Absence Of Magnesium Research Articles

Cloning, expression and characterization of two putative calcium-binding sites in human non-erythroid alpha-spectrin.

The C-terminus of alpha-spectrins contains two putative calcium-binding sites or EF-hands. To characterize the binding, we have isolated clones from a human fetal liver cDNA library and expressed several fragments comprising either one or both of these sites. When the isolated clones were sequenced, we found that three consecutive nucleotides differed compared to the published sequence. The discrepancy affected two codons in the first of the two putative calcium sites. These codons translated into glutamate and phenylalanine, which are identical to the residues present at the same position in other alpha-spectrins. In the presence of magnesium, only recombinant peptides comprising the second putative site bound calcium as determined by a calcium overlay assay. Although the first putative EF-hand appeared to bind some calcium in the absence of magnesium, no binding could be detected under stringent conditions. Therefore, it is likely that the second EF-hand constitutes the only functional calcium-binding site in the C-terminus of human non-erythroid alpha-spectrin. Since peptides comprising the second EF-hand bound calcium nearly as well as intact spectrin, it is also apparent that the second EF-hand constitutes the major binding site for calcium in spectrin. The relative change in negative ellipticity, induced by the binding of calcium, indicates a dissociation constant of approximately 120 microM.

A pivotal role for the structure of the Holliday junction in DNA branch migration.

Branch migration of a DNA Holliday junction is a key step in genetic recombination that affects the extent of transfer of genetic information between homologous DNA sequences. We previously observed that the rate of spontaneous branch migration is exceedingly sensitive to metal ions and postulated that the structure of the cross-over point might be one critical determinant of the rate of branch migration. Other investigators have shown that in the presence of divalent metal ions like magnesium, the Holliday junction assumes a folded conformation in which base stacking is retained through the cross-over point. This base stacking is disrupted in the absence of magnesium. Here we measure the rate of branch migration as a function of Mg2+ concentration. The rate of branch migration increases dramatically at MgCl2 concentrations below 500 microM, with the steepest acceleration occurring between 300 and 100 microM MgCl2. This increase in the rate of branch migration coincides with the loss of base stacking in the four-way junction over this same interval of magnesium concentration, as measured by the susceptibility of junction residues to modification by osmium tetroxide and diethyl pyrocarbonate. We conclude that at physiological concentrations of intracellular Mg2+, base stacking in the Holliday junction constitutes one kinetic barrier to branch migration and that disruption of base stacking at the cross-over relieves this constraint.

Rapid adaptation of cardiac ryanodine receptors: modulation by Mg2+ and phosphorylation.

Channel adaptation is a fundamental feature of sarcoplasmic reticulum calcium release channels (called ryanodine receptors, RyRs). It permits successive increases in the intracellular concentration of calcium (Ca2+) to repeatedly but transiently activate channels. Adaptation of RyRs in the absence of magnesium (Mg2+) and adenosine triphosphate is an extremely slow process (taking seconds). Photorelease of Ca2+ from nitrophenyl-EGTA, a photolabile Ca2+ chelator, demonstrated that RyR adaptation is rapid (milliseconds) in canine heart muscle when physiological Mg2+ concentrations are present. Phosphorylation of the RyR by protein kinase A increased the responsiveness of the channel to Ca2+ and accelerated the kinetics of adaptation. These properties of the RyR from heart may also be relevant to other cells in which multiple agonist-dependent triggering events regulate cellular functions.

