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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
-Induced Inflammation with Extraordinary PotencyDepartment of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana
Received for publication
July 11, 2008
Accepted
August 14, 2008.
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Abstract |
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-mediated inflammation. 5-HT2A receptor stimulation with the agonist (R)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(R)-DOI] rapidly inhibits a variety of TNF--mediated proinflammatory markers, including intracellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), and interleukin (IL)-6 gene expression, nitric-oxide synthase activity, and nuclear translocation of nuclear factor 
B, with IC50 values of only 10 to 20 pM. It is significant that proinflammatory markers can also be inhibited by (R)-DOI hours after treatment with TNF-. With the exception of a few natural toxins, no current drugs or small molecule therapeutics demonstrate a comparable potency for any physiological effect. TNF--mediated inflammatory pathways have been strongly implicated in a number of diseases, including atherosclerosis, rheumatoid arthritis, psoriasis, type II diabetes, depression, schizophrenia, and Alzheimer's disease. Our results indicate that activation of 5-HT2A receptors represents a novel, and extraordinarily potent, potential therapeutic avenue for the treatment of disorders involving TNF--mediated inflammation. Note that because (R)-DOI can significantly inhibit the effects of TNF- many hours after the administration of TNF-, potential therapies could be aimed not only at preventing inflammation but also treating inflammatory injury that has already occurred or is ongoing.
). These behaviors are mediated through interactions at seven different receptor families (5-HT1–7) comprised of 14 distinct subtypes (Nichols and Nichols, 2008). Each of these are G protein-coupled receptors, with the exception of the 5-HT3 receptor, which is a ligand-gated ion channel. Of all the serotonin receptors, the 5-HT2A receptor, which is known to primarily couple to the Gq effector pathway (Roth et al., 1986), has been the one most closely linked to complex behaviors. There is a high level of expression within the frontal cortex, with significant localization to the apical dendrites of cortical pyramidal cells (Willins et al., 1997), and further expression at lower levels throughout the brain (Nichols and Nichols, 2008). Extensive work has been performed to establish the role of 5-HT2A receptors within the brain, where there they have been shown to participate in processes such as cognition and working memory, been implicated in affective disorders such as schizophrenia, and mediate the primary effects of hallucinogenic drugs (Nichols, 2004).
Significantly, many peripheral tissues also express the 5-HT2A receptor. Within the vasculature, 5-HT2A receptors are known to modulate vasoconstriction (Nagatomo et al., 2004). Its role in other tissues such as mesangial cells of the kidney, fibroblasts, liver, and lymphocytes remains less defined but has been linked to cellular proliferation and differentiation.
The presence of 5-HT2A receptor mRNA (along with the mRNAs of other serotonin receptor subtypes) in many immune system-related tissues (Stefulj et al., 2000) suggests a possible role for this receptor in the immune response. However, the role of 5-HT2A receptors in inflammatory processes is unclear, with only a very few published and inconsistent reports. Some studies report that blockade of 5-HT2A receptor function with the selective antagonist sarpogrelate can decrease expression of proinflammatory markers (Marconi et al., 2003; Akiyoshi et al., 2006), whereas others indicate that sarpogrelate can increase expression of proinflammatory markers (Ito et al., 2000). Cloëz-Tayarani et al. (2003) have alleged that the 5-HT2 receptor-specific agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) represses interleukin (IL)-1β expression and production of tumor necrosis factor- (TNF-) through 5-HT2A receptor activation. Yet, their studies are not informative because they used nonselective receptor antagonists and extremely high nonpharmacologically relevant drug doses.
