Sphingosine 1-phosphate (S1P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S1P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used a subunit-selective [35 S]GTP␥S binding assay to investigate whether the variety of actions exerted through Edg-1, a recently identified receptor for S1P, might be achieved through multiple G proteins. We found, employing both Sf9 and HEK293 cells, that Edg-1 activates only members of the G i family, and not G s , G q , G 12 , or G 13 . We additionally established that Edg-1 activates G i in response not only to S1P but also sphingosylphosphorylcholine; no effects of lysophosphatidic acid through Edg-1 were evident. Our assays further revealed a receptor(s) for S1P endogenous to HEK293 cells that mediates activation of G 13 as well as G i . Because several of the biological actions of S1P are assumed to proceed through the G 12/13 family, we tested whether Edg-3 and H218/Edg-5, two other receptors for S1P, might have a broader coupling profile than Edg-1. Indeed, Edg-3 and H218/Edg-5 communicate not only with G i but also with G q and G 13 . These studies represent the first characterization of S1P receptor activity through G proteins directly and establish fundamental differences in coupling.Sphingolipid metabolites, including sphingosine 1-phosphate (S1P), 1 regulate many aspects of cell growth and differentiation. S1P is a mitogen (1-4), opposes ceramide-induced apoptosis (5, 6), inhibits cell motility (2, 7), activates platelets (8), and causes retraction of neurites (9, 10). S1P also elicits diverse biochemical responses, including activation of mitogenactivated protein (MAP) kinases (2, 11), phospholipase C (12-14), phospholipase D (15-17), and I k(ACh) (18,19), inhibition of adenylyl cyclase (16,19), and mobilization of Ca 2ϩ (1, 12, 20 -24).Although postulated to function as an intracellular messenger in some cases (21,22,25,26), S1P exerts many of its effects through cell surface receptors (9,11,16,18,19). Recently, the former orphan receptor Edg-1 (endothelial differentiation gene 1) was identified as a high affinity receptor for S1P (27)(28)(29). In cells overexpressing Edg-1, S1P promotes activation of MAP kinase and inhibition of adenylyl cyclase (28,30,31). Additionally, S1P causes a mobilization of calcium in Chinese hamster ovary cells, although not in Sf9, HEK293, or COS-7 cells, overexpressing Edg-1 (29 -31). The sensitivity of these S1P/Edg-1-induced responses to a pertussis toxin (PTX) indicates that members of the G i family of G proteins mediate them (28 -32). In addition to the functional coupling of Edg-1 with G i proteins, physical interaction of Edg-1 with members of this family has been demonstrated. In transfected HEK293 cells, all four PTXsensitive G ␣ subunits, ␣ i1 , ␣ i2 , ␣ i3 , and ␣ o , associate with the third intracellular loop of Edg-1 in a GTP␥S-sensitive manner, and ␣ i1 and ␣ i3 co-immunop...

Selective delta opioid receptor agonists are promising potential therapeutic agents for the treatment of various types of pain conditions. A spirocyclic derivative was identified as a promising hit through screening. Subsequent lead optimization identified compound 20 (ADL5859) as a potent, selective, and orally bioavailable delta agonist. Compound 20 was selected as a clinical candidate for the treatment of pain.

N,N-diethyl-4-(5-hydroxyspiro[chromene-2,4Ј-piperidine]-4-yl) benzamide (ADL5859) and N,N-diethyl-3-hydroxy-4-(spiro-[chromene-2,4Ј-piperidine]-4-yl)benzamide (ADL5747) are novel ␦-opioid agonists that show good oral bioavailability and analgesic and antidepressive effects in the rat and represent potential drugs for chronic pain treatment. Here, we used genetic approaches to investigate molecular mechanisms underlying their analgesic effects in the mouse. We tested analgesic effects of ADL5859 and ADL5747 in mice by using mechanical sensitivity measures in both complete Freund's adjuvant and sciatic nerve ligation pain models. We examined their analgesic effects in ␦-opioid receptor constitutive knockout (KO) mice and mice with a conditional deletion of ␦-receptor in peripheral voltage-gated sodium channel (Nav)1.8-expressing neurons (cKO mice). Both ADL5859 and ADL5747, and the prototypical ␦ agonist 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethyl-piperazin-1-yl]-(3-methoxyphenyl)methyl]-N,Ndiethyl-benzamide (SNC80) as a control, significantly reduced inflammatory and neuropathic pain. The antiallodynic effects of all three ␦-opioid agonists were abolished in constitutive ␦-receptor KO mice and strongly diminished in ␦-receptor cKO mice. We also measured two other well described effects of ␦ agonists, increase in locomotor activity and agonist-induced receptor internalization by using knock-in mice expressing enhanced green fluorescence protein-tagged ␦ receptors. In contrast to SNC80, ADL5859 and ADL5747 did not induce either hyperlocomotion or receptor internalization in vivo. In conclusion, both ADL5859 and ADL5747 showed efficient pain-reducing properties in the two models of chronic pain. Their effects were mediated by ␦-opioid receptors, with a main contribution of receptors expressed on peripheral Nav1.8-positive neurons. The lack of in vivo receptor internalization and locomotor activation, typically induced by SNC80, suggests agonist-biased activity at the receptor for the two drugs.

