Inhibitor Sphingosine Research Articles

Evaluation of potential drugs against leishmaniasis targeting catalytic subunit of Leishmania donovani nuclear DNA primase using ligand based virtual screening, docking and molecular dynamics approaches

Leishmania donovani, causes leishmaniasis, a global health trouble with around 89 different countries and its population under its risk. Replication initiation events have been instrumental in regulating the DNA duplication and as the small subunit of L. donovani nuclear DNA primase (Ld-PriS) inherits the catalytic site, it plays a vital role in DNA replication. In this study we have aimed Ld-PriS for the first time as a prospective target for the application of drug against Leishmania parasite. 3-D structures of Ld-PriS were built and ligand-based virtual screening was performed using hybrid similarity recognition techniques. Ligands from the ZINC database were used for the screening purposes based on known DNA primase inhibitor Sphingosine as a query. Top 150 ligands were taken into consideration for molecular docking against the query protein (Ld-PriS) using PyRx and iGEMDOCK softwares. Top five compounds with the best docking score were selected for pharmacokinetic investigation and molecular dynamic simulation. These top five screened inhibitors showed very poor binding affinity toward the catalytic subunit of human primase indicating their safety toward the host normal replication mechanism. The top five compounds showed good pharmacokinetic profiles and ADMET predictions revealed good absorption, solubility, permeability, uniform distribution, proper metabolism, minimal toxicity and good bioavailability. Simulation studies upto 50 ns revealed the three leads ZINC000009219046, ZINC000025998119 and ZINC000004677901 bind with Ld-PriS throughout the simulation and there were no huge variations in their backbone suggesting that these three may play as potential lead compounds for developing new drug against leishmaniasis. Communicated by Ramaswamy H. Sarma

A novel role for sphingolipid metabolism in oxidant-mediated skeletal muscle fatigue. Focus on “Sphingomyelinase stimulates oxidant signaling to weaken skeletal muscle and promote fatigue”

sphingolipids are a chemically diverse group of biological lipids typified by a sphingoid base backbone derived from condensation of a fatty acyl-CoA to an amino acid (typically, but not always, serine) ([24][1], [27][2]). These lipids occur throughout eukaryotic species, thus reflecting their

The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens α-toxin

Clostridium perfringens alpha-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. alpha-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and 1H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.

Activation of Mitogen-Activated Protein Kinase by Hepatocyte Growth Factor Is Stimulated by Both α1- and β2-Adrenergic Agonists in Primary Cultures of Adult Rat Hepatocytes

We investigated the effects of α1- and β2-adrenergic agonists on hepatocyte growth factor (HGF)-stimulated mitogen-activated protein kinase (MAPK) isoforms in primary cultures of adult rat hepatocytes. Hepatocytes were isolated and cultured with HGF (5 ng/ml) and/or α- and β-adrenergic agonists. Phosphorylated MAPK isoforms (p42 and p44 MAPK) were detected by Western blotting analysis using anti-phospho-MAPK antibody. The results show that HGF increased phosphorylation of p42 MAPK by 2.2-fold within 3 min. The HGF-induced MAPK activation was abolished by AG1478 treatment (10−7 M). The MEK (MAPK kinase) inhibitor 10−M) completely inhibited the HGF-dependent increase in MAPK activity. Phenylephrine (10−6 M alone had no effect in the absence of HGF, but significantly increased p42 MAPK induction by HGF. Moreover, the cell-permeable cAMP analog, 8-bromo cAMP (10−7 M), and phorbol 12-myristate 13 acetate (10−7 M) potentiated HGFinduced MAPK phosphorylation. The effects of these analogs were antagonized by the protein kinase A (PKA) inhibitor H-89 (10−7 M) and the protein kinase C (PKC) inhibitor sphingosine (10−6 M), respectively. These results suggest that direct or indirect activation of both PKA and PKC represent a positive regulatory mechanism for stimulating MAPK induction by HGF.

