Identification of Tyr504 as an alternative tyrosyl radical site in human prostaglandin H synthase-2

Hydroperoxides induce formation of a tyrosyl radical on Tyr385 in prostaglandin H synthase (PGHS). The Tyr385 radical initiates hydrogen abstraction from arachidonic acid, thereby mechanistically connecting the peroxidase and cyclooxygenase activities. In both PGHS isoforms the tyrosyl radical undergoes a time-dependent transition from a wide doublet to a wide singlet species; pretreatment with cyclooxygenase inhibitors results in a third type of signal, a narrow singlet [Tsai, A.-L.; Kulmacz, R. J. (2000) Prost. Lipid Med. 62, 231-254]. These transitions have been interpreted as resulting from Tyr385 ring rotation, but could also be due to radical migration from Tyr385 to another tyrosine residue. PATHWAYS analysis of PGHS crystal structures identified four tyrosine residues with favorable predicted electronic coupling: residues 148, 348, 404, and 504 (ovine PGHS-1 numbering). We expressed recombinant PGHS-2 proteins containing single Tyr --> Phe mutations at the target residues, a quadruple mutant with all four tyrosines mutated, and a quintuple mutant, which also contains a Y385F mutation. All mutants bind heme and display appreciable peroxidase activity, and with the exception of the quintuple mutant, all retain cyclooxygenase activity, indicating that neither of the active sites is significantly perturbed. Reaction of the Y148F, Y348F, and Y404F mutants with EtOOH generates a wide singlet EPR signal similar to that of native PGHS-2. However, reaction of the Y504F and the quadruple mutants with peroxide yields persistent wide doublets, and the quintuple mutant is EPR silent. Nimesulide pretreatment of Y504F and the quadruple mutant results in an abnormally small amount of wide doublet signal, with no narrow singlet being formed. Therefore, the formation of an alternative tyrosine radical on Tyr504 probably accounts for the transition from a wide doublet to a wide singlet in native PGHS-2 and for formation of a narrow singlet in complexes of PGHS-2 with cyclooxygenase inhibitors.     

A novel genetic model of selective COX-2 inhibition: comparison with COX-2 null mice

Prostaglandin H Synthase (PGHS) is a bi-functional enzyme with a cyclooxygenase (COX) activity and a functionally linked peroxidase (POX) activity that exists in two isoforms (COX-1, COX-2). Non-steroidal anti-inflammatory drugs (NSAIDs), including the selective COX-2 inhibitors, block COX activity while leaving POX activity unscathed. Recently, some selective COX-2 inhibitors were withdrawn from the market due to elevated cardiovascular risk in placebo-controlled trials. Mice deficient for PGHS2 were developed in 1995 and through numerous subsequent studies have revealed significant roles in renal development, ductus arteriosus patency/closure, skin carcinogenesis and cardiovascular function. In this short review, we compare a novel genetic COX-2 selective inhibition mouse model with the originally described COX-2 null mice in these different physiological functions.     

Different suicide inactivation processes for the peroxidase and cyclooxygenase activities of prostaglandin endoperoxide H synthase-1.

Prostaglandin endoperoxide H synthases (PGHSs)-1 and -2 have a cyclooxygenase (COX) activity involved in forming prostaglandin G2 (PGG2) from arachidonic acid and an associated peroxidase (POX) activity that reduces PGG2 to PGH2. Suicide inactivation processes are observed for both POX and COX reactions. Here we report COX reaction conditions for PGHS-1 under which complete COX inactivation occurs but with > or = 60% retention of POX activity. The rates of POX inactivation were compared for native oPGHS-1 versus Y385F oPGHS-1, a mutant that cannot form the Tyr385 radical of COX Intermediate II; the rates were the same for both native and Y385F oPGHS-1. Our data indicate that a COX Intermediate II/acyl or product complex is the precursor in COX inactivation. However, another species, probably an Intermediate II-like species but with a radical centered on a tyrosine other than Tyr385, is the immediate precursor for POX inactivation.     

