Rapid computerized analysis of Na+/H+ exchange flux

The regulation of intracellular pH (pHi) is mediated by membrane transporters whose activity is directly controlled by pHi. Therefore, transport rates must be compared at identical pHi values in functional studies of these transporters. This is conventionally performed using scatter plots showing initial rates of proton flux versus intracellular pH. We present justification for determining proton flux over a wide range of pHi, by digitally smoothing a pHi trace and then directly taking the first derivative versus time of smoothed data. Compared to conventional least-squares analysis of initial rates, the derivative method generates much more information per experiment. Compared to other methods which fit pHi traces to a defined equation prior to rate calculation, the new method does not require that the pHi trace be well fit by any given mathematical function. The derivative technique is illustrated in an analysis of Na+/H+ exchange in Caco-2 cells. Intracellular pH is measured fluorometrically in cells loaded with BCECF (2',7'-bis[2-carboxyethyl]-5-(and-6)carboxyfluorescein). To validate the analysis of Na+/H+ exchange over an extended time range, we demonstrate that cellular acidification with NH4Cl does not change steady state Na+ content. We find that proton flux rates analyzed by the derivative method are equivalent to initial rates measured by least-squares analysis.     

Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance

There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10(-10) mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2+. Omission of extracellular Ca2+ also prevented aldosterone-induced alkalinization. Instead, aldosterone led to a Zn(2+)-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel acitivity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion.     

A novel antagonist, No. 7943, of the Na+/Ca2+ exchange current in guinea-pig cardiac ventricular cells

1. The effects of No. 7943 on the Na+/Ca2+ exchange current and on other membrane currents were investigated in single cardiac ventricular cells of guinea-pig with the whole-cell voltage-clamp technique. 2. No. 7943 at 0.1-10 microM suppressed the outward Na+/Ca2+ exchange current in a concentration-dependent manner. The suppression was reversible and the IC50 value was approximately 0.32 microM. 3. No. 7943 at 5-50 microM suppressed also the inward Na+/Ca2+ exchange current in a concentration-dependent manner but with a higher IC50 value of approximately 17 microM. 4. In a concentration-response curve, No. 7943 raised the K(m)Ca2+ value, but did not affect the Imax value, indicating that No. 7943 is a competitive antagonist with external Ca2+ for the outward Na+/ Ca2+ exchange current. 5. The voltage-gated Na+ current, Ca2+ current and the inward rectifier K+ current were also inhibited by No. 7943 with IC50S of approximately 14, 8 and 7 microM, respectively. 6. In contrast to No. 7943, 3', 4'-dichlorobenzamil (DCB) at 3-30 microM suppressed the inward Na+/Ca2+ exchange current with IC50 of 17 microM, but did not affect the outward exchange current at these concentrations. 7. We conclude that No. 7943 inhibits the outward Na+/Ca2+ exchange current more potently than any other currents as a competitive inhibitor with external Ca2+. This effect is in contrast to DCB which preferentially inhibits the inward rather than the outward Na+/Ca2+ exchange current.     

Hoe 694, a new Na+/H+ exchange inhibitor and its effects in cardiac ischaemia

1. The benzoylguanidine derivative Hoe 694 ((3-methylsulphonyl-4- piperidino-benzoyl) guanidine methanesulphonate) was characterized as an inhibitor of Na+/H+ exchange in rabbit erythrocytes, rat platelets and bovine endothelial cells. The potency of the compound was slightly lower or comparable to ethylisopropyl amiloride (EIPA). 2. To investigate a possible cardioprotective role of the Na+/H+ exchange inhibitor Hoe 694, rat isolated working hearts were subjected to ischaemia and reperfusion. In these experiments all untreated hearts suffered ventricular fibrillation on reperfusion. Addition of 10(-7) M Hoe 694 to the perfusate almost abolished reperfusion arrhythmias in the rat isolated working hearts. 3. Hoe 694 reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK), which are indicators of cellular damage during ischaemia, into the venous effluent of the hearts by 60% and 54%, respectively. 4. The tissue content of glycogen at the end of the experiments was increased by 60% and the high energy phosphates ATP and creatine phosphate were increased by 240% and 270% respectively in the treated hearts as compared to control hearts. 5. Antiischaemic effects of the Na+/H+ exchange inhibitor, Hoe 694, were investigated in a second experiment in anaesthetized rats undergoing coronary artery ligation. In these animals, pretreatment with Hoe 694 caused a dose-dependent reduction of ventricular premature beats and ventricular tachycardia as well as a complete suppression of ventricular fibrillation down to doses of 0.1 mg kg-1, i.v. Blood pressure and heart rate remained unchanged. 6. We conclude that the new Na+/H+ exchange inhibitor, Hoe 694, shows cardioprotective and antiarrhythmic effects in ischaemia and reperfusion in rat isolated hearts and in anaesthetized rats. In view of the role which Na+/H+ exchange seems to play in the pathophysiology of cardiac ischaemia these effects could probably be attributed to Na+/H+ exchange inhibition.     