Modulation by Sphingosine of Phosphorylation of Substrate Proteins by Protein Kinase C in Nuclei from Cow Mammary Gland

Protein kinase C (PKC) is an enzyme activated by diacylglycerols such as 1-oleoyl-2-acetyl-sn-glycerol (OAG), phospholipids (in particular phosphatidylserine; PS) and Ca2+, which regulate a wide variety of intracellular functions by phosphorylating multiple substrate proteins and enzymes. The effect of sphingosine, the backbone moiety of sphingolipids, on PKC activity and phosphorylation of endogenous proteins catalyzed by PKC was investigated in nuclei of cow mammary gland. Sphingosine inhibited nuclear PKC activity when lysine-rich histone was used as the substrate. The sphingosine inhibition of the PKC activity was reversed by the excess addition of PS, but not by OAG or Ca2+. Several nuclear proteins, including 56-kDa, 43-kDa, 38-kDa and 36-kDa proteins, were shown to be substrates for PKC. Of the substrate proteins, the 38-kDa and 36-kDa proteins were identified as annexin I, the Ca2+/phospholipid-binding protein; the 56-kDa and 43-kDa proteins have not yet been identified. Sphingosine inhibited phosphorylation of the 56-kDa protein and the 36-kDa annexin I, whereas it enhanced that of the 43-kDa protein. The 38-kDa annexin I species was unaffected by sphingosine. As with the PKC activity, inhibition by sphingosine of phosphorylation of the 56-kDa protein and 36-kDa annexin I was reversed by the excess addition of PS, but not by OAG or Ca2+. In addition, by the excess addition of PS and not by OAG or Ca2+, the sphingosine-enhanced phosphorylation of the 43-kDa protein was reversed and returned to near the level in the absence of sphingosine. It is suggested that sphingosine is involved in the regulation of PKC-dependent phosphorylation in the nucleus by modulating the association of PKC or its substrates, particularly annexin I, with membrane phospholipids in cow mammary gland.

Effects of endotoxin exposure on cationic amino acid transporter function in ovine peripheral blood mononuclear cells.

Rodent models of sepsis differ from clinical human disease in that humans make substantially less whole-body nitric oxide and have different cellular responses to endotoxin. Sheep, when exposed to endotoxin, behave in a manner more similar to humans. Many studies of rodent peripheral blood mononuclear cells (PBMCs) exposed to endotoxin demonstrate increased cationic amino acid transporter function (particularly through the y+ transporter) to supply arginine substrate to upregulated nitric oxide synthase. Whether this is true in sheep is not known. We have studied cationic amino acid transport in sheep PBMCs stimulated with endotoxin, using labelled lysine. PBMCs stimulated both in vitro and in vivo show an initial reduction in total and y+ lysine transport (after 1-2 h exposure to endotoxin): a previously undescribed effect of endotoxin. In in vitro activated cells, the reduction in y+ transport was prevented by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), and the phospholipase inhibitor 4-bromophenacyl bromide (4-BPAB), but not cyclohexamide or a number of other inhibitors of intracellular second-messenger pathways. In contrast after 14 h incubation, the expected increase in total and y+ lysine transport was seen. The increase in y+ transport could be prevented by cyclohexamide, dexamethasone, ibuprofen, the protein kinase C inhibitor sphingosine, NDGA and 4-BPAB. These results suggest that in response to endotoxin exposure there is an initial decrease in y+ activity mediated by a lipoxygenase product, followed by a substantial increase in y+ activity mediated by the products of either cyclo-oxygenase or lipoxygenase. Cyclo-oxygenase and/or lipoxygenase inhibition might be useful in reducing arginine transport, and hence nitric oxide production, in these cells.

Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms.

Serotonin (5-HT), a major neurotransmitter, has a large number of G protein-coupled receptors in mammals. On activation by exposure to their ligand, 5-HT(2) receptor subtypes increase IP(3) levels and undergo desensitization and internalization. To visualize the receptor in cells during these processes, we have constructed a 5-HT(2A)-enhanced GFP (SR2-GFP) fusion receptor. We show that this fusion receptor undergoes internalization on exposure to its natural ligand, 5-HT. Because 5-HT(2A) receptors activate the phospholipase C pathway, we studied the effect of protein kinase C (PKC) on the internalization process and found that activation of PKC by its specific activator phorbol 12-myristate 13-acetate, in the absence of 5-HT, leads to internalization of the receptor. Moreover, inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, suggesting that activation of PKC is sufficient and necessary for the internalization of 5-HT(2A) receptors. We also show that SR2-GFP recycles back to the plasma membrane after 5-HT-dependent internalization, suggesting a mechanism for resensitization. In addition, receptors that have been internalized on addition of phorbol 12-myristate 13-acetate in the absence of 5-HT also recycle to the surface, with a time course similar to that seen after activation of the receptors by 5-HT. Our study suggests that 5-HT(2A) receptors internalize and return to the surface after both serotonin- and PKC-mediated processes. This study reveals a role for PKC in receptor internalization and also shows that 5-HT(2A) receptors are recycled.

