Podocalyxin enhances the adherence of cells to platelets.

Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Delta) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin alphaVbeta3/alphaVbeta5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid.

Cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme gene. Regulatory role of Sp1 and AP-2.

This work reports the molecular cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme (mME) gene. The proximal promoter region has features of housekeeping genes like high G + C-content and absence of TATA or CCAAT boxes. Deletion analysis of the 5' region of the mME showed that maximal transcriptional activity is located within the -205/+86 region. Footprinting analysis showed two protected regions, one comprising potential overlapped AP-1, CREB, and AP-4 sites and a second one encompassing AP-2 and several Sp1 ci-acting elements. Mutation of putative AP-1/AP-4/CREB sites reduced basal promoter activity to less than 50%. Supershift assays demonstrated the specific binding of Sp1 and AP-2 proteins. Moreover, experiments in Drosophila SL2 cells lacking endogenous Sp1 demonstrated that the Sp1 site(s) is essential to maintain a normal basal rate of transcription of this gene. A low-level expression of AP-2 enhanced the activity of a mME promoter construct in HepG2 cells and this effect was prevented by disruption of the putative AP-2 element. In contrast, higher levels of expression of AP-2 induced a DNA-independent inhibitory response. A biphasic regulation of endogenous mME gene is also shown in HepG2 cells transfected with an AP-2 expression plasmid, suggesting that availability of AP-2 protein may control this gene under physiological conditions. A recombinant lambda genomic clone containing a mME pseudogene was also isolated. The high degree of sequence conservation seems to indicate a recent emergency of this human pseudogene.

Competition between normal [674C] and mutant [674R] subunits: role of the molecular chaperone BiP in the processing of GPIIb-IIIa complexes.

This work aimed at investigating the function of the [C674R] mutation in GPIIb that disrupts the intramolecular 674 to 687 disulfide bridge. Individuals heterozygous for this mutation show a platelet GPIIb-IIIa content approximately 30% of normal controls, which is less than expected from one normal functioning allele. Coexpression of normal [674C]GPIIb and mutant [674R]GPIIb with normal GPIIIa produced a [674R]GPIIb concentration-dependent inhibition of surface exposure of GPIIb-IIIa complexes in Chinese hamster ovary (CHO) cells, suggesting that [674R]GPIIb interferes with the association and/or intracellular trafficking of normal subunits. Mutation of either 674C or 687C had similar effects in reducing the surface exposure of GPIIb-IIIa. However, substitution of 674C for A produced a much lesser inhibition than R, suggesting that a positive-charged residue at that position renders a less efficient subunit conformation. The mutant [674R]GPIIb but not normal GPIIb was found associated with the endoplasmic reticulum chaperone BiP in transiently transfected CHO cells. BiP was also found associated with [674R]GPIIb-IIIa heterodimers, but not with normal GPIIIa or normal heterodimers. Overexpression of BiP did not increase the surface exposure of [674R]GPIIb-IIIa complexes, indicating that its availability was not a limiting step. Platelets from the thrombasthenic patient expressing [674R]GPIIb-IIIa were found to bind soluble fibrinogen in response to physiologic agonists or dithiothreitol treatment. Thus, the [674R]GPIIb mutation leads to a retardation of the secretory pathway, most likely related to its binding to the molecular chaperone BiP, with the result of a defective number of functional GPIIb-IIIa receptors in the cell surface.

Enhanced proliferation of lymphoblasts from patients with Alzheimer dementia associated with calmodulin-dependent activation of the na+/H+ exchanger.

We have recently reported that lymphoblasts from late onset Alzheimer's disease (AD) patients show distinct intracellular pH homeostatic features than those obtained from age-matched healthy donors. Here we report that another distinct feature of AD lymphoblasts is their increased rate of proliferation in serum containing medium, suggesting a different responsiveness of AD cells to serum activators. The increased proliferation of AD cells was accompanied by intracellular alkalinization and was prevented by blockers of the plasma membrane Na+/H+ antiporter (NHE), indicating that the exchanger had to be activated to elicit the cellular responses. The activity of this exchanger can be controlled through several signaling pathways, but only the inhibition of calmodulin activity impeded the serum-induced intracellular alkalinization and enhanced proliferation of AD cells. In contrast, the inhibition of calmodulin did not alter the rate of proliferation of normal cells. Thus, it seems plausible to conclude that the enhanced proliferation of AD cells is the result of a surface receptor-mediated activation of the Ca(2+)-calmodulin signaling pathway. Our observations add further support in favor that AD may be considered a systemic disease which underlying etiopathogenic mechanism may be an altered responsiveness to cell activating agents. Thus, the use of lymphoblastoid cells from AD patients may be a useful model to investigate cell biochemical aspects of this disease.

