Muscarine receptors on the rat phrenic nerve,evidence for positive and negative muscarinic feedback mechanisms

Summary Neuronal transmitter stores of the rat phrenic nerve were labelled by incubation with [³H]choline. Release of [³H]acetylcholine was elicited by electrical nerve stimulation (100 or 1500 pulses, 5 or 25 Hz) or by high potassium (27 mmol/l) and the effects of the muscarine receptor agonist oxotremorine and the antagonist scopolamine were investigated. Neither oxotremorine nor scopolamine affected the basal tritium efflux. A low concentration of oxotremorine (10 nmol/l) enhanced and a high concentration of oxotremorine (1 ol/l) reduced the electrically evoked [³H]acetylcholine release. Likewise, the high potassium-evoked [³H]acetylcholine release was reduced by a high concentration of oxotremorine. Both effects of oxotremorine, increase and decrease, were abolished by a pretreatment (30 min before the first stimulation period) with 0.1 mol/l scopolamine. Scopolamine (0.1 ol/l) alone, enhanced [³H]acetylcholine release evoked by 100 pulses (5 Hz) or by high potassium. Scopolamine, however, reduced [³H]acetylcholine release evoked by 1500 pulses (5 Hz or 25 Hz). The concentration-response curves obtained for scopolamine under these latter stimulation conditions were flat-running and biphasic which might indicate the involvement of two opposite effects (increase and decrease) of scopolamine under the present stimulation conditions. Both effects of scopolamine were reduced in the presence of 10 gmol/l neostigmine. It is concluded that muscarine receptors are present within the endplate region of motor nerves. Transmitter release from motor nerves appears to be regulated by two muscarinic feedback mechanisms. The negatively operating system is activated during short stimulation periods and the positively operating system becomes additionally apparent during long stimulation periods. Blockade of cholinesterase can hide presynaptic muscarinic mechanisms on motor nerves. Send offprint requests to I. Wessler at the above address     

A presynaptic excitatory M1 muscarine receptor at postganglionic cardiac noradrenergic nerve fibres that is activated by endogenous acetylcholine

Summary Rabbit atria were isolated with the extrinsic right vagus and sympathetic nerves intact and perfused with Tyrode solution. Noradrenaline overflow evoked by sympathetic nerve stimulation (SNS) at 3 Hz for 3 min was determined before, during, and after vagus nerve stimulation (VNS), also at 3 Hz and for 3 min. The VNS pulses preceded the SNS pulses by 3, 100 and 233 ms. Acetylcholine overflow was determined after labelling of the transmitter stores with [¹⁴C]choline.Pirenzepine 80 nmol/l failed to alter the muscarinic inhibition of noradrenaline overflow when the vago-sympathetic impulse intervals were 3 and 233 ms. At an interval of 100 ms VNS did not significantly inhibit noradrenaline overflow in the absence of pirenzepine but produced an inhibition in the presence of the drug. When the pirenzepine concentration was varied (0.4–300 nmol/l) the largest inhibition of noradrenaline overflow was observed at 5.7 nmol/l whereas 300 nmol/l fully antagonized the inhibition. Acetylcholine overflow evoked by VNS was not altered by pirenzepine 0.4–300 nmol/l.AF-DX 116 (11-[{2[oi(diethylamino)methyl]-1-piperidinyl}-acetyl]-5,11-dihydro-6H-pyrido-[2,3-b]-[1,4]benzodiazepine-6-one), an M₂ receptor selective antagonist, concentration-dependently (100–800 nmol/l) inhibited the decrease of tension development elicited by VNS. At the 100 ms vago-sympathetic impulse interval noradrenaline overflow was enhanced in the presence of AF-DX 116 400 and 800 nmol/l. However, already 100 nmol/l of the drug caused a maximum (fourfold) increase of acetylcholine overflow.It is concluded that acetylcholine released onto noradrenergic nerve fibres causes a small facilitation of noradrenaline overflow at a vago-sympathetic impulse interval of 100 ms. This response is mediated by an M₁ receptor and is superimposed on the well-known M₂ receptor mediated inhibition of noradrenaline release which is obtained at vago-sympathetic impulse intervals ranging between 3 and 233 ms. The M₂ autoreceptor regulating acetylcholine release is activated by lower synaptic concentrations of the transmitter than the M₂ heteroreceptor regulating noradrenaline release.Abbreviations SNS sympathetic nerve stimulation - VNS vagus nerve stimulation Send offprint requests to: E. Muscholl at the above address     

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.     

