Peroxiredoxin VI oxidation in cerebrospinal fluid correlates with traumatic brain injury outcome.

Traumatic brain injury (TBI) patients would benefit from the identification of reliable biomarkers to predict outcomes and treatment strategies. In our study, cerebrospinal fluid (CSF) from patients with severe TBI was evaluated for oxidant stress-mediated damage progression after hospital admission and subsequent ventriculostomy placement. Interestingly, substantial levels of peroxiredoxin VI (Prdx6), a major antioxidant enzyme normally found in astrocytes, were detected in CSF from control and TBI patients and were not associated with blood contamination. Functionally, Prdx6 and its associated binding partner glutathione S-transferase Pi (GSTP1-1, also detected in CSF) act in tandem to detoxify lipid peroxidation damage to membranes. We found Prdx6 was fully active in CSF of control patients but becomes significantly inactivated (oxidized) in TBI. Furthermore, significant and progressive oxidation of "buried" protein thiols in CSF of TBI patients (compared to those of nontrauma controls) was detected over a 24-h period after hospital admission, with increased oxidation correlating with severity of trauma. Conversely, recovery of Prdx6 activity after 24h indicated more favorable patient outcome. Not only is this the first report of an extracellular form of Prdx6 but also the first report of its detection at a substantial level in CSF. Taken together, our data suggest a meaningful correlation between TBI-initiated oxidation of Prdx6, its specific phospholipid hydroperoxide peroxidase activity, and severity of trauma outcome. Consequently, we propose that Prdx6 redox status detection has the potential to be a biomarker for TBI outcome and a future indicator of therapeutic efficacy.

Inhibition of thromboxane synthase activity modulates bladder cancer cell responses to chemotherapeutic agents.

Recently, we reported prognostic significance of thromboxane synthase (TXAS) gene expression in invasive bladder cancer. The positive correlation between elevated TXAS expression and shorter patient survival supports a potential role for TXAS-regulated pathways in tumor metastases. In this study, using immunohistochemical analysis, we found an increased expression of TXAS protein in bladder cancer. Treatment of T24 and transitional cell carcinoma TCC-SUP bladder cancer cells with the TXAS inhibitors furegrelate or ozagrel induced an apoptotic effect measured as an increase in caspase-3 activation and cell death, and decreased survivin expression. Pharmacological inhibition of TXAS using the TXAS inhibitor furegrelate increased sensitivity to the chemotherapeutic agents cisplatin and paclitaxel. Molecular inhibition of TXAS expression by siRNA significantly decreased cell growth and migration. In concordance with the pharmacological data, siRNA-mediated reduction of TXAS expression increased sensitivity to cisplatin and paclitaxel in T24 and TCC-SUP cells. In summary, the data support a role for the thromboxane A(2) pathway in the pathogenesis of bladder cancer and the potential utility of modulation of this signaling pathway for cancer chemotherapy.

Castration reduces platelet thromboxane A2 receptor density and aggregability.

BACKGROUND: Exogenously administered testosterone upregulates platelet thromboxane A2 (TXA2) receptors and increases aggregation response to thromboxane mimetics in healthy male volunteers. However, the biological impact of endogenous testosterone on platelet TXA2 receptor expression, especially in older men at risk of coronary artery disease, is unclear. AIM: To investigate the impact of reduction in circulating testosterone on platelet TXA2 receptor expression in older men. DESIGN: Cross-sectional case-control study. METHODS: We studied surgically and/or medically castrated men with prostate cancer (group A, n = 8, aged 71 +/- 8 years) and age-matched, uncastrated urology patients (group B, n = 7, aged 67 +/- 9 years). Plasma testosterone was measured by radioimmunoassay. Platelet TXA2 receptor expression was assessed by radioligand binding studies using radioactive 125I-BOP. Platelet aggregation responses to TXA2-mimetic I-BOP, and to thrombin, were also studied. RESULTS: Group A had significantly lower plasma testosterone than group B (16 +/- 5 ng/dl vs. 308 +/- 47 ng/dl, p<0.001). Platelet TXA2 receptor density (B(max)) but not affinity (K(d)) was lower in group A (0.50 +/- 0.12 vs. 1.01 +/- 0.17 pmol/mg protein, p = 0.03). Maximum platelet aggregation response to I-BOP (E(max)), but not sensitivity (EC50) was lower in group A (53 +/- 2% vs. 63 +/- 2%, p = 0.003 ANOVA). In vitro, high concentrations of hydroxyflutamide (100 microM) competitively inhibited U46619-induced platelet aggregation in washed platelets, without affecting the binding of 125I-BOP to platelet TXA2 receptors. DISCUSSION: Endogenous testosterone regulates platelet TXA2 receptor B(max) and the E(max) aggregation response to thromboxane mimetic I-BOP. Blockade of androgen receptors or inhibition of testosterone production may reduce platelet aggregation responses. Preliminary evidence suggests the presence of functional androgen receptors on human platelets, which may regulate TXA2 receptor expression.

