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PubMed1987

The cholecystokinin-induced Ca2+ shuttle from the inositol trisphosphate-sensitive and ATP-dependent pool, and initial pepsinogen release connected with cytoskeleton of the chief cell.

In guinea pig chief cells, inositol 1,4,5-trisphosphate (IP3) caused release of Ca2+, which was accumulated by ATP, from an endoplasmic reticulum-enriched fraction in both the permeable system and the cell-free system. This was mimicked with the Ca2+ ionophores A23187 and ionomycin on a large scale since an IP3-sensitive Ca2+ pool might be a subset of the Ca2+ ionophore-sensitive Ca2+ pool. The permeable chief cells, but not the cell-free system, retained the ability to react to synthetic cholecystokinin octapeptide (CCK-OP) with Ca2+ release from an IP3-sensitive pool due to of the non-additive but constant effect in exerting Ca2+ release from the store(s) induced by the combination with IP3 and CCK-OP. The increase in the cytosolic free Ca2+ concentration of intact chief cells responding to CCK-OP or the Ca2+ ionophore, ionomycin, comprised two components, namely, that by the Ca2+ entry from the extracellular space, and that by the Ca2+ release from the intracellular space(s) (as measured by fura-2). When CCK-OP or ionomycin was added, there was a biphasic response of pepsinogen secretion. An initial but transient response reaching a peak in 5 min was followed by a sustained response reaching a peak in 30 min. The initial pepsinogen release was independent of medium Ca2+, whereas the sustained one was dependent on medium Ca2+. The results suggest that the intracellular Ca2+ release from the store(s), presumably endoplasmic reticulum, may trigger the initial pepsinogen release, whereas the sustained pepsinogen secretion may be caused by acting in concert with the initial response and external Ca2+ entry. On the other hand, the disruption of the microtubular-microfilamentous system by colchicine or cytochalasin D failed to cause the Ca2+ release evoked by either IP3, CCK-OP or Ca2+ ionophores and to cause the CCK-OP- or ionomycin-induced initial pepsinogen release. These findings suggest that the IP3-sensitive pool is the same Ca2+ store which is completely or partially sensitive to CCK-OP and Ca2+ ionophores, respectively, and that the assembly of the cytoskeletal system is involved in initial intracellular Ca2+ metabolism and the following initial pepsinogen release. The assembly of the cytoskeletal system may be an early event in mediating the CCK-OP-induced initial pepsinogen release, perhaps by causing the Ca2+ release from an IP3-sensitive pool of the chief cell. The translocation or attachment of the IP3-sensitive pool brought about by cytoskeletal system might be necessary to cause Ca2+ release after the cell stimulation with CCK-OP.

Adenosine Triphosphate↗
PubMed1988

Mobilization of intracellular calcium by extracellular ATP and by calcium ionophores in the Ehrlich ascites-tumour cell.

We have studied the changes of the intracellular free calcium concentration ([Ca2+]i) effected by external ATP, which induces formation of inositol trisphosphate, and by the divalent cation ionophores ionomycin and A23187. Both, ATP (40 microM) and ionophores (1-80 mumol/l cells ionomycin; 20-400 mumol/l cells A23187), produced a transient rise of [Ca2+]i which reached its maximum within 15-30 s and declined near resting values (about 200 nM) within 1-3 min. When the [Ca2+]i peak surpassed 500 nM a transient cell shrinkage due to simultaneous activation of Ca2+-dependent K+ and Cl- channels was also observed. The cell response was similar in medium containing 1 mM Ca2+ and in Ca2+-free medium, suggesting that the Ca mobilized to the cytosol comes preferently from the intracellular stores. Treatment with low doses of ionophore (1 mumol/l cells for ionomycin; 20 mumol/l cells for A23187) depressed the response to a subsequent treatment, either with ionophore or with ATP. Treatment with ATP did also inhibit the subsequent response to ionophore, but in this case the inhibition was dependent on time, the stronger the shorter the interval between both treatments. This result suggests that the permeabilization of Ca stores by ATP is transient and that Ca can be taken up again by the intracellular stores. Refill was most efficient when Ca2+ was present in the incubation medium. Addition of either ATP or ionomycin (1-25 mumol/l cells) to cells incubated in medium containing 1 mM Ca2+ decreased drastically the total cell Ca content during the following 3 min of incubation. In the case of ATP the total cell levels of Ca returned to the initial values after 7-15 min, whereas in the case of the ionophore they remained decreased during the whole incubation period. These results indicate that Ca released from the intracellular stores by either ATP or ionophores is quickly extruded by active mechanisms located at the plasma membrane. They also suggest that, under the conditions studied here, with 1 mM Ca2+ outside, the Ca-mobilizing effect of ionophores is stronger in endomembranes than in the plasma membrane.