Compound heterozygosity of the GPIbalpha gene associated with Bernard-Soulier syndrome.

We report the molecular genetic analysis of the Bernard-Soulier syndrome (BSS) phenotype in two related patients showing absence of glycoprotein (GP) Ibalpha and detectable amounts of GPIX on the platelet surface, and a truncated form of GPIbalpha in solubilized platelets and plasma. They both were compound heterozygotes for the GPIbalpha gene: a maternal allele with a T insertion at position 1418 causing a translational frameshift and premature polypeptide termination, and a paternal allele with a T715A substitution chan-ino Cys209 to Ser. Heterozygotes for either one of these mutations were asymptomatic. Transient transfection of cells coexpressing GPIbbeta and GPIX failed to detect surface expression of the GPIbalpha mutants. Cells transfected with [1418insT]GPIbalpha-cDNA showed a truncated protein of the predicted size in both cell lysate and conditioned medium, indicating the inability of the mutant protein to anchor the plasma membrane. In contrast. transfection of [T715A]GPIbalpha-cDNA yield a mutated protein barely detectable in the cell lysate and absent in the medium, indicating that the loss of Cys209 renders GPIbalpha more vulnerable to proteolysis and unable to undergo the normal secretory pathway. Our findings indicate that the additive effects of both mutations are responsible for the BSS phenotype of the patients.

Cloning and functional characterization of the 5' regulatory region of the human mitochondrial glycerol-3-phosphate dehydrogenase gene. Lack of 3,5,3'-triiodothyronine responsiveness in adipose tissue.

We report data on the structural and functional characterization of the 5' flanking region of the human mitochondrial glycerol-3-phosphate dehydrogenase (mtGPDH) gene. We found two regions upstream of 5'-untranslated sequences exhibiting promoter activity in transient transfection assays. Transcription start sites and potential regulatory sites in both promoter regions were defined. The proximal promoter was approximately sevenfold more active than the distal one in most cell lines, but it was only twice as active in a neuroblastoma cell line. These observations seem to indicate that the rate of transcription, as well as the tissue-specific expression of the human mtGPDH gene, is the result of a combinatorial effect of transcription factors on at least two promoters. 3,5,3'-Triiodothyronine failed to alter the transcriptional activity of human mtGPDH promoter(s) constructs in transient transfection assays. Although this finding seems to be in conflict with the reported effect of 3,5,3'-triiodothyronine in rodents, it is consistent with our observation of 3,5, 3'-triiodothyronine stimulation of mtGPDH activity in primary cultures of rat adipocytes, but not human cultured adipocytes, suggesting distinct regulation of this gene in both species.

A 1063G-->A mutation in exon 12 of glycoprotein (GP)IIb associated with a thrombasthenic phenotype: mutation analysis of [324E]GPIIb.

We report the molecular, genetic and functional analysis of a case of thrombasthenic phenotype. The proband showed absence of platelet glycoprotein (GP)IIb and very low content of GPIIIa, and both his parents showed a marked reduction in the levels of platelet GPIIb-IIIa. Single-stranded conformational polymorphism-polymerase chain reaction (SSCP-PCR) analysis and direct sequencing of PCR-amplified GPIIb exon-12 revealed the presence of a G-->A transition at position 1063 with the expected substitution of glutamate 324 with lysine (K). This mutation did not alter the level of GPIIb mRNA. Co-expression of normal or mutant [324K] GPIIb with normal human GPIIIa in Chinese hamster ovary (CHO) cells failed to show surface exposure of [324K]GPIIb-IIIa complexes. Pulse-chase and immunoprecipitation analysis demonstrated that [324K]GPIIb cDNA was translated into proGPIIb, but neither mutant GPIIb heavy chain (GPIIbH) nor [324K]GPIIb-GPIIIa complexes were detected, suggesting that this mutation is the underlying molecular basis for the thrombasthenic phenotype. Mutation analysis demonstrated that 324E of GPIIb could be replaced by other negatively charged or polar amino acids (AAs) without impairing the surface expression of GPIIb-IIIa. However, substitution of 324E of GPIIb for a positively charged AA other than K prevented the expression of GPIIb-IIIa complexes. These observations suggest that a domain encompassing 324E of GPIIb is essential for heterodimerization with GPIIIa and its substitution for a positively charged residue precludes normal subunit association.

