Cytoplasmic Free Calcium Levels Research Articles

Cyclic nucleotide-gated ion channel 6 is involved in extracellular ATP signaling and plant immunity.

Extracellular ATP (eATP) is known to act as a danger signal in both plants and animals. In plants, eATP is recognized by the plasma membrane (PM)-localized receptor P2K1 (LecRK-I.9). Among the first measurable responses to eATP addition is a rapid rise in cytoplasmic free calcium levels ([Ca2+ ]cyt ), which requires P2K1. However, the specific transporter/channel proteins that mediate this rise in [Ca2+ ]cyt are unknown. Through a forward genetic screen, we identified an Arabidopsis ethylmethanesulfonate (EMS) mutant impaired in the [Ca2+ ]cyt response to eATP. Positional cloning revealed that the mutation resided in the cngc6 gene, which encodes cyclic nucleotide-gated ion channel 6 (CNGC6). Mutation of the CNGC6 gene led to a notable decrease in the PM inward Ca2+ current in response to eATP. eATP-induced mitogen-activated protein kinase activation and gene expression were also significantly lower in cngc6 mutant plants. In addition, cngc6 mutant plants were also more susceptible to the bacterial pathogen Pseudomonas syringae. Taken together, our results indicate that CNGC6 plays a crucial role in mediating eATP-induced [Ca2+ ]cyt signaling, as well as plant immunity.

Nicorandil attenuates high glucose-induced insulin resistance by suppressing oxidative stress-mediated ER stress PERK signaling pathway

IntroductionGlucose-induced insulin resistance is a typical character of diabetes. Nicorandil is now widely used in ischemic heart disease. Nicorandil shows protective effects against oxidative and endoplasmic reticulum (ER) stress, which...

Cortisol rapidly stimulates calcium waves in the developing trunk muscle of zebrafish

Glucocorticoids (GCs) play a role in stress coping by activating the glucocorticoid receptor (GR), a ligand-bound transcription factor. GCs also exert rapid effects that are nongenomic by modulating second messenger signaling, including Ca2+. However, the mechanism of action of GCs in modulating cytoplasmic free calcium level ([Ca2+]i) is unclear. We hypothesized that cortisol increases ([Ca2+]i) in zebrafish (Danio rerio) muscle, and this is independent of GR activation. Indeed, cortisol rapidly stimulated ([Ca2+]i) rise in the developing trunk muscle (DTM), and this response was not abolished in the GR knockout zebrafish. The rapid cortisol-induced ([Ca2+]i) rise was reduced with EGTA, and completely abolished by the pharmacological inhibition of the calcium release-activated calcium channel (CRACC). Also, cortisol stimulation rapidly increased the expression of Orai1, the pore forming protein subunit of CRACC, in the DTM. Altogether, rapid nongenomic action of cortisol on muscle function may involve Ca2+ signaling by CRACC gating in zebrafish.

Nicorandil Attenuates High Glucose-Induced Insulin Resistance by Suppressing Oxidative Stress-Mediated Endoplasmic Reticulum Stress Protein Kinase RNA-Like Endoplasmic Reticulum Kinase Singling Pathway

