Ana Barat

Guanine nucleotides protect against kainate toxicity in an ex vivo chick retinal preparation

Ex vivo preparations of chick neural retina have been successfully used in the assessment of excitotoxicity and in the evaluation of the protective effects of glutamate antagonists. Using a variation of this approach, and measuring the acute and delayed toxic effects of kainate (KA) in terms of lactate dehydrogenase release, we have shown that guanine nucleotides behave as effective neuroprotecting agents. The anti‐excitotoxic potency of guanine nucleotides (in the case of GMP and GDPβS it is about 100 times lower than that of DNQX, a powerful kainate antagonist) correlates well with their ability to displace KA from retinal KA receptors.

Molecular Forms of Acetylcholinesterase in the Developing Chick Visual System

The developmental profiles of the enzyme acetylcholinesterase, and of some of its quaternary structural forms, characterized by discrete sedimentation coefficients, have been comparatively analyzed in chick retina and optic tectum, between embryonic day 8 and day 10 after hatching. Four molecular species of AChE have been characterized in both retina and tectum during this developmental period: two of them with sedimentation coefficients of 11S and 6S, accounting together for 94-99% of the AChE activity in the initial homogenate, can be easily extracted by homogenization in a buffer containing 1% Triton X-100 and 1 M NaC1, at 4 degrees C. The other two, however, are not extractable by such treatment, but can be released by collagenase from the residue left after the detergent-salt extraction; they have apparent sedimentation coefficients of 21.5S and 16.5S and represent, together, less than 2% of activity in the initial homogenate. All four forms of the enzyme show distinctive patterns of change during the developmental period considered, with significant differences between retina and tectum. These differences are discussed in the context of the specific roles of retina and tectum in the visual process.

Molecular forms of acetylcholinesterase in the chick visual system: Collagenase-released 21.5 S and 16.5 S species

Acetyl~ho~nesterase (a~etylcho~ne hydrolase, EC 3.1.1.7) is the enzyme thought to be responsible for the termination of the action of the neurotransmitter acetylcholine, at the cholinergic synapses [ 11. Nevertheless, its presence is not limited to the cholinergic postsynaptic membrane, being widely distributed in nerve and muscle cell membranes, electrogenic tissue from electric fish, and erythrocyte membranes. This circumstance suggests the possibility that acetylcholinesterase may be involved in other membrane functions as well [ 1,2] and explains the difficulties observed when trying to use the enzyme as a marker for the presence of cholinergic synapses [3,4]. The identification of several structural forms of the enzyme, characterized by definite sedimentation coefficients [S-13], has opened the way for the assignment of different molecular forms to different localizations and/or potential functions [7,9,10,12]. Special attention has been paid to the so-called asymmetric or tailed forms of the enzyme composed of l-3 tetramers of the catalytic subunit (mol. wt 280 000) associated to a filamentous, collagen-like tail, probably implicated in the anchorage of the enzyme to the cell membrane matrix 112-l 51. The specific association of a 16 S form of acetylchol~esterase to the muscle junctional membrane (the postsynaptic element of the neuromuscular cholinergic synapse) was detected [7]. Other fast-sedimenting forms of the enzyme, associated to cholinergic

Solubilization of collagen-tailed molecular forms of acetylcholinesterase from several brain areas in different vertebrate species.

The relative efficiency of a buffered medium containing a high salt concentration and EDTA as a means to solubilize collagen-tailed molecular forms of acetylcholinesterase has been examined in four brain areas of several species belonging to different vertebrate classes. This extraction procedure has proved successful in most cases, with the yield of tailed enzyme varying between less than 1 and 26% of the total tissue activity. The solubilization values are consistently higher in more primitive vertebrates than in mammals and, for a given species, are usually lower in the telencephalon than in other brain structures. Our results confirm that the vertebrate central nervous system contains collagen-tailed quaternary structural forms of acetylcholinesterase.

Heparin and the solubilization of asymmetric acetylcholinesterase

Heparin solubilizes asymmetric acetylcholinesterase, from chick skeletal muscle and retina, as a 24 S complex which is quantitatively converted to conventional asymmetric molecular forms of the enzyme (A12 and A8, either class I or class II) upon exposure to high salt. The simultaneous presence of salt and heparin in the homogenization medium selectively prevents, however, the release of class II A‐forms in both muscle and retina. Heparin may generally act by displacing native proteoglycans involved in the attachment of the enzyme tail to the extracellular matrix, or its neural equivalent, being in turn removed by salt to yield typical asymmetric enzyme forms. Heparin would also appear to displace some other molecules specifically involved in the EDTA‐sensitive attachment of class II tailed forms, this effect being antagonized by salt.

