| 1. |
- Agren, Johan, et al.
(författare)
-
Antenatal Corticosteroids and Postnatal Fluid Restriction Produce Differential Effects on AQP3 Expression, Water Handling, and Barrier Function in Perinatal Rat Epidermis
- 2010
-
Ingår i: Dermatology research and practice. - : Hindawi Limited. - 1687-6113 .- 1687-6105.
-
Tidskriftsartikel (refereegranskat)abstract
- Loss of water through the immature skin can lead to hypothermia and dehydration in preterm infants. The water and glycerol channel aquaglyceroporin-3 (AQP3) is abundant in fetal epidermis and might influence epidermal water handling and transepidermal water flux around birth. To investigate the role of AQP3 in immature skin, we measured in vivo transepidermal water transport and AQP3 expression in rat pups exposed to clinically relevant fluid homeostasis perturbations. Preterm (E18) rat pups were studied after antenatal corticosteroid exposure (ANS), and neonatal (P1) rat pups after an 18 h fast. Transepidermal water loss (TEWL) and skin hydration were determined, AQP3 mRNA was quantified by RT-PCR, and in-situ hybridization and immunocytochemistry were applied to map AQP3 expression. ANS resulted in an improved skin barrier (lower TEWL and skin hydration), while AQP3 mRNA and protein increased. Fasting led to loss of barrier integrity along with an increase in skin hydration. These alterations were not paralleled by any changes in AQP3. To conclude, antenatal corticosteroids and early postnatal fluid restriction produce differential effects on skin barrier function and epidermal AQP3 expression in the rat. In perinatal rats, AQP3 does not directly determine net water transport through the skin.
|
|
| 2. |
|
|
| 3. |
- Akkuratov, Evgeny E., et al.
(författare)
-
Ouabain Modulates the Functional Interaction Between Na,K-ATPase and NMDA Receptor.
- 2020
-
Ingår i: Molecular Neurobiology. - : Springer Science and Business Media LLC. - 0893-7648 .- 1559-1182. ; 57:10, s. 4018-4030
-
Tidskriftsartikel (refereegranskat)abstract
- The N-methyl-D-aspartate (NMDA) receptor plays an essential role in glutamatergic transmission and synaptic plasticity and researchers are seeking for different modulators of NMDA receptor function. One possible mechanism for its regulation could be through adjacent membrane proteins. NMDA receptors coprecipitate with Na,K-ATPase, indicating a potential interaction of these two proteins. Ouabain, a mammalian cardiotonic steroid that specifically binds to Na,K-ATPase and affects its conformation, can protect from some toxic effects of NMDA receptor activation. Here we have examined whether NMDA receptor activity and downstream effects can be modulated by physiological ouabain concentrations. The spatial colocalization between NMDA receptors and the Na,K-ATPase catalytic subunits on dendrites of cultured rat hippocampal neurons was analyzed with super-resolution dSTORM microscopy. The functional interaction was analyzed with calcium imaging of single hippocampal neurons exposed to 10 μM NMDA in presence and absence of ouabain and by determination of the ouabain effect on NMDA receptor-dependent long-term potentiation. We show that NMDA receptors and the Na,K-ATPase catalytic subunits alpha1 and alpha3 exist in same protein complex and that ouabain in nanomolar concentration consistently reduces the calcium response to NMDA. Downregulation of the NMDA response is not associated with internalization of the receptor or with alterations in its state of Src phosphorylation. Ouabain in nanomolar concentration elicits a long-term potentiation response. Our findings suggest that ouabain binding to a fraction of Na,K-ATPase molecules that cluster with the NMDA receptors will, via a conformational effect on the NMDA receptors, cause moderate but consistent reduction of NMDA receptor response at synaptic activation.
|
|
| 4. |
- Akkuratov, Evgeny E.
(författare)
-
The Biophysics of Na+,K+-ATPase in neuronal health and disease
- 2020
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Na+,K+-ATPase is one of the most important proteins in the mammalian cell. It creates sodium and potassium gradients which are fundamental for the membrane potential and sodium-dependent secondary active transport. It has a second role in the cell as a receptor that by binding chemicals from the cardiotonic steroids family, the most knowledgeable of them is ouabain, triggers various signaling pathways in the cell which regulate gene activation, proliferation, apoptosis, etc. It has been shown that several severe neurological diseases are associated with mutations in the Na+,K+-ATPase encoding genes. Although Na+,K+-ATPase was discovered already in 1957 by the Danish scientist Jens Skou, the knowledge about the function of this enzyme is still not complete. In the studies included in the thesis, we have learned more about the function of Na+,K+-ATPase in different aspects of health and disease. In study I we showed a mechanism of ouabain-dependent regulation of the NMDA receptor, one of the most important receptors in the nervous system, via binding with Na+,K+-ATPase. This allows us to look at the Na+,K+-ATPase as regulator via protein-protein interaction. In study II we investigated a different aspect of Na+,K+-ATPase functioning – to look at how binding of ouabain to Na+,K+-ATPase activates a number of signaling cascades by looking at the phosphoproteome status of the cells. This allows us to see the whole picture of ouabain-mediated cascades and further characterize them. In study III we focused on the role of Na+,K+-ATPase in severe epileptic encephalopathy caused by a mutation in the ATP1A1 gene. We performed a molecular and cellular study to describe how mutations affects protein structure and function and found that this mutation converts the ion pump to a nonspecific leak channel. In study IV we performed a translational study of the most common mutation for rapid-onset dystonia-parkinsonism. We studied how this mutation affects the nervous system on the protein-, cellular-, and organism level and found that the complete absence of ultraslow afterhyperpolarization (usAHP) could explain gait disturbances found in patients. In the on-going study we showed that Na+,K+-ATPase can oligomerize and that this effect is triggered by ouabain binding to the Na+,K+-ATPase. In this study, we utilized a novel fluorescence labelling approach and used biophysical techniques with single molecule sensitivity to track Na+,K+-ATPase interactions. In summary, we applied biophysical and molecular methods to study different aspects of the function of Na+,K+-ATPase, and gained insights that could be helpful not only for answering fundamental questions about Na+,K+-ATPase but also to find a treatment for patients with diseases associated with mutations in this protein.
|
|
| 5. |
- Aperia, Anita Chatarina, et al.
(författare)
-
Na+, K+-ATPase, a new class of plasma membrane receptors
- 2016
-
Ingår i: American Journal of Physiology - Cell Physiology. - : American Physiological Society. - 0363-6143 .- 1522-1563. ; 310:7, s. C491-C495
-
Tidskriftsartikel (refereegranskat)abstract
- The Na(+), K(+)-ATPase (NKA) differs from most other ion transporters not only in its capacity to maintain a steep electrochemical gradient across the plasma membrane but also as a receptor for a family of cardiotonic steroids, to which ouabain belongs. Studies from many groups, performed during the last fifteen years, have demonstrated that ouabain, a member of the cardiotonic steroid family, can activate a network of signaling molecules and that NKA will also serve as a signal transducer that can provide a feed back loop between NKA and the mitochondria. This brief review summarizes the current knowledge and controversies with regard to the understanding of NKA signaling.
|
|
| 6. |
- Aperia, Anita, et al.
