ATP Synthase Inhibitor Research Articles

In vitro reduction of bovine oocyte ATP production with oligomycin affects embryo epigenome.

Epigenetic programming is a crucial process during early embryo development that can have a significant impact on the results of assisted reproductive technology (ART) and offspring health. Here we show evidence using a bovine in vitro experiment that embryo epigenetic programing is dependent on oocyte mitochondrial bioenergetic activity during maturation. This study investigated if oocyte and early embryo epigenetic programming are dependent on oocyte mitochondrial ATP production. A bovine in vitro experiment was performed in which oocyte mitochondrial ATP production was reduced using 5 nmol/L oligomycin A (OM; ATP synthase inhibitor) during in vitro maturation (IVM) compared to control (CONT). OM exposure significantly reduced mitochondrial ATP production rate in MII oocytes (34.6% reduction, P = 0.018) and significantly decreased embryo cleavage rate at 48 h post insemination (7.6% reduction, P = 0.031). Compared to CONT, global DNA methylation (5mC) levels were decreased in OM-exposed MII oocytes (9.8% reduction, P = 0.019) while global histone methylation (H3K9me2) was increased (9.4% increase, P = 0.024). In zygotes, OM exposure during IVM increased 5mC (22.3% increase, P < 0.001) and histone acetylation (H3K9ac, 17.3% increase, P = 0.023) levels, while H3K9me2 levels were not affected. In morulae, 5mC levels were increased (10.3% increase, P = 0.041) after OM exposure compared to CONT, while there was no significant difference in H3K9ac and H3K9me2 levels. These epigenetic alterations were not associated with any persistent effects on embryo mitochondrial ATP production rate or mitochondrial membrane potential (assessed at the four-cell stage). Also, epigenetic regulatory genes were not differentially expressed in OM-exposed zygotes or morulae. Finally, apoptotic cell index in blastocysts was increased after OM exposure during oocyte maturation (41.1% increase, P < 0.001). We conclude that oocyte and early embryo epigenetic programming are dependent on mitochondrial ATP production during IVM.

Synergistic lethality in chronic myeloid leukemia – targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

High-Throughput Screen of Microbial Metabolites Identifies F1FO ATP Synthase Inhibitors as New Leads for Naegleria fowleri Infection.

Primary amebic meningoencephalitis (PAM), a brain infection caused by a free-living ameba Naegleria fowleri, leads to an extensive inflammation of the brain and death within 1-18 (median 5) days after symptoms begin. Although natural products have played a significant role in the development of drugs for over a century, research focusing on identifying new natural product-based anti-N. fowleri agents is limited. We undertook a large-scale ATP bioluminescence-based screen of about 10,000 unique marine microbial metabolite mixtures against the trophozoites of N. fowleri. Our screen identified about 100 test materials with >90% inhibition at 50 μg/mL and a dose-response study found 20 of these active test materials exhibiting an EC50 ranging from 0.2 to 2 μg/mL. Examination of four of these potent metabolite mixtures, derived from our actinomycete strains CNT671, CNT756, and CNH301, resulted in the isolation of a pure metabolite identified as oligomycin D. Oligomycin D exhibited nanomolar potency on multiple genotypes of N. fowleri, and it was five- or 850-times more potent than the recommended drugs amphotericin B or miltefosine. Oligomycin D is fast-acting and reached its EC50 in 10 h, and it was also able to inhibit the invasiveness of N. fowleri significantly when tested on a matrigel invasion assay. Since oligomycin is known to manifest inhibitory activity against F1FO ATP synthase, we tested different F1FO ATP synthase inhibitors and identified a natural peptide leucinostatin as a fast-acting amebicidal compound with nanomolar potency on multiple strains.

