眼睛蛇毒心脏毒素对大鼠心肌细胞收缩及细胞内钙离子的作用

目的研究眼睛蛇毒心脏毒素(Cardiotoxin,CTX)对心肌细胞的形态、收缩幅度和细胞内钙离子([Ca²⁺]_i)的作用。方法应用荧光计量法(以Fura-2/AM为荧光染料)及光学成像系统来测定单个心肌细胞[Ca²⁺]_i和收缩幅度。结果0.001～1μmol/L的CTX使心肌细胞由杆状变成圆形,药物的作用从第1分钟时开始,到第20分钟时趋于稳定。在电刺激存在的情况下,1μmol/L的CTX最初导致电诱导的[Ca²⁺]_i和收缩幅度瞬间增加,接下来[Ca²⁺]_i时程延长,最终细胞对电刺激不敏感、突然收缩、[Ca²⁺]_i持续增高。在缺乏电刺激的情况下,1μmol/L的CTX可诱导Ca²⁺震荡波、持续性[Ca²⁺]_i增高,这种作用与40mmol/L的KC l和10mmol/L咖啡因所引起的[Ca²⁺]_i瞬间增加不同。结论CTX作用初期使[Ca²⁺]_i增高,使细胞[Ca²⁺]_i超载,同时伴随细胞形状的改变。     

Multiple effects of 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca2+ signaling in MDCK cells: depletion of thapsigargin-sensitive Ca2+ store followed by capacitative Ca2+ entry, activation of a direct Ca2+ entry, and inhibition of thapsigargin-induced capacitative Ca2+ entry

The effect of 1-[β-[3-(4-methoxyphenyl)pro- poxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was examined. SKF 96365 at 25–100 μM evoked a robust [Ca²⁺]_i transient in a dose-dependent manner, measured by fura-2 fluorimetry. A concentration of 10 μM SKF 96365 did not have an effect. The transient consisted of a slow rise, a gradual decay, and a sustained plateau in physiological Ca²⁺ medium. Removal of extracellular Ca²⁺ reduced the Ca²⁺ signals evoked by 50–100 μM SKF 96365 by nearly half in the area under the curve, suggesting that SKF 96365 induced intracellular Ca²⁺ release and also extracellular Ca²⁺ influx. A concentration of 100 μM SKF 96365 caused significant Mn²⁺ quench of fura-2 fluorescence, which was partly inhibited by La³⁺ (1 mM) or Gd³⁺ (0.1 mM), indicating that the SKF 96365-induced Ca²⁺ influx had two components: one is sensitive to La³⁺ (1 mM) or Gd³⁺ (0.1 mM), the other is not. The internal Ca²⁺ source for the SKF 96365-induced [Ca²⁺]_i transient was the endoplasmic reticulum Ca²⁺ store because, pretreatment with thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca²⁺ pump nearly abolished the SKF 96365-induced [Ca²⁺]_i increase in Ca²⁺-free medium. In contrast, pretreatment with 100 μM SKF 96365 only partly depleted the thapsigargin-sensitive Ca²⁺ store. Addition of 10 mM Ca²⁺ induced a significant [Ca²⁺]_i increase after prior incubation with 100 μM SKF 96365 in Ca²⁺-free medium, demonstrating that SKF 96365 induced capacitative Ca²⁺ entry. This capacitative Ca²⁺ entry was about 40% of that induced by 1 μM thapsigargin. Additional to inducing its own capacitative Ca²⁺ entry, 100 μM SKF 96365 partly inhibited thapsigargin- or uridine trisphos-phate (UTP)-induced capacitative Ca²⁺ entry. We also investigated the mechanisms underlying the decay of the SKF 96365-induced [Ca²⁺]_i transient. Inhibition of the plasma membrane Ca²⁺ pump with La³⁺ or Gd³⁺, or lowering extracellular Na⁺ level to 0.35 mM, significantly increased the SKF 96365-induced [Ca²⁺]_i transient. In contrast, the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone had little effect. In Ca²⁺-free medium, the thapsigargin-induced [Ca²⁺]_i increase was greatly reduced by pretreatment with SKF 96365. Collectively, we have found that besides its well-known inhibitory action on capacitative Ca²⁺ entry in many cell types, in MDCK cells SKF 96365 exerted multiple and complex effects on Ca²⁺ signaling. It induced a considerable increase in [Ca²⁺]_i by releasing Ca²⁺ from the endoplasmic reticulum store followed by capacitative Ca²⁺ entry. It also caused a direct Ca²⁺ entry. The decay of the SKF 96365 response was significantly governed by efflux via the plasma membrane Ca²⁺ pump or Na⁺/Ca²⁺ exchange. Sequestration by mitochondria or the endoplasmic reticulum played a minor role. We caution use of SKF 96365 as an inhibitor of capacitative Ca²⁺ entry. Received: 21 September 1998 / Accepted: 2 December 1998     