(författare)
-
Mending Fences : Na,K-ATPase signaling via Ca2+ in the maintenance of epithelium integrity
- 2020
-
Ingår i: Cell Calcium. - : Elsevier BV. - 0143-4160 .- 1532-1991. ; 88
-
Tidskriftsartikel (refereegranskat)abstract
- Na,K-ATPase is a ubiquitous multifunctional protein that acts both as an ion pump and as a signal transducer. The signaling function is activated by ouabain in non-toxic concentrations. In epithelial cells the ouabain-bound Na,K-ATPase connects with the inositol 1,4,5-trisphosphate receptor via a short linear motif to activate low frequency Ca2+ oscillations. Within a couple of minutes this ouabain mediated signal has resulted in phosphorylation or dephosphorylation of 2580 phospho-sites. Proteins that control cell proliferation and cell adhesion and calmodulin regulated proteins are enriched among the ouabain phosphor-regulated proteins. The inositol 1,4,5-trisphosphate receptor and the stromal interaction molecule, which are both essential for the initiation of Ca2+ oscillations, belong to the ouabain phosphor-regulated proteins. Downstream effects of the ouabain-evoked Ca2+ signal in epithelial cells include interference with the intrinsic mitochondrial apoptotic process and stimulation of embryonic growth processes. The dual function of Na,K-ATPase as an ion pump and a signal transducer is now well established and evaluation of the physiological and pathophysiological consequences of this universal signal emerges as an urgent topic for future studies.
|
|
| 7. |
- Azarias, Guillaume, et al.
(författare)
-
A Specific and Essential Role for Na,K-ATPase alpha 3 in Neurons Co-expressing alpha 1 and alpha 3
- 2013
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:4, s. 2734-2743
-
Tidskriftsartikel (refereegranskat)abstract
- Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous alpha 1, and the more selectively expressed alpha 3. Although neurological syndromes are associated with alpha 3 mutations, the specific role of this isoform is not completely understood. Here, we used electrophysiological and Na+ imaging techniques to study the role of alpha 3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of alpha 3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na+ concentration ([Na+](i)) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na+](i), similar to what could be expected following suprathreshold neuronal activity, selective inhibition of alpha 3 almost completely abolished the capacity to restore [Na+](i) in soma and dendrite. Recordings of Na, K-ATPase specific current supported the notion that when [Na+](i) is elevated in the neuron, alpha 3 is the predominant isoform responsible for rapid extrusion of Na+. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of alpha 3 function in the central nervous system. The alpha isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na+ binding site. Following activation of protein kinase A, both the alpha 3-dependent current and restoration of dendritic [Na+](i) were significantly attenuated, indicating that alpha 3 is a target for phosphorylation and may participate in short term regulation of neuronal function.
|
|
| 8. |
- Bernhem, Kristoffer, et al.
(författare)
-
AT(1)-receptor response to non-saturating Ang-II concentrations is amplified by calcium channel blockers
- 2017
-
Ingår i: BMC Cardiovascular Disorders. - Stockholm : BioMed Central. - 1471-2261 .- 1471-2261. ; 17:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Blockers of angiotensin II type 1 receptor (AT 1 R) and the voltage gated calcium channel 1.2 (Ca V 1.2) are commonly used for treatment of hypertension. Yet there is little information about the effect of physiological concentrations of angiotensin II (AngII) on AT 1 R signaling and whether there is a reciprocal regulation of AT 1 R signaling by Ca V 1.2.Methods: To elucidate these questions, we have studied the Ca 2+ signaling response to physiological and pharmacological AngII doses in HEK293a cells, vascular smooth muscle cells and cardiomyocytes using a Ca 2+ sensitive dye as the principal sensor. Intra-cellular calcium recordings were performed in presence and absence of Ca V 1.2 blockers. Semi- quantitative imaging methods were used to assess the plasma membrane expression of AT 1 R and G-protein activation.Results: Repeated exposure to pharmacological (100 nM) concentrations of AngII caused, as expected, a down-regulation of the Ca 2+ response. In contrast, repeated exposure to physiological (1 nM) AngII concentration resulted in an enhancement of the Ca 2+ response. The up-regulation of the Ca 2+ response to repeated 1 nM AngII doses and the down- egulation of the Ca 2+ response to repeated 100 nM Angll doses were not accompanied by a parallel change of the AT 1 R plasma membrane expression. The Ca 2+ response to 1 nM of AngII was amplified in the presence of therapeutic concentrations of the Ca V 1.2 blockers, nifedipine and verapamil, in vascular smooth muscle cells, cardiomyocytes and HEK293a cells. Amplification of the AT 1 R response was also observed following inhibition of the calcium permeable transient receptor potential cation channels, suggesting that the activity of AT 1 R is sensitive to calcium influx.Conclusions: Our findings have implications for the understanding of hyperactivity of the angiotensin system and for use of Ca 2+ channel blockers as mono-therapy in hypertension.
|
|
| 9. |
|
|
| 10. |
- Bernhem, Kristoffer
(författare)
-
Quantitative bioimaging in single cell signaling
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Imaging of cellular samples has for several hundred years been a way for scientists to investigate biological systems. With the discovery of immunofluorescence labeling in the 1940’s and later genetic fluorescent protein labeling in the 1980’s the most important part in imaging, contrast and specificity, was drastically improved. Eversince, we have seen a increased use of fluorescence imaging in biological research, and the application and tools are constantly being developed further.Specific ion imaging has long been a way to discern signaling events in cell systems. Through use of fluorescent ion reporters, ionic concentrations can be measured inliving cells as result of applied stimuli. Using Ca2+ imaging we have demonstrated that there is a inverse influence by plasma membrane voltage gated calcium channels on angiotensin II type 1 receptor (a protein involved in blood pressure regulation). This has direct implications in treatment of hypertension (high blood pressure),one of the most common serious diseases in the western civilization today with approximately one billion afflicted adults world wide in 2016.Extending from this more lower resolution live cell bioimaging I have moved into super resolution imaging. This thesis includes works on the interpretation of super resolution imaging data of the neuronal Na+, K+ - ATPase α3, a receptor responsible for maintaining cell homeostasis during brain activity. The imaging data is correlated with electrophysiological measurements and computer models to point towards possible artefacts in super resolution imaging that needs to be taken into account when interpreting imaging data. Moreover, I proceeded to develop a software for single-molecule localization microscopy analysis aimed for the wider research community and employ this software to identify expression artifacts in transiently transfected cell systems.In the concluding work super-resultion imaging was used to map out the early steps of the intrinsic apoptotic signaling cascade in space and time. Using superresoultion imaging, I mapped out in intact cells at which time points and at which locations the various proteins involved in apoptotic regulation are activated and interact.
|
|
| 11. |
- Bernhem, Kristoffer, et al.
