Vascular hepoxilin and trioxilins mediate vasorelaxation through TP receptor inhibition in mouse arteries.

AIM: 12/15-lipoxygenase (12/15-LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzyme's 15-LO metabolites 12(S)-hydroxyeicosatetraenoic acid (HETE), 15-HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15-trihydroxyeicosatrienoic acids (11,12,15-THETAs) as endothelium-derived relaxing factors. However, the observed 12-LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzyme's 12-LO activity. METHODS: Metabolites extracted from aortas of C57BL/6 male mice were separated using a series of reverse and normal phase chromatographic steps and identified as hepoxilin A3 , trioxilin A3 and trioxilin C3 by mass spectrometry. Activities of these natural compounds were tested on isometric tension and intracellular calcium release. The role of thromboxane (TP) receptor was determined in HEK293 cells overexpressing TPα receptor (TPα -HEK). RESULTS: All identified vascular 12-LO metabolites were biologically active. In mouse mesenteric arteries, trioxilin A3 , C3 and hepoxilin A3 (3 µm) relaxed arteries constricted with the thromboxane mimetic, U46619-constricted arteries (maximum relaxations of 78.9 ± 3.2, 29.7 ± 4.6, 82.2 ± 5.0 and 88.0 ± 2.4% respectively), but not phenylephrine-constricted arteries. In TPα-HEK cells, trioxilin A3 , C3 and hepoxilin A3 (10 µm) inhibited U46619 (10 nM)-induced increases in intracellular calcium by 53.0 ± 7.2%, 32.8 ± 5.0% and 37.9 ± 13.5% respectively. In contrast, trioxilin B3 and hepoxilin B3 were not synthesized in arteries and exhibited little biological activity. CONCLUSION: Trioxilin A3 and C3 and hepoxilin A3 are endogenous vascular relaxing factors. They are not endothelium-derived hyperpolarizing factors but mediate vascular relaxation by inhibiting TP agonist-induced increases in intracellular calcium. Thus, they regulate vascular homeostasis by acting as endogenous TP antagonists.

In vitro analyses are not reliable predictors of the plant growth promotion capability of bacteria; a Pseudomonas fluorescens strain that promotes the growth and yield of wheat.

AIMS: In this study, we set out to identify bacteria that can be used to promote the growth of cereals, while concurrently investigating the merits of using a range of such tests to preselect bacteria for glasshouse studies. METHODS AND RESULTS: A panel of 15 strains isolated from the rhizosphere and phyllosphere of cereals was tested for the ability to improve the germination of wheat seeds and for production of a range of factors associated with plant growth promotion. In parallel, all bacteria were tested for their ability to improve biomass and grain yield when applied as a soil amendment in glasshouse trials. CONCLUSIONS: There was no significant correlation between growth promotion potential in the glasshouse and the results of either the phenotypic or the germination tests. Glasshouse tests identified that only one strain, Pseudomonas fluorescens strain MKB37, gave a significant increase in head weight and grain yield. SIGNIFICANCE AND IMPACT OF THE STUDY: While this study has identified a candidate for further field tests, it has also highlighted the fact that the modes of action for plant growth-promoting bacteria (PGPB) are still not fully understood, and that there is no efficient and effective screening method for identifying PGPB by laboratory tests.

Glycosylation of the human prostacyclin receptor: role in ligand binding and signal transduction.

Prostacyclin, a potent vasodilator and inhibitor of platelet aggregation, acts through a cell-surface G protein-coupled receptor [prostacyclin (IP)]. The human (h) IP contains two consensus sites for N-linked glycosylation (N(7) and N(78)). However, the role of glycosylation is unknown. Mutant receptors (N(7)-Q(7),N(78)-Q(78) and N(7),N(78)-Q(7),Q(78)) were generated by replacing N(7) and/or N(78) with Q's. Receptor glycosylation was similar in the wild-type and N(7)-Q(7) and was inhibited with tunicamycin. N(78)-Q(78) and N(7),N(78)-Q(7),Q(78) demonstrated little or no glycosylation. Membrane localization was reduced for each mutant concomitant with impaired glycosylation. Partial localization to the plasma membrane allowed direct examination of the effect of glycosylation on IP function. High-affinity binding to N(7)-Q(7) was similar (K(d) = 21.7 +/- 1.7 nM, n = 4) to that of the wild-type receptor (K(d) = 24.3 +/- 3.6 nM, n = 4), despite a reduced value for B(max) (0.35 +/- 0.03 fmol/mg of protein versus 3.34 +/- 0.52 fmol/mg of protein, n = 4). Binding to N(78)-Q(78) (B(max) = 0.27 +/- 0.03 fmol/mg of protein, n = 3; K(d) = 149.1 +/- 11.1, n = 3) and N(7),N(78)-Q(7),Q(78) (no specific binding) was further impaired. Agonist-induced adenylyl cyclase activation was reduced in N(7)-Q(7) cells, whereas N(78)-Q(78) cells responded only to high concentrations of iloprost and N(7),N(78)-Q(7),Q(78) were unresponsive. Inositol phosphate generation was evident only with the wild-type. Only the wild-type and N(7)-Q(7) receptors underwent agonist-induced sequestration. Our findings demonstrate greater glycosylation at N(78) compared with N(7). The extent of N-linked glycosylation of hIP may be important for membrane localization, ligand binding, and signal transduction.

