14β-Chlorocinnamoylamino derivatives of metopon: long-term μ-opioid receptor antagonists

The affinity, selectivity and antinociceptive properties of 5β-methyl-14β-(p-chlorocinnamoylamino)-7,8-dihydromorphinone (MET-Cl-CAMO) and N-cyclopropyl-methyl-5β-methyl-14β-(p-chlorocinnamoylamino)-7,8-dihydronormorphinone (N-CPM-MET-Cl-CAMO) for the multiple opioid receptors were characterized. In competition binding assays using bovine striatal membranes, both compounds inhibited the binding of 0.25 nM [³H][

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-Ala²,(Me)-Phe⁴,Gly(ol)⁵]enkephalin (DAMGO) with IC₅₀ values of less than 2 nM. Preincubation of membranes with MET-Cl-CAMO and N-CPM-MET-Cl-CAMO produced a concentration-dependent, wash-resistant inhibition of μ-opioid receptor binding. Saturation binding experiments with N-CPM-MET-Cl-CAMO showed a reduction in the number of μ-opioid binding sites without a change in affinity. In the mouse 55°C warm-water tail-flick assay, neither MET-Cl-CAMO nor N-CPM-MET-Cl-CAMO at doses up to 100 nmol produced antinociception after intracerebroventricular administration, but morphine-induced antinociception was antagonized in a time- and dose-dependent manner by both compounds. The antagonism produced by 1 nmol of either MET-Cl-CAMO or N-CPM-MET-Cl-CAMO reached a maximal effect after 24 h, and lasted up to 48 h. Analgesia mediated by δ- or κ-opioids was not altered by either compound. In summary, the data suggest that MET-Cl-CAMO and N-CPM-MET-Cl-CAMO are long-term, μ-opioid receptor antagonists, devoid of agonist properties in the mouse tail-flick assay, and that N-CPM-MET-Cl-CAMO may produce its antagonistic effects by binding irreversibly to the μ-opioid receptor.     

Arachidonic acid products-mediated contraction induced by bradykinin in relaxed mesenteric arterial rings from Holtzman rats

This study describes the contractile action of bradykinin on rat isolated mesenteric arterial rings and a possible mechanism responsible for this action. Bradykinin induced dose-dependent contraction of relaxed mesenteric arterial rings from Holtzman rats, but not from Wistar rats. A second bradykinin challenge in the same ring induced a very small effect or no effect at all. Destruction of the endothelium did not modify the response to bradykinin. des-Arg⁹-[Leu⁸]bradykinin failed to antagonize bradykinin's action. HOE 140 (

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-Arg-[Hyp³,Thi⁵,

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-Tic⁷,Oic⁸]bradykinin) reduced bradykinin-induced contractions. Indomethacin abolished the contractile response to bradykinin; prostaglandin F₂ induced a long-lasting contraction, dissimilar from that induced by bradykinin; L-655,240 (3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]-2,2-dimethyl propanoic acid), an antagonist of the thromboxane receptor, inhibited bradykinin-induced contractions. These results suggest that bradykinin-induced contraction in mesenteric arterial rings is indirect, through activation of bradykinin B₂ receptors, resulting in liberation of prostanoids from outside the endothelium. Thromboxane A₂ is probably an intermediate in this response but we cannot exclude the participation of other prostanoids.     

Vasodilator effects of C-type natriuretic peptide on cerebral arterioles in rats

The vasodilator effects of C-type natriuretic peptide (CNP) were investigated in isolated rat cerebral arterioles. CNP caused dose-dependent vasodilation, maximally by 10.0±2.2% at 10⁻⁶ M. The median effective concentration (EC₅₀) was 5.2×10⁻¹⁰ M. In contrast, atrial natriuretic peptide and B-type natriuretic peptide, other members of the natriuretic peptide family, produced little or no vasodilation. Pretreatment with methylene blue (10⁻⁴ M) abolished CNP-induced vasodilation, whereas pretreatment with N^G-monomethyl-

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-arginine or indomethacin did not inhibit vasodilation. Thus, CNP is suggested to cause significant vasodilation in cerebral arterioles via a cyclic guanosine monophosphate-dependent mechanism. © 1997 Elsevier Science B.V. All rights reserved.     

