The serum- and glucocorticoid-inducible kinase-1 (SGK1) is ubiquitously expressed and under genomic control by cell stress (including cell shrinkage) and hormones (including gluco- and mineralocorticoids). Similar to its isoforms SGK2 and SGK3, SGK1 is activated by insulin and growth factors via phosphatidylinositol 3-kinase and the 3-phosphoinositide-dependent kinase PDK1. SGKs activate ion channels (e.g., ENaC, TRPV5, ROMK, Kv1.3, KCNE1/KCNQ1, GluR1, GluR6), carriers (e.g., NHE3, GLUT1, SGLT1, EAAT1-5), and the Na+-K+-ATPase. They regulate the activity of enzymes (e.g., glycogen synthase kinase-3, ubiquitin ligase Nedd4-2, phosphomannose mutase-2) and transcription factors (e.g., forkhead transcription factor FKHRL1, beta-catenin, nuclear factor kappaB). SGKs participate in the regulation of transport, hormone release, neuroexcitability, cell proliferation, and apoptosis. SGK1 contributes to Na+ retention and K+ elimination of the kidney, mineralocorticoid stimulation of salt appetite, glucocorticoid stimulation of intestinal Na+/H+ exchanger and nutrient transport, insulin-dependent salt sensitivity of blood pressure and salt sensitivity of peripheral glucose uptake, memory consolidation, and cardiac repolarization. A common ( approximately 5% prevalence) SGK1 gene variant is associated with increased blood pressure and body weight. SGK1 may thus contribute to metabolic syndrome. SGK1 may further participate in tumor growth, neurodegeneration, fibrosing disease, and the sequelae of ischemia. SGK3 is required for adequate hair growth and maintenance of intestinal nutrient transport and influences locomotive behavior. In conclusion, the SGKs cover a wide variety of physiological functions and may play an active role in a multitude of pathophysiological conditions. There is little doubt that further targets will be identified that are modulated by the SGK isoforms and that further SGK-dependent in vivo physiological functions and pathophysiological conditions will be defined.

The vagus, the 10th cranial nerve, contains pathways that contribute to the regulation of the internal viscera, including the heart. Vagal efferent fibers do not originate in a common brainstem structure. The Polyvagal Theory is introduced to explain the different functions of the two primary medullary source nuclei of the vagus: the nucleus ambiguus (NA) and the dorsal motor nucleus (DMNX). Although vagal pathways from both nuclei terminate on the sinoatrial node, it is argued that the fibers originating in NA are uniquely responsible for respiratory sinus arrhythmia (RSA). Divergent shifts in RSA and heart rate are explained by independent actions of DMNX and NA. The theory emphasizes a phylogenetic perspective and speculates that mammalian, but not reptilian, brainstem organization is characterized by a ventral vagal complex (including NA) related to processes associated with attention, motion, emotion, and communication. Various clinical disorders, such as sudden infant death syndrome and asthma, may be related to the competition between DMNX and NA.

