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Depression by a phorbol ester of Cl− currents generated through cloned glycine receptors
S86
2-64
ACTIVITY-DEPENDENT
DEPRESSION
OF PARALLEL FIBER-PURKINJE
CELL
SYNAPSES UNDER INFLUENCES OF A PROTEIN PHOSPHATASE INHIBITOR. AYAKO AJIMA, YOSHIHIDE TAMORI AND MASAO ITO, Frontier Research Program, Wako,
Saitama
351-01,
In slices fibers induced sively depressed under perfusion rate of decline suggesting depression which is constant depression
RIKEN,
Japan
of guinea pig cerebellum, electric stimulation of parallel monosynaptic EPSPs in Purkinje cells. These EPSPs were progres in magnitude when parallel fibers were continuously stimulated of a protein phosphatase inhibitor, calyculin A at 0. 5,oM. The was dependent on the rate of parallel fiber stimulation,
that the depression was due to a cumulative effect of a small induced at each transmission. However, the dependence was nonlinear, explicable by assuming that each small depression developed with a time of some seconds. The depression so demonstrated mimics longterm (LTD),
suggesting
a role
of
a protein
phosphatase
inhibitor
in
LTD.
2-65
PROTEIN KINASE C INHIBITORS SUPPRESS CYCLIC GMP RISE MEDIATED BY METABOTROPIC GLUTAMATE RECEPTORS IN RAT CEREBELLAR SLICES. DAISUKE OKADA Laboratory Frontier Research Program, RIKEN, 2-l Hirosawa, for Neural Networks, Wako, Saitama, JAPAN 351-01 My previous work showed that a specific agonist of metabotropic glutamate receptor, trans-ACPD (ACPD), raised cyclic GMP (cGMP) levels in the cerebellar molecular layer. ACPD raised the cGMP level through activation of nitric oxide synthase. This activation was independent of calcium influx, and depends on phosphatidylinositol metabolism which generates a protein kinase C (PKC) activator, diacylglycerol. However, the possible involvement of PKC in the ACPD-induced cGMP rise has not been investigated. The present work was conducted to elucidate the involvement of PKC in the ACPD-induced cGMP rise. The cGMP levels in rat cerebellar slices were measured by radioimmunoassay, and the effects of two PKC inhibitors were studied. Calphostin C is a specific PKC inhibitor acting on the diacylglycerol binding site and staurosporine is a general protein kinase inhibitor acting on the catalytic domain. Both inhibitors reduced cGMP rise induced by ACPD at 0.3 mM (calphostin C at 1 uM for 30 min: 75% inhibition, n=5 and staurosporine at 0.3 uM for 30 min: 98% inhibition, n=5), without affecting the basal cGMP levels. These results indicate that phosphorylation by PKC regulates the cerebellar cGMP level.
DEPRESSION BY A PHORBOL ESTER OF Cl- CURRENTS GENERATED THROUGH CLONED GLYCINE RECEPTORS. MUTSUMI UCHIYAMA' HIROYUKI AKAGI'.KEIKO HIRAI'AND FUMIO HISHINUMA'. Departmentsof 'Anesthesiology mmacology, Gunma UniversitySchool of Medicine,Maebashi,Gunma 371 and 'Mitsubishi&& Instituteof Life Sciences.Machida.Tokyo 194, Japan.
Z-66
We have previouslyreportedon isolation and sequence of cDNAs encoding rat glycinereceptor subunits,al and a2 (Akagi,H. et al.,Neurosci.Res. 11, 28-40,1991;FEBS Lett.281, 160-166, 1991).Both al and a2 homomeric receptorsexpressedin Xenopus oocytespossessedan abilityto form functionalCl- channels.In the present experiments, we examined whether the propertiesof clonedglycinereceptoris affectedby phorbol 12-myristate 13-acetate(PMA),a potentactivatorof nroteinkinase C (PKC).Messenger RNA was transcribedin vitro from the al or a2 subunit cDNA ind injectedintokenipus oocyt&, which were submittedto electrophysiological study. The oocytes elicited inward currents in response to glycine (0.01mM to 3 mM) when oocytes were voltageclamped at -70 mV. PMA appliedby superfusioncaused depressionof the glycine-gatedcurrents through al and a2 receptorsin a concentration-dependent manner (1 to 30 nM). By the treatment with PMA (10 nM for 5 min),the glycine-gated currentswere reduced to 30-60 % of the controlresponse, and the effectpersistedfor longer than 60 min afterwashing out the drug. The same concentrations of PMA did not affectkainate-induced currentsin oocytesinjectedwith rat brain mRNAs. Saurosporine(a PKC inhibitor, l-5 PM) partially blockedthe actionof PMA.