Anorectic action of bombesin requires receptor for corticotropin-releasing factor but not for oxytocin

The marked functional similarities between pharmacological effects of bombesin and of corticotropin-releasing factor (CRF), prompted the formulation and testing of our working hypothesis that BN may elicit its biological effects through the release of CRF. Central pretreatment with CRF receptor antagonists, -helical CRF-(9–41) (-CRF-(9–41)) or [

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-Phe¹², CMeLeu³⁷]CRF-(12–41) (CMeCRF), blocked the effects of centrally administered bombesin on food intake and related behaviors and partially attenuated the satiety effects of systemically administered bombesin. We also attempted to characterize the specificity of this interaction through the combined use of bombesin with the oxytocin antagonist, [d(CH₂)₅, Tyr(OMe)², Orn⁸]vasotocin (vasotocin). Central pretreatment with vasotocin failed to alter bombesin-induced behaviors, suggesting the absence of a pharmacological interaction between these two peptidergic systems. Finally, the CRF antagonist failed to reverse the oxytocin-induced suppression of food intake, indicating that CRF does not have a direct role in the mediation and/or modulation of the effects of oxytocin on food intake. Thus, the present experiments support the contention that bombesin partly mediates its feeding-suppressant effects through interactions with CRF. The specificity of this interaction is supported by the lack of interaction between bombesin and/or CRF with oxytocin.     

Characteristics of the NMDA receptor modulating hypoxia/hypoglycaemia-induced rat striatal dopamine release in vitro

We investigated the functional characteristics of the NMDA receptor that modulates hypoxia/hypoglycaemia-induced striatal dopamine release. Dopamine release was detected by fast cyclic voltammetry in rat neostriatal slices. Four variables were measured: T_on — time from initiation of hypoxia/hypoglycaemia to the onset of dopamine release, T_pk — time from onset to maximum, δDA/δt — rate of dopamine release and DA_max — maximum extracellular dopamine concentration. In controls, T_on=164.9±1.7 s, T_pk=20.9±0.9 s, δDA/δt=5.31±0.44 μM/s and DA_max=79.1±2.5 μM (means±S.E.M., n=203). Cis-4-(phosphonomethyl)piperidine-2-carboxylic acid (CGS 19755, 20 μM) lengthened, while N-methyl-

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-aspartate (NMDA) (100 μM) shortened T_on. (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK 801, 1 and 10 μM) and dextromethorphan (10 and 100 μM) increased T_pk and decreased DA_max. Neither glycine (100 μM), 7-chlorokynurenic acid (50 μM) nor 5-nitro-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione (ACEA 1021, 100 μM) had any effect although 7-chlorokynurenic acid blocked the effect of NMDA. Increasing [Mg²⁺] from 1.3 to 3.7 mM, increased T_pk and decreased δDA/δt. Dithiothreitol (1 mM) accelerated T_on while 5,5-dithio-bis-(2-nitrobenzoic acid) (1 mM) delayed T_on. Neither drug affected T_pk, DA_max or δDA/δt. Neither spermidine (100 μM) nor arcaine (100 μM) affected T_on, T_pk or δDA/δt although arcaine decreased DA_max. In conclusion, hypoxia/hypoglycaemia-induced dopamine release was influenced by an NMDA receptor although modulation of the glycine recognition site of the receptor was ineffective, as were agents acting at polyamine modulatory zones. These findings highlight differences between recombinant and native NMDA receptors and suggest caution in extrapolating molecular biology to functional studies.     

