Release of [3H]serotonin from brain slices

1. Labelled serotonin ([³H]5-HT) accumulated by slices of rat brain either in vivo or in vitro is released by depolarizing procedures such as electrical stimulation or high external potassium concentrations. Electrical stimulation predominantly affects the liberation of the unchanged amine, rather than of its principal metabolite, 5-HIAA. 2. Release of [³H]5-HT does not appear to be calcium-dependent. 3. Amount of release parallels the density of serotonin-containing nerve terminals in each of several cerebral regions tested. Release from several extracerebral tissues was similar to that obtained from cerebral tissues having relatively little endogenous 5-HT. 4. Electrically induced release of [³H]5-HT is markedly inhibited by desipramine, chlorpromazine, LSD, lithium and ouabain.     

Modulation of GABA-stimulated chloride influx into membrane vesicles from rat cerebral cortex by triazolobenzodiazepines

The effects of triazolobenzodiazepines on GABA-stimulated 36Cl- uptake by membrane vesicles from rat cerebral cortex were examined. Triazolam and alprazolam showed a significant enhancement of GABA-stimulated 36Cl- uptake at 0.01-10 microM. On the other hand, adinazolam showed a small enhancement at 0.1-1 microM followed by a significant inhibition of GABA-stimulated 36Cl- uptake at 100 microM. The enhancement of GABA-stimulated 36Cl- uptake by 1 microM alprazolam was antagonized by Ro15-1788, a benzodiazepine antagonist, but the inhibition of this response by 30 microM adinazolam was not antagonized by Ro15-1788. These results indicate that triazolobenzodiazepines enhanced GABA-stimulated 36Cl- uptake through benzodiazepine receptors. High concentrations of adinazolam inhibit GABA-stimulated 36Cl- uptake which may be due to the direct blockade of GABA-gated chloride channel.     

Uptake of L-[3H] glutamic acid by crude and purified synaptic vesicles from rat brain

Rat brain synaptic vesicles suspended in a medium comprised of potassium tartrate displayed saturable accumulation of L-[³H] glutamic acid at 37° (Km 2.0 × 10^?4M; 311±13 pmol/mg protein), which was stable for periods up to 60 min. The accumulation was temperature sensitive and partially ATP-dependent, uptake levels being reduced to 18.7±0.8 pmol/mg protein at 4°, and to 141±4 pmol/mg protein in the absence of ATP. Fractionation of a crude vesicle preparation on a discontinuous sucrose gradient demonstrated the accumulation to be specifically associated with the synaptic vesicle fraction.     

Regulatory sites and effectors ofd-[3H]aspartate release from rat cerebral cortex

To study the effect of agents interfering with the biosynthesis and/or the K⁺-evoked Ca²⁺-dependent release of neurotransmitter glutamate, rat cerebral slices were preincubated with Krebs-Ringer-HEPES-glucose-glutamine buffer (KRH buffer), loaded withd-[³H]aspartate and superfused with the preincubation medium in the presence or in the absence of Ca²⁺. The difference in radioactivity release divided by the basal release per min under the two conditions represented the K⁺-evoked Ca²⁺-dependent release. The agents used were: 1) Aminooxyacetic acid (AOAA), the inhibitor of transaminases, 2) Leucine (Leu), the inhibitor of phosphate activated glutaminase (PAG), 3) NH₄ ⁺, the inhibitor of PAG, 4) Phenylsuccinic acid (Phs), the inhibitor of the mitochondrial ketodicarboxylate carrier, 5) ketone bodies, the inhibitors of glycolysis, 6) the absence of glutamine, the substrate of PAG. The results show that Leu, NH₄ ⁺, Phs and the absence of Gln significantly increase the K⁺-evoked Ca²⁺-dependent release of radioactivity by 64%, 200%, 95% and 147% respectively, indicating that these agents are inhibitors of the K⁺-evoked Ca²⁺-dependent release of glutamate. Ketone bodies and AOAA had no effect. These results indicate that the major if not the exclusive biosynthetic pathway of neurotransmitter glutamate in rat cerebral cortex is through the PAG reaction and support a model for the pathway followed by neurotransmitter glutamate i.e. glutamate formed outside the inner mitochondrial membrane has to enter the mitochondrial matrix or is formed within it from where it can be extruded to supply the transmitter pool in exchange of GABA.     

