Stereostructure activity relationships of catecholamines on human platelet function

The concentration-dependent effects of clonidine, isomers of epinephrine, norepinephrine (NE), isoproterenol, cobefrin and alpha-methyldopamine, and related desoxy analogs (epinine, dopamine, N-isopropyldopamine) were examined on human platelets. The rank order of aggregatory potency (pD2 values) was R(-)-epinephrine (6.3) greater than R(-)-NE (5.9) greater than (+/-)-erythro-cobefrin (5.3) greater than S(+)-epinephrine (4.7) = S(+)-NE (4.7) = clonidine (4.7) = dopamine (4.6) greater than epinine (4.4) greater than S(+)-alpha-methyldopamine (4.3) = R(-)-alpha-methyldopamine (4.3) greater than (+/-)-threo-cobefrin (3.7). The isoproterenol isomers and N-isopropyl-dopamine were inactive as agonists. In 9 of 16 platelet-rich plasma preparations, R(-)-epinephrine, R(-)-NE, and (+/-)erythro-cobefrin were agonists and the remaining analogs blocked R(-)-NE-induced aggregation with a rank order of inhibitory potencies (pKB values) of clonidine (6.2) greater than S(+)-alpha-methyldopamine (5.0) greater than dopamine (4.6) = R(-)-alpha-methyldopamine (4.4) greater than or equal to S(+)-NE (4.3) greater than N-isopropyldopamine (4.1) greater than S(+)-isoproterenol (3.7) = R(-)-isoproterenol (3.5). Each compound was also able to reverse prostaglandin E1 (PGE1) (0.1 microM)-induced blockade of the maximal aggregation response to ADP. At high concentrations, R(-)-isoproterenol was more potent than either the S(+)-isomer or desoxy analog, N-isopropyldopamine, in the reversal of PGE1 inhibition of ADP aggregation. Phentolamine blocked these alpha 2-adrenoceptor-mediated actions against PGE1 on ADP aggregation. The rank order of potency for the reversal of PGE1-mediated inhibition of ADP aggregation by these catecholamines was similar to that observed for platelet aggregation. Our results indicate that (i) the stereochemical requirements for the interaction of catecholamines with platelet alpha 2-adrenoceptors are in agreement with the Easson-Stedman hypothesis and other alpha-adrenoceptor tissues; (ii) catecholamines lacking a benzylic hydroxyl group in the R-configuration and/or possessing an N-isopropyl group were alpha 2-adrenoceptor antagonists; (iii) clonidine gave quantitatively different responses compared with catecholamines for interaction with alpha 2-adrenoceptors; and (iv) inhibition of platelet adenylate cyclase is correlated to the inhibition of epinephrine-induced aggregation response for this series of compounds.     

Platelet aggregation induced by platelet-activating factor is suppressed by cystine protease inhibitor

The effect of NCO-700, a cystine protease inhibitor, on platelet-activating factor-induced platelet aggregation was determined. A newly synthesized cystine protease inhibitor (calcium-activated neutral protease and cathepsin B inhibitor), NCO-700 (bis[ethyl (2R, 3R)-3-[(S)-methyl-1-[4-(2,3,4- trimethoxyphenylmethyl) piperazine-1-ylcarbonyl]butylcarbonyl]oxiran-2-carboxy late]sulfate), inhibited platelet-activating factor-induced platelet aggregation. The inhibition was dependent on the preincubation time with NCO-700 and on the concentration of the inhibitor. The release of serotonin was also inhibited almost completely by the 20-min preincubation with 10(-4) M NCO-700. Leupeptin also inhibited platelet-activating factor-induced platelet aggregation. But calcium-activated neutral protease inhibitor did not inhibit it. These observations suggest that NCO-700-sensitive protease(s) such as cystine protease may contribute to platelet aggregation induced by platelet-activating factor.     

