Differences in intraoperative and surgical outcomes between normotensive pheochromocytomas and sympathetic paragangliomas (PPGLs) and hypertensive PPGLs: results from the PHEO-RISK STUDY.

PURPOSE: To compare the intraoperative and surgical outcomes of normotensive pheochromocytomas and sympathetic paragangliomas (PPGLs), hypertensive PPGLs and non-PPGL adrenal lesions. METHODS: This a retrospective multicenter cohort study of patients with PPGLs from 18 tertiary hospitals. A control group of histologically confirmed adrenocortical adenomas (non-PPGL group) was selected to compare intraoperative and surgical outcomes with of the normotensive PPGLs. RESULTS: Two hundred and ninety-six surgeries performed in 289 patients with PPGLs were included. Before surgery, 209 patients were classified as hypertensive PPGLs (70.6%) and 87 as normotensive PPGLs. A higher proportion of normotensive PPGLs than hypertensive PPGLs did not receive alpha presurgical blockade (P = 0.009). When we only considered those patients who received presurgical alpha blockers (200 hypertensive PPGLs and 76 normotensive PPGLs), hypertensive PPGLs had a threefold higher risk of intraoperative hypertensive crisis (OR 3.0 [95% 1.3-7.0]) and of hypotensive episodes (OR 2.9 [95% CI 1.2-6.7]) than normotensive PPGLs. When we compared normotensive PPGLs (n = 76) and non-PPGLs (n = 58), normotensive PPGLs had a fivefold higher risk of intraoperative complications (OR 5.3 [95% CI 1.9-14.9]) and a six times higher risk of postoperative complications (OR 6.1 [95% CI 1.7-21.6]) than non-PPGLs. CONCLUSION: Although the risk of intraoperative hypertensive and hypotensive episodes in normotensive PPGLs is significantly lower than in hypertensive PPGLs, normotensive PPGLs have a greater risk of intraoperative and postoperative complications than non-PPGL adrenal lesions. Therefore, it is recommended to follow the standard of care for presurgical and anesthetic management of PPGLs also in normotensive PPGLs.

Efficacy and safety of ossein-hydroxyapatite complex versus calcium carbonate to prevent bone loss.

Objective: This study aimed to compare the efficacy and safety of ossein-hydroxyapatite complex (OHC) versus calcium carbonate (CC) for preventing bone loss during perimenopause in current clinical practice.Methods: The prospective, comparative, non-randomized, open-label study included 851 perimenopausal women with basal bone mineral density (BMD) T-score ≥-2 standard deviations (SDs). Participants received either OHC (712 mg calcium/day) or CC (1000 mg calcium/day) over 3 years. BMD was evaluated by dual-energy X-ray absorptiometry at the lumbar spine (L2-L4) at baseline and after 18 and 36 months of follow-up. Adverse drug reactions (ADRs) were also recorded.Results: In women receiving OHC, BMD at the L2-L4 site remained stable over the 3-year follow-up period (mean [SD] change 0.00 [0.11] g/cm2). BMD in the CC arm decreased -3.1% (mean [SD] - 0.03 [0.11] g/cm2). Between-group differences were statistically significant (p < 0.001) and favored OHC. ADRs were more frequent in the CC group (7.7% vs. 2.7% in the OHC group; p = 0.001), affecting primarily the gastrointestinal system.Conclusion: OHC showed greater efficacy and tolerability than CC for bone loss prevention in perimenopausal women in real-world practice. As the daily dose of calcium was higher in the CC group, the differences might be linked to the ossein compound in OHC.

Temporal expression of cytokines and signal transducer and activator of transcription factor 3 activation after neonatal hypoxia/ischemia in mice.