Adenosine Triphosphate↗
PubMed1986

Inhibition of macrophage secretion by tetanus toxin is not directly linked to cytosolic calcium homeostasis.

Tetanus toxin (TT) inhibits secretion of neurotransmitters from neurons and lysozyme from human macrophages (Mphi). Because these secretory events are associated with changes in cytosolic free calcium [Ca2+]i, we examined the effect of TT on Mphi calcium homeostasis and secretion in response to ionomycin and phorbol myristate acetate (PMA). Using Quin 2 to report [Ca2+]i, basal [Ca2]i was similar for control cells (133 nM) and Mphi treated with TT (127 nM). In response to ionomycin (50 nM) [Ca2+]i increased to 548 +/- 74 nM in control cells and to 357 +/- 36 nM in TT-treated Mphi (p less than 0.02, N = 12). Despite this rise in [Ca2+]i, neither control Mphi nor TT-treated Mphis secreted the lysosomal enzyme lysozyme in response to this concentration of ionomycin (50 nM). In both control and TT-treated Mphi, stimulation with a higher concentration of ionomycin (1000 nM) caused saturation of the quin 2 fluorescence signal. However, lysozyme secretion from TT-Mphi was inhibited. In response to the phorbol ester, PMA (3 uM), [Ca2+]i did not increase in either control Mphi or TT-treated Mphi. However, secretion of lysozyme from TT-treated Mphi was also inhibited in response to this stimulus (70.8% of control, p less than 0.02, N = 3). These data indicate that the ability of TT to inhibit secretion from Mphi is not directly linked to alterations of cytosolic calcium homeostasis.

Aminoquinolines↗
PubMed1994

Ca2+ ionophore-induced apoptosis on cultured embryonic rat cortical neurons.

The neurotoxicity of ionomycin, a Ca2+ ionophore, was investigated in cultured cortical neurons from embryonic rats. While about 90% of neurons survived 2 h after exposure to ionomycin, the surviving neurons had decreased by about 30 to 40% at 16 h. Both RNA and protein synthesis inhibitors blocked this neurotoxicity. Furthermore, c-Fos immunoreactive neurons increased not only in number but also in the intensity of immunoreactivity. These results suggest that ionomycin-induced neuronal cell death is an active process which requires de novo transcription and translation. In addition, the ultrastructural changes, such as shrinkage of cell body, compaction of nucleus, condensation of chromatin, and membrane blebbing, were observed by electron microscopy. These morphological changes are indexes of apoptosis. Furthermore, DNA fragmentation, a biochemical marker of apoptosis, was also observed. All the results suggest that ionomycin-induced neuronal cell death is apoptotic.

Animals↗
PubMed1988

Activation of human T cells with the physiological regulator of protein kinase C.

