Maria Rosa Accomazzo

CysLT1 receptor is a target for extracellular nucleotide-induced heterologous desensitization: a possible feedback mechanism in inflammation.

Both 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 CysLT1 receptor constitutively and transiently expressed in the human monocyte/macrophage-like U937 or COS-7 cells in response to LTD4 or nucleotides. Exposure to agonist induced a rapid homologous desensitization of the CysLT1 receptor [as measured by the reduction in the maximal agonist-induced intracellular cytosolic Ca2+ ([Ca2+]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 CysLT1 receptor. Conversely, LTD4-induced CysLT1 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 CysLT1 receptor desensitization was unable to cause receptor internalization, induced a faster recovery of CysLT1 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 CysLT1 functionality. Hence, CysLT1 receptor desensitization and trafficking are differentially regulated by the CysLT1 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.

Thromboxane prostanoid receptor in human airway smooth muscle cells: a relevant role in proliferation

Thromboxane A(2) 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 A(2) 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 alpha and beta isoforms of the thromboxane A(2) receptor in smooth muscle cells from human bronchi correlates with protein expression evaluated by radioligand binding of the antagonist, SQ29,548 ([1S-[1alpha,2alpha(Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic-acid) (K(d)=3.4 nM+/-44%CV, coefficient of variation, B(max)=41 fmol/mg prot+/-38%CV). The receptor is functional, as the agonist, U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic-acid), induced a concentration-dependent Ca(2+) transient (EC(50)=0.12 microM+/-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.

Involvement of prenylated proteins in calcium signaling induced by LTD4 in differentiated U937 cells

We investigated signal transduction pathways for LTD4 in the human promonocytic cell line U937 known, upon differentiation, to express CysLT1 receptors. We confirmed the presence of high-affinity binding sites for 3H-LTD4, which, in functional studies, displayed the features of CysLT1 receptor. In fact, three potent and selective CysLT1 receptor antagonists were able to completely inhibit LTD4-induced response. In turn, cytosolic Ca2+ ([Ca2+]i) increase (EC50 = 3.4 nM +/- 27% CV) was only partially sensitive to pertussis toxin (PTx) as well as to the prenylation inhibitor fluvastatin and to the specific geranylgeranylation and farnesylation inhibitors BAL 9504 and FPT II. Finally, Clostridium sordellii lethal toxin, inhibitor of the Ras family of GTPases, and FTS, a potent methyltransferase inhibitor, were both able to partially inhibit LTD4-induced [Ca2+] increase, suggesting a role for a Ras family member in [Ca2+]i regulation. In conclusion, in dU937 LTD4 signal transduction involves: (a) at least two pathways, one sensitive and one insensitive to PTx; (b) isoprenylated proteins, such as betagamma subunits and, possibly, a small G protein of the Ras family.

Synthesis of cysteinyl leukotrienes in human endothelial cells: subcellular localization and autocrine signaling through the CysLT2 receptor

The purpose of this study was to characterize enzyme, receptor, and signaling involved in the synthesis and the activity of cysteinyl leukotrienes (cys‐LTs) in human umbilical vein endothelial cells (HUVECs). We used primary cultures of HUVECs and evaluated the formation of cys‐LTs by RP‐HPLC. Suicide inactivation and subcellular localization of the enzyme responsible for the conversion of leukotriene (LT) A4 into LTC4 were studied by repeated incubations with LTA4 and immunogold electron microscopy. The CysLT2 receptor in HUVECs was characterized by equilibrium binding studies, Western blot analysis, and immunohistochemistry. Concentration‐response curves in HUVECs and in transfected COS‐7 cells were used to characterize a novel specific CysLT2 receptor antagonist (pA2 of 8.33 and 6.79 against CysLT2 and CysLT1 receptors, respectively). The results obtained provide evidence that the mGST‐II synthesizing LTC4 in HUVECs is pharmacologically distinguishable from the LTC4‐synthase (IC50 of MK886 <5 μM for LTC4‐synthase and >30 μM for mGST‐II), is not suicide‐inactivated and is strategically located on endothelial transport vesicles. The CysLT2 receptor is responsible for the increase in intracellular Ca2+ following exposure of HUVECs to cys‐LTs and is coupled to a pertussis toxin‐insensitive Gq protein. The synthesis of cys‐LTs from LTA4 by endothelial cells is directly associated with the activation of the CysLT2 receptor (EC50 0.64 μM) in a typical autocrine fashion.—Carnini, C., Accomazzo, M. R., Borroni, E., Vitellaro‐Zuccarello, L., Durand, T., Folco, G., Rovati, G. E., Capra, V., Sala, S. Synthesis of cysteinyl leukotrienes in human endothelial cells: subcellular localization and autocrine signaling through the CysLT2 receptor. FASEB J. 25, 3519–3528 (2011). www.fasebj.org

