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Biomedical subjects

S L Alper

Publications and source records attributed to S L Alper.

At least 19 recordsLinked to original sources
PubMed2001

Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump.

Transgenic plants overexpressing the vacuolar H(+)-pyrophosphatase are much more resistant to high concentrations of NaCl and to water deprivation than the isogenic wild-type strains. These transgenic plants accumulate more Na(+) and K(+) in their leaf tissue than the wild type. Moreover, direct measurements on isolated vacuolar membrane vesicles derived from the AVP1 transgenic plants and from wild type demonstrate that the vesicles from the transgenic plants have enhanced cation uptake. The phenotypes of the AVP1 transgenic plants suggest that increasing the vacuolar proton gradient results in increased solute accumulation and water retention. Presumably, sequestration of cations in the vacuole reduces their toxic effects. Genetically engineered drought- and salt-tolerant plants could provide an avenue to the reclamation of farmlands lost to agriculture because of salinity and a lack of rainfall.

Abscisic Acid↗
PubMed2001

A dominant negative mutant of the KCC1 K-Cl cotransporter: both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity.

K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunct strategy for the treatment of sickle cell anemia. However, structure-function relationships among the polypeptide products of the four K-Cl cotransporter (KCC) genes are little understood. We have investigated the importance of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl cotransport function expressed in Xenopus oocytes. Truncation of as few as eight C-terminal amino acids (aa) abolished function despite continued polypeptide accumulation and surface expression. These C-terminal loss-of-function mutants lacked a dominant negative phenotype. Truncation of the N-terminal 46 aa diminished function. Removal of 89 or 117 aa (Delta(N)117) abolished function despite continued polypeptide accumulation and surface expression and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant Delta(N)117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. Delta(N)117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2.

Animals↗
PubMed2001

Differential expression and regulation of AE2 anion exchanger subtypes in rabbit parietal and mucous cells.

1. The anion exchanger isoform 2 (AE2) gene encodes three subtypes (AE2a, b and c), which have different N-termini and tissue distributions. AE2 is expressed at high levels in the stomach, where it is thought to mediate basolateral base exit during acid production. The present study investigated if the three AE2 subtypes are differentially expressed and regulated in different cell types within the gastric mucosa. 2. The cloning strategy to obtain rabbit AE2a, b and c cDNAs combined genomic PCR and RT-PCR based on primers deduced from the rat sequences. Semiquantitative RT-PCR using homologous primers revealed much higher AE2 mRNA expression in rabbit parietal cells (PCs) than in mucous cells (MCs). The subtype expression pattern was AE2b >> AE2c > or = AE2a in PCs and AE2a >AE2b >> AE2c in MCs. Sequence analysis revealed the presence of a highly conserved protein kinase C (PKC) consensus sequence in the AE2a alternative N-terminus. 3. Maximal Cl(-)-HCO(3)(-) exchange rates, measured fluorometrically in BCECF-loaded cultured gastric cells, were much higher in PCs than MCs. PKC activation by phorbol ester stimulated maximal Cl(-)-HCO(3)(-) exchange rates in MCs but not in PCs, whereas forskolin had no effect in each cell type. 4. In summary, rabbit PCs and MCs, which originate from the same gastric stem cell population, display a completely different AE2 subtype expression pattern. Therefore, AE2 subtype expression is not organ specific but cell type specific. The different regulation of anion exchange in parietal and mucous cells suggests that AE2 subtypes may be differentially regulated.

Amino Acid Sequence↗
PubMed2001

VHL tumor suppressor regulates Cl-/HCO3- exchange and Na+/H+ exchange activities in renal carcinoma cells.

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are thought to play a critical role in the pathogenesis of both sporadic and VHL disease-associated clear-cell renal carcinomas (RCC). Differential display-PCR identified the AE2 anion exchanger as a candidate VHL target gene. AE2 mRNA and polypeptide levels were approximately threefold higher in 786-O VHL cells than in 786-O Neo cells. In contrast, Cl(-)/HCO(3)(-) exchange activity in 786-O VHL cells was 50% lower than in 786-O Neo cells. Since resting intracellular pH (pH(i)) values were indistinguishable, we postulated that Na(+)/H(+) exchange activity (NHE) might be similarly reduced in 786-O VHL cells. NHE-mediated pH(i) recovery from acid load was less than 50% that in 786-O Neo cells, whereas hypertonicity-stimulated, amiloride-sensitive NHE was indistinguishable in the two cell lines. The NHE3 mRNA level was higher in 786-O VHL than 786-O Neo cells, but NHE1 mRNA levels did not differ. AE2 and NHE3 are the first transcripts reported to be upregulated by pVHL. Elucidation of mechanisms responsible for downregulation of both ion exchange activities will require further investigation.

