Biological oxidation and the mobilization of mitochondrial calcium during the differentiation of Physarum polycephalum

We have previously reported that calcium is required for the starvation-induced differentiation of the slime mold, Physarum polycephalum. With the exception of calcium, each component of the complex starvation medium may be withheld and the organism will still differentiate into spherules. The results of the present study reveal that spherulation will proceed normally when the microplasmoidal cells are transferred from nutrient medium to a citrate buffer containing only 8 mM CaCl2. Electron microscopy and X-ray microprobe analysis reveal that there is an initial increase in the population of calcium-containing mitochondrial granules when the microplasmodia are induced to differentiate. However, as differentiation proceeds, these granules decrease in number and are virtually absent from the mitochondria of mature spherules. The accumulation and depletion of calcium-containing granules is not observed in a nondifferentiating strain of Physarum cultured under standard conditions, but is observed when this strain is first treated with a calcium-enriched nutrient medium that conditions it for spherulation. Changes in the cellular concentrations of NADH and lipid peroxides, and in the activity of superoxide dismutase, correspond temporally to the pattern of increase and depletion of the calcium-containing inclusions. The oxidative stress associated with starvation-induced spherulation may be a consequence of the active accumulation of calcium; the mobilization of this calcium may then be the event that initiates differentiation.     

Effects of the Free Radical Generator Paraquat On Differentiation, Superoxide Dismutase, Glutathione and Inorganic Peroxides In Microplasmodia of Physarum Polycephalum

Abstract. The herbicide paraquat was used to investigate the effects of oxidative stress on the spherulation of Physarum polycephalum microplasmodia. the responses of a white non-differentiating strain of Physarum were compared with those of a common yellow strain that readily spherulates in salts-only starvation medium. the addition of paraquat to the salts medium increased the specific activity of superoxide dismutase in both strains; it also induced an increase in the intracellular inorganic peroxide concentration in both strains. Glutathione concentration was higher in the paraquat-treated yellow strain than in the controls. Paraquat had no effect on glutathione concentration in white microplasmodia. Paraquat accelerated spherulation in yellow microplasmodia. the white microplasmodia responded to the herbicide by cleaving into structures similar to immature spherules; however, these structures were not viable. the results of this study support the hypothesis that free radicals are involved in cell state transitions.     

Effects of the free radical generator paraquat on differentiation, superoxide dismutase, glutathione and inorganic peroxides in microplasmodia of Physarum polycephalum

The herbicide paraquat was used to investigate the effects of oxidative stress on the spherulation of Physarum polycephalum microplasmodia. The responses of a white non-differentiating strain of Physarum were compared with those of a common yellow strain that readily spherulates in salts-only starvation medium. The addition of paraquat to the salts medium increased the specific activity of superoxide dismutase in both strains; it also induced an increase in the intracellular inorganic peroxide concentration in both strains. Glutathione concentration was higher in the paraquat-treated yellow strain than in the controls. Paraquat had no effect on glutathione concentration in white microplasmodia. Paraquat accelerated spherulation in yellow microplasmodia. The white microplasmodia responded to the herbicide by cleaving into structures similar to immature spherules; however, these structures were not viable. The results of this study support the hypothesis that free radicals are involved in cell state transitions.     

Electron microscopic and microprobe analysis of calciuminduced differentiation of the white mutant (LU887 x LU897) strain of Physarum polycephalum

Differentiation of the white mutant (LU887 x LU897) strain of Physarum polycephalum leading to spherule formation can be induced by CaCl(2) if the concentration in the nutrient medium is increased by 5mM prior to the transfer to a non-nutrient salts medium. All stages previously reported for the typical (M(3)cVII) strain of Physarum polycephalum from microplasmodia to spherules are seen but the mutant lacks the synchrony that the replacement technique induces in the typical strain. X-ray microanalyses locate calcium and phosphorus in granules in mitochondria and in the cytoplasm of specimens fixed without osmium. Mitochondria accumulate calcium-containing granules during early differentiation and appear to be essentially without granules in mature spherules. Mobilization of mitochondrial calcium is implicated in the initiation of differentiation. A longitudinally striated cytoplasmic inclusion is abundant in microplasmodia grown in media that have not been supplemented with additional calcium and is seen more rarely during calcium-induced spherulation. Whether or not this inclusion represents cytoplasmic contractile elements is unknown. The calcium-treated mutant strain, previously considered non-differentiating, may prove to be a good alternate model for the study of factors influencing differentiation. It was employed earlier as a control in studies of strains that readily spherulate in response to routine procedures.     