Role of Tyr348 in Tyr385 radical dynamics and cyclooxygenase inhibitor interactions in prostaglandin H synthase-2

Both prostaglandin H synthase (PGHS) isoforms utilize a radical at Tyr385 to abstract a hydrogen atom from arachidonic acid, initializing prostaglandin synthesis. A Tyr348-Tyr385 hydrogen bond appears to be conserved in both isoforms; this hydrogen bonding has the potential to modulate the positioning and reactivity of the Tyr385 side chain. The EPR signal from the Tyr385 radical undergoes a time-dependent transition from a wide doublet to a wide singlet species in both isoforms. In PGHS-2, this transition results from radical migration from Tyr385 to Tyr504. Localization of the radical to Tyr385 in the recombinant human PGHS-2 Y504F mutant was exploited in examining the effects of blocking Tyr385 hydrogen bonding by introduction of a further Y348F mutation. Cyclooxygenase and peroxidase activities were found to be maintained in the Y348F/Y504F mutant, but the Tyr385 radical was formed more slowly and had greater rotational freedom, as evidenced by observation of a transition from an initial wide doublet species to a narrow singlet species, a transition not seen in the parent Y504F mutant. The effect of disrupting Tyr385 hydrogen bonding on the cyclooxygenase active site structure was probed by examination of cyclooxygenase inhibitor kinetics. Aspirin treatment eliminated all oxygenase activity in the Y348F/Y504F double mutant, with no indication of the lipoxygenase activity observed in aspirin-treated wild-type PGHS-2. Introduction of the Y348F mutation also strengthened the time-dependent inhibitory action of nimesulide. These results suggest that removal of Tyr348-Tyr385 hydrogen bonding in PGHS-2 allows greater conformational flexibility in the cyclooxygenase active site, resulting in altered interactions with inhibitors and altered Tyr385 radical behavior.     

Antiproliferative activity of guava leaf extract via inhibition of prostaglandin endoperoxide H synthase isoforms

Prostaglandin endoperoxide H synthase (PGHS) is a key enzyme for the synthesis of prostaglandins (PGs) which play important roles in inflammation and carcinogenesis. Because the extract from Psidium guajava is known to have a variety of beneficial effects on our body including the anti-inflammatory, antioxidative and antiproliferative activities, we investigated whether the extract inhibited the catalytic activity of the two PGHS isoforms using linoleic acid as an alternative substrate. The guava leaf extract inhibited the cyclooxygenase reaction of recombinant human PGHS-1 and PGHS-2 as assessed by conversion of linoleic acid to 9- and 13-hydroxyoctadecadienoic acids (HODEs). The guava leaf extract also inhibited the PG hydroperoxidase activity of PGHS-1, which was not affected by nonsteroidal anti-inflammatory drugs (NSAIDs). Quercetin which was one of the major components not only inhibited the cyclooxygenase activity of both isoforms but also partially inhibited the PG hydroperoxidase activity. Overexpression of human PGHS-1 and PGHS-2 in the human colon carcinoma cells increased the DNA synthesis rate as compared with mock-transfected cells which did not express any isoforms. The guava leaf extract not only inhibited the PGE₂ synthesis but also suppressed the DNA synthesis rate in the PGHS-1- and PGHS-2-expressing cells to the same level as mock-transfected cells. These results demonstrate the antiproliferative activity of the guava leaf extract which is at least in part caused by inhibition of the catalytic activity of PGHS isoforms.     

Prostaglandin endoperoxide H synthases: peroxidase hydroperoxide specificity and cyclooxygenase activation

The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.     

COX-2: Where are we in 2003? - Specific cyclooxygenase-2 inhibitors and aspirin-exacerbated respiratory disease

The use of analgesic anti-inflammatory agents in patients with asthma is clinically challenging because of the prevalence (10-20%) of aspirin hypersensitivity. Aspirin-exacerbated respiratory disease (AERD), or aspirin-induced asthma, is characterized by asthma and rhinitis triggered by the ingestion of aspirin and non-steroidal anti-inflammatory drugs. AERD is associated with upper and lower respiratory-tract mucosal inflammation, progressive sinusitis, nasal polyposis, and asthma regardless of whether patients avoid triggering drugs. The mechanism underlying the propensity of aspirin and non-steroidal anti-inflammatory drugs to cause this reaction is thought to involve inhibition of the synthesis of protective prostaglandins (PGs), resulting in an increase in the synthesis of cysteinyl leukotrienes by eosinophils and mast cells. Clinical data suggest that protective PGs are derived from cyclooxygenase (COX)-1 because studies have now confirmed that drugs specifically inhibiting COX-2 are not cross-reactive with aspirin in patients with AERD.     

