the absence of an alpha 2-adrenergic effect on [Ca2+]i in the PE. The onset, as well as the peak of the Ca2+ responses in PE cells frequently exhibited a small delay with respect to adjacent NPE cells. No such time difference was observed between adjacent NPE cells. 5. Pre-incubation of the ciliary body in Ca(2+)-free solution under conditions known to elicit overt NPE-PE separation abolished the alpha 2-adrenocholinergic response in the PE. 6. Addition of heptanol to the perfusate, to block gap-junctional communication, caused a small [Ca2+]i decrease in the NPE and a slight increase in PE[Ca2+]i. Subsequently, the Ca2+ mobilization in the Pe in response to the brimonidine and carbachol combination was either blocked or showed a substantial delay. The Ca2+ mobilization in the NPE, in contrast, remained unchanged. 7. We conclude that the heterocellular syncytium exhibits rectificatory behaviour with respect to Ca2+ mobilization; responses originating within the NPE are easily transferred to the PE, while the reverse does not occur.

Role of ion channels in aqueous humor formation

The aqueous humor is secreted by the ciliary epithelium, a bilayered syncytial epithelium comprising a pigmented (PE) cell layer abutting the stroma and a nonpigmented (NPE) cell layer facing the aqueous phase. As in other epithelia, secretion depends on the transfer of solute, with water passively following. Na+, K+, and Cl- enter the syncytium principally through a Na(+)-K(+)-2Cl- symport, diffusing to the aqueous surface of the NPE cells. The Na+, K+, and Cl- are secreted into the aqueous humor through the Na+/K+ exchange pump, K+ channels, and Cl- channels, respectively. Na+ is also secreted between the cells in response to a small transepithelial potential. The K+ channels are critical not only for K+ release but also for hyperpolarizing the membrane, providing an electrical driving force for Cl- secretion. Some of the K+ channels are Ca2+ sensitive and can be activated by Ca2+ entry through T- and L-type Ca2+ channels. The roles of the ciliary epithelial nonselective and Na+ channels are less clear. This review describes the ion channels thus far identified in the ciliary epithelium in terms of the activation and inactivation of their macroscopic currents, the open probabilities and conductances of the single channels, and their locations and regulation. The review relates each class of channel to known families of channels and indicates how those channels can contribute to the secretion of the aqueous humor.