Oxytocin analogues with combined high smooth muscle and negligible antidiuretic activities. Investigation of position 7 in neurohypophyseal hormones

The proline residue in position 7 of oxytocin occupies one of the four corner positions in the two beta turns proposed for the preferred conformation of the pituitary hormone. It has been suggested that synthetic modifications of the residues in these corner positions will yield analogues in which one or more of the biological activities of the parent hormone is highly accentuated in terms of potency relative to other activities. In a continued effort to test this hypothesis the following analogues of oxytocin were prepared: [7-glycine]oxytocin, [1-beta-mercaptopropionic acid,7-glycine]oxytocin, [7-alanine]oxytocin, and [1-beta-mercaptopropionic acid,7-alanine]oxytocin. These peptides were found to possess the following specific activities, respectively: rat uterotonic, 65 +/- 2, 355 +/- 3, 22 +/- 1, 123 +/- 4; avian vasodepressor, 5.3 +/- 0.8, 17 +/- 0.4, 4.8 +/- 0.1, 9.8 +/- 0.5; rat antidiuretic, less than0.01, 0.062, 0.081 +/- 0.01, 0.17 +/- 0.01; rat pressor, 0.3, 0.5, 0.4, 0.5 unit/mg. Thus the analogues retain high uterotonic activity but exhibit strongly diminished renal and vascular activities relative to oxytocin. Especially noteworthy is [1-beta-mercaptopropionic acid,7-glycine]oxytocin with its high uterotonic activity but very low antidiuretic and pressor activities. The activity profile of this analogue combined with the fact that it is only slowly enzymatically degraded warrants further investigations of this peptide for clinical applications.     

Heteroaryl analogues of AMPA. 2. Synthesis, absolute stereochemistry, photochemistry, and structure-activity relationships

We have previously shown that (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA, 2] is a weak agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, specifically activated by (S)-AMPA (1), whereas (S)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-4-isoxazolyl]propionic acid [(S)-2-Py-AMPA, 5] and (RS)-2-amino-3-[3-hydroxy-5-(2-thiazolyl)-4-isoxazolyl]propionic acid (4) are potent AMPA agonists. On the other hand, (R)-APPA (3) and (R)-2-Py-AMPA (6) have been shown to be weak AMPA antagonists. We now report the synthesis of 2-Py-AMPA (7a) and the isomeric compounds 3-Py-AMPA (7b) and 4-Py-AMPA (7c) as well as the 7a analogues, (RS)-2-amino-3-[3-hydroxy-5-(6-methyl-2-pyridyl)-4-isoxazolyl]p ropion ic acid (7d) and (RS)-2-amino-3-[3-hydroxy-5-(2-quinolinyl)-4-isoxazolyl]propionic acid (7e). Furthermore, (RS)-2-amino-3-[3-hydroxy-5-(2-furyl)-4-isoxazolyl]propionic acid (2-Fu-AMPA, 7f) and its 5-bromo-2-furyl derivative (7g) were synthesized, and (S)-2-Fu-AMPA (8) and (R)-2-Fu-AMPA (9) were prepared by semipreparative chiral HPLC resolution of 7f. HPLC analyses and circular dichroism spectroscopy indicated the absolute stereochemistry of 8 and 9 to be S and R, respectively. This was confirmed by an X-ray crystallographic analysis of 9.HCl. In receptor binding (IC50 values) and rat cortical wedge electrophysiological (EC50 values) studies, 7c (IC50 = 5.5 +/- 0.6 microM; EC50 = 96 +/- 5 microM) was shown to be markedly weaker than 7a (IC50 = 0.57 +/- 0.16 microM; EC50 = 7.4 +/- 0.2 microM) as an AMPA agonist, whereas 7b,d,e were inactive. The very potent AMPA agonist effect of 7f (IC50 = 0.15 +/- 0.03 microM; EC50 = 1.7 +/- 0. 2 microM) was shown to reside exclusively in 8 (IC50 = 0.11 +/- 0.01 microM; EC50 = 0.71 +/- 0.11 microM), whereas 9 did not interact significantly with AMPA receptors, either as an agonist or as an antagonist. 8 was shown to be photochemically active and is a potential photoaffinity label for the recognition site of the AMPA receptors. Compound 7g turned out to be a very weak AMPA receptor agonist (IC50 = 12 +/- 0.7 microM; EC50 = 160 +/- 15 microM). None of these new compounds showed detectable effects at N-methyl-d-aspartic acid (NMDA) or kainic acid receptors in vitro. The present studies have emphasized that the presence of a heteroatom in the 2-position of the heteroaryl 5-substituent greatly facilitates AMPA receptor agonist activity.     