Oxidative Phosphorylation for Aerobic Survival, but Not for Growth: The Peculiar ‘Make-Accumulate-Consume’ Strategy in Zymomonas mobilis

Understanding the energy metabolism and its regulation is one of the clues to metabolic engineering of stress-resistant lignocellulose-converting microbial strains, also including the promising ethanologen Zymomonas mobilis. Z. mobilis is an obligately fermentative, facultatively anaerobic bacterium, carrying an active respiratory chain with low energy-coupling efficiency. Its respiration does not supply energy to aerobically growing cultures on sugary media, yet oxidative phosphorylation has been demonstrated in non-growing cells with ethanol. Here, we show, for the first time, that in respiring, non-growing Z. mobilis cells receiving regular small amounts of ethanol, oxidative phosphorylation significantly contributes to the maintenance of their viability. No improvement of viability is seen in the NADH dehydrogenase (ndh)-deficient respiratory mutant, which is unable to oxidize ethanol. The ethanol effect is also hampered by the protonophoric uncoupler CCCP, or the inhibitor of ATP synthase, DCCD. At higher concentrations (6% v/v), ethanol causes stress that slows down culture growth. By monitoring the activity of several respiratory gene promoters under ethanol stress with the green fluorescent protein reporter system, we demonstrate downregulation of these promoters, in particular the ndh promoter. We speculate that the decrease in respiratory chain activity in response to stress conditions mitigates the production of reactive oxygen species.

From garden to lab: C-3 chemical modifications of tomatidine unveil broad-spectrum ATP synthase inhibitors to combat bacterial resistance

Antibiotic resistance is escalating alarmingly worldwide. Bacterial resistance mechanisms are surfacing and proliferating across the globe, jeopardizing our capacity to manage prevalent infectious illnesses. Without drastic measures, we risk entering a post-antibiotic era, where even trivial infections and injuries can cause death again. In this context, we have developed a new class of antibiotics based on tomatidine (TO), a natural product derived from tomato plants, with a novel mode of action by targeting bacterial ATP synthases. The first generation of compounds proved highly specific for small-colony variants (SCVs) of Staphylococcus aureus. However, optimization of this scaffold through extensive structure-activity relationship studies has enabled us to broaden its effectiveness to include both Gram-positive and Gram-negative bacteria. Notably, the results showed that specific C3-modification of TO could improve ATP synthase inhibition and also bypass the outer membrane barrier of Gram-negative bacteria to gain substantial growth inhibition including against multi-resistant strains.

Oxidative phosphorylation rather than glycolysis is the primary energy source for sperm motility in the mussels Mytilus edulis

Broadcast-spawning marine mussels rely on high sperm motility for successful fertilization in the dynamic seawater environment. Mitochondria are typically considered the primary source of ATP generation via oxidative phosphorylation (OXPHOS); however, the ATP generation pathways of mussel sperm have not been fully characterized. To better understand the importance of both OXPHOS and glycolysis for mussel sperm function, we conducted experiments inhibiting these pathways in sperm from Mytilus edulis. Our results indicate that oligomycin, an inhibitor of the mitochondrial ATP synthase, immediately decreased sperm motility rate, velocity, and ATP content, while 2-deoxy-d-glucose, a glycolysis inhibitor, had no effect. The OXPHOS inhibitor rotenone also partially reduced sperm motility rate and velocity. Interestingly, no evidence was found for the inhibitors' effects on the content of energy-rich compounds (lipids, carbohydrates, and proteins) in the mussels' sperm, indicating only modest energy demand to fuel sperm motility. Based on these findings, we conclude that OXPHOS is the primary energy source for sperm motility in marine mussels. Our study sheds light on the intricacies of mussel sperm physiology and highlights the importance of understanding the energy requirements for successful fertilization in broadcast-spawning marine invertebrates.

