Developmental Differences in Platelet Inhibition Response to Prostaglandin E1.

BACKGROUND: The mechanisms underlying neonatal platelets hyporesponsiveness are not fully understood. While previous studies have demonstrated developmental impairment of agonist-induced platelet activation, differences in inhibitory signaling pathways have been scarcely investigated. OBJECTIVE: To compare neonatal and adult platelets with regard to inhibition of platelet reactivity by prostaglandin E1 (PGE1). METHODS: Platelet-rich plasma from umbilical cord (CB) or adult blood was incubated with PGE1 (0-1 µM). We assessed aggregation in response to adenosine diphosphate (ADP), collagen, and thrombin receptor activating peptide as well as cyclic adenosine 3'5'-monophosphate (cAMP) levels (ELISA). Gαs, Gαi2, and total- and phospho-protein kinase A (PKA) were evaluated in adult and CB ultrapure and washed platelets, respectively, by immunoblotting. RESULTS: Neonatal (vs. adult) platelets display hypersensitivity to inhibition by PGE1 of platelet aggregation induced by ADP and collagen (PGE1 IC50: 14 and 117 nM for ADP and collagen, respectively, vs. 149 and 491 nM in adults). They also show increased basal and PGE1-induced cAMP levels. Mechanistically, PGE1 acts by binding to the prostanoid receptor IP (prostacyclin receptor), which couples to the Gαs protein-adenylate cyclase axis and increases intracellular levels of cAMP. cAMP activates PKA, which phosphorylates different target inhibitor proteins. Neonatal platelets showed higher basal and PGE1-induced cAMP levels, higher Gαs protein expression, and a trend to increased PKA-dependent protein phosphorylation compared to adult platelets. CONCLUSION: Neonatal platelets have a functionally increased PGE1-cAMP-PKA axis. This finding supports a downregulation of inhibitory when going from neonate to adult contributing to neonatal platelet hyporesponsiveness.

Heat-shock protein 72 promotes platelet aggregation induced by various platelet activators in rats.

Increase of thrombus in the coronary arteries is positively correlated with the level of heat-shock protein 72 (HSP72) in the blood of patients with acute myocardial infarction (AMI). Platelet aggregation participates in thrombus formation on ruptured plaque in AMI. In this study, we aimed to clarify the role of HSP72 in thrombus formation by evaluating the effects of HSP72 on platelet aggregation. Platelet aggregation activities were measured in platelet-rich plasma obtained from male Sprague-Dawley rats with or without the platelet activators, such as adenosine diphosphate (ADP), collagen, thrombin receptor-activating peptide-6 (TRAP-6), ristocetin, and arachidonic acid. Changes in aggregation were estimated by the co-addition of recombinant HSP72 and anti-HSP72 antibodies. Our results showed that addition of HSP72 increased platelet aggregation in the presence of low concentrations of ADP, collagen, TRAP-6, ristocetin, and arachidonic acid. Increased platelet aggregation stimulated by ADP and HSP72 was reduced by the co-addition of anti-HSP72 antibodies. Thus, these findings suggested that HSP72 was released extracellularly in response to stress, promoting thrombus formation and AMI. Additionally, treatment with anti-HSP72 antibodies may control platelet aggregation induced by extracellular HSP72.

[Research on Relation of Platelet Aggregation Rate with Platelet Concentration in Platelet-Rich Plasma'].

