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PubMed2006

Evidence of calcium-dependent pathway in the regulation of human beta1,3-glucuronosyltransferase-1 (GlcAT-I) gene expression: a key enzyme in proteoglycan synthesis.

The importance of heparan- and chondroitin-sulfate proteoglycans in physiological and pathological processes led to the investigation of the regulation of beta1,3-glucuronosyltransferase I (GlcAT-I), responsible for the completion of glycosaminoglycan-protein linkage tetrasaccharide, a key step prior to polymerization of chondroitin- and heparan-sulfate chains. We have cloned and functionally characterized GlcAT-I 5'-flanking regulatory region. Mutation analysis and electrophoretic mobility shift assays demonstrated the importance of Sp1 motif located at -65/-56 position in promoter activity. Furthermore, we found that elevation of intracellular calcium concentration by the calcium ionophore ionomycin stimulated GlcAT-I gene expression as well as glycosaminoglycan chain synthesis in HeLa cells. Bisanthracycline, an anti-Sp1 compound, inhibited GlcAT-I basal promoter activity and suppressed ionomycin induction, suggesting the importance of Sp1 in calcium induction of GlcAT-I gene expression. Nuclear protein extracts from ionomycin-induced cells exhibited an increased DNA binding of Sp1 factor to the consensus sequence at position -65/-56. Signaling pathway analysis and MEK inhibition studies revealed the important role of p42/p44 MAPK in the stimulation of GlcAT-I promoter activity by ionomycin. The present study identifies, for the first time, GlcAT-I as a target of calcium-dependent signaling pathway and evidences the critical role of Sp1 transcription factor in the activation of GlcAT-I expression.

Binding Sites↗
PubMed2005

Cell shrinkage evoked by Ca2+-free solution in rat alveolar type II cells: Ca2+ regulation of Na+-H+ exchange.

The effects of intracellular Ca2+ concentration, [Ca2+]i, on the volume of rat alveolar type II cells (AT-II cells) were examined. Perfusion with a Ca2+-free solution induced shrinkage of the AT-II cell volume in the absence or presence of amiloride (1 microm, an inhibitor of Na+ channels); however, it did not in the presence of 5-(N-methyl-N-isobutyl)-amiloride (MIA, an inhibitor of Na+-H+ exchange). MIA decreased the volume of AT-II cells. Inhibitors of Cl(-)-HCO3- exchange, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) also decreased the volume of AT-II cells. This indicates that the cell shrinkage induced by a Ca2+-free solution is caused by a decrease in NaCl influx via Na+-H+ exchange and Cl(-)-HCO3- exchange. Addition of ionomycin (1 microm), in contrast, induced cell swelling when AT-II cells were pretreated with quinine and amiloride. This swelling of the AT-II cells is not detected in the presence of MIA. Intracellular pH (pHi) measurements demonstrated that the Ca2+-free solution or MIA decreases pHi, and that ionomycin increases it. Ionomycin stimulated the pHi recovery after an acid loading (NH4+ pulse method), which was not noted in MIA-treated AT-II cells. Ionomycin increased [Ca2+]i in fura-2-loaded AT-II cells. In conclusion, the Na+-H+ exchange activities of AT-II cells, which maintain the volume and pHi, are regulated by [Ca2+]i.

Animals↗
PubMed1997

Expression of NFAT-family proteins in normal human T cells.

