Pflugers Archiv : European journal of physiology最新文献

We previously hypothesized that the inflection point of the oxygen dissociation curve (ODC) is linked to the gas exchange threshold (GET) during cardiopulmonary exercise testing. This hypothesis was supported by femoral venous blood gas data sampled during constant exercise below and above the GET, which showed that the ODC shifts rightward at the GET. What had gone unnoticed since these original observations in 1994 was that this rightward shift begins slightly earlier, precisely when the oxygen saturation crosses the ODC inflection point. To investigate this phenomenon, we analyzed the 1994 femoral venous blood gas data obtained during cardiopulmonary exercise testing using a modern validated mechanistic biochemical model of oxygen (O₂), carbon dioxide (CO₂), and proton binding to hemoglobin (Hb). We constructed the ODC for each data point, as well as the in vivo ODC-a composite curve reflecting changes in dynamic blood chemistry during exercise-to assess its alignment with the GET. The model revealed that, at the in vitro ODC inflection point (36% O₂Hb saturation), the amounts of CO₂ bound to Hb equalized with HbNH₃⁺ eventually predominating. This equilibrium apparently triggered the Bohr shift, steepening the in vivo ODC to improve O₂ unloading to the tissues. Shortly afterwards, the in vivo ODC reached its inflection point, matching the measured GET. Our findings support that the GET is mechanistically linked to the in vivo ODC inflection point. These results highlight the physiological relevance of determining the ODC inflection point and its alignment with HbNH₃⁺ and CO₂ binding as critical factors in understanding ODC shifts during cardiopulmonary exercise testing.

Domiphen bromide (DMP) and benzethonium chloride (BZT) are synthetic quaternary ammonium compounds widely used as disinfectants. Both agents are potent inhibitors of the human ether-à-go-go-related gene (HERG) potassium channel, a key contributor to cardiac repolarization. Dysfunction of HERG is associated with long QT syndrome and arrhythmias, yet the molecular mechanisms underlying DMP and BZT inhibition remain incompletely understood. In this study, we employed site-directed mutagenesis and whole-cell patch-clamp recording to identify key residues mediating DMP and BZT binding. Wild-type and nine mutant HERG channels were expressed in HEK-293 T cells, targeting residues in the pore helix (T623A, S624A, V625A), S6 helix (G648A, Y652A, F656A), S5-pore linker (S631A), and S5-S6 connector (N588K), including a double mutant (N588K/S631A). DMP exhibited strong dependence on S624, V625, Y652, N588, and S631, whereas BZT primarily involved S624, V625, and Y652. Computational docking revealed that DMP forms hydrogen bonds, π-cation, and π-π interactions with S624 and Y652, while BZT interacts through π-cation and π-π stacking with Y652 and hydrophobic contacts with S624. Importantly, our data highlight the quaternary ammonium group as a critical pharmacophore, mediating strong interactions with serine and aromatic residues via π-cation, electrostatic, and hydrogen bonding mechanisms, contributing to high-affinity channel blockade. In conclusion, this study defines the molecular determinants underlying DMP and BZT binding to the HERG channel and provides mechanistic insight that may guide the design of safer therapeutics with minimized HERG liability.

IRBIT1 and IRBIT2 (collectively, the IRBITs) are signaling molecules with great universality in their expression (ubiquitous distribution in all major tissues in animals) and considerable versatility in their biological functions. Structurally, the IRBITs are highly homologous to S-adenosyl-L-homocysteine hydrolase (AHCY). However, the IRBITs had lost the catalytic activity during the evolution but gained new functions by the addition of a unique N-terminal IRBIT domain. By direct protein interaction, the IRBITs modulate the functions of an array of target proteins of distinct biological functions, ranging from membrane channels and transporters to cytosolic protein kinase, lipid kinases, ribonucleotide reductase, etc. The interaction of the IRBITs with specific target proteins is modulated by the redox couple NAD⁺/NADH. The IRBITs are involved in the regulation of many cellular processes, such as Ca²⁺ signaling, intracellular pH regulation, transepithelial transport of electrolytes and fluid, apoptosis, and DNA metabolism. However, what we have known about the IRBITs is likely just the tip of the iceberg. The present review covers the expression and distribution, physiological and pathological roles, and the structural organization of the IRBITs. It provides a comprehensive review on the binding partners of the IRBITs. Finally, the review addresses the evolution of the IRBITs in reference to the evolution of AHCY.

