Muscarinic M2 and M3 receptor function in smooth muscle.
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Animals↗
Biomedical subjects
R M Eglen
Publications and source records attributed to R M Eglen.
Muscarinic receptor subtypes and smooth muscle function.
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Animals↗
Characterization of muscarinic receptor and beta-adrenoceptor interactions in guinea-pig oesophageal muscularis mucosae.
Smooth muscles of a number of species contain both muscarinic M2 and M3 receptors in differing proportions and while muscarinic M3 receptors mediate contraction, the role of muscarinic M2 receptors is unclear. Muscarinic M2 receptor-mediated inhibition of adenylyl cyclase activity has been demonstrated in smooth muscle and since beta-adrenoceptor relaxation of this tissue is mediated via stimulation of adenylyl cyclase, an interaction between muscarinic M2 receptors and beta-adrenoceptors in smooth muscle has been postulated. Such an interaction has been demonstrated in guinea-pig ileum and trachea using two different approaches. The present study investigates whether interactions between muscarinic M2 receptors and beta-adrenoceptors also occur in guinea-pig oesophageal muscularis mucosae. Using the technique of selective muscarinic M3 receptor alkylation, we were unable to demonstrate muscarinic M2 receptor-mediated re-contractions in oesophageal smooth muscle, as described previously in ileum. In addition, while increased functional antagonism of relaxant responses to isoprenaline could be demonstrated in tissues pre-contracted with oxotremorine M compared to histamine, muscarinic M2 receptor activation did not contribute to this effect, as described previously in trachea. These data suggest a lack of interaction between muscarinic M2 receptors and beta-adrenoceptors in guinea-pig oesophageal smooth muscle, but suggest an interaction between muscarinic M3 receptors and beta-adrenoceptors.
Alkylation↗
Anandamide hydroxylation by brain lipoxygenase:metabolite structures and potencies at the cannabinoid receptor.
Anandamide (arachidonyl ethanolamide) is a compound that was identified from porcine brain lipids by its ability to bind to the brain cannabinoid receptor. This study assessed anandamide as a substrate for a brain lipoxygenase and characterised the brain metabolite 12-hydroxyanandamide. Anandamide was also compared with arachidonic acid as a lipoxygenase substrate by examining enzyme kinetics in the presence of either of the two compounds. In addition, a non-mammalian enzyme was used to generate 11- and 15-hydroxy-anandamide in order to compare the cannabinomimetic properties of a range of anandamide derivatives. A ligand displacement assay indicated a large variation in the affinity of anandamide metabolites for the brain cannabinoid receptor. The brain metabolite, 12-hydroxyanandamide had an affinity twice that of anandamide, although the 11- and 15- hydroxy-metabolites were considerably poorer ligands of this receptor. Consistent with the receptor binding data, 12-hydroxyanandamide (unlike 15-hydroxyanandamide) inhibited forskolin-stimulated cAMP synthesis, indicating it to be a functional agonist at the brain cannabinoid receptor. Pharmacological studies of the capacity of anandamide and its metabolites to inhibit the murine vas deferens twitch response indicated the 12-hydroxy-metabolite to be less active than the parent compound, but a better cannabinomimetic than 15-hydroxyanandamide.
Animals↗
Characterization of the interaction of zamifenacin at muscarinic receptors in vitro.
The interaction of zamifenacin ((3R)-(+)-diphenylmethoxy-1-(3,4)-methylenedioxyphenethyl)pi peridine) at muscarinic receptor subtypes was studied using radioligand binding and functional techniques, in vitro. In radioligand binding studies, zamifenacin acted as a competitive antagonist, with the following pKi values; rat cerebral cortex (M1) 7.90 +/- 0.08, myocardium (M2) 7.93 +/- 0.13, submaxillary gland (M3) 8.52 +/- 0.04 and rabbit lung (M4) 7.78 +/- 0.04. In functional studies zamifenacin acted as a surmountable antagonist, exhibiting the following apparent affinity values; canine saphenous vein (putative M1) 7.93 +/- 0.09, guinea-pig left atria (M2) 6.60 +/- 0.04, guinea-pig ileum (M3) 9.31 +/- 0.06, guinea-pig oesophageal muscularis mucosae (M3) 8.84 +/- 0.04, guinea-pig trachea (M3) 8.16 +/- 0.04, and guinea-pig urinary bladder (M3) 7.57 +/- 0.15. Therefore, zamifenacin is selective for muscarinic M3 receptors in guinea-pig ileum, oesophageal muscularis mucosae, trachea and bladder over muscarinic M2 receptors in atria. The degree of muscarinic M3/M2 receptor selectivity depends upon the muscarinic M3 receptor preparation studied.
