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R Colonna

Publications and source records attributed to R Colonna.

At least 37 records · Page 2Linked to original sources
PubMed1995

Lipid interaction of the 37-kDa and 58-kDa fragments of the Helicobacter pylori cytotoxin.

Helicobacter pylori cytotoxin vacA (95 kDa) causes a vacuolar degeneration of epithelial cells. There is evidence that this protein toxin acts inside cells, and hence has to cross a cell membrane. This cytotoxin is frequently obtained as two fragments of 58 kDa (p58) and 37 kDa (p37) and it is available only in minute amounts. Here, its membrane interaction was studied with the two fragments, produced in Escherichia coli. Light scattering and energy transfer experiments show that p37 and p58 cause aggregation and fusion of small unilamellar lipid vesicles; only a reversible aggregation is induced at neutral pH, whereas at acid pH fusion also takes place. p58, but not p37, causes potassium efflux from liposomes and this occurs only at acid pH. Hydrophobic photolabelling with photoactivatable phosphatidylcholines inserted into liposomes shows that both fragments are labelled at neutral pH. The amount of labelling of the two fragments is much higher at acid pH, consistent with a further penetration into the hydrophobic core of the lipid bilayer. Tryptophan fluorescence measurements indicate that the two fragments undergo a pH-driven conformational change. These data are consistent with cytotoxin entry in the cell cytosol via an intracellular acidic compartment.

Bacterial Proteins↗
PubMed1995

Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition.

1. The relation between mitochondrial membrane potential (delta psi m) and cell function was investigated in single adult rat cardiac myocytes during anoxia and reoxygenation. delta psi m was studied by loading myocytes with JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetra-ethylbenzimidazolylcarbocyanine iodide), a fluorescent probe characterized by two emission peaks (539 and 597 nm with excitation at 490 nm) corresponding to monomer and aggregate forms of the dye. 2. De-energizing conditions applied to mitochondria, cell suspensions or single cells decreased the aggregate emission and increased the monomer emission. This latter result cannot be explained by changes of JC-1 concentration in the aqueous mitochondrial matrix phase indicating that hydrophobic interaction of the probe with membranes has to be taken into account to explain JC-1 fluorescence properties in isolated mitochondria or intact cells. 3. A different sensitivity of the two JC-1 forms to delta psi m changes was shown in isolated mitochondria by the effects of ADP and FCCP and the calibration with K+ diffusion potentials. The monomer emission was responsive to values of delta psi m below 140 mV, which hardly modified the aggregate emission. Thus JC-1 represents a unique double sensor which can provide semi-quantitative information in both low and high potential ranges. 4. At the onset of glucose-free anoxia the epifluorescence of individual myocytes studied in the single excitation (490 nm)-double emission (530 and 590 nm) mode showed a gradual decline of the aggregate emission, which reached a plateau while electrically stimulated (0.2 Hz) contraction was still retained. The subsequent failure of contraction was followed by the rise of the emission at 530 nm, corresponding to the monomer form of the dye, concomitantly with the development of rigor contracture. 5. The onset of the rigor was preceded by the increase in intracellular Mg2+ concentration ([Mg2+]i) monitored by mag-indo-1 epifluorescence. Since under these experimental conditions intracellular [Ca2+] and pH are fairly stable, the increase in [Mg2+]i was likely to be produced by a decrease in ATP content. 6. The inhibition of mitochondrial ATPase induced by oligomycin during anoxia was associated with a rapid and simultaneous change of both the components of JC-1 fluorescence, suggesting that delta psi m, instead of producing ATP, is generated by glycolytic ATP during anoxia. 7. The readmission of oxygen induced a rapid decrease of the monomer emission and a slower increase of the aggregate emission. These fluorescence changes were not necessarily associated with the recovery of mechanical function.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1995

On the effects of paraquat on isolated mitochondria. Evidence that paraquat causes opening of the cyclosporin A-sensitive permeability transition pore synergistically with nitric oxide.

