Time course of red blood cell intracellular pH recovery following short-circuiting in relation to venous transit times in rainbow trout, Oncorhynchus mykiss.

Accumulating evidence is highlighting the importance of a system of enhanced hemoglobin-oxygen (Hb-O2) unloading for cardiovascular O2 transport in teleosts. Adrenergically stimulated sodium-proton exchangers (ß-NHE) create H+ gradients across the red blood cell (RBC) membrane that are short-circuited in the presence of plasma-accessible carbonic anhydrase (paCA) at the tissues; the result is a large arterial-venous pH shift that greatly enhances O2 unloading from pH-sensitive Hb. However, RBC intracellular pH (pHi) must recover during venous transit (31-90 s) to enable O2 loading at the gills. The halftimes ( t1/2) and magnitudes of RBC ß-adrenergic stimulation, short-circuiting with paCA and recovery of RBC pHi, were assessed in vitro, on rainbow trout whole blood, and using changes in closed-system partial pressure of O2 as a sensitive indicator for changes in RBC pHi. In addition, the recovery rate of RBC pHi was assessed in a continuous-flow apparatus that more closely mimics RBC transit through the circulation. Results indicate that: 1) the t1/2 of ß-NHE short-circuiting is likely within the residence time of blood in the capillaries, 2) the t1/2 of RBC pHi recovery is 17 s and within the time of RBC venous transit, and 3) after short-circuiting, RBCs reestablish the initial H+ gradient across the membrane and can potentially undergo repeated cycles of short-circuiting and recovery. Thus, teleosts have evolved a system that greatly enhances O2 unloading from pH-sensitive Hb at the tissues, while protecting O2 loading at the gills; the resulting increase in O2 transport per unit of blood flow may enable the tremendous athletic ability of salmonids.

SLC9B1 methylation predicts fetal intolerance of labor.

Fetal intolerance of labor is a common indication for delivery by Caesarean section. Diagnosis is based on the presence of category III fetal heart rate tracing, which is an abnormal heart tracing associated with increased likelihood of fetal hypoxia and metabolic acidemia. This study analyzed data from 177 unique women who, during their prenatal visits (7-15 weeks and/or 24-32 weeks) to Atlanta area prenatal care clinics, consented to provide blood samples for DNA methylation (HumanMethylation450 BeadChip) and gene expression (Human HT-12 v4 Expression BeadChip) analyses. We focused on 57 women aged 18-36 (mean 25.4), who had DNA methylation data available from their second prenatal visit. DNA methylation patterns at CpG sites across the genome were interrogated for associations with fetal intolerance of labor. Four CpG sites (P value <8.9 × 10-9, FDR <0.05) in gene SLC9B1, a Na+/H+ exchanger, were associated with fetal intolerance of labor. DNA methylation and gene expression were negatively associated when examined longitudinally during pregnancy using a linear mixed-effects model. Positive predictive values of methylation of these four sites ranged from 0.80 to 0.89, while negative predictive values ranged from 0.91 to 0.92. The four CpG sites were also associated with fetal intolerance of labor in an independent cohort (the Johns Hopkins Prospective PPD cohort). Therefore, fetal intolerance of labor could be accurately predicted from maternal blood samples obtained between 24-32 weeks gestation. Fetal intolerance of labor may be accurately predicted from maternal blood samples obtained between 24-32 weeks gestation by assessing DNA methylation patterns of SLC9B1. The identification of pregnant women at elevated risk for fetal intolerance of labor may allow for the development of targeted treatments or management plans.

Rac1 as a Potential Pharmacodynamic Biomarker for Thiopurine Therapy in Inflammatory Bowel Disease.

