A snake venom phospholipase A2 with high affinity for muscarinic acetylcholine receptors acts on guinea pig ileum

A group of small proteins, designated as muscarinic toxins (MTs), have been isolated from the venom of African green mamba (Dendroaspis angusticeps) and documented to bind selectively to individual muscarinic acetylcholine receptor (mAChR) subtypes. These components have less been reported to be isolated from other snake venoms. In this study, we have isolated a snake factor with high affinity for mAChR from the venom of Naja atra (Chinese cobra) by column chromatography on Sephadex G-50, Sephadex G-150, CM-Sepharose Fast Flow and Poros^®CM 4.6/100 Perfusion Chromatography Column. The final preparation was homogeneous as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and HPLC. The isolated active component, which was designated muscarinic protein (MP), was found to displace [³H]quinuclidinyl benzilate binding to rat cortex synaptosomes in a dose-dependent manner, and the K_i value estimated was 10.1 nM. The isolated MP was determined to have a molecular weight of 13.3 kDa and an N-terminal amino acid sequence of NLYQFKNMIQCTVPSR, which is highly homologous with phospholipase A₂ from the venoms of genus Naja. The N. atra MP could hydrolyze phosphatidylcholine in a dose-dependent manner. In guinea-pig ileum, MP produced an onset and dose-dependent contraction, which could be reversed by atropine indicating the involvement of mAChR. The EC₅₀ value of MP for guinea-pig ileum contraction was estimated as 30 nM, and the maximum contraction caused by MP was approximately 43% of that obtained from carbachol. These results seem to suggest that the snake venom phospholipase A₂ may not only have high affinity for mAChRs but also have the ability to activate mAChRs. However, it is possible that the toxin caused the contraction in the guinea-pig ileum by inducing acetylcholine release via another mechanism.     

Activation of acetylcholine receptors and 5-HT2 receptors have additive effects in the suppression of neocortical high-voltage spindles in aged rats

We investigated if activation of the muscarinic or nicotinic acetylcholine receptors and serotonin (5-hydroxytryptamine; 5-HT) subtype 2 receptors would have additive or synergistic effects on the suppression of thalamocortically generated rhythmic neocortical high-voltage spindles (HVSs) in aged rats. The 5-HT₂ receptor antagonist, ketanserin, at a moderate dose (5 mg/kg) prevented the ability of a muscarinic acetylcholine receptor agonist, (oxotremorine 0.1 mg/kg), and a nicotinic acetylcholine receptor agonist (nicotine 0.1 mg/kg), to decrease HVSs. At a higher dose (20 mg/kg), ketanserin completely blocked the decrease in HVSs produced by moderate doses of muscarinic acetylcholine receptor agonists (pilocarpine 1 mg/kg and oxotremorine 0.1 mg/kg), and by a high dose of nicotine (0.3 mg/kg), though not that produced by high doses of pilocarpine (3 mg/kg) and oxotremorine (0.9 mg/kg). The ability of a 5-HT₂ receptor agonist, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.1–1.0 mg/kg), to suppress HVSs was non-significantly modulated by the nicotinic acetylcholine receptor antagonist, mecamylamine (1–15 mg/kg), and the muscarinic acetylcholine receptor antagonist, scopolamine (0.03–0.3 mg/kg). The effects of the drugs on behavioral activity could be separated from their effects on HVSs. The results suggest that activation of the muscarinic or nicotinic acetylcholine receptors plus 5-HT₂ receptors has additive effects in the suppression of thalamocortical oscillations in aged rats. Received: 2 November 1996 /Final version: 7 February 1997     

The role of muscarinic M1 and M2 receptors in airway constriction in the cat.

