The influence of substrate elastic modulus on retinal pigment epithelial cell phagocytosis

To better understand if a complex process such as phagocytosis is influenced by substrate stiffness, we investigated the influence of substrate elastic modulus on phagocytosis in the retinal pigment epithelial (RPE) cell line ARPE-19. RPE cells lie on Bruch?s membrane, directly under the retina, and phagocytose the shed photoreceptor outer segments. Bruch?s membrane is known to increase in stiffness by an order of magnitude with age and thus, this study has potential relevance in explaining retinal changes in age-related macular degeneration.     

A newly discovered pathway of melanin formation in cultured retinal pigment epithelium of cattle

According to a recent hypothesis the melanin granules in the retinal pigment epithelium of mammals originate from photosensory membrane degradation. To test this hypothesis the retinal pigment epithelium of cattle was kept in tissue culture and exposed to gold-labelled rod outer segments. Gold granules were later detected inside phagosomes, melanosomes and mature melanin granules. Tyrosinase, the key enzyme in melanogenesis, was additionally localized inside phagosomes. These results indicate that in cultured retinal pigment epithelium the matrix of the melanosome can originate from phagosomes. therefore, the melanosome is a specialized lysosome.     

Microtubules and 10 nm-filaments in the retinal pigment epithelium during the diurnal light-dark-cycle

Summary Microtubules and 10 nm-filaments appear to be involved in the functions of the retinal pigment epithelium (RPE). The presence of microtubules in the RPE of light-adapted eyes, but not in dark-adapted eyes, suggests that they may be involved primarily in organelle movement. On the other hand, the random and constant presence of 10 nm-filaments within the basal portion of the PE implies a cytoskeletal role for these filaments.The authors thank their colleagues Pierre Couillard and Michel Anctil for helpful advice and criticism during the course of this study. Financial support was provided by the C.R.S.N.G. du Canada and the Ministère de l'Education du Québec (F.C.A.C.)     

The Ca 2+ pumps and the Na + / Ca 2+ exchangers

The Ca 2+ ATPases or Ca 2+ pumps transport Ca 2+ ions out of the cytosol, by using the energy stored in ATP. The Na + / Ca 2+ exchanger uses the chemical energy of the Na + gradient (the Na + concentration is much higher outside than inside the cell) to remove Ca 2+ from the cytosol. Ca 2+ pumps are found in the plasma membrane and in the endoplasmic reticulum of the cells. The pumps are probably present in the membrane of other organelles, but little experimental information is available on this matter. The Na + / Ca 2+ exchangers are located on the plasma membrane. A Na + / Ca 2+ exchanger was found in the mitochondria, but very little is known on its structure and sequence. These transporters control the Ca 2+ concentration in the cytosol and are vital to prevent Ca 2+ overload of the cells. Their activity is controlled by different mechanisms, that are still under investigation. A number of the possible isoforms for both types of proteins has been detected.© Kluwer Academic Publishers     

Sirtuin 6 protects human retinal pigment epithelium cells from LPS-induced inflammation and apoptosis partly by regulating autophagy

ABSTRACT

Lipopolysaccharides (LPS)-induced retinal inflammation is an important factor in retinal diseases. This study was aimed to investigate the effect of Sirt6 on LPS-induced retinal injury. ARPE-19 cells were incubated with LPS to induce inflammation. The cell viability was determined using CCK-8 assay. The mRNA level and protein expression of corresponding genes was detected using qRT-PCR and western blot, respectively. The production of inflammatory cytokines was measured using ELISA kit. The levels of oxidative stress-related factors were measured using their detection kits. Cell apoptosis was observed using TUNEL assay. The results showed that Sirt6 was downregulated after LPS treatment. Sirt6 strengthened LPS-induced autophagy by promoting the expression of LC3II/I, beclin1 and ATG5. Sirt6 treatment significantly inhibited LPS-induced inflammation, oxidative stress and cell apoptosis, which was then partly abolished by 3 MA. These results suggest Sirt6 to be an important regulator for LPS-induced inflammation, oxidative stress, and apoptosis partly by regulating cell autophagy.     