Animals↗
Characterization of a postjunctional 5-HT receptor mediating relaxation of guinea-pig isolated ileum.
The 5-HT receptor mediating postjunctional relaxation of precontracted guinea-pig ileum has been characterized using several agonists and antagonists. Substance P precontracted tissues were potently relaxed by 5-HT (5-hydroxytryptamine, serotonin), 5-CT (5-carboxamidotryptamine) and several other indoles. The rank order of potency, with pEC50 values in parentheses, was 5-CT (7.6) > 5-methoxytryptamine (5.7) > 5-HT (5.5) > alpha-methyl-5-HT (4.7) > 2-methyl-5-HT (< 4.0) = tryptamine (< 4.0) = N,N-dimethyl-tryptamine (< 4.0) = N,N-dimethyl-5-HT (< 4.0) = dipropyl-5-CT (< 4.0) = sumatriptan (< 4.0). 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)-tetralin) acted as a potent (6.3), but partial, agonist with respect to 5-HT. The responses to 5-CT were antagonized by several compounds with the following rank order of affinity, with pKB values in parentheses: LSD (lysergic acid diethylamide; 8.1) = mesulergine (7.8) > methysergide (7.6) = spiperone (7.6) > clozapine (7.3) >> (-)-pindolol (< 6.0) > ketanserin (< 6.0) = ondansetron (< 6.0) = GR 113808 ([1-(2-methane-sulphonamido-ethyl)-piperidin-4-yl]-methyl-in dole-3- carboxylate maleate; < 6.0). The relaxant responses to 5-HT were also resistant to tetrodotoxin. These data are consistent with a functional 5-HT receptor, mediating relaxation of guinea-pig ileum, which exhibits an operational profile similar to that of the cloned guinea-pig 5-ht7 receptor. This study, therefore, provides evidence for a functional correlate of the 5-ht7 gene product.
5-Methoxytryptamine↗
Role of muscarinic M2 and M3 receptors in guinea-pig trachea: effects of receptor alkylation.
Muscarinic M2 receptors account for more than half the muscarinic receptor population in smooth muscles of a number of species and yet it is the smaller M3 receptor population that mediates contraction of many of these tissues. The role of the majority of M2 receptors in the control of smooth muscle tone is unclear. In guinea-pig ileal smooth muscle, an indirect contractile role (re-contraction) for M2 receptors has been demonstrated in tissues subjected to M3 receptor alkylation and stimulation of adenylyl cyclase. The present studies have employed the technique of irreversible receptor alkylation in order to investigate the role of muscarinic M2 and M3 receptors in the control of guinea-pig tracheal smooth muscle tone. Experiments were performed to determine (i) whether an indirect contractile role for M2 receptors can be demonstrated in tracheal smooth muscle as described for ileum, and (ii) whether stimulation of M2 receptors can inhibit isoprenaline-induced relaxations of histamine pre-contracted trachea after selective M3 receptor alkylation. Our results suggest (i) that there is no evidence of M2 receptor-mediated re-contraction of tracheal smooth muscle after M3 receptor alkylation and stimulation of adenylyl cyclase, but (ii) that activation of M2 receptors, after M3 receptor alkylation, has a small inhibitory effect on relaxant responses to isoprenaline in guinea-pig tracheal smooth muscle. Therefore, it appears that the major role of postjunctional muscarinic M2 receptors in guinea-pig trachea remains to be determined.
Alkylation↗
The identification of heterogeneity of 5-HT3 receptors with [3H]RS-42358-197.
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Animals↗
5-HT4 receptor mediated stimulation of gastric emptying in rats.