Prolactin signaling in porcine adrenocortical cells: involvement of protein kinases

Prolactin (PRL) was found to have a stimulatory effect on adrenal steroidogenesis in vivo and in vitro in several species including pigs. PRL signal transduction pathways, however, in adrenocortical cells are poorly recognized. Therefore, the goal of this paper is to ascertain the involvement of protein kinase C (PKC) and tyrosine kinases in PRL signaling in porcine adrenal cortex. Adrenals were harvested from locally slaughtered mature gilts. Cortical cells were dispersed by sequential treatment with collagenase. The cells were seeded into 24-well culture plates at a density of 3×10 5/mL. Cells were incubated with or without PRL (500 ng/mL), ACTH (5 nM—a positive control), tyrosine kinase inhibitor—genistein (1; 2.5 or 5 μM), PKC inhibitor—sphingosine (20–1000 nM) and PKC activators—diacylglycerol (DiC8; 10–100 μM) and phorbol ester (PMA; 1–1000 nM). All incubations were performed for 8 h (95% air and 5% CO 2, 37°C). PRL and ACTH ( P<0.05) increased cortisol and androstenedione (A 4) secretion. DiC8 and PMA mimicked the stimulatory effect of PRL. Sphingosine ( P<0.05) suppressed basal and PRL-stimulated steroid secretion. Genistein inhibited ( P<0.05) PRL-stimulated cortisol secretion and enhanced ( P<0.05) basal and PRL-stimulated A 4 secretion. Moreover, PKC activation was assessed by measuring the specific association of [ 3 H] phorbol dibutyrate ( [ 3 H] PDBu) with adrenocortical cells after treatment with PRL or ionomycin (a positive control). PRL (within 2–3 min) and ionomycin (within 2–5 min) increased ( P<0.05) specific binding of [ 3 H] PDBu to the porcine adrenocortical cells. In addition, PRL did not augment the cortisol and A 4 secretion by PKC-deficient adrenocortical cells. In conclusion, presented results support the hypothesis that PKC and tyrosine kinases are involved in PRL signaling in adrenocortical cells in pigs. Moreover, activation of PKC is associated with the increased secretion of cortisol and A 4.

Regulation of phospholipase A 2 activity in cockroach ( Periplaneta americana) fat body by hypertrehalosemic hormone: evidence for the participation of protein kinase C

Phospholipase A 2 (PLA 2) associated with the membrane fraction of trophocytes from Periplaneta americana fat body increases by as much as 100% when the cells are incubated with hypertrehalosemic hormone (HTH-II). Activation with HTH-II is approximately halved by inclusion of the PKC inhibitor sphingosine in the incubation medium. Because activation of PLA 2 by HTH-II is blocked by the GDP analogue GDP-β-S, and the unactivated enzyme is activated by the GTP analogue GTP-γ-S it is likely that a G protein is involved in activation of the enzyme. Activation of PLA 2 was also achieved by treating the trophocytes with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol in the presence of thapsigargin. This supports the view that protein kinase C is also involved in the activation process.

Protein kinase C-induced phosphorylation modulates the Na +-ATPase activity from proximal tubules

This study describes the modulation of the ouabain-insensitive Na +-ATPase activity from renal proximal tubule basolateral membranes (BLM) by protein kinase C (PKC). Two PKC isoforms were identified in BLM, one of 75 kDa and the other of 135 kDa. The former correlates with the PKC isoforms described in the literature but the latter seems to be a novel isoform, not yet identified. Both PKC isoforms of BLM are functional since a protein kinase C activator, TPA, increased the total hydroxylamine-resistant 32P i incorporation from [γ- 32P]ATP into the BLM. In parallel, TPA stimulated the Na +-ATPase activity from BLM in a dose-dependent manner, the effect being reversed by the PKC inhibitor sphingosine. The stimulatory effect of TPA on Na +-ATPase involved an increase in the V max (from 13.4±0.6 nmol P i mg −1 min −1 to 25.2±1.4 nmol P i mg −1 min −1, in the presence of TPA, P<0.05) but did not change the apparent affinity for Na + ( K 0.5=14.5±2.1 mM in control and 10.0±2.1 mM in the presence of TPA, P>0.07). PKC involvement was further confirmed by stimulation of the Na +-ATPase activity by the catalytic subunit of PKC (PKC-M). Finally, the phosphorylation of an approx. 100 kDa protein in the BLM (the suggested molecular mass of Na +-ATPase [1]) was induced by TPA. Taken together, these findings indicate that PKCs resident in BLM stimulate Na +-ATPase activity which could represent an important mechanism of regulation of proximal tubule Na + reabsorption.

