A new topological model of the cardiac sarcolemmal Na+-Ca2+ exchanger

The current topological model of the Na+-Ca2+ exchanger consists of 11 transmembrane segments with extracellular loops a, c, e, g, i, and k and cytoplasmic loops b, d, f, h, and j. Cytoplasmic loop f, which plays a role in regulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutating residues near putative transmembrane segments to cysteine and then examining the effects of intracellular and extracellular applications of sulfhydryl-modifying reagents on exchanger activity. To aid in our topological studies, we also constructed a cysteineless Na+-Ca2+ exchanger. This mutant is fully functional in Na+ gradient-dependent 45Ca2+ uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 endogenous cysteine residues are not essential for either activity or regulation of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracellular surface. However, the data also support placing Ser-788 of loop h at the extracellular surface and Gly-837 of loop i at the intracellular surface. To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose that putative transmembrane segment 9 does not span the membrane, but may form a "P-loop"-like structure.     

Signal transduction mechanism for the stimulation of the sarcolemmal Na(+)-Ca2+ exchanger by insulin

The changes of opioid peptide reactivity in seizure activity have been well studied in animals. Increased enkephalin and dynorphin immunoreactivity in the hippocampi of animals are interpreted as the result of seizure induced mossy fibre sprouting. We studied the hippocampi of six patients with a history of long-standing grand mal seizures and six age-matched control patients with no history of epilepsy or neurologic disease, using frozen sections which were immunostained with antibodies against Leu-enkephalin and Met-enkephalin. The staining intensity in the CA3, CA4 and internal molecular layer of the dentate fascia in each case was quantified using optical densitometry image analysis. The CA3 and CA4 of the epileptic hippocampi showed highly significant increase in Leu-enkephalin-like immunoreactivity compared to the controls (P < 0.005) while the inner molecular layer showed only significant increase (P < 0.05). Met-Enkephalin-like immunoreactivity was only significantly increased in CA4 of the epileptic hippocampi (P < 0.05).     

Rapid charge translocation by the cardiac Na(+)-Ca2+ exchanger after a Ca2+ concentration jump

The kinetics of Na(+)-Ca2+ exchange current after a cytoplasmic Ca2+ concentration jump (achieved by photolysis of DM-nitrophen) was measured in excised giant membrane patches from guinea pig or rat heart. Increasing the cytoplasmic Ca2+ concentration from 0.5 microM in the presence of 100 mM extracellular Na+ elicits an inward current that rises with a time constant tau 1 < 50 microseconds and decays to a plateau with a time constant tau 2 = 0.65 +/- 0.18 ms (n = 101) at 21 degrees C. These current signals are suppressed by Ni2+ and dichlorobenzamil. No stationary current, but a transient inward current that rises with tau 1 < 50 microseconds and decays with tau 2 = 0.28 +/- 0.06 ms (n = 53, T = 21 degrees C) is observed if the Ca2+ concentration jump is performed under conditions that promote Ca(2+)-Ca2+ exchange (i.e., no extracellular Na+, 5 mM extracellular Ca2+). The transient and stationary inward current is not observed in the absence of extracellular Ca2+ and Na+. The application of alpha-chymotrypsin reveals the influence of the cytoplasmic regulatory Ca2+ binding site on Ca(2+)-Ca2+ and forward Na(+)-Ca2+ exchange and shows that this site regulates both the transient and stationary current. The temperature dependence of the stationary current exhibits an activation energy of 70 kj/mol for temperatures between 21 degrees C and 38 degrees C, and 138 kj/mol between 10 degrees C and 21 degrees C. For the decay time constant an activation energy of 70 kj/mol is observed in the Na(+)-Ca2+ and the Ca(2+)-Ca2+ exchange mode between 13 degrees C and 35 degrees C. The data indicate that partial reactions of the Na(+)-Ca2+ exchanger associated with Ca2+ binding and translocation are very fast at 35 degrees C, with relaxation time constants of about 6700 s-1 in the forward Na(+)-Ca2+ exchange and about 12,500 s-1 in the Ca(2+)-Ca2+ exchange mode and that net negative charge is moved during Ca2+ translocation. According to model calculations, the turnover number, however, has to be at least 2-4 times smaller than the decay rate of the transient current, and Na+ inward translocation appears to be slower than Ca2+ outward movement.     