Expanding the squaramide library as mycobacterial ATP synthase inhibitors: Innovative synthetic pathway and biological evaluation

Mycobacterial ATP synthase is a validated therapeutic target for combating drug-resistant tuberculosis. Inhibition of this enzyme has been featured as an efficient strategy for the development of new antimycobacterial agents against drug-resistant pathogens. In this study, we synthesised and explored two distinct series of squaric acid analogues designed to inhibit mycobacterial ATP synthase. Among the extensive array of compounds investigated, members of the phenyl-substituted sub-library emerged as primary hits. To gain deeper insights into their mechanisms of action, we conducted advanced biological studies, focusing on the compounds displaying a direct binding of a nitrogen heteroatom to the phenyl ring, resulting in the highest potency. Our investigations into spontaneous mutants led to the validation of a single point mutation within the atpB gene (Rv1304), responsible for encoding the ATP synthase subunit a. This genetic alteration sheds light on the molecular basis of resistance to squaramides. Furthermore, we explored the possibility of synergy between squaramides and the reference drug clofazimine using a checkerboard assay, highlighting the promising avenue for enhancing the effectiveness of existing treatments through combined therapeutic approaches. This study contributes to the expansion of investigating squaramides as promising drug candidates in the ongoing battle against drug-resistant tuberculosis.

Shikonin and Alkannin inhibit ATP synthase and impede the cell growth in Escherichia coli

Naturally occurring naphthoquinones, shikonin and alkannin, are important ingredients of traditional Chinese medicine Zicao. These constituents are reported to have many therapeutic uses, such as wound healing; scar treatment; and anti-inflammation, anti-acne, anti-ulcer, anti-HIV, anticancer, and antibacterial properties. The primary objective of this investigation was to explore the effect of shikonin and alkannin on Escherichia coli ATP synthase and its cell growth. Shikonin caused complete (100 %) inhibition, and alkannin caused partial (79 %) inhibition of wild-type E. coli ATP synthase. Both caused partial (4 %–27 %) inhibition of ATP synthase with genetically modified phytochemical binding site. The growth inhibition of strains expressing normal, deficient, and mutant ATP synthase by shikonin and alkannin, corroborated the inhibition observed in isolated normal wild-type and mutant ATP synthase. Trivial inhibition of mutant enzymes indicated αR283D, αE284R, βV265Q, and γT273A are essential for formation of the phytochemical binding site where shikonin and alkannin bind. Further, shikonin was a potent inhibitor of ATP synthase than alkannin. The antimicrobial properties of shikonin and alkannin were tied to the binding at phytochemical site of microbial ATP synthase. Selective targeting of bacterial ATP synthase by shikonin and alkannin may be an advantageous alternative to address the antibiotic resistance issue.

αβ-T cell receptor transduction gives superior mitochondrial function to γδ-T cells with promising persistence

Adoptive cell therapy using allogeneic γδ-T cells is a promising option for off-the-shelf Tcell products with a low risk of graft-versus-host disease (GVHD). Long-term persistence may boost the clinical development of γδ-T cell products. In this study, we found that genetically modified Vγ9+Vδ2+ Tcells expressing a tumor antigen-specific αβ-TCR and CD8 coreceptor (GMC) showed target-specific killing and excellent persistence. To determine the mechanisms underlying these promising effects, we investigated metabolic characteristics. Cytokine secretion by γδ-TCR-stimulated nongene-modified γδ-T cells (NGMCs) and αβ-TCR-stimulated GMCs was equally suppressed by a glycolysis inhibitor, although the cytokine secretion of αβ-TCR-stimulated GMCs was more strongly inhibited by ATP synthase inhibitors than that of γδ-TCR-stimulated NGMCs. Metabolomic and transcriptomic analyses, flow cytometry analysis using mitochondria-labeling dyes and extracellular flux analysis consistently suggest that αβ-TCR-transduced γδ-T cells acquire superior mitochondrial function. In conclusion, αβ-TCR-transduced γδ-T cells acquire superior mitochondrial function with promising persistence.

Cirsiliol and Quercetin Inhibit ATP Synthesis and Decrease the Energy Balance in Methicillin-Resistant Staphylococcus aureus (MRSA) and Methicillin-Resistant Staphylococcus epidermidis (MRSE) Strains Isolated from Patients.