In two other studies, it was reported that DOI partially blocked lipopolysaccharide and TNF--stimulated nitrite accumulation in C6 glioma cells (Miller et al., 1997; Miller and Gonzalez, 1998). Whereas the authors claimed that this effect was mediated by 5-HT2A receptor stimulation, receptor-selective antagonists were not used. In earlier reports, the synthesis of TNF- in response to lipopolysaccharide was shown to be inhibited by 5-HT through 5-HT2 receptors in monocytes (Arzt et al., 1991). The use of ketanserin as the antagonist to block 5-HT2 receptor activation in many of these studies is problematic. Ketanserin, which was described as a selective 5-HT2A receptor antagonist, only has weak selectivity (
20-fold) for 5-HT2A receptors over 5-HT2C receptors, high affinity for 1-adrenergic receptors, and, significantly, is equipotent at blocking histamine H1 receptors (Ki = 1.8 nM) (Psychoactive Drug Screening Program Ki database: http://pdsp.med.unc.edu), which are well known to regulate inflammatory processes and immune system components. Therefore, at the very least, ketanserin cannot be used to discriminate reliably between the effects of agonists acting at 5-HT2A or 5-HT2C receptors, and furthermore, it seems likely that the observed effects of ketanserin in combination with agonists may be a complex interaction between serotonin, histamine, and adrenergic systems in modulating inflammatory and immune processes.
Nevertheless, the presence of serotonin itself has been demonstrated to be necessary for expression of the inflammatory markers IL-6 and TNF-, with lower serotonin levels inducing, and higher levels decreasing, expression of these markers (Kubera et al., 2005). This inverted U-shaped response clearly indicates that in vivo serotonin plays an important role in modulating molecular components of the inflammatory process. Despite the claims of previous reports, described above, the identity of the serotonin receptor mediating these processes has not been reliably established. In this study, we show through a comprehensive analysis examining proinflammatory gene expression, nitric-oxide synthase (NOS) activity, and activation and translocation of nuclear factor B (NF-B), using relevant doses of selective agents, that activation of 5-HT2A receptors potently inhibits TNF--induced inflammatory markers in a relevant model system: primary aortic smooth muscle cells.
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(rat) was purchased from Pepro-Tech (Rocky Hill, NJ). 5-HT2C receptor antagonist RS102221, HT2B receptor antagonist SB204741, protein kinase C (PKC) inhibitor Gö6976, and PKC inhibitor chelerythrine (1,2-dimethoxy-12-methyl[1,3]benzodioxolo[5,6-c]phenanth ridinium chloride) were purchased from Tocris Bioscience (Ellisville, MO). PKC activator phorbol 12-myristate 13-acetate (PMA) and PKA inhibitor fragment 6–22 (F-22) amide were purchased from Sigma-Aldrich (St. Louis, MO). (R)-DOI, LA-SS-Az, 2C-BCB, and MDL100907 were gifts from Dr. David Nichols (Purdue University, West Lafayette, IN). Lysergic acid diethylamide (LSD) was provided by the National Institute on Drug Abuse.
Rat Aortic Smooth Muscle Cells and Treatment. Rat aortic smooth muscle (RASM) cells were isolated from adult, 180-g male Sprague-Dawley rats and provided by the Cell and Molecular Core Facility in the Department of Pharmacology at LSUHSC. Isolated RASM cells were grown in M-199 medium containing 10% fetal bovine serum (FBS; Invitrogen), 100 units/ml penicillin, and 100 g/ml streptomycin, and incubated at 37°C in 5% CO2. Cells for all assays were used between passages 3 and 5. Before treatments, cells were grown in M-199 medium with 10% FBS until 30 to 50% confluent. For most assays, cells were treated with (R)-DOI, or other drugs as indicated, at specific concentrations for 24 h, followed by TNF- (10 ng/ml) and (R)-DOI at the same pretreatment concentrations in fresh M-199 medium. After another 24 h, the cells were scraped, pelleted, and processed for RNA. Pretreatment and treatment times varied with assays as indicated under Results.
RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction. RNA was extracted from cells using Illustra RNAspin Mini kits from GE Healthcare Life Sciences (Piscataway, NJ) following protocols supplied by the manufacturer. First-strand cDNA was generated using the ImProm-II cDNA synthesis kit (Promega, Madison, WI) following the manufacturer's protocols. Quantitative real-time polymerase chain reaction (PCR) was performed using the ProbeLibrary system from Roche Diagnostics (Indianapolis, IN) in combination with the HotStart-IT Probe qPCR Master Mix from USB (Cleveland, OH) following the manufacturer's protocols.
