A novel Toxoplasma gondii calcium-dependent protein kinase.

Toxoplasma gondii is an obligate intracellular parasite that infects all types of cells in humans. A family of calcium-dependent protein kinases (CDPKs), previously identified as important in the development of plants and protists, was recently shown to play a role in the infectivity of apicomplexans, and in motility and host cell invasion in particular. We report here the isolation of a new calcium-dependent protein kinase gene from the human toxoplasmosis parasite, Toxoplasma gondii. The gene consists of 12 exons. The encoded protein, TgCDPK4, consists of the four characteristic domains of members of the CDPK family and is most similar to PfCDPK2 from Plasmodium falciparum. We measured TgCDPK4 activity, induced by calcium influx, using a kinase assay. A calcium chelator (EGTA) inhibited this activity. These findings provide evidence of signal transduction involving members of the CDPK family in T. gondii.

Dopamine receptors and groups I and II mGluRs cooperate for long-term depression induction in rat prefrontal cortex through converging postsynaptic activation of MAP kinases.

Tetanic stimuli to layer I-II afferents in rat prefrontal cortex induced long-term depression (LTD) of layer I-II to layer V pyramidal neuron glutamatergic synapses when tetani were coupled to bath application of dopamine. This LTD was blocked by the following metabotropic glutamate receptor (mGluR) antagonists coapplied with dopamine: (S)-alpha-methyl-4-carboxyphenylglycine (MCPG; group I and II antagonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; group I antagonist), or (RS)-alpha-methylserine-O-phosphate monophenyl ester (MSOPPE; group II antagonist). This suggests that the dopamine-facilitated LTD requires synaptic activation of groups I and II mGluRs during tetanus. LTD could also be induced by coupling tetani to bath application of groups I and II mGluR agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). In the next series of experiments, coapplication of dopamine and 1S,3R-ACPD, but not application of either drug alone, consistently induced LTD without tetani or even single test stimuli during drug application, suggesting that coactivation of dopamine receptors and the mGluRs is sufficient for LTD induction. Immunoblot analyses with anti-active mitogen-activated protein kinases (MAP-Ks) revealed that D1 receptors, D2 receptors, group I mGluRs, and group II mGluRs all contribute to MAP-K activation in prefrontal cortex, and that combined activation of dopamine receptors and mGluRs synergistically or additively activate MAP-Ks. Consistently, LTD by dopamine + 1S, 3R-ACPD coapplication, as well as the two other forms of LTD (LTD by dopamine + tetani and LTD by 1S,3R-ACPD + tetani), was blocked by bath application of MAP-K kinase inhibitor PD98059. LTD by dopamine + 1S,3R-ACPD coapplication was also blocked by postsynaptic injection of synthetic MAP-K substrate peptide. Our results suggest that dopamine receptors and groups I and II mGluRs cooperate to induce LTD through converging postsynaptic activation of MAP-Ks.

Purification and reconstitution of the 32Pi-ATP exchange activity of bovine chromaffin granule membrane.

Ghosts derived from bovine chromaffin granules have a 32Pi-ATP exchange activity which is associated with the H+ pump of that membrane. This activity was low when compared to bacteria, chloroplasts or submitochondrial particles, but had similar properties (Km for ATP and Pi, ATP/Mg2+ ratio, pH profile, inhibition by dicyclohexylcarbodiimide and tributyltin) to the ATPase from above membranes. The 32Pi-ATP exchange activity was solubilized by cholate/octylglucoside mixtures. The soluble extract was lipid depleted by ammonium sulfate fractionation and partially purified by sucrose gradient centrifugation. The purified preparation was reconstituted with phospholipids by freeze-thawing. The reconstituted vesicles had a 32Pi-ATP exchange sensitive to dicyclohexylcarbodiimide and trybutyltin and an ATPase with a sensitivity to the inhibitors which varied with the reconstitution conditions. The alpha- and beta-subunits of F1-ATPase were major components of the preparation.

Nucleosidediphosphate kinase of Escherichia coli, a periplasmic enzyme.

