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

Summary 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 mol/l but unaffected by the agonist (±)-methacholine (50 mol/l). Thus, under the present stimulation conditions, the autoinhibition of acetylcholine release on the guinea-pig iris cholinergic nerves was nearly maximally activated. Isotonic contractions of the irides upon field stimulation consisted of a rapid, atropine (0.1 mol/l). peak phase followed by a sustained contraction which involved a cholinergic and a non-cholinergic stimulation of the sphincter muscle. The M₂-selective antagonists methoctramine (10 mol/l) and gallamine (100 µmol/l). increased both the ¹⁴Goverflow and the peak contractions evoked by field stimulation. In contrast, the M₃-selective antagonist hexahydrosiladifenidol (0.1–10 mol/l) failed to affect the evoked ¹⁴C-release but concentration-dependently (1–10 mol/l) reduced the iris contractions. Pirenzepine (10 mol/l) enhanced the evoked ¹⁴C-overflow and inhibited the peak contractions (0.1–10 mol/l; maximal effect at 10 mol/l). The low potency of the antagonist at both receptor sites indicates that an M₁ muscarine receptor is not involved. The results are consistent with the idea of M₂ muscarine receptors mediating autoinhibition of acetylcholine release in the guinea-pig iris and M₃-like receptors inducing the contraction of the sphincter muscle. Send offprint requests to I. T. Bognar at the above address     

The M2 muscarinic receptor antagonist methoctramine activates mast cells via pertussis toxin-sensitive G proteins

Methoctramine, a selective M₂ muscarinic cholinergic receptor antagonist, has been reported to activate phosphoinositide breakdown at high concentrations. Its polyamine structure suggests a putative activation of guanine nucleotide-binding proteins (G proteins). Incubation of methoctramine with rat peritoneal mast cells resulted in a dose-dependent noncytotoxic histamine release, with an EC₅₀ of 20μM and a maximum effect at 1mM. Atropine, pirenzepine and HHSiD neither inhibited methoctramine-induced histamine release nor stimulated histamine release. Histamine release and inositol phosphates generation induced by methoctramine were both inhibited by pertussis toxin pretreatment. Benzalkonium chloride, a selective inhibitor of histamine secretion induced by basic secretagogues, inhibited the secretory response to methoctramine. [p-Glu⁵, d-Trp^7,9,10]-SP_5–11 (GPAnt-2), a well-characterized antagonist of G proteins, blocked the methoctramine-induced histamine release when the antagonist was allowed to reach its intracellular target by streptolysin O-permeabilization. The response to methoctramine was prevented by the hydrolysis of sialic acid residues of the cell surface by neuraminidase. The response of mast cells was restored by permeabilization of the plasma membrane. These results demonstrate that methoctramine, following its entry into the cell and the involvement of pertussis toxin-sensitive G proteins, activates phosphoinositide hydrolysis leading to mast cell exocytosis. Received: 26 September 1997 / Accepted: 1 December 1997     

Blockade of hippocampal M1 muscarinic receptors impairs working memory performance of rats

In order to clarify the roles of hippocampal M₁ and M₂ muscarinic receptors in working and reference memory performance of rats, the effects of intrahippocampal injections of selective antagonists at both receptors on this behavior were examined with a three-panel runway task. In the working memory task, the M₁ muscarinic receptor antagonist pirenzepine, injected bilaterally at 0.32 and 1.0 μg/side into the dorsal hippocampus, significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). This effect of intrahippocampal pirenzepine (1.0 μg/side) on working memory was attenuated by concurrent injection of 10 μg/side AF102B, the selective M₁ muscarinic receptor agonist. Intrahippocampal injection of the M₂ muscarinic receptor antagonist methoctramine at doses up to 1.0 μg/side had no significant effect on the number of working memory errors. Intrahippocampal methoctramine injection at 3.2 μg/side produced a significant increase in working memory errors, an effect that was reversed by concurrent injection of 10 μg/side AF102B. Concurrent injection of 0.32 μg/side methoctramine significantly reduced the increase in working memory errors induced by intrahippocampal pirenzepine (1.0 μg/side). In the reference memory task, neither pirenzepine nor methoctramine affected the number of errors when injected into the hippocampus at doses up to 1.0 and 3.2 μg/side, respectively. These results suggest that processes mediated by M₁ muscarinic receptors in the hippocampus are involved in working memory, but not in reference memory, and that blockade of hippocampal M₂ muscarinic receptors ameliorates working memory deficits produced by M₁ muscarinic blockade, possibly by increasing acetylcholine release.     

