Augmentation of human neutrophil and alveolar macrophage LTB4 production by N-acetylcysteine: role of hydrogen peroxide

1. The actions of N-acetylcysteine (NAC) on hydrogen peroxide (H₂O₂) and leukotriene B₄ (LTB₄) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated.
2. At a concentration of 100 μM, NAC significantly (P<0.01) suppressed the accumulation of H₂O₂ in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml⁻¹)- or N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 μM)-stimulated neutrophils and of resting and OZ-stimulated macrophages. At concentrations of 10 μM and above, NAC augmented significantly the level of LTB₄ in the supernatants of OZ- and fMLP-stimulated neutrophils (P<0.01 and P<0.05, respectively) and OZ-stimulated macrophages (P<0.05 at 10 μM, P<0.01 at 100 μM NAC).
3. NAC (100 μM) caused a significant (P<0.01) reduction in the quantity of measurable H₂O₂ when incubated with exogenous H₂O₂ concentrations equivalent to those released from OZ-stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB₄ when incubated with exogenous LTB₄.
4. Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H₂O₂ had no significant effect on LTB₄ production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H₂O₂ to H₂O and O₂, caused a pronounced, statistically significant (P<0.01) increase in the levels of LTB₄ measured in the supernatants of OZ- and fMLP-stimulated neutrophils.
5. H₂O₂ (12.5 μM and 25 μM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB₄ (P=0.051 and P<0.05 at 12.5 μM and 25 μM, respectively) that was inhibited by NAC (100 μM) but not by catalase (400 u ml⁻¹).
6. In conclusion, the anti-oxidant drug, NAC, increases LTB₄ production by human neutrophils and alveolar macrophages, probably through the elimination of cell-derived H₂O₂. LTB₄ undergoes a H₂O₂-dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB₄, suggesting that NAC may increase LTB₄ production by blocking the H₂O₂-dependent inhibition of a synthetic enzyme, such as 5-lipoxygenase.

     

In vitro Antibacterial Activity of Aqueous and Ethanol Extracts of Aristolochia indica and Toddalia asiatica Against Multidrug-Resistant Bacteria

Bacteria have developed multidrug resistance against available antimicrobial agents. Infectious diseases caused by these multidrug-resistant bacteria are major causes of morbidity and mortality in human beings. Synthetic drugs are expensive and inadequate for the treatment of diseases, causing side effects and ineffective against multidrug-resistant bacteria. The medicinal plants are promising to have effective antimicrobial property due to presence of phytochemical compounds like alkaloids, flavanoids, tannins and phenolic compounds. The present study aimed to find the antimicrobial activity of medicinal plants against multidrug-resistant bacteria. Multidrug-resistant bacteria were identified by Kirby-Bauer disc diffusion method. Production of β-lactamases (extended spectrum β-lactamases, metallo β-lactamase and AmpC β-lactamase) were identified by combination disc method. Antibacterial activity of aqueous and ethanol extract of Aristolochia indica and Toddalia asiatica were detected by agar well diffusion assay and minimum inhibitory concentration. All bacteria used in this study showed antibiotic resistance to ≥3 antibiotics. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis and Vibrio cholerae were found to be positive for β-lactamase production. Ethanol extract of Aristolochia indica showed more significant antibacterial activity against multidrug-resistant bacteria than Toddalia asiatica. Ethanol extracts of Aristolochia indica and Toddalia asiatica showed minimum inhibitory concentration values of 50-100 μg/ml and 100-200 μg/ml, respectively against multidrug-resistant bacteria. From this study, it was concluded that Aristolochia indica has more potential to treat multidrug-resistant bacteria than Toddalia asiatica.     

Bioactive Terpenoids and Flavonoids from Daucus littoralis Smith subsp. hyrcanicus Rech.f,an Endemic Species of Iran

Background

Daucus littoralis Smith subsp. hyrcanicus Rech.f. (Apiaceae) is an endemic species in northern parts of Iran where it is commonly named Caspian carrot. The fruits have been used as condiment.

Methods

In a series of in vitro assays, antioxidant (DPPH and FRAP assays), cytotoxic and antimicrobial activities of different extracts of roots and fruits were evaluated for the first time. The separation and purification of the compounds were carried out on the most potent extracts using various chromatographic methods and identified by spectroscopic data (¹H and ¹³C NMR).

