Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Ionomycin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources
PubMed1994

Attenuation of Ca(2+)-induced increase in oxidative metabolism by cooling and calmodulin antagonist in mammalian brain neurons: a flow-cytometric study.

Effects of cooling and calmodulin antagonist on ionomycin-induced increase in oxidative metabolism (or formation of reactive oxygen species) of rat cerebellar neurons was examined using a flow cytometer and 2',7'-dichlorofluorescin diacetate, a fluorescent dye for intracellular hydrogen peroxide. Cooling neurons to temperatures below 16 degrees C greatly attenuated ionomycin-induced augmentation of oxidative metabolism without affecting the Ca2+ influx produced by ionomycin. Rewarming neurons to 36 degrees C in presence of ionomycin increased the oxidative metabolism, indicating a temperature-sensitive metabolic process. Substitution of Ca2+ with Ba2+ or Sr2+ completely abolished an ionomycin-induced increase in the oxidative metabolism. Pretreatment with W-7, a calmodulin antagonist, at concentrations of 10 microM or higher (up to 100 microM) produced a dose-dependent attenuation of ionomycin-induced increase in oxidative metabolism. Results suggest that calmodulin is involved in the ionomycin-induced increase in oxidative metabolism of dissociated cerebellar neurons.

Animals↗
PubMed1988

Human neutrophil protein kinase C: calcium-induced changes in the solubility of the enzyme do not always correlate with enzymatic activity.

We hypothesized that calcium and 1,2-diacylglycerols stimulated human neutrophil (PMN) protein kinase C (EC 2.7.1.37) in a two-step mechanism. The proposed mechanism entails (1) increased insoluble protein kinase C activity and (2) endogenous protein phosphorylation, events which have not been biochemically dissociated. PMN which were treated with 100 nM ionomycin shifted protein kinase C activity from being mostly soluble to insoluble. Concentrations of ionomycin greater than 300 nM stimulated a doubling of total cellular (soluble + insoluble) protein kinase activity and stimulated increased endogenous phosphorylation of PMN proteins. Intracellular calcium (measured with fura-2) increased from 65 nM (basal) to 680 nM using 500 nM ionomycin; calcium increases were dose-dependent. The anti-inflammatory agents acetylsalicylic acid and sodium salicylate (but not ibuprophen, indomethacin or acetaminophen) inhibited ionomycin-induced protein kinase C activation and protein phosphorylation in a dose-dependent manner by inhibiting the production of diacylglycerols. 1-Oleoyl-2-acetylglycerol reversed the inhibitory effect of salicylates. In contrast to the effect of acetylsalicylates on protein kinase C functional activity the distribution of phorbol receptors was unaffected in acetylsalicylate-treated, ionomycin-stimulated PMN using a phorbol-binding assay. Our results show that ionomycin increased intracellular diacylglycerol levels 3.5-fold over those present in control PMN, while acetylsalicylate decreased diacylglycerol production in ionomycin-stimulated PMN below baseline values. These results support the hypothesis that increased intracellular calcium activated protein kinase C leading to protein phosphorylation in two distinct dissociable events: (1) increased intracellular calcium; and (2) increased 1,2-diacylglycerol levels.

Algorithms↗
PubMed1997

Phosphorylation and calcium influx are not sufficient for the activation of cytosolic phospholipase A2 in U937 cells: requirement for a Gi alpha-type G-protein.

