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CysLT1 signal transduction in differentiated U937 cells involves the activation of the small GTP-binding protein Ras
No full textWe investigated the signal transduction pathway(s) of leukotriene D4 (LTD4) in the human promonocytic U937 cells, a cell line known to constitutively express CysLT1 receptors. Herein, we demonstrate that LTD4 specifically acts on a CysLT1 receptor to dose-dependently increase (3-5 fold over basal) RasGTP through a Gi/o protein. In fact, while cytosolic Ca2+ ([Ca2+]i) increase was only partially sensitive to pertussis toxin (PTx), Ras activation was almost completely inhibited by the same toxin. Furthermore, the phospholipase C (PLC) inhibitor U73122 completely inhibited both [Ca2+]i and RasGTP increase, suggesting that in these cells PLC is the point of convergence for both PTx insensitive and sensitive pathways leading to [Ca2+]i release and Ras activation. Indeed, chelating intracellular Ca2+ strongly (>70%) prevented LTD4-induced Ras activation, indicating that this ion plays an essential role for CysLT1-induced downstream signaling in differentiated U937 (dU937) cells. In addition, while Src did not appear to be substantially involved in CysLT1-induced signaling, genistein was able to partially inhibit LTD4-induced [Ca2+]I transient (~34%) and almost completely prevented Ras activation (>90%), suggesting a potential role for other Ca2+-dependent tyrosine kinases in LTD4-induced signaling. Finally, agonist-induced CysLT1 stimulation was followed by a specific extracellular regulated kinase (ERK) 1/2 phosphorylation, an event with a pharmacological profile similar to that of Ras activation, partially (~40%) sensitive to Clostridium sordellii lethal toxin and totally blocked by PTx. In conclusion, LTD4-induced CysLT1 receptor activation in dU937 cells leads to Ras activation and ERK phosphorylation mostly through a PTx-sensitive Gi/o protein, PLC, and Ca2+-dependent tyrosine kinase(s)
CysLT1 receptor is a target for extracellular nucleotides-induced heterologous desensitization : a possible feed-back mechanism in inflammation
No full textCysLT1 receptor is a target for extracellular nucleotide-induced heterologous desensitization: a possible feedback mechanism in inflammation
No full textBoth cysteinyl-leukotrienes and extracellular nucleotides mediate inflammatory responses via specific G-protein-coupled receptors, the CysLT and the P2Y receptors, respectively. Since these mediators accumulate at sites of inflammation, and inflammatory cells express both classes of receptors, their responses are likely to be crossregulated. We investigated the molecular basis of desensitization and trafficking of the CysLT(1) receptor constitutively and transiently expressed in the human monocyte/macrophage-like U937 or COS-7 cells in response to LTD(4) or nucleotides. Exposure to agonist induced a rapid homologous desensitization of the CysLT(1) receptor [as measured by the reduction in the maximal agonist-induced intracellular cytosolic Ca(2+) ([Ca(2+)](i)) transient], followed by receptor internalization (as assessed by equilibrium binding and confocal microscopy). Activation of P2Y receptors with ATP or UDP induced heterologous desensitization of the CysLT(1) receptor. Conversely, LTD(4)-induced CysLT(1) receptor activation had no effect on P2Y receptor responses, which suggests that the latter have a hierarchy in producing desensitizing signals. Furthermore, ATP/UDP-induced CysLT(1) receptor desensitization was unable to cause receptor internalization, induced a faster recovery of CysLT(1) functionality and was dependent upon protein kinase C. By contrast, homologous desensitization, which is probably dependent upon G-protein-receptor kinase 2 activation, induced a fast receptor downregulation and, accordingly, a slower recovery of CysLT(1) functionality. Hence, CysLT(1) receptor desensitization and trafficking are differentially regulated by the CysLT(1) cognate ligand or by extracellular nucleotides. This crosstalk may have a profound physiological implication in the regulation of responses at sites of inflammation, and may represent just an example of a feedback mechanism used by cells to fine-tune their responses
Cys-leukotriene receptors, their antagonists and transduction mechanism in human airways
No full textA role for inflammatory mediators in heterologous desensitization of CysLT(1) receptor in human monocytes
No full textCysteinyl-leukotrienes (cysteinyl-LT) are rapidly generated at sites of inflammation and, in addition to their role in asthma, rhinitis, and other immune disorders, are increasingly regarded as significant inflammatory factors in cancer, gastrointestinal, cardiovascular diseases. We recently demonstrated that in monocyte/macrophage-like U937 cells, extracellular nucleotides heterologously desensitize CysLT(1) receptor (CysLT(1)R)-induced Ca2+ transients. Given that monocytes express a number of other inflammatory and chemoattractant receptors, this study was aimed at characterizing transregulation between these different stimuli. We demonstrate that in U937 cells and in primary human monocytes, a series of inflammatory mediators activating G(i)-coupled receptor (FPR1, BLT1) desensitize CysLT(1)R-induced Ca2+ response unidirectionally through activation of PKC. Conversely, PAF-R, exclusively coupled to G(q), cross-desensitizes CysLT(1)R without the apparent involvement of any kinase. Interestingly, G(s)-coupled receptors (beta(2)AR, H1/2R, EP2/4R) are also able to desensitize CysLT(1)R response through activation of PKA. Heterologous desensitization seems to affect mostly the G(i)-mediated signaling of the CysLT(1)R. The hierarchy of desensitization among agonists may be important for leukocyte signal processing at the site of inflammation. Considering that monocytes/macrophages are likely to be the major source of cysteinyl-LT in many immunological and inflammatory processes, shedding light on how their receptors are regulated will certainly help to better understand the role of these cells in orchestrating this complex network of integrated signals.-Capra, V., M. R. Accomazzo, F. Gardoni, S. Barbieri, and G. E. Rovati. A role for inflammatory mediators in heterologous desensitization of CysLT1 receptor in human monocytes