Comparative molecular analysis of Na+/H+ exchangers: a unified model for Na+/H+ antiport?

There is increasing evidence to suggest that free radical generation is central to a variety of pathological processes, including drug toxicity. Studies demonstrating the ability of gentamicin to facilitate the generation of radical species suggest that this process plays an important role in aminoglycoside-induced ototoxicity. Because transition metals, particularly iron, play an important role in the production of free radicals and the generation of reactive oxygen species, we sought to determine whether gentamicin-induced ototoxicity is exacerbated by increases in serum iron levels. To this end, we assessed the effects of supplemental iron administration (2 mg/kg/day and 6 mg/kg/day) on changes in auditory function induced by co-administration of gentamicin (100 mg/kg/day for 30 days). Experiments were carried out on pigmented guinea pigs initially weighing 250-300 g. Changes in cochlear function were characterized as shifts in compound action potential (CAP) thresholds, estimated every third day throughout the treatment period by use of chronic indwelling electrodes implanted at the round window, vertex, and contralateral mastoid. Results showed that animals receiving iron in combination with gentamicin demonstrated a more rapid and profound elevation in CAP thresholds compared with animals receiving gentamicin alone. This effect occurred in a dose-dependent manner. Animals receiving supplemental iron alone maintained normal CAP thresholds throughout the treatment period. There was no statistically significant difference in serum gentamicin levels between groups receiving gentamicin alone or gentamicin plus iron. These results provide further evidence of the recently reported intrinsic role of iron in aminoglycoside ototoxicity, and highlight a potential risk of aminoglycoside administration in patients with elevated serum iron.     

Identification of a mitochondrial Na+/H+ exchanger

The electroneutral exchange of protons for Na+ and K+ across the mitochondrial inner membrane contributes to organellar volume and Ca2+ homeostasis. The molecular nature of these transporters remains unknown. In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na+/H+ exchanger gene family. Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4',6-diamidino-2-phenylindole staining of mitochondrial DNA. To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22Na+ uptake into mitochondria was abolished in the mutant strain. It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism. Sequence comparisons revealed that Nha2 has highest identity to a putative Na+/H+ exchanger homologue (KIAA0267; renamed NHE6) in humans. Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart. Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells. These data indicate that NHA2 and NHE6 encode homologous Na+/H+ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively.     

Interaction of a thrombin inhibitor and a platelet GP IIb/IIIa antagonist in vivo: evidence that thrombin mediates platelet aggregation and subsequent thromboxane A2 formation during coronary thrombolysis

We examined the effect of a specific thrombin inhibitor, Ro 46-6240, alone and combined with an antagonist of the platelet GP IIb/IIIa, Ro44-9883, on the response to tissue-type plasminogen activator in a canine model of thrombolysis. Platelet activity was determined by measuring the excretion of 2,3-dinorthromboxane (TX)B2, an enzymatic metabolite of TXA2. Ro 46-6240 administered before tissue-type plasminogen activator induced a dose-dependent prolongation of the activated partial thromboplastin time and prothrombin time. The time to reperfusion decreased dose-dependently (P < .01) to 10 +/- 6 min vs. 52 +/- 5 min in controls. Ro 46-6240 also prevented reocclusion, which occurred in every case in control experiments. Urinary excretion of 2,3-dinor-TXB2 increased from 3 +/- 1 to 37 +/- 9 ng/mg creatinine in controls after reperfusion. This increase was reduced in a dose-dependent fashion by Ro 46-6240, such that at the highest dose, urinary 2,3-dinor-TXB2 after reperfusion was 5.6 +/- 1 ng/mg creatinine. Similar functional and biochemical effects were seen when a subthreshold dose of Ro 46-6240 was combined with Ro 44-9883. At the dose used, Ro 44-9883 alone abolished platelet aggregation ex vivo but failed to modify the response to tissue-type plasminogen activator or the excretion of 2,3-dinor-TXB2 after reperfusion (51 +/- 6 ng/mg creatinine, n = 3). However, the combination of Ro 44-9883 and Ro 46-6240 reduced the time to reperfusion (40 +/- 8 vs. 68 +/- 15 min; n = 7, P < .05), prevented reocclusion and abolished the rise in urinary 2,3-dinor-TXB2 (5 +/- 1 ng/mg creatinine, n = 4). These findings suggest that thrombin mediates platelet activation during coronary thrombolysis. The increased platelet activity results in platelet aggregation and a subsequent increase in TXA2 formation.     