We investigated whether sn-1,2-dioctanoylglycerol (diC8) activates highly purified human T cells. diC8's signaling activity was also compared with that of 12-O-tetradecanoylphorbol-13-acetate (TPA). diC8 and ionomycin were synergistic in promoting T-cell proliferation. The proliferative response was dependent upon an operational interleukin-2 (IL-2) system and exhibited a high degree of specificity; sn-1,2-diC8 was twice as active as racemic-1,2-diC8, and diC8 and TPA were not synergistic. diC8's signaling activity differed from that of TPA. diC8, unlike TPA, failed to elicit IL-2 receptors or proliferation, independently of ionomycin. diC8 also failed to promote the proliferation of T cells signaled with anti-CD3 or -CD2 monoclonal antibodies. Two different inhibitors of PKC, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine or staurosporine, inhibited T-cell proliferation induced with diC8 and ionomycin, but not with TPA and ionomycin. These observations, in addition to demonstrating the differential activity of diC8 and TPA, document a signaling role for diacylglycerol in the activation of normal T cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

A time-course study on superoxide generation and protein kinase C activation in human neutrophils.

The time course of superoxide generation and membrane association of protein kinase C was studied in human neutrophils stimulated by PMA, FMLP, ionomycin and A23187. The initiation of superoxide generation in PMA; ionomycin- and A23187-stimulated neutrophils was characterized by a lag period of at least 30 s in contrast to a lag period of 10-15 s in FMLP-stimulated cells. The time course of membrane association of protein kinase C in PMA-stimulated neutrophils was highly dependent upon the PMA concentration used for stimulation. However, membrane association of protein kinase C preceded superoxide generation in cells stimulated by 10-300 ng/ml PMA. FMLP, ionomycin and A23187 induced membrane association of protein kinase C in a few seconds and always before superoxide generation. It is concluded that membrane association of protein kinase C in PMA-, FMLP-, ionomycin- and A23187-stimulated neutrophils precedes superoxide generation, and thereby may be part of the mechanism initiating NADPH-oxidase activity. A simple correlation between the two parameters could not be proven, indicating that also other activation mechanisms are decisive in the activation of NADPH-oxidase.

Calcimycin↗
PubMed1994

Effects of Ca2+ on the activation of conventional and new PKC isozymes and on TPA and endothelin-1 induced translocations of these isozymes in intact cells.

The effects of Ca2+ on the translocation of conventional and new protein kinase C isozymes in intact cells were studied by using C6 glioma cells as a model system. Two conditions which monitor intracellular Ca2+ were performed: one is extracellular Ca(2+)-depletion by treating the cells with physiological saline solution (PSS) without Ca2+ but containing 0.5 mM EGTA, the other is treating the cells with 1 microM ionomycin to induce Ca(2+)-influx. In addition, the TPA and endothelin-1 induced translocations of conventional and new PKC isozymes under these two conditions were also comparatively studied. When the intact cells were treated with Ca(2+)-free, EGTA containing PSS, the membrane-bound conventional PKC alpha (cPKC alpha) was greatly reduced and cytosolic cPKC alpha was slightly increased. However, neither membrane bound nor cytosolic new PKC delta (nPKC delta) was affected by extracellular Ca(2+)-depletion. On the other hand, when the cells were treated with 1 microM ionomycin, the translocation of cPKC alpha itself was observed while nPKC delta was not affected. In extracellular Ca(2+)-depletion, the translocation of cPKC alpha induced by 100 nM TPA still occurred although the extent of translocation was smaller than that induced by TPA under normal Ca2+ conditions; however, that induced by 30 nM ET-1 was blocked. After the cells were treated with 1 microM ionomycin, the translocation of cPKC alpha induced by 30 nM TPA was further increased compared to 1 microM ionomycin or 30 nM TPA alone, while that induced by ET-1 was only slightly further increased. All these results suggested that in intact cells, the activation of cPKC alpha was operated by both the intracellular Ca2+ level and diacylglycerol and that of nPKC delta was operated by diacylglycerol alone as predicted by their properties from purified enzyme or cDNA. In addition, the translocation of cPKC alpha induced by the natural activator ET-1 seemed to be more dependent on Ca2+ than TPA in intact cells.

