Thrombin activation of PI3K/PDK1/Akt signaling promotes cyclin D1 upregulation and RPE cell proliferation.

The retinal pigment epithelium (RPE) plays an essential role in the survival and function of the neural retina. RPE uncontrolled proliferation leads to the development of proliferative ocular pathologies, among which proliferative vitreoretinopathy (PVR) is the main cause of retinal surgery failure. Upon the breakdown of the BRB due to trauma or metabolic imbalance the contact of RPE with serum-contained thrombin has been shown to stimulate the proliferation of otherwise quiescent RPE cells. Although the molecular mechanisms involved in this effect are still undetermined, thrombin proteolytic activation of protease-activated G protein coupled receptor-1 (PAR-1) activates PI3K and Akt, known to play an essential role in proliferation. The present study demonstrates that: 1) thrombin stimulates Ser 473 Akt phosphorylation without affecting Thr 308 basal phosphorylation in RPE cells; 2) thrombin-induced Akt stimulation promotes cyclin D1 accumulation through the phosphorylation/ inhibition of GSK-3ß, thus preventing Thr 286 cyclin D1 phosphorylation, nuclear export and degradation; 3) Akt signaling requires the upstream activation of PI3K and PLC. Since the pharmacological inhibition of these pathways or the silencing of cyclin expression prevent thrombin-induced RPE cell proliferation, these results contribute relevant evidence for establishing the mechanism involved in the development of proliferative eye diseases.

FAK phosphorylation plays a central role in thrombin-induced RPE cell migration.

The migration of retinal pigment epithelial (RPE) cells is an important step in various pathologic conditions including subretinal neovascularization (SRN), proliferative vitreoretinopathy (PVR) and, importantly, as a consequence of retinal surgery. Therefore, the elucidation of the mechanisms underlying RPE trans-differentiation and migration is essential for devising effective treatments aimed to the prevention of these disorders. A common event in these pathologies is the alteration of the blood-retina barrier (BRB), which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Our previous work has demonstrated that thrombin induces RPE cell cytoskeletal remodeling and migration, hallmark processes in the development of PVR; however, the molecular mechanisms involved are still unclear. Cell migration requires the disassembly of focal adhesions induced by Focal Adhesion Kinase (FAK) phosphorylation, together with the formation of actin stress fibers. The aim of the present work was to identify thrombin-activated signaling pathways leading to FAK phosphorylation and to determine FAK participation in thrombin-induced RPE cell migration. Results demonstrate that the activation of PAR1 by thrombin induces FAK autophosphorylation at Y397 and the subsequent phosphorylation of Y576/577 within the activation loop. FAK phosphorylation was shown to be under the control of c/nPKC and PI3K/PKC-ζ, as well as by Rho/ROCK, since the inhibition of these pathways prevented thrombin-induced FAK phosphorylation and the consequent disassembly of focal adhesions, in parallel to FAK-dependent actin stress fiber formation and RPE cell migration. These findings demonstrate, for the first time, that thrombin stimulation of RPE cell transformation and migration are regulated by FAK tyrosine phosphorylation. Thus, targeting FAK phosphorylation may provide a strategical basis for PVR treatment.

Sea urchin sperm head plasma membranes: characteristics and egg jelly induced Ca2+ and Na+ uptake.

Sea urchin sperm respond to egg factors with changes in the ionic permeability of their plasma membrane. It has been previously shown that plasma membranes isolated preferentially from sea urchin sperm flagella respond to egg jelly increasing their Ca2+ and Na+ uptake (Darszon et al. (1984) Eur. J. Biochem. 144, 515-522). However, the egg jelly induced acrosome reaction occurs in the sperm head, and there is evidence for an heterogeneous distribution of plasma membrane components within the various regions of this cell. We here report a method for purifying sperm head membranes using positively charged beads according to Jacobson (1977) Biochim. Biophys. Acta 471, 331-335). Under the transmission electron microscope these membranes appeared homogeneous and apparently free of internal membranes. The yield of the preparation was 0.9% of the total protein in the sperm homogenate. The preparation contained less than 5% of the mitochondrial marker cytochrome oxidase, and 10% of the total DNA/mg protein. Surface labeling with 125I indicated a 2.5-3-fold enrichment in specific activity of the head membranes with respect to whole sperm. The SDS band pattern and the lipid composition of this preparation were different from those of isolated flagellar membranes. Phosphatidylcholine was higher in the head membranes, while phosphatidylserine and phosphatidylethanolamine were lower. The head membranes displayed a 1.7-2.3-fold higher Ca2+-ATPase activity and a 2.5-fold lower Na+/K+-ATPase activity, than the flagellar membranes. These results are consistent with a heterogeneous distribution of membrane components along the sea urchin sperm plasma membranes. Isolated head membranes sonicated in the presence of soybean phospholipid liposomes responded to egg jelly with a species-specific increase in Ca2+ and Na+ uptake. As in whole sperm, Ca2+ uptake was inhibited by the Ca2+ channel blocker nisoldipine. A close analog of this compound, [3H]nitrendipine, binds with high affinity to head membranes in a saturable, reversible manner, showing a Kd and Bmax of 31 nM and 5.3 pmol/mg protein, respectively.

