Contractile activity-induced adaptations in the mitochondrial protein import system

We previouslydemonstrated that subsarcolemmal (SS) and intermyofibrillar (IMF)mitochondrial subfractions import proteins at different rates. Thisstudy was undertaken to investigate1) whether protein import is alteredby chronic contractile activity, which induces mitochondrialbiogenesis, and 2) whether these two subfractions adapt similarly. Using electrical stimulation (10 Hz, 3 h/day for 7 and 14 days) to induce contractile activity, we observedthat malate dehydrogenase import into the matrix of the SS and IMFmitochondia isolated from stimulated muscle was significantly increasedby 1.4- to 1.7-fold, although the pattern of increase differed for eachsubfraction. This acceleration of import may be mitochondrialcompartment specific, since the import of Bcl-2 into the outer membranewas not affected. Contractile activity also modified the mitochondrialcontent of proteins comprising the import machinery, as evident fromincreases in the levels of the intramitochondrial chaperone mtHSP70 aswell as the outer membrane import receptor Tom20 in SS and IMFmitochondria. Addition of cytosol isolated from stimulated or controlmuscles to the import reaction resulted in similar twofold increases inthe ability of mitochondria to import malate dehydrogenase, despiteelevations in the concentration of mitochondrial import-stimulatingfactor within the cytosol of chronically stimulated muscle. Theseresults suggest that chronic contractile activity modifies the extra- and intramitochondrial environments in a fashion that favors the acceleration of precursor protein import into the matrix of the organelle. This increase in protein import is likely an important adaptation in the overall process of mitochondrial biogenesis.

     

The Effects of Gibberellic Acid on Organelle Biogenesis in the Endosperm of Germinating Castor Bean Seeds

Mature seeds of castor bean (Ricinus communis L.) were imbibedin tap water or 0.3 mM GA₃, planted in vermiculite moistenedwith tap water or 0.3 mM GA₃, and incubated at 32 ?C. Duringthe course of germination, GA₃ promoted marked increases inthe activities of the glyoxysomal marker enzyme isocitrate lyaseand certain mitochondrial marker enzymes, but did not affectthe ER marker enzyme choline phosphotransferase. Glyoxysomaland ER protein and phospholipid were not increased in amountby GA₃, whereas mitochondrial protein and phospholipid were.SDS-polyacrylamide gels of the glyoxysomal matrix polypeptidesfrom GA₃-treated beans exhibited two polypeptides additionalto those found to be common to both GA₃-treated and controltissue. Incorporation of CDP-(methyl ¹⁴C)-choline into intactendosperm tissue and the distribution amongst the glyoxysomes,mitochondria, and ER of newly synthesized phosphatidyl-(methyl¹⁴C)-choline was unchanged by GA₃.     

Enzymatic Conversion of Pheophorbide a to the Precursor of Pyropheophorbide a in Leaves of Chenopodium album

Soluble proteins extracted from leaves of Chenopodium albumcatalyzed the conversion of pheophorbide a to a precursor ofpyropheophorbide a, putatively identified as C-13²-carboxyl-pyropheophorbidea. The precursor was then decarboxylated non-enzymatically toyield pyropheophorbide a. Soluble proteins and pheophorbidea, as the substrate, were required for the formation of theprecursor, and boiled proteins were enzymatically inactive.The maximum rate of conversion of pheophorbide a to the precursoroccurred at pH 7.5. The K_m for pheophorbide a was 12.5 µMat pH 7.0. Both pheophorbide b and bacteriopheophorbide a couldserve as substrates, but protopheophorbide a could not. Formationof methanol was detected during the enzymatic reaction, an indicationthat the enzyme is an esterase. Among seven alcohol analogstested, only methanol inhibited the enzymatic activity uncompetitively,with a K₁ of 71.6 mM. Mass-spectrometric (MS) analysis of theprecursor yield a peak at m/z 579 that indicated the releaseof a methyl group from pheophorbide a. It appears thereforethat the enzyme catalyzes the demethylation of the carbomethoxygroup at C-13² of pheophorbide a by hydrolysis to yield methanoland the precursor, C-13²-carboxyl-pyropheophorbide a, whichis converted to pyropheophorbide a by spontaneous decarboxylation.We have tentatively designated the enzyme "pheophorbidase".The presence of the enzyme was dependent on plant species andit was expressed constitutively. ¹Present address: Faculty of Science, Shizuoka University, Ohya,Shizuoka, 422 Japan     

