Hypoxia-induced [(3)H]D-aspartate release from isolated bovine retina: modulation by calcium-channel blockers and glutamatergic agonists and antagonists

PURPOSE: The aim of the present study was two-fold: (a) to examine the effect of hypoxia on [(3)H]D-aspartate release from isolated bovine and human retinae, and (b) to investigate the regulation of hypoxia-induced neurotransmitter release by glutamate receptor agonists and antagonists. METHODS: Isolated neural retinae were incubated in oxygenated Krebs buffer solution containing [(3)H]D-aspartate and then prepared for studies of neurotransmitter release using the superfusion method. Release of [(3)H]D-aspartate was evoked by K(+) (50 mM) applied at 90 minutes (S(1)) and hypoxia (induced by exposure of tissues to solutions pregassed with 95%N(2): 5% CO(2) for 60 minutes) at 108 minutes (S(2)) after onset of superfusion. RESULTS: Under hypoxic conditions, pO(2) in normal Krebs buffer solution was reduced from 14.53 +/- 0.26 ppm (n = 6) to 0.54 +/- 0.04 ppm (n = 9) after one hour of gassing with 95% N(2): 5% CO( 2). Exposure to hypoxia elicited an overflow of [(3)H]D-aspartate yielding S(2)/S(1) ratios of 0.62 +/- 0.06 (n = 12) and 0.54 +/- 0.03 (n = 8) in bovine and human tissues respectively. In isolated bovine retinae, L- and N-calcium-channel antagonists diltiazem, nitrendipine, verapamil and omega-conotoxin significantly (p < 0.01 or higher) attenuated hypoxia-induced [(3)H]D-aspartate release. L-glutamate (30 microM) significantly (p < 0.001) potentiated hypoxia-induced [(3)H]D-aspartate release whereas kainate (30 microM) inhibited this response. NMDA (in concentrations up to 1 mM) had no effect on hypoxia-induced [(3)H]D-aspartate release. Antagonists of glutamate receptors and the polyamine site on the NMDA receptor inhibited hypoxia-induced release of [(3)H]D-aspartate in bovine retina with the following rank order of activity: ifenprodil congruent with MCPG > L-AP3 > MK-801. At an equimolar concentration (10 microM), L-AP3 but not ifenprodil, MCPG, MK 801 or arcaine, caused a significant (p < 0.001) inhibition of hypoxia-induced [(3)H]D-aspartate release from human retinae. CONCLUSIONS: Hypoxia can induce the release of [( 3)H]D-aspartate from isolated bovine retinae by a calcium-dependent process. Hypoxia-induced [(3)H]D-aspartate release from isolated bovine retinae can be regulated by glutamate receptor agonists/antagonists and blockers of polyamine site on the NMDA receptor.     

Arachidonic acid metabolites and peroxide-induced inhibition of [3H]D-aspartate release from bovine isolated retinae

We have previously shown that hydrogen peroxide (H2O2) can inhibit K+-depolarization-evoked [3H]D-aspartate release from bovine isolated retinae. In the present study, we investigated the role of arachidonic acid metabolites in the inhibitory response elicited by H2O2 in the bovine retinae. Furthermore, we examined the direct effect of H2O2 on the production of prostaglandins and isoprostanes in this tissue. Isolated bovine retinae were prepared for studies of [3H]D-aspartate release using the Superfusion Method. Release of [3H]D-aspartate was elicited by Krebs solution containing an iso-osmotic concentration of KCl (50 mM). A direct action of H2O2 on prostaglandin E2 (PGE2) and 8-isoprostane F2alpha (8-iso-PGF2alpha) was also measured by enzyme-linked immunosorbant assay (ELISA). The cyclooxygenase inhibitor, flurbiprofen (3 microM), or the thromboxane-receptor antagonist, SQ 29548 (10 microM) had no significant (p > 0.05) effect on K+-evoked [3H]D-aspartate release. On the other hand, both flurbiprofen (3 microM) and SQ 29548 (10 microM) blocked the inhibition of K+-evoked [3H]D-aspartate induced by H2O2 (30 microM). In concentrations up to 100 microM, H2O2 caused an increase in PGE2 and 8-iso-PGF2alpha over basal levels. For instance, H2O2 (100 microM) increased PGE2 and 8-iso-PGF2alpha over basal levels by 348 +/- 41% and 185 +/- 26 (n = 4), respectively. We conclude that the peroxide-mediated inhibition of [3H]D-aspartate may involve the production of prostaglandins and isoprostanes in the bovine isolated retinae.     

