Effects of quinine on the intracellular calcium level and membrane potential of PC 12 cultures

The mechanism for the perception of bitterness appears to be quite complicated, even for quinine, which is a model bitter substance, and thus has yet to be completely elucidated. To investigate the possibility of being able to predict the bitterness of quinine solutions, we examined the effects of quinine on intracellular calcium ion concentration ([Ca(2+)]i) and membrane potentials in PC 12 cultures. [Ca(2+)]i and membrane potentials were analysed by fluorescence confocal microscopic imaging using the Ca(2+)-sensitive probe Calcium Green 1/AM and the membrane potential-sensitive probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)). Quinine elicited an increase in the membrane potential along with a concentration-dependent increase in [Ca(2+)]i. These increases were inhibited by extracellular Ca(2+)-free conditions, thapsigargin, which is a Ca(2+)-pump inhibitor, and U73122, which is a phospholipase C inhibitor. The quinine-induced increase in [Ca(2+)]i levels was inhibited by nifedipine, an L-type Ca(2+)-channel blocker, omega-conotoxin, a T-type Ca(2+)-channel blocker, and BMI-40, which is a bitterness-masking substance. These results suggest that responses in PC 12 cultures may be used as a simple model of bitterness perception.     

Gatifloxacin induces augmented insulin release and intracellular insulin depletion of pancreatic islet cells

Many hypoglycemic and hyperglycemic episodes associated with clinical use of gatifloxacin (GFLX), a novel fluoroquinolone antimicrobial agent, have been reported in recent years. Some have reported hypoglycemia induced by fluoroquinolones, indicating that these agents may stimulate insulin secretion from pancreatic islet cells. In this study, we investigated the effect of GFLX on insulin homeostasis in islet cells using the insulin secreting cell line, HIT-T15. After 1 h incubation with over 100 microM of GFLX, insulin secretion from the cells was significantly augmented. However, the augmentation of insulin release induced by GFLX subsequently reached a plateau. Coincidentally, cellular insulin was decreased by 120 h incubation, and reactivity to re-stimulation by sulfonylurea was suppressed. The GFLX insulin depletion effect was stronger than the effects produced by such other fluoroquinolones as levofloxacin and ciprofloxacin. This study suggests that GFLX should induce insulin oversecretion from pancreatic islet cells in the short-term, and decrease insulin productivity or increase insulin disintegration in the long-term. These results are consistent with the clinical results of GFLX finding that hypoglycemic episodes were seen after a first single administration, and most hyperglycemic episodes were seen more than 2 d after the start of administration.     

Mercuric chloride activates latent, anion-dependent cation transport systems in the plasma membrane of Ehrlich ascites tumour cells

Electronic cell sizing of Ehrlich ascites tumour cells is presented as a biological test system for assessment of membrane associated effects of toxic compounds. Ehrlich ascites tumour cells readjust their cell volume after osmotic swelling in hypotonic media. This regulatory process (Regulatory Volume Decrease, RVD) involves a net loss of KCl from the cells. Addition of HgCl2 (1 microM) results in a Cl- -dependent acceleration of RVD in hypotonic medium. Cells in isotonic Cl- -containing medium shrink upon addition of HgCl2 due to a Cl- -dependent net loss of K+. In addition, a Cl- -dependent net uptake of Na+ was also seen in the presence of HgCl2. It is concluded that HgCl2 activates a latent K+, Cl- cotransport as well as Na+, Cl- cotransport in Ehrlich cells.     

Inhibitory effects of melatonin on free intracellular calcium in mouse brain cells

目的：研究褪黑激素（Ｍｅｌ）对老年小鼠大脑皮层突触体内钙含量以及激动剂诱发的新生小鼠脑细胞［Ｃａ２＋］ｉ升高的影响,以探讨Ｍｅｌ抗衰老的作用机理．方法：钙离子荧光染料Ｆｕｒａ２ＡＭ负载已制备的突触体或细胞,用ＲＦ５０００型双波长荧光分光光度计测定［Ｃａ２＋］ｉ．结果：长期使用Ｍｅｌ抑制老年小鼠大脑皮层Ｃａ２＋超负荷,Ｍｅｌ也降低钙通道激活剂ＢａｙＫ８６４４,高浓度氯化钾（ＫＣｌ）和谷氨酸钠诱发的分离的新生小鼠脑细胞［Ｃａ２＋］ｉ升高．结论：Ｍｅｌ对中枢神经元［Ｃａ２＋］ｉ超载的抑制作用与其抗衰老作用有关．     

