Role of the Ca2+-activated Cl- channels bestrophin and anoctamin in epithelial cells

2011 ◽  
Vol 392 (1-2) ◽  
Author(s):  
Karl Kunzelmann ◽  
Patthara Kongsuphol ◽  
Krongkarn Chootip ◽  
Caio Toledo ◽  
Joana R. Martins ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Control Cell ◽  
Cell Types ◽  
Cell Swelling ◽  
Physiological Importance ◽  
Proliferation And Apoptosis ◽  
Intracellular Ca ◽  
Cl Channels

Abstract Two families of proteins, the bestrophins (Best) and the recently cloned TMEM16 proteins (anoctamin, Ano), recapitulate properties of Ca2+-activated Cl- currents. Best1 is strongly expressed in the retinal pigment epithelium and could have a function as a Ca2+-activated Cl- channel as well as a regulator of Ca2+ signaling. It is also present at much lower levels in other cell types including epithelial cells, where it regulates plasma membrane localized Cl- channels by controlling intracellular Ca2+ levels. Best1 interacts with important Ca2+-signaling proteins such as STIM1 and can interact directly with other Ca2+-activated Cl- channels such as TMEM16A. Best1 is detected in the endoplasmic reticulum (ER) where it shapes the dynamic ER structure and regulates cell proliferation, which could be important for renal cystogenesis. Ca2+-activated Cl- channels of the anoctamin family (TMEM16A) show biophysical and pharmacological properties that are typical for endogenous Ca2+-dependent Cl- channels. TMEM16 proteins are abundantly expressed and many reports demonstrate their physiological importance in epithelial as well as non-epithelial cells. These channels are also activated by cell swelling and can therefore control cell volume, proliferation and apoptosis. To fully understand the function and regulation of Ca2+-activated Cl- currents, it is necessary to appreciate that Best1 and TMEM16A are embedded in a protein network and that they probably operate in functional microdomains.

scholarly journals Multiocular organoids from human induced pluripotent stem cells displayed retinal, corneal, and retinal pigment epithelium lineages

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Helena Isla-Magrané ◽  
Anna Veiga ◽  
José García-Arumí ◽  
Anna Duarri
Keyword(s):  
Stem Cells ◽  
Retinal Pigment Epithelium ◽  
Epithelial Cells ◽  
Pluripotent Stem Cells ◽  
Eye Development ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cell Types ◽  
Induced Pluripotent ◽  
Different Cell Types

Abstract Background Recently, great efforts have been made to design protocols for obtaining ocular cells from human stem cells to model diseases or for regenerative purposes. Current protocols generally focus on isolating retinal cells, retinal pigment epithelium (RPE), or corneal cells and fail to recapitulate the complexity of the tissue during eye development. Here, the generation of more advanced in vitro multiocular organoids from human induced pluripotent stem cells (hiPSCs) is demonstrated. Methods A 2-step method was established to first obtain self-organized multizone ocular progenitor cells (mzOPCs) from 2D hiPSC cultures within three weeks. Then, after the cells were manually isolated and grown in suspension, 3D multiocular organoids were generated to model important cellular features of developing eyes. Results In the 2D culture, self-formed mzOPCs spanned the neuroectoderm, surface ectoderm, neural crest, and RPE, mimicking early stages of eye development. After lifting, mzOPCs developed into different 3D multiocular organoids composed of multiple cell lineages including RPE, retina, and cornea, and interactions between the different cell types and regions of the eye system were observed. Within these organoids, the retinal regions exhibited correct layering and contained all major retinal cell subtypes as well as retinal morphological cues, whereas the corneal regions closely resembled the transparent ocular-surface epithelium and contained of corneal, limbal, and conjunctival epithelial cells. The arrangement of RPE cells also formed organoids composed of polarized pigmented epithelial cells at the surface that were completely filled with collagen matrix. Conclusions This approach clearly demonstrated the advantages of the combined 2D-3D construction tissue model as it provided a more ocular native-like cellular environment than that of previous models. In this complex preparations, multiocular organoids may be used to model the crosstalk between different cell types in eye development and disease. Graphical abstract

scholarly journals Fine structure of the retinal pigment epithelium and cones of Antarctic fish Notohenia coriiceps Richardson in light and dark-conditions

