Biomedical subjects

Michael R Duchen

Publications and source records attributed to Michael R Duchen.

At least 19 recordsLinked to original sources

PubMed2025

Two genomes, one destiny: Mitophagy at the crossroads of inheritance and disease.

Mechanisms ensuring mito-nuclear compatibility are poorly understood. In a recent study published in Science,1 Frison et al. found that a mouse mitochondrial DNA (mtDNA) mutation can escape mitochondrial surveillance in embryogenesis by repressing the ubiquitin-proteasome system. Inhibition of USP30 restored ubiquitin-mediated mitophagy and reduced mutant burden, suggesting a potential therapeutic target for mtDNA disorders.

Humans↗

PubMed2006

Regulation of redox metabolism in the mouse oocyte and embryo.

Energy homeostasis of the oocyte is a crucial determinant of fertility. Following ovulation, the oocyte is exposed to the unique environment of the Fallopian tube, and this is reflected in a highly specialised biochemistry. The minute amounts of tissue available have made the physiological analysis of oocyte intermediary metabolism almost impossible. We have therefore used confocal imaging of mitochondrial and cytosolic redox state under a range of conditions to explore the oxidative metabolism of intermediary substrates. It has been known for some time that the early mouse embryo metabolises external pyruvate and lactate but not glucose to produce ATP. We now show at the level of single oocytes, that supplied glucose has no effect on the redox potential of the oocyte. Pyruvate is a cytosolic oxidant but a mitochondrial reductant, while lactate is a strong cytosolic reductant via the activity of lactate dehydrogenase. Unexpectedly, lactate-derived pyruvate appears to be diverted from mitochondrial oxidation. Our approach also reveals that the level of reduced glutathione (GSH) in the oocyte is maintained by glutathione reductase, which oxidises intracellular NADPH to reduce oxidised glutathione. Surprisingly, NADPH does not seem to be supplied by the pentose phosphate pathway in the unfertilised oocyte but rather by cytosolic NADP-dependent isocitrate dehydrogenase. Remarkably, we also found that the oxidant action of pyruvate impairs development, demonstrating the fundamental importance of redox state on early development.

Animals↗

PubMed2006

Calcium microdomains and oxidative stress.

The phenomenon of calcium microdomains is firmly established in the field of subcellular physiology. These regions of localized, transient calcium increase are exemplified by the spontaneous 'sparks' released through the ryanodine receptor in myocytes, but include subplasmalemmal microdomains, focal calcium oscillations and microdomains enclosed within organelles, such as the endoplasmic reticulum, golgi and mitochondria. Increasing evidence suggests that oxidative stress regulates both the formation and disappearance of microdomains. Calcium release channels and transporters are all modulated by redox state, while several mechanisms that generate oxidative or nitrosative stress are regulated by calcium. Here, we discuss the evidence for the regulation of calcium microdomains by redox state, and, by way of example, demonstrate that the frequency of calcium sparks in cardiomyocytes is increased in response to oxidative stress. We consider the evidence for the existence of analogous microdomains of reactive oxygen and nitrogen species and suggest that the refinement of imaging techniques for these species might lead to similar concepts. The interaction between Ca(2+) microdomains and proteins that modulate their formation results in a complex and dynamic, spatial signaling mechanism, which is likely to be broadly applicable to different cell types, adding new dimensions to the calcium signaling 'toolkit'.

Animals↗

PubMed2006

FCCP is cardioprotective at concentrations that cause mitochondrial oxidation without detectable depolarisation.

