Rectification properties and Ca2+ permeability of glutamate receptor channels in hippocampal cells.

Excitatory amino acids exert a depolarizing action on central nervous system cells through an increase in cationic conductances. Non-NMDA receptors have been considered to be selectively permeable to Na+ and K+, while Ca2+ influx has been thought to occur through the NMDA receptor subtype. Recently, however, the expression of cloned non-NMDA receptor subunits has shown that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are permeable to Ca2+ whenever the receptor lacks a particular subunit (edited GluR-B). The behaviour of recombinant glutamate receptor channels predicts that Ca2+ would only permeate through receptors that show strong inward rectification and vice versa, i.e. AMPA receptors with linear current-voltage relationships would be impermeable to Ca2+. Using the whole-cell configuration of the patch-clamp technique, we have studied the Ca2+ permeability and the rectifying properties of AMPA receptors, when activated by kainate, in hippocampal neurons kept in culture or acutely dissociated from differentiated hippocampus. Cells were classified according to whether they showed outward rectifying (type I), inward rectifying (type II) or almost linear (type III) current-voltage relationships for kainate-activated responses. AMPA receptors of type I cells (52.2%) were mostly Ca(2+)-impermeable (PCa/PCs = 0.1), while type II cells (6.5%) expressed Ca(2+)-permeable receptors (PCa/PCs = 0.9). Type III cells (41.3%) showed responses with low but not negligible Ca2+ permeability (PCa/PCs = 0.18). The degree of Ca2+ permeability and inward rectification were well correlated in cultured cells, i.e. more inward rectification corresponded to higher Ca2+ permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Characteristics of excitatory amino acid uptake in cultures from neurons and glia from the retina.

3H-D-Aspartate uptake was biochemically characterized in cultures from chick retina enriched in glial (Müller) cells or neurons during progressive days in vitro (DIV). In the neuronal cultures a high-affinity, Na(+)-dependent system was found with Km = 8-13 microM and pharmacological characteristics in agreement with those of reuptake systems in other regions of the CNS. The uptake system in glial cells showed a lower affinity, with Km = 100-135 microM. In both cases, uptake was temperature and energy dependent. A sharp increase in the Vmax of uptake was observed in both neuronal and glial cultures at 5 DIV, at which time morphologically mature synapses have been shown to be present in retinal cultures. A parallel increase in the pharmacological specificity of the uptake system in neuronal cultures was observed, with a rise in the efficiency of D-Asp, L-Asp, L-Glu, and DL-asp- beta-hydroxamate for inhibiting 3H-D-Aspartate uptake. Results suggest the possibility of reuptake participating in the regulation of extracellular glutamate concentration during development.

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.

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.

Effect of selective kainate lesions on the release of glutamate and aspartate from chick retina.

In order to contribute evidence leading to establishing the excitatory pathways in the vertebrate retina, we selectively lesioned chick retinas by intraocular injection of 6, 60, 120, and 200 nmol of kainate, which selectively damages OFF-bipolars, amacrines, horizontals, and ON-bipolars, and measured the K+-stimulated, Ca++-dependent release of L-(3H)-glutamate and L-(3H)-aspartate. We also measured (3H)-GABA release as a marker for horizontal cells and a population of amacrines, as well as (14C)-glycine release as a tracer of a different subpopulation of amacrines. All four amino acids were released from control retinas by a depolarizing K+ concentration in a Ca++-dependent fashion. GABA and glycine, however, showed an additional Ca++-independent component of release. Lesion induced by 6 nmol of kainate decreased by 50% the release of glutamate and by 20% that of aspartate; glycine release was reduced 40% while GABA release was unaffected. Injection of 60 nmol of kainate reduced glutamate release a further 20% and significantly decreased GABA (50%) and glycine (75%) release; aspartate release remained unmodified; 120 nmol of kainate caused a further 30% reduction in aspartate and GABA release. Neither compound was significantly released after treatment with 200 nmol of kainate. These results seem to suggest that while OFF-bipolars could release glutamate as transmitter, aspartate is released from a different cell population which is less sensitive to kainate, probably ON-bipolars.

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.

Characterization of [L-3H]aspartate binding to chick retinal subcellular fractions.

Binding of [L-3H]aspartate to synaptic receptors was examined in membranes from whole chick retina and subcellular fractions enriched with photoreceptor terminals (P1) or terminals from the inner plexiform layer (P2), Na+-independent, stereospecific, high affinity binding was concentrated in the P1 fraction (Kb = 40 nM). P2 fraction also showed a high affinity binding system (KB = 11.8 nM) with lower capacity than in the P1 fraction. Comparative studies with [L-3H]-aspartate, [L-3H]-glutamate and [H3]-kainate showed that L-aspartate and L-glutamate are the most potent inhibitors of the binding of the three ligands. Aspartate and glutamate binding were effectively displaced by N-methyl-DL-aspartate and alpha-amino adipate, whereas only [3H]-glutamate binding was significantly inhibited by glutamate-diethyl-ester. Kainic acid exhibited negligible affinity for aspartate and glutamate binding sites. Results indicate the presence of different receptors for glutamate and aspartate in both plexiform layers of the retina.