Properties of [3H]taurine release from crude synaptosomal fractions of rat cerebral cortex

The release of previously accumulated [³H]taurine and [¹⁴C]GABA from crude synaptosomal (P₂) fractions isolated from rat cerebral cortex was studied using a superfusion system. The spontaneous efflux of [³H]taurine and [¹⁴C]GABA was stimulated by elevated concentrations of K⁺ (15–133 mM) in a concentration-dependent manner. This K⁺-stimulated release of [¹⁴C]GABA but not of [³H]taurine was enhanced in the presence of Ca²⁺. However, addition of 3 mM Ca²⁺ to the superfusion medium in the presence of the ionophore A 23187 resulted in a stimulation of the release of both [³H]taurine and [¹⁴C]GABA. These results are discussed in connection with the cellular localization of tourine in the central nervous system.     

Transport of 2-deoxy-d-[3H]glucose in microvessels isolated from bovine cerebral cortex

Microvessels were isolated from a bovine cortex and the transport of glucose was investigated by using 2-deoxy-d-[³H]glucose (2-DG). The apparentK _m for 2-DG transport was 118 M and therefore indicates a significant high affinity for the substrate. The inhibition of 2-DG uptake byd-glucose showed an apparentK _i of 222 M. Other sugars, e.g., 3-methyl-d-glucose andd-fructose, also inhibited the 2-DG uptake by 60.6 and 36.0%, respectively. Phloretin (1×10^–3 M) inhibited the 2-DG transport more than phlorizin (83.7 vs. 53.8%). Ouabain (1 and 5×10^–4 M) did not inhibit the uptake of 2-DG but 2,4-dinitrophenol (1×10^–4 M) did (78.0%). The uptake of 2-DG could not be demonstrated in homogenized microvessels. Adenine nucleotides (conc. 2 mM) had various effects on the 2-DG uptake by microvessels. ATP inhibited the uptake by 20.7%, ADP was virtually without effect, and AMP stimulated the uptake of 2-DG by 8.5%. It was also found that the decrease of adenylate energy charge favors the uptake of 2-DG. All these findings suggest that in cerebral microvessels of a bovine cortex, 2-DG is apparently transported by a specific, carrier-mediated transport system.Dedicated to Prof. Dr. R. Sammet on the occasion of his 60th birthday.     

Distribution of [3H]noradrenaline and [3H]serotonin in photophores of Porichthys notatus

Summary Photophores of Porichthys notatus were examined by electron-microscopic radioautography following incubation in tritiated noradrenaline ([³H]NA) or serotonin ([³H]5-HT). Nerve varicosities surrounding the photocytes were found to accumulate [³H]NA but not [³H]5-HT, providing compelling evidence for the catecholaminergic nature of the monoaminergic innervation of photophores. The photocytes themselves appeared selectively labelled with both tracers, but the intensity of labelling after [³H]5-HT incubation was considerably greater than after [³H]NA. Stereological sampling of organelle content in photocytes showed ultrastructural differences between [³H]NA- and [³H]5-HT-labelled cells, probably related to light emission induced by NA. The main changes noted after incubation with [³H]NA were mitochondrial swelling and disorganization, increased coalescence of photocytic vesicles and extrusion of vesicular material into the extracellular matrix. With respect to the subcellular localization of [³H]NA and [³H]5-HT within the photocytes, statistical analysis of the distribution of silver grains disclosed a preferential affinity of both labels for appositional zones between mitochondria and coalescent vesicles. Moreover, in the case of 5-HT, selective affinity was also exhibited by sites comprising vesicular membrane and adjacent cytoplasm, suggesting binding of this biogenic amine to the entire membrane of photocytic vesicles.Supported by grants from the Natural Sciences and Engineering Research Council (M.A.), and Medical Research Council of Canada (L.D.). Dr. Pierre Legendre kindly provided advice on statistical methods     

Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes

Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.     