Pharmacological characterization of metabotropic glutamate receptors linked to the inhibition of adenylate cyclase activity in rat striatal slices

The pharmacological profile of mGlu receptors negatively linked to adenylyl cyclase was characterized in adult rat striatal slices. Among the mGlu agonists tested, (+)-2-aminobicyclo-[3.1.0]-hexane-2,6-di carboxylate (LY354740), was the most potent inhibitor of forskolin-stimulated cAMP formation (EC50 = 11 +/- 2 nM). Inhibition of forskolin stimulation by the group III agonist L-2-amino-4-phosphono-butanoate (L-AP4) was biphasic, the two parts of the concentration curve having EC50 values of 6 +/- 1 microM and 260 +/- 4 microM, suggesting a sequential recruitment of mGlu4/8 and mGlu7. The effects of several new phenylglycine derivative antagonists were tested on the inhibition of forskolin cAMP response by (2S,1'S,2'S)-2-(carboxy-cyclopropyl)-glycine (L-CCG I) and L-AP4. At 500 microM, (RS)-alpha-methyl-3-carboxy-methyl-pheny lglycine was unable to antagonize the effect of L-CCG I or L-AP4 but (S)-alpha-methyl-3-carboxy-phenylalanine inhibited the effect of L-AP4 with a low potency. Finally, (RS)-alpha-methyl-4-tetrazolylphenylglyc ine and particularly (RS)-alpha-methyl-4-phosphonophenylglyci ne, appeared to be the most potent and selective antagonists of L-AP4 induced inhibition of forskolin-stimulated cAMP production in adult rat striatal slices.     

Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.     

Duration of analgesia in mice after heroin by two testing methods

1 The apparent dissociation constants (Kd) of four competitive antagonists of oxytocin were estimated from their ability to compete with [3H]-oxytocin for binding sites in particulate fractions from rat uterine homogenates. 2 These apparent Kd values were not significantly different from the Kd values calculated from the published potency of each compound as an antagonist of oxytocin-induced uterine contractions. 3 These results support the conclusion that the binding sites for oxytocin are part of the receptor complex. Furthermore, 'spare receptors' for oxytocin do not appear to be present in significant quantities, and the relative potency of each antagonist appears to depend upon its affinity for the receptor site rather than its intrinsic activity. 4 The antagonists used in these studies were [N-acetyl, 2-O-methyltyrosine]oxytocin, [1-(beta-mercapto-beta,beta-diethylpropionic acid)]oxytocin, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid)]oxytocin, and [1-(deaminopenicillamine), 4-threonine]oxytocin.     

In-vitro characterization of YM872, a selective, potent and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist

The in-vitro pharmacological properties of (2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl)-acetic acid monohydrate, YM872, a novel and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist were investigated. YM872 is highly water soluble (83 mg mL(-1) in Britton-Robinson buffer) compared with 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). YM872 potently inhibits [3H]AMPA binding with a Ki (apparent equilibrium dissociation constant) value of 0.096 +/- 0.0024 microM. However, YM872 had very low affinity for other ionotropic glutamate receptors, as measured by competition with [3H]kainate (high-affinity kainate binding site, concentration resulting in half the maximum inhibition (IC50) = 4.6 +/- 0.14 microM), [3H]glutamate (N-methyl-D-aspartate (NMDA) receptor glutamate binding site, IC50 > 100 microM) and [3H]glycine (NMDA receptor glycine-binding site, IC50 > 100 microM). YM872 competitively antagonized kainate-induced currents in Xenopus laevis oocytes which express rat AMPA receptors, with a pA2 value of 6.97 +/- 0.01. In rat hippocampal primary cultures, YM872 blocked a 20-microM AMPA-induced increase of intracellular Ca2+ concentration with an IC50 value of 0.82 +/- 0.031 microM, and blocked 300-microM kainate-induced neurotoxicity with an IC50 value of 1.02 microM. These results show that YM872 is a potent and highly water-soluble AMPA antagonist with great potential for treatment of neurodegenerative disorders such as stroke.     