Ca2+-Regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis

We have used chlortetracycline (CTC) analysis to investigate mechanisms that may play important roles during bull sperm capacitation in a culture medium (containing glucose, heparin, and caffeine) known to promote capacitation and fertilization in vitro. In initial experiments employing the Ca²⁺ ionophore A23187, we identified three discrete CTC patterns so similar to those described for mouse and human sperm that we have employed the same nomenclature: “F,” characteristic of uncapacitated, acrosome-intact cells; “B,” characteristic of capacitated, acrosome-intact, cells; “AR,” characteristic of capacitated, acrosome-reacted cells. Over a 60-min period, A23187 stimulated significant increases in B and AR pattern cells, with concomitant decreases in F pattern cells, suggesting a very rapid transition from the uncapacitated to the capacitated state and then on to exocytosis. Without ionophore, significant changes in the proportions of F and B pattern cells were also observed, but the maximum responses required 4 hr; the proportion of AR cells was consistently ～ 15% throughout, indicating a low incidence of spontaneous acrosome loss. Analysis of cells in media with altered composition indicated that the inclusion of either heparin or caffeine significantly promoted capacitation to about the same extent, but together, heparin plus caffeine had an even more stimulatory effect. Despite this, none of these treatments triggered acrosome loss above the levels seen in media lacking these constituents. In the presence of caffeine, with or without heparin, the inclusion of glucose had little effect on responses, but in the presence of heparin there were fewer B cells. In the presence of either quercetin, a Ca-ATPase inhibitor used at 50–200 μM, or W-7, a calmodulin antagonist used at 5–125 μM, capacitation per se was accelerated, as evidenced by significant decreases in F and significant increases in B pattern cells; only the highest concentration of each caused significant increases in AR cells. In addition, 25 and 125 μM W-7 markedly stimulated motility, both quantitatively and qualitatively. Finally the Na⁺ ionophore monensin at 500 μM significantly accelerated both capacitation and acrosomal exocytosis. The addition of the dihydropyridine calcium channel blocker nifedipine at 10 nM, just prior to monensin, did not inhibit capacitation (F to B transition) but blocked acrosomal exocytosis (B to AR transition). We suggest that Ca²⁺ is required for functional changes in bull sperm, with a Ca²⁺-ATPase modulating intracellular Ca²⁺ during capacitation and calcium channels controlling the Ca²⁺ influx required for acrosomal exocytosis. © 1995 Wiley-Liss, Inc.     

Activation of KGFR-Akt-mTOR-Nrf2 signaling protects human retinal pigment epithelium cells from Ultra-violet

Ultra-violet (UV) radiation causes oxidative injuries to human retinal pigment epithelium (RPE) cells. We tested the potential effect of keratinocyte growth factor (KGF) against the process. KGF receptor (KGFR) is expressed in ARPE-19?cells and primary human RPE cells. Pre-treatment with KGF inhibited UV-induced reactive oxygen species (ROS) production and RPE cell death. KGF activated nuclear-factor-E2-related factor 2 (Nrf2) signaling in RPE cells, causing Nrf2 Ser-40 phosphorylation, stabilization and nuclear translocation as well as expression of Nrf2-dependent genes (HO1, NOQ1 and GCLC). Nrf2 knockdown (by targeted shRNAs) or S40T mutation almost reversed KGF-induced RPE cell protection against UV. Further studies demonstrated that KGF activated KGFR-Akt-mTORC1 signaling to mediate downstream Nrf2 activation. KGFR shRNA or Akt-mTORC1 inhibition not only blocked KGF-induced Nrf2 Ser-40 phosphorylation and activation, but also nullified KGF-mediated RPE cell protection against UV. We conclude that KGF-KGFR activates Akt-mTORC1 downstream Nrf2 signaling to protect RPE cells from UV radiation.     

L-type voltage-operated Ca(+2) channels modulate transient Ca(+2) influx triggered by activation of Sertoli cell surface L-selectin

Near the base of mammalian seminiferous epithelium, Sertoli cells are joined by tight junctions, which constitute the blood-testis barrier. Differentiating germ cells are completely enveloped by Sertoli cells and must traverse the tight junctions during spermatogenic cycle. Following the specific ligand activation of L-selectin, the up-regulated Rho family small G-proteins have been implicated as important modulators of tight junctional dynamics. Although the activation of L-selectin transmits subsequent intracellular signals in a Ca(+2)-dependent fashion in various cell types, little is understood regarding the signaling pathways utilized by L-selectin in Sertoli cells. Therefore, we have examined the possible resultant calcium influx triggered by specific ligand-activation of cell surface L-selectin receptors or by cross-linking of L-selectin with anti-L-selectin. Spectrofluorimetric studies demonstrate increase of intracellular Ca(+2) levels immediately after the treatment of the L-selectin ligands, fucoidan and sialyl Lewis-a, or after treatment with anti-L-selectin antibody. We then determined the mechanism of Ca(+2) influx by investigating L- and T-type voltage-operated Ca(+2) channels, which have been suggested to present in the membranes of Sertoli cells. Data demonstrate that Sertoli cells treated with L-type voltage-operated Ca(+2) channel antagonists, nifedipine, diltiazem, or verapamil, lead to dose-dependent blockage of L-selectin-induced Ca(+2) influx. Cells treated with mibedradil, a T-type voltage-operated Ca(+2) channel antagonist, results in little or no blocking effect. Therefore, we conclude that activation of Sertoli cell L-selectin induces Ca(+2) influx, which is at least partially regulated by L-type voltage-operated Ca(+2) channels.     

