Biomedical subjects

M Nikinmaa

Publications and source records attributed to M Nikinmaa.

At least 19 recordsLinked to original sources

PubMed2005

Hypoxic responses of Na+/K+ ATPase in trout hepatocytes.

Reduction in oxygenation induces inhibition of Na+/K+ ATPase in a number of cells and tissues, including hepatocytes. When not reversed, decrease in Na+/K+ pump activity leads to a gradual Na+ accumulation, cell swelling and death. However, when accompanied by suppression of dissipative cation pathways, it has also been shown to be a beneficial adaptive strategy used by some hypoxia-tolerant species to reduce ATP consumption during prolonged periods of anoxia. This study aims to investigate acute hypoxic responses of the Na+/K+ ATPase in primary cultures of trout hepatocytes. Gradual decrease in oxygenation was followed by an instantaneous transient dose-dependent downregulation of the Na+/K+ ATPase transport activity, but was without an effect on hydrolytic function of the enzyme. Hypoxia-induced inhibition of active K+ influx was reversed spontaneously when hypoxic incubation time exceeded 20 min. The stimulating effect of prolonged hypoxic exposure on the Na+/K+ pump is most probably secondary to hypoxia-induced activation of the Na+/H+ exchanger with the following Na+ accumulation leading to Na+/K+ ATPase activation. Hypoxia-induced inhibition of the Na+/K+ pump was not caused by ATP depletion or global oxidative stress. However, local controlled production of reactive oxygen species seems to play an important role in hypoxia-induced regulation of the Na+/K+ ATPase. Treatment of cells with mercaptopropionyl glycine (MPG), a scavenger of OH*-, abolished hypoxia-induced inhibition of the Na+/K+ ATPase. Earlier on we have shown that activation of Na+/H+ exchanger under hypoxic conditions can be opposed by MPG treatment as well. Taken together our results suggest that regulation of both oxygen-sensitive transporters may be accomplished by local changes in free radical production.

Adenosine Triphosphate↗

PubMed2003

Intracellular pH regulation in rainbow trout (Oncorhynchus mykiss) hepatocytes: the activity of sodium/proton exchange is oxygen-dependent.

We studied pH regulation in freshly isolated rainbow trout hepatocytes using microspectrofluorometry with the fluorescent dye BCECF. In accordance with earlier data on rainbow trout hepatocytes, ion substitution (N-methyl D-glucamine for sodium and gluconate for chloride) and transport inhibitor [10 microM M methyl isobutyl amiloride (MIA) to inhibit sodium/proton exchange and 100 microM DIDS to inhibit bicarbonate transport] studies in either Hepes-buffered or bicarbonate/carbon dioxide-buffered media (extracellular pH 7.6) indicated a role for sodium/proton exchange, sodium-dependent bicarbonate transport, and sodium-independent anion exchange in the regulation of hepatocyte pH. In Hepes-buffered medium, the activity of the sodium/proton exchanger (i.e. proton extrusion inhibited by MIA) was greater at 1% than at 21% oxygen. The oxygen dependency of the sodium/proton exchange is not caused by hydroxyl radicals, which appear to mediate the oxygen sensitivity of potassium-chloride cotransport in erythrocytes.

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

PubMed2003

Oxygen dependency of the adrenergic Na/H exchange in rainbow trout erythrocytes is diminished by a hydroxyl radical scavenger.

AIM: Potassium transport via the potassium chloride cotransporter in rainbow trout erythrocytes is increased by high oxygen tension. It appears that the effect of oxygen is mediated by reactive oxygen species, especially hydroxyl radicals. In contrast, the activity of adrenergically stimulated sodium proton exchange decreases with increasing oxygen tension. As available data suggest that the two transporters are regulated reciprocally, the present study was undertaken to evaluate, if hydroxyl radicals may inhibit sodium transport via the adrenergically stimulated sodium proton exchanger. METHODS: The effects of the hydroxyl radical scavenger, 2 mm mercaptopropionyl glycine (MPG), on the activity of the adrenergically activated sodium proton exchange in rainbow trout erythrocytes were examined by measuring unidirectional sodium flux, using radioactive isotope, and cellular water content. RESULTS: The activity of the sodium proton exchange increased with decreasing oxygen tension after adrenergic stimulation. When MPG was present during incubation, there was no statistically significant effect of oxygen tension on the adrenergically stimulated sodium proton exchange, whereby the activity of the transporter at atmospheric oxygen tension was markedly higher in the presence than in the absence of MPG. In the absence of adrenergic stimulation, MPG did not influence the transporter activity significantly at any oxygen tension. CONCLUSION: The data suggest that hydroxyl radicals are involved in the inhibition of the adrenergically stimulated sodium proton exchange at elevated oxygen tensions.