NMDA receptors in cultured radial glia.

The expression of the NMDA subtype of glutamate receptors was investigated by Western blot analysis and RT-PCR in cultured chick Bergmann and Müller glial cells. Using subunit-specific antibodies directed to the carboxy terminus of the rat NMDAR2A/B we detected the expression of the NMDAR2 subunit in both kinds of culture. The functional subunit of the NMDA receptor, NMDAR1, was detected by means of RT-PCR. These results, together with our previous functional characterization of NMDA receptors in radial glia, provide conclusive evidence for the expression of functional NMDA receptor/channels in Bergmann and Muller glia cells. Our findings strengthen the notion of a modulatory role of glial cells in synaptic transmission.

N-methyl-D-aspartate receptors in the retina: 3-[(+/-)-2-carboxypiperazin- 4-yl]-propyl-1-phosphonic acid (CPP) binding studies.

3-[(RS)-2-Carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) has been known for some years as one of the most selective antagonists at the N-methyl-D-aspartate (NMDA) receptor in the brain. The characteristics of the binding of [3H]CPP to chick retinal membranes were studied from the biochemical and pharmacological point of view. Magnesium induced a dose-dependent increase in the binding of [3H]CPP (EC50 = 4 microM). In the absence of this ion, a single population of receptors was found (KB = 431 nM; Bmax = 9.5 pmol/mg protein), which was not modified by the addition of 1 mM MgCl2. An additional, high affinity site (KB = 59 nM; Bmax = 2.2 pmol/mg protein) became evident in the latter condition. Saturation curves of the binding of [3H]CPP, using 1 mM AMPA [(RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate] or L-aspartate, as displacers in the presence of Mg2+, showed a KB = 200 and 395 nM, respectively. The relative potency of some analogues of excitatory amino acids, for displacing bound CPP in the absence of Mg2+, was AMPA = APH greater than L-glutamate = CPP; Mg2+ significantly increased the potency of AMPA, APH and L-glutamate. These results showed that CPP bound to high affinity (Mg(2+)-dependent) and low affinity sites and that AMPA and L-aspartate compete with this compound only at the low affinity sites. These findings suggest that either the binding of CPP in the retina shows different properties from those described in the brain or alternatively, that AMPA is not as specific for the quisqualate receptor in this organ.

Taurine receptors in membranes from retinal pigment epithelium cells in culture.

[3H]Taurine-specific binding to membranes from retinal pigment epithelium was demonstrated. A single saturable system was found, with KB = 237 nM and Bmax = 2.8 pmol/mg protein. Binding to freshly prepared membranes showed partial Na(+)-dependence while in frozen/thawed membranes, binding remained unchanged in the absence or presence of this ion. A 30-40% increase in binding was observed at physiological temperature (37 degrees C) compared to 4 degrees C in fresh but not in frozen membranes. Accumulation of taurine was followed during differentiation in vitro; results showed that changes in uptake and receptor binding to frozen membranes are not parallel, discarding the possibility of an interaction with uptake sites. Pharmacology of these binding sites suggests that they could be common to other amino acids, since displacement experiments showed that glycine, beta-alanine and strychnine were as potent as taurine itself in displacing [3H]taurine. Our data open the possibility of taurine being involved in the communication between the retina and the retinal pigment epithelium through an interaction with specific receptors.

Changes in GluR4 expression induced by metabotropic receptor activation in radial glia cultures.