The sequences of primers used are as follows: 5-HT2AR, forward 5'-TGATGTCACTTGCCATAGCTG-3' and reverse 5'TCGCACAGAGCTTGCTAGG-3'; ICAM-1, forward 5'-TTCTGCCACCATCACTGTGT-3' and reverse 5'-AGCGCAGGATGAGGTTCTT-3'; VCAM-1, forward 5'-CAAATGGAGTCTGAACCCAAA-3' and reverse 5'-GGTTCTTTCGGAGCAACG-3'; IL-6, forward 5'-CCTGGAGTTTGTGAAGAACAACT-3' and reverse 5'-GGAAGTTGGGGTAGGAAGGA-3'; and cyclophilin B (control amplicon), forward 5'-ACGTGGTTTTCGGCAAAGT-3' and reverse 5'-CTTGGTGTTCTCCACCTTCC-3'. Primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). ProbeLibrary probes from Roche Diagnostics were as follows: U3, U74, U13, U106, U79 for 5-HT2AR, ICAM-1, VCAM-1, IL-6, and cyclophilin B, respectively. Quantitative determination of gene expression levels using a two-step cycling protocol was performed on a MyIQ-5 Cycler (Bio-Rad, Hercules CA). Relative gene expression levels were calculated using the 2[-
C(T)] method. Levels of all targets from the test samples were normalized to rat cyclophilin B expression.
NOS Activity. NOS activity was determined by detection of nitrite levels in the cell culture medium after (R)-DOI/TNF- treatments using the Nitrate Detection kit from Assay Designs (Ann Arbor, MI) following the manufacturer's protocols. Absorbances were detected at 539 nM on a Molecular Dynamics SpectraMax M2 plate reader (Sunnyvale, CA).
Nuclear Translocation of p65. RASM cells were grown in M-199 medium + 10% FBS until 50% confluent in 8-well chamber slides. Cells were treated with 1 nM (R)-DOI for various times as indicated, and TNF- (10 ng/ml) was added. Thirty minutes after TNF- treatment, cells were fixed and processed with rabbit anti-p65 primary and Alexa Fluor 488-conjugated goat secondary antibodies as described in Zerfaoui et al. (2008). Fluorescent signal was visualized on a Leica DMRA2 fluorescent microscope (Leica, Wetzlar, Germany).
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-Induced Expression of Proinflammatory Genes ICAM-1, VCAM-1, and IL-6 in Primary RASM Cells. First, we verified that primary RASM cells express 5-HT2A mRNA by quantitative real-time PCR using primer sequences and probes as described under Materials and Methods (data not shown). To examine the effects of 5-HT2A receptor activation on TNF--mediated proinflammatory gene expression, RASM cells were pretreated with (R)-DOI, a selective 5-HT2 receptor agonist, as described under Materials and Methods. Dose-response curves were determined for the effects on ICAM-1, VCAM-1, and IL-6 gene expression for 12 different concentrations of (R)-DOI ranging from 0.1 pM to 100 nM, with each experiment repeated in triplicate. We were surprised to discover that (R)-DOI superpotently inhibited expression of each of these genes with an IC50 in the 10 to 20 pM range (Fig. 1). Given this unusually high potency, the experiments were repeated with fresh dilutions of drug and different batches of RASM cells, with the same results: a superpotent effect. TNF- alone consistently increased baseline mRNA expression of these genes 8 to 10-fold, whereas (R)-DOI had no effect by itself (data not shown).
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-induced ICAM1 expression in cells that were serum starved for 8 h. A small reduction in potency was observed (IC50 = 360 pM) (Fig. 1).
Inhibition of Proinflammatory Markers Is Mediated through 5-HT2A Receptor Activation. Because (R)-DOI is an agonist at all three 5-HT2 receptor isoforms, we used receptor-selective antagonists to determine which was mediating its effect. Pretreatment of the cells with 100 nM of either the 5-HT2C receptor-selective antagonist RS102221 or the 5-HT2B receptor-selective antagonist SB204741 for 30 min before the addition of 1 nM (R)-DOI (a dose that completely inhibits TNF--induced proinflammatory gene expression) had no effect (Fig. 2). Pretreatment with 100 nM of the 5-HT2A receptor-selective antagonist MDL100907 for 30 min, however, completely blocked the inhibitory effects of 1 nM (R)-DOI on TNF--mediated ICAM-1, VCAM-1, and IL-6 gene expression changes (Fig. 2), indicating that the effects of (R)-DOI on these processes are being mediated exclusively through activation of 5-HT2A receptors.
