Changes In Extracellular Acidification Rate Research Articles

Hypoxia exposure blunts angiogenic signaling and upregulates the antioxidant system in endothelial cells derived from elephant seals

BackgroundElephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia.ResultsSeal and human endothelial cells exposed to 1% O2 for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure.ConclusionsWe found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

Glutamate Prevents Altered Mitochondrial Function Following Recurrent Low Glucose in Hypothalamic but Not Cortical Primary Rat Astrocytes.

Astrocytes contribute to glutamatergic signalling, which is required for hypoglycaemia counterregulation and is impaired by recurrent insulin-induced hypoglycaemia. This study examined the glutamate response of astrocytes when challenged with acute and recurrent low glucose (RLG) exposure. The metabolic responses of cortical (CRTAS) and hypothalamic (HTAS) primary rat astrocytes were measured in acute and recurrent low glucose using extracellular flux analyses. RLG caused mitochondrial adaptations in both HTAS and CRTAS, many of which were attenuated by glutamate exposure during low glucose (LG) treatments. We observed an increase in capacity of HTAS to metabolise glutamine after RLG exposure. Demonstrating astrocytic heterogeneity in the response to LG, CRTAS increased cellular acidification, a marker of glycolysis in LG, whereas this decreased in HTAS. The directional change in intracellular Ca2+ levels of each cell type, correlated with the change in extracellular acidification rate (ECAR) during LG. Further examination of glutamate-induced Ca2+ responses in low glucose treated CRTAS and HTAS identified sub-populations of glucose-excited- and glucose-inhibited-like cells with differing responses to glutamate. Lastly, release of the gliotransmitter ATP by HTAS was elevated by RLG, both with and without concurrent glutamate exposure. Therefore, hypothalamic astrocytes adapt to RLG by increasing glutamate uptake and oxidation in a manner that prevents RLG-induced mitochondrial adaptations.

Intermittent exposure of cultured endothelial cells to physiologically relevant fructose concentrations has a profound impact on nitric oxide production and bioenergetics.

Hyperglycaemia is known to induce endothelial dysfunction and changes in metabolic function, which could be implicated in diabetes-induced cardiovascular disease. To date, however, little is known about the impact of physiologically relevant concentrations of fructose on endothelial cells. A novel in vitro model was devised to establish the impact of substitution of a small proportion of glucose with an equal concentration (0.1 mM or 1 mM) of fructose on EA.hy926 endothelial cells during periodic carbohydrate “meals” superimposed on a normoglycaemic (5.5 mM) background. Parallel experiments were conducted using meals consisting of normoglycaemic glucose, intermediate glucose (12.5 mM) or profound hyperglycaemia (25 mM), each delivered for 2 h, with and without substituted fructose over 50 h. Outcome measures included nitrite as a surrogate marker of the mediator of healthy endothelial function, nitric oxide (NO), and a range of bioenergetic parameters using a metabolic analyser. Despite its relatively low proportion of carbohydrate load, intermittent fructose induced a substantial reduction (approximately 90%) in NO generation in cells treated with either concentration of fructose. Cell markers of oxidative stress were not altered by this treatment regimen. However, the cells experienced a marked increase in metabolic activity induced by fructose, irrespective of the glucose concentration delivered simultaneously in the “meals”. Indeed, glucose alone failed to induce any metabolic impact in this model. Key metabolic findings were a 2-fold increase in basal oxygen consumption rate and a similar change in extracellular acidification rate–a marker of glycolysis. Non-metabolic oxygen consumption also increased substantially in cells exposed to fructose. There was no difference between results with 0.1 mM fructose and those with 1 mM fructose. Low, physiologically relevant concentrations of fructose, delivered in a pattern that mimics mealtime consumption, had a profound impact on endothelial function and bioenergetics in an in vitro cell model. The results suggest that endothelial cells are exquisitely sensitive to circulating fructose; the potential ensuing dysfunction could have major implications for development of atherosclerotic disease associated with high fructose consumption.

