脂多糖对大鼠肺微血管内皮细胞[Ca~(2+)]_i和Gq蛋白的影响及山莨菪碱的干预

目的 :观察脂多糖对大鼠肺微血管内皮细胞 (RPMVECs) [Ca2 + ]i 和Gq蛋白的影响及山莨菪碱的干预作用。方法 :分离、培养并鉴定Wistar大鼠RPMVECs;应用Fura - 2 /AM法测定RPMVECs[Ca2 + ]i;流式细胞仪技术测定RPMVECsGq蛋白。结果 :①LPS作用于RPMVECs 30min和 90min后 ,[Ca2 + ]i 显著高于对照组 ;Gq蛋白显著低于对照组。②山莨菪碱可抑制LPS的上述作用。结论 :①LPS致RPMVECs[Ca2 + ]i 增加和Gq蛋白下降 ;②山莨菪碱通过抑制LPS诱导RPMVECs[Ca2 + ]i 增加和Gq蛋白下降的作用而保护其内皮屏障功能。     

噻庚啶和山莨菪碱拮抗TNFα诱导的内皮细胞[Ca2+]i增高

目的：研究噻庚啶（Cyp）和山莨菪碱（Ani）对肿瘤坏死因子（TNFα）诱导单个内皮细胞内Ca²⁺浓度（[Ca²⁺]_i）变化的影响，以探TNFα介导休克和Cyp、Ani的抗休克的机制。方法：人脐静脉内皮细胞株（ECV304）接种于35 mm含2 mL DMEM培养基的组织培养盘中培养。Fluo-3/AM负载细胞，激光扫描共聚焦显微技术（LSCM）测定单个内皮细胞[Ca²⁺]_i。结果：TNFα使单个内皮细胞[Ca²⁺]_i呈剂量依赖性升高，在60 s内达到峰值，然后下降并保持在基础水平之上。共聚焦扫描图像显示细胞核区[Ca²⁺]_i升高比胞浆区明显，下降比胞浆区慢。Cyp（3×10^-5 mol/L或6×10^-5 mol/L）、Ani（2×10^-5 mol/L或4×10^-5 mol/L）均能显著抑制由TNFα（1.2×10^-9 mol/L）诱导的单个内皮细胞[Ca²⁺]_i升高。结论：TNFα诱导内皮细胞[Ca²⁺]_i升高可能是TNFα介导休克的重要机制；Cyp和Ani抑制TNFα诱导的[Ca²⁺]_i升高可能是其抗休克作用的机制之一。     

钾通道对大鼠肺动脉平滑肌细胞[Ca2+]i的调节

目的:探讨在常氧、低氧条件下钾通道对大鼠肺动脉平滑肌细胞(PASMCs)[Ca²⁺]_i的调节。方法:采用钙荧光探针(Fura-2/AM)负载培养的大鼠PASMCs,观察常氧、低氧培养后3种钾通道抑制剂(4AP,TEA、Glib)对PASMCs[Ca²⁺]_i的调节,同时用四唑盐(MTT)比色法比较4AP、TEA、Glib对大鼠PASMCs增殖的影响。结果:(1)常氧状态下,PASMCs[Ca²⁺]_i为(156.91±8.60)nmol/L,低氧时为(294.01±16.81)nmol/L(P＜0.01)。(2)常氧状态下,4AP可引起PASMCs[Ca²⁺]_i升高,达(280.52±23.21)nmol/L(P＜0.01),而TEA、Glib无此作用。(3)低氧时,4AP和TEA都可引起PASMCs[Ca²⁺]_i的升高,分别为(422.41±24.28)nmol/L、(380.84±11.02)nmol/L(P<0.01),Glib无作用。(4)MTT比色法中,常氧和低氧状态下4AP均引起吸光度(A)值升高,分别是0.582±0.062,0.873±0.043(P＜0.01)。TEA仅在低氧时A值升高(0.729±0.041,P＜0.05),而Glib无论常氧还是低氧均无影响。结论:无论常氧还是低氧条件下,电压依赖性钾通道(K_V)对PASMCs[Ca²⁺]_i及其增殖起主要作用。钙激活的钾通道(K_Ca)在常氧条件下对[Ca²⁺]_i不起调节作用,而在低氧下使[Ca²⁺]_i降低,反应性地调节PASMCs增殖。ATP敏感性钾通道(K_ATP)无论在常氧还是低氧情况下对[Ca²⁺]_i的调节不起作用。     