It is well documented that certain substituted benzamides, such as cisapride, and benzimidazolones, such as BIMU 8, enhance gastric emptying in rats. As these compounds possess 5-HT3 antagonistic and 5-HT4 agonistic properties, the precise mechanisms (5-HT3 or 5-HT4) underlying their gastroprokinetic effects is still unclear. In the present study, we used SC 49518 (a benzamide and selective 5-HT4 receptor agonist) and two selective 5-HT4 receptor antagonists (RS 23597-190 and SB 204070) to elucidate the role of 5-HT4 receptors in gastroprokinesis. SC 49518 (1-316 micrograms/kg; ip) produced significant and dose-dependent stimulation of gastric emptying in rats (ED50 = 2.3 micrograms/kg; ip). SC 49518 also produced dose-dependent inhibition of bradycardia induced by 2-methyl 5-HT (von Bezold-Jarisch reflex) but with a 156 fold lower potency (ID50 = 0.36 mg/kg; ip). The gastroprokinetic effects of SC 49518 (3-316 micrograms/kg; ip) were significantly antagonized by the selective 5-HT4 receptor antagonist RS 23597-190 (0.1 mg/kg/min; iv). SB 204070 (0.003-1 mg/kg; ip), another selective 5-HT4 receptor antagonist, produced dose-dependent inhibition of the gastroprokinetic effects of SC 49518 (10 micrograms/kg; ip), the inhibition attaining statistical significance at the dose of 0.1 mg/kg; ip. RS 23597-190 had no effects on gastric emptying per se whereas SB 204070 significantly increased gastric emptying by itself at 1 mg/kg; ip but not at 0.1 mg/kg; ip. These findings show, for the first time, that SC 49518, a selective 5-HT4 receptor agonist, produces potent stimulation of gastric emptying in rats via a mechanism involving activation of 5-HT4 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Analysis of Variance↗
Investigation of the 5-hydroxytryptamine receptor mediating the "transient" short-circuit current response in guinea-pig ileal mucosa.
5-Hydroxytryptamine (5-HT) stimulated an increase in short-circuit current (ISC) in guinea-pig isolated ileal mucosa over a wide concentration range (0.1 nM-0.1 mM). The concentration-response relationship was biphasic, consisting of a high potency phase (0.1 nM-1 microM) and a low potency phase (3-10 microM). Stimulation of ISC observed at the high potency phase tended to be sustained while responses at the low potency phase (3-10 microM) contained two components, an initial "transient" response followed by a "maintained" response. Both the high potency phase (maximum stimulation approximately 30 microA cm-2) and the low potency phase (maximum stimulation approximately 80 microA cm-2) 5-HT response were antagonized by tetrodotoxin (TTX, 0.3 microM) and atropine (1 microM). However, another low potency (3 microM-0.1 mM, maximum stimulation approximately 30 microA cm-2) component of the 5-HT response was revealed in the presence of TTX or atropine. In the presence of methysergide (1 microM), the concentration-response relationship of 5-HT was still biphasic and tropisetron (0.1 and 10 microM) antagonized both phases of the 5-HT response. In the presence of methysergide, the high potency phase 5-HT response was mimicked by 5-methoxytryptamine (5-MeOT) and the selective 5-HT4 agonist SC-53116 but not by BIMU 8. The potent 5-HT4 antagonist GR 113808 antagonized the response to 5-MeOT in a surmountable manner with an affinity estimate of 9.6 +/- 0.3 (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Enhancement of adenosine A1 receptor functions by benzoylthiophenes in guinea pig tissues in vitro.
Previous reports on a series of benzoylthiophenes, including PD 81,723 [2-amino-4,5-dimethyl-3-(3-trifluoromethyl-benzoyl) thiophene], have shown specific enhancement of agonist binding at the adenosine A1 receptor. We have studied the effects of two substituted benzoylthiophenes, PD 78,416 (thieno[2,3-c]pyridine-6(5H)-carboxylic acid, 2-amino-3-benzoyl-4,7-dihydro-ethyl ester) and RS-74513-000 [2-amino-4-ethyl-5-methyl-3-(3-trifluoro-methyl-benzoyl) thiophene] on response elicited by adenosine A1 receptors in isolated guinea pig left atrium and ileum. In the electrically paced left atrium, PD 78,416 antagonized negative inotropic effect elicited by the agonist CPA [N6-cyclopentyladenosine] with a pKB value of 6.2 +/- 0.2 (n = 4). At a low concentration which had no antagonistic effect (0.1 microM), PD 78,416 enhanced the effect of CPA. The concentration-response curve to CPA was shifted leftward by 5.1 fold (95% confidence limits 2.4-11.2). In field stimulated isolated ileum, PD 78,416 (0.1, 0.3, 1 microM) did not enhance or antagonize effects of CPA. At concentrations above 1 microM, PD 78,416 decreased electrically induced contraction. This effect was not sensitive to adenosine deaminase and was not antagonized by the A1 antagonist CPX [8-cyclopentyl-1,3-dipropyl-xanthine] (1 microM). Unlike PD 78,416, RS-74513-000 (0.01, 0.1, 1, 3, 10 microM) did not antagonize or enhance effects of CPA in the left atrium. However, effects of CPA in ileum were enhanced by RS-74513-000 (1 and 3 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine↗
Quantitative autoradiography demonstrates selective modulation of rat brain regional dopamine (D1 and D2) receptor subtypes after chronic manipulation of dietary salt.
