Dominant Expression of the CysLT2 Receptor Accounts for Calcium Signaling by Cysteinyl Leukotrienes in Human Umbilical Vein Endothelial Cells
Objective— The objective of the present study was to identify and characterize the cell-surface receptors on human umbilical vein endothelial cells (HUVECs) that transduce calcium transients elicited by cysteinyl leukotrienes (CysLTs), potent spasmogenic and proinflammatory agents with profound effects on the cardiovascular system.
Methods and Results— Using quantitative reverse transcription–polymerase chain reaction, we found that HUVECs abundantly express CysLT2R mRNA in vast excess (>4000-fold) of CysLT1R mRNA. Lipopolysaccharide, tumor necrosis factor-α, or interleukin-1β caused a rapid (within 30 minutes) and partially reversible suppression of CysLT2R mRNA levels. Challenge of HUVECs with BAY u9773, a specific CysLT2R agonist, triggered diagnostic Ca2+ transients. LTC4 and LTD4 are equipotent agonists, and their actions can be blocked by the dual-receptor antagonist BAY u9773, but not by the CysLT1R-selective antagonist MK571.
Conclusions— HUVECs almost exclusively express the CysLT2R. Furthermore, Ca2+ fluxes elicited by CysLT in these cells emanate from perturbation of the CysLT2R, rather than the expected CysLT1R. Hence, signaling events involving CysLT2R might trigger functional responses involved in the critical components of LT-dependant vascular reactions, which in turn have implications for ischemic heart disease and myocardial infarction.
The cysteinyl leukotrienes (CysLTs) are a family of powerful lipid mediators, typically formed by eosinophils, basophils, monocytes, and mast cells, which are involved in critical steps of inflammatory and allergic diseases, particularly in the respiratory tract and cardiovascular system.1 The CysLTs are biosynthesized from arachidonic acid and its oxygenation product, LTA4 [5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid], generated by the enzyme 5-lipoxygenase. LTA4 is further conjugated with reduced glutathione by LTC4 synthase to form LTC4 [5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid), ie, the parent compound of the CysLTs (LTC4, LTD4, and LTE4). The biologic effects of CysLTs are signaled through 2 principal G protein–coupled receptors, designated CysLT1R and CysLT2R.2,3⇓ The biologic role of the CysLT2R is presently not known, but its mRNA expression profile suggests that it might have important functions in the cardiovascular system.3–6⇓⇓⇓
Endothelial cells are strategically located at the interphase between the blood and parenchymal cells, where they take active part in physiologic and pathologic processes involving the vessel wall. Under inflammatory conditions, these cells are directly exposed to LTs that are formed by activated, adhering leukocytes or via transcellular routes involving platelets or endothelial cells themselves.7–9⇓⇓ The CysLTs in turn trigger a number of specific functional responses in endothelial cells, eg, synthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), secretion of von Willebrand factor, and expression of P-selectin, all of which might contribute to vascular inflammation.10,11⇓ Here we report that human umbilical vein endothelial cells (HUVECs) abundantly express the CysLT2R in vast excess over the CysLT1R. Moreover, CysLT2R transduces Ca2+ signals elicited by CysLTs in these cells, which in turn might be linked to pathologic processes of the vessel wall.
LTC4 and LTD4 were from BioMol, whereas MK571 and BAY u9773 were kind gifts from Dr Sven-Erik Dahlén, Karolinska Institutet, Stockholm, Sweden. Oligonucleotides were from Cybergene AB. Lipopolysaccharide (LPS) from Escherichia coli serotype O55:B5, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and fura 2-AM were from Sigma Chemical Co.
HUVECs were obtained from vessels by collagenase treatment and were then cultivated and identified, as described.12,13⇓ The cell viability was >95%, as judged by cell morphology, trypan blue exclusion, and analysis of lactate dehydrogenase release. Glass dishes were seeded with 104 HUVECs per well and grown to confluence.
