共查询到20条相似文献,搜索用时 15 毫秒
1.
Microbubble facilitated ultrasound (US) application can enhance intracellular delivery of drugs and genes in endothelial cells cultured in static condition by transiently disrupting the cell membrane, or sonoporation. However, endothelial cells in vivo that are constantly exposed to blood flow may exhibit different sonoporation characteristics. This study investigates the effects of shear stress cultivation on sonoporation of endothelial cells in terms of membrane disruption and changes in the intracellular calcium concentration ([Ca2+]i). Sonoporation experiments were conducted using murine brain microvascular endothelial (bEnd.3) cells and human umbilical vein endothelial cells (HUVECs) cultured under static or shear stress (5 dyne/cm2 for 5 days) condition in a microchannel environment. The cells were exposed to a short US tone burst (1.25 MHz, 8 μs duration, 0.24 MPa) in the presence of DefinityTM microbubbles to facilitate sonoporation. Membrane disruption was assessed by propidium iodide (PI) and changes in [Ca2+]i measured by fura-2AM. Results from this study show that shear stress cultivation significantly reduced the impact of ultrasound-driven microbubbles activities on endothelial cells. Cells cultured under shear stress condition exhibited much lower percentage with membrane disruption and changes in [Ca2+]i compared to statically cultured cells. The maximum increases of PI uptake and [Ca2+]i were also significantly lower in the shear stress cultured cells. In addition, the extent of [Ca2+]i waves in shear cultured HUVECs was reduced compared to the statically cultured cells. 相似文献
2.
《Cell calcium》2015,58(5-6):366-375
In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca2+]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca2+]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca2+]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca2+]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca2+]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca2+]i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca2+]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca2+]i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion. 相似文献
3.
Although pulsed electromagnetic field (PEMF) exposure has been reported to promote neuronal differentiation, the mechanism is still unclear. Here, we aimed to examine the effects of PEMF exposure on brain-derived neurotrophic factor (Bdnf) mRNA expression and the correlation between the intracellular free calcium concentration ([Ca2+]i) and Bdnf mRNA expression in cultured dorsal root ganglion neurons (DRGNs). Exposure to 50 Hz and 1 mT PEMF for 2 h increased the level of [Ca2+]i and Bdnf mRNA expression, which was found to be mediated by increased [Ca2+]i from Ca2+ influx through L-type voltage-gated calcium channels (VGCCs). However, calcium mobilization was not involved in the increased [Ca2+]i and BDNF expression, indicating that calcium influx was one of the key factors responding to PEMF exposure. Moreover, PD098059, an extracellular signal-regulated kinase (Erk) inhibitor, strongly inhibited PEMF-dependant Erk1/2 activation and BDNF expression, indicating that Erk activation is required for PEMF-induced upregulation of BDNF expression. These findings indicated that PEMF exposure increased BDNF expression in DRGNs by activating Ca2+- and Erk-dependent signaling pathways. 相似文献
4.
A novel staining and quantification method to investigate changes in intracellular calcium levels [Ca2+]i and morphology in filamentous fungus is presented. Using a simple protocol, two fluorescent dyes, Fluo-4-AM and Cell trace calcein red-orange-AM were loaded into the filamentous fungus Penicillium chrysogenum. The present study investigates the applicability of using Ca2+-sensitive dye to quantify and image [Ca2+]i in P. chrysogenum cultures chosen for its potential as an experimental system to study Ca2+ signalling in elicited cultures. The dye loading was optimised and investigated at different pH loading conditions. It was observed that the fluorophore was taken up throughout the hyphae, retaining cell membrane integrity and no dye compartmentalisation within organelles was observed. From the fluorescent plate-reader studies a significant rise (p < 0.001) in the relative fluorescence levels corresponding to [Ca2+]i levels in the hyphae was observed when challenged with an elicitor (mannan oligosaccharide, 150 mg L?1) which was dependent upon extracellular calcium. Concurrently a novel application of dye-loaded hyphae for morphological analysis was also examined using the imaging software Filament Tracer (Bitplane). Essential quantitative mycelial information including the length and diameter of the segments and number of branch points was obtained using this application based on the three-dimensional data. 相似文献
5.
