A microplate technique to simultaneously assay calcium accumulation in endoplasmic reticulum and SERCA release of inorganic phosphate

McMullen, David C.; Kean, William S.; Verma, Ajay; Cole, John T.; Watson, William D.

doi:10.1186/1480-9222-14-4

Cited by 9 publications

(10 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Activity of sarco/endoplasmic Ca 2+ ATPase (SERCA), the main SR Ca 2+ pump of skeletal muscle, was estimated in SR isolated membranes from gastrocnemius by using a colorimetric assay that quantifies the amount of inorganic phosphate (P i ) that complexes with ammonium molybdate and malachite green following release from SERCA-mediated ATP hydrolysis [ 43 , 44 ]. Briefly, the reagent to quantify P i was prepared by mixing 1 vol of 10% (w/v) ammonium molybdate in 4 M HCl with 3 vol 0.2% (w/v) malachite green in 4 M HCl.…”

Section: Methodsmentioning

confidence: 99%

Aerobic Training Prevents Heatstrokes in Calsequestrin‐1 Knockout Mice by Reducing Oxidative Stress

Guarnier

Michelucci

Serano

et al. 2018

Oxidative Medicine and Cellular Longevity

View full text Add to dashboard Cite

Calsequestrin-1 knockout (CASQ1-null) mice suffer lethal episodes when exposed to strenuous exercise and environmental heat, crises known as exertional/environmental heatstroke (EHS). We previously demonstrated that administration of exogenous antioxidants (N-acetylcysteine and trolox) reduces CASQ1-null mortality during exposure to heat. As aerobic training is known to boost endogenous antioxidant protection, we subjected CASQ1-null mice to treadmill running for 2 months at 60% of their maximal speed for 1 h, 5 times/week. When exposed to heat stress protocol (41°C/1 h), the mortality rate of CASQ1-null mice was significantly reduced compared to untrained animals (86% versus 16%). Protection from heatstrokes was accompanied by a reduced increase in core temperature during the stress protocol and by an increased threshold of response to caffeine of isolated extensor digitorum longus muscles during in vitro contracture test. At cellular and molecular levels, aerobic training (i) improved mitochondrial function while reducing their damage and (ii) lowered calpain activity and lipid peroxidation in membranes isolated from sarcoplasmic reticulum and mitochondria. Based on this evidence, we hypothesize that the protective effect of aerobic training is essentially mediated by a reduction in oxidative stress during exposure of CASQ1-null mice to adverse environmental conditions.

show abstract

Section: Methodsmentioning

confidence: 99%

Aerobic Training Prevents Heatstrokes in Calsequestrin‐1 Knockout Mice by Reducing Oxidative Stress

Guarnier

Michelucci

Serano

et al. 2018

Oxidative Medicine and Cellular Longevity

View full text Add to dashboard Cite

show abstract

“…Underneath, a carefully placed clean microplate will gather the filtrate dripping from above, being that microsomes that accumulated 45 Ca 2+ are incapable of passing through the filter, due to their charge and size. added to the filter wells and 45 Ca 2+ accumulation is determined using a Wallac microbeta liquid scintillation counter [8].…”

Section: Radiolabeled Calcium Measuringmentioning

confidence: 99%

Methods for Studying Endoplasmic Reticulum Stress

Silva¹,

Valentão²,

Andrade³

et al. 2019

Preprint

View full text Add to dashboard Cite

The endoplasmic reticulum (ER) comprises a network of tubules and vesicles that constitutes the largest organelle of the eukaryotic cell. Being the location where most proteins are synthesized and folded, it is crucial for the upkeep of cellular homeostasis. In addition, it is the largest ionic calcium reservoir in cells, tightly regulating the levels of this second messenger according to cellular necessities. Disturbed ER homeostasis triggers the activation of an intricate and conserved molecular machinery, termed the unfolded protein response (UPR). Given the impact of this signaling network upon an extensive list of cellular processes, ER stress is involved in the onset and progression of multiple diseases, including cancer and neurodegenerative disorders. There is, for this reason, an increasing number of publications focused on characterizing and/or modulating ER stress, which have resulted in a wide array of techniques employed to study ER-related molecular events. This review aims to sum up the tools available design a study of this nature.

show abstract

“…Intraluminal calcium is the prime regulator for ER function, which has also been studied concomitantly in microsomes by researchers in last decades. Using non-fluorescent methods such as radioactive labelling of calcium ions, extensive studies have been reported using rat brain microsomes (12,13), hepatic microsomes (14) and rabbit skeletal microsomes (15). All the aforementioned radioactive Ca 2+ uptake and release studies have been performed only for native channels and pumps present in the ER such as Sarcoplasmic reticulum Calcium ATPase (SERCA) and leak channels such as Ryanodine receptors (RyR1 and RyR2).…”

Section: Introductionmentioning

confidence: 99%

Targeted Esterase induced Dye loading supports Calcium Imaging in Eukaryotic Cell-Free Systems

Dhandapani

Dondapati

Zemella

et al. 2020

Preprint

View full text Add to dashboard Cite

Calcium imaging is an important functional tool for addressing ion channels, transporters and pumps for drug screening in living cells. Depicted eukaryotic cell-free systems utilize microsomes, derived from endoplasmic reticulum to incorporate the synthesized membrane proteins. Absence or inadequate amount of carboxylesterase in the endoplasmic reticulum of eukaryotic cells, which is necessary to cleave the acetoxymethyl ester moiety of the chemical calcium indicators, advocates the hindrance to perform calcium imaging in microsomes. In this work, we try to overcome this drawback and adapt the cell-based calcium imaging principle to a cell-free protein synthesis platform. Carboxylesterase synthesized in a Spodoptera frugiperda Sf21 lysate translation system is established as a viable calcium imaging tool and hTRPV1 is used as a model channel protein to demonstrate the realization of this concept.

show abstract

A microplate technique to simultaneously assay calcium accumulation in endoplasmic reticulum and SERCA release of inorganic phosphate

Cited by 9 publications

References 35 publications

Aerobic Training Prevents Heatstrokes in Calsequestrin‐1 Knockout Mice by Reducing Oxidative Stress

Aerobic Training Prevents Heatstrokes in Calsequestrin‐1 Knockout Mice by Reducing Oxidative Stress

Methods for Studying Endoplasmic Reticulum Stress

Targeted Esterase induced Dye loading supports Calcium Imaging in Eukaryotic Cell-Free Systems

Contact Info

Product

Resources

About