Chloroquine and Chemotherapeutic Compounds in Experimental Cancer Treatment.

Chloroquine (CQ) and its derivate hydroxychloroquine (HCQ), the compounds with recognized ability to suppress autophagy, have been tested in experimental works and in clinical trials as adjuvant therapy for the treatment of tumors of different origin to increase the efficacy of cytotoxic agents. Such a strategy can be effective in overcoming the resistance of cancer cells to standard chemotherapy or anti-angiogenic therapy. This review presents the results of the combined application of CQ/HCQ with conventional chemotherapy drugs (doxorubicin, paclitaxel, platinum-based compounds, gemcitabine, tyrosine kinases and PI3K/Akt/mTOR inhibitors, and other agents) for the treatment of different malignancies obtained in experiments on cultured cancer cells, animal xenografts models, and in a few clinical trials. The effects of such an approach on the viability of cancer cells or tumor growth, as well as autophagy-dependent and -independent molecular mechanisms underlying cellular responses of cancer cells to CQ/HCQ, are summarized. Although the majority of experimental in vitro and in vivo studies have shown that CQ/HCQ can effectively sensitize cancer cells to cytotoxic agents and increase the potential of chemotherapy, the results of clinical trials are often inconsistent. Nevertheless, the pharmacological suppression of autophagy remains a promising tool for increasing the efficacy of standard chemotherapy, and the development of more specific inhibitors is required.

Optimal Reference Genes for RT-qPCR Experiments in Hippocampus and Cortex of Rats Chronically Exposed to Excessive Fluoride.

Normalization of the quantitative real-time PCR (RT-qPCR) data to the stably expressed reference genes is critically important for obtaining reliable results. However, all previous studies focused on F- toxicity for brain tissues used a single, non-validated reference gene, what might be a cause of contradictory or false results. The present study was designed to analyze the expression of a series of reference genes to select optimal ones for RT-qPCR analysis in cortex and hippocampus of rats chronically exposed to excessive fluoride (F-) amounts. Six-week-old male Wistar rats randomly assigned to four groups consumed regular tap water with 0.4 (control), 5, 20, and 50 ppm F- (NaF) for 12 months. The expression of six genes (Gapdh, Pgk1, Eef1a1, Ppia, Tbp, Helz) was compared by RT-qPCR in brain tissues from control and F--exposed animals. The stability of candidate reference genes was evaluated by coefficient of variation (CV) analysis and RefFinder online program summarizing the results of four well-acknowledged statistical methods (Delta-Ct, BestKeeper, NormFinder, and GeNorm). In spite of some discrepancies in gene ranking between these algorisms, Pgk1, Eef1a1, and Ppia were found to be most valid in cortex, while Ppia, Eef1a1, and Helz showed the greatest expression stability in hippocampus. Tbp and Helz were identified as the least stable genes in cortex, whereas Gapdh and Tbp are unsuitable for hippocampus. These data indicate that reliable mRNA quantification in the cortex and hippocampus of F--poisoned rats is possible using normalization to geometric mean of Pgk1+Eef1a1 or Ppia+Eef1a1 expression, respectively.

Fli1 and Tissue Fibrosis in Various Diseases.

Being initially described as a factor of virally-induced leukemias, Fli1 (Friend leukemia integration 1) has attracted considerable interest lately due to its role in both healthy physiology and a variety of pathological conditions. Over the past few years, Fli1 has been found to be one of the crucial regulators of normal hematopoiesis, vasculogenesis, and immune response. However, abnormal expression of Fli1 due to genetic predisposition, epigenetic reprogramming (modifications), or environmental factors is associated with a few diseases of different etiology. Fli1 hyperexpression leads to malignant transformation of cells and progression of cancers such as Ewing's sarcoma. Deficiency in Fli1 is implicated in the development of systemic sclerosis and hypertensive disorders, which are often accompanied by pronounced fibrosis in different organs. This review summarizes the initial findings and the most recent advances in defining the role of Fli1 in diseases of different origin with emphasis on its pro-fibrotic potential.