Effect of cross-tolerance between endotoxin and TNF-alpha or IL-1beta on cellular signaling and mediator production.

Endotoxin [lipopolysaccharide (LPS)] tolerance suppresses macrophage/monocyte proinflammatory-mediator production. This phenomenon also confers cross-tolerance to other stimuli including tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta. Post-receptor convergence of signal transduction pathways might occur after LPS, IL-1beta, and TNF-alpha stimulation. Therefore, it was hypothesized that down-regulation of common signaling molecules induces cross-tolerance among these stimuli. LPS tolerance and cross-tolerance were examined in THP-1 cells. Phosphorylation of MAP kinases and degradation of inhibitor kappaBalpha (IkappaBalpha) DNA binding of nuclear factor-kappaB (NF-kappaB), and mediator production were examined. In naive cells, LPS, TNF-alpha, and IL-1beta induced IkappaBalpha degradation, kinase phosphorylation, and NF-kappaB DNA binding. LPS stimulation induced production of TNF-alpha or TxB2 and degradation of IRAK. However, neither TNF-alpha nor IL-1beta induced IRAK degradation or stimulated TNF-alpha or TxB2 production in naive cells. Pretreatment with each stimulus induced homologous tolerance to restimulation with the same agonist. LPS tolerance also suppressed LPS-induced TxB2 and TNF-alpha production. LPS pretreatment induced cross-tolerance to TNF-alpha or IL-1beta stimulation. Pretreatment with TNF-alpha induced cross-tolerance to LPS-induced signaling events and TxB2 production. Although pretreatment with IL-1beta did not induce cross-tolerance to LPS-induced signaling events, it strongly inhibited LPS TNF-alpha and TxB2 production. These data demonstrate that IL-1beta induces cross-tolerance to LPS-induced mediator production without suppressing LPS-induced signaling to MAP kinases or NF-kappaB activation.

Changes in vascular responsiveness to a thromboxane mimetic in endotoxin tolerance.

Our previous studies have demonstrated that peritoneal macrophages obtained from endotoxin-tolerant rats exhibit altered cellular activation by endotoxin, possibly involving changes in guanine nucleotide regulatory (G) protein-coupled signal transduction pathways. Endotoxin-tolerant rats also exhibit cross tolerance and altered hemodynamic responses to thromboxane (Tx)A2 mimetics, suggesting potential changes in vascular responsiveness. We tested the hypothesis that endotoxin tolerance results in vascular hyporesponsiveness to a TxA2 mimetic via alterations in the TxA2 receptor, G protein function, and/or second messenger production. Rats were rendered endotoxin tolerant by increasing sublethal consecutive doses of Salmonella enteritidis endotoxin (100 to 5000 micrograms/kg, i.p.) for 4 days. The animals were sacrificed 2 days after the final dose of endotoxin for removal of aortas. Contractile responses of aortic rings to U46619, a TxA2 agonist, were assessed in control and tolerant rats. The EC50 values for U46619 were 14.8 +/- 6.6 nM and 32.3 +/- 3.1 nM (n = 5-7), (P < 0.05) for control and tolerant rats, respectively. Crude membranes were prepared from aortas of control and tolerant rats, and binding of I-BOP TxA2/PGH2 receptor agonist, [1S-(1 alpha, 2 beta (5Z), 3 alpha (1E, 3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7- oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), a TxA2 agonist, was assessed by Scatchard analysis. I-BOP binding to the TxA2 receptor was saturable and revealed a single class of TxA2 receptors for both groups. There was no significant difference in control (n = 7) compared with tolerant (n = 5) Kd values (2.1 +/- 0.2 vs. 2.4 +/- 0.9 nM, respectively), or Bmax (31 +/- 6 vs. 28 +/- 12 fmol/mg protein, respectively). To assess potential changes in G protein function, aortic membrane GTpase activity was determined. GTPase activity in tolerant membranes was significantly reduced (P < 0.05) compared with control membranes (309 +/- 23 (n = 5) vs. 440 +/- 32 (n = 7) pmol/mg/protein/min, respectively). However, U46619-stimulated phosphoinositide production was similar in vascular tissue from control and tolerant rats. These observations suggest that the decreased contractile response to TxA2 mimetics in endotoxin tolerance does not result from a change in receptor number, affinity of TxA2 receptors, or changes in phosphatidylinositol metabolism but is associated with decreased vascular G protein function.