Hypoxia/ischemia (HI) is a prevalent reason for neonatal brain injury with inflammation being an inevitable phenomenon following such injury; but there is a scarcity of data regarding the signaling pathway involved and the effector molecules. The signal transducer and activator of transcription factor 3 (STAT3) is known to modulate injury following imbalance between pro- and anti-inflammatory cytokines in peripheral and central nervous system injury making it a potential molecule for study. The current study investigates the temporal expression of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, IL-1ra, IL-4, IL-10, IL-13 and phosphorylated STAT3 (pSTAT3) after carotid occlusion and hypoxia (8% O2, 55 min) in postnatal day 7 C57BL/6 mice from 3 h to 21 days after hypoxia. Protein array illustrated notable changes in cytokines expressed in both hemispheres in a time-dependent manner. The major pro-inflammatory cytokines showing immediate changes between ipsi- and contralateral hemispheres were IL-6 and IL-1ß. The anti-inflammatory cytokines IL-4 and IL-13 demonstrated a delayed augmentation with no prominent differences between hemispheres, while IL-1ra showed two distinct peaks of expression spread over time. We also illustrate for the first time the spatiotemporal activation of pSTAT3 (Y705 phosphorylation) after a neonatal HI in mice brain. The main regions expressing pSTAT3 were the hippocampus and the corpus callosum. pSTAT3+ cells were mostly a subpopulation of activated astrocytes (GFAP+) and microglia/macrophages (F4/80+) seen only in the ipsilateral hemisphere at most time points studied (till 7 days after hypoxia). The highest expression of pSTAT3+ cells was observed to be around 24-48 h, where the presence of pSTAT3+ astrocytes and pSTAT3+ microglia/macrophages was seen by confocal micrographs. In conclusion, our study highlights a synchronized expression of some pro- and anti-inflammatory cytokines, especially in the long term not previously defined. It also points towards a significant role of STAT3 signaling following micro- and astrogliosis in the pathophysiology of neonatal HI-related brain injury. In the study, a shift from pro-inflammatory to anti-inflammatory cytokine profile was also noted as the injury progressed. We suggest that while designing efficient neuroprotective therapies using inflammatory molecules, the time of intervention and balance between the pro- and anti-inflammatory cytokines must be considered.

A modulation of glutamate-induced phosphoinositide breakdown by intracellular pH changes.

The influence of intracellular pH (pHi) changes on the formation of inositol phosphate metabolites (IPs) produced by glutamatergic stimulation was studied in 8-day-old rat brain synaptoneurosomes. For this purpose pHi was measured using 2',7'-bis-(2-carboxyl)-5,6-carboxyfluorescein (BCECF) fluorimetric assay in parallel with the basal and receptor-mediated formations of inositol monophosphate (IP1) and inositol bisphosphate (IP2). We found that glutamate (1 mM), which induces a transient acidification (delta pH = -0.05), produces an identical accumulation of IP1 and IP2. K+ (30 mM), which provokes an alkalinization of the internal medium (delta pH = +0.22), mainly leads to the formation of IP1 metabolites. Paired combinations of glutamate with 1, 5 and 10 mM NH4+ finally result in an alkalinization of the intrasynaptoneurosomal medium. These combinations produce a strong decrease of the IP2 level concomitant with an increase of the IP1 formation, compared to the levels of IP1 and IP2 evoked by glutamate alone. The total amount of IPs (IP1 + IP2) produced by these combinations is not different from that obtained with glutamate alone. Paired combinations of carbachol with NH4+ produce an identical alkalinization to that produced by NH4+ alone. These combinations produce an increased IP1 accumulation, while the IP2 formation is slightly decreased. When the internal medium is acidified by diminishing the external concentration of Na+, the ratio IP1/IP2 produced after metabotropic glutamate receptor (mGluR) activation is shifted to lower values, while it is not affected for the muscarinic stimulation. These data suggest that the mGluR-associated pathway in synaptoneurosomes is sensitive to pHi shifts, while the muscarinic receptor-associated pathway is less altered when pHi is manipulated. It may be proposed that pH-sensitive inositol phosphate dephosphorylating systems, i.e. phosphatases, are associated with mGluRs in this preparation.