Pre-junctional muscarinic autoreceptors in bovine airways

We searched for pre-junctional inhibitory muscarinic receptors in isolated bovine trachealis strips and bronchial rings. Electric stimulation (ES)-induced tritiated acetylcholine ([³H]-ACh)-release and isometric contractions were determined in muscles incubated with the non-selective muscarinic agonist pilocarpine, the non-selective muscarinic antagonist atropine, the selective M₂-receptor antagonists methoctramine and gallamine, or the selective M₄-receptor antagonist PD102807. Electric field stimulation (EFS)-induced isometric contractile responses were assessed in trachealis strips and bronchial rings treated with 10⁻⁹-10⁻⁵ M methoctramine, gallamine or PD102807. Pilocarpine (10⁻⁶ and 10⁻⁵ M) and atropine (10⁻⁷ M) significantly decreased and increased ES-evoked [³H]-ACh-release, respectively. The enhancing effect of atropine on [³H]-ACh-release prevailed over the inhibitory effect of pilocarpine. M₂- and M₄-receptor antagonists did not increase EFS-induced contraction or ES-induced [³H]-ACh-release. However, 10⁻⁷ M methoctramine, gallamine or PD102807 significantly attenuated the inhibitory effects of pilocarpine 10⁻⁵ M on ES-induced [³H]-ACh-release. Conclusions: Muscarinic autoregulation is present in bovine airways but is not fully accounted for by M₂- and M₄-receptor subtypes.     

Involvement of cholinergic neurons in the release of dopamine elicited by stimulation of μ-opioid receptors in striatum

The involvement of striatal cholinergic neurons in the release of dopamine (DA) elicited by the (μ-opioid receptor agonist DAGO [d-Ala², NMePhe⁴-Gly⁵(ol)]enkephalin) was explored. The striatal release of DA was measured by microdialysis in rats anesthetized with chloral hydrate. When infused in the striatum, through the microdialysis probe, DAGO increased the extracellular levels of DA. The previous injection in striatum of AF 64-A, a toxin for cholinergic neurons, or the concomitant infusion of the M₂-muscarinic antagonist methoctramine abolished the effect of DAGO on the DA release. It is concluded that stimulation of μ-opioid receptors, by inhibiting the acetylcholine release which stimulates tonically M₂-muscarinic receptors likely associated with dopaminergic nerve endings, indirectly increases the striatal DA release.     

Cholinergic sensitivity of irides from donors with various pathological conditions and lens implants

Summary In vitro, iris contractions after muscarinic agonists were measured in mg of tension change and the concentration producing 50% of the response was expressed as EC₅₀ mol/1. Although the average EC₅₀ value of carbachol in the iris sphincter of the donors with diabetes or Parkinson's disease did not change significantly when compared with the control, the maximum contraction of the tissue from the diseased state was increased significantly. Thus, in addition to the well known denervation supersensitivity of the iris-dilator, the iris-sphincter also develops adaptive sensitivity changes. Antimuscarinic drug treatment in some Parkinson's patients interfered with the estimation of supersensitivity in vitro studies. The enhanced response of carbachol at the low temperatures or the relative potency of carbachol and pilocarpine in the tissue obtained from the diseased donors was not significantly different from that of controls. Based on EC₅₀ values, the potency of arecoline on the iris was 1/3 that of carbachol. Significantly lower EC₅₀ values of carbachol were found in irides which were in contact with open loop type anterior chamber lens implants compared with those in contact with the closed loop anterior chamber lens implants. Maximum responses of irides to carbachol were less when the tissue was in contact with open loop lens compared with those in contact with closed loop anterior chamber implants.Irides from many donors having unilateral or bilateral replacement of the artificial lenses responded with EC₅₀ of carbachol which was approximately equal to that of the contralateral eye. The maximum difference between EC₅₀ values of the left and right iris was less than 5 fold. Paired irides with asymmetric surgical procedures responded unequally to carbachol.The dissociation constant KB of atropine (1 nmol/1) at 17°C was equal to that observed at 37.5°C. The K_B values of himbacine, methoctramine and pirenzepine were 120, 1500, 120 nmol/1, respectively. From one tissue to another, there was a spread in the dissociation constant value of pirenzepine indicating that human iris sphincter may contain heterogenous population of muscarinic receptors.A part of this study was presented in the symposium on Ocular Toxicology at the Second International Congress of Toxicology in Developing Countries, New Delhi, (India), November 24–28, 1991Correspondence to P. N. Patil, College of Pharmacy, Lloyd M. Parks Hall, 500 W. 12th Avenue, Columbus, OH 43210, USA     