Additional 5-HT2A Receptor Agonists Inhibit Proinflammatory Marker Expression. To examine whether these effects were exclusive for (R)-DOI acting at 5-HT2A receptors, we tested the ability of other 5-HT2A agonists to inhibit proinflammatory marker expression. These included an additional phenethylamine, 2C-BCB, and two indolealkylamines, LA-SS-Az and LSD. All three have high affinity for rat 5-HT2A receptors (Ki of 2C-BCB = 0.73 nM; LA-SS-Az = 8.3 nM; LSD = 3.5 nM) as well as high potency for activating phosphotidylinositide turnover (EC50 of 2C-BCB = 36 nM; LA-SS-Az = 19 nM; LSD = 15 nM) (Nichols et al., 2002; McLean et al., 2006). Studies examining the effects at both 1 and 50 nM pretreatment on gene expression show that they also have potent effects (Fig. 3). Whereas the effects are potent (predicted IC50 values in the low nanomolar range), they are not extraordinarily potent as is the case for (R)-DOI. However, other untested 5-HT2A receptor ligands may yet prove to be as superpotent as (R)-DOI at blocking proinflammatory marker expression.
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-Induced Proinflammatory Marker Expression Involves PKC. It has been well established that ICAM-1 gene expression can be induced through pathways involving PKC (Roebuck and Finnegan, 1999). Furthermore, PKC can be activated through 5-HT2A receptor stimulation (Roth et al., 1986). To test for PKC involvement in mediating the effects of (R)-DOI on proinflammatory gene expression in our assays, we pretreated RASM cells with 100 nM chelerythrine, a pan-PKC isoform inhibitor, 30 min before the addition of (R)-DOI. Chelerythrine (100 nM) completely inhibited the effects of 1 nM (R)-DOI on TNF--induced ICAM-1 gene expression (Fig. 4), indicating that PKC plays a critical role in the mechanism of action of 5-HT2A receptor-mediated inhibition of proinflammatory markers. To further delineate the role of specific isoforms of PKC in this process, we also pretreated cells with the conventional isoform inhibitor Gö6976 (100 nM) and tested the ability of (R)-DOI to inhibit TNF--induced proinflammatory marker expression. Only 50% of the effect of (R)-DOI was blocked, indicating that the 5-HT2A receptor-stimulated anti-inflammatory effects are mediated through at least two isoforms of PKC: one conventional and one nonconventional (Fig. 4). Finally, we examined the ability of exogenous activation of PKC with PMA in RASM cells to block the effects of TNF- on proinflammatory marker expression. In the absence of (R)-DOI, PMA (100 nM) was found to completely block TNF--mediated expression of ICAM1, VCAM1, and IL-6 (Fig. 4). If the effects are completely mediated through PKC, then we predict that inhibiting other G protein-coupled receptor-initiated effector pathways like PKA would have no effect. Indeed, when we tested the effects of blocking PKA with the inhibitor F-22 amide (100 nM) on the ability of (R)-DOI to block TNF- on proinflammatory marker expression, we observed no effect (Fig. 4).
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The Effects of (R)-DOI at Blocking Proinflammatory Gene Expression Is Rapid and Present Many Hours after Addition of TNF-. Our initial dose-response experiments examined the effects of 24-h pretreatment with (R)-DOI. To determine whether this length of pretreatment was necessary to block the effects of TNF-, we performed a time-course analysis and examined the ability of (R)-DOI (1 nM) to block the effects of TNF- (10 ng/ml) on ICAM-1 gene expression with 24- and 1-h pretreatments, simultaneous treatment, and various time points after treatment with TNF-. The 24- and 1-h pretreatments and simultaneous treatment with (R)-DOI completely blocked the effects of TNF- (data not shown). These data indicate that (R)-DOI very rapidly blocks proinflammatory marker expression. Significantly, we also found that the addition of (R)-DOI after TNF- treatment substantially blocked ICAM-1 expression with a 50% effect at approximately 4 h (Fig. 5).