Involvement of Glutathione in the Differentiation of the Slime Mold Physarum polycephalum

The effects of experimentally-altered glutathione concentration on differentiation of the slime mold, Physarum polycephalum were examined. Spherulation was induced by transfer of Physarum from growth medium to a salts-only starvation medium. As differentiation proceeded, superoxide dismutase (SOD) activity in control cultures increased by as much as 21-fold. This increase in SOD activity paralleled the rate of differentiation. Glutathione (GSH) concentration decreased during differentiation by more than 80% in all cultures, regardless of the initial concentration. The rate of differentiation was inversely related to the initial GSH concentration and directly proportional to the SOD activity. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

Calcium-mediated regulation of the low density lipoprotein receptor and intracellular cholesterol synthesis in human epidermal keratinocytes

Unlike cells cultured under physiological Ca2+ concentrations (1-2 mM), keratinocytes cultured in media containing Ca2+ in low concentrations (less than 0.1 mM) do not stratify. The latter cells also differ with respect to several features of the regulation of cholesterol synthesis. In keratinocytes cultured in medium containing high Ca2+ concentrations (1.6 mM) and fetal calf serum, the rate of cholesterol synthesis was 20-30 times higher than in keratinocytes exposed to a low Ca2+ concentration. The rate of cholesterol synthesis did not change when high-calcium cells were deprived of extracellular sources of cholesterol but increased (8-10 fold) in deprived low-calcium cells. Furthermore, the addition of low density lipoprotein (LDL) reduced cholesterol synthesis markedly in low-calcium cells but had no effect on high-calcium cells. Finally, in keratinocytes cultured at low calcium concentrations the association and degradation of 125I-LDL was 20-30 times higher than in keratinocytes cultured under high-calcium conditions. Switching of the cells from the low-calcium to the high-calcium medium resulted in the induction of terminal differentiation within 15 hours and was accompanied by increased cholesterol and protein synthesis, increased competence of cells to form cornified envelopes, and reduced association of 125I-LDL. A gradual increase of the extracellular Ca2+ concentration was accompanied by a corresponding increase of cholesterol and protein synthesis and a decrease of the response of intracellular cholesterol synthesis to changes in the extracellular concentrations of lipoprotein. Various morphological techniques showed virtually no binding and internalization of LDL by keratinocytes cultured at the high-calcium level, whereas both were observed at the low-calcium level. Once internalized, the LDL was delivered to dense bodies representing lysosomes. It is concluded that in human epidermal keratinocytes, the expression of the LDL receptor and the endogenous synthesis of cholesterol are regulated by the conditions determined by the differentiation stage of the cells.     

Change in the activity of calcium ions during illumination of a suspension of visual cell outer segment fragments]

Changes in the activity of calcium ions in the medium containing outer fragments suspension of bovine eye retina rods have been studied by the method of calcium-selective electrodes. Illumination of the suspension increases calcium ion activity in the incubation medium. Photoinduced yield of calcium ions depends on Ca+2 concentration: it equals 0.11+/-0.015 M Ca2+/1m rodopsin in the medium containing 0.1 mM CaCl2 and 0.046+/-0.002Ca2+/1M rodopsin in the medium containing 0.05 mM CaCl2. In the medium containing more than 10(-4) M CaCl2 both an increase and a decrease of Ca2+ ions have been observed.     

Activation of the calcium permeability of erythrocyte membranes by perfringolysin O

Calcium ions inhibited perfringolysin O-induced hemolysis at a concentration lower than 1 mM, but not the hemolysis by digitonin at 10 mM. The introduction of calcium ions into ghosts inhibited the lysis more strongly than the addition of calcium ions outside ghosts. When erythrocytes were treated with perfringolysin O in the presence of 1 mM CaCl2 containing 45CaCl2, the radioactivities inside cells rapidly increased during incubation. On the other hand, when perfringolysin O-treated erythrocytes were incubated in a calcium-free medium, the erythrocytes released calcium ions at a 3.3-fold higher rate than untreated cells. These results suggested that perfringolysin O accelerated both the calcium influx into and efflux from erythrocytes.     

