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Internal free calcium concentrations ([Ca2+]i) have been monitored in synaptoneurosomes from 8-d-old rat whole brain previously loaded with the calcium-sensitive fluorescent probe Fura 2. Under basal conditions, [Ca2+]i was around 200 nM, this concentration increasing only slowly during storage of the synaptoneurosomes at room temperature (40% increase 2 hr after loading). Opening of sodium channels with veratridine- (10 microM) or KCl- (30 mM) induced depolarization caused rapid increases in synaptoneurosomal [Ca2+]i. [Ca2+]i was also markedly increased by addition of the Ca2+ ionophore A23187 (10-100 nM). The effect of veratridine, but not of KCl was prevented by previous addition of TTX (1 microM). KCl-induced [Ca2+]i increase was dependent on external Ca2+ and was partially blocked by the dihydropyridine derivative PN 200-110 (IC50 0.15 microM, maximal inhibition 55% at 3 microM). L-Glutamate elicited a concentration-dependent fast increase in synaptoneurosomal [Ca2+]i in the 8-d-old (but not in the adult) rat brain (EC50 = 2 microM). The effect of glutamate was stereospecific, the EC50 of the D-isomer being 47 times higher than that of L-isomer. The magnitude of the L-glutamate response differed in several brain regions, being highest in the cerebral cortex and lowest in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

NADH oxidase activity has been detected in luminal membrane derived from the proximal tubules of rat kidney (brush border membranes). The cytochromes associated with these membranes [García, M.L., Benavides, J., Valdivieso, F., Mayor, F., & Giménez-Gallego, G. (1978) Biochem. Biophys. Res. Commun. 82, 738-744] were reduced by NADH, the extent of this reduction being affected by the presence of oxygen and by inhibitors of this NADH oxidase activity. These results suggest that the cytochromes act as an electron transport system between NADH and oxygen. The possibility of mitochondrial contamination is excluded on the basis of differential sensitivity to inhibitors and marker enzyme activities.

Circadian rhythms in both the number of peripheral type binding sites for benzodiazepines in platelet membranes and the microviscosity of the erythrocyte membrane were demonstrated in 7 healthy men. Neither variable appeared to be linked to each other, or regulated by the plasma concentrations of total or free cortisol, testosterone, potassium, magnesium, calcium, cAMP, cGMP or proteins or by the erythrocytic concentration of magnesium or potassium or by the plasma cAMP:cGMP ratio or by the ratio of intra-erythrocyte:plasma concentrations of potassium or magnesium. A highly significant negative correlation was found between the microviscosity of the erythrocyte membrane and the activity of the membrane-bound enzyme, methyltransferase I. Such a correlation was validated both on raw data and on 24 hr-means (r = 0.84; P less than 0.01). A circadian rhythm in the activity of this enzyme was also demonstrated. Moreover, a highly significant correlation was also found between plasma transcortin concentration (TRC) and microviscosity (r = 0.50, P less than 0.01), and between TRC and methyltransferase I activity (r = 0.61, P less than 0.01). Such findings may constitute clues towards the understanding of the regulation of the circadian rhythm in the fluidity of the red blood cell membrane in man and guide future steps with regard to the role of this rhythm upon the availability of drug binding sites at the cell surface.

The effect of chronic administration of the anticonvulsive drug di-n-propylacetate (DPA) on the glycine cleavage enzyme system was studied. Glycine concentrations were monitored in blood, liver, brain and spinal cord of 10-day-old rats. DPA treatment decreases glycine cleavage activity by approximately 50% in liver, and 35% in brain. The decreased cleavage activity correlates with an increase of glycine levels in blood, liver and brain. Failure to cleave glycine characterizes a metabolic disorder known as non-ketotic hyperglycinemia, which is associated with elevated concentrations of glycine in biological fluids. The inhibitory effect of DPA may provide an experimental approach to study the biochemical and pathogenic mechanisms of non-ketotic hyperglycemia.

