Emerging roles of calcium-activated K channels and TRPV4 channels in lung oedema and pulmonary circulatory collapse

It has been suggested that the transient receptor potential cation (TRP) channel subfamily V (vanilloid) type 4 (TRPV4) and intermediate conductance calcium-activated potassium (KCa3.1) channels contribute to endothelium-dependent vasodilation. Here, we summarize very recent evidence for a synergistic interplay of TRPV4 and KCa3.1 channels in lung disease. Among the endothelial Ca2+-permeable TRPs, TRPV4 is best characterized and produces arterial dilation by stimulating Ca2+-dependent nitric oxide synthesis and endothelium-dependent hyperpolarization. Besides these roles, some TRP channels control endothelial/epithelial barrier functions and vascular integrity, while KCa3.1 channels provide the driving force required for Cl- and water transport in some cells and most secretory epithelia. The three conditions, increased pulmonary venous pressure caused by left heart disease, high inflation pressure and chemically induced lung injury, may lead to activation of TRPV4 channels followed by Ca2+ influx leading to activation of KCa3.1 channels in endothelial cells ultimately leading to acute lung injury. We find that a deficiency in KCa3.1 channels protects against TRPV4-induced pulmonary arterial relaxation, fluid extravasation, haemorrhage, pulmonary circulatory collapse and cardiac arrest in vivo. These data identify KCa3.1 channels as crucial molecular components in downstream TRPV4 signal transduction and as a potential target for the prevention of undesired fluid extravasation, vasodilatation and pulmonary circulatory collapse

Treatment with 24 h-delayed normo- and hyperbaric oxygenation in severe sepsis induced by cecal ligation and puncture in rats

Abstract Background Septic shock remains a leading cause of death worldwide. Hyperbaric oxygen treatment (HBO2) has been shown to alter the inflammatory response during sepsis and to reduce mortality. A therapeutic window of HBO2 treatment has been demonstrated experimentally, but optimal timing remains uncertain. We investigated the effects of 24 h delayed normobaric oxygen (NBO2) and HBO2 treatment on the endogenous production of the inflammatory markers interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-10, and on mortality in rats with cecal ligation and puncture (CLP) induced sepsis. Method Fifty-five male Sprague-Dawley rats underwent CLP and were randomized to the following groups: 1) HBO2 2.5 bar absolute pressure (pabs); 2) NBO2 1.0 bar pabs; 3) Control (no-treatment), and they were individually monitored for 72 h with intermittent blood sampling. Results IL-6, TNF-α, and IL-10 were increased 24 h after the procedure, and IL-6 was significantly higher in non-survivors than in survivors. The level of IL-10 was significantly higher at hour 48 in the HBO2 group compared to control (p = 0.01), but this was not the case at other time points. No other significant differences in cytokine levels were found for any group comparisons. Delayed NBO2 and HBO2 treatment failed to change the mortality in the animals. Conclusion High levels of IL-6 in non-surviving animals with sepsis suggest that IL-6 is a potential biomarker. We found a significantly higher concentration of IL-10 in the HBO2 group at hour 48 vs. control animals. However, 24 h–delayed treatment with HBO2 did not change the levels of pro-inflammatory cytokines and survival, suggesting that earlier intervention may be required to obtain an anti-inflammatory effect

Enzyme prodrug therapy achieves site-specific, personalized physiological responses to the locally produced nitric oxide

Nitric oxide (NO) is a highly potent but short-lived endogenous radical with a wide spectrum of physiological activities. In this work, we developed an enzymatic approach to the site-specific synthesis of NO mediated by biocatalytic surface coatings. Multilayered polyelectrolyte films were optimized as host compartments for the immobilized β-galactosidase (β-Gal) enzyme through a screen of eight polycations and eight polyanions. The lead composition was used to achieve localized production of NO through the addition of β-Gal–NONOate, a prodrug that releases NO following enzymatic bioconversion. The resulting coatings afforded physiologically relevant flux of NO matching that of the healthy human endothelium. The antiproliferative effect due to the synthesized NO in cell culture was site-specific: within a multiwell dish with freely shared media and nutrients, a 10-fold inhibition of cell growth was achieved on top of the biocatalytic coatings compared to the immediately adjacent enzyme-free microwells. The physiological effect of NO produced via the enzyme prodrug therapy was validated ex vivo in isolated arteries through the measurement of vasodilation. Biocatalytic coatings were deposited on wires produced using alloys used in clinical practice and successfully mediated a NONOate concentration-dependent vasodilation in the small arteries of rats. The results of this study present an exciting opportunity to manufacture implantable biomaterials with physiological responses controlled to the desired level for personalized treatment

