Ibudilast attenuates astrocyte apoptosis via cyclic GMP signalling pathway in an in vitro reperfusion model.

We examined the effect of 3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine (ibudilast), which has been clinically used for bronchial asthma and cerebrovascular disorders, on cell viability induced in a model of reperfusion injury. Ibudilast at 10 - 100 microM significantly attenuated the H(2)O(2)-induced decrease in cell viability. Ibudilast inhibited the H(2)O(2)-induced cytochrome c release, caspase-3 activation, DNA ladder formation and nuclear condensation, suggesting its anti-apoptotic effect. Phosphodiesterase inhibitors such as theophylline, pentoxyfylline, vinpocetine, dipyridamole and zaprinast, which increased the guanosine-3',5'-cyclic monophosphate (cyclic GMP) level, and dibutyryl cyclic GMP attenuated the H(2)O(2)-induced injury in astrocytes. Ibudilast increased the cyclic GMP level in astrocytes. The cyclic GMP-dependent protein kinase inhibitor KT5823 blocked the protective effects of ibudilast and dipyridamole on the H(2)O(2)-induced decrease in cell viability, while the cyclic AMP-dependent protein kinase inhibitor KT5720, the cyclic AMP antagonist Rp-cyclic AMPS, the mitogen-activated protein/extracellular signal-regulated kinase inhibitor PD98059 and the leukotriene D(4) antagonist LY 171883 did not. KT5823 also blocked the effect of ibudilast on the H(2)O(2)-induced cytochrome c release and caspase-3-like protease activation. These findings suggest that ibudilast prevents the H(2)O(2)-induced delayed apoptosis of astrocytes via a cyclic GMP, but not cyclic AMP, signalling pathway.

Possible dysfunction of ionotropic glutamate receptors in cerebellum of epileptic E1 mouse brain.

Binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten- 5,10-imine (MK-801) to an ion channel domain on the N-methyl-D-aspartate (NMDA)-sensitive subclass of brain glutamate (Glu) receptors was highest in the hippocampus of the hereditary epileptogenic mutant El as well as its parent ddY strain mice, when determined before and at equilibrium in the presence of 3 different agonists at the respective domains on the NMDA receptor complex, including Glu, glycine (Gly) and spermidine (SPD). Cerebellar [3H]MK-801 binding before equilibrium was significantly lower in El mice than in ddY mice, while the binding was not significantly different from each other in other brain structures of both strains of mice. Kinetic analysis revealed that the association rate was significantly lower with [3H]MK-801 binding in the cerebellum of El mice than of ddY mice. In contrast to ddY mice, furthermore, Gly failed to potentiate cerebellar [3H]MK-801 binding before equilibrium in El mice, with SPD being active in significantly inhibiting the binding. However, saturation analysis revealed that the affinity and density of cerebellar [3H]MK-801 binding at equilibrium in El mice were not significantly different from those in ddY mice. In addition, epileptogenic El mice had significantly higher levels of [3H]SPD binding in all brain structures examined than ddY mice, whereas [3H]DL-alpha-amino-3-hydroxy-5- methylisoxazole-4-propionate binding was significantly lower in the cerebellum of El mice than of ddY mice. These results suggest that dysfunction of cerebellar Glu receptors may be at least in part responsible for a variety of abnormal symptoms observed in epileptic El mice.

Binding of [3H]MK-801, NMDA-displaceable [3H]glutamate, [3H]glycine, [3H]spermidine, [3H]kainate and [3H]AMPA to regionally discrete brain membranes of the gerbil: a biochemical study.

Bindings of glutamate receptor agonists and related modulators were investigated in 10 discrete tissues from gerbil brain using a biochemical technique. There appeared considerable discrepancies, in respect of intrahippocampal profiles, from reported data by autoradiography on rat brain. In the gerbil, an almost equivalent level of N-methyl-D-aspartate (NMDA)-displaceable [3H]glutamate binding was found in field CA1 and the dentate gyrus, while approx 30% less in field CA3, a profile which was strikingly similar to that of (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cycloheptene-5,10-imin e maleate (MK-801) or of [3H]glycine. [3H]Kainate binding was highest in the dentate gyrus followed by field CA3 and then field CA1, the ratio of the highest to the lowest being 3 to 2. Binding of [3H]DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) differed, to a certain extent, from that of [3H]kainate and showed the dentate gyrus followed by field CA1 and then field CA3 in the rank order of decreasing binding. Taking together, intrahippocampal localization of glutamate receptor subtypes in the gerbil, when analyzed with a biochemical binding assay, looks to be less region selective than the distribution obtained on autoradiography in the rat. Thus, it is likely that these different distribution profiles show different status of receptor function respectively, or are due merely to species difference.