Animals↗

PubMed2001

Haemoglobin function in vertebrates: evolutionary changes in cellular regulation in hypoxia.

The evolution of erythrocytic hypoxia responses is reviewed by comparing the cellular control of haemoglobin-oxygen affinity in agnathans, teleost fish and terrestrial vertebrates. The most ancient response to hypoxic conditions appears to be an increase in cell volume, which increases the haemoglobin-oxygen affinity in lampreys. In teleost fish, an increase of cell volume in hypoxic conditions is also evident. The volume increase is coupled to an increase in erythrocyte pH. These changes are caused by an adrenergic activation of sodium/proton exchange across the erythrocyte membrane. The mechanism is important in acute hypoxia and is followed by a decrease in cellular adenosine triphosphate (ATP) and guanosine triphosphate (GTP) concentrations in continued hypoxia. In hypoxic bird embryos, the ATP levels are also reduced. The mechanisms by which hypoxia decreases cellular ATP and GTP concentrations remains unknown, although at least in bird embryos cAMP-dependent mechanisms have been implicated. In mammals, hypoxia responses appear to occur mainly via modulation of cellular organic phosphate concentrations. In moderate hypoxia, 2,3-diphosphoglycerate levels are increased as a result of alkalosis caused by increased ventilation.

Animals↗

PubMed2001

Characterization of a hypoxia-inducible factor (HIF-1alpha ) from rainbow trout. Accumulation of protein occurs at normal venous oxygen tension.

The mammalian hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that controls the induction of several genes involved in glycolysis, erythropoiesis, and angiogenesis when cells are exposed to hypoxic conditions. Until now, the expression and function of HIF-1alpha have not been studied in fish, which experience wide fluctuations of oxygen tensions in their natural environment. Using electrophoretic mobility shift assay, we have ascertained that a hypoxia-inducible factor is present in rainbow trout cells. We have also cloned the full-length cDNA (3605 base pairs) of the HIF-1alpha from rainbow trout with a predicted protein sequence of 766 amino acids that showed a 61% similarity to human and mouse HIF-1alpha. Polyclonal antibodies against the N-terminal part (amino acids 12-363) and the C-terminal part (amino acids 330-730) of rainbow trout HIF-1alpha protein recognized rainbow trout and chinook salmon HIF-1alpha protein in Western blot analysis. Also, the human and mouse HIF-1alpha proteins were recognized by the N-terminal rainbow trout anti-HIF-1alpha antibody but not by the C-terminal HIF-1alpha antibody. The accumulation of HIF-1alpha was studied by incubating rainbow trout and chinook salmon cells at different oxygen concentrations from 20 to 0.2% O(2) for 1 h. The greatest accumulation of HIF-1alpha protein occurred at 5% O(2) (38 torr), a typical oxygen tension of venous blood in normoxic animals. The protein stability experiments in the absence or presence of a proteasome inhibitor, MG-132, demonstrated that the inhibitor is able to stabilize the protein, which normally is degraded via the proteasome pathway both in normoxia and hypoxia. Notably, the hypoxia response element of oxygen-dependent degradation domain is identical in mammalian, Xenopus, and rainbow trout HIF-1alpha proteins, suggesting a high degree of evolutionary conservation in degradation of HIF-1alpha protein.

Amino Acid Sequence↗

PubMed2001

Reactive oxygen species regulate oxygen-sensitive potassium flux in rainbow trout erythrocytes.