This paper reports an investigation on the effects of the bipyridylium herbicide, paraquat, on rat liver mitochondria in vitro. We show that paraquat induces a Ca(2+)-dependent permeability increase of the inner mitochondrial membrane leading to membrane depolarization, uncoupling and matrix swelling. The permeability increase is not observed in the absence of Ca2+ accumulation, and is not due to a direct effect of paraquat on the membrane energy level, as assessed by measurements of membrane potential, respiration and mitochondrial permeability to solutes at high concentrations of paraquat in the presence of excess ethylene-bis(oxoethylenenitrilo)tetraacetic acid (EGTA), a Ca2+ chelator. The Ca(2+)-dependent permeability increase is due to inappropriate opening of the endogenous permeability transition pore (MTP), a regulated, voltage-dependent channel of the inner mitochondrial membrane. The pore is primarily affected by paraquat through a shift of the gating potential to more negative values, allowing pore opening at physiological membrane potential. This effect apparently involves oxidation of a critical dithiol in the pore voltage sensor, while other regulatory aspects of the MTP (matrix pH and Ca2+) are unaffected by paraquat, which is not transported inside the mitochondrial matrix. The effects of paraquat on MTP opening depend on inhibition of electron transfer at Site I by rotenone, or by respiratory chain inhibition by nitric oxide, one of the proposed endogenous mediators of paraquat toxicity to the lung (Berisha, H.I., Hedayatollah, P., Absood, A., and Said, S.I. (1994) Proc. Natl. Acad. Sci. USA 91, 7445-7449). Taken together, these data provide an additional biochemical mechanism by which paraquat may affect cell function, and support the idea that mitochondrial damage is an important determinant in paraquat toxicity (Hirai, K.-I., Ikeda, K., and Wang, G.-Y. (1992) Toxicology 72, 1-16).

Animals↗
PubMed1994

Regulation of the permeability transition pore, a voltage-dependent mitochondrial channel inhibited by cyclosporin A.

Mitochondria from a variety of sources possess a regulated inner membrane channel, the permeability transition pore (MTP), which is responsible for the 'permeability transition', a sudden permeability increase to solutes with molecular masses < or = 1500 Da, most easily observed after Ca2+ accumulation. The MTP is a voltage-dependent channel blocked by cyclosporin A with Ki in the nanomolar range. The MTP open probability is regulated by both the membrane potential and matrix pH. The probability of pore opening increases as the membrane is depolarized, while it decreases as matrix pH is decreased below 7.3 through reversible protonation of histidine residues. Many physiological and pathological effectors, including Ca2+ and ADP, modulate MTP operation directly through changes of the gating potential rather than indirectly through changes of the membrane potential (Petronilli, V., Cola, C., Massari, S., Colonna, R. and Bernardi, P. (1993) J. Biol. Chem. 268, 21939-21945). Here we present recent work from our laboratory indicating that (i) the voltage sensor comprises at least two vicinal thiols whose oxidation-reduction state affects the MTP gating potential; as the couple becomes more oxidized the gating potential increases; conversely, as it becomes more reduced the gating potential decreases; (ii) that MTP opening is fully reversible, as mitochondria maintain volume homeostasis through several cycles of pore opening/closure; and (iii) that the mechanism of MTP inhibition by cyclosporin A presumably involves a mitochondrial cyclophilin but does not utilize a calcineurin-dependent pathway.

Amino Acid Isomerases↗
PubMed1994

The voltage sensor of the mitochondrial permeability transition pore is tuned by the oxidation-reduction state of vicinal thiols. Increase of the gating potential by oxidants and its reversal by reducing agents.

