Exocytic Insertion Research Articles

Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion.

SummaryCandida albicans is both a commensal and an opportunistic fungal pathogen. Invading hyphae of C. albicans secrete candidalysin, a pore-forming peptide toxin. To prevent cell death, epithelial cells must protect themselves from direct damage induced by candidalysin and by the mechanical forces exerted by expanding hyphae. We identify two key Ca2+-dependent repair mechanisms employed by epithelial cells to withstand candidalysin-producing hyphae. Using camelid nanobodies, we demonstrate candidalysin secretion directly into the invasion pockets induced by elongating C. albicans hyphae. The toxin induces oscillatory increases in cytosolic [Ca2+], which cause hydrolysis of PtdIns(4,5)P2 and loss of cortical actin. Epithelial cells dispose of damaged membrane regions containing candidalysin by an Alg-2/Alix/ESCRT-III-dependent blebbing process. At later stages, plasmalemmal tears induced mechanically by invading hyphae are repaired by exocytic insertion of lysosomal membranes. These two repair mechanisms maintain epithelial integrity and prevent mucosal damage during both commensal growth and infection by C. albicans.

Calcium-Dependent ESCRT Recruitment and Lysosome Exocytosis Maintain Epithelial Integrity During <i>Candida albicans</i> Invasion

Candida albicans is both a commensal and an opportunistic fungal pathogen. Invading hyphae of C. albicans secrete candidalysin, a pore-forming peptide toxin. To prevent cell death, epithelial cells must protect themselves from direct damage induced by candidalysin and by the mechanical forces exerted by expanding hyphae. We identified two key Ca2+-dependent repair mechanisms employed by epithelial cells to withstand candidalysin-producing hyphae. Using camelid nanobodies we demonstrate candidalysin secretion directly into the invasion pockets induced by elongating C. albicans hyphae. The toxin induced oscillatory increases in cytosolic [Ca2+], which caused hydrolysis of PtdIns(4,5)P2 and loss of cortical actin. Epithelial cells dispose of damaged membrane regions containing candidalysin by an Alg-2/Alix/ESCRT-III-dependent blebbing process, and repair plasmalemmal tears induced mechanically by invading hyphae by exocytic insertion of lysosomal membranes. These two repair mechanisms maintain epithelial integrity and prevent mucosal damage during both commensal growth and infection by C. albicans.

Interaction of NKCC2 With SNARE Proteins in the Thick Ascending Limb

NaCl absorption by the apical Na/K/2 Cl cotransporter (NKCC2) in thick ascending limbs (TAL) is important for blood pressure regulation and urinary concentration. NKCC2 levels in the apical membrane are maintained via exocytic insertion and endocytic retrieval. Inserted NKCC2 may derive from a newly synthesized pool or from those retrieved and then recycled. These processes are called trafficking, and involve several fusion events. Vesicle fusion is mediated by SNARE proteins from the VAMP, SNAP and syntaxin families. NKCC2 may directly interact with these proteins during trafficking into the apical membrane. We showed that VAMP2 and 3 are involved in NKCC2 trafficking in TALs. We hypothesized that NKCC2 directly interacts with VAMP 2, 3, and other SNAREs in TALs. We first studied SNARE expression and localization. We found that TALs express VAMP 2 and 3, SNAP 23 and syntaxins 3 and 4. VAMP 2 and 3, syntaxin 3 and SNAP 23 were localized to the apical membrane and subapical space similar to NKCC2. Syntaxin 4 was distributed throughout the cell. Next we performed co‐immunoprecipitation studies study protein interactions. VAMP 2 and 3 co‐immunoprecipitated with NKCC2 but the other SNAREs did not. Antibody specificity was tested with peptides and brain lysates. We conclude that NKCC2 interacts with VAMP 2 and 3 in TALs. SNAP23 and syntaxin 3 may be involved in NKCC2 trafficking without directly binding NKCC2. Funding: NIH, AHA

Luminal flow modulates H+-ATPase activity in the cortical collecting duct (CCD).