The role of prejunctional inhibitory and facilitatory muscarinic receptors was investigated in cats with tracheal hyperresponsiveness to vagal stimulation. Intrathoracic airway caliber (total lung resistance (R_L) and dynamic compliance (C_dyn)) and the diameter of tracheal ring 4 were measured during vagal stimulation and local acetylcholine (ACh) injection before and after administration of the M₁ receptor antagonist pirenzepine or the M₂ receptor antagonist gallamine. The responses of tracheal ring 4, R_L, and C_dyn to ACh were unaltered by gallamine or pirenzepine. Changes in R_L and C_dyn during vagal stimulation were enhanced by gallamine, but the magnitude of tracheal constriction was unchanged. Vagally induced tracheal constriction was decreased by pirenzepine in hyperresponsive but not in control cats. The M₂ receptors limit intrathoracic airway constriction, but a functional role for M₂ receptors in the cervical trachea could not be demonstrated. However, these data suggest that M₁ excitatory receptors may play a role in vagally mediated tracheal hyperreactivity.     

Repeated ketamine administration produces up-regulation of muscarinic acetylcholine receptors in the forebrain,and reduces behavioral sensitivity to scopolamine in mice

To study the effects of repeated ketamine administration on central muscarinic acetylcholine receptors (mAchRs), ddY male mice were administered subcutaneous doses of 25 mg/kg ketamine every 3 days for a total of five times. Receptor binding assays of mAchR were carried out in the forebrain (FB), cerebellum (CB) and brainstem (BS), using [³H]quinuclidinyl benzilate ([³H]QNB) as a ligand. In addition, we examined whether repeated ketamine (12.5, 25 and 50 mg/kg) or saline (five times) could modify the hyperlocomotion induced by scopolamine (0.5 mg/kg, SC) (a muscarinic antagonist), using a behavior-pharmacological technique. Repeating the ketamine administration resulted in a significant increase in the receptor density value (B_max) for [³H]QNB only in FB, dependent on the numbers of administrations (1270 ± 33 fmol/mg protein for a single dose, 1620 ± 59 for four treatments, 1738 ± 70 for five treatments without any change in apparent affinity (defined as the reciprocal of the dissociation constant) (Kd). A competitive inhibition study of repeated (5 times) administration of ketamine failed to detect any subtype-specific changes in mAchRs. Repeated ketamine administration reduced the scopolamine-induced hyperlocomotion in a doserelated way, and the changes were significant at 50 mg/kg. Our results suggest that repeated ketamine administration produces an up-regulation of mAchRs, and this change may be associated with altered Ach transmission in the central nervous system.     

Effects of estrogen on intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors and on acetylcholinesterase activity in rat hippocampus.

The aim of the present study was to investigate the effects of estrogen lack and estrogen replacement on the production of total [3H]inositol phosphate ([3H]IP) induced by the activation of muscarinic acetylcholine receptors (mAChRs) and on the mechanisms for inactivation of acetylcholine. Hippocampi were obtained from rats in proestrus (PE), ovariectomized for 15 days (C15), ovariectomized for 15 days and then treated with 17beta-estradiol for 7 days (E7) and ovariectomized and immediately treated with 17beta-estradiol for 21 days (E21). Ovariectomy did not change the basal level of total [3H]IP in the hippocampus. 17beta-Estradiol replacement (E7 and E21) reduced the basal level of total [3H]IP. In all experimental groups, carbachol (CCh) caused a concentration-dependent rise in total [3H]IP. The maximum effect was reached with 10(-4) M CCh. The response to 10(-4) M CCh in the hippocampi from C15 and E7 rats was twofold higher than in hippocampi from PE and E21 animals and was blocked by pirenzepine, but not by methoctramine. Ovariectomy or 17beta-estradiol treatment for 7 days did not change neither the total acetylcholinesterase (AChE) activity nor the relative amount of mono- and dimeric G1/G2 and tetrameric G4 globular forms. Conversely, hormonal treatment for 21 days induced an increase in AChE activity of G1/G2 and G4 forms, indicating that 17beta-estradiol stimulates both synthesis and assembly of AChE molecular forms. The present results suggest that the duration and/or a critical period with regard to the initiation of estrogen therapy are important to regulate the function of mAChRs and AChE activity in female rat hippocampus.     

