Muscle Sodium Research Articles

Sodium, hypertension, and an explanation of the “lag phenomenon” in hemodialysis patients

Sodium balance is precisely regulated by intake and output. The kidneys are responsible for adjusting sodium excretion to maintain balance at varying intakes. Our distant ancestors were herbivores. Their diet contained little sodium, so they developed powerful mechanisms for conserving sodium and achieving low urinary excretion. About 10,000 years ago, early humans became villagers and discovered that food could be preserved in brine. This led to increased consumption of salt. High salt intake increases extracellular volume (ECV), blood volume, and cardiac output resulting in elevation of blood pressure. High ECV induces release of a digitalis-like immunoreactive substance and other inhibitors of Na(+)-K(+)-ATPase. As a consequence, intracellular sodium and calcium concentrations increase in vascular smooth muscles predisposing them to contraction. Moreover, high ECV increases synthesis and decreases clearance of asymmetrical dimethyl-l-arginine leading to inhibition of nitric oxide (NO) synthase. High concentration of sodium and calcium in vascular smooth muscles, and decreased synthesis of NO lead to an increase in total peripheral resistance. Restoration of normal ECV and blood pressure are attained by increased glomerular filtration and decreased sodium reabsorption. In some individuals, the kidneys have difficulty in excreting sodium, so the equilibrium is achieved at the expense of elevated blood pressure. There is some lag time between reduction of ECV and normalization of blood pressure because the normal levels of Na(+)-K(+)-ATPase inhibitors and asymmetrical dimethyl-l-arginine are restored slowly. In dialysis patients, all mechanisms intended to increase renal sodium removal are futile but they still operate and elevate blood pressure. The sodium balance must be achieved via dialysis and ultrafiltration. Blood pressure is normalized a few weeks after ECV is returned to normal, i.e., when the patient reaches dry body weight. This is called the "lag phenomenon."

The Dominant Cold-SensitiveOut-ColdMutants ofDrosophila melanogasterHave Novel Missense Mutations in the Voltage-Gated Sodium Channel Geneparalytic

Here we report the molecular characterization of Out-cold (Ocd) mutants of Drosophila melanogaster, which produce a dominant, X-linked, cold-sensitive paralytic phenotype. From its initial 1.5-Mb cytological location within 13F1-16A2, P-element and SNP mapping reduced the Ocd critical region to <100 kb and to six candidate genes: hangover, CG9947, CG4420, eIF2a, Rbp2, and paralytic (para). Complementation testing with para null mutations strongly suggests Ocd and para are allelic, as does gene rescue of Ocd semilethality with a wild-type para transgene. Pesticide resistance and electrophysiological phenotypes of Ocd mutants support this conclusion. The para gene encodes a voltage-gated sodium channel. Sequencing the Ocd lines revealed mutations within highly conserved regions of the para coding sequence, in the transmembrane segment S6 of domain III (I1545M and T1551I), and in the linker between domains III and IV (G1571R), the location of the channel inactivation gate. The G1571R mutation is of particular interest as mutations of the orthologous residue (G1306) in the human skeletal muscle sodium channel gene SCN4A are associated with cases of periodic paralysis and myotonia, including the human cold-sensitive disorder paramyotonia congenita. The mechanisms by which sodium channel mutations cause cold sensitivity are not well understood. Therefore, in the absence of suitable vertebrate models, Ocd provides a system in which genetic, molecular, physiological, and behavioral tools can be exploited to determine mechanisms underlying sodium channel periodic paralyses.

High‐affinity blockade of voltage‐operated skeletal muscle and neuronal sodium channels by halogenated propofol analogues

Voltage-operated sodium channels constitute major target sites for local anaesthetic-like action. The clinical use of local anaesthetics is still limited by severe side effects, in particular, arrhythmias and convulsions. These side effects render the search for new local anaesthetics a matter of high interest. We have investigated the effects of three halogenated structural analogues of propofol on voltage-operated human skeletal muscle sodium channels (Na(V)1.4) and the effect of one compound (4-chloropropofol) on neuronal sodium channels (Na(V)1.2) heterologously expressed in human embryonic kidney cell line 293. 4-Iodo-, 4-bromo- and 4-chloropropofol reversibly suppressed depolarization-induced whole-cell sodium inward currents with high potency. The IC(50) for block of resting channels at -150 mV was 2.3, 3.9 and 11.3 microM in Na(V)1.4, respectively, and 29.2 microM for 4-chloropropofol in Na(V)1.2. Membrane depolarization inducing inactivation strongly increased the blocking potency of all compounds. Estimated affinities for the fast-inactivated channel state were 81 nM, 312 nM and 227 nM for 4-iodopropofol, 4-bromopropofol and 4-chloropropofol in Na(V)1.4, and 450 nM for 4-chloropropofol in Na(V)1.2. Recovery from fast inactivation was prolonged in the presence of drug leading to an accumulation of block during repetitive stimulation at high frequencies (100 Hz). Halogenated propofol analogues constitute a novel class of sodium channel-blocking drugs possessing almost 100-fold higher potency compared with the local anaesthetic and anti-arrhythmic drug lidocaine. Preferential drug binding to inactivated channel states suggests that halogenated propofol analogues might be especially effective in suppressing ectopic discharges in a variety of pathological conditions.