Carbon dioxide is the major metabolite of quercetin in humans.

A previous study in ileostomy patients indicated that dietary glucosides of the flavonoid quercetin are hydrolyzed efficiently in the intestinal lumen, followed by absorption of a large fraction of the quercetin aglycone. To determine the fate of quercetin, we administered 1.85 MBq (50 microCi) of (14)C-quercetin both orally (100 mg, 330 micromol) and intravenously (iv; 0.3 mg, 1 micromol) to healthy volunteers. Serial plasma samples, urines and stools were collected for 72 h. Total radioactivity was determined by liquid scintillation spectrometry directly in plasma and urine and after repeated methanol extraction of stool homogenate samples. The oral absorption, based on total radioactivity, was surprisingly high, ranging from 36.4 to 53.0%. The biological half-life was very long, ranging from 20 to 72 h. The urinary recovery of total radioactivity ranged from 18.4 to 26.8% after the iv dose and from 3.3 to 5.7% after the oral dose. The corresponding fecal recoveries were only 1.5-5.0% and 1.6-4.6%, respectively. Thus, the total recovery of the (14)C-quercetin doses, in particular after oral administration, was very low. In search for the unaccounted for fraction of the (14)C-quercetin dose, we performed (14)CO(2) recovery studies in three volunteers (3 iv and 3 oral doses). At timed intervals, (14)CO(2) in expired air was trapped in hyamine hydroxide/thymolphthalein and analyzed for radioactivity. As much as 23.0-81.1% of the quercetin dose was recovered as (14)CO(2) in the expired air from these volunteers, after both oral and iv doses. The disposition of quercetin in humans is thus highly complex, requiring further studies.

Progesterone regulation of vascular thromboxane A(2) receptors in rhesus monkeys.

We hypothesized that progesterone regulates thromboxane A(2) receptor (TxA(2)R) density in primate vascular muscle and that TxA(2)R density correlates with coronary reactivity in vivo and in vitro. Reactivity to serotonin + U-46619 was determined by angiography in surgically postmenopausal [ovariectomized (Ovx)] rhesus monkeys without progesterone replacement and after 2-wk progesterone treatment (1-2 ng/ml). In untreated Ovx animals, 100 micromol/l serotonin + 1 micromol/l U-46619 (syringe concentrations) provoked vasospasm-like constrictions in six of six monkeys; zero of six progesterone-treated monkeys developed vasospasms. Sustained Ca(2+) responses in vascular muscle cells isolated from Ovx coronaries (208 +/- 63% of basal 20 min after stimulation) treated with serotonin + U-46619 contrasted with transient Ca(2+) responses (143 +/- 18% of basal and decreasing 5 min after stimulation) in progesterone-treated monkeys. The maximum density of [1S-(1I,2J(5Z),3I(1E,3R*),4I)]-7-[3-(3-hydroxy-4-(4'-[(125)I]iodophenoxy)- 1-butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5-heptenoic acid ([(125)I]-BOP) binding was greater (P < 0.01) in carotid arteries and aortic membranes from Ovx (109 +/- 11 fmol/mg) compared with progesterone-treated (43 +/- 15 fmol/mg) monkeys. TxA(2)R immunolabeling revealed greater coronary TxA(2)R labeling in Ovx compared with progesterone-treated monkeys. The results suggest that progesterone can decrease arterial TxA(2)R in Ovx monkeys.

