Forskolin-stimulated Cyclic AMP Production Research Articles

Succinate, increased in metabolic syndrome, activates GPR91 receptor signaling in urothelial cells

Metabolic syndrome is associated with overactive bladder syndrome (OAB) and increased circulating levels of succinate, an intermediate of the Krebs cycle. The urothelium is an essential regulator of bladder muscle contraction. This study aimed to determine if GPR91, the succinate receptor, is expressed and functional in the bladder. Urothelial and smooth muscle cells (SMCs) were cultured and characterized. PCR revealed that urothelial cells express GPR91, twice as much as SMCs. Incubation of cells with succinate stimulated phosphorylation of ERK and JNK in urothelial cells. Succinate also potently inhibited forskolin-stimulated cyclic AMP production in urothelial cells, an effect prevented by a protein Gi inhibitor. ERK phosphorylation stimulated by succinate was abolished by inhibitors of protein Gq, phospholipase C, MAPK pathway and PKC. Incubation of urothelial cells with succinate potently increased iNOS synthesis and secretion of nitric oxide (NO), and decreased secretion of prostaglandin E2 (PGE2). Finally, succinate triggered entry of calcium in urothelial cells. GPR91 knockdown by shRNA abolished most of these signaling effects. We conclude that in the bladder, urothelial cells are a primary target of succinate through its receptor GPR91. Its activation leads to signaling via phospholipase C, MAPK, PKC pathway and protein Gq and Gi. Succinate binding to GPR91 triggers a rise in intracellular calcium, an increase in secretion of NO and a decrease in the release of PGE2. Succinate might be essential in the understanding of OAB that occurs in metabolic syndrome.

A new cannabinoid CB2 receptor agonist HU‐910 attenuates oxidative stress, inflammation and cell death associated with hepatic ischaemia/reperfusion injury

Cannabinoid CB(2) receptor activation has been reported to attenuate myocardial, cerebral and hepatic ischaemia-reperfusion (I/R) injury. We have investigated the effects of a novel CB(2) receptor agonist ((1S,4R)-2-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-7,7-dimethylbicyclo[2.2.1]hept-2-en-1-yl)methanol (HU-910) on liver injury induced by 1 h of ischaemia followed by 2, 6 or 24 h of reperfusion, using a well-established mouse model of segmental hepatic I/R. Displacement of [(3) H]CP55940 by HU-910 from specific binding sites in CHO cell membranes transfected with human CB(2) or CB(1) receptors (hCB(1/2) ) yielded K(i) values of 6 nM and 1.4 µM respectively. HU-910 inhibited forskolin-stimulated cyclic AMP production by hCB(2) CHO cells (EC(50) = 162 nM) and yielded EC(50) of 26.4 nM in [(35) S]GTPγS binding assays using hCB(2) expressing CHO membranes. HU-910 given before ischaemia significantly attenuated levels of I/R-induced hepatic pro-inflammatory chemokines (CCL3 and CXCL2), TNF-α, inter-cellular adhesion molecule-1, neutrophil infiltration, oxidative stress and cell death. Some of the beneficial effect of HU-910 also persisted when given at the beginning of the reperfusion or 1 h after the ischaemic episode. Furthermore, HU-910 attenuated the bacterial endotoxin-triggered TNF-α production in isolated Kupffer cells and expression of adhesion molecules in primary human liver sinusoidal endothelial cells stimulated with TNF-α. Pretreatment with a CB(2) receptor antagonist attenuated the protective effects of HU-910, while pretreatment with a CB(1) antagonist tended to enhance them. HU-910 is a potent CB(2) receptor agonist which may exert protective effects in various diseases associated with inflammation and tissue injury. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

ChemInform Abstract: (-)-5-Methyl-8-hydroxy-(di-n-propylamino)tetralin: A New 5-HT1A Receptor Antagonist.