OBJECTIVE: To explore the effect of platelet concentration in platelet-rich plasma (PRP)' on platelet aggregation rate, to determine the allowed range of inducer adenosine diphosphate (ADP: 5.0 µmol/L, ADP: 1.5 µmol/L) and arachidonic acid (AA: 500.0 µg/ml) for platelet concentration in platelet aggregation rate. METHODS: The venous blood samples of 72 healthy persons with citrate anticoagulation were selected and divided into groups, every 12 cases were mixed for each group. After the samples were mixed, different levels of platelet concentrations were prepared, and platelet aggregation rate of 6 groups with different platelet concentrations by ADP and AA inducer were detected by Helena platelet aggregation analyzer respectively. RESULTS: When the concentration of the inducer increased, the platelet aggregation rate of different platelet concentrations increased. With the platelet concentration increased, AA and ADP (15 µmol/L) induced platelet aggregation rate showed a rapid increase at first and then showed slowing, when the Plt concentration is less than 200×109/L, AA induced platelet aggregation rate decreased with the steep drop, and ADP induced platelet aggregation rate increased slightly slowly along with platelet concentration increasing. When platelet count lower than 200×109/L and AA 500 µg/ml and ADP 15 µmol/L were used for induction, the changes of platelet aggregation rate of different concentrations of platelet was slow, less volatile and more stable. CONCLUSION: When the platelet aggregation rate was detected, the appropriate platelet concentration of AA and ADP inducing agents should be greater than 200×109/L.

Association of α2A-Adrenergic Receptor Genetic Variants with Platelet Reactivity in Chinese Patients on Dual Antiplatelet Therapy Undergoing Percutaneous Coronary Intervention.

OBJECTIVE: The alpha 2A-adrenergic receptor gene (ADRA2A) polymorphism in individuals modifies the antiplatelet response to sympathetic stimulation. The aim of this study was to investigate the effect of ADRA2A variants on platelet reactivity in Chinese patients on dual antiplatelet therapy (DAPT) after undergoing percutaneous coronary intervention (PCI). METHODS: From March 2011 to March 2013, 1,024 patients were enrolled in this prospective, single-center, observational study in China. Four single nucleotide polymorphisms (SNPs) of ADRA2A gene (rs11195419, rs3750625, rs13306146, and rs553668) and CYP2C19*2 were detected by ligase detection reaction (LDR), and adenosine diphosphate (ADP) inhibition was detected by thromboelastography (TEG®). RESULTS: The minor allele frequencies of ADRA2A SNPs were common. Platelet ADP inhibition was significantly different among patients carrying rs11195419 (adjusted P = 0.022) and rs3750625 (adjusted P = 0.016). The homozygous allele carriers had the lowest ADP inhibition. However, ADP inhibition was not significantly different in rs553668 and rs13306146. At the multivariate analysis, rs11195419 (P = 0.033), rs3750625 (P = 0.020) and CYP2C19*2 (P = 0.002) were independent predictors of ADP inhibition. Subgroups analysis based on sex showed rs11195419 (P = 0.003) and rs3750625 (P = 0.002) were significantly associated with ADP inhibition in males, but not in females. CONCLUSION: ADRA2A genetic variations were associated with ADP-induced platelet aggregation during DAPT in Chinese patients undergoing PCI, and the effect was particularly more pronounced in males.

Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms.

The γ subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different γ isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the γ1, γ2 or γ3 isoform. When assayed at a physiological ATP concentration (5 mM), γ1- and γ2-containing complexes were allosterically activated almost 10-fold by AMP, with EC50 values one to two orders of magnitude lower than the ATP concentration. By contrast, γ3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr(172) phosphorylation, when cells were incubated with mitochondrial inhibitors that increase cellular AMP. With γ1 complexes, activation and Thr(172) phosphorylation induced by the upstream kinase LKB1 [liver kinase B1; but not calmodulin-dependent kinase kinase (CaMKKß)] in cell-free assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the γ2 and γ3 complexes were small or insignificant. Binding of AMP or ADP protected all three γ subunit complexes against inactivation by Thr(172) dephosphorylation; with γ2 complexes, ADP had similar potency to AMP, but with γ1 and γ3 complexes, ADP was less potent than AMP. Thus, AMPK complexes containing different γ subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles.

[Hetero-oligomerization and Functional Interaction between Purinergic Receptors Expressed in Platelets to Regulate Platelet Shape Change].

