Na+/H+ exchange and its inhibition in cardiac ischemia and reperfusion

Summary The characterization of various ion transport systems has led to a better understanding of the effects, which seem to take part in the impairment of ischemic and reperfused cardiac tissue. This review discusses the role of the Na⁺/H⁺ exchange system in the pathophysiology of ischemia and reperfusion and the beneficial effects of its inhibition.At the onset of ischemia intracellular pH (pH_i) decreases due to anaerobic metabolism and ATP hydrolysis, leading to an activation of Na⁺/H⁺ exchange. This in turn increases intracellular Na⁺ (Na⁺ _i) and activates Na⁺/K⁺ ATPase, with a consecutive increase of energy consumption. Since cellular Na⁺ and Ca⁺⁺ transport are coupled by the Na⁺/Ca⁺⁺ exchange system, which depends on the Na⁺ gradient, the high Na⁺ _i leads to increased intracellular Ca⁺⁺ (Ca⁺⁺ _i). After a certain period, Na⁺/H⁺ exchange is inactivated by a decrease of extracellular pH.In case of reperfusion the acid extracellular fluid is washed out, which reactivates Na⁺/H⁺ exchange, leading to an unfavourably fast restoration of pH_i and a second time to Na⁺ and Ca⁺⁺ _i overflow.High Ca⁺⁺ _i is assumed to be one of the main reasons for ischemic and reperfusion injury, like arrhythmias, myocardial contracture, stunning and necrosis.It seems that the inhibition of Na⁺/H⁺ exchange can interrupt this process at an early phase and prevent or delay the consequences of ischemia and reperfusion as demonstrated by numerous investigators.     

Inhibition of Na+/H+ exchange preserves viability,restores mechanical function,and prevents the pH paradox in reperfusion injury to rat neonatal myocytes

Summary Rat neonatal myocytes exposed to 2.5 mM CaCN and 20 mM 2-deoxyglucose at pH 6.2 (chemical hypoxia) quickly lose viability when pH is increased to 7.4, with or without washout of inhibitors — a pH paradox. In this study, we evaluated the effect of two Na⁺/H⁺ exchange inhibitors (dimethylamiloride and HOE694) and a Na⁺/Ca²⁺ exchange inhibitor (dichlorobenzamil) on pH-dependent reperfusion injury. Intracellular free Ca²⁺ and electrical potential were monitored by laser scanning confocal microscopy of rat neonatal cardiac myocytes grown on coverslips and co-loaded with Fluo-3 and tetramethylrhodamine methylester. After 30–60 min of chemical hypoxia at pH 6.2, mitochondria depolarized and Ca²⁺ began to increase uniformly throughout the cell. Free Ca²⁺ reached levels estimated to exceed 2 M by 4h. Washout of inhibitors at pH 7.4 (reperfusion), with or without dichlorobenzamil, killed most cells within 60 min, despite a marked reduction of Ca²⁺ in dichloroben zamil-treated cells. Reperfusion at pH 7.4 in the presence of 75 M dimethylamiloride or 20 M HOE694, or at pH 6.2, prevented cell death. HOE694-treated cells placed into culture medium recovered mitochondrial membrane potential. In most cells, this occurred before normal Ca²⁺ was restored. Contracted myocytes re-extended over a 24-h-period. By 48 hours, most cells contracted spontaneously and showed normal Ca²⁺ transients. Our results indicate that Na⁺/H⁺ exchange inhibition protects against pH-dependent reperfusion injury and facilitates full recovery of cell function.Abbreviations DCB dichlorobenzamil - DMA dimethylamiloride - 2-DOG 2-deoxy-D-glucose - HOE694 3-methylsulfonyl-4-piperidinobenzoyl guanidine hydrochloride - KRH Krebs-Ringer-HEPES buffer containing 115 mM NaCl, 5 mM KCl, 1 mM KH₂PO₄, 1,2 mM MgSO₄, 2 mM CaCl₂, and 25 mM Na-HEPES buffer - PI propidium iodide - TMRM tetramethylrhodamine methylester - electrical potential difference - electric potential This work was supported, in part, by Grant N00014-89-J-1433 from the Office of Naval Research and Grants AG07218 an DK37034 from the National Institutes of Health. I.S.H. was the recipient of a Post-Doctoral Scholarship from the Medical Research Council of South Africa. A preliminary report of portions of this work was presented at Experimental Biology '93, New Orleans, March 28–April 1, 1993 (15)     