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     

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     

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     

Modulation of noradrenaline release by presynaptic alpha-2 and beta adrenoceptors in rat atria

Summary The effect of pretreatment with the beta-2-selective adrenoceptor agonist, (+-)-clenbuterol (0.3 mg/kg, twice daily, 14 days) on prejunctional alpha-2- and beta-adrenoceptors was studied in rat atria. When atria from non-pre-treated rats had been preincubated with (³H)-noradrenaline, (-)-isoprenaline (0.02 to 4.0 M) did not affect tritium overflow evoked by stimulation of the cardioaccelerant nerves, but a higher concentration (40 M) decreased it. Blockade of prejunctional inhibitory alpha-2-adrenoceptors by yohimbine (0.03, 0.3 and 0.8 M) enhanced the overflow of tritium. In the presence of yohimbine, isoprenaline (1.2 M) significantly increased stimulation-induced transmitter overflow, suggesting that in rat atria the facilitatory effect of isoprenaline mediated via prejunctional beta-adrenoceptors, is masked by the dominant influence of inhibitory alpha-2-adrenoceptors. (-)-Propranolol (0.1 M) prevented the isoprenaline-induced increase in atrial rate and the isoprenaline-induced enhancement of transmitter release in the presence of yohimbine (0.3 M), but did not modify by itself the stimulation-induced efflux of tritium, suggesting that neuronally released noradrenaline failed to activate facilitatory prejunctional beta-adrenoceptors. When atria from clenbuterol-pretreated rats had been preincubated with ³H-noradrenaline, the facilitatory effect of yohimbine 0.03 and 0.3 M was markedly enhanced and, in this case, isoprenaline (1.2 and 12.0 M) failed to cause its facilitatory effect in the presence of the alpha-2-adrenoceptor antagonist. Propranolol did not modify the facilitatory effect of yohimbine. No changes in the isoprenaline-induced increase in atrial rate were observed in clenbuterol-treated rats. In addition, the treatment reduced the positive chronotropic effect of nerve stimulation without affecting the response to exogenous noradrenaline, suggesting a reduction in the transmitter release induced by nerve stimulation in clenbuterol-treated rats. These results suggest that chronic treatment with clenbuterol desensitizes facilitatory prejunctional beta-adrenoceptors, without affecting the postsynaptic beta-adrenoceptors, thus implying that prejunctional beta-adrenoceptors possess properties of the beta-2-subtype. Send offprint requests to M. A. Enero 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.     

Estimation of the biophase concentration of noradrenaline at presynaptic α2-adrenoceptors in brain slices

