Helicobacter pylori-induced Prostaglandin E2 Synthesis Involves Activation of Cytosolic Phospholipase A2 in Epithelial Cells

Helicobacter pylori initiates an inflammatory response and gastric diseases, which are more common in patients infected with H. pylori strains carrying the pathogenicity island, by colonizing the gastric epithelium. In the present study we investigated the mechanism of prostaglandin E2 (PGE2) synthesis in response to H. pylori infection. We demonstrate that H. pyloriinduces the synthesis of PGE2 via release of arachidonic acid predominately from phosphatidylinositol. In contrast to H. pylori wild type, an isogenic H. pylori strain with a mutation in the pathogenicity island exerts only weak arachidonic acid and PGE2 synthesis. The H. pylori-induced arachidonic acid release was abolished by phospholipase A2(PLA2) inhibitors and by pertussis toxin (affects the activity of Gαi/Gαo). The role of phospholipase C, diacylglycerol lipase, or phospholipase D was excluded by using specific inhibitors. An inhibitor of the stress-activated p38 kinase (SB202190), but neither inhibitors of protein kinase C nor an inhibitor of the extracellular-regulated kinase pathway (PD98059), decreased the H. pylori-induced arachidonic acid release.H. pylori-induced phosphorylation of p38 kinase and cytosolic PLA2 was blocked by SB202190. These results indicate that H. pylori induces the release of PGE2 from epithelial cells by cytosolic PLA2activation via Gαi/Gαo proteins and the p38 kinase pathway. Helicobacter pylori initiates an inflammatory response and gastric diseases, which are more common in patients infected with H. pylori strains carrying the pathogenicity island, by colonizing the gastric epithelium. In the present study we investigated the mechanism of prostaglandin E2 (PGE2) synthesis in response to H. pylori infection. We demonstrate that H. pyloriinduces the synthesis of PGE2 via release of arachidonic acid predominately from phosphatidylinositol. In contrast to H. pylori wild type, an isogenic H. pylori strain with a mutation in the pathogenicity island exerts only weak arachidonic acid and PGE2 synthesis. The H. pylori-induced arachidonic acid release was abolished by phospholipase A2(PLA2) inhibitors and by pertussis toxin (affects the activity of Gαi/Gαo). The role of phospholipase C, diacylglycerol lipase, or phospholipase D was excluded by using specific inhibitors. An inhibitor of the stress-activated p38 kinase (SB202190), but neither inhibitors of protein kinase C nor an inhibitor of the extracellular-regulated kinase pathway (PD98059), decreased the H. pylori-induced arachidonic acid release.H. pylori-induced phosphorylation of p38 kinase and cytosolic PLA2 was blocked by SB202190. These results indicate that H. pylori induces the release of PGE2 from epithelial cells by cytosolic PLA2activation via Gαi/Gαo proteins and the p38 kinase pathway. prostaglandin pathogenicity island arachidonic acid bisindolylmaleimide I diacylglycerol haloenol lactone suicide substrate methyl arachidonylfluorophosphonate multiplicity of infection phosphatidic acid phosphatidylcholine 2-(2-amino-3-methoxyphenyl)-oxanaphthalen-4-one phosphatidylethanolamine phosphatidylethanol phosphatidylinositol phospholipase A2 Ca2+-independent PLA2 cytosolic PLA2 phospholipase C phospholipase D phosphatidylserine 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl) imidazole sphingomyelin pertussis toxin The Helicobacter pylori infection induces the release of a number of proinflammatory cytokines and chemokines from the gastric epithelium (1Bodger K. Crabtree JE. Br. Med. Bull. 1998; 54: 139-150Crossref PubMed Scopus (161) Google Scholar) and plays a critical role in the development of gastritis, peptic ulcer disease, and rarely, in gastric carcinoma and B-cell mucosa-associated lymphoid tissue-associated carcinoma (2Blaser M.J. Gastroenterology. 1987; 93: 371-383Abstract Full Text PDF PubMed Google Scholar, 3Parsonnet J. Hansen S. Rodriguez L. Gelb A.B. Warnke R.A. Jellum E. Orentreich N. Vogelman J.H. Friedman G.D. N. Engl. J. Med. 1994; 330: 1267-1271Crossref PubMed Scopus (1729) Google Scholar). Evidence has been presented that an increase of prostaglandins (PGs)1 in gastric tissue from patients may play a crucial role in H. pylori infection (4Wakabayashi H. Orihara T. Nakaya A. Miyamoto A. Watanabe A. J. Gastroenterol. Hepatol. 1998; 13: 566-571Crossref PubMed Scopus (12) Google Scholar). In gastrointestinal epithelia, PGE2 is implicated in maintaining the normal function and structure of the gastric mucosa by modulating diverse cellular functions such as secretion of fluid and electrolytes, mucosal blood flow, and cell proliferation (5Eberhart C.E. Dubois R.N. Gastroenterology. 