Differentiation with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U937 cell line toward a monocyte/granulocyte-like cell results in the cell acquiring an ability to release arachidonate upon stimulation. In contrast, the calcium ionophore ionomycin was able to stimulate phospholipase C, as measured by inositol 1,4,5-trisphosphate formation, to equal extents in both undifferentiated and dBcAMP-differentiated U937 cells. The role and regulation of cytosolic phospholipase A2 (cPLA2) in the production of arachidonate in these cells when either the chemotactic peptide fMLP or ionomycin are used as stimulus were investigated. The ionomycin- and fMLP-stimulated release of arachidonate were sensitive to the cPLA2 inhibitor arachidonyl trifluoromethylketone (IC50 values of 32 and 18 microM, respectively), but were not inhibited by E-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2 H-pyran-2-one, a bromoenol lactone inhibitor of the calcium-independent phospholipase A2. These results, coupled with the inhibition of ionomycin-induced arachidonate production by electroporation of differentiated cells to introduce an anti-cPLA2, demonstrate that the cPLA2 is the enzyme responsible for arachidonate release in differentiated cells. SDS-PAGE and immunoblot analysis of differentiated cells showed the cells to contain both phosphorylated and unphosphorylated forms of cPLA2 (ratio of about 2: 3). Surprisingly, undifferentiated cells contain 30% more enzyme than differentiated cells and contain a higher percentage (approximately 75%) of the phosphorylated in the absence of stimulation. The inability of undifferentiated cells to produce arachidonate is not due to insufficient intracellular calcium concentrations since ionomycin induces large (820-940 nM) influxes of intracellular calcium in both differentiated and undifferentiated cells. This demonstrates that phosphorylation of cPLA2 andan influx of intracellular calcium are not sufficient to activate the enzyme to produce arachidonate. Instead, activation of a pertussis toxin-sensitive Gi alpha-type G-protein is required as evidenced by the production of arachidonate in undifferentiated cells stimulated with mastoparan, an activator of Gi alpha subunits, in combination with ionomycin. This activation of a Gi alpha-type G-protein is independent of modulations of adenylyl cyclase activity since cellular cAMP levels were not modulated upon treatment with mastoparan and ionomycin.

Acyltransferases↗
PubMed2000

Ca2+-induced redistribution of Ca2+/calmodulin-dependent protein kinase II associated with an endoplasmic reticulum stress response in vascular smooth muscle.

The relation between CaM kinase II activity and high Ca2+-mediated stress responses was studied in cultured vascular smooth muscle cells. Treatment with ionomycin (1 microM) for 5 min caused a significant loss of CaM kinase II activity in whole cell homegenates and prominent vesiculation of the endoplasmic reticulum (ER). Similar losses of CaM kinase II activity were observed in the soluble lysate as assessed by activity measurements and Western blotting. Examination of the post-lysate particulate fraction showed that the loss of CaM kinase II from the soluble lysate was accompanied by a redistribution of CaM kinase II to this fraction. The ionomycin-mediated response was limited to this concentration (1 microM); lower concentrations of ionomycin as well as stimulation with angiotensin II (1 microM) orATP (100 microM) did not cause a shift in CaM kinase II distribution. Treatment with neither the CaM kinase II inhibitor KN-93 nor the phosphatase inhibitor okadaic acid altered the ionomycin-induced redistribution indicating that CaM kinase II activation and/or phosphorylation was not part of the mechanism. The response, however, was eliminated when the cells were treated in Ca2+-free medium. Washout of ionomycin led to only a partial restoration of the kinase activity in the soluble fraction after 10 min. Immunofluorescence microscopy of resting cells indicated colocalization of antibodies to CaM kinase II and an ER protein marker. ER vesiculation induced by ionomycin coincided with a parallel redistribution of CaM kinase II and ER marker proteins. These data link ionomycin-induced ER restructuring to a progressive redistribution of CaM kinase II protein to an insoluble particulate fraction and loss of cellular CaM kinase II activity. We propose that redistribution of CaM kinase II and loss of cellular activity are components of a common Ca2+-overload induced cellular stress response in cells.

Animals↗
PubMed1992

Mechanisms of activation of Na+/H+ exchange in human osteoblast-like SaOS-2 cells.

Because of the importance of pH homeostasis in bone and the current uncertainty about the mechanisms by which intracellular pH (pHi) is regulated in this tissue, we have investigated the roles of cytosolic free Ca2+ concentrations ([Ca2+]i) and protein kinase C on the activation of Na+/H+ exchange in human osteoblast-like SaOS-2 cells. [Ca2+]i and pHi were measured using Fura-2 and 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) respectively. The basal pHi in HCO3(-)-free buffer was 7.36 +/- 0.04 units (mean +/- S.D.). Addition of ionomycin in Ca(2+)-containing buffer did not cause a rise in basal pHi; however, addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) did cause a slowly developing rise in resting pHi of 0.14 +/- 0.02 unit over 4-5 min. Nigericin, a K+/H+ ionophore, caused an abrupt fall in pHi to 6.70 +/- 0.07 units. In nigericin-pretreated cells, PMA caused a rapid rise in pHi without changing the [Ca2+]i. In acidified cells, ionomycin increased [Ca2+]i and pHi in a parallel concentration-dependent (30-500 nM) manner. This action of ionomycin occurred in both the presence and the nominal absence of extracellular Ca2+. Ionomycin-induced alkalinization depended on extracellular Na+ and was inhibited in cells incubated with hexamethylene amiloride. When the incremental increase in [Ca2+]i induced by ionomycin was blocked by preincubation with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA)/AM, the effect on pHi was inhibited. Staurosporine, a protein kinase C inhibitor, blocked the action of PMA on pHi, but it had no effect on the ionomycin-induced increase in pHi. The action of ionomycin was not due to osmotic shock. We conclude that SaOS-2 cells have a protein kinase C-activatable Na+/H+ antiporter that is also stimulated, in acidified cells, in a concentration-dependent fashion by transients in [Ca2+]i which act via a non-protein kinase C pathway.

