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S Halpain

Publications and source records attributed to S Halpain.

27 records · Page 2Linked to original sources
PubMed1989

Characterization and partial purification of a chloride- and calcium-dependent glutamate-binding protein from rat brain.

A glutamate-binding protein was solubilized from rat brain synaptic plasma membranes using sodium cholate. Its properties were characterized after addition of exogenous phospholipids and formation of proteoliposomes. Glutamate binding was dependent on calcium and chloride ions with maximal binding at concentrations of 10(-5) M calcium and 10 mM chloride ions. The effects of the two ions were synergistic rather than additive. In addition, glutamate binding was not affected by inhibitors specific for N-methyl-D-aspartate and kainate receptor subtypes, but was inhibited by quisqualate (Ki = 50 microM) and DL-2-amino-4-phosphonobutyrate (Ki = 1.3 mM). Furthermore, binding was abolished by 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1 mM dithiothreitol. These properties resemble those of the chloride- and calcium-dependent binding site. Starting from the detergent extract, the glutamate-binding protein was purified 123-fold using fractionated ammonium sulfate precipitation, chromatography on hydroxyapatite and on DEAE-Sephacel as sequential purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein fraction showed two major bands migrating with Mr values of 51,000 and 105,000. The properties of the partially purified binding protein were similar to those of the detergent extract. Glutamate binding to the partially purified protein is not due to a sequestration process or product binding to N-acetylated alpha-linked dipeptidase. Thus, the functional role of the binding protein remains to be established.

Animals↗
PubMed1988

Corticosterone decreases 3H-glutamate binding in rat hippocampal formation.

The effect of in vivo corticosterone manipulation on binding sites for the excitatory neurotransmitter glutamate was determined using radioligand binding to rat brain cryostat sections and crude synaptic membranes. 3H-glutamate binding to regions of the dorsal hippocampal formation was significantly decreased in adrenalectomized animals following 5-10 days of corticosterone treatment. Corticosterone did not alter 3H-glutamate binding in several other brain regions. The loss in 3H-glutamate binding appeared to be due to a decrease in the maximal number of binding sites, with little change in binding affinity. Both chloride-dependent and chloride-independent 3H-glutamate binding sites were decreased by corticosterone treatment. results indicate that corticosterone can selectively alter binding sites for excitatory amino acids in hippocampal tissue.

Adrenalectomy↗
PubMed1986

Features of ligand binding in homogenate and section preparations.

The binding of [3H]N-methyl scopolamine (NMS) to muscarinic binding sites in rat forebrain was compared in two types of preparations: homogenates and slide-mounted sections. Under standard assay conditions, [3H]NMS bound to the muscarinic binding site with an apparent Kd that was almost one order of magnitude lower in homogenates (Kd = 0.15 nM) than sections (Kd = 1.25 nM). In addition, the muscarinic agonist carbachol inhibited [3H]NMS binding more effectively in homogenates (Ki = 5 microM) than sections (Ki = 100 microM). The higher Kd observed in sections was dependent upon the thickness of the section since the apparent Kd decreased as the thickness of the section was reduced. A slower equilibration rate may account, in part, for the higher apparent Kd seen in thicker sections. The results support previous evidence that certain ligands exhibit different binding profiles in homogenate and section preparations.

Animals↗
PubMed1986

Loss of 3H-corticosterone binding capacity in hippocampal slices maintained in vitro.

Nuclear uptake of 3H-corticosterone was studied in hippocampal slices subjected to varying periods of preincubation before application of steroid. Uptake capacity declined rapidly toward nonspecific levels over the course of 3 hours from the time of cutting. Glucocorticoid receptors in the cytosol fraction of hippocampal slices also were found to decline, and Scatchard analysis indicated a loss of corticosterone binding sites rather than a change in binding affinity. These results are discussed in terms of the reported effects of corticosterone in vitro.

Adrenalectomy↗
PubMed1984

Quantitative autoradiography of binding sites for [3H]AMPA, a structural analogue of glutamic acid.