Inhibition of potassium- and ischemia-evoked [3H] D-aspartate release from isolated bovine retina by cannabinoids

We investigated the effect of cannabinoids on potassium chloride (K+)- and ischemia-induced [3H]D-aspartate release from isolated bovine retinae. The superfusion method was employed for studies of [3H]-neurotransmitter release. Cannabinoid receptor CB1 agonists, but not the CB2 agonist JWH 015, inhibited K+ -induced [3H]D-aspartate release from bovine retinae with the following rank order of activity: anandamide > ACEA > methanandamide > WIN 55,212-2. In the ischemic model, the rank order of activity was as follows: methanandamide > ACEA > WIN 55,212-2. The CB1 receptor antagonist AM 251 blocked inhibitory responses produced by cannabinoids in both experimental conditions. In conclusion, cannabinoids inhibit evoked [3H]D-aspartate release from isolated bovine retinae via an effect on CB1 receptors.     

Factors affecting the spontaneous release of (3H)glycine from rabbit retina

The efflux of [3H]glycine was studied in superfused rabbit retina in the presence of various amino acids, ouabain, or high K+ or low Ca2+ concentrations in the superfusion medium. Unlabelled glycine evoked an accelerated efflux as did the structurally similar neutral alpha-amino acids. beta-alanine and GABA were ineffective. The results demonstrate a homoexchange of glycine, and a heteroexchange with the neutral alpha-amino acids. A low concentration of glutamic acid (10(-5) M) will release glycine from the retina. This is an ATPase dependent process which is partially blocked by a high Mg2+/Ca2+ ratio and which may be related to a retinal transmitter function of glutamic acid. A high concentration of K+ or the presence of ouabain in the superfusing medium greatly increases the rate at which glycine is lost from the retina.     

Role of catalase in pre- and postjunctional responses of mammalian irides to hydrogen peroxide.

In the present study, we examined the effect of inhibition of catalase with 3-aminotriazole (3-AT) on hydrogen peroxide (H2O2)-induced enhancement of sympathetic neurotransmission in bovine irides and on the inhibitory effect of this oxidant on norepinephrine (NE) release from human irides, in vitro. Furthermore, we investigated the effect of 3-AT on H2O2-induced attenuation of contractile responses to carbachol in the bovine isolated irides. Isolated mammalian irides were prepared for studies of [3H]NE release using the superfusion method and for contractile studies using isolated organ baths. At concentrations less than 100 microM, H2O2 had no significant effect on field-stimulated [3H]NE release from bovine or human irides. In bovine irides, 3-AT caused significant (P < 0.001) leftward shifts of concentration-response curves to H2O2 (10-300 microM). 3-AT also increased H2O2-induced attenuation of evoked [3H]NE release from human isolated irides. Low concentrations of H2O2 (< 100 microM) had no effect on carbachol contractions. However, 3-AT unmasked an inhibitory effect of low concentrations of H2O2 (3-100 microM) on carbachol-induced contractions. We conclude that inhibition of catalase causes both pre- and postjunctional responses of isolated mammalian irides to be more susceptible to oxidative stress induced by H2O2.     

