The intracellular calcium antagonist TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] inhibits mitochondrial ATP production in rat thymocytes.
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ABSTRACT:
SUBMITTER: Brand MD
PROVIDER: S-EPMC1144544 | biostudies-other | 1984 Dec
REPOSITORIES: biostudies-other
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Project description:The mechanism of 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) action was evaluated in isolated adrenal glomerulosa cells. TMB-8 inhibits both angiotensin II- and K+-stimulated aldosterone secretion in a dose-dependent manner. The ID50 for angiotensin II- and K+-stimulated aldosterone secretion is 46 and 28 microM, respectively. In spite of the fact that 100 microM-TMB-8 inhibits angiotensin II-stimulated aldosterone secretion almost completely, TMB-8 (100 microM) does not inhibit angiotensin II-induced 45Ca2+ efflux from prelabelled cells nor does it affect inositol 1,4,5-trisphosphate-induced calcium release from non-mitochondrial pool(s) in saponin-permeabilized cells. TMB-8 has no inhibitory effect on A23187-induced aldosterone secretion, but 12-O-tetradecanoylphorbol 13-acetate-induced aldosterone secretion is completely abolished. TMB-8 effectively inhibits both angiotensin II- and K+-induced increases in calcium influx but has no effect on A23187-induced calcium influx. TMB-8 inhibits the activity of protein kinase C dose-dependently. These results indicate that TMB-8 inhibits aldosterone secretion without inhibiting mobilization of calcium from an intracellular pool. The inhibitory effect of TMB-8 is due largely to an inhibition of plasma membrane calcium influx, but this drug also inhibits the activity of protein kinase C directly.
| S-EPMC1152843 | biostudies-other
Project description:Regulation of ATP production by mitochondria, critical to multicellular life, is poorly understood. Here we investigate the molecular controls of this process in heart and provide a framework for its Ca2+-dependent regulation. We find that the entry of Ca2+ into the matrix through the mitochondrial calcium uniporter (MCU) in heart has neither an apparent cytosolic Ca2+ threshold nor gating function and guides ATP production by its influence on the inner mitochondrial membrane (IMM) potential, ??m. This regulation occurs by matrix Ca2+-dependent modulation of pyruvate and glutamate dehydrogenase activity and not through any effect of Ca2+ on ATP Synthase or on Electron Transport Chain Complexes II, III or IV. Examining the ??m dependence of ATP production over the range of -60 mV to -170 mV in detail reveals that cardiac ATP synthase has a voltage dependence that distinguishes it fundamentally from the previous standard, the bacterial ATP synthase. Cardiac ATP synthase operates with a different ??m threshold for ATP production than bacterial ATP synthase and reveals a concave-upwards shape without saturation. Skeletal muscle MCU Ca2+ flux, while also having no apparent cytosolic Ca2+ threshold, is substantially different from the cardiac MCU, yet the ATP synthase voltage dependence in skeletal muscle is identical to that in the heart. These results suggest that while the conduction of cytosolic Ca2+ signals through the MCU appears to be tissue-dependent, as shown by earlier work1, the control of ATP synthase by ??m appears to be broadly consistent among tissues but is clearly different from bacteria.
| S-EPMC6964030 | biostudies-literature
Project description:ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events), the phase 3 morbidity and mortality trial of torcetrapib, a cholesteryl ester transfer protein (CETP) inhibitor, identified previously undescribed changes in plasma levels of potassium, sodium, bicarbonate, and aldosterone. A key question after this trial is whether the failure of torcetrapib was a result of CETP inhibition or of some other pharmacology of the molecule. The direct effects of torcetrapib and related molecules on adrenal steroid production were assessed in cell culture using the H295R as well as the newly developed HAC15 human adrenal carcinoma cell lines. Torcetrapib induced the synthesis of both aldosterone and cortisol in these two in vitro cell systems. Analysis of steroidogenic gene expression indicated that torcetrapib significantly induced the expression of CYP11B2 and CYP11B1, two enzymes in the last step of aldosterone and cortisol biosynthesis pathway, respectively. Transcription profiling indicated that torcetrapib and angiotensin II share overlapping pathways in regulating adrenal steroid biosynthesis. Hormone-induced steroid production is mainly mediated by two messengers, calcium and cAMP. An increase of intracellular calcium was observed after torcetrapib treatment, whereas cAMP was unchanged. Consistent with intracellular calcium being the key mediator of torcetrapib’s effect in adrenal cells, calcium channel blockers completely blocked torcetrapib-induced corticoid release and calcium increase. A series of compounds structurally related to torcetrapib as well as structurally distinct compounds were profiled. The results indicate that the pressor and adrenal effects observed with torcetrapib and related molecules are independent of CETP inhibition.