神经肽DGAVP和Org2766对神经细胞内游离Ca2+的影响

运用Ca²⁺指示剂Fura-2作为细胞内钙离子的荧光探针,利用AR—CM—MIC阳离子测定系统,检测了分离的神经细胞内游离钙及其变化,并观测了DGAVP和Org2766对蛋白质合成抑制剂茴香霉素(ANI)引起细胞内钙离子浓度([Ca²⁺]_i)变化的影响。结果表明茴香霉素可使[Ca²⁺]_i显著升高,且有量效关系;DGAVP本身并不引起[Ca²⁺]_i发生显著变化,但适当剂量的DGAVP可显著对抗一定剂量范围内ANI升高[Ca²⁺]_i的作用,提示DGAVP对抗ANI的蛋白质合成抑制效应可能是通过拮抗ANI升高[Ca²⁺]_i这一途径实现的,另一神经肽Org2766则可能不是通过这一机制发生作用。从细胞内Ca²⁺的角度看,这两种肽的作用机理显然是不同的。     

马尾松花粉硫酸酯化多糖对大鼠主动脉平滑肌细胞 [Ca2+]i调控及增殖的影响

目的 研究马尾松花粉多糖PPM60-A及其硫酸酯化物SPPM60-A对大鼠动脉平滑肌细胞 [Ca²⁺]_i调控及增殖的影响。方法 常规水提醇沉法制备马尾松花粉多糖,Sephacryl S-400HR色谱分离得PPM60-A,氯磺酸-吡啶法得硫酸酯化物SPPM60-A,酯化度为1.28。酶解法分离制备大鼠动脉平滑肌细胞,测定酯化前后多糖对其胞内 [Ca²⁺]_i和细胞增殖的影响。结果 PPM60-A和SPPM60-A均可以降低 [Ca²⁺]_i,抑制高K⁺和去甲肾上腺素（NE）诱导的钙离子升高,降低高K⁺引起的钙离子水平上升,对NE诱导的血管主动脉平滑肌细胞增殖具有显著的抑制作用。PPM60-A作用效果好于SPPM60-A。结论 PPM60-A及SPPM60-A均能抑制细胞外Ca²⁺内流,抑制血管平滑肌细胞增殖。     

毒毛旋花子苷元对豚鼠心室肌细胞内钙浓度的影响

观察毒毛旋花子苷元(strophanthidin, Str)对分离豚鼠心室肌细胞内游离钙浓度(［Ca²⁺］_i)的影响。酶解分离豚鼠心室肌细胞, 用Fluo 3-AM负载, 激光共聚焦显微镜法测定单个豚鼠心室肌细胞［Ca²⁺］_i的荧光密度。Str可浓度依赖性地升高［Ca²⁺］_i, Str (10 μmol·L^-1)在［Ca²⁺］_i升高达峰值时, 可使细胞挛缩, 而Str (1和10 nmol·L^-1)对细胞形态无影响。TTX、 尼索地平或升高细胞外钙可影响Str (1和100 nmol·L^-1)对［Ca²⁺］_i的升高作用,而对Str (10 μmol·L^-1)无明显影响。在外液中加入ryanodine或去除细胞外钙, 则3个检测浓度的Str升高［Ca²⁺］_i作用均被明显抑制。在无K⁺、 无Na⁺液中, 10 μmol·L^-1 Str升高［Ca²⁺］_i的作用减弱, 而Str (1和100 nmol·L^-1)升高［Ca²⁺］_i的作用无明显影响。加入TTX、 尼索地平或增加细胞外的钙离子浓度, 则3个检测浓度Str的作用均受到影响。提示低浓度Str对［Ca²⁺］_i的升高作用与抑制Na⁺、K⁺-ATP酶活性无关, 而与促进L-型钙通道和TTX敏感性钠通道的“slip-mode”钙电导有关; 高浓度Str升高［Ca²⁺］_i的作用则是抑制Na⁺、K⁺-ATP酶的结果。此外, Str对［Ca²⁺］_i的升高作用还与直接作用于ryanodine受体促进内钙释放有关。     