Ferulic acid dehydrodimers from wheat bran: isolation, purification and antioxidant properties of 8-O-4-diferulic acid

Wheat bran contains several ester-linked dehydrodimers of ferulic acid, which were detected and quantified after sequential alkaline hydrolysis. The major dimers released were: trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2, 3- dihydrobenzofuran-3-carboxylic acid (5-8-BendiFA), (Z)-beta-[4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy]-4-hydroxy-3-methox ycinnamic acid (8-O-4-diFA) and (E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid (5-5-diFA). trans-7-hydroxy-1-(4-hydroxy-3methoxyphenyl)-6-methoxy-1,2-dihydro - naphthalene-2,3-dicarboxylic acid (8-8-diFA cyclic form) and 4,4'-dihydroxy-3,3'-dimethoxy-beta,beta'-bicinnamic acid (8-8-diFA non cyclic form) were not detected. One of the most abundant dimers, 8-O-4-diFA, was purified from de-starched wheat bran after alkaline hydrolysis and preparative HPLC. The resultant product was identical to the chemically synthesised 8-O-4-dimer by TLC and HPLC as confirmed by 1H-NMR and mass spectrometry. The absorption maxima and absorption coefficients for the synthetic compound in ethanol were: lambda max: 323 nm, lambda min: 258 nm, epsilon lambda max (M-1 cm-1): 24,800 +/- 2100 and epsilon 280 (M-1 cm-1): 19,700 +/- 1100. The antioxidant properties of 8-O-4-diFA were assessed using: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes and; (b) scavenging of the radical cation of 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue, Trolox C. The 8-O-4-diFA was a better antioxidant than ferulic acid in both lipid and aqueous phases. This is the first report of the antioxidant activity of a natural diferulate obtained from a plant.     

Effects of gavage versus dosed feed administration on the toxicokinetics of benzyl acetate in rats and mice

8-epi-prostaglandin F2 alpha stimulated contraction of human myometrial strips obtained from five different donors at the time of hysterectomy with a pEC50 value of 6.3 +/- 0.5. In paired strips from the same donors the pEC50 value for the selective TP receptor agonist U46619 ([1R-[1a,4a,5b(Z),6a(1E,3S*)]]-7-[6-(3- hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid) was 8.3 +/- 0.4. In strips from four other donors 8-epi-prostaglandin F2 alpha was ineffective whereas the pEC50 for U46619 was 6.9 +/- 0.3. Responses to 8-epi-prostaglandin F2 alpha were unaffected by the selective DP receptor antagonist BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2- cyclohexyl-2-hydroxyethylamino)hydantoin) at 50 nM but were blocked by the selective TP receptor antagonist L670596 ((-)6,8-difluoro-9-p-methylsulfonyl benzyl-1,2,3,4- tetrahydrocarbazol-1-yl-acetic acid) at 50 nM. The pIC50 values obtained when the TP receptor antagonists GR 32191 ([1R- [1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-[[(1,1'-biphenyl)-4- yl]methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid), ICI D1542 ((4(Z)-6-[(2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]- 4-(3-pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid), ICI 192605 (4(Z)-6-[(2,4,5-cis)-2-(2-chlorophenyl)-4-(2-hydroxyphenyl)-1,3- dioxan-5-yl]hexenoic acid), L670596 and SQ 29548 ([1S-(1 alpha,2 beta(5Z),3 beta,4 alpha]]-7- [3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7- oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid) were added cumulatively to strips pre-contracted with an EC80 concentration of 8-epi-prostaglandin F2 alpha were not significantly different from those obtained when an EC80 concentration of U46619 was used. The effects of 8-epi-prostaglandin F2 alpha on strips pre-contracted with an EC80 concentration of U46619 were not different from those of U46619 itself. It is concluded that in the non-pregnant human myometrium 8-epi-prostaglandin F2 alpha is a medium potency contractile agonist acting predominantly at the TP receptor.     

