Nitric oxide-dependent and -independent mechanisms in the relaxation elicited by acetylcholine in fetal rat aorta

The aim of the present study was to analyze the mechanisms involved in the relaxation induced by 1 microM acetylcholine (ACh) in aortic segments from fetal rats at term precontracted with 3 microM prostaglandin F2alpha (PGF2alpha) and incubated with 1 microM indomethacin. The endothelium-dependent relaxation caused by ACh was reduced by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.1 mM), such an effect was reversed by 0.1 mM L-arginine (L-Arg). After precontraction of segments with 50 mM KCl the relaxant response to ACh was smaller than that after precontraction with PGF2alpha; this reduction was increased by L-NMMA, whereas L-NMMA plus L-Arg potentiated the relaxation. Thiopentone sodium (0. 1 mM), ouabain (10 microM), tetraethylammonium (TEA, 0.5 mM) and apamin (1 microM), inhibitors of cytochrome P450 monooxygenases, Na+ pump, Ca2+-activated (KCa) and small-conductance (SKCa) K+ channels, respectively, reduced the relaxation to ACh, which was unaffected by charybdotoxin (0.1 microM) and glibenclamide (1 microM), inhibitors of large-conductance BKCa and ATP-sensitive K+ channels. The L-NMMA/indomethacin-resistant relaxation to ACh was markedly reduced by thiopentone sodium, and similarly decreased by either ouabain or TEA. The endothelium-independent relaxation induced by exogenous NO (10 microM) in segments precontracted with PGF2alpha was unaltered by ouabain, glibenclamide, TEA and after precontraction with 50 mM KCl, and potentiated by L-NMMA. The potentiation of NO responses by L-NMMA was also observed in segments precontracted with KCl. These results suggest that ACh relaxes the fetal rat aorta by endothelial release of both NO and endothelium-derived hyperpolarizing factor (EDHF), a metabolite derived from cytochrome P450 monooxygenases, that hyperpolarizes smooth muscle cells by activation of KCa, essentially SKCa channels, and Na+ pump. It seems that when the effect of EDHF is abolished, the formation of NO could be increased.     

Inhibition by sodium bromide of acetylcholine release and synaptic transmission in the superior cervical ganglion of the rat

In the superior cervical ganglion (SCG) of rats, the interaction of sodium bromide (NaBr) with various drugs which interfere with the GABA system, such as 3-(4-chlorophenyl)-4-aminobutyrate [( + )baclofen, Bac], ( + )bicuculline (Bic), picrotoxin (Pic) and chlorpromazine (CPZ), and the effects of NaBr on the K⁺-induced release of [³H]acetylcholine ([³H]ACh) were studied in vitro. The effects on the evoked potentials induced by preganglionic stimulation were analysed in situ. The in vitro experiments revealed that 1 mM NaBr inhibits both the basal and the K⁺-induced release of [³H]ACh in a Ca²⁺-dependent manner. This NaBr effect was additive with the similar effect of the GABA agonist Bac, but it could not be blocked with any of the drugs applied. In vivo, 1 mM NaBr depressed the amplitude of the evoked potentials in the SCG. It is concluded that, in the SCG of rats, NaBr interacts with the presynaptic and postsynaptic membranes. The inhibitory effects of NaBr on both the [³H]ACh release and the potentials evoked by preganglionic stimulation cannot be attributed to a direct interference with GABA receptor complexes; some other binding site/s on the presynaptic and postsynaptic membranes might be responsible for the bromide-induced reduction of the synaptic transmission in the SCG of rats.     