Regulatory role of translocation of Na+-K+ pumps in skeletal muscle: hypothesis or reality?

to the editor: The Perspectives article by Benziane and Chibalin ([1][1]) in this issue comments on part of the literature regarding the possible role of translocation in the regulation of Na+-K+ pumps in skeletal muscle. I have been invited to submit a letter to the editor about this review article

α-Scorpion Toxin Impairs a Conformational Change that Leads to Fast Inactivation of Muscle Sodium Channels

α-Scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an α-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence–voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

Poster Abstract #13: Interactive Voice Response Diary and Objective Myotonia Measurement as Endpoints for Clinical Trials in Nondystrophic Interactive Voice Response Diary and Objective Myotonia Measurement as Endpoints for Clinical Trials in Nondystrophic Myotonia

Background Nondystrophic myotonia (NDM) is a heterogeneous group of neuromuscular disorders caused by mutations in skeletal muscle sodium and chloride channels. There are no established treatments for myotonia, despite the availability of agents that deserve careful study. An outcome measure of myotonia is important for clinical trials. Methods 45 subjects were enrolled from six academic centers across the United States, England, and Canada. Patients were categorized as myotonia congenita (MC), paramyotonia congenita (PMC), and other myotonic disorders (OMD) Three possible myotonia (symptoms/signs) measures were assessed: relaxation time after maximum voluntary isometric contraction of the finger flexors (QMA); myotonic discharges on needle EMG; an Interactive Voice Response Diary (IVR), for which participants called in once a week for up to 8 weeks to rank symptom severity on a scale of 1 to 9 (stiffness, pain, weakness, fatigue) and frequency (1–7 days). Results Initial clinical diagnosis was 20 MC; 16 PMC; 9 OMD. Myotonic discharge potentials were seen in all subjects with no difference in their degree and location among the subtypes. Of 33 participants for whom QMA test results were available, 15 showed a delay in relaxation; however, for subjects with e 2's hand grip opening on clinical exam, 14/16 showed a delay in relaxation on QMA. From IVR data, frequency of symptoms for the total population was stiffness 91%, pain 60%, weakness 56%, and fatigue 65%. Average severity for each symptom was stiffness, 4.1; pain, 4.0; weakness, 4.2; and fatigue, 4.2. Standard deviation of reported symptom severity within subject was stiffness, 1.2; pain, 1.1; weakness, 1.3; and tiredness, 0.9. For participants reporting symptoms for a given week, the average number of days they experienced symptoms was stiffness, 5.2; pain, 5.2; weakness, 4.4; and fatigue, 4.5. Conclusions The IVR data showed consistency in patient self-rated clinical symptoms related to myotonia, which makes this a potentially useful endpoint for future clinical trials. Quantitive myotonia measure is a less sensitive outcome measure, because only minority patients showed abnormal results. This project is supported by NIH grant 5U54RRO19482.

Frontiers: Skeletal muscle sodium pump regulation: a translocation paradigm

The skeletal muscle sodium pump plays a major role in the removal of K(+) ions from the circulation postprandial, or after a physical activity bout, thereby preventing the development of hyperkalemia and fatigue. Insulin and muscle contractions stimulate Na(+)-K(+)-ATPase activity in skeletal muscle, at least partially via translocation of sodium pump units to the plasma membrane from intracellular stores. The molecular mechanism of this phenomenon is poorly understood. Due to the contradictory reports in the literature, the very existence of the translocation of Na(+)-K(+)-ATPase to the skeletal muscle cell surface is questionable. This review summarizes more than 30 years work on the skeletal muscle sodium pump translocation paradigm. Furthermore, the methodological caveats of major approaches to study the sodium pump translocation in skeletal muscle are discussed. An understanding of the molecular regulation of Na(+)-K(+)-ATPase in skeletal muscle will have important clinical implications for the understanding of the development of complications associated with the metabolic syndrome, such as cardiovascular diseases or increased muscle fatigue in diabetic patients.