Background: Glucose- induced insulin resistance (IR) is a typical character of diabetes. Nicorandil is widely used in ischemic heart disease. Nicorandil shows protective effects against oxidative and endoplasmic reticulum (ER) stress which are involved in insulin resistance. Here we investigated mechanisms of nicorandil’s novel pharmacological activity on IR in diabetes. Methods: Nicorandil was administrated to diabetic animals and high-glucose exposed skeletal muscle cells. IR and glucose tolerance were evaluated. Molecular mechanisms concerning oxidative stress, ER stress and glucose uptake were assessed. Findings: Nicorandil attenuated high- glucose induced IR without affecting fasting blood glucose and glucose tolerance in whole body and skeletal muscle in diabetic rats. Nicorandil treatment suppressed protein kinase C(PKC)/ nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) system activities by reducing cytoplasmic free calcium level in skeletal muscle cells exposed to high glucose. As a result, the oxidative stress- mediated ER stress protein kinase RNA- like endoplasmic reticulum kinase (PERK)/ eukaryotic initiation factor (EIF) 2α/ activating transcription factor (ATF)4/CEBP homologous protein (CHOP)/tribbles homolog (TRB)3 signaling pathway activation was inhibited. Nicorandil down-regulated expression of TRB3 and thus facilitated Akt phosphorylation in response to insulin stimulation, leading to glucose transporter (GLUT)4 plasma membrane translocation which promoted glucose uptake capability of skeletal muscle cells. Interpretation: By reducing cytoplasmic calcium, nicorandil alleviated high-glucose induced IR by inhibiting oxidative stress- mediated ER stress PERK pathway. This study indicated nicorandil as a promising agent in treatment of patients with ischemic heart disease complicated with diabetes. Funding Statement: This study was supported by Innovative Talents Promotion Project of Shaanxi Province (2019KJXX-019); Fundamental Scientific Research Foundation of Xi’an Jiaotong University (xzy012019131); Health Scientific Foundation of Shaanxi Province (2018E11); Natural Science Basic Research Foundation of Shaanxi Province (2020JQ-941). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: The experimental protocols were reviewed and approved by Institutional Animal Care and Use Committee of Medical Research Institute of Northwestern Polytechnical University.

Ginsenoside Rg5 induces apoptosis in human esophageal cancer cells through the phosphoinositide‑3 kinase/protein kinase B signaling pathway.

The role of ginsenoside in the prevention of cancer has been well established. Ginsenoside Rg5 is one of the main components isolated from red ginseng, which has been demonstrated to have anti-tumor effects by inhibiting cell proliferation and causing DNA damage. However, the role of ginsenoside Rg5 and its molecular mechanisms remain unclear in human esophageal cancer. In the present study, Rg5 was investigated as a novel drug for the chemotherapy of esophageal cancer in in vitro experiments. Esophageal cancer Eca109 cells were exposed to various concentrations of ginsenoside Rg5 (0–32 µΜ) for 24 h. Subsequent cell proliferation assays demonstrated that treatment with ginsenoside Rg5 resulted in the dose-dependent inhibition of proliferation, while a significant increase in apoptotic rate and increased activities of caspase-3, −8 and −9 were observed. In addition, the mitochondrial membrane potential was decreased and the cytoplasmic free calcium level increased following treatment with ginsenoside Rg5. Furthermore, the expression of B-cell lymphoma 2 and phosphorylated-protein kinase B (p-Akt) decreased. The specific phosphoinositide-3 kinase (PI3K) inhibitor LY294002 promoted this effect, while insulin-like growth factor-1, a specific PI3K activator, inhibited this action. Taken together, the results suggested that ginsenoside Rg5 may have a tumor-suppressive effect on esophageal cancer by promoting apoptosis and may be associated with the downregulation of the PI3K/Akt signaling pathway.

Calcium influx kinetics, and the features of potassium channels of peripheral lymphocytes in primary Sjögren’s syndrome

ObjectiveThe transient increase of the cytoplasmic free calcium level plays a key role in the process of lymphocyte activation. Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx and present a possible target for selective immunomodulation. DesignCase–control study. Subjects and methodsWe took peripheral blood samples from 8 healthy individuals and 15 primary Sjögren’s syndrome (pSS) patients. We evaluated calcium influx kinetics following activation in peripheral T lymphocytes. We also assessed the sensitivity of T lymphocytes to specific inhibition of the Kv1.3 and IKCa1 potassium channels, and the Kv1.3 channel expression. ResultsThe basal cytoplasmatic calcium levels were lower in both Th1 and Th2 lymphocytes in pSS compared to controls. The peak of calcium influx in lymphocytes isolated from pSS patients is reached later, indicating that they respond more slowly to stimulation compared to controls. In healthy individuals, the inhibition of the IKCa1 channel decreased calcium influx in Th2 and CD4 cells to a lower extent than in Th1 and CD8 cells. On the contrary, the inhibition of Kv1.3 channels resulted in a larger decrease of calcium entry in Th2 and CD4 than in Th1 and CD8 cells. In the pSS group, neither of the inhibitors induced alteration in calcium influx. Expression of Kv1.3 channels on CD4, Th2 and CD8 lymphocytes in pSS was significantly higher compared to controls. ConclusionThe altered expression and specific inhibition of potassium channels seem to be related to altered calcium influx kinetics in pSS which distinguish pSS either from healthy controls or other systemic autoimmune diseases.

