Parallel in vivo and in vitro transcriptomics analysis reveals calcium and zinc signalling in the brain as sensitive targets of HBCD neurotoxicity.

Hexabromocyclododecane (HBCD) is a brominated flame retardant (BFR) that accumulates in humans and affects the nervous system. To elucidate the mechanisms of HBCD neurotoxicity, we used transcriptomic profiling in brains of female mice exposed through their diet to HBCD (199 mg/kg body weight per day) for 28 days and compared with those of neuronal N2A and NSC-19 cell lines exposed to 1 or 2 µM HBCD. Similar pathways and functions were affected both in vivo and in vitro, including Ca2+ and Zn2+ signalling, glutamatergic neuron activity, apoptosis, and oxidative stress. Release of cytosolic free Zn2+ by HBCD was confirmed in N2A cells. This Zn2+ release was partially quenched by the antioxidant N-acetyl cysteine indicating that, in accordance with transcriptomic analysis, free radical formation is involved in HBCD toxicity. To investigate the effects of HBCD in excitable cells, we isolated mouse hippocampal neurons and monitored Ca2+ signalling triggered by extracellular glutamate or zinc, which are co-released pre-synaptically to trigger postsynaptic signalling. In control cells application of zinc or glutamate triggered a rapid rise of intracellular [Ca2+]. Treatment of the cultures with 1 µM of HBCD was sufficient to reduce the glutamate-dependent Ca2+ signal by 50%. The effect of HBCD on zinc-dependent Ca2+ signalling was even more pronounced, resulting in the reduction of the Ca2+ signal with 86% inhibition at 1 µM HBCD. Our results show that low concentrations of HBCD affect neural signalling in mouse brain acting through dysregulation of Ca2+ and Zn2+ homeostasis.

The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon.

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn(2+) are not well understood. Here, we determined a role of the colonocytic Zn(2+) sensing receptor, ZnR/GPR39, in mediating Zn(2+)-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn(2+)-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn(2+) by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases.

Effects of long-term treatment with pioglitazone on cognition and glucose metabolism of PS1-KI, 3xTg-AD, and wild-type mice.

In this study, we investigated the effects of long-term (9-month) treatment with pioglitazone (PIO; 20 mg/kg/d) in two animal models of Alzheimer's disease (AD)-related neural dysfunction and pathology: the PS1-KI(M146V) (human presenilin-1 (M146V) knock-in mouse) and 3xTg-AD (triple transgenic mouse carrying AD-linked mutations) mice. We also investigated the effects on wild-type (WT) mice. Mice were monitored for body mass changes, fasting glycemia, glucose tolerance, and studied for changes in brain mitochondrial enzyme activity (complexes I and IV) as well as energy metabolism (lactate dehydrogenase (LDH)). Cognitive effects were investigated with the Morris water maze (MWM) test and the object recognition task (ORT). Behavioral analysis revealed that PIO treatment promoted positive cognitive effects in PS1-KI female mice. These effects were associated with normalization of peripheral gluco-regulatory abnormalities that were found in untreated PS1-KI females. PIO-treated PS1-KI females also showed no statistically significant alterations in brain mitochondrial enzyme activity but significantly increased reverse LDH activity.PIO treatment produced no effects on cognition, glucose metabolism, or mitochondrial functioning in 3xTg-AD mice. Finally, PIO treatment promoted enhanced short-term memory performance in WT male mice, a group that did not show deregulation of glucose metabolism but that showed decreased activity of complex I in hippocampal and cortical mitochondria. Overall, these results indicate metabolically driven cognitive-enhancing effects of PIO that are differentially gender-related among specific genotypes.

A zinc-sensing receptor triggers the release of intracellular Ca2+ and regulates ion transport.

Changes in extracellular zinc concentration participate in modulating fundamental cellular processes such as proliferation, secretion, and ion transport in a mechanism that is not well understood. Here, we show that a micromolar concentration of extracellular zinc triggers a massive release of calcium from thapsigargin-sensitive intracellular pools in the colonocytic cell line HT29. Calcium release was blocked by a phospholipase-C inhibitor, indicating that formation of inositol 1,4,5-triphosphate is required for zinc-dependent calcium release. Zinc influx was not observed, indicating that extracellular zinc triggered the release. The Ca(i)2+ release was zinc specific and could not be triggered by other heavy metals. Furthermore, zinc failed to activate the Ca(2+)-sensing receptor heterologously expressed in HEK293 cells. The zinc-induced Ca(i)2+ rise stimulated the activity of the Na(+)/H(+) exchanger in HT29 cells. Our results indicate that a previously uncharacterized extracellular, G protein-coupled, Zn(2+)-sensing receptor is functional in colonocytes. Because Ca(i)2+ rise is known to regulate key cellular and signal-transduction processes, the zinc-sensing receptor may provide the missing link between extracellular zinc concentration changes and the regulation of cellular processes.

