Small molecule inhibitors of intestinal epithelial anion exchanger SLC26A3 (DRA) with a luminal, extracellular site of action.

The anion exchanger protein SLC26A3 (down-regulated in adenoma, DRA) is expressed in the luminal membrane of intestinal epithelial cells in colon, where it facilitates the absorption of Cl- and oxalate. We previously identified a 4,8-dimethylcoumarin class of SLC26A3 inhibitors that act from the SLC26A3 cytoplasmic surface, and demonstrated their efficacy in mouse models of constipation and hyperoxaluria. Here, screening of 50,000 new compounds and 1740 chemical analogs of active compounds from the primary screen produced five novel classes of SLC26A3-selective inhibitors (1,3-dioxoisoindoline-amides; N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamides; thiazolo-pyrimidin-5-ones; 3-carboxy-2-phenylbenzofurans and benzoxazin-4-ones) with IC50 down to 100 nM. Kinetic washout and onset of action studies revealed an extracellular site of action for the thiazolo-pyrimidin-5-one and 3-carboxy-2-phenylbenzofuran inhibitors. Molecular docking computations revealed putative binding sites for these inhibitors. In a loperamide model of constipation in mice, orally administered 7-(2-chloro-phenoxymethyl)-3-phenyl-thiazolo [3,2-a]pyrimidin-5-one (3a) significantly increased stool weight, pellet number and water content. SLC26A3 inhibitors with an extracellular site of action offer the possibility of creating non-absorbable, luminally acting inhibitors with minimal systemic exposure following oral administration. Our findings also suggest that inhibitors of related SLC26 anion transporters with an extracellular site of action might be identified for pharmacological modulation of selected epithelial ion transport processes.

Selenium Increased Arsenic Accumulation by Upregulating the Expression of Genes Responsible for Arsenic Reduction, Translocation, and Sequestration in Arsenic Hyperaccumulator Pteris vittata.

Selenate enhances arsenic (As) accumulation in As-hyperaccumulator Pteris vittata, but the associated molecular mechanisms are unclear. Here, we investigated the mechanisms of selenate-induced arsenic accumulation by exposing P. vittata to 50 µM arsenate (AsV50) and 1.25 (Se1.25) or 5 µM (Se5) selenate in hydroponics. After 2 weeks, plant biomass, plant As and Se contents, As speciation in plant and growth media, and important genes related to As detoxification in P. vittata were determined. These genes included P transporters PvPht1;3 and PvPht1;4 (AsV uptake), arsenate reductases PvHAC1 and PvHAC2 (AsV reduction), and arsenite (AsIII) antiporters PvACR3 and PvACR3;2 (AsIII translocation) in the roots, and AsIII antiporters PvACR3;1 and PvACR3;3 (AsIII sequestration) in the fronds. The results show that Se1.25 was more effective than Se5 in increasing As accumulation in both P. vittata roots and fronds, which increased by 27 and 153% to 353 and 506 mg kg-1. The As speciation analyses show that selenate increased the AsIII levels in P. vittata, with 124-282% more AsIII being translocated into the fronds. The qPCR analyses indicate that Se1.25 upregulated the gene expression of PvHAC1 by 1.2-fold, and PvACR3 and PvACR3;2 by 1.0- to 2.5-fold in the roots, and PvACR3;1 and PvACR3;3 by 0.6- to 1.1-fold in the fronds under AsV50 treatment. Though arsenate enhanced gene expression of P transporters PvPht1;3 and PvPht1;4, selenate had little effect. Our results indicate that selenate effectively increased As accumulation in P. vittata, mostly by increasing reduction of AsV to AsIII in the roots, AsIII translocation from the roots to fronds, and AsIII sequestration into the vacuoles in the fronds. The results suggest that selenate may be used to enhance phytoremediation of As-contaminated soils using P. vittata.

Tumor bud-derived CCL5 recruits fibroblasts and promotes colorectal cancer progression via CCR5-SLC25A24 signaling.

