Tonicity inversely modulates lipocalin-2 (Lcn2/24p3/NGAL) receptor (SLC22A17) and Lcn2 expression via Wnt/ß-catenin signaling in renal inner medullary collecting duct cells: implications for cell fate and bacterial infection.

BACKGROUND: We have previously evidenced apical expression of the 24p3/NGAL/lipocalin-2 receptor (Lcn2-R; SLC22A17) in inner medullary collecting duct (IMCD) cells, which are present in vivo in a hyperosmotic/-tonic environment that activates canonical Wnt/ß-catenin signaling. The localization of Lcn2-R in the inner medulla is intriguing considering local bacterial infections trigger toll-like receptor-4 (TLR-4)-mediated secretion of the bacteriostatic Fe3+-free (apo-)Lcn2. AIM: To determine the effects of osmolarity/tonicity changes, Wnt/ß-catenin and TLR-4 activation on Lcn2-R and Lcn2 expression and cell viability in rat primary IMCD and mouse (m)IMCD3 cells. METHODS: Normosmolarity/-tonicity was 300 mosmol/l whereas hyperosmolarity/-tonicity was induced by adding 100 mmol/l NaCl + 100 mmol/l urea (600 mosmol/l, 1-7 days). Lcn2-R and Lcn2 expression were determined by qPCR, immunoblotting, flow cytometry and immunofluorescence microscopy. ß-catenin was silenced by RNAi. Cell viability/death was determined with MTT and LDH release assays. TLR-4 was activated by bacterial lipopolysaccharides (LPS). RESULTS: Hyperosmotic/-tonic media upregulated Lcn2-R by ~4-fold and decreased Lcn2 expression/secretion, along with Wnt/ß-catenin activation, in IMCD cells. These effects of hyperosmotic/-tonic media on Lcn2-R/Lcn2 expression were reverted by normosmolarity/-tonicity, ß-catenin silencing and/or LPS. Exposure of cells with endogenous or stably overexpressing Lcn2-R to apo-Lcn2 or LPS decreased cell viability. CONCLUSIONS: Lcn2-R upregulation and Lcn2 downregulation via Wnt/ß-catenin may promote adaptive osmotolerant survival of IMCD cells in response to hyperosmolarity/-tonicity whereas Lcn2 upregulation and Lcn2-R downregulation via TLR-4 and/or normosmolarity/-tonicity may protect IMCD cells against bacterial infections and prevent autocrine death induction by Lcn2.

Initial autophagic protection switches to disruption of autophagic flux by lysosomal instability during cadmium stress accrual in renal NRK-52E cells.

The renal proximal tubule (PT) is the major target of cadmium (Cd2+) toxicity where Cd2+ causes stress and apoptosis. Autophagy is induced by cell stress, e.g., endoplasmic reticulum (ER) stress, and may contribute to cell survival or death. The role of autophagy in Cd2+-induced nephrotoxicity remains unsettled due to contradictory results and lack of evidence for autophagic machinery damage by Cd2+. Cd2+-induced autophagy in rat kidney PT cell line NRK-52E and its role in cell death was investigated. Increased LC3-II and decreased p62 as autophagy markers indicate rapid induction of autophagic flux by Cd2+ (5-10 µM) after 1 h, accompanied by ER stress (increased p-PERK, p-eIF2α, CHOP). Cd2+ exposure exceeding 3 h results in p62/LC3-II accumulation, but diminished effect of lysosomal inhibitors (bafilomycin A1, pepstatin A +E-64d) on p62/LC3-II levels, indicating decreased autophagic flux and cargo degradation. At 24 h exposure, Cd2+ (5-25 µM) activates intrinsic apoptotic pathways (Bax/Bcl-2, PARP-1), which is not evident earlier (≤6 h) although cell viability by MTT assay is decreased. Autophagy inducer rapamycin (100 nM) does not overcome autophagy inhibition or Cd2+-induced cell viability loss. The autophagosome-lysosome fusion inhibitor liensinine (5 µM) increases CHOP and Bax/Bcl-2-dependent apoptosis by low Cd2+ stress, but not by high Cd2+. Lysosomal instability by Cd2+ (5 µM; 6 h) is indicated by increases in cellular sphingomyelin and membrane fluidity and decreases in cathepsins and LAMP1. The data suggest dual and temporal impact of Cd2+ on autophagy: Low Cd2+ stress rapidly activates autophagy counteracting damage but Cd2+ stress accrual disrupts autophagic flux and lysosomal stability, possibly resulting in lysosomal cell death.

