Activated PKCδ and PKC? Inhibit Epithelial Chloride Secretion Response to cAMP via Inducing Internalization of the Na+-K+-2Cl? Cotransporter NKCC1

The basolateral Na⁺-K⁺-2Cl⁻ cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (α, δ, ϵ) (shPKCs) or LacZ (shLacZ, non-targeting control). After 72 h of adenoviral transduction, protein levels of the PKC isoforms in shPKCs-T84 cells were decreased by ∼90% compared with the shLacZ-control. Activation of PKCs by phorbol 12-myristate 13-acetate (PMA) caused a redistribution of NKCC1 immunostaining from the basolateral membrane to intracellular vesicles in both shLacZ- and shPKCα-T84 cells, whereas the effect of PMA was not observed in shPKCδ- and shPKCϵ- cells. These results were further confirmed by basolateral surface biotinylation. Furthermore, activation of PKCs by PMA inhibited cAMP-stimulated chloride secretion in the uninfected, shLacZ- and shPKCα-T84 monolayers, but the inhibitory effect was significantly attenuated in shPKCδ- and shPKCϵ-T84 monolayers. In conclusion, the activated novel isoforms PKCδ or PKCϵ, but not the conventional isoform PKCα, inhibits transepithelial chloride secretion through inducing internalization of the basolateral surface NKCC1. Our study reveals that the novel PKC isoform-regulated NKCC1 surface expression plays an important role in the regulation of chloride secretion.     

cAMP Stimulates Apical Exocytosis of the Renal Na+-K+-2Cl? Cotransporter NKCC2 in the Thick Ascending Limb: ROLE OF PROTEIN KINASE A*

The apical renal Na⁺-K⁺-2Cl⁻ cotransporter NKCC2 mediates NaCl absorption by the thick ascending limb (TAL) of Henle''s loop. cAMP stimulates NKCC2 by enhancing steady-state apical membrane levels of this protein; however, the trafficking and signaling mechanisms by which this occurs have not been studied. Here, we report that stimulation of endogenous cAMP levels with either forskolin/3-isobutyl-1-methylxanthine (IBMX) or the V2 receptor agonist [deamino-Cys¹,d-Arg⁸]vasopressin increases steady-state surface NKCC2 and that the protein kinase A (PKA) inhibitor H-89 blocks this effect. Confocal imaging of apical surface NKCC2 in isolated perfused TALs confirmed a stimulatory effect of cAMP on apical trafficking that was blocked by PKA inhibition. Selective stimulation of PKA with the agonist N⁶-benzoyl-cAMP (500 μm) stimulated steady-state surface NKCC2, whereas the Epac-selective agonist 8-p-chlorophenylthio-2′-O-methyl-cAMP (100 and 250 μm) had no effect. To explore the trafficking mechanism by which cAMP increases apical NKCC2, we measured cumulative apical membrane exocytosis and NKCC2 exocytic insertion in TALs. By monitoring apical FM1–43 fluorescence, we observed rapid stimulation of apical exocytosis (2 min) by forskolin/IBMX. We also found constitutive exocytic insertion of NKCC2 in TALs over time, which was increased by 3-fold in the presence of forskolin/IBMX. PKA inhibition blunted cAMP-stimulated exocytic insertion but did not affect the rate of constitutive exocytosis. We conclude that cAMP stimulates steady-state apical surface NKCC2 by stimulating exocytic insertion and that this process is highly dependent on PKA but not Epac.The renal-specific Na⁺-K⁺-2Cl⁻ cotransporter NKCC2 is expressed at the apical membrane and in subapical vesicles in the thick ascending limb (TAL)² of Henle''s loop, where it mediates NaCl reabsorption (1). Hormonal stimulation of intracellular cAMP by arginine vasopressin enhances NaCl absorption in the TAL by stimulating NKCC2-dependent transport (2–5).As NKCC2 must be in the plasma membrane to mediate NaCl absorption, vesicle trafficking of NKCC2, including exocytic insertion, endocytic retrieval, and recycling to and from the plasma membrane, is likely to play a major role in NKCC2 regulation. Despite its importance, the regulation of NKCC2 trafficking by cAMP has not been thoroughly studied.We showed previously that cAMP stimulates NKCC2-dependent NaCl reabsorption by increasing steady-state surface NKCC2 in rat TALs (6). In addition, others have shown that the V2 receptor agonist [deamino-Cys¹,d-Arg⁸]vasopressin (dDAVP) increases apical membrane NKCC2 labeling in mouse TALs in vivo (7). These data indicate that enhanced steady-state apical surface NKCC2 levels are involved in the stimulation of NKCC2 activity and NaCl absorption caused by cAMP. However, the signaling cascade involved in the stimulation of NKCC2 trafficking has not been studied in polarized TAL cells.In other epithelial cells, cAMP stimulates protein trafficking by activating protein kinase A (PKA) and/or Epac (guanine exchange protein activated by cAMP) (8–13). PKA is expressed in TALs and binds cAMP in response to arginine vasopressin stimulation (14). In addition to PKA, the Epac isoforms Epac1 and Epac2 are expressed in TALs (15), but their role in NKCC2 trafficking has not been addressed. In the collecting duct epithelium, arginine vasopressin and cAMP stimulate aquaporin-2 exocytic insertion into the apical membrane and enhance water permeability (16) in a process mediated by PKA (17–20). However, Epac-selective agonists also enhance aquaporin-2 trafficking and apical exocytosis in this renal epithelium, suggesting a role for Epac1 (10, 21). In addition, in other cells, Epac-dependent signaling exerts opposite effects compared with PKA (22, 23). We hypothesized that cAMP increases steady-state surface NKCC2 expression in native TALs by stimulating apical exocytosis and that PKA mediates this process. Our data show for the first time that cAMP stimulates the rate of NKCC2 exocytosis via PKA and that this trafficking step mediates the increase in steady-state surface NKCC2 in native TALs.     

