Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells

The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning and in vivo genetic complementation to demonstrate that Mertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.     

Antagonism of L-type Ca2+ channels CaV1.3 and CaV1.2 by 1,4-dihydropyrimidines and 4H-pyrans as dihydropyridine mimics

The L-type calcium channel (LTCC) Ca_V1.3 is regarded as a new potential therapeutic target for Parkinson’s disease. Calcium influx through Ca_V1.3 LTCC during autonomous pacemaking in adult dopaminergic neurons of the substantia nigra pars compacta is related to the generation of mitochondrial oxidative stress in animal models. Development of a Ca_V1.3 antagonist selective over Ca_V1.2 is essential because Ca_V1.2 pore-forming subunits are the predominant form of LTCCs and are abundant in the central nervous and cardiovascular systems. We have explored 1,4-dihydropyrimidines and 4H-pyrans to identify potent and selective antagonists of Ca_V1.3 relative to Ca_V1.2 LTCCs. A library of 36 dihydropyridine (DHP)-mimic 1,4-dihydropyrimidines and 4H-pyrans was synthesized, and promising chiral compounds were resolved. The antagonism studies of Ca_V1.3 and Ca_V1.2 LTCCs using DHP mimic compounds showed that dihydropyrimidines and 4H-pyrans are effective antagonists of DHPs for Ca_V1.3 LTCCs. Some 1,4-dihydropyrimidines are more selective than isradipine for Ca_V1.3 over Ca_V1.2, shown here by both calcium flux and patch-clamp electrophysiology experiments, where the ratio of antagonism is around 2–3. These results support the hypothesis that the modified hydrogen bonding donor/acceptors in DHP-mimic dihydropyrimidines and 4H-pyrans can interact differently with DHP binding sites, but, in addition, the data suggest that the binding sites of DHP in Ca_V1.3 and Ca_V1.2 LTCCs are very similar.     

Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain

Low voltage activation of Ca(V)1.3 L-type Ca(2+) channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Ca(V)1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in Ca(V)1.4 L-type Ca(2+) channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Ca(V)1.3 alpha1 subunit C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), we investigated if a C-terminal modulatory mechanism also controls Ca(V)1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with beta3 and alpha2delta1 subunits in HEK-293 cells. Activation of calcium current through Ca(V)1.3(42A) channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Ca(V)1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Ca(V)1.3(DeltaC116) channels showed gating properties similar to Ca(V)1.3(42A) that were reverted by co-expression of the corresponding C-terminal peptide C(116). Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Ca(V)1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Ca(V)1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Ca(V)1.3 channel activation at lower voltages expected to favor Ca(V)1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.     

Antagonism of 4-substituted 1,4-dihydropyridine-3,5-dicarboxylates toward voltage-dependent L-type Ca2+ channels CaV1.3 and CaV1.2

L-type Ca²⁺ channels in mammalian brain neurons have either a Ca_V1.2 or Ca_V1.3 pore-forming subunit. Recently, it was shown that Ca_V1.3 Ca²⁺ channels underlie autonomous pacemaking in adult dopaminergic neurons in the substantia nigra pars compacta, and this reliance renders them sensitive to toxins used to create animal models of Parkinson’s disease. Antagonism of these channels with the dihydropyridine antihypertensive drug isradipine diminishes the reliance on Ca²⁺ and the sensitivity of these neurons to toxins, pointing to a potential neuroprotective strategy. However, for neuroprotection without an antihypertensive side effect, selective Ca_V1.3 channel antagonists are required. In an attempt to identify potent and selective antagonists of Ca_V1.3 channels, 124 dihydropyridines (4-substituted-1,4-dihydropyridine-3,5-dicarboxylic diesters) were synthesized. The antagonism of heterologously expressed Ca_V1.2 and Ca_V1.3 channels was then tested using electrophysiological approaches and the FLIPR Calcium 4 assay. Despite the large diversity in substitution on the dihydropyridine scaffold, the most Ca_V1.3 selectivity was only about twofold. These results support a highly similar dihydropyridine binding site at both Ca_V1.2 and Ca_V1.3 channels and suggests that other classes of compounds need to be identified for Ca_V1.3 selectivity.     