In the present study, we have investigated if reactive oxygen species are involved in the oxygen-dependent regulation of potassium-chloride cotransport activity in trout erythrocyte membrane. An increase in the oxygen level caused an increase in chloride-sensitive potassium transport (K(+)-Cl(-) cotransport). 5 mM hydrogen peroxide caused an increase in K(+)-Cl(-) cotransport at 5% oxygen. The increase in flux could be inhibited by adding extracellular catalase in the incubation. Pretreatment of the cells with mercaptopropionyl glycine (MPG), a scavenger of reactive oxygen species showing preference for hydroxyl radicals, abolished the activation of the K(+)-Cl(-) cotransporter by increased oxygen levels. The inhibition by MPG was reversible, and MPG could not inhibit the activation of transporter by the sulfhydryl reagent, N-ethylmaleimide, indicating that the effect of MPG was due to the scavenging of reactive oxygen species and not to the reaction of MPG with the cotransporter. Copper ions, which catalyze the production of hydroxyl radicals in the Fenton reaction, activated K(+)-Cl(-) cotransport significantly at hypoxic conditions (1% O(2)). These data suggest that hydroxyl radicals, formed from O(2) in close vicinity to the cell membrane, play an important role in the oxygen-dependent activation of the K(+)-Cl(-) cotransporter.

Animals↗

PubMed2000

O(2)-dependent K(+) fluxes in trout red blood cells: the nature of O(2) sensing revealed by the O(2) affinity, cooperativity and pH dependence of transport.

The effects of pH and O(2) tension on the isotonic ouabain-resistant K(+) (Rb+) flux pathway and on haemoglobin O2 binding were studied in trout red blood cells (RBCs) in order to test for a direct effect of haemoglobin O(2) saturation on K(+) transport across the RBC membrane. At pH values corresponding to in vivo control arterial plasma pH and higher, elevation of the O(2) partial pressure (PO(2)) from 7.8 to 157 mmHg increased unidirectional K(+) influx across the RBC membrane several-fold. At lower extracellular pH values, stimulation of K(+) influx by O(2) was depressed, exhibiting an apparent pK(a) (pK'(a)) for the process of 8.0. Under similar conditions the pK'(a) for acid-induced deoxygenation of haemoglobin (Hb) was 7.3. When trout RBCs were exposed to PO(2) values between 0 and 747 mmHg, O(2) equilibrium curves typical of Hb O(2) saturation were also obtained for K(+) influx and efflux. However, at pH 7.9, the PO(2) for half-maximal K(+) efflux and K(+) influx (P50) was about 8- to 12-fold higher than the P(50) for Hb-O(2) binding. While K(+) influx and efflux stimulation by O(2) was essentially non-cooperative, Hb-O(2) equilibrium curves were distinctly sigmoidal (Hill parameters close to 1 and 3, respectively). O(2)-stimulated K(+) influx and efflux were strongly pH dependent. When the definition of the Bohr factor for respiratory pigments (Phi = delta logP50 x delta pH(-1)) was extended to the effect of pH on O(2)-dependent K(+) influx and efflux, extracellular Bohr factors (Phi(o) of -2.00 and -2.06 were obtained, values much higher than that for Hb (Phi(o) = -0.49). The results of this study are consistent with an O(2) sensing mechanism differing markedly in affinity and cooperativity of O(2) binding, as well as in pH sensitivity, from bulk Hb.

Acetates↗

PubMed2000

Influence of band 3 protein absence and skeletal structures on amphiphile- and Ca(2+)-induced shape alterations in erythrocytes: a study with lamprey (Lampetra fluviatilis), trout (Onchorhynchus mykiss) and human erythrocytes.

