Activation of nicotinic acetylcholine receptors patterns network activity in the rodent hippocampus.
(1/121)
1. Intracellular and extracellular recordings from area CA3 of rat and mouse hippocampal slices revealed two distinct modes of synchronous network activity in response to continuous application of muscarinic acetylcholine receptor (mAChR) agonists. At low concentrations (e.g. 0.1-1 microM oxotremorine-M), 'burst-mode' activity comprised regular individual AMPA receptor-mediated depolarizing events, each generating several action potentials. At higher concentrations (5-50 microM), 'theta-mode' prevailed in which ordered clusters of depolarizing theta-frequency oscillations occurred. 2. Whilst theta-mode activity was abolished by the mAChR antagonist atropine (5 microM), the nicotinic acetylcholine receptor (nAChR) antagonists tubocurarine (100 microM), mecamylamine (100-500 microM) and dihydro-beta-erythroidine (250 microM) converted this mode of activity to burst-mode. 3. Likewise, disruption of synaptically available ACh using inhibitors of choline uptake (hemicholinium-3; 20-50 microM) or vesicular ACh transport (vesamicol; 50 microM) converted theta-mode into burst-mode activity. 4. Hippocampal slices prepared 2-3 weeks after transection of the primary cholinergic efferent pathway from the medial septum exhibited reduced vesicular ACh transporter immunoreactivity but still supported nAChR-dependent theta-mode activity suggesting that ACh released from this pathway was not critical for the activation of these receptors. 5. In summary, ACh-mediated activation of nAChRs tailors the pattern of network activity into theta-frequency depolarizing episodes as opposed to synchronized individual events at much lower frequencies. (+info)
Extracellular calcium stimulates DNA synthesis in synergism with zinc, insulin and insulin-like growth factor I in fibroblasts.
(2/121)
In serum-starved mouse NIH 3T3 fibroblasts cultured in 1.8 mM Ca2+-containing medium, addition of 0.75-2 mM extra Ca2+ stimulated DNA synthesis in synergism with zinc (15-60 microM), insulin and insulin-like growth factor I. Extra Ca2+ stimulated phosphorylation/activation of p42/p44 mitogen-activated protein kinases by an initially (10 min) zinc-independent mechanism; however, insulin, and particularly zinc, significantly prolonged Ca2+-induced mitogen-activated protein kinase phosphorylation. In addition, extra Ca2+ activated p70 S6 kinase by a zinc-dependent mechanism and enhanced the stimulatory effect of zinc on choline kinase activity. Insulin and insulin-like growth factor I also commonly increased both p70 S6 kinase and choline kinase activities. In support of the role of the choline kinase product phosphocholine in the mediation of mitogenic Ca2+ effects, cotreatments with the choline kinase substrate choline (250 microM) and the choline kinase inhibitor hemicholinium-3 (2 mM) enhanced and inhibited, respectively, the combined stimulatory effect of extra Ca2+ (3.8 mM total) and zinc on DNA synthesis. In various human skin fibroblast lines, 1-2 mM extra Ca2+ also stimulated DNA synthesis in synergism with zinc and insulin. The results show that in various fibroblast cultures, high concentrations of extracellular Ca2+ can collaborate with zinc and certain growth factors to stimulate DNA synthesis. Considering the high concentration of extracellular Ca2+ in the dermal layer, Ca2+ may promote fibroblast growth during wound healing in concert with zinc, insulin growth factor-I insulin, and perhaps other growth factors. (+info)
The effects of prolonged repetitive stimulation in hemicholinium on the frog neuromuscular junction.
