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PubMed2004

Calcium entry into keratinocytes induces exocytosis of lysosomes.

During the final steps of epidermal differentiation, extracellular calcium ions enter keratinocytes and induce transglutaminase activity and cornified envelope formation. In other cell types, entry of calcium mediated by ionophores has been reported to induce exocytosis of lysosomes. In this study, we investigated whether lysosomes of keratinocytes might exhibit a similar behaviour. Ionomycin treatment induced cornified envelope formation in keratinocytes, but also morphological changes including plasma membrane blebbing, although no immediate alteration in cell viability could be detected. The activity of the soluble lysosomal enzymes cathepsin C and beta-galactosidase in the culture medium was increased upon ionomycin treatment. Cell leakage did not seem to be responsible for this phenomenon, as suggested by measurements of the cytosolic enzymes adenylate kinase and dipeptidylpeptidase III in the culture medium. Metabolic labelling followed by immunoprecipitation showed that ionomycin induced release of cathepsin D into the culture medium. Simultaneously, lysosome-associated membrane proteins (Lamps) 1 and 2 were detected at the cell surface of ionomycin-treated keratinocytes by biochemical and morphological approaches. These results suggest that upon ionomycin treatment, calcium entry stimulates exocytosis of lysosomes in keratinocytes.

Adult↗
PubMed2005

Calcium homeostasis change in CD4+ T lymphocytes from human peripheral blood during differentiation in vivo.

Resting naïve CD4+(CD45R0-)CD45RA+ T cells are sensitive to ionomycin. In contrast, resting CD4+(CD45RA-)CD45R0+ memory T cells show resistance to this Ca2+ ionophore. In the present study, the ability of activated T lymphocytes to respond to ionomycin during the transition from naïve precursors into memory T cells has been analyzed. Activated CD4+(CD45RA+)CD45R0+ T cells are always present both in human peripheral blood (HPB) and in the ionomycin-resistant (IR) fraction. Therefore, some activated T cells are resistant toward the Ca2+ ionophore. CD69 molecules are markers of the very early stage of T cell activation. However, CD4+CD69+ T cells have never been found in the IR fraction. Thus, the majority of CD4+ T lymphocytes at the early stage of activation are ionomycin-sensitive cells. The proportion of CD4+CD25+ T cells did not differ significantly in HPB and in the IR fraction. The presence of CD4+CD25+ T lymphocytes in the IR fraction reflects changes in the Ca2+-signaling pathway at this differentiation step of activated cells. Depending on the expression level of CD25 molecules, the population of CD4+CD25+ cells is divided in T-regulatory (CD25high) and proliferating (CD25low) subpopulations. The action of ionomycin results in a decrease in the portion of the CD4+CD25low T-cells, but it leads to an increase in the proportion of the CD4+CD25high T lymphocytes. Consequently, greater portion of CD4+CD25high T lymphocytes and smaller portion of CD4+CD25low T cells are IR cells. Expression of HLA-DR molecules can be used as the marker for the late activation step. The IR fraction is significantly rich in CD4+HLA-DR+ T lymphocytes in comparison to the blood of the same donor. The link between different differentiation steps of CD4+ T-lymphocytes and alterations in calcium ion homeostasis is discussed.

Antigens, CD↗
PubMed1994

Differential role of extra- and intracellular calcium in bradykinin and interleukin 1 alpha stimulation of arachidonic acid release from A549 cells.