Microbial transformation of (+)-nootkatone and the antiproliferative activity of its metabolites

Six metabolites were obtained as a result of microbial transformation of (+)-nootkatone (1) by the fungal strains: Botrytis, Didymosphaeria, Aspergillus, Chaetomium and Fusarium. Their structure were established as (+)-(4R,5S,7R,9R)-9α-hydroxynootkatone (2), (+)-(4R,5S,7R)-13-hydroxynootkatone (3) and (+)-(4R,5S,7R,9R,11S)-11,12-epoxy-9α-hydroxynootkatone (4), (+)-(4R,5S,7R,11S)-11,12-epoksynootkatone (5), (+)-(4R,5S,7R)-11,12-dihydroxynootkatone (6) and (+)-(4R,5S,7R)-7,11,12-trihydroxynootkatone (7) on the basis of their spectral data. Two products: (4) and (7) were not previously reported in the literature. The antiproliferative activity of (+)-nootkatone (1) and isolated metabolites (2-7) of its biotransformation has been evaluated.     

The response to thromboxane A2 analogues in human platelets. Discrimination of two binding sites linked to distinct effector systems

Thromboxane A2 (TXA2) induces platelet shape change, secretion, and aggregation. Using a novel TXA2/prostaglandin endoperoxide receptor antagonist, [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 hydrochloride (GR32191), we demonstrate that these responses are mediated by at least two receptor-effector systems. GR32191 non-competitively inhibited platelet aggregation to the TXA2 mimetics, (15S)-hydroxy-11,9-(epoxymethano) prostadienoic acid (U46619) and [1S-(1 alpha,2 beta(5Z),3 alpha (1E,-3S), 4 alpha)]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)7- oxabicyclo[2.2.1]hept-2yl]-5-heptenoic acid by binding irreversibly to a TXA2/prostaglandin endoperoxide receptor. Dissociation of [3H]GR32191 from human platelets demonstrated two specific binding sites, one which was rapidly dissociating and a site to which binding was essentially irreversible. Stimulation by U46619 of platelets incubated with GR32191 and subsequently washed to expose the reversible binding site failed to aggregate or to secrete [3H]5-hydroxy-tryptamine; formation of inositol phosphates and activation of protein kinase C were markedly suppressed. In contrast, platelet shape change and calcium stimulation remained at 90% of control. Furthermore, stimulation of the reversible binding site with U46619 induced aggregation in the presence of ADP, demonstrating its functional importance in amplifying the response to other agonists. These data suggest that TXA2 mediates platelet activation through at least two receptor-effector systems; one linked to phospholipase C activation, resulting in platelet aggregation and secretion and a second site mediating an increase in cytosolic calcium and platelet shape change.     

Dihydroflavonols from Lannea coromandelica

The dihydroflavonols, (2R,3S)-(+)-3',5-dihydroxy-4',7-dimethoxydihydroflavonol and (2R,3R)-(+)-4',5,7-trimethoxydihydroflavonol were isolated from the stem bark of Lannea coromandelica, along with the known (2R,3R)-(+)-4',7-di-O-methyldihydroquercetin, (2R,3R)-(+)-4',7-di-O-methyldihydrokaempferol and (2R,3R)-(+)-4'-O-methyldihydroquercetin. All five compounds were isolated for the first time from the genus Lannea; furthermore, (2R,3S)-(+)-3',5-dihydroxy-4',7-dimethoxydihydroflavonol, was a rare cis-type isomer. The structures of all compounds were elucidated by spectroscopic methods including 2D NMR and CD analysis.     

Pharmacologic antagonism of thromboxane A2 receptors by trimetoquinol analogs in vitro and in vivo.

