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Modulation of adenylyl cyclase activity by noradrenaline and serotonin in human platelets
P.6 Other topics
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][3.6.051[ Modulation of adenylyl cyclase activity by noradrenaline and sarotonln in human platelets
Identification of activated protein kinase BIAkt positive neurons in the rat brain: Increment of the number in the CA1 hippocampai area with aging
M . K u b o t a I , M . Kishi I , K. Kobayasi I , M . N i w a I , H. Sugiyama
$403
I,
K. Kudo2, J. Arita2, S. Kanba1. 1Department of Neuropsychiatry,"
2Department of Physiology, Yamanashi Medical University, YamanashL Japan One of programmed cell death (PCD) suppressive factors, PKB/Akt, was first identified as an oncogene and is known to be activated by a variety of growth factors. Activated PKB/Akt (phospho-Akt, pAkt) activates glycogen synthesis, protein synthesis and most importantly survival/proliferation of cells. On the other hand, the hippocampus has long been known to be vulnerable to neuronal damage or loss by aging, ischemia and stress. We have first examined whether or not pAkt is present in the in vioo rat brain using immunohistochemieal methods. Then, to investigate in vivo role of pAkt in aging, we have compared the rate of pAkt-immunoreactive cells between 8 and 70-week-old rats. Material and Methods: Young (7 week old) and aged (70 week old) Wistar strain rats were used in this study. Rats were transcardially perfused with 4% paraforrnaldehyde in phosphate buffer under sodium pentobarbital anesthesia (50 mg/kg). The brains were post-fixed in a solution having the same composition as their perfusate for 24 h at 4°C, 10% sucrose in PBS and 20% sucrose in PBS solution one after another. The brains were placed in powdered dry ice, thereafter 40 ~m coronal brain sections were cut on an tissue slicer into a bath of phosphate-buffered saline (PBS). These sections were quenched with 3% 1-1202 in PBS for 10 rain. and pre-incubated in cocktails with 10% horse serum and a 0.1% TritonX-100 (TBS-T), then incubated in primary polyclonal rabbit-anti-phospho-Akt antibody or in the cocktail for 48 hours. The section were rinsed with TBS-T and then in goat biotinylated anti-rabbit IgG (Vector lab.;l:400) for 1 hour. Following this, the sections were again rinsed in TBS-T and incubated with avidinbiotin-horseradish peroxidase (ABC kit, Vector., USA) in TBST for 1 hour. After another rinse in TBS-T the sections were reacted in diaminobenzidine and hydrogen peroxide. Following a final rinse with TBS-T and PBS, the sections were mounted onto gelatinized glass slides. Results and Discussion: We have observed pAktimmunoreactive cells densely distributed in the hippocampus and the arcuate hypothalamic nucleus. These pAkt-immunoreactive cells were erased or reduced their signals with absorption test as well as wortmannin treatment in young rats. Young and aged rats displayed age-related increase in the number of pAktimmunoreactive cells in CAI (p < 0.05), nonetheless no significant age-related change was observed in the other areas of the hippocampus. PAkt was reported to induce Bcl-2 and suppress PCD. Therefore rich distribution of pAkt within the hippocampus is beneficial from the viewpoint of cell protection.
L. Palego, A. Giromella, M.R. Mazzoni, G. Giaunaccini, A. Lueacchini, G.B. Cassano, D. Marazziti. Dipartimento di
Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Unioersitd di Pisa, Italy We aimed at simultaneously investigating the effects o f noradrenaline (NA) and serotonin (5-HT) on adenylyl eyclase activity in platelets from 10 drug-free healthy volunteers. Adenylyl cyclase was measured both in inhibitory or stimulatory conditions through a double-step Dowex-alumina chromatographic method. Results revealed that either NA or 5-HT inhibited forskolin-stimulated adenylyl cyclase activity in platelet membranes. The two neurotransmitters were also able to stimulate basal AC activity. Basal enzyme velocities were h!gher in the stimulatory experimental conditions than the inhibitory ones. This finding may be due to the different assay temperatures (30°C vs. 24"C). When comparison between potency and efficacy of NA and 5-HT was made, no significant difference was observed. Further, NA stimulated AC with a greater efficacy than that observed in enzyme inhibitionexperiments. Conversely, 5-HT was able to either inhibit or stimulate enzyme activity with similar efficacy values. Our results could contribute to a better understanding of the interactions between NA and 5-HT-mediated signal transduction pathways in human platelets.