The effects of chronic dietary sodium chloride (NaCl) consumption on renal function and brain dopamine receptors were studied in adult, male normotensive rats. Compared to rats maintained on the normal NaCl (0.33%) diet, animals maintained on the low NaCl (0%) diet for 4 weeks exhibited significant increases in plasma aldosterone, chloride and changes in urinary electrolyte excretion. In contrast, rats maintained on the high NaCl (8%) diet for 4 weeks demonstrated significant increases in urine volume and urinary sodium, chloride and dopamine excretions and water intake. Rats fed the high NaCl diet displayed a 42-59% decrease (p < 0.001-0.05) in D1 binding in the nucleus accumbens (NA), olfactory tubercle (OT) and the striatum (STM), without any effects on D2 binding in these brain regions. Rats maintained on the low NaCl diet also demonstrated decreased D1 binding in the ventral (24%, p < 0.02) and lateral (29%, p < 0.01) STM, but not in the OT, NA, entopeduncular nucleus and substantia nigra. Rats fed low or high NaCl diets exhibited a 35-180% increase (p < 0.01-0.05) in D2 binding in several mid-brain areas (e.g. hypothalamus, thalamus and hippocampus) and hindbrain regions (e.g. superior colliculus and nucleus tractus solitarius) without affecting the D1 binding. These data indicate that chronic modification of dietary salt intake profoundly affects the renal handling of sodium/water excretion and leads to selective up- and/or down-regulation of DA receptor subtypes in different areas of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
M3 muscarinic receptors on murine HSDM1C1 cells: further functional, regulatory, and receptor binding studies.
In the present studies, the pharmacology and regulation of the functional muscarinic receptors on HSDM1C1 cells were probed using phosphoinositide (PI) turnover assays. In addition, the receptor binding of the putative M3-selective radioligand, [3H]4-DAMP, to cell homogenates was characterized. Carbachol (EC50 = 9 microM), (+)muscarine (EC50 = 4.5 microM) and cis-dioxolane (EC50 = 0.72 microM) were full agonists which stimulated PI turnover by 13.3 +/- 1.0 fold above basal values. The potencies of numerous agonists in this assay system were relatively similar to their affinities in receptor binding assays. Exposure of HSDM1C1 cells to 10 nM-10 microM muscarine during the last 24h of [3H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cis-dioxolane affinity and maximal PI response induced by subsequent treatment with cis-dioxolane. Pertussis toxin (5-2000 ng/ml) caused a partial reduction in the cis-dioxolane-induced PI turnover. Likewise, exposure of the HSDM1C1 cells to an active phorbol ester (TPA) resulted in a partial inhibition of the cis-dioxolane-induced (100 microM) PI turnover. The half-maximal effect of TPA was produced at 1.8 +/- 0.3 nM. [3H]4-DAMP binding to cell homogenates was of high affinity (Kd = 0.19 +/- 0.04 nM) and moderate capacity (Bmax = 201 +/- 22 fmol/mg protein). The pharmacological specificity (4-DAMP > p-FHHSiD > dicyclomine > pirenzepine > methoctramine > AFDX-116 > gallamine) resembled that for [3H]NMS binding and correlated well with that observed for inhibition of PI turnover. These studies further support the identification of M3 receptors on HSDM1C1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Ondansetron improves cognitive performance in the Morris water maze spatial navigation task.