Methoctramine selectively blocks cardiac muscarinic M2 receptors in vivo

Summary The antimuscarinic effects of methoctramine (N, N- bis[6-[(2-methoxybenzyl)amino]hexyl]-1,8-octanediamine tetrahydrochloride), a polymethylene tetraamine endowed with high cardioselectivity in vitro, were assessed in two in vivo preparations. Methoctramine (300 g/kg i.v.) strongly inhibited the methacholine- and muscarine-induced bradycardia in the anaesthetized and pithed rat, respectively. The same dose of methoctramine did not significantly affect the depressor action of methacholine in the anaesthetized rat mediated by vascular M₂ receptors. Furthermore, even high doses of methoctramine (up to 1 mg/kg i. v.) did not reduce the ganglionic M₁ receptor-mediated tachycardia and pressor response to muscarine or McN-A-343 in the pithed rat. These data suggest that methoctramine while showing high affinity for cardiac M₂ receptors has rather low affinity for ganglionic M₁ and vascular M₂ receptors. This in vivo study thus provides further evidence to support the view that methoctramine is a potent and highly selective antagonist of cardiac M₂ receptors. Send offprint requests to: G. Lambrecht at the above address     

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

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

Cardiovascular effects elicited by central administration of physostigmine via M2 muscarinic receptors in conscious cats

The cardiovascular effects of an intracerebroventricular (i.c.v.) injection of physostigmine were studied using conscious cats. Physostigmine (5–25 μg: 5 μl) caused a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR). The highest dose (25 μg) increased MAP and HR by 32 ± 3 mmHg and 45 ± 5 beats/min, respectively (n = 5). Pre-administration of the muscarinic receptor antagonist, atropine (25 μg; i.c.v.) blocked the effects of physostigmine (25 μg; i.c.v.). Also, the pre-administration of the M₂ muscarinic antagonist, methoctramine (25 μg; i.c.v.), antagonized the cardiovascular effects of physostigmine without altering the baseline variables. However, the M₁ muscarinic antagonist, pirenzepine (100 μg; i.c.v.) did not alter baseline MAP or HR, and also failed to inhibit the cardiovascular responses to physostigmine. Similarly, the M₃ muscarinic blocker, 4-diphenyl-acetoxy-N-methylpiperidine methiodide (50 μg; i.c.v.), neither changed baseline cardiovascular variables nor blocked the effects of physostigmine. When the same cats were anesthetized with intravenous injection of sodium pentobarbital (25–30 mg/kg), physostigmine (25 μg; i.c.v.) evoked a decrease in MAP and HR of 13 ± 6 mmHg and 15 ± 6 bpm, respectively (n = 5). These results demonstrate that the increases in MAP and HR to the i.c.v. administration of physostigmine in conscious cats arepossibly mediated through stimulation of central M₂ muscarinic receptors. In addition, anesthesia reverses the effects elicited by the central administration of physostigmine to a decrease in MAP and HR.     

Methoctramine and hexahydrodifenidol antagonise two muscarinic responses on the rat superior cervical ganglion with opposite selectivity

Two novel muscarinic antagonists, methoctramine and hexahydrodifenidol, have been assessed for their action against two muscarinic agonist-induced responses on the rat superior cervical ganglion in vitro. DC recordings were made between the desheathed ganglion and its internal carotid nerve using the grease-gap technique. Hexahydrodifenidol and methoctramine antagonised the muscarine-induced M₁-mediated depolarisation of this preparation with estimated pA₂ values of 7.5 and 6.5, respectively. In 0.3 μM pirenzepine and 0.1 mM CaCl₂, 1 μM muscarine evoked a hyperpolarisation mediated by cardiac-like M₂ receptors. Hexahydrodifenidol and methoctramine antagonised this response with pIC₅₀ values (−log₁₀IC₅₀) of 5.7 and 7.4, respectively. The selectivity of methoctramine for cardiac-like M₂ receptors over M₁ receptors is therefore confirmed and extended to these two neuronal responses. The selectivity of hexahydrodifenidol was opposite to, and greater than, that seen with methoctramine.