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The plant cannabinoid Δ9‐tetrahydrocannabivarin can decrease signs of inflammation and inflammatory pain in mice

The phytocannabinoid, Delta(9)-tetrahydrocannabivarin (THCV), can block cannabinoid CB(1) receptors. This investigation explored its ability to activate CB(2) receptors, there being evidence that combined CB(2) activation/CB(1) blockade would ameliorate certain disorders. We tested the ability of THCV to activate CB(2) receptors by determining whether: (i) it inhibited forskolin-stimulated cyclic AMP production by Chinese hamster ovary (CHO) cells transfected with human CB(2) (hCB(2)) receptors; (ii) it stimulated [(35)S]GTPgammaS binding to hCB(2) CHO cell and mouse spleen membranes; (iii) it attenuated signs of inflammation/hyperalgesia induced in mouse hind paws by intraplantar injection of carrageenan or formalin; and (iv) any such anti-inflammatory or anti-hyperalgesic effects were blocked by a CB(1) or CB(2) receptor antagonist. THCV inhibited cyclic AMP production by hCB(2) CHO cells (EC(50)= 38 nM), but not by hCB(1) or untransfected CHO cells or by hCB(2) CHO cells pre-incubated with pertussis toxin (100 ng.mL(-1)) and stimulated [(35)S]GTPgammaS binding to hCB(2) CHO and mouse spleen membranes. THCV (0.3 or 1 mg.kg(-1) i.p.) decreased carrageenan-induced oedema in a manner that seemed to be CB(2) receptor-mediated and suppressed carrageenan-induced hyperalgesia. THCV (i.p.) also decreased pain behaviour in phase 2 of the formalin test at 1 mg.kg(-1), and in both phases of this test at 5 mg.kg(-1); these effects of THCV appeared to be CB(1) and CB(2) receptor mediated. THCV can activate CB(2) receptors in vitro and decrease signs of inflammation and inflammatory pain in mice partly via CB(1) and/or CB(2) receptor activation.

Lack of effect of the α2C‐adrenoceptor Del322‐325 polymorphism on inhibition of cyclic AMP production in HEK293 cells

The alpha(2C)-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human alpha(2C) polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells. Human alpha(2C) wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200-2320 fmol.mg(-1) protein) was measured following agonist treatment. Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325. The alpha(2C) WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the alpha(2C)-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.

Muscarinic Inhibition of Hippocampal and Striatal Adenylyl Cyclase is Mainly Due to the M4 Receptor

The five muscarinic acetylcholine receptors (M(1)-M(5)) are differentially expressed in the brain. M(2) and M(4) are coupled to inhibition of stimulated adenylyl cyclase, while M(1), M(3) and M(5) are mainly coupled to the phosphoinositide pathway. We studied the muscarinic receptor regulation of adenylyl cyclase activity in the rat hippocampus, compared to the striatum and amygdala. Basal and forskolin-stimulated adenylyl cyclase activity was higher in the striatum but the muscarinic inhibition was much lower. Highly selective muscarinic toxins MT1 and MT2-affinity order M(1) > or = M(4) >> others-and MT3-highly selective M(4) antagonist-did not show significant effects on basal or forskolin-stimulated cyclic AMP production but, like scopolamine, counteracted oxotremorine inhibition. Since MTs have negligible affinity for M(2), M(4) would be the main subtype responsible for muscarinic inhibition of forskolin-stimulated enzyme. Dopamine stimulated a small fraction of the enzyme (3.1% in striatum, 1.3% in the hippocampus). Since MT3 fully blocked muscarinic inhibition of dopamine-stimulated enzyme, M(4) receptor would be responsible for this regulation.

New Pansomatostatin Ligands and Their Chelated Versions: Affinity Profile, Agonist Activity, Internalization, and Tumor Targeting

