Comparative studies of the postjunctional activities of some very potent muscarinic agonists

Summary This study was undertaken to determine the potenties of seven muscarinic agonists (methylfurtrethonium, dioxolane, oxathiolane, carbachol, muscarine, muscarone and oxotremorine) on the postjunctional muscarinic receptors of seven isolated preparations (guinea pig taenia-coli, ileum, jejunum, trachea and atria and rat jejunum and urinary bladder).The results indicate that the rank order of sensitivity of the preparations varies independently of the potency of the agonist used and it is almost the same for all the compounds with the exception of oxotremorine.Muscarone was the most potent compound in all the tissues. Intergroup comparisons in each preparation and the evaluation of the equieffective molar ratios relative to muscarone revealed that carbachol possesses a certain degree of cardioselectivity and oxathiolane, on the other hand, is much less active on the cardiac tissue than on the others.Oxotremorine is a peculiar compound endowed with cardioselectivity.     

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     

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     

Different muscarine receptors mediate the prejunctional inhibition of [3H]-noradrenaline release in rat or guinea-pig iris and the contraction of the rabbit iris sphincter muscle

Summary To investigate the muscarine receptor type mediating inhibition of [³H]-noradrenaline release from the isolated rat and guinea-pig iris we have determined the potency of antimuscarinic drugs to antagonize the methacholine-induced inhibition of [³H]-noradrenaline overflow evoked by field stimulation (3 Hz, 2 min). The prejunctional apparent affinities were compared with those obtained for postjunctional muscarine receptors mediating the methacholine-induced contraction of the isolated rabbit iris sphincter muscle.Prejunctional apparent affinity constants of pirenzepine (6.67), himbacine (8.51), methoctramine (7.92), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 8.00), hexahydro-difenidol enantiomers (6.92, (R); 5.77, (S)) in the rat iris and methoctramine (7.58) in the guinea-pig iris indicate the presence of M₂ receptors. Although the post-junctional affinity constants in the rabbit iris sphincter of methoctramine (5.93), gallamine (3.92), and 4-DAMP (9.07) confirm our previous suggestions of the presence of M₃-like receptors, the results obtained with the hexahydro-difenidol enantiomers do not agree with that concept. The post-junctional affinity constants of the hexahydro-difenidol enantiomers were not different from the prejunctional values (6.86, (R); 5.55, (S)), indicating a similar and low degree of stereoselectivity for these stereoisomers at both receptor sites (14 and 17, (R)/(S)-ratios, respectively). Hence, the postjunctional muscarine receptor in the rabbit iris sphincter fails to exhibit the high degree of stereo selectivity observed for hexahydro-difenidol enantiomers at M₃ receptors on other smooth muscles.This study was supported by the Deusche Forschungsgemeinschaft (Fu 163/2) Send offprint requests to H. Fuder at the above address     

Determination of dissociation constants and relative efficacies of some potent muscarinic agonists at postjunctional muscarinic receptors

Summary This study was undertaken to determine dissociation constants (K _A) and relative efficacies (e _r) of seven muscarinic agonists (methylfurtrethonium; dioxolane, oxathiolane, carbachol, muscarine, muscarone and oxotremorine) in three isolated tissues (guinea-pig ileum and atria and rat urinary bladder).The rank order of affinities (-log K _A) of the various compounds varied depending on the tissue used. e _r values for the different agonists did not differ significantly from each other in any of the three tissues, except that the e _r of muscarine in the guinea-pig ileum was higher than those of the other compounds and that of oxotremorine in the rat urinary bladder was lower than those of the other agonists.Comparisons among tissues show that K _A and e _r values were the same in different tissues for some compounds (muscarone, muscarine and methylfurtrethonium), while significant differences were found for the other compounds. This suggests the existence of a discrete receptor population recognized by some but not all agonists.For oxotremorine er as well as -log K _A, is greater in atria than in smooth muscle: these factors combine to determine the cardioselectivity of this compound which can now ascribed to receptor selectivity. Send offprint requests to E. Grana at the above address     

Comparison of affinities of muscarinic antagonists to pre- and postjunctional receptors in the guinea-pig ileum

The myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum was preincubated with [3H]choline, then superfused and stimulated electrically (1 Hz 120 pulses). Oxotremorine reduced the evoked outflow of [3H]acetylcholine in a concentration-dependent manner. Each of the six antagonists (scopolamine, methylatropine, trihexyphenidyl, 4-DAMP, clozapine, pirenzipine) produced parallel shifts of the concentration-response curves for the prejunctional effects of oxotremorine. Similarly, in contraction experiments, the antagonists competitively antagonized the postjunctional responses to oxotremorine. The pre- and postjunctional pA2 values did not differ significantly for any of the antagonists. It is concluded that pre- and postjunctional muscarinic receptors in the guinea-pig ileum are pharmacologically similar.     

