Presynaptic effects of scopolamine,oxotremorine, noradrenaline and morphine on [3H] acetylcholine release from the myenteric plexus at different stimulation frequencies and calcium concentrations

Summary The inhibition by three modulators (oxotremorine, noradrenaline, morphine) of acetylcholine release from the myenteric plexus preincubated with [³H]choline was investigated at different stimulation frequencies and calcium concentrations. Moreover, [³H]acetylcholine release evoked by a low (0.1 Hz) or a high (10 Hz) stimulation rate was investigated at different calcium concentrations either in the absence or presence of scopolamine. A reduced calcium concentration (0.6 mmol/l) inhibited acetylcholine release more at 0.1 Hz (74% ± 3%) than at 10 Hz (44% ± 8%). Scopolamine enhanced the stimulated acetylcholine release at a calcium concentration of 1.8 mmol/l. At calcium concentrations higher than 1.8 mmol/l scopolamine failed to enhance transmitter release markedly. A reduction of the calcium concentration (< 1.8 mmol/l) significantly enhanced the effect of scopolamine, when acetylcholine release was evoked at 0.1 Hz. Oxotremorine (10 mol/l) completely suppressed acetylcholine release at 1 Hz (120 pulses). When 120 pulses were applied at 10 Hz the maximal effect was only a 64% inhibition and the concentration-response curve was significantly shifted to the right. However, after a reduction of both the train length or the calcium concentration oxotremorine produced a complete inhibition of acetylcholine release evoked at 10 Hz. In contrast to the effect of oxotremorine, the concentration-response curves for morphine and noradrenaline were similar at 1 Hz and 10 Hz. Following conclusions can be drawn: 1. The present findings fit into the concept that residual calcium accumulates in the nerve terminal during 10 Hz stimulation. 2. The results obtained with scopolamine and oxotremorine are consistent with the view that muscarine autoreceptor activation triggers a reduction of the intraneuronal availability of calcium for the stimulus-secretion coupling. 3. The presynaptic effect of morphine and partly that of noradrenaline might be mediated by a different mechanism, probably by a reduction of release sites. Send offprint requests to I. Wessler at the above address     

Facilitatory and inhibitory muscarine receptors on the rat phrenic nerve: effects of pirenzepine and dicyclomine

Summary Neuronal transmitter stores of the rat phrenic nerve were labelled by an incubation with [³H]choline. Release of [³H]acetylcholine was elicited either by a short (100 pulses, 5 Hz) or by a long (1500 pulses, 5 or 25 Hz) period of electrical nerve stimulation. Pirenzepine and dicyclomine enhanced transmitter release evoked by the short stimulation period. Both antagonists reduced transmitter release evoked by the long stimulation period. Pirenzepine reduced transmitter release at low concentrations (1 nmol/l) whereas a higher concentration was necessary for the enhancing effect; the opposite pattern was found for dicyclomine. A low concentration of oxotremorine (10 nmol/l) enhanced and a high concentration (1 mol/l) reduced transmitter release evoked by the short stimulation period. Both effects could be prevented by a low concentration of pirenzepine (10 nmol/l). It is concluded that facilitatory and inhibitory muscarine receptors are present on the motor nerve. A short stimulation period activates predominantly the negative muscarinic feedback, whereas during a long period of continuous nerve stimulation the positive muscarinic feedback mechanism is additionally activated. Both the facilitatory and inhibitory receptors might be regarded as M1-receptors but differences in the pharmacological properties between both receptor populations appear possible.This work was supported by the Deutsche Forschungsgemeinschaft. The paper contains part of the Dr. med. thesis of A. D. and M. O. Send offprint requests to I. Wessler at the above address     

Release of [3H]acetylcholine from the isolated rat or guinea-pig trachea evoked by preganglionic nerve stimulation; a comparison with transmural stimulation

