Sensitisation of TRPV4 by PAR2 is independent of intracellular calcium signalling and can be mediated by the biased agonist neutrophil elastase

Silvia Sostegni, Alexei Diakov, Peter McIntyre, Nigel Bunnett, Christoph Korbmacher, Silke Haerteis

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.
Original languageEnglish
Pages (from-to)687-701
Number of pages15
JournalPflugers Archiv-European Journal of Physiology
Volume467
Issue number4
DOIs
Publication statusPublished - 2015

Keywords

  • Elastase
  • PAR2
  • Proteases
  • Proteolytic activation
  • Rho-kinase
  • TRPV4
  • Two-electrode voltage clamp
  • Xenopus laevis oocytes

Cite this

Sostegni, Silvia ; Diakov, Alexei ; McIntyre, Peter ; Bunnett, Nigel ; Korbmacher, Christoph ; Haerteis, Silke. / Sensitisation of TRPV4 by PAR2 is independent of intracellular calcium signalling and can be mediated by the biased agonist neutrophil elastase. In: Pflugers Archiv-European Journal of Physiology. 2015 ; Vol. 467, No. 4. pp. 687-701.
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abstract = "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.",
keywords = "Elastase, PAR2, Proteases, Proteolytic activation, Rho-kinase, TRPV4, Two-electrode voltage clamp, Xenopus laevis oocytes",
author = "Silvia Sostegni and Alexei Diakov and Peter McIntyre and Nigel Bunnett and Christoph Korbmacher and Silke Haerteis",
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Sensitisation of TRPV4 by PAR2 is independent of intracellular calcium signalling and can be mediated by the biased agonist neutrophil elastase. / Sostegni, Silvia; Diakov, Alexei; McIntyre, Peter; Bunnett, Nigel; Korbmacher, Christoph; Haerteis, Silke.

In: Pflugers Archiv-European Journal of Physiology, Vol. 467, No. 4, 2015, p. 687-701.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Sensitisation of TRPV4 by PAR2 is independent of intracellular calcium signalling and can be mediated by the biased agonist neutrophil elastase

AU - Sostegni, Silvia

AU - Diakov, Alexei

AU - McIntyre, Peter

AU - Bunnett, Nigel

AU - Korbmacher, Christoph

AU - Haerteis, Silke

PY - 2015

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AB - 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.

KW - Elastase

KW - PAR2

KW - Proteases

KW - Proteolytic activation

KW - Rho-kinase

KW - TRPV4

KW - Two-electrode voltage clamp

KW - Xenopus laevis oocytes

UR - http://link.springer.com.ezproxy.lib.monash.edu.au/content/pdf/10.1007%2Fs00424-014-1539-6.pdf

U2 - 10.1007/s00424-014-1539-6

DO - 10.1007/s00424-014-1539-6

M3 - Article

VL - 467

SP - 687

EP - 701

JO - Pflugers Archiv-European Journal of Physiology

JF - Pflugers Archiv-European Journal of Physiology

SN - 0031-6768

IS - 4

ER -