Results

The results showed that among the extracts only fruit methanol extract (FME) has significant antioxidant activity (IC₅₀ = 145.93 μg.ml^-1 in DPPH assay and 358 ± 0.02 mmol FeII/g dry extract in FRAP assay). The radical scavenging activity of FME at 400 μg.ml^-1 was comparable with α-tocopherol (40 μg.ml^-1) and with BHA (100 μg.ml^-1) (p > 0.05). FME did not show any toxicity against cancerous and normal cell lines. Fruit ethyl acetate extract (FEE) had cytotoxic activity against breast carcinoma and hepatocellular carcinoma cells (IC₅₀ 168.4 and 185 μg.ml^-1, respectively), while it did not possess antioxidant activity in comparison with α-tocopherol and BHA as standard compounds. Ethyl acetate and methanol extract of fruits showed antimicrobial activity against Staphylococcus aureus (MIC: 3.75 mg.ml^-1) and Candida albicans (MIC: 15.6 and 7.8 mg.ml^-1, respectively). Four terpenoids were isolated form FEE including: β-sitosterol (1), stigmasterol (2), caryophyllene oxide (3), β-amyrin (4). Also, three flavonoids namely quercetin 3-O-β-glucoside (5), quercetin 3-O-β-galactoside (6) and luteolin (7) were isolated from FME.

Conclusion

This study showed that FEE and FME of D. littoralis Smith subsp. hyrcanicus Rech.f. had the highest biological activities which may be correlated with in vitro cytotoxic, antimicrobial and antioxidant activities of terpenoids and flavonoids components of the extracts.     

Combating Drug Resistant Pathogenic Bacteria Isolated from Clinical Infections,with Silver Oxide Nanoparticles

The antibiogram study of methicillin resistant Staphylococcus aureus isolates revealed 100% resistance to vancomycin, bacitracin, erythromycin, ciprofloxacin and nalidixic acid. Eight isolates (53.3%) showed resistance to co-trimoxazole and one isolate to rifampicin, which was the drug of choice. An effort was made to evaluate the antimethicillin resistant Staphylococcus aureus activity of silver oxide (Ag₂O) nanoparticles synthesized from Aspergillus terreus VIT 2013. Production of Ag₂O nanoparticles was confirmed by color change of fungal filtrate and UV light absorption at 450 nm. X-ray diffraction pattern showed 2θ values at 27, 32, 38 and 57°, which corresponded to the cubic structure of Ag₂O nanocrystals. Fourier transform infrared spectroscopy indicated the presence of primary amine, carbonyl group, NO₂ and silver, revealing protein mediated nanoparticle production. The scanning electron microscope image showed freely dispersed Ag₂O nanoparticles. The nanoparticles were active against all methicillin resistant isolates and hence can be used as antibacterial agents against drug resistant bacteria.     

Development and Validation of a Reversed-Phase High-performance Liquid Chromatography Method for Determination of Desmopressin in Chitosan Nanoparticles

A simple isocratic reversed-phase high performance liquid chromatographic method was developed for determination of released desmopressin from chitosan nanoparticles in the in vitro media. The chromatographic separation was achieved with acetonitrile/water (25:75, v/v), in which water contained 0.1% v/v trifluoroacetic acid with pH=2.5 as mobile phase, a Chromolith^® Performance RP-18e column (150×4.6 mm; 5 μm) kept at 40° and ultraviolet detection at 220 nm. The compound was eluted isocritically at a constant flow rate of 1.6 ml/min. The method was validated according to the International Conference on Harmonisation guidelines. The validation characteristics included accuracy, precision, linearity rang, selectivity, limit of detection, limit of quantitation and robustness. The calibration curve was linear (r>0.9999) over the concentration rang 0.5-100 μg/ml. The limit of detection and limit of quantitation in the release media were 0.05 and 0.5 μg/ml, respectively. The proposed method had an accuracy of and intra- and inter-day precision <4.2. Furthermore, to evaluate the performance of the proposed method, it was used in the analysis of desmopressin level in real samples containing chitosan nanoparticles in the in vitro media.     

Different effects of gadolinium on IKR,IKS and IK1 in guinea-pig isolated ventricular myocytes

1. Using the whole cell configuration of the patch clamp technique, we studied the potential blocking effects of gadolinium (1 μM to 1 mM) on potassium currents: I_KR, I_KS and I_K1. The study was performed on guinea-pig isolated ventricular myocytes.
2. The background current, I_K1 was insensitive to Gd³⁺. Thus, we found that no obvious screening of surface charges was visible with concentrations of Gd³⁺ up to 100 μM.
3. By use of three different protocols: tail currents fitting, analysis of envelope of tails and electrophysiological dissection, we found that I_KR was the only component of I_K that was sensitive to Gd³⁺. The sensitivity was apparently different depending on the protocol used.
4. Comparison of the results obtained with the different protocols revealed that the rapid component of I_KR is more sensitive to Gd³⁺ than the slow one.
5. Of the different protocols used to distinguish between I_KR and I_KS, the electrophysiological dissection seems to be the more accurate.