1995; 109: 285-301Abstract Full Text PDF PubMed Scopus (407) Google Scholar, 6Kobayashi K. Arakawa T. J. Clin. Gastroenterol. 1995; 21: 12-17PubMed Google Scholar). Studies have shown that H. pylori strains differ in their virulence and in their ability to trigger the induction of inflammatory mediators in gastric epithelial cell lines (1Bodger K. Crabtree JE. Br. Med. Bull. 1998; 54: 139-150Crossref PubMed Scopus (161) Google Scholar). The response is more intense to strains carrying the cagA gene. The analysis of the genomic region containing the cagA gene revealed a 40-kilobase DNA region, which represents a pathogenicity island (PAI) and codes for 31 genes (7Covacci A. Telford J.L. Del Giudice G. Parsonnet J. Rappuoli R. Science. 1999; 284: 1328-1333Crossref PubMed Scopus (957) Google Scholar). Upon contact with the gastric epithelium, PAI-encoded components contribute in a specialized type IV secretion machinery that translocates the CagA protein into the eukaryotic target cell where it is phosphorylated on tyrosine residues (8Segal E.D. Cha J. Lo J. Falkow S. Tompkins L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14559-14564Crossref PubMed Scopus (672) Google Scholar, 9Stein M. Rappuoli R. Covacci A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1263-1268Crossref PubMed Scopus (520) Google Scholar, 10Asahi M. Azuma T. Ito S. Ito Y. Suto H. Nagai Y. Tsubokawa M. Tohyama Y. Maeda S. Omata M. Suzuki T. Sasakawa C. J. Exp. Med. 2000; 191: 593-602Crossref PubMed Scopus (426) Google Scholar, 11Odenbreit S. Püls J. Sedlmaier B. Gerland E. Fischer W. Haas R. Science. 2000; 287: 1497-1500Crossref PubMed Scopus (1083) Google Scholar, 12Backert S. Ziska E. Brinkmann V. Zimny-Arndt U. Fauconnier A. Jungblut P. Naumann M. Meyer T.F. Cell. Microbiol. 2000; 2: 155-162Crossref PubMed Scopus (371) Google Scholar). H. pylori infection triggers by unknown bacterial factors multiple biochemical pathways in host cells including activation of transcription factors NF-κB and AP-1 (13Keates S. Hitti Y.S. Upton M. Kelly C.P. Gastroenterology. 1997; 113: 1099-1109Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 14Keates S. Keates A.C. Warny M. Peek R. Murray P. Kelly C. J. Immunol. 1999; 163: 5552-5559PubMed Google Scholar, 15Münzenmaier A. Lange C. Glocker E. Covacci A. Moran A. Bereswill S. Baeuerle P.A. Kist M. Pahl H.L. J. Immunol. 1997; 159: 6140-6147PubMed Google Scholar, 16Naumann M. Wessler S. Bartsch C. Wieland B. Covacci A. Haas R. Meyer T.F. J. Biol. Chem. 1999; 274: 31655-31662Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17Wessler S. Höcker M. Fischer W. Wang T. Rosewicz S. Haas R. Wiedenmann B. Meyer T. Naumann M. J. Biol. Chem. 2000; 275: 3629-3636Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), phospholipase C (PLC) (18Pucciarelli M.G. Ruschkowski S. Trust T.J. Finlay B.B. FEMS Microbiol. Lett. 1995; 129: 293-299PubMed Google Scholar), and the increase of the cytosolic free calcium concentration as well as the generation of adenosine 3′,5′-cyclic monophosphate and guanosine 3′,5′-cyclic monophosphate (19Chan E.C. Chen C.T. Lin Y.L. Chen K.T. Int. J. Mol. Med. 1999; 3: 421-425PubMed Google Scholar). The activation of the PGE2 signaling pathway in H. pylori-colonized gastric cells has not been studied so far. One of the mechanisms for increased PG production in response to H. pylori infection is an induction of COX-2 expression (20Eckmann L. Stenson W.F. Savidge T.C. Lowe D.C. Barrett K.E. Fierer J. Smith J.R. Kagnoff M.F. J. Clin. Invest. 1997; 100: 296-309Crossref PubMed Scopus (174) Google Scholar,21Romano M. Ricci V. Memoli A. Tuccillo C. Di Popolo A. Sommi P. Acquaviva A.M. Del Vecchio B. Bruni C.B. Zarrilli R. J. Biol. Chem. 1998; 273: 28560-28563Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Another rate-limiting step in the control of PG production is the release of AA from membrane phospholipids, which is known to occur via a number of different pathways. One involves the activation of phospholipase A2 (PLA2), others involve the action of PLC or phospholipase D (PLD) (22Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, 23Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4231) Google Scholar, 24Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (924) Google Scholar). In this report, we studied the control of PGE2 and AA production in response to H. pylori infection of epithelial cells after specific labeling of potential phospholipid precursors and selective inhibition of enzymes involved in the pathways of AA production. The presented results provide evidence that colonization of epithelial cells by H. pylori induces a release of PGE2 and AA by activation of the cytosolic PLA2 (cPLA2) via pertussis toxin-sensitive heterotrimeric Gαi/Gαo proteins and the p38 stress-activated kinase cascade. This process does not seem to involve PLC or PLD pathways. The identification of H. pylori-specific signaling pathways leading to the induction of putative anti-inflammatory immune response mediators is of substantial interest for therapeutic intervention to overcome H. pylori-induced diseases. The isogenic H. pylori strains P12 wild type, cagA (mutation affect cagA with a probable polar effect in the PAI), and vacA (25Schmitt W. Haas R. Mol. Microbiol. 