Renal cells exposed to cadmium in vitro and in vivo: normalizing gene expression data.

Cadmium (Cd) is a toxic metal with a long half-life in biological systems. This half-life is partly as a result of metallothioneins (MTs), metal-binding proteins with a high affinity for Cd. The high retention properties of the kidneys reside in proximal tubular cells that possess transport mechanisms for Cd-MT uptake, ultimately leading to more Cd accumulation. Researchers have studied MT-metal interactions using various techniques including quantitative real-time PCR (qPCR), an efficient tool for quantifying gene expression. Often a poor choice of reference genes, which is represented by their instability and condition dependency, leads to inefficient normalization of gene expression data and misinterpretations. This study demonstrates the importance of an efficient normalization strategy in toxicological research. A selection of stable reference genes was proposed in order to acquire reliable and reproducible gene quantification under metal stress using MT expression as an example. Moreover, in vitro and in vivo setups were compared to identify the influence of toxicological compounds in function of the experimental design. This study shows that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine monooxygenase/tryptophan5-monooxygenase activation-protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) are the most stable reference genes in a kidney proximal tubular cell line exposed to moderate and high Cd concentrations, applied as CdCl2 . A slightly different sequence in reference gene stability was found in renal cells isolated from rats in vivo exposed to Cd. It was further shown that three reference genes are required for efficient normalization in this experimental setup. This study demonstrates the importance of an efficient normalization strategy in toxicological research.

The role of Wnt/beta-catenin signaling in renal carcinogenesis: lessons from cadmium toxicity studies.

Wnt/beta-catenin signaling plays a crucial role during embryogenesis. However, this signaling pathway also plays a role in normal adult tissues and in carcinogenesis, including cadmium (Cd2+) induced nephrocarcinogenesis, which is the topic of this review. Wnt/beta-catenin signaling is tightly regulated in mature epithelia to balance cell proliferation, differentiation and death. This is accomplished by modulating phosphorylation of the multifunctional protein beta-catenin which in turn determines its preference for a particular fate, i.e. cell-cell adhesion by binding to E-cadherin, proteasomal degradation, or co-activation of the transcription factor Tcf/Lef. The pivotal role of beta-catenin is not limited to Wnt signaling, but can be challenged by other transcription factors under stress conditions (e.g. FOXO, HIF-1alpha, NF-kappaB, c-jun), where beta-catenin acts as a molecular switch in response to the cellular redox status. Aberrant Wnt/beta-catenin signaling can contribute to carcinogenesis of intestinal, lung or kidney epithelia, either by mutations of its signaling components and/or disruption of linked signaling networks. The nephrotoxic metal Cd2+ causes renal cancer in humans. Because it is not genotoxic Cd2+ is thought to induce mutations and carcinomas indirectly: Possible mechanisms include oxidative stress, inhibition of DNA repair, aberrant gene expression, deregulation of cell proliferation, resistance to apoptosis, and/or disruption of cell adhesion. Wnt signaling may contribute to Cd2+ carcinogenesis because Cd2+ disrupts the junctional E-cadherin/beta-catenin complex, resulting in excessive nuclear translocation of beta-catenin and activation of Tcf4. Up-regulation of target genes of the beta-catenin/Tcf4 complex, such as c-myc, cyclin D1 and the multidrug transporter P-glycoprotein (MDR1/ABCB1), leads to increased proliferation, evasion of apoptosis, adaptation to Cd2+ toxicity and thereby promotes the selection of mutated and pre-neoplastic cells.