5-hydroxytryptamine: its facilitative action on [3H]dopamine release from the retina

5-Hydroxytryptamine (5-HT) at 5 X 10(-4) M caused a two-fold increase in [3H]dopamine (DA) release from retinal particulate fractions of the carp (Cyprinus carpio). The 5-HT action was dose- and Ca2+-dependent. The three 5-HT agonists examined (5-methoxytryptamine, 5-methoxy-N,N-dimethyltryptamine and tryptamine) were more effective than 5-HT on [3H]DA release. 5,6-Dihydroxytryptamine was the strongest competitor among drugs tested for [3H ]DA uptake, but did not evoke any significant release of [3H]DA. Noradrenaline (or DA) at 5 X 10(-4) M produced a large increase in [3H]DA release from the particulate fractions, but its action was Ca2+-independent. The 5-HT-induced DA release could be seen in the frog retina but not in the rat retina, which does not contain indoleamine-accumulating cells. The results obtained strongly suggest that 5-HT stimulates [3H ]DA release through a receptor mechanism on DAergic terminals and modifies the DA-cell's function in the retina.     

A human lens model of cortical cataract: Ca2+-induced protein loss, vimentin cleavage and opacification

PURPOSE: Cortical cataract in humans is associated with Ca2+ overload and protein loss, and although animal models of cataract have implicated Ca2+-activated proteases in this process, it remains to be determined whether the human lens responds in this manner to conditions of Ca2+ overload. The purpose of these experiments was to investigate Ca2+-induced opacification and proteolysis in the organ-cultured human lens. METHODS: Donor human lenses were cultured in Eagle's minimum essential medium (EMEM) for up to 14 days. The Ca2+ ionophore ionomycin was used to induce a Ca2+ overload. Lenses were loaded with [3H]-amino acids for 48 hours. After a 24-hour control efflux period, lenses were cultured in control EMEM (Ca2+ 1.8 mM), EMEM + 5 microM ionomycin, or EMEM + 5 microM ionomycin + 5 mM EGTA (Ca2+ < 1 microM). Efflux of proteins and transparency were monitored daily. Protein distribution and cytoskeletal proteolysis were analyzed at the end of the experiment. Cytoskeletal proteins were isolated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analyses were probed with anti-vimentin antibody (clone V9) and detected by enhanced chemiluminescence. RESULTS: Lenses cultured under control conditions remained transparent for 14 days in EMEM with no added supplements or serum. The lenses synthesized proteins and had a low rate of protein efflux throughout the experimental period. Ionomycin treatment resulted in cortical opacification, which was inhibited when external Ca2+ was chelated with EGTA. Exposure to ionomycin also led to an efflux of [3H]-labeled protein, amounting to 41% of the labeled protein over the 7-day experimental period, compared with 12% in ionomycin + EGTA-treated lenses. Efflux was accounted for by loss from the lens soluble protein (crystallin) fraction. Western blot analysis of the cytoskeletal protein vimentin (56 kDa) revealed a distinct breakdown product of 48 kDa in ionomycin-treated lenses that was not present when Ca2+ was chelated with EGTA. In addition, high-molecular-weight proteins (approximately 115 kDa and 235 kDa) that cross-reacted with the vimentin antibody were observed in ionomycin-treated lenses. The Ca2+-induced changes were not age dependent. CONCLUSIONS: Human lenses can be successfully maintained in vitro, remaining transparent for extended periods. Increased intracellular Ca2+ induces cortical opacification in the human lens. Ca2+-dependent cleavage and cross-linking of vimentin supports possible roles for calpain and transglutaminase in the opacification process. This human lens calcium-induced opacification (HLCO) model enables investigation of the molecular mechanisms of opacification, and the data help to explain the loss of protein observed in human cortical cataractous lenses in vivo.     