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). NOS activity can regulate NF-B activation (Ckless et al., 2007), and cytokine pathway-activated NF-B can transcriptionally regulate inducible NOS gene expression (Guo et al., 2007). To examine the effects of 5-HT2A receptor activation on this component of inflammatory mechanisms, we performed assays in primary RASM cells to examine the ability of 1 nM (R)-DOI to block TNF--mediated increases in nitrite levels. TNF- alone increased nitrite levels in the medium 8-fold over control levels 24 h after treatment. Pretreatment with 1 nM (R)-DOI 24 h before treatment with TNF- completely inhibited the TNF--mediated increase in nitrite production (Fig. 6). Pretreatment with the pan-isoform PKC inhibitor chelerythrine (100 nM) for 30 min before (R)-DOI completely blocked the effects of (R)-DOI on nitrite accumulation (Fig. 6), indicating that PKC activation is upstream of NOS activity in this process.
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B Subunit p65 Is Blocked by 5-HT2A Receptor Activation with (R)-DOI. Both ICAM-1 and VCAM-1 gene transcription during inflammatory processes is regulated by the transcription factor NF-B (Collins et al., 1995). During this process, NF-B must be activated and then translocate from the cytoplasm to the nucleus, where it promotes gene transcription. To investigate whether 5-HT2A receptor-stimulated inhibition of NF-B activation and translocation might be a possible mechanism for blockade of proinflammatory gene expression changes, we performed a series of immunohistochemical experiments examining p65 translocation in RASM cells. TNF- treatment for 30 min caused the expected dramatic shift in localization of the p65 subunit of NF-B from the cytoplasm to the nucleus (Fig. 7). Pretreatment with 1 nM (R)-DOI for 24 h (data not shown), 1 h, and together (data not shown) with TNF- (10 ng/ml) completely blocked p65 nuclear translocation (Fig. 7). Furthermore, pretreatment of the 1-h time point with the PKC inhibitor chelerythrine blocked the (R)-DOI-induced inhibition of p65 translocation (data not shown), indicating that PKC activation is upstream of this process. The ability of (R)-DOI to block translocation when administered simultaneously with TNF-, and not as a pretreatment, in agreement with our time-course gene expression studies, further support that the effects of 5-HT2A receptor stimulation on inhibiting inflammatory processes are very rapid.
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when added as a pre- or cotreatment, but also when added many hours after TNF- treatment with a half-maximal effect at 4 h after TNF-.
Primary cultures of RASM cells are a well established system to study inflammatory processes. Aortic smooth muscle cells normally form the media of the aorta, and they serve to provide and regulate vascular tone of the artery. It is noteworthy that the pathophysiological status of vascular smooth muscle cells is a crucial determinant of vascular disease (Hansson et al., 2006). During the development of atherosclerosis, which is believed to have a major inflammatory component, levels of cytokines such as TNF- are elevated, leading to increased expression of genes and proteins, e.g., ICAM-1 and VCAM-1, in the aortic smooth muscle (Blankenberg et al., 2003; Hansson et al., 2006). ICAM-1 (CD54) belongs to the IgG superfamily and is expressed in many cell types, including vascular endothelial cells, epithelial cells, fibroblasts, and macrophages (Hughes et al., 2000). Cytokine-mediated increased levels of intracellular adhesion molecules like ICAM-1 in the aortic smooth muscle serve as an important component of atherosclerotic plaque formation.
Our most surprising finding is the extreme potency with which (R)-DOI inhibits proinflammatory markers. Nonsteroidal anti-inflammatory drugs typically have IC50 values in the micromolar range for their targets, whereas steroidal anti-inflammatory drugs typically have IC50 values in the low nanomolar range (Huntjens et al., 2005). The IC50 values in the low picomolar range for (R)-DOI to block proinflammatory markers show that (R)-DOI activation of 5-HT2A receptors is 300-fold more potent than the more effective current anti-inflammatory agents. With the exception of a few natural toxins (e.g., botulinum toxin), no current drugs or small molecule therapeutics demonstrate a comparable potency for any physiological effect.
