Na+/H+ exchanger mediates TNF-α-induced hepatocyte apoptosis via the calpain-dependent degradation of Bcl-xL

Background and Aim:  It is well known that tumor necrosis factor-α (TNF-α) induces hepatocyte apoptosis and contributes to liver diseases. However, the exact mechanisms are not well understood.
Methods:  In the present study, we reported that Na⁺/H⁺ exchanger (NHE) is involved in TNF-α-induced hepatocyte apoptosis.
Results:  TNF-α time dependently induced an increase in NHE activity in hepatocytes, but cariporide, an NHE inhibitor, blocked the TNF-α-induced increase of NHE activity in a dose-dependent manner. Increased NHE activity induced by TNF-α was associated with increased intracellular calcium (Ca²⁺_i) concentration and calpain activity. Cariporide reversed these effects induced by TNF-α. In addition, TNF-α downregulated Bcl-xL, an anti-apoptotic protein, but not mRNA levels. The inhibition of either calpain or NHE blocked the TNF-α-induced decrease of the Bcl-xL protein. TNF-α did not change the pro-apoptotic Bax and Bak protein levels. Cariporide, calcium remover 1,2-bis (2-aminophenoxy) ethane-N,N,N0,N0–tetraacetic acid, or calpain inhibitor benzyloxycarbonyl-leucyl-leucinal attenuated TNF-α-induced hepatocyte apoptosis.
Conclusion:  TNF-α via NHE results in hepatocyte apoptosis through the calcium/calpain/Bcl-xL pathway.     

Energetics of the Na+ Pump in the Heart

Movement of ions across the cell membrane is driven by the free energy released from ATP hydrolysis. Here we will briefly review the chemistry of Na⁺, K⁺-ATPase reaction; we will also review some recent results defining the relationship between the energetic driving force and Na⁺ efflux by the Na⁺ pump in the normal myocardium and then discuss this relationship for pathophysiologic states in the heart.     

Na+/Ca2+ Exchanger Expression and Function in a Rabbit Model of Myocardial Infarction

Introduction: In general, sarcolemmal Na⁺/Ca²⁺ exchanger (NCX) protein and activity is increased in hearts with ventricular dysfunction. However, in a subset of studies, reduced activity of NCX has been reported. Left ventricular dysfunction (LVD) was induced in the rabbit eight weeks after an apical myocardial infarction.
Methods: Using single microelectrode voltage clamp to assess the NCX activity in isolated ventricular cells, a decrease in NCX activity by ∼30% was observed. Immunoblot analysis indicated increased NCX protein levels by ∼20% in the LVD group. The cause of this paradox is unknown. Overexpression of the protein sorcin increased the activity of NCX without affecting NCX protein levels.
Results: Sorcin protein (dimer) levels were significantly lower in the LVD group (0.67 ± 0.05 n = 15, P < 0.05) compared to sham (1.0 ± 0.16, n = 15). Sorcin monomer levels were not significantly different (sham: 1.0 ± 0.26, LVD: 0.83 ± 0.13). Mathematical modeling of NCX suggests that a reduction of NCX activity during diastole to that in LVD could be achieved by holding the diastolic membrane potential at −60 mV instead of −80 mV. Holding E_m at −60 mV decreased NCX-mediated Ca²⁺ efflux rates to values comparable to those seen in LVD and increased SR Ca²⁺ content and peak systolic [Ca²⁺] in sham and LVD cardiomyocytes.
Conclusions: In conclusion, reduced sorcin expression may be linked to the lower NCX activity in the rabbit model of LVD. Reduced NCX activity during diastole increases SR Ca²⁺ content and Ca²⁺ transient amplitude.     

Cytoprotective effect of epidermal growth factor on acid- and pepsininduced damage to rat gastric epithelial cells: Roles of Na+/H+ exchangers

We have previously established a cell damage model, with damage induced by either acid or pepsin treatment for 30 min, involving a rat gastric epithelial cell line (RGM1). In the present study, pretreatment of cells with epidermal growth factor (EGF; 0.1–10ng/mL) or sucralfate (0.1–3 mg/mL) for 4 h prevented such cell damage in a concentration-dependent manner. Protection of cells by these drugs was not affected by pretreatment with indomethacin (10⁻⁵ mol/L) for 4 h. Removal of Na⁻, but not Ca²⁺, from the acidified medium totally abolished the inhibitory effect of EGF, but not that of sucralfate. Genistein (a tyrosine kinase inhibitor) apparently reduced the inhibitory effect of EGF. DNA synthesis by RGM1 cells did not increase when cells were incubated with EGF for 4 h. We conclude that both EGF and sucralfate protect RGM1 cells from acid- and pepsin-induced damage and that the mechanism of protection by EGF against acid-induced damage seems to be via activation of Na⁺/H⁺ exchangers.     