Polyphenols have attracted attention in the fight against antibiotic-resistant bacteria, as they show antibacterial action. Considering that polyphenols inhibit F1Fo-ATP synthase (ATP synthase) and that bacteria need a constant energy production to maintain their homeostasis, we evaluated the effect of two flavones, cirsiliol (tri-hy-droxy-6,7-dimethoxyflavone) and quercetin (3,3,4,5,7-pentahydroxyflavone), on energy production and intracellular ATP content in a methicillin-resistant Staphylococcus aureus (MRSA) strain and a methicillin-resistant Staphylococcus epidermidis (MRSE) strain isolated from patients, comparing the results to those obtained by treating the bacteria with oligomycin, a specific ATP synthase Fo moiety inhibitor. Real-time quantitative ATP synthesis and total ATP content of permeabilized Gram-positive bacteria were assayed by luminometry. The results showed that cirsiliol and quercetin inhibited ATP synthase and decreased the intracellular ATP levels in both strains, although the effect was higher in MRSE. In addition, while cirsiliol and quercetin acted immediately after the treatment, oligomycin inhibited ATP synthesis only after 30 min of incubation, suggesting that the different responses may depend on the different permeability of the bacterial wall to the three molecules. Thus, cirsiliol and quercetin could be considered potential additions to antibiotics due to their ability to target ATP synthase, against which bacteria cannot develop resistance.

Abstract P3183: Cardiac Troponin I Directly Binds Mitochondrial Proteins And Inhibits Mitochondrial Functions

The heart relies on mitochondria for its extensive energetic demands, and some cytoskeletal elements can regulate mitochondrial function. Cardiac troponin I (cTnI) is a structural protein involved in sarcomeric contraction. Here we demonstrate that some cTnI localizes to mitochondria, inhibits mitochondrial functions, and increases opening of the mitochondrial permeability transition pore under oxidative stress. Purified cTnI directly binds immune-captured ATP synthase in saturable fashion and doubles "reverse" F 1 F 0 -ATPase activity in vitro , whereas skeletal troponin I (ssTnI) does not. Relevant to human disease, several pathogenic variants of cTnI associated with hypertrophic cardiomyopathy do not increase F 1 F 0 -ATPase activity. Using a rational search, we identified the d subunit of ATP synthase as a potential cTnI-binding partner, and a ten-amino acid peptide inhibitor of cTnI’s interaction with ATP synthase, peptide P888, inhibited cTnI binding to ATP synthase and abolished the cTnI-induced increase in ATPase activity. P888 treatment greatly reduced cardiac LDH release and improved fractional shortening in vivo after transient coronary artery occlusion in rats, indicating that mitochondrial cTnI exacerbates cardiac ischemia-reperfusion injury. We explore the mechanisms governing cTnI’s mitochondrial localization and additional mitochondrial binding partners for cTnI. Together, our studies suggest that mitochondria-associated cTnI inhibits ATP synthase under basal conditions. Preventing cTnI from binding to ATP synthase during cardiac ischemia may increase the reservoir of functional mitochondria to mitigate post-ischemic cardiac injury. We also suggest that cTnI has additional roles in regulating mitochondrial functions. Finally, pathogenic cTnI mutations associated with hypertrophic cardiomyopathy do not affect ATP synthase activity, suggesting a potential mechanism that contributes to the diverse pathologies associated with these mutations.

Transcriptome Analysis Revealed That Hydrogen Peroxide-Regulated Oxidative Phosphorylation Plays an Important Role in the Formation of Pleurotus ostreatus Cap Color.

Pleurotus ostreatus is widely cultivated in China. H2O2, as a signaling molecule, can regulate the formation of cap color, but its regulatory pathway is still unclear, severely inhibiting the breeding of dark-colored strains. In this study, 614 DEGs specifically regulated by H2O2 were identified by RNA-seq analysis. GO-enrichment analysis shows that DEGs can be significantly enriched in multiple pathways related to ATP synthesis, mainly including proton-transporting ATP synthesis complex, coupling factor F(o), ATP biosynthetic process, nucleoside triphosphate metabolic processes, ATP metabolic process, purine nucleoside triphosphate biosynthetic and metabolic processes, and purine ribonuclease triphosphate biosynthetic metabolic processes. Further KEGG analysis revealed that 23 DEGs were involved in cap color formation through the oxidative phosphorylation pathway. They were enriched in Complexes I, III, IV, and V in the respiratory chain. Further addition of exogenous uncoupling agents and ATP synthase inhibitors clarifies the important role of ATP synthesis in color formation. In summary, H2O2 may upregulate the expression of complex-encoding genes in the respiratory chain and promote ATP synthesis, thereby affecting the formation of cap color. The results of this study lay the foundation for the breeding of dark-colored strains of P. ostreatus and provide a basis for the color-formation mechanism of edible fungi.

