Acrolein induces vasodilatation of rodent mesenteric bed via an EDHF-dependent mechanism.

Acrolein is generated endogenously during lipid peroxidation and inflammation and is an environmental pollutant. Protein adducts of acrolein are detected in atherosclerotic plaques and neurons of patients with Alzheimer's disease. To understand vascular effects of acrolein exposure, we studied acrolein vasoreactivity in perfused rodent mesenteric bed. Acrolein induced endothelium-dependent vasodilatation that was more robust and more sensitive than dilation induced by 4-hydroxy-trans-2-nonenal, trans-2-hexenal, or propionaldehyde. Acrolein-induced vasodilatation was mediated by K(+)-sensitive components, e.g., it was abolished in 0 [K(+)](o) buffer or in 3 mM tetrabutylammonium, inhibited 75% in 50 microM ouabain, and inhibited 64% in 20 mM K(+) buffer. Moreover, combined treatment with the Ca(2+)-activated K(+) channel inhibitors 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34, 100 nM) and apamin (5 microM) significantly reduced vasodilatation without altering sensitivity to acrolein. However, acrolein-induced % dilation was unaffected by l-NAME or indomethacin pretreatment indicating mechanistic independence of NO and prostaglandins. Moreover, acrolein induced vasodilatation in cirazoline-precontracted mesenteric bed of eNOS-null mice confirming eNOS independence. Pretreatment with 6-(2-propargyloxyphenyl) hexanoic acid (PPOH 50 microM), an epoxygenase inhibitor, or the superoxide dismutase mimetic Tempol (100 microM) significantly attenuated acrolein-induced vasodilatation. Collectively, these data indicate that acrolein stimulates mesenteric bed vasodilatation due to endothelium-derived signal(s) that is K(+)-, ouabain-, PPOH-, and Tempol-sensitive, and thus, a likely endothelium-derived hyperpolarizing factor (EDHF). These data indicate that low level acrolein exposure associated with vascular oxidative stress or inflammation stimulates vasodilatation via EDHF release in medium-sized arteries--a novel function.

Fibrinogen interactions with ICAM-1 (CD54) regulate endothelial cell survival.

We tested hypothesis that the interaction of fibrinogen (Fg) with intercellular adhesion molecule 1 (ICAM-1) mediates cellular adhesion and cell proliferation. Our results demonstrate that Fg : ICAM-1 ligation mediates endothelial cell survival and has an anti-apoptotic effect via activation of the MAP kinase pathway. Fg : ICAM-1 ligation in endothelial cells treated with tumor necrosis factor (TNF)alpha resulted in the hyperphosphorylation of extracellular signal-regulated kinase (ERK)-1/2 (eightfold to 10-fold) at 5-30 min. The specificity of ERK-1/2 phosphorylation was verified using the recognition peptides Fg-gamma-(117-133) and ICAM-1(8-22). ERK-1/2 hyperphosphorylation was dependent on intact cytoskeleton, as treatment with cytochalasin B and nocodazole blocked this activity. The attachment of TNFalpha-treated endothelial cells to fibrinogen or Fg-gamma-(117-133) resulted in cell survival, as assessed by an annexin V binding assay. ICAM-1(8-22) blocked the survival process. The MEK-1 inhibitor PD 98059 blocked ERK-1/2 phosphorylation, and treatment of endothelial cells with PD 98059 resulted in apoptosis even upon Fg : ICAM-1 ligation. Cells transfected with dominant-negative ERK-1/2 underwent apoptosis upon Fg : ICAM-1 ligation. Cell survival factor A1 was specifically upregulated upon adhesion of TNFalpha-stimulated endothelial cells to Fg. A1 expression was blocked by ICAM-1(8-22) and PD 98059. The Fg : ICAM-1 endothelial cell survival pathway appears to be mediated via the activation and upregulation of ERK-1/2 and A1.

Src homology domain 2-containing tyrosine phosphatase 2 associates with intercellular adhesion molecule 1 to regulate cell survival.