A M3 muscarinic receptor coupled to inositol phosphate formation in the rat cochlea?

Various neuroactive substances, including excitatory and inhibitory amino acids, biogenic amines and neuropeptides, were tested for their ability to stimulate the inositol phosphate (IPs) cascade in the presence of lithium in the rat cochlea. Among them, only the muscarinic agonists (carbachol and oxotremorine M) were able to stimulate the IPs formation in 12-day-old rat cochleas. The carbachol-elicited IPs formation was inhibited by muscarinic antagonists with the following relative order of potency: atropine greater than 4-DAMP much greater than pirenzepine greater than methoctramine = AF-DX 116. This pharmacological profile suggests that the activation of the M3 muscarinic receptor subtype is responsible for the increase in IPs synthesis in the rat cochlea. However, an interaction with a m5 receptor subtype could not be completely excluded. The unusual link of only one receptor subtype with the phosphoinositide breakdown in the cochlea, as opposed to the usual existence of several receptors coupled to this transduction system in other organs such as the brain, suggest a unique role for muscarinic agonists in the cochlea.

White coat hypertension in type 1 diabetic patients without nephropathy.

The aim of our study was to determine the prevalence of white coat hypertension (WCH) in type 1 diabetic patients. Therefore, ambulatory blood pressure monitoring (ABPM) and 24-h urinary albumin excretion (UAE) were determined in 47 patients with type 1 diabetes mellitus (27 with new diagnosis of hypertension by office blood pressure (BP) measurement and 20 with normotension). WCH was diagnosed in 20 patients (74%). Patients with WCH presented higher values of systolic and diastolic BP and UAE than normotensive patients. The results indicate that in type 1 diabetes mellitus WCH is very frequent. Thus, WCH may represent a potential risk for the development of diabetic complications, mainly diabetic nephropathy.

Protein kinase C differently regulates quisqualate- and 1S,3R-trans aminocyclopentane dicarboxylate-induced phosphoinositide hydrolysis during in vitro development of hippocampal neurons.

Transient peaks of quisqualate (QA)-, but not 1S,3R-1-amino-3-cyclopentane dicarboxylate (1S,3R-ACPD)- and carbachol-induced inositol phosphate formation occur between 2 and 5 days in vitro (DIV) in hippocampal neurons in culture. In order to elucidate the putative origin of such developmental activity differences, the effect of PKC on metabotropic glutamate receptor (mGluR) and muscarinic receptor responses was investigated at 3 and 10 DIV. (i) Stimulation of PKC by phorbol-12,13-dibutyrate inhibited QA, 1S,3R-ACPD and carbachol responses at 3 DIV. At 10 DIV, only 1S,3R-ACPD response was still inhibited by phorbol esters. (ii) Inhibition of PKC by staurosporine at 3 DIV potentiated 1S,3R-ACPD-induced inositol phosphate formation, but had no effect on QA and carbachol responses. At 10 DIV, all responses were potentiated by staurosporine. These data strongly suggest that PKC differently modulates 1S,3R-ACPD- and QA-induced inositol phosphate accumulations during in vitro development. The specific activity of mGluRs during development, vs that of muscarinic receptor, and the peculiar modes of regulation by PKC of these two mGluR activities further suggest their particular involvement in the maturation of neuronal culture.

Potassium and veratrine-stimulated l-[(3)H]cysteine sulfinate and l-[(3)H]glutamate release from rat brain slices.