Effects of osmotic shrinkage on voltage-gated Ca2+ channel currents in rat anterior pituitary cells

Increased extracellular osmolarity ([Os]_e) suppresses stimulated hormone secretion from anterior pituitary cells. Ca²⁺ influx may mediate this effect. We show that increase in [Os]_e (by 18–125%) differentially suppresses L-type and T-type Ca²⁺ channel currents (I_L and I_T, respectively); I_L was more sensitive than I_T. Hyperosmotic suppression of I_L depended on the magnitude of increase in [Os]_e and was correlated with the percent decrease in pituitary cell volume, suggesting that pituitary cell shrinkage can modulate L-type currents. The hyperosmotic suppression of I_L and I_T persisted after incubation of pituitary cells either with the actin-disrupter cytochalasin D or with the actin stabilizer phalloidin, suggesting that the actin cytoskeleton is not involved in this modulation. The hyperosmotic suppression of Ca²⁺ influx was not correlated with changes in reversal potential, membrane capacitance, and access resistance. Together, these results suggest that the hyperosmotic suppression of Ca²⁺ influx involves Ca²⁺ channel proteins. We therefore recorded the activity of L-type Ca²⁺ channels from cell-attached patches while exposing the cell outside the patch pipette to hyperosmotic media. Increased [Os]_e reduced the activity of Ca²⁺ channels but did not change single-channel conductance. This hyperosmotic suppression of Ca²⁺ currents may therefore contribute to the previously reported hyperosmotic suppression of hormone secretion. L-type Ca²⁺ channels; osmosensitivity; mechanosensitivity; osmolarity; hyperosmolarity     

Characterization of ganglioside associated with the thyrotrophin receptor

The receptor protein for thyrotrophin (thyroid-stimulating hormone;TSH) is associated with a glycosphingolipid moiety. The proteinbelongs to the family of receptors that couple to guanine nucleotidebinding proteins; the glycosphingolipid contains sialic acidand belongs to the family of gangliosides. This report definesthe structure of the receptor ganglioside in the Fisher ratthyroid cell line (FRTL-5). Receptor protein was purified byTSH affinity chromatography from FRTL-5 cells, biosyntheticallylabelled with [³H]galactose and [³H]glucosamine, and resolvedby SDS-PAGE. A single radiolabelled band of M_r     

Effects of Calmodulin Antagonists on the Mitochondrial and Microsomal Ca2$ Uptake in Apple Fruit

In apple fruit, active ATP-dependent microsomal Ca^2$ uptakeand respiration-dependent mitochondrial Ca^2$ uptake were observed. The mitochondrial Ca^2$ uptake was depressed by the calmodulinantagonists chlorpromazine hydrochloride (CPZ) and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamidehydrochloride (W-7). The Ca^2$-ATPase from apple mitochondriawas also inhibited by CPZ or W-7. The apparent K_m value forCa^2$ in mitochondrial Ca^2$ uptake (K_m=0.35 mM) was similar tothat of mitochondrial Ca^2$-ATPase (K_m=0.32 mM). The inhibitoryeffect of W-7 on the activity of the mitochondrial Ca^2$ uptakewas closely correlated with the inhibition by W-7 of mitochondrialCa^2$-ATPase (r=0.996). These findings indicate that the mitochondrialuptake of Ca^2$ in apple fruit depends on the calmodulin-mediatedactivation of Ca^2$-ATPase. The microsomal Ca^2$ uptake was depressed by CPZ, suggestingthat the microsomal Ca^2$ uptake may also be modulated by calmodulin. ¹ Contribution No. C-72, Fruit Tree Research Station. (Received June 7, 1982; Accepted October 19, 1982)     

Defective coupling of apical PTH receptors to phospholipase C prevents internalization of the Na+-phosphate cotransporter NaPi-IIa in Nherf1-deficient mice