Physiological and ecological significance of biomineralization in plants

Biomineralization is widespread in the plant kingdom. The most common types of biominerals in plants are calcium oxalate crystals, calcium carbonate, and silica. Functions of biominerals may depend on their shape, size, abundance, placement, and chemical composition. In this review we highlight advances in understanding physiological and ecological significance of biomineralization in plants. We focus on the functions of biomineralization in regulating cytoplasmic free calcium levels, detoxifying aluminum and heavy metals, light gathering and scattering to optimize photosynthesis, aiding in pollen release, germination, and tube growth, the roles it plays in herbivore deterrence, biogeochemical cycling of carbon, calcium, and silicon, and sequestering atmospheric CO2.

Characterization of Apoptosis Induced by Emodin and Related Regulatory Mechanisms in Human Neuroblastoma Cells

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Recent studies have shown that emodin can induce or prevent cell apoptosis, although the precise molecular mechanisms underlying these effects are unknown. Experiments from the current study revealed that emodin (10–20 μM) induces apoptotic processes in the human neuroblastoma cell line, IMR-32, but exerts no injury effects at treatment doses below 10 μM. Treatment with emodin at concentrations of 10–20 μM led to a direct increase in the reactive oxygen species (ROS) content in IMR-32 cells, along with significant elevation of cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. Pretreatment with nitric oxide (NO) scavengers suppressed the apoptotic biochemical changes induced by 20 μM emodin, and attenuated emodin-induced p53 and p21 expression involved in apoptotic signaling. Our results collectively indicate that emodin at concentrations of 10–20 μM triggers apoptosis of IMR-32 cells via a mechanism involving both ROS and NO. Based on the collective results, we propose a model for an emodin-triggered apoptotic signaling cascade that sequentially involves ROS, Ca2+, NO, p53, caspase-9 and caspase-3.

AB0047 The effects of KV1.3 and IKCA1 potassium channel inhibition on calcium influx of human peripheral T lymphocytes in rheumatoid arthritis

BackgroundThe transient increase of the cytoplasmic free calcium level plays a key role in the process of lymphocyte activation. Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance...

Analysis by flow cytometry of calcium influx kinetics in peripheral lymphocytes of patients with rheumatoid arthritis

The transient increase of the cytoplasmic free calcium level in T lymphocytes plays a key role in initiating and maintaining the autoimmune reaction in rheumatoid arthritis (RA). Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation. We aimed to compare peripheral T lymphocyte calcium influx kinetics upon activation in patients with recently diagnosed and established RA, and to demonstrate the differences in analysis of kinetic flow cytometry data when using two different algorithms. We took peripheral blood samples from nine patients with recently diagnosed and six patients with established RA. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2, and CD8 cells applying an approach based on smoothing of median fluorescence values (FlowJo) and an algorithm based on function fitting (FacsKin). We assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels. Th2 cells of patients with established RA react slower to activating stimuli, whereas CD8 cells show a faster reaction than in patients with recently diagnosed RA. While initially Th1 cells are less sensitive to the inhibition of Kv1.3 and IKCa1 channels in RA, their sensitivity increases along with the duration of the disease. With the algorithm of function fitting instead of smoothing, more statistically significant differences of potassium channel inhibition between the two RA groups could be demonstrated. The function fitting algorithm applied by FacsKin is suitable to provide a common basis for evaluating and comparing flow cytometry kinetic data.

