阿托伐他汀的肝毒性机制研究

目的 研究阿托伐他汀肝毒性损伤作用及机制。方法 将24只Wistar han雄鼠分为对照组和阿托伐他汀低（68.5mg/kg）、高剂量组（205.5 mg/kg）,按照10 mL/kg的药液体积给药,溶媒对照组ig等体积5% CMC-Na,连续ig 28 d。检测血清中天门冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、碱性磷酸酶（ALP）、尿素氮（BUN）和血肌酐（CRE）的含量,HE染色观察肝组织病理。在体外,HepG2细胞经传代培养后,给予阿托伐他汀干预24 h,检测细胞存活率,丙二醛（MDA）水平、Na⁺-K⁺-ATP酶和Ca²⁺-Mg²⁺-ATP酶活性及线粒体膜电位。结果 与对照组比较,阿托伐他汀高剂量组大鼠肝细胞弥漫性肿胀,核分裂多见,部分肝细胞极性消失,排列紊乱（P<0.05）。与对照组比较,阿托伐他汀高剂量组给药后血清中ALT和AST显著升高（P<0.05、0.01）。在体外,与对照组比较,阿托伐他汀125、250、500 μmol/L能明显抑制细胞存活率（P<0.05、0.001）。与对照组比较,阿托伐他汀500 μmol/L HepG2细胞MDA含量明显升高（P<0.01）。与对照组比较,阿托伐他汀125 μmol/L能使Na⁺-K⁺-ATP酶活性增强,500 μmol/L使Na⁺-K⁺-ATP酶活性降低（P<0.001）。与对照组比较,阿托伐他汀125、250、500 μmol/L均能使能使Ca²⁺-Mg²⁺-ATP酶活性降低（P<0.01,0.001）。与对照组比较,阿托伐他汀125、250、500 μmol/L均能降低线粒体膜电位（P<0.001）。结论 阿托伐他汀高剂量可导致肝组织损伤,其毒性作用通过破坏细胞的线粒体膜电位,抑制Na⁺-K⁺-ATP酶、Ca²⁺-Mg²⁺-ATP酶活性,细胞膜脂质过氧化,从而破坏细胞内微环境的平衡,导致细胞凋亡和坏死。     

不同生境人参茎叶总皂苷对心律失常小鼠的作用比较

目的 探讨园参茎叶总皂苷(GSLS)和林下山参茎叶总皂苷(MSLS)对心律失常小鼠的改善作用.方法 将80只SPF级BALB/c小鼠随机分为正常组、模型组、园参茎叶总皂苷低(GSLS-L)、中(GSLS-M)、高(GSLS-H)剂量组、林下山参茎叶总皂苷低(MSLS-L)、中(MSLS-M)、高(MSLS-H)剂量组....     

Effects of presynaptic modulators on Ca2+-induced noradrenaline relase from cardiac sympathetic nerves

Summary In order to elucidate the mode of action of the Ca²⁺-antagonistic inhibitor nifedipine, its effect on Ca²⁺-mediated action potentials and transmembrane slow inward current in papillary muscles of guinea pigs and cats was studied.Nifedipine (0.5 mg/l1.4×10^–6M) depressed upstroke velocity and overshoot of the Ca²⁺-mediated action potential and reduced the transmembrane slow inward current by about 50%, but the kinetics of inactivation and recovery from inactivation were not affected. The decrease of upstroke velocity was accompanied by a proportional diminution of isometric contractile force. This indicates that nifedipine exerts its Ca²⁺-antagonistic effect on excitation-contraction coupling in mammalian ventricular myocardium by inhibition of the transmembrane Ca²⁺ inward current. The inhibitory action of nifedipine on contractile tension development could be neutralized by an augmentation of the extracellular Ca²⁺ concentration from 2 mM to 4 mM or by -receptor stimulation (isoproterenol) that promotes the transmembrane Ca²⁺-rich medium or under the influence of isoproterenol the upstroke velocity of the Ca²⁺-mediated action potentials rose even above the initial values which were measured prior to the nifedipine administration.     