Adaptation to excess acetylcholine by downregulation of adrenoceptors and muscarinic receptors in lungs of acetylcholinesterase knockout mice

The acetylcholinesterase knockout mouse has elevated acetylcholine levels due to the complete absence of acetylcholinesterase. Our goal was to determine the adaptive changes in lung receptors that allow these animals to tolerate excess neurotransmitter. The hypothesis was tested that not only muscarinic receptors but also α₁-adrenoceptors and β-adrenoceptors are downregulated, thus maintaining a proper balance of receptors and accounting for lung function in these animals. The quantity of α_1A, α_1B, α_1D, β₁, and β₂-adrenoceptors and muscarinic receptors was determined by binding of radioligands. G-protein coupling was assessed using pseudo-competition with agonists. Phospholipase C activity was measured by an enzymatic assay. Cyclic AMP (cAMP) content was measured by immunoassay. Muscarinic receptors were decreased to 50%, α₁-adrenoceptors to 23%, and β-adrenoceptors to about 50% of control. Changes were subtype specific, as α_1A, α_1B, and β₂-adrenoceptors, but not α_1D-adrenoceptor, were decreased. In contrast, receptor signaling into the cell as measured by coupling to G proteins, cAMP content, and PI-phospholipase C activity was the same as in control. This shows that the nearly normal lung function of these animals was explained by maintenance of a correct balance of adrenoceptors and muscarinic receptors. In conclusion, knockout mice have adapted to high concentrations of acetylcholine by downregulating receptors that bind acetylcholine, as well as by downregulating receptors that oppose the action of muscarinic receptors. Tolerance to excess acetylcholine is achieved by reducing the levels of muscarinic receptors and adrenoceptors.     

Differential calcium signalling by m2 and m3 muscarinic acetylcholine receptors in a single cell type

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca²⁺ concentration [Ca²⁺]_i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca²⁺]_i with EC₅₀ values of about 2 M and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP₃) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca²⁺]_i were 300–350 nM and mainly, almost 90%, due to influx of extracellular Ca²⁺. The efficacy of pilocarpine for stimulating InsP andCa²⁺ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca²⁺]_i with EC₅₀ values of about 20 M and 7 M, respectively. Maximal InsP formation was only 10–15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca²⁺]_i were similar in both cell types. Formation of InsP₃ was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca²⁺]_i increases induced by m2 mAChR activation were exclusively due to Ca²⁺ mobilization from intracellular stores.The efficacy of pilocarpine for stimulating InsP and Ca²⁺ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca²⁺]_i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca²⁺ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.     

The inhibitory modulation of guinea-pig intestinal peristalsis caused by capsaicin involves calcitonin gene-related peptide and nitric oxide

The effect of capsaicin-induced stimulation of afferent neurons on peristalsis and the possible neural mediators involved in this action were examined in the guinea-pig isolated ileum. The intraluminal pressure threshold for eliciting peristaltic waves was used to quantify facilitation (decrease in threshold) or inhibition (increase in threshold) of peristalsis. Capsaicin (0.1–1 M) caused an initial short-lasting stimulation of peristalsis followed by a prolonged inhibition of peristaltic activity. Capsaicin (1 M) was ineffective when the gut segments had been pretreated with 3.3 M capsaicin, which is indicative of an afferent neuron-dependent action of the drug. In contrast, the abolition of peristalsis caused by a high concentration of capsaicin (33 M) was fully reversible on removal and reproducible on readministration of capsaicin, a feature characteristic of a nonspecific depression of smooth muscle excitability. Baseline peristalsis and the excitatory/inhibitory effect of capsaicin (1 M) on peristalsis remained unaltered by a combination of the tachykinin NK₁ receptor antagonist ( + )-(2S, 3S)-3-(2-methoxybenzylamino)-2-phenyl piperidine (CP-99,994; 0.3 M) and the tachykinin NK₂ receptor antagonist L(-)-N-methyl-N[4-acetylamino-4-phenyl-piperidino-2-(3,4-dichlorophenyl)butyl]-benzamide (SR-48,968; 0.1 M). Further experiments, performed in the presence of a low concentration of atropine (10 nM) showed that the catcitonin gene-related peptide (CGRP) antagonist human -catcitonin gene-related peptide (8–37) [hCGRP (8–37); 10 M] attenuated the delayed inhibitory effect of capsaicin on peristalsis, but did not influence baseline peristaltic activity and the capsaicin-induced facilitation of peristalsis. Blockade of nitric oxide (NO) synthesis by N ^G-nitro-l-arginine methylester (l-NAME, 300 M) facilitated baseline peristaltic activity and reduced the delayed inhibition of peristalsis caused by capsaicin (1 M) without affecting the initial peristalsis-stimulating action of capsaicin. The effects of l-NAME were prevented by l-arginine (1 mM). The data of the current study indicate that capsaicin-sensitive afferent neurons do not participate in the neural pathways subserving peristalsis in the guinea-pig small intestine, but modulate peristaltic activity upon stimulation with capsaicin. The initial stimulant action of capsaicin on peristalsis is independent of tachykinins acting via NK₁ or NK₂ receptors, while the delayed capsaicin-induced depression of peristalsis involves CGRP and NO.     

