黄体酮对豚鼠结肠平滑肌及细胞膜钙依赖的钾通道电流的影响

目的　观察黄体酮 (Progesterone)对豚鼠结肠平滑肌肌条和细胞的作用 ,借此探讨肠易激综合征 (IBS)的病理生理机制。方法　急性分离豚鼠结肠平滑肌肌条和单个平滑肌细胞。采用TD 112S型等张传感器测量肌条收缩与舒张的幅值、频率 ,并用Axopatch 1 D膜片钳放大器测全细胞模式下的单个平滑肌细胞大电导的钙离子依赖钾通道电流(BKCa)。结果　 6 4 8μmol·L-1黄体酮可抑制结肠带肌条的收缩 (0 1792± 0 0 873) g(n =6 ,P <0 0 5 ) ,而低浓度黄体酮仅抑制结肠环行平滑肌肌条的收缩 (16 2pmol·L-10 2 36 0g± 0 15 78g ,n =6 ,P <0 0 5 ;1 6 2nmol·l-10 4 332 g±0 2 111g ,n =6 ,P <0 0 1;3 2 4nmol·l-1部分肌条完全抑制P <0 0 1) ,其效应呈剂量依赖的趋势。细胞实验中 12 96μmol·L-1的黄体酮可抑制BKCa的幅值约 6 0 %± 17% (n =10 ,P <0 0 1) ,而灌流液含 5 μmol·L-1尼卡地平 (nicardip ine)时抑制BKCa的作用不明显。结论　高浓度黄体酮对纵行和环行平滑肌均有抑制作用 ,而低浓度主要抑制环行平滑肌的收缩 ,作用机制与减少细胞外钙离子内流及抑制BKCa有关 ,这种作用可部分解释在临床上IBS妇女患病更普遍的现象 ,对女性IBS患者的激素治疗有一定的提示作用     

HMJ-53A accelerates slow inactivation gating of voltage-gated K+ channels in mouse neuroblastoma N2A cells

Voltage-gated K⁺ (Kv) channels are important in repolarization of excitable cells such as neurons and endocrine cells. Kv channel gating exhibits slow inactivation (slow current decay) during continuous depolarization. The molecular mechanism involved in such slow inactivation is not completely understood, but evidence has suggested that it involves a restriction of the outer channel pore surrounding the selectivity filter. Pharmacological tools probing this slow inactivation process are scarce. In this work we reported that bath application of HMJ-53A (30 μM), a novel compound, could drastically speed up the slow decay (decay τ = 1677 ± 120 ms and 85.6 ± 7.7 ms, respectively, in the absence and presence of HMJ-53A) of Kv currents in neuroblastoma N2A cells. HMJ-53A also significantly left-shifted the steady-state inactivation curve by 12 mV. HMJ-53A, however, did not affect voltage-dependence of activation and the kinetics of channel activation. Intracellular application of this drug through patch pipette dialysis was ineffective at all in accelerating the slow current decay, suggesting that HMJ-53A acted extracellularly. Blockade of currents by HMJ-53A did not require an open state of channels. In addition, the inactivation time constants and percentage block of Kv currents in the presence of HMJ-53A were independent of the (i) degree of depolarization and (ii) intracellular K⁺ concentration. Therefore, this drug did not appear to directly occlude the outer channel pore during stimulation (depolarization). Taken together, our results suggest that HMJ-53A selectively affected (accelerated) the slow inactivation gating process of Kv channels, and could thus be a selective and novel probe for the inactivation gate.     

