Cyclosporine A up-regulates angiotensin II receptors and calcium responses in human vascular smooth muscle cells.
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BACKGROUND: The most widely used immunosuppressive drug for preventing graft rejection and treating autoimmune diseases is currently cyclosporine A (CsA). However, CsA also causes vasoconstriction, which is considered to be at the origin of CsA-induced nephrotoxicity and hypertension. To evaluate the cellular basis for these side effects, we studied the influence of CsA on the regulation of the free cytosolic Ca2+ concentration ([Ca2+]c) in cultured human vascular smooth muscle cells (SMCs). METHODS: SMCs were isolated from the medial layer of human aorta. [Ca2+]c regulation was studied by fluorimetry with fura 2 and by measuring 45Ca2+ effluxes. Angiotensin II (Ang II) receptors were detected by [125I]Ang II binding. RESULTS: Pretreatment of human SMCs for 24 hours with CsA in its therapeutic concentration range (0. 1 to 10.0 microM) had no effect on basal [Ca2+]c, but increased the [Ca2+]c elevation and 45Ca2+ efflux when cells were stimulated with Ang II. Half-maximal effects occurred at approximately 1 microM CsA. The CsA effects on [Ca2+]c were accompanied by a nearly twofold increase in Ang II receptor number, whereas no change in affinity to Ang II was observed. CsA did not alter endothelin-1- or thapsigargin-induced 45Ca2+ efflux. Increases in both Ca2+ responses and [125I]Ang II binding were attenuated by the transcriptional inhibitor actinomycin D. The effects of CsA did not appear to be mediated by calcineurin inhibition because cyclosporine H, which is not immunosuppressive, also increased the Ang II-induced 45Ca2+ efflux. CONCLUSION: These data suggest that CsA preferentially up-regulates the transcription of Ang II receptors, which very likely leads to vasoconstriction in vivo and could be at the origin of CsA-induced hypertension and nephrotoxicity in humans. (+info)
Effects of pH on kinetic parameters of the Na-HCO3 cotransporter in renal proximal tubule.
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The effects of pH on cotransporter kinetics were studied in renal proximal tubule cells. Cells were grown to confluence on permeable support, mounted in an Ussing-type chamber, and permeabilized apically to small monovalent ions with amphotericin B. The steady-state, dinitrostilbene-disulfonate-sensitive current (DeltaI) was Na+ and HCO3- dependent and therefore was taken as flux through the cotransporter. When the pH of the perfusing solution was changed between 6.0 and 8.0, the conductance attributable to the cotransporter showed a maximum between pH 7.25 and pH 7.50. A similar profile was observed in the presence of a pH gradient when the pH of the apical solutions was varied between 7.0 and 8.0 (basal pH lower by 1), but not when the pH of the basal solution was varied between 7.0 and 8.0 (apical pH lower by 1 unit). To delineate the kinetic basis for these observations, DeltaI-voltage curves were obtained as a function of Na+ and HCO3- concentrations and analyzed on the basis of a kinetic cotransporter model. Increases in pH from 7.0 to 8.0 decreased the binding constants for the intracellular and extracellular substrates by a factor of 2. Furthermore, the electrical parameters that describe the interaction strength between the electric field and substrate binding or charge on the unloaded transporter increased by four- to fivefold. These data can be explained by a channel-like structure of the cotransporter, whose configuration is modified by intracellular pH such that, with increasing pH, binding of substrate to the carrier is sterically hindered but electrically facilitated. (+info)
Genistein inhibits the regulation of active sodium-potassium transport by dopaminergic agonists in nonpigmented ciliary epithelium.
