The effect of some antidepressants on prejunctional muscarinic receptors on the sympathetic nerves of the isolated rabbit ear artery

Summary The antimuscarinic activity of amitriptyline, desipramine, iprindole, mianserin and viloxazine on prejunctional sympathetic nerve endings were compared in the isolated rabbit ear artery. In the presence of cocaine (10 M) and yohimbine (1 M), amitriptyline (0.5–1 M), desipramine (1–3 M) and iprindole (5–10 M), desipramine (1–3 M) and iprindole (5–10 M) produced parallel rightward shifts of the concentration-response curve for the inhibitory effect of carbachol (CCh) on responses to electrical stimulation of the preparation at 3 Hz. Mianserin (3 M) produced some inhibition but altered the slope of the concentration-responses curve to CCh while viloxazine (10 M) produced no inhibition.The depression of tritium efflux by CCh from arteries preincubated in ³H-noradrenaline was inhibited significantly (P<0.05) by amitriptyline (0.1 M) and desipramine (1 M) and not by iprindole (17 M), mianserin (3 M) or viloxazine (10 M). Amitriptyline was 10-fold more active than desipramine and at least 30-fold more active than the other antidepressants as a muscarine receptor blocking drug in this preparation.Thus, mianserin, viloxazine and iprindole exhibit much weaker antimuscarinic activity relative to amitriptyline on prejunctional muscarine receptors on sympathetic nerve endings compared with that observed by others for excitatory muscarine receptors in sympathetic ganglia. The findings support an earlier suggestion that these receptors differ.     

Involvement of muscarinic and nicotinic receptors in behavioral tolerance to DFP

Effects of various cholinergic agents on the free operant responding and single alternation behavior of rats were examined following two regimens of chronic treatment with diisopropylfluorophosphate (DFP), an irreversible anticholinesterase, which lowered brain cholinesterase to 45% and 30% of normal, respectively. Reduction to 45% produced no observable changes in behavior; reduction to 30% gave rise to a decrease in the number of reinforced responses and an increase in the number of nonreinforced responses. Tolerance for the former measure developed within 10 days, whereas tolerance for the latter was not observed. Subsequent challenges were carried out using anticholinesterase agents, and muscarinic and nicotinic agonists and antagonists. The results suggest that the sensitivity og both muscarinic and nicotinic receptors to acetylcholine may be reduced during chronic treatment with DFP, but that muscarinic receptors may be more labile than nicotinic receptors. It is hypothesized that this reduction in sensitivity is one mechanism underlying the development of behavioral tolerance to DFP.     

Radioligand binding characteristics of the chicken cardiac muscarinic receptor

Summary The muscarinic receptor present in chicken cardiac membranes was characterised using a ligand binding approach and compared to the M1, M2 and M3 receptors that can be identified in ligand binding studies at present. [³H]N-methylscopolamine and [³H]pirenzepine appeared to label the same population of muscarinic receptors in chicken cardiac membranes since the density of sites labeled by the two radioligands was similar. Furthermore, affinity estimates of 8 muscarinic receptor antagonists for chicken cardiac muscarinic receptors were the same irrespective of whether [³H]N-methylscopolamine or [³H]pirenzepine was used as the radioligand. The chicken cardiac muscarinic receptor displayed high affinity for pirenzepine (pK _i = 7.9) and so did not appear to represent an M2 receptor. Despite the high affinity of chicken cardiac muscarinic receptors for pirenzepine, affinity estimates for dicyclomine (pK _i = 8.0), CPPS (pK _i = 8.4) and 4DAMP (pK _i = 8.6) in chicken heart were not consistent with the presence of M1 receptors. The chicken cardiac muscarinic receptor also displayed significant differences to the M3 receptor since it displayed high affinity for AF-DX 116 (pK _i = 7.1) and methoctramine (pK _i = 8.4). Finally, chicken heart muscarinic receptors displayed high affinity for gallamine (pK _i = 7.0) and pirenzepine suggesting that the receptor was different to the M4 muscarinic receptor of the NG108-15 cell line. These findings suggest that chicken heart expresses a novel muscarinic receptor subtype distinct from the M1, M2, M3, and M4 subtypes already described. Send offprint requests to A. Michel.     