Structure–activity relationships of astemizole derivatives for inhibition of store operated Ca channels and exocytosis

The effects of a series of analogues of the antiallergic drug astemizole on the exocytosis of the enzyme β-hexosaminidase were studied in a mast cell model, the rat basophilic leukemia (RBL-2H3) cell. Besides differences in the effects on FcεRI receptor-stimulated exocytosis, changes were also observed in Ca²⁺ influx and in the perturbation of the cell membrane. A strong correlation was found between the effects on antigen- and thapsigargin-stimulated

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influx. Furthermore, the inhibition of

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influx was correlated with the inhibition of β-hexosaminidase release and membrane stabilization. It is concluded that the astemizole analogues are capable of inhibiting mast cell β-hexosaminidase release through inhibition of Ca²⁺-store-operated Ca²⁺ channels (SOC). Compounds with high lipophilicity also released Ca²⁺ from intracellular stores. Lowering of the hydrophobicity by introduction of nitrogens or truncation at different sites in the astemizole structure decreased inhibitory activity on SOC channels. The inhibition of SOC channels cannot completely be ascribed to non-specific membrane effects. The piperidinyl–benzimidazole moiety was found to be important for inhibition of SOC channels. The observed differences in activity possibly depend on the way the compounds penetrate the membrane bilayer. Astemizole is an interesting new tool to study SOC channels and can be a lead for the design of mast cell-stabilizing antiallergic drugs.     

Inhibition of N-, P/Q- and other types of Ca channels in rat hippocampal nerve terminals by the adenosine A1 receptor

The effects of the adenosine A₁ receptor agonist, N⁶-cyclopentyladenosine (CPA), on both the increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and on the release of endogenous glutamate in rat hippocampal synaptosomes were studied. The inhibitory effect of CPA on the increase in [Ca²⁺]_i stimulated with 4-aminopyridine was neutralized by the adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The inhibitory effect of CPA was greater in synaptosomes from the CA1 subregion than in whole hippocampal synaptosomes. The inhibitory effects of both CPA and of the Ca²⁺ channel blockers, ω-conotoxin GVIA, ω-conotoxin MVIIC or ω-conotoxin GVIA plus ω-conotoxin MVIIC, were greater than those caused by the Ca²⁺ channel blockers. The release of endogenous glutamate was inhibited by 41% by CPA. The inhibition observed when CPA and ω-conotoxin GVIA or CPA and ω-conotoxin MVIIC were present was also greater than the inhibition by the Ca²⁺ channel blockers alone. The presence of both ω-conotoxin GVIA and ω-conotoxin MVIIC did not completely inhibit the release of glutamate, and CPA significantly enhanced this inhibition. The membrane potential and the accumulation of [

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]tetraphenylphosphonium of polarized or depolarized synaptosomes was not affected by CPA, suggesting that adenosine did not increase potassium conductances. The present results suggest that, in hippocampal glutamatergic nerve terminals, adenosine A₁ receptor activation partly inhibits P/Q- and other non-identified types of Ca²⁺ channels.     

Differential effects of chronic imipramine and fluoxetine on basal and amphetamine-induced extracellular dopamine levels in rat nucleus accumbens

The effect of chronic treatment with the tricyclic antidepressant drug, imipramine (10 mg/kg per day), the selective serotonin (5-HT) reuptake inhibitor, fluoxetine hydrochloride (10 mg/kg per day), and vehicle, in drinking water for 24–28 days followed by 3–5 days withdrawal, on extracellular dopamine levels was studied in rat nucleus accumbens by in vivo microdialysis. Basal extracellular dopamine levels in the nucleus accumbens were increased after chronic imipramine (12.7±1.5 fmol/20 μl per 30 min, P=0.019), and moderately decreased after chronic fluoxetine (6.5±0.6, P=0.047), as compared to the vehicle controls (9.1±0.7), determined by one-way analysis of variance (ANOVA). Repeated measure ANOVA indicated that the

