Effect of maitotoxin on sea urchin egg fertilization and on Ca2+ permeabilities of eggs and intracellular stores.

Maitotoxin (MTX), a potent marine toxin involved in ciguatera poisoning, inhibited sea urchin egg fertilization in a dose-dependent manner with an IC50 of 7.5 x 10(-3) MU (mouse-unit)/ml. It did not affect male gametes fertilizing capabilities but provoked exocytosis in female gametes. It induced a K+ loss simultaneously with a Na+ entry into unfertilized eggs and increased the Ca2+ influx at higher concentrations. On isolated cortex preparations, high concentrations of MTX reduced the rate of ATP-dependent Ca2+ accumulation into reticulum compartments and caused a leakage of Ca2+ from a preparation pre-loaded with 45Ca2+. Verapamil (10(-4) M) similarly blocked the increase of egg permeability to Ca2+ and the effect on Ca2+ sequestering into intracellular compartment, induced by MTX. Ion transport perturbations which evolved relatively slowly are probably not the direct cause of fertilization inhibition which could be related to a modification of the plasma membrane of the female gametes by this hydrophilic toxin.     

Fertilization envelope formation induced by melittin in sea urchin eggs does not depend on Ca2+ signalling

In sea urchin eggs, 10 μg/mL melittin was found to induce fertilization envelope formation without any increase in [Ca²⁺]_i (the intracellular free Ca²⁺ level). On the other hand, 10 μmol/L Br-A23187 and 100 μg/mL SDS induced fertilization envelope formation associated with [Ca²⁺]_i increase. If EGTA was injected into eggs to make an intracellular concentration of 2 mmol/L, [Ca²⁺]_i became quite low and was not altered by melittin, or by Br-A23187 and SDS. In eggs containing EGTA, fertilization envelope formation was induced by melittin even in Ca²⁺-free artificial sea water, but not by Br-A23187 or SDS. Thus [Ca²⁺]_i is essential for induction of a fertilization envelope in sea urchin eggs by Br-A23187 or SDS but not by melittin. Melittin probably activates some Ca²⁺-independent reaction downstream of Ca²⁺-dependent reactions in a sequential reaction system that finally results in fertilization envelope formation.     

Behavior of Na+/K+-ATPase α-subunit alleles in sea urchin eggs upon fertilization

The allelic division of the Na⁺/K⁺-ATPase α-subunit gene was found in eggs of the sea urchin Hemicentrotus pulcherrimus by polymerase chain reaction (PCR). Two PCR products of different lengths were detected from a genome in one embryo derived from a fertilized egg, although only one product in one embryo derived from an artificially activated egg by parthenogenesis was detected, indicating one copy of the gene in a haploid genome. One of the two PCR products from each fertilized egg was identical in size to the product from an artificially activated egg in the same batch. The other PCR product was the same in length as one of the products from the sperm with which the eggs were fertilized. These results indicate that recombination of the heteromeric alleles of the Na⁺/K⁺-ATPase α-subunit gene occurs in the sea urchin egg due to meiosis and fertilization. The sequencing of these products demonstrated that their exon sequences were identical and that the intron, inserted in the PCR products, generated polymorphism in length due to the frequency of the repeating 53 bp sequence and insertion/deletion of other two segments.     

Sphingosine releases Ca2+ from intracellular stores via the ryanodine receptor in sea urchin egg homogenates

Various reports have demonstrated that the sphingolipids sphingosine and sphingosine-1-phosphate are able to induce Ca2+ release from intracellular stores in a similar way to second messengers. Here, we have used the sea urchin egg homogenate, a model system for the study of intracellular Ca2+ release mechanisms, to investigate the effect of these sphingolipids. While ceramide and sphingosine-1-phosphate did not display the ability to release Ca2+, sphingosine stimulated transient Ca2+ release from thapsigargin-sensitive intracellular stores. This release was inhibited by ryanodine receptor blockers (high concentrations of ryanodine, Mg2+, and procaine) but not by pre-treatment of homogenates with cADPR, 8-bromo-cADPR or blockers of other intracellular Ca2+ channels. However, sphingosine rendered the ryanodine receptor refractory to cADPR. We propose that, in the sea urchin egg, sphingosine is able to activate the ryanodine receptor via a mechanism distinct from that used by cADPR.     

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A₂ inhibitor) blocked fertilization envelope formation and transient CN⁻-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca²⁺]_i, (cytosolic Ca²⁺ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN⁻-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN⁻-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A₂, induced the envelope formation, acid production, augmentation of CN⁻-insensitive and sensitive respiration, but did not cause any increase in [Ca²⁺]_i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A₂ induced by Ca²⁺ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN⁻-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca²⁺-triggered sequential reactions other than Ca²⁺-caused activation of phospholipase A₂.     