Sequence-specific DNA Recognition by the SmaI Endonuclease

SmaI endonuclease recognizes and cleaves the sequence CCC decreases GGG. The enzyme requires magnesium for catalysis; however, equilibrium binding assays revealed that the enzyme binds specifically to DNA in the absence of magnesium. A specific association constant of 0.9 x 10(8) M-1 was determined for SmaI binding to a 22-base duplex oligonucleotide. Furthermore, the KA was a function of the length of the DNA substrate and the enzyme exhibited an affinity of 1.2 x 10(9) M-1 for a 195-base pair fragment and which represented a 10(4)-fold increase in affinity over binding to nonspecific sequences. A Km of 17.5 nM was estimated from kinetic assays based on cleavage of the 22-base oligonucleotide and is not significantly different from the KD estimated from the thermodynamic analyses. Footprinting (dimethyl sulfate and missing nucleoside) analyses revealed that SmaI interacts with each of the base pairs within the recognition sequence. Ethylation interference assays suggested that the protein contacts three adjacent phosphates on each strand of the recognition sequence. Significantly, a predicted protein contact with the phosphate 3' of the scissile bond may have implications in the mechanism of catalysis by SmaI.

Proton Exchange and Basepair Kinetics of Yeast tRNAPhe and tRNAASP1

Nuclear magnetic resonance measurements of proton exchange were performed on yeast tRNA(Phe) and yeast tRNA(Asp), at temperatures from 20 to 45 degrees C, in the presence of various levels of salt, phosphate, added magnesium, and pH. The dynamical changes of the tRNA molecule were interpreted, with the aid of firmly established assignments and the use of the saturation recovery technique. In tRNA(Phe), the exchange rates in zero magnesium indicated early melting of the acceptor stem, tertiary structure, and D stem. However, in the presence of even low levels of magnesium the D stem remained intact up to high temperature, stabilized by a Mg2+ ion. A similar unfolding sequence was observed in tRNA(Asp). The difference between these two tRNAs was the thermal behavior of the tertiary resonance U8-A14. In tRNA(Phe), this base pair showed sharp rate increases between 32 and 39 degrees C. However, in tRNA(Asp), it remained intact up to 36 degrees C and disappeared at 39 degrees C, even if there was not important kinetic broadening. By measuring the temperature dependence of the exchange rates, we obtained an activation energy of 40-60 kcal/mol for all the imino protons of yeast tRNA(Phe) in zero magnesium. The same activation energy was obtained for tRNA(Phe) with equimolar concentration of magnesium. By investigation of the dependence of the exchange rates of these imino protons on solution conditions, we observed the transition from kop rate limiting in the absence of magnesium to kex rate limiting in the presence of magnesium.

Melatonin protects primary cultures of cerebellar granule neurons from kainate but not from N-methyl- d-aspartate excitotoxicity

The antiexcitotoxic efficacy of melatonin, a putative endogenous hydroxyl radical scavenger, was studied in primary cultures of rat cerebellar granule neurons. Excitotoxicity was induced in 7- to 9-day-old cultures by an exposure to glutamate (15 min in the absence of magnesium) or to glutamate receptor agonists, kainate (30 min), and N-methyl- d-aspartate (60 min in the absence of magnesium). Thereafter, cultures were returned to the culture-conditioned medium for 18 h at the end of which time viability was assessed by quantitative staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. Cotreatment with melatonin (500 μ M protected the neurons completely from the toxicity of kainate (up to 1 m M) and shifted the ED 50 for glutamate from 55 ± 2.6 to 97 ± 3.6 μ M. Melatonin cotreatment was ineffective in protecting the neurons from N-methyl- d-aspartate toxicity. When melatonin was added to the cultures only before or after kainate treatment, there was no resultant protection from kainate toxicity. The neuroprotective effect of melatonin does not appear to be related to the direct action of melatonin on ionotropic glutamate receptors. That is, the kainate-stimulated inward currents measured by a patch-clamp technique in voltage clamped neurons and the kainate-stimulated increase in free cytosolic calcium measured at the single-cell level using digital imaging fluorescent microscopy with fura-2 were not affected by melatonin. Moreover, the binding of [ 3H]glutamate to rat cerebellar membranes was not competed off by melatonin. Further studies are needed to evaluate the pharmacologic relevance of the neuroprotective action of melatonin.