Silencing of Fli1 Gene Mimics Effects of Preeclampsia and Induces Collagen Synthesis in Human Umbilical Arteries.

BACKGROUND: Previously we demonstrated that in patients with preeclampsia elevated levels of endogenous Na/K-ATPase inhibitor, marinobufagenin, cause inhibition of Friend leukemia virus integration 1 (Fli1), a negative regulator of collagen-1 synthesis. We hypothesized that in vitro silencing of Fli1 in healthy human umbilical arteries would be associated with an increase in collagen-1 output, similar to the effect of preeclampsia in rat and human tissues. METHODS: The isolated segments of healthy human umbilical arteries were tested for sensitivity to MBG and Fli1 silencing with Fli1 siRNA or control siRNA. RESULTS: Following 24-hour incubation of arteries with nanomolar concentrations of marinobufagenin, Fli1 expression was inhibited 5-fold (P < 0.001), and synthesis of collagen-1 increased 3 times (P < 0.01). Twenty-four-hour incubation of umbilical artery fragments with Fli1 siRNA caused a dramatic decrease of Fli1 (7-fold; P < 0.001) and cytoplasmic PKC δ (4-fold; P < 0.001) expression in comparison to control siRNA or untreated control, followed by elevation in procollagen (3-fold; P < 0.001) and collagen-1 (3-fold; P < 0.001) levels in vascular tissue. CONCLUSIONS: Our results show that after silencing the Fli1 gene in healthy human umbilical arteries a new phenotype emerges which is typical for preeclampsia and is associated with vascular fibrosis.

Canrenone Restores Vasorelaxation Impaired by Marinobufagenin in Human Preeclampsia.

Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Recently, we demonstrated that (i) MBG induces fibrosis in rat tissues via a mechanism involving Fli1, a negative regulator of collagen-1 synthesis, and (ii) MBG sensitive Na/K-ATPase inhibition is reversed by mineralocorticoid antagonists. We hypothesized that in human PE elevated MBG level is associated with the development of fibrosis of the umbilical arteries and that this fibrosis can be attenuated by canrenone. Fifteen patients with PE (mean BP = 118 ± 4 mmHg; 34 ± 2 years; 38 ± 0.3 weeks gest. age) and twelve gestational age-matched normal pregnant subjects (mean BP = 92 ± 2 mmHg; 34 ± 1 years; 39 ± 0.2 weeks gest. age) were enrolled in the study. PE was associated with a higher plasma MBG level, with a four-fold decrease in Fli1 level and a three-fold increase in collagen-1 level in the PE umbilical arteries vs. those from the normal subjects (p < 0.01). Isolated rings of umbilical arteries from the subjects with PE exhibited impaired responses to the relaxant effect of sodium nitroprusside vs. control vessels (EC50 = 141 nmol/L vs. EC50 = 0.9 nmol/L; p < 0.001). The effects of PE on Fli1 and collagen-1 were blocked by the in vitro treatment of umbilical arteries by 10 µmol/L canrenone. Similar results were obtained for umbilical arteries pretreated with MBG. These data demonstrate that elevated MBG level is implicated in the development of the fibrosis of umbilical arteries in PE, and that this could be blocked by mineralocorticoid antagonists.

Differential protein expression of mitogen-activated protein kinases in erythrocytes and liver of lamprey Lampetra fluviatilis on the course of prespawning starvation.