Fertilization induced release of glycogen from bound state in sea urchin eggs

Glycogen in sea urchin eggs is found in both the precipitate and the supernatant fractions obtained by adding perchloric acid to the egg homogenate. Glycogen in the acid-insoluble fraction is apparently protein-bound (bound glycogen) while the acid-extractable form (free glycogen) seems to bind with less protein. The greatest amount of bound glycogen is found in the particulate fraction obtained by centrifugation of the egg homogenate at 10,000g for 30 minutes. The supernatant fraction obtained by centrifugation at 105,000g for two hours contained the largest amount of free glycogen of all the fractions obtained. The bound glycogen decreases and the free glycogen increases markedly following fertilization, while the total level of glycogen does not change. The glycogen release from the bound state occurs in vitro and the rate of release is higher in fertilized eggs than in unfertilized eggs. Polyamines (putrecine, spermidine, and spermine) cause an increase in the rate of glycogen release in the egg homogenate. cAMP, AMP, and ADP exert no effect on glycogen release in vitro, whereas ATP slightly enhances the rate of glycogen release. Na⁺ and K⁺ hardly accelerate the rate of glycogen release, and divalent cations, such as Ca²⁺ and Mg²⁺, cause an increase in the rate of glycogen release.     

Sea urchin fertilization stimulates CaM kinase-II (multifunctional [type II] Ca2+/CaM kinase) activity and association with p34cdc2

Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca²⁺ dependence to M-phase onset. A potential target of this Ca²⁺ dependence may be CaM kinase-II (the multifunctional [type II] Ca²⁺/calmodulin [CaM]-dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK-II is activated after fertilization and whether it interacts with other known M-phase regulators, such as p34^cdc2. We report that total CaMK-II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK-II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK-II phosphoproteins after fertilization. Finally, CaMK-II activity and only the 62 kDa phosphoprotein physically associate with p34^cdc2, but again only after fertilization. These changes in CaMK-II activity and p34^cdc2-association after fertilization may ensure that Ca²⁺ signals are targeted to the M-phase machinery at the appropriate developmental times.     

Exocytosis reconstituted from the sea urchin egg is unaffected by calcium pretreatment of granules and plasma membrane

Micromolar calcium ion concentrations stimulate exocytosis in a reconstituted system made by recombining in the plasma membrane and cortical secretory granules of the sea urchin egg. The isolated cortical granules are unaffected by calcium concentrations up to 1 mM, nor do granule aggregates undergo any mutual fusion at this concentration. Both isolated plasma membrane and cortical granules can be pretreated with 1 mM Ca before reconstitution without affecting the subsequent exocytosis of the reconstituted system in response to micromolar calcium concentrations. On reconstitution, aggregated cortical granules will fuse with one another in response to micromolar calcium provided that one of their number is in contact with the plasma membrane. If exocytosis involves the generation of lipid fusogens, then these results suggest that the calcium-stimulated production of a fusogen can occur only when contiguity exists between cortical granules and plasma membrane. They also suggest that a substance involved in exocytosis can diffuse and cause piggy-back fusion of secretory granules that are in contact with the plasma membrane. Our results are also consistent with a scheme in which calcium ions cause a reversible, allosteric activation of an exocytotic protein.     

'Nectosome': a novel cytoplasmic vesicle containing nectin in the egg of the sea urchin, Temnopleurus hardwickii

Localization of an extracellular matrix protein, Th-nectin, in the eggs and embryos of the sea urchin Temnopleurus hardwickii was examined by both immunofluorescence and immunoelectron microscopy. The protein is associated with a tubular structure packaged in rod-shaped vesicles that were designated as 'nectosomes'. In unfertilized eggs, nectosomes are distributed uniformly throughout the cytoplasm, but after fertilization, they gradually translocate to the cortical zone where they are arranged perpendicular to the plasma membrane. The migration of the nectosomes was strongly inhibited by cytochalasin B, which suggested that microfilaments play an important role in this process. Immunocytochemical and immunoblotting analyses both ascertained that nectin is secreted into the hyaline layer. Some nectosomes remain in the apical cytoplasm of dermal cells until the gastrula stage. Ultrastructural examination revealed that the accumulation of nectosomes in the oocyte cytoplasm begins quite early in oogenesis, concomitant with the accumulation of cortical vesicles.     

