Toll-like receptor-4 (TLR4) has been identified in primary sensory neurons, both in vivo and in vitro, but is reportedly absent from satellite glial cells (SGCs). Herein we reveal that, in rat dorsal root ganglia (DRG), SGCs do express TLR4 but this expression is inhibited by direct contact with neurons. Thus, TLR4 mRNA and protein is strongly up-regulated in isolated DRG glial cells in the absence of neurons. Lipopolysaccharide (LPS) increased cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNFα) mRNA expression with greater efficacy in DRG glial cell cultures than in mixed DRG cell cultures containing TLR4-positive neurons. Using an insert co-culture system, we have shown that neuronal inhibition of glial cell TLR4 is likely to be dependent on cell-cell contact rather than diffusible factors from neurons. LPS stimulated prostaglandin E2 (PGE2) production from DRG glial cells in a TLR4- and COX-2-dependent manner. In addition, exogenous PGE2 potentiated LPS-stimulated COX-2 mRNA while inhibiting TNFα mRNA expression by DRG cells, suggestive of a complex regulatory system to control inflammation within the DRG. In addition to LPS, conditioned medium from heat-shocked DRG neurons also increased COX-2 mRNA expression in DRG glial cells in a partially TLR4-dependent manner. We therefore hypothesize that neuronal suppression of glial TLR4 activity is a protective mechanism to prevent uncontrolled inflammation within the DRG. Under conditions where DRG neuronal viability is compromised, DRG glial cells become responsive to PAMPs (pathogen-associated molecular patterns) and DAMPs (danger-associated molecular patterns) and generate a range of classical inflammatory responses.

We have examined the functional expression of Toll-like receptor 4 (TLR4) in adult male rat dorsal root ganglion (DRG) cells in culture by studying changes in pro-inflammatory cytokines and cyclooxygenase (COX)-dependent prostanoid production. In the mixed population of DRG neurons and glial cells, only DRG neurons expressed cell surface TLR4 along with MD-2 and CD14. This classical TLR4 signaling complex on DRG neurons responded to lipopolysaccharide (LPS) with a TLR4-dependent and time-dependent increase in interleukin-1β and tumor necrosis factor-α mRNA expression which was entirely dependent on NF-κB activity. In contrast, after 2-h incubation with DRG cells, LPS-stimulated COX-2 was regulated by both NF-κB and transactivation of epidermal growth factor receptor (EGFR) with potential downstream activation of ERK1/2 and p38 kinase. In contrast to this evidence for myeloid differentiation primary response gene-88 (MyD88)-dependent signaling, no evidence was obtained for TIR-domain-containing adaptor-inducing interferon-ß (TRIF)-dependent signaling from TLR4 in DRG neurons. LPS surprisingly produced a time-dependent decrease in COX-1 protein which likely facilitates the COX-2-dependent production of prostaglandin E2 and prostacyclin. Our study is the first to demonstrate the activation of TLR4-dependent production of prostaglandin E2 and prostacyclin in DRG cell cultures. Our findings support the concept that the activation of TLR4 on primary sensory neurons by endogenous ligands may underlie neuropathic and inflammatory pain states.

Background and PurposeHyper‐nociceptive PGE2 EP4 receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell‐specific expression of two other Gs‐protein coupled hyper‐nociceptive receptor systems: β‐adrenoceptors and calcitonin gene‐related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone–glial cell interactions in regulating adenylyl cyclase (AC) activity.Experimental ApproachAgonist‐stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone‐enriched cell cultures and pure DRG glial cell cultures.Key ResultsPharmacological analysis showed the presence of Gs‐coupled β2‐adrenoceptors and CGRP receptors, but not β1‐adrenoceptors, in all three DRG cell preparations. Agonist‐stimulated AC activity was weakest in DRG neurone‐enriched cell cultures. DRG neurones inhibited IP receptor‐stimulated glial cell AC activity by a process dependent on both cell–cell contact and neurone‐derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation.Conclusions and ImplicationsGs‐coupled hyper‐nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP4 and IP receptors, but not β2‐adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper‐nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses.

