Muscarinic modulation of endogenous noradrenaline release from adrenergic terminals in the guinea-pig colon

1 The present study examined the role of muscarinic receptors in the modulation of noradrenaline (NA) release in the guinea-pig isolated distal colon. The spontaneous endogenous NA overflow assayed by HPLC-ED was taken as an index of NA release from enteric noradrenergic nerve terminals. 2 Physostigmine (10 microM) significantly enhanced spontaneous endogenous NA overflow. Hyoscine (muscarinic antagonist), (R)-(-)-trihexyphenidyl and telenzepine (M1-selective antagonists), and 11[[2-[(diethylamino)methyl]-1-piperydil]acetyl]-5,11 -dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116, M2-selective antagonist) inhibited NA overflow in a concentration dependent manner, with the following EC50 values: 131.74 (18.19-953.96), 101.62 (58.83-175.60), 150 (60-330), 30 (5-170) nM, respectively. 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M1- and M3-selective antagonist) had no significant effect up to 100 microM. 3 The muscarinic agonist oxotremorine inhibited NA overflow in a concentration dependent manner, with an EC50 value of 0.67 (0.30-1.51) microM. The response to oxotremorine was inhibited by muscarinic antagonists with the following order of potency: hyoscine = (R)-(-)-trihexyphenidyl = telenzepine > 4-DAMP > AF-DX 116. 4 In the presence of 3 microM tetrodotoxin (TTX), the effect of oxotremorine and 4-DAMP was unchanged, while hyoscine, (R)-(-)-trihexyphenidyl, telenzepine and AF-DX 116, instead of inhibiting, significantly enhanced NA overflow. 5 The present results indicate that, in the guinea-pig colon, endogenous acetylcholine sustains spontaneous NA release by activating muscarinic receptors possibly located on interneurones. In addition, inhibitory muscarinic receptors may exist on adrenergic terminals.     

In vitro electro-pharmacological and autoradiographic analyses of muscarinic receptor subtypes in rat hypothalamic ventromedial nucleus: implications for cholinergic regulation of lordosis

Muscarinic agonists can act through the hypothalamic ventromedial nucleus (VMN) to facilitate lordosis. To elucidate the neuronal mechanism(s) underlying this muscarinic facilitation, effects of muscarinic agents on the single-unit activity of VMN neurons recorded in brain tissue slices of estrogen-primed female rats were analyzed. All the agonists tested, including acetylcholine (ACh), oxotremorine-M (OM), carbachol (CCh) and McN-A-343 (McN), evoked primarily excitation (80-100%), some inhibition (0-20%) and occasional biphasic responses (0-8%). By comparing the response magnitude and the effectiveness in evoking a response, the rank order for evoking excitation, the primary response, was found to be: OM > CCh > ACh approximately McN, which is consistent with that (OM > CCh > McN) for facilitating lordosis reported by others. This consistency and the frequency of its occurrence suggest that the excitatory electric action of the muscarinic agonists is related to their facilitatory behavioral effect. Experiments with antagonists selective for M1 (pirenzepine), M2 (AF-DX 116) and M3 (4-DAMP and p-F-HHSiD) indicate that muscarinic excitations are mediated by M1 and/or M3, but not M2. Since M1 receptors have been shown to be neither sufficient nor necessary to mediate the muscarinic facilitation, M3 receptor may be crucially involved in this behavioral effect. Autoradiographic assays of binding to [3H]4-DAMP with or without pirenzepine and AF-DX 116, also indicate the presence of M3 receptors in the VMN. Quantitative analyses show that the M3 binding was not affected by the in vivo estrogen priming required to permit muscarinic agonists to facilitate lordosis. Thus, while the excitation mediated by M3 is likely to be involved in muscarinic facilitation of lordosis, the regulation of M3 receptor density does not seem to be involved in the permissive     

The use of controlled-release oxycodone for the treatment of chronic cancer pain: a randomized, double-blind study

