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 CysLT(1) receptor (CysLT(1)R)-induced Ca(2+) 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, BLT(1)) desensitize CysLT(1)R-induced Ca(2+) 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, H(1/2)R, EP(2/4)R) 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.

fi ne tuning of CysLT 1 R function by a series of mediators that largely accumulate at sites of infl ammation might shed light on a new network of integrated signaling in infl ammatory responses.

Isolation of primary human monocytes
Blood from healthy donors was collected into sodium citrate 3.8% or in citrate phosphate dextrose (CPD) and centrifuged at 200 g for 15 min. The obtained plasma reach platelet (PRP) was discharged and the blood was diluted (1:2) with RPMI-1640 culture medium or PBS, layered on a cushion of Ficoll Paque (3:1), and then centrifuged at 500 g for 20 min. The mononuclear lymphocyte cells layer was transferred and washed twice in PBS +0.5% BSA + 0.1% glucose + 5 mM EDTA, once in PBS + 0.1% glucose + 5 mM EDTA and then resuspended in RPMI-1640 culture medium. The cells were seeded (2-3 × 10 6 /mL) on coverslips coated with poly-D-lysine and incubated for 2 h at 37°C. The lymphocytes, which do not adhere to coverslip, were washed with PBS and the adherent monocytes were incubated overnight in RPMI-1640 culture medium at 37°C.

