Neuronal intracellular pH directly mediates nitric oxide‐induced programmed cell death

Neuronal injury is intricately linked to the activation of three distinct neuronal endonucleases. Since these endonucleases are exquisitely pH dependent, we investigated in primary rat hippocampal neurons the role of intracellular pH (pH_i) regulation during nitric oxide (NO)‐induced toxicity. Neuronal injury was assessed by both a 0.4% Trypan blue dye exclusion survival assay and programmed cell death (PCD) with terminal deoxynucleotidyl transferase nick‐end labeling (TUNEL) 24 h following treatment with the NO generators sodium nitroprusside (300 μM), 3‐morpholinosydnonimine (300 μM), or 6‐(2‐hyrdroxy‐1‐methyl‐2‐nitrosohydrazino)‐N‐methyl‐1‐hexanamine (300 μM). The pH_i was measured using the fluorescent probe BCECF. NO exposure yielded a rapid intracellular acidification during the initial 30 min from pH_i 7.36 ± 0.01 to approximately 7.00 (p < .0001). Within 45 min, a biphasic alkaline response was evident, with pH_i reaching 7.40 ± 0.02, that was persistent for a 6‐h period. To mimic the effect of NO‐induced acidification, neurons were acid‐loaded with ammonium ions to yield a pH_i of 7.09 ± 0.02 for 30 min. Similar to NO toxicity, neuronal survival decreased to 45 ± 2% (24 h) and DNA fragmentation increased to 58 ± 8% (24 h) (p < .0001). Although neuronal caspases did not play a dominant role, neuronal injury and the induction of PCD during intracellular acidification were dependent upon enhanced endonuclease activity. Furthermore, maintenance of an alkaline pH_i of 7.60 ± 0.02 during the initial 30 min of NO exposure prevented neuronal injury, suggesting the necessity for the rapid but transient induction of intracellular acidification during NO toxicity. Through the identification of the critical role of both NO‐induced intracellular acidification and the induction of the neuronal endonuclease activity, our work suggests a potential regulatory trigger for the prevention of neuronal degeneration. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 171–184, 1999     

Nitric Oxide Induction of Neuronal Endonuclease Activity in Programmed Cell Death

Neuronal survival is intricately linked to the maintenance of intact DNA. In contrast, neuronal degeneration following nitric oxide (NO) exposure is dependent, in part, on the degradation of DNA through programmed cell death (PCD). We therefore investigated in primary rat hippocampal neurons the role of endogenous deoxyribonucleases, enzymes responsible for metabolically derived DNA cleavage, during NO-induced neurodegeneration. Twenty-four hours following exposure to the NO generators sodium nitroprusside (300 μM) and SIN-1 (300 μM), neuronal survival was reduced from approximately 88 to 23%. Treatment with aurintricarboxylic acid (1–100 μM), an endonuclease inhibitor, during NO exposure increased neuronal survival from 23 to 80% and decreased DNA fragmentation from 70 to 30% over a 24-h period. Enhancement of endonuclease activity alone with zinc chelation actively decreased neuronal survival from approximately 80% to approximately 34%. DNA digestion assays identified not only two constitutively active endonucleases, an acidic endonuclease (pH 4.0–7.0) and a calcium/magnesium-dependent endonuclease (pH 7.2–8.0), but also a NO-inducible magnesium-dependent endonuclease (pH 8.0). In the absence of endonuclease activity, DNA degradation did not occur during NO application, suggesting that endonuclease activity was a requisite pathway for NO-induced PCD. In addition, NO independently altered intracellular pH in ranges that were physiologically relevant for the activity of the endonucleases responsible for DNA degradation. Our identification and characterization of specific neuronal endonucleases suggest that the constitutive endonucleases may play a role in the initial stages of NO-induced PCD, but the subsequent “downstream” degradation of DNA may ultimately be dependent upon the NO-inducible endonuclease.     