四肽FMRFa对大鼠心室肌Na+/Ca2+交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na⁺/Ca²⁺交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na⁺/Ca²⁺交换电流(I_Na⁺/Ca²⁺)和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞I_Na⁺/Ca²⁺呈浓度依赖性抑制,100μmol·L^-1浓度时抑制内向和外向I_Na⁺/Ca²⁺密度分别达60.1%和56.5%,对内向电流及外向电流的IC₅₀分别为20μmol·L^-1和34μmol·L^-1。FMRFa5μmol·L^-1抑制I_Na⁺/Ca²⁺内向和外向电流密度分别为38.7%和34.9%,但FMRFa5μmol·L^-1及20μmol·L^-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na⁺/Ca²⁺交换抑制剂。     

银杏叶提取物对低氧复氧、H2O2和谷氨酸损伤时谷氨酸引起的大鼠星形胶质细胞［Ca2+］i变化的影响

目的研究银杏叶提取物对低氧复氧、H₂O₂和L-谷氨酸损伤时谷氨酸升高大鼠星形胶质细胞［Ca²⁺］_i的影响。方法钙荧光探针Fluo-3/AM标记胞浆内游离钙离子，激光扫描共聚焦显微镜测定［Ca²⁺］_i的变化。结果 在低氧复氧、H₂O₂以及高浓度的L-谷氨酸损伤后，外源性谷氨酸（27 μmol·L^-1）均不能引起培养乳大鼠星形胶质细胞正常的［Ca²⁺］_i升高，反而使［Ca²⁺］_i分别降低(3.3±1.6)%，(81±11)%和(81±7)%；损伤前预先给予GbE（10 mg·L^-1）不能明显改善星形胶质细胞的谷氨酸反应，但预先给予GbE（100 mg·L^-1）后，27 μmol·L^-1谷氨酸可使损伤的星形胶质细胞［Ca²⁺］_i分别升高(135±98)%，(117±93)%和(89±36)%。结论低氧复氧、H₂O₂以及高浓度的L-谷氨酸均能损伤星形胶质细胞的谷氨酸反应，影响神经细胞与胶质细胞的双向交流。GbE能明显逆转不同损伤后谷氨酸诱导星形胶质细胞［Ca²⁺］_i的异常变化，使星形胶质细胞在不同损伤时能维持正常功能，该作用可能与GbE的脑保护作用有关。     

小檗碱对培养大鼠心肌细胞胞内游离Ca2+的作用

利用Fura-2技术和AR-CM-MIC阳离子测定系统,直接观察了小檗碱对培养大鼠心肌细胞胞内[Ca²⁺]i的影响。结果显示:小檗碱可明显升高心肌细胞静息[Ca²⁺]i且具饱合性,维拉帕米和CoCl₂对其有一定的抑制作用;小檗碱与高K⁺,高Ca²⁺,去甲肾上腺素,哇巴因合用比单用上述激动剂更能明显增高[Ca²⁺]i;维拉帕米对其有抑制作用;在胞外无外Ca²⁺和无外Ca²⁺,外K⁺,外Na⁺时,小檗碱30～200μmol·L^-1仍能升高静息[Ca²⁺]i,维拉帕米只对前者有一定抑制作用。结果提示:小檗碱可能通过促胞外Ca²⁺内流和胞内Ca²⁺释放等途径有限度地增高心肌细胞内游离Ca²⁺浓度,显示强心作用。     

间硝苯地平对老龄肾性高血压大鼠心肌和脑微粒体Na+,K+-ATP酶,Ca2+-ATP酶和Mg2+-ATP酶活性的影响

间硝苯地平(m-Nif,ig20mg·kg^-1·d^-1持续给药9周)可显著降低老龄肾性高血压大鼠(RVHR)血压和左室重量(P<0.01),增高心、脑微粒体Na⁺,K⁺-ATP酶和Ca²⁺-ATP酶活性(P<0.01),降低Mg²⁺-ATP酶活性,体外量效关系研究发现,m-Nif在较高剂量(10～1000μmol·L^-1)时可增高RVHR心脑微粒体Na⁺,K⁺-ATP酶和Ca²⁺-ATP酶活性,且随剂量增加而增高。上述结果表明,m-Nif可改善老龄RVHR心脑微粒体Na⁺,K⁺泵和Ca²⁺泵功能。     