Summary The aim of the present study was to determine the local concentrations of noradrenaline existing at presynaptic ₂-adrenoceptors during electrical pulse train stimulation of brain slices at different frequencies. The experiments are based on the assumption that the concentration of released noradrenaline at the ₂-adrenoceptors exerting a certain autoinhibition should be equal to the concentration of exogenous noradrenaline causing the same inhibition under conditions in which any influence of the released transmitter is excluded. In order to avoid autoinhibition, hippocampus and cortex slices of the rabbit and the rat, prelabelled with [³H]noradrenaline and superfused in presence of an uptake inhibitor, were electrically stimulated using 4 pulses delivered at 100 Hz (POP stimulation). Exogenous noradrenaline diminished the overflow of tritium elicited by POP stimulation in a concentration-dependent manner. In rabbit brain tissues the EC₅₀ value and maximum inhibition of noradrenaline release were found to be approximately 6 nmol/l and more than 95%, respectively, whereas in rat tissues the corresponding values were between 20 and 30 nmol/l and approximately 90%. When electrical stimulation was performed with trains of 36 pulses delivered at 0.1, 0.3 or 3 Hz in absence or presence of an uptake inhibitor, the ₂-adrenoceptor antagonist yohimbine (1 or 10 mol/l) enhanced the evoked tritium overflow in a manner which was dependent on the frequency of stimulation and on blockade of the re-uptake mechanism. The facilitatory effects of yohimbine reflected an extent of autoinhibition which was between 53% (36 pulses/0.1 Hz, no uptake inhibitor) and 85% (36 pulses/3 Hz, uptake inhibitor present) in rabbit and between 16% (36 pulses/0.3 Hz, no uptake inhibitor) and 71% (36 pulses/3 Hz, uptake inhibitor present) in rat brain slices. Accordingly, the corresponding estimated biophase concentrations of noradrenaline were generally higher in rat than in rabbit tissues (they were between 32.5 and 74.5 or 5.1 and 51.6 nmol/l in the presence or absence of an uptake inhibitor, respectively, in the rat, and between 15 and 23.1 or 6.1 and 18.6 nmol/l in the rabbit). The observed frequency dependence of the effect of re-uptake blockade on the calculated biophase concentrations of noradrenaline would be compatible with the idea of a dependence of the effectiveness of the re-uptake mechanism on the firing rate of the neurone in being more effective at lower frequencies. Moreover, the stikingly low biophase concentrations of noradrenaline suggest that also in brain tissue noradrenaline causes lateral inhibition of release as has recently been shown for guinea-pig vas deferens. Send offprint requests to C. Allgaier at the above address     

RILMENIDINE ACTS AS A PARTIAL AGONIST AT PREJUNCTIONAL α2-ADRENOCEPTORS IN GUINEA-PIG ATRIA

1. The present study was carried out to determine whether rilmenidine, a recently introduced antihypertensive agent which acts on alpha 2-adrenoceptors, has partial agonist activity on prejunctional alpha 2-adrenoceptors in guinea-pig atria. 2. Isolated preparations of guinea-pig atria were incubated with [3H]-noradrenaline and the efflux of radioactivity induced by stimulation of intramural sympathetic nerves was used as an index of release of transmitter noradrenaline. 3. Rilmenidine (1 mumol/l) inhibited noradrenaline release evoked by short trains (five, 20 and 50 pulses) of sympathetic nerve stimulation and this inhibitory effect of rilmenidine was antagonized by the alpha 2-adrenoceptor antagonists, idazoxan (0.1 and 0.3 mumol/l) and rauwolscine (0.3 mumol/l) whereas it was not affected by the alpha 1-adrenoceptor antagonist prazosin (0.1 mumol/l). 4. On the other hand, rilmenidine (1 mumol/l) enhanced noradrenaline release evoked by long trains (150 and 300 pulses) of stimulation and this effect was also abolished by idazoxan (0.1 mumol/l). 5. These findings suggest that the effects of rilmenidine on transmitter release depend on the degree of auto-inhibition: when the concentration of noradrenaline in the biophase of the prejunctional alpha 2-adrenoceptors is low, rilmenidine acts as an agonist, but when the concentration is high it acts as an antagonist. Thus, rilmenidine, like clonidine, is a partial agonist on prejunctional alpha 2-adrenoceptors in guinea-pig atria.     

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.     

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     

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     

Subtypes of muscarinic receptor on cholinergic nerves and atrial cells of chicken and guinea-pig hearts.

