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PubMed2005

Evaluation of activation treatments for blastocyst production and birth of viable calves following bovine intracytoplasmic sperm injection.

The objective of this study was to compare the effectiveness of different methods of bovine oocyte activation following intracytoplasmic sperm injection (ICSI) in terms of oocyte cleavage and blastocyst rates, and calf production. Oocytes were harvested, post mortem, from the ovaries of Japanese Black heifers or cows. ICSI was carried out using a piezo-electric actuator. The injected or sham-injected oocytes that were assigned to three activation treatments, each replicated three times, were studied: (1) exposure to 5 microM ionomycin for 5 min (ionomycin); (2) exposure to 5 microM ionomycin for 5 min followed by culture in TCM199 for 3 h and a further 3h culture in 1.9 mM 6-dimethylaminopurine (DMAP-ionomycin+DMAP); (3) exposure to 7% ethanol in TCM199 for 5 min, 4 h after ICSI (ethanol). One or two blastocysts from the ionomycin+DMAP (8 recipients) and ethanol (17 recipients) oocyte activation treatments were non-surgically transferred into Holsteins for the study of calf production. The highest cleavage and blastocyst production rates were observed in the ionomycin+DMAP treatment (83.9% and 40.1%) by the ICSI. These rates were significantly (P<0.05) higher than those for the ionomycin oocyte activation treatment (57.6% and 18.2%) but did not differ from the ethanol treatment (75.6% and 29.4%). In the sham-injected, the highest blastocyst production rates were observed for the ionomycin+DMAP and ethanol treatments (10.7% and 11.3%). Pregnancy and birth rates for blastocysts derived from the ethanol oocyte activation treatment (58.8% and 47.4%) were significantly higher (P<0.05) than those of the ionomycin+DMAP treatment (12.5% and 9.2%). The results showed that post-ICSI oocyte activation with ethanol is more effective than activation with ionomycin alone or with ionomycin+DMAP for the production of viable blastocysts and calves.

Adenine↗
PubMed1998

Expression and function of Fas and Fas ligand on peripheral blood lymphocytes in normal subjects.

We investigated the expression and function of Fas and Fas ligand (FasL) on peripheral blood lymphocytes (PBLs). The cells were stimulated with various cytokines or 12-0-tetradecanoyl phorbol 13-acetate (PMA) plus ionomycin. About 30% of unstimulated PBLs expressed Fas, and the expression was augmented by interleukin-1beta (IL-1beta), IL-2, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or PMA plus ionomycin. Although only minimal FasL expression was detected on unstimulated PBLs, FasL expression was markedly induced by IL-2 or PMA plus ionomycin, suggesting that Fas and FasL were both expressed on IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs. Although IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs were positive for both Fas and FasL, no significant increase in apoptosis was demonstrated in these activated PBLs. In addition, treatment of PBLs with IL-2 or PMA plus ionomycin did not change anti-Fas-induced apoptosis, although these activated PBLs expressed Fas strongly when compared with unstimulated PBLs. Only IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs killed Fas+ target cells efficiently via the interaction of Fas on target cells with FasL of PBLs. Bcl-2 was constitutively expressed on unstimulated PBLs, but its expression was significantly augmented by IL-2 or PMA plus ionomycin. The expression of Bax was clearly induced only on IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs and that of other Bcl-2 family proteins such as Bcl-x and Bad could not be detected on human PBLs, including IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs. Our results suggest that PBLs activated by IL-2 or PMA plus ionomycin express both Fas and FasL and that they kill Fas+ target cells by using FasL on the surface. The resistance of these activated PBLs to Fas-mediated apoptosis may be due to the augmented Bcl-2 expression or the presence of Bcl-2:Bax heterodimers on these cells.

Adult↗
PubMed1995

Ca2+ x calmodulin prevents myristoylated alanine-rich kinase C substrate protein phosphorylation by protein kinase Cs in C6 rat glioma cells.

