Extracellular pH changes in the superfused cat carotid body during hypoxia and hypercapnia

Extracellular pH changes were measured in the superfused cat carotid body with double barreled pH glass microelectrodes, under constant pH (7.45 ± 0.02), temperature (35°C) and flow (3.6 ml/min) of the superfusion medium. Changes of pO₂ in the medium from about 188 Torr (30% O₂) to 35 or 12 Torr (5% and 2% respectively) called hypoxia, induced a change of the pH signal of about 0.1 units indicating acidification of the tissue. Medium pH monitored with pH macroelectrode did not change during hypoxic stimulation. An increase of pCO₂ in the medium from about 20 Torr (3%CO₂, pH7.45 ± 0.02) to 70 Torr (12%CO₂, pH6.98 ± 0.01) called hypercapnia, under constant pO₂ (188 ± 2Torr), temperature (35°C) and flow (3.6 ml/min) resulted in acidification of the tissue of about 0.3 pH units. Extracellular pH changes during hypoxia did not occur when the superfusion medium had no glucose; however, pH changes during hypercapnia persisted under these conditions. The hypoxic and hypercapnic chemosensory response of the sinus nerve were decreased or abolished during glucose deprivation in a time-dependent manner. Replacement of glucose with 2-deoxyglucose in the medium led to a similar pattern, i.e. inhibition of the hypoxic and hypercapnic chemosensory nerve response and of the extracellular hypoxic pH changes. These results indicate that glycolysis takes place and contributes to O₂ and CO₂-chemoreception in the carotid body.     

M2型丙酮酸激酶(PKM2)在肿瘤代谢和发生中的作用

肿瘤细胞即使在氧气充足的情况下也会通过糖酵解方式来进行代谢,消耗大量葡萄糖并最终生成乳酸,这种现象被称为肿瘤的有氧糖酵解,也叫做Warburg效应.在这一过程中存在一个重要的调节因子,即M2型丙酮酸激酶(PKM2),该酶催化其上游底物磷酸烯醇式丙酮酸(PEP)生成丙酮酸.PKM2在增殖的细胞尤其是肿瘤细胞中高表达.PKM2经常在四聚体和二聚体的形式之间变换以决定葡萄糖转化为丙酮酸后是用于供能还是参与生物合成过程.在肿瘤细胞中PKM2通常以二聚体形式存在.EDTA血浆和粪便中的PKM2含量检测可作为一些癌症的诊断标记物.PKM2是一个磷酸酪氨酸(pTyr)结合蛋白,受多种转录因子如HIF1α和一些代谢中间产物如FBP的调节.随着对其调节机制越来越深入的研究,PKM2在肿瘤代谢和发生中的重要作用使它成为临床上肿瘤治疗的一个新靶点,具有广泛的应用前景.     

Highly Selective Inhibitors of Class II Microbial Fructose Bis-phosphate Aldolases

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An RNA-directed nucleoside anti-metabolite, 1-(3-C-ethynyl-beta-d-ribo-pentofuranosyl)cytosine (ECyd), elicits antitumor effect via TP53-induced Glycolysis and Apoptosis Regulator (TIGAR) downregulation

1-(3-C-ethynyl-beta-d-ribo-pentofuranosyl)cytosine (ECyd) is a ribose-modified nucleoside analog of cytidine with potent anticancer activity in several cancers. The main antitumor mechanism of this promising RNA-directed nucleoside anti-metabolite is efficient blockade of RNA synthesis in cancer cells. Here, we examined the therapeutic potential of this RNA-directed anti-metabolite in in vitro models of nasopharyngeal cancer (NPC). In a panel of 6 NPC cell lines, ECyd effectively inhibited cellular proliferation at nM concentrations (IC₅₀:∼13-44 nM). Moreover, cisplatin-resistant NPC cells were highly sensitive to ECyd (at nM concentration). The ECyd-mediated growth inhibition was associated with G₂/M cell cycle arrest, PARP cleavage (a hallmark of apoptosis) and Bcl-2 downregulation, indicating induction of apoptosis by ECyd in NPC cells. Unexpectedly, ECyd-induced significant downregulation of TIGAR, a newly described dual regulator of apoptosis and glycolysis. More importantly, this novel action of ECyd on TIGAR was accompanied by marked depletion of NADPH, the major reducing power critically required for cell proliferation and survival. We hypothesized that ECyd-induced TIGAR downregulation was crucially involved in the antitumor activity of ECyd. Indeed, overexpression of TIGAR was able to rescue NPC cells from ECyd-induced growth inhibition, demonstrating a novel mechanistic action of ECyd on TIGAR. We demonstrated for the first time that an RNA-directed nucleoside analog, ECyd, exerts its antitumor activity via downregulation of a novel regulator of apoptosis, TIGAR. Moreover, ECyd may represent a novel therapy for NPC.     