Bronchoconstrictor and hypotensive effects in relation to pharmacokinetics of tachykinins in the guinea-pig —evidence for extraneuronal cleavage of neuropeptide K to neurokinin A

Summary 1. The biological effects of the tachykinins substance P (SP), neurokinin A (NKA) and neuropeptide K (NPK) were studied in relation to their pharmacokinetic properties in the guinea-pig in vivo. 2. NKA and NPK exerted a considerably larger bronchoconstrictor effect than SP. The effect of NPK was slow in onset and had a long duration. The three tachykinins showed similar hypotensive effects although NPK had a longer duration of action than SP and NKA. 3. The disappearance of NPK-like immunoreactivity (-LI) from plasma after i. v. infusion of synthetic NPK was biphasic with apparent half-lives of 0.9 min and 6 min. The plasma half-life of NKA-LI was less than 2 min, while plasma SP-LI was degraded before biochemical analysis could be performed. 4. In guinea-pig plasma at 37°C in vitro, NKA- and NPK-LI were stable for 10 min, while SP-LI disappeared with a half-life of 10 s. 5. Reversed phase HPLC analysis of plasma collected after an i. v. infusion of NPK for 25 min, indicated a partial cleavage of NPK into NKA. 6. It is concluded that potency of the biological effects of SP, NKA and NPK in the guinea-pig in vivo, may not only be attributed to activation of multiple tachykinin receptors but must also be related to the marked differences in pharmacokinetical properties between the tachykinins. Furthermore, whereas SP is rapidly degraded in plasma, NKA and NPK seem to be metabolized in other compartments. Send offprint requests to C.-R. Martling at the above address     

Xanthine‐analog,KMUP‐2, enhances cyclic GMP and K+ channel activities in rabbit aorta and corpus cavernosum with associated penile erection

The pharmacological properties of KMUP‐2 were examined in isolated rabbit aorta and corpus cavernosum smooth muscle (CCSM). KMUP‐2 caused relaxations that were attenuated by removed endothelium, high K⁺, and pretreatment with the soluble guanylate cyclase (sGC) inhibitors methylene blue (10 μM) and ODQ (1 μM), a NOS inhibitor, L‐NAME (100 μM), a K⁺ channel blocker TEA (10 mM), a K_ATP channel blocker glibenclamide (1 μM), a voltage‐dependent K⁺ channel blocker 4‐AP (100 μM), and the Ca²⁺‐dependent K⁺ channel blockers apamin (1 μM) and charybdotoxin (ChTX, 0.1 μM). The relaxant responses of KMUP‐2 (0.01, 0.05, 0.1 μM) together with a PDE inhibitor, IBMX (0.5 μM), had additive effects on rabbit aorta and CCSM. Additionally, KMUP‐2 (100 μM) also affected cGMP metabolism, due to its inhibiting activity on PDE in human platelets. KMUP‐2 (0.1–100 μM) further induced an increase of intracellular cGMP levels in the primary cultured rabbit aortic and CCSM cells. These increases in cGMP content were abolished in the presence of methylene blue (100 μM) and ODQ (10 μM). Obviously, the relaxant effects of KMUP‐2 on rabbit isolated tissues are more sensitive in CCSM than in aorta. Moreover, KMUP‐2 also stimulated NO/sGC/cGMP pathway and subsequent elevation of cGMP by blockade of PDE and enhanced opening of K⁺ channels in rabbit aorta and CCSM. KMUP‐2 (0.2, 0.4, 0.6 mg/kg), similar to KMUP‐1 and sildenafil, caused increases of intracavernous pressure (ICP) and duration of tumescene (DT) in a dose‐dependent manner. It is concluded that both the increases of cGMP and the opening activity of K⁺ channels play prominent roles in KMUP‐2‐induced aortic smooth muscle and CCSM relaxation and increases of ICP in rabbits. Drug Dev. Res. 55:162–172, 2002. © 2002 Wiley‐Liss, Inc.     