Adenosine and its precursors, ATP and ADP, exert various physiological effects via binding to purinergic receptors. We previously used co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and immunoelectron microscopy to demonstrate the hetero-oligomerization of purinergic receptor subtypes. Furthermore, pharmacological studies found significant changes in receptor-mediated signaling in human embryonic kidney (HEK) 293T cells co-transfected with these receptors. These findings suggest that heterodimers of purinergic receptors may have distinct pharmacological profiles, possibly due to dimerization-induced conformational changes, further suggesting that hetero-dimerization may be employed to "fine-tune" purinergic receptor signaling. Adenosine A(2A) receptor (A(2A)R), P2Y1 receptor (P2Y1R) and P2Y12 receptor (P2Y12R) are predominantly expressed on human platelets. ADP activates human platelets by stimulating both P2Y1R and P2Y12R, which act sequentially and in concert to achieve complete platelet aggregation. In contrast, adenosine stimulates Gs-coupled A(2A)R, followed by activativation of adenylate cyclase, leading to an increase in intracellular cAMP levels, which potently inhibits platelet activation. We examined the hetero-oligomerization and functional interactions of A(2A)R, P2Y1R, and P2Y12R. In HEK293T cells triply expressing all three receptors, hetero-oligomerization was observed among the three receptors. Additionally, P2Y1R agonist-evoked Ca(2+) signaling was significantly inhibited by co-treatment with an A(2A)R antagonist in HEK293T cells. In human platelets, we identified endogenous A(2A)R/P2Y1R and A(2A)R/P2Y12R heterodimers. We also observed functional Ca(2+)-signaling-related cross-talk similar to those found in HEK293T cells, and found that they appeared to affect platelet shape. These results collectively suggest that intermolecular signal transduction and specific conformational changes occur among components of the hetero-oligomers formed by these three receptors.

Spatiotemporal regulation of coagulation and platelet activation during the hemostatic response in vivo.

The hemostatic response requires the tightly regulated interaction of the coagulation system, platelets, other blood cells and components of the vessel wall at a site of vascular injury. The dysregulation of this response may result in excessive bleeding if the response is impaired, and pathologic thrombosis with vessel occlusion and tissue ischemia if the response is overly robust. Extensive studies over the past decade have sought to unravel the regulatory mechanisms that coordinate the multiple biochemical and cellular responses in time and space to ensure that an optimal response to vascular damage is achieved. These studies have relied in part on advances in in vivo imaging techniques in animal models, allowing for the direct visualization of various molecular and cellular events in real time during the hemostatic response. This review summarizes knowledge gained with these in vivo imaging and other approaches that provides new insights into the spatiotemporal regulation of coagulation and platelet activation at a site of vascular injury.

Blood cells: an historical account of the roles of purinergic signalling.

The involvement of purinergic signalling in the physiology of erythrocytes, platelets and leukocytes was recognised early. The release of ATP and the expression of purinoceptors and ectonucleotidases on erythrocytes in health and disease are reviewed. The release of ATP and ADP from platelets and the expression and roles of P1, P2Y(1), P2Y(12) and P2X1 receptors on platelets are described. P2Y(1) and P2X(1) receptors mediate changes in platelet shape, while P2Y(12) receptors mediate platelet aggregation. The changes in the role of purinergic signalling in a variety of disease conditions are considered. The successful use of P2Y(12) receptor antagonists, such as clopidogrel and ticagrelor, for the treatment of thrombosis, myocardial infarction and stroke is discussed.

Synergistic role of ADP and Ca(2+) in diastolic myocardial stiffness.

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin-related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca(2+) ] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction. Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca(2+) ] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca(2+) handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca(2+) ] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca(2+) in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca(2+) both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca(2+) ] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca(2+) overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca(2+) ]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca(2+) , and thereby increase myocardial stiffness.

Disabled-2 is required for efficient hemostasis and platelet activation by thrombin in mice.