Selective, nonpeptidic delta opioid receptor agonists have been the subject of great interest as potential novel analgesic agents. The discoveries of BW373U86 (1) and SNC80 (2) contributed to the rapid expansion of research in this field. However, poor drug-like properties and low therapeutic indices have prevented clinical evaluation of these agents. Doses of 1 and 2 similar to those required for analgesic activity produce convulsions in rodents and nonhuman primates. Recently, we described a novel series of potent, selective, and orally bioavailable delta opioid receptor agonists. The lead derivative, ADL5859 (4), is currently in phase II proof-of-concept studies for the management of pain. Further structure activity relationship exploration has led to the discovery of ADL5747 (36), which is approximately 50-fold more potent than 4 in an animal model of inflammatory pain. On the basis of its favorable efficacy, safety, and pharmacokinetic profile, 36 was selected as a clinical candidate for the treatment of pain.

The purpose of the present study was to determine the relative ontogeny of mu- and kappa-opiate receptor control of the hypothalamo-pituitary-adrenal (HPA) axis in rats. The ability of the mu-agonist morphine and the kappa-agonist U-50,488 to stimulate the HPA axis was determined by evaluating ACTH and corticosterone (CS) secretion in developing rat pups. Morphine elicited marked rises in both ACTH and CS secretion in 10-day-old rats, and these increases were maximal from 30-60 min after drug administration. Both morphine and U50,488H caused a dose-related rise in CS secretion that was blocked by the synthetic glucocorticoid dexamethasone. The mu-opiate antagonist beta-funaltrexamine blocked the morphine-induced rise in CS secretion, and the kappa-antagonist norbinaltorphimine blocked the action of U50,488H. While a maximal dose of U50,488H (1 mg/kg) elicited a significant rise in CS secretion as early as postnatal day 2, significant effects of a maximal dose of morphine (5 mg/kg) were not observed until day 5. The effects of both drugs were significantly blunted during the stress-hyporesponsive period from days 5-15. The results of this study demonstrate that significant opiate receptor control of HPA function can be demonstrated early in postnatal development, even before the onset of the stress-hyporesponsive period. In addition, these data suggest that kappa-receptor control is functional before mu-receptor control of HPA function.

Sphingosine 1-phosphate (S1P) exerts a variety of actions as a second messenger or as an agonist that binds to one or more members of the Edg family of G proteincoupled receptors. By using human embryonic kidney 293 cells, we show that S1P activates nuclear factor-B (NF-B) in a receptor-dependent fashion. Edg-3 and Edg-5, which are coupled to G i , G q , and G 13 , affect activation of NF-B, whereas Edg-1, which is coupled to G i alone, does not. We find that the activation of NF-B requires protein kinase C and Ca 2؉ , probably downstream of G q , but that the activation of Rho alone by S1P, whether through G q or G 13 , does not translate into the activation of NF-B. G␤␥ has little effect of its own but potentiates the activation of NF-B achieved through other G proteins. We conclude that the activation of NF-B by S1P is a receptor-mediated process that relies primarily on the activation of a phospholipase C by G q and secondarily on effector regulation through other G proteins.

Many of the ␣ subunits of heterotrimeric GTP-binding regulatory proteins (G proteins) are palmitoylated, a modification proposed to play a key role in the stable anchorage of the subunits to the plasma membrane. Palmitoylation of ␣ subunits from the G i family is preceded by N-myristoylation, which alone or together with ␤␥ probably supports a reversible interaction of the ␣ subunit with membrane as a prerequisite to the eventual incorporation of palmitate. Previous studies have not addressed, however, the question of whether membrane association alone, carried out through N-myristoylation, interaction with ␤␥, or other events, is sufficient for palmitoylation. We report here for ␣ o that it is not. We found that N-myristoylation is required for palmitoylation at least in part because it supports events subsequent to membrane attachment. Mutants of ␣ o designed to target the subunit to membrane without an N-myristoyl group are unable to be palmitoylated as evaluated by incorporation of [ 3 H]palmitate. Mutants of ␣ o unable to interact normally with ␤␥ yet still attach to membrane demonstrate that ␤␥, in contrast, is not required for palmitoylation. ␤␥ becomes necessary, however, when the N-myristoyl group is absent. Our results suggest that N-myristoylation and ␤␥, while almost certainly relevant to the reversible interaction of ␣ o with membrane, also play at least partly overlapping, postanchorage roles in palmitoylation.Fatty acid acylation is one of the major covalent modifications of heterotrimeric GTP-binding regulatory protein (G protein) ␣ subunits (1-4). For most ␣ subunits of the G i family, including ␣ i , ␣ o , and ␣ z , two distinct acylation events occur. N-Myristoylation takes place at the amino terminus and represents the attachment of a myristate through an amide bond to Gly 2 following cleavage of the initiator methionine (5, 6). Palmitoylation occurs at the adjacent Cys 3 and represents the attachment of a palmitate (primarily) through a thioester bond (7,8). ␣ s , ␣ q , and ␣ 12 family members are palmitoylated but not N-myristoylated (8 -11), whereas ␣ t is N-myristoylated but not palmitoylated (12). The functional consequences of N-myristoylation and palmitoylation have been studied intensively. NMyristoylation plays an important role in the attachment of G i family ␣ subunits to membrane (5, 6), targeting of the subunits to caveolin-enriched membrane domains (13), and interactions with the ␤␥ heterodimer (14) and effectors such as adenylyl cyclase (15). Palmitoylation is relevant to the attachment of G protein ␣ subunits to membrane (10, 16) and interaction of subunits with ␤␥ (17) and RGS proteins (18).The palmitoylation of G protein ␣ subunits has drawn much attention recently, as this modification, unlike N-myristoylation, is reversible and subject to regulation. Several groups have demonstrated that the activation of G s is accompanied by an increased rate of palmitate exchange on ␣ s , probably reflecting depalmitoylation and repalmitoylation of the subunit released from ␤␥ (19 -21)....

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