斑蝥素对A549细胞增殖抑制作用的研究

目的研究斑蝥素对人肺癌A549细胞增殖的抑制作用和机制。方法采用MTT法检测斑蝥素对A549细胞增殖的抑制作用;流式细胞仪分析A549细胞周期及斑蝥素对细胞周期的影响。结果斑蝥素对A549细胞的增殖有抑制作用;其抑制作用机制为使A549细胞周期出现明显的G2/M期阻滞。结论斑蝥素对人肺癌A549细胞的增殖有抑制作用。     

A re-appraisal of the nature of the atropine-resistant contraction to electrical field stimulation in the human isolated detrusor muscle

We investigated whether in human isolated detrusor strips the atropine-resistant contractile response to electrical field stimulation was mediated by ATP (or a related purine), as previously shown in the urinary bladder of other mammalian species. Electrical stimulation (1–50Hz for 5s at 1min intervals, 0.1ms pulse width, 60V) elicited reproducible, frequency-dependent twitch contractions, which were markedly reduced by atropine (10μM). Tetrodotoxin (TTX: 1μM) inhibited contractile responses to a similar degree. When applied together, atropine and TTX caused an inhibition which was superimposable to that caused by either drug alone. The TTX-resistant contractions were totally unaffected by omega-conotoxin GVIA (ω-CTX: 0.1μM). The atropine-resistant contractions were unaffected by the P₂-purinoceptor antagonists suramin (300μM) and PPADS (30μM), at concentrations which virtually suppressed the contractile response induced by applied ATP (10μM–1mM). As previously described, antagonism of the ATP-induced contractions by suramin (30, 100, 300μM) and PPADS (3, 10, 30μM) was insurmountable, with apparent ‘pA₂’ values (calculated at the lowest antagonist concentrations) of 4.9 and 5.2, respectively. It is concluded that, under our experimental conditions, the non-cholinergic (atropine-resistant) component of the excitatory transmission in the human detrusor is not mediated by neural release of ATP, in spite of the presence of excitatory P₂-purinoceptors on the effector cells. The TTX- and ω-CTX-resistant, non-cholinergic component might be related to the release of unknown transmitter(s) through a mechanism independent of both Na⁺- and N-type Ca²⁺-channels. More likely, the atropine-resistant component may reflect direct smooth muscle excitation since the human detrusor has a very short chronaxie (Sibley 1984). Received: 26 June 1997 / Accepted: 26 August 1997     

Histone demethylase JHDM2A regulates H3K9 dimethylation in response to arsenic-induced DNA damage and repair in normal human liver cells

Long-term arsenic exposure is a worldwide public health problem that causes serious harm to human health. The liver is the main target organ of arsenic toxicity; arsenic induces disruption of the DNA damage repair pathway, but its mechanisms remain unclear. In recent years, studies have found that epigenetic mechanisms play an important role in arsenic-induced lesions. In this study, we conducted experiments in vitro using normal human liver cells (L-02) to explore the mechanism by which the histone demethylase JHDM2A regulates H3K9 dimethylation (me2) in response to arsenic-induced DNA damage. Our results indicated that arsenic exposure upregulated the expression of JHDM2A, downregulated global H3K9me2 modification levels, increased the H3K9me2 levels at the promoters of base excision repair (BER) genes (N-methylpurine-DNA glycosylase [MPG], XRCC1 and poly(ADP-ribose)polymerase 1) and inhibited their expression levels, causing DNA damage in cells. In addition, we studied the effects of overexpression and inhibition of JHDM2A and found that JHDM2A can participate in the molecular mechanism of arsenic-induced DNA damage via the BER pathway, which may not be involved in the BER process because H3K9me2 levels at the promoter region of the BER genes were unchanged following JHDM2A interference. These results suggest a potential mechanism by which JHDM2A can regulate the MPG and XRCC1 genes in the process of responding to DNA damage induced by arsenic exposure and can participate in the process of DNA damage repair, which provides a scientific basis for understanding the epigenetic mechanisms and treatments for endemic arsenic poisoning.     

Metformin attenuates hepatic insulin resistance in type-2 diabetic rats through PI3K/Akt/GLUT-4 signalling independent to bicuculline-sensitive GABAA receptor stimulation

Context: Metformin attenuates type-2 diabetes mellitus (T2DM)-induced hepatic dysfunction and altered PI3K/Akt/GLUT-4 signalling in experimental studies. However, its effect on bicuculline-sensitive gamma amino butyric acid (GABA)-A receptor (GABA_AR)-mediated calcium-dependent PI3K/Akt/GLUT-4 signalling in liver challenged to T2DM has not been established.

