Kyoko Akita

Non-NMDA receptor mediates cytoplasmic Ca2+ elevation in cultured hippocampal neurones

Microscopic fluorometry of cultured rat hippocampal neurones revealed that the intracellular concentration of Ca2+ ([Ca2+]i) rises in response to the activation of excitatory amino acid receptor (EAA-R), which included not only N-methyl-D-aspartate subspecies but also kainate and quisqualate subspecies of EAA-R. Each EAA-R mediated [Ca2+]i rise consisted of the components dependent on and independent of the activity of the voltage-dependent Ca2+ channel. The receptor-mediated voltage-independent [Ca2+]i rise may be related to the modulation of synaptic transmission efficacy.

Mode of blockade by MK-801 of N-methyl-D-aspartate-induced increase in intracellular Ca2+ in cultured mouse hippocampal neurons.

Microfluorometry with fura-2 was applied to study the action of the anticonvulsant (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) on N-methyl-D-aspartate (NMDA)-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured mouse hippocampal neurons. MK-801 caused a potent and long-lasting blockade of the NMDA-activated [Ca2+]i elevation in a selective manner, not affecting the [Ca2+]i rise induced by quisqualate or kainate. Blockade and recovery from the blockade by MK-801 showed use dependency; the degree of blockade was dependent on the presence of NMDA. The use-dependent onset of antagonism was, however, highly sensitive to the bath temperature. MK-801 applied in the absence of NMDA had no effect on the response to subsequent application of NMDA at 22 degrees C, whereas it reduced the subsequent response to NMDA significantly at 37 degrees C. MK-801 interacted with the receptor-ion channel complex even when Mg2+, which is considered to block the open channel, had already blocked the NMDA-induced [Ca2+]i. The recovery from blockade by MK-801 was not accelerated by the application of 10 mM Mg2+ for 5 min. These results suggest that MK-801 can gain access to its binding site in the absence of NMDA at physiological temperature, and that this binding site is distinct from that for Mg2+.

A significant increase in intracellular Ca2+ concentration induced by (2S,3R,4S)-2-(car☐ycyclopropyl)glycine, a new potent NMDA agonist, in cultured rat hippocampal neurons

The increase in intracellular Ca2+ concentration, [Ca2+]i, induced by isomers of 2-(carboxycyclopropyl)glycine (CCG) was examined in cultured rat hippocampal neurons. Some CCG isomers and N-methyl-D-aspartate (NMDA) increased [Ca2+]i in a concentration dependent manner. The 2S,3R,4S isomer of CCG (L-CCG-IV) was the most potent in elevating [Ca2+]i, and its activity was more than 100 times higher than that of NMDA and about 10 times higher than that of L-glutamate. The increase in [Ca2+]i was effectively blocked by NMDA blockers and Mg2+, and was markedly augmented by the addition of a low concentration of glycine. L-CCG-IV would be a useful tool for elucidation of functions of NMDA receptors.

MK-801 blocked the functional NMDA receptors in identified cerebellar neurons

In order to clarify the nature of N-methyl-D-aspartate (NMDA) receptors in cerebellum, where heterogeneity of the NMDA receptor has been suggested, we investigated the action of MK-801 on the NMDA-induced [Ca2+]i rise in cultured cerebellar neurons using video-assisted microfluorometry. MK-801 caused a potent and selective blockade of the NMDA-activated [Ca2+]i elevation. The blockade caused by MK-801 was dependent on the presence of NMDA, i.e., use-dependent. There was no difference in the mode of blockade between immunocytochemically identified Purkinje and non-Purkinje cells, although the relative size of the NMDA-induced [Ca2+]i rise was significantly less in Purkinje cells. These results indicate that the NMDA receptors in cultured cerebellar neurons are coupled with the same channels as those in other brain regions.

Binding characteristics of [3H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCl: the recovery of binding activity by dilution

This paper describes the binding properties of [3H]peptidergic opioid ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [3H]-[D-Ala2-, Met5]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl2 and 100 mM NaCl at 0 degree C (on ice) for 3 h. With this assay condition, the significant binding of following [3H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin (mu-type), [3H]-[D-Ala2, D-Leu5]enkephalin (delta-type) and [3H]dynorphin1-9 (kappa-type). The number of binding sites in solubilized preparations for each [3H]ligand corresponded to 40-50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [3H]ligand. Apparent dissociation constants between solubilized receptors and [3H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycodeoxycholate and NaCl and the dilution process.