Primary cilium has emerged as mechanosensor to subtle flow variations in epithelial cells, but its role in shear stress detection remains controversial. To probe the function of this non-motile organelle in shear stress detection by cells, we compared calcium signalling responses induced by shear stress in ciliated and unciliated MDCK cells. Cytosolic free Ca2+ ([Ca2+]i) was measured using Fura-PE3 video imaging fluorescence microscopy in response to shear stress due to laminar flow (385 μl s?1). Our results show that both unciliated and ciliated MDCK cells are shear stress sensitive via ATP release and autocrine feedback through purinergic receptors. However, purinergic calcium signals differed in response intensity and receptor subtypes. In unciliated cells, shear stress-induced elevation in [Ca2+]i was predominantly mediated through P2X receptors (P2XR). In contrast, calcium mobilization in ciliated MDCK cells resulted from P2YRs and store-operated Ca2+-permeable channels besides P2XRs. These findings lend support to the hypothesis that ATP release in response to shear stress is independent of the primary cilium and that transduction of mechanical strain into a specific biochemical responses stems on the mobilization of different sets of purinergic receptors. 相似文献
6.
《Biochemical and biophysical research communications》2014,445(3):629-632
Elevation of glucose induces transient inhibition of insulin release by lowering cytoplasmic Ca2+ ([Ca2+]i) below baseline in pancreatic β-cells. The period of [Ca2+]i decrease (phase 0) coincides with increased glucagon release and is therefore the starting point for antisynchronous pulses of insulin and glucagon. We now examine if activation of adrenergic α2A and muscarinic M3 receptors affects the initial [Ca2+]i response to increase of glucose from 3 to 20 mM in β-cells situated in mouse islets. In the absence of receptor stimulation the elevation of glucose lowered [Ca2+]i during 90–120 s followed by rise due to opening of voltage-dependent Ca2+ channels. The period of [Ca2+]i decrease was prolonged by activation of the α2A adrenergic receptors (1 μM epinephrine or 100 nM clonidine) and shortened by stimulation of the muscarinic M3 receptors (0.1 μM acetylcholine). The latter effect was mimicked by the Na/K pump inhibitor ouabain (10–100 μM). The results indicate that prolonged initial decrease (phase 0) is followed by slow [Ca2+]i rise and shorter decrease followed by fast rise. It is concluded that the period of initial decrease of [Ca2+]i regulates the subsequent β-cell response to glucose. 相似文献
7.
Braga-Sobrinho C Leite-Dellova DC Mello-Aires M 《The Journal of steroid biochemistry and molecular biology》2012,128(3-5):89-97
The rapid (2 min) nongenomic effects of aldosterone (ALDO) and/or spironolactone (MR antagonist), RU 486 (GR antagonist), atrial natriuretic peptide (ANP) and dimethyl-BAPTA (BAPTA) on the intracellular pH recovery rate (pHirr) via NHE1 (basolateral Na+/H+ exchanger isoform), after the acid load induced by NH4Cl, and on the cytosolic free calcium concentration ([Ca2+]i) were investigated in the proximal S3 segment isolated from rats, by the probes BCECF-AM and FLUO-4-AM, respectively. The basal pHi was 7.15 ± 0.008 and the basal pHirr was 0.195 ± 0.012 pH units/min (number of tubules/number of tubular areas = 16/96). Our results confirmed the rapid biphasic effect of ALDO on NHE1: ALDO (10?12 M) increases the pHirr to approximately 59% of control value, and ALDO (10?6 M) decreases it to approximately 49%. Spironolactone did not change these effects, but RU 486 inhibited the stimulatory effect and maintained the inhibitory effect. ANP (10?6 M) or BAPTA (5 × 10?5 M) alone had no significant effect on NHE1 but prevented both effects of ALDO on this exchanger. The basal [Ca2+]i was 104 ± 3 nM (15), and ALDO (10?12 or 10?6 M) increased the basal [Ca2+]i to approximately 50% or 124%, respectively. RU 486, ANP and BAPTA decreased the [Ca2+]i and inhibited the stimulatory effect of both doses of ALDO. The results suggest the involvement of GR on the nongenomic effects of ALDO and indicate a pHirr-regulating role for [Ca2+]i that is mediated by NHE1, stimulated/impaired by ALDO, and affected by ANP or BAPTA with ALDO. The observed nongenomic hormonal interaction in the S3 segment may represent a rapid and physiologically relevant regulatory mechanism in the intact animal under conditions of volume alterations. 相似文献
8.