Na/K‐ATPase Alpha1 Isoform Signaling in Cardiac Metabolism

Understanding cardiac metabolic signaling and its relation to pathological hypertrophy is critical to develop new therapeutic intervention in heart failure (HF). Decreased Na/K‐ATPase (NKA) activity and expression have long been associated with HF in experimental models and in patients. Classically known for its crucial role in transmembrane ion transport, electrical excitability, and contractility, NKA also supports non‐enzymatic receptor functions that differ among major cardiac isoforms (α1 and α2 in rodents). Studies from our group and others have suggested a role of these receptor functions in cardiac myocyte hypertrophy. Using epithelial (LLC‐PK1)‐derived cell lines, we have previously observed a NKA α1‐specific ability to modulate metabolic reserve and capacity. Mechanistically, this property is highly dependent upon NKA α1 ability to regulate the signaling partner Src kinase and is not supported by NKA α2, which lacks the Src binding domain. In this work, our goal was to assess the impact of NKA α1 ablation in the heart and demonstrate the importance of NKA α1 signaling function for proper cardiomyocyte metabolic function. We used a Myh6‐Cre/Lox approach to delete NKA α1 in the mouse heart (cardio α1‐/‐). These animals (3‐month‐old male mice) presented a normal cardiac structure, reduced NKA α1 expression (more than 100‐fold; n = 6‐7; p < 0.01) and increased NKA α2 expression (10‐fold; n = 6‐7; p < 0.01) by Western blot. RNA sequencing followed by gene set enrichment analysis revealed that cardio α1‐/‐ hearts have downregulated pathways related to mitochondrial metabolism, such as cytochrome complex, NADH dehydrogenase complex, respiratory chain, and mitochondrial fatty acid β‐oxidation (n = 3; FDR < 0.05). We next generated human induced pluripotent stem cells (hiPSCs) with a mutant form of the NKA α1 isoform that can pump ions but have blunted Src signaling (A420P mutant) using a CRISPR/Cas9‐mediated knock‐in approach. Genotyping and sequencing confirmed the substitution of alanine to proline in the NaKtide sequence to generate the desired mutation. These cells were differentiated into cardiomyocytes, as confirmed by overexpression of the cardiac markers myosin heavy chain 6 (MYH6) and cardiac muscle troponin T (TNNT2) (more than 10‐fold; n = 3; p < 0.05). Metabolic phenotyping using the Agilent Seahorse Extracellular Flux technology suggested that the A420P mutant hiPSC‐derived cardiomyocytes (A420P iCM) have decreased basal respiration, maximal respiration, spare respiratory capacity, and ATP production compared to the controls (WT iCM) based on changes in oxygen consumption rate (OCR) using the Agilent Seahorse XF Cell Mito Stress Test Kit. In addition, A420P iCM have reduced glycolytic capacity and reserve, based on changes in extracellular acidification rate (ECAR) using the Agilent Seahorse XF Glycolysis Stress Test Kit. Taken together, these results suggest that NKA α1 isoform signaling modulates the cardiomyocyte metabolism. Further studies may reveal a link between NKA/Src signaling, metabolic flexibility, and structural cardiac remodeling in the context of HF and cardiac hypertrophy.

Real-Time Assessment of Mitochondrial Toxicity in HepG2 Cells Using the Seahorse Extracellular Flux Analyzer.

The liver is the primary organ responsible for drug detoxification. Drug-induced liver injury (DILI) is a leading cause of attrition during drug development and is one of the main reasons that drugs are withdrawn from the market. Hence, the prevention of DILI plays a central role in the overall drug-discovery process. Most of the liver's energy supply comes in the form of adenosine triphosphate (ATP), which is largely generated by mitochondria. This article describes the evaluation of drug-induced mitochondrial dysfunction using the Seahorse Extracellular Flux Analyzer (Agilent). The described protocols detail the accurate measurement of ATP production rate in HepG2 cells after exposure to a panel of potentially toxic compounds. This assay measures changes in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indicators of glycolysis and mitochondrial respiration-the two major energy-generating pathways in a cell. This assay provides a useful model to predict mitochondrial dysfunction-mediated DILI. © 2021 Wiley Periodicals LLC. Basic Protocol: Measurement of cellular ECAR, OCR, and ATP production in live HepG2 cells Support Protocol 1: Culturing and maintaining of HepG2 cells Support Protocol 2: Determining optimal cell density per well.