Signal transduction events in Chinese hamster ovary cells expressing human CD14; effect of endotoxin desensitization.

Previous studies suggest that endotoxin (LPS) stimulation of CD14 receptors may be coupled to heterotrimeric G proteins. However, characterization of the G protein-coupled signaling pathways is incomplete. Also, specific changes in the transduction pathways occur in a phenomenon known as LPS tolerance or desensitization induced by prior exposure to LPS. In the present study, we examined potential CD14-dependent G protein-coupled signaling events in response to LPS, and changes in signaling in these pathways during LPS desensitization in Chinese Hamster Ovary (CHO) cells. LPS stimulated inhibitory kappa B alpha (IkappaB alpha) degradation and p38 phosphorylation in CHO cells transfected with human CD14 receptor (CHO-CD14), but not in CHO cells transfected with vector only. However, activation of these signaling events diverged early in the signal transduction pathways. Pretreatment with pertussis toxin, which inactivates inhibitor G protein (G alpha i) function, significantly inhibited LPS-induced p38 phosphorylation, but not LPS-induced IkappaB alpha degradation. Mastoparan, a putative G alpha i agonist, synergized with LPS to induce p38 phosphorylation. Thus, LPS stimulation of p38 phosphorylation is, in part, G alpha i coupled, whereas IkappaB alpha degradation is not. In subsequent studies, CHO-CD14 cells were desensitized by prior LPS exposure. LPS-desensitized cells exhibited augmented IkappaB alpha content and were refractory to LPS-induced IkappaB alpha degradation and p38 phosphorylation. Pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the effect of LPS desensitization on augmenting cellular IkappaB alpha content and its refractoriness to LPS-induced degradation. However, cycloheximide pretreatment did not prevent impaired p38 phosphorylation in desensitized cells. IkappaB alpha upregulation in LPS tolerance may occur through increased synthesis and/or induction of protein that suppress IkappaB alpha degradation. The latter protein synthesis-dependent mechanisms may be distinct from mechanismis inhibiting p38 phosphorylation in tolerance. These findings suggest that LPS tolerance induces CD14-dependent signaling alterations in G alpha i-coupled pathways leading to mitogen-activated (MAP) kinase activation as well as G alpha i-independent pathways inducing IkappaB alpha degradation.

Disposition and metabolism of the flavonoid chrysin in normal volunteers.

AIMS: To describe the oral disposition of the dietary flavonoid chrysin in healthy volunteers. METHODS: Oral 400 mg doses of chrysin were administered to seven subjects. Chrysin and metabolites were assayed in plasma, urine and faeces by h.p.l.c. RESULTS: Peak plasma chrysin concentrations were only 3-16 ng ml(-1) with AUCs of 5-193 ng ml(-1) h. Plasma chrysin sulphate concentrations were 30-fold higher (AUC 450-4220 ng ml(-1) h). In urine, chrysin and chrysin glucuronide accounted for 0.2-3.1 mg and 2-26 mg, respectively. Most of the dose appeared in faeces as chrysin. Parallel experiments in rats showed high bile concentrations of chrysin conjugates. CONCLUSIONS: These findings, together with previous data using Caco-2 cells, suggest that chrysin has low oral bioavailability, mainly due to extensive metabolism and efflux of metabolites back into the intestine for hydrolysis and faecal elimination.

Prostaglandin J(2) inhibition of mesangial cell iNOS expression.