Somatostatin receptor (sst) targeting is an established method to image and treat sst-positive tumors. Particularly, neuroendocrine tumors express the receptor subtype 2 in high density, but sst1, sst3, sst4, and sst5 are also expressed to some extent in different human tumors. Currently used targeting peptides mainly have sst2 affinity. We aimed at developing (radio)peptides that bind with high affinity to all receptor subtypes. Carbocyclic octapeptides were coupled with macrocyclic chelators for radiometal labeling. Affinity, internalization, and agonist potencies were determined on sst1- to sst5-expressing cell lines. Biodistribution was determined on nude mice bearing HEK-sst2 or AR4-2J and HEK-sst3 tumors. High affinity to all receptor subtypes was found. Y(III)-KE88 showed agonistic properties at all five sst receptor subtypes as it inhibits forskolin-stimulated cyclic AMP production. Surprisingly, very low or even absent sst2 receptor internalization was found compared with currently clinically established octapeptides, whereas the sst3 internalization was very efficient. Biodistribution studies of [(111)In]KE88 and [(67)Ga]KE88/[(68)Ga]KE88 reflected the in vitro data. In nude mice with s.c. implanted sst2 (HEK-sst2, AR4-2J)-expressing and sst3 (HEK-sst3)-expressing tumors, high and persistent uptake was found in sst3-expressing tumors, whereas the uptake in the sst2-expressing tumors was lower and showed fast washout. The kidney uptake was high but blockable by coinjection of lysine. This peptide family shows pansomatostatin potency. As radiopeptides, they are the first to show a full pansomatostatin profile. Despite some drawback, they should be useful for imaging sst2-expressing tumors with short-lived radiometals, such as (68)Ga, at early time points and for sst3-expressing tumors at later time points with longer-lived radiometals, such as (64)Cu or (86)Y.

The intrinsic relative activity of an agonist and its correlation to affinity and efficacy

We investigated a method for estimating the receptor-dependent component of agonist activity called intrinsic relative activity (RAi). This parameter is calculated from the agonist concentration-response curve, and it represents the product of observed affinity and intrinsic efficacy of an agonist expressed relative to that of a standard agonist. Consequently, RAi should be dependent mainly on the agonist-receptor-G protein interaction and unaffected by downstream signaling elements. To validate the RAi analysis, we estimated the affinities and relative efficacies of a series of agonists using Furchgott's method of partial receptor inactivation, and compared the respective product of these two parameters with the corresponding RAi estimate for each agonist. In these experiments, we explored agonist–mediated inhibition of forskolin-stimulated cyclic AMP production in Chinese hamster ovary cells stably expressing the human M2 muscarinic receptor. The log product of affinity and efficacy for the agonists, oxotremorine-M, oxotremorine and S-aceclidine, expressed relative to carbachol, were 0.782 ± 0.06, 0.641 ± 0.08 and -0.369 ± 0.05, respectively. The corresponding log RAi values were 0.653 ± 0.05, 0.748 ± 0.17 and -0.183 ± 0.07, respectively. Our results indicate that RAi is a relative measure of the product of affinity and efficacy of an agonist-receptor complex. Supported by NIH grant 69829.

Blockade of excitatory amino acid transporters in the rat hippocampus results in enhanced activation of group I and group III metabotropic glutamate receptors

The idea that excitatory amino acid transporters (EAATs) can control the activation of specific metabotropic glutamate receptors (mGluRs) was investigated in rat hippocampal slices. Using the accumulation of inositol phosphates as a measure of group I mGluR activity, we have shown that the broad spectrum, non-transportable EAAT blocker, TBOA, produces a significant shift to the left of agonist concentration–response curves. Moreover, this increase in potency did not occur if endogenous glutamate was enzymatically removed, suggesting a glutamate-dependent mechanism. This shift in potency was shown to be NMDA and group II mGlu receptor independent. Additionally, experiments with selective antagonists indicated that the group I receptor responsible for the stimulation of inositol phosphate production in this preparation is likely to be mGluR5. Inhibition of forskolin-stimulated cyclic AMP (cAMP) production was used as an index of group II/III mGluR activity. TBOA produced a rightward shift of the forskolin concentration–response curve. A group III, but not a group II, mGluR agonist also produced this effect, suggesting that the TBOA-mediated increase in glutamate activates a receptor, which appears to be a member of the group III mGluR subset. This was confirmed by the observation that an antagonist of group III mGluRs, prevented the TBOA-induced rightward shift in forskolin potency. These results provide evidence of a role for EAATs in the regulation of mGluR5 and group III mGluRs in the rat hippocampus, which may have therapeutic implications.

Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of G-protein coupled receptors

The G-protein coupled receptor (GPCR) human mu opioid receptor (hMOR) fused to the carboxy-terminus of the enhanced green fluorescent protein (EGFP) has been successfully and stably expressed in Drosophila Schneider 2 cells under the control of an inducible metallothionein promoter. Polyclonal cells expressing EGFPhMOR display high-affinity, saturable, and specific binding sites for the opioid antagonist diprenorphine. Competition studies with opioid agonists and antagonists defined the pharmacological profile of a mu opioid receptor similar to that observed in mammalian cells, suggesting proper folding of EGFPhMOR in a high-affinity state in Drosophila cells. The functionality of the fusion protein was demonstrated by the ability of agonist to reduce forskolin-stimulated cyclic AMP production and to induce [ 35S]GTPγS incorporation. The EGFPhMOR protein had the expected molecular weight (70 kDa), as demonstrated by protein immunoblotting with anti-EGFP and anti-C-terminus hMOR antibodies. However, quantitative EGFP fluorescence intensity analysis revealed that the total level of expressed EGFPhMOR is 8-fold higher than the level of diprenorphine binding sites, indicating that part of the receptor is not in a high-affinity state. This may in part be due to a population of receptors localized in intracellular compartments, as shown by the distribution of fluorescence between the plasma membrane and the cell interior. This study shows that EGFP is a valuable and versatile tool for monitoring and quantifying expression levels as well as for optimizing and characterizing an expression system. Optimization of the Drosophila Schneider 2 cell expression system will allow large-scale purification of GPCRs, thus enabling structural studies to be undertaken.

2-Chloro- N6-cyclopentyladenosine, adenosine A 1 receptor agonist, antagonizes the adenosine A 3 receptor

The potent adenosine A 1 receptor agonists, N 6-cyclopentyladenosine (CPA) and 2-chloro- N 6-cyclopentyladenosine (CCPA), were studied in Chinese hamster ovary (CHO) cells expressing the human adenosine A 3 receptor. CPA, but not CCPA, induced phosphoinositide turnover. CPA inhibited forskolin-stimulated cyclic AMP production (EC 50 value of 242±47 nM). CCPA competitively antagonized the effects of agonist Cl-IB-MECA (2-chloro- N 6-(3-iodobenzyl)-5′- N-methylcarbamoyladenosine) with K B value of 5.0 nM. CPA competition curves versus the A 3 antagonist radioligand [ 3H]PSB-11 (8-ethyl-4-methyl-2-phenyl-(8 R)-4,5,7,8-tetrahydro-1 H-imidazo[2.1- i]purin-5-one) were right-shifted four-fold by 100 μM GTP, which had no effect on binding of CCPA or the antagonist MRS 1220 ( N-[9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5- c]quinazolin-5-yl]benzene-acetamide). Thus, CCPA is a moderately potent antagonist ( K i=38 nM) of the human A 3 adenosine receptor.

Association of dopamine D 3 receptors with actin-binding protein 280 (ABP-280)

Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D 2 receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D 3 receptors, but not with D 4 receptors. Similar to the dopamine D 2 receptor, the D 3/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D 3 receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D 4 receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D 3/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D 3 receptor third loop, which is conserved in the D 2 receptor but not in the D 4 receptor. Our results demonstrate a subtype-specific regulation of dopamine D 2-family receptor signaling by the cytoskeletal protein ABP-280.

Agonist and antagonist activities on human NPFF(2) receptors of the NPY ligands GR231118 and BIBP3226.

Neuropeptide FF (NPFF) is a part of a neurotransmitter system acting as a modulator of endogenous opioid functions. At this time, no non-peptide or peptide NPFF-antagonists have been discovered. Here, we demonstrate that Neuropeptide Y (NPY) ligands, in fact possess significant ability to interact with the human NPFF(2) receptors. NPY Y(1) antagonist BIBP3226 and mixed Y(1) antagonist/Y(4) agonist GR231118 are able to displace with low affinity, 50 -- 100 nM, the specific binding on NPFF receptors expressed in CHO cells as well as in rat dorsal spinal cord, an affinity however superior to those determined against Y(2), Y(4) or Y(5) receptors. Furthermore, BIBP3226 which is unable to inhibit the forskolin-stimulated cyclic AMP production mediated by NPFF(2) receptors, antagonizes the effect of NPFF, revealing the first antagonist of NPFF receptors. These properties of NPY ligands on Neuropeptide FF receptors must be considered when evaluating pharmacological activities of these drugs.

O-1057, a potent water-soluble cannabinoid receptor agonist with antinociceptive properties.