The ATP-ADP exchange activity previously described in a membrane farction of Escherichia coli appeared after a cold osmotic shock according to Neu and Heppel ((1965) J. Biol. Chem. 240, 3685--3692) in the shock fluid. Membranes derived from shocked cells had no activity. The enzyme responsible for this activity has been purified 125-fold and catalyzed the transfer of a phosphoryl radical from ribonucleosidetriphosphates (NTPs) to ribonucleosidediphosphates (NDPs); this is, therefore, a non-specific nucleosidediphosphate kinase (ATP:nucleosidediphosphate phosphotransferase, EC 2.7.4.6). The activity required the presence of a divalent cation, Mg2+, Mn2+ or Ca2+ at a unity mol/mol ratio of nucleotide for maximal activation. The enzyme exhibited simple saturation kinetics with respect to the phosphate donor but inhibition by excess substrate was observed upon increasing phosphate acceptor. The kinetics of the reaction indicated an ordered bi-molecular ping-pong reaction mechanism. Differential heat sensitivity of the enzyme whether it is heated alone with ATP, ADP or Mg2+ opens possibilities to study different enzyme-substrate complexes.

Mutants of Escherichia coli K12 unable to grow on non-fermentable carbon substrates.

Among a number of mutants unable to utilize non-fermentable carbon substrates, scoring for membrane ATPase and for ATP-driven transhydrogenase activity permitted to distinguish two phenotypes: (A) mutants lacking ATPase and ATP-driven transhydrogenase; (B) one mutant with an ATPase which behaved according to several criteria as released into solution instead of being membrane bound, a.o it exhibited no ATP-driven transhydrogenase activity. All A and B mutants exhibited a common nutritional pattern. The ATPase-deficient group, when scored for ATPase-binding sites on its membrane particles revealed three different subgroups: (1) mutants having free ATPase-binding sites, (2) mutants with ATPase-binding sites made available by the procedure which releases ATPase from wild-type membrane, and (3) mutants with no detectable ATPase-binding sites. Membranes of the mutant B with unbound ATPase also exhibited a deficiency in ATPase-binding sites, but its soluble ATPase was also found unable to bind to ATPase-binding sites of wild type membranes. The double alteration, namely abnormal or inactive ATPase and absence of ATPase-binding sites on the membrane is compatible with a single mutational defect.

[Molecular pharmacology of the catecholamine transporter of chromaffin granules from the bovine adrenal medulla]. / Pharmacologie moléculaire du transporteur des catécholamines des granules chromaffines de la médullo-surrénale bovine.

Tetrabenazine (TBZ) and reserpine are two inhibitors of the catecholamine uptake system of the chromaffin granule membrane. They are structural analogs of the substrates dopamine and serotonin and they inhibit the monoamine transporter, which catalyzes a H+/neutral amine antiport. [3H]Dihydrotetrabenazine ([3H]TBZOH) is bound by chromaffin granule membranes on one class of site (T sites, KD = 3 nM); [3H]reserpine is bound on T sites and a second class of site (R1 sites, KD = 0.7 nM). The two sites are involved in monoamine translocation. The substrates displace the ligands with different efficiency: noradrenaline (Km = 10 microM) displaces reserpine efficiently (EC50 = 30 microM), but TBZOH poorly (EC50 = 2000 microM); m-iodobenzylguanidine, which has recently been shown to be a substrate of the monoamine uptake system (Km = 5 microM), displaces TBZOH efficiently (EC50 = 25 microM), but reserpine inefficiently (EC50 = 300 microM). Since both substrates are translocated by the same transporter, this result confirms the existence of two sites with different properties. T sites are characterized by a linear relationship between the reciprocal of the dissociation constants of various drugs displacing [3H]TBZOH and their partition coefficient in octanol/H2O mixtures. This relationship, which indicates a hydrophobic environment of T sites, does not exist for R1 sites. T sites have been identified by covalent labeling with a derivative of TBZ coupled to an arylazido group. The labeled sites are borne by a 65,000 dalton protein. The kinetics of reserpine binding are accelerated in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term potentiation in the hippocampus of the anaesthetized rat is not associated with a sustained enhanced release of endogenous excitatory amino acids.

The relationship between long-term potentiation of synaptic transmission and the release of endogenous glutamate and aspartate has been investigated in the CA1 region of the hippocampus and in the fascia dentata of the anaesthetized rat. A high-frequency train of electrical stimulation of afferent pathways produced a long lasting (greater than 2 h) enhancement of the field excitatory postsynaptic potential in CA1 and of the population spike in the fascia dentata. In both regions, this was not associated with a significant long lasting increase in the release of glutamate and aspartate. It is concluded that the maintenance of long-term potentiation is not associated with a sustained increase in the release of excitatory amino acids.

Enkephalins are associated with adrenergic granules in bovine adrenal medulla.

