Characterization of two putative mechanosensitive channel proteins of Campylobacter jejuni involved in protection against osmotic downshock.

Acute hypotonic stress becomes a threat to the survival of bacteria in the environment. Mechanosensitive channels play an essential role in the maintenance of bacterial cell integrity during hypoosmotic shock. A database search suggested that Campylobacter jejuni, a major worldwide cause of bacterial gastroenteritis in humans, possesses two putative mechanosensitive channels, designated Cjj0263 and Cjj1025, in C. jejuni strain 81-176. Osmotic downshock experiments demonstrated that a mutant lacking Cjj0263 showed a severe defect in survival of hypoosmotic shock, while a mutant lacking Cjj1025 exhibited the same survival capacity as the wild type. We further examined the colonization ability of each mutant using the one-day old chick model. Cjj0263 or Cjj1025 mutants were able to colonize chick ceca at the same level as the wild type, but a Cjj0263 Cjj1025 double mutant revealed significantly reduced ability to colonize chick ceca. To examine whether C. jejuni that have grown in the digestive tract of chicks are protected against acute hypotonic stress, bacteria in ceca were directly exposed to water. The wild type was able to survive acute osmotic downshift, but the Cjj0263 mutant suffered a substantial loss of viability when subjected to a rapid osmotic downshock. Immunoblot analysis suggested that both Cjj0263 and Cjj1025 were glycosylated via the N-linked protein glycosylation pathway, but glycan modification of these proteins was unlikely to have a major effect on their function and stability. Our data suggest that Cjj0263, a mechanosensitive channel, has a pivotal role in protection against hypoosmotic stress experienced during environmental transmission.

Newly synthetic ceramide-1-phosphate analogs; their uptake, intracellular localization, and roles as an inhibitor of cytosolic phospholipase A(2)α and inducer of cell toxicity.

Ceramide-1-phosphate (C1P) regulates cellular functions including arachidonic acid (AA) metabolism and modulates cell fate. The mechanism by which C1P is taken up is unclear, and the development of lipophilic analogs may be useful for regulating C1P's actions. We synthesized new mono- and di-methyl-ester (MM and DM, respectively) analogs of C1P with N-acyl chains of different lengths, and examined their effects on AA release and cell toxicity. Short-N-acyl-DM-C1P analogs including C5- and C6-DM-C1P, but not long-N-acyl-DM-C1P analogs, inhibited the release of AA mediated by α type cytosolic phospholipase A(2) (cPLA(2)α) in Chinese hamster ovary (CHO) cells and the enzymatic activity. Short-N-acyl-DM-C1P analogs including C6-DM-C1P caused morphological changes with cell toxicity 24h after the treatment in three cells lines (CHO, L929, and RLC-18 cells), although the role of AA in the toxicity was not clear. Neither long-N-acyl-DM-C1P analogs nor MM-C1P analogs including C6-MM-C1P affected cPLA(2)α activity and cell toxicity. Similar to C6-ceramide having a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) group on a C6-N-acyl chain (NBD-C6-ceramide), NBD-C6-DM-C1P and C6-DM-C1P-NBD (with a C6-N-acyl chain and an NBD-labeled C14-alkyl chain) were accumulated in the Golgi complex, although less C6-DM-C1P-NBD than NBD-C6-DM-C1P was taken up. NBD-C6-ceramide was converted to various metabolites including NBD-C6-sphingomyelin, but both NBD-C6-DM-C1P and C6-DM-C1P-NBD were stable in cells within 2h. The short-N-acyl-DM-C1P analogs acted directly as an inhibitor of cPLA(2)α and an inducer of cell toxicity, and may be useful for the regulation of ceramide/C1P-regulated responses.

Sphingosine 1-phosphate (S1P) regulates vascular contraction via S1P3 receptor: investigation based on a new S1P3 receptor antagonist.