A novel (288delC) mutation in exon 2 of GPIIb associated with type I Glanzmann's thrombasthenia.

This work reports the molecular genetic analysis of two patients who suffer mucocutaneous haemorrhages, prolonged bleeding time and failure of platelets to aggregate, either spontaneously or in response to agonists. The absence of platelet surface glycoprotein (GP)IIb-IIIa complexes confirmed the clinical diagnosis of Glanzmann's thrombasthenia (GT). Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of exon 2 of GPIIb showed polymorphic bands caused by the homozygous deletion of a cytosine at position 288 relative to the translation start site. causing a shifting of the reading frame and appearance of a premature termination codon. The heterozygous relatives showed a reduced platelet content of GPIIb-IIIa, and a correlation was found between the levels of GPIIb mRNA and surface expression of GPIIb-IIIa complexes. Unlike other mRNAs carrying a nonsense mutation, (288Cdel)GPIIb does not force alternative splicing of GPIIb mRNA. As expected, co-transfection of Chinese hamster ovary (CHO) cells with cDNAs encoding GPIIIa and (288delC)GPIIb failed to enhance the surface exposure of GPIIIa. It is concluded that the (288delC)GPIIb mutation is responsible for the thrombasthenic phenotype of the patients. In addition, it has also been determined that heterodimerization of GPIIb-IIIa requires the integrity of exons 2 and 3 of GPIIb.

Molecular genetic analysis of a compound heterozygote for the glycoprotein (GP) IIb gene associated with Glanzmann's thrombasthenia: disruption of the 674-687 disulfide bridge in GPIIb prevents surface exposure of GPIIb-IIIa complexes.

This work was aimed at elucidating the molecular genetic lesion(s) responsible for the thrombasthenic phenotype of a patient whose low platelet content of glycoprotein (GP) IIb-IIIa indicated that it was a case of type II Glanzmann's thrombasthenia (GT). The parents did not admit consanguinity and showed a reduced platelet content of GPIIb-IIIa. Polymerase chain reaction (PCR)-single-stranded conformational polymorphism analysis of genomic DNA showed no mutations in the patient's GPIIIa and two novel mutations in the GPIIb gene: one of them was a heterozygous splice junction mutation, a C-->A transversion, at position +2 of the exon 5-intron 5 boundary [IVS5(+2)C-->A] inherited from the father. The predicted effect of this mutation, insertion of intron 5 (76 bp) into the GPIIb-mRNA, was confirmed by reverse transcription-PCR analysis of platelet mRNA. The almost complete absence of this mutated form of GPIIb-mRNA suggests that it is very unstable. Virtually all of the proband's GPIIb-mRNA was accounted for by the allele inherited from the mother showing a T2113-->C transition that changes Cys674-->Arg674 disrupting the 674-687 intramolecular disulfide bridge. The proband showed a platelet accumulation of proGPIIb and minute amounts of GPIIb and GPIIIa. Moreover, transfection and immunoprecipitation analysis demonstrated that [Arg674]GPIIb is capable of forming a heterodimer complex with GPIIIa, but the rate of subunit maturation and the surface exposure of GPIIb-IIIa are strongly reduced. Thus, the intramolecular 674-687 disulfide bridge in GPIIb is essential for the normal processing of GPIIb-IIIa complexes. The additive effect of these two GPIIb mutations provides the molecular basis for the thrombasthenic phenotype of the proband.

AP-1 and T3RE cis elements operate as a functional unit in the transcriptional control of the human malic enzyme gene.

The human malic enzyme (hME) promoter contains an inverted palindromic (IP4) 3,5,3'-triiodo-thyronine (T3) response element (T3RE) 15bp downstream from an activating protein-1 (AP-1) site. The purpose of this study was to analyze the functional relationship between both cis-acting elements. The following observations indicate that these two elements operate as a functional unit in controlling the human ME gene:T3 failed to stimulate transcription above the basal levels in cells overexpressing either TRb or TRb/retinoid acid receptor (RXR), indicating that TRbeta acts primarily as a transcriptional repressor in the context of the hME. Moreover, the finding of a repressive effect of TRbeta without DNA binding suggests the existence of both DNA-dependent and independent mechanisms of TRbeta-induced repression of transcription.