The study was designed to identify the types of mitogen-activated protein kinases (MAPKs) in erythrocytes and liver tissues of river lamprey Lampetra fluviatilis and monitor the changes in protein expression levels of found enzymes on the course of prespawning starvation (from November to the end of May). Immunoreactivity of the native and phosphorylated forms of ERK1/2, JNK and p38 was examined in the cytosolic and membrane cell fractions. Both lamprey erythrocytes and liver were found to highly express ERK1/2 and JNK, whereas only trace amounts of p38 were revealed in hepatic tissues. ERK1/2 was identified in cytosolic and membrane fractions, whereas JNK and p38 were predominantly cytosolic enzymes. Total cellular amounts of ERK1/2 and phospho-ERK1/2 in both erythrocytes and liver tissues appeared to be relatively stable on the course of prespawning starvation. However, before spawning ERK1/2 translocated from cytosol to membranes, with partial decline of its cytoplasmic expression being compensated by increases in membrane-bound pool. Immunoreactivity of cytoplasmic JNK, phospho-JNK and p38 were stable from November to March, but sharply decreased before spawning exhibiting almost negligible levels in May, which suggests the depletion of their cellular fractions. Most probably, ERK1/2 plays more important role in mediating adaptive responses of erythrocytes and liver tissues to conditions of natural starvation and maintenance of cell viability before spawning and death of animals in May.

Preeclampsia: Cardiotonic Steroids, Fibrosis, Fli1 and Hint to Carcinogenesis.

Despite prophylaxis and attempts to select a therapy, the frequency of preeclampsia does not decrease and it still takes the leading position in the structure of maternal mortality and morbidity worldwide. In this review, we present a new theory of the etiology and pathogenesis of preeclampsia that is based on the interaction of Na/K-ATPase and its endogenous ligands including marinobufagenin. The signaling pathway of marinobufagenin involves an inhibition of transcriptional factor Fli1, a negative regulator of collagen synthesis, followed by the deposition of collagen in the vascular tissues and altered vascular functions. Moreover, in vitro and in vivo neutralization of marinobufagenin is associated with the restoration of Fli1. The inverse relationship between marinobufagenin and Fli1 opens new possibilities in the treatment of cancer; as Fli1 is a proto-oncogene, a hypothesis on the suppression of Fli1 by cardiotonic steroids as a potential anti-tumor therapeutic strategy is discussed as well. We propose a novel therapy of preeclampsia that is based on immunoneutralization of the marinobufagenin by monoclonal antibodies, which is capable of impairing marinobufagenin-Na/K-ATPase interactions.

Antibody against Na/K-ATPase Inhibitor Lowers Blood Pressure and Increases Vascular Fli1 in Experimental Preeclampsia.

BACKGROUND: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). We demonstrated that MBG induces fibrosis via mechanism involving inhibition of Fli1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. We hypothesized that PE blockade of increased MBG with antibody would lessen the fibrosis of umbilical arteries and lower the blood pressure in rats with PE. METHODS: We tested 36 pregnant Sprague-Dawley rats in which 12 were made hypertensive by 1.8% Na supplementation (days 6-19 of gestation), 12 pregnant rats served controls. At day 19, PE rats received one intraperitoneal injection of polyclonal anti-MBG-4 antibody (0.5 ug/ml) for 4 hours. RESULTS: PE was associated with higher blood pressure (117 ± 2 vs. 107 ± 2 mm Hg; P < 0.01), plasma MBG levels (1.54 ± 0.34 vs. 0.49 ± 0.11 nmol/L; P < 0.01), protein excretion (26 vs. 12 mg/24 hours), sFlt-1 (3-fold), decrease in Fli1 (7-fold) and increase in collagen-1 in aorta (4-fold) vs. control rats (all P < 0.01). In 12 rats treated with polyclonal anti-MBG-4 antibody blood pressure dropped (93 ± 3 mm Hg) and Fli1 was decreased much less (2-fold; P < 0.01 vs. nontreated rats). CONCLUSIONS: These results demonstrate that in experimental PE elevated MBG level is implicated in umbilical fibrosis via suppression of Fli1.

Cognitive Decline of Rats with Chronic Fluorosis Is Associated with Alterations in Hippocampal Calpain Signaling.

