Kinetic studies of K-Cl cotransport in cultured rat vascular smooth muscle cells.

During aging, and development of atherosclerosis and cardiovascular disease (CVD), aortic vascular smooth muscle cells (VSMCs) transition from healthy contractile to diseased synthetic phenotypes. K-Cl cotransport (KCC) maintains cell volume and ion homeostasis in growth and differentiation, and hence is important for VSMC proliferation and migration. Therefore, KCC activity may play a role in the contractile-to-synthetic VSMC phenotypic transition. Early, medium, and late synthetic passage VSMCs were tested for specific cytoskeletal protein expression. KCC-mediated ouabain- and bumetanide-insensitive Rb+ (a K+ congener) influx was determined as Cl--dependent Rb+ influx at different external Rb+ and Cl- ion concentrations, [Rb+]o and [Cl-]o. Expressions of the cytoskeletal proteins α-actin, vimentin, and desmin fell from early through late synthetic VSMCs. KCC kinetic parameters, such as maximum velocity ( Vm), and apparent Cl- and Rb+ affinities ( Km), were calculated with Lineweaver-Burk, Hanes-Woolf, and Hill approximations. Vm values of both Rb+- and Cl--dependent influxes were of equal magnitude, commensurate with a KCC stoichiometry of unity, and rose threefold from early to late synthetic VSMCs. Hill coefficients for Rb+ and Cl- correlated with cell passage number, suggesting increased KCC ligand cooperativity. However, Km values for [Cl-]o were strikingly bimodal with 60-80 mM in early, ~20-30 mM in medium, and 60 mM in late passage cells. In contrast, Km values for [Rb+]o remained steady at ~17 mM. Since total KCC isoform expression was similar with cell passage, structure/function changes of the KCC signalosome may accompany the transition of aortic VSMCs from a healthy to a diseased phenotype.

Assessment of Silver-Nanoparticles-Induced Erythrocyte Cytotoxicity through Ion Transport Studies.

BACKGROUND/AIMS: Silver nanoparticles (AgNPs) are the most frequently used nanomaterials in industrial and biomedical applications. Their functionalization significantly impacts their properties and potential applications. Despite the need to produce, investigate and apply them, not much is known about the toxicity of silver nanoparticles to and their interaction with blood components, such as erythrocytes. Here, we report on the effect of two negatively charged AgNPs (Creighton, and Lee-Meisel) on ion transport in human red blood cells (HRBCs). METHODS: HRBCs were obtained from blood of adult donors, which was either expired, fresh or refrigerated for variable lengths of time, and from fresh or refrigerated cord blood. Rb+ and K+ ions were measured by atomic emission and absorption spectrophotometry, respectively. Erythrocyte hemoglobin optical density (Hbc OD), was determined at 527 nm to estimate RBC volume in the same tubes in which Rb+ and K+ were measured. Cellular Rb+ uptake and intracellular K+ concentrations, [K]i, were calculated in mmol/L of original cells (LOC) per time. Rubidium, a potassium ion (K+) congener used to measure K+ influx, [K]i, and Hbc ODs were determined in the presence and absence of several concentrations (0-150 µg mL-1) of spherical AgNPs of an average diameter of 10 nm, at different time points (0-60 min). RESULTS: Creighton AgNPs inhibited Rb+ influx and depleted the cells of K+ independently of the source and in a time and dose-dependent manner. In contrast, Lee-Meisel AgNPs caused ~ 50 % Rb+ influx inhibition and ~ 15 % K+ loss with larger interindividual variability than Creighton AgNPs. The loss of K+ from HRBCs was entirely accounted for by an increase in extracellular K+ concentration, [K]o. Enhanced dark field optical microscopy in conjunction with CytoViva® hyperspectral imaging helped visualize AgNPs internalized by HRBCs, thus pointing to a potential cause for their cytotoxic effects. CONCLUSION: These findings indicate that HRBC K+ homeostasis is an early and sensitive biomarker for AgNPs toxicity and is a function of their surface functionalization.

Interaction between Na-K-ATPase and Bcl-2 proteins BclXL and Bak.

