The inhibitory effect of propolis and caffeic acid phenethyl ester on cyclooxygenase activity in J774 macrophages

The effect of an ethanolic extract of propolis, with and without CAPE, and some of its components on cyclooxygenase (COX-1 and COX-2) activity in J774 macrophages has been investigated. COX-1 and COX-2 activity, measaured as prostaglandin E2 (PGE2) production, were concentration-dependently inhibited by propolis (3 x 10(-3) - 3 x 10(2) microgml(-1)) with an IC50 of 2.7 microgml(-1) and 4.8 x 10(-2) microgml(-1), respectively. Among the compounds tested pinocembrin and caffeic, ferulic, cinnamic and chlorogenic acids did not affect the activity of COX isoforms. Conversely, CAPE (2.8 x 10(-4) - 28 microgml(-1); 10(-9) - 10(-4) M) and galangin (2.7 x 10(-4) - 27 microgml(-1); 10(-9) - 10(-4) M) were effective, the last being about ten-twenty times less potent. In fact the IC50 of CAPE for COX-1 and COX-2 were 4.4 x 10(-1) microgml(-1) (1.5 x 10(-6) M) and 2 x 10(-3) microgml(-1) (6.3 x 10(-9) M), respectively. The IC50 of galangin were 3.7 microgml(-1) (15 x 10(-6) M) and 3 x 10(-2) microgml(-1) (120 x 10(-9) M), for COX-1 and COX-2 respectively. To better investigate the role of CAPE, we tested the action of the ethanolic extract of propolis deprived of CAPE, which resulted about ten times less potent than the extract with CAPE in the inhibition of both COX-1 and COX-2, with an IC50 of 30 microgml(-1) and 5.3 x 10(-1) microgml(-1), respectively. Moreover the comparison of the inhibition curves showed a significant difference (p < 0.001).These results suggest that both CAPE and galangin contribute to the overall activity of propolis, CAPE being more effective.     

Chalcogenopyrylium dyes as inhibitors/modulators of P-glycoprotein in multidrug-resistant cells

A series of chalcogenopyrylium dyes were evaluated as modulators/inhibitors of P-glycoprotein (Pgp). Their ability to inhibit verapamil (VER)-dependent ATPase activity (IC(50) values) in lipid-activated, mouse Cys-less mdr3 Pgp was determined. Their ability to promote calcein-AM (CAM) uptake in MDCKII-MDR1 cells and their capacity to be transported by Pgp in monolayers of MDCKII-MDR1 cells were also evaluated. The chalcogenopyrylium dyes promoted CAM uptake with values of EC(50) between 5 x 10(-6) and 3.5 x 10(-5)M and 7 of the 9 dyes examined in transport studies were substrates for Pgp with efflux ratios (P(BA/AB)) between 14 and 390. Binding of three compounds (1-S, 3-S, and 4-S) to Pgp was also assessed by fluorescence. These three thiopyrylium dyes showed increased fluorescence upon binding to Pgp, giving apparent binding constants, K(app), on the order of 10(-7) to 10(-6)M. Compound 8-Te was particularly intriguing since it appeared to influence Pgp at low micromolar concentrations as evidenced by its influence on VER-stimulated ATPase activity (IC(50) of 1.2 x 10(-6)M), CAM uptake (EC(50) of 5.4 x 10(-6)M), as well as [(3)H]-vinblastine transport by Pgp in cells (IC(50) of 4.3 x 10(-6)M) and within inside-out membrane vesicles (IC(50) of 9.6 x 10(-6)M). Yet, Pgp did not influence the distribution of 8-Te in MDCKII-MDR1 monolayers suggesting that 8-Te may bind to an allosteric site.     

