Abstract
Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases. Although these receptors have been described in human, bovine and rat cerebral microvascular tissue, a subtype functional characterization in mouse brain endothelium is lacking. Here, we show that all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells. The mRNA expression of M2, M3, and M5 correlates with their respective protein abundance, but a mismatch exists for M1 and M4 mRNA versus protein levels. Acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors. Moreover, although M1 and M3 are the most abundant receptors, only a small fraction of M1 is present in the plasma membrane and functions in ACh-induced Ca2+ signaling. Bioinformatic analyses performed on eukaryotic muscarinic receptors demonstrate a high degree of conservation of the orthosteric binding site and a great variability of the allosteric site. In line with previous studies, this result indicates muscarinic acetylcholine receptors as potential pharmacological targets in future translational studies. We argue that research on drug development should especially focus on the allosteric binding sites of the M1 and M3 receptors.
Highlights
Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases
All muscarinic acetylcholine receptor subtypes are expressed by mouse brain microvascular endothelial cells
Quantitative real-time PCR analyses using both β-actin (Actb) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) as reference genes indicated that both brain microvascular endothelial cells (BMVECs) and brain endothelial cells express mRNA transcripts ffoorlloCwhrsm: C1h–r5m, w3 h>i cChhernmc4o>d∼eCmhArmC1h>R∼sCMhr1-mM55 >
Summary
Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases These receptors have been described in human, bovine and rat cerebral microvascular tissue, a subtype functional characterization in mouse brain endothelium is lacking. While it is widely known that acetylcholine (ACh) is released by axonal terminals of cholinergic neurons, cholinergic receptors are not found solely on target neurons Rather, these receptor subtypes are expressed by multiple non-neuronal cell types, both in the CNS (central nervous system) and peripherally, including endothelial, epithelial, mesothelial and immune cells[1]. These receptor subtypes are expressed by multiple non-neuronal cell types, both in the CNS (central nervous system) and peripherally, including endothelial, epithelial, mesothelial and immune cells[1] This is not surprising, since cholinergic regulatory mechanisms have been described even in organisms lacking nervous systems, including plants, algae, and bacteria[2]. Endothelial cells present a high degree of heterogeneity in the expression of surface markers, carrier proteins, and intracellular enzymes, across and within tissues[17]
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