BACKGROUND: Azathioprine and mercaptopurine (MP) are effective in treating patients with inflammatory bowel disease (IBD). Immunosuppressive effects of thiopurines involve T-cell apoptosis after inhibition of GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1). This study aimed to assess whether expression and activity of Rac1 or phosphorylated ezrin-radixin-moesin (pERM) in patients with IBD could provide a useful biomarker for the pharmacodynamic thiopurine effect and might be related to clinical effectiveness. METHODS: This was a 2-stage study: stage 1 concerned a cross-sectional cohort of patients with IBD clinically in remission and treated with (n = 10) or without stable weight-based thiopurine therapy (n = 11) and healthy controls (n = 6); stage 2 concerned a prospective study regarding IBD patients with clinically active disease who initiated MP therapy (n = 11) compared with healthy controls (n = 11). Expression and activity of Rac1 and ERM and pERM were determined. RESULTS: The median Rac1 expression was statistically significantly reduced by thiopurine maintenance therapy {0.54 [interquartile range (IQR) 0.47-0.88] versus 0.80 arbitrary units [IQR 0.64-1.46]} compared with patients without immunosuppressive therapy (P = 0.042), but not Rac1 activity and pERM. In responders to MP therapy (n = 6), both median active Rac1 [93 (IQR 81-151) to 76 ng Rac1/mg protein (IQR 62-98)] and Rac1 expression [16.2 (8.8-29.4) to 1.5 arbitrary units (0.9-5.3)] decreased (P = 0.028). In nonresponders (n = 3), Rac1 expression and activity increased. CONCLUSIONS: IBD patients treated with thiopurines had a lower expression of Rac1 compared with those not treated with thiopurine. Effective MP therapy led to decreasing concentrations of Rac1-GTP and Rac1 expression. Therefore, Rac1-GTP and expression of Rac1, but not phosphorylation of ERM, form potentially pharmacodynamic markers of therapeutic thiopurine effectiveness in patients with IBD.

mGluR1 interacts with cystic fibrosis transmembrane conductance regulator and modulates the secretion of IL-10 in cystic fibrosis peripheral lymphocytes.

Cystic fibrosis (CF) is caused by the mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. CFTR dysfunction in T cells could lead directly to aberrant immune responses. The action of glutamate on the secretion of IL-8 and IL-10 by lymphocytes derived from healthy subjects and cystic CF patients, as well as the expression of metabotropic glutamate receptor subtype 1 (mGluR1) in the membrane fractions of lymphocytes was investigated. Our results have shown that CF-derived T-cells in the presence of IL-2 produce more IL-8 and IL-10, than T-cell from healthy control. However, only in normal lymphocytes a significant increase (144%) in the IL-10 secretion during exposure to high concentration of glutamate (10(-4)M) was detected. Glutamate-dependent secretion of IL-10 was not inhibited either by NMDA-receptor (NMDAR), or by AMPA-receptor (AMPAR) antagonist. Only mGluR1 antagonist, LY367385, strongly decreases the production of IL-10. Furthermore, the content of mGluR1, as well as cystic fibrosis transmembrane conductance regulator-associated ligand (CAL), Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), was analyzed in plasma membrane of lymphocytes after immunoprecipitation of CFTR. We have found that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with metabotropic mGluR1, but the level of surface exposed mGluR1 in CF-lymphocytes was much lower than in normal cells. Besides, our results have shown that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with NHERF-1 and CAL; however in lymphocytes with CFTR mutation the amount of cell-surface expressed CFTR-CAL complex was greatly decreased. We have concluded that CFTR and mGluR1 could compete for binding to CAL, which in turn downregulates the post-synthetic trafficking of mGluR1 and decreases the synthesis of IL-10.

Comparative proteomics reveals deficiency of SLC9A1 (sodium/hydrogen exchanger NHE1) in ß-adducin null red cells.

Spherocytosis is one of the most common inherited disorders, yet presents with a wide range of clinical severity. While several genes have been found mutated in patients with spherocytosis, the molecular basis for the variability in severity of haemolytic anaemia is not entirely understood. To identify candidate proteins involved in haemolytic anaemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in red blood cells (RBCs) from normal and ß-adducin (Add2) knock-out mice. We detected seven proteins that were decreased and 48 proteins that were increased in ß-adducin null RBC ghosts. Since haemolytic anaemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Among the 48 proteins increased in Add2 knockout RBCs, only 11 were also increased in reticulocytes. Of the proteins decreased in Add2 knockout RBCs, α-adducin showed the greatest intensity difference, followed by SLC9A1, the sodium-hydrogen exchanger previously termed NHE1. We verified these mass spectrometry results by immunoblot. This is the first example of SLC9A1deficiency in haemolytic anaemia and suggests new insights into the mechanisms leading to fragile RBCs.

