Affinity and efficacy of racemic, (+)-, and (−)-methacholine in muscarinic inhibition of [3H]-noradrenaline release

The right postganglionic sympathetic nerves of rat isolated perfused hearts (previously loaded with [3H]-noradrenaline) were stimulated electrically with 10 trains of 10 pulses at 10 Hz. The inhibition by methacholine of stimulation-evoked [3H]-noradrenaline overflow into the perfusate (determined in the presence of corticosterone, desipramine, phentolamine, and propranolol) was taken as a measure for activation of presynaptic muscarinic receptors. The evoked [3H]-noradrenaline overflow was inhibited by (+)-, racemic, and (-)-methacholine in a reversible and concentration-dependent manner. The concentration causing 50% inhibition (IC50) was 0.1, 0.26, and 65 microM, respectively, resulting …

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

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-d…

Inhibition by interleukin-1 beta of noradrenaline release in rat spleen: involvement of lymphocytes, NO and opioid receptors.

Effects of indomethacin, N omega-nitro-L-arginine (NNA) and naloxone, and of pretreatment with cyclophosphamide (CY), on the interleukin (IL)-1 beta induced inhibition of exocytotic noradrenaline release were investigated in the isolated, vascularly perfused spleen of the rat. Neurotransmitter release was evoked by perivascular electrical stimulation (4 Hz) and the overflow of endogenous noradrenaline was determined by HPLC with electrochemical detection. Perfusion of the spleen with Tyrode's solution containing IL-1 beta (100 pg/ml) for 90 min caused an inhibition of the stimulation-evoked noradrenaline overflow which persisted for at least 20 min after washout of the IL. The evoked overfl…

Characterization of the prejunctional muscarinic receptors mediating inhibition of evoked release of endogenous noradrenaline in rabbit isolated vas deferens

The aim of the present study was to characterize the prejunctional modulation of evoked release of endogenous noradrenaline in rabbit vas deferens by the use of muscarinic receptor agonists and subtype-preferring antagonists. Vasa deferentia of the rabbit were stimulated electrically by trains of 120 pulses delivered at 4 Hz or trains of 30 pulses at 1 Hz. The inhibition by muscarinic agonists of the stimulation-evoked overflow of endogenous noradrenaline in the absence and presence of antagonists was used to determine affinity constants for antagonists. These values were compared with those observed at putative M1 receptors inhibiting neurogenic twitch contractions in the rabbit vas defere…

A muscarinic receptor different from the M1, M2, M3 and M4 subtypes mediates the contraction of the rabbit iris sphincter

In order to analyse the subtype of muscarinic receptors involved in the methacholine-induced contraction of the rabbit iris sphincter we have determined equilibrium dissociation constants (KB) of various antagonists in the sphincter muscle. The values were compared with those observed at M1 (rabbit vas deferens), M2 (heteroreceptors in rat iris) and M3 receptors (guinea-pig ileum), or at the muscarinic receptors in the guinea-pig uterus. The methacholine-induced contraction of the uterus from immature guinea-pigs was competitively antagonized by pirenzepine (6.64, -log KB), 4-DAMP (8.39), hexahydrodifenidol (HHD; 7.00 for the (R)- and 5.40 for the (S)-enantiomer), p-fluoro-hexahydrosiladife…

Cholinesterase activity and exposure time to acetylcholine as factors influencing the muscarinic inhibition of [3H]-noradrenaline overflow from guinea- pig isolated atria

Guinea-pig isolated atria were incubated and loaded with [3H]-noradrenaline. The release of 3H and of [3H]-noradrenaline was induced by field stimulation (6-9 trains of 150 pulses at 5 Hz). The stimulation-evoked overflows of 3H and of [3H]-noradrenaline were determined. In the absence of an inhibitor of acetylcholinesterase, acetylcholine (12 min preincubation before nerve stimulation, up to 10 microM) failed to inhibit the evoked [3H]-noradrenaline overflow. In the presence of atropine, an increase by acetylcholine of evoked release was observed in the same atria. In contrast, the selective muscarinic agonist methacholine significantly decreased the evoked overflow. The inhibition was ant…

Muscarinic inhibition of [3H]-noradrenaline release on rabbit iris in vitro: effects of stimulation conditions on intrinsic activity of methacholine and pilocarpine

1. Rabbit isolated irides were loaded with [3H]-noradrenaline and superfused with Tyrode solution. The inhibition by the muscarinic agonists (+/-)-methacholine and pilocarpine of the [3H]-noradrenaline overflow into the superfusate evoked by field stimulation (pulses of 1 ms duration, 75 mA) was measured as an index of activation of presynaptic muscarinic receptors. 2. The fractional rate of release per pulse during the first stimulation period (S1) was low with 360 pulses at 3 Hz, intermediate with 360 pulses at 10 Hz and high with 1200 pulses at 10 Hz. Upon repetitive stimulation (7 periods at 20 min intervals), the fractional rates of release per pulse during S7 no longer differed, sugge…

The effect of dopamine on the overflow of endogenous noradrenaline from the perfused rabbit heart evoked by sympathetic nerve stimulation.