Quantification of Functional Selectivity at the Human α1A-Adrenoceptor

Although G protein-coupled receptors are often categorized in terms of their primary coupling to a given type of Gα protein subunit, it is now well established that many show promiscuous coupling and activate multiple signaling pathways. Furthermore, some agonists selectively activate signaling pathways by promoting interaction between distinct receptor conformational states and particular Gα subunits or alternative signaling proteins. We have tested the capacity of agonists to stimulate Ca(2+) release, cAMP accumulation, and changes in extracellular acidification rate (ECAR) at the human α(1A)-adrenoceptor. Signaling bias factors were determined by novel application of an operational model of agonism and compared with the reference endogenous agonist norepinephrine; values significantly different from 1.0 indicated an agonist that promoted receptor conformations distinct from that favored by norepinephrine. Oxymetazoline was a full agonist for ECAR and a partial agonist for Ca(2+) release (bias factor 8.2) but failed to stimulate cAMP production. Phenylephrine showed substantial bias toward ECAR versus Ca(2+) release or cAMP accumulation (bias factors 21 and 33, respectively) but did not display bias between Ca(2+) and cAMP pathways. Cirazoline and N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide (A61603) displayed bias toward cAMP relative to Ca(2+) release (bias factors of 7.4 and 8.6). It is noteworthy that epinephrine, a second endogenous adrenoceptor agonist, did not display bias relative to norepinephrine. Our finding that phenylephrine displayed significant signaling bias, despite being highly similar in structure to epinephrine, indicates that subtle differences in agonist-receptor interaction can affect conformational changes in cytoplasmic domains and thereby modulate the repertoire of effector proteins that are activated.

Identification of functional bradykinin B 2 receptors endogenously expressed in HEK293 cells

The human embryonic kidney (HEK) 293 cell line is widely used in cell biology research. Although HEK293 cells have been meticulously studied, our knowledge about endogenous G protein-coupled receptors (GPCR) in these cells is incomplete. While studying the effects of bradykinin (BK), a potent growth factor for renal cells, we unexpectedly discovered that BK activates extracellular signal-regulated protein kinase 1 and 2 (ERK) in HEK293 cells. Thus, we hypothesized that HEK293 cells possess endogenous BK receptors. RT-PCR demonstrated the presence of mRNAs for BK B 1 and BK B 2 receptors in HEK293 cells. Western blotting with BK B 1 and BK B 2 receptor antibodies confirmed this result at the protein level. To establish that BK receptors are functional, we employed fluorescent measurements of intracellular Ca 2+, measured changes in extracellular acidification rate (ECAR) as a reflection of the Na +/H + exchange (NHE) with a Cytosensor™ microphysiometer, and assessed ERK activation by Western blotting with a phospho-specific ERK antibody. Exposure of HEK293 cells to BK produced a concentration-dependent rise in intracellular Ca 2+ (EC 50 = 36.5 ± 8.0 × 10 −9 M), a rapid increase in tyrosine phosphorylation of ERK (EC 50 = 9.8 ± 0.4 × 10 −9 M), and elevation in ECAR by ∼20%. All of these signals were blocked by HOE-140 (B 2 receptor antagonist) but not by des-Arg 10-HOE-140 (B 1 receptor antagonist). We conclude that HEK293 cells express endogenous functional BK B 2 receptors, which couple to the mobilization of intracellular Ca 2+, increases in ECAR and increases in ERK phosphorylation.

Insulin‐like growth factor‐1‐dependent maintenance of neuronal metabolism through the phosphatidylinositol 3‐kinase–Akt pathway is inhibited by C2‐ceramide in CAD cells

Ceramide is a lipid second-messenger generated in response to stimuli associated with neurodegeneration that induces apoptosis, a mechanism underlying neuronal death in Parkinson's disease. We tested the hypothesis that insulin-like growth factor-1 (IGF-1) could mediate a metabolic response in CAD cells, a dopaminergic cell line of mesencephalic origin that differentiate into a neuronal-like phenotype upon serum removal, extend processes resembling neurites, synthesize abundant dopamine and noradrenaline and express the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase, and that this process was phosphatidylinositol 3-kinase (PI 3-K)-Akt-dependent and could be inhibited by C(2)-ceramide. The metabolic response was evaluated as real-time changes in extracellular acidification rate (ECAR) using microphysiometry. The IGF-1-induced ECAR response was associated with increased glycolysis, determined by increased NAD(P)H reduction, elevated hexokinase activity and Akt phosphorylation. C(2)-ceramide inhibited all these changes in a dose-dependent manner, and was specific, as it was not induced by the inactive C(2)-ceramide analogue C(2)-dihydroceramide. Inhibition of the upstream kinase, PI 3-K, also inhibited Akt phosphorylation and the metabolic response to IGF-1, similar to C(2)-ceramide. Decreased mitochondrial membrane potential occurred after loss of Akt phosphorylation. These results show that IGF-1 can rapidly modulate neuronal metabolism through PI 3-K-Akt and that early metabolic inhibition induced by C(2)-ceramide involves blockade of the PI 3-K-Akt pathway, and may compromise the first step of glycolysis. This may represent a new early event in the C(2)-ceramide-induced cell death pathway that could coordinate subsequent changes in mitochondria and commitment of neurons to apoptosis.