Uptake of [3H]ouabain from the cell surface into the lysosomal compartment of HeLa cells

[3H]Ouabain specifically bound at sublethal concentrations to Na,K-ATPase on the surface of HeLa cells is taken up (internalized) by the cells at a rate of three membrane equivalents of labeled sites per generation. Immediately following a pulse label with the glycoside, codistribution of radioactivity with the surface marker 5'-nucleotidase is found in both conventional sucrose-gradient fractionation and in fractionation following a digitonin treatment. At appropriate concentrations digitonin increases the buoyant density of the HeLa surface membrane and solubilizes the lysosomal marker beta-hexosaminidase (Tulkens et al., 1974). After internalization, [3H]ouabain is also solubilized by digitonin. A shear analysis is described which shows internalized ouabain and beta-hexosaminidase to be codistributed in a particulate fraction that is homogeneous with respect to shear; extrapolation to zero-shear shows that little or none of either marker is found in the soluble fraction of the cytosol. Both markers are coreleased from the particulate fraction by osmotic shock. Although internalized ouabain is subsequently released from these cells with a half-time of about 70 hr, apparently by exocytosis, the shear sensitivity of the remaining cell-associated ouabain does not change for up to 72 hr. Thus ouabain (together with Na,K-ATPase?) appears to be taken up from the surface into a lysosomal compartment and, by at least one criterion, this compartment does not change its physical properties with time, i.e., does not "age."     

Incorporation of [3H]Leucine and [3H]Valine into Protein of Freshwater Bacteria: Uptake Kinetics and Intracellular Isotope Dilution

Incorporation of [³H]leucine and [³H]valine into proteins of freshwater bacteria was studied in two eutrophic lakes. Incorporation of both amino acids had a saturation level of about 50 nM external concentration. Only a fraction of the two amino acids taken up was used in protein synthesis. At 100 nM, the bacteria respired 91 and 78% of leucine and valine taken up, respectively. Respiration of ³H and ¹⁴C isotopes of leucine gave similar results. Most of the nonrespired leucine was recovered in bacterial proteins, while only up to one-half of the nonrespired valine occurred in proteins. In intracellular pools of the bacteria, [³H]leucine reached an isotope saturation of 88 to 100% at concentrations of >40 nM. For [³H]valine, an isotope equilibrium of about 90% was obtained at concentrations of >80 nM. Within an incubation period of typically 1 h, tritiated leucine and valine incorporated into proteins of the bacteria reached an isotope saturation of 2 to 6%. In a 99-h batch experiment, bacterial protein synthesis calculated from incorporation of leucine and valine corresponded to 31 and 51% (10 nM) and 89 and 97% (100 nM), respectively, of the chemically determined protein production. Measured conversion factors of 100 nM leucine and valine were 6.4 × 10¹⁶ and 6.6 × 10¹⁶ cells per mol, respectively, and fell within the expected theoretical values. The present study demonstrates that incorporation of both valine and leucine produces realistic measurements of protein synthesis in freshwater bacteria and that the incorporation can be used as a measure of bacterial production.     

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.     

H2 histaminic receptors in rat cerebral cortex. 1. Binding of [3H]histamine

Saturable binding of [3H]histamine in equilibrium with homogenates of rat cerebral cortex reveals Hill coefficients between 0.4 and 1.0, depending upon the conditions. Data from individual experiments are well described assuming one or two classes of sites. Only the sites of higher affinity (KP1 = 3.9 +/- 0.5 nM) are observed when binding is measured by isotopic dilution at a low concentration of the radioligand (less than 1.5 nM) in the presence of magnesium or by varying the concentration of the radioligand. The sites of lower affinity (KP2 = 221 +/- 26 nM) appear during isotopic dilution at higher concentrations of the radioligand or at lower concentrations either upon the addition of guanylyl imidodiphosphate (GMP-PNP) or upon the removal of magnesium. Estimates of the second- and first-order rate constants for association and dissociation of [3H]histamine agree well with KP1. Apparent capacities corresponding to KP1 and KP2 are of the order of 100 ([R1]t) and 1300 pmol/g of protein ([R2]t), respectively. Simple interconversion cannot account for the changes in binding that occur upon adding GMP-PNP or removing magnesium, since the increase in [R2]t exceeds the decrease in [R1]t. Moreover, the apparent amount of high-affinity complex exhibits a biphasic dependence on the concentration of [3H]histamine; an increase at low concentrations is offset by a decrease that occurs at higher concentrations. The latter appears to be positively cooperative and concomitant with formation of the low-affinity complex. These and other observations indicate that the binding of histamine is inconsistent with models commonly invoked to rationalize the binding of agonists to neurohumoral receptors. GMP-PNP and magnesium reciprocally alter capacity at the sites of higher affinity, however, and the reduction caused by GMP-PNP reflects a substantial increase in the rate constant for dissociation at the sites that appear to be lost. The sites labeled by [3H]histamine thus reveal the properties of neurohumoral receptors linked to a nucleotide-specific G/F protein.     