COX-2: Where are we in 2003? - Specific cyclooxygenase-2 inhibitors and aspirin-exacerbated respiratory disease

The use of analgesic anti-inflammatory agents in patients with asthma is clinically challenging because of the prevalence (10–20%) of aspirin hypersensitivity. Aspirin-exacerbated respiratory disease (AERD), or aspirin-induced asthma, is characterized by asthma and rhinitis triggered by the ingestion of aspirin and non-steroidal anti-inflammatory drugs. AERD is associated with upper and lower respiratory-tract mucosal inflammation, progressive sinusitis, nasal polyposis, and asthma regardless of whether patients avoid triggering drugs. The mechanism underlying the propensity of aspirin and non-steroidal anti-inflammatory drugs to cause this reaction is thought to involve inhibition of the synthesis of protective prostaglandins (PGs), resulting in an increase in the synthesis of cysteinyl leukotrienes by eosinophils and mast cells. Clinical data suggest that protective PGs are derived from cyclooxygenase (COX)-1 because studies have now confirmed that drugs specifically inhibiting COX-2 are not cross-reactive with aspirin in patients with AERD.     

Structural and catalytic insights into the algal prostaglandin H synthase reveal atypical features of the first non-animal cyclooxygenase

Prostaglandin H synthases (PGHSs) have been identified in the majority of vertebrate and invertebrate animals, and most recently in the red alga Gracilaria vermiculophylla. Here we report on the cloning, expression and characterization of the algal PGHS, which shares only about 20% of the amino acid sequence identity with its animal counterparts, yet catalyzes the conversion of arachidonic acid into prostaglandin-endoperoxides, PGG₂ and PGH₂. The algal PGHS lacks structural elements identified in all known animal PGHSs, such as epidermal growth factor-like domain and helix B in the membrane binding domain. The key residues of animal PGHS, like catalytic Tyr-385 and heme liganding His-388 are conserved in the algal enzyme. However, the amino acid residues shown to be important for substrate binding and coordination, and the target residues for nonsteroidal anti-inflammatory drugs (Arg-120, Tyr-355, and Ser-530) are not found at the appropriate positions in the algal sequences. Differently from animal PGHSs the G. vermiculophylla PGHS easily expresses in Escherichia coli as a fully functional enzyme. The recombinant protein was identified as an oligomeric (evidently tetrameric) ferric heme protein. The preferred substrate for the algal PGHS is arachidonic acid with cyclooxygenase reaction rate remarkably higher than values reported for mammalian PGHS isoforms. Similarly to animal PGHS-2, the algal enzyme is capable of metabolizing ester and amide derivatives of arachidonic acid to corresponding prostaglandin products. Algal PGHS is not inhibited by non-steroidal anti-inflammatory drugs. A single copy of intron-free gene encoding for PGHS was identified in the red algae G. vermiculophylla and Coccotylus truncatus genomes.     

Computer-aided, rational design of a potent and selective small peptide inhibitor of cyclooxygenase 2 (COX2)

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are the mediators of inflammation. This enzyme exists mainly in two isoforms, COX1 and COX2. Prostaglandins responsible for the inflammatory process could be sufficiently controlled with the conventional non-steroidal anti-inflammatory drugs (NSAIDs). These drugs, however, had adverse gastrointestinal side-effects and, therefore, drugs that selectively inhibit COX2, such as the coxibs, were developed. Recent reports on the harmful cardiovascular and renal side-effects of the conventional NSAIDs as well as the COX2 selective inhibitors valdecoxib and rofecoxib have once again led to the quest for a novel class of COX2 selective inhibitors. Keeping this in mind, we have used the available X-ray crystal structures of the complexes of COX1 and COX2 with the known inhibitors to carry out a structure-based, rational, molecular modeling approach to design a small peptide inhibitor, which is both potent and selective for COX2. Docking studies using SYBYL 6.81 (Tripos, Inc.) and AutoDock 3.0, indicate that the designed peptides inhibit COX2 with potency in the nanomolar range. Furthermore, it is found to be a million-fold selective for COX2 as compared with COX1. Thus, the small peptide inhibitor is a suitable lead compound for the design of a new class of anti-inflammatory drugs.     