Biological Transport↗
PubMed1996

Ligation of CD40 rescues Ramos-Burkitt lymphoma B cells from calcium ionophore- and antigen receptor-triggered apoptosis by inhibiting activation of the cysteine protease CPP32/Yama and cleavage of its substrate PARP.

The new and growing family of interleukin-1beta-converting enzyme (ICE) cysteine proteases are now recognised to be major effectors of cellular death by apoptosis. Like other members of this family, the CPP32/Yama proform is activated by processing to its active heterodimeric enzyme or apopain when it likely contributes to the process of apoptosis by cleaving poly(ADP-ribose) polymerase (PARP) and thereby inhibiting much of its DNA repair activity. Apoptosis plays a fundamental role in the regulation of the immune system where it is involved in the selection of both T and B lymphocytes bearing antigen receptor (AgR) for non-self. Cells of the Ramos Epstein-Barr virus (EBV)-genome-negative Burkitt lymphoma (BL) B cell line (Ramos-BL) can be triggered into growth arrest and apoptosis by treating with the calcium ionophore ionomycin or by crosslinking their surface AgR with antibodies directed against immunoglobulin (Ig)M (anti-IgM). Ionomycin- and AgR-triggered growth arrest and apoptosis are arrested by signals transduced through the surface CD40 of Ramos-BL B cells. Both ionomycin and anti-IgM trigger activation of CPP32 and cleavage of PARP prior to the onset of apoptosis; this process is abrogated by treatment with anti-CD40 and is independent of Bcl-2 expression. A tripeptide inhibitor of ICE family cysteine proteases, Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) inhibits ionomycin- and AgR-triggered CPP32 activation, PARP cleavage and apoptosis, but not growth arrest, in Ramos-BL B cells. Thus, in this report we demonstrate that in a physiological system, activation of endogenous members of the ICE family, including CPP32, and cleavage of the death substrate PARP act as major effectors of apoptotic death.

Amino Acid Chloromethyl Ketones↗
PubMed1994

Effect of ganglioside GM1 on arachidonic acid release in bovine aortic endothelial cells.

A role for the ganglioside GM1 in arachidonic acid release in bovine aortic endothelial cells (BAEC) was investigated. [3H]Arachidonic acid-labeled BAEC were preincubated with GM1 and incubated with one of four different stimulators. GM1 inhibited arachidonic acid release when stimulated with maitotoxin or melittin but not with ionomycin or thapsigargin. A 10 microM GM1 concentration achieved a 50% and 100% inhibition of the maitotoxin and melittin responses, respectively. The selective inhibition displayed by GM1 on the maitotoxin and melittin responses was not due to its ability to bind calcium since all four drugs, maitotoxin, melittin, ionomycin, and thapsigargin, required extracellular calcium. The effect of GM1 was not specific to arachidonic acid release. GM1 at 50 microM inhibited phosphatidylinositol polyphosphate (PIP) hydrolysis mediated by melittin, but did not affect hydrolysis mediated by ionomycin. Moreover, the activity of GM1 was not restricted to phospholipid metabolism since it also inhibited calcium influx that was stimulated by maitotoxin or melittin but not by ionomycin. We conclude that GM1 is not a specific inhibitor of phospholipases in bovine aortic endothelial cells, but rather its activity is dependent on the type of stimulant used to activate the cell.

Animals↗
PubMed1988

Functional responses of aequorin-loaded human neutrophils. Comparison with fura-2-loaded cells.

