Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages.

Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.

Influence of sex hormone disturbances on the internal structure of the mandible in newborn mice.

It has not yet been clarified how sex hormones affect craniofacial bone development immediately after birth. The purpose of this study was to examine the effects of sex hormone deficiency on craniofacial bone development immediately after birth, in terms of the internal structure of the mandible in newborn mice with orchiectomy (ORX) and ovariectomy (OVX). ORX, OVX and a sham-operation were performed on 40 five-day-old C57BL/6J mice. Eight weeks after surgery, each mandible was subjected to histomorphometric analysis of trabecular (Tr) and cortical (Ct) bone mineral density (BMD) by peripheral quantitative computed tomography (pQCT). In the experimental groups, a significant reduction in BMD was found in comparison with the control groups. In histomorphometric analysis, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the condyle and the thickness of the condylar cartilage layer was significantly greater in the experimental mice than in the controls. Trabecular bone volume of the condyle measured on azocarmine-aniline blue (AZAN) sections was significantly less in the experimental mice than in the controls. These results indicate that mandibular growth is inhibited by sex hormone disturbances and the relevant internal structures changed. The findings show that sex hormones are one of the key determinants of mandibular growth and development immediately after birth.

Amyloid beta protein deposition in osteopetrotic (op/op) mice is reduced by injections of macrophage colony stimulating factor.

The deposition of amyloid beta (Abeta) protein is a neuropathological change that characterizes Alzheimer's disease. Animals with the osteopetrosis (op/op) mutation suffer from a general skeletal sclerosis, a significantly reduced number of macrophages and osteoclasts in various tissues, and have no systemic macrophage colony stimulating factor (M-CSF). This study examined the effect that M-CSF injections had on Abeta deposition and microglial cell distribution in the brains of normal and op/op mice. Abeta-positive plaques were detected in the cerebral cortex of op/op mice, but not in normal mice. M-CSF reduced the numbers of Abeta-positive plaques in op/op mice. The microglial cell population was reduced in op/op mice compared with normal mice, and M-CSF increased the numbers to 65.8% of that observed in normal mice. Our results suggest that a clearer understanding of the role that microglial cells play in Abeta deposition may help determine the mechanisms involved in the pathogenesis of Alzheimer's disease.

Effects of cyclic tensile forces on the expression of vascular endothelial growth factor (VEGF) and macrophage-colony-stimulating factor (M-CSF) in murine osteoblastic MC3T3-E1 cells.

It has been reported that vascular endothelial growth factor (VEGF), expressed by osteoblasts, can induce osteoclast recruitment and thus affects bone remodeling. The purpose of this study was to investigate the effects of cyclic tensile forces on the expression of VEGF and macrophage-colony-stimulating factor (M-CSF) in osteoblastic MC3T3-E1 cells. VEGF and M-CSF gene expression and protein concentration were determined by real-time PCR and enzyme-linked immunoassay. The expression of VEGF and M-CSF mRNA in the experimental group was higher than in the control group. The increase in the concentration of VEGF and M-CSF protein in the experimental group was time-dependent. Moreover, gadolinium (an S-A channel inhibitor), but not nifedipine (L-Type Ca2+ channel blocker), treatment reduced the concentration of VEGF and M-CSF mRNA and protein in the experimental groups. These findings suggest that cyclic tensile forces increase the expression of VEGF and M-CSF in osteoblastic MC3T3-E1 cells via a stretch-activated channel (S-A channel).

Effects of sex hormone disturbances on craniofacial growth in newborn mice.

It is well-known that sex hormones influence bone metabolism. However, it remains unclear as to how sex hormones affect bone growth in newborn mice. In this study, we performed orchiectomy (ORX) and ovariectomy (OVX) on newborn mice, and examined the effects on craniofacial growth morphometrically. ORX and OVX were performed on five-day-old C57BL/6J mice. Four weeks after surgery, lateral cephalograms were taken of all of the mice, with the use of a rat and mouse cephalometer. Cephalometric analysis of the craniofacial skeleton was performed by means of a personal computer. Inhibition of craniofacial growth was found in the experimental groups but not in the sham-operated groups. In the nasomaxillary bone and mandible, the amount of growth was significantly reduced. These results suggest that craniofacial growth is inhibited by sex hormone disturbances not only in puberty but also immediately after birth.