Cannabinoids have low water solubility, necessitating the use of a solubilizing agent. In this paper we investigated whether a novel water-soluble cannabinoid, 3-(5'-cyano-1', 1'-dimethylpentyl)-1-(4-N-morpholinobutyryloxy)-Delta(8)- tetrahydroca nnabinol hydrochloride (O-1057), would interact with cannabinoid receptors when water or saline were used as the only vehicle. O-1057 displaced [(3)H]-CP55940 from specific binding sites on Chinese hamster ovary (CHO) cell membranes expressing CB(1) or CB(2) cannabinoid receptors, with pK(i) values of 8.36 and 7.95 respectively. It also displaced [(3)H]-CP55940 from specific binding sites on rat brain membranes (pK(i) = 7.86). O-1057 inhibited forskolin-stimulated cyclic AMP production by both CB(1)- and CB(2)-transfected CHO cells (pEC(50) = 9.16 and 9.72 respectively), its potency matching that of CP55940 and exceeding that of Delta(9)-tetrahydrocannabinol. In the mouse isolated vas deferens, O-1057 inhibited electrically-evoked contractions with pEC(50) and E(max) values of 9.73 and 76.84% respectively. It was antagonized by 100 nM SR141716A, the pK(B) of SR141716A against O-1057 (8.90) approximating to that against CP55940 (8.97). O-1057 also behaved as a CB(1) receptor agonist in vivo, reducing mouse spontaneous activity and rectal temperature when injected intravenously and inducing antinociception in the mouse tail flick test when given intravenously (ED(50) = 0.02 mg kg(-1)), intrathecally, intracerebroventricularly or by gavage. In all these assays, O-1057 was more potent than Delta(9)-tetrahydrocannabinol and, at 0.1 mg kg(-1) i.v., was antagonized by SR141716A (3 mg kg(-1) i.v.). These data demonstrate the ability of the water-soluble cannabinoid, O-1057, to act as a potent agonist at CB(1) and CB(2) receptors and warrant investigation of the clinical potential of O-1057 as an analgesic.

Pharmacological characterization of human recombinant melatonin mt(1) and MT(2) receptors.

We have pharmacologically characterized recombinant human mt(1) and MT(2) receptors, stably expressed in Chinese hamster ovary cells (CHO-mt(1) and CHO-MT(2)), by measurement of [(3)H]-melatonin binding and forskolin-stimulated cyclic AMP (cAMP) production. [3H]-melatonin bound to mt(1) and MT(2) receptors with pK(D) values of 9.89 and 9.56 and B(max) values of 1.20 and 0.82 pmol mg(-1) protein, respectively. Whilst most melatonin receptor agonists had similar affinities for mt(1) and MT(2) receptors, a number of putative antagonists had substantially higher affinities for MT(2) receptors, including luzindole (11 fold), GR128107 (23 fold) and 4-P-PDOT (61 fold). In both CHO-mt(1) and CHO-MT(2) cells, melatonin inhibited forskolin-stimulated accumulation of cyclic AMP in a concentration-dependent manner (pIC(50) 9.53 and 9.74, respectively) causing 83 and 64% inhibition of cyclic AMP production at 100 nM, respectively. The potencies of a range of melatonin receptor agonists were determined. At MT(2) receptors, melatonin, 2-iodomelatonin and 6-chloromelatonin were essentially equipotent, whilst at the mt(1) receptor these agonists gave the rank order of potency of 2-iodomelatonin>melatonin>6-chloromelatonin. In both CHO-mt(1) and CHO-MT(2) cells, melatonin-induced inhibition of forskolin-stimulated cyclic AMP production was antagonized in a concentration-dependent manner by the melatonin receptor antagonist luzindole, with pA(2) values of 5.75 and 7.64, respectively. Melatonin-mediated responses were abolished by pre-treatment of cells with pertussis toxin, consistent with activation of G(i)/G(o) G-proteins. This is the first report of the use of [(3)H]-melatonin for the characterization of recombinant mt(1) and MT(2) receptors. Our results demonstrate that these receptor subtypes have distinct pharmacological profiles.

HU-308: a specific agonist for CB(2), a peripheral cannabinoid receptor.