Na+ Current in Human Ventricle: Implications for Sodium Loading and Homeostasis

The Na current (I_Na) in human ventricle is carried through a specific isoform of the voltage gated Na channel in heart. The pore forming α-subunit is encoded by the gene SCN5A . Up to four β-subunits may be associated, and the larger macromolecular complex may include attachments to cytoskeleton and scaffolding proteins, all of which may affect the gating kinetics of the current. I_Na underlies initiation and propagation of action potentials in the heart and plays a prominent role in cardiac electrophysiology and arrhythmia. In addition, I_Na also loads the ventricular cell with Na⁺ ions and plays an important role in intracellular Na homeostasis. This review considers the structure and function of the human cardiac Na channel that carries I_Na with a particular consideration of the implications of alterations in I_Na in acquired cardiac diseases such as hypertrophy, failure, and ischemia, which affect Na loading.     

pH-Regulated Na(+) influx into the mammalian ventricular myocyte: the relative role of Na(+)-H(+) exchange and Na(+)-HCO Co-transport

In the heart, intracellular Na(+) concentration (Na(+) (i)) is a controller of intracellular Ca(2+) signaling, and hence of key aspects of cell contractility and rhythm. Na(+) (i) will be influenced by variation in Na(+) influx. In the present work, we consider one source of Na(+) influx, sarcolemmal acid extrusion. Acid extrusion is accomplished by sarcolemmal H(+) and HCO(3) (-) transporters that import Na(+) ions while exporting H(+) or importing HCO(3) (-). The capacity of this system to import Na(+) is enormous, up to four times the maximum capacity of the Na(+)-K(+) ATPase to extrude Na(+) ions from the cell. In this review we consider the role of Na(+)-H(+) exchange (NHE) and Na(+)-HCO(3) (-)co-transport (NBC) in mediating Na(+) influx into cardiac myocytes. We consider, in particular, the role of NBC, as so little is known about Na(+) influx through this transporter. We show that both proteins mediate significant Na(+) influx and that although, in the ventricular myocyte, NBC-mediated Na(+) influx is less than through NHE, the proportions may be altered under a variety of conditions, including exposure to catecholamines, membrane depolarization, and interference with activity of the enzyme, carbonic anhydrase.     

Membrane Time Constant During Internal Defibrillation Strength Shocks in Intact Heart: Effects of Na+ and Ca2+ Channel Blockers

Introduction: We assessed defibrillation strength shock-induced changes of the membrane time constant (τ) and membrane potential (ΔVm) in intact rabbit hearts after administration of lidocaine, a sodium (Na⁺) channel blocker, or nifedipine, a L-type calcium (Ca²⁺) channel blocker.
Methods and Results: We optically mapped anterior, epicardial, electrical activity during monophasic shocks (±100, ±130, ±160, ±190, and ±220 V; 150 μF; 8 ms) applied at 25%, 50%, and 75% of the action potential duration via a shock lead system in Langendorff-perfused hearts. The protocol was run twice for each heart under control and after lidocaine (15 μM, n = 6) or nifedipine (2μM, n = 6) addition. τ in the virtual electrode area away from the shock lead was approximated with single-exponential fits from a total of 121,125 recordings. The same data set was used to calculate ΔVm. We found (1) Under all conditions, there is inverse relationship between τ and ΔVm with respect to changes of shock strength, regardless of shock polarity and phase of application: a stronger shock resulted in a larger ΔVm, which corresponded to a smaller τ (faster cellular response); (2) Lidocaine did not cause appreciable changes in either τ or ΔVm versus control, and (3) Nifedipine significantly increased both τ and ΔVm in the virtual cathode area; in contrast, in the virtual anode area, this effect depended on the phase of shock application.
Conclusion: τ and ΔVm are inversely related. Na⁺ channel blocker has minimal impact on either τ or ΔVm. Ca²⁺ blocker caused polarity and phase-dependent significant changes in τ and ΔVm.     