Synthesis and antioxidant properties of novel N-methyl-1,3,4-thiadiazol-2-amine and 4-methyl-2H-1,2,4-triazole-3(4H)-thione derivatives of benzimidazole class

Some novel 1-methyl-4-(2-(2-substitutedphenyl-1H-benzimidazol-1-yl)acetyl)thiosemicarbazides (16a-20a), 5-[(2-(substitutedphenyl)-1H-benzimidazol-1-yl)methyl]-N-methyl-1,3,4-thiadiazol-2-amines (17b-20b), and 5-[(2-(substitutedphenyl)-1H-benzimidazol-1-yl)methyl-4-methyl-2H-1,2,4-triazole-3(4H)-thiones (16c-20c) were synthesized and tested for antioxidant properties by using various in vitro systems. Compounds 16a-20a were found to be a good scavenger of DPPH radical (IC(50), 26 microM; IC(50), 30 microM; IC(50), 43 microM; IC(50), 55 microM; IC(50), 74 microM, respectively) when compared to BHT (IC(50), 54 microM). Noteworthy results could not be found on superoxide radical. Compound 19b, which is the most active derivative inhibited slightly lipid peroxidation (28%) at 10(-3)M concentration. Compound 17c inhibited the microsomal ethoxyresorufin O-deethylase (EROD) activity with an IC(50)=4.5 x 10(-4)M which is similarly better than the specific inhibitor caffeine IC(50)=5.2 x 10(-4)M.     

Binding sites for alpha-bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the alpha-subunit of the nicotinic acetylcholine receptor.

The binding of the competitive antagonist alpha-bungarotoxin (alpha-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the alpha-subunit of the Torpedo acetylcholine receptor has been characterized. 125I-alpha-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by alpha-Btx (IC50 = 5.0 x 10(-8) M), d-tubocurarine (IC50 = 5.9 X 10(-5)M), and NaCl (IC50 = 7.9 x 10(-2)M). In the presence of 0.02% sodium dodecyl sulfate, 125I-alpha-Btx bound to the 56-residue peptide with a KD of 3.5 nM, as determined by equilibrium saturation binding studies. Because alpha-Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, [3H]PCP was bound to the 172-227 peptide. [3H]PCP binding was inhibited by chlorpromazine (IC50 = 6.3 x 10(-5)M), tetracaine (IC50 = 4.2 x 10(-6)M), and dibucaine (IC50 = 2.7 x 10(-4)M). Equilibrium saturation binding studies in the presence of 0.02% sodium dodecyl sulfate showed that [3H]PCP bound at two sites, a major site of high affinity with an apparent KD of 0.4 microM and a minor low-affinity site with an apparent KD of 4.6 microM. High -affinity binding occurred at a single site on peptide 205-227 (KD = 0.27 microM) and was competed by chlorpromazine but not by alpha-Btx.(ABSTRACT TRUNCATED AT 250 WORDS)     

The monoterpene alkaloid cantleyine from Strychnos trinervis root and its spasmolytic properties.

Cantleyine, a monoterpene alkaloid isolated from the root bark of Strychnos trinervis, was submitted to a broad spectrum pharmacological screening, in which the principal effect observed was a nonspecific relaxation of isolated smooth muscles. Cantleyine relaxed (IC50 2.1 x 10(-4) M) the guinea-pig trachea, pre-contracted by carbachol and antagonized in a nonspecific manner; carbachol (IC50 2.1 x 10(-4) M) and histamine (IC50 1.4 x 10(-4) M) induced contractions in the guinea-pig ileum; and phenylephrine (IC50 3.8 x 10(-4) M) responses in the rat aorta. Cantleyine antagonized (pD'2, 3.82) cumulative concentration response curves to histamine in the ileum in a noncompetitive, reversible (slope, 4.84) and concentration dependent manner. The tonic contractions induced by histamine and KCl were also inhibited in a concentration-dependent and reversible manner (IC50 7.2 x 10(-5) and 1.8 x 10(-4) M, respectively), suggesting that cantleyine should be acting on voltage-dependent Ca2+ channels. This hypothesis was confirmed by the observation that cantleyine inhibited (pD'2, 3.35), in a concentration dependent manner, the CaCl2 induced contraction in depolarizing medium. These results suggest that cantleyine produces nonspecific spasmolytic effects in smooth muscle and that in guinea-pig ileum this effect is, in part, due to the inhibition of Ca+2 influx through voltage-dependent Ca2+ channels.     