Characterization of Na+/H+ exchange in a rabbit corneal epithelial cell line (SIRC)

Continuous intracellular pH (pHi) measurements were performed in SIRC rabbit corneal epithelial cells using the pH-sensitive absorbance of intracellularly trapped 5(and 6)-carboxy-4',5'-dimethylfluorescein. Steady-state pHi in nominally bicarbonate free Ringer's solution averaged 6.87 +/- 0.02 (mean +/- S.E., n = 53). After intracellular acidification induced by the NH4Cl-prepulse technique, there was a sodium-dependent pHi recovery towards the normal steady-state pHi. The initial pHi recovery rate was a saturable function of extracellular sodium concentration with an apparent Km for external sodium of about 25 mM and a Vmax of about 0.28 pH units/min. Virtually no pHi recovery was observed in the absence of extracellular sodium. Sodium removal during steady state acidified the cells by 0.36 +/- 0.05 pH units (mean +/- S.E., n = 13) within 5 min. There was a dose-dependent inhibition of pHi recovery after NH4Cl prepulse by amiloride with an IC50 of about 15 microM. Amiloride in a concentration of 1 mM almost completely abolished pHi recovery. Amiloride (1 mM) applied during steady state induced an intracellular acidification of 0.2 +/- 0.03 pH units (mean +/- S.E., n = 7) within 5 min. These findings suggest that a Na+/H+ exchange is present in SIRC rabbit corneal epithelial cells. Na+/H+ exchange seems to be the major process involved in pHi recovery in SIRC cells after an intracellular acid load. Na+/H+ exchange also plays a role in the maintenance of steady-state pHi.     

Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation

The regulation of intracellular pH (pHi) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2',7'-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO3(-)-containing medium (25 mM NaHCO3/5% CO2; pH 7.4) have a steady-state pHi of 7.25 +/- 0.02. Acid loading of acinar cells using the NH4+/NH3 prepulse technique resulted in a Na(+)-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, Ki approximately 0.42 microM) pHi recovery, the kinetics of which were not influenced by the absence of extracellular Cl-. The rate and magnitude of the pHi recovery were dependent on the extracellular Na+ concentration, indicating that Na+/H+ exchange plays a critical role in maintaining pHi above the pH predicted for electrochemical equilibrium. When the NH4+/NH3 concentration was varied, the rate of pHi recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na+/H+ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl(-)-free medium did not significantly alter resting pHi, suggesting the absence of Cl-/HCO3- exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t 1/2 < 30 sec; 0.16 +/- 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37 +/- 0.05 pH unit) or in the absence of extracellular Na+ (0.34 +/- 0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO3(-)-free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl-. The Ca2+ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid) to chelate intracellular Ca2+ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na+/H+ exchange activity; (ii) is most likely mediated by HCO3- efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca2+].     

Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts.

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.     

The Tyrosine Phosphatase SHP2 Modulates MAP Kinase p38 and Caspase 1 and 3 to Foster Neuronal Survival

1. The Src homology protein tyrosine phosphatase SHP2 is associated with cytoskeletal maintenance, cell division, and cell differentiation, but the role of SHP2 during central nervous system injury requires further definition. We therefore characterized the role of SHP2 during nitric oxide (NO)-induced programmed cell death (PCD).2. Employing primary hippocampal neurons from mice with a dominant negative SHP2 mutant to render the phosphatase site of the SHP2 protein biologically inactive, but functionally capable of binding substrate, neuronal injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidyl serine (PS) exposure, mitogen-activated protein (MAP) kinase phosphorylation, and cysteine protease activity. NO was administered through the NO generators SIN-1 (300 M) or NOC-9 (300 M).3. Following NO exposure, neuronal survival decreased from 89 ± 3% in untreated controls to 37 ± 2% in wild-type neurons and to 21 ± 4% in SHP2 mutant neurons. In sister cultures following NO exposure, this increased susceptibility to neuronal injury paralleled enhanced genomic DNA degradation and membrane PS exposure with PCD induction increasing in SHP2 mutant neurons by approximately 42% during specified time periods when compared to wild-type neurons. Interestingly, modulation of the MAP kinase p38 appears to represent an initial level of neuronal protection employed by SHP2. In addition, both the rate and degree of caspase 1- and caspase 3-like activities in SHP2 mutant neurons were significantly increased over a 24-h course when compared to wild-type neurons. Inhibition of caspase 1- and caspase 3-like activities reversed the progression of neuronal PCD, suggesting that inhibition of cysteine protease activity is a downstream mechanism for SHP2 to afford neuronal protection.4. Our work supports the premise that the tyrosine phosphatase SHP2 plays a dominant role during NO-induced PCD and may offer a potential molecular checkpoint against neurodegenerative disease.     

