Prednisolone Targets Claudins in Mouse Brain Blood Vessels

Endothelial cells in brain capillaries are crucial for the function of the blood–brain barrier (BBB), and members of the tight junction protein family of claudins are regarded to be primarily responsible for barrier properties. Thus, the analysis of bioactive substances that can affect the BBB’s permeability is of great importance and may be useful for the development of new therapeutic strategies for brain pathologies. In our study, we tested the hypothesis that the application of the glucocorticoid prednisolone affects the murine blood–brain barrier in vivo. Isolated brain tissue of control and prednisolone-injected mice was examined by employing immunoblotting and confocal laser scanning immunofluorescence microscopy, and the physiological and behavioral effects were analyzed. The control tissue samples revealed the expression of barrier-forming tight junction proteins claudin-1, -3, and -5 and of the paracellular cation and water-channel-forming protein claudin-2. Prednisolone administration for 7 days at doses of 70 mg/kg caused physiological and behavioral effects and downregulated claudin-1 and -3 and the channel-forming claudin-2 without altering their localization in cerebral blood vessels. Changes in the expression of these claudins might have effects on the ionic and acid–base balance in brain tissue, suggesting the relevance of our findings for therapeutic options in disorders such as cerebral edema and psychiatric failure

Circulating Ouabain Modulates Expression of Claudins in Rat Intestine and Cerebral Blood Vessels

The ability of exogenous low ouabain concentrations to affect claudin expression and therefore epithelial barrier properties was demonstrated previously in cultured cell studies. We hypothesized that chronic elevation of circulating ouabain in vivo can affect the expression of claudins and tight junction permeability in different tissues. We tested this hypothesis in rats intraperitoneally injected with ouabain (1 μg/kg) for 4 days. Rat jejunum, colon and brain frontal lobes, which are variable in the expressed claudins and tight junction permeability, were examined. Moreover, the porcine jejunum cell line IPEC-J2 was studied. In IPEC-J2-cells, ouabain (10 nM, 19 days of incubation) stimulated epithelial barrier formation, increased transepithelial resistance and the level of cSrc-kinase activation by phosphorylation, accompanied with an increased expression of claudin-1, -5 and down-regulation of claudin-12; the expression of claudin-3, -4, -8 and tricellulin was not changed. In the jejunum, chronic ouabain increased the expression of claudin-1, -3 and -5 without an effect on claudin-2 and -4 expression. In the colon, only down-regulation of claudin-3 was observed. Chronic ouabain protected the intestine transepithelial resistance against functional injury induced by lipopolysaccharide treatment or by modeled acute microgravity; this regulation was most pronounced in the jejunum. Claudin-1 was also up-regulated in cerebral blood vessels. This was associated with reduction of claudin-3 expression while the expression of claudin-5 and occludin was not affected. Altogether, our results confirm that circulating ouabain can functionally and tissue-specifically affect barrier properties of epithelial and endothelial tissues via Na,K-ATPase-mediated modulation of claudins expression

Human APP Gene Expression Alters Active Zone Distribution and Spontaneous Neurotransmitter Release at the Drosophila Larval Neuromuscular Junction

This study provides further insight into the molecular mechanisms that control neurotransmitter release. Experiments were performed on larval neuromuscular junctions of transgenic Drosophila melanogaster lines with different levels of human amyloid precursor protein (APP) production. To express human genes in motor neurons of Drosophila, the UAS-GAL4 system was used. Human APP gene expression increased the number of synaptic boutons per neuromuscular junction. The total number of active zones, detected by Bruchpilot protein puncta distribution, remained unchanged; however, the average number of active zones per bouton decreased. These disturbances were accompanied by a decrease in frequency of miniature excitatory junction potentials without alteration in random nature of spontaneous quantal release. Similar structural and functional changes were observed with co-overexpression of human APP and β-secretase genes. In Drosophila line with expression of human amyloid-β42 peptide itself, parameters analyzed did not differ from controls, suggesting the specificity of APP effects.These results confirm the involvement of APP in synaptogenesis and provide evidence to suggest that human APP overexpression specifically disturbs the structural and functional organization of active zone and results in altered Bruchpilot distribution and lowered probability of spontaneous neurotransmitter release

