Incompetence of preovulatory mouse oocytes to undergo cortical granule exocytosis following induced calcium oscillations.
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Immature oocytes of many species are incompetent to undergo cortical granule (CG) exocytosis upon fertilization. In mouse eggs, CG exocytosis is dependent primarily on an inositol 1,4,5-trisphosphate (IP3)-mediated elevation of intracellular calcium ([Ca2+]i). While deficiencies upstream of [Ca2+]i release are known, this study examined whether downstream deficiencies also contribute to the incompetence of preovulatory mouse oocytes to release CGs. The experimental strategy was to bypass upstream deficiencies by inducing normal, fertilization-like [Ca2+]i oscillations in fully grown, germinal vesicle (GV) stage oocytes and determine if the extent of CG exocytosis was restored to levels observed in mature, metaphase II (MII)-stage eggs. Because IP3 does not stimulate a normal Ca2+ response in GV-stage oocytes, three alternate methods were used to induce oscillations: thimerosal treatment, electroporation, and sperm factor injection. Long-lasting oscillations from thimerosal treatment resulted in 64 and 10% mean CG release at the MII and GV stages, respectively (P < 0.001). Three electrical pulses induced mean [Ca2+]i elevations of approximately 730 and 650 nM in MII- and GV-stage oocytes, respectively, and 31% CG release in MII-stage eggs and 9% in GV-stage oocytes (P < 0.001). Sperm factor microinjection resulted in 86% CG release in MII-stage eggs, while similarly treated GV-stage oocytes exhibited < 1% CG release (P < 0.001). Taken together, these results demonstrate a deficiency downstream of [Ca2+]i release which is developmentally regulated in the 12 h prior to ovulation. (+info)
Redox modulation of intracellular free calcium concentration in thyroid FRTL-5 cells: evidence for an enhanced extrusion of calcium.
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Redox modulation is involved in the regulation of the intracellular free calcium concentration ([Ca2+]i) in several cell types. In thyroid cells, including thyroid FRTL-5 cells, changes in [Ca2+]i regulate important functions. In the present study we investigated the effects of the oxidizing compounds thimerosal and t-butyl hydroperoxide on [Ca2+]i in thyroid FRTL-5 cells. Thimerosal mobilized sequestered calcium, and evoked modest store-dependent calcium entry. Both compounds potently attenuated the increase in [Ca2+]i when store-operated calcium entry was evoked with thapsigargin. The entry of barium was not attenuated. Experiments performed with high extracellular pH, in sodium-free buffer and in the presence of vanadate suggested that thimerosal decreased [Ca2+]i by activating a calcium extrusion mechanism, probably a plasma membrane Ca2+-ATPase. All the observed effects were abrogated by the reducing agent beta-mercaptoethanol. The mechanism of action was apparently mediated via activation of protein kinase C, as thimerosal potently stimulated binding of [3H]phorbol 12, 13-dibutyrate, and was without effect on store-operated calcium entry in cells treated with staurosporine or in cells with down-regulated protein kinase C. Thimerosal did not depolarize the membrane potential, as evaluated using patch-clamp in the whole-cell mode. In immunoprecipitates obtained with an antibody against plasma membrane Ca2+-ATPase, we observed several phosphorylated bands in cells stimulated with thimerosal. In conclusion, we have shown that thimerosal attenuates an increase in [Ca2+]i, probably by activating a plasma membrane Ca2+-ATPase. (+info)
Calcium release and subsequent development induced by modification of sulfhydryl groups in porcine oocytes.
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The mechanism of Ca2+ release induced by modification of sulfhydryl groups and the subsequent activation of porcine oocytes were investigated. Thimerosal, a sulfhydryl-oxidizing compound, induced Ca2+ oscillation in matured oocytes. In thimerosal-preincubated oocytes, the amount of Ca2+ released after microinjection of inositol 1,4,5-trisphosphate (InsP3) or ryanodine increased strikingly, indicating that thimerosal potentiated both InsP3- and ryanodine-sensitive Ca2+ release pathways. Thimerosal also enhanced the sensitivity of oocytes to microinjected Ca2+ so that in pretreated oocytes a Ca2+ injection triggered a larger transient. Heparin at concentrations that normally block the InsP3-induced Ca2+ release were without effect; higher doses significantly increased the time leading up to the first spike. The thimerosal-induced Ca2+ release could not be blocked by procaine, and it did not require the formation of InsP3 since preinjection with neomycin did not prevent the oscillation. Immunocytochemistry revealed that thimerosal treatment destroyed the meiotic spindle, preventing further development, an effect that could be reversed by dithiothreitol. The combined thimerosal/dithiothreitol treatment triggered second polar body extrusion in 50% of the oocytes, and as a result of this activation scheme approximately 15% of the in vitro- and approximately 60% of the in vivo-matured oocytes developed to blastocyst during a 7-day culture in vitro. (+info)
Thimerosal in vaccines: a joint statement of the American Academy of Pediatrics and the Public Health Service.
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The Food and Drug Administration (FDA) Modernization Act of 1997 called for FDA to review and assess the risk of all mercury-containing food and drugs. In line with this review, U.S. vaccine manufacturers responded to a December 1998 and April 1999 FDA request to provide more detailed information about the thimerosal content of their preparations that include this compound as a preservative. Thimerosal has been used as an additive to biologics and vaccines since the 1930s because it is very effective in killing bacteria used in several vaccines and in preventing bacterial contamination, particularly in opened multidose containers. Some but not all of the vaccines recommended routinely for children in the United States contain thimerosal. (+info)
Caspase-induced proteolysis of the cyclin-dependent kinase inhibitor p27Kip1 mediates its anti-apoptotic activity.