Contribution of anion transporters to the acidosis-induced swelling and intracellular acidification of glial cells

This study examines the contribution of anion transporters to the swelling and intracellular acidification of glial cells from an extracellular lactacidosis, a condition well-known to accompany cerebral ischemia and traumatic brain injury. Suspended C6 glioma cells were exposed to lactacidosis in physiological or anion-depleted media, and different anion transport inhibitors were applied. Changes in cell volume and intracellular pH (pH(i)) were simultaneously quantified by flow cytometry. Extracellular lactacidosis (pH 6.2) led to an increase in cell volume to 125.1 +/- 2.5% of baseline within 60 min, whereas the pH(i) dropped from the physiological value of 7.13 +/- 0.05 to 6.32 +/- 0.03. Suspension in Cl(-)-free or HCO(3)(-)/CO(2)-free media or application of anion transport inhibitors [0.1 mM bumetanide or 0.5 mM 4, 4'-diisothio-cyanatostilbene-2,2'-disulfonic acid (DIDS)] did not affect cell volume during baseline conditions but significantly reduced cell swelling from lactacidosis. In addition, the Cl(-)-free or HCO(3)(-)/CO(2)-free media and DIDS attenuated intracellular acidosis on extracellular acidification. From these findings it is concluded that besides the known activation of the Na(+)/H(+) exchanger, activation of the Na(+)-independent Cl(-)/HCO(3)(-) exchanger and the Na(+)-K(+)-Cl(-) cotransporter contributes to acidosis-induced glial swelling and the intracellular acidification. Inhibition of these processes may be of interest for future strategies in the treatment of cytotoxic brain edema from cerebral ischemia or traumatic brain injury.     

Evidence that Na+/H+ exchange regulates angiotensin II-stimulated diacylglycerol accumulation in vascular smooth muscle cells

Angiotensin II stimulation of vascular smooth muscle cells results in initial, rapid diacylglycerol (DG) formation from the polyphosphoinositides accompanied by intracellular acidification, as well as a more sustained DG accumulation which is accompanied by a prolonged intracellular alkalinization. To determine whether intracellular pH (pHi) modulates DG accumulation, NH4Cl and potassium acetate were used to alter pHi and DG formation was measured. NH4Cl (10 mM) increased pHi from 7.15 +/- 0.05 to 7.34 +/- 0.02 pH units and markedly enhanced the sustained (5 min), but not the initial (15 s), phase of DG formation in response to 100 nM angiotensin II (65 +/- 13% increase). Conversely, intracellular acidification with Na+-free buffer and potassium acetate (20 mM) decreased pHi to 6.93 +/- 0.08 and reduced subsequent angiotensin II-induced sustained DG formation by 82 +/- 9%. In intact cells, inhibition of angiotensin II-stimulated alkalinization by incubation in Na+-free buffer or by addition of the Na+/H+ exchange inhibitor dimethylamiloride (10 microM) decreased the ability of the cell to sustain DG formation, suggesting that active Na+/H+ exchange is necessary for continued DG formation. Thus, it seems that sustained, angiotensin II-induced diacylglycerol accumulation is regulated by intracellular alkalinization secondary to Na+/H+ exchange in cultured vascular smooth muscle cells.     

Response of membrane potential and intracellular pH to hypercapnia in neurons and astrocytes from rat retrotrapezoid nucleus

We compared the response to hypercapnia (10%) in neurons and astrocytes among a distinct area of the retrotrapezoid nucleus (RTN), the mediocaudal RTN (mcRTN), and more intermediate and rostral RTN areas (irRTN) in medullary brain slices from neonatal rats. Hypercapnic acidosis (HA) caused pH(o) to decline from 7.45 to 7.15 and a maintained intracellular acidification of 0.15 +/- 0.02 pH unit in 90% of neurons from both areas (n = 16). HA excited 44% of mcRTN (7/16) and 38% of irRTN neurons (6/16), increasing firing rate by 167 +/- 75% (chemosensitivity index, CI, 256 +/- 72%) and 310 +/- 93% (CI 292 +/- 50%), respectively. These responses did not vary throughout neonatal development. We compared the responses of mcRTN neurons to HA (decreased pH(i) and pH(o)) and isohydric hypercapnia (IH; decreased pH(i) with constant pH(o)). Neurons excited by HA (firing rate increased 156 +/- 46%; n = 5) were similarly excited by IH (firing rate increased 167 +/- 38%; n = 5). In astrocytes from both RTN areas, HA caused a maintained intracellular acidification of 0.17 +/- 0.02 pH unit (n = 6) and a depolarization of 5 +/- 1 mV (n = 12). In summary, many neurons (42%) from the RTN are highly responsive (CI 248%) to HA; this may reflect both synaptically driven and intrinsic mechanisms of CO(2) sensitivity. Changes of pH(i) are more significant than changes of pH(o) in chemosensory signaling in RTN neurons. Finally, the lack of pH(i) regulation in response to HA suggests that astrocytes do not enhance extracellular acidification during hypercapnia in the RTN.     