1. We have studied the effects of muscarinic cholinoceptor agonists and subtype-preferring antagonists on the isometric contraction of smooth muscle strips from dog prostate. 2. Acetylcholine and carbachol induced contraction of prostate strips from the peripheral zone, ('the capsule'). Bethanechol contracted the tissue but not at lower doses. McN-A-343 and oxotremorine-M showed the same effects. 3. Blocking alpha- and beta-adrenoceptors with phentolamine and propranolol, respectively, did not modify carbachol-induced contractions. 4. The nicotinic receptor blocker, hexamethonium (10(-6)-10(-4) M) did not affect the contractile response evoked by a single dose of carbachol (10(-5) M), whilst the muscarinic receptor antagonist, atropine (10(-11)-10(-9) M), inhibited it in a competitive manner. 5. The muscarinic M1 (pirenzepine), M2 [AF-DX 116, himbacine (M2/M4) and methoctramine], M3 (HHSID and f-F-HHSID), and putative M4 (tropicamide) antagonists reduced significantly the carbachol-induced contractions. The pIC50 values were: atropine (10.01) > himbacine (8.3) > methoctramine (7.85) > AF-DX 116 (7.60) > HHSID (7.21) > p-F-HHSID (7.10) > pirenzepine (7.30) > tropicamide (7.00). 6. The antagonist profile indicates that an predominant M2 receptor subtype could mediate the muscarinic contraction in the canine prostate.     

Neuromuscular facilitation induced by muscarinic antagonists in the rat isolated diaphragm

1. Atropine (EC50 = 87 microM), pirenzepine (447 microM), and AF-DX 116 (95.5 microM), but not 4-DAMP (at concentrations of up to 110 microM), produced neuromuscular facilitation and antagonized the oxotremorine-induced neuromuscular blockade in the rat isolated diaphragm. 2. Atropine, pirenzepine, and AF-DX 116 did not change the responses of curarized diaphragms to direct stimulation, or the twitch tension produced by retrograde injection of acetylcholine. 3. These results indicate that neuromuscular facilitation induced by muscarinic antagonists may depend on drug interaction with the M2 subtype of muscarinic autoreceptors to increase acetylcholine output in the neuromuscular junction.     

NO does not mediate inhibitory neural responses in sheep airway and bronchial vascular smooth muscle

Endogenous nitric oxide (NO) influences acetylcholine-induced bronchovascular dilation in sheep and is a mediator of the airway smooth muscle inhibitory nonadrenergic, noncholinergic neural response in several species. This study was designed to determine the importance of NO as a neurally derived modulator of ovine airway and bronchial vascular smooth muscle. We measured the response of pulmonary resistance (RL) and bronchial blood flow (Qbr) to vagal stimulation in 14 anesthetized, ventilated, open-chest sheep during the following conditions: 1) control; 2) infusion of the alpha-agonist phenylephrine to reduce baseline Qbr by the same amount as would be produced by infusion of Nomega-nitro-L-arginine (L-NNA), a NO synthase inhibitor; 3) infusion of L-NNA (10(-2) M); and 4) after administration of atropine (1.5 mg/kg). The results showed that vagal stimulation produced an increase in RL and Qbr in periods 1, 2, and 3 (P < 0.01) that was not affected by L-NNA. After atropine was administered, there was no increase in Qbr or RL. In vitro experiments on trachealis smooth muscle contracted with carbachol showed no effect of L-NNA on neural relaxation but showed a complete blockade with propranolol (P < 0.01). In conclusion, the vagally induced airway smooth muscle contraction and bronchial vascular dilation are not influenced by NO, and the sheep's trachealis muscle, unlike that in several other species, does not have inhibitory nonadrenergic, noncholinergic innervation.     

Exocytotic release from neuronal cell bodies, dendrites and nerve terminals in sympathetic ganglia of the rat, and its differential regulation