Alkaloids↗
PubMed1991

Mechanism of calcium ionophore and phorbol ester-induced T-cell activation. Accessory cell requirement for T-cell activation.

We examined the role of monocytes in T-cell activation induced by phorbol myristate acetate (PMA) and calcium ionophore ionomycin. Depletion of monocytes from peripheral blood mononuclear cells (PBMC) was associated with the loss of interleukin-2 (IL-2) production, IL-2 receptor (IL-2R) expression and proliferation, in response to either PMA or ionomycin. Addition of monocytes to highly purified T cells resulted in the complete reconstitution of IL-2 production, IL-2R expression and proliferation by PMA-stimulated lymphocytes. Exogenous IL-2, but not interleukin-1 (IL-1), could reconstitute the T-cell responsiveness. Addition of monocytes to highly purified T cells stimulated with ionomycin resulted in partial reconstitution of IL-2 production, IL-2R expression and proliferation. Similarly, the addition of exogenous IL-2 to ionomycin-stimulated T cells only partially reconstituted the response compared with PBMC. These results suggest that monocyte-T-cell interactions contribute to IL-2 production and IL-2R expression and are crucial events for PMA-induced T-cell proliferation. With ionomycin, monocytes play a role, in part, in inducing IL-2 production, IL-2R expression and proliferation. However, IL-2 is not a sufficient signal to induce T-cell proliferative response to ionomycin, suggesting that an IL-2-independent mechanism may exist in ionomycin-induced T-cell proliferation.

Cell Division↗
PubMed1992

Priming of human polymorphonuclear leukocytes with granulocyte-macrophage colony-stimulating factor involves protein kinase C rather than enhanced calcium mobilisation.

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N-formyl-methionyl-leucyl-phenylalanine, fMLP) or a Ca(2+)-ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5-lipoxygenase. Preincubation of polymorphonuclear leukocytes with GM-CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca(2+)-threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350-400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells. Protein biosynthesis inhibitors like actinomycin D (10 micrograms/ml) and cycloheximide (50 micrograms/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However, staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM-CSF after stimulation with ionomycin. The priming effect of GM-CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultaneous pretreatment with PMA and GM-CSF and stimulation with either fMLP or ionomycin. These results provide evidence that the enhanced arachidonic acid release in GM-CSF-primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.

Alkaloids↗
PubMed1987

Ryanodine releases calcium from sarcoplasmic reticulum in calcium-tolerant rat cardiac myocytes.

1. The hypothesis tested in this study is that ryanodine depletes sarcoplasmic reticulum (s.r.) Ca2+ loading in suspensions of single adult rat cardiac myocytes by effecting Ca2+ release into the myoplasm resulting in an increase in myoplasmic free [Ca2+] ([Ca2+]i). The latter was monitored by the fluorescent dye, quin2. 2. The competency of the technique to detect s.r. Ca2+ release was tested by using caffeine to induce Ca2+ release. The addition of 5-10 mM-caffeine to myocytes loaded with quin2 and incubated in a medium containing 1 mM-Ca2+ gives a large, transient increase in fluorescence, which is interpreted as indicating an increase in [Ca2+]i. If the chelating agent EGTA is added to the cell suspension 1-5 min prior to the caffeine, to a concentration sufficient to decrease extracellular Ca2+ to 0.1-0.15 microM, then caffeine again gives a large, transient increase in fluorescence, indicative of the fact that sarcolemmal Ca2+ transport is not necessary for this response. The ionophore ionomycin also raises [Ca2+]i in a transient manner when added after EGTA. The addition of caffeine prior to ionomycin largely diminishes the response to the latter; however, addition of ionomycin prior to caffeine totally abolishes its effect to increase [Ca2+]i. This is taken to indicate that the intracellular store which is releasable by caffeine--and which presumably reflects the s.r.--is also releasable by ionomycin: ionomycin, however, also gives access to another, minor intracellular pool. 3. The plant alkaloid, ryanodine, at concentrations of 10(-8) to 10(-6) M, consistently causes a slow and prolonged increase in [Ca2+]i when added to cell suspensions incubated with 1 mM-extracellular Ca2+. Under conditions precluding net entry of Ca2+ into the cell, viz. 0.1 microM-extracellular Ca2+, ryanodine causes a more limited, partially reversible, increase in [Ca2+]i. 4. When added prior to EGTA, ryanodine attenuates, or prevents, the subsequent response to caffeine: efficacy depends upon the time of pre-incubation (1-10 min) and the concentration of ryanodine (10(-8) to 10(-6) M). When the response to caffeine is largely prevented by ryanodine, the response to ionomycin is also severely attenuated, i.e. there is no evidence that ryanodine causes sequestration of Ca2+ within an ionomycin-sensitive pool.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaloids↗
PubMed1996

