Oxygen consumption rate and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in early developmental stages of the sea urchin <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> Lam.

Changes in oxygen consumption rate and Na⁺/K⁺-ATPase activity during early development were studied in the sea urchin Paracentrotus lividus Lam. The oxygen consumption rate increased from 0.12 μmol O₂ mg protein⁻¹ h⁻¹ in unfertilized eggs to 0.38 μmol O₂ mg protein⁻¹ h⁻¹ 25 min after fertilization. Specific activity of the Na⁺/K⁺-ATPase was significantly stimulated after fertilization, ranging up to 1.07 μmol P_i h⁻¹ mg protein⁻¹ in the late blastula stage and slightly lower values in the early and late pluteus stages.     

Cardiotonic steroid-resistant α1-Na+,K+-ATPase rescues renal epithelial cells from the cytotoxic action of ouabain: evidence for a Na i+ ,K i+ -independent mechanism

Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood. This study examines the role of Na⁺,K⁺-ATPase α subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing a variant of the α1 isoform, CTS-sensitive α1S, were stably transfected with a cDNA encoding CTS-resistant α1R-Na⁺,K⁺-ATPase, whose expression was confirmed by RT–PCR. In mock-transfected and α1R-cells, maximal inhibition of ⁸⁶Rb influx was observed at 10 and 1000 μM ouabain, respectively, thus confirming high abundance of α1R-Na⁺,K⁺-ATPase in these cells. Six-hour treatment of α1R-cells with 1000 μM ouabain led to the same elevation of the [Na⁺]_i/[K⁺]_i ratio that was detected in mock-transfected cells treated with 3 μM ouabain. However, in contrast to the massive death of mock-transfected cells exposed to 3 μM ouabain, α1R-cells survived after 24-h incubation with 1000 μM ouabain. Inversion of the [Na⁺]_i/[K⁺]_i ratio evoked by Na⁺,K⁺-ATPase inhibition in K⁺-free medium did not affect survival of α1R-cells but increased their sensitivity to ouabain. Our results show that the α1R subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na⁺,K⁺-ATPase-mediated Na⁺ and K⁺ fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio.     

Changes in Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity and Alpha 3 Subunit Expression in CNS After Administration of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitors

The expression of Na⁺, K⁺-ATPase α3 subunit and synaptosomal membrane Na⁺, K⁺-ATPase activity were analyzed after administration of ouabain and endobain E, respectively commercial and endogenous Na⁺, K⁺-ATPase inhibitors. Wistar rats received intracerebroventricularly ouabain or endobain E dissolved in saline solution or Tris–HCl, respectively or the vehicles (controls). Two days later, animals were decapitated, cerebral cortex and hippocampus removed and crude and synaptosomal membrane fractions were isolated. Western blot analysis showed that Na⁺, K⁺-ATPase α3 subunit expression increased roughly 40% after administration of 10 or 100 nmoles ouabain in cerebral cortex but remained unaltered in hippocampus. After administration of 10 μl endobain E (1 μl = 28 mg tissue) Na⁺, K⁺-ATPase α3 subunit enhanced 130% in cerebral cortex and 103% in hippocampus. The activity of Na⁺, K⁺-ATPase in cortical synaptosomal membranes diminished or increased after administration of ouabain or endobain E, respectively. It is concluded that Na⁺, K⁺-ATPase inhibitors modify differentially the expression of Na⁺, K⁺-ATPase α3 subunit and enzyme activity, most likely involving compensatory mechanisms.     