Phosphate reabsorption in the renal proximal tubule occurs mostly via the type IIa Na⁺-phosphate cotransporter (NaP_i-IIa) in the brush border membrane (BBM). The activity and localization of NaP_i-IIa are regulated, among other factors, by parathyroid hormone (PTH). NaP_i-IIa interacts in vitro via its last three COOH-terminal amino acids with the PDZ protein Na⁺/H⁺-exchanger isoform 3 regulatory factor (NHERF)-1 (NHERF1). Renal phosphate reabsorption in Nherf1-deficient mice is altered, and NaP_i-IIa expression in the BBM is reduced. In addition, it has been proposed that NHERF1 and NHERF2 are important for the coupling of PTH receptors (PTHRs) to phospholipase C (PLC) and the activation of the protein kinase C pathway. We tested the role of NHERF1 in the regulation of NaP_i-IIa by PTH in Nherf1-deficient mice. Immunohistochemistry and Western blotting demonstrated that stimulation of apical and basolateral receptors with PTH-(1–34) led to internalization of NaP_i-IIa in wild-type and Nherf1-deficient mice. Stimulation of only apical receptors with PTH-(3–34) failed to induce internalization in Nherf1-deficient mice. Expression and localization of apical PTHRs were similar in wild-type and Nherf1-deficient mice. Activation of the protein kinase C- and A-dependent pathways with 1,2-dioctanoyl-sn-glycerol or 8-bromo-cAMP induced normal internalization of NaP_i-IIa in wild-type, as well as Nherf1-deficient, mice. Stimulation of PLC activity due to apical PTHRs was impaired in Nherf1-deficient mice. These data suggest that NHERF1 in the proximal tubule is important for PTH-induced internalization of NaP_i-IIa and, specifically, couples the apical PTHR to PLC. phosphate cotransporter; PDZ protein; parathyroid hormone; proximal tubule     

意大利蜜蜂蜂毒磷脂酶A2基因在杆状病毒-昆虫细胞系统中的表达

利用Bac to Bac系统将意大利蜜蜂蜂毒磷脂酶A₂（AmPLA₂）基因cDNA克隆至转移载体pFastBacHTa中,得到pBacHT-AmPLA₂,再将其转化入含穿梭载体Bacmid的受体大肠杆菌DH10Bac中,通过转座作用,得到含AmPLA₂基因的重组病毒rBacmid-AmPLA₂的DNA。提取其基因组DNA,用脂质体介导转染粉纹夜蛾细胞Tn-5B1-4,得到重组病毒rACV-Bac-AmPLA₂。用此重组病毒感染Tn-5B1-4细胞, 在细胞中表达AmPLA₂。SDS-PAGE电泳结果显示,与6×His Tag融合表达的产物蛋白分子量约为18 kD左右,表达量约占细胞总蛋白的5.35%。Western blot印迹显示,融合表达产物能与意大利蜜蜂蜂毒AmPLA₂抗血清发生免疫反应。生物活性测定显示,含表达产物的细胞蛋白粗提物对底物蛋黄的酶活力约为6.13 μmol·min^-1·mg^-1。     

Interaction of hyperthyroidism and hindlimb suspension on skeletal myosin heavy chain expression

We examined the novel interaction ofhyperthyroidism and hindlimb suspension on the pattern of myosin heavychain (MHC) expression (mRNA and protein) in skeletal muscles. FemaleSprague-Dawley rats were assigned to four groups:1) normal control (Con);2) thyroid hormone treated[150 µg 3,5,3'-triiodothyronine(T₃) · kg¹ · day¹](T₃);3) hindlimb suspension (HS); or4)T₃-treated and HS(T₃ + HS). Results show for thefirst time the novel observation that the combinationT₃ + HS induces a rapid andsustained, marked (80-90%) downregulation of type I MHC geneexpression that is mirrored temporally by concomitant markedupregulation of type IIb MHC gene expression, as evidenced by the denovo synthesis of type IIb MHC protein in the soleus. The fast type IIxMHC isoform showed a differential response among the experimentalgroups, generally increasing with the separate and combined treatments in both the soleus and vastus intermedius muscles while decreasing inthe plantaris muscles. The fast type IIa MHC was the least responsiveto suspension of the MHCs and reflected its greatest responsiveness toT₃ treatment while also undergoingdifferential adaptations in slow vs. fast muscle (increases vs.decreases, respectively). These results confirm previous findings thatall four adult MHC genes are sensitive toT₃ and suspension in amuscle-specific manner. In addition, we show thatT₃ + HS can interactsynergistically to create novel adaptations in MHC expression thatcould not be observed when each factor was imposed separately.