Mesangial cells from MRL/lpr mice, a model of lupus, overproduce nitric oxide (NO) compared to controls. J series prostaglandins (PG) and thiazolidinediones block LPS stimulation of NO production via the activation of peroxisome proliferator-activator receptor-gamma (PPAR-gamma) in macrophages but utilize an alternative mechanism in microglial cells. We investigated the mechanism by which PGJ(2) inhibits NO production in LPS/IFN-gamma-stimulated MRL/lpr mesangial cells. Our results demonstrated that LPS/IFN-gamma addition to MRL/lpr mesangial cells stimulated iNOS activation, expression of p-38 kinase and p44/42 MAPK, and NF-kappaB translocation to the nucleus. Both pioglitazone, a specific PPAR-gamma agonist, and PGJ(2) blocked NO production, iNOS protein expression, and iNOS mRNA transcription. PGJ(2) failed to inhibit nuclear NF-kappaB translocation or p44/42 MAPK or p-38 kinase induction in stimulated mesangial cells. These data suggest that PGJ(2) blocks iNOS expression and subsequent NO production in mesangial cells via a PPAR-gamma-mediated mechanism either by interfering with NF-kappaB transcriptional activity or by an NF-kappaB-independent mechanism.

Suppression of Cox-2 and TNF-alpha mRNA in endotoxin tolerance: effect of cycloheximide, antinomycin D, and okadaic acid.

Lipopolysaccharide (LPS)-tolerant human promonocytic THP-1 cells produce decreased levels of inflammatory mediators such as eicosanoids and tumor necrosis factor alpha (TNFalpha) in response to LPS. We hypothesized that transcriptional repression by newly synthesized proteins may be a mechanism for the reduced cellular response to a secondary challenge with LPS. THP-1 cells were desensitized after a 3.5 h or 20 h pre-exposure to LPS (1 microg/mL) and subsequently challenged with LPS (10 microg/mL). In cells rendered tolerant by exposure to LPS for 20 h, LPS-induced expression of cyclooxygenase (Cox)-2 and TNFalpha mRNA was suppressed. Cycloheximide (10 microM) prevented the transcriptional down-regulation of Cox-2 mRNA and to a lesser extent, TNFalpha mRNA, in LPS-tolerant cells. Transcriptional arrest with actinomycin D stabilized steady-state expression of Cox-2 mRNA in naive and tolerant cells but destabilized TNFalpha mRNA expression in LPS-tolerant cells. The observation that in naive cells Cox-2 and TNFalpha mRNA levels subside at 3 to 4 h after LPS (10 microg/mL or 1 microg/mL) suggested that LPS tolerance may occur earlier. Therefore, in subsequent experiments, the effect of LPS pretreatment for only 3.5 h was examined. This abbreviated tolerance regimen diminished secondary LPS-induced Cox-2 mRNA expression but had a lesser effect on TNFalpha mRNA expression. However, cycloheximide augmented both Cox-2 and TNFalpha mRNA expression in this group. Also, the serine/threonine phosphatase inhibitor okadaic acid augmented Cox-2 and TNFalpha mRNA expression in the LPS-tolerant cells. Although LPS-induced TNFalpha production in LPS-tolerant cells was suppressed relative to the naive cells, okadaic acid induced comparable levels of TNFalpha in tolerant and naive cells. These findings support the concept that LPS tolerance is associated with induction of proteins that alter expression of certain genes. Expression of Cox-2 mRNA appears to be particularly sensitive to down-regulation and, to a lesser extent, TNFalpha mRNA. However, this seems to vary depending on the LPS pretreatment regimen. The ability of a phosphatase inhibitor to induce TNFalpha and expression of Cox-2 and TNFalpha mRNA in LPS tolerance suggests that there may be alterations in phosphorylation status of signaling pathways, transcriptional mechanisms, or post-transcriptional mRNA stability.

Inhibition of mesangial cell nitric oxide in MRL/lpr mice by prostaglandin J2 and proliferator activation receptor-gamma agonists.