The expression of neurotransmitter receptors in glial cells has suggested a regulatory role of these cells in synaptic function. In radial glia, glutamate receptors elicit a cascade from the membrane to the nucleus and a consequent change in gene expression. In order to gain insight into this process, we address the question of whether receptor activation leads to changes in the repertoire of AMPA/KA glutamate receptor subunits in Bergmann and Müller glial cells. Of the subunits investigated, only GluR4 was up-regulated in Bergmann glial cells both at mRNA and protein levels. In contrast, in Müller glial cells Glu treatment leads to a reduction in GluR4 mRNA and protein expression. Both effects are receptor-mediated and must probably involve group I of metabotropic glutamate receptors. Accordingly, using Northern blot analysis and RT-PCR we detected the expression of both mGluR1 and mGluR5 transcripts in the cultured cells. Our results confirm that glutamate receptors in Bergmann and Müller cells modulate gene expression and further strengthen a plausible role of glial cells in long-lasting changes in the central nervous system.

AMPA/KA receptor expression in radial glia.

The expression of four genes (GluR 1; 2; 3; 4) encoding functional subunits of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/low affinity kainate (KA) subtype of glutamate receptors was investigated in chick radial glia, namely Bergmann and Müller glial cells, using Northern blot analysis with oligonucleotide probes. Both cell types expressed the transcripts GluR 1; 3; 4, whereas the GluR 2 mRNA could not be detected. The synaptic localization of these receptors, their ion-channel properties and their regulation further strengthen the putative role of glial cells in the modulation of synaptic efficacy and plasticity.

Serum affects the characteristics of excitatory amino acid-binding sites on Müller cells.

The binding of [3H]L-aspartate to membranes obtained from primary cultures of chick retinal Müller cells (glia) was studied Cells seeded in low-serum-containing medium (1%) and maintained in this condition showed an increased number of binding site from 1 to 5 days in vitro (DIV), when compared with controls cultured in medium containing 10% serum; these changes were not reversed by the addition of 10% serum after 48 h in vitro. Increased binding at this age was due to the expression of a low affinity binding system, competitively inhibited by the glutamate uptake blocker L-aspartate-beta-hydroxamate, suggesting that high serum might inhibit the expression of uptake sites at precise maturation stages. Experiments showed the effect was due to a thermolabile serum component. The increase in binding sites is parallel in time to both an increase in aspartate uptake and the initiation of synaptogenesis in the whole retina. Our results suggest that the presence of serum at defined stages in retinal development, could result in the elevation of extracellular glutamate and the concomitant excitotoxic death of neuronal cells, due to a decreased glutamate uptake by glial cells.

Characterization of quisqualate-type L-glutamate receptors in the retina.

In the vertebrate retina excitatory transmission seems to be mediated mainly by excitatory amino acids; glutamate and/or aspartate are the most viable candidates to subserve this function. Postsynaptic receptors for N-methyl-D-aspartate (NMDA), kainate (KA), quisqualate (QA) and 2-amino-4-phosphonobutyric acid have been electrophysiologically identified. In this work we have tried to identify and characterize QA receptors through the binding of the most specific analogue available for this receptor: [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA). AMPA binding to retinal membranes was sodium- and temperature-independent, with optimum pH at 6-7. Ligand-receptor interaction was reversible and saturable. Pharmacologically, glutamate analogues were more active displacers than NMDA analogues: AMPA greater than (RS)-3-hydroxy-4,5,6,7-tetrahydro-isoxazolo-(5,4-C)-pyridine-7-car boxylic acid = L-Glu = QA; with IC50 in the low microM range. Glutamic acid diethylester was uneffective while KA and cis-2,3-piperidine dicarboxylate were potent inhibitors of binding. Binding was stereospecific, L-isomers being more effective displacers than D-forms. Subcellular distribution showed binding concentrated in the inner plexiform layer (IPL), but also present in the outer plexiform layer (OPL). Kinetics of [3H]AMPA binding showed a high affinity kB = 1-2 microM in membranes from complete retina, IPL and OPL, with binding sites concentrated in P2 (Bmax = 16.2 pmol/mg protein). Our results provide biochemical evidence for the presence and distribution of physiologically relevant QA receptors in the chick retina which is in agreement with previous physiological findings.

Localization of L-glutamate and L-aspartate synaptic receptors in chick retinal neurons.