Epithelial Na(+) channel (ENaC)-mediated Na(+) absorption and BK channel-mediated K(+) secretion in the cortical collecting duct (CCD) are modulated by flow, the latter requiring an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. As axial flow modulates HCO(3)(-) reabsorption in the proximal tubule due to changes in both luminal Na(+)/H(+) exchanger 3 and H(+)-ATPase activity (Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, Wang T. Am J Physiol Renal Physiol 290: F289-F296, 2006), we sought to test the hypothesis that flow also regulates H(+)-ATPase activity in the CCD. H(+)-ATPase activity was assayed in individually identified cells in microperfused CCDs isolated from New Zealand White rabbits, loaded with the pH-sensitive dye BCECF, and then subjected to an acute intracellular acid load (NH(4)Cl prepulse technique). H(+)-ATPase activity was defined as the initial rate of bafilomycin-inhibitable cell pH (pH(i)) recovery in the absence of luminal K(+), bilateral Na(+), and CO(2)/HCO(3)(-), from a nadir pH of ∼6.2. We found that 1) an increase in luminal flow rate from ∼1 to 5 nl·min(-1)·mm(-1) stimulated H(+)-ATPase activity, 2) flow-stimulated H(+) pumping was Ca(2+) dependent and required microtubule integrity, and 3) basal and flow-stimulated pH(i) recovery was detected in cells that labeled with the apical principal cell marker rhodamine Dolichos biflorus agglutinin as well as cells that did not. We conclude that luminal flow modulates H(+)-ATPase activity in the rabbit CCD and that H(+)-ATPases therein are present in both principal and intercalated cells.

VAMP2 and VAMP3 mediate different pathways in NKCC2 trafficking to the surface in thick ascending limbs (TALs)

NKCC2 mediates NaCl absorption by the TAL in a process stimulated by cAMP. The mediator proteins involved in constitutive and cAMP stimulated NKCC2 trafficking are unknown. VAMP2 and 3 are expressed in TALs. However, it is not known which isoform mediates NKCC2 trafficking or whether they are involved in different pathways to the surface. We hypothesized that VAMP2 but not VAMP3 mediates cAMP-stimulated NKCC2 exocytosis. We silenced VAMP2 or VAMP3 in TALs by in vivo delivery of adenovirus carrying shRNAs into the outer medulla of rat kidneys. We measured steady-state surface NKCC2 and the rate of exocytosis with 2 different biotinylation protocols in isolated TALs. Silencing VAMP2 blocked cAMP-stimulated surface NKCC2 expression by 43 ± 6 % (p<0.05) without affecting baseline surface NKCC2. In control TALs the rate of constitutive NKCC2 exocytic insertion averaged 1.5 ± 0.3 au/min and cAMP enhanced this rate to 2.8 ± 0.5 au/min (p<0.05). Silencing VAMP2 completely blocked cAMP-stimulated NKCC2 insertion (p<0.05) without affecting constitutive delivery. In contrast, silencing VAMP3 did not block cAMP-stimulated NKCC2 insertion but eliminated constitutive delivery of NKCC2 (0.05 ± 0.04 au/min, p<0.05) and decreased surface NKCC2 expression by 53 ± 13% (p<0.05). We concluded that VAMP2 mediates cAMP-stimulated NKCC2 trafficking while VAMP3 is involved in constitutive delivery of NKCC2 to the surface. Funding: NIH

Secretory Carrier Membrane Protein 2 Regulates Exocytic Insertion of NKCC2 into the Cell Membrane

The renal-specific Na-K-2Cl co-transporter, NKCC2, plays a pivotal role in regulating body salt levels and blood pressure. NKCC2 mutations lead to type I Bartter syndrome, a life-threatening kidney disease. Regulation of NKCC2 trafficking behavior serves as a major mechanism in controlling NKCC2 activity across the plasma membrane. However, the identities of the protein partners involved in cell surface targeting of NKCC2 are largely unknown. To gain insight into these processes, we used a yeast two-hybrid system to screen a kidney cDNA library for proteins that interact with the NKCC2 C terminus. One binding partner we identified was SCAMP2 (secretory carrier membrane protein 2). Microscopic confocal imaging and co-immunoprecipitation assays confirmed NKCC2-SCAMP2 interaction in renal cells. SCAMP2 associated also with the structurally related co-transporter NCC, suggesting that the interaction with SCAMP2 is a common feature of sodium-dependent chloride co-transporters. Heterologous expression of SCAMP2 specifically decreased cell surface abundance as well as transport activity of NKCC2 across the plasma membrane. Co-immunolocalization experiments revealed that intracellularly retained NKCC2 co-localizes with SCAMP2 in recycling endosomes. The rate of NKCC2 endocytic retrieval, assessed by the sodium 2-mercaptoethane sulfonate cleavage assay, was not affected by SCAMP2. The surface-biotinylatable fraction of newly inserted NKCC2 in the plasma membrane was reduced by SCAMP2, demonstrating that SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytotic trafficking. Finally, a single amino acid mutation, cysteine 201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, which is believed to regulate exocytosis, abolished SCAMP2-mediated down-regulation of the co-transporter. Taken together, these data are consistent with a model whereby SCAMP2 regulates NKCC2 transit through recycling endosomes and limits the cell surface targeting of the co-transporter by interfering with its exocytotic trafficking.