Stimulant-induced exocytosis has been demonstrated in sympathetic ganglia of the rat by in vitro incubation of excised ganglia in the presence of tannic acid, which stabilizes vesicle cores after their exocytotic release. Sites of exocytosis were observed along non-synaptic regions of the surfaces of neuron somata and dendrites, including regions of dendrosomatic and dendrodendritic apposition, as well as along the surfaces of nerve terminals About half the exocytoses associated with nerve terminals were parasynaptic or synaptic, and these appeared mostly to arise from the presynaptic terminal, but occasionally from the postsynaptic element. The results demonstrated that the neurons of sympathetic ganglia release materials intraganglionically in response to stimulation, that release from different parts of the neuron is subject to independent regulation, at least via cholinergic receptors, and that release is partly diffuse, potentially mediating autocrine or paracrine effects, and partly targeted toward other neurons, but that the latter mode is not necessarily, and not evidently, synaptic. Specifically, exocytosis from all locations increased significantly during incubation in modified Krebs' solution containing 56 nm potassium. Observation of the effects of cholinergic agonists (nicotine, carbachol, oxotremorine) and antagonists (atropine, AF-DX 116) showed that nicotinic and muscarinic excitation each, independently, increased the incidence of exocytosis from somata and dendrites. Exocytosis from nerve endings was not altered by nicotine, but was enhanced or, at high initial rates of exocytosis, decreased, by muscarinic stimulation. Evidence was obtained for muscarinic auto-inhibition of exocytosis from nerve terminals, occurring under basal incubation conditions, and for a muscarinic excitatory component of somatic exocytosis, elicitable by endogenous acetylcholine. The M2-selective muscarinic antagonist AF-DX 116 was found to modify the exocytotic response of the dendrites to oxotremorine, widening the range of its variation; this effect is consistent with recent evidence for the presence of M2-like muscarinic binding sites, in addition to M1-like binding, upon these dendrites [Ramcharan E. J. and Matthews M. R. (1996) Neuroscience 71, 797-832]. Over all conditions, disproportionately more sites of somatic and dendritic exocytosis were found to be located in regions of dendrosomatic and dendrodendritic apposition than would be expected from the relative extent of the neuronal surface occupied by these relationships. Such mechanisms of intraganglionic release may be expected to contribute to the regulation and integration of the behaviour of the various functionally distinctive populations of neurons in these ganglia, by autocrine, paracrine, and focal, neuroneuronal, routes of action. Similar phenomena of exocytotic soma-dendritic release might prove to subserve integrative neuroneuronal interactions more widely throughout the nervous system.     

Muscarinic receptor subtype involvement in brain cholinergic stimulation by intracerebroventricular neostigmine in sinoaortic denervated rats

1. The present studies evaluated the participation of central muscarinic receptors in the cardiovascular effects of centrally injected neostigmine, a quaternary anticholinesterase, in conscious, sham-operated rats and in sinoaortic denervated animals. 2. The dose-dependent pressor effect of neostigmine (0.1 to 1 microg i.c.v.) was greater in sinoaortic denervated rats than in sham-operated animals, but only a dose-dependent bradycardic effect was seen in sham-operated rats. 3. Doses of 3.3 nmol (i.c.v.) of both the M1 muscarinic antagonist, pirenzepine, and the M3 muscarinic antagonist, 4-DAMP, prevented the pressor response to 1 microg of neostigmine in sham-operated rats and in sinoaortic denervated animals; however, the M2 muscarinic antagonist, AF-DX116, partially blocked this response in sham-operated rats while failing to do so in sinoaortic denervated rats. In sham rats, doses of 3.3 nmol (i.c.v.) of both pirenzepine and 4-DAMP prevented the bradycardic response to 1 microg (i.c.v.) of neostigmine, whereas AF-DX116 induced a partial blockade. 4. 4-DAMP, at the dose of 0.3 nmol (i.c.v.), but not pirenzepine at the same dose, prevented the pressor effect of neostigmine (0.1 to 1 microg i.c.v.) in both groups of rats. Both muscarinic antagonists at this dose prevented the bradycardia elicited by the anticholinesterase (0.1 to 1 microg i.c.v.), but 4-DAMP showed a greater antagonistic action on this cardiac effect than pirenzepine. In sham-operated rats, i.c.v. injection of 0.3 nmol of AF-DX116 failed to modify the cardiovascular responses to 0.3 microg of neostigmine. 5. Results suggest mainly an involvement of brain M3-subtype muscarinic receptors in the cardiovascular effect of intracerebroventricular administration of anticholinesterase neostigmine in both groups of rats.     

