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 homeostasisand accordingly onset of chill coma has been hypothesized 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 [K + ] o , 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 [K + ] o from 10 mmol l −1 to 30 mmol l −1 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. As [K + ] also has a large effect on tetanic force production, it is hypothesized that recovery of [K + ] o following chill coma could be important for the time to recovery of normal neuromuscular function.

Human MCF-7 breast cancer cells were exposed to a Random Positioning Machine (RPM). After 24 hours (h) the cells grew either adherently within a monolayer (AD) or within multicellular spheroids (MCS). AD and MCS populations were separately harvested, their cellular differences were determined performing qPCR on genes, which were differently expressed in AD and MCS cells. Gene array technology was applied to detect RPM-sensitive genes in MCF-7 cells after 24 h. Furthermore, the capability to form multicellular spheroids in vitro was compared with the intracellular distribution of NF-kappaB (NFκB) p65. NFκB was equally distributed in static control cells, but predominantly localized in the cytoplasm in AD cells and nucleus in MCS cells exposed to the RPM. Gene array analyses revealed a more than 2-fold change of only 23 genes including some whose products are affected by oxygen levels or regulate glycolysis. Significant upregulations of the mRNAs of enzymes degrading heme, of ANXA1, ANXA2, CTGF, CAV2 and ICAM1, as well as of FAS, Casp8, BAX, p53, CYC1 and PARP1 were observed in MCS cells as compared with 1g-control and AD cells. An interaction analysis of 47 investigated genes suggested that HMOX-1 and NFκB variants are activated, when multicellular spheroids are formed.

Donors of H2S may be beneficial in treating cardiovascular diseases where the plasma levels of H2S are decreased. Therefore, we investigated the mechanisms involved in relaxation of small arteries induced by GYY4137 [(4-methoxyphenyl)-morpholin-4-yl-sulfanylidene-sulfido-λ5-phosphane;morpholin-4-ium], which is considered a slow-releasing H2S donor. Sulfides were measured by use of 5,5′-dithiobis-(2-nitro benzoic acid), and small rat mesenteric arteries with internal diameters of 200–250 µm were mounted in microvascular myographs for isometric tension recordings. GYY4137 produced similar low levels of sulfides in the absence and the presence of arteries. In U46619-contracted small mesenteric arteries, GYY4137 (10−6–10–3 M) induced concentration-dependent relaxations, while a synthetic, sulfur-free, GYY4137 did not change the vascular tone. L-cysteine (10−6–10–3 M) induced only small relaxations reaching 24 ± 6% at 10–3 M. Premixing L-cysteine (10–3 M) with Na2S and GYY4137 decreased Na2S relaxation and abolished GYY4137 relaxation, an effect prevented by an nitric oxide (NO) synthase inhibitor, L-NAME (Nω-nitro-L-arginine methyl ester). In arteries without endothelium or in the presence of L-NAME, relaxation curves for GYY4137 were rightward shifted. High extracellular K+ concentrations decreased Na2S and abolished GYY4137 relaxation suggesting potassium channel-independent mechanisms are also involved Na2S relaxation while potassium channel activation is pivotal for GYY4137 relaxation in small arteries. Blockers of large-conductance calcium-activated (BKCa) and voltage-gated type 7 (KV7) potassium channels also inhibited GYY4137 relaxations. The present findings suggest that L-cysteine by reaction with Na2S and GYY4137 and formation of sulfides, inhibits relaxations by these compounds. The low rate of release of H2S species from GYY4137 is reflected by the different sensitivity of these relaxations towards high K+ concentration and potassium channel blockers compared with Na2S. The perspective is that the rate of release of sulfides plays an important for the effects of H2S salt vs. donors in small arteries, and hence for a beneficial effect of GYY4137 for treatment of cardiovascular disease.

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 °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.

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, we examined the possible vasorelaxant effect of GLP‐1 and its underlying mechanisms. Experimental Approach Rat mesenteric arteries (diameter ≈ 200–400 μm) and human s.c. arteries were mounted in microvascular myographs for isometric tension recordings. The effect of GLP‐1 on vascular responses was examined under normoglycaemic conditions and at high glucose concentrations. Key Results In rat mesenteric arteries and human s.c. 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 the concentration relaxation curves for bradykinin in s.c. arteries from patients with peripheral arterial disease, an effect resistant to exendin‐(9‐39). Under normoglycaemic conditions, neither GLP‐1 nor liraglutide affected ACh relaxation in rat mesenteric arteries. In arteries exposed to 40 mM glucose, GLP‐1, in contrast to liraglutide, potentiated ACh‐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. Conclusions and Implications GLP‐1 receptors are involved in the liraglutide‐induced relaxation of branched arteries, under normoglycaemic conditions, while GLP‐1 inhibition of vascular superoxide levels contributes to GLP‐1 receptor‐independent potentiation of endothelium‐dependent vasodilatation in hyperglycaemia.