Effect of oxytocin as a partial agonist at vasoconstrictor vasopressin receptors on the human isolated uterine artery

1. The effect of oxytocin on endothelium-intact and endothelium-denuded segments of the human uterine artery rings was investigated. 2. In both types of preparation oxytocin induced contraction of human uterine artery with similar potency and efficacy (pEC50 values: 6.95 +/- 0.05 vs 7.06 +/- 0.01; maximal response values: 61 +/- 4.1% vs 63 +/- 5.1% for arteries with and without endothelium, respectively). 3. In contrast, human uterine arteries, both intact and denuded of endothelium, did not respond to the addition of the selective oxytocin receptor agonist, [Thr4, Gly7]oxytocin (10 nM(-1) microM). 4. The vasopressin receptor antagonists, [d(CH2)5Tyr(Me)]AVP (10-100nM) and [d(CH2)5,D-Ile2,Ile4]AVP (300 nM-3 microM) produced parallel rightward shifts of the curves for oxytocin. The Schild plots constrained to a slope of unity gave the following -log K(B) values: [d(CH2)5Tyr(Me)] AVP vs [d(CH2)5,D-Ile2,Ile4] AVP 9.24 vs 6.91 and 9.26 vs 6.84 for human uterine artery with intact and those denuded of endothelium, respectively. In contrast, in both types of preparations the oxytocin receptor antagonist, [d(CH2)5Tyr(OMe), 2Orn8]vasotocin (1 microM), did not significantly affect oxytocin-induced contractions. 5. The calculated pK(A) values for oxytocin itself also did not differ between preparations: 6.56 and 6.43 for human uterine artery with and without endothelium, respectively. In both types of preparations, the receptor reserve (K(A)/EC50) was close to unity (intact vs denuded: 3.9 vs 3.0). 6. It is concluded that, in human uterine artery, oxytocin induces contractions that are not modulated by the endothelium. It is likely that oxytocin acts as a partial agonist on human uterine artery, regardless of the endothelial condition. On the basis of differential antagonists affinity and affinity of oxytocin itself, it is probable that receptors involved in oxytocin-induced contraction in human uterine arteries belong to the V(1A) vasopressin receptors.     

Pharmacology and toxicology of ATOA, an AMPA receptor antagonist and a partial agonist at GluR5 receptors

(RS)-2-Amino-3-[3-(carboxymethoxy)-5-tert-butyl-4-isoxazolyl]propi onic acid (ATOA) has previously been described as an antagonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors with an IC50 value of 150 microM towards AMPA-induced depolarisation in the rat cortical wedge preparation. ATOA has now been shown also to be a partial agonist at recombinant GluR5 receptors, expressed in Xenopus oocytes, with an EC50 value of 170 microM and a relative efficacy of 0.17 +/- 0.04 compared with responses produced by kainic acid (1.0). Using cultured cerebral cortical neurones as a test system and leakage of lactate dehydrogenase (LDH) as an indicator of cell damage, ATOA was shown to be cytotoxic (ED50 > 300 microM), though much less toxic than the structurally related dual AMPA and GluR5 agonist, (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid (ATPA) (ED50 = 14 +/- 2 microM). The toxic effect of ATPA was sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX) but was not significantly reduced by the selective AMPA receptor antagonist, (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA). The toxicity of ATOA (1 mM) could not be significantly attenuated by co-administration of AMOA (300 microM) or DNQX (25 microM). A structure-activity analysis indicates that the tert-butyl group of ATPA and ATOA facilitates the interaction of these compounds with GluR5 receptors.     

Expression, characterization, and purification of C-terminally hexahistidine-tagged thromboxane A2 receptors

