Role of the Na+-H+ exchanger (NHE1) in heart muscle function during transient acidosis. A study in papillary muscles from rat and guinea pig hearts

The sodium-hydrogen exchanger (NHE) helps the cell to recover from intracellular acidosis. In this study, we have investigated the effect of HOE 642 (a specific NHE1 blocker) on papillary muscles from rats and guinea pigs during transient acidosis and PKC activation by recording developed force (DF), action potential characteristics, and electrical conductance (stimulus-response interval). Two protocols were used, with or without HOE 642 (10(-5) mol/L): papillary muscle was exposed (i) for 15 min to a glucose-free, nonoxygenated HEPES buffer containing lactate (20 mmol/L) (pH 6.8) followed by 15 min recovery or (ii) to a PKC activator (phorbolmyristate acetate (PMA) (10(-9) mol/L)) for 30 min. The DF after acidification remained significantly decreased in the NHE-blocked papillary muscles. During recovery from acidosis, papillary muscles exposed to HOE 642 remained at a higher electrical resistance. The present study shows that post-acidotic continued depression of DF and change in tissue electrophysiological properties might occur as a result of blocking the NHE. During infarct development, the tissue-protecting effect of NHE blockade has been well documented. When acidosis or reduced contractile function is present, however, blocking NHE by HOE 642 might not improve the situation.     

Adenovirus-Mediated Gene Transfer of Inhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons

Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 m M potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 m M potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N -acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, not at later time points suggesting that IAPS delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 μ M or 1 m M glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

Blockade of calcium entry accelerates arsenite-mediated apoptosis in rat cerebellar granule cells

Arsenical exposure can cause defects in the central nervous system, yet the underlying cellular and molecular mechanisms are largely unknown. We have recently demonstrated that sodium arsenite induces apoptosis of cultured cortical and cerebellar neurons, suggesting that arsenite-induced neuronal apoptosis may contribute to at least some of its neurotoxic effects. Here we investigated the effect of Ca2+ on arsenite-mediated cerebellar granule neuron death. Sodium arsenite induced apoptosis in cerebellar neurons which were maintained in the presence of serum and depolarizing concentrations of potassium chloride (25 mM KCI). Under these conditions, inhibition of calcium entry by N-methyl-D-aspartate (NMDA) receptor blocker DL-aminophosphonovalerate (APV) or calcium channel antagonist nifedipine increased arsenite-induced apoptosis, while APV or nifedipine alone had little effect on cell viability. In cortical neurons or cerebellar neurons maintained at low potassium (5 mM), arsenite also induced apoptosis. However, the addition of APV or nifedipine did not alter levels of arsenite-induced apoptosis. These data suggest that arsenite-mediated apoptosis is regulated by intracellular calcium levels.     

Apoptosis-inducing and -preventing signal transduction pathways in cultured cerebellar granule neurons

1. Cultured cerebellar granule neurons maintained in medium containing 26 mM potassium (high K+ or HK+) undergo cell death when switched to medium with 5 mM potassium (low K+ or LK+). This low K(+)-induced cell death has typical features of apoptosis. The intracellular signaling pathway of low K(+)-induced apoptosis has been investigated. 2. Cerebellar granule neurons become committed to undergo apoptosis between 2 and 5 h after K+ deprivation, judging from the inability of high K+ to rescue them after this time. Although the levels of most mRNAs decrease markedly concomitant with commitment, expression of c-jun mRNA increases 2-3 h after K+ deprivation. Among the family of caspases, a caspase-3-like protease is activated within 4 h of lowering the K+ concentration. A caspase-1-like protease is also activated within 2 h of K+ deprivation. 3. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity by LY294002 or wortmannin also induces apoptosis in cerebellar granule neurons. The intracellular signaling pathway of LY294002-induced apoptosis has been investigated. The activity of c-Jun N-terminal kinase (JNK) increases 8 h after addition of LY294002 to high K+ medium or low K+ medium containing BDNF. Expression of c-Jun protein also increases almost simultaneously. 4. The low K(+)-induced apoptosis of cerebellar granule neurons is prevented by high K+ (membrane depolarization by high K+), BDNF, IGF-1, bFGF or cAMP. The intracellular signaling pathways by which these agents prevent low K(+)-induced apoptosis have been investigated. Agents other than cAMP prevent apoptosis through PI3-K and a Ser/Thr kinase, Akt/PKB. The survival-promoting effect of cAMP does not depend on the PI3-K-Akt pathway.     