Expression of a sodium bicarbonate cotransporter in human parotid salivary glands.

The human parotid gland secretes much of the bicarbonate that enters the mouth. Prompted by studies of animal models, this study sought evidence for the expression of a functional Na(+)-HCO(3)(-) cotransporter (NBC) in human parotid acinar cells. Microfluorometric measurements of intracellular pH in isolated acini showed that the recovery from an acid load was achieved in part by HCO(3)(-) uptake via a Na(+)-dependent, DIDS-sensitive mechanism. By reverse transcriptase-polymerase chain reaction, a full-length NBC1 clone was obtained showing more than 99% homology with the human pancreatic isoform hpNBC1. Expressed in Xenopus oocytes, the electrogenicity of the transporter was detected as an inwardly directed, Na(+)- and HCO(3)(-)-dependent flux of negative charge. Immunohistochemistry using antibodies raised to NBC1 showed strong staining of the basolateral membrane of the acinar cells. Therefore, it was concluded that a functional electrogenic Na(+)-HCO(3)(-) cotransporter is expressed in the human parotid gland, and that it contributes to pH regulation in the acinar cells and could play a significant part in salivary secretion.

Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification.

ATP-dependent priming of the secretory granules precedes Ca(2+)-regulated neuroendocrine secretion, but the exact nature of this reaction is not fully established in all secretory cell types. We have further investigated this reaction in the insulin-secreting pancreatic B-cell and demonstrate that granular acidification driven by a V-type H(+)-ATPase in the granular membrane is a decisive step in priming. This requires simultaneous Cl(-) uptake through granular ClC-3 Cl(-) channels. Accordingly, granule acidification and priming are inhibited by agents that prevent transgranular Cl(-) fluxes, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and an antibody against the ClC-3 channels, but accelerated by increases in the intracellular ATP:ADP ratio or addition of hypoglycemic sulfonylureas. We suggest that this might represent an important mechanism for metabolic regulation of Ca(2+)-dependent exocytosis that is also likely to be operational in other secretory cell types.

Immortalized bovine pancreatic duct cells become tumorigenic after transfection with mutant k-ras.

Mutation of the K-ras gene is thought to be an early and important event in pancreatic carcinogenesis. In order to study the role of this molecular alteration in the transition from the normal to the neoplastic pancreatic cell, bovine pancreatic duct cells were first immortalized by SV40 large T antigen (Ag) complementary (c)DNA transfection and then transfected with a mutated K-ras gene. As did primary duct cells, the immortalized duct cells (more than 100 passages) expressed cytokeratins, carbonic anhydrase type-II, cystic fibrosis transmembrane conductance regulator (CFTR), and multidrug resistance (mdr). They grew as a single layer after transplantation under plastic domes and formed three-dimensional structures resembling ducts when grown on Matrigel. Cell growth was stimulated by insulin, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, but cells did not respond to gastrin and CCK-8. They did not form colonies in soft agar nor did they form tumors in nude mice. Immortalized cells transfected with mutated K-ras acquired the ability to form tumors after orthotopic injection into the nude mouse pancreas. It is concluded that SV 40 immortalized bovine pancreatic

Expression of multidrug resistance P-glycoprotein in kidney allografts from cyclosporine A-treated patients.

