苄普地尔对豚鼠甲亢性肥厚心肌中延迟整流钾电流的抑制作用

目的　研究苄普地尔(bepridil)对肥厚心肌细胞延迟整流钾电流(I_K)中快激活成份(I_Kr)和慢激活成份(I_Ks)及内向整流钾电流(I_K1)的作用。方法　全细胞膜片钳技术。结果　在肥厚心肌细胞中,Bepridil 30 μmol·L- 1 对I_Kr及I_Ks有阻断作用,抑制率分别为20.9% (0 mV)和27.2 % (+50 mV)。“Envelopeoftail”显示bepridil对I_Ks的阻断作用大于I_Kr。Bepridil(1 - 100 μmol·L^-1 )浓度依赖性的阻断I_Ks及I_Kr,其IC₅₀ 分别为23.8μmol·L^-1 和46.7μmol·L^-1 。Bepridil 30 μmol·L^-1 也能阻断I_K1 ,抑制率为15.1% (- 100 mV) ,但不影响其反转电位。结论　Bepridil对甲亢性豚鼠肥厚心肌中I_Ks,I_Kr及I_K1有阻断作用     

MECHANISMS FOR REGULATION OF ARTERIAL TONE BY Ca2+- DEPENDENT K+ CHANNELS IN HYPERTENSION

1. The membrane potential and reactivity of arterial smooth muscle cells is regulated by a variety of K⁺ channels, which are highly expressed in vascular smooth muscle meuscle membrances. 2. Of these K⁺ channel types, the high-conductance, Ca²⁺-ependent K⁺ channel appears to be up-regulated in arterial smooth muscle membrances from hypertensive animals. 3. Patch-clamp studies show that whole-cell membrances and membrane patches of arterial smooth muscle obtained from rats with genetic or renal hypertension show an increased macroscopic and single-channel Ca²⁺-activated K⁺ current. Pharmacological block of this K⁺ current profoundly constricts aortic, renal, mesenteric and femoral arteries obtained from the same hypertensive animals, suggesting that Ca²⁺-dependent K⁺ current is a critical determinant of resting membrane potential in arterial muscle exposed to elevated blood pressure. 4. Thus, K⁺ efflux through Ca²⁺-dependent K⁺ channels appears to constitute an important homeostatic mechanism for buffering increases in arterial reactivity in hypertension.     

INTERACTIONS OF Na+, H2O2 AND THE Na+-Ca2+ EXCHANGER STIMULATE Ca2+ RELEASE IN CK1.4 CELLS

1. The present study aimed to demonstrate that interactions of cations, hydrogen peroxide (H₂O₂) and the Na⁺-Ca²⁺exchanger stimulate Ca²⁺ release and oscillations of cytosolic Ca²⁺ [Ca²⁺]i in non-transfected Chinese Hamster Ovary (CHO) C1 cells and in transfected CHO (CK1.4) cells that contained an expression vector coding the Na⁺-Ca²⁺ exchanger sequence. 2. The [⁴⁵Ca²⁺] uptake assay, fura-2 fluorescence imaging and 2² and 2³ factorial orthogonal statistics provide comparative, direct, efficient, quantitative and transient methods to delineate the effects of such interactions on Ca²⁺ influx, Ca²⁺release and [Ca²⁺]i in C1 and CK1.4 cells. 3. In contrast to the control of either Na⁺-, Ca²⁺- or H₂O₂-free or CI cells, an elevated [⁴⁵Ca²⁺] uptake was induced by Ca²⁺, Na⁺ and H₂O₂ individually and in combination, intracellular Ca²⁺ release was activated by H₂O₂ and by combinations of either H₂O₂ and Na⁺, H₂O₂ and the Na⁺-Ca²⁺ exchanger, Na⁺ and the Na⁺-Ca²⁺ exchanger or by H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger and a rise in [Ca²⁺]i was triggered by H₂O₂, Na⁺ and a combination of Na⁺ and the Na⁺-Ca²⁺exchanger. 4. These results indicate that interactions between H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger stimulate intracellular Ca²⁺mobilization via Ca²⁺-induced Ca²⁺ release mechanisms, ATP-activated G-protein coupled P_2y-purinoceptor-sensitive pathways, Na⁺-Ca²⁺ exchanger-mediated Ca²⁺ influx and cation-π interaction (a strong non-covalent force between the cation and the π face of an aromatic structure in the transmembrane protein). 5. The present findings provide important clues for understanding Ca²⁺ signal transduction mechanisms from the plasma membrane to the endoplasmic reticulum.     