Regulation of K–Cl cotransport in erythrocytes of frog Rana temporaria by commonly used protein kinase and protein phosphatase inhibitors

Recently (Agalakova and Gusev in J Comp Physiol 179:443–450, 2009), we demonstrated that the activity of K–Cl cotransport (KCC) in frog red blood cells is inhibited under stimulation of protein kinase C (PKC) with phorbol ester PMA (12-myristate-13-acetate). Present work was performed to uncover possible implication of protein kinases and protein phosphatases (PPs) in the regulation of baseline and volume-dependent KCC activity in these cells. K⁺ influx was estimated as ⁸⁶Rb uptake by the cells in isotonic or hypotonic media in the presence of ouabain, K⁺ efflux was determined as the difference between K⁺ loss by the cells incubated in parallel in isotonic or hypotonic K⁺-free Cl⁻- and NO₃ ⁻-media. Swelling of the cells in hypotonic medium was accompanied by approximately 50% activation of Cl-dependent K⁺ influx and efflux. Protein tyrosine kinase (PTK) inhibitor genistein (0.1 mM) stably and considerably (up to 89%) suppressed both baseline and volume-dependent KCC activity in each direction. Other PTK blockers (tyrphostin 23 and quercetin) had no influence on KCC activity in frog erythrocytes. PKC inhibitor chelerythrine (20 μM) and both PP inhibitors, fluoride (5 mM) and okadaic acid (1 μM), reduced KCC activity by 25–70%. Neither basal nor swelling-activated KCC in frog erythrocytes was affected by PKC inhibitor staurosporine (1 μM). Based on the previous and present results, we can suggest that the main role in the maintenance of basal and volume-dependent KCC activity in frog erythrocytes belongs to PTKs and PPs, whereas PKC is a negative regulator of this ion system.     

Local Loperamide Inhibits Thermal Hyperalgesia But Not Mechanical Allodynia Induced by Intratibial Inoculation of Melanoma Cells in Mice

The stimulation of peripheral opioid receptors counteracts thermal hyperalgesia produced by the intratibial inoculation of NCTC 2472 cells in mice, through the activation of the nitric oxide/cGMP/ATP-sensitive K⁺-channels (NO/cGMP/K⁺ _ATP) cascade (Menéndez et al. 2007, Neuropharmacology 53:71–80). We aimed to elucidate whether this peripheral opioid antihyperalgesic effect is exclusive to this model or might also occur in other types of bone neoplastic processes. In C57BL/6 mice intratibially inoculated with B16-F10 melanoma cells, the progressive tumoral damage was accompanied by the establishment of thermal hyperalgesia (unilateral hot plate test) and mechanical allodynia (von Frey test). Intraplantar administration of loperamide (15 μg, 30 min before) inhibited thermal hyperalgesia, but did not modify the intense mechanical allodynia. The fact that the coadministration of naloxone-methiodide (5 μg) completely suppressed the thermal antihyperalgesic effect induced by loperamide indicates its production through the stimulation of peripheral opioid receptors. Furthermore, its prevention by the coadministration of the non-selective inhibitor of the NO synthase, N^G-monomethyl-L-arginine (L-NMMA, 10 μg), the selective inhibitor of neural NOS, N-ω-propyl-L-arginine (1–10 μg), or the K⁺ _ATP channel blocker, glibenclamide (10 μg) demonstrated the involvement of the NO/cGMP/K⁺ _ATP pathway in the antihyperalgesic effect induced by loperamide. Overall, the present results show that the intratibial inoculation of B16-F10 cells to C57BL/6 mice evokes thermal hyperalgesia and mechanical allodynia and that, as occurred in the osteosarcoma model, the stimulation of peripheral opioid receptors is not effective in modifying neoplastic allodynia but completely inhibits thermal hyperalgesia through the activation of the NO/cGMP/K⁺ _ATP cascade.     

