• The Korean Journal of Pharmacology
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• v.30 no.3
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• pp.263-272
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• 1994

Since it was been reported that the depolarization-induced ACh release is inhibited by activation of presynaptic $A_1-adenosine$ heteroreceptor in hippocampus, a large body of experimental data on the post-receptor mechanism of this process has been accumulated. But, the post-receptor mechanism of presynaptic $A_1-adenosine$ receptor on the ACh release has not been clearly elucidated yet. Therefore, it was attempted to clarify the post-receptor mechanisms of the $A_1-adenosine$ receptor-mediated control of ACh release in this study. Slices from rat hippocampus were equilibrated with $^3H-choline$ and the release of the labelled products was evoked by electrical stimulation (3 Hz, 5 $VCm^{-1}$, 2ms, rectangular pulses), and the influence of various agents on the evoked tritium-outflow was investigated. Adenosine, in concentrations ranging from $0.3{\sim}300\;{\mu}M$, decreased the ACh release in a dose-dependent manner, without affecting the basal rate of release. The adenosine effects were significantly inhibited by $DPCPX\;(2\;{\mu}M)$, a selective $A_1-receptor$ antagonist. The responses to N-ethylmaleimide $(10&30{\mu}M)$, a SH-alkylating agent of G-protein, were characterized by increments of the evoked ACh-release and the basal release, and the adenosine effects were completely abolished by NEM pretreatment. PDB $(1{\sim}10\;{\mu}M)$, a specific protein kinase C (PKC) activator, increased, whereas PMB $(0.03{\sim}1\;mg)$, a PKC inhibitor, decreased the evoked ACh-release, and the adenosine effects were not affected by these agents. Nifedipine $(1\;{\mu}M)$, a $Ca^{2+}\;-channel$ blocker of dihydropyridine analogue, significantly inhibited the adenosine effect, but glibenclamide, a $K^+-channel$ blocker, did not. Finally, 8-bromo cyclic AMP $(100\;&\;300{\mu}M)$, a membrane-permeable analogue of cAMP, did not alter the ACh release, but adenosine effects were inhibited by pretreatment with large dose of 8-br-cAMP $(300\;{\mu}M)$. These results indicate that the decrement of the evoked ACh-release by $A_1-adenosine$ receptor is mediated by the G-protein, and nifedipine-sensitive $Ca^{2+}-channel$ and adenylate cyclase system are coupled partly to this effect, and that protein kinase C and glibenclamide-sensitive $K{^+}-channel$ are not involved in this process.

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.1-11
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• 1996

Since it has been reported that the depolarization-induced norepinephrine (NE) release is inhibited by activation of presynaptic $A_1-adenosine$ heteroreceptor in hippocampus, a large body of experimental data on the post-receptor mechanism of this process has been accumulated. But, the post-receptor mechanism of presynaptic $A_1-adenosine$ receptor on the NE release has not been clearly elucidated yet. Therefore, it was attempted to clarify the post-receptor mechanisms of the $A_1-adenosine$ receptor-mediated control of NE release in this study. Slices from rat hippocampus were equilibrated with $^3H-norepinephrine$ and the release of the labelled products was evoked by electrical stimulation (3 Hz, 5 $Vcm^{-1}$, 2 ms, rectangular pulses), and the influence of various agents on the evoked tritium-outflow was investigated. Adenosine, in concentrations ranging from $1{\sim}30{\mu}M$, decreased the NE release in a dose-dependent manner, without affecting the basal rate of release. The adenosine effects were significantly inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, $2{\mu}M$), a selective $A_1-receptor$ antagonist. The responses to N-ethylmaleimide (NEM, 10 & $30{\mu}M$), a SH-alkylating agent of G-protein, were characterized by increments of the evoked NE-release and the basal release, and the adenosine effects were completely abolished by NEM pretreatment. $4{\beta}-Phorbol$ 12,13-dibutyrate (PDB, $1{\mu}M$), a specific protein kinase C (PKC) activator, increased the evoked NE release, whereas polymyxin B sulfate (PMB,0.1 mg), a PKC inhibitor, decreased the release, and the adenosine effects were inhibited by these agents. Nifedipine $(1{\mu}M)$, a $Ca^{2+}-channel$ blocker of dihydropyridine analogue, did not affect the adenosine effect. Tetraethylammonium (TEA, 3 mM) increased the evoked NE release, and inhibited the adenosine effects, but glibenclamide, a ATP dependent $K^+-channel$ blocker, did not. Finally, 8-bromo cyclic AMP (100 & $300{\mu}M$), a membrane-permeable analogue of cAMP, did not alter the NE release, but adenosine effects were inhibited by pretreatment with 8br-cAMP. These results suggest that the decrement of the evoked NE-release by $A_1-adenosine$ receptor is mediated by the C-protein, which is coupled to protein kinase C, adenylate cyclase system and TEA sensitive $K^+-channel$, and that nifedipine-sensitive $Ca^{2+}-channel$ and glibenclamide-sensitive $K^+-channel$ are not involved in this process.

• The Korean Journal of Pharmacology
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• v.31 no.1 s.57
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• pp.11-15
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• 1995

Serotonergic neurons in medulla oblongata play an important role in the endogenous descending pain inhibitory system. To illucidate the factors involved in the regulation of medullary serotonergic neurons, we studied the effects of cholecystokinin (CCK) and agents acting on various second messenger systems on 5-hydroxytryptamine (5-HT) release from cultured neurons of rat fetal (gestational age 14th day) medulla oblongata. Cultured cells maintained for 10 days in vitro were stimulated for 48 hours with CCK or other neuropeptides at 10 micromolar concentration. CCK ($10{\mu}M$) and substance P ($10{\mu}M$) significantly increased. 5-HT release. However, somatostatin, proctolin, thyrotropin releasing hormone, and interleukin-6 did not have any effects on 5-HT release. Nimodipine ($1{\mu}M$), a calcium channel blocker, almost completely, and calmidazolium ($1{\mu}M$), a calmodulin antagonist, significantly inhibited the CCK-induced 5-HT release. The total 5-HT content (intracellular 5-HT plus released 5-HT) was significantly increased by CCK. However, the intracellular 5-HT content was not significantly changed by CCK. Forskolin ($5{\mu}M$), an adenylate cyclase activiator, but not $2{\mu}M$ phorbol myristate acetate (PMA), a protein kinase C activator, significantly enhanced 5-HT release. The total 5-HT content (intracellular 5-HT plus released 5-HT) was significantly increased by forskolin. However, the intracellular 5-HT content was not significantly changed by forskolin. PMA had no effect on intracellular 5-HT levels. These results suggest that CCK regulates serotonergic neurons in the medulla oblongata by enhancing 5-HT secretion through calcium influx and caimodulin, and that cyclic AMP system but not protein kinase C system is involved in 5-HT release.