Ruthenium red protects HepG2 cells overexpressing CYP2E1 against acetaminophen cytotoxicity

We studied the mechanisms of acetaminophen (APAP) cytotoxicity in HepG2 cells overexpressing cytochrome p4502E1, particularly the role of oxidative/nitrosative stress and ryanodine Ca²⁺ channel. Cells were grown for 24 h with APAP in the presence or absence of 4-methylpyrazole (4MP), l-arginine methyl ester (L-NAME), superoxide dismutase (SOD), or ruthenium red (RuR). Drug cytotoxicity was also tested in cells pretreated overnight with V-PYRRO/NO. APAP was without effect on empty vector-transfected cells, but damaged CYP2E1-transfected cells and this was abolished by RuR, reduced by 4MP, or V-PYRRO/NO but affected by L-NAME or SOD. APAP increased microsomal [³H]-ryanodine binding, while microsomal Ca²⁺ uptake was significantly lowered. RuR increased net microsomal Ca²⁺ uptake and normalized cytosolic Ca²⁺ levels. We can conclude that neither oxidative nor nitrosative stress is relevant to APAP cytotoxicity in cultured HepG2 cells, but our results point to ryanodine receptors as a potential crucial protein in the early stages of APAP cytotoxicity.     

Influence of kaempferol,a flavonoid compound,on membrane-bound ATPases in streptozotocin-induced diabetic rats

Abstract

Context: Kaempferol is a flavonoid found in many edible plants (e.g. tea, cabbage, beans, tomato, strawberries, and grapes) and in plants or botanical products commonly used in traditional medicine. Numerous preclinical studies have shown that kaempferol have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and antidiabetic activities.

Objective: The present study investigates the effect of kaempferol on membrane-bound ATPases in erythrocytes and in liver, kidney, and heart of streptozotocin (STZ)-induced diabetic rats.

Materials and methods: Diabetes was induced into adult male albino rats of the Wistar strain, by intraperitoneal administration of STZ (40?mg/kg body weight (BW)). Kaempferol (100?mg/kg BW) or glibenclamide (600?µg/kg BW) was administered orally once daily for 45?d to normal and STZ-induced diabetic rats. The effects of kaempferol on membrane-bound ATPases (total ATPase, Na⁺/K⁺-ATPase, Ca²⁺-ATPase, and Mg²⁺-ATPase) activity in erythrocytes and in liver, kidney, and heart were determined.

Results: In our study, diabetic rats had significantly (p?<?0.05) decreased activities of total ATPases, Na⁺/K⁺-ATPase, Ca²⁺-ATPase, and Mg²⁺-ATPase in erythrocytes and tissues. Oral administration of kaempferol (100?mg/kg BW) or glibenclamide (600?µg/kg BW) for a period of 45?d resulted in significant (p?<?0.05) reversal of these enzymes' activities to near normal in erythrocytes and tissues when compared with diabetic control rats.

Discussion and conclusion: Thus, obtained results indicate that administration of kaempferol has the potential to restore deranged activity of membrane-bound ATPases in STZ-induced diabetic rats. Further detailed investigation is necessary to discover kaempferol’s action mechanism.     

Lead enters Rcho-1 trophoblastic cells by calcium transport mechanisms and complexes with cytosolic calcium-binding proteins

Within the placenta, a specialized Ca(2+) transport pathway develops in trophoblasts to promote growth of the fetus and hypothetically to enhance fetal uptake of Pb(2+). This hypothesis could not be tested until a method to monitor Pb(2+) influx by indo-1 fluorescence quench became available. We have applied this new method to cultured undifferentiated and differentiated Rcho-1 trophoblastic cells. Pb(2+) concentrations of 1 and 10 microM are equivalent to blood levels of 20 and 200 microg/dl in pregnant women. Over this range, Pb(2+) uptake increased with time and concentration in medium containing 1 mM Ca(2+) but was greater in Ca(2+)-omitted solutions. Activation of capacitative Ca(2+) entry (CCE) with thapsigargin, an endoplasmic reticulum (ER) Ca(2+) pump inhibitor, increased Pb(2+) uptake, while inhibition of CCE by La(3+) decreased influx. Parathyroid hormone-related peptide (PTHrP) stimulates the synthesis of Ca(2+)-binding proteins (CaBPs), as well as Ca(2+) transporters, during trophoblastic differentiation. Pretreatment for 72 h with PTHrP increased Pb(2+) uptake by undifferentiated Rcho-1 cells but had little effect on the quench in differentiated cells, probably due to their greater content of CaBPs which competed for Pb(2+)-binding with indo-1. This competition was most evident in differentiated cells when 1 microM Pb(2+) caused an initial quench, followed by a rise in fluorescence. This rise was not inhibited by thapsigargin, thereby ruling out sequestration into the ER and leaving complexation of Pb(2+) by CaBPs as the most plausible interpretation. We conclude that trophoblasts have the ability to clear Pb(2+) from the maternal circulation and deliver it to the fetus.     