β-淀粉样蛋白对大鼠海马细胞内钙浓度和线粒体膜电位的影响

目的 研究β-淀粉样蛋白(Aβ25-35)对SD大鼠脑海马细胞内Ca2+-ATPase、游离钙离子浓度和线粒体膜电位的影响,探讨Aβ25-35对大鼠神经毒性作用的机制.方法 将90只SD大鼠按体重随机分为6组,采用海马内注射染毒方式,剂量分别为Aβ10、5和1 μg/只组,设立生理盐水组、假手术组和正常对照组.分别于术后7、14和21 d处死实验大鼠,取海马检测神经细胞内Ca2+-ATP活力、细胞内游离钙离子浓度和线粒体膜电位的改变.结果在术后7、14和21 d时,Aβ10 μg/只组海马细胞内Ca2+-ATPase分别为(0.24±0.05)、(0.14±0.01)和(0.09±0.01)U/mg,显著低于生理盐水组(P<0.01),同时具有明显的剂量-时间依赖关系;Aβ25-35可以增高细胞内Ca2+浓度,与生理盐水组相比,差异有统计学意义(P<0.01),并且具有明显的剂量-反应关系与剂量-时间关系,差异有统计学意义(P<0.01);同时,Aβ25-35染毒组实验动物细胞内线粒体膜电位随时间延长和剂量升高而降低,差异有统计学意义(P<0.01);假手术组、生理盐水组与正常对照组之间各指标检测差异无统计学意义.结论 Aβ25-35可以通过增加细胞内钙浓度,破坏线粒体功能而发挥神经毒性作用.     

褪黑激素对小鼠脑细胞内游离钙浓度的抑制作用

目的 研究褪黑激素（Ｍｅｌ）对老年小鼠大脑皮层突触体内钙含量以及激动剂动诱发的新生小鼠脑细胞（Ｃａ＾２＋）升高的影响,以探讨Ｍｅｌ抗衰老的作用机理。方法 钙离子荧光染料Ｆｕｒａ－２ＡＭ负载已制备的突触体或细胞,ＲＦ－５０００型双波长荧光分光光度计测定（Ｃａ＾２＋）ｉ,结果：长期使用Ｍｅｌ抑制老年小鼠大脑皮层Ｃａ＾２＋超负荷,Ｍｅｌ也降低钙通道激活剂Ｂａｙ－Ｋ８６４４,高浓度氯化钾（ＫＣｌ）和谷氨酸     

Mercuric chloride induces apoptosis via a mitochondrial-dependent pathway in human leukemia cells

Mercurial compounds modulate immunologic functions by inducing cytotoxicity. Although mercury chloride (HgCl(2)) is known to induce apoptosis in various immune system cells, the mechanism of the induction of apoptosis is poorly understood. In this study, we examined the activation of caspase-3, an important cysteine aspartic protease, during HgCl(2)-induced apoptosis in a human leukemia cell line (HL-60 cells). Both DNA fragmentation, a characteristic of apoptotic cells, and proteolysis of poly(ADP-ribose) polymerase (PARP), a substrate of caspase-3, occurred at 6 h after HgCl(2) treatment in HL-60 cells. These results suggest that the activation of caspase-3 was involved in HgCl(2)-induced apoptosis. The release of cytochrome c (Cyt c) from mitochondria into the cytosol, which is an initiator of the activation of caspase cascades, was also observed in HgCl(2)-treated HL-60 cells. Moreover, the release of Cyt c from mitochondria was observed in HgCl(2)-treated mitochondria isolated from mice liver, and this was followed by mitochondrial permeability transition (PT). The PT was inhibited by cyclosporin A (CsA), a potent inhibitor of PT. CsA also suppressed the occurrence of DNA fragmentation induced by HgCl(2) treatment in HL-60 cells. Taken together, these findings indicate that HgCl(2) is a potent inducer of apoptosis via Cyt c release from the mitochondria in HL-60 cells.     