Coordinated control of volume regulatory Na+/H+ and K+/H+ exchange pathways in Amphiuma red blood cells.

The Na(+)/H(+) and K(+)/H(+) exchange pathways of Amphiuma tridactylum red blood cells (RBCs) are quiescent at normal resting cell volume yet are selectively activated in response to cell shrinkage and swelling, respectively. These alkali metal/H(+) exchangers are activated by net kinase activity and deactivated by net phosphatase activity. We employed relaxation kinetic analyses to gain insight into the basis for coordinated control of these volume regulatory ion flux pathways. This approach enabled us to develop a model explaining how phosphorylation/dephosphorylation-dependent events control and coordinate the activity of the Na(+)/H(+) and K(+)/H(+) exchangers around the cell volume set point. We found that the transition between initial and final steady state for both activation and deactivation of the volume-induced Na(+)/H(+) and K(+)/H(+) exchange pathways in Amphiuma RBCs proceed as a single exponential function of time. The rate of Na(+)/H(+) exchange activation increases with cell shrinkage, whereas the rate of Na(+)/H(+) exchange deactivation increases as preshrunken cells are progressively swollen. Similarly, the rate of K(+)/H(+) exchange activation increases with cell swelling, whereas the rate of K(+)/H(+) exchange deactivation increases as preswollen cells are progressively shrunken. We propose a model in which the activities of the controlling kinases and phosphatases are volume sensitive and reciprocally regulated. Briefly, the activity of each kinase-phosphatase pair is reciprocally related, as a function of volume, and the volume sensitivities of kinases and phosphatases controlling K(+)/H(+) exchange are reciprocally related to those controlling Na(+)/H(+) exchange.

Oral calcium supplementation ambulatory blood pressure and relation to changes in intracellular ions and sodium-hydrogen exchange.

BACKGROUND: Calcium (Ca2+) supplementation has been shown paradoxically to reduce intracellular Ca2+ and induce vascular relaxation. The aim of the study was to assess 24-h blood pressure (BP) change after Ca2+ supplementation and to investigate its relation to changes in intracellular ions and the activity of the first isoform of sodium-hydrogen exchange (NHE-1) in subjects with hypertension and type 2 diabetes. METHODS: This parallel, randomized controlled, single-blinded trial, consisted of 31 patients with type 2 diabetes, and hypertension who were allocated to receive 1,500 mg of Ca2+ per day (n = 15) or no treatment (n = 16) for 8 weeks. RESULTS: In the Ca2+ group a decrease of 1.7 +/- 2.7 mm Hg (mean +/- SE) P = 0.52 for mean 24-h systolic BP (SBP) and 2.1 +/- 1.5 mm Hg, P = 0.19 for mean 24-h diastolic BP (DBP) was recorded. Whereas in the control group an increase of 1.4 +/- 2.7 mm Hg, P = 0.59 for mean 24-h SBP and 1.2 +/- 2.8 mm Hg, P = 0.83 for mean 24-h DBP was observed. Intraplatelet Ca2+ decreased whereas intraplatelet magnesium (Mg2+) and erythrocyte K+ increased in the intervention group. Change in mean 24-h SBP in the pooled group correlated with both change in intraplatelet Ca2+ (r = 0.49, P < 0.05) and NHE-1 activity (r = 0.6, P < 0.001). The contribution of intraplatelet Ca2+ was attenuated when both parameters were entered in a multivariate regression model. CONCLUSIONS: The present study shows a weak, statistically nonsignificant trend towards association of Ca2+ supplementation on 24-h BP in hypertensive subjects with type 2 diabetes. However, our results indicated an interrelation of [Ca2+]i levels and NHE-1 activity on BP in patients with hypertension and type 2 diabetes.

Oxidative stress and intracellular pH in the young and old erythrocytes of rat.