The release of l-[(3)H]cysteine sulfinic acid, l-[(3)H]glutamatic acid and [(3)H]GABA from preloaded slices of various rat brain regions in response to either 30 mM K(+) or veratrin was investigated. All these aminoacids were released by both depolarizing agents, which did not produce any changes in the spontaneous efflux of [(3)H]lysine. The K(+) stimulated cysteine sulfinate release from superfused slices was found partly Ca(2+)-dependent in the subiculum, and mainly Ca(2+)-independent in the hippocampus whereas the K(+)-elicited glutamate release was partly Ca(2+)-dependent in both regions. The veratrine-induced release of both cysteine sulfinate and glutamate was blocked by verapamil in a dose-dependent way, although a small verapamil concentration independent release remained. The release pattern of both amino acids was heterogeneous, but roughly correlated among brain regions, except in the subiculum and hypothalamus. These findings demonstrate the releasability of both substances from various brain regions and suggest that those releases occur from different pools, being probably mainly of neuronal origin. They give further evidence that cysteine sulfinate as well as glutamate may serve a neurotransmitter role in the CNS.

Intra- vs extracellular calcium regulation of neurotransmitter-stimulated phosphoinositide breakdown.

The dependence on Ca2+ of basal, glutamate- and carbachol-stimulated phosphoinositide (PI) turnover was studied on 8-day old rat brain synaptoneurosomes. For that purpose, intracellular and extracellular Ca2+ concentrations were buffered by bis-(alpha-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, in its tetra(acetoxymethyl)-ester form (BAPTA-AM) and in its free acid form (BAPTA), respectively. The effects of both forms of the calcium chelator intracellular and extracellular Ca2+ buffering on intracellular and extracellular Ca2+ concentration ([Ca2+]i and [Ca2+]e) were determined with fluorimetric assay using fura2, either in its acetoxymethyl ester form (fura2-AM) or in its free acid form. Intracellular chelation of Ca2+ ions with BAPTA-AM induced a dose-dependent reduction of the [Ca2+]i. Basal inositol phosphate (IP) formation was slightly affected by this [Ca2+]i buffering, while glutamate and carbachol stimulations of PI hydrolysis were similarly diminished. Chelation of extracellular Ca2+ ions with BAPTA produced a reduction of both [Ca2+]e and [Ca2+]i. Basal IP accumulation was maximally inhibited by 50%. The carbachol-induced PI hydrolysis was completely inhibited in the presence of 200 microM BAPTA, while a substantial residual glutamate-elicited IP response remained (40% of the control response). It is concluded that [Ca2+]i of synaptoneurosomes is not critical for basal and neurotransmitter-stimulated IP formation, whilst [Ca2+]e is critical. Glutamate may, in part, stimulate PI breakdown in a Ca(2+)-insensitive way.

Dithiotreitol specifically inhibits metabotropic responses of glutamate and depolarizing agents in rat brain synaptoneurosomes.

Dithiotreitol (DTT), a sulfhydryl reducing agent inhibits in a dose-dependent manner the inositol phosphates (IPs) accumulation responses evoked by glutamate and potassium without affecting that of carbachol in rat forebrain synaptoneurosomes. Furthermore, DTT neither provokes a depolarization of the membrane, nor increases the internal calcium concentration. Depolarization and internal calcium rise are known to stimulate IPs production. Moreover, DTT does not modify the depolarizing effect and the calcium rise elicited by glutamate and potassium. In addition, the antioxidant compounds 2-aminoethylisothiouronium bromide (AET) and ascorbic acid have no effect on the basal and stimulated IPs accumulation. Thus, it is concluded that: (1) two distinct transduction pathways exist, one stimulated by glutamate and depolarizing agents and the other one by cholinergic agonists; (2) DTT produces its inhibition by reducing disulfide bridges likely at the level of proteins of the phosphoinositide transduction mechanism.

A new quisqualate receptor subtype (sAA(2)) responsible for the glutamate-induced inositol phosphate formation in rat brain synaptoneurosomes.