A cluster of cytoplasmic histidine residues specifies pH dependence of the AE2 plasma membrane anion exchanger.

Intracellular pH is maintained by a dynamic equilibrium balancing the opposing forces of proton loading and proton extrusion. By providing an efflux pathway for base, anion exchangers constitute a key component of the plasma membrane proton-loading machinery. The data in this paper identify a histidine-rich sequence within the cytoplasmic domain of the nonerythroid anion exchanger, AE2, that serves as an intracellular pH "sensor" that modulates anion exchange activity within the physiological range of cytoplasmic pH. These data reveal an interaction between the two major domains of the anion exchanger and suggest a novel substrate feedback mechanism by which intracellular protons directly control the activity of an acid-loading plasma membrane ion transporter.

The product of HUM1, a novel yeast gene, is required for vacuolar Ca2+/H+ exchange and is related to mammalian Na+/Ca2+ exchangers.

Calcineurin, or PP2B, plays a critical role in mediating Ca2+-dependent signaling in many cell types. In yeast cells, this highly conserved protein phosphatase regulates aspects of ion homeostasis and cell wall synthesis. We show that calcineurin mutants are sensitive to high concentrations of Mn2+ and identify two genes, CCC1 and HUM1, that, at high dosages, increase the Mn2+ tolerance of calcineurin mutants. CCC1 was previously identified by complementation of a Ca2+-sensitive (csg1) mutant. HUM1 (for "high copy number undoes manganese") is a novel gene whose predicted protein product shows similarity to mammalian Na+/Ca2+ exchangers. hum1 mutations confer Mn2+ sensitivity in some genetic backgrounds and exacerbate the Mn2+ sensitivity of calcineurin mutants. Furthermore, disruption of HUM1 in a calcineurin mutant strain results in a Ca2+-sensitive phenotype. We investigated the effect of disrupting HUM1 in other strains with defects in Ca2+ homeostasis. The Ca2+ sensitivity of pmc1 mutants, which lack a P-type ATPase presumed to transport Ca2+ into the vacuole, is exacerbated in a hum1 mutant strain background. Also, the Ca2+ content of hum1 pmc1 cells is less than that of pmc1 cells. In contrast, the Ca2+ sensitivity of vph1 mutants, which are specifically defective in vacuolar acidification, is not significantly altered by disruption of Hum1p function. These genetic interactions suggest that Hum1p may participate in vacuolar Ca2+/H+ exchange. Therefore, we prepared vacuolar membrane vesicles from wild-type and hum1 cells and compared their Ca2+ transport properties. Vacuolar membrane vesicles from hum1 mutants lack all Ca2+/H+ antiport activity, demonstrating that Hum1p catalyzes the exchange of Ca2+ for H+ across the yeast vacuolar membrane.

A conserved glutamate is responsible for ion selectivity and pH dependence of the mammalian anion exchangers AE1 and AE2.

The erythrocyte anion exchanger AE1 (band 3) serves as an important model for the study of the mechanism of ion transport. Chemical modification of human erythrocyte AE1 has previously suggested that glutamic acid residue 681 lies within the transport pathway and can cross the permeability barrier. This glutamate is conserved in all anion exchangers sequenced to date. We examined the effect on divalent (sulfate) and monovalent (chloride and bicarbonate) anion transport of mutating the corresponding glutamates in mouse AE1 and the closely related anion exchanger, AE2. Substitution of this conserved glutamate with uncharged or basic amino acids had a negligible effect on the maximal rate of sulfate-sulfate exchange in AE-reconstituted proteoliposomes, but largely abolished the steep pH dependence of sulfate transport observed in wild-type AE1 and AE2. In contrast, exchange of monovalent anions was undetectable in cells expressing these mutants. Replacement of the conserved glutamate with aspartate abolished both monovalent and divalent anion transport. These data suggest that the conserved glutamate residue plays a dual role in determining anion selectivity and in proton coupling to sulfate transport. A model explaining the role of the conserved glutamate in promoting ion selectivity and pH regulation is discussed.