Co-ordinated Increase of Sodium Leak and Sodium Pump in Hereditary Spherocytosis

The presence of an increased sodium leak into hereditary spherocytes up to three times normal has been confirmed from measurement of ²²Na⁺ influx into red cells. ²²Na⁺ influx was not identical to the net inward leak of Na⁺ but the two kept a constant relationship since influx was about double the net Na⁺ gain in both normal and abnormal cells incubated with ouabain. The intracellular concentration of Na⁺ ions in fresh cells from hereditary spherocytes was identical to that in normal red cells. Hereditary spherocytes showed increased maximal activity of a membrane ATP-ase inhibited by ouabain and the Na⁺ concentration giving half-maximal activation of this ATP-ase was the same for spherocytes and normal cells. When the ²²Na⁺ influx into red cells from nine patients with hereditary spherocytosis was compared with maximal activity of cation pump ATP-ase in the same cells a significant correlation was obtained ( r = 0.78, P > 0.01). The results indicate some linkage between the increase in the Na⁺ leak and functional number of active cation pumps in the membrane of hereditary spherocytes.     

Chronic myelogenous leukaemia CD34+ cells exit G0/G1 phases of cell cycle more rapidly than normal marrow CD34+ cells

To investigate the mechanisms behind the leukaemic expansion of chronic myelogenous leukaemia (CML), we examined the cell cycle status and activation kinetics of purified subpopulations of CD34⁺ cells from normal and CML bone marrow (BM). Propidium iodide staining was used to assess cell cycle status of fresh cells or those stimulated with cytokines. Although the cell cycle status of fresh low-density cells from CML and normal BM was similar, a larger percentage of CML CD34⁺ cells were cycling than those from normal BM. The HLA-DR⁻ compartment of CML CD34⁺ cells, a fraction enriched for normal, non-leukaemic progenitors, contained a higher percentage of quiescent cells than the CD34⁺ HLA-DR⁺ fraction. When the activation of CD34⁺ cells was examined in response to SCF or IL-3 alone, or SCF+IL-3+IL-6, CML CD34⁺ cells exited G₀/G₁ more rapidly than normal CD34⁺ cells. Interestingly, although normal BM CD34⁺ cells failed to cycle in response to IL-6 alone, or in the absence of exogenous cytokines, 30% of CML cells cycled under these conditions. No differences in the degree of apoptosis were documented among CML and normal CD34⁺ cells in these cultures. These data suggest that enhanced cell cycle activation of CML CD34⁺ cells, by either autocrine stimuli or via enhanced sensitivity to exogenous stimuli, may be partially responsible for the pronounced cellular expansion characteristic of CML.     

Function, Regulation, Pharmacology, and Molecular Structure of ATP-Sensitive K+ Channels in the Cardiovascular System

K_ATP Channels in Cardiovascular System. ATP-sensitive K⁺ (K_ATP) channels are inhibited by intracellular ATP and activated by intracellular nucleoside diphosphates, and thus provide a link between cellular metabolism and excitability. K_ATP channels are widely distributed in various tissues and may be associated with diverse cellular functions. In the heart, the K_ATP channel appears to be activated during ischemic or hypoxic conditions and may be responsible for the increase of K⁺ efflux and shortening of the action potential duration. Therefore, opening of this channel may result in cardioprotective as well as proarrhythmic effects. In the vascular smooth muscle, the K_ATP channel is believed to mediate the relaxation of vascular tone. Thus, K_ATP channels play important regulatory roles in the cardiovascular system. Furthermore, K_ATP channels are the targets of two important classes of drugs, i.e., the antidiabetic sulfonylureas, which block the channels, and a series of vasorelaxants called "K⁺ channel openers," which tend to maintain the channels in an open conformation. Recently, the molecular structure of K_ATP channels has been clarified. The K_ATP channel in pancreatic β-cells is a complex composed of at least two subunits, a member of inwardly rectifying K⁺ channels and a sulfonylurea receptor. Subsequently, two additional homologs of the sulfonylurea receptor, which form cardiac and smooth muscle type K_ATP channels, respectively, have been reported. Further works are now in progress to understand the molecular mechanisms of K_ATP channel function.     