ATP-dependent Ca2+ Transport and Na+/Ca2+ Exchange System in Renal Basolateral Plasmamembranes of Spontaneously Hypertensive Rats

A common abnormality of cellular Ca²⁺ handling in most tissues of spontaneously hypertensive rats (SHR) has been suggested. Therefore we investigated the ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange system in basolateral membrane vesicles (BLMV) of renal cortices from SHR and normotensive Wistar-Kyoto rats (WKY) at 12 and 20 weeks of age. In WKY the maximal transport rate of the ATP-dependent Ca²⁺ transport was 5.7 nmol/min/mg prot with an affinity for Ca²⁺ of 0.14 µM. These values were not significantly different in SHR at both ages studied. High concentrations of Na⁺ inhibited ATP-dependent Ca²⁺ uptake by 40% in BLMV of SHR and WKY. Low concentrations of Na⁺ stimulated ATP-dependent Ca²⁺ transport 20% in both rats. These findings suggest equal Na⁺/Ca²⁺ exchange activity in WKY and SHR. The present study failed to show a significant change in ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange activity in renal BLMV in SHR, suggesting that the Ca²⁺ homeostasis of the cortical cells is normal in SHR as far as the plasmamembrane is concerned.     

Na+/H+ exchange mediates postprandial ileal water and electrolyte transport

Feeding stimulates fluid and electrolyte absorption in the small intestine. Previous studies have suggested that Na⁺/glucose cotransport is important in initiating this response in the jejunum. The purpose of this study was to determine whether Na⁺/H⁺ exchange plays a role in meal-induced absorption. Exteriorized, neurovascularly intact jejunal and ileal loops (25 cm) were constructed in dogs. Following a two-week period of postoperative recovery, the loops of awake dogs were perfused with standard buffer alone or with increasing concentrations of amiloride, a Na⁺/H⁺ exchange inhibitor. Water, sodium, and chloride fluxes were calculated following a meal using [¹⁴C]PEG as a volume marker. The meal significantly increased absorption in both the jejunum (P<0.001) and ileum (P<0.01) in those animals perfused with buffer alone. More significantly, amiloride suppressed the increased absorption seen following a meal in the ileum (P<0.001) but not the jejunum. The response in the ileum was dose dependent. These findings suggest that a major mediator of postprandial sodium and water absorption in the ileum is the Na⁺/H⁺ exchanger.Supported by NIH R29-DK-47326 (S.W.A.), R01-DK-39879 (M.J.Z.), and a VA Merit Review (D.W.M.).Portions of this work were presented at the Annual Meeting of the American Gastroenterological Association, May 1993, Boston, Massachusetts.     

Evidence for apical Na+/H+ exchanger in bovine main pancreatic duct

The finding of a high Pco ₂ in basally secreted pancreatic juice of man and dog raises the hypothesis of proton secretion from ductal epithelial cells presumably through a Na⁺/H⁺ exchanger. To test this possibility, H⁺ luminal secretion and Na⁺ movements were measuredin vitro on samples of bovine pancreatic ducts mounted in Ussing-type chambers. The rate of luminal acidification measured by the pH stat method, using bicarbonate-free media gassed with 100% O₂, reached 2.75 Eq/cm²/hr. Proton secretion was blocked in the presence of 1 mM amiloride or in the absence of Na⁺ (replaced by choline) in the mucosal solution. Study of transepithelial²²Na fluxes in short-circuited tissue, bathed on both sides by control Ringer solution, gassed by 95% O₂-5% CO₂ demonstrated a net sodium transport from the mucosal to the interstitial side of the duct (net²²Na flux=3.23±0.8 Eq/cm²/hr). This net sodium transport was electroneutral and blocked by mucosal amiloride (0.5–1 mM/liter) or by interstitial ouabain (1 mM/liter). These results are consistent with the existence of a Na⁺/H⁺ exchanger on the luminal side of the bovine main pancreatic duct.     