Role of the alpha-subunit 326GRV sequence in the surface expression of fibrinogen and vitronectin receptors.

The platelet GPIIb-GPIIIa heterodimer (integrin alphaIIbbeta3) binds fibrinogen with high affinity in response to activation by agonists, leading to platelet aggregation and formation of a hemostatic plug. The 326GRV motif in GPIIb is highly conserved in the alpha-subunit of other integrins, suggesting that it might play an important functional role. Moreover, Arg327-->His substitution in GPIIb has been associated with defective platelet surface expression of GPIIb-IIIa and thrombasthenic phenotype. This work aimed at elucidating whether the absence of Arg327 or its substitution by His was responsible for the impaired surface expression of GPIIb-IIIa complexes. Transfection of cDNA encoding [Ala327]GPIIb, [Gln327]GPIIb, or [Phe327]GPIIb into Chinese hamster ovary cells inherently expressing GPIIIa permitted surface exposure of GPIIb-IIIa complexes, whereas [Glu327]GPIIb did not. These observations indicate that it is not the loss of [Arg327]GPIIb but the presence of His327 or a negatively charged residue like Glu at position 327 of GPIIb that prevents the surface exposure of GPIIb-IIIa heterodimers. In contrast, changing Gln344, the homologue to Arg327 in the alpha-subunit of the vitronectin receptor, to His did not prevent the surface expression of alphav-GPIIIa complexes. Thus the conformational constraint imposed by His327 seems to be rather specific for the heterodimerization and/or processing of GPIIb-IIIa complexes.

Distinct pH homeostatic features in lymphoblasts from Alzheimer's disease patients.

Epstein-Barr-transformed lymphocytes from Alzheimer's disease patients showed the following distinct features in controlling the intracellular pH compared with cells from normal age-matched controls: (1) The alphaIgM-induced intracellular acidification was more pronounced in Alzheimer's disease than control cells and this effect appears to be associated with a loss of effectiveness of a Ca2+/calmodulin-dependent mechanism in controlling the activity of the Na+/H+ exchanger; and (2) the intracellular H+-buffering capacity and the rate of proton efflux in response to an acid load were both decreased in Alzheimer's disease cells. It is concluded that the amplitude of the intracellular pH changes under acid-loading conditions will always be greater in Alzheimer's disease than in control cells.

Mutation analysis of chromosome 19 calmodulin (CALM3) gene in Alzheimer's disease patients.

The calcium buffering capacity of lymphoblasts from patients suffering of late onset Alzheimer's disease (AD) has been reported to be diminished. Calmodulin is a calcium binding protein codified by three genes, one of them (CALM3) maps to chromosome 19, nearby a gene, apoE, associated with late onset AD. In this study we screened for structural changes in the CALM3 gene from AD patients by PCR-SSCP analysis. We observed several point mutations in the intronic flanking regions of exons 3 and 4 of CALM 3 gene. However, we failed to detect any structural changes in the regions encoding the calcium binding domains of this gene. Similar results were obtained by RT-PCR analysis of CALM3 transcripts from AD patients carrying apoE epsilon4 allele. It is concluded that structural alterations in the CALM3 gene are not associated with the altered Ca2+ homeostasis shown by lymphoblasts from these patients.

Molecular cloning and functional characterization of the human cytosolic malic enzyme promoter: thyroid hormone responsiveness.

We report the structural and functional features of the 5'-flanking region of the human cytosolic malic enzyme (ME) gene. A 2.2-kb subclone, comprising 1.5 kb upstream of the translation initiation codon, the first exon, and 0.7 kb of flanking intronic region, was sequenced and mapped to chromosome 6. The proximal promoter region is rich in G + C, lacks TATA or CCAAT boxes, and shows multiple transcription start sites, the major one 106 nucleotides upstream the ATG codon. Sequences -59/-13 and -137/-103 conferred maximal promoter activity. Deletional analysis revealed the presence of two regions positively regulated by 3,5,3'-triiodo-L-thyronine (T3). The proximal region confers the strongest T3 inducibility to the human ME as well as to a heterologous promoter. Thyroid hormone receptor beta (TRbeta) binds to an inverted palindromic T3 response element (TRE) at position -105/-87 in a manner that is prevented by T3. Nuclear extracts or in vitro-translated retinoid acid receptor alpha (RXR alpha) shifted the TRbeta retarded band to slower-mobility complexes, which are unaffected by T3. In the absence of T3, overexpression of TRbeta repressed the ME promoter activity, most probably, through binding of TRbeta homodimers to the TRE. Thus, T3 seems to control ME transcription by inducing the dissociation of TRbeta homodimers and the functional activation of liganded heterodimers.