Na(+)-Ca2+ exchange currents in cortical neurons: concomitant forward and reverse operation and effect of glutamate

Na(+)-Ca2+ exchanger-associated membrane currents were studied in cultured murine neocortical neurons, using whole-cell recording combined with intracellular perfusion. A net inward current specifically associated with forward (Na+(o)-Ca2+(i)) exchange was evoked at -40 mV by switching external 140 mM Li+ to 140 mM Na+. The voltage dependence of this current was consistent with that predicted for 3Na+:1Ca2+ exchange. As expected, the current depended on internal Ca2+, and could be blocked by intracellular application of the exchanger inhibitory peptide, XIP. Raising internal Na+ from 3 to 20 mM or switching the external solution from 140 mM Li+ to 30 mM Na+ activated outward currents, consistent with reverse (Na+(i)-Ca2+(o)) exchange. An external Ca2(+)-sensitive current was also identified as associated with reverse Na(+)-Ca2+ exchange based on its internal Na+ dependence and sensitivity to XIP. Combined application of external Na+ and Ca2+ in the absence of internal Na+ triggered a 3.3-fold larger inward current than the current activated in the presence of 3 mM internal Na+, raising the intriguing possibility that Na(+)-Ca2+ exchangers might concurrently operate in both the forward and the reverse direction, perhaps in different subcellular locations. With this idea in mind, we examined the effect of excitotoxic glutamate receptor activation on exchanger operation. After 3-5 min of exposure to 100-200 microM glutamate, the forward exchanger current was significantly increased even when external Na+ was reduced to 100 mM, and the external Ca2(+)-activated reverse exchanger current was eliminated.     

Subcellular distribution of ankyrin in developing rabbit heart--relationship to the Na+-Ca2+ exchanger

Ankyrins are a multigene family of proteins that function as adapters between the cytoskeleton and trans-membrane proteins, such as ion channels. Previous studies have shown the linkage between ankyrin and ionic transport proteins such as Na+-K+ ATPase, voltage-dependent Na+ channels and Ca2+ channels. In the present study, we have investigated the subcellular distribution of ankyrin and its relationship to the Na+-Ca2+ exchange protein in immature and adult rabbit ventricular myocytes. Isolated single cardiomyocytes from neonatal, juvenile and adult rabbit hearts were examined by immunofluorescence labeling techniques, using antibodies against ankyrin and the Na+-Ca2+ exchanger. We found that in neonatal rabbit cardiac myocytes, ankyrin labeling was mainly present at the Z disk, whereas the Na+-Ca2+ exchanger was only present on the peripheral sarcolemma. At 2 weeks of age, ankyrin labeling was still predominantly observed at the level of the Z disks as well as in the partially developed T-tubules. In the adult cells, however, ankyrin and the Na+-Ca2+ exchanger seem to be co-localized within T-tubules and at the costamere region of the peripheral sarcolemma. Immunogold labeling studies at the higher resolution electron microscopic level using cyrosection tissues of rabbit heart at different ages confirm these findings. These results indicate that the distribution pattern of ankyrin and the Na+-Ca2+ exchanger changes with development in rabbit ventricular myocytes.     

Parameters of lymphocyte Na+-Ca2+ regulation and blood pressure: the gender effect

Alterations in cellular Ca2+ and Na+ regulation play a role in the pathogenesis of essential hypertension. Using peripheral lymphocytes from 68 normal persons, we observed the following relationships for major cellular Ca2+ regulatory parameters. Among men and women, Na+-Ca2+ exchanger activity was positively correlated with the resting cytosolic free Ca2+ ([Ca2+]c) (r=0.43, P=0.0003), and the resting [Ca2+]c was positively correlated with cytosolic Na+ ([Na+]c) (r=0.50, P=0.0001). For men only, store-operated Ca2+ entry was positively correlated with Na+-Ca2+ exchanger activity (r=0.63, P=0.0001). In addition, systolic and diastolic blood pressures were positively correlated with [Na+]c in men (r=0.53, P=0.001, and r=0. 41, P=0.017, respectively) but not in women (r=0.30, P=0.088, and r=0.24, P=0.17, respectively). Some of the relationships between cellular and blood pressure parameters were confounded by serum triglycerides. These observations indicate a gender effect on cellular Ca2+-Na+ regulation and its relationship with blood pressure.     