Inappropriate mineralization of soft tissues, also called ectopic calcification, is a well-known pathology in chronic kidney disease (CKD) that is associated with increases in systemic phosphate levels. Vascular calcification is a major contributor to cardiovascular injury and high mortality rates in CKD patients. Therefore, most animal and human studies have focused on the vasculature when describing ectopic calcifications and on the pathologic actions of elevated phosphate on vascular smooth muscle cells in this process. The extent of calcifications within soft tissues beyond the vasculature is not well described, and the involvement of cell types other than vascular smooth muscle cells is not clear. Here we provide a summary of CKD-associated extravascular calcifications in various tissues, which includes the lung, the gastrointestinal system, the liver, the skin, and the brain. Since phosphate elevations and widespread ectopic calcifications do not only occur in the context of CKD, but also in rare genetic disorders that affect the regulators of phosphate metabolism, the cellular transporters of phosphate and the factors protecting from mineral depositions outside of bone, we also discuss these pathologic scenarios. We describe different types of ectopic calcification to flesh out common aspects as well as differences in the potential mechanisms and target cell types. We postulate that phosphate elevations might act in various ways and on various tissues, which together causes a wide spectrum of phosphate-induced pathologies in CKD.

Hepatic injury is one of the most critical problems in major liver surgeries, trauma, sepsis or shock. The novel Elabela (ELA) peptide was shown to exert protective effects against cardiac and renal injury. We hypothesized that ELA could also have protective effects in hepatic ischemia-reperfusion (HI/R) injury and associated remote organ injury. Male (n = 37) and female (n = 37) Sprague-Dawley rats were used. Rats were divided into short-term and long-term HI/R injury groups. Each group was then divided into saline-treated, N-acetylcysteine-treated (NAC, 150 mg/kg) and ELA-treated (40 μg/kg) subgroups. Immediately before hepatic ischemia and during reperfusion, rats were subcutaneously injected with saline, NAC or ELA, while injections in long-term groups were continued twice a day for four days. Short-term and long-term sham-operation groups received saline injections. Hepatic blood flow was measured via laser Doppler flowmetry. Intracardiac blood was obtained for analyses of aminotransferase, alanine aminotransferase, bilirubin, urea, creatinine and interleukin (IL)-6. Caspase-3 and 8-hydroxy-2'-deoxyguanosine levels were determined and histopathological analyses (hematoxylin-eosin and alpha-smooth muscle actin (SMA) immunohistochemical staining) were performed in hepatic tissues. Levels of malondialdehyde, antioxidant glutathione, myeloperoxidase activity, luminol and lucigenin-enhanced chemiluminescence were measured in liver, lung, and kidney. Significant improvement in hepatic blood flow was observed in both short- and long-term ELA-treated groups. HI/R-induced elevations in reactive oxygen species in all the studied tissues were decreased by ELA, indicating its efficient radical scavenging function similar to NAC treatment. ELA treatment improved hepatic function tests and alleviated liver fibrosis, as detected by increased alpha-SMA-immunoreactivity. Serum IL-6 levels were increased by ELA treatment, suggesting its role in the activation of IL-6-dependent intracellular pathways which may contribute to hepatocyte proliferation and liver regeneration. Similar to the common use of NAC in hepatic surgery, Elabela appears to have a therapeutic potential in alleviating the consequences of hepatic postreperfusion injury.

