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Endothelins (ETs), potent vasoconstrictors, are also neuropeptides in mammalian brain. The objectives of this study were to identify and characterize the receptors for ETs in rat cerebellum, using radioligand binding techniques and affinity cross-linking method. Iodinated ET-1 bound specifically to receptors in rat cerebellar membrane in a dose-dependent manner. Scatchard plot indicated a single class of high-affinity binding sites. Apparent K(subscript d) value was 110 pM, and the B(subscript max) value was 4.85pmol/mg of membrane protein. Through experiments of affinity cross-linking of ET receptors with 125 I-ET-1, two forms of endothelin receptors with molecular masses of 47 KD and 32 KD were identified. Electrophoresis conducted in the presence of reducing reagents did not affect the mobilities of specifically labeled bands, suggesting that endothelin receptors exist as a single polypeptide chain. The order of potency for E1 and sarafotoxin 6b (S6b) in displacing the specifically bound iodinated ET-1 from cerebellar membrane was ET-1=ET-2=ET-3＞S6b. Result from receptor specificity indicated that endothelin receptors in rat cerebellum belong to endothelin receptor subtype B.

Opioid analgesics remain the first choice for the treatment of moderate to severe pain, but they are also notorious for their respiratory depression and addictive effects. This study focused on the pharmacology of a novel opioid receptor mixed agonist DPI-125 and attempted to elucidate the relationship between the δ-, μ- and κ-receptor potency ratio and respiratory depression and abuse liability. Five diarylmethylpiperazine compounds (DPI-125, DPI-3290, DPI-130, KUST202 and KUST13T02) were selected for this study. PKA fluorescence redistribution assays in CHO cells individually expressing δ-, μ- or κ-receptors were used to measure the agonist potency. The respiratory safety profiles were estimated in rats by the ratio of ED50 (pCO2 increase)/ED50 (antinociception). The abuse liability of DPI-125 was evaluated with a self-administration model in rhesus monkeys. The observed agonist potencies of DPI-125 for δ-, μ- and κ-opioid receptors were 4.29±0.36, 11.10±3.04, and 16.57±4.14 nmol/L, respectively. The other four compounds were also mixed agonists with varying potencies. DPI-125 exhibited a high respiratory safety profile, clearly related to its high δ-receptor potency. The ratio of the EC50 potencies for the μ- and δ-receptors was found to be positively correlated with the respiratory safety ratio. DPI-125 has similar potencies for μ- and κ-receptors, which is likely the reason for its reduced abuse potential. Our results demonstrate that the opioid receptor mixed agonist DPI-125 is safer and less addictive than traditional μ-agonist analgesics. These findings suggest that the development of δ>μ∼κ opioid receptor mixed agonists is feasible, and such compounds could represent a promising class of potent analgesics with wider therapeutic windows.

The discovery of the TRAIL protein and its death receptors DR4/5 changed the horizon of cancer research because TRAIL specifically kills cancer cells. However, the validity of TRAIL-based cancer therapies has yet to be established, as most cancer cells are TRAIL-resistant. In this report, we demonstrate that TRAIL-resistance of many cancer cell lines can be overcome after siRNA- or rocaglamide-mediated downregulation of c-FLIP expression and simultaneous inhibition of IAPs activity using AT406, a pan-antagonist of IAPs. Combined triple actions of the TRAIL, the IAPs inhibitor, AT406, and the c-FLIP expression inhibitor, rocaglamide (ART), markedly improve TRAIL-induced apoptotic effects in most solid cancer cell lines through the activation of an extrinsic apoptosis pathway. Furthermore, this ART combination does not harm normal cells. Among the 18 TRAIL-resistant cancer cell lines used, 15 cell lines become sensitive or highly sensitive to ART, and two out of three glioma cell lines exhibit high resistance to ART treatment due to very low levels of procaspase-8. This study provides a rationale for the development of TRAIL-induced apoptosis-based cancer therapies.

The binding affinity of a series of benzhydrylpiperazine δ opioid receptor agonists were pooled and evaluated by using 3D-QSAR and homology modeling/molecular docking methods. Ligand-based CoMFA and CoMSIA 3D-QSAR analyses with 46 compounds were performed on benzhydrylpiperazine analogues by taking the most active compound BW373U86 as the template. The models were generated successfully with q2 value of 0.508 and r2 value of 0.964 for CoMFA, and q2 value of 0.530 and r2 value of 0.927 for CoMSIA. The predictive capabilities of the two models were validated on the test set with R2pred value of 0.720 and 0.814, respectively. The CoMSIA model appeared to work better in this case. A homology model of active form of δ opioid receptor was established by Swiss-Model using a reported crystal structure of active μ opioid receptor as a template, and was further optimized using nanosecond scale molecular dynamics simulation. The most active compound BW373U86 was docked to the active site of δ opioid receptor and the lowest energy binding pose was then used to identify binding residues such as s Gln105, Lys108, Leu125, Asp128, Tyr129, Leu200, Met132, Met199, Lys214, Trp274, Ile277, Ile304 and Tyr308. The docking and 3D-QSAR results showed that hydrogen bond and hydrophobic interactions played major roles in ligand-receptor interactions. Our results highlight that an approach combining structure-based homology modeling/molecular docking and ligand-based 3D-QSAR methods could be useful in designing of new opioid receptor agonists.

