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PubMed1984

Relationship of thyrotropin-releasing hormone-induced spike and plateau phases in cytosolic free Ca2+ concentrations to hormone secretion. Selective blockade using ionomycin and nifedipine.

In clonal rat pituitary cells (GH cells), thyrotropin-releasing hormone (TRH) induced a pattern of changes in cytosolic free calcium concentrations [( Ca2+]i) composed of two phases: an acute spike phase to micromolar levels which decayed (t1/2 = 8 s) to a near-basal concentration and then rose to a prolonged plateau phase of elevated [Ca2+]i (as measured using Quin 2). Closely following these changes in [Ca2+]i, TRH stimulated a rapid "spike phase" of pronounced, but brief, enhancement of the rate of prolactin and growth-hormone secretion and then a "plateau phase" of prolonged enhancement. These two phases were dissociated using two classes of pharmacologic agents: the ionophore ionomycin, and a calcium channel antagonist nifedipine. Ionomycin (100 nM) specifically blocked (less than 90%) the spike phase of TRH action by rapidly emptying the TRH-regulated reservoir of cellular Ca2+ to generate a TRH-like spike in [Ca2+]i; nifedipine inhibited (less than 50%) the plateau phase of TRH-induced changes in [Ca2+]i and hormone secretion by preventing Ca2+ influx through voltage-dependent Ca2+ channels. These agents demonstrated that the TRH-induced spike in [Ca2+]i in GH cells is caused by release of an ionomycin-sensitive pool of cellular Ca2+ with a small component (10%) due to influx of extracellular Ca2+. The TRH-induced plateau in [Ca2+]i is due to influx of extracellular Ca2+, about half of which enters through voltage-dependent calcium channels and half of which enters via nifedipine/verapamil-insensitive influx. The TRH-induced spike in [Ca2+]i led to a burst in hormone secretion, and the plateau in [Ca2+]i produced a prolonged enhancement of secretion; the spike and plateau phases were generated independently by TRH. A spike in [Ca2+]i is necessary, but not sufficient, to induce burst release of hormone, while the prolonged rate of hormone secretion is intimately related to the steady-state [Ca2+]i.

Animals↗
PubMed1994

Regulation by sphingomyelinase and sphingosine of Ca2+ signals elicited by CD3 monoclonal antibody, thapsigargin, or ionomycin in the Jurkat T cell line.

Sphingomyelinase induces a marked rapid decrease of cytosolic Ca2+ concentration in Jurkat T cells treated with either CD3 monoclonal antibody; the Ca(2+)-ATPase inhibitor, thapsigargin; or the Ca2+ ionophore, ionomycin. Sphingomyelinase treatment of Jurkat cells results in a net decrease of cellular sphingomyelin content. Among the products generated by the catabolism of sphingomyelin, sphingosine displayed exactly the same effect as sphingomyelinase. Sphingosine decreases the cytosolic Ca2+ concentration in cells treated with CD3, thapsigargin, or ionomycin. Studying the effect of sphingosine in CD3-activated cells showed that this compound does not modify Ca2+ mobilization from intracellular stores but strongly inhibited the Ca2+ influx induced by the monoclonal antibody. The fact that sphingosine inhibits Ca2+ influx generated by thapsigargin and ionomycin supports the hypothesis that the drug activates the Ca2+ extrusion process. Derivatives of sphingosine such as erythrosphinganine and threosphinganine share the same inhibitory properties.

Antibodies, Monoclonal↗
PubMedDate not supplied

Ionomycin stimulates mast cell histamine secretion by forming a lipid-soluble calcium complex.