Effects of Ca2+ on flavin-linked complex enzymes in mitochondria isolated from eggs and embryos of sea urchin

Mitochondria isolated from sea urchin embryos in early development show almost the same activities of cytochrome c oxidase and flavin-linked complex enzymes, which are estimated by cytochrome c reductases as in those isolated from unfertilized eggs. The activities of these cytochrome c reductases are inhibited by Ca2+ at above 10-5 M more strongly than cytochrome c oxidase. To investigate the changes in intramitochondrial Ca2+ concentration at fertilization, the activity of pyruvate dehydrogenase, another mitochondrial enzyme, was measured. The activity of this enzyme was controlled by phosphorylation and Ca2+-dependent dephosphorylation of the catalytic unit. The enzyme activity increased for 30 min after fertilization, decreased and became close to zero within ~60 min. Then, the activity appreciably increased again after hatching. This seems to reflect changes in the intramitochondrial Ca2+ concentration. The enzyme activity was enhanced by pre-incubation with Ca2+ at concentrations up to 10-5 M but was made quite low at above 10-4 M Ca2+ and 10-3 M adenosine triphosphate. Although the changes in pyruvate dehydrogenase activity observed at fertilization will reflect the changes in the intramitochondrial calcium concentration, the intramitochondrial Ca2+ concentration of unfertilized eggs cannot be estimated from these results because high (> 10-4 M) or low (10-6 M) Ca2+ can inhibit the enzyme. Measurement of respiration of a single egg showed that injection of ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid released the mitochondrial electron transport in the unfertilized egg. The possibility that changes in intramitochondrial calcium concentration occur at fertilization is discussed in relation to activation of both mitochondrial respiration and pyruvate dehydrogenase.     

Intracellular Ca2+ Homeostasis in Trypomastigotes of Trypanosoma cruzi

ABSTRACT Trypomastigotes of Trypanosoma cruzi maintain an intracellular Ca²⁺ concentration([Ca²⁺]_i) of 64 ± 30 nM. Equilibration of trypomastigotes in an extracellular buffer containing 0.5 mM [Ca²⁺]_o (preloaded cells) increased [Ca²⁺]_i < 20 nM whereas total cell Ca²⁺ increased by 1.5 to 2.0 pmole/cell. This amount of Ca²⁺ would be expected to increase [Ca²⁺]_i to > 10 μM suggesting active sequestration of Ca²⁺. We tested the hypothesis that maintenance of [Ca²⁺]_i involved both the sequestration into intracellular storage sites and extrusion into the extracellular space. Pharmacological probes known to influence [Ca²⁺]_i through well characterized pathways in higher eukaryotic cells were employed. [Ca²⁺], responses in the presence or absence of [Ca²⁺]o were measured to asses the relative contribution of sequestration or extrusion processes in [Ca²⁺]_i homeostasis. In the presence of 0.5 mM [Ca²⁺]_o, the ability of several agents to increase [Ca²⁺]_i was magnified in the order ionomycin ? nigericin > thapsigargin > monensin > valinomycin. In contrast, preloading markedly enhanced the increase in [Ca²⁺], observed only in response to monensin. Manoalide, an inhibitor of phospholipase A₂, enhanced the accumulation of [Ca²⁺]_i due to all agents tested, particularly ionomycin and thapsigargin. Our results suggest that sequestration of [Ca²⁺]_i involved storage sites sensitive to monensin and ionomycin whereas extrusion of Ca²⁺ may involve phospholipase A₂ activity. A Na⁺/Ca²⁺ exchange mechanism did not appear to contribute to Ca²⁺ homeostasis.     

Chronic inhibition of cortex microsomal Mg2+/Ca2+ ATPase-mediated Ca2+ uptake in the rat pilocarpine model following epileptogenesis

In the rat pilocarpine model, 1 h of status epilepticus caused significant inhibition of Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in cortex endoplasmic reticulum (microsomes) isolated immediately after the status episode. The rat pilocarpine model is also an established model of acquired epilepsy. Several weeks after the initial status epilepticus episode, the rats develop spontaneous recurrent seizures, or epilepsy. To determine whether inhibition of Ca(2+) uptake persists after the establishment of epilepsy, Ca(2+) uptake was studied in cortical microsomes isolated from rats displaying spontaneous recurrent seizures for 1 year. The initial rate and total Ca(2+) uptake in microsomes from epileptic animals remained significantly inhibited 1 year after the expression of epilepsy compared to age-matched controls. The inhibition of Ca(2+) uptake was not due to individual seizures nor an artifact of increased Ca(2+) release from epileptic microsomes. In addition, the decreased Ca(2+) uptake was not due to either selective isolation of damaged epileptic microsomes from the homogenate or decreased Mg(2+)/Ca(2+) ATPase protein in the epileptic microsomes. The data demonstrate that inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in the pilocarpine model may underlie some of the long-term plasticity changes associated with epileptogenesis.     