To study effects of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) inhibition by RNA interference (RNAi) on sensitivity of U2OS cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, plasmid pSUPER-c-FLIP-siRNA was constructed and then transfected into U2OS cells. A stable transfection cell clone U2OS/pSUPER-c-FLIP-siRNA was screened from the c-FLIP-siRNA transfected cells. RT-PCR and Western blotting were applied to measure the expression of c-FLIP at the levels of mRNA and protein. The results indicated that the expression of c-FLIP was significantly suppressed by the c-FLIP-siRNA in the cloned U2OS/pSUPER-c-FLIP-siRNA as compared with the control cells of U2OS/pSUPER. The cloned cell line of U2OS/pSUPER-c-FLIP-siRNA was further examined for TRAIL- induced cell death and apoptosis in the presence of a pan-antagonist of inhibitor of apoptosis proteins (IAPs) AT406, with or without 4 hrs pretreatment with rocaglamide, an inhibitor of c-FLIP biosynthesis, for 24 hrs. Cell death effects and apoptosis were measured by the methods of MTT assay with 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide and flow cytometry, respectively. The results indicated that TRAIL-induced cell death in U2OS/pSUPER-c-FLIP-siRNA was increased compared with control cells U2OS/pSUPER in the presence or absence of AT406. Flow cytometry indicated that TRAIL-induced cell death effects proceeded through cell apoptosis pathway. However, in the presence of rocaglamide, cell death or apoptotic effects of TRAIL were similar and profound in both cell lines, suggesting that the mechanism of action for both c-FLIP-siRNA and rocaglamide was identical. We conclude that the inhibition of c-FLIP by either c-FLIP-siRNA or rocaglamide can enhance the sensitivity of U2OS to TRAIL-induced apopotosis, suggesting that inhibition of c-FLIP is a good target for anti-cancer therapy.

We report the first oxidative condensation between N,N-dimethylenaminones with amines to form 1,4-dihydropyridines in moderate to good yields, promoted by oxone and trifluoroacetic acid in PEG-400. This reaction features an unusual oxidation of in situ formed dimethylamine to efficiently provide the 4-methylene of 1,4-dihydropyridines, and a highly environment-friendly reaction condition.

An epimerization–lactamization cascade of functionalized (2S,4R)-4-aminoproline methyl esters is developed and applied in synthesizing (1R,4R)-2,5-diazabicyclo[2.2.1]heptane (DBH) derivatives. (2S,4R)-4-Aminoproline methyl esters are likely to undergo 2-epimerization under basic conditions, followed by an intramolecular aminolysis of the (2R)-epimer to form the bridged lactam intermediates. Key factors identified for this cascade reaction include the electron-withdrawal N-protective group in the substrates and a strong base as the promoter.

A modified Weix's reductive coupling for alkylation of amidoaryl bromides based on Ni(COD)2 precatalyst and 2,2′-dipyridyl ligand was developed. This reaction is reliable for amidophenyl bromides and gives yields up to 87%, and is potentially useful in the synthesis of amidophenyl-containing molecules.

A novel arylnitration of alkenes by nitration and C–H functionalization cascade process has been developed. This methodology provides an efficient way to construct a variety of nitro-containing oxindoles and dihydroquinolin-2(1H)-ones. In addition, the process exhibits significant functional group tolerance. Moreover, the use of inexpensive and readily available starting materials makes this practical and atom-economical approach particularly attractive.

A novel metal-free oxidative arylphosphination of activated N -substituted- N -arylacrylamide derivatives by phosphorylation and C–H functionalization cascade process has been developed. This methodology provides an efficient way to construct a variety of phosphorus-containing oxindole moieties.

Ring-closing metathesis (RCM) and intramolecular Horner–Wadsworth–Emmons (HWE) reactions were assessed for synthesis of cis-5-methyl-6-phenylethyl-5,6-dihydro-α-pyrone. The RCM reaction employing Grubbs’ second generation catalyst afforded a 96% yield at 80 °C in toluene. The HWE reaction under modified Masamune–Roush conditions delivered only 23% yield due to a competitive elimination reaction.

The title compounds are efficiently obtained by radical mediated cascade reaction which shows significant functional group tolerance.

Ring-closing metathesis (RCM) and intramolecular Horner–Wadsworth–Emmons (HWE) reactions were assessed for synthesis of cis-5-methyl-6-phenylethyl-5,6-dihydro-α-pyrone. The RCM reaction employing Grubbs’ second generation catalyst afforded a 96% yield at 80 °C in toluene. The HWE reaction under modified Masamune–Roush conditions delivered only 23% yield due to a competitive elimination reaction.

The oxidative three-component reaction between N,N-dimethylenaminones and amines features an unusual oxidation of in situ formed dimethyl amine to provide the 4-methylene bridge of the dihydropyridines formed.

A novel and facile Cu-catalyzed addition/cyclization cascade of o-cyanoarylacrylamide was developed. The process exhibits significant functional group tolerance, and provides an efficient and straightforward pathway for the synthesis of various phosphonylated quinoline-2,4(1H,3H)-diones.

