Novel glycine transporter type-2 reuptake inhibitors. Part 1: alpha-amino acid derivatives

A variety of alpha-amino acid derivatives were prepared as glycine transport inhibitors and their ability to block the uptake of [(14)C]-glycine in COS7 cells transfected with human glycine transporter-2 (hGlyT-2) was evaluated. An array of substituents at the chiral center was studied and overall, L-phenylalanine was identified as the preferred amino acid residue. Compounds prepared from l-amino acids were more potent GlyT-2 inhibitors than analogs derived from the corresponding d-amino acids. Introducing an achiral amino acid such as glycine, or incorporating geminal substitution in the alpha-position, led to a significant reduction in GlyT-2 inhibitory properties.     

Effect of inorganic cations and metabolic inhibitors on putrescine transport in roots of intact maize seedlings

The specificity and regulation of putrescine transport was investigated in roots of intact maize (Zea mays L.) seedlings. In concentration-dependent transport studies, the kinetics for putrescine uptake could be resolved into a single saturable component that was noncompetitively inhibited by increasing concentrations of Ca²⁺ (50 micromolar to 5 millimolar). Similarly, other polyvalent cations, including Mg²⁺ (1.8 millimolar) and La³⁺ (200 micromolar), almost completely abolished the saturable component for putrescine uptake. This suggests that putrescine does not share a common transport system with other divalent or polyvalent inorganic cations. Further characterization of the putrescine transport system indicated that 0.3 millimolar N-ethyl-maleimide had no effect on putrescine uptake, and 2 millimolar p-chloromercuribenzene sulfonic acid only partially inhibited transport of the diamine (39% inhibition). Metabolic inhibitors, including carbonylcyanide-m-chlorphenylhydrazone (20 micromolar) and KCN (0.5 millimolar), also partially inhibited the saturable component for putrescine uptake (V_max reduced 48-60%). Increasing the time of exposure to carbonylcyanide-m-chlorphenylhydrazone from 30 minutes to 2 hours did not significantly increase the inhibition of putrescine uptake. Electrophysiological evidence indicates that the inhibitory effect on putrescine uptake by these inhibitors is correlated to a depolarization of the membrane potential, suggesting that the driving force for putrescine uptake is the transmembrane electrical potential across the plasmalemma.     

The reaction of bisulfite with liposomal membranes

Bisulfite has been shown to induce leakage of encapsulated substances from liposomal vesicles. The bisulfite induced leakage of either DNP-tyrosine, potassium ferricyanide, or [³H]glycine was observed to be greater with lipsomes composed of phospholipids containing unsaturated fatty acids. The leakage of encapsulated substances from liposomes was found to be concentration dependent when incubated for a constant time interval and time dependent when incubated at a constant bisulfite concentration. In addition, bisulfite caused the leakage of approximately 5 times more [³H]glycine from unilamellar liposomes than from multilamellar liposomes. These findings are consistent with the interaction of bisulfite with liposomal membranes via reaction with sites of unsaturation.     

Inhibitory action of phosphatidylinositol on synaptosomal (Na+ + K+)-ATPase activity

Phosphatidylinositol and several other phospholipids were tested for their ability to influence the (Na+ + K+)-ATPase activity of the cortical synaptic membrane from rats at various levels of free Ca2+. Phosphatidylinositol, but not phosphatidylethanolamine, phosphatidylcholine nor phosphatidylserine, markedly inhibited this enzyme activity, when the free Ca2+ concentration in the incubation media was less than 2.5 X 10(-6) M. This result suggests that phosphatidylinositol may play a role in the depolarization and/or the release of neurotransmitters or intracellular substances in the brain.     