Intercellular adhesion molecule-1 (ICAM-1) binds to the plasma protein fibrinogen (Fg) to mediate leukocyte/endothelial cell interactions. In our studies, the ligation of Fg to ICAM-1 on tumor necrosis factor-alpha-stimulated endothelial cells resulted in the tyrosine phosphorylation of Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). The ICAM-1 cytoplasmic sequence IKKYRLQ conforms poorly to the concensus immunoreceptor tyrosine-based inhibition motifs found in receptors that bind SHP-2. Nevertheless, the tyrosine phosphorylated sequence (IKKpYRLQ) bound specifically to the SH2 domain proximal to the NH(2)-terminal of SHP-2 (SHP-2-N) but not to the SH2 domain proximal on the COOH-terminal side (SHP-2-C). Phosphorylated ICAM-1 bound SHP-2-N. In immunoprecipitation experiments, SHP-2 associated with phosphorylated ICAM-1. Cells expressing truncated ICAM-1 that lacked the cytoplasmic sequence (ICAM-1(TR)) failed to associate with SHP-2. ICAM-1 containing the tyrosine to alanine substitution at position 485 (ICAM-1(Y485A)) associated weakly with SHP-2. Cells expressing ICAM-1(TR) and ICAM-1(Y485A) underwent apoptosis upon adhesion to Fg, whereas the wild type ICAM-1 maintained cell survival. These results indicate that ICAM-1 interactions with SHP-2 allow better cellular survival mediated through Fg-ICAM-1 ligation.

Sequences within fibrinogen and intercellular adhesion molecule-1 (ICAM-1) modulate signals required for mitogenesis.

The interaction of fibrinogen (Fg) with intercellular adhesion molecule-1 (ICAM) on B-lymphoid Raji cells results in mitogenesis (Gardiner, E. E., and D'Souza, S. E. (1997) J. Biol. Chem. 272, 15474-15480). Incubation of Raji with Fg resulted in the increased tyrosine phosphorylation of the receptor-associated tyrosine kinase, pp60(Src) and extracellular signal-regulated kinase-1 (ERK). The increase in ERK-1 phosphorylation was blocked by a peptide with sequence matching ICAM-1-(8-22) and corresponded to a decrease in ERK-1 enzymatic activity. 100 microM amounts of Fg peptide gamma-(117-133) caused an increase in tyrosine phosphorylation of ERK-1. These results are consistent with our previous report wherein ICAM-1-(8-22) blocked Fg-induced mitogenesis and Fg-gamma-(117-133) induced proliferation in Raji. The specific inhibitor of MEK, PD98059 (25 microM), abrogated the increased phosphorylation of ERK-1 and blocked Raji mitogenesis by >50%. Inhibitors of pp60(Src), geldanamycin (62 nM), and herbimycin A (2.5 microM) blocked >50% of Raji proliferation. These results indicate that the proliferation induced by Fg interactions with ICAM-1 is mediated in part by receptor-associated tyrosine kinases and ERK-1, and that the recognition sequences within Fg and ICAM-1 participate in the signaling process.

Adaptive response of Haloferax mediterranei to low concentrations of NaCl (< 20%) in the growth medium.

Halobacteria require 20-25% NaCl for optimal growth and lyse when the salt concentration falls below 10%. The response of Haloferax mediterranei cells to low concentrations of NaCl (< 20%) in the medium was studied. The cells adapted to and grew in concentrations of NaCl as low as 10% and survived in concentrations lower than 5%. The cells synthesised a red pigment, bacterioruberin, in response to stress caused by a low concentration of NaCl (< 20%).

A mitogenic action for fibrinogen mediated through intercellular adhesion molecule-1.