前胡丙素对培养大鼠心肌细胞内游离Ca2+的影响

用Fura-2/AM技术直接观察前胡丙素(Pra-C)对培养大鼠心室肌细胞内游离钙的影响。结果显示Pra-C浓度为0.1～1.0μmol·L^-1可明显抑制CaCl₂,高K⁺和Bay K 8644引起[Ca²⁺]i增加,并且有剂量—效应关系,对ouabain引起的[Ca²⁺]i增加无明显作用。结果提示Pra-C降低心肌细胞[Ca²⁺]i的作用与抑制电压依赖性钙通道有关。     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

Callipeltin A: sodium ionophore effect and tension development in vascular smooth muscle

Callipeltin A is a cyclic depsidecapeptide isolated from the marine sponges Callipelta sp. and Latrunculia sp. that has been previously shown to increase the force of contraction of guinea-pig atria through the inhibition of Na+/Ca2+ exchanger (NCX). We investigated the effect of callipeltin A on guinea-pig aortic rings contracted by procedures that activate NCX in "calcium entry mode". Callipeltin A did not inhibit these contractions. Resting aorta responded to callipeltin A with a remarkable contraction that was concentration-dependent (EC50 0.44microM). This contraction was not inhibited by the calcium channel blocker verapamil and was not mediated by the activation of alpha-adrenergic or endothelin-1 receptors. Pre-incubation of aortic rings with 0.5mM amiloride, an inhibitor of NCX, completely prevented callipeltin A-induced contraction. Furthermore, callipeltin A (EC50 0.51microM) increased Na+ efflux of Na-loaded erythrocytes. 1H and 13C NMR resonances of callipeltin A revealed small but significant changes in the titration with K+ and Na+ salts. It is suggested that the effect of callipeltin A on cardiac and vascular preparations is linked to a Na-ionophore action.     

Protective Effect of Liriodendrin Isolated from Kalopanax pictus against Gastric Injury

In this study, we investigated the inhibitory activities on gastritis and gastric ulcer using liriodendrin which is a constituent isolated from Kalopanax pictus. To elucidate its abilities to prevent gastric injury, we measured the quantity of prostaglandin E₂ (PGE₂) as the protective factor, and we assessed inhibition of activities related to excessive gastric acid be notorious for aggressive factor and inhibition of Helicobacter pylori (H. pylori) colonization known as a cause of chronic gastritis, gastric ulcer, and gastric cancer. Liriodendrin exhibited higher PGE₂ level than rebamipide used as a positive control group at the dose of 500 μM. It was also exhibited acid-neutralizing capacity (10.3%) and H⁺/K⁺-ATPase inhibition of 42.6% (500 μM). In pylorus-ligated rats, liriodendrin showed lower volume of gastric juice (4.38 ± 2.14 ml), slightly higher pH (1.53 ± 0.41), and smaller total acid output (0.47 ± 0.3 mEq/4 hrs) than the control group. Furthermore liriodendrin inhibited colonization of H. pylori effectively. In vivo test, liriodendrin significantly inhibited both of HCl/EtOH-induced gastritis (46.9 %) and indomethacin-induced gastric ulcer (46.1%). From these results, we suggest that liriodendrin could be utilized for the treatment and/or protection of gastritis and gastric ulcer.     

Gastroprotective Activities of Sennoside A and Sennoside B via the Up-Regulation of Prostaglandin E2 and the Inhibition of H+/K+-ATPase

Sennoside A (erythro) and sennoside B (threo) are dianthrone glycosides and diastereomers. We investigated their abilities to prevent the gastric lesions associated with diseases, such as, gastritis and gastric ulcer. To elucidate their gastroprotective effects, the inhibitions of HCl•EtOH-induced gastritis and indomethacin-induced gastric ulcers were assessed in rats. It was observed that both sennoside A and sennoside B increased prostaglandin E₂ (PGE₂) levels and inhibited H⁺/K⁺-ATPase (proton pump). In a rat model, both compounds reduced gastric juice, total acidity and increased pH, indicating that proton pump inhibition reduces gastric acid secretion. Furthermore, sennoside A and B increased PGE₂ in a concentration-dependent manner. In a gastric emptying and intestinal transporting rate experiment, both sennoside A and sennoside B accelerated motility. Our results thus suggest that sennoside A and sennoside B possess significant gastroprotective activities and they might be useful for the treatment of gastric disease.     