Kainic acid (KA) produces lesions in the chick retina when injected intravitreally. In order to ascribe L-glutamate and L-aspartate receptors to specific neuronal populations, we measured L-[3H]glutamate and L-[3H]aspartate specific binding to membranes from retinas treated with different doses of KA. A 20% reduction in glutamate and aspartate receptors was observed when amacrine cells were eliminated (50 nmol KA). When 120 nmol KA were injected, horizontal cells were highly decreased and so were glutamate (48%) and aspartate (22%) receptors. 200 nmol KA killed most of the bipolar cells in addition to the horizontal and amacrine cells; this lesion was associated with an additional 21% decrease in the specific binding of aspartate but not glutamate. Receptors for both compounds which remained after 200 nmol KA (30%) could be located in ganglion cells, which were spared by KA.

The adenylate cyclase inhibitor MDL-12330A has a non-specific effect on glycine transport in Müller cells from the retina.

Müller glial cells express two transport systems for glycine (Gly): one with low affinity and another identified as GLYT1 with high affinity. The latter colocalizes with NMDA receptors in the CNS. Gly is considered as an obligatory coagonist at NMDA receptors, and, hence, the Gly transport system could contribute to the modulation of glutamate (Glu) excitatory transmission in the vertical pathways of the retina. For this reason, the regulation of Gly transport by cAMP was studied. We report here a non-specific effect of MDL-12330A, a compound reported to inhibit adenylate cyclase (AC), on Gly transport in Müller glia. This effect might be due to a toxic action on the cells, decreasing cell viability, and not to a specific inhibition of the adenylate cyclase. Non-specific effects of this drug should be considered when the participation of cAMP in any biological process is studied. We have clearly demonstrated that cAMP does not participate in the regulation of Gly transport in Müller glia.

Chloramphenicol modifies benzodiazepine receptor rhythm in the pontomesencephalic formation of the rat.

Pontomesencephalic benzodiazepine (BZ) receptors were measured at 4 h intervals throughout a 24 h day, and compared with those in frontal cortex, using [3H]diazepam binding. Animals were treated with saline, chloramphenicol (CAP) or thiamphenicol (TAP). An ultradian rhythm of receptors was observed in both cases, which was abolished by CAP but not by TAP. Saturation curves and Scatchard analysis indicated decreased binding was due to a decrease in the number of receptors. CAP effect on REM sleep, could be mediated by a general decrease of neurotransmitter receptors at precise periods of the wake-sleep rhythm.

Maturational changes in retinal excitatory amino acid receptors.

The appearance, kinetics and pharmacological properties of receptors for n-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate (KA), L-glutamate (Glu) and L-aspartate (Asp) was investigated using 3H-ligand binding during the development of chick embryo retina. Receptors for AMPA are maximally concentrated at embryonic day 7 (ED 7) and decline 50% in subsequent days; L-Glu receptors are low until ED 11, and the same is true for Asp and NMDA receptors which increase at ED 14 and 18 respectively. All receptors studied underwent an increase in pharmacological specificity, whereas only AMPA-receptors showed an important change in affinity during ontogeny. Results demonstrate that receptors for excitatory amino acids in the retina suffer maturational changes and suggest that while NMDA and aspartate could interact with the same receptor, AMPA and glutamate seem to bind to different sites.

Recovery of taste aversion learning induced by fetal neocortex grafts: correlation with in vivo extracellular acetylcholine.

Rats showing disrupted taste aversion due to insular cortex lesions, received either homotopic or heterotopic (occipital) cortical fetal brain grafts. Behavioral results showed that the recovery of the ability to acquire conditioned taste aversions induced by fetal grafts depended on post-graft time (45 but not at 15 days) and tissue specificity (homotopic but not heterotopic). In vivo analysis of acetylcholine (ACh) release revealed that only the group receiving homotopic grafts and tested 45 days post graft had a release of ACh after KCl stimulation similar to that in the control group. Furthermore, homotopic grafts and lesioned groups showed significantly weaker specific receptor binding of [3H]L-glutamate compared with controls. These results suggest that ACh is specifically involved in the process of behavioral recovery induced by homotopic cortical transplants.

Calcium-independent release of [3H]spermine from chick retina.

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [3H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [3H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca2+-independent manner. Inhibition of Na+/K+-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na+ electrochemical gradients, since nigericin and veratrine did not induce release in Na+ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.