氢氟酸烧伤中毒对兔外周血单个核细胞凋亡与胞内钙浓度的影响

目的: 研究氢氟酸烧伤中毒对兔外周血单个核细胞(PBMC)早期凋亡百分率和胞内游离钙浓度([Ca²⁺]_i)的影响。方法: 用流式细胞仪检测兔烧伤染毒前后外周血单个核细胞的Annexin V变化, 以观察其早期凋亡百分率。用Fluo-3/Am荧光探针观察烧伤染毒前后外周血单个核细胞内Ca²⁺平均荧光强度值的变化, 以观察细胞内[Ca²⁺]_i的变化。 结果: 12只烧伤中毒兔外周血单个核细胞早期凋亡百分率显著增加, 烧伤染毒前后比较P<0.01。而12只烧伤中毒兔中8只染毒后1 h外周血单个核细胞内[Ca²⁺]_i显著降低, 烧伤染毒前后比较P<0.05。其余4只却表现[Ca²⁺]_i染毒前后比较P>0.05。 结论: 本实验氢氟酸烧伤中毒使兔外周血单个核细胞早期凋亡百分率显著增加, 外周血单个核细胞内[Ca²⁺]_i却显著降低。提示氢氟酸烧伤中毒引导的细胞凋亡并非细胞内[Ca²⁺]_i增加所引发。     

阿魏酸钠拮抗氧化型脂蛋白(a)对人动脉平滑肌细胞的作用

目的:研究脂蛋白(a)氧化前后致人动脉平滑肌细胞(SMC)增殖及细胞内游离钙浓度([Ca²⁺]_i)的变化,观察阿魏酸钠(SF)对其的影响。方法:Lp(a)经体外Cu²⁺氧化法氧化,硫代巴比妥酸(TBARS)比色法检测氧化程度,培养的人动脉SMC中分别加入不同浓度SF,作用12h后再与天然和氧化型Lp(a)共同孵育,以MTT比色法、流式细胞仪检测细胞增殖状况,采用荧光探针Fura-2/AM检测细胞[Ca²⁺]_i。结果:氧化型Lp(a)促人动脉SMC增殖的同时亦明显增加了[Ca²⁺]_i水平,作用较天然Lp(a)明显,SF(40,80mg/L)可显著抑制氧化型Lp(a)所致的细胞增殖和[Ca²⁺]_i增加,并呈剂量效应关系,而对天然Lp(a)所致的细胞增殖和[Ca²⁺]_i增加无明显影响。结论:氧化型Lp(a)通过升高[Ca²⁺]_i而显著促动脉SMC增殖可能是其致动脉粥样硬化的机制之一,SF拮抗这种作用可能与其抗氧化能力有关。     

Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats

 In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca²⁺]_i) is determined by Ca²⁺ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however, has not been established. We therefore examined the effect of the sarcolemmal Ca²⁺ pump inhibitor, carboxyeosin, on the rate of the [Ca²⁺]_i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp technique to measure [Ca²⁺]_i simultaneously with transmembrane calcium current (I _Ca). [Ca²⁺]_i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity. The rate of [Ca²⁺]_i removal was calculated from the falling phase of the [Ca²⁺]_i transient. Pre-treatment of the cells with 2 μM carboxyeosin led to a marked decrease in the rate of [Ca²⁺]_i transient decay, suggesting that the sarcolemmal Ca²⁺ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca²⁺]_i decay, indicating an important role for the Na⁺/Ca²⁺ exchange. When both the sarcolemmal Ca²⁺ pump and Na⁺/Ca²⁺ exchange were inhibited the cell failed to restore [Ca²⁺]_i after the stimulation. Comparison of the rate constants of [Ca²⁺]_i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca²⁺]_i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na⁺/Ca²⁺ exchanger and the intracellular calcium stores. Received: 17 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