Summary Basal and stimulated outflow of radioactive acetylcholine, phosphorylcholine and choline from rat and guinea-pig isolated tracheae were measured by reverse phase HPLC followed by liquid-scintillation-spectrometry. Tracheae were stimulated either by an electrical field (transmural stimulation) or by a local stimulation of the innervating parasympathetic nerves (preganglionic stimulation). Epithelium was removed in most experiments, as the epithelium inhibits acetylcholine release.The basal tritium efflux (1,600 dpm/3min) from rat isolated tracheae incubated with [³H]choline consisted of 56% [³H]phosphorylcholine and 38% [³H]choline. Preganglionic stimulation (15 Hz, 1,200 pulses) caused a 2-fold increase in tritium outflow that was abolished by the removal of extracellular calcium or by the addition of tetrodotoxin. The stimulated outflow of tritium induced by preganglionic nerve stimulation was caused by an exclusive release of [³H]acetylcholine, whereas the efflux of [³H]phosphorylcholine and [³H]choline remained unaffected by this stimulation mode. Transmural stimulation of the rat or guinea-pig trachea, however, caused, in addition to the release of [³H]acetylcholine, the outflow of [³H]phosphorylcholine. Hexamethonium (300 mol/l) or tubocurarine (100 mol/l) inhibited (80%) the increase in tritium outflow evoked by preganglionic stimulation, but did not affect tritium outflow evoked by transmural stimulation. Oxotremorine reduced [³H]acetylcholine release evoked by both stimulation modes, but oxotremorine was less potent with transmural stimulation. Scopolamine (0.3 mol/l) enhanced (120%) the release of [³H]acetylcholine evoked by preganglionic nerve stimulation indicating the blockade of an endogenous negative muscarinic feedback mechanism. Epithelium-dependent inhibition of [³H]acetylcholine release was evident with both preganglionic and transmural stimulation.The present experiments demonstrate the release of [³H]acetylcholine evoked from the isolated trachea by stimulation of the preganglionic trunk of the parasympathetic cholinergic nerves. Qualitative and quantitative differences were observed in comparison to transmural stimulation. Preganglionic nerve stimulation allows a selective excitation of pulmonary, parasympathetic nerve fibres, mimics the physiological excitation of intramural neurones and is not followed by the liberation of phosphorylcholine from non-neuronal cells. Send offprint requests to I. Wessler at the above address     

Evaluation by reverse phase HPLC of [3H]acetylcholine release evoked from the myenteric plexus of the rat

Summary Myenteric plexus-longitudinal muscle strips isolated from the small intestine of rats were incubated with [³H]choline to measure the synthesis and the release of [³H]acetylcholine. To separate different radioactive compounds (acetylcholine, choline, phosphorylcholine) from both the tissue and the overflow a new method, the reverse phase HPLC, was used.The radiochromatogram following the injection of a [³H]choline-standard and a [¹⁴C]acetylcholine-standard onto the HPLC showed a clear separation of both isotopes with a recovery rate of roughly 100%. Incubation of the muscle strips with [³H]choline caused the synthesis of [³H]acetylcholine (30,000 dpm/preparation) that increased 2-fold, when the electrical field stimulation during labelling was increased from 0.2 Hz to 1 Hz. Electrical field stimulation (3 Hz, 2 min) caused an increase in tritium efflux that was abolished by the removal of extracellular calcium or by the addition of tetrodotoxin. Analysis by reverse phase HPLC of the overflow showed that the stimulated increase in tritium overflow was balanced by the enhanced release of [³H]acetylcholine, whereas the overflow of [³H]choline was not affected by the electrical field stimulation. Oxotremorine (1 mol/l) suppressed the release of [³H]acetylcholine by 60%. Scopolamine (0.1 mol/l) prevented this inhibition and, given alone, enhanced the release of [³H]acetylcholine by 43%. The release of [³H]acetylcholine evoked at 0.2, 2 or 20 Hz did not consistently decline at increasing frequencies.The present experiments show the synthesis and the calcium-dependent release of [³H]acetylcholine from the myenteric plexus-longitudinal muscle preparation of rats correspondingly to the same in-vitro preparation isolated from guinea-pigs. Muscarinic autoinhibition operates also in the small intestine of rats. However, some differences (frequency-dependency of [³H]acetylcholine release, spontaneous neuronal activity) are evident between both species. Reverse phase HPLC is a useful method to separate radioactive choline and acetylcholine with a high recovery rate.Send offprint requests to I. Wessler at the above address     

Effects of (+)-tubocurarine on [3H]acetylcholine release from the rat phrenic nerve at different stimulation frequencies and train lengths

Summary The effect of (+)-tubocurarine (TC) on the release of [³H]acetylcholine from the rat phrenic nerve-hemidiaphragm preincubated with [3H]choline was investigated at different stimulation frequencies and train lengths.At 0.5 Hz (100 pulses) TC failed to modulate the evoked acetylcholine release. A slight (30%) inhibition was observed at 1 Hz (100 pulses). Release of acetylcholine evoked at 5, 25 and 50 Hz (100 pulses) or 100 Hz (200 pulses) was markedly reduced by TC. The degree of inhibition (60%) was similar between 5 Hz and 100 Hz. A concentration of 1 mol/l TC was a maximal effective concentration at 5 Hz whilst at all higher stimulation frequencies a 10-fold higher concentration was necessary for the maximal effect. When 300 pulses were continuously applied at 5 Hz or 50 Hz TC caused only a slight inhibition (20%). Additionally, the phrenic nerve was stimulated intermittently. Trains of 15 pulses were repeated 10 times with an interval of 3 s between each train. Under this latter stimulation condition TC failed to reduce acetylcholine release.It is concluded that nicotinic autofacilitation of acetylcholine release from the motor nerve operates at frequencies and stimulation conditions similar to the pattern of nerve activity under in vivo conditions. At least more than 15 pulses are required before the nicotinic autofacilitation becomes apparent. It appears unlikely that the TC induced fading of end-organ responses can only be attributed to a blockade of the presynaptic nicotine receptors. Send offprint requests to I. Wessler at the above address     