OBJECTIVE: The role of mitochondria and in particular of mitochondrial uncoupling in the mechanism of cardioprotection is not defined. In the accompanying paper we have shown that pretreatment of isolated rat hearts with a low concentration (100 nM) of FCCP, prior to global ischaemia, is cardioprotective, while 300 nM FCCP exacerbates injury. Here we define the mitochondrial responses to increasing concentrations of FCCP and also to explore the equivalence of the cardioprotective doses of diazoxide. METHODS: Changes in mitochondrial respiration in response to FCCP and diazoxide were determined in isolated rat ventricular myocytes. In addition, mitochondrial state was monitored using confocal microscopy to record mitochondrial potential (TMRM) and redox state (NADH) during FCCP and diazoxide treatment. Myocytes were also voltage-clamped and whole cell currents recorded in response to 100 nM FCCP. RESULTS: FCCP (10-1000 nM) caused significant dose-dependent increase in oxygen consumption. Diazoxide (30 microM) failed to cause any measurable change in mitochondrial function. FCCP at 100 nM caused mitochondrial oxidation, but no change in mitochondrial membrane potential or (sarc)K(ATP) channel current, while at 300 nM, FCCP caused significant mitochondrial depolarisation. Diazoxide failed to induce any mitochondrial oxidation or depolarisation. CONCLUSIONS: Concentrations of FCCP that cause mitochondrial oxidation without depolarisation are cardioprotective. Higher FCCP concentrations dissipate mitochondrial membrane potential and exacerbate injury. This establishes the principle that mild mitochondrial uncoupling activates a protective mechanism. Diazoxide did not cause mitochondrial oxidation or mitochondrial depolarisation, suggesting it induces protection via another mechanism.

Animals↗

PubMed2006

Mitochondrial uncoupling, with low concentration FCCP, induces ROS-dependent cardioprotection independent of KATP channel activation.

OBJECTIVE: Both K(ATP) channel opening drugs and ischaemic preconditioning have been suggested to protect the ischaemic heart by acting on K(ATP) channels in the inner mitochondrial membrane, uncoupling the proton gradient and partially dissipating the mitochondrial membrane potential. The aim of these studies was to use low concentrations of FCCP, a mitochondrial protonophore, to bypass the mitochondrial K(ATP) channel and partially uncouple the mitochondria and establish whether this activates protective pathways within the rat heart analogous to K(ATP) channel openers or preconditioning. METHODS: Isolated, Langendorff-perfused rat hearts were subjected to 25 min global zero-flow ischaemia and functional recovery assessed. Hearts were pretreated with FCCP (30-300 nM) in the presence or absence of glibenclamide (1 microM), 5-hydroxydecanoate (5-HD: 100 microM), N-acetyl cysteine (4 mM), or N-2-mercaptopropionyl glycine (1 mM). The metabolic consequences of FCCP perfusion in isolated hearts were studied using (31)P NMR, and reactive oxygen species (ROS) production was measured using DCF fluorescence in isolated rat ventricular myocytes. RESULTS: FCCP exerted a dose-dependent cardioprotective effect, with 100 nM FCCP being the optimal concentration. This effect could not be blocked by glibenclamide or 5-HD, but was completely attenuated by N-acetyl cysteine and N-2-mercaptopropionyl glycine. Perfusion with FCCP (100 nM) did not deplete bulk ATP during the pretreatment period but significantly depleted phosphocreatine. In ventricular myocytes, FCCP caused an antioxidant-sensitive increase in ROS production but diazoxide was without effect. CONCLUSIONS: In the isolated rat heart, partial mitochondrial uncoupling with low-dose FCCP significantly improves post-ischaemic functional recovery via a ROS-dependent pathway. This cardioprotection is not mediated via the depletion of cellular ATP or mitochondrial K(ATP) channel activation.

Acetylcysteine↗

PubMed2006

Effects of NO on mitochondrial function in cardiomyocytes: Pathophysiological relevance.

Although the specific roles of nitric oxide (NO) in the heart in general and on cardiac mitochondria in particular remain controversial, it is now clear that both endogenous and exogenous sources of NO exert important modulatory effects on mitochondrial function. There is also growing evidence that NO can be produced within the mitochondria themselves. NO can influence respiratory activity, both through direct effects on the respiratory chain or indirectly via modulation of mitochondrial calcium accumulation. At pathological concentrations, NO can cause irreversible alterations in respiratory function and can also interact with reactive oxygen species (ROS) to form reactive nitrogen species, which may further impair mitochondrial respiration and can even lead to opening of the mitochondrial permeability transition pore and cell death. Diabetes, aging, myocardial ischemia, and heart failure have all been associated with altered ROS generation, which can alter the delicate regulatory balance of effects of NO in the mitochondria. As NO competes with oxygen at cytochrome oxidase, it can be argued that experiments exploring the roles of NO on mitochondrial respiration should be performed at physiological (i.e. relatively low) oxygen tensions. Improvements in techniques, and a gradual appreciation of the many potential pitfalls in studying mitochondrial NO, are leading to a recognition of the role of NO in the regulation of mitochondrial function in the heart in health and disease.