Mingzhen Mao Yuxin Li Qiaoxia Liu Yunyun Zhou Xiulan Zhang Lixia Xiong Yongqiang Li Zhengming Li 《Bioorganic & medicinal chemistry letters》2013,23(1):42-46
In an attempt to search for potent insecticides targeting the ryanodine receptor (RyR), a series of novel N-pyridylpyrazolecarboxamides containing cyano substituent in the ortho-position were designed and synthesized. Their insecticidal activities of target compounds against oriental armyworm (Mythimna separata) and diamondback moth (Plutella xylostella) indicated that most of the compounds showed moderate to high activities at the tested concentrations. In particular, compound 6l and 6o showed 86% larvicidal activities against Plutella xylostella at the concentration of 0.1 mg/L, while the activity of compound 6h against Mythimna separate was 80% at 1 mg/L. The calcium imaging technique was applied to investigate the effects of some title compounds on the intracellular calcium ion concentration ([Ca2+]i), experimental results demonstrated that compound 6h stimulates a transient elevation in [Ca2+]i in the absence of external calcium after the central neurons dye loading with fluo-3 AM. However, when the central neurons were dyed with fluo-5 N and incubated with 2-APB, [Ca2+]i decreased transiently by treated of compound 6h. All of the calcium imaging technique experiments demonstrated that these novel compounds deliver calcium from endoplasmic reticulum to cytoplasm, which proved that the title compounds were the possible activators of insect RyR. 相似文献
9.
AimsThis study examined whether the essential oil component carvacrol altered cytosolic free Ca2+ level ([Ca2+]i) and viability in human glioblastoma cells.Main methodsThe Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry.Key findingsCarvacrol at concentrations of 400–1000 μM induced a [Ca2+]i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca2+. Carvacrol-induced Ca2+ signal was not altered by nifedipine, econazole, SK&;F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca2+ was removed, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca2+]i rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca2+]i rise. At concentrations of 200–800 μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N–-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that carvacrol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600 μM, carvacrol induced production of ROS.SignificanceIn human glioblastoma cells, carvacrol induced a [Ca2+]i rise by inducing phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via protein kinase C-sensitive, non store-operated Ca2+ channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis. 相似文献
10.
《Journal of biomechanics》2014,47(16):3903-3908
Intracellular calcium transient ([Ca2+]i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca2+]i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca2+]i transient in osteoblasts. Fluorescent dyes DiBAC4(3) and fura-2 AM were respectively used to detect membrane potential and [Ca2+]i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca2+]i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca2+]i transients. Replacing extracellular Na+ with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca2+]i transients. However, voltage-activated K+ channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca2+ or blocking of L-type voltage-sensitive Ca2+ channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca2+]i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was −43.3 mV and the depolarization induced by FSS was about 44 mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca2+ mobilization wherein FSS activated SACs to promote Na+ entry to depolarize membrane that, in turn, activated L-VSCCs and Ca2+ influx though L-VSCCs switched on [Ca2+]i response in osteoblasts. 相似文献
11.
