Sodium-hydrogen Exchange Inhibition Research Articles

Cardioprotection with sodium-hydrogen exchange inhibition: An archetype of "Bench to bedside"

Cardioprotection with sodium-hydrogen exchange inhibition: An archetype of "Bench to bedside"

Hydrogen peroxide induced impairment of post-ischemic ventricular function is prevented by the sodium-hydrogen exchange inhibitor HOE 642 (cariporide).

Sodium-hydrogen exchange (NHE) activation is a major mechanism of cardiac injury produced by ischemia and reperfusion. In addition, NHE may mediate the direct effects of hydrogen peroxide (H2O2) in normally perfused hearts. The present study was done to determine whether H2O2 at low concentrations producing mild myocardial depression affects post-ischemic recovery of function and to determine the ability of the NHE inhibitor HOE 642 to modulate this effect. Isolated Langendorff-perfused rat hearts with a left ventricular balloon inflated to an initial end-diastolic pressure of 5 mmHg were subjected to 90 min of global zero-flow ischemia followed by 60 min reperfusion. In Study 1, hearts were randomized for perfusion with or without H2O2 (20 microM) for 15 min before ischemia and throughout reperfusion. In Study 2, identical experiments were done except that the hearts were pretreated with the NHE inhibitor HOE 642 (5 microM). Function was assessed by determining intraventricular pressures. Recovery of developed pressure in Study 1 after 10 min reperfusion was 60.3 +/- 8% of pre-ischemic values in control hearts whereas this was reduced to 29.9 +/- 10% in hearts treated with H2O2 (P < 0.05). After 60 min of reperfusion recovery of developed pressure was 80.3 +/- 5.2% and 60.7 +/- 7% in control and H2O2-treated hearts, respectively (P < 0.05). Recovery of rates of pressure development (+dP/dt) and relaxation (-dP/dt) paralleled the effects seen with developed pressure. Moreover, these effects were associated with significantly elevated end-diastolic pressure during the last 20 min of reperfusion. In Study 2, HOE 642 completely prevented the deleterious effect of H2O2, both with respect to ventricular recovery and to the elevation in end-diastolic pressure during reperfusion. Our results show that very low concentrations of H2O2 significantly impair recovery of function in this rat model of myocardial ischemia-reperfusion. Moreover, our results suggest that this effect is likely dependent on NHE activity and can be prevented by treatment with the NHE inhibitor HOE 642.

Interaction between anesthetics and the sodium-hydrogen exchange inhibitor HOE 642 (cariporide) in ischemic and reperfused rat hearts.

Sodium (Na+)-hydrogen (H+) exchange (NHE) inhibitors are effective cardioprotective agents. The potent NHE inhibitor HOE 642 (cariporide) is being evaluated clinically in high-risk patients, including those having coronary artery bypass. Volatile anesthetics are also cardioprotective, most likely via different mechanisms. The potential interaction between anesthetics and HOE 642 was investigated. Electrically paced isolated rat hearts were perfused at constant flow. Left ventricular developed pressure and end-diastolic pressure were monitored as determinants of function. Hearts were subjected to 60 min each of total ischemia followed by reperfusion. Isoflurane (0.93 minimum alveolar concentration [MAC]), sevoflurane (1.03 MAC), or sufentanil (1.2 nM) was added 15 min before ischemia and throughout reperfusion, either alone or in combination with HOE 642 (5 microM). The effect of HOE 642 alone was also studied. At the end of reperfusion, hearts were freeze-clamped for subsequent determination of tissue metabolites. In control hearts, left ventricular developed pressure recovered to 40% of preischemia values, whereas left ventricular end-diastolic pressure increased by 650% after reperfusion. Sevoflurane, isoflurane, or HOE 642 alone significantly enhanced left ventricular developed pressure recovery to more than 90%, although recovery with HOE 642 was more rapid and accompanied by significantly reduced left ventricular end-diastolic pressure. HOE 642 plus volatile anesthetics produced additive effects, with left ventricular developed pressure recovering by more that 100%, although left ventricular end-diastolic pressure was not further reduced. Sufentanil had no effect in terms of developed pressure, but protection with HOE 642 was maintained. HOE 642 with or without volatile anesthetics also preserved adenosine triphosphate levels. Isoflurane, sevoflurane, and HOE 642 enhance ventricular recovery, but the effect of HOE 642 is also associated with reduced contracture and adenosine triphosphate preservation. A combination of the NHE inhibitor and either volatile agent confers additive and superior protection, which could be relevant for the establishment of ideal cardioprotective strategies during surgery.