Induction of GLUT-1 mRNA in response to inhibition of oxidative phosphorylation: role of increased [Ca2+]i.

Exposure of Clone 9 cells (a rat liver cell line expressing only the GLUT-1 isoform) to 5 mM azide or to 3 microM ionomycin for 12 h results in 3.7 +/- 0.3- and 4.9 +/- 0.4-fold increases in GLUT-1 mRNA content, respectively, suggesting the hypothesis that a rise in cytosolic free calcium concentration ([Ca2+]i) mediates the induction of GLUT-1 mRNA by azide. Five lines of evidence were employed to test this hypothesis. 1) Exposure of cells to 0-3 microM of ionomycin increased [Ca2+]i from 83 +/- 9 to 504 +/- 20 nM (half-maximal effect at 0.1 microM ionomycin), whereas half-maximal increase in GLUT-1 mRNA occurred at 1 microM ionomycin, with the increase in the mRNA being negligible at [Ca2+]i below 400 nM. Exposure of cells to 5 mM azide, however, increased [Ca2+]i to maximal value of 174 +/- 22 nM at 15 s, suggesting that the magnitude of the increase in [Ca2+]i by azide may not be adequate for the response. 2) The increase in GLUT-1 mRNA content by azide was fully preserved in cells preloaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). 3) GLUT-1 mRNA content increased within 30 min of exposure to ionomycin, whereas the mRNA increased after a "delay" period of 2 h in cells exposed to 5 mM azide. 4) A brief (2-min) rise in [Ca2+]i by ionomycin was sufficient to increase GLUT-1 mRNA content, whereas continuous exposure to azide for > 1 h was necessary for a subsequent induction of the mRNA. 5) Treatment with ionomycin, A-23187, and thapsigargin caused larger increases in glucose-regulated protein 78 and 94 and in 70-kDa heat shock protein mRNAs than in GLUT-1 mRNA, whereas treatment with azide resulted in greater induction of GLUT-1 mRNA. These results strongly suggest that, whereas increased [Ca2+]i enhances GLUT-1 mRNA expression and azide increases [Ca2+]i, the rise in [Ca2+]i does not mediate the induction of GLUT-1 mRNA in response to inhibition of oxidative phosphorylation.

Adenosine Triphosphate↗
PubMed1991

Physiological and cytotoxic effects of Ca(2+) ionophores on Caco-2 paracellular permeability: relationship of 45Ca(2+) efflux to 51 Cr release.

The human intestinal cell line, Caco-2, and the Ca2+ ionophores, A23187 and ionomycin, were used to determine the interrelationships of 45Ca(2+) efflux, transepithelial electrical resistance (Rt), and [3H]-mannitol flux to changes in 51Cr release and lactate dehydrogenase (LDH) activity. Treatment of Caco-2 monolayers with ionomycin at concentrations of between 0.25 and 2.50 mumol/l showed similar 45Ca(2+) efflux rate constants and coefficients. Analysis of the control and ionomycin-induced 45Ca(2+) efflux values showed the data to best fit a three Ca(2+) compartmental model. All changes in Caco-2 Rt and [3H]-mannitol flux were reversible with no significant increases in 51Cr release with ionomycin concentrations of less than or equal to 2.5 mumols/l. Caco-2 monolayers treated with ionomycin at concentrations of between 5.0 and 50.0 mumols/l showed rapid non-exponential 45Ca(2+) effluxes with irreversible changes in Rt, [3H]-mannitol flux, and significant increases in 51Cr release. There was no changes in media LDH activity using either ionomycin or A23187 at concentrations of up to 50 mumols/l for 60 min. The results of our study show that: (1) disruption of Ca(2+) homeostasis in Caco-2 cells will occur with the addition of Ca(2+) ionophores at concentrations of greater than 2.50 mumols/l; (2) high concentrations (greater than 2.5 mumols/l) of ionomycin will cause non-exponential 45Ca(2+) efflux rates with irreversible changes in intracellular Rt and 14C-mannitol flux, and (3) early signs of Ca(2+) ionophore-induced damage can be detected by 51Cr release from Caco-2 cells into the media and not by changes in LDH media activity.