小鼠腹腔巨噬细胞基态游离钙离子浓度的不均一性及其和细胞反应性的关系

目的:在单细胞水平上研究小鼠腹腔巨噬细胞(PM)基态游离钙离子浓度([Ca²⁺]_i)的不均一性及其和细胞反应性的关系。方法:用荧光指示剂Fura-2/AM结合荧光显微镜成像系统检测单个PM基态的及用激动剂刺激细胞后的[Ca²⁺]_i;同时结合NBT染色法定量检测单个PM产超氧阴离子(O₂^-)水平。结果:对7只正常小鼠共392个PM基态[Ca²⁺]_i的研究表明小鼠PM的基态[Ca²⁺]_i呈正态分布[(54±24)nmol/L,n=392],但波动范围较大(从10nmol/L到高于100nmol/L),以[Ca²⁺]_i在40-60nmol/L的细胞数量最多(约占50%)。用PMA、fMLP刺激后PM[Ca²⁺]_i升高,且受刺激后[Ca²⁺]_i升高的峰值和基态[Ca²⁺]_i之间呈正相关(PMA刺激组:r=052,P<0.01,n=58;fMLP刺激组:r=0.59,P<0.01,n=44。此两组实验均以不同的小鼠重复3次,其它两只小鼠的结果与上同。下面的表述方法同此)。另外小鼠PM的基态[Ca²⁺]_i与其受PMA刺激后产生O₂^-的量也呈显著正相关(r=0.42,P<0.01,n=43,重复4次)。结论:小鼠PM的基态[Ca²⁺]_i是不均一的,且基态[Ca²⁺]_i的高低和该细胞对致炎因子的反应性密切相关。     

ERKs及细胞内游离钙在内皮素-1诱导心肌细胞肥大反应中的作用

目的:研究细胞外信号调节激酶(ERKs)及细胞内游离钙(i)在内皮素-1(ET-1)介导心肌细胞肥大反应中的作用及机制。方法:利用培养的新生大鼠心肌细胞,①以蛋白合成速率、蛋白含量及细胞表面积为心肌肥大反应的指标;②用滤纸法测定ERKs活性;③用Fura-2/AM作为钙荧光指示剂测定心肌[Ca²⁺]i浓度。结果:①ET-1浓度依赖性增加新生大鼠心肌细胞蛋白质含量和心肌细胞表面积、ERKs活性及[Ca²⁺]i浓度,以上作用可被ET_A受体拮抗剂BQ123所完全抑制,被百日咳毒素(PTX)部分抑制,而ET_B受体拮抗剂BQ788则无效;②ERKs激酶特异性抑制剂PD98059可完全抑制ET-1激活ERKs的作用,钙通道拮抗剂硝苯地平可明显抑制ET-1介导的[Ca²⁺]i浓度增加,但二者皆仅部分抑制ET-1介导的心肌细胞肥大反应;③蛋白激酶C(PKC)选择性抑制剂staurosporine并不能明显抑制ET-1介导的ERKs激活,但可抑制ET-1介导的的[Ca²⁺]i浓度增加及心肌细胞肥大反应。结论:ET-1主要通过ET_A受体并经PTX敏感的G-蛋白介导心肌细胞肥大反应,该作用至少涉及两条途径:①通过PKC介导的心肌[Ca²⁺]i浓度增加;②不通过PKC介导的ERKs激活。     

低氧后心肌钙瞬变变化与β-肾上腺素受体脱敏的关系

目的:慢性低氧时,心脏对β-肾上腺素受体激动的反应出现脱敏现象,细胞内钙[Ca²⁺]_i瞬变的幅度降低、时程延长,本研究观察上述变化在低氧时发生和发展的时间过程和对应关系。方法:在年龄对等的正常及慢性低氧1d、3d、1周、2周、3周、4周和8周的大鼠,分别分离正常及慢性低氧条件下的心室肌细胞,以Fura-2为[Ca²⁺]_i的指示剂,用光谱荧光法测定心肌细胞的[Ca²⁺]_i瞬变及其对心肌β-受体激动后反应的变化。结果:在低氧1d、3d、1周等时间内,电刺激引起的[Ca²⁺]_i瞬变、咖啡因引起的[Ca²⁺]_i瞬变及电刺激引起的[Ca²⁺]_i瞬变对β-受体激动剂异丙肾上腺素的增加反应无明显变化;在低氧2周以上时,电刺激引起的[Ca²⁺]_i瞬变的幅度开始降低,而时程开始延长,其对异丙肾上腺素的反应也开始降低。咖啡因引起的[Ca²⁺]_i瞬变幅度也开始降低;在低氧3周和4周时,上述变化程度逐渐加重;在低氧8周时各参数的变化有所恢复,但与4周时的变化程度无明显差异。结论:低氧2-4周时,心脏的β-肾上腺素受体发生脱敏现象,脱敏的机制与3种调节[Ca²⁺]_i瞬变的蛋白质:L-型钙通道、ryanodine受体操纵的钙通道和钙泵等活性的降低有关,后者也可能是低氧时心脏功能降低的重要机制。低氧4-8周时,心脏对低氧产生了适应和代偿。     