Comparative conformational analysis and in vitro pharmacological evaluation of three cyclic hexapeptide NK-2 antagonists

The conformational analysis of three cyclic hexapeptides is presented. Cyclo-(-Gln⁶-Trp⁷-Phe⁸-Gly⁹-Leu¹⁰-d -Met¹¹-) (1) and cyclo-(-Gln⁶-Trp⁷-Phe⁸-Gly⁹-Leu¹⁰-Met¹¹-) (2) are NK-2 antagonists in the hamster trachea assay, whereas cyclo-(-Gln⁶-Trp⁷-Phe⁸-(R)-Gly⁹-[ANC-2]Leu¹⁰-Met¹¹-) (3), where Gly⁹[ANC-2]Leu¹⁰ represents (2S)-2-((3R)-3-amino-2-oxo-1-pyrrolidinyl)-4-methylpentanoyl, is inactive as agonist and antagonist in this assay. In DMSO, the NMR results cannot be interpreted as being consistent with a single conformation. However, the combined interpretation of results from NMR spectroscopy, restrained molecular dynamics simulations with application of proton–proton distance information from ROESY spectra, and pharmacological results leads to a reduced number of conformational domains for each peptide, which can be compared with each other and may be classified as responsible for their biological activity. Trying to match the conformational domains approximately with regular β- and γ-turns, we find a γⁿ-turn at the position of the methionine occuring in all peptides. For the active peptides 1 and 2 we arrive at an inverse γⁱ-turn at Phe⁸, and βI′- or βII-turns with Gly⁹ and Leu¹⁰ at the corner positions, these β-turns having a similar topology with respect to the linking peptide unit. Other conformational domains common to only 1 and 2 support their classification as responsible for the biological activity.     

Inhibitory and excitatory muscarinic receptors modulating the release of acetylcholine from the postganglionic parasympathetic neuron of the chicken heart