Aequorin-loaded human neutrophils in response to chemotactic peptides and ionomycin showed a sharp rise in their intracellular Ca2+ concentration which decayed within 2 min. Depletion of extracellular Ca2+ suppressed only the ionomycin-induced increase. Fura-2-loaded cells also showed a sharp rise in the intracellular Ca2+ concentration in response to each stimulator, while the decline was extremely slow in the ionomycin-induced Ca2+ increase. Depletion of extracellular Ca2+ reduced the duration of ionomycin-induced Ca2+ increase. Cytochalasin B almost equally potentiated the rise in the intracellular Ca2+ concentration induced by each stimulator. Aequorin-loaded cells showed impaired phagocytotic activity, while degranulation and oxygen radical production were not affected.

Aequorin↗
PubMed1989

Difference in hydrogen peroxide release between human neutrophils and neutrophil cytoplasts following calcium ionophore activation. A role of the subcellular granule in activation of the NADPH-oxidase in human neutrophils?

The role of subcellular granule in the capacity to generate reactive oxygen metabolites in human granulocytes was studied using normal cells and organell-free neutrophil cytoplasts. The cytoplasts are devoid of granules but have an intact ligand-receptor coupling mechanism. Both the chemotactic peptide formylmethionylleucylphenylalanine (FMLP) and the ionophore ionomycin induced a chemiluminescence response in normal cells, but only FMLP stimulation was associated with any notable hydrogen peroxide production. However, in the presence of azide, a potent inhibitor of the hydrogen peroxide-consuming enzymes, catalase and myeloperoxidase, a pronounced release of hydrogen peroxide was also induced by ionomycin. The response of cytoplasts to FMLP proceeded with a rate and time-course similar to those seen in intact cells, whereas in response to ionomycin they produced very low quantities of hydrogen peroxide, even in the presence of azide. Analysis of the data presented in this study leads to the following conclusion: (i) FMLP, which acts through cell surface receptors, causes the cells to produce oxygen metabolites, which, to a large extent, are released from the cells, a process that is not dependent on subcellular granule; and (ii) ionomycin, which bypasses cell surface receptors, is also capable of stimulating hydrogen peroxide formation that is granule-dependent and that is retained inside the cells.

Azides↗
PubMed1992

Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells.

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca2+]i) and the intracellular level of inositol 1,4,5-trisphosphate (IP3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP3 levels, [Ca2+]i and PGI2 release. An increase in [Ca2+]; and PGI2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca2+]i or PGI2 release. The inability of ATP to cause an increase in [Ca2+]i or PGI2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca2+]i and PGI2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca2+]i and PGI2 release, further evidence that PGI2 release is dependent upon an increase in [Ca2+]i. The increase in [Ca2+]i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A2, prevented the stimulation of PGI2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI2 synthesis, prior to the release of PGI2 from BPAE cells. These findings suggest that the increase in PGI2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca2+]i and the subsequent activation of a phosphatidylcholine-specific phospholipase A2. ATP analogs modified in the adenine base or phosphate moiety caused PGI2 release with a rank order of agonist potency of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) greater than 2-methylthioATP (2-MeSATP) greater than ATP, whereas alpha, beta methyleneATP and beta, gamma methyleneATP had no effect on PGI2 release.

Adenosine Triphosphate↗
PubMed1995

Rapamycin inhibits the in vitro release of soluble interleukin-2 receptor by activated peripheral blood mononuclear cells (PBMC) independently of the mode of activation.

In the present study we compared the effect of rapamycin to that of CsA on the in vitro responses of lectin (PHA), phorbol-ester (PMA) and CA2+ ionophore (ionomycin)-activated peripheral blood mononuclear cells by measuring the release of soluble IL-2R (sIL-2R), high levels of which have been detected in clinical syndromes characterized by an ongoing immune activation. PHA was the stimulant associated with high sIL-2R release, whereas ionomycin-induced sIL-2R release only exceeded the background response and the sensitivity of the ELISA kit. The highest sIL-2R release, however, was obtained when PMA was used in combination with either PHA or ionomycin. Rapamycin inhibited the release of sIL-2R in response to all activators, whereas CsA only abolished the ionomycin-induced sIL-2R release. In parallel experiments rapamycin inhibited cell proliferation in response to all stimulants with the exception of PMA/ionomycin, whereas CsA inhibited all proliferation. Our study clearly shows that for optimal sIL-2R release both Ca+ and protein kinase C-triggered signals are required and that rapamycin has a distinct advantage over CsA in inhibiting the release of sIL-2R, which has been shown to be a reliable marker of lymphocyte activation either in vivo or in vitro.