Role of perinuclear mitochondria in the spatiotemporal dynamics of spontaneous Ca2+ waves in interstitial cells of Cajal-like cells of the rabbit urethra

BACKGROUND AND PURPOSE

Although spontaneous Ca²⁺ waves in interstitial cells of Cajal (ICC)-like cells (ICC-LCs) primarily arise from endoplasmic reticulum (ER) Ca²⁺ release, the interactions among mitochondrial Ca²⁺ buffering, cellular energetics and ER Ca²⁺ release in determining the spatiotemporal dynamics of intracellular Ca²⁺ remain to be elucidated.

EXPERIMENTAL APPROACH

Spontaneous Ca²⁺ transients in freshly isolated ICC-LCs of the rabbit urethra were visualized using fluo-4 Ca²⁺ imaging, while the intracellular distribution of mitochondria was viewed with MitoTracker Red.

KEY RESULTS

Spontaneous Ca²⁺ waves invariably originated from the perinuclear region where clusters of mitochondria surround the nucleus. Perinuclear Ca²⁺ dynamics were characterized by a gradual rise in basal Ca²⁺ that preceded each regenerative Ca²⁺ transient. Caffeine evoked oscillatory Ca²⁺ waves originating from anywhere within ICC-LCs. Ryanodine or cyclopiazonic acid prevented Ca²⁺ wave generation with a rise in basal Ca²⁺, and subsequent caffeine evoked a single rudimentary Ca²⁺ transient. Inhibition of glycolysis with 2-deoxy-glucose or carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial protonophore, increased basal Ca²⁺ and abolished Ca²⁺ waves. However, caffeine still induced oscillatory Ca²⁺ transients. Mitochondrial Ca²⁺ uptake inhibition with RU360 attenuated Ca²⁺ wave amplitudes, while mitochondrial Ca²⁺ efflux inhibition with {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 suppressed the initial Ca²⁺ rise to reduce Ca²⁺ wave frequency.

CONCLUSIONS AND IMPLICATIONS

Perinuclear mitochondria in ICC-LCs play a dominant role in the spatial regulation of Ca²⁺ wave generation and may regulate ER Ca²⁺ release frequency by buffering Ca²⁺ within microdomains between both organelles. Glycolysis inhibition reduced mitochondrial Ca²⁺ buffering without critically disrupting ER function. Perinuclear mitochondria may function as sensors of intracellular metabolites.     

Digitoxin as an anticancer agent with selectivity for cancer cells: possible mechanisms involved

Accumulating preclinical and clinical data suggest that the cardiac drug digitoxin might be used in cancer therapy. Recent reports have shown that digitoxin can inhibit the growth and induce apoptosis in cancer cells at concentrations commonly found in the plasma of cardiac patients treated with this drug. Several mechanisms have been associated with the anticancer activity of digitoxin, yet at present it is unknown why malignant cells are more susceptible to this cardiac glycoside than non-malignant cells. This report analyses the possible anticancer mechanisms of digitoxin and proposes that the inhibition of glycolysis may be a key mechanism by which this natural product selectively targets cancer cells. Finally, whether or not there is enough evidence to support the clinical evaluation of digitoxin in patients with cancer is discussed.     