The activation of cellular function following the direct introduction of Ca2+ into the cytosol by the use of a Ca2+-transporting ionophore has served to confirm the widely held idea that Ca2+ has the status of a second messenger in many cell types. However, this evidence has been obtained largely from the use of a single ionophore, the antibiotic A23187; experiments with X537A, which is another ionophorous antibiotic capable of transporting Ca2+ (ref. 3), have failed to show the expected characteristics. For example, histamine release from rat mast cells mediated by X537A is neither dependent on extracellular Ca2+ nor prevented by metabolic inhibitors. Ionomycin is a recently described polyether antibiotic produced by Streptomyces conglobatus ATCC 31005, and is active against Gram-positive bacteria. The antibiotic action is presumably due to its ionophorous properties, as it extracts Ca2+ from an aqueous phase into an organic phase with a stoichiometry of 1:1 (ref. 6). The ionophore is also capable of transporting 45Ca2+ across biological membranes (our unpublished results). Here we report the application of ionomycin to rat mast cells. We show that ionomycin stimulates mast cell secretion solely through its ability to form a lipid-soluble calcium complex, and thus to convey Ca2+ across the hydrocarbon region of the cell membrane.

Animals↗
PubMed1991

Effect of Glutamate and Ionomycin on the Release of Arachidonic Acid, Prostaglandins and HETEs from Cultured Neurons and Astrocytes.

The release of arachidonic acid (ArA) metabolites from mouse neurons and astrocytes in primary culture has been studied in response to ionomycin or glutamate stimulation. Cells were preincubated with [3H]ArA for 24 h and the radioactivity released was examined by HPLC. In striatal, cortical and hippocampal neurons, glutamate and ionomycin strongly stimulated the release of ArA, but neither prostaglandins (PGs) nor hydroxyeicosatetraenoic acids (HETEs) could be detected. If they were released, these latter compounds represented < 0.02% of the amount of ArA. In contrast, in astrocyte cultures, ionomycin (but not glutamate) strongly stimulated the release of PGs and HETEs as well as ArA. Reversed- and straight-phase HPLC analysis revealed the presence of PGD2, PGE2, PGF2alpha, 12-hydroxyheptadeca-5,8,10-trienoic acid (HHT) and HETEs (15-HETE, 11-HETE and 5-HETE). Indomethacin inhibited the release of PGs and HHT, but also that of 11- and 15-HETE, indicating that these two HETEs may be produced through the cyclooxygenase pathway. Metabolism of [3H]ArA was also examined in cellular homogenates. Although > 50% of the [3H]ArA was metabolized to PGF2alpha, PGE2, PGD2, HHT, 15- and 11-HETE in cultured astrocyte homogenates, no [3H]ArA metabolism could be detected in cultured striatal neuron homogenates. Moreover, neuronal homogenates did not inhibit the metabolism of [3H]ArA observed in either astrocyte or platelet homogenates. These results indicate that central neurons in primary culture possess very low lipoxygenase and cyclooxygenase activities. They emphasize the need to identify the cellular source of ArA metabolites in the brain, particularly when considering the multiple new messenger roles proposed for these molecules, such as that of retrograde messengers involved in synaptic plasticity phenomena.

Journal Article↗
PubMed1991

Intracellular production of reactive oxygen species in human neutrophils following activation by the soluble stimuli FMLP, dioctanoylglycerol and ionomycin.

The stimuli, sn-1, 2-dioctanoylglycerol; (DG8) the calcium specific ionophore, ionomycin, and the chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) can interact with normal human neutrophils and activate their superoxide/hydrogen peroxide generating NADPH-oxidase. In response to the peptide as well as DG8, the neutrophils produced both superoxide (O2-) and hydrogen peroxide (H2O2). Since interaction between the cells and ionomycin was not associated with any notable superoxide production and hydrogen peroxide was induced only in the presence of azide, a potent inhibitor of the hydrogen peroxide-consuming enzymes catalase and myeloperoxidase, we conclude that this stimulus can generate oxygen metabolites intracellularly. Since the DG8-induced production of hydrogen peroxide was increased in the presence of azide, whereas the FMLP-induced response was largely unaffected, we concluded that the three stimuli differ in their capacity to generate oxygen metabolites intracellularly. The use of sn-1,2-didecanoylglycerol (DG10) as stimulating agent did not result in any detectable activation of the NADPH-oxidase. However, preincubation caused an increased (primed) response during stimulation with the chemotactic peptide FMLP. The response of primed neutrophils to FMLP proceeds with a time-course different from that seen in normal cells. From the results presented on FMLP-induced activity in the presence of azide, we conclude that FMLP causes normal cells to produce oxygen radicals which are released from the cells. However, the primed cells are also capable of generating oxygen metabolites that are retained inside the cells. In fact, measurement of the intracellularly generated metabolites discloses this to be the predominant part of the response.