Modulation of WI-38 cell proliferation by elevated levels of CaCl2

Elevating the level of extracellular calcium (CaEx2+) increases the saturation density achieved by the normal human diploid cell line, WI-38, but does not change the growth rate. Day 7 cell yields remain unchanged when [CaEx2+] is between 0.5 mM and 3.0 mM, decrease when [CaEx2+] less than 0.5 mM, and increase when [CaEx2+] greater than 3.0 mM. Combining hydrocortisone with additional CaCl2 results in an additive effect on the saturation density relative to that obtained with each treatment separately. The stimulatory effect of elevated [CaCl2] is independent of serum concentration but is lost when WI-38 cells are grown in conditioned medium. Stimulation is recovered when conditioned medium is diluted with serum-free medium. In the case of young cultures grown in conditioned medium, stimulation can also be recovered when higher than usual levels of additional CaCl2 are used (2-3 mM). A glutamine supplementation to the conditioned medium potentiates cell response to elevated [CaCl2]. These results indicate that the loss of an enhanced saturation density when cells are grown in conditioned medium is not due to serum depletion but is more likely the effect of metabolites and/or nutrient depletion. When older or less vigorously growing cultures are grown in conditioned medium, additions of up to 3 mM CaCl2 only lead to inhibition, suggesting an age-related change in proliferative regulation. Elevated levels of CaEx2+ also enhance the proliferative response of quiescent monolayers to serum stimulation. This finding, along with the increase in saturation density of Ca2+-treated cultures, suggests that an elevated level of CaEx2+ affects cell entry into and exit from quiescence brought on by density-dependent inhibition.     

Use of strontium to separate calcium-dependent pathways for proliferation and differentiation in human keratinocytes

The role of extracellular calcium (Caex) in modulating keratinocyte differentiation has been well documented, but its role in proliferation has been harder to define due to the confounding effect of terminal differentiation. Because strontium (Sr) does not induce terminal differentiation in murine keratinocytes but does mimic the stimulatory effect of Caex on DNA synthesis in chick fibroblasts, experiments were undertaken to determine if Sr could be used to separate the presumably opposing effects of Caex on the proliferation and differentiation of cultured human keratinocytes. In response to additions of SrCl2, keratinocytes in a serum-free hormone-supplemented basal medium containing 0.03 mM Ca showed a dose-dependent increase in day 7 cell yields. Cell yield in the optimal concentration of SrCl2 (1.8 mM) was approximately twice that obtained in any concentration of CaCl2. Maximally stimulatory additions of CaCl2 varied from 0.05 to 1.8 mM, but 0.03 and 0.05 mM additional CaCl2 always increased cell yield relative to unsupplemented controls. Keratinocytes grown in low levels of CaCl2 or any level of SrCl2 have minimal contact with each other regardless of cell density in contrast to the colonies of tightly apposed and stratified cells grown in 1.8 mM CaCl2. Transmission electron micrographs of vertically sectioned confluent cultures in low or high levels of SrCl2 or in low levels of CaCl2 revealed abundant ribosomes and keratin filaments but no stratification or desmosomes, while cultures in 1.8 mM CaCl2 were stratified with numerous desmosomes. These results suggest that Caex may separately stimulate keratinocyte proliferation and terminal differentiation and that Srex can substitute for Caex in the former but not the latter process.     

Effect of exogenous glutathione on the in vitro fertilization of bovine oocytes

Increased amounts of reactive oxygen species (ROS) during in vitro culture may cause cytotoxic damage to gametes and embryos. The main purpose of this study was to investigate the effect of glutathione (GSH), a ROS scavenger, supplemented during IVF of bovine oocytes on embryo development using spermatozoa from different bulls. The following experiments were performed: 1) matured COCs were fertilized in the absence or presence of 1 mM GSH using semen from 4 bulls (Bulls A, B, C and D); 2) matured COCs were fertilized in the absence or presence of 1 mM GSH using semen from Bull C to examine sperm penetration, pronuclear formation and apposition; 3) COCs were fertilized with in the presence of either 0, 0.1, 1.0 or 10 mM GSH to examine the effect of GSH concentration using sperm from Bull C; 4) concentrations of GSH were measured both in the medium and in the oocytes during IVF. Glutathione at 1 mM in IVF medium affected the blastocyst formation, but not the cleavage rate. The effect on blastocyst formation was bull dependent: semen from Bull B and D had a negative, that from Bull C a positive and the one from Bull A no effect. The positive effect of Bull C semen increased the rate of blastocyst formation from 20.1 to 27.3% in control and GSH-treated samples, respectively. The increased rate was due to more zygotes reaching the 8-cell or greater stage by Day 4 after IVF. There was no change in the fertilization or cleavage rates. The GSH was still stable after 18 h incubation in IVF medium, and there was a dose-dependent increase in the GSH concentration in the oocytes. It is concluded that the effect of GSH during IVF on the proportion of blastocysts is dependent on both bull and GSH concentration.     