Although (-)-(S)-trimetoquinol [1-(3,4,5-trimethoxy-benzyl)- 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TMQ] is recognized as a potent bronchodilator, (+)-(R)-TMQ is a selective antagonist of human platelet aggregation and serotonin secretion induced by thromboxane A2 (TXA2) agonists. To confirm the pharmacological actions of TMQ analogs, the interaction of the drugs with TXA2 receptors was examined in human platelets and in a mouse sudden death model. The inhibitory potencies of TMQ analogs (pIC50 values) for displacement of [3H]SQ 29,548 binding to platelets showed excellent correlation with the respective pIC50 (-log IC50) values for U46619-induced aggregation (r = 0.99, P less than 0.01) and serotonin secretion (r = 0.99, P less than 0.01) in human platelet-rich plasma and for whole blood aggregation (r = 0.99, P less than 0.01). In each system, the rank order of inhibitory potencies was rac-iodoTMQ greater than or equal to (+)-(R)-TMQ greater than rac-TMQ much greater than (-)-(S)-TMQ. Antithrombotic effects of TMQ analogs were evaluated in a mouse sudden death model. In vivo antithrombotic potencies of these compounds were consistent with the in vitro potencies as TXA2 receptor antagonists in platelet systems. Administration of rac-iodoTMQ, (+)-(R)-TMQ and rac-TMQ 15 min before the injection of U46619 (800 micrograms/kg, iv) protected mice against U46619-induced sudden death. On the other hand, (-)-(S)-TMQ did not protect animals against death. Protection of U46619-induced cardiopulmonary thrombosis by TMQ analogs was seen at doses of 3-100 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resolution and adrenergic activities of the optical isomers of 4-[1-(1-naphthyl)ethyl]-1H-imidazole.

Recently we synthesized a naphthalene analog of medetomidine, 4-[1-(1-naphthyl)ethyl]-1H-imidazole hydrochloride (1), and found it to be highly potent in adrenergic systems. The separation of optical isomers of this naphthalene analog was achieved by using the isomers of tartaric acid. The optical purities of the isomers were determined by HPLC using a chiral column. Using X-ray analysis the (+)-isomer was determined to have the S absolute configuration. It has been reported that the (+)-isomer of medetomidine (2) is the most potent enantiomer on alpha 2-adrenergic receptors. There were both qualitative and quantitative differences in biological activities of the optical isomers of 1 in alpha 1- and alpha 2-adrenergic receptor systems of guinea pig ileum and human platelets. (+)-(S)-1, but not (-)-(R)-1 was a selective agonist of alpha 2-mediated responses in ileum whereas (-)-(R)-1 was more potent than (+)-(S)-1 as an inhibitor of alpha 2-mediated platelet aggregation.     

The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB₁and CB₂receptors in the mouse hippocampus

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.     

Novel, not adenylyl cyclase-coupled cannabinoid binding site in cerebellum of mice

In this study we report data suggesting the presence of a non-CB1, non-CB2 cannabinoid site in the cerebellum of CB1-/- mice. We have carried out [(35)S]GTPgammaS binding experiments in striata, hippocampi, and cerebella of CB1-/- and CB1(+/+) mice with Delta(9)-THC, WIN55,212-2, HU-210, SR141716A, and SR144528. In CB1-/- mice Delta(9)-THC and HU-210 did not stimulate [(35)S]GTPgammaS binding. However, WIN55,212-2 was able to stimulate [(35)S]GTPgammaS binding in cerebella of CB1-/- mice. The maximal effect of this stimulation was 31% that of wild type animals. This effect was reversible neither by CB1 nor CB2 receptor antagonists. Similar results were obtained with the endogenous cannabinoid, anandamide. However, adenylyl cyclase was not inhibited by WIN55,212-2 or anandamide in the CB1(minus sign/minus sign) animals. In striata and hippocampi of CB1-/- mice no [(35)S]GTPgammaS stimulation curve could be obtained with WIN55,212. Our findings suggest that there is a non-CB1 non-CB2 receptor present in the cerebellum of CB1-/- mice.     

