Neurosecretory granules: evidence from an aging process within the neurohypophysis.

When cysteine labeled with sulfur-35 is injected into the third ventricle of the rat brain, it is first incorporated into only one of the two populations of neurosecretory granules that can be isolated on an isosmotic gradient. The second population of granules is labeled much later. Stimulation of hormone release from isolated labeled neural lobes and subsequent isolation of neurosecretory granules at different times after the injection of labeled cysteine shows that the radioactivity decreases in only one population of granules. One of the fractions of the gradient represents the granules found near the release site; the second population is probably located deeper in the nerve endings or in the nerve swellings. Whereas neurophysins are found in both populations, smaller proteins can only be detected in one. Thus it appears that neurosecretory granules undergo an aging process and that isosmotic gradients can separate the aged granules from those newly formed.

Binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase from bovine pancreas under pH conditions of maximum activity. Analysis by ultracentrifugation, fluorescence quenching and chemical modification.

The binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase (EC 6.1.1.2) from beef is examined by three approaches, under pH conditions of maximum activity (pH 8.0). (1) Analytical ultracentrifugation evidences the binding of a single mol of tRNATrp in a 2.5-10 microM concentration range. (2) tRNATrp quenches the fluorescence of the enzyme. The dependence of this fluorescence quenching on the tRNATrp concentration (0.1-4 microM) reflects also the binding of 1 mol of tRNA per mol of enzyme, with a Kd value of 0.19 +/- 0.02 microM. (3) tRNATrp protects the enzyme against derivatization by oxidized ATP. Out of the two fast-reacting lysine residues of the native enzyme, only one is prevented from reacting by tRNATrp in the 0.5-110 microM concentration range. This protection can be significantly analyzed only by assuming a one-to-one complex between the enzyme and tRNA. These results, obtained at pH 8.0 and 25 degrees C, are in contrast with the stoichiometry of 2 mol of tRNA to 1 mol of enzyme, previously observed at pH 6.0 and 4 degrees C.

Signal transduction by membrane receptors in viable electropermeabilized cells: isoproterenol-stimulated cyclic AMP synthesis in C6 glioma cells.

The activity of beta-adrenergic receptors at the plasma membrane level was investigated in viable, electropermeabilized C6 glioma cells. Electric field pulses were applied directly to the plated cells without any previous proteinase treatment. The affinity for isoproterenol and the density of the beta-adrenergic receptors, as judged from the number of [3H]CGP-12177 binding sites, were not affected by the electropermeabilization whereas the isoproterenol-stimulated cAMP accumulation was transiently impaired. This decrease in activity is due to an electropermeabilization-induced GTP leak. Normal activity could be obtained either by treating the cells by the electric field in a GTP-containing buffer, or by spontaneous recovery of the cells after the resealing of the plasma membrane, with a delay depending on the temperature. The activity of the receptors was not affected by the structural organization of the membrane associated to its electropermeabilization.

Primary structure of bovine liver tRNATrp.

Purified tRNATrp from bovine liver, accepting 1700 pmol tryptophan per A260nm unit, was completely digested with pancreatic ribonuclease and T1 ribonuclease. The sequences of the resulting oligonucleotides were determined and the primary structure of the tRNA was deduced. These analyses showed numerous incomplete post-transcriptional modifications, and several positions heterogenously occupied by two different nucleotides, which lead us to think that in bovine liver there exist a mixture of several tRNATrp.

A quenched-flow study of a receptor-triggered second messenger response: cyclic AMP burst elicited by isoproterenol in C6 glioma cell membranes.

Fast kinetic studies of cAMP accumulation in C6 cell membranes show a burst of cAMP after beta-adrenergic receptor stimulation by isoproterenol. This burst is no longer observed when the ATP present in membrane preparations is hydrolyzed, but can be restored by their preincubation in the presence of ATP-Mg. The size of the burst is much larger than the number of beta-adrenergic receptors and is of the same order of magnitude as the value reported for G proteins. Further characterization of the burst will allow studies of the functional interaction of receptor-adenylate cyclase components in C6 membranes.

Haloperidol-succinylglycyl[125I]iodotyrosine, a novel iodinated ligand for dopamine D2 receptors.