2009-05-02 | GSE15918 | GEO
Project description:Inflammatory bowel disease (IBD) has a close association with transketolase (TKT) that links glycolysis and the pentose phosphate pathway (PPP). However, how TKT functions in the intestinal epithelium remains to be elucidated. To address this question, we specifically delete TKT in intestinal epithelial cells (IECs). IEC TKT-deficient mice are growth retarded and suffer from spontaneous colitis. TKT ablation brings about striking alterations of the intestine, including extensive mucosal erosion, aberrant tight junctions, impaired barrier function, and increased inflammatory cell infiltration. Mechanistically, TKT deficiency significantly accumulates PPP metabolites and decreases glycolytic metabolites, thereby reducing ATP production, which results in excessive apoptosis and defective intestinal barrier. Therefore, our data demonstrate that TKT serves as an essential guardian of intestinal integrity and barrier function as well as a potential therapeutic target for intestinal disorders.
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Project description:P2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATP?S or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.
| S-EPMC5979340 | biostudies-literature
Project description:The CB(1) cannabinoid receptor mediates many of the psychoactive effects of Delta(9)THC, the principal active component of cannabis. However, ample evidence suggests that additional non-CB(1)/CB(2) receptors may contribute to the behavioral, vascular, and immunological actions of Delta(9)THC and endogenous cannabinoids. Here, we provide further evidence that GPR55, a G protein-coupled receptor, is a cannabinoid receptor. GPR55 is highly expressed in large dorsal root ganglion neurons and, upon activation by various cannabinoids (Delta(9)THC, the anandamide analog methanandamide, and JWH015) increases intracellular calcium in these neurons. Examination of its signaling pathway in HEK293 cells transiently expressing GPR55 found the calcium increase to involve G(q), G(12), RhoA, actin, phospholipase C, and calcium release from IP(3)R-gated stores. GPR55 activation also inhibits M current. These results establish GPR55 as a cannabinoid receptor with signaling distinct from CB(1) and CB(2).
| S-EPMC2268199 | biostudies-literature
Project description:The endogenous metabolite itaconate has emerged as a regulator of macrophage function that limits inflammation. However, its effect on cell differentiation and osteoclast-related diseases is unclear. Here, for the first time, we explored the effect of itaconate and its cell-permeable itaconate derivative, 4-octyl itaconate (OI) on osteoclast differentiation in vitro and in vivo. Firstly, we demonstrated that itaconate concentration was lower in estrogen-deficient mice. OI released itaconate and induced the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) in bone marrow-derived macrophages during osteoclastogenesis. Furthermore, OI significantly suppressed the early, middle, and late stages of osteoclastogenesis induced by receptor activator of NF-?B ligand in vitro, as confirmed by tartrate-resistant acid phosphatase staining. Moreover, it significantly inhibited fibrous actin ring formation and bone resorption in vitro. Mechanistically, we observed that OI enhanced Nrf2 expression by suppressing its association with ubiquitin via inhibition of the E3 ubiquitin ligase (Hrd1). OI also inhibited LPS-induced the reactive oxygen species and inflammatory responses via Hrd1. An estrogen deficiency (via ovariectomy)-induced osteoporosis model was also established. Here, on micro-computed tomography and histologic analysis showed that OI effectively suppressed ovariectomy-induced bone loss. In summary, OI, an itaconate derivative, can inhibit osteoclastogenesis in vitro and in vivo, indicating that OI could be a potential drug to treat osteoclast-related diseases; our results also link itaconate to the development of osteoporosis.-Sun, X., Zhang, B., Pan, X., Huang, H., Xie, Z., Ma, Y., Hu, B., Wang, J., Chen, Z., Shi, P. Octyl itaconate inhibits osteoclastogenesis by suppressing Hrd1 and activating Nrf2 signaling.