EFFECTS OF ATRIAL NATRIURETIC FACTOR ON CYCLIC GMP CONTENT IN THE RAT AORTIC SMOOTH MUSCLE: STUDIES ON THE ROLE OF MEMBRANE Na+,K+-ATPase

1. These studies were conducted to determine whether preservation of the functional integrity of the membrane, Na+,K+-stimulated ATPase is essential for the atrial natriuretic factor (r-ANF-8-33) to enhance guanosine 3',5'-monophosphate (cGMP) content in the rat aortic smooth muscle. In freshly dissected rat aortic tissues, levels of cGMP were estimated using radioimmunoassay. 2. ANF (0.1 mumol/L in Krebs-Henseleit media) produced significant elevation in cGMP levels in the aortic smooth muscle when compared with that incubated in the control media, whereas suppression of Na+-pump with ouabain (1.0 mmol/L) and/or K+-free media did not produce any significant changes in the basal cGMP level; these two experimental manoeuvres did not prevent enhancement of cGMP by ANF. 3. Incubation of the tissues in the media containing ouabain plus vasoconstrictor concentrations of norepinephrine (0.3 mumol/L) also did not alter basal cGMP levels and did not prevent the ability of ANF to elevate cGMP. 4. These studies demonstrate that the antagonism by ouabain, of vasorelaxant effects of ANF (as reported in the literature) are not due to the prevention of the ability of ANF to enhance cGMP levels in the arterial smooth muscle. It is proposed that such an antagonism may be related to the actions of ouabain and ANF on diverse, and perhaps independent, mechanisms which affect Ca2+-fluxes across the cell membrane.     

NEUROPROTECTION IN VITRO AND IN VIVO BY CELL MEMBRANE-PERMEANT Ca2+ CHELATORS

1. A method of attenuating excitotoxic and hypoxic/ ischaemic neurodegeneration in vitro and in vivo using cell-permeant Ca²⁺-chelating agents is described. 2. The mechanism of neuroprotection may depend on both pre- and post-synaptic effects on intracellular Ca²⁺ dynamics. 3. This method is unique, because it targets Ca²⁺ ions, the presumed triggers of neurodegeneration, rather than a specific cellular receptor.     

RELATIVE CONTRIBUTIONS OF EXTRACELLULAR Ca2+ AND Ca2+ STORES TO SMOOTH MUSCLE CONTRACTION IN ARTERIES AND ARTERIOLES OF RAT, GUINEA-PIG DOG AND RABBIT

1. These studies describe the functional effects of modulation of the sarcoplasmic reticulum (SR) Ca²⁺ stores at three levels of the vasculature: (i) large arteries (rat and guinea-pig aorta); (ii) small resistance arteries (rat tail artery, rabbit mesenteric artery, dog mesenteric artery); and (iii) arterioles (guinea-pig submucosal arterioles of the small intestine). 2. All tissues responded to phenylephrine (PE; 10 μmol/L) with a transient contraction in Ca²⁺-free Krebs', reflecting Ca²⁺ release from PE-sensitive Ca²⁺ stores. After pretreatment with cyclopiazonic acid (CPA; 30 μmol/L) or thapsigargin (TSG; 1 μmol/L), putative SR Ca²⁺ pump inhibitors, the PE-induced contraction in a Ca²⁺-free medium was significantly inhibited in arterial tissues at all levels of the vasculature. Similarly, ryanodine (RYA; 30 μmol/L), an agonist that enhances Ca²⁺ release from the SR, also reduced the PE contraction in a Ca²⁺-free solution. 3. CPA or TSG alone in the presence of extracellular Ca²⁺, caused marked and sustained contraction in the rat and guinea-pig aorta and marked but transient or no contraction in the resistance arteries. In the rat and guinea-pig aorta, RYA caused a slowly developing tension. Little increase in basal tension was produced by RYA in resistance arteries and arterioles. 4. The findings show that an agonist-releasable Ca²⁺ pool is present at all levels of the vasculature that is independent of the size of the vessels and suggest that under normal physiological conditions there is an intimate balance between the roles of the plasma membrane and of the SR in the maintenance of vascular contractility. It appears that the role of the SR diminishes as the arteries become smaller, while Ca²⁺ fluxes across the plasma membrane predominates.     