Bell-shaped curves for prostaglandin-induced modulation of adenylate cyclase: two mutually opposing effects

Each of the natural prostanoid is at least one order of magnitude more potent for its specific receptor (DP, EP, FP, IP and TP) than any of the other prostanoids. However, they are able to interact also with one or more of the other classes of prostanoid receptors. The concentration-response curves for modulation of adenylate cyclase activity in rabbit mesenteric artery smooth muscle cells by different prostaglandins are not always monotonic, i.e. simple sigmoidal curves in logarithmic scale, but they are often biphasic. Prostacyclin, iloprost and prostaglandin E(1) showed a convex bell-shaped curve, i.e. adenylate cyclase activity is stimulated at lower concentrations and inhibited at higher concentrations, while the curve of prostaglandin E(2) showed a concave bell-shaped curve, i.e. adenylate cyclase is inhibited at lower concentrations and stimulated at higher concentrations. By selectively inhibiting one of the transduction mechanisms present in mesenteric smooth muscle cells, we have demonstrated that the observed responses to these prostanoids are likely due to two mutually opposing effects. Thus, the data previously published by our laboratory on a prostacyclin analog, 5(Z)-carbacyclin, might be reinterpreted more correctly in the light of this new possibility.

Heterotrimeric G proteins demonstrate differential sensitivity to β-arrestin dependent desensitization

G15 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 neurotransmitters.

Iloprost binding and inhibition of aggregation in platelet rich plasma: Differences between normal and type IIa hypercholesterolemic subjects

Platelets from type IIa hypercholesterolemic subjects have been previously shown to be less sensitive than normal platelets to the antiaggregatory effect of PGI2. We demonstrate here that these platelets display a reduced response to iloprost, a chemically stable analogue of PGI2, as well. In fact, the concentration of iloprost yielding 50% inhibition of PRP aggregation was higher in type IIa patients (0.77 +/- 0.08 nM) than in controls (0.51 +/- 0.06 nM, P less than 0.01). In addition, an inverse relationship existed between the threshold aggregatory concentration for collagen and the concentration of iloprost yielding 50% inhibition of PRP aggregation, both in type IIa and normal individuals. In order to elucidate the mechanism of the different sensitivity of platelets to prostacyclin and its analogue, we characterized the binding of 3H-iloprost to platelet rich plasma from single individuals. The binding was rapid, reversible, inhibited by iloprost, PGI2 and PGE1 (Kd = 50.7; 346.2 and 7500 nM, respectively); no heterogeneity of sites could be demonstrated in the PRP from a single individual. When binding studies were carried out in PRP of type IIa patients and controls, it appeared that the amount of 3H-iloprost bound at a fixed (300 nM) concentration was significantly lower in platelets from type IIa individuals (0.94 +/- 0.17 vs. 1.77 +/- 0.27 fmol/10(6) platelets, for patients and controls, respectively). It is concluded that such difference in binding might represent the mechanism underlying the reduced response to PGI2 and iloprost observed in platelets from type IIa patients.

A role for inflammatory mediators in heterologous desensitization of CysLT1 receptor in human monocytes

Cysteinyl-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 CysLT1 receptor (CysLT1R)-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 Gi-coupled receptor (FPR1, BLT1) desensitize CysLT1R-induced Ca2+ response unidirectionally through activation of PKC. Conversely, PAF-R, exclusively coupled to Gq, cross-desensitizes CysLT1R without the apparent involvement of any kinase. Interestingly, Gs-coupled receptors (β2AR, H1/2R, EP2/4R) are also able to desensitize CysLT1R response through activation of PKA. Heterologous desensitization seems to affect mostly the Gi-mediated signaling of the CysLT1R. 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.

Receptors and Second Messengers for Cys-Leukotrienes

It is well known that leukotrienes (LTs) can be divided into two classes, which differ for both the chemical structure and the pattern of biological activities: one class encompasses LTB4 only, an hydroxyacid that, mainly by means of its chemotactic action, plays a fundamental role in inflammation. The second class comprises LTC4, LTD4 and LTE4, cysteine containing leukotrienes (Cys-LTs), which can be interconverted into one another. They are potent smooth muscle constrictors, cause mucus hypersecretion in the airways and contribute to the onset of inflammation by their ability to cause plasma extravasation. Given these differences, it is not unexpected that, although both classes have been demonstrated to elicit their actions through the interaction with specific receptors, the receptors for LTB4 are totally different from those for Cys-LTs and no cross-reactivity has been shown among the two classes.

Full and Partial Agonists of Thromboxane Prostanoid Receptor Unveil Fine Tuning of Receptor Superactive Conformation and G Protein Activation

The intrahelical salt bridge between E/D3.49 and R3.50 within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of E3.49/6.30 in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow “resistant” to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.