Thromboxane A2 (TxA2) receptors belong to the class of G-protein-coupled receptors. Knowledge of the relationship of structure to function for TxA2 receptors is limited because of their low levels of expression, lengthy purification procedures and poor recoveries. A C-terminal hexahistidine-tag (C-His) was ligated to the alpha-isoform of TxA2 receptors and expressed in COS-7 and Chinese hamster ovary cells. The C-His-TxA2 receptors bound the radioligands 125I-7-[(1R,2S,3S,5R)-6, 6-dimethyl-3-(4-benzenesulfonylamino)bicyclo[3.1. 1]hept-2-yl]-5(Z)-heptenoic acid, an antagonist, and 125I-[1S-1alpha, 2beta(5Z),3alpha(1E,3S*), 4alpha]-7-[3[(3-hydroxy-4-(4'-phenoxy)-1butenyl)-7-oxabicycl o-[2.2. 1]heptan-2-yl]-5-heptanoic acid, an agonist, with affinities not significantly different from those of the wild type (wt)-TxA2 receptors. LipofectAMINE transfection of the cDNAs resulted in high levels of expression (Bmax = 95 +/- 6 pmol/mg) of the C-His-TxA2 receptors. In competition binding studies the IC50 values of five different ligands were not significantly different between C-His-TxA2 and wt-TxA2 receptors. Agonist-induced stimulation of cAMP and total inositol phosphate formation were not significantly different between the two receptors. Purification on a Ni2+-NTA column resulted in a rapid (within 4 h) purification with a 36 +/- 2% recovery and a 30 +/- 6-fold purification (n = 5). The partially purified receptors were resolved on SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, dissolved in acetone/trifluoroacetic acid/hexafluoroisopropanol/sinapinic acid, and successfully subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results suggest that the combination of a high level of expression of C-His-TxA2 receptors and a rapid purification procedure followed by SDS- polyacrylamide gel electrophoresis may provide a useful approach for mass-spectrometry based structure-function and other studies of TxA2 receptors.     

Synthesis and some pharmacological properties of deamino(4-threonine,8-D-arginine)vasopressin and deamino(8-D-arginine)vasopressin, highly potent and specific antidiuretic peptides, and (8-D-arginine)vasopressin and deamino-arginine-vasopressin

Deamino[4-threonine,8-D-arginine]vasopressin (dTDAVP), deamino[8-D-arginine]vasopressin (dDAVP), [8-D-arginine[vasopressin (DAVP), and deamino-arginine-vasopressin (dAVP) were synthesized by the solid-phase method and tested for their biological activities. dTDAVP has an antidiuretic potency of 793+/-95 units/mg and undetectable vasporessor activity, less than 0.02unit/mg. The antidiuretic-pressor (A/P) ratio of dTDAVP is greater than 39 000. dDAVP has an antidiuretic potency of 1200+/-126 units/mg and a vasopressor potency of 0.39+/-0.02; its A/P ratio is thus 3000. DAVP has an antidiuretic potency of 253+/-44 units/mg, a vasopressor potency of 1.1+/-0.04 units/mg, and an A/P ratio of 240. The A/P ratios of dDAVP and DAVP are much higher than those originally reported. dAVP has an antidiuretic potency of 1745+/-385 units/mg, a vasopressor potency of 346+/-13, and an A/P ratio of 5; values are in general agreement with those in the literature. Threonine subsitution has thus brought about a significant enhancement in antidiuretic specificity, a finding entirely consistent with earlier observations that enhancement of lipophilicity at position 4 alone or in combination in arginine-vasopressin can lead to enhanced antidiuretic specificity.     

Characterization of 5-HT1A receptor-mediated [35S]GTPgammaS binding in rat hippocampal membranes

Stimulation of [35S]GTPgammaS binding by serotonin (5-hydroxytryptamine, 5-HT) receptor ligands was characterized in rat hippocampal membranes. The optimized assay contained 30-50 microg protein, 300 microM GDP and 0.1 nM [35S]GTPgammaS, incubated at 37 degrees C for 20 min. At 10 microM, the 5-HT1A receptor agonist R(+)-8-hydroxy-2-(di-n-propylamino)tetralin [R(+)-8-OH-DPAT] stimulated GTPgammaS binding from 27.1 +/- 2.5 to 45.7 +/- 4.2 fmol/mg protein. Increasing the protein concentration did not affect the absolute difference between basal and maximal GTPgammaS binding nor the EC50, but decreased the percent stimulation. The non-selective agonists serotonin and 5-carboxamidotryptamine were 30-35% more efficacious, whereas the partial agonists buspirone and S(-)-8-hydroxy-2-(di-n-propylamino)tetralin stimulated GTPgammaS binding by 19 +/- 1 and 43 +/- 3%, respectively, compared to R(+)-8-OH-DPAT. Neither the 5-HT2 receptor agonist [(+/-)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl] (DOI) nor the 5-HT1A receptor antagonists WAY 100,635 (n-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-n-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride) and spiperone altered basal GTPgammaS binding. WAY 100,635 abolished the effect of R(+)-8-OH-DPAT, but only reduced the effect of serotonin by 88 +/- 3%. Finally, methiothepin antagonized R(+)-8-OH-DPAT-stimulated GTPgammaS binding and reduced basal GTPgammaS binding by itself. The reduction was not affected by WAY 100,635. We have characterized a method to assess functional activity at 5-HT1A receptors in rat hippocampal membranes by measuring agonist-induced [35S]GTPgammaS binding.     