A modified Weix′s reductive coupling of amidoaryl bromides and octyl bromide is developed to give products (III) in moderate to good yields.

For the last decade, progresses in the understanding of opioid actions are enormous. The X-ray crystalline structures of all four receptor proteins were successfully obtained and analyzed in the antagonist bound stable conformation. Among all four opioid receptors cloned previously, we believe that MOR is the major player in mediating the good pain-killing effects of opioids as well as the bad adverse side effects of opioid analgesics. DOR, KOR and ORL-1 all seem to play modulating functions on MOR-mediating activities including good and bad effects in addition to their own independent activities. Recently, several new concepts have been evolved. These are biased mechanisms, opioid receptor heteromerization, signaling, internalization, trafficking and recycling. This Chapter will focus on the DOR related development during the last decade.

A novel delta-receptor selective compound, ARD-353 [4-((2R,5S)-4-(R)-(4-diethylcarbamoylphenyl)(3-hydroxyphenyl)methyl)-2, 5-dimethylpiperazin-1-ylmethyl)benzoic acid], was evaluated for activity on infarct size in a rat model of acute myocardial infarction. ARD-353 was characterized as having delta receptor selectivity using radioligand binding and had no apparent selectivity between delta receptor subtypes as determined by [(3)H] cyclic [D-Pen(2),D-Pen(5)]enkephalin (delta(1)) and [(3)H]Deltorphin II (delta(2)) competition binding. ARD-353 also showed selective delta receptor agonist activity in mouse-isolated vas deferens. There was no evidence of any seizure-like convulsions when ARD-353 was administered to mice either i.v. or p.o., implying minimal penetration of the blood-brain barrier. ARD-353 decreased infarct size in a left anterior descending coronary artery (LAD) occlusion model of myocardial infarction. In animals pretreated with ARD-353 (i.v.) and then subjected to 30 min of LAD occlusion followed by 90 min of reperfusion, infarct size was reduced in a dose-dependent manner compared with vehicle-treated controls. The effects of ARD-353 on infarct size were blocked by the delta(1)-opioid selective antagonist 7-benzylidenenaltrexone, indicating a significant role for the delta(1)-opioid receptor in the cardioprotective mechanism of ARD-353. ARD-353 (0.3 mg/kg i.v.) produced significant protection when administered 5 min and 12 and 48 h before ischemic insult or when given immediately after the ischemic insult (at the start of reperfusion). Given the lack of central nervous system effects and beneficial efficacy in the rat model of myocardial ischemia, it is felt that ARD-353 is the first nonpeptide delta-receptor agonist with true potential for clinical use before surgically induced ischemia or in an emergency setting.

Allyl-2,5-dimethyl-1-piperazines have been of interest as analgesic agents for the management of moderate-to-severe pain. In this study, we compared the antinociceptive properties and respiratory depressant activity of one such agent, (+)-3-((alpha-R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-(3-fluorophenyl)-N-methylbenzamide (DPI-3290), with those of established narcotic analgesics, morphine and fentanyl. Intravenous administration of DPI-3290 in conscious laboratory rats increased antinociception in a dose-dependent manner with a corresponding ED(50) value of 0.05 +/- 0.0072 mg/kg. Simultaneous measurement of arterial blood gas in animals treated with DPI-3290 demonstrated dose-dependent increases in pCO2 with an ED(50) value of 0.91 +/- 0.22 mg/kg. In comparison, morphine and fentanyl increased antinociception in rats with ED(50) values of 2.01 +/- 0.0005 and 0.0034 +/- 0.00024 mg/kg, respectively, and the ED(50) value for morphine-induced changes in pCO2 was 4.23 +/- 0.72 mg/kg, whereas the ED(50) value for fentanyl-induced changes in pCO2 was 0.0127 +/- 0.0035 mg/kg. A separate series of experiments were designed to examine the effects of DPI-3290 on mu-opioid receptor induced antinociception and hypercapnia. Intravenous bolus doses of DPI-3290 that ranged from 0.2 to 1.0 mg/kg had no effect on antinociception mediated by alfentanil (2 microg/kg/min i.v.) but reduced hypercapnia by approximately 50%. Results from these studies demonstrate the equivalent antinociceptive efficacy of DPI-3290, morphine, and fentanyl but dramatic differences in the hypercapnia that antinociceptive doses of these drugs produce. When measured simultaneously, DPI-3290 had an 18.2-fold difference in the ratio comparing the ED(50) value for antinociception with the ED(50) value for changes in pCO2; this ratio was 2.1 for morphine and 3.7 for fentanyl. Furthermore, DPI-3290 reduced the alfentanil-mediated hypercapnia without any effect on antinociception. Together, the balanced opioid agonist activity of DPI-3290 may provide a means of powerful analgesia while mitigating the mu-opioid receptor-mediated hypercapnia.