Binding sites for the potent glutamate agonist [3H] alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were localized in rat brain frozen sections by quantitative autoradiography. Highest levels of binding were seen in stratum radiatum and stratum oriens of the CA1 hippocampal subfield and in the dorsal subiculum. Substantially less but still high amounts of [3H]AMPA binding occurred in other hippocampal subfields and in rostral forebrain structures. The heterogeneous nature of [3H]AMPA binding is discussed in relation to [3H]glutamate binding visualized by similar methods. From these data it is suggested that [3H]AMPA may label a particular subclass of the glutamate receptor population which exhibits a high affinity for quisqualic acid.

Animals↗
PubMed1984

Localization of L-glutamate receptors in rat brain by quantitative autoradiography.

In vitro autoradiography was used to characterize and quantitatively map the sodium-independent binding of [3H]glutamate in rat brain. Measured in the presence of chloride, glutamate binding to frozen brain sections was specific, saturable, and reversible, with a Kd in the low micromolar range. At least two distinct binding sites were detected which had different affinities for quisqualic acid (0.7 microM and 1.2 mM). Autoradiograms revealed very high levels of binding in rostral forebrain areas, especially olfactory structures and frontal cortex. High levels of binding were found in sensory cortex, certain hippocampal subfields, caudate, lateral septum, and other limbic structures. Lowest levels were seen in globus pallidus, preoptic area, brainstem reticular formation, and spinal trigeminal nucleus. All other regions, including midbrain, thalamus, hypothalamus, and cerebellar cortex exhibited moderate levels of [3H]glutamate binding. Within the hippocampus glutamate binding was greatest in the inner two-thirds of the dentate molecular layer, subiculum, and CA1 stratum radiatum. CA3 was much less densely labeled; CA2 and CA4 were intermediate. Unlike cell layers in the cerebellum, hippocampal pyramidal and granule cell layers appeared mostly devoid of binding. The relationship of these binding sites to putative glutamate receptor subclasses and glutamatergic pathways is discussed.

Animals↗
PubMed1983

Biochemical effects of high-frequency synaptic activity studied with in vitro slices.

Brain slices have a number of features that may be of value in the analysis of how physiological events affect neuronal chemistry. This paper discusses this topic and describes slice experiments concerned with the chemical events responsible for long-term potentiation (LTP) of synaptic responses found in hippocampus after brief episodes of high-frequency stimulation. Work with two variants of the slice procedure indicated that LTP is accompanied by an increase in the sodium-independent binding of [3H]glutamate to partially purified synaptic membranes; this effect very likely results from an increase in the numbers of a particular postsynaptic receptor. Stimulation that produces long-term potentiation also causes a significant change in the endogenous phosphorylation of pyruvate dehydrogenase (PDH), a key mitochondrial enzyme. Inasmuch as the phosphorylated state of PDH is strongly correlated with calcium sequestration by mitochondria, it is possible that LTP is triggered by a transient perturbation of the calcium buffering function provided by mitochondria. Low micromolecular levels of calcium increase glutamate binding to purified membranes apparently via the activation of a calcium-sensitive thiol proteinase. This mechanism could account for the increase in glutamate binding found in slices exhibiting LTP. These experiments suggest a possible explanation for long-term potentiation and indicate that slices can be used to detect at least some of the biochemical consequences of repetitive synaptic activity.

Animals↗
PubMed1982

Effects of high-frequency synaptic stimulation on glumate receptor binding studied with a modified in vitro hippocampal slice preparation.

Slices of the field CA1 minus the stratum moleculare were prepared from the rat hippocampus and maintained in an in vitro recording chamber. The physiological properties of these "minislices" were similar to those reported for non-dissected hippocampal slices. Slices receiving various patterns of electrical stimulation through multiple electrodes were subsequently homogenized and crude membrane fractions prepared; the binding of [3H]glutamate was measured by a rapid filtration assay. Binding in membranes prepared from control slices exhibited kinetic properties and sensitivities to pharmacological and ionic manipulations which were comparable to those found in previous studies using conventional fractionation and assay techniques. Brief bursts of high-frequency stimulation increased [3H]glutamate binding compared to non-stimulated controls in 3 separate experiments. Stimulation at low frequency or at high frequency in low calcium medium did not produce this effect. In addition to suggesting that glutamate binding sites are regulated by patterns of afferent activity, these findings indicate that the minislice preparation should be of general utility in relating synaptic physiology to synaptic chemistry.

Animals↗