Impaired phosphate transport in SLC34A2 variants in patients with pulmonary alveolar microlithiasis

Background: Variants in SLC34A2 encoding the sodium-dependent phosphate transport protein 2b (NaPi-IIb) cause the rare lung disease pulmonary alveolar microlithiasis (PAM). PAM is characterised by the deposition of calcium-phosphate concretions in the alveoli usually progressing over time. No effective treatment is available. So far, 30 allelic variants in patients have been reported but only a few have been functionally characterised. This study aimed to determine the impact of selected SLC34A2 variants on transporter expression and phosphate uptake in cellular studies. Methods: Two nonsense variants (c.910A > T and c.1456C > T), one frameshift (c.1328delT), and one in-frame deletion (c.1402_1404delACC) previously reported in patients with PAM were selected for investigation. Wild-type and mutant c-Myc-tagged human NaPi-IIb constructs were expressed in Xenopus laevis oocytes. The transport function was investigated with a 32Pi uptake assay. NaPi-IIb protein expression and localisation were determined with immunoblotting and immunohistochemistry, respectively. Results: Oocytes injected with the wild-type human NaPi-IIb construct had significant 32Pi transport compared to water-injected oocytes. In addition, the protein had a molecular weight as expected for the glycosylated form, and it was readily detectable in the oocyte membrane. Although the protein from the Thr468del construct was synthesised and expressed in the oocyte membrane, phosphate transport was similar to non-injected control oocytes. All other mutants were non-functional and not expressed in the membrane, consistent with the expected impact of the truncations caused by premature stop codons. Conclusions: Of four analysed SLC34A2 variants, only the Thr468del showed similar protein expression as the wild-type cotransporter in the oocyte membrane. All mutant transporters were non-functional, supporting that dysfunction of NaPi-IIb underlies the pathology of PAM

Soluble guanylate cyclase mediates the relaxation of healthy and inflamed bladder smooth muscle by aqueous nitric oxide

Introduction: Due to its chemical properties, functional responses to nitric oxide (NO) are often difficult to examine. In the present study, we established a method to produce NO in an aqueous solution and validated its capacity to evoke functional responses in isolated rat bladders. Furthermore, we compared the NO responses to the commonly used NO donor sodium nitroprusside (SNP). We also investigated the impact of ongoing inflammation on the involvement of soluble guanylate cyclase (sGC) dependent signaling in NO relaxation.Methods: A setup to produce an aqueous NO solution was established, allowing the production of an aqueous solution containing a calculated NO concentration of 2 mM. Sixty male Sprague-Dawley rats received either no treatment (controls) or cyclophosphamide (CYP; 100 mg*kg−1 i.p., 60 h prior to the experiment) to induce experimental cystitis. Bladder strip preparations were mounted in organ baths and studied at basal tension or pre-contracted with methacholine (3 μM). Aqueous NO solution (40–400 μL; 2 mM corresponding to 4–40 μM) or SNP (1–1,000 μM) was added cumulatively in increasing concentrations. Relaxation to aqueous NO was also studied in the presence of the sGC inhibitor ODQ (0.25–25 μM). The expression of sGC was investigated by immunohistochemical analysis.Results: The NO solution caused functional relaxations in both controls and inflamed bladder preparations. NO-induced relaxations were significantly greater in inflamed bladder strips at basal tension, whereas no differences were seen in methacholine pre-contracted strips. In the presence of the sGC inhibitor ODQ in a high concentration, the NO-evoked relaxations were abolished in both control and inflamed preparations. At a lower concentration of ODQ, only NO relaxations in inflamed preparations were attenuated. Immunohistochemical analysis showed that sGC was expressed in the detrusor and mucosa, with a significantly lower expression in the inflamed detrusor.Conclusion: In the present study, we found that aqueous NO solution induces relaxation of the rat detrusor by activating soluble guanylate cyclase in both control and inflamed bladder strips. Induction of inflammation conceivably leads to decreased sGC expression in the detrusor, which may explain the different susceptibility towards inhibition of sGC in inflamed versus control tissue. The use of an aqueous NO solution should be further considered as a valuable complement to the pharmacological tools currently used