Roles of Ca2+ and Protein Tyrosine Kinase in Insulin Action on Cell Volume via Na+ and K+ Channels and Na+/K+/2Cl− Cotransporter in Fetal Rat Alveolar Type II Pneumocyte

The aim of the present study was to investigate the roles of Ca²⁺ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca²⁺ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca²⁺ (<1 nm). This insulin action in a Ca²⁺-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na⁺/K⁺/2Cl⁻ cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na⁺ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca²⁺-free solution was completely blocked by quinine (1 mm, a blocker of Ca²⁺-activated K⁺ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm extracellular Ca²⁺, that the stimulatory action of insulin on an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter requires Ca²⁺, and that in a Ca²⁺-free solution insulin activates a quinine-sensitive K⁺ channel but not in the presence of 1 mm Ca²⁺. The insulin action on cell volume in a Ca²⁺-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca²⁺-ATPase which depletes the intracellular Ca²⁺ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca²⁺-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K⁺ channel is mediated through PTK activity in a cytosolic Ca²⁺-dependent manner. Lavendustin A, further, completely blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in a Ca²⁺-free solution, but only partially blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm Ca²⁺. This observation suggests that the activity of the Na⁺/K⁺/2Cl⁻ cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca²⁺-independent pathway and the other is a PTK-independent, Ca²⁺-dependent pathway. Further, we observed that removal of extracellular Ca²⁺ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na⁺ channel and the bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter, and that removal of extracellular Ca²⁺ abolished the activity of the quinine-sensitive K⁺ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca²⁺ results from diverse effects on the cotransporter and Na⁺ and K⁺ channels. Received: 2 September 1998/Revised: 30 November 1998     

Identification of Regulatory Phosphorylation Sites in a Cell Volume– and Ste20 Kinase–dependent ClC Anion Channel

Changes in phosphorylation regulate the activity of various ClC anion transport proteins. However, the physiological context under which such regulation occurs and the signaling cascades that mediate phosphorylation are poorly understood. We have exploited the genetic model organism Caenorhabditis elegans to characterize ClC regulatory mechanisms and signaling networks. CLH-3b is a ClC anion channel that is expressed in the worm oocyte and excretory cell. Channel activation occurs in response to oocyte meiotic maturation and swelling via serine/threonine dephosphorylation mediated by the type I phosphatases GLC-7α and GLC-7β. A Ste20 kinase, germinal center kinase (GCK)-3, binds to the cytoplasmic C terminus of CLH-3b and inhibits channel activity in a phosphorylation-dependent manner. Analysis of hyperpolarization-induced activation kinetics suggests that phosphorylation may inhibit the ClC fast gating mechanism. GCK-3 is an ortholog of mammalian SPAK and OSR1, kinases that bind to, phosphorylate, and regulate the cell volume–dependent activity of mammalian cation-Cl⁻ cotransporters. Using mass spectrometry and patch clamp electrophysiology, we demonstrate here that CLH-3b is a target of regulatory phosphorylation. Concomitant phosphorylation of S742 and S747, which are located 70 and 75 amino acids downstream from the GCK-3 binding site, are required for kinase-mediated channel inhibition. In contrast, swelling-induced channel activation occurs with dephosphorylation of S747 alone. Replacement of both S742 and S747 with glutamate gives rise to kinase- and swelling-insensitive channels that exhibit activity and biophysical properties similar to those of wild-type CLH-3b inhibited by GCK-3. Our studies provide novel insights into ClC regulation and mechanisms of cell volume signaling, and provide the foundation for studies aimed at defining how conformational changes in the cytoplasmic C terminus alter ClC gating and function in response to intracellular signaling events.     