Summary The effects of muscarinic receptor antagonists on ACh release were studied in the absence or presence of cholinesterase (ChE) inhibition using the isolated perfused chicken heart. Presynaptic inhibitory muscarinic autoreceptor were characterized by determining the potency of various antagonists to enhance [³H]-ACh release evoked by field stimulation (3 Hz, 1 min). The order of potencies was: (±)-telenzepine > atropine > 4-DAMP > silahexocyclium > pirenzepine > hexahydro-siladifenidol > AF-DX 116. The comparison with known pA₂ values for M₁-, M₂- and M₃-receptors revealed that the presynaptic autoreceptor meets the criteria of an M₁-receptor. Basal, not electrically evoked overflow of unlabelled ACh into the perfusate was caused by leakage release (non-exocytotic), as it was independent of extracellular Ca²⁺ . Muscarinic receptor antagonists failed to enhance basel overflow. In contrast, when ChE activity was inhibited by 10^–6M tacrine or pretreatment with 10^–4M DFP, the ACh overflow was partially Ca²⁺-dependent and was reduced by tetrodotoxine. Moreover, block of the inhibitory muscarinic autoreceptors by (±)-telenzepine or pirenzepine caused a several-fold enhancement of the ACh release. The potencies of these antagonists were identical to those found for the electrically evoked [³H]-ACh release. The rate of ACh release enhanced by ChE inhibition plus telenzepine corresponds to about 12% of the total ACh pool per min, which is about the maximum amount of ACh that is available for any kind of stimuli. The release was dependent on the presence of exogenous choline. Hence elevation of ACh release led to a correspondingly enhanced ACh synthesis. The dramatic enhancement of ACh release by the ChE inhibition in combination with a block of presynaptic muscarinic autoinhibition was not inhibited by (+)-tubocurarine but by atropine (10^–9 to 10^–7 M) or 10^–6 M telenzepine. It is concluded that basal release of ACh in the heart was due to non-exocytotic leakage release. Inhibition of ChE led to a marked stimulation of excitatory muscarinic receptors of the intrinsic parasympathetic neuron with a consecutive postganglionic release of ACh. The strong postganglionic excitation was obvious when the inhibitory muscarinic autoreceptors were selectively blocked. Of the two described muscarinic receptors found in the parasympathetic postganglionic neuron of the chicken heart only the inhibitory was classified as being M₁, whereas the subtype of the excitatory one is unlike M₁ and remains to be identified.Preliminary results have been presented at the Spring meeting of the German Pharmacological Society in 1992 (Brehm and Lindmar 1992) Correspondence to R. Lindmar at the above address     

In vivo modulation of histamine release by autoreceptors and muscarinic acetylcholine receptors in the rat anterior hypothalamus

The modulation of histamine release by histamine and muscarinic acetylcholine receptors was investigated by using the push-pull technique. The anterior hypothalamic area of the conscious, freely moving rat was superfused through the push-pull cannula with CSF or with CSF containing drugs and the release of endogenous histamine was determined in the superfusate.Hypothalamic superfusion with tetrodotoxin (10 mol/1) led to a pronounced and sustained decrease in the histamine release rate. Superfusion with compound 48/80 (100 mg/1) was ineffective. Hypothalamic superfusion with the H₃ agonist (R)--methylhistamine inhibited, while superfusion with the H₃ antagonist thioperamide enhanced the release of histamine. The release of histamine was inhibited on hypothalamic superfusion with the muscarinic receptor agonists carbachol or oxotremorine. Histamine release was enhanced by atropine, and this release-enhancing effect was abolished by oxotremorine. The selective M₁ antagonist pirenzepine (100 mol/I) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 10 ol/1), which blocks M₁ and M₃ receptors, also enhanced the release rate of histamine. On the other hand, 50 and 100 moI/I methoctramine (M₂ receptor antagonist) 10 and 100 moI/l p-fluoro-hexahydro-siladifenidol (p-F-HHSiD, a M₃ receptor antagonist) were ineffective.It is concluded that histamine released in the hypothalamus originates predominantly from neurons. The release of histamine is modulated by H₃ autoreceptors. The histamine release is also modulated by cholinergic neurons which modify histamine release from histaminergic neurons by stimulating M₁ muscarinic acetylcholine heteroreceptors probably located on histaminergic neurons.Supported by the Fonds zur Förderung der wissenschaftlichen Forschung Correspondence to: H. Prast at the above address     

P2X7 receptors contribute to the currents induced by ATP in guinea pig intestinal myenteric neurons