     

Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells

A sustained increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) can cause cell death. In this study, we found that, in cultured porcine aortic smooth muscle cells, endoplasmic reticulum (ER) stress, triggered by depletion of Ca²⁺ stores by thapsigargin (TG), induced an increase in the [Ca²⁺]_i and cell death. However, the TG-induced death was not related to the [Ca²⁺]_i increase but was mediated by targeting of activated Bax to mitochondria and the opening of mitochondrial permeability transition pores (PTPs). Once the mitochondrial PTPs had opened, several events, including collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, occurred and the cells died. TG-induced cell death was completely inhibited by the pan-caspase inhibitor Z-VAD-fmk and was enhanced by the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting the existence of a Ca²⁺-dependent anti-apoptotic mechanism. After TG treatment, Ca²⁺-sensitive mitogen-activated protein kinase (MAPK) activation was induced and acted as a downstream effector of phosphatidylinositol 3-kinase (PI 3-kinase). The protective effect of Z-VAD-fmk on TG-induced cell death was reversed by BAPTA, PD-098059 (an MAPK kinase inhibitor), or LY-294002 (a PI 3-kinase inhibitor). Taken together, our data indicate that ER stress simultaneously activate two pathways, the mitochondrial caspase-dependent death cascade and the Ca²⁺-dependent PI 3-kinase/MAPK anti-apoptotic machinery. The Bax activation and translocation, but not the [Ca²⁺]_i increase, may activate mitochondrial PTPs, which, in turn, causes activation of caspases and cell death, whereas Ca²⁺-dependent MAPK activation counteracts death signaling; removal of Ca²⁺ activated a second caspase-independent death pathway. sarco(endo)plasmic reticulum calcium ion adenosine triphosphatase; cytosolic calcium ion concentration; mitogen-activated protein kinase     

Acclimation of Lolium temulentum to enhanced carbon dioxide concentration

Acclimation of single plants of Lolium temulentum to changing[CO₂] was studied on plants grown in controlled environmentsat 20°C with an 8 h photoperiod. In the first experimentplants were grown at 135 µ;mol m^–2 s^–1 photosyntheticphoton flux density (PPFD) at 415µl l^–1 or 550µll^–1 [CO₂] with some plants transferred from the lowerto the higher [CO₂] at emergence of leaf 4. In the second experimentplants were grown at 135 and 500 µmol m^–2 s^–1PPFD at 345 and 575 µl l^–1 [CO₂]. High [CO₂] during growth had little effect on stomatal density,total soluble proteins, chlorophyll a content, amount of Rubiscoor cytochrome f. However, increasing [CO₂] during measurementincreased photosynthetic rates, particularly in high light.Plants grown in the higher [CO₂] had greater leaf extension,leaf and plant growth rates in low but not in high light. Theresults are discussed in relation to the limitation of growthby sink capacity and the modifications in the plant which allowthe storage of extra assimilates at high [CO₂]. Key words: Lolium, carbon dioxide, photosynthesis, growth, stomatal density     

cAMP-independent phosphorylation activation of CFTR by G proteins in native human sweat duct