NFAT proteins constitute a family of transcription factors involved in mediating signal transduction. Using a panel of specific antisera in immunoprecipitation assays, we found that NFATp (135 kDa) is constitutively expressed in normal human T cells, while synthesis of NFATc (predominant form of 86 kDa) is induced by ionomycin treatment. NFAT4/x was very weakly expressed in unstimulated cells, and its level did not increase upon treatment with activating agents. NFAT3 protein was not observed under any conditions. Higher-molecular-weight species of NFATc (of 110 and 140 kDa) were also detected. In addition, translation of NFATc mRNA apparently initiates at two different AUG codons, giving rise to proteins that differ in size by 36 amino acids. Additional size heterogeneity of both NFATc and NFATp results from phosphorylation. In contrast to ionomycin treatment, exposure of cells to phorbol myristate acetate (PMA) plus anti-CD28 did not induce NFATc, indicating that under these conditions, interleukin-2 synthesis by these cells is apparently independent of NFATc. In DNA binding assays, both PMA plus anti-CD28 and PMA plus ionomycin resulted in nuclear NFAT. Surprisingly, the PMA-ionomycin-induced synthesis of NFATc that was detected by immunoprecipitation was not mirrored in the DNA binding assays: nearly all of the activity was due to NFATp. This is the first study of expression of all family members at the protein level in normal human T cells.

CD28 Antigens↗
PubMed1997

Insulin enhances endothelial alpha2-adrenergic vasorelaxation by a pertussis toxin mechanism.

To investigate whether insulin effect on endothelium is related to a specific signal transduction pathway or reflects a more generalized action of the hormone, we studied in aortic rings of Wistar-Kyoto (WKY) rats the effects of the hormone on endothelium-dependent relaxations generated by acetylcholine, adenosine diphosphate, the selective alpha2-adrenergic agonist UK 14,304, and the calcium ionophore ionomycin. The responses were evaluated both in control conditions and after 30 minutes of exposure to three different levels of insulin (30, 100, and 500 microU/mL). Insulin failed to modify the phenylephrine aortic contractions and the relaxations induced by acetylcholine, adenosine diphosphate, and ionomycin. In contrast, both 100 and 500 microU/mL insulin were able to potentiate the UK 14,304-induced vasorelaxation (+96+/-19% and +91+/-12%, respectively). Pertussis toxin, which causes alpha2-adrenergic receptor Gi uncoupling, reduced the alpha2-adrenergic vasorelaxation and prevented the insulin potentiation of the response to UK 14,304. Furthermore, in primary cultured aortic endothelial cells from WKY, we evaluated the conversion of [3H]arginine to [3H]citrulline in response to acetylcholine, ionomycin, and UK 14,304, both in control conditions and during insulin exposure. Again, insulin did not affect basal citrulline production or the increase induced by acetylcholine and ionomycin, whereas it potentiated the response to UK 14,304. Finally, in aortic rings of spontaneously hypertensive rats, insulin treatment (100 and 500 microU/mL) was unable to enhance the alpha2-adrenergic vasodilator response; in vascular endothelial cells from spontaneously hypertensive rats, insulin did not potentiate the increase in citrulline production evoked by UK 14,304. In conclusion, insulin selectively enhances alpha2-adrenergic endothelial vasorelaxation through a pertussis toxin-sensitive mechanism, by potentiating endothelial nitric oxide production. This vasorelaxant mechanism is altered in spontaneously hypertensive rats.

Adrenergic alpha-Agonists↗
PubMed1995

Cl- secretion by trachea of CFTR (+/-) and (-/-) fetal mouse.

The absence of pathologic changes in newborn cystic fibrosis (CF) lung suggests that the fetal CF lung is inflated with a normal volume of liquid and that Cl- is secreted through paths other than the cystic fibrosis transmembrane conductance regulator (CFTR)-associated Cl- channel. We studied liquid content of distal lung and transepithelial electrical potential difference (PD) of cultured cystic tracheal explants from 16 to 19 day gestation fetal mice of CFTR (+/-)(heterozygous) females that were mated with CFTR (-/-) "knockout" males. Distal lung water content was not affected by fetal genotype. Basal PDs were not different (CFTR (+/-), 8.6 mV, and CFTR (-/-), 9.1 mV), and PDs of both groups were inhibited by intraluminal injection of amiloride (10(-4) M) (-25%) and after addition of bumetanide (10(-4) M) to the bath (-40%). Terbutaline (3 x 10(-5) M) induced a similar increase in PD (about 65%) in both groups. Intraluminal injection of ionomycin (2 x 10(-5) and 5 x 10(-6) M) raised PD in both groups (CFTR (+/-) by 32 and 27% and CFTR (-/-) by 41 and 11%). All of the increase in PD induced by terbutaline and ionomycin was inhibited by bumetanide. The PD response to terbutaline was not attenuated by pretreatment with ionomycin or the Ca2+ chelator BAPTA (10(-4) M). Ionomycin or ATP, but not terbutaline, increased intracellular Ca2+ concentration of isolated cultured tracheal epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Staurosporine mobilizes Ca(2+) from secretory granules by inhibiting protein kinase C in rat submandibular acinar cells.