Aging↗

PubMed2005

The role of an astrocytic NADPH oxidase in the neurotoxicity of amyloid beta peptides.

Amyloid beta peptide (Abeta) accumulates in the CNS in Alzheimer's disease. Both the full peptide (1-42) or the 25-35 fragment are toxic to neurons in culture. We have used fluorescence imaging technology to explore the mechanism of neurotoxicity in mixed asytrocyte/neuronal cultures prepared from rat or mouse cortex or hippocampus, and have found that Abeta acts preferentially on astrocytes but causes neuronal death. Abeta causes sporadic transient increases in [Ca2+]c in astrocytes, associated with a calcium dependent increased generation of reactive oxygen species (ROS) and glutathione depletion. This caused a slow dissipation of mitochondrial potential on which abrupt calcium dependent transient depolarizations were superimposed. The mitochondrial depolarization was reversed by mitochondrial substrates glutamate, pyruvate or methyl succinate, and by NADPH oxidase (NOX) inhibitors, suggesting that it reflects oxidative damage to metabolic pathways upstream of mitochondrial complex I. The Abeta induced increase in ROS and the mitochondrial depolarization were absent in cells cultured from transgenic mice lacking the NOX component, gp91phox. Neuronal death after 24 h of Abeta exposure was dramatically reduced both by NOX inhibitors and in gp91phox knockout mice. Thus, by raising [Ca2+]c in astrocytes, Abeta activates NOX, generating oxidative stress that is transmitted to neurons, causing neuronal death.

Amyloid beta-Peptides↗

PubMed2005

Signalling via the reperfusion injury signalling kinase (RISK) pathway links closure of the mitochondrial permeability transition pore to cardioprotection.

Post-ischemic interventions that activate phosphatidylinositol-3-OH kinase (PI3K)-Akt or ERK1/2 pro-survival kinases (the so-called "reperfusion injury signalling kinase (RISK) pathway") during the first few minutes of reperfusion protect against lethal reperfusion-induced injury. We have previously shown that insulin protects against reperfusion-induced injury via activation of the PI3K-Akt pathway. In addition, opening of the mitochondrial permeability transition pore (mPTP) at the time of reperfusion is a major determinant of lethal reperfusion-induced injury, and pharmacologically inhibiting it is cardioprotective. In this study, we examined the relationship between the pro-survival kinase pathways and mPTP opening. Specifically, we tested the hypothesis that activation of the pro-survival kinase pathway by insulin protects cardiomyocytes by reducing the probability of mPTP opening upon reperfusion. Laser illumination of the fluorophore, tetramethyl rhodamine methyl ester (TMRM), was used to induce oxidative stress in the preparation of adult rat ventricular cardiomyocytes. Maintained illumination ultimately induces mPTP opening, detected as a global mitochondrial depolarization, followed by ATP depletion and rigor contracture. Insulin significantly delayed mPTP opening by a factor of approximately 1.7-fold (P<0.001). The effect of insulin was prevented by Wortmannin and by LY-294002, inhibitors of the PI3K pathway, by SH-6, a selective inhibitor of Akt, and by L-NAME, an inhibitor of nitric oxide production. The expression of a dominant negative construct of Akt eliminated the effect of insulin in delaying mPTP opening in a cardiac cell line. Furthermore, the overexpression of constitutively active Akt was sufficient to maximally delay mPTP opening. These results indicate that activation of the PI3K-Akt pro-survival kinase pathway inhibits opening of the mPTP, and demonstrate an important link between the survival kinases and the mPTP.

Animals↗

PubMed2005

IL-6 induces PI 3-kinase and nitric oxide-dependent protection and preserves mitochondrial function in cardiomyocytes.

