Changes in Cytosolic Ca2+ Levels Regulate Bcl-xS and Bcl-xL Expression in Spermatogenic Cells during Apoptotic Death

Mishra, Durga Prasad; Pal, Rajarshi; Shaha, Chandrima

doi:10.1074/jbc.m508648200

Cited by 49 publications

(51 citation statements)

References 43 publications

(61 reference statements)

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“…The day preceding the end of the experiment, HUVECs were plated at a density of 7,000 cells (70% confluence) on to white 96-well microplates with clear bottoms (3610; Costar, Cambridge, MA, USA) in 5 mmol/l with or without inhibitors. At the end of the experiment, medium was removed from the cells and 2 μg/ml of the cell-permeable CM-H 2 DCFDA probe (Molecular Probes-Invitrogen, Eugene, OR, USA) in Hank's Balanced Salt Solution was added to each well of the plates and incubated for 15 min at 37°C as previously described [22]. Fluorescence intensity was then measured using a plate reader (BMG Labtech, Durham, NC, USA) using 488 nm excitation and 530 nm emission filters.…”

Section: Methodsmentioning

confidence: 99%

Reactive oxygen species mediate a cellular ‘memory’ of high glucose stress signalling

et al. 2007

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Aims/hypothesis A long-term 'memory' of hyperglycaemic stress, even when glycaemia is normalised, has been previously reported in endothelial cells. In this report we sought to duplicate and extend this finding. Materials and methods HUVECs and ARPE-19 retinal cells were incubated in 5 or in 30 mmol/l glucose for 3 weeks or subjected to 1 week of normal glucose after being exposed for 2 weeks to continuous high glucose. HUVECs were also treated in this last condition with several antioxidants. Similarly, four groups of rats were studied for 3 weeks: (1) normal rats; (2) diabetic rats not treated with insulin; (3) diabetic rats treated with insulin during the last week; and (4) diabetic rats treated with insulin plus α-lipoic acid in the last week. Results In human endothelial cells and ARPE-19 retinal cells in culture, as well as in the retina of diabetic rats, levels of the following markers of high glucose stress remained induced for 1 week after levels of glucose had normalised: protein kinase C-β, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3-nitrotyrosine, fibronectin, poly(ADPribose) Blockade of reactive species using different approaches, i.e. the mitochondrial antioxidant α-lipoic acid, overexpression of uncoupling protein 2, oxypurinol, apocynin and the poly(ADP-ribose) polymerase inhibitor PJ34, interrupted the induction both of high glucose stress markers and of the fluorescent reactive oxygen species (ROS) probe CM-H 2 DCFDA in human endothelial cells. Similar results were obtained in the retina of diabetic rats with α-lipoic acid added to the last week of normalised glucose. Conclusions/interpretation These results provide proofof-principle of a ROS-mediated cellular persistence of vascular stress after glucose normalisation.

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Section: Methodsmentioning

confidence: 99%

Reactive oxygen species mediate a cellular ‘memory’ of high glucose stress signalling

et al. 2007

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“…Fmax represents the maximum fluorescence (obtained by treating cells with 10 μM calcium ionophore A23187), and Fmin corresponds to the minimum fluorescence (obtained from ionophore-treated cells in the presence of 3 mM EGTA). The fluorescence intensities were expressed as the increase in fluorescence with respect to baseline fluorescence intensity before stimulation (62). The experiments were repeated 3 times per biopsy.…”

Section: Figurementioning

confidence: 99%

Role for protease activity in visceral pain in irritable bowel syndrome

Cenac¹,

Andrews²,

Holzhausen³

et al. 2007

J. Clin. Invest.

506

551

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Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-κB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR 2 ). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR 2 antagonist and were absent in PAR 2 -deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR 2 .

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“…Changes in intracellular free Ca 2ϩ concentration were monitored using the Ca 2ϩ binding fluorescent probe fluo-3-AM as previously described (36). Briefly, 10 6 cells/ml loaded with 0.5 M fluo-3-AM containing 0.5 M pluronic acid F-127 were used for different experiments, and free Ca 2ϩ was monitored at an excitation of 480 nm and emission of 520 nm with a Fluostar Optima spectrofluorometer (BMG Technologies).…”

Section: Intracellular Free Ca 2ϩ Assaymentioning

confidence: 99%

Up-Regulation of Bcl-2 through ERK Phosphorylation Is Associated with Human Macrophage Survival in an Estrogen Microenvironment

Subramanian

Shaha

2007

The Journal of Immunology

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Estrogen is a known immunomodulator with pleiotropic effects on macrophage function that partly accounts for the gender bias observed in numerous autoimmune, cardiovascular, and neurodegenerative disorders. The effect of estrogen on the survival of human macrophages is largely unknown, and in this study we demonstrate that 17β-estradiol (E2) provokes a death response in human THP-1 macrophages by initiating Bax translocation from cytosol to the mitochondria; however, a concomitant up-regulation of Bcl-2 creates a Bax to Bcl-2 ratio favorable for Bcl-2, thus ensuring cell survival. Both Bcl-2 up-regulation and Bax translocation are estrogen receptor-dependent events; however, Bcl-2 augmentation but not Bax translocation is dependent on Ca2+ increase, activation of protein kinase C, and ERK phosphorylation. This estrogen-induced Bcl-2 increase is crucial for the survival of THP-1 macrophages as well as that of human peripheral blood monocyte-derived macrophages, which is evident from E2-induced cell death under small interfering RNA-mediated Bcl-2 knockdown conditions. Hence, this study demonstrates that E2-induced Bcl-2 up-regulation is a homeostatic survival mechanism necessary for the manifestation of immunomodulatory effect of estrogen on human macrophages.

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Changes in Cytosolic Ca2+ Levels Regulate Bcl-xS and Bcl-xL Expression in Spermatogenic Cells during Apoptotic Death

Cited by 49 publications

References 43 publications

Reactive oxygen species mediate a cellular ‘memory’ of high glucose stress signalling

Reactive oxygen species mediate a cellular ‘memory’ of high glucose stress signalling

Role for protease activity in visceral pain in irritable bowel syndrome

Up-Regulation of Bcl-2 through ERK Phosphorylation Is Associated with Human Macrophage Survival in an Estrogen Microenvironment

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