9 alpha-homo-9,11-epoxy-5,13-prostadienoic acid analogues: specific stable agonist (SQ 26, 538) and antagonist (SQ 26, 536) of the human platelet thromboxane receptor

A newly synthesized 9 alpha-homo-9,11-epoxy-5,13-prostadienoic acid analogue, SQ 26, 536, (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z), 13(E)-15S-hydroxyprostadienoic acid) inhibited arachidonic acid (AA)-induced platelet aggregation with an I50 value of 1.7 microM. SQ 26,536 did not inhibit prostaglandin (PG) synthetase activity of bovine seminal vesicle microsomes or thromboxane (Tx) synthetase activity of lysed human blood platelets. SQ 26,536 also inhibited platelet aggregation induced by epinephrine (secondary phase), 9,11-azoPGH2 and collagen but did not inhibit the primary phase of epinephrine-induced aggregation or ADP-induced platelet aggregation. SQ 26,538 (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z),13(E)-15R-hydroxyprostadienoic acid), a 15-epimer of SQ 26,536, induced platelet aggregation with an A50 value of 2.5 microM. SQ 26,536 competitively inhibited SQ 26,538-induced platelet aggregation with a Ki value of 3 microM. Neither indomethacin, a PG synthetase inhibitor, nor SQ 80,338 (1-(3-phenyl-2-propenyl)-1H-imidazole), a Tx synthetase inhibitor, inhibited SQ 26,538- or 9,11-azoPGH2-induced platelet aggregation. These data indicate that SQ 26,536 and SQ 26,538 are stable antagonist and agonist, respectively, of the human blood platelet thromboxane receptor.     

Revision of the structure of the major aggregation pheromone of the broad-horned flour beetle (Gnatocerus cornutus) to (1S,4R,5R)-alpha-acoradiene by its synthesis

(1S,4R,5R)-(+)-alpha-acoradiene [1,8-dimethyl-4-(1-methylethenyl)spiro[4.5]dec-7-ene] (1) is shown by its synthesis to be the major component of the male-produced aggregation pheromone of the broad-horned flour beetle (Gnatocerus cornutus).     

Novel chiral isoxazole derivatives: synthesis and pharmacological characterization at human beta-adrenergic receptor subtypes

Isoxazole derivative (+/-)-4 and the three pairs of stereoisomeric 3-bromo-isoxazolyl amino alcohols (S,R)-(-)-7a/(R,R)-(+)-7b, (S,R)-(-)-8a/(R,R)-(+)-8b, and (S,R)-(-)-9a/(R,R)-(+)-9b were synthesized and assayed for their affinity and efficacy at human beta(1)-, beta(2)-, and beta(3)-adrenergic receptors (beta-ARs) in membranes from Chinese hamster ovary (CHO) cells stably transfected with the respective receptor subtype. Whereas derivative (+/-)-4 did not bind at all three beta-ARs, stereoisomers (S,R)-7a-(S,R)-9a behaved as high-affinity ligands at beta(1)- and, particularly, at beta(2)-ARs (K(i) 2.82-66.7 nM). The K(i) values of isomers (R,R)-7b-(R,R)-9b at beta(1)- and beta(2)-subtypes were about 30-100 times higher than those of their (S,R)-7a-9a counterparts, indicating a sizable stereochemical effect. The affinity at beta(3)-ARs was negligible for all the investigated compounds. When submitted to a functional assay, the three stereoisomeric pairs showed a comparable pattern of efficacy at all three beta-AR subtypes. The highest value of efficacy (75-90%) was observed at beta(2)-ARs, whereas all compounds behaved as partial agonists (30-60%) at the beta(3)-subtype. The lowest degree of efficacy (15-35%) was found at beta(1)-ARs. The affinity/efficacy profile of the derivatives under study has been compared with that of the two model compounds, Broxaterol [(+/-)-1] and BRL 37344 [(+/-)-6].     