Pharmacological characterization of metabotropic glutamate receptors coupled to phospholipase D in the rat hippocampus

1. Phospholipase D (PLD) is the key enzyme in a signal transduction pathway leading to the formation of the second messengers phosphatidic acid and diacylglycerol. In order to define the pharmacological profile of PLD-coupled metabotropic glutamate receptors (mGluRs), PLD activity was measured in slices of adult rat brain in the presence of mGluR agonists or antagonists. Activation of the phospholipase C (PLC) pathway by the same agents was also examined. 2. The mGluR-selective agonist (1S,3R)-l-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] induced a concentration-dependent (10-300 microM) activation of PLD in the hippocampus, neocortex, and striatum, but not in the cerebellum. The effect was particularly evident in hippocampal slices, which were thus used for all subsequent experiments. 3. The rank order of potencies for agonists stimulating the PLD response was: quisqualate > ibotenate > (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine > (1S,3R)-ACPD > L-cysteine sulphinic acid > L-aspartate > L-glutamate. L-(+)-2-Amino-4-phosphonobutyric acid and the ionotropic glutamate receptor agonists N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate failed to activate PLD. (RS)-3,5-dihydroxyphenylglycine (100300 microM), an agonist of mGluRs of the first group, stimulated PLC but inhibited the PLD response elicited by 100 microM (1S,3R)-ACPD. 4. (+)-alpha-Methyl-4-carboxyphenylglycine (0.1-1 mM), a competitive antagonist of mGluRs of the first and second group, elicited a significant PLD response. L-(+)-2-Amino-3-phosphonopropionic acid (1 mM), an antagonist of mGluRs of the first group, inhibited the 100 microM (1S,3R)-ACPD-induced PLC response but produced a robust stimulation of PLD. 5. 12-O-Tetradecanoylphorbol 13-acetic acid and phorbol 12,13-dibutyrate (PDBu), activators of protein kinase C, at 1 microM had a stimulatory effect on mGluRs linked to PLD but depressed (1S,3R)-ACPD-induced phosphoinositide hydrolysis. The protein kinase C inhibitor, staurosporine (1 and 10 microM) reduced PLD activation induced by 1 microM PDBu but not by 100 microM (1S,3R)-ACPD. 6. Our results suggest that PLD-linked mGluRs in rat hippocampus may be distinct from any known mGluR subtype coupled to PLC or adenylyl cyclase. Moreover, they indicate that independent mGluRs coupled to the PLC and PLD pathways exist and that mGluR agonists can stimulate PLD through a PKC-independent mechanism.     

In vivo pharmacology of an angiotensin AT1 receptor antagonist with balanced affinity for AT2 receptors

L-163,017 (6-[benzoylamino]-7-methyl-2-propyl-3-[[2'-(N-(3-methyl-1-butoxy) carbonylaminosulfonyl)[1,1']-biphenyl-4-yl]methyl]-3H-imidazo[4,5- b]pyridine) is a potent, orally active, nonpeptide angiotensin II receptor antagonist. Conscious rats and dogs were dosed p.o. and i.v.; in both species the plasma bioequivalents are similar at the angiotensin AT1 and AT2 receptor sites indicating balanced activity is maintained in vivo. L-163,017 prevents the pressor response to intravenous (i.v.) angiotensin II in the conscious rat, dog, and rhesus monkey. L-163,017 also significantly reduces blood pressure in a renin-dependent model of hypertension, similar to an angiotensin converting enzyme inhibitor (Enalapril) and an angiotensin AT1 receptor-selective antagonist (L-159,282). These studies indicate that neither the angiotensin AT2 receptor nor bradykinin is important in the acute antihypertensive activity of angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists.     

