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.     

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.     

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.     

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.     

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).     

Cytopathic effect, plaque formation, and lysis of Ehrlichia chaffeensis grown on continuous cell lines

The present study describes some unexpected receptor mediated effects of N-methylcarbamylcholine on mouse M1 muscarinic receptor gene transfected cell line (M1Y1) that were not evident from biochemical studies with mouse and rat brain tissue where N-methylcarbamylcholine exhibited only nicotinic properties. Although N-methylcarbamycholine was devoid of muscarinic properties in mouse and rat brain preparations, as determined by phosphoinositide turnover and inhibition of [3H]QNB binding, it exhibited significant muscarinic characteristics in the transfected M1Y1 cell line. At a concentration of 10(-6) M or greater, N-methylcarbamycholine caused a transient increase in intracellular Ca2+ of 50 s duration that was reversible by atropine or pirezepine. The Ca(2+)-transient was not elicited by other nicotinic agents such as nicotine and N,N-dimethylcarbamylcholine, a close analogue of N-methylcarbamylcholine, with comparable affinity for nicotinic receptors and devoid of muscarinic activity. N-Methylcarbamylcholine also stimulated phosphoinositide turnover in M1Y1 cells with an estimated EC50 value 10 times greater than that of carbachol, and the effect was blocked by atropine. Both carbachol and N-methylcarbamycholine inhibited [3H]QNB binding in a concentration-dependent manner; however, the IC50 for carbachol was over two orders of magnitude greater than that observed in mouse and rat brain membranes. In considering possible explanations for the differential characteristics of N-methylcarbamylcholine in mouse and rat brain as compared to the transfected M1Y1 cells, it was concluded that the difference may be attributable to differences in the receptor-transduction coupling efficiency and the microenvironment of the muscarinic receptors.     

Contrasting effects of carbachol, McN-A-343 and AHR-602 on Ca(2+)-mobilization and Ca(2+)-influx pathways in taenia caeci

1. We compared the binding profiles and contractile mechanisms of putative muscarinic M1 agonists McN-A-343 and AHR-602 with those of carbachol in smooth muscle of guinea-pig taenia caeci. 2. McN-A-343 and AHR-602, as well as carbachol, completely displaced the atropine-sensitive binding of [3H]-quinuclidinyl benzilate to muscarinic receptors present in the membrane preparation. The potency order for the affinity of these agents for muscarinic receptors was carbachol > McN-A-343 > AHR-602. 3. In the presence of 2.2 mM extracellular Ca2+, McN-A-343 and AHR-602 induced contraction corresponding to 79 and 85%, respectively, of the maximal contraction to 0.1 mM carbachol. Contractions induced by these agents were mediated via activation of the muscarinic receptor subtype that had a high affinity for 4-DAMP (M3 selective) but a low affinity for pirenzepine (M1 selective) and AF-DX 116 (M2 selective). These contractions were inhibited by an L-type Ca2+ channel blocker, verapamil. 4. In Ca(2+)-free solution containing 2 mM EGTA, carbachol elicited a transient contraction whereas no contraction was observed in response to McN-A-343 and AHR-602. Application of McN-A-343 or AHR-602 inhibited the carbachol-induced contraction in Ca(2+)-free solution, and this inhibition was surmounted by a higher concentration of carbachol. 5. The EC50 value for carbachol-induced contraction in the presence of extracellular Ca2+ was approximately 175 times lower than that in the absence of Ca2+. After treatment with propylbenzilylcholine mustard, carbachol induced contraction only in the presence of extracellular Ca2+. 6. The results suggest that in the taenia caeci there is a greater receptor reserve for muscarinic M3 receptor-mediated Ca2+ influx than for M3 mediated Ca2+ release. The compounds McN-A-343 and AHR-602 are agonists of the Ca2+ influx pathway, but do not appear to stimulate the Ca2+ release pathway.     