A Rapid Ischemia-induced Apoptosis in Isolated Rat Hearts and its Attenuation by the Sodium–Hydrogen Exchange Inhibitor HOE 642 (Cariporide)

Apoptosis is a potentially important myocardial response to pathology including ischemia and reperfusion. Na–H exchange (NHE) represents an important mechanism for mediating such injury. The present study was done to determine if NHE inhibition can affect early apoptosis in an acute model of ischemia and reperfusion. Isolated rat hearts were subjected to zero-flow ischemia for various durations with or without subsequent 30 min of reperfusion. Nick-end-labelling of biotin-dUTP (TUNEL staining), as well as DNA extraction followed by agarose gel electrophoresis, were used to semiquantify apoptotic cells and identify DNA laddering, respectively. Apoptosis first appeared after 10 min of ischemia and reached a maximum level after 30 min. The number of apoptotic cells after 30 min of ischemia was 31 ± 3 per 100 high power microscopic fields, whereas in reperfused hearts the number of cells was 34 ± 3. To determine the effect of NHE inhibition, hearts were pretreated 15 min prior to ischemia with HOE 642, a potent and specific inhibitor of the isoform (NHE-1) found in myocardium. HOE 642 significantly reduced the number of apoptotic cells in the ischemic and reperfused heart to 2 ± 1 and 6 ± 1, respectively (P<0.05 from untreated hearts). DNA laddering was not observed with electrophoretic DNA analysis, likely owing to the small number of apoptotic cells involved. Hearts recovered nearly 100% of function in both groups, although there was a significantly higher recovery after 1 and 2 min of reperfusion in those hearts treated with HOE 642. Our study shows that apoptosis, albeit very mild in nature, can be rapidly induced in isolated hearts by a relatively brief period of ischemia without reperfusion, which can be markedly attenuated by the NHE inhibitor HOE 642. The ability of HOE 642 to markedly attenuate apoptosis may be important in terms of understanding the drug's cardioprotective properties as well as the overall role of NHE in heart disease.

Effect of sodium-hydrogen exchange inhibition on functional and metabolic impairment produced by oxidative stress in the isolated rat heart

Sodium-hydrogen exchange (NHE) represents an important process mediating myocardial ischemic and reperfusion injury, and NHE inhibitors have been shown to be effective cardioprotective agents against this form of injury. The precise mechanisms by which NHE inhibition protect the heart are not known and we therefore postulated that attenuation of oxidative stress could contribute to such protection. Accordingly, we examined whether the potent and specific NHE inhibitor 4-isopropyl-3-methylsulphonylbenzoyl-guanidine methanesulphonate (HOE 642, 5 microM) can protect isolated rat hearts against mechanical and biochemical impairment produced by either hydrogen peroxide (150 or 200 microM) or a free radical generating system consisting of purine (4.6 or 9.2 mM) and xanthine oxidase (20 or 40 U/L). HOE 642 significantly delayed and attenuated both the depression in left ventricular developed pressure (LVDP) as well as the elevation in left ventricular end-diastolic pressure (LVEDP) produced by both concentrations of hydrogen peroxide, although greater protection was generally seen against the lower hydrogen peroxide concentration, particularly with respect to LVEDP. Hydrogen peroxide, at both concentrations, significantly reduced high energy phosphate and glycogen contents and elevated lactate levels, all of which were significantly attenuated by HOE 642. In contrast, HOE 642 had no effect on functional impairment produced by either concentration of the free radical generating system. At its lower concentration, the combination of purine plus xanthine oxidase had no effect on energy metabolites, although a significant reduction in high energy phosphate stores was seen with the higher concentration. However, this was unaffected by HOE 642. The protective effect of HOE 642 was mimicked by another NHE inhibitor, methylisobutylamiloride (MIA, 5 microM). Our study therefore shows that NHE inhibition selectively protects against functional and metabolic impairment produced by hydrogen peroxide. Since hydrogen peroxide formation has been implicated in the development of ischemic and reperfusion injury, it is possible that the protective effect of NHE inhibition against this form of oxidative stress may explain in part the basis for the well-established salutary actions of NHE inhibitors in the ischemic and reperfused myocardium. Since HOE 642 failed to modify the response to free radical generators, it is unlikely that the protective effects of NHE inhibitors can be explained by a free radical scavenging mechanism.