Zn2+ inhibits glycine transport by glycine transporter subtype 1b

In the central nervous system, glycine is a co-agonist with glutamate at the N-methyl-D-aspartate subtype of glutamate receptors and also an agonist at inhibitory, strychnine-sensitive glycine receptors. The GLYT1 subtypes of glycine transporters (GLYTs) are responsible for regulation of glycine at excitatory synapses, whereas a combination of GLYT1 and GLYT2 subtypes of glycine transporters are used at inhibitory glycinergic synapses. Zn2+ is stored in synaptic vesicles with glutamate in a number of regions of the brain and is believed to play a role in modulation of excitatory neurotransmission. In this study we have investigated the actions of Zn2+ on the glycine transporters, GLYT1b and GLYT2a, expressed in Xenopus laevis oocytes and we demonstrate that Zn2+ is a noncompetitive inhibitor of GLYT1 but has no effect on GLYT2. We have also investigated the molecular basis for these differences and the relationship between the Zn2+ and proton binding sites on GLYT1. Using site-directed mutagenesis, we identified 2 histidine residues, His-243 in the large second extracellular loop (ECL2) and His-410 in the fourth extracellular loop (ECL4), as two coordinates in the Zn2+ binding site of GLYT1b. In addition, our study suggests that the molecular determinants of proton regulation of GLYT1b are localized to the 2 histidine residues (His-410 and His-421) of ECL4. The ability of Zn2+ and protons to regulate the rate of glycine transport by interacting with residues situated in ECL4 of GLYT1b suggests that this region may influence the substrate translocation mechanism.     

Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts

Ethylene production in apple fruit and protoplasts and in leaf tissue was inhibited by spermidine or spermine. These polyamines, as well as putrescine, inhibited auxin-induced ethylene production and the conversion of methionine and 1-aminocyclopropane-1-carboxylic acid to ethylene. Polyamines were more effective as inhibitors of ethylene synthesis at the early, rather than at the late, stages of fruit ripening. Ca²⁺ in the incubation medium reduced the inhibitory effect caused by the amines. A possible mode of action by which polyamines inhibit ethylene production is discussed.     

Inhibition of phospholipase A2 and phospholipase C by polyamines

The polyamines spermine, spermidine, and putrescine inhibit the activity of phospholipase A₂ (Naja naja) and phospholipase C (Clostridium welchii) on phospholipid vesicles and mitochondrial membranes as sources of substrate phospholipids. The inhibitory effect is highest for spermine and lowest for putrescine. With both enzymes, inhibition is stronger when phospholipid vesicles rather than mitochondrial membranes are used as the substrate. No clear competition of polyamines with Ca²⁺, which is required for the activity of both enzymes, has been observed. The inhibition appears to be due to steric hindrance of enzyme-substrate interaction due to the binding of the organic polycations to the phospholipid bilayer.     

Consumption of dissolved amino acids and carbohydrates by limnetic bacterioplankton according to molecular weight fractions and proportions bound to humic matter

We investigated the bacterial decomposition of dissolved amino acids (DAA) and carbohydrates (DCHO) bound to humic substances and in the nonhumic fraction, and of low (< 3 KDA) AND HIGH (> 3 kDa) molecular weight (MW). Experiments were conducted in mesotrophic Lake Constance, Germany, in October and November 1997 and June 1998 during periods of low phytoplankton biomass. Dilution cultures, inoculated with bacterioplankton of the < 1 mm size fraction from 3 m, were run over 5 to 8 days to determine bacterial consumption of the various fractions of DAA and DCHO. In all experiments consumption rates of DCHO were higher than that of DAA. On average, 70% of the DAA and DCHO consumed were associated with the humic fraction, which, however, also consisted of a substantial recalcitrant component. In contrast, there was no preferential consumption of DAA and DCHO in either of the two MW fractions. Overall, we found a highly significant positive correlation between the consumption of the various fractions of DAA and DCHO and their initial concentrations. The consumed DAA, i.e, their labile pool, was dominated by serine and glutamate and that of DCHO by arabinose. In contrast, the residual DAA, i.e., the recalcitrant pool, comprised highest proportions of glycine + threonine and that of DCHO of mannose + xylose. These results indicate that the bacterial consumption of DAA and DCHO was mainly a function of their concentrations, irrespective of the molecular weight and whether they are bound to humic substances or not and despite the fact that they consist of a more labile and a more refractory pool.     

