Lipopolysaccharide pretreatment increases the sensitivity of the TRPV1 channel and promotes an anti-inflammatory phenotype of capsaicin-activated macrophages.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) is well-established in neuronal function, yet its role in immune reactions remains enigmatic. The conflicting data on its inflammatory role, suggesting both pro-inflammatory and anti-inflammatory effects upon TRPV1 stimulation in immune cells, adds complexity. To unravel TRPV1 immunomodulatory mechanisms, we investigated how the TRPV1 agonist capsaicin influences lipopolysaccharide (LPS)-induced pro-inflammatory macrophage phenotypes. RESULTS: Changes in the surface molecules, cytokine production, and signaling cascades linked to the phenotype of M1 or M2 macrophages of the J774 macrophage cell line and bone marrow-derived macrophages, treated with capsaicin before or after the LPS-induced inflammatory reaction were determined. The functional capacity of macrophages was also assessed by infecting the stimulated macrophages with the intracellular parasite Leishmania mexicana. CONCLUSION: Our findings reveal that TRPV1 activation yields distinct macrophage responses influenced by the inflammatory context. LPS pre-treatment followed by capsaicin activation prompted increased calcium influx, accompanied by a shift toward an anti-inflammatory M2b-like polarization state.

Spatiotemporal GPCR signaling illuminated by genetically encoded fluorescent biosensors.

G protein-coupled receptors (GPCRs) are ligand-activated cell membrane proteins and represent the most important class of drug targets. GPCRs adopt several active conformations that stimulate different intracellular G proteins (and other transducers) and thereby modulate second messenger levels, eventually resulting in receptor-specific cell responses. It is increasingly accepted that not only the type of active signaling protein but also the duration of its stimulation and the subcellular location from where receptors signal distinctly contribute to the overall cell response. However, the molecular principles governing such spatiotemporal GPCR signaling and their role in disease are incompletely understood. Genetically encoded, fluorescent biosensors-in particular for the GPCR/cAMP signaling axis-have been pivotal to the discovery and molecular understanding of novel concepts in spatiotemporal GPCR signaling. These include GPCR priming, location bias, and receptor-associated independent cAMP nanodomains. Here, we review such technologies that we believe will illuminate the spatiotemporal organization of other GPCR signaling pathways that define the complex signaling architecture of the cell.

ß-Arrestin 1 and 2 similarly influence µ-opioid receptor mobility and distinctly modulate adenylyl cyclase activity.

ß-Arrestins are known to play a crucial role in GPCR-mediated transmembrane signaling processes. However, there are still many unanswered questions, especially those concerning the presumed similarities and differences of ß-arrestin isoforms. Here, we examined the roles of ß-arrestin 1 and ß-arrestin 2 at different levels of µ-opioid receptor (MOR)-regulated signaling, including MOR mobility, internalization of MORs, and adenylyl cyclase (AC) activity. For this purpose, naïve HEK293 cells or HEK293 cells stably expressing YFP-tagged MOR were transfected with appropriate siRNAs to block in a specific way the expression of ß-arrestin 1 or ß-arrestin 2. We did not find any significant differences in the ability of ß-arrestin isoforms to influence the lateral mobility of MORs in the plasma membrane. Using FRAP and line-scan FCS, we observed that knockdown of both ß-arrestins similarly increased MOR lateral mobility and diminished the ability of DAMGO and endomorphin-2, respectively, to enhance and slow down receptor diffusion kinetics. However, ß-arrestin 1 and ß-arrestin 2 diversely affected the process of agonist-induced MOR endocytosis and exhibited distinct modulatory effects on AC function. Knockdown of ß-arrestin 1, in contrast to ß-arrestin 2, more effectively suppressed forskolin-stimulated AC activity and prevented the ability of activated-MORs to inhibit the enzyme activity. Moreover, we have demonstrated for the first time that ß-arrestin 1, and partially ß-arrestin 2, may somehow interact with AC and that this interaction is strongly supported by the enzyme activation. These data provide new insights into the functioning of ß-arrestin isoforms and their distinct roles in GPCR-mediated signaling.

ß-Arrestin 2 and ERK1/2 Are Important Mediators Engaged in Close Cooperation between TRPV1 and µ-Opioid Receptors in the Plasma Membrane.

The interactions between TRPV1 and µ-opioid receptors (MOR) have recently attracted much attention because these two receptors play important roles in pain pathways and can apparently modulate each other's functioning. However, the knowledge about signaling interactions and crosstalk between these two receptors is still limited. In this study, we investigated the mutual interactions between MOR and TRPV1 shortly after their activation in HEK293 cells expressing these two receptors. After activation of one receptor we observed significant changes in the other receptor's lateral mobility and vice versa. However, the changes in receptor movement within the plasma membrane were not connected with activation of the other receptor. We also observed that plasma membrane ß-arrestin 2 levels were altered after treatment with agonists of both these receptors. Knockdown of ß-arrestin 2 blocked all changes in the lateral mobility of both receptors. Furthermore, we found that ß-arrestin 2 can play an important role in modulating the effectiveness of ERK1/2 phosphorylation after activation of MOR in the presence of TRPV1. These data suggest that ß-arrestin 2 and ERK1/2 are important mediators between these two receptors and their signaling pathways. Collectively, MOR and TRPV1 can mutually affect each other's behavior and ß-arrestin 2 apparently plays a key role in the bidirectional crosstalk between these two receptors in the plasma membrane.