Effects of Na+-H+ exchange blocker amiloride on left ventricular remodeling after anterior myocardial infarction in rats

Summary We investigated the effects of amiloride, a Na⁺-H⁺ exchange blocker, on ventricular remodeling in an infarcted rat model. In the amiloride group, the left descending coronary artery was ligated and rats were given amiloride (1 mg/kg/day, n=11) in their drinking water for 4 weeks. In the control group, rats were given water for 4 weeks (n=8) after myocardial infarction. The rats were killed on day 28. Both the ratio of heart weight to body weight and that of left ventricular weight to body weight were significantly less in the amiloride group (p<0.05). The diameter of a myocardial fiber in the region adjacent to the operated area was significantly reduced in the amiloride group compared with the control group (p<0.05). Left ventricular cavity dimension was significantly smaller in the amiloride group than that in control group (p<0.05). Our findings suggest that amiloride prevents ventricular remodeling after myocardial infarction.     

Reduced infarct size in the rabbit heartin vivo by ethylisopropyl-amiloride. A role for Na+/H+ exchange

Inhibition of Na⁺/H⁺ exchange with amiloride analogues has been shown to offer functional protection during ischemia and reperfusion and reduce infarct size in isolated rat hearts and intact pigs. The aim of the present study was to examine if pre- or postischemic treatment with ethylisopropylamiloride (EIPA), a selective Na⁺/H⁺ exchange inhibitor, could reduce infarct size in anin situ rabbit model of regional ischemia and reperfusion. Anesthetized, open-chest rabbits were subjected to 30 min of regional ischemia and 180 min of reperfusion. The risk zone was determined by fluorescent particles, and infarct size was determined by TTC staining. Preischemic treatment with EIPA (0.65 mg/kg) significantly reduced infarct size from 45.8±3.5% of the risk zone in the control group to 10.6±3.1% (p<0.01). EIPA-treatment during the first part of the reperfusion period did not reduced infarct size compared to controls (41.9±3.5%). We conclude that EIPA, when administered prior to ischemia, reduces infarct size in the rabbit heartin situ, a protection most likely due to inhibition of Na⁺/H⁺ exchange.     

Quantification in crude homogenates of rat myocardial Na+, K+-and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes

Assays for complete quantification of Na⁺, K⁺-and Ca²⁺-ATPase in crude homogenates of rat ventricular myocardium by determination of K⁺-and Ca²⁺-dependentp-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K⁺-and Ca²⁺-dependentpNPPase activities in ventricular myocardium of 11–12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 mol×min^–1×g^–1 wet wt. (mean±SEM) (n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na⁺, K⁺-and Ca²⁺-ATPase concentrations were estimated to 2 and 10 nmol×g^–1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca²⁺-dependentpNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K⁺-dependentpNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K⁺-and Ca²⁺-dependentpNPPase activity gradually increased to a plateau. The present assay for Na⁺, K⁺-ATPase quantification has the advantage over [³H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K⁺-dependent 3-0-methylfluorescein phosphatase (3-0-MFPase) in crude tissue homogenates. Furthermore, with few modifications thepNPPase assay allows quantification of Ca²⁺-ATPase on crude myocardial homogenates. Age-dependent changes in K⁺-and Ca²⁺-dependentpNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na⁺, K⁺-and Ca²⁺-ATPase in excitable tissues.Abbreviations Na⁺, K⁺-ATPase sodium, potassium-dependent ATPase - Ca²⁺-ATPase caldium-dependent ATPase - pNP p-nitrophenyl - pNPP p-nitrophenyl phosphate - 3-0-MFP 3-0 methylfluorescein phosphate - DOC sodium deoxycholate     