H2 histaminic receptors in rat cerebral cortex. 3. Inhibition of [3H]histamine by H2 agonists

The binding of [3H]histamine to H2 receptors in homogenates of rat cerebral cortex is inhibited by 11 H2 agonists in a characteristic and unique manner. At low concentrations of the radioligand (less than 1.5 nM), the inhibitory profiles of individual agonists (A) are distinctly biphasic; specific binding is well described in most cases by the empirical expression Y = F1K1/(K1 + [A]) + F2K2/(K2 + [A]), in which F1 and F2 sum to 1. Maximal inhibition is the same for all agonists. Since values of F2 vary from 0.42 to 0.90, the agonist appears to determine the equilibrium distribution of receptors between two states of affinity. Ratios of apparent affinity (K2/K1) vary from 204 to 3 090 000, and there is no correlation between values of K1 and K2. Compounds lacking H2 activity, including structural analogues of histamine and dimaprit, reveal a Hill coefficient of 1 and inhibit the radioligand only weakly. For six agonists, values of K2 agree and correlate well (P = 0.00047) with H2 pharmacological potency (EC50) in the guinea pig right atrium; for the others, K2 is less than EC50 by 15-61-fold. Four observations suggest that the inhibition corresponding to F1 is allosteric and cooperative: the dissociation constant of the radioligand appears to vary in the presence of an unlabeled agonist, absolute levels of binding corresponding to F1, as defined by dimaprit, decrease at higher concentrations of [3H]histamine, F1 for dimaprit is reduced from 0.48 to 0.32 by 2-methylhistamine (F1 = 0.27) at a concentration of 20 nM (approximately K1(0.5) K2(0.5) for 2-methylhistamine), but the increase in K1 for dimprit is at least 100-fold less than expected from competitive effects, and 1 equiv of some agonists appears to preclude access of [3H]histamine to more than 1 equiv of receptors, with no evidence that an appreciable fraction of the unlabeled drug is bound. Noncompetitive effects also may account in part for the inhibition corresponding to F2.     

Functional significance of nitric oxide in ionomycin-evoked [3H]GABA release from mouse cerebral cortical neurons

We investigated a role of nitric oxide (NO) on ionomycin-evoked [3H]GABA release using mouse cerebral cortical neurons. lonomycin dose-dependently released [3H]GABA up to 1 microM. The extent of the release by 0.1 microM ionomycin was in a range similar to that by 30 mM KCl. The ionomycin (0.1 microM)-evoked [3H]GABA release was dose-dependently inhibited by NO synthase inhibitors and hemoglobin, indicating that the ionomycin-evoked [3H]GABA release is mediated through NO formation. The inhibition of cGMP formation by 1H-[1,2,4] oxodizao [4,3-a] quinoxalin-1-one (ODQ), a selective inhibitor for NO-sensitive guanylate cyclase, showed no affects on the ionomycin-evoked [3H]GABA release. Tetrodotoxin and dibucaine significantly suppressed the ionomycin-evoked [3H]GABA release and ionomycin increased fluorescence intensity of bis-oxonol, suggesting the involvement of membrane depolarization in this release. The ionomycin-evoked [3H]GABA release was maximally reduced by about 50% by GABA uptake inhibitors. The concomitant presence of nifedipine and omega-agatoxin VIA (omega-ATX), inhibitors for L- and P/Q-type voltage-dependent calcium channels, respectively, caused the reduction in the ionomycin-evoked release by about 50%. The simultaneous addition of nifedipine, omega-ATX and nipecotic acid completely abolished the release. Although ionomycin released glutamate, (+)-5-methyl-1-,11-dihydro-5H-dibenzo-[a,d]cycloheptan-5,10-imine (MK-801) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) showed no effects on the ionomycin-induced [3H]GABA release. Based on these results, it is concluded that NO formed by ionomycin plays a critical role in ionomycin-evoked [3H]GABA release from the neurons.     

