Natalia I. Agalakova

Molecular Mechanisms of Cytotoxicity and Apoptosis Induced by Inorganic Fluoride

Fluoride (F) is ubiquitous natural substance and widespread industrial pollutant. Although low fluoride concentrations are beneficial for normal tooth and bone development, acute or chronic exposure to high fluoride doses results in adverse health effects. The molecular mechanisms underlying fluoride toxicity are different by nature. Fluoride is able to stimulate G-proteins with subsequent activation of downstream signal transduction pathways such as PKA-, PKC-, PI3-kinase-, Ca2

Fluoride induces oxidative stress and ATP depletion in the rat erythrocytes in vitro.

The present study was designed to examine an ability of inorganic fluoride (F) to induce oxidative stress and energy depletion in the rat erythrocytes in vitro. Accumulation of ROS and alterations in glutathione (GSH) and ATP contents were estimated in the cells incubated with 0.1-10mM NaF for 1, 5 and 24h. Exposure of the rat erythrocytes to NaF was accompanied by progressive accumulation of peroxides, while superoxide (O(2)(-)) production was insignificant. Intracellular GSH content was reduced following 5-h incubation, but considerably elevated after 24h, although GSH/GSSG ratio decreased in both cases. ATP concentration in the NaF-treated cell exhibited a dose- and time-dependent decline, diminishing to extremely low levels within 24h. Thus, exposure of the rat erythrocytes to NaF leads to impairment of the cellular antioxidant system and severe energy depletion, the latter probably being the primary toxic effect.

Fluoride-induced death of rat erythrocytes in vitro

Although fluoride (F) in low concentrations is essential for teeth and bone development, its excessive consumption causes numerous deleterious abnormalities in cellular metabolism and physiology often leading to cell death. The present study was performed to establish the toxic F effects inducing the death of rat erythrocytes in vitro. The cells were cultured in the presence of 0.5-16 mM NaF for 1, 5 and 24 h. The progression of erythrocyte death was monitored by cell viability (calcein assay), membrane integrity (hemolysis assay), alterations in the cell morphology (light microscopy) and size (flow cytometry forward scatter), plasma membrane scrambling (annexin V binding). To elucidate the molecular mechanisms underlying F-induced cell death, the cytosolic Ca2+ activity (Fluo-3 fluorescence) and ceramide formation (binding of FITC-labeled antibodies) were determined. Exposure of the rat erythrocytes to NaF considerably suppressed their viability and caused partial cell hemolysis within 24 h. The cells underwent dramatic morphological alterations resulted in appearance of shrunken echinocytes after 1h and swollen spherocytes within 24 h. The development of NaF-induced erythrocyte death was accompanied by progressive PS externalization at the outer cell membrane, ∼45% of the cells were annexin V-positive in response to 16 mM NaF within 24 h with a small cell population exhibiting necrotic features. The cell death was preceded by considerable accumulation of the free cytosolic Ca2+, with statistically significant increase in the number of Fluo-3-positive erythrocytes observed as early as during 1-h incubation with 0.5 mM NaF. NaF also induced moderate ceramide formation. Overall, exposure of the rat erythrocytes to NaF triggers rapid progression of their death in a dose- and time-dependent manner, with appearance of apoptotic cells after 1 and 5 h and transition to necrosis within 24 h. An increase in intracellular [Ca2+] appears to be crucial mechanism implicated in development of NaF-induced apoptosis in rat erythrocytes.

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 NO-3medium. 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 NO-3medium. 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 NO-3. 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. These results indicate that about two-thirds of the total K+ influx in frog erythrocytes is mediated by a K−Cl cotransport which is only partially blocked by furosemide.

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+

Abstract K + and Na + influx into frog erythrocytes incubated in standard saline was studied using 86 Rb and 22 Na as tracers. 10 μM isoproterenol (1SP) 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-l-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 + 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 μ.M. The effects of ISP (10 −8 –10 −58 M) on K + transport were completely abolished with 10 μM of the βadrenergic blocker propranolol, but α-adrenergic antagonists (phentolamine, prazosin, and yohimbine) did not alter the ISP-induced increase in K + influx. The drugs tested had no effect on 22 Na 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 + ( 86 Rb) 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 + pump.

z.sbnd;-k+ pump in frog erythrocytes by catecholamines and phosphodiesterase blockers