Comparison of Haematological Features of the β0 and β+ Thalassaemia Traits in Jamaican Negroes

Haematological characteristics have been compared in 29 subjects with heterozygous β⁰ thalassaemia and in 33 subjects with heterozygous β⁺ thalassaemia, identified by the type of sickle cell-β thalassaemia among close relatives, in a Jamaican Negro population. Total haemoglobin, MCV and MCH were significantly lower in the β⁰ type but the level of Hb A₂ was not significantly different. Individual values for MCV, MCH and Hb A₂ in the β⁺ type occasionally overlapped those in the normal population casting doubt on the adequacy of these criteria in identifying all cases of heterozygous β⁺ thalassaemia. The haematological differences are those which would be expected on theoretical grounds. The inability to confidently differentiate the two types of heterozygous β thalassaemia has implications for genetic counselling. The inability to distinguish heterozygous β⁺ thalassaemia from normals on any single haematological index suggests that surveys depending on estimations of Hb A₂ or on MCV alone may have underestimated the prevalence of the β⁺ thalassaemia gene.     

Eosinophilia associated with proliferation of CD3+4−8−αβ+ T cells with chromosome 16 anomalies

We describe a patient with eosinophilia and an abnormal CD3⁺4⁻8⁻αβ⁺ T-cell population. Chromosomal analysis of sorted CD3⁺4⁻8⁻ cells revealed abnormal karyotypes on chromosome 16. In the presence of IL-2 the production of IL-5 from CD3⁺4⁻8⁻ cells was higher than that from CD3⁺4⁺/8⁺ cells. Eosinophil survival-enhancing activity in the patient serum was inhibited by a combination of anti-IL-5 and anti-GM-CSF monoclonal antibodies. These data suggest that increased production of IL-5 and GM-CSF from the abnormal CD3⁺4⁻8⁻ cells might cause eosinophilia.     

CD3+ CD8+ CD56− clonal large granular lymphocyte leukaemia and HIV infection

High-grade malignant lymphomas associated with HIV infection are usually derived from B lymphocytes. Although a broad spectrum of T-cell-derived malignancies has been described, no case of monoclonal T large granular lymphocyte leukaemia has been reported to date. We report a case of clonal T-LGL (CD3⁺, CD4⁻, CD8⁺, CD56⁻, CD57⁺) in an HIV-infected, HTLV1/2-negative individual. Large granular lymphocytes are thought to represent activated cytotoxic T lymphocytes. HIV infection, as previously reported for HTLV1/2, may represent a pathway of antigen activation and lead to clonal expansion of T large granular lymphocytes.     

M9+ WINS Blood Group Phenotype: Further Observations

Red cells carrying the low-frequency MNS antigen M⁸ reacted with the only example of anti-DANE, an antibody which had previously defined the GP.Dane (Mi.IX) phenotype. Furthermore, M^g + cells reacted with the original anti-Mur (serum of Mrs. Murrell), but with none of 14 other anti-Mur. Therefore, M⁸ + cells carry both DANE antigen and an atypical Mur antigen. Immunoblotting of membranes from M^g + cells with anti-M, and with eluates prepared from anti-M^g and Mrs. Murrell's serum demonstrated a glycophorin A (GPA) molecule whose mobility was increased by an apparent M_r of about 3,000 presumably due to the loss of the three O-glycans known to be absent from M^g -active GPA.     

Homozygous deletional α+ thalassaemia associated with unequal expression of the two remaining α1 genes (α1A and α1Q)

S ummary . A Cambodian family presenting several haemoglobinopathies, Hb E, Hb Q and α⁺ thalassaemia, has been investigated. DNA analysis showed that the thalassaemia syndrome corresponds to a leftward type (4.2 kb) deletional from of α⁺ thalassaemia. Genotypes found in the family are: propositus -α^A/-α^Q, β^A/β^E, mother and older sister α^Aα^A/ -α^Q, β^A/β^E; father α^Aα^A/-α^A, β^A/β^A. The propositus consistently presents an α^Q/α^A chain ratio of 60/40 although both chains are products of α₁ loci. The relatively higher expression of the α^Q chain is not observed in the mother and therefore makes it unlikely to reflect anything other than differential expression of the maternal -α^Q/ and paternal -α^A/ haplotypes. This observation raises the possibility that both haplotypes are not strictly identical and that the region of the cross-over event is important for α gene expression.     