EFFECTS OF INTRACELLULAR Ca2+ CHELATING ON NORADRENALINE RELEASE IN NORMOXIC AND ANOXIC HEARTS

1. Ischaemia and anoxia induce excessive noradrenaline (NA) release in the heart by a mechanism independent of both nerve activity and extracellular Ca²⁺. The present study was designed to examine the potential role of intracellular Ca²⁺ mobilization in anoxic NA release in the heart by chelating intracellular free Ca²⁺. 2. In normoxic hearts, preloading with an intracellular fre. Ca²⁺ chelator (BAPTA) reduced neuronal NA release by 65%, confirming the effectiveness of the loading protocol. Release of NA independent of nerve activity occurred in hearts subjected to a 40 min period of anoxic, substrate-free and nominal Ca²⁺-free perfusion. Loading hearts with BAPTA prior to anoxia failed to reduce NA overflow (1561 ± 147 vs 1496 ± 206 pmol/g over 40 min). Infusion with BAPTA (20 μmol/L) during the first 25 min of the anoxic period reduced the quantity of anoxic NA release by approximately 25% from 2013 ± 124 to 1476 ± 207 pmol/g (P < 0.05). 3. Our results confirm that anoxic NA release is predominantly. Ca²⁺-independent process with Ca²⁺ mobilization from endogenous storage playing only a minor contributing role.     

INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF Na+/Ca2+ EXCHANGER IN BOVINE ADRENAL CHROMAFFIN CELLS

• 1 The Na⁺/Ca²⁺ exchanger (NCX) exchanges Na+ and Ca²⁺ bidirectionally through the forward mode (Ca²⁺ extrusion) or the reverse mode (Ca²⁺ influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na⁺‐loaded cells were prepared by treatment with 100 µmol/L ouabain and 50 µmol/L veratridine. Incubation of Na⁺‐loaded cells with Na⁺‐free solution in the presence of the Ca²⁺ channel blockers nicardipine (3 µmol/L) and ω‐conotoxin MVIIC (0.3 µmol/L) caused Ca²⁺ uptake and catecholamine release.
• 2 The Na⁺‐dependent Ca²⁺ uptake and catecholamine release were inhibited by 2‐[4‐[(2,5‐difluorophenyl)methoxy]phenoxy]‐5‐ethoxyaniline (SEA0400; 1 µmol/L) and 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]isothiourea (KB‐R7943; 10 µmol/L), both NCX inhibitors. These results indicate that the Na⁺‐dependent responses are mostly due to activation of the NCX working in the reverse mode.
• 3 In addition, we examined the effects of PKC inhibitors and an activator on the NCX‐mediated Ca²⁺ uptake and catecholamine release. Bisindolylmaleimide I (0.3–10 µmol/L) and chelerythrine (3–100 µmol/L), both PKC inhibitors, inhibited NCX‐mediated responses. In contrast, phorbol 12,13‐dibutyrate (0.1–10 µmol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na⁺‐dependent catecholamine release, had a little or no effect on high K⁺‐induced catecholamine release in intact cells or on Ca²⁺‐induced catecholamine release in β‐escin‐permeabilized cells.
• 4 These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.

     

STUDIES ON AGE-RELATED CHANGES IN VASCULAR SMOOTH MUSCLE AND THEIR Ca2+ MECHANISMS

1. Increases in cell proliferation and DNA synthesis were observed in vascular smooth muscle cells (VSMC) from old rats. These effects were significantly enhanced by noradrenaline but were inhibited by nifedipine. 2. Ginsenosides, traditional Chinese drugs, inhibited the proliferation and DNA synthesis in VSMC from old rats. Cytosolic and nuclear Ca2+ levels, as well as calmodulin activity, were clearly higher in old rats compared with young rats. 3. Both Ca2+ and calmodulin levels rose abruptly in the late G1 phase in the VSMC cell cycle in old rats. 4. The increase in inositol phosphate levels stimulated by phenylephrine in VSMC was greater in old than in young rats. 5. The platelet-derived growth factor (PDGF) gene was overexpressed in old rats. The results indicate that vascular ageing is related to enhanced proliferation of VSMC. Abnormal Ca2+ homeostasis as well as overexpression of the PDGF gene may be responsible. 6. Nifedipine and ginsenosides may inhibit VSMC proliferation with age.     