Seizure Sensitivity Is Ameliorated by Targeted Expression of K+–Cl? Cotransporter Function in the Mushroom Body of the Drosophila Brain

The kcc^DHS1 allele of kazachoc (kcc) was identified as a seizure-enhancer mutation exacerbating the bang-sensitive (BS) paralytic behavioral phenotypes of several seizure-sensitive Drosophila mutants. On their own, young kcc^DHS1 flies also display seizure-like behavior and demonstrate a reduced threshold for seizures induced by electroconvulsive shock. The product of kcc shows substantial homology to KCC2, the mammalian neuronal K⁺–Cl⁻ cotransporter. The kcc^DHS1 allele is a hypomorph, and its seizure-like phenotype reflects reduced expression of the kcc gene. We report here that kcc functions as a K⁺–Cl⁻ cotransporter when expressed heterologously in Xenopus laevis oocytes: under hypotonic conditions that induce oocyte swelling, oocytes that express Drosophila kcc display robust ion transport activity observed as a Cl⁻-dependent uptake of the K⁺ congener ⁸⁶Rb⁺. Ectopic, spatially restricted expression of a UAS-kcc⁺ transgene was used to determine where cotransporter function is required in order to rescue the kcc^DHS1 BS paralytic phenotype. Interestingly, phenotypic rescue is largely accounted for by targeted, circumscribed expression in the mushroom bodies (MBs) and the ellipsoid body (EB) of the central complex. Intriguingly, we observed that MB induction of kcc⁺ functioned as a general seizure suppressor in Drosophila. Drosophila MBs have generated considerable interest especially for their role as the neural substrate for olfactory learning and memory; they have not been previously implicated in seizure susceptibility. We show that kcc^DHS1 seizure sensitivity in MB neurons acts via a weakening of chemical synaptic inhibition by GABAergic transmission and suggest that this is due to disruption of intracellular Cl⁻ gradients in MB neurons.Mushroom body (MB) expression of the kazachoc (kcc) K⁺–Cl⁻ cotransporter is shown here to rescue seizure-sensitive phenotypes in Drosophila through an effect on GABAergic fast synaptic inhibition. Heretofore, considerable interest has focused on the MB because of its essential role in olfactory learning and memory (Heisenberg 2003; Davis 2005; Keene and Waddell 2007; Berry et al. 2008). The MB occupies a central position in the fly nervous system, integrating incoming olfactory, mechanical, taste, and visual sensory signals and then sorting the distribution of outgoing motor signals (Heisenberg 2003). Short- and long-term alteration of individual nerve cell physiology in the MB is thought to form the basis of learning and memory (Davis 2005; Keene and Waddell 2007; Berry et al. 2008). A role for the MB in seizure susceptibility has not previously been suspected. Here we suggest that the orderly arrangements of axons and neuropile of MB Kenyon cells (KCs) not only facilitate learning and memory, but also provide the type of anatomical substrate in flies that is thought to be essential for seizure spread in the mammalian brain (Hauser and Hesdorffer 1990; Traub and Miles 1991).Inhibitory synaptic transmission in Drosophila is thought to be mediated primarily by GABAergic neurons found throughout the CNS at all stages of development (Buchner et al. 1988; Jackson et al. 1990; Harrison et al. 1996; Yasuyama et al. 2002). γ-aminobutyric acid (GABA) is synthesized from glutamate via a conserved glutamic acid decarboxylase encoded by the Drosophila Gad1 gene (Jackson et al. 1990; Buchner 1991). GABAergic activity is limited by sequestering extracellular GABA back into presynaptic neurons by GABA transporters that are sensitive to inhibition by dl-2,4-diaminobutyric acid, nipecotic acid, and valproic acid (Neckameyer and Cooper 1998; Leal et al. 2004). Three ionotropic GABA_A receptor subunits have been identified in Drosophila and are encoded by the Rdl, LCCH3, and GRD loci (Hosie et al. 1997). When expressed heterotopically in Xenopus oocytes, the best studied of these, Rdl, forms GABA-gated Cl⁻ channels that are sensitive to block by picrotoxin (ffrench-Constant et al. 1991, 1993; Zhang et al. 1995). Inhibitory Cl⁻ currents are dependent on maintenance of Cl⁻ gradients, particularly in low intracellular Cl⁻ concentrations. In the fly, Cl⁻ gradients appear to be maintained by the kcc K⁺–Cl⁻ cotransporter (Hekmat-Scafe et al. 2006).Chemical synaptic transmission onto MB neurons has been examined in dissociated KCs in primary culture (Su and O''dowd 2003). Spontaneous miniature excitatory postsynaptic currents (EPSCs) are mediated mainly by nicotinic acetylcholine (ACh) receptors. Miniature inhibitory postsynaptic currents (IPSCs) appear to be mediated primarily by picrotoxin-sensitive GABA_A receptors, probably encoded by Rdl (Su and O''dowd 2003; Harrison et al. 1996). In vivo, cholinergic inputs to the MB are thought to arise primarily from antennal lobe projection neurons (Yasuyama et al. 2002). Two antennal lobe neurons that project to the MB, the anterior paired lateral (APL) neurons, were recently shown to be GABAergic (Liu and Davis, 2009). Additional GABAergic inputs to the Drosophila MB seem likely; in locust they appear to come from a poorly understood region of the brain called the lateral horn, which is itself also driven by antennal lobe projection neurons (Perez-Orive et al. 2002).Previously, we identified the kcc^DHS1 partial loss-of-function mutation as a seizure enhancer that also causes increased seizure sensitivity in young flies (Hekmat-Scafe et al. 2006). The kcc product shows homology to the mammalian KCC2 K⁺–Cl⁻ cotransporter, and we inferred that a decrease in inhibitory synaptic strength is responsible for causing the seizure phenotypes. In this study, we describe our search for identifying the source of these vulnerable inhibitory synapses and report that they appear to lie primarily in the MBs of the Drosophila brain. Further, we speculate on the possibility of their involvement in synaptic plasticity functions of the MB.     