The ammonium salts of Eccles' anions are inhibitors of 3H-strychnine binding. By contrast, the binding is activated by the sodium salts of these anions. This activation is due to an increase in the affinity for 3H-strychnine. Pretreatment of the membranes with acetic anhydride abolishes the effect of sodium salt, whereas ammonium salts remain unaffected. In the presence of chloride the inhibitory effect of ammonium predominates over the effect of sodium. These results suggest that the effect of Eccles' anions is profoundly modified by their counter-ions and that Na+ and ammonium are acting through distinct sites on the glycine receptor.

Plasma membranes from erythrocytes were used to study various parameters: phospholipid methylation, viscosity and fragility. These parameters were analysed in 57 normal subjects (52 +/- 3 years old) and 14 patients with idiopathic Parkinson's disease without previous treatment (71 +/- 2 years old). No significant correlations were observed between the various parameters and sex or age in the normal group. No correlation was observed between fragility and methylation or viscosity. A statistically significant correlation was found between viscosity and phospholipid methylation. In the patients with Parkinson's disease, fragility is identical to the normal group but the viscosity is increased by 25 p. 100. The increase of viscosity is more obvious among the patients with a pure or predominantly akinetic form of the disease. These results favour the previously described correlation between phospholipid methylation and viscosity of the plasma membrane and suggest a modification of these parameters in Parkinson's disease. They also suggest that the increased viscosity observed in Parkinson patients could render the receptors inaccessible to their endogenous ligands.

The biochemical characteristics of a patient with most of the clinical symptoms of nonketotic-hyperglycinemia have been studied. Despite the extremely low plasma/cerebrospinal fluid glycine ratio of the patient, typical of the nonketotic syndrome, at autopsy we found no enhancement of glycine levels in brain and glycine cleavage enzyme activities in the brain and liver that were similar to those of a control newborn. In order to ascertain the basic defect responsible for glycine accumulation in the cerebrospinal fluid and for the neurological damage, we examined the possible existence of altered glycine transport systems in nervous cells. Our findings indicate that (1) low and high affinity, Na+-dependent transport systems for glycine can be detected in both rat and control human postmortem tissue and (2) the low affinity glycine transport system is absent in the brain of the patient, whereas an alteration of transport systems occurs in the patient's spinal cord, probably involving the high-affinity component. These alterations could account for the clinical features of the patient. Since the feasibility of determining glycine transport parameters in postmortem tissue has been established, we think it would be of interest to investigate such systems in cases where the etiology of hyperglycinemia is not clear.

Glycine degradation in the CNS is catalyzed by the glycine cleavage system (GCS). We have tested several compounds with a structural analogy to glycine for their inhibition of the GCS. Compounds having an inhibitory activity, such as aminoacetonitrile and propargylamine, are characterized by having a primary amine group and a pi-electron system present in the group substituting for the carboxylic function. The inhibition by these compounds of glycine degradation may be of pharmacological interest given the neurotransmitter role of glycine.

The effect of propionate and methylmalonate on the transport of glycine into rat liver and brain mitochondria was investigated. Both propionate and methylmalonate markedly inhibited mitochondrial glycine transport. These compounds also inhibited 14CO2 production from [14C]glycine by isolated brain and liver mitochondria and glycine metabolism in rat brain cortex slices. These results are discussed with reference to hyperglycinaemia associated wtih propionic acidaemia and methylmalonic acidaemia and as a possible contributory factor to the pathological mechanisms in these conditions.

The anatomical distribution of omega 3 (peripheral type benzodiazepine binding) sites in the immune system organs of the rat has been studied autoradiographically at both macroscopic and microscopic levels of resolution using either reversible or irreversible (UV irradiation) labeling with [3H]PK 14105. In thymus sections, [3H]PK 14105 labeled with high affinity (Kd, derived from saturation experiments = 10.8 nM) a single population of sites which possessed the pharmacological characteristics of omega 3 sites. In the thymus gland, higher omega 3 site densities were detected in the cortex than in the medulla; in these subregions, silver grains were associated to small (10-18 microns diameter) cells. In the spleen, omega 3 sites were more abundant in the white than in the red pulp. In the white pulp, silver grains were denser in the marginal zone than in the vicinity of the central artery and labeling was, as in the thymus, associated to small cytoplasm-poor cells. In the red pulp, omega 3 site associated silver grains were observed mainly in the Bilroth cords. In the lymph nodes, the medullary region showed a higher labeling than the surrounding follicles and paracortex. A significant accumulation of silver grains was observed in the lymph node medullary cords. In the intestine, Peyer patches were particularly enriched in omega 3 sites (especially in the periphery of the follicles). The distribution of omega 3 sites in the immune system organs suggests a preferential labeling of cells of T and monocytic lineages. This is consistent with the proposed immunoregulatory properties of some omega 3 site ligands.