Identification and characterization of muscarinic receptors potentiating the stimulation of adenylyl cyclase activity by corticotropin-releasing hormone in membranes of rat frontal cortex

In membranes of the rat frontal cortex, acetylcholine (ACh) and other cholinergic agonists were found to potentiate the stimulation of adenylyl cyclase activity elicited by corticotropin-releasing hormone (CRH). Oxotremorine-M, carbachol and methacholine were as effective as ACh, whereas oxotremorine and arecoline were much less effective. The facilitating effect of Ach was potently blocked by the M1 antagonists R-trihexyphenidyl, telenzepine and pirenzepine and by the M3 antagonists hexahydro-sila-difenidol and p-fluorohexahydro-sila-difenidol, whereas the M2 and M4 antagonists himbacine, methoctramine, AF-DX 116 and AQ-RA 741 were less potent. The mamba venom toxin MT-1, which binds with high affinity to M1 receptors, was also a potent blocker. The pharmacological profile of the muscarinic potentiation of CRH receptor activity was markedly different from that displayed by the muscarinic inhibition of forskolin-stimulated adenylyl cyclase, which could be detected in the same membrane preparations. Moreover, the intracerebral injection of pertussis toxin impaired the muscarinic inhibition of cyclic AMP formation and reduced the Ach stimulation of [35S]GTPgammaS binding to membrane G proteins but failed to affect the facilitating effect on CRH receptor activity. The latter response was also insensitive to the phospholipase C inhibitor U-73122, the protein kinase inhibitor staurosporine and to the inhibitors of arachidonic acid metabolism indomethacin and nordihydroguaiaretic acid. These data demonstrate that in the rat frontal cortex, muscarinic receptors of the M1 subtype potentiate CRH transmission by interacting with pertussis toxin-insensitive G proteins.     

Muscarinic M1 receptor mediated inhibition of GABA release from rat cerebral cortex

Presynaptic modulation of [3H]GABA release was examined using rat cerebral cortical slices. In vitro addition of carbachol, a muscarinic receptor agonist, resulted in a significant suppression of the release of [3H]GABA evoked by high potassium (50 mM) stimulation in a dose dependent manner, while noradrenaline, isoproterenol, dopamine, 5-hydroxytryptamine, histamine and glutamic acid had no significant effect on the evoked release of [3H]GABA. This suppressive effect of carbachol was antagonized invariably by atropine. Furthermore, it was found that the suppressive action of carbachol could be antagonized by pirenzepine, a selective M1 muscarinic receptor antagonist, but not by AF-DX 116 and 4-DAMP, M2 and M3 receptor antagonists, respectively. These results suggest that the release of GABA from cerebral cortical GABA neurons may be modulated by presynaptic M1 muscarinic receptor.     

Pharmacological profiles of the subtypes of muscarinic cholinoceptors that mediate aggregation of pigment in the melanophores of two species of catfish

Using selective antagonists, including pirenzepine, adiphenine, AF-DX 116, gallamine, and 4-DAMP, we attempted to characterize the muscarinic cholinoceptors on the melanophores of the translucent glass catfish Kryptopterus bicirrhis and the mailed catfish Corydoras paleatus. The M3 receptor-selective antagonist, 4-DAMP, potently inhibited the acetylcholine-induced aggregation of pigment in both species. It appeared, therefore, that the receptors that mediated the cholinergically evoked aggregation of melanosomes in these species were of the M3 muscarinic subtype.     

Pharmacological characterization of the muscarinic receptor subtype mediating contraction of human peripheral airways

The postjunctional muscarinic receptors mediating contraction of human bronchial smooth muscle have been characterized using four nonselective muscarinic receptor agonists and eight subtype selective and nonselective muscarinic antagonists. Carbachol, methacholine, oxotremorine M and (+)-cis-dioxolane all caused concentration-related contractions of human bronchial smooth muscle with a rank order of potency (pD2) of (+)-cis-dioxolane (7.3 +/- 0.2) > oxotremorine M (6.7 +/- 0.2) > carbachol (6.4 +/- 0.1) > methacholine (5.8 +/- 0.2, n = 5 for all). Maximum contractions were not significantly different between agonists, whether expressed as absolute my tension changes or as a percentage of the maximum response to 0.3 mM histamine. Antagonist apparent affinities (pKB) were determined against carbachol-induced contractions and the following rank order was obtained; 4-DAMP (9.4 +/- 0.3) > or = atropine (9.1 +/- 0.1) > zamifenacin (7.6 +/- 0.1) > hexahydrosiladifenidol (HHSiD; 7.1 +/- 0.1) > or = himbacine (7.0 +/- 0.3) > or = pirenzepine (6.8 +/- 0.2) > para-fluoro-hexahydrosiladifenidol (p-F-HHSiD; 6.7 +/- 0.1) > methoctramine (5.3 +/- 0.2). This rank order of antagonist affinities is consistent with activation of M3 receptors. The affinities of HHSiD, p-F-HHSiD and zamifenacin were, however, lower than those reported in guinea pig trachea.     