Staurosporine was previously shown to mobilize Ca(2+) from the thapsigargin-insensitive Ca(2+) store in rat submandibular acinar cells. However, the nature of the store is not yet known. Therefore, in the present study, the staurosporine-releasable intracellular Ca(2+) store was characterized. Staurosporine increased the cytosolic Ca(2+) concentration ([Ca(2+)](c)) after the inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) store was depleted. Ionomycin caused only small increases in [Ca(2+)](c) after the depletion of the IP(3)-sensitive Ca(2+) store, whereas ionomycin+monensin caused large increases. However, ionomycin+monensin did not increase [Ca(2+)](c) when added after [Ca(2+)](c) was increased by staurosporine, indicating that the acidic Ca(2+) store was the main source of Ca(2+). The acidic Ca(2+) store appeared to be associated with secretory granules, since ionomycin+monensin- and staurosporine-induced [Ca(2+)](c) increases were significantly reduced when the acinar cells were degranulated. The effect of staurosporine on [Ca(2+)](c) was mimicked by other protein kinase C inhibitors. Therefore, we conclude that staurosporine mobilizes Ca(2+) from secretory granules, probably through the inhibition of protein kinase C in rat submandibular acinar cells.

Animals↗
PubMed1999

Multiple intracellular pathways for regulation of chloride secretion in cultured pig tracheal submucosal gland cells.

Tracheal submucosal glands are of great relative importance in the secretion of chloride and water to the airway lumen. This study aimed to examine whether the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in cyclic adenosine monophosphate (cAMP) or Ca2+-activated Cl- secretion. Regulation of Cl- secretion in cell cultures derived from pig tracheal submucosal gland acini was investigated by X-ray microanalysis. With or without preincubation with CFTR antisense oligodeoxynucleotide (5 microM). A significant decrease in cellular Cl and K concentration was induced by 5 mM 8-bromo-adenosine 3': 5'-cyclic monophosphate (8-bromo-cAMP), 3 microM calcium ionophore ionomycin, 200 microM 5'-uridine triphosphate (UTP) and 200 microM 5'-adenosine triphosphate (ATP), respectively. The decrease in cellular Cl content was significantly inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB; 50 microm). Preincubation of the cells with CFTR antisense oligodeoxynucleotide significantly inhibited the 8-bromo-cAMP-induced decrease in Cl, whereas CFTR sense oligodeoxynucleotide had no effect. The effects of ionomycin, ATP or UTP were not blocked by either CFTR antisense oligodeoxynucleotide or CFTR sense oligodeoxynueleotide. To measure the cytosolic free calcium concentration ([Ca2+]i) the cells grown on glass coverslips were loaded with fura-2 tetraoxymethylester (fura-2 AM; 5 microM). The [Ca2+]i was measured as the fluorescence ratio of emission (340/380 nm). Ionomycin (3 microM) caused a rapid increase in [Ca2+]i followed by a sustained plateau, but 8-bromo-cAMP had a more complex effect on [Ca2+]i. Exposure to ATP or UTP caused a rapid increase in [Ca2+]i followed by a decrease. In conclusion, cystic adenosine monophosphate and ionomycin induced Cl- secretion through different intracellular pathways. Adenosine triphosphate and uridine triphosphate also induced Cl- secretion probably with Ca as an intracellular messenger. The cystic fibrosis transmembrane conductance regulator is not involved in Cl- secretion activated by extracellular adenosine triphosphate and uridine triphosphate.