Adult↗
PubMed1990

Enhancement of thrombin- and ionomycin-stimulated prostacyclin and platelet-activating factor production in cultured endothelial cells by a tumor-promoting phorbol ester.

Tumor-promoting phorbol esters such as 4 beta-phorbol 12-myristate 13-acetate (PMA) have been shown to act synergistically with Ca2+ ionophores in cell activation, including stimulation of arachidonic acid metabolism. The effects of PMA on unstimulated and Ca2+ ionophore- or thrombin-stimulated PGI2 and platelet-activating factor (PAF) production in cultured bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were investigated. Incubation of BAEC or HUVEC for 5-10 min with 100 nM PMA alone slightly increased basal PGI2 production. PGI2 production was rapidly stimulated in BAEC and HUVEC treated with the Ca2+ ionophore ionomycin. Preincubation of BAEC or HUVEC with 100 nM PMA for 5-10 min followed by ionomycin for up to 60 min enhanced PGI2 production up to 2.5-fold. Pretreatment with 100 nM PMA for 5 min also caused a 2-fold enhancement of thrombin-stimulated (1 U/ml) PGI2 production in HUVEC. The production of other prostaglandins, PGF2 alpha, PGE2, and PGD2, was also enhanced. In contrast, PMA had no effect on PGI2 synthesized directly from exogenous arachidonic acid or PGH2. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. Since the biosyntheses of both PGI2 and PAF share a common first step, the hydrolysis of their respective phospholipid precursors by phospholipase A2, we investigated whether PMA preincubation could also enhance PAF biosynthesis. Incubation of HUVEC with 100 nM PMA alone had a negligible effect on PAF production. However, thrombin-stimulated (1 U/ml) PAF production was enhanced 2.6-fold by preincubation with 100 nM PMA. The protein kinase C inhibitors H-7 and staurosporine ablated the enhancing effect of PMA on thrombin-stimulated PGI2 and PAF biosynthesis. These results demonstrate that PMA can significantly alter the production of PGI2 and PAF in vascular endothelial cells, and suggest that protein kinase C activation modulates phospholipase A2 activity in this cell type.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Transforming growth factor beta 1 inhibits interleukin-1-induced but enhances ionomycin-induced interferon-gamma production in a T cell lymphoma: comparison with the effects of rapamycin.

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine whose potent immunomodulatory activity is well documented. To explore the mechanisms of this activity we examined the effect of TGF-beta 1 on the production of IFN-gamma measured at the mRNA and protein levels in the YAC-1 T cell lymphoma. In previous studies, this model proved useful to characterize the mode of action of the immunosuppressant rapamycin (RAP). Here, we found that when induced by IL-1 or IL-1 + PMA, the production of IFN-gamma is suppressed by both TGF-beta 1 (ED50 = 1.9 pM) and RAP (ED50 = 0.2 nM). In contrast, when induced by the calcium ionophore ionomycin, in the absence or in the presence of PMA, this production is enhanced up to 10-fold by TGF-beta 1 (ED50 = 1.8 pM) and 1.5-3-fold by RAP. Therefore, in YAC-1 cells, TGF-beta 1 exerts opposite effects on IFN-gamma production depending on the mode of activation, and these effects parallel those of RAP. To further analyze the mode of action of TGF-beta 1 in this system, we used okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases. Treatment with OA rendered the expression of IFN-gamma mRNA induced by IL-1 insensitive to TGF-beta 1 or RAP, indicating that activation of a phosphatase may play a role in the suppressive effect of both agents. However, OA did not prevent the augmentation of ionomycin-mediated induction of IFN-gamma mRNA by either TGF-beta 1 or RAP. Hence, the up-regulation of IFN-gamma production by TGF-beta 1 and RAP may involve a different biochemical mechanism than that mediating their suppressive action. These observations also favor the hypothesis that the two agents act on the same regulatory pathways. This was further supported by the finding that TGF-beta 1 and RAP modulate IFN-gamma production in an additive rather than synergistic fashion. However, their effects could be dissociated in mutants of YAC-1 cells selected for resistance to the inhibition of IL-1-mediated IFN-gamma induction by RAP. Moreover, the IFN-gamma modulatory action of RAP in YAC-1 cells was accompanied by an antiproliferative effect, whereas TGF-beta 1 failed to alter the growth of these cells. Therefore, the immunomodulatory action of TGF-beta 1 may result from the disruption of biochemical processes related to, although distinct from, those affected by RAP.