Effect of nicotine and tacrine on acetylcholine release from rat cerebral cortical slices

Summary The effect of nicotine (1–10 M) and tacrine (9-amino-1,2,3,4-tetrahydroacridine; THA) on stimulation evoked release of [³H]acetylcholine from the rat brain slice preparation preincubated with [³H]choline was investigated.In these preparations, nicotine enhanced while tacrine inhibited evoked [³H]acetylcholine release. These effects were blocked by (+)tubocurarine (1 M) and atropine (0.1 M) respectively. In the presence of idazoxan (0.3 M) plus atropine (0.1 M), nicotine (3 M) continued to enhance evoked [³H]acetylcholine release while the inhibitory effect of tacrine (1 M) on evoked [³H]acetylcholine release was reversed to an enhancement. Under these circumstances the effects of both nicotine and tacrine were blocked by (+)tubocurarine (1 M).These findings demonstrate that tacrine can both inhibit or enhance [³H]acetylcholine release, most likely through its activity as a cholinesterase inhibitor. Under normal circumstances following tacrine the predominant effect of the elevated levels of acetylcholine will be activation of inhibitory presynaptic muscarine receptors on cholinergic nerves and an inhibition of evoked [³H]acetylcholine release. Under conditions where both presynaptic inhibitory muscarine and ₂-adrenoceptors are blocked, the elevated levels of acetylcholine produced by tacrine will lead to the activation of facilitatory presynaptic nicotine cholinoceptors on cholinergic nerves and an enhancement of evoked [³H]acetylcholine release. Send offprint requests to R. Loiacono at the above address     

Muscarinic inhibition of endogenous noradrenaline release from rabbit isolated trachea: receptor subtype and receptor reserve

The aim of the present study was to characterize putative muscarine receptors on sympathetic nerve terminals in the rabbit trachea. Release of endogenous noradrenaline from in vitro incubated rabbit tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses) and quantified by high performance liquid chromatography with electrochemical detection.The muscarine receptor agonist oxotremorine inhibited the evoked release of noradrenaline completely at 1 ol/l (EC₅₀: 64 nmol/l). The concentration response curve was very steep (Hill coefficient of 2.3). Scopolamine shifted the concentration response curve of oxotremorine to the right (–log K_B 8.48) demonstrating specific, inhibitory muscarine receptors. Several subtype-preferring muscarine receptor antagonists also shifted the concentration response curve of oxotremorine to the right. The rank order of potency was (–log K_B or pA^* ₂): scopolamine (8.48) > AF DX 384 (7.88^*; slope of Schild plot 1.1) > (R)-trihexyphenidyl (7.87) > 4-DAMP (7.85) > AQ-RA 741 (7.77) methoctramine 6.18 > pirenzepine (6.0) >p-fluoro-hexahydrosiladifenidol (p-FHHSiD, 5.68). When these affinity constants were plotted against reported –log K_i values determined in binding studies on human cloned muscarine receptor subtypes (m₁-m₅), the best correlation was obtained for m₂. Indomethacin (3 mol/l), which on its own increased the evoked noradrenaline release by about 45%, affected neither the inhibitory effect of oxotremorine nor the antagonistic potency of methoctramine or p-FHHSiD. After preincubation for 48 min with 300 mol/l phenoxybenzamine, which has been shown to inactivate muscarine receptors irreversibly, the concentration response curve of oxotremorine was shifted 5.2 fold to the right and the maximal inhibition was reduced by 50%, whereas the slope remained steep (Hill coefficient 2.6). These experiments indicated that a fraction of about 22% of the muscarine receptors has to be occupied by oxotremorine to produce half-maximum inhibition of noradrenaline release; the dissociation constant of oxotremorine at the prejunctional muscarine receptors was 0.33 mol/l.In conclusion, the sympathetic nerve terminals in the rabbit trachea are endowed with inhibitory M₂-like muscarine receptors for which methoctramine displayed a low affinity. Since a large receptor reserve could be excluded, the steep concentration response curve of oxotremorine suggests that activation of muscarine receptors has to reach a threshold level before the onset of an inhibitory effect. Correspondence to: K. Racké at the above address     