Hydrogen peroxide decreases pHi in human aortic endothelial cells by inhibiting Na+/H+ exchange

Postischemic endothelial dysfunction may occur as a result of the effects of endogenous oxidants like hydrogen peroxide. Since endothelium-dependent vasodilator function may be affected by pHi, the effect of hydrogen peroxide on endothelial pHi was examined. Hydrogen peroxide (100 micromol/L for 10 minutes) decreased pHi from 7.24+/-0.01 to 7.02+/-0.02 and inhibited recovery from an ammonium chloride-induced intracellular acid load in carboxy SNARF 1 (c-SNARF 1)-loaded human aortic endothelial cells in bicarbonate-free solution. Prior inhibition of Na+/H+ exchange with 5-(N-ethyl-N-isopropyl)amiloride (10 micromol/L), by removal of extracellular Na+, or by glycolytic inhibition with iodoacetic acid blocked the subsequent effect of hydrogen peroxide on pHi. A 2-minute exposure to 100 micromol/L H2O2 decreased intracellular ATP levels by approximately 40%; this was prevented by 3-aminobenzamide and nicotinamide (1 mmol/L each), inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase. Both 3-aminobenzamide and nicotinamide significantly inhibited the hydrogen peroxide-induced intracellular acidification and the effect of hydrogen peroxide on recovery from an intracellular acid load. Hydrogen peroxide decreases pHi in human endothelial cells by inhibiting Na+/H+ exchange. This appears to be mediated by activation of the DNA repair enzyme poly(ADP-ribose) polymerase and subsequent depletion of intracellular ATP. Since a decrease in pHi in this range may alter the activity of NO synthase or affect the synthesis of vasodilator prostaglandins, the effect of hydrogen peroxide on the endothelial Na+/H+ exchanger may be important in the pathogenesis of postischemic endothelial dysfunction.     

Moxonidine inhibits Na+/H+ exchange in proximal tubule cells and cortical collecting duct

The lateral arm free flap can be harvested as a fascial flap or fasciocutaneous flap. In this report we describe the use of the lateral arm fascial flap for degloving injuries of the fingers and for skin loss on the dorsum of the hand with exposure of tendons and bones. Concomitant reconstruction of a missing phalanx with a portion of the distal humerus is also described. The use of the fascial flap allows a large area of tissue to be harvested, and still, the donor site can be closed primarily. The fascia is thin and pliable and so conforms well to the contour of the fingers. Its bulk does not interfere with finger motion, and its undersurface creates a gliding surface for tendons. Complications in the reported cases were negligible.     

Effects of suramin on human platelet aggregation and Ca2+ mobilization induced by thrombin and other agonists

The purpose of this study was to investigate the effect of suramin, a polyanionic napthalene sulfonic acid, on human platelet aggregation and Ca2+ mobilization induced by various agonists. Our results show that suramin completely inhibited aggregation by thrombin, platelet activating factor (PAF), alkyllysophosphatidic acid (ALPA), or arachidonic acid in a concentration-dependent manner. The IC50 values of suramin for inhibition of aggregation by PAF, arachidonic acid, and thrombin were 76.7, 239, and 1.49 microg/ml, respectively. Ca2+ mobilization induced by thrombin was inhibited by suramin with an approximate IC50 value of 20 microg/ml. This concentration of suramin had no effect on PAF or oleic acid-induced Ca2+ mobilization. The mechanism by which suramin inhibits aggregation is not clear, but our results suggest that suramin inhibits the ligand-receptor interaction.     

Intracellular pH regulation in bovine aortic endothelial cells: evidence of both Na+/H+ exchange and Na+-dependent Cl-/HCO3- exchange

Regulation of intracellular pH (pHi) was studied in cultured bovine aortic endothelial cells, an important cell system for cardiovascular research. Suspended cells were acidified by the NH4Cl prepulse technique as well as by exposure to CO2/HCO3-. Subsequent rates of pHi recovery were monitored using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF). In HCO3(-)-free solutions, an EIPA-sensitive, Na+-dependent mechanism fully accounted for realkalinization, namely the Na+/H+ exchanger (NHE). In the presence of HCO3-, an additional acid efflux mechanism was found. This one was dependent on Na+ and intracellular Cl-, EIPA-insensitive but DIDS-sensitive, and therefore represented a Na+-dependent Cl-/HCO3- exchanger (NCBE). In summary, two acid-extruding mechanisms were identified in bovine aortic endothelial cells: NHE and NCBE.     