The effect of the anticonvulsive di-n-propylacetate (DPA) and some structural analogous, all branched-chain organic acids, on the glycine cleavage system in rat liver and brain mitochondria has been studied. DPA and n-propyl, prop-2-enylacetic, 4,4-di-n-propylbutyric, 2-ethyl, 3-methylvaleric acids inhibit this enzymatic activity corresponding the greatest degree of inhibition to DPA (70% and 86% inhibition in liver and brain mitochondria respectively at 10 mM DPA). 2-methyl, 3-ethyl caproic acid has an inhibitory effect on glycine cleavage activity only in liver mitochondria, but showing no effect on this activity in brain mitochondria. The inhibitory effect of DPA can explain the hyperglycinemia and hyperglycinuria in DPA-treated patients. This effect may provide also an experimental approach for the study of biochemical and pathogenia mechanisms of non-ketotic hyperglycinemia.

The "in vivo" effects of L-phenylalanine on the gluconeogenic pathway in the liver of fasted rats with experimentally induced phenylketonuria-like characteristics have been investigated. Significant increases of the fructose 6-phosphate, glucose 6-phosphate and glucose concentrations were observed. The study of the effect of L-phenylalanine on the cytoplasmic and mitochondrial redox state and energy charge showed an increase in the mitochondrial NAD+/NADH ratio while the energy charge was virtually unchanged. The effects of phenylalanine and its metabolic derivatives (phenylacetate, phenylethylamine, phenyl-lactate, o-hydroxyphenylacetate and phenylpyruvate) on the activity of lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in rat liver have been also investigated. Phenylpyruvate inhibited the lactate dehydrogenase activity with a Ki of 5.3 mM. Phenylpyruvate also inhibited both the mitochondrial (Ki = 4 mM) and cytoplasmic (Ki = 5 mM) malate dehydrogenase activities. Phenylpyruvate, phenylacetate and o-hydroxyphenylacetate inhibited the 3-hydroxybutyrate dehydrogenase activity with Ki values of 0.7, 6.0 and 9.5 mM respectively.

'Peripheral type' benzodiazepine binding sites in several rat tissues were labelled by intravenous injection of [3H]PK 11195 and [3H] RO5 -4864. Binding was saturable in all tissues studied and regional distribution paralleled the in vitro binding. A similar potency order of displacing compounds was found in vivo and in vitro PK 11195 greater than PK 11211 greater than RO5 -4864 greater than diazepam greater than dipyridamole greater than clonazepam. These results demonstrate the feasibility of using this technique to examine the effects of pharmacological manipulation on the binding sites in their native state. However some properties (broader maximum during time course, higher percentage of particulate binding in the brain and independence of temperature) make [3H]PK 11195 the most suitable ligand for this kind of studies.

The precise neuronal localization of D1 receptors in the substantia nigra has been studied autoradiographically in the rat by measuring the alterations of [3H]SCH 23390 binding site densities in this brain area after 6-hydroxydopamine (6-OHDA) induced destruction of nigrostriatal dopaminergic neurons and after ibotenate-induced lesion of striatal afferents. 6-OHDA-induced nigral lesion provoked a total loss of [3H]SCH 23390 binding sites in the pars compacta and pars lateralis (but not in the pars reticulata) of the substantia nigra. In contrast, ibotenate-induced striatal lesion caused a large diminution of the [3H]ligand binding site density in the pars reticulata but not in the pars compacta and pars lateralis of the substantia nigra. These results suggest that D1 receptors in the pars compacta or pars lateralis of the substantia nigra are located on the dopaminergic perikarya whereas those D1 receptors present in the pars reticulata of the substantia nigra lie on the terminals of nigral afferents of striatal origin.