Histamine increases vascular tone and intracellular calcium level using both intracellular and extracellular calcium in porcine coronary arteries

Effects of histamine on the tone and intracellular calcium level (Ca2+i) in porcine coronary arteries were simultaneously investigated by use of the fura-2 microscopic fluorometric method. Histamine (10(-6)-10(-4) M) induced concentration-dependent increases in tone and Ca2+i, but these responses were not sustained. Histamine induced a larger contraction than did KCl with a similar increase in Ca2+i. Depletion of the caffeine-sensitive Ca2+ store with ryanodine (3 x 10(-5) M) and repetitive applications of caffeine (2.5 x 10(-2) M) scarcely affected contractile and Ca2+i responses to histamine. In Ca2(+)-free medium or in the presence of verapamil (10(-6) M), histamine produced a briefer increase in Ca2+i and a smaller contraction than in normal medium. When histamine or caffeine was repetitively applied in Ca2(+)-free medium, the first application produced an increase in Ca2+i but the second application produced no increase. Although caffeine increased Ca2+i after repetitive histamine applications, histamine failed to increase Ca2+i after repetitive caffeine applications in Ca2(+)-free medium. These results indicate that vascular contraction induced by histamine may involve the following mechanisms: an increase in Ca2+ influx through Ca2+ channels, release of Ca2+ from the intracellular Ca2+ store which has an interaction with the caffeine-sensitive Ca2+ store, and sensitization of contractile proteins to Ca2+.     

The kappa-opioid agonist U-69593 affects intracellular calcium level in R1.1 mouse thymoma cell line

In this study, the effect of the kappa-opioid agonist U-69593 on the intracellular calcium level in R1.1. cells was investigated using FURA 2-AM dye. In the previous study, calcium transport into R1.1 cells was not affected by the kappa-opioid agonist (-)U50,488 [Int. J. Immunopharmacol. 21 (1999) 133]. In this study, the kappa-opioid agonist U-69593 (10(-10)-10(-6) M), decreased intracellular calcium level in unstimulated cells. This decrease could not be reversed by the kappa-opioid antagonist NBI (10(-5) or 10(-6) M). Ionophore A23187 was used to increase intracellular calcium level. Stimulation of intracellular calcium level by Ionophore A23187 was potentiated by the kappa-opioid agonist U-69593. Thus, we have shown that basal intracellular calcium level was decreased in R1.1 by the kappa-opioid agonist U-69593 and increased in R1.1 cells stimulated by Ionophore A23187.     

Effects of secretagogues on intracellular free calcium and magnesium concentrations in rat pancreatic acinar cells.

1. Rat pancreatic acinar cells were loaded with Fura 2 AM or Magfura AM and levels of cytosolic Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were observed. 2. Addition of acetylcholine (ACh) evoked a transient rise in [Ca2+]i. The component of the rise dependent on extracellular Ca2+ sources, but not intracellular sources, was seen to be enhanced when both ACh and 5 mM Ca2+ were present in the medium. In the presence of elevated extracellular Mg2+ (10 mM) and ACh both components of the Ca2+ transient were inhibited. 3. Both GTP gamma S and fluoroaluminate, which can directly stimulate G-proteins, evoked a transient rise in [Ca2+]i in acinar cells. These responses were inhibited in the presence of elevated Mg2+. 4. Resting [Mg2+]i was seen to be 1.36 mM +/- 0.08 (n = 29) for cells in normal medium, 1.8 mM +/- 0.08 (n = 6) in elevated Mg2+ medium and 0.93 mM +/- 0.02 (n = 5) in cells bathed and Mg(2+)-free medium. Addition of ACh led to reductions in [Mg2+]i in cells bathed in normal medium and Mg(2+)-free medium but not elevated Mg2+ medium. 5. It is concluded that levels of extracellular Mg2+ strongly influence [Mg2+]i and [Ca2+]i mobilization during ACh-evoked responses. Mg2+ does not appear to be exerting its effects by influencing receptor-agonist interactions or by competing with Ca2+ at extracellular sites of Ca2+ uptake.     