(författare)
-
Super resolution imaging reveals details in hyperglycemic induced apoptosis in kidney cells
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The role of the Bcl-family proteins in the mitochondrial apoptotic process is well described with biochemical and molecular methods in studies of isolated mitochondria and transfected cell lines. There is however little knowledge about the mechanisms for Bcl protein interaction leading to apoptosis in intact cells. In particular, the time sequence and location for Bcl protein interaction has so far only been described in hypothetical models.Here we have used Stimulated Emission Depletion (STED) microscopy and Single Molecule Localization Microscopy (SMLM) to study the apoptotic process in immune-stained rat renal epithelia cells exposed to 20 mM glucose (HG) and to study its rescue by ouabain. To assess distance between Bcl-2 proteins, we used the nearest-neighbor algorithm. The anti-apoptotic protein Bcl-xLl was predominantly expressed on mitochondria in control cells, and remained so throughout the process, although its abundance decreased. After 2h HG the apoptosis-inducing protein BAD had translocated from the cytoplasm to the mitochondria where it clustered with Bcl-xL. This occurred before an increase in reactive oxygen species and was dependent on activation of the PI3K –AKT pathway. According to current concepts, Bcl-xL interacts with the apoptotic protein Bax on the mitochondria under control conditions to translocate Bax back to the cytosol1. We found that Bax started to accumulate on the mitochondria after 4h HG and, surprisingly, that the interaction between Bcl-xL and Bax became more pronounced during the course of the apoptotic process. After 6h HG Bax also interacted with the non-specific ion transporter VDAC; an interaction described to lead to penetration of the inner mitochondrial membrane and mark the point of no return.
|
|
| 12. |
- Bernhem, Kristoffer, et al.
(författare)
-
Super-resolution microscopy reveals that Na+/K+-ATPase signaling protects against glucose-induced apoptosis by deactivating Bad
- 2021
-
Ingår i: Cell Death and Disease. - : Springer Nature. - 2041-4889. ; 12:8
-
Tidskriftsartikel (refereegranskat)abstract
- Activation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na+,K+-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca2+/calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.
|
|
| 13. |
- Blom, Hans, et al.
(författare)
-
Nearest neighbor analysis of dopamine D1 receptors and Na plus -K plus -ATPases in dendritic spines dissected by STED microscopy
- 2012
-
Ingår i: Microscopy research and technique (Print). - : Wiley. - 1059-910X .- 1097-0029. ; 75:2, s. 220-228
-
Tidskriftsartikel (refereegranskat)abstract
- Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super-resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual-color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact. In this study we thus introduced nearest neighbor analysis to quantify the spatial relations between two important proteins in neurons, the dopamine D1 receptor and Na+,K+-ATPase. The analysis gave new information on how dense the D1 receptor and Na+,K+-ATPase constituting nanoclusters are located both with respect to the homogenous (self to same) and the heterogeneous (same to other) topology. The STED dissected nanoscale topologies provide evidence for both a joint as well as a separated confinement of the D1 receptor and the Na+,K+-ATPase in the postsynaptic areas of dendritic spines. This confined topology may have implications for generation of local sodium gradients and for structural and functional interactions modulating slow synaptic transmission processes. Microsc. Res. Tech., 2011.
|
|
| 14. |
- Blom, Hans, et al.
(författare)
-
Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy
- 2011
-
Ingår i: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 12, s. 16-
-
Tidskriftsartikel (refereegranskat)abstract
- Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (alpha 3 isoform) in the postsynaptic region of the spine. Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.
|
|
| 15. |
|
|
| 16. |
- Burlaka, Ievgeniia, et al.
(författare)
-
Ouabain Protects against Shiga Toxin-Triggered Apoptosis by Reversing the Imbalance between Bax and Bcl-xL
- 2013
-
Ingår i: Journal of the American Society of Nephrology. - 1046-6673 .- 1533-3450. ; 24:9, s. 1413-1423
-
Tidskriftsartikel (refereegranskat)abstract
- Hemolytic uremic syndrome, a life-threatening disease often accompanied by acute renal failure, usually occurs after gastrointestinal infection with Shiga toxin 2 (Stx2)-producing Escherichia coli. Stx2 binds to the glycosphingolipid globotriaosylceramide receptor, expressed by renal epithelial cells, and triggers apoptosis by activating the apoptotic factor Bax. Signaling via the ouabain/Na,K-ATPase/IP3R/NF-B pathway increases expression of Bcl-xL, an inhibitor of Bax, suggesting that ouabain might protect renal cells from Stx2-triggered apoptosis. Here, exposing rat proximal tubular cells to Stx2 in vitro resulted in massive apoptosis, upregulation of the apoptotic factor Bax, increased cleaved caspase-3, and downregulation of the survival factor Bcl-xL; co-incubation with ouabain prevented all of these effects. Ouabain activated the NF-B antiapoptotic subunit p65, and the inhibition of p65 DNA binding abolished the antiapoptotic effect of ouabain in Stx2-exposed tubular cells. Furthermore, in vivo, administration of ouabain reversed the imbalance between Bax and Bcl-xL in Stx2-treated mice. Taken together, these results suggest that ouabain can protect the kidney from the apoptotic effects of Stx2.
|
|
| 17. |
- Burlaka, Ievgeniia, et al.
(författare)
-
Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease
- 2016
-
Ingår i: Kidney International. - : Nature Publishing Group. - 0085-2538 .- 1523-1755. ; 90:1, s. 135-148
-
Tidskriftsartikel (refereegranskat)abstract
- There is a great need for treatment that arrests progression of chronic kidney disease. Increased albumin in urine leads to apoptosis and fibrosis of podocytes and tubular cells and is a major cause of functional deterioration. There have been many attempts to target fibrosis, but because of the lack of appropriate agents, few have targeted apoptosis. Our group has described an ouabain-activated Na,K-ATPase/IP3R signalosome, which protects from apoptosis. Here we show that albumin uptake in primary rat renal epithelial cells is accompanied by a time-and dose-dependent mitochondrial accumulation of the apoptotic factor Bax, down-regulation of the antiapoptotic factor Bcl-xL and mitochondrial membrane depolarization. Ouabain opposes these effects and protects from apoptosis in albumin-exposed proximal tubule cells and podocytes. The efficacy of ouabain as an antiapoptotic and kidney-protective therapeutic tool was then tested in rats with passive Heymann nephritis, a model of proteinuric chronic kidney disease. Chronic ouabain treatment preserved renal function, protected from renal cortical apoptosis, up-regulated Bax, down-regulated Bcl-xL, and rescued from glomerular tubular disconnection and podocyte loss. Thus we have identified a novel clinically feasible therapeutic tool, which has the potential to protect from apoptosis and rescue from loss of functional tissue in chronic proteinuric kidney disease.
|
|
| 18. |
|
|
| 19. |
- Fontana, Jacopo M., et al.