Peroxide-induced radical formation at TYR385 and TYR504 in human PGHS-1

Prostaglandin H synthase isoforms 1 and -2 (PGHS-1 and -2) react with peroxide to form a radical on Tyr385 that initiates the cyclooxygenase catalysis. The tyrosyl radical EPR signals of PGHS-1 and -2 change over time and are altered by cyclooxygenase inhibitor binding. We characterized the tyrosyl radical dynamics using wild type human PGHS-1 (hPGHS-1) and its Y504F, Y385F, and Y385F/Y504F mutants to determine whether the radical EPR signal changes involve Tyr504 radical formation, Tyr385 radical phenyl ring rotation, or both. Reaction of hPGHS-1 with peroxide produced a wide singlet, whereas its Y504F mutant produced only a wide doublet signal, assigned to the Tyr385 radical. The cyclooxygenase specific activity and K_M value for arachidonate of hPGHS-1 were not affected by the Y504F mutation, but the peroxidase specific activity and the K_M value for peroxide were increased. The Y385F and Y385F/Y504F mutants retained only a small fraction of the peroxidase activity; the former had a much-reduced yield of peroxide-induced radical and the latter essentially none. After binding of indomethacin, a cyclooxygenase inhibitor, hPGHS-1 produced a narrow singlet but the Y504F mutant did not form a tyrosyl radical. These results indicate that peroxide-induced radicals form on Tyr385 and Tyr504 of hPGHS-1, with radical primarily on Tyr504 in the wild type protein; indomethacin binding prevented radical formation on Tyr385 but allowed radical formation on Tyr504. Thus, hPGHS-1 and -2 have different distributions of peroxide-derived radical between Tyr385 and Tyr504. Y504F mutants in both hPGHS-1 and -2 significantly decreased the cyclooxygenase activation efficiency, indicating that formation of the Tyr504 radical is functionally important for both isoforms.     

Computer-Aided,Rational Design of a Potent and Selective Small Peptide Inhibitor of Cyclooxygenase 2 (COX2)

Abstract

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are the mediators of inflammation. This enzyme exists mainly in two isoforms, COX1 and COX2. Prostaglandins responsible for the inflammatory process could be sufficiently controlled with the conventional non-steroidal anti-inflammatory drugs (NSAIDs). These drugs, however, had adverse gastrointestinal side-effects and, therefore, drugs that selectively inhibit COX2, such as the coxibs, were developed. Recent reports on the harmful cardiovascular and renal side-effects of the conventional NSAIDs as well as the COX2 selective inhibitors valdecoxib and rofecoxib have once again led to the quest for a novel class of COX2 selective inhibitors.

Keeping this in mind, we have used the available X-ray crystal structures of the complexes of COX' and COX2 with the known inhibitors to carry out a structure-based, rational, molecular modeling approach to design a small peptide inhibitor, which is both potent and selective for COX2. Docking studies using SYBYL 6.81 (Tripos, Inc.) and AutoDock 3.0, indicate that the designed peptides inhibit COX2 with potency in the nanomolar range. Furthermore, it is found to be a million-fold selective for COX2 as compared with COX1. Thus, the small peptide inhibitor is a suitable lead compound for the design of a new class of anti-inflammatory drugs.     