Activation of Ca2+-sensitive Cl- current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

We investigated how Ca2+-sensitive transient outward current, Ito(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na+ (Na+i) using the whole-cell patch-clamp technique at 36 degreesC. In cells dialysed with Na+-free solutions, the application of nicardipine (5 microM) to block L-type Ca2+ current (ICa) completely inhibited Ito(Ca). In cells dialysed with a [Na+]i>/=5 mM, however, Ito(Ca) could be observed after blockade of ICa, indicating the activity of an ICa-independent component. The amplitude of ICa-independent Ito(Ca) increased with voltage in a [Na+]i-dependent manner. The block of Ca2+ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked ICa-independent Ito(Ca). In Ca2+-free bath solution Ito(Ca) was completely abolished. The application of 2 mM Ni2+ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na+/Ca2+ exchange, or perfusion with pipette solution containing XIP (10 microM), a selective blocker of the exchanger, blocked ICa-independent Ito(Ca). From these results we conclude that, in the presence of Na+i, Ito(Ca) can be activated via Ca2+-induced Ca2+ release triggered by Na+/Ca2+ exchange operating in the reverse mode after blockade of ICa.     

Role of Na+/H+ exchange in the recovery of contractility during hypercapnia in cat papillary muscles

Cholesterol reduction reduces ischaemic cardiovascular morbidity and mortality in the asymptomatic healthy population as well as in those with known coronary artery disease. Angiographic studies have also demonstrated regression of atherosclerotic plaques as well as retardation of new atheroma formation with such therapy. Yet, there is a consistent inability to reduce overall mortality in cholesterol-lowering drug trials. An excess of suicide, homicide and violence has been attributed to cholesterol reduction interfering with membrane lipids and receptors, leading to aggressive behaviour. The risk and benefits of cholesterol reduction must thus be weighed in the individual patient; it is more useful in those with known coronary artery disease who are at high risk of subsequent ischaemic cardiovascular events.     

Glucose metabolism, H+ production and Na+/H+-exchanger mRNA levels in ischemic hearts from diabetic rats

CD105 (endoglin) is a receptor for transforming growth factor beta (TGFbeta). Although methods to measure soluble forms of TGFbeta and CD105 have been published, no assay is available to quantify the receptor-ligand complexes. We describe both an indirect enzyme-linked immunosorbent assay for the quantitation of soluble CD105-TGFbeta1 and the characterization of the complexes by immunoprecipitation and immunoblotting. Mab E9, specifically reactive with CD105, was utilised as the capture reagent in the ELISA system. Detection of complexes was achieved using chicken antibody against TGFbeta1 and the subsequent detection of bound antibody demonstrated by the addition of anti-species antiserum conjugated to horseradish peroxidase (HRP). By using enhanced chemiluminescence and optimised antibodies, the assay was made sufficiently sensitive and reproducible to detect low levels of circulating complexes. Whether the assay had any practical applications was evaluated in breast cancer patients. Plasma levels of CD105-TGFbeta1 were significantly elevated in 59 patients with breast cancer compared to 52 age matched normal women (p < 0.001). Immunoprecipitation using a rabbit anti-CD105 antibody, which reacts with both dimeric and monomeric CD105, and immunoblotting showed that three molecular forms of CD105-TGFbeta1 complexes > 200, 195, and 125 kDa existed in the plasma. We believe these represent the oligomer, dimer and probably the protease degraded form of CD105 complexed to TGFbeta1. The resistance to hypertonic solution, SDS and heat treatment suggested that the soluble CD105-TGFbeta1 complex may be linked by covalent bonds. The measurement of CD105-TGFbeta complexes in the circulation may have important clinical applications not only in cancer but also in patients with other angiogenic diseases such as rheumatoid arthritis, myocardial infarction and stroke.     

Inhibition of platelet aggregation by acetylsalicylic acid and other inhibitors

Effects on platelet aggregation were examined of acetylsalicylic acid (ASA), indomethacin and a number of other agents including dipyridamole, phenylbutazone and sulfinpyrazone under standardized conditions. The Born turbidometric method of measuring platelet aggregation was used with collagen as the stimulus for aggregation. ASA and indomethacin were shown to be among the most potent inhibitors of aggregation, being active at minimal effective concentrations of 1-3 mug/ml using a 10 min time of pre-incubation with the platelet-rich plasma (degree of aggregation inhibition was time dependent). Most of the other agents tested were also active in vitro and both prostaglandin E1 and adenosine were more potent than ASA or indomethacin. However, these agents were shown not to exert significant inhibitory effects when administered orally to rats (dose 10 and 30 mg/kg). ASA proved to be effective in doses as low as 3 mg/kg, and indomethacin in doses as low as 1 mg/kg orally. The inhibitory effects of ASA on aggregation remained for several days after a single oral dose, whereas the effects of indomethacin disappeared within 24 h.