Inhibition of NHE protects reoxygenated cardiomyocytes independently of anoxic Ca(2+) overload and acidosis

We investigated the question of whether inhibition of the Na(+)/H(+) exchanger (NHE) during ischemia is protective due to reduction of cytosolic Ca(2+) accumulation or enhanced acidosis in cardiomyocytes. Additionally, the role of the Na(+)-HCO(3)(-) symporter (NBS) was investigated. Adult rat cardiomyocytes were exposed to simulated ischemia and reoxygenation. Cytosolic pH [2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)], Ca(2+) (fura 2), Na(+) [sodium-binding benzolfuran isophthatlate (SBFI)], and cell length were measured. NHE was inhibited with 3 micromol/l HOE 642 or 1 micromol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and NBS was inhibited with HEPES buffer. During anoxia in bicarbonate buffer, cells developed acidosis and intracellular Na and Ca (Na(i) and Ca(i), respectively) overload. During reoxygenation cells underwent hypercontracture (44.0 +/- 4.1% of the preanoxic length). During anoxia in bicarbonate buffer, inhibition of NHE had no effect on changes in intracellular pH (pH(i)), Na(i), and Ca(i), but it significantly reduced the reoxygenation-induced hypercontracture (HOE: 61.0 +/- 1.4%, EIPA: 68.2 +/- 1.8%). The sole inhibition of NBS during anoxia was not protective. We conclude that inhibition of NHE during anoxia protects cardiomyocytes against reoxygenation injury independently of cytosolic acidification and Ca(i) overload.     

Distinct requirements for p38alpha and c-Jun N-terminal kinase stress-activated protein kinases in different forms of apoptotic neuronal death

The stress-activated protein kinases c-Jun-activated kinase (JNK) and p38 are implicated in neuronal apoptosis. Early studies in cell lines suggested a requirement for both in the apoptosis induced by withdrawal of nerve growth factor. However, studies in neuronal cells typically implicate JNK but not p38 in apoptosis. In some cases, p38 is implicated, but the role of JNK is undefined. It remains unclear whether p38 and JNK have differing roles dependent on cell type, apoptotic stimulus, or mechanism of cell death or whether they are redundant and each sufficient to induce identical forms of cell death. We investigate the relative roles of these protein kinases in different death mechanisms in a single system, cultured cerebellar granule neurons. Apoptosis induced by withdrawal of trophic support and glutamate are mechanistically different in terms of caspase activation, DNA fragmentation profile, chromatin morphology, and dependence on de novo gene expression. Caspase-independent apoptosis induced by glutamate is accompanied by strong activation of p38, and dominant negatives and inhibitors of the p38 pathway prevent this apoptosis. In contrast, withdrawal of trophic support induces caspase-dependent death accompanied by JNK-dependent phosphorylation of c-Jun, and inhibition of JNK is sufficient to prevent the death induced by withdrawal of trophic support. Inhibition of p38 does not block withdrawal of trophic support-induced death, nor does inhibition of JNK block glutamate-induced death. We propose that mechanistically different forms of apoptosis have differing requirements for p38 and JNK activities in neurons and demonstrate that only inhibition of the appropriate kinase will prevent neurons from undergoing apoptosis.     

Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca²⁺ current through both enhancement of Ca²⁺ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na⁺–H⁺ exchange (NHE), we examined the possible role of NHE in the regulation of I_Ca during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16 dyn/cm²) fluid was applied onto single rat ventricular myocytes, and the I_Ca was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the I_Ca (IC₅₀ = 0.97 ± 0.12 μM) without altering current–voltage relationships and inactivation time constants. In contrast, the I_Ca in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the I_Ca under FP was not altered by intracellular high Ca²⁺ buffering. Replacement of external Cl⁻ with aspartate to inhibit the Cl⁻-dependent acid loader eliminated the inhibitory effect of HOE642 on I_Ca. These results suggest that NHE may attenuate FP-induced I_Ca suppression by preventing intracellular H⁺ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca²⁺-dependent inhibition of the I_Ca during FP exposure.     