1994; 12: 307-319Crossref PubMed Scopus (283) Google Scholar) for colonization of epithelial cell the in on containing and for in a by H. pylori was in with and and epithelial cells and cells in with and in a of and The cells into or for infection. the was by with the the in using and to the multiplicity of infection as in the for different of in the or of the the host cell the epithelial cell for with H. pylori was by of the cells with or was for the of in with of AA activity of or of acid in of containing for of the AA of the and of the acid to the was into the cells this was studied in cells with of in of containing for the was and the cells with containing and in the for In inhibitors of or enzymes to the cells this the the was for and from the cells by the of acid to the In the of the was to a The to the of and J. PubMed Scopus Google Scholar). The was and on of the cells and with The and to The in the which with and by phospholipid the in by for the of phosphatidylinositol and phosphatidylserine the in by for the The of was by using The for on and on a The by The in in with W. J. PubMed Scopus Google Scholar) and to The by with was as in and J. Biol. Chem. Full Text PDF PubMed Google was by PLA2 of and with common such as or for on the analysis of the release was into the and with the cells was in of the and the cell using a The of PGE2 in the was by to the by the analysis of the of cells in for in of containing and of with and with this for In PLC inhibitor was to the cells this with the for or with acid or as The was by of the and the of to the of the was with as in A. T. R. G. T. Mol. 1999; 13: PubMed Scopus Google Scholar). the analysis of p38 the cells infected with H. pylori for the of and the cells in an using a p38 The was with an to indicate protein in an was The inhibitors in this study as methyl arachidonylfluorophosphonate haloenol lactone suicide substrate acid pertussis toxin (PD98059), 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl) imidazole (SB202190), bisindolylmaleimide I of and in and and in was in the effect of the was H. pylori release of AA for PGE2 release was in and cells infected with H. pylori wild type and an isogenic vacA but not by an isogenic not and cells for different of in the or of different H. pylori the AA and in the was in the of from cells and their and by shown in colonization of and cells by the P12 strain increased the and release of free AA as with a increase of free AA and the release was a of which not increase of the of cells with isogenic vacA a increase in AA release the with the isogenic cagA a increase in AA known to AA release by activation of PLA2 Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), was as a control and a increase of AA The analysis of in the cellular revealed that the AA release by H. pylori was by a substantial in the of the AA in the The of the from to the In of the of AA that from the phospholipid in H. pylori-colonized cells the of phospholipid which from revealed that was the of AA in the The of decreased only after after H. a increase after in the of by of and of AA into In cells the decreased and of cells to P12 strain or with in a but increase and the production of diacylglycerol via activation of PLC and phosphorylation by kinase H. K. Sci. Full Text PDF PubMed Scopus Google of H. pylori on AA release and phospholipid in are as a of and the of for H. pylori and for with the control by in a are as a of and the of for H. pylori and for with the control by shown in H. release of AA was by and a inhibitor of the PubMed Scopus Google Scholar), and and a inhibitor of and Ca2+-independent E. K. E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). In with inhibition of AA inhibitors abolished the in the of the not that of not that this may as a for the type of PLA2 that is involved in the H. pylori-induced AA the effect of the inhibitor E. K. E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) and the PLA2 inhibitor acid 12: PubMed Scopus Google Scholar) was H. pylori-induced AA release from the cells was not by acid not not affect the AA and H. pylori-induced AA release not by in the J. N. M. J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). These a role of in the AA H. pylori induces an increase of cytosolic colonizing epithelial cells (19Chan E.C. Chen C.T. Lin Y.L. Chen K.T. Int. J. Mol. Med. 1999; 3: 421-425PubMed Google Scholar), and we studied the role of in AA calcium by of cells with and abolished release of AA in response to H. pylori results that AA release by H. pylori is The release of AA from membrane occur PLC activation by the action of the Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4231) Google Scholar). pylori-induced AA release from the cells was to inhibition by and an inhibitor of the PLC Smith S. J. Exp. Google Scholar). an increase in AA the AA in H. the H. pylori a and increase of and in and which was by the PLC inhibitor not that the of the inhibitor to H. pylori-induced AA release is not the to and an inhibitor of the J. E. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the release of AA from cells by but effect on H. pylori-induced AA release PLC and signaling pathways are not involved in the H. pylori-induced AA Another potential pathway of AA release involves activation of PLD J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (924) Google Scholar). this the release of into the was in cells that been with the into and was in and The of known to PLD in cells Y.S. W. Murray Lett. PubMed Scopus Google Scholar), in a release of into the of cells with H. pylori to not increase the release of as with control cells In of in cells with H. pylori was These results indicate that H. pylori cells not of by evidence that H. pylori does not affect the PLD the effect of known to activation of PLD R. N. R. R. G. H. L. Br. J. PubMed Scopus Google Scholar), was a concentration of effect on H. pylori-induced AA release not of H. pylori on the release of in of of is as a of with cells and The are the of for H. pylori and the of in for in a The of is as a of with cells and The are the of for H. pylori and the of in for was in response to H. pylori in cells with for not In the of PLD a in the production of and a in a cells with AA or acid for with in the of This an of and a of production. In of cells with H. pylori to the of and was only that H. pylori cells not not H. pylori-induced AA the of PLD in H. pylori-induced AA that activation of is involved in H. pylori-induced AA the generation of was in cells with or cells with of and was with release of AA from and via the PLA2 pathway cells with P12 the with increased the H. pylori not the of in The of in the of H. pylori is in with the substantial in the of the AA in the cells where infected with H. the role of in modulating the H. pylori on AA cells with for not have effect on AA but the H. pylori-induced AA release These results that are involved in the effect of H. pylori on AA we investigated protein kinase C protein are involved in the H. pylori-induced AA colonization with H. cells with inhibitors of protein kinase T. H. Y. M. Biophys. PubMed Scopus Google Scholar), V. R. W. C. J. K. C. K. H. Br. J. PubMed Scopus Google Scholar), and shown in and to affect the AA release by H. of effect on H. pylori-induced AA and increased the AA release and the AA in H. cells not with a specific inhibitor of the H. K. S. Y. K. M. C. A. M. Biophys. 1987; PubMed Scopus Google Scholar, L. S. A. A. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), effect on the AA release by H. pylori We have shown that blocked the activation of the kinase pathway in response to H. S. Höcker M. Fischer W. Wang T. Rosewicz S. Haas R. Wiedenmann B. Meyer T. Naumann M. J. Biol. Chem. 2000; 275: 3629-3636Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In of cells with and a specific inhibitor of the p38 stress-activated kinase Y. Chen C. W. J. Lin S. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), decreased the release of AA by H. pylori In with the inhibition of the AA release by H. pylori-induced activation of p38 kinase and phosphorylation of blocked in the of the a cells with known to increase AA release phosphorylation of via p38 kinase M. Gelb M. S. A. J. 1999; PubMed Scopus Google Scholar). of epithelial cells by H. pylori a generation of AA and the production of The of by that the release of AA by H. pylori was by a substantial in the in to that of that as a for AA in the PGE2 synthesis. with this which is for the of by C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar), was the cells with H. the of the has a role in the release of AA from cell to as substrate for the production of This has a for AA the of and for activation of the concentration of and a phosphorylation step (22Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, M. Lin J.L. A. Cell. Full Text PDF PubMed Scopus Google Scholar). We with that of cells with the inhibitor it is selective for known E. K. E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), blocked the release of In the H. pylori-induced AA release was not by or that and PLA2 are not The effect of the calcium