Reaction of isolated mitochondria with a variety of agents that lead to oxidation or cross-linking of sulfhydryl groups leads to an increased "open" probability of the permeability transition pore, a cyclosporin A-sensitive channel. We have investigated the mechanism by which the pore is induced by menadione, diamide, arsenite, and tert-butylhydroperoxide. We find that these inducers increase the probability of pore opening by shifting its gating potential to higher values. Furthermore, the induced shift was prevented by treatment with N-ethylmaleimide or dithiothreitol. At moderate levels of depolarization an apparent I50 for N-ethylmaleimide of bout 5 microM can be defined, while the N-ethylmaleimide or dithiothreitol effects are overcome by maximal depolarization. We conclude that the oxidation-reduction state of vicinal thiols in cysteinyl residues plays a critical role in tuning the voltage sensor of the transition pore, with an increase of gating potential (i.e. an increase in the probability of pore opening despite a high transmembrane potential difference) as the couple is poised to a more oxidized state. These findings may have implications for the mechanism of cell damage under oxidative stress.

Animals↗
PubMed1994

Plasma membrane block to sperm entry occurs in mouse eggs upon parthenogenetic activation.

The ability of parthenogenetically activated mouse eggs to establish a plasma membrane (PM) block to sperm penetration was studied. Zona-free eggs preloaded with Hoechst 33342 were activated by exposure to ethanol or OAG (1-oleoyl-2-acetyl-sn-glycerol) and inseminated after different periods. Eggs challenged with sperm at 30- or 60-min postactivation displayed a fertilization frequency significantly lower than that of control eggs. Conversely, when insemination was carried out at 120-min postactivation, the proportion of fertilized eggs was equivalent to that observed in the control group. Moreover, we report that when the eggs were induced to resume meiosis without any notable loss of CGs (egg exposure to OAG at 100 microM external Ca2+ or to heat shock), a normal ability to be penetrated was recorded at 30-min postactivation. Similar behaviour was exhibited by eggs that underwent a CG exocytosis close to that triggered by sperm in absence of nuclear activation (microinjection of inositol 1,4,5-trisphosphate into the egg at 1 microM cytosolic concentration). Present data support the conclusion that parthenogenetically activated mouse eggs are capable of a transitory PM block response that requires both CG exocytosis and meiosis resumption to occur.

Animals↗
PubMed1994

Follicle-stimulating hormone-induced phospholipase A2 activity and eicosanoid generation in rat Sertoli cells.

The possibility that FSH stimulates the phospholipase A2 (PLA2) pathway was studied in cultured immature Sertoli cells. FSH induced [3H]-arachidonic acid (AA) release from prelabeled cells in a time- and concentration-dependent fashion (ED50 = 21.8 +/- 1.9 ng/ml). This response could be fully prevented by pretreatment of cells with the PLA2 inhibitor, mepacrine. That PLA2 was the main enzyme responsible for cleavage of AA from membrane phospholipids was directly shown by PLA2 activity assay using vesicles of radiolabeled phosphatidylcholine (PC) as substrate. Furthermore, FSH stimulated eicosanoid generation in a time-dependent manner through the cyclooxygenase but not the lipoxygenase pathway. In fact, higher levels of prostaglandin (PG) E2, F2 alpha, and the stable products of PGI2 and thromboxane A2 (6-keto PGF1 alpha and thromboxane B2, respectively) were generated by the gonadotropin-treated cells as compared to control cells. The effect was inhibited by mepacrine, further supporting the pivotal role of PLA2 in the release of the eicosanoid precursor, AA. Finally, the effect of the main product of FSH-induced AA metabolism, i.e., PGE2, was studied. Intracellular cAMP accumulation in Sertoli cells was stimulated by the prostanoid in a dose-dependent manner (ED50 = 2.3 +/- 0.37 nM). PGE2 also significantly stimulated aromatase activity, a specific marker of Sertoli cell functions, measured as 17 beta-estradiol production (ED50 = 4.7 +/- 0.8 nM). Similar results were obtained with PGF2 alpha. Our findings show that FSH, through the activation of PLA2, leads to AA release with consequent metabolism by the cyclooxygenase pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Physiological effectors modify voltage sensing by the cyclosporin A-sensitive permeability transition pore of mitochondria.