Characteristics and possible mechanisms of low - Na+ induced contractions in rat aorta

The influence of reducing external Na⁺ concentration ([Na⁺]^ex) upon vascular smooth muscle contractility was investigated using the rat isolated aorta. NaCl from the physiological saline solution (PSS) was replaced with either choline-Cl, sucrose, or LiCl to give the following [Na⁺]_ex (mM): 115, 85, 55, and 25 (115NaPSS to 25NaPSS). Small reductions in [Na⁺]_ex (115NaPSS) induced a biphasic contraction, comparable in amplitude with the control one induced by phenylephrine 10^–6 M. Elimination of the endogenous catecholamine participation using either phentolamine 10^–5 M or guanethidine 3.10^–6 M similarly reduces these contractions to 25% (sucrose replacement). A similar relaxing effect was obtained with D600 10^–5 M, an antagonist of the voltage operated Ca²⁺ channels (25–30% residual tension for all the substitutes). Large reductions in [Na⁺]_ex (25NaPSS) induced contractions comparable in amplitude and shape, but less sensitive to phentolamine and guanethidine (residual tension 65–75 %, sucrose replacement) and insensitive to D600 (all the substitutes). The Na⁺/K⁺ ATPase inhibitor ouabain (10^–4 M) elicited slowly developing contractions, the amplitude being 115% of the phenylephrine 10^–6 M control.Phenylephrine further contracted the 115NaPSS precontracted preparations, but was significantly less effective in 25NaPSS, although the precontraction levels were similar for the same substitute used. The amplitude of the superimposed phenylephrine contractions exhibited [Na⁺]_ex dependence. Phenylephrine 10^–6 M failed to further contract the ouabain 10^–4 M precontracted rings.We conclude that relatively small reductions in [Na⁺]_ex are able to induce contractions of rat aorta primarily through release of endogenous catecholamines, probably through neural Na⁺/Ca²⁺ exchange. Larger reductions in [Na⁺]_ex appear to cause contraction through muscular Na⁺/Ca²⁺ exchange.     

Influence of Haplophyllum tuberculatum on the Cardiovascular System

The aqueous extract of H. tuberculatum significantly decreased the contractility and the heart rate but did not affect the flow rate of isolated perfused rabbit heart. This effect was not blocked by atropine; however, the muscarinic antagonist blocked the fall in blood pressure seen when the extract was administered to anaesthetized cats. The extract also stimulated rabbit aortic strip, rat vas deferens, and rat anococcygeus muscles. These adrenergic effects were largely reduced by phentolamine. The extract may contain a mixture of pharmacologically active ingredients that necessiate its separation.     

Effects of mibefradil on uterine contractility

Mibefradil, a benzimidazolyl tetralol derivative, is a new Ca(2+) channel antagonist which is structurally distinct from other Ca(2+) channel antagonists such as nifedipine, verapamil and diltiazem. It is a very effective antihypertensive agent that is thought to achieve its action via a higher affinity block for low-voltage activated (T) than for high-voltage-activated (L) Ca(2+) channels. Nevertheless, it blocks L-type Ca(2+) channels in several tissues. In the present study, the effects of mibefradil on spontaneous rhythmic contractions and on contractions elicited by CaCl(2) (K(+)-depolarized preparations) and oxytocin (in low Ca(2+)/Ca(2+)-free solutions) were investigated on uterus strips from pregnant and non-pregnant rats. Mibefradil (10(-8)-3 x 10(-6) M) caused concentration-dependent inhibition of spontaneous contractions of uterus strips from pregnant and non-pregnant rats with the IC(50) values of 8.83 x 10(-7) M; 5.94 x 10(-7) M (amplitude) and 1.03 x 10(-6) M; 5.48 x 10(-7) M (frequency), respectively. Mibefradil (3 microM) caused a rightward shift in the concentration-response curves for CaCl(2) in K(+) (40 mM)-depolarized uterus strips taken from both pregnant and non-pregnant rats. Mibefradil (3 microM) was, however, more potent for antagonising CaCl(2) responses in uterus strips obtained from pregnant rats than in those from non-pregnant rats. Mibefradil (3 microM) had no effect on oxytocin-induced contraction in Ca(2+)-free physiological salt solution (PSS) on uterus strips from non-pregnant rats. However, it markedly inhibited oxytocin-induced contraction of pregnant rat uterus strips in Ca(2+)-free PSS. Thus, mibefradil probably antagonizes L-type Ca(2+) channels as well as interferes with the intracellular Ca(2+) release mechanism, which would be helpful in the development of a tocolytic agent.     