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-amphetamine sulfate (0.5 mg/kg, s.c.)-induced increase in extracellular dopamine levels in the nucleus accumbens was potentiated after chronic imipramine (P=0.002), but unchanged after chronic fluoxetine (P=0.83). The difference in the effect of amphetamine could be influenced by the significant differences in basal levels. However, these results were also confirmed by analysis of the net area under the curve (net-AUC) for a 180-min period (six samples): for chronic imipramine (337±45 fmol/180 min, P=0.005) and chronic fluoxetine (249±38, P=0.57), as compared to the vehicle controls (178±29), determined by one-way ANOVA. We suggest that the effect of treatment with these agents on mesolimbic dopamine is unlikely to be involved in their shared antidepressant action, but may be relevant to other aspects of the therapeutic profile of these two drugs, e.g. the switch into mania which is more common after treatment with imipramine than fluoxetine and exacerbation of positive symptoms in patients with schizophrenia or schizoaffective disorder.     

An Assessment of Nicotine Dependence Among Pregnant Adolescents

Studies have reported that between 28 and 62% of pregnant teenagers smoke (Cornelius and Trollestrup). Because smoking is prevalent among pregnant teenagers, the purpose of this research is to assess nicotine dependence in this high-risk group. This study analyzed baseline data from a sample of pregnant teen smokers who had volunteered to participate in a smoking cessation study (N = 94). Nicotine dependence was measured by adapting the Fagerstrom Tolerance Questionnaire (FTQ; Prokhorov, Pallonen, Fava, Ding, & Niaura, 1996), and by a 6-item withdrawal symptom scale. The overall FTQ score found among pregnant adolescents was 3.10 (SD = 2.3) compared to the mean overall FTQ score among vocational-technical students of 4.27 (SD = 2.2) (Prokhorov et al., 1996). Duration of smoking in years was significantly correlated with the overall FTQ score (r = 0.43, p < .01). Quantity of smoking, as measured by average number of cigarettes smoked, significantly correlated with overall FTQ scores (r = 0.67, p < .01). Lighter smokers were more likely to have previously attempted to quit, however, among the quit attempters, those who smoked 10 + cigarettes per day reported greater severity of withdrawal symptoms than those who smoked less per day. Prenatal education and smoking cessation programs for pregnant teenagers, and pregnant women in general, need to consider that nicotine dependence is an important issue. Early pregnancy may be an opportune time to intervene among pregnant smokers; incentives may be necessary to attract those women who are the heaviest smokers, and possibly the most dependent on nicotine.     

Myocardial oxidative stress contributes to transgenic β₂-adrenoceptor activation-induced cardiomyopathy and heart failure

BACKGROUND AND PURPOSE

While maintaining cardiac performance, chronic β-adrenoceptor activation eventually exacerbates the progression of cardiac remodelling and failure. We examined the adverse signalling pathways mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and reactive oxygen species (ROS) after chronic β₂-adrenoceptor activation.

EXPERIMENTAL APPROACH

Mice with transgenic β₂-adrenoceptor overexpression (β₂-TG) and non-transgenic littermates were either untreated or treated with an antioxidant (N-acetylcysteine, NAC) or NADPH oxidase inhibitors (apocynin, diphenyliodonium). Levels of ROS, phosphorylated p38 mitogen-activated protein kinase (MAPK), pro-inflammatory cytokines and collagen content in the left ventricle (LV) and LV function were measured and compared.

KEY RESULTS

β₂-TG mice showed increased ROS production, phosphorylation of p38 MAPK and heat shock protein 27 (HSP27), expression of pro-inflammatory cytokines and collagen, and progressive ventricular dysfunction. β₂-adrenoceptor stimulation similarly increased ROS production and phosphorylation of p38 MAPK and HSP27 in cultured cardiomyocytes. Treatment with apocynin, diphenyliodonium or NAC reduced phosphorylation of p38 MAPK and HSP27 in both cultured cardiomyocytes and the LV of β₂-TG mice. NAC treatment (500 mg·kg⁻¹·day⁻¹) for 2 weeks eliminated the up-regulated expression of pro-inflammatory cytokines and collagen in the LV of β₂-TG mice. Chronic NAC treatment to β₂-TG mice from 7 to 10 months of age largely prevented progression of ventricular dilatation, preserved contractile function (fractional shortening 37 ± 5% vs. 25 ± 3%, ejection fraction 52 ± 5% vs. 32 ± 4%, both P < 0.05), reduced cardiac fibrosis and suppressed matrix metalloproteinase activity.