OBJECTIVE: Interleukin-6 (IL-6) is a pro-inflammatory cytokine which is a prognostic marker associated with left ventricular contractile dysfunction and heart failure. On the other hand, IL-6 activates signalling pathways which mediate delayed ischemic preconditioning. We have therefore studied the cellular mechanisms of IL-6-induced cardioprotection. METHODS: Inducible nitric oxide synthase (iNOS) expression, cardiomyocyte calcium handling, mitochondrial energetics, and the activation of protective signalling pathways in response to IL-6 were studied in a model of simulated ischemia/reperfusion (sI/R) in isolated neonatal rat ventricular cardiomyocytes. RESULTS: Reperfusion after sI/R induced a rise in cytosolic [Ca2+], a loss of cell morphology and integrity, and a transient increase in mitochondrial potential (Deltapsi m), followed by mitochondrial swelling and collapse of Deltapsi m. Pre-treatment of cardiomyocytes with 10 ng/ml IL-6 for 6 h, 24 h prior to sI/R prevented the secondary rise in cytosolic [Ca2+] and induced expression of iNOS and NO-dependent protection against sI/R injury. The protection against sI/R was concomitant with a NO-dependent reduction in the amplitude of cytosolic Ca2+ transients. IL-6 induced an increase in inner mitochondrial membrane polarisation and increased mitochondrial Ca2+ loading (rhod-2 fluorescence) at baseline, but prevented the reperfusion-induced changes in mitochondrial function. IL-6 pre-treatment also resulted in activation of the phosphatidylinositol (PI) 3-kinase/Akt pathway, and both iNOS induction and IL-6-dependent protection were blocked by the PI 3-kinase inhibitor wortmannin. CONCLUSION: IL-6 induces a PI 3-kinase and NO-dependent protection of cardiomyocytes, which is associated with alterations in mitochondrial Ca2+ handling, inhibition of reperfusion-induced mitochondrial depolarisation, swelling and loss of structural integrity, and suppression of cytosolic Ca2+ transients.

Androstadienes↗

PubMed2005

Expression and modulation of an NADPH oxidase in mammalian astrocytes.

Amyloid beta peptides generate oxidative stress in hippocampal astrocytes through a mechanism sensitive to inhibitors of the NADPH oxidase [diphenylene iodonium (DPI) and apocynin]. Seeking evidence for the expression and function of the enzyme in primary hippocampal astrocytes, we confirmed the expression of the subunits of the phagocyte NADPH oxidase by Western blot analysis and by immunofluorescence and coexpression with the astrocyte-specific marker glial fibrillary acidic protein both in cultures and in vivo. Functional assays using lucigenin luminescence, dihydroethidine, or dicarboxyfluorescein fluorescence to measure the production of reactive oxygen species (ROS) demonstrated DPI and apocynin-sensitive ROS generation in response to the phorbol ester PMA and to raised [Ca2+]c after application of ionomycin or P2u receptor activation. Stimulation by PMA but not Ca2+ was inhibited by the protein kinase C (PKC) inhibitors staurosporine and hispidin. Responses were absent in transgenic mice lacking gp91phox. Expression of gp91phox and p67phox was increased in reactive astrocytes, which showed increased rates of both resting and stimulated ROS generation. NADPH oxidase activity was modulated by intracellular pH, suppressed by intracellular alkalinization, and enhanced by acidification. The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone suppressed basal ROS generation but markedly increased PMA-stimulated ROS generation. This was independent of mitochondrial ROS production, because it was unaffected by mitochondrial depolarization with rotenone and oligomycin. Thus, the NADPH oxidase is expressed in astrocytes and is functional, activated by PKC and intracellular calcium, modulated by pHi, and upregulated by astrocyte activation. The astrocytic NADPH oxidase is likely to play important roles in CNS physiology and pathology.

Animals↗

PubMed2005

Induction of mitochondrial oxidative stress in astrocytes by nitric oxide precedes disruption of energy metabolism.

