Abstract P148: Muscarinic Acetylcholine Receptors are Localized to Primary Endothelial Cilia and Involved in Nitric Oxide Signaling

Abstract

Primary cilia are considered sensory hubs housing a variety of mechanosensory proteins, chemosensory receptors, and ion channels to translate extracellular stimuli into an intracellular biochemical signals such as the synthesis and release of nitric oxide (NO). Malformations in cilia structure or defects in cilia function lead to ciliopathies such as Polycystic Kidney Disease (PKD) and hypertension. Muscarinic receptors 1 and 3 play an essential role in regulating cardiovascular function by mediating both dilation and constriction in the vasculature. However, nothing is known about the relationship between primary cilia and muscarinic receptors. Supported by our exciting discovery of the mAChRs in the cilia, we hypothesize that primary cilia in the vascular system play important roles in regulating blood pressure through NO biosynthesis. Our objective is to unravel the mechanism by which cilia dysfunction contribute to hypertension and to introduce cilia as a novel therapeutic target for PKD. To explore the relationship between mAChRs and primary cilia, the effects of muscarinic modulators on cilia length and function in wildtype, and cilia mutant endothelial cells, Pkd1 – / – (dysfunctional cilia) and Tg737 orpk/orpk (cilia-less) were examined. We show that both AChM1R and AChM3R localize to primary endothelial cilia. AChM1R and AChM3R activation lead to a significant increase in cilia length in endothelial cells treated with cdd0102a, an AChM1R and AChM3R agonist (2.84±0.02 vs. 3.47± 0.04 for wildtype, 2.31± 0.03 vs. 2.53± 0.03 for Pkd1 – / – , and 0.024 ± 0.005 vs. 0.3 ± 0.004 for Tg737 orpk/orpk ) compared to control cells. Treatment with pirenzepine, an AChM1R antagonist, led to a significant decrease of cilia length (2.66± 0.02 vs. 2.48± 0.03 in wildtype, and 2.12± 0.02 vs. 1.93± 0.02 in Pkd1 – / – ) compared to control cells. Treatment with cdd0102a also significantly upregulated the expression of AChM1R, AChM3R and phosphorylated eNOS in wildtype and Pkd1 - / - cells. More importantly, cdd0102a treatment rescued NO response in Pkd1 - / - cells in response to fluid shear stress. Our data clearly suggest that modulating cilia sensory function could have a positive influence on nitric oxide generation and blood pressure regulation.