Intercellular adhesion molecule-1 (ICAM-1) is a cell surface ligand for alphaLbeta2 and alphaMbeta2 integrins and has a key role in leukocyte adhesion to the vascular endothelium. The plasma protein fibrinogen has also been shown to interact with ICAM-1. We have investigated the effect of fibrinogen binding to ICAM-1-expressing cells on cell proliferation. The inclusion of 200-800 nM fibrinogen but not fibronectin to the culture medium of Raji induced a 2-4-fold increase in [3H]thymidine incorporation after 8 h. Cell proliferation in cultures containing fibrinogen was also confirmed by direct cell counting. The proliferative response in Raji was abrogated by an anti-ICAM-1 mAb 84H10 which maps to the first Ig domain of ICAM-1. A purified truncated form of ICAM-1 containing the first two Ig-like domains and a peptide with amino acid sequence corresponding to ICAM-1 (8-22) was also able to block the proliferative action of fibrinogen on Raji. 200 nM fibrinogen induced a 3-fold increase in [3H]thymidine incorporation by 293 cells transfected with ICAM-1 cDNA but not control non-transfected 293 cells. Comparable mitogenic effects were achieved with fibrinogen fragments X and D100, and with a synthetic peptide with an amino acid sequence matching fibrinogen gamma chain (117-133). These results indicate that interaction between discrete sequences within ICAM-1 and fibrinogen result in cellular proliferation.

Identification of an active sequence within the first immunoglobulin domain of intercellular cell adhesion molecule-1 (ICAM-1) that interacts with fibrinogen.

Monocytic cells bind fibrinogen (fg) through integrin alphaMbeta2. fg-bound monocytic cells demonstrate an enhanced adhesion to endothelial cells, which is dependent on intercellular adhesion molecule-1 (ICAM-1). Our studies differentiate fg interactions with stimulated and resting endothelial cells, which are ICAM-1 dependent and independent, respectively. This report documents a direct interaction between fg and intact ICAM-1 and with a two-Ig domain form of ICAM-1. A small region within the first Ig domain of ICAM-1, ICAM-1-(8-21) (KVILPRGGSVLVTC), was identified to interact with fg in a specific and selective manner. ICAM-1-(8-21) bound to plasmin-derived fg fragments X, D100, and D80 but not to fragment E. Consistent with this finding, fg gamma-chain peptide, fg-gamma-117-133, blocked fg interaction with ICAM-1-(8-2 1. ICAM-1-(8-21) peptide and antibodies directed against ICAM-1-(8-21) also blocked the adhesion and binding of ICAM-1-bearing Raji cells with fg. ICAM-1-(8-21) and fg-gamma-117-133 are likely to be one of the contact pairs mediating fg-ICAM-1 interactions.

Ligand and cation binding are dual functions of a discrete segment of the integrin beta 3 subunit: cation displacement is involved in ligand binding.

The alpha IIb beta 3 integrin binds Arg-Gly-Asp-containing (RGD-containing) ligands in a cation-dependent interaction. A fourteen amino acid sequence, beta 3 (118-131), and an antibody to it, inhibited ligand binding functions of alpha IIb beta 3, and a 1:1 stoichiometric beta 3 (118-131)-RGD complex was detected by mass spectroscopy. Cation binding to beta 3 (118-131) was demonstrated by terbium luminescence and mass spectroscopy. Notably, ligand displaced cation from the beta 3(118-131) peptide and also from purified alpha IIb beta 3. Thus, beta 3 (118-131), a highly conserved region in integrin beta subunits, binds both ligand and cation. Formation of a ternary complex between cation, ligand, and receptor, with subsequent displacement of cation from beta 3 (118-131) and a second site within the receptor, may be central to the mechanism of ligand recognition by integrins.

A novel technique for the preparation of osmotically stabilized and permeabilized cells of extremely halophilic bacteria.

The cells of Haloferax mediterannei were stabilized by cross-linking with 0.5% glutaraldehyde for 10 min. Such cells were found to be osmotically stable even when suspended in water. The stabilized cells could be permeabilized by treatment with chloroform without leakage of intracellular components. No significant difference in the properties of an intracellular enzyme aldolase was observed, using either cell-free extract or the osmotically stabilized and permeabilized cells. This novel technique can serve as a useful tool for studying in situ regulatory characteristics of intracellular functions in halobacteria and can also help in their re-use under more stabilized conditions for biotechnological applications.

Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif.