The release of arachidonic acid in A549 cells was stimulated in a time- and dose-dependent manner by the Ca2+ ionophore ionomycin (t1/2 = 4 min), thapsigargin (t1/2 = 8 min), bradykinin (t1/2 = 12 min, EC50 = 3 nM), and interleukin 1 alpha (t1/2 = 28 min, EC50 = 0.3 ng/ml). Bradykinin (10 nM) and interleukin 1 alpha (1 ng/ml) stimulation was blocked by the bradykinin B2 receptor antagonist, D-Arg,[Hyp3,Thi5,8, D-Phe7]bradykinin and interleukin 1 receptor antagonist (IC50 = 30 mM and 20 ng/ml, respectively), suggesting receptor mediation. Diacylglycerol release was < 10% of total arachidonic acid release in all cases, suggesting activation of phospholipase A2 activity was greater than phospholipase C activation by these agents. The effects of ionomycin (3 microM) and thapsigargin (0.3 microM) were abolished in Ca(2+)-free buffer with and without 0.5 mM EGTA. Bradykinin (10 nM) stimulation was reduced by 50% in Ca(2+)-free buffer whereas interleukin 1 alpha (1 ng/ml) stimulation remained unaffected. However, the presence of EGTA completely abolished bradykinin stimulation and partially blocked the effect of interleukin 1 alpha (43% inhibition). In the presence of extracellular Ca2+, ionomycin (3 mM), thapsigargin (0.3 mM), bradykinin (10 nM), and interleukin 1 alpha (1 ng/ml) stimulation of arachidonic acid release was blocked by the Ca2+ influx blocker LaCl3 (29, 44, 35, and 41% inhibition, respectively). Nifedipine also blocked ionomycin and thapsigargin stimulation but only partially blocked bradykinin and interleukin 1 alpha stimulation. These results suggest that following B2 receptor activation, cytosolic phospholipase A2 is stimulated by a rise in intracellular Ca2+ levels which are sensitive to the action of EGTA, whereas interleukin 1 alpha stimulation of cytosolic phospholipase A2 is mediated by a rise in intracellular Ca2+ from both EGTA-sensitive and resistant pools. Furthermore the results of ionomycin and thapsigargin indicate that extracellular Ca2+ is important for activation of cytosolic phospholipase A2 in A549 cells.

Arachidonic Acid↗
PubMed1991

Cytosolic free Ca2+ in daunorubicin and vincristine resistant Ehrlich ascites tumor cells. Drug accumulation is independent of intracellular Ca2+ changes.

The possible role of intracellular calcium on daunorubicin (DNR) accumulation in wild-type (EHR2) and multi-drug resistant (MDR) Ehrlich ascites tumor cell subline was investigated. DNR accumulation was not enhanced either by increasing the concentration of cellular calcium with the calcium ionophore ionomycin nor by chelating the cytosolic free Ca2+ by the membrane permeable Ca2(+)-buffering agents BAPTA or MAPTAM. No effect was observed in the presence of extremely low extracellular calcium concentration that prevent transmembrane calcium influx or when the cells were calcium depleted using EGTA and ionomycin. Using the fluorescent Ca2+ indicator fura-2 it is further shown that both drug-resistant daunorubicin (EHR2/DNR+) and vincristine (EHR/VCR+) sublines had lower (50-80 nM) concentration of cytosolic free calcium ([Ca2+]i) compared to their corresponding wild-type parenteral tumors (140-180 nM). In calcium free medium, however, no significant difference was found, all cell lines having a [Ca2+]i of 60-80 nM. Furthermore, the total amount of Ca2+ released to the cytosol with 10 microM ionomycin and 5 mM EGTA was 3-4-fold higher in EHR2 than in EHR2/DNR+ or EHR2/VCR+. Mobilization of Ca2+ with 1 microM ionomycin was almost identical in the presence and absence of Ca2+ in the extracellular medium in EHR2 as well as in EHR2/DNR+ suggesting that the increase in [Ca2+]i is mainly due to discharge of Ca2+ from intracellular stores. Furthermore, the total cell calcium [Ca2+]t concentration was slightly higher in EHR2/DNR+ and EHR2/VCR+ cells compared to EHR2. Incubation of the cells with the Ca2(+)-channel blocker verapamil or the intracellular Ca2(+)-antagonist TMB-8 causes depression of the Ca2(+)-response in terms of rise in [Ca2+]i caused by ionomycin. Sorcin, a major calcium-binding protein (Mr 22 kDa), is shown to be overproduced in EHR2/DNR+ cells. The overproduction of this protein in resistant cells may be related to the difference in the intracellular calcium observed in this study. Thus, though handling of Ca2+ is different in wild-type and MDR cell lines, our data suggest that calcium is not involved directly in drug transport processes and the level of Ca2+ per se have no influence on drug accumulation.

