Prolyl-hydroxylase inhibition preserves endothelial cell function in a rat model of vascular ischemia reperfusion injury.

Storage protocols of vascular grafts need further improvement against ischemia-reperfusion (IR) injury. Hypoxia elicits a variety of complex cellular responses by altering the activity of many signaling pathways, such as the oxygen-dependent prolyl-hyroxylase domain-containing enzyme (PHD). Reduction of PHD activity during hypoxia leads to stabilization and accumulation of hypoxia inducible factor (HIF) 1α. We examined the effects of PHD inhibiton by dimethyloxalylglycine on the vasomotor responses of isolated rat aorta and aortic vascular smooth muscle cells (VSMCs) in a model of cold ischemia/warm reperfusion. Aortic segments underwent 24 hours of cold ischemic preservation in saline or DMOG (dimethyloxalylglycine)-supplemented saline solution. We investigated endothelium-dependent and -independent vasorelaxations. To simulate IR injury, hypochlorite (NaOCl) was added during warm reperfusion. VSMCs were incubated in NaCl or DMOG solution at 4°C for 24 hours after the medium was changed for a supplied standard medium at 37°C for 6 hours. Apoptosis was assessed using the TUNEL method. Gene expression analysis was performed using quantitative real-time polymerase chain reaction. Cold ischemic preservation and NaOCl induced severe endothelial dysfunction, which was significantly improved by DMOG supplementation (maximal relaxation of aortic segments to acetylcholine: control 95% ± 1% versus NaOCl 44% ± 4% versus DMOG 68% ± 5%). Number of TUNEL-positive cell nuclei was significantly higher in the NaOCl group, and DMOG treatment significantly decreased apoptosis. Inducible heme-oxygenase 1 mRNA expressions were significantly higher in the DMOG group. Pharmacological modulation of oxygen sensing system by DMOG in an in vitro model of vascular IR effectively preserved endothelial function. Inhibition of PHDs could therefore be a new therapeutic avenue for protecting endothelium and vascular muscle cells against IR injury.

Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor in dental pulp-derived cells.

INTRODUCTION: Prolyl hydroxylase (PHD) inhibitors can induce a proangiogenic response that stimulates regeneration in soft and hard tissues. However, the effect of PHD inhibitors on the dental pulp is unclear. The purpose of this study was to evaluate the effects of PHD inhibitors on the proangiogenic capacity of human dental pulp-derived cells. METHODS: To test the response of dental pulp-derived cells to PHD inhibitors, the cells were exposed to dimethyloxalylglycine, desferrioxamine, L-mimosine, and cobalt chloride. To assess the response of dental pulp cells to a capping material supplemented with PHD inhibitors, the cells were treated with supernatants from calcium hydroxide. Viability, proliferation, and protein synthesis were assessed by formazan formation, (3)[H]thymidine, and (3)[H]leucine incorporation assays. The effect on the proangiogenic capacity was measured by immunoassays for vascular endothelial growth factor (VEGF). RESULTS: We found that all 4 PHD inhibitors can reduce viability, proliferation, and protein synthesis at high concentrations. At nontoxic concentrations and in the presence of supernatants from calcium hydroxide, PHD inhibitors stimulated the production of VEGF in dental pulp-derived cells. When calcium hydroxide was supplemented with the PHD inhibitors, the supernatants from these preparations did not significantly elevate VEGF levels. CONCLUSIONS: These results show that PHD inhibitors can stimulate VEGF production of dental pulp-derived cells, suggesting a corresponding increase in their proangiogenic capacity. Further studies will be required to understand the impact that this might have on pulp regeneration.

Dimethyloxalyglycine stimulates the early stages of gastrointestinal repair processes through VEGF-dependent mechanisms.