Ca2+-conductances in cultured rat retinal pigment epithelial cells

Membrane conductances for Ca²⁺ in cultured rat pigment epithelial cells were studied in the whole-cell configuration of the patch-clamp technique using barium (10 mM) as a charge carrier. Two types of voltage-dependent and verapamiland diltiazem-sensitive Ba²⁺ currents were observed. First, a nearly sustained current was activated by depolarization to potentials more positive than — 30mV and blocked by nifedipine (1 μM). This current was observed in cells of primary cultures less than 13 days old. Second, a transient nifedipine (1 μM) insensitive current was activated by depolarization to potentials more positive than — 55mV in cultures which were more than 13 days old. This current was not carried by sodium and blocked by 1 μM tetrodotoxin (TTX). In summary, cultured rat retinal pigment epithelial cells in younger primary cultures express Ba²⁺ currents indicating the presence of L-type Ca²⁺ channels. In order primary cultures a low-voltage activated channel was observed with properties different from T-type calcium channels or TTX-sensitive calcium conducting sodium channels. © 1994 Wiley-Liss, Inc.     

Light-induced oxidation of photoreceptor outer segment phospholipids generates ligands for CD36-mediated phagocytosis by retinal pigment epithelium: a potential mechanism for modulating outer segment phagocytosis under oxidant stress conditions

Clearance by the retinal pigment epithelium (RPE) of shed photoreceptor outer segments (OSs), a tissue with one of the highest turnover rates in the body, is critical to the maintenance and normal function of the retina. We hypothesized that there is a potential role for photo-oxidation in OS uptake by RPE via scavenger receptor-mediated recognition of structurally defined lipid peroxidation products. We now demonstrate that specific structurally defined oxidized species derived from arachidonyl, linoleoyl, and docosahexanoyl phosphatidylcholine may serve as endogenous ligands on OSs for uptake by RPE via the scavenger receptor CD36. Mass spectrometry studies of retinal lipids recovered from dark-adapted rats following physiological light exposure demonstrate in vivo formation of specific oxidized phosphatidylcholine molecular species possessing a CD36 recognition motif, an oxidatively truncated sn-2 acyl group with a terminal gamma-hydroxy(or oxo)-alpha,beta-unsaturated carbonyl. Cellular studies using RPE isolated from wild-type versus CD36 null mice suggest that CD36 plays a role in engulfment, but not initial binding, of OSs via these oxidized phospholipids. Parallel increases in OS protein-bound nitrotyrosine, a post-translational modification by nitric oxide (NO)-derived oxidants, were also observed, suggesting a possible role for light-induced generation of NO-derived oxidants in the initiation of OS lipid peroxidation. Collectively, these studies suggest that intense light exposure promotes "oxidative tagging" of photoreceptor outer segments with structurally defined choline glycerophospholipids that may serve as a physiological signal for CD36-mediated phagocytosis under oxidant stress conditions.     

Fibroblast growth factor receptor 2 (FGFR2) in brain neurons and retinal pigment epithelial cells act via stimulation of neuroendocrine L-type channels (Ca(v)1.3).

In contrast to the fibroblast growth factor receptor 1 (FGFR1), little is known about intracellular signaling of FGFR2. The signaling cascade of FGFR2 was studied using the perforated patch configuration of the patch-clamp technique in cultured rat retinal pigment epithelial (RPE) cells that express both FGFR1 and FGFR2. Interaction of signaling proteins was studied using immunoprecipitation techniques with membrane proteins from RPE cells and freshly isolated rat brain. When Ba(2+) currents through L-type channels were studied, extracellular application of bFGF (10 ng/ml) led to a shift of the steady-state activation to more negative values. In 50% of cells, an additional increase in maximal current amplitude was observed. This effect was blocked by the tyrosine kinase inhibitor lavendustin A (10(-5) M) but was not influenced by the FGFR1 blocker SU5402 (2 x 10(-5) M) or by the blocker for src-kinase herbimycin A (10(-5) M). Immunoprecipitation of FGFR2 led to coprecipitation of alpha 1D Ca(2+) channel subunits and precipitation of alpha 1D subunits led to coprecipitation of FGFR2. Immunoprecipitation of FGFR1 did not result in the coprecipitation with alpha 1D Ca(2+) channel subunits. The coprecipitation results were comparable when using brain tissue and RPE cells. The alpha 1D subunit-specific band were stained with antiphosphotyrosine antibodies. We conclude that FGFR2 acts via a different signaling cascade than FGFR1. This cascade involves an src-kinase-independent, close functional interaction of FGFR2 and the alpha subunit of neuroendocrine L-type channels.     