The whole-cell configuration, several pharmacological tools, and single-cell RT-PCR were used to investigate the contribution of P2X7 subunits to the ATP-induced currents (I_ATP) in guinea pig myenteric neurons. I_ATP was recorded in the great majority of tested neurons. ATP concentration-response curve (0.01–10 mM) showed two phases, the first mediated by high-sensitive P2X receptors (hsP2X receptors), observed between 0.01–0.3 mM and the second mediated by low-sensitive P2X receptors (lsP2X receptors). The calculated EC₅₀ values of these phases were 38 and 1759 μM, respectively. 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP) concentration-response curve was monophasic (0.01–1 mM), and less potent (EC₅₀ 142 μM) than ATP to activate hsP2X receptors. A strong inward rectification was noticed when hsP2X receptors were activated with ATP (0.1 mM) and for BzATP-induced currents (0.1 mM; I_BzATP) but a significant lower rectification was noticed when lsP2X receptors were activated (5 mM). Brilliant blue G (BBG) at a concentration of 0.3 μM (known to inhibit only P2X7 receptors) reduced I_ATP when lsP2X receptors contributed to it but neither affect hsP2X receptors nor I_BzATP. However, hsP2X receptors and I_BzATP were both inhibited by concentrations ≥ 1 μM of this antagonist. BzATP inhibited hsP2X receptors and therefore, it behaves as partial agonist on these receptors. Using the single-cell RT-PCR technique P2X7 mRNA was detectable in 7 out of 13 myenteric neurons exhibiting P2X2 mRNA. Altogether, our results show that low-sensitive P2X receptors are likely P2X7, whereas, the high-sensitive P2X channels are probably constituted, at least in part, by P2X2 subunits.     

Effect of low dose metronomic therapy on MCF-7 tumor cells growth and angiogenesis. Role of muscarinic acetylcholine receptors

The non-neuronal cholinergic system refers to the presence of acetylcholine, choline acetyltransferase, acetylcholinesterase and cholinergic receptors, nicotinic and muscarinic (mAChRs) expressed in non-neuronal cells. The presence of mAChRs has been detected in different type of tumor cells and they are linked with tumorigenesis. We had previously documented the expression of mAChRs in murine and human mammary adenocarcinomas and the absence of these receptors in normal mammary cells of the same origins. We also demonstrated that mAChRs are involved in breast cancer progression, pointing to a main role for mAChRs as oncogenic proteins. Since the long term treatment of breast cancer cells with the muscarinic agonist carbachol promoted cell death, here we investigated the ability of low doses of this agonist combined with paclitaxel (PX), a taxane usually administered to treat breast cancer, to inhibit the progression of human MCF-7 tumor cells. We demonstrated that PX plus carbachol reduced cell viability and tumor growth in vitro probably due to a down-regulation in cancer stem cells population and in the expression of ATP “binding cassette” G2 drug extrusion pump; also a reduction in malignant-induced angiogenesis was produced by the in vivo administration of the mentioned combination in a metronomic schedule to MCF-7 tumor-bearing NUDE mice. Our results confirm that mAChRs could be considered as therapeutic targets for metronomic therapy in breast cancer as well as the usefulness of a muscarinic agonist as repositioning drug in the treatment of this type of tumors.     

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

Summary 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 antagonists acted competitively and their effects were reversible. Furthermore, for none of the drugs did the pA₂ (pre) differ from the pA₂ (post).With QNB (3-quinuclidinyl benzilate) a pA₂ (post) of 11.65 was obtained. However, the affinity to presynaptic receptors could not be determined as a pA₂ value due to the very prolonged exposure time required for the equilibrium with QNB and for that with methacholine in the presence of QNB.It is concluded that the antagonists employed do not reveal differences between pre- and postsynaptic muscarine receptors of the rabbit heart, in spite of their greatly varying chemical structure and their individual affinities ranging over 5 orders of magnitude. The findings confirm the view of a homogeneous muscarine receptor population characterized by functional parameters.     