1. The effect of L-NG-nitro arginine (L-NOARG) was compared with that of L-NG-monomethyl arginine (L-NMMA) on vasodilatation of the isolated aorta of the rabbit and perfused mesentery of the rat in response to acetylcholine (ACh) and sodium nitroprusside (NP). 2. L-NOARG (1.5-100 microM) and L-NMMA (3-100 microM) produced concentration-related contraction of the rabbit aorta precontracted with phenylephrine (700-900 nM). Similarly, L-NOARG (10-200 microM) and L-NMMA (30-100 microM) elevated perfusion pressure of the noradrenaline (NA, 0.6-2.5 mM)-preconstricted rat mesentery preparation. 3. L-NOARG (1.5-100 microM) and L-NMMA (3-100 microM) caused concentration-related inhibition of the vasodilator effect of ACh (0.01-1.0 microM) on the rabbit aorta without influencing responses to NP (0.03-0.5 microM). L-NOARG methyl ester (30 microM) also inhibited ACh-induced vasorelaxation with similar potency to NOARG. L-arginine (30-150 microM) but not D-arginine (100 microM) caused graded reversal of the inhibitory effect of both L-NOARG (15 microM) and L-NMMA (30 microM). Complete reversal of the effect of both inhibitors was achieved with 150 microM L-arginine. L-Alanine (50 microM), L-arginosuccinic acid (5 microM), L-citrulline (50 microM), L-methionine (50 microM) and L-ornithine (50 microM) failed to reverse the inhibitory effect of L-NOARG (15 microM). 4. L-NOARG (10-200 microM) and L-NMMA (30-100 microM) inhibited the vasodilator effect of ACh (0.006-18.0 nmol) in the rat mesentery without affecting vasodilatation due to NP (1.1-11.1 nmol). L-Arginine (100 microM) but not D-arginine (100 microM) produced partial reversal of the effect of L-NOARG (30 microM) and L-NMMA (30 microM). 5. L- and D-N'-butyloxycarbonyl No-nitro arginine (100 microM) produced modest (approximately 20%) inhibition of the effect of ACh on the rabbit aorta; this effect was not reversible with L-arginine (100 microM). L-Namonocarbobenzoxy arginine (L-NMCA, 5O microM), L-N-NG-dicarbobenzoxy arginine (L-NDCA, 5 microM) and L-NG-tosyl arginine (50 microM) were inactive. 6. These results identify L-NOARG as a potent, L-arginine reversible inhibitor of endothelium-dependent vasodilatation. The available data suggests that L-NOARG, like L-NMMA, inhibits endothelial nitric oxide (NO) biosynthesis.

The most commonly used signal transduction pathway for receptor-mediated N-type Ca2+-channel modulation involves activation of a heterotrimeric G protein to produce voltage-dependent inhibition. Although it is widely assumed that Galpha mediates this effect, experiments to address this hypothesis directly are lacking. Here I show that transient overexpression of Gbetagamma in sympathetic neurons mimics and occludes the voltage-dependent Ca2+ channel modulation produced by noradrenaline (NA). Conversely, over-expression of Galpha produces minimal effects on basal Ca2+ channel behaviour but attenuates NA-mediated inhibition in a manner consistent with the buffering of Gbetagamma. These observations indicate that it is Gbetagamma, and Galpha, that mediates voltage-dependent inhibition of N-type Ca2+ channels. The identification of Gbetagamma as the mediator of this pathway has broad implications as G-protein-coupled receptors, many of which are implicated in disease or are targets of therapeutic agents, couple to N-type Ca2+ channels and may modulate synaptic transmission by this mechanism.

The cellular mechanisms by which excess exposure to the excitatory neurotransmitter glutamate can produce neuronal injury are unknown. More than a decade ago it was hypothesized that glutamate neurotoxicity (GNT) is a direct consequence of excessive neuronal excitation ("excitotoxicity" hypothesis); more recently, it has been hypothesized that a Ca influx triggered by glutamate exposure might mediate GNT (Ca hypothesis). A basic test to discriminate between these hypotheses would be to determine the dependence of GNT on the extracellular ionic environment. The excitotoxicity hypothesis predicts that GNT should depend critically on the presence of extracellular Na, since that ion appears to mediate glutamate neuroexcitation in the CNS; the Ca hypothesis predicts that GNT should depend critically on the presence of extracellular Ca. The focus of the present experiments was to determine the effects of several alterations in the extracellular ionic environment upon the serial morphologic changes that occur after mouse neocortical neurons in cell culture receive toxic exposure to glutamate. The results suggest that GNT in cortical neurons can be separated into 2 components distinguishable on the basis of differences in time course and ionic dependence. The first component, marked by neuronal swelling, occurs early, is dependent on extracellular Na and Cl, can be mimicked by high K, and is thus possibly "excitotoxic." The second component, marked by gradual neuronal disintegration, occurs late, is dependent on extracellular Ca, can be mimicked by A23187, and is thus possibly mediated by a transmembrane influx of Ca. While either component alone is ultimately capable of producing irreversible neuronal injury, the Ca-dependent mechanism predominates at lower exposures to glutamate. Glutamate exposure likely leads to a Ca influx both through glutamate-activated cation channels and through voltage-dependent Ca channels activated by membrane depolarization. Addition of 20 mM Mg, however, did not substantially block GNT; this finding, together with the observation that GNT is largely preserved in sodium-free solution, supports the notion that the activation of voltage-dependent Ca channels may not be required for lethal Ca entry. The possibility that N-methyl-D-aspartate receptors may play a dominant role in mediating glutamate-induced lethal Ca influx is discussed.