MRL/Mp-lpr/lpr (MRL/lpr) mice develop immune complex glomerulonephritis similar to human lupus. Glomerular mesangial cells are key modulators of the inflammatory response in lupus nephritis. When activated, these cells secrete inflammatory mediators including NO and products of cyclooxygenase perpetuating the local inflammatory response. PGJ2, a product of cyclooxygenase, is a potent in vitro inhibitor of macrophage inflammatory functions and is postulated to function as an in vivo inhibitor of macrophage-mediated inflammatory responses. We hypothesized that in lupus, a defect in PGJ2 production allows the inflammatory response to continue unchecked. To test this hypothesis, mesangial cells were isolated from MRL/lpr and BALB/c mice and stimulated with IL-1beta or LPS plus IFN-gamma. In contrast to the 2- to 3-fold increase in PGJ2 production by stimulated BALB/c mesangial cells, supernatant PGJ2 did not increase in MRL/lpr mesangial cell cultures. NO production in stimulated MRL/lpr and BALB/c mesangial cells, was blocked by PGJ2 and pioglitazone. These studies suggest that abnormalities in PGJ2 production are present in MRL/lpr mice and may be linked to the heightened activation state of mesangial cells in these mice.

Coupling of thromboxane A2 receptor isoforms to Galpha13: effects on ligand binding and signalling.

Previous subtyping of thromboxane A2 (TXA2) receptors in platelets and vascular smooth muscle cells was based on pharmacological criteria. Two distinct carboxy-terminal splice variants for TXA2 receptors exist and they couple to several different G protein alpha subunits including Galpha13, but it has not been established whether either or both isoforms interact with and signal through it. We sought to determine: (1) which TXA2 receptor isoforms exist in vascular smooth muscle, (2) if Galpha13 is present in vascular smooth muscle and (3) if Galpha13 interacts with either or both of the two TXA2 receptor isoforms as determined by changes in ligand binding properties and generation of intracellular signals. Both TXA2 receptor isoforms and Galpha13 were found in vascular smooth muscle cells. Both the alpha and beta isoforms of the TXA2 receptors were transiently transfected with or without Galpha13 into COS-7 (radioligand binding assays) or CHO cells (agonist induced Na+/H+ exchange). Co-expression of each receptor isoform with Galpha13 significantly (P<0.05) increased the affinity of each receptor for the two agonists, I-BOP and ONO11113, and decreased the affinity of the receptor for the antagonists, SQ29,548 and L657,925. I-BOP stimulated Na+/H+ exchange in vascular smooth muscle cells. Co-expression of Galpha13 with each TXA2 receptor isoform in CHO cells resulted in a significant (P<0.04) agonist induced increase in Na+/H+ exchange compared to cells not transfected with Galpha13. The results support the possibility that the previous classification of TXA2 receptor subtypes based on pharmacological criteria reflect unique interactions with specific G protein alpha subunits.

Tolerance to LPS decreases macrophage G protein content.

The effects of tolerizing doses of LPS on mRNA and protein levels of three different G protein subunits were investigated to understand the mechanism(s) responsible for the reduction in Gialpha protein content in LPS tolerance. Tolerance was induced in rats using Salmonella enteritidis LPS (intraperitoneal route) with a single dose of 100 microg/kg. Peritoneal macrophages were harvested 6 and 24 h later. In some studies, a second dose of LPS 500 microg/kg was given on the following day, and peritoneal macrophages were harvested 5 days after the first injection. Macrophage RNA or a crude membrane fraction was prepared from macrophages, and the mRNA level or the protein content for Gialpha3, Gialpha2, and Gsalpha was analyzed using Northern or Western blots, respectively. Compared with the control levels, the message for Gialpha3 was reduced (p < .025) at 6 and 24 h and 5 day time periods after LPS treatment. The Gialpha2 mRNA was increased relative to the control levels (p < .05) at 6 h and 5 days after LPS treatment, respectively, and Gsalpha message was not significantly changed. The half-life of Gialpha3 mRNA was not significantly different in control versus tolerant macrophages. The Gialpha3 mRNA and membrane protein were not significantly changed by incubation with LPS for intervals up to 6 h in vitro. Macrophage membrane Gialpha3 and Gialpha1 and 2 protein content from tolerant rats were reduced compared with the controls at 6 and 24 h, respectively (p < .05). These studies are consistent with our previous observations of selective changes in macrophage Gialpha protein content in LPS tolerance and raise the possibility that this may affect signal transduction events in these cells.

Expression, characterization, and purification of C-terminally hexahistidine-tagged thromboxane A2 receptors.