BACKGROUND: Tumor budding is included in the routine diagnosis of colorectal cancer (CRC) and is considered a tumor prognostic factor independent of TNM staging. This study aimed to identify the fibroblast-mediated effect of tumor bud-derived C-C chemokine ligand 5 (CCL5) on the tumor microenvironment (TME). METHODS: Recruitment assays and a human cytokine array were used to detect the main cytokines that CRC tumor buds secrete to recruit fibroblasts. siRNA transfection and inhibitor treatment were used to investigate the role of fibroblast CCL5 receptors in fibroblast recruitment. Subsequently, transcriptome sequencing was performed to explore the molecular changes occurring in fibroblasts upon stimulation with CCL5. Finally, clinical specimens and orthotopic xenograft mouse models were studied to explore the contribution of CCL5 to angiogenesis and collagen synthesis. RESULTS: Hematoxylin-eosin staining and immunochemistry revealed a higher number of fibroblasts at the invasive front of CRC tissue showing tumor budding than at sites without tumor budding. In vitro experiments demonstrated that CCL5 derived from tumor buds could recruit fibroblasts by acting on the CCR5 receptors on fibroblasts. Tumor bud-derived CCL5 could also positively regulate solute carrier family 25 member 24 (SLC25A24) expression in fibroblasts, potentially activating pAkt-pmTOR signaling. Moreover, CCL5 could increase the number of α-SMAhigh CD90high FAPlow fibroblasts and thus promote tumor angiogenesis by enhancing VEGFA expression and making fibroblasts transdifferentiate into vascular endothelial cells. Finally, the results also showed that CCL5 could promote collagen synthesis through fibroblasts, thus contributing to tumor progression. CONCLUSIONS: At the invasive front of CRC, tumor bud-derived CCL5 can recruit fibroblasts via CCR5-SLC25A24 signaling, further promoting angiogenesis and collagen synthesis via recruited fibroblasts, and eventually create a tumor-promoting microenvironment. Therefore, CCL5 may serve as a potential diagnostic marker and therapeutic target for tumor budding in CRC.

Separating the contributions of SLC26A9 and CFTR to anion secretion in primary human bronchial epithelia.

Aberrant anion secretion across the bronchial epithelium is associated with airway disease, most notably in cystic fibrosis. Although the cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as the primary source of airway anion secretion, alternative anion transport mechanisms play a contributing role. An alternative anion transporter of growing interest is SLC26A9, a constitutively active chloride channel that has been shown to interact with CFTR and may also contribute to bicarbonate secretion. Interest in SLC26A9 has been fueled by genome-wide association studies that suggest it is a significant modifier of CF disease severity. Despite this growing evidence that SLC26A9 plays an important role in the airway, its presence and function in bronchial epithelia remain poorly understood, in part, because its activity is difficult to separate from the activity of CFTR. Here, we present results using primary human bronchial epithelia (HBE) from multiple patient sources to confirm that SLC26A9 mRNA is present in HBE and that its constitutive channel activity is unaffected by knockdown of CFTR. Furthermore, SLC26A9 and CFTR show differential responses to common inhibitors of anion secretion. Finally, we assess the impact of bicarbonate on the activity of SLC26A9 and CFTR. These results confirm that SLC26A9 is the primary source of constitutive anion secretion across HBE, and should inform future studies focused on activation of SLC26A9 as an alternative anion channel in CF. These results should provide a strong foundation to investigate how single-nucleotide polymorphisms in SLC26A9 modulate airway disease.

Increased brain penetration of diphenhydramine and memantine in rats with adjuvant-induced arthritis.

Brain penetration of cationic drugs is an important determinant of their efficacy and side effects. However, the effects of alterations in the activity of uptake transporters in the brain under inflammatory conditions on the brain penetration of cationic drugs are not fully understood. The aim of this study was to examine changes in brain penetration of cationic drugs, including diphenhydramine (DPHM), memantine (MMT), and cimetidine (CMD), and changes in the expression of uptake transporters such as organic cation transporter (Oct) in brain microvascular endothelial cells (BMECs) under inflammatory conditions. To clarify the effects of inflammation on the brain penetration of DPHM, MMT, and CMD, we performed brain microdialysis studies in a rat model of adjuvant-induced arthritis (AA). Further, differences in transporter mRNA expression levels between BMECs from control and AA rats were evaluated. Brain microdialysis showed that the unbound brain-to-plasma partition coefficient (Kp,uu,brain) for DPHM and MMT was significantly lower in AA rats compared with control rats. OCT mRNA levels were increased and proton-coupled organic cation (H+/OC) antiporter mRNA levels were decreased in AA rats compared with control rats. Taken together, our findings suggest that inflammation decreases the brain penetration of H+/OC antiporter substrates such as DPHM and MMT.