Effects of interactions of EGCG and Cd(2+) on the growth of PC-3 cells and their mechanisms.

The preventive and therapeutic effects of a major component of catechins of green tea, epigallocatechin-3-gallate (EGCG), on prostate cancer have been demonstrated in many studies. It is well known that metal ions are necessary for human health, but an imbalance in metal ions metabolism can lead to many diseases including prostate cancer. Understanding the interactions of EGCG with metal ions might elucidate its mechanism in preventing and curing prostate cancer. The present study focused on the effects of Cd(2+) and EGCG on the growth of androgen-insensitive prostate cancer cell PC-3 investigated by MTT assay, the effects of EGCG and Cd(2+) on absorption of Cd(2+) and Zn(2+) by PC-3 cells were detected by atomic absorption spectroscopy (AAS), and the interactions of EGCG with Cd(2+) were determined by distribution coefficient and UV-Vis spectroscopy detection. The results showed that Cd(2+) suppressed viability of PC-3 cells in concentration- and time-dependent manner, and EGCG enhanced the effect of Cd(2+) on PC-3 cells. EGCG was shown to decrease the absorption Cd(2+) and increase the absorption of Zn(2+) by PC-3 cells, while the effects of Cd(2+) on the absorption of Cd(2+) and Zn(2+) were opposite to that of EGCG. In the presence of both EGCG and Cd(2+), absorption of Cd(2+) and Zn(2+) by PC-3 cells was dependent on concentrations of EGCG, Cd(2+) and its order of addition. Results from the distribution coefficient determination and UV-Vis spectroscopy analysis indicated that Cd(2+) might affect conformation of EGCG, while no complex of EGCG with Cd(2+) was observed in the system.     

Use of ion channel blockers in the exploration of possible mechanisms involved in the myopathy of diabetic mice

Summary Changes in the muscle contractions of the phrenic nerve-diaphragm preparation from the diabetic mouse were investigated by means of K⁺- and Cl^–-channel blockers and the Ca²⁺-mobilizing agent, selenite. The K⁺-channel blockers (UO₂ ²⁺ and 4-aminopyridine) cooperated synergistically with the Cl^–-channel blockers (Cd²⁺ and 9-anthracenecarboxylic acid) in increasing normal muscle contraction as described previously, but failed to induce this effect in the diaphragm of the diabetic mouse. Treatment with a Cl^–-channel blocker alone in 0.25 mmol/1 Ca²⁺ Krebs solution induced a myotonic activity accompanied by stimulus-bound repetitive action potential firings. This effect was also diminished in the diaphragm from diabetic mice. The membrane potential of the muscle cells in the diaphragm of the diabetic mouse was slightly but significantly decreased. The membrane input resistance was also increased and was refractory to being further increased by either a Cl^–-channel blocker or a low Cl^–-medium. Furthermore, the membrane chloride conductance was found to be decreased, but the membrane K+ conductance remained unchanged in the muscle from diabetic mice. These changes of membrane properties in the muscles from diabetic mice were shown to be similar to those induced by either Cl^–-channel blockers or a low Cl^–-medium. In addition, the combined treatment of the diaphragm from diabetic mice with Cd²⁺ Plus UO₂ ²⁺ in 0.25 mmol/l Ca²⁺ Krebs solution and then stepwise replenishment of Ca²⁺ led to a greater restoration of muscle contractions at a lower cumulative Ca²⁺ concentration than that was found with the normal diaphragm. The sustained muscle contracture of the mouse diaphragm induced by U0₂ ²⁺ plus selenite was partially inhibited in the diaphragm from diabetic mice, indicating that the Ca²⁺ mobilizing mechanism of the diaphragm of the diabetic mouse was also altered. All of these observations obtained with the diaphragm of the diabetic mouse can be attributed to the diabetic state, because most of them could be normalized by insulin administration in vivo. Therefore, it is concluded that diabetes-induced changes of sarcolemmal ion channels and ion transporters may cause inhibition of muscle contraction and eventually lead to diabetic myopathy. Correspondence to: S. Y. Lin-Shiau at the above address     