Ammonium Chloride↗
PubMed2001

Structure and complex transcription pattern of the mouse SK1 K(Ca) channel gene, KCNN1.

Small conductance calcium-gated K(+) channels (SK channels) are encoded by the three SK genes, SK1, SK2, and SK3. These channels likely contribute to slow synaptic afterhyperpolarizations of apamin-sensitive and apamin-insensitive types. SK channels are also widely expressed outside the nervous system. The mouse SK1 gene comprises at least 12 exons extending across 19.8 kb of genomic DNA. This gene encodes a complex pattern of alternatively spliced SK1 transcripts widely expressed among mouse tissues. These transcripts exhibit at least four distinct 5'-nucleotide sequence variants encoding at least two N-terminal amino acid sequences. Optional inclusion of exons 7 and 9, together with two alternate splice donor sites in exon 8, yields transcripts encoding eight variant C-terminal amino acid sequences for SK1. These include an altered putative S6 transmembrane span, modification of the C-terminal cytoplasmic domain binding site for calmodulin, and generation of two alternate predicted binding sites for PDZ domain-containing proteins. 20 of the 32 predicted mouse SK1 transcripts are expressed in brain at levels sufficient to allow consistent detection, and encode 16 SK1 polypeptide variants. Only four of these 16 polypeptides preserve the ability to bind calmodulin in a Ca(2+)-independent manner. Mouse SK1 also exhibits novel, strain-specific, length polymorphism of a polyglutamate repeat in the N-terminal cytoplasmic domain. The evolutionary conservation of this complex transcription pattern suggests a possible role in the tuning of SK1 channel function.

3' Untranslated Regions↗
PubMed2001

Intracellular Ca(2+) signaling in endothelial cells by the angiogenesis inhibitors endostatin and angiostatin.

Intracellular signaling mechanisms by the angiogenesis inhibitors endostatin and angiostatin remain poorly understood. We have found that endostatin (2 microg/ml) and angiostatin (5 microg/ml) elicited transient, approximately threefold increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Acute exposure to angiostatin or endostatin nearly abolished subsequent endothelial [Ca(2+)](i) responses to carbachol or to thapsigargin; conversely, thapsigargin attenuated the Ca(2+) signal elicited by endostatin. The phospholipase C inhibitor U-73122 and the inositol trisphosphate (IP(3)) receptor inhibitor xestospongin C both inhibited endostatin-induced elevation in [Ca(2+)](i), and endostatin rapidly elevated endothelial cell IP(3) levels. Pertussis toxin and SB-220025 modestly inhibited the endostatin-induced Ca(2+) signal. Removal of extracellular Ca(2+) inhibited the endostatin-induced rise in [Ca(2+)](i), as did a subset of Ca(2+)-entry inhibitors. Peak Ca(2+) responses to endostatin and angiostatin in endothelial cells exceeded those in epithelial cells and were minimal in NIH/3T3 cells. Overnight pretreatment of endothelial cells with endostatin reduced the subsequent acute elevation in [Ca(2+)](i) in response to vascular endothelial growth factor or to fibroblast growth factor by approximately 70%. Intracellular Ca(2+) signaling may initiate or mediate some of the cellular actions of endostatin and angiostatin.

Angiogenesis Inhibitors↗
PubMed2001

Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH(2)-terminal cytoplasmic domain.

The role of intracellular pH (pH(i)) in regulation of AE2 function in Xenopus oocytes remains unclear. We therefore compared AE2-mediated (36)Cl(-) efflux from Xenopus oocytes during imposed variation of extracellular pH (pH(o)) or variation of pH(i) at constant pH(o). Wild-type AE2-mediated (36)Cl(-) efflux displayed a steep pH(o) vs. activity curve, with pH(o(50)) = 6.91 +/- 0.04. Sequential NH(2)-terminal deletion of amino acid residues in two regions, between amino acids 328 and 347 or between amino acids 391 and 510, shifted pH(o(50)) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocyte pH(i) at constant pH(o) and were shown to be neither substrates nor inhibitors of AE2-mediated Cl(-) transport. At constant pH(o), AE2 was inhibited by intracellular acidification and activated by intracellular alkalinization. Our data define structure-function relationships within the AE2 NH(2)-terminal cytoplasmic domain, which demonstrates distinct structural requirements for AE2 regulation by intracellular and extracellular protons.