Inhibition of hypoxia-induced [(3)H]glycine release from chicken retina by the glycine transporter type-1 (GlyT-1) inhibitors NFPS and Org-24461

Chicken posterior eyecup lined by the retina were prepared, loaded with [(3)H]glycine and superfused in order to determine its release in various experimental conditions. Electrical field stimulation of the retina evoked [(3)H]glycine release with a voltage- and frequency-dependent manner and this release may be originated from glycinergic amacrine cell processes of the inner plexiform layer of the retina. Glycine released from an abundance of different amacrine cells may modulate retinal circuitry by activation of inhibitory glycine receptors and by acting as a coagonist on N-methyl-d-aspartate receptors on AII amacrine cells and retinal ganglion cells. The latter effect of glycine may be modulated by glycine transporter type-1. Cells with glycine transporter type-1 immunopositive staining were visualized in the inner nuclear layer and dens immunolabeling was also detected throughout the inner plexiform layer of chicken retina. Glycine and the substrate-type glycine transporter type-1 inhibitor sarcosine increased [(3)H]glycine release from glycinergic amacrine cells and/or glial cells by extrusion of glycine from cytoplasmic pools by homo- and heteroexchange mechanisms. Deprivation of oxygen and glucose from the buffer used for superfusion evoked a marked increase in [(3)H]glycine efflux, an effect probably due to reverse mode operation of glycine transporter type-1. The non-transportable glycine transporter type-1 inhibitors NFPS and Org-24461, which did not alter [(3)H]glycine efflux from isolated chicken retina by themselves in normoxic condition, inhibited oxygen and glucose deprivation-induced [(3)H]glycine release. It is concluded that reduction of the N-methyl-d-aspartate receptor coagonist glycine concentrations in hypoxic conditions by glycine transporter type-1 inhibitors may decrease N-methyl-d-aspartate receptor-mediated neuronal toxicity and cell death in retinal tissue.     

Similar properties of taurine release induced by potassium and hyposmolarity in the rat retina

Increasing external K+ concentration to 56, 75 or 100 mM stimulated taurine release by 3-, 7- and 11-fold, respectively. The K(+)-evoked release of [3H]taurine was markedly delayed, sustained and Ca(2+)-independent, in clear contrast to the usual neurotransmitter release pattern. These high K+ concentrations caused a marked increase in retinal cell volume which was prevented by removal of Cl-, or in hyperosmotic solutions. In these conditions [3H]taurine release also was abolished, suggesting an association of taurine efflux and cell swelling. Taurine release was also markedly increased 10- and 20-fold upon reduction of external osmolarity by 25 and 50%, respectively. Both, K(+)- and hyposmolarity-induced release were markedly inhibited by DIDS and quinidine. Total inhibition of the K(+)-evoked release was observed at 200 microM DIDS or 1 mM quinidine, whereas the drugs inhibited the hyposmolarity-evoked release by 50 and 68% respectively, at these concentrations. It is suggested that swelling is the signal for the K(+)-induced taurine release from rat retina.     

Guanosine 5'-O-thiotriphosphate and NaF stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in bovine corneal epithelium

The role of a GTP-binding protein in activation of phospholipase C in bovine corneal epithelium was determined by investigating the effects of non-hydrolyzable GTP analog, GTP-gamma-S, and NaF on breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) in this tissue. GTP-gamma-S (2-50 microM), when introduced into the permeabilized corneal epithelial cells labeled with myo-[3H]inositol, dose-dependently increased the formation of myoinositol trisphosphate (IP3). Other guanine nucleotides and ATP were ineffective. Incubation of 32P-prelabeled corneal epithelium with NaF (2-50 mM) resulted in increased breakdown of PIP2 and increased synthesis of phosphatidic acid. In myo-[3H]inositol-labeled tissue, NaF dose-dependently increased the accumulation of IP3. Microsomal membrane fraction from corneal epithelium was found to contain phospholipase C activity towards endogenous phosphatidylinositol 4-phosphate and PIP2. The enzyme activity was stimulated by Ca2+ (100 microM). Addition of GTP-gamma-S to microsomal fraction containing phosphoinositides which were radiolabeled with 32Pi in situ or with [gamma-32P]ATP in vitro caused a dose dependent hydrolysis of PIP2. These data, taken collectively, suggest that a GTP-binding protein is involved in activation of phospholipase C towards PIP2 in bovine corneal epithelium, and that this guanine nucleotide regulatory protein may serve to couple norepinephrine and 5-hydroxytryptamine receptors to phospholipase C during transmembrane signalling.     