Adenosine Triphosphate↗
PubMed1987

The control of mediator release from RBL-2H3 cells: roles for Ca2+, Na+, and protein kinase C1.

Antigen-stimulated rat basophilic leukemia (RBL-2H3) cells release serotonin and other inflammatory mediators by a process that requires Ca2+ influx and increased cytoplasmic Ca2+ levels, and is mimicked by Ca2+ ionophores. We report here that the Ca2+ response to antigen and to ionomycin has two components, a Ca2+ spike and a Ca2+ plateau. In nominally Ca2+-free medium, both components of the Ca2+ response are inhibited and secretion does not occur. In Na+-free medium, the initial Ca2+ spike induced by antigen or ionomycin occurs, but the plateau is again absent and secretion is inhibited by 30 to 50%. Secretion is also reduced by 10(-4) M amiloride, an inhibitor of Na+ transport pathways, and by 10(-5) M concentrations of two amiloride analogs with greater activity than amiloride, respectively, against Na+ channels and Na+/Ca2+ exchange. Phorbol esters, which stimulate protein kinase C, enhance the Ca2+ plateau and secretion caused by suboptimal amounts of both antigen and ionomycin; this enhancement depends on extracellular Na+. The Na+ ionophore, monensin, mimics the Ca2+ plateau. From these data, we infer that the Ca2+ spike and plateau reflect separate responses of RBL-2H3 cells to antigen or ionomycin. We propose that the Ca2+ plateau results at least in part from the activation of a Na+-dependent Ca2+ influx pathway. One possible mechanism is that antigen binding stimulates a protein kinase C-regulated Na+ transport system. The resulting influx of Na+ may activate a Na+/Ca2+ antiporter that supports the Ca2+ plateau and mediator release.

Amiloride↗
PubMed1981

Phosphatidate and oxidized fatty acids are calcium ionophores. Studies employing arsenazo III in liposomes.

Liposomes which have entrapped the metallochromic dye, arsenazo III, constitute a sensitive assay system for ionophoresis of divalent cations. By this means we have compared known calcium ionophores (A23187, ionomycin) with membrane phospholipids, fatty acids, prostanoids, and retinoids. Added at micromolar concentrations to preformed multilamellar liposomes (phosphatidylcholine 7:dicetyl phosphate 2: cholesterol 1) both A23187 and ionomycin, as well as phosphatidic acid and products derived from linoleic acid, linolenic acid, and two eicosatrienoic acids provoked Ca influx (e.g. phosphatidic acid: 0.13 mol of Ca2+/mol of membrane lipid/5 min). A variety of other phospholipids (e.g. phosphatidylinositol), fatty acids (e.g. arachidonic acid), prostanoids (e.g. PGE1) retinoids (e.g. retinoic acid), and glyceryl ether phosphorylcholines ("platelet-activating factors") were without effect. Phosphatidic acid and oxidized fatty acids translocated divalent cations selectively, demonstrating the same rank order as A23187 or ionomycin: Mn greater than Ca greater than Sr much greater than Mg. Membrane lysis did not contribute to the perceived translocation; the liposomes remained impermeable to EDTA, EGTA, arsenazo III, or Mg. Liposomes with phosphatidic acid or oxidized trienoic acids preincorporated at 1-5 mole % of total lipids also permitted translocation of Ca but not Mg. Reduction of ionophoretic fatty acids or ionomycin with stannous chloride abolished their ionophoretic activity. Release of Ca from liposomes which had entrapped arsenazo III-Ca complexes into a medium rich in EGTA permitted calculation of efflux induced by ionophores, whether these were added to the outside of liposomes or preincorporated. Data suggest that phosphatidic acid and oxidized di- and trienoic fatty acids, which act as calcium ionophores in model bilayers, could serve as "endogenous ionophores" in cells.