Characterization of muscarinic receptors mediating the contraction of the urinary detrusor muscle in cynomolgus monkeys and guinea pigs

We have characterized in vitro the muscarinic receptors mediating the contraction of the detrusor muscle in Cynomolgus monkeys and guinea pigs using carbachol as the agonist and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M3-selective), methoctramine (M2-selective) and pirenzepine (M1-selective) as the antagonists. Carbachol induced a concentration-dependent contraction of the detrusor muscle of monkey and guinea pig yielding similar pD2 values of 6.67+/-0.03 (n=50) and 6.77+/-0.06 (n=36), respectively. In the detrusor muscle of Cynomolgus monkey, all antagonists produced a concentration-dependent inhibition of carbachol-induced contractions, without decreasing the maximal response. Schild plot analysis yielded slopes not different from unity for all antagonists. The order of antagonist potency was: 4-DAMP (pA2=8.96)>pirenzepine (pA2=6.66)>methoctramine (pA2=6.03), suggesting that M3 receptors have a dominant role in mediating detrusor contraction. In the detrusor muscle of the guinea pig, 4-DAMP and pirenzepine, but not methoctramine, produced a concentration-dependent inhibition of the carbachol-induced contractions, without decreasing the maximal response. Schild plot analysis yielded a slope not different from unity for 4-DAMP and pirenzepine. 4-DAMP (pA2=9.07) had a higher potency than pirenzepine (pA2=6.66), a finding consistent with previously published data. The present study shows that in Cynomolgus monkey stimulation of the M3 subtype is dominant in mediating detrusor contraction upon carbachol stimulation.     

Higher touch and kinesthetic functions in children with former speech/language development disorders: a neuropsychological study

1. The affinities of 10 selective muscarinic receptor antagonists against [3H]-quinuclidinyl benzilate (QNB) binding were determined to characterize the muscarinic receptors present in guinea-pig gallbladder smooth muscle. The highest correlation was obtained for the comparison between the pKi values for the gallbladder smooth muscle and M2 sites. Pirenzepine revealed two binding sites with affinities indicating the presence of muscarinic M2 receptors in abundance and a minor population of an additional site(s). 2. Carbachol produced gallbladder contractions, stimulated phosphoinositide (PI) hydrolysis and inhibited cAMP formation concentration-dependently with pD2 values of 6.12 +/- 0.11, 5.18 +/- 0.33 and 7.19 +/- 0.15, respectively. 3. Pirenzepine, 4-DAMP, HHSiD, pF-HHSiD, AF-DX 116, methoctramine, AQ-RA 741, guanylpirenzepine and AF-DX 384 showed competitive antagonism against carbachol-induced gallbladder contractions. There was no correlation between the pA2 values for the gallbladder and pKi values for the M2 sites, whereas significant correlations were found for the M1, M3 and M4 sites, the best correlation being between the pA2 values for the gallbladder and M4 subtypes. 4. Finally, the presence of both m2 and m4 receptor proteins were demonstrated by Western blot analysis. It is concluded that guinea-pig gallbladder smooth muscle has both muscarinic M2 and M4 receptors, which are coupled to adenylate cyclase inhibition and PI hydrolysis. 5. Although it seems likely that M2 receptors do not play a primary role in carbachol-induced guinea-pig gallbladder contraction, the characterization of the muscarinic subtypes which mediate these contractile responses needs further evidence.     

