Inorganic fluoride and functions of brain.

Although actively disputed and questioned, it has been proposed that chronic exposure to inorganic fluoride (F-) is toxic for brain. The major question for this review was whether an excessive F- intake is causally related to adverse neurological and cognitive health conditions in human beings and animals. The paper systematically and critically summarizes the findings of the studies showing positive associations between F- intoxication and various intellectual defects, as well as of those which attempted to clarify the nature of F- neurotoxicity. Many works provide support for a link between pre- and postnatal F- exposure and structural and functional changes in the central nervous system responsible for neurological and cognitive disorders. The mechanisms suggested to underlie F- neurotoxicity include the disturbances in synaptic transmission and synaptic plasticity, premature death of neurons, altered activities of components of intracellular signaling cascades, impaired protein synthesis, deficit of neurotrophic and transcriptional factors, oxidative stress, metabolic changes, inflammatory processes. However, the majority of works have been performed on laboratory rodents using such F- doses which are never exist in the nature even in the regions of endemic fluorosis. Thus, this kind of treatment is hardly comparable with human exposure even taking into account the higher rate of F- clearance in animals. Of special importance are the data collected on humans chronically consuming excessive F- doses in the regions of endemic fluorosis or contacting with toxic F- compounds at industrial sites, but those works are scarce and often criticized due to low quality. New, expertly performed studies with repeated exposure assessment in independent populations are needed to prove an ability of F- to impair neurological and intellectual development of human beings and to understand the molecular mechanisms implicated in F--induced neurotoxicity.

[Effect of inorganic fluorine on living organisms of different phylogenetic level].

The presented review summarizes literature data on pathways of the inorganic fluoride intake into the plant, animal, and human organisms, on its metabolism, distribution, and accumulation in the organism, forms of fluoride in biological tissues, toxic effects of fluoride on physiological and reproductive functions of living organisms of various phylogenetic groups, as well as clinical symptoms of deficient and excessive fluoride intake into the human organism.

Marinobufagenin, an endogenous alpha-1 sodium pump ligand, in hypertensive Dahl salt-sensitive rats.

Dahl salt-sensitive rats (DS), which have a mutation in the alpha-1 subunit of Na(+)/K(+)-ATPase, exhibit impaired pressure natriuresis and on a high-salt diet, retain Na(+) and exhibit increased blood pressure. Recently, we have shown that mammalian tissues contain a bufadienolide Na(+)/K(+)-ATPase inhibitory factor, marinobufagenin (MBG), that exhibits greater affinity for the alpha-1 than alpha-3 sodium pump isoform. The present study investigated the possible role of MBG in hypertension in DS on a high NaCl intake. Eight DS and 8 Dahl salt-resistant rats (DR) were placed on an 8% NaCl diet. Within 2 weeks, systolic blood pressure increased in DS (162+/-9 mm Hg at week 2 versus 110+/-2 mm Hg in baseline, P<0.01), and increased less in DR (124+/-3 mm Hg at week 2 versus 112+/-2 mm Hg in baseline). Renal excretion of MBG increased 4-fold (38.9+/-7.6 pmol versus 9.1+/-1.3 pmol in baseline, P<0.01) in DS, but by only 25% in DR (13.2+/-0.9 pmol versus 10.3+/-0.7 pmol in baseline). Excretion of endogenous ouabain did not change in either strain. MBG-immunoreactive material was purified from the urine of hypertensive DS by means of 2 steps of reverse-phase high performance liquid chromatography (HPLC) and compared with plant ouabain and amphibian MBG for its ability to inhibit the Na(+)/K(+)-ATPase from rat kidney (which expresses only alpha-1 Na(+)/K(+)-ATPase isoform). Unlike ouabain (IC(50)=248 micromol/L), serially diluted, HPLC-purified MBG immunoreactivity from DS and authentic MBG potently inhibited rat kidney Na(+)/K(+)-ATPase (IC(50)=70 and 78 nmol/L, respectively). Our results suggest that an alpha-1 Na(+)/K(+)-ATPase ligand, MBG, is elaborated to promote natriuresis in hypertensive DS. MBG acts as a selective inhibitor of the ouabain-resistant alpha-1 Na(+)/K(+)-ATPase subunit, ie, the major sodium pump isoform of the kidneys, as would be expected of a putative natriuretic hormone.

Effects of fluoride and vanadate on K+ transport across the erythrocyte membrane of Rana temporaria.

