Accumulation and nuclear import of HIF1 alpha during high and low oxygen concentration in skeletal muscle cells in primary culture

AbstractThe hypoxia-inducible-factor-1 (HIF1) mediates the transcriptional upregulation of several target genes during hypoxia. HIF1 itself is known to be regulated essentially by ubiquitinylation and proteolytic degradation of the subunit HIF1α of the dimeric transcription factor HIF1. In contrast to other tissues, skeletal muscle expresses high amounts of HIF1α in normoxia as well as in hypoxia. In view of this, we aimed to investigate HIF1α accumulation and subcellular localization as well as the transcriptional activity of the HIF1α-regulated gene of glyceraldehyde dehydrogenase (GAPDH) in skeletal muscle cells exposed to low oxygen concentration (3% O2), normoxia (20% O2) or high oxygen concentration (42% O2). Immunofluorescence analysis reveals that under normoxic and high oxygen conditions, significant amounts of HIF1α can be found exclusively in the cytoplasm of the myotubes. Muscle cells treated with CoCl2, a known inhibitor of HIF1α degradation, show even higher levels of HIF1α, again exclusively in the cytoplasm. Under conditions of low oxygen, HIF1α in controls as well as in CoCl2-treated cells is found in the nuclei. CdCl2 inhibits nuclear import of HIF1α at low oxygen concentration and leads to a transcriptional downregulation of the marker enzyme of anaerobic glycolysis GAPDH. Immunoprecipitation with anti-HIF1α antibody co-precipitates HSP90 in an oxygen-dependent manner, more at high pO2 than at low pO2. Cadmium-treated samples also show high amounts of co-immunoprecipitated HSP90, independent of oxygen concentration. We conclude that in skeletal muscle cells, HIF1α, in contrast to other tissues, may, in addition to its regulation by degradation, also be regulated by binding to HSP90 and subsequent inhibition of its import into the nuclei

CO2 and HCO3- Permeability of the Rat Liver Mitochondrial Membrane

Background/Aims: Across the mitochondrial membrane an exceptionally intense exchange of O2 and CO2 occurs. We have asked, 1) whether the CO2 permeability, PM,CO2, of this membrane is also exceptionally high, and 2) whether the mitochondrial membrane is sufficiently permeable to HCO3- to make passage of this ion an alternative pathway for exit of metabolically produced CO2. Methods: The two permeabilities were measured using the previously published mass spectrometric 18O exchange technique to study suspensions of mitochondria freshly isolated from rat livers. The mitochondria were functionally and morphologically in excellent condition. Results: The intramitochondrial CA activity was exclusively localized in the matrix. PM,CO2 of the inner mitochondrial membrane was 0.33 (SD ± 0.03) cm/s, which is the highest value reported for any biological membrane, even two times higher than PM,CO2 of the red cell membrane. PM,HCO3- was 2· 10-6 (SD ± 2· 10-6) cm/s and thus extremely low, almost 3 orders of magnitude lower than PM,HCO3- of the red cell membrane. Conclusion: The inner mitochondrial membrane is almost impermeable to HCO3- but extremely permeable to CO2. Since gas channels are absent, this membrane constitutes a unique example of a membrane of very high gas permeability due to its extremely low content of cholesterol

The Most Recently Discovered Carbonic Anhydrase, CA XV, Is Expressed in the Thick Ascending Limb of Henle and in the Collecting Ducts of Mouse Kidney

BACKGROUND: Carbonic anhydrases (CAs) are key enzymes for physiological pH regulation, including the process of urine acidification. Previous studies have identified seven cytosolic or membrane-bound CA isozymes in the kidney. Recently, we showed by in situ hybridization that the mRNA for the most novel CA isozyme, CA XV, is present in the renal cortex. CA XV is a unique isozyme among mammalian CAs, because it has become a pseudogene in primates even though expressed in several other species. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we raised a polyclonal antibody against recombinant mouse CA XV that was produced in a baculovirus/insect cell expression system, and the antibody was used for immunohistochemical analysis in different mouse tissues. Positive immunoreactions were found only in the kidney, where the enzyme showed a very limited distribution pattern. Parallel immunostaining experiments with several other anti-CA sera indicated that CA XV is mainly expressed in the thick ascending limb of Henle and collecting ducts, and the reactions were most prominent in the cortex and outer medulla. CONCLUSION/SIGNIFICANCE: Although other studies have proposed a role for CA XV in cell proliferation, its tightly limited distribution may point to a specialized function in the regulation of acid-base homeostasis