Opioid analgesics with both micro and delta opioid receptor activation represent a new approach to the treatment of severe pain with an improved safety profile. Compounds with this profile may exhibit strong analgesic properties due to micro agonism, with a reduced side effect profile resulting from delta agonism. Replacing the p-diethylamide of the known potent delta opioid receptor selective agonist BW373U86 with a m-diethylamide resulted in a compound with agonist activity at both the micro and delta opioid receptors. Modifying the amide to an N-methyl-N-phenylamide increased agonist potency at both receptors. A series of 3-(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-alkyl-N-arylbenzamides have been made to explore the structure-activity relationship (SAR) around the N-methyl-N-phenylamide. Several potent agonists of both the micro and delta opioid receptors have been identified, including (+)-3-((alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-(4-fluorophenyl)-N-methylbenzamide (23), which has EC50 values of 0.67 and 1.1 nM at the micro (guinea pig ileum assay) and delta (mouse vas deferens assay) opioid receptors, respectively.

A novel arylnitration of alkenes by nitration and C–H functionalization cascade process has been developed. This methodology provides an efficient way to construct a variety of nitro-containing oxindoles and dihydroquinolin-2(1H)-ones. In addition, the process exhibits significant functional group tolerance. Moreover, the use of inexpensive and readily available starting materials makes this practical and atom-economical approach particularly attractive.

A novel metal-free oxidative arylphosphination of activated N -substituted- N -arylacrylamide derivatives by phosphorylation and C–H functionalization cascade process has been developed. This methodology provides an efficient way to construct a variety of phosphorus-containing oxindole moieties.

A tritiated form of the non-peptide delta-opioid receptor agonist (+)-BW373U86 ((+)-4-((alpha-R)-alpha-((2S,5R)-4-allyl-2, 5-dimethyl-l-piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide) was synthesized and its binding characteristics studied. [3H](+)-BW373U86 bound with subnanomolar affinity to rat brain membranes and was displaced most effectively by ligands selective for delta-opioid receptors. Naltrindole, naltriben, and 7-benzylidenenaltrexone exhibited apparent inhibition constants of 0.06, 1.54, and 4.49 nM, respectively, while mu- or kappa-selective ligands showed little affinity for this site. [3H](+)-BW373U86 binding was sensitive to the presence of guanine nucleotides; GDP caused a 3-fold decrease and 5'-guanylyl-imidodiphosphate (Gpp[NH]p) caused a 25% increase in binding affinity.

The synthesis of a series of heterocyclic analogues of (±)-4-((αR*)-α-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazmyl)-3-hydroxybenzyl)-N,N-diethylbenzarrude (BW373U86) for screening against opioid receptors is described. The intermediate α-heterocyclic benzyl alcohols 24 were synthesized either by low temperature reaction of lithioheterocycles with 3-((tert-butyldimethylsilyl)oxy)benzaldehyde (10) or by reaction of 3-((tert-butyldimethylsilyl)oxy)phenylmagnesium bromide (19) with heterocyclic carbaldehydes. The α-heterocyclic benzyl alcohols 24 were converted to chloromethines (25) with thionyl chloride and used to alkylate with trans-1-allyl-2,5-dimethylpiperazine (5) to give diastereomeric pairs of the target compounds. The bromoheterocycles were then derivatized to produce amides. Compounds that are potent and selective for the 5 or μ opioid receptors and some mixed δ/μ analogues are reported.

BW 373U86 is a novel, non-peptidic δ-opioid receptor ligand with agonist properties in mouse brain and in the mouse isolated vas deferens. The sensitivity of BW 373U86, and of the peptidic δ-opioid agonists [ d -Pen 2 , D-Pen 5 ]enkephalin (DPDPE) and [ d -Ala 2 ,Glu 4 ]deltorphin, to regulation by guanine nucleotides and sodium was evaluated in competition studies against the 5 selective radioligand [ 3 H]naltrindole. The IC 50 values for DPDPE and [ d -Ala 2 ,Glu 4 ]deltorphin were significantly increased in brain and mouse vas deferens in the presence of Gpp(NH)p and NaCl. In contrast, the IC 50 values for BW 373U86 were not altered in the presence of Gpp(NH)p and NaCl in either tissue. The data indicate that the agonist properties of BW 373U86 may not be affected by the supposed uncoupling of the α-subunit of the G-protein from a receptor thought to be G-protein linked.

Rap2B, a member of the ras superfamily of low-molecular-mass GTP-binding proteins, induced a characteristic rearrangement of the pigment granules in Xenopus oocytes following its microinjection, resulting in numerous unpigmented spots on the animal hemisphere. This phenomenon, termed ‘mottling‘, was also induced by microinjection of in vitro-transcribed Rap2B RNA or of purified recombinant Rap2A. Following the microinjection of Rap2B, more than 90% of the oocytes showed signs of mottling within 10 h. The time course of mottling paralleled the association of the recombinant Rap2B with an oocyte membrane fraction. Like other members of the ras superfamily, Rap2B possesses a C-terminal CAAX motif that serves as a signal for post-translational processing. Mutation of the cysteine residue in the CAAX motif to serine prevents the association of Rap2B with oocyte membranes, and also prevents mottling. This result suggests that post-translational processing of Rap2B is required for the observed effect. Mottling was blocked by boiling Rap2B prior to its microinjection or by co-injection of the cytoskeletal reagent phalloidin.