Targeting mitochondria in the treatment of human cancer: a coordinated attack against cancer cell energy metabolism and signalling

Mitochondria have major roles in bioenergetics and vital signalling of the mammalian cell. Consequently, these organelles have been implicated in the process of carcinogenesis, which includes alterations of cellular metabolism and cell death pathways. Multiple molecular routes of malignant transformation appear to result in the common ability of many tumours to take up large amounts of glucose. This metabolic twist has been explained by phenomena such as aerobic glycolysis and impaired mitochondrial function, and is linked to tumour growth potential via major cellular signalling pathways. This paper reviews the literature on central mechanisms through which energy metabolism merges with growth, proliferation and death signalling, which tend to include mitochondria at some level. These processes can potentially be targeted by pharmacological agents for therapeutic and chemosensitising purposes.     

Evaluation of liver glycogen catabolism during hypercortisolism induced by the administration of dexamethasone in rats

BackgroundThe contribution of liver glycogen catabolism to hyperglycemia and glucose intolerance induced by pharmacological hypercortisolism were investigated.MethodsFor this purpose, adult maleWistar rats that received 1.0 mg/kg dexamethasone (DEX) ip at 8:00 a.m. (DEX group) or saline (CON group) once a day for 5 consecutive days were compared.ResultsExperimental hypercortisolism was confirmed by higher (p < 0.05) glycemia, lower (p < 0.05) body weight and glucose intolerance. In the fed state, the basal glycogen catabolism and the glucagon (1 nM) and epinephrine (2 μM) induced glycogen catabolism were similar between the groups. The activation of glycogen catabolism induced by phenylephrine (2 μM) and isoproterenol (20 μM) were increased (p < 0.05) and decreased (p < 0.05), respectively, in DEX rats. Furthermore, DEX rats exhibited higher (p < 0.05) glycogen catabolism during the infusion of cAMP (3 μM). However, during the infusion of cAMP (15 μM), 6MBcAMP (3 μM) or cyanide (0.5 mM), the intensification of glycogen breakdown was similar. Thus, in general, hypercortisolism does not influence the basal glycogen catabolism and the liver responsiveness to glycogenolytic agents in the fed state. In contrast with fed state, fasted rats (DEX group) showed a more intense (p < 0.05) basal glycogen catabolism.ConclusionThe contribution of glycogen catabolism to hyperglycemia during hypercortisolism depends of the nutritional status, starting from a negligible participation in the fed state up to a significant contribution in the fasted state.     

Melittin ameliorates inflammation in mouse acute liver failure via inhibition of PKM2-mediated Warburg effect

Acute liver failure (ALF) is a fatal clinical syndrome with no special drug. Recent evidence shows that modulation of macrophage to inhibit inflammation may be a promising strategy for ALF treatment. In this study we investigated the potential therapeutic effects of melittin, a major peptide component of bee venom both in mice model of ALF and in LPS-stimulated macrophages in vitro, and elucidated the underlying mechanisms. ALF was induced in mice by intraperitoneal injection of d-galactosamine/LPS. Then the mice were treated with melittin (2, 4, and 8 mg/kg, ip). We showed that melittin treatment markedly improved mortality, attenuated severe symptoms and signs, and alleviated hepatic inflammation in d-galactosamine/LPS-induced ALF mice with the optimal dose being 4 mg/kg. In addition, melittin within the effective doses did not cause significant in vivo toxicity. In LPS-stimulated RAW264.7 macrophages, melittin (0.7 μM) exerted anti-oxidation and anti-inflammation effects. We showed that LPS stimulation promoted aerobic glycolysis of macrophages through increasing glycolytic rate, upregulated the levels of Warburg effect-related enzymes and metabolites including lactate, LDHA, LDH, and GLUT-1, and activated Akt/mTOR/PKM2/HIF-1α signaling. Melittin treatment suppressed M2 isoform of pyruvate kinase (PKM2), thus disrupted the Warburg effect to alleviate inflammation. Molecular docking analysis confirmed that melittin targeted PKM2. In LPS-stimulated RAW264.7 macrophages, knockdown of PKM2 caused similar anti-inflammation effects as melittin did. In d-galactosamine/LPS-induced ALF mice, melittin treatment markedly decreased the expression levels of PKM2 and HIF-1α in liver. This work demonstrates that melittin inhibits macrophage activation-mediated inflammation via inhibition of aerobic glycolysis by targeting PKM2, which highlights a novel strategy of using melittin for ALF treatment.