The effects of oxidative stress (OS) on the rat erythrocytes (RBCs) that were fractionated on the percoll/BSA gradient into young and old cells were studied to find out if the altered Na+/H+ and Cl⁻/ HCO3⁻ antiporters and in turn the intracellular pH (pHi) could act as one of the promoters of cell death. Old cells were more spherical with lesser surface area, more fragile osmotically and had lesser protein sulphydryl content than the young cells. OS was induced in RBCs by 2,20-azobis (2-amidinopropane) dihydrochloride (AAPH). AAPH increased the superoxide dismutase (SOD) activity and MDA level and, the changes between the young and old. Interestingly, vitamin C was effective in reducing MDA in the old. Further, in the old a rapid Na+-dependent acidification in the presence of AAPH and a marginal acidosis in the presence of vitamin C were evident. Old RBCs exhibited higher acidosis and vitamin C was less effective in lowering the stress-induced acidosis compared to the young. Our studies suggest that increased acidosis followed by low intracellular pH could be one of the determinant factors for the disappearance of old RBCs from circulation, and perhaps of the young too under OS.

Vasopressin acts on platelets to generate procoagulant activity.

The aim of our study was to examine whether arginine vasopressin (AVP) is able to evoke in human platelets a procoagulant response due to activation of an Na+/H+ exchanger. It was found that treatment of platelets with AVP (20-100 nmol/l) results in generation of a weak calcium signal, activation of Na+/H+ exchanger, aggregation, and development of a procoagulant response. The AVP-evoked procoagulant response was dose and time dependent, weaker than that produced by collagen or monensin (mimics Na+/H+ exchanger), and less pronounced following the inhibition of Na+/H+ exchanger by 5-(N-ethyl-N-isopropyl) amiloride or genistein. Flow cytometry studies reveal that in-vitro platelet treatment with AVP results in an unimodal left shift in the forward and side scatter of the entire platelet population, indicating morphological changes on the plasma membrane. The shift was dose related, weaker than that evoked by collagen, similar to that produced by monensin and strongly reduced in the presence of 5-(N-ethyl-N-isopropyl) amiloride or genistein. Using flow cytometry, we demonstrated enhanced expression of phosphatidylserine on the AVP-treated platelets. AVP-evoked phosphatidylserine exposure was dose dependent, inhibited by 5-(N-ethyl-N-isopropyl) amiloride or genistein and weaker than that produced by collagen. AVP in a dose-dependent manner produced a rise in platelet volume. The swelling was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, and its kinetics was similar to that observed in the presence of monensin. We conclude that prolonged treatment of platelets with AVP results in a procoagulant response, which may occur as a consequence of Na+ influx mediated by Na+/H+ exchanger.

Familial pure proximal renal tubular acidosis--a clinical and genetic study.

BACKGROUND: Inherited proximal renal tubular acidosis (pRTA) is commonly associated with more generalized proximal tubular dysfunctions and occasionally with other organ system defects. Inherited combined pRTA and distal RTA with osteopetrosis and pure pRTA associated with ocular abnormalities, a rare disease which has been recently described. Only one family with pure isolated pRTA has been reported so far and the genetic cause for this disease is unknown. Objectives. We report a unique family with isolated pRTA. The aim of the project was to define the phenotype and to try to find the gene defect causing the disease. METHODS: Clinical and metabolic evaluation of all family members was performed and a family pedigree was constructed. DNA was extracted from blood samples of affected and unaffected family members. We amplified by PCR and sequenced the coding areas and splice-sites of the genes that contribute to HCO(-)(3) reclamation in the proximal tubule. The genes studied were as follows: CA II, CA IV, CA XIV, NCB1, Na(+)/H(+) exchanger (NHE)-3, NHE-8, the regulatory proteins of NHE3, NHRF1 and NHRF2 and the Cl(-)/HCO(-)(3) exchanger, SLC26A6. RESULTS: The father and all four children had RTA with blood HCO(-)(3) levels of 11-14 meq/l and urine pH of 5.3-5.4. Increased HCO(-)(3) fractional excretion after bicarbonate loading to 40-60% confirmed the diagnosis pRTA. No other tubular dysfunction was found, and no organ system dysfunction was detected, besides short stature. No mutation was found in all candidate genes studied. CONCLUSIONS: We presented a second family in the literature with familial isolated pure pRTA. The mode of inheritance is compatible with an autosomal dominant disease. Because of the small size of the family, wide genome search was not applicable and the gene candidate approach was chosen. Nine important candidate genes were extensively studied but the molecular basis of the disease was not yet found and genotyping nine important gene candidates were negative.

The Na+/H+ exchange inhibitor cariporide is washed out of the myocardium by crystalloid cardioplegia.

