Discrimination by added ions of ligands at ionotropic excitatory amino acid receptors insensitive to N-methyl-D-aspartate in rat brain using membrane binding techniques.

The addition of potassium thiocyanate almost quadrupled binding of [3H]DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) to an AMPA-sensitive subclass of brain excitatory amino acid receptors in rat brain synaptic membranes, treated with Triton X-100. Among several ligands tested, quisqualic acid (QA) was the most potent displacer of [3H]AMPA binding in the absence of added SCN- ions, followed by AMPA, 6,7-dinitroquinoxaline-2,3-dione (DNQX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), glutamic (Glu) and kainic (KA) acids in a rank order of decreasing potency. The addition of SCN- ions was effective in significantly reducing the potencies of antagonists such as DNQX and CNQX, without affecting those of agonists including QA, AMPA, Glu and KA.

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.

Differential profiles of binding of a radiolabeled agonist and antagonist at a glycine recognition domain on the N-methyl-D-aspartate receptor ionophore complex in rat brain.

Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [3H]5,7-dichlorokynurenic acid ([3H]-DCKA) a Gly recognition domain on the N-methyl-D-aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [3H]DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (+/-)-alpha-(4-chlorophenyl)-4-[(4-fluorophenyl) methyl]-1-piperidine ethanol, with [3H]Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil.

Potentiation by polyamines of an interaction of noncompetitive antagonists at the N-methyl-D-aspartate receptor ionophore complex with phosphatidylserine.

The addition of phosphatidylserine induced a significant interaction with radioligands widely used for labeling an ion channel associated with an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, such as [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) and [3H]N-[1-(2-thienyl)cyclohexyl]piperidine, in the absence of brain synaptic membranes irrespective of the addition of either L-glutamic acid or glycine when determined in the presence of the polyamine spermidine. The interaction with [3H]MK-801 increased in proportion to increasing concentrations of phosphatidylserine added at concentrations from 0.01 to 0.

Further evidence for multiple forms of an N-methyl-D-aspartate recognition domain in rat brain using membrane binding techniques.

Pretreatment with sulfhydryl-reactive agents, such as N-ethylmaleimide and p-chloromercuriphenylsulfonic acid, invariably resulted in marked inhibition of the binding of DL-(E)-2-amino-4-[3H]propyl-5-phosphono-3-pentenoic acid ([3H]CGP 39653), a competitive antagonist at an N-methyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors, in brain synaptic membranes extensively washed and treated with Triton X-100, but did not significantly affect the binding of L-[3H]-glutamic acid ([3H]Glu), an endogenous agonist. The pretreatment was effective in reducing the binding of [3H]-CGP 39653 at equilibrium, without altering the initial association rate, and decreased the affinity for the ligand. Pretreatment with sulfhydryl-reactive agents also enhanced the potencies of NMDA agonists to displace [3H]-CGP 39653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [3H]Glu binding.

Selectively high expression of the transcription factor AP1 in telencephalic structures of epileptic E1 mice.

In ddY mouse brain, the transcription factors AP1 and CREB were rich in the cerebral cortex, hippocampus and cerebellum but relatively poor in the striatum, hypothalamus and midbrain. In contrast, the transcription factor Myc was rather poorly distributed in mouse brain under the conditions employed. Among these 3 transcription factors examined, DNA binding activities of only AP1 were invariably higher in telencephalic structures, such as the cortex, hippocampus and striatum, of the epileptic El mice than in those of parent ddY mice.

Multiplicity of [3H]1,3-di-o-tolylguanidine binding sites with low affinity for haloperidol in rat brain.

Specific binding of [3H]1,3-di-o-tolylguanidine (DTG) was found not only in synaptic membrane fractions but also in subcellular fractions enriched of microsomes, nuclei and mitochondria/myelins, with different sensitivities to displacement by the antipsychotic haloperidol. The highest binding was detected in microsomal fractions followed by, in order of decreasing binding, fractions enriched in nuclei, synaptic membranes, mitochondria/myelins and homogenates. [3H]DTG binding was completely abolished by prior treatment of the synaptic membranes with a low concentration of Triton X-100.

Excitatory amino acid receptor binding in hippocampus of gerbils with transient global brain ischemia.

Binding of a variety of ligands for brain excitatory amino acid receptors was examined in membrane preparations extensively washed and treated with Triton X-100 that were obtained from the hippocampus and cerebral cortex of gerbils that survived for different periods after transient global brain ischemia. Bilateral occlusion of the carotid arteries for 5 min did not affect the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) to an open ion channel associated with the N-methyl-D-aspartate (NMDA)-sensitive subclass in both central structures of gerbils that survived for 1 to 4 weeks after the injury when determined at equilibrium in the presence of 3 different endogenous agonists including L-glutamic acid (Glu), glycine (Gly) and spermidine at maximally effective concentrations. In contrast, the ischemic occlusion significantly diminished [3H]MK-801 binding when determined before equilibrium in the presence of the 3 stimulants in hippocampal membranes without altering that in cortical membrane 2 weeks after the insult, so that the initial association rates were invariably reduced by more than 60%.