Two cannabinoid receptors have been identified: CB(1), present in the central nervous system (CNS) and to a lesser extent in other tissues, and CB(2), present outside the CNS, in peripheral organs. There is evidence for the presence of CB(2)-like receptors in peripheral nerve terminals. We report now that we have synthesized a CB(2)-specific agonist, code-named HU-308. This cannabinoid does not bind to CB(1) (K(i) > 10 microM), but does so efficiently to CB(2) (K(i) = 22.7 +/- 3.9 nM); it inhibits forskolin-stimulated cyclic AMP production in CB(2)-transfected cells, but does so much less in CB(1)-transfected cells. HU-308 shows no activity in mice in a tetrad of behavioral tests, which together have been shown to be specific for tetrahydrocannabinol (THC)-type activity in the CNS mediated by CB(1). However, HU-308 reduces blood pressure, blocks defecation, and elicits anti-inflammatory and peripheral analgesic activity. The hypotension, the inhibition of defecation, the anti-inflammatory and peripheral analgesic effects produced by HU-308 are blocked (or partially blocked) by the CB(2) antagonist SR-144528, but not by the CB(1) antagonist SR-141716A. These results demonstrate the feasibility of discovering novel nonpsychotropic cannabinoids that may lead to new therapies for hypertension, inflammation, and pain.

Structural determinants of the partial agonist-inverse agonist properties of 6'-azidohex-2'-yne-delta8-tetrahydrocannabinol at cannabinoid receptors.

1. We have extended previous investigations of four analogues of Delta8-tetrahydrocannabinol (Delta8-THC): 6'-azidohex-2'-yne-Delta8-THC (O-1184), 6'-azidohex-cis-2'-ene-Delta8-THC (O-1238) and octyl-2'-yne-Delta8-THC (O-584) and its 1-deoxy-analogue (O-1315). 2. O-1184, O-1238 and O-584 displaced [3H]-CP55940 from specific binding sites on Chinese hamster ovary (CHO) cell membranes expressing CB1 or CB2 cannabinoid receptors, with pKi values of 8.28 to 8.45 (CB1) and 8.03 to 8.13 (CB2). The pKi values of O-1315 were significantly less, 7.63 (CB1) and 7.01 (CB2). 3. All the analogues inhibited forskolin-stimulated cyclic AMP production by CB1-transfected CHO cells (pEC50=9.16 to 9.72). Only O-1238 behaved as a full agonist in this cell line. 4. In mouse vasa deferentia, O-1238 inhibited electrically-evoked contractions (pEC50=10.18 and Emax=70.5%). Corresponding values for O-1184 were 9.08 and 21.1% respectively. At 1 nM, O-1184 produced surmountable antagonism of the cannabinoid receptor agonist, CP55940. However, at 0.1 nM, O-1184 did not attenuate CP55940-induced inhibition of cyclic AMP production by CB1-transfected CHO cells. 5. In CB2-transfected CHO cells, cyclic AMP production was inhibited by CP55940 (pEC50=8.59), enhanced by O-1184 and O-584 (pEC50=8.20 and 6.86 respectively) and not significantly affected by O-1238 or O-1315. 6. At 100 nM, O-1184 and O-1238 produced surmountable antagonism of CP55940 in CB2 cells, decreasing the pEC50 of CP55940 from 8.61 to 7.42 (O-1184) or from 8. 54 to 7.44 (O-1238). 7. These data support the hypothesis that increasing the degree of unsaturation of the aliphatic side-chain of Delta8-THC analogues has little effect on CB1 or CB2 receptor affinity but can reduce CB1 receptor efficacy and reverse the direction of responses elicited at CB2 receptors.

A conserved arginine in the distal third intracellular loop of the mu-opioid receptor is required for G protein activation.

In the present study, the functional significance of the intracellular C-terminal loop of the mu-opioid receptor in activating Gi proteins was determined by constructing a C-terminal deletion mutant mu(C delta 45) receptor, which lacks the carboxyl 45 amino acids. When the truncated mu(C delta 45) receptor was stably expressed in human embryonic kidney (HEK) 293 cells, the efficacy and the potency of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO), a specific mu-opioid receptor agonist, to inhibit forskolin-stimulated adenylate cyclase activity were not significantly affected. Similar to other G-coupled receptors, the third cytoplasmic loop of the mu-opioid receptor contains conserved basic residues (R276/R277/R280) at the C-terminal segment. Mutating these basic residues to neutral amino acids (L276/M277/L280) greatly impaired the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP formation. Replacing R276/R277 with L276/M277 did not affect the efficacy and potency by which DAMGO inhibits the adenylate cyclase activity. In HEK 293 cells stably expressing mutant (R280L) mu-opioid receptors, the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP production was greatly reduced. These results suggest that the intracellular carboxyl tail of the mu-opioid receptor does not play a significant role in activating Gi proteins and that the arginine residue (R280) at the distal third cytoplasmic loop is required for Gi activation by the mu-opioid receptor.