Increased Spinal Cord Na+-K+-2Cl? Cotransporter-1 (NKCC1) Activity Contributes to Impairment of Synaptic Inhibition in Paclitaxel-induced Neuropathic Pain

Microtubule-stabilizing agents, such as paclitaxel (Taxol), are effective chemotherapy drugs for treating many cancers, and painful neuropathy is a major dose-limiting adverse effect. Cation-chloride cotransporters, such as Na⁺-K⁺-2Cl⁻ cotransporter-1 (NKCC1) and K⁺-Cl⁻ cotransporter-2 (KCC2), critically influence spinal synaptic inhibition by regulating intracellular chloride concentrations. Here we show that paclitaxel treatment in rats significantly reduced GABA-induced membrane hyperpolarization and caused a depolarizing shift in GABA reversal potential of dorsal horn neurons. However, paclitaxel had no significant effect on AMPA or NMDA receptor-mediated glutamatergic input from primary afferents to dorsal horn neurons. Paclitaxel treatment significantly increased protein levels, but not mRNA levels, of NKCC1 in spinal cords. Inhibition of NKCC1 with bumetanide reversed the paclitaxel effect on GABA-mediated hyperpolarization and GABA reversal potentials. Also, intrathecal bumetanide significantly attenuated hyperalgesia and allodynia induced by paclitaxel. Co-immunoprecipitation revealed that NKCC1 interacted with β-tubulin and β-actin in spinal cords. Remarkably, paclitaxel increased NKCC1 protein levels at the plasma membrane and reduced NKCC1 levels in the cytosol of spinal cords. In contrast, treatment with an actin-stabilizing agent had no significant effect on NKCC1 protein levels in the plasma membrane or cytosolic fractions of spinal cords. In addition, inhibition of the motor protein dynein blocked paclitaxel-induced subcellular redistribution of NKCC1, whereas inhibition of kinesin-5 mimicked the paclitaxel effect. Our findings suggest that increased NKCC1 activity contributes to diminished spinal synaptic inhibition and neuropathic pain caused by paclitaxel. Paclitaxel disrupts intracellular NKCC1 trafficking by interfering with microtubule dynamics and associated motor proteins.     