A mechanism of Ca2+ release from Ca2+ stores coupling to the Na+/Ca2+ exchanger in cultured smooth muscle cells.

We previously observed Ca2+ release from intracellular Ca2+ stores caused by reduction in extracellular Na+ concentration ([Na+]o). The purpose of this study was to determine whether lowering [Na+]o can elicit Ca2+ release from Ca2+ stores via the Na+/Ca2+ exchanger and to elucidate the mechanisms related to the Ca2+ release pathway in cultured longitudinal smooth muscle cells obtained from guinea pig ileum. Low [Na+]o-induced Ca2+ release was inhibited by antisense oligodeoxynucleotides for Na+/Ca2+ exchanger type 1 (anti-NCX). Application of anti-NCX to cells attenuated both the number of Ca2+ responding cells and the expression of the exchanger. Moreover, microinjection of heparin, a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, into the cells inhibited low [Na+]o-induced Ca2+ release. These findings suggest that low [Na+]o-induced Ca2+ release occurs through an IP3-induced Ca2+ release mechanism due to changes in the Ca2+ flux regulated by the Na+/Ca2+ exchanger.

NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell.

The NHE6 protein is a unique Na(+)/H(+) exchanger isoform believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. When green fluorescent protein was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the endoplasmic reticulum (ER) membrane. The localization pattern was the same as that of fusion proteins in which green fluorescent protein was fused after H2 of NHE1. In an in vitro system, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H-resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.

Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters.

Deficiency of delta-sarcoglycan (delta-SG), a component of the dystrophin-glycoprotein complex, causes cardiomyopathy and skeletal muscle dystrophy in Bio14.6 hamsters. Using cultured myotubes prepared from skeletal muscle of normal and Bio14.6 hamsters (J2N-k strain), we investigated the possibility that the delta-SG deficiency may lead to alterations in ionic conductances, which may ultimately lead to myocyte damage. In cell-attached patches (with Ba(2+) as the charge carrier), an approximately 20-pS channel was observed in both control and Bio14.6 myotubes. This channel is also permeable to K(+) and Na(+) but not to Cl(-). Channel activity was increased by pressure-induced stretch and was reduced by GdCl(3) (>5 microM). The basal open probability of this channel was fourfold higher in Bio14.6 myotubes, with longer open and shorter closed times. This was mimicked by depolymerization of the actin cytoskeleton. In intact Bio14.6 myotubes, the unidirectional basal Ca(2+) influx was enhanced compared with control. This Ca(2+) influx was sensitive to GdCl(3), signifying that stretch-activated cation channels may have been responsible for Ca(2+) influx in Bio14.6 hamster myotubes. These results suggest a possible mechanism by which cell damage might occur in this animal model of muscular dystrophy.

Stretch-induced cell damage in sarcoglycan-deficient myotubes.

Sarcoglycans (SGs) are components of the dystrophin-glycoprotein complex, genetic defects in which cause skeletal muscle dystrophy and cardiomyopathy in humans and animals. To obtain insight into the roles of SGs, we characterized properties of myotubes prepared from cells of the rat L6 line or primary myoblast cultures of rat gastrocnemius muscle that were made SG-deficient by treatment with antisense oligodeoxynucleotides (AS-ODNs). Immunoblot and immunoprecipitation analyses revealed that dystrophin and its remaining associated proteins were tightly associated in these cells despite SG deficiency. 45Ca2+ influx into SG AS-ODN-treated L6 myotubes under resting conditions was significantly higher (1.7-fold at 6 min) than in controls, suggesting that Ca2+ influx is activated in these SG-deficient myotubes. When these cells were subjected to cyclic elongation of up to 20% for 1 h, a marked increase in creatine phosphokinase (CK) release into the medium was observed. Nifedipine, tranilast, FK506 and E64 or intracellular loading with 1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid, tetrakis(acetoxymethyl)ester (BAPTA/AM) reduced the stretch-induced CK release; a raised extracellular [Ca2+] increased CK release. The stretch-induced damage to SG-deficient myotubes thus appears to be caused by alterations in cell Ca2+ homeostasis. A similar abnormality in Ca2+ handling has been reported for myoctes from mdx mice or dystrophin-deficient patients, in whom SGs are also greatly reduced or absent. Thus it is possible that SG deficiency may play a critical role in the pathology of dystrophin-deficient muscle.