Syntaxin 3 is necessary for cAMP- and cGMP-regulated exocytosis of CFTR: implications for enterotoxigenic diarrhea

Enterotoxins elaborated by Vibrio cholerae and Escherichia coli cannot elicit fluid secretion in the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. After enterotoxin exposure, CFTR channels are rapidly recruited from endosomes and undergo exocytic insertion into the apical plasma membrane of enterocytes to increase the number of channels on the cell surface by at least fourfold. However, the molecular machinery that orchestrates exocytic insertion of CFTR into the plasma membrane is largely unknown. The present study used immunofluorescence, immunoblotting, surface biotinylation, glutathione S-transferase (GST) pulldown assays, and immunoprecipitation to identify components of the exocytic soluble N-ethylmaleimide (NEM)-sensitive factor attachment receptor (SNARE) vesicle fusion machinery in cyclic nucleotide-activated exocytosis of CFTR in rat jejunum and polarized intestinal Caco-2(BB)e cells. Syntaxin 3, an intestine-specific SNARE, colocalized with CFTR on the apical domain of enterocytes in rat jejunum and polarized Caco-2(BB)e cells. Coimmunoprecipitation and GST binding studies confirmed that syntaxin 3 interacts with CFTR in vivo. Moreover, heat-stable enterotoxin (STa) activated exocytosis of both CFTR and syntaxin 3 to the surface of rat jejunum. Silencing of syntaxin 3 by short hairpin RNA (shRNA) interference abrogated cyclic nucleotide-stimulated exocytosis of CFTR in cells. These observations reveal a new and important role for syntaxin 3 in the pathophysiology of enterotoxin-elicited diarrhea.

5-Nitro-2-(3-phenylpropylamino)benzoic Acid (NPPB) Stimulates Cellular ATP Release through Exocytosis of ATP-enriched Vesicles

Cells release ATP in response to physiologic stimuli. Extracellular ATP regulates a broad range of important cellular functions by activation of the purinergic receptors in the plasma membrane. The purpose of these studies was to assess the role of vesicular exocytosis in cellular ATP release. FM1-43 fluorescence was used to measure exocytosis and bioluminescence to measure ATP release in HTC rat hepatoma and Mz-Cha-1 human cholangiocarcinoma cells. Exposure to a Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (50-300 microM) stimulated a 5-100-fold increase in extracellular ATP levels within minutes of the exposure. This rapid response was not a result of changes in cell viability or Cl(-) channel activity. NPPB also potently stimulated ATP release in HEK293 cells and HEK293 cells expressing a rat P2X7 receptor indicating that P2X7 receptors are not involved in stimulation of ATP release by NPPB. In all cells studied, NPPB rapidly stimulated vesicular exocytosis that persisted many minutes after the exposure. The kinetics of NPPB-evoked exocytosis and ATP release were similar. Furthermore, the magnitudes of NPPB-evoked exocytosis and ATP release were correlated (correlation coefficient 0.77), indicating that NPPB may stimulate exocytosis of a pool of ATP-enriched vesicles. These findings provide further support for the concept that vesicular exocytosis plays an important role in cellular ATP release and suggest that NPPB can be used as a biochemical tool to specifically stimulate ATP release through exocytic mechanisms.

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.

Modulation of adenosine receptor expression in the proximal tubule: a novel adaptive mechanism to regulate renal salt and water metabolism

about 180 liters of filtrate are produced by the human kidneys every day, with more than 99% of the filtered salt and water being subsequently reabsorbed along the nephron. In view of this high level of renal filtration, even slight alterations in the balance between filtration and reabsorption will