Patrizia Rubini Javorina Milosevic Johannes Engelhardt Mahmoud Al-Khrasani Heike Franke Attilla Heinrich Beata Sperlagh Sigrid C. Schwarz Johannes Schwarz Wolfgang Nörenberg Peter Illes 《Cell calcium》2009,45(5):485-498
Nucleotides play an important role in brain development and may exert their action via ligand-gated cationic channels or G protein-coupled receptors. Patch-clamp measurements indicated that in contrast to AMPA, ATP did not induce membrane currents in human midbrain derived neuronal progenitor cells (hmNPCs). Various nucleotide agonists concentration-dependently increased [Ca2+]i as measured by the Fura-2 method, with the rank order of potency ATP > ADP > UTP > UDP. A Ca2+-free external medium moderately decreased, whereas a depletion of the intracellular Ca2+ storage sites by cyclopiazonic acid markedly depressed the [Ca2+]i transients induced by either ATP or UTP. Further, the P2Y1 receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y1,2,12 antagonistic suramin only slightly blocked the action of ATP, but strongly inhibited that of UTP. In agreement with this finding, UTP evoked the release of ATP from hmNPCs in a suramin-, but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of P2Y1,2,4-immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y2 receptor-activation and thereby causes [Ca2+]i transients by stimulating a P2Y1-like receptor. 相似文献
12.
Michel Ronjat José Pablo Finkelstein Paola Llanos Luis Montecinos Hicham Bichraoui Michel De Waard Cecilia Hidalgo Ricardo Bull 《Cell calcium》2013,53(5-6):357-365
The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca2+ release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca2+ uptake completion prevented this response. Maurocalcine enhanced equilibrium [3H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent Ki for Mg2+ inhibition of [3H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca2+] from 10 μM to 0.1 μM or at cytoplasmic [Mg2+] ≥ 30 μM. At 0.1 μM [Ca2+], only channels that displayed poor activation by Ca2+ were readily activated by 5 nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions. 相似文献
13.
Rebecca R. Foster Maryam A.H. Zadeh Gavin I. Welsh Simon C. Satchell Yi Ye Peter W. Mathieson David O. Bates Moin A. Saleem 《Cell calcium》2009,45(4):384-390
Mutations in the cation channel TRPC6 result in a renal-specific phenotype of familial nephrotic syndrome, affecting intracellular calcium ([Ca2+]i) signalling in the glomerular podocyte. Tools to study native TRPC6 activity are scarce, although there has been recent success with flufenamic acid (FFA). We confirm the specificity of FFA for TRPC6 both in an artificial expression system and in a human conditionally immortalised podocyte cell line (ciPod).Cells were loaded with fura-2AM and changes in intracellular calcium ([Ca2+]i) were calculated. 200 μM FFA induced an increase in [Ca2+]i in HEK293 cells with native TRPC6 expression, which was enhanced by overexpression of TRPC6 and completely blocked in the absence of extracellular calcium. Expressed TRPC7 did not significantly affect the response to FFA whereas expressed TRPC3 reduced it. FFA also induced an increase ciPod in [Ca2+]i, which was inhibited using SKF96365 and 2-APB, but not indomethacin. In ciPod, adenovirus (Ad-v) wild type (WT) TRPC6 increased [Ca2+]i activity to FFA compared to native TRPC6, whereas activity was significantly reduced with Ad-v dominant negative (DN) TRPC6. The niflumic acid (NFA) induced increase in [Ca2+]i in ciPod was not affected by Ad-v TRPC6 DN, and in HEK293 cells was not affected by WT TRPC6.In conclusion, FFA activates TRPC6 [Ca2+]i signalling in both ciPod and HEK293 cells independently of TRPC3 and TRPC7, and independently of properties of the fenamate family. 相似文献
14.