Thromboxane A2 (TxA2) receptors belong to the class of G-protein-coupled receptors. Knowledge of the relationship of structure to function for TxA2 receptors is limited because of their low levels of expression, lengthy purification procedures and poor recoveries. A C-terminal hexahistidine-tag (C-His) was ligated to the alpha-isoform of TxA2 receptors and expressed in COS-7 and Chinese hamster ovary cells. The C-His-TxA2 receptors bound the radioligands 125I-7-[(1R,2S,3S,5R)-6, 6-dimethyl-3-(4-benzenesulfonylamino)bicyclo[3.1. 1]hept-2-yl]-5(Z)-heptenoic acid, an antagonist, and 125I-[1S-1alpha, 2beta(5Z),3alpha(1E,3S*), 4alpha]-7-[3[(3-hydroxy-4-(4'-phenoxy)-1butenyl)-7-oxabicycl o-[2.2. 1]heptan-2-yl]-5-heptanoic acid, an agonist, with affinities not significantly different from those of the wild type (wt)-TxA2 receptors. LipofectAMINE transfection of the cDNAs resulted in high levels of expression (Bmax = 95 +/- 6 pmol/mg) of the C-His-TxA2 receptors. In competition binding studies the IC50 values of five different ligands were not significantly different between C-His-TxA2 and wt-TxA2 receptors. Agonist-induced stimulation of cAMP and total inositol phosphate formation were not significantly different between the two receptors. Purification on a Ni2+-NTA column resulted in a rapid (within 4 h) purification with a 36 +/- 2% recovery and a 30 +/- 6-fold purification (n = 5). The partially purified receptors were resolved on SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, dissolved in acetone/trifluoroacetic acid/hexafluoroisopropanol/sinapinic acid, and successfully subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results suggest that the combination of a high level of expression of C-His-TxA2 receptors and a rapid purification procedure followed by SDS- polyacrylamide gel electrophoresis may provide a useful approach for mass-spectrometry based structure-function and other studies of TxA2 receptors.

Increased prostacyclin and PGE2 stimulated cAMP production by macrophages from endotoxin-tolerant rats.

Sublethal administration of lipopolysaccharide (LPS) renders rats tolerant to multiple lethal stimuli. Tolerant macrophages exhibit differential alterations in LPS-stimulated cytokine and inflammatory mediator release. Increased cAMP levels stimulated by PGE2 or prostacyclin (PGI2) result in differential effects on LPS-induced cytokine release and protect against the pathophysiological changes of endotoxemia. In the present studies, we sought to determine whether PGE2- and PGI2-stimulated cAMP levels are altered in tolerant macrophages. Incubation of macrophages with cicaprost or 11-deoxy-PGE1 in the presence of phosphodiesterase inhibitors resulted in significantly higher (2.5- to 6.5-fold) cAMP concentrations in tolerant macrophages compared with control. In contrast, isoproterenol-stimulated cAMP levels were not significantly different between control and tolerant cells. Also, incubation of tolerant macrophages with LPS did not result in significantly elevated cAMP levels. Prostacyclin (IP) receptor mRNA levels were significantly increased in tolerant cells compared with controls, whereas [3H]PGE2 binding and PGE2 EP4 receptor mRNA levels were not significantly changed. These studies suggest that LPS tolerance induces selective alterations in eicosanoid regulation of cAMP formation.

Mechanism of platelet inhibition by nitric oxide: in vivo phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase.

