Participation of phospholipase D and alpha/beta-protein kinase C in growth factor-induced signalling in C3H10T1/2 fibroblasts.
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We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the involvement of PLD in extracellularly regulated kinase 1 (MAPK) (ERK1) activation and c-fos mRNA expression in C3H/10T1/2 (Cl8) fibroblasts. In these cells, the PLD activity was significantly increased by porcine platelet-derived growth factor (PDGF-BB), phorbol 12-myristate 13-acetate (PMA), and epidermal growth factor (EGF). PLD activation by PDGF-BB and PMA, but not EGF, was inhibited in Cl8 cells expressing the HAbetaC2-1 peptide (Cl8 HAbetaC2-1 cells), with a sequence (betaC2-1) shown to bind receptor for activated C kinase 1 (RACK1) and inhibit c-PKC-mediated cell functions [Science 268 (1995) 247]. A role of alpha-PKC in PLD activation is further underscored by co-immunoprecipitation of alpha-PKC with PLD1 and PLD2 in non-stimulated as well as PMA- and PDGF-BB-stimulated Cl8 cells. However, only PKC in PLD1 precipitates was activated by these agonists, while the PKC in the PLD2 precipitates was constitutively activated. The c-fos mRNA levels in Cl8 cells increased more than 30-fold in response to either PDGF-BB, EGF, or PMA. Approximately 60% inhibition of this increase in c-fos mRNA levels was observed in Cl8 HAbetaC2-1 cells. Formation of phosphatidylbutanol (PtdBut) at the expense of phosphatidic acid (PtdH) in the presence of n-butanol inhibited ERK1 activation and c-fos mRNA expression in PDGF-BB-treated Cl8 cells. ERK activation by PMA was unaffected by n-butanol in Cl8 cells but almost abolished by n-butanol in Cl8 HAbetaC2-1 cells, showing that ERK activation by PMA is heavily dependent on PKC and PLD1. In contrast, ERK activation by EGF in both cell types was not sensitive to n-butanol. These results indicate (1) a role of a functional interaction between the RACK1 scaffolding protein and a alphaPKC-PLD complex for achieving full PLD activity in PDGF-BB- and PMA-stimulated Cl8 cells; (2) PLD-mediated PtdH formation is needed for optimal ERK1 activation by PDGF-BB and maximal increase in c-fos mRNA expression. These findings place PLD as an important component in PDGF-BB- and PMA-stimulated intracellular signalling leading to gene activation in Cl8 cells, while EGF does not require PLD. (+info)
Inhibition of experimental allergic rhinitis by the n-butanol fraction from the anomalous fruits of Gleditsia sinensis.
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This study was conducted to clarify the effect of the n-butanol fraction from the anomalous fruits of Gleditsia sinensis LAM. (NBGS) on experimental allergic rhinitis. NBGS (100, 200, 400 mg/kg, p.o.) dose-dependently inhibited nasal symptoms (sneezing and nasal rubbing) and dye leakage induced by antigen challenge into the nasal cavity of actively sensitized rats. Significant effects were observed at doses of 200 and 400 mg/kg. NBGS (200, 400 mg/kg) also showed a clear inhibition of sneezing and an inhibitory tendency on nasal rubbing induced by histamine in normal rats. At 400 mg/kg, it significantly reduced dye leakage induced by histamine into the nasal cavity of rats. Terfenadine (10 mg/kg, p.o.), an antihistaminic drug, clearly inhibited the nasal symptoms and the amount of dye leakage induced by antigen or histamine. Furthermore, NBGS significantly reduced in vitro histamine release from rat peritoneal mast cells triggered by compound 48/80 at concentrations of 30 and 100 microg/ml. These results suggest that NBGS may be clinically effective in alleviating the nasal symptoms of allergic rhinitis, probably by inhibiting both histamine release from mast cells and nasal vascular permeability. (+info)
Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide.