Ionomycin stimulated membrane-associated protein kinase Cs (PKCs) activity in C6 rat glioma cells as much as the potent PKCs stimulator 12-O-tetradecanoyl phorbol 13-acetate (TPA). However, while TPA, as expected, powerfully stimulated the phosphorylation of the PKCs' 85-kDa myristoylated alanine-rich protein kinase C substrate (MARCKS) protein, ionomycin unexpectedly did not. Instead, ionomycin reduced the basal MARCKS phosphorylation. Pretreating the glioma cells with ionomycin prevented TPA-stimulated PKCs from phosphorylating the MARCKS protein. The stimulation of membrane PKCs activity and the prevention of MARCKS phosphorylation by ionomycin required external Ca2+ because they were both abolished by adding 5 mM EGTA to the culture medium. Recently (Chakravarthy, B. R., Isaacs, R. J., Morley, P., Durkin, J. P., and Whitfield, J. F. (1995) J. Biol. Chem. 270, 1362-1368), we proposed that Ca2+ x calmodulin complexes block MARCKS phosphorylation by the activated PKCs in keratinocytes stimulated by raising the external Ca2+ concentration. In the present experiments calmodulin prevented MARCKS phosphorylation by TPA-stimulated PKCs in glioma cell lysates, and this blockade was lifted by a calmodulin antagonist, the calmodulin-binding domain peptide. But, physiologically more significant, pretreating intact glioma cells with a cell-permeable calmodulin antagonist, calmidazolium, prevented ionomycin from blocking MARCKS phosphorylation by PKCs in unstimulated and TPA-stimulated cells. The effect of ionomycin on MARCKS phosphorylation was not due to the stimulation of Ca2+ x calmodulin-dependent phosphoprotein phosphatase, calcineurin, because cyclosporin A, a potent inhibitor of this phosphatase, did not stop ionomycin from preventing MARCKS phosphorylation. The ability of ionomycin to prevent TPA-stimulated PKCs from phosphorylating MARCKS depended on whether ionomycin was added before, with, or after TPA. Maximum blockade occurred when ionomycin was added before TPA but was less effective when added with or after TPA. These results indicate that Ca2+ x calmodulin can profoundly affect PKCs' signaling at the substrate level.

Amino Acid Sequence↗
PubMed1993

Elevation of cytosolic calcium is sufficient to induce growth inhibition in a B cell lymphoma.

Recently, we have described that anti-IgM antibodies profoundly inhibited the growth of BKS-2, an immature B cell lymphoma. In this report, we demonstrated that ionomycin alone at very low concentrations (20 nM) inhibited the growth of BKS-2 cells completely. The levels of intracellular Ca2+ induced by the inhibitory concentrations of ionomycin were comparable to those in anti-IgM-treated cells. The growth inhibition caused by ionomycin was reversed by phorbol 12-myristate 13-acetate and lipopolysaccharide. In addition, the immunosuppressants, cyclosporin A and FK506 conferred significant protection from the negative signal induced by ionomycin. However, either cyclosporin A, FK506 or lipopolysaccharide was not found to have direct effect on ionomycin-induced Ca2+ mobilization in BKS-2 cells. Also, ionomycin augmented the anti-IgM-induced growth arrest in these cells. Furthermore, BKS-2 cells that were exposed to anti-IgM or ionomycin underwent apoptosis as characterized by DNA fragmentation. Thus, the characteristics of growth inhibition induced by ionomycin and anti-IgM appeared to be similar in that phorbol 12-myristate 13-acetate, lipopolysaccharide, cyclosporin A and FK506 caused significant reversal from such negative signals and both ionomycin and anti-IgM induced apoptosis in these cells. Altogether, these results showed that the elevation of intracellular Ca2+ alone was sufficient to inhibit the growth of some B lymphoma cells.

Animals↗
PubMed1993

Activation of the respiratory burst and tyrosine phosphorylation of proteins in human neutrophils: no direct relationship and involvement of protein kinase C-dependent and -independent signaling pathways.

Human neutrophils maximally stimulated with the optimal concentration (100 ng/ml) of phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), for 5 min at 37 degrees C did not respond with superoxide (O2-) release to the later addition of PMA itself or the Ca2+ ionophore ionomycin. However, these cells did respond with enhanced release of O2- to the later addition of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A (Con A). In these PMA-pretreated cells, an increase in cytoplasmic free Ca2+ ([Ca2+]i) induced by ionomycin was unaffected, whereas that induced by FMLP was inhibited by 50-60% and that induced by Con A was completely abolished. A 42-kDa protein was predominantly and consistently tyrosine-phosphorylated by FMLP, PMA and ionomycin with the different kinetics according to the stimuli. The dose-response curves showed that tyrosine phosphorylation and O2- release were stimulated in parallel by PMA, whereas tyrosine phosphorylation and an increase in [Ca2+]i, but not O2- release, were stimulated in parallel by FMLP or ionomycin. The potency of inducing tyrosine phosphorylation was ionomycin > FMLP = PMA, whereas the potency of triggering of O2- release was PMA > ionomycin = FMLP. UCN-01, a PKC inhibitor, inhibited O2- release and tyrosine phosphorylation induced by PMA, but not by FMLP or ionomycin. In contrast, pertussis toxin inhibited O2- release and tyrosine phosphorylation induced by FMLP, but not by PMA. Tyrosine kinase inhibitors (erbstatin and genistein) inhibited O2- release induced by FMLP, but not by PMA. However, both tyrosine kinase inhibitors did not impair FMLP- or PMA-induced tyrosine phosphorylation of a 42-kDa protein. Increased tyrosine phosphorylation of a 42-kDa protein was also detected in immature myeloid cells (HL-60 cells) stimulated by PMA, but not by ionomycin. These findings suggest that FMLP and Con A trigger the respiratory burst in human neutrophils by activating the definite pathway which include other signals than activation of PKC and an increase in [Ca2+]i; tyrosine phosphorylation of a 42-kDa protein is induced by the PKC-dependent and independent mechanisms according to the stimuli, and the PKC-independent and ionomycin-sensitive mechanism is inoperative in HL-60 cells; and tyrosine phosphorylation of a 42-kDa protein is unlikely to be causally related to activation of the respiratory burst.