Alternative chloride transport pathways as pharmacological targets for the treatment of cystic fibrosis.

Cystic fibrosis is a hereditary disease that originates from mutations in the epithelial chloride channel CFTR. Whereas established therapies for the treatment of cystic fibrosis target CFTR to repair its function, alternative therapeutic strategies aim for the restoration of chloride transport by the activation of other chloride transport proteins such as TMEM16A or SLC26A9 or by the application of synthetic anionophores. TMEM16A is an anion-selective channel that is activated by the binding of Ca2+ from the cytoplasm. Pharmacological efforts aim for the increase of its open probability at resting Ca2+ concentrations. SLC26 is an uncoupled chloride transporter, which shuttles chloride across the membrane by an alternate-access mechanism. Its activation requires its mobilization from intracellular stores. Finally, anionophores are small synthetic molecules that bind chloride to form lipid-soluble complexes, which shuttle the anion across the membrane. All three approaches are currently pursued and have provided promising initial results.

4,8-Dimethylcoumarin Inhibitors of Intestinal Anion Exchanger slc26a3 (Downregulated in Adenoma) for Anti-Absorptive Therapy of Constipation.

The chloride/bicarbonate exchanger SLC26A3 (downregulated in adenoma) is expressed mainly in colonic epithelium, where it dehydrates the stool by facilitating the final step of chloride and fluid absorption. SLC26A3 inhibition has predicted efficacy in various types of constipation including that associated with cystic fibrosis. We previously identified, by high-throughput screening, 4,8-dimethylcoumarin inhibitors of murine slc26a3 with IC50 down to ∼150 nM. Here, we synthesized a focused library of forty-three 4,8-dimethylcoumarin analogues. Structure-activity studies revealed the requirement of 4,8-dimethylcoumarin-3-acetic acid for activity. The most potent inhibitors were produced by replacements at C7, including 3-iodo- (4az) and 3-trifluoromethyl- (4be), with IC50 of 40 and 25 nM, respectively. Pharmacokinetics in mice showed predicted therapeutic concentrations of 4az for >72 h following a single 10 mg/kg oral dose. 4az at 10 mg/kg fully normalized stool water content in a loperamide-induced mouse model of constipation. The favorable inhibition potency, selectivity within the SLC26 family, and pharmacological properties of 4az support its further preclinical development.

SLC26A3 inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation.

SLC26A3 (downregulated in adenoma; DRA) is a Cl-/anion exchanger expressed in the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. SLC26A3 loss of function in humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors in a screen of 50,000 synthetic small molecules done in Fischer rat thyroid (FRT) cells coexpressing slc26a3 and a genetically encoded halide sensor. Structure-activity relationship studies were done on the most potent inhibitor classes identified in the screen: 4,8-dimethylcoumarins and acetamide-thioimidazoles. The dimethylcoumarin DRAinh-A250 fully and reversibly inhibited slc26a3-mediated Cl- exchange with HCO3-, I-, and thiocyanate (SCN-), with an IC50 of ~0.2 µM. DRAinh-A250 did not inhibit the homologous anion exchangers slc26a4 (pendrin) or slc26a6 (PAT-1), nor did it alter activity of other related proteins or intestinal ion channels. In mice, intraluminal DRAinh-A250 blocked fluid absorption in closed colonic loops but not in jejunal loops, while the NHE3 (SLC9A3) inhibitor tenapanor blocked absorption only in the jejunum. Oral DRAinh-A250 and tenapanor comparably reduced signs of constipation in loperamide-treated mice, with additive effects found on coadministration. DRAinh-A250 was also effective in loperamide-treated cystic fibrosis mice. These studies support a major role of slc26a3 in colonic fluid absorption and suggest the therapeutic utility of SLC26A3 inhibition in constipation.