Amphiphiles which induce either spiculated (echinocytic) or invaginated (stomatocytic) shapes in human erythrocytes, and ionophore A23187 plus Ca(2+), were studied for their capacity to induce shape alterations, vesiculation and hemolysis in the morphologically and structurally different lamprey and trout erythrocytes. Both qualitative and quantitative differences were found. Amphiphiles induced no gross morphological changes in the non-axisymmetric stomatocyte-like lamprey erythrocyte or in the flat ellipsoidal trout erythrocyte, besides a rounding up at higher amphiphile concentrations. No shapes with large broad spicula were seen. Nevertheless, some of the 'echinocytogenic' amphiphiles induced plasma membrane protrusions in lamprey and trout erythrocytes, from where exovesicles were shed. In trout erythrocytes, occurrence of corrugations at the cell rim preceded protrusion formation. Other 'echinocytogenic' amphiphiles induced invaginations in lamprey erythrocytes. The 'stomatocytogenic' amphiphiles induced invaginations in both lamprey and trout erythrocytes. Surprisingly, in trout erythrocytes, some protrusions also occurred. Some of the amphiphiles hemolyzed lamprey, trout and human erythrocytes at a significantly different concentration/membrane area. Ionophore A23187 plus Ca(2+) induced membrane protrusions and sphering in human and trout erythrocytes; however, the lamprey erythrocyte remained unperturbed. The shape alterations in lamprey erythrocytes, we suggest, are characterized by weak membrane skeleton-lipid bilayer interactions, due to band 3 protein and ankyrin deficiency. In trout erythrocyte, the marginal band of microtubules appears to strongly influence cell shape. Furthermore, the presence of intermediate filaments and nuclei, additionally affecting the cell membrane shear elasticity, apparently influences cell shape changes in lamprey and trout erythrocytes. The different types of shape alterations induced by certain amphiphiles in the cell types indicates that their plasma membrane phospholipid composition differs.

Abietanes↗

PubMed2000

Tissue-specific expression of zebrafish (Danio rerio) heat shock factor 1 mRNAs in response to heat stress.

All organisms respond to environmental, chemical and physiological stresses by enhanced synthesis of an evolutionarily conserved family of proteins known as heat shock proteins (HSPs) or stress proteins. Certain HSPs are also expressed constitutively during cell growth and development, and they function as molecular chaperones. The transcriptional regulation of hsp genes is mediated by the heat shock transcription factor (HSF). The stress response has been studied mostly in mammalian cell lines or organisms normally maintained under constant laboratory conditions. There is much less information on the regulation of the stress response of animals, such as fish, that have to tolerate large fluctuations in environmental and internal conditions. To characterize the regulation of the heat shock response in fish, we have cloned the first heat shock transcription factor from fish, zebrafish Danio rerio. Phylogenetic analysis confirms that the isolated zebrafish HSF belongs to the HSF1 family and is therefore designated zHSF1. Analysis by reverse transcriptase polymerase chain reaction (RT-PCR) shows the presence of two zHSF1 mRNA forms that are expressed in a tissue-specific fashion upon exposure to heat stress. Both forms are expressed in gonads under all conditions; in liver and to a lesser extent in the gills, the longer splice form of zHSF1 disappears upon heat shock. We present evidence for a unique tissue-specific regulation of HSF1 upon exposure to elevated temperature.

Amino Acid Sequence↗

PubMed2000

The adrenergic volume changes of immature and mature rainbow trout (Oncorhynchus mykiss) erythrocytes.

In this study, we examined whether the adrenergic volume response of teleost erythrocytes is related to cell maturity. Rainbow trout (Oncorhynchus mykiss) were made anaemic by reducing their haematocrit to approximately 50 % of the original value. After 3-4 weeks, small, young erythrocytes were seen in the circulation. By measuring the volume distribution of blood samples from anaemic fish before and after noradrenaline stimulation (10 min, 10(-5)mol l(-1) final concentration), we were able to show that the volume response of young, immature erythrocytes to catecholamine stimulation was greater than that of mature erythrocytes. In addition, the membrane fluidity, measured using the steady-state fluorescence polarisation method, was greater in anaemic fish after 24 days of recovery from bleeding than in control fish. Since blood from anaemic fish contained a large fraction of immature erythrocytes, this result indicates that the fluidity of the membrane of immature erythrocytes is greater than that of mature erythrocytes.

Anemia↗

PubMed1999

Copper effects on ion transport across lamprey erythrocyte membrane: Cl(-)/OH(-) exchange induced by cuprous ions.