Phospholipid actions on PGHS-1 and -2 cyclooxygenase kinetics

Cyclooxygenase (COX) catalysis by prostaglandin H synthase (PGHS) is a key control step for regulation of prostanoid biosynthesis. Both PGHS isoforms are integral membrane proteins and their substrate fatty acids readily partition into membranes, but the impact of phospholipids and lipid membranes on COX catalysis and the actions of COX inhibitors are not well understood. We have characterized the COX kinetics and ibuprofen inhibition of the purified PGHS isoforms in the presence of phosphatidylcholine (PC) with varying acyl chain structure and physical state. PC was found to directly inhibit COX activity, with non-competitive inhibition by PC monomers binding away from the COX active site and competitive inhibition by micellar/bilayer forms of PC due to sequestration of the arachidonate substrate. Competitive inhibition by native membranes was observed in a comparison of COX kinetics in sheep seminal vesicle microsomes before and after solubilization of PGHS-1. PC liposomes significantly increase the inhibitory potency of ibuprofen against both PGHS isoforms without changing the reversible character of ibuprofen action or requiring binding of PGHS to the liposomes. These results suggest a useful conceptual framework for analyzing the complex interactions among the PGHS proteins, substrates, inhibitors and phospholipid.     

Substitution of tyrosine for the proximal histidine ligand to the heme of prostaglandin endoperoxide synthase 2: implications for the mechanism of cyclooxygenase activation and catalysis

Prostaglandin H(2) synthesis by prostaglandin endoperoxide synthase (PGHS) requires the heme-dependent activation of the protein's cyclooxygenase activity. The PGHS heme participates in cyclooxygenase activation by accepting an electron from Tyr385 located in the cyclooxygenase active site. Two mechanisms have been proposed for the oxidation of Tyr385 by the heme iron: (1) ferric enzyme oxidizes a hydroperoxide activator and the incipient peroxyl radical oxidizes Tyr385, or (2) ferric enzyme reduces a hydroperoxide activator and the incipient ferryl-oxo heme oxidizes Tyr385. The participation of ferrous PGHS in cyclooxygenase activation was evaluated by determining the reduction potential of PGHS-2. Under all conditions tested, this potential (<-135 mV) was well below that required for reactions leading to cyclooxygenase activation. Substitution of the proximal heme ligand, His388, with tyrosine was used as a mechanistic probe of cyclooxygenase activation. His388Tyr PGHS-2, expressed in insect cells and purified to homogeneity, retained cyclooxygenase activity but its peroxidase activity was diminished more than 300-fold. Concordant with this poor peroxidase activity, an extensive lag in His388Tyr cyclooxygenase activity was observed. Addition of hydroperoxides resulted in a concentration-dependent decrease in lag time consistent with each peroxide's ability to act as a His388Tyr peroxidase substrate. However, hydroperoxide treatment had no effect on the maximal rate of arachidonate oxygenation. These data imply that the ferryl-oxo intermediates of peroxidase catalysis, but not the Fe(III)/Fe(II) couple of PGHS, are essential for cyclooxygenase activation. In addition, our findings are strongly supportive of a branched-chain mechanism of cyclooxygenase catalysis in which one activation event leads to many cyclooxygenase turnovers.     

Cyclooxygenase-2 modulates cellular growth and promotes tumorigenesis

Cyclooxygenase (COX)-2 and the prostaglandins resulting from its enzymatic activity have been shown to play a role in modulating cell growth and development of human neoplasia. Evidence includes a direct relationship between COX-2 expression and cancer incidence in humans and animal models, increased tumorigenesis after genetic manipulation of COX-2, and significant anti-tumor properties of non-steroidal anti-inflammatory drugs in animal models and in some human cancers. Recent data showed that COX-2 and the derived prostaglandins are involved in control of cellular growth, apoptosis, and signal through a group of nuclear receptors named peroxisome proliferator-activated receptors (PPARs). In this article we will review some of the findings suggesting that COX-2 is involved in multiple cellular mechanisms that lead to tumorigenesis.     

N-Acetylcysteine enhances the action of anti-inflammatory drugs as suppressors of prostaglandin production in monocytes

The anti-inflammatory effect of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with inhibition of cyclooxygenase (COX), the rate-limiting enzyme responsible for the synthesis of prostaglandins. Since oxygen free radicals can act as second cellular messengers, especially to modulate the metabolism of arachidonic acid and the prostaglandin tract, it seems plausible that antioxidants might affect the production of prostaglandin by activated cells. This research is focused on the effect of the antioxidant N-acetylcysteine (NAC) on the inhibition of prostaglandin E(2) formation in activated monocytes by specific and non-specific COX inhibitors. We found that lipopolysaccharide-induced prostaglandin E(2) formation was significantly reduced by rofecoxib and by diclofenac, two NSAIDs. Addition of NAC to each of these drugs enhanced the effect of the NSAIDs. These results suggest that one might expect either a potentiation of the anti-inflammatory effect of COX inhibitors by their simultaneous administration with NAC, or obtaining the same anti-inflammatory at lower drug levels.     