BACKGROUND: The multidrug resistance (MDR) gene product P-glycoprotein (P-gp) is a transmembrane efflux pump for hydrophobic, potentially toxic compounds, including the immunosuppressant cyclosporine A (CsA). We have previously shown that CsA increases P-gp expression in proximal tubule and endothelial cells in vitro. The aim of the present study was to investigate the in vivo relevance of these observations in renal allograft biopsies from CsA-treated patients. METHODS: P-gp expression was determined by immunohistochemistry of paraffin sections using two different monoclonal antibodies (UIC2 and MRK16). Biopsies were taken from CsA-treated renal transplant patients with different histopathological diagnoses (N = 79) and were compared with biopsies from normal human kidneys (N = 13) or with allograft biopsies from patients under a CsA-free immunosuppression (N = 15). Moreover, biopsies from 10 donor kidneys before implantation and during rejection episodes ("zero biopsies") were investigated. RESULTS: P-gp expression in biopsies with acute tubular necrosis (ATN; N = 10) after CsA treatment was significantly higher in arterial endothelia, proximal tubules, and epithelial cells of Bowman's capsule (BC), whereas P-gp was sparsely induced in CsA nephrotoxicity (N = 19) compared with controls. Acute cellular (N = 30) and vascular rejection (N = 10) or chronic allograft nephropathy (N = 10) after CsA was associated with strong P-gp expression in infiltrating leukocytes and increased P-gp expression in arterial endothelia, proximal tubules, and BC. In contrast, biopsies of patients treated with a CsA-free immunosuppression regimen did not show increases in P-gp expression compared with controls. Zero biopsies showed a weak, homogeneous, nonpolarized expression of P-gp in tubules and an increased expression of P-gp after CsA therapy in the brush border, arterial endothelia, and BC. CONCLUSIONS: CsA treatment was associated with increased P-gp expression in parenchymal cells of kidney transplants with ATN, acute or chronic transplant rejection, but P-gp was not increased in patients with CsA nephrotoxicity. This indicates that CsA induces its own detoxification by P-gp and that inadequate up-regulation of P-gp in renal parenchymal cells contributes to CsA nephrotoxicity. Increased expression of P-gp in infiltrating leukocytes correlated with the severity of allograft rejection, suggesting that P-gp may decrease the immunosuppressive efficacy of CsA. Thus, individual differences in the P-gp induction response of CsA-exposed renal parenchymal cells and/or infiltrating leukocytes may predispose to either CsA nephrotoxicity or rejection, respectively.

Immunolocalization of anion exchanger AE2, Na(+)/H(+) exchangers NHE1 and NHE4, and vacuolar type H(+)-ATPase in rat pancreas.

We have studied the expression and localization of several H(+) and HCO(3)(-) transporters, whose presence in the rat pancreas is still unclear. The Cl(-)/HCO(3)(-) exchanger AE2, the Na(+)/H(+) exchangers NHE1 and NHE4, and the 31-kD and 70-kD vacuolar H(+)-ATPase (V-ATPase) subunits were detected by immunoblotting and immunocytochemical techniques. Immunoblotting of plasma membranes with transporter-specific antibodies revealed protein bands at approximately 160 kD for AE2, at approximately 90 kD and approximately 103 kD for NHE1 and NHE4, respectively, and at 31 kD and 70 kD for V-ATPase. NHE1 and NHE4 were further identified by amplification of isoform-specific cDNA using RT-PCR. Immunohistochemistry revealed a basolateral location of AE2, NHE1, and NHE4 in acinar cells. In ducts, NHE1 and NHE4 were basolaterally located but no AE2 expression was detected. V-ATPase was detected in zymogen granules (ZGs) by immunogold labeling, and basolaterally in duct cells by immunohistochemistry. The data indicate that NHE1 and NHE4 are co-expressed in rat pancreatic acini and ducts. Basolateral acinar AE2 could contribute to Cl(-) uptake and/or pH regulation. V-ATPase may be involved in ZG fusion/exocytosis and ductal HCO(3)(-) secretion. The molecular identity of the ductal Cl(-)/HCO(3)(-) exchanger remains unclear.

Molecular characterisation of pancreatic zymogen granule ion channel and regulator proteins involved in exocytosis.