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain enzymes. Its potential value and mechanism of actions in preventing/treating gastrointestinal injury are, however, poorly understood. We, therefore, examined the effect of DMOG on influencing gut injury and repair using a variety of in vitro and in vivo models. We performed in vitro studies utilising pro-migratory (wounded monolayer) and proliferation (using DNA quantitation) assays of human stomach (AGS) and colonic (HT29) carcinoma cells. Time course studies examined changes in hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF) levels, a growth factor known to be regulated via HIF. In vivo studies utilised a rat gastric (indomethacin, 20 mg/kg and 3 h restraint) damage model. DMOG stimulated migration in a dose-dependent manner, increasing migration twofold when added at 25µM (P<0.01). Additive effects were seen when DMOG was added to cells in hypoxic conditions. DMOG stimulated proliferation dose dependently, increasing proliferation threefold when added at 70 µM (P<0.01). DMOG caused upregulation of both HIF and VEGF within 4 h of administration. Addition of VEGF neutralising antibody truncated migratory and proliferative activity of DMOG by about 70%. Both oral and subcutaneous administration of DMOG decreased gastric injury without influencing intragastric pH (50% reduction in injury when 1 ml gavaged at 0.57 mM, P < 0.01). Indomethacin reduced tissue HIF and VEGF levels but this was prevented if DMOG was present. In conclusion, DMOG stimulates the early phases of gut repair and VEGF-dependent processes appear relevant. Non-peptide factors such as this may be useful to stabilise or repair gut mucosa.

HIF1alpha synergizes with glucocorticoids to promote BFU-E progenitor self-renewal.

With the aim of finding small molecules that stimulate erythropoiesis earlier than erythropoietin and that enhance erythroid colony-forming unit (CFU-E) production, we studied the mechanism by which glucocorticoids increase CFU-E formation. Using erythroid burst-forming unit (BFU-E) and CFU-E progenitors purified by a new technique, we demonstrate that glucocorticoids stimulate the earliest (BFU-E) progenitors to undergo limited self-renewal, which increases formation of CFU-E cells > 20-fold. Interestingly, glucocorticoids induce expression of genes in BFU-E cells that contain promoter regions highly enriched for hypoxia-induced factor 1α (HIF1α) binding sites. This suggests activation of HIF1α may enhance or replace the effect of glucocorticoids on BFU-E self-renewal. Indeed, HIF1α activation by a prolyl hydroxylase inhibitor (PHI) synergizes with glucocorticoids and enhances production of CFU-Es 170-fold. Because PHIs are able to increase erythroblast production at very low concentrations of glucocorticoids, PHI-induced stimulation of BFU-E progenitors thus represents a conceptually new therapeutic window for treating erythropoietin-resistant anemia.

Group I, II, and III mGluR compounds affect rhythm generation in the gastric circuit of the crustacean stomatogastric ganglion.

We have studied the effects of group I, II, and III metabotropic glutamate receptor (mGluR) agonists on rhythm generation by the gastric circuit of the stomatogastric ganglion (STG) of the Caribbean spiny lobster Panulirus argus. All mGluR agonists and some antagonists we tested in this study had clear and distinct effects on gastric rhythm generation when superfused over combined oscillating or blocked silent STG preparations. A consistent difference between group I agonists and group II and III agonists was that group I agonists acted excitatory. The group I-specific agonists L-quisqualic acid and (S)-3,5-dihydroxyphenylglycine, as well as the nonspecific agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid accelerated ongoing rhythms and could induce gastric rhythms in silent preparations. The group II agonist (2S,1'S, 2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) and the group III agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) slowed down or completely blocked ongoing gastric rhythms and were without detectable effect on silent preparations. The action of L-CCG-I was blocked partially by the group-II-specific antagonist, (RS)-1-amino-5-phosphonoindan-1-carboxylic acid [(RS)APICA], and the group-III-specific antagonist (RS)-alpha-methyl-4-phosphonophenylglycine completely blocked the action of L-AP4. Besides its antagonistic action, the group-II-specific antagonist (RS)APICA had a remarkably strong apparent inverse agonist action when applied alone on oscillating preparations. The action of all drugs was dose dependent and reversible, although recovery was not always complete. In our experiments, the effects of none of the mGluR-specific agonists were antagonized or amplified by the N-methyl-D-aspartate (NMDA)-receptor-specific antagonist D(-)-2-amino-5-phosphonopentanoic acid, excluding the contamination of responses to mGluR agonists by nonspecific cross-reactivity with NMDA receptors. Picrotoxin did not prevent the inhibitory action of L-CCG-I and L-AP4. We conclude that mGluRs, probably similar to those belonging to groups I, II, and III described in mammals, may play a role as modulators of gastric circuit rhythm generation in vivo.

