Pflugers Archiv : European journal of physiology最新文献

SARS-CoV-2 virus infects cells by engaging with ACE2 requiring protease TMPRSS2. ACE2 is highly expressed in kidneys. Predictors for severe disease are high age and male sex. We hypothesized that ACE2 and TMPRSS2 proteins are more abundant (1) in males and with increasing age in kidney and (2) in urine and extracellular vesicles (EVs) from male patients with COVID-19 and (3) SARS-CoV-2 is present in urine and EVs during infection. Kidney cortex samples from patients subjected to cancer nephrectomy (male/female; < 50 years/˃75 years, n = 24; ˃80 years, n = 15) were analyzed for ACE2 and TMPRSS2 protein levels. Urine from patients hospitalized with SARS-CoV-2 infection was analyzed for ACE2 and TMPRSS2. uEVs were used for immunoblotting and SARS-CoV-2 mRNA and antigen detection. Tissue ACE2 and TMPRSS2 protein levels did not change with age. ACE2 was not more abundant in male kidneys in any age group. ACE2 protein was associated with proximal tubule apical membranes in cortex. TMPRSS2 was observed predominantly in the medulla. ACE2 was elevated significantly in uEVs and urine from patients with COVID-19 with no sex difference compared with urine from controls w/wo albuminuria. TMPRSS2 was elevated in uEVs from males compared to female. ACE2 and TMPRSS2 did not co-localize in uEVs/apical membranes. SARS-CoV-2 nucleoprotein and mRNA were not detected in urine. Higher kidney ACE2 protein abundance is unlikely to explain higher susceptibility to SARS-CoV-2 infection in males. Kidney tubular cells appear not highly susceptible to SARS-CoV-2 infection. Loss of ACE2 into urine in COVID could impact susceptibility and angiotensin metabolism.

Pancreatic stellate cells (PSCs) are central in the development of acute pancreatitis and tumor fibrosis in pancreatic ductal adenocarcinoma (PDAC). Fibrosis and a unique pH landscape represent characteristic properties of the PDAC microenvironment. Mechanosensitive ion channels are involved in the activation of PSCs. Among these channels, K_2P2.1 has not yet been studied in PSCs. K_2P2.1 channels are pH- and mechanosensitive. We confirmed K_2P2.1 expression in PSCs by RT-qPCR and immunofluorescence. PSCs from K_2P2.1^+/+ and K_2P2.1^-/- mice were studied under conditions mimicking properties of the PDAC microenvironment (acidic extracellular pH (pH_e), ambient pressure elevated by + 100 mmHg). Migration and the cell area were taken as surrogates for PSC activation and evaluated with live cell imaging. pH_e-dependent changes of the membrane potential of PSCs were investigated with DiBAC₄(3), a voltage-sensitive fluorescent dye. We observed a correlation between morphological activation and progressive hyperpolarization of the cells in response to changes in pH_e and pressure. The effect was in part dependent on the expression of K_2P2.1 channels because the membrane potential of K_2P2.1^+/+ PSCs was always more hyperpolarized than that of K_2P2.1^-/- PSCs. Cell migration velocity of K_2P2.1^+/+ cells decreased upon pressure application when cells were kept in an acidic medium (pH_e 6.6). This was not the case in K_2P2.1^-/- PSCs. Taken together, our study highlights the critical role of K_2P2.1 channels in the combined sensing of environmental pressure and pH_e by PSCs and in coordinating cellular morphology with membrane potential dynamics. Thus, K_2P2.1 channels are important mechano-sensors in murine PSCs.

Gestational diabetes mellitus is one of the most common complications during pregnancy. Its prevalence is rapidly increasing worldwide. Gestational diabetes mellitus is leading to an elevated risk for the development of endothelial dysfunction and cardiovascular diseases both in the mother and the child in later life. The underlying pathophysiological mechanisms are not well-understood. Therefore, we aimed to characterize the endothelial function in fetal placental vessels from mothers with gestational diabetes mellitus. In this study, we distinguished between insulin-treated and diet-controlled gestational diabetes mothers and compared them to a normoglycemic control group. The clinical data confirmed pre-conceptional overweight as a risk factor in women with insulin-treated gestational diabetes mellitus. The insulin-treated gestational diabetes group was also characterized by a recent family history of diabetes compared to mothers of the control or diet-controlled gestational diabetes group. Analyses of blood serum from umbilical cords suggested a reduced fetal insulin metabolism in the insulin-treated gestational diabetes group. Vascular function analysis in fetal placental vessels revealed an altered substance P-induced vasorelaxation in vessels from patients with insulin-dependent gestational diabetes. Inhibition of nitric oxide synthase affected only fetal vessel segments from the control group or diet-controlled gestational diabetes group, but not from insulin-dependent gestational diabetes. Finally, we found a significantly decreased substance P receptor (TACR1) mRNA expression in fetal vessel segments from patients with insulin-treated gestational diabetes. In conclusion, we provide evidence that different pathophysiological mechanisms might be responsible for the development of insulin-treated versus diet-controlled gestational diabetes. Only in fetal vessels from patients with insulin-treated gestational diabetes were we able to detect an endothelial dysfunction and a reduced fetal insulin conversion. This provides novel insights into the pathophysiology of the subtypes of gestational diabetes.