Anti-Bacterial Agents↗
PubMed1990

Effects of protein kinase C activation on inositol phosphate generation and intracellular Ca2+ mobilization in bovine parathyroid cells.

Activators of protein kinase C, such as phorbol myristate acetate (PMA) and the synthetic diacylglycerol dioctanoylglycerol (diC8), either stimulate or inhibit PTH release depending on the extracellular Ca2+ concentration. By increasing PTH release at high extracellular Ca2+, these agents, in effect, block high Ca2(+)-induced inhibition of secretion. Since raising extracellular Ca2+ increases intracellular free Ca2+ ([Ca2+]i) and inositol trisphosphate (InsP3) formation in parathyroid cells, we assessed the effects of PMA pretreatment on [Ca2+]i and InsP3 to ascertain whether these second messengers might be altered by protein kinase C activation. Preincubation of parathyroid cells with PMA (10(-6) M) significantly lowered the intracellular Ca2+ response to raising extracellular Ca2+ from 0.5-2.0 mM. The peak increase in [Ca2+]i averaged 475 +/- 11 nM in PMA-treated cells compared to 703 +/- 44 nM in control cells. High extracellular Ca2(+)-induced InsP3 accumulation was also reduced after incubating the cells with PMA. To determine whether intracellular Ca2+ stores and/or transmembrane Ca2+ uptake were affected by activating protein kinase C, we examined intracellular Ca2+ responses to the Ca2+ ionophore ionomycin after PMA pretreatment. At 0.5 mM Ca2+, ionomycin (10(-6) M) increased [Ca2+]i to an initial peak of 738 +/- 49 nM followed by a sustained increase to 501 +/- 30 nM in control cells (n = 15). After exposure to PMA (greater than or equal to 20 min), however, peak and sustained increments in [Ca2+]i were significantly lower at 550 +/- 32 and 394 +/- 16 nM, respectively (P less than 0.02, n = 8). In the absence of extracellular Ca2+, basal [Ca2+]i was 197 +/- 5 and peaked at 323 +/- 15 nM with ionomycin (10(-6) M) in PMA-treated cells (n = 16). The latter value was significantly less than the peak increase in [Ca2+]i to 461 +/- 19 nM observed with ionomycin (10(-6) M) in control cells (P less than 0.001, n = 15). With respect to secretion, either of the protein kinase C agonists (i.e. PMA or diC8) or the Ca2+ ionophore ionomycin inhibited PTH release at 0.5 mM Ca2+. To determine whether the concomitant activation of protein kinase C- and Ca2(+)-dependent pathways could additively suppress PTH release, we assessed the effects of ionomycin and either PMA or diC8 on secretion. PTH release at 0.5 mM Ca2+ was reduced in an additive manner by either of these protein kinase C agonists plus ionomycin. At 2 mM Ca2+, protein kinase C agonists stimulated PTH release.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2004

The effect of activation protocols on the development of cloned goat embryos.

