Human Mas-related G protein-coupled receptors-X1 induce chemokine receptor 2 expression in rat dorsal root ganglia neurons and release of chemokine ligand 2 from the human LAD-2 mast cell line

Primate-specific Mas-related G protein-coupled receptors-X1 (MRGPR-X1) are highly enriched in dorsal root ganglia (DRG) neurons and induce acute pain. Herein, we analyzed effects of MRGPR-X1 on serum response factors (SRF) or nuclear factors of activated T cells (NFAT), which control expression of various markers of chronic pain. Using HEK293, DRG neuron-derived F11 cells and cultured rat DRG neurons recombinantly expressing human MRGPR-X1, we found activation of a SRF reporter gene construct and induction of the early growth response protein-1 via extracellular signal-regulated kinases-1/2 known to play a significant role in the development of inflammatory pain. Furthermore, we observed MRGPR-X1-induced up-regulation of the chemokine receptor 2 (CCR2) via NFAT, which is considered as a key event in the onset of neuropathic pain and, so far, has not yet been described for any endogenous neuropeptide. Up-regulation of CCR2 is often associated with increased release of its endogenous agonist chemokine ligand 2 (CCL2). We also found MRGPR-X1-promoted release of CCL2 in a human connective tissue mast cell line endogenously expressing MRGPR-X1. Thus, we provide first evidence to suggest that MRGPR-X1 induce expression of chronic pain markers in DRG neurons and propose a so far unidentified signaling circuit that enhances chemokine signaling by acting on two distinct yet functionally co-operating cell types. Given the important role of chemokine signaling in pain chronification, we propose that interruption of this signaling circuit might be a promising new strategy to alleviate chemokine-promoted pain

Multiple agonist-affinity states of opioid receptors: Regulation of binding by guanyl nucleotides in guinea pig cortical, NG108-15, and 7315c cell membranes

Multiple affinity states of opioid receptors of the μ and δ types have been identified in membranes prepared from cells which bear only one type of opioid receptor (μ receptors in 7315c cells, δ receptors in NG 108-15 cells), and in guinea pig cortical membranes where both types of receptors were present in the membrane preparations. States of μ and δ receptors which have agonist affinities too low to be identified by radiolabeled agonist have been measured indirectly by agonist competition for sites labeled by radioactive antagonist. Using analogues of guanyl nucleotides, we have examined the competition of the μ and δ agonists DAGO and DSLET against [3H]DIP or [3H]NAL binding to opioid receptors and identified several agonist affinity states. In the absence of added nucleotide, competition of DSLET for [3H]DIP binding to δ opioid receptors revealed the presence of two binding sites with differing apparent agonist affinities. Addition of GDPβS produced a steep monophasic curve which was best fit by a one-site model. In contrast, in the presence of added GTP or GTPγS, two affinity states were again apparent for DSLET competition at the δ receptor. The competition curve with GTP was shifted to the right relative to that produced in the absence of added guanyl nucleotide, indicating the presence of a lower apparent affinity state than any observed under other treatment conditions. DAGO competed against [3H]DIP or [3H]NAL binding to μ receptors over a wide concentration range in the absence of added guanyl nucleotide, consistent with the occupation by this ligand of more than one agonist affinity state of the μ receptor. However, when GDPβS was added to the incubation mixture, only a single binding site was identified. Two μ receptor affinity states were again observed in the presence of added GTP or GTPγS. One of these had significantly lower apparent affinity than those states detected in the absence of added nucleotide or with GDPβS. Pertussis toxin treatment resulted in a monophasic agonist competition curve which was best fitted by a single-site model in both 7315c and NG108-15 cell membranes. Addition of 100 μM GTP did not affect the agonist K(app) or B(max) after pertussis toxin treatment, suggesting that sites labeled under these conditions were not functionally associated with a G protein. In general, the effects of guanyl nucleotides were qualitatively similar at μ and δ receptors. The multiple apparent affinity states of each type of receptor probably reflect the preferential occurrence of different forms of agonist-receptor-G protein-guanyl nucleotide complex depending on the agonist or antagonist properties of the ligand and the guanyl nucleotides present

Effects of chronic morphine exposure on opioid inhibition of adenylyl cyclase in 7315c cell membranes: A useful model for the study of tolerance at μ opioid receptors