Microsomal prostaglandin E2 synthase: a safer target than cyclooxygenases?

Non-steroidal anti-inflammatory drugs (NSAIDs) are inhibitors of the cyclo-oxygenase (COX)-1 and -2 activities of prostaglandin G/H synthase-1 and -2, respectively. They have been extensively used in the treatment of prostaglandin E(2)-mediated chronic inflammatory diseases. Selective COX-2 inhibitors (coxibs), which were developed to provide an alternative with reduced gastrointestinal risk for the traditional NSAIDs, have been associated with an increased incidence of major adverse cardiovascular events. Could the targeting of microsomal prostaglandin E(2) synthase (mPGES-1) lead to novel anti-inflammatory drugs with possibly reduced risks of gastrointestinal and cardiovascular side effects?     

COX-2 inhibition attenuates anorexia during systemic inflammation without impairing cytokine production

Anorexia and weight loss are frequent complications of acute and chronic infections and result from induction of cytokines, prostaglandins, and other inflammatory mediators that are critical for pathogen elimination. Selective attenuation of the hypophagic response to infection and maintenance of the production of factors essential for infection control would be a useful addition to antimicrobial therapy in the treatment of human disease. Here, we evaluate the relative contribution of cyclooxygenase (COX)-1- and COX-2-derived prostaglandins to anorexia and weight loss precipitated by systemic immune activation by lipopolysaccharide (LPS). Using COX isoform-selective pharmacological inhibitors and gene knockout mice, we found that COX-2 inhibition during LPS-induced inflammation results in preserved food intake and maintenance of body weight, whereas COX-1 inhibition results in augmented and prolonged weight loss. Regulation of neuropeptide Y, corticotropin-releasing hormone, leptin, and interleukin-6 does not change as a function of COX-2 inhibition after LPS administration. Our data implicate COX-2 inhibition as a therapeutic target to maintain nutritional status while still allowing a normal cytokine response during infection.     

Cardiovascular expression of inflammatory signaling molecules, the kinin B1 receptor and COX2, in the rabbit: effects of LPS, anti-inflammatory and anti-hypertensive drugs

We first aimed to test the effect of anti-inflammatory drugs, etanercept and dexamethasone sodium phosphate (DSP), on the expression of inducible inflammatory signaling molecules (the bradykinin [BK] B(1) receptor [B(1)R], cyclooxygenase [COX]-2) in lipopolysaccharide (LPS)-treated rabbits. Preliminary experiments mostly based on a novel cellular model, rabbit dermis fibroblasts, showed that etanercept inhibited TNF-alpha-induced B(1)R expression ([(3)H]Lys-des-Arg(9)-BK binding), but that DSP also inhibited cytokine-induced B(1)R upregulation with less selectivity. LPS (100 microg/kg i.v.) induced the expression of the B(1)R (aortic contractility ex vivo, mRNA in hearts) and COX2 (immunoblots, heart extracts). However, the function of the BK B(2) receptor was unchanged (jugular vein contractility ex vivo). DSP pre-treatment profoundly reduced the induction of the B(1)R and COX2 whereas etanercept significantly inhibited only COX2 expression. The second aim was to verify whether chronic angiotensin converting enzyme (ACE) blockade in rabbits would induce B(1)R expression, as reported in other species. 14-Day enalapril oral dosing, but not treatment with the angiotensin receptor antagonist losartan, significantly increased aortic contractions mediated by B(1)Rs, however much less than LPS. Enalapril treatment did not increase COX2 expression but increased the ex vivo relaxation of the mesenteric artery mediated by endogenous prostaglandins. Chronic ACE inhibition recruits inflammatory signaling systems.