Protein kinase C-mediated modulation of glutamate transporter activity in rat retina

It has previously been shown that inhibitors of protein kinase C (PKC) attenuate retinal glutamate uptake in situ. The aim of the current study was to determine whether PKCdelta-mediated inhibition differentially reduces the transport of glutamate into retinal Müller cells when compared with retinal neurons. The influence of two different types of PKC inhibitors on the uptake of [3H]D-aspartate was therefore compared in the intact retina, mixed retinal cultures, and Müller cell-enriched retinal cultures. It was found that 25 microM of the pan-isoform PKC inhibitor, chelerythrine, reduced [3H]D-aspartate uptake by 78%, 71%, and 68% in isolated retinas, mixed neuronal/glial cultures, and Müller cell-enriched cultures, respectively. Importantly, 20 microM of the PKCdelta-selective inhibitor rottlerin also reduced the uptake of D-aspartate to similar extents in all three systems, and the reductions were statistically similar to those found for the pan-specific PKC inhibitor. Neither pan-isoform nor PKCdelta-selective activators stimulated glutamate uptake in either culture system or the intact retina. The current results suggest that specific PKC inhibitors are quantitatively similar in reducing the uptake of glutamate into retinal neurons and Müller cells.     

Glycine stimulates calcium-independent release of 3H-GABA from isolated retinas of Xenopus laevis

A perfusion system was used to monitor the release of [3H]-GABA from isolated retinas of Xenopus laevis. Measurable release was stimulated by glycine at concentrations as low as 200 microM. Glycine-stimulated release was blocked by strychnine, and was not reduced in "calcium-free" Ringer's solution (0 Ca2+/20 mM Mg2+). Glutamate also stimulated calcium-independent release, using concentrations as low as 100 microM. In contrast, release stimulated by 25 mM potassium was reduced by 80% in calcium-free medium. In most experiments, agonists were applied in six consecutive 4-min pulses separated by 10-min washes with Ringer's solution. Under these conditions, the release stimulated by 0.5 mM glutamate or 25 mM potassium decreased by at least 50% from the first to the second pulse, and then gradually decreased with successive applications. In contrast, the response to 0.5 mM glycine at first increased and then only gradually decreased with successive pulses. These patterns of response to different agonists were similar in calcium-free medium. Somatostatin (-14 or -28) also stimulated release, and this effect was inhibited by AOAA, an inhibitor of GABA degradation. In the presence of AOAA, somatostatin had little effect, except at high concentrations of somatostatin (5 microM), which increased both basal and glycine-stimulated release. In contrast to somatostatin, glycine-stimulated release was much larger in the presence of AOAA. Autoradiography was used to investigate which cell types released [3H]-GABA under our conditions. Autoradiograms showed that horizontal cells and a population of apparent "off" bipolar cells were well-labeled by [3H]-GABA high-affinity uptake. In addition, light labeling was seen over numerous amacrine cells. After application of glycine, glutamate, or potassium, there was a decrease in label density over horizontal cells.     

Somatostatin inhibits calcium influx into rat rod bipolar cell axonal terminals

In the retina, somatostatin (SST), an inhibitory peptide that influences neuronal activity, is predominantly expressed by sparsely occurring amacrine cells. The SST subtype 2A receptor is expressed by rod bipolar cells, including their axonal terminals. We used Ca2+-imaging techniques and the ratiometric Ca2+ indicator dye fura-2 AM to investigate Ca2+ dynamics in rod bipolar cell terminals. Depolarization of rod bipolar cells by the addition of high K+ (50 or 100 mM) elicited a sustained increase in [Ca2+]i in rod bipolar terminals that returned to basal levels following K+ removal. The Ca2+ response was dependent on extracellular Ca2+, and was inhibited by the Ca2+ channel blocker Cd2+ and by the selective L-type Ca2+ channel blocker, nimodipine, SST inhibited a K+ depolarization-induced [Ca2+]i response in rod bipolar terminals. This inhibition was observed with 1 nM SST and was maximal with 1 microM SST. These findings indicate that SST may regulate transmitter release from rod bipolar terminals by activating the SST subtype 2A receptor through modulation of intracellular Ca2+.     