Neuronal control of heart rate in isolated mouse atria

A novel mouse isolated atrial preparation with intact postganglionic autonomic innervation was used to investigate the neuronal control of heart rate. To establish whether autonomic activation was likely to alter heart rate by modulating the hyperpolarization-activated current (If), the L-type Ca2+ current (ICa,L), or the ACh-activated K+ current (IK,ACh), the effects of nerve stimulation (right stellate ganglion or right vagus, 1-30 Hz) and autonomic agonists (0.1 microM norepinephrine or 0.3 microM carbachol) on heart rate were investigated in the presence of inhibitors of these currents, cesium chloride (Cs+, 1 mM), nifedipine (200 nM), and barium chloride (Ba2+, 0.1 mM), respectively. The positive chronotropic response to stellate ganglion stimulation was reduced by approximately 20% with Cs+ and nifedipine (P < 0.05), whereas the heart rate response to norepinephrine was only reduced with Cs+ (P < 0.05). Ba2+ attenuated the decrease in heart rate with vagal stimulation and carbachol by approximately 60% (P < 0.05). These results are consistent with the idea that sympathetic nerve stimulation modulates If to increase heart rate in the mouse. Activation of ICa,L also appears to contribute to the sympathetic heart rate response. However, the decrease in heart rate with vagal stimulation or carbachol is likely to result primarily from the activation of IK,ACh.     

Choline and acetylcholine metabolism in rat neostriatal slices

Choline (Ch) uptake and release and acetylcholine (ACh) synthesis and release have been studied by gas chromatography mass spectrometry (GCMS) in slices of rat neostriatum in vitro to assess the effects of depolarization by 25 mM K+ and the influence of elevated concentrations of Ch in the incubation medium. During the first 60 min after preparation, 25 mM K+ increased ACh release by 182% and reduced ACh levels by 40%. The rate of ACh synthesis was unchanged. After a 1-h equilibration period, the rate of ACh synthesis was considerably less (2.41 nmol mg-1 h-1, compared to 9.78 nmol mg-1 h-1). Exposure to 25 mM K+ during the second hour increased the rate to 6.47 nmol mg-1 h-1. During the first 10 min of exposure to 25 mM K+, ACh synthesis was reduced, regardless of incubation. Increasing concentrations of external [2H4]Ch apparently favored initial rates of net ACh synthesis, since the rank order of initial net ACh synthesis rates is the same as the rank order of external [2H4] Ch concentration under both normal and depolarized conditions. However, the only significant effect of external [2H4]Ch on ACh metabolism was that it increased ACh release during the initial 10 min, when the preparation was depolarized with K+. The efflux of endogenous [2H0]Ch was increased initially (10 min) and slowed over a 60-min period by 25 mM K+, and increased when [2H4]Ch in the medium was increased. Changes in ACh synthesis and release were dependent upon the time exposure of slices to high K+, and the results suggest that Ch favors initial rates of ACh synthesis, but that Ch influences ACh release primarily under conditions of stress (i.e., depolarization).     

K+-induced alterations in airway muscle responsiveness to electrical field stimulation

We investigated possible pre- and postsynaptic effects of K+-induced depolarization on ferret tracheal smooth muscle (TSM) responsiveness to cholinergic stimulation. To assess electromechanical activity, cell membrane potential (Em) and tension (Tm) were simultaneously recorded in buffer containing 6, 12, 18, or 24 mM K+ before and after electrical field stimulation (EFS) or exogenous acetylcholine (ACh). In 6 mM K+, Em was -58.1 +/- 1.0 mV (mean +/- SE). In 12 mM K+, Em was depolarized to -52.3 +/- 0.9 mV, basal Tm did not change, and both excitatory junctional potentials and contractile responses to EFS at short stimulus duration were larger than in 6 mM K+. No such potentiation occurred at a higher K+, although resting Em and Tm increased progressively above 12 mM K+. The sensitivity of ferret TSM to exogenous ACh appeared unaffected by K+. To determine whether the hyperresponsiveness in 12 mM K+ was due, in part, to augmented ACh release from intramural airway nerves, experiments were done using TSM preparations incubated with [3H]choline to measure [3H]ACh release at rest and during EFS. Although resting [3H]ACh release increased progressively in higher K+, release evoked by EFS was maximal in 12 mM K+ and declined in higher concentrations. We conclude that small elevations in the extracellular K+ concentration augment responsiveness of the airways, by increasing the release of ACh both at rest and during EFS from intramural cholinergic nerve terminals. Larger increases in K+ appear to be inhibitory, possibly due to voltage-dependent effects that occur both pre- and postsynaptically.     

