Activation of Na+/H+ exchanger-directed protein kinases in the ischemic and ischemic-reperfused rat myocardium

The activity of the Na(+)/H(+) exchanger has been implicated as an important contributing factor in damage to the myocardium that occurs during ischemia and reperfusion. We examined regulation of the protein in ischemic and reperfused isolated hearts and isolated ventricular myocytes. In isolated myocytes, extracellular signal-regulated kinases were important in regulating activity of the exchanger after recovery from ischemia. Ischemia followed by reperfusion caused a strong inhibitory effect on NHE1 activity that abated with continued reperfusion. Four major protein kinases of size 90, 55, 44, and 40 kDa phosphorylated the Na(+)/H(+) exchanger. The Na(+)/H(+) exchanger-directed kinases demonstrated dramatic increases in activity of 2-10-fold that was induced by 3 different models of ischemia and reperfusion in intact hearts and isolated myocytes. p90(rsk) was identified as the 90-kDa protein kinase activated by ischemia and reperfusion while ERK1/2 was identified as accounting for some of the 44-kDa protein kinase phosphorylating the Na(+)/H(+) exchanger. The results demonstrate that MAPK-dependent pathways including p90(rsk) and ERK1/2 and are important in regulating the Na(+)/H(+) exchanger and show their dramatic increase in activity toward the Na(+)/H(+) exchanger during ischemia and reperfusion of the myocardium. They also show that ischemia followed by reperfusion have important inhibitory effects on Na(+)/H(+) exchanger activity.     

Relative contributions of Na+/H+ exchange and Na+/HCO3- cotransport to ischemic Nai+ overload in isolated rat hearts

The Na(+)/H(+) exchanger (NHE) and/or the Na(+)/HCO(3)(-) cotransporter (NBC) were blocked during ischemia in isolated rat hearts. Intracellular Na(+) concentration ([Na(+)](i)), intracellular pH (pH(i)), and energy-related phosphates were measured by using simultaneous (23)Na and (31)P NMR spectroscopy. Hearts were subjected to 30 min of global ischemia and 30 min of reperfusion. Cariporide (3 microM) or HCO(3)(-)-free HEPES buffer was used, respectively, to block NHE, NBC, or both. End-ischemic [Na(+)](i) was 320 +/- 18% of baseline in HCO(3)(-)-perfused, untreated hearts, 184 +/- 6% of baseline when NHE was blocked, 253 +/- 19% of baseline when NBC was blocked, and 154 +/- 6% of baseline when both NHE and NBC were blocked. End-ischemic pH(i) was 6.09 +/- 0.06 in HCO(3)(-)-perfused, untreated hearts, 5.85 +/- 0.02 when NHE was blocked, 5.81 +/- 0.05 when NBC was blocked, and 5.70 +/- 0.01 when both NHE and NBC were blocked. NHE blockade was cardioprotective, but NBC blockade and combined blockade were not, the latter likely due to a reduction in coronary flow, because omission of HCO(3)(-) under conditions of NHE blockade severely impaired coronary flow. Combined blockade of NHE and NBC conserved intracellular H(+) load during reperfusion and led to massive Na(+) influx when blockades were lifted. Without blockade, both NHE and NBC mediate acid-equivalent efflux in exchange for Na(+) influx during ischemia, NHE much more than NBC. Blockade of either one does not affect the other.     

HOE-642 (cariporide) alters pH(i) and diastolic function after ischemia during reperfusion in pig hearts in situ

The specific Na(+)/H(+) exchange inhibitor HOE-642 prevents ischemic and reperfusion injury in the myocardium. Although this inhibitor alters H(+) ion flux during reperfusion in vitro, this action has not been confirmed during complex conditions in situ. Myocardial intracellular pH (pH(i)) and high-energy phosphates were monitored using (31)P magnetic resonance spectroscopy in open-chest pigs supported by cardiopulmonary bypass during 10 min of ischemia and reperfusion. Intravenous HOE-642 (2 mg/kg; n = 8) administered before ischemia prevented the increases in diastolic stiffness noted in control pigs (n = 8), although it did not alter the postischemic peak-elastance or pressure-rate product measured using a distensible balloon within the left ventricle. HOE-642 induced no change in pH(i) during ischemia but caused significant delays in intracellular realkalinization during reperfusion. HOE-642 did not alter phosphocreatine depletion and repletion but did improve ATP preservation. Na(+)/H(+) exchange inhibition through HOE-642 delays intracellular alkalinization in the myocardium in situ during reperfusion in association with improved diastolic function and high-energy phosphate preservation.     