In silico analysis predicts interaction between Na-K-ATPase (NKA) and Bcl-2 protein canonical BH3- and BH1-like motifs, consistent with NKA inhibition by the benzo-phenanthridine alkaloid chelerythrine, a BH3 mimetic, in fetal human lens epithelial cells (FHLCs) (Lauf PK, Heiny J, Meller J, Lepera MA, Koikov L, Alter GM, Brown TL, Adragna NC. Cell Physiol Biochem 31: 257-276, 2013). This report establishes proof of concept: coimmunoprecipitation and immunocolocalization showed unequivocal and direct physical interaction between NKA and Bcl-2 proteins. Specifically, NKA antibodies (ABs) coimmunoprecipitated BclXL (B-cell lymphoma extra large) and BAK (Bcl-2 antagonist killer) proteins in FHLCs and A549 lung cancer cells. In contrast, both anti-Bcl-2 ABs failed to pull down NKA. Notably, the molecular mass of BAK1 proteins pulled down by NKA and BclXL ABs appeared to be some 4-kDa larger than found in input monomers. In silico analysis predicts these higher molecular mass BAK1 proteins as alternative splicing variants, encoding 42 amino acid (aa) larger proteins than the known 211-aa long canonical BAK1 protein. These BAK1 variants may constitute a pool separate from that forming mitochondrial pores by specifically interacting with NKA and BclXL proteins. We propose a NKA-Bcl-2 protein ternary complex supporting our hypothesis for a special sensor role of NKA in Bcl-2 protein control of cell survival and apoptosis.

Synopsis of the 48 annual meeting of the Lake Cumberland Biological Transport Group and the second biannual meeting of the Pendrin Consortium.

Ion transporters are the molecular basis for ion homeostasis of the cell and the whole organism. The anion exchanger pendrin is only one of a number of examples where a complete or partial loss of function and/or deregulation of expression of ion transporters may lead or contribute to pathological conditions in humans. A complete understanding of the function of ion transporters in health and disease may pave the way for the identification of new and focused therapeutic approaches. Exchange of knowledge and connectivity between the experts in the feld of transport physiology is essential in facing these challenging tasks. The Lake Cumberland Biological Transport Group and the Pendrin Consortium are examples of scientific forums where investigators combine their efforts towards a better understanding of molecular pathophysiology of ion transport. This issue discusses the versatility of ion transporters involved in the regulation of cellular volume and other functions, such as the solute carrier (SLC) 12A gene family members SLC12A4-7, encoding the Na(+)-independent cation-chloride cotransporters commonly known as the K(+)-Cl(-) cotransporters KCC1-4, and the betaine/γ-aminobutyric acid transport system (BGT1, SLC6A12), just to name a few. The issue further addresses the pathophysiology of intestinal and respiratory epithelia and related therapeutic tools and techniques to investigate interactions between proteins and proteins and small compounds. Finally, the current knowledge and new findings on the expression, regulation and function of pendrin (SLC26A4) in the inner ear, kidney, airways and blood platelets are presented.

Sea water acidification affects osmotic swelling, regulatory volume decrease and discharge in nematocytes of the jellyfish Pelagia noctiluca.

BACKGROUND: Increased acidification/PCO2 of sea water is a threat to the environment and affects the homeostasis of marine animals. In this study, the effect of sea water pH changes on the osmotic phase (OP), regulatory volume decrease (RVD) and discharge of the jellyfish Pelagia noctiluca (Cnidaria, Scyphozoa) nematocytes, collected from the Strait of Messina (Italy), was assessed. METHODS: Isolated nematocytes, suspended in artificial sea water (ASW) with pH 7.65, 6.5 and 4.5, were exposed to hyposmotic ASW of the same pH values and their osmotic response and RVD measured optically in a special flow through chamber. Nematocyte discharge was analyzed in situ in ASW at all three pH values. RESULTS: At normal pH (7.65), nematocytes subjected to hyposmotic shock first expanded osmotically and then regulated their cell volume within 15 min. Exposure to hyposmotic ASW pH 6.5 and 4.5 compromised the OP and reduced or totally abrogated the ensuing RVD, respectively. Acidic pH also significantly reduced the nematocyte discharge response. CONCLUSION: Data indicate that the homeostasis and function of Cnidarians may be altered by environmental changes such as sea water acidification, thereby validating their use as novel bioindicators for the quality of the marine environment.