OBJECTIVE

We prospectively investigated the prevalence of curable forms of primary aldosteronism (PA) in newly diagnosed hypertensive patients.

BACKGROUND

The prevalence of curable forms of PA is currently unknown, although retrospective data suggest that it is not as low as commonly perceived.

METHODS

Consecutive hypertensive patients referred to 14 hypertension centers underwent a diagnostic protocol composed of measurement of Na+ and K+ in serum and 24-h urine, sitting plasma renin activity, and aldosterone at baseline and after 50 mg captopril. The patients with an aldosterone/renin ratio >40 at baseline, and/or >30 after captopril, and/or a probability of PA (by a logistic discriminant function)>> or =50% underwent imaging tests and adrenal vein sampling (AVS) or adrenocortical scintigraphy to identify the underlying adrenal pathology. An aldosterone-producing adenoma (APA) was diagnosed in patients who in addition to excess autonomous aldosterone secretion showed: 1) lateralized aldosterone secretion at AVS or adrenocortical scintigraphy, 2) adenoma at surgery and pathology, and 3) a blood pressure decrease after adrenalectomy. Evidence of excess autonomous aldosterone secretion without such criteria led to a diagnosis of idiopathic hyperaldosteronism (IHA).

RESULTS

A total of 1,180 patients (age 46 +/- 12 years) were enrolled; a conclusive diagnosis was attained in 1,125 (95.3%). Of these, 54 (4.8%) had an APA and 72 (6.4%) had an IHA. There were more APA (62.5%) and fewer IHA cases (37.5%) at centers where AVS was available (p = 0.002); the opposite occurred where AVS was unavailable.

CONCLUSIONS

In newly diagnosed hypertensive patients referred to hypertension centers, the prevalence of APA is high (4.8%). The availability of AVS is essential for an accurate identification of the adrenocortical pathologies underlying PA.

Halophytes, plants that survive to reproduce in environments where the salt concentration is around 200 mm NaCl or more, constitute about 1% of the world's flora. Some halophytes show optimal growth in saline conditions; others grow optimally in the absence of salt. However, the tolerance of all halophytes to salinity relies on controlled uptake and compartmentalization of Na+, K+ and Cl- and the synthesis of organic 'compatible' solutes, even where salt glands are operative. Although there is evidence that different species may utilize different transporters in their accumulation of Na+, in general little is known of the proteins and regulatory networks involved. Consequently, it is not yet possible to assign molecular mechanisms to apparent differences in rates of Na+ and Cl- uptake, in root-to-shoot transport (xylem loading and retrieval), or in net selectivity for K+ over Na+. At the cellular level, H+-ATPases in the plasma membrane and tonoplast, as well as the tonoplast H+-PPiase, provide the trans-membrane proton motive force used by various secondary transporters. The widespread occurrence, taxonomically, of halophytes and the general paucity of information on the molecular regulation of tolerance mechanisms persuade us that research should be concentrated on a number of 'model' species that are representative of the various mechanisms that might be involved in tolerance.

