Pp.13.10: distribution of cholinergic system components gene expression in intact rat aorta

Objective: Acetylcholine-induced (ACh) relaxation is routinely used to evaluate endothelial function. Via muscarinic M3 receptors (M3MR), ACh stimulates synthesis of NO and EDHF resulting in endothelium-dependent relaxation. Vascular cholinergic system remains practically unexplored. The objective of this project was to determine the localization of mRNA of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), M3 MR, cholinesterase anchoring proteins PRiMA1, and ColQ1 within the endothelium and smooth muscle (SM) of freshly isolated rat aorta. Design and method: Thoracic aortas of adult male Wistar rats were used. Expression of the cholinergic markers was analyzed by RT-PCR in endothelium-intact aortic rings (AoE+), endothelial RNA fractions obtained by aorta perfusion with Tri-Reagent (E1-E3) and in aorta fraction without endothelium (AoE-). Endothelial and SM origin of fractions was evaluated endothelial markers (endothelial nitric oxide synthase, NOS3; preproendothelin-1, ET1), SM markers (L-type calcium channel, Cav1.2; endothelin receptor type A, ETA; smooth muscle actin, ACTA2) and genes reported to be expressed both in the endothelium and SM (endothelin receptor type B, ETB). Cholinergic markers -AChE, AChE-T, BChE, M3MR, PRiMA1, and ColQ1 mRNA were assayed in all obtained fractions. Results: E1-E3 were strongly enriched with NOS3, ET1, while SM markers (Cav1.2, ETA) were scarce, slightly increasing from E1 to E3 and abundant in AoE- and AoE+. ETB mRNA was similar in all fractions. All cholinergic markers were detected in AoE+. Expression of BChE was 30-fold higher than AChE. AChE, BChE, PRiMA1 and ColQ1 mRNAs all had an expression pattern similar to that of the SM markers. M3MR mRNA was present in both endothelial and SM fractions. Conclusions: Despite the lack of parasympathetic innervation, cholinergic markers are abundant in rat aorta. M3MR distribution suggest an action of Ach on both endothelium and SM. Expression of cholinesterase in SM could prevent the diffusion of ACh into the media layers and thus control its vasoconstrictor action. The absence of cholinesterases in the endothelium could allow ACh-induced vasorelaxation.