Objective: The effectiveness of metformin on bicuculline-sensitive GABA_AR-mediated hepatic insulin signalling was carried out in presence or absence of bicuculline (2.0?mg/kg, i.p.) in experimental T2DM rats.

Materials and methods: The whole experimental design was divided into three independent sets of experiments. Each set comprised seven groups of six male rats each. T2DM was induced in the animals by administering streptozotocin (45?mg/kg, i.p.) and nicotinamide (110?mg/kg, i.p.) at a time lag of 15?min except control group rats in three experiments. Metformin and/or bicuculline or wortmannin were administered once daily for one week from seventh day of streptozotocin injection in all the experimental sets.

Results: Metformin attenuated T2DM-induced hyperglycaemia in glucose (40%) and insulin (50%) tolerance tests in rats. Metformin also attenuated T2DM-induced hyperglycaemia (40%), hyperinsulinaemia (30%), insulin resistance (50%) and β-cell dysfunction (300%) in the animals. Metformin did not attenuate T2DM-induced decrease in rat hepatic intracellular calcium. Further, metformin mitigated T2DM-induced decrease in hepatic phosphorylated Akt and GLUT-4 translocation in the animals. The anti-diabetic activity of metformin was abolished by wortmannin but not with bicuculline co-administration in T2DM animals.

Discussion and conclusion: These results suggest that metformin ameliorated T2DM-induced hepatic insulin resistance through bicuculline-sensitive GABA_A receptor-independent PI3K/Akt/GLUT-4 signalling pathway in animals.     

Adenovirus-mediated expression of 5-HT1B receptors in cardiac ventricle myocytes; coupling to inwardly rectifying K channels

The 5-HT_1B receptor is expressed on nerve terminals where it inhibits neurotransmitter release. When expressed ectopically in fibroblasts, the 5-HT_1B receptor inhibits adenylyl cyclase. However, in the central nervous system, the effect of this receptor on neurotransmitter release appears to be cAMP-independent. We therefore investigated alternative effector systems that might be activated by the 5-HT_1B receptor. We constructed a recombinant adenovirus that allows expression of high levels of the 5-HT_1B receptor in a variety of cells. We chose cardiac ventricle myocytes because they express a muscarinic-gated, inwardly rectifying K⁺ channel (i_KACh). In infected ventricle cells, both 5-HT and the muscarinic receptor agonist, carbachol, elicited a similar inwardly rectifying K⁺ current. The currents elicited by these agonists were pertussis-toxin sensitive and were not additive. These results suggest a common signal transduction pathway for 5-HT_1B and muscarinic receptors in ventricle cells.     

Toxicity of ZnO nanoparticles (NPs) to A549 cells and A549 epithelium in vitro: Interactions with dipalmitoyl phosphatidylcholine (DPPC)

Once inhaled, nanoparticles (NPs) will first interact with lung surfactant system, which may influence the colloidal aspects of NPs and consequently the toxic potential of NPs to pulmonary cells. In this study, we investigated the effects of dipalmitoyl phosphatidylcholine (DPPC), the major component in lung surfactant, on stability and toxicity of ZnO NPs. The presence of DPPC increased the UV–vis spectra, hydrodynamic size, Zeta potential and dissolution rate of ZnO NPs, which indicates that DPPC might interact with NPs and affect the colloidal stability of NPs. Exposure to ZnO NPs induced cytotoxicity associated with increased intracellular Zn ions but not superoxide in A549 cells. In A549 epithelium model, exposure to ZnO NPs induced cytotoxicity and decreased the release of interleukin 6 (IL-6) without a significant effect on epithelial permeability rate. Co-exposure of A549 cells or A549 epithelium model to DPPC and ZnO NPs induced a higher release of lactate dehydrogenase (LDH) and interleukin-6 (IL-6) compared with the exposure of ZnO NPs alone. We concluded that the presence of DPPC could influence the colloidal stability of ZnO NPs and increase the damage of NPs to membrane probably due to the increased positive surface charge.     