Regulation of acrosomal matrix dispersion in digitonin-permeabilized guinea pig spermatozoa

Digitonin-permeabilized guinea pig spermatozoa undergo acrosomal matrix dispersion in response to 2.0 mM CaCl2. In this report, the effects of pH and metal ions on matrix dispersion in permeabilized spermatozoa are examined. Calcium-induced dispersion of the acrosomal matrix was dependent on the calcium concentration; the response was not observed at concentrations of CaCl2 less than 50 microM. Magnesium could not substitute for calcium and, in fact, had a retarding effect on the calcium-induced response. Matrix dispersion was also found to be pH-dependent. The induction of matrix dispersion was inhibited at pH 5.6 and pH 9.5 relative to the responses observed at pH 6.3 and pH 7.8. Nigericin induced acrosomal matrix dispersion in the absence of added calcium, indicating a possible role of Na+/H+ exchange across the outer acrosomal membrane in initiating the matrix modification. Sodium was required for the action of nigericin; the ionophore was ineffective in medium in which choline chloride or sucrose was substituted for NaCl. In contrast, the calcium-induced dispersion of the acrosomal matrix occurred in the absence of sodium. Furthermore, low concentrations of calcium inhibited an adenosine triphosphatase activity associated with isolated acrosomal apical segments. These data are consistent with the hypothesis that calcium induces alkalinization of the acrosome, leading to matrix dispersion. However, permeabilized spermatozoa incubated at either pH 9.5 or in the presence of 50 mM NH4Cl at pH 7.5 failed to undergo spontaneous matrix dispersion, suggesting that elevated intraacrosomal pH alone was not sufficient to initiate the reaction. The proposed alternative hypothesis is that calcium initiates matrix dispersion by a mechanism in which elevated intraacrosomal pH may be a secondary response.     

The effects of the ionophore A23187 on pattern formation in the alga Micrasterias

We have used the divalent cation ionophore A23187 to investigate the hypothesis that cytoplasmic localization of Ca2+ is responsible for localized growth in the alga Micrasterias. In a preliminary study we found that, of the major salts contained in the cell's medium, only CaCl2 was needed for normal development. In cells developing in the presence of A23187 and extracellular Ca2+, we postulated that the ionophore would induce a spatially uniform influx of Ca2+ that would overwhelm endogenous Ca2+ gradients. When developing cells were treated with A23187 and 2 mM CaCl2, we observed a delocalization of the cell's normal pattern of wall deposition. This effect was less pronounced when cells were exposed to A23187 and 2 mM MgCl2. These results support the hypothesis that localized regions of high Ca2+ concentration normally mediate localized expansion of tip-growing lobes in Micrasterias.     

Alterations in superoxide dismutase, glutathione, and peroxides in the plasmodial slime mold Physarum polycephalum during differentiation

Changes in the level of antioxidant defenses and the concentration of free radical by-products were examined in differentiating (M3cVII and LU897 X LU863), non-differentiating (LU887 X LU897), and heterokaryon microplasmodia of the slime mold Physarum polycephalum during spherulation in salts-only medium. As differentiation proceeded, superoxide dismutase activity increased by as much as 46 fold; glutathione concentration and the rate of oxygen consumption decreased; cyanide-resistant respiration, hydrogen peroxide, and organic peroxide concentrations increased. The non-differentiating culture failed to exhibit any of these changes. A heterokaryon obtained by the fusion of differentiating and non-differentiating strains was observed to differentiate at a very retarded rate and to exhibit the changes observed in the spherulating strains at a correspondingly slower rate. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

Effect of magnesium on the contractile behavior of actomyosin aggregates isolated from Physarum plasmodia

The dependence on calcium concentration of the contractile behavior of actomyosin isolated from Physarum plasmodia according to Kohama & Kendrick-Jones (1986) was investigated under different magnesium conditions. The inhibitory calcium sensitivity is reduced at magnesium concentrations above or below 1 mM, i.e., contraction of actomyosin aggregates is most effectively inhibited in the presence of 1 mM calcium in combination with physiological magnesium concentrations. In the absence of calcium reactivation optimum is obtained at 8.5 mM Mg2+.     