Activation of proteolytic enzymes and depression of the sarcolemmal Na+/K+-ATPase in ischemia-reperfused heart may be mediated through oxidative stress

We tested whether the activation of proteolytic enzymes, calpain, and matrix metalloproteinases (MMPs) during ischemia-reperfusion (I/R) is mediated through oxidative stress. For this purpose, isolated rat hearts were subjected to a 30?min global ischemia followed by a 30?min reperfusion. Cardiac function was monitored and the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, calpain, and MMP were measured. Depression of cardiac function and Na(+)/K(+)-ATPase activity in I/R hearts was associated with increased calpain and MMP activities. These alterations owing to I/R were similar to those observed in hearts perfused with hypoxic medium, H(2)O(2) and xanthine plus xanthine oxidase. The I/R-induced changes were attenuated by ischemic preconditioning as well as by perfusing the hearts with N-acetylcysteine or mercaptopropionylglycine. Inhibition of MMP activity in hearts treated with doxycycline depressed the I/R-induced changes in cardiac function and Na(+)/K(+)-ATPase activity without affecting the calpain activation. On the other hand, inhibition of calpain activity upon treatment with leupeptin or MDL 28170 significantly reduced the MMP activity in addition to attenuating the I/R-induced alterations in cardiac function and Na(+)/K(+)-ATPase activity. These results suggest that the I/R-induced depression in Na(+)/K(+)-ATPase and cardiac function may be a consequence of the increased activities of both calpain and MMP because of oxidative stress in the heart.     

Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.     

Hyposmotic shock: effects on rubidium/potassium efflux in normal and ischemic rat hearts,assessed by 87Rb and 31P NMR

The study evaluated effects of hyposmotic shock on the rate of Rb(+)/K(+) efflux, intracellular pH and energetics in Langendorff-perfused rat hearts with the help of 87Rb- and 31P-NMR. Two models of hyposmotic shock were compared: (1) normosmotic hearts perfused with low [NaCl] (70 mM) buffer, (2) hyperosmotic hearts equilibrated with additional methyl alpha-D-glucopyranoside (Me-GPD, 90 or 33 mM) or urea (90 mM) perfused with normosmotic buffer. Four minutes after hyposmotic shock, Rb(+) efflux rate constant transiently increased approximately two-fold, while pH transiently decreased by 0.08 and 0.06 units, in the first and the second models, respectively, without significant changes in phosphocreatine and ATP. Hyposmotic shock (second model) did not change the rate of Rb(+)/K(+) uptake, indicating that the activity of Na(+)/K(+) ATPase was not affected. Dimethylamiloride (DMA) (10 microM) abolished activation of the Rb(+)/K(+) efflux in the second model; however, Na(+)/H(+) exchanger was not involved, because intracellular acidosis induced by the hyposmotic shock was not enhanced by DMA treatment. After 12 or 20 min of global ischemia, the rate of Rb(+)/K(+) efflux increased by 120%. Inhibitor of the ATP-sensitive potassium channels, glibenclamide (5 microM), partially (40%) decreased the rate constant; however, reperfusion with hyperosmolar buffer (90 mM Me-GPD) did not. We concluded that the shock-induced stimulation of Rb(+)/K(+) efflux occurred, at least partially, through the DMA-sensitive cation/H(+) exchanger and swelling-induced mechanisms did not considerably contribute to the ischemia-reperfusion-induced activation of Rb(+)/K(+) efflux.     

Role of Na+-Ca2+ exchanger in myocardial ischemia/reperfusion injury: evaluation using a heterozygous Na+-Ca2+ exchanger knockout mouse model

We used Na(+)-Ca(2+) exchanger (NCX) knockout mice to evaluate the effects of NCX in cardiac function and the infarct size after ischemia/reperfusion injury. The contractile function in NCX KO mice hearts was significantly better than that in wild type (WT) mice hearts after ischemia/reperfusion and the infarct size was significantly small in NCX KO mice hearts compared with that in WT mice hearts. NCX is critically involved in the development of ischemia/reperfusion-induced myocardial injury and therefore the inhibition of NCX function may contribute to cardioprotection against ischemia/reperfusion injury.     