Differential effects of SH-reactive agents on [(3)H](+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate and [(3)H]glutamate binding in brain synaptic membranes treated with triton X-100.

Specific binding of [(3)H](+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an antagonist highly selective for the N- methyl- d -aspartate (NMDA)-sensitive subclass of the brain excitatory amino acid receptors, was examined in rat brain synaptic membranes treated with a low concentration of Triton X-100 using a filtration assay method. The binding was displaced by l-glutamic acid (Glu) and its analogous amino acids in a concentration-dependent manner at a concentration range of 10 nM to 0.1 mM. Agonists as well as competitive antagonists for the subclass invariably displaced the binding, while noncompetitive antagonists were ineffective as displacers of the binding. Agonists selective for the other subclasses did not affect the binding, d-2-Amino-5-phosphonovaleric and d-2-amino-7-phosphonoheptanoic acids exhibited more potent inhibition of the binding than their respective l-isomers, with the other aminophosphonates being inactive. Preincubation of synaptic membranes with different SH-reactive agents invariably resulted in a significant reduction of [(3)H]CPP binding, without significantly affecting NMDA-sensitive [(3)H]Glu binding. These results suggest that a filtration assay method is also applicable to detecting the binding of an antagonist highly selective for the NMDA-sensitive receptors to the NMDA recognition sites, in addition to a centrifugation assay method. Heterogeneity of the NMDA recognition sites is also suggested.

Differential potentiation by spermidine of abilities of a variety of displacers for [3H]MK-801 binding in hippocampal synaptic membranes.

Binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) to an ion channel associated with the N-methyl-D-aspartate (NMDA)-sensitive subtype of brain excitatory amino acid receptors was studied in Triton-treated preparations of synaptic membranes of rat brain. The initial association rate of the binding measured at 30 min after onset of incubation was markedly potentiated by the addition of either L-glutamic acid (Glu) alone or both Glu and glycine (Gly) in a concentration-dependent manner at 10 nM to 0.1 mM. Potentiation occurred to a significantly greater extent in the hippocampus and cerebral cortex than in the cerebellum. In the presence of both Glu and Gly, the endogenous polyamine spermidine (SPD) further potentiated binding in hippocampal and cortical membranes at concentrations above 10 microM without significantly affecting that in cerebellar membranes. The binding of [3H]MK-801 was slowly equilibrated in 16 h. When examined in hippocampal synaptic membranes, the binding at equilibrium was markedly displaced by numerous noncompetitive antagonists for the NMDA receptor. The addition of SPD markedly enhanced potencies of those displacers having a high affinity to [3H]MK-801 binding sites, without affecting other displacers having a low affinity. These results suggest that SPD promotes transition of sites responsible for mediating NMDA responses within the channel to a state with higher affinity for noncompetitive blockers.

Competitive inhibition of NMDA-mediated responses by guanine nucleotides in brain synaptic membranes treated with Triton X-100.

The effect of guanine nucleotides on physiological responses mediated by the N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors was examined by using NMDA-sensitive [3H]L-glutamic acid (Glu) binding as well as [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) binding in rat brain synaptic membranes treated with a low concentration of Triton X-100. The NMDA-sensitive [3H]Glu binding was significantly inhibited by the addition of some guanine nucleotides such as GTP, GDP, 5'-guanylylimidodiphosphate and guanosine 5'-O-(3-thiotriphosphate), but not by other nucleotides or nucleosides such as guanosine, cyclic GMP, adenosine, AMP, ADP, ATP, CTP, ITP and UTP. Inclusion of GTP not only attenuated the ability of NMDA to displace [3H]Glu binding in a concentration-dependent manner, but also lowered the affinity of the binding sites for [3H]Glu without altering their densities. The inhibitory potency of an antagonist highly selective to the NMDA receptors (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate on [3H]Glu binding also deteriorated with GTP at concentrations above 10 microM. Addition of Glu induced a concentration-dependent potentiation of [3H]MK-801 binding through an activation of the NMDA-sensitive receptors, and the potency of Glu to potentiate the binding was markedly reduced by the afore-mentioned positive guanine nucleotides in a competitive manner. In contrast, GTP at 0.1 mM non-competitively weakened the stimulatory property of glycine to additionally enhance the binding found in the presence of Glu alone. These results suggest that some guanine nucleotides may have a relatively high affinity for NMDA recognition sites within the NMDA receptor complex in the brain.

