Structure of mammalian steroid receptors: evolving concepts and methodological developments.
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Dissociation of TNF-alpha from endotoxin-induced nitric oxide and acute-phase hypotension.
We tested the concept that tumor necrosis factor-alpha (TNF-alpha) or platelet-activating factor (PAF) mediated Escherichia coli endotoxin lipopolysaccharide (LPS)-induced upregulation of nitric oxide (NO) and acute-phase hypotension (APH) in the rat. LPS (0.5 mg/kg i.v.) given to rats treated with saline or nonimmune goat-derived gamma-globulin (immunoglobulin G, 22 mg/kg i.m.) produced APH and increased plasma concentrations of TNF-alpha and nitrate and nitrite anions (reactive nitrogen intermediates; RNI) and NO in ex vivo incubates of polymorphonuclear neutrophils (PMN) and inducible NO synthase (iNOS) mRNA in PMN. Pretreatment of rats with a polyclonal TNF-alpha antibody (TNF-Ab, 22 mg/kg i.m.) abolished LPS-mediated increases in plasma TNF-alpha but failed to inhibit APH or the NO system. TNF-alpha (8.2 micrograms/kg i.v.) produced transient hypertension and sustained tachycardia and increased plasma TNF-alpha and PMN iNOS mRNA but not RNI. LPS and TNF-alpha decreased spontaneous and calcimycin (Ca2+ ionophore, 1 microM)- and PAF (1 microM)-mediated increases in head-space NO production by rings of mesenteric artery incubated ex vivo. TNF-Ab abolished all effects of TNF-alpha. PAF (25, 50, and 100 ng/kg) produced APH without increasing plasma TNF-alpha, RNI, or PMN iNOS mRNA. The PAF receptor antagonist BN-50730 (80 micrograms/kg i.v.) abolished PAF-induced APH and attenuated LPS-induced increases in RNI. We conclude that 1) LPS produces parallel but unrelated changes in TNF-alpha and RNI in plasma and PMN during the APH of endotoxemia; and 2) endogenous TNF-alpha is not required for LPS-mediated induction of iNOS mRNA, and PAF mediates LPS-induced APH.
Animals↗
Mechanisms of fibrin formation and lysis by human lung fibroblasts: influence of TGF-beta and TNF-alpha.
Fibrin gels form within the alveolar and interstitial compartments of the injured lung, and fibroblasts invade and facilitate organization of these transitional gels. We studied the effects of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) on fibrinolytic and procoagulant activities of human lung fibroblasts (HLF) to determine their capacity to regulate pulmonary fibrin deposition. Fibrinolytic activity of cell lysates and media (n = 6 HLF cultures) were uniformly depressed by TGF-beta or TNF-alpha. In dose and time-course studies, HLF plasminogen activator inhibitor-1 (PAI-1) was increased by TGF-beta, whereas TNF-alpha induced release of PAI-1 into the media. HLF and media urokinase concentrations were depressed by TGF-beta, whereas urokinase was unchanged or increased by TNF-alpha. Tissue plasminogen activator was mainly cell associated and unchanged by TGF-beta or TNF-alpha. HLF antiplasmin activity was not detected. Plasma recalcification times of HLF media were decreased by TNF-alpha but unchanged by TGF-beta. These studies suggest that TGF-beta and TNF-alpha impair the ability of HLF to degrade fibrin by disturbing the balance of HLF plasminogen activators and PAI and that these cytokines concurrently leave unchanged or increase the capacity of HLF to initiate fibrin formation. Cytokines likely to occur in the injured lung induce abnormalities of fibrinolysis in HLF from adults; such abnormalities favor extravascular fibrin deposition, a characteristic feature of alveolitis.
Base Sequence↗
The anti-platelet agent, ticlopidine, upregulates interleukin-1-Beta-stimulated nitric oxide production in cultured rat vascular smooth muscle cells.
