Na+ overload during ischemia and reperfusion in rat hearts: Comparison of the Na+/H+ exchange blockers EIPA,cariporide and eniporide

Intracellular myocardial Na⁺ overload during ischemia is an important cause of reperfusion injury via reversed Na⁺/Ca²⁺ exchange. Prevention of this Na⁺ overload can be accomplished by blocking the different Na⁺ influx routes. In this study the effect of ischemic inhibition of the Na⁺/H⁺ exchanger (NHE) on [Na⁺]_i, pHi and post-ischemic contractile recovery was tested, using three different NHE-blockers: EIPA, cariporide and eniporide. pHi and [Na⁺]_i were measured using simultaneous ³¹P and ²³Na NMR spectroscopy, respectively, in paced (5 Hz) isolated, Langendorff perfused rat hearts while contractility was assessed by an intraventricular balloon. NHE-blockers (3 M) were administered during 5 min prior to 30 min of global ischemia followed by 30 min drug-free reperfusion. NHE blockade markedly reduced ischemic Na⁺ overload; after 30 min of ischemia [Na⁺]_i had increased to 293 ± 26, 212 ± 6, 157 ± 5 and 146 ± 6% of baseline values in untreated and EIPA (p < 0.01 vs. untreated), cariporide (p < 0.01 vs. untreated) and eniporide (p < 0.01 vs. untreated) treated hearts, respectively. Ischemic acidosis did not differ significantly between groups. During reperfusion, however, recovery of pHi was significantly delayed in treated hearts. The rate pressure product recovered to 12.0 ± 1.9, 12.1 ± 2.1, 19.5 ± 2.8 and 20.4 ± 2.5 × 103 mmHg/min in untreated and EIPA, cariporide (p < 0.01 vs. untreated) and eniporide (p < 0.01 vs. untreated) treated hearts, respectively. In conclusion, blocking the NHE reduced ischemic Na⁺ overload and improved post-ischemic contractile recovery. EIPA, however, was less effective and exhibited more side effects than cariporide and eniporide in the concentrations used.     

Na+-H+ exchange inhibition at reperfusion is cardioprotective during myocardial ischemia-reperfusion; 31P NMR studies

To help resolve the controversy as to whether or not Na⁺-H⁺ exchange is functioning during reperfusion of the ischemic myocardium we assessed the effects of dimethylamiloride (DMA, an amiloride analogue possessing selectivity for inhibition of the Na⁺-H⁺ exchanger) on cardiac function and intracellular pH during ischemia-reperfusion. Studies were performed in the presence of bicarbonate (modified Krebs-Henseleit buffer) or in the nominal absence of bicarbonate (HEPES buffer) in order to determine if similar cardioprotection and effects on intracellular pH were observed in the presence and absence of bicarbonate dependent transport processes. Isovolumic rat hearts were perfused in the Langendorff mode at a constant pressure of 80 mm Hg and subjected to 28 min total global ischemia at 37°C. Intracellular pH was determined from the pH dependent shift of the inorganic phosphate peak in ³¹P nuclear magnetic resonance spectra. DMA (20 µM) was infused for either 2.5 min before ischemia, for the initial 5 min of reperfusion, or at both time intervals. DMA had no effect on the intracellular pH during ischemia. Intracellular pH returned to pre-ischemic levels within 2.5 min of reperfusion in bicarbonate buffer. This normalization of pH was slower in HEPES perfusate. In both bicarbonate and HEPES perfused hearts all drug dosing regimens caused a significant increase in the recovery of mechanical function after reperfusion and slowed the recovery of intracellular pH during reperfusion. These results suggest that the Na⁺-H⁺ exchanger is activated during reperfusion of the ischemic myocardium, that this activation of the exchanger contributes to ischemia-reperfusion induced cardiac dysfunction and that administration of an inhibitor of Na⁺-H⁺ exchange at reperfusion significantly attenuates the deleterious effects of exchanger activation.     

Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

Summary We have investigated the kinetic properties of the human red blood cell Na⁺/H⁺ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H⁺ (H _i ) and external Na⁺ (Na _o ) in red blood cells of normal subjects.Red blood cells with different cell Na⁺ (Na _i ) and pH (pH _i ) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na⁺ influx were measured by varying pH _i (from 5.7 to 7.4), external pH (pH _o ), Na _i and Na _o and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na⁺ influx (Na _i <2.0 mmol/liter cell, Na _o = 150mm) increased sigmoidally (Hill coefficient 2.5) when pH _i fell below 7.0 and the external pH _o was 8.0, but increased linearly at pH _o 6.0. The net Na⁺ influx driven by an outward H⁺ gradient was estimated from the difference of Na⁺ influx at the two pH _o levels (pH _o 8 and pH _o 6). The H⁺-driven Na⁺ influx reached saturation between pH _i 5.9 and 6.1. TheV _max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK _m for H _i to activate H⁺-driven Na⁺ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na⁺/H⁺ exchange. The stoichiometric ratio between H⁺-driven Na⁺ influx and Na⁺-driven H⁺ efflux was 11. The dependence of Na⁺ influx from Na _o was studied at pH _i 6.0, and Na _i lower than 2 mmol/liter cell at pH _o 6.0 and 8.0. The meanK _m for Na _o of the H⁺-gradient-driven Na⁺ influx was 55±7mm.An increase in Na _i from 2 to 20 mmol/liter cell did not change significantly H⁺-driven net Na⁺ influx as estimated from the difference between unidirectional²²Na influx and efflux. Na⁺/Na⁺ exchange was negligible in acid-loaded, DIDS-treated cells. Na⁺ and H⁺ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na⁺ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na⁺/H⁺ exchanger.It is concluded that human red cell Na⁺/H⁺ exchange performs 11 exchange of external Na⁺ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H⁺ and external Na⁺ is similar to other cells, but it displays a larger variability in theV _max between individuals.     

Importance of Ca2+ influx by Na+/Ca2+ exchange under normal and sodium-loaded conditions in mammalian ventricles

Na⁺/Ca²⁺ exchange (NCX) is a major Ca²⁺ extrusion system in cardiac myocytes, but can also mediate Ca²⁺ influx and trigger sarcoplasmic reticulum Ca²⁺ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na⁺]_i may lead to a rise in [Ca²⁺]_i through NCX, causing Ca²⁺ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca²⁺ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca²⁺ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca²⁺ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na⁺-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca²⁺]_i and abolished spontaneous Ca²⁺ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca²⁺ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca²⁺ influx via NCX may be critical in causing myocardial Ca²⁺ overload and triggered activities induced by cardiac glycoside or reoxygenation.     

Amiloride-sensitive Na+ transport in human red cells: Evidence for a Na/H exchange system

Summary The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na⁺ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 m) and methazolamide (400 m). Red cells with different internal pH (pH _i =6.4, 7.0 and 7.8) were prepared and Na⁺ influx was measured at different external pH (pH _o =6.0, 7.0, 8.0). Na⁺ influx into acid-loaded cells (pH _i =6.4) markedly increased when pH _o was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na⁺/H⁺ exchange systems blocked about 60% of the H⁺-induced Na⁺ entry, while showing small inhibitory effects in the absence of pH gradients. When pH₀ was kept at 8.0, the amiloride-sensitive Na⁺ entry was abolished as pH _i was increased from 6.4 to 7.8. Moreover, measurements of H⁺ efflux into lightly buffered media indicated that the imposition of an inward Na⁺ gradient stimulated a net H⁺ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na⁺ (Na _i ⁺ =Na ₀ ⁺ =15mm,Em=+6.7 mV), an outward H⁺ gradient (pH _i =6.4, pH₀=8.0) promoted a net amiloride-sensitive Na⁺ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na⁺/H⁺ exchange system in human red cells.     

Acetylcholine-Induced Na+ influx in the mouse lacrimal gland acinar cells: Demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements

Summary In the isolated, superfused mouse lacrimal gland, intracellular Na⁺ activities (aNa _i ) of the acinar cells were directly measured with double-barreled Na⁺-selective microelectrodes. In the nonstimulated conditionaNa _i was 6.5±0.5 mM and membrane potential (V _m ) was –38.9±0.4 mV. Addition of 1 mM ouabain or superfusion with a K⁺-free solution slightly depolarized the membrane and caused a gradual increase inaNa _i . Stimulation with acetylcholine (ACh, 1 M) caused a membrane hyperpolarization by about 20 mV and an increase inaNa _i by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase inaNa _i by approximately 50%, without affectingV _m and input resistance in both nonstimulated and ACh-stimulated conditions. Acid loading the acinar cells by an addition/withdrawal of 20 mM NH₄Cl or by replacement of Tris⁺-buffer saline solution with HCO ₃ ^– /CO₂-buffered solution increasedaNa _i by a few mM. Superfusion with a Cl^–-free NO ₃ ^– solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the restingaNa _i levels, however, it reduced the ACh-induced increase inaNa _i by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the restingaNa _i and the ACh-induced increase inaNa _i .The present results suggest the presence of multiple Na⁺ entry mechanisms activated by ACh, namely, Na⁺/H⁺ exchange, Na-K-Cl cotransport and organic substrate-coupled Na⁺ transport mechanisms.     