CONCLUSION AND IMPLICATIONS

β₂-adrenoceptor stimulation provoked NADPH oxidase-derived ROS production in the heart. Elevated ROS activated p38 MAPK and contributed significantly to cardiac inflammation, remodelling and failure.

LINKED ARTICLE

This article is commented on by Di Lisa et al., pp. 1009–1011 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01130.x     

Contrasting effects of estradiol and 17 beta-aminoestrogens on blood clotting time in rats and mice

Estrogens have been associated with thromboembolic events. Our group has described the anticoagulant effect of 17β-aminoestrogens in rodents, potentially new alternative estrogenic agents without thrombogenic risk. This work compares the contrasting effects of estradiol and the 17β-aminoestrogens (prolame, butolame, and pentolame) on blood clotting time. Ovariectomized CD1 mice received a single injection of 17β-aminoestrogens, estradiol (20 to 80 mg/kg), or vehicle. Estradiol decreased blood clotting time from −10% to −25% (48 h; P<0.01) and 17β-aminoestrogens increased it, differing in latency (12 h; +48%, P<0.01) and duration (72 h +58%, P<0.01). In male Wistar rats, similar effects (pentolame +45%; estradiol −31%; P<0.01) were observed 48 h after five consecutive daily injections of 1000 μg/animal/day. The maximum procoagulant effect of estradiol was obtained after 72 h with 10 μg/animal/day (−45%; P<0.01). 17β-Aminoestrogens always produced opposite effects to those of estradiol on blood coagulation.     

The β-adrenoceptors of the lung mediating inhibition of antigen-induced histamine release

The β-adrenoceptor stimulants, isoprenaline (IPR), orciprenaline (OPR), terbutaline (TRB), and ITP^** were studied for effects on antigen-induced release of histamine from guinea-pig lung tissue and for effects on guinea-pig isolated trachea and heart. The order of potency for the agents in the four functions studied were: (a) inhibition of histamine release, IPR > OPR ≈ TRB ITP = 0; (b) heart stimulation, chronotropic effect, IPR > OPR > ITP ≈ TRB; (c) heart stimulation, inotropic effect, IPR > OPR > ITP > TRB; (d) trachea relaxation: IPR > TRB > OPR ITP. These findings suggest that the β-adrenoceptors mediating inhibition of antigen-induced release of histamine are more related to those mediating trachea relaxation (β₂) than those mediating cardiac stimulation (β₁).     

Intrinsic sympathomimetic activity of beta-adrenoceptor antagonists: down-regulation of cardiac beta 1- and beta 2-adrenoceptors

Prolonged treatment of cultured rat heart muscle cells containing β₁- and non-muscle cells containing β₂-adrenoceptors with β-adrenoceptor antagonists devoid of intrinsic sympathomimetic activity had no effect on β-adrenoceptor density. In contrast, antagonists with intrinsic sympathomimetic activity decreased β-adrenoceptor density and response (adenylate cyclase stimulation) in both heart muscle (β₁) and non-muscle cells (β₂) by a maximum of about 50%. An even larger down-regulation of β-adrenoceptors and loss of receptor-stimulated adenylate cyclase activity was induced by the full endogenous agonist, noradrenaline, with the β-adrenoceptors of heart muscle cells (β₁) being much more sensitive to the β₁-selective noradrenaline than the heart non-muscle cell β₂-adrenoceptors. When combined with noradrenaline, the antagonists with intrinsic sympathomimetic activity prevented the action of noradrenaline at both β₁- and β₂-adrenoceptors, thereby leading to an apparent up-regulation of receptor density and response. This apparent reversal from an agonist to an antagonist action was observed at much lower concentrations of noradrenaline at β₁- than at β₂-adrenoceptors. The data presented indicate that the β-adrenoceptor antagonists with intrinsic sympathomimetic activity, but not those without, upon prolonged treatment decrease the density and responsiveness of both β₁- and β₂-adrenoceptors in cultured rat heart cells. This suggests that the intrinsic sympathomimetic activity of these agents is not a subtype-selective component. Furthermore, the agonist and antagonist activity of these agents apparently depends on the concomitant presence of an endogenous full agonist and an its own affinity and that of the partial agonist for the β-adrenoceptor subtype.     

