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Calcium-dependent chloride current is activated during spike after-hyperpolarization in rabbit parasympathetic neurons
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CALCIUM-DEPENDENT CHLORIDE CURRENT IS ACTIVATED DURING SPIKE AFTER-HYPERPOLARIZATION IN RABBIT PARASYMPATHETIC NEURONS. TAKASHI AKASU~ TOSHIHIKO NISHIMURA~ AND TAKAyLrKI TOKIMASA, Department of Physiolosy, Kurume University School of Medicine~ 67 Asahi-machi, Kurume 830~ Japan Intracellular and voltage-clamp recordings were made from neurons in rabbit pelvic (parasympathetic) ganglia (VPG) by using a single microelectrode filled with either 2 M CsCI or 2 M KCI. In a Krebs solution containing TEA (50 mM) and TTX (300 riM), depolarizing voltage jumps activated inward calcium currents followed by calcium-dependent inward tail currents at -30 to +30 mV, which were accompanied by a large increase in membrane conductance. Substitution of barium for calcium selectively reduced the tail current. Lowering external chloride activity increased the tail current amplitude without affecting the calcium current. The reversal potentials of the tail current measured using a twin-pulse protocol were -18 ± 5 (mean ± sem) (n=8) and 5 ± 3 mV (n=5) in Krebs solution and low C1 (62 mM) solution, respectively, suggesting a calcium-dependent chloride current. A stilbene derivative (SITS, i00 pM) blocked the tail current. Transient (T) and sustained (N and L) types of calcium currents are likely to co-exist in the rabbit VPG neurons. Calcium-dependent chloride current was activated by N- and L-type calcium currents but not by T-type current. Substantial activation of the calcium-dependent chloride conductance could occur during a post-tetanic after-potential when VPG neurons fire~ action potentials repetitively. It is suggested that calcium-dependent potassium and chloride currents contribute to the spike after-hyperpolarization of the action potential.
VOLTAGE-DEPENDENCE OF ACTIVATION OF CALCIUM CURRENTS IN PRESYNAPTIC TERMINALS OF CHICK CILIARY GANGLION. HIROMU YAWO, Department of Physiology~ Kyoto University Faculty of Medicine, YoshidaKonoe-cho, Sakyo-ku, Kyoto 606, Japan. Transmitter release from nerve terminals is triggered2bY Ca 2+ influx through voltage-gated Ca 2+ channels. The whole-cell recording of presynaptic Ca + currents was attempted from presynaptic calyces of chick ciliary ganglia. The ciliary ganglion of a chick embryo (12-14 days) was sliced. The calyces were identified under the microscope. When sulforhodamine 101 was applied at the cut end of the oculomotor nerve, the fluorescence accumulated in the calyciform nerve terminal enwrapping the ganglionic neuron. The Inactivating and non-inactivating subpopulatio~s of high-voltage-activated Ca + channels were present while the low-voltageactivated Ca + chan~el subtype was absent. Both inactivating and non-inactivating components were blocked by 50 ~M Cd + and by i0 ~M c0-conotoxin GVIA. The non-inactivating component was suppressed by D600 (100-250 pM) while the inactivating component was resistant. The relaxation of tail currents revealed the presence of 2 components in the non-inactivating Ca 2+ 2current, fastdeactivating and slow-deactivating. Therefore, 3 high-voltage activated Ca + channel subtypes are suggested to be present in the presynaptic terminal. The major component is the non-inactivating, fast-deactivating type which is very rapid in activation. The second component is fast in both activation and inactivation. A small component of Ca + current is classified as the noninactivating, slow-deactivating type which has a slightly lower threshold for a~tivation than the two other types. The slow-deactivating type may participate in subthreshold Ca L+ influx, while fast-deactivating types are suggested to be involved in excitation-secretion coupling.