Animals↗
PubMed1990

Physiologic signaling in normal human T-cells: mRNA phenotyping by northern blot analysis and reverse transcription-polymerase chain reaction.

Synergy between ionomycin and sn-1,2-dioctanoylglycerol (diC8) was shown at the level of lymphokine gene transcription. Transcriptional activation of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and the protooncogene H-ras was accomplished by signaling highly purified normal human resting T-lymphocytes (T-cells) with diC8, a physiologic regulator of protein kinase C, and the calcium ionophore, ionomycin. Northern blot analysis of mRNA for early T-cell activation genes demonstrated the synergism between diC8 and ionomycin at the gene induction level. To amplify very low levels of IL-2 mRNA, sequential reverse transcription and polymerase chain reaction (RT-PCR) of T cell mRNA were used to demonstrate the capacity of the calcium signal (ionomycin) to promote low-level IL-2 transcription in normal human T-cells without additional signals. Cyclosporine (CsA) prevented diC8 and ionomycin-induced expression of IL-2, IFN-gamma, and H-ras genes. The completeness of its inhibitory effect was evident by the absence of IL-2 transcripts in CsA-treated cultures screened by the RT-PCR technique. CsA also prevented IL-2 and IFN-gamma gene expression in accessory cell-depleted T-cells stimulated by cross-linking the CD2 and CD3 antigens on the cell surface. Our observations demonstrate that a physiologic regulator of PKC, diC8, and cell surface crosslinking of the CD2 and CD3 antigen, promote gene expression in normal human quiescent T-cells independently of accessory cells, and that CsA prevents gene expression via a direct effect on T-cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

The proliferative response of rat T cells to calcium ionophores increases with age.

Splenocytes and T cells from both old and young rats proliferate to A23187 and ionomycin, and this response increases 3- to 10-fold in aged animals. Augmented responsiveness to ionomycin occurs in the absence of any defect in Con A-induced proliferation of T lymphocytes of aged rats and is dependent upon the addition of thiol compounds to the tissue culture medium. Augmented proliferative responses to ionomycin precede the significant but much smaller decline (30 to 40%) in Con A-induced proliferative responsiveness of splenocytes, which is evident only when rats reach 24 months of age. Heightened proliferation to calcium ionophores is not caused by a greater ability of T lymphocytes from aged rats to increase [Ca2+]i in response to ionomycin. The increased proliferative response of lymphocytes from aged rats to ionomycin occurs in the absence of detectable amounts of secreted IL 2 or IL 4. The ionophore response is a much more sensitive biomarker of age than the decline in Con A-induced proliferative responses of lymphocytes and identifies an activity of T lymphocytes that increases rather than decreases during the aging process.

Aging↗
PubMed1993

The role of calcium in cell shrinkage and intracellular alkalinization by bradykinin in Ha-ras oncogene expressing cells.