Pharmacological heterogeneity of NMDA receptors in cerebellar granule cells in immature rat slices. A microfluorimetric study with the [Ca 2+] i sensitive dye indo-1

We have developed a technique for monitoring the internal calcium concentration—[Ca 2+] i— in a single selected cell in cerebellar slices of 8-day-old rats. In post-migratory granule cells NMDA induced a rapid, reversible and concentration-dependent increase in [Ca 2+] i (+ 22% at 10 μM and +210% at 100 μM). This effect was dependent on the presence of Ca 2+ in the extracellular medium. When Mg 2+ was absent from the perfusion buffer the sensitivity to NMDA was greatly increased (+108% at 10 μM). The NMDA response was not affected by glycine site agonists or by tetrodotoxin, a sodium channel blocker. In the absence of magnesium, the NMDA (10 μM)-induced increase in [Ca 2+] i was prevented in a monophasic manner by the recognition site antagonist 2-amino-phosphonovalerate (2-APV; IC 50 =13 μM), and in a biphasic manner by the glycine site antagonist 7-chlorokynurenate (IC 50s = 25nM and 5.9 μM) and by the channel blocker dizocilpine (IC 50s = 5 nM and 3 μM). In contrast, this NMDA response was only partially antagonized by the polyamine site antagonists ifenprodil and eliprodil (maximal inhibition ~50% at concentrations ⩾ 10 μM, IC 50 1 and 2 μM, respectively). These results demonstrate the presence in granule cells from immature rat cerebellum of at least two NMDA receptor populations which can be differentiated by their sensitivity to inhibitors acting upon the different sites of the NMDA receptor complex.

DNA determinants in sequence-specific recognition by XmaI endonuclease.

The XmaI endonuclease recognizes and cleaves the sequence C decreases CCGGG. Magnesium is required for catalysis, however, the enzyme forms stable, specific complexes with DNA in the absence of magnesium. An association constant of 1.2 x 10(9)/M was estimated for the affinity of the enzyme for a specific 195 bp fragment. Competition assays revealed that the site-specific association constant represented an approximately 10(4)-fold increase in affinity over that for non-cognate sites. Missing nucleoside analyses suggested an interaction of the enzyme with each of the cytosines and guanines within the recognition site. Recognition of each of the guanines was also indicated by dimethylsulfate interference footprinting assays. The phosphates 5' to the guanines within the recognition site appeared to be the major sites of interaction of XmaI with the sugar-phosphate backbone. No significant interaction of the protein was observed with phosphates flanking the recognition sequence. Comparison of the footprinting patterns of XmaI with those of the neoschizomer SmaI (CCC decreases GGG) revealed that the two enzymes utilize the same DNA determinants in their specific interaction with the CCCGGG recognition site.

Structure-mapping of the Hairpin Ribozyme: Magnesium-dependent Folding and Evidence for Tertiary Interactions within the Ribozyme-Substrate Complex

We have used chemical modification analysis to probe the solution structure of the hairpin ribozyme. The modifiying reagents dimethylsulfate, 1-cyclohexyl- N′-[2-( N-methylmorpholino) ethyl-carbodiimide- p-toluenesulfonate, kethoxal, diethylpyrocarbonate and (2, 12-dimethyl-3,7,11,17,-tetraazabicyclo [11.3.1.] heptadeca-1(17),2,11,13,15-pentaenato) nickel(II) perchlorate were used to probe functional groups that participate in Watson-Crick and non-canonical base-pairs. Our results confirm the existence of four short helices (3 to 6 bp) within the ribozyme-substrate complex, and demonstrate that one intramolecular helix (helix 4) is comprised of three base-pairs rather than the previously suggested five. In the absence of magnesium, the ribozyme is observed to fold into its secondary structure. Upon additional of magnesium, a striking difference in chemical modification is observed, particularly at sites within the ribozyme's large internal loop (loop B) that are essential for catalytic function (bases 21 to 26). Moreover, magnesium-dependent folding clearly destabilizes an A-U base-pair in a region where a proposed bend is required to juxtapose the catalytically essential loop A and B. Upon addition of substrate, no changes are observed in the structure of helix 3, loop B or helix 4. However, strong protection of bases in the substrate-binding domain is observed, including those located across internal loop A. The modification data are consistent with the formation of a previously proposed tertiary structure motif within loop B that includes non-canonical G-A, A-U and A-A base-pairs, and that is identical with those identified by NMR analysis of loop E of 5 S rRNA and the sarcin/ricin loop of 28 S rRNA. Our results indicate that the hairpin ribozyme adopts a stable magnesium-dependent tertiary structure to which the substrate binds without including major conformational changes, and that substrate recognition is likely to involve non-canonical base-pairs between the ribozyme and substrate sequences adjacent to the cleavage site.