Sphingosine 1-phosphate (S1P) induces diverse biological responses in various tissues by activating specific G protein-coupled receptors (S1P(1)-S1P(5) receptors). The biological signaling regulated by S1P(3) receptor has not been fully elucidated because of the lack of an S1P(3) receptor-specific antagonist or agonist. We developed a novel S1P(3) receptor antagonist, 1-(4-chlorophenylhydrazono)-1-(4-chlorophenylamino)-3,3-dimethyl- 2-butanone (TY-52156), and show here that the S1P-induced decrease in coronary flow (CF) is mediated by the S1P(3) receptor. In functional studies, TY-52156 showed submicromolar potency and a high degree of selectivity for S1P(3) receptor. TY-52156, but not an S1P(1) receptor antagonist [(R)-phosphoric acid mono-[2-amino-2-(3-octyl-phenylcarbamoyl)-ethyl] ester; VPC23019] or S1P(2) receptor antagonist [1-[1,3-dimethyl-4-(2-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-4-(3,5-dichloro-4-pyridinyl)-semicarbazide; JTE013], inhibited the decrease in CF induced by S1P in isolated perfused rat hearts. We further investigated the effect of TY-52156 on both the S1P-induced increase in intracellular calcium ([Ca(2+)](i)) and Rho activation that are responsible for the contraction of human coronary artery smooth muscle cells. TY-52156 inhibited both the S1P-induced increase in [Ca(2+)](i) and Rho activation. In contrast, VPC23019 and JTE013 inhibited only the increase in [Ca(2+)](i) and Rho activation, respectively. We further confirmed that TY-52156 inhibited FTY-720-induced S1P(3) receptor-mediated bradycardia in vivo. These results clearly show that TY-52156 is both sensitive and useful as an S1P(3) receptor-specific antagonist and reveal that S1P induces vasoconstriction by directly activating S1P(3) receptor and through a subsequent increase in [Ca(2+)](i) and Rho activation in vascular smooth muscle cells.

Effects of synthetic sphingosine-1-phosphate analogs on cytosolic phospholipase A2alpha-independent release of arachidonic acid and cell toxicity in L929 fibrosarcoma cells: the structure-activity relationship.

Sphingolipid metabolites including ceramide, sphingosine, and their phosphorylated products [sphingosine-1-phosphate (S1P) and ceramide-1-phosphate] regulate cell functions including arachidonic acid (AA) metabolism and cell death. The development of analogs of S1P may be useful for regulating these mediator-induced cellular responses. We synthesized new analogs of S1P and examined their effects on the release of AA and cell death in L929 mouse fibrosarcoma cells. Among the analogs tested, several compounds including DMB-mC11S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMB-mC9S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-nonyl)phenylpropyl phosphate] released AA within 1 h and caused cell death 6 h after treatment. The release of AA was observed in C12 cells [a L929 variant lacking a type alpha cytosolic phospholipase A(2) (cPLA(2)alpha)] and L929-cPLAalpha-siRNA cells (L929 cells treated with small interference RNA for cPLA(2)alpha). Treatment with pharmacological inhibitors of secretory and Ca(2+)-independent PLA(2)s decreased the DMB-mC11S-induced release of AA. The effect of the S1P analogs tested on the release of AA was comparable to that on cell death in L929 cells, and a high correlation coefficient was observed. Two analogs lacking a butoxycarbonyl moiety [DMAc-mC11S (dimethyl (2S,3R)-2-acetamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMAm-mC11S [dimethyl (2S,3R)-2-amino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate)] had inhibitory effects on the release of AA and cell toxicity induced by DMB-mC11S. Synthetic phosphorylated lipid analogs may be useful for studying PLA(2) activity and its toxicity in cells. [Supplementary Fig. 1: available only at http://dx.doi.org/10.1254/jphs.08284FP].

Pharmacophore-based design of sphingosine 1-phosphate-3 receptor antagonists that include a 3,4-dialkoxybenzophenone scaffold.

