Zosuquidar: An Effective Molecule for Intracellular Ca2+ Measurement in P-gp Positive Cells.

Intracellular calcium, as a second messenger, is involved in multilevel cellular regulatory pathways and plays a role (among other processes) in switching between survival and initiation of cell death in neoplastic cells. The development of multidrug resistance (MDR) in neoplastic cells is associated with the ability of cells to escape programmed cell death, in which dysregulation of intracellular calcium may play an important role. Therefore, reliable monitoring of intracellular calcium levels is necessary. However, such a role might be limited by a real obstacle since several fluorescent intracellular calcium indicators are substrates of membrane ABC drug transporters. For example, Fluo-3/AM is a substrate of P-glycoprotein (ABCB1 member of the ABC family), whose overexpression is the most frequent cause of MDR. The overexpression of ABCB1 prevents MDR cell variants from retaining this tracer in the intracellular space where it is supposed to detect calcium. The solution is to use a proper inhibitor of P-gp efflux activity to ensure the retention of the tracer inside the cells. The present study showed that Zosuquidar and Tariquidar (P-gp inhibitors) are suitable for monitoring intracellular calcium, either by flow cytometry or confocal microscopy, in cells overexpressing P-gp.

Identifying subcutaneous tissue microcalcification by Fluo-3 AM imaging in cutaneous calciphylaxis.

Calciphylaxis, also known as calcific uremic arteriolopathy (CUA), is typically characterized by subcutaneous tissue calcification and excruciatingly painful cutaneous lesions with high mortality. It is critical for dermatologists to make early diagnosis and appropriate management, yet currently only 56% of calciphylaxis cases are correctly diagnosed by conventional histological stain1. Specially, the identification of subtle calcium deposits of subcutaneous can be challenging but is believed crucial for early diagnosis of calciphylaxis2. More sensitive calcification staining is in high demand. In this study, Fluo-3 AM was found to be a rapid, sensitive and reliable fluorescent probe for the detection of calcium deposits and could be a promising diagnostic tool for calciphylaxis.

Role of JAK-STAT pathway in reducing cardiomyocytes hypoxia/reoxygenation injury induced by S1P postconditioning.

This experiment was designed to explore the protection of sphingosine1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation acting via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal pathway. The data showed that S1P could significantly increase cell viability, lower the rate of apoptosis, decrease the content of lactate dehydrogenase (LDH) and caspase3 activity in the culture medium, increase the activity of total superoxide dismutase (T-SOD) and manganese superoxide dismutase (Mn-SOD), reduce the loss of mitochondrial membrane potential and the fluorescence intensity of intracellular calcium, as well as increase the phosphorylation of JAK2 and STAT3 in comparison with the H/R group. When the JAK inhibitor AG490 or the STAT inhibitor stattic were added, the effects of S1P were inhibited. Our date shows that S1P protects H9c2 cells from hypoxia/reoxygenation injury and that the protection by S1P was inhibited by AG490 and stattic. Therefore S1P protects H9c2 cells against hypoxia/reoxygenation injury via the JAK-STAT pathway.

Activation of autophagy via Ca(2+)-dependent AMPK/mTOR pathway in rat notochordal cells is a cellular adaptation under hyperosmotic stress.

Nucleus pulposus (NP) cells experience hyperosmotic stress in spinal discs; however, how these cells can survive in the hostile microenvironment remains unclear. Autophagy has been suggested to maintain cellular homeostasis under different stresses by degrading the cytoplasmic proteins and organelles. Here, we explored whether autophagy is a cellular adaptation in rat notochordal cells under hyperosmotic stress. Hyperosmotic stress was found to activate autophagy in a dose- and time-dependent manner. SQSTM1/P62 expression was decreased as the autophagy level increased. Transient Ca(2+) influx from intracellular stores and extracellular space was stimulated by hyperosmotic stress. Activation of AMPK and inhibition of p70S6K were observed under hyperosmotic conditions. However, intercellular Ca(2+) chelation inhibited the increase of LC3-II and partly reversed the decrease of p70S6K. Hyperosmotic stress decreased cell viability and promoted apoptosis. Inhibition of autophagy led to SQSTM1/P62 accumulation, reduced cell viability, and accelerated apoptosis in notochordal cells under this condition. These evidences suggest that autophagy induction via the Ca(2+)-dependent AMPK/mTOR pathway might occur as an adaptation mechanism for notochordal cells under hyperosmotic stress. Thus, activating autophagy might be a promising approach to improve viability of notochordal cells in intervertebral discs.

Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells.

Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca(2+)]c) and nuclear calcium ([Ca(2+)]n) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca(2+)]n detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca(2+)]n and [Ca(2+)]c in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.

Acute application of ApoE4 increases the neuronal resting [Ca~(2+)] i in rat cortex / 基础医学与临床

Objective To investigate the acute effect of apolipoprotein E(apoE)on the intracellular free Ca~(2+) of rat cortical neurons. Methods The intracellular resting calcium level in cultured primary rat cortical neurons was measured by using confocal fluorescent imaging technique. MK-801, an N-methyl-D-aspartate (NMDA) receptor noncom-petitive antagonist, was employed to test potential function of apoE4 through blocking NMDN receptor. Results Acute application of apoE4, but not apoE3, significantly increased the resting [Ca~(2+)] i in a dose-and time-dependent manner (P <0. 01 or P <0. 05) , and MK-801 partly blocked the apoE4-induced elevation of resting [Ca~(2+)]i (P <0. 05 or P <0. 01). Conclusion Acute administration of ApoE4 disturbs calcium homeostasis, and the activation of NMDA receptor may play a critical role in the intracellular calcium overload induced by apoE4 and neurotoxicity.