Effect of sodium-hydrogen exchange inhibition on functional and metabolic impairment produced by oxidative stress in the isolated rat heart

Sodium-hydrogen exchange (NHE) represents an important process mediating myocardial ischemic and reperfusion injury, and NHE inhibitors have been shown to be effective cardioprotective agents against this form of injury. The precise mechanisms by which NHE inhibition protect the heart are not known and we therefore postulated that attenuation of oxidative stress could contribute to such protection. Accordingly, we examined whether the potent and specific NHE inhibitor 4-isopropyl-3-methylsulphonylbenzoyl-guanidine methanesulphonate (HOE 642, 5 microM) can protect isolated rat hearts against mechanical and biochemical impairment produced by either hydrogen peroxide (150 or 200 microM) or a free radical generating system consisting of purine (4.6 or 9.2 mM) and xanthine oxidase (20 or 40 U/L). HOE 642 significantly delayed and attenuated both the depression in left ventricular developed pressure (LVDP) as well as the elevation in left ventricular end-diastolic pressure (LVEDP) produced by both concentrations of hydrogen peroxide, although greater protection was generally seen against the lower hydrogen peroxide concentration, particularly with respect to LVEDP. Hydrogen peroxide, at both concentrations, significantly reduced high energy phosphate and glycogen contents and elevated lactate levels, all of which were significantly attenuated by HOE 642. In contrast, HOE 642 had no effect on functional impairment produced by either concentration of the free radical generating system. At its lower concentration, the combination of purine plus xanthine oxidase had no effect on energy metabolites, although a significant reduction in high energy phosphate stores was seen with the higher concentration. However, this was unaffected by HOE 642. The protective effect of HOE 642 was mimicked by another NHE inhibitor, methylisobutylamiloride (MIA, 5 microM). Our study therefore shows that NHE inhibition selectively protects against functional and metabolic impairment produced by hydrogen peroxide. Since hydrogen peroxide formation has been implicated in the development of ischemic and reperfusion injury, it is possible that the protective effect of NHE inhibition against this form of oxidative stress may explain in part the basis for the well-established salutary actions of NHE inhibitors in the ischemic and reperfused myocardium. Since HOE 642 failed to modify the response to free radical generators, it is unlikely that the protective effects of NHE inhibitors can be explained by a free radical scavenging mechanism.