Recent studies have shown that the mitochondrial DNA mutations are involved in the pathogenesis of hypertension. Our previous study identified mitochondrial tRNAIle A4263G mutation in a large Chinese Han family with maternally-inherited hypertension. This mutation may contribute to mitochondrial Ca2+ cycling dysfuntion, but the mechanism is unclear. Lymphoblastoid cell lines were derived from hypertensive and normotensive individuals, either with or without tRNAIle A4263G mutation. The mitochondrial calcium ([Ca2+]m) in cells from hypertensive subjects with the tRNAIle A4263G mutation, was lower than in cells from normotension or hypertension without mutation, or normotension with mutation (P < 0.05). Meanwhile, cytosolic calcium ([Ca2+]c) in hypertensive with mutation cells was higher than another three groups. After exposure to caffeine, which could increase the [Ca2+]c by activating ryanodine receptor on endoplasmic reticulum, [Ca2+]c/[Ca2+]m increased higher than in hypertensive with mutation cells from another three groups. Moreover, MCU expression was decreased in hypertensive with mutation cells compared with in another three groups (P < 0.05). [Ca2+]c increased and [Ca2+]m decreased after treatment with Ru360 (an inhibitor of MCU) or an siRNA against MCU. In this study we found decreased MCU expression in hypertensive with mutation cells contributed to dysregulated Ca2+ uptake into the mitochondria, and cytoplasmic Ca2+ overload. This abnormality might be involved in the underlying mechanisms of maternally inherited hypertension in subjects carrying the mitochondrial tRNAIle A4263G mutation. 相似文献
15.
《Cytokine》2011,53(3):210-214
To examine the roles of intracellular calcium in RANKL-induced bone marrow macrophages (BMMs) differentiation, the effects of intracellular calcium chelator BAPTA-AM on RANKL-induced BMMs differentiation, and the activation of its relating signal proteins (MAPKs, and the PI3K/Akt) were studied. BMMs were cultured with various concentrations of BAPTA-AM in the presence of M-CSF (25 ng/ml) and RANKL (25 ng/ml) for 7 days, osteoclastogenic ability, cytosolic free Ca2+ concentration, osteoclast survival and the expression of phosphorylated ERK1/2, SAPK/JNK, Akt and p38 MAPK were measured by TRAP staining, spectrofluorometer and Western blotting. BAPTA-AM inhibited osteoclastogenesis and osteoclast survival of BMMs by RANKL induction. In osteoclasts without the pretreatment of BAPTA-AM, the increased response of [Ca2+]i was observed within 15 min and the maximum was about 1.2 times that of control. This response was sustained for 30 min and returned to the control level at 1 h after RANKL-inducing, and the increased response of [Ca2+]i was completely abolished and sustained to at least 8 h by BAPTA-AM. Although immunoblotting data revealed that RANKL could activate the phosphorylation of ERK1/2, SAPK/JNK, Akt and p38 MAPK, the expression of ERK1/2, Akt and p38 MAPK phosphorylation was inhibited by BAPTA-AM dose-dependently. These results revealed that BAPTA-AM inhibit osteoclastogenic ability of BMMs via suppressing the increase of [Ca2+]i which lead to inhibit RANKL-induced the phosphorylation of ERK, Akt and p38 MAPK, but not JNK. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors. 相似文献
16.
Giuseppe Cassano Giuseppe Gasparre Mauro Niso Marialessandra Contino Vito Scalera Nicola Antonio Colabufo 《Cell calcium》2009,45(4):340-345
We demonstrate that F281, a synthetic agonist of the sigma-2 receptor (s2R), induces a non transient increase in intracellular [Ca2+] ([Ca2+]i) and cell death in SK-N-SH cells. Sigma receptors are classified into two subtypes, with different molecular weight and tissue distribution. While the sigma-1 receptor has been cloned, the s2r is less characterized and its physiological ligand and role need further investigation. In tumour cell lines, synthetic agonists of the s2R trigger apoptosis and modulate [Ca2+]i. In particular, CB-64D induces a Ca2+ response while PB28 supresses Ca2+ signalling. We have recently synthesized F281, by replacing the 5-methoxytetraline moiety of PB28 with a carbazole nucleus. Although this bioisosteric substitution should not affect the ligand affinity at the receptor, F281 (after 24 h incubation) was more cytotoxic than PB28 (EC50 values 65.4 nM and 8.13 μM, respectively) in SK-N-SH cells. We used the fluorescent probes fura-2, rhod-2 and JC-1. F281 mobilizes Ca2+ from mitochondria and from the endoplasmic reticulum, by opening its inositol 1,4,5-trisphosphate receptor; Ca2+-entry through the channels activated by store depletion was also observed. After the increase in [Ca2+]i and within 10 min, we observed a sudden drop in metabolic activity and intracellular [ATP] leading to cell death. 相似文献
17.