Cooperativity and stoichiometry of substrate binding to the catalytic sites of Escherichia coli F1-ATPase. Effects of magnesium, inhibitors, and mutation.

The fluorescence of residue Trp beta 331 in beta Y331W mutant Escherichia coli F1-ATPase was used as reporter probe to investigate the effects of magnesium ions, inhibitors, and mutation on substrate (ATP) binding stoichiometry and cooperativity. It was found that Mg2+ is required for catalytic site binding cooperativity. In the absence of magnesium, ATP bound to three independent catalytic sites, each with Kd = 76 microM. In contrast, MgATP bound to three catalytic sites with Kd1 < 50 nM, Kd2 = 0.5 microM, and Kd3 = 25 microM. There was no significant ATPase activity in the absence of Mg2+. Catalysis is therefore correlated with substrate binding cooperativity and the formation of the high-affinity catalytic site 1. Catalytic site 3 had properties similar to those of the isolated beta-subunit nucleotide-binding site. The inhibitors dicyclohexylcarbodiimide and N-ethylmaleimide (in alpha S373C/beta Y331W mutant F1) gave potent inhibition of multisite ATPase activity without significantly affecting MgATP binding stoichiometry or cooperativity. Therefore each seems to selectively attenuate positive catalytic cooperativity. The same conclusions held for the alpha S373F mutation (in alpha S373F/beta Y331W mutant F1). 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole, however, reduced the catalytic site MgATP binding stoichiometry from three to two, and appears to inhibit catalysis by sterically blocking catalytic site 3.

Presynaptic control of dopamine synthesis and release by excitatory amino acids in rat striatal synaptosomes

Purified striatal synaptosomes were continuously superfused with l,3,5[3H]tyrosine in order to estimate the synthesis ([3H]water) and release of newly formed [3H]dopamine. In the presence of magnesium, l-glutamate, d,l-α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) and kainate, but not N-methyl-d-aspartate (NMDA) and 1-aminocyclopentane-1S,3R-dicarboxylate (t-ACPD), stimulated the release of [3H]dopamine, in a dose-dependent manner. When magnesium was omitted or in the presence of AMPA, NMDA also increased the release of [3H]dopamine. The effects of AMPA and kainate were competitively inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 6,7-dinitro-quinoxaline-2,3-dione (DNQX), whereas those of NMDA were reduced by 2-amino-5-phosphonovalerate (APV) or (+)-5-methyl-10,11-dihydro-5-H-dibenzo(a,d)cyclo-hepten-5,10-imine maleate (MK801). The stimulation of [3H]dopamine release by a high concentration of glutamate resulted from the concomitant activation of AMPA and NMDA receptors since this effect was potentiated by glycine and reduced by 2-amino-5-phosphonovalerate or MK801. This reduction was almost complete in the combined presence of DNQX and MK801. Surprisingly, glutamate and NMDA (in the absence of magnesium) reduced the efflux of [3H]water. The reduction of [3H]dopamine synthesis was blocked by 2-amino-5-phosphonovalerate indicating the involvement of NMDA receptors. Neither AMPA nor kainate affected dopamine synthesis. The inhibition of [3H]dopamine synthesis resulting from the stimulation of NMDA receptors was prevented when synaptosomes were continuously superfused with adenosine deaminase and quinpirole, a combined treatment known to markedly reduce the phosphorylation of tyrosine hydroxylase by cAMP-dependent protein kinase. The opposite effects of a high concentration of glutamate on [3H]dopamine synthesis and release were mimicked by ionomycin. As a working hypothesis, it is proposed that the NMDA-triggered calcium influx could lead to a reduction of tyrosine hydroxylase phosphorylation, possibly through an activation of calcineurin.