     

Evaluation of In Vitro Antioxidant Potential of Cordia retusa

The present study was carried out to investigate the antioxidant potential, total flavonoid and phenolic content in extracts of aerial parts of Cordia retua (Vahl.) Masam. The samples such as ethyl acetate and ethanol extracts were tested using six in vitro models such as 2,2-diphenyl-1-picrylhydrazyl, nitric oxide radical, iron chelating, hydroxyl radical, superoxide radical scavenging activity and total antioxidant activity to evaluate the in vitro antioxidant potential of C. retusa by spectrophotometrically. Total flavonoid and phenolic content in samples were estimated using aluminum chloride colorimetric and Folin-Ciocalteu method. The results were analyzed statistically by the regression method. Half maximal inhibitory concentration (IC₅₀) of the ethanol extract was found to be 596 μg/ml for DPPH, 597 μg/ml for nitric oxide radical, 554 μg/ml for iron chelating, 580 μg/ml for hydroxyl radical, 562 μg/ml for superoxide radical and 566 μg/ml for total antioxidant capacity. Furthermore, the total flavonoid content and total phenolic content of the ethanol extract were found to be 2.71 mg gallic acid equivalent per gram of extract and 1.86 mg quercetin equivalent per gram of extract, respectively. In all the testing, a significant correlation existed between concentrations of the extract and percentage inhibition of free radicals. The results of the present comprehensive analysis demonstrated that C. retusa possess potent antioxidant activity, high flavonoid and phenolic content. The antioxidant property may be related to the polyphenols and flavonoids present in the extract. These results clearly indicated that C. retusa is effective against free radical mediated diseases as a natural antioxidant.     

Evaluation of Parmotrema reticulatum Taylor for Antibacterial and Antiinflammatory Activities

Lichens produce variety of secondary metabolites including depsides, depsidones and dibenzofurans having multifunctional activity in response to external environmental condition. Present study provides evidence for in vitro antibacterial and in vivo antiinflammatory activity of acetone and ethanol extracts of Parmotrema reticulatum. In vitro antibacterial activity was investigated against gram positive and gram negative bacteria. Cotton pellet-induced granuloma, xylene-induced ear swelling, carragennan-induced edema, histamine-induced and carboxymethylcellulose sodium-induced leukocyte emigration in mice models were used to quantify the antiinflammatory activity. Acetone and ethanol extracts were showed antibacterial activity against Bacillus subtilis (minimal inhibitory concentration: 100 and 150 μg/ml) and Staphylococcus aureus (minimal inhibitory concentration: 100 and 200 μg/ml), Escherichia coli (minimal inhibitory concentration: 200 and 250 μg/ml), and Pseudomonasa eruginosa (minimal inhibitory concentration: 200 and 300 μg/ml). Acetone extract was inhibited edema significantly at 200 mg/kg with xylene, cotton pellet, carragennan and histamine induced edema in vivo models. Ethanol extract was found effective at dose of 300 mg/kg with all in vivo antiinflammatory models. The results showed significant (P<0.01) antiinflammatory effects at 200 and 300 mg/kg dose of acetone and ethanol extracts, respectively, which can be concluded that significant activity may be due to presence of flavanoids in ethanol extract and usnic acid in acetone extract.     

Biological activity and microscopic characterization of Lythrum salicaria L

Background

There are several plants have been used worldwide in the folk medicine with high incidence for treatment of human disorders, of which Lythrum salicaria belongs to the Lythraceae family has traditionally reputation for some medicinal usage and recently many biological and pharmacological activity of the plant have been studied.

Methods

In this study, microscopic characterizations of the aerial parts of the plant were determined. Moreover, the plant extract (aqueous methanol 80%) was subjected to an anti-diabetic activity test (in a rat model of streptozocin induced diabetes), anti-Helicobacter pylori (using disc diffusion method) along with antioxidant activity against DPPH (stable free radical) tests. Besides, total flavonoids, phenols, tannins, as well as polysaccharides contents have been assessed using spectroscopic methods.

Results

The microscopic properties of the plant fragments revealed anomocytic stomata, conical shape trichomes, and abundant spherical pollen grains as a characteristic pattern for the aerial parts of the plant. The extract of the plant at concentration of 15 g/kg showed mild lowering activity on blood glucose level to 12.6% and 7.3% after 2 and 3 h of administration. Additionally, clinically isolated H. pylori strain was inhibited with the plant extract at concentration of 500 mg/mL (zone of inhibition: 17 ± 0.08 mm). Moreover, IC₅₀ values for DPPH inhibition of the plant extract, vitamin E, BHA were examined as 13.5, 14.2, and 7.8 μg/mL, respectively. Total flavonoids, phenols, tannin, and polysaccharides contents of the extract were successfully evaluated as 5.8 ± 0.4 μg QE/mg EXT, 331 ± 3.7 μg GAE/mg EXT, 340 ± 2.3 μg TAE/mg EXT, 21 ± 0.2 μg GE/mg EXT, respectively.