It is generally believed thatcAMP-dependent phosphorylation is the principle mechanism foractivating cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. However, we showed that activating Gproteins in the sweat duct stimulated CFTR Cl conductance(G_Cl) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR G_Cl is independent ofprotein kinase A. We activated G proteins and monitored CFTRG_Cl in basolaterally permeabilized sweat duct.Activating G proteins with guanosine5'-O-(3-thiotriphosphate) (10-100 µM) stimulated CFTRG_Cl in the presence of 5 mM ATP alone withoutcAMP. G protein activation of CFTR G_Cl requiredMg²⁺ and ATP hydrolysis (5'-adenylylimidodiphosphate couldnot substitute for ATP). G protein activation of CFTRG_Cl was 1) sensitive to inhibition bythe kinase inhibitor staurosporine (1 µM), indicating that theactivation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM)and SQ-22536 (100 µM); and 3) independent ofCa²⁺, suggesting that Ca²⁺-dependent proteinkinase C and Ca²⁺/calmodulin-dependent kinase(s) are notinvolved in the activation process. Activating AC with10⁶ M forskolin plus 10⁶ M IBMX (in thepresence of 5 mM ATP) did not activate CFTR, indicating that cAMPcannot accumulate sufficiently to activate CFTR in permeabilized cells.We concluded that heterotrimeric G proteins activate CFTR G_Cl endogenously via a cAMP-independent pathwayin this native absorptive epithelium.

     

Proteomic analysis of pathogenesis-related proteins (PRs) induced by compatible and incompatible interactions of pepper mild mottle virus (PMMoV) in Capsicum chinense L3 plants

Resistance conferred by the L³ gene is active against most ofthe tobamoviruses, including the Spanish strain (PMMoV-S), aP_1,2 pathotype, but not against certain strains of pepper mildmottle virus (PMMoV), termed P_1,2,3 pathotype, such as the Italianstrain (PMMoV-I). Both viruses are nearly identical at theirnucleotide sequence level (98%) and were used to challenge Capsicumchinense PI159236 plants harbouring the L³ gene in order tocarry out a comparative proteomic analysis of PR proteins inducedin this host in response to infection by either PMMoV-S or PMMoV-I.PMMoV-S induces a hypersensitive reaction (HR) in C. chinensePI159236 plant leaves with the formation of necrotic local lesionsand restriction of the virus at the primary infection sites.In this paper, C. chinense PR protein isoforms belonging tothe PR-1, β-1,3-glucanases (PR-2), chitinases (PR-3), osmotin-likeprotein (PR-5), peroxidases (PR-9), germin-like protein (PR-16),and PRp27 (PR-17) have been identified. Three of these PR proteinisoforms were specifically induced during PMMoV-S-activationof C. chinense L³ gene-mediated resistance: an acidic β-1,3-glucanaseisoform (PR-2) (M_r 44.6; pI 5.1), an osmotin-like protein (PR-5)(M_r 26.8; pI 7.5), and a basic PR-1 protein isoform (M_r 18;pI 9.4–10.0). In addition, evidence is presented for adifferential accumulation of C. chinense PR proteins and mRNAsin the compatible (PMMoV-I)–C. chinense and incompatible(PMMoV-S)–C. chinense interactions for proteins belongingto all PR proteins detected. Except for an acidic chitinase(PR-3) (M_r 30.2; pI 5.0), an earlier and higher accumulationof PR proteins and mRNAs was detected in plants associated withHR induction. Furthermore, the accumulation rates of PR proteinsand mRNA did not correlate with maximal accumulation levelsof viral RNA, thus indicating that PR protein expression mayreflect the physiological status of the plant. Key words: Capsicum chinense, compatible interaction, incompatible interaction, HR-induction, PMMoV, PR proteins Received 5 December 2007; Revised 21 January 2008 Accepted 22 January 2008     

Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway

N-Linked protein glycosylation in most eukaryotic cells initiateswith the transfer of the oligosaccharide Glc₃Man₉GlcNAc₂ fromthe lipid carrier dolichyl pyrophosphate to selected asparagineresidues. In the yeast Saccharomyces cerevisiae, alg mutationswhich affect the assembly of the lipid-linked oligosaccharideat the membrane of the endoplasmic reticulum result in the accumulationof lipid-linked oligosaccharide intermediates and a hypoglycosylationof proteins. Exploiting the synthetic growth defect of alg mutationsin combination with mutations affecting oligosaccharyl transferaseactivity (Stagljar et al., 1994), we have isolated the ALG6locus. alg6 mutants accumulate lipid-linked Man₉GlcNAc₂, suggestingthat this locus encodes an endoplasmic glucosyltransferase.Alg6p has sequence similarity to Alg8p, a protein required forglucosylation of Glc₁Man⁹GlcNAc². Saccharomyces cerevisiae endoplasmic reticulum glycosyltransferase dolichol     

Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation

Pulmonary vasoconstriction and vascularmedial hypertrophy greatly contribute to the elevated pulmonaryvascular resistance in patients with pulmonary hypertension. A rise incytosolic free Ca²⁺ ([Ca²⁺]_cyt)in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential (E_m) regulates[Ca²⁺]_cyt by governing Ca²⁺influx through voltage-dependent Ca²⁺ channels. Thusintracellular Ca²⁺ may serve as a shared signaltransduction element that leads to pulmonary vasoconstriction andvascular remodeling. In PASMC, activity of voltage-gated K⁺(Kv) channels regulates resting E_m. In thisstudy, we investigated whether changes of Kv currents[I_K(V)], E_m, and[Ca²⁺]_cyt affect cell growth by comparingthese parameters in proliferating and growth-arrested PASMC. Serumdeprivation induced growth arrest of PASMC, whereas chelation ofextracellular Ca²⁺ abolished PASMC growth. Resting[Ca²⁺]_cyt was significantly higher, andresting E_m was more depolarized, inproliferating PASMC than in growth-arrested cells. Consistently, wholecell I_K(V) was significantly attenuated in PASMCduring proliferation. Furthermore, E_mdepolarization significantly increased resting[Ca²⁺]_cyt and augmented agonist-mediatedrises in [Ca²⁺]_cyt in the absence ofextracellular Ca²⁺. These results demonstrate that reducedI_K(V), depolarized E_m, and elevated [Ca²⁺]_cyt may play a criticalrole in stimulating PASMC proliferation. Pulmonary vascular medialhypertrophy in patients with pulmonary hypertension may be partlycaused by a membrane depolarization-mediated increase in[Ca²⁺]_cyt in PASMC.

     

Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity

The human Na⁺-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na⁺-sulfate cotransporter (hSUT-1) and the Na⁺-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide-N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn⁵⁹¹ is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced V_max, with no change in K_m. These results suggest that Asn⁵⁹¹ and/or N-glycosylation is critical for transport activity in NaSi-1. antifusion protein antibodies; Xenopus oocytes; sulfate; immunofluorescence     

PLA2 stimulation of Na+/H+ antiport and proliferation in rat aortic smooth muscle cells

The proliferative properties and the ability to stimulate theNa⁺/H⁺antiport activity of a secretory phospholipaseA₂ were studied in rat aorticsmooth muscle cells in culture. The requirement of the enzymaticactivity of phospholipase A₂ toelicit mitogenesis was assessed by the use of ammodytin L, aSer⁴⁹ phospholipaseA₂ from the venom ofVipera ammodytes, devoid of hydrolyticactivity. We propose that the proliferative effect is mediated by thesame transduction pathway for both proteins. In particular,1) both secretory phospholipaseA₂ and ammodytin L stimulatedthymidine incorporation in a dose-dependent manner; 2) both proteins affected the cellcycle, as assessed by cell growth and fluorescence-activated cellsorting experiments; 3) bothphospholipase A₂ and ammodytin Lincreased intracellular pH, a permissive factor for cell proliferation,through activation of theNa⁺/H⁺antiport; 4) ammodytin L was able todisplace the ¹²⁵I-labeledphospholipase A₂ from specificbinding sites in a concentration range consistent with that capable ofeliciting a cellular response; and5) the inhibition by heparin wassimilar for both proteins, taking into account the ratio of heparin toprotein. In conclusion, the enzymatic activity of phospholipaseA₂ is not required for thestimulation of mitogenesis. The inhibitory effect of heparin combinedwith its therapeutic potential could help to clarify the role ofphospholipase A₂ in thepathogenesis of several preinflammatory situations.

