In-situ generation of peroxynitrite from 3-morpholinosydnonimine decay in pulmonary cell culture medium

Background: Peroxynitrite (ONOO-) is a strong oxidising and nitrating agent generally implicated in oxidative stress. Its cellular action is linked with pulmonary artery cell hyper-proliferation and vascular remodelling seen in pulmonary hypertensive diseases. It is thus vital to elucidate the biological actions of peroxynitrite; however, working with the anion is challenging. Whether supplied commercially or prepared extemporaneously, ONOO- is stabilised and stored under strongly alkaline conditions and the exposure of cells to this form of ONOO- will in tandem increase culture media pH. Accordingly, increasing number of studies are seeking alternative means of generating peroxynitrite and have utilised 3-morpholinosydnonimine (SIN-1) the active metabolite of the vasodilatory drug molisdomine to generate peroxynitrite in-situ. Even so, it is not clear how much authentic ONOO- is generated under these conditions and for how long. Aim: To establish the formation of peroxynitrite and to determine its decay kinetics following SIN-1 decomposition in a medium formulated for the culture of primary human and bovine pulmonary artery cells. Results: The half-life of authentic peroxynitrite was determined to be 1.38s in pulmonary artery cell culture medium. Formation of peroxynitrite during 3-morpholinosydnonimine (SIN-1) decomposition was continuously monitored from the loss in fluorescence associated with the ONOO- oxidation of nicotinamide adenine dinucleotide (NADH) to NAD+. SIN-1 decayed by 1st order kinetics and 20μM SIN-1 generated ONOO- at the rate of 0.11 μM min-1. SIN-1 decomposition in cell culture medium was associated with the formation of a stable intermediate product SIN-1C with absorbance (λmax) at 279±2nm. The SIN-1 ➝ SIN-1C transformation was oxygen dependent and the result of -OH catalyzed hydrolytic decomposition. Stopped-flow spectroscopic evidence revealed that SIN-1 can be deprotonated in a pH dependent manner during the phase of the reaction leading up to SIN-1A formation. Conclusion: The formation of ONOO- was demonstrated by the qualitative and quantitative determination of its insitu generation from the decay of 3-morpholinosydnonimine (SIN-1). This will enable relevant correlations of the life of peroxynitrite in culture conditions to its actions in pulmonary cells.Keywords: Peroxynitrite, from SIN-1, culture mediumBiokemistri 28(1): 1–1

Oxidative stress induced pulmonary endothelial cell proliferation is not mediated by superoxide

Cellular hyper-proliferation, endothelial dysfunction and oxidative stress are hallmarks of the pathobiology of pulmonary hypertension. Indeed, pulmonary endothelial cells proliferation is susceptible to redox state modulation. Some studies suggest that superoxide stimulates endothelial cell proliferation while others have linked the proliferative response to an up-regulation of peroxynitrite in lungs under oxidative stress. Given the divergence of opinion on the subject, it is important to establish the agents mediating cellular hyper-proliferation under oxidative stress. Using the combination of xanthine and xanthine oxidase, the current study demonstrates that neither superoxide nor hydrogen peroxide stimulated pulmonary endothelial cell proliferation. Alone, low level superoxide (100 RLU/s) did not alter DNA synthesis in endothelial cell and high concentration (500 RLU/s) superoxide decreased DNA synthesis to 31.8\ub13%, 30.4\ub12%, and 53.8\ub15% control at 0.1, 0.5 and 2.5% basal growth stimulation, respectively. Nonetheless, the formation of peroxynitrite under this condition stimulated proliferation to 49.2\ub19%, 51.1\ub18% and 71.2\ub12%, respectively. Taken together, pulmonary endothelial cell proliferation occurred only under conditions producing nitric oxide and superoxide in concert

Threshold of peroxynitrite cytotoxicity in bovine pulmonary artery endothelial and smooth muscle cells

Peroxynitrite is widely reported as highly cytotoxic; yet recent evidence indicates that at certain concentrations, it can induce pulmonary cell hyper-proliferation and tissue remodelling. This study aimed to establish the threshold concentration of peroxynitrite to induce functional impairment of bovine pulmonary artery endothelial (PAEC) and smooth muscle cells (PASMC). PAEC or PASMC were exposed to solution of peroxynitrite or 3-morpholinosydnonimine (SIN-1). Twenty-four hour cell viability, DNA synthesis, and protein biochemistry were assessed by trypan blue dye exclusion, [(3)H] thymidine incorporation and western blot analysis, respectively. Threshold concentration of peroxynitrite to significantly impair viability of PAEC and PASMC was 2μM peroxynitrite. In PASMC and PAEC, low concentrations of peroxynitrite (2nM-0.2μM) increased cell proliferation and did not activate p38 MAP kinase. The decrease in DNA synthesis and cell viability caused by 2μM peroxynitrite was associated with caspase-3 cleavage but not p38 activation. Also, 2-20μM peroxynitrite significantly activated poly ADP ribose polymerase and stress activated kinase JNK in PAEC. However, the higher concentration of 20μM peroxynitrite did cause a threefold increase in p38 activation. In conclusion, the threshold for the cytotoxic effects of peroxynitrite was 2μM; which caused apoptotic cell death independent of p38 MAP kinase activation in pulmonary artery cells

Membrane Ballooning in Aggregated Platelets is Synchronised and Mediates a Surge in Microvesiculation:Synchronised ballooning and microvesiculation