The interfacial calcium ion concentration as modulator of the latency phase in the hydrolysis of dimyristoylphosphatidylcholine liposomes by phospholipase A2.

Analysis of the time course of hydrolysis of dimyristoylphosphatidylcholine liposomes catalyzed by porcine pancreatic phospholipase A2 at 18 degrees C shows that, in the presence of 10 mM NaCl, the length of the latency period in the presteady-state phase increases from 3 to 10.5 min when the CaCl2 concentration is reduced from 15 to 1 mM. This inverse dependence of the lag period on calcium ion concentration is seen more readily at 1 M NaCl, where the induction time changes from 13.5 to 42 min by decreasing the concentration of CaCl2 from 15 to 1 mM. To interpret these results, we took into account the small amount of fatty acid that is produced during the latency phases. The fatty acid generates a negative surface electrostatic potential and makes the interfacial concentration of calcium ions different from the concentration in the bulk solvent. Variations in the analytical concentrations of NaCl and CaCl2 affect both the interfacial calcium ion concentration and electrostatic potential, as estimated theoretically from Grahame and Boltzmann equations. According to these estimates, the length of the latency period diminishes with the increase of the interfacial calcium concentration, but does not show any logical dependence on the change in surface electrostatic potential.     

In vitro fertilization in inbred BALB/c mice I: isotonic osmolarity and increased calcium-enhanced sperm penetration through the zona pellucida and male pronuclear formation

To optimize IVF conditions for BALB/c mice, which are known to have poor in vitro fertilizability, the requirements for sperm-ova interaction were studied by use of modified simplex optimization medium (mKSOM) as a basic medium. Modified human tubal fluid (mHTF) was used for sperm preincubation and acted as a positive control. When the two media were compared, neither capacitation nor fertilization was supported in mKSOM. Increasing the calcium concentration in mKSOM to 5 mM or more during sperm: ova coincubation improved zona penetration but not male pronuclear (MPN) formation to the same level as those cells incubated in mHTF. When medium osmolarity was varied from 230-305 mOsmol by NaCl at 5 mM CaCl2, MPN formation improved at 280 mOsmol or higher osmolarity to the same level as that found when using mHTF. When NaCl equivalent to 25-75 mOsmol was substituted with trehalose, no significant reduction in fertilization was observed. Substitution of NaCl equivalent to 75 mOsmol with other osmotic reagents (sucrose, choline chloride and sorbitol) resulted in similar levels of fertilization as found with mHTF, except for sorbitol, which reduced fertilization significantly caused by its detrimental effect on sperm viability. At isotonic osmolarity (305 mOsmol), maximum fertilization was observed at 5 mM CaCl2; lower or higher concentrations of CaCl2 resulted in reduced fertilization. Calcium and osmolarity, therefore, are important for sperm : ova interaction in BALB/c mice and the increases in calcium to 5 mM and osmolarity to 305 mOsmol are optimal for BALB/c sperm to penetrate through the zona and to form MPN.     

Superoxide dismutase induces differentiation in microplasmodia of the slime mold Physarum polycephalum

Evidence is presented that supports a role for the enzyme superoxide dismutase (SOD) in the differentiation of the slime mold, Physarum polycephalum. SOD activity increases 46-fold during differentiation. A strain of Physarum that does not differentiate exhibits no change in SOD activity. Addition of SOD, via liposomes, to the nondifferentiating strain induces differentiation; this effect is enhanced by an inhibitor of glutathione synthesis. Other antioxidants selected for study failed to induce differentiation. Conversely, oxidative treatments including introduction of D-amino acid oxidase, via liposomes, induced differentiation. Cellular oxidation is the probable cause of the SOD effect.