The effects of Kv1.3 and IKCa1 potassium channel inhibition on calcium influx of human peripheral T lymphocytes in rheumatoid arthritis

ObjectiveThe transient increase of the cytoplasmic free calcium level plays a key role in the process of lymphocyte activation. Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation. DesignCase–control study. Subjects and methodsWe took peripheral blood samples from 10 healthy individuals and 9 recently diagnosed rheumatoid arthritis (RA) patients receiving no anti-rheumatic treatment. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2 and CD8 cells applying a novel flow cytometry approach. We also assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels. ResultsThe peak of calcium influx in lymphocytes isolated from RA patients is reached more rapidly, indicating that they respond more quickly to stimulation compared to controls. In healthy individuals, the inhibition of the IKCa1 channel decreased calcium influx in Th2 and CD4 cells to a lower extent than in Th1 and CD8 cells. On the contrary, the inhibition of Kv1.3 channels resulted in a larger decrease of calcium entry in Th2 and CD4 than in Th1 and CD8 cells. No difference was detected between Th1 and Th2 or CD4 and CD8 cells in the sensitivity to IKCa1 channel inhibition among lymphocytes of RA patients. However, specific inhibition of the Kv1.3 channel acts differentially on calcium influx kinetics in RA lymphocyte subsets. Th2 and particularly CD8 cells are inhibited more dominantly than Th1 and CD4 cells. ConclusionThe inhibition of Kv1.3 channels does not seem to be specific enough in peripheral RA lymphocytes, since anti-inflammatory Th2 cells are also affected to a noteworthy extent.

Human Th1 and Th2 lymphocytes are distinguished by calcium flux regulation during the first 10 min of lymphocyte activation

Preliminary data suggest different intracellular calcium handling of Th1 and Th2 lymphocytes that may contribute to distinct cytokine production patterns. In this study we explored the contribution of the main mechanisms in charge of the elevation and decrease of cytoplasmic free calcium levels, i.e., the endoplasmic calcium release, the calcium release activated calcium (CRAC) channel, the mitochondrial calcium uniporter (MCU), the sarco/endoplasmic reticulum calcium ATPase (SERCA), and the plasma membrane calcium ATPase (PMCA) during the first 10min of activation in human Th1 and Th2 lymphocytes applying a kinetic flow cytometry approach. We isolated peripheral blood mononuclear cells from 10 healthy individuals. Cells were stained with CD4, CXCR3 and CCR4 cell surface markers to identify Th1 and Th2 cells, respectively and loaded with Fluo-3/AM calcium sensitive dye. Cells were activated with phytohemagglutinine and alterations of cytoplasmic free calcium levels were monitored for 10min after specific inhibition of the above mechanisms. Our results revealed delicate differences in calcium flux kinetics of Th1 and Th2 lymphocytes. The lower activity of MCU, and therefore of CRAC channels, along with the higher activity of the SERCA pump account for the notion that Th2 cells go through a lower level of lymphocyte activation compared with Th1 cells upon identical activating stimuli. The observed differences in calcium flux of Th1 and Th2 cells may contribute to different calcium handling kinetics and, hence, to distinct cytokine production patterns by these subsets.

Rottlerin Exhibits Antiangiogenic Effects In vitro

Rottlerin, a natural product purified from Mallotus philippinensis, has a number of target molecules and biological effects. We recently found that Rottlerin caused growth arrest in MCF-7 breast cancer cells and human immortalized keratinocytes, through inhibition of NFκB and downregulation of cyclin D-1. To evaluate whether this effect could be generalized to primary cells, human microvascular endothelial cells were treated with Rottlerin. In this study, we demonstrated that Rottlerin prevents basal and TNFα-stimulated NFκB nuclear migration and DNA binding also in human microvascular endothelial cell, where NFκB inhibition was accompanied by the downregulation of NFκB target gene products, such as cyclin D-1 and endothelin-1, which are essential molecules for endothelial cell proliferation and survival. Rottlerin, indeed, inhibited human microvascular endothelial cells proliferation and tube formation on Matrigel. Rottlerin also increases cytoplasmic free calcium and nitric oxide levels and downregulates endothelin converting enzyme-1 expression, thus contributing to the drop in endothelin-1 and growth arrest. These results suggest that Rottlerin may prove useful in the development of therapeutic agents against angiogenesis.