Inhibition of the mitochondrial electron transport chain (ETC) ultimately limits ATP production and depletes cellular ATP. However, the individual complexes of the ETC in brain mitochondria need to be inhibited by approximately 50% before causing significant depression of ATP synthesis. Moreover, the ETC is the key site for the production of intracellular reactive oxygen species (ROS) and inhibition of one or more of the complexes of the ETC may increase the rate of mitochondrial ROS generation. We asked whether partial inhibition of the ETC, to a degree insufficient to perturb oxidative phosphorylation, might nonetheless induce ROS production. Chronic increase in mitochondrial ROS might then cause oxidative damage to the ETC sufficient to produce prolonged changes in ETC function and so compound the defect. We show that the exposure of astrocytes in culture to low concentrations of nitric oxide (NO) induces an increased rate of O2*- generation that outlasts the presence of NO. No effect was seen on oxygen consumption, lactate or ATP content over the 4-6 h that the cells were exposed to NO. These data suggest that partial ETC inhibition by NO may initially cause oxidative stress rather than ATP depletion, and this may subsequently induce irreversible changes in ETC function providing the basis for a cycle of damage.

Adenosine Triphosphate↗

PubMed2005

Prostaglandin F2alpha potentiates the calcium dependent activation of mitochondrial metabolism in luteal cells.

Cytoplasmic Ca2+ signals are transferred to the mitochondria and activate the Krebs cycle. We have compared the efficiency of this process for two Ca2+ mobilising agonists, PGF2alpha and ATP (acting at metabotropic P2 receptors) in rat luteal cells. [Ca2+]c, [Ca2+]m and mitochondrial NAD(P)H were monitored by means of microspectrofluorimetry and confocal microscopy. While both agonists caused similar elevations of [Ca2+]c, changes in NAD(P)H were larger in response to PGF2alpha than to ATP. PGF2alpha more effectively increased NAD(P)H level also in mouse luteal cells. PGF2alpha caused a faster rate of rise of NAD(P)H fluorescence than ATP when reoxidation was prevented with rotenone, suggesting a faster rate of NAD(P)+ reduction. The NAD(P)H response to both agonists was dependent on the mobilisation of stored Ca2+. We found no difference in the efficacy of transmission of the [Ca2+]c signal to mitochondria in response to PGF2alpha and ATP. Raising [Ca2+]c with ionomycin increased the NAD(P)H signal, which was further raised by PGF2alpha but not by ATP. These data suggest that PGF2alpha potentiates the Ca2+-induced stimulation of mitochondrial metabolism by a Ca2+-independent mechanism and shows that agonists may modulate mitochondrial function differentially through a novel process beyond the simple transfer of Ca2+ from ER to mitochondria.

Adenosine Triphosphate↗

PubMed2005

Effects of beauvericin on the metabolic state and ionic homeostasis of ventricular myocytes of the guinea pig.

Beauvericin, a cyclic hexadepsipeptide with antibiotic properties, has been shown to reduce contraction force and to affect action potential parameters of guinea pig papillary muscles. Its potential to form cation-selective channels in mammalian membranes has been demonstrated. Patch clamp and fluorescence imaging techniques were used to investigate its effects in enzymatically isolated ventricular myocytes. Application of 10 microM beauvericin caused a large [Ca2+]i increase in Fura 2AM-loaded cardiomyocytes leading to cell shortening. The effect could be partially inhibited by ryanodine pretreatment and was largely dependent on external Ca2+ and blocked by 5 mM Ni2+. Beauvericin initiated a progressive increase in [Mg2+]i, the time course of which developed similarly upon increasing the external chemical gradient of Mg2+ 10-fold, to produce an ionophoric challenge. Monitoring of pH(i) with BCECF showed that beauvericin caused cytosolic acidification. Confocal microscopy revealed mitochondrial depolarization in TMRM-loaded cardiomyocytes, which resembled the effect of classical mitochondrial uncouplers. However, the NADH autofluorescence signal followed a biphasic pattern, in contrast to the NADH response to the uncouplers FCCP and the K+-ionophore valinomycin. These results suggest that beauvericin, possibly via its ionophoric properties, acts as an atypical mitochondrial uncoupler, greatly disturbs the physiological ionic balance and pH, challenges cellular metabolism, and causes ATP depletion.

Adenosine Triphosphate↗

PubMed2005

Mitochondrial permeability transition pore as a target for cardioprotection in the human heart.