Calcium-Independent Release of Acetylcholine from Electric Organ Synaptosomes and Its Changes by Depolarization and Cholinergic Drugs

Chemiluminescent detection was applied to measure the continuous spontaneous Ca2+-independent liberation of acetylcholine (ACh) from Torpedo electric organ synaptosomes. Differentiation between the release of ACh and choline was achieved by inhibiting cholinesterases with phospholine, and a way to quantify the continuous release was devised. The method permitted measurements during short time intervals from minute amounts of tissue and without an accumulation of ACh in the medium. Synaptosomes continuously liberated small amounts of ACh during incubations in the presence of 3 mM K+ and in the absence of Ca2+. The spontaneous liberation of ACh was similar both quantitatively and qualitatively at pH values of 8.6 and 7.8. It was unaltered by MgCl2 (10.4 mM), 2-(4-phenylpiperidino)cyclohexanol (10 microM), ouabain (104 microM), atropine (10 microM), and valinomycin (102 nM). Carbamoylcholine brought about a decrease, which could be partially reversed by atropine. The Ca2+-independent output of ACh was increased considerably when the concentration of K+ ions was raised (eightfold at 103 and 35-fold at 203 mM K+). Carbamoylcholine (104 microM) blocked the increase in ACh release produced by high K+; this effect of carbamoylcholine was not reversed by atropine (10 microM). When Ca2+ was added to synaptosomes depolarized by a high concentration of K+, the amount of ACh released during the first 1-3 min after the addition of Ca2+ was at least 20 times higher than in the absence of Ca2+, but the release returned rapidly to predepolarization values. Similarly high values of ACh release could be achieved by adding Ca2+ plus the ionophore A23187 and even higher values by adding Ca2+ plus gramicidin.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the Mechanism of Ouabain-Induced Release of Acetylcholine from Synaptosomes

Ouabain (5 x 10(-8)-5 x 10(-4) M) was confirmed to cause a dose-dependent increase in [3H]acetylcholine ([3H]ACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose-dependent decrease in membrane potential. In a low-Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10(-6) M) had no effect on the ouabain-induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10(-6)-5 x 10(-4) M) inhibited the ouabain-induced increase in both [3H]ACh release and [Ca2+]i in a dose-dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mM Mg2+ blocked ouabain-induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.     

The influence of aminopyridines on Ca2+-dependent evoked release of acetylcholine from rat cortex slices

The release of acetylcholine (ACh) elicited by electrical stimulation was investigated in rat brain cortical slices preloaded with³H-choline. Decreasing the [Ca²⁺]o from 2.5 to 0.3 mM caused a progressive reduction of the evoked release of ACh. 4-Aminopyridine (4AP) or LF14 [(1,1-dimethyl-3-(4-amino-3-pyridyl)], 4×10^–5 M doubled the evoked release of ACh when the [Ca²⁺]o was 2.5 mM and quadrupled it when it was 0.3 mM, to levels higher than those obtained with 2.5 mM [Ca²⁺]o alone. This indicates that both 4AP and LF14 decrease the Ca²⁺ requirements for the evoked release of ACh. The findings of this study indicate that LF14 may be suitable for the symptomatic treatment of senile dementia of Alzheimer's type, presumably caused by dysfuntion of cholinergic transmission in the brain.     

Calcium dependent release of acetylcholine and gamma-aminobutyric acid from the rabbit retina.

The concurrent release of endogenous ACh and GABA from the retina (in the presence of physostigmine) was measured using either an eye-cup preparation in rabbits anaesthetized with urethane or isolated rabbit retinas. There was a spontaneous resting release of ACh and GABA from the dark adapted retina of ca 5 and 160 pmol min-1 respectively. Stimulation of the initially dark adapted retina in vivo with flickering light (0.1-20 Hz) increased the release of ACh by up to 5 times the spontaneous resting release but did not cause a detectable increase in GABA release. The maximum light-evoked release of ACh was about 24 pmol min-1/retina and occurred at a frequency of 10 Hz. However, the maximum release of ACh per flash occurred at 0.1 Hz at which frequency the average ACh release per flash from one amacrine cell was ca 2.35 x 10(-18) mol. Exposure of the retina to the potent inhibitors of GABA uptake, SKF89976A and SKF100330A markedly reduced the resting release of ACh and abolished the light-evoked release of ACh but did not enable a light-evoked release of GABA to be detected. Bicuculline blocked the inhibitory actions of both SKF89976A and SKF100330A on ACh release but the combination of bicuculline and uptake inhibitor did not result in a light-evoked release of GABA. In contrast, KCl (20 mM) applied locally to the retina in vivo resulted in the release of both ACh and GABA (61 and 2.6-fold respectively). KCl (20 mM) also evoked large increases in ACh and GABA release from isolated rabbit retinas in room light (13.5 and 3.4-fold respectively). The K-evoked release of ACh and GABA from the rabbit retina both in vivo and in vitro was calcium dependent. These experiments are the first in which endogenous ACh and GABA release from the retina have been simultaneously measured and suggest that the release mechanisms for these transmitters are fundamentally similar.     