Inhibitory prejunctional muscarinic receptors at sympathetic nerves do not operate through a cyclic AMP dependent pathway

Summary In mouse atria previously incubated with [³H]-noradrenaline, carbachol (1.0 μmol/l) significantly inhibited the fractional stimulation-induced (S-I) outflow of radioactivity. The inhibitory effect of carbachol was greater in the presence of the α-adrenoceptor antagonist phentolamine (1.0 μmol/l), which by itself significantly increased the S-I outflow of radioactivity. In both cases the inhibitory effect of carbachol was blocked by atropine (0.3 μmol/l), suggesting that the effect was mediated through muscarinic receptors. 8-Bromo cyclic AMP (270 μmol/l) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX,100 μmol/l), was used to maximally enhance the S-I outflow of radioactivity through the cyclic AMP mechanism. The inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was not reduced in the presence of 8-bromo cyclic AMP and IBMX. Similar results with carbachol in the presence of 8-bromo cyclic AMP and IBMX were also found in rat right atrial strips which had been incubated with [3H]-noradrenaline. These results suggest that the effects through inhibitory prejunctional muscarinic receptors are not mediated by cyclic AMP. The protein kinase inhibitor, staurosporine (0.1 μmol/l), significantly blocked the enhancing effects of 8-bromo cyclic AMP (270 μmol/l) plus IBMX (100 μmol/l) on the S-I outflow of radioactivity from rat atrial strips. The inhibitory effect of carbachol (1.0 μmol/l) however, was not reduced in the presence of staurosporine, suggesting that protein kinases affected by staurosporine (protein kinase A, protein kinase C) are not involved in the postreceptor mechanism for inhibitory prejunctional muscarinic receptors. This finding further rules out the involvement of cyclic AMP in muscarinic inhibition. The inhibitory effect of carbachol either by itself or in the presence of phentolamine, was not reduced in atria from mice that had been pretreated with pertussis toxin (1.5 or 3.0 μg). Furthermore, in rat atrial strips, the inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was also not altered by pretreating the rats with pertussis toxin (8.4 μg). The results suggest that in both tissues the major mechanism for inhibition of noradrenaline release through muscarinic receptors does not involve a pertussis toxin sensitive G protein. Send offprint requests to M. Costa at the above address     

Mixed nicotinic–muscarinic properties of the α9 nicotinic cholinergic receptor

The rat α9 nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus laevis oocytes and tested for its sensitivity to a wide variety of cholinergic compounds. Acetylcholine (ACh), carbachol, choline and methylcarbachol elicited agonist-evoked currents, giving maximal or near maximal responses. Both the nicotinic agonist suberyldicholine as well as the muscarinic agonists McN-A-343 and methylfurtrethonium behaved as weak partial agonists of the receptor. Most classical cholinergic compounds tested, being either nicotinic (nicotine, epibatidine, cytisine, methyllycaconitine, mecamylamine, dihydro-β-erythroidine), or muscarinic (muscarine, atropine, gallamine, pilocarpine, bethanechol) agonists and antagonists, blocked the recombinant α9 receptor. Block by nicotine, epibatidine, cytisine, methyllycaconitine and atropine was overcome at high ACh concentrations, suggesting a competitive type of block. The present results indicate that α9 displays mixed nicotinic–muscarinic features that resemble the ones described for the cholinergic receptor of cochlear outer hair cells (OHCs). We suggest that α9 contains the structural determinants responsible for the pharmacological properties of the native receptor.     

Inhibition by ethanol of excitatory amino acid receptors and nicotinic acetylcholine receptors at rat locus coeruleus neurons