Heparin and related glycosaminoglycans are important modulators of vascular smooth muscle cell growth, and may be involved in pathological processes such as atherosclerosis. Since polyphosphoinositide metabolism is a major mechanism for regulating cellular activities, including proliferation, the effects of glycosaminoglycans and polyanionic compounds on the activities of phosphoinositide kinases were characterized. Heparin and heparan sulphate caused dose-dependent inhibitions of rat brain cytosolic phosphatidylinositol 4-phosphate (PIP) kinase activity, with half-maximal inhibitory concentrations of approx. 0.5 and 5 microM respectively. PIP kinase was also inhibited by several dextran sulphates, but was not sensitive to inhibition by keratin sulphate, chondroitin sulphate or hyaluronic acid. Polynucleotides and acidic polypeptides were only weakly inhibitory. Heparin did not alter either the PIP- or the Mg(2+)-dependence of PIP kinase. Addition of heparin to brain membranes suppressed PIP kinase activity without affecting phosphatidylinositol (PI) kinase activity. Heparin interfered with the ability of a GTP analogue to stimulate PIP kinase activity in these membranes, suggesting that it uncouples the kinase from an activating guanine-nucleotide-binding protein. In cultured A-10 vascular smooth muscle cells, heparin caused dose- and time-dependent inhibition of [3H]thymidine incorporation into DNA. Similar treatments with heparin decreased cellular levels of phosphatidylinositol 4,5-bisphosphate (PIP2) without changing PI and PIP levels. Therefore heparin-mediated inhibition of PIP kinase appears to lead to decreases in PIP2 levels which may attenuate cellular proliferation.

The effects of fibroblast growth factor (FGF), insulin, insulin-like growth factor-1 (IGF-1), and dibutyryl cAMP (Bt 2 cAMP) on cell proliferation and differentiation, opioid receptor binding activity, and expression of fos, myc , and ras proto-oncogenes were studied in N4TG1 neuroblastoma cells. IGF-1 and insulin increased thymidine incorporation and cell number, but Bt 2 cAMP inhibited incorporation and FGF did not change thymidine incorporation after 24 h of treatment. Bt 2 cAMP profoundly extended and IGF-1 slightly extended neurite-like processes, a morphologic marker of neuronal differentiation. A combination of IGF-1 and Bt 2 cAMP potentiated neurite process extension. Thus, IGF-1 could induce both proliferation and differentiation of N4TG1 neuroblastoma cells. FGF did not cause neurite extension. N4TG1 neuroblastoma cells have a high density of α-opioid receptors. IGF-1 and Bt 2 cAMP, but not FGF and other growth factors, decreased δ-opioid receptor binding activity with a reduced B max value and a normal apparent K d value. Using Northern blot and hybridization, both acidic and basic FGF elicited stimulatory effects on fos expression. Cycloheximide (CHX), a protein synthesis inhibitor, potentiated and prolonged the effect of FGF on fos expression. However, FGF had no effect on myc or ras expression. Neither IGF-1 nor Bt 2 cAMP significantly changed fos, myc , or ras expression. The results suggest that fos, myc , or ras proto-oncogene expression is independent from or insufficient for IGF-1-induced and Bt 2 cAMP-induced differentiation and regulation of opioid receptor synthesis in N4TG 1 neuroblastoma cells.

A cDNA clone (Krev-1) has recently been identified that possesses the ability to reverse the transformed phenotype when introduced into a K-ras-transformed NIH 3 T 3 cell line. The Krev-1 protein, also known as rap-1A, was found to share 50% homology with the ras proteins. The rap-1A protein has also been shown to block the interaction of ras with its GTPase activating protein in vitro, leading to speculation regarding its role in vivo. A closely related protein, rap-1B, has also been identified in platelets, human erythroleukemia cells, neutrophils, and aortic smooth muscle cells. Unlike rap-1A, rap-1B has been shown to be phosphorylated in platelets. Given the high degree of similarity between the amino acid sequences of rap-1A and rap-1B, we sought to investigate the effect of microinjected rap-1B on H-ras(Val12)-induced germinal vesicle breakdown in Xenopus laevis oocytes. In this assay system, equimolar concentrations of rap-1B were found to block germinal vesicle breakdown triggered by the oncogenic ras protein. However, in the presence of IGF-1, this inhibition was not observed. Moreover, rap-1B is readily phosphorylated in the oocyte.

We describe an updated and improved protocol to perform urea gradient/SDS-PAGE in which proteins are electrophoresed through 9% polyacrylamide gel slabs in the presence of a linear 4 M to 8 M gradient of urea using Laemmli's separation buffers. We provide examples of this technique to separate PTX labeled G protein alpha subunits, as well as unlabeled alpha and beta subunits of G proteins. Applications of the technique are exemplified in which (1) the chromatographic separations of G proteins in DEAE-Toyopearl and MonoQ columns are compared, (2) the complexity of PTX substrates expressed in human erythrocytes, bovine brain, dog ventricle, FRTL-5 cells, HIT cells, GH4C1 cells and RIN cells are compared, and (3) the polypeptide composition of G protein beta gamma subunits, as expressed in several tissues and found in three distinct G proteins from a single cell population, are analyzed.