In pancreatic acinar cells Ca(2+)-dependent secretagogues promote the fusion of zymogen granules (ZG) with the apical plasma membrane (PM) and exocytosis of digestive enzymes. In addition to exocytotic fusion complexes between SNARE proteins in the ZG membrane (ZGM) and the apical PM, enzyme secretion elicited by Ca(2+)-dependent secretagogues requires cytosolic Cl and K+ and is inhibited by blockers of Cl- and K+-channels. We have identified a Cl-conductance activated by ATP, and a K+-conductance (with properties similar to ATP-sensitive K+-channels), regulated by the granule matrix protein Zg-16p in the ZGM. Both conductances are inversely regulated by a 65-kD mdr1 gene product. We have also identified a novel Ca(2+)-activated anion conductance in ZGM, the Ca(2+)-sensitivity of which increases 50-fold when Cl is replaced by 1. This conductance is blocked by micromolar H2-DIDS or DTT, reminiscent of a family of epithelial Ca(2+)-activated Cl -channels (CaCC). Expression of a CaCC in exocrine pancreas has been confirmed by RT-PCR analysis, and by immunoblotting and immunogold labeling of ZG membranes. These data suggest that ion channels in the ZGM are essential elements in pancreatic exocytosis.

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation.

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis¿2-aminophenoxyethane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.

Up-regulation of multidrug resistance P-glycoprotein via nuclear factor-kappaB activation protects kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis.

Cadmium-mediated toxicity of cultured proximal tubule (PT) cells is associated with increased production of reactive oxygen species (ROS) and apoptosis. We found that cadmium-dependent apoptosis (Hoechst 33342 and annexin V assays) decreased with prolonged CdCl(2) (10 microM) application (controls: 2.4 +/- 1.6%; 5 h: +5.1 +/- 2.3%, 20 h: +5.7 +/- 2.5%, 48 h: +3.3 +/- 1.0% and 72 h: +2.1 +/- 0.4% above controls), while cell proliferation was not affected. Reduction of apoptosis correlated with a time-dependent up-regulation of the drug efflux pump multidrug resistance P-glycoprotein (mdr1) in cadmium-treated cells ( approximately 4-fold after 72 h), as determined by immunoblotting with the monoclonal antibody C219 and measurement of intracellular accumulation of the fluorescent probe calcein +/- the mdr1 inhibitor PSC833 (0.5 microM). When mdr1 inhibitors (PSC833, cyclosporine A, verapamil) were transiently added to cells with mdr1 up-regulation by pretreatment for 72 h with cadmium, cadmium-induced apoptosis increased significantly and to a percentage similar to that obtained in cells with no mdr1 up-regulation (72-h cadmium: 5.2 +/- 0.9% versus 72-h cadmium + 1-h PSC833: 7.2 +/- 1.4%; p < or = 0.001). Cadmium-induced apoptosis and mdr1 up-regulation depended on ROS, since co-incubation with the ROS scavengers N-acetylcysteine (15 mM) or pyrrolidine dithiocarbamate (0.1 mM) abolished both responses. Moreover, cadmium- and ROS-associated mdr1 up-regulation was linked to activation of the transcription factor NF-kappaB; N-acetylcysteine, pyrrolidine dithiocarbamate, and the IkappaB-alpha kinase inhibitor Bay 11-7082 (20 microM) prevented both, mdr1 overexpression and degradation of the inhibitory NF-kappaB subunit, IkappaB-alpha, induced by cadmium. The data show that 1) cadmium-mediated apoptosis in PT cells is associated with ROS production, 2) ROS increase mdr1 expression by a process involving NF-kappaB activation, and 3) mdr1 overexpression protects PT cells against cadmium-mediated apoptosis. These data suggest that mdr1 up-regulation, at least in part, provides anti-apoptotic protection for PT cells against cadmium-mediated stress.

Photoaffinity labeling and purification of ZG-16p, a high-affinity dihydropyridine binding protein of rat pancreatic zymogen granule membranes that regulates a K(+)-selective conductance.