Arsenazo III↗
PubMed1993

P2-purinergic receptors in human breast tumor cells: coupling of intracellular calcium signaling to anion secretion.

ATP increases intracellular Ca++ ([Ca++]i) by activating different P2-purinoreceptors. Because ATP increases Cl- secretion in cystic fibrosis (CF)-affected epithelia, the current study was designed to establish the link between these two events. Studies were done in epithelial, human MCF-7 breast tumor cells in which the presence of mRNA transcripts encoding CF transmembrane conductance regulator was initially established. Changes in [Ca++]i were measured in single cells by fluorescence microscopy; anion transport was measured by 125I efflux. ATP stimulated concentration-dependent increases in [Ca++]i and 125I efflux from MCF-7 cells. The relative order of agonist potency of various selective P2-purinoreceptor agonists in increasing [Ca++]i and 125I efflux was: UTP > or = ATP > ADP = AMP; 2-chloro-ATP, 2-methylthio-ATP and AMP-phencyclidine were considerably less potent than ATP. The Ca++ ionophore ionomycin increased both intracellular [Ca++]i and 125I secretion. Exposing cells to the intracellular chelator ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetra-acetic acid (EGTA)-acetoxymethylester decreased (AM) decreased ATP- and ionomycin-stimulated 125I efflux. Extracellular EGTA did not alter the Ca++ response to ATP, but inhibited the response to ionomycin. The chelator inhibited both ATP- and ionomycin-induced 125I secretion. Exposure of cells to nifedipine did not affect the responsiveness of MCF-7 cells to ATP. The anion transport antagonist 4,4'-diisothiocyananatostilbene-2,2'-disulfonic acid partially inhibited ATP- and cationophore-stimulated increases in [Ca++]i and 125I secretion. The data suggest that activation of P2 receptors in MCF-7 cells leads to an increase in anion transport as a result of the ability of ATP to increase [Ca++]i; moreover, anion channel antagonists may produce their inhibitory effect on 125I secretion, in part, by blocking agonist-induced intracellular Ca++ signaling.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Suppression of TNF-alpha secretion by azelastine in a rat mast (RBL-2H3) cell line: evidence for differential regulation of TNF-alpha release, transcription, and degranulation.

The mast cell plays a pivotal role in initiating allergic inflammation by secreting several cytokines including TNF-alpha, in addition to granule mediators such as histamine. Anti-allergic drugs including azelastine prevent immediate-type hypersensitivity by inhibiting mast cell degranulation, as well as blocking histamine H1 receptors. However, their effects on cytokine release from mast cells remain unknown. In a rat mast RBL-2H3 cell line, azelastine inhibited Ag- and ionomycin-induced TNF-alpha release with IC50 values of 25.7 +/- 3.4 microM and 1.66 +/- 0.45 microM, respectively. These effects were observed at lower concentrations than needed for the inhibition of degranulation. In Ag-stimulated cells, azelastine also inhibited TNF-alpha mRNA expression, TNF-alpha protein synthesis and release, and, possibly related to these effects, Ca2+ influx. In ionomycin-stimulated cells, however, azelastine inhibited TNF-alpha release to a greater extent than mRNA expression/protein synthesis and Ca2+ influx, suggesting that azelastine inhibits the release process more potently than transcription or production of TNF-alpha by interfering with a signal other than Ca2+. Azelastine added 1 h after ionomycin stimulation also immediately blocked subsequent release of TNF-alpha, which had been produced in the cells, without affecting Ca2+ influx. Pretreatment with 1 microM azelastine inhibited ionomycin-induced, but not Ag-induced, protein kinase C translocation to the membranes. These results suggest that the release process of TNF-alpha in mast cells is regulated by a mechanism distinct from that of degranulation, and that in Ca2+-ionophore-stimulated cells, it is also different from that of transcription/production, and possibly involves protein kinase C activation.