High level expression, partial purification, and functional reconstitution of the human AE1 anion exchanger in Saccharomyces cerevisiae.

Human erythroid anion exchanger AE1 (Band 3) was expressed in the yeast Saccharomyces cerevisiae under the control of the constitutive promoter and transcriptional terminator of the yeast phosphoglycerate kinase gene. AE1 expression in stable yeast transformants was estimated to be approximately 0.7 mg AE1 per liter. Density gradient sedimentation analysis indicated that the AE1 protein was associated with a membrane fraction distinct from plasma membrane, most likely the endoplasmic reticulum. AE1 protein was solubilized from yeast membranes with lysophosphatidyl choline, and the protein, tagged with six histidines at its amino terminus, was purified to 35% homogeneity by metal chelation affinity chromatography. Size-exclusion chromatography in the presence of octaethylene glycol monododecyl ether indicated that the solubilized yeast-expressed AE1, like endogenous erythroid AE1, eluted at a stokes radius of 77 A, consistent with a dimeric oligomeric state. Binding of partially purified yeast-expressed AE1 to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate resin was competitive with the transportable substrate chloride but not the nontransported anion citrate, suggesting that the structure of the anion binding site is preserved. The specific activity of sulfate transport by partially purified yeast AE1 was determined in proteoliposomes to be similar to that of authentic AE1 purified from erythrocyte membranes. These data show that this expression system has the capacity to produce functional mammalian plasma membrane anion exchangers at levels sufficient for biochemical and biophysical analysis.

Sulfate transport mediated by the mammalian anion exchangers in reconstituted proteoliposomes.

The kinetic properties of sulfate transport mediated by the anion exchangers AE1 and AE2 have been examined. Microsomes isolated from HEK cells transiently overexpressing either protein were reconstituted in unilamellar, 200-600-nm diameter proteoliposomes. Transport mediated by the exchangers was monitored by loading the reconstituted proteoliposomes with the slowly transportable anion SO4(2-) using [35S]SO4(2-) as a tracer and performing [35S]SO4(2-)/SO4(2-) exchange. The following data suggest that AE1 and AE2 have been functionally reconstituted: (i) the rate of SO4(2-) transport in AE1 and AE2 containing proteoliposomes was 10-20 times higher than in proteoliposomes derived from control microsomes; (ii) the transport of SO4(2-) was strongly dependent on the presence of a trans anion; and (iii) the anion exchanger inhibitors, 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 4,4'-dinitrostilbene-2,2'-di-sulfonate (DIDS) totally abolished SO4(2-) transport. furthermore, DIDS inhibits SO4(2-) transport only when occluded inside the vesicles, indicating a uniform, asymmetrical, inside-out orientation of the reconstituted exchangers. The Ki values of the stilbene disulfonate compound DNDS were 2.5 and 4 microM for AE1 and AE2, respectively, suggesting that the two exchangers possess similar high affinity sites for stilbene compounds. Both AE1 and AE2 showed the same steep pH dependence of sulfate transport, which was maximal at pH 5.5 and reduced to less than 10% (of the value at pH 5.5) at pH 8.5, suggesting that an acidic residue shared by AE1 and AE2 participates in the pH regulation of sulfate transport.

Activation of the Dunaliella acidophila plasma membrane H(+)-ATPase by trypsin cleavage of a fragment that contains a phosphorylation site.

Trypsin treatment of purified H(+)-ATPase from plasma membranes of the extreme acidophilic alga Dunaliella acidophila enhances ATP hydrolysis and H+ pumping activities. The activation is associated with an alkaline pH shift, an increase in Vmax, and a decrease in Km(ATP). The activation is correlated with cleavage of the 100-kD ATPase polypeptide to a fragment of approximately 85 kD and the appearance of three minor hydrophobic fragments of 7 to 8 kD, which remain associated with the major 85-kD polypeptide. The N-terminal sequence of the small fragments has partial homology to residues 713 to 741 of Arabidopsis thaliana plasma membrane H(+)-ATPases. Incubation of cells with 32P-labeled orthophosphate (32Pi) results in incorporation of 32P into the ATPase 100-kD polypeptide. Trypsin treatment of the 32Pi-labeled ATPase leads to complete elimination of label from the approximately 85-kD polypeptide. Cleavage of the phosphorylated enzyme with endoproteinase Glu-C (V-8) yields a phosphorylated 12-kD fragment. Peptide mapping comparison between the 100-kD and the trypsinized 85-kD polypeptides shows that the 12-kD fragment is derived from the trypsin-cleaved part of the enzyme. The N-terminal sequence of the 12-kD fragment closely resembles a C-terminal stretch of an ATPase from another Dunaliella species. It is suggested that trypsin activation of the D. acidophila plasma membrane H(+)-ATPase results from elimination of an autoinhibitory domain at the C-terminal end of the enzyme that carries a vicinal phosphorylation site.