OBJECTIVE: The essential role of platelet activation in hemostasis and thrombotic diseases focuses attention on unveiling the underlying intracellular signals of platelet activation. Disabled-2 (Dab2) has been implicated in platelet aggregation and in the control of clotting responses. However, there is not yet any in vivo study to provide direct evidence for the role of Dab2 in hemostasis and platelet activation. APPROACH AND RESULTS: Megakaryocyte lineage-restricted Dab2 knockout (Dab2(-/-)) mice were generated to delineate in vivo functions of Dab2 in platelets. Dab2(-/-) mice appeared normal in size with prolonged bleeding time and impaired thrombus formation. Although normal in platelet production and granule biogenesis, Dab2(-/-) platelets elicited a selective defect in platelet aggregation and spreading on fibrinogen in response to low concentrations of thrombin, but not other soluble agonists. Investigation of the role of Dab2 in thrombin signaling revealed that Dab2 has no effect on the expression of thrombin receptors and the outside-in signaling. Dab2(-/-) platelets stimulated by low concentrations of thrombin were normal in Gαq-mediated calcium mobilization and protein kinase C activation, but were defective in Gα12/13-mediated RhoA-ROCKII activation. The attenuated Gα12/13 signaling led to impaired ADP release, Akt-mammalian target of rapamycin and integrin αIIbß3 activation, fibrinogen binding, and clot retraction. The defective responses of Dab2(-/-) platelets to low concentrations of thrombin stimulation may contribute to the impaired hemostasis and thrombosis of Dab2(-/-) mice. CONCLUSIONS: This study sheds new insight in platelet biology and represents the first report demonstrating that Dab2 is a key regulator of hemostasis and thrombosis by functional interplay with Gα12/13-mediated thrombin signaling.

Human neutrophil α-defensins are associated with adenosine diphosphate-inducible neutrophil-platelet aggregate formation and response to clopidogrel in patients with atherosclerosis.

Human neutrophil α-defensins (HNPs) are antimicrobial peptides stored primarily in the azurophilic granules of polymorphonuclear leukocytes. Recently, it was shown that HNPs act as platelet agonists. We hypothesized that HNP levels are associated with the formation of neutrophil-platelet aggregates, and that they influence the response to clopidogrel therapy. HNP levels were determined by a commercially available enzyme-linked immunosorbent assay in 305 patients undergoing angioplasty and stenting for atherosclerotic cardiovascular disease. Neutrophil-platelet aggregates were measured by flow cytometry, and on-treatment platelet reactivity was determined using the VerifyNow P2Y12 and aspirin assays. HNP levels did not correlate with the formation of neutrophil-platelet aggregates in vivo (r = 0.05, P = 0.4). In contrast, HNP levels correlated significantly with adenosine diphosphate (ADP)-inducible neutrophil-platelet aggregate formation (r = 0.13, P = 0.04). On-treatment platelet reactivity by the VerifyNow P2Y12 assay was significantly more pronounced in patients with high HNP levels compared with patients with low HNP levels (211 P2Y12 reaction units [PRU; range, 143-293 PRU] vs 181 PRU [range, 129-237 PRU], P = 0.009). This association remained significant after adjusting for high-sensitivity C-reactive protein and interleukin 6 by multivariate regression analysis (P = 0.007). Moreover, high on-treatment residual platelet reactivity by the VerifyNow P2Y12 assay was more frequent in patients with high HNP levels than in patients with low HNP levels (40% vs 26.6%, P = 0.01). In conclusion, HNP levels are associated with ADP-inducible neutrophil-platelet aggregate formation and clopidogrel-mediated platelet inhibition. High levels of HNPs may, in part, be responsible for the observed response variability to clopidogrel.

Submaximal ADP-stimulated respiration is impaired in ZDF rats and recovered by resveratrol.

Mitochondrial dysfunction and reactive oxygen species (ROS) have been implicated in the aetiology of skeletal muscle insulin resistance, although there is considerable controversy regarding these concepts. Mitochondrial function has been traditionally assessed in the presence of saturating ADP, but ATP turnover and the resultant ADP is thought to limit respiration in vivo. Therefore, we investigated the potential link between submaximal ADP-stimulated respiration rates, ROS generation and skeletal muscle insulin sensitivity in a model of type 2 diabetes mellitus, the ZDF rat. Utilizing permeabilized muscle fibres we observed that submaximal ADP-stimulated respiration rates (250-2000 µm ADP) were lower in ZDF rats than in lean controls, which coincided with decreased adenine nucleotide translocase 2 (ANT2) protein content. This decrease in submaximal ADP-stimulated respiration occurred in the absence of a decrease in electron transport chain function. Treating ZDF rats with resveratrol improved skeletal muscle insulin resistance and this was associated with elevated submaximal ADP-stimulated respiration rates as well as an increase in ANT2 protein content. These results coincided with a greater ability of ADP to attenuate mitochondrial ROS emission and an improvement in cellular redox balance. Together, these data suggest that mitochondrial dysfunction is present in skeletal muscle insulin resistance when assessed at submaximal ADP concentrations and that ADP dynamics may influence skeletal muscle insulin sensitivity through alterations in the propensity for mitochondrial ROS emission.