This study was conducted to compare the developmental competence of somatic nuclear transfer (NT) embryos, after either ionomycin or ethanol activation, in locally bred goats. Donor cells were prepared from the ear skin fibroblasts of a female goat. Cells, at passage 3-8, starved by culturing in 0.5% FCS for 4-8 d, were used for NT. Immature oocytes were obtained from FSH-stimulated goats and matured for 22 hr before enucleation and NT. After fusion, the reconstructed embryos were activated with either ionomycin or ethanol followed by culturing in 6-dimethylaminopurine (6-DMAP) and cytochalasin B (CB), for 3 hr. In experiment I, the fused NT embryos (n=63, ionomycin and n=68, ethanol treatments, respectively) were cultured in B2 with a Vero co-culture system and their developmental competence was evaluated through to Day 9. In experiment II, the NT embryos at the 2-4 cell stage on Day 2 derived from each treatment (ionomycin n=46, and ethanol n=37), were transferred into 10 synchronous recipients. There were no significant differences between the NT embryos derived from the ionomycin and ethanol groups, in fusion (86.3% versus 82.9%), cleavage (90.5% versus 82.4%) and for morula/blastocyst development rates (9.5% versus 5.9%). Sixty percent (3/5) of the recipients from ionomycin became pregnant by midterm (2.5 mts) while only 20% (1/5) from ethanol treatment was pregnant by Day 45. The results demonstrate that activation with either ionomycin or ethanol in combination with 6-DMAP-CB treatment does not affect the development of cloned goat embryos.

Adenine↗
PubMed1986

Forskolin enhances calcium-evoked prolactin release from 7315c tumor cells without increasing the cytosolic calcium concentration.

The 7315c prolactin-secreting tumor cell was used as a model of a normal pituitary cell in order to study the enhancement by adenosine 3',5'-cyclic monophosphate (cAMP) of calcium-evoked hormone release. Forskolin and, by implication, cAMP had little effect on basal hormone release during a 10-min incubation period. Ionomycin and a high potassium concentration, treatments which enhanced the cytosolic calcium concentration, increased hormone release. When cells were exposed to forskolin prior to and during a challenge with either ionomycin or high potassium, a synergistic effect on prolactin release was observed. 8-Bromoadenosine 3',5'-cyclic monophosphate mimicked forskolin in enhancing ionomycin-evoked prolactin release while having little effect of its own on hormone release. Forskolin did not alter the increase in cytosolic calcium concentration elicited by either ionomycin or high potassium, nor did it increase the potency of ionomycin in enhancing prolactin release. The calcium channel antagonist, D-600, did not alter ionomycin-induced release or its enhancement by forskolin; D-600 blocked potassium-induced prolactin release. Ionomycin had no effect on basal cAMP synthesis by tumor cells and inhibited slightly the forskolin-induced increase in nucleotide synthesis. The results suggest that cAMP acts, at a site distal to the entry of calcium into the cytosol, to enhance the amount of prolactin released in response to an increase in the cytosolic calcium concentration.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Alteration of human lymphokine-activated killer cell activity by manipulation of protein kinase C and cytosolic Ca2+.

We have examined the effects of protein kinase C (PK-C) stimulation and cytosolic Ca2+ elevation on the in vitro induction of non-histocompatibility-restricted tumoricidal activity from human peripheral blood lymphocytes. The tumor cytolytic activity, as well as the number of cells recovered from interleukin 2 (IL-2)-stimulated cultures, was enhanced by the addition of the PK-C stimulator, phorbol dibutyrate (PDBu), but not non-PK-C-activating phorbol ester analogues while the Ca2+ ionophore, ionomycin, did not significantly alter development of IL-2-induced tumor cytolytic activity nor enhance cell yield. Neither PDBu nor ionomycin, alone or in combination, induced tumoricidal activity. The addition of both PDBu and ionomycin to recombinant interleukin 2 (rIL-2)-exposed cultures produced a strong mitogenic response and high cell yield, although Daudi cell killing measured at Day 5 was completely abolished. This abrogation of lymphokine-activated killer cell activity was seen as early as 24 h following exposure to PDBu and ionomycin, reaching 50% following 2 days of exposure. When lymphocytes mitogenically expanded by primary exposure to PDBu and ionomycin and then washed free of these agents were further cultured with rIL-2 alone, proliferation continued, and substantial cytolytic activity for Daudi cells was induced. The development of this postexpansion cytotoxic activity was not dependent on the addition of exogenous rIL-2 during the primary cultures. Fractionation of cells into large granular lymphocytes and small T-lymphocytes indicated that only the large granular lymphocytes proliferate in response to rIL-2 alone. Both large granular lymphocytes and small T-lymphocytes proliferate in response to the addition of PDBu and ionomycin, and both populations of cells developed tumor cytolytic activity following removal of PDBu and ionomycin and subsequent culture in rIL-2. These data suggest that PK-C and Ca2+ signals play key roles in the regulation and/or proliferation of tumor cytotoxic lymphocytes or their precursors and that manipulation of those signals can be utilized to produce substantially more tumoricidal activity from lymphocyte populations than can be achieved with rIL-2 alone.