We studied the effects of prelytic copper concentrations on cell volume, intracellular pH, and ion transport in lamprey erythrocytes. Ion fluxes and pH were measured by radioactive tracer technique, patch clamp, and flame photometry. Prelytic CuSO(4) concentration of 100 microM caused anion-dependent intracellular acidification and increase in Cl(-) influx after 2 min lag-phase. In the presence of ascorbate copper effect was amplified and lag-phase was skipped. Pretreatment of the cells with N-phenyl maleimide abolished copper-induced changes completely. Copper treatment caused an increase in Na(+) fluxes in both directions and a net Na(+) uptake. Copper-induced Na(+) transport was partially amiloride(MIA)-sensitive representing Na(+)/H(+) exchange. The nature of the amiloride-insensitive fraction of copper-activated Na(+) influx remains unknown. Cell swelling after 15 min of copper exposure induced regulatory volume decrease response involving KCl extrusion via K(+) and Cl(-) volume-sensitive channels. We suggest that the effects of copper on ion transport fit the following sequence of events: (i) cupric ions are reduced to cuprous state on the membrane surface, (ii) electroneutral pairs CuCl and CuOH mediate chloride/hydroxyl exchange, as shown before for trialkyltin, dissipating transmembrane pH gradient, and (iii) changes in intracellular pH result in the activation of the Na(+)/H(+) exchange and consecutive volume changes cause the RVD response.

Animals↗

PubMed1999

Developmental changes in 7-ethoxyresorufin-O-deethylase (EROD) and delta-aminolevulinic acid dehydratase (ALA-D) activities in three passerines.

In this study, we report changes in 7-ethoxyresorufin-O-deethylase (EROD) and delta-aminolevulinic acid dehydratase (ALA-D) enzyme activities during development of three wild passerine bird species: pied flycather (Ficedula hypoleuca), great tit (Parus major) and blue tit (P. caeruleus). Activities were determined from late embryos, newly hatched, 1-week-old and 15-day-old nestlings and adult birds. EROD activity from hepatic microsomes and ALA-D activity from liver tissue and erythrocytes were measured. In all species investigated EROD and ALA-D activities were increased during the first posthatching week and decreased thereafter. Also, differences between activities in late-term nestlings and adults were seen. Based on these results, we suggest that the developmental status of an animal must always be taken into account when evaluating the results obtained in the biomarker studies.

Animals↗

PubMed1999

Seasonal and temperature effects on the adrenergic responses of Arctic charr (Salvelinus alpinus) erythrocytes.

In the present study, we have examined the adrenergic responses of Arctic charr (Salvelinus alpinus) erythrocytes acclimated to different temperatures (2, 8 and 14 degrees C) during different seasons. We measured the changes in cellular water and ion contents after noradrenaline stimulation using different noradrenaline concentrations and external pH values. Furthermore, the effects of acute temperature changes on the magnitude of the adrenergic response were studied. The adrenergic response of Arctic charr erythrocytes showed pronounced seasonal variation. The [Na(+)]/[Cl(-)] accumulation ratio after adrenergic stimulation was greatest in May, indicating an enhanced activity of the Na(+)/H(+) exchanger. The noradrenaline-induced change in [Na(+)](i) was greatest in spring. In addition to a seasonal effect, the exchanger seemed to be most active in erythrocytes from charr acclimated to low temperature (2 degrees C) early in May: the EC(50) value was lower and the calculated maximal increase in [Na(+)](i) was greater in the 2 degrees C-acclimated group than in the other acclimation groups. In contrast, acclimation to different temperatures did not affect these responses (measured at a constant temperature) in February. An acute temperature change has a smaller effect on the adrenergic response of Arctic charr erythrocytes than on rainbow trout (Oncorhynchus mykiss) erythrocytes.