Acids↗
PubMed2001

K-Cl cotransport modulation by intracellular Mg in erythrocytes from mice bred for low and high Mg levels.

Mg is an important determinant of erythrocyte cation transport system(s) activity. We investigated cation transport in erythrocytes from mice bred for high (MGH) and low (MGL) Mg levels in erythrocytes and plasma. We found that K-Cl cotransport activity was higher in MGL than in MGH erythrocytes, and this could explain their higher mean corpuscular hemoglobin concentration, median density, and reduced cell K content. Although mouse KCC1 protein abundance was comparable in MGL and MGH erythrocytes, activities of Src family tyrosine kinases were higher in MGH than in MGL erythrocytes. In contrast, protein phosphatase (PP) isoform 1 alpha (PP1 alpha) enzymatic activity, which has been suggested to play a positive regulatory role in K-Cl cotransport, was lower in MGH than in MGL erythrocytes. Additionally, we found that the Src family kinase c-Fgr tyrosine phosphorylates PP1 alpha in vitro. These findings suggest that in vivo downregulation of K-Cl cotransport activity by Mg is mediated by enhanced Src family kinase activity, leading to inhibition of the K-Cl cotransport stimulator PP1.

Animals↗
PubMed2001

Role of JNK in hypertonic activation of Cl(-)-dependent Na(+)/H(+) exchange in Xenopus oocytes.

In the course of studying the hypertonicity-activated ion transporters in Xenopus oocytes, we found that activation of endogenous oocyte Na(+)/H(+) exchange activity (xoNHE) by hypertonic shrinkage required Cl(-), with an EC(50) for bath [Cl(-)] of approximately 3mM. This requirement for chloride was not supported by several nonhalide anions and was not shared by xoNHE activated by acid loading. Hypertonicity-activated xoNHE exhibited an unusual rank order of inhibitory potency among amiloride derivatives and was blocked by Cl(-) transport inhibitors. Chelation of intracellular Ca(2+) by injection of EGTA blocked hypertonic activation of xoNHE, although many inhibitors of Ca(2+)-related signaling pathways were without inhibitory effect. Hypertonicity activated oocyte extracellular signal-regulated kinase 1/2 (ERK1/2), but inhibitors of neither ERK1/2 nor p38 prevented hypertonic activation of xoNHE. However, hypertonicity also stimulated a Cl(-)-dependent increase in c-Jun NH(2)-terminal kinase (JNK) activity. Inhibition of JNK activity prevented hypertonic activation of xoNHE but not activation by acid loading. We conclude that hypertonic activation of Na(+)/H(+) exchange in Xenopus oocytes requires Cl(-) and is mediated by activation of JNK.

Amiloride↗
PubMed2001

AE anion exchangers in atrial tumor cells.

Intracellular pH homeostasis and intracellular Cl(-) concentration in cardiac myocytes are regulated by anion exchange mechanisms. In physiological extracellular Cl(-) concentrations, Cl(-)/HCO(3)(-) exchange promotes intracellular acidification and Cl(-) loading sensitive to inhibition by stilbene disulfonates. We investigated the expression of AE anion exchangers in the AT-1 mouse atrial tumor cell line. Cultured AT-1 cells exhibited a substantial basal Na(+)-independent Cl(-)/HCO(3)(-) (but not Cl(-)/OH(-)) exchange activity that was inhibited by DIDS but not by dibenzamidostilbene disulfonic acid (DBDS). AT-1 cell Cl(-)/HCO(3)(-) activity was stimulated two- to threefold by extracellular ATP and ANG II. AE mRNAs detected by RT-PCR in AT-1 cells included brain AE3 (bAE3), cardiac AE3 (cAE3), AE2a, AE2b, AE2c1, AE2c2, and erythroid AE1 (eAE1), but not kidney AE1 (kAE1). Cultured AT-1 cells expressed AE2, cAE3, and bAE3 polypeptides, which were detected by immunoblot and immunocytochemistry. An AE1-like epitope was detected by immunocytochemistry but not by immunoblot. Both bAE3 and cAE3 were present in intact AT-1 tumors. Cultured AT-1 cells provide a useful system for the study of mediators and regulators of Cl(-)/HCO(3)(-) exchange activity in an atrial cell type.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

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.

Animals↗
PubMed2001

Physiological and molecular characterization of urea transport by the gills of the Lake Magadi tilapia (Alcolapia grahami).