Surface-enhanced Raman spectroscopy (SERS) tracking of chelerythrine, a Na(+)/K(+) pump inhibitor, into cytosol and plasma membrane fractions of human lens epithelial cell cultures.

BACKGROUND/AIMS: The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na(+)/K(+) pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). METHODS: Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 µM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm(-1) marker band as a function of CET concentration. RESULTS: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. CONCLUSION: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET(+)) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.

Canonical Bcl-2 motifs of the Na+/K+ pump revealed by the BH3 mimetic chelerythrine: early signal transducers of apoptosis?

BACKGROUND/AIMS: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na+/K+ pump/ATPase [NKP, NKA], Na+-K+-2Cl+ [NKCC] and K+-Cl- [KCC] cotransporters, and channel-supported K+ loss) in human lens epithelial cells [LECs]. METHODS: K+ loss and K+ uptake, using Rb+ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl- replacement by NO3ˉ to determine KCC. 3H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures. RESULTS: CET inhibited NKP and NKCC by >90% (IC50 values ~35 and ~15 µM, respectively) without significant KCC activity change, and stimulated K+ loss by ~35% at 10-30 µM. Neither ATP levels nor phosphorylation of the NKA α1 subunit changed. 3H-ouabain was displaced from pig renal NKA only at 100 fold higher CET concentrations than the ligand. Sequence alignments of NKA with BH1- and BH3-like motifs containing pro-survival Bcl-2 and BclXl proteins showed more than one BH1-like motif within NKA for interaction with CET or with BH3 motifs. One NKA BH1-like motif (ARAAEILARDGPN) was also found in all P-type ATPases. Also, NKA possessed a second motif similar to that near the BH3 region of Bcl-2. CONCLUSION: Findings support the hypothesis that CET inhibits NKP by binding to BH1-like motifs and disrupting the α1 subunit catalytic activity through conformational changes. By interacting with Bcl-2 proteins through their complementary BH1- or BH3-like-motifs, NKP proteins may be sensors of normal and pathological cell functions, becoming important yet unrecognized signal transducers in the initial phases of apoptosis. CET action on NKCC1 and K+ channels may involve PKC-regulated mechanisms; however, limited sequence homologies to BH1-like motifs cannot exclude direct effects.

KCC2a expression in a human fetal lens epithelial cell line.

The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K(+)Cl(-) (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define the KCC2 expression profile in FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins of molecular weights ranging from 95 to 135 kDa were found by Western blotting using non-variant specific anti-KCC2 antibodies directed against two different regions of the KCC2 proteins, and by biotinylation suggesting membrane expression. Immunofluorescence revealed membrane and punctate cytoplasmic staining for KCC2. Low levels of cytosolic αA and αB crystallines, and neuron-specific enolase were also detected contrasting with the strong membrane immunofluorescence staining for the Na/K ATPase α1 subunit. Since the lack of neuron-specific expression of the KCC2b variant in non-neuronal tissues has been proposed under control of a neuron-restrictive silencing element in the KCC2 gene, we hypothesize that this control may be lifted for the KCC2a variant in the FHL124 epithelial cell culture, a non-neuronal tissue of ectodermal origin.

Evidence for aquaporin-mediated water transport in nematocytes of the jellyfish Pelagia noctiluca.

Nematocytes, the stinging cells of Cnidarians, have a cytoplasm confined to a thin rim. The main cell body is occupied by an organoid, the nematocyst, containing the stinging tubule and venom. Exposed to hypotonic shock, nematocytes initially swell during an osmotic phase (OP) and then undergo regulatory volume decrease (RVD) driven by K(+), Cl(-) and obligatory water extrusion mechanisms. The purpose of this report is to characterize the OP. Nematocytes were isolated by the NaSCN/Ca(2+) method from tentacles of the jellyfish Pelagia noctiluca, collected in the Strait of Messina, Italy. Isolated nematocytes were subjected to hyposmotic shock in 65% artificial seawater (ASW) for 15 min. The selective aquaporin water channel inhibitor HgCl(2) (0.1-25 µM) applied prior to osmotic shock prevented the OP and thus RVD. These effects were attenuated in the presence of 1mM dithiothreitol (DTT), a mercaptide bond reducing agent. AgNO(3) (1 µM) and TEA (tetraethylammonium, 100 µM), also reported to inhibit water transport, did not alter the OP but significantly diminished RVD, suggesting different modes of action for the inhibitors tested. Based on estimates of the nematocyte surface area and volume, and OP duration, a relative water permeability of ~10(-7) cm/sec was calculated and the number of putative aquaporin molecules mediating the OP was estimated. This water permeability is 3-4 orders of magnitude lower in comparison to higher order animals and may constitute an evolutionary advantage for Cnidarian survival.