Mitochondrial uncoupling in skeletal muscle by UCP1 augments energy expenditure and glutathione content while mitigating ROS production.

Enhancement of proton leaks in muscle tissue represents a potential target for obesity treatment. In this study, we examined the bioenergetic and physiological implications of increased proton leak in skeletal muscle. To induce muscle-specific increases in proton leak, we used mice that selectively express uncoupling protein-1 (UCP1) in skeletal muscle tissue. UCP1 expression in muscle mitochondria was ∼13% of levels in brown adipose tissue (BAT) mitochondria and caused increased GDP-sensitive proton leak. This was associated with an increase in whole body energy expenditure and a decrease in white adipose tissue content. Muscle UCP1 activity had divergent effects on mitochondrial ROS emission and glutathione levels compared with BAT. UCP1 in muscle increased total mitochondrial glutathione levels ∼7.6 fold. Intriguingly, unlike in BAT mitochondria, leak through UCP1 in muscle controlled mitochondrial ROS emission. Inhibition of UCP1 with GDP in muscle mitochondria increased ROS emission ∼2.8-fold relative to WT muscle mitochondria. GDP had no impact on ROS emission from BAT mitochondria from either genotype. Collectively, these findings indicate that selective induction of UCP1-mediated proton leak in muscle can increase whole body energy expenditure and decrease adiposity. Moreover, ectopic UCP1 expression in skeletal muscle can control mitochondrial ROS emission, while it apparently plays no such role in its endogenous tissue, brown fat.

Single KATP channel opening in response to stimulation of AMPA/kainate receptors is mediated by Na+ accumulation and submembrane ATP and ADP changes.

Excessive stimulation of glutamatergic receptors (GluRs) can overexcite neurons. This can be dampened by KATP channels linking metabolic and neuronal activities, but the cross-talk has not yet been examined on the single channel level. In the brainstem and hippocampal neurons, GluR agonists augmented the open state probability (Popen) of KATP channels with relative efficacy: kainate AMPA > NMDA > t-ACPD. Inhibition of calcium influx and chelation of intracellular calcium did not modify the effects. Kainate did not augment production of reactive oxygen species measured with roGFP1. H2O2 slightly increased Popen, but GluR effects were not modified. GluR actions were abolished in Na(+)-free solutions and after blockade of Na(+)-K(+)-ATPase. KATP channels in open-cell patch-clamp measurements were inhibited by ATP, stimulated by ADP, and kainate was effective only in the presence of ATP. GluR stimulation enhanced ATP consumption that decreased submembrane ATP levels, whereas metabolic poisoning diminished bulk ATP. Modelling showed strong ATP depletion and ADP accumulation near the membrane, and both effects contributed to Popen increases after GluR stimulation. Kainate and hypoxia activated KATP channels in the functional brainstem slices. Inhibition of aerobic ATP production and GluR stimulation were about equally effective in KATP channel opening during hypoxia. Induction of seizure-like activity in hippocampal slices with Mg(2+)-free solutions was accompanied by ATP decrease and KATP channel opening. We propose that KATP channels and GluRs are functionally coupled that can regulate long-lasting changes of neuronal activity in the CNS neurons.

Energy cost of force production is reduced after active stretch in skinned muscle fibres.