Copper modifies the activity of sodium-transporting systems in erythrocyte membrane in patients with essential hypertension

The aim of the study was to verify the hypothesis if copper could influence the activity of sodium-transporting systems in erythrocyte membrane that could be related to essential hypertension. The examined group of patients consisted of 15 men with hypertension. The control group was 11 healthy male volunteers. The Na⁺/H⁺ exchanger (NHE) activity in erythrocytes was determined according to Orlov et al. The activity of transporting systems (ATP-Na⁺/K⁺; co-Na⁺/K⁺/Cl⁻; ex-Na⁺/Li⁺; free Na⁺ and K⁺ outflow [Na⁺, K⁺-outflow]) was determined according to Garay's method. The concentration of copper in plasma was assessed using atomic absorption spectrometry. The activity of ATP-Na⁺/K⁺ (μmol/L red blood cells [RBCs]/h) in hypertensive patients was 2231.5±657.6 vs 1750.5±291 in the control (p<0.05), the activity of co-Na⁺/K⁺/Cl⁻ (μmol/L RBCs/h) in hypertensives was 171.3±77.9 vs 150.7±53.9 in the control (NS). Na⁺-outflow (μmol/L RBCs/h) in hypertensives was 118.3±51.6 vs 113.3±24.4 in the control (NS). The K⁺-outflow (μmol/L RBCs/h) in hypertensives was 1361.7±545.4 vs 1035.6±188.3 in the control (NS). The activity of ex-Na⁺/Li⁺ (μmol/L RBCs/h) in hypertensive patients was 266.1±76.1 vs 204.1±71.6 in the control (p<0.05). NHE activity (mmol/L RBCs/h) in hypertensives was 9.7±2.96 vs 7.7±1.33 in the control (p<0.05). In hypertensive patients, negative correlation was found between the activity of Na⁺/K⁺/Cl⁻ co-transport and plasma copper concentration (R _s=−0.579, p <0.05) and between the activity of ex-Na⁺/Li⁺ and plasma copper concentration (R _s=−0.508, p<0.05). Plasma copper concentration significantly influences the activity of sodium transporting systems in erythrocyte membrane. Copper supplementation could be expected to provide therapeutic benefits for hypertensive patients.     

Fluoxetine Inhibits K+ Transport Pathways (K+ Efflux, Na+-K+-2Cl− Cotransport, and Na+ Pump) Underlying Volume Regulation in Corneal Endothelial Cells

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K⁺ efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 μm by 26%, and 50 μm totally. Fluoxetine had a triphasic effect on K⁺ efflux; from 20 to 100 μm it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 μm by 25%, and 50 μm completely. Bumetanide (1 μm) inhibited RVI by 43%. In a Cl⁻-free medium, fluoxetine (50–500 μm) progressively inhibited bumetanide-insensitive K⁺ influx. The inhibitions of RVI and K⁺ influx induced by fluoxetine 20 to 50 μm were similar to those induced by 1 μm bumetanide and by Cl⁻-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K⁺ channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K⁺ efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 μm inhibits NKCC, and at 100–1000 μm inhibits the Na⁺ pump. One possible explanation for these findings is that fluoxetine could interact with K⁺-selective sites in K⁺ channels, the NKC cotransporter and the Na⁺ pump.     

Light-Dependency of Growth and Secondary Metabolite Production in the Captive Zooxanthellate Soft Coral Sinularia flexibilis

The branching zooxanthellate soft coral Sinularia flexibillis releases antimicrobial and toxic compounds with potential pharmaceutical importance. As photosynthesis by the symbiotic algae is vital to the host, the light-dependency of the coral, including its specific growth rate (μ day⁻¹) and the physiological response to a range of light intensities (10–1,000 μmol quanta m⁻² s⁻¹) was studied for 12 weeks. Although a range of irradiances from 100 to 400 μmol quanta m⁻² s⁻¹ was favorable for S. flexibilis, based on chlorophyll content, a light intensity around 100 μmol quanta m⁻² s⁻¹ was found to be optimal. The contents of both zooxanthellae and chlorophyll a were highest at 100 μmol quanta m⁻² s⁻¹. The specific budding rate showed almost the same pattern as the specific growth rate. The concentration of the terpene flexibilide, produced by this species, increased at high light intensities (200–600 μmol quanta m⁻² s⁻¹).     

