RESUMO
Mitochondrial respiration extends beyond ATP generation, with the organelle participating in many cellular and physiological processes. Parallel changes in components of the mitochondrial electron transfer system with respiration render it an appropriate hub for coordinating cellular adaption to changes in oxygen levels. How changes in respiration under functional hypoxia (i.e., when intracellular O2 levels limit mitochondrial respiration) are relayed by the electron transfer system to impact mitochondrial adaption and remodeling after hypoxic exposure remains poorly defined. This is largely due to challenges integrating findings under controlled and defined O2 levels in studies connecting functions of isolated mitochondria to humans during physical exercise. Here we present experiments under conditions of hypoxia in isolated mitochondria, myotubes and exercising humans. Performing steady-state respirometry with isolated mitochondria we found that oxygen limitation of respiration reduced electron flow and oxidative phosphorylation, lowered the mitochondrial membrane potential difference, and decreased mitochondrial calcium influx. Similarly, in myotubes under functional hypoxia mitochondrial calcium uptake decreased in response to sarcoplasmic reticulum calcium release for contraction. In both myotubes and human skeletal muscle this blunted mitochondrial adaptive responses and remodeling upon contractions. Our results suggest that by regulating calcium uptake the mitochondrial electron transfer system is a hub for coordinating cellular adaption under functional hypoxia.
Assuntos
Cálcio , Consumo de Oxigênio , Humanos , Cálcio/metabolismo , Consumo de Oxigênio/fisiologia , Respiração Celular , Hipóxia/metabolismo , Músculo Esquelético/metabolismo , Oxigênio/metabolismoRESUMO
The molecular assembly of cells depends not only on the balance between anabolism and catabolism but to a large degree on the building blocks available in the environment. For cultured mammalian cells, this is largely determined by the composition of the applied growth medium. Here, we study the impact of lipids in the medium on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum-free and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins strongly depends on the lipid environment in cultured cells and favors the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This raises the question on a link between membrane composition and respiratory control. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. This underlines the importance of considering these factors when using and establishing cell culture models in biomedical research. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium.
Assuntos
Cardiolipinas/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Células HeLa , Humanos , Suínos , Espectrometria de Massas em Tandem , Células Tumorais CultivadasRESUMO
ANG II has many biological effects in renal physiology, particularly in Ca2+ handling in the regulation of fluid and solute reabsorption. It involves the systemic endocrine renin-angiotensin system (RAS), but tissue and intracrine ANG II are also known. We have shown that ANG II induces heterodimerization of its AT1 and AT2 receptors (AT1R and AT2R) to stimulate sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity. Thus, we investigated whether ANG II-AT1R/AT2R complex is formed and internalized, and also examined the intracellular localization of this complex to determine how its effect might be exerted on renal intracrine RAS. Living cell imaging of LLC-PK1 cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with AT1R/AT2R heterodimers as a complex in a microtubule-dependent and clathrin-independent manner, since colchicine-but not Pitstop2-blocked this process. This result was confirmed by an increase of ß-arrestin phosphorylation after ANG II treatment, clathrin-mediated endocytosis being dependent on dephosphorylation of ß-arrestin. Internalized ANG II colocalized with an endoplasmic reticulum (ER) marker and increased levels of AT1R, AT2R, and PKCα in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT1/AT2 complex to target ER, where it might trigger intracellular Ca2+ responses.
Assuntos
Angiotensina II/metabolismo , Membrana Celular/metabolismo , Endocitose , Retículo Endoplasmático/metabolismo , Rim/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Rim/efeitos dos fármacos , Células LLC-PK1 , Microtúbulos/metabolismo , Complexos Multiproteicos , Fosforilação , Proteína Quinase C-alfa/metabolismo , Transporte Proteico , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Suínos , beta-Arrestinas/metabolismoRESUMO
Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, É, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)-ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10(-8) M), a PKC activator, increased Cu(I)-ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 µM Ca(2+), whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCÉ to levels comparable with those found after phosphatase λ treatment, showing that the É isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced Vmax without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCÉ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling.