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PubMed1993

Costimulatory signals are required for optimal proliferation of human natural killer cells.

CD56dim NK cells, which comprise approximately 90% of human peripheral blood NK cells, respond to IL-2 with cytokine production, up-regulation of functionally relevant surface molecules, and augmented cytolytic activity. Nevertheless, CD56dim NK cells proliferate poorly in response to IL-2 alone. We found that other NK cell mitogens, including IL-4, IL-7, and IL-12, also induced little proliferation of CD56dim NK cells. Indeed, IL-2 stimulated at least 10-fold more NK cell proliferation than did IL-4, IL-7, or IL-12. In contrast, leukocyte-conditioned medium (LCM) induced two- to threefold greater proliferation of CD56dim NK cells than did optimal concentrations of IL-2. Although the calcium ionophore ionomycin did not stimulate proliferation by itself, it markedly augmented LCM-induced proliferation of CD56dim NK cells. Proliferation in response to either LCM alone or LCM together with ionomycin was almost completely abrogated by anti-IL-2R antibodies. Thus, IL-2 appears to be necessary but not sufficient for optimal proliferation of CD56dim NK cells. LCM-induced proliferation of ionomycin-activated CD56dim NK cells was inhibited 24% by anti-IL-1 heteroantisera and 57% by anti-TNF antisera; a combination of both antisera inhibited proliferation by 73%. Furthermore, although rIL-1 and TNF did not induce proliferation by themselves, both cytokines could augment IL-2-induced proliferation of resting or ionomycin-activated NK cells. Hence IL-1 and TNF do not appear to be primary NK cell mitogens, but rather accessory factors that can enhance IL-2-dependent NK cell proliferation. Stimulation through CD2 or CD16 Ag did not enhance LCM-induced NK cell proliferation. However, stimulation with NK-sensitive K562 cells strongly augmented CD56dim NK cell proliferation to LCM or to IL-2, IL-1, and TNF in combination. NK-resistant Daudi cells did not promote the proliferation of highly purified NK cells. Thus, NK cell proliferation may be enhanced by triggering through putative receptors for natural killing, and ionomycin may mimic such triggering. Although IL-2 by itself can induce NK cell proliferation, most NK cells resemble T and B lymphocytes in that they require multiple signals for optimal proliferation.

Animals↗

PubMed1994

Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H(+)-sensitive and Ca2+ regulation-defective.

The ubiquitous plasma membrane Na+/H+ exchanger (NHE1) is rapidly activated in response to various extracellular signals. To understand how the intracellular Ca2+ is involved in this activation process, we investigated the effect of Ca2+ ionophore ionomycin on activity of the wild-type or mutant NHE1 expressed in the exchanger-deficient fibroblasts (PS120). In wild-type transfectants, a short (up to 1 min) incubation with ionomycin induced a significant alkaline shift (approximately 0.2 pH unit) in the intracellular pH (pHi) dependence of the rate of 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ uptake, without changes in the cell volume and phosphorylation state of NHE1. Mutations that prevented calmodulin (CaM) binding to a high affinity binding region (region A, amino acids 636-656) rendered NHE1 constitutively active by inducing a similar alkaline shift in pHi dependence of Na+/H+ exchange. These same mutations abolished the ionomycin-induced NHE1 activation. These data suggest that CaM-binding region A functions as an "autoinhibitory domain" and that Ca2+/CaM activates NHE1 by binding to region A and thus abolishing its inhibitory effect. Furthermore, we found that a short stimulation with thrombin and ionomycin had apparently no additive effects on the alkaline shift in the pHi dependence of Na+/H+ exchange and that deletion of region A also abolished such an alkaline shift induced by a short thrombin stimulation. The results strongly suggest that the early thrombin response and the ionomycin response share the same activation mechanism. Based on these data and the results shown in the accompanying paper (Bertrand, B., Wakabayashi, S., Ikeda, T., Pouysségur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13703-13709), we propose that CaM is one of the major "signal transducers" that mediate distinct extracellular signals to the "pHi sensor" of NHE1.