Dynamic localization of hepatocellular transporters in health and disease

Vesicle-based trafficking of hepatocellular transporters involves delivery of the newly-synthesized carriers from the rough endoplasmic reticulum to either the plasma membrane domain or to an endosomal, submembrane compartment, followed by exocytic targeting to the plasma membrane. Once delivered to the plasma membrane, the transporters usually undergo recycling between the plasma membrane and the endosomal compartment, which usually serves as a reservoir of pre-existing transporters available on demand. The balance between exocytic targeting and endocytic internalization from/to this recycling compartment is therefore a chief determinant of the overall capability of the liver epithelium to secrete bile and to detoxify endo and xenobiotics. Hence, it is a highly regulated process. Impaired regulation of this balance may lead to abnormal localization of these transporters, which results in bile secretory failure due to endocytic internalization of key transporters involved in bile formation. This occurs in several experimental models of hepatocellular cholestasis, and in most human cholestatic liver diseases. This review describes the molecular bases involved in the biology of the dynamic localization of hepatocellular transporters and its regulation, with a focus on the involvement of signaling pathways in this process. Their alterations in different experimental models of cholestasis and in human cholestatic liver disease are reviewed. In addition, the causes explaining the pathological condition (e.g. disorganization of actin or actin-transporter linkers) and the mediators involved (e.g. activation of cholestatic signaling transduction pathways) are also discussed. Finally, several experimental therapeutic approaches based upon the administration of compounds known to stimulate exocytic insertion of canalicular transporters (e.g. cAMP, tauroursodeoxycholate) are described.

Real-Time Imaging of Discrete Exocytic Events Mediating Surface Delivery of AMPA Receptors

We directly resolved discrete exocytic fusion events mediating insertion of AMPA-type glutamate receptors (AMPARs) to the somatodendritic surface of rat hippocampal pyramidal neurons, in slice and dissociated cultures, using protein tagging with a pH-sensitive GFP (green fluorescent protein) variant and rapid (10 frames/s) fluorescence microscopy. AMPAR-containing exocytic events occurred under basal culture conditions in both the cell body and dendrites; potentiating chemical stimuli produced an NMDA receptor-dependent increase in the frequency of individual exocytic events. The number of AMPARs inserted per exocytic event, estimated using single-molecule analysis, was quite uniform but individual events differed significantly in kinetic properties affecting the subsequent surface distribution of receptors. "Transient" events, from which AMPARs dispersed laterally immediately after surface insertion, generated a pronounced but short-lived (dissipating within approximately 1 s) increase in surface AMPAR fluorescence extending locally (2-5 microm) from the site of exocytosis. "Persistent" events, from which inserted AMPARs dispersed slowly (typically over 5-10 s), affected local surface receptor concentration to a much smaller degree. Both modes of exocytic insertion occurred throughout the dendritic shaft, but remarkably, neither mode of insertion was observed directly into synaptic spines. AMPARs entered spines preferentially from transient events occurring in the adjoining dendritic shaft, driven apparently by mass action and short-range lateral diffusion, and locally delivered AMPARs remained mostly in the mobile fraction. These results suggest a highly dynamic mechanism for both constitutive and activity-dependent surface delivery of AMPARs, mediated by kinetically distinct exocytic modes that differ in propensity to drive lateral entry of receptors to nearby synapses.

Cellular and subcellular distribution of the type-2 vasopressin receptor in the kidney

Arginine vasopressin (AVP) is essential for maintaining body fluid homeostasis. The antidiuretic effects of AVP are initialized by binding of AVP to the type-2 vasopressin receptor (V2R) in the kidney collecting duct (CD), resulting in the exocytic insertion of aquaporin-2 (AQP-2) water channels into the apical plasma membrane. In this study, we describe the generation and characterization of a polyclonal antibody targeted against the NH2 terminus of the rat V2R. HEK-293 cells overexpressing the rat, mouse, or human V2R showed strong intracellular immunolabeling. Additionally, immunostaining of M-1 kidney cells expressing a V2R-green fluorescent protein (GFP) fusion construct showed colocalization between GFP and antibody-specific V2R labeling. Immunoblots of rat kidney showed 43- and 47-kDa proteins in all zones that were both reduced to 34-kDa by N-glycosidase F. Protein solubilization with nonionic detergents or the use of homobifunctional cross-linkers demonstrated that the rat V2R exists as a protein complex in native kidney. Immunohistochemistry of rat and mouse kidney revealed abundant labeling of the CD. Double-labeling confocal immunofluorescence microscopy [using distal convoluted tubule/connecting tubule (CNT)-specific marker calbindin and CNT/CD-specific marker AQP-2] showed V2R labeling in both CD and CNT. There was a complete absence of labeling in vascular structures and other renal tubules, including the thick ascending limb (TAL), although RT-PCR of microdissected tubules showed expression of V2R mRNA in TAL. Confocal microscopy demonstrated that at the subcellular level, V2R labeling was predominantly intracellular in normal kidneys, although some staining was apparent in basolateral membrane domains. Confocal microscopy of isolated inner medullary collecting duct tubules showed that the V2R is expressed both intracellularly and in basolateral membrane domains.