Nitric oxide (NO) is a potent vasodilator and inhibitor of platelet activation. NO stimulates production of cGMP and activates cGMP-dependent protein kinase (G kinase), which by an unknown mechanism leads to inhibition of Galphaq-phospholipase C-inositol 1, 4,5-triphosphate signaling and intracellular calcium mobilization for several important agonists, including thromboxane A2 (TXA2). To explore the mechanism of platelet inhibition by NO, activation of platelet TXA2 receptors in the presence of cGMP was studied. The nonhydrolyzable analog 8-bromo-cyclic GMP (8-Br-cGMP) potently inhibited activation of the TXA2-specific GTPase in platelet membranes in a concentration-dependent fashion, suggesting that G kinase catalyzes the phosphorylation of some proximal component of the receptor-G protein signaling pathway. Nanomolar concentrations of G kinase were found to catalyze the phosphorylation of platelet TXA2 receptors in vitro, but not Galphaq copurifying with the TXA2 receptors in these experiments. Using immunoaffinity methods, in vivo phosphorylation of TXA2 receptors by cyclic GMP was demonstrated from 32P-labeled cells treated with 8-Br-cGMP. Peptide mapping studies of in vivo phosphorylated TXA2 receptors demonstrated cGMP mediates phosphorylation of the carboxyl terminus of the TXA2 receptor. G kinase also catalyzed the phosphorylation of peptides corresponding to the cytoplasmic tails of both alpha and beta forms of the receptor but not control peptide or a peptide corresponding to the third intracytoplasmic loop of the TXA2 receptor. These data identify TXA2 receptors as cGMP-dependent protein kinase substrates and support a novel mechanism for the inhibition of cell function by NO in which activation of G kinase inhibits signaling by G protein-coupled receptors by catalyzing their phosphorylation.

Cloning and characterization of an endogenous COS-7 cell thromboxane A2 receptor.

A cDNA for a thromboxane A2 (TXA2) receptor was cloned from an SV40 transformed African Green Monkey kidney cell line (COS-7). The sequence is 98% homologous with the isoform of the human TXA2 receptor and has agonist and antagonist ligand binding characteristics that are not significantly different from the human receptor. Stimulation of the COS-7 cells with the TXA2 receptor agonist, ONO 11113 resulted in a significant increase in cAMP formation that was blocked by a receptor antagonist. The results raise the question of the utility of the COS-7 cell line for studies of cloned and expressed TXA2 receptor signalling mechanisms.

Endotoxin tolerance alters macrophage membrane regulatory G proteins.

Administration of sublethal doses of endotoxin (LPS) or tumor necrosis factor-alpha (TNF alpha) renders rats tolerant to supralethal doses of LPS. Peritoneal macrophages from tolerant rats are refractory to LPS induced arachidonic acid (AA) metabolism and cytokine production in vivo, and exhibit reduced membrane GTPase activity and GTP gamma S binding. Since LPS stimulated AA metabolism is mediated by Gi alpha proteins, we sought to determine whether Gi alpha and/or other G proteins are reduced in LPS tolerance. Rats were rendered tolerant by two daily sublethal doses of Salmonella enteritidis LPS, 100 micrograms/kg and 500 micrograms/kg administered intraperitoneally. Animals were allowed to rest for 72 hours. Alternatively, tolerance to LPS was induced by sublethal administration of human recombinant TNF alpha (10 micrograms/kg) intraperitoneally 24 hrs before the experiments. Macrophage membrane G protein content was determined by immunoblot analysis with specific antisera to Gi1,2 alpha, Gi3 alpha, Gs alpha and the G protein beta subunits (G beta). Membrane G proteins were differentially decreased in tolerant macrophages. In macrophages from rats rendered tolerant by sublethal doses of LPS, Gi3 alpha was reduced the most to 48 +/- 8% of control (n = 3, P < 0.05) and this reduction was significant compared to those of other G proteins. Gi1,2 alpha and G beta were reduced to 73 +/- 5% (n = 3, P < 0.05) and 65 +/- 4% (n = 3, P < 0.05) of control respectively. Gs alpha(L) and Gs alpha(H) were also reduced to 61 +/- 5% (n = 3, P < 0.05) and 68 +/- 3% (n = 3, P < 0.05) of control, respectively. In contrast, only Gi3 alpha was reduced in macrophage membranes from rats pretreated with TNF alpha. Gi3 alpha was reduced to 57 +/- 11% of control (n = 4, P < 0.05) whereas Gi1,2 alpha and G beta were not significantly affected. These results demonstrate selective changes in tolerant macrophage membrane G proteins and suggest a potential role for Gi3 alpha in mediating LPS tolerance. The molecular mechanisms underlying these changes and their significance in LPS tolerance merit further investigation.