Binding Sites↗

PubMed1996

Regulation of gamma T-cell antigen receptor expression by intracellular calcium in acute lymphoblastic leukemia cell line DND41.

The calcium ionophore, ionomycin, promotes an increase of intracellular calcium and regulates mRNA expression of gamma/delta-TcR gene in human T lymphocytes. The mechanism of this regulation is not yet clear. Thus, the regulation by intracellular calcium requires elucidation. We studied the gamma-TcR gene expression in acute lymphoblastic leukemia cell line DND41 (CD4- CD8-) by Northern blot and flow cytometric analysis. The mRNA levels of gamma-TcR increased by ionomycin, anti-CD3, and with TPA. TPA had an antagonistic effect to both ionomycin and anti-CD3. Also, TPA inhibits the increased intracellular calcium promoted by ionomycin but not the increase promoted by anti-CD3 and ionomycin. Our results suggest that intracellular calcium induces mRNA and protein expression of gamma-TcR chain. This effect is antagonized by protein kinase C-activation. Thus, we conclude that the target cells of the differential regulation on gamma-TcR mRNA expression by intracellular calcium modulators are the CD4- CD8- cells, and this is due to cytosolic calcium mobilization.

Calcium↗

PubMed1997

Mechanism of tissue factor activation on HL-60 cells.

Tissue factor (TF) procoagulant activity (PCA) on the surface of intact HL-60 cells is encrypted. This latent TF PCA was activated by exposing the cells to ionomycin, a calcium ionophore. Within seconds an increase in TF PCA of greater than 100-fold was observed. The ionomycin effect was blocked by pretreating the cells with calmidazolium, a calmodulin inhibitor. Changes in TF structure and function, coincident with the ionophore-induced increase in TF PCA, were identified. TF-factor VIIa complexes formed on both untreated and ionophore-treated cells, but pseudosubstrate inhibitors only bound to TF-factor VIIa on the ionophore-treated cells. TF PCA was inhibited by reacting cells with sulfosuccinimidyl-6-(biotinamido)hexanoate, and the rate of this reaction increased twofold after cells were exposed to ionomycin. When proteins on the surface of untreated cells, expressing minimal TF PCA, were cross-linked with 3-3'-dithiobis(sulfosuccinimidylpropionate), cross-linked TF dimers were produced. TF cross-linking was prevented by first treating the cells with ionomycin. These results suggest a mechanism for the ionomycin-induced increase in TF PCA. TF activation appears to be a calmodulin-dependent process, which exposes an essential macromolecular substrate binding site on TF, possibly as the result of a change in TF quaternary structure.

Anticoagulants↗

PubMed1997

Changes in nitric oxide synthase activity during exposure to hydrogen peroxide in cultured endothelial cells.

This study clarified the changes in maximum nitric oxide synthase (NOS) activity in cultured bovine aortic endothelial cells during exposure to hydrogen peroxide (H2O2). NOS activity was determined by measuring the conversion of [3H]arginine to [3H]citrulline. Ionomycin, a Ca2+ ionophore, was used to stimulate NOS activity. Addition of ionomycin (10(-5) M) increased the level of L-citrulline formation, and the ionomycin-induced increase in L-citrulline formation was stimulated by H2O2 pretreatment (15-90 min). Longer exposure to H2O2 (> or = 180 min) markedly inhibited the ionomycin-induced L-citrulline formation. The stimulation of ionomycin-induced L-citrulline formation by H2O2 was completely inhibited by N(G)-nitro-L-arginine (L-NNA) or N(G)-methyl-L-arginine (L-NMA), both blockers of NOS. Cell death which was determined by lactate dehydrogenase (LDH) release was induced from 120 min after the addition of H2O2. These findings suggest that the maximum L-citrulline formation from L-arginine, coupled with NOS activity, was increased by H2O2 treatment before cell death. The stimulation of NOS activity may be implicated in H2O2-induced endothelial cell death.

Animals↗

PubMed1996

Disruption of inositol phosphate accumulation in cerebellar granule cells by polychlorinated biphenyls: a consequence of altered Ca2+ homeostasis.

The present study examined the activation of protein kinase C (PKC) and disruption of Ca2+ homeostasis as potential mechanisms underlying effects of the polychlorinated biphenyl (PCB) congener 2,2'-dichlorobiphenyl (DCB) on inositol phosphate (IP) signaling in cerebellar granule cells. DCB (100 microM) increased basal IP accumulation in cerebellar granule cells when the extracellular free Ca2+ concentration ([Ca2+]e) was 0.75 mM but not when [Ca2+]e was 1 microM. Ionomycin (0.1 to 30 microM), a Ca2+ ionophore, also increased basal IP accumulation and [Ca2+]i in a concentration-dependent manner in cerebellar granule cells in the absence of DCB; increases in basal IP accumulation induced by 100 microM DCB were not additive with ionomycin. Ionomycin also disrupted carbachol (CARB, 1 mM)-stimulated IP accumulation. A 30-min preincubation with 0.3 or 1.0 microM ionomycin decreased CARB-stimulated IP accumulation, whereas simultaneous addition of 1.0 and 10 microM ionomycin with CARB increased and decreased, respectively, IP accumulation. DCB caused concentration-dependent increases in intracellular free Ca2+ concentration ([Ca2+]i) in cerebellar granule cells under experimental conditions identical to those used to measure IP accumulation. Following a one-half hour exposure to DMSO, 50 or 100 microM DCB, the [Ca2+]i was 36, 103, and 453 nM, respectively. We examined whether direct or indirect activation of PKC underlies DCB-induced inhibition of agonist-stimulated IP accumulation. DCB (100 microM) did not alter PKC activity in cytosolic or membrane fractions of granule cell homogenates. In intact cells, 50 nM phorbol 12-myristate, 13-acetate (PMA) inhibited CARB-stimulated IP accumulation by 80%, an effect which was blocked completely by the PKC inhibitor bisindolylmaleimide (2 microM; BIM). However, inhibition of CARB-stimulated IP accumulation (90%) induced by 100 microM DCB was not relieved by BIM. These results suggest that (1) perturbations of Ca2+ homeostasis may underlie DCB effects on IP accumulation, (2) at a time which corresponds to addition of agonists in IP accumulation assays, [Ca2+]i is elevated in cerebellar granule cells exposed to DCB, and (3) activation of PKC is not a mechanism by which DCB inhibits agonist-stimulated IP accumulation.

Animals↗

PubMed2005

Pathways for K+ efflux in isolated surface and crypt colonic cells. Activation by calcium.

K+ -conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring (86)Rb efflux. In crypt cells, basal K+ efflux (rate constant: 0.24 +/- 0.044 min(-1), span: 24 +/- 1.3%) was inhibited by 30 mM TEA and 5 mM Ba2+ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 microM) stimulated K+ efflux, increasing the rate constant to 0.65 +/- 0.007 min(-1) and the span to 83 +/- 3.2%. Ionomycin-induced K+ efflux was inhibited by clotrimazole (IC(50) of 25 +/- 0.4 microM) and charybdotoxin (IC(50) of 65 +/- 5.0 nM) and was insensitive to TEA, Ba2+, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca2+-activated intermediate-conductance K+ channels (IK(ca)). Absence of extracellular Ca2+ did neither affect basal nor ionomycin-induced K+ efflux. However, intracellular Ca2+ depletion totally inhibited the ionomycin-induced K+ efflux, indicating that the activation of these K+ channels mainly depends on intracellular calcium liberation. K+ efflux was stimulated by intracellular Ca(2+) with an EC(50) of 1.1 +/- 0.04 microM. In surface cells, K+ efflux (rate constant: 0.17 +/- 0.027 min(-1); span: 25 +/- 3.4%) was insensitive to TEA and Ba2+. However, ionomycin induced K+ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK(Ca) channels but only crypt cells have TEA- and Ba2+-sensitive conductive pathways, which would determine their participation in colonic K+ secretion.

Animals↗

PubMed1982

Arachidonic acid metabolism in rat pancreatic acinar cells: calcium-mediated stimulation of the lipoxygenase system.