The renin-angiotensin-aldosterone system (RAAS) is critical in ethanol-induced vascular dysfunction. Mineralocorticoid receptors (MR) trigger ethanol-induced vascular hypercontractility through pro-oxidative and pro-inflammatory effects. However, the contribution of MR to ethanol-induced perivascular adipose tissue (PVAT) dysfunction is unknown. Appreciating the importance of MR to PVAT dysfunction in distinctive pathological conditions, we investigated whether MR would play a role in ethanol-induced PVAT dysfunction. With this purpose, male Wistar Hannover rats were treated with ethanol 20% (in volume ratio) and/or potassium canrenoate [a MR antagonist (MRA); 30 mg/kg/day, gavage] for 5 weeks. Ethanol increased the circulating levels of aldosterone and impaired acetylcholine-induced relaxation of mesenteric arteries with, but not without PVAT. Antagonism of MR prevented ethanol-induced impairment in acetylcholine relaxation as well as the reduction of leptin levels and reactive oxygen species (ROS) overproduction in the mesenteric PVAT (mPVAT) from ethanol-treated rats. Ethanol promoted neutrophil accumulation and augmented the concentration of tumor necrosis factor (TNF)-α in the mPVAT and these responses were prevented by the MRA. Functional assays showed that tiron [a scavenger of superoxide (O₂^•-)] and etanercept (an antibody anti-TNF-α) failed to reverse the impairment of acetylcholine-induced relaxation promoted by ethanol. In mesenteric arteries, antagonism of MR prevented ROS generation, lipoperoxidation, and increased TNF-α levels induced by ethanol. In conclusion, our findings suggest that MR is involved in ethanol-induced dysfunction of mPVAT. This study enhances our understanding of how ethanol exerts harmful effects on the cardiovascular system, highlighting PVAT as a target for these detrimental effects.

Hyponatremia is the most common electrolyte disturbance in hospitalized patients. Symptoms of hyponatremia include attention deficits and cognitive impairments. The cause of such abnormalities may be disturbances in the regulation of microcirculation. Previous studies have shown that increased vasopressin (AVP) concentration to 15 pg/ml in the presence of decreased Na⁺ concentration to 121 mM, which mimics AVP-associated hyponatremia in vivo leads to dysfunction, i.e., constriction and impaired endothelial regulation of small intracerebral blood vessels-parenchymal arterioles (PA). One of the possible causes of this dysfunction may be excessive production of superoxide anion (O2^•-). The superoxide anion binds nitric oxide (NO) in a reaction that produces aggressive nitrogen-free radical, peroxynitrite (ONOO^-), which simultaneously reduces the bioavailability of NO. The present studies were performed in the organ chamber on isolated, perfused, and pressurized rats' PA in low sodium environment in the presence of AVP. These studies aimed to investigate the mechanism leading to PA dysfunction, i.e., constriction and disturbed endothelial regulation. L-NAME (N(ω)-nitro-L-arginine methyl ester) did not elicit constriction of PA, indicating reduced involvement of NO in maintaining basal tone of PA. Vasopressin V_1a receptor antagonist (SR 49059), endothelin ET_A/ET_B receptors antagonist (PD 142,893), peroxynitrite decomposition catalyst (FeTMPyP) and ROS scavengers: superoxide dismutase (SOD) and catalase (CAT) improved studied responses. Dihydroethidium (DHE) staining confirmed the increased superoxide anion formation in low sodium environment in the presence of AVP. Thromboxane A₂/prostaglandin H₂ receptor blocker (SQ 29,548), an inhibitor of the production of 20-HETE (HET0016), and L-arginine, a precursor of NO, did not improve dysfunctions of PA. Thus, in studied conditions, endothelial dysfunction occurs due to oxidative/nitrosative stress. These findings provide novel insight into the detrimental effects of decreased Na⁺ concentration in the presence of increased AVP concentration that mimic hyponatremia, on the regulation of cerebral microcirculation.