     

Preconditioning of cortical neurons by oxygen-glucose deprivation: tolerance induction through abbreviated neurotoxic signaling

Transient exposure of rat cortical cultures to nonlethal oxygen-glucose deprivation (OGD preconditioning) induces tolerance to otherwise lethal oxygen-glucose deprivation (OGD) or N-methyl-D-aspartate 24 h later. This study evaluates the role of cytosolic and mitochondrial Ca²⁺-dependent cellular signaling. Mechanistic findings are placed in context with other models of ischemic preconditioning or known neurotoxic pathways within cortical neurons. Tolerance to otherwise lethal OGD is suppressed by performing OGD preconditioning in the presence of the broad-scope catalytic antioxidants Mn(III)tetra(4-carboxyphenyl)porphyrin (MnTBAP) or Zn(II)tetra(4-carboxyphenyl)porphyrin [Zn(II)TBAP], but not by a less active analog, Mn(III)tetra(4-sulfonatophenyl)porphyrin, or a potent superoxide scavenger, Mn(III)tetra(N-ethyl-2-pyridyl)porphyrin chloride. Inhibitors of adenosine A₁ receptors, nitric oxide synthase, mitogen-activated protein kinase, and poly(ADP-ribose) polymerase fail to suppress OGD preconditioning despite possible links with reactive oxygen species in other models of ischemic preconditioning. Preconditioning is suppressed by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which has been ascribed elsewhere to inhibition of superoxide transport to the cytosol through mitochondrial anion channels. However, although it induces mitochondrial Ca²⁺ uptake, neuronal preconditioning is largely insensitive to mitochondrial uncoupling with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone or 2,4-dinitrophenol. Un-couplers will prevent production of mitochondrial reactive oxygen species, implying nonmitochondrial targets by MnTBAP, Zn(II)TBAP, and DIDS. Emphasizing the importance of an increase in cytosolic Ca²⁺ during preconditioning, a Ca²⁺/calmodulin-dependent protein kinase II inhibitor, KN-62, suppresses development of subsequent tolerance. Summarizing, only those cellular transduction pathways that have the potential to be neurotoxic may be activated by preconditioning in cortical neurons. Finally, a marked decrease in extracellular glutamate is observed during otherwise lethal OGD in preconditioned cultures, suggesting that this end effector may represent a point of convergence across different preconditioning models. N-methyl-D-aspartate; Ca²⁺; antioxidants; mitochondria     

Role of Na+-K+-Cl- cotransport and Na+/Ca2+ exchange in mitochondrial dysfunction in astrocytes following in vitro ischemia

Na⁺-K⁺-Cl^– cotransporter isoform 1 (NKCC1) and reverse mode operation of the Na⁺/Ca²⁺ exchanger (NCX) contribute to intracellular Na⁺ and Ca²⁺ overload in astrocytes following oxygen-glucose deprivation (OGD) and reoxygenation (REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca²⁺ (Ca_m²⁺) overload and dysfunction. OGD/REOX caused a doubling of mitochondrial-releasable Ca²⁺ (P < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial-releasable Ca²⁺ was reduced by 42% (P < 0.05). Genetic ablation of NKCC1 also reduced Ca_m²⁺ accumulation. Moreover, OGD/REOX in NKCC1^+/+ astrocytes caused dissipation of the mitochondrial membrane potential (_m) to 42 ± 3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of _m was attenuated significantly (66 ± 10% of controls, P < 0.05). Cells were also subjected to severe in vitro hypoxia by superfusion with a hypoxic, acidic, ion-shifted Ringer buffer (HAIR). HAIR/REOX triggered a secondary, sustained rise in intracellular Ca²⁺ that was attenuated by reversal NCX inhibitor KB-R7943. The hypoxia-mediated increase in Ca_m²⁺ was accompanied by loss of _m and cytochrome c release in NKCC1^+/+ astrocytes. Bumetanide or genetic ablation of NKCC1 attenuated mitochondrial dysfunction and astrocyte death following ischemia. Our study suggests that NKCC1 acting in concert with NCX causes a perturbation of Ca_m²⁺ homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia. intracellular calcium ion; mitochondrial membrane potential; sodium ion influx; bumetanide; cytochrome c; glial cell death