慢性低氧对肺动脉内皮及平滑肌细胞急性低氧时胞浆游离钙变化的影响

目的和方法:以Fura-2/AM荧光指示剂负载,检测常氧(PO2213kPa)及慢性低氧[PO2(53±07)kPa]培养的大鼠肺内动脉平滑肌细胞及猪肺动脉内皮细胞胞浆游离钙的水平及其对急性低氧刺激反应的变化。结果:慢性低氧条件培养的第6代肺内动脉平滑肌细胞在急性低氧时[Ca²⁺]_i升高的程度明显降低(P<0.05);而慢性低氧条件培养的第5代肺动脉内皮细胞对急性低氧引起的[Ca²⁺]_i升高程度明显增加(P<0.05)。结论:慢性低氧可以减弱肺内动脉平滑肌细胞对急性低氧所致[Ca²⁺]_i升高的反应而增强肺动脉内皮细胞低氧性[Ca²⁺]_i升高的反应。这可能在慢性低氧时肺血管对低氧的反应性降低中起重要作用。     

当归与硝苯吡啶对慢性支气管炎肺泡巨噬细胞胞浆游离钙水平的影响

目的:探讨当归与硝苯吡啶对慢性支气管炎(慢支)肺泡巨噬细胞胞浆游离钙水平的影响。方法:对慢支缓解期患者7例和正常对照者6例进行支气管肺泡灌洗获得的肺泡巨噬细胞经分离、纯化后,加Fura-2/AM负载,以Fura-2荧光比值法测定加当归、硝苯吡啶及LPS后胞浆游离钙的水平。结果:慢支组肺泡巨噬细胞胞浆中基础钙水平(189.47±23.69)nmol/L较正常对照组(99.65±32.21)nmol/L明显增高(P＜0.01);LPS促进慢支组肺泡巨噬细胞包括胞内钙库释放引起的胞浆游离钙水平升高(基础钙189.47±23.69nmol/L;LPS组235.53±30.30nmol/L)(静息钙228.41±27.36nmol/L;氯化钙+LPS组288.47±43.68nmol/L)(P均＜0.01);当归及硝苯吡啶均抑制LPS对慢支组肺泡巨噬细胞胞浆游离钙水平的升高作用(静息钙228.41±27.36nmol/L;氯化钙+当归+LPS组236.68±28.60nmol/L;氯化钙+硝苯吡啶+LPS组252.64±37.05nmol/L)(P均＞0.05)。结论:当归与硝苯吡啶通过抑制慢支患者肺泡巨噬细胞胞浆游离钙水平升高,影响肺泡巨噬细胞的活化,对于慢支气道内的非特异性炎症可能具有抑制作用。     

Role for protein kinase in Ca2+-dependent feedback modulation of divalent cation influx in internal-Ca2+-store-depleted rat parotid gland cells

 Divalent cation (Ca²⁺ and Mn²⁺) influx, stimulated by internal Ca²⁺ store depletion, into rat parotid acinar cells is inhibited by conditions which increase protein phosphorylation [T. Sakai and I.S. Ambudkar (1996) Am J Physiol 271:C284–C294]. The present study examines the involvement of this protein phosphorylation and Ca²⁺ in the store-dependent inactivation of divalent cation entry. Internal Ca²⁺ store depletion, achieved by incubation (30 min) of cells in nominally Ca²⁺-free medium containing either carbachol or thapsigargin, stimulated Ca²⁺, and Mn²⁺, influx into cells. In either case, inclusion of 1.5 mM Ca²⁺ for the last 5 min of incubation resulted in a decrease in Ca²⁺ (33–41%) and Mn²⁺ (50%) influx, which could not be accounted for by internal Ca²⁺ store refill. The inhibition was prevented when internal-store-depleted cells were treated (prior to incubation with Ca²⁺) with either staurosporine or K-252a, but not with H-7 or KN-93. Refilling of internal Ca²⁺ store(s) in carbachol-treated cells (incubation with Ca²⁺+atropine) induced complete inhibition of divalent cation influx, which was not prevented by treatment with protein kinase inhibitors. These data suggest the staurosporine-sensitive (and K-252a-sensitive) protein phosphorylation is not involved in Ca²⁺-store-refilling-dependent inactivation of Ca²⁺ influx but mediates a Ca²⁺-dependent feedback modulation of divalent cation influx in rat parotid gland acinar cells. Received: 24 July 1996 / Received after revision: 26 September 1996 / Accepted: 2 October 1996     

Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae

 Rat ventricular trabeculae were mounted for isometric tension recording, and then permeabilized with saponin. The Ca²⁺ concentration ([Ca²⁺]) within the permeabilized preparation (cytosolic [Ca²⁺]) was monitored continuously using Indo-1 and the integrals of Ca²⁺ transients resulting from brief caffeine application used as an index of the sarcoplasmic reticulum (SR) Ca²⁺ content. The relationship between SR Ca²⁺ content and cytosolic [Ca²⁺] was studied within the reported physiological range (i.e. 50–250 nmol · l^–1 Ca²⁺). Increasing cytosolic [Ca²⁺] from 50 nmol · l^–1 to 250 nmol · l^–1 increased the steady-state SR Ca²⁺ content about threefold. However, increasing [Ca²⁺] above 250 nmol · l^–1 typically resulted in spontaneous SR Ca²⁺ release, with no further increase in SR Ca²⁺ content. The SR Ca²⁺ content increased only slowly when cytosolic [Ca²⁺] was increased; it was unchanged 20 s after a rapid increase in cytosolic [Ca²⁺], but increased progressively to a new steady-state level during the following 1–2 min. In a parallel series of experiments using intact papillary muscles, increasing extracellular [Ca²⁺] (from 0.5 to 5 mmol · l^–1) significantly increased twitch tension within 20 s of the solution change. These results support previous suggestions that the SR Ca²⁺ content may increase when diastolic cytosolic [Ca²⁺] rises during inotropic interventions such as increased stimulus rate or extracellular [Ca²⁺]. However, the rate at which SR Ca²⁺ responds to changes in cytoplasmic [Ca²⁺] within the diastolic range does not appear rapid enough to explain the early potentiation of twitch tension in intact preparations after an increase in extracellular [Ca²⁺]. Received: 26 August 1997 / Accepted: 28 October 1997     

Green tea extract given before regional myocardial ischemia–reperfusion in rats improves myocardial contractility by attenuating calcium overload

There is evidence for a negative correlation between green tea consumption and cardiovascular diseases. The aim of the present study was to examine whether green tea extract (GTE) given before regional myocardial ischemia could improve depression of myocardial contractility by preventing cytosolic Ca²⁺ overload. Regional ischemia–reperfusion (IR) was induced in rats by ligating the left anterior descending branch for 20 min, then releasing the ligature. Ligation induced ventricular arrhythmias in rats without GTE pretreatment, but decreased arrhythmogenesis was seen in rats pretreated 30 min earlier with GTE (400 mg/kg). During reperfusion, arrhythmias only occurred during the initial 5 min, and GTE pretreatment had no effect. After overnight recovery, serum cTnI levels were greatly increased in control post-IR rats but only slightly elevated in GTE-pretreated post-IR rats. Myocardial contractility measured by echocardiography was still depressed after 3 days in control post-IR rats, but not in GTE-pretreated post-IR rats. No myocardial ischemic injury was seen in post-IR rats with or without GTE pretreatment. Using freshly isolated single heart myocytes, GTE was found to attenuate the post-IR injury-associated cytosolic Ca²⁺ overload and modulate changes in the levels and distribution of myofibril, adherens junction, and gap junction proteins. In summary, GTE pretreatment protects cardiomyocytes from IR injury by preventing cytosolic Ca²⁺ overload, myofibril disruption, and alterations in adherens and gap junction protein expression and distribution.     