Biotransformation↗
PubMed1992

Potentiation by Ca2+ ionophores and inhibition by extracellular KCl of endothelin-induced phosphoinositide turnover in C6 glioma cells.

Interactions between endothelin-1 (ET)-induced phosphoinositide (PI) hydrolysis and agents that increase Ca2+ influx (i.e. A23187 and ionomycin) or induce depolarization (i.e. KCl) were investigated using C6 glioma. A23187 dose-dependently potentiated ET (30 nM)- and ATP (100 microM)-induced [3H]inositol phosphate (IP) accumulation. This potentiation was associated with an increase in the maximal stimulation elicited by both ET and ATP but their EC50 values were unchanged. This effect of A23187 occurred at concentrations that did not affect basal PI turnover; i.e. 10 nM-3 microM. Ionomycin within the range of 1 nM-1 microM also significantly enhanced ET-induced PI breakdown and this effect was associated with an increase of [Ca2+]i. KCl in a concentration-dependent manner (14.7-54.7 mM) markedly inhibited PI breakdown elicited by ET and ATP, but had much less inhibition on basal activity and no effect on A23187- and ionomycin-induced responses. In parallel, KCl added before or after ET, sharply attenuated the increase of ET-induced [Ca2+]i but did not affect basal level or ionomycin-induced [Ca2+]i response. Neither the potentiation by A23187 nor the inhibition by KCl of ET-induced PI turnover was observed in cultured cerebellar astrocytes. Our results suggest that the cell type-specific regulation by Ca2+ ionophores and KCl on ET-induced PI metabolism is closely related to perturbation of [Ca2+]i.

Adenosine Diphosphate↗
PubMed1987

Inhibitory effect of polymyxin B on catecholamine secretion from cultured bovine adrenal medullary cells.

Incubation of cultured bovine adrenal medullary cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), was associated with increased secretion of catecholamine (CA) from the cells. Polymyxin B (PMB, 30-300 microM), a preferential inhibitor of protein kinase C, inhibited the TPA-induced secretion of CA. PMB also inhibited CA secretion induced by other secretagogues, the Ca2+ ionophore ionomycin (10 microM), 56 mM K+ or acetylcholine (ACh). Ionomycin, 56 mM K+ or ACh increased the concentration of intracellular free Ca2+ ([Ca2+]i) (measured using the fluorescent calcium indicator quin2), whereas TPA did not increase [Ca2+]i. PMB blocked the increase in [Ca2+]i induced by 56 mM K+ or ACh at concentrations similar to those inhibiting the secretion of CA. In contrast, PMB did not affect ionomycin-induced increase in [Ca2+]i. These results strongly suggest that CA secretion induced by TPA or ionomycin is mediated via activation of protein kinase C. The results further indicate that in 56 mM K+- or ACh-evoked CA secretion, PMB inhibits the secretion by blocking Ca2+ influx into the cells.

Acetylcholine↗
PubMed2006

Involvement of Gi/o in the PAR-4-induced NO production in endothelial cells.