Inositol phosphate synthesis elicited by excitatory amino acids was measured in rat forebrain synaptoneurosomes in presence of Li(+). Quisqualate (QA) was the most potent excitatory amino acid inducing inositol phosphate formation. This QA action was not blocked by any of the usual antagonists [glutamate-amino-methyl-sulphonate (GAMS); glutamate-diethyl-ester (GDEE); ?-d-glutamyl-glycine (?-DGG)] known to inhibit the QA-induced depolarization. The same was found for the most potent and selective QA antagonist reported so far [6-nitro-7-cyanoquinoxaline-2,3-dion (FG 9065)]. In addition, dl-?-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) a potent depolarizing agonist at the quisqualate receptor subtype was about 300 times less potent than quisqualate in increasing inositol phosphate accumulation. Our results provide the first pharmacological evidence indicating that a new quisqualate receptor subtype, tentatively termed sAA(2) is responsible for inositol phosphate formation.

Cadmium rapidly and irreversibly blocks presynaptic phospholipase C-linked metabotropic glutamate receptors.

Calcium ions (Cd2+) inhibit inositol phosphate (IP) formation elicited by glutamate (GLU) or K+ ions, without affecting carbachol (Carb)-induced IP response in 8-day-old rat forebrain synaptoneurosomes and synaptosomes. On the contrary, Cd2+ was almost ineffective in blocking GLU- and K(+)-responses in hippocampal neurones in culture. The mechanism of Cd2+ inhibition was thus examined in synaptoneurosomes. Extensive washing of synaptoneurosomes pretreated for 1, 5, 15, or 30 min by 100 microM Cd2+ did not modify the inhibitory effect of Cd2+ on GLU-, K(+)- and A23187-evoked IP formation or its lack of effect on Carb response. The later addition of a high affinity Cd2+ chelator (100 microM), N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) also did not reverse the inhibitory effect. TPEN, however, penetrates into synaptoneurosomes and efficiently displaces Cd2+ from the Fura-2-Cd2+ complex as shown by Fura-2 fluorescence recordings. TPEN is not easily removed from the intracellular space, as demonstrated by its ability to still block Cd(2+)-induced Fura-2 fluorescence increase after extensive washing. Pretreatment of synaptoneurosomes by this chelator did not prevent Cd2+ inhibition of GLU-induced IP formation. These data indicate that Cd2+ ions rapidly, irreversibly and extracellularly inhibit GLU-elicited IP formation in synaptoneurosomes or synaptosomes, but not in hippocampal neurones in culture. It is speculated that Cd2+ ions could allow one to distinguish the activity of presynaptic metabotropic glutamate receptors (mGLURs) linked to phosphoinositide metabolism from that of mGLURs located postsynaptically.

Transient increase in the high affinity [3H]-L-glutamate uptake activity during in vitro development of hippocampal neurons in culture.

The glial GLAST and GLT-1 glutamate transporters are transiently expressed in hippocampal neurons as shown by immunocytochemistry (Plachez et al., 2000. J. Neurosci. Res., 59, 587-593). In order to test if this transient expression is associated to a transient glutamate uptake activity, [3H]-glutamate uptake was studied during the in vitro development of embryonic hippocampal neurons cultured in a defined (serum free) medium. In these cultures, the ratio of the number of glial cells to the number of neurons increased from 1.7 to 11.3% during the first 10 days of culture, while 77% of the neurons died. The number of neurons then remains stable up to 23 days of culture. The initial glutamate uptake velocity at 20 and 200 microM [3H]-glutamate usually increased about five times between 1 and 10 days in vitro (DIV). Interestingly, at 2 microM [3H]-glutamate, the uptake initial velocity showed a biphasic pattern, with a transient peak between 1 and 6 DIV, the maximum being reached at 2 DIV and a delayed regular increase from 8 to 23 DIV. The concentration-dependent curves were best fitted with two saturable sites high and low affinities, at both 2 and 10 DIV. To pharmacologically characterize the transient increased glutamate uptake activity, four uptake inhibitors, L-threo-3-hydroxy-aspartic acid (THA), L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-2,4-PDC), dihydrokainate (DHK), and DL-threo-beta-benzyloxyaspartate (TBOA) were tested. THA, L-trans-2,4-PDC and DL-TBOA inhibited glutamate uptake both at 2 and 10 DIV, while the GLT-1 selective uptake inhibitor DHK neither strongly affected the uptake at 2, nor at 10 DIV. These data indicated that, besides the regular increase in the glial-dependent glutamate uptake activity, a transient high-affinity, DHK insensitive, glutamate transport activity in hippocampal neurons in culture is present. This latter activity could potentially be related to the transient expression of the glial GLAST transporter in neurons.