Lack of anticholinergic effect of N-nitro- -arginine methylester in the small intestine

The nitric oxide (NO)-synthase inhibitor, N^G-nitro- -arginine methylester ( -NAME), has been reported to have an atropine-like action. We compared the effects of -NAME (1 mM) and atropine on isolated small intestinal preparations of the guinea-pig, rat, rabbit and mouse. Half-maximal longitudinal contractions in response to acetylcholine (50–100 nM) were not influenced by -NAME, but were strongly suppressed by atropine (1 nM). Cholinergic ‘twitch' contractions of the guinea-pig ileum were slightly enhanced by -NAME; this effect was prevented by pretreatment with N^G-nitro- -arginine ( -NOARG, 100 μM), another NO synthase inhibitor. ‘Twitch' contractions were concentration dependently inhibited by atropine (1–100 nM). We conclude that -NAME is free of atropine-like activity in isolated intestinal preparations.     

Effects of water deprivation and angiotensin II intracerebroventricular administration on brain nitric oxide synthase activity

Intracranial administration of -arginine causes a reduction of the water intake induced by water deprivation or by intracerebroventricular (i.c.v.) injection of angiotensin II (angiotensin II), through the release of nitric oxide (NO) in the central nervous system. We studied the effects of i.c.v. angiotensin II (120 ng/rat) in association with i.c.v. -arginine (2.5–10 μg/rat) on blood pressure. We also studied the effects of both peripheral and central angiotensin II injection (1.5–6 mg kg⁻¹ i.p. and 30–120 ng rat⁻¹ i.c.v., respectively) on NO synthase activity in the cortex, diencephalon and brainstem, after water deprivation (24 h), conditions producing activation of the renin-angiotensin system. -arginine dose dependently antagonized the increase in blood pressure induced by i.c.v. angiotensin II (P<0.001). Peripheral administration of angiotensin II produced a dose-dependent reduction of NO synthase activity in the brainstem and cortex (P<0.001), but not in the diencephalon. Water deprivation produced similar effects on brain NO synthase activity. Angiotensin II i.c.v. injection caused NO synthase activity reduction in all brain regions studied (P<0.001). Our findings suggest that NO and angiotensin II could play opposite roles in brain regulation of blood pressure and drinking behaviour.     

Hyperpolarization-activated inward current in embryonic chick cardiac myocytes: developmental changes and modulation by isoproterenol and carbachol