Inorganic polyphosphate's role in energy production and mitochondrial permeability transition pore opening in tick mitochondria.

Inorganic polyphosphate (polyP) is a biopolymer composed of phosphoanhydride-linked orthophosphate molecules. PolyP is engaged in a variety of cellular functions, including mitochondrial metabolism. Here, we examined the effects of polyP on electron transport chain enzymes and F1 Fo ATP synthase in tick embryos during embryonic development. The study found that polyPs containing medium and long chains (polyP15 and polyP65 ) enhanced the activity of complex I, complex II, complex III, and F1 Fo ATP synthase, while short polyP chains (polyP3 ) had no effect. The study also examined the activity of exopolyphosphatases (PPX) in various energy-demand situations. PPX activity was stimulated when ADP concentrations are high, characterizing a low-energy context. When complexes I-III and F1 Fo ATP synthase inhibitors were added in energized mitochondria, PPX activity decreased, whereas the mitochondrial uncoupler FCCP had no impact on PPX activity. Additionally, the study investigated the effect of polyP on mitochondrial swelling, finding that polyP causes mitochondrial swelling by increasing calcium effects on the mitochondrial permeability transition pore. The findings presented here to increase our understanding of the function of polyP in mitochondrial metabolism and its relationship to mitochondrial permeability transition pore opening in an arthropod model.

Molecular docking-based interaction studies on imidazo[1,2-a] pyridine ethers and squaramides as anti-tubercular agents

ABSTRACT Development of new anti-tubercular agents is required in the wake of resistance to the existing and newly approved drugs through novel-validated targets like ATP synthase, etc. The major limitation of poor correlation between docking scores and biological activity by SBDD was overcome by a novel approach of quantitatively correlating the interactions of different amino acid residues present in the target protein structure with the activity. This approach well predicted the ATP synthase inhibitory activity of imidazo[1,2-a] pyridine ethers and squaramides (r = 0.84) in terms of Glu65b interactions. Hence, the models were developed on combined (r = 0.78), and training (r = 0.82) sets of 52, and 27 molecules, respectively. The training set model well predicted the diverse dataset (r = 0.84), test set (r = 0.755), and, external dataset (r ext = 0.76). This model predicted three compounds from a focused library generated by incorporating the essential features of the ATP synthase inhibition with the pIC50 values in the range of 0.0508–0.1494 µM. Molecular dynamics simulation studies ascertain the stability of the protein structure and the docked poses of the ligands. The developed model(s) may be useful in the identification and optimization of novel compounds against TB.

Haploinsufficiency of progranulin causes cell type specific impairments in PINK1/Parkin mitophagy