In ras oncogene expressing cells, bradykinin leads to intracellular alkalinization by activation of the Na+/H+ exchanger. This effect is paralleled by oscillatory increase of intracellular calcium activity and cell shrinkage. Staurosporine (1 mumol/l) is not sufficient to prevent bradykinin induced intracellular alkalinization, thus pointing to a protein kinase C independent pathway for the activation of Na+/H+ exchange. The present study has been performed to elucidate, whether the increase of intracellular calcium contributes to cell shrinkage and activation of the Na+/H+ exchanger. To this end, the effects of the calcium ionophore ionomycin have been tested. Ionomycin leads to a dose dependent increase of intracellular calcium activity. At 100 nmol/l ionomycin intracellular calcium is increased from 114 +/- 17 nmol/l to 342 +/- 24 nmol/l (n = 9), a value within the range of intracellular calcium concentrations following application of bradykinin. The calcium increase is paralleled by a decrease of cell volume by 12 +/- 2% (n = 5) and an increase of intracellular pH from 6.78 +/- 0.02 to 6.90 +/- 0.03 (n = 11), values similar to those following application of bradykinin. The alkalinizing effect of ionomycin is completely abolished in the presence of the novel Na+/H+ exchange inhibitor HOE 694 (10 mumol/l), but is not inhibited by 1 mumol/l staurosporine. Inhibition of K+ and Cl- channels by barium (5 mmol/l) and ochratoxin-A (5 mumol/l) prevents both ionomycin induced cell shrinkage and protein kinase C independent intracellular alkalinization. It is concluded that bradykinin leads to intracellular alkalinization mainly by increasing intracellular calcium concentration. Calcium triggers calcium sensitive K+ channels, and presumably Cl- channels, the subsequent loss of cellular KCl leads to cell shrinkage which, in turn, activates Na+/H+ exchange.

3T3 Cells↗
PubMed1982

Inhibition of 45Ca2+ uptake and catecholamine secretion by phenothiazines and pimozide in adrenal medulla cells cultures.

The inhibition by several phenothiazine drugs and pimozide of the uptake of 45Ca2+ and secretion of catecholamines by cultured adrenal medulla cells stimulated with nicotine, veratridine, 50 mM K+, ionomycin and Ba2+ was studied. The inhibition of 45Ca2+ uptake, except for ionomycin, closely parallelled the inhibition of catecholamine secretion. The nicotine-and veratridine-stimulated effects were several fold more sensitive to inhibition by the drugs than were those stimulated by 50mM K+, ionomycin and Ba2+; the ionomycin-stimulated effects were least sensitive to inhibition. These studies indicate that the drugs have multiple effects on stimulus-secretion coupling in adrenal medulla cells. It is suggested that inhibition of the veratridine- and nicotine-stimulated events is due to membrane perturbations caused by the drugs, that inhibition of the 50mM K+- and Ba2+-stimulated events is due to alterations in the voltage sensitive membrane Ca2+ channel, and that inhibition of secretion elicited by ionomycin may be due to inhibition of Ca2+-calmodulin reactions or to more profound non specific membrane effects.

Adrenal Medulla↗
PubMed1991

Platelet responses promoted by the activation of protein kinase C or the increase of cytosolic Ca2+ are potentiated by adrenaline. Effects of cAMP and staurosporine.

We studied the action of the alpha 2 adrenergic agonist adrenaline on the platelet responses evoked by the activation of protein kinase C or by the ionophore induced increase of cytosolic Ca2+. Both the phorbol ester and ionomycin-induced aggregation are strongly potentiated by adrenaline which per se does not behave as an activating agonist. The potentiation by adrenaline is observed both when added before and after the aggregating agent; in the latter case the effect increases on increasing the delay of adrenaline addition. Adrenaline also reverses the inhibition by cAMP of the PMA (or ionomycin) induced aggregation. It also has a strong potentiating effect (over 100%) on the phorbol ester induced ATP secretion and a weaker effect on the secretion induced by ionomycin. The effect on secretion is visible only when adrenaline is added prior to the stimulus. The inhibition by cAMP of the PMA or ionomycin induced secretion is also counteracted by adrenaline. In no case adrenaline modifies the pattern of platelet phosphoproteins. Ionomycin induces some platelet aggregation also in the presence of the protein kinase inhibitor staurosporine; also this phosphoprotein independent aggregation is strongly stimulated by adrenaline.

Alkaloids↗
PubMed1993

Intracellular calcium mobilization in rat platelets is adversely affected by copper deficiency.