Naloxone Is a Potential Binding Ligand and Activator of the Capsaicin Receptor TRPV1.

The receptor channel transient receptor potential vanilloid 1 (TRPV1) functions as a sensor of noxious heat and various chemicals. There is increasing evidence for a crosstalk between TRPV1 and opioid receptors. Here we investigated the effect of the prototypical TRPV1 agonist capsaicin and selected opioid ligands on TRPV1 movement in the plasma membrane and intracellular calcium levels in HEK293 cells expressing TRPV1 tagged with cyan fluorescent protein (CFP). We observed that lateral mobility of TRPV1 increased after treatment of cells with capsaicin or naloxone (a nonselective opioid receptor antagonist) but not with DAMGO (a µ-opioid receptor agonist). Interestingly, both capsaicin and naloxone, unlike DAMGO, elicited intracellular calcium responses. The increased TRPV1 movement and calcium influx induced by capsaicin and naloxone were blocked by the TRPV1 antagonist capsazepine. The ability of naloxone to directly interact with TRPV1 was further corroborated by [3H]-naloxone binding. In conclusion, our data suggest that besides acting as an opioid receptor antagonist, naloxone may function as a potential TRPV1 agonist.

The day/night difference in the circadian clock's response to acute lipopolysaccharide and the rhythmic Stat3 expression in the rat suprachiasmatic nucleus.

The circadian clock in the suprachiasmatic nucleus (SCN) regulates daily rhythms in physiology and behaviour and is an important part of the mammalian homeostatic system. Previously, we have shown that systemic inflammatory stimulation with lipopolysaccharide (LPS) induced the daytime-dependent phosphorylation of STAT3 in the SCN. Here, we demonstrate the LPS-induced Stat3 mRNA expression in the SCN and show also the circadian rhythm in Stat3 expression in the SCN, with high levels during the day. Moreover, we examined the effects of LPS (1mg/kg), applied either during the day or the night, on the rhythm in locomotor activity of male Wistar rats. We observed that recovery of normal locomotor activity patterns took longer when the animals were injected during the night. The clock genes Per1, Per2 and Nr1d1, and phosphorylation of kinases ERK1/2 and GSK3ß are sensitive to external cues and function as the molecular entry for external signals into the circadian clockwork. We also studied the immediate changes in these clock genes expressions and the phosphorylation of ERK1/2 and GSK3ß in the suprachiasmatic nucleus in response to daytime or night-time inflammatory stimulation. We revealed mild and transient changes with respect to the controls. Our data stress the role of STAT3 in the circadian clock response to the LPS and provide further evidence of the interaction between the circadian clock and immune system.

TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins.

OBJECTIVES: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to Gq/11 proteins. METHODS: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP). RESULTS: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of Gq/11α, Gß, ß-arrestin2 and phospholipase Cß1, but not of Giα1, ß-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of Giα1 and ß-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling. CONCLUSION: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.

Biased µ-opioid receptor agonists diversely regulate lateral mobility and functional coupling of the receptor to its cognate G proteins.

There are some indications that biased µ-opioid ligands may diversely affect µ-opioid receptor (MOR) properties. Here, we used confocal fluorescence recovery after photobleaching (FRAP) to study the regulation by different MOR agonists of receptor movement within the plasma membrane of HEK293 cells stably expressing a functional yellow fluorescent protein (YFP)-tagged µ-opioid receptor (MOR-YFP). We found that the lateral mobility of MOR-YFP was increased by (D-Ala2,N-MePhe4,Gly5-ol)-enkephalin (DAMGO) and to a lesser extent also by morphine but decreased by endomorphin-2. Interestingly, cholesterol depletion strongly enhanced the ability of morphine to elevate receptor mobility but significantly reduced or even eliminated the effect of DAMGO and endomorphin-2, respectively. Moreover, the ability of DAMGO and endomorphin-2 to influence MOR-YFP movement was diminished by pertussis toxin treatment. The results obtained by agonist-stimulated [35S]GTPγS binding assays indicated that DAMGO exhibited higher efficacy than morphine and endomorphin-2 did and that the efficacy of DAMGO, contrary to the latter agonists, was enhanced by cholesterol depletion. Overall, our study provides clear evidence that biased MOR agonists diversely affect receptor mobility in plasma membranes as well as MOR/G protein coupling and that the regulatory effect of different ligands depends on the membrane cholesterol content. These findings help to delineate the fundamental properties of MOR regarding their interaction with biased MOR ligands and cognate G proteins.