Endotoxin reduces availability of voltage-gated human skeletal muscle sodium channels at depolarized membrane potentials*

Critical illness myopathy is a common cause for difficulties in weaning from the respirator and prolonged rehabilitation of patients recovering from sepsis. Several studies have shown that the primary cause of acute generalized muscle weakness is loss of muscle membrane excitability. This study was designed to investigate a potential direct interaction of lipopolysaccharides from Escherichia coli with voltage-gated human skeletal muscle sodium channels (NaV1.4) in vitro. In vitro laboratory investigation. University laboratory. NaV1.4 sodium channel alpha-subunits stably expressed in human embryonic kidney (HEK293) cells. We investigated the effect of lipopolysaccharide on voltage-dependent sodium channel gating by using two distinct modes of application: 1) acute perfusion (pharmacologic lipopolysaccharide concentrations between 5 ng/mL and 50 microg/mL) in order to establish a concentration-effect relationship; and 2) incubation with a clinically relevant concentration of lipopolysaccharide (300 pg/mL). Lipopolysaccharide did not alter the kinetics of sodium current activation or inactivation when depolarizations were started from hyperpolarized holding potentials. However, when either fast or slow inactivation was induced by membrane depolarization before the test pulse, lipopolysaccharide reversibly reduced channel availability during the test pulse at concentrations of > or = 50 ng/mL revealed by a maximum hyperpolarizing shift of -25 mV in the voltage dependence of fast and slow inactivation, respectively. Incubation with a lipopolysaccharide concentration of 300 pg/mL for 1 hr reproduced the effects on slow but not on fast inactivation. After 20 hrs of low-dose lipopolysaccharide, the peak sodium current was significantly reduced. Our results show that lipopolysaccharide interacts with voltage-gated sodium channels, reducing channel availability at depolarized membrane potentials during acute application, independent of the membrane potential after chronic exposure. These effects may contribute to reduced muscle membrane excitability in sepsis.

Oxidation of multiple methionine residues impairs rapid sodium channel inactivation

Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel Na(V)1.4 expressed in mammalian cells was studied by applying the Met-preferring oxidant chloramine-T or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of Met1470 in the putative receptor of the inactivation lid also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues in intracellular linkers connecting the membrane-spanning segments (442, 1139, 1154, 1316, 1469) were of minor importance. The results of mutagenesis, assays of other Na(V) channel isoforms (Na(V)1.2, Na(V)1.5, Na(V)1.7), and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of Na(V) channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion.

Slow inactivation

Skeletal muscle membrane excitability resides on a razor’s edge. Increasing excitability leads to myotonia. Reducing excitability can lead to paralysis due to membrane inexcitability. By complexly altering membrane excitability, channelopathies can produce phenotypes that are characterized by both hyperexcitability and paralysis. Detailed physiologic studies can sometimes explain the complex phenotypes produced by specific channelopathies. In this issue of Neurology ®, Webb and Cannon1 provide a cogent explanation for the genesis of a phenotype of myotonia at warm temperature and cold-induced paralysis that is associated with the P1158S mutation of the skeletal muscle sodium channel, NaV1.4. Skeletal muscle channelopathies are fascinating because they illuminate the connection between basic physiology and clinical neurology. The channelopathies are natural experiments that enable us to explore how altering the function of a channel changes membrane excitability. Nobel laureates Hodgkin and Huxley2 opened the modern age of membrane physiology more than a half-century ago through a series of elegant experiments that demonstrated that the action potential in the giant axon of a squid was produced by an interplay between voltage-gated …

Targeted mutation of mouse skeletal muscle sodium channel produces myotonia and potassium-sensitive weakness

Hyperkalemic periodic paralysis (HyperKPP) produces myotonia and attacks of muscle weakness triggered by rest after exercise or by K+ ingestion. We introduced a missense substitution corresponding to a human familial HyperKPP mutation (Met1592Val) into the mouse gene encoding the skeletal muscle voltage-gated Na+ channel NaV1.4. Mice heterozygous for this mutation exhibited prominent myotonia at rest and muscle fiber-type switching to a more oxidative phenotype compared with controls. Isolated mutant extensor digitorum longus muscles were abnormally sensitive to the Na+/K+ pump inhibitor ouabain and exhibited age-dependent changes, including delayed relaxation and altered generation of tetanic force. Moreover, rapid and sustained weakness of isolated mutant muscles was induced when the extracellular K+ concentration was increased from 4 mM to 10 mM, a level observed in the muscle interstitium of humans during exercise. Mutant muscle recovered from stimulation-induced fatigue more slowly than did control muscle, and the extent of recovery was decreased in the presence of high extracellular K+ levels. These findings demonstrate that expression of the Met1592ValNa+ channel in mouse muscle is sufficient to produce important features of HyperKPP, including myotonia, K+-sensitive paralysis, and susceptibility to delayed weakness during recovery from fatigue.

