Nuclei contain two differentially regulated pools of diacylglycerol.
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A number of recent studies have highlighted the presence of a nuclear pool of inositol lipids [1] [2] that is regulated during progression through the cell cycle [1] [3], differentiation [1] [2] and after DNA damage [2], suggesting that a number of different regulatory pathways impinge upon this pool of lipids. It has been suggested that the downstream consequence of the activation of one of these nuclear phosphoinositide (PI) regulatory pathways is the generation of nuclear diacylglycerol (DAG) [1] [3] [4], which is important in the activation of nuclear protein kinase C (PKC) [5] [6] [7]. Activation of PKC in turn appears to regulate the progression of cells through G1 and into S phase [4] and through G2 to mitosis [3] [8] [9] [10] [11]. Although the evidence is enticing, there is as yet no direct demonstration that nuclear PIs can be hydrolysed to generate nuclear DAG. Previous data in murine erythroleukemia (MEL) cells have suggested that nuclear phosphoinositidase Cbeta1 (PIC-beta1) activity is important in the generation of nuclear DAG. Here, we demonstrate that the molecular species of nuclear DAG bears little resemblance to the PI pool and is unlikely to be generated directly by hydrolysis of these inositol lipids. Further, we show that there are in fact two distinct subnuclear pools of DAG; one that is highly disaturated and mono-unsaturated (representing more than 90% of the total nuclear DAG) and one that is highly polyunsaturated and is likely to be derived from the hydrolysis of PI. Analysis of these pools, either after differentiation or during cell-cycle progression, suggests that the pools are independently regulated, possibly by the regulation of two different nuclear phospholipase Cs (PLCs). (+info)
Regulatory role of cAMP in transport of Na+, Cl- and short-chain fatty acids across sheep ruminal epithelium.
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Sodium is absorbed in considerable amounts across the ruminal epithelium, whilst its transport is strongly interrelated with the permeation of chloride and short-chain fatty acids (SCFAs). However, regulation of ruminal Na+, Cl-, and SCFA absorption is hardly understood. The present study was therefore performed to characterize the influence of cAMP on sodium and sodium-coupled transport mechanisms in short-circuited, stripped ruminal epithelia of sheep. Elevation of intracellular cAMP concentrations by theophylline (10 mM) or theophylline in combination with forskolin (0.1 mM) significantly reduced mucosal-to-serosal sodium transport, leading to a reduction of net transport. The theophylline- or theophylline-forskolin-induced reduction of sodium transport was accompanied by a decrease in chloride net transport but revealed no effect on propionate flux. Short-chain fatty acids stimulated Na+ transport but their stimulatory effect was almost completely blocked by theophylline-forskolin. In solutions with and without SCFAs, the inhibitory effect of 1 mM amiloride on sodium transport was strongly reduced after theophylline-forskolin pretreatment of the tissues. Blocking the production of endogenous prostaglandins by addition of indomethacin (10 microM) led to a theophylline-sensitive stimulation of unidirectional and net fluxes of sodium. The findings indicate that apical, amiloride-sensitive Na+-H+ exchange and/or basolateral Na+-K+-ATPase can effectively be blocked by cAMP, leading to a decrease in sodium and chloride transport. In the ruminal epithelium, cAMP is a second messenger of prostaglandins, which are released spontaneously under in vitro conditions. (+info)
Interleukin-1 stimulates Jun N-terminal/stress-activated protein kinase by an arachidonate-dependent mechanism in mesangial cells.