赛庚啶对离体犬心肌肌质网Ca2+,Mg2+—ATP酶活性和45Ca2+摄取功能的影响

当赛庚啶浓度在8×10^-6mol/L～2×10^-4mol/L之间时,该药对正常犬心肌肌质网Ca²⁺,Mg²⁺—ATP酶活性几乎没有影响,仅在10^-3mol/L时对该酶活性才有一定的抑制作用(抑制率为39.85%,P<0.01)。正常犬心肌肌质网的⁴⁵Ca²⁺摄取过程有明显的时间依赖性,至第30 min,其⁴⁵Ca²⁺摄取量可达312.79±22.25 nmol/mg protein.赛庚啶对心肌肌质网的~(45)Ca²⁺摄取有一定的抑制作用,其IC₅₀为1.94×10^-4mol/L。     

Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: > 800 U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91^phox−/− mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury.     

The glutathione reductase inhibitor carmustine induces an influx of Ca2+ in PC12 cells

We studied the effects of carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in PC12 cells using fura-2 fluorescence imaging. Carmustine (100 microM) caused a delayed increase in [Ca(2+)](i) that developed within approximately 3 h. This effect was enhanced in cells that were pretreated with an inhibitor of glutathione (GSH) synthesis, buthionine sulfoximine (BSO, 200 microM, 24 h), and was suppressed in cells that were treated with an antioxidant deferoxamine (50 microM). The carmustine-induced increase in [Ca(2+)](i) was absolutely dependent on the presence of extracellular Ca(2+) and could be inhibited by dihydropyridine blockers of L-type voltage-gated Ca(2+) channels (nimodipine or nitrendipine, 10 microM). The increase in [Ca(2+)](i) was also suppressed in Cl(-)-free solution and in the presence of the Cl(-) channel blockers, indanyloxyacetic acid 94 (IAA-94, 100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM). The inhibition was complete when the blockers were applied simultaneously with carmustine and was partial when the blockers were applied after the initial increase in [Ca(2+)](i). We conclude that carmustine induces an influx of extracellular Ca(2+) through L-type Ca(2+) channels and that this effect is mediated by oxidative stress that results from the depletion of GSH following the inhibition by carmustine of glutathione reductase.     

Effects of propylthiouracil and methylthiouracil on cyclic AMP and ion movements in rat pancreatic islets

Summary Propylthiouracil and methylthiouracil have been shown to potentiate glucose-induced insulin secretion from rat pancreatic islets: the effect of methylthiouracil being less pronounced than that of propylthiouracil. In this study the effects of these substances on cAMP levels, ⁸⁶Rb⁺ efflux, ⁴⁵Ca²⁺ net uptake, and ⁴⁵Ca²⁺ efflux were tested in isolated rat islets in order to obtain information on their possible mechanism of action. Propylthiouracil and to a lesser extent methylthiouracil increased islet cyclic AMP in a concentration-related manner. Maximum increases at the highest concentrations tested were 261% and 190% respectively. In the presence of 3 mM glucose propylthiouracil and methylthiouracil led to a decrease in the ⁸⁶Rb efflux rate. With 5.6 mM glucose, both thiourea derivatives produced an increase in the ⁸⁶Rb⁺ efflux rate which was independent of the presence or absence of calcium in the medium. Propylthiouracil and methylthiouracil augmented the ⁴⁵Ca²⁺ efflux rate in the presence as well as in the absence of external calcium at various glucose concentrations. Propylthiouracil did not change, and methylthiouracil only slightly augmented, ⁴⁵Ca²⁺ net uptake into the isolated islets. It is suggested that the synergistic effect of propylthiouracil and methylthiouracil on glucose-induced insulin release is at least in part due to an increase in islet cAMP levels. Whether the two substances have additional direct effects on ionic fluxes which contribute to their insulinotropic action or whether the observed changes in ion movements are secondary to the elevation of cAMP levels remains to be unclear and needs further investigation. Send offprint requests to H. P. T. Ammon at the above address     