AbstractBackgroundGRN mutations that result in progranulin haploinsufficiency cause frontotemporal dementia (FTD). Mitophagy, the selective autophagy of damaged mitochondria, is impaired in several neurodegenerative diseases. A number of genes linked to FTD (e.g. OPTN, SQSTM, VCP and TBK1) are also known to play a role in mitophagy. Xenophagy, the selective autophagy of non‐host pathogens, relies on some of the same proteins as mitophagy (Parkin and TBK1) and is reduced in GRN knockout mice. Progranulin has also been found to play a role in mitophagy in mouse kidney cells. We therefore hypothesised that loss of progranulin could lead to defective mitophagy in neurons, astrocytes and microglia.MethodsWe investigated PINK1/Parkin mitophagy in astrocytic‐like H4 cells and neuron‐like Parkin overexpressing SHSY5Y cells (PoE‐5Ys) +/‐ siRNA against GRN. We also examined induced pluripotent stem cells (iPSCs) from four controls, three patients with FTD‐associated GRN mutations (R493X and C31fs) and a CRISPR series from the human iPSC Neurodegenerative Disease Initiative1,2,3 (iNDI) with an isogenic control, heterozygous and homozygous R493X mutation lines. The iPSCs were differentiated to cortical neurons, astrocytes and microglia. PINK1/Parkin mitophagy was induced using Antimycin A (respiratory complex III inhibitor) and oligomycin (ATP synthase inhibitor). PINK1 accumulation, levels of S65 phosphorylated ubiquitin (pUb), other mitophagy markers as well as proteins hypothesised to play a role in the mechanism were examined using western blotting and immunofluorescence (ICC).ResultsLower levels of pUb were detected in PoE‐5Ys treated with oligomycin/antimycin following GRN knockdown. There was a significant reduction in mitophagy in GRN siRNA treated H4 cells, detected by ICC and western blotting of mitophagy markers. Initial results from iPSC neurons found no significant difference in mitophagy between control and patient lines. Work is ongoing in a larger set of iPSC neurons, astrocytes and microglia. Preliminary results suggest that reducing progranulin affects mitophagy in astrocytes but has a limited effect on mitophagy in neurons.ConclusionsThe results suggest that progranulin has a cell specific role in mitophagy with progranulin regulating stability and/or activity of PINK1. Current work aims to understand the mechanisms of this process and to dissect cell‐type specific contributions of progranulin to mitophagy.

Study of mitochondrial function in thawed bull spermatozoa using selective electron transfer chain inhibitors

Bull spermatozoa depend equally on glycolysis and oxidative phosphorylation for the maintenance of the energy necessary for their proper functioning. The aim of the present work was to delineate the mitochondrial activity of bull spermatozoa after incubation with specific inhibitors of the different mitochondrial complexes and evaluate their ROS production. Thawed bull sperm cells (30 × 106 mL−1 in Tyrode's extender) were incubated 1 and 3h at 37 °C with rotenone 5 μM (ROT), complex I inhibitor; dimethyl-malonate 10 mM (DMM), complex II inhibitor; carbonyl cyanide m-chlorophenyl hydrazine 5 μM (CCCP), uncoupling agent; antimycin A 1 μg/mL (ANTI), complex III inhibitor; oligomycin 5 μM (OLIGO), ATP synthase inhibitor, and 0.5% DMSO, vehicle (CTR). Sperm motility and kinematics were assessed by Hamilton Thorn IVOS 12.0. Mitochondrial membrane potential, mitochondrial O2•– production and H2O2 intracellular content were evaluated by BD FACSCalibur flow cytometer, and sperm viability (SYBR-14/PI) and mitochondrial activity (JC-1/SYBR-14/PI) were assessed by epifluorescence microscopy. A multivariate analysis was performed on the results. In addition, sperm kinematic features, registered for each motile spermatozoon, were studied by cluster analysis. The incubation during 1 or 3 h in presence of the inhibitors of mitochondrial functionality only had a minor effect on motility parameters, decreasing the proportion of the SP1 (fast progressive) subpopulation after 3 h of incubation with ROT, ANTI or OLIGO. The percentage of live spermatozoa with active mitochondria was reduced under the effect of ANTI and CCCP both at 1 and 3 h. In conclusion, mitochondrial function is somehow impaired in frozen thawed bull sperm as not all live cells showed active mitochondria. These results support the findings that bull spermatozoa can alternatively rely on oxidative phosphorylation or glycolysis for energy obtainment and that their mitochondria are less affected by ETC inhibitors.

Cell surface ATP synthase-released H+ and ATP play key roles in cocoa butter intake-mediated regulation of gut immunity through releases of cytokines in rat.