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [14C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromatography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8-10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonate derivative was stimulated by the calcium ionophore ionomycin. Stimulation of the lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF1 alpha, PGF2 alpha, and PGE2 were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.

Animals↗

PubMed1990

Cyclic AMP-dependent and calcium-dependent signals in parathyroid hormone function.

Previous work demonstrated that parathyroid hormone (PTH) activates the Ca2+/protein kinase C (PKC) system in addition to cAMP production. Therefore, the authors explored the role of cAMP-dependent and Ca2(+)-dependent signals in the regulation of osteoblastic growth and bone resorption. In exponentially growing UMR 106-01 osteogenic sarcoma cells, PTH (10(-7) M) inhibited [3H] thymidine incorporation by 80%. This effect was reproduced by maximal doses of both dibutyryl-cAMP (dbcAMP) and forskolin. The Ca2+ ionophore ionomycin (10(-7) M) had no effect, whereas phorbol 12-myristate 13-acetate (PMA) was slightly mitogenic. The antimitogenic action of dbcAMP was dose-dependent, with ED0.5 at about 3 X 10(-5) M. Ionomycin enhanced this dbcAMP effect at submaximal doses of the cAMP analog. PMA used in combination with both dbcAMP and ionomycin induced further depression of cell proliferation, indicating synergism with cAMP. Both dbcAMP (10(-4) M) and ionomycin (10(-7) M) stimulated 45Ca release from fetal rat limb bones after five days in culture, although the Ca2+ ionophore was less potent. 1-Oleoyl 2-acetyl-glycerol (2 X 10(-6) M) was ineffective alone, and slightly inhibited the 45Ca release produced by the other second messenger analogs in all combinations. The combination of dbcAMP and ionomycin showed a synergistic effect, and fully reproduced PTH effect. In conclusion, PTH signal transduction for control of cell proliferation and bone resorption is mediated mainly by cAMP. Activation of the Ca2+/PKC message system is nevertheless necessary to express a full hormonal response in both cell and organ culture systems.

Animals↗

PubMed1992

Activation of bovine endothelial thromboxane receptors triggers release of prostacyclin but not EDRF.

OBJECTIVE: The aim was to examine the capacity of U46619 (a stable thromboxane A2 mimetic) to mediate release of endothelium derived relaxing factor (EDRF) from bovine aortic endothelial cells, and compare the response to the U46619 dependent release of prostacyclin (PGI2). METHODS: Bovine aortic endothelial cells (AG4762) were cultured in vitro on microcarrier beads, which were then loaded onto a column and perfused. The cells were challenged with U46619, bradykinin, or the Ca2+ ionophore ionomycin in the perfusate, and measurements made of the release of 6-oxo-PGF1 alpha (the stable hydrolysis product of PGI2, measured by radioimmunoassay) and EDRF (bioassay). Cells were also cultured on glass cover slips, loaded with Fura 2-AM, and measurements made of the rise in intracellular Ca2+ after challenge with U46619, bradykinin or ionomycin. RESULTS: U46619 triggered release of 6-oxo-PGF1 alpha but not EDRF from AG4762 cells, contrasting with bradykinin which released both 6-oxo-PGF1 alpha and EDRF. Ionomycin had little or no capacity to mimic and trigger release of 6-oxo-PGF1 alpha, although ionomycin mediated large increases in intracellular Ca2+. In contrast, staurosporine (a putative inhibitor of protein kinase C) substantially inhibited the U46619 and bradykinin dependent release of 6-oxo-PGF1 alpha. CONCLUSIONS: In contrast to bradykinin linked receptors on AG4762 endothelial cells, which are coupled to the release of both prostacyclin and EDRF, activation of thromboxane A2 receptors on these cells selectively triggers release of PGI2 but not EDRF. Further, based on the distinct effects of ionomycin and staurosporine, it appears that agonist stimulated PGI2 release from these cells is mediated predominantly by protein kinase C, rather than by rises in intracellular Ca2+. This observation contrasts with previously described mechanisms of PGI2 release from endothelium obtained from other sources.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1994

The regulation of membrane 125I- and 86Rb+ permeability in a virally transformed cell line (NCL-SG3) derived from the human sweat gland epithelium.

We have explored the factors that may regulate membrane permeability in a cell line (NCL-SG3) derived from the human sweat gland epithelium. Ionomycin increased the rate of 125I-efflux from preloaded cells and this action appeared to be due to an increase in intracellular free calcium ([Ca2+]i). The ionomycin-evoked increase in 125I- efflux was reduced in cells that were exposed either to barium or to valinomycin in the presence of a high concentration of external potassium. It thus appears that a fraction of the ionomycin-evoked increase in 125I- efflux is due to the activation of potassium channels and experiments using 86Rb+ also suggested that ionomycin increased the rate of potassium efflux, an effect which was totally abolished by barium. Blockade of Na(+)-K(+)-2Cl- cotransport and of Cl- -HCO3- exchange reduced the basal rate of 125I- efflux and the ionomycin-evoked increase in 125I-efflux from control cells and from cells depolarized by valinomycin. These transport systems thus contribute to anion efflux, although [Ca2+]i-dependent chloride channels also appear to be present. Acetylcholine increases [Ca2+]i in the secretory cells of human sweat glands, but this neurotransmitter did not increase [Ca2+]i in NCL-SG3 cells and so membrane permeability was not under cholinergic control. Adrenaline did not increase [Ca2+]i, but this hormone did evoke cyclic-3',5'-adenosine monophosphate (cyclic AMP) production. However, membrane permeability was not under adrenergic control, as the cells did not appear to express functional, cyclic AMP-dependent anion channels. This may be because they were not fully differentiated under the culture conditions. ATP consistently evoked a dose-dependent increase in anion efflux that appeared to be mediated by [Ca2+]i. The increase in [Ca2+]i was initiated by the release of calcium from a limited internal store and was subsequently sustained by calcium influx. UTP and ADP also increased [Ca2+]i, whereas adenosine, AMP and alpha,beta-methylene ATP were without effect. These data thus suggest that a subclass of type 2 purine receptor, which is functionally coupled to phosphoinositidase C, is present in these cells.

Calcium↗

PubMed1993

Intracellular Ca2+ and PKC activation do not inhibit Na+ and water transport in rat CCD.

Experiments examined the effects of elevation of intracellular calcium concentration ([Ca2+]i) or activation of protein kinase C (PKC) on Na+ and water transport in the rat cortical collecting duct (CCD). We measured the lumen-to-bath 22Na+ flux (J1-->b), transepithelial voltage (VT), and water permeability (Pf) in CCD from deoxycorticosterone (DOC)-treated rats. Ionomycin (0.5 and 1 microM) and thapsigargin (1 and 2 microM) were used to increase [Ca2+]i. Phorbol 12-myristate 13-acetate (PMA; 0.3 and 1 microM) and oleoyl-acetyl-glycerol (OAG; 100 microM) were used as activators of PKC. [Ca2+]i was measured in isolated perfused tubules using the fluorescent dye fura 2. When added to the bathing solution, 220 pM arginine vasopressin (AVP) failed to affect [Ca2+]i, whereas 1 microM ionomycin increased [Ca2+]i by 103 +/- 15% and 2 microM thapsigargin increased [Ca2+]i by 24 +/- 4%. In flux studies, neither ionomycin nor thapsigargin affected J1-->b or Pf, although ionomycin caused marked morphological changes. Ionomycin also failed to alter either parameter in tubules from non-DOC-treated rats. Neither 100 microM OAG nor 1 microM PMA affected J1-->b or Pf. OAG at 50 microM had no effect on VT or transepithelial resistance, indicating no inhibition of conductive Na+ transport. We conclude that increased [Ca2+]i and PKC activation do not affect J1--b or Pf in the rat CCD. These findings may account for the sustained increase in J1--b produced in the rat CCD by AVP.

Animals↗

PubMed1998

Withdrawal of 2-mercaptoethanol induces apoptosis in a B-cell line via Fas upregulation.