After an episode of myocardial ischemia, opening of the mitochondrial permeability transition pore (mPTP), at the onset of reperfusion, is a critical determinant of myocyte death. We investigated the role of the mPTP as a target for cardioprotection in the human heart. We subjected human atrial tissue, harvested from patients undergoing cardiac surgery, to a period of lethal hypoxia and investigated the effect of suppressing mPTP opening at the onset of reoxygenation. We found that suppressing mPTP opening at the onset of reoxygenation with known mPTP inhibitors cyclosporin A (CsA, 0.2 micromol/l) and sanglifehrin A (SfA, 1.0 micromol/l) 1) improved recovery of baseline contractile function from 29.4 +/- 2.0% under control conditions to 48.7 +/- 2.2% with CsA and 46.1 +/- 2.3% with SfA (P < 0.01) and 2) improved cell survival from 62.8 +/- 5.3% under hypoxic control conditions to 91.4 +/- 4.1% with CsA and 87.2 +/- 6.2% with SfA (P < 0.001). Furthermore, with a cell model in which oxidative stress was used to induce mPTP opening in human atrial myocytes, we demonstrated directly that CsA and SfA mediated their cardioprotective effects by inhibiting mPTP opening, as evidenced by an extension in the time required to induce mPTP opening from 116 +/- 8 s under control conditions to 189 +/- 10 s with CsA and 183 +/- 12 s with SfA (P < 0.01). We report that suppressing mPTP opening at the onset of reoxygenation protects human myocardium against lethal hypoxia-reoxygenation injury. This suggests that, in the human heart, the mPTP is a viable target for cardioprotection.

Cardiotonic Agents↗

PubMed2004

Calcium signals induced by amyloid beta peptide and their consequences in neurons and astrocytes in culture.

In Alzheimer's disease, amyloid beta (Abeta) peptide is deposited in neuritic plaques in the brain. The Abeta peptide 1-42 or the fragment 25-35 are neurotoxic. We here review our recent explorations of the mechanisms of Abeta toxicity in hippocampal cultures. Abeta had no effect on intracellular calcium in neurons but caused striking changes in nearby astrocytes. The [Ca(2+)](c) signals started approximately 5-15 min after Abeta application and consisted of sporadic [Ca(2+)](c) pulses. These were entirely dependent on extracellular Ca(2+), independent of ER Ca(2+) stores and resulted from Ca(2+) influx, probably through Abeta-induced membrane channels. The Ca(2+) signals were closely associated with transient, episodic acidification which may reflect displacement of protons from binding sites or Ca(2+)/2H(+) exchange. Abeta caused an increased rate of generation of reactive oxygen species (ROS), also seen in astrocytes and not in neurons. The increased ROS generation was blocked by inhibitors of the NADPH oxidase, strongly suggesting that this enzyme, normally associated with immune cells, is expressed in astrocytes. ROS generation was also Ca(2+)-dependent, suggesting that Abeta activation of the enzyme may be secondary to the increase in [Ca(2+)](c). Abeta caused delayed neuronal death despite the fact that all responses were seen only in astrocytes. Neurons could not be protected by glutamate receptor antagonists, but were rescued by inhibition of the NADPH oxidase, by antioxidants and by increasing glutathione. These data suggest that Abeta causes Ca(2+)-dependent oxidative stress by activating an astrocytic NADPH oxidase, and that neuronal death follows through a failure of antioxidant support.

Amyloid beta-Peptides↗

PubMed2004

Vascular endothelial growth factor (VEGF)-D and VEGF-A differentially regulate KDR-mediated signaling and biological function in vascular endothelial cells.