The influence of copper deficiency on the mobilization of Ca2+ from intracellular stores following ionomycin treatment or thrombin activation of rat platelets was examined using the fluorescent indicator, fura-2, to measure changes in cytosolic Ca2+ concentration ([Ca2+]i). Platelets, obtained from copper-deficient and control rats and loaded with fura-2, were suspended in medium containing 1 mM EGTA and no added Ca2+. The size of the internal Ca2+ pools in the suspended platelets was estimated from the rise in [Ca2+]i following maximal discharge of stored Ca2+ by treatment with 1 microM ionomycin. Peak [Ca2+]i following ionomycin treatment was lower in platelets from copper-deficient rats compared to control rats (148 +/- 27 nM vs. 188 +/- 17 nM), suggesting that the size of the Ca2+ storage pools was decreased by copper deficiency. Furthermore, once internal Ca2+ stores were discharged by ionomycin, [Ca2+]i remained elevated in platelets from copper-deficient rats, but decreased in control rats. These data indicate that copper deficiency may inhibit the efflux of Ca2+ from platelets after its release from internal stores by ionomycin treatment. In platelets from copper-deficient and control rats, stimulation with 0.1 U/ml thrombin led to rapid rise followed by a slow decay in [Ca2+]i. However, peak [Ca2+]i was lower in platelets from copper-deficient rats than in control rats (94 +/- 19 nM vs. 131 +/- 16 nM). These findings imply that by reducing the amount of Ca2+ available for release from intracellular stores, copper deficiency also reduces [Ca2+]i following thrombin activation in the absence of external Ca2+.

Animals↗
PubMed1994

Second messenger regulation of mouse gonadotropin-releasing hormone gene expression in immortalized mouse hypothalamic GT1-3 cells.

Using a transgenic mouse derived GnRH expressing neuronal cell line, GT1-3, we studied the effects of activation of cAMP, Ca2+ and protein kinase C pathways by forskolin, ionomycin and the phorbol ester phorbol 12-myristate 13-acetate (PMA), respectively, upon gonadotropin-releasing hormone (GnRH) secretion, cellular peptide content, mRNA and RNA primary transcript levels. Forskolin, ionomycin and phorbol ester all caused an increase in GnRH secretion in GT1-3 cells in a time and dose-dependent manner during a short-term (1 h) static incubation. Prolonged treatment with forskolin (10 microM), ionomycin (1 microM) and PMA (10 nM) for 12 or 24 h resulted in significant decreases in GnRH mRNA levels. Time-course studies showed that the increases in GnRH secretion stimulated by forskolin, ionomycin and PMA were gradually attenuated over time in parallel with the decreases in mRNA expression. In contrast, there were only small and variable changes in the GnRH cellular content. Studies using a GnRH antagonist (100 microM) suggested that the released GnRH has a negative feedback effect on its own secretion. However, co-incubation with the GnRH antagonist did not alter the inhibitory effects on GnRH mRNA levels by the secretagogues. Further studies on the transcriptional effects of forskolin, ionomycin and PMA on GnRH gene expression in GT1-3 cells revealed that all three secretagogues suppressed GnRH RNA primary transcript levels, with forskolin having a slower time course of action. Thus, the inhibition of cytoplasmic GnRH mRNA, and presumably its synthesis, after 12-24 h of secretagogue treatment may be due at least in part to a suppression of GnRH gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Developmental rate and ploidy of embryos produced by nuclear transfer with different activation treatments in cattle.

