Sildenafil reduces polyuria in rats with lithium-induced NDI

Sanches TR, Volpini RA, Massola Shimizu MH, de Bragan a AC, Oshiro-Monreal F, Seguro AC, Andrade L. Sildenafil reduces polyuria in rats with lithium-induced NDI. Am J Physiol Renal Physiol 302: F216-F225, 2012. First published October 12, 2011; doi:10.1152/ajprenal.00439.2010.-Lithium (Li)-treated patients often develop urinary concentrating defect and polyuria, a condition known as nephrogenic diabetes insipidus (NDI). In a rat model of Li-induced NDI, we studied the effect that sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, has on renal expression of aquaporin-2 (AQP2), urea transporter UT-A1, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), epithelial Na channel (ENaC; alpha-, beta-, and gamma-subunits), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase. We also evaluated cGMP levels in medullary collecting duct cells in suspension. For 4 wk, Wistar rats received Li (40 mmol/kg food) or no treatment (control), some receiving, in weeks 2-4, Sil (200 mg/kg food) or Li and Sil (Li+Sil). In Li+Sil rats, urine output and free water clearance were markedly lower, whereas urinary osmolality was higher, than in Li rats. The cGMP levels in the suspensions of medullary collecting duct cells were markedly higher in the Li+Sil and Sil groups than in the control and Li groups. Semiquantitative immunoblotting revealed the following: in Li+Sil rats, AQP2 expression was partially normalized, whereas that of UT-A1, gamma-ENaC, and eNOS was completely normalized; and expression of NKCC2 and NHE3 was significantly higher in Li rats than in controls. Inulin clearance was normal in all groups. Mean arterial pressure and plasma arginine vasopressin did not differ among the groups. Sil completely reversed the Li-induced increase in renal vascular resistance. We conclude that, in experimental Li-induced NDI, Sil reduces polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1.

Influence of Genomic Ancestry on the Distribution of SLCO1B1, SLCO1B3 and ABCB1 Gene Polymorphisms among Brazilians

The frequency distribution of SNPs and haplotypes in the ABCB1, SLCO1B1 and SLCO1B3 genes varies largely among continental populations. This variation can lead to biases in pharmacogenetic studies conducted in admixed populations such as those from Brazil and other Latin American countries. The aim of this study was to evaluate the influence of self-reported colour, geographical origin and genomic ancestry on distributions of the ABCB1, SLCO1B1 and SLCO1B3 polymorphisms and derived haplotypes in admixed Brazilian populations. A total of 1039 healthy adults from the north, north-east, south-east and south of Brazil were recruited for this investigation. The c.388A>G (rs2306283), c.463C>A (rs11045819) and c.521T>C (rs4149056) SNPs in the SLCO1B1 gene and c.334T>G (rs4149117) and c.699G>A (rs7311358) SNPs in the SLCO1B3 gene were determined by Taqman 5'-nuclease assays. The ABCB1 c.1236C>T (rs1128503), c.2677G>T/A (rs2032582) and c.3435C>T (rs1045642) polymorphisms were genotyped using a previously described single-base extension/termination method. The results showed that genotype and haplotype distributions are highly variable among populations of the same self-reported colour and geographical region. However, genomic ancestry showed that these associations are better explained by a continuous variable. The influence of ancestry on the distribution of alleles and haplotype frequencies was more evident in variants with large differences in allele frequencies between European and African populations. Design and interpretation of pharmacogenetic studies using these transporter genes should include genomic controls to avoid spurious conclusions based on improper matching of study cohorts from Brazilian populations and other highly admixed populations.

Co-expression of monocarboxylate transporter 1 (MCT1) and its chaperone (CD147) is associated with low survival in patients with gastrointestinal stromal tumors (GISTs)

Monocarboxylate transporters (MCTs) have been described to play an important role in cancer, but to date there are no reports on the significance of MCT expression in gastrointestinal stromal tumors (GISTs). The aim of the present work was to assess the value of MCT expression, as well as co-expression with the MCT chaperone CD147 in GISTs and evaluate their clinical-pathological significance. We analyzed the immunohistochemical expression of MCT1, MCT2, MCT4 and CD147 in a series of 64 GISTs molecularly characterized for KIT, PDGFRA and BRAF mutations. MCT1, MCT2 and MCT4 were highly expressed in GISTs. CD147 expression was associated with mutated KIT (p = 0.039), as well as a progressive increase in Fletcher's Risk of Malignancy (p = 0.020). Importantly, co-expression of MCT1 with CD147 was associated with low patient's overall survival (p = 0.037). These findings suggest that co-expression of MCT1 with its chaperone CD147 is involved in GISTs aggressiveness, pointing to a contribution of cancer cell metabolic adaptations in GIST development and/or progression.

