Modulation by CertainConserved AspartateResidues of the Allosteric Interaction of Gallamine at the ml Muscarinic Receptor1

ABBREVIATiONS:Asp. aspartic acid; Asn, asparagine; CHO, Chinese hamster ovary; NMS, N-methylscopolamine; Kd, equilibriumdissociation constant; KA,eqliilibnum dissociationconstant ofallosteric antagonIstfor the unhigandedreceptor; a, cooperativity factor; lC@,concentration required to inhibit 50%of ligand binding ordissociation. ABSTRACT Muscarinic acetyicholine receptors belong to a superfamily of 6-protein coupled receptors and contain within their structure several conserved aspartate residues. These residues have been implicated to play important roles in the interaction of agonists and their competitive antagonists with the receptor. In the pres entwork,we investigated whether thesameresidues might also serve as Importantcontact points for allostencantagonistsof Recently, much attention has been focused on the localiza tion of the binding site for muscarinicreceptor agonistsand their competitive antagonists. Site-directed mutagenesis of the ml muscarinic receptor indicated that four aspartate residues, which are conserved among all muscarmnicreceptor subtypes, play an important role in ligand binding and/or receptor acti vation 1989). It is believed that the primary (competitive) binding site Received for publication January 6, 1992

Mixed Competitive and Allosteric Antagonism by Gallamine of Muscarinic Receptor‐Mediated Second Messenger Responses in N1E‐115 Neuroblastoma Cells

Abstract: The antagonistic effects of gallamine on muscarinic receptor‐linked responses were investigated in N1E‐115 neuroblastoma cells. M1 muscarinic receptor‐mediated phos‐phoinositide hydrolysis induced by carbamylcholine was antagonized by gallamine, with a Ki value of 33 μM. By comparison, gallamine was four‐ to fivefold less potent in blocking noncardiac M2 muscarinic receptor‐mediated inhibition of cyclic AMP formation, with a Ki value of 144 μM. The resulting Arunlakshana‐Schild plots of the antagonism of both responses by gallamine were linear and exhibited slopes not differing from 1, a result indicative of a competitive mechanism. To elucidate further the nature of gallamine\u27s inhibitory actions, experiments were performed where the effects of gallamine in combination with the known competitive muscarinic antagonist, N‐methylscopolamine (NMS), were studied. In the presence of both antagonists, a supraadditive shift in the carbamylcholine dose‐response curve was demonstrated for the two responses, a result suggestive of an allosteric mode of interaction between gallamine and NMS binding sites. Confirmation that gallamine allosterically modifies the muscarinic receptor was provided by radioligand binding studies. Gallamine competition curves with either [N‐methyl‐3H]scopolamime methyl chloride ([3H]NMS) or [N‐methyl‐3H]quinuclidinyl benzilate methyl chloride ([3H]NMeQNB) were unusually shallow. Furthermore, gallamine decelerated the rate of dissociation of receptor‐bound [3H]NMS \u3e [3HJNMeQNB in a dose‐dependent manner. The present study demonstrates that whereas gallamine antagonizes carbamylcholine‐mediated responses in N1E‐115 cells in a competitive manner, an allosteric component of its action is revealed in the presence of muscarinic antagonists such as NMS. Copyright © 1989, Wiley Blackwell. All rights reserve

The allosteric binding profile of himbacine: a comparison with other cardioselective muscarinic antagonists

The possibility of an allosteric interaction by himbacine, a cardioselective antagonist, with rat cardiac muscarinic receptors was studied. Himbacine allosterically decelerated the dissociation of bound [3H]N-methylscopolamine ([3H]NMS) in a concentration-dependent manner with an IC50 value of 103.7 μM. When compared to the 3C50 values of other cardiovascular antagonists, the rank order of potencies was: methoctramine \u3e gallamine \u3e himbacine \u3e AF-DX 116. In contrast, the potencies of these compounds to displace [3H]NMS binding were: himbacine \u3e methoctramine \u3e AF-DX 116 \u3e gallamine. The allosteric potencies were found not to be correlated with binding potencies (correlation coefficient). = -0.15. A striking common fefeature of the cardioselective anatagonists is their ability to bind to an allosteric site on cardiac muscarinic receptors. © 1990