NMDA and carbachol but not AMPA affect differently the release of [3H]GABA in striosome- and matrix-enriched areas of the rat striatum

The effects of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 10−3 M),N-methyl-d-aspartate (10−3 M, in the absence of magnesium or presence of AMPA) and carbachol (10−3 M) on the release of preloaded [3H]γ-aminobutyric acid ([3H]GABA) from microdiscs of tissue punched out from sagittal brain slices in striosome- or matrix-enriched areas of the rat striatum have been compared. Although AMPA stimulated similarly the release of [3H]GABA in both striatal compartments, the release of [3H]GABA evoked by eitherN-methyl-d-aspartate (in the presence of AMPA) or carbachol was more pronounced in matrix- than in striosome-enriched areas. AMPA- andN-methyl-d-aspartate- (in the absence of magnesium) evoked responses were reduced but not abolished in the presence of tetrodotoxin (10−6 M) in both compartments while the carbachol-evoked release of [3H]GABA was decreased by tetrodotoxin only in the matrix. The interruption of cholinergic transmission by the combined application of atropine (10−5 M) and pempidine (10−4 M) was without effect on the AMPA-evoked release of [3H]GABA, but it reduced theN-methyl-d-aspartate- (in the absence of magnesium or presence of AMPA) evoked release of [3H]GABA in both compartments, these reductions being of similar amplitude than those observed with tetrodotoxin.

Mitochondrial adenosine triphosphatase from human placenta--inhibition by free magnesium ions of ITP hydrolysis.

The effects of Mg2+ and bicarbonate on the kinetics of ITP hydrolysis by soluble ATPase (F1) from human placental mitochondria were studied. Increasing amounts of Mg2+ at fixed ITP concentration, caused a marked activation of F1 followed by inhibition at higher Mg2+ concentration. The appropriate substrate for the mitochondrial F1 seems to be the MgITP complex as almost no ITP was hydrolysed in the absence of magnesium. Mg2+ behaved as a competitive inhibitor towards the MgITP complex. In this respect the human placental enzyme differ from that from other sources such as yeast, beef liver or rat liver. The linearity of the plot presenting competitive inhibition by free Mg2+ of MgITP hydrolysis (in the presence of activating bicarbonate anion) suggests that both Mg2+ and MgITP bind to the same catalytic site (Km(MgITP) = 0.46 mM, Ki(Mg) = 4 mM). When bicarbonate was absent in the ITPase assay, placental F1 exhibited apparent negative cooperativity in the presence of 5 mM Mg2+, just as it did with MgATP as a substrate under similar conditions. Bicarbonate ions eliminated the negative cooperativity with respect to ITP (as the Hill coefficient of 0.46 was brought to approx. 1), and thus limited inhibition by free Mg2+. The results presented suggest that the concentration of free magnesium ions may be an important regulatory factor of the human placental F1 activity.

The kinetics of spontaneous DNA branch migration.