Concanavalin A↗
PubMed1991

Importance of transients in cytosolic free calcium concentrations on activation of Na+/H+ exchange in GH4C1 pituitary cells.

In GH4C1 cells, TRH and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), have been shown to activate Na+/H+ exchange, probably via stimulation of protein kinase C. In the present study, the dependence of changes in intracellular pH (pHi) on transients in the cytosolic free calcium concentration [( Ca2+]i) was investigated using BCECF and fura-2, respectively. In buffer containing 0.4 mM extracellular Ca2+, both TRH and ionomycin induced rapid cytosolic alkalinization in GH4C1 cells acid loaded with nigericin. The action of ionomycin on pHi was abolished by preincubating the cells with 100 microM amiloride or by replacing extracellular Na+ with choline+, indicating that the change in pHi was probably due to activation of Na+/H+ exchange. The actions of both TRH and ionomycin on pHi were blunted in Ca2(+)-free buffer. When acid-loaded cells were stimulated first with ionomycin, to deplete intracellular Ca2+ stores, and then incubated with TRH, the TRH-induced alkalinization was blunted; thus, an increase in [Ca2+]i is needed for full activation of Na+/H+ exchange. To study further the importance of agonist-induced changes in [Ca2+]i on the activation of Na+/H+ exchange, acid-loaded cells were incubated first with TPA, and then with either TRH or ionomycin. TPA induced a rise in pHi, which was further enhanced by TRH, but not ionomycin. The actions of both TRH and ionomycin on Na+/H+ exchange were attenuated, but not abolished, in cells pretreated with TPA for 36 h. Acidification of the cytosol with nigericin increased the resting [Ca2+]i level from 125 +/- 29 to 200 +/- 25 nM (P less than 0.01). The increase in [Ca2+]i was greatly attenuated when extracellular Ca2+ was chelated with EGTA before the addition of nigericin. Both the TRH- and ionomycin-induced increases in [Ca2+]i were blunted in acid-loaded cells. We conclude that in GH4C1 cells, a transient increase in [Ca2+]i can enhance Na+/H+ exchange and cause a rise in pHi, but that to obtain full activation of exchange, protein kinase C activity must also be stimulated. Furthermore, pHi is important in maintaining an adequate store of sequestered intracellular Ca2+ and in the release of Ca2+ from that store in response to TRH and ionomycin.

Animals↗
PubMed1989

Diacylglycerol and calcium induce rapid enhancement of A-system amino acid transport by independent mechanisms in human T-lymphocytes.

Sn-1,2-diacylglycerols (DAG) and ionized-free calcium can act as intracellular second messengers for cell activation. Traditionally, T-lymphocyte activation is assessed by measurements of DNA synthesis or lymphokine production, but these responses require several days to occur and involve multiple intermediary regulatory steps. In contrast, we have found that T-lymphocytes demonstrate rapid enhancement of A-(alanine-favoring) system amino acid uptake when treated with DAG or ionomycin. A 30-40% increase in the initial velocity of uptake (vi) of the synthetic A-system specific amino acid, methylamino-isobutyric acid (MeAIB), was measured following 5 min of exposure to DAG or ionomycin. The vi was enhanced 60% from 12 to 19 mumol/liter cell water per min after 30 min exposure of T-cells to optimal concentrations of dioctanoylglycerol (30 microM), oleoylacetylglycerol (30 microM), or ionomycin (5 microM) (P less than .01 for each agent). A 50-fold excess of non-radioactive MeAIB inhibited 80% of [14C]MeAIB uptake in both unstimulated and stimulated cells, indicating that uptake remained largely carrier-mediated on treatment with these agents. Cycloheximide, 100 micrograms/ml, inhibited protein synthesis but did not block the A-system amino acid transport enhancement induced by DAG or ionomycin. The DAG-induced increase in the vi was blocked 40% with 100 microM H-7, an inhibitor of protein kinase C. H-7 treatment did not inhibit the ionomycin-induced A-system enhancement. A marked increase in cytoplasmic free calcium was measured when T-lymphocytes were exposed to ionomycin but not on DAG exposure, and the A-system effect of ionomycin but not DAG was blocked by extracellular EGTA. These data are compatible with two pathways for rapid enhancement of A-system amino acid uptake in T-lymphocytes. DAG stimulation is mediated via protein kinase C whereas ionomycin produces an A-system effect of similar magnitude independent of protein kinase C by an increase in cytoplasmic calcium.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Endothelium-dependent relaxations in sheep pulmonary arteries and veins: resistance to block by NG-nitro-L-arginine in pulmonary hypertension.