9α-homo-9,11-epoxy-5,13-prostadienoic acid analogues: Specific stable agonist (SQ 26,538) and antagonist (SQ 26,536) of the human platelet thromboxane receptor

A newly synthesized 9α-homo-9,11-epoxy-5,13-prostadienoic acid analogue, SQ 26,536, (8(R)9(S)11(R)12(S)-9α-homo-9,11-epoxy-5(Z), 13(E)-15S-hydroxyprostadienoic acid) inhibited arachidonic acid (AA)-induced platelet aggregation with an I₅₀ value of 1.7 μ . SQ 26,536 did not inhibit prostaglandin (PG) synthetase activity of bovine seminal vesicle microsomes or thromboxane (Tx) synthetase activity of lysed human blood platelets. SQ 26,536 also inhibited platelet aggregation induced by epinephrine (secondary phase), 9,11-azoPGH₂ and collagen but did not inhibit the primary phase of epinephrine-induced aggregation or ADP-induced platelet aggregation. SQ 26,538 (8(R)9(S)11(R)12(S)-9α-homo-9-, 11-epoxy-5(Z),13(E)-15R-hydroxyprostadienoic acid), a 15-epimer of SQ 26,536, induced platelet aggregation with an A₅₀ value of 2.5 μ . SQ 26,536 competitively inhibited SQ 26,538-induced platelet aggregation with a K_i value of 3 μ . Neither indomethacin, a PG synthetase inhibitor, nor SQ 80,338 (1-(3-phenyl-2-propenyl)-1H-imidazole), a Tx synthetase inhibitor, inhibited SQ 26,538- or 9,11-azoPGH₂-induced platelet aggregation. These data indicate that SQ 26,536 and SQ 26,538 are stable antagonist and agonist, respectively, of the human blood platelet thromboxane receptor.     

Anandamide uptake by human endothelial cells and its regulation by nitric oxide

Anandamide (AEA) has vasodilator activity, which can be terminated by cellular re-uptake and degradation. Here we investigated the presence and regulation of the AEA transporter in human umbelical vein endothelial cells (HUVECs). HUVECs take up AEA by facilitated transport (apparent K(m) = 190 +/- 10 nm and V(max) = 45 +/- 3 pmol. min(-1).mg(-1) protein), which is inhibited by alpha-linolenoyl-vanillyl-amide and N-(4-hydroxyphenyl)-arachidonoylamide, and stimulated up to 2.2-fold by nitric oxide (NO) donors. The NO scavenger hydroxocobalamin abolishes the latter effect, which is instead enhanced by superoxide anions but inhibited by superoxide dismutase and N-acetylcysteine, a precursor of glutathione synthesis. Peroxynitrite (ONOO(-)) causes a 4-fold activation of AEA transport into cells. The HUVEC AEA transporter contributes to the termination of a typical type 1 cannabinoid receptor (CB(1)) -mediated action of AEA, i.e. the inhibition of forskolin-stimulated adenylyl cyclase, because NO/ONOO(-) donors and alpha-linolenoyl-vanillyl-amide/N-(4-hydroxyphenyl)-arachidonoylamide were found to attenuate and enhance, respectively, this effect of AEA. Consistently, activation of CB(1) cannabinoid receptors by either AEA or the cannabinoid HU-210 caused a stimulation of HUVEC inducible NO synthase activity and expression up to 2.9- and 2. 6-fold, respectively. Also these effects are regulated by the AEA transporter. HU-210 enhanced AEA uptake by HUVECs in a fashion sensitive to the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester. These findings suggest a NO-mediated regulatory loop between CB(1) cannabinoid receptors and AEA transporter.     

Stereoselective synthesis of 2,3,7-trimethylcyclooctanone and related compounds and determination of their relative and absolute configurations by the MalphaNP acid method