Internalization and sequestration of the human prostacyclin receptor.

Prostacyclin (PGI(2)), the major product of cyclooxygenase in macrovascular endothelium, mediates its biological effects through its cell surface G protein-coupled receptor, the IP. PKC-mediated phosphorylation of human (h) IP is a critical determinant of agonist-induced desensitization (Smyth, E. M., Hong Li, W., and FitzGerald, G. A. (1998) J. Biol. Chem. 273, 23258-23266). The regulatory events that follow desensitization are unclear. We have examined agonist-induced sequestration of hIP. Human IP, tagged at the N terminus with hemagglutinin (HA) and fused at the C terminus to the green fluorescent protein (GFP), was coupled to increased cAMP (EC(50) = 0.39 +/- 0.09 nm) and inositol phosphate (EC(50) = 86. 6 +/- 18.3 nm) generation when overexpressed in HEK 293 cells. Iloprost-induced sequestration of HAhIP-GFP, followed in real time by confocal microscopy, was partially colocalized to clathrin-coated vesicles. Iloprost induced a time- and concentration-dependent loss of cell surface HA, indicating receptor internalization, which was prevented by inhibitors of clathrin-mediated trafficking and partially reduced by cotransfection of cells with a dynamin dominant negative mutant. Sequestration (EC(50) = 27.6 +/- 5.7 nm) was evident at those concentrations of iloprost that induce PKC-dependent desensitization. Neither the PKC inhibitor GF109203X nor mutation of Ser-328, the site for PKC phosphorylation, altered receptor sequestration indicating that, unlike desensitization, internalization is PKC-independent. Deletion of the C terminus prevented iloprost-induced internalization, demonstrating the critical nature of this region for sequestration. Internalization was unaltered by cotransfection of cells with G protein-coupled receptor kinases (GRK)-2, -3, -5, -6, arrestin-2, or an arrestin-2 dominant negative mutant, indicating that GRKs and arrestins do not play a role in hIP trafficking. The hIP is sequestered in response to agonist activation via a PKC-independent pathway that is distinct from desensitization. Trafficking is dependent on determinants located in the C terminus, is GRK/arrestin-independent, and proceeds in part via a dynamin-dependent clathrin-coated vesicular endocytotic pathway although other dynamin-independent pathways may also be involved.

Phosphorylation of the prostacyclin receptor during homologous desensitization. A critical role for protein kinase c.