Stimulation of phospholipase D by phorbol esters and ionomycin in bovine corneal epithelial cells.

This study was performed to determine the effects of phorbol esters and ionomycin on phospholipase D (PLD) activity in bovine corneal epithelial cells (BCEC). The cells were prelabeled with [3H]myristic acid and incubated for specific time intervals with various test agents in the presence and absence of ethanol. The PLD activity was assayed by monitoring the formation of labeled phosphatidylethanol ([3H]PEt) in [3H]myristate labeled cells. In the absence of ethanol, 1 microM phorbol 12-myristate 13-acetate (PMA) increased the formation of labeled phosphatidic acid ([3H]PA) with no significant effect on the radioactivity of [3H]PEt. In the presence of 85 mM ethanol, whereas there was only a small further increase in [3H]PA, the formation of [3H]PEt was increased by several-fold, demonstrating activation of PLD by the phorbol ester. The effects of PMA were time- and dose-dependent, and were mimicked by phorbol 12,13-dibutyrate. The inactive phorbol derivatives, 4-alpha-phorbol, 4-alpha-phorbol 12,13-didecanoate, 4-alpha-phorbol 12-myristate 13-acetate and 4-alpha-phorbol 12,13-dibutyrate, were without effect. Short-time (30 min) incubation of BCEC with staurosporine or H-7, or prolonged (20 hours) incubation with PMA rendered the cells less sensitive to subsequent treatment with PMA, suggesting that activation of PLD in the cells is mediated by protein kinase C (PKC). Addition of 20 microM ionomycin in the presence of ethanol also increased the formation of [3H]PA and [3H]PEt in a time- and dose-dependent manner. Co-presence of ionomycin and PMA at submaximal concentrations in the incubation medium resulted in increased formation of [3H]PA and [3H]PEt which was less than their individual effects combined, indicating a lack of synergism between Ca2+ and PMA in activating PLD. Incubation of BCEC with staurosporine resulted in significant inhibition of ionomycin-induced production of [3H]PEt, suggesting that in addition to direct activation of PLD by Ca2+, the enzyme is probably stimulated by sequential activation of PLC (producing diacylglycerol) and PKC following the ionomycin addition. We conclude that BCEC possess PLD which is stimulated by PKC as well as elevated intracellular Ca2+.     

The inhibition of CA2+ influx induced by hypericin in cultured human retinal pigment epithelial cells analyzed by confocal imaging

PURPOSE: Hypericin, a specific inhibitor of protein kinase C, has been reported to have potential as a therapeutic drug for proliferative vitreoretinopathy (PVR) in vitro and in vivo. In the present studies, we analyzed the dynamic changes in Ca2+ influx and free intracellular Ca2+ concentration ([Ca2+]i) of cultured human retinal pigment epithelial (RPE) cells after stimulation with hypericin in an attempt to elucidate its mechanism as a therapeutic drug for PVR. METHODS: RPE cells were plated in a special plastic dish and then stimulated with 100 nM phorbol-12-myristate-13-acetate (PMA) and/or 6 hypericin concentrations (0.5, 1, 2, 3, 4 and 5 microM), after which Ca2+ influx and [Ca2+]i were determined using the fluorescence Ca2+ dye fluo-3 AM and laser scanning confocal microscopy. RESULTS: The fluorescence in resting RPE cells was strong and distributed throughout the cells. The nucleus appeared more fluorescent than the cytoplasm. After stimulation with 0.5 microM hypericin, no obvious change of Ca2+ influx and [Ca2+]i was observed. In contrast, stimulation with higher concentrations of hypericin (1-5 microM) led to a rapid decrease in Ca2+ influx and [Ca2+]i, which was significantly different from those detected without hypericin (control experiments). In addition, no significant differences in [Ca2+]i were found between 1 and 5 microM hypericin used. Stimulation with hypericin, which was applied immediately after preincubation with PMA for 24 h did not further change Ca2+ influx and [Ca2+]i. CONCLUSION: In RPE cells, high concentrations of hypericin (1-5 microM) significantly inhibit Ca2+ influx and induce a decrease in [Ca2+]i. Therefore, hypericin has potential as a therapeutic drug for PVR maybe through its inhibition of the Ca2+ influx pathway.     