Animals↗
PubMed1999

Regulation of delta opioid receptor expression by anti-CD3-epsilon, PMA, and ionomycin in murine splenocytes and T cells.

Previous studies have shown that low levels of delta opioid receptor (DOR) mRNA were detectable by reverse transcription polymerase chain reaction (RT-PCR) in unstimulated splenocytes from BALB/c female mice. This study demonstrates that DOR transcripts can be detected in freshly obtained splenocytes froin CD 1 female mice as well. The results of studies using quantitative competitive RT-PCR showed that DOR transcripts in splenic T cells increased from < 1 copy/cell to 22 and 42 copies/cell, respectively, after stimulation with anti-CD3-epsilon for 24 and 48 h compared to the level in freshly obtained T cells. In the presence of actinomycin D, anti-CD3-epsilon did not affect the rate of degradation of DOR mRNA, suggesting that its stability is not altered by anti-CD3-epsilon. After incubation with phorbol myristate acetate (PMA) and ionomycin, the expression of DOR mRNA in splenocytes and T cells was significantly reduced compared with unstimulated cells in culture. In addition, the inhibitory effect of PMA prevented anti-CD3-epsilon-stimulated DOR expression. These data suggest that signaling through the T cell receptor complex by anti-CD3-epsilon regulates DOR expression differently than PMA and ionomycin.

Animals↗
PubMed1993

Induction of CD45 isoform switch in murine B cells by antigen receptor stimulation and by phorbol myristate acetate and ionomycin.

In this study, we examined whether CD45 isoform can be switched in murine mature B cells and what signals are responsible for the process. Stimulation of murine splenic B cells with lipopolysaccharide did not reduce the expression of CD45RA-, B-, and C-exon-dependent epitopes or a CD45 common epitope, but rather enhanced the expression. Stimulation with goat antimouse IgM antibody did not significantly reduce CD45 expression but caused a partial reduction in the expression of CD45RA-, B-, and C-exon-dependent epitopes. Phorbol myristate acetate (PMA) alone did not significantly alter the expression of CD45 but the combination of PMA and ionomycin induced a strong reduction in the expression of CD45RA-, B-, and C-exon-dependent epitopes without affecting the level of CD45 common epitope expression. Reverse transcription and polymerase chain reaction analysis demonstrated that CD45 isoform switch induced by anti-IgM or PMA plus ionomycin is indeed mediated by alternative splicing of A-, B-, and C-exon-derived mRNA. These results suggest that CD45 isoform of murine mature B cells can be switched by antigen receptor-mediated signals, and the process seems to be regulated at least in part by protein kinase C activation and mobilization of calcium ions.

Alternative Splicing↗
PubMed1993

Activation of ion transport by combined effects of ionomycin, forskolin and phorbol ester on cultured HT-29cl.19A human colonocytes.

