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Background and Purpose: Acute respiratory distress syndrome (ARDS) is characterized by pulmonary oedema and severe hypoxaemia. We investigated whether genetic deficit or blockade of calcium-activated potassium (K Ca3.1) channels would counteract pulmonary oedema and hypoxaemia in ventilator-induced lung injury, an experimental model for ARDS. Experimental Approach: K Ca3.1 channel knockout (Kccn4 -/-) mice were exposed to ventilator-induced lung injury. Control mice exposed to ventilator-induced lung injury were treated with the K Ca3.1 channel inhibitor, senicapoc. The outcomes were oxygenation (PaO 2/FiO 2 ratio), lung compliance, lung wet-to-dry weight and protein and cytokines in bronchoalveolar lavage fluid (BALF). Key Results: Ventilator-induced lung injury resulted in lung oedema, decreased lung compliance, a severe drop in PaO 2/FiO 2 ratio, increased protein, neutrophils and tumour necrosis factor-alpha (TNF-α) in BALF from wild-type mice compared with Kccn4 -/- mice. Pretreatment with senicapoc (10–70 mg·kg −1) prevented the reduction in PaO 2/FiO 2 ratio, decrease in lung compliance, increased protein and TNF-α. Senicapoc (30 mg·kg −1) reduced histopathological lung injury score and neutrophils in BALF. After injurious ventilation, administration of 30 mg·kg −1 senicapoc also improved the PaO 2/FiO 2 ratio and reduced lung injury score and neutrophils in the BALF compared with vehicle-treated mice. In human lung epithelial cells, senicapoc decreased TNF-α-induced permeability. Conclusions and Implications: Genetic deficiency of K Ca3.1 channels and senicapoc improved the PaO 2/FiO 2 ratio and decreased the cytokines after a ventilator-induced lung injury. Moreover, senicapoc directly affects lung epithelial cells and blocks neutrophil infiltration in the injured lung. These findings indicate that blocking K Ca3.1 channels is a potential treatment in ARDS-like disease.

Background Senicapoc is a potent and selective blocker of KCa3.1, a calcium-activated potassium channel of intermediate conductance. In the present study, we investigated whether there is a beneficial effect of senicapoc in a large animal model of acute respiratory distress syndrome (ARDS). The primary end point was the PaO2/FiO2 ratio. Methods ARDS was induced in female pigs (42–49 kg) by repeated lung lavages followed by injurious mechanical ventilation. Animals were then randomly assigned to vehicle (n = 9) or intravenous senicapoc (10 mg, n = 9) and received lung-protective ventilation for 6 h. Results Final senicapoc plasma concentrations were 67 ± 18 nM (n = 9). Senicapoc failed to change the primary endpoint PaO2/FiO2 ratio (senicapoc, 133 ± 23 mmHg; vehicle, 149 ± 68 mmHg). Lung compliance remained similar in the two groups. Senicapoc reduced the level of white blood cells and neutrophils, while the proinflammatory cytokines TNFα, IL-1β, and IL-6 in the bronchoalveolar lavage fluid were unaltered 6 h after induction of the lung injury. Senicapoc-treatment reduced the level of neutrophils in the alveolar space but with no difference between groups in the cumulative lung injury score. Histological analysis of pulmonary hemorrhage indicated a positive effect of senicapoc on alveolar–capillary barrier function, but this was not supported by measurements of albumin content and total protein in the bronchoalveolar lavage fluid. Conclusions In summary, senicapoc failed to improve the primary endpoint PaO2/FiO2 ratio, but reduced pulmonary hemorrhage and the influx of neutrophils into the lung. These findings open the perspective that blocking KCa3.1 channels is a potential treatment to reduce alveolar neutrophil accumulation and improve long-term outcome in ARDS.