Characterization of brimonidine transport in retinal pigment epithelium

PURPOSE: To investigate the involvement of carrier-mediated transport mechanisms in brimonidine transport in retinal pigment epithelium (RPE). METHODS: The transport of [3H]-brimonidine in bovine RPE-choroid explants was evaluated in a modified Ussing chamber. The uptake of [3H]brimonidine was evaluated in differentiated ARPE-19 cells cultured on permeable transwell filters. RESULTS: The transport of brimonidine into (choroid-to-retina transport [inward]) and out of (retina-to-choroid transport [outward]) the eye in bovine RPE-choroid explants was temperature dependent. Both inward and outward brimonidine transport decreased at 5 microM compared with 10 nM. The melanin pigmentation of RPE did not significantly affect tissue permeability at either brimonidine dose. A saturable component was identified for the inward transport with the apparent Michaelis-Menten constant and a maximum transport rate of 51 microM and 148 pmol/(cm2 x h), respectively. Both apical (representing retina-to-choroid transport) and basolateral (representing choroid-to-retina transport) brimonidine uptake in ARPE-19 cells showed temperature dependence. Apical uptake was higher than basolateral uptake at 37 degrees C and was decreased to 70% in the presence of NaN3 or in the absence of extracellular Na+. Besides alpha2-agonists, apical uptake was inhibited by verapamil, desipramine, and quinidine, but not by MPP+ (1-methyl-4-phenylpyridinium), TEA (tetraethylammonium), decynium-22, carnitine, PHA (p-aminohippurate), alanine, or inosine. Basolateral brimonidine uptake increased by 35% at extracellular pH of 6 and decreased by 50% under cell-depolarized conditions of high medium K+ and 1 microM valinomycin. Temperature-dependent components of basolateral uptake were not saturated at doses up to 2 mM. CONCLUSIONS: A carrier-mediated transport process for brimonidine in RPE was demonstrated in bovine RPE-choroid explants and polarized ARPE-19 cells. This transport system may play a significant role in modulating the movement of brimonidine into and out of the eye.     

Glutamate regulates the viability of retinal cells in culture

In this study, we show that glutamate regulates the viability of cultured retinal cells upon transient glucose deprivation. At low concentrations (10-100 microM) glutamate decreased MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction to about 50% of control and decreased intracellular ATP levels (about 4-fold) after transient glucose removal. Under these conditions, the decrease in MTT reduction was associated with the activation of NMDA (N-methyl-D-aspartate) receptors. Upon exposure to high (10 mM) glutamate and transient glucose deprivation, the intracellular levels of glutamate increased. High glutamate significantly counteracted the decrease in MTT reduction and ATP production observed in the presence of low glutamate concentrations. AOAA (aminooxyacetic acid), a non-specific inhibitor of mitochondrial transaminases, enhanced the intracellular glutamate levels, but did not largely affect glutamate-mediated changes in MTT reduction or ATP production. Furthermore, the intracellular levels of pyruvate were not significantly altered, suggesting that changes in ATP production were not due to an increase in glycolysis. Thus, the recovery from glucose deprivation seems to be facilitated in retinal neuronal cells that had been exposed to high glutamate, in comparison with low glutamate, suggesting a role for high glutamate and glucose in maintaining retinal cell function following conditions of glucose scarcity.     