BACKGROUND: Hemodialysis patients who had been treated with anti-platelet aggregation drugs, including ticlopidine, sometimes developed hypotension. The mechanism by which ticlopidine lowers the blood pressure in hemodialysis patients is unclear. To elucidate the mechanism of the action of this drug, we investigated cytokine-stimulated nitric oxide (NO) metabolism by ticlopidine in cultured rat vascular smooth muscle cells (VSMC). METHODS: Nitrite, a stable metabolite of NO, and intracellular cAMP and cGMP contents were assayed by the Griess method and enzyme immunoassay, respectively. iNOS mRNA and protein expressions were analyzed by Northern blotting and Western blotting. RESULTS: Ticlopidine enhanced interleukin-1beta (IL-1beta)-induced nitrite production in a dose- and time-dependent manner. The mRNA and protein expressions of inducible NO synthase were upregulated by ticlopidine in a dose- and time-dependent manner. IL-1beta alone stimulated both intracellular cAMP and cGMP contents, and the addition of ticlopidine further enhanced their contents. KT 5720, a selective inhibitor of protein kinase A, but not KT 5823, a selective inhibitor of protein kinase G, abolished the enhancement of IL-1beta-induced nitrite production by ticlopidine. In addition, a phosphodiesterase inhibitor, isobutylmethylxanthine, enhanced IL-1beta and ticlopidine induced nitrite production. CONCLUSION: We concluded that ticlopidine enhanced the IL-1beta-induced NO production via cAMP and subsequent activation of protein kinase A in cultured rat VSMC.
Animals↗
Platelet activation induces cell-surface immunoreactive tissue factor expression, which is modulated differently by antiplatelet drugs.
OBJECTIVE: Tissue factor (TF) is the main activator of the coagulation cascade occurring in physiologic and pathologic conditions. Recent data suggest that human platelets might contain TF that is possibly derived from leukocytes. In this study, we investigated whether intraplatelet TF can be exposed on the membrane by platelet agonists. The modulation of this process by antiplatelet drugs has been evaluated as well. METHODS AND RESULTS: Flow cytometric analysis of unstimulated platelets showed a small amount of membrane-associated immunoreactive TF (irTF) in whole blood, platelet-rich plasma, and washed platelets isolated from healthy subjects. ADP, thrombin receptor-activating peptide, and epinephrine significantly increased functionally active, membrane-associated irTF. ADP induced irTF exposure in a concentration- and time-dependent fashion. Agonist-induced irTF expression was completely inhibited by iloprost but not by aspirin. Interestingly, glycoprotein IIb/IIIa antagonists did not inhibit but rather potentiated the stimulatory effect of ADP on platelet irTF expression. Real-time polymerase chain reaction experiments showed detectable amounts of TF mRNA in unstimulated platelets. CONCLUSIONS: These findings indicate that platelet agonists and antiplatelet drugs might modulate platelet-associated irTF expression. Regulated TF expression establishes the potential for a previously unrecognized role for platelets in sustaining thrombus formation and growth via coagulation-mediated mechanisms.
Antigens, Surface↗
High-dose heparin decreases nitric oxide production by cultured bovine endothelial cells.
BACKGROUND: Abrupt cessation of heparin therapy can lead to a recrudescence of thrombosis and acute ischemia. Endothelial NO is an important endogenous inhibitor of platelet-mediated thrombosis, yet biochemical studies examining the effect of heparin on NO production by the endothelium have heretofore been lacking. METHODS AND RESULTS: In an attempt to address the effect of heparin on endothelial cell production of NO, confluent bovine aortic endothelial cells (BAECs) on microcarrier beads were incubated in the presence or absence of heparin. Results indicate that BAECs incubated with heparin were less able to inhibit platelet aggregation than control cells (P<.005 by ANOVA) and that this effect correlated with a decrease in NO production (36% decrease for heparin compared with control, P<.05). Dextran sulfate evoked the same response (67% decrease, P<.0001 compared with control), suggesting that the decrease in NO after heparin treatment is secondary to its negative charge rather than to a specific polysaccharide sequence. The decrease in NO production by heparin was accompanied by a 72% decrease in steady-state Nos 3 mRNA as well as a 49% decrease in immunodetectable endothelial NO synthase (eNOS) protein. CONCLUSIONS: These data show that high-dose heparin at concentrations achieved in some acute cardiovascular settings increases in vitro platelet aggregation in media conditioned by endothelial cells by decreasing endothelial NO production through a mechanism that involves a decrease in steady-state Nos 3 mRNA and eNOS protein. These observations suggest a possible mechanism by which to explain in part the prothrombotic effects of heparin.