1. The effect of the nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NOARG), on endothelium-dependent relaxation to a receptor-independent agent, ionomycin, was examined in isolated pulmonary arteries and veins from control, short-term and chronic pulmonary hypertensive sheep. All vessel segments were contracted to optimal levels of active force with endothelin-1 to record endothelium-dependent relaxation. 2. Pulmonary hypertension was induced by continuous pulmonary artery air embolization for 1 day (short-term) and 14 days (chronic) and was associated with a 2 and 3 fold increase in pulmonary vascular resistance respectively. 3. L-NOARG (0.1 mM) reduced the maximum relaxation (Rmax) to ionomycin in large and medium-sized pulmonary arteries from control sheep by approximately 70%. By contrast, L-NOARG (0.1 mM) did not inhibit the Rmax to ionomycin in matched vessels from short-term and chronic pulmonary hypertensive sheep. 4. Resistance of ionomycin-induced relaxations to inhibition by L-NOARG, was confined to the arterial vasculature in chronic pulmonary hypertensive animals, as relaxations to ionomycin in large and medium-sized chronic pulmonary hypertensive veins were, like those in control veins, abolished by L-NOARG. Both large and medium-sized pulmonary veins from short-term pulmonary hypertensive sheep, however, were resistant to block by L-NOARG. 5. Neither sensitivity (pEC50) nor Rmax to ionomycin in large, short-term pulmonary hypertensive arteries was affected when the extracellular concentration of K+ was increased isotonically to 30 mM. Nifedipine (0.3 microM) was present throughout to prevent high K(+)-induced smooth muscle contraction. In the presence of this high extracellular K+, however, L-NOARG (0.1 mM) caused complete inhibition of the relaxation to ionomycin, whereas in normal extracellular K+ (4.7 mM), L-NOARG only weakly inhibited ionomycin relaxations. 6. In conclusion, the onset of pulmonary hypertension in sheep following air embolization, is associated with the development of resistance of endothelium-dependent relaxations to block by L-NOARG. The mechanism of L-NOARG resistance appears to be due to the up-regulation of a K+ channel-mediated backup vasodilator mechanism which can compensate for the loss of nitric oxide (NO)-mediated relaxation. Although this mechanism remains functionally 'silent' in the presence of NO it is able to maintain adequate endothelium-dependent vasodilatation during pulmonary hypertension if NO synthesis is compromised.

Animals↗
PubMed2003

Role of Ca2+-activated K+ channels in human erythrocyte apoptosis.

Exposure of erythrocytes to the Ca2+ ionophore ionomycin has recently been shown to induce cell shrinkage, cell membrane blebbing, and breakdown of phosphatidylserine asymmetry, all features typical of apoptosis of nucleated cells. Although breakdown of phosphatidylserine asymmetry is thought to result from activation of a Ca2+-sensitive scramblase, the mechanism and role of cell shrinkage have not been explored. The present study was performed to test whether ionomycin-induced activation of Ca2+-sensitive Gardos K+ channels and subsequent cell shrinkage participate in ionomycin-induced breakdown of phosphatidylserine asymmetry of human erythrocytes. According to on-cell patch-clamp experiments, ionomycin (1 microM) induces activation of inwardly rectifying K+-selective channels in the erythrocyte membrane. Fluorescence-activated cell sorter analysis reveals that ionomycin leads to a significant decrease of forward scatter, reflecting cell volume, an effect blunted by an increase of extracellular K+ concentration to 25 mM and exposure to the Gardos K+ channel blockers charybdotoxin (230 nM) and clotrimazole (5 microM). As reflected by annexin binding, breakdown of phosphatidylserine asymmetry is triggered by ionomycin, an effect again blunted, but not abolished, by an increase of extracellular K+ concentration and exposure to charybdotoxin (230 nM) and clotrimazole (5 microM). Similar to ionomycin, glucose depletion leads (within 55 h) to annexin binding of erythrocytes, an effect again partially reversed by an increase of extracellular K+ concentration and exposure to charybdotoxin. K-562 human erythroleukemia cells similarly respond to ionomycin with cell shrinkage and annexin binding, effects blunted by antisense, but not sense, oligonucleotides against the small-conductance Ca2+-activated K+ channel isoform hSK4 (KCNN4). The experiments disclose a novel functional role of Ca2+-sensitive K+ channels in erythrocytes, i.e., their participation in regulation of erythrocyte apoptosis.