Cardiac Na(+)-Ca(2+) exchange: molecular and pharmacological aspects.

The Na(+)-Ca(2+) exchanger (NCX) is one of the essential regulators of Ca(2+) homeostasis in cardiomyocytes and thus an important modulator of the cardiac contractile function. The purpose of this review is to survey recent advances in cardiac NCX research, with particular emphasis on molecular and pharmacological aspects. The NCX function is thought to be regulated by a variety of cellular factors. However, data obtained by use of different experimental systems often appear to be in conflict. Where possible, we endeavor to provide a rational interpretation of such data. We also provide a summary of current work relating to the structure and function of the cardiac NCX. Recent molecular studies of the NCX protein are beginning to shed light on structural features of the ion translocation pathway in the NCX membrane domain, which seems likely to be formed, at least partly, by the phylogenetically conserved alpha-1 and alpha-2 repeat structures and their neighboring membrane-spanning segments. Finally, we discuss new classes of NCX inhibitors with improved selectivity. One of these, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), appears to exhibit unique selectivity for Ca(2+)-influx-mode NCX activity. Data obtained with these inhibitors should provide a basis for designing more selective and clinically useful drugs targeting NCX.

Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers.

The Na+/H+ exchangers (NHEs) comprise a family of transporters that catalyze cell functions such as regulation of the pH and volume of a cell and epithelial absorption of Na+ and bicarbonate. Ubiquitous calcineurin B homologous protein (CHP or p22) is co-localized and co-immunoprecipitated with expressed NHE1, NHE2, or NHE3 independently of its myristoylation and Ca2+ binding, and its binding site was identified as the juxtamembrane region within the carboxyl-terminal cytoplasmic domain of exchangers. CHP binding-defective mutations of NHE1-3 or CHP depletion by injection of the competitive CHP-binding region of NHE1 into Xenopus oocytes resulted in a dramatic reduction (>90%) in the Na+/H+ exchange activity. The data suggest that CHP serves as an essential cofactor, which supports the physiological activity of NHE family members.

Inhibitory effect of 2,3-butanedione monoxime (BDM) on Na(+)/Ca(2+) exchange current in guinea-pig cardiac ventricular myocytes.

1. The effect of 2,3-butanedione monoxime (BDM), a 'chemical phosphatase', on Na(+)/Ca(2+) exchange current (I(NCX)) was investigated using the whole-cell voltage-clamp technique in single guinea-pig cardiac ventricular myocytes and in CCL39 fibroblast cells expressing canine NCX1. 2. I(NCX) was identified as a current sensitive to KB-R7943, a relatively selective NCX inhibitor, at 140 mM Na(+) and 2 mM Ca(2+) in the external solution and 20 mM Na(+) and 433 nM free Ca(2+) in the pipette solution. 3. In guinea-pig ventricular cells, BDM inhibited I(NCX) in a concentration-dependent manner. The IC(50) value was 2.4 mM with a Hill coefficients of 1. The average time for 50% inhibition by 10 mM BDM was 124+/-31 s (n=5). 4. The effect of BDM was not affected by 1 microM okadaic acid in the pipette solution, indicating that the inhibition was not via activation of okadaic acid-sensitive protein phosphatases. 5. Intracellular trypsin treatment via the pipette solution significantly suppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM. 6. PAM (pralidoxime), another oxime compound, also inhibited I(NCX) in a manner similar to BDM. 7. Isoprenaline at 50 microM and phorbol 12-myristate 13-acetate (PMA) at 8 microM did not reverse the inhibition of I(NCX) by BDM. 8. BDM inhibited I(NCX) in CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines. 9. We conclude that BDM inhibits I(NCX) but the mechanism of inhibition is not by dephosphorylation of the Na(+)/Ca(2+) exchanger as a 'chemical phosphatase'.