In the present studies we investigated the actions of ondansetron, a prototypic 5-hydroxytryptamine3 (5-HT3) receptor antagonist, on performance in a complex spatial navigation/memory task in rats. Specifically, we compared the activity of ondansetron to that of the cholinesterase inhibitor physostigmine in attenuating two distinct cognitive deficits in the Morris water maze. In the first model, rats treated with the muscarinic receptor antagonist atropine (30 mg/kg) had significantly longer latencies to find the submerged platform across two days of testing. Physostigmine (0.03, 0.1 and 0.3 mg/kg) and ondansetron (0.03-1 mg/kg) significantly reduced the latencies to find the submerged platform in atropine-treated animals, suggesting an increase in cognitive performance. There was little evidence of a dose-response relationship for either compound, and a loss of efficacy for ondansetron was seen at 3 mg/kg. In the second model, pre-screened, aged (23 months), cognition-impaired and nonimpaired rats were tested. Ondansetron (0.1 mg/kg), but not physostigmine (0.1 mg/kg), decreased the latencies to find the submerged platform in the aged-impaired rats, while neither compound improved performance of aged-nonimpaired rats. These data suggest that ondansetron may have cognition enhancing properties in animal models of aging and cholinergic hypofunction.
Animals↗
Chronic manipulation of dietary salt modulates renal physiology and kidney dopamine receptor subtypes: functional and autoradiographic studies.
1. Compared to rats maintained on the normal NaCl (0.33%) diet, animals maintained on the low NaCl (0%) diet for 4 weeks exhibited increased plasma aldosterone and chloride and decreased urinary sodium excretion. 2. Rats maintained on the high NaCl (8%) diet for 4 weeks showed increased systolic blood pressure, water intake, urine volume, sodium and dopamine excretion and decreased plasma aldosterone and glomerular filtration rate. 3. Administration of SCH 23390 (10 mg/kg, po), but not domperidone to the high salt diet rats attenuated the diuretic effect, indicating the involvement of DA1 rather than DA2 receptors. The dopamine decarboxylase inhibitor, carbidopa (30 mg/kg, i.p.), also reduced the high salt-induced diuresis. 4. Kidney sections from rats fed the low NaCl diet showed a 63-100% decrease (P < 0.001-0.02) in cortical and medullary DA1 and DA2 binding sites, while rats fed the high NaCl diet demonstrated only a 70% decrease (P < 0.01-0.02) in cortical DA1 binding, without affecting DA2 binding. 5. These data indicate that chronic modification of dietary salt profoundly affects the sodium, water and dopamine excretion and leads to selective modulation of renal dopamine receptor subtypes.
Animals↗
Central 5-HT4 receptors.
Activation of the 5-HT4 receptor mediates widespread effects in central and peripheral nervous systems. Recent developments, such as the identification of novel, selective agonists and antagonists, as well the cloning of the receptor, have provided insights into the physiological role of the receptor. In this article, Richard Eglen and colleagues assess the emerging evidence relating to the function of the 5-HT4 receptor in the brain. The cerebral distribution of the receptor, along with neurochemical and electrophysiological data, suggests a role in cognition. The role of the receptor in modulation of dopamine transmission and anxiolysis is also addressed.
Animals↗
Allosteric interactions among agonists and antagonists at 5-hydroxytryptamine3 receptors.
Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine3 (5-HT3) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT3 receptors. The affinities of competitive 5-HT3 receptor antagonists were similar regardless of whether the receptors were labeled with [3H]RS-42358, [3H]granisetron, or 1-(m-[3H]chlorophenyl)biguanide ([3H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [3H]mCPG. The dissociation of [3H]mCPG, [3H]RS-42358, and [3H]RS-25259, but not [3H]granisetron, from both cloned and native 5-HT3 receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT3 receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.
Allosteric Regulation↗
Pharmacological characterization of the muscarinic receptors mediating contraction of canine saphenous vein.
1. The muscarinic receptor subtype mediating contraction of the canine saphenous vein has been characterized using a range of muscarinic agonists and subtype-selective antagonists. 2. Oxotremorine M and (+)-cis-dioxolane behaved as full agonists, while in comparison L-660,863 ((+/-)-3-(3-amino-1,2,4-oxadiazole-5-yl)quinuclidine) acted as a partial agonist. SDZ ENS 163 (thiopilocarpine), pilocarpine and McN-A-343 (0.1 microM-0.3 mM) did not elicit a response. The profile of agonist potencies suggests a low receptor reserve for contraction. 3. The rank order of antagonist apparent affinities was 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide; 8.41) > pirenzepine (8.10) > himbacine (7.34) > or = p-F-HHSiD (para-fluoro-hexahydrosiladifenidol; 7.15) > methoctramine (6.23). This antagonist apparent affinity profile is consistent with the activation of muscarinic M1 receptors.
Animals↗