Cytoprotection by pyruvate through an anti-oxidative mechanism in cultured rat calvarial osteoblasts.

Although we have previously shown drastic cell death by pyruvate deficiency in osteoblasts at the proliferative stage, the exact mechanism remains unclear so far. Cell survivability was significantly decreased in rat calvarial osteoblasts cultured for 0 to 3 days in vitro (DIV) following replacement of the eutrophic alpha-modified minimum essential medium (alpha-MEM) with Dulbecco's modified eagle medium (DMEM) for cultivation. The addition of pyruvate enriched in alpha-MEM, but not in MEM, entirely prevented cell death induced by the medium replacement throughout a culture period from 0 to 3 DIV. Both cysteine and reduced glutathione protected cell death in cells cultured for 3 DIV without significantly affecting that in cells cultured for 1 DIV, however, while none of lactate, acetate and insulin significantly prevented the cell death irrespective of the culture period up to 3 DIV. A marked increase was detected in intracellular reactive oxygen species (ROS) levels 4 h after the medium replacement. In osteoblasts cultured in alpha-MEM for 3 DIV, but not in those for 7 DIV, hydrogen peroxide (H2O2) markedly decreased cell survivability when exposed for 2 to 24 h. Furthermore, H2O2 was effective in significantly decreasing cell survivability in osteoblasts cultured in DMEM for 7 DIV. Pyruvate at 1 mM not only prevented cell death by H2O2, but also suppressed the generation of intracellular ROS in osteoblasts exposed to H2O2. These results suggest that pyruvate could be cytoprotective through a mechanism associated with the anti-oxidative property rather than an energy fuel in cultured rat calvarial osteoblasts.

Possible expression of functional glutamate transporters in the rat testis.

Neither expression nor functionality is clear in peripheral tissues with the molecular machineries required for excitatory neurotransmitter signaling by L-glutamate (Glu) in the central nervous system, while a recent study has shown that several Glu receptors are functionally expressed in the rat testis. This fact prompted us to explore the possible functional expression in the rat testis of the Glu transporters usually responsible for the regulation of extracellular Glu concentrations in the brain. RT-PCR revealed the expression, in the rat testis, of mRNA for five different subtypes of Glu transporters, in addition to that for particular subtypes of ionotropic and metabotropic Glu receptors. Glutamate transporter-1 (GLT-1) was different in the brain from that in the testis in terms of molecular sizes on Northern and Western blot analyses. In situ hybridization as well as immunohistochemical analysis showed localized expression of glutamate aspartate transporter at interstitial spaces and GLT-1 at elongated spermatids in the rat testis respectively. The expression of mRNA was localized for excitatory amino acid transporter-5 at the basal compartment of the seminiferous tubule in the rat testis. [(3)H]Glu was accumulated in testicular crude mitochondrial fractions in a temperature- and sodium-dependent saturable manner with pharmacological profiles similar to those shown in brain crude mitochondrial fractions. These results suggested that particular subtypes of central Glu transporters for the regulation of extracellular Glu concentrations in the rat testis could be constitutively and functionally expressed.

Expression of GluR6/7 subunits of kainate receptors in rat adenohypophysis.

We have previously demonstrated the presence of unidentified [3H]glutamate (Glu) binding sites with stereo-selectivity, high affinity and saturability in rat peripheral excitable tissues such as the pituitary (Yoneda, Y., Ogita, K., 1986a. [3H]Glutamate binding sites in the rat pituitary. Neurosci. Res. 3, 430--435) and adrenal (Yoneda, Y., Ogita, K., 1986b. Localization of [3H]glutamate binding sites in rat adrenal medulla. Brain Res. 383, 387--391, 1986). In this study, peripheral binding sites were further evaluated for the ionotropic Glu receptor subtype insensitive to N-methyl-D-aspartate by using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, in addition to receptor binding using radiolabeled ligands other than [3H]Glu. Binding of [3H]kainate (KA) and [3H]DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate was detected in membrane preparations obtained from the rat pituitary and adrenal irrespective of prior treatment with Triton X-100. An RT-PCR analysis revealed constitutive expression of mRNA for GluR1, GluR3, GluR5, KA1 and KA2 subunits in the rat adrenal and pituitary, as well as the brain and retina. The pituitary also expressed mRNA for GluR2, GluR4, GluR6 and GluR7 subunits in contrast to the adrenal. Under our experimental conditions employed, however, Western blotting assays failed to confirm the expression of receptor proteins for GluR1, GluR2/3 and GluR4 subunits in the adrenal cortex, adrenal medulla, adenohypophysis and neurohypophysis. Immunoreactive GluR6/7 subunits were only detectable in the adenohypophysis, but not in the adrenal cortex, adrenal medulla and neurohypophysis. An intraperitoneal injection of KA doubled DNA binding activity of the nuclear transcription factor activator protein-1 in the rat pituitary, with concomitant more potent potentiation of that in the hippocampus. These results suggest that GluR6/7 subunits of KA receptors may be constitutively expressed with responsiveness to the systemic administration of an agonist at least in the rat adenohypophysis.