In rat pancreatic zymogen granules (ZG), an ATP-sensitive K(+) conductance and a Cl(-) conductance have been characterized that are inversely regulated by an approximately 65-kDa multidrug resistance P-glycoprotein (mdr1) gene product. In search of a label for purification of this protein, we found that the dihydropyridine derivative (-)-[(3)H]BZDC-DHP, a recently developed high-affinity ligand for Mdr1, binds with similar affinity to ZG membranes (ZGM) (K(d) = 6.2 nM). Binding was inhibited by nanomolar concentrations of the L-type Ca(2+) channel blockers azidopine and verapamil and by micromolar concentrations of the K(+) channel blockers glibenclamide and quinidine. Inhibition by glibenclamide was noncompetitive. The Mdr1 modulators cyclosporin A and vinblastine did not inhibit binding, which is different from Mdr1. In addition, only (+/-)-BZDC-DHP, azidopine, and verapamil selectively inhibited the K(+) conductance in ZGs, whereas the Cl(-) conductance was not affected. In photoaffinity labeling experiments, (-)-[(3)H]BZDC-DHP surprisingly specifically and selectively labeled a approximately 19-kDa protein in ZGM with a pharmacological profile identical with the high-affinity binding site but did not label a 65-kDa protein. The 19-kDa protein was purified by ion exchange chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The sequence obtained corresponds to ZG-16p, a recently cloned ZG protein with no apparent homology to Mdr1. The identity of the 19-kDa protein was confirmed by immunoprecipitation of (-)-[(3)H]BZDC-DHP-labeled ZGM with an anti-ZG-16p antibody. Furthermore, it is shown that ZG-16p is associated with the ZGM. We propose that ZG-16p, as part of the submembranous granule matrix, regulates the ATP-sensitive K(+) conductance of ZGs.

Immunolocalization of anion exchanger AE2 and Na(+)-HCO(-)(3) cotransporter in rat parotid and submandibular glands.

Salivary glands secrete K(+) and HCO(-)(3) and reabsorb Na(+) and Cl(-), but the identity of transporters involved in HCO(-)(3) transport remains unclear. We investigated localization of Cl(-)/HCO(-)(3) exchanger isoform AE2 and of Na(+)-HCO(-)(3) cotransporter (NBC) in rat parotid gland (PAR) and submandibular gland (SMG) by immunoblot and immunocytochemical techniques. Immunoblotting of PAR and SMG plasma membranes with specific antibodies against mouse kidney AE2 and rat kidney NBC revealed protein bands at approximately 160 and 180 kDa for AE2 and approximately 130 kDa for NBC, as expected for the AE2 full-length protein and consistent with the apparent molecular mass of NBC in several tissues other than kidney. Immunostaining of fixed PAR and SMG tissue sections revealed specific basolateral staining of PAR acinar cells for AE2 and NBC, but in SMG acinar cells only basolateral AE2 labeling was observed. No AE2 expression was detected in any ducts. Striated, intralobular, and main duct cells of both glands showed NBC expression predominantly at basolateral membranes, with some cells being apically stained. In SMG duct cells, NBC staining exhibited a gradient of distribution from basolateral localization in more proximal parts of the ductal tree to apical localization toward distal parts of the ductal tree. Both immunoblotting signals and immunostaining were abolished in preabsorption experiments with the respective antigens. Thus the mechanisms of fluid and anion secretion in salivary acinar cells may be different between PAR and SMG, and, because NBC was detected in acinar and duct cells, it may play a more important role in transport of HCO(-)(3) by rat salivary duct cells than previously believed.

Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K(+)-ATPase through proteasomal and endo-/lysosomal proteolytic pathways.