1. The effects of dopamine and two dopamine receptor antagonists (pimozide, flupenthixol) on the release of endogenous noradrenaline evoked by electrical stimulation of the postganglionic sympathetic nerves and their influence on cardiac performance were measured in isolated perfused rabbit hearts. 2. Dopamine 0.2μM decreased noradrenaline overflow and ventricular tension development in response to nerve stimulation. 3. Dopamine 2μM increased spontaneous noradrenaline output and tension development. The noradrenaline overflow in response to nerve stimulation was greatly enhanced. This action was only partly reversed by wash out of exogenous dopamine, indicating de novo synthesis and facilit…

Characterization of sensory neurotransmission and its inhibition via alpha 2B-adrenoceptors and via non-alpha 2-receptors in rabbit iris.

To find out whether, and which type of, adrenoceptors mediate prejunctional inhibition of sensory neurotransmitter release from trigeminal fibres, the modulation of twitch response to electrical field stimulation on rabbit isolated iris was investigated. Evoked iris sphincter contractions consisted of a minor fast cholinergic and a large slow component. The latter was unaffected by atropine and guanethidine, hence nonadrenergic noncholinergic in nature (NANC), but nearly completely abolished by capsaicin pretreatment and by the neurokinin receptor antagonist spantide. The response was probably not mediated by NK2 receptors as SR 48,968, an NK2 selective nonpeptide antagonist, failed to redu…

On the opioid receptor subtype inhibiting the evoked release of 3H-noradrenaline from guinea-pig atria in vitro

1. Guinea-pig isolated atria were incubated and loaded with 3H-(−)-noradrenaline. The intrinsic nerves were stimulated with trains of 5 or 35 field pulses (4 Hz), and the evoked efflux of 3H-noradrenaline and of total tritium was determined in the presence of atropine, corticosterone, desipramine, and phentolamine by liquid scintillation spectrometry. 2. Ethylketocyclazocine (1.4 nmol/l, IC50), MR 2033 (9.1 nmol/l), dynorphin A (1–13) (25 nmol/l, peptidase inhibitors present), etorphine (71 nmol/l), and [d-Ala2, d-Leu5]-enkephalin (>10 μmol/l, peptidase inhibitors present) inhibited the stimulation-evoked efflux of 3H-noradrenaline in a concentration-dependent manner, but not morphine up to…

Purinoceptor-mediated modulation by endogenous and exogenous agonists of stimulation-evoked [3H]noradrenaline release on rat iris.

To investigate whether endogenous purinoceptor agonists affect the sympathetic neurotransmission in the rat isolated iris, and to classify the purinoceptors modulating exocytotic [3H]-noradrenaline release, we have determined the effect of adenosine receptor antagonists on, and the relative potency of selected agonists in modulating, the field stimulation-evoked (3 Hz, 2 min) [3H]-noradrenaline overflow. In addition, the apparent affinity constants of 8-phenyltheophylline (8-PT) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in antagonizing the prejunctional effects of purinoceptor agonists were estimated. The relatively A1-selective DPCPX 10 and 100 nmol/l increased the evoked [3H]-noradre…

The effects of methacholine and calcium deprivation on the release of the false transmitter, ?-methyladrenaline, from the isolated rabbit heart

1. Anaesthetized rabbits were infused for 20 min with 85 μg · kg−1 · min−1 (±)-α-methyladrenaline. The hearts dissected 15 min after the infusion contained 1.49 μg/g α-methyladrenaline; the endogenous noradrenaline content was correspondingly decreased. 2. Hearts from α-methyladrenaline-infused animals were isolated with the right sympathetic nerves intact and perfused. Ventricular rate, right atrial and right ventricular tensions were recorded using the transverse method. 3. Electrical stimulation (10 Hz, 1 ms, 1 min) of sympathetic nerves, perfusion with the nicotinic drug, p-aminophenethyltrimethylammonium (PAPETA) or perfusion with 54 mM KCl (high K+) solution evoked an output of both α…

ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors.

Effects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [3H]-noradrenaline overflow were investigated in the rat isolated iris. ATP and adenosine inhibited the evoked overflow of [3H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (-log KB = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 mumol/l) and DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 50 mumol/l) but was antagonized by the P2Y-rece…

Desensitization of inhibitory prejunctional alpha 2-adrenoceptors and putative imidazoline receptors on rabbit heart sympathetic nerves.

