Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function. It is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and backwards. The integrity of the BBB is compromised in many disorders of the human CNS; therapeutical strategies for several of these diseases include treatment with glucocorticoids, but the molecular basis of how glucocorticoids regulate BBB permeability is not understood. Here, we report the generation and characterization of a murine immortalized brain (cerebral) capillary endothelial (cEND) cell line which expresses the BBB marker occludin at intercellular tight junctions (TJ). Hydrocortisone at physiological concentrations induced upregulation of occludin, accompanied by a threefold enhancement of transendothelial electrical resistance to values up to 1000 Ωcm 2 . Insulin enhanced the glucocorticoid response. At the molecular level, hydrocortisone induces increase of occludin at protein and mRNA levels by activation of the glucocorticoid receptor (GR) and its binding to putative glucocorticoid responsive elements in the occludin promoter. At the same time, insulin potentiated the ligand-dependent GR transactivation via induction of the GR in this in vitro system. This study thus provides insights into the molecular processes of barrier genesis, and may help to elucidate mechanisms of brain pathology at the microvascular level.

Seven members of the small heat shock protein (sHSP) family are exceptional with respect to their constitutive high abundance in muscle tissue. It has been suggested that sHSPs displaying chaperone-like properties may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. In the present study five sHSPs (alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP) were investigated with respect to similarities and differences of their expression in heart and skeletal muscle under normal and ischemic conditions. In ischemic heart and skeletal muscle these five sHSPs translocated from cytosol to the Z-/I-area of myofibrils. Myofibrillar binding of all sHSPs was very tight and resisted for the most part extraction with 1 M NaSCN or 1 M urea. MKBP and HSP20 became extracted by 1 M NaSCN to a significant extent indicating that these two sHSPs may bind partially to actin-associated proteins which were completely extracted by this treatment. Ultrastructural localization of alphaB-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes alphaB-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of alphaB-crystallin in cardiomyocytes versus skeletal muscle fibers. Further differences of the properties of sHSPs could be observed regarding fiber type distribution of sHSPs. Thus sHSPs form a complex stress-response system in striated muscle tissue with some common as well as some distinct functions in different muscle types.

Regulation of actin dynamics is critical for endothelial barrier functions. We provide evidence that the actin-binding protein vasodilator-stimulated phosphoprotein (VASP) is required for endothelial barrier maintenance. Baseline permeability was significantly increased in VASP-deficient (VASP(-/-)) microvascular myocardial endothelial cells (MyEnd) in the absence of discernible alterations of immunostaining for adherens and tight junctions. We tested whether VASP is involved in the endothelium-stabilizing effects of cAMP or Rac 1. Forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 (CNF-1) to activate Rac 1 were equally efficient to stabilize barrier functions in VASP(-/-) and wild-type (wt) cells. In wt cells, VASP was phosphorylated in response to F/R but did not localize to intercellular junctions. In contrast, CNF-1 and expression of constitutively active Rac 1 induced translocation of VASP to cell borders in wt cells, where it colocalized with active Rac 1. In VASP(-/-) cells, Rac 1 activity was reduced to 0.4 of wt levels in controls and increased approximately 20-fold in response to CNF-1 compared with 7-fold activation in wt cells. Moreover, inactivation of Rac 1 by lethal toxin led to a greater increase of permeability compared with wt cells. All these data suggest that VASP is involved in the regulation of Rac 1 activity. Taking these findings together, our study indicates that VASP at least in part stabilizes endothelial barrier functions by control of Rho-family GTPases.

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world's experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12-15, 2016).

In endothelial monolayers agonist-induced influx of Ca2+ and activities of the actin cytoskeleton have been shown to be crucially involved in regulation of barrier properties. By laser tweezer application we demonstrated that the strength of adhesion of VE-cadherin-coated microspheres to the surface of cultured endothelial monolayers is significantly reduced by treatment with two well-established permeability-increasing compounds,cytochalasin D and the Ca2+-ionophore A23187, which shows that both compounds directly affect cadherin-mediated adhesion. Cytochalasin D and A23187 caused considerable decay of F-actin (30-60%). Stabilisation of F-actin by jasplakinolide completely blocked drug-induced weakening of bead adhesion showing that attenuation of cadherin-cadherin trans-interaction induced by cytochalasin D and A23187 depends largely on downregulation of F-actin. Single molecule fluorescence microscopy demonstrated that drug-induced weakening of adhesion is accompanied by an increase in lateral mobility of cadherins as well as by dispersal of cadherin-enriched plasmalemmal microdomains. However,the lifetime (≈700 milliseconds, koff≈1.4 second–1) and apparent on-rate of cadherin trans-interaction(relative frequency of binding) remained unchanged in response to cytochalasin D and A23187 indicating that cadherin-mediated adhesion is not modulated by inside-out changes of the affinity but, rather, appears to be controlled by actin-dependent tethering and compartmentalization of cadherins.

Porcine arterial endothelial cells (PAECs)Pulmonary arterial endothelial cells were obtained from freshly slaughtered pigs by exposure of the excised pulmonary artery to 0.1 % collagenase for 12-15 min. Cells were cultured in medium 199 containing 10 % fetal calf serum (GIBCO, Karlsruhe, Germany), passaged and characterized as described by Suttorp et al. (1988). Only cultures of passages 2-5 were used.Cell culture studies Cells were grown on coverslips, coated with gelatin cross-linked with glutaraldehyde (Schnittler et al. 1993). Toxin B was added to R. H. Adamson and others 296

There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10-10 mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2 . Omission of extracellular Ca2+ also prevented aldosteroneinduced alkalinization. Instead, aldosterone led to a Zn2+-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel activity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion.The classical genomic mechanism of steroid hormone action involves binding to intracellular receptors, transcription, translation, and protein synthesis. Thus, the steroid hormoneinduced response following genomic activation should be preceded by a latent period in the range of hours. Yet it was shown as long as four decades ago that there is a rapid action of aldosterone, with a latency of -5 min, on renal electrolyte excretion (1). This and other findings (2) indicate that aldosterone is not only responsible for chronic but also for acute regulation of water and electrolyte balance. The underlying mechanisms leading to rapid changes in Na+ and K+ excretion are not completely understood.Aldosterone can induce a so-called early response by interfering with mechanisms of regulation of intracellular pH (3-5) and intracellular generation of inositol-1,4,5-trisphosphate (InsP3) (6) with a latency shorter than 1 h. These evidently nongenomic actions of aldosterone are thought to be mediated by a plasma membrane receptor with high affinity for aldosterone but low affinity for glucocorticoids (7). Furthermore, there is some evide...

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