Recently we have demonstrated that
Recently we have demonstrated that the synthesis of cysteinyl-LTs from LTA4 by human umbilical vein endothelial cells (HUVECs) is directly associated with the activation of the CysLT2 receptor (CysLT2R) in a typical autocrine fashion, and that solely the CysLT2R is responsible for the increase in intracellular Ca2+ following exposure of HUVECs to LTC4 or LTD4 . In the present work we focus on ECs as the privileged target of the effects of cysteinyl-LTs, as well as one of their main biosynthetic sources. Cysteinyl-LTs (and LTA4) increase HUVEC monolayers contraction and permeability to macromolecules, as well as formation of stress fibers and phosphorylation of myosin light-chain (MLC), an event that may represent the molecular mechanism of increased vascular permeability. Finally, in a complex organ model of cerebral vasculature with an intact intima, perfused with human primed PMNL, selective neutrophil challenge leads to significant formation of cysteinyl-LTs and edema. Pretreatment with a selective CysLT2R antagonist prevents HUVEC contraction, and formation of edema in isolated brain preparations, while leaving the synthesis of cysteinyl-LTs unaffected, effects that are further supporting a key role for CysLT2R in the cerebrovascular activities of cysteinyl-LTs.
Materials and methods
Discussion Cysteinyl-LTs activities in the CV system, including constriction and permeability alteration of the microvasculature, reduction of coronary blood flow, decrease in myocardial contractility, as well as reduction of cardiac output, have been well documented in the 1980s, leading to hypothesis that they might be important mediators of ischemia and shock (see Capra et al.  for a recent review). In addition, cysteinyl-LTs have been observed to induce contractions of human atherosclerotic coronary arteries, whereas non-atherosclerotic CCT137690 are unresponsive , potentially involving these inflammatory mediators in response to the atherosclerotic injury. Interestingly, the CysLT2 also showed an exquisite CV distribution, with significant expression in the heart, particularly in Purkinje fibers , myocytes and fibroblasts derived from atrium and ventricle, in human coronary artery smooth muscle cells  and in ECs , , ; in these latter cellular types, activation by LTC4 or LTD4 increased [Ca2+]i, an effect that was not blocked by the selective CysLT1R antagonist montelukast , . Indeed, endothelium-targeted over-expression of the human CysLT2R in mice was shown to increase endothelial permeability , thus worsening myocardial ischemia-reperfusion injury, leading to accelerated left ventricular remodeling and impaired cardiac performance . Again, these effects were significantly attenuated by CysLT2R antagonists , in agreement with the observation of decreased alterations in vascular permeability observed upon CysLT2R gene disruption , suggesting a critical role for this receptor in vascular pathophysiological responses. In the present paper we report unequivocal evidence that the conversion of the unstable intermediate LTA4 into LTC4 carried out by ECs through a microsomal glutathione S-transferase type II (mGST-II) , results in clear-cut autocrine effects mediated by the activation of CysLT2R on the same cells, leading to stress fibers formation, cellular contraction, and macromolecule permeability alterations (Fig. 9). No evidence is available about the possibility that LTA4 itself could bind and/or activate any receptor, and its extremely short half-life at physiological pH  certainly contributes to rule out this possibility. According to the selective expression of the CysLT2R by ECs, a specific CysLT2R antagonist, CysLT2cpd, significantly inhibited EC contraction induced by LTC4; screening of different compounds showed that several putative CysLT1R antagonists also had the ability to prevent cysteinyl-LTs-induced EC contraction, an effect that clearly points to their affinity for the CysLT2R in addition to that for the CysLT1R.