Ion transport in a human lens epithelial cell line exposed to hyposmotic and apoptotic stress.

Membrane transport changes in human lens epithelial (HLE-B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium content, K(i), uptake of the K congener rubidium, Rb(i), and water content were measured after hyposmotic stress induced by hypotonicity, and apoptotic stress by the protein-kinase inhibitor staurosporine (STP). Cell water increased in hyposmotic (150 mOsm) as compared to isosmotic (300 mOsm) balanced salt solution (BSS) by >2-fold at 5 min and decreased within 15 min to baseline values accompanied by a 40% K(i) loss commensurate with cell swelling and subsequent cell shrinkage likely due to regulatory volume decrease (RVD). Loss of K(i), and accompanying water, and Rb(i) uptake in hyposmotic BSS were prevented by clotrimazole (CTZ) suggesting water shifts associated with K and Rb flux via intermediate conductance K (IK) channels, also detected at the mRNA and protein level. In contrast, 2 h after 2 microM STP exposure, the cells lost approximately 40% water and approximately 60% K(i), respectively, consistent with apoptotic volume decrease (AVD). Indeed, water and K(i) loss was at least fivefold greater after hyposmotic than after apoptotic stress. High extracellular K and 2 mM 4-aminopyridine (4-AP) but not CTZ significantly reduced apoptosis. Annexin labeling phosphatidylserine (PS) at 15 min suggested loss of lipid asymmetry. Quantitative PCR revealed significant IK channel expression during prolonged hyposmotic stress. Results suggest in HLE-B3 cells, IK channels likely partook in and were down regulated after RVD, whereas pro-apoptotic STP-activation of 4-AP-sensitive voltage-gated K channels preceded or accompanied PS externalization before subsequent apoptosis.

Mechanisms of hyposmotic volume regulation in isolated nematocytes of the anthozoan Aiptasia diaphana.

The nature and role of potassium (K) and water transport mediating hyposmotically-induced regulatory volume decrease (RVD) were studied in nematocytes dissociated with 605 mM thiocyanate from acontia of the Anthozoan Aiptasia diaphana. Cell volume and hence RVD were calculated from the inverse ratios of the cross sectional areas of nematocytes (A/A(o)) measured before (A(o)) and after (A) challenge with 65% artificial sea water (ASW). To distinguish between K channels and K-Cl cotransport (KCC), external sodium (Na) and chloride (Cl) were replaced by K and nitrate (NO(3)), respectively. Inhibitors were added to identify K channels (barium, Ba), and putative kinase (N-ethylmaleimide, NEM) and phosphatase (okadaic acid, OA) regulation of KCC. In 65% NaCl ASW, nematocytes displayed a biphasic change in A/A(o), peaking within 4 min due to osmotic water entry and thereafter declining within 6 min due to RVD. Changing NaCl to KCl or NaNO(3) ASW did not affect the osmotic phase but attenuated RVD, consistent with K channel and KCC mechanisms. Ba (3 mM) inhibited RVD. NEM and OA, applied separately, inhibited the osmotic phase and muted RVD suggesting primary action on water transport (aquaporins). NEM and OA together reduced the peak A/A(o) ratio during the osmotic phase whereas RVD was inhibited when OA preceded NEM. Thus, both K channels and KCC partake in the nematocyte RVD, the extent of which is determined by functional thiols and dephosphorylation of putative aquaporins facilitating the preceding osmotic water shifts.

Lithium fluxes indicate presence of Na-Cl cotransport (NCC) in human lens epithelial cells.