The study was designed to evaluate an influence of excessive fluoride (F-) intake on cognitive capacities of adult rats and on proteins of memory-related calpain signaling in hippocampus. Control animals were given water with natural F- content of 0.4 ppm; rats from other groups consumed the same water supplemented with 5, 20, and 50 ppm F- (as NaF) for 12 months. The efficiency of learning and memory formation was evaluated by novel object recognition (NOR) and Morris water maze tests. The expression of enzymes of calpain-1 and calpain-2 signaling in hippocampus was detected by Western blotting. Excessive F- consumption had moderate impact on short-term memory, but impaired spatial learning and long-term memory of animals. Intoxication of rats with 5-50 ppm F- led to stimulation of calpain-1 in hippocampal cells and its translocation from cytosol to membranes, accompanied by activation of GTPase RhoA. Exposure to 20-50 ppm F- resulted in proteolytic cleavage of phosphatase PHLPP1 and increased expression of phospho-ERK1/2 kinase with insignificant decline of total ERK1/2 activity. In contrast, F- did not change the expression of calpain-2 and its substrates-phosphatase PTEN and kinase mTOR. However, F- intake led to downregulation of cAMP-response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF). Thus, altered expression of calpain-1 and its downstream effectors at a background of stable activity of calpain-2 indicates overstimulation of signaling pathways of early LTP phase and disrupted link between early and late LTP phases, most probably due to altered activity of transcriptional and neurotrophic factors.

Cardiotonic Steroids Induce Vascular Fibrosis Via Pressure-Independent Mechanism in NaCl-Loaded Diabetic Rats.

Endogenous cardiotonic steroid, marinobufagenin (MBG), induces Fli1-dependent tissue fibrosis. We hypothesized that an increase in MBG initiates the development of aortic fibrosis in salt-loaded rats with type 2 diabetes mellitus (DM2) via pressure-independent mechanism. DM2 was induced by a single intraperitoneal administration of 65 mg/kg streptozotocin to neonatal (4-5 days) male Wistar rats. Eight-week-old DM2 rats received water or 1.8% NaCl (DM-NaCl) solution for 4 weeks (n = 16); half of DM-NaCl rats were treated with anti-MBG monoclonal antibody (mAb) (DM-NaCl-AB) during week 4 of salt loading; control intact rats received water (n = 8/group). Blood pressure, MBG, erythrocyte Na/K-ATPase activity, aortic weights, levels of fibrosis markers (Fli1, protein kinase Cδ, transforming growth factor-ß1, receptors of the transforming growth factor beta5, fibronectin, collagen-1), and sensitivity of the aortic explants to the vasorelaxant effect of sodium nitroprusside were assessed. No changes in systolic blood pressure were observed while erythrocyte Na/K-ATPase was inhibited by 30%, plasma MBG was doubled, and aortic markers of fibrosis became elevated in DM-NaCl rats versus control. Treatment of DM-NaCl rats with anti-MBG mAb activated Na/K-ATPase, prevented increases in aortic weights, and the levels of fibrosis markers returned to the control levels. The responsiveness of the aortic rings from DM-NaCl rats to the relaxant effect of sodium nitroprusside was reduced (half maximal effective concentration (EC50) = 29 nmol/L) versus control rings (EC50 = 7 nmol/L) and was restored by anti-MBG mAb (EC50 = 9 nmol/L). Our results suggest that in salt-loaded diabetic rats, MBG stimulates aortic collagen synthesis in a pressure-independent fashion and that 2 profibrotic mechanisms, Fli1 dependent and transforming growth factor-ß dependent, underlie its effects.

Endogenous Bufadienolides, Fibrosis and Preeclampsia.

Frequency of preeclampsia has no tendency to decrease, and it still takes the leading position in the structure of maternal mortality and morbidity worldwide. In this review, we present the "fibrotic concept" of the etiology and pathogenesis of preeclampsia which involves system consisting of Na/K-ATPase and its endogenous ligands including marinobufagenin. New therapy of preeclampsia includes modulation of the Na/K-ATPase system by immunoneutralization of the marinobufagenin and use of mineralocorticoid antagonists which are capable to impair marinobufagenin-Na/K-ATPase interactions.