"Peripheral type" benzodiazepine binding sites have been solubilized with digitonin. Binding site density for the solubilized material is increased 1.7 times compared to membranes. A decrease in the affinity for [3H]-PK 11195 (a new ligand for the peripheral type benzodiazepine binding sites) was also observed. Pharmacological specificity of displacing agents was conserved during solubilization. The apparent molecular weight determined by gel filtration was 215,000 +/- 20,000. The high Bmax value of the solubilized preparation (greater than 50 pmole/mg protein) makes it advantageous as the starting point for a purification procedure.

The topography and magnitude of increase in peripheral type benzodiazepine binding sites (omega 3 sites) was investigated autoradiographically in the brains of patients with ischemic cerebrovascular disease, with multiple sclerosis, and with malignant glioma. 3H-PK 11195, a selective omega 3 site ligand, was employed. A manyfold increase in omega 3 site density was observed in all these disease states; this increase reflects macrophage invasion or glial proliferation or both as demonstrated by neuropathological studies carried out in parallel. There was an excellent spatial correlation between increased omega 3 site densities and extent of the lesion histologically. Specifically, an elevated density of omega 3 sites was observed in the plaques of demyelination in multiple sclerosis patients, in the periphery of infarcted zones in stroke patients, and throughout tumor in patients with grade IV astrocytomas. As our approach is applicable to both tomographic (in vivo) and autoradiographic investigations, imaging of omega 3 sites may be considered for the detection and monitoring of the natural evolution of many disorders of the human central nervous system.

Peripheral type of benzodiazepine binding sites were labelled in the kidney, the heart and the brain with [3H] RO5-4864 following intravenous injection in mice. The regional distribution of this in vivo binding parallels the in vitro binding: heart and kidney were more labelled than brain. Benzodiazepine potencies in reducing [3H] RO5-4864 binding in vivo parallel relative affinities for [3H] RO5-4864 binding sites in isolated organs membranes: RO5-4864 greater than diazepam greater than clonazepam. PK 11195 a new compound, chemically unrelated to benzodiazepines, which is a potent inhibitor of [3H] RO5-4864 in vitro is also very effective (more than RO5-4864) after I.P. injection and oral administration. These results emphasize the feasibility of using this technique to examine the effects on various pharmacological and physiological manipulations of these binding sites in vivo. Moreover the fact that PK 11195 binds to these sites in vivo might indicate that this compound could help to elucidate the physiological relevance of the peripheral type of benzodiazepine binding sites.

The effects of excitotoxic lesions on the neuronal marker enzymes choline acetyltransferase and glutamate decarboxylase and on the levels of 'peripheral type' benzodiazepine binding sites (PTBBS) (a putative glial marker) have been compared to see whether PTBBS provide a suitable if indirect quantitative index of neuronal damage. Intrastriatal injection of excitotoxic compounds provoked a dose-dependent increase in the levels of PTBBS. The potency order was the following: kainate greater than AMPA greater than N-methyl-D-aspartate (NMDA) greater than quisqualate. The maximal increases in this parameter were 400, 470, 320 and 210% for kainate (12 nmol), AMPA (100 nmol), NMDA (500 nmol) and quisqualate (250 nmol), respectively. 2-Amino-5-phosphonovalerate (100 nmol)--an antagonist of the NMDA receptor subtype--completely blocked the increase in PTBBS induced by NMDA (250 nmol), but was without effect against the other excitotoxins. Increases in binding levels were in general mirrored by a decrease in choline acetyltransferase and glutamate decarboxylase activity. However, PTBBS were a more sensitive indirect index of neuronal damage than neuronal enzymes because the alterations in binding were statistically significant at doses of excitotoxins lower than those causing a loss of marker enzymes. It is concluded that PTBBS are a suitable and sensitive means of detecting discrete neurotoxic changes and that its measurement will help in the study of other pathological and experimental models.