Journal Article↗

PubMed1998

Regulation of ion transport across lamprey (Lampetra fluviatilis) erythrocyte membrane by oxygen tension

We have measured the effects of oxygen tension on the transport of Na+, K+ and Cl- across the erythrocyte membrane of the lamprey Lampetra fluviatilis. The transport of each ion was affected by the oxygen tension of the medium. Hypoxic conditions (PO2 2 kPa) caused an increase in the acidification-induced influx of Na+ via Na+/H+ exchange. The influx of K+ was only slightly affected by the oxygenation of the medium. In contrast, the basal K+ efflux, measured using the radioactive isotope 43K, was markedly reduced by decreasing the oxygen tension of the medium, whereas the K+ flux in hypotonic medium was not affected. Only minor effects of hypoxic conditions on the influx of Cl- were observed in either isotonic or hypotonic conditions (there was a tendency for the isotonic influx to increase) or on the efflux in isotonic conditions. However, deoxygenation caused a marked reduction in the Cl- efflux in hypotonic conditions. The results show that oxygen tension has a marked effect on the pH and volume regulatory transport pathways of lamprey erythrocytes. For K+ and Cl-, the regulation appears to be asymmetric, i.e. influx and efflux are affected differently.

Journal Article↗

PubMed1998

Two distinct K+ channels in lamprey (Lampetra fluviatilis) erythrocyte membrane characterized by single channel patch clamp.

Two channels, distinguished by using single-channel patch-clamp, carry out potassium transport across the red cell membrane of lamprey erythrocytes. A small-conductance, inwardly rectifying K(+)-selective channel was observed in both isotonic and hypotonic solutions (osmolarity decreased by 50%). The single-channel conductance was 26 +/- 3 pS in isotonic (132 mM K+) solutions and 24 +/- 2 pS in hypotonic (63 mM K+) solutions. No outward conductance was found for this channel, and the channel activity was completely inhibited by barium. Cell swelling activated another inwardly rectifying K+ channel with a larger inward conductance of 65 pS and outward conductance of 15 pS in the on-cell configuration. In this channel, rectification was due to the block of outward currents by Mg2+ and Ca2+ ions, since when both ions were removed from the cytosolic side in inside-out patches the conductance of the channel was nearly ohmic. In contrast to the small-conductance channel, the swelling-activated channel was observed also in the presence of barium in the pipette. Neither type of channel was dependent on the presence of Ca2+ ions on the cytosolic side for activity.

Animals↗

PubMed1998

Effects of heat shock and hypoxia on protein synthesis in rainbow trout (Oncorhynchus mykiss) cells.

We examined the effects of heat stress (from 18 degreesC to 26 degreesC) and low oxygen tension (1% O2=1 kPa) on protein synthesis in primary cultures of hepatocytes, gill epithelial cells and fibroblast-like RTG-2 cells of rainbow trout Oncorhynchus mykiss. All these cell types displayed elevated levels of 67, 69 and 92 kDa proteins, whereas a 104 kDa protein was induced only in RTG-2 cells. Hypoxia induced a cell-type-specific response, increasing the synthesis of 36, 39 and 51 kDa proteins in the gill epithelial cells. The regulation of the heat-shock response in fish hepatocytes showed that an HSF1-like factor is involved in the transcriptional induction of the hsp70 gene. Consequently, there was a pronounced accumulation of hsp70 mRNA. Furthermore, the kinetics of activation of DNA binding and the increase in hsp70 gene expression showed a remarkable correlation, indicating that hsp70 expression is regulated at the transcriptional level in these trout cells.

Animals↗

PubMed1998

Regulation of ion transport across lamprey (Lampetra fluviatilis) erythrocyte membrane by oxygen tension.

Animals↗

PubMed1997

River water with high iron concentration and low pH causes mortality of lamprey roe and newly hatched larvae.

The effects of prevailing water quality of Perhonjoki River in Western Finland on the hatchability of lamprey roe and survival of newly hatched larvae were investigated. The river flows through sulfide-rich soils, and is characterized by high average total iron (1.5-3 mg liter-1) and aluminum (0.45-0.6 mg liter-1) concentrations and relatively low pH commonly 5.5 to 6.0, with acidification peaks below pH 5. The hatchability of lamprey roe was markedly reduced under these conditions as compared with ground water with low iron (0.002 mg liter-1) and aluminum (0.11 mg liter-1) concentrations but at the same pH. Furthermore, the hatchability of roe and survival of newly hatched larvae was especially affected by an increase in total iron concentration to values that may be observed after heavy rains and during the snow-melt period (4-6 mg liter-1). The results suggest that increases in total iron concentration may play a role in the reproductive failures of lampreys observed in Finnish rivers.

Aluminum↗