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Effect of remving [Ca2+]o on anoxia-induced [Ca2+]i increase in cultured carotid body glomus cells of newborn rabbits
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ACTIVATION OF THE cALCIUM CEANNEL IN DOPAMINERGIC NEURONS BY CR-L-ERYTHROTETRABYDROBIOPTBRIN.KUNIO KOSHIMVRA', TAKUMA SHIRAKI', SEIGEO KOBAYASHIz, SOICHI MIWA'L TOMOH MASAKI' AND YUZDRU KATO','lst. Div., Dept. of Med., Shimane Med. Univ., Izumo 693, 'Graduate Sch. of Human and EnvironmentStudies and 'Dept. of Pharmacol.,Fat. of l4ed.,Kyoto Univ., I(yoto 606-01, Japan.
6R-L-erythro-Tetrahydrobiopterin (6R-BH,) is a cofactor for aromatic amino acid hydroxylases and for nitric oxide (NO) synthase. Recently we have shown that 6R-BH, acts from the outside of the cell to enhance dopamine release independently of its cofactor activities. Here, to clarify the mechanism of action of 6R-BH4, we examined the effects of 6R-BE, on voltage-activated Ca'* channels in dopaminergic neurons of the rat dorsal motor nucleus of vagus using whole-cell patch clamp technique. Two types of Ca" currents were identified; T-type channel current which was rapidly inactivated, sensitive to w-conotoxin but resistant to Cd'+ and nicardipine and N-type current which was slowly inactivated, sensitive to w-conotoxin and Cd'*but resistant to nicardipine. Bath application of 6R-BH, (25 MM) inactivated the T-type channel but activated the N-type channel which plays a key role in dopamine release mechanism. 6S-BH,, a diastereoisomer of 6R-BE,, had no effect on Ca'*channels. Administration of nitroprusside, an NO generator, or L-DOPA had no effect on the Ca'+ channels. These results indicate that LR-BA. activates Ca'* channel independentlv of its cofactor activity to enhance dopamine release.
115
Kinetics of Calcium Release Mediated by Immunoaffity-Purified Inositol 1,4,5Trisphosphate Receptor (Type I) in Reconstituted Lipid Vesicles JuniiJ3iro@l~2,TakavukiMichikawa1 , Atsushi Mivawakil, Ichiro Okura2,TeiichiFuruichil andKatwhikoMikoshibal 13 Minato-ku Tokvo 108. Jaoan, 2Dept. of Bioenzineerin? Tokyo Inst. of Tech.. Midori-ku Yokohama 227. Jaoan Inositol 1,45trisphosphate (IP3) receptor (IP3R) is an IP3-gated Ca 2+ releasing channel. IP3 induced Ca2+ release (IICR) may play crucial role in the intracellular signal transduction. Most interesting phenomenon in IICR is quanta1 Ca2+ release, originally described by Muallem et al., where sub-maximal concentrations of IP3 cause the Ca2+ release of a small fraction of Ca2+ stores. Since the successive applications of equal sub-maximal doses of IP3 release the same amount of Ca2+ (called incremental detection) described by Meyer and Stryer, they suggested that the partial Ca2+ release is not a result of channelinactivation. No work,however, has been studied on the kinetics of IICR including “quanta1 release” using a purified single type of IP3R. We have isolated a single type of mouse IP3R (type I) by using immunoaffinity chromatography and investigated the kinetics of IICR mediated by immunoaffinity-purified IP3R fluo-3. The Ca2+ releasing profiles fromthepurified IP3R in lipid vesicles usingCa2+ indicator reconstituted inlipid vesicles was found to be biexponential with fast and slow phase, indicating that IP3R has two states to release Ca2+. The applications of sub-maximal doses of IP3 show quantal, but not incremental Ca2+ release.
116
EFFECT OF REMVING [CA'+]0 ON ANOXIA-INDUCED [CA'+]x INCREASE IN CULTURED CAROTID BODY GLOMUS CELLS OF NEWBORN RABBITS. MINORU SAT0 AND MASAHITO KAWATANI, Dept. of Physiol, Akita Univ. Sch. of Med.,Hondo 1-1-1,Akita O1O,Japan.
Furamicrofluorometry was used to measure cytosolic Ca" ([Ca"]i) in cultured carotid body glomus cells of newborn rabbits. We examined the effect of removing extracellular Ca2+ ([Ca2+lo) on an increase in CCa2'li produced by anoxia (induced by Na,S,O,). Removal of [Ca2']o eliminated the anoxia-induced [Ca2'li changes in 85% of the tested cells (n=67), and depressed it in the rest, suggesting that the increased [Ca"]i was, in most cells, coming from extracellular sources and, in a few cells, from both intracellular stores and from extracellular sources. But alternatively re-introducing [Ca2']o, in a normoxic solution, immediately produced a large and transient increase in [Ca2+li. This rebound, post-stimulus increase in [Ca2']i (PSCI), was directly proportional to the suppression of the anoxia-induced [Ca"]i changes by [Ca2'lo removal, indicating that the [Ca2']i changes were shifted to PSCI by [Ca"]o removal. The PSCI was mostly inhibited by L-type Ca2' channel blockers (nifedipine and D600), but was not affected by tetrodotoxin. Furthermore, the PSCI, which also occurred in the case of ouabain (sodium pump inhibitor) used as a stimulus, was inhibited by D600. It is concluded that the PSCI may be modification of the [Ca2']i response by [Ca2+lo removal through Ca" channels and/or cytosolic Na'.