There is a wealth of information from animal models and clinical opioid-analgesic use that indicates a significant role for opioid receptors in the modulation of bladder activity. The novel benzhydrylpiperazine compound DPI-221 [4-((alpha-S)-alpha-((2S,5R)-2,5-dimethyl-4-(3-fluorobenzyl)-1-piperazinyl)benzyl)-N,N-diethylbenzamide] was characterized as having delta receptor selectivity using radioligand binding (K(i) = 2.0 +/- 0.7 nM, delta receptor; 1800 +/- 360 nM, mu receptor; and 2300 +/- 680 nM, kappa receptor), and agonist activity was demonstrated in the mouse isolated vas deferens where DPI-221 inhibited electrically induced contractions with an IC(50) value of 88 +/- 7.5 nM. In the guinea pig isolated ileum, DPI-221 had no effect on electrically induced contractions at concentrations as high as 1 microM. Sterile saline was infused (7 ml/h) into the bladder of Sprague-Dawley rats, via a transmural catheter; DPI-221 (1.0 to 20 mg/kg p.o.) significantly increased the interval between micturition events, whereas peak void pressure was not significantly decreased by any dose of DPI-221. The micturition effects of 10 mg/kg p.o. DPI-221 were blocked by naltrindole, indicating a delta receptor mechanism of action. In isolated rat bladder strips, DPI-221 was ineffective at relaxing detrusor muscle precontracted with carbachol. The most crucial safety aspect of delta agonist administration is the incidence of seizure-like convulsions in rodents. DPI-221 produced no convulsions at doses up to 100 mg/kg p.o. in mice, although rapid bolus i.v. injection of 5 mg/kg produced convulsions in 3% of mice tested. These findings indicate a good safety profile for DPI-221 administered orally, with potent efficacy in modifying bladder activity.

The Delta Receptor: History of Delta Opioid Receptors, Wen-Jen Chang Cloning of Delta Opioid Receptors, Arnaud Lacoste and Christopher J. Evans Cloning and Expression of the Human Delta Opioid Receptor, Richard J. Knapp, Ewa Malatynska, Eva V. Varga, William R. Roeske, and Henry I. Yamamura In Vitro and In Vivo Mutagenesis - Insights into Delta Receptor Structure and Function, F.M. Dicaillot and Brigiitte L. Kieffer Delta Opioid Receptor Signaling and Trafficking, P.Y. Law Delta Opioid Receptors and G Proteins, Mary J. Clark and John R. Traynor Transcriptional Regulation of Delta Opioid Receptor, Gene Ping Sun and Horace Loh. Delta Ligands: Benzhydrylpiperazines as Nonpeptidic Delta Opioid Receptor Ligands, Michael J. Bishop and Robert W. McNutt Delta-Selective Ligands Related to Naltrindole. D.J. Daniels and P.S. Portoghese Endogenous Peptides for Delta Opioid Receptors and Analogues, Victor J. Hruby and Henry I. Mosberg Deltorphins, Lucia Negri and Elisa Giannini Opioid Peptide--Derived Delta Antagonists, Inverse Agonists and Mixed Mu Agonists/Delta Antagonists. Peter W. Schiller Inverse Agonism at the Delta Opioid Receptors, Eva V. Varga, Keiko Hosohata, Yoshioko Hosohata, Jennifer Tsang, Thomas Burkey, Josue Alfaro-Lopez, Xuejun Tang, Victor R. Hruby, William R. Roeske and Henry I. Yamamura Mixed Opioid Receptor Agonists as a New Class of Agents for the Treatment of Moderate-to-Severe Pain, Peter J. Gengo and Kwen-Jen Chang Biphalin - A Multireceptor Opioid Ligand Binding and Activity of Opioid Ligands at the Cloned Human Delta, Mu and Kappa Receptors Andrez W. Lipkowski, Daniel B. Carr, Iwona Bonney and Aleksandra Misicka Kemal Payza Inhibitors of Enkephalin-Inactivating Enzymes and Delta Opioid Responses, Bernard P. Roques and Florence Noble. Pharmacology And Physiology: Delta Opioid Receptor Subtypes and Pain Modulation, Michael H. Ossipov, Josephine Lai, Todd W. Vanderah and Frank Porreca Delta Opioid Receptor-Mediated Antinociception/Analgesia Minoru Narita and Tsutomu Suzuki Antidepressant-like Effects of Delta Opioid Receptor Agonists, Emily M. Jutkiewicz and James H. Woods Mu-Delta Interactions In Vitro and In Vivo, Richard B. Rothman and Heng Xu Delta Opioids and Immune Function, Richard J. Weber and Ricardo Gomez-Flores Delta Opioids and Substance Abuse, S. Stevens Negus Delta Opioid Receptors in the Gastrointestinal Tract, DeWayne Townsend IV and David R. Brown Cardioprotection and Delta Opioid Receptors, Garrett J. Gross, Ryan M. Fryer, Hemal H. Patel and Joel J. Schultz The Delta Opioid Receptor and Brain Pain-Modulating Circuits, Mary M. Heinricher and Howard L. Fields.