Proper food intake is important for maintaining good health in humans. Chocolate is known to exert anti-inflammatory effects; however, the mechanisms remain unclear. In this study, we aimed to investigate the effects of cocoa butter intake on gut immunity in rats and rabbits. Cocoa butter intake increased the lymph flow, cell density, and IL-1β, IL-6 and IL-10 levels in mesenteric lymph. Clodronate, a macrophage depletion compound, significantly enhanced the release of all cytokines. The immunoreactivities of macrophage markers CD68 and F4/80 in the jejunal villi were significantly decreased with clodronate. Piceatannol, a selective cell surface ATP synthase inhibitor significantly reduced the cocoa butter intake-mediated releases of IL-1β, IL-6 and IL-10. The immunoreactivities of cell surface ATP synthase were observed in rat jejunal villi. Shear stress stimulation on the myofibroblast cells isolated from rat jejunum released ATP and carbon dioxide depended with H+ release. In rabbit in vivo experiments, cocoa butter intake increased the concentrations of ATP and H+ in the portal vein. The in vitro experiments with isolated cells of rat jejunal lamina propria the pH of 3.0 and 5.0 in the medium released significantly IL-1β and IL-6. ATP selectively released IL-10. These findings suggest that cocoa butter intake regulates the gut immunity through the release and transport of IL-1β, IL-6, and IL-10 into mesenteric lymph vessels in a negative feedback system. In addition, the H+ and ATP released from cell surface ATP synthase in jejunal villi play key roles in the cocoa butter intake-mediated regulation of gut immunity.

Mitochondria and renal microvascular dysfunction following ischemia-reperfusion in rats

The pathophysiological mechanisms underlying ischemia-reperfusion (IR) induced acute kidney injury (AKI) remain poorly understood. Preglomerular microvessels (PGMV), specifically afferent arterioles, possess an intrinsic autoregulatory ability that is vital for ensuring stable renal hemodynamics. Our recent study indicates that renal autoregulation is impaired in IR rats due to reactive oxygen species (ROS) accumulation. We hypothesized that mitochondria-derived ROS (mtROS) in preglomerular microvessels contribute to renal autoregulatory impairment in IR rats. Blood-perfused juxtamedullary nephron experiments were performed on kidneys of male Sprague-Dawley rats 24 hours after IR induction (60-min, bilateral renal artery clamping). Afferent arteriolar autoregulation was assessed as perfusion pressure (PP) was increased from 65 to 170 mmHg in 15 mmHg increments. Afferent arterioles of sham-operated control rats (n=7) established pressure-mediated autoregulatory responses. Baseline diameter (100 mmHg) averaged 13.2±0.4 μm and increased to 114±4% of baseline when PP decreased from 100 to 65 mmHg. Diameter decreased to 65±3% of baseline when PP increased to 170 mmHg. In contrast, afferent arterioles from IR rats lost autoregulatory capability and remained between 90 and 100% of baseline diameter over 65-170 mmHg (n=8). Addition of Mito-Tempo (mitochondria-targeted antioxidant, 0.1 mM, n=3) to the perfusate restored autoregulation in IR rats at lower PP (65-125 mmHg, P&gt;0.05 vs sham) and partially restored it over 140-170 mmHg. Superfusion with oligomycin (ATP synthase inhibitor, 2.5 μM) in normal kidneys evoked vasodilation to 178±13% baseline and blunting of autoregulatory reactivity. Renal autoregulation is linked to ATP-sensitive P2X1-receptor signaling. Changes in ATP-induced intracellular calcium concentration ([Ca2+]i) were assessed in isolated PGMV myocytes using Fura-2 fluorescent dye. IR markedly attenuated [Ca2+]i responses to 10 and 100 μM ATP by 46±11% and 57±15%, respectively (P&lt;0.05, n=3-4 rats/group), and blunted ATP-induced afferent arteriolar vasoconstriction ( P&lt;0.05 vs. sham, n=3/group). IR markedly increased the fluorescence intensity of mitoROS (MitoSOX™ kit) in PGMV compared to sham rats. Mitochondrial respiratory function was assessed using High Resolution Respirometry in freshly isolated PGMV (n=3/group). IR significantly reduced basal respiration (20±4 vs. 58±9 pmol/s·ml in sham, P&lt;0.05) and oxidative phosphorylation capacity (51±10 vs. 219±37 pmol/s·ml in sham, P&lt;0.05). The percentage of ATP-linked respiration was also reduced in PGMV of IR (60±2% vs. 75±2% in sham, P&lt;0.05). In conclusion, mitochondria of PGMV are involved in regulating renal microvascular tone and autoregulation. IR leads to autoregulatory dysfunction by causing mitochondrial dysfunction in PGMV, increased mtROS production and suppression of ATP-dependent calcium signaling. This study is supported by grants R01DK106500. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Estimation of energy pathway fluxes in cancer cells - Beyond the Warburg effect