Comparative studies on binding of 3 different ligands to the N-methyl-D-aspartate recognition domain in brain synaptic membranes treated with Triton X-100.

Treatment with a low concentration of Triton X-100 almost tripled the binding of [3H]D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653), a novel competitive antagonist at an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, in synaptic membranes of the rat brain. The binding linearly increased with increasing protein concentrations of up to 0.4 mg/ml and also increased in proportion to incubation time with a plateau within 60 min after the initiation of incubation at 2 degrees C in Triton-treated membranes.

Cellular and intracellular localization of epsilon-subspecies of protein kinase C in the rat brain; presynaptic localization of the epsilon-subspecies.

The cellular and intracellular localization of the epsilon-subspecies of protein kinase C (PKC) in the rat brain was demonstrated by immunocytochemistry using specific antibodies against epsilon-PKC. The epsilon-PKC-specific immunoreactivity was most abundant in the hippocampal formation, olfactory tubercle and Calleja's islands, was moderate in the cerebral cortex, anterior olfactory nuclei, accumbens nucleus, lateral septal nuclei and caudate-putamen and low in the thalamus and medulla. The epsilon-PKC-immunoreactivity was scanty in the perikarya, except for the pyramidal cells of CA3 region of the hippocampus and the immunoreactivity was mainly present in neuropils and nerve fibers.

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.

Selective increase of the alpha subspecies of protein kinase C and inhibition of melanogenesis induced by retinoic acid in melanoma cells.

Retinoic acid (RA) has been shown to inhibit melanogenesis in B16 mouse melanoma cells (B16 cells). On the other hand, it has been reported that RA increases protein kinase C (PKC) activity in these cells. Further investigation was carried out to identify the PKC subspecies expressed in B16 cells and to examine the changes in the level of each PKC subspecies by RA treatment.

Support for radiolabeling of a glycine recognition domain on the N-methyl-D-aspartate receptor ionophore complex by 5,7-[3H]dichlorokynurenate in rat brain.

Pretreatment with Triton X-100 more than doubled the binding of radiolabeled 5,7-dichlorokynurenic acid (DCKA), a proposed antagonist at a glycine (Gly) recognition domain on the N-methyl-D-aspartate (NMDA) receptor ionophore complex, in rat brain synaptic membranes. The binding exhibited an inverse temperature dependency, reversibility, and saturability, the binding sites consisting of a single component with a high affinity (27.5 nM) and a relatively low density (2.

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.

Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-delta subspecies.

Several lines of CHO cells stably overexpressing protein kinase C (PKC) subspecies to various extents were established by the DNA-mediated transfer. Upon treatment with phorbol 12-myristate 13-acetate, the growth of the cells expressing the PKC-delta subspecies was markedly inhibited, whereas cell lines expressing PKC-alpha, PKC-beta II, and PKC-zeta subspecies were not significantly affected. Flow cytometric analysis indicated that all cell lines overexpressing PKC-delta subspecies accumulated in G2/M phase in response to phorbol 12-myristate 13-acetate.

Enzymatic properties of ubiquitously expressed delta-subspecies of protein kinase C differing from other members of protein kinase C family.

The delta-subspecies of protein kinase C (PKC) was purified to near homogeneity from the Triton X-100 extract of the rat brain particulate fraction by successive chromatographies on S-Sepharose Fast Flow, Phenyl 5PW, Heparin 5PW, hydroxyapatite, and Mono Q columns. The purified enzyme was doublet with molecular weight of 78 kDa and 76 kDa on SDS-PAGE. This doublet proteins were separated partially by Mono Q column chromatography, both of which were recognized by the antibodies raised against synthetic oligopeptides, parts of the deduced amino acid sequence of the rat delta PKC.

Expression of protein kinase C subspecies in rat retina.

An extract of rat retina was subjected to Mono Q followed by chromatography on hydroxyapatite, and the protein kinase C (PKC) subspecies were identified by immunoblot and biochemical analysis. It was found that, although the relative activities assayed with myelin basic protein as a common phosphate acceptor vary greatly with one another, the alpha-, beta I-, beta II-, gamma-, delta-, epsilon-, zeta-, and another structurally unknown PKC subspecies are expressed in this tissue. Thus, the retina is a unique tissue which expresses most of the PKC subspecies so far identified in mammals.

Inhibition by calmodulin antagonists of [3H]MK-801 binding in brain synaptic membranes.