Human APP

This study provides further insight into the molecular mechanisms that control neurotransmitter release. Experiments were performed on larval neuromuscular junctions of transgenic Drosophila melanogaster lines with different levels of human amyloid precursor protein (APP) production. To express human genes in motor neurons of Drosophila, the UAS-GAL4 system was used. Human APP gene expression increased the number of synaptic boutons per neuromuscular junction. The total number of active zones, detected by Bruchpilot protein puncta distribution, remained unchanged; however, the average number of active zones per bouton decreased. These disturbances were accompanied by a decrease in frequency of miniature excitatory junction potentials without alteration in random nature of spontaneous quantal release. Similar structural and functional changes were observed with co-overexpression of human APP and β-secretase genes. In Drosophila line with expression of human amyloid-β42 peptide itself, parameters analyzed did not differ from controls, suggesting the specificity of APP effects. These results confirm the involvement of APP in synaptogenesis and provide evidence to suggest that human APP overexpression specifically disturbs the structural and functional organization of active zone and results in altered Bruchpilot distribution and lowered probability of spontaneous neurotransmitter release

Chronic Nicotine Modifies Skeletal Muscle Na,K-ATPase Activity through Its Interaction with the Nicotinic Acetylcholine Receptor and Phospholemman

Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21–31 days. Chronic nicotine produced a steady membrane depolarization of ∼3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV) while the activity of the α1 isoform decreased (−4.4 mV). Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM), measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser63 and Ser68. Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM

Specialized functional diversity and interactions of the Na,K-ATPase

Na,K-ATPase is a protein ubiquitously expressed in the plasma membrane of all animal cells and vitally essential for their functions. A specialized functional diversity of the Na,K-ATPase isozymes is provided by molecular heterogeneity, distinct subcellular localizations and functional interactions with molecular environment. Studies over the last decades clearly demonstrated complex and isoform-specific reciprocal functional interactions between the Na,K-ATPase and neighboring proteins and lipids. These interactions are enabled by a spatially restricted ion homeostasis, direct protein-protein/lipid interactions and protein kinase signaling pathways. In addition to its ‘classical’ function in ion translocation, the Na,K-ATPase is now considered as one of the most important signaling molecules in neuronal, epithelial, skeletal, cardiac and vascular tissues. Accordingly, the Na,K-ATPase forms specialized sub-cellular multimolecular microdomains which act as receptors to circulating endogenous cardiotonic steroids triggering a number of signaling pathways. Changes in these endogenous cardiotonic steroid levels and initiated signaling responses have significant adaptive values for tissues and whole organisms under numerous physiological and pathophysiological conditions. This review discusses recent progress in the studies of functional interactions between the Na,K-ATPase and molecular microenvironment, the Na,K-ATPase-dependent signaling pathways and their significance for diversity of cell function

AMP-Activated Protein Kinase as a Key Trigger for the Disuse-Induced Skeletal Muscle Remodeling

Molecular mechanisms that trigger disuse-induced postural muscle atrophy as well as myosin phenotype transformations are poorly studied. This review will summarize the impact of 5′ adenosine monophosphate -activated protein kinase (AMPK) activity on mammalian target of rapamycin complex 1 (mTORC1)-signaling, nuclear-cytoplasmic traffic of class IIa histone deacetylases (HDAC), and myosin heavy chain gene expression in mammalian postural muscles (mainly, soleus muscle) under disuse conditions, i.e., withdrawal of weight-bearing from ankle extensors. Based on the current literature and the authors’ own experimental data, the present review points out that AMPK plays a key role in the regulation of signaling pathways that determine metabolic, structural, and functional alternations in skeletal muscle fibers under disuse