Calcium↗
PubMedDate not supplied

Effects of Elevated Cytosolic Calcium on ACh-Induced Swine Tracheal Smooth Muscle Contraction.

Increased intracellular calcium concentration ([Ca(2+)](i)) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca(2+)](i) are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca(2+) on tension maintenance, [Ca(2+)](i) was elevated using ionomycin, a Ca(2+) ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K(Ca)) blocker prior to or during exposure of tracheal smooth muscle strips to ACh (10(-9) to 10(-4) M). Ionomycin (5 &mgr;M) in resting muscles induced increases in [Ca(2+)](i) to 500 +/- 230 nM and small increases in force of 2.6 +/- 2.3 N/cm(2). This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca(2+)](i) or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC(50) = 0.07 +/- 0.05 &mgr;M ionomycin; 0.17 +/- 0.07 &mgr;M, control, p < 0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca(2+)](i) induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca(2+)](i) when compared to muscles stimulated with ACh. Steady-state [Ca(2+)](i) limits tension development induced by submaximal concentrations of ACh. The activity of K(Ca) moderates the response of the muscle to ACh at concentrations less than 1 &mgr;M. Copyright 1996 S. Karger AG, Basel

Journal Article↗
PubMed1998

Insulin inhibits vascular smooth muscle contraction at a site distal to intracellular Ca2+ concentration.

Several hypertensive states are associated with resistance to insulin-induced glucose disposal and insulin-induced vasodilation. Insulin can inhibit vascular smooth muscle (VSM) contraction at the level of the VSM cell, and resistance to insulin's inhibition of VSM cell contraction may be of pathophysiological importance. To understand the VSM cellular mechanisms by which insulin resistance leads to increased VSM contraction, we sought to determine how insulin inhibits contraction of normal VSM. It has been shown that insulin lowers the contractile agonist-stimulated intracellular Ca2+ (Ca2+i) transient in VSM cells. In this study, our goal was to see whether insulin inhibits VSM cell contraction at steps distal to Ca2+i and, if so, to determine whether the mechanism is dependent on nitric oxide synthase (NOS) and cGMP. Primary cultured VSM cells from canine femoral artery were bathed in a physiological concentration of extracellular Ca2+ and permeabilized to Ca2+ with a Ca2+ ionophore, either ionomycin or A-23187. The resultant increase in Ca2+i contracted individual cells, as measured by photomicroscopy. Preincubating cells with 1 nM insulin for 30 min did not affect basal Ca2+i or the ionomycin-induced increase in Ca2+i, as determined by fura 2 fluorescence measurements, but it did inhibit ionomycin- and A-23187-induced contractions by 47 and 51%, respectively (both P < 0.05). In the presence of 1.0 microM ionized Ca2+, ionomycin-induced contractions were inhibited by insulin in a dose-dependent manner. In the presence of ionomycin, insulin increased cGMP production by 43% (P < 0.05). 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM), a selective inhibitor of guanylate cyclase that blocked cGMP production in these cells, completely blocked the inhibition by insulin of ionomycin-induced contraction. It was found that the cells expressed the inducible isoform of NOS. NG-monomethyl-L-arginine or NG-nitro-L-arginine methyl ester (0.1 mM), inhibitors of NOS, did not affect ionomycin-induced contraction but prevented insulin from inhibiting contraction. We conclude that insulin stimulates cGMP production and inhibits VSM contraction in the presence of elevated Ca2+i. This inhibition by insulin of VSM contraction at sites where Ca2+i could not be rate limiting is dependent on NOS and cGMP.

Animals↗
PubMed1996

Increases in HSF1 translocation and synthesis in human epidermoid A-431 cells: role of protein kinase C and [Ca2+]i.