Conclusions

The results suggested that L. salicaria has low anti-diabetic and anti-Helicobacter pylori effects, but high antioxidant activity, just the same as positive standard (vitamin E), which might be attributed to the high content of phenolic compounds in the extract.     

The interaction of diadenosine polyphosphates with P2X-receptors in the guinea-pig isolated vas deferens

1. The site(s) at which diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (AP₄A) and diadenosine 5′, 5′′′-P¹,P⁵-pentaphosphate (AP₅A) act to evoke contraction of the guinea-pig isolated vas deferens was studied by use of a series of P₂-receptor antagonists and the ecto-ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
2. Pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (300 nM–30 μM), suramin (3–100 μM) and pyridoxal-5′-phosphate (P-5-P) (3–1000 μM) inhibited contractions evoked by equi-effective concentrations of AP₅A (3 μM), AP₄A (30 μM) and α,β-methyleneATP (α,β-meATP) (1 μM), in a concentration-dependent manner and abolished them at the highest concentrations used.
3. PPADS was more potent than suramin, which in turn was more potent than P-5-P. PPADS inhibited AP₅A, AP₄A and α,β-meATP with similar IC₅₀ values. No significant difference was found between IC₅₀ values for suramin against α,β-meATP and AP₅A or α,β-meATP and AP₄A, but suramin was more than 2.5 times more potent against AP₄A than AP₅A. P-5-P showed the same pattern of antagonism.
4. Desensitization of the P_2X1-receptor by α,β-meATP abolished contractions evoked by AP₅A (3 μM) and AP₄A (30 μM), but had no effect on those elicited by noradrenaline (100 μM).
5. ARL 67156 (100 μM) reversibly potentiated contractions evoked by AP₄A (30 μM) by 61%, but caused a small, significant decrease in the mean response to AP₅A (3 μM).
6. It is concluded that AP₄A and AP₅A act at the P_2X1-receptor, or a site similar to the P_2X1-receptor, to evoke contraction of the guinea-pig isolated vas deferens. Furthermore, the potency of AP₄A, but not AP₅A, appears to be inhibited by an ecto-enzyme which is sensitive to ARL 67156.

     

In vitro Antioxidant and Antibacterial Activities of Methanol Extract of Kyllinga nemoralis

The present study was designed to evaluate the antioxidant and antibacterial activity of methanol extract of Kyllinga nemoralis. Six different in vitro antioxidant assays including 2,2-diphenyl-1-picrylhydrazyl, hydroxyl radical, superoxide anion radical, hydrogen peroxide radical, ferric reducing antioxidant power assay and reducing power were carried out to ensure the scavenging effect of the plant on free radicals. In addition, total antioxidant capacity assay, total phenolic contents, tannins, flavonoids and flavonol contents of the plant were also analysed by the standard protocols. Kyllinga nemoralis exhibited high antioxidant activity on 2,2-diphenyl-1-picrylhydrazyl assay (IC₅₀= 90 μg/ml), superoxide radical scavenging assay (IC₅₀= 180 μg/ml) and hydrogen peroxide radical scavenging assay (IC₅₀= 200 μg/ml), compared with standards. These observations provide comprehensible supporting evidence for the antioxidant potential of the plant extract. Reducing power (IC₅₀= 213.16 μg/ml) and hydroxyl radical scavenging activity (IC₅₀= 223 μg/ml) of the plant extract was remarkable. The methanol extract of K. nemoralis exhibited significant antimicrobial activity against Gram-positive human pathogenic bacteria. Standard in vitro antioxidant assays assessed the electron donating ability of the plant extract in scavenging free radicals. The inhibitory effect of the plant extract against bacterial pathogens may be due to the presence of phytochemicals. Thus, the results suggest that Kyllinga nemoralis is a potential source of antioxidants and could serve as the base for drug development.     

Occurrence of Deoxynivalenol and Zearalenone in Commercial Fish Feed: An Initial Study

The control of mycotoxins is a global challenge not only in human consumption but also in nutrition of farm animals including aquatic species. Fusarium toxins, such as deoxynivalenol (DON) and zearalenone (ZEN), are common contaminants of animal feed but no study reported the occurrence of both mycotoxins in fish feed so far. Here, we report for the first time the occurrence of DON and ZEN in samples of commercial fish feed designed for nutrition of cyprinids collected from central Europe. A maximal DON concentration of 825 μg kg⁻¹ feed was found in one feed whereas average values of 289 μg kg⁻¹ feed were noted. ZEN was the more prevalent mycotoxin but the concentrations were lower showing an average level of 67.9 μg kg⁻¹ feed.     