Defining the proteome of human iris,ciliary body,retinal pigment epithelium,and choroid

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS‐PAGE. After in‐gel digestion, peptides were analyzed using LC‐MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false‐positive rates of <0.1% and <1%, respectively. Forty‐three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.     

Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

Ca2+ sparks are the elementary events of intracellular Ca2+ release from the sar-coplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca2+ channel activation contributes to the genesis of spontaneous Ca2+ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca2+ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI2 which inhibits spontaneous L-type Ca2+ channel activation, the rate of occurrence of spontaneous Ca2+ sparks was halved from 4.20 to 2.04 events/(100 μm·s), with temporal and spatial properties of individual Ca2+ sparks unchanged. Analysis of the Cd2+-sensitive spark production revealed an open probability of-10-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca2+ channels in resting heart cells contribute significantly to the production of spontaneous Ca2+ sparks.     

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.     

Insulin secretion and intracellular Ca rises in monolayer cultures of neonatal rat β-cells

Glucose-induced insuline release, glucose-induced rises in intracellular free Ca²⁺ concentration ([Ca²⁺]_i), and voltage-dependent Ca²⁺ channel activity were assessed in monolayer cultures of β-vells 3–5 day-old rats. The glucose-stimulated insulin secretory responses and [Ca²⁺]_i rises were like those in adult rat β-cells rather than fetal rat β-cells. Voltage-dependent Ca²⁺ channel antagonists decreased glucose-induced insulin secretion, aborted the [Ca²⁺]₂ rise and, like deprivation of extracellular Ca²⁺, prevented the glucose-induced rise in [Ca²⁺]_i when added before the glucose challenge. The presence of nifedipine-sensitive, voltage-dependent Ca²⁺ channels was demonstrated directly by measuring Ca²⁺ currents using the whole-cell configuration of the patch-clamp technique and indirectly by measuring [Ca²⁺]₁ after membrane depolarization by 45 mMm K⁺ or 200 μM tolbutamide. Thus, in cultured β-cells of 3–5 day-old rats the coupling of glucose stimulation to Ca²⁺ influx is essentially mature, in contrast to what has been reported for fetal or very early neonatal cells.     

Phosphorylation/inactivation of PTEN by Akt-independent PI3K signaling in retinal pigment epithelium

Retinal pigment epithelium (RPE) plays a critical role in vertebrate vision by providing functional and structural support to the retina. Degeneration of RPE by cumulative oxidative stresses or acute injury frequently results in retinal degenerative diseases, notably age-related macular degeneration (AMD). Moreover, it has been shown that phosphorylation-mediated inactivation of PTEN (phosphatase and tensin homolog) in RPE is closely linked to AMD-like retinal degeneration in mice [1]. In this study, we used AMD mouse models, in which chemokine (C–C motif) ligand 2 (Ccl2) or chemokine (C–C motif) receptor 2 (Ccr2) were genetically ablated, to examine mechanisms linking reactive oxygen species (ROS) to phosphorylation/inactivation of PTEN in RPE. We found that ROS levels were increased in these RPE cells in association with phosphorylation/inactivation of PTEN. Both PTEN phosphorylation/inactivation and consequent Akt activation in the RPE of AMD model mice were inhibited by antioxidant treatment, indicating a functional role for elevated intracellular ROS. We further discovered that PTEN phosphorylation in oxidatively stressed RPE was repressed by a phosphoinositide 3-kinase (PI3K) inhibitor, but not by an Akt inhibitor. Taken together, these results suggest that ROS-activated PI3K potentiates AMD-related RPE pathogenesis through phosphorylation/inactivation of PTEN.     