The cystine/glutamate antiporter system xc- drives breast tumor cell glutamate release and cancer-induced bone pain.

Bone is one of the leading sites of metastasis for frequently diagnosed malignancies, including those arising in the breast, prostate and lung. Although these cancers develop unnoticed and are painless in their primary sites, bone metastases result in debilitating pain. Deeper investigation of this pain may reveal etiology and lead to early cancer detection. Cancer-induced bone pain (CIBP) is inadequately managed with current standard-of-care analgesics and dramatically diminishes patient quality of life. While CIBP etiology is multifaceted, elevated levels of glutamate, an excitatory neurotransmitter, in the bone-tumor microenvironment may drive maladaptive nociceptive signaling. Here, we establish a relationship between the reactive nitrogen species peroxynitrite, tumor-derived glutamate, and CIBP. In vitro and in a syngeneic in vivo model of breast CIBP, murine mammary adenocarcinoma cells significantly elevated glutamate via the cystine/glutamate antiporter system xc. The well-known system xc inhibitor sulfasalazine significantly reduced levels of glutamate and attenuated CIBP-associated flinching and guarding behaviors. Peroxynitrite, a highly reactive species produced in tumors, significantly increased system xc functional expression and tumor cell glutamate release. Scavenging peroxynitrite with the iron and mangano-based porphyrins, FeTMPyP and SRI10, significantly diminished tumor cell system xc functional expression, reduced femur glutamate levels and mitigated CIBP. In sum, we demonstrate how breast cancer bone metastases upregulate a cystine/glutamate co-transporter to elevate extracellular glutamate. Pharmacological manipulation of peroxynitrite or system xc attenuates CIBP, supporting a role for tumor-derived glutamate in CIBP and validating the targeting of system xc as a novel therapeutic strategy for the management of metastatic bone pain.

Perenosins: a new class of anion transporter with anti-cancer activity.

A new class of anion transporter named 'perenosins' consisting of a pyrrole linked through an imine to either an indole, benzimidazole or indazole is reported. The indole containing members of the perenosin family function as effective transmembrane Cl(-)/NO3(-) antiporters and HCl cotransporters in a manner similar to the prodigiosenes. The compounds reduce the viability of MDA-MB-231 and MCF-7.

Proton-Coupled Organic Cation Antiporter Contributes to the Hepatic Uptake of Matrine.

Matrine is the major bioactive alkaloid found in certain Sophora plants and has been used for the treatment of liver diseases and protection of liver function. The aim of this study was to investigate the human liver uptake mechanism of matrine by using HepG2 cells as the in vitro model. Matrine was transported into HepG2 cells in a time- and temperature-dependent manner. The cellular uptake was saturable and was significantly reduced by the metabolic inhibitors, such as sodium azide and rotenone. Furthermore, the uptake of matrine was found to be regulated by a protonophore (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) and pH, indicating that this influx transporter may be a proton-coupled antiporter. The uptake of matrine was sensitive to inhibition by the cationic drugs including pyrilamine, quinidine, verapamil, amantadine, diphenhydramine, and cimetidine but insensitive to other typical substrates or inhibitors of well-known organic cation transport systems. The present study reveals that, for the first time, in HepG2 cells, the existence of a proton-coupled organic cation antiporter that contributes substantially to the hepatic uptake of matrine.

Neutropenia in type Ib glycogen storage disease.

