Sostegni Silvia, Diakov Alexei, McIntyre Peter, Bunnett Nigel, Korbmacher Christoph, Haerteis Silke
Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Waldstr. 6, 91054, Erlangen, Germany.
Pflugers Arch. 2015 Apr;467(4):687-701. doi: 10.1007/s00424-014-1539-6. Epub 2014 Jun 8.
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.
蛋白酶激活受体2（PAR2）的蛋白水解激活可能是在与蛋白酶激活相关的病理生理条件下（如在炎症期间）调节瞬时受体电位香草酸亚型4（TRPV4）非选择性阳离子通道的主要机制。为了提供PAR2介导的TRPV4调节的电生理证据，我们在存在和不存在共表达的人PAR2的情况下，对非洲爪蟾卵母细胞中异源表达的人TRPV4的特性进行了表征。在表达TRPV4的卵母细胞的外向膜片中，我们检测到TRPV4典型的单通道活性，外向电流的单通道电导约为100 pS，内向电流的单通道电导约为55 pS。合成的TRPV4激活剂GSK1016790A主要通过将先前沉默的通道转化为开放概率接近1的活性通道来刺激TRPV4。在共表达TRPV4和PAR2的卵母细胞中，胰蛋白酶或特异性PAR2激动剂SLIGRL-NH2对PAR2的激活使GSK1016790A刺激的TRPV4全细胞电流增强了几倍，表明通道敏化。用钙螯合剂1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）-AM对卵母细胞进行预孵育并没有降低PAR2激活对TRPV4的刺激作用，这表明该作用与细胞内钙信号传导无关。中性粒细胞弹性蛋白酶是PAR2的一种偏向性激动剂，不诱导细胞内钙信号传导，它也导致TRPV4的PAR2依赖性敏化。Rho激酶抑制剂Y27362消除了弹性蛋白酶刺激的TRPV4敏化，这表明Rho激酶信号传导在PAR2介导的由偏向性激动剂中性粒细胞弹性蛋白酶引起的TRPV4敏化中起关键作用。在急性炎症期间，中性粒细胞弹性蛋白酶可能通过一种涉及PAR2的偏向性激动作用和Rho激酶激活机制使TRPV4敏化。