Thimerosal induces calcium mobilization, fructose 2,6-bisphosphate synthesis and cytoplasmic alkalinization in rat thymus lymphocytes

The effect of thimerosal on intracellular calcium ([Ca2+]i), pH (pHi) and fructose 2,6-bisphosphate (Fru 2,6-P2) in thymus lymphocytes was investigated. The effect of thimerosal on cell growth was also examined. Thimerosal produced a dose-dependent increase in [Ca2+]i, pHi and in the level of fructose 2,6-bisphosphate. Thimerosal was, however, unable to produce cell proliferation and inhibited [3H]thymidine incorporation when cells were challenged with PHA and costimulator. In the absence of external calcium, thimerosal produced only a slight increase in [Ca2+]i. In Na(+)-containing buffer, thimerosal induced an initial acidification (0.05 +/- 0.01 pH units), followed by an alkalinization of 0.08 pH units/min, whereas in Na(+)-free media, pHi decreased 0.2 +/- 0.02 units and this acidification was maintained for more than 40 min. When external calcium was removed the initial acidification was unchanged and no further increase in pHi was observed. Polymyxin B, an inhibitor of protein kinase C, did not modify the initial thimerosal-induced acidification although pH returned to basal levels after 10 min. It was concluded that alkalinization induced by thimerosal is probably due to activation of the Na+/H+ exchanger and that changes in internal Ca2+, pH and metabolic rate are not sufficient to induce cellular proliferation. The mechanism by which thimerosal inhibits thymocyte proliferation remains to be clarified.     

Role of G proteins and modulation of p38 MAPK activation in the protection by nitric oxide against ischemia-reoxygenation injury

Protein kinase C (PKC)-mediated regulation of the mitogen-activated protein kinases (MAPK) may play a role in the protection afforded by ischemic preconditioning (PC). Nitric oxide (NO) can influence MAPK activation via interaction with PKC or farnesylation of low-molecular-weight (LMWT) G proteins. However, we have recently reported the mechanism of NO-induced cardioprotection to be a PKC-independent process. Therefore, we investigated the role of LMWT G proteins and MAPK signaling in NO-induced cardioprotection against simulated ischemia-reoxygenation (SI-R) injury. Neonatal rat cardiomyocytes treated for 90 min with the NO donor S-nitroso-N-acetyl-l,l-penicillamine (SNAP) 1 mM were protected against 6 h of SI (hypoxic conditions at 37 degrees C with 20 mM lactate, 16 mM KCl at pH 6.2) and 24 h reoxygenation under normal culture conditions. NO-induced protection was blocked by the G protein inhibitor alpha-hydroxyfarnesylphosphonic acid (alphaHFP) 10 microM. We studied the time course of p42/44 and p38 MAPK dual-phosphorylation hourly during SI using phospho-specific antibodies. p38 was phosphorylated during SI and the peak phosphorylation was significantly delayed by SNAP pretreatment. The p38 inhibitor SB203580 1 microM, given during SI, protected against injury. Thus the delay in peak p38 activation may contribute to, rather than be the effect of, NO-induced cardioprotection. We have shown that p38beta does not contribute to the total p38 signal in our extracts. Thus there is no detectable beta isoform. We conclude that the main isoform present in these cells and thought to be responsible for the observed phenomenon, is the alpha isoform.     