Effects of canine endotoxin shock on lymphocytic beta-adrenergic receptors

To determine whether beta-adrenergic receptors on circulating lymphocytes are impaired during endotoxemia and the precise role of catecholamines in this process, we allocated 16 dogs to three groups: I) control-saline vehicle (n = 5), II) endotoxin-Escherichia coli endotoxin 1.0 mg/kg iv bolus (n = 6), and III) endotoxin + propranolol-E. coli endotoxin 1.0 mg/kg after pretreatment with propranolol, 1.5 mg/kg iv bolus followed by a continuous infusion, 30 μg/kg per min, (n = 5). Five hours after endotoxin injection, lymphocytic beta-adrenergic receptor number and sodium fluoride (NaF)-stimulated cyclic AMP accumulation were reduced to 41 ± 6% and 25 ± 7% of baseline values, respectively, which were significantly different from those observed in the control group (both P \u3c .01). Propranolol pretreatment prevented the endotoxin-induced reduction in lymphocytic beta-adrenergic receptor number (P \u3c .02 compared with the endotoxin group), but not the decrease in NaF-stimulated cyclic AMP accumulation (P \u3c .01 compared with the control group). Myocardial beta-adrenergic receptor number was reduced in the endotoxin group compared with that observed in the control group (P \u3c .06). These changes were associated with a decreased chronotropic response to isoproterenol in the endotoxin group compared with the control group (P \u3c .05). We conclude that decreased lymphocytic beta-adrenergic receptor number in endotoxin shock is caused by circulating catecholamines, whereas alterations distal to the receptors may be due to other mechanisms

Methoctramine, a cardioselective muscarinic antagonist, stimulates phosphoinositide hydrolysis in rat cerebral cortex

The cardioselective muscarinic antagonist methoctramine antagonized carbamylcholine-mediated phosphoinositide (PI) hydrolysis in a concentration-dependent fashion in dissociated rat cerebrocortical cells. However, as the concentration of methoctramine was increased above 5 μM, there was a reversal of the antagonism of the PI response. In the absence of carbamylcholine, methoctramine by itself significantly increased PI hydrolysis with a maximal effect at 30 μM. Various classes of receptor antagonists, including atropine, and ion-channel blockers were unable to block methoctramine-stimulated PI hydrolysis. © 1989

Modulation by certain conserved aspartate residues of the allosteric interaction of gallamine at the m1 muscarinic receptor

Muscarinic acetylcholine receptors belong to a superfamily of G-protein coupled receptors and contain within their structure several conserved aspartate residues. These residues have been implicated to play important roles in the interaction of agonists and their competitive antagonists with the receptor. In the present work, we investigated whether the same residues might also serve as important contact points for allosteric antagonists of muscarinic receptors, because the majority of these compounds are cationic in nature, or if such residues are involved in modification of receptor conformation by these antagonists. Gallamine was used as a prototype for these antagonists. Site-directed mutagenesis of the m1 muscarinic receptor subtype was utilized to define some of the molecular determinants involved in cooperative allosteric interactions. We report that substitution of the aspartate residue at position 71, but not at positions 99 and 122 with asparagine, affected the affinity of gallamine for the unliganded m1 receptor. A similar substitution at positions 71 and 99 decreased the magnitude of its cooperative effects on the binding of [3H]N- methylscopolamine. Our data suggest that these residues are implicated in cooperative interactions. At present, however, we cannot discount a more pivotal role of other residues on the receptor sequence in allosteric interactions. The data also support the notion that different molecular entities are required for the binding of allosteric antagonists as compared to the interaction of agonists and competitive antagonists at the receptor

Effects of tacrine on brain muscarinic-receptor-mediated second-messenger signals

The purpose of this study was to investigate the effects of 9- amino-l,2,3,4-tetrahydroacridine(THA; Tacrine®) on musca- rinic-receptor-linkcd second-messenger systems in rat brain and to determine the selectivity and mechanisms of these effects. Both competitive and noncompetitive antagonism was revealed in saturation radioligand binding studies performed in cortical and striatal tissue, depending on THA concentration. Micromolar THA concentrations blocked muscarinic- receptor-mediated inhibition of cAMP formation and stimulation of phosphoinositide (PI) hydrolysis with poor selectivity between the two responses. While both responses were blocked in the same concentration range (4-60 μmol/l), noncompetitive antagonism of PI hydrolysis occurred at THA concentrations greater than 10 μmol/l while competitive antagonism was displayed for the cAMP response at concentrations of THA up to 40 μmol/l. THA was equally effective at inhibiting PI hydrolysis stimulated by histamine, phenylephrine or oxotremorine-M, when these agonists were employed in concentrations equal to their EC50S for the response. THA did not antagonize PI hydrolysis mediated by the quisqualate receptor at any agonist concentration used. Furthermore, THA blocked carbachol- but not morphine-induced inhibition of forskolin-stimulated cAMP formation in the striatum. © 1993 S. Karger AG, Basel

Effect of aging on the interaction of quinuclidinyl benzilate, N-methylscopolamine, pirenzepine, and gallamine with brain muscarinic receptors

The objective of the present study was to investigate the effects of senescence on the binding characteristics of muscarinic receptors by using [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]NMS) as ligands in young (3months), middle-age (10months) and old (24 months) male Fischer 344 rats. Muscarinic receptor density was found to decrease significantly with aging in certain brain regions, depending on the ligand employed. Moreover, the relative proportions of M1 and M2 muscarinic receptor subtypes was not significantly altered by aging, except in the aged striatum. Furthermore, the dissociation kinetics of [3H]NMS in the cerebral cortex and their allosteric modulation by gallamine were only slightly influenced by age. © 1988 Plenum Publishing Corporation