Effect of celecoxib on Ca2+ movement and cell proliferation in human osteoblasts

In human osteoblasts, the effect of the widely prescribed cyclooxygenase-2 inhibitor celecoxib on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cell proliferation was explored by using fura-2 and the tetrazolium assay, respectively. Celecoxib at concentrations greater than 1microM caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner ( EC 50= 10 microM). Celecoxib-induced [Ca(2+)](i) rise was reduced by 90% by removal of extracellular Ca(2+), and by 30% by l-type Ca(2+) channel blockers. Celecoxib-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that celecoxib-induced extracellular Ca(2+) influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of celecoxib on [Ca(2+)](i) was greatly inhibited. Conversely, pretreatment with celecoxib to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). U73122, an inhibitor of phoispholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not celecoxib-induced, [Ca(2+)](i) rise. Pretreatment with phorbol 12-myristate 13-acetate and forskolin to activate protein kinase C and adenylate cyclase, respectively, partly inhibited celecoxib-induced [Ca(2+)](i) rise in Ca(2+)-containing medium. Separately, overnight treatment with 1-100microM celecoxib inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human osteoblasts, celecoxib increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing intracellular Ca(2+) release from the endoplasmic reticulum via a phospholiase C-independent manner. Celecoxib may be cytotoxic at higher concentrations.     

Effects of ouabain on human bronchial muscle in vitro

The effects of ouabain, an inhibitor of the plasmalemmal Na⁺/K⁺-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with –logEC₅₀=7.16±0.11 and maximal effect of 67±4% of the response to acetylcholine (1 mM). Ouabain (10 M)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H₁-receptors and -adrenoceptors, or neuronal Na⁺ channel blockade. The inhibition of ouabain contraction in tissues bathed in K⁺-free medium, and the inhibition by ouabain of the K⁺-induced relaxation confirm that the contractile action of ouabain is mediated by inhibition of Na⁺/K⁺-ATPase. Furthermore, depolarization (16.4±0.9 mV) was observed in human isolated bronchus by intracellular microelectrode recording. Ouabain (10 M)-induced contractions were abolished by a Ca²⁺-free solution but not by blockers of L-type Ca²⁺ channels. In human cultured bronchial smooth muscle cells, ouabain (10 M) produced a sustained increase in [Ca²⁺]_i (116±26 nM) abolished in Ca²⁺-free medium. Incubation with a Na⁺-free medium or amiloride (0.1 mM) markedly inhibited the spasmogenic effect of ouabain thus suggesting the role of Na⁺/Ca²⁺ exchange in ouabain contraction while selective inhibitors of Na⁺/H⁺-antiport, Na⁺/K⁺/Cl^–-antiport, or protein kinase C had no effect. Ouabain (10 M) failed to increase inositol phosphate accumulation in human bronchus. Ouabain (10 M) did not alter bronchial responsiveness to acetylcholine or histamine but inhibited the relaxant effects of isoprenaline, forskolin, levcromakalim, or sodium nitroprusside. These results indicate that ouabain acts directly to produce contraction of human airway smooth muscle that depends on extracellular Ca²⁺ entry unrelated to L-type channels and involving the Na⁺/Ca²⁺-antiporter.     

Effects of SEA0400 and KB-R7943 on Na+/Ca2+ exchange current and L-type Ca2+ current in canine ventricular cardiomyocytes

SEA0400 and KB-R7943 are compounds synthesised to block transsarcolemmal Na⁺/Ca²⁺ exchange current (I_Na/Ca); however, they Have also been shown to inhibit L-type Ca²⁺ current (I_Ca). The potential value of these compounds depends critically on their relative selectivity for I_Na/Ca over I_Ca. In the present work, therefore, the concentration-dependent effects of SEA0400 and KB-R7943 on I_Na/Ca and I_Ca were studied and compared in canine ventricular cardiomyocytes using the whole-cell configuration of the patch clamp technique. SEA0400 and KB-R7943 decreased I_Na/Ca in a concentration-dependent manner, having EC₅₀ values of 111±43 nM and 3.35±0.82 M, when suppressing inward currents, while the respective EC₅₀ values were estimated at 108±18 nM and 4.74±0.69 M in the case of outward current block. SEA0400 and KB-R7943 also blocked I_Ca, having comparable EC₅₀ values (3.6 M and 3.2 M, respectively). At higher concentrations (10 M) both drugs accelerated inactivation of I_Ca, retarded recovery from inactivation and shifted the voltage dependence of inactivation towards more negative voltages. The voltage dependence of activation was slightly modified by SEA0400, but not by KB-R7943. Based on the relatively good selectivity of submicromolar concentrations of SEA0400—but not KB-R7943—for I_Na/Ca over I_Ca, SEA0400 appears to be a suitable tool to study the role of I_Na/Ca in Ca²⁺ handling in canine cardiac cells. At concentrations higher than 1 M, however, I_Ca is progressively suppressed by the compound.     