The mechanisms of cadmium (Cd)-dependent nephrotoxicity were studied in a rat proximal tubule (PT) cell line. CdCl(2) (5 microM) increased the production of reactive oxygen species (ROS), as determined by oxidation of dihydrorhodamine 123 to fluorescent rhodamine 123. The levels of ubiquitin-conjugated cellular proteins were increased by Cd in a time-dependent fashion (maximum at 24-48 h). This was prevented by coincubation with the thiol antioxidant N-acetylcysteine (NAC, 15 mM). Cd also increased apoptosis (controls: 2.4+/-1.6%; Cd: 8.1+/-1.9%), but not necrosis (controls: 0.5 +/- 0.3%; Cd: 1.4+/- 2.5%). Exposure of PT cells with Cd decreased protein levels of the catalytic subunit (alpha1) of Na+/K(+)-ATPase, a long-lived membrane protein (t(1/2)>48 h) that drives reabsorption of ions and nutrients through Na(+)-dependent transporters in PT. Incubation of PT cells for 48 h with Cd decreased Na+/K(+)-ATPase alpha1-subunit, as determined by immunoblotting, by approximately 50%, and NAC largely prevented this effect. Inhibitors of the proteasome such as MG-132 (20 microM) or lactacystin (10 microM), as well as lysosomotropic weak bases such as chloroquine (0.2 mM) or NH(4)Cl (30 mM), significantly reduced the decrease of Na(+)/K(+)-ATPase alpha1-subunit induced by Cd, and in combination abolished the effect of Cd on Na+/K(+)-ATPase. Immunofluorescence labeling of Na+/K(+)-ATPase showed a reduced expression of the protein in the plasma membrane of Cd-exposed cells. After addition of lactacystin and chloroquine to Cd-exposed PT cells, immunoreactive material accumulated into intracellular vesicles. The data indicate that micromolar concentrations of Cd can increase ROS production and exert a toxic effect on PT cells. Oxidative damage increases the degradation of Na+/K(+)-ATPase through both the proteasomal and endo-/lysosomal proteolytic pathways. Degradation of oxidatively damaged Na+/K(+)-ATPase may contribute to the 'Fanconi syndrome'-like Na(+)-dependent transport defects associated with Cd-nephrotoxicity.

Cloning and immunolocalization of a rat pancreatic Na(+) bicarbonate cotransporter.

In the rat, pancreatic HCO(-)(3) secretion is believed to be mediated by duct cells with an apical Cl(-)/HCO(-)(3) exchanger acting in parallel with a cAMP-activated Cl(-) channel and protons being extruded through a basolateral Na(+)/H(+) exchanger. However, this may not be the only mechanism for HCO(-)(3) secretion by the rat pancreas. Recently, several members of electrogenic Na(+)/HCO(-)(3) cotransporters (NBC) have been cloned. Here we report the cloning of a NBC from rat pancreas (rpNBC). This rpNBC is 99% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas, prostate, and a minor clone in kidney]. The longer NBC isoforms are identical to the rat and human kidney-specific forms (kNBC; 1035 aa; 116 kDa) at the approximately 980 C-terminal aa's and are unique (with different lengths) at the initial N-terminus. Using polyclonal antibodies to the common N- and C-termini of rat kidney NBC, a approximately 130-kDa protein band was labeled by immunoblotting of rat pancreas homogenate and was enriched in the plasma membrane fraction. Immunofluorescence and immunoperoxidase light microscopy of rat pancreatic tissue with both antibodies revealed basolateral labeling of acinar cells. Labeling of both apical and basolateral membranes was found in centroacinar cells, intra- and extralobular duct, and main duct cells. The specificity of the antibody labeling was confirmed by antibody preabsorption experiments with the fusion protein used for immunization. The data suggest that rpNBC likely plays a more important role in the transport of HCO(-)(3) by rat pancreatic acinar and duct cells than previously believed.

The stimulatory action of tolbutamide on Ca2+-dependent exocytosis in pancreatic beta cells is mediated by a 65-kDa mdr-like P-glycoprotein.

Intracellular application of the sulfonylurea tolbutamide during whole-cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca2+ concentration of 0.17 microM. This effect was not detectable in the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl--channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl- conductance. We speculate that the activation of granular Cl- fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydrostatic pressure.

Distribution of V-ATPase in rat salivary glands.