AimsThis study was designed to investigate the effects of sodium ferulate (SF) on rat isolated thoracic aortas and the possible mechanisms.Main methodsIsometric tension was recorded in response to drugs in organ bath. Cytosolic free Ca2+ concentration ([Ca2+]i) was measured using Fluo-3 in cultured rat aortic smooth muscle cells (RASMC).Key findingsSF (0.1–30 mM) relaxed the isolated aortic rings precontracted with phenylephrine (PE) and high-K+ in a concentration-dependent manner with respective pD2 of 2.7 ± 0.02 and 2.6 ± 0.06. Mechanical removal of endothelium did not significantly modify the SF-induced relaxation. In Ca2+-free solution, SF noticeably inhibited extracellular Ca2+-induced contraction in high-K+ and PE pre-challenged rings, and suppressed the transient contraction induced by PE and caffeine. The vasorelaxant effect of SF was unaffected by various K+ channel blockers such as tetraethylammonium, glibenclamide, 4-aminopyridine, and barium chloride. In addition, SF concentration-dependently reduced the contraction induced by phorbol-12-myristate-13-acetate, an activator of protein kinase C (PKC), in the absence of extracellular Ca2+, with the pD2 of 2.9 ± 0.03. In RASMC, SF had no effect on PE- or KCl-induced [Ca2+]i increase either in the presence or in the absence of external Ca2+.SignificanceThese results indicate that SF acts directly as a non-selective relaxant to vascular smooth muscle. The direct inhibition of the common pathway after [Ca2+]i increase may account for the SF-induced relaxation in Ca2+-dependent contraction, while the blockage of the PKC-mediated contractile mechanism is likely responsible for the SF-induced relaxation in Ca2+-independent contraction. 相似文献
18.
19.
《Biochemical and biophysical research communications》2014,443(3):888-893
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 ([Ca2+]c) and nuclear calcium ([Ca2+]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 [Ca2+]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 [Ca2+]n and [Ca2+]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. 相似文献
20.
Soo-Jin Park Kyoung-Pil Lee Saeromi Kang Hae-Young Chung Yoe-Sik Bae Fumikazu Okajima Dong-Soon Im 《Cellular signalling》2013,25(11):2147-2154
Lysophosphatidylethanolamine (LPE) is a lyso-type metabolite of phosphatidylethanolamine (a plasma membrane component), and its intracellular Ca2 + ([Ca2 +]i) increasing actions may be mediated through G-protein-coupled receptor (GPCR). However, GPCRs for lysophosphatidic acid (LPA), a structurally similar representative lipid mediator, have not been implicated in LPE-mediated activities in SK-OV3 or OVCAR-3 ovarian cancer cells or in receptor over-expression systems. In the present study, LPE-induced [Ca2 +]i increase was observed in MDA-MB-231 cells but not in other breast cancer cell lines. In addition, LPE- and LPA-induced responses showed homologous and heterologous desensitization. Furthermore, VPC32183 and Ki16425 (antagonists of LPA1 and LPA3) inhibited LPE-induced [Ca2 +]i increases, and knockdown of LPA1 by transfection with LPA1 siRNA completely inhibited LPE-induced [Ca2 +]i increases. Furthermore, the involvement of CD97 (an adhesion GPCR) in the action of LPA1 in MDA-MB-231 cells was demonstrated by siRNA transfection. Pertussis toxin (a specific inhibitor of Gi/o proteins), edelfosine (an inhibitor of phospholipase C), or 2-APB (an inhibitor of IP3 receptor) completely inhibited LPE-induced [Ca2 +]i increases, whereas HA130, an inhibitor of autotaxin/lysophospholipase D, did not. Therefore, LPE is supposed to act on LPA1-CD97/Gi/o proteins/phospholipase C/IP3/Ca2 + rise in MDA-MB-231 breast cancer cells. 相似文献