Altered Ca2+ homeostasis in lymphoblasts from patients with late-onset Alzheimer disease.

The authors report calcium (Ca2+) homeostasis features of transformed lymphocytes from patients with late-onset Alzheimer disease and healthy age-matched controls. Alzheimer lymphoblasts show higher basal cytosolic-free [Ca2+] than controls. The antibodies anti-immunoglobulin M or the beta-amyloid (beta-amyloid) peptide fragment 25-35-induced elevation of cytosolic-free [Ca2+] was higher in Alzheimer disease lymphoblasts than in control cells. However, the kinetics of Ca2+ replenishment of Ca(2+)-depleted cells shows a higher accumulation of cytosolic Ca2+ in Alzheimer disease than in control lymphoblasts, which is better appreciated when the Ca2+ efflux is inhibited. Thus, the authors concluded that Alzheimer disease lymphoblasts have a lower Ca2+ buffering capacity than normal cells, probably because of changes in availability or intrinsic functional properties of the intracellular Ca(2+)-binding structures. Aging alters the kinetics of the Ca2+ replenishment in lymphoblasts in a manner that resembles Alzheimer disease. However, unlike Alzheimer disease, aging does not change the maximum cytosolic-free [Ca2+], suggesting that the mechanisms underlying the altered Ca2+ homeostasis in aging and late-onset Alzheimer disease are different.

A mutant (Arg327-->His) GPIIb associated to thrombasthenia exerts a dominant negative effect in stably transfected CHO cells.

This work reports the structural and functional characterization of the platelet glycoprotein complex GPIIb-IIIa (integrin alpha IIb beta 3) in a patient of type II Glanzmann thrombasthenia, bearing a homozygous G-->A base transition at position 1074 of GPIIb that results in an Arg327-->His substitution. CHO cells stably transfected with cDNA encoding His327GPIIb showed a drastic reduction in the surface expression of alpha IIb beta 3 complex relative to control cells transfected with wild type GPIIb. Immunoprecipitation analysis demonstrated that GPIIb synthesis, heterodimerization, and short term maturation were not impeded, suggesting that conformational changes dependent on Arg327 of GPIIb may play an essential role in either the rate of maturation and/or transport of heterodimers to the cell surface. Cotransfection of CHO cells with equimolar amounts of cDNAs encoding wild type and mutant His327-GPIIb led to a marked reduction in the surface expression of alpha IIb beta 3. This novel observation of a dominant-negative effect of the mutant His 327 alpha IIb subunit provides a molecular basis for the reduced platelet alpha IIb beta 3 content observed in the heterozygous offspring.

Modulation of the hepatic alpha 1-adrenoceptor responsiveness by colchicine: dissociation of free cytosolic Ca(2+)-dependent and independent responses.

1. The cytoskeletal depolymerizing agent, colchicine, prevents the hepatic alpha 1-adrenoceptor-mediated stimulation of respiration, H+ and Ca2+ release to the effluent perfusate, intracellular alkalosis, and glycogenolysis. Unlike the other parameters, colchicine does not perturb the alpha 1-agonist-induced stimulation of gluconeogenesis or phosphorylase 'a' activation, and enhances the increase in portal pressure response. The lack of effect of colchicine on the hepatic alpha 2-adrenoceptor-mediated effects indicates that its actions are alpha 1-specific. 2. Colchicine enhances the acute alpha 1-adrenoceptor-mediated intracellular Ca2+ mobilization and prevents the activation of protein kinase C. This differential effect on the two branches of the alpha 1-adrenoceptor signalling pathway is a distinctive feature of the colchicine action. 3. The lack of effect of colchicine in altering the alpha 1-adrenoceptor ligand binding affinity suggests that it might interact with some receptor-coupled regulatory element(s). 4. The acuteness of the colchicine effect and the ability of its isomer beta-lumicolchicine to prevent all the alpha 1-adrenoceptor-mediated responses but the increase in vascular resistance, indicate that its action cannot be merely ascribed to its effects in depolymerizing tubulin. 5. Colchicine perturbs the hepatic responses to vasoactive peptides. It enhances the vasopressin-induced rise of cytosolic free Ca2+ in isolated hepatocytes and prevents the sustained decrease of Ca2+ in the effluent perfusate. It also inhibits the stimulation of glycogenolysis, without altering the stimulation of gluconeogenesis. 6. It is concluded that there are at least two major alpha 1-adrenoceptor signalling pathways. One is colchicine-sensitive, independent of variations in free cytosolic Ca2+, and protein kinase C-dependent; the other one is colchicine-insensitive, dependent on variations in free cytosolic Ca2+, and protein kinase C-independent.