Obligatory, coupled cotransport of Na(+), K(+), and Cl(-) by cell membranes has been reported in nearly every animal cell type. This review examines the current status of our knowledge about this ion transport mechanism. Two isoforms of the Na(+)-K(+)-Cl(-) cotransporter (NKCC) protein (approximately 120-130 kDa, unglycosylated) are currently known. One isoform (NKCC2) has at least three alternatively spliced variants and is found exclusively in the kidney. The other (NKCC1) is found in nearly all cell types. The NKCC maintains intracellular Cl(-) concentration ([Cl(-)](i)) at levels above the predicted electrochemical equilibrium. The high [Cl(-)](i) is used by epithelial tissues to promote net salt transport and by neural cells to set synaptic potentials; its function in other cells is unknown. There is substantial evidence in some cells that the NKCC functions to offset osmotically induced cell shrinkage by mediating the net influx of osmotically active ions. Whether it serves to maintain cell volume under euvolemic conditons is less clear. The NKCC may play an important role in the cell cycle. Evidence that each cotransport cycle of the NKCC is electrically silent is discussed along with evidence for the electrically neutral stoichiometries of 1 Na(+):1 K(+):2 Cl- (for most cells) and 2 Na(+):1 K(+):3 Cl(-) (in squid axon). Evidence that the absolute dependence on ATP of the NKCC is the result of regulatory phosphorylation/dephosphorylation mechanisms is decribed. Interestingly, the presumed protein kinase(s) responsible has not been identified. An unusual form of NKCC regulation is by [Cl(-)](i). [Cl(-)](i) in the physiological range and above strongly inhibits the NKCC. This effect may be mediated by a decrease of protein phosphorylation. Although the NKCC has been studied for approximately 20 years, we are only beginning to frame the broad outlines of the structure, function, and regulation of this ubiquitous ion transport mechanism.

Receptor-operated Ca2+ entry has been proposed as a signalling mechanism in many cells. Receptor-operated Ca2+ channels (ROCs) were first postulated in smooth muscle by Bolton, van Breemen and Somlyo and Somlyo, but recordings of directly ligand-gated Ca2+ current are lacking. Here we describe receptor-operated Ca2+ current evoked in arterial smooth muscle cells by ATP, a sympathetic neurotransmitter. ATP activates channels with approximately 3:1 selectivity for Ca2+ over Na+ at near-physiological concentrations and with a unitary conductance of approximately 5 pS in 110 mM Ca2+ or Ba2+. The channels can be opened even at very negative potentials and resist inhibition by cadmium or nifedipine, unlike voltage-gated Ca2+ channels; they are not blocked by Mg2+, unlike NMDA (N-methyl-D-aspartate)-activated channels; they are directly activated by ligand, without involvement of readily diffusible second messengers, unlike cation channels in neutrophils and T lymphocytes. Thus, the ATP-activated channels provide a distinct mechanism for excitatory synaptic current and Ca2+ entry in smooth muscle.

The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.

The membrane of tissue-cultured chick pectoral muscle contains an ionic channel which is activated by membrane stretch. Nicotinic channels and Ca2+-activated K+ channels are not affected by stretch. In 150 mM-external K+ and 150 mM-internal Na+ the channel has a conductance of 70 pS, linear current-voltage relationship between -50 and -140 mV and a reversal potential of +30 mV. Kinetic analysis of single-channel records indicates that there are one open (O) and three closed (C) states. The data can be fitted by the reaction scheme: C1-C2-C3-O. Only the rate constant that governs the C1-C2 transition (k1,2) is stretch-sensitive. None of the rates are voltage-sensitive. The rate constant k1,2 varies with the square of the tension as k1, 2 = k0 X e alpha T2, where alpha is a constant describing the sensitivity to stretch and T is the tension. A typical value of alpha is 0.08 (dyn cm-1)-2. Following exposure to cytochalasin B the channel becomes more sensitive to stretch. The stretch-sensitivity constant, alpha, increases from 0.08 to 2.4 (dyn cm-1)-2. The probability of the channel being open is strongly dependent upon the extracellular K+ concentration. With a suction of 2 cmHg the probability increases from 0.004 in normal saline (5 mM-K+) to 0.26 in 150 mM-K+. The channel appears to gather force from a large area of membrane (greater than 3 X 10(5) A2), probably by a cytochalasin-resistant cytoskeletal network.