Inhibition of nitric oxide synthase slows heart rate recovery from cholinergic activation

The role of nitric oxide (NO) in the cholinergic regulation of heart rate (HR) recovery from an aspect of simulated exercise was investigated in atria isolated from guinea pig to test the hypothesis that NO may be involved in the cholinergic antagonism of the positive chronotropic response to adrenergic stimulation. Inhibition of NO synthesis with NG-monomethyl-L-arginine (L-NMMA, 100 micro M) significantly slowed the time course of the reduction in HR without affecting the magnitude of the response elicited by bath-applied ACh (100 nM) or vagal nerve stimulation (2 Hz). The half-times (t1/2) of responses were 3.99 +/- 0.41 s in control vs. 7. 49 +/- 0.68 s in L-NMMA (P < 0.05). This was dependent on prior adrenergic stimulation (norepinephrine, 1 micro M). The effect of L-NMMA was reversed by L-arginine (1 mM; t1/2 4.62 +/- 0.39 s). The calcium-channel antagonist nifedipine (0.2 micro M) also slowed the kinetics of the reduction in HR caused by vagal nerve stimulation. However, the t1/2 for the reduction in HR with antagonists (2 mM Cs+ and 1 micro M ZD-7288) of the hyperpolarization-activated current were significantly faster compared with control. There was no additional effect of L-NMMA or L-NMMA+L-arginine on vagal stimulation in groups treated with nifedipine, Cs+, or ZD-7288. We conclude that NO contributes to the cholinergic antagonism of the positive cardiac chronotropic effects of adrenergic stimulation by accelerating the HR response to vagal stimulation. This may involve an interplay between two pacemaking currents (L-type calcium channel current and hyperpolarization-activated current). Whether NO modulates the vagal control of HR recovery from actual exercise remains to be determined.     

M2 muscarinic autoreceptors modulate acetylcholine release in the medial pontine reticular formation

Muscarinic autoreceptors regulate acetylcholine (ACh) release in several brain regions, including the medial pontine reticular formation (mPRF). This study tested the hypothesis that the muscarinic cholinergic receptor mediating mPRF ACh release is the pharmacologically defined M2 subtype. In vivo microdialysis was used to deliver muscarinic cholinergic receptor (MAChR) antagonists to the feline mPRF while simultaneously measuring endogenously released ACh. The lowest concentration of each antagonist that caused a significant increase in mPRF ACh release was determined and defined as the minimum ACh-releasing concentration. Data obtained from 41 mPRF dialysis sites in 10 animals showed that the order of potency (followed by the minimum ACh-releasing concentration) was scopolamine (1 nM) > AF-DX 116 (3 nM) > pirenzepine (300 nM). Comparison of these minimum ACh-releasing concentrations to the known affinities of the antagonists for the five mAChR subtypes is consistent with the conclusion that the autoreceptor regulating mPRF ACh release is the M2 subtype. Considerable evidence supports a role for cholinergic neurotransmission and postsynaptic M2 receptors in the mPRF in regulating levels of arousal. The present data suggest that presynaptic M2 receptors contribute to the regulation of arousal states by modulating mPRF ACh release.     

The use of irreversible ligands to inactivate receptor subtypes: 4-DAMP mustard and muscarinic receptors in smooth muscle

Irreversible ligands are useful tools for investigating the function of receptor subtypes in various physiological processes. The mechanism for alkylation involves the formation of a reversible receptor complex followed by a covalent reaction. The extent of receptor alkylation is determined by the dissociation constant of the reversible complex and the rate constant for conversion to the covalent complex. Selectivity can be achieved if the irreversible ligand exhibits a difference in its dissociation constants for receptor subtypes. Selective alkylation can also be achieved using a selective competitive inhibitor to protect the desired receptor subtype. By using the non-M2-selective irreversible antagonist, 4-DAMP mustard, in combination with the competitive M2-selective antagonist, AF-DX 116, it has been possible to achieve a highly selective inactivation of all non-M2 subtypes of the muscarinic receptors in smooth muscle and has enabled the discovery of the functional role of M2 receptors in smooth muscle.     

Age-related alterations in pre-synaptic and receptor-mediated cholinergic functions in rat brain

Fractional [3H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [3H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [3H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 microM) significantly decreased stimulation-induced fractional [3H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [3H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [3H]ACh release from striatum of both age groups is modulated mainly by M3 muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo-3H]inositol decreased with aging, carbachol-stimulated [3H]myo inositol-1-fosfat (IP1) accumulation was found similar in striatal, cortical and hippocampal slices.     