1. Electrically driven chicken and guinea-pig atria were used to investigate the negative inotropic effects of the muscarinic agonists methacholine and acetylcholine (ACh). The release of ACh from isolated hearts into the perfusate in response to (preganglionic) vagal or (pre- and postganglionic) field stimulation was bioassayed on the guinea-pig ileum or determined by labelling with [3H]-choline. 2. Concentration-response curves for the negative inotropic effect of methacholine were shifted to the right by pirenzepine in various concentrations (0.03 to 10 mumol l-1). The pA2 values were 7.76 in chicken atria and 6.53 in guinea-pig atria. Pirenzepine and atropine antagonized the negative inotropic response to 0.3 mumol l-1 ACh. The half-maximally effective concentrations (IC50) of pirenzepine (Pz) and atropine were 40 and 5.4 nmol l-1 in chicken atria and 330 and 3.5 nmol l-1, respectively, in guinea-pig atria. Thus, the respective potency ratios (IC50Pz/IC50atropine) were 7.4 and 94.3 in the two species. 3. Pirenzepine in low concentrations increased the release of unlabelled and 3H-labelled ACh from isolated hearts evoked by vagal and field stimulation only in chicken, but not in guinea-pigs. The half-maximally-effective concentration of pirenzepine was about 30 nmol l-1 in the chicken heart, whereas, in the guinea-pig heart, an increased release was observed at 300 nmol l-1. 4. (+)-Tubocurarine [(+)-Tc; 100 mumol l-1] reduced the release of ACh evoked by (preganglionic) vagal stimulation to a (+)-Tc-resistant release of about 30%. The time-course of the neuronal release of [3H]-ACh was markedly altered: the onset was delayed and the termination was extended beyond the period of stimulation (1 min or 5s) by several seconds. The (+)-Tc-resistant release was nearly abolished by 30 nmol l-1 pirenzepine. 5. In conclusion, the pre- and post-synaptic muscarinic receptors of the parasympathetic neuroeffector junction of the heart both belong to the M1-subtype in the chicken and to an M2-subtype in the guinea-pig. Block of the nicotinic ganglionic transmission in the chicken heart by (+)-Tc unmasked a muscarinic transmission, which presumably was mediated through M1-receptors stimulating a low and prolonged postganglionic release of ACh.     

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     

Transmitter release patterns of noradrenergic,dopaminergic and cholinergic axons in rabbit brain slices during short pulse trains,and the operation of presynaptic autoreceptors

Summary Slices of rabbit brain were field-stimulated either by single electrical pulses or by trains of 4 or 8 pulses at 1 or 100 Hz in order to study transmitter release patterns and the autoinhibition of transmitter release. The slices were preincubated with ³H-noradrenaline (cortex), ³H-dopamine (caudate nucleus) or ³H-choline (caudate nucleus).Slices preincubated with ³ H-noradrenaline were superfused with medium containing desipramine 1 gmol/l. The overflow of tritium elicited by single pulses amounted to 0 .19% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was similar, whereas that elicited by 4 pulses/100 Hz was 5.1-fold higher. Yohimbine 101000 nmol/l increased up to 2.5-fold the overflow evoked by 4 pulses/1 Hz but did not change the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with ³ H-dopamine were superfused with medium containing nomifensine 1 mol/l. The overflow of tritium elicited by single pulses was 0.39% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was 1.3-fold and the overflow elicited by 4 pulses/100 Hz 1.4-fold higher. Domperidone 1–100 nmol/l and sulpiride 10–1000 nmol/1 increased up to 2.4-fold the overflow evoked by 4 pulses/ 1 Hz but increased only slightly the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with ³ H-choline were superfused either with physostigmine-free medium or with medium containing physostigmine 1 mol/l. In physostigmine-free medium, atropine did not increase the evoked overflow of tritium at any stimulation condition. In physostigmine-containing medium, the overflow elicited by single pulses was 0.18% of the tritium content of the tissue. The overflow elicited by 8 pulses/1 Hz was 2.0-fold and the overflow elicited by 8 pulses/100 Hz 2.2-fold higher. Atropine 2–200 nmol/1 increased up to 2.4-fold the overflow evoked by 8 pulses/1 Hz but increased only slightly the overflow evoked bysingle pulses or 8 pulses/100 Hz. In physostigmine-free medium, sulpiride 10–1000 nmol/1 did not change the single-pulse-evoked overflow of tritium in the absence but increased it in the presence of nomifensine 1 mol/l.Single pulses elicit a large release of ³H-noradrenaline, ³H-dopamine and ³H-acetylcholine under the conditions of these experiments. Release elicited by single pulses is not subject to autoinhibition except for a small inhibition by spontaneously released transmitter in the case of dopaminergic and cholinergic axons. When 3 or 7 further pulses follow the first one at intervals of 1 s, they elicit much smaller release. At least a great part of the fall is due to autoreceptor mediated inhibition (for 3H-acetylcholine release in the presence of physostigmine only). When 3 or 7 further pulses follow at intervals of 10 ms, they elicit release that is either similar to that evoked by the first pulse (³H-noradrenaline) or much smaller (³H-dopamine, ³H-acetylcholine). However, the fall is not due to stimulation-dependent, auto-receptor-mediated inhibition; autoinhibition does not develop in these short high-frequency trains. Overall, the results are in accord with the autoreceptor theory. They demonstrate the role of autoinhibition in determining the transmitter release patterns of central noradrenergic, dopaminergic and cholinergic neurones. Send offprint requests to N. Limberger at the above address     