In 1997, 18 confirmed cases of human influenza arising from multiple independent transmissions of H5N1 viruses from infected chickens were reported from Hong Kong. To identify possible phenotypic changes in the hemagglutinin (HA) and neuraminidase (NA) of the H5 viruses during interspecies transfer, we compared the receptor-binding properties and NA activities of the human and chicken H5N1 isolates from Hong Kong and of H5N3 and H5N1 viruses from wild aquatic birds. All H5N1 viruses, including the human isolate bound to Sia2-3Gal-containing receptors but not to Sia2-6Gal-containing receptors. This finding formally demonstrates for the first time that receptor specificity of avian influenza viruses may not restrict initial avian-to-human transmission. The H5N1 chicken viruses differed from H5 viruses of wild aquatic birds by a 19-amino-acid deletion in the stalk of the NA and the presence of a carbohydrate at the globular head of the HA. We found that a deletion in the NA decreased its ability to release the virus from cells, whereas carbohydrate at the HA head decreased the affinity of the virus for cell receptors. Comparison of amino acid sequences from GenBank of the HAs and NAs from different avian species revealed that additional glycosylation of the HA and a shortened NA stalk are characteristic features of the H5 and H7 chicken viruses. This finding indicates that changes in both HA and NA may be required for the adaptation of influenza viruses from wild aquatic birds to domestic chickens and raises the possibility that chickens may be a possible intermediate host in zoonotic transmission.

Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.

Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The approximately 3.0 angstrom structure contains 14 transmembrane (TM) helices in an inward-facing conformation with a core structure of inverted repeats of 5 TM helices (TM2 to TM6 and TM7 to TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to that of the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.

The sodium channel, one of the family of structurally homologous voltage-gated ion channels, differs from other members, such as the calcium and the potassium channels, in its high selectivity for Na+. This selectivity presumably reflects a distinct structure of its ion-conducting pore. We have recently identified two clusters of predominantly negatively charged amino-acid residues, located at equivalent positions in the four internal repeats of the sodium channel as the main determinants of sensitivity to the blockers tetrodotoxin and saxitoxin. All site-directed mutations reducing net negative charge at these positions also caused a marked decrease in single-channel conductance. Thus these two amino-acid clusters probably form part of the extracellular mouth and/or the pore wall of the sodium channel. We report here the effects on ion selectivity of replacing lysine at position 1,422 in repeat III and/or alanine at position 1,714 in repeat IV of rat sodium channel II (ref. 3), each located in one of the two clusters, by glutamic acid, which occurs at the equivalent positions in calcium channels. These amino-acid substitutions, unlike other substitutions in the adjacent regions, alter ion-selection properties of the sodium channel to resemble those of calcium channels. This result indicates that lysine 1,422 and alanine 1,714 are critical in determining the ion selectivity of the sodium channel, suggesting that these residues constitute part of the selectivity filter of the channel.

Recent studies have identified that the activation of protein kinase C (PKC) and increased diacylglycerol (DAG) levels initiated by hyperglycemia are associated with many vascular abnormalities in retinal, renal, and cardiovascular tissues. Among the various PKC isoforms, the beta- and delta-isoforms appear to be activated preferentially in the vasculatures of diabetic animals, although other PKC isoforms are also increased in the renal glomeruli and retina. The glucose-induced activation of PKC has been shown to increase the production of extracellular matrix and cytokines; to enhance contractility, permeability, and vascular cell proliferation; to induce the activation of cytosolic phospholipase A2; and to inhibit Na+-K+-ATPase. The synthesis and characterization of a specific inhibitor for PKC-beta isoforms have confirmed the role of PKC activation in mediating hyperglycemic effects on vascular cells, as described above, and provide in vivo evidence that PKC activation could be responsible for abnormal retinal and renal hemodynamics in diabetic animals. Transgenic mice overexpressing PKC-beta isoform in the myocardium developed cardiac hypertrophy and failure, further supporting the hypothesis that PKC-beta isoform activation can cause vascular dysfunctions. Interestingly, hyperglycemia-induced oxidative stress may also mediate the adverse effects of PKC-beta isoforms by the activation of the DAG-PKC pathway, since treatment with D-alpha-tocopherol was able to prevent many glucose-induced vascular dysfunctions and inhibit DAG-PKC activation. Clinical studies are now in progress to determine whether PKC-beta inhibition can prevent diabetic complications.