Boric acid inhibits stored Ca<Superscript>2+</Superscript> release in DU-145 prostate cancer cells

Boron (B) is a developmental and reproductive toxin. It is also essential for some organisms. Plants use uptake and efflux transport proteins to maintain homeostasis, and in humans, boron has been reported to reduce prostate cancer. Ca²⁺ signaling is one of the primary mechanisms used by cells to respond to their environment. In this paper, we report that boric acid (BA) inhibits NAD⁺ and NADP⁺ as well as mechanically induced release of stored Ca²⁺ in growing DU-145 prostate cancer cells. Cell proliferation was inhibited by 30% at 100μM, 60% at 250μM, and 97% at 1,000μM BA. NAD⁺-induced Ca²⁺ transients were partly inhibited at 250μM BA and completely at 1,000μM BA, whereas both NADP⁺ and mechanically induced transients were inhibited by 1,000μM BA. Expression of CD38 protein increased in proportion to BA exposure (0–1,000μM). In vitro mass spectrometry analysis showed that BA formed adducts with the CD38 products and Ca²⁺ channel agonists cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Vesicles positive for the Ca²⁺ fluorophore fluo-3 acetoxymethyl ester accumulated in cells exposed to 250 and 1,000μM BA. The BA analog, methylboronic acid (MBA; 250 and 1,000μM), did not inhibit cell proliferation or NAD⁺, NADP⁺, or mechanically stimulated Ca²⁺ store release. Nor did MBA increase CD38 expression or cause the formation of intracellular vesicles. Thus, mammalian cells can distinguish between BA and its synthetic analog MBA and exhibit graded concentration-dependent responses. Based on these observations, we hypothesize that toxicity of BA stems from the ability of high concentrations to impair Ca²⁺ signaling.     

Sodium-dependent and -independent Choline Uptake by Type II Epithelial Cells from Rat Lung

The uptake of ³H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium. Uptake was linear for 4 min at both 0.1 μm and 5.0 μm medium choline; at 5 μm, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1 μm), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected: (i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when Na⁺ was replaced by Li⁺, K⁺ or Mg²⁺, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na⁺ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na⁺ concentration. Kinetic properties of the uptake of 0.1 μm ³H-choline in the presence and absence of medium Na⁺ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5–100 μm) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na⁺; a Na⁺-dependent portion (a mean of 0.52 of the total) had a K _m for choline of 1.5 μm while K _m in the absence of Na⁺ (Li⁺ substituting) was 18.6 μm. (b) Inhibition by HC-3 (0.3–300 μm) gave K_i values of 1.7 μm and 5.0 μm HC-3 for the Na⁺-dependent and -independent fractions. The apparent K _m of the Na⁺-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K _m values reported for high-affinity, Na⁺-dependent choline uptake by neuronal cells. Received: 18 February 1997/Revised: 7 December 1997     

The occurrence of killer,sensitive, and neutral yeasts in Brazilian Riesling Italico grape must and the effect of neutral strains on killing behaviour