Possible involvement of presynaptic α1-adrenoceptors in the effects of idazoxan and prazosin on 3H-noradrenaline release from tail arteries of SHR

Summary The effects of several -adrenoceptor antagonists have been examined on tritium release elicited by electrical stimulation from isolated perfused SHR tail artery preparations prelabelled with ³H-noradrenaline (³H-NA). Phentolamine and yohimbine potently facilitated the stimulation evoked release of tritium at low frequencies of stimulation, but the ₂-adrenoceptor antagonist idazoxan was only weakly active at 1 mol/l, despite antagonising the clonidine-evoked inhibition of ³H-release at a lower concentration of 0.1 mol/l. The ₁-adrenoceptor antagonists prazosin and corynanthine also increased stimulation evoked tritium release in this preparation, suggesting the presence of prejunctional ₁-adrenoceptors. Furthermore, the ₁-adrenoceptor agonist methoxamine (3 mol/l) caused a significant inhibition of tritium-evoked release, an effect which was blocked by prazosin (10 nmol/l).When ₁-adrenoceptors were blocked in the presence of prazosin, idazoxan (0.1 mol/l) produced a significant facilitatory effect on the electrically-evoked release of ³H-transmitter. On the other hand, when ₂-adrenoceptors were blocked in the presence of yohimbine, exposure to idazoxan (0.1 mol/l) reduced significantly the stimulation-evoked release of tritium elicited by electrical stimulation.The results indicate that in the SHR tail arteries, idazoxan has a partial agonist inhibitory activity on transmitter release, which can mask the facilitatory effects due to blockade of presynaptic ₂-adrenoceptors. The inhibitory effects of idazoxan appear to involve presynaptic ₂-adrenoceptors, which when stimulated, reduce ³H-NA release in SHR tail arteries.     

Frequency dependence of muscarinic facilitation of transmitter release in urinary bladder strips from neurally intact or chronic spinal cord transected rats

1. Electrical stimulation evoked release of ³H-noradrenaline (NA) and ¹⁴C-acetylcholine (ACh), as well as neurally evoked contractions were measured at various (1–40 Hz, 100 shocks) stimulation frequencies in bladder strips from neurally intact (NI) and spinal cord transected (SCT) rats.
2. The frequency response curves for ACh and NA release were shifted to the left in SCT bladder strips as compared to NI bladder strips.
3. Atropine (1 μM) depressed ACh release in NI bladder strips at high frequency stimulation (10 and 40 Hz) but not at low frequency stimulation (2–5 Hz). However, in SCT bladders, atropine depressed ACh release both at low and high frequencies of stimulation, indicating that muscarinic facilitation occurs at lower frequencies.
4. Atropine depressed the release of NA in NI bladders at only 40 Hz stimulation, but depressed release at all frequencies in SCT bladders.
5. The amplitude of neurally evoked contractions of bladder strips from NI rats was enhanced as the frequency of stimulation was increased from 1 to 40 Hz (80 shocks). The frequency response curve was shifted to the left in SCT bladders. Atropine blocked the neurally evoked contractions in SCT bladder strips to a greater extent than the contractions in NI strips indicating a cholinergic dominance in the SCT bladders.
6. Maximal contractile force of SCT bladder strips evoked by neural stimulation at 20 Hz 10 shocks and 80 shocks was significantly lower than that of NI bladder strips, whereas the release of ACh was significantly higher in SCT than NI bladders indicating a postjunctional defect in the SCT preparations.
7. It is suggested that presynaptic muscarinic facilitatory mechanisms are upregulated in the cholinergic and adrenergic nerve terminals in SCT bladders leading to a larger relative contractile response at lower frequencies of stimulation (2–5 Hz). Thus the hyperreflexic bladder occurring after spinal cord injury may be due in part to an enhancement of transmitter release at bladder postganglionic nerve terminals.