ROLE OF EXTRACELLULAR Na+, Ca2+-ACTIVATED Cl- CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE-DEPLETED TRACHEAL SMOOTH MUSCLE

• 1 In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re‐addition of Ca²⁺ in an in vitro experimental model in which Ca²⁺ stores had been depleted and their refilling had been blocked by thapsigargin.
• 2 Mean (±SEM) contraction was diminished by: (i) inhibitors of store‐operated calcium channels (SOCC), namely 100  µ mol/L SKF‐96365 and 100  µ mol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca²⁺ channels Ca_V1.2 channels, namely 1  µ mol/L nifedipine and 10  µ mol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20  µ mol/L niflumic acid, a non‐selective inhibitor of Ca²⁺‐dependent Cl^? channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3‐fold by 100 nmol/L iberiotoxin, a blocker of the large‐conductance Ca²⁺‐activated K⁺ (BK) channels.
• 3 Furthermore, contraction was significantly inhibited when Na⁺ in the bathing solution was replaced by N‐methyl–d ‐glucamine (NMDG⁺) to 39.9 ± 7.2% of control, but not when it was replaced by Li⁺ (114.5 ± 24.4% of control). In addition, when Na⁺ had been replaced by NMDG⁺, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na⁺ had been replaced by Li⁺ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control).
• 4 In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca_V1.2 channels in the contractions induced by the re‐addition of Ca²⁺ to the solution bathing guinea‐pig tracheal rings under conditions of Ca²⁺‐depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na⁺, suggesting a role for SOCC in mediating the Na⁺ influx.

     

苄基四氢巴马汀对表达于非洲爪蟾卵母细胞及中华大蟾蜍卵母细胞的延迟整流钾电流的抑制作用

苄基四氢巴马汀对表达于非洲爪蟾卵母细胞及中华大蟾蜍卵母细胞的延迟整流钾电流的抑制作用童秋生，夏国瑾，姚伟星，江明性，白小川，包永德（同济医科大学基础医学院药理学教研室，武汉４３００３０；中国科学院上海生理学研究所，上海２０００３１）苄基四氢巴马汀（ｂ...     

EFFECTS OF CA2+ ANTAGONISTS ON CARDIOVASCULAR RESPONSES TO POSTERIOR HYPOTHALAMIC STIMULATION IN THE RAT

The effects of methoxyverapamil or hydralazine on pressor responses to posterior hypothalamic stimulation and injected pressor agents were studied in normotensive male Wistar rats. Methoxyverapamil inhibited both phases of pressor responses to hypothalamic stimulation and pressor responses to injected noradrenaline or angiotensin II. Hydralazine inhibited the secondary phase (due to adrenomedullary catecholamine) and not the primary phase (due to increased sympathetic vasomotor activity) of pressor response to hypothalamic stimulation. However, it inhibited the pressor responses to exogenous noradrenaline or angiotensin II. The data indicate that hydralazine is ineffective in inhibiting the pressor response elicited by noradrenaline endogenously released at the sympathetic nerve endings.     