Intracerebral administration of metabotropic glutamate receptor agonists disrupts prepulse inhibition of acoustic startle in Sprague-Dawley rats.

The functional role of striatal metabotropic glutamate receptors (mGluRs) was examined by measuring prepulse inhibition (PPI) of an acoustic startle response following the intracerebral administration of selective agonists in male Sprague-Dawley rats prepared with bilateral cannulae aimed at either the nucleus accumbens or dorsal striatum. mGluR subtypes (1-8) are classed in three groups based on sequence homology, signal transduction mechanism and pharmacology. Intra-accumbens IS,3R-ACPD, an agonist at group 1 and 2 mGluRs (0.5-1.0 micromol/2 microl), caused a dose-dependent loss of PPI. The effect of 1S,3R-ACPD was diminished when injected into dorsal striatum. Intra-accumbens infusion of the group 1 selective agonist 3,5-DHPG (1 micromol) and the group 2 selective agonist L-CCG-I (100 nmol) also led to statistically significant disruptions of PPI, while the group 3 selective agonist L-AP4 (0.4-1.0 micromol) had no significant effect. Although the group 1/2 mGluR antagonist (+) MCPG (0.5 micromol) had no significant effect of its own on PPI, co-administration with IS,3R-ACPD (1 micromol) blocked ACPD-induced loss of PPI. In addition, pretreatment (30 min) with haloperidol (0.3 mg/kg IP) attenuated the PPI disruption induced by 1 micromol 1S,3R-ACPD, suggesting dopamine may play a role in mGluR agonist induced loss of PPI. These results support a role for group 1 and group 2 mGluRs in the nucleus accumbens in the regulation of PPI, a measure of sensory gating. As PPI is abnormal in some patient populations, such as Huntington's and schizophrenia, mGluRs may have potential as novel therapeutic targets for these diseases.

Activation of group I mGluRs increases spontaneous IPSC frequency in rat frontal cortex.

The effect of metabotropic glutamate receptor (mGluR) activation on inhibitory synaptic transmission was examined by using whole cell patch-clamp recordings. Spontaneous (s) and miniature (m) inhibitory postsynaptic currents (IPSCs) were recorded from visually identified layer II/III pyramidal neurons in rat neocortex in vitro. Excitatory postsynaptic currents (EPSCs) were blocked by using bath application of 20 microM D(-)2-amino-5-phosphonovaleric acid and 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione. In the presence of 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (30-100 microM), Lp4-quisqualate (5 microM), and the group I selective mGluR agonist (S)-3,5-dihydroxyphenylglycine (100 microM), the frequency of sIPSCs was increased. Decay kinetics of sIPSCs were unaffected. No enhancement of mIPSCs was observed. Bath application of group II (2S,3S,4S-alpha-carboxycyclopropyl-glycine; 5 microM) and group III selective mGluR agonists (L-2-amino-4-phosphonobutyric acid; 100 microM) had no detectable effects on the frequency or amplitude of sIPSCs. These findings indicate that activation of group I mGluRs (mGluR1 and/or mGluR5) enhances gamma-aminobutyric acid-mediated synaptic inhibition in layer II/III pyramidal neurons in neocortex. The lack of effect on mIPSCs suggests a presynaptic action via excitation of inhibitory interneurons.

G-protein activation by metabotropic glutamate receptors reduces spike frequency adaptation in neocortical neurons.