Characterisation and uptake of paraquat by rat renal proximal tubular cells in primary culture

1 Uptake of the herbicide paraquat (PQ), by rat proximal tubular cells (PTC) in primary culture grown on a collagen coated support was investigated. 2 The uptake of PQ by PTC was predominantly from the basolateral side. The basolateral uptake of PQ was saturable with time and increasing concentrations, energy dependent and could be inhibited by certain organic cations. Using Michaelis Menten kinetics, the apparent K(m) was 778 +/- 241 microM and Vmax was 0.97 +/- 0.24 pmol/microgram protein/15 min for the basolateral uptake of PQ. Cimetidine (5.7 +/- 0.4 pg/microgram protein/ 30 min, P < 0.001) was the most potent inhibitor of PQ uptake, followed by quinine (6.5 +/- 0.4 pg/microgram protein/30 min, P < 0.01) and then tetraethylammonium (8.2 +/- 0.5 pg/microgram protein/30 min, P < 0.05) when compared with control (11 +/- 1 pg/microgram protein/30 min). N-methylnicotinamide, p-aminohippurate and putrescine did not inhibit the basolateral uptake of PQ. The sodium hydrogen exchange inhibitors, amiloride and its analogue, 5-(N,N hexamethylene) amiloride (HMA) inhibited both the apical and basolateral uptake of PQ. 3 The apical uptake of PQ was not saturable with increasing concentrations and was not inhibited by 2,4-dinitrophenol, but it was reduced by cimetidine (P < 0.01), quinine (P < 0.05) and a sodium potassium ATPase inhibitor, ouabain (P < 0.01). 4 It is concluded that PQ was taken up from the basolateral side of primary cultured rat PTC by an energy dependent transport system.

Editorial: Sodium-hydrogen exchange inhibition—superior cardioprotective strategy

Editorial: Sodium-hydrogen exchange inhibition—superior cardioprotective strategy

Inhibition of sodium-hydrogen exchange reduces infarct size in the isolated rat heart — a protective additive to ischaemic preconditioning

Inhibition of sodium-hydrogen exchange reduces infarct size in the isolated rat heart — a protective additive to ischaemic preconditioning

Inhibition of sodium-hydrogen exchange reduces infarct size in the isolated rat heart -- a protective additive to ischaemic preconditioning

Inhibition of Na+/H+ exchange with amiloride analogues has been shown to protect the ischaemic and reperfused heart. The aim of this study was to examine if preischaemic or postischaemic treatment with the selective Na+/H+ exchange inhibitor ethyl-isopropyl amiloride (EIPA, 1 microM) influenced infarct size in an isolated rat heart model of regional ischaemia and reperfusion, and if any such protection was additive to the protection afforded by ischaemic preconditioning. Langendorff perfused rat hearts were subjected to 30 or 45 min of regional ischaemia and 120 min of reperfusion. The risk zone was determined by fluorescent particles and infarct size was determined by staining with triphenyltetrazolium chloride. Treatment with EIPA for 20 min before 30 min regional ischaemia significantly reduced infarct size (in % of the risk zone) compared to untreated controls [3.1 (SEM 1.0)% v 38.1(5.8)%, P < 0.001], a protection similar to that afforded by ischaemic preconditioning [6.1(2.5)%]. Combination of preischaemic EIPA treatment and ischaemic preconditioning also reduced infarct size [5.2(2.0)%, P < 0.01 v control group]. When EIPA was added to the buffer only during the first 30 min of reperfusion, no protection was observed [infarct size = 37.8(5.8)%, NS v control group]. In order to clarify if the protection observed with EIPA treatment was additive to protection by ischaemic preconditioning, another set of experiments was performed. In these experiments regional ischaemia was extended to 45 min. Preischaemic EIPA treatment reduced infarct size also in this model compared to controls [15.3(2.9)% v 64.3(2.9)%, P < 0.001], as did ischaemic preconditioning [23.5(4.2)%, P < 0.001 v controls, NS v EIPA treated hearts]. Combination of ischaemic preconditioning and preischaemic EIPA treatment further reduced infarct size significantly [3.9(0.6)%, P < 0.05 v all other groups with 45 min regional ischaemia]. Inhibition of Na+/H+ exchange reduces infarct size in the isolated rat heart infarct model if the exchanger is inhibited during the ischaemic period, and this protection is additive to the protection afforded by ischaemic preconditioning.