Analysis of mRNA by RT-PCR and Real-Time RT-PCR
Preparation of total RNA and reverse transcription–polymerase chain reaction (RT-PCR) were performed essentially as described.14 In nested PCR analyses, 12 +20 cycles (CysLT2R mRNA) or 40 +40 cycles (CysLT1R mRNA) were used for the first set (5′-ATGGAG-AGAAAATTTATGTCC; 5′-AATAGAGCAGAGGATTGAAGC) and second set (5′-ACCTTCAGCAATAACAACAGC; 5′-CTTTA- TGCAGTCTGTCTTTGC) of primers, respectively (Figure 1). One of 2 housekeeping genes (glyceraldehyde 3-phosphate dehydrogenase and β-actin) was amplified as an internal standard and tested for potential responsiveness to the cell stimulus before calculations. For semiquantitative analysis of PCR products, a fluorescent dye (PicoGreen, Molecular Probes) was used according to the manufacturer’s instructions.15 The fluorescence was measured (excitation wavelength=485 nm; emission wavelength=538 nm) in a fluorometer (SpectraMax GeminiXS). For comparative RT-PCR, total RNA was diluted until the PCR signal for CysLT2R was comparable to that of CysLT1R in undiluted RNA, with the use of 40 +40 cycles. To correct for differences in template efficiency, equal amounts of CysLT1R and CysLT2R DNA standards were subjected to PCR amplification, and the relative signal intensity was used for normalization. Real-time RT-PCR was performed on a commercially available amplification system (Rotor Gene 2000, Corbett Research Mortlake) with SYBRgreen (Roche Diagnostics) as the detection dye.16
Ca2+ Mobilization Experiments
Mobilization of cytosolic calcium, Ca2+i, was monitored spectrophotometrically with the use of fura 2-AM, essentially as described.13 The results are given as the ratio of fluorescence between 340 and 380 nm, calibrated and calculated with commercially available software (Miracal, Life Science Resources Ltd), according to the recommendations of the manufacturer.
Results and Discussion
Dominant Expression of CysLT2R mRNA in HUVECs
Nested RT-PCR analyses (12 +20 cycles) of total RNA from HUVECs generated a robust signal, corresponding to CysLT2R mRNA (793 bp), whereas no signal for CysLT1R could be detected (Figure 1). However, when the number of cycles was increased to 40 +40, a weak signal for CysLT1R mRNA (947 bp) was obtained, as previously reported.14 To quantify the difference in mRNA levels, we made serial dilutions of the total RNA until the signal for CysLT2R was comparable to that of CysLT1R in undiluted RNA and then corrected the signal intensities for differences in template efficiency (Figure 1). With this technique, the ratio of CysLT2R mRNA to CysLT1R mRNA was calculated as >4000:1. In addition, real-time RT-PCR detected 2130 molecules of CysLT2R mRNA and 4 molecules of CysLT1R mRNA, indicating a 500-fold difference in mRNA levels (Figure 1). However, cautious interpretation is warranted, because the value for CysLT2R transcripts was obtained by extrapolation outside the linear portion of the standard curve.
Rapid Suppression of CysLT2R mRNA Levels by LPS, IL-1β, and TNF-α
Treatment of HUVECs with LPS (100 ng/mL), IL-1β (5 U/mL), or TNF-α (10 ng/mL) for 30, 60, and 120 minutes led to rapid (30 to 60 minutes) suppression (30% to 60%) of the CysLT2R mRNA level (Figure 2). The effects of LPS were reversible, whereas those of IL-1β and TNF-α persisted after 120 minutes, with mRNA levels 20% and 40%, respectively, below those of controls, suggesting that these cytokines reduce the expression of CysLT2R. The levels of CysLT1R mRNA remained very low, and no significant alterations could be detected (data not shown).
Challenge of HUVECs With LTC4, LTD4, and BAY u9773 Elicits Ca2+ Transients That Are Diagnostic for CysLT2R
In the absence of reliable antibodies against CysLT2R, we used the natural agonists LTC4 and LTD4, together with the specific CysLT2R agonist BAY u9773, to identify and functionally characterize CysLT2R on HUVECs. Thus, stimulation of HUVECs with either LTC4 or LTD4 (100 nmol/L) elicited a Ca2+ response that peaked after ≈5 to 10 seconds and lasted for nearly 1 minute (Figure 3). The 2 ligands were almost equipotent, in agreement with the typical ligand specificity of the CysLT2R (LTC4≈LTD4)3–5⇓⇓ and exhibited kinetics, which closely resembled that of thrombin and other G protein–coupled surface receptors on HUVECs.13 Furthermore, stimulation of HUVECs with BAY u97734 elicited a significant Ca2+ response of almost the same strength as that of LTC4 and LTD4, thus providing direct evidence for the presence of functionally intact CysLT2R (Figure 3). Moreover, an initial challenge with LTD4 blocked the Ca2+ response to subsequent (1-minute) stimulation with BAY u9773 (Figure 3), indicating CysLT2R occupancy or desensitization.