The distribution of the central omega 1 (benzodiazepine1; BZD1) and omega 2 (BZD2) receptor subtypes has been studied autoradiographically in monkey and human brain sections using [3H]flunitrazepam (which binds indiscriminately to omega 1 and omega 2 subtypes) and [3H]zolpidem (which recognizes selectively the omega 1 subtype). Both ligands labeled an homogeneous population of binding sites (Kd values approximately equal to 1.5 nM and approximately equal to 5 nM, respectively) whose pharmacological characteristics were similar to those of central omega (BZD) receptors. Regional displacement studies in monkey brain showed that zolpidem was a more effective displacer of [3H]flunitrazepam from omega 1-than from omega 2-enriched areas. For example, it was 73-fold more potent in the cerebellum (omega 1-enriched) than in the dentate gyrus (omega 2-enriched). Zolpidem thus selectively recognizes omega 1 sites in primate brain. The autoradiographic distribution of [3H]flunitrazepam (omega 1 + omega 2) binding sites in primate brain was highly heterogeneous. Very high densities were observed in lamina IV of the neocortex (with higher densities in the occipital than in the frontal pole), the substantia innominata and molecular layer of the dentate gyrus. Intermediate densities were found in other neocortical laminae, cerebellum (molecular layer), claustrum, globus pallidus, caudate-putamen, nucleus accumbens, dentate gyrus (granular layer) and the majority of thalamic nuclei. Structures displaying low binding densities included the substantia nigra and the subthalamic nucleus. The regional distribution pattern of [3H]zolpidem binding sites was qualitatively similar to that of [3H]flunitrazepam but the relative density of the 3H-ligands differed in several brain regions. The relative density of omega 1 and omega 2 subtypes in each particular primate brain region was evaluated by measuring 1) the ratio of [3H]zolpidem to [3H]flunitrazepam binding; 2) [3H]flunitrazepam binding in the presence and in the absence of 100 nM zolpidem. With both approaches, a preferential enrichment in omega 1 sites was observed in lamina IV of sensorimotor cortical regions and in the extrapyramidal motor system (globus pallidus, ventral thalamic complex, subthalamic nucleus, substantia nigra and cerebellum). In contrast, omega 2 sites predominated in limbic areas (e.g., the dentate gyrus, amygdala, nucleus accumbens, cingulate and parahippocampal gyri) and in the anterior thalamic nucleus and caudate nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Glycine synthase from rat brain mitochondria shows an apparent Km for glycine of 2.6 mM. Glycine synthase from rat brain homogenate has a maximum of activity at the 10th day of life. However, each area of the central nervous system shows a different developmental pattern of glycine synthase activity. Both the 14CO2 production from (U-14C)-glycine and the incorporation of the radioactivity from the (U-14C)-glycine to the acid-insoluble fraction by cortex slices has a developmental profile similar to the glycine synthase activity in this area, having a maximum on the 10th day.

The Na+-independent binding of L-[3H]cysteine sulfinate and L-[3H]cysteine sulfinate uptake were investigated in rat brain membranes and vesicles. Specific binding of L-[3H]cysteine sulfinate was saturable and occurred by a single high affinity process with a Kb of 100 nM +/- 9 and a capacity (Bmax) of 2.4 +/- 0.22 pmol/mg protein. Sodium ions were found to have a biphasic effect; low concentrations (in the range of 0.1-3 mM) induced a marked inhibition of the binding whereas higher concentrations (10-300 mM) resulted in a dose-dependent stimulation of binding. The inhibition potency, expressed as the Ki values of a wide range of compounds with known pharmacological activities was tested. L-Cysteine sulfinate was the most potent inhibitor being 3-fold more potent than L-glutamate and 80 times more potent than L-aspartate. The regional distribution of the binding of L-[3H]cysteine sulfinate in the brain was found to be heterogeneous. These results provide the first evidence for an interaction of cysteine sulfinate with specific receptor sites on the synaptic membrane. The rate of L-[3H]cysteine sulfinate uptake shows a biphasic dependence on the concentration of L-cysteine sulfinate, corresponding to a high affinity (27.2 microM) and a low affinity (398 microM) transport system. The maximum L-[3H]cysteine sulfinate uptake is reached at 2 min. The reversibility of this transport was demonstrated. The L-[3H]cysteine sulfinate uptake increases as a function of the sodium concentration. Chloride and potassium ions stimulate the uptake. The decrease or increase in the electrical membrane potential (delta psi) caused by replacing the chloride ions by the sulfate or sulfocyanate ions respectively leads to a decrease or increase in the rate of uptake. Increase in the extravesicular osmolarity leads to a decrease in the extent of L-[3H]cysteine sulfinate accumulation. Amino acids with an acidic group in position omega were found to be potent inhibitors (the most potent being L-aspartate). The length of the carbon chain also has a bearing on the inhibitory effect. The regional distribution of L-[3H]cysteine sulfinate uptake in the brain was heterogeneous. These results demonstrate the existence of a high affinity system which may correspond to the transmitter inactivation. Binding and uptake sites are distinguishable as evidenced by the affinity constants, the ionic and pharmacological effects and the different regional distributions in the brain. Finally, these results give further evidence for a neurotransmitter role of L-cysteine sulfinate.