We examined the effects of three other known 5-HT2A receptor agonists belonging to two different chemical classes in this process. 2C-BCB is a phenethylamine selective for 5-HT2 receptors, whereas LA-SS-Az and LSD are indolealkylamines with high affinity for 5-HT2 receptors, as well as affinity for most other 5-HT receptors. Whereas these drugs can potently block TNF--induced proinflammatory marker expression with predicted IC50 values in the low nanomolar range, they are not superpotent as (R)-DOI. In this context, it might be noted that the powerful hallucinogenic drug LSD is believed to exert its hallucinogenic effects through agonist interactions at the 5-HT2A receptor (Nichols, 2004). A typical clinically effective dose of LSD would be approximately 0.1 mg, although the effects of doses as low as 0.020 to 0.025 mg are detectable in some individuals. A comparably effective hallucinogenic dose of DOI, with a similar molecular weight, would be approximately 2 to 4 mg (Shulgin and Shulgin, 1991), yet neither the Ki of DOI at the 5-HT2A receptor (which is similar to LSD) nor its efficacy for activation of traditional Gq-coupled signal transduction pathways (which is greater than for LSD) can explain why it is so much less potent a hallucinogen than LSD.
Why then is (R)-DOI more potent at blocking proinflammatory components than other 5-HT2A receptor agonists? The answer may lie in the concept of functional selectivity. Functional selectivity is now a well established phenomenon, where different drugs acting at the same receptor have the ability to differentially activate individual effector pathways, presumably through stabilization of different conformational states of the receptor (Urban et al., 2007). This concept with respect to 5-HT2A receptor signaling was first proposed by Roth and Chuang (1987). It is noteworthy that both (R)-DOI and LSD have been shown to exhibit functional selectivity at 5-HT2A receptors. Whereas (R)-DOI preferentially activates the phospholipase C (PLC)β pathway over the phospholipase A2 pathway, LSD preferentially activates the phospholipase A2 pathway over the PLC pathway (Kurrasch-Orbaugh et al., 2003). However, in addition to these two pathways, the 5-HT2A receptor couples to many other effector systems, and it may be one or more of these "nontraditional" effector pathways that underlies the superpotency of both (R)-DOI at blocking proinflammatory markers and LSD at altering behaviors through 5-HT2A receptor stimulation. Our results indicate that PKC activity is necessary for the anti-inflammatory effects of (R)-DOI and that more than one isoform is involved, probably at least one conventional isoform and at least one nonconventional isoform. Furthermore, we have shown that PKC activation is upstream of NOS activity in this process. However, more work remains to be performed to determine whether PKC activity is entirely mediated through traditional activation pathways (e.g., PLC and diacylglycerol) or novel undefined pathways.
One consideration to take into account is our use of 10% FBS in the media for each of our experiments. Earlier work has shown that a certain minimal level of serotonin is required for expression of some inflammatory pathway components (Kubera et al., 2005). This percentage of FBS should provide a final 5-HT concentration of approximately 0.36 µM (Little et al., 2002), which is almost 3-fold below the average Ki value of 1.1 µM for 5-HT at endogenous rat 5-HT2A receptors (Psychoactive Drug Screening Program Ki database: http://pdsp.med.unc.edu). This relatively low amount of 5-HT in the media would not be anticipated to have many functional consequences. Indeed, significant repression of proinflammatory markers by 5-HT itself is not observed until a concentration of 85 µM (Kubera et al., 2005). Although one might imagine the presence of 5-HT in the growth media would augment the effects of (R)-DOI, it might be more likely to inhibit the effects. Continued exposure of cells in culture, like 3T3 cells, to normal 5-HT levels in the serum results in both significant desensitization (>75%) and down-regulation (60%) of the 5-HT2A receptor (Saucier et al., 1998). We observed that in the absence of serum, the EC50 value for (R)-DOI to inhibit TNF--induced ICAM1 expression only shifted to 0.36 nM, which is still very potent. One possible explanation for this slight rightward shift in the dose-response curve is that serotonin may be activating additional 5-HT receptors that, whereas not necessary for the effects, enhance pathway activation leading to a facilitation of proinflammatory marker inhibition. If this were true, one might predict that a drug like LSD, which is an agonist at almost all 5-HT receptors, would be as potent or more potent than a 5-HT2 receptor-selective agonist like (R)-DOI. However, LSD is the weakest drug tested with respect to blockade of TNF--induced ICAM1 expression. Therefore, it seems more likely that the receptor desensitization and down-regulation process that has occurred in media containing serotonin has altered the physiology or conformation of the receptor so that it couples much more efficiently to the pathway(s) mediating the anti-inflammatory effects. This, combined with potential functional selectivity for (R)-DOI at 5-HT2A receptors, may result in the observed superpotent anti-inflammatory effects. Nevertheless, the presence of physiologically relevant levels of 5-HT in the media more closely mimics what would normally be seen in an intact animal, making our results more relevant with respect to potential in vivo inflammatory therapeutic mechanisms.