Comparative study of oestrogenic properties of eight phytoestrogens in MCF7 human breast cancer cells

Previous studies have compared the oestrogenic properties of phytoestrogens in a wide variety of disparate assays. Since not all phytoestrogens have been tested in each assay, this makes inter-study comparisons and ranking oestrogenic potency difficult. In this report, we have compared the oestrogen agonist and antagonist activity of eight phytoestrogens (genistein, daidzein, equol, miroestrol, deoxymiroestrol, 8-prenylnaringenin, coumestrol and resveratrol) in a range of assays all based within the same receptor and cellular context of the MCF7 human breast cancer cell line. The relative binding of each phytoestrogen to oestrogen receptor (ER) of MCF7 cytosol was calculated from the molar excess needed for 50% inhibition of 3H]oestradiol binding (IC50), and was in the order coumestrol (35x)/8-prenylnaringenin (45x)/deoxymiroestrol (50x)>miroestrol (260x)>genistein (1000x)>equol (4000x)>daidzein (not achieved: 40% inhibition at 10(4)-fold molar excess)>resveratrol (not achieved: 10% inhibition at 10(5)-fold molar excess). For cell-based assays, the rank order of potency (estimated in terms of the concentration needed to achieve a response equivalent to 50% of that found with 17beta-oestradiol (IC50)) remained very similar for all the assays whether measuring ligand ability to induce a stably transfected oestrogen-responsive ERE-CAT reporter gene, cell growth in terms of proliferation rate after 7 days or cell growth in terms of saturation density after 14 days. The IC50 values for these three assays in order were for 17beta-oestradiol (1 x 10(-11)M, 1 x 10(-11)M, 2 x 10(-11)M), and in rank order of potency for the phytoestrogens, deoxymiroestrol (1 x 10(-10)M, 3 x 10(-11)M, 2 x 10(-11)M)>miroestrol (3 x 10(-10)M, 2 x 10(-10)M, 8 x 10(-11)M)>8-prenylnaringenin (1 x 10(-9)M, 3 x 10(-10)M, 3 x 10(-10)M)>coumestrol (3 x 10(-8)M, 2 x 10(-8)M, 3 x 10(-8)M)>genistein (4 x 10(-8)M, 2 x 10(-8)M, 1 x 10(-8)M)/equol (1 x 10(-7)M, 3 x 10(-8)M, 2 x 10(-8)M)>daidzein (3 x 10(-7)M, 2 x 10(-7)M, 4 x 10(-8)M)>resveratrol (4 x 10(-6)M, not achieved, not achieved). Despite using the same receptor context of the MCF7 cells, this rank order differed from that determined from receptor binding. The most marked difference was for coumestrol and 8-prenylnaringenin which both displayed a relatively potent ability to displace [3H]oestradiol from cytosolic ER compared with their much lower activity in the cell-based assays. Albeit at varying concentrations, seven of the eight phytoestrogens (all except resveratrol) gave similar maximal responses to that given by 17beta-oestradiol in cell-based assays which makes them full oestrogen agonists. We found no evidence for any oestrogen antagonist action of any of these phytoestrogens at concentrations of up to 10(-6)M on either reporter gene induction or on stimulation of cell growth.     

A facile synthesis of D-glucose-type gem-diamine 1-N-iminosugars: a new family of glucosidase inhibitors.

gem-Diamine 1-N iminosugars of D-glucose-type, a new type of glycosidase inhibitors, have been synthesized from siastatin B, isolated from Streptomyces culture. 2-Trifluoroacetamido-1-N-iminosugar, (2S,3R,4R,5R)-2-trifluoroacetamido-5-hydroxymethylpiperidine -3,4-diol was proved to be a potent inhibitor for alpha-D- and beta-D-glucosidases (IC50 1.9x10(-7) and 4.2x10(-7) M, respectively). 2-Acetamido-1-N-iminosugar, (2S,3R,4R,5R)-2-acetamido-5-hydroxymethylpiperidine-3,4-diol also affected these enzymes (IC50 2.9x10(-6) and 5.4x10(-6) M, respectively).     

Effect of KATP channel openers on myogenic and neurogenic responses in goat urinary bladder