Determination of cytosolic free Ca 2+ levels
Determination of free [Ca 2+ ] i in U937 cells was performed as previously described ( 16,29,30 ). Briefl y, cells were incubated for 30 min at 30°C in the dark with 2 µM Fluo3/AM. After loading, Fluo3/AM was removed and cells were further incubated for 30 min at 30°C to complete the hydrolysis of Fluo3/AM. Then cells were centrifuged, resuspended, diluted to the concentration of 10 6 cells/mL, transferred to the spectrofl uorimeter (Perkin Elmer LS50), and fl uorescence monitored at 37°C (506 nm excitation, 530 nm emission). Primary monocytes were incubated for 1 h at 25°C in the dark with 2µM Fura2/AM. After loading, cells were washed twice with a saline solution, transferred to the spectrofl uorimeter, and fl uorescence monitored at 37°C (505 nm emission, 340 and 380 nm excitation). Calibration was performed by adding 2 µM ionomycin and 100 µM digitonin (F max , maximal fl uorescence of the system) and by adding 5 mM EGTA and 60 mM Trisbase (F min minimal fl uorescence of the system). [Ca 2+ ] i elevation has been expressed as the ratio of stimulated over basal (s/b).
The activation of a number of receptors does not always lead to a direct effect on a particular signaling pathway but, rather, to amplifi cation ( 11,12 ) or, more often, to inhibition of the response produced by distinct but convergent signals (13)(14)(15)(16). In this respect, heterologous desensitization (i.e., the reduced response to activation of an unrelated or apparently unrelated receptor that has not been exposed to an agonist) plays an important role ( 17,18 ). This latter process is generally ascribed to the action of different kinases, such as PKA and PKC, that participate as a feedback mechanisms in which the second messenger activates a kinase that, in turn, modulates the activity of other receptors.
Monocytes/macrophages, besides providing innate immune surveillance for every tissue in the body, are generally believed to sustain and precipitate pathological states. Cytokines, 5-LO metabolites, and several other agents selectively home monocytes to the parenchymal tissue where they participate in the innate immune response and lipid homeostasis ( 19 ). Of particular interest is the ability of macrophages to produce large amounts of proinfl ammatory mediators including cytokines ( 20 ), prostaglandins, and LTs ( 2 ), as well as to express infl ammatory surface receptors such as CysLT 1 ( 3 ). This correlates with the recent fi nding that cysteinyl-LTs might contribute to infl ammatory reactions by induction of MCP-1 ( 21 ) and increasing CCR2B expression in human monocytes/macrophages ( 22 ).
The past decade has revealed that macrophage-derived foam cells are integral to the development and progression of atherosclerosis ( 23 ), while a very recent paper demonstrated that montelukast (Singulair), a specifi c CysLT 1 R antagonist widely used for asthma treatment, prevented ROS production and reduced atherosclerotic plaque formation in apolipoprotein E-defi cient knockout mice in vivo ( 24 ). Monocyte/macrophage lineage may play a potential role in the pathophysiology of COPD ( 25 ) as well in tumor initiation and various key steps in growth and metastasis ( 26 ). Therefore, elucidation of molecular mechanisms of macrophage function and their pharmacological modulation represents an important strategy for the modulation of immune response, prevention, and treatment of diseases.
We have recently demonstrated that in monocytes/macrophages like U937 cells, a CysLT 1 R-induced increase in cytosolic free Ca ] i ) can be modulated by extracellular nucleotides, such as ATP and UDP that, following activation of different P2Y receptors, heterologously desensitize the CysLT 1 R through PKC ( 16 ). Given that monocytes/macrophages besides CysLT 1 R and P2Y-R express a number of other infl ammatory and chemoattractant receptors, such as those for LTB 4 (BLT 1 R), fMLF (previously known as fMLP, [ 27 ]), FPR1, platelet activating factor (PAF-R), histamine H (H 1/2 R), and prostaglandin E 2 (EP 2/4 R) or infl ammatory-related receptors, such as ␤ 2 adrenoreceptor ( ␤ 2 AR) ( 20 ), this study was aimed at characterizing transregulation between these different stimuli ( 13,28 ). Although much has been learned about cellular activation and regulation by single receptors, mechanisms of receptor cross-regulation leading to priming or desensitization are only beginning to be unraveled. The ] i transient following a fi rst challenge with fMLF. Fig.  1A shows the concentration-dependent desensitization curve of fMLF on the response elicited by 10 nM LTD 4 in dU937 cells. Maximum degree of desensitization (77%, P < 0.01) was obtained starting from 3 M fMLF ( Fig. 1A ). This effect can be largely prevented by a 5 min preincubation with increasing concentration of the PKC inhibitor GFX ( Fig. 1B ), suggesting the involvement of the second messenger-activated PKC in the heterologous desensitization of CysLT 1 R. Pretreatment of cells with the PKC inhibitor only marginally increase LTD 4 -induced response, suggesting a minimal tonic desensitization of the CysLT 1 R by PKC in resting conditions (data not shown). Overnight pretreatment of cells with 300 ng/mL PTX completely abolished fMLF-induced desensitization, demonstrating specifi city of the fMLF effect ( Fig. 1B ).
Similar results have been obtained with the chemotactic lipid mediator LTB 4 . Fig. 1C shows that a fi rst challenge with 1 M LTB 4 provoked a reduction in CysLT 1 R-induced Ca 2+ response, reaching a maximal CysLT 1 R desensitization of 50% ( P < 0.01). It is also clear that this effect can be prevented by preincubation with increasing concentrations of GFX. Pretreatment of cells with PTX strongly impaired, but did not abolish, LTB 4 -induced desensitization, in agreement with the effect of PTX on LTB 4 signaling (Table 1). LTB 4 effects can be fully ascribed to the activation of the BLT 1 R, as the specifi c BLT 2 antagonist LY255283 up to 1 M was completely ineffective in inhibiting LTB 4 -induced response in dU937 cells (data not shown). The same is also true for primary human monocytes (HM) (see below).

Effect of a G q -coupled receptor on CysLT 1 -induced [Ca 2+ ] i transient
We also examined the possible effect of a "pure" G q/11coupled receptor, such as PAF-R, on the regulation of Cys-LT 1 R activity. Fig. 2A shows that a fi rst challenge with 36 nM PAF affected LTD 4 -induced response up to 35% ( P < 0.01) and that this effect cannot be substantially prevented by increasing concentration of GFX. In addition, none of the following were able to completely revert PAF-induced CysLT 1 desensitization: pretreatment with 10 M H89, a PKA inhibitor; pretreatment with 10 M KN93, a Ca 2+ / calmodulin-dependent kinase II (CaMKII) inhibitor; and The heterologous desensitization protocol was performed as previously described ( 16 ). Briefl y, the second challenge was dispensed 3 min after the fi rst challenge or when [Ca 2+ ] i levels returned to baseline without washing the sample.

Statistical analysis
Statistical analysis of concentration-response curves was performed using GraphPad Prism version 4, which use the four parameters logistic model as described in the ALLFIT program ( 31 ). All curves are computer generated. Statistical comparison of multiple groups was performed using one-way ANOVA followed by Bonferroni's multiple comparison test. Data are expressed as mean ± SEM.