Increased tolerance to oxygen and glucose deprivation in astrocytes from Na(+)/H(+) exchanger isoform 1 null mice

The ubiquitously expressed Na(+)/H(+) exchanger isoform 1 (NHE1) functions as a major intracellular pH (pH(i)) regulatory mechanism in many cell types, and in some tissues its activity may contribute to ischemic injury. In the present study, cortical astrocyte cultures from wild-type (NHE1(+/+)) and NHE1-deficient (NHE1(-/-)) mice were used to investigate the role of NHE1 in pH(i) recovery and ischemic injury in astrocytes. In the absence of HCO(3)(-), the mean resting pH(i) levels were 6.86 +/- 0.03 in NHE1(+/+) astrocytes and 6.53 +/- 0.04 in NHE1(-/-) astrocytes. Removal of extracellular Na(+) or blocking of NHE1 activity by the potent NHE1 inhibitor HOE-642 significantly reduced the resting level of pH(i) in NHE1(+/+) astrocytes. NHE1(+/+) astrocytes exhibited a rapid pH(i) recovery (0.33 +/- 0.08 pH unit/min) after NH(4)Cl prepulse acid load. The pH(i) recovery in NHE1(+/+) astrocytes was reversibly inhibited by HOE-642 or removal of extracellular Na(+). In NHE1(-/-) astrocytes, the pH(i) recovery after acidification was impaired and not affected by either Na(+)-free conditions or HOE-642. Furthermore, 2 h of oxygen and glucose deprivation (OGD) led to an approximately 80% increase in pH(i) recovery rate in NHE1(+/+) astrocytes. OGD induced a 5-fold rise in intracellular [Na(+)] and 26% swelling in NHE1(+/+) astrocytes. HOE-642 or genetic ablation of NHE1 significantly reduced the Na(+) rise and swelling after OGD. These results suggest that NHE1 is the major pH(i) regulatory mechanism in cortical astrocytes and that ablation of NHE1 in astrocytes attenuates ischemia-induced disruption of ionic regulation and swelling.     

Phospholemman expression is high in the newborn rabbit heart and declines with postnatal maturation

Phospholemman (PLM) is a small sarcolemmal protein that modulates the activities of Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger (NCX), thus contributing to the maintenance of intracellular Na(+) and Ca(2+) homeostasis. We characterized the expression and subcellular localization of PLM, NCX, and the Na(+)/K(+)-ATPase alpha1-subunit during perinatal development. Western blotting demonstrates that PLM (15kDa), NCX (120kDa), and Na(+)/K(+)-ATPase alpha-1 (approximately 100kDa) proteins are all more than 2-fold higher in ventricular membrane fractions from newborn rabbit hearts (1-4-day old) compared to adult hearts. Our immunocytochemistry data demonstrate that PLM, NCX, and Na(+)/K(+)-ATPase are all expressed at the sarcolemma of newborn ventricular myocytes. Taken together, our data indicate that PLM, NCX, and Na(+)/K(+)-ATPase alpha-1 proteins have similar developmental expression patterns in rabbit ventricular myocardium. Thus, PLM may have an important regulatory role in maintaining cardiac Na(+) and Ca(2+) homeostasis during perinatal maturation.     

Mitogen-activated protein kinase-dependent activation of the Na+/H+ exchanger is mediated through phosphorylation of amino acids Ser770 and Ser771

We investigated regulation of the type 1 isoform of the Na(+)/H(+) exchanger by phosphorylation. Four specific groups of serine and threonine residues in the regulatory carboxyl-terminal tail were mutated to alanine residues: group 1, S693A; group 2, T718A and S723A/S726A/S729A; group 3, S766A/S770A/S771A; and group 4, T779A and S785A. The proteins were expressed in Na(+)/H(+) exchanger-deficient cells, and the activity was characterized. All of the mutants had proper expression, localization, and normal basal activity relative to wild type NHE1. Sustained intracellular acidosis was used to activate NHE1 via an ERK-dependent pathway that could be blocked with the MEK inhibitor U0126. Immunoprecipitation of (32)P-labeled Na(+)/H(+) exchanger from intact cells showed that sustained intracellular acidosis increased Na(+)/H(+) exchanger phosphorylation in vivo. This was blocked by U0126. The Na(+)/H(+) exchanger activity of mutants 1 and 2 was stimulated similar to wild type Na(+)/H(+) exchanger. Mutant 4 showed a partially reduced level of activation. However, mutant 3 was not stimulated by sustained intracellular acidosis, and loss of stimulation of activity correlated to a loss of sustained acidosis-mediated phosphorylation in vivo. Mutation of the individual amino acids within mutant 3, Ser(766), Ser(770), and Ser(771), showed that Ser(770) and Ser(771) are responsible for mediating increases in NHE1 activity through sustained acidosis. Both intact Ser(770) and Ser(771) were required for sustained acidosis-mediated activation of NHE1. Our results suggest that amino acids Ser(770) and Ser(771) mediate ERK-dependent activation of the Na(+)/H(+) exchanger in vivo.     