A novel radioiodinated ligand of the butyrophenone type has been synthesized for the quantification and characterization of dopamine D2 receptors. This haloperidol-derived ligand, haloperidol-succinylglycyl[125I]iodotyrosine ([125I]HSGTI), binds rapidly (equilibrium is reached within 30 min, at 10 pM and 37 degrees C) and with high affinity (Kd = 0.3 nM) to bovine striatal membranes. Its pharmacology, determined by competitive displacement with dopaminergic and non-dopaminergic drugs, is characteristic of binding to dopamine D2 receptors.

Comparative 31P and 1H NMR studies on rat astrocytes and C6 glioma cells in culture.

Rat astroglial cells in primary culture (95% enrichment) and C6 glioma cells were adapted to grow on microcarrier beads. In vivo 31P NMR spectra were collected from cell-covered beads perfused in the NMR tube. The NMR-visible phosphorylated metabolite contents of both cell types were determined using saturation factors calculated from the values of longitudinal relaxation times determined for C6 cells using progressive saturation experiments. On the other hand, the amounts of phosphorylated metabolites in cells were determined from proton decoupled 31P NMR spectra of cell perchloric acid extracts. The results indicate that the NTP and Pi contents of the normal and tumoral cells were similar, whereas the PCr level was higher in C6 cells and the NDP and phosphomonoester levels higher in astrocytes. The comparison of 1H NMR spectra of cell perchloric acid extracts evidenced larger inositol and alanine contents in C6 cells, whereas larger taurine and choline (and choline derivatives) contents were found in astrocytes. The Glu/Gln ratio was very different, 3.5 and 1 in C6 cells and astrocytes, respectively. In both cases, the more intense resonance in the 1H NMR spectrum was assigned to glycine. Based on the comparison of the metabolite content of a tumoral and a normal cell of glial origin, this work emphasizes the usefulness of a multinuclear NMR study in characterizing intrinsic differences between normal and tumoral cells.

Magnetic resonance spectroscopy and metabolism. Applications of proton and 13C NMR to the study of glutamate metabolism in cultured glial cells and human brain in vivo.

Nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of cells from the central nervous system both in vitro on perchloric acid extracts obtained either from cultured tumoral cells (C6 rat glioma) or rat astrocytes in primary culture, and in vivo within the human brain. Analysis of carbon 13 NMR spectra of perchloric acid extracts prepared from cultured cells in the presence of NMR [1-13C] glucose as substrate allowed determination of the glutamate and glutamine enrichments in both normal and tumoral cells. Preliminary results indicated large changes in the metabolism of these amino acids (and also of aspartate and alanine) in the C6 cell as compared to its normal counterpart. Localized proton NMR spectra of the human brain in vivo were obtained at 1.5 T, in order to evaluate the content of various metabolites, including glutamate, in peritumoral edema from a selected volume of 2 x 2 x 2 cm3. N-acetyl aspartate, glutamate, phosphocreatine, creatine, choline and inositol derivative resonances were observed in 15 min spectra. N-acetyl-aspartate was found to be at a lower level in contrast to glutamate which was detected at a higher level in the injured area as compared to the contralateral unaffected side.

Epitopes common to trypanosomes (T. cruzi, T. dionisii and T. vespertilionis (Schizotrypanum)): astrocytes and neurons.

An autoimmune mechanism is commonly invoked to explain the occurrence of neuronal destruction in the chronic phase of Chagas' disease. Monoclonal antibodies raised against T. dionisii (DION) and T. vespertilionis (VESP), and cross-reactive with T. cruzi recognize antigens in cultured cerebellar cells from embryonic and postnatal mice, as revealed by indirect immunofluorescence. Astrocytes (labelled with rabbit anti-GFAP antibody) showed positive reactions with DION 12.7, VESP 8.2 and VESP 9.3 while neurons (labelled with either tetanus toxin or anti-neuron-specific enolase antibody) reacted with the monoclonal antibody DION 10.1b. VESP 6.2 reacted with living cells of a subpopulation of neuronal or unidentifiable cell type. These cross-reactions may explain why not only neurons but also astrocytes may be involved in the autoimmune damage.

Beta-adrenergic receptors of cerebellar astrocytes in culture: intact cells versus membrane preparation.