Interaction of Asymmetric and Globular Acetylcholinesterase Species with Glycosaminoglycans

Abstract: Chicken muscle and retina, and rat muscle asymmetric acetylcholinesterase (AChE) species were bound to immobilized heparin at 0.4 M NaCl. Binding efficiency was between 50 and 80% for crude fraction I A‐forms (A1; muscle), and nearly 100% for fraction II A‐forms (A11; muscle and retina). Antibody‐affinity‐purified A1‐forms (chicken) were, however, quantitatively bound to heparin–agarose gels, whereas diisopropylfluorophosphate‐inactivated high‐salt extracts partially prevented the binding of both A1 and A11 AChE forms, thus suggesting the presence in crude A1 extracts of heparin‐like molecules interfering with the tail–heparin interaction. All bound A‐forms were progressively displaced from the heparin–agarose columns by increasing salt concentrations, with maximal release at about 0.6 M. They were also efficiently eluted by heparin solutions (1 mg/ml), other glycosaminoglycans being much less effective. Chicken globular AChE forms (G‐forms, both low‐salt‐soluble and detergent‐soluble) also bound to immobilized heparin in the absence of salt. Stepwise elution with increasing NaCl concentrations showed maximal release of G‐forms at 0.15 M, all globular forms being totally displaced from the column at 0.4 M NaCl. Heparin (1 mg/ml) had the same eluting capacity as 0.4 M NaCl, whereas other glycosaminoglycans were only marginally effective. We conclude that the molecular forms of AChE in these vertebrate species interact with heparin, at salt concentrations that are characteristic for asymmetric and globular forms. Within the A and G molecular form groups, no differences were found in the behavior of the different fractions or subtypes, provided that the enzyme samples were free of interfering molecules. If heparin affinity reflects the ability of AChE forms to interact with extracellular matrix components, not only asymmetric but also some globular enzyme forms could be bound to basal laminae under physiological ionic strength conditions.

Solubilization of 20S acetylcholinesterase from the chick central nervous system

The ionic detergent sodium cholate, in the presence of 1 M NaCl, solubilizes a 20S acetylcholinesterase from chick retina and other brain tissues previously extracted with a buffered solution containing 1% Triton X-100 and 1 M NaCl. This 20S acetylcholinesterase appears to be a tailed form of the enzyme which, upon collagenase digestion, is converted to a 22S (mainly) form. This finding suggests that the vertebrate central nervous system does contain asymmetric, collagen-tailed forms of acetylcholinesterase, as is the case in skeletal muscle and cholinergic ganglia.

Solubilization of 20S acetylcholinesterase from chick retina

Abstract A 20S form of acetylcholinesterase has been solubilized from young chick retinas by means of a buffered salt-detergent solution containing EDTA. The release of this fast-sedimenting form of the enzyme is selectively blocked by the presence of even small amounts of Ca ++ in the homogenization medium. The collagen-tailed nature of this molecular species of acetylcholinesterase has been ascertained by collagenase digestion. This finding suggests that the avian central nervous system contains asymmetric, collagen-tailed quaternary structural forms of acetylcholinesterase as is the case in skeletal muscle and cholinergic ganglia.

Chondroitinases release acetylcholinesterase from chick skeletal muscle

Bacterial chondroitinases (both ABC and AC types) release asymmetric and globular forms of AChE from chick skeletal muscle samples. Heparinases, however, including heparitinase I, fail to do so under different incubation conditions. These results do not support the direct implication of the heparin/heparan sulfate family of GAGs in the interaction of the different AChE molecular forms with the muscle ECM. GAGs of the chondroitin/dermatan sulfate group could however be involved, either directly or indirectly, in the attachment of the AChE collagen‐like tail to the muscle basal lamina.

Asymmetric acetylcholinesterase is absent from chick retina, but present in choroid, ciliary muscles and iris

Freshly dissected chick neural retina and pigmented epithelium do not apparently contain asymmetric molecular forms (A-forms) of acetylcholinesterase (AChE). The neighboring choroid, and the ciliary muscles and iris, are however rich in type II A-forms (high salt/EDTA-extractable). Most if not all the asymmetric AChE activity detected in chick whole retina preparations could then be explained in terms of contamination by non-retinal eye tissues.