Synthesis and Na+/H+ Exchanger‐1 Inhibitory Activity of Substituted (Quinolinecarbonyl)guanidine Derivatives

The Na⁺/H⁺ exchanger (NHE) is a protein expressed in many mammalian cell types. It is involved in intracellular pH (pH_i) homeostasis by exchanging extracellular Na⁺ for intracellular H⁺. To date, nine NHE isoforms (NHE1–NHE9) have been identified. NHE1 is the most predominant isoform expressed in mammalian cardiac muscle. A novel series of substituted (quinolinecarbonyl)guanidine derivatives were designed and synthesized as NHE inhibitors. Most compounds can inhibit NHE1‐mediated platelet swelling in a concentration‐dependent manner, among which compound 7f was the most active and more potent than cariporide. Furthermore, compound 7f has also been demonstrated to exhibit the in vivo cardioprotective effects against SD rat myocardial ischemic‐reperfusion injury superior to those of cariporide.     

Interactions of external and internal H+ and Na+ with Na+/Na+ and Na+/H+ exchange of rabbit red cells: Evidence for a common pathway

Summary We have studied the kinetic properties of rabbit red cell (RRBC) Na⁺/Na⁺ and Na⁺/H⁺ exchanges (EXC) in order to define whether or not both transport functions are conducted by the same molecule. The strategy has been to determine the interactions of Na⁺ and H⁺ at the internal (i) and external (o) sites for both exchanges modes. RRBC containing varying Na _i and H _l were prepared by nystatin and DIDS treatment of acid-loaded cells. Na⁺/Na⁺ EXC was measured as Na _o -stimulated Na⁺ efflux and Na⁺/H⁺ EXC as Na _o -stimulated H⁺ efflux and pH _o -stimulated Na⁺ influx into acid-loaded cells.The activation of Na⁺/Na⁺ EXC by Na _o at pH _i 7.4 did not follow simple hyperbolic kinetics. Testing of different kinetic models to obtain the best fit for the experimental data indicated the presence of high (K _m 2.2 mM) and low affinity (K _m 108 mM) sites for a single- or two-carrier system. The activation of Na⁺/H⁺ EXC by Na _o (pH _i 6.6, Na _i <1 mM) also showed high (K _m 11 mM) and low (K _m 248 mM) affinity sites. External H⁺ competitively inhibited Na⁺/Na⁺ EXC at the low affinity Na _o site (K _H 52 nM) while internally H⁺ were competitive inhibitors (pK 6.7) at low Na _i and allosteric activators (pK 7.0) at high Na _i .Na⁺/H₊ EXC was also inhibited by acid pH _o and allosterically activated by H _i (pK 6.4). We also established the presence of a Na _i regulatory site which activates Na⁺/H⁺ and Na⁺/Na⁺ EXC modifying the affinity for Na _o of both pathways. At low Na _i , Na⁺/Na⁺ EXC was inhibited by acid pH _i and Na⁺/H⁺ stimulated but at high Na _i , Na⁺/Na⁺ EXC was stimulated and Na⁺/H⁺ inhibited being the sum of both pathways kept constant. Both exchange modes were activated by two classes of Na _o sites,cis-inhibited by external H _o , allosterically modified by the binding of H⁺ to a H _i regulatory site and regulated by Na _i . These findings are consistent with Na⁺/Na⁺ EXC being a mode of operation of the Na⁺/H⁺ exchanger.Na⁺/H⁺ EXC was partially inhibited (80–100%) by dimethyl-amiloride (DMA) but basal or pH _i -stimulated Na⁺/Na⁺ EXC (pH _i 6.5, Na _i 80 mM) was completely insensitive indicating that Na⁺/Na⁺ EXC is an amiloride-insensitive component of Na⁺/H⁺ EXC. However, Na⁺ and H⁺ efflux into Na-free media were stimulated by cell acidification and also partially (10 to 40%) inhibited by DMA: this also indicates that the Na⁺/H⁺ EXC might operate in reverse or uncoupled modes in the absence of Na⁺/Na⁺ EXC.In summary, the observed kinetic properties can be explained by a model of Na⁺/H⁺ EXC with several conformational states, H _i and Na _i regulatory sites and loaded/unloaded internal and external transport sites at which Na⁺ and H⁺ can compete. The occupancy of the H⁺ regulatory site induces a conformational change and the occupancy of the Na _i regulatory site modulates the flow through both pathways so that it will conduct Na⁺/H⁺ and/or Na⁺/Na⁺ EXC depending on the ratio of internal Na⁺:H⁺.     