Erythrocytes are the most numerous cells in human body and their function of oxygen transport is pivotal to human physiology. However, being enucleated, they are often referred to as a sac of molecules and their cellularity is challenged. Interestingly, their programmed death stands a testimony to their cell-hood. They are capable of self-execution after a defined life span by both cell-specific mechanism and that resembling the cytoplasmic events in apoptosis of nucleated cells. Since the execution process lacks the nuclear and mitochondrial events in apoptosis, it has been referred to as quasi-apoptosis or eryptosis. Several studies on molecular mechanisms underlying death of erythrocytes have been reported. The data has generated a non-cohesive sketch of the process. The lacunae in the present knowledge need to be filled to gain deeper insight into the mechanism of physiological ageing and death of erythrocytes, as well as the effect of age of organism on RBCs survival. This would entail how the most numerous cells in the human body die and enable a better understanding of signaling mechanisms of their senescence and premature eryptosis observed in individuals of advanced age.

Understanding quasi-apoptosis of the most numerous enucleated components of blood needs detailed molecular autopsy

The treatment of frog erythrocytes incubated in standard nitrate medium with 100 nM phorbol ester (PMA) induced a sharp increase in the 22Na uptake by the cells and intracellular Na(+) concentration. The PMA-induced enhancement in 22Na uptake was stimulated by the addition of 0.1 mM ouabain to the incubation medium and completely blocked by 1 mM amiloride. The time course of 22Na uptake by frog red cells in the presence of PMA showed a lag phase ( approximately 5 min), after which was linear within 5-15 min. The calculated Na(+) influx in erythrocytes treated with PMA was 49.4+/-3.7 mmol l(-1) cells h(-1) as compared with 1.2+/-0.25 mmol l(-1) h(-1) for control cells. 5-(N-ethyl-N-isopropyl)-amiloride, selective blocker of NHE1, caused a dose-dependent inhibition of the PMA-induced Na(+) influx with IC(50) of 0.27 microM. The PMA-induced Na(+) influx was almost completely inhibited by 0.1 microM staurosporine, protein kinase C blocker. Pretreatment of frog red blood cells for 5, 10 or 15 min with 10 mM NaF, non-selective inhibitor of protein phosphatase, led to a progressive stimulation of the PMA effect on Na(+) influx. Both amiloride and NaF did not affect the basal Na(+) influx in frog erythrocytes. The data indicate that the Na(+)-H(+) exchanger in the frog erythrocytes is quiescent under basal conditions and can be markedly stimulated by PMA.

Effect of protein kinase C activation on Na+-H+ exchange in erythrocytes of frog Rana temporaria.

Abstract 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-C1 cotransport, the frog erythrocytes were incubated in nitrate media at 20 °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.

DIFFERENTIAL EFFECTS OF GLYCOLYTIC AND OXIDATIVE METABOLISM BLOCKERS ON THE NA-K PUMP IN ERYTHROCYTES OF THE FROG, RANA TEMPORARIA

Role of PKC in fluoride-induced apoptosis of rat erythrocytes was studied in vitro and in vivo. Treatment of erythrocytes with 5 mM NaF for 1-24 h caused progressive accumulation of cytosolic Ca2+ and PS exposure at outer membrane surface. After 1 h, these processes were suppressed by PKC inhibitors staurosporine, GF 109203X and chelerythrine, but increased by PKC activator PMA. Following 24 h, NaF-induced Ca2+ uptake and PS externalization were partly prevented by PMA or staurosporine, but not by GF 109293X and chelerythrine. Application of PP inhibitor OA augmented NaF-induced cell responses within 1 h, but not after 24 h. Incubation of erythrocytes with 0.1-10 mM NaF for 1 h produced a dose-dependent PKCα translocation from cytosol to membranes with appearance of active PKM fragment. 24 h NaF exposure led to complete loss of cytosolic PKCα and proteolysis of membrane PKCα. Besides, NaF weakly stimulated membrane PKCζ, although its subcellular distribution was not altered. Thus, transient PKCα activation/translocation positively contributes to NaF-induced apoptosis in vitro. Consumption of 2-20 ppm fluoride by the rats for 12 months also induced dose-dependent PKCα translocation to membranes and activation of membrane PKCζ, what indicates that PKC stimulation is an important physiological mechanism of fluoride toxicity.