Endothelial cell dysfunction and fibrosis are associated with worsening of the prognosis in patients with cardiovascular disease. Pirfenidone has a direct antifibrotic effect, but vasodilatation may also contribute to the effects of pirfenidone. Therefore, in a first study we investigated the mechanisms involved in the relaxant effect of pirfenidone in rat intrapulmonary arteries and coronary arteries from normal mice. Then in a second study, we investigated whether pirfenidone restores endothelial function in the aorta and mesenteric arteries from diabetic animals. From 16–18-week old normal male C57BL/6 mice and normoglycemic (db/db+), and type 2 diabetic (db/db) male and female mice, arteries were mounted in microvascular isometric myographs for functional studies, and immunoblotting was performed. In rat pulmonary arteries and mouse coronary arteries, pirfenidone induced relaxations, which were inhibited in preparations without endothelium. In mouse coronary arteries, pirfenidone relaxation was inhibited in the presence of a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine (L-NOARG), a blocker of large-conductance calcium-activated potassium channels (BKCa), iberiotoxin, and a blocker of KV7 channels, XE991. Patch clamp studies in vascular smooth muscle revealed pirfenidone increased iberiotoxin-sensitive current. In the aorta and mesenteric small arteries from diabetic db/db mice relaxations induced by the endothelium-dependent vasodilator, acetylcholine, were markedly reduced compared to db/db + mice. Pirfenidone enhanced the relaxations induced by acetylcholine in the aorta from diabetic male and female db/db mice. An opener of KV7 channels, flupirtine, had the same effect as pirfenidone. XE991 reduced the effect of pirfenidone and flupirtine and further reduced acetylcholine relaxations in the aorta. In the presence of iberiotoxin, pirfenidone still increased acetylcholine relaxation in aorta from db/db mice. Immunoblotting for KV7.4, KV7.5, and BKCa channel subunits were unaltered in aorta from db/db mice. Pirfenidone failed to improve acetylcholine relaxation in mesenteric arteries, and neither changed acetylcholine-induced transient decreases in blood pressure in db/db+ and db/db mice. In conclusion, pirfenidone vasodilates pulmonary and coronary arteries. In coronary arteries from normal mice, pirfenidone induces NO-dependent vasodilatation involving BKCa and KV7 channels. Pirfenidone improves endothelium-dependent vasodilatation in aorta from diabetic animals by a mechanism involving voltage-gated KV7 channels, a mechanism that may contribute to the antifibrotic effect of pirfenidone.

The ability of many reptilian hemoglobins (Hbs) to form high-molecular weight polymers, albeit known for decades, has not been investigated in detail. Given that turtle Hbs often contain a high number of cysteine (Cys), potentially contributing to the red blood cell defense against reactive oxygen species, we have examined whether polymerization of Hb could occur via intermolecular disulfide bonds in red blood cells of freshwater turtle Trachemys scripta, a species that is highly tolerant of hypoxia and oxidative stress. We find that one of the two Hb isoforms of the hemolysate HbA is prone to polymerization in vitro into linear flexible chains of different size that are visible by electron microscopy but not the HbD isoform. Polymerization of purified HbA is favored by hydrogen peroxide, a main cellular reactive oxygen species and a thiol oxidant, and inhibited by thiol reduction and alkylation, indicating that HbA polymerization is due to disulfide bonds. By using mass spectrometry, we identify Cys5 of the αA-subunit of HbA as specifically responsible for forming disulfide bonds between adjacent HbA tetramers. Polymerization of HbA does not affect oxygen affinity, cooperativity, and sensitivity to the allosteric cofactor ATP, indicating that HbA is still fully functional. Polymers also form in T. scripta blood after exposure to anoxia but not normoxia, indicating that they are of physiological relevance. Taken together, these results show that HbA polymers may form during oxidative stress and that Cys5αAof HbA is a key element of the antioxidant capacity of turtle red blood cells.

The clinically tested KCa3.1 channel blocker, senicapoc, has been proven to have excellent pharmacological properties and prior clinical trials found it to be safe for use in patients with sickle cell anaemia. Currently, several preclinical projects are aiming to repurpose senicapoc for other indications, but well-described analytical methods in the literature are lacking. Our aim was to develop a sensitive, rapid and accurate ultra-high-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation (UHPLC-ESI-MS/MS) suitable for the determination of senicapoc in plasma samples. Unfortunately, direct analysis of senicapoc in crude acetonitrile extracts of human plasma samples by UHPLC-ESI-MS/MS was subjected to significant and variable ion suppression from coeluting phospholipids (PLs). The interferences were mainly caused by the presence of phosphatidylcholine and phosphatidylethanolamine classes of PLs, including their lyso-products. However, the PLs were easily removed from crude extracts by filtration through a sorbent with Lewis acid properties which decreased the total ion suppression effect to approximately 5%. Based on this technique, a simple high-throughput UHPLC-MS/MS method was developed and validated for the determination of senicapoc in 100-μL plasma samples. The lower limit of quantification was 0.1 ng/mL. The mean true extraction recovery was close to 100 %. The relative intra-laboratory reproducibility standard deviations of the measured concentrations were 8% and 4% at concentrations of 0.1 ng/mL and 250 ng/mL, respectively. The trueness expressed as the relative bias of the test results was within ± 2% at concentrations of 1 ng/mL or higher.