The Responsiveness of Bee Venom Phospholipase A2 on Regulatory T Cells Correlates with the CD11c+CD206+Population in Human Peripheral Blood Mononuclear Cells

Bee venom phospholipase A2 (bvPLA2) has been reported to have therapeutic effects such as neuroprotection, anti-inflammation, anti-nociception, anti-cancer properties, caused by increasing regulatory T cells (Tregs). The mechanism of Tregs modulation by bvPLA2 has been demonstrated by binding with the mannose receptor, CD206 in experimental models of several diseases. However, it remains unknown whether this mechanism can also be applied in human blood. In this study, we collected peripheral blood samples from healthy donors and analyzed the percentages of monocyte-derived dendritic cells with CD206 (CD206⁺ DCs) before expansion, the proportion of Tregs, and the subpopulations after expansion treated with bvPLA2 or PBS using flow cytometry and the correlations among them. The percentage of Tregs tended to be higher in the bvPLA2 group than in the control group. There were significant positive correlations between the CD206 population in hPBMC and the proportions of Tregs treated with bvPLA2, especially in the Treg fold change comparing the increase ratio of Tregs in bvPLA2 and in PBS. These findings indicate that bvPLA2 increased the proportion of Tregs in healthy human peripheral blood and the number of CD206⁺ DCs could be a predictor of the bvPLA2 response of different individuals.     

甲型H1N1流感儿童发生呼吸衰竭的危险因素分析

目的探讨儿童甲型H1N1流感患者发生呼吸衰竭（acute respiratory failure,ARF）的危险因素,为针对性预防其发生提供临床依据。方法对2009年9月至2010年1月我院住院51例甲型H1N1流感儿童的资料进行回顾性分析。结果51例儿童甲型H1N1流感患儿,5例发生呼吸衰竭,发生率为9．80％,多元回归分析最近有呼吸道感染或使用免疫抑制剂、中性粒细胞计数〉70％,具有统计学意义（P〈0．05）,均为独立的危险因素。结论甲型H1N1流感儿童必须预防或积极治疗其合并的细菌感染,以防呼吸衰竭的发生。     

A TRH analog (DN-1417): Motor stimulation with rearing related to catecholaminergic mechanisms in rats

The effect of an analog of TRH, γ-butyrolactone-γ-carbonyl-histidyl-prolinamide citrate (DN-1417) on motor activity was studied in rats. Peripheral administration of DN-1417 (0.2–20 $\text{mg}\text{kg}$, i.p.) caused a significant, dose-dependent increase in total spontaneous motor activity, with a definite increase in rearing behaviour. Both increases in spontaneous motor activity and rearing behaviour were markedly inhibited by pretreatment with chlorpromazine (1, 5 $\text{mg}\text{kg}$, i.p.), haloperidol (0.1, 0.5 $\text{mg}\text{kg}$, i.p.), pimozide (1 $\text{mg}\text{kg}$, i.p.) or α-methyltyrosine (250 $\text{mg}\text{kg}$, i.p.). Only stimulation of rearing behaviour was selectively attenuated by phenoxybenzamine (5 $\text{mg}\text{kg}$, i.p.) or FLA-63 (25 $\text{mg}\text{kg}$, i.p.) at doses producing no significant effect on spontaneous motor activity. Although propranolol (10 $\text{mg}\text{kg}$, i.p.) and methysergide (10 $\text{mg}\text{kg}$, i.p.) had no effect, atropine (10 $\text{mg}\text{kg}$, i.p.) and mecamylamine (10$\text{mg}\text{kg}$, i.p.) respectively potentiated and counteracted the effects of DN-1417. Concerning the stimulation of spontaneous motor activity, the nucleus accumbens and lateral hypothalamic area were most sensitive to DN-1417, and the lateral hypothalamic area was the most sensitive site for the stimulation of rearing. Furthermore, DN-1417 (5 × 10^?5 M) significantly enhanced the spontaneous release of [³H]dopamine from the rat nucleus accumbens slices in vitro. These findings indicate that the motor stimulatory action of DN-1417 appears to be mediated primarily via a dopaminergic mechanism by enhancing the release of dopamine from nerve terminals, including the nucleus accumbens in the mesolimbic dopamine system, and, in turn, the rearing may be mediated via noradrenergic mechanism.     