Experiments were carried out to assess: the influence of culture conditions on the expression of beta-adrenergic receptors in intact glial cells from the central nervous system; and the extent to which quantitation of receptor sites in membrane preparations reflects the receptor population of the whole cells they are derived from. Cerebellar astrocytes were chosen for this study since essentially one receptor subtype, the beta 2 one, is present in adult cerebellum. Intact, attached cerebellar astrocytes exhibit only one class of binding sites for the beta-adrenergic antagonist, [3H]CGP 12177. Replating of the astrocytes after a few days of culture in vitro induces an up-regulation of the receptors. This effect is particularly important when astrocytes are maintained for 6 days in the presence of horse serum, a condition that favors cellular differentiation. Only 30-50% of the beta-adrenergic receptors of the intact cells can be detected on membrane preparations. When membranes are prepared from astrocytes grown either in the presence of horse serum or under chemically controlled medium (i.e. under differentiation promoting conditions) two classes of binding sites for [125I](-)-iodocyanopindolol are revealed. Several hypotheses, mainly related to the morphology of the cells, may provide an explanation for such differences. Studies of the pharmacological specificity of receptors of membrane fractions show that cerebellar astrocytes cultured in vitro exhibit both beta 1 and beta 2 receptor subtypes. The beta 1 subtype receptors are slightly more abundant when astrocytes are grown in fetal calf serum (FCS), a condition under which they exhibit a polygonal, poorly differentiated morphology. When culture conditions favor cellular differentiation, more receptors of the beta 2 subtype are seen, which can be related to what is observed in the adult in vivo where the astrocytes exhibit a differentiated morphology.

Differential expression of heat shock 70 proteins in primary cultures from rat cerebellum.

While a number of studies have described the heat shock response in established cell lines and in primary cultures of cells derived from the nervous system, there has been no systematic analysis comparing expression and localization of the inducible heat shock 70 (hsp70) proteins and the constitutively synthesized members of the family (hsc70) in neurons and glia. In the present communication, we utilized specific probes to compare the expression of hsp70 and hsc70 mRNAs and proteins in two types of primary cultures, astroglial and neuro-astroglial, from postnatal rat cerebellum. Conditions were adjusted to maintain physiological numbers of microglia in both types of culture, and cultures were analyzed at a number of different time points following a precisely defined heat shock. The northern, in situ hybridization and immunohistochemical analyses resulted in a number of novel observations concerning the nature of the heat shock response in these neuronal and glial cells. In postnatal day 4-5 cultures, hsp70 mRNA levels were elevated for at least 10 h in both types of culture, but in situ hybridization analysis showed no evidence for hsp70 mRNAs in neurons. Microglia were the only cell type in which hsp70 was detected in non-stressed cultures and this cell type contained the highest concentrations of hsp70 proteins in stressed cultures. Hsc70 mRNA levels were also increased after heat shock, but the increase was more transient. Hsc70 mRNAs and proteins were present in all cell types, again with the highest concentrations being present in microglia. Hsc70 mRNAs and proteins were localized in the cytoplasm at all time points examined, with hsc70 protein also being localized in nucleoli. Hsp70 mRNAs and proteins were diffusely localized over nuclei of astrocytes, as well as of most microglia. Hsp70, but not hsc70, was localized on chromosomes in glia once they had resumed cell division after heat shock, suggesting a role for hsp70 either in targeting damaged chromosomal proteins or in cell division. Some cytoplasmic hsp70 was observed in astrocytes of the mixed neuro-astroglial cultures and a delayed hsp70 immunoreactivity was observed in granule neurons in these cultures, suggesting either that translation of low levels of hsp70 mRNAs was more efficient in neurons, or that glial-neuronal translocation of hsp70 proteins had taken place. These results suggest that metabolism and functions of different heat shock protein family members may not always be identical and that care must be taken in extrapolation of results from one cell type to another.

Bovine serum albumin--haloperidol as a tool for the study of dopaminergic transmission: behavioural and neurochemical effects following a single injection in the nucleus accumbens.

Bovine serum albumin haloperidol (BSA-Hal) is a macromolecular complex with 14 molecules of haloperidol immobilized on the BSA protein backbone. This compound produces a selective, long lasting and reversible blockade of dopamine receptor activity. Its action was demonstrated by the ability to trigger a high rate of ipsilateral amphetamine-induced rotation up to 6 days after a single unilateral injection of the conjugate into the striatum. In the present study, the effect of the blockade of dopamine transmission in the nucleus accumbens (n.Acc) on spontaneous and learned behaviors was tested. The results indicate that the specific and long lasting blockade of dopamine receptors by bilateral injection of BSA-Hal in the n.Acc (1.6 micrograms/2 microliters) induced deficits in spontaneous alternation in a Y-maze on the 2nd and 5th days after the injection but not on the 11th day of the experiment, impaired acquisition but not retention in a radial 8-arm maze, increased latency to escape during learning in the place navigation task. These findings confirm the involvement of the n.Acc system in processes that have been generally attributed to the limbic system.