Intracellular recordings were obtained from neocortical brain slices of adult rats maintained in vitro. The effect of metabotropic glutamate receptor activation on spike frequency adaptation in regular spiking layer II and III neurons was determined. Putative metabotropic glutamate receptor agonists and antagonists, as well as inhibitors of intracellular signaling systems, were tested. Activation of metabotropic glutamate receptors by bath applied (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD; 50-200 microM) reduced the first interspike interval and increased action potential frequency at all current intensities. This effect was not blocked by ionotropic glutamate receptor antagonists. Under these recording conditions, quisqualate (1-10 microM) similarly reduced spike frequency adaptation. Neither 1R,3S-ACPD, L-2-carboxycyclopropylglycine-I nor the putative presynaptic metabotropic glutamate receptor agonist, L-2-amino-4-phosphonobutyrate, mimicked the effects of 1S,3R-ACPD or quisqualate. Bath application of the putative metabotropic glutamate receptor antagonist, alpha-methyl-4-carboxyphenylglycine, competitively antagonized the excitatory actions of 1S,3R-ACPD. Another putative antagonist, L-2-amino-3-phosphonopropionate, failed to antagonize the reduction in spike frequency adaptation. Intracellular injection of guanosine-5'-O-(2-thiodiphosphate), a non-hydrolysable analog of GTP, inhibited the postsynaptic metabotropic glutamate receptor-mediated effects. However, the depression of synaptic transmission by 1S,3R-ACPD was not antagonized by this compound. The decrease in spike frequency adaptation by 1S,3R-ACPD was not prevented by prior exposure to the non-specific protein kinase inhibitors H-7 or H-8 (10 microM), the protein kinase A inhibitor H-89 (0.25 microM) or the protein kinase C inhibitor staurosporine (0.10 microM). These data suggest that the metabotropic glutamate receptor-mediated reduction in spike adaptation requires the activation of specific G-protein-coupled metabotropic glutamate receptor subtypes located on postsynaptic sites. The increase in neuronal excitability observed in the adult neocortex may be mediated either by an unidentified G-protein-coupled second messenger or via a membrane-delimited G-protein action.

Antagonism of the presumed presynaptic action of L-AP4 on GABAergic transmission in the ventrobasal thalamus by the novel mGluR antagonist MPPG.

The metabotropic glutamate receptor (mGluR) agonists CCG-I and L-AP4, acting at Group II and Group III mGluRs respectively, can reduce GABAergic synaptic inhibition on single neurones in the rat thalamus in vivo via a presumed presynaptic mechanism. The actions of L-AP4 were antagonized by (+/-)-alpha-methyl-4-phosphonophenylglycine (MPPG), whereas CCG-I was significantly less affected. Thus MPPG may be a useful tool for detecting physiological roles for Group III mGluRs.

Inhibition by folded isomers of L-2-(carboxycyclopropyl)glycine of glutamate uptake via the human glutamate transporter hGluT-1.

The effects of isomers of 2-(carboxycyclopropyl)glycine (CCG) on uptake of L-glutamate were investigated in COS-7 cells that expressed a cloned human glutamate transporter (hGluT-1). The (2S, 3S, 4R)-isomer (L-CCG-III) and the (2S, 3R, 4S)-isomer (L-CCG-IV) markedly inhibited glutamate uptake with a 50% inhibitory concentration of 290 nM and 1.1 microM, respectively. The (2S, 3S, 4S)-isomer (L-CCG-I) and the (2S, 3R, 4R)-isomer (L-CCG-II) did not inhibit glutamate uptake at concentrations of < or = 10 microM. Thus, hGluT-1 showed a markedly higher affinity for L-CCG-III and L-CCG-IV with a folded conformation of the glutamate skeleton, than for L-CCG-I or L-CCG-II with an extended conformation.

Excitatory action of a plant extract, stizolobic acid, in the isolated spinal cord of the rat.

Actions of stizolobic acid, stizolobinic acid and their derivatives were examined on the isolated spinal cord of the newborn rat. The responses were recorded from the ventral root. Stizolobic acid and its bromo-derivatives caused a depolarizing response in a dose-dependent manner. Stizolobinic acid was considerably less potent than stizolobic acid. Depolarizing responses to stizolobic acid and its bromo-derivatives were not affected by the existence of Mg2+ and specific N-methyl-D-aspartate (NMDA) antagonists. Kynurenate depressed responses to stizolobic acid. These results suggest that stizolobic acid is a new excitatory amino acid in the mammalian central neurons which binds preferably to other receptors than the NMDA-type receptor.