Structural domains influencing sensitivity to isothiourea derivative inhibitor KB-R7943 in cardiac Nna(+)/Ca(2+) exchanger.

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) is a potent and selective Na(+)/Ca(2+) exchange (NCX) inhibitor that is 3-fold more inhibitory to NCX3 than to NCX1 or NCX2. Here we searched for amino acid residues that may form the KB-R7943 receptor in the exchanger by analyzing the function of chimeras between NCX1 and NCX3 as well as of their site-directed mutants. We found that the highly conserved alpha-2 repeat of the exchanger is almost exclusively responsible for the difference in drug response of the isoforms. Such difference was mostly reproduced by single substitutions of residues in the alpha-2 repeat (V820G or Q826V in NCX1 and A809V or A809I in NCX3), suggesting their importance in drug sensitivity. Cysteine scanning mutagenesis of the alpha-2 repeat of NCX1 identified one residue (Gly833) that caused a large (> or = 30-fold) reduction in drug sensitivity. We found that the Gly-to-Thr substitution caused even larger reduction in drug sensitivity. Interestingly, extracellularly applied KB-R7943 at 0.8 microM markedly inhibited the whole-cell outward exchange current, whereas the drug applied intracellularly at 30 microM did not. These results suggest that KB-R7943 inhibits the exchanger from the external side in intact cells and that a region of the alpha-2 repeat of NCX1 containing Gly833 may participate in the formation of the drug receptor. Because we suggested previously that Gly833 is accessible from the inside of a cell, the results raised an interesting possibility that this residue may alter its position during Na(+)/Ca(2+) exchange in such a way that it becomes accessible to external drug.

[Pharmaceutical agents for calcium-induced cell damage (Na+/Ca2+ exchange inhibitor)].

Recently, a novel selective inhibitor of the Ca(2+) influx mode of the Na(+)/Ca(2+) exchanger, KB-R7943, has been developed. This compound is expected to be a pharmaceutical agent that offers effective protection against ischemia/reperfusion-associated injury in several organs such as heart and kidney. Here, we summarize pharmacological profiles of KB-R7943, the molecular mechanism of its action, and its future prospect as a novel pharmaceutical agent.

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts.

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.

The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity.

We examined the membrane topology and functional importance of residues in regions of the Na(+)/Ca(2+) exchanger NCX1 encompassing the conserved internal alpha repeats by substituted cysteine scanning analysis and kinetic analysis of site-directed mutants. The results suggest that both the alpha-1 repeat and a region encompassing the alpha-2 repeat and its immediately C-terminal segment contain reentrant loop domains, each oriented in an opposite direction with respect to the membrane. We found that single or multiple mutations of six residues including Asn-125 and conserved aspartates Asp-130, Asp-825, and Asp-829 in the alpha repeat reentrant domains reduce the apparent affinity of the exchanger for extracellular Ca(2+) by up to 6-fold. In contrast, the triple cysteine mutation D130C/D825C/D829C did not influence the current-voltage (I-V) relationship of the exchange current. Cysteine accessibility scanning with different thiol modifiers suggested that N125C, D130C, and D825C may be located in a restricted aqueous space in the membrane accessible only to ions when examined with external probes, although N125C and D825C were previously shown to be internally accessible during exchange reaction. The results suggest that these reentrant domains in the alpha repeats may participate in the formation of the ion transport pathway in the exchanger with some of the aspartates possibly lining it or located close to it.

Targeted disruption of Na+/Ca2+ exchanger gene leads to cardiomyocyte apoptosis and defects in heartbeat.