Isolated goat detrusor muscle exhibited spontaneous contractility with an irregular amplitude and frequency. The spontaneity of detrusor muscle exhibited a mean amplitude as 11.99 +/- 0.83 mm and frequency as 1.37 +/- 0.16/min. KATP-channel openers namely, cromakalim or pinacidil (10(-7) - 10(-4) M) added cumulatively, elicited a concentration-related inhibition of both amplitude and rate of spontaneous contractions. The mean IC50 values for both amplitude and frequency for cromakalim were 3.3 x 10(-6) M and 2.9 x 10(-6) M, respectively; and for pinacidil were 2.0 x 10(-5) M and 1.5 x 10(-5) M, respectively. Glibenclamide, a KATP-channel blocker inhibited the cromakalim-induced concentration-related relaxation of spontaneous contractions with a significant increase in its mean IC50. ACh-induced concentration-related contractile response was inhibited in the presence of either cromakalim (10(-4) M) or pinacidil (10(-4) M). The mean EC50 value of ACh, in the presence of cromakalim (2.5 x 10(-3) M) was significantly increased as compared to the control (1.2 x 10(-6) M). In the presence of glibenclamide (10(-5) M) the inhibitory effect of cromakalim was significantly reduced with consequent decrease in the EC50 value (1.9 x 10(-5) M). Application of EFS (30 V and 5 ms) on goat urinary bladder strips at 1, 2, 5, 10, 20 and 30 Hz elicited frequency-related contractile responses. Both cromakalim and pinacidil caused a rightward shift in the frequency-related contractile response curve with significant increase in the mean EF25 and EF50 values, respectively. In the presence of glibenclamide (10(-4) M), the frequency-related inhibitory response curve was shifted to left with significant (P < 0.001) increase in the mean EF25, EF50 and EF75. The present results suggest that in the goat detrusor muscle, agonist and EFS-induced contractile responses were more potently inhibited by cromakalim than pinacidil with activation of glibenclamide sensitive KATP channels.     

Binding sites for 3H-LTC4 in membranes from guinea pig ileal longitudinal muscle

Leutriene (LTC4) is one of the components of Slow Reacting Substance of Anaphylaxis (SRS-A) and is a potent constrictor of guinea pig ilea. The contraction is likely to be a receptor-mediated process. Here we report the existence of specific binding sites for 3H-LTC4 in a crude membrane preparation from guinea pig ileal longitudinal muscle. At 4 degrees C in the presence of 20 mM Serine-borate, binding increases linearly with protein concentration, reaches equilibrium in 10 minutes, and is reversible upon addition of 3 x 10(-5) M unlabelled LTC4. The dissociation curve is consistent with the existence of more than one class of binding site. Ca++ and Mg++ greatly enhance the binding of 3H-LTC4 at equilibrium. In the presence of 5 mM CaCl2 and MgCl2 not only LTC4 (IC50 10(-7)M), but also LTD4 (albeit with much lower affinity, IC50 = 6 x 10(-5) M) and the SRS-A antagonist FPL 55712 (IC50 = 10(-5) M) can compete with 3H-LTC4 for its binding sites. FPL 55712 only displaces 60-70% of the total amount bound, while LTC4 displaces 90-95%. These studies indicate that multiple classes of binding sites exist for 3H-LTC4 in guinea pig ileal longitudinal muscle, and that at least part of these binding sites might be related to the ability of LTC4 to contract guinea pig ilea.     

Hypothyroid state reduces calcium channel function in 18-day pregnant rat uterus

Hypothyroidism significantly reduced the mean amplitude and increased the mean frequency of spontaneous rhythmic contractions in 18 day pregnant rat uterus. Nifedipine (10(-12)-10(-9) M) and diltiazem (10(-10)-10(-6) M) caused concentration related inhibition of the myogenic responses of the uterine strips obtained from both pregnant and hypothyroid state. However, nifedipine was less potent (IC50:2.11 x 10(-11) M) in pregnant hypothyroid state as compared to pregnant control (IC50: 3.1 x 10(-12) M). Similarly, diltiazem was less potent (IC50: 3.72 x 10(-9) M) in inhibiting the uterine spontaneous contractions in hypothyroid than in pregnant rat uterus (IC50:5.37 x 10(-10) M). A similar decrease in the sensitivity to nifedipine and diltiazem for reversal of K+ (100 mM)-induced tonic contraction and K(+)-stimulated 45Ca2+ influx was observed with these calcium channel antagonists in uterus obtained from hypothyroid pregnant rats compared to the controls. Nifedipine-sensitive influx of 45Ca(2+)-stimulated either by K+ (100 mM) or by Bay K8644 (1,4-dihydro-2,6-methyl-5-nitro-4-[2'-(trifluromethyl)phenyl]-3-pyridine carboxylic acid methyl ester) (10(-9) M) was significantly less in uterine strips from hypothyroid rats compared to controls. The results suggest that the inhibition of uterine rhythmic contractions may be attributable to a reduction in rat myometrial Ca2+ channel function in the hypothyroid state.     

