Reiko Fujita

Blockade of ionotropic receptor responses by progesterone in the ganglion cells of Aplysia.

To compare nongenomic effects of progesterone on various receptor responses of neurons, Aplysia ganglion cells were pretreated with 30 microM progesterone for 5 min and various receptor responses were tested using a conventional voltage-clamp method. Progesterone reduced nicotinic receptor-activated Na(+)-currents, nicotinic receptor-activated Cl(-)-currents, gamma-aminobutyric acid receptor-activated Cl(-)-currents, and dopamine receptor-activated Na(+)-currents. These depressant effects are similar at two different agonist concentrations. On the other hand, progesterone affected neither muscarinic receptor-activated K(+)-currents nor dopamine receptor-activated K(+)-currents. The former four types of receptors are known to be ionotropic while the latter two types of receptors are known to be metabotropic. Therefore, progesterone selectively inhibited all the types of ionotropic receptor responses, presumably in a noncompetitive manner.

Blocking effect of serotonin on β-adrenoceptor activity in follicle-enclosed Xenopus oocytes

The effects of serotonin (5-HT) on a beta-adrenergic response were studied, using the voltage clamp technique, in Xenopus laevis oocytes surrounded by their follicular cells. noradrenaline induced marked hyperpolarization, with a specific increase in the permeability of the membrane toward K+. Application of 10 microM 5-HT had little effect on the resting membrane, but significantly depressed the response to 0.1 microM noradrenaline. The dose-response curve for noradrenaline showed a parallel shift to the right in the presence of 5-HT, suggesting that 5-HT competes with noradrenaline for common binding sites at the beta-adrenoceptor.

Depressing Effects of Protein Kinases a & c on the Receptor-Induced K+-Current Responses in the Ganglion Cells of Aplysia

Application of either dopamine (DA), acetylcholine (ACh), histamine (HA), or Phe-Met-Arg-Phe-NH2 (FMRFamide) induces a K+-current response in the ganglion cells of Aplysia under voltage clamp. We have previously reported that these responses are all mediated by a pertussis toxin (PTX)-sensitive G-protcin Gi or Go. Intracellular application of cAMP, an activator of protein kinase A (PKA), or extracellular application of 30 μM phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), markedly suppressed these transmitter-induced K+-current responses. The depressing effect of cAMP was reversible while that of PDBu was irreversible as observed for 1 hour. Intracellular injection of the catalytic subunit of either PKA or PKC mimicked the effects of cAMP or PDBu. Pretreatment of the ganglion cells with 100 μM H-7, an inhibitor of PKC, significantly suppressed the effect of PDBu. Furthermore, an intraccllular injection of okadaic acid (OA), an inhibitor of phosphatases, facilitated the blocking effects of both cAMP and PDBu. The dose-response curve obtained by each transmitter-receptor system shifted downward by application of either cAMP or PDBu without affecting the affinity of the agonist to each receptor. K+-channel opening directly induced by guanosine thiotriphosphate (GTPγS) or by raising the temperature was not depressed by cither cAMP or 100 μM PDBu. From these results, we postulated that the acting sites of both PKA and PKC might be somewhere between the receptors and G-protein, and that the phosphorylation of these sites would impair the functional coupling efficiency between the receptors and G-protein.

THE INFLUENCE OF HYDROGEN PEROXIDE AND PH ON THE MOHR TITRATION

The official Japanese method for determination of chloride in hot-spring waters requires any H(2)S present to be oxidized with hydrogen peroxide in ammoniacal medium. When this was done, and the solution was titrated with silver nitrate at pH 9.7, the indicator colour changed from yellow to brownish grey, owing to reduction of silver ions to the metal by hydrogen peroxide. The effect can be eliminated by adjusting the pH to 7.0-7.5 before the titration.

Blocking effect of insulin on the K+ current response induced by adenosine and FSH in the follicle-enclosed Xenopus oocytes

in DKO mice. On the other hand, the reduction of GluA4 in -7-KO granular layer reflected its loss at mossy fiber-granule cell synapses, whereas the reduction of GluA1 and GluA4 in -7-KO molecular layer was caused, at least partly, by their loss in Bergmann glia. These findings suggest -2 and -7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and -7 alone promotes glial expression.

Facilitation of dopamine-induced Na+ current response by Rho GTPase and phospholipase D in Aplysia neurons

In the terrestrial slug Limax, NO is necessary for the synchronous oscillation of the local field potential in the procerebrum, which is thought to be involved in the odor discrimination and/or odor aversion learning. But there is no description about the genomic structure of the molluscan NO synthase, nor has the evolutionary origin of neuronal NO synthase (NOS) of mammals been clarified. Here we identified two types of NOS mRNAs of the slug, which show differential expression patterns within the brain. One is expressed broadly in the brain but in the low level (limNOS1), whereas, interestingly, the other is almost exclusively in the procerebrum (limNOS2). We also determined the whole genomic structure for limNOS1. It is composed of as many as 32 exons comparable to human nNOS gene, and has very similar exon–intron structure to that of human nNOS. Our results indicated that Limax NOS and human nNOS share the prototypical gene structure of NOS, and that their organization is highly conserved during the evolutionary history.