Animals↗
PubMed1998

Selective coupling of mu-calpain activation with the NMDA receptor is independent of translocation and autolysis in primary cortical neurons.

Excessive mu-calpain activation has been linked to several cellular pathologies including excitotoxicity and ischemia. In erythrocytes and other non-central nervous system (CNS) cells, calpain activation is thought to occur following a Ca2+-induced translocation of inactive cytosolic enzyme to membranes and subsequent autolysis. In the present report, we show that transiently exposing primary rat cortical neurons to lethal (50 microM) N-methyl-D-aspartic acid (NMDA) caused protracted calpain activation, measured as increased spectrin hydrolysis, but this was independent of translocation or autolysis of the protease. An anti-mu-calpain antibody showed that calpain was largely membrane associated in cortical neurons, and, consequently, neither translocation nor autolysis of the protease was observed following ionomycin or lethal NMDA treatment. By contrast, in rat erythrocytes, calpain was largely cytosolic and underwent rapid translocation and autolysis in response to ionomycin. Calpain-mediated spectrin hydrolysis was specifically coupled to Ca2+ entry through the NMDA receptor because nonspecific Ca2+ influx via ionomycin or KCl-mediated depolarization failed to activate the enzyme. Thus, calpain appears selectively linked to glutamate receptors in cortical neurons and regulated by mechanisms distinct from that occurring in many non-CNS cells. The data suggest that intracellular signals coupled to the NMDA receptor are responsible for activating calpain already associated with cellular membranes in cortical cells.

Animals↗
PubMed1999

Modulation of Ca2+ mobilization by protein kinase C in rat submandibular acinar cells.

The effects of protein kinase C (PKC) activation and inhibition on the inositol 1,4,5-trisphosphate (IP3) and cytosolic Ca2+ ([Ca2+]i) responses of rat submandibular acinar cells were investigated. IP3 formation in response to acetylcholine (ACh) was not affected by the PKC activator phorbol 12-myristate 13-acetate (PMA), nor by the PKC inhibitor calphostin C (CaC). The ACh-elicited initial increase in [Ca2+]i in the absence of extracellular Ca2+ was not changed by short-term (0.5 min) exposure to PMA, but significantly reduced by long-term (30 min) exposure to PMA, and also by pre-exposure to the PKC inhibitors CaC and chelerythrine chloride (ChC). After ACh stimulation, subsequent exposure to ionomycin caused a significantly (258%) larger [Ca2+]i increase in CaC-treated cells than in control cells. However, pre-exposure to CaC for 30 min did not alter the Ca2+ release induced by ionomycin alone. These results suggest that the reduction of the initial [Ca2+]i increase is due to an inhibition of the Ca2+ release mechanism and not to store shrinkage. The thapsigargin (TG)-induced increase in [Ca2+]i was significantly reduced by short-term (0.5 min), but not by long-term (30 min) exposure to PMA, nor by pre-exposure to ChC or CaC. Subsequent exposure to ionomycin after TG resulted in a significantly (70%) larger [Ca2+]i increase in PMA-treated cells than in control cells, suggesting that activation of PKC slows down the Ca2+ efflux or passive leak seen in the presence of TG. Taken together, these results indicate that inhibition of PKC reduces the IP3-induced Ca2+ release and activation of PKC reduces the Ca2+ efflux seen after inhibition of the endoplasmic Ca2+-ATPase in submandibular acinar cells.

Acetylcholine↗
PubMed1998

Intracellular calcium requirements for beta1 integrin activation.