2007 ◽  
Vol 24 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Lucélia Donatti ◽  
Edith Fanta
Keyword(s):  
Epithelial Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Antarctic Fish ◽  
Basal Region ◽  
Dense Cytoplasm ◽  
Transmission Electron ◽  
Notothenia Coriiceps ◽  
The Antarctic ◽  
Pigment Epithelial Cells

The Antarctic fish Notothenia coriiceps Richardson, 1844 lives in an environment of daily and annual photic variation and retina cells have to adjust morphologically to environmental luminosity. After seven day dark or seven day light acclimation of two groups of fish, retinas were extracted and processed for light and transmission electron microscopy. In seven day dark adapted, retina pigment epithelium melanin granules were aggregated at the basal region of cells, and macrophages were seen adjacent to the apical microvilli, between the photoreceptors. In seven day light adapted epithelium, melanin granules were inside the apical microvilli of epithelial cells and macrophages were absent. The supranuclear region of cones adapted to seven day light had less electron dense cytoplasm, and an endoplasmic reticulum with broad tubules. The mitochondria in the internal segment of cones adapted to seven day light were larger, and less electron dense. The differences in the morphology of cones and pigment epithelial cells indicate that N. coriiceps has retinal structural adjustments presumably optimizing vision in different light conditions.

scholarly journals Concise Review: Considering Optimal Temperature for Short-Term Storage of Epithelial Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Ayyad Zartasht Khan ◽  
Tor Paaske Utheim ◽  
Catherine Joan Jackson ◽  
Kim Alexander Tønseth ◽  
Jon Roger Eidet
Keyword(s):  
Epithelial Cells ◽  
Storage Temperature ◽  
Freezing Point ◽  
Retinal Pigment ◽  
Cell Types ◽  
Retinal Pigment Epithelial Cells ◽  
Storage Medium ◽  
Short Term ◽  
Short Term Storage ◽  
Term Storage

Transplantation of novel tissue-engineered products using cultured epithelial cells is gaining significant interest. While such treatments can readily be provided at centralized medical centers, delivery to patients at geographically remote locations requires the establishment of suitable storage protocols. One important aspect of storage technology is temperature. This paper reviews storage temperature for above-freezing point storage of human epithelial cells for regenerative medicine purposes. The literature search uncovered publications on epidermal cells, retinal pigment epithelial cells, conjunctival epithelial cells, corneal/limbal epithelial cells, oral keratinocytes, and seminiferous epithelial cells. The following general patterns were noted: (1) Several studies across different cell types inclined toward 4 and 16°C being suitable short-term storage temperatures. Correspondingly, almost all studies investigating 37°C concluded that this storage temperature was suboptimal. (2) Cell death typically escalates rapidly following 7–10 days of storage. (3) The importance of the type of storage medium and its composition was highlighted by some of the studies; however, the relative importance of storage medium vs. storage temperature has not been investigated systematically. Although a direct comparison between the included investigations is not reasonable due to differences in cell types, storage media, and storage duration, this review provides an overview, summarizing the work carried out on each cell type during the past two decades.

Degradation of extracellular ATP by the retinal pigment epithelium

2005 ◽  
Vol 289 (3) ◽  
pp. C617-C624 ◽  
Author(s):  
David Reigada ◽  
Wennan Lu ◽  
Xiulan Zhang ◽  
Constantin Friedman ◽  
Klara Pendrak ◽  
...  
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Adenosine Diphosphate ◽  
Cell Types ◽  
P2y Receptors ◽  
Extracellular Atp ◽  
Pcr Analysis ◽  
Subretinal Space ◽  
Rpe Cells ◽  
Mrs 2179

Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156 and βγ-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca2+. Both responses were inhibited by thapsigargin and P2Y1 receptor inhibitor MRS 2179. Message for both P2Y1 and P2Y12 receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca2+ signaling in the RPE.