(3/121)
1. Cutaneous pectoris nerve-muscle preparations from the frog were stimulated for prolonged periods in solutions with curare alone, curare and hemicholinium no. 3 (HC-3), or curare and glucose plus choline. End-plate potentials (e.p.p.s) and miniature end-plate potentials (m.e.p.p.s) were recorded intracellularly. Black widow spider venom (BWSV) was applied to determine the degree of depletion of the transmitter stores. 2. The ultrastructure of the neuromuscular junctions was studied in the electron microscope. Some of the preparations were fixed immediately at the end of the period of stimulation and others were fixed about an hour after BWSV had been applied. In some experiments horseradish peroxidase (HRP) was present during the period of stimulation and the fixed tissue was treated to reveal the distribution of the tracer. 3. The amplitude of the e.p.p. fell rapidly to almost zero during 2 hr of stimulation at 2/sec in 100 muM HC-3 and little recovery occurred during a subsequent hour of rest. About 2-7 times 10-5 quanta were secreted. The e.p.p.s usually persisted throughout the period of stimulation in the other solutions and 2-2-6 times as much transmitter was secreted. 4. When BWSV was applied immediately at the end of the period of stimulation in HC-3, almost no m.e.p.p.s were discharged and only small m.e.p.p.s were discharged when the venom was applied after an hour of rest. 5. When BWSV was applied to unstimulated terminals that had been bathed in HC-3, or to terminals that had been stimulated and rested for an hour in glucose plus choline, m.e.p.p.s of nearly normal amplitude were discharged. 6. Terminals stimulated for 2 hr at 2/sec in 100 muM HC-3 contained a normal complement of synaptic vesicles and a large proportion of vesicles were labelled with HRP when the tracer was present during the period of stimulation. 7. BWSV induced the almost complete depletion of vesicles from terminals that had been stimulated in HC-3. 8. Depletion of vesicles also occurred when terminals were stimulated for 20 min at 10/sec after they had been previously stimulated for 2 hr at 2/sec in HC-3. These terminals showed extensive infolding of the axolemma and they contained swollen mitochondria. 9. These results indicate that stimulation in HC-3 depletes terminals of their store of transmitter but not of their population of vesicles and that vesicles empty of transmitter can fuse with and reform from the axolemma of the nerve terminal. (+info)
Effects of perinatal nicotine exposure on development of [3H]hemicholinium-3 binding sites in rat neonate brain.
(4/121)
In this study, [3H]hemicholinium-3 ([3H]HC-3) binding, which labels the presynaptic high affinity-choline transport sites, was examined in two brain regions, cerebral cortex and midbrain, of nicotine-treated and -untreated rat neonates. In nicotine-untreated neonates, [3H]HC-3 binding sites of cerebral cortex increased from 64 fmol/mg protein at postnatal day 7 to 142 fmol/mg protein at postnatal day 35. In nicotine-treated neonates, the development of [3H]HC-3 binding sites in cerebral cortex was significantly retarded, compared with control neonates on the 7th, 14th and 21st postnatal days. In parallel with this, the development of muscarinic receptor in cerebral cortex, which was detected by [3H]quinuclidinyl benzylate ([3H]QNB) binding, was also retarded by nicotine treatment. However, in midbrain, neither [3H]HC-3 nor [3H]QNB binding sites at postnatal day 14 was affected by nicotine treatment. These results strongly suggest that perinatal treatment with nicotine inhibits presynaptic and postsynaptic development of the cholinergic pathway in cerebral cortex but not in midbrain of rat neonate. (+info)
Repetitive nerve stimulation decreases the acetylcholine content of quanta at the frog neuromuscular junction.
(5/121)
We investigated how elevated quantal release produced by motor nerve stimulation affects the size of the quanta. The motor nerve was stimulated at 10 Hz in preparations in which excitation-contraction coupling was disrupted. Two hundred stimuli reduced the size of the time integrals of the miniature endplate currents ([integral]MEPCs), measured at the same junction immediately after stimulation, by 16 %. Three thousand stimuli reduced size by 23 %. When the solution contained 10 microM neostigmine (NEO) 3000 stimuli reduced [integral]MEPCs by 60 %, because with acetylcholinesterase (AChE) inhibited, [integral]MEPC size is more sensitive to changes in acetylcholine (ACh) content. Similar decreases in miniature endplate potential size ([integral]MEPP) followed repetitive stimulation of contracting preparations. The depolarization produced by iontophoretic pulses of ACh was scarcely changed by 3000 nerve stimuli at 10 Hz, suggesting that the decreases in miniature sizes are largely due to less ACh released per quantum. Following 3000 stimuli at 10 Hz the sizes of the [integral]MEPCs increased back to pre-stimulus values with a half-time of 8-10 min. Recovery was blocked by (-)-vesamicol (VES), by hemicholinium-3 (HC3) and by nicotinic cholinergic agonists - all of which inhibit ACh loading into synaptic vesicles. The number of quanta in the total store was estimated by releasing them with carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP releases fewer quanta after stimulation than from unstimulated controls. After resting for hours following stimulation, the releasable number increased, even when ACh loading inhibitors were present. We conclude that the inhibitors do not block a significant fraction of the ACh loading into reformed reserve vesicles and propose that ACh can be loaded in a series of steps. (+info)
Transport of choline and its relationship to the expression of the organic cation transporters in a rat brain microvessel endothelial cell line (RBE4).