(författare)
-
Calcium oscillations triggered by cardiotonic steroids
- 2013
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 280:21, s. 5450-5455
-
Forskningsöversikt (refereegranskat)abstract
- Na+, K+-ATPase (NKA) is well known for its function as an ion pump. Studies during the last decade have revealed an additional role for NKA as a signal transducer. In this brief review, we describe how cardiotonic steroids, which are highly specific NKA ligands, trigger slow Ca2+ oscillations by promoting the interaction between NKA and the inositol trisphosphate receptor, and how this Ca2+ signal activates the NF-B subunit p65 and increases the expression of the antiapoptotic factor Bcl-xL. The potential tissue-protective effects of this signal are discussed.
|
|
| 20. |
|
|
| 21. |
- Fontana, Jacopo M., et al.
(författare)
-
Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney
- 2019
-
Ingår i: The FASEB Journal. - : FEDERATION AMER SOC EXP BIOL. - 0892-6638 .- 1530-6860. ; 33:3, s. 4089-4096
-
Tidskriftsartikel (refereegranskat)abstract
- The central role of calcium signaling during development of early vertebrates is well documented, but little is known about its role in mammalian embryogenesis. We have used immunofluorescence and time-lapse calcium imaging of cultured explanted embryonic rat kidneys to study the role of calcium signaling for branching morphogenesis. In mesenchymal cells, we recorded spontaneous calcium activity that was characterized by irregular calcium transients. The calcium signals were dependent on release of calcium from intracellular stores in the endoplasmic reticulum. Down-regulation of the calcium activity, both by blocking the sarco-endoplasmic reticulum Ca2+-ATPase and by chelating cytosolic calcium, resulted in retardation of branching morphogenesis and a reduced formation of primitive nephrons but had no effect on cell proliferation. We propose that spontaneous calcium activity contributes with a stochastic factor to the self-organizing process that controls branching morphogenesis, a major determinant of the ultimate number of nephrons in the kidney.Fontana, J. M., Khodus, G. R., Unnersjo-Jess, D., Blom, H., Aperia, A., Brismar, H. Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney.
|
|
| 22. |
- Fontana, Jacopo, et al.
(författare)
-
Temporal calcium activity in metanephric mesenchyme cells regulates kidney branching morphogenesis
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The role of calcium signaling for development of early vertebrates is well documented, but little is known about its role in mammalian embryogenesis. We have used explanted embryonic rat kidneys to study the role of calcium for branching morphogenesis, a process that depends on reciprocal interaction between mesenchymal and epithelial ureteric bud cells. We recorded a spontaneous calcium activity characterized by stochastic and irregular calcium spikes, in the mesenchymal cells. This activity is due to calcium release from the endoplasmic reticulum (ER). Depletion of ER calcium stores results in down-regulation of the calcium activity, retardation of branching morphogenesis and formation of primitive nephrons, but has no effect on cell proliferation. We propose that the excretion of morphogenic factors that mediate the interaction between 26 the mesenchymal and epithelial cells, which initiate branching morphogenesis, is calcium dependent. In support of this we demonstrate expression of the calcium dependent excretory protein synaptotagmin1.
|
|
| 23. |
|
|
| 24. |
|
|
| 25. |
|
|
| 26. |
- Gunnarson, Eli, et al.
(författare)
-
Erythropoietin modulation of astrocyte water permeability as a component of neuroprotection.
- 2009
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490. ; 106:5, s. 1602-7
-
Tidskriftsartikel (refereegranskat)abstract
- Disturbed brain water homeostasis with swelling of astroglial cells is a common complication in stroke, trauma, and meningitis and is considered to be a major cause of permanent brain damage. Astroglial cells possess the water channel aquaporin 4 (AQP4). Recent studies from our laboratory have shown that glutamate, acting on group I metabotropic glutamate receptors (mGluRs), increases the permeability of astrocyte AQP4, which, in situations of hypoxia-ischemia, will increase astrocyte water uptake. Here we report that erythropoietin (EPO), which in recent years has emerged as a potent neuro-protective agent, antagonizes the effect of a group I mGluR agonist on astrocyte water permeability. Activation of group I mGluRs triggers fast and highly regular intracellular calcium oscillations and we show that EPO interferes with this signaling event by altering the frequency of the oscillations. These effects of EPO are immediate, in contrast to the neuroprotective effects of EPO that are known to depend upon gene activation. Our findings indicate that EPO may directly reduce the risk of astrocyte swelling in stroke and other brain insults. In support of this conclusion we found that EPO reduced the neurological symptoms in a mouse model of primary brain edema known to depend upon AQP4 water transport.
|
|
| 27. |
- Gunnarson, Eli, et al.
(författare)
-
Identification of a molecular target for glutamate regulation of astrocyte water permeability
- 2008
-
Ingår i: Glia. - : Wiley. - 0894-1491 .- 1098-1136. ; 56:6, s. 587-596
-
Tidskriftsartikel (refereegranskat)abstract
- Astrocytes play a key role for maintenance of brain water homeostasis, but little is known about mechanisms of short-term regulation of astrocyte water permeability. Here, we report that glutamate increases astrocyte water permeability and that the molecular target for this effect is the aquaporin-4 (AQP4) serine 111 residue, which is in a strategic position for control of the water channel gating. The glutamate effect involves activation of group I metabotropic glutamate receptors (mGluR), intracellular calcium release, and activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and nitric oxide synthase (NOS). The physiological impact of our results is underlined by the finding that mGluR activation increases the rate of hypoosmotic tissue swelling in acute rat hippocampal slices. Cerebral ischemia is associated with an excessive release of glutamate, and in postischemic cerebral edema ablation of AQP4 attenuates the degree of damage. Thus, we have identified AQP4 as the molecular target for drugs that may attenuate the development of brain edema.
|
|
| 28. |
- Gunnarson, E, et al.