The Lake Magadi tilapia (Alcolapia grahami) is an unusual fish, excreting all its nitrogenous waste as urea because of its highly alkaline and buffered aquatic habitat. Here, using both physiological and molecular studies, we describe the mechanism of branchial urea excretion in this species. In vivo, repeated short-interval sampling revealed that urea excretion is continuous. The computed urea permeability of A. grahami gill is 4.74x10(-)(5)+/-0.38x10(-)(5 )cm s(-)(1) (mean +/- s.e.m., N=11), some 10 times higher than passive permeability through a lipid bilayer and some five times higher than that of even the most urea-permeable teleosts studied to date (e.g. the gulf toadfish). Transport of urea was bidirectional, as demonstrated by experiments in which external [urea] was elevated. Furthermore, urea transport was inhibited by classic inhibitors of mammalian and piscine urea transporters in the order thiourea>N-methylurea>acetamide. A 1700 base pair cDNA for a putative Magadi tilapia urea transporter (mtUT) was cloned, sequenced and found to display high homology with urea transporters from mammals, amphibians and other fishes. When cRNA transcribed from mtUT cDNA was injected into Xenopus laevis oocytes, phloretin-inhibitable urea uptake was enhanced 3.4-fold relative to water-injected controls. Northern analysis of gill, red blood cells, liver, muscle and brain using a portion of mtUT as a probe revealed that gill is the only tissue in which mtUT RNA is expressed. Magadi tilapia gill pavement cells exhibited a trafficking of dense-cored vesicles between the well-developed Golgi cisternae and the apical membrane. The absence of this trafficking and the poor development of the Golgi system in a non-ureotelic relative (Oreochromis niloticus) suggest that vesicle trafficking could be related to urea excretion in Alcolapia grahami. Taken together, the above findings suggest that the gills of this alkaline-lake-adapted species excrete urea constitutively via the specific facilitated urea transporter mtUT.

Amino Acid Sequence↗
PubMed2000

The cytoplasmic C-terminal fragment of polycystin-1 regulates a Ca2+-permeable cation channel.

The cytoplasmic C-terminal portion of the polycystin-1 polypeptide (PKD1(1-226)) regulates several important cell signaling pathways, and its deletion suffices to cause autosomal dominant polycystic kidney disease. However, a functional link between PKD1 and the ion transport processes required to drive renal cyst enlargement has remained elusive. We report here that expression at the Xenopus oocyte surface of a transmembrane fusion protein encoding the C-terminal portion of the PKD1 cytoplasmic tail, PKD1(115-226), but not the N-terminal portion, induced a large, Ca(2+)-permeable cation current, which shifted oocyte reversal potential (E(rev)) by +33 mV. Whole cell currents were sensitive to inhibition by La(3+), Gd(3+), and Zn(2+), and partially inhibited by SKF96365 and amiloride. Currents were not activated by bath hypertonicity, but were inhibited by acid pH. Outside-out patches pulled from PKD1(115-226)-expressing oocytes exhibited a 5.1-fold increased NP(o) of endogenous 20-picosiemens cation channels of linear conductance. PKD1(115-226)-injected oocytes also exhibited elevated NP(o) of unitary calcium currents in outside-out and cell-attached patches, and elevated calcium permeability documented by fluorescence ratio and (45)Ca(2+) flux experiments. Both Ca(2+) conductance and influx were inhibited by La(3+). Mutation of candidate phosphorylation sites within PKD1(115-226) abolished the cation current. We conclude that the C-terminal cytoplasmic tail of PKD1 up-regulates inward current that includes a major contribution from Ca(2+)-permeable nonspecific cation channels. Dysregulation of these or similar channels in autosomal dominant polycystic kidney disease may contribute to cyst formation or expansion.

Amino Acid Sequence↗
PubMed2000

Structure and genetic polymorphism of the mouse KCC1 gene.

The KCC1 K-Cl cotransporter is a major regulator of erythroid and non-erythroid cell volume, and the KCC1 gene is a candidate modifier gene for sickle cell disease and other hemoglobinopathies. We have cloned and sequenced the mouse KCC1 (mKCC1) gene, defined its intron-exon junctions, and analyzed (AC)/(TG) intragenic polymorphisms. A highly polymorphic (AC) repeat of mKCC1 intron 1 was characterized in musculus strains, and used to prove lack of linkage between the mKCC1 gene and the rol (resistant to osmotic lysis) locus. The intron 1 (AC) repeat in CAST/Ei and SPRET/Ei was not only more divergent in length but also underwent additional sequence variation. A dimorphic (TG) repeat in intron 2 distinguished CAST/Ei from other strains, and an intron 17 B1 Alu-like SINE present in all musculus strains was found to be absent from intron 17 in SPRET/Ei. These and additional described strain-specific polymorphisms will be useful mapping and genetic tools in the study of mouse models of sickle cell disease.