Bovine oocyte activation is one of the essential elements that determine the success of nuclear transfer and the subsequent development of cloned embryos. Three methods for oocyte activation, including 5 microM ionomycin (5 min, Group 1) alone, ionomycin+1.9 mM 6-dimethylaminopurine (DMAP, 3h, Group 2), and ionomycin+10 microg/ml cycloheximide (CHX, 3h, Group 3) were compared for the development of embryos produced by somatic nuclear transfer (SCNT) to parthenotes and IVF counterparts. At 19-h post-activation/insemination (hpa/hpi), 27.5% of oocytes in Group 2 cleaved and this rate was greater (P<0.05) than other groups (Group 1, 2.1%; Group 3, 3.0%). None of the oocytes in the IVF control group cleaved at 19-22 hpi. At 24 hpa, the rates of cleavage of oocytes in Group 2 (52.1%) were greater (P<0.05) than those in Groups 1 and 3 (7 and 38.3%, respectively). Only six oocytes (3.3%) in the IVF control group cleaved at 24 hpi. The overall cleavage rates of oocytes in Group 2 (85.5%) at 48 hpa were greater (P<0.05) than other treatments, but it did not show any difference when compared with the IVF control group (75.0%). The development rate to two-cell stage embryos of Group 2 was consistently greater at all observation points followed by Groups 3 and 1. Similar results were obtained in SCNT embryos, but the rates of cleavage at 48 hpi and blastocyst development in Group 2 (68.4 and 16.3%, respectively) did not differ from Group 3 (63.0 and 13.1%, respectively). The chromosomal composition in the parthenotes and SCNT embryos differed (P<0.05) among treatments. In Groups 1 and 3, greater percentages of haploid parthenotes (86 and 71%, respectively) were observed. In contrast, 84% of parthenotes in Group 2 had abnormal ploidy (44% polyploid and 40% mixoploid). In the case of SCNT embryos, Groups 1 and 3 had greater percentages of diploid chromosomal sets (77 and 70%, respectively), whereas 54% in Group 2 were polyploid or mixoploid. These results indicate that DMAP treatment after ionomycin greatly increases the developmental rates of parthenotes, but did not differ in blastocyst development compare with CHX treatment. However, DMAP treatment increased the time-dependent cleavage rate to two-cell stage embryos. Further, it greatly enhanced the incidence of chromosomal abnormalities in parthenotes and SCNT embryos. Hence, it is concluded that CHX combined with ionomycin is more desirable than DMAP for oocyte activation during nuclear transfer in cattle.

Adenine↗
PubMed2005

Differential regulation of NPR-B/GC-B by protein kinase c and calcium.

C-type natriuretic peptide (CNP) activation of the guanylyl cyclase-linked natriuretic peptide receptor-B (NPR-B) stimulates vasorelaxation and bone growth. Hormones and phorbol esters (PMA) inhibit NPR-B in calcium and protein kinase c-dependent manners, respectively. Here, we characterize the kinetic properties of NPR-B in membranes from cells exposed to PMA, the calcium ionophore, ionomycin, or sphingosine-1-phosphate (S1P). PMA and ionomycin primarily increased the K(m) and decreased the V(max) of NPR-B for GTP, respectively, whereas S1P caused modest changes in both parameters. PMA and S1P treatment increased the EC50 for CNP activation by eight- and three-fold, whereas ionomycin was ineffective. All three agents caused NPR-B dephosphorylation, but the basis for the loss of phosphate differed between treatments. In vitro phosphorylation of NPR-B in membranes was markedly diminished by prior whole cell PMA or S1P exposure, whereas ionomycin pretreatment had no effect. The involvement of the known phosphorylated residues in each process was tested with a mutant receptor containing glutamates substituted for these sites. While the effect of PMA was lost on this receptor, the effects of S1P and ionomycin were only partially blocked. Our data suggest that the molecular bases for PMA- and calcium-dependent inhibition of NPR-B are unique. The former results from reduced phosphorylation of a known site and primarily affects the affinity of NPR-B for CNP and GTP. The latter is associated with reductions in maximal velocities by a mechanism that does not involve inhibition of NPR-B phosphorylation and requires a process in addition to the dephosphorylation of the known sites.

Animals↗
PubMed1997

Promotion of HL-60 cell differentiation by 1,25-dihydroxyvitamin D3 regulation of protein kinase C levels and activity.

The hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] promotes differentiation of a number of cell types including HL-60 promyelocytic leukemia cells. It is now established that protein kinase Cbeta (PKCbeta) plays a critical role in HL-60 cell maturation to a monocyte/macrophage phenotype. In the present study, we investigated the importance of PKCbeta levels and activation in 1,25-(OH)2D3-mediated differentiation of HL-60 cells. Cell differentiation promoted by 1,25-(OH)2D3 at 48 hr was 39 +/- 3% (mean +/- SEM) nitroblue tetrazolium (NBT) positive and at 72 hr it was 35 +/- 2% NBT positive and 70% CD14 positive. Thus, promotion of cell differentiation by 20 nM 1,25-(OH)2D3 treatment was maximal at 48-72 hr. When PKCbeta levels and cell differentiation were assayed at 72 hr, treatment with 20 nM 1,25-(OH)2D3 for the initial 6 hr increased PKCbeta levels by 175% but had little effect on cell differentiation (7 +/- 2% NBT positive; 11% CD14 positive). The effect of ionomycin, a calcium ionophore, on PKCbeta levels and cell differentiation also was examined. Alone, 5 microM ionomycin promoted few cells (3% CD14 positive) to differentiate. In contrast, cells treated with 5 microM ionomycin for 66 hr after a 6-hr pretreatment with 20 nM 1,25-(OH)2D3 resulted in 34 +/- 5% NBT positive cells and 73% CD14 positive cells. Quantitatively, this induction of differentiation was identical to that observed in cultures continuously treated with 1,25-(OH)2D3 (35 +/- 2% NBT positive; 70% CD14 positive). Therefore, ionomycin seemed to replace the requirement for the continuous presence of 1,25-(OH)2D3. Chelerythrine chloride (3 microM), a specific PKC inhibitor, blocked differentiation promoted by 1,25-(OH)2D3 alone (82 +/- 2% inhibition) or in sequence with ionomycin (86 +/- 3% inhibition). Taken together, our data show that the capacity of 1,25-(OH)2D3 to both increase PKCbeta levels and activate PKC is utilized to promote HL-60 cell differentiation. These data further suggest that 1,25-(OH)2D3 has a genomic action to increase PKCbeta levels and also a nongenomic action requiring its continuous presence to promote HL-60 cell differentiation.

Calcitriol↗
PubMed2003

Intracytoplasmic sperm injection of bovine oocytes with stallion spermatozoa.

Five experiments were designed to study the fertilizability and development of bovine oocytes fertilized by intracytoplasmic sperm injection (ICSI) with stallion spermatozoa. Experiment 1 determined the time required for pronuclear formation after ICSI. Equine sperm head decondensation began 3 h after ICSI; 42% were decondensed 6 h after ICSI. Male pronuclei (MPN) began to form 12 h after ICSI. Female pronuclei (FPN), however, formed as early as 6 h after ICSI. In Experiment 2, ionomycin, ionomycin plus 6-dimethylaminopurine (DMAP), and thimerosal were used to activate ICSI ova. None of the ICSI ova cleaved after treatment with thimerosal. Ionomycin activation after 24 and 30 h of oocyte maturation resulted in 29 and 48% cleavage rates, respectively. Ionomycin combined with DMAP resulted in 49, 6 and 3% cleavage, morula and blastocyst rates, respectively, when oocytes were activated after 24 h maturation. In Experiment 3, rates of cleavage (45-60%) and development to morulae (4-13%) and blastocysts (1-5%) stages following ICSI were not different (P>0.05) among three stallions. Treatment of stallion spermatozoa with ionomycin did not affect cleavage or development of ova fertilized by ICSI. The chromosomal constitution of blastocysts derived from ICSI was bovine, not bovine and equine hybrids. In Experiment 4, to make male and FPN form synchronously, colchicine and DMAP were used for 4 h to inhibit oocytes at metaphase during activation; 63% of oocytes were still at metaphase 8h after ICSI when treated with colchicine, and 50% of sperm nuclei were decondensed. About 18 h after ICSI, 21 and 50% male and FPN had formed, respectively, but cleavage rates were low, and only 1% developed to morulae. In Experiment 5, to test if capacitated equine sperm could fuse with the bovine oolemma, capacitated spermatozoa were injected subzonally (SUZI). Of the 182 SUZI oocytes, 49 (27%) contained extruded second polar bodies. After activation of oocytes with second polar bodies, 44, 22 and 15% developed to 2-, 4- and 8-cell stages, respectively, but development stopped at the 8-cell stage. None of the unactivated oocytes cleaved. In conclusion, equine spermatozoa can decondense and form MPN in bovine oocytes after ICSI, but subsequent embryonic development is parthenogenetic with only bovine chromosomes being found.