(författare)
-
Regulation of brain aquaporins
- 2004
-
Ingår i: Neuroscience. - : Elsevier BV. - 0306-4522 .- 1873-7544. ; 129:4, s. 947-955
-
Tidskriftsartikel (refereegranskat)abstract
- Emerging evidence suggests that brain aquaporins (AQP) play important roles for the dynamic regulation of brain water homeostasis and for the regulation of cerebrospinal fluid production. This review deals with the short- and long-term regulation of AQP4 and AQP9, both expressed in astrocytes, and of AQP1, expressed in the choroid plexus. AQP1 and 4 have in other cell types been shown to be regulated by phosphorylation. Phosphorylation affects the gating of AQP4 and the trafficking and insertion into membrane of AQP1. Mercury inhibits the water permeability of AQP1 and AQP9, but not AQP4. The permeability of AQP4 is increased by lead. AQP4 is also regulated by protein-protein interaction. The assembly between AQP4 and syntrophin is required for the proper localization of AQP4 in the astrocyte plasma membrane that faces capillaries. There is evidence from studies on peripheral tissues that steroid hormones regulate the expression of AQP1, AQP4 and AQP9. There is also evidence that the expression of AQP1 can be regulated by ubiquitination, and that osmolality can regulate the expression of AQP1, AQP4 and AQP9. Further insight into the mechanisms by which brain AQPs are regulated will be of utmost clinical importance, since perturbed water flow via brain AQPs has been implicated in many neurological diseases and since, in brain edema, water flow via AQP4 may have a harmful effect.
|
|
| 29. |
|
|
| 30. |
- Illarionava, Nina B., et al.
(författare)
-
Role of Na,K-ATPase alpha 1 and alpha 2 Isoforms in the Support of Astrocyte Glutamate Uptake
- 2014
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:6, s. e98469-
-
Tidskriftsartikel (refereegranskat)abstract
- Glutamate released during neuronal activity is cleared from the synaptic space via the astrocytic glutamate/Na+ co-transporters. This transport is driven by the transmembrane Na+ gradient mediated by Na,K-ATPase. Astrocytes express two isoforms of the catalytic Na, K-ATPase alpha subunits; the ubiquitously expressed alpha 1 subunit and the alpha 2 subunit that has a more specific expression profile. In the brain alpha 2 is predominantly expressed in astrocytes. The isoforms differ with regard to Na+ affinity, which is lower for alpha 2. The relative roles of the alpha 1 and alpha 2 isoforms in astrocytes are not well understood. Here we present evidence that the presence of the alpha 2 isoform may contribute to a more efficient restoration of glutamate triggered increases in intracellular sodium concentration [Na+](i). Studies were performed on primary astrocytes derived from E17 rat striatum expressing Na, K-ATPase alpha 1 and alpha 2 and the glutamate/Na+ co-transporter GLAST. Selective inhibition of alpha 2 resulted in a modest increase of [Na+](i) accompanied by a disproportionately large decrease in uptake of aspartate, an indicator of glutamate uptake. To compare the capacity of alpha 1 and alpha 2 to handle increases in [Na+](i) triggered by glutamate, primary astrocytes overexpressing either alpha 1 or alpha 2 were used. Exposure to glutamate 200 mM caused a significantly larger increase in [Na+](i) in alpha 1 than in alpha 2 overexpressing cells, and as a consequence restoration of [Na+](i), after glutamate exposure was discontinued, took longer time in alpha 1 than in alpha 2 overexpressing cells. Both alpha 1 and alpha 2 interacted with astrocyte glutamate/Na+ co-transporters via the 1st intracellular loop.
|
|
| 31. |
|
|
| 32. |
- Kamali-Zare, Padideh, 1980-, et al.
(författare)
-
Diffusion limited space contributes to K+ siphoning by regulation of K+ and water homeostasis in astrocytes
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Diffusion Limited Space (DLS) is defined as a region where diffusion is limited by the geometry. Two examples of DLS in the brain are the neuronal synapse, and the narrow region between astrocyte endfeet and blood capillaries. In a series of geometrical models we show that DLS plays a role in regulation of water and K+ homeostasis in the brain by an indirect functional coupling of aquaporins (AQPs) and inward rectifying K+ (Kir) channels in a membrane microdomain. 1. Simulations in geometrical models of a synapse region show that following a step increase in synaptic [K+], both K+ and water are taken up by astrocytes via AQPs and Kir channels lining the synapse. This uptake creates a transient depletion of water in the synapse region that, enhanced by the DLS, facilitates K+ uptake and an efficient clearance of excess K+ from the synapse. 2. Simulations in a geometrical model of astrocytes show that the DLS formed between astrocyte endfeet and blood capillaries, facilitate the siphoning of accumulated K+ into the extracellular space facing the blood capillaries. The DLS geometry creates an efficient coupling between AQPs and Kir channels. 3. Furthermore, the models show that a local coupling between water and K+ transport is important for the maintenance of membrane potential and the net K+ spatial buffering capacity in the astrocytes. 4. In the full geometrical model of K+ spatial buffering we show that the geometry of the extracellular space both in the synapse region and in the endfeet is an essential component for the cell volume regulation. Our results suggest that for regulation of K+ and water homeostasis in astrocytes, not only the classical aspects of functional couplings between proteins, but also the geometry of the cell and the microdomains are important. Further, our results suggest a central role for AQPs in the astrocyte endfeet and identify their contribution to K+ siphoning.
|
|
| 33. |
|
|
| 34. |
|
|
| 35. |
- Kowalewski, Jacob, 1978-
(författare)
-
Modeling and Data Analysis in Cellular Biophysics
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular biophysics deals with the physical aspects of cell biology. This thesis presents a number of studies where mathematical models and data analysis can increase our understanding of this field. During recent years development in experimental methods and mathematical modeling have driven the amount of data and complexity in our understanding of cellular biology to a new level. This development has made it possible to describe cellular systems quantitatively where only qualitative descriptions were previously possible. To deal with the complex data and models that arise in this kind of research a combination of tools from physics and cell biology has to be applied; this constitutes a field we call cellular biophysics. The aim of this doctoral thesis is to develop novel approaches in this field. I present eight studies where quantitative modeling and analysis are involved. The first two studies concern cells interacting with their surrounding environment in the kidney. These cells sense fluid flow and respond with calcium (Ca2+) signals. The interaction between fluid and cells in renal tubular epithelium can be described by biomechanical models. This thesis describes a mathematical model of flow sensing by cilia with focus on the flow frequency response and time delay between the mechanical stress and the Ca2+ signaling response. Intracellular Ca2+ is kept at a very low level compared to the extracellular environment, while several intracellular compartments have higher Ca2+ concentration than the cytoplasm. This makes Ca2+ an efficient messenger for intracellular signaling, the process whereby signals are transduced from an extracellular stimulus to an intracellular activity such as gene expression. An important type of Ca2+ signaling is oscillations in intracellular Ca2+ concentration which occur due to the concerted interplay between different transport mechanisms within a cell. A study in this thesis examines ways to explain these mechanisms in terms of a mathematical model. Another study in the thesis reports that erythropoietin can regulate the water permeability of astrocytes and that it alters the pattern of Ca2+ oscillations in astrocytes. In this thesis the analysis of this Ca2+ signaling is described. Simulations described in one of the studies show how different geometries can affect the fluorescence recovery and that geometrically constrained reactions can trap diffusing receptors in dendritic spines. When separate time scales are present in a fluorescence revovery after photobleaching (FRAP) experiment the reaction and diffusion components can be studied separately. Applying single particle tracking methods to the migration trajectories of natural killer cells shows that there is a correlation between the formation of conjugates and transient confinement zones (TCZs) in these trajectories in vitro. TCZs are also present in in vivo experiments where they show strong similarities with the in vitro situation. This approach is a novel concept in data analysis methods for tracking immune cells.
|
|
| 36. |
|
|
| 37. |
|
|
| 38. |
- Li, Juan, et al.