Infl ammatory-related stimuli elicited second messenger generation in DMSO-differentiated U937 cells
It is widely known that LTD 4 elicits a cytosolic Ca 2+ transient ([Ca 2+ ] i ) in DMSO-differentiated U937 (dU937) cells through the activation of the CysLT 1 R promiscuously coupled to both G q and G i ( 16,29,30 ). In the same cells, the ability to increase [Ca 2+ ] i is shared also by other infl ammatory stimuli through different molecular signaling pathways. Indeed, LTB 4 , fMLF, UTP, and PAF were found to increase [Ca 2+ ] i through the activation of specifi c receptors exclusively coupled either to G q/11 (PAF-R) or G i (FPR1) or promiscuously coupled to both G proteins (BLT 1 R and P2Y 2/6 R) ( Table 1 ). In addition, a number of other infl ammatory or infl ammatory-related stimuli (e.g., PGE 2 , histamine and isoproterenol) were found to increase cAMP levels in dU937 cells through activation of their G s protein-coupled receptors (EP 2/4 R, H 1/2 R, and ␤ 2 AR, respectively) (Table 1). Therefore, these heterogeneous stimuli were studied to expand the previous notion of extracellular nucleotide regulation of CysLT 1 R activity ( 16 ), examining the possibility that they might participate in Cys-LT 1 R regulation through specifi c molecular mechanisms and possibly specifi c hierarchical organization.

Effect of G i -coupled receptors on CysLT 1 R-induced [Ca 2+ ] i transient
We examined modulation of CysLT 1 R activation by a "pure" G i -coupled receptor measuring LTD 4 -induced pretreatment with 10 M KT-5823, a PKG inhibitor, alone or in addition to GFX (data not shown). Specifi city of PAF response was assessed by demonstrating that 10 M of the PAF-R antagonist WEB 2086 completely prevented PAF signal (data not shown) and PAF-induced CysLT 1 R desensitization ( Fig. 2B ). ] i stimulation over basal (s/b) ± SEM of at least three independent experiments. Statistical comparison of multiple groups was performed using one-way ANOVA followed by Bonferroni's multiple comparison test (** P < 0.01, versus control, unless otherwise indicated). GFX, GF109203X.

Effect of YM-254890 on PAF-induced CysLT 1 R heterologous desensitization
We previously reported that use of YM-254890 to selectively inhibit the G q signaling pathway produced important augmentation of P2Y-induced CysLT 1 desensitization compared with untreated cells, suggesting that the majority of P2Y-derived desensitization might preferentially affect the G i signaling pathway of the Cys-LT 1 R ( 16 ). Here we found that the increase in the extent of PAF-induced desensitization of the CysLT 1 R directly correlated with the concentration of YM-254890 (administered in between the fi rst challenge with PAF and the second challenge with LTD 4 ), again indicating a preferential desensitization of the G i -coupled pathway of the CysLT 1 R ( Fig. 2C ). As a control, 10 nM of YM-254890 did not statistically affect fMLF receptor signaling (a pure G i coupled receptor), while it completely inhibited PAF signaling (data not shown).

Effect of LTD 4 pretreatment on fMLF-, LTB 4 -, and PAF-induced [Ca 2+ ] i transient
To verify a possible infl uence of CysLT 1 activation on FPR1, BLT 1 R, and PAF-R responses, we measured the fMLF-, LTB 4 -, and PAF-induced [Ca 2+ ] i transient subse-quent to a fi rst challenge with LTD 4 . Fig. 3A and 3B show that fMLF-and LTB 4 -induced responses were only marginally ( P > 0.05) affected by a prior challenge with 10 nM LTD 4 , suggesting that transregulation between Cys-LT 1 R and FPR1 or BLT 1 R occurs only in a unidirectional way. At variance, LTD 4 was able to partially desensitize PAF response (Fig. 3C) (44% reduction, P < 0.01), indicating the existence of cross-desensitization between Cys-LT 1 R and PAF-R in dU937 cells. Moreover, Fig. 3A and 3B clearly demonstrate that after a fi rst challenge with LTD 4 , cells can still be responsive to the second challenge with a different agonist, ruling out the possibility that the heterologous desensitization observed might depend upon an altered ability of the cells to mobilize calcium.