In brain synaptic membranes not extensively washed, (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine ([3H]MK-801) binding was markedly inhibited in a concentration-dependent manner (at concentrations above 1 microM) by several compounds having antagonistic activity at the Ca(2+)-binding protein calmodulin. Scatchard analysis revealed that N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the binding through a significant decrease in the density of binding sites without affecting the affinity at 10 microM. In membranes extensively washed and treated with a low concentration of Triton X-100, L-glutamic acid (Glu) drastically accelerated the initial association rate of [3H]MK-801 binding with glycine (Gly), almost doubling the initial association rate found in the presence of Glu alone.

Differential modulation by divalent cations of [3H]MK-801 binding in brain synaptic membranes.

Endogenous divalent cations, such as Mg2+, Ca2+, and Zn2+, differentially affected the binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801) to an ion channel associated with an N-methyl-D-aspartate-sensitive subclass of excitatory amino acid receptors in different preparations of brain synaptic membranes. Both Mg2+ and Ca2+ were weak inhibitors of the binding in membranes which had not been extensively washed (nonwashed membranes), over a concentration range effective in markedly potentiating the binding in the absence of any added stimulants in membranes which had been extensively washed, but not treated with a detergent (untreated membranes). In membranes extensively washed and treated with Triton X-100 (Triton-treated membranes), both cations significantly potentiated the binding in the presence of added glutamate alone.

Ionotropic excitatory amino acid receptors in discrete brain regions of kindled rats.

A study was performed to examine the specific binding of excitatory amino acid (EAA) receptor subtypes in 5 brain regions of rats kindled from the amygdala or hippocampus, using extensively washed and Triton X-100-treated membranes. Seven days after the last amygdala kindled seizure, [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine maleate ([3H]MK-801) binding, which labels N-methyl-D-aspartate (NMDA)-sensitive receptor-linked cation channels, decreased significantly only in the amygdala of kindled rats compared to that of controls under equilibrium assay conditions. There was no significant change in [3H]MK-801 binding in the amygdala or hippocampus 7 days after the last hippocampal kindled seizure, or 28 days after the last amygdala kindled seizure.

Catalytic properties of yeast protein kinase C: difference between the yeast and mammalian enzymes.

With bovine myelin basic protein as a model common substrate, protein kinases C (PKC) purified from yeast (Saccharomyces cerevisiae) and mammalian tissue (rat brain) were shown to exhibit clearly different catalytic properties. The major sites of phosphorylation in bovine myelin basic protein by the yeast PKC were identified: Thr-19, Thr-34, and Thr-65. These sites are distinctly different from those for the mammalian PKC: Ser-8, Ser-46, Ser-55, Ser-110, Ser-132, Ser-151, and Ser-161, which were previously identified [Kishimoto, A.

Effects of ifenprodil on the N-methyl-D-aspartate receptor ionophore complex in rat brain.

The effects of a cerebral anti-ischemic drug ifenprodil on the receptor ionophore complex of an N-methyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors were examined using [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine (MK-801) binding in rat brain synaptic membrane preparations as a biochemical measure. The binding in membrane preparations not extensively washed was markedly inhibited not only by competitive NMDA antagonists such as (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic, D-2-amino-5-phosphonovaleric and D-2-amino-7-phosphonoheptanoic acids, but also by competitive antagonists at the strychnine-insensitive glycine (Gly) site including 7-chlorokynurenic acid and 6,7-dichloroquinoxaline-2,3-dione. Among several proposed ligands for alpha-adrenergic receptors tested, ifenprodil most potently inhibited the binding in these membrane preparations due to a decrease in the density of the binding sites without significantly affecting the affinity.

Excitatory amino acid receptors in brains of rats with methylazoxymethanol-induced microencephaly.

We used methylazoxymethanol-acetate (MAM), a potent alkylating agent, to produce microencephaly in offspring by injecting it into pregnant rats on day 15 of gestation. Binding activities of central excitatory amino acid receptors were examined in Triton-treated membranes prepared from brains of adult offspring with MAM-induced microencephaly (MAM rats). MAM rats exhibited approximately 40-50% reductions of the wet weights of the cerebral cortex, hippocampus and striatum compared to those in controls.

Presence of the binding of a variety of ligands related to ionotropic excitatory amino acid receptors in rat retina.

The binding of [3H]L-glutamic (Glu), [3H](+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic and [3H]D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acids was detected in rat retinal membranes extensively washed and treated with a low concentration of Triton X-100, in addition to the binding of both [3H]glycine (Gly) and [3H]5,7-dichlorokynurenic acid. Furthermore, retinal membranes exhibited the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne in the presence of Glu, Gly and spermidine irrespective of the incubation period employed. Rat retina also contained the binding of [3H]kainic acid as well as the binding of [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid which was sensitive to potentiation by potassium thiocyanate.