Agonist-induced phosphorylation of an epitope-tagged prostacyclin receptor (HAhIP) is mediated primarily by PKC (Smyth, E. M., Nestor, P. V., and FitzGerald G. A. (1996) J. Biol. Chem. 271, 33698-33704). Based on the two consensus sites for protein kinase C (PKC) phosphorylation in the C-terminal region mutant HAhIPs were generated: S328A and S374A, in which an alanine replaced Ser-328 or Ser-374, respectively, S328A/S374A and C-DEL, in which the C-terminal portion was truncated at amino acid 313. Mutant receptors, stably expressed in HEK293 cells, coupled normally to cAMP production. Substantially less coupling to inositol phosphate was apparent with S328A, S328A/S374A, and C-DEL compared with HAhIP or S374A. Point mutants resolved by SDS-polyacrylamide gel electrophoresis as a broad band with a molecular mass of 44-62, indicating that the receptors are glycosylated, and immunofluoresence staining demonstrated their membrane localization. C-DEL demonstrated a substantial reduction in glycosylation; bands with molecular masses of 38-54 (glycosylated), 30, and 27 kDa (unglycosylated) were apparent. Although membrane localization was evident, cellular localization was more diffuse. HAhIP and S374A underwent iloprost- and PMA-induced phosphorylation (1 and 5 microM, respectively, for 10 min). S328A and S328A/S374A showed a markedly less iloprost- and no PMA-induced phosphorylation. Phosphorylation of C-DEL was completely absent with either agonist. Electrospray mass spectrometry indicated that a peptide, including Ser-328, was phosphorylated in vitro by PKC, whereas one including Ser-374 was not. Iloprost (1 microM, 10 min) desensitized HAhIP- and S374A-mediated adenylyl cyclase activation. A less impressive desensitization was evident with S328A and S328A/S374A, and no desensitization of C-DEL coupling was apparent. Exposure of transfected cells to iloprost (1 microM) for increasing times induced a rapid desensitization of subsequent iloprost-induced (1 microM) HAhIP and S374A adenylyl cyclase coupling. In contrast, no significant time-dependent desensitization of S328A, S328A/S374A, or C-DEL coupling was evident. These results indicate that PKC-dependent phosphorylation is of critical importance to homologous regulation of hIP. Ser-328 is a primary site for PKC phosphorylation of hIP.

Agonist-dependent phosphorylation of an epitope-tagged human prostacyclin receptor.

An epitope-tagged human prostacyclin receptor (HAhIP) was constructed and stably transfected into human embryonic kidney 293 cells. The receptor exhibited high (Kd = 0.4 +/- 0.08 nM, Bmax = 0.7 +/- 0.2 pmol/mg protein; n = 4) and low (Kd = 75 +/- 27.4 nM, Bmax = 7.1 +/- 3.6 pmol/mg protein; n = 4) affinity for iloprost and coupled to both cAMP (EC50 = 0.1 +/- 0.03 nM) and inositol phosphate (EC50 = 43.1 +/- 10 nM) production. The receptor resolved on SDS-polyacrylamide gel electrophoresis as a broad complex with a molecular mass of 44-62 kDa and is glycosylated and phosphorylated. Stimulation of transfected cells with iloprost induced a rapid time- and concentration-dependent phosphorylation of HAhIP. Pretreatment of cells with a protein kinase C (PKC) inhibitor (GF109203X; 5 microM) abolished basal phosphorylation and dramatically reduced iloprost-induced HAhIP phosphorylation. A protein kinase A (PKA) inhibitor (H89) was largely ineffective under the same conditions. HAhIP phosphorylation was stimulated by receptor-dependent (thrombin, 2 units/ml) or receptor-independent (phorbol 12-myristate 13-acetate, 5 microM) PKC activation; both were abolished by pretreatment of cells with GF109203X. In contrast, receptor-independent (forskolin (5 microM) or dibutyryl cAMP (1 microM)) activation of PKA did not induce HAhIP phosphorylation. These results indicate that the human prostacyclin receptor may be regulated by agonist-dependent phosphorylation. This appears to be mediated, in part, by activation of PKC but not by PKA.

Evidence for modulation of hydrogen peroxide-induced endothelial barrier dysfunction by nitric oxide in vitro.

Acute effects of the nitric oxide (NO) donors sodium nitroprusside and glyceryl trinitrate on hydrogen peroxide (H2O2)-induced increases in endothelial monolayer permeability to trypan blue-labelled bovine serum albumin have been investigated in vitro. Exposure of bovine pulmonary artery endothelial cell monolayers to 0.2 mM H2O2 for 20 min caused a significant increase in percentage trypan blue-labelled albumin transfer from the lumenal to the ablumenal compartment (basal 6.0 +/- 0.6 to 25.4 +/- 0.9%, n = 4, P < 0.0005). In separate experiments 100 microM sodium nitroprusside significantly enhanced the effect of 0.2 mM H2O2 (from 7.4 +/- 1.4 to 11.9 +/- 1.5%, n = 9, P < 0.0001) but did not alter albumin transfer in the absence of H2O2. This additive effect appeared to be due to NO release from sodium nitroprusside, since nitrite concentration in the medium overlying cells treated with 100 microM sodium nitroprusside was 19.9 +/- 1.8 microM (n = 12). Significantly less nitrite (3.5 +/- 0.5 microM, n = 12, P < 0.0001) was found in the medium overlying cells treated with 100 microM glyceryl trinitrate, which in contrast to sodium nitroprusside, inhibited the permeability increase caused by H2O2 (from 15.6 +/- 3.3 to 13.8 +/- 3.1%, n = 6, P < 0.001). Furthermore 10 microM sodium nitroprusside, which released comparable amount of nitrite (4.5 +/- 0.4 microM, n = 6) to 100 microM glyceryl trinitrate, also inhibited the permeability increase caused by H2O2 (from 20.7 +/- 0.4 to 19.4 +/- 0.3%, n = 9, P < 0.01). We conclude that relatively large amounts of NO released from 100 microM sodium nitroprusside exacerbate the barrier dysfunction caused by H2O2, while lower amounts of NO give a small amount of cytoprotection.