Activation of Ca2+-dependent K+ channels by cyanide in guinea pig adrenal chromaffin cells

The effects ofcyanide (CN) on whole cell current measured with the perforated-patchmethod were studied in adrenal medullary cells. Application of CNproduced initially inward and then outward currents at 52 mV ormore negative. As the membrane potential was hyperpolarized, amplitudeand latency of the outward current (I_o) by CNbecame small and long, respectively. A decrease in the externalNa⁺ concentration did not affectthe latency for CN-inducedI_o but enhancedthe amplitude markedly. The CNI_o reversedpolarity at 85 mV, close to the Nernst potential forK⁺, and was suppressed by theK⁺ channel blockers curare andapamin but not by glibenclamide, suggesting thatI_o is due to theactivation of Ca²⁺-dependentK⁺ channels. Consistent with thisnotion, the Ca²⁺-mobilizingagents, muscarine and caffeine, also producedI_o. Exposure toCN in a Ca²⁺-deficient medium for4 min abolished caffeine- or muscarine-induced I_o withoutdevelopment ofI_o, and additionof Ca²⁺ to the CN-containingsolution inducedI_o. We concludethat exposure to CN producesCa²⁺-dependentK⁺ currents in an externalCa²⁺-dependent manner, probablyvia facilitation of Ca²⁺ influx.

     

Membranes of retinal pigment epithelial cells in vitro are damaged in the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions

The ferrous ions released from haemoglobin and storage-transferrin ions cause oxidative stress in the eyes. We observed the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions in the retinal pigment epithelial (RPE) cells in vitro, and investigated how the ferrous ions influenced RPE in vitro and the photoreceptor outer segment discs. We obtained isolated photoreceptor outer segment discs using sucrose gradient of specific gravity after homogenizing porcine retinas. After bovine RPE cells were cultured with isolated photoreceptor outer segment discs containing FeCl2 for 5 and 24 h, we incubated the specimens with rhodamine phalloidin, antimouse alpha-tubulin antibody and antimouse Ig G (FITC and rhodamine labelled). We observed the specimens by a laser scanning microscopy, and made the ultrathin sections with or without 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy. Actin filaments and microtubules of specialized cells such as RPE cells were actively involved in phagocytosis of the photoreceptor outer segment discs. Microtubules were damaged during the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions. The peroxidation increased the granular and aggregated autofluorescence of the photoreceptor outer segment discs. The membranes of the disc and the phagosomes, and lysosomes in RPE cells were damaged by ferrous ions and had fine particles with high electron density staining without uranium acetate and lead citrate. The cytoskeletons such as actin filaments and microtubules, and the membranes of the phagosomes and the lysosomes in RPE cells in vitro were damaged during the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions.     

Inhibition of red cell Ca2+-dependent K+ channels by snake venoms

We have investigated the effects of several snake venoms on the Ca2+-dependent K+ channels of human red cells. A heat-resistant component of the venom of the snake Notechis scutatus irreversibly inhibited Ca2+-dependent K+ transport with a Ki value of 0.1-0.2 micrograms/ml. Metabolic changes of the cells modified the maximal effect of the venom. Binding of the venom required extracellular Ca2+ and was quick, but development of full inhibition required additional time. The effects of the venoms from Notechis scutatus and Leiurus quinquestriatus were additive, suggesting that both venoms act through different mechanisms. Venoms of the snakes Vipera russelli russelli and Oxyuranus scutellatus also inhibited Ca2+-dependent K+ transport with the same characteristics as the Notechis scutatus venom.     

Endogenous Gas6 and Ca2+ -channel activation modulate phagocytosis by retinal pigment epithelium

Mutation or loss of MerTK as well as deficiency of vβ5-integrins, gives rise to retinal-degeneration due to inefficient phagocytosis of photoreceptor outer-segment fragments by the retinal pigment epithelium (RPE). This study shows that Gas6 expressed endogenously by human RPE promotes phagocytosis. The RPE expresses Gas6 more highly in vivo and in serum-reduced conditions in vitro than in high-serum conditions, suggesting a negative-feedback control. An antibody-blockage approach revealed that Gas6-expressing RPE phagocytizes photoreceptor outer-segment fragments due to stimulation of MerTK by endogenous Gas6 in vitro. MerTK- and Gas6-antibodies reduced phagocytosis. Blocking L-type Ca²⁺-channels with nifedipine inhibited MerTK dependent phagocytosis in vitro. Application of integrin inhibitory, soluble, RGD-containing peptides or soluble vitronectin reduced L-type Ca²⁺-channel currents in RPE. Herbimycin A, which reduces phosphorylation of integrin receptor-associated proteins and decreases L-type Ca²⁺-channel currents in RPE, eliminates the inhibiting vitronectin effect and abolishes phagocytosis. Thus, Gas6-promoted phagocytosis was inhibited by L-type Ca²⁺-channel blockage, which in turn may be activated by integrin receptor stimulation. These results suggest that L-type Ca²⁺-channels could be regulated downstream of both MerTK and vβ5-integrin, indicating that the binding and uptake mechanisms of phagocytosis are part of a converging pathway.     