Relative contributions of Na+/H+ exchange and Na+/HCO3- cotransport to ischemic Nai+ overload in isolated rat hearts

The Na(+)/H(+) exchanger (NHE) and/or the Na(+)/HCO(3)(-) cotransporter (NBC) were blocked during ischemia in isolated rat hearts. Intracellular Na(+) concentration ([Na(+)](i)), intracellular pH (pH(i)), and energy-related phosphates were measured by using simultaneous (23)Na and (31)P NMR spectroscopy. Hearts were subjected to 30 min of global ischemia and 30 min of reperfusion. Cariporide (3 microM) or HCO(3)(-)-free HEPES buffer was used, respectively, to block NHE, NBC, or both. End-ischemic [Na(+)](i) was 320 +/- 18% of baseline in HCO(3)(-)-perfused, untreated hearts, 184 +/- 6% of baseline when NHE was blocked, 253 +/- 19% of baseline when NBC was blocked, and 154 +/- 6% of baseline when both NHE and NBC were blocked. End-ischemic pH(i) was 6.09 +/- 0.06 in HCO(3)(-)-perfused, untreated hearts, 5.85 +/- 0.02 when NHE was blocked, 5.81 +/- 0.05 when NBC was blocked, and 5.70 +/- 0.01 when both NHE and NBC were blocked. NHE blockade was cardioprotective, but NBC blockade and combined blockade were not, the latter likely due to a reduction in coronary flow, because omission of HCO(3)(-) under conditions of NHE blockade severely impaired coronary flow. Combined blockade of NHE and NBC conserved intracellular H(+) load during reperfusion and led to massive Na(+) influx when blockades were lifted. Without blockade, both NHE and NBC mediate acid-equivalent efflux in exchange for Na(+) influx during ischemia, NHE much more than NBC. Blockade of either one does not affect the other.     

Magnetic resonance study of the transmembrane nitrite diffusion.

Nitrite (NO(2)-), being a product of metabolism of both nitric oxide (NO(*)) and nitrate (NO(3)-), can accumulate in tissues and regenerate NO() by several mechanisms. The effect of NO(2)- on ischemia/reperfusion injury was also reported. Nevertheless, the mechanisms of intracellular NO(2)- accumulation are poorly understood. We suggested significant role of nitrite penetration through biological membranes in the form of undissociated nitrous acid (HNO(2)). This hypothesis has been tested using large unilamellar phosphatidylcholine liposomes and several spectroscopic techniques. HNO(2) transport across the phospholipid bilayer of liposomes facilitates proton transfer resulting in intraliposomal acidification, which was measured using pH-sensitive probes. NO(2)(-)-mediated intraliposomal acidification was confirmed by EPR spectroscopy using membrane-impermeable pH-sensitive nitroxide, AMC (2,2,5,5-tetramethyl-1-yloxy-2,5-dihydro-1H-imidazol-3-ium-4-yl)-aminomethanesulfonic acid (pK 5.25), and by (31)P NMR spectroscopy using inorganic phosphate (pK 6.9). Nitrite accumulates inside liposomes in concentration exceeding its concentration in the bulk solution, when initial transmembrane pH gradient (alkaline inside) is applied. Intraliposomal accumulation of NO(2)- was observed by direct measurement using chemiluminescence technique. Perfusion of isolated rat hearts with buffer containing 4 microM NO(2)- was performed. The nitrite concentrations in the effluent and in the tissue, measured after 1 min perfusion, were close, supporting fast penetration of the nitrite through the tissue. Measurements of the nitrite/nitrate showed that total concentration of NO(x) in myocardium increased from initial 7.8 to 24.7 microM after nitrite perfusion. Physiological significance of passive transmembrane transport of NO(2)- and its coupling with intraliposomal acidification are discussed.     

Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function

Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.     

Critical Temporal Modulation of Neuronal Programmed Cell Injury

1. As a free radical, nitric oxide (NO) may be toxic to neurons through mechanisms that directly involve DNA damage. Lubeluzole, a novel benzothiazole compound, has recently been demonstrated to be neuroprotective through the signal transduction pathways of NO. We therefore examined whether neuroprotection by lubeluzole was dependent upon the molecular pathways of programmed cell death (PCD).2. In primary hippocampal neurons, evidence of PCD was determined by hematoxylin and eosin (H&E) stain, transmission electron microscopy, and annexin-V binding. NO administration with the NO generators sodium nitroprusside (300 M) or SIN-1 (300 M) directly induced PCD.3. Neurons positive for PCD increased from 22 ± 3% (untreated) to 72 ± 3% (NO) over a 24-hr period. Coadministration of NO and lubeluzole (750 nM), a neuroprotective concentration, actively decreased PCD expression on H&E stain from 72 ± 3% (NO only) to 25 ± 3% (NO and lubeluzole). Significant reduction in DNA fragmentation by lubeluzole also was evident on electron microscopy. Application of lubeluzole in concentrations that were not neuroprotective or administration of the biologically inactive R-isomer did not significantly alter NO-induced PCD, suggesting that neuroprotection by lubeluzole was intimately linked to the modulation of PCD. Lubeluzole also was able to prevent the initial stages of cellular membrane inversion labeled with annexin-V binding, an early and sensitive indicator of PCD. Interestingly, the critical period for lubeluzole to reverse PCD induction appeared to be within the first 4 hr following NO exposure.4. Further investigation into the neuroprotective pathways that alter PCD may provide greater insight into the molecular mechanisms that ultimately determine neuronal injury.     