Two models have been chosen to study the effect of 2-amino-6-trifluoromethoxy benzothiazole (PK 26124) on excitatory amino acid neurotransmission: the pool of cyclic guanosine monophosphate (cGMP) in the cerebellum and the release of acetylcholine in the striatum and olfactory tubercles. The release of acetylcholine induced by N-methyl-DL-aspartate in the striatum and olfactory tubercles was antagonized by PK 26124 which was less potent on the release of acetylcholine induced electrically. The increase in levels of cGMP in the cerebellum induced by excitatory amino acids such as glutamate and quisqualate was antagonized by PK 26124, but the drug was inactive against N-methyl-DL-aspartate, L-aspartate, kainate and cysteine sulphinate. In vivo it antagonized the increases of cGMP in the cerebellum elicited by all these excitatory compounds. All these results are compatible with a possible antagonism by PK 26124 of the excitatory amino acid neurotransmission and may explain its anticonvulsant properties.

Zolpidem is a novel hypnotic drug which possesses preferential affinity, under in vitro conditions, for the omega 1 (BZD1) subtype of BZD binding sites. In the present study the in vivo interaction of zolpidem with mouse brain BZD binding sites, as labeled by i.v. injection of [3H]Ro 15-1788, has been investigated. Intraperitoneal administration of zolpidem (30 min before sacrifice) decreased in a dose-dependent manner, the retention of [3H]Ro 15-1788 in the cerebral cortex (ED50 = 8.9 mg/kg i.p.); the inhibition by zolpidem was maximal (70%) at 5 to 10 min postinjection and of only 10% 1 hr later. These kinetics are in agreement with its short lasting hypnotic properties. CGS 9896, CL 218,872 and flunitrazepam also prevented the cortical accumulation of [3H]Ro 15-1788 with ED50 values of 12.5, 24 and 0.17 mg/kg i.p., respectively. Zolpidem, like flunitrazepam, diminishes exploratory activity and possesses anticonvulsant and myorelaxant effects in the mouse. However, in contrast to flunitrazepam, the sedative action of zolpidem can be evidenced at a much lower recognition site occupancy (35%) than that needed for myorelaxant or anticonvulsant effects (50-56%). The regional selectivity of zolpidem as an inhibitor of [3H]Ro 15-1788 in vitro and in vivo binding in the mouse brain has been assessed by quantitative autoradiography.(ABSTRACT TRUNCATED AT 250 WORDS)

Our study included children up to 15 years of age who were selected from an open population. We found a frequency of 3.4% and it ranked 3rd among the most common dermatoses. The age range varied between 1 year 9 months to 15 years. In only one case we found an important predisposing factor (lupus erythematosus which was treated with systemic steroids). It is concluded that this dermatosis is not as infrequent as it seems.

Ketone body uptake by renal brush border vesicles has been investigated. Ketone bodies enter into the brush border vesicles by a carrier-mediated process. The uptake is dependent on an Na+ gradient ([Na+]outside>[Na+]inside) and is electroneutral. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. A pH gradient (alkaline inside) also stimulates the ketone body uptake. Acetoacetate and 3-hydroxybutyrate share the same carrier as demonstrated by the accelerated exchange diffusion and mutual inhibitory effects.

