The aims of this study were to determine the cysteinyl leukotriene (CysLT) receptors expressed in the human saphenous vein, to examine contractile response to LTC4 and LTD4, to evaluate antagonist activity of montelukast, a specific CysLT1 receptor antagonist used in asthma, and to characterize the CysLT receptors involved in the contractile response. The analysis by reverse-transcriptase polymerase chain reaction indicated that CysLT1 and CysLT2 receptors are expressed by saphenous veins. In varicose vein rings, the potencies (pD2) of LTC4 and LTD4 were similar: 7.4 ± 0.2 and 7.4 ± 0.1, respectively. Pretreatment with acivicin, a γ-glutamyl transpeptidase (γ-GT) inhibitor, to prevent potential metabolism of LTC4 to LTD4, did not alter the response to LTC4. In nondistended vein rings from patients undergoing arterial bypass, the LTC4 pD2 was 7.8 ± 0.1, and pretreatment with S-hexyl-GSH, a potent γ-GT inhibitor, caused a fourfold rightward shift of the LTC4 concentration-response curve. In varicose and nondistended saphenous vein rings, montelukast (10-8 and 10-7M) exerted a potent activity against LTD4 and LTC4, in the presence or absence of γ-GT inhibitors. In varicose vein rings, the two active metabolites of montelukast also exerted antagonist activities with potencies similar to montelukast. BAY u9773 (CysLT2 agonist/dual CysLT1/CysLT2 antagonist) did not cause contraction and inhibited the LTC4- and LTD4-induced contractions. In conclusion, human saphenous veins express CysLT1 and CysLT2 receptors, but only CysLT1 receptors are implicated in the contraction.
Cysteinyl leukotrienes (leukotriene C4 [LTC4], LTD4, and LTE4) are potent proinflammatory mediators and smooth muscle constrictors and have been shown to activate receptors (CysLT receptors) in human tissues. In human lung, the cysteinyl leukotrienes cause airways smooth muscle constriction and proliferation and secretion of mucus, promote eosinophil recruitment and activation, and increase microvascular permeability. The receptor involved in the effects of cysteinyl leukotrienes in human airways is referred to as CysLT1. 1,2 For these reasons, cysteinyl leukotrienes are considered to be pivotal inflammatory mediators that contribute to the pathogenesis of asthma. Three systemic CysLT1 receptor antagonists are currently in clinical use (montelukast, pranlukast, and zafirlukast) for the treatment of asthma. 2,3 Exploring the in vitro effects of cysteinyl leukotrienes on human vascular preparations and investigating whether such actions are affected by the currently used CysLT1 receptor antagonists may contribute to our understanding of the CysLT receptors involved and the potential vascular side effects of these drugs. Our current understanding of the role of cysteinyl leukotrienes in the cardiovascular system, although far from complete, seems to indicate enough potential for an involvement of these mediators in cardiac, hemodynamic, and microcirculatory pathophysiology, and may therefore justify antileukotriene therapy. 4-7 In a number of vascular preparations, cysteinyl leukotrienes exert paradoxic effects, namely contraction and relaxation. 7 These paradoxic effects of cysteinyl leukotrienes depend on the vascular bed and on the species investigated. In human preparations, LTC4 and LTD4 did not produce effective contractions in internal mammary arteries, 8,9 nonatherosclerotic coronary arteries, 10 gastroepiploic arteries, and femoral arteries, 9 whereas effective contractions were reported in atherosclerotic coronary arteries, 10 saphenous veins, 8,9,11 and pulmonary arteries and veins. 12-14 In the human pulmonary vein, functional studies have shown that endothelial CysLT1 receptors and smooth muscle CysLT2 receptors could be involved in the contractile response to cysteinyl leukotrienes. 7,12 In human pulmonary artery, the contractile response to cysteinyl leukotrienes was resistant to a CysLT1 receptor antagonist (MK-571) and to the dual CysLT1/CysLT2 receptor antagonist activity of BAY u9773, suggesting that a third type of CysLT receptor (CysLT3?) may be involved. 13,14 In human saphenous veins, the CysLT receptors have not been fully characterized, but preliminary results suggest that CysLT1 and CysLT2 receptors might be involved in the contractile response. 8
The CysLT1 and CysLT2 receptors have recently been cloned, 15-18 and therefore the expression of these two types of receptors can be explored by reverse-transcription polymerase chain reaction (RT-PCR). Binding studies on cloned human CysLT1 and CysLT2 receptors have demonstrated that montelukast is a potent and selective CysLT1 receptor antagonist. 17,18 After oral administration, this drug is extensively metabolized into six derivatives. Only the 21- and 36-hydroxylated metabolites (named M5 and M6, respectively) were detected in plasma after administration of montelukast to healthy volunteers. M6 binds to the CysLT1 receptor on guinea pig lung membranes nearly as avidly as montelukast, whereas M5 binds approximately 10-fold less (MSD preclinical data). No functional studies have been performed on human isolated saphenous veins to evaluate the antagonist activity of montelukast or the two hydroxylated metabolites, M5 and M6, against cysteinyl leukotriene-induced contractions.
The main objectives of the present study were therefore to determine the cysteinyl leukotriene receptors expressed in human saphenous vein by RT-PCR studies, to examine the contractile response to LTC4 and LTD4, and to characterize the type of receptors involved in the contractile response to cysteinyl leukotrienes by functional studies using the CysLT1 receptor antagonist montelukast, as well as its two active metabolites, and the dual CysLT1/CysLT2 receptor antagonist BAY u9773. 15