Gating of Ca2+-activated K+ channels controls fast inhibitory synaptic transmission at auditory outer hair cells

Fast inhibitory synaptic transmission in the central nervous system is mediated by ionotropic GABA or glycine receptors. Auditory outer hair cells present a unique inhibitory synapse that uses a Ca2+-permeable excitatory acetylcholine receptor to activate a hyperpolarizing potassium current mediated by small conductance calcium-activated potassium (SK) channels. It is shown here that unitary inhibitory postsynaptic currents at this synapse are mediated by SK2 channels and occur rapidly, with rise and decay time constants of approximately 6 ms and approximately 30 ms, respectively. This time course is determined by the Ca2+ gating of SK channels rather than by the changes in intracellular Ca2+. The results demonstrate fast coupling between an excitatory ionotropic neurotransmitter receptor and an inhibitory ion channel and imply rapid, localized changes in subsynaptic calcium levels.     

Purification and kinetic characteristics of strombine dehydrogenase from the foot muscle of the hard clam (Meretrix lusoria)

Strombine dehydrogenase (SDH, EC 1.5.1.22) from the foot of the hard clam Meretrix lusoria was purified over 470-fold to apparent homogeneity. It has a monomeric structure with a relative molecular mass of 46,000. Two isoenzymes were identified with isoelectric points of 6.83 and 6.88. SDH is heat labile, and has pH and temperature optima of 7.4–7.6 and 45–46 °C, respectively. l-Alanine, glycine, and pyruvate are the preferred substrates. l-Serine is the third preferred amino acid. Iminodiacetate with the lowest K_i of SDH at both pH 6.5 and 7.5 was the strongest inhibitor among succinate, acetate, iminodiacetate, oxaloacetate, and l-/d-lactate. The inhibitory activities of succinate at pH 6.5, and iminodiacetate and oxaloacetate at pH 7.5 on the SDH were higher. These inhibitors are either competitive or mixed-competitive inhibitors. Half of the enzymatic activity of SDH was inhibited by 0.2 mM Fe³⁺ and 0.6 mM Zn²⁺.     

GLYCINE UPTAKE IN RAT CENTRAL NERVOUS SYSTEM SLICES AND HOMOGENATES: EVIDENCE FOR DIFFERENT UPTAKE SYSTEMS IN SPINAL CORD AND CEREBRAL CORTEX

Abstract— Evidence is presented that glycine is taken up by two different transport systems in rat CNS tissue slices; one system has relatively low affinity for glycine (K_m = 300 μ m ) and predominates in cerebral cortex, cerebellum and mid-brain, the other has a higher affinity for glycine (K_m = 40 μ m ) and is detectable only in spinal cord, medulla and pons. The low affinity transport system appears to be shared by other small neutral amino acids, whereas the high affinity system is very specific for glycine. Both transport systems were shown to be present in particles in homogenates of CNS tissue by incubation with glycine in vitro , and subcellular fractionation studies suggested that synaptosomes were partly responsible for such uptake. Various substances were tested as inhibitors of the high affinity uptake system for glycine in spinal cord slices; the most potent inhibitors were p -chloro-mercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine, imipramine, desipramine, hydrazinoacetic acid and haloperidol. No competitive inhibitors of the high affinity glycine uptake were found. It is suggested that the high affinity transport system is associated with inhibitory synapses where glycine is a transmitter.     

Polyamines and neomycin inhibit the purified plasma-membrane Ca2+ pump by interacting with associated polyphosphoinositides.

We investigated the effect of spermine, spermidine, putrescine and neomycin on the activity of the plasma-membrane Ca2+ pump and on its stimulation by negatively charged phospholipids and calmodulin. Millimolar concentrations of spermine and to a lesser extent of spermidine decreased the ATPase activity in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2), without affecting the stimulation by phosphatidylinositol 4-phosphate (PIP). Sub-millimolar concentrations of neomycin inhibited the stimulation of the ATPase by PIP and by PIP2. Neomycin was more effective at the higher concentrations of PIP and PIP2. We discuss that these findings are compatible with the hypothesis that PIP and PIP2 bind to the ATPase and that several of these molecules have to be available to stimulate the ATPase.     