The differentiated clone 19A of the HT-29 human colon carcinoma cell line was used as a model to study the intracellular electrophysiological effects of interaction of the cAMP, the protein kinase C (PKC) and the Ca2+ pathways. (a) A synergistic effect between ionomycin and forskolin was observed. From intracellular responses it was concluded that the synergistic effect is caused by activation of an apical Cl- conductance by protein kinase A and a basolateral K+ conductance by Ca2+. (b) A transient synergistic effect of ionomycin and the phorbol ester phorbol dibutyrate (PDB) was found. The decrease of the response appeared to be due to PKC-dependent inactivation of the basolateral K+ conductance. The synergism is caused by PKC-dependent increase of the apical Cl- conductance and Ca(2+)-dependent increase of the basolateral K+ conductance. (c) The effects of carbachol and PDB were not fully additive presumably because of their convergence on PKC activation. (d) Forskolin and PDB, when added in this order, had a less than additive effect. Results of cell-attached patch-clamp studies, presented in the accompanying paper, showed a synergistic effect of forskolin and PDB on non-rectifying small-conductance Cl- channels. Assuming that these channels are involved in the transepithelial responses it is suggested that forskolin and PDB induce a modulatory, synergistic increase of the apical Cl- conductance when both pathways are activated simultaneously. (e) The HT-29cl.19A cells differ from T84 cells in that the latter did n ot respond with an increase of the short-circuit current to addition of phorbol ester. this may be due to a very low expression of PKA alpha.

Biological Transport↗
PubMed1992

The antioxidant butylated hydroxytoluene (BHT) inhibits the dioctanoylglycerol-evoked platelet response but potentiates that elicited by ionomycin.

Preincubation of aspirin-treated human platelets with butylated hydroxytoluene (BHT) inhibits secretion, aggregation, and protein phosphorylation induced by dioctanoylglycerol or phorbol 12-myristate 13-acetate (PMA). BHT alone elicits a rapid and transient phosphorylation of a 47-kDa protein, which is indistinguishable from the well-recognized major substrate of protein kinase C (PKC). Inhibition of diacylglycerol- or PMA-induced platelet activation is also observed after decay to the basal level of the BHT-evoked phosphorylation of the 47-kDa protein. By contrast BHT potentiates platelet responses elicited by the calcium ionophore ionomycin. In the presence of the PKC inhibitor staurosporine BHT fails to increase the ionomycin-promoted platelet aggregation, indicating that its effect occurs through a PKC activation, even if no correlation with the 47-kDa protein phosphorylation is observed. BHT does not significantly modify the affinity of protein kinase C purified from calf brain for Ca2+ or dioctanoylglycerol. It is concluded that: (a) a short exposure of platelets to BHT induces an activation, whereas a long exposure an inhibition of PKC, (b) at variance with diacylglycerols BHT decreases the platelet responses promoted by subsequent challenge with PKC activators themselves, and (c) similarly to other PKC activators BHT potentiates the cellular response elicited by calcium ionophores most likely by activating the phospholipase A2.

Adenosine Triphosphate↗
PubMed1992

Cyclic AMP modulation of human B cell proliferative responses: role of cAMP-dependent protein kinases in enhancing B cell responses to phorbol diesters and ionomycin.

The ability of cyclic AMP (cAMP) to modulate human B cell proliferative responses and the possible role of cAMP-dependent kinases (PKA) in cAMP modulation of proliferative responses were investigated. The addition of dibutyl cAMP (Bt2 cAMP) or the cAMP-elevating agent forskolin to B cells stimulated by crosslinking surface immunoglobulins (sIg) resulted in a concentration-dependent inhibition of proliferative responses. By contrast, Bt2 cAMP or forskolin enhanced the proliferative responses of B cells after direct stimulation by phorbol myristate acetate (PMA) and the calcium ionophore ionomycin. The inhibition and enhancement of B cell proliferative responses by Bt2 cAMP were observed at different incubation intervals and were not due to temporal shifts of optimal responses. Also, Bt2 cAMP caused only small changes in B cell RNA synthesis compared to modulation of proliferative responses. Exposure of B cells to Bt2 cAMP rapidly activated PKA. Blocking Bt2 cAMP activation of PKA with the kinase inhibitor HA1004 prevented Bt2 cAMP enhancement of B cell responses after direct stimulation by PMA and ionomycin. In reciprocal experiments, the kinase inhibitor H7 resulted in some inhibition of PKC activation but did not inhibit Bt2 cAMP activation of PKA or Bt2 cAMP enhancement of proliferative responses. Other experiments demonstrated that B cells treated with Bt2 cAMP had selective increases in the de novo phosphorylations of two endogenous substrates which reflected PKA activation. Furthermore, concentrations of HA1004 or H8 which inhibited Bt2 cAMP enhancement of proliferative responses also inhibited PKA phosphorylations of these substrates whereas H7 did not. Thus, elevations of cAMP can enhance or inhibit human B cell proliferative responses to different stimuli and the activation of PKA is important for cAMP enhancement of certain responses.