BACKGROUND: It is known that heat shock increases both heat shock protein 70 kd (HSP-70) mRNA synthesis, and intracellular cytosolic free calcium concentration ([Ca2+]i). The latter enhances the heat inducible form of HSP-70 production by increasing the complex formation between heat shock transcriptional factor (HSF) and heat shock elements (HSE). In this study, we investigated the effect of agonists (PMA; ionomycin) and antagonists (BAPTA; staurosporine) of protein kinase C (PKC), and calcium channel on translocation and synthesis of HSF1, and activation of HSP-70 gene in human epidermoid A-431 cells. METHODS: Cells were incubated with poly 12-myristate 13-acetate (PMA) or ionomycin at different concentrations for various periods of time. Messenger RNAs of HSF and HSP-70 were measured with RT-PCR. The HSP-70 protein was determined with Western blots, and HSF protein was measured by gel mobility retardation assay. RESULTS: Significant increases in HSF binding to [32P]labeled HSE were found at 30 minutes in nuclear extract and at 4 hours in both nuclear and cytosol extracts. The PMA- and ionomycin-induced increases in HSF were in a concentration-dependent manner with a maximal increase at 10(-6) mol/L of each drug. Meanwhile, the mRNAs encoded for HSF1 and HSP-70, but not HSF2, were significantly increased and reached the maximum at 1 hour after the treatment. The PMA increased [Ca2+]i by 92% because of Ca2+ influx. The increases in mRNA of HSF1 and HSP-70 induced by treatment with 1 mumol/L PMA were completely blocked by preincubating cells with either 2 mumol/L staurosporine in the presence of extracellular Ca2+ or 100 mumol/L BAPTA-am in absence of extracellular Ca2+. Like PMA, the increases induced by ionomycin were also inhibited by 100 mumol/L BAPTA-am in absence of extracellular Ca2+. Furthermore, Western blots show that 1 mumol/L PMA or ionomycin induced maximal increase in HSP-70 after 7 hours of continuous incubation with either agent. When cells were simultaneously treated with 1 mumol/L PMA and ionomycin together for 1 hour, the increase in HSP-70 and HSF1 mRNAs reached a greater level than the level stimulated by either drug alone. CONCLUSIONS: These results indicate that both PMA and ionomycin stimulate HSF1, but not HSF2, translocation and synthesis leading to the HSP-70 expression and that their effects are Ca(2+)-dependent.

Calcium↗
PubMed1996

Characterization of intracellular calcium pools and their desensitization in thermotolerant human A-431 cells.

BACKGROUND: This study characterizes the intracellular Ca2+ pools in nonthermotolerant and thermotolerant human A-431 cells and the reduced cytotoxicity using the inhibitors of Ca2+ mobilizations. METHODS: Nonthermotolerant and thermotolerant cells were treated with different Ca2+ mobilizers in the absence of external Ca2+. The cytosolic Ca2+ concentration using fura-2 fluorescence probe was measured to identify the presence of intracellular Ca2+ pools. The cytotoxicity of the increase in [Ca2+]i was studied using the colony forming efficiency assay. RESULTS: The resting intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Ca2+ was 42 +/- 9 nm, determined by fura-2. Bradykinin (10 mumol/L), monensin (200 mumol/L), and ionomycin (1 mumol/L) sequentially treated to cells mobilized Ca2+ and increased [Ca2+]i by 64 +/- 23, 40 +/- 6, and 59 +/- 21 nm, respectively. The bradykinin effect was blocked by 5 mumol/L U-73122 (an inhibitor of inositol trisphosphate production); the ionomycin effect was inhibited by increasing intracellular pH (pHi) or treatment with 100 mumol/L ryanodine while the monensin effect was enhanced by increasing pHi, but was not inhibited by ryanodine. Cells that were made tolerant to lethal temperatures also responded to bradykinin, monensin, and ionomycin, but the magnitude of the response was diminished. Subsequent treatments with bradykinin, monensin, and ionomycin increased [Ca2+]i in thermotolerant cells to levels 68 +/- 8, 44 +/- 5, and 45 +/- 5%, respectively, of values found in nonthermotolerant cells. Higher concentrations of these agents did not further increase [Ca2+]i. The bradykinin-induced increase in inositol trisphosphates in thermotolerant cells was also reduced, which perhaps accounts for the attenuation in Ca2+ mobilization. Unlike nonthermotolerant cells, the monensin effect was not enhanced when pHi was increased. However, the ionomycin effect was still dependent on pHi and was blocked by ryanodine at a higher concentration. CONCLUSIONS: These results show that there are bradykinin-, monensin-, and ryanodine-sensitive pools and that thermotolerance attenuates Ca2+ mobilization stimulated by these three agents. Ionomycin at 10 mumol/L or NaCN at 10 mM for 1 hour demonstrated cytotoxicity. Pretreatment with 100 mumol/L ryanodine and/or 5 mumol/L U-73122 reduced cytotoxicity produced by either NaCN or ionomycin. These results suggest that an attenuation of [Ca2+]i increases can diminish cytotoxicity.

Adaptation, Physiological↗