 The occurrence of killer toxins amongst yeasts in Brazilian Riesling Italico grape must was investigated by using the sensitive strain EMBRAPA-26B as a reference strain at 18°C and 28°C. From a total of 85 previously isolated yeasts, 21 strains showed ability to kill the sensitive strain on unbuffered grape must/agar (MA-MB) and 0.1 M citrate/phosphate-buffered yeast extract/peptone/dextrose/agar (YEPD-MB) media both supplemented with 30 mg/l methylene blue. The killer activity of only four yeasts depended on the incubation temperature rather than the medium used. At 28°C, the strains 11B and 53B were not able to show killer action. On the other hand, strains 49B and 84B did not kill the sensitive yeast at 18°C. The killer strain EMBRAPA-91B and a commercial wine killer yeast K-1 were employed to examine the sensitivity of the isolated yeasts on YEPD-MB and MA-MB at 18°C. The sensitivity and neutral characteristics of yeasts were shown to be dependent on the medium and the killer strain. Interactions, including K^- R^-, K^- R⁺ and K⁺ R⁺ strains, simultaneously, have revealed that some K^-R⁺ strains appear to protect the K^- R^- strain against the killer toxin. Sensitive dead cells, although to a less extent, also exhibited similar protection. Kinetic studies have shown that the maximum specific growth rates were higher for the 20B YEPD-MB-sensitive strain (μ_max=0.517 h^-1) than for both the 91B (μ_max=0.428 h^-1) and K-1 (μ_max= 0.466 h^-1) killer strains. The protective capacity of neutral or sensitive cells that contaminate a fermentation, as well as the higher maximum specific growth rate of sensitive yeasts, besides other factors, may preclude the dominance of a killer strain. This protective capacity may also reduce the risk of a sensitive inoculum being killed by wild-type killer yeasts in open non-sterile fermentation. Received: 3 November 1995/Received revision: 11 March 1996/Accepted: 15 April 1996     

Effects of phorbol 12-myristate 13-acetate on potassium transport in the red blood cells of frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Phorbol 12-myristate 13-acetate (PMA), a stimulator of PKC, was examined for its influence on K⁺ (⁸⁶Rb) influx in the frog erythrocyte. PMA, 0.1 μM, was found to accelerate ouabain-sensitive K⁺ influx, which was suppressed by 73% with 1 mM amiloride, indicating secondary activation of the Na⁺–K⁺-pump due to stimulation of Na⁺ /H⁺ exchange. PMA-induced stimulation of the sodium pump was completely inhibited with 1 μM staurosporine and by ~50% with 20 μM chelerythrine. In contrast to Na⁺–K⁺-pump, an activity of Cl⁻-dependent K⁺ transport (K–Cl cotransport, KCC), calculated as the difference between K⁺ influxes in Cl⁻ and NO₃ ⁻-media, was substantially decreased under the influence of PMA. Staurosporine fully restored the PMA-induced inhibition of KCC, whereas chelerythrine did not exert any influence. Osmotic swelling of the frog erythrocytes was accompanied by approximately twofold stimulation of KCC. Swelling-activated KCC was inhibited by ~50 and ~83% in the presence of PMA and genistein, respectively, but not chelerythrine. Exposure of the frog erythrocytes to 5 mM fluoride (F⁻) also reduced the KCC activity in isotonic and hypotonic media, with maximal suppression of K⁺ influx in both media being observed upon simultaneous addition of PMA and F⁻. Furosemide and [(dihydronindenyl)oxy] alkanoic acid inhibited the K⁺ influx in both the media by ~50–60%. The results obtained show both the direct and indirect effects of PMA on the K⁺ transport in frog erythrocytes and a complicated picture of KCC regulation in frog erythrocytes with involvement of PKC, tyrosine kinase and protein phosphatase.     

Vasotocin has the potential to inhibit basolateral Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-pump current across isolated skin of tree frog in vitro<Emphasis Type="Italic">,</Emphasis> via its V<Subscript>2</Subscript>-type receptor/cAMP pathway

Adult frog skin transports Na⁺ from the apical to the basolateral side across the skin. Antidiuretic hormone (ADH) is involved in the regulation of Na⁺ transport in both mammals and amphibians. We investigated the effect of arginine vasotocin (AVT), the ADH of amphibians, on the short-circuit current (SCC) across intact skin and on the basolateral Na⁺/K⁺-pump current across apically nystatin-permeabilized skin of the tree frog, Hyla japonica, in which the V₂-type ADH receptor is expressed in vitro. In intact skin, 1 pM AVT had no effect on the SCC, but 10 nM AVT was sufficient to stimulate the SCC since 10 nM and 1 μM of AVT increased the SCC 3.2- and 3.4-fold, respectively (P > 0.9). However, in permeabilized skin, AVT (1 μM) decreased the Na⁺/K⁺-pump current to 0.79 times vehicle control. Similarly, 500 μM of 8Br-cAMP increased the SCC 3.2-fold, yet 1 mM of 8Br-cAMP decreased the Na⁺/K⁺-pump current to 0.76 times vehicle control. Arachidonic acid (10⁻⁵ M) tended to decrease the Na⁺/K⁺-pump current. To judge from these in vitro experiments, AVT has the potential to inhibit the basolateral Na⁺/K⁺-pump current via the V₂-type receptor/cAMP pathway in the skin of the tree frog.     