Preconditioning Prevents the Inhibition of Na+,K+-ATPase Activity after Brain Ischemia

Application of single transient forebrain ischemia (ISC) in adult Wistar rats, lasting 2 or 10 min, caused inhibition of Na⁺,K⁺-ATPase activity in cytoplasmic membrane fractions of hippocampus and cerebral cortex immediately after the event. In the 2-min ISC group followed by 60 min of reperfusion, the enzyme inhibition was maintained in the cortex, while there was an increase in hippocampal enzyme activity; both effects were over 1 day after the event. However, in the 10-min ISC group enzyme inhibition had been maintained for 7 days in both cerebral structures. Interestingly, ischemic preconditioning (2-min plus 10-min ISC, with a 24-hour interval in between) prevented the inhibitory effect of ischemia/reperfusion on Na⁺,K⁺-ATPase activity observed either after a single insult of 2 min or 10 min ischemia. We suggest that the maintenance of Na⁺,K⁺-ATPase activity afforded by preconditioning be related to cellular neuroprotection.     

Regulation of intracellular pH in capacitated human spermatozoa by a Na+/H+ exchanger

We previously demonstrated that the progesterone‐ (P) initiated human sperm acrosome reaction (AR) was dependent on the presence of extracellular Na⁺ (Na⁺_o). Moreover, Na⁺_o depletion resulted in a decreased cytosolic pH (pH_i), suggesting involvement of a Na⁺‐dependent pH_i regulatory mechanism during the P‐initiated AR. We now report that the decreased pH_i resulting from Na⁺_o depletion is reversible and mediated by a Na⁺/H⁺ exchange (NHE) mechanism. To determine the role of an NHE in the regulation of pH_i, capacitated spermatozoa were incubated in Na⁺‐deficient, bicarbonate/CO₂‐buffered (0NaB) medium for 15–30 min, which resulted in an intracellular acidification as previously reported. These spermatozoa were then transferred to Na⁺‐containing, bicarbonate/CO₂‐buffered (NaB) medium; Na⁺‐containing, Hepes‐buffered (NaH) medium; or maintained in the 0NaB medium. Included in the NaH medium was the NHE inhibitor 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA). The steady‐state pH_i was then determined by spectrofluorometric measurement of bis(carboxyethyl)‐5(6)‐carboxyfluoroscein (BCECF) fluorescence. EIPA (0.1 μM) significantly (P < 0.05) inhibited the pH_i recovery produced by NaH medium. Moreover, the pH_i in NaH medium was not significantly (P < 0.05) different than NaB medium. These results indicate that a Na⁺‐dependent, bicarbonate‐independent pH_i regulatory mechanism, with a pharmacological characteristic consistent with an NHE, is present in capacitated spermatozoa. In support of the involvement of a sperm NHE, we also demonstrated specific immunoreactivity for a 100 kDa porcine sperm protein using an NHE‐1 specific monoclonal antibody. Interestingly, no significant (P = 0.79) effect was seen on the P‐initiated AR when EIPA was included in either the NaH or NaB medium. While these findings suggest that inhibition of NHE‐dependent pH_i regulation in capacitated spermatozoa is not sufficient to block initiation of the AR by P, they do not preclude the possibility that an NHE mediates the regulation of capacitation or sperm motility. Mol. Reprod. Dev. 52:189–195, 1999. © 1999 Wiley‐Liss, Inc.     