Annexins↗
PubMed1993

Cl- secretion by cultured shark rectal gland cells. III. Ca2+ regulation of apical membrane Cl- conductance.

Calcium ionophores (ionomycin and A-23187) were employed to assess the effects of increased intracellular Ca2+ concentration ([Ca2+]i) on apical membrane Cl- conductance (GaCl) and rate of transepithelial Cl- secretion in cultured shark rectal gland (SRG) cells. Apical 2 microM ionomycin induced dramatic changes in cellular electrophysiological properties: the apical membrane electrical potential difference (V(a)) depolarized from -66 mV to -46 mV, the fractional resistance of the apical membrane (fRa) decreased from 0.88 to 0.23, and the transepithelial electrical potential difference (Vab) increased slightly from +1.2 mV to +1.4 mV. These effects result from increased GaCl because apical low-Cl- shark Ringer (SR) depolarized V(a) by 32 mV and increased fRa from 0.23 to 0.36. Ionomycin-stimulated Vab or short-circuit current (Isc) results largely from increased Cl- secretion because approximately 80% of the increase in Isc is Cl- dependent. Establishing the Ca2+ dependence of ionophore activation of GaCl was confounded because apical low-Ca2+ SR [Ca2+ concentration ([Ca2+]) 1 mM to 0.1 microM] alone activated this conductive pathway. To establish the Ca2+ dependence of ionophore action, we assessed the effect of ionomycin on Isc in low-Ca2+ SR ([Ca2+] = 0.1 microM) and in SR. In low-Ca2+ SR, apical ionomycin stimulated Isc by 14.0 microA/cm2. In SR (normal [Ca2+]), ionomycin increased Isc further by 27.0 microA/cm2. Superfusing the basolateral surface with 2 microM ionomycin for 8-16 min failed to activate GaCl. In every case, subsequent superfusion of the apical surface with ionophore for 1.5-2 min activated GaCl. Bilateral 4 microM indomethacin (45-min superfusion) failed to block the ionomycin-induced GaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

A human lens model of cortical cataract: Ca2+-induced protein loss, vimentin cleavage and opacification.

PURPOSE: Cortical cataract in humans is associated with Ca2+ overload and protein loss, and although animal models of cataract have implicated Ca2+-activated proteases in this process, it remains to be determined whether the human lens responds in this manner to conditions of Ca2+ overload. The purpose of these experiments was to investigate Ca2+-induced opacification and proteolysis in the organ-cultured human lens. METHODS: Donor human lenses were cultured in Eagle's minimum essential medium (EMEM) for up to 14 days. The Ca2+ ionophore ionomycin was used to induce a Ca2+ overload. Lenses were loaded with [3H]-amino acids for 48 hours. After a 24-hour control efflux period, lenses were cultured in control EMEM (Ca2+ 1.8 mM), EMEM + 5 microM ionomycin, or EMEM + 5 microM ionomycin + 5 mM EGTA (Ca2+ < 1 microM). Efflux of proteins and transparency were monitored daily. Protein distribution and cytoskeletal proteolysis were analyzed at the end of the experiment. Cytoskeletal proteins were isolated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analyses were probed with anti-vimentin antibody (clone V9) and detected by enhanced chemiluminescence. RESULTS: Lenses cultured under control conditions remained transparent for 14 days in EMEM with no added supplements or serum. The lenses synthesized proteins and had a low rate of protein efflux throughout the experimental period. Ionomycin treatment resulted in cortical opacification, which was inhibited when external Ca2+ was chelated with EGTA. Exposure to ionomycin also led to an efflux of [3H]-labeled protein, amounting to 41% of the labeled protein over the 7-day experimental period, compared with 12% in ionomycin + EGTA-treated lenses. Efflux was accounted for by loss from the lens soluble protein (crystallin) fraction. Western blot analysis of the cytoskeletal protein vimentin (56 kDa) revealed a distinct breakdown product of 48 kDa in ionomycin-treated lenses that was not present when Ca2+ was chelated with EGTA. In addition, high-molecular-weight proteins (approximately 115 kDa and 235 kDa) that cross-reacted with the vimentin antibody were observed in ionomycin-treated lenses. The Ca2+-induced changes were not age dependent. CONCLUSIONS: Human lenses can be successfully maintained in vitro, remaining transparent for extended periods. Increased intracellular Ca2+ induces cortical opacification in the human lens. Ca2+-dependent cleavage and cross-linking of vimentin supports possible roles for calpain and transglutaminase in the opacification process. This human lens calcium-induced opacification (HLCO) model enables investigation of the molecular mechanisms of opacification, and the data help to explain the loss of protein observed in human cortical cataractous lenses in vivo.