Evaluation of extra‐ and intracellular apparent diffusion coefficient of sodium in rat skeletal muscle: Effects of prolonged ischemia

The mechanism of water and sodium apparent diffusion coefficient (ADC) changes in rat skeletal muscle during global ischemia was examined by in vivo 1H and 23Na magnetic resonance spectroscopy (MRS). The ADCs of Na+ and water are expected to have similar characteristics because sodium is present as an aqua-cation in tissue. The shift reagent, TmDOTP5(-), was used to separate intra- and extracellular sodium (Na+i and Na+e, respectively) signals. Water, total tissue sodium (Na+t), Na+i, and Na+e ADCs were measured before and 1, 2, 3, and 4 hr after ischemia. Contrary to the general perception, Na+i and Na+e ADCs were identical before ischemia. Thus, ischemia-induced changes in Na+e ADC cannot be explained by a simple change in the size of relative intracellular or extracellular space. Na+t and Na+e ADCs decreased after 2-4 hr of ischemia, while water and Na+i ADC remained unchanged. The correlation between Na+t and Na+e ADCs was observed because of high Na+e concentration. Similarly, the correlation between water and Na+i ADCs was observed because cells occupy 80% of the tissue space in the skeletal muscle. Ischemia also caused an increase in the Na+i and an equal decrease in Na+e signal intensity due to cessation of Na+/K+-ATPase function.

Constrained analogues of tocainide as potent skeletal muscle sodium channel blockers towards the development of antimyotonic agents

1-Benzyl- N-(2,6-dimethylphenyl)piperidine-3-carboxamide and 4-benzyl- N-(2,6-dimethylphenyl)piperazine-2-carboxamide, two conformationally restricted analogues of tocainide, were designed and synthesized as voltage-gated skeletal muscle sodium channel blockers. They showed, with respect to tocainide, a marked increase in both potency and use-dependent block.

Mechanism of sodium loss with muscle sodium deficiency in sodium supplemented and unsupplemented subjects during hypokinesia

This study aims at showing the effect of hypokinesia (HK) on sodium (Na+) loss with different muscle Na+ deficiency and different Na+ intake. Muscle Na+ content, plasma Na+ level and Na+ loss with and without Na+ supplementation were measured. This study was conducted on 40 healthy male volunteers during a pre-experimental and an experimental period. Subjects were equally divided into four groups: unsupplemented active control subjects (UACS), unsupplemented hypokinetic subjects (UHKS), supplemented active control subjects (SACS) and supplemented hypokinetic subjects (SHKS). A daily supplementation of 3.21 mmol of sodium chloride (NaCl) per kg body weight was given to subjects in the SACS and SHKS groups. Muscle Na+ content levels decreased and plasma Na+ levels, and levels of Na+ loss in urine and feces increased (p<0.05) in the SHKS and UHKS groups compared to their pre-experimental values and the values in the respective active control groups (SACS and UACS). However, muscle Na+ content levels decreased more (p<0.05), and plasma Na+ levels and levels of Na+ loss in urine and feces increased more (p<0.05) in the SHKS group than in the UHKS group. The greater muscle Na+ deficiency with higher than lower Na+ consumption shows that the risk of greater muscle Na+ deficiency is directly related to Na+ consumption. The higher Na+ loss with higher than lower muscle Na+ deficiency shows that the risk of greater muscle Na+ loss is directly related to muscle Na+ deficiency. It is concluded that muscle Na+ deficiency is more evident when Na+ consumption is higher and that muscle Na+ loss was more exacerbated with higher than lower muscle Na+ deficiency indicating that during prolonged HK the muscle Na+ deficiency is due to the inability of the body to use Na+, but not to Na+ shortage in diet.