Profiles of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine binding in brain synaptic membranes treated with Triton X-100.

Binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) was examined using rat brain synaptic membranes treated with a low concentration of Triton X-100. This compound is assumed to be a non-competitive antagonist for the N-methyl-D-aspartate(NMDA)-sensitive subclass of central excitatory amino acid receptors. Binding was quite low but detectable in Triton-treated membranes irrespective of the incubation temperature, and the temperature-dependent portion of the binding was greatly reduced in these Triton-treated membranes. However, binding was drastically potentiated by the inclusion of L-glutamate and its analogous amino acids in a concentration-dependent manner at a concentration range of 10 nM to 0.1 mM. Agonists for the NMDA-sensitive subclass also potentiated binding, with agonists for the other subclasses being ineffective. Glycine at a concentration above 10 nM was not only effective as a stimulant of potentiated binding by glutamate, but was also active in enhancing binding in the absence of added glutamate. Glycine increased both the association and dissociation rates without significantly affecting the dissociation constant. Pharmacological profiles of binding in Triton-treated membranes were not significantly different from those in untreated membranes, except for that of haloperidol. Haloperidol is proposed to be highly selective for brain sigma-receptors on the basis of a potent inhibition of sigma-receptor binding. The inhibitory potency of this sigma-ligand was markedly attenuated in the presence of both glutamate and glycine in Triton-treated membranes, as compared with that in untreated membranes. These results suggest that [3H]TCP binding in Triton-treated membranes is a useful biochemical tool to evaluate predominantly the activated state of ion channels associated with the NMDA-sensitive receptors in terms of freedom from the confounding effects of endogenous amino acids.

Supporting evidence for negative modulation by protons of an ion channel associated with the N-methyl-D-aspartate receptor complex in rat brain using ligand binding techniques.

The addition of L-glutamic acid (Glu) alone, both Glu and glycine (Gly) or Glu/Gly/spermidine (SPD) was effective in potentiating [3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine (MK-801) binding before equilibrium to an ion channel associated with the N-methyl-D-aspartate (NMDA) receptor complex in brain synaptic membranes extensively washed and treated with Triton X-100. The binding dependent on Glu almost linearly increased in proportion to decreasing proton concentrations at a pH range of 6.0 to 9.0 in external incubation medium, while a Gly-dependent portion of the binding increased with decreasing proton concentrations up to a pH of 7.5 with a plateau thereafter. In contrast, the SPD-dependent binding increased in proportion to decreasing proton concentrations up to a pH of 7.0 with a gradual decline thereafter. Similar profiles were also obtained with [3H]MK-801 binding at equilibrium, with an exception that significant binding of [3H]MK-801 was detected in the absence of any added agonists. The potency of SPD to potentiate [3H]MK-801 binding before equilibrium increased in proportion to decreasing proton concentrations, with those of both Glu and Gly being unchanged. In contrast, the ability of (+)MK-801 to displace [3H]MK-801 binding at equilibrium was not significantly affected by a decrement of external proton concentrations from pH 7.5 to pH 8.5 in the presence of Glu/Gly and Glu/Gly/SPD added. However, similar changes in external proton concentrations did not similarly affect binding of several radioligands for the NMDA and Gly domains on the receptor complex. Decreasing proton concentrations were effective in exponentially potentiating binding of [3H]SPD at a pH range of 6.0 to 9.0 without virtually altering [3H]D,L-alpha-amino-3- hydroxy-5-methyl-isoxazole-4-propionic acid binding. In addition, [3H]kainic acid binding markedly decreased with decreasing proton concentrations only in the presence of Ca2+ ions. These results suggest that protons negatively modulate neuronal responses mediated by the NMDA receptor ionophore complex through interference with opening mechanisms of the channel domain without disturbing association processes of the endogenous agonists with the respective recognition domains in rat brain. Moreover, possible modulation by protons of responses mediated by the kainate receptor in the presence of Ca2+ ions at concentrations that occur in vivo is also suggested.