Adolescent↗
PubMed1991

Differential sensitivity of virgin and memory T lymphocytes to calcium ionophores suggests a buoyant density separation method and a model for memory cell hyporesponsiveness to Con A.

Previous work from this laboratory has indicated that murine memory T cells differ from virgin T cells in that the former are more resistant to agents that alter intracellular [Ca]i. We have used this difference to devise a method for separating virgin from memory T cells by centrifugation over an ionomycin-containing Percoll step gradient after brief exposure to 2 microM ionomycin. Under these conditions, those T cells that are most sensitive to ionomycin-induced changes in [Ca]i become more dense and therefore travel further into the Percoll/ionomycin gradient than cells that are more resistant to ionomycin. We show that the ionomycin-resistant cell population is enriched for cells that express high levels of Pgp-1 (CD44), and low levels of CD45RB, and thus appears to consist largely of memory T cells. Both CD4 and CD8 cells can be divided into Pgp-1hi and Pgp-1lo subsets in this way. Cells recovered from such a gradient and washed to remove the ionomycin appear normally functional, i.e., neither more nor less responsive to mitogens and costimuli than untreated cells. Limiting dilution methods show that the ionomycin-sensitive (virgin) subset contains most of the Con A-responsive precursors for cytotoxicity, and most of the cells able to produce IL-2 in responses to Con A or staphylococcal enterotoxin B. Ag-specific helper memory cells are, however, found predominantly in the ionomycin-resistant fraction of the spleen and draining lymph nodes of mice infected with Schistosoma mansoni. Changes in resistance to calcium signal development may represent a fundamental distinction between virgin and memory T cells, and could contribute to differences in activation requirements between these two cell subsets.

Animals↗
PubMed1996

Signal requirements for interleukin 4 promoter activation in human T cells.

We have studied the signal requirements for human IL-4 promoter activation in Jurkat T cells by the use of DNA transfection assays with vectors carrying the IL-4 promoter linked to a reporter gene. Stimulation with calcium (Ca2+) ionophores (ionomycin), but not with phorbol esters (phorbol myristate acetate, PMA) or cyclic AMP elevating agents (prostaglandin E2, PGE2), induced the transcriptional activity of the IL-4 promoter by approximately 3-fold. Costimulation with ionomycin and PGE2 resulted in the same level of IL-4 promoter activity as the stimulation with ionomycin alone. In contrast, costimulation with ionomycin and PMA decreased the activity of the IL-4 promoter by approximately 40% compared to stimulation with ionomycin alone. Induction of Il-4 promoter by ionomycin was partially inhibited (approximately 50% inhibition) in the presence of as high as 2 microgram/ml cyclosporin A (CsA), an inhibitor of the Ca+/calmodulin-dependent phosphatase calcineurin. Under the same conditions, only 0.1 microgram/ml of CsA inhibited by >95% the transactivation of the IL-2 promoter in response to ionomycin and PMA. Transfection of a deletion mutant of the calcineurin catalytic subunit (delta CaM-AI) known to have Ca2+-independent, constitutive phosphatase activity increased IL-4 promoter activity by approximately 14-fold. Stimulation with ionomycin of cells transfected with low doses of delta CaM-AI, further induced IL-4 promoter activity by approximately 2-fold. These results identify the Ca2+-signaling system as a key component of the signal transduction pathway leading to IL-4 promoter activation in Jurkat T cells and suggest a major role of calcineurin in its regulation.

Calcineurin↗
PubMed1992

Age-associated changes in mitogen-induced protein phosphorylation in murine T lymphocytes.