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2

1. In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels.
2. Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 μM, the highest concentration tested, increasing the rate to 153±4% of control (n=9).
3. Repetitive 5 min applications of substance P (1 μM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively.
4. The competitive antagonist of tachykinin receptors, spantide (5 μM) and the specific NK₁ receptor antagonist, WIN51708 (10 μM) both prevented the enhancement of constriction rate induced by 1 μM substance P.
5. Endothelial cells loaded with the Ca²⁺ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca²⁺]_i upon application of 1 μM substance P.
6. Inhibition of nitric oxide synthase by N^G nitro-L-arginine (L-NOARG; 100 μM) had no significant effect on the response induced by 1 μM substance P.
7. The enhancement of constriction rate induced by 1 μM substance P was prevented by the cyclo-oxygenase inhibitor, indomethacin (3 μM), the thromboxane A₂ synthase inhibitor, imidazole (50 μM), and the thromboxane A₂ receptor antagonist, SQ29548 (0.3 μM).
8. The stable analogue of thromboxane A₂, U46619 (0.1 μM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μM).
9. Treatment with pertussis toxin (PTx; 100 ng ml⁻¹) completely abolished the response to 1 μM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate.
10. Application of the phospholipase A₂ inhibitor, antiflammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 μM), without inhibiting the response to either U46619 (0.1 μM) or acetylcholine (10 μM).
11. The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK₁ receptors coupled by a PTx sensitive G-protein to phospholipase A₂ and resulting in generation of the arachidonic acid metabolite, thromboxane A₂, this serving as the diffusible activator.

     

In vitro and in vivo characterization of NK3 receptors in the rabbit eye by use of selective non-peptide NK3 receptor antagonists

1. Inhibition of NK₃ receptor agonist-induced contraction in the rabbit isolated iris sphincter muscle was used to assess the in vitro functional activity of three 2-phenyl-4-quinolinecarboxamides, members of a novel class of potent and selective non-peptide NK₃ receptor antagonists. In addition, an in vivo correlate of this in vitro response, namely NK₃ receptor agonist-induced miosis in conscious rabbits, was characterized with some of these antagonists.
2. In vitro senktide (succinyl-[Asp⁹,MePhe⁸]-substance P (6-11) and [MePhe⁷]-neurokinin B ([MePhe⁷]-NKB) were potent contractile agents in the rabbit iris sphincter muscle but exhibited quite different profiles. Senktide produced monophasic log concentration-effect curves with a mean pD₂=9.03±0.06 and mean n_H=1.2±0.02 (n=14). In contrast, [MePhe⁷]-NKB produced shallow log concentration-effect curves which often appeared biphasic (n_H=0.54±0.04, n=8), preventing the accurate determination of pD₂ values.
3. The contractile responses to the NK₃ receptor agonist senktide were antagonized in a surmountable and concentration-dependent manner by SB 223412 ((−)-(S)-N-(α-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide; 3–30 nM, pA₂=8.4, slope=1.8±0.3, n=4), SB 222200 ((−)-(S)-N-(α-ethylbenzyl)-3-methyl-2-phenylquinoline-4-carboxamide; 30–300 nM, pA₂=7.9, slope=1.4±0.06, n=4) and SB 218795 ((−)-(R)-N-(α-methoxycarbonylbenzyl)-2-phenylquinoline-4-carboxamide; 0.3 and 3 μM apparent pK_B=7.4±0.06, n=6).
4. Contractile responses to the NK₃ receptor agonist [MePhe⁷]-NKB in the rabbit iris sphincter muscle were unaffected by SB 218795 (0.3 and 3 μM, n=8). In contrast, SB 223412 (30 and 300 μM, n=4) and SB 222200 (0.3 and 3 μM, n=4) inhibited responses to low concentrations (⩽1 nM), to a greater extent than higher concentrations (>1 nM) of [MePhe⁷]-NKB. Furthermore, log concentration-effect curves to [MePhe⁷]-NKB became steeper and monophasic in the presence of each antagonist.
5. SB 218795 (3 μM, n=4) had no effect on contractions induced by transmural nerve stimulation (2 Hz) or substance P, exemplifying the selectivity of this class of antagonist for functional NK₃ receptors over NK₁ receptors in the rabbit.
6. In vivo, senktide (1, 10 and 25 μg i.v., i.e. 1.2, 11.9 and 29.7 nmol, respectively) induced concentration-dependent bilateral miosis in conscious rabbits (maximum pupillary constriction=4.25±0.25 mm; basal pupillary diameter 7.75±0.48 mm; n=4). The onset of miosis was within 2–5 min of application of senktide and responses lasted up to 30 min. Responses to two i.v. administrations of 25 μg senktide given 30 min apart revealed no evidence of tachyphylaxis. Topical administration of atropine (1%) to the eye enhanced pupillary responses to 25 μg senktide. This was probably due to the mydriatic effect of atropine since it significantly increased baseline pupillary diameter from 7.0±0.4 mm to 9.0±0.7 mm (n=4), thereby increasing the maximum capacity for miosis. Senktide-induced miosis was inhibited by SB 222200 (1 and 2 mg kg⁻¹, i.v., i.e. 2.63 and 5.26 μmol kg⁻¹; maximum inhibition 100%; n=3–4), SB 223412 (0.5 and 1 mg kg⁻¹, i.v., i.e. 1.31 and 2.61 μmol kg⁻¹; maximum inhibition 100%; n=3), SB 218795 (0.5 and 1 mg kg−1, i.v., i.e. 1.26 and 2.52 μmol kg−1; maximum inhibition 78%; n=3), and the structurally distinct NK₃ receptor antagonist SR 142801 ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylepipiperidin-4-yl)-N-methylacetamide; 1.5 mg kg⁻¹, i.v., i.e. 2.47 μmol kg⁻¹, maximum inhibition 92%; n=3).
7. Topical administration of senktide (25 μg; 29.7 nmol) to the eye induced unilateral miosis in the treated eye only. At this dose there was no significant difference (P<0.05) between pupillary constriction obtained by topical or i.v. senktide, and topically administered atropine had no significant effect on responses to topical senktide (n=4).
8. [MePhe⁷]-NKB (125, 250 and 500 μg, i.v., i.e. 98.31, 196.62 and 393.24 nmol, respectively) also induced bilateral miosis in conscious rabbits (maximum pupillary constriction=4.13±0.30 mm; n=4), but in contrast to in vitro studies this agonist was approximately 100 fold less potent than senktide. [MePhe⁷]-NKB-induced miosis was inhibited by SB 222200 (5 mg kg⁻¹, i.v., i.e. 13.14 μmol kg⁻¹; maximum inhibition 69%; n=3).
9. In summary, SB 223412, SB 222200 and SB 218795 are potent and selective antagonists of NK₃ receptor-mediated contraction in the rabbit isolated iris sphincter muscle. In addition, NK₃ receptor agonist-induced miosis in conscious rabbits is a good in vivo correlate of the in vitro rabbit iris sphincter muscle preparation and appears to be a useful model for characterizing the pharmacodynamic profile and efficacy of structurally distinct NK₃ receptor antagonists, such as SB 222200, SB 223412, SB 218795 and SR 142801.