Using immunohistochemistry we have investigated the presence and cellular distribution of the 31-kDa subunit of vacuolar-type H+-ATPase (V-ATPase) in secretory endpieces and the duct system of rat major salivary glands. In all three salivary glands studied the 31-kDa subunit of V-ATPase was not expressed in secretory endpieces. In rat parotid gland V-ATPase was luminally located in main excretory and striated duct cells. In contrast, both rat submandibular and sublingual glands showed a diffuse intracellular V-ATPase distribution. The differences in V-ATPase immunolocalization in rat salivary glands probably reflect the structural heterogeneity of the different glands. The data also suggest that the duct systems of major salivary glands may modify the H+ and HCO3- concentration of the final saliva in different ways.

Therapeutic concentrations of cyclosporine A, but not FK506, increase P-glycoprotein expression in endothelial and renal tubule cells.

BACKGROUND: The immunosuppressive drugs cyclosporine A (CsA) and tacrolimus (FK506) are extruded from cells by the multidrug resistance P-glycoprotein (P-gp), an efflux pump for drugs and xenobiotics, which may limit their therapeutic effectiveness and/or incidence of toxic side effects. In the present study, we investigated the effect of therapeutic concentrations of CsA and FK506 on the expression of P-gp in cultured endothelial and proximal tubule cells. METHODS: P-gp expression in human arterial endothelial (HAEC) and rat proximal tubule cells (RPTC) was determined by immunoblotting and immunocytochemistry, and correlated with P-gp-mediated transport by measuring the intracellular accumulation of the fluorescent probe calcein. RESULTS: Following incubation of HAEC with therapeutic concentrations of 0.1 to 1.6 microM CsA up to seven days, P-gp expression increased in a time- and concentration-dependent manner, maximally to 291 +/- 42% of controls with 0.8 microM CsA for seven days. Similar effects of CsA were observed in RPTC. In contrast, therapeutic concentrations of FK506 (0.01 to 0.2 microM up to 7 days) did not change P-gp expression in either cell type, though at higher, supratherapeutic concentrations of FK506 (0.6 to 1.2 microM) P-gp expression was also increased. Immunocytochemistry revealed increased P-gp expression in the plasma membrane of HAEC and RPTC treated with 0.8 microM CsA, which was reflected by a decrease of P-gp-mediated accumulation of calcein in both cell types. CONCLUSIONS: The data suggest that the induction of P-gp expression in HAEC and RPTC at concentrations of CsA or FK506 above 0.5 microM is part of the protective answer of cells to toxic concentrations of the drugs and could therefore interfere with the therapeutic effectiveness of CsA in vivo.

Expression of NHE1 and NHE4 in rat pancreatic zymogen granule membranes.

We previously characterized a Na+/H+ exchange activity in rat pancreatic zymogen granules [Anderie, I., and Thévenod, F. (1996) J. Membrane Biol, 152, 195-205]. Here we have identified the Na+/H+ exchanger (NHE) isoforms present in zymogen granules by functional studies with NHE inhibitors. The NHE1 specific blocker HOE 694 [3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate] inhibited zymogen granule Na+/H+ exchange in a concentration dependent manner, maximally to 53 +/- 5% of controls at 100nM. The remaining Na+/H+ exchange activity was inhibitable by EIPA [5-(N-ethyl-N-isopropyl)amiloride] (EC50 approximately 25 microM) or benzamil (EC50 approximately 100 microM). Amiloride inhibited weakly suggesting that "amiloride-resistant" and "amiloride-sensitive" NHE are expressed in zymogen granules. cDNA sequences encoding NHE1- and NHE4-specific transmembrane domains were detected by RT-PCR in rat pancreatic tissue and in the rat pancreatic acinar cell line AR4-2J. The presence of NHE1 and NHE4 in zymogen granule membranes was confirmed by immunoblots of zymogen granule membranes and by pre-embedding immunogold labeling of purified rat pancreatic zymogen granules with polyclonal NHE1 and NHE4 antibodies. Therefore, we propose that NHE1 and NHE4 are expressed in zymogen granule membranes of rat exocrine pancreas.