3H-noradrenaline release from mouse iris–ciliary body: role of presynaptic muscarinic heteroreceptors

Sympathetic neurotransmitter release and its modulation by presynaptic muscarinic heteroreceptors were studied in mouse iris–ciliary bodies. Tissue preparations were preincubated with ³H-noradrenaline and then superfused and stimulated electrically. Firstly, experimental conditions were defined, allowing study of presynaptic sympathetic inhibition in mouse iris–ciliary body. If tissue was stimulated four times with 36 pulses/3 Hz, tritium overflow peaks were reliably and reproducibly measured. As expected, these stimulation conditions led to marked ₂-autoinhibition as indicated by the release-enhancing effect of the ₂-antagonists phentolamine and rauwolscine. To ensure autoinhibition-free ³H-noradrenaline release, which is optimal for studying presynaptic sympathetic inhibition, ₂-receptors were blocked in all subsequent experiments. Under these conditions, evoked tritium overflow was almost completely abolished in the presence of the sodium channel blocker tetrodotoxin, indicating a neuronal origin of ³H-noradrenaline release. Secondly, muscarinic inhibition of ³H-noradrenaline release was characterized using the conditions described above (36 pulses/3 Hz; phentolamine 1 M and rauwolscine 1 M throughout). The muscarinic receptor agonist oxotremorine M decreased evoked tritium overflow in a concentration-dependent manner with an IC₅₀ of 0.33 M and maximal inhibition of 51%. The concentration–response curve of oxotremorine M was shifted to the right by the muscarinic antagonists ipratropium and methoctramine, whereas pirenzepine was ineffective. The observed rank order of antagonist potencies, ipratropium > methoctramine > pirenzepine, which is typical for the M₂ subtype, indicates that presynaptic muscarinic receptors on sympathetic axons of mouse iris–ciliary bodies are predominantly M₂. Finally, inhibition of ³H-noradrenaline release by endogenously secreted acetylcholine was investigated. Longer pulse trains, 120 pulses/3 Hz and 600 pulses/5 Hz, were used and the cholinesterase inhibitor physostigmine was added to the superfusion medium to increase synaptic levels of endogenous acetylcholine. Under these conditions, ipratropium approximately doubled the evoked overflow of tritium, indicating that endogenously released acetylcholine can activate presynaptic muscarinic heteroreceptors. In conclusion, the present experiments establish measurement of the electrically induced release of ³H-noradrenaline from mouse iris–ciliary bodies. As in other species, noradrenaline release in this preparation was subject to presynaptic muscarinic inhibition. Our results also indicate that the presynaptic muscarinic receptors on sympathetic axons in mouse iris–ciliary body are predominantly M₂. Moreover, these receptors can be activated by both exogenous agonists and endogenously released acetylcholine and, hence, may operate physiologically in the interplay between the parasympathetic and sympathetic nervous system.     

Differential effects of calcium channel antagonists (ω-conotoxin GVIA,nifedipine, verapamil) on the electrically-evoked release of [3H]acetylcholine from the myenteric plexus,phrenic nerve and neocortex of rats

Summary Electrically-evoked release of [³H]acetylcholine from autonomic neurons (myenteric plexus), motoneurons (phrenic nerve) and the central nevous system (neocortex) was investigated in the presence and absence of the calcium channel antagonists -conotoxin GVIA, nifedipine and verapamil, whereby the same species (rat) was used in all experiments. Release of [³H]acetylcholine was measured after incubation of the tissue with [³H]choline.-Conotoxin GVIA markedly reduced (70%) the evoked release of [³H]acetylcholine from the myenteric plexus of the small intestine (IC₅₀: 0.7 nmol/l) with a similar potency at 3 and 10 Hz stimulation. An increase in the extracellular calcium concentration attenuated the inhibitory effect of -conotoxin GVIA. Release of [³H]acetylcholine from the rat neocortex was also inhibited (90%) by -conotoxin GVIA, but the potency was 19-fold lower (IC₅₀: 13 nmol/l). However, the release of [³H]acetylcholine from the phrenic nerve was not reduced by -conotoxin GVIA (100 nmol/l) at 1.8 mmol/l calcium (normal concentration), whereas -conotoxin GVIA inhibited evoked [³H]acetylcholine release by 47% at 0.9 mmol/l calcium. Neither nifedipine (0.1 and 1 mol/l) nor verapamil (0.1, 1 and 10 mol/l) modified the evoked release of [3H]acetylcholine from the myenteric plexus and the phrenic nerve.Acetylcholine release from different neurons appears to be regulated by different types of calcium channels. N-type channels play the dominant role in regulating acetylcholine release from both the myenteric plexus and the neocortex, whereas acetylcholine release from motor nerves is regulated by calcium channel(s) not yet characterized. Send offprint requests to I. Wessler at the above address     