Residual force enhancement has been observed consistently in skeletal muscles. Despite an abundance of experimental observations, there has been no information about the metabolic cost of the force observed after stretch. Our aim was to investigate the energy cost of force production after active stretch in skinned fibres isolated from rabbit psoas muscle, by quantifying the ATPase activity using an enzyme-coupled assay. Fibres were actively stretched from an average sarcomere length of 2.4 µm to average sarcomere lengths of 2.8 and 3.2 µm. Purely isometric reference contractions were performed at average sarcomere lengths of 2.8 and 3.2 µm. Simultaneously with the force measurements, the ATP cost per unit of force produced was measured during the last 40s of isometric contraction. Results showed that ATPase activity per unit of force was reduced by 17.2±4.1% in the isometric contractions after active stretch, compared to the purely isometric contraction at the corresponding lengths for both stretch magnitudes. Fibres stretched to an average sarcomere length of 3.2 µm showed a higher reduction in ATPase activity per unit of force compared to fibres stretched to an average sarcomere length of 2.8 µm (20.7±4.4 versus 12.4±3.2% respectively). Passive force enhancement was observed in all fibres and was correlated with the decrease in ATPase activity. No difference in stiffness was observed between reference and active stretch contractions. These results suggest that skeletal muscles become more efficient after stretch, either by increasing the amount of force produced per cross bridge or by engaging a passive element.

Deciphering of ADP-induced, phosphotyrosine-dependent signaling networks in human platelets by Src-homology 2 region (SH2)-profiling.

Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets.

Calmodulin and calmodulin kinase II mediate emergent bursting activity in the brainstem respiratory network (preBötzinger complex).

Emergence of persistent activity in networks can be controlled by intracellular signalling pathways but the mechanisms involved and their role are not yet fully explored. Using calcium imaging and patch-clamp we examined the rhythmic activity in the preBötzinger complex (preBötC) in the lower brainstem that generates the respiratory motor output. In functionally intact acute slices brief hypoxia, electrical stimulation and activation of AMPA receptors transiently depressed bursting activity which then recovered with augmentation. The effects were abrogated after chelation of intracellular calcium, blockade of L-type calcium channels and inhibition of calmodulin (CaM) and CaM kinase (CaMKII). Rhythmic calcium transients and synaptic drive currents in preBötC neurons in the organotypic slices showed similar CaM- and CaMKII-dependent responses. The stimuli increased the amplitude of spontaneous and miniature excitatory synaptic currents indicating postsynaptic changes at glutamatergic synapses. In the acute and organotypic slices, CaM stimulated and ADP inhibited calcium-dependent TRPM4 channels and CaMKII augmented synaptic drive currents. Experimental data and simulations show the role of ADP and CaMKII in the control of bursting activity and its relation to intracellular signalling. I propose that CaMKII-mediated facilitation of glutamatergic transmission strengthens emergent synchronous activity within preBötC that is then maintained by periodic surges of calcium during the bursts. This may find implications in restoration and consolidation of autonomous activity in the respiratory disorders.

High Ca2+ load promotes hydrogen peroxide generation via activation of α-glycerophosphate dehydrogenase in brain mitochondria.

H(2)O(2) generation associated with α-glycerophosphate (α-GP) oxidation was addressed in guinea pig brain mitochondria challenged with high Ca(2+) load (10 µM). Exposure to 10 µM Ca(2+) induced an abrupt 2.5-fold increase in H(2)O(2) release compared to that measured in the presence of a physiological cytosolic Ca(2+) concentration (100 nM) from mitochondria respiring on 5 mM α-GP in the presence of ADP (2 mM). The Ca(2+)-induced stimulation of H(2)O(2) generation was reversible and unaltered by the uniporter blocker Ru 360, indicating that it did not require Ca(2+) uptake into mitochondria. Enhanced H(2)O(2) generation by Ca(2+) was also observed in the absence of ADP when mitochondria exhibited permeability transition pore opening with a decrease in the NAD(P)H level, dissipation of membrane potential, and mitochondrial swelling. Furthermore, mitochondria treated with the pore-forming peptide alamethicin also responded with an elevated H(2)O(2) generation to a challenge with 10 µM Ca(2+). Ca(2+)-induced promotion of H(2)O(2) formation was further enhanced by the complex III inhibitor myxothiazol. With 20 mM α-GP concentration, stimulation of H(2)O(2) formation by Ca(2+) was detected only in the presence, not in the absence, of ADP. It is concluded that α-glycerophosphate dehydrogenase, which is accessible to and could be activated by a rise in the level of cytosolic Ca(2+), makes a major contribution to Ca(2+)-stimulated H(2)O(2) generation. This work highlights a unique high-Ca(2+)-stimulated reactive oxygen species-forming mechanism in association with oxidation of α-GP, which is largely independent of the bioenergetic state and can proceed even in damaged, functionally incompetent mitochondria.