During regulatory volume decrease (RVD) of human lens epithelial cells (hLECs) by clotrimazole (CTZ)-sensitive K fluxes, Na-K-2Cl cotransport (NKCC) remains active and K-Cl cotransport (KCC) inactive. To determine whether such an abnormal behavior was caused by RVD-induced cell shrinkage, NKCC was measured in the presence of either CTZ or in high K media to prevent RVD. NKCC transports RbCl + NaCl, and LiCl + KCl; thus ouabain-insensitive, bumetanide-sensitive (BS) or Cl-dependent (ClD) Rb and Li fluxes were determined in hyposmotic high NaCl media with CTZ, or in high KCl media alone, or with sulfamate (Sf) or nitrate as Cl replacement at varying Rb, Li or Cl mol fractions (MF). Unexpectedly, NKCC was inhibited by 80% with CTZ (IC(50) = 31 microM). In isosmotic (300 mOsM) K, Li influx was approximately 1/3 of Rb influx in Na, 50% lower in Sf, and bumetanide-insensitive (BI). In hypotonic (200 mOsM) K, only the ClD but not BS Li fluxes were detected. At Li MFs from 0.1-1, Li fluxes fitted a bell-shaped curve maxing at approximately 0.6 Li MF, with the BS fluxes equaling approximately 1/4 of the ClD-Li influx. The difference, i.e. the BI/ClD Li influx, saturated with increasing Li and Cl MFs, with K(ms) for Li of 11 with, and 7 mM without K, and of approximately 46 mM for Cl. Inhibition of this K-independent Li influx by thiazides was weak whilst furosemide (<100 microM) was ineffective. Reverse transcription polymerase chain reaction and Western blots verified presence of both NKCC1 and Na-Cl cotransport (NCC). In conclusion, in hyposmotic high K media, which prevents CTZ-sensitive K flux-mediated RVD in hLECs, NKCC1, though molecularly expressed, was functionally silent. However, a K-independent and moderately thiazide-sensitive ClD-Li flux, i.e. LiCC, likely occurring through NCC was detected operationally and molecularly.

K-Cl cotransport function and its potential contribution to cardiovascular disease.

K-Cl cotransport is the coupled electroneutral movement of K and Cl ions carried out by at least four protein isoforms, KCC1-4. These transporters belong to the SLC12A family of coupled cotransporters and, due to their multiple functions, play an important role in the maintenance of cellular homeostasis. Significant information exists on the overall function of these transporters, but less is known about the role of the specific isoforms. Most functional studies were done on K-Cl cotransport fluxes without knowing the molecular details, and only recently attention has been paid to the isoforms and their individual contribution to the fluxes. This review summarizes briefly and updates the information on the overall functions of this transporter, and offers some ideas on its potential contribution to the pathophysiological basis of cardiovascular disease. By virtue of its properties and the cellular ionic distribution, K-Cl cotransport participates in volume regulation of the nucleated and some enucleated cells studied thus far. One of the hallmarks in cardiovascular disease is the inability of the organism to maintain water and electrolyte balance in effectors and/or target tissues. Oxidative stress is another compounding factor in cardiovascular disease and of great significance in our modern life styles. Several functions of the transporter are modulated by oxidative stress, which in turn may cause the transporter to operate in either "overdrive" with the purpose to counteract homeostatic changes, or not to respond at all, again setting the stage for pathological changes leading to cardiovascular disease. Intracellular Mg, a second messenger, acts as an inhibitor of K-Cl cotransport and plays a crucial role in regulating the activity of protein kinases and phosphatases, which, in turn, regulate a myriad of cellular functions. Although the role of Mg in cardiovascular disease has been dealt with for several decades, this chapter is evolving nowadays at a faster pace and the relationships between Mg, K-Cl cotransport, and cardiovascular disease is an area that awaits further experimentation. We envision that further studies on the role of K-Cl cotransport, and ideally on its specific isoforms, in mammalian cells will add missing links and help to understand the cellular mechanisms involved in the pathophysiology of cardiovascular disease.

PDGF activates K-Cl cotransport through phosphoinositide 3-kinase and protein phosphatase-1 in primary cultures of vascular smooth muscle cells.