BACKGROUND AND PURPOSE Adenosine is believed to participate in the pathological development of asthma through a mast cell‐dependent mechanism. Our study aimed to pharmacologically characterize the functions of adenosine receptor (AR) subtypes (A 1 , A 2A , A 2B and A 3 ) in primary human cultured mast cells (HCMC). EXPERIMENTAL APPROACH HCMC were derived from progenitor stem cells in buffy coat and the effects of adenosine receptor ligands on basal and IgE‐dependent histamine release were evaluated. KEY RESULTS Adenosine and analogues alone did not induce HCMC degranulation. When HCMC were activated by anti‐IgE after 10 min pre‐incubation with adenosine, a biphasic effect on histamine release was observed with enhancement of HCMC activation at low concentrations of adenosine (10 −9 –10 −7 mol·L −1 ) and inhibition at higher concentrations (10 −6 –10 −4 mol·L −1 ). The potentiating action was mimicked by A 1 AR agonists CCPA and 2'MeCCPA, and inhibited by the A 1 AR antagonist PSB36. In contrast, the inhibitory action of adenosine was mimicked by the non‐specific A 2 AR agonist CV1808 and attenuated by A 2B AR antagonists PSB1115 and MRS1760. The non‐selective AR antagonist CGS15943 attenuated both the potentiating and inhibitory actions. CONCLUSIONS AND IMPLICATIONS We have defined for the first time the contribution of A 1 and A 2B ARs, respectively, to the potentiating and inhibitory action of adenosine on human mast cell activation. With reference to the current trend of developing novel anti‐asthmatic agents from AR ligands, our results suggest that inhibition of human mast cell activation would be a mechanism for A 1 AR antagonists, but not A 2B AR antagonists.

Rat pheochromocytoma (PC12) cells characteristically undergo differentiation when cultured with nerve growth factor (NGF). Here we show that NGF dramatically increased the adenylyl cyclase-activating property of forskolin in PC12 cells. This effect of NGF was well maintained even when NGF was removed after 4 days, even though the morphological features of neuronal differentiation were rapidly lost on removal of NGF. The enhanced cAMP production in response to forskolin could be due to a synergistic interaction between forskolin and endogenously released agonists acting on Gs-coupled receptors. However, responses to forskolin were not attenuated by antagonists of adenosine A2 receptors or pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, suggesting that adenosine and PACAP were not involved. Adenylyl cyclases 3, 6 and 9 were the predominant isoforms expressed in PC12 cells, but we found no evidence for NGF-induced changes in expression levels of any of the 9 adenylyl cyclase isoforms, nor in the expression of Gαs. These findings highlight that NGF has a subtle influence on adenylyl cyclase activity in PC12 cells which may influence more than the neurite extension process classically associated with neuronal differentiation.

Cyclooxygenase-1 (COX-1) behaves as a delayed response gene in rat pheochromocytoma (PC12) cells exposed to nerve growth factor (NGF). To investigate the possible targets for COX-1 generated prostanoids in the early stages of neuronal differentiation, we have examined the expression of prostanoid receptors by PC12 cells using functional assays. Prostanoid receptor-specific agonists failed to activate adenylyl cyclase in undifferentiated and NGF-treated PC12 cells; neither did they stimulate phospholipase C activity. EP3 receptor agonists and PGF2α were the only active ligands, able to inhibit forskolin-stimulated adenylyl cyclase activity. PC12 cells expressed EP3 and FP receptor mRNA, but only the responses to EP3 receptor agonists were inhibited by the EP3 receptor antagonist ONO-AE3-240. The functional role of NGF-stimulated COX-1 remains to be determined since we found no strong evidence of a role for EP3 receptors in the morphological changes induced by NGF during the early stages of differentiation of PC12 cells.