References [1] Menninger JA, Tabakoff B, Forskolin-stimulatedadenylylcyclase activity is lower in persons with major depression. Biol Psychiatry, 1997, 42. 30-38.
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Alfa2 and beta adrenocept~rs in the human brain: Activation of the adenylyl cyclase system
L. Palego, A. Giromella, D. Marazziti, G. G-iannaccini, A. Lucacchini, G.B. Cassano, M.R. Mazzoni. Dipartimento di
Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universitd di Pisa, Italy This study aimed at measuring the effects of clonidine and isoproterenol, two agonists at ~2 and 13 adrenoceptors respectively, on adenylyl cyclase activity in the prefrontal cortex and hippocampus from non-diseased human brains postmortem. Adenylyl cyclase was measured both in inhibitory or stimulatory conditions through a double-step Dowex-alumina chromatographic method. Results revealed that clonidine inhibited forskolin-sfirnulated adenylyl cyclase activity in the prefrontal cortex and hippoeampus. The clonidine effect was sensitive to the action of the ct2 antagonist rauwolscine in the two brain areas under investigation. In the adenylyl cyclase stimulatory conditions, isoproterenol was able to stimulate adenylyl cyclase activity in the cortex as well as in the hippocampus. The 13adrenergic antagonist pindolol was able to shift towards fight the dose-response curve of isoproterenol in the cortex but not in the hippocampus, where it markedly enhanced enzyme activity. The 131 selective compound atenolol was able to reverse isoproterenol effects in the hippocampus. These findings report that the activation of the adenylyl cyclase system via ct2 or [3 adrenergic receptors is reproducible in postmortem human
•
][3.6.051[ Modulation of adenylyl cyclase activity by noradrenaline and sarotonln in human platelets
Identification of activated protein kinase BIAkt positive neurons in the rat brain: Increment of the number in the CA1 hippocampai area with aging
M . K u b o t a I , M . Kishi I , K. Kobayasi I , M . N i w a I , H. Sugiyama
$403
I,
K. Kudo2, J. Arita2, S. Kanba1. 1Department of Neuropsychiatry,"
2Department of Physiology, Yamanashi Medical University, YamanashL Japan One of programmed cell death (PCD) suppressive factors, PKB/Akt, was first identified as an oncogene and is known to be activated by a variety of growth factors. Activated PKB/Akt (phospho-Akt, pAkt) activates glycogen synthesis, protein synthesis and most importantly survival/proliferation of cells. On the other hand, the hippocampus has long been known to be vulnerable to neuronal damage or loss by aging, ischemia and stress. We have first examined whether or not pAkt is present in the in vioo rat brain using immunohistochemieal methods. Then, to investigate in vivo role of pAkt in aging, we have compared the rate of pAkt-immunoreactive cells between 8 and 70-week-old rats. Material and Methods: Young (7 week old) and aged (70 week old) Wistar strain rats were used in this study. Rats were transcardially perfused with 4% paraforrnaldehyde in phosphate buffer under sodium pentobarbital anesthesia (50 mg/kg). The brains were post-fixed in a solution having the same composition as their perfusate for 24 h at 4°C, 10% sucrose in PBS and 20% sucrose in PBS solution one after another. The brains were placed in powdered dry ice, thereafter 40 ~m coronal brain sections were cut on an tissue slicer into a bath of phosphate-buffered saline (PBS). These sections were quenched with 3% 1-1202 in PBS for 10 rain. and pre-incubated in cocktails with 10% horse serum and a 0.1% TritonX-100 (TBS-T), then incubated in primary polyclonal rabbit-anti-phospho-Akt antibody or in the cocktail for 48 hours. The section were rinsed with TBS-T and then in goat biotinylated anti-rabbit IgG (Vector lab.;l:400) for 1 hour. Following this, the sections were again rinsed in TBS-T and incubated with avidinbiotin-horseradish peroxidase (ABC kit, Vector., USA) in TBST for 1 hour. After another rinse in TBS-T the sections were reacted in diaminobenzidine and hydrogen peroxide. Following a final rinse with TBS-T and PBS, the sections were mounted onto gelatinized glass slides. Results and Discussion: We have observed pAktimmunoreactive cells densely distributed in the hippocampus and the arcuate hypothalamic nucleus. These pAkt-immunoreactive cells were erased or reduced their signals with absorption test as well as wortmannin treatment in young rats. Young and aged rats displayed age-related increase in the number of pAktimmunoreactive cells in CAI (p < 0.05), nonetheless no significant age-related change was observed in the other areas of the hippocampus. PAkt was reported to induce Bcl-2 and suppress PCD. Therefore rich distribution of pAkt within the hippocampus is beneficial from the viewpoint of cell protection.