Differential in vitro inhibition of polyphenoloxidase from a wild edible mushroom Lactarius salmonicolor

The polyphenol oxidase (LsPPO) from a wild edible mushroom Lactarius salmonicolor was purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. At the optimum pH and temperature, the K(M) and V(Max) values of LsPPO towards catechol, 4-methylcatechol and pyrogallol were determined as 0.025 M & 0.748 EU/mL, 1.809 x 10(- 3) M & 0.723 EU/mL and 9.465 x 10(- 3) M & 0.722 EU/mL, respectively. Optimum pH and temperature values of LsPPO for the three substrates above ranged between the pH 4.5-11.0 and 5-50 degrees C. Enzyme activity decreased due to heat denaturation with increasing temperature. Effects of a variety of classical PPO inhibitors were investigated opon the activity of LsPPO using catechol as the substrate. IC(50) values for glutathione, p-aminobenzenesulfonamide, L-cysteine, L-tyrosine, oxalic acid, beta-mercaptoethanol and syringic acid were determined as 9.1 x 10(- 4), 2.3 x 10(- 4) M, 1.5 x 10(- 4) M, 3.8 x 10(- 7) M, 1.2 x 10(- 4) M, 4.9 x 10(- 4) M, and 4 x 10(- 4) M respectively. Thus L-tyrosine was by far the most effective inhibitor. Interestingly, sulfosalicylic acid behaved as an activator of LsPPO in this study.     

Synthesis, antioxidant and radical scavenging activities of novel benzimidazoles

The synthesis and antioxidant evaluation of some novel benzimidazole derivatives (10-24) are described. Antioxidant properties of the compounds were investigated employing various in vitro systems viz., microsomal NADPH-dependent inhibition of lipid peroxidation (LP), interaction of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and scavenging of superoxide anion radical. Compounds 12 and 13 showed very good antioxidant capacity and were 17-18-fold more potent than BHT (IC50 2.3 x 10(-4) M) with 1.3 x 10(-5) M and 1.2 x 10(-5) M IC50 values, respectively, by interaction of the stable DPPH free radical.     

Effects of specific alpha-adrenoceptive agents on extraneuronal uptake (uptake2) of isoproterenol in perfused rat heart

Effects of specific alpha-adrenoceptive agents (alpha 1-agonist, alpha 1-antagonist, alpha 2-agonist and alpha 2-antagonist) on the extraneuronal accumulation of 3H-isoproterenol in the perfused rat heart were examined. The extraneuronal accumulation of 3H-isoproterenol in the hearts perfused with 3H-isoproterenol (10(-6)M) under COMT inhibition by tropolone (10(-4)M) was about 6 times higher than that of intact COMT. The increase in the accumulation by COMT inhibition was regarded as 100% and the effects of specific alpha-adrenoceptive agents on the accumulation was evaluated. alpha 1-agonists, methoxamine and phenylephrine, did not affect the accumulation. alpha 1-antagonists, prazosin, bunazosin and YM-12617, significantly decreased the accumulation of 3H-isoproterenol and these IC50 values were 2 x 10(-6)M, 3.5 x 10(-6)M and 2.3 x 10(-5)M, respectively. alpha 2-agonists, clonidine and guanabenz, significantly reduced the accumulation and these IC50 values were 3.4 x 10(-5)M and 2.9 x 10(-7)M, respectively. The alpha 2-antagonist, yohimbine, did not affect the accumulation. The present experiments clearly demonstrated that the tested alpha 1-antagonists and alpha 2-agonists inhibited uptake2 in rat heart but the tested alpha 1-agonists and an alpha 2-antagonist did not inhibit it.     

Aldose reductase inhibitors from the leaves of Myrciaria dubia (H. B. & K.) McVaugh

Ellagic acid (1) and its two derivatives, 4-O-methylellagic acid (2) and 4-(alpha-rhamnopyranosyl)ellagic acid (3) were isolated as inhibitors of aldose reductase (AR) from Myrciaria dubia (H. B. & K.) McVaugh. Compound 2 was the first isolated from the nature. Compound 3 showed the strongest inhibition against human recombinant AR (HRAR) and rat lens AR (RLAR). Inhibitory activity of compound 3 against HRAR (IC50 value = 4.1 x 10(-8) M) was 60 times more than that of quercetin (2.5 x 10(-6) M). The type of inhibition against HRAR was uncompetitive.     