To find out whether sympathetic nerves of the rabbit heart possess pharmacologically relevant prejunctional imidazoline receptors different from α-autoreceptors, the inhibition by oxymetazoline, aganodine and BDF 6143 (4-chloro-2-[2-imidazoline-2-ylamino]-isoindoline hydrochloride) of endogenous noradrenaline overflow evoked by stimulation of extrinsic postganglionic sympathetic nerves (0.66 Hz, 80 pulses) was investigated. In addition we wanted to find out whether either type of these prejunctional receptors undergoes desensitization upon pre-exposure to respective agonists. The α2-adrenoceptor agonist oxymetazoline inhibited the evoked noradrenaline overflow (2.9 nmol/l, IC50; about 90010…

The effects of several muscarinic antagonists on pre- and postsynaptic receptors in the isolated rabbit heart

In order to reveal possible differences between pre- and postsynaptic muscarine receptors, seven antagonists were tested for their affinities on these receptor sites in the rabbit isolated perfused heart. Methacholine was used as an agonist to inhibit the noradrenaline overflow evoked by electrical stimulation (3 Hz, 3 min) of the sympathetic nerves (presynaptic parameter) and to decrease the systolic tension development of the right atrium (postsynaptic parameter). The affinity of an antagonist was expressed as pA2. A decreasing order of potency was obtained with ipratropium, scopolamine, atropine, trihexyphenidyl, amitriptyline, and gallamine, both for pre- and postsynaptic responses. The…

Nicotine receptors do not modulate the 3H-noradrenaline release from the isolated rat heart evoked by sympathetic nerve stimulation.

Isolated rat hearts with the right sympathetic nerves attached were perfused at a constant flow rate of 7 ml/min with Tyrode's solution. (-)-3H-Noradrenaline (final concentration 10–13.9 nM) was infused for 10 min to label the noradrenaline stores. After wash-out the sympathetic nerves were stimulated electrically (3 Hz, 180 impulses, 1 ms, 20–30 mA) three times (S1–S3) at intervals of 15 min. 3H-Noradrenaline and its metabolites were determined by liquid scintillation counting according to Graefe et al. (1973). Both, nicotine 50 μM and p-aminophenethyltrimethylammonium (PAPETA) 30 μM, enhanced the 3H-noradrenaline overflow in the absence of nerve stimulation. The effect of PAPETA was bipha…

An ?-Adrenoceptor Facilitating the Stimulation-Evoked Acetylcholine Release in the Rat Perfused Heart

AF-DX 116 differentiates between prejunctional muscarine receptors located on noradrenergic and cholinergic nerves.

Prejunctional affinity constants of the cardioselective muscarine receptor antagonist AF-DX 116 (11-[(2-[(diethyl-amino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6 H-pyrido [2,3-b] [1,4] benzodiazepine-6-one) were determined for muscarine autoreceptors on cholinergic nerves of the guinea-pig ileum and for heteroreceptors on noradrenergic nerves of the rat heart and guinea-pig iris. AF-DX 116 antagonized with low affinity the muscarinic inhibition induced by arecaidine propargyl ester of the stimulation-evoked [3H]acetylcholine overflow (pA2 6.74) from the guinea-pig ileum. In contrast, AF-DX 116 was more potent in antagonizing the methacholine-induced inhibition of the stimulation-evoked […

Glycopyrronium bromide blocks differentially responses mediated by muscarinic receptor subtypes.

To analyse the potency of glycopyrronium bromide in blocking responses mediated via subtypes of muscarinic receptors in vitro, we tried to determine its equilibrium dissociation constants at prejunctional muscarinic receptors inhibiting the twitch response of rabbit vas deferens (presumed M1 type), at M2 (paced at left atria), M3 (guinea pig ileum) muscarinic receptor subtypes and at the muscarinic receptor of the rabbit iris sphincter (not M1-M4, not m5). Glycopyrronium bromide shifted to the right the curve for inhibition of the twitch response induced by the agonist McN-A-343, and the methacholine-induced curves for inhibition of rat atrial contraction, and for tonic contraction of guine…

Muscarine receptor types mediating autoinhibition of acetylcholine release and sphincter contraction in the guinea-pig iris.

The potencies of several muscarine receptor antagonists in blocking either the autoinhibition of acetylcholine release or the muscarinic contraction of the sphincter muscle upon acetylcholine release were investigated in the guinea-pig iris. The agonist at pre- or postjunctional muscarine receptors was acetylcholine released upon field stimulation (5.5 Hz, 2 min) of the irides preloaded with 14C-choline. The stimulation-evoked 14C-overflow was doubled in the presence of atropine 0.1 mumol/l but unaffected by the agonist (+/-)-methacholine (50 mumol/l). Thus, under the present stimulation conditions, the autoinhibition of acetylcholine release on the guinea-pig iris cholinergic nerves was ne…

Autoinhibition of noradrenaline release from the rat heart as a function of the biophase concentration. Effects of exogenous alpha-adrenoceptor agonists, cocaine, and perfusion rate.