The subcellular localization of enkephalins was studied in the bovine adrenal medulla. In the adrenal medulla enkephalins (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg6-Phe7 and Met-enkephalin-Arg6-Gly7-Leu8) are found free and in the form of cryptic peptides included in larger precursors. Total Met-enkephalin immunoreactivity, which includes free and cryptic peptides, was determined after a sequential enzymatic treatment with trypsin and carboxypeptidase B. Total Met-enkephalin immunoreactivity, dopamine beta-hydroxylase and catecholamines were found to have a parallel distribution in the various subcellular fractions. The bulk of the total Met-enkephalin immunoreactivity (42%) was recovered in the large granule fraction. The large granule fraction also contained 38% of the total dopamine beta-hydroxylase activity, and 42% of the total catecholamines. Enkephalins are thus concentrated in the chromaffin granules. Chromaffin granules were also separated according to the method of Terland & coworkers into two fractions: one containing the dense noradrenergic vesicles and the other containing lighter adrenergic vesicles. Total Met-enkephalin immunoreactivity was restricted to the fractions containing the lighter adrenergic vesicles. In these fractions the molar ratio of adrenaline to total Met-enkephalin immunoreactivity was 97. This study is in accord with immunocytochemical observations which have indicated that enkephalins are located in adrenergic and not in the noradrenergic cells in the bovine adrenal medulla.

Long-term potentiation in the rat hippocampus induced by the mast cell degranulating peptide: analysis of the release of endogenous excitatory amino acids and proteins.

Using a push-pull device, we have analysed, in vivo, the release of endogenous excitatory amino acids and proteins induced by the mast cell degranulating peptide in the CA1 region of the hippocampus. Local application of the mast cell degranulating peptide (20 microM) for 5 or 10 min produced a long-term potentiation of the slope of the field excitatory postsynaptic potential (70 +/- 40%, 3 h after the drug application). This long-term potentiation was associated with (i) a transient increase (10 min) in the release of endogenous glutamate and aspartate and (ii) a late transient enhanced release of proteins and newly secreted proteins. In cases in which the mast cell degranulating peptide induced recurrent interictal activity, there was a sustained enhanced release of glutamate. These observations suggest that mast cell degranulating peptide induced long-term potentiation is not associated with a sustained enhanced release of excitatory amino acids.

Characterization of the monoamine uptake system in catecholamine storage vesicles isolated from a pheochromocytoma taken from a child.

The catecholamine storage vesicles of a pheochromocytoma taken from a child have been isolated and characterized. The tumor contained almost exclusively noradrenaline and a large proportion of this amine was vesicle-bound. The noradrenaline-containing vesicles showed great resemblance to bovine chromaffin granules. Their catecholamine and dopamine beta-hydroxylase contents were that of chromaffin granules; their morphology and density were similar to those of the subpopulation of these granules that contain noradrenaline. The pheochromocytoma vesicles contained in their membranes an abundant polypeptide of mol. wt 110,000, which was not apparent in bovine adrenal medulla vesicle membranes. Monoamine uptake by pheochromocytoma noradrenaline vesicles did not differ significantly from that observed in bovine chromaffin granules. The time-course, plateau level and KM for noradrenaline were similar for both types of organelles. Both had an oligomycin-resistant ATPase with similar properties. Investigations using the tetrabenazine derivative [2-3H]dihydrotetrabenazine (2-hydroxy-3-isobutyl-9,10-dimethoxy-1,2,3,4,6, 7-hexahydro-11b-H-benzo[a]quinolizine), which binds specially to the bovine chromaffin granule monoamine carrier indicated that granule membranes from the tumor have a 10-fold increased number of [2-3H]dihydrotetrabenazine binding sites, with no change in dissociation constant. As in the case of bovine chromaffin granules, [2-3H]dihydrotetrabenazine can be totally displaced by noradrenaline and serotonin. To account for the discrepancy observed between the uptake data (which indicated no difference with bovine chromaffin granules) and the [2-3H]dihydrotetrabenazine binding studies (which showed a large excess of binding sites in the tumor membranes), we propose that granules in the investigated tumor contained a large amount of inactive monoamine carrier.

Differential expression of PKC isoforms in hippocampal neuronal cultures: modifications after basic FGF treatment.