3-Alpha-chloro-imperialine, a potent blocker of cholinergic presynaptic modulation of glutamatergic afferents in the rat neostriatum

Cortico-thalamic glutamatergic afferents control neuronal activity in the neostriatum. Cholinergic interneurons modulate the activity of medium spiny neurons through both pre- and post-synaptic actions via the activation of muscarinic receptors. The muscarinic pre-synaptic modulation was analyzed electrophysiologically. The transmitter release, induced by 4-AP, was studied and the block of paired pulse facilitation (PPF) by different muscarinic receptor antagonists was analyzed. The GABA(A) antagonist bicuculline isolated the glutamatergic transmission. Muscarinic agonists decreased the frequency of random synaptic potentials induced by 4-AP in about 60% of the cases without changes in input resistance (RN) of the post-synaptic neuron or in the mean amplitude of the synaptic events; indicating a presynaptic action. The administration of both 1 microM carbachol or 20 nM muscarine increased PPF. Muscarinic receptor antagonists blocked this action with a potency order: 3-alpha-chloroimperialine > 4-DAMP>AFDX-116 > or = gallamine > pirenzepine. The IC50's for the first three antagonists were (nM): 0.65, 1.1, and 3.0. Their respective Hill coefficients were: 1.9, 1.4, and 1.3. 3-alpha-Chloroimperialine reduced the PPF almost completely. The M3 and the M2 muscarinic receptor antagonists 4-DAMP and AFDX-116, given at saturating concentrations, consistently blocked only a part of the PPF but had additive effects when given together. These data are consistent with the existence of both M2 and M3 muscarinic receptors in striatal glutamatergic afferents.     

Functional role of M2 and M3 muscarinic receptors in the urinary bladder of rats in vitro and in vivo

1. Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M2 subtype whereas the remaining minority belong to the M3 subtype. The objective of the present study was to assess the functional role of M2 and M3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. 2. In the isolated bladder of rat, (+)-cis-dioxolane produced concentration-dependent contractions (pEC50 = 6.3) which were unaffected by tetrodotoxin (0.1 microM). These contractions were antagonized by muscarinic antagonists with the following rank order of affinity (pA2) estimates: atropine (9.1) > 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) > darifenacin (8.5) > para fluoro hexahydrosiladifenidol (p-F-HHSiD) (7.4) > pirenzepine (6.8) > methoctramine (5.9). These pA2 estimates correlated most favourably (r = 0.99, P < 0.001) with the binding affinity (pKi) estimates of these compounds at human recombinant muscarinic m3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)-cis-dioxolane equates with the pharmacologically defined M3 receptor. 3. As M2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline-induced relaxation of KCl pre-contracted tissues). Muscarinic M3 receptors were preferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 microM) to protect M2 receptors. Under these conditions, (+)-cis-dioxolane produced concentration-dependent reversal (re-contraction) of isoprenaline-induced relaxation (pEC50 = 5.8) but had marginal effects on pinacidil-induced, adenosine 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yielding pA2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M2 and M3 receptors and were consistent with the involvement of both of these subtypes. 4. In urethane-anaesthetized rats, the cholinergic component (approximately 55%) of volume-induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID35%inh, nmol kg-1, i.v.): 4-DAMP (8.1) > atropine (20.7) > methoctramine (119.9) > darifenacin (283.3) > pirenzepine (369.1) > p-F-HHSiD (1053.8). These potency estimates correlated most favourably (r = 0.89, P = 0.04) with the pKi estimates of these compounds at human recombinant muscarinic m2 receptors. This is consistent with a major contribution of M2 receptors in the generation of volume-induced bladder contractions, although the modest potency of darifenacin does not exclude a role of M3 receptors. Pretreatment with propranolol (1 mg kg-1, i.v.) increased the ID35%inh of methoctramine significantly from 95.9 to 404.5 nmol kg-1 but had no significant effects on the inhibitory responses to darifenacin. These data suggest an obligatory role of beta-adrenoceptors in M2 receptor-mediated bladder contractions in vivo. 5. The findings of the present study suggest that both M2 and M3 receptors can cause contraction of the rat bladder in vitro and may also mediate reflex bladder contractions in vivo. It is proposed that muscarinic M3 receptor activation primarily causes direct contraction of the detrusor whereas M2 receptor activation can contract the bladder indirectly by reversing sympathetically (i.e. beta-adrenoceptor)-mediated relaxation. This dual mechanism may allow the parasympathetic nervous system, which is activated during voiding, to cause more efficient and complete emptying of the bladder.     

Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein

This investigation was undertaken to characterize the muscarinic receptor subtypes involved in methacholine-induced vasodilation, vagal bradycardia, neurally-evoked sudomotor responses and sympathetic muscarinic ganglionic transmission in anesthetized cats. Dose-response curves were constructed using the putatively selective antagonists pirenzepine (M1), gallamine (M2) and 4-DAMP (M3: 4-diphenyl-acetoxy-N-methylpiperidine) and compared with the non-selective blocker, atropine. Methacholine hypotension and evoked sudomotor responses exhibited an M3 muscarinic receptor profile with the following potency relationships: atropine > or = 4-DAMP > pirenzepine > gallamine. Vagal bradycardia (M2) was antagonized by gallamine and exhibited a lower relative sensitivity to 4-DAMP when corrected for atropine effect. Pirenzepine was inactive in inhibition of bradycardia but was highly potent against transmission in the sympathetic ganglion (M1) with the following potency relationships: atropine > or = pirenzepine > 4-DAMP > gallamine. In comparison with atropine, 4-DAMP exhibited a significantly lower potency for M1 and M2 muscarinic receptors as compared to its effect on the M3 muscarinic receptor subtypes.     

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     

Increased active stress generation of denervated rat intestinal smooth muscle: functional analysis of muscarinic receptor population

The effects of denervation on the active stress production by the longitudinal muscle (LM) layer of rat jejunum were examined. Extrinsic and myenteric denervation of a segment of rat jejunum was accomplished by the serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC). Isolated muscle contraction experiments revealed that the LM of the jejunum taken from rats treated with BAC 15 days before developed significantly increased active stress in response to bethanechol and carbachol, but not in response to potassium chloride. No change in -log EC50 values of any of the agonists was observed in the denervated LM layer, although a significant increase in the slope of the carbachol and bethanechol concentration-response curves was observed in the denervated LM. Schild analysis of several muscarinic antagonists revealed a 3-fold increase in the apparent dissociation constant of the M2 antagonist methoctramine in BAC-treated LM. These results suggest that the increased responsiveness of the denervated LM may originate in the muscarinic receptor population of the myocytes.     

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.     

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.     

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.     

Relationship between agonist binding, phosphorylation and immunoprecipitation of the m3-muscarinic receptor, and second messenger responses

1. Phosphoinositidase C-linked m3-muscarinic receptors expressed in Chinese hamster ovary cells (CHO-m3 cells) are phosphorylated on serine following agonist stimulation. 2. m3-Muscarinic receptor phosphorylation is concentration-dependent requiring a carbachol concentration of 13.2 microM for half maximal stimulation. 3. The phosphorylation concentration-response curve lies to the left of the curve for carbachol binding to muscarinic receptors (KD = 100 microM) in membranes from CHO-m3 cells. In contrast, receptor phosphorylation closely correlates with receptor-mediated phosphoinositidase C activation (EC50 for inositol 1,4,5 trisphosphate accumulation during the peak and plateau phases were 7.14 microM and 5.92 microM respectively) but not with rapid agonist-mediated calcium elevation (EC50 = 0.32 microM) measured in fura-2-AM loaded cells. 4. These data suggest a dissociation of receptor phosphorylation from agonist occupation. Such an apparent 'receptor reserve' for m3-muscarinic receptor phosphorylation may be indicative of a mechanism that is dependent on a small amplification of the receptor signal, though probably dissociated from the calcium signal.     

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.     

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.