     

Evaluation of Cellular Antioxidant and Antiproliferative Activities of Five Main Phyllanthus Emblica L. Cultivars in China

The cell-based antioxidant activity assay as more biological relevant assay was considered to be more accurate to predict antioxidant activity in vivo than chemical activity assays. In the present study, the five main Phyllanthus emblica L. cultivars in China were subjected for cellular antioxidant activity based on HepG₂ cells as well as antiproliferative activity. Total phenolics, total flavonoids and oxygen radical absorbance capacity were also measured. The results showed that Qingyougan, Binggan and Boligan (832±100, 774±52 and 704±28 μmol of quercetin equivalents/100 g) had higher cellular antioxidant activity than Tianyougan and Yougan (553±50 and 457±24 μmol of quercetin equivalents/100 g) in phosphate buffered saline wash protocol whereas, Boligan (3735±217 μmol of quercetin equivalents/100 g) had the highest cellular antioxidant activity and Tianyougan (2025±171 μmol of quercetin equivalents/100 g) had the lowest cellular antioxidant activity in no phosphate buffered saline wash protocol. The highest and lowest antiproliferative activities were observed in Binggan and Tianyougan (median effective dose: 6.95±0.11 and 14.03±0.10 mg/ml), respectively. The significant correlation was only observed between total flavonoids and cellular antioxidant activity from no phosphate buffered saline wash protocol (R² =0.908, P<0.05), and total flavonoids and antiproliferative activity (R² =0.887, P<0.05), suggesting the major contribution of flavonoids to the bioactivities of emblica. Overall, the data obtained revealed that different Phyllanthus emblica L. cultivars had strong cellular antioxidant and antiproliferative activities, thus should be recommended to increase consumption for health.     

