Ca2+-induced changes in energy metabolism and viability of melanoma cells.
(65/1922)
Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. We show here that a rise in intracellular-free calcium ion (Ca2+), induced by Ca2+-ionophore A23187, exerted a deleterious effect on glycolysis and viability of B16 melanoma cells. Ca2+-ionophore caused a dose-dependent detachment of phosphofructokinase (EC 2.7.1.11), one of the key enzymes of glycolysis, from cytoskeleton. It also induced a decrease in the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis. All these changes occurred at lower concentrations of the drug than those required to induce a reduction in viability of melanoma cells. We also found that low concentrations of Ca2+-ionophore induced an increase in adenosine 5'-triphosphate (ATP), which most probably resulted from the increase in mitochondrial-bound hexokinase, which reflects a defence mechanism. This mechanism can no longer operate at high concentrations of the Ca2+-ionophore, which causes a decrease in mitochondrial and cytosolic hexokinase, leading to a drastic fall in ATP and melanoma cell death. The present results suggest that drugs which are capable of inducing accumulation of intracellular-free Ca2+ in melanoma cells would cause a reduction in energy-producing systems, leading to melanoma cell death. (+info)
Kinetics of prostanoid synthesis by macrophages is regulated by arachidonic acid sources.
(66/1922)
The dependence of prostanoid synthesis on the nature of free arachidonic acid (AA) appearance was investigated in mouse peritoneal macrophages. AA delivery from intracellular sources to the constitutive prostaglandin (PG)H synthase was provided by action of calcium-ionophore A23187; and from extracellular sources by AA addition to the culture medium. It was found that the kinetics of prostanoid synthesis dramatically depends on the sources of AA. Free AA concentration used for prostanoid synthesis is either a constant or a variable value depending upon the sources. The kinetics of cellular prostanoid synthesis can be regulated by the following processes: (a) the irreversible inactivation of PGH-synthase in the course of the reaction (kin), (b) prostanoid metabolism (kr), and (c) incorporation of exogenous AA into cellular membranes (ka). From our experiments and mathematical calculation these parameters were found to be kin = 0.20 +/- 0.02 min-1, kr = 0.17 +/- 0.03 min-1 in the case of stimulation with A23187, and kin = 0.0156 min-1, kr = 0. 0134 min-1, ka = 0.0025 min-1 in the case of exogenous AA addition. The studies of prostanoid biosynthesis by macrophage microsomes led to independent determination of kin = 0.20 +/- 0.02 min-1. This value perfectly fits the kinetics of the prostanoid cell synthesis under endogenous AA supply but shows a 10-fold decrease in the case of exogenous AA supply. Our study on the kinetics of prostanoid synthesis by mouse peritoneal macrophages clearly demonstrate that AA is able to regulate cellular prostanoid synthesis in the presence of constitutive PGH-synthase only. A regulation mechanism based on the co-operation of the constitutive PGH-synthase isoform and the availability of free AA is proposed and could be confirmed by mathematical modelling. (+info)
Biosynthesis and turnover of anandamide and other N-acylethanolamines in peritoneal macrophages.
(67/1922)
Polyunsaturated N-acylethanolamines (NAEs), including anandamide (20:4n-6 NAE), elicit a variety of biological effects through cannabinoid receptors, whereas saturated and monounsaturated NAEs are inactive. Arachidonic acid mobilization induced by treatment of intact mouse peritoneal macrophages with Ca2+ ionophore A23187 had no effect on the production of NAE or its precursor N-acylphosphatidylethanolamine (N-acyl PE). Addition of exogenous ethanolamine resulted in enhanced NAE synthesis by its N-acylation with endogenous fatty acids, but this pathway was not selective for arachidonic acid. Incorporation of (18)O from H2 (18)O-containing media into the amide carbonyls of both NAE and N-acyl PE demonstrated a rapid, constitutive turnover of both lipids. (+info)
Characterization of RNA binding proteins associated with CD40 ligand (CD154) mRNA turnover in human T lymphocytes.
(68/1922)
CD154 (CD40 ligand (CD40L)) has been demonstrated to play an essential role in the development of humoral and cellular immunity through its interaction with CD40. While earlier studies have examined the regulation of CD154 expression by transcriptional and posttranslational pathways, scant data exist on its regulation at a posttranscriptional level. In this report we demonstrate that CD154 mRNA is rapidly turned over in primary culture of activated human T lymphocytes. Moreover, we demonstrate that CD154 mRNA is unstable, but can be stabilized by treatment with either phorbol esters or calcium ionophores. To address this lability of CD154 mRNA, we examined the ability of cytoplasmic proteins to bind to its 3' untranslated region (3'UTR). Two major proteins (p25 and p50) capable of binding the 3'UTR of CD154 were identified. The p25 binding activity was associated with polysomes and appeared to correlate with CD154 mRNA instability. Intriguingly, these proteins did not appear to bind to the AU-rich elements present in the 3'UTR of CD154. Rather, their binding was localized to unique sites between nt 471-811 of the 3'UTR, which lack any classical AU-rich elements. These data suggest that these proteins interact with distinct cis-acting elements that are important in the posttranscriptional regulation of CD154 expression. As such, identifying these proteins will help us understand the signals that are necessary for CD154 expression by activated T cells. (+info)
Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions.