The mechanism of potentiation of the glutamate-induced neurotoxicity by serum albumin. A possible role of nitric oxide

Potentiation of the delayed (Glu)-induced neurotoxicity by serum albumin (SA) was studied in experiments with cultured cerebellar granule cells. The delayed neuronal death (DND) was evaluated by counting neurons containing or excluding Trypan Blue 4 h after treatment with Glu. Cytoplasmic Ca2+ ([Ca2+]i) was measured in individual Fura-2-loaded neurons. It was shown that a 15-min application of bovine SA (4 mg/ml) together with Glu (100 microM, 10 microM glycine, Mg2+-free solution) enhanced DND in the culture 1.7 times (43.1+/-3.1%) with respect to the effect induced by Glu alone (24.6+/-0.6%). The bovine SA application did not change the dynamics of [Ca2+]i response during a short-term (1 min) and long-term (15 min) Glu-treatment. DND was prevented by simultaneous application of Glu and inhibitor of NO-synthase N omega-nitro-L-arginine methyl ester (L-NAME), 100 microM) (10.8+/-1.0%) as well as by the application of Glu with SA and L-NAME (9.8+/-1.2%). In order to evaluate the role of nitric oxide (NO) in the SA effect, the cells were incubated for 15 min with the NO-donors sodium nitroprusside (SNP, 10 and 100 microM) and sodium nitrite (NaNO2, 10 and 100 microM) together with SA and in its absence. SA also greatly enhanced the DND induced by SNP and NaNO2. Thus, the DND after simultaneous treatment with SA and SNP was 16.3+/-2.5% (10 microM) or 29.6+/-2.1% (100 microM), and 9.6+/-0.8% (10 microM) and 19.7+/-2.1% after treatment with SNP alone. Exposure to SA together with NaNO2 led to the DND increase up to 26.5+/-1.9% (10 microM) and 37.7+/-3.5% (100 microM) in comparison with 7.4+/-2.0% (10 microM) and 18.9+/-0.8% (100 microM) in experiments with NaNO2 alone. Taking into account the ability of NO and NO2 to oxidize unsaturated fatty acids and the ability of SA to bind them after their hydrolytic removal, we suggested that the SA-induced potentiation of Glu neurotoxicity resulted from exacerbation of the toxic effects of NO and other trace radicals on the neuronal membranes. This hypothesis was supported by the finding that SA also enhanced the neurotoxicity of the lipid prooxidant FeCl2. The simultaneous 15-min application of FeCl2 (10 microM) and SA caused a 51.5+/-4.0% increase in DND, which exceeded 2.4 times the effect produced by FeCl2 alone (21.3+/-2.3%).     

Isolation and properties of fibroblast mutants overexpressing an altered Na+/H+ antiporter

A new method based on the toxicity of low intracellular pH (pHi) was developed to isolate fibroblast variants overexpressing Na+/H+ antiport activity. Chinese hamster lung fibroblasts (CCL39) were incubated for 60 min in medium containing 50 mM NH4Cl. Removal of external NH+4 induced a rapid and lethal intracellular acidification when the Na+/H+ antiporter was inhibited during the 60 min of the pHi recovery phase. The inhibition was provoked either by adding 5-(N-methyl,N-propyl)amiloride (MPA, LD50 = 0.3 microM) or by reducing external [Na+] (LD50 = 25 mM). Progressively increasing the MPA concentration during the acid-load selection led to the isolation of two stable variants: AR40 and AR300, resistant, respectively, to 40 and 300 microM MPA. In response to an acid-load, these variants display a much higher rate of pHi recovery due to an overexpression of Na+/H+ antiport activity. In addition, AR40 and AR300 have an altered Na+/H+ antiporter: in AR300 cells K0.5 of MPA for inhibiting Na+/H+ exchange is shifted from 5 X 10(-8) to 1.5 X 10(-6) M, Km (Na+) is decreased 2-fold, and Vmax is increased 4.5-fold. Alternatively reducing Na+ concentration of the pHi recovery saline medium in a stepwise manner led to the selection of another class of variants (DD8 and DD12) also characterized by an altered Na+/H+ antiporter and an increased expression level. The 10-fold increased rate of amiloride-sensitive Na+ influx of DD12 is accounted for by a 4-fold increase in Vmax and a 2.5-fold increase in affinity for Na+ or Li+ at the external site. Interestingly, the affinity for the amiloride analog MPA and for external H+ is unchanged in DD12. In conclusion, the genetic approach presented here: provides a general and specific method for selecting variants of the Na+/H+ antiporter with increased expression levels and/or with structural alterations and demonstrates that the external Na+- and amiloride-binding sites are not identical, since they can be genetically altered independently of each other.     

Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca++

Gap junctional conductance (gj) between cardiac ventricular myocyte pairs is rapidly, substantially, and reversibly reduced by sarcoplasmic acidification with CO2 when extracellular calcium activity is near physiological levels (1.0 mM CaCl2 added; 470 microM Ca++). Intracellular calcium concentration (Cai), measured by fura-2 fluorescence in cell suspensions, was 148 +/- 39 nM (+/- SEM, n = 6) and intracellular pH (pHi), measured with intracellular ion-selective microelectrodes, was 7.05 +/- 0.02 (n = 5) in cell pair preparations bathed in medium equilibrated with air. Cai increased to 515 +/- 12 nM (n = 6) and pHi decreased to 5.9-6.0 in medium equilibrated with 100% CO2. In air-equilibrated low-calcium medium (no added CaCl2; 2-5 microM Ca++), Cai was 61 +/- 9 nM (n = 13) at pHi 7.1. Cai increased to only 243 +/- 42 nM (n = 9) at pHi 6.0 in CO2-equilibrated low-calcium medium. Junctional conductance, in most cell pairs, was not substantially reduced by acidification to pHi 5.9-6.0 in low-calcium medium. Cell pairs could still be electrically uncoupled reversibly by the addition of 100 microM octanol, an agent which does not significantly affect Cai. In low-calcium low-sodium medium (choline substitution for all but 13 mM sodium), acidification with CO2 increased Cai to 425 +/- 35 nM (n = 11) at pHi 5.9-6.0 and gj was reduced to near zero. Junctional conductance could also be reduced to near zero at pHi 6.0 in low-calcium medium containing the calcium ionophore, A23187. The addition of the calcium ionophore did not uncouple cell pairs in the absence of acidification. In contrast, acidification did not substantially reduce gj when intracellular calcium was low. Increasing intracellular calcium did not appreciably reduce gj at pHi 7.0. These results suggest that, although other factors may play a role, H+ and Ca++ act synergistically to decrease gj.     

Killing of Klebsiella pneumoniae by human alveolar macrophages

We investigated putative mechanisms by which human surfactant protein A (SP-A) effects killing of Klebsiella pneumoniae by human alveolar macrophages (AMs) isolated from bronchoalveolar lavagates of patients with transplanted lungs. Coincubation of AMs with human SP-A (25 microg/ml) and Klebsiella resulted in a 68% decrease in total colony forming units by 120 min compared with AMs infected with Klebsiella in the absence of SP-A, and this SP-A-mediated effect was abolished by preincubation with N(G)-monomethyl-L-arginine. Incubation of transplant AMs with SP-A increased intracellular Ca(2+) concentration ([Ca(2+)](i)) by 70% and nitrite and nitrate (NO(x)) production by 45% (from 0.24 +/- 0.02 to 1.3 +/- 0.21 nmol small middle dot 10(6) AMs(-1).h(-1)). Preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester inhibited the increase in [Ca(2+)](i) and abrogated the SP-A-mediated Klebsiella phagocytosis and killing. In contrast, incubation of AMs from normal volunteers with SP-A decreased both [Ca(2+)](i) and NO(x) production and did not result in killing of Klebsiella. Significant killing of Klebsiella was also seen in a cell-free system by sustained production of peroxynitrite (>1 microM/min) at pH 5 but not at pH 7.4. These findings indicate that SP-A mediates pathogen killing by AMs from transplant lungs by stimulating phagocytosis and production of reactive oxygen-nitrogen intermediates.