Significant correlation between the acceleration of platelet aggregation and phosphorylation of HSP27 at Ser-78 in diabetic patients.

To clarify the mechanism underlying a high risk of thrombotic complications in diabetic patients, we investigated the relationship between HSP27 phosphorylation and the platelet activation induced by adenosine diphosphate (ADP) in diabetic patients. Platelet-rich plasma was prepared from the blood of type 2 diabetes mellitus (DM) patients. By measuring the dose response of platelet aggregation to ADP, an individual ED50 was determined. Based on the normal range identified in non-DM controls, the subjects were divided into a hyper-aggregate (Group 1) and a normo- or hypo-aggregate group (Group 2). The protein phosphorylation was analyzed by western blotting. The release of PDGF-AB and sCD40 ligand (sCD40L) was measured by ELISA. In both groups, ADP induced HSP27 phosphorylation at Ser-78 and Ser-82. The phosphorylation at Ser-78 and the release of both PDGF-AB and sCD40L induced by a low dose of ADP (1 µM) in Group 1 were significantly higher than these values in Group 2. There was a significant relationship between the ADP-induced HSP27 phosphorylation level at Ser-78 and the ADP ED50 value of platelet aggregation. The ADP (1 µM)-induced phosphorylation of HSP at Ser-78 observed in the platelets from Group 1 was inhibited by PD98059 or SB203580. The use of aspirin ameliorated the accelerated microaggregation of platelets in Group 1, and the low-dose ADP-induced phosphorylation of HSP27 at Ser-78 was no longer observed. These results strongly suggest that the phosphorylation of HSP27 at Ser-78 is correlated with the acceleration of platelet aggregation induced by ADP in type 2 DM patients.

Hepatic mitochondrial dysfunction induced by fatty acids and ethanol.

Understanding the key aspects of the pathogenesis of alcoholic fatty liver disease particularly alterations to mitochondrial function remains to be resolved. The role of fatty acids in this regard requires further investigation due to their involvement in fatty liver disease and obesity. This study aimed to characterize the early effects of saturated and unsaturated fatty acids alone on liver mitochondrial function and during concomitant ethanol exposure using isolated liver mitochondria and VA-13 cells (Hep G2 cells that efficiently express alcohol dehydrogenase). Liver mitochondria or VA-13 cells were treated with increasing concentrations of palmitic or arachidonic acid (1 to 160 µM) for 24 h with or without 100 mM ethanol. The results showed that in isolated liver mitochondria both palmitic and arachidonic acids significantly reduced state 3 respiration in a concentration-dependent manner (P<0.001), implicating their ionophoric activities. Increased ROS production occurred in a dose-dependent manner especially in the presence of rotenone (complex I inhibitor), which was significantly more prominent in arachidonic acid at 80 µM (+970%, P<0.001) than palmitic acid (+40%, P<0.01). In VA-13 cells, ethanol alone and both fatty acids (40 µM) were able to decrease the mitochondrial membrane potential and cellular ATP levels and increase lipid formation. ROS production was significantly increased with arachidonic acid (+110%, P<0.001) exhibiting a greater effect than palmitic acid (+39%, P<0.05). While in the presence of ethanol, the drop in the mitochondrial membrane potential, cellular ATP levels, and increased lipid formation were further enhanced by both fatty acids, but with greater effect in the case of arachidonic acid, which also correlated with significant cytotoxicity (P<0.001). This study confirms the ability of fatty acids to promote mitochondrial injury in the development of alcoholic fatty liver disease.