The tripeptide Arg-Gly-Asp (RGD) was originally identified as the sequence within fibronectin that mediates cell attachment. The RGD motif has now been found in numerous other proteins and supports cell adhesion in many, but not all, of these. The integrins, a family of cell-surface proteins, act as receptors for cell adhesion molecules. A subset of the integrins recognize the RGD motif within their ligands, the binding of which mediates both cell-substratum and cell-cell interactions. RGD peptides and mimetics, in addition to providing insights into the fundamental mechanisms of cell adhesion, are potential therapeutic agents for the treatment of diseases such as thrombosis and cancer.

A discrete sequence in a platelet integrin is involved in ligand recognition.

Platelet membrane glycoprotein IIb-IIIa (gpIIb-IIIa; alpha IIb-beta 3), the most prominent member of the integrin family of adhesion receptors on these cells, mediates platelet aggregation by binding fibrinogen and is critical in thrombosis and haemostasis. A short amino-acid sequence at the carboxy terminus of the gamma chain of fibrinogen is recognized by gpIIb-IIIa and peptides containing this sequence are selectively crosslinked to residues 294-314 of gpIIb. Here we show that an 11-residue peptide from this region of gpIIb inhibits platelet aggregation and binding of fibrinogen to platelets and to purified gpIIb-IIIa, and that it interacts directly with fibrinogen. These results implicate this segment of gpIIb-IIIa in the ligand-binding function of the receptor. Moreover, as this region is highly conserved among integrins, it may have a general function in ligand recognition by this broadly distributed family of adhesion receptors.

The ligand binding site of the platelet integrin receptor GPIIb-IIIa is proximal to the second calcium binding domain of its alpha subunit.

The extreme carboxyl-terminal amino acid sequence of the gamma chain of fibrinogen is involved in the binding of this adhesive protein to the platelet integrin glycoprotein (GP) IIb-IIIa, and synthetic peptides corresponding to this region inhibit fibrinogen as well as fibronectin and von Willebrand factor binding to platelets. A chemical cross-linking approach was used to characterize the interaction of a 16-amino acid fibrinogen gamma chain peptide with platelets and to localize the site of its binding to GPIIb-IIIa. This peptide became specifically cross-linked to GPIIb, and platelet stimulation selectively enhanced its cross-linking to this alpha subunit. The cross-linking reaction was specifically inhibited by fibrinogen and an Arg-Gly-Asp peptide but not by an unrelated protein or a substituted peptide. Utilizing a combination of immunochemical mapping, enzymatic and chemical digestions, and amino acid sequencing, the cross-linking site of the gamma chain peptide in GPIIb was localized to a stretch of 21 amino acids. The identified region, GPIIb 294-314, contains the second putative calcium binding domain within GPIIb. The primary structure of this region is highly conserved among alpha subunits of other integrin adhesion receptors. These results identify a discrete region of GPIIb that resides in close proximity to a ligand binding site within GPIIb-IIIa. The homologous region may be involved in the functions of other integrin receptors.

Turnover of proteoglycans in articular-cartilage cultures. Characterization of proteoglycans released into the medium.

By using an e.l.i.s.a. method it was demonstrated that the majority of proteoglycans released into the medium of both control and retinoic acid-treated explant cultures of bovine articular cartilage did not contain a hyaluronate-binding region. This supports our previous findings [Campbell & Handley (1987) Arch. Biochem. Biophys. 258, 143-155] that proteoglycans released into the medium of both cultures were of smaller hydrodynamic size, more polydisperse and unable to form aggregates with hyaluronate. Analysis of 35S-labelled core proteins associated with proteoglycans released into the medium of both cultures by using SDS/polyacrylamide-gel electrophoresis and fluorography indicated the presence of a series of core-protein bands (Mr approx. 300,000, 230,000, 215,000, 200,000, 180,000, 140,000, 135,000, 105,000, 85,000 and 60,000) compared with three core proteins derived from the proteoglycans remaining in the matrix (Mr 300,000, 230,000 and 215,000). Further analysis of the core proteins released into the medium indicated that the larger core proteins associated with medium proteoglycans contain both chondroitin sulphate and keratan sulphate glycosaminoglycans whereas the smaller core proteins contain only chondroitin sulphate chains. These experiments provide definitive evidence that the loss of proteoglycans from the matrix involves proteolytic cleavage at various sites along the proteoglycan core protein.