Interaction of the Na(+)-Ca2+ exchanger with small molecules on cell Ca2+ signaling

BACKGROUND: Evaluation of outcome after CPR in severe hypothermic patients. DESIGN: Perspective study from October 1995 to April 1996. SETTING: First aid team of Italian Red Cross, Busto Arsizio (Varese), Italy. METHODS: A population of 22 patients in cardiac arrest in which CPR was performed immediately after rescue team's arrival is studied. ECG, core temperature, SpO2 and MAP were monitored whereas vital parameters were present during Basic Life Support. Outcome after CPR was evaluated with GOS scale. RESULTS: It has been observed that severe hypothermia and time of cardiac arrest impact on the clinical outcome after CPR. The high mortality rate after CPR with BLS standard is worsened by a core temperature < or = 33 degrees C. CONCLUSIONS: Severe hypothermia seems to have a dangerous effect upon outcome after cardiopulmonary resuscitation; heating systems for body temperature could prevent this situation improving CPR results.     

Relationship between Na+-Ca2+-exchanger protein levels and diastolic function of failing human myocardium

BACKGROUND: In the failing human heart, sarcoplasmic reticulum (SR) calcium handling is impaired, and therefore, calcium elimination and diastolic function may depend on the expression of sarcolemmal Na+-Ca2+ exchanger. METHODS AND RESULTS: Force-frequency relations were studied in ventricular muscle strip preparations from failing human hearts (n=29). Protein levels of Na+-Ca2+ exchanger and SR Ca2+-ATPase were measured in the same hearts. Hearts were divided into 3 groups by discriminant analysis according to the behavior of diastolic function when stimulation rate of muscle strips was increased from 30 to 180 min-1. At 180 compared with 30 min-1, diastolic force was increased by 160%, maximum rate of force decline was decreased by 46%, and relaxation time was unchanged in group III. In contrast, in group I, diastolic force and maximum rate of force decline did not change, and relaxation time decreased by 20%. Na+-Ca2+ exchanger was 66% higher in group I than in group III. Na+-Ca2+ exchanger was inversely correlated with the frequency-dependent rise of diastolic force when stimulation rate was increased (r=-0.74; P<0.001). Compared with nonfailing human hearts (n=6), SR Ca2+-ATPase was decreased and Na+-Ca2+ exchanger unchanged in group III, whereas Na+-Ca2+ exchanger was increased and SR Ca2+-ATPase unchanged in group I. Results with group II hearts were between those of group I and group III hearts. CONCLUSIONS: By discriminating failing human hearts according to their diastolic function, we identified different phenotypes. Disturbed diastolic function occurs in hearts with decreased SR Ca2+-ATPase and unchanged Na+-Ca2+ exchanger, whereas increased expression of the Na+-Ca2+ exchanger is associated with preserved diastolic function.     

Differential regulation of Na+ and Cl- conductances by PTX-sensitive G proteins in fetal lung apical membrane vesicles

In late 1990 the National Institute on Drug Abuse (NIDA) initiated the Cooperative Agreement (CA) for AIDS Community-Based Outreach/Intervention Research Program. The goal of this program was to prevent the further spread of HIV among out-of-treatment drug users, in particular injection drug users (IDUs) and crack cocaine users, their sexual partners, and those at risk for initiating injection behavior. To accomplish this goal, the CA set out to monitor drug use and HIV risk behaviors, assess the efficacy of various HIV risk reduction interventions, and develop and refine outreach and intervention strategies. Twenty-three research sites, 21 rural and urban sites in the United States and one each in Puerto Rico and Brazil, were included in the CA program. This article presents an overview of the CA as well as a synopsis of the studies covered in this special issue examining the total CA database.     