K-Cl cotransport activity in frog erythrocytes was estimated as a Cl- -dependent component of K+ efflux from cells incubated in Cl- - or NO3- -containing medium at 20 degrees C. Decreasing the osmolality of the medium resulted in an increase in K+ efflux from the cells in a Cl- medium but not in an NO3- medium. Treatment of red cells with 5 mM NaF caused a significant decrease (approximately 50%) in K+ loss from the cells in iso- and hypotonic Cl- media but only a small decrease in K+ loss in isotonic NO3- medium. Addition of 1 mM vanadate to an isotonic Cl- medium also led to a significant reduction in K+ efflux. Similar inhibitory effects of NaF and vanadate on K+ efflux in a Cl- medium, but not in an NO3- medium were observed when the incubation temperature was decreased from 20 to 5 degrees C. Thus, under various experimental conditions, NaF and vanadate inhibited about 50% of Cl- -dependent K+ efflux from frog red cells probably due to inhibition of protein phosphatases. Cl- -dependent K+ (86Rb) influx into frog erythrocytes was nearly completely blocked (approximately 94%) by 5 mM NaF. In a NO3- medium, K+ influx was mainly mediated by the Na+,K+ pump and was unchanged in the presence of 5 mM NaF, 0.03 mM Al3+ or their combination. These data indicate that G proteins or cAMP are not involved in the regulation of Na+,K+ pump activity which is activated by catecholamines and phosphodiesterase blockers in these cells.

Kinetics of K-Cl cotransport in frog erythrocyte membrane: effect of external sodium.

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO(3)) has been shown to mediate a large fraction of the total K(+) transport. In the present study, Cl(-)-dependent and Cl(-)-independent K(+) fluxes via frog erythrocyte membranes were investigated as a function of external and internal K(+) ([K(+)](e) and [K(+)](i)) concentration. The dependence of ouabain-resistant Cl(-)-dependent K(+) ((86)Rb) influx on [K(+)](e) over the range 0-20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K(m)) of 8.2 +/- 1.3 mm and maximal velocity (V(max)) of 10.4 +/- 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl(-)-dependent K(+) influx increased both K(m) (12.8 +/- 1.7 mm, P < 0.05) and V(max) (20.2 +/- 2.9 mmol/l/hr, P < 0.001). Raising [K(+)](e) above 20 mm in isotonic media significantly reduced the Cl(-)-dependent K(+) influx due to a reciprocal decrease of the external Na(+) ([Na(+)](e)) concentration below 50 mm. Replacing [Na(+)](e) by NMDG(+) markedly decreased V(max) (3.2 +/- 0.7 mmol/l/hr, P < 0.001) and increased K(m) (15.7 +/- 2.1 mm, P < 0.03) of Cl(-)-dependent K(+) influx. Moreover, NMDG(+) Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb(+) uptake and K(+) loss from red cells. Cell swelling did not affect the Na(+)-dependent changes in Rb(+) uptake and K(+) loss. In a nominally K(+)(Rb(+))-free medium, net K(+) loss was reduced after lowering [Na(+)](e) below 50 mm. These results indicate that over 50 mm [Na(+)](e) is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K(+), Cl(-)-dependent K(+) loss in K(+)-free media was a linear function of [K(+)](i), with a rate constant of 0.11 +/- 0.01 and 0.18 +/- 0.008 hr(-1) (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K(+) ions. The residual, ouabain-resistant K(+) fluxes in NO(3) were only 5-10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K(+) effluxes in isotonic ( approximately 0. 014 hr(-1)) and hypotonic ( approximately 0.022 hr(-1)) media, but cell swelling resulted in a significant increase in the rate constants.

Inhibition and stimulation of K+ transport across the frog erythrocyte membrane by furosemide, DIOA, DIDS and quinine.