Animals↗
Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin.
Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT-cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin-stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca2+]i was strongly attenuated by the cGMP-PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca2+ and endothelial permeability.(ABSTRACT TRUNCATED AT 250 WORDS)
Aorta↗
PPAR gamma ligand troglitazone lowers cholesterol synthesis in HepG2 and Caco-2 cells via a reduced concentration of nuclear SREBP-2.
Cholesterol synthesis in animal cells is regulated by sterol regulatory element-binding protein (SREBP)-2. The objective of this study was to investigate whether activation of peroxisome proliferator-activatedreceptor (PPAR)-gamma influences the SREBP-2 dependent cholesterol synthesis in liver and intestinal cells. Therefore, HepG2 and Caco-2 cells were incubated with and without 10 or 30 microM of troglitazone, a synthetic PPAR gamma agonist, for 4 hrs. Incubation with 10 or 30 microM of troglitazone caused a significant, dose-dependent reduction of cholesterol synthesis in both HepG2 and Caco-2 cells (P < 0.05). HepG2 and Caco-2 cells incubated with 10 or 30 microM of troglitazone had also lower mRNA concentrations and lower nuclear protein concentrations of SREBP-2 than untreated control cells (P < 0.05). mRNA concentrations of the SREBP-2 target genes HMG-CoA reductase and LDL receptor were also reduced in HepG2 and Caco-2 cells treated with 30 microM of troglitazone compared to control cells (P < 0.05). In conclusion, this study shows that PPAR gamma activation by troglitazone lowers the cholesterol synthesis in HepG2 and Caco-2 cells by reducing the concentration of nuclear SREBP-2 and successive downregulation of its target genes involved in cholesterol synthesis.
Blotting, Western↗
Protease-activated receptors and platelet function.
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Animals↗
Short-term inhibition of ADP-induced platelet aggregation by clopidogrel ameliorates radiation-induced toxicity in rat small intestine.
Endothelial dysfunction and increased platelet aggregation may be involved in the pathogenesis of normal tissue radiation toxicity. This study assessed clopidogrel, an inhibitor of ADP-induced platelet aggregation, as a modulator of intestinal radiation injury (radiation enteropathy). Rat small intestine was exposed to 21 Gy X-radiation. Clopidogrel (20 mg/kg/day) or vehicle was administered from 2 days before to 10 days after irradiation. Structural radiation injury, neutrophil infiltration, smooth muscle cell proliferation, collagen content, and TGF-beta1 expression were assessed 2 weeks (early phase) and 26 weeks (delayed phase) after irradiation, using quantitative histology and immunohistochemistry, morphometry, and real-time fluorogenic probe RT-PCR. Irradiated intestine exhibited significant histopathologic injury, reduced mucosal surface area, vascular sclerosis, intestinal wall fibrosis, increased collagen content, and increased TGF-beta1 expression. Clopidogrel reduced ADP-induced platelet aggregation by 93% and significantly attenuated the severity of post-radiation vascular sclerosis (p = 0.004 and p = 0.02) and the loss of mucosal surface area (p = 0.0008 and p = 0.003) at both 2 and 26 weeks. Clopidogrel also ameliorated overall histopathologic injury (p = 0.02), relative intestinal collagen content (p = 0.03), and collagen III immunoreactivity levels 2 weeks after irradiation, and caused a borderline reduction in the radiation-induced increase in extracellular matrix-associated TGF-beta immunoreactivity at 26 weeks (p = 0.04). The effects of clopidogrel on steady-state TGF-beta1 mRNA levels and neutrophil infiltration were not statistically significant. Short-term clopidogrel administration affords protection against early and, to a lesser extent, delayed radiation enteropathy. Modulation of platelet aggregation should be subject to further studies as a potential method to increase safety and efficacy of radiation therapy.