Different muscarinic receptors mediate autoinhibition of acetylcholine release and vagally-induced vasoconstriction in the rat isolated perfused heart

Summary Experiments were carried out on rat isolated perfused hearts with both vagus nerves attached. The acetylcholine stores were labelled with [¹⁴C]-choline. The effects of muscarinic receptor antagonists on the [14C]overflow and increase in perfusion pressure evoked by vagus nerve stimulation (10 Hz, 4–10 mA) were studied in order to determine the muscarinic receptor type involved in autoinhibition of acetylcholine release and vagally-induced vasoconstriction in the rat heart.Stimulation of the vagus nerves (1200 pulses) caused an increase in [¹⁴C]-overflow and in perfusion pressure which was significantly reduced by hexamethonium 500 mol/l and abolished by tetrodotoxin 0.3 mol/l or perfusion with Ca²⁺-free solution. The fractional rate of evoked [¹⁴C]-overflow per pulse upon stimulation at 10 Hz (720 pulses) was doubled in the presence of the non-selective antagonist atropine (0.01–1 mol/l) as well as in that of the M₂-selective compounds methoctramine (0.1 mol/l) and AF-DX 116 (0.1–1 mol/l), but remained unaffected by the M3-selective hexahydrosiladifenidol (0.1 mol/l). The increase in perfusion pressure upon nerve stimulation was reduced by atropine (0.01 mol/l) or hexahydrosiladifenidol (0.1 mol/l) to approximately 50% and increased by about 50% in the presence of AF-DX 116 (0.1 mol/l).The results show that the autoinhibition of acetylcholine release in the rat heart is mediated by M₂ receptors. On the other hand, the increase in perfusion pressure upon vagus nerve stimulation is caused by a different muscarinic receptor, more sensitive to hexahydrosiladifenidol than to M₂-selective antagonists. Send offprint requests to I. T. Bognar at the above address     

Prejunctional M1 and postjunctional M3 muscarinic receptors in the circular muscle of the guinea-pig ileum

The effects of subtype-selective muscarinic receptor antagonists on electrically evoked release of acetylcholine and muscle contraction were compared in circular muscle preparations of the guinea-pig ileum. Incubation of the preparation with [³H]choline resulted in the formation of [³H]acetylcholine. Electrical stimulation caused the release of [³H]acetylcholine which was abolished by tetrodotoxin and omission of calcium from the medium. 5-Hydroxytryptamine (10 M) and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (300 M) did not change acetylcholine release. The muscarinic antagonists pirenzepine (M1 selective), AF-DX 116 (M2 selective) and hexahydrosiladifenidol (M3 selective) caused concentration-dependent increases in the evoked release of acetylcholine, and inhibitions of the circular muscle contraction. The postjunctional affinity constants (pA₂ values) obtained for hexahydrosiladifenidol (8.06), pirenzepine (6.95) and AF-DX 116 (6.60) identified the muscular receptor as an M3 subtype. Pirenzepine was more potent in facilitating the evoked release than hexahydrosiladifenidol and AF-DX 116. These findings suggest that the release of acetylcholine in the circular muscle is inhibited by M1 muscarinic autoreceptors whereas muscle contraction is mediated by M3 receptors.     

Epithelium-derived inhibition of [3H]acetylcholine release from the isolated guinea-pig trachea