Allosteric Activation off Brain Mitochondrial Ca2+ Uptake by Spermine and by Ca2+: Developmental Changes

Kinetic analysis of 45Ca2+ uptake by rat brain mitochondria in Ca2+ - 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid buffers indicated that spermine both increased the apparent affinity for Ca2+ and decreased the cooperativity of uptake. Both effects are consistent with an allosteric activation of uptake by spermine. The stimulating effect of spermine on 45Ca2+ uptake was maximal with mitochondria from postnatal day 10 animals and then steadily decreased with increasing age to reach adult values by approximately 30 postnatal days; this was observed independently of the substrates used to fuel mitochondria. Mitochondrial Ca2+ buffering was also analyzed by use of a Ca2+-selective electrode. Addition of a large bolus of Ca2+ produced a decrease in the subsequent equilibrium extramitochondrial Ca2+ concentration (or a "rebound overshoot") under some conditions. It is proposed that this effect is the result of an allosteric activation of Ca2+ uptake by Ca2+. This effect was slowly reversible, or hysteretic, and was blocked by spermine. The overshoot was increased in the presence of higher concentrations of Mg2+ and was absent when mitochondria were incubated with 0.3 mM Mg2+. It was maximal in mitochondria prepared from early postnatal brain, and changes in the magnitude of the effect during development paralleled those obtained with spermine stimulation of 45Ca2+ uptake. The data suggest that spermine produces an allosteric activation of Ca2+ uptake by binding to the same regulatory sites that are involved in the Ca2+-induced activation. The results as a whole suggest that spermine could modulate mitochondrial buffering of the intracellular Ca2+ concentration in brain, particularly during the early postnatal period.     

Activation of newt eggs in the absence of Ca2+ activity by treatment with cycloheximide or D2O

Unfertilized eggs of urodeles that exhibit physiological polyspermy are difficult to activate by ordinary egg-activating agents, such as pricking and Ca²⁺ ionophores, that easily activate monospermic anuran eggs. Therefore, we have tested the effects of other agents that cause egg activation in non-amphibian species in order to investigate the mechanism of egg activation in urodeles. We have found that cycloheximide (a protein synthesis inhibitor), D₂O (that induces microtubule polymerization) and 6-DMAP (a protein kinase inhibitor) caused activation of unfertilized eggs of the newt, Cynops pyrrhogaster . The cell cycle, arrested at meiotic metaphase II, was resumed to form the second polar body accompanied by a loss of maturation promoting factor and cytostatic factor activity. The treated eggs underwent abnormal cleavage. These results indicate that protein synthesis followed by protein phosphorylation is necessary to maintain M phase in unfertilized Cynops eggs. Unfertilized eggs failed to be activated by pricking, but were activated by the ionophore A23187, but only at a concentration 30 times higher than that required to activate Xenopus eggs. Eggs whose intracellular Ca²⁺ ions had been chelated by BAPTA could also be activated by either cycloheximide or D₂O. Cycloheximide- as well as 6-DMAP-induced egg activations were not inhibited by nocodazole, a microtubule-depolymerizing agent. These results suggest that the inhibition of synthesis and phosphorylation of short-lived proteins acts as an egg activation mechanism, downstream of the site of Ca²⁺ action and independently of microtubule polymerization.     

Ca2+ and peroxidase derived from cultured peanut cells

A 5% increase of Ca²⁺ content of the incubation medium for cultured peanut ( Arachis hypogaea L.) cells caused a rise of peroxidase (EC 1.11.1.7) activity in the medium, which could be abolished by the addition of the chelator EGTA [eth-yleneglycol-bis-(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. However, the determination of in vivo peroxidase synthesis in these cells showed that Ca²⁺ had a direct effect on the biosynthesis rather than on transport alone. This concept was re-enforced by the lack of effect by the ionophore A23187 on the transport. The Ca²⁺ content was 2 and 5 mol (mol protein⁻¹) for the cationic and anionic peanut peroxidase, respectively. The latter is different from the value reported for the anionic peroxidase from horseradish.     