Human polymorphonuclear leukocytes (PMNs) express beta1 integrins that mediate adhesion to extracellular matrix proteins following stimulation with agonists that induce an increase in intracellular calcium. The purpose of these studies was to determine the contribution made by alterations in intracellular calcium ([Ca++]i) to inside-out activation of beta1 integrins using dimethyl sulfoxide (DMSO)-differentiated granulocytic HL60 cells as a model of human PMNs. Activation of beta1 integrins was determined by measuring the expression of an activation-dependent epitope on the beta1 subunit that is recognized by monoclonal antibody (mAb) 15/7. Exposure of granulocytic HL60 cells to calcium ionophore ionomycin (800 nM) alone did not increase the binding of mAb 15/7 to the cell surface, nor did it increase beta1 integrin-mediated adhesion of the cells to fibronectin. Similarly, exposure of the cells to the direct protein kinase C (PKC) activator, dioctanoylglycerol (di-C8) at 100 microM, neither increased binding of mAb 15/7 to these cells nor adhesion to fibronectin. Simultaneous addition of di-C8 and ionomycin, however, caused a significant increase in the expression of the 15/7 epitope and cell adhesion, suggesting synergy between elevating [Ca++]i and stimulating PKC in beta1 integrin activation. Chelation of [Ca++]i with Quin-2 and EGTA reduced both basal (unstimulated) expression of the 15/7 epitope and basal adhesion of granulocytic HL60 cells to fibronectin. In addition, chelation of [Ca++]i caused a significant decrease in 15/7 binding and adhesion stimulated by low (1 ng/ml) concentrations of phorbol myristate acetate (PMA). The inhibitory effect of [Ca++]i chelation on beta1 integrin activation was reversed by repleting [Ca++]i with ionomycin in a Ca++-containing buffer, or by the addition of higher concentrations of PMA (10 ng/ml). These data suggest a role for [Ca++]i in inside-out activation of beta1 integrins, probably through a synergistic effect with PKC activation.

Antibodies, Monoclonal↗
PubMed2000

Activation of bovine oocytes by specific inhibition of cyclin-dependent kinases.

Activation of bovine oocytes by experimental procedures that closely mimic normal fertilization and allow to obtain haploid oocytes is essential both for intracytoplasmic sperm injection (ICSI) and for nuclear transfer. Therefore, with the goal of producing haploid activated oocytes, this study evaluated whether bohemine, either alone or in combination with ionomycin, is able to activate young matured bovine oocytes. Furthermore, the effect of bohemine on the patterns of DNA synthesis after pronuclear formation as well as changes in histone H1 kinase and MAP kinase activities during the process of activation were studied. Our results with bohemine show that the specific inhibition of CDKs in metaphase II bovine oocytes induces parthenogenetic activation in a dose-dependent manner (25, 50, and 100 microM, respectively), either alone (3%, 30%, and 50%) or in combination with ionomycin (30%, 70%, and 87.5%). A single pronucleus and extrusion of the second polar body was observed (97%) when Ca(2+) influx was stimulated in the presence of bohemine, although pronuclear formation without polar body extrusion was observed when bohemine was used alone. Bohemine-activated oocytes started to synthesize DNA in the first hour (37%) after their removal from bohemine-supplemented medium (6-7 hr post-activation; hpa). A high synchrony in the S-phase was registered with more than 85% of parthenotes actively synthesizing DNA 8 hpa. By contrast, DNA synthesis was absent in oocytes cultured for 4, 6, and 8 hpa in the presence of bohemine and a low rate was observed by those cultured for 18 hr (30%) in bohemine-supplemented medium. This confirms the ability of the inhibitor to arrest the cell cycle in the G1/S boundary for at least 8 hr. A drop in histone H1 kinase activity was observed in bohemine-activated oocytes. The activity of MBP kinase decreased later than histone H1 kinase and even 4 hr after inomycin-bohemine treatment at least half of this activity was still detectable. Then, the MBP kinase activity decreased and the lowest level could be seen 6-8 hpa. In summary, our study shows that in vitro matured bovine oocytes can be successfully activated by a synthetic inhibitor of CDKs. This effect can be improved by combination with ionomycin. The targeting of CDKs in the way to activate bovine oocytes can be an approach to improve the efficiency of mammalian oocyte activation.