A Case of Hypothyroidism with Brugada Electrocardiographic Waveforms

A 77-year-old man was diagnosed as having hypothyroidism. An electrocardiogram obtained at the time of the first examination revealed Brugada electrocardiographic waveforms in leads V1 to V3. When pilsicainide hydrochloride loading changed the waveforms of the electrocardiographic ST segment, this result suggested an abnormality of the cardiac muscle sodium channels. The Brugada electrocardiographic waveforms disappeared with the normalization of thyroid function. This case is the first report ever of hypothyroidism that presented Brugada electrocardiographic waveforms. The results obtained in this case suggested that thyroid functions changed the waveforms of the electrocardiogram because of its effect on myocardial ion channels.

ChemInform Abstract: Pilsicainide and Its Oxymethylene Analogue: Facile Alternative Syntheses and in vitro Testing on Human Skeletal Muscle Sodium Channels.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Design of a Specific Activator for Skeletal Muscle Sodium Channels Uncovers Channel Architecture

Gating modifiers of voltage-gated sodium channels (Na(v)s) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion beta-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNa(v)1.2a) and skeletal muscle (rNa(v)1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4(F14A/E15A/E28R), of rNa(v)1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4(F14A/E15A/E28R) was tested using an rNa(v)1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4(F14A/E15A/E28R) and residues whose equivalents in the rat brain channel rNa(v)1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1-2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Na(v)s, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNa(v)1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Na(v)s resembles that of voltage-gated potassium channels.

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

Skeletal-muscle channelopathies: periodic paralysis and nondystrophic myotonias

To provide a current review of clinical phenotypes, genetics, molecular pathophysiology, and electro-diagnostic testing strategies of periodic paralysis and nondystrophic myotonias. The number of pathogenic mutations causing periodic paralysis and nondystrophic myotonias continues to increase. Important insight into the molecular pathogenesis of muscle sodium channelopathies has been revealed by the finding of 'leaky' closed sodium channels. Previously, alterations in sodium-channel activation or inactivation have been identified as important disease mechanisms. The recent discovery that substitutions of key arginine residues in the voltage-sensing segment of the channel may lead to a 'pore leak' when the channel is closed suggests a new mechanism. Since similar mutations exist in corresponding positions of other channels, this mechanism may apply to other channel diseases. The recognition of different electrophysiological patterns that are specific to muscle ion-channel genotypes will be useful in diagnosis and in guiding genetic testing. Recent studies demonstrate that magnetic resonance imaging may be used to detect intramuscular accumulation of sodium during episodes of weakness. Recent advances have refined our ability to make a precise molecular diagnosis in muscle channelopathies. The description of a pore leak with voltage-sensor mutations may represent a new disease mechanism.

The toxicity of copper to crucian carp (Carassius carassius) in soft water

Crucian carp (Carassius carassius) were exposed to a Cu rich medium (pH 6.6, conductivity 25 microS/cm, 2.91 mg Ca(2+)/l, approximately 300 microg Cu(2+)/l). Untreated department water (pH 6.6, conductivity 25 microS/cm, 2.91 mg Ca(2+)/l) acted as control. Mortality in crucian carp was first observed after 13 days of exposure to the Cu rich medium. There were, however, significant changes in haematocrit, plasma chloride, plasma sodium and water content in muscle in fish exposed to the Cu rich medium after two days. After 14 days of exposure to copper, haematocrit increased to 52+/-2% (control: between 37 and 40%), plasma chloride decreased to 45+/-5 mmol/l (control: 99-106 mmol/l), plasma sodium decreased to 81+/-6 mmol/l (control: 116-137 mmol/l), and water content in muscle increased to 83.0+/-0.3% (control: 78.7-79.9%). No apparent changes in blood ethanol, and minor changes in plasma lactate were observed in copper exposed fish. Analyses of the gills revealed an increasing concentration of copper on the gills from fish exposed to Cu rich water. After 14 days, the concentration of copper accumulated in the gill was 12.8+/-4.1 microg Cu/g wet weight (control: 0.91-1.19 microg Cu/g wet weight). A reduction of the respiratory area in fish exposed to copper was observed, in terms of both lamellar and filamental fusion. The normoxic O(2) uptake did not change, but the critical oxygen tension was elevated to 6.12+/-1.04 mg O(2)/l after a 6 day exposure to copper (control: 1.03+/-0.05 mg O(2)/l). This study shows that crucian carp has a higher tolerance to copper compared to other freshwater fish species. Our results suggest that this tolerance is based on the ability of crucian carp to avoid becoming hypoxic as well as an extreme tolerance to severe loss of plasma ions.