Frog erythrocytes were incubated in iso- or hypotonic media containing 10 mmol/l Rb+ and 0.1 mmol/l ouabain and both Rb+ uptake and K+ loss were measured simultaneously. Rb+ uptake by frog red cells in iso- and hypotonic media was reduced by 30-60% in the presence of 0.01-0.1 mmol/l [(dihydroindenyl)oxy] alkanoic acid (DIOA) or 0.5-1.0 mmol/l furosemide. Furosemide inhibited K+ loss from frog erythrocytes incubated in hypotonic media but did not affect it in isotonic media. DIOA at a concentration of 0.05 mmol/l inhibited of K+ loss from frog erythrocytes in both iso- and hypotonic media. At the concentrations of 0.01 and 0.02 mmol/l DIOA significantly suppressed K+ loss in a K+-free chloride medium but not in a K+-free nitrate medium. The Cl(-)-dependent K+ loss was completely blocked at a concentration of 0.1 mmol/l DIOA and the concentration required for 50% inhibition of K-Cl cotransport was approximately 0.015 mmol/l. However, the inhibitory effect of DIOA on K-Cl cotransport was masked by an opposite stimulatory effect on K+ transport which was also observed in nitrate medium. Quinine in a concentration of 0.2-1.0 mmol/l was able to inhibit Rb+ uptake and K+ loss only in hypotonic media. In isotonic media, quinine produced a stimulation of Rb+ uptake and K+ loss. A three to five-fold activation of Rb+ uptake and K+ loss was consistently observed in frog erythrocytes treated with 0.05-0.2 mmol/l 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). In contrast, another stilbene derivative 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) had no effect on K+ transport in the cells. Thus, of these drugs tested in the present study only DIOA at low concentrations may be considered as a selective blocker of the K-Cl cotransporter in the frog red blood cells.

Differential effects of glycolytic and oxidative metabolism blockers on the Na-K pump in erythrocytes of the frog, Rana temporaria.

This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content of frog erythrocytes. To eliminate K-Cl cotransport, the frog erythrocytes were incubated in nitrate media at 20 degrees C. Incubation of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K+ influx measured as 86Rb uptake for 60 min. The Na(+)-K(+)-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose or adenosine. Unexpectedly, the treatment of red cells with 1-2 mM glycolytic blocker iodoacetate produced a 2-fold increase in the ouabain-sensitive K+ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K+ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K+ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast, 0.01-0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation were without effect on K+ influx into frog erythrocytes. These results indicate that about one-half of the Na(+)-K(+)-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate "membrane-associated" ATP pool.

Temperature effects on ion transport across the erythrocyte membrane of the frog Rana temporaria.

Unidirectional K+ and Na+ influxes in the frog erythrocytes incubated in Cl- or NO(3)- media with 2.7 mM K+ were measured using 86Rb and 22Na as tracers. K+ influx was inhibited by 35-55% in the presence of 0.2-1.0 mM furosemide but it was unaffected by 0.1-0.2 mM bumetanide. Furosemide at a concentration of 0.5 mM had no effect on K+ loss from the frog red cells incubated in a nominally K(+)-free medium. Together with our previous studies the data support the existence of K-Cl cotransport and the absence of Na-K-2Cl cotransport in the frog erythrocyte membrane. Cell cooling from 20 to 5 degrees C caused a decrease in K+ influx and K+ efflux via the K-Cl cotransporter (3.2- and 3.7-fold, respectively) giving an apparent energy of activation (EA) of about 60 kJ/mol and Q10 value of 2.5. Only small decline (approximately 30%) in the ouabain-sensitive K+ influx was found as temperature was changed from 20 to 5-10 degrees C. Low values of Q10 (approximately 1.5) and EA (27.3 kJ/mol) were obtained for passive K+ influx in the frog erythrocytes (ouabain-insensitive in NO(3)- medium) at temperature within 5-20 degrees C. However, the temperature coefficients were greater for passive Na+ influx and passive K+ efflux (Q10 approximately 2.4-2.5 and EA approximately 56-58 kJ/mol). The temperature dependence of all ion transport components displayed discontinuities showing no changes at temperature between 5 and 10 degrees C. Thus, cooling of the frog red cells is associated with a greater decrease of Na+ influx and K+ efflux than passive and active K+ influx. These data indicate that the preservation of a relative high activity of the Na,K-pump during cell cooling and also the temperature-induced changes in the K-Cl cotransport activity and ion passive diffusion contribute to maintenance of ion concentration gradients in the frog erythrocytes at decreased temperature.

Activation of the Na(+)-K+ pump in frog erythrocytes by catecholamines and phosphodiesterase blockers.