Since T lymphocyte proliferation declines with age, and since activation of a still only partially defined set of protein kinases is thought to play a critical role in T cell activation, we have carried out a systematic survey of age-related changes in protein phosphorylation in T lymphocytes. We used two-dimensional electrophoretic analysis to examine lysates prepared from T cells after 10 min of exposure to mitogens [anti-CD3, concanavalin A (Con A) and phorbol 12-myristate 13-acetate (PMA) plus ionomycin] and nonmitogenic activators (PMA or ionomycin used separately). Our results show a progressive, life-long decline in the levels of anti-CD3-induced phosphorylation of all 16 phosphoproteins that respond vigorously in T cells of young mice. Mice 10-18 months of age showed levels of response that were clearly below those induced in young mice, while responses of mice 22-24 months of age were even more severely diminished. Responses to Con A, PMA, ionomycin and a combination of PMA plus ionomycin were equally blunted in old mice, except for 2 (of 12) phosphoproteins that continued to respond to PMA even in the old animals. None of the phosphoproteins which were ionomycin responsive in young mice continued to respond in old mice, although 1 (of 3) ionomycin-inhibitable phosphoproteins remained inhibitable in old mice. We also found three phosphoproteins which became phosphorylated in response to anti-CD3, PMA and Con A (but not ionomycin) only in old mice, and a pair of phosphoproteins which were unresponsive to all mitogens but showed a higher "baseline" phosphorylation in T cells from old mice. Comparing phosphoprotein patterns between CD8- and CD8-CD45RB- T cells from young mice allowed us to identify nine phosphoproteins that were strongly responsive to anti-CD3 only when CD45RB+ (i.e. virgin) cells were present. Although the immune system of old mice consists largely of memory T cells, the change in phosphoprotein phosphorylation patterns cannot simply be explained by the accumulation of this cell type. We conclude that aging leads to a global impairment of several distinct protein kinase pathways in both virgin and memory T cell sets.

Adenosine Triphosphate↗
PubMed2003

Potentiation of the osmosensitive taurine release and cell volume regulation by cytosolic Ca2+ rise in cultured cerebellar astrocytes.

Hyposmolarity (-30%) in cultured cerebellar astrocytes raised cytosolic Ca2+ concentration ([Ca2+]i) from 160 to 400 nM and activated the osmosensitive taurine release (OTR) pathway. Although OTR is essentially [Ca2+]i-independent, further increase in [Ca2+]i by ionomycin strongly enhanced OTR, with a more robust effect at low and mild osmolarity reductions. Ionomycin did not affect isosmotic taurine efflux. OTR was decreased by tyrphostin A25 and increased by ortho-vanadate, suggesting a modulation by tyrosine kinase or phosphorylation state. Inhibition of phosphatidylinositol-3-kinase activity by wortmannin markedly decreased OTR and the ionomycin increase. Conversely, OTR and the ionomycin effect were independent of ERK1/ERK2 activation. OTR and its potentiation by ionomycin differed in their sensitivity to CaM and CaMK blockers and in the requirement of an intact cytoskeleton for the ionomycin effect, but not for normal OTR. Changes in the actin cytoskeleton organization elicited by hyposmolarity were not observed in ionomycin-treated cells, which may permit the operation of CaM/CaMK pathways involved in the OTR potentiation by [Ca2+]i rise. OTR potentiation by [Ca2+]i requires the previous or simultaneous activation/operation of the taurine release mechanism and is not modifying its set point, but rather increasing the effectiveness of the pathway, resulting in a more efficient volume regulation. This may have a beneficial effect in pathological situations with concurrent swelling and [Ca2+]i elevation in astrocytes.

Animals↗
PubMed1993

Extracellular calcium does not contribute to cryopreservation-induced cytotoxicity.

The possible role of extracellular calcium ([Ca+2]e) in cryopreservation-induced cytotoxicity was tested using Madin-Darby canine kidney (MDCK) cells and a fluorescent multiple endpoint assay. MDCK cells maintained in 2 mM [Ca+2]e and treated with the calcium ionophore, ionomycin, increased their intracellular calcium ([Ca+2]i) as revealed by the calcium indicator dye, Fluo3 and the bottom-reading spectrofluorometer, CytoFluor 2300. The addition of 10 mM [ethylene bis (oxyethylenenitrilo)]-tetraacetic acid (EGTA) to the extracellular medium before treatment with ionomycin blocked this ionomycin-dependent increase in [Ca+2]i. A number of site and activity-specific fluorescent probes were surveyed to determine which indicator dye might best reveal the ionomycin-induced cytotoxic events during this increase in [Ca+2]i. Although most dyes changed their emission profiles in response to calcium, neutral red was found to best reflect the loss of [Ca+2]i homeostasis. The NR50 for a 15-min exposure to ionomycin in the presence of 2 mM [Ca+2]e was approximately 2 microM ionomycin, but ionomycin had little apparent effect on neutral red retention when 10 mM EGTA was added to the extracellular medium. Thus it was clear that an increase in [Ca+2]i could be cytotoxic to MDCK cells and that neutral red could monitor this cytotoxic episode. To test if [Ca+2]e was similarly cytotoxic during cryopreservation, MDCK cells were subjected to cryopreservation in the presence of dimethylsulfoxide (DMSO). In contrast to previous studies, plasma membrane integrity, not lysosomal function, seemed to best correlate with cell survival subsequent to cryopreservation. In addition, decreasing [Ca+2]e had no discernable effect on the retention of plasma membrane indicator dyes, neutral red, or cell survival. It is concluded that a) plasma membrane indicator dyes, not neutral red, might be better indicators of cytotoxicity occurring during cryopreservation; b) DMSO might be toxic to lysosomes during cryopreservation of cultured cells; and c) although [Ca+2]e can contribute to cytotoxicity, the presence of [Ca+2]e might not influence cryopreservation-induced cytotoxicity.