Taurine effects on 45Ca2+ transport in retinal subcellular fractions.

The effect of taurine on 45Ca2+ transport by subcellular fractions from the chick retina was examined. An inhibitory action of taurine on 45Ca2+ uptake was observed in retinal fractions incubated for 1--5 min in a Krebs--bicarbonate medium, pH 7.4. In the crude nuclear fraction, 25 mM taurine produced a decrease of 50% in 45Ca2+ uptake; in the crude synaptosomal fraction, taurine reduced 45Ca2+ accumulation by 70%; the maximum inhibitory effect of taurine on 45Ca2+ uptake (80%) was observed in a fraction containing outer segments and pigment epithelium cells. Taurine effect was specific, dose-dependent and related to osmotically sensitive particles. The results suggest a role for taurine in the regulation of calcium fluxes in the retina.

[3H]Spermine binding to synaptosomal membranes from the chick retina.

The binding of [3H]spermine to synaptosomal membranes from chick retina was examined. Saturable specific binding of [3H]spermine to synaptosomal membranes from plexiform layers of retina (P1 and P2) has been characterized, and found to concentrate in the inner plexiform layer compared to the outer plexiform layer (Bmax=9.3 and 37 pmol/mg protein for P1 and P2, respectively). Kinetics of specific [3H]spermine binding yield a sigmoidal saturation curve, indicating positive cooperativity (nH: 2.4 and 3.2 for P1 and P2, respectively) with high affinity: Kapp=61 and 67 nM for P1 and P2. The time required to attain equilibrium at room temperature was less than 5 min in both fractions. Dose-response curves for spermine, spermidine, and diethylene-triamine (DET) show different potencies for inhibiting [3HDET. Our results support a role for polyamines (PA) as neurotransmitters or neuromodulators in the vertebrate retina.

Strychnine-insensitive [3H] glycine binding to synaptosomal membranes from the chick retina.

The pharmacology and kinetics of strychnine-insensitive [3H] glycine binding to synaptic membranes from the outer (P1) and the inner (P2) plexiform layers of chick retina was studied. Inhibition curves for glycine, D-serine, 1-amincyclopropanecarboxylic acid (ACPC) and strychnine were analyzed by non-linear regression. Hill slopes for glycine and D-serine were not different from unity, whereas those for ACPC were < 1 in both fractions, revealing heterogeneity of binding sites in these membranes. Non-linear regression analysis of time course and saturation experiments strengthen the idea that [3H] glycine binds to more than one class of sites, with similar affinities at equilibrium. Antagonists of strychnine-insensitive glycine receptors in the CNS did not inhibit [3H] glycine binding to these membranes, which demonstrates that NMDA receptors in the retina have different structural requirements for ligand interaction at these sites. pH affected the specific binding, in agreement with the participation of specific amino acid residues at glycine binding sites on NMDA receptors, and also with functional studies in which the modulation of affinity at this site by protons has been observed. These results support previous studies regarding CPP and MK-801 binding, and provide evidence which indicates that the pharmacophore for glycine and other NMDA-related ligands is distinct for the retina, compared to the CNS, mainly regarding the effects of glycine-site antagonists.

Effect of excitatory amino acid analogues on the release of D-[3H]aspartate from chick retina.

There is good evidence to suggest that L-glutamate (L-Glu) and/or L-aspartate (L-Asp) might function as excitatory neurotransmitters in the vertebrate retina. Postsynaptic receptors for these compounds have been identified in both plexiform layers by means of physiological and biochemical techniques. However, the presence of excitatory amino acid receptors which could regulate the release of these compounds has not previously been demonstrated. We have now shown that the K+-stimulated, Ca2+-dependent release of [3H]D-aspartate from superfused chick retina is inhibited by L-Glu, N-methyl-D-aspartate (NMDA), kainate (KA) and several other neuroactive analogues. While alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and (+/-)2-amino-5-phosphonovaleric acid both exhibited agonist activity when tested alone, the former substance reversed the inhibition observed with L-Glu and KA whereas the latter effectively antagonized the NMDA-induced inhibition of release, possibly acting as partial agonists. The results demonstrate an interaction of NMDA and AMPA with KA probably at receptors in the chick retina that are involved in the regulation of glutamate/aspartate release.