Release of Acetylcholine from Tissue Slices of the Rat Nucleus Basalis Magnocellularis

We investigated the release of acetylcholine (ACh) from tissue slices obtained from the nucleus basalis magnocellularis (nbM) of the rat brain. Potassium (35 mM) depolarization produced a 10- to 12-fold increase in the release of endogenous ACh above spontaneous release. Potassium-evoked ACh release was Ca2+ dependent. Injection of the excitotoxin quinolinic acid into the nbM produced a 72.8 +/- 13.0% decrease in spontaneous ACh release and a 60.4 +/- 8.2% decrease in potassium-evoked release. A fourfold increase in ACh release was observed following perfusion of the tissue with 1 mM 3,4-diaminopyridine (3,4-DAP) whereas 10 mM 3,4-DAP caused a sevenfold increase. The increase in ACh release caused by 3,4-DAP was inhibited by tetrodotoxin. Tissue slices accumulated [3H]choline by high-affinity choline uptake and this could be inhibited by hemicholinium-3. These results indicate that ACh can be released from tissue slices of the nbM by a calcium-dependent process and that a part of this release appears to be from the cholinergic neurons of the nbM.     

Developmental switch in the pharmacology of Ca2+ channels coupled to acetylcholine release.

The pharmacological specificity of Ca2+ channel-secretion coupling in acetylcholine (ACh) and somatostatin (SOM) release was studied in the chick eye choroid neuromuscular junctions and in dissociated ciliary ganglion (CG) neurons. ACh secretion changes in development from stage (St) 40, when release is dihydropyridine (DHP) and partially omega-conotoxin (omega-CgTX) sensitive, to posthatch, when release is insensitive to DHPs but sensitive to omega-CgTX. St 40 CG neurons cultured with striated muscle have release properties similar to those of St 40 iris and choroid but different from those of St 34 neurons, which are neither DHP nor omega-CgTX sensitive. SOM (also coreleased from posthatch choroid terminals) can inhibit ACh release in both posthatch and St 40 choroids, suggesting that the SOM receptor interacts with both DHP-sensitive and -insensitive channels.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

Cholinergic control of membrane conductance and intracellular free Ca2+ in outer hair cells of the guinea pig cochlea

We have studied the action of cholinergic agonists on outer hair cells, both in situ and isolated from the cochlea of the guinea pig, combining new fast CCD technology for Ca2+ imaging and conventional patch-clamp methods. Carbachol (1 mM) activated a current with a reversal potential near -70 mV and a bell-shaped I-V curve, suggesting that it was a Ca2+ activated K+ current. In a few cells, this current was preceded by a transient inward current, probably owing to an influx of Ca2+ and other cations through the acetylcholine (ACh) receptors. The amplitude of the Ca2+ signal was maximal in a circumscribed region at the basal pole of the cell and decreased steeply towards the apical pole, compatible with Ca2+ influx and/or Ca2+ induced Ca2+ release at the cells base. The time course of the Ca2+ rise was fastest at the base, but it was still slightly slower, and more rounded, than that of the K+ current. In some recordings the K+ current was observed without any measurable change of intracellular Ca2+. The K+ current was potentiated (18%) by caffeine (5 mM), and decreased (19%) by ryanodine (0.1 mM) in the majority of cells tested. The results are discussed in terms of a labile intracellular Ca2+ store located at the base of the cell, close to the Ca2+ permeable ACh receptor channels and Ca2+ activated K+ channels, whose contribution to the Ca2+ rise occurring in the region of the channels is variable, and probably dependent on its ability to refill with Ca2+.     