scholarly journals Volume-regulated Cl− current: contributions of distinct Cl− channels and localized Ca2+ signals

2019 ◽  
Vol 317 (3) ◽  
pp. C466-C480 ◽  
Author(s):  
Yani Liu ◽  
Huiran Zhang ◽  
Hongchao Men ◽  
Yuwei Du ◽  
Ziqian Xiao ◽  
...  
Keyword(s):  
Anion Channel ◽  
Fluorescent Imaging ◽  
Hek293 Cells ◽  
Cell Swelling ◽  
Anoctamin 1 ◽  
Component Cell ◽  
Intracellular Ca ◽  
Cl Channels ◽  
A Cell ◽  
Necessary And Sufficient

The swelling-activated chloride current ( ICl,swell) is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of ICl,swell is the volume-regulated anion channel (VRAC). Leucine-rich repeat containing 8A (LRRC8A; SWELL1) was recently identified as an essential component of VRAC, but the mechanisms of VRAC activation are still largely unknown; moreover, other Cl− channels, such as anoctamin 1 (ANO1), were also suggested to contribute to ICl,swell. In this present study, we investigated the roles of LRRC8A and ANO1 in activation of ICl,swell; we also explored the role of intracellular Ca2+ in ICl,swell activation. We used a CRISPR/Cas9 gene editing approach, electrophysiology, live fluorescent imaging, selective pharmacology, and other approaches to show that both LRRC8A and ANO1 can be activated by cell swelling in HEK293 cells. Yet, both channels contribute biophysically and pharmacologically distinct components to ICl,swell, with LRRC8A being the major component. Cell swelling induced oscillatory Ca2+ transients, and these Ca2+ signals were required to activate both the LRRC8A- and ANO1-dependent components of ICl,swell. Both ICl,swell components required localized rather than global Ca2+ for activation. Interestingly, while intracellular Ca2+ was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca2+ transients linked to the ICl,swell activation were mediated by the G protein-coupled receptor-independent PLC isoforms.

PGE2, Ca2+, and cAMP mediate ATP activation of Cl− channels in pigmented ciliary epithelial cells

2001 ◽  
Vol 281 (5) ◽  
pp. C1614-C1623 ◽  
Author(s):  
Johannes C. Fleischhauer ◽  
Claire H. Mitchell ◽  
Kim Peterson-Yantorno ◽  
Miguel Coca-Prados ◽  
Mortimer M. Civan
Keyword(s):  
Intraocular Pressure ◽  
Epithelial Cells ◽  
Aqueous Humor ◽  
Second Messengers ◽  
Atp Release ◽  
Channel Activity ◽  
Atp Assay ◽  
Intracellular Ca ◽  
Cl Channels ◽  
Nonpigmented Ciliary Epithelial

Purines regulate intraocular pressure. Adenosine activates Cl− channels of nonpigmented ciliary epithelial cells facing the aqueous humor, enhancing secretion. Tamoxifen and ATP synergistically activate Cl− channels of pigmented ciliary epithelial (PE) cells facing the stroma, potentially reducing net secretion. The actions of nucleotides alone on Cl− channel activity of bovine PE cells were studied by electronic cell sorting, patch clamping, and luciferin/luciferase ATP assay. Cl−channels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 μM. UTP triggered ATP release. The second messengers Ca2+, prostaglandin (PG)E2, and cAMP activated Cl− channels without enhancing effects of 100 μM ATP. Buffering intracellular Ca2+activity with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′- tetraacetic acid or blocking PGE2 formation with indomethacin inhibited ATP-triggered channel activation. The Rp stereoisomer of 8-bromoadenosine 3′,5′-cyclic monophosphothioate inhibited protein kinase A activity but mimicked 8-bromoadenosine 3′,5′-cyclic monophosphate. We conclude that nucleotides can act at >1 P2Y receptor to trigger a sequential cascade involving Ca2+, PGE2, and cAMP. cAMP acts directly on Cl−channels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure.