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PURPOSE: To determine whether dopamine receptor stimulation regulates Na,K-ATPase-mediated ion transport in cultured nonpigmented ciliary epithelium (NPE). METHODS: Using a rabbit NPE cell line, active Na-K transport activity was determined by measuring ouabain-sensitive potassium (86Rb) uptake in cell monolayers. Western blot analysis of membrane material obtained from cell homogenates was conducted to examine tyrosine phosphorylation of membrane proteins. RESULTS: Ouabain-sensitive potassium (86Rb) uptake was inhibited in the presence of either dopamine or the D1-selective agonist SKF82958. The response was suppressed by SCH23390, a D1 antagonist, but not by sulpiride, a D2-selective antagonist. Quinpirole, a D2-selective agonist, did not cause inhibition of ouabain-sensitive potassium (86Rb) uptake. Cyclic adenosine monophosphate (cAMP) was detectably increased in SKF82958-treated cells, although the concentration of SKF required to elevate cell cAMP was higher than the concentration needed to inhibit ouabain-sensitive potassium (86Rb) uptake. The protein kinase A inhibitor H89 prevented the 86Rb uptake response to SKF82958. Genistein, an inhibitor of tyrosine kinases, also prevented the 86Rb uptake response to SKF82958. Membrane material isolated from cells exposed to SKF82958 showed an increase in the density of several phosphotyrosine bands. These changes in phosphotyrosine immunoblot density were not observed in material isolated from cells that received either genistein or SCH23390 before SKF82958 treatment. CONCLUSIONS: The results of this study suggest D1 agonists cause a reduction of Na,K-ATPase-mediated ion transport by a mechanism that could involve a tyrosine kinase step. (+info)
Mild spherocytosis and altered red cell ion transport in protein 4. 2-null mice.
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Protein 4.2 is a major component of the red blood cell (RBC) membrane skeleton. We used targeted mutagenesis in embryonic stem (ES) cells to elucidate protein 4.2 functions in vivo. Protein 4. 2-null (4.2(-/-)) mice have mild hereditary spherocytosis (HS). Scanning electron microscopy and ektacytometry confirm loss of membrane surface in 4.2(-/-) RBCs. The membrane skeleton architecture is intact, and the spectrin and ankyrin content of 4. 2(-/-) RBCs are normal. Band 3 and band 3-mediated anion transport are decreased. Protein 4.2(-/-) RBCs show altered cation content (increased K+/decreased Na+)resulting in dehydration. The passive Na+ permeability and the activities of the Na-K-2Cl and K-Cl cotransporters, the Na/H exchanger, and the Gardos channel in 4. 2(-/-) RBCs are significantly increased. Protein 4.2(-/-) RBCs demonstrate an abnormal regulation of cation transport by cell volume. Cell shrinkage induces a greater activation of Na/H exchange and Na-K-2Cl cotransport in 4.2(-/-) RBCs compared with controls. The increased passive Na+ permeability of 4.2(-/-) RBCs is also dependent on cell shrinkage. We conclude that protein 4.2 is important in the maintenance of normal surface area in RBCs and for normal RBC cation transport. (+info)
Uptake of bromosulfophthalein via SO2-4/OH- exchange increases the K+ conductance of rat hepatocytes.
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In confluent primary cultures of rat hepatocytes, micromolar concentrations of bromosulfophthalein (BSP) lead to a sizeable hyperpolarization of membrane voltage. The effect is a saturable function of BSP concentration yielding an apparent value of 226 micromol/l and a Vmax of -10.3 mV. The BSP-induced membrane hyperpolarization is inhibited by the K+ channel blocker Ba2+, and in cable-analysis and ion-substitution experiments it becomes evident that the effect is due to a significant increase in cell membrane K+ conductance. Voltage changes were attenuated by the simultaneous administration of SO2-4, succinate, and cholate (cis-inhibition) and increased after preincubation with SO2-4 and succinate (trans-stimulation), suggesting that the effect occurs via BSP uptake through the known SO2-4/OH- exchanger. Microfluorometric measurements reveal that BSP-induced activation of K+ conductance is not mediated by changes in cell pH, cell Ca2+, or cell volume. It is concluded that K+ channel activation by BSP (as well as by DIDS and indocyanine green) may reflect a physiological mechanism linking the sinusoidal uptake of certain anions to their electrogenic canalicular secretion. (+info)
Chemoattractant- and mitogen-induced generation of reactive oxygen species in human lymphocytes: the role of calcium.