Transport Pathways for Therapeutic Concentrations of Lithium in Rat Liver

Although both amiloride- and phloretin-sensitive Na⁺/Li⁺ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li⁺ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li⁺ by hepatocytes, preloaded with Na⁺ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K _m 1.2 mm Li⁺, V _max 40 μmol · (kg dry wt · min)⁻¹, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H⁺ was curvilinear suggesting a modifier role of [H⁺] _i . This system persisted in Na⁺-depleted cells, although with apparent K _m 3.8 mm. (b) A phloretin-sensitive component, with K _m 1.2 mm, V _max 21 μmol · (kg · min)⁻¹, was unaffected by pH but was inactive in Na⁺-depleted cells. Phloretin inhibited Li⁺ uptake and Na⁺ efflux in parallel. (c) A residual uptake increased linearly with the external Li⁺ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li⁺ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H⁺]. Received: 8 April 1999/Revised: 19 July 1999     

Effects of tellurite on growth of Saccharomyces cerevisiae

The effects of potassium tellurite on growth and survival of rho⁺ and rho⁰ Saccharomyces cerevisiae strains were investigated. Both rho⁺ and rho⁰ strains grew on a fermentable carbon source with up to 1.2 mM K₂TeO₃, while rho⁺ yeast cells grown on a non-fermentable carbon source were inhibited at tellurite levels as low as 50 μM suggesting that this metalloid specifically inhibited mitochondrial functions. Growth of rho⁺ yeast cells in the presence of increasing amount of tellurite resulted in dose-dependent blackening of the culture, a phenomenon not observed with rho⁰ cultures. Transmission electron microscopy of S. cerevisiae rho⁺ cells grown in the presence of tellurite showed that blackening was likely due to elemental tellurium (Te⁰) that formed large deposits along the cell wall and small precipitates in both the cytoplasm and mitochondria.     

Effect of Lead Ion on the Function of the Human Ether-À-Go-Go-Related Gene K<Superscript>+</Superscript> Channel

Lead (Pb) is a trace metal element in the human body. In order to understand the hazard mechanism of the elevated blood lead level on the human body, the effect of Pb²⁺ on the human ether-à-go-go-related gene (hERG) K⁺ channel in the HEK 293 cell was investigated for the first time using whole-cell patch clamp technique, molecular dynamics simulation, and quantum chemistry calculation methods. We found that Pb²⁺ obviously inhibits the current of the hERG K⁺ channel, and delays the “activation” and “deactivation” of the hERG K⁺ channel, indicating that Pb²⁺ evidently decreases the function of the K⁺ channel in the cell. The effect is increased with increasing the concentration of Pb²⁺. When the concentration of Pb²⁺ is 400 μg L⁻¹, the function of the K⁺ channel is entirely lost. The results from the molecular dynamics simulation and quantum chemistry calculation indicated that Pb²⁺ can coordinate with the oxygen/sulfur atoms in the K⁺ channel protein, leading to the decrease in the function of the K⁺ channel. According to the experimental results, we suggested that once the K⁺ channel in the human body was irreversibly inactivated by Pb²⁺, it would affect the treatment and prognosis of Pb²⁺ intoxication.     

