Diego de la Rosa

The Serum and Glucocorticoid Kinase sgk Increases the Abundance of Epithelial Sodium Channels in the Plasma Membrane of Xenopus Oocytes

The serum- and glucocorticoid-induced kinase (sgk) is a serine and threonine kinase that stimulates amiloride-sensitive sodium transport in Xenopus oocytes. Because aldosterone induces phosphorylation on serine/threonine (Ser/Thr) residues in the carboxyl termini of β and γ subunits of epithelial sodium channels (ENaCs) and causes an increase in thesgk transcript in mammalian and amphibian renal epithelial cells, it seems likely that sgk mediates the action of aldosterone to stimulate sodium transport. Experiments were performed in Xenopus oocytes to determine the mechanism by whichsgk increases sodium conductance by examining its effect on phosphorylation, kinetics, and membrane abundance of ENaC. Our results demonstrate that deletions of the carboxyl termini of the three subunits do not inhibit sgk-induced sodium current, indicating that the effect of sgk is not mediated via phosphorylation within the carboxyl termini of ENaC. They also show no evidence that sgk reduces the removal of ENaC from the plasma membrane because mutations of tyrosine residues in the sequences necessary for endocytosis and degradation did not affect the response to sgk. Further studies performed with the patch-clamp technique indicated that sgk did not increase the open probability or changed the kinetics of ENaC. These studies, however, showed a 3-fold increase in the abundance of ENaC in the plasma membrane in the presence of sgk compared with control. Together, the experiments indicate that sgk stimulates electrogenic sodium transport by increasing the number of ENaCs at the cell surface and suggest that sgk may mediate the early increase in aldosterone-induced sodium current.

Functional implications of the localization and activity of acid-sensitive channels in rat peripheral nervous system

Acid-sensitive ion channels (ASIC) are proton-gated ion channels expressed in neurons of the mammalian central and peripheral nervous systems. The functional role of these channels is still uncertain, but they have been proposed to constitute mechanoreceptors and/or nociceptors. We have raised specific antibodies for ASIC1, ASIC2, ASIC3, and ASIC4 to examine the distribution of these proteins in neurons from dorsal root ganglia (DRG) and to determine their subcellular localization. Western blot analysis demonstrates that all four ASIC proteins are expressed in DRG and sciatic nerve. Immunohistochemical experiments and functional measurements of unitary currents from the ASICs with the patch–clamp technique indicate that ASIC1 localizes to the plasma membrane of small-, medium-, and large-diameter cells, whereas ASIC2 and ASIC3 are preferentially in medium to large cells. Neurons coexpressing ASIC2 and ASIC3 form predominantly heteromeric ASIC2–3 channels. Two spliced forms, ASIC2a and ASIC2b, colocalize in the same population of DRG neurons. Within cells, the ASICs are present mainly on the plasma membrane of the soma and cellular processes. Functional studies indicate that the pH sensitivity for inactivation of ASIC1 is much higher than the one for activation; hence, increases in proton concentration will inactivate the channel. These functional properties and localization in DRG have profound implications for the putative functional roles of ASICs in the nervous system.

Distribution, subcellular localization and ontogeny of ASIC1 in the mammalian central nervous system

The acid‐sensitive ion channel ASIC1 is a proton‐gated ion channel from the mammalian nervous system. Its expression in sensory neurons and activation by low extracellular pH suggest that ASIC is involved in transmitting nociceptive impulses produced by the acidification caused by injury or inflammation. However, ASIC1 expression is not restricted to sensory neurons. To understand the functional role of ASIC1 in the CNS we investigated its expression and subcellular distribution therein. In particular, we examined the presence of ASIC1 in domains where the local pH may drop sufficiently to activate ASIC1 under physiological conditions. Immunostaining with specific antibodies revealed broad expression of ASIC1 in many areas of the adult rat brain including the cerebral cortex, hippocampus and cerebellum. Within cells, ASIC1 was found predominantly throughout the soma and along the branches of axons and dendrites. ASIC1 was not enriched in the microdomains where pH may reach low values, such as in synaptic vesicles or synaptic membranes. Pre‐ or postsynaptic ASIC1 was not gated by synaptic activity in cultured hippocampal neurons. Blockage or desensitization of ASIC1 with amiloride or pH 6.7, respectively, did not modify postsynaptic currents. Finally, the ontogeny of ASIC1 in mouse brain revealed constant levels of expression of ASIC1 protein from embryonic day 12 to the postnatal period, indicating an early and almost constant level of expression of ASIC1 during brain development.