The frequency of spontaneous action potentials of locus coeruleus (LC) neurons was recorded extracellularly in pontine slices of the rat brain. Ethanol (1–100 mM) elevated the firing rate in most neurons; this effect was concentration-dependent. (S)--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 0.03–1 M), kainate ( 0.1–3 M), N-methyl-D-aspartate (NMDA; 1–30 M), substance P 0.01–1 M, nicotine 0.1–10 M and ,-methylene ATP ,-meATP; 0.3–30 M, all increased the firing. Application of ethanol g10–100 mM to the superfusion medium for 10 min, reproducibly and concentration-dependently inhibited the facilitatory effect of NMDA g10 M. However, the inhibitory effect of ethanol (100 mM) decreased during a 30-min superfusion period and after the washout of ethanol the sensitivity of LC neurons to NMDA (10 M) tended to overshoot above their initial level. Although NMDA was more potent in the absence than in the presence of external Mg²⁺, ethanol (100 mM) continued to depress the facilitatory effect of a low concentration of NMDA (EM) in a Mg²⁺-free medium. By contrast, in a medium containing normal Mg²⁺, ethanol (100 mM) failed to significantly interfere with the increase in firing rate induced by a high concentration of NMDA (30 M). The effects of kainate (0.5 M), AMPA (0.3 M) and nicotine (1 M) were also depressed by ethanol (100 M), while the effects of substance P (0.03 M) and ,-meATP (30 M) were not changed. In conclusion, ethanol selectively counteracts the opening of cationic channels caused by excitatory amino acid (EAA) receptor agonists and nicotinic acetylcholine receptor agonists. During a longer lasting incubation with ethanol, the inhibition of the NMDA-induced excitatory effect declines, indicating the development of tolerance. Correspondence to: P. Illes at the above address     

Potentiation of sympathetic neuromuscular transmission mediated by muscarinic receptors in guinea pig isolated vas deferens

In guinea-pig isolated vasa deferentia, purinergic neurogenic contractions and responses to applied adenosine 5-triphosphate (ATP) were potentiated by carbachol; responses to adrenergic transmission and applied noradrenaline were not. Following blockade of P2 receptors and -adrenoceptors, the residual neurogenic response was massively potentiated by carbachol, suggesting the presence of a non-purinergic, non-adrenergic component. In the presence of guanethidine, carbachol had no significant effect, indicating that sympathetic transmission was the only element involved. Use of oxotremorine and selective muscarinic receptor antagonists suggested that the potentiating effect of carbachol and oxotremorine was mediated via M3 muscarinic receptors without involvement of nicotinic receptors.     

Nicotinic and muscarinic cholinergic receptors coexist on GABAergic nerve endings in the mouse striatum and interact in modulating GABA release

Muscarinic cholinergic receptors (mAChRs) and nicotinic cholinergic receptors (nAChRs) regulating GABA release from striatal nerve endings were studied by monitoring release of previously accumulated [³H]GABA or endogenous GABA from superfused mouse striatal synaptosomes. Oxotremorine inhibited the release of [³H]GABA elicited by depolarization with 4-aminopyridine (4-AP), an effect antagonized by atropine. Agonists at nAChRs, including the α₄β₂^∗ subunit-selective RJR2403, provoked the release of [³H]GABA as well as of the endogenous transmitter; these effects also were prevented by oxotremorine and pilocarpine suggesting coexpression of functional mAChRs and α₄β₂^∗ nAChRs on GABAergic nerve endings. The inhibitory effects of oxotremorine on the release of [³H]GABA evoked by 4-AP or by RJR2403 were: (i) prevented by the M₂/M₄ mAChR antagonist himbacine; (ii) insensitive to the M2 antagonist AFDX116; (iii) blocked by the selective M₄ mAChR antagonists MT3, thus indicating the involvement of receptors of the M₄ subtype. In conclusion, in the corpus striatum, acetylcholine released from cholinergic interneurons can activate α₄β₂^∗ nAChRs mediating release of GABA; this evoked release can be negatively modulated by M₄ mAChRs coexpressed on the same GABAergic terminals.     