A glial toxin reduces effects of gold thioglucose on the hypothalamus and area postrema.

To see if DL-alpha amino adipate (DL-AA) an agent known to cause transient swelling and dysfunction of hypothalamic glia, would affect the ability of goldthioglucose (GTG) to cause hypothalamic lesions, groups of male, 100 g rats were given saline, 0.5 mg/g of DL-AA, or 0.75 mg/g of DL-AA 30 min before injection of 0.4 mg/g of GTG. The incidence of GTG lesions was significantly less in the DL-AA treated groups than in the saline treated group. Histochemical staining of brain tissue revealed the existence of a population of specialized iron-containing glia with an anatomical distribution similar to the specific areas in the hypothalamus area postrema most sensitive to GTG. The results suggest that GTG lesion formation may result from an effect of GTG on glia.

Growth inhibition by alpha-aminoadipate and reversal of the effect by specific amino acid supplements in Saccharomyces cerevisiae.

The growth of Saccharomyces cerevisiae wild-type strain X2180 in minimal medium was inhibited by the addition of higher-than-supplementary levels of alpha-aminoadipate. This inhibitory effect was reversed by the addition of arginine, asparagine, aspartate, glutamine, homoserine, methionine, or serine as single amino acid supplements. Mutants belonging to the lys2 and lys14 loci were able to grow in lysine-supplemented alpha-aminoadipate medium, although not as well as when selected amino acids were added. Growth in alpha-aminoadipate medium by all strains was accompanied by an accumulation of alpha-ketoadipate. Glutamate:keto-adipate transaminase levels were derepressed two- to fivefold in lys2 mutants using alpha-aminoadipate as a nitrogen source. Wild-type strain X2180 growing in amino acid-supplemented AA medium exhibited higher levels of alpha-aminoadipate reductase. Mutants unable to use alpha-aminoadipate without amino acid supplementation were obtained by treatment of lys2 strain MW5-64 and were shown to have glutamate: ketoadipate transaminase activity and to lack alpha-aminoadipate reductase activity. Altered cell morphologies, including increased size, multiple buds, pseudohyphae, and germ tubes, evidenced by cells grown in alpha-aminoadipate medium suggest that higher-than-supplementary levels of alpha-aminoadipate result in an impairment of cell division.

On the configuration of the receptors for excitatory amino acids.

Separate receptors are recognized for the excitation of mammalian neurones by (a) L-glutamic and quisqualic acids and (b) N-methyl-D-aspartic (NMDA), and other amino acids which have conformationally restricted molecules. Several other compounds, both agonists and antagonists, have been examined, and it is concluded that (i) the NMDA receptor reacts preferentially with substances in a relatively extended configuration, (ii) the glutamate/quisqualate receptor prefers folded molecules and (iii) the distance separating the amino group from the distal anionic function is the critical one determining receptor preference.

D-aminophosphonovalerate is 100-fold more powerful than D-alpha-aminoadipate in blocking N-methylaspartate neurotoxicity.

Here we report that the D-isomers of 2-amino-5-phosphonovalerate (D-APV) and alpha-amino-adipate (D-alpha AA) protect arcuate hypothalamic neurons from the potent excitotoxic activity of N-methylaspartate (NMA). Consistent with evidence that APV is much more powerful than alpha AA in antagonizing the neuroexcitatory activity of NMA, we found D-APV nearly 100 times more powerful than D-alpha AA in preventing NMA from destroying arcuate neurons.

Ranking of excitatory amino acids by the antagonists glutamic acid diethylester and D-alpha-aminoadipic acid.

A separation of the excitatory actions of the amino acids upon thalamic neurones of rats anaesthetized with urethane has been accomplished through the use of two antagonists. It has been possible to rank the excitatory compounds in their order of susceptibility to D-alpha-aminoadipic acid (DalphaAA) and L-glutamic acid diethylester (GDEE). The observation that the ranking orders of the excitants differ for these two antagonists permits an analysis of the types of receptors with which the amino acid excitants react. The results support the proposition that more than one neuronal receptor sensitive to the amino acids exists.