Characterization of the K+-channel-coupled adenosine receptor in guinea pig atria

Summary In the present work we studied the pharmacological profile of adenosine receptors in guinea pig atria by investigating the effect of different adenosine analogues on⁸⁶Rb⁺-efflux from isolated left atria and on binding of the antagonist radioligand 8-cyclopentyl-1,3-[³H]dipropylxanthine ([³H]DPCPX) to atrial membrane preparations. The rate of⁸⁶Rb⁺-efflux was increased twofold by the maximally effective concentrations of adenosine receptor agonists. The EC₅₀-values for 2-chloro-N⁶-cyclopentyladenosine (CCPA), R-N⁶-phenylisopropyladenosine (R-PIA), 5-N-ethylcarboxamidoadenosine (NECA), and S-N⁶-phenylisopropyladenosine (S-PIA) were 0.10, 0.14, 0.24 and 12.9 M, respectively. DPCPX shifted the R-PIA concentration-response curve to the right in a concentration-dependent manner with a K_B-value of 8.1 nM, indicating competitive antagonism. [³H]DPCPX showed a saturable binding to atrial membranes with a B_max-value of 227 fmol/mg protein and a K_D-value of 1.3 nM. Competition experiments showed a similar potency for the three agonists CCPA, R-PIA and NECA. S-PIA is 200 times less potent than R-PIA. Our results suggest that the K⁺ channel-coupled adenosine receptor in guinea pig atria is of an A₁ subtype.Abbreviations CCPA 2-chloro-N⁶-cyclopentyladenosine - DPCPX 8-cyclopentyl-1,3-dipropylxanthine - NECA 5-N-ethylcarboxami-doadenosine - PIA N⁶-phenylisopropyladenosine Send offprint requests to H. Tawfik-Schlieper at the above address     

The Cl-HCO3- exchanger slows the recovery of acute pHi changes in rat mast cells

The CO(2)/HCO(3)(-) buffering system is one of the main mechanisms implicated in cytosolic pH (pH(i)) regulation. We studied this pH(i)-regulatory system in rat mast cells using a fluorescent dye. Mast cells had a more alkaline pH(i) in the presence of HCO(3)(-) than in its absence. The recovery from an acid load was faster in HCO(3)(-)-free conditions than in HCO(3)(-)-containing media. In HCO(3)(-)-buffered conditions the increase of the recovery rate of an acidification in 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-incubated cells suggested the implication of a Na(+)-independent Cl(-)/HCO(3)(-) exchanger. This HCO(3)(-) transport acidified the cytosol and was also partially responsible for the recovery of intracellular alkalinizations. Moreover, regulation of the recovery rate of an acidification by protein kinase C and calcium signaling pathways depended on the presence or absence of HCO(3)(-). The presence of HCO(3)(-) limits the recovery of acute intracellular acidifications probably through the Na(+)-independent Cl(-)/HCO(3)(-) exchanger and modulates the regulation of pH(i) by protein kinase C and calcium.     

Reduction of myocardial infarct size by SM-198110, a novel Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange inhibitor,in rabbits

