Suppression of Na+/H+ exchanger isoform-3 in human inflammatory bowel disease: lack of reversal by 5'-aminosalicylate treatment.

OBJECTIVE: Na+/H+ exchanger isoform 3 (NHE-3) is responsible for net uptake of NaCl and water from the gastrointestinal (GI) tract. However, its status in human inflammatory bowel diseases (IBDs) such as ulcerative colitis(UC) and Crohn's disease (CD) remains poorly understood. The aim of this study was to investigate the underlying mechanism of NHE-3 isoform expression and its modulation by 5'-aminosalicylate in human CD and UC. MATERIAL AND METHODS: Subjects were divided into three groups: 1) controls; 2) untreated/new IBD cases (n = 13) and 3) 5'-aminosalicylate-treated IBD patients (n = 13). Subjects presenting with abdominal pain but with endoscopically normal colons served as normal controls. Inflammation was confirmed by the level of myeloperoxidase (MPO) activity, malondialdehyde (MDA) concentrations and by histologic evaluation. Expressions of NHE-3 protein and mRNA, sodium pump activity and IL-1beta and TNF-alpha mRNA were estimated in the colonic biopsies using ECL-Western blot analysis,reverse transcription-polymerase chain reaction (RT-PCR) and enzyme assays. RESULTS: The level of NHE-3 protein and sodium pump activity was reduced (p < 0.05) in both the untreated and treated CD and UC patients. NHE-3 mRNA was reduced only in CD patients but not in those with UC. The treatment reversed the symptoms, but levels of MPO activity, MDA concentration, IL-1beta, TNF-alpha and infiltration of inflammatory cells remained high with the exception of IL-1beta mRNA in the treated patients. CONCLUSIONS: NHE-3 suppression is regulated differentially in CD and UC, which together with suppression of sodium pump activity will reduce NaCl and water uptake from the colonic lumen. These findings suggest a role of TNF-a in the regulation of NHE-3 expression in IBD.

Targeting Na+/H+ exchanger regulation for cardiac protection: a RSKy approach?

Extensive pre-clinical work indicates that inhibition of the Na(+)/H(+) exchanger (NHE) affords significant protection to myocardium subjected to ischaemia and reperfusion. By contrast, clinical studies with the NHE inhibitors cariporide, eniporide and zoniporide, in patients with evolving myocardial infarction and those at risk of myocardial infarction, have provided largely disappointing data. Nevertheless, some of these studies have confirmed that, in certain settings, NHE inhibition does indeed protect human myocardium. Furthermore, pre-clinical work suggests that NHE inhibition may provide therapeutic benefit in heart failure also. As an alternative to direct and global NHE inhibition, which may trigger non-cardiac adverse effects, the molecular mechanisms that stimulate cardiac NHE activity in disease may be targeted to attenuate such activity selectively in jeopardized tissue. Many factors associated with cardiac pathology activate RSK, an established NHE kinase, and several selective RSK inhibitors have been described recently. The role of RSK as a potential therapeutic target for indirectly suppressing cardiac NHE activity warrants further investigation.

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.

Impairment of sodium pump and Na/H exchanger in erythrocytes from non-insulin dependent diabetes mellitus patients: effect of tea catechins.

BACKGROUND: Tea catechins (EGCG, EGC, ECG and EC) possess many important biological properties. We evaluated the effect of tea catechins on erythrocyte membrane Na(+)/K(+)-ATPase and sodium/hydrogen exchanger (NHE) activity in normal (control) and NIDDM subjects. METHODS: Erythrocyte membrane Na(+)/K(+)-ATPase and NHE activity were determined in normal and non-insulin dependent diabetes mellitus (NIDDM) patients. In vitro effect of tea catechins was studied by incubating membrane/intact erythrocytes in assay medium prior to Na(+)/K(+)-ATPase/NHE activity determination. RESULTS: A 24.2% decrease in the activity of Na(+)/K(+)-ATPase (p<0.001) and 39.37% increase in activity of NHE (p<0.02) were observed in NIDDM subjects compared to normal. Tea catechins inhibited the activity of Na(+)/K(+)-ATPase and NHE in both normal and NIDDM erythrocytes, the effect was concentration-dependent. The inhibitory effect of EGCG and ECG at micromolar concentrations was greater compared to EGC and EC on Na(+)/K(+)-ATPase. On NHE the inhibition of tea catechins was in the order: EC>EGC>ECG>EGCG at concentrations up to 10 micromol/l. CONCLUSIONS: This data may help to explain the anti-carcinogenic and cardioprotective effects of tea catechins. The effect of tea catechins on Na(+)/K(+)-ATPase and NHE may be explained due to a direct effect of these compounds on plasma membrane leading to a change in membrane fluidity.