(69/1922)
Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca(2+)-calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca(2+)-calmodulin or Ca(2+)-S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation. (+info)
Suppression or induction of apoptosis by opposing pathways downstream from calcium-activated calcineurin.
(70/1922)
Ca(2+)-mobilizing compounds such as the Ca(2+) ionophore A23187 or the endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin can suppress or induce apoptosis in the same cells. The use of different calcineurin inhibitors has shown that both suppression and induction of apoptosis by the Ca(2+)-mobilizing compounds were mediated by calcineurin activation. Ca(2+)-mobilizing compounds activated p38 and p44/42 mitogen-activated protein kinases (MAPKs). Induction of apoptosis by the Ca(2+)-mobilizing compounds was suppressed by an inhibitor of p38 MAPK but not by an inhibitor of p44/42 MAPK. These MAPK inhibitors did not suppress apoptosis induction by wild-type p53 or by withdrawal of IL-6 from IL-6-dependent cells that are mediated by calcineurin-independent pathways. These MAPK inhibitors also did not affect the ability of Ca(2+)-mobilizing compounds to suppress apoptosis. The results indicate that (i) Ca(2+)- mobilizing compounds activate different and opposing pathways that diverge downstream from calcineurin activation that can either suppress or induce apoptosis in the same cells; (ii) p38 MAPK but not p44/42 MAPK is involved in induction of apoptosis but not in its suppression by the Ca(2+)-mobilizing compounds; and (iii) neither p38 nor p44/42 MAPKs mediate induction of apoptosis by some calcineurin-independent pathways. (+info)
Human keratinocytes and tumor-derived cell lines express alternatively spliced forms of transforming growth factor-alpha mRNA, encoding precursors lacking carboxyl-terminal valine residues.
(71/1922)
The human transforming growth factor-alpha (TGF-alpha) gene is thought to contain five introns and six exons, encoding a transmembrane precursor (proTGF-alpha) from which the mature polypeptide is released by proteolytic cleavage. We identified a novel 32-nucleotide exon (exon alpha) within intron 5 and an alternative splice acceptor site in exon 6, splitting exon 6 into two segments: 6A and 6B. Therefore, in addition to wild type (wt) proTGF-alpha mRNA, which skips exon alpha, two novel proTGF-alpha variants are produced: Variant I (VaI), skipping exons alpha and 6A, and Variant II (VaII) which includes exon alpha and skips exon 6A. The only significant difference between variant and wt proTGF-alpha proteins is that the two wt carboxyl-terminal valines are replaced in the variants by five or four other amino acids, respectively. Both variant TGF-alpha mRNAs were readily detected in human keratinocytes and tumor-derived cell lines. Their protein products were cleaved as efficiently as wt TGF-alpha in response to the calcium ionophore A23187. However, both variants (but not wt) reduced serum requirements for proliferation in CHO cells. In addition, VaII-expressing CHO cells (not VaI or wt) formed foci in monolayer cultures. These results suggest that variant TGF-alpha precursors induce autonomous growth. (+info)
Prompt recovery of damaged adrenal medullae induced by salinomycin.
(72/1922)
The morphologic changes in the adrenal medullae of rats treated with an ionophore antibiotic, salinomycin, are described. Male rats of approximately 7 wk of age were treated orally with a single dose of salinomycin at 80 mg/kg body weight. Following this treatment, the adrenal glands were examined, using immunohistochemistry, for neurofilament, laminin, fibronectin, and S-100 protein; the glands were also examined using transmission electron microscopy. One hour after the treatment, a karyopyknosis was observed in the clusters of affected chromaffin cells in which the neurofilament, laminin, and fibronectin were present. The lesions became progressively conspicuous between hours 5 and 10. Ultimately, the outcome was cell lysis. Five hours after salinomycin treatment, unaffected chromaffin cells strongly stained to tyrosine hydroxylase. At 10 hr, new chromaffin cells, which were irregular in shape with electron-dense cytoplasm (dark cell), that were strongly stained for tyrosine hydroxylase appeared at the basement membrane site of the necrotic clusters, and these cells contained very few immature catecholamine granules of less than 80 nm. At 17 hr, the catecholamine granules increased in number and size to about 200 nm. The newly formed chromaffin cells grew within the clusters to fill in the medulla by 24 hr, and cytoplasmic granules progressively increased in number and size. The interstitial tissue was seen to be edematous at 5 hr. New capillaries were found in the adrenal medullae of both control and salinomycin-treated rats. The protruding chromaffin cells (protruding cells), which we previously described in normal rats, were also observed in salinomycin-treated rats, which suggests that holocrine secretion is performed in the adrenal medullae. The results indicated that the rat adrenal medullae have the ability to make a rapid recovery after an insult by salinomycin. (+info)