BACKGROUND: Inhibition of the Na (+)/H (+) exchanger (NHE) is cardioprotective, but dosage and timing of NHE-inhibitors are critical for their efficacy. We studied the effect of a new dosing regime of the NHE-inhibitor cariporide on myocardial function and damage after cardioplegic arrest (CPA) and determined its myocardial and serum concentrations. METHODS: 3 pigs received a bolus of 180 mg cariporide intravenously (i. v.) and were sacrificed shortly thereafter to allow measurement of the myocardial concentrations of cariporide. Subsequently, 10 pigs were randomized to receive either i. v. cariporide (bolus followed by an infusion of 40 mg/h) or placebo. Cardiopulmonary bypass was initiated, and the heart was arrested for 60 minutes by infusion of St. Thomas Hospital solution. Left ventricular (LV) function was studied using microsonometry. Myocardial damage was assessed by troponin T. Serum concentrations of cariporide were measured throughout the study, and myocardial concentrations were measured before the end of CPA and 180 minutes thereafter. RESULTS: Cariporide was present in all myocardial specimens (median: 1.4 ng/mg) studied previously. In the main study, LV function or myocardial damage did not differ significantly between the groups at any time point. Stable serum cariporide concentrations were achieved (3.4 +/- 0.5 microg/ml). Cariporide was detectable in only one of the myocardial biopsies obtained before the end of CPA, but 180 minutes thereafter, the myocardial cariporide concentration was 2.5 +/- 0.3 ng/mg. CONCLUSION: We observed no effect of i. v. cariporide on LV function or myocardial damage after cardioplegic arrest. Our data suggest that cariporide is washed out of the myocardium by repeated application of crystalloid cardioplegia. Thus, the mode of delivery also appears to be critical for cardioprotection with NHE-inhibitors.

Involvement of the Na+/H+ exchanger in membrane phosphatidylserine exposure during human platelet activation.

Platelet membrane phosphatidylserine (PS) exposure that regulates the production of thrombin represents an important link between platelet activation and the coagulation cascade. Here, we have evaluated the involvement of the Na+/H+ exchanger (NHE) in this process in human platelets. PS exposure induced in human platelets by thrombin, TRAP, collagen or TRAP+ collagen was abolished in a Na+ -free medium. Inhibition of the Na+/H+ exchanger (NHE) by 5-(N-Ethyl-N-Isopropyl) Amiloride (EIPA) reduced significantly PS exposure, whereas monensin or nigericin, which mimic or cause activation of NHE, respectively, reproduced the agonist effect. These data suggest a role for Na+ influx through NHE activation in the mechanism of PS exposure. This newly identified pathway does not discount a role for Ca2+, whose cytosolic concentration varies together with that of Na+ after agonist stimulation. Ca2+ deprivation from the incubation medium only attenuated PS exposure induced by thrombin, measured from the uptake of FM1-43 (a marker of phospholipid scrambling independent of external Ca2+). Surprisingly, removal of external Ca2+ partially reduced FM1-43 uptake induced by A23187, known as a Ca2+ ionophore. The residual effect can be attributed to an increase in [Na+]i mediated by the ionophore due to a lack of its specificity. Finally, phosphatidylinositol 4,5-bisphosphate (PIP2), previously reported as a target for Ca2+ in the induction of phospholipid scrambling, was involved in PS exposure through a regulation of NHE activity. All these results would indicate that the mechanism that results in PS exposure uses redundant pathways inextricably linked to the physio-pathological requirements of this process.

Physiology and pathophysiology of Na+/H+ exchange and Na+ -K+ -2Cl- cotransport in the heart, brain, and blood.