Heat-induced changes in intracellular Na+, pH and bioenergetic status in superfused RIF-1 tumour cells determined by 23Na and 31P magnetic resonance spectroscopy.

The acute effects of hyperthermia on intracellular Na+ (Nai+), bioenergetic status and intracellular pH (pHi) were investigated in superfused Radiation Induced Fibrosarcoma-1 (RIF-1) tumour cells using shift-reagent-aided 23Na and 31P nuclear magnetic resonance (NMR) spectroscopy. Hyperthermia at 45 degrees C for 30 min produced a 50% increase in Na, a 0.42 unit decrease in pHi and a 40-45% decrease in NTP/P(i). During post-hyperthermia superfusion at 37 degrees C, pHi and NTP/P(i) recovered to the baseline value, but Na initially decreased and then increased to the hyperthermic level 60 min after heating. Hyperthermia at 42 degrees C caused only a 15-20% increase in Nai+. In the presence of 3 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na+/H+ exchanger, the increase in Nai+ during 45 degrees C hyperthermia was attenuated, suggesting that the heat-induced increase in Nai+ was mainly due to an increase in Na+/H+ anti-porter activity. EIPA did not prevent hyperthermia-induced acidification. This suggests that pHi is controlled by other ion exchange mechanisms in addition to the Na+/H+ exchanger. EIPA increased the thermo-sensitivity of the RIF-1 tumour cells only slightly as measured by cell viability and clonogenic assays. The hyperthermia-induced irreversible increase in Nai+ suggests that changes in transmembrane ion gradients play an important role in cell damage induced by hyperthermia.

Mechanism of garlic (Allium sativum) induced reduction of hypertension in 2K-1C rats: a possible mediation of Na/H exchanger isoform-1.

Garlic causes reduction in blood pressure (BP), however the role of Na/H exchanger (NHE) which mediates hypertension and related tissue-damage is poorly understood. In this study the effect of an established dose of raw garlic extract was investigated on the expression of NHE-1 and -3 and sodium pump activity in a 2K-1C model of hypertension in rats. 2K-1C animals showed high BP, increased serum concentration of PGE2 and TxB2, hypertrophy of the unclipped kidneys, but not in the clipped kidneys In addition, NHE-1 and NHE-3 isoforms were increased in both the 2K-1C kidneys, whereas alpha-actin was increased in the clipped but not in unclipped kidneys. Sodium pump activity was decreased in the clipped kidneys, but remained unchanged in the unclipped kidneys. Garlic treatment reduced the induction of NHE-1 only in the unclipped 2K-1C kidneys, whereas garlic treatment increased the sodium pump activity in both the 2K-1C kidneys. These findings demonstrate that the antihypertensive action of garlic is associated with a reversal of NHE-1 induction in the unclipped kidneys. Induction of NHE isoforms together with a reduced sodium pump activity might cause necrosis in the 2K-1C clipped kidneys due to cellular retention of Na+. On the other hand, activation of sodium pump by garlic extract in the kidneys should reduce intracellular Na+ concentration and normalize BP. These findings signify the use of garlic in the treatment of hypertension.

Elicitor-activated phospholipase A(2) generates lysophosphatidylcholines that mobilize the vacuolar H(+) pool for pH signaling via the activation of Na(+)-dependent proton fluxes.