Glutamate release from cerebellar granule cells differentiating in culture: Modulation of the K+-stimulated release by inhibitory amino acids

The properties of l-[³H]glutamate release with an emphasis on the modulation by inhibitory amino acids of the potassium-induced release were studied with cerebellar granule cells from 7-day-old rats cultured for 7 or 14 days. Spontaneous glutamate release from cells grown for 7 days was fast, being slightly enchanced in Na⁺-free medium. l-Glutamate, kainate and quisqualate stimulated the release whereas N-methyl-d-aspartate and taurine were without any effect. The potassium-evoked glutamate release was Ca²⁺-dependent and potentiated by l-glutamate and quisqualate. Stimulated release was strongly depressed by glutamatediethylester. This inhibition was antagonized by GABA but not by taurine. GABA and its structural analogues taurine, hypotaurine, β-alanine and glycine were all equally effective in depressing stimulated glutamate release. The inhibition by GABA could be blocked by GABA antagonist. Both K⁺-evoked release and the kainate-induced release of glutamate were significantly greater in 14-day-old than in 7-day-old cultures, but the other properties of release were similar. The demonstration of calcium-dependent and potassium-stimulated glutamate release from cerebellar granule cells is consonant with the proposed neurotransmitter role of glutamate in these cells. The release could be modulated by both glutamatergic substances and inhibitory amino acids, the effect of GABA probably being mediated by GABAergic receptors.     

Regulation and biochemical characterization of the glutamine synthetase of azotobacter vinelandii

We have investigated the regulation of the activity and synthesis of the glutamine synthetase (l-glutamate:ammonia ligase (ADP-forming), EC (6.3.1.2) of Azotobacter vinelandii. Synthesis of the enzyme was not repressed by NH+4 and/or a number of amino acids in the growth medium; however, biosynthetic activity was rapidly lost through adenylylation in response to ammonium ion. The enzyme could be prepared as a 'relaxed, divalent-cation-free form which was catalytically inactive. The 'taut', active form could be restored with 1-5 mM Mg2+, Mn2+, Ca2+ or CO2+ and taut-vs.-relaxed difference spectra unique to each divalent cation were generated. Mg2+ and CO2+ each supported biosynthetic catalysis, but with different substrate Km and Vmax values. L-Alanine, glycine and L-aspartate were the most potent of several inhibitors of the biosynthetic and the gamma-glutamyl transferase activities; only aspartate and AMP behaved differentially toward glutamine synthetase adenylylation state: the more highly adenylylated enzyme was more severely affected. Any two of alanine, glycine or AMP showed cumulative inhibition, while the inhibitory effects of groups of three effectors were not cumulative. The Co2+-supported biosynthetic activity of Al vinelandii glutamine synthetase was markedly less sensitive to inhibition my glycine and alanine and was stimulated up to 50% by 1-10 mM aspartate.     

Antagonistic action between spermidine and putrescine on association and dissociation of purified, run-off ribosomes from Escherichia coli.

The effects of polyamines on the equilibrium between prokaryotic ribosomal subunits and 70 S ribosomes have been studied as a function of concentration of Mg2+ from 2.5 to 7.5 mM. Run-off ribosomes were obtained from Escherichia coli and were washed with buffered 1 M NH4C1. Spermidine at 1 mm favors association of subunits at all concentrations of Mg2+. Putrescine, at concentrations above 8 mM, favors net dissociation at concentrations of Mg2+ below 4.5 mM. Streptomycin behaves like spermidine, while putrescine behaves like initiation factor 1 and initiation factor 3. The effect of putrescine on dissociation is time-dependent and appears to have a half-life of about 3.5 min at 30 degrees. When added after the effects of spermidine or streptomycin on association have occurred, putrescine still causes dissociation. The data suggests that putrescine may reduce net formation of vacant 70 S ribosomes. Another possibility is that putrescine and spermidine may act antagonistically to maintain a labile equilibrium between ribosomal subunits and vacant 70 S ribosomes. It may be significant that the putrescine effect is observed at the concentration of Mg2+ found to be optimum for initiation.     