Pertussis toxin-sensitive pathway inhibits glucose-stimulated Ca2+ signals of rat islet beta-cells by affecting L-type Ca2+ channels and voltage-dependent K+ channels

A role of pertussis toxin (PTX)-sensitive pathway in regulation of glucose-stimulated Ca2+ signaling in rat islet beta-cells was investigated by using clonidine as a selective agonist to alpha2-adrenoceptors which link to the pathway. An elevation of extracellular glucose concentration from 5.5 to 22.2 mM (glucose stimulation) increased the levels of [Ca2+]i of beta-cells, and clonidine reversibly reduced the elevated levels of [Ca2+]i. This clonidine effect was antagonized by yohimbine, and abolished in beta-cells pre-treated with PTX. Clonidine showed little effect on membrane currents including those through ATP-sensitive K+ channels induced by voltage ramps from -90 to -50 mV. Clonidine showed little effect on the magnitude of whole-cell currents through L-type Ca2+ channels (ICa(L)), but increased the inactivation process of the currents. Clonidine increased the magnitude of the voltage-dependent K+ currents (IVK). These clonidine effects on ICa(L) and IVK were abolished in beta-cells treated with PTX or GDP-betaS. These results suggest that the PTX-sensitive pathway increases IVK activity and decreases ICa(L) activity of islet beta-cells, resulting in a decrease in the levels of [Ca2+]i elevated by depolarization-induced Ca2+ entry. This mechanism seems responsible at least in part for well-known inhibitory action of PTX-sensitive pathway on glucose-stimulated insulin secretion from islet beta-cells.     

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.     

L-type Ca2+ channels access multiple open states to produce two components of Bay K 8644-dependent current in GH3 cells

To determine the number of L-channel populations responsible for producing the two components of whole-cell L-type Ca2+ channel current revealed by Bay K 8644 (Fass, D.M., and E.S. Levitan. 1996. J. Gen. Physiol. 108:1-11), L-type Ca2+ channel activity was recorded in cell- attached patches. Ensemble tail currents from most (six out of nine) single-channel patches had double-exponential time courses, with time constants that were similar to whole-cell tail current decay values. Also, in single-channel patches subjected to two different levels of depolarization, ensemble tail currents exactly reproduced the voltage dependence of activation of the two whole-cell components: The slow component is activated at more negative potentials than the fast component. In addition, deactivation of Bay K 8644-modified whole-cell L-current was slower after long (100-ms) depolarizations than after short (20-ms) depolarizations, and this phenomenon was also evident in ensemble tail currents from single L-channels. Thus, a single population of L-channels can produce the two components of macroscopic L-current deactivation. To determine how individual L-channels produce multiple macroscopic tail current components, we constructed ensemble tail currents from traces that contained a single opening upon repolarization and no reopenings. These ensemble tails were biexponential. This type of analysis also revealed that reopenings do not contribute to the slowing of tail current deactivation after long depolarizations. Thus, individual L-channels must have access to several open states to produce multiple macroscopic current components. We also obtained evidence that access to these open states can vary over time. Use of several open states may give L-channels the flexibility to participate in many cell functions.     

Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle

The patch-clamptechnique was used to determine the ionic conductances activated by ATPin murine colonic smooth muscle cells. Extracellular ATP, UTP, and2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increasedoutward currents in cells with amphotericin B-perforated patches. ATP(0.5-1 mM) did not affect whole cell currents of cells dialyzedwith solutions containing ethylene glycol-bis(-aminoethylether)-N,N,N',N'-tetraaceticacid. Apamin (3 × 10⁷M) reduced the outward current activated by ATP by 32 ± 5%. Single channel recordings from cell-attached patches showed that ATP, UTP, and2-MeS-ATP increased the open probability of small-conductance, Ca²⁺-dependentK⁺ channels with a slopeconductance of 5.3 ± 0.02 pS. Caffeine (500 µM) enhanced the openprobability of the small-conductance K⁺ channels, and ATP had no effectafter caffeine. Pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS,10⁴ M), a nonselectiveP₂ receptor antagonist, preventedthe increase in open probability caused by ATP and 2-MeS-ATP. PPADS hadno effect on the response to caffeine. ATP-induced hyperpolarization inthe murine colon may be mediated byP_2y-induced release of Ca²⁺ from intracellular stores andactivation of the 5.3-pSCa²⁺-activatedK⁺ channels.