Nicotinic receptor-mediated activation by the tobacco-specific nitrosamine NNK of a Raf-1/MAP kinase pathway, resulting in phosphorylation of c-myc in human small cell lung carcinoma cells and pulmonary neuroendocrine cells.

Small cell lung carcinoma (SCLC) expresses phenotypic features of pulmonary neuroendocrine cells and demonstrates a strong etiologic association with smoking. SCLC cell lines express a Raf-1-dependent mitogenic signal transduction pathway, which is thought to transduce the mitogenic signals initiated by neuropeptide autocrine growth factors. Recent studies have identified the tobacco-specific carcinogenic nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as a site-selective high-affinity agonist for the alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), which regulates the growth of a significant subset of SCLC in vitro by stimulating the release of the autocrine growth factor serotonin. The purpose of this study was to identify signaling events initiated by binding of NNK to the alpha7 nAChR. We have used a human SCLC cell line and fetal hamster pulmonary neuroendocrine cells with in vitro kinase activation assays and western blots to assess the levels of expression and activation of Raf-1, MAPK and c-myc to address this issue. Our data show that NNK activates the Raf-1, MAP kinase pathway, resulting in phosphorylation of c-myc. The activation of this signal transduction pathway by NNK was inhibited by the site-selective antagonist for the alpha7 nAChR alpha-bungarotoxin (alpha-BTX) or by the serotonin reuptake inhibitor imipramine, suggesting that the responses to NNK were mediated by nicotinic receptor-initiated release of serotonin. Accordingly, NNK-induced 5-HT release was blocked by alpha-BTX while NNK-induced DNA synthesis was inhibited by alpha-BTX, imipramine, the PKC inhibitor sphingosine or the MEK inhibitor PD98059. SCLC cells demonstrated high basal levels of 5-HT release, DNA synthesis, and over-expressed Raf-1 and MAPK protein suggesting the constitutive activation of an upstream regulator such as the alpha7 nAChR. Our findings link, for the first time, the stimulation of a nicotinic acetylcholine receptor by a cancer-causing agent with the activation of a Raf-1/MAPK/c-myc signaling pathway. Furthermore, our data suggest that serotonin uptake inhibitors may protect against the development or be useful in the clinical management of SCLC.

Chlorpromazine induces apoptosis in activated human lymphoblasts: a mechanism supporting the induction of drug-induced lupus erythematosus?

Drug-induced lupus erythematosus is a serious side effect of certain medications, such as procainamide, quinidine, hydralazine, chlorpromazine, and isoniazid, the underlying pathogenesis of which is unresolved. In this study, we examined the influence of these drugs on the regulation of apoptosis, or programmed cell death, in quiescent and activated human lymphocytes. We also discuss the dysregulation of apoptosis as a pathogenetic factor in systemic lupus erythematosus. Peripheral blood mononuclear cells or activated lymphoblasts from normal donors were incubated with different concentrations of each of the above-mentioned drugs. We did not find induction of apoptosis in quiescent cells over a broad concentration range. In contrast, lymphoblasts readily underwent apoptosis when cultured with chlorpromazine, but not any of the other drugs, after stimulation with interleukin-2 (IL-2) in a dose-, time- and cell cycle-dependent manner. By several lines of evidence, toxicity was ruled out. Characteristic features of apoptosis-like incorporation of propidium iodide (PI), such as increased annexin V binding, changes in mitochondrial membrane potential, and induction of DNA breaks (as evidenced by TUNEL techniques), could be induced in lymphoblasts after chlorpromazine treatment. Chlorpromazine did not cause apoptosis by inhibition of cytokine binding or blockade of early intracellular signaling. The protease inhibitor Z-VAD and the ceramide inhibitor sphingosine 1-phosphate effectively blocked chlorpromazine-induced apoptosis (by PI staining and by externalization of phosphatidylserine), in contrast to the caspase 3/CPP32 inhibitor DEVD, which had only minor effects. Western blot analysis revealed IL-2-mediated phosphorylation of extracellular signal-regulated kinase, which was sensitive to chlorpromazine. Using lymphoblasts from a patient with Canale-Smith syndrome, we found that chlorpromazine-mediated apoptosis is Fas/ APO-1 independent. These data suggest that chlorpromazine mediates apoptosis in human lymphoblasts through specific activation of intracellular proapoptotic signaling cascades. This mechanism might lead to an unsynchronized inflow of apoptotic break-down products and thereby to the induction of (auto)immunity against nuclear components.