Characterization of prejunctional 5-HT1 receptors that mediate the inhibition of pressor effects elicited by sympathetic stimulation in the pithed rat

1. A study was made of the effects of 5-carboxamidotryptamine (5-CT) on pressor responses induced in vivo by electrical stimulation of the sympathetic outflow from the spinal cord of pithed rats. All animals had been pretreated with atropine. Sympathetic stimulation (0.1, 0.5, 1 and 5 Hz) resulted in frequency-dependent increases in blood pressure. Intravenous infusion of 5-CT at doses of 0.01, 0.1 and 1 μg kg⁻¹ min⁻¹ reduced the pressor effects obtained by electrical stimulation. The inhibitory effect of 5-CT was significantly more pronounced at lower frequencies of stimulation. In the present study we characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-CT.
2. The inhibition induced by 0.01 μg kg⁻¹ min⁻¹ of 5-CT on sympathetically-induced pressor responses was partially blocked after i.v. treatment with methiothepin (10  μg kg⁻¹), WAY-100,635 (100 μg kg⁻¹) or GR127935T (250 μg kg⁻¹), but was not affected by cyanopindolol (100 μg kg⁻¹).
3. The selective 5-HT_1A receptor agonist 8-OH-DPAT and the selective 5-HT_1B/1D receptor agonists sumatriptan and L-694,247 inhibited the pressor response, whereas the 5-HT_1B receptor agonists CGS-12066B and CP-93,129 and the 5-HT_2C receptor agonist m-CPP did not modify the pressor symapthetic responses.
4. The selective 5-HT_1A receptor antagonist WAY-100,635 (100 μg kg⁻¹) blocked the inhibition induced by 8-OH-DPAT and the selective 5-HT_1B/1D receptor antagonist GR127935T (250 μg kg⁻¹) abolished the inhibition induced either by L-694,247 or sumatriptan.
5. None of the 5-HT receptor agonists used in our experiments modified the pressor responses induced by exogenous noradrenaline (NA).
6. These results suggest that the presynaptic inhibitory action of 5-CT on the electrically-induced pressor response is mediated by both r-5-HT_1D and 5-HT_1A receptors.

     

Analogues of γ-aminobutyric acid (GABA) and trans-4-aminocrotonic acid (TACA) substituted in the 2 position as GABAC receptor antagonists

1. γ-Aminobutyric acid (GABA) and trans-4-aminocrotonic acid (TACA) have been shown to activate GABA_C receptors. In this study, a range of C2, C3, C4 and N-substituted GABA and TACA analogues were examined for activity at GABA_C receptors.
2. The effects of these compounds were examined by use of electrophysiological recording from Xenopus oocytes expressing the human ρ₁ subunit of GABA_C receptors with the two-electrode voltage-clamp method.
3. trans-4-Amino-2-fluorobut-2-enoic acid was found to be a potent agonist (K_D=2.43 μM). In contrast, trans-4-amino-2-methylbut-2-enoic acid was found to be a moderately potent antagonist (IC₅₀=31.0 μM and K_B=45.5 μM). These observations highlight the possibility that subtle structural substitutions may change an agonist into an antagonist.
4. 4-Amino-2-methylbutanoic acid (K_D=189 μM), 4-amino-2-methylenebutanoic acid (K_D=182 μM) and 4-amino-2-chlorobutanoic acid (K_D=285 μM) were weak partial agonists. The intrinsic activities of these compounds were 12.1%, 4.4% and 5.2% of the maximal response of GABA, respectively. These compounds more effectively blocked the effects of the agonist, GABA, giving rise to K_B values of 53 μM and 101 μM, respectively.
5. The sulphinic acid analogue of GABA, homohypotaurine, was found to be a potent partial agonist (K_D=4.59 μM, intrinsic activity 69%).
6. It was concluded that substitution of a methyl or a halo group in the C2 position of GABA or TACA is tolerated at GABA_C receptors. However, there was dramatic loss of activity when these groups were substituted at the C3, C4 and nitrogen positions of GABA and TACA.
7. Molecular modelling studies on a range of active and inactive compounds indicated that the agonist/competitive antagonist binding site of the GABA_C receptor may be smaller than that of the GABA_A and GABA_B receptors. It is suggested that only compounds that can attain relatively flat conformations may bind to the GABA_C receptor agonist/competitive antagonist binding site.