(författare)
-
Ouabain protects against adverse developmental programming of the kidney
- 2010
-
Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 1, s. 42-
-
Tidskriftsartikel (refereegranskat)abstract
- The kidney is extraordinarily sensitive to adverse fetal programming. Malnutrition, the most common form of developmental challenge, retards the formation of functional units, the nephrons. The resulting low nephron endowment increases susceptibility to renal injury and disease. Using explanted rat embryonic kidneys, we found that ouabain, the Na, K-ATPase ligand, triggers a calcium-nuclear factor-kappa B signal, which protects kidney development from adverse effects of malnutrition. To mimic malnutrition, kidneys were serum deprived for 24 h. This resulted in severe retardation of nephron formation and a robust increase in apoptosis. In ouabain-exposed kidneys, no adverse effects of serum deprivation were observed. Proof of principle that ouabain rescues development of embryonic kidneys exposed to malnutrition was obtained from studies on pregnant rats given a low-protein diet and treated with ouabain or vehicle throughout pregnancy. Thus, we have identified a survival signal and a feasible therapeutic tool to prevent adverse programming of kidney development.
|
|
| 39. |
- Li, Yanhong, et al.
(författare)
-
Urinary aquaporin-2 excretion during ibuprofen or indomethacin treatment in preterm infants with patent ductus arteriosus
- 2011
-
Ingår i: Acta Paediatrica. - : Wiley-Blackwell. - 0803-5253 .- 1651-2227. ; 100:1, s. 59-66
-
Tidskriftsartikel (refereegranskat)abstract
- Aim: Water channel AQP2 is the target for vasopressin (AVP) and a major determinant of urinary concentrating capacity. In mature kidneys, prostaglandins counteract the effect of AVP on AQP2 expression at functional sites. We investigated whether disturbances in water homeostasis in infants with patent ductus arteriosus (PDA) treated with prostaglandin inhibitors can be attributed to activation of AQP2. Methods: In 53 infants with symptomatic PDA (gestational age 24-33 weeks), 30 receiving ibuprofen and 23 indomethacin starting at 2-15 days of life, clinical and biochemical data were collected before treatment and after each dose of the drugs. Urinary AQP2 was determined by dot immunoblotting. Results: Urinary AQP2 level and osmolality were decreased in both groups. Urinary osmolality was overall low and correlated inversely with fluid uptake. In ibuprofen group, there was no correlation of AQP2 level with urinary osmolality. Conclusion: There was no AQP2 upregulation in the infants. The low urinary osmolality and dissociation between urinary osmolality and urinary AQP2 level indicate that the fluid retention sometimes observed in PDA infants treated with prostaglandin inhibitors is not caused by increased levels of functional AQP2. Thus, knowledge about the renal physiology of the adult cannot always be transferred to the infant kidney.
|
|
| 40. |
- Liebmann, Thomas, et al.
(författare)
-
A Noncanonical Postsynaptic Transport Route for a GPCR Belonging to the Serotonin Receptor Family
- 2012
-
Ingår i: Journal of Neuroscience. - 0270-6474 .- 1529-2401. ; 32:50, s. 17998-18008
-
Tidskriftsartikel (refereegranskat)abstract
- Postsynaptic receptor trafficking plays an essential role in tuning neurotransmission and signal plasticity and has emerged as a potential therapeutic target in neuropsychiatric disease. Using a novel application of fluorescence recovery after photobleaching in rat hippocampal neurons, we examined transport from the soma to dendrites of seven G-protein-coupled receptors (GPCRs) implicated in mood disorders. Most GPCRs were delivered to dendrites via lateral diffusion, but one GPCR, the serotonin 1B receptor (5-HT1B), was delivered to the dendrites in secretory vesicles. Within the dendrites, 5-HT1B were stored in a reservoir of accessible vesicles that were recruited to preferential sites in plasma membrane, as observed with superecliptic pHluorin labeling. After membrane recruitment, 5-HT1B transport via lateral diffusion and temporal confinement to inhibitory and excitatory synapses was monitored by single particle tracking. These results suggest an alternative mechanism for control of neuronal activity via a GPCR that has been implicated in mood regulation.
|
|
| 41. |
- Liebmann, Thomas, et al.
(författare)
-
Regulation of Neuronal Na,K-ATPase by Extracellular Scaffolding Proteins
- 2018
-
Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 19:8
-
Tidskriftsartikel (refereegranskat)abstract
- Neuronal activity leads to an influx of Na+ that needs to be rapidly cleared. The sodium-potassium ATPase (Na,K-ATPase) exports three Na+ ions and imports two K+ ions at the expense of one ATP molecule. Na,K-ATPase turnover accounts for the majority of energy used by the brain. To prevent an energy crisis, the energy expense for Na+ clearance must provide an optimal effect. Here we report that in rat primary hippocampal neurons, the clearance of Na+ ions is more efficient if Na,K-ATPase is laterally mobile in the membrane than if it is clustered. Using fluorescence recovery after photobleaching and single particle tracking analysis, we show that the ubiquitous alpha 1 and the neuron-specific alpha 3 catalytic subunits as well as the supportive beta 1 subunit of Na,K-ATPase are highly mobile in the plasma membrane. We show that cross-linking of the beta 1 subunit with polyclonal antibodies or exposure to Modulator of Na,K-ATPase (MONaKA), a secreted protein which binds to the extracellular domain of the beta subunit, clusters the alpha 3 subunit in the membrane and restricts its mobility. We demonstrate that clustering, caused by cross-linking or by exposure to MONaKA, reduces the efficiency in restoring intracellular Na+. These results demonstrate that extracellular interactions with Na,K-ATPase regulate the Na+ extrusion efficiency with consequences for neuronal energy balance.
|
|
| 42. |
- Liu, Xiao, et al.