Amino Acid Sequence↗
PubMed1988

Peripheral demyelination and remyelination initiated by the calcium-selective ionophore ionomycin: in vivo observations.

Incubation of nerve with Ca2+ and the divalent cation ionophores A23187 or ionomycin, causes a prompt vesiculation of the myelin at the paranodes and Schmidt-Lanterman incisures. The vesiculation appears to be dependent upon a rise in the intracellular calcium concentration of the affected Schwann cells. To determine whether a similar vesiculation might occur in vivo, and to examine the long term consequences of ionophore exposure, the sciatic nerves of rats and mice were injected with ionomycin and examined histologically after intervals of 1 h to 75 days. A prompt myelin vesiculation was again observed, and this spread from the paranodes and incisures to invade regions of formerly compact myelin: segmentally demyelinated axons were common 7 days post-injection. The lesions were large, and involved up to 95% of the fibres in the mouse. There was little evidence of either Schwann cell necrosis, or axonal degeneration in either species. The appearance and spatio-temporal progression of the lesion was strikingly similar to that seen after the intraneural injection of lysophosphatidylcholine or phospholipase A2. However, in the ionophore lesion, not all the demyelination was effected by myelin vesiculation, for up to 30% of the affected fibres showed evidence of macrophage-mediated stripping. Macrophage stripping was confined to internodes already affected by vesiculation. Most of the myelin debris was removed by macrophages, and myelin debris was only rarely observed within Schwann cells. Remyelination was initiated in nearly all the affected fibres by 21 days, and by 2 months all affected fibres were invested with thin, new myelin sheaths. These findings may be relevant to immune-mediated demyelination in the peripheral nervous system.

Animals↗
PubMed1990

The effect of taurine on age-related immune decline in mice: the effect of taurine on T cell and B cell proliferative response under costimulation with ionomycin and phorbol myristate acetate.

Proliferative responses to the costimulation with phorbol-12-myristate-13-acetate (PMA) and suboptimal doses of ionomycin in the purified T and B cells from old mice were lower than those from young mice. The degree of the age-related decline was more significant in T cells than in B cells. Taurine, a sulfur containing amino acid, augmented the proliferative responses of T cells from both young and old mice. The augmentation of the proliferative response by taurine was more marked in old T cells than in young ones. The concentration of intracellular free calcium ion ([Ca2+]i) was significantly lower in the old T cells under the stimulation with PMA and ionomycin than that in the young ones. In the presence of taurine, the concentration of [Ca2+]i in the old T cell significantly increased under the stimulation. The results indicate that taurine improved the proliferative response of old T cells by the restoration of the increment of the concentration of [Ca2+]i under the stimulation.

Aging↗
PubMed1991

Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells.