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Project description:Endothelial cells are reported to be glycolytic and to minimally rely on mitochondria for ATP generation. Rather than providing energy, mitochondria in endothelial cells may act as signaling organelles that control cytosolic Ca2+ signaling or modify reactive oxygen species (ROS). To control Ca2+ signaling, these organelles are often observed close to influx and release sites and may be tethered near Ca2+ transporters. In this study, we used high-resolution, wide-field fluorescence imaging to investigate the regulation of Ca2+ signaling by mitochondria in large numbers of endothelial cells (∼50 per field) in intact arteries from rats. We observed that mitochondria were mostly spherical or short-rod structures and were distributed widely throughout the cytoplasm. The density of these organelles did not increase near contact sites with smooth muscle cells. However, local inositol trisphosphate (IP3)-mediated Ca2+ signaling predominated near these contact sites and required polarized mitochondria. Of note, mitochondrial control of Ca2+ signals occurred even when mitochondria were far from Ca2+ release sites. Indeed, the endothelial mitochondria were mobile and moved throughout the cytoplasm. Mitochondrial control of Ca2+ signaling was mediated by ATP production, which, when reduced by mitochondrial depolarization or ATP synthase inhibition, eliminated local IP3-mediated Ca2+ release events. ROS buffering did not significantly alter local Ca2+ release events. These results highlight the importance of mitochondrial ATP production in providing long-range control of endothelial signaling via IP3-evoked local Ca2+ release in intact endothelium.
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Project description:Partitioning of ATP generation between glycolysis and oxidative phosphorylation is central to cellular bioenergetics but cumbersome to measure. We describe here how rates of ATP generation by each pathway can be calculated from simultaneous measurements of extracellular acidification and oxygen consumption. We update theoretical maximum ATP yields by mitochondria and cells catabolizing different substrates. Mitochondrial P/O ratios (mol of ATP generated per mol of [O] consumed) are 2.73 for oxidation of pyruvate plus malate and 1.64 for oxidation of succinate. Complete oxidation of glucose by cells yields up to 33.45 ATP/glucose with a maximum P/O of 2.79. We introduce novel indices to quantify bioenergetic phenotypes. The glycolytic index reports the proportion of ATP production from glycolysis and identifies cells as primarily glycolytic (glycolytic index > 50%) or primarily oxidative. The Warburg effect is a chronic increase in glycolytic index, quantified by the Warburg index. Additional indices quantify the acute flexibility of ATP supply. The Crabtree index and Pasteur index quantify the responses of oxidative and glycolytic ATP production to alterations in glycolysis and oxidative reactions, respectively; the supply flexibility index quantifies overall flexibility of ATP supply; and the bioenergetic capacity quantifies the maximum rate of total ATP production. We illustrate the determination of these indices using C2C12 myoblasts. Measurement of ATP use revealed no significant preference for glycolytic or oxidative ATP by specific ATP consumers. Overall, we demonstrate how extracellular fluxes quantitatively reflect intracellular ATP turnover and cellular bioenergetics. We provide a simple spreadsheet to calculate glycolytic and oxidative ATP production rates from raw extracellular acidification and respiration data.
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Project description:TOM40 is a channel-forming subunit of translocase, which is essential for the movement of proteins into the mitochondria. We found that TOM40 was highly expressed in epithelial ovarian cancer (EOC) cells at both the transcriptional and translational levels; its expression increased significantly during the transformation from normal ovarian epithelial cells to EOC (p < 0.001), and TOM40 expression negatively correlated with disease-free survival (Hazard ratio = 1.79, 95% Confidence inerval 1.16-2.78, p = 0.009). TOM40 knockdown decreased proliferation in several EOC cell lines and reduced tumor burden in an in vivo xenograft mouse model. TOM40 expression positively correlated with intracellular adenosine triphosphate (ATP) levels. The low ATP and high reactive oxygen species (ROS) levels increased the activity of AMP-activated protein kinase (AMPK) in TOM40 knockdown EOC cells. However, AMPK activity did not correlate with declined cell growth in TOM40 knockdown EOC cells. We found that metformin, first-line therapy for type 2 diabetes, effectively inhibited the growth of EOC cell lines in an AMPK-independent manner by inhibiting mitochondria complex I. In conclusion, TOM40 positively correlated with mitochondrial activities, and its association enhances the proliferation of ovarian cancer. Also, metformin is an effective therapeutic option in TOM40 overexpressed ovarian cancer than normal ovarian epithelium.
| S-EPMC7281007 | biostudies-literature