Effects of Aldosterone on Biosynthesis, Traffic, and Functional Expression of Epithelial Sodium Channels in A6 Cells

The collecting duct regulates Na+ transport by adjusting the abundance/activity of epithelial Na+ channels (ENaC). In this study we have investigated the synthesis, degradation, endocytosis, and activity of ENaC and the effects of aldosterone on these processes using endogenous channels expressed in the A6 cell line. Biochemical studies were performed with a newly raised set of specific antibodies against each of the three subunits of the amphibian ENaC. Our results indicate simultaneous transcription and translation of α, β, and γ subunits and enhancement of both processes by aldosterone: two- and fourfold increase, respectively. The biosynthesis of new channels can be followed by acquisition of endoglycosidase H–resistant oligosacharides in α and β subunits and, in the case of α, by the appearance of a form resistant to reducing agents. The half-life of the total pool of subunits (t 1/2 40–70 min) is longer than the fraction of channels in the apical membrane (t 1/2 12–17 min). Aldosterone induces a fourfold increase in the abundance of the three subunits in the apical membrane without significant changes in the open probability, kinetics of single channels, or in the rate of degradation of ENaC subunits. Accordingly, the aldosterone response could be accounted by an increase in the abundance of apical channels due, at least in part, to de novo synthesis of subunits.

A land evaluation decision support system (MicroLEIS DSS) for agricultural soil protection with special reference to the Mediterranean region

Soil protection requires the improvement of agricultural land use, its planning and its management, in order to achieve a sustainable development. Land evaluation analysis is considered an interface between land resources survey and land use planning and management. The application of information and communication technology has exerted an impact on sustainable land use decision support. Since the early 1990s and following this trend, MicroLEIS (Mediterranean Land Evaluation Information System) has evolved towards an agro-ecological decision support system. The original project corresponds to a set of qualitative land evaluation methods converted to PC computer programs to automate their application. Today, MicroLEIS DSS is a set of useful tools for decision-making which in a wide range of agro-ecological schemes. The design philosophy follows a toolkit approach, integrating many software tools: databases, statistics, expert systems, neural networks, Web and GIS applications, and other information technologies. The MicroLEIS DSS system, in English and Spanish simultaneously, is available ready-for-use from the following website address: www.microleis.com. The new, re-designed MicroLEIS DSS will provide considerable opportunities for greater cooperation in interdisciplinary research and in the application of knowledge to solve problems of soil protection. # 2003 Elsevier Ltd. All rights reserved.

The Crucial Role of Chromogranins in Storage and Exocytosis Revealed Using Chromaffin Cells from Chromogranin A Null Mouse

Chromogranins (Cgs) are the major soluble proteins of dense-core secretory vesicles. Chromaffin cells from Chga null mice [chromogranin A knock-out (CgA-KO)] exhibited ∼30% reduction in the content and in the release of catecholamines compared with wild type. This was because of a lower secretion per single exocytotic event, rather than to a lower frequency of exocytotic events. Cell incubation with l-DOPA produced an increase in the vesicular amine content of wild-type, but not CgA-KO vesicles. In contrast, intracellular electrochemistry showed that l-DOPA produced a significantly larger increase in cytosolic amines in CgA-KO cells than in the wild type. These data indicate that the mechanisms for vesicular accumulation in CgA-KO cells were fully saturated. Patch-amperometry recordings showed a delayed initiation of the amperometric signal after vesicle fusion, whereas no changes were observed in vesicle size or fusion pore kinetics despite the smaller amine content. We conclude that intravesicular proteins are highly efficient systems directly implicated in transmitter accumulation and in the control of neurosecretion.