Vascular endothelial growth factor (VEGF)-D binds to VEGF receptors (VEGFR) VEGFR2/KDR and VEGFR3/Flt4, but the signaling mechanisms mediating its biological activities in endothelial cells are poorly understood. Here we investigated the mechanism of action of VEGF-D, and we compared the signaling pathways and biological responses induced by VEGF-D and VEGF-A in endothelial cells. VEGF-D induced KDR and phospholipase C-gamma tyrosine phosphorylation more slowly and less effectively than VEGF-A at early times but had a more sustained effect and was as effective as VEGF-A after 60 min. VEGF-D activated extracellular signal-regulated protein kinases 1 and 2 with similar efficacy but slower kinetics compared with VEGF-A, and this effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase. In contrast to VEGF-A, VEGF-D weakly stimulated prostacyclin production and gene expression, had little effect on cell proliferation, and stimulated a smaller and more transient increase in intracellular [Ca(2+)]. VEGF-D induced strong but more transient phosphatidylinositol 3-kinase (PI3K)-mediated Akt activation and increased PI3K-dependent endothelial nitric-oxide synthase phosphorylation and cell survival more weakly. VEGF-D stimulated chemotaxis via a PI3K/Akt- and endothelial nitric-oxide synthase-dependent pathway, enhanced protein kinase C- and PI3K-dependent endothelial tubulogenesis, and stimulated angiogenesis in a mouse sponge implant model less effectively than VEGF-A. VEGF-D-induced signaling and biological effects were blocked by the KDR inhibitor SU5614. The finding that differential KDR activation by VEGF-A and VEGF-D has distinct consequences for endothelial signaling and function has important implications for understanding how multiple ligands for the same VEGF receptors can generate ligand-specific biological responses.

Animals↗

PubMed2004

Preconditioning protects by inhibiting the mitochondrial permeability transition.

Mitochondrial permeability transition (mPT) is a crucial event in the progression to cell death in the setting of ischemia-reperfusion. We have used a model system in which mPT can be reliably and reproducibly induced to test the hypothesis that the profound protection associated with the phenomenon of myocardial preconditioning is mediated by suppression of the mPT. Adult rat myocytes were loaded with the fluorescent probe tetramethylrhodamine methyl ester, which generates oxidative stress on laser illumination, thus inducing the mPT (indicated by collapse of the mitochondrial membrane potential) and ATP depletion, seen as rigor contracture. The known inhibitors of the mPT, cyclosporin A (0.2 microM) and N-methyl-4-valine-cyclosporin A (0.4 microM), increased the time taken to induce the mPT by 1.8- and 2.9-fold, respectively, compared with control (P < 0.001) and rigor contracture by 1.5-fold compared with control (P < 0.001). Hypoxic preconditioning (HP) and pharmacological preconditioning, using diazoxide (30 microM) or nicorandil (100 microM), also increased the time taken to induce the mPT by 2.0-, 2.1-, and 1.5-fold, respectively (P < 0.001), and rigor contracture by 1.9-, 1.7-, and 1.5-fold, respectively, compared with control (P < 0.001). Effects of HP, diazoxide, and nicorandil were abolished in the presence of mitochondrial ATP-sensitive K(+) (K(ATP)) channel blockers glibenclamide (10 microM) and 5-hydroxydecanoate (100 microM) but were maintained in the presence of the sarcolemmal K(ATP) channel blocker HMR-1098 (10 microM). In conclusion, preconditioning protects the myocardium by reducing the probability of the mPT, which normally occurs during ischemia-reperfusion in response to oxidative stress.

Animals↗

PubMed2004

The large-conductance Ca2+-activated K+ channel is essential for innate immunity.

Neutrophil leukocytes have a pivotal function in innate immunity. Dogma dictates that the lethal blow is delivered to microbes by reactive oxygen species (ROS) and halogens, products of the NADPH oxidase, whose impairment causes immunodeficiency. However, recent evidence indicates that the microbes might be killed by proteases, activated by the oxidase through the generation of a hypertonic, K+-rich and alkaline environment in the phagocytic vacuole. Here we show that K+ crosses the membrane through large-conductance Ca2+-activated K+ (BK(Ca)) channels. Specific inhibitors of these channels, iberiotoxin and paxilline, blocked oxidase-induced 86Rb+ fluxes and alkalinization of the phagocytic vacuole, whereas NS1619, a BK(Ca) channel opener, enhanced both. Characteristic outwardly rectifying K+ currents, reversibly inhibited by iberiotoxin, were demonstrated in neutrophils and eosinophils and the expression of the alpha-subunit of the BK channel was confirmed by western blotting. The channels were opened by the combination of membrane depolarization and elevated Ca2+ concentration, both consequences of oxidase activity. Remarkably, microbial killing and digestion were abolished when the BK(Ca) channel was blocked, revealing an essential and unexpected function for this K+ channel in the microbicidal process.

Calcium↗