Antibody to Marinobufagenin Reverses Placenta-Induced Fibrosis of Umbilical Arteries in Preeclampsia.

BACKGROUND: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. OBJECTIVES AND METHODS: We hypothesized that in PE, elevated placental MBG levels are associated with development of fibrosis in umbilical arteries. Eleven patients with PE (mean BP 124 ± 4 mmHg; age 29 ± 2 years; 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 ± 2 mmHg; controls) were enrolled in the clinical study. RESULTS: PE was associated with a higher placental (0.04 ± 0.01 vs. 0.49 ± 0.11 pmol/g; p < 0.01) and plasma MBG (0.5 ± 0.1 vs. 1.6 ± 0.5 nmol/L; p < 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 ± 0.2 vs. 1.5 ± 0.2 µmol Pi/mL/hr; p < 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae (p < 0.01) vs. control. Incubation of umbilical arteries from control patients with 1 nmol/L MBG was associated with four-fold decrease in Fli-1 level and two-fold increase in collagen-1 level vs. those incubated with placebo (p < 0.01), i.e., physiological concentration of MBG mimicked effect of PE in vitro. Collagen-1 abundance in umbilical arteries from PE patients was 4-fold higher than in control arteries, and this PE-associated fibrosis was reversed by monoclonal anti-MBG antibody ex vivo. CONCLUSION: These results demonstrate that elevated placental MBG level is implicated in the development of fibrosis of the placenta and umbilical arteries in PE.

Apoptotic death in erythrocytes of lamprey Lampetra fluviatilis induced by ionomycin and tert-butyl hydroperoxide.

The work examined the effects of Ca2+ overload and oxidative damage on erythrocytes of river lamprey Lampetra fluvialtilis. The cells were incubated for 3h with 0.1-5µM Ca2+ ionophore ionomycin in combination with 2.5mM Ca2+ and 10-100µM pro-oxidant agent tert-butyl hydroperoxide (tBHP). The sensitivity of lamprey RBCs to studied compounds was evaluated by the kinetics of their death. Both toxicants induced dose- and time dependent phosphatidylserine (PS) externalization (annexin V-FITC labeling) and loss of membrane integrity (propidium iodide uptake). Highest doses of ionomycin (1-2µM) increased the number of PS-exposed erythrocytes to 7-9% within 3h, while 100µM tBHP produced up to 50% of annexin V-FITC-positive cells. Caspase inhibitor Boc-D-FMK (50µM), calpain inhibitor PD150606 (10µM) and broad protease inhibitor leupeptin (200µM) did not prevent ionomycin-induced PS externalization, whereas tBHP-triggered apoptosis was blunted by Boc-D-FMK. tBHP-dependent death of lamprey erythrocytes was accompanied by the decrease in relative cell size, loss of cell viability, activation of caspases 9 and 3/7, and loss of mitochondrial membrane potential, but all these processes were partially attenuated by Boc-D-FMK. None of examined death-associated events were observed in ionomycin-treated erythrocytes except activation of caspase-9. Incubation with ionomycin did not alter intracellular K+ and Na+ content, while exposure to tBHP resulted in 80% loss of K+ and 2.8-fold accumulation of Na+. Thus, lamprey erythrocytes appear to be more susceptible to oxidative damage. Ca2+ overload does not activate the cytosolic death pathways in these cells.

ATP-consuming processes in hepatocytes of river lamprey Lampetra fluviatilis on the course of prespawning starvation.