Glycolytic and respiratory fluxes were analyzed in cancer and non-cancer cells. The steady-state fluxes in energy metabolism were used to estimate the contributions of aerobic glycolytic and oxidative phosphorylation (OxPhos) pathways to the cellular ATP supply. The rate of lactate production - corrected for the fraction generated by glutaminolysis - is proposed as the appropriate way to estimate glycolytic flux. In general, the glycolytic rates estimated for cancer cells are higher than those found in non-cancer cells, as originally observed by Otto Warburg. The rate of basal or endogenous cellular O2 consumption corrected for non-ATP synthesizing O2 consumption, measured after inhibition by oligomycin (a specific, potent and permeable ATP synthase inhibitor), has been proposed as the appropriate way to estimate mitochondrial ATP synthesis-linked O2 flux or net OxPhos flux in living cells. Detecting non-negligible oligomycin-sensitive O2 consumption rates in cancer cells has revealed that the mitochondrial function is not impaired, as claimed by the Warburg effect. Furthermore, when calculating the relative contributions to cellular ATP supply, under a variety of environmental conditions and for different types of cancer cells, it was found that OxPhos pathway was the main ATP provider over glycolysis. Hence, OxPhos pathway targeting can be successfully used to block in cancer cells ATP-dependent processes such as migration. These observations may guide the re-design of novel targeted therapies.

Expansion of the intraepithelial lymphocyte (IEL) compartment results in an increased bioenergetic profile and reduced IFNγ production in γδ IELs.

Abstract Intestinal γδ intraepithelial lymphocytes (γδ IEL) serve as the first line of defense against pathogen invasion. We recently identified a transmissible hyperproliferative γδ IEL (γδ HYP) phenotype that protects against enteric infection, and these mice have no signs of overt intestinal pathology. Given that IELs are metabolically constrained to prevent aberrant activation, we hypothesized that the bioenergetics of γδ HYPIELs may regulate their effector function. Electron microscopy revealed a 70% increase in mitochondrial number (p=0.005) and 24% increase in mitochondrial area (p=0.04) in γδ HYPIELs relative to WT. Seahorse mitochondrial stress assays on γδ IELs showed a 50% increase in spare respiratory capacity in γδ HYPIELs compared to WT (p=0.014). Mitochondrial metabolism was shown to influence γδ T cell cytokine production, and similarly, we found that stimulation with PMA and ionomycin reduced the frequency of IFNγ +γδ IELs by 59% in γδ HYPrelative to WT (p&amp;lt;0.0001). We also observed that γδ HYPIELs exhibit higher CD39 expression compared to WT (p&amp;lt;0.0001), and a CD39 hisubset produces less IFNγ compared to CD39 negand CD39 intcells (p&amp;lt;0.0001) The addition of the ATP-synthase inhibitor oligomycin during IEL stimulation increased IFNγ production by both WT and γδ HYPIELs by 66% and 44%, respectively (p&amp;lt;0.0001). Together, our data demonstrate that the shift towards high CD39 expression and elevated mitochondrial metabolism contribute to decreased IFNγ production in γδ HYPIELs. Further understanding of the mechanisms regulating γδ IEL homeostasis and effector function may ultimately allow fine tuning of mucosal surveillance to confer protection against intestinal injury or infection while limiting aberrant cytotoxicity. Supported by NJCCR (COCR23PRF024)