Identification and characterization of specific binding sites of [3H]spermidine in synaptic membranes of rat brain.

Synaptic membranes of rat brain contained specific binding sites of [3H]spermidine (SPD) that exhibited an inverse temperature dependency, structure selectivity, reversibility, saturability, low affinity and high density with an uneven distribution profile. The affinities were not significantly different from each other in the rodent brain, while the highest density was found in the medulla-pons among the central structures examined with progressively lower densities in the midbrain, striatum, cerebellum, hypothalamus, hippocampus and cerebral cortex. The binding was insensitive to digestion by various proteases and glycosidases but sensitive to potentiation by phospholipases. A clear correlation was seen between the abilities of several natural and synthetic polyamines to displace [3H]SPD binding and to potentiate [3H] (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine binding to open cation channels associated with an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors. Treatment of brain membranes with deoxycholic acid resulted in a significant solubilization of [3H]SPD binding sites. Furthermore, [3H]SPD markedly associated with the acidic phospholipid phosphatidylserine irrespective of the presence of synaptic membranes in a manner sensitive to inhibition by a variety of calmodulin antagonists. These results suggest that endogenous polyamines may play a stimulatory role in neuronal responses mediated by the NMDA receptor ionophore complex through an interaction between their positive charges and negative charges of membranous phosphatidylserine in rat brain.

Simultaneous determination of binding of a variety of radioligands related to ionotropic excitatory amino acid receptors in fetal and neonatal rat brains.

Expression of ionotropic excitatory amino acid receptors was assessed by membrane binding assays using a variety of radioligands in fetal and neonatal rat brains. In fetal rat brain, receptors sensitive to N-methyl-D-aspartate (NMDA) exhibited delayed onset of expression during the last 7 days before birth as compared with those insensitive to NMDA. In addition, developmental increases in agonist-preferring sites preceded those in antagonist-preferring sites within the first 7 postnatal days in particular brain structures with respect to each domain on the NMDA receptor complex. Growth of animals led to drastic increments of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801) binding to the NMDA channel in telencephalic regions until 21 to 28 days after birth, with concomitant desensitization to inhibition by protons of [3H]MK-801 binding in cortical membranes. By contrast, three different agonists were invariably effective in more potently potentiating [3H]MK-801 binding in cortical membranes of 14- and 28-day-old rats than in those of 5-day-old rats. These results suggest that the NMDA-sensitive subclass may play more critical roles in mechanisms underlying postnatal development of rat telencephalon than do the NMDA-insensitive subclasses.

Search for novel ligands selective at a polyamine recognition domain on the N-methyl-D-aspartate receptor complex using membrane binding techniques.

Among over 60 polyamine derivatives tested, only N-(3-aminopropyl)octanediamine and bis-(3-aminopropyl)nonanediamine (TE393) markedly inhibited [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) binding at equilibrium in the presence of added spermidine (SPD) in "non-washed" rat brain synaptic membranes, without affecting that in the absence of added SPD. Although TE393 significantly potentiated [3H]MK-801 binding before equilibrium in the presence of L-glutamic acid (Glu) alone or both Glu and glycine (Gly) added in "Triton-treated" membranes, the putative polyamine antagonists 1,10-decanediamine (DA10) and arcaine invariably inhibited binding irrespective of the addition of agonists. In the absence of added SPD, in addition, TE393 markedly enhanced abilities of both Glu and Gly to potentiate [3H]MK-801 binding before equilibrium. However, TE393 induced a rightward shift of the concentration-response curve of SPD for [3H]MK-801 binding before equilibrium. Moreover, TE393 was effective in potentiating binding of an antagonist but not an agonist radioligand to the NMDA domain and in inhibiting binding of an antagonist but not an agonist radioligand to the Gly domain. The potentiation of NMDA antagonist binding by TE393 occurred in a manner sensitive to prevention by arcaine but not by DA10. These results suggest that TE393 may be a novel ligand at the polyamine domain with an ability to interact with both the NMDA and Gly recognition domains in antagonist-preferring forms.