Use of thapsigargin to study Ca2+ homeostasis in cardiac cells

Rogers, Terry B.; Inesi, Giuseppe; Wade, Robert; Lederer, W. Jonathan

doi:10.1007/bf01788366

Cited by 128 publications

(90 citation statements)

References 16 publications

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“…On stimulation with the calcium ionophore A23187, CSE translocates from the cytosol into mitochondria, and H 2 S is now produced inside mitochondria. The promotion of CSE translocation by A23187 is due to an increased intracellular calcium level ( (1,7,8). Thapsigargin also may affect protein and DNA synthesis, DNA fragmentation apoptosis (9), and ER stress (10).…”

Section: Discussionmentioning

confidence: 99%

Hydrogen sulfide (H ₂ S) metabolism in mitochondria and its regulatory role in energy production

Zhang²,

Wu³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

417

325

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Although many types of ancient bacteria and archea rely on hydrogen sulfide (H 2 S) for their energy production, eukaryotes generate ATP in an oxygen-dependent fashion. We hypothesize that endogenous H 2 S remains a regulator of energy production in mammalian cells under stress conditions, which enables the body to cope with energy demand when oxygen supply is insufficient. Cystathionine γ-lyase (CSE) is a major H 2 S-producing enzyme in the cardiovascular system that uses cysteine as the main substrate. Here we show that CSE is localized only in the cytosol, not in mitochondria, of vascular smooth-muscle cells (SMCs) under resting conditions, revealed by Western blot analysis and confocal microscopy of SMCs transfected with GFP-tagged CSE plasmid. After SMCs were exposed to A23187, thapsigargin, or tunicamycin, intracellular calcium level was increased, and CSE translocated from the cytosol to mitochondria. CSE was coimmunoprecipitated with translocase of the outer membrane 20 (Tom20) in mitochondrial membrane. Tom20 siRNA significantly inhibited mitochondrial translocation of CSE and mitochondrial H 2 S production. The cysteine level inside mitochondria is approximately three times that in the cytosol. Translocation of CSE to mitochondria metabolized cysteine, produced H 2 S inside mitochondria, and increased ATP production. Inhibition of CSE activity reversed A23187-stimulated mitochondrial ATP production. H 2 S improved mitochondrial ATP production in SMCs with hypoxia, which alone decreased ATP production. These results suggest that translocation of CSE to mitochondria on specific stress stimulations is a unique mechanism to promote H 2 S production inside mitochondria, which subsequently sustains mitochondrial ATP production under hypoxic conditions. mitochondrion | oxygen sensing | evolution | sulfur metabolism | phenylephrine M any photoautotrophic and chemoautotrophic bacteria and certain animals, such as the lugworm Arenicola marina, use sulfide as an energetic substrate. Mitochondria are the powerhouse of eukaryotic cells, where ATP is produced via oxidative phosphorylation. Considering mitochondria as the evolutionary trait of bacteria in eukaryotes, the metabolism of hydrogen sulfide (H 2 S) in mitochondria may serve as a means for energy supplementation. It has been demonstrated that H 2 S can drastically reduce metabolic demand (1). Similar to nitric oxide, H 2 S exerts protective effects on mitochondrial function and respiration (2). However, a conventional belief is that H 2 S is produced in the cytoplasm resulting from the cytosol localization of H 2 Sgenerating enzymes and is consumed through oxidation in mitochondria (3). It was recently demonstrated that mitochondria of human colon adenocarcinoma cells use sulfide as an energetic substrate at low micromolar concentrations, well below toxic levels (4). The foregoing observations call for reevaluation of the metabolism of H 2 S and its role in mitochondrial energization of eukaryotes. In the present study, we explored whether H 2 S can be prod...

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Section: Discussionmentioning

confidence: 99%

Hydrogen sulfide (H ₂ S) metabolism in mitochondria and its regulatory role in energy production

Zhang²,

Wu³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

417

325

View full text Add to dashboard Cite

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“…3F). Thapsigargin is a tight-binding inhibitor of sarcoplasmic and=or endoplasmic reticulum calcium ATPase (SERCA) (35). Cells preincubated in thapsigargin did not respond to peroxide with increased cytosolic calcium levels (Fig.…”

Section: Calcium Responses Induced By H 2 O 2 In Ht22 Cells Measuredmentioning

confidence: 99%

The Neuroprotective Functions of Selenoprotein M and its Role in Cytosolic Calcium Regulation