The work was performed to establish which of the major ATP-consuming processes is the most important for surviving of hepatocytes of female lampreys on the course of prespawning starvation. The requirements of protein synthesis and Na(+)-K(+)-ATPase for ATP in the cells were monitored by the changes in mitochondrial membrane potential (MMP) in the presence of corresponding inhibitors from the peak of metabolic depression (January-February) to the time of recovery from it (March-April) and spawning (May). Integrity of lamprey liver cells was estimated by catalytic activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in blood plasma. In January-February, the share of ATP necessary for protein synthesis was 20-22%, whereas before spawning it decreased to 8-11%. Functioning of Na(+)-K(+)-pump required 22% of cellular ATP at the peak of metabolic depression, but 38% and 62% of ATP in March-April and May, respectively. Progression of prespawning period was accompanied by 3.75- and 1.6-fold rise of ALT and AST activities in blood plasma, respectively, whereas de Ritis coefficient decreased from 2.51±0.34 to 0.81±0.08, what indicates severe damage of hepatocyte membranes. Thus, the adaptive strategy of lamprey hepatocytes to develop metabolic depression under conditions of energy limitation is the selective production of proteins necessary for spawning, most probably vitellogenins. As spawning approaches, the maintenance of transmembrane ion gradients, membrane potential and cell volume to prevent premature cell death becomes the priority cell function.

Transient activation of protein kinase C contributes to fluoride-induced apoptosis of rat erythrocytes.

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 1­24 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 0.1­10 mM NaF for 1 h produced a dose-dependent PKCa translocation from cytosol to membranes with appearance of active PKM fragment. 24 h NaF exposure led to complete loss of cytosolic PKCa and proteolysis of membrane PKCa. Besides, NaF weakly stimulated membrane PKCf, although its subcellular distribution was not altered. Thus, transient PKCa activation/translocation positively contributes to NaF-induced apoptosis in vitro. Consumption of 2­20 ppm fluoride by the rats for 12 months also induced dose-dependent PKCa translocation to membranes and activation of membrane PKCf, what indicates that PKC stimulation is an important physiological mechanism of fluoride toxicity.

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.

Endogenous cardiotonic steroids and differential patterns of sodium pump inhibition in NaCl-loaded salt-sensitive and normotensive rats.

BACKGROUND: Endogenous sodium pump inhibitors promote sodium excretion in normotensives and contribute to vasoconstriction in NaCl-sensitive hypertension. Marinobufagenin (MBG), an endogenous bufadienolide inhibitor of alpha-1 sodium pump, contributes to hypertension in Dahl salt-sensitive rats (DS). We hypothesized that in NaCl-loaded DS and normotensive Sprague-Dawley rats (S-D), MBG would elicit different patterns of sodium pump inhibition. METHODS: We compared systolic blood pressure (SBP), renal sodium excretion, activity of the sodium pump in aorta and renal medulla, and levels of MBG, atrial natriuretic peptide (ANP), and cyclic guanosine monophosphate (cGMP) in salt-loaded DS and S-D (20% NaCl, 2.5 ml/kg, intraperitoneally). RESULTS: NaCl loading produced sustained elevations in renal MBG excretion in both DS (2.41 +/- 0.24 vs. 0.79 +/- 0.08 pmol/h/kg, P < 0.01) and S-D (1.97 +/- 0.37 vs. 0.60 +/- 0.07 pmol/h/kg, P < 0.01) vs. that at baseline (n = 10 for each group). In NaCl-loaded DS, SBP rose by 18 mm Hg (P < 0.01) and aortic sodium pump was inhibited by 22% (P < 0.05 vs. control), while in S-D, SBP and activity of aortic sodium pump did not change. NaCl-loaded S-D excreted twice as much sodium as DS; in S-D, renal sodium pump was inhibited by 24% vs. 14% inhibition in DS (P < 0.05). NaCl loading elicited increases in plasma ANP and in renal cGMP excretion in S-D but not in DS. CONCLUSIONS: Our present observations demonstrate that in NaCl-loaded S-D and DS, a comparable MBG response is associated with preferential inhibition of the sodium pump in the kidney and in vascular smooth muscle, respectively, resulting in an adaptive natriuresis in S-D but sodium retention and pressor response in DS.