Hydrogen Peroxide Activation of Ca2+-Independent Phospholipase A2 in Uterine Stromal Cells

In rat uterine stromal cells (UIII cells), an oxidative stress induced by H2O2 caused a dose-dependent release of arachidonic acid (AA) that was independent of intracellular Ca2+ concentration and was not inhibited by Ca2+-dependent phospholipase A2 (cPLA2) inhibitors, nor by protein kinase C (PKC) inhibitors or by PKC down-regulation. H2O2 treatment did not impair AA esterification but significantly increased Ca2+-independent PLA2 (iPLA2) activity. Since iPLA2 specific inhibitor bromoenollactone almost completely suppressed the release of AA induced by H2O2, we conclude that iPLA2 activity represents the major mechanism by which H2O2 increases the availability of non-esterified AA in UIII cells. Moreover, PKC inhibitors sphingosine and calphostin C markedly potentiated the release of AA trigger by H2O2, suggesting a regulatory mechanism of iPLA2 by PKC that remains to be clarified.

Sphingosine inhibits nitric oxide synthase from cerebellar granule cells differentiated in vitro

The effects of different bioactive sphingoid molecules on NOS activity of differentiated cerebellar granule cells were investigated by measuring the conversion of [ 3H]arginine to [ 3H]citrulline. Cytosolic Ca 2+-dependent NOS activity was strongly inhibited in a dose-dependent manner by sphingosine in concentrations of 1–40 μM. This inhibition seems to be peculiar to sphingosine in that ceramide, N-acetylsphingosine, sphingosine-1P, sphinganine and tetradecylamine have no effect on the cytosolic enzyme at the considered concentrations, suggesting that it is the bulk of the sphingosine hydrophilic portion that is critical for cytosolic NOS inhibition. This inhibition of cytosolic NOS is not reversed by increasing the arginine concentration, so a competitive mechanism can be excluded. Instead, increasing the concentrations of calmodulin led to loss of sphingosine inhibition, suggesting that sphingosine interferes with the calmodulin-dependent activation of the enzyme by a competitive mechanism. Sphingosine and related compounds had no effect on the particulate Ca 2+-independent NOS activity. The data obtained suggest that sphingosine could be involved in the regulation of NO production in neurons.

Influence of probes for calcium–calmodulin and protein kinase C signalling on the plasma membrane Ca2+–ATPase activity of rat synaptosomes and leukocyte membranes

The influence of selected inhibitors of calcium signalling on the plasma membrane Ca2+-ATPase activity of rat synaptosomes and peritoneal leukocyte membranes was studied. The calmodulin inhibitor calmidazolium was an efficient inhibitor (50%) of the synaptosomal Ca2+-ATPase activity in a manner competitive with phosphatidylserine. The inhibition by CGS 9343B (30%) was not counteracted by phosphatidylserine. The intracellular calcium antagonist TMB-8 and the protein kinase inhibitor staurosporine and the derivatives CGP 41251 and CGP 42700 hardly affected the synaptosomal Ca2+-ATPase activity. The flavonoid quercetin was a more effective inhibitor of the ATPase activity of synaptosomal than of leukocyte membranes. Phloretin, at relatively high concentrations, caused only a modest inhibition of synaptosomes. The protein kinase C inhibitor sphingosine was a weak inhibitor of the synaptosomal but an effective inhibitor of the leukocyte membrane Ca2+-ATPase activity. The antineoplastic ether phospholipids BM 41.440 (ilmofosine) and ET-18-OCH3 (edelfosine) effectively inhibited the leukocyte membranes whereas the ATPase activity of synaptosomes was significantly increased by 20 μM and slightly inhibited by higher concentrations of these agents. The analogue hexadecylphosphocholine (miltefosine) did not affect the ATPase activity of the synaptosomes and only inhibited that of the leukocyte membranes at concentrations above 20 μM. These results show that several test substances of current interest affect the activity of the plasma membrane Ca2+-ATPase. The effects depend on the origin of the membranes. The investigation does not permit a distinction between direct effects on the enzyme and an interference with its membrane environment although the latter is indicated for the ether phospholipids.