Insulin does not regulate vascular smooth muscle Na+, K+-ATPase activity in rabbit aorta

Summary To determine whether insulin regulates vascular smooth muscle Na⁺, K⁺-ATPase activity and if impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity could be a cause of increased vascular reactivity to norepinephrine and angiotensin II in diabetic states, the effects of insulin on Na⁺, K⁺-ATPase activity were examined in normal rabbit aortic intima-media incubated with normal plasma glucose and myo-inositol levels for 30 min. Insulin at 100 U/ml (600 pmol/l) had no effect on Na⁺, K⁺-ATPase activity. At 250 U/ml it caused a 4.2±0.8% increase, and at 500 U/ml insulin caused a 17.7±1.4% increase in Na⁺, K⁺-ATPase activity that was completely inhibited by amiloride (1 mmol/l). Human insulin-like growth factor I (600 pmol/l) caused an 18.0±1.0% increase in Na⁺, K⁺-ATPase activity that was inhibited by amiloride. Insulin does not regulate (stimulate) aortic vascular smooth muscle Na⁺, K⁺-ATPase activity. Supraphysiological insulin concentrations, probably acting through an insulin-like growth factor I receptor, stimulate Na⁺/H⁺ exchange in aortic vascular smooth muscle and cause small secondary increases in Na⁺, K⁺-ATPase activity. In aortic intima-media incubated with normal plasma glucose and myo-inositol levels, endogenously released adenosine stimulates and maintains a component of resting Na⁺, K⁺-ATPase activity and stimulates acute increases in activity when norepinephrine (1 mol/l) or angiotensin II (100 nmol/l) is added. These adenosine-stimulated components of Na⁺, K⁺-ATPase activity are selectively inhibited when the medium glucose is raised to 30 mmol/l during a 30-min equilibration and 30-min incubation. Insulin (100 U/ml) added during the incubation had no effect on the alterations in Na⁺, K⁺-ATPase activity induced by glucose at an elevated plasma level. Impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity is not a possible cause for alterations in vascular reactivity in diabetes.     

The role of Na+/H+ exchange in ischemia-reperfusion

In ischemia the cytosol of cardiomyocytes acidifies; this is reversed upon reperfusion. One of the major pH_i-regulating transport systems involved is the Na⁺/H⁺ exchanger. Inhibitors of the Na⁺/H⁺ exchanger have been found to more effectively protect ischemic-reperfused myocardium when administered before and during ischemia than during reperfusion alone. It has been hypothesized that the protection provided by pre-ischemic administration is due to a reduction in Na⁺ and secondary Ca²⁺ influx. Under reperfusion conditions Na⁺/H⁺ exchange inhibition also seems protective since it prolongs intracellular acidosis which can prevent hypercontracture. In detail, however, the mechanisms by which Na⁺/H⁺ exchange inhibition provides protection in ischemic-reperfused myocardium are still not fully identified.     

Cardiovascular remodelling and red blood cell Li+/Na+ countertransport in patients with type 1 diabetes and essential hypertension