PURPOSE OF REVIEW: Glycogen storage disease type Ib, characterized by disturbed glucose homeostasis, neutropenia, and neutrophil dysfunction, is caused by a deficiency in a ubiquitously expressed glucose-6-phosphate transporter (G6PT). G6PT translocates glucose-6-phosphate (G6P) from the cytoplasm into the lumen of the endoplasmic reticulum, in which it is hydrolyzed to glucose either by a liver/kidney/intestine-restricted glucose-6-phosphatase-alpha (G6Pase-alpha) or by a ubiquitously expressed G6Pase-beta. The role of the G6PT/G6Pase-alpha complex is well established and readily explains why G6PT disruptions disturb interprandial blood glucose homeostasis. However, the basis for neutropenia and neutrophil dysfunction in glycogen storage disease type Ib is poorly understood. Recent studies that are now starting to unveil the mechanisms are presented in this review. RECENT FINDINGS: Characterization of G6Pase-beta and generation of mice lacking either G6PT or G6Pase-beta have shown that neutrophils express the G6PT/G6Pase-beta complex capable of producing endogenous glucose. Loss of G6PT activity leads to enhanced endoplasmic reticulum stress, oxidative stress, and apoptosis that underlie neutropenia and neutrophil dysfunction in glycogen storage disease type Ib. SUMMARY: Neutrophil function is intimately linked to the regulation of glucose and G6P metabolism by the G6PT/G6Pase-beta complex. Understanding the molecular mechanisms that govern energy homeostasis in neutrophils has revealed a previously unrecognized pathway of intracellular G6P metabolism in neutrophils.

Anion exchanger 2 mediates the action of arsenic trioxide.

Anion exchanger 2 (AE2) mediates the exchange of C1-/HCO3- across the plasma membrane and plays a role in the regulation of intracellular pH. The present study showed that AE2 protein expression was upregulated immediately after exposure to either low (0.5 micromol/l) or high (1 and 2 micromol/l) concentrations of arsenic trioxide. This suggests that arsenic trioxide may act via regulation of intracellular pH. Changing the culture pH in NB4 cells modulated the degradation of promyelocytic leukaemia-retinoic acid receptor-alpha (PML-RARalpha), PML and RARalpha, which supported this hypothesis. DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulphonic acid) inhibited AE2 function, preventing the arsenic trioxide-induced degradation of RARalpha and low concentration showed synergistic effects on the expression of CD11c, which is related with cell differentiation. In addition, DIDS rescued the cells from 1 micromol/l arsenic trioxide-induced apoptosis. In conclusion, AE2 mediated the action of arsenic trioxide via regulation of intracellular pH and a novel pathway for the mechanism of action of arsenic trioxide is reported.

Ion exchangers mediating Na+, HCO3 - and Cl- transport in the renal proximal tubule.

Most of the Na+ , Cl- and HCO3 - filtered by the kidney is reabsorbed in the proximal tubule. Several lines of evidence indicate that NHE3 is the principal Na+-H+ exchanger isoform involved in mediating acid secretion across the apical membrane. NHE8 is a newly identified isoform also expressed on the brush border in the proximal tubule, but its function remains unknown. A significant fraction of Cl - is reabsorbed via apical membrane Cl- -base exchange: Cl--formate exchange in parallel with Na+-H+ exchange and H+-formate cotransport, and Cl--oxalate exchange in parallel with oxalate-sulfate exchange and Na+-sulfate cotransport. Apical membrane Cl --OH-/HCO3-exchange has also been observed. SLC26 family members have emerged as candidates to mediate proximal tubule Cl--base exchange. Pendrin (SLC26A4) expression is not detected in the proximal tubule, and there is no change in transtubular NaCl absorption in pendrin null mice. In contrast, SLC26A6 (CFEX, PAT1) is expressed on the brush border of proximal tubule cells. Functional expression studies indicate that SLC26A6 is capable of mediating all of the modes of Cl--base exchange described to take place across the brush border membrane. In SLC26A6 null mice the effects of formate or oxalate to stimulate NaCl absorption in microperfused proximal tubules are reduced or absent, respectively, but there is no change in baseline NaCl absorption measured in the absence of formate and oxalate. These findings suggest that SLC26A6 primarily mediates proximal tubule Cl- absorption by Cl--oxalate exchange and Cl--formate exchange rather than by Cl--HCO3- or Cl--OH-exchange. Differential regulation of Cl--base exchange mediated by SLC26A6, and Na+-H+ exchange mediated by NHE3, may act as a switch to govern the ratio of transcellular NaHCO3 to NaCl reabsorption in the proximal tubule.