Antioxidative compounds isolated from safflower (Carthamus tinctorius L.) oil cake

Seven antioxidative serotonin derivatives were isolated from safflower (Carthamus tinctorius L.) oil cake. Their structures were established as N-[2-(5-hydroxy-1H-indol-3-yl)ethyl]ferulamide (1), N-[2-(5-hydroxy-1H-indol-3-yl)ethyl]-p-coumaramide (2), N,N'-[2,2'-(5,5'-dihydroxy-4,4'-bi-1H-indol-3,3'-yl)diethyl]- di-p-coumaramide (3), N-[2-[3'-[2-(p-coumaramido)ethyl]-5,5'-dihydroxy- 4,4'-bi-1H-indol-3-yl]ethyl]ferulamide (4), and N,N'-[2,2'-(5,5'-dihydroxy-4,4'-bi-1H-indol-3,3'-yl)diethyl]- diferulamide (5), N-[2-[5-(beta-D-glucosyloxy)-1H-indol-3-yl)ethyl]- p-coumaramide (6), and N-[2-[5-(beta-D-glucosyloxy)-1H-indol-3-yl)ethyl]ferulamide (7). Antioxidative activities of the compounds were measured by the ferric thiocyanate method and the alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) method, and compounds 1-5 were found to have relatively strong antioxidative activity.     

Rates of lysine catabolism are inversely related to rates of protein synthesis when measured concurrently in adult female rats induced to grow at different rates

To test the effect of changes in the rate of protein synthesis on amino acid oxidation, both were studied concurrently in individual 200-g female Sprague-Dawley rats. In a growth trial (Experiment 1), recombinant bovine somatotropin (rbST) was injected subcutaneously (0, 2 or 12 mg/d) over 6 d (n = 4 rats per rbST level). Weight gain increased with rbST level (P < 0.01); 1.96 +/- 0.8, 4.24 +/- 0.8 and 8.67 +/- 0.8 g/d, respectively. After treatment with rbST (0 or 12 mg/d) for 4 d (Experiment 2), rats were injected via a tail vein catheter with valine (400 mmol, 4.07 mBq L-[3,4(n)-3H]valine) at 0, 4, 10, 13 or 16 h after the daily rbST injection and killed 20 min later. This flooding dose was 5 to 6 times, not 10 times, the free pool as hoped. Protein synthesis in rbST-treated rats increased 46% in muscle (P < 0.001) and 36% in liver (P < 0.01). The ks was unaltered with time after rbST injection (0-16 h, P > 0.05). When 600 mmol valine (4.4 mBq L-[3,4(n)-3H]valine) was used in Experiment 3, specific activity (SA) of free valine was constant over 20 min and was 94 +/- 4% of that injected. Finally, in Experiment 4, protein synthesis and amino acid oxidation rates measured in the same rat revealed a 35% increase (P < 0.01) in protein synthesis in hind leg muscle and a 29% increase in liver (P < 0.05) from rbST-injected (12 mg/d) rats (n = 6). Lysine oxidation was estimated by continuous (12 h) infusion of L-[1-14C]lysine via the opposite tail vein catheter. Expired CO2 was collected over 20-min intervals and SA at plateau was estimated by fitting an exponential model. Lysine oxidation was reduced (P < 0.05) by 44% in rbST-treated rats. The idea that an increase in protein synthesis results in decreased amino acid oxidation remains tenable.     

The co-culture of dermal fibroblasts with human epidermal keratinocytes induces increased prostaglandin E2 production and cyclooxygenase 2 activity in fibroblasts