Patients with type 1 (insulin-dependent) diabetes may develop a specific cardiac disease characterized by functional and structural abnormalities. The pathogenesis of the cardiac disease is poorly understood but cardiac and renal complications may coexist. Patients with overt diabetic nephropathy have increased red cell Li⁺/Na⁺ countertransport (CT), which reflects abnormal kinetic properties of the red cell membrane Na⁺/H⁺ exchange. Since the activation of Na⁺/H⁺ exchange has a key role in cell proliferation and cell growth, as well as in the regulation of cell sodium and cell pH and in the renal reabsorption of Na⁺ and bicarbonate, we have looked for relationships between red cell Li⁺/Na⁺ CT, Na⁺/H⁺ exchange and cardiovascular remodeling in patients with type 1 diabetes, essential hypertension and idiopathic familiar cardiomyopathy. In type 1 diabetes the maximal velocity of Li⁺/Na⁺ CT is positively correlated with interventricular septum thickness and the left ventricular wall to lumen ratio. Similar results were obtained in patients with essential hypertension. In these patients an increased Li⁺/Na⁺ CT is also associated with severe and drug-resistant hypertension and with significant vascular remodelling, estimated by the minimal post-ischaemic vascular resistance in the calf. Finally, Li⁺/Na⁺ CT is negatively correlated with diastolic relaxation of the left ventricle in familiar hypertrophic cardiomyopathy. From these data it appears that widespread abnormal kinetic properties of Na⁺/H⁺ exchange, estimated by increased red cell Li⁺/Na⁺ CT, may have epistatic effects on the pathogenesis of cardiac complications of type 1 diabetes and essential hypertension.     

Role of Na+-H+-antiport in restitution of isolated guinea pig gastric epithelium after superficial injury

In addition to its pH_i regulatory function Na⁺-H⁺-antiport is also involved in volume regulation of epithelial cells, particularly in neutral conditions. It is also known that the antiport is activated after ligand binding following growth factor receptor activation. The aim of the present study was to evaluate the role of the antiport in restitution of gastric mucosa and whether its activity is dependent on the type of superficial injury. Therefore the fundic epithelium of guinea pig stomach was perfused in an Ussing chamber in neutral conditions. Na⁺-H⁺- and Cl^–-HCO ₃ ^– -antiports were inhibited with 1.0 mM amiloride, 1.0 mM SITS, or with HCO ₃ ^– removal and Na⁺-K⁺-2Cl^2–-cotransporter with 0.3 M furosemide during 4 hr of restitution after superficial injury induced either by 1.25 M NaCl or by 1.0% Triton. Luminal exposure of the epithelium to amiloride had no effect on restitution but serosal application abolished the process completely. The inhibitory effect of amiloride was similar after both NaCl and Triton injury. The inhibition of Cl^–-HCO ₃ ^– -antiport with SITS interfered with the process as well, while HCO ₃ ^– removal had no significant inhibitory effect, nor did the inhibition of Na⁺-K⁺-2Cl^–-cotransporter. The morphologic findings were in accordance with the electrophysiologic measurements in each pair of tissues. It is concluded that the Na⁺-H⁺-antiport is essential for the epithelial cells during restitution even in neutral conditions, but a functional Cl^–-HCO ₃ ^– -antiport is also required. The activity of Na⁺-H⁺-antiport is sensitive to basolateral amiloride and is necessary regardless of the type of chemical injury.This study was supported by grants from Astra Co., the Orion Corporation Research Foundation, the Sigrid Juselius Foundation, the Academy of Finland, the Research Foundation of Gastrointestinal Diseases, the Clinical Research Institute of Helsinki University Central Hospital, Helsinki, Finland.     

Na+/H+ antiporter properties in peripheral blood lymphocytes from normotensive obese and type 2 diabetic patients do not differ significantly from controls

It has been hypothesized that some genetic factors link different conditions characterized by the presence of insulin resistance: among them, obesity, type 2 (non-insulin-dependent) diabetes mellitus and arterial hypertension. A good candidate could be the Na⁺/H⁺ exchanger, the increased activity of which is considered a genetic marker of essential hypertension. In this study we looked at whether the Na⁺ dependence of the Na⁺/H⁺ antiporter is modified in obese and type 2 diabetic patients, in the absence of arterial hypertension. The activity of this ion exchanger was measured in peripheral blood lymphocytes by acidifying them in Na⁺-free buffer and then monitoring the recovery of intracellular pH after Na⁺ addition. Quiescent lymphocytes were used because they do not have insulin receptors, thus ruling out the effects of the elevated insulin concentrations on the Na⁺/H⁺ exchanger activity. Antiport activity, measured as the ability to extrude H⁺ in the presence of external Na⁺, showed no differences in normotensive obese and type 2 diabetic patients when compared with healthy subjects. Our data therefore suggest that an altered Na⁺/H⁺ exchange activity cannot be considered a common feature of insulin-resistant states.     

Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation

Ca²⁺ efflux by Ca²⁺ cation antiporter (CaCA) proteins is important for maintenance of Ca²⁺ homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na⁺/Ca²⁺ exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca²⁺ efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca²⁺/H⁺ antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca²⁺-binding site. A hydrophobic “seal” closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca²⁺/H⁺ exchange pathway. In the Ca²⁺-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na⁺-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca²⁺/H⁺ exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.     

Na+-K+ pump inhibition caused by chronic amiodarone in guinea pig myocardium

Summary Although it is known that amiodarone inhibits myocardial Na⁺-K⁺ pump activity, the potency and the time course of this inhibition are unknown. The aim of this study was to investigate these aspects with reference to digoxin, using guinea pigs treated with either intraperitoneal amiodarone (20mg/kg per week, up to 12 weeks,n = 26) or the same amount of vehicle as a control (n = 24). ECG recording and microelectrode experiments were conducted every 2 weeks. QT interval corrected by heart rate and action potential duration were prolonged as a function of the time of exposure to amiodarone. Hyperpolarization observed immediately after the overdrive (1.0Hz) termination or K⁺-replenishment following K⁺-depletion in the presence of 0.1mM Ba²⁺ was compared in the amiodarone-treated and untreated groups, as an index of the Na⁺-K⁺ pump activity. The resting membrane potential recovery from overdrive-induced depolarization was slower and the amplitude of K⁺-induced hyperpolarization was smaller in the amiodarone-treated group than in the untreated group. These changes were evident as the chronic amiodarone treatment progressed, although the changes in these parameters were greater in the case of acute application of 50µM digoxin. In conclusion, this study indicates that treatment with amiodarone for longer than several weeks moderately inhibits the myocardial Na⁺-K⁺ pump.This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (11877125).     

PKC mediates GnRH activation of a Na/H exchanger in goldfish somatotropes

Previous results suggest that gonadotropin-releasing hormone (GnRH) stimulation of somatotropin secretion in goldfish involves activation of Na⁺/H⁺ exchange (NHE). We tested the hypothesis that GnRH alkalinizes intracellular pH (pH_i) via protein kinase C (PKC) activation of NHE. Two types of alkalinization responses were observed in identified goldfish somatotropes preloaded with the pH-sensitive dye BCECF; the rate of pH_i changes went from a neutral or slightly negative slope to either a positive or a less negative slope relative to control. Two GnRHs, the PKC-activating TPA, and dioctanoyl glycerol each caused an alkalinization in 70-90% of somatotropes. The PKC inhibitors, Bis II and Gö6976, the NHE inhibitor amiloride, or Na⁺-free solution attenuated TPA and GnRHs actions, suggesting that PKC mediates GnRH activation of NHE. Since amiloride and Na⁺-free solution caused acidification in somatotropes at rest, regulation of basal pH_i in these cells likely involves Na⁺ flux through amiloride-sensitive NHE.     

Na+ Channel Activators as Positive Inotropic Agents for the Treatment of Chronic Heart Failure

The Na⁺ channel agonists DPI 201-106, BDF 9148 and BDF 9198 are a new group of positive inotropic agents which increase cardiac contractility in a cAMP independent manner. The most likely mechanism by which positive inotropy is mediated is an enhancement of Na⁺/Ca²⁺ exchange activity in response to a Na⁺ channel agonist induced increase in the cardiac myocyte intracellular Na⁺ concentration. While the positive inotropic effect of drugs which exert their effects in a cAMP dependent manner is blunted in failing compared to nonfailing myocardium, the efficacy and potency of Na⁺ channel agonists is not only maintained, but enhanced in failing myocardium. This finding makes these substances interesting for the treatment of patients with heart failure. The positive inotropic effects of the Na⁺ channel agonists, however, are accompanied by a potential increase in the incidence of cardiac arrhythmias. These side effects might limit the clinical use of Na⁺ channel agonists and demand future development of Na⁺ channel modulators without significant arrhythmogenic effects.     