Sphingosine 1-phosphate (S1P) receptors are G-protein-coupled receptors. Among the five identified subtypes S1P1-5, the S1P3 receptor expressed on vascular endothelial cells has been shown to play an important role in cell proliferation, migration, and inflammation. A pharmacophore-based database search was used to identify a potent scaffold for an S1P3 receptor antagonist by common feature-based alignment and further validated using the Güner-Henry (GH) scoring method. Assumed excluded volumes were merged into this model to evaluate the steric effect with the S1P3 receptor. Three commercially available compounds were identified as S1P3 receptor antagonists, with IC50 values <5 microM. The synthesis of further derivatives revealed that the 3,4-dialkoxybenzophenone scaffold is a potent component of an S1P3 receptor antagonist. Our results indicate that pharmacophore-based design of S1P3 receptor antagonists can be used to expand the possibility of structural modification through scaffold-hopping based on a database search.

Topics on the Na+/Ca2+ exchanger: pharmacological characterization of Na+/Ca2+ exchanger inhibitors.

Using the whole-cell voltage clamp, we examined acute effects of various agents on Na(+)/Ca(2+) exchange current (I(NCX)) in guinea-pig cardiac ventricular cells and transfected cells. Among the antiarrhythmic drugs, amiodarone, bepridil, dronedarone, cibenzoline, azimilide, and aprindine inhibited I(NCX) in a concentration-dependent manner. We also investigated the effects on NCX of 2,3-buanedione monoxim (BDM) and selective NCX inhibitors such as KB-R7943, SEA0400, and SN-6. The presence of trypsin in the pipette solution attenuated the inhibitory effects on NCX of amiodarone, bepridil, and BDM, suggesting that these drugs inhibit NCX from the cytosolic side. In contrast, the trypsin-insensitive NCX inhibitors were aprindine, azimilide, dronedarone, cibenzoline, KB-R7943, SEA0400, and SN-6. KB-R7943, SEA0400, and SN-6 suppressed the uni-directional outward I(NCX) more potently than the uni-directional inward I(NCX). The mechanism of this mode-dependency is unknown, but is suggested to be related to intracellular Na(+) concentration.

Involvement of p38 MAP kinase-mediated cytochrome c release on sphingosine-1-phosphate (S1P)- and N-monomethyl-S1P-induced cell death of PC12 cells.

d-erythro-Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, affects various neuronal functions including cell fate. S1P appears to have contradictory effects in PC12 cells, a neuronal model cell line; neurite retraction and cell survival/differentiation. In the present study, we examined whether S1P induces cell death in undifferentiated PC12 cells. Culture with S1P at 20 microM for 4 h caused lactate dehydrogenase leakage 24 h later. The response was reduced by an inhibitor of caspases and accompanied by the release of cytochrome c and DNA fragmentation. S1P caused the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) within 10 min. An inhibitor of p38 MAPK (10 microM SB203580) inhibited both the release of cytochrome c and DNA fragmentation induced by S1P. Treatment with nerve growth factor or pertussis toxin (PTX) decreased S1P-induced phosphorylation of p38 MAPK and cell death. These findings suggest that S1P-activated p38 MAPK acts as a death signal upstream of the release of cytochrome c. N-Monomethyl-S1P (MM-S1P), a weak agonist in cells expressing S1P1 receptors, had marked effects (phosphorylation of p38 MAPK, release of cytochrome c and DNA fragmentation) at lower concentrations than S1P and in a PTX-sensitive manner. These findings show that the activation of S1P receptors by S1P and MM-S1P causes cell death accompanied by DNA fragmentation via the p38 MAPK pathway-mediated release of cytochrome c in PC12 cells. The potential of S1P and MM-S1P to act as agonists of S1P receptors and as intracellular messengers is discussed.

Effects of synthetic sphingosine-1-phosphate analogs on arachidonic acid metabolism and cell death.