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This study examined the role of calcium in the generation of reactive oxygen species (ROS) in human lymphocytes activated by the chemoattractant formyl-Met-Leu-Phe (fMLP) and the T-cell mitogen phytohaemagglutinin (PHA). The concentrations of cytosolic calcium ([Ca2+]i) and ROS were monitored simultaneously with a fluorescence spectrophotometer after the cells had been incubated in fura-2 (calcium-sensitive dye) and 2',7'-dichlorofluorescein diacetate (DCF-DA, ROS-sensitive dye). The lymphocytes were stimulated with fMLP (200 nmol l-1) or PHA (10 micromol l-1) in the absence and presence of extracellular calcium. A dose-response test was also conducted for extracellular calcium. fMLP and PHA significantly increased both [Ca2+]i (P < 0.001) and ROS concentrations (P < 0. 001) above the control levels in the presence of extracellular calcium. However, such increases were abolished in the absence of extracellular calcium, suggesting total dependence of the responses to both fMLP and PHA on transplasma-membrane calcium influx. There were also graded increases in ROS with increasing concentrations of extracellular calcium. The results show that transplasma-membrane calcium influx is essential for fMLP- and PHA-induced generation of reactive oxygen species in human lymphocytes. (+info)
The role of Na+-H+ exchange in fluid and solute transport in the rat efferent ducts.
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In vivo microperfusion techniques were used to investigate the role of Na+-H+ exchange in the efferent ducts of the rat. Individual efferent ducts were perfused with a Krebs-Ringer bicarbonate solution (KRB) containing 0, 1, 3, 5 or 7.5 mM amiloride. Concentrations of 1-5 mM amiloride inhibited fluid reabsorption from the efferent ducts in a linear dose-dependent manner with an apparent Km of 3 mM. Inhibition was maximal at 5 mM with reabsorption reduced by about 70 %. The effects of amiloride were completely reversible and there was little effect of amiloride on luminal osmolality and concentrations of Na+, Cl- or K+. It is concluded that Na+-H+ exchange is one of the principal mechanisms responsible for fluid and electrolyte reabsorption in the efferent ducts and offers a means by which the efferent ducts are able to achieve flow-dependent, autoregulated fluid reabsorption. (+info)
The extracellular matrix, interstitial fluid and ions as a mechanical signal transducer in articular cartilage.
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OBJECTIVE: (1) Provide an overview of the biomechanical factors that are required to analyze and interpret biological data from explant experiments; (2) Present a description of some of the mechano-electrochemical events which occur in cartilage explants during loading. DESIGN: A thorough and provocative discussion on the effects of loading on articular cartilage will be presented. Five simplest loading cases are considered: hydrostatic pressure, osmotic pressure, permeation (pressure loading), confined compression and unconfined compression. Details of how such surface loadings are converted or transduced by the extracellular matrix (ECM) to pressure, fluid, solute and ion flows, deformation and electrical fields are discussed. RESULTS: Similarities and differences in these quantities for the five types of loading are specifically noted. For example, it is noted that there is no practical mechanical loading condition that can be achieved in the laboratory to produce effects that are equal to the effects of osmotic pressure loading within the ECM. Some counter-intuitive effects from these loadings are also described. Further, the significance of flow-induced compression of the ECM is emphasized, since this frictional drag effect is likely to be one of the major effects of fluid flow through the porous-permeable ECM. Streaming potentials arising from the flow of ions past the fixed charges of the ECM are discussed in relation to the flow-induced compaction effect as well. CONCLUSION: Understanding the differences among these explant loading cases is important; it will help to provide greater insights to the mechano-electrochemical events which mediate metabolic responses of chondrocytes in explant loading experiments. (+info)