Ca2+ dependence of flow-stimulated K secretion in the mammalian cortical collecting duct

Apical low-conductance SK and high-conductance Ca(2+)-activated BK channels are present in distal nephron, including the cortical collecting duct (CCD). Flow-stimulated net K secretion (J(K)) in the CCD is 1) blocked by iberiotoxin, an inhibitor of BK but not SK channels, and 2) associated with an increase in [Ca(2+)](i), leading us to conclude that BK channels mediate flow-stimulated J(K). To examine the Ca(2+) dependence and sources of Ca(2+) contributing to flow-stimulated J(K), J(K) and net Na absorption (J(Na)) were measured at slow (approximately 1) and fast (approximately 5 nl.min(-1).mm(-1)) flow rates in rabbit CCDs microperfused in the absence of luminal Ca(2+) or after pretreatment with BAPTA-AM to chelate intracellular Ca(2+), 2-aminoethoxydiphenyl borate (2-APB), to inhibit the inositol 1,4,5-trisphosphate (IP(3)) receptor or thapsigargin to deplete internal stores. These treatments, which do not affect flow-stimulated J(Na) (Morimoto et al. Am J Physiol Renal Physiol 291: F663-F669, 2006), inhibited flow-stimulated J(K). Increases in [Ca(2+)](i) stimulate exocytosis. To test whether flow induces exocytic insertion of preformed BK channels into the apical membrane, CCDs were pretreated with 10 microM colchicine (COL) to disrupt microtubule function or 5 microg/ml brefeldin-A (BFA) to inhibit delivery of channels from the intracellular pool to the plasma membrane. Both agents inhibited flow-stimulated J(K) but not J(Na) (Morimoto et al. Am J Physiol Renal Physiol 291: F663-F669, 2006), although COL but not BFA also blocked the flow-induced [Ca(2+)](i) transient. We thus speculate that BK channel-mediated, flow-stimulated J(K) requires an increase in [Ca(2+)](i) due, in part, to luminal Ca(2+) entry and ER Ca(2+) release, microtubule integrity, and exocytic insertion of preformed channels into the apical membrane.

Efficient Trafficking of MDR1/P-Glycoprotein to Apical Canalicular Plasma Membranes in HepG2 Cells Requires PKA-RIIα Anchoring and Glucosylceramide

In hepatocytes, cAMP/PKA activity stimulates the exocytic insertion of apical proteins and lipids and the biogenesis of bile canalicular plasma membranes. Here, we show that the displacement of PKA-RIIalpha from the Golgi apparatus severely delays the trafficking of the bile canalicular protein MDR1 (P-glycoprotein), but not that of MRP2 (cMOAT), DPP IV and 5'NT, to newly formed apical surfaces. In addition, the direct trafficking of de novo synthesized glycosphingolipid analogues from the Golgi apparatus to the apical surface is inhibited. Instead, newly synthesized glucosylceramide analogues are rerouted to the basolateral surface via a vesicular pathway, from where they are subsequently endocytosed and delivered to the apical surface via transcytosis. Treatment of HepG2 cells with the glucosylceramide synthase inhibitor PDMP delays the appearance of MDR1, but not MRP2, DPP IV, and 5'NT at newly formed apical surfaces, implicating glucosylceramide synthesis as an important parameter for the efficient Golgi-to-apical surface transport of MDR1. Neither PKA-RIIalpha displacement nor PDMP inhibited (cAMP-stimulated) apical plasma membrane biogenesis per se, suggesting that other cAMP effectors may play a role in canalicular development. Taken together, our data implicate the involvement of PKA-RIIalpha anchoring in the efficient direct apical targeting of distinct proteins and glycosphingolipids to newly formed apical plasma membrane domains and suggest that rerouting of Golgi-derived glycosphingolipids may underlie the delayed Golgi-to-apical surface transport of MDR1.