Separate binding sites in rat brain synaptic membranes for l-cysteine sulfinate and for l-glutamate.

Binding of l-[(3)H]cysteine sulfinic acid (CSA) and l-[(3)H]glutamate were compared in various subcellular fractions and in the presence of a variety of pharmacological and ionic manipulations in order to test the possibility that the two amino acids possessed separate binding sites. The specific l-[(3)H]cysteine sulfinate binding was found to be enriched maximally in medium and high density synaptic membranes, while the crude mitochondrial synaptosomal fraction displayed the highest l-[(3)H]glutamate binding. The ratio of l-[(3)H]cysteine sulfinate binding/l-[(3)H]glutamate binding was variable across brain regions. Several compounds differentially affected l-[(3)H]cysteine sulfinate and l-[(3)H]glutamate binding. l-cysteine sulfinate was the most potent displacer regardless of the binding considered. Finally, while cations produced qualitatively similar effects on the binding of the two amino acids, quantitative differences were evident. In sum, these data revealed the complexity of l-[(3)H]cysteine sulfinate and l-[(3)H]glutamate binding. They suggest the existence of several binding sites and that some of these are shared by both substances. However, the results also indicate that separate binding sites for the two amino acids exist in synaptic membrane, giving further support to the hypothesis that cysteine sulfinate serves a neurotransmitter role in the central nervous system.

Immunological properties and immunohistochemical localization of cysteine sulfinate or aspartate aminotransferase-isoenzymes in rat CNS.

Organ and interspecies specificities of cysteine sulfinate transaminase isoenzymes were studied by double immunodiffusion. Antisera were produced in rabbits against purified mitochondrial and cytosolic enzymes of rat brain. No cross-reaction was found between the two isoenzymes. The enzymes present in crude mitochondrial fractions prepared from rat brain, heart, kidney and liver are immunologically identical. Similar results were obtained with the enzymes present in cytosolic fractions. Mouse, chicken, frog and rat crude mitochondrial and soluble fractions were tested, but only mouse isoenzymes reacted. The localizations of mitochondrial and cytosolic cysteine sulfinate transaminases were studied in the rat olfactory bulb and retina. The mitochondrial enzyme was found essentially in the plexiform, glomeruli and mitral cell layers of the olfactory bulb, and in the inner segment and external plexiform layer of the retina, whereas the cytosolic enzyme was located mainly in the granule cell areas of the olfactory bulb and in the nuclear layer of the retina. These results revealed a lack of organ specificity, an interspecies specificity, and a specific localization indicating specific roles for both isoenzymes.

The binding of acidic amino acids to snail, Helix aspersa, periesophagic ring membranes reveals a single high-affinity glutamate/kainate site.

The characterization of specific acidic amino acid binding sites to snail, Helix aspersa, ganglia membranes has been assayed using tritiated glutamate (L-[3H]Glu), aspartate (L-[3H]Asp), cysteine sulfinate (L-[3H]CSA) and kainate. At 2 degrees C, only L-[3H]Glu and [3H]kainate specific binding could be measured using a filtration procedure to separate bound from free ligand. The analysis of L-[3H]Glu specific binding reveals the presence of one class of high-affinity binding sites with Kd = 0.12 microM and Bmax = 30 pmol/mg protein. This L-[3H]Glu binding was specific, reversible and saturable. The order of potency of different substances, agonists or antagonists of the rat brain excitatory amino acid receptors, has been determined. Kainate was the best displacing agent, followed by ibotenate = L-Glu greater than L-alpha-aminoadipate (L-alpha-AA) greater than homocysteate (HCA). Using 10 nM [3H]kainate, a single class of binding site was detected. Its pharmacological properties indicate that it is likely identical to the L-[3H]Glu binding site. This L-Glu-kainate site possesses most of the properties expected for a specific receptor. However, whereas L-[3H]Glu binding could be detected on purified neuronal membranes, the major component of specifically bound L-[3H]Glu appeared to be located on the sheaths surrounding neuronal cell bodies. These findings suggest that Glu or another endogenous acidic amino acid may function as a transmitter at neuromuscular junctions in Helix periesophagic ring, acting at a receptor distinct from those on nerve cells.