Increased clopidogrel response is associated with ABCC3 expression: A pilot study

Background: The aim of this study was investigate the relationship between ABCB1 and ABCC3 gene expressions in peripheral blood cells (PBC) and the response to clopidogrel in patients with coronary arterial disease (CAD). Methods: Twenty-six male CAD patients (50-70 years) under treatment with clopidogrel (75 mg/day) for at least 5 days were selected. Blood samples were obtained to evaluate platelet reactivity and ABCB1 and ABCC3 mRNA expression. Platelet reactivity was measured in P2Y12 Reaction Units (PRU) using VerifyNow. RNA was extracted from PBC and mRNA levels were measured by qPCR, using GAPD as a reference gene. Results: Platelet response to clopidogrel was categorized in to PRU quartiles. Individuals with PRU values within the first quartile (Q1, <151 units) were considered good responders, while those who had PRU within the fourth quartile (Q4. PRU>260) were considered non-responders. ABCC3 was 1.7 times more expressed in Q4 than in Q1 PRU group (p=0.048). Moreover, CAD patients with low ABCC3 expression (Qe1, <2.5x10(-3)) had higher probability to have a good response to clopidogrel (OR: 18.00, 95%CI: 1.90-169.99, p=0.001). Univariate linear regression analysis demonstrated that low ABCC3 mRNA expression contributed with a reduction of 73 PRU in relation to the patients with expression value higher than 2.5x10(-3) (p=0.027). Neither ABCB1 mRNA levels nor clinical variables studied influenced PRU values. Conclusions: Low ABCC3 mRNA expression in peripheral blood cells is associated with increased clopidogrel response, but further studies are needed to describe the functional relationship of clopidogrel with the ABCC3. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.

Effects of 3 weeks GMP oral administration on glutamatergic parameters in mice neocortex

Overstimulation of the glutamatergic system (excitotoxicity) is involved in various acute and chronic brain diseases. Several studies support the hypothesis that guanosine-5'-monophosphate (GMP) can modulate glutamatergic neurotransmission. The aim of this study was to evaluate the effects of chronically administered GMP on brain cortical glutamatergic parameters in mice. Additionally, we investigated the neuroprotective potential of the GMP treatment submitting cortical brain slices to oxygen and glucose deprivation (OGD). Moreover, measurements of the cerebrospinal fluid (CSF) purine levels were performed after the treatment. Mice received an oral administration of saline or GMP during 3 weeks. GMP significantly decreases the cortical brain glutamate binding and uptake. Accordingly, GMP reduced the immunocontent of the glutamate receptors subunits, NR2A/B and GluR1 (NMDA and AMPA receptors, respectively) and glutamate transporters EAAC1 and GLT1. GMP treatment significantly reduced the immunocontent of PSD-95 while did not affect the content of Snap 25, GLAST and GFAP. Moreover, GMP treatment increased the resistance of neocortex to OGD insult. The chronic GMP administration increased the CSF levels of GMP and its metabolites. Altogether, these findings suggest a potential modulatory role of GMP on neocortex glutamatergic system by promoting functional and plastic changes associated to more resistance of mice neocortex against an in vitro excitotoxicity event.

Effects of Continuous Erythropoietin Receptor Activator in Sepsis-Induced Acute Kidney Injury and Multi-Organ Dysfunction

Background: Despite advances in supportive care, sepsis-related mortality remains high, especially in patients with acute kidney injury (AKI). Erythropoietin can protect organs against ischemia and sepsis. This effect has been linked to activation of intracellular survival pathways, although the mechanism remains unclear. Continuous erythropoietin receptor activator (CERA) is an erythropoietin with a unique pharmacologic profile and long half-life. We hypothesized that pretreatment with CERA would be renoprotective in the cecal ligation and puncture (CLP) model of sepsis-induced AKI. Methods: Rats were randomized into three groups: control; CLP; and CLP+CERA (5 mu g/kg body weight, i.p. administered 24 h before CLP). At 24 hours after CLP, we measured creatinine clearance, biochemical variables, and hemodynamic parameters. In kidney tissue, we performed immunoblotting-to quantify expression of the Na-K-2Cl cotransporter (NKCC2), aquaporin 2 (AQP2), Toll-like receptor 4 (TLR4), erythropoietin receptor (EpoR), and nuclear factor kappa B (NF-kappa B)-and immunohistochemical staining for CD68 (macrophage infiltration). Plasma interleukin (IL)-2, IL-1 beta, IL-6, IL-10, interferon gamma, and tumor necrosis factor alpha were measured by multiplex detection. Results: Pretreatment with CERA preserved creatinine clearance and tubular function, as well as the expression of NKCC2 and AQP2. In addition, CERA maintained plasma lactate at normal levels, as well as preserving plasma levels of transaminases and lactate dehydrogenase. Renal expression of TLR4 and NF-kappa B was lower in CLP+CERA rats than in CLP rats (p<0.05 and p<0.01, respectively), as were CD68-positive cell counts (p<0.01), whereas renal EpoR expression was higher (p<0.05). Plasma levels of all measured cytokines were lower in CLP+CERA rats than in CLP rats. Conclusion: CERA protects against sepsis-induced AKI. This protective effect is, in part, attributable to suppression of the inflammatory response.

Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -beta and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The Delta pmcA and Delta pmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the Delta calA and Delta crzA mutant strains. However, only the A. fumigatus Delta pmcA was avirulent in the murine model of invasive pulmonary aspergillosis.

Atorvastatin Prevents the Downregulation of Aquaporin-2 Receptor After Bilateral Ureteral Obstruction and Protects Renal Function in a Rat Model

OBJECTIVE To assess the effects of atorvastatin (ATORV) on renal function after bilateral ureteral obstruction (BUO), measuring inulin clearance and its effect on renal hemodynamic, filtration, and inflammatory response, as well as the expression of Aquaporin-2 (AQP2) in response to BUO and after the release of BUO. METHODS Adult Munich-Wistar male rats were subjected to BUO for 24 hours and monitored during the following 48 hours. Rats were divided into 5 groups: sham operated (n = 6); sham + ATORV (n = 6); BUO (n = 6); BUO + ATORV (10 mg/kg in drinking water started 2 days before BUO [n = 5]; and BUO + ATORV (10 mg/kg in drinking water started on the day of the release of BUO [n = 5]). We measured blood pressure (BP, mm Hg); inulin clearance (glomerular filtration rate [GFR]; mL/min/100 g); and renal blood flow (RBF, mL/min, by transient-time flowmeter). Inflammatory response was evaluated by histologic analysis of the interstitial area. AQP2 expression was evaluated by electrophoresis and immunoblotting. RESULTS Renal function was preserved by ATORV treatment, even if initiated on the day of obstruction release, as expressed by GFR, measured by inulin clearance. Relative interstitial area was decreased in both BUO + ATORV groups. Urine osmolality was improved in the ATORV-treated groups. AQP2 protein expression decreased in BUO animals and was reverted by ATORV treatment. CONCLUSION ATORV administration significantly prevented and restored impairment in GFR and renal vascular resistance. Furthermore, ATORV also improved urinary concentration by reversing the BUO-induced downregulation of AQP2. These findings have significant clinical implication in treating obstructive nephropathy. UROLOGY 80: 485.e15-485.e20, 2012. (c) 2012 Elsevier Inc.

Global transcriptional response of Caulobacter crescentus to iron availability

Abstract Background In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. Results In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identified 42 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB-dependent receptor gene clusters, and feoAB), riboflavin biosynthesis and genes encoding hypothetical proteins. Most of these genes are associated with predicted Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by β-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 27 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, included in this group are also TonB-dependent receptors genes and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of 113 more genes, including induction of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as repression of genes implicated in amino acid metabolism, chemotaxis and motility. Conclusions Using a global transcriptional approach, we determined the C. crescentus iron stimulon. Many but not all of iron responsive genes were directly or indirectly controlled by Fur. The iron limitation stimulon overlaps with other regulatory systems, such as the RpoH and FixK regulons. Altogether, our results showed that adaptation of C. crescentus to iron limitation not only involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins, but also includes novel genes and regulatory mechanisms.

Identification of differentially expressed genes from Trichoderma harzianum during growth on cell wall of Fusarium solani as a tool for biotechnological application

Background: The species of T. harzianum are well known for their biocontrol activity against many plant pathogens. However, there is a lack of studies concerning its use as a biological control agent against F. solani, a pathogen involved in several crop diseases. In this study, we have used subtractive library hybridization (SSH) and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum genes expression during growth on cell wall of F. solani (FSCW) or glucose. RT-qPCR was also used to examine the regulation of 18 genes, potentially involved in biocontrol, during confrontation between T. harzianum and F. solani. Results: Data obtained from two subtractive libraries were compared after annotation using the Blast2GO suite. A total of 417 and 78 readable EST sequence were annotated in the FSCW and glucose libraries, respectively. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on FSCW or glucose. We identified various genes of biotechnological value encoding to proteins which function such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. Fifteen genes were up-regulated and sixteen were down-regulated at least at one-time point during growth of T. harzianum in FSCW. During the confrontation assay most of the genes were up-regulated, mainly after contact, when the interaction has been established. Conclusions: This study demonstrates that T. harzianum expressed different genes when grown on FSCW compared to glucose. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against F. solani. The identification and evaluation of these genes may contribute to the development of an efficient biological control agent.

Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production

Abstract Background Despite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus Aspergillus nidulans. Results Eleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA: GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA:GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose. Conclusions Collectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion.

Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads

Abstract Background Xanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences. Results Comparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations. Conclusions This work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness.