Requirement of Cl− and Na+ for the ouabain-resistant control of cell volume in slices of rat liver

Summary The ability of liver cells to control their volume in the presence of ouabain has been studied in tissue slices that were recovering at 38°C from a period of swelling at 1°C. Morphological observations were made in conjunction with measurements of the net movements of water and ions. Extrusion of water in the presence of ouabain (2mm) was accompanied by a net loss of Na⁺ and Cl^– and by the formation of characteristic, rounded vesicles in the peri-canalicular regions of the hepatocytes; bile canaliculi were patent. When incubation was carried out in a medium in which either NO ₃ ^– or SO ₄ ^2– replaced Cl^–, ouabain-resistant water extrusion was prevented and the cytoplasmic vesicles normally found with ouabain were almost totally absent. When these slices were subsequently transferred to Cl^– medium with oubain, extrusion of intracellular water was initiated and cytoplasmic vesicles reappeared. Replacement of medium Na⁺ by Li⁺ mimicked the effects of ouabain on water and ion movements and ultrastructure. In addition, the ouabain-resistant extrusion of water and Cl^– was reduced and there was some diminution in the number of vesicles induced by ouabain. Furosemide (2mm) had little effect on water movement or ultrastructure in the absence of ouabain, but it slowed the net water loss and substantially reduced the formation of cytoplasmic vesicles in the presence of ouabain. The results show a close relationship between ouabain-resistant water extrusion and the formation of the cytoplasmic vesicles that are characteristic of treatment with ouabain. They further suggest that a cotransport of Na⁺ and Cl^– forms an important part of the mechanism underlying ouabain-resistant water extrusion and, specifically, that this cotransport may take place across the membranes of the cytoplasmic vesicles.     

The Detailed Localization Pattern of Na+/K+/2Cl? Cotransporter Type 2 and Its Related Ion Transport System in the Rat Endolymphatic Sac

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na⁺/K⁺/2Cl⁻ cotransporter type 2 (NKCC-2) and Na⁺/K⁺-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na⁺/K⁺-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na⁺/K⁺-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules. (J Histochem Cytochem 58:759–763, 2010)     

Volume regulation in human fibroblasts: role of Ca2+ and 5-lipoxygenase products in the activation of the Cl− efflux

Trypsinized human skin fibroblasts in suspension perform regulatory volume decrease (RVD) after cell swelling in hypotonic medium. During RVD, ³⁶Cl^– efflux is dramatically increased and the cell membrane is depolarized, indicating the activation of Cl^– channels. This activation of Cl^– channels depends on extracellular as well as on intracellular Ca²⁺. The swelling-induced Cl^– efflux and the RVD response are inhibited by the 5-lipoxygenase inhibitor ETH 615-139. Finally, following hypotonic treatment, cellular pH decreases. The pH decrease does not involve the Cl^–/HCO ₃ ^– exchange because it is independent of the external Cl^– concentration.T. Mastrocola was recipient of a scientific fellowship from the Italian Consiglio Nazionale delle Ricerche (C.N.R.). This work was supported by Progetto Finalizzato Ingegneria Genetica, C.N.R., Roma, and by the Danish Natural Research Council.     

Cell Swelling Activates the K+ Conductance and Inhibits the Cl? Conductance of the Basolateral Membrane of Cells from a Leaky Epithelium

Necturus gallbladder epithelial cells bathed in 10 mM HCO₃/1% CO₂ display sizable basolateral membrane conductances for Cl⁻ (G_Cl ^b) and K ⁺ (G_K ^b). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K ⁺/Cl⁻ selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca²⁺]_i) increased transiently during hyposmotic swelling (peak at ∼30 s, return to baseline within ∼90 s), but chelation of cell Ca²⁺ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10–12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (V_cs) produced a significant increase in the change in V_cs elicited by lowering basolateral solution [Cl⁻], whereas hyperpolarization of V_cs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases G_K ^b and decreases G _Cl ^b. These two effects appear to be linked, i.e., the increase in G _K ^b produces membrane hyperpolarization, which in turn reduces G _Cl ^b. ( b) Hyperosmotic shrinkage has the opposite effects on G_K ^b and G _Cl ^b. ( c) Cell swelling causes a transient increase in [Ca²⁺]_i, but this response may not be necessary for the increase in G_K ^b during cell swelling.     