This paper reports an investigation on the modulation of the mitochondrial permeability transition pore (MTP) by the membrane potential. Energized rat liver mitochondria loaded with a small Ca2+ pulse in sucrose medium supplemented with phosphate favor a high MTP "closed" probability because of the high membrane potential and therefore maintain a low permeability to sucrose. Upon depolarization by the addition of fully uncoupling concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) mitochondria favor a high MTP "open" probability and rapidly undergo a process of osmotic swelling following sucrose diffusion toward the matrix. A titration with FCCP reveals that discrete subpopulations of mitochondria with different gating potentials for MTP opening may exist, since increasing concentrations of FCCP increase the fraction of mitochondria undergoing osmotic swelling. We show that physiological effectors (Ca2+, Mg2+, ADP, palmitate) modify pore opening in a mitochondrial population by shifting the fraction of mitochondria with a functionally open pore at any given membrane potential. Many inducers and inhibitors may therefore affect the pore directly through an effect on the MTP voltage sensing rather than indirectly through an effect on the membrane potential. Thus, many effectors may induce pore opening by shifting the MTP gating potential to higher levels, whereas many inhibitors may induce pore closure by shifting the MTP gating potential to lower levels.

Animals↗
PubMed1993

Protein kinase C-dependent and independent events in mouse egg activation.

The involvement of calcium- or protein kinase C (PKC)-dependent pathways in cortical granule exocytosis (CGE) and pronucleus formation was examined in mouse eggs using the specific PKC stimulator OAG (1-oleyl-2-acetyl-sn-glycerol) at different external calcium concentrations ([Ca2+]e) ranging from 1.7 mM to 0.1 microM. A 10 min exposure of eggs to 150 microM OAG in the presence of 1.7 mM [Ca2+]e caused a large calcium influx, cortical granule release and 82% activation. The increased permeability of the egg membrane to Ca2+ ions after OAG treatment lasted 20 min. At [Ca2+]e lower than 1.7 mM, both OAG-induced calcium influx and CGE decreased, reaching a non-detectable level at 0.1 microM and 100 microM [Ca2+]e, respectively. Resumption of meiosis was not affected by [Ca2+]e above 200 microM but it was reduced at any lower [Ca2+]e, with a minimum activation frequency of 46% at 0.1 microM [Ca2+]e. Loading of eggs with > or = 3 microM of the calcium chelator BAPTA AM (1,2-bis(o-aminophenoxy)ethane-N',N',N',N'-tetraacetic acid-acetoxymethyl ester) prior to OAG treatment caused a reduction in meiosis resumption with 50% of eggs forming pronuclei. Potent inhibitors of PKC, such as acridine orange and sphingosine, did not interfere with OAG-induced CGE. Conversely, these compounds prevented OAG-induced pronucleus formation in a dose-dependent manner with an IC50 (inhibiting concentration, 50%) of 5 microM and 30 microM for acridine orange and sphingosine, respectively. Microinjection of inositol 1,4,5-trisphosphate into eggs at 0.1 microM elicited Ca2+ release from intracellular stores and the cortical reaction, but failed to stimulate pronucleus formation. These results indicate that, in mouse eggs, CGE is a PKC-independent event, and that the transition from M-phase to interphase may require PKC activity for stimulation.

Acridine Orange↗
PubMed1992

Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations.