Impact of dihydrolipoic acid on mouse embryonic stem cells and related regulatory mechanisms

α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet, taken up by cells and tissues, and subsequently reduced to dihydrolipoic acid (DHLA). Recently, DHLA has been used as the hydrophilic nanomaterial preparations, and therefore, determination of its bio-safety profile is essential. In this article, we show that DHLA (50-100 μM) induces apoptotic processes in mouse embryonic stem cells (ESC-B5), but exerts no injury effects at treatment dosages below 50 μM. Higher concentrations of DHLA (50-100 μM) directly increased the reactive oxygen species (ROS) content in ESC-B5 cells, along with a significant increase in cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. Pretreatment with NO scavengers suppressed the apoptotic biochemical changes induced by 100 μM DHLA and promoted the gene expression levels of p53 and p21 involved in apoptotic signaling. Our results collectively indicate that DHLA at concentrations of 50-100 μM triggers apoptosis of ESC-B5 cells, which involves both ROS and NO. Importantly, at doses of less than 50 μM (0-25 μM), DHLA does not exert hazardous effects on ESC-B5 cell properties, including viability, development and differentiation. These results provide important information in terms of dosage safety and biocompatibility of DHLA to facilitate its further use as a precursor for biomaterial preparation.

Golgi Calcium Pump Secretory Pathway Calcium ATPase 1 (SPCA1) Is a Key Regulator of Insulin-like Growth Factor Receptor (IGF1R) Processing in the Basal-like Breast Cancer Cell Line MDA-MB-231

Calcium signaling is a key regulator of pathways important in tumor progression, such as proliferation and apoptosis. Most studies assessing altered calcium homeostasis in cancer cells have focused on alterations mediated through changes in cytoplasmic free calcium levels. Here, we show that basal-like breast cancers are characterized by an alteration in the secretory pathway calcium ATPase 1 (SPCA1), a calcium pump localized to the Golgi. Inhibition of SPCA1 in MDA-MB-231 cells produced pronounced changes in cell proliferation and morphology in three-dimensional culture, without alterations in sensitivity to endoplasmic reticulum stress induction or changes in global calcium signaling. Instead, the effects of SPCA1 inhibition in MDA-MB-231 cells reside in altered regulation of calcium-dependent enzymes located in the secretory pathway, such as proprotein convertases. Inhibition of SPCA1 produced a pronounced alteration in the processing of insulin-like growth factor receptor (IGF1R), with significantly reduced levels of functional IGF1Rβ and accumulation of the inactive trans-Golgi network pro-IGF1R form. These studies identify for the first time a calcium transporter associated with the basal-like breast cancer subtype. The pronounced effects of SPCA1 inhibition on the processing of IGF1R in MDA-MB-231 cells independent of alterations in global calcium signaling also demonstrate that some calcium transporters can regulate the processing of proteins important in tumor progression without major alterations in cytosolic calcium signaling. Inhibitors of SPCA1 may offer an alternative strategy to direct inhibitors of IGF1R and attenuate the processing of other proprotein convertase substrates important in basal breast cancers.