Effects of Acetylethylcholine Mustard on [3H]Quinuclidinyl Benzilate Binding and Acetylcholine Release in Rat Brain Synaptosomes

The effects of acetylethylcholine mustard and its aziridinium derivative (AMMA) on acetylcholine (ACh) release and [3H]quinuclidinyl benzilate (QNB) binding were studied in rat cortical synaptosomes. After incubation for 5 min at 37 degrees C, AMMA reduced [3H]QNB binding with an IC50 of 9 microM. Following incubation for 5 min with 50 microM AMMA and washing, there was a 62% reduction in the [3H]QNB binding capacity with no change in the KD value for the remaining receptors, a result indicating the irreversibility of the AMMA binding. AMMA and oxotremorine both reduced the basal and 30 mM K+-induced release of newly synthesized [3H]ACh in dose-dependent manners over a 2.5-min period. At identical 50 microM concentrations, AMMA produced a much longer inhibition of basal [3H]ACh release than oxotremorine did. The inhibition of basal and 30 mM K+-induced [3H]ACh release by AMMA (10-250 microM) was blocked by 2 microM atropine during a 2.5-min release incubation, but not during a 30-min release incubation. After synaptosomes were treated with 50 microM AMMA for 5 min and the unbound drug was washed out from the tissue, [3H]ACh release (basal and K+-induced) was reduced. AMMA (50 microM) reduced high-affinity choline uptake and ACh synthesis by greater than 90% in this tissue, but these effects did not account for the [3H]ACh release inhibition, because they were not atropine sensitive and hemicholinium-3 had no effect on [3H]ACh release under the conditions used in these studies, i.e., after extracellular [3H]choline was washed out. Taken together, these results suggest that AMMA may be an irreversible agonist at presynaptic muscarinic autoreceptors.     

Effect of Lanthanum on the Release of Acetylcholine from the Myenteric Plexus and on Its Activation by Ouabain and Electrical Stimulation

The effect of lanthanum ions (La3+) on the release of acetylcholine (ACh) from longitudinal muscle strips of the guinea pig ileum with the myenteric plexus attached was investigated. After an exposure of the tissue to 2 mM LaCl3 for 18 min the rate of ACh release was increased approximately eightfold and the increased release lasted for more than 100 min. The augmented release of ACh was accompanied by enhanced synthesis. At the end of the experiments (102 min after LaCl3 had been removed), when the release of ACh was still more than six times higher than in controls, the content of ACh was the same in La3+-treated and untreated tissues. Electrical field stimulation failed to cause a further increase in the release of ACh from La3+-pretreated preparations whereas ouabain released considerable more ACh when compared to controls. It is concluded from this difference that electrical stimulation and ouabain release ACh from different pools.     

硝苯吡啶抑制豚鼠交感神经元的钙依赖性电位

应用细胞内记录技术观察了钙通道阻滞剂硝苯吡啶(nifedlpine)对离体豚鼠腹腔神经节细胞三种钙依赖性电位的可逆性作用。硝苯吡啶(0.1—1mmol/L)可剂量依赖式地抑制动作电位后超极化、强直后膜电位的变化,在无钠高钙加 TEA 溶液中,硝苯吡啶(0.1μmol/L)能抑制钙锋电位。结果表明,大剂量的硝苯吡啶可继发性抑制钙依赖性钾电导,临床治疗剂量的硝苯吡啶还直接减少钙电导。以上作用是硝苯吡啶调节交感节后神经元的兴奋性,阻滞突触前膜 ACh 的量子性释放的基础。     

The spatial relationship between Ca2+ channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.     

The K(+)-evoked release of [3H]acetylcholine from slices of rat globus pallidus: modulation by delta-opioid receptors.