Mg2+-malate co-transport, a mechanism for Na+-independent Mg2+ transport in neurons of the leech Hirudo medicinalis.

Mg2+-extrusion from Mg2+-loaded neurons of the leech, Hirudo medicinalis, is mediated mainly by Na+/Mg2+ antiport. However, in a number of leech neurons, Mg2+ is extruded in the nominal absence of extracellular Na+, indicating the existence of an additional, Na+-independent Mg2+ transport mechanism. This mechanism was investigated using electrophysiological and microfluorimetrical techniques. The rate of Na+-independent Mg2+ extrusion from Mg2+-loaded leech neurons was found to be independent of extracellular Ca2+, K+, NO3-, HCO3-, SO4(2-), HPO4(2-), and of intra- and extracellular pH. Na+-independent Mg2+ extrusion was not inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), furosemide, ouabain, vanadate, iodoacetate, 4-amino-hippurate, or alpha-cyano-4-hydroxycinnamate and was not influenced by changes in the membrane potential in voltage-clamp experiments. Na+-independent Mg2+ extrusion was, however, inhibited by the application of 2 mM probenecid, a blocker of organic anion transporters, suggesting that Mg2+ might be co-transported with organic anions. Extracellularly, of all organic anions tested (malate, citrate, lactate, alpha-ketoglutarate, and 4-amino-hippurate) only high, but physiological, concentrations of malate (30 mM) had a significant inhibitory effect on Na+-independent Mg2+ extrusion. Intracellularly, iontophoretically injected malate, citrate, or fura-2, but not Cl-, alpha-ketoglutarate, glutamate, succinate, or urate, were stimulating Na+-independent Mg2+ extrusion from those neurons that initially did not extrude Mg2+ in Na+-free solutions. Our data indicate that Mg2+ is co-transported with organic anions, preferably with malate, the predominant extracellular anion in the leech. The proposed model implies that, under experimental conditions, malate drives Mg2+ extrusion, whereas under physiological conditions, malate is actively taken up, driven by Mg2+, so that malate can be metabolized.

Chloride conductance of CFTR facilitates basal Cl-/HCO3- exchange in the villous epithelium of intact murine duodenum.

Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated Cl(-)/HCO(3)(-) exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the Cl(-)/HCO(3)(-) exchange rate was reduced by approximately 35% in CF compared with WT villous epithelium. Cl(-)/HCO(3)(-) exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of Cl(-)(out)/HCO(3)(-)(in) exchange in both epithelia. In anion substitution studies, anion(in)/HCO(3)(-)(out) exchange rates were greater in WT epithelium using Cl(-) or NO(3)(-), but decreased to the level of the CF epithelium using the CFTR-impermeant anion, SO(4)(2-). Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the Cl(-)/HCO(3)(-) exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of Cl(-)/HCO(3)(-) exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) Cl(-) channel activity of CFTR facilitates apical membrane Cl(-)(in)/HCO(3)(-)(out) exchange by providing a Cl(-) "leak" under basal conditions.

Characterization of basolateral chloride/bicarbonate exchange in macula densa cells.

Functional and immunohistological studies were performed to identify basolateral chloride/bicarbonate exchange in macula densa cells. Using the isolated, perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney, macula densa intracellular pH (pH(i)) was measured with fluorescence microscopy and BCECF. For these experiments, basolateral chloride was reduced, resulting in reversible macula densa cell alkalinization. Anion exchange activity was assessed by measuring the maximal net base efflux on readdition of bath chloride. Anion exchange activity required the presence of bicarbonate, was independent of changes in membrane potential, did not require the presence of sodium, and was inhibited by high concentrations of DIDS. Inhibition of macula densa anion exchange activity by basolateral DIDS increased luminal NaCl concentration-induced elevations in pH(i). Immunohistochemical studies using antibodies against AE2 demonstrated expression of AE2 along the basolateral membrane of macula densa cells of rabbit kidney. These results suggest that macula densa cells functionally and immunologically express a chloride/bicarbonate exchanger at the basolateral membrane. This transporter likely participates in the regulation of pH(i) and might be involved in macula densa signaling.