Aniline Compounds↗
PubMed1988

Positive correlation between cytosolic free calcium and surfactant secretion in cultured rat alveolar type II cells.

To determine whether increases in the cytosolic free Ca2+ concentration ([Ca2+]i) accompany agonist-stimulated surfactant secretion by cultured alveolar type II cells, we measured the [Ca2+]i of quin2-loaded cells isolated from adult rats before and after cells were stimulated with ionomycin, terbutaline or tetradecanoylphorbol acetate (TPA). To determine whether increases in [Ca2+]i are necessary for stimulated surfactant secretion to occur, we measured secretion in cells after [Ca2+]i had been reduced by loading cells with quin2 in medium containing low [Ca2+]. Ionomycin increased [Ca2+]i and stimulated surfactant secretion in a dose-dependent manner. Reductions in [Ca2+]i correlated with reductions in secretion stimulated by ionomycin, terbutaline or TPA. Ionomycin-stimulated secretion was most sensitive to reductions in [Ca2+]i; terbutaline-stimulated secretion was more sensitive than TPA-stimulated secretion. When [Ca2+]i was less than 65 nM, all stimulated secretion was blocked. Restoration of [Ca2+]i to greater than 100 nM restored ionomycin-stimulated secretion. We conclude that ionomycin increases [Ca2+]i and stimulates surfactant secretion in cultured alveolar type II cells, and that increased [Ca2+]i appears to be necessary for ionomycin-stimulated secretion to occur. Terbutaline-stimulated surfactant secretion seems to be more easily inhibited by a reduction in [Ca2+]i than does TPA-stimulated secretion.

Aminoquinolines↗
PubMed1991

Differential effects of propranolol on the IgE-dependent, or calcium ionophore-stimulated, phosphoinositide hydrolysis and calcium mobilization in a mast (RBL 2H3) cell line.

Our previous studies demonstrated that propranolol, an inhibitor of phosphatidic acid phosphohydrolase (PAPase) (EC 3.1.3.4) blocks the IgE-dependent mediator release from a rat mast (RBL 2H3) cell line. To continue these studies, we examined the ability of propranolol to inhibit the IgE-dependent or ionomycin-mediated phosphoinositide hydrolysis and calcium mobilization in RBL 2H3 cells. RBL 2H3 cells, sensitized with mouse monoclonal anti-trinitrophenol IgE (anti-TNP IgE), were stimulated to release both histamine and peptidoleukotrienes (LT) in response to a suboptimal concentration of trinitrophenol-ovalbumin conjugate (TNP-OVA) or ionomycin. Preincubation of the cells with d,l-propranolol (300 microM) significantly (P less than 0.05) inhibited the effects of both TNP-OVA and ionomycin on histamine and LT release. There was no difference in potency for the different isomers of propranolol, indicating that these effects were not a consequence of an effect on beta 2-adrenergic receptors. TNP-OVA produced a rapid hydrolysis of phosphoinositides resulting in a time-dependent increase in mono- (IP1), di- (IP2), tri- (IP3), and total inositol phosphate production. Ionomycin also produced a rapid increase in total inositol phosphate production; however, this largely reflected an accumulation of IP1. Both secretagogues produced a rapid elevation in cytosolic free calcium ([Ca2+]i); however, the effect of ionomycin maximized within a much shorter time frame than the effect of TNP-OVA. The effects of TNP-OVA on phosphoinositide hydrolysis and increase in [Ca2+]i were inhibited by propranolol over exactly the same concentration range as the effects of this compound on TNP-OVA-stimulated mediator release. In contrast, propranolol had no effect on the increase in [Ca2+]i and phosphoinositide hydrolysis in response to ionomycin. Taken together, these results suggest that PAPase/phospholipase D (PLD) (EC 3.1.4.4) activation may be a prerequisite for both IgE-dependent and ionomycin-stimulated mediator release from RBL 2H3 cells. Although other explanations are possible, the data further suggest that receptor-mediated, but not ionophore-stimulated, phosphoinositide hydrolysis and [Ca2+]i in RBL 2H3 cells may be regulated by a propranolol-sensitive pathway involving possible activation of PAPase.

Animals↗