Previous investigations have shown that the activation of delta-opioid receptors depresses the release of acetylcholine (ACh) in the rat caudate putamen. This finding raised the possibility that the release of ACh is similarly modulated in the globus pallidus, a region containing a distinct population of cholinergic neurons and enriched in enkephalinergic nerve terminals. In the present study the pallidal release of ACh was characterized and the effects of delta-opioid receptor activation on this release were examined. The results show that this release is stimulated by high K+ in a concentration- and Ca(2+)-dependent manner. D-Pen2,L-Pen5-enkephalin (0.1-10 microM), a selective delta-opioid receptor agonist, produced a dose-related inhibition of the 25 mM K(+)-evoked tritium release. The maximal inhibitory effect, representing a 34% decrease in the K(+)-induced tritium release, was observed at a concentration of 1 microM. This opioid effect was attenuated by the selective delta-opioid receptor antagonist, ICI 174864 (1 microM). These findings support the role of a delta-opioid receptor in the modulation of ACh release in the rat globus pallidus.     

Ca2+ UPTAKE BY SYNAPTOSOMES AND ITS EFFECT ON THE INHIBITION OF ACETYLCHOLINE RELEASE BY BOTULINUM TOXIN

Abstract— ⁴⁵Ca²⁺ uptake by cerebral cortex synaptosomes was determined by gel filtration, glass fibre disc filtration under suction and by centrifugation with EGTA present. The filtration methods gave comparable results which were higher than values obtained by the centrifugation method. Uptake was increased by 25mM-K⁺ at all times investigated. The accumulated ⁴⁵Ca²⁺ was bound within the synaptosome. ⁴⁵Ca²⁺-ionophore A23187 stimulated uptake only during the first min; levels of intra-synaptosomal ⁴⁵Ca²⁺ then returned to control values. A23187 also increased intra-synaptosomal Na⁺ and Cl⁻ contents. Botulinum toxin inhibits the K.⁺-stimulated release of [¹⁴C]ACh from synaptosomes but the ionophore released [¹⁴C]ACh from both normal and botulinum-treated preparations in a Ca²⁺-dependent manner. However, it also elicited Ca²⁺-dependent release of [choline. Increased extracellular Ca²⁺ (10 mM and 20 mM) released [¹⁴C]ACh (but not [¹⁴C]choline) from both normal and botulinum-treated synaptosomes. It is concluded that botulinum toxin interferes with the provision of Ca²⁺ essential for the mechanism of ACh release.     

Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, G-protein activation, internal application of inositol trisphosphate or Ca2+: simultaneous microfluorimetry and Ca2+ dependent Cl- current recording in single pancreatic acinar cells.

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP-gamma-S, inositol trisphosphate [Ins (1,4,5) P3] or Ca2+ on the cytoplasmic free Ca2+ concentration [( Ca2+]i) were assessed by simultaneous microfluorimetry (fura-2) and measurement of the Ca2(+)-dependent Cl- current (patch-clamp whole-cell recording) in single internally perfused mouse pancreatic acinar cells. ACh (0.1-0.2 microM) evoked an oscillating increase in [Ca2+]i measured in the cell as a whole (microfluorimetry) which was synchronous with oscillations in the Ca2(+)-dependent Cl- current reporting [Ca2+]i close to the cell membrane. In the same cells a lower ACh concentration (0.05 microM) evoked shorter repetitive Cl- current pulses that were not accompanied by similar spikes in the microfluorimetric recording. When cells did not respond to 0.1 microM ACh, caffeine (1 mM) added on top of the sustained ACh stimulus resulted in [Ca2+]i oscillations seen synchronously in both types of recording. CCK (10 nM) also evoked [Ca2+]i oscillations, but with much longer intervals between slightly broader Ca2+ pulses. Internal perfusion with 100 microM GTP-gamma-S evoked [Ca2+]i oscillations with a similar pattern. Ins (1,4,5) P3 (10 microM) evoked repetitive shortlasting spikes in [Ca2+]i that were only seen in the Cl- current traces, except in one small cell where these spikes were also observed synchronously in the microfluorimetric recording. Caffeine (1 mM) broadened these Ca2+ pulses. [Ca2+]i was also directly changed, bypassing the normal signalling process, by infusion of a low or high Ca2+ solution into the pipette.(ABSTRACT TRUNCATED AT 250 WORDS)