1. Rat isolated perfused hearts with the right sympathetic nerves intact were loaded with 3H-(-)-noradrenaline. The nerves were stimulated with trains of 180 pulses at 3 Hz and at 10 min intervals. The overflow of 3H-noradrenaline and 3H-metabolites was determined by liquid scintillation spectrometry. 2. Clonidine (IC50 17 nM), oxymetazoline (IC50 63 nM), and α-methylnoradrenaline (apparent IC50 35 nM, determined in the presence of cocaine and propranolol) decreased the stimulation-evoked overflow of 3H-noradrenaline by 26, 49, and 78%, respectively, but not methoxamine up to 100 μM (propranolol present). Oxymetazoline and α-methyl-noradrenaline did not cause desensitization of the presynap…

Heteroreceptor-mediated modulation of noradrenaline and acetylcholine release from peripheral nerves

The effect of methacholine on noradrenaline release from the rabbit heart perfused with indometacin

The experiments were undertaken in order to study the effect of inhibition of prostaglandin synthesis on the muscarinic inhibition of noradrenaline release evoked by sympathetic nerve stimulation. The right sympathetic nerves of the perfused rabbit heart were stimulated electrically. The noradrenaline output was enhanced after perfusion of the hearts with indometacin 3×10−5 M indicating blockade of the prostaglandin-mediated negative feedback control. Both in the presence and in the absence of indometacin methacholine 4×10−5 M decreased the noradrenaline output by a similar percentage. It is concluded that the muscarinic inhibition of noradrenaline release does not require the functional in…

Effects of human recombinant interleukins on stimulation-evoked noradrenaline overflow from the rat perfused spleen

Experiments were carried out in the isolated spleen of the rat to study in a lymphoid organ the influence of interleukins (ILs) on noradrenaline release. Spleens were perfused with Tyrode's solution and the overflow of endogenous noradrenaline was determined by HPLC with electrochemical detection. Perivascular electrical stimulation (4 or 10 Hz, 20-28 mA, 2 min) caused an increase in noradrenaline overflow and in perfusion pressure, both of which were markedly reduced by perfusion with Ca(2+)-free solution, abolished by tetrodotoxin, unaffected by hexamethonium, and subject to alpha 2-adrenoceptor- and muscarinic receptor-mediated modulation as shown by the effects of rauwolscine and methac…

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

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 mumol/l and abolished by tetrodotoxin 0.3 mumol/l or perfu…

The determination of presynaptic pA2 values of yohimbine and phentolamine on the perfused rat heart under conditions of negligible autoinhibition

Abstract 1 Rat isolated perfused hearts with the right sympathetic nerves attached were loaded with [3H]-(-)-noradrenaline. The nerves were stimulated with up to 40 trains of 10 pulses every min at 1 Hz, and the evoked increases of [3H-]noradrenaline overflow into the perfusate, of right atrial tension development and ventricular beating frequency were measured. 2 Oxymetazoline inhibited the evoked transmitter overflow (IC50: 10 nM) and decreased the postsynaptic responses in a concentration-dependent manner. It behaved as a full against in abolishing the evoked transmitter overflow. 3 Yohimbine up to 1 microM neither enhanced the evoked [3H]-noradrenaline overflow nor the postsynaptic para…

Sympathetic Nerve Stimulation on the perfused rat heart. Affinities of N-methylatropine and pirenzepine at pre- and postsynaptic muscarine receptors.

Rat isolated hearts with the sympathetic nerves attached were perfused with (-)-3H-noradrenaline in order to label the storage vesicles of the adrenergic nerves. Release was induced either by electrical stimulation of the nerves (3 Hz, 1 min) or by perfusion with high K+ solution (54 mM). The overflow of 3H-noradrenaline and its metabolites was determined by liquid scintillation counting after separation of the compounds by column chromatography. The experimental conditions ensured a minor contribution of 3H-metabolites to the evoked total tritium overflow. The release of 3H-noradrenaline evoked by nerve stimulation or high K+ solution was decreased in the presence of the muscarinic agonist…

Functional consequences of prejunctional receptor activation or blockade in the iris.

The iris is innervated by nerves of the sympathetic, parasympathetic, and sensory nervous systems. The terminal nerve fibres are endowed with prejunctional receptors which modulate neurotransmitter release. Activation or blockade of prejunctional receptors by drugs may have an influence on iris smooth muscle tone. Several findings are in favour of the hypothesis that prejunctional receptors may be involved in regulation of iris smooth muscle tone and/or pathophysiological events. (i). Release of acetylcholine from parasympathetic nerves of guinea-pig iris sphincter evoked by electrical stimulation is subject to autoinhibition via prejunctional M2 muscarinic receptors, and the release can be…