Exposure of human skin to benzo[a]pyrene: Role of CYP1A1 and aryl hydrocarbon receptor in oxidative stress generation

Exposure to benzo[a]pyrene (BaP) can induce inflammatory skin diseases and skin cancer, which are both associated to oxidative stress. BaP is known to bind with high specificity to the aryl hydrocarbon receptor (AhR), modifying the expression of CYP1A1, involved both in cancer and inflammation.     

Activation of the unfolded protein response contributed to the selective cytotoxicity of oroxylin A in human hepatocellular carcinoma HepG2 cells

Hepatocellular carcinoma (HCC) is a refractory malignancy with a high incidence and large mortality. Current strategy for the chemotherapy of HCC focuses on developing agents with better efficacy and lower toxicity. In this study, we demonstrated that the natural flavonoid oroxylin A preferentially inhibited the viability of HCC cell line HepG2 but not the normal hepatic cell line L02. In HepG2 but not L02 cells, oroxylin A induced substantial production of intracellular H₂O₂ and inordinate activation of the PERK-eIF2α-ATF4-CHOP branch of the unfolded protein response (UPR) pathway, which resulted in the induction of TRB3 and causal reduction of p-AKT1/2/3 (Ser473). Moreover, these effects were eliminated by either the stable knockdown of CHOP or the pretreatment and then co-incubation with the specific H₂O₂ scavenger catalase. These results indicated that the H₂O₂-triggered overactivation of the UPR pathway and causal inactivation of AKT signaling contributed to the preferential cytotoxicity of oroxylin A in malignant HepG2 cells. Therefore, present study proposed an underlying molecular mechanism that implicated the selective antitumor effect of oroxylin A and recommended oroxylin A as a prospect for improving the current chemotherapeutic strategy for the treatment of HCC.     

Human adenosine A(3) receptor leads to intracellular Ca(2+) mobilization but is insufficient to activate the signaling pathway via phosphoinositide 3-kinase gamma in mice

Selective antagonists for the adenosine A(3) receptor (A3AR), a member of the G protein-coupled receptors, have been indicated as potential drugs for anti-asthma or anti-inflammation. However, potent antagonists for the rodent A3AR have not been identified. To evaluate the pharmacological effects of human A3AR antagonists in mice, we here generated A3AR-humanized mice, in which the mouse A3AR gene was replaced by its human counterpart. The expression levels of human A3AR in the A3AR-humanized mice were equivalent to those of mouse A3AR in wild-type mice. Elevation of the intracellular Ca(2+) concentration induced by an A3AR agonist was observed in bone marrow-derived mast cells from the A3AR-humanized mice and this Ca(2+) mobilization was completely antagonized by a human A3AR antagonist. However, antigen-dependent degranulation was not potentiated by the A3AR agonist in the mast cells from A3AR-humanized mice. The agonist-stimulated human A3AR did not lead to the phosphorylation of either extracellular signal-regulated kinase 1/2 or protein kinase B in A3AR-humanized mice. The rate of human A3AR internalization in the mast cells was also markedly decreased compared with that of mouse A3AR in the mast cells. These results demonstrate that the human A3AR is insufficient to activate phosphoinositide 3-kinase gamma-dependent signaling pathways in mice, probably due to the uncoupling of member(s) of the G proteins, which are capable of activating phosphoinositide 3-kinase gamma, to the human A3AR, despite the mouse G protein(s) responsible for the Ca(2+) elevation are coupled with the human A3AR.