Global transcriptional response of Caulobacter crescentus to iron availability

BACKGROUND: In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. RESULTS: In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identified 42 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB-dependent receptor gene clusters, and feoAB), riboflavin biosynthesis and genes encoding hypothetical proteins. Most of these genes are associated with predicted Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by β-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 27 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, included in this group are also TonB-dependent receptors genes and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of 113 more genes, including induction of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as repression of genes implicated in amino acid metabolism, chemotaxis and motility. CONCLUSIONS: Using a global transcriptional approach, we determined the C. crescentus iron stimulon. Many but not all of iron responsive genes were directly or indirectly controlled by Fur. The iron limitation stimulon overlaps with other regulatory systems, such as the RpoH and FixK regulons. Altogether, our results showed that adaptation of C. crescentus to iron limitation not only involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins, but also includes novel genes and regulatory mechanisms

Renal GLUT1 reduction depends on angiotensin-converting enzyme inhibition in diabetic hypertensive rats

Aims: Angiotensin-converting enzyme (ACE) inhibitors are used in diabetic kidney disease to reduce systemic/intra-glomerular pressure. The objective of this study was to investigate whether reducing blood pressure (BP) could modulate renal glucose transporter expression, and urinary markers of diabetic nephropathy in diabetic hypertensive rats treated with ramipril or amlodipine. Main methods: Diabetes was induced in spontaneously-hypertensive rats (~210 g) by streptozotocin (50 mg/kg). Thirty days later, animals received ramipril 15 μg/kg/day (R, n =10), or amlodipine 10 mg/kg/day (A, n= 8,) or water (C, n = 10) by gavage. After 30-day treatment, body weight, glycaemia, urinary albumin and TGF-β1 (enzyme-linked immunosorbent assay) and BP (tail-cuff pressure method) were evaluated. Kidneys were removed for evaluation of renal cortex glucose transporters (Western blotting) and renal tissue ACE activity (fluorometric assay). Key findings: After treatments, body weight (p = 0.77) and glycaemia (p = 0.22) were similar among the groups. Systolic BP was similarly reduced (p < 0.001) in A and R vs. C (172.4 ± 3.2; 186.7 ± 3.7 and 202.2 ± 4.3 mm Hg; respectively). ACE activity (C: 0.903 ± 0.086; A: 0.654 ± 0.025, and R: 0.389 ± 0.057 mU/mg), albuminuria (C: 264.8 ± 15.4; A: 140.8 ± 13.5 and R: 102.8 ± 6.7 mg/24 h), and renal cortex GLUT1 content (C: 46.81 ± 4.54; A: 40.30 ± 5.39 and R: 26.89 ± 0.79 AU) decreased only in R (p < 0.001, p < 0.05 and p < 0.001; respectively). Significance:We concluded that the blockade of the renin–angiotensin systemwith ramipril reduced earlymarkers of diabetic nephropathy, a phenomenon that cannot be specifically related to decreased BP levels.

Human skeletal muscle and erythrocyte proteins involved in acid-base homeostasis: adaptations to chronic hypoxia

[EN] Chronic hypoxia is accompanied by changes in blood and skeletal muscle acid-base control. We hypothesized that the underlying mechanisms include altered protein expression of transport systems and the enzymes involved in lactate, HCO3- and H+ fluxes in skeletal muscle and erythrocytes. Immunoblotting was used to quantify densities of the transport systems and enzymes. Muscle and erythrocyte samples were obtained from eight Danish lowlanders at sea level and after 2 and 8 weeks at 4100 m (Bolivia). For comparison, samples were obtained from eight Bolivian natives. In muscle membranes there were no changes in fibre-type distribution, lactate dehydrogenase isoforms, Na+,K+-pump subunits or in the lactate-H+ co-transporters MCT1 and MCT4. The Na+-H+ exchanger protein NHE1 was elevated by 39 % in natives compared to lowlanders. The Na+-HCO3- co-transporter density in muscle was elevated by 47-69 % after 2 and 8 weeks at altitude. The membrane-bound carbonic anhydrase (CA) IV in muscle increased in the lowlanders by 39 %, whereas CA XIV decreased by 23-47 %. Levels of cytosolic CA II and III in muscle and CA I and II in erythrocytes were unchanged. The erythrocyte lactate-H+ co-transporter MCT1 increased by 230-405 % in lowlanders and was 324 % higher in natives. The erythrocyte inorganic anion exchanger (Cl--HCO3- exchanger AE1) was increased by 149-228 %. In conclusion, chronic hypoxia induces dramatic changes in erythrocyte proteins, but only moderate changes in muscle proteins involved in acid-base control. Together, these changes suggest a hypoxia-induced increase in the capacity for lactate, HCO3- and H+ fluxes from muscle to blood and from blood to erythrocytes.