Na+/Na+ exchange and Na+/H+ antiport in rabbit erythrocytes: Two distinct transport systems

Summary Rabbit erythrocytes are well known for possessing highly active Na⁺/Na⁺ and Na⁺/H⁺ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na⁺ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethylmaleimide (NEM) were used. Na⁺/Na⁺ exchange activity, determined as the Na _o ⁺ -dependent²²Na efflux or Na _i ⁺ -induced²²Na entry was completely abolished by NEM. This inhibitor, however, did not affect the H _i ⁺ -induced Na⁺ entry sensitive to amiloride (Na⁺/H⁺ exchange activity). Similarly, EIPA, a strong inhibitor of the Na⁺/H⁺ exchanger, did not inhibit Na⁺/Na^– countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na⁺/Na⁺ exchanger could be involved in Na⁺/H⁺ countertransport was suggested by studies in which the net Na⁺ transport sensitive to NEM was determined. As expected, net Na⁺ transport through this transport system was zero at different [Na⁺] _i /[Na⁺] _o ratios when intracellular pH was 7.2. However, at pH _i =6.1, net Na⁺ influx occurred when [Na⁺] _i was lower than 39mm. Valinomycin, which at low [K⁺] _o was lower than 39mm. Valinomycin, which at low [K⁺] _o clamps the membrane potential close to the K⁺ equilibrium potential, did not affect the net NEM-sensitive Na⁺ entry but markedly stimulated, the EIPA-and NEM-resistant Na⁺ uptake. This suggest that the net Na⁺ entry through the NEM-sensitive pathway at low pH _i , is mediated by an electroneutral process possibly involving Na⁺/H⁺ exchange. In contrast, the EIPA-sensitive Na⁺/H⁺ exchanger is not involved in Na⁺/Na⁺ countertransport, because Na⁺ transport through this mechanism is not affected by an increase in cell Na^– from 0.4 to 39mm. Altogether, these findings indicate that both transport systems: the Na⁺/Na⁺ and Na⁺/H⁺ exchangers, are mediated by distinct transport proteins.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Na+/H+ exchanger and reperfusion-induced ventricular arrhythmias in isolated perfused heart: possible role of amiloride

The roles of the Na⁺/H⁺ exchange system in the development and cessation of reperfusion induced ventricular arrhythmias were studied in the isolated perfused rat heart. The hearts were perfused in the working heart mode with modified Krebs Henseleit bicarbonate (KHB) buffer and whole heart ischemia was induced by a one-way ball valve with 330 beat/min pacing. Ischemia was continued for 15 min followed by 20 min of aerobic reperfusion (control). Amiloride (1.0mM), an inhibitor of the Na⁺/H⁺ exchange system, was added to the KHB buffer only during reperfusion (group B) or only during ischemic periods (group C). Electrocardiographic and hemodynamic parameters were monitored throughout the perfusion. Coronary effluent was collected through pulmonary artery cannulation and PO₂, PCO₂, HCO ₃ ^– and pH were measured by blood-gas analyzer.The incidence of reperfusion induced ventricular arrhythmias was 100%, 100% and 0% in control, group B and group C, respectively. The mean onset time of termination of reperfusion arrhythmias was significantly shorter in group B than in control. PCO₂ increased from 39.0±0.9 to 89.3±6.0 mmHg at the end of ischemia in control and from 40.6±0.4 to 60.5±5.8 in group C, the difference between groups was statistically significant. HCO ₃ ^– level decreased from 21.8±0.1 to 18.3±0.5 mmol/l in control, however, this decrease was significantly inhibited in group C (from 22.0±0.5 to 20.3±0.2). The increase in PCO₂ and the decrease in HCO ₃ ^– in group B were similar over time to those observed in control. The decrease in pH produced by ischemia was marked in control (from 7.35±0.01 to 6.92±0.04) and group B (from 7.34±0.01 to 6.94±0.02), whereas a decrease in pH was significantly prevented in group C (from 7.34±0.01 to 7.15±0.04). There were no significant differences in PCO₂, HCO ₃ ^– or pH among the three groups during reperfusion.These experiments provide evidence that amiloride significantly prevented the incidence of reperfusion arrhythmias when added only during ischemia and significantly terminated reperfusion arrhythmias when added only during reperfusion. Amiloride may prevent a decrease in pH, due to alterations in PCO₂ and/or HCO ₃ ^– . These changes in PCO₂ and HCO ₃ ^– might be indirectly influenced by inhibition of the Na⁺/H⁺ exchange system via Cl^–/HCO ₃ ^– exchange. The mechanism by which amiloride terminates reperfusion arrhythmias seems to involve electrophysiological effects which were not directly addressed in this experiment.     