Reeves

Bellinger

Berry

2010

Antioxidants & Redox Signaling

136

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Selenoproteins contain the trace element selenium incorporated as selenocysteine, the 21 st amino acid. Some members of the selenoprotein family, such as the glutathione peroxidases, have well-characterized antioxidant activity, functioning in enzymatic breakdown of hydroperoxides to protect cells against oxidative stress. However, the functions of many of the 25 human selenoproteins, including the brain-enriched selenoprotein M, are unknown. We investigated selenoprotein M function by manipulating expression in murine hippocampal HT22 cells, cerebellar astrocyte C8-D1A cells, and primary neuronal cultures. Overexpression of the protein resulted in a reduction in reactive oxygen species and apoptotic cell death in response to oxidative challenge with hydrogen peroxide. In contrast, knock-down of selenoprotein M using shRNA in primary neuronal cultures caused apoptotic cell death comparable to levels resulting from addition of hydrogen peroxide. Calcium measurements with the indicator cameleon demonstrated that overexpression of selenoprotein M decreased calcium influx in response to hydrogen peroxide. Additionally, knock-down of selenoprotein M expression in cortical cultures caused higher baseline levels of cytosolic calcium than in control cells. These results suggest that selenoprotein M may have an important role in protecting against oxidative damage in the brain and may potentially function in calcium regulation. Antioxid. Redox Signal. 12, 809-818.

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“…46 Thapsigargin blocks SERCA, which normally pumps Ca 2þ from the cytosol into the lumen of the sarco-ER, [48][49][50] thereby resulting in the depletion of intracellular stores. Remaining Ca 2þ is eventually cleared by plasma membrane Ca 2þ pumps.…”

Section: Intracellular Ca 2+ After Infrared Exposurementioning

confidence: 99%

Ryanodine and IP₃ receptor-mediated calcium signaling play a pivotal role in neurological infrared laser modulation

et al. 2017

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Abstract. Pulsed infrared (IR) laser energy has been shown to modulate neurological activity through both stimulation and inhibition of action potentials. While the mechanism(s) behind this phenomenon is (are) not completely understood, certain hypotheses suggest that the rise in temperature from IR exposure could activate temperature-or pressure-sensitive ion channels or create pores in the cellular outer membrane, allowing an influx of typically plasma-membrane-impermeant ions. Studies using fluorescent intensity-based calcium ion (Ca 2þ ) sensitive dyes show changes in Ca 2þ levels after various IR stimulation parameters, which suggests that Ca 2þ may originate from the external solution. However, activation of intracellular signaling pathways has also been demonstrated, indicating a more complex mechanism of increasing intracellular Ca 2þ concentration. We quantified the Ca 2þ mobilization in terms of influx from the external solution and efflux from intracellular organelles using Fura-2 and a high-speed ratiometric imaging system that rapidly alternates the dye excitation wavelengths. Using nonexcitable Chinese hamster ovarian (CHO-hM 1 ) cells and neuroblastomaglioma (NG108) cells, we demonstrate that intracellular IP 3 receptors play an important role in the IR-induced Ca 2þ , with the Ca 2þ response augmented by ryanodine receptors in excitable cells.

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Use of thapsigargin to study Ca2+ homeostasis in cardiac cells

Cited by 128 publications

References 16 publications

Hydrogen sulfide (H ₂ S) metabolism in mitochondria and its regulatory role in energy production

Hydrogen sulfide (H ₂ S) metabolism in mitochondria and its regulatory role in energy production

The Neuroprotective Functions of Selenoprotein M and its Role in Cytosolic Calcium Regulation

Ryanodine and IP₃ receptor-mediated calcium signaling play a pivotal role in neurological infrared laser modulation

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Use of thapsigargin to study Ca2+ homeostasis in cardiac cells

Cited by 128 publications

References 16 publications

Hydrogen sulfide (H 2 S) metabolism in mitochondria and its regulatory role in energy production

Hydrogen sulfide (H 2 S) metabolism in mitochondria and its regulatory role in energy production

The Neuroprotective Functions of Selenoprotein M and its Role in Cytosolic Calcium Regulation

Ryanodine and IP3 receptor-mediated calcium signaling play a pivotal role in neurological infrared laser modulation

Contact Info

Product

Resources

About

Hydrogen sulfide (H ₂ S) metabolism in mitochondria and its regulatory role in energy production

Hydrogen sulfide (H ₂ S) metabolism in mitochondria and its regulatory role in energy production

Ryanodine and IP₃ receptor-mediated calcium signaling play a pivotal role in neurological infrared laser modulation