OSR1-sensitive regulation of Na+/H+ exchanger activity in dendritic cells

The oxidative stress-responsive kinase 1 (OSR1) is activated by WNK (with no K kinases) and in turn stimulates the thiazide-sensitive Na-Cl cotransporter (NCC) and the furosemide-sensitive Na-K-2Cl cotransporter (NKCC), thus contributing to transport and cell volume regulation. Little is known about extrarenal functions of OSR1. The present study analyzed the impact of decreased OSR1 activity on the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs were cultured from bone marrow of heterozygous WNK-resistant OSR1 knockin mice (osr(KI)) and wild-type mice (osr(WT)). Cell volume was estimated from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, and Na(+)/H(+) exchanger activity from Na(+)-dependent realkalinization following ammonium pulse and migration utilizing transwell chambers. DCs expressed WNK1, WNK3, NCC, NKCC1, and OSR1. Phosphorylated NKCC1 was reduced in osr(KI) DCs. Cell volume and pH(i) were similar in osr(KI) and osr(WT) DCs, but Na(+)/H(+) exchanger activity and ROS production were higher in osr(KI) than in osr(WT) DCs. Before LPS treatment, migration was similar in osr(KI) and osr(WT) DCs. LPS (1 μg/ml), however, increased migration of osr(WT) DCs but not of osr(KI) DCs. Na(+)/H(+) exchanger 1 inhibitor cariporide (10 μM) decreased cell volume, intracellular reactive oxygen species (ROS) formation, Na(+)/H(+) exchanger activity, and pH(i) to a greater extent in osr(KI) than in osr(WT) DCs. LPS increased cell volume, Na(+)/H(+) exchanger activity, and ROS formation in osr(WT) DCs but not in osr(KI) DCs and blunted the difference between osr(KI) and osr(WT) DCs. Na(+)/H(+) exchanger activity in osr(WT) DCs was increased by the NKCC1 inhibitor furosemide (100 nM) to values similar to those in osr(KI) DCs. Oxidative stress (10 μM tert-butyl-hydroperoxide) increased Na(+)/H(+) exchanger activity in osr(WT) DCs but not in osr(KI) DCs and reversed the difference between genotypes. Cariporide virtually abrogated Na(+)/H(+) exchanger activity in both genotypes and blunted LPS-induced cell swelling and ROS formation in osr(WT) mice. In conclusion, partial OSR1 deficiency influences Na(+)/H(+) exchanger activity, ROS formation, and migration of dendritic cells.     

Gender-specific effects of exercise on cardiac pathology in Na+/H+ exchanger overexpressing mice

The Na(+)/H(+) exchanger isoform 1 (NHE1) has been implicated in various cardiac pathologies including ischemia/reperfusion damage to the myocardium and cardiac hypertrophy. It is known that NHE1 levels increase in cardiac disease and we have recently demonstrated that expression of an activated NHE1 protein promotes cardiac hypertrophy in the mouse myocardium. We examined the gender-specific effects of exercise in combination with elevated cardiac expression of NHE1 on the myocardium in mice. Control mice and transgenic mice expressing elevated levels of wild type NHE1 and activated NHE1 were examined. There were gender-specific differences in the effects of NHE1 with exercise. Exercised wild type male mice showed a tendency toward increased heart weight. This was not apparent in female mice expressing elevated NHE1 levels. In some transgenic female mice, there was a significant decrease in the size of the exercised hearts, which was different from what occurred with male mice. Body weight was maintained in exercised control and transgenic male mice; however, it decreased in female mice with exercise more so in transgenic female mice expressing elevated levels of NHE1. Female mice expressing activated NHE1 had elevated HW/BW ratios compared to males, and this was exaggerated by exercise. These results suggest that gender-specific activation of NHE1 may be critical and that NHE1 plays a more critical role in promoting some types of hypertrophy in females in comparison with males.     