The copper/chiral phosphoramidite (L(1))-catalyzed conjugate addition of dimethylzinc to cycloocta-2,7-dienone 4, followed by the methylation of the intermediate enolate, yielded a single isomer of 7,8-dimethylcyclooct-2-enone (+)-5. Compound (+)-5 was subjected to the second conjugate addition with ent-L(1) giving only one stereoisomer of 2,3,7-trimethylcyclooctanone (+)-6, which was converted to 2,3,7-trimethylcyclooctanol 7. To determine the relative and absolute configurations of these compounds, the (1)H NMR anisotropy method using (S)-(+)-2-methoxy-2-(1-naphthyl)propionic acid {(S)-(+)-MalphaNP acid} 1 was applied. Racemic alcohol (+/-)-7 was esterified with (S)-(+)-MalphaNP acid 1 yielding diastereomeric esters, which were efficiently separated by HPLC on silica gel affording the first-eluted MalphaNP ester (-)-10a and the second-eluted one (-)-10b. The relative and absolute configurations of ester (-)-10a were determined to be (S;1R,2S,3R,7S) by analyzing the (1)H and (13)C NMR spectra of (-)-10a and (-)-10b, especially their HSQC-TOCSY and NOESY spectra, and by applying the MalphaNP anisotropy method. The alcohol 7 formed from (+)-6 was similarly esterified with (S)-(+)-MalphaNP acid 1 yielding an MalphaNP ester, which was identical with (-)-10a, and the relative and absolute configurations of 2,3,7-trimethylcyclooctanone (+)-6 were determined to be (2S,3R,7S).     

Effects of atrial natriuretic factor on human platelet function

We examined the hypothesis that atrial natriuretic factor (ANF), a substance with known vasorelaxant activities, shares with other vasodilators the property of inhibiting platelet function. Aggregation of citrated platelet-rich plasma (PRP) from 23 healthy volunteers induced by ADP, adrenaline, arachidonic acid, collagen, gamma-thrombin, the endoperoxide analogue U-44069, serotonin, the calcium ionophore A-23187 or platelet aggregating factor was measured after incubation of PRP with ANF for 3 minutes at concentrations of 4 X 10(-9), 4 X 10(-8) and 4 X 10(-7) M or vehicle as control. ANF decreased ADP-induced aggregation significantly (P less than 0.02), but only at the highest concentration used and to a minor extent (control: 73.6 +/- 11.2%; after ANF 4 X 10(-7) M: 60.0 +/- 17.1%, mean +/- S.D., n = 39) by a selective inhibitory effect on the secondary wave; neither aggregation by all other agents tested nor thromboxane B2 generation induced by ADP and adrenaline was altered by incubation with ANF. Although ANF thus has detectable effects on ADP-induced platelet aggregation in vitro, these data suggest that ANF is unlikely to be a physiologically significant modulator of platelet function.     

Absolute configuration of the alpha-methylbutyryl residue in longipinene derivatives from Stevia pilosa

The absolute configuration of the alpha-methylbutyryl residue in (4R,5S,7S,8S,9S,10R,11R,2'S)-7-angeloyloxy-9-hydroxy-8-(alpha-methylbutyryloxy)-longipin-2-en-L-one and (4R,5S,7S,8R,10R,11R,2'S)-7-angeloyloxy-8-(alpha-methylbutyryloxy)- longipin-2-en-L-one was determined by chemical correlation with (S)-(+)-benzyl alpha-methylbutyrate prepared from authentic (S)-(+)-alpha-methylbutyric acid. Both compounds were isolated from the hexane extracts of roots of Stevia pilosa Lag. together with four other longipinene derivatives. The developed correlation method is useful to ascertain the chirality of natural alpha-methylbutyryl esters found in nature and to reinforce the hypotheses on the biogenetic origin of these residues.     