CD3−CD4−CD8−CD56−CD19−CD14− cells and CD3+CD8 dull-positive cells produce IL-4 in AIDS patients

We used flow cytometry to identify the presence of intracellular cytokines (cytoflow) and analyse the production of IL-4 in peripheral blood from AIDS patients who have practically no CD4⁺ T cells. We found that IL-4 was produced by CD3⁻CD4⁻CD8⁻CD56⁻CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells in AIDS patients. Moreover, CD3⁻CD4⁻ CD8⁻CD56⁻CD19⁻CD14⁻ cells had helper activity for immunoglobulin synthesis. These findings indicate that instead of CD4⁺ T helper cells, C3⁻CD4⁻CD8⁻CD56⁻ CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells may be an important source of IL-4 in a variety of immune responses for AIDS patients.     

Analysis of clinical AIDS-free interval after CD4+ cell counts fall below 200 × 106 L−1 in Japanese Haemophiliacs infected with HIV-1

We analysed the time from the date CD4+ cell counts fell below 200 × 10⁶ L⁻¹, defined as t _i, to the onset of clinical AIDS, according to the 1987 Centers for Disease Control and Prevention case definition, in 129 Japanese Haemophilia patients infected with HIV-1. The cumulative onset of clinical AIDS was analysed by the Kaplan–Meier method and proportional hazard model. Incorporated covariates were age of each patient at time t _i, as well as CD4+ and CD8+ cell counts, serum levels of IgG, IgA, IgM, GOT and GPT at t _i. The time of antiretroviral treatment initiation was also considered. The 50% AIDS-free interval after t _i was 3.00 years (95% confidence interval (CI), range 0.49–5.51) and 1.71 years (95% CI, range 0.66–2.76) for the patients at CDC stage II and stage III, respectively (significantly different, P = 0.0013). Among the patients at CDC stage II at t _i, higher levels of IgA were tightly associated with a shorter period from t _i to onset of clinical AIDS ( P < 0.0001), and relative hazard was 1.35 (95% CI, 1.11–1.64) with increase of IgA level by 1.0 g L⁻¹. Thus there is a broad distribution in the time to onset of clinical AIDS in Japanese Haemophiliacs even after CD4+ cell counts fall below 200 × 10⁶ L⁻¹. This should be taken into consideration in deciding upon the therapy and care of HIV-1 infected people.     

In vivo and in vitro effects of the pineal gland and melatonin on [Ca2++ Mg2+]-dependent ATPase in cardiac sarcolemma

Abstract: The possible diurnal variation in cardiac [Ca²⁺+ Mg²⁺]-dependent ATPase (Ca²⁺ pump) activity and the influence of pinealectomy and melatonin on this enzyme in rat heart have been studied. Lowest levels of cardiac sarcolemma] membrane [Ca²⁺+ Mg²⁺]-dependent ATPase activity were measured in late afternoon in rats kept under a 14:10 light:dark cycle. Late in the dark phase the enzyme activity began to increase with the rise continuing until 0900, 3 hr after light onset. These time-dependent changes in [Ca²⁺+ Mg²⁺]-dependent ATPase activity did not occur in either pinealectomized or light-exposed rats suggesting that melatonin, secreted from the pineal gland during the night, induces the change in [Ca²⁺+ Mg²⁺]-dependent ATPase activity. In vitro studies in which cardiac tissue was incubated in the presence of melatonin over a wide range of doses showed that this indole stimulated the Ca²⁺ pump. The half-maximal effect of melatonin was observed at a melatonin concentration of 28 ng/ml. These findings suggest that the daily change in [Ca²⁺+ Mg²⁺]-dependent ATPase activity in the sarcolemma of heart tissue is a result of the circadian rhythm in pineal melatonin production and secretion. These findings may be applicable to normal cardiac physiology.     

Identification of biclonal (duplex) leukaemic cells expressing either CD4+/CD8- or CD4-/CD8+ from a patient with adult T-cell leukaemia/lymphoma

A 24-year-old Japanese woman was admitted to our hospital in 1987 with a chief complaint of skin eruptions, and was diagnosed as having chronic ATLL. In 1993 the leucocyte count increased gradually to 126.0x10⁹/l with 91.5% abnormal lymphocytes expressing two different types of antigenicity, either CD4⁺/CD8^- or CD4^-/CD8⁺. Monoclonal integration of human T-cell lymphotropic virus type-I proviral DNA was detected at different sites of the genomic DNA in each cell type. These studies clearly indicate that CD4⁺/CD8^- and CD4^-/CD8^- leukaemic cells originated from two independent clones.