BACKGROUND AND PURPOSE: Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates insulin biosynthesis and secretion in a glucose-dependent manner, and has been reported to induce vasodilatation. Here, the aim was to examine the possible vasorelaxant effect of GLP-1 and the underlying mechanisms.EXPERIMENTAL APPROACH: Rat mesenteric arteries (diameter ≈ 200-400 μm) and human subcutaneous arteries were mounted in microvascular myographs for isometric tension recordings. The effect of GLP-1 on vascular responses was examined at normoglycemic conditions and at high glucose.KEY RESULTS: In rat mesenteric arteries and human subcutaneous arteries without branches, physiological concentrations (1-100 nM) of GLP-1(7-36) and liraglutide failed to cause relaxation, or affect contractions evoked by electrical field stimulation. In contrast to GLP-1(7-36), liraglutide induced relaxations antagonized by the GLP-1 receptor antagonist, exendin-(9-39) in branched mesenteric arteries. In contrast to liraglutide, GLP-1 leftward shifted concentration relaxation curves for bradykinin in subcutaneous arteries from patients with peripheral arterial disesase, an effect insensitive to exendin-(9-39). In normoglycemic conditions neither GLP-1 nor liraglutide changed acetylcholine relaxation in rat mesenteric arteries. In arteries exposed to 40 mM glucose, GLP-1, in contrast to liraglutide, potentiated acetylcholine-induced relaxation by a mechanism that was not antagonized by exendin-(9-39). GLP-1 decreased superoxide levels measured with dihydroethidium in rat mesenteric arteries exposed to 40 mM glucose.CONCLUSION AND IMPLICATIONS: Our findings suggest that a GLP-1 receptor-dependent mechanism is involved in liraglutide relaxation in branched arteries in normoglycemic conditions, while GLP-1 inhibition of vascular superoxide levels contributes to GLP-1 receptor-independent potentiation of endothelium-dependent vasodilatation in hyperglycemia.

To prepare the ESA (European Space Agency) spaceflight project "Wound healing and Sutures in Unloading Conditions", we studied mechanisms of apoptosis in wound healing models based on ex vivo skin tissue cultures, kept for 10 days alive in serum-free DMEM/F12 medium supplemented with bovine serum albumin, hydrocortisone, insulin, ascorbic acid and antibiotics at 32 degrees C. The overall goal is to test: (i) the viability of tissue specimens; (ii) the gene expression of activators and inhibitors of apoptosis and extracellular matrix components in wound and suture models; and (iii) to design analytical protocols for future tissue specimens after post-spaceflight download. Hematoxylin-Eosin and Elastica-van-Gieson staining showed a normal skin histology with no signs of necrosis in controls and showed a normal wound suture. TdT-mediated dUTP-biotin nick end labeling for detecting DNA fragmentation revealed no significant apoptosis. No activation of caspase-3 protein was detectable. FASL, FADD, CASP3, CASP8, CASP10, BAX, BCL2, CYC1, APAF1, LAMA3 and SPP1 mRNAs were not altered in epidermis and dermis samples with and without a wound compared to 0 day samples (specimens investigated directly post-surgery). BIRC5, CASP9, and FN1 mRNAs were downregulated in epidermis/dermis samples with and/or without a wound compared to 0 day samples. BIRC2, BIRC3 were upregulated in 10 day wound samples compared to 0 day samples in epidermis/dermis. RELA/FAS mRNAs were elevated in 10 day wound and no wound samples compared to 0 day samples in dermis. In conclusion, we demonstrate that it is possible to maintain live skin tissue cultures for 10 days. The viability analysis showed no significant signs of cell death in wound and suture models. The gene expression analysis demonstrated the interplay of activators and inhibitors of apoptosis and extracellular matrix components, thereby describing important features in ex vivo sutured wound healing models. Collectively, the performed methods defining analytical protocols proved to be applicable for post-flight analyzes of tissue specimens after sample return.

When exposed to low temperatures, many insect species enter a reversible comatose state (chill coma), which is driven by a failure of neuromuscular function. Chill coma and chill coma recovery have been associated with a loss and recovery of ion-homeostasis (particularly extracellular [K+]) and accordingly onset of chill coma has been hypothesised to result from depolarization of membrane potential caused by loss of ion-homeostasis. Here we examined whether onset of chill coma is associated with a disturbance in ion balance by examining the correlation between disruption of ion homeostasis and onset of chill coma in locusts exposed to cold at varying rates of cooling. Chill coma onset temperature changed maximally 1°C under different cooling rates and marked disturbances of ion homeostasis were not observed at any of the cooling rates. In a second set of experiments we used isolated tibial muscle to determine how temperature and extracellular [K+], independently and together, affect tetanic force production. Tetanic force decreased by 80% when temperature was reduced from 23°C to 0.5°C, while an increase in extracellular [K+] from 10 mM to 30 mM at 23°C caused a 40% reduction in force. Combining these two stressors almost abolished force production. Thus, low temperature alone may be responsible for chill coma entry, rather than a disruption of extracellular K+ homeostasis. Since [K+] also has a large effect on tetanic force production, it is hypothesized that recovery of extracellular [K+] following chill coma could be important for the time to recovery of normal neuromuscular function.