A novel molecular determinant for cAMP-dependent regulation of the frog heart Na+-Ca2+ exchanger

Na+-Ca2+ exchanger is one of the major sarcolemmal Ca2+ transporters of cardiac myocytes. In frog ventricular myocytes the exchanger is regulated by isoproterenol via a beta-adrenoreceptor/adenylate-cyclase/cAMPdependent signaling pathway providing a molecular mechanism for the relaxant effect of the hormone. Here, we report on the presence of a novel exon of 27-base pair insertion, which generates a nucleotide binding motif (P-loop) in the frog cardiac Na+-Ca2+ exchanger. To examine the functional role of this motif, we constructed a full-length frog heart Na+-Ca2+ exchanger cDNA (fNCX1a) containing this exon. The functional expression of fNCX1a in oocytes showed characteristic voltage dependence, divalent (Ni2+, Cd2+) inhibition, and sensitivity to cAMP in a manner similar to that of native exchanger in frog myocytes. In oocytes expressing the dog heart NCX1 or the frog mutant (DeltafNCX1a) lacking the 9-amino acid exon, cAMP failed to regulate Na+-dependent Ca2+ uptake. We suggest that this motif is responsible for the observed cAMP-dependent functional differences between the frog and the mammalian hearts.     

Hydrogen peroxide activates mitogen-activated protein kinases and Na+-H+ exchange in neonatal rat cardiac myocytes

Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na+-H+ exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 micromol/L hydrogen peroxide (H2O2) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H2O2 (100 micromol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H2O2. Pretreatment with chelerythrine (10 micromol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 micromol/L, 45 minutes) or herbimycin A (3 micromol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H2O2-stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H2O2 also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 micromol/L, 60 minutes). These results suggest that low doses of H2O2 induce MAP kinase-dependent pathways that regulate NHE activity during reperfusion injury.     

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

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

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

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

The Na+-responsive ntp operon is indispensable for homeostatis of K+ and Na+ in Enterococcus hirae at limited proton potential

Enterococcus hirae ATCC 9790 grew well in Na+-deficient, low-K+ medium, but growth was inhibited by carbonylcyanide m-chlorophenylhydrazone (CCCP). Growth inhibition and decrease of cellular K+ levels in the presence of CCCP were relieved by the addition of Na+ and a high concentration of K+. In contrast, in the mutant defective in Na+-ATPase or the NtpJ component of the KtrII K+ uptake system, CCCP-induced growth inhibition was rescued by a high concentration of K+ but not of Na+. These transporters are thus indispensable for homeostatis of K+ and Na+ at low proton potential.     

Ca2+ flux through promiscuous cardiac Na+ channels: slip-mode conductance

The tetrodotoxin-sensitive sodium ion (Na+) channel is opened by cellular depolarization and favors the passage of Na+ over other ions. Activation of the beta-adrenergic receptor or protein kinase A in rat heart cells transformed this Na+ channel into one that is promiscuous with respect to ion selectivity, permitting calcium ions (Ca2+) to permeate as readily as Na+. Similarly, nanomolar concentrations of cardiotonic steroids such as ouabain and digoxin switched the ion selectivity of the Na+ channel to this state of promiscuous permeability called slip-mode conductance. Slip-mode conductance of the Na+ channel can contribute significantly to local and global cardiac Ca2+ signaling and may be a general signaling mechanism in excitable cells.     

Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites

Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na+i- and Ca2+i-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that microM levels of Ca2+i inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. Mutations within the putative regulatory Ca2+i binding site of CALX1. 1, like corresponding alterations in NCX1.1, led to reduced ability (i.e. D516V and D550I) or inability (i.e. G555P) of Ca2+i to inhibit Na+-Ca2+ exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Delta310-313) of Na+i-dependent inactivation. These results indicate that the respective regulatory roles of the Ca2+i binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca2+i. We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca2+i regulatory phenotypes was feasible. With one chimera (i.e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca2+i. This result indicates that the regulatory Ca2+i binding site of NCX1.1 retains function in a CALX1. 1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca2+i binding signal.     

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

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