Effect of isoproterenol challenge on CysLT 1 -induced [Ca 2+ ] i transient
As previously mentioned, dU937 cells express a number of receptors for infl ammatory-related stimuli that, at variance with the stimuli previously examined, are not coupled to G q/11 or G i/o , but rather to G s (Table 1). To verify whether cAMP increase could affect LTD 4 -induced response through activation of PKA, we challenged dU937 cells with increasing concentrations of the ␤ 2 AR agonist isoprotere- (53% recovery, P < 0.01) ( Fig. 4B ). It is also clear from Fig.  4B that while GFX at a concentration of 1 M (a concentration at which the compound is specifi c for PKC) had no effect on the isoproterenol-induced CysLT 1 R desensitization, at 10 M (a concentration at which GFX start to inhibit also PKA [ 32 ]), it had an effect similar to that of H89. Pretreatment of cells with the PKA inhibitor did not alter the LTD 4 -induced response (data not shown). Finally, to assess the specifi city of the observed responses, we demonstrated that pretreatment with propranolol, a competitive antagonist at the ␤ 2 AR, prevented isoproterenol-induced CysLT 1 R desensitization ( Fig. 4C ). As expected, PTX pretreatment was totally ineffective on isoproterenol-induced CysLT 1 R desensitization (data not shown).

2+
] i transient Similar to isoproterenol, PGE 2 and histamine also, through their specifi c receptors coupled to cAMP production (EP 2/4 and H 1/2 receptors, respectively) ( Table 1), induced desensitization of the CysLT 1 R up to 61% and 56%, respectively ( P < 0.01) ( Fig. 5A , B ). Again, both effects could only be partially reverted by 10 M GFX or 10 H89. Finally, similar to isoproterenol data, there was no effect of PTX pretreatment on PGE 2 and histamine-induced desensitization (data not shown).

Effect of forskolin pretreatment on CysLT 1 -induced [Ca 2+ ] i transient
To confi rm cAMP-induced CysLT 1 transregulation, we investigated the effect of a non-specifi c activator of adenylate cyclase. 100 M forskolin completely desensitized the CysLT 1 R ( Fig. 6 ), confi rming the involvement of cAMP in regulation of the activity of CysLT 1 R and strongly indicating that also the second messenger-activated PKA is able to heterologously desensitize the CysLT 1 R.

Heterologous desensitization of CysLT 1 receptor in primary human monocytes
To be sure that dU937 cells accurately represented the spectrum of receptors expressed by monocytes/macrophages, we repeated some key experiments in monocytes. Fig. 7 shows that the same results as obtained in dU937 were achieved in freshly isolated primary HM. Agonist and antagonist concentrations were adjusted to the different sensitivity of adherent cells. fMLF, PAF, and isoproterenol specifi cally desensitize 100 nM LTD 4 -induced [Ca 2+ ] i transient (91%, 79%, and 59%, respectively; P < 0.01). fMLF and PAF used the same molecular mechanism observed in dU937 cells; i.e., unidirectional for fMLF and complete cross-desensitization for PAF (34% inhibition, data not shown). Finally, as previously observed, the PAF effect was not prevented by pretreatment with 10 M GFX (data not shown).