Expression of P2Y 2 purinoceptors in MCG 101 murine sarcoma cells, and HT-29 human colon carcinoma cells

We investigated how agonists at purinoceptors may affect tumour cell metabolism. This was investigated in vitro in tumour cell lines by microphysiometry, which method monitors extracellular acidification rate (ECAR), on-line. The cell lines investigated were the murine sarcoma, MCG 101, and the human colon cancer, HT-29. In MCG 101, adenosine-5′-triphosphate (ATP) or uridine-5′-triphosphate (UTP) caused a concentration-dependent increase in ECAR, most likely due to the ligation of P2Y 2 receptors, which response was blocked by suramin. In HT-29, ATP or UTP elicited a concentration-dependent, biphasic change in ECAR (increase/decrease). The pharmacological analysis suggests the involvement of P2Y 2 receptors, although other P2 receptor subtypes cannot be entirely excluded. This biphasic response to UTP or ATP was resistant to suramin. The expression of P2Y 2 receptors was demonstrated in both cell lines by immunocytochemistry and Western blot. The current study, thus, shows the functional and morphological expression of a purinoceptor subtype with partly different effects on metabolism in two different tumour cell lines.

Cellular responses of NG108-15 and SK-N-MC lines to sweet and bitter tastants as measured by extracellular acidification rates.

The Cytosensor microphysiometer device (Molecular Devices, Sunnyvale, CA) is capable of detecting small changes in cellular metabolism in response to specific bioactive ligands by measuring the extracellular acidification rate (ECAR). By measuring the ECAR we were able to detect responses of tissue culture cell lines to a variety of sweet- and bitter-tasting compounds. We examined in detail the responses of the NG108-15 (mouse neuroblastoma x rat glioma hybrid) and SK-N-MC (human neuroepithelioma) cell lines. We determined that NG108-15 cells were consistently very responsive to several potent sweeteners and bitter compounds, such as sodium saccharin, guanidino- sweeteners, denatonium benzoate, quinine, and ranitidine. These compounds could evoke changes in cellular metabolism (measured as ECAR) that were rapid in onset, saturable with respect to ligand concentration, and sensitive to several inhibitors of G-protein-coupled receptor signaling pathways. In sharp contrast, the neuroepithelioma SK-N-MC did not respond to any of the sweet or bitter compounds. Rapid changes in ECAR were easily detectable in both cell lines with the calcium ionophore A23187. Bradykinin elicited changes in the ECAR only in the NG108-15 cell line, which is known to express the B2 receptor. The changes in ECAR of the NG108-15 cell line in response to sweet and bitter taste compounds suggest these cells may expresses a receptor(s) specific for small sapid molecules.

Monocyte chemoattractant protein-2 is a potent agonist of CCR2B.

The binding and functional activity of the CC chemokines monocyte chemoattractant protein-1 (MCP-1), MCP-2, and MCP-3 have been characterized using Chinese hamster ovary DXB-11 cells transfected with the chemokine receptor CCR2B. Receptor binding studies demonstrated that 125I-labeled MCP-1 bound to a single class of high-affinity receptors with a Kd of 0.14 (0.07-0.32) nM. In competition studies MCP-1, MCP-2, and MCP-3 completely inhibited 125I-labeled MCP-1 binding with Ki values of 0.3 (0.16-0.46), 8.8 (3.4-26), and 12.2 (0.6-22) nM, respectively. In calcium mobilization studies, MCP-1 and MCP-3 induced robust elevations in intracellular calcium concentrations, whereas MCP-2 was only weakly active. In contrast, using changes in extracellular acidification rate as a functional readout, all three chemokines were identified as potent agonists of CCR2B. These data demonstrate that MCP-2, in addition to MCP-1 and MCP-3, is a potent agonist of CCR2B and furthermore that MCP-2 activates either different or a subset of the signaling pathways activated by MCP-1 and MCP-3.