BACKGROUND: We recently demonstrated that inhibition of nitric oxide (NO) production ameliorated acute pulmonary allograft rejection. This study examined whether inducible NO synthase (iNOS) was expressed in the transplanted lung during acute rejection. METHODS: With a rat left lung transplant model, tissue from syngeneic (Fischer 344 to Fischer 344) and allogeneic (Brown Norway to Fischer 344) transplants were harvested on postoperative day 4 and analyzed for iNOS mRNA expression (ribonuclease protection assay), iNOS enzyme activity (conversion of L-[3H]-arginine to NO and L-[3H]-citrulline), and serum nitrite/nitrate levels. RESULTS: The iNOS mRNA was expressed in allograft lungs but was not detected in isografts or controls. The iNOS protein was present in allograft lungs, as demonstrated by high levels of L-[3H]-citrulline production compared with minimal iNOS enzyme activity in isograft and control lungs (10.1 +/- 2.4 vs 0.6 +/- 0.2 and 0.7 +/- 0.2 pmol L-[3H]-citrulline.mg-1.min-1, respectively; n = 6, p < 0.001). Allografts had significantly elevated systemic serum nitrite/nitrate levels compared with isografts and controls (38 +/- 6 vs 18 +/- 2 and 16 +/- 1 mumol/L, respectively; n = 6; p < 0.005). CONCLUSIONS: These results, together with our previous demonstration that iNOS inhibition ameliorated lung allograft rejection, suggest that (1) iNOS expression and increased NO production contributed to acute rejection of the transplanted lung, (2) iNOS inhibition may offer an alternative in management of acute lung allograft rejection, and (3) increased NO production, detected by the presence of iNOS mRNA or protein or noninvasively by measuring serum nitrite/nitrate levels, may serve as an early marker of acute allograft rejection.     

The paradoxical hedonic valence of acute ethanol withdrawal (hangover) states in rats: place and taste conditioning

We describe the synthesis and pharmacological properties of two series of analogues: one which consists of three peptides having L-1-naphthylalanine in position 3 and the second composed of analogues substituted in position 3 with L-2-naphthylalanine. All peptides were tested in bioassays for pressor and antidiuretic activities. We also checked the uterotonic activity in vitro. We observed that the activity of counterparts in both series is, in two cases, strikingly different. One of the new analogues, [(L-2-Nal)3,(D-Arg)8]VP is among the most potent antagonists of the vasopressor response to AVP. Moreover, it is the first potent V1 antagonist devoid of antiuterotonic activity. This analogue was designed without modification of position 1, which was previously thought to be essential for substantial pressor antagonism. Two other peptides, [Mpa1;(L-2-Nal)3;(D-Arg)8]VP and [Mpa1,(L-1-Nal)3,D-Arg)8]VP, are highly potent V2 agonists. The second analogue is highly selective. With the exception of [(L-2-Nal)3]AVP, which showed weak antioxytocic activity, (L-Nal)3 modification resulted in the almost complete removal of interaction of our analogues with oxytocic receptors in vitro. Our results suggest that position 3 in AVP and its analogues is important not only for binding and recognition as previously though, but also for pressor, antidiuretic and uterotonic activities. We also assume that the hindering effect caused by bulky naphthyl moiety has a significant impact on the bioactive conformations of molecules which contain Nal residue, and can thus influence their interaction with V1, V2 and oxytocic receptors.     

PYY-preferring receptor in the dorsal vagal complex and its involvement in PYY stimulation of gastric acid secretion in rats

1. Microinjection of peptide YY (PYY, 7-46 pmol) into the dorsal vagal complex (DVC) stimulated gastric acid secretion in urethane-anaesthetized rats. Using a variety of neuropeptide Y (NPY) and PYY derivatives, we characterized the pharmacological profile of the receptor mediating the acid secretory response to PYY. 2. [Pro34]rat(r)/porcine(p)PYY and [Pro34]human(h)PYY (23-117 pmol), microinjected unilaterally into the DVC resulted in a similar maximal increase in net acid secretion reaching 68+/-11 and 89+/-31 micromol 90 min(-1) respectively. 3. Rat/hNPY and pNPY (47 pmol) microinjected into the DVC induced a similar net gastric acid secretion (27+/-8 and 23+/-8 micromol 90 min(-1) respectively) and a higher dose (116 pmol) tended to reduce the response. 4. Pancreatic polypeptide (PP, 4-46 pmol), [Leu31,Pro34]r/hNPY (47 and 117 pmol) and the Y2 selective agonists, hPYY3-36, pNPY5-36 and PNPY13-36 (25-168 pmol) microinjected into the DVC failed to influence basal gastric acid secretion. 5. The rank order of potency of PYY > or = [Pro34]r/pPYY = [Pro34]hPYY> r/hNPY = pNPY to stimulate gastric acid secretion upon injection into the DVC and the ineffectiveness of PP, [Leu31,Pro34]NPY and C-terminal NPY/PYY fragments suggest that a PYY-preferring receptor subtype may be involved in mediating the stimulating effect.