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BACKGROUND: We have studied interleukin-1 (IL-1)-stimulated signals and gene expression in mesangial cells (MCs) to identify molecular mechanisms of MC activation, a process characteristic of glomerular inflammation. The JNK1 pathway has been implicated in cell fate decisions, and IL-1 stimulates the Jun N-terminal/stress-activated protein kinases (JNK1/SAPK). However, early postreceptor mechanisms by which IL-1 activates these enzymes remain unclear. Free arachidonic acid (AA) activates several protein kinases, and because IL-1 rapidly stimulates phospholipase A2 (PLA2) activity release AA, IL-1-induced activation of JNK1/SAPK may be mediated by AA release. METHODS: MCs were grown from collagenase-treated glomeruli, and JNK/SAPK activity in MC lysates was determined using an immunocomplex kinase assay. RESULT: Treatment of MCs with IL-1 alpha induced a time-dependent increase in JNK1/SAPK kinase activity, assessed by phosphorylation of the activating transcription factor-2 (ATF-2). Using similar incubation conditions, IL-1 also increased [3H]AA release from MCs. Pretreatment of MCs with aristolochic acid, a PLA2 inhibitor, concordantly reduced IL-1-regulated [3H]AA release and JNK1/SAPK activity, suggesting that cytosolic AA in part mediates IL-1-induced JNK1/SAPK activation. Addition of AA stimulated JNK1/SAPK activity in a time- and concentration-dependent manner. This effect was AA specific, as only AA and its precursor linoleic acid stimulated JNK1/SAPK activity. Other fatty acids failed to activate JNK1/SAPK. Pretreatment of MCs with specific inhibitors of AA oxidation by cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase had no effect on either IL-1- or AA-induced JNK1/SAPK activation. Furthermore, stimulation of MCs with the exogenous cyclooxygenase-, lipoxygenase-, phosphodiesterase-, and epoxygenase-derived arachidonate metabolites, in contrast to AA itself, did not activate JNK1/SAPK. CONCLUSION: We conclude that IL-1-stimulated AA release, in part, mediates stimulation of JNK1/SAPK activity and that AA activates JNK1/SAPK by a mechanism that does not require enzymatic oxygenation. JNK1 signaling pathway components may provide molecular switches that mediate structural rearrangements and biochemical processes characteristic of MC activation and could provide a novel target(s) for therapeutic intervention. (+info)
Effect of cAMP on the activity and the phosphorylation of Na+,K(+)-ATPase in rat thick ascending limb of Henle.
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BACKGROUND: In rat kidney medullary thick ascending limb of Henle's loop (MTAL), activation of protein kinase A (PKA) was previously reported to inhibit Na+,K(+)-ATPase activity. This is paradoxical with the known stimulatory effect of cAMP on sodium reabsorption. Because this inhibition was mediated by phospholipase A2 (PLA2) activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on cAMP action on Na+,K(+)-ATPase in MTAL. METHODS: Ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity were measured in isolated MTALs. Cellular ATP content and the phosphorylation level of Na+,K(+)-ATPase were determined in suspensions of outer medullary tubules. Experiments were carried out under nonoxygenated or oxygenated conditions in the absence or presence of PKA activators. RESULTS: cAMP analogues or forskolin associated with 3-isobutyl-1-methylxanthine (IBMX) inhibited ouabain-sensitive 86Rb uptake in nonoxygenated MTALs. In contrast, when oxygen supply was increased, cAMP stimulated ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity. Improved oxygen supply was associated with increased intracellular ATP content. The phosphorylation level of the Na+,K(+)-ATPase alpha subunit was increased by cAMP analogues or forskolin associated with IBMX in oxygenated as well as in nonoxygenated tubules. Under nonoxygenated conditions, the inhibition of Na+,K(+)-ATPase was dissociated from its cAMP-dependent phosphorylation, whereas under oxygenated conditions, the stimulatory effect of cAMP analogues on ouabain-sensitive 86Rb uptake was linearly related and cosaturated with the level of phosphorylation of the Na+,K(+)-ATPase alpha subunit. CONCLUSION: In oxygenated MTALs, PKA-mediated stimulation of Na+,K(+)-ATPase likely participates in the cAMP-dependent stimulation of sodium reabsorption. Under nonoxygenated conditions, this stimulatory pathway is likely overridden by the PLA2-mediated inhibitory pathway, a possible adaptation to protect the cells against hypoxic damage. (+info)
Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination. (+info)
Studies of the role of group VI phospholipase A2 in fatty acid incorporation, phospholipid remodeling, lysophosphatidylcholine generation, and secretagogue-induced arachidonic acid release in pancreatic islets and insulinoma cells.