The elicitation of phytoalexin biosynthesis in cultured cells of California poppy involves a shift of cytoplasmic pH via the transient efflux of vacuolar protons. Intracellular effectors of vacuolar proton transport were identified by a novel in situ approach based on the selective permeabilization of the plasma membrane for molecules of < or = 10 kD. Subsequent fluorescence imaging of the vacuolar pH correctly reported experimental changes of activity of the tonoplast proton transporters. Lysophosphatidylcholine (LPC) caused a transient increase of the vacuolar pH by increasing the Na(+) sensitivity of a Na(+)-dependent proton efflux that was inhibited by amiloride. In intact cells, yeast elicitor activated phospholipase A(2), as demonstrated by the formation of LPC from fluorescent substrate analogs, and caused a transient increase of endogenous LPC, as determined by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. It is suggested that LPC generated by phospholipase A(2) at the plasma membrane transduces the elicitor-triggered signal into the activation of a tonoplast H(+)/Na(+) antiporter.

Effects of antihypertensive therapy on cardiac sodium/hydrogen ion exchanger activity and hypertrophy in spontaneously hypertensive rats.

BACKGROUND: Sodium/hydrogen ion exchange is hyperactive in hypertension. Myocardial sodium/hydrogen ion exchange hyperactivity accompanies the regression of cardiac hypertrophy in spontaneously hypertensive rats (SHR) after long term control of blood pressure with enalapril. OBJECTIVES: To explore whether this effect is shared by other antihypertensive agents or is specific to angiotensin-converting enzyme inhibition. ANIMALS AND METHODS: SHR and normotensive Wistar Kyoto (WKY) rats were treated for five weeks with enalapril (20 mg/kg/day), nifedipine (10 mg/kg/day) or losartan (40 mg/kg/day). Sodium/hydrogen ion exchange activity was estimated in terms of both steady intracellular pH in HEPES buffer and the rate of intracellular pH recovery from intracellular acid loads in isolated superfused 2'-7'-bis(2-carboxyethyl)-5,-(and-6)-carboxyfluorescein, acetoxymethyl ester form-loaded papillary muscles. RESULTS: Enalapril, nifedipine and losartan decreased systolic blood pressure in SHR to about the same value (140 3, 140 2 and 146 3 mmHg, respectively, at the end the treatment). However, the index of cardiac hypertrophy (heart weight to body weight ratio) was decreased to a smaller value with losartan than with nifedipine or enalapril (2.66 0.09, 3.06 0.05 and 2.86 0.04 mg/g respectively; P<0.05 ANOVA). For the untreated SHR, the index of cardiac hypertrophy was 3.30 0.04 mg/g. Myocardial sodium/hydrogen ion exchange hyperactivity in SHR was normalized by all treatments. CONCLUSIONS: The three treatments regressed cardiac hypertrophy and normalized sodium/hydrogen ion exchange exchange activity in SHR, and losartan was the most effective treatment for reversing cardiac hypertrophy, despite producing effects on blood pressure and sodium/hydrogen exchange activity similar to that of other antihypertensive drugs.

Effect of caffeic acid on apical transporters' dysfunction of renal proximal tubule cells under oxidative stress in vitro.

The protective effect of caffeic acid (CA) against oxidative stress-induced inhibition of proximal tubule apical transporter was investigated. In the present study, 10 (-4) M H2O2 did not affect cell viability regardless of incubation time. However, it decreased apical transporters' activity such as Na (+)/glucose cotransporter, Na (+)/Pi cotransporter, and Na (+)/H(+) antiporter in the proximal tubule cells. CA (>10(-6) M) prevented H2O2-induced inhibition of apical transporters. Thus, we investigated its action mechanism. CA also prevented H2O2-induced lipid peroxides formation, arachidonic acid (AA) release, and Ca(2+) uptake. In conclusion, CA, in part, prevented H2O2-induced inhibition of apical transporter activity via decrease of AA release and Ca(2+) uptake in primary cultured renal proximal tubule cells.

Profiling of drugs for membrane activity using liposomes as an in vitro model system.