An important step in genetic recombination is DNA branch migration, the movement of the Holliday junction or exchange point between two homologous duplex DNAs. We have determined kinetic parameters of spontaneous branch migration as a function of temperature and ionic conditions. The branch migration substrates consist of two homologous duplex DNAs each having two single-strand tails at one end that are complementary to the corresponding single-strand tails of the other duplex. Upon rapid annealing of the two duplex DNAs, a four-stranded intermediate is formed that has a Holliday junction at one end of the duplexes. Branch migration to the opposite end of the duplexes results in complete strand exchange and formation of two duplex products. The rate of branch migration is exceedingly sensitive to the type of metal ions present. In magnesium, branch migration is quite slow with a step time, tau, equal to 300 msec at 37 degrees C. Surprisingly, branch migration in the absence of magnesium was 1000 times faster. Despite this difference in rates, apparent activation energies for the branch migration step in the presence and absence of magnesium are similar. Since metal ions have a profound effect on the structure of the Holliday junction, it appears that the structure of the branch point plays a key role in determining the rate of spontaneous DNA branch migration. We discuss the role of proteins in promoting the branch migration step during homologous recombination.

Interaction of 4-azido-2-nitrophenyl phosphate, a pseudosubstrate, with the sarcoplasmic reticulum Ca-ATPase.

In the dark, 4-azido-2-nitrophenyl phosphate (ANPP) is a phosphate analog which behaves like a simple energy-rich phosphate donor for the sarcoplasmic reticulum (SR) Ca-ATPase. Like p-nitrophenyl phosphate (pNPP), ANPP is hydrolyzed by the enzyme only in the presence of calcium and magnesium (K0.5 and Vmax are 0.3 mM and 60 nmol mg-1 min-1, respectively). After photoirradiation in the absence of magnesium, SR Ca-ATPase is specifically labeled by [32P]ANPP in the presence and in the absence of calcium. The presence of nucleotide in the medium provides some protection against photolabeling but less than phosphorylation by inorganic phosphate. The maximal stoichiometry of covalently bound ANPP extrapolates to 0.8 mol/mol of Ca-ATPase in the absence of magnesium. Autoradiography of a sodium dodecyl sulfate-polyacrylamide gel, after controlled trypsin digestion of the photolabeled protein, reveals that the radioactivity is incorporated in the B subfragment. Three radioactive polypeptides with approximate molecular masses of 55, 25, and 6 kDa are obtained depending on the digestion conditions. N-Terminal sequence analysis of the 50- and 25-kDa peptides reveals the same sequence beginning at Ala-506, whereas two different sequence beginning at Ala-506 and Phe-584 are observed in the 6-kDa peptides.

Role of the Divalent Metal Ion in Sugar Binding, Ring Opening, and Isomerization by D-Xylose Isomerase: Replacement of a Catalytic Metal by an Amino Acid

The distinct roles of the two magnesium ions essential to the activity of D-xylose isomerase from Streptomyces olivochromogenes were examined. The enzyme-magnesium complex was isolated, and the stoichiometry of cation binding determined by neutron activation analysis to be 2 mol of magnesium per mole of enzyme. A plot of Mg2+ added versus Mg2+ bound to enzyme is consistent with apparent KD values of < or = 0.5-1.0 mM for one Mg2+ and < or = 2-5 mM for the second. A site-directed mutant of D-xylose isomerase was designed to remove the tighter, tetracoordinated magnesium binding site (site 1, Mg-1); Glu180 was replaced with Lys180. The stoichiometry of metal binding to this mutant, E180K, is 1 mol of magnesium per mole of enzyme. Ring-opening assays with 1-thioglucose (H2S released upon ring opening) show E180K catalyzes the opening of the sugar ring at 20% the rate of the wild-type, but E180K does not catalyze isomerization of glucose to fructose. Thus, the magnesium bound to Glu180 is essential for isomerization but not essential for ring opening. The X-ray crystallographic structures of E180K in the absence of magnesium and in the presence and absence of 250 mM glucose were obtained to 1.8-A resolution and refined to R factors of 17.7% and 19.7%, respectively. The wild-type and both E180K structures show no significant structural differences, except the epsilon-amino group of Lys180, which occupies the position usually occupied by the Mg-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Linkages between the dissociation of .alpha..beta. tubulin into subunits and ligand binding: The ground state of tubulin is the GDP conformation