CysLT(1) leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors
No full textMontelukast and pranlukast are orally active leukotriene receptor antagonists selective for the CysLT1 receptor. Conversely, the hP2Y1,2,4,6,11,12,13,14 receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937). Montelukast and pranlukast inhibited the effects of UTP with IC50 values of 7.7 and 4.3 mM, respectively, and inhibited the effects of UDP with IC50 values of 4.5 and 1.6 mM, respectively, in an insurmountable manner. Furthermore, ligand binding studies using [3H]LTD4 excluded the possibility of orthosteric nucleotide binding to the CysLT1 receptor. dU937 cells were shown to express P2Y2, P2Y4, P2Y6, P2Y11, P2Y13 and P2Y14 receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing human P2Y1,2,4,6 receptors, CysLT1 antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization. IC50 values at P2Y1 and P2Y6 receptors were <1 mM. In control astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10 mM montelukast had no effect on the carbachol-induced rise in intracellular Ca2+. These data demonstrated that CysLT1 receptor antagonists interact functionally with signaling pathways of P2Y receptors, and this should foster the study of possible implications for the clinical use of these compounds in asthma or in other inflammatory conditions
The new calcium antagonist lercanidipine and its enantiomers affect major processes of atherogenesis in vitro: is calcium entry involved?
No full textAtherosclerosis results from multiple factors and involves several mechanisms, including endothelial monocyte and smooth muscle cell (SMC) changes, cholesterol accumulation, plaque rupture and thromboembolism. Calcium ions play a role in the initial and chronic development of atherosclerotic lesions. Several studies in experimental animal models have demonstrated the potential direct antiatherosclerotic effects of calcium antagonists. In this study the antiatherogenic activity of lercanidipine, a new lipophilic, second-generation calcium antagonist, was investigated. Lercanidipine and its enantiomers inhibited the replication and migration of arterial myocytes in concentrations ranging from 10 to 50 microM. The antiproliferative effect of lercanidipine was dose dependent, with a potency similar to that of lacidipine and nifedipine, and was unrelated to the stereoselectivity of enantiomers to bind L-type calcium channels. Lercanidipine and its enantiomers (25 microM) decreased the serum-induced elevation of [Ca2+]i in SMC, with the (S)-enantiomer (69% inhibition) being 2.4-fold more active than the (R)-counterpart (29% inhibition). The studies performed with enantiomers of lercanidipine suggest that the observed effects are not related to the blockade of voltage-dependent Ca2+ channels and confirm, at least in vitro, the pharmacological potential of the compound to influence negatively the process of atherogenesis
Thromboxane prostanoid receptor in human airway smooth muscle cells : a relevant role in proliferation
No full textThromboxane A2 has been implicated as a mediator of bronchial hyperresponsiveness in asthma. Modulating agents are currently marketed in Japan and under clinical evaluation in the US, but full characterization of the thromboxane A2 receptor and the signaling pathways that link it to the proliferative events taking place during airways structural remodeling has not been achieved. Here, we report that the presence of mRNA for both α and β isoforms of the thromboxane A2 receptor in smooth muscle cells from human bronchi correlates with protein expression evaluated by radioligand binding of the antagonist, SQ29,548 ([1S-[1α,2α(Z),3α,4α]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic-acid) (Kd= 3.4 nM ± 44%CV, coefficient of variation, Bmax= 41 fmol/mg prot ± 38%CV). The receptor is functional, as the agonist, U46619 (9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic-acid), induced a concentration-dependent Ca2+ transient (EC50= 0.12 μM ± 27%CV). Furthermore, U46619 concentration dependently increased DNA synthesis and markedly potentiated the epidermal growth factor mitogenic effect. Both events were specifically inhibited by SQ29,548, independently from transactivation of the epidermal growth factor receptor and partially sensitive to pertussis toxin
Heterotrimeric G proteins demonstrate differential sensitivity to beta-arrestin dependent desensitization
No full textG15 is a heterotrimeric G protein of the Gq/11 family. In this study, we describe its exceptional poor sensitivity to the general regulatory mechanism of G protein-coupled receptor (GPCR) desensitization. Enhancing beta2 adrenergic receptor desensitization by arrestin overexpression, did not affect signalling to G15. Similarly, increased levels of arrestin did not affect G15 signalling triggered by the activation of V2 vasopressin and delta opioid receptors. Furthermore, co-immunoprecipitation experiments showed that G15 alpha subunit (as opposed to Galphaq and Galphas) is recruited to a V2 vasopressin receptor mutant that is constitutively desensitized by beta-arrestin. Interestingly, co-expression of Galpha15 partially rescued cell surface localization and signalling capabilities of the same mutant receptor and reduced beta2 adrenergic receptor internalization. Taken together, these findings provide evidence for a novel mechanism whereby GPCR desensitization can be bypassed and G15 can support sustained signalling in cells chronically exposed to hormones or neurotransmitter
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