The increasing size of chemical libraries being analyzed by high-throughput screening results in a growing number of active compounds that need to be assessed before moving forward in the drug development process. As a consequence, more rapid and highly sensitive strategies are required to accelerate the process of drug discovery without increasing the cost. Due to the fact that significant numbers of compounds from combinatorial libraries are hydrophobic in nature, approaches are needed to evaluate the potentialfor these compounds to interfere with the functions of biological membranes. The liposome system was used to detect agents that act as follows: (i) ionophores able to induce specific ion permeability, e.g., valinomycin for K+ and protonophoric uncouplers for H+; (ii) ion antiporters which exchange H+ for other ions, e.g., nigericin; (iii) agents that form low specificity ion channels in the membrane, e.g., gramicidin; and (iv) detergents and other membrane-disrupting agents. We propose using this liposome assay during the drug development process to identify compounds that have membrane activity and, as a consequence, produce a biological effect by altering the physico-chemical properties of the cell membrane rather than interacting with a protein target. Screening of a representative set of biologically-active compounds (198) indicated that the majority of systemic antimicrobial drugs, but not topical drugs, lack membrane activity in this model system.

Na+/Li+ and Na+/H+ countertransport activity in hypertensive non-insulin-dependent diabetic patients: role of insulin resistance and antihypertensive treatment.

We measured erythrocyte Na+/Li+ and Na+/H+ countertransport (CT) activity (millimoles per liter per cell per hour) in 10 healthy control subjects (age, 38 +/- 4 years; body mass index, 25 +/- 1 kg/m2) and in 25 hypertensive patients with non-insulin-dependent diabetes mellitus ([NIDDM] age, 49 +/- 3 years; body mass index, 29 +/- 1 kg/m2; fasting plasma glucose, 157 +/- 12 mg/dL) 4 weeks after discontinuation of previous antihypertensive treatment. Na+/Li+ CT was significantly increased in hypertensive NIDDM patients compared with controls (0.56 +/- 0.04 v 0.30 +/- 0.03, P < .01), whereas Na+/H+ CT was similar to control levels (21 +/- 1 v 20 +/- 2). A positive correlation was found between Na+/Li+ CT and the severity of insulin resistance (r = .69, P < .01), mean arterial pressure ([MAP] r = .64, P < .01), plasma triglyceride concentration (r = .46, P < .05), and plasma total cholesterol (r = .41, P < .05). An inverse correlation was found between Na+/Li+ CT activity and plasma insulin concentration (r = -.47, P < .05). No relationship was observed between Na+/Li+ CT activity and either creatinine clearance or proteinuria. Stepwise multiple regression analysis for all metabolic variables and blood pressure showed that only the severity of insulin resistance was positively correlated with increased Na+/Li+ CT activity. Na+/H+ and Na+/Li+ CT activity were not altered by 3 hours of euglycemic physiologic hyperinsulinemia (84 +/- 3 microU/mL). Hypertensive NIDDM subjects were treated for 3 months with captopril, nifedipine, or doxazosin. After captopril, a reduction of Na+/H+ CT was observed (22 +/- 4 v 13 +/- 2, P < .05); Na+/Li+ CT decreased after doxazosin (0.56 +/- 0.06 v 0.45 +/- 0.05, P < .05) and nifedipine (0.52 +/- 0.06 v 0.42 +/- 0.05, P < .05). In conclusion, in hypertensive NIDDM subjects, (1) Na+/Li+ CT is increased and is correlated with the level of insulin resistance and the MAP; (2) acute physiologic hyperinsulinemia does not affect Na+/Li+ or Na+/H+ CT activity; and (3) Na+/H+ CT activity is reduced by captopril, and Na+/Li+ CT is decreased by doxazosin and nifedipine.

The mechanism underlying the cardiotoxic effect of the toxin from the jellyfish Chironex fleckeri.