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An 84-kDa group VI phospholipase A2 (iPLA2) that does not require Ca2+ for catalysis has been cloned from Chinese hamster ovary cells, murine P388D1 cells, and pancreatic islet beta-cells. A housekeeping role for iPLA2 in generating lysophosphatidylcholine (LPC) acceptors for arachidonic acid incorporation into phosphatidylcholine (PC) has been proposed because iPLA2 inhibition reduces LPC levels and suppresses arachidonate incorporation and phospholipid remodeling in P388D1 cells. Because islet beta-cell phospholipids are enriched in arachidonate, we have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1 insulinoma cells. Inhibition of iPLA2 with a bromoenol lactone (BEL) suicide substrate did not suppress and generally enhanced [3H]arachidonate incorporation into these cells in the presence or absence of extracellular calcium at varied time points and BEL concentrations. Arachidonate incorporation into islet phospholipids involved deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular glucose concentrations and by examining [14C]glucose incorporation into phospholipids. BEL also inhibited islet cytosolic phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor propranolol did not affect arachidonate incorporation into islet or INS-1 cell phospholipids. Inhibition of islet iPLA2 did not alter the phospholipid head-group classes into which [3H]arachidonate was initially incorporated or its subsequent transfer from PC to other lipids. Electrospray ionization mass spectrometric measurements indicated that inhibition of INS-1 cell iPLA2 accelerated arachidonate incorporation into PC and that inhibition of islet iPLA2 reduced LPC levels by 25%, suggesting that LPC mass does not limit arachidonate incorporation into islet PC. Gas chromatography/mass spectrometry measurements indicated that BEL but not propranolol suppressed insulin secretagogue-induced hydrolysis of arachidonate from islet phospholipids. In islets and INS-1 cells, iPLA2 is thus not required for arachidonate incorporation or phospholipid remodeling and may play other roles in these cells. (+info)
Relaxin stimulates glycodelin mRNA and protein concentrations in human endometrial glandular epithelial cells.
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Human endometrium is the major organ that produces glycodelin A (GdA). The production of endometrial GdA causes a fluctuation of the peripheral glycodelin concentrations in women during the menstrual cycle and pregnancy. It has recently been reported that the rise of plasma concentrations of glycodelin is correlated with relaxin during the late luteal phase and early pregnancy. In addition, administration of relaxin increases glycodelin plasma concentrations, suggesting that relaxin induces GdA production in endometrium. To investigate whether relaxin regulates the GdA synthesis, human endometrial glandular epithelial cells were isolated and cultured with or without relaxin for up to 4 days. Western blot showed that GdA synthesized and secreted from epithelial glands had a major molecular weight of 28 kDa, i.e. the same as the GdA isolated from amniotic fluid. Cells incubated with relaxin consistently increased in GdA production rate (2-6-fold). The GdA mRNA concentrations increased 2-11-fold in cells incubated with relaxin for 2-4 days, as determined by solution hybridization/ribonuclease protection assay. The increase of the mRNA concentration indicates that relaxin activates GdA transcription. (+info)
Effect of U-73122 on calcium channel activity in porcine myometrial cells and pancreatic beta-cell line RINm5F.
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AIM: To study the effect of U-73122, putative phospholipase C (PLC) inhibitor, on cytosolic Ca2+ concentration ([Ca2+]i) and voltage-dependent calcium channels (VDCC). METHODS: To record whole cell calcium current (ICa) by perforated patch-clamp and to measure cytosolic free calcium concentration ([Ca2+]i) with Fura-2. RESULTS: U-73122 inhibited KCl-induced and Bay-K-8644-induced increase in [Ca2+]i dose-dependently. U-73122 decreased markedly depolarization-induced ICa both in RINm5F and porcine myometrial cells in a concentration-dependent manner. The effect of U-73122 was more potent in porcine myometrial cells than that in RINm5F cells. U-73343 (an analog of U-73122), that does not inhibit PLC, also reduced the ICa by 50%. CONCLUSION: U-73122 inhibited VDCC and [Ca2+]i in both RINm5F and porcine myometrial cells. (+info)