Agonist-inverse agonist characterization at CB1 and CB2 cannabinoid receptors of L759633, L759656, and AM630.

We have tested our prediction that AM630 is a CB2 cannabinoid receptor ligand and also investigated whether L759633 and L759656, are CB2 receptor agonists. Binding assays with membranes from CHO cells stably transfected with human CB1 or CB2 receptors using [3H]-CP55940, confirmed the CB2-selectivity of L759633 and L759656 (CB2/CB1 affinity ratios = 163 and 414 respectively) and showed AM630 to have a Ki at CB2 receptors of 31.2 nM and a CB2/CB1 affinity ratio of 165. In CB2-transfected cells, L759633 and L759656 were potent inhibitors of forskolin-stimulated cyclic AMP production, with EC50 values of 8.1 and 3.1 nM respectively and CB1/CB2 EC50 ratios of > 1000 and > 3000 respectively. AM630 inhibited [35S]-GTPgammaS binding to CB2 receptor membranes (EC50 = 76.6 nM), enhanced forskolin-stimulated cyclic AMP production in CB2-transfected cells (5.2 fold by 1 microM), and antagonized the inhibition of forskolin-stimulated cyclic AMP production in this cell line induced by CP55940. In CB1-transfected cells, forskolin-stimulated cyclic AMP production was significantly inhibited by AM630 (22.6% at 1 microM and 45.9% at 10 microM) and by L759633 at 10 microM (48%) but not 1 microM. L759656 (10 microM) was not inhibitory. AM630 also produced a slight decrease in the mean inhibitory effect of CP55940 on cyclic AMP production which was not statistically significant. We conclude that AM630 is a CB2-selective ligand that behaves as an inverse agonist at CB2 receptors and as a weak partial agonist at CB1 receptors. L759633 and L759656 are both potent CB2-selective agonists.

δ-Opioid Suppression of Human Immunodeficiency Virus-1 Expression in T Cells (Jurkat)

δ-opioid receptor (DOR) transcripts and binding sites are expressed by lymphocytes and lymphoid cell lines from several species. Direct modulation of lymphocyte function through DORs affects T cell proliferation, interleukin-2 production, chemotaxis, and intracellular signaling. Moreover, in human DOR-transfected T cells (DOR-Ju.1), δ-opioids have been shown previously to mobilize intracellular calcium rapidly, to inhibit forskolin-stimulated cyclic AMP production, and to activate the mitogen-activated protein kinases ERKs 1 and 2. These observations led us to consider whether δ agonists modify T cell functions, thus affecting the expression of human immunodeficiency virus-1 (HIV-1) by CD4 + T cells. To test this hypothesis, DOR-Ju.1 cells, derived from Jurkat cells stably transfected with a cDNA encoding the neuronal DOR, were stimulated with deltorphin or benzamide, 4-[[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl] N-,[2 S[( S∗),2α,5β]]-(9Cl) (SNC-80) prior to the addition of HIV-1. Both deltorphin and SNC-80 concentration-dependently inhibited the production of p24 antigen, an index of HIV-1 expression. Inhibition was maximal with 10 −13–10 −9 M SNC-80 (>60% reduction) or 10 −15–10 −11 M deltorphin (>50% reduction). At higher concentrations, less inhibition of p24 antigen production was found. Naltrindole (NTI, 10 −11 M), a selective DOR antagonist, abolished the inhibitory effects of 10 −9 M SNC-80, whereas 10 −13 M NTI partially reversed the effect of SNC-80. Thus, activation of DORs expressed by CD4 + T cells significantly ( P < 0.05) reduced the expression of HIV-1 by these cells. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard antiviral approaches to HIV-1 infection.