Neurons in central nervous systems receive multiple synaptic inputs and transform them into a largely standardized output to their target cells-the action potential. A simplified model posits that synaptic signals are integrated by linear summation and passive propagation towards the axon initial segment, where the threshold for spike generation is either crossed or not. However, multiple lines of research during past decades have shown that signal integration in individual neurons is much more complex, with important functional consequences at the cellular, network, and behavioral-cognitive level. The interplay between concomitant excitatory and inhibitory postsynaptic potentials depends strongly on the relative timing and localization of the respective synapses. In addition, dendrites contain multiple voltage-dependent conductances, which allow scaling of postsynaptic potentials, non-linear input processing, and compartmentalization of signals. Together, these features enable a rich variety of single-neuron computations, including non-linear operations and synaptic plasticity. Hence, we have to revise over-simplified messages from textbooks and use simplified computational models like integrate-and-fire neurons with some caution. This concept article summarizes the most important mechanisms of dendritic integration and highlights some recent developments in the field.

An autaptic synapse (or 'autapse') is a functional connection between a neuron and itself, commonly used in studying the molecular mechanisms underlying synaptic transmission and plasticity in central neurons. Most previous studies on autonomic synaptic functions have relied on spontaneous connections among neurons in mass cultures. However, growing evidence supports the utility of microcultures cultivating autaptic neurons for examining cholinergic transmission within sympathetic ganglia. Despite these advancements, standardized protocols for culturing autaptic sympathetic neurons have yet to be established. Drawing on historical literature, this study delineates optimal experimental conditions to efficiently and reliably produce cholinergic synapses in sympathetic neurons within a short time frame. Our research emphasizes five key factors: (i) the generation of uniformly sized microislands of growth permissive substrates; (ii) the addition of nerve growth factor, ciliary neurotrophic factor (CNTF), and serum to the culture medium; (iii) independence from specific serum and neuronal medium types; (iv) the reciprocal roles of CNTF and glial cells; and (v) the promotion of cholinergic synaptogenesis in SCG neurons through indirect glia co-cultures, rather than direct glial feeder layer cultures. In conclusion, glia-free monocultures of SCG neurons are relatively simple to prepare and yield robust and reliable synaptic currents. This makes them an effective model system for straightforwardly addressing fundamental questions about neurogenic mechanisms involved in cholinergic synaptic transmission in autonomic ganglia. Furthermore, autaptic culture experiments could eventually be implemented to investigate the roles of functional neuron-satellite glia units in regulating cholinergic functions under physiological and pathological conditions.

Appropriate composition of oral saliva is essential for a healthy milieu that protects mucosa and teeth. Only few studies, with small sample numbers, investigated physiological saliva ion composition in humans. We determined saliva ion composition in a sufficiently large cohort of healthy adults and analyzed the effect of physiological stimulation. We collected saliva from 102 adults under non-stimulated and physiologically stimulated conditions (chewing). Individual flow rates, pH, osmolality, Na⁺, K⁺, Cl^-, and HCO₃^- concentrations under both conditions as well as the individual changes due to stimulation (Δvalues) were determined. Non-stimulated saliva was hypoosmolal and acidic. Na⁺, Cl^-, and HCO₃^- concentrations remained well below physiological plasma values, whereas K⁺ concentrations exceeded plasma values more than twofold. Stimulation resulted in a doubling of flow rates and substantial increases in pH, HCO₃^-, and Na⁺ concentrations. Overall, stimulation did not considerably affect osmolality nor K⁺ or Cl^- concentrations of saliva. An in-depth analysis of stimulation effects, using individual Δvalues, showed no correlation of Δflow rate with Δion concentrations, indicating independent regulation of acinar volume and ductal ion transport. Stimulation-induced Δ[Na⁺] correlated with Δ[HCO₃^-] and Δ[Cl^-] but not with Δ[K⁺], indicating common regulation of ductal Na⁺, Cl^-, and HCO₃^- transport. We present a robust data set of human oral saliva ion composition in healthy adults and functional insights into physiological stimulation. Our data show (i) that flow-dependence exists for Na⁺ and HCO₃^- but not for K⁺ and Cl^- concentrations, (ii) osmolality is flow-independent, (iii) regulation of Na⁺, Cl^-, and HCO₃^- transport is coupled, (iv) regulation of flow rate and ion concentrations are independent and (v) spatially separated between acini and ducts, respectively.