Clinical and in vivo antiarrhythmic potential of sodium-hydrogen exchange inhibitors

Intracellular acidification may be the initial stimulus for a cascade of events contributing to intracellular calcium overload. Ischaemia results in the accumulation of lactate and other proton donors causing intracellular acidification. During reperfusion the activity of the Na+/H+ exchanger recovers, allowing extrusion of protons at the expense of increases in intracellular sodium. The rise in intracellular sodium decreases the gradient required for sodium calcium exchange, resulting in accumulation of intracellular calcium. These data are in keeping with the pathophysiological model that the Na+/H+ exchanger is an important part of a cascade leading from intracellular acidosis to intracellular sodium loading followed by calcium overload. From this model we predicted that amiloride would be antiarrhythmic. In 1988, we reported that low concentration of amiloride (0.1-0.3 microM) suppresses the induction of sustained ventricular tachyarrhythmias in dogs late following infarction. Amiloride suppressed inducible ventricular tachycardia in approximately 50% of the animals. In an extension of this work we assessed the efficacy of amiloride in suppressing inducible ventricular tachycardia in humans who presented with symptomatic ventricular tachycardia. In that study, amiloride manifested antiarrhythmic activity, but not to the degree that was observed in our dog model. Six of 31 patients (19%) had complete suppression of induced ventricular tachycardia. In an extension of this work, we assessed in our in vivo dog model which of the pharmacological effects of amiloride were associated with antiarrhythmic efficacy.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-hydrogen exchange inhibitors improve postischaemic recovery of function in the perfused rabbit heart

Sodium-hydrogen exchange inhibitors improve postischaemic recovery of function in the perfused rabbit heart

Sodium-hydrogen exchange inhibitors improve postischaemic recovery of function in the perfused rabbit heart

The aim was to examine the effects of the Na+/H+ exchange inhibitors amiloride and methylisobutyl amiloride (MIA) in buffer perfused rabbit hearts subjected to one hour of normothermic ischaemia (37 degrees C) followed by reperfusion. Experiments were carried out in five groups of Langendorff perfused rabbit hearts: (1) control, (2) amiloride, and (3) MIA (agents in both the preischaemic and reperfusion perfusate), (4) amiloride-R and (5) MIA-R (agents added at reperfusion only). Functional evaluation included serial measurement of resting tension, force, rates of ventricular force development and relaxation, and coronary perfusion pressure. Samples of coronary effluent were obtained for creatine kinase assay and hearts were freeze clamped for metabolite assays. Reperfusion resulted in a marked increase in resting tension in group (1) which was statistically significant compared to groups (2) and (3). Groups (2) and (3) also showed significantly improved recovery of ventricular force, rate of force development, and rate of ventricular relaxation. Addition of either agent only during reperfusion failed to produce a significant beneficial effect. There were no significant differences among the groups with respect to postreperfusion creatine kinase release or end reperfusion metabolite levels. This study shows for the first time that both of the Na+/H+ exchange inhibitors amiloride and MIA produce improved recovery of ventricular function in rabbit hearts subjected to ischaemia and reperfusion, although the beneficial effect was not obtained with drug administration at the time of reperfusion only.

Clinical and in vivo antiarrhythmic potential of sodium-hydrogen exchange inhibitors

Clinical and in vivo antiarrhythmic potential of sodium-hydrogen exchange inhibitors

Potential of selective sodium-hydrogen exchange inhibitors in cardiovascular therapy

Potential of selective sodium-hydrogen exchange inhibitors in cardiovascular therapy

Potential of selective sodium-hydrogen exchange inhibitors in cardiovascular therapy

Potential of selective sodium-hydrogen exchange inhibitors in cardiovascular therapy

Na +/H + exchange in granulosa cells of the three largest preovulatory follicles of the domestic hen