The diagnosis of nonalcoholic steatohepatitis (NASH) is defined by the presence and pattern of specific histological abnormalities on liver biopsy. A separate system of scoring the features of nonalcoholic fatty liver disease (NAFLD) called the NAFLD Activity Score (NAS) was developed as a tool to measure changes in NAFLD during therapeutic trials. However, some studies have used threshold values of the NAS, specifically NAS ≥5, as a surrogate for the histologic diagnosis of NASH. To evaluate whether this unintended use of the NAS is valid, biopsy and clinical data from the 976 adults in NASH Clinical Research Network (CRN) studies were reviewed. Biopsies were evaluated centrally by the NASH CRN Pathology Committee. Definite steatohepatitis (SH) was diagnosed in 58.1%, borderline SH in 19.5% and "not SH" in 22%. The NAS was ≥5 in 50% and ≤4 in 49%; in this cohort only 75% of biopsies with definite SH had an NAS ≥5, whereas 28% of borderline SH and 7% of "not SH" biopsies had NAS ≥5. Of biopsies with an NAS ≥5, 86% had SH and 3% "not SH". NAS ≤4 did not indicate benign histology; 29% had SH and only 42% had "not SH." Higher values of the NAS were associated with higher levels of alanine aminotransferase and aspartate aminotransferase, whereas the diagnosis of SH was associated with features of the metabolic syndrome.

CONCLUSIONS

The diagnosis of definite SH or the absence of SH based on evaluation of patterns as well as individual lesions on liver biopsies does not always correlate with threshold values of the semiquantitative NAS. Clinical trials and observational studies should take these different performance characteristics into account.

Cardiac Na+,Ca2+ exchange is activated by a mechanism that requires hydrolysis of adenosine triphosphate (ATP) but is not mediated by protein kinases. In giant cardiac membrane patches, ATP acted to generate phosphatidylinositol-4,5-bisphosphate (PIP2) from phosphatidylinositol (PI). The action of ATP was abolished by a PI-specific phospholipase C (PLC) and recovered after addition of exogenous PI; it was reversed by a PIP2-specific PLC; and it was mimicked by exogenous PIP2. High concentrations of free Ca2+ (5 to 20 microM) accelerated reversal of the ATP effect, and PLC activity in myocyte membranes was activated with a similar Ca2+ dependence. Aluminum reversed the ATP effect by binding with high affinity to PIP2. ATP-inhibited potassium channels (KATP) were also sensitive to PIP2, whereas Na+,K+ pumps and Na+ channels were not. Thus, PIP2 may be an important regulator of both ion transporters and channels.

High concentrations of Na+ in saline soils inhibit plant growth and reduce agricultural productivity. We report here that CaMV 35S promoter driven overexpression of the Arabidopsis thaliana SOS1 gene, which encodes a plasma membrane Na+/H+ antiporter, improves plant salt tolerance in A. thaliana. Transgenic plants showed substantial upregulation of SOS1 transcript levels upon NaCl treatment, suggesting post-transcriptional control of SOS1 transcript accumulation. In response to NaCl treatment, transgenic plants overexpressing SOS1 accumulated less Na+ in the xylem transpirational stream and in the shoot. Undifferentiated callus cultures regenerated from the transgenic plants were also more tolerant of salt stress, which was correlated with reduced Na+ content in the transgenic cells. These results show that improved salt tolerance could be achieved by limiting Na+ accumulation in plant cells.