Aging does not affect calcium response to CCL2 and LPS in human monocytes

Monocytes play important roles in anti-microbial and anti-viral responses and chronic inflammatory diseases. Monocytes' functions are altered by aging. We investigated age-changes in calcium (Ca²⁺) response to CCL2 and LPS in human monocytes. CCL2 and LPS induced a slow increase of the cytosolic Ca²⁺ level, with a maximum response at ～360 s and ～300 s, respectively, in monocytes of young and older adults. No difference was observed in the magnitude and in the Ca²⁺ kinetic with both stimuli. Furthermore, store-operated Ca²⁺ entry and plasma membrane expression of ORAI1 showed no difference between both groups. In summary, monocytes from older adults maintained the capacity to mobilize calcium as their counterparts in young adults suggesting that the mechanisms underlying the dysfunctions in monocytes in aging might not involve alterations in Ca²⁺ flow through the plasma membrane.     

Calcium mobilization in C5a-stimulated adult and newborn bovine neutrophils

Calcium (Ca²⁺) is an important second messenger central to many neutrophil (PMN) functional activities. Impaired Ca²⁺ mobilization in newborn PMNs following membrane perturbation could be one of the mechanisms underlying observed abnormalities in neonatal PMN function. To compare Ca²⁺ mobilization in bovine newborn and adult PMNs, cytosolic Ca²⁺ responses after stimulation with recombinant human C5a (rHC5a) were measured. PMNs from normal newborn calves (N=6) and adult cows (N=5) were loaded with fura-2/AM for 60 min at room temperature and the fluorescence changes monitored following stimulation with 0.1, 1, 10, or 50 nM rHC5a in Ca²⁺-containing buffer. Resting levels of Ca²⁺ in both newborn (54.6±1.9 nM) and adult (57.3±1.8 nM) bovine PMNs were comparable. After stimulation with rHC5a, a rapid rise of cytosolic Ca²⁺ was observed, which peaked within 20 sec and was followed by a sustained phase of elevated Ca²⁺ lasting up to 20 min. There were no significant differences (P > 0.05) in peak levels of cytosolic Ca²⁺ obtained by newborn and adult PMNs at 0.1, 10, and 50 nM rHC5a. At 1 nM rHC5a, newborn PMNs reached significantly (P < 0.05) higher levels of cytosolic Ca²⁺ (217.9 ± 21.7 nM) than did adult PMNs (158.7 ± 7.9 nM). At 1 nM rHC5a, newborn PMNs also sustained higher levels of cytosolic Ca²⁺ for 3 min following the peak. At all concentrations of rHC5a tested, the time required to reach the peak and the duration of the peak were comparable in both populations. In the absence of extracellular Ca²⁺ (Ca²⁺-free buffer with 1 mM EGTA), resting levels of cytosolic Ca²⁺ were lower in both newborn (33.3 ± 2.9 nM) and adult PMNs (27.9 ± 2.4 nM) and the magnitude of the peak response to rHC5a was diminished at all concentrations of agonist. Additionally, in the absence of extracellular Ca²⁺, the return to basal cytosolic Ca²⁺ levels occurred rapidly and the sustained phase of increased cytosolic Ca²⁺ seen with rHC5a-stimulated PMNs in Ca²⁺-containing buffer was virtually eliminated. These results indicate that bovine PMNs respond well to rHC5a, that stimulated newborn bovine PMNs can mobilize Ca²⁺ as efficiently as adult PMNs, and that the sustained cytosolic Ca²⁺ response to rHC5a in both age groups requires both release of Ca²⁺ from intracellular stores as well as influx of extracellular Ca²⁺. Such data suggest that observed functional deficits in newborn bovine PMNs are probably not related to improper mobilization of Ca²⁺ following stimulation.     

Effects of high-intensity training and acute exercise on in vitro function of rat sarcoplasmic reticulum

To evaluate the effects of high-intensity training and/or a single bout of exercise on in vitro function of the sarcoplasmic reticulum (SR), the rats were subjected to 8 weeks of interval running program (final training: 2.5-min running × 4 sets per day, 50 m/min at 10% incline). Following training, SR function, i.e., Ca²⁺-ATPase activity and Ca²⁺-uptake and release rates, was examined in homogenates of the superficial region of the vastus lateralis muscle from rats subjected to a single bout of treadmill running (50 m/min at 10% incline) for 2.5 min or to exhaustion. Training brought about a 12.4% increase (P < 0.05) in SR Ca²⁺-uptake rate in rested muscles. This change was not accompanied by alterations in Ca²⁺-ATPase activity, Ca²⁺-release rate, Ca²⁺ dependence of enzyme and protein contents of Ca²⁺-ATPase and ryanodine receptor. A single bout of high-intensity exercise to exhaustion evoked significant reductions (P < 0.05) in SR function, irrespective of whether or not the animals were trained. For 2.5-min run and exhausted rats, no differences existed between SR functions of untrained and trained muscles. These data suggest that high-intensity training may be capable of enhancing SR Ca²⁺-sequestering ability, and may not protect against decreasing SR function with high-intensity exercise.     