The effects of ligands on the dissociation of the alpha beta tubulin dimer into the two subunits were investigated using calf brain tubulin. Sedimentation equilibrium studies showed a number of linkages. In the absence of magnesium in the medium, tubulin-GTP, tubulin-GDP, and tubulin with the exchangeable site unoccupied associate with essentially the same strength (K alpha beta = 1 x 10(7) M-1). This indicates that the ground state of tubulin (i.e., in the absence of magnesium) is not affected by occupancy of the exchangeable nucleotide binding site (E site). The alpha beta association is enhanced by magnesium ions. The association of tubulin with GDP in the E site is linked to the uptake of twice as many magnesium ions as that of tubulin with GTP in the E site. This suggests that magnesium binding is linked to an E-site-related conformational change. Consideration of the linkages between the binding of magnesium ions, E-site occupancy, and tubulin conformation in terms of the model [Howard, W. D., & Timasheff, S. N. (1986) Biochemistry 25, 8292-8300] in which the tubulin alpha beta dimer exists in an equilibrium between two conformations, a microtubule-forming ("straight") state favored by GTP and a double-ring-forming ("curved") state favored by GDP, leads to the conclusion that the ground state of tubulin is the ring-forming or "curved" conformation. Thus, in the absence of magnesium, the tubulin heterodimer exists in the ring-forming conformation, whether the E site is occupied by GTP or GDP.(ABSTRACT TRUNCATED AT 250 WORDS)

NMDA regulation of dopamine release from proximal and distal dendrites in the cat substantia nigra

The NMDA regulation of the dendritic release of [ 3H]dopamine ([ 3H]DA) synthesized from [ 3H]tyrosine was investigated in vitro using a microsuperfusion procedure in the pars compacta (SNC) and the pars reticulata (SNR) of the cat substantia nigra. The spontaneous release of [ 3H]DA was threefold higher in the SNC than in the SNR and amphetamine (1 μM) enhanced similarly [ 3H]DA release in both nigral areas. In the absence of magnesium, NMDA (50 μM) stimulated markedly the release of [ 3H]DA in the SNC and SNR, these effects being completely prevented by MK 801 (1 μM), the NMDA receptor antagonist. The DA uptake inhibitor, nomifensine (5 μM), increased the amount of [ 3H]DA recovered in SNC (×2) and SNR (×3) superfusates but did not significantly modify the NMDA-evoked responses. The effects of NMDA seen in the absence or presence of nomifensine persisted when the two nigral areas were continuously superfused with tetrodotoxin (1 μM). These results are in favor of the presence of NMDA receptors on dopaminergic dendritic arborizations and indicate that the stimulation of these receptors facilitates in a similar way the release of DA from proximal and distal dendrites.

Local GABAergic regulation of the N-methyl- d-aspartate-evoked release of dopamine is more prominent in striosomes than in matrix of the rat striatum

Using an in vitro microsuperfusion device we have previously demonstrated that in the absence of magnesium, the N-methyl- d-aspartate-evoked release of [ 3H]dopamine (continuously synthesized from [ 3H]tyrosine) is more prominent in matrix- than in striosome-enriched areas of the rat striatum and that in the matrix, the response is partially tetrodotoxin-sensitive. Since the medium-sized GABAergic neurons are the main targets of the corticostriatal glutamatergic fibers, the involvement of local GABAergic regulation in the N-methyl- d-aspartate-evoked release of [ 3H]dopamine was investigated in both striatal compartments using the same experimental approach. Firstly, bicuculline alone (5 μM, 25-min application) was shown to enhance the release of [ 3H]dopamine similarly in both compartments revealing the existence of a tonic GABAergic control of the spontaneous release of [ 3H]dopamine. Secondly, the N-methyl- d-aspartate (50 μM, 25-min application)-evoked release of [ 3H]dopamine was markedly amplified in the presence of bicuculline (5 μM, continuous delivery). This effect being more important in striosome- than in matrix-enriched areas (5.5- and two-times the N-methyl- d-aspartate-evoked response observed in the absence of the GABA A antagonist, respectively). Thirdly, the tetrodotoxin (1 μM, continuous delivery) resistant N-methyl- d-aspartate-evoked responses were also enhanced in the presence of bicuculline, but in this case, the amplification of the N-methyl- d-aspartate-evoked release of [ 3H]dopamine was less marked than in the absence of tetrodotoxin and identical in both compartments (about two-times the tetrodotoxin-resistant N-methyl- d-aspartate-evoked responses observed in the absence of bicuculline). Altogether, these results indicate that GABAergic neurons exert locally an important inhibitory regulation of the N-methyl- d-aspartate-evoked release of dopamine and that this effect is more prominent in the striosome-enriched area. Both tetrodotoxin-sensitive (striosome) and tetrodotoxin-resistant (striosome and matrix) processes intervene in this inhibitory GABAergic presynaptic regulation of dopamine release.