Anemia, Sickle Cell↗
PubMed2000

Na+/H+ and CI-/HCO3-antiporters of bovine pigmented ciliary epithelial cells.

Medical therapy of glaucoma commonly aims at slowing aqueous humor formation by the ocular ciliary epithelial bilayer, but underlying mechanisms are poorly understood. The first step in secretion is NaCl uptake from the stroma into the pigmented ciliary epithelial (PE) cell layer by electroneutral transporters. After crossing gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, solute is released into the aqueous humor. Published data have indicated that both paired Na+/H+ and Cl-/HCO3- antiporters and the Na+-K+-2Cl- symporter are involved in net uptake. The molecular identities of the paired antiporters have not been elucidated. We have studied continuously cultured bovine PE cells. Acid-activated 22Na+ uptake was inhibited by cariporide, EIPA (ethyl-isopropyl-amiloride) and amiloride, at concentrations characteristic of the NHE-1 isoform. Videomicroscopy of BCECF-loaded PE cells verified the presence of an EIPA-inhibitable Na+/H+ antiporter. Removing external Cl- also triggered an alkalinization, which was Na+-independent and could be inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Application of hypotonicity followed by return to isotonicity triggered a regulatory volume increase, which was pharmacologically similar to the uptake mechanisms described for intact rabbit ciliary epithelium. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of RNA from the human ciliary body detected expression of the AE2 Cl-/HCO3- exchanger, but not of AE1, cAE3 or bAE3. Immunostaining of bovine PE cells also revealed the presence of AE2 epitope. We conclude that paired NHE-1 Na+/H+ and AE2 Cl-/HCO3- antiporters are important components in the initial step in aqueous humor formation.

Animals↗
PubMed2000

Polarization of Na(+)/H(+) and Cl(-)/HCO (3)(-) exchangers in migrating renal epithelial cells.

Cell migration is crucial for processes such as immune defense, wound healing, or the formation of tumor metastases. Typically, migrating cells are polarized within the plane of movement with lamellipodium and cell body representing the front and rear of the cell, respectively. Here, we address the question of whether this polarization also extends to the distribution of ion transporters such as Na(+)/H(+) exchanger (NHE) and anion exchanger in the plasma membrane of migrating cells. Both transporters are required for locomotion of renal epithelial (Madin-Darby canine kidney, MDCK-F) cells and human melanoma cells since their blockade reduces the rate of migration in a dose-dependent manner. Inhibition of migration of MDCK-F cells by NHE blockers is accompanied by a decrease of pH(i). However, when cells are acidified with weak organic acids, migration of MDCK-F cells is normal despite an even more pronounced decrease of pH(i). Under these conditions, NHE activity is increased so that cells are swelling due to the accumulation of organic anions and Na(+). When exclusively applied to the lamellipodium, blockers of NHE or anion exchange inhibit migration of MDCK-F cells as effectively as when applied to the entire cell surface. When they are directed to the cell body, migration is not affected. These data are confirmed immunocytochemically in that the anion exchanger AE2 is concentrated at the front of MDCK-F cells. Our findings show that NHE and anion exchanger are distributed in a polarized way in migrating cells. They are consistent with important contributions of both transporters to protrusion of the lamellipodium via solute uptake and consequent volume increase at the front of migrating cells.

Amiloride↗
PubMed2000

Inherited renal tubular acidosis.

The past few years have witnessed great progress in elucidating the molecular basis of inherited renal tubular acidosis. Consistent with the physiologically defined importance of multiple gene products in urinary acidification, heritable renal tubular acidosis is genetically heterogeneous. Autosomal dominant distal renal tubular acidosis has been associated with a small number of mutations in the AE1 Cl-/HCO3- exchanger although the pathophysiologic mechanisms behind these mutations remain unclear. Rarely, autosomal recessive distal RTA is caused by homozygosity or compound heterozygosity for the loss-of-function mutation AE1 G701D. A larger proportion, often accompanied by hearing loss, is associated with mutations in the ATP6B1 gene encoding the 58 kDa B1 subunit of the vacuolar H+-ATPase. Mutations in the gene encoding the Na+/HCO3- cotransporter, NBC1, have recently been identified in proximal renal tubular acidosis with corneal calcification.

Acidosis, Renal Tubular↗