Sphingolipid metabolites such as sphingosine regulate cell functions including cell death and arachidonic acid (AA) metabolism. D-erythro-C18-Sphingosine-1-phosphate (D-e-S1P), a sphingolipid metabolite, acts as an intracellular messenger in addition to being an endogenous ligand of some cell surface receptors. The development of S1P analogs may be useful for studying and/or regulating S1P-mediated cellular responses. In the present study, we found that several synthetic S1P analogs at pharmacological concentrations stimulated AA metabolism and cell death in PC12 cells. D-erythro-N,O,O-Trimethyl-C18-S1P (D-e-TM-S1P), L-threo-O,O-dimethyl-C18-S1P (L-t-DM-S1P) and L-threo-O,O-dimethyl-3O-benzyl-C18-S1P (L-t-DMBn-S1P) at 100 microM stimulated [(3)H]AA release from the prelabeled PC12 cells. L-t-DMBn-S1P at 20 microM increased prostanoid formation in PC12 cells. L-t-DMBn-S1P-induced AA release was inhibited by D-e-sphingosine, but not by the tested PLA(2) inhibitors. L-t-DMBn-S1P did not stimulate the activity of cytosolic phospholipase A(2alpha) (cPLA(2alpha)) in vitro and the translocation of cPLA(2alpha) in the cells, and caused AA release from the cells lacking cPLA(2alpha). These findings suggest that L-t-DMBn-S1P stimulated AA release in a cPLA(2alpha)-independent manner. In contrast, D-e-S1P and D-erythro-N-monomethyl-C18-S1P caused cell death without AA release in PC12 cells, and the effects of D-e-TM-S1P, L-t-DM-S1P and L-t-DMBn-S1P on cell death were limited. Synthetic S1P analogs may be useful tools for studying AA metabolism and cell death in cells.

Structure-activity relationship of benzo[b]thiophene-2-sulfonamide derivatives as novel human chymase inhibitors.

We have identified a new class of chymase inhibitor through a substituent analysis of MWP00965, which we previously discovered by in silico screening. TY-51076 (7) showed high potency (IC(50)=56 nM) and excellent selectivity for chymase compared to chymotrypsin and cathepsin G (>400-fold). The synthesis and structure-activity relationship of this class are described.

Identification of a stable chymase inhibitor using a pharmacophore-Based database search.

In general, serine protease chymase inhibitors readily decompose in plasma. We previously found that thiazolidine-2,4-dione and thiadiazole derivatives are also unstable. Using a pharmacophore-based database search, we identified a benzo[b]thiophen-2-sulfonamide derivative as a stable chymase inhibitor. Finding a lead compound with adequate activity and stability by a pharmacophore-based approach is more efficient than modifying an unstable compound to reduce its instability without simultaneously decreasing its inhibitory activity. Our pharmacophore model of chymase inhibitors suggests that the two hydrophobic interactions in the S1 and S1' regions and the two H-bonding interactions between them play important roles in chymase inhibitors.

Development of novel EDG3 antagonists using a 3D database search and their structure-activity relationships.

Sphingosine-1-phosphate (S1P) is an intracellular second messenger and an extracellular mediator through endothelial differentiation gene (EDG) receptors, which are a novel class of G-protein-coupled receptors. Although EDG has attracted much attention because of its various roles, no selective agonists or antagonists have yet been developed. This could account for the delay in clarifying the physiological roles of members of the EDG family. Because precise structural information on EDG receptors is not yet available, pharmacophore models were generated based on structural information for S1P using the rational drug design software Catalyst. Novel antagonists, 2-alkylthiazolidine-4-carboxylic acids, were retrieved from a three-dimensional database search using the pharmacophore models, and these showed activity for EDG3. On the basis of their nonphosphoric acid structure, more potent antagonists, 2-(m- or p-heptylphenyl)thiazolidine-4-carboxylic acid, were developed.