Modulation of GABA release by α-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl- d-aspartate receptors in matrix-enriched areas of the rat striatum

Using a new in vitro superfusion device, the release of preloaded [ 3H]GABA was examined in microdiscs of tissues taken from sagittal slices in matrix-enriched areas of the rat striatum. Potassium (9 mM, 15 mM) stimulated the release of [ 3H]GABA in a concentration- and calcium-dependent manner and the veratridine (1 μM)-evoked release of [ 3H]GABA was completely abolished in the presence of tetrodotoxin (1 μM). The selective glutamatergic agonist α-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM) enhanced the potassium-evoked release of [ 3H]GABA as well as the basal outflow of [ 3H]GABA. This latter effect was found to be calcium-dependent, partially diminished by tetrodotoxin (1 μM), completely blocked by 6,7-dinitro-quinoxaline-2,3-dione (0.1 mM), which is generally used as an antagonist of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors, but not affected by ( + )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801, 10μM), a specific antagonist of N-methyl- d-aspartate receptors. Similarly, N-methyl- d-aspartate (1 mM) enhanced both the potassium (9mM) and the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM )-evoked release of [ 3H]GABA but when used alone, due to the presence of magnesium in the superfusion medium, was ineffective on the basal efflux of [ 3H]GABA. A stimulatory effect of N-methyl- d-aspartate (1 mM) on the basal outflow of [ 3H]GABA was observed, however, when magnesium was omitted from the superfusion medium. The stimulatory effect of N-methyl- d-aspartate (1 mM) observed in the presence of α-amino-3-hydroxy-5-methylisoxazole-4-propionate was not potentiated by glycine (1 μM, in the presence of strychnine 1 gmM) and the N-methyl- d-aspartate-evoked response seen in the absence of magnesium was not enhanced by d-serine (1 mM). suggesting that endogenous glycine is already acting on N-methyl- d-aspartate receptors. In fact, in the absence of magnesium, 7-chloro-kynurenate (1 mM) completely abolished the stimulatory effect of N-methyl- d-aspartate on the release of [ 3H]GABA confirming that under our conditions, the glycine site of the N-methyl- d-aspartate receptor is saturated. N-methyl- d-aspartate-evoked responses were all blocked by MK801 (10 μM). Finally, the N-methyl- d-aspartate-evoked response seen in the absence of magnesium was markedly reduced in the presence of tetrodotoxin (1 μM). While potassium (9 mM) markedly stimulated the release of [ 3H]GABA from purified synaptosomes from the rat striatum, neither α-amino-3-hydroxy-5-methylisoxazole-4-propionate alone nor N-methyl- d-aspartate in the presence of α-amino-3-hydroxy-5-methylisoxazole-4-propionate or in the absence of magnesium enhanced the release of [ 3H]GABA. A slight but not significant stimulatory effect was observed with N-methyl- d-aspartateand d-serine using a magnesium-free superfusion medium. Altogether, these results strongly suggest that α-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl- d-aspartate receptors are present on dendrites and/or cell bodies of efferent GABAergic neurons but not on their nerve terminals in matrix-enriched areas of the rat striatum.