     

Potentiation of adenosine A1 receptor-mediated inositol phospholipid hydrolysis by tyrosine kinase inhibitors in CHO cells

1. The effect of protein tyrosine kinase inhibitors on human adenosine A₁ receptor-mediated [³H]-inositol phosphate ([³H]-IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO-A1) cells.
2. In agreement with our previous studies the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated the accumulation of [³H]-IPs in CHO-A1 cells. Pre-treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 μM; 30 min) potentiated the responses elicited by 1 μM (199±17% of control CPA response) and 10 μM CPA (234±15%). Similarly, tyrphostin A47 (100 μM) potentiated the accumulation of [³H]-IPs elicited by 1 μM CPA (280±32%).
3. Genistein (EC₅₀=13.7±1.2 μM) and tyrphostin A47 (EC₅₀=10.4±3.9 μM) potentiated the [³H]-IP response to 1 μM CPA in a concentration-dependent manner.
4. Pre-incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 μM; 30 min) and tyrphostin A1 (100 μM; 30 min), respectively, had no significant effect on the accumulation of [³H]-IPs elicited by 1 μM CPA.
5. Genistein (100 μM) had no significant effect on the accumulation of [³H]-IPs produced by the endogenous thrombin receptor (1 u ml⁻¹; 100±10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [³H]-IP response (148±13%).
6. Genistein (100 μM) had no effect on the [³H]-IP response produced by activation of the endogenous G_q-protein coupled CCK_A receptor with the sulphated C-terminal octapeptide of cholecystokinin (1 μM CCK-8; 96±6% of control). In contrast, tyrphostin A47 (100 μM) caused a small but significant increase in the response to 1 μM CCK-8 (113±3% of control).
7. The phosphatidylinositol 3-kinase inhibitor LY 294002 (30 μM) and the MAP kinase kinase inhibitor PD 98059 (50 μM) had no significant effect on the [³H]-IP responses produced by 1 μM CPA and 1 μM CCK-8.
8. These observations suggest that a tyrosine kinase-dependent pathway may be involved in the regulation of human adenosine A₁ receptor mediated [³H]-IP responses in CHO-A1 cells.

     

Inhibition of Ca2+ channel current by μ- and κ-opioid receptors coexpressed in Xenopus oocytes: desensitization dependence on Ca2+ channel α1 subunits

1. Desensitization of μ- and κ-opioid receptor-mediated inhibition of voltage-dependent Ca²⁺ channels was studied in a Xenopus oocyte translation system.
2. In the oocytes coexpressing κ-opioid receptors with N- or Q-type Ca²⁺ channel α₁ and β subunits, the κ-agonist, U50488H, inhibited both neuronal Ca²⁺ channel current responses in a pertussis toxin-sensitive manner and the inhibition was reduced by prolonged agonist exposure.
3. More than 10 min was required to halve the inhibition of Q-type channels by the κ-agonist. However, the half-life for the inhibition of N-type channels was only 6±1 min. In addition, in the oocytes coexpressing μ-opioid receptors with N-type or Q-type channels, the uncoupling rate of the μ-receptor-mediated inhibition of N-channels was also faster than that of Q-type channels.
4. In the oocytes coexpressing both μ- and κ-receptors with N-type channels, stimulation of either receptor resulted in a cross-desensitization of the subsequent response to the other agonist. Treatment of oocytes with either H-8 (100 μM), staurosporine (400 nM), okadaic acid (200 nM), phorbol myristate acetate (5 nM) or forskolin (50 μM) plus phosphodiesterase inhibitor did not affect either the desensitization or the agonist-evoked inhibition of Ca²⁺ channels.
5. These results suggest that the rate of rapid desensitization is dependent on the α₁ subtype of the neuronal Ca²⁺ channel, and that a common phosphorylation-independent mechanism underlies the heterologous desensitization between opioid receptor subtypes.

     

Effect of trans-resveratrol,a natural polyphenolic compound,on human polymorphonuclear leukocyte function

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD).
2. Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more effective than other alcoholic beverages in reducing the risk of CHD mortality.
3. The aim of the present study was to investigate the effects of trans-resveratrol (3,4′,5-trihydroxy-trans-stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation.
4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 μM formyl methionyl leucyl phenylalamine (fMLP) (IC₅₀ 1.3±0.13 μM, mean±s.e.mean), as evaluated by luminol-amplified chemiluminescence.
5. trans-Resveratrol prevented the release of elastase and β-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 μM, IC₅₀ 18.4±1.8 and 31±1.8 μM), and C5a (0.1 μM, IC₅₀ 41.6±3.5 and 42±8.3 μM), and also inhibited elastase and β-glucuronidase secretion (IC₅₀ 37.7±7 and 25.4±2.2 μM) and production of 5-lipoxygenase metabolites leukotriene B₄ (LTB₄), 6-trans-LTB₄ and 12-trans-epi-LTB₄ (IC₅₀ 48±7 μM) by PMN stimulated with the calcium ionophore A23187 (5 μM).
6. trans-Resveratrol significantly reduced the expression and activation of the β₂ integrin MAC-1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation-dependent epitope on MAC-1. Consistently, PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by trans-resveratrol.
7. These results, indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions, may provide some biological plausibility to the protective effect of red wine consumption against CHD.