Background and purpose: The current study examined the utility of the recently described prostacyclin (prostanoid IP) receptor antagonist RO1138452 (2-(4-(4-isopropoxybenzyl)-phenylamino) imidazoline) as a tool for classifying prostanoid receptors.Experimental approach: pA(2) values were determined on isolated smooth muscle and platelet preparations.Key results: RO1138452 antagonized relaxation of human pulmonary artery, guinea-pig aorta and rabbit mesenteric artery induced by the selective IP agonist cicaprost. Schild plots had slopes close to unity, generating pA(2) values of 8.20, 8.39 and 8.12 respectively. Non-surmountable antagonism was sometimes found with the higher concentrations of RO1138452, attributable to the EP3 contractile action of cicaprost. RO1138452 did not block relaxation of guinea-pig trachea induced by the EP2-selective agonist butaprost. In contrast, there was a modest inhibition of butaprost-induced relaxation of human pulmonary artery by RO1138452, implying activation of both EP2 and IP receptors by butaprost. RO1138452 did not affect relaxation induced by PGE(2) (EP4 agonist) and substance P (NK1/ endothelium-dependent agonist) in rabbit mesenteric artery. In human and rat platelet-rich plasmas, RO1138452 antagonized cicaprost-induced inhibition of platelet aggregation in a surmountable manner; pA(2) values may have been affected by binding of RO1138452 to plasma protein. RO1138452 did not affect the inhibitory actions of PGD(2) (DP1 agonist) and NECA (adenosine A(2A) agonist) in human platelets.Conclusions and implications: The data indicate that RO1138452 is a potent and selective IP receptor antagonist. RO1138452 represents an important addition to our armoury of prostanoid receptor antagonists and a potential clinical agent in situations where prostacyclin has a pathophysiological function.

In the present study, a phage-displayed random peptide library was used to identify surrogate peptide ligands for orphan GPCR mas. Sequence analysis of the isolated phage clones indicated a selective enrichment of some peptide sequences. Moreover, multiple alignments of the isolated phage clones gave two conserved peptide motifs from which we synthesized peptide MBP7 for further evaluation. Characterization of the representative phage clones and the synthetic peptide MBP7 by immunocytochemistry revealed a strong punctate cell surface staining in CHO cells expressing mas-GFP fusion protein. The isolated phage clones and synthetic peptide MBP7 induced mas internalization in a stable CHO cell clone (MC0M80) over-expressing mas. In addition, MBP7-stimulated phospholipase C activity and intracellular calcium mobilization in these same cells. In summary, we have demonstrated a systematic approach to derive surrogate peptide ligands for orphan GPCRs. With this technique, we have identified two conserved peptide motifs which allow us to identify potential protein partners for mas, and have generated a peptide agonist MBP7 which will be invaluable for functional characterization of the mas oncogene.

The hyperalgesic response to prostaglandin E2 (PGE2) is thought to be mediated by activation of the cAMP/protein kinase A pathway in primary sensory neurones. The aim of this study was to investigate the relative contribution of different PGE2 (EP) receptor subtypes to the overall activity of adenylyl cyclase in adult rat isolated dorsal root ganglion (DRG) cells, in vitro. PGE2 and the prostanoid EP4 receptor agonist ONO-AE1-329 increased [3H]cAMP production with EC50 values of 500 nM and 70 nM, respectively, and showed similar efficacies. No combination of prostanoid EP1, EP2, EP3 or EP4 receptor selective agonists produced synergistic increases in [3H]cAMP. The prostacyclin mimetic cicaprost increased [3H]cAMP production with an EC50 value of 42 nM and produced a significantly greater maximal response compared with PGE2. No evidence for prostanoid EP3 receptor-dependent inhibition of adenylyl cyclase activity could be obtained to account for the relatively weak effect of PGE2 compared with prostacyclin receptor agonists. Interestingly, sulprostone (prostanoid EP3/EP1 receptor agonist) caused a Rho-kinase-dependent retraction of neurites, suggesting an alternative role for prostanoid EP3 receptors in DRG cells. In conclusion, PGE2 mediated increases in adenylyl cyclase activity in primary sensory neurones is likely to be mediated by activation of prostanoid EP4 receptors, and is not under inhibitory control by prostanoid EP3 receptors.