ADP and, indirectly, ATP are potent inhibitors of cAMP production in intact isoproterenol-stimulated C6 glioma cells.

When added to intact C6 glioma cells in the micromolar range of concentrations, ADP and ATP induce an inhibition of the isoproterenol-elicited cAMP responses. ATP is rapidly hydrolyzed by the ectonucleotidases present on these cells, with an apparent Km of 50 microM and a Vmax of 1.1 nmol/min/10(6) cells. cAMP responses are also inhibited by millimolar concentrations of either ATP in the presence of an ATP-regenerating system to prevent ADP accumulation or AMP-PCP. These observations show that, in C6 glioma cells, ADP is a more potent inhibitor of cAMP production than ATP, the latter acting indirectly, via its rapid hydrolysis to ADP. The additive inhibition of isoproterenol-elicited cAMP responses induced, on one hand, by the treatment of the cells with a phorbol ester and by addition of ADP to the cells, and, on the other hand, by the progressive disappearance of the effects of ADP and ATP when cells are treated with increasing concentrations of Pertussis toxin, demonstrate that ADP and ATP exert their action in C6 glioma cells via a P2 purinoceptor probably negatively coupled to adenylate cyclase and a G regulatory protein.

Reaction of tryptophanyl-tRNA synthetase from beef pancreas with periodate-oxidized ATP.

Tryptophanyl-tRNA synthetase from beef pancreas reacts with periodate-oxidized ATP according to biphasic kinetics. A rapid phase involves two groups of the protein, presumably lysine side-chains. The slow phase corresponds to the reaction of a larger number of groups. The time-course of the partial losses of the ATP-PPi isotopic exchange and of the aminoacylation activities of the enzyme follow the labelling of the two fast-reacting groups. However, the ability of the enzyme to form a bis(tryptophanyladenylate)-enzyme complex is not lost after reaction of these two groups with the reagent. The affinity for ATP is also unaffected by this initial labelling of the protein, as seen from the Km values of this substrate in the ATP-PPi isotopic exchange reaction. These data suggest that, in this fast initial reaction, oxidized ATP reacts neither with specific ATP-binding groups of the enzyme nor with any major catalytic residue of the tryptophan-activation site. In contrast with this first step, the further slow labelling of lysine residues leads to a disappearance of the aminoacylation ability of the enzyme, while it does not further affect the ATP-PPi exchange activity. The behaviour of beef tryptophanyl-tRNA synthetase during derivatization with oxidized ATP is therefore at variance with that which has been described for the homologous E. coli enzyme.

The conformational oscillation of delta-chymotrypsin involvement of methionine-192.

In delta-chymotrypsin the reactivity of methionine-192 towards p-nitrophenacyl bromide is strongly reduced when the alpha-amino group of isoleucine-16 has been acetylated. Since acetylation of isoleucine-16 brings delta-chymotrypsin to a conformation similar to its alkaline one this suggests that methionine-192 should present an impaired reactivity in the alkaline conformation of the protein. It is indeed observed that its chemical reactivity as a function of pH depends on the ionization state of the alpha-amino group of isoleucine-16 (pKapp 9 at 15 degrees C) as does the structure of the enzyme. Reciprocally, after chemical reaction of methionine-192 with hydrogen peroxide, isoleucine-16 presents a slower rate of reaction with fluorescamine than when methionine-192 is free. As a result of methionine-192 oxidation the apparent pK of the alkaline transition is shifted from 9 to about 11 at 15 degrees C. This is reflected in the disappearance of the lag phase previously observed for the initial activity of the enzyme when it is incubated at alkaline pH [Eur. J. Biochem. (1973) 39,293-300]. The absence of chemical reactivity of methionine-192 in the alkaline state of the enzyme is confirmed by the appearance of a lag phase in the reaction of the protein with iodoacetate after an incubation at alkaline pH. Such a lag phase does not appear when this incubation is carried out at neutral pH. Since this lag phase is similar to that which shows up in the activity during the isomerization of the enzyme from its alkaline to its neutral state, the present data are interpreted as implying a concerted movement of isoleucine-16 and methionine-192 during this isomerization process. They also indicate that in the alkaline form of the enzyme methionine-192 has moved back into the interior of the protein. Since the spectroscopic properties of the zymogen and of the high-pH form of the enzyme are similar they suggest that methionine-192 occupies in the alkaline conformation of the enzyme a similar position as it does in the zymogen.