Acute application of ApoE4 increases the neuronal resting[Ca~(2+)]i in rat cortex / 基础医学与临床

Objective To investigate the acute effect of apolipoprotein E(apoE)on the intracellular free Ca2+ of rat cortical neurons.Methods The intracellular resting calcium level in cultured primary rat cortical neurons was measured by using confocal fluorescent imaging technique. MK-801,an N-methyl-D-aspartate (NMDA) receptor noncompetitive antagonist,was employed to test potential function of apoE4 through blocking NMDN receptor. ResultsAcute application of apoE4,but not apoE3,significantly increased the resting [Ca2+]i in a dose-and time-dependent manner (P

Effects and mechanisms of compound G004 on experimental thrombosis / 中国临床药理学与治疗学

AIM:To study the effects and mechanisms of a novel sulfonylureous compound 1 ｛4 [2 (4 bromobenzenesulfonaminoethyl)phenylsufonyl｝ 3 (trans 4 methylcyclohexyl) urea, G004, on antithrombosis. METHODS: The influence of compound G004 on the vasoconstrictor action, platelet aggregation and thrombosis formation was studied. The effects of compound G004 on the tail vein bleeding time in mice was examined. The influence of compound G004 on the release of prostanglandin I 2 and thromboxan A 2 from ECV304 cells was investigated. The measurement of cytosolic free Ca 2+ in attached ECV304 cells loaded with Fluo3/AM was carried out. RESULTS: Compound G004 did not inhibit the contraction of rat aorta rings induced by norepinephrine or potassium chloride, but potently inhibit human platelet aggregation challenged by arachidonic acid and adenosine diphosphate. Compound G004 significantly prolonged the tail vein bleeding time in mice and occlusion time of carotid artery in experimentally thrombotic rats. Compound G004 reduced mice mortality induced by the collagen plus epinephrine in a dose dependent manner. Compound G004 enhanced PGI 2 release and reduced TXA 2 secretion from ECV304 cells. G004 had no effect on the increase of cytosolic free Ca 2+ induced by patassium chloride. CONCLUSION: The compound G004 has a remarkable antithrombotic effect in vivo. Its active mechanism may be attributed to inhibition of platelet aggregation, enhancing PGI 2 generation and decreasing TXA 2 secretion from human umbilical vein endothelium.

Effect of quateranary ammonium salt derivative (F_2) of haloperidol on calcium in vascular smooth muscle cells / 中国药理学通报

AIM To observe the effect of quateranary ammonium salt derivative (F 2) of haloperidol on calcium level in vascular smooth muscle cells (VSMCs) isolated from thoracic aortas of rat. METHODS VSMCs were loaded with Fluo 3 AM, a calcium sensitive fluorescent dye, and [Ca 2+ ] i was determined by the use of laser scanning confocal microscope (LCSM). RESULTS In Ca 2+ (1 26 mmol?L -1 ) bath solution, intracellular calcium fluorescent intensity (FI) in VSMCs was increased when application with 30 mmol?L -1 KCl, and then was inhibited after addition with F 2. The FI of the ASMCs with different concentrations of F 2 (0 1,1,10,100 ?mmol?L -1 )within three minutes were 64%?9%( n =16, P

Mechanism of 5-hydroxytryptamine induced calcium signaling in cultured rat stomach fundus smooth muscle cells / 中国药理学通报

AIM To get an insight into intracellular signaling steps, a very early step in the signaling cascade, the biphasic Ca 2+ elicited by 5 HT in rat stomach fundus smooth muscle cells was investigated. METHODS Cells were cultured and loaded with Fluo 3 AM. [Ca 2+ ] i was measured by fluorescent intensity (FI) in each cell with confocal microscopy. RESULTS The resting FI level of SFSMC was 264?15. Stimulation of SFSMCs by 5 HT produced an elevation of [Ca 2+ ] i; Depletion of external Ca 2+ by addition of EGTA led to a significant attenuation of [Ca 2+ ] i change induced by 5 HT; Pre treatment of SFSMCs with ryanodine (10 ?mol?L -1 , 5 min) in D Hanks, the effect of 5 HT was completely inhibited; The stimulation of SFSMCs by 5 HT was partly attenuated by miaserin(10 ?mol?L -1 ), however, L type Ca 2+ channel antagonist lacidipine and G protein inhibitor NEM completely abolished the increase of [Ca 2+ ] i mediated by 5 HT; 5 HT mediated Ca 2+ release was reduced by phospholipase C specific inhibitor compound 48/80(1 2 ?g?ml -1 ); When protein kinase C was activated by phorbol 12 myristate 13 acetate (PMA 0 1 ?mol?L -1 , 5 min) the effect of 5 HT was inhibited, and the inhibitory effect of PMA was reversed by D sphingosine, a PKC inhibitor. CONCLUSION Our data suggest that G protein coupled 5 HT 2B receptor in the rat stomach fundus modulates 5 HT stimulated Ca 2+ increase, and it is coupled to calcium influx through L type calcium channels, and also intracellular calcium release by the opening of ryanodine receptor. The 5 HT 2B receptor mediated signal of 5 HT is transduced by PLC and PKC.