虎杖苷对休克大鼠微血管平滑肌细胞内钙、pH和膜电位的影响

目的探讨虎杖苷（ＰＤ）改善休克动物微循环的作用机制。方法股动脉放血复制休克模型，取肠系膜微动脉用链霉蛋白酶Ｅ消化以分离单个血管平滑肌细胞（ＶＳＭＣ），再用不同荧光染料分别标记细胞，在激光共聚焦显微镜上测定细胞内钙（［Ｃａ２＋］ｉ）、ｐＨ和膜电位的变化。结果ＰＤ（０４ｍｍｏｌ·Ｌ－１）使休克大鼠ＶＳＭＣ［Ｃａ２＋］ｉ浓度在１０ｍｉｎ内显著下降至加药前的７８４％±５６％、ｐＨ下降至原来的７２８％±８２％；而正常对照组ＶＳＭＣ［Ｃａ２＋］ｉ升高１７５％±６３％、ｐＨ上升３４％±１０１％。当ＰＤ加入前１０ｍｉｎ用钙通道阻断剂维拉帕米（５０μｍｏｌ·Ｌ－１）预处理后，则动物不论休克与否，其［Ｃａ２＋］ｉ、ｐＨ都显著下降。另外ＰＤ使正常及休克大鼠ＶＳＭＣ膜电位负值下降，出现去极化反应。加入ＥＧＴＡ和维拉帕米预处理不能阻断ＰＤ作用，但加入钠通道阻断剂河豚毒素（１μｍｏｌ·Ｌ－１）则可完全阻断ＰＤ的去极化作用。结论ＰＤ对细胞内钙、ｐＨ有双向调节作用，正常情况下ＰＤ增加细胞内游离钙及升高ｐＨ以提高血管张力，休克时ＰＤ降低细胞内钙浓度及降低细胞内ｐＨ以降低血管张力，使血管扩张。此外ＰＤ还可能通过促进细胞外钠离子?     

Influence of the carcinogenic oestrogen diethylstilboestrol on the intracellular calcium level in C6 rat glioma cells

The synthetic oestrogen diethylstilboestrol (DES) causes a dose-dependent elevation of the cytoplasmic Ca²⁺ concentration in C6 rat glioma cells. This Ca²⁺ rise is caused neither by Ca²⁺ influx nor by release from the Ca²⁺ stores of the endoplasmic reticulum. Therefore it seems likely that DES mobilizes Ca²⁺ from a mitochondrial source. The DES-induced Ca²⁺ signal is remarkably similar to the one induced by the tumour promotor thapsigargin. As this compound causes leakage of calcium from the endoplasmic reticulum it seems possible that DES induces a similar leakage from mitochondrial Ca²⁺ stores. It remains to be established whether the DES-mediated rise in intracellular calcium is causally related to the tumour-promoting properties of this compound.     

Thioperamide reduces intracellular calcium in mouse brain synaptosomes.

Intracellular calcium regulation is vital for cells, especially for neurons; raised levels are associated with cytotoxicity and neuronal death. In this report, we present the first experimental evidence showing a concentration-dependent reduction of free calcium in the mouse brain synaptosomes by thioperamide (THP), an H3 receptor antagonist. This is interesting in view of the recent reports on the anticonvulsant and cognition facilitating effects of THP. A neuroprotective potential of THP is suggested.     

Pharmacological evaluation of the role of cytochrome P450 in intracellular calcium signalling in rat pancreatic acinar cells

We have investigated whether the cytochrome P450 system is involved in Ca(2+) signalling in rat pancreatic acinar cells. Intracellular free [Ca(2+)] ([Ca(2+)](i)) was measured in collagenase-isolated cells using fura-2 microspectrofluorimetry and imaging. The imidazole P450 inhibitor ketoconazole (5 - 50 microM) inhibited [Ca(2+)](i) oscillations induced by cholecystokinin octapeptide (CCK). However, ketoconazole also raised baseline [Ca(2+)](i) when applied in the absence of CCK. These effects were mimicked by 5 - 50 microM SKF96365, an imidazole widely used as an inhibitor of Ca(2+) entry. The non-imidazole P450 inhibitor proadifen (SKF525A) inhibited CCK-induced [Ca(2+)](i) oscillations at a concentration of 10 - 50 microM. Proadifen alone caused intracellular Ca(2+) release at 25 or 50 microM, but not at 10 microM. Octadecynoic acid and 1-aminobenzotriazole, structurally-unrelated non-imidazole P450 inhibitors, did not alter baseline [Ca(2+)](i) or CCK-evoked oscillations. We compared cumulative CCK dose-response relationship in control cells and in cells where P450 had been induced by prior injection of animals with beta-naphthoflavone. Only minor differences were apparent, with induced cells showing some decrease in responsiveness at moderate and higher concentration of CCK (30 pM - 3 nM). Direct assessment of depletion-activated Ca(2+) entry showed no clear differences between control and induced cells. In conclusion, we could find no compelling evidence for a role of P450 in controlling Ca(2+) signalling generally, or Ca(2+) entry in particular, in pancreatic acinar cells. Induction of P450 is therefore probably toxic to acinar cells via a Ca(2+)-independent mechanism.     