Inhibition of the plasma-membrane H(+)-ATPase from Dunaliella adidophila by omeprazole.

The acid-activated sulfhydryl reagent omeprazole inhibits light-induced H+ secretion at pH 1 in cells of the halotolerant alga Dunaliella acidophila. Plasma-membrane vesicles, prepared from omeprazole-treated cells, have impaired vanadate-sensitive ATPase and ATP-induced H+ uptake activities. Omeprazole inhibits ATPase activity also in isolated plasma-membrane vesicles. The inhibition is enhanced at acidic pH and can be prevented by protonophores indicating that it is promoted by internal acidification of the vesicles. Mercaptoethanol partially reverses omeprazole inhibition. ADP does not afford protection against omeprazole but it does protect against inhibition by N-ethylmaleimide, indicating that these reagents modify different sulfhydryl groups. It is suggested that omeprazole blocks SH groups of the D. acidophila plasma-membrane H(+)-ATPase, which face the outer side of the cell.

Purification and Properties of a Plasma Membrane H+-ATPase from the Extremely Acidophilic Alga Dunaliella acidophila.

This paper describes partial purification and characterization of a vanadate-sensitive H+-ATPase from plasma membranes of Dunaliella acidophila, an extremely acidophilic unicellular alga (I. Sekler, H.U. Glaser, U. Pick [1991] J Membr Biol 121: 51-57). Purification is based on the insolubility in and stability of the enzyme in Triton X-100. The purified enzyme is highly enriched in a polypeptide of molecular mass 100 kD, which cross-reacts with antibodies against the plant plasma membrane H+-ATPase. Upon reconstitution into proteoliposomes, the enzyme catalyzes an ATP-dependent electrogenic H+ uptake. ATP hydrolysis is stimulated by lipids, is inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide, erythrosine, and mercurials, and shows a sharp optimum at pH 6. Unusual properties of this enzyme, by comparison with plant plasma membrane H+-ATPases, are a higher affinity for ATP (Km = 40 [mu]M) and a larger stimulation by K+, which interacts with the enzyme from its cytoplasmic side. Comparative studies with cross-reacting antibodies, prepared against different domains of the plant H+-ATPase, suggest that the central hydrophilic domain containing the catalytic site is more conserved than the C- and N-terminal ends. The high abundance and stability of the plasma membrane H+-ATPase from D. acidophila make it an attractive model system for studies of the structure-function relations and regulation of this crucial enzyme.

Characterization of a plasma membrane H(+)-ATPase from the extremely acidophilic alga Dunaliella acidophila.

Dunaliella acidophila is an unicellular green alga which grows optimally at pH 0-1 while maintaining neutral internal pH. A plasma membrane preparation of this algae has been purified on sucrose density gradients. The preparation exhibits vanadate-sensitive ATPase activity of 2 mumol Pi/mg protein/min, an activity 15 to 30-fold higher than that in the related neutrophilic species D. salina. The following properties suggest that the ATPase is an electrogenic plasma membrane H+ pump. (i) ATP induces proton uptake and generates a positive-inside membrane potential as demonstrated with optical probes. (ii) ATP hydrolysis and proton uptake are inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide and erythrosine but not by molybdate, azide or nitrate. (iii) ATP hydrolysis and proton uptake are stimulated by fussicoccin in a pH-dependent manner as found for plants plasma membrane H(+)-ATPase. Unusual properties of this enzyme are: (i) the Km for ATP is around 60 microM, considerably lower than in other plasma membrane H(+)-ATPases, and (ii) the ATPase activity and proton uptake are stimulated three to fourfold by K+ and to a smaller extent by other monovalent cations. These results suggest that D. acidophila possesses a vanadate-sensitive H(+)-ATPase with unusual features enabling it to maintain the large transmembrane pH gradient.