K+ and Na+ influx into frog erythrocytes incubated in standard saline was studied using 86Rb and 22Na as tracers. 10 microM isoproterenol (ISP) produced a significant increase in K+ influx for the first 15 min, which was sustained during the entire 60 min of cell incubation. Treatment of red cells with the phosphodiesterase (PDE) blockers theophylline (THEO, 1 and 5 mM) or 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM) was also accompanied by an enhancement in K+ influx. A distinct additive effect on K+ influx into red cells was found when ISP and THEO or IBMX were added together. The increase in K+ transport induced by ISP plus IBMX was totally abolished by pretreatment of red cells with 0.1 mM ouabain. The ouabain-sensitive K+ influx in frog erythrocytes was elevated in the presence of ISP plus IBMX to 2.05 +/- 0.45, as compared with the control level of 0.39 +/- 0.11 mmol/L cells/hr (P < 0.001). Similar effects of ISP and IBMX on K+ influx were observed after chloride was replaced by nitrate. A dose-related increase in K+ influx into frog erythrocytes was observed at ISP concentrations of 10(-8)-10(-6) M, with a half-maximal stimulatory concentration of approximately 0.02 microM. The effects of ISP (10(-8)-10(-5) M) on K+ transport were completely abolished with 10 microM of the beta-adrenergic blocker propranolol, but alpha-adrenergic antagonists (phentolamine, prazosin, and yohimbine) did not alter the ISP-induced increase in K+ influx. The drugs tested had no effect on 22Na influx in frog red cells, but ISP produced a small decline (13%) in intracellular Na+ concentration. Thus, our study indicates that catecholamines and PDE blockers enhance K+ (86Rb) transport in frog erythrocytes mediated by Na(+)-K+ pump activity. The frog erythrocyte membrane may serve as a convenient model to investigate the hormonal modulation of the Na(+)-K+.

[Potassium ion transport in the erythrocytes of the frog Rana ridibunda]. / Transport ionov kaliia v éritrotsitakh liagushki Rana ridibunda.

The K+ transport in isolated erythrocytes from the frog Rana ridibunda has been studied using 86Rb as a tracer at the temperature of 18-20 degrees C. At physiological K+ concentration (3 mM) in C1- medium ouabain and furosemide inhibited K+ influx by approximately 42 and 47%, respectively. Furosemide had no effect on the Na+ (22Na) transport under the same conditions. The replacement of C1- by NO-3 in medium resulted in significant decrease of total K+ influx, which was not inhibited by furosemide. The ouabain- and furosemide-sensitive components of K+ influx hyperbolically increased as a function of external K+e concentration (1-90 mM) in C1- medium and calculated values of Vmax were 2.2 +/- 0.14 and 5.8 +/- 1.2 mmol/1 cells/h, respectively. K+ influx into frog erythrocytes in the presence of both ouabain and furosemide in NO-3 medium was significantly lower compared with C1- medium. C1--dependent, furosemide-insensitive component of K+ influx increased in a saturable fashion in the range of K+e concentration from 1 to 90 mM. Residual component of K+ transport in NO3 medium in the presence of the blockers was a linear function of K+e concentration and is characterized by the constant of K+ influx rate equal to 0.028 +/- 0.002 h-1. The data obtained indicate that in parallel with the Na, K-pump specific K, C1-transporter participates in K+-transport in the frog erythrocyte membrane. The latter mechanism was only partially blocked by 1 mM furosemide.

Potassium transport in red blood cells of frog Rana temporaria: demonstration of a K-Cl cotransport.

Pathways of K+ movement across the erythrocyte membrane of frog Rana temporaria were studied using 86Rb as a tracer. The K+ influx was significantly blocked by 0.1 mmol.l-1 ouabain (by 30%) and 1 mmol.l-1 furosemide (by 56%) in the red cells incubated in saline at physiological K+ concentration (2.7 mmol.l-1). Ouabain and furosemide had an additive effect on K+ transport in frog red cells. The ouabain-sensitive and furosemide-sensitive components of K+ influx saturated as f(K+)e with apparent Km values for external Ke+ concentration of 0.96 +/- 0.11 and 4.6 +/- 0.5 mmol.l-1 and Vmax of 0.89 +/- 0.04 and 2.8 +/- 0.4 mmol.l cells-1.h-1, respectively. The residual ouabain-furosemide-resistant component was also a saturable function of Ke+ medium concentration. Total K+ influx was significantly reduced when frog erythrocytes were incubated in NO3- medium. Furosemide did not affect K+ transport in frog red cells in NO3- media. At the same Ke+ concentration the ouabain-furosemide-insensitive K+ influx in Cl- medium was significantly greater than that in NO3- medium. We found no inhibitory effect of 1 mmol.l-1 furosemide on Na+ influx in frog red cells in Cl- medium. K+ loss from the frog erythrocytes in a K(+)-free medium was significantly reduced (mean 58%) after replacement of Cl- with NO3-. Furosemide (0.5 mmol.l-1) did not produce any significant reduction in the K+ loss in both media. The Cl(-)-dependent component of K+ loss from frog red cells was 5.7 +/- 1.2 mmol.l-1.h-1.(ABSTRACT TRUNCATED AT 250 WORDS)