Protein kinase C (PKC) is present in high concentrations in neuronal tissues and participates in various neuronal functions. Ten isoforms have so far been identified and each PKC isoform may be activated by a variety of stimuli. By immunoblot analysis the presence of PKC isoforms was examined in dissociated cell cultures of the hippocampus and during the development in vivo. As soon as embryonic day 17 the hippocampus contains detectable amounts of PKC epsilon and zeta and low levels of PKC alpha. PKC beta and gamma appear during the first and second post-natal week. All isoforms progressively increase until the adult age. Cultures of hippocampal neurons derived from rat embryos express PKC alpha, epsilon and zeta whereas PKC beta and gamma are undetectable; a distinct pattern is observed in cultures of hippocampal glial cells. The neuronal levels of PKC alpha, epsilon and zeta increase during the period in culture and are enhanced when hippocampal neurons are exposed to the continuous presence of basic fibroblast growth factor. Immunofluorescence of PKC epsilon and zeta occupies all the cytoplasmic neuronal compartment. The early expression of some PKC isoforms in cultures of post-mitotic hippocampal neurons suggest their involvement in morphological events that occur during this period; in particular the neuritic outgrowth.

A fraction enriched in rat hippocampal mossy fibre synaptosomes contains trophic activities.

Subcellular fractions prepared from the rat hippocampus, were assessed for the presence of trophic activities. The cytosol of synaptosomal fractions induced mitotic reinitiation of confluent 3T3 fibroblasts. The synaptosomal fraction, enriched in mossy fibre terminals, contained the highest mitotic activity. The mitogenic activity was heat and trypsin sensitive, suggesting that polypeptides are involved. The cytosol of the mossy fibre synaptosomal fraction promoted neuritic outgrowth of PC 12 cells and embryonic hippocampal neurones in primary cultures. These results suggest that mossy fibres contain both mitogenic and neurotrophic activities. These factors could participate in mossy fibre sprouting that occur following brief seizures or experimental lesions.

A new method for the measurement of endogenous transmitter release in localized regions of hippocampal slices.

We describe here a method that allows measurement of the release of endogenous amino acids from localized regions of brain slices combined with conventional electrophysiological experiments. Hippocampal slices were placed in fully submerged chambers and a cannula was positioned just above the dendritic layers of CA1. The cannula was connected to a peristaltic pump and the content of amino acids in the perfusate was measured by HPLC. Extracellular field potentials were concomitantly recorded. Stable levels of aspartate and glutamate were found above the stratum radiatum of CA1. No detectable release was found when the cannula was located above the alveus, the fimbria or in the effluent of the slice. A pulse of K+ (50 mM) produced a brief 3-fold increase in glutamate, aspartate and a detectable release of GABA in CA1. Brief high frequency trains (10 Hz) also increased significantly the release. This method will be useful in determining alterations in transmitter release in the slice in relation to anoxia, epilepsy and long term potentiation.

Identification and cellular localization of protein kinase C isoforms in cultures of rat type-1 astrocytes.

We have examined which isoforms of protein kinase C were present in rat brain astrocytes and found that: (1) the total of calcium-independent isoforms was greater than the total of calcium-dependent isoforms; (2) there were differences in the intracellular distribution of different isoforms; and (3) the abundance of total protein kinase C was greater in astrocytes from cortex than astrocytes from diencephalon.

Implication of protein kinase C in mechanisms of potassium-induced long-term potentiation in rat hippocampal slices.

The involvement of Ca2+/phospholipid-dependent (alpha, beta, gamma, PKCs) and Ca(2+)-independent PKC (epsilon and zeta isoforms) in mechanisms of long-term potentiation was investigated in CA1 hippocampal slices, using a brief high potassium pulse (50 mM, 40 s) to induce long-term potentiation (K+/LTP). The K+ pulse induced first, in 15 s a translocation of PKC activity to the membrane. This was rapidly followed, from 1 to 60 min after the pulse, by a selective activation of PKC in the cytosol. This activation, which could be blocked by the NMDA (N-methyl-D-aspartate) receptor antagonist 2-amino-5-phosphonovalerate (APV), was associated with a significant increase n immunoreactivity for gamma PKC in he cytosol, and also to a less degree for beta PKC. In contrast, application of the phorbol ester PMA (phorbol 12-mirystate 13 acetate) to other slices induced a rapid and persistent translocation to the membrane of alpha, beta, epsilon and zeta PKCs. A major role for the activation role for the activation of cytosolic gamma PKC in the maintenance of LTP is discussed.

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: evoked release of glutamate, GABA, aspartate and glutamate decarboxylase activity in control and degranulated rat hippocampus.