Mouse lymphoid cell cultures are dependent on reducing agents in their culture medium to allow proliferation and survival of the cells. In the case of the mouse CD5+-pre-B cell line SPGM-1, withdrawal of 2-mercaptoethanol (2-ME) resulted in rapid inhibition of proliferation and subsequent cell death by apoptosis. The pathways leading to cell death by withdrawal of 2-ME or by incubation with ionomycin, a known inducer of apoptosis, were compared. Both kinds of stimulation resulted in apoptosis of the whole population, but cell death occurred with different kinetics. Only apoptosis induced by ionomycin was inhibited by coincubation with the phorbol ester PMA, while apoptosis induced by withdrawal of 2-ME was not. Overexpression of the human bcl-2 proto-oncogene in these cells delayed the death process induced by either method. SPGM-1xbcl-2 cells accumulated in the G0/G1 and G2/M cell cycle phases after removal of 2-ME from the medium, whereas treatment with ionomycin resulted in an arrest only in the G0/G1 transition. Interestingly, both stimuli induced the expression of the Fas receptor, but with different kinetics, while the Fas ligand (FasL) was expressed constitutively in SPGM-1 cells. These data demonstrate that withdrawal of 2-ME and incubation with ionomycin both induce rapid cell death by apoptosis, possibly mediated by an autocrine Fas/FasL loop. Although the initial pathways activated by the two forms of treatment must be different, they converge on a common level controlled by the anti-apoptotic gene product Bcl-2.

Animals↗

PubMed1998

Activation regimens to prepare bovine oocytes for intracytoplasmic sperm injection.

Activation of bovine oocytes to produce a single haploid pronucleus in preparation for intracytoplasmic sperm injection (ICSI) has been investigated with various combinations of ionomycin and 6-dimethylaminopurine (DMAP). Effects were evaluated by immunocytochemical staining, chromosomal analysis and assessment of development in vitro. Oocytes matured in vitro were exposed to: ionomycin alone (single or repeated treatments, Groups 1 and 2 respectively), ionomycin followed by DMAP (immediately or after a 3-h delay, Groups 3 and 4), or no treatment (control, Group 5). They were then co-cultured in M199 with bovine oviductal epithelial cells. Activation rates were not significantly different among groups but significantly fewer oocytes in Group 3 extruded a second polar body than in Groups 1, 2, and 4. Most parthenotes (60% to 80%) in Groups 1, 2, and 4 were haploid, whereas 82% in Group 3 were mixoploid or polyploid. Most of the parthenotes (88%) in Group 4 formed a single pronucleus besides extruding the second polar body and were therefore more suitable for ICSI than those of Groups 1 and 2 in which condensed chromosomes predominated. The respective rates of oocyte cleavage in Groups 1 to 4 were 24%, 36%, 70%, and 75%; corresponding blastocyst rates were 1%, 5%, 17%, and 8%. There were significantly fewer cells in the parthenotes of Groups 1, 2, and 4 than of Group 3, or of embryos produced by in vitro fertilization. Thus, delaying the addition of DMAP after ionomycin decreases chromosomal abnormalities and produces a high proportion of activated oocytes suitable for ICSI.

Adenine↗

PubMed1991

Down-regulation of protein kinase C activation in human lamina propria T lymphocytes: influence of intestinal mucosa on T cell reactivity.

Human lamina propria T lymphocytes (LPL-T) were shown to have lower proliferative responses to CD3 triggering than autologous peripheral blood T lymphocytes (PBL-T), yet preserved their responsiveness to CD2 stimulation. In order to elucidate the basis of these differences, freshly recovered human LPL-T and autologous PBL-T were stimulated with CD2 monoclonal antibodies anti-T11(2/3) plus sheep red blood cells and phorbol 12,13-dibutyrate (PBu2) plus ionomycin, respectively. LPL-T showed invariably lower responses to PBu2 plus ionomycin than PBL-T. In contrast, LPL-T still preserved proliferation to CD2 activation even when their responses to PBu2 plus ionomycin were decreased almost to background levels. Preincubation of PBL-T with intestinal mucosa supernatant led to a similar reactivity as observed in fresh LPL-T. Moreover, the protein kinase C (PKC) inhibitor sphinganine was able to inhibit DNA synthesis to stimulation with PBu2 plus ionomycin but not to CD2 triggering. This study suggests that CD2-induced proliferation is not dependent on PKC activation and that down-regulation of PKC activation may be one of the mechanisms for inhibition of the CD3-Ti-dependent activation pathway in LPL-T by intestinal mucosa-derived influences in vivo.