Tapasin decreases immune responsiveness to a model tumor antigen.

The T-cell response against cancer is dependent on the cell surface presentation of tumor-associated or tumor-specific peptides by major histocompatibility complex (MHC) class I molecules. We found that tapasin, a chaperone protein that normally assists in the assembly of MHC class I molecules, is undetectable in an unstimulated pancreatic tumor cell line, Panc02, and only very weakly expressed after gamma-interferon stimulation. Transfection of tapasin into the Panc02 cells did not quantitatively increase MHC class I surface expression or detectably affect MHC class I association with peptide and beta(2)-microglubulin (beta(2)m). However, we found that transfected tapasin downregulated immune reactivity against a model tumor antigen, MUC1. Although tapasin has been previously shown by others to increase immune recognition of particular antigens, our results suggest that tapasin has a negative impact on the presentation of an immunodominant epitope from a specific model tumor antigen.

An evaluation of detergents for NMR structural studies of membrane proteins.

Structural information on membrane proteins lags far behind that on soluble proteins, in large part due to difficulties producing homogeneous, stable, structurally relevant samples in a membrane-like environment. In this study 25 membrane mimetics were screened using 2D (1)H-(15)N heteronuclear single quantum correlation NMR experiments to establish sample homogeneity and predict fitness for structure determination. A single detergent, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LPPG), yielded high quality NMR spectra with sample lifetimes greater than one month for the five proteins tested - R. sphaeroides LH1 alpha and beta subunits, E. coli and B. pseudofirmus OF4 ATP synthase c subunits, and S. aureus small multidrug resistance transporter - with 1, 2, or 4 membrane spanning alpha-helices, respectively. Site-specific spin labeling established interhelical distances in the drug transporter and genetically fused dimers of c subunits in LPPG consistent with in vivo distances. Optical spectroscopy showed that LH1 beta subunits form native-like complexes with bacteriochlorophyll a in LPPG. All the protein/micelle complexes were estimated to exceed 100 kDaltons by translational diffusion measurements. However, analysis of (15)N transverse, longitudinal and (15)N[(1)H] nuclear Overhauser effect relaxation measurements yielded overall rotational correlation times of 8 to 12 nsec, similar to a 15-20 kDalton protein tumbling isotropically in solution, and consistent with the high quality NMR data observed.

cPrG-HCl a potential H+/Cl- symporter prevents acidification of storage vacuoles in aleurone cells and inhibits GA-dependent hydrolysis of storage protein and phytate.

The putative H+/Cl- symporter cycloprodigiosin-HCl (cPrG-HCl) was used to investigate the role of vacuole acidification in cereal aleurone cell function. The protein storage vacuole (PSV) becomes acidified rapidly when aleurone cells are treated with gibberellic acid (GA) but not abscisic acid (ABA). We show that cPrG prevents PSV acidification in aleurone layers and prevents synthesis of secretory proteins such as alpha-amylase. Our data support the hypothesis that decreased hydrolase synthesis is a consequence of decreased hydrolysis of storage proteins in PSV. Support for this hypothesis comes from experiments showing that breakdown of barley 7S globulins and phytate is inhibited by cPrG in GA-treated aleurone layers. Decreased mobilization of PSV reserves is accompanied by reductions in the free amino acid pool size and in the amount of ions released from the aleurone layer. Vacuolation of the aleurone cell is a diagnostic feature of the response to GA, and vacuolation is also inhibited by cPrG. Evidence that cPrG acts as a potential H+/Cl- symporter in aleurone is presented. We show that cPrG does not inhibit the synthesis and secretion of alpha-amylase when Cl- ions are omitted from the incubation medium. Although cPrG blocks many GA-induced responses of aleurone layers, it does not affect early steps in GA signaling. The SLN1 protein, a negative regulator of GA signaling, is turned over in GA-treated cells in the presence and absence of cPrG. Similarly, synthesis of the transcriptional activator GAMYB is unaffected by the presence of cPrG in GA-treated cells.