The postnatal development of glycine synaptic receptors has been studied. Strychnine binding to the synaptic membrane fraction is very low at birth, increases thereafter, and reaches adult values at the 15th day in the brain, and at the 30th day in the spinal cord. Throughout postnatal development, there are more glycine receptors in the spinal cord than in the brain. The development of receptors in the spinal cord displays a pattern similar to that reported previously for the glycine reuptake system in spinal cord slices and in the activity of spinal cord glycine synthase. In rats with experimental hyperglycinemia strychnine binding to spinal cord glycine receptors increases much more rapidly, reaching a level 1.5 times the control value by day 10. When the hyperglycinemia was induced after the 10th postnatal day, however, no effect on the glycine receptors was observed. This increased number of receptors could be explained by an effect of glycine on the synaptic stabilisation process. No changes in the KD for strychnine were observed either during postnatal development or in hyperglycinemic rats. The KD remained approximately 10 nM in the spinal cord and 50 nM in the brain. Results are discussed with respect to the ontogeny of glycinergic synapses and the pathogenesis of nonketotic hyperglycinemia.

Human urine and extracts of rat liver mitochondria contain apparently identical agents capable of inhibiting the precipitation or crystallization of calcium phosphate. Its general properties, as well as 1H NMR and mass spectra, have suggested that the agent is phosphocitric acid. This paper reports the synthesis of phosphocitric acid via the phosphorylation of triethyl citrate with o-phenylene phosphochloridate, hydrogenolysis of the product to yield triethyl phosphocitrate, hydrolytic removal of the blocking ethyl groups and also chromatographic purification. An enzymatic assay of phosphocitrate is described. Synthetic phosphocitrate was found to be an exceedingly potent inhibitor of the growth of hydroxylapatite seed crystals in a medium supersaturated with respect to Ca2+ and phosphate. Comparative assays showed phosphocitrate to be much more potent than the most active precipitation-crystallization inhibitors previously reported, which include pyrophosphate and ATP. 14C-Labeled phosphocitrate was bound very tightly to hydroxylapatite crystals. Such binding appeared to be essential for its inhibitory activity on crystal growth. Citrate added before but not after, phosphocitrate greatly enhanced the inhibitory potency of the latter. This enhancement effect was not given by other tricarboxylic acids. The monoethyl ester of phosphocitrate had no inhibitory effect on hydroxylapatite crystal growth.

Transport of glycine by rat brain and liver mitochondria has been investigated by both [14C]glycine uptake and swelling experiments. Glycine enters mitochondria passively down its concentration gradient by a respiratory-independent carrier-mediated process. This view is supported by the following observations: (a) glycine inside the mitochondria reaches the incubation medium concentration; (b) mitochondria swell in the presence of isoomotic solutions of glycine in a concentration-dependent fashion; (c) the uptake of glycine is not influenced by respiratory inhibitors such as KCN or by uncouplers such as carbonylcyanide p-trifluoromethoxyphenylhydrazone; (d) initial rates of uptake approach saturation kinetics, the apparent Km of the rat brain mitochondria for glycine being 1.7 mM and that of the liver mitochondria being 5.7 mM; (e) the rate of swelling is inhibited by methylmalonate, propionate and, at pH 6.5, by mersalyl, and (f) uptake is inhibited by phosphoserine, methylmalonate and propionate, but not by alanine or proline.

A series of 4-amino-6-chloro-2-piperazinopyrimidines were synthesized and evaluated for their ability to interact with alpha 1- and alpha 2-adrenoceptors in vitro in binding assays using [3H]WB-4101, [3H]clonidine, and [3H]idazoxan as radioligands. Some compounds were also tested as inhibitors of [3H]spiroperidol binding. Several members of this series showed high and selective affinity for alpha 2-adrenoceptors. The nature of the 4-amino substituent seems to be the most critical factor in determining the potency at these receptors.

The [3H]indalpine binding sites have been characterized in slide-mounted cat brain sections. This inhibitor of 5-HT reuptake binds with a very high affinity to sites which have the pharmacological properties of the serotonin carrier. These sites can, however, be differentiated from the [3H]imipramine binding sites by their Na+ dependency and competitive inhibition by serotonin. Quantitative autoradiographic studies demonstrate that indalpine binding sites are localized in structures rich in serotonergic neurons. The widespread distribution of indalpine binding sites in limbic and associative areas is consisted with its well characterized antidepressant activity in human.