Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors.

Methoctramine (N,N'-bis[6-[[(2-methoxyphenyl)-methyl]hexyl]-1,8-octane] diamine) is an M(2)-selective competitive antagonist of muscarinic acetylcholine receptors and exhibits allosteric properties at high concentrations. To reveal the molecular mechanisms of methoctramine binding and selectivity we took advantage of reciprocal mutations of the M(2) and M(3) receptors in the second and third extracellular loops that are involved in the binding of allosteric ligands. To this end we performed measurements of kinetics of the radiolabeled antagonists N-methylscopolamine (NMS) in the presence of methoctramine and its precursors, fluorescence energy transfer between green fluorescent protein-fused receptors and an Alexa-555-conjugated precursor of methoctramine, and simulation of molecular dynamics of methoctramine association with the receptor. We confirm the hypothesis that methoctramine high-affinity binding to the M(2) receptors involves simultaneous interaction with both the orthosteric binding site and the allosteric binding site located between the second and third extracellular loops. Methoctramine can bind solely with low affinity to the allosteric binding site on the extracellular domain of NMS-occupied M(2) receptors by interacting primarily with glutamate 175 in the second extracellular loop. In this mode, methoctramine physically prevents dissociation of NMS from the orthosteric binding site. Our results also demonstrate that lysine 523 in the third extracellular loop of the M(3) receptors forms a hydrogen bond with glutamate 219 of the second extracellular loop that hinders methoctramine binding to the allosteric site at this receptor subtype. Impaired interaction with the allosteric binding site manifests as low-affinity binding of methoctramine at the M(3) receptor.

Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.

Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

Mechanisms of M(1) muscarinic receptor-mediated up-regulation of neuronal nitric oxide synthase in N1E-115 neuroblastoma cells.

The neuronal form of nitric oxide synthase (nNOS) was generally assumed to be constitutively expressed at a constant level. However, it is now becoming recognized that its expression can be modulated by a number of physiological and pathophysiological conditions. Previously, we reported that nNOS expression is up-regulated after prolonged muscarinic M(1) receptor stimulation. In this work, we report that muscarinic receptor activation signals the up-regulation of nNOS via multiple pathways in N1E-115 mouse neuroblastoma cells. These include protein kinase C (PKC) activation, cytosolic calcium mobilization and NO production. Further characterization showed that the half-life of nNOS is slightly, but significantly, increased in agonist-pretreated cells compared with vehicle-treated control cells. Based on these data, it appears that the level of nNOS expression is modulated in a complex manner by a number of mechanisms that include, but might not be limited to, those described here.

Endogenous expression of nNOS protein in several neuronal cell lines.

Several neuronal cell lines were screened for endogenous expression of neuronal nitric oxide synthase (nNOS) protein using Western blot analysis. Detectable levels of the nNOS protein were evident in the SK-N-SH, SH-SY5Y, and N1E-115 neuroblastoma cell lines, as well as the NG108-15 neuroblastoma x glioma hybrid. Only trace amounts were visible in Neuro2A human neuroblastoma cells. The presence of endogenously expressed nNOS in these cells may allow for the study of the interaction between nNOS and the endogenous receptor systems expressed in the same cells.

Microglial derived nitric oxide decreases serotonin content in rat basophilic leukemia (RBL-2H3) cells.

Nitric oxide (NO) and serotonin (5-hydroxytryptamine; 5-HT) are important neuromodulators that are involved in a myriad of biochemical reactions. In this work, we describe a novel model co-culture system to study the interactions between NO and 5-HT. NO derived from cytokine stimulated Bv2 microglial cells depleted 5-HT from RBL-2H3 cells. Reduction of 5-HT content by NO derived from the NO donor S-nitroso-N-acetylpenicillamine (SNAP) was concentration-dependent, independent of intracellular Ca(2+) and inhibited by reduced glutathione (GSH). Collectively, these data indicate that this cell co-culture system is a viable model to study the mechanisms of interaction between nitrergic and serotonergic pathways.