2-64
ACTIVITY-DEPENDENT
DEPRESSION
OF PARALLEL FIBER-PURKINJE
CELL
SYNAPSES UNDER INFLUENCES OF A PROTEIN PHOSPHATASE INHIBITOR. AYAKO AJIMA, YOSHIHIDE TAMORI AND MASAO ITO, Frontier Research Program, Wako,
Saitama
351-01,
In slices fibers induced sively depressed under perfusion rate of decline suggesting depression which is constant depression
RIKEN,
Japan
of guinea pig cerebellum, electric stimulation of parallel monosynaptic EPSPs in Purkinje cells. These EPSPs were progres in magnitude when parallel fibers were continuously stimulated of a protein phosphatase inhibitor, calyculin A at 0. 5,oM. The was dependent on the rate of parallel fiber stimulation,
that the depression was due to a cumulative effect of a small induced at each transmission. However, the dependence was nonlinear, explicable by assuming that each small depression developed with a time of some seconds. The depression so demonstrated mimics longterm (LTD),
suggesting
a role
of
a protein
phosphatase
inhibitor
in
LTD.
2-65
PROTEIN KINASE C INHIBITORS SUPPRESS CYCLIC GMP RISE MEDIATED BY METABOTROPIC GLUTAMATE RECEPTORS IN RAT CEREBELLAR SLICES. DAISUKE OKADA Laboratory Frontier Research Program, RIKEN, 2-l Hirosawa, for Neural Networks, Wako, Saitama, JAPAN 351-01 My previous work showed that a specific agonist of metabotropic glutamate receptor, trans-ACPD (ACPD), raised cyclic GMP (cGMP) levels in the cerebellar molecular layer. ACPD raised the cGMP level through activation of nitric oxide synthase. This activation was independent of calcium influx, and depends on phosphatidylinositol metabolism which generates a protein kinase C (PKC) activator, diacylglycerol. However, the possible involvement of PKC in the ACPD-induced cGMP rise has not been investigated. The present work was conducted to elucidate the involvement of PKC in the ACPD-induced cGMP rise. The cGMP levels in rat cerebellar slices were measured by radioimmunoassay, and the effects of two PKC inhibitors were studied. Calphostin C is a specific PKC inhibitor acting on the diacylglycerol binding site and staurosporine is a general protein kinase inhibitor acting on the catalytic domain. Both inhibitors reduced cGMP rise induced by ACPD at 0.3 mM (calphostin C at 1 uM for 30 min: 75% inhibition, n=5 and staurosporine at 0.3 uM for 30 min: 98% inhibition, n=5), without affecting the basal cGMP levels. These results indicate that phosphorylation by PKC regulates the cerebellar cGMP level.
DEPRESSION BY A PHORBOL ESTER OF Cl- CURRENTS GENERATED THROUGH CLONED GLYCINE RECEPTORS. MUTSUMI UCHIYAMA' HIROYUKI AKAGI'.KEIKO HIRAI'AND FUMIO HISHINUMA'. Departmentsof 'Anesthesiology mmacology, Gunma UniversitySchool of Medicine,Maebashi,Gunma 371 and 'Mitsubishi&& Instituteof Life Sciences.Machida.Tokyo 194, Japan.
Z-66
We have previouslyreportedon isolation and sequence of cDNAs encoding rat glycinereceptor subunits,al and a2 (Akagi,H. et al.,Neurosci.Res. 11, 28-40,1991;FEBS Lett.281, 160-166, 1991).Both al and a2 homomeric receptorsexpressedin Xenopus oocytespossessedan abilityto form functionalCl- channels.In the present experiments, we examined whether the propertiesof clonedglycinereceptoris affectedby phorbol 12-myristate 13-acetate(PMA),a potentactivatorof nroteinkinase C (PKC).Messenger RNA was transcribedin vitro from the al or a2 subunit cDNA ind injectedintokenipus oocyt&, which were submittedto electrophysiological study. The oocytes elicited inward currents in response to glycine (0.01mM to 3 mM) when oocytes were voltageclamped at -70 mV. PMA appliedby superfusioncaused depressionof the glycine-gatedcurrents through al and a2 receptorsin a concentration-dependent manner (1 to 30 nM). By the treatment with PMA (10 nM for 5 min),the glycine-gated currentswere reduced to 30-60 % of the controlresponse, and the effectpersistedfor longer than 60 min afterwashing out the drug. The same concentrations of PMA did not affectkainate-induced currentsin oocytesinjectedwith rat brain mRNAs. Saurosporine(a PKC inhibitor, l-5 PM) partially blockedthe actionof PMA.