The effects of NH4Cl on cytoplasmic free calcium concentration ([Ca2+]i) and pH (pHi) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH4Cl (10-40 mM) in the presence of 1 mM extracellular calcium ([Ca2+]e) increased [Ca2+]i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH4Cl. In medium containing 0.1 microM [Ca2+]e, or in 1 mM [Ca2+]e medium containing 0.1 microM nitrendipine, the plateau was absent leaving only a transient [Ca2+]i spike. NH4Cl also increased pHi and this, like the [Ca2+]i plateau, remained elevated during the continued presence of NH4Cl. In medium containing only 0.1 microM [Ca2+]e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH4Cl induced repeated [Ca2+]i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20-500 nM) caused a [Ca2+]i rise but, after an additional exposure to NH4CI, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abolished the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH4Cl. Neither acetate removal nor methylamine increased [Ca2+]i in medium containing 0.1 microM [Ca2+]e, although in both cases pHi increased. We conclude that in bovine anterior pituitary cells NH4Cl raises [Ca2+]i by two independent pathways, increasing net calcium entry and mobilizing Ca2+ from a TRH-insensitive calcium store.

Ammonium Chloride↗
PubMed1992

Activation of the oxygen-radical-generating system in granules of intact human neutrophils by a calcium ionophore (ionomycin).

Subcellular fractionation studies were performed on human neutrophils stimulated with ionomycin (a Ca(2+)-specific ionophore). The results of these studies revealed NADPH-oxidase activity, without any additive, both in the plasma membrane and in the specific granule fractions. After comparing these results with the NADPH oxidase activity induced by the ionophore in intact neutrophils, in differentiated HL-60 cells and in neutrophil cytoplasts, we conclude that ionomycin preferentially activates the NADPH oxidase pool located in the membrane of specific granules. Furthermore, we suggest that incorporation of granule membrane into the plasma membrane makes the associated NADPH oxidase less sensitive to activation induced by a rise in [Ca(2+)]i.

Adult↗
PubMed2004

Ionomycin, but not physiologic doses of epinephrine, stimulates skeletal muscle interleukin-6 mRNA expression and protein release.

It has been hypothesized that epinephrine may stimulate interleukin (IL)-6 gene expression in skeletal muscle. The aim of the present study was to examine the effect of epinephrine on IL-6 gene expression within, and protein release from, skeletal muscle. We hypothesized that physiologic epinephrine would neither result in an increase in IL-6 mRNA nor protein release from skeletal muscle. Soleus muscle was excised from 4-week-old anesthetized Sprague Dawley rats and incubated in a Krebs buffer with the addition of either saline (CON), epinephrine, at concentrations of 1,000 nmol/L (EPI 1,000), 100 nmol/L (EPI 100), or 10 nmol/L (EPI 10), or the calcium ionophore, ionomycin (IONO), a positive control. After a 1-hour incubation, muscle was collected and extracted for RNA, reverse transcribed, and IL-6 gene expression was determined by real-time polymerase chain reaction (PCR). An aliquot of incubation medium was also collected and analyzed for IL-6 protein by enzyme-linked immunosorbent (ELISA). EPI 1,000 and IONO increased (P < .05) IL-6 mRNA, whereas EPI 100 and EPI 10 were without effect. IL-6 protein release from skeletal muscle was increased in IONO (P < .05), but not in CON or EPI at any concentration. These data demonstrate that while pharmacologic concentrations of epinephrine activate IL-6 mRNA, supraphysiologic and high-physiologic doses appear to have little, if any, effect on IL-6 gene transcription in skeletal muscle. In addition, ionomycin can stimulate IL-6 gene expression and protein release after only 1 hour of exposure.

Animals↗
PubMed1986

No synergism between ionomycin and phorbol ester in fatty acid synthesis by isolated rat hepatocytes.

With hepatocytes in suspension, freshly isolated from meal-fed rats, no significant effect of ionomycin on the rate of de novo fatty acid synthesis was observed, whereas phorbol myristate acetate (PMA) was strongly stimulatory. The combination of ionomycin and PMA produced the same stimulation as was seen with PMA alone. Stimulation of fatty acid synthesis by vasopressin was comparable and not additive to that observed with PMA, indicating that activation of protein kinase C is solely responsible for this metabolic effect of vasopressin. Both vasopressin and PMA increased acetyl-CoA carboxylase activity in isolated rat hepatocytes.

Acetyl-CoA Carboxylase↗