Modulation of the hyperpolarization-activated inward current (I_f) in embryonic chick ventricular myocytes was examined using whole-cell voltage-clamp. Long (3 s) hyperpolarizing pulses were applied from a holding potential of −30 mV to steps of −40 to − 120 mV. I_f was marked in 3-day-old cells, diminished at 10 days, and was almost completely gone at 17 days. I_f current density (at −120 mV) was −6.7 ± 1.3 (3 days), −3.3 ± 1.0 (10 days), and −2.0 ± 0.5 pA/pF (17 days). I_f reduction paralleled the decrease in spontaneous activity. In 3-day cells, the threshold potential was −50 to −60 mV, and the reversal potential was −13.4 ± 1.3 mV. The time course of activation was fitted by a single exponential and was temperature dependent: τ was 1.3 ± 0.4 s at 20°C(and 0.7 ± 0.4 s at 30°C (at−120 mV). I_f amplitude was enhanced by 12.1 ± 1.8% at 30 °C compared with 20°C. Cs⁺ (3 mM) blocked I_f and had a negative chronotropic effect (rate decreased by 61%). Isoproterenol (1 μM) caused a positive chronotropic effect (17.1 ± 2.9%) and increased I_f by 65.2 ± 5.6%. Carbachol (0.1 μM) had a negative chronotropic effect (26.3 ± 3.4%), and decreased I_f by 41.2 ± 1.3%; it also reversed the enhancement produced by isoproterenol. Intracellular application of 100 μM GTP-γS decreased basal I_f by 35.2 ± 5.0%, but potentiated the stimulant effect of isoproterenol (by 37.8 ± 4.7%) and the inhibitory effect of carbachol (21.2 ± 4.3%). These results indicate that the I_f current may contribute to the pacemaker potential of young embryonic chick heart cells and decreases during development. β-Adrenoceptor agonists stimulate I_f, whereas muscarinic cholinergic agonists inhibit I_f and reverse β-adrenoceptor stimulation. G proteins may directly and indirectly couple autonomic receptors to I_f channels.     

Z-ligustilide attenuates lipopolysaccharide-induced proinflammatory response via inhibiting NF-KB pathway in primary rat microglia

Aim:

To investigate the anti-inflammatory effect of Z-ligustilide (LIG) on lipopolysaccharide (LPS)-activated primary rat microglia.

Methods:

Microglia were pretreated with LIG 1 h prior to stimulation with LPS (1 μg/mL). After 24 h, cell viability was tested with MTT, nitric oxide (NO) production was assayed with Griess reagent, and the content of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein (MCP-1) was measured with ELISA. Protein expression of the nuclear factor-κB (NF-κB) p65 subunit, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) was detected with immunocytochemistry 1 h or 24 h after LPS treatment.

Results:

LIG showed a concentration-dependent anti-inflammatory effect in LPS-activated microglia, without causing cytotoxicity. Pretreatment with LIG at 2.5, 5, 10, and 20 μmol/L decreased LPS-induced NO production to 75.9%, 54.4%, 43.1%, and 47.6% (P<0.05 or P< 0.01), TNF-α content to 86.2%, 68.3%, 40.1%, and 39.9% (P<0.01, with the exception of 86.2% for 2.5 μmol/L LIG), IL-1β content to 31.5%, 27.7%, 0.6%, and 0% (P<0.01), and MCP-1 content to 84.4%, 50.3%, 45.1%, and 42.2% (P<0.05 or P<0.01), respectively, compared with LPS treatment alone. LIG (10 μmol/L) significantly inhibited LPS-stimulated immunoreactivity of activated NF-κB, COX-2, and iNOS (P<0.01 vs LPS group).

Conclusion:

LIG exerted a potent anti-inflammatory effect on microglia through inhibition of NF-κB pathway. The data provide direct evidence of the neuroprotective effects of LIG and the potential application of LIG for the treatment of the neuroinflammatory diseases characterized by excessive microglial activation.     

Metabolic differences and their impact on human disease Sulfotransferase and colorectal cancer

Gene–environment interaction is an important aspect of human cancer risk. Genetic polymorphisms in acetylation and N-oxidation have previously been described regarding their impact on the heterocyclic amine-induced risk for colon cancer. Here, we report that another enzyme involved in the metabolism of food-borne carcinogens, sulfotransferase (ST1A3 measured by 2-naphthol activity), may function as a potential protective factor for colon cancer in humans. Initially characterized in human liver and colon (Chou et al., 1995), TS-PST activity can also be measured in platelets. A simple microtiter-based colorimetric technique was developed for use in this case-control study. African-Americans had a higher mean ST activity than Caucasians (2.32±0.24 versus 1.77±0.09 nmols/min per mg cytosolic protein, P=0.036). Furthermore, the slow ST phenotype (ST≤1.53) was more frequently associated with colon cancer than controls (57 versus 40%, P=0.026). These data suggest that the ST1A3 isoform may play a role in the differential risk for colorectal cancer.     