Bradykinin-evoked Ca 2+ mobilization in Madin Darby canine kidney cells

We studied the mechanisms underlying the bradykinin-evoked changes in intracellular calcium concentration ([Ca 2+] i) in Madin Darby canine kidney (MDCK) cells. Bradykinin evoked a [Ca 2+] i transient in a dose-dependent manner, measured by fura-2 fluorimetry and digital video imaging. The transient consisted of a rise and a decay and [Ca 2+] i returned to baseline without oscillations. External Ca 2+ influx occurred, as demonstrated by Mn 2+ quench and external Ca 2+ removal measurements. Bradykinin acted by stimulating bradykinin B 2 receptors as evidenced by blockade by d-arginyl- l-arginlyl- l-prolyl- trans-4-hydroxy- l-prolylglycyl-3-(2-thienyl)- l-alanyl- l-seryl- d-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl- l-(2α,3β,7aβ)-octahydro-1 H-indole-2-carbonyl- l-arginine (HOE 140) but not by d-arginyl- l-arginlyl- l-prolyl- trans-4-hydroxy- l-prolylglycyl-3-(2-thienyl)- l-alanyl- l-seryl- d-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl- l-(2α,3β,7aβ)-octahydro-1 H-indole-2-carbonyl ([Des–Arg]HOE 140). The [Ca 2+] i signal was abolished by 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dione (U73122) and partially inhibited by neomycin, implying mediation by phospholipase C. The transient was initiated by a release of Ca 2+ from internal stores since it was abolished by pretreatment with thapsigargin or cyclopiazonic acid. The mobilization of the internal Ca 2+ store subsequently triggered a 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1 H-imidazole hydrochloride (SKF 96365)-insensitive Ca 2+ entry. Pretreatment with carbonylcyanide m-chlorophynylhydrozone and gly-phe-β-naphthylamide did not alter the transient, thus excluding the participation of mitochondria and lysosomes. Efflux via Ca 2+ pumps contributed to the decay of the transient. Efflux via Na +/Ca 2+ exchange or sequestration by mitochondria and lysosomes was insignificant. The transient was blunted by the protein kinase C activator phorbol 12-myristate 13-acetate, and was enhanced by the protein kinase C inhibitors sphingosine and chelerythrine, the protein kinase A inhibitor 2,5-di-( t-butyl)-1,4-hydroquinone, N-[2-( p-bromocinnamylamino)ethyl]5-isoquinolinesulfonamide (H-89), the agent 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), and agents that elevated levels of 3′,5′-cyclic guanosine monophosphate. The transient did not heterologously desensitize with that evoked by ATP, ADP or UTP.

The naturally occurring PKC inhibitor sphingosine and tumor promoter phorbol ester potentially induce tyrosine phosphorylation/activation of oncogenic proline-directed protein kinase FA/GSK-3alpha in a common signalling pathway.

When serum-starved A431 cells were treated with 200 nM phorbol ester TPA for 15 min, the cellular activity of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) could be decreased to approximately 25% of control. Conversely, when treated with 1 microM TPA for 24 hr, the activity could be reversibly increased to approximately 200% of Control. The naturally occurring protein kinase C (PKC) inhibitor sphingosine at a concentration of 27 microM could also induce activation of kinase FA/GSK-3alpha to approximately 200% of control within 60 min. Further, when cells were chronically treated with 1 microM TPA for 24 hr and then with 27 microM sphingosine for 60 min, the activity of kinase FA/GSK-3alpha could only be increased to approximately 200% of control. Furthermore, when cells were pretreated with sphingosine and then acutely treated with TPA, the acute TPA effect on kinase FA/GSK-3alpha activity could be abolished by genistein or tyrosine phosphorylation, which could be blocked by genistein or tyrosine phosphatase, but could be reversed by orthovanadate. Taken together, the results demonstrate that TPA/sphingosine induce tyrosine phosphorylation and concurrent activation of kinase FA/GSK-3alpha in a common signalling pathway. Since TPA and sphingosine are potent PKC modulators, the results further suggest a potential role of PKC in modulating tyrosine phosphorylation/activation of kinase FA/GSK-3alpha. Kinetic studies on seven subtypes of PKC further demonstrate a specific involvement of PKCE in this tyrosine phosphorylation/activation process. This provides a new mode of signal transduction between these two important serine/threonine kinases in cells.