Maintenance of a stable cell volume and intracellular pH is critical for normal cell function. Arguably, two of the most important ion transporters involved in these processes are the Na+/H+ exchanger isoform 1 (NHE1) and Na+ -K+ -2Cl- cotransporter isoform 1 (NKCC1). Both NHE1 and NKCC1 are stimulated by cell shrinkage and by numerous other stimuli, including a wide range of hormones and growth factors, and for NHE1, intracellular acidification. Both transporters can be important regulators of cell volume, yet their activity also, directly or indirectly, affects the intracellular concentrations of Na+, Ca2+, Cl-, K+, and H+. Conversely, when either transporter responds to a stimulus other than cell shrinkage and when the driving force is directed to promote Na+ entry, one consequence may be cell swelling. Thus stimulation of NHE1 and/or NKCC1 by a deviation from homeostasis of a given parameter may regulate that parameter at the expense of compromising others, a coupling that may contribute to irreversible cell damage in a number of pathophysiological conditions. This review addresses the roles of NHE1 and NKCC1 in the cellular responses to physiological and pathophysiological stress. The aim is to provide a comprehensive overview of the mechanisms and consequences of stress-induced stimulation of these transporters with focus on the heart, brain, and blood. The physiological stressors reviewed are metabolic/exercise stress, osmotic stress, and mechanical stress, conditions in which NHE1 and NKCC1 play important physiological roles. With respect to pathophysiology, the focus is on ischemia and severe hypoxia where the roles of NHE1 and NKCC1 have been widely studied yet remain controversial and incompletely elucidated.

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+ (micromol/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- (micromol/L RBCs/h) in hypertensives was 171.3 +/- 77.9 vs 150.7 +/- 53.9 in the control (NS). Na+-outflow (micromol/L RBCs/h) in hypertensives was 118.3 +/- 51.6 vs 113.3 +/- 24.4 in the control (NS). The K+-outflow (micromol/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+ (micromol/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 (Rs = -0.579, p < 0.05) and between the activity of ex-Na+/Li+ and plasma copper concentration (Rs = -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.

Do trace elements modify the activity of erythrocyte sodium-proton exchanger in hemodialyzed patients?

The kinetics (Vmax and Km) of the erythrocyte Na+-H+ exchanger was studied in a group of 21 patients undergoing regular hemodialysis (HD) and in 21 control subjects. The activity of antioxidative enzymes--superoxide dismutase and glutathione peroxidase--as well as the concentrations of their cofactors--zinc, copper, and selenium--in plasma and in erythrocytes were determined. The thiobarbituric acid-reactive substances (TBARS) concentration served as an indicator of oxidative stress intensity in plasma and erythrocytes. It was found that in the control group the concentration of copper in erythrocytes was positively correlated with Km and Vmax. When the concentration of copper increased, the shape of the kinetic curve changed from sigmoidal to hyperbolic. In the control group, the concentration of zinc in erythrocytes also correlated with Km. However, the results obtained for the group of hemodialyzed patients were the opposite: when the erythrocyte concentration of copper increased, a Km decline was observed and the shape of the curve changed from hyperbolic to sigmoidal. In the group of hemodialyzed patients, we also found a positive correlation between Km and the concentration of selenium in erythrocytes, and a negative correlation between Km and erythrocyte TBARS.

Evolution of oxygen secretion in fishes and the emergence of a complex physiological system.

We have reconstructed the events that led to the evolution of a key physiological innovation underpinning the large adaptive radiation of fishes, namely their unique ability to secrete molecular oxygen (O2). We show that O2 secretion into the swimbladder evolved some 100 million years after another O2-secreting system in the eye. We unravel the likely sequence in which the functional components of both systems evolved. These components include ocular and swimbladder countercurrent exchangers, the Bohr and Root effects, the buffering power and surface histidine content of hemoglobins, and red blood cell Na+/H+ exchange activity. Our synthesis reveals the dynamics of gains and losses of these multiple traits over time, accounting for part of the huge diversity of form and function in living fishes.

Involvement of Na+/H+ exchanger in desmopressin-induced platelet procoagulant response.