Antigens↗

PubMed2006

PKC and cAMP positively modulate alkaline-induced exocytosis in the human mast cell line HMC-1.

We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3',5'-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used Gö6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.

Alkalies↗

PubMed1994

Engagement of the T-cell antigen receptor by anti-CD3 monoclonal antibody causes a rapid increase in lymphocyte F-actin.

Activation of protein kinase C (PKC) causes a rapid and sustained increase in the F-actin of T lymphocytes. Because the phosphatidylinositol pathway and the cytoskeleton play a role in lymphocyte activation, we examined the relationship between signal transduction and the F-actin increase in human blood T cells. Anti-CD3 monoclonal antibodies (mAbs) initiate signals which result in activation of T lymphocytes through the T-cell receptor (TCR), involving the phosphatidylinositol pathway, activation of PKC, and increasing intracellular calcium (Cai2+). The fluorescent probe NBD-phallacidin was used to examine the conformational state of actin following stimulation of T lymphocytes with anti-CD3 mAb. Each of three different murine anti-CD3 mAbs caused rapid increases in lymphocytic F-actin content, which was enhanced by cross-linking with a goat anti-mouse IgG. A maximally effective dose of the mAb Leu 4 caused a rise in cellular F-actin of 1.8-fold at 2 minutes and a three-fold increase in Cai2+. Ionomycin, 100 nM, caused a Cai2+ rise similar in magnitude to that caused by anti-CD3 mAb but had no effect on F-actin content. Inhibitors of PKC, 1(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), sphingosine, and sphinganine lowered the resting cellular F-actin and partially blocked the increase in F-actin caused by either anti-CD3 mAb or ionomycin; however, they had no effect on the rise in Cai2+. Cells leached of Ca2+ with EGTA and ionomycin exhibited no Cai2+ increase in response to anti-CD3 mAb or ionomycin; such cells retained the F-actin increase caused by anti-CD3 mAb. We conclude that stimulation of human T lymphocytes via the TCR causes an early rapid increase in F-actin content. Activation of PKC may play a role but the concomitant Cai2+ increase is neither sufficient nor necessary for the F-actin increase.

Actins↗

PubMed2003

Protein synthesis is required for the transition to Ca(2+)-dependent regulated secretion in progesterone-matured Xenopus oocytes.

Calcium (Ca) ionophores trigger cortical granule exocytosis in progesterone-matured Xenopus oocytes (eggs), but not in immature oocytes. Prior work suggested that this secretory transition involved a Ca-dependent isoform of protein kinase C (PKC). To address this possibility, we treated eggs with several different inhibitors of Ca-dependent PKCs. Although these agents (eg., staurosporine, Ro31-8220) completely blocked cortical granule exocytosis that is triggered in eggs by phorbol esters, they had no impact on ionomycin-evoked secretion of cortical granule lectin. These data suggest that Ca-dependent PKCs do not mediate secretory triggering in eggs. Instead, further investigation revealed that protein synthesis (but not RNA synthesis) was required for eggs to secrete in response to ionomycin. Moreover, we observed that when oocytes were matured by injection of maturation promoting factor (MPF), they failed to secrete in response to ionomycin. Collectively, these results suggest that the progesterone-dependent maturation pathway induces these cells either to synthesize de novo, a protein that mediates Ca-dependent secretory triggering, or that intrinsic Ca-sensing machinery is modified in a protein-synthesis-dependent fashion. Initial efforts to distinguish between these possibilities (using Ca overlay, pharmacological and immunoblot strategies) revealed that such Ca-binding proteins as calmodulin, synaptotagmin1, CAPS, rabphilin-3A and calcineurin were unlikely to transduce the secretory effects of ionomycin in eggs. Thus, the cortical reaction in these cells may rely on a novel mechanism for initiating Ca-dependent exocytosis.

Animals↗