Adenine↗
PubMed1999

Calcium ionophores can induce either apoptosis or necrosis in cultured cortical neurons.

Cultured cortical neurons exposed for 24 h to low concentrations of the Ca2+ ionophores, ionomycin (250 nM) or A-23187 (100 nM), underwent apoptosis, accompanied by early degeneration of neurites, cell body shrinkage, chromatin condensation and internucleosomal DNA fragmentation. This death could be blocked by protein synthesis inhibitors, as well as by the growth factors brain-derived neurotrophic factor or insulin-like growth factor I. If the ionomycin concentration was increased to 1-3 microM, then neurons underwent necrosis, accompanied by early cell body swelling without DNA laddering, or sensitivity to cycloheximide or growth factors. Calcium imaging with Fura-2 suggested a possible basis for the differential effects of low and high concentrations of ionomycin. At low concentrations, ionomycin induced greater increases in intracellular Ca2+ concentration in neurites than in neuronal cell bodies, whereas at high concentrations, ionomycin produced large increases in intracellular Ca2+ concentration in both neurites and cell bodies. We hypothesize that the ability of low concentrations of Ca2+ ionophores to raise intracellular Ca2+ concentration preferentially in neurites caused early neurite degeneration, leading to loss of growth factor availability to the cell body and consequent apoptosis, whereas high concentrations of ionophores produced global cellular Ca2+ overload and consequent necrosis.

Animals↗
PubMed1991

Influx of extracellular calcium is required for the membrane translocation of 5-lipoxygenase and leukotriene synthesis.

Our studies assessed the effects of increases in intracellular calcium concentrations [( Ca2+]i) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]i. Leukotriene C4 production associated with [Ca2+]i in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]i level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin- and thapsigargin-induced rises in [Ca2+]i were transient, indicating that the prolonged [Ca2+]i elevation is due to an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peak rises in [Ca2+]i that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1,4,5-inositol triphosphate (IP3), 1,3,4-IP3, and 1,3,4,5-inositol tetraphosphate (IP4).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Stimulation of leukotriene production and membrane translocation of 5-lipoxygenase by cross-linking of the IgE receptors in RBL-2H3 cells.

Recent studies in rat basophilic leukemia cells (RBL-2H3) have shown that two pharmacological agents, ionomycin and thapsigargin, induce leukotriene C4 production and translocation of 5-lipoxygenase from cytosol to membrane, primarily by causing an influx of extracellular calcium. In the present study, we investigate the induction of these events by receptor activation. Cross-linking of high-affinity IgE receptors (Fc epsilon RI) by antigen in RBL-2H3 cells leads to leukotriene C4 production and membrane translocation of 5-lipoxygenase. As in the ionomycin-stimulated cells, leukotriene C4 production in antigen-stimulated cells is calcium-dependent since the amount of leukotriene C4 produced correlates quantitatively with the increase in intracellular free calcium concentration ([Ca2+]i). However, the increase in [Ca2+]i required for equivalent leukotriene C4 production by antigen is not as high as it is using ionomycin. In addition, no threshold [Ca2+]i level is required for leukotriene production by antigen, which is in contrast to the ionomycin stimulation that a [Ca2+]i level of 300-400 nM is required. Furthermore, antigen causes an additive increase in leukotriene C4 production in cells stimulated by the ionomycin. These results suggest that another as yet unidentified intracellular pathway acts in conjunction with Ca2+ for leukotriene synthesis in antigen-stimulated cells. Antigen stimulation causes 20-30% of the total cell 5-lipoxygenase to associate with membranes (compared with 10% in unstimulated cells) as demonstrated by enzyme activity assay and by Western Blot using antibodies to 5-lipoxygenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