Impact of Methylglyoxal and High Glucose Co-treatment on Human Mononuclear Cells

Hyperglycemia and elevation of methylglyoxal (MG) are symptoms of diabetes mellitus (DM). In this report, we show that co-treatment of human mononuclear cells (HMNCs) with MG (5 μM) and high glucose (HG; 15 – 30 mM) induces apoptosis or necrosis. HG/MG co-treatment directly enhanced the reactive oxygen species (ROS) content in HMNCs, leading to decreased intracellular ATP levels, which control cell death via apoptosis or necrosis. Concentrations of 5 μM MG and 15 mM glucose significantly increased cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. In contrast, no apoptotic biochemical changes were detected in HMNCs treated with 5 μM MG and 25 mM glucose, which appeared to undergo necrosis. Pretreatment with nitric oxide (NO) scavengers inhibited apoptotic biochemical changes induced by 5 μM MG/15 mM glucose, and increased the gene expression levels of p53 and p21 involved in apoptotic signaling. The results collectively suggest that the treatment dosage of MG and glucose determines the mode of cell death (apoptosis vs. necrosis) of HMNCs, and that both ROS and NO play important roles in MG/HG-induced apoptosis.

Effect of salicylic acid and calcium on mitochondrial functions.

The rapid mitochondrial uptake of calcium followed by slow release in certain pathophysiological states associated with an increase in intracellular calcium, to normalize the cytoplasmic levels of free calcium, provides an important protective mechanism against calcium cellular toxicity. Salicylic acid, an in vivo metabolite of aspirin, inhibits the uptake and enhances the release of calcium by mitochondria, thereby increasing the levels of cytoplasmic free calcium. The Ca2+ induced mitochondrial swelling is enhanced in the presence of salicylic acid and in which turn leads to loss of biosynthesis of ATP. These results suggest that salicylic acid may promote cellular damage in pathophysiological states associated with increase in intracellular free calcium.

Citrinin induces apoptosis in mouse embryonic stem cells

The mycotoxin citrinin (CTN) is a natural contaminant in foodstuffs and animal feeds, and exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis. However, its precise regulatory mechanisms of action, particularly in stem cells and embryos, are currently unclear. Recent studies show that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Experiments with the embryonic stem cell line, ESC-B5, disclose that CTN induces apoptosis via several mechanisms, including ROS generation, increased cytoplasmic free calcium levels, intracellular nitric oxide production, enhanced Bax/Bcl-2 ratio, loss of mitochondrial membrane potential, cytochrome c release, activation of caspase-9 and caspase-3, and p21-activated protein kinase 2 and c-Jun N-terminal protein kinase activation. Additional studies show that CTN promotes cell death via inactivation of the HSP90/multi-chaperone complex and subsequent degradation of Ras and Raf-1, further inhibiting anti-apoptotic processes such as the Ras-->ERK signal transduction pathway. On the basis of these findings, we propose a model for CTN-induced cell injury signalling cascades in embryonic stem cells and blastocysts.

Methylglyoxal and high glucose co‐treatment induces apoptosis or necrosis in human umbilical vein endothelial cells

Hyperglycemia and elevation of methylglyoxal (MG) are symptoms of diabetes mellitus (DM). We previously showed that high glucose (HG; 30 mM) or MG (50-400 microM) could induce apoptosis in mammalian cells, but these doses are higher than the physiological concentrations of glucose and MG in the plasma of DM patients. The physiological concentration of MG and glucose in the normal blood circulation is about 1 microM and 5 mM, respectively. Here, we show that co-treatment with concentrations of MG and glucose comparable to those seen in the blood circulation of DM patients (5 microM and 15-30 mM, respectively) could cause cell apoptosis or necrosis in human umbilical vein endothelial cells (HUVECs) in vitro. HG/MG co-treatment directly increased the reactive oxygen species (ROS) content in HUVECs, leading to increases in intracellular ATP levels, which can control cell death through apoptosis or necrosis. Co-treatment of HUVECs with 5 microM MG and 20 mM glucose significantly increased cytoplasmic free calcium levels, activation of nitric oxide synthase (NOS), caspase-3 and -9, cytochrome c release, and apoptotic cell death. In contrast, these apoptotic biochemical changes were not detected in HUVECs treated with 5 microM MG and 30 mM glucose, which appeared to undergo necrosis. Pretreatment with nitric oxide (NO) scavengers could inhibit 5 microM MG/20 mM glucose-induced cytochrome c release, decrease activation of caspase-9 and caspase-3, and increase the gene expression and protein levels of p53 and p21, which are known to be involved in apoptotic signaling. Inhibition of p53 protein expression using small interfering RNA (siRNA) blocked the activation of p21 and the cell apoptosis induced by 5 microM MG/20 mM glucose. In contrast, inhibition of p21 protein expression by siRNA prevented apoptosis in HUVECs but had no effect on p53 expression. These results collectively suggest that the treatment dosage of MG and glucose could determine the mode of cell death (apoptosis vs. necrosis) in HUVECs, and both ROS and NO played important roles in MG/HG-induced apoptosis of these cells.