Sodium-dependent intracellular pH (pH i) regulation was compared in granulosa cells from the three largest avian ovarian follicles by monitoring the pH i with biscarboxyethyl-carboxyfluorescein, a dye whose fluorescence increases with alkalinity. Collagenase-dispersed granulosa cells obtained from the largest (F 1), second largest (F 2), and third largest (F 3) preovulatory follicles about 2–3 hr prior to expected ovulation of F 1 were used in the present study. The resting pH i measured in nominally bicarbonate free buffer with extracellular Na + ( Na o + = 144 m M ) and external pH (pH o) of 7.3 was about 6.8 in cells from F 1, F 2, and F 3. There was no correlation between the stage of follicular development and the pH i whether the follicles were removed in the early or late preovulatory period. After acute cytoplasmic acidification by exposure of cells to nigericin in choline + buffer, or by the abrupt removal of ammonium chloride, complete recovery of pH i occurred in 4–5 min. The rate and magnitude of the recovery were dependent upon the concentration of Na o + and were abolished when Na o + was replaced completely by choline +. Recovery in the presence of Na o + was inhibited dose-dependently by amiloride (sodium-hydrogen exchange inhibitor). There was no difference between the rate and the extent of pH i recovery in acid-loaded cells obtained from F 1, F 2, and F 3. Furthermore, by varying the concentration of Na o + between 0 and 144 m M both young and matured granulosa cells extruded acid at the same rate. In addition, amiloride inhibited the Na o + dependency of pH i recovery to a similar degree in F 1, F 2, and F 3 cells. Our observations demonstrate in avian granulosa cells the existence of a Na o +-dependent , amiloride-sensitive pH i regulatory system that is equally effective in cells obtained from the three largest yolk-filled follicles.

Ionic regulation of intracellular pH in rat calvarial osteoblasts.

1. Intracellular pH of cultured rat calvarial osteoblasts was monitored continuously using the pH-sensitive fluorescent probe bis-carboxyethyl carboxyfluorescein. 2. Recovery from an intracellular acid load brought about by exposure to ammonium chloride was dependent on external sodium and blocked by the sodium-hydrogen exchange inhibitor amiloride (1 mM), indicating the presence of a plasma membrane sodium-hydrogen exchanger. 3. A SITS- (4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid) sensitive alkalinization occurred on the isosmotic replacement of external chloride by gluconate, suggesting the presence of chloride-bicarbonate exchange. 4. The dependence of the rate of sodium-hydrogen exchange on external sodium followed first-order kinetics, but the rate of exchange appeared to be sensitive to intracellular pH. 5. The rate of alkalinization brought about by the isosmotic replacement of chloride was sensitive to external bicarbonate concentration, but independent of external sodium. 6. Sodium-hydrogen exchange appeared to be inhibited and chloride-bicarbonate exchange stimulated by 1-34 parathyroid hormone.

Control of intracellular pH in rat calvarial osteoblasts: coexistence of both chloride-bicarbonate and sodium-hydrogen exchange.

Intracellular pH was monitored continuously in cultured rat calvarial osteoblasts using the pH-sensitive fluorescent dye bis carboxyethyl carboxyfluorescein (BCECF), loaded into the cells as its membrane permeant ester. Recovery from an intracellular acid load generated by exposure to NH4Cl was unaffected by the anion exchange inhibitors 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disuphonic acid (SITS) and 4,4'-diisothiocyanato-stilbene 2,2'-disuphonic acid (DIDS) (100 microM), but blocked by the sodium-hydrogen exchange inhibitor amiloride (1 mM) and dependent on external sodium, suggesting that recovery is brought about by a sodium-hydrogen exchanger in the plasma membrane. The cells do, however, possess a SITS-sensitive chloride-bicarbonate exchanger, because iso-osmotic replacement of chloride by gluconate leads to intracellular alkalinization, that is inhibited by SITS, but independent of external sodium. Parathyroid hormone brings about an intracellular acidification, which may be due to an inhibiton of sodium-hydrogen exchange.