Evidence that catecholamines increase acetylcholine release from neuromuscular junction through stimulation of alpha-1 adrenoceptors

Summary 1. The effects of alpha-1 adrenoceptor agonists on ³H-acetylcholine release from mouse phrenic nerve-hemidiaphragm preparation were studied. 2. In preparation which had been incubated with ³H-choline, electrical stimulation (50 Hz, 0.8 s trains every 10 s) released ³H-acetylcholine. Neurochemical evidence was obtained that noradrenaline, adrenaline and phenylephrine facilitated the stimulation evoked release of ³H-acetylcholine. This effect was much more marked when the prejunctional nicotinic receptors mediating positive feedback modulation were blocked by (+)-tubocurarine. In the presence of this drug, when the release of ³H-acetylcholine was reduced, alpha-1 adrenoceptor agonists enhanced the release in a prazosin-sensitive manner. 3. The rank order of potency of alpha-1 adrenoceptor agonists was adrenaline > noradrenaline > phenylephrine. By contrast, methoxamine had no effect on the release of ³H-acetylcholine. It is suggested, therefore, that circulating catecholamines may be able to affect neuromuscular transmission through stimulation of presynaptic alpha-1 adrenoceptors. Send offprint requests to E. S. Vizi at the above address     

Protein kinase C and presynaptic modulation of acetylcholine release in rabbit hippocampus.

1. The involvement of protein kinase C in the presynaptic modulation of stimulated acetylcholine release was investigated in rabbit hippocampus. 2. Slices of the rabbit hippocampus, labelled with [3H]-acetylcholine, were superfused with medium and stimulated electrically during superfusion. 3. The protein kinase C activating phorbol ester 4 beta-phorbol 12,13-dibutyrate (4 beta-PDB) enhanced the electrically evoked tritium overflow in a concentration-dependent manner. Its biologically inactive 4 alpha-isomer was without any effect on transmitter release. 4. The protein kinase C inhibitor polymyxin B decreased the stimulation-evoked tritium overflow and counteracted the enhancement of release caused by 4 beta-PDB. 5. The stimulation-evoked tritium overflow was facilitated when the muscarine receptor antagonist atropine was present. The effects of both atropine and 4 beta-PDB, given in combination, were additive. 6. The net inhibition of the evoked tritium overflow caused by the muscarine receptor agonists carbachol and oxotremorine was similar, irrespective of whether 4 beta-PDB was present or not. 7. Similar results to those for muscarine autoreceptor-mediated inhibition, were obtained for inhibition of the stimulated tritium overflow caused by the adenosine receptor agonist (-)-N6-(R-phenylisopropyl)-adenosine ((-)-PIA) and the opioid receptor agonist ethylketocyclazocine (EKC). The net inhibition of both agonists was independent of the presence of the phorbol ester. 8. The above results provide further evidence for participation of a presynaptically located protein kinase C in the modulation of acetylcholine release. However, the modulatory mechanisms which are coupled to presynaptic receptors and mediate inhibition of release seem not to be directly affected by protein kinase C.     

Pharmacological characterization of muscarine receptors of PC 12 (rat phaeochromocytoma) cells

Summary The muscarinereceptors of PC12 (rat phaeochromocytoma) cells were studied in functional and binding experiments. The catecholamine stores of PC 12 cells were labelled by incubation of the cells with tritiated noradrenaline. Muscarinic agonists elicited concentration-dependent release of tritium which consisted overwhelmingly of unchanged ³H-noradrenaline. The rank order of potency was: oxotremorine > acetylcholine > muscarine = methacholine > carbachol > bethanechol. The release evoked by carbachol (0.1 mmol/l) was inhibited with high potency by the M₁-selective antagonist telenzepine (pK _i = 8.82), with intermediate potency by pirenzepine (pK _i = 7.00) and with low potency by the M₂-selective antagonist AF-DX 116 (pK _i = 5.74).The binding of 3H-N-methylscopolamine to PC 12 membranes was inhibited by various non-selective and subtype-selective muscarinic antagonists with the following rank order of potency: telenzepine = atropine > 4-DAMP > dicyclomine > pirenzepine > HHSiD > AF-DX 116. A similar rank order was obtained for the inhibition by these compounds of 3H-telenzepine binding to Mi-receptors in membranes of the cerebral cortex of the guinea pig. The Hill coefficients for inhibition of 3H-N-methylscopolamine binding (to PC 12 membranes) by pirenzepine, telenzepine and AF-DX 116 were below unity. Specific binding of both ³H-telenzepine and 3H-N-methylscopolamine to muscarine receptors of PC 12 membranes was saturable and of high affinity; the maximal number of binding sites was higher for ³H-N-methylscopolamine than for 3H-telenzepine (calculated for the active (+)enantiomer).PC 12 cells are presumably endowed with more than one subtype of muscarine receptors. The predominant receptor is an atypical receptor; it is neither a M₂- nor a M₃-receptor, and in spite of the high affinity of telenzepine for this receptor it is probably also not an M₁-receptor.     