INVOLVEMENT OF PROTEIN KINASE C IN MUSCARINIC EXCITATION OF RAT ADRENAL CHROMAFF1N CELLS. AKINORI AKAIKE 1,2, MASASHI SASA1 , HISAMITSU UJIHARA* 1, AND SHUJI TAKAORI 1, 1Department of Pharmacology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan, and ~Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshims 729-02, Japan. We investigated the second messenger system mediating cellular responses to muscarinic stimulation in adrenal chromaffin cells. Whole-cell currents were recorded from chromaffin cells freshly dissociated from the rat adrenal medulla. Application of either acetylcholine (ACh), muscarine or MeN-A-343, a selective M1 agonist, at 10-s M elicited firing of the cells recorded extracellularly. Phorbol dibutyrate, an activator of protein kinase C, at 10-~ M, increased the rate of ACh-indueed firing. ACh produced biphasic depolarization with fast and slow components, while muscarine, MeN-A-343 and phorbol dibutyrate induced persistent depolarization with slow onset. To examine the effects of a muscarinic agonist and phorbol dibutyrate on resting K + channels, inward rectifying current was elicited by passing a slow ramp command voltage from -140 to -40 mV. Currents induced by the ramp command voltage were markedly decreased with Cs + (2 mM} applied to the outside of the cell. MeN-A-343 and phorbol dibutyrate also decreased the inward current induced by the ramp command voltage. These results suggest that protein kinase C participates in the MI receptor-mediated suppression of the inward rectifying K channel. •
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CALCIUM-DEPENDENT CHLORIDE CURRENT IS ACTIVATED DURING SPIKE AFTER-HYPERPOLARIZATION IN RABBIT PARASYMPATHETIC NEURONS. TAKASHI AKASU~ TOSHIHIKO NISHIMURA~ AND TAKAyLrKI TOKIMASA, Department of Physiolosy, Kurume University School of Medicine~ 67 Asahi-machi, Kurume 830~ Japan Intracellular and voltage-clamp recordings were made from neurons in rabbit pelvic (parasympathetic) ganglia (VPG) by using a single microelectrode filled with either 2 M CsCI or 2 M KCI. In a Krebs solution containing TEA (50 mM) and TTX (300 riM), depolarizing voltage jumps activated inward calcium currents followed by calcium-dependent inward tail currents at -30 to +30 mV, which were accompanied by a large increase in membrane conductance. Substitution of barium for calcium selectively reduced the tail current. Lowering external chloride activity increased the tail current amplitude without affecting the calcium current. The reversal potentials of the tail current measured using a twin-pulse protocol were -18 ± 5 (mean ± sem) (n=8) and 5 ± 3 mV (n=5) in Krebs solution and low C1 (62 mM) solution, respectively, suggesting a calcium-dependent chloride current. A stilbene derivative (SITS, i00 pM) blocked the tail current. Transient (T) and sustained (N and L) types of calcium currents are likely to co-exist in the rabbit VPG neurons. Calcium-dependent chloride current was activated by N- and L-type calcium currents but not by T-type current. Substantial activation of the calcium-dependent chloride conductance could occur during a post-tetanic after-potential when VPG neurons fire~ action potentials repetitively. It is suggested that calcium-dependent potassium and chloride currents contribute to the spike after-hyperpolarization of the action potential.
VOLTAGE-DEPENDENCE OF ACTIVATION OF CALCIUM CURRENTS IN PRESYNAPTIC TERMINALS OF CHICK CILIARY GANGLION. HIROMU YAWO, Department of Physiology~ Kyoto University Faculty of Medicine, YoshidaKonoe-cho, Sakyo-ku, Kyoto 606, Japan. Transmitter release from nerve terminals is triggered2bY Ca 2+ influx through voltage-gated Ca 2+ channels. The whole-cell recording of presynaptic Ca + currents was attempted from presynaptic calyces of chick ciliary ganglia. The ciliary ganglion of a chick embryo (12-14 days) was sliced. The calyces were identified under the microscope. When sulforhodamine 101 was applied at the cut end of the oculomotor nerve, the fluorescence accumulated in the calyciform nerve terminal enwrapping the ganglionic neuron. The Inactivating and non-inactivating subpopulatio~s of high-voltage-activated Ca + channels were present while the low-voltageactivated Ca + chan~el subtype was absent. Both inactivating and non-inactivating components were blocked by 50 ~M Cd + and by i0 ~M c0-conotoxin GVIA. The non-inactivating component was suppressed by D600 (100-250 pM) while the inactivating component was resistant. The relaxation of tail currents revealed the presence of 2 components in the non-inactivating Ca 2+ 2current, fastdeactivating and slow-deactivating. Therefore, 3 high-voltage activated Ca + channel subtypes are suggested to be present in the presynaptic terminal. The major component is the non-inactivating, fast-deactivating type which is very rapid in activation. The second component is fast in both activation and inactivation. A small component of Ca + current is classified as the noninactivating, slow-deactivating type which has a slightly lower threshold for a~tivation than the two other types. The slow-deactivating type may participate in subthreshold Ca L+ influx, while fast-deactivating types are suggested to be involved in excitation-secretion coupling.
INVOLVEMENT OF PROTEIN KINASE C IN MUSCARINIC EXCITATION OF RAT ADRENAL CHROMAFF1N CELLS. AKINORI AKAIKE 1,2, MASASHI SASA1 , HISAMITSU UJIHARA* 1, AND SHUJI TAKAORI 1, 1Department of Pharmacology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan, and ~Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshims 729-02, Japan. We investigated the second messenger system mediating cellular responses to muscarinic stimulation in adrenal chromaffin cells. Whole-cell currents were recorded from chromaffin cells freshly dissociated from the rat adrenal medulla. Application of either acetylcholine (ACh), muscarine or MeN-A-343, a selective M1 agonist, at 10-s M elicited firing of the cells recorded extracellularly. Phorbol dibutyrate, an activator of protein kinase C, at 10-~ M, increased the rate of ACh-indueed firing. ACh produced biphasic depolarization with fast and slow components, while muscarine, MeN-A-343 and phorbol dibutyrate induced persistent depolarization with slow onset. To examine the effects of a muscarinic agonist and phorbol dibutyrate on resting K + channels, inward rectifying current was elicited by passing a slow ramp command voltage from -140 to -40 mV. Currents induced by the ramp command voltage were markedly decreased with Cs + (2 mM} applied to the outside of the cell. MeN-A-343 and phorbol dibutyrate also decreased the inward current induced by the ramp command voltage. These results suggest that protein kinase C participates in the MI receptor-mediated suppression of the inward rectifying K channel. •
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