Epinephrine potentiates calcium mobilization and activation of protein kinases in platelets stimulated by ADP through a mechanism unrelated to phospholipase C

ADP, added to suspensions of aspirinized 32P-prelabelled washed platelets, induced reversible platelet aggregation, the rapid elevation of cytosolic Ca2+ (maximum at 2 s), 20 kDa myosin light chain phosphorylation (maximum faster than 3 s), 40 kDa protein phosphorylation (maximum at 3-10 s) and phosphatidic acid formation (maximum at 30 s). Prior addition of epinephrine potentiated platelet aggregation, cytosolic Ca2(+)-elevation, 20 and 40 kDa protein phosphorylation evoked by ADP, but it did not enhance phosphatidic acid formation induced by ADP. The potentiating effect of epinephrine on aggregation, cytosolic Ca2(+)-increase and 20 and 40 kDa protein phosphorylation induced by ADP was also observed in the presence of EGTA. Ethylisopropylamiloride, an inhibitor of Na+/H(+)-exchange, did not affect the potentiation of ADP-induced platelet aggregation by epinephrine. We conclude that epinephrine primes platelets to increase Ca2(+)-influx and Ca2(+)-mobilization in response to ADP. The potentiation of cytosolic Ca2(+)-elevation by epinephrine leads to further stimulation of myosin light chain phosphorylation and protein kinase C activation and ultimately to enhanced platelet aggregation. These effects of epinephrine do not seem to take place at the level of phospholipase C.     

Cannabinoids in Models of Chronic Inflammatory Conditions

Cannabis sativa has been used as an anti-inflammatory plant for millennia. However until the elucidation of the chemistry of its constituents and the discovery of the endogenous cannabinoid system only a limited amount of research had been done on the effects of the plant or its constituents on inflammation. In the present overview we summarize our work on the effects of the non-psychotropic cannabidiol (CBD) and of a synthetic cannabidiol-derived acid (HU-320) in animal models of arthritis. Both compounds block progression of the disease, when administered after its onset. Cannabidiol was equally effective was administered i.p. or orally. Significant protection of the joints against severe damage was noted. In vitro cannabidiol reduced lymphocyte proliferation, and TNF-α formation and blocked zymosan-triggered production of reactive oxygen intermediates (ROI). Ex vivo lymph node cells from CBD-treated mice showed a decrease of collagen II-specific proliferation and IFN-γ production. A decreased release of TNF by knee synovial cells was also noted. A synthetic cannabidiol derivative, HU-320 also inhibited production of TNF and ROI by mouse macrophages in vitro and suppressed in vivo rise in serum TNF following endotoxin challenge. HU-320 showed no activity in a standard assay for THC-type psychotropic effects. These results suggest that CBD and HU-320 hold promise as potential novel anti-inflammatory agents.     

(1S,3S,7R)-3-methyl-alpha-himachalene from the male sandfly Lutzomyia longipalpis (Diptera: Psychodidae) induces neurophysiological responses and attracts both males and females

Lutzomyia longipalpis adult males form leks on or near hosts and release (1S,3S,7R)-3-methyl-alpha-himachalene from their tergal glands to lure females to the same site for mating and feeding. Here we have examined whether the male-produced attractant could also serve as a male aggregation stimulus. High resolution chiral capillary gas chromatography analysis of male tergal gland extracts, synthetic (1S,3S,7R)-3-methyl-alpha-himachalene, and a synthetic mixture of all isomers of 3-methyl-alpha-himachalene, was coupled to electrophysiological recordings from ascoid sensillum receptor cells in antennae of male and female sandflies. Receptor cells of both sexes responded only to the main component of the male tergal gland extract that eluted at the same retention time as (1S,3S,7R)-3-methyl-alpha-himachalene. Furthermore, of the eight 3-methyl-alpha-himachalene isomers in the synthetic mixture only the fraction containing (1S,3S,7R)-3-methyl-alpha-himachalene, co-eluting with an isomer of (1S*,3S*,7S*)-3-methyl-alpha-himachalene, elicited an electrophysiological response from male and female ascoid sensillum receptor cells. Both males and females flew upwind in a wind tunnel towards a filter paper disk treated with either 4-6 male equivalents of the tergal gland extract, pure (1S,3S,7R)-3-methyl-alpha-himachalene or the synthetic mixture of eight isomers. This indicates that (1S,3S,7R)-3-methyl-alpha-himachalene derived from L. longipalpis males may have a dual function in causing male aggregation as well as serving as a sex pheromone for females.