We have investigated the mechanisms underlying the cardiac effects of the toxin from the box jellyfish Chironex fleckeri. Papillary muscles isolated from the hearts of ferrets and ventricular myocytes isolated from the hearts of ferrets and rats were used. Force, intracellular [Ca2+], and membrane potential were monitored in the papillary muscles; contraction, intracellular [Ca2+], intracellular [Na+], and membrane currents were monitored in the isolated myocytes. Application of the toxin to these preparations resulted in a large increase in intracellular [Ca2+] and the adverse symptoms of Ca2+ overload (aftercontractions, spontaneous contractions, a decrease in developed force, and an increase in resting force). The response of papillary muscles to the toxin was not inhibited by blockers of Ca2+ or Na+ channels or by inhibitors of the sarcoplasmic reticulum, Na+/K+ ATPase, or Na+/H+ exchange. The response to the toxin was, however, blocked by prior exposure to a solution which contained no Na+ and by Ni2+. In the isolated myocytes, as well as an increase in intracellular [Ca2+], the toxin also caused an increase in intracellular [Na+] and the appearance of a current which was inward at negative potentials and reversed at about -10 mV. These data can be explained by the toxin increasing Na+ influx into the cell. The increase in intracellular [Na+] will then increase intracellular [Ca2+] via the Na+/Ca2+ exchange mechanism, thus producing the observed Ca2+ overload.

Methyl isobutyl amiloride delays normalization of brain intracellular pH after cardiac arrest in rats.

OBJECTIVE: The sodium/hydrogen ion (Na+/H+) antiporter system of brain cells is responsible for reducing intracellular acid loads and regulating cellular volume. Activation of this system during reperfusion following cardiac arrest may contribute to cerebral edema and subsequent brain damage. Therefore, we wished to determine whether administration of methyl isobutyl amiloride, a known inhibitor of the Na+/H+ antiporter system, would cross the blood brain barrier and delay the return of brain intracellular pH to normal values during reperfusion after cardiac arrest in rats. DESIGN: a) Prospective sequential evaluation of the regional brain blood flow and 3H-methyl isobutyl amiloride extraction fraction in rats; b) prospective sequential evaluation of brain intracellular pH in rats treated with methyl isobutyl amiloride compared with untreated control rats. SETTING: A research laboratory. SUBJECTS: Thirteen male Wistar rats: a) three rats to study regional brain blood flow and 3H-methyl isobutyl amiloride cerebral extraction; and b) ten rats to study the effect of methyl isobutyl amiloride on brain intracellular pH after cardiac arrest and reperfusion. INTERVENTIONS: a) Rats were injected with 14C iodoantipyrine and 3H-methyl isobutyl amiloride, and their brains were subsequently analyzed to determine regional cerebral blood flow and percent of cerebral extraction of methyl isobutyl amiloride. b) Cardiac arrest was induced with potassium chloride followed by resuscitation 7 mins later in untreated control rats and rats treated with methyl isobutyl amiloride. MEASUREMENTS AND MAIN RESULTS: a) Regional cerebral blood flow (mL/100 g/min) determined with 14C iodoantipyrine and percent of cerebral extraction of 3H-methyl isobutyl amiloride were evaluated in various regions of the brain. Mean +/- SD values were 167 +/- 15 and 7 +/- 1 for the frontal cerebral cortex; 159 +/- 10 and 7 +/- 2 for the parietal cerebral cortex, 130 +/- 17 and 8 +/- 1 for the hippocampus, 154 +/- 33 and 13 +/- 4 for the cerebellum and 166 +/- 27 and 6 +/- 1 for the striatum (mL/100 g/min). These values were determined by a dual label indicator fractionation method. b) Brain intracellular pH was measured by neutral red histophotometry after 15 mins of reperfusion following cardiac arrest. As compared with untreated control rats, methyl isobutyl amiloride-treated animals had significantly lower brain intracellular pH values after 15 mins of reperfusion. Mean +/- SD pH values were 6.78 +/- 0.18 for the rats treated with methyl isobutyl amiloride vs. normal intracellular pH of 7.11 +/- 0.07 for the untreated control rats. CONCLUSIONS: a) Methyl isobutyl amiloride crosses the blood brain barrier of rats. b) The Na+/H+ antiporter system is operative during reperfusion after cardiac arrest in rats.