Animals↗
PubMedDate not supplied

Effects of gestational age on prostaglandin production and its regulation in human myometrial cells.

The objective of this study was to evaluate the effects of known stimulants of prostaglandin production on cultured myometrial cells from women who are at various gestational ages. To evaluate this, myometrial segments were obtained from 12 patients at 27-40 weeks gestation at cesarean delivery. Myometrial cells were then isolated and grown to confluence in culture. Incubations were conducted in quadruplicate for 16 hours with interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA), or ionomycin. Prostaglandins E2, F2 alpha, and 6-keto F1 alpha (the stable metabolite of prostacyclin) were measured by radioimmunoassay and cellular protein determined. The results revealed that cultured myometrial cells produced prostaglandins in response to IL-1 beta, TNF-alpha, EGF, PMA, and ionomycin at all gestational ages tested at a significantly increased rate (P < 0.05 vs controls at and above 10 ng/ml of IL-1 beta, TNF alpha, and EGF; 1 microM for PMA, and 5 microM for ionomycin). The major prostaglandin produced in response to each stimulant was 6-keto-PGF1 alpha. Cultured human myometrial cells respond to known stimulants of prostaglandin production throughout the third trimester of pregnancy in the absence of labor. The myometrium may therefore be a source of prostaglandins that influences uterine activity.

6-Ketoprostaglandin F1 alpha↗
PubMed1996

Differential induction of helper and killer T cells from isolated CD4+CD8+ thymocytes in suspension culture.

Thymocytes of T cell receptor transgenic mice with nonselecting and RAG-2 -/- backgrounds were developmentally arrested at the CD4+CD8+ stage before positive selection. These thymocytes underwent lineage commitment upon transient stimulation with a combination of ionomycin, a calcium ionophore, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, in suspension culture. The effective drug doses were limited within narrow ranges and much lower than those which induce proliferation of mature T cells. The doses corresponded to those which inhibit glucocorticoid-induced apoptosis in these thymocytes. CD4 lineage commitment required longer duration, higher intensity of the stimulation, or both, than CD8 lineage commitment. Functional helper T cells (Th1 and Th2) were induced from the CD4 lineage-committed cells upon secondary stimulation with a combination of ionomycin and PMA followed by lymphokine treatment. Cytotoxic T cells were induced from the CD8 lineage-committed cells upon incubation with concanavalin A and irradiated splenic dendritic cells, but not with the combination of ionomycin and PMA. These results indicate that positive selection is mimicked by the pharmacological stimulation in the absence of other cell types, but that final maturation of CD8 T cells may require a different signal.

Animals↗
PubMed2006

Lipopolysaccharide sensitizes microglia toward Ca(2+)-induced cell death: mode of cell death shifts from apoptosis to necrosis.

Little is known about the effect of microglial activation on cell death. This study examines the effects of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), triggers of microglial activation, on cell death induced by several agents in cultured rat microglia. For comparison, the effect of LPS on cell death is also examined in cultured astrocytes. LPS or IFN-gamma enhanced cell death induced by thapsigargin or ionomycin, an agent that increases intracellular Ca2+ concentration, although LPS or IFN-gamma alone did not affect cell viability. Thapsigargin or ionomycin induced apoptosis in LPS-untreated microglia, while they induced necrosis in LPS-treated microglia, which were partially reversed by O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM, an intracellular Ca2+ chelator). In contrast, LPS treatment did not affect tunicamycin- or staurosporine-induced apoptosis, while it inhibited S-nitroso-N-acetylpenicillamine-induced apoptosis. The effect of LPS on thapsigargin or ionomycin-induced apoptosis was not observed in astrocytes. These results indicate that microglial activation sensitizes the cells toward cell death induced by the change in intracellular Ca2+ concentration and shifts the mode of cell death from apoptosis to necrosis.

Animals↗