Using discontinuous density gradient centrifugation in isotonic Percoll sucrose, we have characterized two subcellular fractions (PII and PIII) enriched in mossy fiber synaptosomes and two others (SII and SIII) enriched in small synaptosomes. These synaptosomal fractions were compared with those obtained from adult hippocampus irradiated at neonatal stage to destroy granule cells and their mossy fibers. Synaptosomes were viable as judged by their ability to release aspartate, glutamate and GABA upon K+ depolarization. After irradiation, compared to the control values, the release of glutamate and GABA was decreased by 57 and 74% in the PIII fraction, but not in the other fractions and the content of glutamate, aspartate and GABA was also decreased in PIII fraction by 62, 44 and 52% respectively. These results suggest that mossy fiber (MF) synaptosomes contain and release glutamate and GABA. Measurement of the GABA synthesizing enzyme, glutamate decarboxylase, exhibited no significant difference after irradiation, suggesting that GABA is not synthesized by this enzyme in mossy fibers.

Two binding sites for [3H]glibenclamide in the rat brain.

The binding of [3H]glibenclamide, a potent sulfonylurea which blocks ATP-sensitive potassium channels, was studied in the rat brain. A Scatchard plot of saturation isotherms suggests that [3H]glibenclamide binds in various brain regions to a high- and a low-affinity binding site (Kd values of 0.21 nM and 111 nM and Bmax values of 41 and 1060 fmol/mg protein, respectively). Competitive binding assays with various unlabelled sulfonylureas showed biphasic displacements of [3H]glibenclamide with pseudo-Hill coefficients significantly different from unity. These data indicate the existence of a heterogeneity of binding sites to [3H]glibenclamide in the rat brain; this may correlate with the variability of effects of sulfonylureas observed from physiological experiments.

Transient increased density of NMDA binding sites in the developing rat hippocampus.

Using quantitative autoradiography and membrane preparations, the density of specific glutamate and N-methyl-D-aspartic acid (NMDA) binding sites have been determined in the developing rat hippocampus. We found an abrupt reduction in the density of NMDA binding sites after P8 (postnatal day) without change in affinity. The transient expression of NMDA receptors during maturation suggests that they may play a particularly important role in synaptogenesis.

Galanin reduces release of endogenous excitatory amino acids in the rat hippocampus.

Galanin is a 29-amino acid peptide widely distributed in the central nervous system where it modulates the release of several neurotransmitters and neurohormones. Because previous data had postulated that galanin could inhibit the release of excitatory amino acids and protect hippocampal neurons against anoxia, we have investigated the effect of galanin on the release of endogenous glutamate and aspartate evoked by potassium depolarization in rat hippocampal slices. Galanin, added to a concentration of 0.3 microM, produced a 50-60% reduction in the release of endogenous glutamate and aspartate as evoked by 40 mM K+. This effect was concentration-dependent with half-maximal effective galanin concentrations (EC50) of 1.7 and 5.9 nM for glutamate and aspartate, respectively. Such an effect was found to occur preferentially in the ventral rather than in the dorsal region of the hippocampus. The inhibitory effect of galanin on the K(+)-evoked release of excitatory amino acids was reversed by the specific galanin antagonist M-15 (0.3 microM), and by the ATP-sensitive potassium channel blocker glibenclamide (10 microM). Furthermore, M-15 alone increased the basal and the K(+)-evoked release of glutamate and aspartate from hippocampal slices. It is concluded that galanin exerts a tonic inhibition of excitatory glutamate/aspartate neurotransmission in the rat ventral hippocampus. The efficacy of glibenclamide in antagonizing the effect of galanin suggests the involvement of ATP-sensitive or -insensitive potassium channels in such a regulation.

Effect of potassium channel modulators on the release of glutamate induced by ischaemic-like conditions in rat hippocampal slices.

The effects of the potassium channel openers lemakalim, RP 52891 and galanin and the potassium channel blockers glibenclamide and gliquidone were evaluated by the release of endogenous glutamate from rat hippocampal slices subjected to a brief period of ischaemia (2-10 min). Ischaemia was mimicked by incubating slices in a glucose free medium equilibrated with 95% N2/5% CO2. These conditions evoked a release of glutamate which was insensitive to tetrodotoxin and Ca2+ indicating a non-vesicular origin. The release of glutamate evoked by a 6- or 8-min period of ischaemia was reduced by 25-40% in the presence of lemakalim (10 microM), RP 52891 (10 microM) or galanin (0.3 microM), whereas it was enhanced by 60 to 100% in the presence of glibenclamide (1 microM) and gliquidone (2 microM). These observations suggest that cellular damage resulting from ischaemia induced excessive release of glutamate in the hippocampus may be partly reduced by potassium channel openers, and conversely increased by sulfonylureas.