Phosphoinositide turnover and Ca2+ mobilization during phagocytosis in cultured chick retinal pigment epithelial cells

Ca2+ mobilization and phosphoinositide turnover were examined during phagocytosis of latex particles in cultured chick retinal pigment epithelial cells (RPE cells). Ca2+ influx into cells and Ca2+ efflux from the cells were enhanced by about 1.5-fold and 3-fold compared to control cells, respectively. A high content of Ca2+ in RPE cells has been reported. Therefore, the Ca2+ efflux observed here may be a reflection of Ca2+ release into cytosol from the intracellular storage site(s). In [3H] inositol-prelabeled RPE cells, addition of latex particles elicited decreases in phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol with the concomitant formation of inositol phosphates including inositol trisphosphate, indicating the hydrolysis of phosphoinositides by phospholipase C. These results suggest that phosphoinositide turnover may be closely coupled with Ca2+ mobilization during phagocytosis in RPE cells.     

EGF suppresses hydrogen peroxide induced Ca2+ influx by inhibiting L-type channel activity in cultured human corneal endothelial cells

Endogenous generated hydrogen peroxide during eye bank storage limits viability. We determined in cultured human corneal endothelial cells (HCEC) whether: (1) this oxidant induces elevations in intracellular calcium concentration [Ca2+]i; (2) epidermal growth factor (EGF) medium supplementation has a protective effect against peroxide mediated rises in [Ca2+]i. Whereas pathophysiological concentrations of H2O2 (10 mM) induced irreversible large increases in [Ca2+]i, lower concentrations (up to 1 mM) had smaller effects, which were further reduced by exposure to either 5 microM nifedipine or EGF (10 ng ml(-1)). EGF had a larger protective effect against H2O2-induced rises in [Ca2+]i than nifedipine. In addition, icilin, the agonist for the temperature sensitive transient receptor potential protein, TRPM8, had complex dose-dependent effects (i.e. 10 and 50 microM) on [Ca2+]i. At 10 microM, it reversibly elevated [Ca2+]i whereas at 50 microM an opposite effect occurred suggesting complex effects of temperature on endothelial viability. Taken together, H2O2 induces rises in [Ca2+]i that occur through increases in Ca2+ permeation along plasma membrane pathways that include L-type Ca2+ channels as well as other EGF-sensitive pathways. As EGF overcomes H2O2-induced rises in [Ca2+]i, its presence during eye bank storage could improve the outcome of corneal transplant surgery.     

Does the neurotransmitter transporter underlie adaptation at a histaminergic photoreceptor synapse?

Using autoradiographic and biochemical techniques, we studied the sodium-dependent forward and reverse transport of the neurotransmitter histamine in an arthropod photoreceptor in order to test whether the transporter plays a central role in visual signal transfer at this synapse. In particular, we asked whether the histamine transporter might be the important factor in synaptic adaptation, the process by which the operating range of the synapse adapts to increasing depolarizations of the photoreceptor in increasing background light. Drugs known from electrophysiological observations to interfere with synaptic adaptation blocked the uptake of [3H]histamine into photoreceptors. These drugs also blocked the sodium (Na)-triggered efflux of [3H]histamine, previously loaded into photoreceptors, via the histamine transporter. Several lines of evidence showed that efflux of [3H]histamine did not occur via calcium-dependent exocytosis. First, efflux occurred when the preparation was bathed in calcium (Ca)-free/EGTA salines or in cobalt (Co)-containing salines. Even more importantly, efflux could be elicited from axons, whose membranes must contain the transporter protein since they take up [3H]histamine independently from the presynaptic terminals. Since both adaptation and the histamine transporter are blocked by the same agents, the transporter may underlie adaptation by maintaining the cleft histamine concentration in a particular range independent of light intensity. We also characterized the transporter further and found that it is partially dependent on chloride ions, and that neither [3H]norepinephrine nor [3H]dopamine are transported (at 20 microM), adding to evidence that the transporter is highly selective for histamine.