Loss of epithelial polarity: A novel hypothesis for reduced proximal tubule Na+ transport following ischemic injury

Summary Ischemia results in the marked reduction of renal proximal tubule function which is manifested by decreased Na⁺ and H₂O reabsorption. In the present studies the possibility that altered Na⁺ and H₂O reabsorption were due to ischemia-induced loss of surface membrane polarity was investigated. Following 15 min of renal ischemia and 2 hr of reperfusion, proximal tubule cellular ultrastructure was normal. However, abnormal redistribution of NaK-ATPase to the apical membrane domain was observed and large alterations in apical membrane lipid composition consistent with loss of surface membrane polarity were noted. These changes were associated with large decreases in Na⁺ (37.4vs. 23.0%,P<0.01) and H₂O (48.6vs. 36.9%,P<0.01) reabsorption at a time when cellular morphology, apical Na⁺ permeability, Na⁺-coupled cotransport, intracellular pH and single nephron filtration rates were normal. We propose that the abnormal redistribution of NaK-ATPase to the apical membrane domain is in part responsible for reduced Na⁺ and H₂O reabsorption following ischemic injury.     

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

Glycolysis uncoupled from glucose oxidation is a major reason for the intracellular acidosis that occurs during severe myocardial ischemia. The imbalance between glycolysis and glucose oxidation, and the resultant H⁺ produced from glycolytically derived ATP hydrolysis in the diabetic rat heart is the focus of this study. Isolated working hearts from 6 week streptozotocin diabetic rat hearts were perfused with 11 mM glucose and 1.2 mM palmitate and subjected to a 25 min period of global ischemia. A second series of experiments were also performed in which hearts from control, diabetic, and islet-transplanted diabetic rats were subjected to a 30 min aerobic perfusion, followed by a 60 min period of low-flow ischemia (coronary flow = 0.5 ml/min) and 30 min of aerobic reperfusion. H⁺ production from glucose metabolism was measured throughout the two protocols by simultaneous measurement of glycolysis and glucose oxidation using perfusate labelled with [5-³H/U-¹⁴C]-glucose. Rates of H⁺ production were calculated by measuring the difference between glycolysis and glucose oxidation. The H⁺ production throughout the perfusion was generally lower in diabetic rat hearts compared to control hearts, while islet-transplantation of diabetic rats increased H⁺ production to rates similar to those seen in control hearts. This occurred primarily due to a dramatic increase in the rates of glycolysis. Despite the difference in H⁺ production between control, diabetic and islet-transplanted diabetic rat hearts, no difference in mRNA levels of the cardiac Na⁺/H⁺-exchanger (NHE-1) was seen. This suggests that alterations in the source of protons (i.e. glucose metabolism) are as important as alterations in the fate of protons, when considering diabetes-induced changes in cellular pH. Furthermore, our data suggests that alterations in Na⁺/H⁺-exchange activity in the diabetic rat heart occur at a post-translational level, possibly due to direct alterations in the sarcolemmal membranes.     

Inhibitory effect of menaquinone-7 (vitamin K2) on the bone-resorbing factors-induced bone resorption in elderly female rat femoral tissues in vitro

A number of data are consistent with the hypothesis that increases in intracellular Na⁺ concentration (Na⁺ _i) during ischemia and early reperfusion lead to calcium overload and exacerbation of myocardial injury. However, the mechanisms underlying the increased Na⁺ _i remain unclear. ²³Na nuclear magnetic resonance spectroscopy was used to monitor Na⁺ _i in isolated rat hearts perfused with a high concentration of fatty acid as can occur under some pathological conditions. Whole-cell patch-clamp experiments were also performed on isolated cardiomyocytes in order to investigate the role of voltage-gated sodium channels. Na⁺ _i increased to substantially above control levels during no-flow ischemia. The results show that a pharmacological reduction of Na⁺ _i increase by cariporide (1 mol/L, a Na⁺/H⁺ exchange blocker) is not the only protection against ischemia-reperfusion damage, but that such protection may also be brought about by metabolic action aimed at reducing fatty acid utilization by myocardial cells. This action was obtained in the presence of etomoxir (0.1 mol/L), an inhibitor of carnitine palmitoyltransferase-1 (the key enzyme involved in fatty acid uptake by the mitochondria) which also decreases long-chain acyl carnitine accumulation. The possibility of Na⁺ channels participating in Na⁺ _i increase as a consequence of alterations in cardiac metabolism was studied in isolated cells. Sustained I_Na was stimulated by the presence of lysophosphatidylcholine (LPC, 10 mol/L) whose accumulation during ischemia is, at least partly, dependent on increased long-chain acyl carnitine. Current activation was particularly significant in the range of potentials between –60 and –20 mV. This may have particular relevance in ischemia. The quantity of charge carried by sustained I_Na was reduced by 24% in the presence of 1 mol/L cariporide. Therefore, limitation of long-chain fatty acid metabolism, and consequent limitation of ischemia-induced long-chain acyl carnitine accumulation, may contribute to reducing intracellular Na⁺ increase during ischemia-reperfusion.     