Summary To investigate presynaptic, regulatory mechanisms on parasympathetic nerve fibres innervating the airways, the release of newly-synthesized [³H]acetylcholine from the isolated trachea was studied. Reverse phase HPLC followed by liquid scintillation spectrometry was used to separate and quantify the radioactive compounds choline, phosphorylcholine and acetylcholine in the incubation medium and the tissue.During the incubation of the tracheae with [³H]choline a significant synthesis of [³H]acetylcholine (35,000 dpm/preparation) and [³H]phosphorylcholine (500,000 dpm/preparation) occurred. In epithelium-deficient tracheae the formation of [³H]phosphorylcholine was enhanced, whereas the content of [³H]acetylcholine remained unchanged. The spontaneous outflow of tritium consisted mainly of [³H]phosphorylcholine (900 dpm/3 min) and [³H]choline (800 dpm/3 min); [³H]acetylcholine was only a minor fraction (50 dpm/3 min). Electrical stimulation of tracheae with intact epithelium caused only a small release of [³H]acetylcholine (460 dpm in the sample obtained during stimulation), but a considerable outflow of [³H]phosphorylcholine (1,900 dpm) without affecting the outflow of [³H]choline. Electrical stimulation of epithelium-deficient tracheae, however, induced a substantial release of [³H]acetylcholine (2,400 dpm), but only a small outflow of [³H]phosphorylcholine. Chemical stimulation (30 mol/1 veratridine) also caused a large release of [³H]acetylcholine (1,700 dpm) without affecting the outflow of [³H]phosphorylcholine or [³H]choline. Indomethacin (3 mol/1) enhanced the electrically-evoked release of [³H]acetylcholine from tracheae with intact epithelium by 89%.The present experiments demonstrate a strong inhibition by the epithelium of the electrically-evoked release of [³H]acetylcholine from the isolated guinea-pig trachea. Cyclooxygenase products of arachidonic acid do not appear as the main mediators of the epithelium-derived inhibition of acetylcholine release. Send offprint requests to I. Wessler at the above address     

Muscarinic inhibition of acetylcholine release from a novel in vitro preparation of the guinea-pig trachea

Summary An isolated preparation of the guinea-pig trachea is described which allows the simultaneous measurement of acetylcholine release and smooth muscle contraction. Incubation of the epithelium-free preparation with [³H]choline resulted in the formation of [³H]acetylcholine. Electrical stimulation caused the release of [³H]acetylcholine and a contractile response. Tetrodotoxin and omission of calcium from the medium abolished both the evoked release and contractions.The muscarinic agonists oxotremorine, carbachol and pilocarpine concentration-dependently inhibited the electrically evoked acetylcholine release and contracted the tracheal smooth muscle. Pre- and postsynaptic EC₅₀ values for a given agonist were not different. Atropine (100 nmol/l) significantly faciliated the evoked acetylcholine release. A concentration of 10 nmol/l atropine did not change the evoked release but antagonized the inhibitory effect of oxotremorine. It is concluded that presynaptic muscarine autoreceptors inhibit the release of acetylcholine from parasympathetic nerves of the guinea-pig trachea.Send offprint requests to G. D'Agostino at the above address     

Activation of dopamine D1 receptors does not affect D2 receptor-mediated inhibition of acetylcholine release in rabbit striatum

Summary The possible involvement of dopamine D₁ receptors in the regulation of acetylcholine release in the rabbit caudate nucleus was investigated. Caudate slices, preincubated with [³H]choline, were superfused continuously and subjected to electrical field stimulation with only a single pulse. In agreement with the view that the release of acetylcholine evoked by a single electrical pulse is not influenced by endogenous transmitters, atropine and domperidone failed to icnrease the evoked release of [³H]acetylcholine, whereas oxotremorine and quinpirole caused a concentration-dependent inhibition of transmitter release. Neither the dopamine D₁ receptor antagonist SCH 23390 nor the Dt agonist SKF 38393 in a concentration range of 0.01–1 mol/l changed the evoked [³H]acetylcholine release. The inhibitory effect of the dopamine D₂ receptor agonist quinpirole was virtually abolished in the presence of 0.1 mol/l domperidone and diminished in the presence of 1 mol/l SCH 23390. It remained unchanged in the presence of 1 mol/l SKF 38393. It is concluded that the inhibition of acetylcholine release by dopamine is mediated exclusively via presynaptic dopamine D₂ receptors and that the antagonistic effect of SCH 23390 on the inhibition of acetylcholine release by quinpirole is due to its interaction with dopamine D₂ rather than D₁ receptors located on cholinergic nerve terminals. Send offprint requests to C. Allgaier at the above address     

Two types of neuronal muscarine receptors modulating acetylcholine release from guinea-pig myenteric plexus