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Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD). This stimulates production of phosphatidic acid (PA), which increases expression of COX-2 mRNA and protein. Inhibition of PC-PLC by D609 (tricyclodecanoyl xanthogenate), and of PC-PLD activity by 1-butanol, reduced LPS-dependent over-production of PA and suppressed the increase of COX-2 mRNA and protein. Activation of PKC, normally seen in LPS-treated cells, was mimicked with phorbol myristic acid (PMA), and this also increased PA production and enhanced COX-2 expression. Propranolol inhibition of phosphatidic acid phosphohydrolase (PPH) increased PA accumulation and enhanced LPS-dependent COX-2 protein synthesis. These results suggest that inhibitors of PC-PLC, PKC and PC-PLD, or activators of PPH could be useful in the management of LPS-induced overproduction of prostaglandins and of vascular dysfunction in septic shock. (+info)
Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell's transcriptional program.
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DNA array and Western analyses were used to examine the effects of groESL overexpression and host-plasmid interactions on solvent production in Clostridium acetobutylicum ATCC 824. Strain 824(pGROE1) was created to overexpress the groESL operon genes from a clostridial thiolase promoter. The growth of 824(pGROE1) was inhibited up to 85% less by a butanol challenge than that of the control strain, 824(pSOS95del). Overexpression of groESL resulted in increased final solvent titers 40% and 33% higher than those of the wild type and plasmid control strains, respectively. Active metabolism lasted two and one half times longer in 824(pGROE1) than in the wild type. Transcriptional analysis of 824(pGROE1) revealed increased expression of motility and chemotaxis genes and a decrease in the expression of the other major stress response genes. Decreased expression of the dnaKJ operon upon overexpression of groESL suggests that groESL functions as a modulator of the CIRCE regulon, which is shown here to include the hsp90 gene. Analysis of the plasmid control strain 824(pSOS95del) revealed complex host-plasmid interactions relative to the wild-type strain, resulting in prolonged biphasic growth and metabolism. Decreased expression of four DNA gyrases resulted in differential expression of many key primary metabolism genes. The ftsA and ftsZ genes were expressed at higher levels in 824(pSOS95del), revealing an altered cell division and sporulation pattern. Both transcriptional and Western analyses revealed elevated stress protein expression in the plasmid-carrying strain. (+info)
Activation of phospholipase D by the small GTPase Sar1p is required to support COPII assembly and ER export.
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The small GTPase Sar1p controls the assembly of the cytosolic COPII coat that mediates export from the endoplasmic reticulum (ER). Here we demonstrate that phospholipase D (PLD) activation is required to support COPII-mediated ER export. PLD activity by itself does not lead to the recruitment of COPII to the membranes or ER export. However, PLD activity is required to support Sar1p-dependent membrane tubulation, the subsequent Sar1p-dependent recruitment of Sec23/24 and Sec13/31 COPII complexes to ER export sites and ER export. Sar1p recruitment to the membrane is PLD independent, yet activation of Sar1p is required to stimulate PLD activity on ER membranes, thus PLD is temporally regulated to support ER export. Regulated modification of membrane lipid composition is required to support the cooperative interactions that enable selective transport, as we demonstrate here for the mammalian COPII coat. (+info)
Effect of surface modification on hydration kinetics of carbamazepine anhydrate using isothermal microcalorimetry.