Distribution and regulation of expression of serum‐ and glucocorticoid‐induced kinase‐1 in the rat kidney

The serum‐ and glucocorticoid‐induced kinase‐1 (sgk1) increases the activity of a number of epithelial ion channels and transporters. The present study examines the distribution and subcellular localization of sgk1 protein in the rat kidney and the regulation of levels of expression induced by steroids. The results indicate that the kidney expresses predominantly the sgk1 isoform with a distribution restricted to the thick ascending limb of Henle, distal convoluted, connecting and cortical collecting tubules. Within cells, sgk1 strongly associates with the microsomal fraction of homogenates and it colocalizes with the Na+,K+‐ATPase to the basolateral membrane. Analysis of the levels of expression of sgk1 by Western blotting and immunohistochemistry indicates constitutive high expression under basal conditions. Approximately half of the basal level is maintained by glucocorticoids whereas physiological fluctuations of aldosterone produce minor changes in sgk1 abundance in adrenal‐intact animals. These results do not support the notion that physiological changes of aldosterone concentration turn the expression of sgk1 ‘on and off’ in the mammalian kidney. Additionally, localization of sgk1 to the basolateral membrane indicates that the effects mediated by sgk1 do not require a direct interaction with the ion channels and transporters whose activity is modulated, since most of these proteins are located in the apical membrane of renal epithelial cells.

Dopamine transporter glycosylation correlates with the vulnerability of midbrain dopaminergic cells in Parkinson's disease

The dopamine transporter (DAT) is a membrane glycoprotein responsible for dopamine (DA) uptake, which has been involved in the degeneration of DA cells in Parkinsons disease (PD). Given that DAT activity depends on its glycosylation status and membrane expression, and that not all midbrain DA cells show the same susceptibility to degeneration in PD, we have investigated a possible relationship between DAT glycosylation and function and the differential vulnerability of DA cells. Glycosylated DAT expression, DA uptake, and DAT V(max) were significantly higher in terminals of nigrostriatal neurons than in those of mesolimbic neurons. No differences were found in non-glycosylated DAT expression and DAT K(m), and DA uptake differences disappeared after deglycosylation of nigrostriatal synaptosomes. The expression pattern of glycosylated DAT in the human midbrain and striatum showed a close anatomical relationship with DA degeneration in parkinsonian patients. This relationship was confirmed in rodent and monkey models of PD, and in HEK cells expressing the wild-type and a partially deglycosylated DAT form. These results strongly suggest that DAT glycosylation is involved in the differential vulnerability of midbrain DA cells in PD.

Activation of serum/glucocorticoid-induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy

Maintaining skeletal muscle mass is essential for general health and prevention of disease progression in various neuromuscular conditions. Currently, no treatments are available to prevent progressive loss of muscle mass in any of these conditions. Hibernating mammals are protected from muscle atrophy despite prolonged periods of immobilization and starvation. Here, we describe a mechanism underlying muscle preservation and translate it to non‐hibernating mammals. Although Akt has an established role in skeletal muscle homeostasis, we find that serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) regulates muscle mass maintenance via downregulation of proteolysis and autophagy as well as increased protein synthesis during hibernation. We demonstrate that SGK1 is critical for the maintenance of skeletal muscle homeostasis and function in non‐hibernating mammals in normal and atrophic conditions such as starvation and immobilization. Our results identify a novel therapeutic target to combat loss of skeletal muscle mass associated with muscle degeneration and atrophy.

Mechanisms of Regulation of Epithelial Sodium Channel by SGK1 in A6 Cells

The serum and glucocorticoid induced kinase 1 (SGK1) participates in the regulation of sodium reabsorption in the distal segment of the renal tubule, where it may modify the function of the epithelial sodium channel (ENaC). The molecular mechanism underlying SGK1 regulation of ENaC in renal epithelial cells remains controversial. We have addressed this issue in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible system. Expression of a constitutively active mutant of SGK1 (SGK1T S425D) induced a sixfold increase in amiloride-sensitive short-circuit current (I sc). Using noise analysis we demonstrate that SGK1 effect on I sc is due to a fourfold increase in the number of functional ENaCs in the membrane and a 43% increase in channel open probability. Impedance analysis indicated that SGK1T S425D increased the absolute value of cell equivalent capacitance by an average of 13.7%. SGK1T S425D also produced a 1.6–1.9-fold increase in total and plasma membrane subunit abundance, without changing the half-life of channels in the membrane. We conclude that in contrast to aldosterone, where stimulation of transport can be explained simply by an increase in channel synthesis, SGK1 effects are more complex and involve at least three actions: (1) increase of ENaC open probability; (2) increase of subunit abundance within apical membranes and intracellular compartments; and (3) activation of one or more pools of preexistent channels within the apical membranes and/or intracellular compartments.