AbstractHuman platelet transformation into balloons is part of the haemostatic response and thrombus architecture. Here we reveal that in aggregates of platelets in plasma, ballooning in multiple platelets occurs in a synchronised manner. This suggests a mechanism of coordination between cells, previously unrecognised. We aimed to understand this mechanism, and how it may contribute to thrombus development. Using spinning-disc confocal microscopy we visualised membrane ballooning in human platelet aggregates adherent to collagen-coated surfaces. Within an aggregate, multiple platelets undergo ballooning in a synchronised fashion, dependent upon extracellular calcium, in a manner that followed peak cytosolic calcium levels in the aggregate. Synchrony was observed in platelets within but not between aggregates, suggesting a level of intra-thrombus communication. Blocking phosphatidylserine, inhibiting thrombin or blocking PAR1 receptor, largely prevented synchrony without blocking ballooning itself. In contrast, inhibition of connexins, P2Y12, P2Y1 or thromboxane formation had no effect on synchrony or ballooning. Importantly, synchronised ballooning was closely followed by a surge in microvesicle formation, which was absent when synchrony was blocked. Our data demonstrate that the mechanism underlying synchronised membrane ballooning requires thrombin generation acting effectively in a positive feedback loop, mediating a subsequent surge in procoagulant activity and microvesicle release.</jats:p

Genotoxic Damage Activates the AMPK-α1 Isoform in the Nucleus via Ca2+/CaMKK2 Signaling to Enhance Tumor Cell Survival

2017 American Association for Cancer Research. Many genotoxic cancer treatments activate AMP-activated protein kinase (AMPK), but the mechanisms of AMPK activation in response to DNA damage, and its downstream consequences, have been unclear. In this study, etoposide activates the a1 but not the a2 isoform of AMPK, primarily within the nucleus. AMPK activation is independent of ataxia-telangiectasia mutated (ATM), a DNA damage-activated kinase, and the principal upstream kinase for AMPK, LKB1, but correlates with increased nuclear Ca2þ and requires the Ca2þ/calmodulin-dependent kinase, CaMKK2. Intriguingly, Ca2þ-dependent activation of AMPK in two different LKB1-null cancer cell lines caused G1-phase cell-cycle arrest, and enhanced cell viability/ survival after etoposide treatment, with both effects being abolished by knockout of AMPK-a1 and a2. The CDK4/6 inhibitor palbociclib also caused G1 arrest in G361 but not HeLa cells and, consistent with this, enhanced cell survival after etoposide treatment only in G361 cells. These results suggest that AMPK activation protects cells against etoposide by limiting entry into S-phase, where cells would be more vulnerable to genotoxic stress. Implications: These results reveal that the a1 isoform of AMPK promotes tumorigenesis by protecting cells against genotoxic stress, which may explain findings that the gene encoding AMPK-a1 (but not -a2) is amplified in some human cancers. Furthermore, a1-selective inhibitors might enhance the anticancer effects of genotoxic-based therapies

Carbonic Anhydrase Inhibitors Suppress Platelet Procoagulant Responses and In Vivo Thrombosis

Carbonic anhydrase (CA) inhibitors have a long history of safe clinical use as mild diuretics, in the treatment of glaucoma and for altitude sickness prevention. In this study, we aimed to determine if CA inhibition may be an alternative approach to control thrombosis. We utilized a high-resolution dynamic imaging approach to provide mechanistic evidence that CA inhibitors may be potent anti-procoagulant agents in vitro and effective anti-thrombotics in vivo. Acetazolamide and methazolamide, while sparing platelet secretion, attenuated intracellular chloride ion entry and suppressed the procoagulant response of activated platelets in vitro and thrombosis in vivo. The chemically similar N-methyl acetazolamide, which lacks CA inhibitory activity, did not affect platelet procoagulant response in vitro. Outputs from rotational thromboelastometry did not reflect changes in procoagulant activity and reveal the need for a suitable clinical test for procoagulant activity. Drugs specifically targeting procoagulant remodeling of activated platelets, by blockade of carbonic anhydrases, may provide a new way to control platelet-driven thrombosis without blocking essential platelet secretion responses

The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin α IIb β 3 Outside-in Signaling

The class I PI3K family of lipid kinases plays an important role in integrin αIIbβ3 function, thereby supporting thrombus growth and consolidation. Here, we identify Ras/Rap1GAP Rasa3 (GAP1IP4BP) as a major phosphatidylinositol 3,4,5-trisphosphate-binding protein in human platelets and a key regulator of integrin αIIbβ3 outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI3K-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin αIIbβ3-expressing CHO cells blocked Rap1 activity and integrin αIIbβ3-mediated spreading on fibrinogen. In contrast, Rap1GAP-deficient (P489V) and Ras/Rap1GAP-deficient (R371Q) Rasa3 had no effect. We furthermore show that two Rasa3 mutants (H794L and G125V), which are expressed in different mouse models of thrombocytopenia, lack both Ras and Rap1GAP activity and do not affect integrin αIIbβ3-mediated spreading of CHO cells on fibrinogen. Platelets from thrombocytopenic mice expressing GAP-deficient Rasa3 (H794L) show increased spreading on fibrinogen, which in contrast to wild-type platelets is insensitive to PI3K inhibitors. Together, these results support an important role for Rasa3 in PI3K-dependent integrin αIIbβ3-mediated outside-in signaling and cell spreading