Compound (+)-3-((alpha-R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-(3-fluorophenyl)-N-methylbenzamide (DPI-3290), is one of a series of novel centrally acting agents with potent antinociceptive activity that binds specifically and with high affinity to opioid receptors. In saturation equilibrium binding studies performed at 25 degrees C using membranes from rat brain or guinea pig cerebellum, the Ki values measured for DPI-3290 at delta-, mu-, and kappa-opioid receptors were 0.18 +/- 0.02, 0.46 +/- 0.05, and 0.62 +/- 0.09 nM, respectively. In vas deferens isolated from laboratory mice, DPI-3290 decreased electrically induced tension development in a concentration-dependent manner with corresponding IC50 values of 1.0 +/- 0.3, 6.2 +/- 2.0, and 25.0 +/- 3.3 nM at delta-, mu-, and kappa-receptors, respectively. The activity of DPI-3290 in isolated vas deferens tissue was approximately 20,000, 175.8, and 1500 times more efficacious than morphine, and 492, 2.5, and 35 times more efficacious than fentanyl at delta-, mu-, and kappa-receptors, respectively. In ileal strips isolated from guinea pigs, DPI-3290 inhibited tension development with a corresponding IC50 value of 3.4 +/- 1.6 nM at mu-opioid receptors and 6.7 +/- 1.6 nM at kappa-opioid receptors. Intravenous administration of 0.05 +/- 0.007 mg/kg DPI-3290 produced a 50% antinociceptive response in rats. The antinociceptive properties of DPI-3290 were blocked by naloxone (0.5 mg/kg s.c.). Compared with morphine, this study demonstrated that DPI-3290 is more potent and elicited a similar magnitude of antinociceptive activity in the rat, actions mediated by its mixed opioid receptor agonist activity. The marked antinociceptive activity of DPI-3290 will likely provide a means for relieving severe pain in patients that require analgesic treatment.

Allyl-2,5-dimethyl-1-piperazines have been of interest as analgesic agents for the management of moderate-to-severe pain. In this study, we compared the antinociceptive properties and respiratory depressant activity of one such agent, (+)-3-((alpha-R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-(3-fluorophenyl)-N-methylbenzamide (DPI-3290), with those of established narcotic analgesics, morphine and fentanyl. Intravenous administration of DPI-3290 in conscious laboratory rats increased antinociception in a dose-dependent manner with a corresponding ED(50) value of 0.05 +/- 0.0072 mg/kg. Simultaneous measurement of arterial blood gas in animals treated with DPI-3290 demonstrated dose-dependent increases in pCO2 with an ED(50) value of 0.91 +/- 0.22 mg/kg. In comparison, morphine and fentanyl increased antinociception in rats with ED(50) values of 2.01 +/- 0.0005 and 0.0034 +/- 0.00024 mg/kg, respectively, and the ED(50) value for morphine-induced changes in pCO2 was 4.23 +/- 0.72 mg/kg, whereas the ED(50) value for fentanyl-induced changes in pCO2 was 0.0127 +/- 0.0035 mg/kg. A separate series of experiments were designed to examine the effects of DPI-3290 on mu-opioid receptor induced antinociception and hypercapnia. Intravenous bolus doses of DPI-3290 that ranged from 0.2 to 1.0 mg/kg had no effect on antinociception mediated by alfentanil (2 microg/kg/min i.v.) but reduced hypercapnia by approximately 50%. Results from these studies demonstrate the equivalent antinociceptive efficacy of DPI-3290, morphine, and fentanyl but dramatic differences in the hypercapnia that antinociceptive doses of these drugs produce. When measured simultaneously, DPI-3290 had an 18.2-fold difference in the ratio comparing the ED(50) value for antinociception with the ED(50) value for changes in pCO2; this ratio was 2.1 for morphine and 3.7 for fentanyl. Furthermore, DPI-3290 reduced the alfentanil-mediated hypercapnia without any effect on antinociception. Together, the balanced opioid agonist activity of DPI-3290 may provide a means of powerful analgesia while mitigating the mu-opioid receptor-mediated hypercapnia.

Our laboratory has previously shown that intracerebroventricular (i.c.v.) administration of beta-endorphin suppresses brain and liver ornithine decarboxylase activity (ODC; a growth regulatory enzyme) in preweanling rats. This investigation examined, in 6-day-old rats, the relative participation of brain mu-, delta- and epsilon-opioid receptors in beta-endorphin's ODC effects, by comparing tissue ODC responses to beta-endorphin given alone i.c.v. and in the presence of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; mu-opioid receptor antagonist), N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI-174,864; delta-opioid receptor antagonist) or beta-endorphin-(1-27) (epsilon-opioid receptor antagonist). Administration of 0.5 microgram of beta-endorphin alone significantly decreased brain and liver ODC activity 4 h after injection, and the effect was completely blocked by coinjection of CTOP (0.075 micrograms) but not by ICI-174,864 (0.75 or 3.75 micrograms) or beta-endorphin-(1-27) (3.75 or 7.5 micrograms). The blockade of endogenous opioid:opioid receptor interactions by either CTOP (at doses > 0.075 microgram) or ICI-174,864 alone was accompanied by increased levels of basal ODC activity. The results obtained demonstrate that i.c.v. beta-endorphin downregulates ODC expression in central as well as in peripheral tissues by interacting with brain mu-opioid receptors, but not with delta- or epsilon-opioid receptors or mu/delta-opioid receptor complexes. Also, they indicate that endogenous opioid systems have a tonic inhibitory influence on ODC activity which is mediated, at least in part, by mu- and delta-opioid receptors.