TNF--mediated inflammatory pathways have been strongly implicated in a number of diseases, including atherosclerosis, rheumatoid arthritis, psoriasis, type II diabetes, irritable bowel syndrome, Crohn's disease, and septicemia (Reimold, 2002; Popa et al., 2007; Williams et al., 2007). It is noteworthy that TNF- and other cytokine-induced inflammatory pathways have also been linked to psychiatric conditions such as depression and bipolar disorder (Dunn et al., 2005; Kim et al., 2007), as well as schizophrenia (Saetre et al., 2007), and neurodegenerative diseases like Alzheimer's and Parkinson's disease and stroke (Tweedie et al., 2007). As such, inhibitors of TNF--mediated proinflammatory pathways represent potential therapeutics for each of these conditions. Currently, the only available therapeutic inhibitors of TNF- pathways are monoclonal antibodies against TNF- (infliximab and adalimumab) and soluble TNF- receptor (enteracept), and the development of small molecules for this purpose is highly desirable (Tracey et al., 2007).
Our results indicate that activation of 5-HT2A receptors by (R)-DOI, as well as additional 5-HT2A receptor agonists, represents a novel and extremely potent therapeutic avenue to explore for the treatment of diseases and disorders involving TNF--mediated inflammation. It is noteworthy that 5-HT2A receptor expression has been detected in most, if not all, of the tissues mediating the inflammatory conditions mentioned above. Given the extremely high potency of (R)-DOI to suppress multiple proinflammatory markers rapidly, ranging from NOS activity, through NF-B translocation, to gene expression of ICAM-1, VCAM-1, and IL-6, the predicted therapeutic dose would be at least two orders of magnitude below that necessary to produce undesirable hallucinogenic side effects. It is noteworthy that because (R)-DOI can significantly inhibit the effects of TNF- many hours after the administration of TNF-, potential therapies could be aimed at not only preventing inflammation, but also treating inflammation or injury that has already occurred or is ongoing.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; DOI, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; IL, interleukin; TNF-, tumor necrosis factor-; NOS, nitric-oxide synthase; NF-B, nuclear factor B; LSUHSC, Louisiana State University Health Sciences Center; RS102221, 8-[5-(2,4-dimethoxy-5-(4-trifluoromethylphenylsulphonamido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione hydrochloride; SB204741, N-(1-methyl-1H-indolyl-5-yl)-N''-(3-methyl-5-isothiazolyl)urea; PKC, protein kinase C; Gö6976, 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-]pyrrolo[3,4-c]carbazole-12-propanenitrile; PMA, phorbol 12-myristate 13-acetate; F-22, fragment 6–22; LA-SS-Az, (2'S,4'S)-(+)-9,10-didehydro-6-methylergoline-8β-(trans-2,4-dimethylazetidide); 2C-BCB, (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl) methylamine; MDL100907, R(+)--(2,3-dimeth-oxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-pipeddine-methanol; LSD, lysergic acid diethylamide; RASM, rat aortic smooth muscle; FBS, fetal bovine serum; PCR, polymerase chain reaction; ICAM-1, intracellular adhesion molecule 1; VCAM-1, vascular adhesion molecule 1; C(T), threshold cycle; PLC, phospholipase C; ANOVA, analysis of variance.
Address correspondence to: Dr. Charles D. Nichols, Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA. E-mail: cnich1{at}lsuhsc.edu
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