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     

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     

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.     

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     

A pharmacological differentiation between postjunctional (AT<Subscript>1A</Subscript>) and prejunctional (AT<Subscript>1B</Subscript>) angiotensin II receptors in the rabbit aorta

The effects of angiotensin II and angiotensin III were compared at prejunctional and postjunctional AT₁ receptors of the rabbit thoracic aorta. Furthermore, the influence of PD123319, losartan and eprosartan on these effects was also compared. To study prejunctional effects, the tissues were preincubated with (³H)-noradrenaline, superfused and electrically stimulated (1 Hz, 2 ms, 50 mA, 5 min). To study postjunctional effects, non-cumulative concentration–response curves were determined. Both angiotensin II and angiotensin III were more potent prejunctionally than postjunctionally. In the case of angiotensin II, the EC₅₀ was 12 times lower at the prejunctional than at the postjunctional level, while that of angiotensin III was 30 times lower prejunctionally. Furthermore, whereas angiotensin II was about 33 times more potent than angiotensin III postjunctionally, it was only 12 times more potent than angiotensin III prejunctionally. Eprosartan did not differentiate between prejunctional and postjunctional effects of both angiotensins. In contrast, PD123319 and losartan did differentiate; however, whereas PD123319 concentration-dependently antagonised the facilitation of tritium release caused by angiotensin II and angiotensin III and had no influence on the contraction of the aortic rings elicited by the peptides, losartan did the opposite: it concentration-dependently antagonised the contractions caused by the peptides on the aortic rings and exerted no influence on the facilitatory effect of angiotensin II and angiotensin III. These results show that prejunctional and postjunctional receptors for angiotensin II and angiotensin III are different and underline the hypothesis that postjunctional AT₁ receptors belong to the AT_1A subtype, while prejunctional AT₁ receptors belong to the AT_1B subtype.     

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     

GABAergic modulation of D-1 dopamine receptor-mediated 3H-acetylcholine release from rabbit retina

Summary Dopamine evokes calcium-dependent release of ³H-acetylcholine from superfused rabbit retina labeled in vitro with ³H-choline, through activation of a D-1 dopamine receptor. This study investigates the activation of this receptor by endogenous dopamine and the modulation of the spontaneous and dopamine-evoked release of ³H-acetylcholine from rabbit retina labeled with ³H-choline by GABAergic agonists and antagonists. Endogenous dopamine, released from dopaminergic amacrine neurons by the indirect amines tyramine or D-amphetamine evoked the calcium-dependent release of ³H-acetylcholine from rabbit retina. The release of ³H-acetylcholine elicited by tyramine (10 M) or D-amphetamine (10 M) was attenuated by the selective D-1 antagonist SCH 23390 (0.1 M) and by the dopamine uptake inhibitor nomifensine (3 M). At concentrations of 1 mM and 1 M respectively, GABA and muscimol inhibited the spontaneous release of tritium from rabbit retina labeled in vitro with ³H-choline. Picrotoxin and bicuculline (10 M) increased the spontaneous release of tritium. GABA and the GABA agonist muscimol (0.01–100 M) inhibited in a concentration-dependent manner the release of ³H-acetylcholine elicited by 100 M dopamine with IC₅₀ values of 4.5 M and 0.02 M respectively. The inhibition of dopamine-evoked ³H-acetylcholine release by GABA (10 M) and muscimol (0.1 M) was antagonized by the GABA antagonists bicuculline and picrotoxin. Picrotoxin and bicuculline (10 M) increased the spontaneous release of tritium, and potentiated the release of ³H-acetylcholine evoked by 100 M dopamine consistant with a tonic, inhibitory GABAergic input to the cholinergic amacrine neurons in rabbit retina. Dopamine-evoked acetylcholine release in rabbit retina may be of physiological importance as D-1 dopamine receptor-mediated increases in ³H-acetylcholine release from rabbit retina can be elicited by endogenous dopamine. In addition, activation of GABA receptor sites modulates the spontaneous and dopamine-evoked acetylcholine release from rabbit retina. Send offprint requests to M. L. Dubocovich 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     