INTERACTION OF THE NON-STEROIDAL ANTIINFLAMMATORY DRUG FLUFENAMIC ACID WITH GASTRIC ACID SECRETION AND H+/K+ -ATPase

1. The effects of the non-steroidal anti-inflammatory drug (NSAID) flufenamic acid on H+ production in isolated and enriched guinea-pig parietal cells and on H+/K(+)-ATPase activity in ion-tight inside-out membrane vesicles from pig gastric mucosa were studied. 2. At low concentrations (0.1 and 1.0 mumol/L), flufenamic acid increased the secretory response of parietal cells to dibutyryl cyclic AMP (dbcAMP). At higher concentrations (10 and 100 mumol/L) it progressively inhibited basal and dbcAMP-stimulated acid production. 3. Flufenamic acid (10 mumol/L) increased K+ (0.5-10.0 mmol/L) and K+ (0.5-1.0 mmol/L) plus gramicidin-stimulated ATPase activity in gastric membrane vesicles. The Km value for K+ (1.6 and 1.0 mmol/L in the absence and presence of gramicidin, respectively) was decreased to 0.8 and 0.5 mmol/L, respectively. At higher concentrations (greater than or equal to 50 mumol/L), flufenamic acid inhibited K+ plus gramicidin-stimulated ATPase activity (inhibited concentration at 50% [IC50] = 186 mumol/L) and reduced the proton concentration (IC50 = 50 mumol/L). 4. It is concluded that flufenamic acid-induced enhancement of dibutyryl cyclic AMP-stimulated H+ production in the parietal cell reflects the stimulation of H+/K(+)-ATPase. We suggest that activation of the enzyme involves increased affinity of K+ towards the K(+)-binding site of the enzyme and/or increased KCl permeability at the vesicle membrane. The inhibitory action of the drug on H+ production in parietal cells results from a detergent and/or protonophoric-like action at the apical parietal cell membrane, and from inhibition of H+/K(+)-ATPase activity.     

人参皂甙Rb1降低细胞内Ca2+作用的机制

使用荧光探针Fura-2/AM,采用双波长荧光分光光度法观察到,人参皂甙Rb₁(10,50,100μmol·L^-1)能剂量依赖性减少新生鼠脑细胞内钙浓度,并能增加由硫酸亚铁及半胱氨酸所降低的膜流动性,Rb₁(10μmol·L^-1)能使离体大鼠尾动脉去甲肾上腺素量—效曲线右移,最大效应降低;Rb₁(10,100μmol·L^-1)能降低离体鼠基底动脉5-HT所引起的收缩。使用全细胞膜片钳技术发现人参皂甙Rb₁(50,100μmol·L^-1)对钙电流无明显影响;Rb₁在低剂量能增加大鼠突触体Na⁺-K⁺ATPase及Ca²⁺-Mg²⁺ATPase活性。从而揭示Rb₁降低胞内钙含量可能通过增加ATP酶活性而产生。     

THE MEASUREMENT OF PLASMA THROMBOXANE B2 AND THE EFFECT OF SMOKING

Plasma thromboxane B2 (TxB2) was measured by radioimmunoassay using an iodinated ligand following extraction and further purification by thin layer chromatography. Venous blood was sampled into a syringe containing the cyclooxygenase inhibitor meclofenamate. Normal levels, 15 pg/ml (s.d. = 8, n = 21), were lower than usually reported and measured values increased several fold over 20 min sampling from an indwelling needle. With appropriate sampling there was a statistically insignificant increase in plasma TxB2 after subjects smoked two cigarettes (n = 11). An average decrease occurred in a control group (n = 10) and the difference between groups was of borderline significance (P less than 0.05). Smoking did not change TxB2 production associated with platelet aggregation induced in vitro by collagen, whereas plasma adrenaline increased significantly. The results emphasize the importance of the technique of sampling and assay in the measurement of plasma TxB2.     

EFFECTS OF HISTAMINE BOLUS INJECTIONS AND CONTINUOUS INFUSIONS ON THE H1- AND H2-RECEPTORS IN THE HINDLIMB VESSELS OF THE RABBIT

1. Hindlimb vascular resistance (HVR) was continuously measured after pharmacological block of the autonomic effectors in unanaesthetized rabbits with previously implanted Doppler ultrasonic flowmeters. 2. Histamine bolus injections caused a dose-related short lived fall in HVR followed by a more sustained rise. The fall was due to H₂-receptor stimulation (blocked by burimamide or metiamide) and the rise to H₁-receptor stimulation (blocked by mepyramine). At the doses of histamine tested the magnitude of the H₁-mediated vasocoiistriction had a larger peak effect than the H₂-mediated vasodilatation. 3. Histamine infusions up to 200 μg kg^?1 min^?1 did not alter HVR significantly but both increases and decreases in HVR were observed after giving H₂- or H₁-antagonists, respectively. 4. From the double reciprocal plots of 1/peak HVR change and 1/dose of histamine the magnitude of the predicted H₁- and H₂-mediated peak HVR effects at large doses were the same. This suggested that the number of H₁-_- and H₂-receptors were similar in the hindlimb vascular bed, in agreement with the infusion data.