Effects of polyamine biosynthesis inhibitors on the progesterone-initiated increase in intracellular free Ca2+ and acrosome reactions in human sperm

This laboratory has previously reported that progesterone can initiate a rapid transient increase in the concentration of intracellular free Ca²⁺([Ca²⁺]_i) and an increase in a Ca²⁺-requiring exocytotic event, the acrosome reaction (AR) in human sperm. Rapid increases in Ca²⁺ fluxes of some mammalian cells caused by another steroid, testosterone, require polyamine biosynthesis. Herein, we tested two polyamine biosynthesis suicide inhibitors for their effects on the progesterone-initiated increase in [Ca²⁺]_i and AR in capacitated human sperm in vitro: DL-α-(difluoromethyl)ornithine hydrochloride (DFMO), an inhibitor of putrescine synthesis by ornithine decarboxylase and (5′-{[(Z))-4-amino-2-butenyl]methylamino}-5′-deoxyadenosine (MDL 73811), an inhibitor of S-adenosylmethionine decarboxylase (required for spermidine and spermine synthesis). Sperm were capacitated in vitro and preincubated 10 min with 4.9 mM DFMO or 9.8 μM MDL 73811 with or without various polyamines (245 μM). Progesterone (3.09 μM final concentration) or progesterone solvent (ethanol, 0.1% final concentration) was then added, sperm fixed 1 min after additions and AR assayed by indirect immunofluorescence or with fluorescein-labeled Con A lectin. DFMO strongly inhibited the AR but putrescine (product of ornithine decarboxylase and precursor of spermidine and spermine) reversed that inhibition. Preincubation for 25 min with DMFO + spermidine also reversed DFMO inhibition. MDL 73811 inhibited the progesterone-initiated AR, and a 10 min preincubation with spermidine, but not putrescine or spermine, reversed that inhibition. Preincubations with putrescine alone or with spermidine alone followed by addition of the progesterone solvent did not initiate the AR, and such preincubations followed by progesterone addition did not increase the AR more than progesterone alone. MDL 73811 and DFMO partially inhibited the rapid progesterone-initiated increase in [Ca²⁺]_i (assayed with fura-2), and those inhibitions were partially reversed by putrescine and spermidine, respectively. Putrescine or spermidine alone did not increase [Ca²⁺]_i nor did preincubation with either polyamine followed by progesterone addition increase [Ca²⁺]_i more than progesterone alone. Neither inhibitor was able to inhibit the AR initiated by the calcium ionophore, ionomycin. Our results suggest that human sperm polyamine biosynthesis is necessary for the progesterone-initiated rapid increase in [Ca²⁺]_i and subsequent membrane events of the AR. © 1993 Wiley-Liss, Inc.     

Comparison of fluorescence characteristics of products of peroxidation of membrane phospholipids with those of products derived from reaction of malonaldehyde with glycine as a model of lipofuscin fluorescent substances

The fluorescence characteristics of product (I), formed during the lipid peroxidation of rat liver phosphatidylcholine liposomes containing glycine, and fluorescent product (II), derived from the reaction of malonaldehyde with glycine, were examined to elucidate the mechanism of fluorescent chromophore formation. Fluorescent product (I) had a fluorescence emission maximum at 430 nm when excited at 360 nm; its fluorescence intensity decreases in alkaline medium, but is restored by readjustment of pH to neutrality. In contrast, fluorescent product (II) exhibited an emission maximum at 458 nm, and the fluorescence was quenched at acidic pH. The fluorescent substances formed during the lipid peroxidation of hemoglobin-free human erythrocyte ghost membranes had similar fluorescence characteristics to product (I). Gel filtration experiments showed that molecular size of fluorescent product (I) was larger than that of fluorescent product (II). The thiobarbituric acid-reactive substances released from peroxidizing liposomal phospholipids had a larger molecular size than malonaldehyde, and produced little or no fluorescence with glycine. It is concluded that the precursor of the fluorescent product formed during the lipid peroxidation of membrane phospholipids differs from malonaldehyde. The mechanism of the formation of blue emitting fluorescent material, believed to be a component of lipofuscin, seems to involve peroxidized phospholipids of the membrane.     