Mesothelin Virus-Like Particle Immunization Controls Pancreatic Cancer Growth through CD8+ T Cell Induction and Reduction in the Frequency of CD4+foxp3+ICOS? Regulatory T Cells

Our previous study has shown that mesothelin (MSLN) is a potential immunotherapeutic target for pancreatic cancer. Here, we further studied the immunogenicity of chimeric murine MSLN-virus-like particles (mMSLN-VLPs), their ability to break tolerance to mMSLN, a self-antigen, and deciphered the mechanism of immune responses elicited by mMSLN-VLP immunization using a pancreatic cancer (PC) mouse model. In addition to what we have found with xenogeneic human MSLN-VLP (hMSLN-VLP), mMSLN-VLP immunization was able to break the tolerance to intrinsic MSLN and mount mMSLN-specific, cytotoxic CD8⁺ T cells which led to a significant reduction in tumor volume and prolonged survival in an orthotopic PC mouse model. Furthermore, CD4⁺foxp3⁺ regulatory T cells (Tregs) were progressively decreased in both spleen and tumor tissues following mMSLN-VLP immunization and this was at least partly due to elevated levels of IL-6 production from activated plasmocytoid dendritic cell (pDC)-like cells following mMSLN-VLP immunization. Moreover, mMSLN-VLP treatment mainly reduced the frequency of the CD4⁺foxp3⁺ICOS⁻ Treg subset. However, mMSLN-VLP induced IL-6 production also increased ICOSL expression on pDC-like cells which supported the proliferation of immunosuppressive CD4⁺foxp3⁺ICOS⁺ Treg cells. This study reveals that mMSLN-VLP immunization is capable of controlling PC progression by effectively mounting an immune response against mMSLN, a tumor self-antigen, and altering the immunosuppressive tumor microenvironment via activation of pDCs-like cells and reduction in the frequency of CD4⁺foxp3⁺ICOS⁻ Treg cells. However, combination therapies will likely need to be used in order to target residual CD4⁺foxp3⁺ICOS⁺ Treg cells.     

Vesicle-associated Membrane Protein 2 (VAMP2) but Not VAMP3 Mediates cAMP-stimulated Trafficking of the Renal Na+-K+-2Cl? Co-transporter NKCC2 in Thick Ascending Limbs

In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na⁺/K⁺/2Cl⁻ co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.     

Pouring Salt on a Wound: Pseudomonas aeruginosa Virulence Factors Alter Na+ and Cl? Flux in the Lung

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen with multiple niches in the human body, including the lung. P. aeruginosa infections are particularly damaging or fatal for patients with ventilator-associated pneumonia, chronic obstructive pulmonary disease, and cystic fibrosis (CF). To establish an infection, P. aeruginosa relies on a suite of virulence factors, including lipopolysaccharide, phospholipases, exoproteases, phenazines, outer membrane vesicles, type III secreted effectors, flagella, and pili. These factors not only damage the epithelial cell lining but also induce changes in cell physiology and function such as cell shape, membrane permeability, and protein synthesis. While such virulence factors are important in initial infection, many become dysregulated or nonfunctional during the course of chronic infection. Recent work on the virulence factors alkaline protease (AprA) and CF transmembrane conductance regulator inhibitory factor (Cif) show that P. aeruginosa also perturbs epithelial ion transport and osmosis, which may be important for the long-term survival of this microbe in the lung. Here we discuss the literature regarding host physiology-altering virulence factors with a focus on Cif and AprA and their potential roles in chronic infection and immune evasion.     

Intracellular compartmentation of Na+, K+ and Cl− in the Ehrlich ascites tumor cell: Correlation with the membrane potential

Summary The intracellular distribution of Na⁺, K⁺, Cl^– and water has been studied in the Ehrlich ascites tumor cell. Comparison of the ion and water contents of whole cells with those of cells exposed to La³⁺ and mechanical stress indicated that La³⁺ treatment results in selective damage to the cell membrane and permits evaluation of cytoplasmic and nuclear ion concentrations. The results show that Na⁺ is sequestered within the nucleus, while K⁺ and Cl^– are more highly concentrated in the cell cytoplasm. Reduction of the [Na⁺] of the incubation medium by replacement with K⁺ results in reduced cytoplasmic [Na⁺], increased [Cl^–] and no change in [K⁺]. Nuclear concentrations of these ions are virtually insensitive to the cation composition of the medium. Concomitant measurements of the membrane potential were made. The potential in control cells was –13.7 mV. Reduction of [Na⁺] in the medium caused significant depolarization. The measured potential is describable by the Cl^– equilibrium potential and can be accounted for in terms of cation distributions and permeabilities. The energetic implications of the intracellular compartmentation of ions are discussed.