Adenosine Diphosphate↗
Dilazep and dipyridamole inhibit tissue factor expression on monocytes induced by IgG from patients with antiphospholipid syndrome.
AIM: To investigate whether antiplatelet agents, dilazep and dipyridamole, inhibit tissue factor (TF) expression on monocytes induced by IgG from patients with antiphospholipid syndrome (APS). METHODS: Freshly isolated peripheral blood monocytes were allowed to adhere on plastic and then cultured in media containing patient or control antibodies and/or other agonists with or without dilazep or dipyridamole. The TF activity on monocytes was investigated by measuring factor VIIa-dependent generation of factor Xa, using a chromogenic substrate and the TF mRNA expression was examined by real-time PCR (TaqMan PCR). RESULTS: The TF activity on monocytes induced by APS IgG (250 mg/L) was inhibited by dilazep (0.15-150 micromol/L) and dipyridamole (0.2-200 micromol/L) in a dose-dependent fashion. But, the TF mRNA expression induced by APS IgG was not inhibited. Theophylline (500 micromol/L), an adenosine receptor antagonist, could counteract the inhibitory effect of dilazep and dipyridamole on TF activity. CONCLUSION: Antiplatelet agents, dilazep and dipyridamole, block APS IgG-induced monocytes TF expression at a post-transcriptional level, partly by adenosine receptor pathway. Pharmacological agents that block monocytes TF activity, such as dilazep and dipyridamole, are a novel therapeutic approach in APS.
Adrenergic Antagonists↗
[Effects of essentiale forte on biosynthesis of nucleic acids and several hemodynamic parameters of the damaged myocardium in rabbits].
Normalization of heart muscle phospholipid metabolism and reactivation of the activity of membrane-bound enzymes participating in the reactions of biosynthesis of nucleic acids have been studied. These investigations were carried out under the conditions of experimental myocardial infarction. The effects of essential forte have been examined when it was combined with both a tocopherol, and sodium nucleinate. It was pointed out that essential forte using without other reagents leads to the depression of nucleic acids biosynthesis in affected heart muscle, while the combinations of it with a-tocopherol, and especially with sodium nucleinate are accompanied by the significant intensification of reactions of formation of desoxynucleic and ribonucleic acids biosynthesis in necrotic areas as well as in peripheral regions of affected myocardial tissue. These positive changes are accompanied also by the pronounced normalization of haemodynamic activity of infarcted myocardium.
Adjuvants, Immunologic↗
[Chemical composition, metabolism and aggregation of thrombocytes].
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Adenosine Diphosphate↗
[Synthetic estrogens--new pharmacological actions and mechanisms].
Many constitutional analogues of estrogen have been reported. In this review, some new synthetic estrogens possessing a different action from natural estrogens are described. Aminoestrogens inhibit platelet aggregation. Estradiol-chlorambucil conjugate (KM2210) decreases the level of estrogen receptor mRNA in the human breast cancer cell MCF-7. The representative synthetic estrogen, diethylstilbestrol, and its derivatives show an inhibitory action against H(+)- and Ca(2+)-ATPase. Moreover, the effect of the diastereometric [1,2-bis(2,6-dihalo-3-hydroxyphenyl)ethylenediamine ] sulfatoplatinum (II) complexes, derivatives of hexesterol-platinum complexes, on estrogenic action and the growth-inhibitory action of hormone dependent breast cancer, are strongly dependent on the halogen atoms and on their position in the aromatic ring. These drugs are expected to be applicable for clinical use.
Animals↗
Protein-protein interactions in high molecular weight forms of the transformation-related phosphoprotein p53.