Cytochemical Ca2+ Distribution in Activated Discoglossus pictus Eggs: A Gradient in the Predetermined Site of Fertilization

The K-pyroantimonate/OsO₄ (PA) cytochemical method coupled with EGTA and X-ray microanalytical controls has been used to localize Ca²⁺ at egg activation in Discoglossus pictus eggs. The results show that: 1) the PA method is able to selectively localize Ca²⁺ pools mobilized by activating stimuli; 2) the smooth endoplasmic reticulum (SER) elements located in the animal dimple region, i.e. in the predetermined site of fertilization, are the first egg components labeled by precipitates; 3) a decreasing gradient of precipitates is present from the center beyond the boundaries of the dimple region; 4) precipitates are lacking in the remainder of the egg even at late times after activation.
The possibilities are discussed that a) SER is the major Ca²⁺-releasing store at activation in Discoglossus , and b) the observed gradient of pyroantimonate-detected Ca²⁺ reflects an ionic Ca²⁺ gradient.     

Internal Ca2+ Mobilization by Muscarinic Stimulation Increases Secretion from Adrenal Chromaffin Cells Only in the Presence of Ca2+ Influx

The cytosolic free Ca2+ concentration ([Ca2+]in) in single cat and bovine adrenal chromaffin cells was measured to determine whether or not there was any correlation between the [Ca2+]in and the catecholamine (CA) secretion caused by muscarinic receptor stimulation. In cat chromaffin cells, methacholine (MCh), a muscarinic agonist, raised [Ca2+]in by activating both Ca2+ influx and intracellular Ca2+ mobilization with an accompanying CA secretion. In bovine cells, MCh elevated [Ca2+]in by mobilizing intracellular Ca2+ but did not cause CA secretion. The MCh-induced rise in [Ca2+]in in cat cells was much higher than that in bovine cells, but when Ca2+ influx was blocked, the rise was reduced, with a concomitant loss of secretion, to a level comparable to that in bovine cells. Intracellular Ca2+ mobilization due to muscarinic stimulation substantially increased secretion from depolarized bovine and cat cells, where a [Ca2+]in elevated above basal values was maintained by a continuous Ca2+ influx. These results show that Ca2+ released from internal stores is not effective in triggering secretion unless Ca2+ continues to enter across the plasma membrane, a conclusion suggesting that secretion depends on [Ca2+]in in a particular region of the cell.     

Parallel pathways for intracellular Ca2+ release from the vacuole of higher plants

A novel pathway for intracellular Ca²⁺ release via a voltage-gated channel has been diered in the tonoplast of Beta vulgaris (sugar beet) tap roots. The channel is characterized electrophysiologically in isolated vacuoles and radiometrically in tonoplast-enriched microsomes. Single channel properties were studied in excised membrane patches. With 5-20 mM Ca²⁺ as a charge carrier on the vacuolar side, the unitary current saturates to a maximal value of 0.59 ± 0.05 pA as membrane voltage approaches +30 to +50 mV. The maximal slope conductance at non-saturating voltages is 12.45 ± 1.06 pS. Open-state probability increases markedly with positive-going voltage changes in the physiological range. Channel activity is also increased by vacuolar, but not by cytosolic Ca²⁺. The lanthanide Gd³⁺ is alone among a large range of Ca²⁺ channel antagonists in acting as an effective inhibitor of the channel, primarily as a result of a dramatic effect on the open-state probability. We conclude that this voltage-gated channel could constitute an alternative, inositol (1,4,5)-trisphosphate-independent pathway for vacuolar Ca²⁺ release during signal transduction in plants.     

Evidence of phospholipase A2 in the sea urchin egg: Its possible involvement in the cortical granule reaction

Fertilization of the sea urchin egg involves an exocytotic event known as the cortical granule reaction (CGR). In many cell systems, phospholipase A₂ is implicated in regulation of the secretory event. Indirect evidence suggests that phospholipase A₂ mediates the CGR; however, there has been no direct demonstration of phospholipase A₂ activity in the sea urchin egg. We report here evidence of phospholipase A₂ activity in egg homogenate of the sea urchin Lytechinus pictus. The enzyme was calcium-dependent and had a pH optimum near the intracellular pH of the unfertilized egg. Neither exogenous calmodulin nor trifluoperazine had any apparent effect on enzyme activity. Quinacrine, a phospholipase A₂ inhibitor, blocked the enzyme activity in the egg homogenate. In intact eggs, quinacrine blocked the CGR in a dose-dependent, egg-concentration-dependent manner. The inhibitory effect of quinacrine on the CGR could not be overcome by the phospholipase A₂ activator melittin or by the calcium ionophore A23187. Quinacrine did not inhibit sperm-egg binding or sperm incorporation. These results lend further support to the hypothesis that phospholipase A₂ is involved in the CGR.