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The purpose of this research was to improve the stability of carbamazepine (CBZ) bulk powder under high humidity by surface modification. The surface-modified anhydrates of CBZ were obtained in a specially designed surface modification apparatus at 60 degrees C via the adsorption of n-butanol, and powder x-ray diffraction, Fourier-Transformed Infrared spectra, and differential scanning calorimetry were used to determine the crystalline characteristics of the samples. The hydration process of intact and surface-modified CBZ anhydrate at 97% relative humidity (RH) and 40 +/-C 1 degrees C was automatically monitored by using isothermal microcalorimetry (IMC). The dissolution test for surface-modified samples (20 mg) was performed in 900 mL of distilled water at 37 +/-C 0.5 degrees C with stirring by a paddle at 100 rpm as in the Japanese Pharmacopoeia XIII. The heat flow profiles of hydration of intact and surface-modified CBZ anhydrates at 97% RH by using IMC profiles showed a maximum peak at around 10 hours and 45 hours after 0 and 10 hours of induction, respectively. The result indicated that hydration of CBZ anhydrate was completely inhibited at the initial stage by surface modification of n-butanol and thereafter transformed into dihydrate. The hydration of surface-modified samples followed a 2-dimensional phase boundary process with an induction period (IP). The IP of intact and surface-modified samples decreased with increase of the reaction temperature, and the hydration rate constant (k) increased with increase of the temperature. The crystal growth rate constants of nuclei of the intact sample were significantly larger than the surface-modified sample's at each temperature. The activation energy (E) of nuclei formation and crystal growth process for hydration of surface-modified CBZ anhydrate were evaluated to be 20.1 and 32.5 kJ/mol, respectively, from Arrhenius plots, but the Es of intact anhydrate were 56.3 and 26.8 kJ/mol, respectively. The dissolution profiles showed that the surface-modified sample dissolved faster than the intact sample at the initial stage. The dissolution kinetics were analyzed based on the Hixon-Crowell equation, and the dissolution rate constants for intact and surface-modified anhydrates were found to be 0.0102 +/-C 0.008 mg(1/3) x min(-1) and 0.1442 +/-C 0.0482 mg(1/3) x min(-1). The surface-modified anhydrate powders were more stable than the nonmodified samples under high humidity and showed resistance against moisture. However, surface modification induced rapid dissolution in water compared to the control. (+info)
Ca(2+)- and phospholipase D-dependent and -independent pathways activate mTOR signaling.
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The mammalian target of rapamycin (mTOR) promotes increased protein synthesis required for cell growth. It has been suggested that phosphatidic acid, produced upon activation of phospholipase D (PLD), is a common mediator of growth factor activation of mTOR signaling. We used Rat-1 fibroblasts expressing the alpha(1A) adrenergic receptor to study if this G(q)-coupled receptor uses PLD to regulate mTOR signaling. Phenylephrine (PE) stimulation of the alpha(1A) adrenergic receptor induced mTOR autophosphorylation at Ser2481 and phosphorylation of two mTOR effectors, 4E-BP1 and p70 S6 kinase. These PE-induced phosphorylations were greatly reduced in cells depleted of intracellular Ca(2+). PE activation of PLD was also inhibited in Ca(2+)-depleted cells. Incubation of cells with 1-butanol to inhibit PLD signaling attenuated PE-induced phosphorylation of mTOR, 4E-BP1 and p70 S6 kinase. By contrast, platelet-derived growth factor (PDGF)-induced phosphorylation of these proteins was not blocked by Ca(2+) depletion or 1-butanol treatment. These results suggest that the alpha(1A) adrenergic receptor promotes mTOR signaling via a pathway that requires an increase in intracellular Ca(2+) and activation of PLD. The PDGF receptor, by contrast, appears to activate mTOR by a distinct pathway that does not require Ca(2+) or PLD. (+info)
Allosteric modulation of a neuronal K+ channel by 1-alkanols is linked to a key residue in the activation gate.
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The selective inhibition of neuronal Shaw2 K+ channels by 1-alkanols is conferred by the internal S4-S5 loop, a region that also contributes to the gating of voltage-gated K+ channels. Here, we applied alanine scanning mutagenesis to examine the contribution of the S5 and S6 segments to the allosteric modulation of Shaw2 K+ channels by 1-alkanols. The internal section of S6 is the main activation gate of K+ channels. While several mutations in S5 and S6 modulated the inhibition of the channels by 1-butanol and others had no effect, a single mutation at a key site in S6 (P410A) converted this inhibition into a dramatic dose-dependent potentiation (approximately 2-fold at 15 mM and approximately 6-fold at 50 mM). P410 is the second proline in the highly conserved PVP motif that may cause a significant alpha-helix kink. The P410A currents in the presence of 1-butanol also exhibited novel kinetics (faster activation and slow inactivation). Internal application of 15 mM 1-butanol to inside-out patches expressing P410A did not significantly affect the mean unitary currents (approximately 2 pA at 0 mV) or the mean open time (5-6 ms) but clearly increased the opening frequency and open probability (approximately 2- to 4-fold). All effects displayed a fast onset and were fully reversible upon washout. The results suggest that the allosteric modulation of the Shaw2 K+ channel by 1-alkanols depends on a critical link between the PVP motif and activation gating. This study establishes the Shaw2 K+ channel as a robust model to investigate the mechanisms of alcohol intoxication and general anesthesia. (+info)