Summary Longitudinal muscle strips of the guinea-pig ileum were incubated with [³H]choline and the effects of muscarinic agonists on smooth muscle contraction and on spontaneous and electrically-evoked outflow of tritium were studied. Muscarine and pilocarpine concentration-dependently increased both muscle contraction and spontaneous outflow of [³H]ACh, and inhibited the electrically-evoked outflow of [³H]ACh. The increase in spontaneous outflow was prevented by tetrodotoxin and scopolamine, but not by hexamethonium. Oxotremorine (1–100 M) did not increase the spontaneous outflow of tritium.Pirenzepine in concentrations of 10 and 100 nM hardly affected the muscle contractions induced by pilocarpine, but significantly antagonized the pilocarpine-evoked increases in [³H]ACh outflow. Likewise, pirenzepine (100 nM) antagonized more effectively the enhancement by muscarine of spontaneous outflow than the inhibitory effect of muscarine on the electrically-evoked release of [³H]ACh. Scopolamine (1 and 10 nM) antagonized to a similar extent the effects of pilocarpine on spontaneous outflow of [³H]ACh and on muscle contraction.The results suggest that the cholinergic nerves of the myenteric plexus are endowed with excitatory (ganglionic) and inhibitory (prejunctional) muscarine receptors which modulate the release of ACh and which differ in their affinities to pirenzepine.     

Cholinergic modulation of the release of 5-hydroxytryptamine from the guinea pig ileum

Summary Isolated segements of the guinea pig ileum were vascularly perfused and the release of 5-HT and its metabolite 5-HIAA into the portal venous effluent determined by HPLC with electrochemical detection. Test substances were applied via the arterial perfusion medium. Oxotremorine inhibited concentration-dependently the release of 5-HT and 5-HIAA (by 47% at 1 mol/l). Scopolamine (0.1 mol/1) did not affect the release of 5-HT and 5-HIAA, but antagonized the effect of oxotremorine. In the presence of TTX (1 mol/1) oxotremorine (1 pmol/1) increased the release of 5-HT by 150% and that of 5-HIAA by 220%. This increase was completely blocked by scopolamine. Hexamethonium (100 pmol/1) and TTX (1 pmol/1) reduced the release of 5-HT by 32 and 40%, respectively. DMPP (10 pmol/1) increased the release of 5-HT by 57%, and this effect was prevented by hexamethonium. Neither DMPP nor hexamethonium significantly affected the release of 5-HIAA. The enhancing effect of DMPP on 5-HT release was increased and prolonged in the presence of TTX or scopolamine. Nicotine (1, 10 or 30 mol/l) alone did not cause a consistent increase in the release of 5-HT. However, in the presence of scopolamine nicotine increased the release of 5-HT by 57%. In conclusion, the release of intestinal 5-HT is facilitated via muscarine and nicotine receptors located on the enterochromaffin cells. Indirect evidence suggests that the release of 5-HT is additionally modulated by an as yet unknown inhibitory neurotransmitter released by muscarine receptor activation.Abbreviations DMPP 1,1-dimethyl-4-phenylpiperazinium - 5-HT 5-hydroxytryptamine - 5-HIAA 5-hydroxyindoleacetic acid - TTX tetrodotoxin Send offprint requests to H. S. at the above address     

Modulation of stimulation-evoked release of newly formed acetylcholine from mouse hemidiaphragm preparation

Summary A radioisotope method has been developed for measuring the stimulation-evoked release of acetylcholine without the use of cholinesterase inhibitors from the mouse hemidiaphragm preparation which had been loaded with ³H-choline. Evidence has been obtained that ³H-choline was taken up by and released from both innervated and non-innervated mouse hemidiaphragm preparations. However, it was released in the form of ³H-acetylcholine in response to electrical field stimulation only from the innervated preparations. Long lasting (51 min) S₁ stimulation of the preparations exhausted the radioactive acetylcholine stores to the extent that S₂ did not evoke any release of ³H. These data suggest that when the labelled acetylcholine stores become exhausted, the labelled choline, still present in the tissue, cannot be released by electrical stimulation. Tetrodotoxin (1 mol/1) administration and Ca withdrawal inhibited, 20–100 mol/l 4-aminopyridine enhanced the release of ³H-acetylcholine in response to electrical stimulation. Activation of the presynaptic muscarinic receptors by the agonist oxotremorine (50 mol/l) decreased the liberation of ³H-acetylcholine. The muscarinic antagonist atropine (1 mol/l) abolished the inhibitory effect of oxotremorine and by itself increased the evoked release of the newly formed ³H-acetylcholine. Adenosine (50 gmol/l) reduced the evoked release of radioactivity. Theophylline (30 mol/l) prevented the inhibitory effect of adenosine and itself enhanced the release. Xylazine (1 mol/l), an alpha₂-adrenoceptor agonist did not affect the release. It is concluded that the stimulation-evoked release of ³H-acetylcholine from the mouse phrenic nerve hemidiaphragm preparation preloaded with ³H-choline is derived from the motor nerves. The release of acetylcholine is modulated by activation of presynaptic muscarinic and adenosine receptors. Send offprint requests to G. T. Somogyi at the above address     

Glycine-evoked release of [3H]acetylcholine from rat striatal slices is independent of the NMDA receptor