Stimulation of phosphoinositide hydrolysis by muscarinic receptor activation in the rat olfactory bulb

The effect of muscarinic receptor activation on phosphoinositide hydrolysis in the rat olfactory bulb was investigated by determining either the inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) mass or the accumulation of [3H]inositol phosphates ([3H]InsPs). In miniprisms of rat olfactory bulb, carbachol produced an atropine-sensitive increase in Ins(1,4,5)P3 concentration. In a membrane preparation, the formation of Ins(1,4,5)P3 was stimulated by guanosine-5'-(3-O-thio) triphosphate (GTP gamma S), but not by carbachol. However, carbachol potentiated the GTP gamma S stimulation when the two agents were combined. In miniprisms prelabelled with [3H]myo-inositol, carbachol increased the accumulation of [3H]InsPs and this effect was significantly reduced by tissue treatment with either 1 microM phorbol 12-myristate 13-acetate or 1 mM dibutyryl cyclic AMP. Analysis of concentration-response curves indicated that carbachol (EC50 = 96 microM) and oxotremorine-M (EC50 = 8.2 microM) behaved like full agonists, whereas oxotremorine, BM5, arecoline and bethanechol were partial agonists. The carbachol stimulation of [3H]InsPs accumulation was counteracted with high affinity by the M1 antagonist pirenzepine (pA2 = 8.26), and less potently by the M3 antagonist para-fluorohexahydro-sila-difenidol (pA2 = 6.7) and the M2 antagonist AF-DX 116 (pA2 = 6.12). The biochemical and pharmacological properties of the muscarinic stimulation of phosphoinositide hydrolysis were compared with those displayed by the muscarinic stimulation of adenylate cyclase in the rat olfactory bulb.     

Effects of muscarinic agents on cultured human trabecular meshwork cells

Intracellular calcium measurements were performed in cultured human trabecular meshwork cells preloaded with the cell permeant dye fura 2-AM. Fluctuations in calcium levels were then monitored with microscope-based ratio fluorometry. Carbachol increased intracellular calcium in a dose-dependent manner; as did oxotremorine-M, aceclidine, and pilocarpine. Carbachol's effect was blocked by the non-selective muscarinic antagonist atropine, as well as by muscarinic receptor subtype-selective antagonists such as pirenzepine (M1-selective), p-fHHSiD (M3-selective), and 4-DAMP (M1, M3 subtypes). Rank order of potencies for the antagonists' effects was atropine = 4-DAMP > p-fHHSiD > pirenzepine, a profile suggesting that the M3 receptor subtype is essential in the carbachol effect. Phospholipase C activity was estimated via measurement of total production of inositol phosphates in cultured human trabecular meshwork cells pre-exposed to 3H-myoinositol. In these cells, carbachol also stimulated phosphoinositide production in a dose-dependent manner, and an antagonist profile similar to that seen for calcium response was obtained when carbachol was used as the effector. The data indicate that muscarinic effects on cultured human trabecular meshwork calcium mobilization and phospholipase C activity are mediated by an M3-like receptor subtype. Therefore, the muscarinic M3 receptor may play a role in trabecular meshwork cell function(s).     

Bronchial reactivity: interaction between vagal stimulation and inhaled histamine

The interaction between the effects of vagal stimulation and inhaled histamine on the bronchi was studied in anesthetized dogs. Reactivity was assessed by measuring changes in bronchial caliber visualized with tantalum bronchograms. In seven vagotomized dogs the bronchoconstrictor response to a combination of electrical stimulation of the vagus nerves and inhaled histamine solution produced a mean reduction in airway diameter (Daw) of 2.21 mm which was significantly greater than the additive results of the two stimuli applied separately (mean decrease in Daw 0.29 +/- 0.91 mm). In three dogs the effect of vagal stimulation was to produce a shift in the dose-response curve to inhaled histamine. These results indicate that the effect of the base-line bronchomotor tone must be considered in the evaluation of the effect of vagal blockade on airway reactivity. An increase in the resting degree of bronchomotor tone may contribute to the hyperreactivity observed in patients with asthma.