T Tubules and Surface Membranes Provide Equally Effective Pathways of Carbonic Anhydrase-Facilitated Lactic Acid Transport in Skeletal Muscle

We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV–CAIX–CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.Public Library of Scienc

Concentration Dependence of the Self-Diffusion of Human and Lumbricus terrestris Hemoglobin

The self-diffusion coefficient of the extracellular hemoglobin of Lumbricus terrestris (mol wt 3.7 × 10(6) daltons) has been measured at protein concentrations ranging from 2 to 25 g/100 ml. The self-diffusion coefficient of human hemoglobin has been measured at concentrations between 10 and 43 g/100 ml. For these measurements, (14)C-labeled hemoglobin was made to diffuse from one Millipore filter into three consecutively arranged Millipore filters containing unlabeled hemoglobin. After a suitable time the filters were separated, and the protein diffusion coefficient was determined from the distribution of radioactivity in the four filters with a table given by Kawalki (1894, Ann. Phys. Chem. 52:166-190.). The following results were obtained. The diffusion coefficient of Lumbricus hemoglobin is 1.2 × 10(-7) cm(2)s(-1) at a protein concentration of 2.1 g/100 ml, and is reduced to about 1/10 of this value when the concentration is 25 g/100 ml (T = 21°C). Between 0 and 16 g/100 ml the logarithm of the diffusion coefficient of Lumbricus hemoglobin falls linearly with concentration. Above 16 g/100 ml a marked increase in the concentration dependence of the diffusion coefficient is observed. Extrapolation of the data to zero hemoglobin concentration yields a limiting value of the diffusion coefficient of Lumbricus hemoglobin of 1.3 × 10(-7) cm(2)s(-1). The diffusion coefficient of human hemoglobin is 4.5 × 10(-7) cm(2)s(-1) at a hemoglobin concentration of 9.7 g/100 ml, and falls to 0.14 × 10(-7) cm(2)s(-1) at a hemoglobin concentration of 43.0 g/100 ml. In addition to diffusivities, the viscosities of human and Lumbricus hemoglobin solutions were measured in a wide range of protein concentrations. The concentration dependence of the diffusivity of Lumbricus hemoglobin is compared to that of myoglobin, ovalbumin, and tetrameric hemoglobin. Proportionality between the diffusion coefficient and the reciprocal of the viscosity of the protein solution is found for all these proteins. It is also shown that an equation proposed by Anderson (1973) gives an excellent description of the diffusivity of the various proteins up to moderate protein concentrations. Above concentrations of 16 g/100 ml for Lumbricus hemoglobin, and 30 g/100 ml for tetrameric hemoglobin, however, protein diffusivity falls much more rapidly with increasing concentration than is predicted by this equation

How does carbon dioxide permeate cell membranes? A discussion of concepts, results and methods

We review briefly how the thinking about the permeation of gases, especially CO2, across cell and artificial lipid membranes has evolved during the last hundred years. We then describe how the recent finding of a drastic effect of cholesterol on CO2 permeability of both biological and artificial membranes fundamentally alters the long-standing idea that CO2 – as well as other gases – permeates all membranes with great ease. This requires revision of the widely accepted paradigm that membranes never offer a serious diffusion resistance to CO2 or other gases. Earlier observations of CO2-impermeable membranes can now be explained by the high cholesterol content of some membranes. Thus, cholesterol is a membrane component that nature can use to adapt membrane CO2 permeability to the functional needs of the cell. Since cholesterol serves many other cellular functions, it cannot be reduced indefinitely. We show, however, that cells that possess a high metabolic rate and/or a high rate of O2 and CO2 exchange, do require very high CO2 permeabilities that may not be achievable merely by reduction of membrane cholesterol. The article then discusses the alternative possibility of raising the CO2 permeability of a membrane by incorporating protein CO2 channels. The highly controversial issue of gas and CO2 channels is systematically and critically reviewed. It is concluded that a majority of the results considered to be reliable, is in favour of the concept of existence and functional relevance of protein gas channels. The effect of intracellular carbonic anhydrase, which has recently been proposed as an alternative mechanism to a membrane CO2 channel, is analysed quantitatively and the idea considered untenable. After a brief review of the knowledge on permeation of O2 and NO through membranes, we present a summary of the 18O method used to measure the CO2 permeability of membranes and discuss quantitatively critical questions that may be addressed to this method