We investigated the involvement of G(i/o) protein in NO production following the activation of proteinase-activated receptor-4 (PAR-4) in cultured bovine aortic endothelial cells. AYPGKF-NH(2) (PAR-4 activating peptide), thrombin, and ionomycin induced a concentration-dependent NO production, with the maximal production seen at 30 microM, 0.1U/ml, and 1 microM, respectively. Ionomycin elevated [Ca(2+)](i) in a concentration-dependent manner. However, AYPGKF-NH(2) and thrombin induced no [Ca(2+)](i) elevation. The loading of cells with BAPTA almost completely inhibited both the NO production and [Ca(2+)](i) elevation induced by 1 microM ionomycin, while it had no significant effect on the AYPGKF-NH(2)-induced NO production. Treatment with pertussis toxin inhibited the AYPGKF-NH(2)-induced NO production, while it had no effect on the ionomycin-induced NO production. Our findings thus demonstrate, for the first time, that PAR-4 activation induced NO production in a manner mostly independent of the Ca(2+) signal and also that G(i/o) is involved in such NO production in vascular endothelial cells.

Animals↗
PubMed2007

Differential regulation of IL-6 and TNF-alpha via calcineurin in human skeletal muscle cells.

Interleukin-6 increases in skeletal muscle during exercise, and evidence points to Ca2+ as an initiator of IL-6 production. However, the signalling pathway whereby this occurs is unknown. One candidate for Ca2+ -mediated IL-6 induction is calcineurin, an activator of NF-AT. Here we investigated whether skeletal myocytes produce IL-6 in a Ca2+/calcineurin-dependent manner, and whether TNF-alpha, an inducer of IL-6, is affected by these stimuli. Human skeletal muscle cell cultures were stimulated with ionomycin time-and dose-dependently to elevate intracellular Ca2+ levels, with or without addition of cyclosporin A (CSA); a calcineurin inhibitor. mRNA was extracted from myocytes and analysed for IL-6 and TNF-alpha gene expression. IL-6 mRNA increased time- and dose-dependently with ionomycin stimulation, an effect that was blunted by approximately 75% in the presence of CSA. In contrast, TNF-alpha gene expression was decreased by approximately 70% in response to ionomycin treatment, but increased in response to addition of CSA. These data demonstrate that IL-6 and TNF-alpha are regulated differentially in skeletal muscle cells in response to a Ca2+ stimulus. Blocking the calcineurin pathway resulted in inhibition of the IL-6 response to ionomycin, whereas TNF-alpha increased by addition of CSA, further indicating a differential regulation of IL-6 and TNF-alpha in human skeletal myocytes.

Calcineurin↗
PubMed2005

In vitro development of bovine oocytes reconstructed with round spermatids.

The timing between round spermatid(s) (RS) injection and oocyte activation are critical for spermatid remodeling and embryo development in intracytoplasmic injection of round spermatid (ROSI) procedure. The objective of the present study was to develop an appropriate oocyte activation method for producing developmentally competent bovine embryos reconstructed with RS. Embryos reconstructed by ROSI were compared with three activation treatments for the rates of pronuclear formation, development and ploidy. RS were isolated from bull testes by Percoll density gradients. Matured oocytes were divided into three activation groups. In Group 1, oocytes were activated with ionomycin (5 microM, 5 min) before ROSI. In Group 2, oocytes were activated with ionomycin after ROSI. In Group 3, oocytes were activated twice with ionomycin before and after ROSI. All the eggs were then incubated in cycloheximide (CHX, 10 microg/mL) for 5 h and cultured in CR1aa medium for up to 8 days. Three methods of oocyte activation were also compared for the activation and development of parthenotes. Activation rates among the groups were 70-79% and did not differ. Cleavage rates in parthenotes were significantly (P < 0.05) higher in Group 3 than in Groups 1 and 2, but blastocyst rates did not differ among the groups. In ROSI embryos, the rates of cleavage and development into blastocysts were significantly (P < 0.05) greater in Group 3 (82.3% and 13.1%) than in Groups 1 and 2 (53.7, 5.8% and 64.2, 1.7%, respectively). Ploidy analysis by examining the metaphase spreads of ROSI blastocysts displayed greater numbers of diploid chromosomal complements. These results suggest that intracytoplasmic RS injection combined with repeated ionomycin activation followed by CHX treatment is more efficient for producing developmentally competent embryos.

Animals↗