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The caspase-mediated cleavage of a limited number of cellular proteins is a common feature of apoptotic cell death. This cleavage usually inhibits the function of the target protein or generates peptides that actively contribute to the death process. In the present study, we demonstrate that the cyclin-dependent kinase inhibitor p27Kip1 is cleaved by caspases in human leukemic cells exposed to apoptotic stimuli. We have shown recently that p27Kip1 overexpression delayed leukemic cell death in response to cytotoxic drugs. In transient transfection experiments, the p23 and the p15 N-terminal peptides generated by p27Kip1 proteolysis demonstrate an anti-apoptotic effect similar to that induced by the wild-type protein, whereas cleavage-resistant mutants have lost their protective effect. Moreover, stable transfection of a cleavage-resistant mutant of p27Kip1 sensitizes leukemic cells to drug-induced cell death. Altogether, these results indicate that proteolysis of p27Kip1 triggered by caspases mediates the anti-apoptotic activity of the protein. (+info)
Spontaneous calcium transients are required for neuronal differentiation of murine neural crest.
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We have shown that cultured mouse neural crest (NC) cells exhibit transient increases in intracellular calcium. Up to 50% of the cultured NC-derived cells exhibited calcium transients during the period of neuronal differentiation. As neurogenic activity declined, so did the percentage of active NC-derived cells and their calcium spiking frequency. The decrease in calcium transient activity correlated with a decreased sensitivity to thimerosal, which sensitizes inositol 1,4,5-triphosphate receptors. Thimerosal increased the frequency of oscillations in active NC-derived cells and induced them in a subpopulation of quiescent cells. As neurogenesis ended, NC-derived cells became nonresponsive to thimerosal. Using the expression of time-dependent neuronal traits, we determined that neurons exhibited spontaneous calcium transients as early as a neuronal phenotype could be detected and continued through the acquisition of caffeine sensitivity, soon after which calcium transient activity stopped. A subpopulation of nonneuronal NC-derived cells exhibited calcium transient activity within the same time frame as neurogenesis in culture. Exposing NC-derived cells to 20 mM Mg(2+) blocked calcium transient activity and reduced neuronal number without affecting the survival of differentiated neurons. Using lineage-tracing analysis, we found that 50% of active NC-derived cells gave rise to clones containing neurons, while inactive cells did not. We hypothesize that calcium transient activity establishes a neuronal competence for undifferentiated NC cells. (+info)
Recommendations regarding the use of vaccines that contain thimerosal as a preservative.
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On October 20, 1999, the Advisory Committee on Immunization Practices (ACIP) reviewed information about thimerosal in vaccines and received updates from CDC's National Immunization Program and several vaccine manufacturers on the current and anticipated availability of vaccines that do not contain thimerosal as a preservative. The review was prompted by a joint statement about thimerosal issued July 8, 1999, by the American Academy of Pediatrics (AAP) and the Public Health Service (PHS) (1) and a comparable statement released by the American Academy of Family Physicians (2). These statements followed a Congressionally mandated Food and Drug Administration (FDA) review of mercury in drugs and food, which included a reassessment of the use of thimerosal in vaccines. (+info)
alpha(2A)-adrenergic receptor stimulation potentiates calcium release in platelets by modulating cAMP levels.
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alpha(2A)-Adrenergic receptor-mediated Ca(2+) signaling and integrin alpha(IIb)beta(3) exposure were investigated in human platelets under conditions where indirect, thromboxane- or ADP-mediated effects were absent. The alpha(2)-adrenergic receptor agonists, UK14304 and epinephrine (EPI), were unable to raise cytosolic levels of inositol 1,4,5-trisphosphate (InsP(3)) or Ca(2+) but potentiated the [Ca(2+)](i) rises evoked by other agonists that act through stimulation of phospholipase C (thrombin or platelet-activating factor) or stimulation of Ca(2+)-induced Ca(2+) release (CICR) in the absence of InsP(3) generation (thimerosal or thapsigargin). In addition, alpha(2)-adrenergic stimulation resulted in a 20% lowering in the cytosolic cAMP level. In platelets treated with G(salpha)-stimulating prostaglandin E(1), EPI increased the Ca(2+) signal evoked by either phospholipase C- or CICR-stimulating agonists mainly through modulation of the cAMP level. The stimulating effects of UK14304 and EPI on platelet Ca(2+) responses, and also on integrin alpha(IIb)beta(3) exposure and platelet aggregation, were abolished by pharmacological stimulation of cAMP-dependent protein kinase, and these effects were mimicked by inhibition of this activity. In permeabilized platelets, UK14304 and EPI potentiated InsP(3)-induced, CICR-mediated mobilization of Ca(2+) from internal stores in a similar way as did inhibition of cAMP-dependent protein kinase. In summary, a G(ialpha)-mediated decrease in cAMP level appears to play a major role in the platelet-activating effects of alpha(2A)-adrenergic receptor stimulation. Thus, in platelets, unlike other cell types, occupation of the G(ialpha)-coupled alpha(2A)-adrenergic receptors does not result in phospholipase C activation but rather in modulation of the Ca(2+) response by relieving cAMP-mediated suppression of InsP(3)-dependent CICR. (+info)