Local amplification of platelet function by 8-Epi prostaglandin F2alpha is not mediated by thromboxane receptor isoforms.

8-epi-Prostaglandin (PG) F2alpha may be formed by cyclooxygenases 1 and 2 or by a free radical catalyzed process as an isoprostane. Concentrations of 8-epi-PGF2alpha in the range 1 nM to 1 microM induce a dose-dependent increase in platelet shape change, in calcium release from intracellular stores [Ca2+]iand in inositol phosphates; it also causes irreversible platelet aggregation, dependent on thromboxane generation, when incubated with subthreshold concentrations of ADP, thrombin, collagen, and arachidonic acid. Much higher concentrations of 8-epi-PGF2alpha (10-20 microM) alone induce weak, reversible aggregation. Although these effects are prevented by pharmacological thromboxane receptor antagonists, they are unlikely to be mediated by thromboxane receptors. Thus, 8-epi-PGF2alpha does not compete for binding at the stably expressed placental or endothelial isoforms of the thromboxane receptor or for binding of thromboxane ligands to human platelets. Furthermore, the response to 8-epi PGF2alpha exhibits structural specificity versus 8-epi PGF3alpha and PGF2alpha. Concentrations in the range that evoke its effects on platelets do not desensitize the aggregation response stimulated by thromboxane or PGH2 analogs. Unlike primary prostaglandins, which are rapidly metabolized to inactive products, 8-epi PGF2alpha circulates in plasma. However, the systemic concentrations found in healthy volunteers (median 48 pmol/liter) and in patients with hepatic cirrhosis (median 147 pmol/liter), a syndrome of oxidant stress in vivo, fall well below those which modulate platelet function. 8-Epi PGF2alpha may amplify the response to platelet agonists in syndromes where oxidant stress and platelet activation coincide. Despite blockade by thromboxane antagonists, 8-epi PGF2alpha does not activate either of the thromboxane receptor isoforms described in platelets. Activation of a distinct receptor would be consistent with the enzymatic formation of 8-epi PGF2alpha by cyclooxygenases. However, incidental activation of such a receptor by systemic concentrations of 8-epi PGF2alpha is unlikely to occur, even in syndromes of excessive free radical generation in vivo.

Effects of the ANF-C receptor ligand des[Cys105,Cys121]rANF(104-126) on ANF internalization and cGMP production by bovine pulmonary artery endothelial cells.

A role for the ANF-C receptor ligand des[Cys105,Cys121]rANF(104-126) in ANF receptor-effector coupling was investigated using cultured bovine pulmonary artery endothelial cells as a model system. The ligand was equipotent with rANF(99-126) in displacement of [125I]des[Cys105,Cys121]rANF(104-126) binding to whole cells and labelled only one population of ANF receptors as shown by affinity crosslinking experiments. In cells pretreated with des[Cys105, Cys121]rANF(104-126), internalization of [125I]rANF(99-126) binding was reduced and enhanced accumulation of cGMP was observed under basal conditions, or in the presence of a low concentration (0.1 nM) of rANF(99-126). These results provide a mechanistic basis for the observed enhancement of ANF-dependent vasodilation by ANF-C receptor selective ligands.

Selection of the type of urinary diversion in conjunction with radical cystectomy.

Several different methods of urinary diversion currently are available for use in conjunction with radical cystectomy. The method of urinary diversion performed at the time of radical cystectomy was reviewed for patients undergoing surgery from April 1984 through March 1986. A total of 91 urinary diversions was performed. Of the patients 74 per cent had a diversion with an external appliance (including 68 per cent with an ileal conduit), 14 per cent had a continent urinary reservoir (Kock pouch) and 11 per cent had an internal reservoir anastomosed to the urethra and, thus, avoided any stoma. There are inherent disadvantages to each form of urinary diversion and patient selection will be important to identify the most appropriate method of diversion for an individual.