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Ca⁽²⁺⁾N It Be Measured? Detection of Extramitochondrial Calcium Movement With High-Resolution FluoRespirometry.

ABSTRACT: Our aim was to develop a method to detect extramitochondrial Ca²⁺ movement and O₂ fluxes simultaneously. Using High-Resolution FluoRespirometry, we also tested whether mitochondrial permeability transition pore (mPTP) inhibition or anoxia affects the mitochondrial Ca²⁺ flux. Ca²⁺ movement evoked by CaCl₂ or anoxia was assessed with CaGreen-5N dye using Blue-Fluorescence-Sensor in isolated liver mitochondria, liver homogenates and duodenal biopsies. Exogenous CaCl₂ (50?µM) resulted in an abrupt elevation in CaGreen-5N fluorescence followed by a decrease (Ca²⁺ uptake) with simultaneous elevation in O₂ consumption in liver preparations. This was followed by a rapid increase in the fluorescence signal, reaching a higher intensity (Ca²⁺ efflux) than that of the initial CaCl₂-induced elevation. Chelation of Ca²⁺ with EGTA completely abolished the fluorescence of the indicator. After pre-incubation with cyclosporin A, a marked delay in Ca²⁺ movement was observed, not only in isolated liver mitochondria, but also in tissue homogenates. In all samples, the transition to anoxia resulted in immediate increase in the level of extramitochondrial Ca²⁺. The results demonstrate that the CaGreen-5N method is suitable to monitor simultaneous O₂ and Ca²⁺ fluxes, and the opening of mPTP in various biological samples. In this system the duration of stimulated Ca²⁺ fluxes may provide a novel parameter to evaluate the efficacy of mPTP blocker compounds.

SUBMITTER: Naszai A

PROVIDER: S-EPMC6917783 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The nutritional status of dairy cows and the metabolism of specific nutrients are critical regulators of immune cell function. Around the time of parturition, mobilization of body lipid and muscle helps compensate for the decrease in nutrient intake and the increased requirements of the mammary gland for lactation. An end-result of these processes is the marked increase in circulating concentrations of fatty acids (FA), which are a major risk factor for immune dysfunction. In food animal species such as dairy cows, any disturbance in nutritional or immunological homeostasis leads to deleterious feedback loops that can further risk health, efficiency of nutrient use, and compromise availability of safe and nutritious dairy foods for humans. Despite substantial progress with respect to regulation of innate immunity, such knowledge for adaptive immunity is scarce. To help bridge this gap in knowledge, we sought to study the role of calcium release-activated calcium modulator ORAI1 activation in T cells systemic immune function in vivo. CD4+ T cells were isolated from peripheral blood of dairy cows diagnosed as healthy or with ketosis, a common metabolic disorder of FA metabolism. Results revealed that levels of intracellular Ca2+ and reactive oxygen species (ROS) along with the abundance of store-operated Ca2+ entry (SOCE) moiety increased during ketosis. Further, plasma concentrations of inflammatory cytokines were elevated, the balance of Th17/Treg cells was disrupted, mitochondrial function impaired, and the abundance of mitophagy-related proteins in CD4+ T cells altered during ketosis. Molecular characterization of the direct effects of FA was evaluated in CD4+ T cells isolated from the spleen of 1-day-old calves. Enhanced supply of FA increased intracellular Ca2+ and ROS concentrations, upregulated the abundance of proteins associated with mitochondrial dynamics and ORAI1. Intermediates of mitophagy accumulated and the balance of Th17/Treg cells also was affected by the supply of FA. These negative effects were attenuated by silencing or inhibition of ORAI1 in CD4+ T cells. Together, data indicated that physiological states that lead to increases in systemic concentrations of FA could impact adaptive immunity negatively through ORAI1 regulated intracellular Ca2+, ROS balance, and increased effector functions of Th17 cells.

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Ca⁽²⁺⁾N It Be Measured? Detection of Extramitochondrial Calcium Movement With High-Resolution FluoRespirometry.

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Ca(2+)N It Be Measured? Detection of Extramitochondrial Calcium Movement With High-Resolution FluoRespirometry.

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Ca⁽²⁺⁾N It Be Measured? Detection of Extramitochondrial Calcium Movement With High-Resolution FluoRespirometry.