the of a PLA2 in the AA In of cells to H. in a in the of a with M. Lin J.L. A. Cell. Full Text PDF PubMed Scopus Google Scholar), which is known to increase the activity of in (22Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). presented in this study the of pathways in the generation of of and cells to H. pylori generation of (18Pucciarelli M.G. Ruschkowski S. Trust T.J. Finlay B.B. FEMS Microbiol. Lett. 1995; 129: 293-299PubMed Google Scholar), inhibition of production by the inhibitor effect on AA that is not release of or a of was cells to H. the activation of PLD by H. This was by the that in the of which for in the by of was not in H. of the of the AA is the for inhibition of H. pylori-induced AA release was not for cells with known to PLD activation in a number of cell R. N. R. R. G. H. L. Br. J. PubMed Scopus Google Scholar), to affect the AA release by H. in the of the inhibitor H. pylori-induced AA release was The H. pylori-induced of which is in the of the membrane A. J. PubMed Scopus Google Scholar), that the host epithelial cell membrane was not by the H. pylori or C J. Clin. 1992; PubMed Scopus Google Scholar, A. Sci. PubMed Scopus Google Scholar, J.H. G. P. M.J. PubMed Scopus Google Scholar) or Y.L. Chen K.T. Chen C.T. E.C. Lett. 1998; PubMed Scopus Google Scholar). by using the isogenic which does not we the that the bacterial are to host cell AA release from the epithelial as has been shown in the of the J. S. H. 1994; PubMed Google Scholar). In a number of including activation of been shown to that of the heterotrimeric Gαi/Gαo proteins are involved in S. Biochim. Biophys. Acta. 1992; PubMed Scopus Google Scholar, R. M. W. J. 1995; PubMed Scopus Google Scholar, S. R.A. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J.R. G. Biochim. Biophys. Acta. 1997; PubMed Scopus Google Scholar). the mechanism for the role of Gαi/Gαo proteins on activation is it has been that the from Gαi/Gαo proteins activity and AA release T. Proc. Natl. Acad. Sci. U. S. A. 1987; PubMed Scopus Google Scholar). it has been shown that protein are involved in activation (22Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, M. Lin J.L. A. Cell. Full Text PDF PubMed Scopus Google Scholar). using specific inhibitors of protein kinase C and the kinase that the ability of H. pylori to a release of AA from epithelial cells does not on the activation of This is by the that H. pylori induces the activation of the kinase in a S. Keates A.C. Warny M. Peek R. Murray P. Kelly C. J. Immunol. 1999; 163: 5552-5559PubMed Google Scholar, 17Wessler S. Höcker M. Fischer W. Wang T. Rosewicz S. Haas R. Wiedenmann B. Meyer T. Naumann M. J. Biol. Chem. 2000; 275: 3629-3636Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), in contrast to AA release by H. pylori is evidence for the of p38 kinase in has been presented M. Gelb M. S. A. J. 1999; PubMed Scopus Google Scholar, E. H. S. Watanabe Y. Biophys. PubMed Scopus Google Scholar, A. J. T. R. R. S. J. 1998; 273: Scholar, S. W. T. J. R. J. Immunol. 1999; Google Scholar). In H. activation of p38 the of and AA release abolished after of cells with the p38 kinase inhibitor SB202190. These indicate the of the p38 kinase in the signaling leading to phosphorylation and AA has been to p38 kinase by S. W. T. J. R. J. Immunol. 1999; Google Scholar, A. S. M. P. Gelb M. S. J. 1999; PubMed Scopus Google Scholar) and shown to phosphorylation of in cells A. J. T. R. R. S. J. 1998; 273: Scholar) as well as to AA release in A. S. M. P. Gelb M. S. J. 1999; PubMed Scopus Google Scholar). are with the that activation of the p38 kinase by H. pylori in epithelial cells is S. Keates A.C. Warny M. Peek R. Murray P. Kelly C. J. Immunol. 1999; 163: 5552-5559PubMed Google Scholar). The release of AA was to and an inhibitor of p38 that Gαi/Gαo proteins involved in p38 kinase is to the signaling of p38 kinase involved in the activation of by H. In in this study we have shown that colonization of epithelial cells by H. pylori a release of AA predominately from for PGE2 production via activation of The signaling pathway a pertussis toxin-sensitive and p38 stress-activated protein kinase but not the activation of protein kinase C, and The H. pylori-induced release of AA and PGE2 from epithelial cells and their role in the or of inflammatory in the to of AA for PG production by activation of play an role in mucosal to the bacterial infection (5Eberhart C.E. Dubois R.N. Gastroenterology. 1995; 109: 285-301Abstract Full Text PDF PubMed Scopus (407) Google Scholar, J.L. 1995; PubMed Scopus Google Scholar) gastric epithelial cells are the of contact with H. the activation of is to to the gastric by of membrane AA and We T. and A. for their in the of and for the of T. for and A. G. for on We and the of C. S. and B. Wieland in