Skeletal Muscle Na,K-ATPase as a Target for Circulating Ouabain

While the role of circulating ouabain-like compounds in the cardiovascular and central nervous systems, kidney and other tissues in health and disease is well documented, little is known about its effects in skeletal muscle. In this study, rats were intraperitoneally injected with ouabain (0.1–10 µg/kg for 4 days) alone or with subsequent injections of lipopolysaccharide (1 mg/kg). Some rats were also subjected to disuse for 6 h by hindlimb suspension. In the diaphragm muscle, chronic ouabain (1 µg/kg) hyperpolarized resting potential of extrajunctional membrane due to specific increase in electrogenic transport activity of the α2 Na,K-ATPase isozyme and without changes in α1 and α2 Na,K-ATPase protein content. Ouabain (10–20 nM), acutely applied to isolated intact diaphragm muscle from not injected rats, hyperpolarized the membrane to a similar extent. Chronic ouabain administration prevented lipopolysaccharide-induced (diaphragm muscle) or disuse-induced (soleus muscle) depolarization of the extrajunctional membrane. No stimulation of the α1 Na,K-ATPase activity in human red blood cells, purified lamb kidney and Torpedo membrane preparations by low ouabain concentrations was observed. Our results suggest that skeletal muscle electrogenesis is subjected to regulation by circulating ouabain via the α2 Na,K-ATPase isozyme that could be important for adaptation of this tissue to functional impairment

Dose- and Segment-Dependent Disturbance of Rat Gut by Ionizing Radiation: Impact of Tight Junction Proteins

The damaging effect of ionizing radiation (IR) exposure results in the disturbance of the gut natural barrier, followed by the development of severe gastrointestinal injury. However, the dose and application segment are known to determine the effects of IR. In this study, we demonstrated the dose- and segment-specificity of tight junction (TJ) alteration in IR-induced gastrointestinal injury in rats. Male Wistar rats were subjected to a total-body X-ray irradiation at doses of 2 or 10 Gy. Isolated jejunum and colon segments were tested in an Ussing chamber 72 h after exposure. In the jejunum, 10-Gy IR dramatically altered transepithelial resistance, short-circuit current and permeability for sodium fluorescein. These changes were accompanied by severe disturbance of histological structure and total rearrangement of TJ content (increased content of claudin-1, -2, -3 and -4; multidirectional changes in tricellulin and occludin). In the colon of 10-Gy irradiated rats, lesions of barrier and transport functions were less pronounced, with only claudin-2 and -4 altered among TJ proteins. The 2-Gy IR did not change electrophysiological characteristics or permeability in the colon or jejunum, although slight alterations in jejunum histology were noted, emphasized with claudin-3 increase. Considering that TJ proteins are critical for maintaining epithelial barrier integrity, these findings may have implications for countermeasures in gastrointestinal acute radiation injury

Isoform-Specific Na,K-ATPase Alterations Precede Disuse-Induced Atrophy of Rat Soleus Muscle

This study examines the isoform-specific effects of short-term hindlimb suspension (HS) on the Na,K-ATPase in rat soleus muscle. Rats were exposed to 24–72 h of HS and we analyzed the consequences on soleus muscle mass and contractile parameters; excitability and the resting membrane potential (RMP) of muscle fibers; the electrogenic activity, protein, and mRNA content of the α1 and α2 Na,K-ATPase; the functional activity and plasma membrane localization of the α2 Na,K-ATPase. Our results indicate that 24–72 h of HS specifically decreases the electrogenic activity of the Na,K-ATPase α2 isozyme and the RMP of soleus muscle fibers. This decrease occurs prior to muscle atrophy or any change in contractile parameters. The α2 mRNA and protein content increased after 24 h of HS and returned to initial levels at 72 h; however, even the increased content was not able to restore α2 enzyme activity in the disused soleus muscle. There was no change in the membrane localization of α2 Na,K-ATPase. The α1 Na,K-ATPase electrogenic activity, protein and mRNA content did not change. Our findings suggest that skeletal muscle use is absolutely required for α2 Na,K-ATPase transport activity and provide the first evidence that Na,K-ATPase alterations precede HS-induced muscle atrophy