We have studied the induction of the mitochondrial cyclosporin A-sensitive permeability transition pore (PTP) by the bifunctional SH group reagent phenylarsine oxide (PhAsO). Addition of nanomolar concentrations of the electroneutral H(+)-K+ ionophore nigericin to nonrespiring mitochondria in sucrose medium determines a dramatic increase of the time required for PTP induction by PhAsO, while no effect of nigericin is apparent in KCl medium. Using mitochondria loaded with the internal pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, we show that the effect of nigericin is mediated by the ionophore-induced acidification of matrix pH. Indeed, experimental manipulation of pHi by a number of treatments indicates that PTP induction is directly related to matrix pH, in that the PTP induction process becomes slower as pHi decreases at constant pHo. PTP induction by PhAsO in respiration-inhibited mitochondria is stimulated by Ca2+ and inhibited by a series of divalent cations. Since PhAsO induces the PTP even in the presence of excess EGTA and in the absence of respiration (Lenartowicz, E., Bernardi, P., and Azzone, G.F. (1991) J. Bioenerg. Biomembr. 23, 679-688), we have been able to study the Ca2+ dependence of the induction process. We show that the apparent Km for Ca2+ activation is about 10(-5) M and that Ca2+, cyclosporin A, and inhibitory Me2+ ions behave as if they were competing for the same binding site(s) on the pore. Since similar results are obtained from patch-clamp experiments on the mitochondrial megachannel (Szabó, I., Bernardi, P., and Zoratti, M. (1992) J. Biol. Chem. 267, 2940-2946), we suggest that (i) the PTP and the mitochondrial megachannel are the same molecular structures and (ii) the same factors affect both the process of pore induction and its open-closed orientation.

Arsenicals↗
PubMed1991

pH-dependent lipid packing, membrane permeability and fusion in phosphatidylcholine vesicles.

We have studied the rate of membrane fusion, the lipid dynamics and order and the membrane permeability of phosphatidylcholine vesicles as a function of pH. Acidification induced very different effects depending on the state of the bilayer. In liquid-crystalline bilayers, acidification decreased the rate of membrane fusion, the acyl chain motion and disorder and the rate of K+ release, whereas in solid bilayers acidification increased the rate of membrane fusion, the lipid acyl chain disorder and the rate of K+ release. These pH-dependent modifications are interpreted in terms of conformational and/or packing changes of the phosphatidylcholine head group in the membrane. In solid bilayers, these changes are not easily accommodated by the rigid structure, and the resulting stress leads to an unstable bilayer.

Cell Fusion↗
PubMed1991

Granulosa cell-oocyte interactions: the phosphorylation of specific proteins in mouse oocytes at the germinal vesicle stage is dependent upon the differentiative state of companion somatic cells.

The role of granulosa cells in the regulation of mouse ovarian oocyte metabolism was investigated. Fully grown antral oocytes, isolated from surrounding cumulus cells, were cultured on monolayers of preantral granulosa cells in the presence of dbcAMP to prevent the resumption of meiosis. Under these conditions metabolic cooperativity was established between the two cell types as early as 1 hr after seeding. Moreover, cocultured oocytes phosphorylated two polypeptides of 74 and 21 kDa which are normally phosphorylated in follicle-enclosed growing oocytes but not in cumulus cell-enclosed fully grown oocytes at the germinal vesicle stage. When cocultured oocytes were allowed to resume meiosis, the 74 and 21 kDa proteins were synthesized but no longer phosphorylated even though intercellular coupling between the two cell types was maintained during radiolabeling. It appears therefore: a) that the different protein kinase activity of growing and fully grown germinal vesicle-stage mouse oocytes is related to the differentiative state of granulosa cells, and b) that the regulation of oocyte protein phosphorylation activity by granulosa cells is dependent on the meiotic stage of the oocyte.

Animals↗
PubMed1989

Effects of protein kinase C stimulation and free Ca2+ rise in mammalian egg activation.