Changes in K<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> balance and K<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K⁺, Na⁺, and Cl⁻ balance and K⁺ (Rb⁺) and ³⁶Cl⁻ fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K⁺ and Cl⁻ and an increase in the Na⁺ content. The rate of ³⁶Cl⁻, Rb⁺ (K⁺), and ²²Na⁺ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb⁺ (K⁺) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb⁺ (K⁺) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl⁻ level is accompanied by a 1.2–1.6-fold decreased flux.     

Death of ouabain-treated renal epithelial cells: evidence for p38 MAPK-mediated Na i + /K i + -independent signaling

Recent studies demonstrate that cytotoxic actions of ouabain and other cardiotonic steroids (CTS) on renal epithelial cells (REC) are triggered by their interaction with the Na⁺,K⁺-ATPase α-subunit but not the result of inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio. This study examined the role of mitogen-activated protein kinases (MAPK) in the death of ouabain-treated REC. Exposure of C7-MDCK cells that resembled principal cells from canine kidney to 3 μM ouabain led to phosphorylation of p38 without significant impact on phosphorylation of ERK and JNK MAPK. Maximal increment of p38 phosphorylation was observed at 4 h followed by cell death at 12 h of ouabain addition. In contrast to ouabain, neither cell death nor p38 MAPK phosphorylation were affected by elevation of the [Na⁺]_i/[K⁺]_i ratio triggered by Na⁺,K⁺-ATPase inhibition in K⁺-free medium. p38 phosphorylation was noted in all other cell types exhibiting death in the presence of ouabain, such as intercalated cells from canine kidney and human colon rectal carcinoma cells. We did not observe any action of ouabain on p38 phosphorylation in ouabain-resistant smooth muscle cells from rat aorta and endothelial cells from human umbilical vein. Both p38 phosphorylation and death of ouabain-treated C7-MDCK cells were suppressed by p38 inhibitor SB 202190 but were resistant to its inactive analogue SB 202474. Our results demonstrate that death of CTS-treated REC is triggered by Na_i⁺,K_i⁺—independent activation of p38 MAPK.     

Muscle Potassium and Potassium Losses During Hypokinesia in Healthy Subjects

Hypokinesia (HK) induces electrolyte losses in electrolyte-deficient tissue, yet the mechanisms of electrolyte losses in electrolyte-deficient tissue remain unknown. Mechanisms of electrolyte deposition could be involved. To determine the effect of prolonged HK on potassium (K⁺) deposition were measured muscle K⁺ content and K⁺ losses. Studies were conducted on 20 physically healthy male volunteers during 30 days pre-experimental period and 364 days experimental period. Subjects were equally divided into two groups: control subjects (CS) and experimental subjects (ES). The CS group was run average distances of 9.8 ± 1.7 km day⁻¹ and the ES group was walked average distances of 2.7 ± 0.6 km day⁻¹. Muscle K⁺ content decreased (p < 0.05) and plasma K⁺ concentration, and K⁺ losses in urine and feces increased (p < 0.05) in the ES group compared to their pre-experimental level and the values in their respective CS group. Muscle K⁺ content, plasma K⁺ level, and urine and fecal K⁺ losses did not show any changes in the CS group compared to their pre-experimental values. The conclusion was that K⁺ losses in K⁺-deficient muscle of healthy subjects could have been attributable to the less efficient K⁺ deposition inherently to prolonged HK.     

Carrier-mediated acetate uptake in Corynebacterium glutamicum

Acetate is effectively taken up by whole cells of Corynebacterium glutamicum via a specific carrier with a pH optimum of 8. The K _m of acetate uptake was 50 μM and the V _max 25–35 nmol/mg dw min. The activation energy was determined to be 70 kJ/mol. Acetate uptake was competitively inhibited by propionate with a K _i of about 30 μM and blocked by addition of sulfhydryl reagents. The transport activity was clearly dependent on the membrane potential, but independent of the presence of Na⁺-ions. It is concluded that uptake of acetate proceeds by a secondary, proton coupled mechanism.