The effects of 3-[2-({[amino(imino)methyl]amino}carbonyl)-4-chloro-1H-indol-1-yl]-1-propanesulphonic acid monohydrate (SM-198110), a novel potent Na⁺/H⁺ exchange inhibitor, and cariporide (Hoe642), another Na⁺/H⁺ exchange inhibitor, were studied in a myocardial ischaemia and reperfusion injury model. Anaesthetized rabbits were subjected to occlusion of the coronary artery for 30 min followed by reperfusion for 5 h. SM-198110 or cariporide was administered before ischaemia and before reperfusion. We also assessed the anti-necrotic effect of SM-198110 when given before reperfusion, both alone and together with glibenclamide, a K_ATP channel blocker, 5-hydroxydecanoate (5-HD), a mitochondrial K_ATP channel-selective blocker and 8-(p-sulphophenyl)-theophylline (8-SPT), an adenosine receptor blocker. The infarct size was reduced dose-dependently by i.v. administration of SM-198110 before ischaemia, with a significant reduction in serum creatine phosphokinase activity. Infarct sizes, normalized to the size of the area-at-risk (means±SE) were: vehicle 56.6±3.7%; low-dose SM-198110 39.2±6.3%; mid-dose 32.8±7.4% (P<0.05); high-dose 22.1±6.7% (P<0.01). This anti-necrotic effect of SM-198110 was achieved without significant haemodynamic changes. Cariporide given before ischaemia also reduced infarct size significantly and dose-dependently. SM-198110 administered before reperfusion also resulted in a dose-dependent reduction in the infarct size. Infarct sizes were: vehicle 56.6±3.7%; low-dose SM-198110 44.5±5.7%; mid-dose 36.3±6.6% (P<0.01); high-dose 34.7±3.8% (P<0.01). In contrast, cariporide given before reperfusion did not reduce infarct sizes significantly. The anti-necrotic effect of SM-198110 was observed even when given 10 min after the beginning of reperfusion. Glibenclamide and 5-HD abolished the anti-necrotic effect of treatment before reperfusion with SM-198110. However, the co-administration of 8-SPT with SM-198110 did not affect infarct size. These results suggest that, in addition to Na⁺/H⁺ exchange inhibition, mitochondrial and/or sarcolemmal K_ATP channels contribute to the anti-necrotic effect of SM-198110 when the latter is given before reperfusion.     

Molecular and functional characterization of the human platelet Na(+) /Ca(2+) exchangers

BACKGROUND AND PURPOSE The Na(+) /Ca(2+) exchanger is a bi-directional transporter that plays an important role in maintaining the concentration of cytosolic Ca(2+) ([Ca(2+) ](i) ) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na(+) /Ca(2+) exchangers: K(+) -independent Na(+) /Ca(2+) exchanger (NCX) and K(+) -dependent Na(+) /Ca(2+) exchanger (NCKX). Platelets have previously been shown to express NCKX1. However, initial studies from our laboratory suggest that NCX may also play a role in platelet activation. The objective of this study was to determine if the human platelet expresses functional NCXs. EXPERIMENTAL APPROACH RT-PCR, DNA sequencing and Western blot analysis were utilized to characterize the human platelet Na(+) /Ca(2+) exchangers. Their function during quiescence and collagen-induced activation was determined by measuring [Ca(2+) ](i) with calcium-green/fura-red in response to: changes in the Na(+) and K(+) gradient, NCX pharmacological inhibitors (CBDMB, KB-R7943 and SEA0400) and antibodies specific to extracellular epitopes of the exchangers. KEY RESULTS Human platelets express NCX1.3, NCX3.2 and NCX3.4. The NCXs operate in the Ca(2+) efflux mode in resting platelets and also during their activation with thrombin but not collagen. Collagen-induced increase in [Ca(2+) ](i) was reduced with the pharmacological inhibitors of NCX (CBDMB, KB-R7943 or SEA0400), anti-NCX1 and anti-NCX3. In contrast, anti-NCKX1 enhanced the collagen-induced increase in [Ca(2+) ](i) . CONCLUSIONS AND IMPLICATIONS Human platelets express K(+) -independent Na(+) /Ca(2+) exchangers NCX1.3, NCX3.2 and NCX3.4. During collagen activation, NCX1 and NCX3 transiently reverse to promote Ca(2+) influx, whereas NCKX1 continues to operate in the Ca(2+) efflux mode to reduce [Ca(2+) ](i) .     

Reduction of the equilibrium binding of cardiac glycosides and related compounds to Na+, K+-ATPase as a possible mechanism for the potassium-induced reversal of their toxicity

Summary The influence of potassium ions on the equilibrium state of the binding of cardiac glycosides and their derivatives to partially purified dog heart and rat brain enzyme preparations was studied in vitro. The addition of potassium to the incubation mixture containing enzyme preparation, ³H-ouabain, Na⁺, Mg²⁺ and ATP, at the time when the binding reaction is close to equilibrium, caused an immediate reduction of the bound drug concentration; the concentration apparently shifting toward a lower equilibrium state. The degree of the potassium-induced reduction in bound drug concentration was dependent on the potassium concentration and on the chemical structure of the compound. The binding of aglycones, pentacetyl-gitoxin and cassaine was affected to a greater extent than that of the glycosides. These data suggest that one of the mechanisms by which potassium antagonizes the toxic actions of digitalis on the heart is to reduce the drug binding to cardiac Na⁺, K⁺-ATPase.This work was supported by a U.S. Public Health Service Grant, HL-16052