Monoclonal antibody to an endogenous bufadienolide, marinobufagenin, reverses preeclampsia-induced Na/K-ATPase inhibition and lowers blood pressure in NaCl-sensitive hypertension.

BACKGROUND: Levels of marinobufagenin (MBG), an endogenous bufadienolide Na/K-ATPase (NKA) inhibitor, increase in preeclampsia and in NaCl-sensitive hypertension. METHODS: We tested a 3E9 monoclonal anti-MBG antibody (mAb) for the ability to lower blood pressure (BP) in NaCl-sensitive hypertension and to reverse the preeclampsia-induced inhibition of erythrocyte NKA. Measurements of MBG were performed via immunoassay based on 4G4 anti-MBG mAb. RESULTS: In hypertensive Dahl-S rats, intraperitoneal administration of 50 microg/kg 3E9 mAb lowered BP by 32 mmHg and activated the Na/K-pump in the thoracic aorta by 51%. NaCl supplementation of pregnant rats (n = 16) produced a 37 mmHg increase in BP, a 3.5-fold rise in MBG excretion, and a 25% inhibition of the Na/K-pump in the thoracic aorta, compared with pregnant rats on a normal NaCl intake. In eight pregnant hypertensive rats, 3E9 mAb reduced the BP (21 mmHg) and restored the vascular Na/K-pump. In 14 patients with preeclampsia (mean BP, 126 +/- 3 mmHg; 26.9 +/- 1.4 years; gestational age, 37 +/- 0.8 weeks), plasma MBG was increased three-fold and erythrocyte NKA was inhibited compared with that of 12 normotensive pregnant women (mean BP, 71 +/- 3 mmHg) (1.5 +/- 0.1 vs. 3.1 +/- 0.2 micromol Pi/ml/h, respectively; P < 0.01). Ex-vivo 3E9 mAb restored NKA activity in erythrocytes from patients with preeclampsia. As compared with 3E9 mAb, Digibind, an affinity-purified antidigoxin antibody, was less active with respect to lowering BP in both hypertensive models and to restoration of NKA from erythrocytes from patients with preeclampsia. CONCLUSION: Anti-MBG mAbs may be a useful tool in studies of MBG in vitro and in vivo and may offer treatment of preeclampsia.

Transport of lithium across the lamprey (Lampetra fluviatilis) erythrocyte membrane.

Lithium, capable of replacing Na+ in various membrane transport processes, was used to investigate Na+ transport pathways across the lamprey erythrocytes membrane. The values of Li+ influxes have ranged from 8 to 24 mmol/l cells/h. Intracellular accumulation of Li+ was associated with loss of cellular Na+, the value of which was less than the value of Li+ influx. Both Li+ influx and Na+ efflux were partially inhibited by amiloride. The amiloride-sensitive Li+ influx was considerably stimulated by hyperosmotic cell shrinkage. The treatment of lamprey erythrocytes with blockers of protein phosphatases (fluoride and cantharidin) also resulted in a considerable increase in Li+ accumulation within the cells. No significant difference was observed between the values of Li+ and Na+ (22Na) influxes measured in red cells incubated simultaneously in isotonic LiCl and NaCl media (9.2 +/- 2.1 and 7.8 +/- 1.3 mmol/l cells/h, respectively). In hypo- and hypertonic media, however, the rate of Na+ influx in lamprey erythrocytes was approximately twice higher as compared to the rate of Li+ influx, what was determined by the difference in the amiloride-sensitive components. In acidified lamprey erythrocytes (intracellular pH 6.0) Li+ and Na+ influxes were considerably increased due to activation of amiloride-sensitive Na+/H+ (Li+/H+) exchange mechanism, although the activity of Na+/H+ exchange was much greater than that of Li+/H+ exchange. The data obtained confirm the hypothesis on the presence of two amiloride-sensitive systems of Na+ transport in the lamprey red blood cells.