Apolipoprotein E genotype in Spanish patients of Alzheimer's or Parkinson's disease.

Blood donors of the Madrid area show a 6% frequency of apolipoprotein E genotype carrying allele epsilon 4. This frequency is smaller than other populations of Caucasian origin. This proportion decreases to 4% in a selected sample of healthy individuals of ages > 60 years. The frequency (34%) of the allele epsilon 4 was significantly increased in patients of late onset Alzheimer's disease, similarly to other populations. An earlier age of onset of the dementia is observed in the patients of late-onset Alzheimer's disease carrying the allele epsilon 4. No increased frequency in allele epsilon 4 frequency was found in patients of early-onset Alzheimer's disease. Patients of Parkinson's disease do not show any differences in the frequency of the alleles of apolipoprotein E when compared with healthy individuals.

Cloning, sequencing and functional expression of a cDNA encoding a NADP-dependent malic enzyme from human liver.

This work reports the structure of a cDNA (ME) encoding a human malic enzyme (ME) (malate NADP oxidoreductase, EC 1.1.1.40) elucidated by joining several overlapping fragments amplified by PCR from human hepatic cDNA or from cDNA libraries. The full-length cDNA has an open reading frame (ORF) of 1719 bp that encodes a 572-amino-acid protein of 64 113 Da, similar to the native monomeric, cytosolic, NADP-dependent ME isolated from human liver. The comparison of the structure of this cDNA with that of the human mitochondrial NAD(P)-dependent ME (EC 1.1.1.39) shows a homology of 63%, suggesting that these two forms originated from the same gene. The expression of the cDNA in Escherichia coli as a translational fusion (glutathione S-transferase::ME) protein yielded a product of the predicted mass. The recombinant protein shows NADP-dependent malate oxidoreductase activity and is virtually inactive with NAD. It also shows other distinct features of the native cytosolic NADP-dependent ME, like Mn2+ dependence, similar substrate (Km = 117 microM) and cofactor affinity (Km = 2 microM) constants, and a lack of allosteric regulation. In human proliferative cells, the NADP-dependent ME activity is poorly expressed and barely inducible by thyroid hormones.

Role of amino acid-induced changes in ion fluxes in the regulation of hepatic protein synthesis.

Alanine is a powerful stimulator of hepatic protein synthesis whose mechanism of action has not yet been ascertained. The present work aimed to elucidate whether rate changes in ion fluxes accompanying the transport of this amino acid could play a role in the stimulation of protein synthesis. In perfused livers, the utilization of alanine produced a net uptake of K+ of 1.5 mumol/min/liver, a progressively increasing efflux of Ca2+ to reach a maximum of 0.9 mumol/min/liver, and alkalization of the extracellular medium. Inhibition of Na+/K+ exchange by ouabain reversed only the uptake of K+, indicating that this is the main way for the efflux of Na+ cotransported with alanine. In isolated hepatocytes, the uptake of alanine increased the intracellular content of K+ and the cell volume. The following observations suggest that these changes, and not an increased intracellular concentration of Na+, are associated with the stimulation of protein synthesis: 1) Ouabain inhibited the alanine stimulation of L-[3H]-valine incorporation into protein without altering the basal rate of protein labeling; 2) ouabain had no effects on alanine uptake indicating that Na+ influx is not involved in the alanine stimulation of protein synthesis; 3) disruption of Na+ gradient across the plasma membrane by specific ionophores, monensin and gramicidin D, inhibited both basal and alanine-stimulated protein synthesis, but substitution of extracellular Na+ by K+ did not prevent the stimulatory action of alanine. The observation that hypotonic buffer enhanced protein synthesis to the same degree than alanine in liver cells indicates that alanine-induced cell swelling could be sufficient to stimulate protein synthesis.