Spasmolytic action of the methanol extract and isojuripidine from Solanum asterophorum Mart. (Solanaceae) leaves in guinea-pig ileum

Solanum asterophorum Mart. (Solanaceae) is a shrub popularly known as "jurubeba-defogo" in the northeast of Brazil. In the present work, the methanol extract (SA-MeOH, 3750 microg/mL) and isojuripidine (10(-7) - 3 x 10(-4) M), a steroidal alkaloid obtained from S. asterophorum Mart. leaves, inhibited phasic contractions induced by both 1 microM histamine [IC50 = (225.8 +/- 47.4), g/mL and (3.5 +/- 0.8) x 10(-5) M] or 1 microm acetylcholine [IC50 = (112.5 +/- 20.6) microg/mL and (2.3 +/- 0.4) x 10(-5) M] in guinea-pig ileum, respectively. The extract and isojuripidine also relaxed the ileum (SA-MeOH, 1-750 microg/mL, and isojuripidine, 10(-9) - 3 x 10(-4) M) pre-contracted with 1 M histamine [EC50 = (101.1 +/- 17.4) microg/mL and (1.2 +/- 0.3) x 10(-6) M] or 1 microM acetylcholine [EC50 = (136.8 +/- 21.1) microg/mL and (1.9 +/- 0.4) x 10(-6) M] or 40 mm KCl [EC50 = (149.4 +/- 19.5) microg/mL and (1.8 +/- 0.7) x 10(-6) M], respectively, in an equipotent and concentration-dependent manner. This effect is probably due to inhibition of calcium influx through voltage-operated calcium (Ca(v)) channels. To confirm this hypothesis, we evaluated their effect on cumulative CaCl2 curves in depolarizing medium nominally without Ca2+. SA-MeOH (27, 243, 500, and 750 microg/mL) and isojuripidine (3 x 10(-8), 10(-6), 3 x 10(-5), and 3 x 10(-4) M) inhibited the contractions induced by CaCl2, in a concentration-dependent manner. The concentration-response curves to CaCl2, in the presence of SA-MeOH and isojuripidine, were shifted downward in relation to a control curve in a non-parallel manner resulting in reduction of the maximum effect [E(max) = (71.2 +/- 9.2); (57.4 +/- 9.2); (43.8 +/- 3.4); (41.5 +/- 2.4) and (90.6 +/- 4.8); (74.7 +/- 8.7); (66.4 +/- 3.9); (31.3 +/- 4.1)%, respectively]. SA-MeOH and isojuripidine present spasmolytic action in guinea-pig ileum due to a partially blockade of calcium influx through Ca(v) channels.     

Tonic regulation of vascular tone by nitric oxide and chloride ions in rat isolated small coronary arteries

We have investigated the involvement of Cl(-) in regulating vascular tone in rat isolated coronary arteries mounted on a small vessel myograph. Mechanical removal of the endothelium or inhibition of nitric oxide (NO) synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) led to contraction of rat coronary arteries, and these contractions were sensitive to nicardipine (10(-6) M). This suggests that release of NO tonically inhibits a contractile mechanism that involves voltage-dependent Ca(2+) channels. In arteries contracted with L-NAME, switching the bathing solution to physiological saline solution with a reduced Cl(-) concentration potentiated the contraction. DIDS (5 x 10(-6)-3 x 10(-4) M) caused relaxation of L-NAME-induced tension (IC(50) = 55 +/- 10 microM), providing evidence for a role of Cl(-). SITS (10(-5)-5 x 10(-4) M) did not affect L-NAME-induced tension, suggesting that DIDS is not acting by inhibition of anion exchange. Mechanical removal of the endothelium led to contraction of arteries, which was sensitive to DIDS (IC(50) = 50 +/- 8 microM) and was not affected by SITS. This study suggests that, in rat coronary arteries, NO tonically suppresses a contractile mechanism that involves a Cl(-) conductance.     

Solubilization of the active vasoactive intestinal peptide receptor from human colonic adenocarcinoma cells

The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.     