(6/121)
The present study was undertaken to elucidate the functional characteristics of choline uptake and deduce the relationship between choline uptake and the expression of organic cation transporters in the rat brain microvessel endothelial cell line RBE4. Confluent RBE4 cells were found to express a high affinity choline uptake system. The system is Na(+)-independent and shows a Michaelis-Menten constant of approx. 20 microM for choline. The choline analogue hemicholinium-3 inhibits choline uptake in these cells with an inhibition constant of approx. 50 microM. The uptake system is also susceptible for inhibition by various organic cations, including 1-methyl-4-phenylpyridinium, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, clonidine, procainamide, and tetramethylammonium. The prototypical organic cation tetraethylammonium shows very little affinity for the choline uptake system in these cells. The inhibition of choline uptake by hemicholinium-3 is competitive. Northern analysis and RT-PCR show that these cells do not express the organic cation transporters OCT2 and OCT3. These cells do express, however, low levels of OCT1, but the functional characteristics of choline uptake in these cells are very different from the known properties of choline uptake via OCT1. The Na(+)-coupled high affinity choline transporter CHT1 is not expressed in these cells as evidenced by RT-PCR. This corroborates the Na(+)-independent nature of choline uptake in these cells. It is concluded that RBE4 cells express an organic cation transporter that is responsible for choline uptake in these cells and that this transporter is not identical to any of the organic cation transporters thus far identified at the molecular level in mammalian cells. (+info)
In vivo assessment of acetylcholine-releasing function at cardiac vagal nerve terminals.
(7/121)
We examined whether the ACh concentration measured by cardiac microdialysis provided information on left ventricular ACh levels under a variety of vagal stimulatory and modulatory conditions in anesthetized cats. Local administration of KCl (n = 5) and ouabain (n = 7) significantly increased the ACh concentration in the dialysate to 4.3 +/- 0.8 and 7.3 +/- 1.3 nmol/l, respectively, from the baseline value of 0.6 +/- 0.5 nmol/l. Intravenous administration of phenylbiguanide (n = 5) and phenylephrine (n = 6) significantly increased the ACh concentration to 5.4 +/- 0.9 and 6.0 +/- 1.5 nmol/l, respectively, suggesting that the Bezold-Jarisch and arterial baroreceptor reflexes affected myocardial ACh levels. Modulation of vagal nerve terminal function by local administration of tetrodotoxin (n = 6), hemicholinium-3 (n = 6), and vesamicol (n = 5) significantly suppressed the electrical stimulation-induced ACh release from 20.4 +/- 3.9 to 0.6 +/- 0.1, 7.2 +/- 1.9, and 2.7 +/- 0.6 nmol/l, respectively. Increasing the heart rate from 120 to 200 beats/min significantly reduced the myocardial ACh levels during electrical vagal stimulation, suggesting a heart rate-dependent washout of ACh. We conclude that ACh concentration measured by cardiac microdialysis provides information regarding ACh release and disposition under a variety of pathophysiological conditions in vivo. (+info)
Spinal endogenous acetylcholine contributes to the analgesic effect of systemic morphine in rats.
(8/121)
BACKGROUND: Systemic morphine is known to cause increased release of acetyicholine in the spinal cord. Intrathecal injection of the cholinergic receptor agonists or acetyicholinesterase inhibitors produces antinociception in both animals and humans. In the present study, we explored the functional importance of spinal endogenous acetylcholine in the analgesic action produced by intravenous morphine. METHODS: Rats were implanted with intravenous and intrathecal catheters. The antinociceptive effect of morphine was determined by the paw-withdrawal latency in response to a radiant heat stimulus after intrathecal treatment with atropine (a muscarinic receptor antagonist), mecamylamine (a nicotinic receptor antagonist), or cholinergic neurotoxins (ethylcholine mustard aziridinium ion [AF64A] and hemicholinium-3). RESULTS: Intravenous injection of 2.5 mg/kg morphine increased significantly the paw-withdrawal latency. Intrathecal pretreatment with 30 microg atropine (n = 7) or 50 microg mecamylamine (n = 6) both attenuated significantly the antinociceptive effect of morphine. The inhibitory effect of atropine on the effect of morphine was greater than that of mecamylanilne. Furthermore, the antinociceptive effect of morphine was significantly reduced in rats pretreated with intrathecal AF64A (n = 7) or hemicholinium-3 (n = 6) to inhibit the high-affinity choline transporter and acetylcholine synthesis. We found that intrathecal AF64A reduced significantly the [3H]hemicholinium-3 binding sites but did not affect its affinity in the dorsal spinal cord. CONCLUSIONS: The data in the current study indicate that spinal endogenous acetylcholine plays an important role in mediating the analgesic effect of systemic morphine through both muscarinic and nicotinic receptors. (+info)