Summary KCl-, NMDA-, and glycine-evoked release of [³H]acetylcholine was studied in superfused rat striatal slices. KCl-evoked release of [³H]acetylcholine was inhibited by 1.2 mM MgC12 and 100 M lidocaine. Similarly, NMDA-evoked release was inhibited by MgCl₂ and lidocaine as well as 10 M CGS 19755, a competitive antagonist at NMDA receptors, and 10 nM MK-801, a noncompetitive antagonist of NMDA-induced responses. Glycine-evoked release was calcium-dependent and was inhibited by 0.1 M strychnine whereas KCl- and NMDA-evoked release were resistant to strychnine. In addition, lidocaine inhibited the glycine-induced response. Cross-tachyphylaxis was not observed between NMDA- and glycine-evoked release. These results indicate that the strychnine-sensitive, glycine-evoked release of [³H]acetylcholine is independent of the NMDA receptor.     

Interaction between presynaptic facilitatory angiotensin II receptors and inhibitory muscarinic cholinoceptors on3H-noradrenaline release in the rabbit heart

Summary The existence of a functional interaction between presynaptic receptors modulating the release of noradrenaline was studied in the rabbit heart. Isolated right atria were prelabelled with³H-noradrenaline and the overflow of tritium was induced by field stimulation (2 Hz, 0.1 ms duration, supramaximal voltage for a total of 180 pulses). In atria superfused with Krebs' solution containing 10 mol/l cocaine and 30 mol/l corticosterone, angiotensin II (10 nmol/l) increased the stimulation-evoked overflow of³H-transmitter by 2.8-fold. The addition of atropine (0.3 mol/l) to the perfusion medium, either in the presence or in the absence of uptake inhibitors, further enhanced the facilitatory effect of angiotension II (³H-transmitter release increased by 3.5-fold). Exposure to 1 mol/l carbachol decreased by 65% the stimulation-evoked release of³H-transmitter while the facilitatory effect of angiotensin II determined in the presence of the muscarinic cholinoceptor agonist was enhanced (³H-transmitter release increased by 6.6-fold). Conversely, during sustained activation of presynaptic angiotensin receptors producing a 2.5-fold increase in the release of³H-transmitter, the inhibitory effect of carbachol remained unchanged. These results suggest a functional interaction between presynaptic inhibitory muscarinic cholinoceptors and the presynaptic facilitatory angiotensin receptor which modulate the release of noradrenaline from cardiac noradrenergic nerves.     

Benzimidazolones and renzapride facilitate acetylcholine release from guinea-pig myenteric plexus via 5-HT4 receptors

The effects of the 5-HT₄ receptor agonists BIMU 8, BIMU 1, renzapride and of the 5-HT_1p receptor agonist 5-hydroxyindalpine on basal and electrically evoked outflow of tritium were studied in guinea-pig longitudinal muscle myenteric plexus preparations preincubated with [³H]choline. Muscle contractions were recorded simultaneously.BIMU 8 caused a calcium dependent and tetrodotoxin sensitive increase in basal [³H]outflow that was assumed to represent release of [³H]acetylcholine. In addition, BIMU 8 enhanced the release of [³H]acetylcholine and twitch contractions evoked by submaximal electrical stimulation. Ondansetron (1 mol/l) did not change the effects of BIMU 8, but DAU 6285 and tropisetron (each 1 mol/l) competitively antagonized the various facilitatory effects of BIMU 8 with pA₂ values of 7.0–7.2 (DAU 6285) and 7.0–7.3 (tropisetron). The phosphodiesterase inhibitors IBMX and rolipram did not increase the effects of BIMU 8. BIMU 1 and renzapride also concentration-dependently increased basal release of acetylcholine, and release and contractions caused by submaximal stimulation. The effects of BIMU 1 and renzapride were competitively antagonized by 1 mol/l tropisetron (pA₂ 6.6–7.1). The EC₅₀ values for the increase in the evoked [³H]acetylcholine release and contractions were closely similar. 5-Hydroxyindalpine did not change basal release and slightly inhibited the evoked release of [³H]acetylcholine. Release of acetylcholine and contractions elicited by submaximal stimulation were strongly inhibited by ( + )-tubocurarine which indicates that nicotinic ganglionic transmission is involved in this kind of release.The results suggest that BIMU 8, BIMU 1 and renzapride stimulate 5-HT₄ receptors at cholinergic interneurones and thereby facilitate nicotinic ganglionic transmission in the myenteric plexus. Cyclic AMP is probably not involved in the 5-HT₄ receptor mediated facilitation of acetylcholine release.