Acetylcholine released by endothelial cells facilitates flow‐mediated dilatation

Abstract

Key points The endothelium plays a pivotal role in the vascular response to chemical and mechanical stimuli.The endothelium is exquisitely sensitive to ACh, although the physiological significance of ACh‐induced activation of the endothelium is unknown.In the present study, we investigated the mechanisms of flow‐mediated endothelial calcium signalling.Our data establish that flow‐mediated endothelial calcium responses arise from the autocrine action of non‐neuronal ACh released by the endothelium. AbstractCirculating blood generates frictional forces (shear stress) on the walls of blood vessels. These frictional forces critically regulate vascular function. The endothelium senses these frictional forces and, in response, releases various vasodilators that relax smooth muscle cells in a process termed flow‐mediated dilatation. Although some elements of the signalling mechanisms have been identified, precisely how flow is sensed and transduced to cause the release of relaxing factors is poorly understood. By imaging signalling in large areas of the endothelium of intact arteries, we show that the endothelium responds to flow by releasing ACh. Once liberated, ACh acts to trigger calcium release from the internal store in endothelial cells, nitric oxide production and artery relaxation. Flow‐activated release of ACh from the endothelium is non‐vesicular and occurs via organic cation transporters. ACh is generated following mitochondrial production of acetylCoA. Thus, we show ACh is an autocrine signalling molecule released from endothelial cells, and identify a new role for the classical neurotransmitter in endothelial mechanotransduction.

The Endothelium Solves Problems That Endothelial Cells Do Not Know Exist

“…Endothelial cells are fixed in physical space and the physical connections between cells are invariant; the arrangement is a mesh. Despite this fixed anatomy, and mesh cell arrangement, the number of functional communication routes in the endothelium is large and gives rise to multiple possible outputs ([48], see Figure 4). Even a casual glance at communication across the endothelium shows the complex varying paths on activation (Video S2).…”

“…In our own studies of endothelial Ca 2+ signalling, we observed Ca 2+ waves that seemingly propagate across clusters of cells in both pressurised and en face arteries. The propagation of Ca 2+ waves among groups of cells was most apparent during prolonged (∼5–10 min) activation with an agonist (GSK1016790A, 30 nM) of transient receptor potential cation channel subfamily V member 4 (TRPV4) (Figure 2 and Video S3 in the supplemental information online) [48]. The coordinated nature of these waves suggests that the endothelium has a modular architecture.…”

Endothelial TRPV4 channels modulate vascular tone by Ca2+‐induced Ca2+ release at inositol 1,4,5‐trisphosphate receptors

“…To block IP 3 Rs, we used both 2‐APB and caffeine. 2‐APB is a broad‐spectrum inhibitor that, although effective in blocking IP 3 Rs in native endothelial cells, may inhibit Ca 2+ entry pathways (see Peppiatt et al, ; Trebak, Bird, McKay, & Putney, ; Voets et al, ; Wilson, Lee, & McCarron, ). However, in the present experimental conditions, 2‐APB did not reduce the slow global Ca 2+ rise evoked by activation of TRPV4 channels suggesting that it is not inhibiting influx in the present experiments.…”

“…Endothelial Ca 2+ signals were extracted automatically from fluorescence recordings using custom written Python software (; Lee et al, ; Wilson, Lee, & McCarron, ; Wilson, Saunter, et al, ). In brief, cellular regions of interest (ROIs) were first generated from average‐intensity projections of each time series recording.…”

“…This procedure effectively flattens the baseline and removes any slow drift from the signal (Figure S1C–E). Peaks in each Ca 2+ signal arising from the waves were then detected automatically, using a zero‐crossing detector on derivative F/F 0 traces (Lee et al, ; Wilson, Lee, & McCarron, ). Various signal metrics (number of peaks, peak amplitudes, peak durations, 10–90% rise time, and 90–10% fall time) were extracted from the corresponding ALS‐smoothed F/F 0 trace.…”