Fas receptor-deficient lpr mice are protected against acetaminophen hepatotoxicity due to higher glutathione synthesis and enhanced detoxification of oxidant stress

Acetaminophen (APAP) overdose is a classical model of hepatocellular necrosis; however, the involvement of the Fas receptor in the pathophysiology remains controversial. Fas receptor-deficient (lpr) and C57BL/6 mice were treated with APAP to compare the mechanisms of hepatotoxicity. Lpr mice were partially protected against APAP hepatotoxicity as indicated by reduced plasma ALT and GDH levels and liver necrosis. Hepatic Cyp2e1 protein, adduct formation and hepatic glutathione (GSH) depletion were similar, demonstrating equivalent reactive metabolite generation. There was no difference in cytokine formation or hepatic neutrophil recruitment. Interestingly, hepatic GSH recovered faster in lpr mice than in wild type animals resulting in enhanced detoxification of reactive oxygen species. Driving the increased GSH levels, mRNA induction and protein expression of glutamate–cysteine ligase (gclc) were higher in lpr mice. Inducible nitric oxide synthase (iNOS) mRNA and protein levels at 6 h were significantly lower in lpr mice, which correlated with reduced nitrotyrosine staining. Heat shock protein 70 (Hsp70) mRNA levels were substantially higher in lpr mice after APAP. Conclusion: Our data suggest that the faster recovery of hepatic GSH levels during oxidant stress and peroxynitrite formation, reduced iNOS expression and enhanced induction of Hsp70 attenuated the susceptibility to APAP-induced cell death in lpr mice.     

Vasorelaxant and antihypertensive effects of formononetin through endothelium-dependent and -independent mechanisms

Aim:

To investigate the mechanisms underlying the vasorelaxant effect of formononetin, an O-methylated isoflavone, in isolated arteries, and its antihypertensive activity in vivo.

Methods:

Arterial rings of superior mesenteric arteries, renal arteries, cerebral basilar arteries, coronary arteries and abdominal aortas were prepared from SD rats. Isometric tension of the arterial rings was recorded using a myograph system. Arterial pressure was measured using tail-cuff method in spontaneously hypertensive rats.

Results:

Formononetin (1–300 μmol/L) elicited relaxation in arteries of the five regions that were pre-contracted by KCl (60 mmol/L), U46619 (1 μmol/L) or phenylephrine (10 μmol/L). The formononetin-induced relaxation was reduced by removal of endothelium or by pretreatment with L-NAME (100 μmol/L). Under conditions of endothelium denudation, formononetin (10, 30, and 100 μmol/L) inhibited the contraction induced by KCl and that induced by CaCl₂ in Ca²⁺-free depolarized medium. In the absence of extracellular Ca²⁺, formononetin (10, 30, and 100 μmol/L) depressed the constriction caused by phenylephrine (10 μmol/L), but did not inhibit the tonic contraction in response to the addition of CaCl₂ (2 mmol/L). The contraction caused by caffeine (30 mmol/L) was not inhibited by formononetin (100 μmol/L). Formononetin (10 and 100 μmol/L) reduced the change rate of Ca²⁺-fluorescence intensity in response to KCl (50 mmol/L). In spontaneously hypertensive rats, formononetin (5, 10, and 20 mg/kg) slowly lowered the systolic, diastolic and mean arterial pressure.

Conclusion:

Formononetin causes vasodilatation via two pathways: (1) endothelium-independent pathway, probably due to inhibition of voltage-dependent Ca²⁺ channels and intracellular Ca²⁺ release; and (2) endothelium-dependent pathway by releasing NO. Both the pathways may contribute to its antihypertensive effect.