DISCUSSION
Here we report that in dU937 cells and in primary HM a series of infl ammatory mediators activating G i -coupled receptors (FPR1, BLT 1 , and P2Y 4 ) desensitize CysLT 1 R re-nol. Indeed, Fig. 4A shows that isoproterenol dose-dependently impaired the CysLT 1 R response up to 70% and that this effect can be only partially reverted by pretreatment of cells with 10 M of the widely used PKA inhibitor H89 in many pathological states. As we and others have previously demonstrated ( 29,30,33,34 ), one of the principal CysLT 1 R-induced responses in these cells is the increase of [Ca 2+ ] i . Classically, G q -coupled receptors are the ones responsible for inositol phosphate (IP)3 and [Ca 2+ ] i generation. However, it is now known that PLC ␤ 2 and PLC ␤ 3 are also activated by the ␤ ␥ subunit of the G i family of G proteins, as is the case for CysLT 1 R ( 16,30 ). sponse in a unidirectional way through activation of PKC and that a hierarchy becomes evident in the desensitization process. Conversely, PAF-R, exclusively coupled to G q in these cellular systems, not only transregulates CysLT 1 R without the apparent involvement of a second messenger-activated kinase but also is cross-desensitized by the CysLT 1 R. Interestingly, G s -coupled receptors ( ␤ 2 AR, H 1/2 R, and EP 2/4 R) are able to desensitize CysLT 1 R response through activation of PKA. As we already suggested for extracellular nucleotides, heterologous desensitization seems to affect mostly the G imediated signaling of the promiscuous (G q /G i ) CysLT 1 R.
The human promonocytic leukemia U937 cell line, known to constitutively express only CysLT 1 Rs ( 16,29,30 ), has been selected to study the regulation of this important infl ammatory receptor in a cellular system that is generally believed to contribute to the innate immune surveillance  ceptors constitutively expressed in dU937 cells and human monocytes but differently coupled to calcium homeostasis through the activation of G i or G q heterotrimeric G proteins. We fi rst examined the effect of the activation of two "pure" G i coupled receptors, such as those activated by fMLF and LTB 4 , and of a promiscuously coupled receptor (G i and G q , at least in leukocytes), such as P2Y 4 , preferentially activated by UTP (data not shown). The CysLT 1 R desensitization had an extent similar to that previously published for ATP and UDP ( 16 ) and was almost completely prevented by the PKC inhibitor GFX, confi rming the ability of this second messengeractivated kinase to heterologously regulate CysLT 1 R func tionality. In agreement with our previous work, none of the [Ca 2+ ] i responses induced by these stimuli could be affected by a fi rst challenge with LTD 4 (unidirectional regulation). This was not totally unexpected, at least for fMLF, because it is known that FPR1 is resistant to this process because of the lack of PKC phosphorylation sites ( 35 ).
Surprisingly, in examining the ability of PAF-R to modulate CysLT 1 R response, we found that PAF-induced desensitization could not be prevented by preincubation of cells not only with GFX but also with inhibitors of PKA, PKG or CaMKII, alone or in combination with GFX. Although we cannot rule out the possibility that another kinase might be involved, these data point to additional mechanism(s) of transregulation besides phosphorylation ( 36 ), either at the receptor level or distal to receptor/G protein coupling. Unfortunately, despite several attempts to study CysLT 1 R phosphorylation in dU937 cells, we could not demonstrate even an agonist-induced phosphorylation (data not shown), likely because of the insuffi cient level of endogenous expression to permit detection of 32 P incorporation in immunoprecipitated receptors, as already reported by other groups ( 37 ).
At this stage we can only speculate about a number of possible phosphorylation-independent mechanisms ( 38 ). One possibility is a reduced activation of PLC ( 39 ) or an IP3-receptor downregulation ( 40 ). Another possible mechanism is a phosphorylation-independent GRK2-mediated sequestration of G ␣ q ( 41 ). Alternatively, because members of the G ␣ q subfamily are known have a slow intrinsic GTPase activity in vitro ( 42 ), their rate is increased dramatically by the so-called regulators of G protein signaling (RGS) proteins. It has been demonstrated that RGS4 is able to inhibit [Ca 2+ ] i mobilization induced by PAF-R, as well as PAF-and PMA-induced PAF-R phosphorylation; it also blocked cross-phosphorylation by fMLF ( 43 ). Furthermore, LTD 4 also cross-desensitized the PAF-R, albeit partially.
Because U937 cells are a leukemia-derived cell line, we confi rmed our observation repeating some key experiments in primary HM that also have been reported to express mainly the CysLT 1 R transcript ( 44 ). We confi rmed that the LTD 4induced [Ca 2+ ] i transient was sensitive to montelukast and PTX (data not shown), indicating that this response is because of a CysLT 1 R activation ( 44 ) similar to the one observed in dU937 cells ( 16,29,30 ). Basically, all the data observed in dU937 cells have been confi rmed in primary HM, validating our observations and demonstrating that our model accurately represents the spectrum of receptors expressed in monocytes.