Acute atrial arrhythmogenesis in murine hearts following enhanced extracellular Ca2+ entry depends on intracellular Ca2+ stores

Aim: To investigate the effect of increases in extracellular Ca²⁺ entry produced by the L-type Ca²⁺ channel agonist FPL-64176 (FPL) upon acute atrial arrhythmogenesis in intact Langendorff-perfused mouse hearts and its dependence upon diastolic Ca²⁺ release from sarcoplasmic reticular Ca²⁺ stores. Methods: Confocal microscope studies of Fluo-3 fluorescence in isolated atrial myocytes were performed in parallel with electrophysiological examination of Langendorff-perfused mouse hearts. Results: Atrial myocytes stimulated at 1 Hz and exposed to FPL (0.1 μm ) initially showed (<10 min) frequent, often multiple, diastolic peaks following the evoked Ca²⁺ transients whose amplitudes remained close to control values. With continued pacing (>10 min) this reverted to a regular pattern of evoked transients with increased amplitudes but in which diastolic peaks were absent. Higher FPL concentrations (1.0 μm ) produced sustained and irregular patterns of cytosolic Ca²⁺ activity, independent of pacing. Nifedipine (0.5 μm ), and caffeine (1.0 mm ) and cyclopiazonic acid (CPA) (0.15 μm ) pre-treatments respectively produced immediate and gradual reductions in the F/F₀ peaks. Such nifedipine and caffeine, or CPA pre-treatments, abolished, or reduced, the effects of 0.1 and 1.0 μm FPL on cytosolic Ca²⁺ signals. FPL (1.0 μm ) increased the incidence of atrial tachycardia and fibrillation in intact Langendorff-perfused hearts without altering atrial effective refractory periods. These effects were inhibited by nifedipine and caffeine, and reduced by CPA. Conclusion: Enhanced extracellular Ca²⁺ entry exerts acute atrial arrhythmogenic effects that is nevertheless dependent upon diastolic Ca²⁺ release. These findings complement reports that associate established, chronic, atrial arrhythmogenesis with decreased overall inward Ca²⁺ current.     

Regulation of Cl–transport by IBMX in renal A6 epithelium

 We studied regulation of Cl^–transport by cAMP and Ca²⁺ in renal epithelial A6 cells. Stimulation of A6 cells by 1 mM 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase), which increased cytosolic cAMP, elicited biphasic increases in short-circuit current (I _sc), i.e., a transient phase followed by a sustained one. Apical application of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, a Cl^–channel blocker) markedly and dose-dependently inhibited the IBMX-induced I _sc. Pretreatment with nifedipine (100 μM, a Ca²⁺ channel blocker) or 1,2-bis (o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA/AM, 10 μM, a Ca²⁺ chelator) partially but markedly inhibited the I _sc. On the other hand, a cAMP-dependent protein kinase inhibitor, H89 (0.5 μM for 1 h), also reduced the IBMX-induced I _sc to a level similar to that following nifedipine or BAPTA pretreatment. Nifedipine had no synergistic effects on the IBMX-induced I _sc in cells treated with H89. Ionomycin (a Ca²⁺ ionophore) could mimic the transient increase dose dependently, and H89 did not block the ionomycin-induced I _sc. Taken together, our observations suggest that: (1) part of the IBMX-stimulated Cl^–release is regulated by an increased cytosolic Ca²⁺ through nifedipine-sensitive Ca²⁺ influx; (2) cAMP-dependent phosphorylation may be required for elevation of the cytosolic Ca²⁺ concentration but not for activation of Cl^–channels, which are directly activated by cytosolic Ca²⁺; and (3) the IBMX-induced sustained Cl^–release requires cAMP elevation in addition to an increase in the cytosolic Ca²⁺ concentration. Received: 9 September 1996 / Received after revision: 14 January 1997 / Accepted: 28 February 1997