Multiple Cl(-)-independent binding sites for the excitatory amino acids: glutamate, aspartate and cysteine sulfinate in rat brain membranes.

As we have recently reported that Cl(-)-dependent glutamate (GLU) binding reflects GLU accumulation into membrane vesicles, the characteristics, kinetics and pharmacological specificities of L-[3H]glutamate (L-[3H]GLU) binding to crude rat brain synaptic membranes, were investigated in Cl(-)-free medium. L-[3H]GLU binding was systematically compared to that of L-[3H]cysteine sulfinate (L-[3H]CSA) and L-[3H]ASP), two other putative excitatory amino acids. A high affinity site was determined for each of these radioactive ligands (L-[3H]GLU: Kd = 0.14 microM, Bm = 3.4 pmol/mg protein; L-[3H]CSA: Kd = 0.07 microM, Bm = 2.2 pmol/mg protein; L-[3H]ASP: Kd = 5.8 microM, Bm = 31.2 pmol/mg protein). The pharmacological specificity of these Cl(-)-independent binding sites indicate the existence of at least 3 distinct high affinity sites, all different from the Cl(-)-dependent GLU binding 'site': one having a similar affinity for GLU and CSA, a second one preferring CSA, and a third one preferring ASP. Among the large quantity of structural analogs of the neuroexcitatory amino acids tested, only endogenous compounds (GLU, ASP and CSA) (except hydroxylamine-o-sulfate) were able to interact efficiently. No inhibition by classical agonists and antagonists (such as N-methyl-D-aspartate, quisqualate, kainate, 2-amino-4-phosphonobutyrate, or 2-amino-5-phosphonovalerate) was found. In addition to their high specificity, these Cl(-)-independent sites possess most other biochemical characteristics of receptor proteins.

Evidence for cysteine sulfinate as a neurotransmitter.

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.

Developmental changes in the chemosensitivity of rat brain synaptoneurosomes to excitatory amino acids, estimated by inositol phosphate formation.

The evolution of excitatory amino acids-(EAA) stimulated inositol phosphates (IPs) turnover during postnatal development was investigated in synaptoneurosomes prepared from rat forebrains. The two main EAA agonists which induce the IPs synthesis were quisqualate (QA) and N-methyl-D-aspartate (NMDA). The QA and NMDA stimulations of IPs formation present a particular developmental pattern, characterized by an active phase during rat synaptogenesis. The QA-evoked IPs accumulation peaked in synaptoneurosomes prepared from 8-day-old rat forebrains while that evoked by NMDA peaked in synaptoneurosomes from 12-day-old rats. These two developmental patterns are specific of the EAA agonists since the other various neuroactive substances tested (carbachol (Carb), noradrenaline, and high concentrations of potassium) induced an IPs accumulation, which increases during development and reaches a maximum in synaptoneurosomes of adult animals. Aging leads to a decrease in the capability of EAAs and muscarinic agonists to stimulate IPs formation in synaptoneurosomes, whereas the stimulation of IPs turnover by noradrenaline remains constant. Taken together, these results suggest that EAAs play a key role during brain development by sequentially activating two receptor subtypes, a new QA receptor, and a NMDA receptor, linked to the phosphoinositide metabolism. They may also indicate that these EAA-induced IPs responses are related to neuronal plastic events, the amplitude of which decreases with aging.