We have previously demonstrated that CysLT 1 receptor is heterologously desensitized by P2Y 2-and P2Y 6 receptors for ATP and UDP, respectively, in a unidirectional way ( 16 ).
Here we expand those observations to different classes of re- ] i stimulation over basal (s/b) ± SEM of at least three independent experiments. Statistical comparison of multiple groups was performed using one-way ANOVA followed by Bonferroni's multiple comparison test (** P < 0.01, vs. control).
leading to fi ne regulation of the ultimate physiological responses ( 48,49 ). Very recently GPR17, an orphan GPCR recently proposed to respond to both cysLTs and extracellular nucleotides ( 8 ), also seems to negatively regulate CysLT 1 R ( 50 ). Another group has also demonstrated intracellular localization of a functional CysLT 1 R, which adds complexity and opens new potential pathways for receptor signaling and transregulation ( 51 ).
Furthermore, comparison of the ability of fMLF, C5a, and IL-8 to desensitize one another has led to the observation of hierarchy in receptor desensitization between these chemoattractant receptors. This hierarchy may be important for leukocyte signal processing when multiple infl ammatory mediators are present at sites of infl ammation. Under these conditions, the "end-target mediators," such as fMLF, that are more capable of activating terminal functions of neutrophils seem to prevail over "intermediary mediators," such as LTD 4 and other chemokines present at the site of infl ammation ( 52 ).
Considering that monocytes/macrophages are likely to be the major source of cysteinyl-LT in many immunological and infl ammatory processes, shedding light on how their receptors are regulated will certainly help to better understand the role of these cells in orchestrating the complex network of integrated signals.
All these observations suggest a different regulation of the CysLT 1 R in U937 cells and in primary HM based on the peculiar signaling pathway used by the different receptors to regulate [Ca 2+ ] i homeostasis. Confi rmation of these data also comes from experiments performed in a recombinant system, namely, HEK293 cells transiently transfected with the CysLT 1 R, where we and others ( 45 ) have demonstrated that this receptor is exclusively coupled to G q . In this system we found a complete cross-desensitization between the CysLT 1 R and P2Y 2 -R that was not reverted by GFX (data not shown). Thus, changing the coupling from a promiscuous G q /G i (dU937) or pure G i (HM) to a pure G q (HEK293) cellular system produces a change also in the regulation of receptor functionality. This observation, therefore, should be a warning about the extrapolation of physiological data from results obtained in recombinant systems.
As we previously suggested, CysLT 1 R heterologous desensitization seems to affect mostly the G i -coupled pathway of this promiscuous receptor ( 16 ). In this work, by dose-dependently inhibiting the G q -coupled pathway of the CysLT 1 R (without affecting the PAF-R response), we were able to demonstrate an increase in receptor desensitization induced by PAF. These data suggest a predominant desensitization of the G i -coupled pathway of the CysLT 1 R in our system.
In an in vitro model of asthma, we have recently demonstrated the potential role of CysLT 1 R in the desensitization of the ␤ 2 AR, highlighting another possible network of cross-talk in infl ammation ( 46 ). Here we demonstrate that not only G i -and G q -coupled receptors but also receptors coupled to G s (i.e., ␤ 2 AR, H 1/2 , and EP 2/4 ) can desensitize the CysLT 1 R in an heterologous way. Elevated cAMP levels through the activation of PKA are known to cause phosphorylation of either receptors or PLC ␤ isoforms blocking G ␤ ␥ -mediated activation ( 47 ). Thus, isoproterenol-, PGE 2 -, and histamine-mediated cAMP increase in dU937 cells desensitized CysLT 1 R either because PKA directly phosphorylates the receptor or because it phosphorylates PLC ␤ . The fact that the PKA inhibitor H89 only partially prevented G s -dependent CysLT 1 R desensitization might depend on the limits of pharmacological inhibition or on the fact that additional mechanisms might be involved. As far as we know, this is the fi rst demonstration that PKA might regulate the activity of CysLT 1 R either in a constitutive or recombinant system. Lipid mediators were historically considered infl ammatory mediators, causing symptoms such as fever, pain, and infl ammation. However, recent studies using gene knockout mice (for both biosynthetic enzymes and receptors) have revealed that lipid mediators play a much more fundamental role in normal physiological processes and in disease. The fact that most of the stimuli tested desensitize the CysLT 1 R in a unidirectional way seems to suggest a hierarchy in the desensitization process. This hierarchy in the circuit of infl ammation might refl ect either a temporal-or a concentration-dependent control of infl ammatory events, thus regulating the amplifi cation of the infl ammatory process.
It is becoming clear that receptor crosstalk is the rule and not the exception in cellular signaling and that an integration and communication network presumably tunes the strength and duration of the signals, thereby