The transformation-related cellular phosphoprotein p53 interacts with a variety of viral and cellular proteins and with itself to form high molecular weight complexes. The formation of high molecular weight complexes correlates with the transformed morphology of the cells whereas in non-transformed cells low molecular weight forms are predominant. Thus, aggregation seems to be involved in the regulation of biological functions of p53. Analyzing wild-type and mutant p53 in the same cellular environment i.e. after an in vitro transcription/translation reaction in rabbit reticulocytes we found high molecular weight forms for wild-type and mutant p53. The sedimentation profile resembled the profile obtained for mutant p53 from transformed cells. As shown by dilution experiments, aggregation of p53 was not due to high p53 protein concentrations. Although p53 is known to bind RNA, treatment with RNAse did not change the aggregation state of p53 suggesting that RNA may not contribute to the quaternary structure of p53. High molecular weight aggregates of p53 were resistant to treatment with 1 M NaCl and also stable in weak acidic conditions. Alkaline pH as well as treatment with 3.5 M NaCl led to a disaggregation of high molecular weight complexes of p53. This treatment resulted in low molecular weight forms consisting probably of dimers to tetramers whereas monomers of p53 are hardly detectable. A nearly complete disaggregation was obtained only with the ionic denaturing detergent sodium dodecyl sulfate. Therefore, one has to assume different types of protein-protein interactions leading to the various quaternary structures of p53.
Animals↗
Basophil histamine release by nuclear components in chronic bronchitis and bronchial asthma of intrinsic type.
The involvement of an allergic reaction directed against nuclear components and aggregated IgG was examined by basophil histamine release technique in chronic bronchitis (CB) in the intrinsic type of bronchial asthma (IA). In the group of 21 CB patients, seven responded to RNA, DNA or histone and three responded to aggregated IgG. In the other group of seven IA patients one responded to RNA and histone. In comparison with healthy blood donors the prevalence of smooth muscle antibodies was increased in CB patients and antinuclear antibodies were increased in both groups of patients. The findings suggest an involvement of an allergic reaction directed against RNA, DNA, histone, and aggregated IgG in some patients with CB and IA. This may play some role in the reversible airway obstruction of these diseases.
Adult↗
Breakdown of rat-liver ergosomes in vivo after actinomycin inhibition of messenger RNA synthesis.
Ribosomes isolated from rat liver occur predominantly in the form of aggregates (ergosomes) corresponding to multiples of 73S particles held together by messenger RNA. After injecting rats with actinomycin, these aggregates gradually break down in vivo to 73S monomers and 113S dimers. We conclude that the observed breakdown results from the degradation of messenger RNA and the prevention by actinomycin of the synthesis of new messenger RNA.
Animals↗
Coat protein interactions involved in tobacco mosaic tobamovirus cross-protection.
To investigate the molecular role of the tobacco mosaic tobamovirus (TMV) coat protein (CP) in conferring cross-protection, a potato X potexvirus (PVX) vector (S. Chapman, Plant J. 2, 549-557, 1992) was used to systemically express a set of TMV mutant CPs in Nicotiana benthamiana prior to challenge inoculation with TMV. PVX-expressed wild-type TMV CP delayed TMV accumulation for up to 2 weeks compared to unprotected plants or plants preinfected with the unmodified PVX vector. Similar delays in TMV accumulation were obtained using TMV CPs that were deficient in virion formation but competent to assemble into helical aggregates. In contrast, TMV CPs that were incapable of helical aggregation or unable to bind viral RNA did not delay the accumulation of TMV. Furthermore, TMV CPs with enhanced intersubunit interactions that favor helical aggregation produced significantly greater delays in the accumulation of challenge TMV than obtained from the wild-type CP. Thus the capabilities of TMV CP to interact with viral RNA and self-associate in a helical fashion appear to be essential to its ability to confer protection. Taken together, these findings support a model for CP-mediated resistance in which the protecting CP recoats the challenge virus RNA as it disassembles.
Capsid Proteins↗