The effects of prolonged morphine exposure on the μ opioid receptor in 7315c pituitary tumor cell membranes have been examined. Since a low concentration of naloxone reversed the inhibition of forskolin-stimulated adenylyl cyclase induced by the μ-selective agonist, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO), and by high concentrations of [D-Pen2-D-Pen5]enkephalin (DPDPE), we suggest that these cells contain a homogeneous population of μ opioid receptors coupled to adenylyl cyclase via a guanyl nucleotide-binding protein. Studies measuring the ability of [D-Ala2-D-Leu5]enkephalin (DADLE), an opioid agonist, to inhibit adenylyl cyclase in cells that had been exposed to 100 μM morphine for varying periods of time, indicated that the agonist no longer inhibited enzyme activity after 5 hr of morphine exposure. Measurements of 3H-antagonist binding in membranes from cells exposed to morphine demonstrated a decreased receptor density after 24 hr of 100 μM morphine exposure with no change in the antagonist affinity. Computer analysis indicated a 20% decrease in the number of μ receptors labeled after 24 hr of morphine exposure and a 60% decrease after 72 hr of exposure. Computer analysis of agonist competition against 3H-antagonist binding confirmed the existence of one binding site with an affinity intermediate between the high and low apparent affinity states observed in membranes from untreated cells. Addition of 10 μM GTPγS did not affect the agonist affinity or receptor density in membranes from morphine-treated cells, suggesting that the receptors were uncoupled from G proteins, as observed in 7315c cell membranes that have been treated with pertussis toxin. Thus chronic morphine treatment induced a rapid loss of opioid μ receptor-mediated inhibition of adenylyl cyclase (desensitization), and a more slowly developing reduction in receptor number. The desensitization was accompanied by a loss of guanyl nucleotide regulation of agonist affinity. These findings are comparable to results reported for the δ opioid receptor and the β-adrenergic receptor upon prolonged agonist exposure

Sodium regulation of agonist binding at opioid receptors. II. Effect of sodium replacement on opioid binding in guinea pig cortical membranes

The authors have examined the effects of sodium on the binding of opioid agonists to μ-, δ-, and κ-receptors in guinea pig cortical membranes. Concentration curves for sodium indicated that maximal inhibition of μ binding by this cation was about 60% and maximal inhibition for δ binding was about 70%, whereas that for κ binding was only about 20%. The concentration of sodium required for half-maximal inhibition of binding to all three sites was about 10-30 mM, corresponding to the intracellular sodium concentration. The nature of the sodium effect was further characterized by saturation analysis of binding to each of the three receptor types by comparing results obtained in the presence of 120 mM sodium with those obtained with equimolar replacement of sodium by another cation. Two radiolabeled agonists with different structural characteristics were tested for each binding site. In the presence of sodium, the affinity of the labeled agonists for μ sites was approximately 2-3-fold less than in its absence, but the density of binding sites was not changed. At κ sites, sodium reduced agonist affinity slightly but, again, did not alter the number of binding sites. In contrast, sodium reduced the apparent density of δ-binding sites while leaving the agonist affinity unchanged. Competition against antagonist binding to δ sites indicated that, in the presence of sodium, a higher proportion of sites was in a lower affinity state, as reflected by the biphasic nature of the agonist displacement curve. In contrast, the effect of sodium on displacement of antagonist from μ sites was to lower the affinity of the agonist. Competition against antagonist binding to κ sites also showed a reduction in agonist affinity by sodium, but no change in numbers of receptors. The results indicate that sodium may differentially regulate agonist binding to opioid receptor types and that this regulation may occur at an intracellular site. The κ site appears to be less sensitive to sodium than the μ and δ sites

N-acetyl-aspartylglutamate modulation of N-methyl-D-aspartate-stimulated [\u3csup\u3e3\u3c/sup\u3eH]norepinephrine release from rat hippocampal slices

The release of preloaded radiolabeled norepinephrine ([3H]NE) from slices of rat hippocampus can be stimulated by excitatory amino acids that interact with the N-methyl-D-aspartate (NMDA) receptor. The acidic dipeptide N- acetyl-L-aspartylglutamate (NAAG) is colocalized with NE in the cell bodies of locus coeruleus (the origin of the noradrenergic projections to the hippocampus) and the hippocampus itself. The function of NAAG in these neurons has not been demonstrated, although evidence exists that it may serve as a neuromodulator in other neuronal pathways. NAAG inhibited the release of [3H]NE stimulated by NMDA and L-glutamate in a concentration-related manner. The maximal inhibition produced by NAAG was about 25% of the control release stimulated by 25 μM NMDA. The effects observed were caused by the intact dipeptide and not the degradation artifacts produced by the enzyme N- acetylated-α-linked-acidic dipeptidase because N-acetyl-L-aspartate had no significant effect on the release and L-glutamate was stimulatory. The activity of this enzyme appears to be suppressed under the assay conditions used. Although the addition of glycine did not enhance NMDA-stimulated release, 7-chlorokynurenate and 1-hydroxy-3-amino-pyrrolidone-2 decreased the release in a concentration-dependent manner. Furthermore, the attenuation produced by NAAG plus 7-chlorokynurenate or 1-hydroxy-3-aminopyrrolidone-2 was greater than the inhibitory actions of either glycine antagonist alone. Similarly, NAAG produced additional inhibition over that produced by either of two different voltage-dependent calcium channel blockers. These findings suggest that NAAG may serve as a modulator of excitatory amino acid-mediated NE release in the hippocampus. The site of action of NAAG is most likely not through the glycine binding site nor the L or N type of voltage-dependent calcium channel