Protein phosphorylation activity, chromosome segregation, and cortical granule exocytosis (CGE) have been studied in mouse eggs activated parthenogenetically by specific PKC stimulators such as 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleyl-2-acetylglycerol (OAG), or by agents inducing an immediate increase in cytosolic calcium concentration ([Ca2+]i) such as ethanol and Ca-ionophore A23187. When protein phosphorylation activity of mouse eggs was analyzed 10 min after different activation treatments, the phosphorylation of a 32 kDa polypeptide was a feature common to all different parthenogenetic agents used. The appearance of such labeling was independent of an increasing [Ca2+]i, as indicated by direct measurements of 1) cytosolic Ca2+ concentration with fura-2 and 2) exogenous Ca2+ entrance into activated eggs. Emission of the second polar body was blocked in PMA-elicited parthenogenones, whereas it was apparently normal in OAG-treated eggs, unless the eggs were continuously exposed to OAG. CGE was almost immediate in ethanol-activated eggs, but in PMA-treated cells, it occurred significantly later, with a timing corresponding to that found for the appearance of sustained Ca2+ oscillations in this system. Here, we propose that in mammalian eggs 1) PKC stimulation represents an early regulatory step in egg activation; 2) this kinase activity is turned off before the second meiotic cleavage; and 3) cytosolic free Ca2+ rise is essential for CGE occurrence.

Animals↗
PubMed1989

Somatic cell-oocyte interactions in mouse oogenesis: stage-specific regulation of mouse oocyte protein phosphorylation by granulosa cells.

The relative rate of synthesis of a number of proteins and the protein phosphorylation pattern of growing and fully grown oocytes were influenced by the presence of granulosa cells. In particular, a 74-kDa phosphorylated protein was detected only in granulosa cell-enclosed growing mouse oocytes. When reaggregated with granulosa cells, the growing oocyte displayed the phosphorylated form of the 74-kDa protein but when oocytes were cultured on Sertoli cell monolayers or in granulosa cell-conditioned medium the 74-kDa protein was not phosphorylated. We propose that (1) granulosa cells regulate protein phosphorylation in mouse oocytes; (2) a 74-kDa protein is phosphorylated only in growing oocytes when surrounded by granulosa cells; and (3) granulosa cells, but not Sertoli cells, are competent to send the appropriate "signal" to the growing oocyte.

Animals↗
PubMed1988

Interaction of the fluorescent probe N-(lissamine Rhodamine B sulfonyl)dipalmitoylphosphatidylethanolamine with phosphatidylcholine bilayers.

The surface density of the fluorescent probe N-(lissamine Rhodamine B sulfonyl)dipalmitoylphosphatidylcholine is the same in the two lipid leaflets of phosphatidylcholine bilayers containing the probe. In the liquid-crystalline state, the probe molecules aggregate above a threshold amount, approximately 0.2 mol/mol phospholipids. Above this threshold value, the surface density of the free probe molecules is constant, and all probe molecules added are incorporated in the aggregated form. The aggregation of the probe increases by approximately 20% when the medium pH is lowered to 4. In the gel state, the probe aggregation is higher than that in the liquid-crystalline state, and the free probe molecules distribute unevenly in the bilayer surface. Even though the results obtained in our model system cannot be directly extrapolated to all model systems, we point out that care is to be taken in the use of the probe. In fact, only in membranes in the liquid-crystalline state in which the amount of probe molecules to phospholipid molecules is lower than 1:7 the fluorescence response of the probe is independent of the pH changes and of the molecular aggregation.

Calcium Chloride↗
PubMed1988

Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes.

The synthesis and intracellular distribution of actin were studied in isolated dictyate and metaphase II mouse oocytes by 1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of newly synthetized oocyte protein and 2) cytochemical F-actin labeling by fluorescent phalloidin. Unpermeabilized, fully grown oocytes bound phalloidin intensely at the level of the zona pellucida (ZP), such ZP-associated actin representing a significant portion of total actin found in these cells. In contrast, phalloidin binding to ZP was very low in growing oocytes and was undetectable in ovulated, metaphase II eggs. When ZP-associated actin of fully grown oocytes was removed by prolongedly exposing oocytes to alpha-chymotrypsin, the amount of newly synthesized actin displayed by cumulus-enclosed oocytes was reduced to a level comparable to that shown by oocytes isolated from granulosa cells. We demonstrate that ZP-associated actin belongs to granulosa cell processes that remain within the ZP as a consequence of oocyte isolation procedures. We conclude that actin synthesis of mouse oocytes is not regulated by granulosa cells.

Actins↗