The synthetic delta-opioid receptor agonist BW373U86 (0.18-0.56 mg/kg s.c.) was studied in rhesus monkeys with a warm-water, tail-withdrawal assay, designed to detect bradykinin (0.1 microgram) and prostaglandin E2 (5-15.8 micrograms)-induced thermal allodynia. BW373U86 dose-dependently reversed bradykinin allodynia, but was ineffective against prostaglandin E2 allodynia. The BW373U86 dose-effect curve was shifted to the right by the delta-opioid receptor-selective antagonist naltrindole (1.0 mg/kg) but not by the mu-opioid receptor-selective antagonist quadazocine (0.1 mg/kg). The present findings add to the conditions in which delta-opioid receptor-mediated behavioral effects have been detected in primates, and suggest that delta-opioid agonists may be of therapeutic interest in the treatment of some types of hyperalgesic conditions.

A cDNA clone encoding bovine muscarinic acetylcholine m3 receptor has been isolated from a bovine brain library. Sequencing of this clone has revealed a single open reading frame encoding a protein of 590 amino acids. Comparison with known muscarinic m3 receptor sequences from other species shows a high degree of conservation (92–98% homology) in the protein sequence. COS cells transfected with this cDNA expressed a single high-affinity ( K d = 43.9 pM) binding site for muscarinic receptor ligand N-[ 3 H ] methylscopolamine .

The effect of pertussis toxin on opioid antinociception was studied in rats. Intrathecal injection of a single dose of pertussis toxin reduced the antinociceptive effect of PL017, a highly selective mu-opioid agonist, in a dose- and time-dependent manner. The maximal effective dose of pertussis toxin was 1 microgram, and the maximal effect was seen at day 3. The effect of the toxin lasted for 2 weeks, and the antinociceptive response recovered partially at the third week. The dose-response curves of the antinociceptive effect of PL017 were shifted to the right with increasing doses of pertussis toxin. Three days after pertussis injection, receptor-binding activity of membranes in the lumbosacral segment of spinal cords decreased to 70% of control as assayed by 125I-FK33824, a highly selective mu-receptor agonist. In experiments using [3H]naloxone as the radiolabeled ligand, displacement curves of FK33824 were shifted to the right after pertussis toxin treatment. The shift also was dose and time dependent. Scatchard analysis of binding data showed that, after pertussis toxin treatment, there was no significant change in the total number of binding sites, but a class of low-affinity binding sites appeared in addition to the high-affinity sites. When spinal membranes were washed in Na+ (100 mM) and guanosine diphosphate (100 microM) and binding was assayed in the presence of Mg2+ (5 mM), all the mu-receptors were in the high-affinity state in control membranes. After the pertussis toxin treatment, the ratio of low-affinity sites to high-affinity sites increased.(ABSTRACT TRUNCATED AT 250 WORDS)

The antibiotic azatyrosine [DL-3-(5-hydroxy-2-pyridyl)alanine] suppressed meiotic maturation in oocytes induced by progesterone or the combination of [Val12]p21Ha-ras microinjection and insulin-like growth factor I. The suppression was dose-dependent in the range of 20-250 microM azatyrosine. In addition, azatyrosine blocked the tyrosine phosphorylation of Xp42, a member of the mitogen-activated protein kinase family, after progesterone or [Val12]p21Ha-ras/insulin-like growth factor I stimulation. Activation of maturation-promoting factor, as shown by a decrease in the tyrosine phosphorylation of the Xenopus homolog of p34cdc2, was also suppressed by azatyrosine. Azatyrosine had no effect in vivo or in vitro on the growth factor-induced autophosphorylation of the oocyte insulin-like growth factor I receptor. Azatyrosine has been shown by others [Shindo-Okada, N., Makabe, O., Nagahara, H. & Nishimura, S. (1989) Mol. Carcinog. 2, 159-167] to inhibit the growth of ras-transformed cells without affecting that of nontransformed cells. In oocytes, the antibiotic exerts an inhibitory action on both a ras-dependent and a ras-independent pathway. Lack of an effect of azatyrosine on germinal vesicle breakdown induced by the microinjection of an extract from mature oocytes, however, suggests that azatryosine is acting upstream of maturation-promoting factor activation.

Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents CHAPS and digitonin in the presence of protease inhibitors and 1 M NaCl. The solubilized receptor bound mu-opioid agonists and antagonists with affinities similar to those of native membrane receptor. The affinity of solubilized receptor for the agonist PL017 was greatly reduced by GTP gamma S, suggesting the receptor is still associated with G-protein. The solubilized material was passed through an opioid antagonist (10cd) affinity column and a wheat germ agglutinin column, set up in series, to obtain a partially purified receptor preparation. This partially purified material bound mu-agonist with low affinity and the binding affinity was no longer affected by GTP gamma S. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. Binding of opioid antagonist [3H]diprenorphine to the partially or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed saturable and stereospecific binding for opioid ligands, was predominantly of the mu-type, and exhibited as a diffuse band with a medium molecular mass of 62 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The average specific binding activity of the purified receptor was 18.8 +/- 2.3 pmol/micrograms protein, a value close to the theoretical estimation.