The role of muscarinic receptors in synaptic transmission and its modulation in the rabbit superior cervical ganglion

The role of muscarinic receptors in synaptic transmission has been reinvestigated using a superfusion bath, a technique allowing recording of postganglionic responses of great constancy. Atropine or hyoscine, 0.26–2.9 μM, depressed transmission by about 10%, implicating muscarinic receptors in the transmission process. Ganglionic P and LN waves elicited by single stimuli were reduced by atropine by 33 and 70%, respectively, and were reduced by hexamethonium, 688 μM, by 92 and 70%, respectively. Late facilitation following a 1 sec train of stimuli increased with frequency of stimulation within the train up to 30 Hz, when 30% facilitation was observed; longer trains resulted in greater post-train facilitation but also enhanced negative after-potentials. About $\text{1}\text{3}$ of post-train facilitation was atropine sensitive. The atropine sensitive component decayed more rapidly than total facilitation and was enhanced when C fibres were excited during the repetitive stimulation. It is argued that in the rabbit superior cervical ganglion facilitation is mainly due to a non-muscarinic mechanism, probably presynaptic in nature, and the LN process primarily allows expression of this facilitation. Sa and Sb post-train facilitation were reciprocally related consistent with convergence of B and C fibres onto a common pool of cells.     

Effects of muscarinic toxins MT2 and MT7, from green mamba venom, on m1, m3 and m5 muscarinic receptors expressed in Chinese Hamster Ovary cells.

Several small proteins called muscarinic toxins (MTs) have been isolated from venom of green mamba (Dendroaspis angusticeps). They have previously been shown in radioligand binding studies to have high selectivity and affinity for individual muscarinic receptor subtypes, but less is known of their functional effects. This study has examined the actions of two of these MTs, MT2 and MT7, using changes in cytosolic Ca(2+) ([Ca(2+)](i)) measured using the fluorescent indicator fura-2 in Chinese Hamster Ovary (CHO) cells stably transfected with individual muscarinic receptor subtypes, m1, m3 and m5. MT2 activated the m1 receptor: at concentrations above 100 nM it caused significant and concentration-dependent increases in [Ca(2+)](i). From 25 to 800 nM MT2 also produced increases in [Ca(2+)](i) by activating m3 receptors, although these increases in [Ca(2+)](i) were not strictly concentration-dependent with only intermittent responses being recorded (i.e. it was not always possible to obtain a response to the agonist with each application of the compound). MT2 (800-1600 nM) also caused significant increases in [Ca(2+)](i) in CHO cells expressing the m5 muscarinic receptor subtype. MT7 (1 microM) displayed no agonist activity at any of the muscarinic receptors but was a potent non-competitive antagonist (at 20 nM) at the m1 muscarinic receptor subtype. It had no antagonist activity at the m3 or m5 subtypes. These results indicate that MT7 is a highly specific antagonist at the m1 muscarinic receptor subtype as suggested by results from radioligand binding studies. However, MT2 is less selective for the m1 muscarinic receptor than previously described as it also exhibits agonist activity at the m3 and m5 muscarinic receptors, which was not detected in radioligand binding studies.     

Effects of guanine nucleotide and sulfhydryl reagent on subpopulations of muscarinic acetylcholine receptors in mammalian hearts: possible evidence for interconversion of super-high and low affinity agonist binding sites

Multiple site models of muscarinic acetylcholine receptors (mAChR) for agonist binding were applied to curves for the inhibition of QNB binding by carbachol by using nonlinear least square regression analysis. The effects of a guanine nucleotide guanyl-5′-yl imidodiphosphate (Gpp(NH)p) and a sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid (DTNB) on the curves were also analyzed. The results suggested that mAChR of dog and guinea pig heart had three types of sites with different affinities for carbachol (super-high (SH), high (H) and low (L)). In the presence of Gpp(NH)p, SH sites were eliminated and L sites increased, indicating conversion of SH sites to L sites. On the contrary, in the presence of DTNB, L sites were converted to SH sites. These results were obtained at both 37°C and 0°C incubation although the affinity of each site was high at 0°C than at 37°C. These data suggest the interconversion of SH and L sites. The possible existence of two subtypes (GTP-regulated mAChR(SH-L type) and GTP-independent mAChR (H type)) is discussed.     

