Faculty Bibliography

BACKGROUND AND PURPOSE/OBJECTIVE:Transient receptor potential vanilloid-4 (TRPV4) is a calcium-permeant ion channel that is known to affect vascular function. The ability of TRPV4 to cause a vasoconstriction in blood vessels has not yet been mechanistically examined. Further in neuronal cells, TRPV4 signalling can be potentiated by GPCR activation. Thus, we studied the mechanisms underlying the vascular contractile action of TRPV4 and the GPCR-mediated potentiation of such vasoconstriction, both of which are as yet unappreciated aspects of TRPV4 function. EXPERIMENTAL APPROACH/METHODS:The mechanisms of TRPV4-dependent regulation of vascular tone in isolated mouse aortae were studied using wire myography. TRPV4-dependent calcium signalling and prostanoid production was studied in cultured human umbilical vein endothelial cells (HUVECs). KEY RESULTS/RESULTS:In addition to the well-documented vasorelaxation response triggered by TRPV4 activation, we report here a TRPV4-triggered vasoconstriction in the mouse aorta that involves a COX-generated Tx receptor (TP) agonist that acts in a MAPK and Src kinase signalling dependent manner. This constriction is potentiated by activation of the GPCRs for angiotensin (AT1 receptors) or proteinases (PAR1 and PAR2) via transactivation of the EGF receptor and a process involving PKC. TRPV4-dependent vascular contraction can be blocked by COX inhibitors or with TP antagonists. Further, TRPV4 activation in HUVECs stimulated Tx release as detected by an elisa. CONCLUSION AND IMPLICATIONS/CONCLUSIONS:We conclude that the GPCR potentiation of TRPV4 action and TRPV4-dependent Tx receptor activation are important regulators of vascular function and could be therapeutically targeted in vascular diseases.

Proteolytic activation of protease-activated receptor 2 (PAR2) may represent a major mechanism of regulating the transient receptor potential vanilloid 4 (TRPV4) non-selective cation channel in pathophysiological conditions associated with protease activation (e.g. during inflammation). To provide electrophysiological evidence for PAR2-mediated TRPV4 regulation, we characterised the properties of human TRPV4 heterologously expressed in Xenopus laevis oocytes in the presence and absence of co-expressed human PAR2. In outside-out patches from TRPV4 expressing oocytes, we detected single-channel activity typical for TRPV4 with a single-channel conductance of about 100 pS for outward and 55 pS for inward currents. The synthetic TRPV4 activator GSK1016790A stimulated TRPV4 mainly by converting previously silent channels into active channels with an open probability of nearly one. In oocytes co-expressing TRPV4 and PAR2, PAR2 activation by trypsin or by specific PAR2 agonist SLIGRL-NH2 potentiated the GSK1016790A-stimulated TRPV4 whole-cell currents several fold, indicative of channel sensitisation. Pre-incubation of oocytes with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM did not reduce the stimulatory effect of PAR2 activation on TRPV4, which indicates that the effect is independent of intracellular calcium signalling. Neutrophil elastase, a biased agonist of PAR2 that does not induce intracellular calcium signalling, also caused a PAR2-dependent sensitisation of TRPV4. The Rho-kinase inhibitor Y27362 abolished elastase-stimulated sensitisation of TRPV4, which indicates that Rho-kinase signalling plays a critical role in PAR2-mediated TRPV4 sensitisation by the biased agonist neutrophil elastase. During acute inflammation, neutrophil elastase may sensitise TRPV4 by a mechanism involving biased agonism of PAR2 and activation of Rho-kinase.

Sensory nerves are equipped with receptors and ion channels that allow them to detect and respond to diverse chemical, mechanical, and thermal stimuli. These sensory proteins include G protein-coupled receptors (GPCRs) and transient receptor potential (TRP) ion channels. A subclass of peptidergic sensory nerves express GPCRs and TRP channels that detect noxious, irritant, and inflammatory stimuli. Activation of these nerves triggers protective mechanisms that lead to withdrawal from danger (pain), removal of irritants (itch, cough), and resolution of infection (neurogenic inflammation). The GPCR-TRP axis is central to these mechanisms. Signals that emanate from the GPCR superfamily converge on the small TRP family, leading to channel sensitization and activation, which amplify pain, itch, cough, and neurogenic inflammation. Herein we discuss how GPCRs and TRP channels function independently and synergistically to excite sensory nerves that mediate noxious and irritant responses and inflammation in the skin and the gastrointestinal and respiratory systems. We discuss the signaling mechanisms that underlie the GPCR-TRP axis and evaluate how new information about the structure of GPCRs and TRP channels provides insights into their functional interactions. We propose that a deeper understanding of the GPCR-TRP axis may facilitate the development of more selective and effective therapies to treat dysregulated processes that underlie chronic pain, itch, cough, and inflammation.