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     

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     

Properties of the muscarinic cholinergic receptors in rat atrium

Summary 1. Properties of the muscarinic cholinergic receptor sites in the rat atrial homogenate and microsomal fraction were studied by the use of tritium labelled 3-quinuclidinyl benzilate ([³H]-QNB), a potent muscarinic antagonist. 2. The specific [³H]-QNB binding to the receptor sites displayed saturability, stereospecificity as well as reversibility. 3. The competition studies showed that muscarinic antagonists were more potent than muscarinic agonists. 4. Certain neuromuscular blocking agents, antipsychotics, antiarrhythmics and antihistamines also were capable of interacting with the [³H]-QNB binding sites. However, - and -adrenergic agents, calcium antagonist (D-600) and ionophore (A-23187) failed to show any effect. 5. Analyses by the double reciprocal plot, Hill plot and Scatchard plot of the dependence of the specific [³H]-QNB binding on the concentration of QNB suggested that binding was occurring to a single population of receptor sites in the atrial homogenate or microsomal fraction. Further, there was no evidence of any detectable site to site interactions (positive or negative cooperative type). From the Scatchard plot, the equilibrium dissociation constant (K_D) of 1.1 nM was calculated and the Hill coefficient was close to 1.0 6. Interaction of the muscarinic antagonists with the [³H]-QNB sites showed the Hill coefficients close to 1.0 whereas for the agonists, the coefficients were much less than 1.0 indicating that agonist-receptor site interactions have some different characteristics from those following antagonist-receptor site interaction. 7. The rate and the maximal level of QNB binding to the receptor sites was markedly influenced by the temperature; various cations, on the other hand, displayed no effect either on the association or dissociation of QNB binding. The specific QNB binding exhibited a broad pH optimum from pH 6.0–8.5. 8. Treatment of the membrane fraction (or homogenate) with either phospholipase A or C and with p-chloromercuribenzoate caused significant inhibition of [³H]-QNB binding. 9. The QNB binding site was resistant to tryptic digestion. Even when about 40% of the membrane proteins were removed by the tryptic proteolysis, the [³H]-QNB binding ability of the membrane remained unaffected; in fact, the removal of tryptic proteolytic products by centrifugation markedly increased the specific QNB binding to the membrane.     

Effects of nicotinic and muscarinic compounds on biting attack in the cat.

Predatory-like biting attack on a rat, as well as hissing, growling, and other threat behaviors, could be induced in normally non-aggressive cats by systemic administration of the muscarinic agonist, arecoline (7-12 mg/kg). In contrast to arecoline, nicotine was found to suppress aggressive behaviors. Systemic administration of nicotine (0.5 mg/kg) prior to arecoline injection resulted in a significant reduction in elicited attack and threat behaviors. Furthermore, nicotine (0.075-0.500 mg/kg) was found to produce a dose-dependent suppression of natural predatory behavior as well. This nicotine-produced suppression of attack did not appear to be due to the induction of general malaise, since attack suppression could be seen in the absence of general behavioral inhibition, and doses of nicotine resulting in complete suppression of attack had little effect on food intake. Results indicate that muscarinic and nicotinic compounds can exert antagonistic control over some types of aggressive behaviors.     

Central activation of the sympathetic nervous system including the adrenals in anaesthetized guinea pigs by the muscarinic agonist talsaclidine

Talsaclidine, a novel M₁-receptor selective muscarinic agonist for cholinergic substitution therapy of Alzheimer’s disease, activates the sympathetic nervous system in guinea pigs and dogs at the orthosympathic ganglia and the paraganglionic adrenals. Results from guinea pigs provide indirect evidence for an additional central site of action. The present investigation in anaesthetized and vagotomized guinea pigs intended to demonstrate central activation of the sympathetic nervous system directly by comparing the blood pressure effects of intracerebroventricular and intravenous injections of small doses of talsaclidine. Increasing doses of 0.2 and 0.6mg/kg talsaclidine were injected alternately into the third cerebral ventricle and intravenously in 6 guinea pigs before and after blockade of peripheral muscarinic receptors with 1mg/kg ipratropium bromide i.v. In another group of 6 animals the injections were given into the cisterna cerebellomedullaris using the same protocol. In both groups central administration of talsaclidine caused dose-related hypertension while intravenous injections were hypotensive. Ipratropium bromide, a peripheral antimuscarinic drug, reversed this hypotensive action of intravenous talsaclidine into hypertension, but did not inhibit the effects of central administration. In contrast, atropine, an antimuscarinic drug which passes the blood-brain barrier, abolished the effect of 0.6mg/kg talsaclidine injected into the cisterna cerebellomedullaris of 8 guinea pigs. The hypertensive effect of a first injection of 0.6mg/kg talsaclidine into the cisterna cerebellomedullaris of 6 guinea pigs was approximately twice as large as that of a second given 90min after bilateral adrenalectomy. Sham operation in another 6 animals was not inhibitory. The results demonstrate that talsaclidine, a selective muscarinic M₁-receptor agonist, activates central parts of the sympathetic nervous system, including central projections of the adrenals by an action mediated by central muscarinic receptors. Received: 11 December 1997/ Accepted: 20 January 1998     

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.     