Can protonated β-blockers interact with biomembranes stronger than neutral isolipophilic compounds?: A chromatographic study on three different phospholipid stationary phases (IAM-HPLC)

The chromatographic capacity factors (k′) of 10 β-adrenoceptor antagonists (“β-blockers”) were measured on three different immobilized artificial membrane-phosphatidylcholine (IAM-PC) HPLC stationary phases, namely IAM-PC-MG, IAM-PC-DD2, and IAM-PC-DD. The two former phases are made of phosphatidylcholine as found in biomembranes and differ each other in end-capping of free propylamino residues whereas the latter is made of single-chain phosphatidylcholine analogues. On IAM-PC-DD2 we found that structurally unrelated neutral compounds give a single correlation between log k′ values and the respective octanol/water partition coefficients (log P), as previously observed on IAM-PC-MG phase. This was not observed on the IAM-PC-DD phase. IAM chromatography was performed at a pH of the aqueous eluent (7.0) close to the physiological pH 7.4. Retention on all IAM phases showed a biphasic pattern, being proportional to log P^N (lipophilicity of neutral forms) for more lipophilic congeners (log P^N > 1.90), and quite constant for the others. The comparison of β-blocker retention with that of neutral compounds on IAM-PC-MG and IAM-PC-DD2 suggests that they can interact with phospholipids as strongly or more strongly than neutral isolipophilic compounds, despite their being more than 98% in their ionized form. Therefore, we hypothesize that electrostatic interactions play a pivotal role in the interactions between β-blockers and membrane phospholipids.     

Cystatin C, beta 2-microglobulin, and retinol-binding protein as indicators of glomerular filtration rate: comparison with plasma creatinine

Background: The aim of this study was to assess the diagnostic accuracy of plasma levels of three low-molecular weight proteins cystatin C, β2-microglobulin, and retinol-binding protein, as indicators of impairment of glomerular filtration rate in comparison with plasma creatinine. Methods: Glomerular filtration rate (GFR) was measured in 110 patients (51 M and 59 F, aged 18–79 years); creatinine (Creat), cystatin C (Cys), β2-microglobulin (β2M), and retinol-binding protein (RBP) were determined on the same day. The correlation coefficients between the different markers and GFR were determined. Receiver-operating characteristics (ROC) analysis was performed to assess their diagnostic accuracy. Furthermore, the relationship between plasma levels of the examined markers of GFR and body weight, height, fat-free mass (FFM) and body cell mass (BCM) was determined. FFM and BCM were calculated by means of total body electrical impedance measurement. Results: Serum concentrations of Cys, β2M and RBP increase progressively with the reduction of GFR. The magnitude of the increase in blood levels of Creat and β2M was higher than the increase of Cys, and much more than that of RBP, in particular in patients with GFR<20 ml/min/1.73 m². The correlation coefficients between GFR and 1/plasma concentrations were 0.647 for Creat, 0.651 for Cys, 0.731 for β2M, and 0.406 for RBP. ROC analysis indicated that the accuracy of β2M and Cys, as indicators of different degrees of GFR impairment (<80, <60, and <40 ml/min per 1.73 m²), was similar to that of Creat, while the diagnostic accuracy of RBP resulted significantly lower than that of Creat for any level of GFR. In patients without renal failure (GFR>40 ml/min per 1.73 m²), plasma concentrations of Creat were positively correlated with body weight (P<0.01), height (P<0.01), FFM (P<0.001) and BCM (P<0.001). Serum concentrations of RBP resulted correlated with FFM (P<0.05) and BCM (P<0.05), while no correlation was found between anthropometric data and Cys and β2M. Conclusion: Cystatin C and β2-microglobulin have a diagnostic accuracy very similar to that of creatinine, while retinol-binding protein is not an adequate marker of glomerular filtration.