K+-dependent cerebellar granule neuron apoptosis. Role of task leak K+ channels

Rat mature cerebellar granule, unlike hippocampal neurons, die by apoptosis when cultured in a medium containing a physiological concentration of K+ but survive under high external K+ concentrations. Cell death in physiological K+ parallels the developmental expression of the TASK-1 and TASK-3 subunits that encode the pH-sensitive standing outward K+ current IKso. Genetic transfer of the TASK subunits in hippocampal neurons, lacking IKso, induces cell death, while their genetic inactivation protects cerebellar granule neurons. Neuronal death of cultured rat granule neurons is also prevented by conditions that specifically reduce K+ efflux through the TASK-3 channels such as extracellular acidosis and ruthenium red. TASK leak K+ channels thus play an important role in K+-dependent apoptosis of cerebellar granule neurons in culture.     

p38 mitogen-activated protein kinase regulates low potassium-induced c-Jun phosphorylation and apoptosis in cultured cerebellar granule neurons

Cultured rat cerebellar granule neurons are widely used as a model system for studying neuronal apoptosis. After maturation by culturing in medium containing 26 mm potassium (high K(+)), changing to medium containing 5 mm potassium (low K(+); LK) rapidly induces neuronal apoptosis. Then over 50% of granule cells die within 24 h. However, the molecular mechanisms by which the LK-induced apoptosis occurs in cultured cerebellar granule cells remain unclear. In the present study, we found that p38 MAP kinase (p38) was an important factor for LK-induced apoptosis. Three hours after changing to LK medium, p38 was markedly activated. In addition, SB203580, a specific inhibitor of p38, strongly inhibited the phosphorylation and expression of c-Jun in LK-induced apoptosis of cultured cerebellar granule cells. In vitro kinase assay using glutathione S-transferase-c-Jun as a substrate showed that p38 directly phosphorylated c-Jun. Furthermore, in the presence of SB203580, about 80% of neurons survived. These results indicate that p38 regulates LK-induced apoptosis of cerebellar granule neurons.     

pH nanoenvironment at the surface of single melanoma cells.

Extracellular pH and the Na(+)/H(+) exchanger (NHE1) modulate tumor cell migration. Yet, the pH nanoenvironment at the outer surface of the cell membrane (pH(em)) where cell/matrix interaction occurs and matrix metalloproteinases work was never measured. We present a method to measure this pH nanoenvironment using proton-sensitive dyes to label the outer leaflet of the plasma membrane or the glycocalyx of human melanoma cells. Polarized cells generate an extracellular proton gradient at their surface that increases from the rear end to the leading edge of the lamellipodium along the direction of movement. This gradient collapses upon NHE1 inhibition by HOE642. NHE1 stimulation by intracellular acidification increases the difference in pH(em) between the tips of lamellipodia and the cell body in a Na(+) dependent way. Thus, cells create a pH nanoenvironment that promotes cell migration by facilitating cell adhesion at their front and the release of cell/matrix contacts at their rear part.     

The Na+/H+ exchanger controls deoxycholic acid-induced apoptosis by a H+-activated, Na+-dependent ionic shift in esophageal cells

Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.     

Mechanisms of activation of NHE by cell shrinkage and by calyculin A in Ehrlich ascites tumor cells

The Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 were all found to be expressed in Ehrlich ascites tumor cells, as evaluated by Western blotting and confocal microscopy. Under unstimulated conditions, NHE1 was found predominantly in the plasma membrane, NHE3 intracellularly, and NHE2 in both compartments. Osmotic cell shrinkage elicited a rapid intracellular alkalinization, the sensitivity of which to EIPA (IC50 0.19 microM) and HOE 642 (IC50 0.85 microM) indicated that it predominantly reflected activation of NHE1. NHE activation by osmotic shrinkage was inhibited by the protein kinase C inhibitors chelerythrine (IC50 12.5 microM), G? 6850 (5 microM), and G? 6976 (1 microM), and by the p38 MAPK inhibitor SB 203580 (10 microM). Furthermore, hypertonic cell shrinkage elicited a biphasic increase in p38 MAPK phosphorylation, with the first significant increase detectable 2 minutes after the hypertonic challenge. Neither myosin light chain kinase-specific concentrations of ML-7 (IC50 40 microM) nor ERK1/2 inhibition by PD 98059 (50 microM) had any effect on NHE activation. Under isotonic conditions, the serine/threonine protein phosphatase inhibitor calyculin A elicited an EIPA- and HOE 642-inhibitable intracellular alkalinization, indicating NHE1 activation. Similarly, shrinkage-induced NHE activation was potentiated by calyculin A. The calyculin A-induced alkalinization was not associated with an increase in the free, intracellular calcium concentration, but was abolished by chelerythrine. It is concluded that shrinkage-induced NHE activation is dependent on PKC and p38 MAPK, but not on MLCK or ERK1/2. NHE activity under both iso- and hypertonic conditions is increased by inhibition of serine/threonine phosphatases, and this effect appears to be PKC-dependent.     