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     

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.     

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     

Comparison of the muscarinic receptors in the coronary artery,cerebral artery and atrium of the pig

Summary The affinity of various muscarinic antagonists for the muscarinic receptors mediating contraction (induced by acetyl-\-methylcholine) of the isolated pig coronary and basilar artery was determined in order to compare the muscarinic receptor subtype involved in the contractile response of these arteries. In order to identify the muscarinic receptor subtype(s) involved, the affinity of the antagonists for the M₂ receptor present in the pig atria was also investigated. The following muscarinic antagonists were used: atropine, pirenzepine, AF-DX 116 (11-2{{2-{(diethyl-amino)methyl} -1- piperidinyl}acetyl} - 5, 11- dihydro - 6H - pyrido {2, 3 - b} {, 4}benzodiazepin - 6 - one), 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide), HHSiD (hexahydrosiladifenidol), methoctramine (N, N- bis{6 - {(2 - methoxybenzyl)amino} hexyl} -1, 8 - octane - diamine tetrahydrochloride) and ipratropium.The order of affinity of the antagonists with respect to the muscarinic receptor in the coronary artery was clearly different from that for the muscarinic receptor in the basilar artery. The order of affinity established on the basilar artery closely resembled that for the M2 receptor in the atria.It is concluded that the muscarinic receptors on smooth muscle of the coronary and basilar arteries are not identical. The muscarinic receptor involved in the contraction of the basilar artery adheres to the M2 receptor subtype. A comparison of the selectivity of the antagonists suggests that the muscarinic receptor involved in the contraction of the coronary artery belongs to the M₃ (like in exocrine glands) or M₄ (as found in ileal smooth muscle) receptor subtype.     

Transmitter release and adrenergic mechanical responses in the heart: effect of papaverine and imidazole.

Chronotropic and inotropic responses were elicited in isolated rabbit hearts by stimulation of the sympathetic nerves or by infusion of noradrenaline or adrenaline and the effects of papaverine and imidazole (10(-7)-10(-6) M) on these responses were studied. The outflow of noradrenaline induced by sympathetic nerve stimulation was assayed in the absence and in the presence of papaverine and imidazole (10(-7)-5 X 10(-7) M). Papaverine increased the outflow of transmitter during nerve stimulation by 45% and potentiated both the chronotropic and inotropic responses induced by nerve stimulation and those induced by infusion of catecholamines. Imidazole inhibited the outflow of transmitter during nerve stimulation by 33%. The data indicate that the "second messenger" cyclic AMP is active in more than one step in adrenergic neurotransmission and receptor activation in the heart. Furthermore, tissue cyclic AMP seems to be involved not only in the inotropy induced by circulating catecholamines but also in the more "physiological" inotropy elicited by sympathetic nerve stimulation.     

Fear conditioning and latent inhibition in mice lacking the high affinity subclass of nicotinic acetylcholine receptors in the brain

Nicotine can enhance performance in several tests of cognition but the specific nicotinic receptor subtypes mediating these effects are largely unknown. Knock out mice lacking the β2 subunit of the nicotinic receptor were evaluated in fear conditioning and latent inhibition tasks to begin to determine which receptor subtypes mediate the cognitive effects of nicotine. Young (2–4 months) knock out and wild type mice did not differ in either contextual or tone-conditioned fear, but aged (9–20 months) knock out males were impaired in freezing to both context and tone compared to aged wild type males. No differences in fear conditioning were observed between aged knock out and wild type females. Latent inhibition of fear to a pre-exposed tone, as measured by behavioral freezing, was also assessed. Both knock out and wild type mice displayed similar levels of latent inhibition, although overall levels of freezing were lower in knock out mice. These results support a previous study showing spatial learning deficits in aged β2 subunit knock out mice [EMBO J. 18 (1999) 1235] and suggest that performance of other cognitive tasks may not be influenced by absence of β2 subunit-containing receptors.