TRPV4 channel participates in receptor-operated calcium entry and ciliary beat frequency regulation in mouse airway epithelial cells

Abstract

The rate of mucociliary clearance in the airways is a function of ciliary beat frequency (CBF), and this, in turn, is increased by increases in intracellular calcium. The TRPV4 cation channel mediates Ca 2؉ influx in response to mechanical and osmotic stimuli in ciliated epithelia. With the use of a TRPV4-deficient mouse, we now show that TRPV4 is involved in the airways' response to physiologically relevant physical and chemical stimuli. Ciliary TRPV4 expression in tracheal epithelial cells was confirmed with immunofluorescence in TRPV4 ؉/؉ mice. Ciliated tracheal cells from TRPV4 ؊/؊ mice showed no increases in intracellular Ca 2؉ and CBF in response to the synthetic activator 4␣-phorbol 12,13-didecanoate (4␣PDD) and reduced responses to mild temperature, another TRPV4-activating stimulus. Autoregulation of CBF in response to high viscosity solutions is preserved in TRPV4 ؊/؊ despite a reduced Ca 2؉ signal. More interestingly, TRPV4 contributed to an ATPinduced increase in CBF, providing a pathway for receptor-operated Ca 2؉ entry but not store-operated Ca 2؉ entry as the former mechanism is lost in TRPV4 ؊/؊ cells. Collectively, these results suggest that TRPV4 is predominantly located in the cilia of tracheal epithelial cells and plays a key role in the transduction of physical and chemical stimuli into a Ca 2؉ signal that regulates CBF and mucociliary transport. Moreover, these studies implicate the participation of TRPV4 in receptor-operated Ca 2؉ entry.ATP ͉ trachea ͉ temperature ͉ transient receptor potential channel ͉ store-operated calcium entry I n mammalian airways, ciliated and mucus-secreting epithelial cells form a structural and functional unit that functions as a conveyor belt system for particle transport. In this analogy, cilia provide the power, whereas the mucus serves as the viscoelastic belt (1). A critical factor regulating the velocity of mucociliary transport is the ciliary beat frequency (CBF), a mechanism in which cytosolic Ca 2ϩ plays a major role (1, 2). Increases in cytosolic Ca 2ϩ are associated with increases in CBF (3, 4), although the ultimate molecular mechanism explaining CBF regulation by Ca 2ϩ remains controversial (3). Both mechanical and chemical (paracrine) stimulation of epithelial ciliated cells can lead to an increase in intracellular Ca 2ϩ concentration and the consequent enhancement of CBF (2). ATP-mediated activation of G protein-coupled receptors, typically P2Y receptors, is one of the strongest signals known to increase CBF (2). As with many other agonists of G proteincoupled receptors, ATP promotes both the release of Ca 2ϩ from inositol trisphosphate (IP 3 )-sensitive intracellular stores and Ca 2ϩ entry across the plasma membrane (4), and the latter process is more evident at micromolar concentrations of ATP where a sustained Ca 2ϩ influx occurs. The stimulation of Ca 2ϩ influx involves signaling from the depleted stores to plasma membrane Ca 2ϩ channels (store-operated calcium entry, SOCE; also known as capacitative Ca 2ϩ entry) and/or through a phospholipase...

Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli

“…For electrophysiological and calcium imaging experiments, HeLa or HEK-293 cells were transiently transfected as described previously (16,48). Primary cultures of trachea and oviduct ciliated cells were obtained as described previously (7,17). Animals were maintained and experiments performed in accordance with guidelines issued by the Institutional Ethics Committee of the Universitat Pompeu Fabra.…”

“…For that purpose, we used primary cultures of ciliated epithelial cells obtained from trachea and oviduct that express functional TRPV4 channels (7,16,17). Fig.…”

“…structure | regulation | thermosensitivity T he transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel that responds to osmotic (1)(2)(3)(4), mechanical (5-7), and temperature stimulation (8), thereby contributing to many different physiological functions, including cellular (4,9) and systemic volume homeostasis (10), vasodilation (11,12), nociception (13), epithelial hydroelectrolyte transport (14), bladder voiding (15), ciliary beat frequency regulation (7,16,17), chondroprotection (18), and skeletal regulation (19). The osmotic (20) and mechanical (7,16) sensitivity of TRPV4 depends on the activation of phospholipase A 2 and subsequent production of the arachidonic acid metabolite 5′-6′-epoxyeicosatrienoic acid (EET), whereas the mechanism leading to temperature-mediated activation (observed only in intact cells) is not known at present (21).…”

IP3 Receptor Binds to and Sensitizes TRPV4 Channel to Osmotic Stimuli via a Calmodulin-binding Site

“…TRPV4 participation in osmo-and mechanotransduction (1,2) contributes to important functions including cellular (3,4) and systemic volume homeostasis (5,6), arterial dilation (7,8), nociception (9), epithelial hydroelectrolyte transport (10,11), bladder voiding (12), and ciliary beat frequency regulation (13)(14)(15). TRPV4 also responds to temperature (16,17), endogenous arachidonic acid (AA) metabolites (18), and phorbol esters including the inactive 4␣-phorbol 12,13-didecanoate (4␣PDD) (19,20) and participates in receptor-operated Ca 2ϩ entry (15), thus showing multiple modes of activation.…”

Endogenous expression of thermo-sensitive ion channels TRPV1 and TRPV4 in immune tissues of avian species (duck, Anas platyrhynchos)

“…This polymodal receptor is also involved in cellular processes such as mechanosensation, osmosensation and thermosensation (Liedtke et al, 2003;Goswami et al, 2010). In higher organisms TRPV4 is endogenously expressed in dorsal root ganglion (DRG) neurons and in several other nonneuronal tissues such as in skin, kidney and corneal epithelial cells (Pan et al, 2008), cerebral microvascular endothelial cells , cortical astrocytes (Benfenati et al, 2007), and in tracheal epithelial cells (Lorenzo et al, 2008). The widespread distribution of TRPV4 is indicative of its involvement in various physiological functions.…”