Structure-activity relationships in tryptophanyl transfer ribonucleic acid synthetase from beef pancreas. Influence of the alkylation of the sulfhydryl groups on the dimer-monomer equilibrium.

Upon reaction with N-ethylmaleimide, tryptophanyl-tRNA synthetase from beef pancreas dissociates into subunits. At pH7, the rate of the dissociation is close to both the reaction rate of the buried--SH groups and the rate of inactivation (Iborra, F., Mourgeon, G., Labouesse B., and Labouesse, J. (1973) Eur. J. Biochem. 39, 547-556). The pH and enzyme concnetration dependences of the reaction rate of the 16 cysteinyl residues of the enzyme as well as that of its inactivation support the idea that inactivation by alkylation of the--SH groups is due essentially to the dissociation of the protein into inactive subunits and not to the chemical blocking of a catalytic residue. This is confirmed by the independence on N-ethylmaleimide concentration of the reaction of the buried--SH groups and of the inactivation of the enzyme at high N-ethylmaleimide concentration. The dissociation becomes in this case the rate-limiting step of the chemical reaction. The monomeric structure is stabilized by the blocking of the--SH groups exposed during the dissociation. The dissociation constant of the dimeric enzyme is progressively increased during the alkylation. The tightness of the associated structure depends on the protonation of groups titrating between pH 7 and pH 9.

Pre-steady state study of beta-adrenergic and purinergic receptor interaction in C6 cell membranes: undelayed balance between positive and negative coupling to adenylyl cyclase.

Interactions between beta-adrenergic and ADP purinergic receptors in C6 glioma cell membrane preparations were investigated under steady state and then pre-steady state conditions of adenylyl cyclase (EC 4.6.1.1) activity, in order to determine how fast the second receptor antagonizes the transduction mechanism of the first. Cell membranes were washed to deplete them as thoroughly as possible of low molecular weight compounds, especially ATP and ADP, and to ensure better control of both substrate and agonist nucleotide concentrations. ATP concentrations were kept constant with the use of an ATP-regenerating system; the C6 cell line exhibited very active ectonucleotidases. The purinergic agonist ADP was replaced by its nonhydrolyzable congener adenosine 5'-O-(2-thio)diphosphate (ADP beta S), which was demonstrated, like ADP, to inhibit isoproterenol-stimulated adenylyl cyclase activity in intact cells (IC50 for ADP, 0.5 +/- 0.1 microM; IC50 for ADP beta S, 25 +/- 2 microM) and in membrane preparations (IC50 for ADP beta S, 79 +/- 20 microM). In the case of membrane preparations, ADP beta S did not compete with ATP, the substrate of the cyclase-catalyzed reaction, and behaved apparently as a non-competitive inhibitor of the enzyme. The pre-steady state kinetics of isoproterenol-stimulated adenylyl cyclase activity measured with a pulsed quenched-flow apparatus have previously been shown to include two steps, the first very rapid (taking place within 1-2 sec) and giving rise to a burst of cAMP synthesis and the second much slower and corresponding to the steady state reaction. ADP beta S inhibited the occurrence of both steps with comparable IC50 values (mean value, 55 +/- 20 microM). In the presence of increasing concentrations of the purinergic receptor agonist, the time constant of the exponential burst reaction was not affected, but its amplitude progressively decreased to zero. These results showed that the extinction of the beta receptor cAMP response by the purinergic ADP receptor occurred within the dead-time of the pulsed quenched-flow apparatus, which was 50 msec. Such a rapid inhibition of cAMP production excluded modulation of isoproterenol-stimulated adenylyl cyclase activity by the ADP receptor by a pathway other than its direct negative coupling to the cyclase via a Gi protein. In this respect, the P2 purinergic ADP receptor of the C6 glioma cell line appears comparable to the P2t receptor of platelets.