Activity coefficients of the electrolyte and the amino acid in water + NaNO(3) + glycine and water + NaCl + DL-methionine systems at 298.15 K

The activity coefficients at 298.15 K of glycine in water + NaNO(3) + glycine system and dl-methionine in water + NaCl + dl-methionine system are reported. The measurements were performed in an electrochemical cell with two ion selective electrodes, a cation and an anion ion selective electrode, each versus a double junction reference electrode. The concentrations of the electrolytes and the amino acids studied covered up to 1.0 molality electrolyte, 2.4 molality glycine and 0.2 molality dl-methionine. The results of the activity coefficients of glycine are compared with the activity coefficients of glycine in water + NaCl + glycine and water + KCl + glycine systems, obtained from the previous studies. The results show that the nature of both the cation and the anion of an electrolyte have significant effects on the activity coefficient of glycine in aqueous electrolyte solutions. The results also show that there are attractive interactions between the molecules of glycine and NaNO(3) and repulsive interactions between the molecules of dl-methionine and NaCl.     

Mutations within the agonist-binding site convert the homomeric alpha1 glycine receptor into a Zn2+-activated chloride channel

The divalent cation Zn2+ has been shown to regulate inhibitory neurotransmission in the mammalian CNS by affecting the activation of the strychnine-sensitive glycine receptor (GlyR). In spinal neurons and cells expressing recombinant GlyRs, low micromolar (<10 microM) concentrations of Zn2+ enhance glycine currents, whereas higher concentrations (>10 microM) have an inhibitory effect. Mutational studies have localized the Zn2+ binding sites mediating allosteric potentiation and inhibition of GlyRs in distinct regions of the N-terminal extracellular domain of the GlyR alpha-subunits. Here, we examined the Zn2+ sensitivity of different mutations within the agonist binding site of the homomeric alpha(1)-subunit GlyR upon heterologous expression in Xenopus oocytes. This revealed that six substitutions within the ligand-binding pocket result in a total loss of Zn2+ inhibition. Furthermore, substitution of the positively charged residues arginine 65 and arginine 131 by alanine (alpha(1)(R65A), alpha(1)(R131A), or of the aromatic residue phenylalanine 207 by histidine (alpha(1)(F207H)), converted the alpha(1) GlyR into a chloride channel that was activated by Zn2+ alone. Dose-response analysis of the alpha(1)(F207H) GlyR disclosed an EC(50) value of 1.2 microM for Zn2+ activation; concomitantly the apparent glycine affinity was 1000-fold reduced. Thus, single point mutations within the agonist-binding site of the alpha(1) subunit convert the inhibitory GlyR from a glycine-gated into a selectively Zn2+-activated chloride channel. This might be exploited for the design of metal-specific biosensors by modeling-assisted mutagenesis.     

Effect of alpha-conotoxin MII and its N-terminal derivatives on Ca2+ and Na+ signals induced by nicotine in neuroblastoma cell line SH-SY5Y

Nicotinic acetylcholine receptors (nAChRs) are implicated in the regulation ofintracellular Ca2+-dependent processes in cells both in normal and pathological states, alpha-Conotoxins isolated from Conus snails venom are a valuable tool for the study of pharmacological properties and functional role of nAChRs. In the present study the alpha-conotoxin MII analogue with the additional tyrosine attached to the N terminus (Y0-MII) was prepared. Also we synthesized analogs with the N-terminal glycine residue labeled with the Bolton- Hunter reagent (BH-MII) or fluorestsein isothiocyanate (FITC-MII). Fluorescence microscopy studies of the neuroblastoma SH-SY5Y cells loaded with Ca2+ indicator Fura-2 or with Ca2+ and Na+ indicators Fluo-4 and SBFI were performed to examine effect of MII modification on its ability to inhibit nicotin-induced increases in intracellular free Ca2+ and Na+ concentrations ([Ca2+] and [Na+]i respectively). Monitoring of individual cell [Ca2+]i and [Na+]i signals revealed different kinetics of [Ca2+]i and [Na+]i rise and decay in responses to brief nicotine (Nic) applications (10-30 microM, 3-5 min), which indicates to different mechanisms of Ca2+ and Na+ homeostasis control in SH-SY5Y cells. MII inhibited in concentration-dependent manner the both [Ca2+]i and [Na+]i increase induced by Nic. Additional tyrosine in the Y0-MII or, especially, more sizeable label in FITC-MII significantly reduced the inhibitory effect of MII. Whereas the efficiency of the Ca2+ response inhibition by BH-MII was found to be close to the efficiency of its inhibition by natural alpha-conotoxin MII, radioiodinated derivatives BH-MII can be used in radioligand assay.