K-Cl cotransport (K-Cl COT, KCC) is an electroneutrally coupled movement of K and Cl present in most cells. In this work, we studied the pathways of regulation of K-Cl COT by platelet-derived growth factor (PDGF) in primary cultures of vascular smooth muscle cells (VSMCs). Wortmannin and LY 294002 blocked the PDGF-induced K-Cl COT activation, indicating that the phosphoinositide 3-kinase (PI 3-K) pathway is involved. However, PD 98059 had no effect on K-Cl COT activation by PDGF, suggesting that the mitogen-activated protein kinase pathway is not involved under the experimental conditions tested. Involvement of phosphatases was also examined. Sodium orthovanadate, cyclosporin A and okadaic acid had no effect on PDGF-stimulated K-Cl COT. Calyculin A blocked the PDGF-stimulated K-Cl COT by 60%, suggesting that protein phosphatase-1 (PP-1) is a mediator in the PDGF signaling pathway/s. In conclusion, our results indicate that the PDGF-mediated pathways of K-Cl COT regulation involve the signaling molecules PI 3-K and PP-1.

Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis.

The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K(+) and Cl(-) efflux via activation of K(+) channels, volume-regulated anion channels (VRACs), and the K(+)-Cl(-) cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter isoform 1 (NKCC1). This results in a rapid (<10 min) and significant (>90%) reduction in intracellular K(+) content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent [DCPIB (VRAC inhibitor)-sensitive] pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD.

K-Cl cotransporters, cell volume homeostasis, and neurological disease.

K(+)-Cl(-) cotransporters (KCCs) were originally characterized as regulators of red blood cell (RBC) volume. Since then, four distinct KCCs have been cloned, and their importance for volume regulation has been demonstrated in other cell types. Genetic models of certain KCCs, such as KCC3, and their inhibitory WNK-STE20/SPS1-related proline/alanine-rich kinase (SPAK) serine-threonine kinases, have demonstrated the evolutionary necessity of these molecules for nervous system cell volume regulation, structure, and function, and their involvement in neurological disease. The recent characterization of a swelling-activated dephosphorylation mechanism that potently stimulates the KCCs has pinpointed a potentially druggable switch of KCC activity. An improved understanding of WNK/SPAK-mediated KCC cell volume regulation in the nervous system might reveal novel avenues for the treatment of multiple neurological diseases.

Incidence of respiratory disorders in neonates born between 34 and 36 weeks of gestation following exposure to antenatal corticosteroids between 24 and 34 weeks of gestation.

We studied the effect of antenatal corticosteroids on the incidence of respiratory disorders in singleton neonates born between 34 and 36 weeks of gestation. Retrospective analysis was conducted of the incidence of respiratory distress syndrome (RDS) and other respiratory disorders (need for mechanical ventilation, continuous positive airway pressure, and prolonged oxygen therapy) among singleton neonates delivered between 34 and 36 weeks of gestation who were exposed to antenatal corticosteroids, compared with neonates who were not exposed. Statistical analyses included two-tailed T tests, two-way analysis of variance for continuous data, and chi-square analysis for ratios. A probability of 0.05 was considered significant. Between January 1, 2000, and December 31, 2004, 1078 neonates were born between 34 and 36 weeks of gestation. Information regarding antenatal corticosteroids was available in 1044: 574 neonates (53.2%) were exposed to antenatal corticosteroids and 470 (43.6%) were not. One thousand and eighteen neonates were admitted to the neonatal intensive care unit. Respiratory disorders were diagnosed in 140 of those exposed to antenatal steroids (24.4%) and in 382 of the nonexposed (81.3%) ( P < 0.0001). Two hundred and ten neonates (20.6%) developed RDS: Of those, 43 were exposed to antenatal corticosteroids and 167 were not (incidence of RDS was 7.5% and 35.5%, respectively; P = 0.0001). The beneficial effects of corticosteroids were similar in both genders. It appears that the exposure of singleton pregnancies to antenatal corticosteroids between 24 and 34 weeks of gestation is associated with a significantly lower incidence of respiratory disorders among neonates born at 34 to 36 weeks of gestation. Further studies are needed to determine whether administering antenatal steroids to women experiencing preterm labor after 34 weeks of gestation would be associated with a similar beneficial effect.