Multivesicular bodies (MVBs) are endocytic structures that contain small vesicles formed by the budding of an endosomal membrane into the lumen of the compartment. Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles termed exosomes. K562 cells are a hematopoietic cell line that releases exosomes. The application of monensin (MON) generated large MVBs that were labeled with a fluorescent lipid. Exosome release was markedly enhanced by MON treatment, a Na+/H+ exchanger that induces changes in intracellular calcium (Ca2+). To explore the possibility that the effect of MON on exosome release was caused via an increase in Ca2+, we have used a calcium ionophore and a chelator of intracellular Ca2+. Our results indicate that increasing intracellular Ca2+ stimulates exosome secretion. Furthermore, MON-stimulated exosome release was completely eliminated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), implying a requirement for Ca2+ in this process. We have observed that the large MVBs generated in the presence of MON accumulated Ca2+ as determined by labeling with Fluo3-AM, suggesting that intralumenal Ca2+ might play a critical role in the secretory process. Interestingly, our results indicate that transferrin (Tf) stimulated exosome release in a Ca2+-dependent manner, suggesting that Tf might be a physiological stimulus for exosome release in K562 cells.

There is compelling evidence that active Cl absorption by a variety of epithelia, widely distributed throughout the animal kingdom, is the result of an electrically neutral Na-coupled transport process at the luminal membrane and that the energy for transcellular Cl movement is derived from the Na gradient across that barrier. These co-transport processes are found predominantly in "leaky" or "moderately leaky" epithelia and permit these tissues to absorb Na and Cl with high degrees of efficacy. In addition, there is a growing body of evidence that cyclic AMP and Ca-induced electrogenic Cl secretion by a wide variety of epithelia may involve electrically neutral, Na-coupled Cl entry across the contraluminal membrane and that the energy for these secretory processes is derived from the Na-gradient across that barrier. A model for electrogenic Cl secretion that accounts for the available data is presented.

The plasma membrane ATPase of plants and fungi is a hydrogen ion pump. The proton gradient generated by the enzyme drives the active transport of nutrients by H+-symport. In addition, the external acidification in plants and the internal alkalinization in fungi, both resulting from activation of the H+ pump, have been proposed to mediate growth responses. This ATPase has a relative molecular mass (Mr) similar to those of the Na+-, K+- and Ca2+-ATPases of animal cells and, like these proteins, forms an aspartylphosphate intermediate. We have cloned, mapped and sequenced the gene encoding the yeast plasma membrane ATPase (PMA1) and report here that it maps to chromosome VII adjacent to LEU1. The strong homology between the amino-acid sequence encoded by PMA1 and those of (Na+ + K+), Na+-, K+- and Ca2+- ATPases is consistent with the notion that the family of cation pumps which form a phosphorylated intermediate evolved from a common ancestral ATPase. The function of the PMA1 gene is essential because a null mutation is lethal in haploid cells.

We have worked out a simplification of the procedure described by Schmitz et al. (Biochim. Biophys. Acta (1973) 323, 98--112) for the preparation of brush border membranes from small intestine. The procedure ultimately adopted is simple, rapid, does not necessarily require scraping and can be started from fresh or frozen material. It can be scaled up easily, allowing a quick production of large amounts of brush border membrane vesicles. These vesicles prove to be excellently suited for transport studies, as suggested by our measurements of D-glucose transport. Using these vesicles, the mode of choline transport across the brush border membrane was also investigated. Choline transport was found to occur by a saturable component with a Km of 83 +/- 4 micrometer (at 20 degrees C) and by a non-saturable component. It is independent of the presence of Na+ and appears to be non-electrogenic.

Excessive sodium (Na+) in salinized soils inhibits plant growth and development. A mutation in the SOS3 gene renders Arabidopsis thaliana plants hypersensitive to Na+-induced growth inhibition. SOS3 encodes a protein that shares significant sequence similarity with the calcineurin B subunit from yeast and neuronal calcium sensors from animals. The results suggest that intracellular calcium signaling through a calcineurin-like pathway mediates the beneficial effect of calcium on plant salt tolerance.