CysLT2R Is the Major Functional Receptor for CysLTs in HUVECs
At present, there is no selective CysLT2R antagonist available. However, BAY u9773 is not only a CysLT2R agonist (see Methods) but also a dual CysLT1R and CysLT2R antagonist.4 Therefore, we used BAY u9773 together with MK571, a selective CysLT1R antagonist, to assess the role of CysLT2R in CysLT-induced Ca2+ signaling. Preincubation of HUVECs for 15 minutes with 1 μmol/L BAY u9773 completely blocked the Ca2+ responses to a subsequent stimulation with 100 nmol/L of either LTC4 or LTD4 (Figure 3). In contrast, MK571 (1 μmol/L, 15 minutes) was a very poor antagonist and could only partially, if at all, prevent subsequent Ca2+ mobilization by 100 nmol/L LTC4 or LTD4 (Figure 3). This pharmacologic profile, together with the similar agonistic potency of LTC4 and LTD4, as well as the high level of mRNA (cf Figure 1), strongly indicates that HUVECs almost exclusively express the CysLT2R and that this receptor accounts for the Ca2+ signals induced by CysLTs in these cells. It should be noted that we and others have previously reported that HUVECs express the CysLT1R, albeit at a low level, unless the cells are subjected to long-term treatment with IL-1β.14,17⇓ In light of the results of the present study, it seems likely that CysLT1R on HUVECs lacks functional integrity and will not contribute significantly to Ca2+ signaling triggered by CysLTs.
Potential Role of CysLT2R in the Cardiovascular System
CysLTs have profound effects in the vascular system and constrict coronary vessels with a negative inotropic effect and reduced cardiac output.18 Furthermore, early metabolic studies have suggested that CysLTs play a role in cardiac ischemia19 and only very recently, pharmacologic, genetic, and immunohistochemical data were presented that indicate that 5-lipoxygenase and LTs are involved in atherosclerosis and ischemic heart disease.16,20,21⇓⇓ Furthermore, CysLTs, formed via transcellular routes along a leukocyte–endothelial cell axis, might elicit coronary vasospasm and inflammatory changes in the vasculature.9 For certain contraction-relaxation responses of the vessel wall, CysLT1R has been implicated in signal transduction, particularly during inflammatory states in the microvasculature,22 and CysLT1R mRNA is expressed in smooth muscle cells.2 Most likely, signaling via CysLT2R also contributes significantly to the cardiovascular effects of CysLTs, because mRNA has been identified in the heart, including Purkinje cells, smooth muscle cells, and heart muscle.3–5,23⇓⇓⇓ Interestingly, Hui et al6 recently reported that in the mouse heart, CysLT2R mRNA was also found in certain endothelial cells, in line with our data for HUVECs. Moreover, CysLTs have been found to elicit several specific functional responses in endothelial cells, eg, synthesis of platelet-activating factor,10 secretion of von Willebrand factor, and surface expression of P-selectin.11 Hence, although the cardiovascular actions of CysLTs are complex and involve both the CysLT1R and CysLT2R, our data demonstrate that CysLT2R is the dominating, functional receptor for CysLTs on HUVECs and might thus be involved in the propagation of local as well as systemic effects of these lipid mediators. Certainly, the CysLT2R appears to be an interesting target for pharmacologic intervention in cardiovascular diseases, in particular, ischemic heart disease and myocardial infarction.
This work was financially supported by the Swedish Research Council (O3X-10350, 19X-05991), the Vårdal Foundation, the European Union (QLG1-CT-2001–01521), Konung Gustav V:s 80-årsfond, the Foundation for Strategic Research, the AFA Health Foundation, the Swedish Association against Rheumatism, the Swedish Heart and Lung Foundation, Systembolagets Research Fund, and the funds of Uggla and Wiberg. The authors thank Andreas Habenicht for technical help and discussions.
↵*These authors contributed equally to this work.
- Received April 2, 2003.
- Accepted May 16, 2003.
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