Cardiac diastolic dysfunction is a hallmark of diabetic cardiomyopathy (DCM), particularly in postmenopausal women where estrogen deficiency exacerbates cardiac remodeling. This study investigated the roles of NLRP3 inflammasome activation and cardiac mast cell (CMC) behavior in diabetic ovariectomized (OVX) rat models. Female Wistar rats were divided into five groups: sham-operated, OVX, diabetic (Sham-DM), OVX-diabetic (OVX-DM), and OVX-DM treated with the NLRP3 inhibitor MCC950. Diabetes was induced using a high-calorie quick fat diet (13.8% crude fat, 24.35% crude protein, 25% sucrose; 406.80 kcal/100 g) followed by a single intraperitoneal injection of streptozotocin (30 mg/kg). MCC950 (10 mg/kg BW, intraperitoneally) was administered daily for 4 weeks. Echocardiography revealed significant diastolic dysfunction in OVX-DM rats, with increased left ventricular internal diameter (LVIDd) and reduced mitral valve E/A ratio, while MCC950 treatment partially restored diastolic function (p < 0.05). Masson's trichrome staining showed increased myocardial fibrosis in OVX-DM rats (2.59 ± 0.20%) compared to Sham-DM (1.94 ± 0.16%, p < 0.05), which was reduced with MCC950 treatment (0.88 ± 0.13%, p < 0.05). Western blot analysis demonstrated elevated expression of NLRP3, cleaved caspase-1, IL-1β, and GSDMD-N in OVX-DM hearts. MCC950 significantly reduced cleaved caspase-1, IL-1β, and GSDMD-N expression without altering NLRP3 protein levels. Additionally, mast cell degranulation was markedly increased in OVX-DM rats (62.14%) compared to controls (P < 0.05) and was attenuated by MCC950 (31.06%, P < 0.05). These findings suggest that NLRP3 inflammasome activation under conditions of estrogen deficiency and diabetes contributes to myocardial pyroptosis and mast cell degranulation, driving cardiac remodeling in postmenopausal DCM. Targeting NLRP3 pathways may provide an effective therapeutic strategy to mitigate diastolic dysfunction, fibrosis, and inflammation in diabetic hearts.

RNA-DNA interactions are fundamental to cellular physiology, playing critical roles in genome integrity, gene expression, and stress responses. This review highlights the diverse structures of RNA-DNA hybrids, including R-loops, RNA-DNA triplexes, and RNA-DNA hybrid G-quadruplexes (hG4s) and their relevance in physiology. R-loops are formed during transcription and replication, which regulate gene expression and chromatin dynamics but can also threaten genome stability. RNA-DNA triplexes, often formed by long noncoding RNAs (lncRNAs) such as FENDRR and MEG3, recruit chromatin modifiers like Polycomb repressive complex 2 to modulate gene expression, influencing organogenesis and cell specification. hG4s, formed by guanine-rich sequences in RNA and DNA, regulate transcription termination and telomere stability. Through this, hG4s can affect gene suppression and replication regulation. RNA-DNA hybrids are tightly regulated by helicases, RNase H enzymes, and topoisomerases, with altered regulation linked to genomic instability and disease. This review discusses the complexity of RNA-DNA interactions and their recently identified contributions to cellular physiology.

The endothelial ENaC (EnNaC) is mainly responsible for maintaining the mechanical properties of the endothelial cell surface, the sensitivity to the shear forces of the streaming blood and thus for vascular function. The correlation between EnNaC surface expression, the dynamics of the actin cortex, the mechanical stiffness, and nitric oxide release indicates a close structure-function relationship. Mechanical flexibility of the endothelial surface has been associated with proper vascular function, while chronic stiffening leads to endothelial dysfunction and the so-called 'stiff endothelial cell syndrome' (SECS). With the help of atomic force microscopy (AFM)-based nanoindentation and immunofluorescence staining in vitro and ex vivo, we investigated the underlying cellular mechanisms and signalling pathways of EnNaC-dependent endothelial behaviour. We were able to show that the interaction between EnNaC and the cortical cytoskeleton is mediated by the small GTPases RhoA, Rac1, and the Arp2/3 complex. The functional inhibition of EnNaC by the drugs amiloride and benzamil led to membrane removal of the channel within minutes. Furthermore, we could observe an involvement of mineralocorticoid receptor, SGK1 and Nedd4-2 in regulation of endothelial cell stiffness. Our study contributes further insights on complex regulation of EnNaC and elucidates its interaction with the actin cytoskeleton, which could be central to its role as a key regulator of vascular function in health and disease.