Apical and basolateral Na+/H+ exchange in the rabbit outer medullary thin descending limb of henle: Role in intracellular pH regulation

Summary The present study was designed to investigate the apical and basolateral transport processes responsible for intracellular pH regulation in the thin descending limb of Henle. Rabbit thin descending limbs of long-loop nephrons were perfused in vitro and intracellular pH (pH _i ) was measured using BCECF. Steady-state pH _i in HEPES buffered solutions (pH 7.4) was 7.18±0.03. Following the removal of luminal Na⁺, pH _i decreased at a rate of 1.96±0.37 pH/min. In the presence of luminal amiloride (1mm), the rate of decrease of pH _i was significantly less, 0.73±0.18 pH/min. Steady-state pH _i decreased 0.18 pH units following the addition of amiloride (1mm) to the lumen (Na⁺ 140mm lumen and bath). When Na⁺ was removed from the basolateral side of the tubule, pH _i decreased at a rate of 0.49±0.05 pH/min. The rate of decrease of pH _i was significantly less in the presence of 1mm basolateral amiloride, 0.29±0.04 pH/min. Addition of 1mm amiloride to the basolateral side (Na⁺ 140mm lumen and bath) caused steady-state pH _i to decrease significantly by 0.06 pH units. When pH _i was acutely decreased to 5.87±0.02 following NH₄Cl removal (lumen, bath), pH _i failed to recover in the absence of Na⁺ (lumen, bath). Addition of 140mm Na⁺ to the lumen caused pH _i to recover at a rate of 2.17±0.59 pH/min. The rate of pH _i recovery was inhibited 93% by 1mm luminal amiloride. When 140mm Na⁺ was added to the basolateral side, pH _i recovered only partially at 0.38±0.07 pH/min. Addition of 1mm basolateral amiloride inhibited the recovery of pH _i , by 97%. The results demonstrate that the rabbit thin descending limb of long-loop nephrons possesses apical and basolateral Na⁺/N⁺ antiporters. In the steady state, the rate of Na⁺-dependent H⁺ flux across the apical antiporter exceeds the rate of Na⁺-dependent H⁺ flux via the basolateral antiporter. Recovery of pH _i following acute intracellular acidification is Na⁺ dependent and mediated primarily by the luminal antiporter.     

Characterization of Na+/H+ Exchanger Isoform (NHE1, NHE2 and NHE3) Expression in Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. Na⁺/H⁺ exchange (NHE) is one of the major Na⁺ absorptive pathways in gallbladder. In this study, we measured gallbladder Na⁺/H⁺ exchange and characterized the NHE isoforms expressed in prairie dogs. Na⁺/H⁺ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na⁺ flux and apical ²²Na⁺ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J ^Na _ms was higher than J ^Na _sm with J ^Na _net absorption. Mucosal DMA inhibited transepithelial Na⁺ flux in a dose-dependent fashion, causing J ^Na _ms equal to J ^Na _sm and blocking J ^Na _net absorption at 100 μm. Basal ²²Na⁺ uptake rate was 10.9 ± 1.0 μmol · cm⁻²· hr⁻¹ which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na⁺/H⁺ exchange accounts for a substantial fraction of gallbladder apical Na⁺ entry and most of net Na⁺ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na⁺ uptake and transepithelial Na⁺ absorption. Received: 9 February 2001/Revised: 11 April 2001     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na⁺/K⁺-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH_i), intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺/K⁺-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca²⁺ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca²⁺]_i (200%). A decrease in pH_i (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca²⁺. Inhibition of Na⁺/H²⁺ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca²⁺ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K⁺/H⁺ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca²⁺ influx via Na⁺/Ca²⁺ exchange, which under hyposmotic conditions activates the K⁺ and Ca²⁺ mitochondrial exchange systems (K⁺/H⁺ and Ca²⁺/H⁺).Deceased