The NHE1 Na+/H+ exchanger recruits ezrin/radixin/moesin proteins to regulate Akt-dependent cell survival

Apoptosis results in cell shrinkage and intracellular acidification, processes opposed by the ubiquitously expressed NHE1 Na(+)/H(+) exchanger. In addition to mediating Na(+)/H(+) transport, NHE1 interacts with ezrin/radixin/moesin (ERM), which tethers NHE1 to cortical actin cytoskeleton to regulate cell shape, adhesion, motility, and resistance to apoptosis. We hypothesize that apoptotic stress activates NHE1-dependent Na(+)/H(+) exchange, and NHE1-ERM interaction is required for cell survival signaling. Apoptotic stimuli induced NHE1-regulated Na(+)/H(+) transport, as demonstrated by ethyl-N-isopropyl-amiloride-inhibitable, intracellular alkalinization. Ectopic NHE1, but not NHE3, expression rescued NHE1-null cells from apoptosis induced by staurosporine or N-ethylmaleimide-stimulated KCl efflux. When cells were subjected to apoptotic stress, NHE1 and phosphorylated ERM physically associated within the cytoskeleton-enriched fraction, resulting in activation of the pro-survival kinase, Akt. NHE1-associated Akt activity and cell survival were inhibited in cells expressing ERM binding-deficient NHE1, dominant negative ezrin constructs, or ezrin mutants with defective binding to phosphoinositide 3-kinase, an upstream regulator of Akt. We conclude that NHE1 promotes cell survival by dual mechanisms: by defending cell volume and pH(i) through Na(+)/H(+) exchange and by functioning as a scaffold for recruitment of a signalplex that includes ERM, phosphoinositide 3-kinase, and Akt.     

Alterations of Na(+) homeostasis in hepatocyte reoxygenation injury

Reperfusion injury represents an important cause of primary graft non-function during liver transplantation. However, the mechanism responsible for cellular damage during reoxygenation has not yet been completely understood. We have investigated whether changes in intracellular Na(+) distribution might contribute to cause hepatocyte damage during reoxygenation buffer after 24 h of cold storage. Hepatocyte reoxygenation resulted in a rapid increase in cellular Na(+) content that was associated with cytotoxicity. Na(+) accumulation and hepatocyte death were prevented by the omission of Na(+) from the incubation medium, but not by the addition of antioxidants. Blocking Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) co-transporter by, respectively, 5-(N,N-dimethyl)-amiloride or omitting HCO(3)(-) from the reoxygenation medium significantly decreased Na(+) overload and cytotoxicity. Stimulation of ATP re-synthesis by the addition of fructose also lowered Na(+) accumulation and cell death during reoxygenation. A significant protection against Na(+)-mediated reoxygenation injury was evident in hepatocytes maintained in an acidic buffer (pH 6.5) or in the presence of glycine. The cytoprotective action of glycine or of the acidic buffer was reverted by promoting Na(+) influx with the Na(+)/H(+) ionophore monensin. Altogether, these results suggest that Na(+) accumulation during the early phases of reoxygenation might contribute to liver graft reperfusion injury.     

Elevated expression of activated Na+/H+ exchanger protein induces hypertrophy in isolated rat neonatal ventricular cardiomyocytes

The plasma membrane protein the Na(+)/H(+) exchanger isoform1 (NHE1) has been implicated in various cardiac pathologies including ischemia/reperfusion damage to the myocardium and cardiac hypertrophy. Levels of NHE1 protein and activity are elevated in cardiac disease; however, the mechanism by which these factors contribute to the accompanying hypertrophy in the myocardium is still not clear. To investigate the mechanism of NHE1-induced hypertrophy in the myocardium we constructed two adenoviral vectors expressing either wild type NHE1 protein or a constitutively active NHE1 protein. Infection of neonatal rat ventricular cardiomyocytes (NRVM) resulted in elevated expression of both wild type NHE1 or constitutively active NHE1. Only expression of activated NHE1 protein resulted in an increase in cell size and in an increase in protein synthesis in isolated cardiomyocyte cells. The results demonstrate that expression of activated NHE1 promotes cardiac hypertrophy in isolated cardiac cells and that simple elevation of levels of wild type NHE1 protein does not have a significant hypertrophic effect in NRVM. The results suggest that regulation of NHE1 activity is a critical direct effector of the hypertrophic effect induced in the myocardium by the NHE1 protein.     

Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加

本文旨在研究Na+/H+交换以及Na+/Ca2 +交换对模拟缺血 /复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血 /复灌处理 ,在缺血期间分别应用Na+/H+交换抑制剂阿米洛利 (amiloride)、Na+/Ca2 +交换抑制剂NiCl2 以及无钙液 ,观察它们对细胞内游离Ca2 +浓度变化的影响。利用Zeiss LSM 5 10激光共聚焦显微镜检测、采集细胞内游离Ca2 +的指示剂Fluo 3 AM的荧光信号 ,计算出相对于正常(缺血前 )的相对荧光强度 ,以表示胞内游离Ca2 +浓度的变化。结果显示 ,模拟缺血引起大鼠心肌细胞内游离Ca2 +持续上升 ,缺血前的相对荧光强度值为 10 0 % ,模拟缺血 5min后为 140 3± 13 0 % (P <0 0 5 ) ,复灌 15min后为 142 8±15 5 % (P <0 0 5 )。经 10 0 μmol/Lamiloride、5mmol/LNiCl2 和无钙液分别预处理 ,模拟缺血 5min后的相对荧光强度分别为 10 1 4± 16 3 % (P <0 0 5 )、110 4± 11 1% (P <0 0 5 )和 10 7 1± 10 8(P <0 0 5 ) ;复灌 15min后则分别为 97 8±14 3 % (P <0 0 5 )、10 6 2± 14 5 % (P <0 0 5 )和 10 6 6± 15 7(P <0 0 5 )。另外 ,与对照组细胞相比 ,再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱 ,amilorid     

Influence of ischemic preconditioning on intracellular sodium,pH, and cellular energy status in isolated perfused heart

The possible relationships between intracellular Na(+) (Na(i)(+)), bioenergetic status and intracellular pH (pH(i)) in the mechanism for ischemic preconditioning were studied using (23)Na and (31)P magnetic resonance spectroscopy in isolated Langendorff perfused rat heart. The ischemic preconditioning (three 5-min ischemic episodes followed by two 5-min and one 10-min period of reperfusion) prior to prolonged ischemia (20 min stop-flow) resulted in a decrease in ischemic acidosis and faster and complete recovery of cardiac function (ventricular developed pressure and heart rate) after 30 min of reperfusion. The response of Na(i) during ischemia in the preconditioned hearts was characterized by an increase in Na(i)(+) at the end of preconditioning and an accelerated decrease during the first few minutes of reperfusion. During post-ischemic reperfusion, bioenergetic parameters (PCr/P(i) and betaATP/P(i) ratios) were partly recovered without any significant difference between control and preconditioned hearts. The reduced acidosis during prolonged ischemia and the accelerated decrease in Na(i)(+) during reperfusion in the preconditioned hearts suggest activation of Na(+)/H(+) exchanger and other ion transport systems during preconditioning, which may protect the heart from intracellular acidosis during prolonged ischemia, and result in better recovery of mechanical function (LVDP and heart rate) during post-ischemic reperfusion.     

Uroguanylin regulates net fluid secretion via the NHE2 isoform of the Na/H+ exchanger in an intestinal cellular model

Uroguanylin (UGN) has been proposed as a key regulator of salt and water intestinal transport. Uroguanylin activates cell-surface guanylate cyclase C receptor (GC-C) and modulates cellular function via cyclic GMP (cGMP), thus increasing electrolyte and net water secretion. It has been suggested that the action of UGN could involve the Na(+)/H(+) exchanger, but the actual contribution of this transporter still remains unclear. The objective of our study was to investigate the putative effects of UGN on some members of the Na(+)/H(+) exchanger family (NHEs), as well as to clarify its consequences on transepithelial fluid flow in T84 cells. In order to do so, transepithelial fluid flow (J(v)) was studied by optic techniques and intracellular pH (pH(i)) was measured with a fluorescence method. Results showed that NHE2 is found at the apical membrane and has a major role in Na(+) absorption; NHE1 and NHE4 are localized at the basolateral membrane with a house-keeping role in steady state pH(i). In the assayed conditions, cell exposure to apical UGN increases net secretory J(v), without changing short-circuit currents nor transepithelial resistance, and reduces NHE2 activity. Therefore, at physiological pH, the effect on net J(v) was produced mainly by a reduction in normal Na(+) absorption through NHE2, rather than by the stimulation of electrolyte secretion. Our study shows that the effect of UGN on pH(i) is GC-C/cGMP-mediated and enhanced by sildenafil, thus involving PDE5 enzyme. Additionally, cell exposure to apical UGN results in intracellular alkalinization, probably due to indirect effects on basolateral NHE1 and NHE4, which have a major role in pH(i) regulation.