An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium

Abstract

Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca ] -dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca ] were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml ) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an antagonist used to block P2X7 receptors, which reduced basal CBF by 85%. Additionally, increasing extracellular ATP levels (0.1-100 μm) increased CBF, maintaining a sustained response that was suppressed in the presence of carbenoxolone. We also show that high levels of ATP (1 mm), associated with inflammatory conditions, lowered basal CBF by reducing [Ca ] and hemichannel functionality. In summary, we provide evidence indicating that airway epithelium ATP release is the molecular autocrine mechanism regulating basal ciliary activity and is also the mediator of the ciliary response to chemical stimulation.

Tracheal brush cells release acetylcholine in response to bitter tastants for paracrine and autocrine signaling

“…Strikingly, the denatonium-induced increase in PTS was absent when the M3R was inhibited with 4-DAMP ( Figure 4D) and not changed upon inhibition of gap junctions with carbenoxolone ( Figure S4). Consistent with previous findings by Droguett et al, 32 the PTS-increase evoked by extracellular ATP applied at the end of each experiment was reduced in the presence of carbenoxolone ( Figure S4). Notably, denatonium did not evoke changes in PTS in Trpm5 −/− mice ( Figure 4E) indicating that the denatonium-induced PTSincrease is dependent on ACh released from BC acting in a paracrine manner on ciliated cells.…”

“…Additionally, the bitter substance denatonium significantly 324 | HOLLENHORST ET aL. Consistent with previous findings by Droguett et al, 32 the PTS-increase evoked by extracellular ATP applied at the end of each experiment was reduced in the presence of carbenoxolone ( Figure S4). This response was completely abolished in the presence of the general cholinergic receptor inhibitors mecamylamine (nAChR) and atropine (MR) ( Figure 4C).…”

Respiratory Cilia as a Therapeutic Target of Phosphodiesterase Inhibitors

“…This explains potential benefit of selective PDE1A inhibition in increasing the CBF ( Figure 2B). Furthermore, the closing connexin/pannexin hemichannels that release ATP and decreasing intracellular Ca 2+ levels may also suppress the CBF (Schmid et al, 2014;Shishikura et al, 2016;Droguett et al, 2017;Zhou et al, 2019).…”

Fueling influenza and the immune response: Implications for metabolic reprogramming during influenza infection and immunometabolism

“…Glutamine can be converted to glutamate and enter the TCA cycle at α-ketoglutarate and be used in the canonical reactions or in the non-canonical reversal to produce citrate the mechanical motion of cilia beating by epithelial cells is energetically costly. 46 Mucociliary clearance depends on the movement of mucus and the periciliary layer of ASL by coordinated ciliary beating.…”