scholarly journals KCNQ5/Kv7.5 potassium channel expression and subcellular localization in primate retinal pigment epithelium and neural retina

2011 ◽  
Vol 301 (5) ◽  
pp. C1017-C1026 ◽  
Author(s):  
Xiaoming Zhang ◽  
Dongli Yang ◽  
Bret A. Hughes
Keyword(s):  
Visual Information ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basal Membrane ◽  
Cell Types ◽  
Neural Retina ◽  
Pcr Analysis ◽  
Rod Photoreceptor ◽  
Rpe Cells ◽  
Channel Expression

Previous studies identified in retinal pigment epithelial (RPE) cells an M-type K+ current, which in many other cell types is mediated by channels encoded by KCNQ genes. The aim of this study was to assess the expression of KCNQ genes in the monkey RPE and neural retina. Application of the specific KCNQ channel blocker XE991 eliminated the M-type current in freshly isolated monkey RPE cells, indicating that KCNQ subunits contribute to the underlying channels. RT-PCR analysis revealed the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in the RPE and all five KCNQ transcripts in the neural retina. At the protein level, KCNQ5 was detected in the RPE, whereas both KCNQ4 and KCNQ5 were found in neural retina. In situ hybridization in frozen monkey retinal sections revealed KCNQ5 gene expression in the ganglion cell layer and the inner and outer nuclear layers of the neural retina, but results in the RPE were inconclusive due to the presence of melanin. Immunohistochemistry revealed KCNQ5 in the inner and outer plexiform layers, in cone and rod photoreceptor inner segments, and near the basal membrane of the RPE. The data suggest that KCNQ5 channels contribute to the RPE basal membrane K+ conductance and, thus, likely play an important role in active K+ absorption. The distribution of KCNQ5 in neural retina suggests that these channels may function in the shaping of the photoresponses of cone and rod photoreceptors and the processing of visual information by retinal neurons.

Release of ATP from retinal pigment epithelial cells involves both CFTR and vesicular transport

2005 ◽  
Vol 288 (1) ◽  
pp. C132-C140 ◽  
Author(s):  
David Reigada ◽  
Claire H. Mitchell
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Vesicular Transport ◽  
Atp Release ◽  
Retinal Pigment Epithelial Cells ◽  
Subretinal Space ◽  
Transport Pathways ◽  
Rpe Cells

The retinal pigment epithelium (RPE) faces the photoreceptor outer segments and regulates the composition of the interstitial subretinal space. ATP enhances fluid movement from the subretinal space across the RPE. RPE cells can themselves release ATP, but the mechanisms and polarity of this release are unknown. The RPE expresses the cystic fibrosis transmembrane conductance regulator (CFTR), and CFTR is associated with ATP release in other epithelial cells. However, an increasing number of reports have suggested that the exocytotic pathway contributes to release. In the present study, we examined the involvement of CFTR and the vesicular pathway in ATP release from RPE cells. Release from cultured human ARPE-19 cells and across the apical membrane of fresh bovine RPE cells in an eyecup was studied. A cAMP cocktail to activate CFTR triggered ATP release from fresh and cultured RPE cells. Release from both RPE preparations was largely prevented by the broad-acting blocker glibenclamide and the specific thiazolidinone CFTR inhibitor CFTR-172. The block by CFTR-172 was enhanced by preincubation and prevented ATP release with 3.5 μM IC50. The rise in intracellular Ca2+ accompanying hypotonic challenge was prevented by CFTR-172. The vesicular transport inhibitor brefeldin A prevented ATP release after stimulation with both hypotonic and cAMP conditions, suggesting vesicular insertion was also involved. These results show an intimate involvement of CFTR in ATP release from RPE cells which can autostimulate receptors on the apical membrane to modify Ca2+ signaling. The requirement for both CFTR and vesicular transport pathways suggests vesicular insertion of CFTR may underlie the release of ATP.

Sign in / Sign up

Export Citation Format

Share Document