(författare)
-
Ankyrin B Modulates the Function of Na,K-ATPase/Inositol 1,4,5-Trisphosphate Receptor Signaling Microdomain
- 2008
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:17, s. 11461-11468
-
Tidskriftsartikel (refereegranskat)abstract
- Na, K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappa B activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na, K-ATPase/ IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na, K-ATPase and IP3R. We identify the N terminus tail of the Na, K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na, K-ATPase and IP3R, reduced the number of cells responding to pM doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappa B. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na, K-ATPase and no effect on the distribution and apparent mobility of Na, K-ATPase in the plasma membrane.
|
|
| 43. |
- Nejsum, Lene N., et al.
(författare)
-
Bidirectional regulation of AQP2 trafficking and recycling : involvement of AQP2-S256 phosphorylation
- 2005
-
Ingår i: American Journal of Physiology: Renal Physiology. - : American Physiological Society. - 1931-857X .- 1522-1466. ; 288:5, s. F930-F938
-
Tidskriftsartikel (refereegranskat)abstract
- The present study examined the role of PKA and serine256 (S256) phosphorylation for AQP2 trafficking and recycling using cells transfected with wild-type AQP2 (AQP2-WT) or mutant AQP2 and high-resolution confocal microscopic techniques. In transiently transfected MDCK-C7 cells, stimulation with forskolin induced translocation of AQP2-WT to the plasma membrane. Treatment of AQP2-WT cells with the PKA inhibitor H-89 following forskolin stimulation resulted in internalization of AQP2-WT. Moreover, H-89 treatment of AQP2-S256D (mimicking constitutively phosphorylated AQP2 and hence localized to the plasma membrane) resulted in redistribution of AQP2-S256D to intracellular vesicles, even in the presence of forskolin. Both PGE2 and dopamine stimulation induced endocytosis of AQP2-WT and AQP2-S256D, respectively, in forskolin-stimulated cells. Consistent with this, dopamine in the presence of vasopressin stimulated endocytosis of AQP2 in slices of rat kidney inner medulla without substantial dephosphorylation. In conclusion, these results strongly suggest that 1) S256 phosphorylation is necessary but not sufficient for AQP2 plasma membrane expression, 2) active PKA is required for AQP2 plasma membrane expression, 3) PGE2 and dopamine induce internalization of AQP2 independently of AQP2 dephosphorylation, and 4) preceding activation of cAMP production is necessary for PGE2 and dopamine to cause AQP2 internalization.
|
|
| 44. |
|
|
| 45. |
- Nilsson, Linnéa, et al.
(författare)
-
Prompt apoptotic response to high glucose in SGLT expressing cells
- 2019
-
Ingår i: American Journal of Physiology - Renal Physiology. - : American Physiological Society. - 1931-857X .- 1522-1466. ; 316:5, s. F1078-F1089
-
Tidskriftsartikel (refereegranskat)abstract
- It is generally believed that cells that are unable to downregulate glucose transport are particularly vulnerable to hyperglycemia. Yet little is known about the relation between expression of glucose transporters and acute toxic effects of high glucose exposure.Here we have, in an ex vivo study on rat renal cells, compared the apoptotic response to a moderate increase in glucose concentration. We have studied the cell types that commonly are targeted in diabetic kidney disease (DKD): proximal tubule cells (PTC) that express SGLT2, mesangial cells (MC) that express SGLT1, and podocytes that lack SGLT and take up glucose via the insulin dependent GLUT4.PTC and MC responded within 4-8 h exposure to 15 mM glucose with translocation of the apoptotic protein Bax to mitochondria and increased apoptotic index. SGLT down-regulation and exposure to SGLT inhibitors abolished the apoptotic response. Onset of overt DKD generally coincides with onset of albuminuria. Albumin had an additive effect on the apoptotic response. Ouabain, which interferes with apoptotic onset, rescued from the apoptotic response. Insulin supplemented podocytes remained resistant to 15 and 30 mM glucose for at least 24 h.Our study points to a previously unappreciated role of SGLT dependent glucose uptake as a risk-factor for diabetic complications and highlights the importance of therapeutic approaches that specifically target the different cell types in DKD.
|
|
| 46. |
- Nilsson, Linnéa
(författare)
-
Studies on the Role of Apoptosis in Kidney Diseases
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Apoptosis is one of the most common types of cell death. Under physiological conditions, it plays an essential role in removal of damaged and potentially harmful cells. Excessive apoptosis has however been linked to a number of diseases including proteinuric kidney disease and DKD, and is believed to enhance the disease progression. Albuminuria and hyperglycemia are common symptoms of these diseases and albumin and high glucose have been seen to trigger intrinsic apoptosis in renal cells. Ouabain, a cardiotonic steroid, has previously been identified as an antiapoptotic agent that in subsaturating concentrations protect from intrinsic apoptosis. The mechanism of the protective effect of ouabain is still not fully understood and it remains to be concluded whether ouabain can protect from albumin and/or glucotoxic-triggered apoptosis.In study I we investigated the protective effects of ouabain in albumin-exposed primary rat PTC and podocytes and in the proteinuric kidney disease animal model passive Heymann nephritis. By reestablishing the balance between the proapoptotic protein BAX and the antiapoptotic protein BCL-XL, ouabain averted the albumin-triggered apoptosis in vitro and in vivo and protected from podocytes loss and glomerular-tubular disconnection.In study II we investigated the relationship between the glucose transporters renal cells express and their susceptibility of glucotoxic-triggered apoptosis. We identified the SGLT expressing cells, PTC and MC, to be more susceptible to high glucose-induced apoptosis than cells without SGLT. The apoptosis was mediated by BAX and BCL-XL imbalance and mitochondrial dysfunction, and was abolished when treated with ouabain or SGLT inhibitors. Podocytes, which lack SGLT, did not respond to short-term high glucose exposure.In study III we used super-resolution microscopy to investigate at which stage of the apoptotic process ouabain start to intervene. Ouabain interfered early in the apoptotic process, where it prevented activation of the sensitizer protein BAD. This allowed BCL-XL to avert BAX activation and translocation to mitochondria and thereby protected from mitochondrial dysfunction and apoptosis.In study IV we investigated differentially expressed genes between renal cortex and primary short-term PTC cultures and between PTC exposed to control and high glucose. The mRNA expression level of most genes was significantly up- or downregulated in PTC compared to renal cortex, with the biggest differences in mitochondria and metabolism related genes. Early state glucotoxicity did not significantly alter mRNA expression levels in PTC.
|
|
| 47. |
- Panizza, Elena, et al.