Change in superfusion rate alters excitatory synaptic transmission in rat hippocampal slice

Cbln1, a member of the C1q and tumor necrosis factor superfamily, plays crucial roles as a cerebellar granule cell-derived transneuronal regulator for synapse integrity and plasticity in Purkinje cells. Although other Cbln family members Cbln2–Cbln4 have distinct spatial and temporal patterns of expression throughout the central nervous system, their biochemical and biological properties have remained largely uncharacterized. Here, we demonstrated that Cbln2 and Cbln4 were secreted glycoproteins like Cbln1. In contrast, Cbln3 was not secreted when expressed alone, and retained in the endoplasmic reticulum or cis-Golgi. In wild-type granule cells expressing Cbln1 and Cbln3, Cbln3 proteins were secreted and reached postsynaptic sites on Purkinje cell dendrites, while Cbln3 was dramatically degraded in cbln1-null granule cells. These results indicate that Cbln2–Cbln4 may also serve as a trans-neuronal regulator of synaptic functions in various brain regions.

Facilitatory Effect of Calmodulin-Dependent Protein Kinase on the K+-Current Responses to Dopamine, Acetylcholine, and Phe-Met-Arg-Phe­ NH2 in the Ganglion Cells of Aplysia

Formation of either long-term potentiation (LTP) or long-term depression (LTD) of the synaptic transmission is thought to be the primary requirement for learning and memory. These LTP and LTD are usually produced by the activation of various enzymes at both pre- and postsynaptic loci after the transsynaptic stimulation of the receptors. We previously reported that acetylcholine (ACh)-, dopamine (DA)-, histamine (HA)-, and Phe-Met-Arg-Phe-NH2 (FMRFamide)-induced K+-current responses are all mediated by common GTP-binding protein Gi or G0, irrespective of their kinds of transmitters or types of receptors (Sasaki and Sato 1987). Furthermore, we recently reported that all these K+-current responses are markedly depressed by the activation of intracellular protein kinase A or C (Sasaki et al. 1997). At present we are considering the possibility that other kinases, such as calmodulin-dependent protein kinase (CaMK) and protein tyrosine kinase, or various protein phosphatases may also be activated simultaneously after the stimulation of these receptors, producing the primary reactions necessary for the formation of LTP or LTD (Kawasaki et al. 1998). This study examines the role of CaMK activation on the DA-, ACh-, and FMRFamideinduced K+-current responses in the ganglion cells of Aplysia, and discusses the possible acting site of this enzyme in the signal-transducing pathway involved in these receptor-induced responses.

Quisqualic acid produces a novel K+-current response in identified neurons of aplysia ganglion

Extracellular application of either quisqualic acid (QA) or Phe-Met-Arg-Phe-NH1 (FMRFamide) to the identified neurons of Aplysia ganglion elicits a slow K’-current response under voltage clamp. The QA-induced K’-current response was markedly depressed in the presence of CNQX, an antagonist for non-NMDA receptors. On the other hand, application of kainate and AMPA, agonists for non-NMDA receptors, did not induce any significant response in the same neurons. QA-induced Kf-current response was not depressed at all by intracellular injection of guanosine S--O-(2-thiodiphosphate) (GDP@), while the FMRFamide-induced response was completely blocked by GDP-OS in the same cell. However. both the QAand FMRFamide-induced K’-current responses decreased markedly when the temperature was lowered to 15 “C from 22 “C. These results indicate that the QA-induced Kt-current response does not conform in its pharmacological characteristics to any of the known glutamate-induced responses in vertebrate.

Regulatory roles of calmodulin-dependent protein kinase and phosphatase on the receptor-induced K+-current responses

009 EGUIJ,TORY ROLES OF CALMODULIN-DEPENDENT PROTEIN KINASE AND PHOSPHATASE ON THE RECEPTOR-INDUCED K+-CURRENT RESPONSES SATOSHI KAWASAKI’, SHINGO KIMURA’, REIKO FUJITA’, KOICHIRO TAKASHInlA’, MITSUHIKO MATSUMOT02, KAZUHIKO SASAKI’ ‘Dept. of Physiology, School of Medicine, Iwate Medical Univ., Morioka 020-8505, ‘Lab. 01’ Physics, School of Allied Medical Sciences, Hirosaki Univ., Hirosaki 036-8203 Application of dopamine, acetylcholine , or Phe-Met-Arg-Phe-NH, to identified neurons of Aplysia ganglia induces identical K’-current responses which are produced by activation of specific G-proteins of the Gi family. These K’-current responses were depressed by intracellular injection of either KN-9.3, an inhibitor for Ca”-calmodulin dependent protein kinase, W-7, a calmodulin inhibitor, or U-73 122, a phospholipase C inhibitor, irrespective of their receptor tj pes. In contrast, intracellular injection of cypermethrin, an inhibitor of calcineurin, significantly augmented the K’-current responses. Howe\ier, all the above inhibitors did not affect at all the K’-current response elicited by direct activation of G-protein via intracellular apphcation of GTPyS. These results suggest that both Ca”-calmodulin dependent protein kinase and calcineurin are physiologically activated by stimulation of these receptors, regulating the opening process of the receptor-operated Kkhannels, and that the acting site of these enzymes might be localized somewhere between the receptors and G-protein.