Long-term administration of 1,3-dipropyl-8-sulphophenylxanthine (DPSPX) alters α2-adrenoceptor-mediated effects at the pre- but not at the postjunctional level

The present investigation was undertaken to see whether a long-term inhibition of adenosine receptors —leading to hypertension — interferes with ₂-adrenoceptor-mediated modulation of noradrenaline release. Rat tail arteries were removed from normal and from hypertensive animals obtained by chronic treatment with intraperitoneally infused DPSPX (1,3,-dipropyl-8-sulphophenylxanthine) or orally administered L-NAME (N^G-Nitro-L-arginine methyl ester). To study prejunctional effects, the influence of UK-14,304 (5-bromo-6(imidazoline-2-ylamino)-quinoxaline) and yohimbine on the overflow of tritium evoked by electrical stimulation (100 V; 1 Hz; 2 ms; 5 min) from tissues preloaded with ³H-noradrenaline was analysed. To study postjunctional effects, concentration-response curves to UK-14,304 were determined. In DPSPX-treated rats there was an enhancement of the prejunctional effects of UK-14,304: its EC_30% was reduced from 381 (250; 579) to 85 (73; 99) nmol.l^–1 (n = 5; P<0.05) and its maximal effect — expressed as percent reduction of tritium overflow-increased from 45 ± 5% to 61 ± 5% (n = 6; P < 0.05). In L-NAME-treated rats there was no change in either of these two parameters. At the postjunctional level, there was no change in the sensitivity to UK-14,304 in tissues from either DPSPX- or L-NAME-treated rats. Yohimbine (10–1000 nmol.l^–1) caused a concentration-dependent increase of tritium overflow evoked by electrical stimulation in both control and hypertensive animals (either DPSPX- or L-NAME-treated). The EC_50%-pre-antagonist values (concentration of the antagonist that increases the evoked overflow by 50%) were not significantly different in the three situations. We conclude that long-term administration of DPSPX increases the sensitivity to the prejunctional effects of UK-14,304 without changing that to its postjunctional effects, showing a specific interaction between -adrenoceptors and adenosine receptors at a prejunctional level. The question arises whether there is any link between that alteration and the development of the hypertensive state. Correspondence to: S. Guimaraes at the above address     

Inhibitory muscarinic receptors modulate the potassium-evoked release of [3H]serotonin from rat hypothalamic slices

The effects of oxotremorine, acetylcholine and nicotine have been investigated on the potassium-evoked release of [³H]serotonin from slices of rat hypothalamus. Oxotremorine and acetylcholine in the presence of physostigmine inhibited potassium-evoked tritium release without affecting the spontaneous release. Nicotine had no effect. The response to oxotremorine was unaffected by tetrodotoxin but was blocked by atropine and hyoscine suggesting that the muscarinic receptor mediating the response to oxotremorine was located on the serotonergic nerve terminal.     

Release of [3H]acetylcholine from a modified rat phrenic nerve-hemidiaphragm preparation

Summary Two different preparations of the rat phrenic nerve-hemidiaphragm (whole nerve-muscle preparation, end-plate preparation) were used for studying synthesis and release of radioactive acetylcholine in the absence and presence of cholinesterase inhibitors.When the whole nerve-muscle preparation (110–180 mg) was incubated with [³H]choline, only small amounts of radioactive acetylcholine were synthesized within the tissue. Electrical nerve stimulation of the whole nerve-muscle preparation produced no increase in tritium outflow.Incubation of the end-plate preparation (16–29 mg) which was obtained after removal of most of the muscle mass led to the formation of large amounts of [³H]acetylcholine. Synthesis depended on nerve activity and increased 13-fold during a high loading stimulation (50 Hz), as compared to the synthesis at rest. In a denervated end-plate preparation the formation of [³H]acetylcholine was reduced to 4% of the control preparation. Electrical nerve stimulation of the end-plate preparation produced a release of tritium that could be attributed entirely to the release of [³H]acetylcholine. The stimulated tritium efflux was completely suppressed in a calcium-free medium or in the presence of tetrodotoxin (300 nM). Release could even be detected during a short train of 50 pulses (5 Hz) with a fractional release of about 0.04% of the [³H]acetylcholine tissue content per pulse.It is concluded that the large muscle mass interferes with nerve labelling by a reduction of the [³H]choline supply to the nerve terminals when the whole nerve-muscle preparation is used. Removal of most of the muscle fibres reduces the possibility for [³H]choline to be captured by them and then more radioactive choline can enter the end-plate region. From this end-plate preparation a calcium-dependent release of radioactive transmitter can be measured in the absence of cholinesterase inhibitors.