Suppression of death receptor signaling in cerebellar Purkinje neurons protects neighboring granule neurons from apoptosis via an insulin-like growth factor I-dependent mechanism

Neuronal apoptosis contributes to the progression of neurodegenerative disease. Primary cerebellar granule neurons are an established in vitro model for investigating neuronal death. After removal of serum and depolarizing potassium, granule neurons undergo apoptosis via a mechanism that requires intrinsic (mitochondrial) death signals; however, the role of extrinsic (death receptor-mediated) signals is presently unclear. Here, we investigate involvement of death receptor signaling in granule neuron apoptosis by expressing adenoviral, AU1-tagged, dominant-negative Fas-associated death domain (Ad-AU1-deltaFADD). Ad-AU1-deltaFADD decreased apoptosis of granule neurons from 65 +/- 5 to 27 +/- 2% (n = 7, p < 0.01). Unexpectedly, immunocytochemical staining for AU1 revealed that <5% of granule neurons expressed deltaFADD. In contrast, deltaFADD was expressed in >95% of calbindin-positive Purkinje neurons ( approximately 2% of the cerebellar culture). Granule neurons in proximity to deltaFADD-expressing Purkinje cells demonstrated markedly increased survival. Both granule and Purkinje neurons expressed insulin-like growth factor-I (IGF-I) receptors, and deltaFADD-mediated survival of granule neurons was inhibited by an IGF-I receptor blocking antibody. These results demonstrate that the selective suppression of death receptor signaling in Purkinje neurons is sufficient to rescue neighboring granule neurons that depend on Purkinje cell-derived IGF-I. Thus, the extrinsic death pathway has a profound but indirect effect on the survival of cerebellar granule neurons.     

Mitochondria are not required for death receptor-mediated cytosolic acidification during apoptosis

In addition to cell shrinkage, membrane blebbing, DNA fragmentation and phosphatidylserine exposure, intracellular acidification represents a hallmark of apoptosis. Although the mechanisms underlying cytosolic acidification during apoptosis remained largely elusive, a pivotal role of mitochondria has been proposed. In order to investigate the involvement of mitochondria in cytosolic acidification during apoptosis, we blocked the mitochondrial death pathway by overexpression of Bcl-2 and subsequently activated the death receptor pathway by anti-CD95 or TRAIL or the mitochondrial pathway by staurosporine. We show that Bcl-2 but not caspase inhibition prevented staurosporine-induced intracellular acidification. Thus, intracellular acidification in mitochondrial apoptosis is a Bcl-2-inhibitable, but caspase-independent process. In contrast, Bcl-2 only slightly delayed, but did not prevent intracellular acidification upon triggering of death receptors. The Na⁺/H⁺ exchanger NHE1 was partially degraded during apoptosis but only to a small extent and and at a delayed time point when cytosolic acidification was almost completed. We therefore conclude that cytosolic acidification is mitochondrially controlled in response to mitochondria-dependent death stimuli, but requires additional caspase-dependent mechanisms during death receptor-mediated apoptosis. Michaela Waibel, Stefan Kramer and Kirsten Lauber share equal first authorship.     

Activation of AMP-activated Protein Kinase Regulates Hippocampal Neuronal pH by Recruiting Na+/H+ Exchanger NHE5 to the Cell Surface

Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H⁺-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na⁺/H⁺ exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress.     

Activation of G Proteins Bidirectionally Affects Apoptosis of Cultured Cerebellar Granule Neurons

Abstract: Cultured cerebellar granule neurons maintained in depolarizing concentrations of K⁺ (25 m M ) and then switched to physiological concentrations of K⁺ (5 m M ) undergo apoptosis. We now report that activation of specific G proteins robustly and bidirectionally affects apoptosis of cultured rat cerebellar granule neurons. Stimulation of G_s with cholera toxin completely blocks apoptosis induced by nondepolarizing concentrations of K⁺, whereas stimulation of G_o/G_i with the wasp venom peptide mastoparan induces apoptosis of cerebellar granule neurons even in high (depolarizing) concentrations of K⁺. Moreover, pretreatment of cerebellar granule neurons with cholera toxin attenuates neuronal death induced by mastoparan. By contrast, pertussis toxin, cell-permeable analogues of cyclic AMP, and activators of protein kinase A do not affect apoptosis of cultured cerebellar granule neurons. These data suggest that G proteins may function as key switches for controlling the programmed death of mammalian neurons, especially in the developing CNS.