Degradation of thymopentin by human lymphocytes: evidence for aminopeptidase activity

Thymopentin (Arg-Lys-Asp-Val-Tyr) was shown to be degraded in vitro by human lymphocytes into two main fragments; the tetrapeptide Lys-Asp-Val-Tyr and the tripeptide Asp-Val-Tyr. Degradation products were identified by HPLC and amino-acid analysis. Analysis of the time-course of degradation revealed a 'stepwise' degradative event beginning at the N-terminal. The degradation of thymopentin after the first 10 min, as well as the formation of the tetrapeptide (5-30 min) were essentially curvilinear. Degradation of the tripeptide, was linear. Upon screening a panel of compounds that inhibit enzymatic activity, bestatin, amastatin and 1,10-phenanthroline were shown to be the most effective. Bestatin and amastatin caused an 85-90% inhibition of thymopentin degrading activity with IC50 values of 7.1 x 10(-6) M and 4.5 x 10(-9) M, respectively. 1,10-Phenanthroline completely inhibited the degradative process with an IC50 of 2 x 10(-4) M. When the tetrapeptide Lys-Asp-Val-Tyr was used as the starting substrate, similar IC50 values were seen for amastatin, bestatin and 1,10-phenanthroline. The importance of divalent metal ions in the degradative event was demonstrated not only by the effect of 1,10-phenanthroline, but also by the ability of Zn2+ and Co2+ to reverse the inhibition of 1,10-phenanthroline (at its IC50) to activities near control values (no inhibitor). These data strongly suggest that an aminopeptidase(s) is responsible for the degradative activity.     

Flunitrazepam binding sites in rat diaphragm. Receptors for direct neuromuscular effects of benzodiazepines?

The evidence for direct muscle relaxant effects of benzodiazepines is controversial. We now show that a crude membrane preparation of rat diaphragm possesses binding sites for [3H]flunitrazepam (FNZ). Scatchard analysis gave a binding site density of 1689 +/- 143 fmol/mg protein (Kd = 25.6 +/- 2.6 nM). These sites are of the "peripheral" type since clonazepam fails to displace [3H]FNZ as effectively as R05-4864 (IC50 values: 7.5 x 10(-6) M and 8 x 10(-9) M, respectively). Diazepam is almost as effective as R05-4864 and potently displaces [3H]FNZ binding (IC50 = 3 x 10(-8) M). We propose that the previously described effects of diazepam on rat diaphragm are mediated through high-affinity binding sites.     

Anti-filarial effects of nine quinoline-containing drugs on adult filariae in vitro

The potencies and efficacies of 9 quinoline-containing anti-malarials including chloroquine, (bis)desethylchloroquine, SN6911, SN12108, amodiaquine, CN-2999-2K, primaquine, quinacrine, and quinine were examined in vitro against adult female Brugia pahangi. Parasite motility and lactate excretion were measured as indicators of drug effects. All of the agents tested showed time-dependent increases in potency over a 24-72-hr incubation period. SN12108 was the most potent at 72 hr, reducing motility by greater than or equal to 50% (IC50) at 1.0 x 10(-7) M. Chloroquine (IC50 2.3 x 10(-6) M), desethylchloroquine (IC50 7.0 x 10(-6) M), quinacrine (IC50 1.9 x 10(-6) M), and quinine (IC50 1.5 x 10(-5) M) were the least potent. All of the drugs caused time-dependent decreases in lactate excretion, except quinine; decreases were found to be dose dependent. A high correlation (r greater than 0.85) was seen between time-dependent effects on motility and lactate excretion. The effects of chloroquine (10 microM) on motility were also examined in female Acanthocheilonema viteae, Dirofilaria immitis, Onchocerca volvulus, and male Onchocerca gutturosa. Dirofilaria immitis was less sensitive to chloroquine than B. pahangi; A. viteae was equally sensitive. Species of Onchocerca were the most sensitive parasites studied. Adult O. gutturosa and O. volvulus were affected by 10 microM chloroquine within 4-6 hr; motility was reduced by 80% within 24 hr. Although the mechanism of anti-filarial activity of the quinoline-containing drugs is not known, their in vitro activity against a variety of adult filariae at clinically relevant concentrations, as well as differential sensitivity seen between the different filariae examined, warrants further study of these compounds.