ACTIVATION OF THE cALCIUM CEANNEL IN DOPAMINERGIC NEURONS BY CR-L-ERYTHROTETRABYDROBIOPTBRIN.KUNIO KOSHIMVRA', TAKUMA SHIRAKI', SEIGEO KOBAYASHIz, SOICHI MIWA'L TOMOH MASAKI' AND YUZDRU KATO','lst. Div., Dept. of Med., Shimane Med. Univ., Izumo 693, 'Graduate Sch. of Human and EnvironmentStudies and 'Dept. of Pharmacol.,Fat. of l4ed.,Kyoto Univ., I(yoto 606-01, Japan.
6R-L-erythro-Tetrahydrobiopterin (6R-BH,) is a cofactor for aromatic amino acid hydroxylases and for nitric oxide (NO) synthase. Recently we have shown that 6R-BH, acts from the outside of the cell to enhance dopamine release independently of its cofactor activities. Here, to clarify the mechanism of action of 6R-BH4, we examined the effects of 6R-BE, on voltage-activated Ca'* channels in dopaminergic neurons of the rat dorsal motor nucleus of vagus using whole-cell patch clamp technique. Two types of Ca" currents were identified; T-type channel current which was rapidly inactivated, sensitive to w-conotoxin but resistant to Cd'+ and nicardipine and N-type current which was slowly inactivated, sensitive to w-conotoxin and Cd'*but resistant to nicardipine. Bath application of 6R-BH, (25 MM) inactivated the T-type channel but activated the N-type channel which plays a key role in dopamine release mechanism. 6S-BH,, a diastereoisomer of 6R-BE,, had no effect on Ca'*channels. Administration of nitroprusside, an NO generator, or L-DOPA had no effect on the Ca'+ channels. These results indicate that LR-BA. activates Ca'* channel independentlv of its cofactor activity to enhance dopamine release.
115
Kinetics of Calcium Release Mediated by Immunoaffity-Purified Inositol 1,4,5Trisphosphate Receptor (Type I) in Reconstituted Lipid Vesicles JuniiJ3iro@l~2,TakavukiMichikawa1 , Atsushi Mivawakil, Ichiro Okura2,TeiichiFuruichil andKatwhikoMikoshibal 13 Minato-ku Tokvo 108. Jaoan, 2Dept. of Bioenzineerin? Tokyo Inst. of Tech.. Midori-ku Yokohama 227. Jaoan Inositol 1,45trisphosphate (IP3) receptor (IP3R) is an IP3-gated Ca 2+ releasing channel. IP3 induced Ca2+ release (IICR) may play crucial role in the intracellular signal transduction. Most interesting phenomenon in IICR is quanta1 Ca2+ release, originally described by Muallem et al., where sub-maximal concentrations of IP3 cause the Ca2+ release of a small fraction of Ca2+ stores. Since the successive applications of equal sub-maximal doses of IP3 release the same amount of Ca2+ (called incremental detection) described by Meyer and Stryer, they suggested that the partial Ca2+ release is not a result of channelinactivation. No work,however, has been studied on the kinetics of IICR including “quanta1 release” using a purified single type of IP3R. We have isolated a single type of mouse IP3R (type I) by using immunoaffinity chromatography and investigated the kinetics of IICR mediated by immunoaffinity-purified IP3R fluo-3. The Ca2+ releasing profiles fromthepurified IP3R in lipid vesicles usingCa2+ indicator reconstituted inlipid vesicles was found to be biexponential with fast and slow phase, indicating that IP3R has two states to release Ca2+. The applications of sub-maximal doses of IP3 show quantal, but not incremental Ca2+ release.
116
EFFECT OF REMVING [CA'+]0 ON ANOXIA-INDUCED [CA'+]x INCREASE IN CULTURED CAROTID BODY GLOMUS CELLS OF NEWBORN RABBITS. MINORU SAT0 AND MASAHITO KAWATANI, Dept. of Physiol, Akita Univ. Sch. of Med.,Hondo 1-1-1,Akita O1O,Japan.
Furamicrofluorometry was used to measure cytosolic Ca" ([Ca"]i) in cultured carotid body glomus cells of newborn rabbits. We examined the effect of removing extracellular Ca2+ ([Ca2+lo) on an increase in CCa2'li produced by anoxia (induced by Na,S,O,). Removal of [Ca2']o eliminated the anoxia-induced [Ca2'li changes in 85% of the tested cells (n=67), and depressed it in the rest, suggesting that the increased [Ca"]i was, in most cells, coming from extracellular sources and, in a few cells, from both intracellular stores and from extracellular sources. But alternatively re-introducing [Ca2']o, in a normoxic solution, immediately produced a large and transient increase in [Ca2+li. This rebound, post-stimulus increase in [Ca2']i (PSCI), was directly proportional to the suppression of the anoxia-induced [Ca"]i changes by [Ca2'lo removal, indicating that the [Ca2']i changes were shifted to PSCI by [Ca"]o removal. The PSCI was mostly inhibited by L-type Ca2' channel blockers (nifedipine and D600), but was not affected by tetrodotoxin. Furthermore, the PSCI, which also occurred in the case of ouabain (sodium pump inhibitor) used as a stimulus, was inhibited by D600. It is concluded that the PSCI may be modification of the [Ca2']i response by [Ca2+lo removal through Ca" channels and/or cytosolic Na'.