Desmopressin (DDAVP) action on platelets is associated with the development of procoagulant response but the underlying mechanism of this phenomenon is not known. We investigated whether this effect of DDAVP might be due to activation of plasma membrane Na+/H+ exchanger. The DDAVP-induced platelet procoagulant response, measured as phospholipid-dependent thrombin generation, was dose dependent and significantly weaker than that produced by collagen or monensin (mimics Na+/H+ antiport). Both the DDAVP- and collagen-produced procoagulant responses were less pronounced in the presence of EIPA, an Na+/H+ exchanger inhibitor. Flow cytometry studies revealed that in vitro treatment of platelets with DDAVP or collagen was associated with the appearance of both degranulated (and fragmented) and swollen cells. The DDAVP-evoked rise in size and granularity heterogeneity was similar to that produced by collagen or monensin and was not observed in the presence of EIPA. Using flow cytometry and annexin V-FITC as a probe for phosphatidylserine (PS) we demonstrated increased and uniform binding of this marker to all subsets of DDAVP-treated platelet population. The DDAVP-evoked PS expression was dose dependent, strongly reduced by EIPA and weaker than that caused by monensin or collagen. As judged by optical swelling assay, DDAVP in a dose dependent manner produced a rise in platelet volume. The swelling was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Electronic cell-sizing measurements showed an increase in mean platelet volume and a decrease in platelet count and platelet crit upon treatment with DDAVP. DDAVP elicited a slow (much slower than collagen) alkalinization of platelet cytosol. Altogether the data indicate an involvement of Na+/H+ exchanger in the generation of procoagulant activity in DDAVP-treated platelets.

[Factors influencing platelet procoagulant activity in type II diabetic patients. The role of sodium-proton exchange]. / Czynniki regulujace aktywnosc prokoagulacyjna plytek krwi u pacjentów z cukrzyca typu 2. Rola antyporterów sodowo-protonowych.

The aim of our study was the estimation of platelet sodium-proton exchanger activity and platelet pro-coagulant activity, expressed as the availability of platelet factor 3 (PF3) and thrombin generation, in 83 type 2 diabetic patients (mean age 56.7 +/- 7.8 years) and 40 healthy subjects (mean age 54.4 +/- 6.2 years). Thrombin generation was measured in platelet rich plasma, using a chromogenic substrate S-2238. The availability of PF3 was estimated in platelet rich plasma, platelet poor plasma and platelet filtrated plasma, to assess procoagulant activity connected with platelets and cell derived microparticles, shedding upon activation (according to Jy and Horstman). The activity of platelet Na+/H+ exchanger was measured using an optical swelling assay. We found that the activity of PF3 and phospholipid dependent thrombin generation were significantly higher in diabetic patients, irrespective of their vascular complications and metabolic control. The highest increase of PF3 activity was observed in platelet poor (p < 0.0001) and platelet filtrated plasma (p < 0.000001). Na+/H+ exchange rate was significantly higher in diabetic patients in comparison to the controls (4.29 +/- 0.71 x 10(-3)/s vs 3.21 +/- 0.64 x 10(-3)/s, p < 0.00001). There was also a positive correlation between Na+/H+ exchanger activity and PF3 activity in all plasma fractions. Our results suggest that increased thrombin generation, enhanced platelet Na+/H+ exchanger activity and raised PF3 availability, connected mainly with cell derived microparticles, may enhance the risk of vascular damage in type 2 diabetic patients.

[Sodium-proton exchanges and platelet procoagulant activity in type 1 diabetic patients]. / Antyportery sodowo-protonowe i aktywnosc prokoagulacyjna plytek krwi u chorych na cukrzyce typu 1.

Platelet sodium-proton exchange rate and phospholipid dependent procoagulant activity were measured in 31 type 1 diabetics (mean age 32.3 +/- 10.1 years) and 35 healthy subjects (mean age 35.4 +/- 9.4 years). The activity of platelet Na+/H+ exchanger was measured in platelet rich plasma, using an optical swelling assay, according to Rosskopf et al. Platelet procoagulant activity was measured in platelet rich plasma, platelet poor plasma and platelet/microparticles filtrated plasma, using Russell's viper venom (according to Jy and Horstman) and calibrated with ship L-alpha-phosphatidylethanolamine. We found that Na+/H+ exchange rate was significantly higher in diabetic patients in comparison to the controls (p = 0.0009). There was also a positive correlation between the activity of Na+/H+ exchanger and phospholipid dependent procoagulant activity in all plasma fractions. We did not find a significant association between Na+/H+ exchanger activity and metabolic parameters studied, however in patients with HbA1c level > 7.5% higher Na+/H+ exchange rates were noted. Total procoagulant activity did not rise significantly in diabetic patients, but was markedly higher in platelet poor and platelet filtrated plasma. It was supposed that it originated from platelet derived microparticles, enriched in phospholipids. Our results suggest that an increased platelet Na+/H+ exchange rate and raised procoagulant activity connected with platelet microparticles may enhance the risk of vascular damage in type 1 diabetic patients.