Ca2+ clearance and contractility in vascular smooth muscle: evidence from gene-altered murine models

The central importance of calcium clearance proteins, and their regulators, in the modulation of myocardial contractility and intracellular Ca²⁺ concentration ([Ca²⁺]_i) has long been established. Key players identified include the Na⁺-Ca²⁺ exchanger, the Na⁺-K⁺ ATPase, the sarco(endo)plasmic reticulum Ca²⁺-ATPase and associated phospholamban. Gene-targeted and transgenic murine models have been critical in the elucidation of their function. The study of these proteins in the regulation of contractile parameters in vascular smooth muscle, on the other hand, is less well studied. More recently, gene-targeted and transgenic models have expanded our knowledge of Ca²⁺ clearance proteins and their role in both tonic and phasic smooth muscle contractility. In this review, we will briefly treat the mechanisms which underlie Ca²⁺ clearance in smooth muscle. These will be addressed in light of studies using gene-modified mouse models, the results of which will be compared and contrasted with those in the cardiomyocyte. The recently identified human mutations in phospholamban, which lead to dilated cardiomyopathy, are also present in vascular and other smooth muscle. Given the importance of these Ca²⁺ clearance systems to modulation of smooth muscle, it is likely that mutations will also lead to smooth muscle pathology.     

The Autocrine/Paracrine Loop After Myocardial Stretch: Mineralocorticoid Receptor Activation

The stretch of cardiac muscle increases developed force in two phases. The first phase, which occurs rapidly, constitutes the well-known Frank-Starling mechanism and it is generally attributed to enhanced myofilament responsiveness to Ca²⁺. The second phase or slow force response (SFR) occurs gradually and is due to an increase in the calcium transient amplitude as a result of a stretch-triggered autocrine/paracrine mechanism. We previously showed that Ca²⁺ entry through reverse Na⁺/Ca²⁺ exchange underlies the SFR, as the final step of an autocrine/paracrine cascade involving release of angiotensin II/endothelin, and a Na⁺/H⁺ exchanger (NHE-1) activation-mediated rise in Na⁺. In the present review we mainly focus on our three latest contributions to the understanding of this signalling pathway triggered by myocardial stretch: 1) The finding that an increased production of reactive oxygen species (ROS) from mitochondrial origin is critical in the activation of the NHE-1 and therefore in the genesis of the SFR; 2) the demonstration of a key role played by the transactivation of the epidermal growth factor receptor; and 3) the involvement of mineralocorticoid receptors (MR) activation in the stretch-triggered cascade leading to the SFR. Among these novel contributions, the critical role played by the MR is perhaps the most important one. This finding may conceivably provide a mechanistic explanation to the recently discovered strikingly beneficial effects of MR antagonism in humans with cardiac hypertrophy and failure.     

Functional and Cellular Regulation of the Myocardial Na+/H+ Exchanger

The Na⁺/H⁺ exchanger is a pH-regulatory protein present in the plasma membrane of cardiomyocytes and other cell types. In response to intracellular acidosis, the protein removes one intracellular proton in exchange for an extracellular sodium. The protein consists of a membrane transporting domain and a regulatory cytosolic domain. The regulatory cytosolic domain mediates the stimulation of the membrane domain. Hormonal stimulation of myocardial cells results in activation of the antiporter, possibly through protein kinases and other regulatory proteins. Several hormones and growth factors have been shown to stimulate the antiporter in the myocardium, including endothelin, thrombin, angiotensin II, and ₁-adrenergic stimulation. The exact mechanisms involved in this stimulation are as yet unclear, and may be important in regulation of the Na⁺/H⁺ exchanger during ischemia and reperfusion.