Group III metabotropic glutamate receptors in rat cultured calvarial osteoblasts.

Reverse transcription polymerase chain reaction revealed expression of mRNAs for particular receptors for the central neurotransmitter l-glutamic acid (Glu) in primary cultures of rat calvarial osteoblastic cells under premature to mature states according to the duration of days in vitro. These included metabotropic Glu receptors (mGluR) such as mGluR4 and mGluR8, in addition to several ionotropic Glu receptor subunits including NR1 and NR2D. Expression of mRNAs was not detected with other mGluR and NR2A-C subunits irrespective of the maturity of cultured cells. The agonist for group III mGluR L-(+)-2-amino-4-phosphonobutyric acid significantly inhibited the forskolin-induced accumulation of cAMP in premature osteoblasts, which occurred in a manner sensitive to prevention by the group III mGluR antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine. These results suggest that Glu may at least in part play a role in mechanisms associated with cellular proliferation and/or differentiation through group III mGluR functionally expressed in rat calvarial osteoblastic cells.

Consolidation of transient ionotropic glutamate signals through nuclear transcription factors in the brain.

Long-lasting alterations of neuronal functions could involve mechanisms associated with consolidation of transient extracellular signals through modulation of de novo synthesis of particular functional proteins in the brain. In eukaryotes, protein de novo synthesis is mainly under the control at the level of gene transcription by transcription factors in the cell nucleus. Transcription factors are nuclear proteins with an ability to recognize particular core nucleotides at the upstream and/or downstream of target genes, and thereby to modulate the activity of RNA polymerase II that is responsible for the formation of mRNA from double stranded DNA. Gel retardation electrophoresis is widely employed for conventional detection of DNA binding activities of a variety of transcription factors with different protein motifs. Extracellular ionotropic glutamate (Glu) signals lead to rapid and selective potentiation of DNA binding of the nuclear transcription factor activator protein-1 (AP1) that is a homo- and heterodimeric complex between Jun and Fos family members, in addition to inducing expression of the corresponding proteins, in a manner unique to each Glu signal in murine hippocampus. Therefore, extracellular Glu signals may be differentially transduced into the nucleus to express AP1 with different assemblies between Jun and Fos family members, and thereby to modulate de novo synthesis of the individual target proteins at the level of gene transcription in the hippocampus. Such mechanisms may be operative on synaptic plasticity as well as delayed neuronal death through consolidation of alterations of a variety of cellular functions induced by transient extracellular signals in the brain.

Characterization with [3H]quisqualate of group I metabotropic glutamate receptor subtype in rat central and peripheral excitable tissues.