Isolation and characterization of a platelet membrane protein related to the vitronectin receptor.

Glycoprotein IIb-IIIa is the most prominent Arg-Gly-Asp (RGD)-binding adhesion receptor on platelets. By affinity chromatography on an immobilized RGD peptide, we have investigated the possible existence of other platelet-associated adhesion receptors that bind RGD peptides. When an octyl glucoside extract of surface-radioiodinated platelets was applied to an affinity matrix of KYGRGDS-coupled Sepharose 4B, a 160-kDa-labeled protein (P160) and GPIIb-IIIa bound and were specifically eluted by soluble GRGDSP peptide, but not by the variant GRGESP peptide. Furthermore, a dodecapeptide corresponding to fibrinogen gamma 400-411 eluted only GPIIb-IIIa but not P160 from the RGD affinity matrix. Characterization of P160 by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the O'Farrell gel electrophoresis system indicated that P160 is a component of platelet GPIc. GoH3, a monoclonal antibody recognizing the alpha subunit of the very late antigen-6, failed to immunoprecipitate P160 from the RGD eluate, indicating that it did not contain the very late antigen-6 alpha subunit. In immunoblots, P160 reacted specifically with a polyclonal anti-peptide antibody recognizing the alpha subunit of the vitronectin receptor (VnR), but not with the monoclonal anti-GPIIb antibody PMI-1, suggesting that P160 is the alpha subunit of platelet VnR. This possibility was further substantiated by the complete identity between the determined amino-terminal sequence of P160 and the known sequence of the VnR alpha subunit. Moreover, direct association of P160 with a beta subunit having an apparent molecular weight similar to that of GPIIIa was demonstrated by immunoprecipitation with LM609, an anti-VnR complex monoclonal antibody. These results indicate that the VnR complex is present on platelets and may play a functional role in platelet adhesive reactions.

Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor.

Many adhesive interactions are mediated by Arg-Gly-Asp (RGD) sequences within adhesive proteins. Such RGD sequences are frequently recognized by structurally related heterodimers that are members of the integrin family of adhesion receptors. A region was found in the platelet RGD receptor, gpIIb/IIIa, to which an RGD peptide becomes chemically cross-linked. This region corresponds to residues 109 to 171 of gpIIIa. This segment is conserved among the beta subunits of the integrins (76 percent identity of sequence), indicating that it may play a role in the adhesive functions of this family of receptors.

Chemical cross-linking of arginyl-glycyl-aspartic acid peptides to an adhesion receptor on platelets.

A chemical cross-linking approach has been used to characterize the interaction of platelets with small peptides of 7 and 14 residues containing the arginyl-glycyl-aspartic acid (RGD) sequence recognized by a variety of cellular adhesion receptors. The radioiodinated peptides were bound to platelets, and chemical cross-linking was attained by subsequent addition of bifunctional reagents. Three different cross-linking reagents coupled the RGD-containing peptides to platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa), and both subunits of this platelet membrane glycoprotein became radiolabeled with the RGD peptides. Platelet stimulation with agonists including thrombin, phorbol myristrate acetate, and ADP increased the extent of cross-linking by predominantly enhancing the coupling of the RGD peptides to the GPIIIa subunit. Cross-linking of the labeled RGD peptides to GPIIb and GPIIIa on stimulated and nonstimulated platelets exhibited structural specificity and was inhibited by excess nonlabeled RGD peptides. The interactions were inhibited by nonlabeled RGD peptides and a peptide with an amino acid sequence corresponding to the carboxyl terminus of the gamma chain of fibrinogen but less effectively by an arginyl-glycyl-glutamic acid peptide. Cross-linking of the RGD peptides to GPIIb-IIIa was divalent ion-dependent and, on stimulated platelets, was inhibited by the adhesive proteins fibrinogen and fibronectin, but not by albumin. These results indicate that the RGD-binding sites on platelets reside in close proximity to both subunits of GPIIb-IIIa and that platelet stimulation alters the topography of these sites such that the peptides become more efficiently cross-linked to GPIIIa.