Mercuric chloride toxicity in rat liver mitochondria and isolated hepatocytes

The effects of mercuric chloride on isolated rat liver mitochondria and freshly isolated rat hepatocytes were investigated. Mercuric chloride (in the μM range) depresses state 3 respiration, suggesting a strong effect at the level of the phosphorylation system. It also stimulates state 4 respiration and decreases mitochondrial membrane potential, suggesting an uncoupling action. In isolated hepatocytes, mercuric chloride causes a dose- and time-dependent cell death, accompanied by depletion of intracellular glutathione. Furthermore, mercuric chloride decreases intracellular ATP and ADP levels simultaneously with increasing AMP concentration, indicating fast hydrolysis of ATP without adequate rephosphorylation. From this study we can conclude that the bioenergetic lesion promoted by mercuric chloride seems to be sufficient to explain lethal hepatocyte injury.     

Developmental regulation of intracellular calcium transients during cardiomyocyte differentiation of mouse embryonic stem cells

AIM: To investigate the developmental regulation of intracellular Ca2+ transients, an essential event in excitation-contraction coupling, during cardiomyocyte differentiation. METHODS: Using the embryonic stem (ES) cell in vitro differentiation system and pharmacological intervention, we investigated the molecular and functional regulation of Ca2+ handling proteins on the Ca2+ transients at early, intermediate and later differentiation stages of ES cell-derived cardiomyocytes (ESCM). RESULTS: Nifedipine, a selective antagonist of L-type Ca2+ channels, totally blocked Ca2+ transients even in the condition of field-electric stimulation in ESCM at three differentiation stages. The Ca2+ transients of ESCM were also inhibited by both ryanodine [an inhibitor of ryanodine receptors (RyRs)] and 2-aminoethoxydipheylborate [2-APB, an inhibitor of inositol-1,4,5-trisphosphate receptors (IP3Rs)]. The inhibitory effect of ryanodine increased with the time of differentiation, while the effect of 2-APB decreased with the differentiation. Thapsigargin, an inhibitor of SR Ca2+-pump ATPase, inhibited Ca2+ transients equally at three differentiation stages that matched the expression profile. Na+ free solution, which inhibits Na+-Ca2+ exchanger (NCX) to extrude Ca2+ from cytosol, did not affect the amplitude of Ca2+ transients of ESCM until the latter differentiation stage, but it significantly enhanced the basal Ca2+ concentration. CONCLUSION: The Ca2+ transients in ESCM depend on both the sarcolemmal Ca2+ entry via L-type Ca2+ channels and the SR Ca2+ release from RyRs and IP3Rs even at the early differentiation stage; but NCX seems not to regulate the peak of Ca2+ transients until the latter differentiation stage.     

Ethanol hyperpolarizes mitochondrial membrane potential and increases mitochondrial fraction in cultured mouse myocardial cells

Cultured mouse heart-derived myocardial and non-muscle cells were exposed to ethanol, stained with cell-permeant fluorescent vital probes, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide) and oxidation-sensitive dihydrorhodamine 123, and analyzed by flow cytometry to elucidate ethanol-induced time-wise alterations in the mitochondrial membrane potential (ΔΨm) and the production of reactive oxygen species (ROS). Ethanol (50 and 200 mM) not only hyperpolarized ΔΨm of both types of cells but also dose-dependently increased ROS production at 24 h, although a 200-mM dose reduced the production until 3 h. These cell pathophysiological reactions suggest the depression of mitochondrial ATPase and mitochondrial respiratory chain. However, differences between these cells appeared after a 24-h exposure to 200 mM ethanol: the increase in ROS production was approximately twice as large for myocardial cells as for non-muscle cells; and the side-scatter parameter of light scattering significantly increased for myocardial cells, but not for non-muscle cells. All these myocyte-specific alterations indicate an increase in the mitochondrial fraction in a cell. This reaction might be a countermeasure against ethanol-induced dysfunction of mitochondrial respiration that is needed to meet the energy requirements of spontaneous myocardial contractions.