Retinal carotenoids can attenuate formation of A2E in the retinal pigment epithelium

A2E, an important constituent of lipofuscin in human retinal pigment epithelium (RPE), is thought to mediate light-induced oxidative damage associated with aging and other ocular disorders. Ocular carotenoids in overlying retinal tissues were measured by HPLC and mass spectrometry and were correlated with levels of RPE A2E. We observed a statistically significant increase in total A2E levels in human RPE/choroid with age, and A2E levels in macular regions were approximately 1/3 lower than in peripheral retinal regions of the same size. There was a statistically significant inverse correlation between peripheral retina carotenoids and peripheral RPE/choroid A2E. Prospective carotenoid supplementation studies in Japanese quail demonstrated nearly complete inhibition of A2E formation and oxidation. These findings support current recommendations to increase dietary intake of xanthophyll carotenoids in individuals at risk for macular degeneration and highlight a new potential mechanism for their protective effects—inhibition of A2E formation and oxidation in the eye.     

Function of caveolae in Ca2+ entry and Ca2+-dependent signal transduction

The correct spatial and temporal control of Ca²⁺ signaling is essential for such cellular activities as fertilization, secretion, motility, and cell division. There has been a long-standing interest in the role of caveolae in regulating intracellular Ca²⁺ concentration. In this review we provide an updated view of how caveolae may regulate both Ca²⁺ entry into cells and Ca²⁺-dependent signal transduction     

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca²⁺ in brain microsomes after Ca²⁺ loading by the Ca²⁺-ATPase or by the Na⁺/Ca²⁺ exchanger. The results show that in microsomes loaded with Ca²⁺ by the Ca²⁺-ATPase, Ins(1,4,5)P₃ (5 μM) release 21±2% of the total Ca²⁺ accumulated, and that in the microsomes loaded with Ca²⁺ by the Na²⁺/Ca²⁺ exchanger, Ins(1,4,5)P₃ released 28±3% of the total Ca²⁺ accumulated. These results suggest that receptors of Ins(1,4,5)P₃ may be co-localized with the Na²⁺/Ca²⁺ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P₃ receptors in the plasma membrane where the Na²⁺/Ca²⁺ exchanger is normally present, or both. We also found that Ins(1,4,5)P₃ inhibited the Ca²⁺-ATPase by 33.7%, but that it had no significant effect on the Na²⁺/Ca²⁺ exchanger.     

Na+-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus

It is concluded that Ca²⁺ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na⁺/Ca²⁺-exchange mechanism that is driven by the (transmembrane) Na⁺ gradient established by Na⁺/K⁺-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na⁺/Ca²⁺-exchanger next to the Ca²⁺-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca²⁺ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na⁺ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca²⁺ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca²⁺ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adaptated to FW. Branchial Na⁺/K⁺-ATPase activity was two times higher in SW adapted fish. Accepted: 29 October 1996     

Na+ and Ca2+ channel expression in cultured newt retinal pigment epithelial cells: Comparison with neuronal types of ion channels

We cultured retinal pigment epithelial (RPE) cells dissociated from adult newt eye and analyzed their voltage-gated ion channels during culture using whole-cell patch-clamp techniques. The results were compared with those of retinal neurons under identical experimental conditions. After 6–9 days in culture (early stage), > 60% of RPE cells developed voltage-gated Na⁺ and Ca²⁺ channels that were not observed in freshly dissociated RPE cells. The number of cells expressing Na⁺ channels and Na⁺ current density were high after 12–15 days in culture (intermediate stage), while the number of Ca²⁺ channel-expressing cells and Ca²⁺ current density were high after 20–30 days in culture (late stage). The activation voltage of the Na⁺ current in the RPE cells was similar to that in neurons. The voltage dependence of Na⁺ current inactivation was somewhat different between two cell types. The steepness of the inactivation curve tended to be less in cultured RPE cells than in neurons, and the half-inactivation voltage was about −54 mV for the RPE cells and −45 mV for neurons. The Ca²⁺ current expressed in cultured RPE cells was too small to detect without replacement of external Ca²⁺ with Ba²⁺. The Ba²⁺ current, like Ca²⁺ current in neurons, was enhanced by Bay-K 8644 and blocked by nicardipine. These results suggest that the RPE cells, like neurons, expressed L-type Ca²⁺ channels in culture. The possibility that the development of both Na²⁺ and Ca²⁺ channels in cultured RPE cells is a manifestation of the transdifferentiation of RPE cells into neurons is discussed. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 377–390, 1997.