     

Effect of ω-conotoxin GVIA on release of 3H-γ-aminobutyric acid from slices of rat neostriatum

Summary -Conotoxin GVIA (-CT) diminished the potassium-induced in vitro release of ³H--aminobutyric acid (³H-GABA) from slices of rat neostriatum in a manner which depended on the concentration of potassium. -CT (0.1 nmol/l) decreased the release of ³H-GABA induced by 25 mmol/l K⁺ from 11.6% to 6.1% of tissue content, ie. by 48%, while it did not affect the release of ³H-GABA caused by 20 mmol/l K⁺, which was 4.8% of tissue content. However, in the presence of a polyclonal antiserum or cysteamine (600 mol/l), both of which diminish the effects of endogenous somatostatin, 0.1–10 nmol/l -CT decreased the release of ³H-GABA induced by 20 mmoles/l K⁺ by 40%. It is concluded that -CT did not only inhibit GABA-neurones, but had an additional inhibitory effect on somatostatin neurones which are known to depress the release of ³H-GABA. It is further concluded that neuronal interactions, which are possible in brain slice preparations, may impede the interpretation of effects of drugs, especially if agents are used which affect basic mechanisms of transmitter release and thus the release of various transmitters from neurones. Send offprint requests to D. K. Meyer at the above address     

Presynaptic action of adrenaline on adrenoceptors modulating stimulation-evoked 3H-noradrenaline release from rabbit isolated aorta

Summary The purpose of this investigation was to study the effect of adrenaline on presynaptic adrenoceptors by recording the release of ³H-noradrenaline evoked by electrical-field stimulation. Adrenaline (10^–10–³ × 10^–9 mol/l) had no effect on the ³H-overflow evoked by stimulation of aorta preloaded with ³H-noradrenaline. At 10^–8 and 3 × 10^–8 mol/l, the ³H-overflow was decreased by up to 47%. The maximum decrease was more marked in the presence of either cocaine (3 × 10^–5 mol/l) plus corticosterone (4 × 10^–5 mol/l), cocaine (3.3 × 10^–6 mol/l) plus normetanephrine (4 × 10^–5 mol/l), or desipramine (10^–6 mol/l) plus normetanephrine (10^–5 mol/l). The relationship between adrenaline-induced decrease and stimulation-frequency was dependent on the experimental design: either the decrease was the same at all frequencies (1–16 Hz) or it was more marked, the lower the frequency (1 > 3 > 8 Hz). Phentolamine and rauwolscine (both 10^–6 mol/l) antagonized the inhibitory effect of adrenaline (10 ^{– 8}–10^–6 mol/l). Phenoxybenzamine (10^–6 mol/l), prevented the inhibitory effect. No enhancing effect of adrenaline (10^–9–10^–6 mol/l) was observed in the presence of these three -adrenoceptor antagonists. Our results suggest that adrenaline activates inhibitory ₂-adrenoceptors, but not facilitatory -adrenoceptors on postganglionic sympathetic nerve terminals in rabbit aorta. Send offprint requests to J. Abrahamsen at the above address