Effects on guanine nucleotide and sulfhydryl reagent on subpopulations of muscarinic acetylcholine receptors in mammalian hearts: Possible evidence for interconversion of super-high and low affinity agonist binding sites

Multiple site models of muscarinic acetylcholine receptors (mAChR) for agonist binding were applied to curves for the inhibition of QNB binding by carbachol by using nonlinear least square regression analysis. The effects of a guanine nucleotide guanyl-5′-yl imidodiphosphate (Gpp(NH)p) and a sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid (DTNB) on the curves were also analyzed. The results suggested that mAChR of dog and guinea pig heart had three types of sites with different affinities for carbachol (super-high (SH), high (H) and low (L)). In the presence of Gpp(NH)p, SH sites were eliminated and L sites increased, indicating conversion of SH sites to L sites. On the contrary, in the presence of DTNB, L sites were converted to SH sites. These results were obtained at both 37°C and 0°C incubation although the affinity of each site was high at 0°C than at 37°C. These data suggest the interconversion of SH and L sites. The possible existence of two subtypes (GTP-regulated mAChR(SH-L type) and GTP-independent mAChR (H type)) is discussed.     

Antimuscarinic, calcium channel blocker and tachykinin NK2 receptor antagonist actions of otilonium bromide in the circular muscle of guinea-pig colon

We have analyzed, by the sucrose gap method, the action of otilonium bromide, a quaternary ammonium derivative in use for the symptomatic therapy of irritable bowel syndrome, on the electrical and mechanical responses initiated by different stimuli in the circular muscle of the guinea-pig proximal colon. Otilonium bromide produced a concentration-dependent inhibition of membrane depolarization (IC₅₀ 4.1 μM), action potentials (APs) and contraction (IC₅₀ 3.7 μM) produced by the muscarinic receptor agonist, methacholine. It also produced a concentration-dependent inhibition of APs and accompanying contraction (IC₅₀ 31 μM) produced by KCl (30 mM), and had a biphasic effect on the cholinergic excitatory junction potential (e.j.p.) produced by single pulse electrical field stimulation: at low concentrations (0.1–0.3 μM) otilonium bromide enhanced the e.j.p. and, at higher concentrations (IC₅₀ 22 μM and 16 μM toward depolarization and contraction), produced a concentration-dependent inhibition. Otilonium bromide eliminated the APs superimposed on the depolarization induced by the tachykinin NK₁ receptor agonist, [Sar⁹]substance P-sulphone and suppressed the corresponding contraction (IC₅₀ 43 μM) but had little effect on the sustained membrane depolarization induced by this agonist. On the other hand, otilonium bromide produced a similar inhibitory effect on both membrane depolarization and contraction (IC₅₀ 38 μM and 45 μM, respectively) induced by the tachykinin NK₂ receptor agonist [βAla⁸]neurokinin A (4–10). When tested in the presence of nifedipine (1 μM), otilonium bromide had no effect on the membrane depolarization induced by [Sar⁹]substance P-sulphone but inhibited in a concentration-dependent manner the depolarization induced by [βAla⁸]neurokinin A (4–10) (IC₅₀ 41 μM). In contrast, the blocker of receptor-operated cation channels, SKF 96365, inhibited with similar potency the depolarization induced by both [Sar⁹]substance P-sulphone and [βAla⁸]neurokinin A (4–10) (IC₅₀ 60 μM and 54 μM, respectively). In radioligand binding experiments otilonium bromide produced a concentration-dependent inhibition of the binding of both an agonist ([¹²⁵I]neurokinin A, K _i 7.2 μM) and an antagonist ([³H]SR 48968, K _i 2.2 μM) to membranes of Chinese hamster ovary cells transfected with the human tachykinin NK₂ receptor. In conclusion, the present findings demonstrate that, in the μM range of concentrations, otilonium bromide acts as a muscarinic and tachykinin NK₂ receptor antagonist and as a calcium channel blocker. The latter property is likely to account for its ability to suppress contraction initiated by the tachykinin NK₁ receptor agonist. Therefore multiple mechanisms of action account for the ability of otilonium bromide to reduce stimulated motility of intestinal smooth muscle. Received: 27 November 1998 / Accepted: 2 February 1999