Involvement of RB kinases and phosphatases in life and death decisions

Previously we found that retinoblastoma protein (RB) became dephosphorylated in an early stage of DNA damage-induced, p53-independent apoptosis. Here, we report that both RB dephosphorylation and apoptosis are regulated by relative levels of RB kinases (cyclin-dependent kinases, or cdks) and phosphatases. Treatment of human Jurkat T cells with roscovitine, a potent and selective synthetic inhibitor of several cdks, rapidly induced RB dephosphorylation, which was followed by induction of apoptosis-associated internucleosomal DNA fragmentation. The roscovitine treatment did not increase levels of the endogenous cdk inhibitor proteins p16(Ink4a), p27(kip1) and p21(Waf1), supporting the idea that the observed RB dephosphorylation was due to a direct inhibition of cdk activities by roscovitine. Treatment with a protein kinase C inhibitor (sphingosine or staurosporine), which leads to suppression of several cdk kinase activities, also induced cellular RB dephosphorylation and apoptosis. Finally, roscovitine- or sphingosine-induced RB dephosphorylation was blocked by a specific inhibitor of protein-serine/threonine phosphatases (calyculin A or okadaic acid). Therefore, RB phosphorylation status and cellular fate are regulated by the ratio of RB kinases to RB phosphatases.

SPONTANEOUS AND INDUCIBLE PRODUCTION OF LEUKAEMIA INHIBITORY FACTOR BY HUMAN BONE MARROW STROMAL CELLS

Leukaemia inhibitory factor (LIF) acts on the growth and differentiation of haematopoietic cells. By using a specific enzyme-linked immunosorbent assay for human LIF, we demonstrate that human bone marrow stromal cells produce LIF. LIF synthesis is enhanced in a dose-dependent manner after stimulation with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMAS). LIF production in response to PMA is PKC-dependent since the two PKC inhibitors sphingosine and staurosporine markedly diminished it. Interleukin 1α (IL-1α), IL-1β, IL-3, IL-6, IL-8, tumour necrosis factor (TNF-α) and SCF (both at 10 ng/ml) stimulate LIF production. By contrast macrophage colony-stimulating factor (M-CSF), granulocyte (G)-CSF, GM-CSF, basic fibroblast growth factor (bFGF), platelet-activating factor (PAF), protaglandin E2(PGE2), leukotriene B4(LTB4), and leukotriene C4(LTC4) did not. These results suggest that bone marrow stromal cells might represent a major source for the cytokine-regulated local production of LIF inside human bone marrow.

Positive and negative regulation of JNK1 by protein kinase C and p42 MAP kinase in adult rat hepatocytes

The role of protein kinase C (PKC) and p42 MAP kinase signaling in the regulation of proliferation and apoptosis was investigated in freshly isolated and primary cultured rat hepatocytes. Acute treatment of freshly isolated hepatocytes with phenylephrine and EGF caused rapid phasic activations of p42 MAP kinase and JNK1. Acute pre-treatment of hepatocytes with the PKC inhibitors sphingosine, chelerythrine and bis-indolylmaleimide abolished the ability of phenylephrine, but not EGF, to activate p42 MAP kinase and JNK1. Acute pre-treatments with all of the PKC inhibitors alone increased JNK1 basal activity ∼2-fold. Acute treatments of primary cultures of hepatocytes with an inhibitor of MEK1 activation (PD98059) also caused inhibition of p42 MAP kinase and a ∼2-fold activation of JNK1. These data demonstrate that PKC can function as both a proximal activator and a distal inhibitor of signaling through the JNK1/SAP kinase pathway. Treatments (4 h) of primary cultured hepatocytes with sphingosine, chelerythrine, bis-indolylmaleimide and PD98059 did not induce apoptosis as judged by propidium iodide staining. Similar acute treatments of HepG2 cells rapidly induced cell death. These data demonstrate that acute inhibition of either PKC or p42 MAP kinase function is sufficient to rapidly induce apoptosis in transformed, but not in non-transformed hepatocytes.