(författare)
-
Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival
- 2019
-
Ingår i: The FASEB Journal. - : FEDERATION AMER SOC EXP BIOL. - 0892-6638 .- 1530-6860. ; 33:9, s. 10193-10206
-
Tidskriftsartikel (refereegranskat)abstract
- The ion pump Na+, K+-ATPase (NKA) is a receptor for the cardiotonic steroid ouabain. Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. However, it is controversial whether ouabain-bound NKA is considered a signal transducer. To address this question, we performed a global analysis of protein phosphorylation in COS-7 cells, identifying 2580 regulated phosphorylation events on 1242 proteins upon 10- and 20-min treatment with ouabain. Regulated phosphorylated proteins include the inositol triphosphate receptor and stromal interaction molecule, which are essential for initiating calcium oscillations. Hierarchical clustering revealed that ouabain triggers a structured phosphorylation response that occurs in a well-defined, time-dependent manner and affects specific cellular processes, including cell proliferation and cell-cell junctions. We additionally identify regulation of the phosphorylation of several calcium and calmodulin-dependent protein kinases (CAMKs), including 2 sites of CAMK type II-gamma (CAMK2G), a protein known to regulate apoptosis. To verify the significance of this result, CAMK2G was knocked down in primary kidney cells. CAMK2G knockdown impaired ouabain-dependent protection from apoptosis upon treatment with high glucose or serum deprivation. In conclusion, we establish NKA as the coordinator of a broad, tightly regulated phosphorylation response in cells and define CAMK2G as a downstream effector of NKA.-Panizza, E., Zhang, L., Fontana, J. M., Hamada, K., Svensson, D., Akkuratov, E. E., Scott, L., Mikoshiba, K., Brismar, H., Lehtio, J., Aperia, A. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival.
|
|
| 48. |
- Ringman, Andreas, et al.
(författare)
-
NKCC-1 and ENaC are down-regulated in nitrofen-induced hypoplastic lungs with congenital diaphragmatic hernia
- 2008
-
Ingår i: Pediatric surgery international (Print). - : Springer Science and Business Media LLC. - 0179-0358 .- 1437-9813. ; 24:9, s. 993-1000
-
Tidskriftsartikel (refereegranskat)abstract
- Congenital diaphragmatic hernia (CDH) is accompanied by pulmonary hypoplasia and pulmonary hypertension. Fetal lung growth is dependent on the secretion of lung liquid, which normally is absorbed at partus. The ion channel NKCC-1 is involved in this secretory process, but has recently also been reported to be implicated in absorption. CDH patients show a disturbed transition from secretion to absorption. alpha- and beta-ENaC are essential for lung liquid absorption. Common for all transcellular ion transport is the need for Na/K-ATPase as a primary driving force. The aim of the study was first to map the normal pulmonary expression of the above proteins during late gestation and secondly to see if the expression was affected in a CDH rat model. Pregnant Sprague-Dawley rat dams were given nitrofen on gestational day 9.5 to induce CDH. The fetuses were removed on gestational days E18 and E21. In addition, newborn rats were harvested postpartum on day P2. The fetuses were put into one of two groups: hypoplastic lungs without CDH (N-CDH) and hypoplastic lungs with CDH (N+CDH). The pulmonary expression of NKCC-1, alpha-/beta-ENaC and Na/K-ATPase was then analyzed using Western blot. We found that the protein levels of NKCC-1 on gestational days E18 and E21 were significantly lower among fetuses with N+CDH as well as N-CDH compared to controls. The expression of beta-ENaC was also significantly down-regulated in both the groups on E18 and E21. The protein levels of alpha-ENaC and Na/K-ATPase were not found to be significantly decreased, but both showed a tendency towards down-regulation. The marked down-regulation of NKCC-1 in fetal hypoplastic lungs with CDH indicates a possibly decreased lung liquid production. This may be one of the mechanisms behind the disturbed pulmonary development in CDH. We also show that beta-ENaC is down-regulated. Down-regulation of beta-ENaC may result in abnormal lung liquid absorption, which could be one of the mechanisms behind the respiratory distress seen in CDH patients postpartum.
|
|
| 49. |
- Ringman Uggla, Andreas, et al.
(författare)
-
Low pulmonary expression of epithelial Na(+) channel and Na(+), K(+)-ATPase in newborn infants with congenital diaphragmatic hernia
- 2011
-
Ingår i: Neonatology. - : S. Karger AG. - 1661-7800 .- 1661-7819. ; 99:1, s. 14-22
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: It has been suggested from several animal studies and clinical observations that congenital diaphragmatic hernia (CDH) with pulmonary hypoplasia is accompanied by a disturbed perinatal ion transport. This could lead to respiratory distress due to slower clearance of fetal lung fluid at birth. OBJECTIVES: The purpose of this study was to determine whether CDH is related to changes in the expression of three rate-limiting transporter proteins in lung epithelium at birth. METHODS: Tracheal aspirate was collected from 12 newborn infants with CDH and from 8 newborn control patients. Sampling was performed at postnatal age 18 and at 43 h in the CDH group and at 18 h in the control group. The protein abundance of α-, β- and γ-epithelial Na(+) channel (ENaC), aquaporin 5 and Na(+), K(+)-ATPase α(1) was analyzed using semiquantitative immunoblotting. RESULTS: The levels of β-ENaC, γ-ENaC and Na(+), K(+)-ATPase α(1) collected at 18 h postnatally were significantly lower in CDH infants compared to control infants. In the CDH group, no significant difference in the expression of the ENaC subunits, Na(+), K(+)-ATPase α(1) or aquaporin 5 could be detected between the two sampling time points. CONCLUSIONS: This downregulation may result in an abnormal lung fluid absorption which could be an important mechanism behind the respiratory distress seen in newborn CDH patients.
|
|
| 50. |
- Scott, Lena, et al.
(författare)
-
Allosteric changes of the NMDA receptor trap diffusible dopamine 1 receptors in spines
- 2006
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 103:3, s. 762-767
-
Tidskriftsartikel (refereegranskat)abstract
- The dopaminergic and glutamatergic systems interact to initiate and organize normal behavior, a communication that may be perturbed in many neuropsychiatric diseases, including schizophrenia. We show here that NMDA, by allosterically modifying NMDA receptors, can act as a scaffold to recruit laterally diffusing dopamine D1 receptors (D1R) to neuronal spines. Using organotypic culture from rat striatum transfected with D1R fused to a fluorescent protein, we show that the majority of dendritic D1R are in lateral diffusion and that their mobility is confined by interaction with NMDA receptors. Exposure to NMDA reduces the diffusion coefficient for D1R and causes an increase in the number of D1R-positive spines. Unexpectedly, the action of NMDA in potentiating D1R recruitment was independent of calcium flow via the NMDA receptor channel. Thus, a highly energy-efficient, diffusion-trap mechanism can account for intraneuronal interaction between the glutamatergic and dopaminergic systems and for regulation of the number of D1R-positive spines. This diffusion trap system represents a molecular mechanism for brain plasticity and offers a promising target for development of antipsychotic therapy
|
|