Cellular and Subcellular Localization of the Type 2 Vasopressin Receptor in Kidney

Arginine vasopressin (AVP) is essential in body fluid homeostasis. The antidiuretic effects of AVP are initialized by binding of AVP to the type 2 vasopressin receptor (V2R), resulting in the exocytic insertion of aquaporin-2 water channels into the apical plasma membrane. Furthermore, V2R has been proposed to play a role in the TAL in the regulation of sodium balance. However, difficulties in obtaining antibodies, defining the exact localization and studying the regulation of the V2R have prevented progress in the understanding of V2R. We have generated and characterized a polyclonal antibody targeted against the 19 NH2-terminal amino acids of the rat V2R. HEK293 cells overexpressing the rat or mouse V2R showed strong intracellular immunolabeling, whereas no labeling was observed in non-transfected cells. In vitro translation of rat V2R cDNA followed by immunoblotting revealed an approximately 60kDa protein product that was not apparent in control reactions. Immunoblots of rat kidney showed an approximately 60kDa band in all regions that was not apparent after peptide pre-absorption. Immunohistochemistry of rat and mouse kidney revealed abundant labeling of the collecting-duct throughout the kidney. In addition, double labeling confocal immunofluorescence microscopy (using DCT and CNT specific marker calbindin and CD principal cell specific marker AQP2) showed that staining was restricted to collecting-duct and connecting tubule principal cells and IMCD cells. There was a complete absence of labeling in vascular structures and other renal tubules, including PT and TAL. At the subcellular level, labeling was predominantly intracellular, although some staining was apparent in basolateral membrane domains.

Isolation and characterization of lipid microdomains from apical and basolateral plasma membranes of rat hepatocytes

Canalicular bile is formed by the osmotic filtration of water in response to osmotic gradients generated by active transport at the apical and basolateral plasma membrane domains of hepatocytes. We recently demonstrated that mixed plasma membrane fractions isolated from rat hepatocyte couplets contain lipid microdomains ("rafts") enriched in cholesterol and sphingolipids and AQP8 and 9. We isolated lipid microdomains from hepatocyte apical and basolateral plasma membrane domains using Triton X-100 as detergent, and characterized their lipid and protein composition. A Triton-insoluble band ("raft fraction") at the 5%/30% sucrose interface in both apical and basolateral fractions was enriched for alkaline phosphatase (apical) and Na/K ATPase (basolateral) and was negative for amino peptidase-N. This detergent-insoluble band was also positive for caveolin-1 (a "raft" associated protein) and negative for clathrin (a "raft" negative protein). Lipid analysis showed that, the Triton-insoluble fraction was highly enriched in cholesterol and sphingolipids. Immunofluorescence staining on hepatocyte couplets for both caveolin-1 and cholera toxin B showed a punctate distribution on both the apical and basolateral plasma membranes, consistent with localized membrane microdomains. Dot blot analysis showed that the "raft" associated ganglioside GM1 was enriched in the detergent-insoluble fraction both domains. Furthermore, exposure of isolated hepatocytes to glucagon, a choleretic agonist, significantly increased the expression of AQP8 associated with the apical microdomain fractions but had no effect on AQP9 expression in the basolateral microdomain fractions. In conclusion, "rafts" represent target microdomains for exocytic insertion and retrieval of "flux proteins", including AQPs, involved in canalicular bile secretion.

Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells

H(+) transport in the collecting duct is regulated by exocytic insertion of H(+)-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H(+)-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H(+)-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of H(+)-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1ADeltaC [amino acids (aa) 1-264] formed complexes with H(+)-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H(+)-ATPase (aa 235-264) and another that bound SNAP23 and VAMP (aa 190-234) to an equivalent degree as full-length syntaxin. However, the aa 235-264 cassette alone without the SNARE N (aa 1-160) does not bind but requires ligation to the SNARE N to bind H(+)-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H(+)-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H(+)-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H(+)-ATPase exocytosis.

Acute ENaC Stimulation by cAMP in a Kidney Cell Line is Mediated by Exocytic Insertion from a Recycling Channel Pool

Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na+ transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, MPKCCD14) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (ISC), membrane capacitance (CT), and conductance (GT) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 ± 6% (n = 31) increase in ISC and a 15.3 ± 1.5% (n = 15) increase in CT. Selective membrane permeabilization demonstrated that the CT increase was due to an increase in apical membrane capacitance. ISC and CT declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (∼1 h each cycle) resulted in response fatigue; ΔISC decreased ∼10% per stimulation–recovery cycle. When channel production was blocked by cycloheximide, ΔISC decreased ∼15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated ISC, and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the ISC responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.

Role of microtubules in estradiol-17beta-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity.

Estradiol-17beta-D-glucuronide (E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 micromol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 micromol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h). Administration of E2-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process.