L. Palego, A. Giromella, M.R. Mazzoni, G. Giaunaccini, A. Lueacchini, G.B. Cassano, D. Marazziti. Dipartimento di
Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Unioersitd di Pisa, Italy We aimed at simultaneously investigating the effects o f noradrenaline (NA) and serotonin (5-HT) on adenylyl eyclase activity in platelets from 10 drug-free healthy volunteers. Adenylyl cyclase was measured both in inhibitory or stimulatory conditions through a double-step Dowex-alumina chromatographic method. Results revealed that either NA or 5-HT inhibited forskolin-stimulated adenylyl cyclase activity in platelet membranes. The two neurotransmitters were also able to stimulate basal AC activity. Basal enzyme velocities were h!gher in the stimulatory experimental conditions than the inhibitory ones. This finding may be due to the different assay temperatures (30°C vs. 24"C). When comparison between potency and efficacy of NA and 5-HT was made, no significant difference was observed. Further, NA stimulated AC with a greater efficacy than that observed in enzyme inhibitionexperiments. Conversely, 5-HT was able to either inhibit or stimulate enzyme activity with similar efficacy values. Our results could contribute to a better understanding of the interactions between NA and 5-HT-mediated signal transduction pathways in human platelets.
References [1] Menninger JA, Tabakoff B, Forskolin-stimulatedadenylylcyclase activity is lower in persons with major depression. Biol Psychiatry, 1997, 42. 30-38.
•
Alfa2 and beta adrenocept~rs in the human brain: Activation of the adenylyl cyclase system
L. Palego, A. Giromella, D. Marazziti, G. G-iannaccini, A. Lucacchini, G.B. Cassano, M.R. Mazzoni. Dipartimento di
Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universitd di Pisa, Italy This study aimed at measuring the effects of clonidine and isoproterenol, two agonists at ~2 and 13 adrenoceptors respectively, on adenylyl cyclase activity in the prefrontal cortex and hippocampus from non-diseased human brains postmortem. Adenylyl cyclase was measured both in inhibitory or stimulatory conditions through a double-step Dowex-alumina chromatographic method. Results revealed that clonidine inhibited forskolin-sfirnulated adenylyl cyclase activity in the prefrontal cortex and hippoeampus. The clonidine effect was sensitive to the action of the ct2 antagonist rauwolscine in the two brain areas under investigation. In the adenylyl cyclase stimulatory conditions, isoproterenol was able to stimulate adenylyl cyclase activity in the cortex as well as in the hippocampus. The 13adrenergic antagonist pindolol was able to shift towards fight the dose-response curve of isoproterenol in the cortex but not in the hippocampus, where it markedly enhanced enzyme activity. The 131 selective compound atenolol was able to reverse isoproterenol effects in the hippocampus. These findings report that the activation of the adenylyl cyclase system via ct2 or [3 adrenergic receptors is reproducible in postmortem human