Ca(2+), which enters cardiac myocytes through voltage-dependent Ca(2+) channels during excitation, is extruded from myocytes primarily by the Na(+)/Ca(2+) exchanger (NCX1) during relaxation. The increase in intracellular Ca(2+) concentration in myocytes by digitalis treatment and after ischemia/reperfusion is also thought to result from the reverse mode of the Na(+)/Ca(2+) exchange mechanism. However, the precise roles of the NCX1 are still unclear because of the lack of its specific inhibitors. We generated Ncx1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous Ncx1-deficient mice died between embryonic days 9 and 10. Their hearts did not beat, and cardiac myocytes showed apoptosis. No forward mode or reverse mode of the Na(+)/Ca(2+) exchange activity was detected in null mutant hearts. The Na(+)-dependent Ca(2+) exchange activity as well as protein content of NCX1 were decreased by approximately 50% in the heart, kidney, aorta, and smooth muscle cells of the heterozygous mice, and tension development of the aortic ring in Na(+)-free solution was markedly impaired in heterozygous mice. These findings suggest that NCX1 is required for heartbeats and survival of cardiac myocytes in embryos and plays critical roles in Na(+)-dependent Ca(2+) handling in the heart and aorta.

Physiological functions of the regulatory domains of the cardiac Na(+)/Ca(2+) exchanger NCX1.

Physiological functions of the intracellular regulatory domains of the Na(+)/Ca(2+) exchanger NCX1 were studied by examining Ca(2+) handling in CCL39 cells expressing a low-affinity Ca(2+) regulatory site mutant (D447V/D498I), an exchanger inhibitory peptide (XIP) region mutant displaying no Na(+) inactivation (XIP-4YW), or a mutant lacking most of the central cytoplasmic loop (Delta246-672). We found that D447V/D498I was unable to efficiently extrude Ca(2+) from the cytoplasm, particularly during a small rise in intracellular Ca(2+) concentration induced by the physiological agonist alpha-thrombin or thapsigargin. The same mutant took up Ca(2+) much less efficiently than the wild-type NCX1 in Na(+)-free medium when transfectants were not loaded with Na(+), although it appeared to take up Ca(2+) normally in transfectants preloaded with Na(+). XIP-4YW and, to a lesser extent, Delta246-672, but not NCX1 and D447V/D498I, markedly accelerated the loss of viability of Na(+)-loaded transfectants. Furthermore, XIP-4YW was not activated by phorbol ester, whereas XIP-4YW and D447V/D498I were resistant to inhibition by ATP depletion. The results suggest that these regulatory domains play important roles in the physiological and pathological Ca(2+) handling by NCX1, as well as in the regulation of NCX1 by protein kinase C or ATP depletion.

Lithium activates mammalian Na+/H+ exchangers: isoform specificity and inhibition by genistein.

Replacement of external NaCl with LiCl induced cytoplasmic alkalinization in CCL-39 cells and rat L6 myoblasts expressing the endogenous Na+/H+ exchanger isoform NHE1. This Li+-induced alkalinization is due to activation of the Na+/H+ exchanger because it was completely inhibited by 100 microM ethylisopropylamiloride (apparent Kd=1 microM) and because it did not occur in exchanger-deficient PS120 cells. The effect of Li+ was not mimicked by Na+, K+, Cs+ and choline+. Li+ caused cytoplasmic alkalinization in PS120 cells expressing NHE1 or NHE2, but not NHE3, when Li+ was added to cells at a concentration high enough to saturate their external transport sites as predicted from Li+ affinities. Li+ did not induce phosphatidylinositol (PI) turnover or intracellular Ca2+ mobilization. Li+-induced alkalinization was not affected by protein kinase C down-regulation, loss of glycogen synthase kinase 3beta caused by antisense oligonucleotide treatment, or pretreatment with calphostin C, pertussis toxin, MEK inhibitor PD98059 and PI3-kinase inhibitor LY294002. However, it was markedly suppressed by the tyrosine kinase inhibitor genistein (10 microM). Thus, externally added Li+ activates NHE1 and NHE2 via a mechanism possibly involving a tyrosine kinase, causing an increase in cytoplasmic pH that could potentially affect various cell functions.