Radioligand binding studies were performed to label metabotropic glutamate receptor (mGluR) in rat brain synaptic membranes using [3H]quisqualic acid (QA) synthesized in our laboratory as a radioligand. In the presence of ionotropic glutamate receptor (iGluR) agonists, including N-methyl-D-aspartic (NMDA), DL-alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic (AMPA) and kainic acids (KA), at concentrations maximally effective in displacing each receptor binding, the agonists for group I mGluR subtype (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) and (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) more potently displaced [3H]QA binding in a concentration-dependent manner than their absence. The addition of these three iGluR agonists did not significantly affect potencies of (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) and L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) to displace [3H]QA binding. Scatchard analysis revealed that [3H]QA binding consisted of a single component with a maximal number of binding sites (B(max)) of 431.6 fmol/mg protein and a dissociation constant (K(d)) of 50.9 nM, in the presence of the three iGluR agonists. [3H]QA binding was markedly inhibited by GTP and its analogues; but not by GDP, GMP and ATP, under these conditions. Inhibition by GTP was seen in all central structures examined, but [3H]QA binding was not detectable in peripheral tissues, such as pituitary and adrenal glands. Neither reverses transcription polymerase chain reaction nor immunoblotting analysis demonstrated the expression of mGluR1 and mGluR5 subunits in the aforementioned two peripheral tissues. These results suggest that [3H]QA indeed labels group I mGluR subtype functionally coupled to GTP binding protein in rat brain synaptic membranes under the experimental conditions employed. Group I mGluR subtype seems to be selectively distributed in central structures but not in pituitary and adrenal glands.

Direct radiolabeling by [3H]quisqualic acid of group I metabotropic glutamate receptor in rat brain synaptic membranes.

[3H]Quisqualic acid (QA) was synthesized and used to label metabotropic glutamate receptor (mGluR) in rat brain synaptic membranes in the presence of three different ionotropic glutamate receptor agonists at respective saturating concentrations. Of several mGluR agonists tested, group I agonists were more potent in displacing [3H]QA binding than group II and group III agonists in the presence of the three ionotropic agonists. [3H]QA binding was markedly inhibited by guanine nucleotide analogues in a concentration-dependent manner at a concentration range of 10 nM to 1 mM. Scatchard analysis revealed that [3H]QA binding consisted of a single component with a K(d) of 50.9+/-5.3 nM and a B(max) of 431. 6+/-18.3 fmol/mg protein. These results suggest that [3H]QA indeed labels group I mGluR functionally coupled to GTP binding protein in rat brain synaptic membranes when determined under the experimental conditions employed.

Ferrous iron modulates quinolinate-mediated [3H]MK-801 binding to rat brain synaptic membranes in the presence of glycine and spermidine.

Quinolinic acid (QUIN) displaced binding of agonist and antagonist ligands for the N-methyl-D-aspartate (NMDA) receptor in rat brain synaptic membranes. Both QUIN and glutamic acid (GLU) potentiated binding of [3H]dizocilpine (MK-801) in the presence of glycine (GLY) alone, whereas the potentiation by QUIN was in a bell-shaped fashion in contrast to that by GLU. However, further addition of spermidine (SPD) induced bell-shaped potentiation by GLU as well as QUIN. The potentiation by QUIN was markedly deteriorated by the further addition of FeCl2 irrespective of the presence of GLY and SPD added. These results suggest that QUIN may potentiate [3H]MK-801 binding to the open NMDA channel in rat brain synaptic membranes through a mechanism different from that underlying the potentiation by GLU.

Differential inhibition by ferrous ions of [3H]MK-801 binding to native N-methyl-D-aspartate channel in neonatal and adult rat brains.

In vitro addition or pretreatment with >/=1 microM ferrous chloride markedly inhibited in a concentration-dependent manner [3H]dizocilpine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in rat brain synaptic membranes. The addition of NMDA agonists invariably attenuated the inhibition of [3H]MK-801 binding in hippocampal synaptic membranes previously treated with ferrous chloride, without significantly affecting that in cerebellar synaptic membranes. In the absence of spermidine, ferrous chloride was more potent in inhibiting binding in the cerebral cortex and hippocampus in adult rats than in those in rats at 3 days after birth, while in the striatum [3H]MK-801 binding was 10 times more sensitive to inhibition by added ferrous chloride in neonatal rats than in adult rats. Addition of spermidine significantly attenuated the potency of ferrous chloride to inhibit binding in the cerebral cortex of adult rats, with facilitation of the inhibition in newborn rats. Moreover, spermidine significantly reduced the inhibitory potency of ferrous chloride in neonatal rat striatum, without markedly affecting that in adult rat striatum. These results suggest that ferrous ions may interfere with opening processes of the native NMDA channel through molecular mechanisms peculiar to neuronal development in a manner associated with the polyamine recognition domain.