Effects of Valeriana Officinalis Extracts on [3H]Flunitrazepam Binding,Synaptosomal [3H]GABA Uptake,and Hippocampal [3H]GABA Release

Extracts of Valeriana officinalis have been used in folkloric medicine for its sedative, hypnotic, tranquilizer and anticonvulsant effects, and may interact with -aminobutyric acid (GABA) and/or benzodiazepine sites. At low concentrations, valerian extracts enhance [³H]flunitrazepam binding (EC₅₀ 4.13 × 10^–10 mg/ml). However, this increased [³H]flunitrazepam binding is replaced by an inhibition at higher concentrations (IC₅₀ of 4.82 × 10^–1 mg/ml). These results are consistent with the presence of at least two different biological activities interacting with [³H]flunitrazepam binding sites. Valerian extracts also potentiate K⁺ or veratridine-stimulated release of radioactivity from hippocampal slices preloaded with [³H]GABA. Finally, inhibition of synaptosomal [³H]GABA uptake by valerian extracts also displays a biphasic interaction with guvacine. The results confirm that valerian extracts have effects on GABA_A receptors, but can also interact at other presynaptic components of GABAergic neurons.     

Uptake and metabolism ofl-[3H]glutamate andl-[3H]glutamine in adult rat cerebellar slices

Using very low concentrations (1 mumol range) of L-2-3-[3H]glutamate, (3H-Glu) or L-2-3-[3H]glutamine (3H-Gln), we have previously shown by autoradiography that these amino acids were preferentially taken up in the molecular layer of the cerebellar cortex. Furthermore, the accumulation of 3H-Glu was essentially glial in these conditions. We report here experiments in which uptake and metabolism of either (3H-Glu) or (3H-Gln) were studied in adult rat cerebellar slices. Both amino acids were rapidly converted into other metabolic compounds: after seven minutes of incubation in the presence of exogenous 3H-Glu, 70% of the tissue accumulated radioactivity was found to be in compounds other than glutamate. The main metabolites were Gln (42%), alpha-ketoglutarate (25%) and GABA (1,4%). In the presence of exogenous 3H-Gln the rate of metabolism was slightly slower (50% after seven minutes of incubation) and the metabolites were also Glu (29%), alpha-ketoglutarate (15%) and GABA (5%). Using depolarizing conditions (56 mM KCl) with either exogenous 3H-Glu or 3H-Gln, the radioactivity was preferentially accumulated in glutamate compared to control. From these results we conclude: i) there are two cellular compartments for the neurotransmission-glutamate-glutamine cycle; one is glial, the other neuronal; ii) these two cellular compartments contain both Gln and Glu; iii) transmitter glutamate is always in equilibrium with the so-called metabolic pool of glutamate; iv) the regulation of the glutamate-glutamine cycle occurs at least at two different levels: the uptake of glutamate and the enzymatic activity of the neuronal glutaminase.     

Kainate-enhanced release of D-[3H]aspartate from cerebral cortex and striatum: reversal by baclofen and pentobarbital

A study was made of the actions of the excitant neurotoxin, kainic acid, on the uptake and the release of D-[2,3-3H]aspartate (D-ASP) in slices of guinea pig cerebral neocortex and striatum. The slices took up D-ASP, reaching concentrations of the amino acid in the tissue which were 14-23 times that in the medium. Subsequently, electrical stimulation of the slices evoked a Ca2+-dependent release of a portion of the D-ASP. Kainic acid (10(-5)-10(-3) M) produced a dose-dependent inhibition of D-ASP uptake. The electrically evoked release of D-ASP was increased 1.6-2.0 fold by 10(-5) and 10(-4)M kainic acid. The kainate-enlarged release was Ca2+-dependent. Dihydrokainic acid, an analogue of kainic acid with little excitatory or toxic action, did not increase D-ASP release but depressed D-ASP uptake. Attempts were made to block the action of kainic acid with baclofen and pentobarbital, compounds which depress the electrically evoked release of L-glutamate (L-GLU) and L-aspartate (L-ASP). Baclofen (4 X 10(-6)M), an antispastic drug, and pentobarbital (10(-4)M), an anesthetic agent, each inhibited the electrically evoked release of D-ASP and prevented the enhancement of the release above control levels usually produced by 10(-4)M kainic acid. It is proposed that 10(-5) and 10(-4)M kainic acid may enhance the synaptic release of L-GLU and L-ASP from neurons which use these amino acids as transmitters. This action is prevented by baclofen and pentobarbital. In view of the possibility that cell death in Huntington's disease could involve excessive depolarization of striatal and other cells by glutamate, baclofen might be effective in delaying the loss of neurons associated with this condition.