ACTIVATION OF DOMESTIC CAT OOCYTES USING THE SULFHYDRYL REAGENT THIMEROSAL

Domestic cats are useful as animal models in comparative medicine programs as they provide insight into disease mechanism and facilitate investigation of new therapeutic options. The application of cloning in these animals is also beneficial in terms of conserving the precious animal models. Although successful cat cloning by nuclear transfer has recently been reported, the technology is still extremely ineffective. One potential reason for the low efficiency is suboptimal embryo development after oocyte activation. During nuclear transfer, the calcium signals that are normally triggered by the fertilizing sperm must be reproduced artificially to initiate the development of the reconstructed embryos. Thimerosal is a chemical which is able to oxidize critical sulfhydryl groups on intracellular calcium release proteins and thus induce the release of calcium from the intracellular stores and trigger oocyte activation. The effects of thimerosal have previously been reported in a number of species; the objective of this study was to evaluate whether thimerosal can induce calcium release and initiate parthenogenetic embryo development in the oocyte of the domestic cat. Cat ovaries were obtained from a local veterinary clinic. Immature oocytes collected from the ovaries were matured in vitro in Feline Optimized Culture Medium (FOCM) supplemented with 0.6 mM cysteine, 0.1 mM cysteamine, 1.0 IU/ml eCG, 2.0 IU/ml hCG, 25 ng/ml EGF, for 24 hours. For intracellular calcium measurements, cat oocytes were incubated in the presence of 2 μM fura-2 AM, a calcium indicator dye and 0.02% pluronic F-127, for 40 minutes. Individual oocytes were then transferred into TL-Hepes medium containing 200 μM thimerosal. Changes in the cytoplasmic free calcium levels were monitored using the InCyt Im2 fluorescence imaging system. Preimplantation embryonic development was also evaluated; for this purpose the oocytes were incubated in TL-Hepes in the presence of 200 μM thimerosal for 30 minutes followed by a 20-min incubation in TL-Hepes with 8 mM dithiothreitol, a sulfhydryl reducing agent. Control oocytes were cultured in TL-Hepes for 50 minutes. After the treatments, the oocytes were held in 7.5 μg/ml cytochalasin B for 4 hours to stimulate the formation of diploid embryos then cultured in FOCM for 6 days. The total number of nuclei in the developing embryos was recorded by staining with Hoechst 33342. After the addition of thimerosal, a series of transient rises in the intracellular free calcium concentration were observed in 81.8% (9 out of 11) of the oocytes during the 30-min incubation period. Furthermore, thimerosal induced cleavage in 21.8% (14/64) of the activated oocytes; 4.7% (3/64) of the oocytes developed to morula or blastocyst stage. The average cell number of those morulae/blastocysts was 19.7±3.2. In the control group, no cleavage was observed (0/43). The results support the hypothesis that thimerosal can induce an oscillation in the intracytoplasmic free calcium levels in oocytes by oxidizing sulfhydryl groups on intracellular calcium release proteins. The series of calcium increases was sufficient to trigger the signal transduction pathway in cat oocytes that resulted in embryonic development up to the blastocyst stage. The results indicate the potential application of thimerosal for oocyte activation during cat nuclear transfer. Additional studies are needed for the optimization of the activation parameters. (poster)