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G Cirino

Publications and source records attributed to G Cirino.

At least 37 records · Page 2Linked to original sources
PubMed2000

In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation.

Caveolin-1, the primary coat protein of caveolae, has been implicated as a regulator of signal transduction through binding of its "scaffolding domain" to key signaling molecules. However, the physiological importance of caveolin-1 in regulating signaling has been difficult to distinguish from its traditional functions in caveolae assembly, transcytosis, and cholesterol transport. To directly address the importance of the caveolin scaffolding domain in vivo, we generated a chimeric peptide with a cellular internalization sequence fused to the caveolin-1 scaffolding domain (amino acids 82-101). The chimeric peptide was efficiently taken up into blood vessels and endothelial cells, resulting in selective inhibition of acetylcholine (Ach)-induced vasodilation and nitric oxide (NO) production, respectively. More importantly, systemic administration of the peptide to mice suppressed acute inflammation and vascular leak to the same extent as a glucocorticoid or an endothelial nitric oxide synthase (eNOS) inhibitor. These data imply that the caveolin-1 scaffolding domain can selectively regulate signal transduction to eNOS in endothelial cells and that small-molecule mimicry of this domain may provide a new therapeutic approach.

Animals↗
PubMed2000

Anti-very late antigen-1 monoclonal antibody modulates the development of secondary lesion and T-cell response in experimental arthritis.

Rats injected in the hind paw with a mixture of Mycobacterium butirricum emulsified in mineral oil (FA) developed a severe polyarthritis that shared some immunological features with human rheumatoid arthritis. After this local administration, rats developed a secondary lesion (edema) in the contralateral paw, which is a hallmark of immune system activation. In vivo intravenous treatment with a monoclonal anti-very late antigen (VLA)-1 antibody (HA31/8) significantly reduced the edema formation in the contralateral paw. T cells isolated from contralateral paw draining lymph nodes of FA rats treated with HA31/8 showed a reduced cell proliferation in vitro, after stimulation with concanavalin A. Furthermore FACS analysis showed that the reduction in proliferation was concomitant to a reduction in the number of T cells positive to surface IL-2 receptor expression. Our data indicate that after in vivo treatment with a monoclonal anti-very late antigen-1 antibody, there is a beneficial effect on the development of the secondary lesion, which correlates to the reduced ability of T cells to proliferate in vitro as well as to a reduced surface expression of IL-2 receptor. The association of this antibody to other drugs interfering at other levels in rheumatoid arthritis may open a new therapeutic window.

Animals↗
PubMed2000

NO-naproxen modulates inflammation, nociception and downregulates T cell response in rat Freund's adjuvant arthritis.

1. Anti-inflammatory non steroidal drugs releasing NO (NO-NSAIDs) are a new class of anti-inflammatory drugs to which has been added an NO-releasing moiety. These compounds have been shown to retain the anti-inflammatory, analgesic and antipyretic activity of the parent compound but to be devoid of gastrointestinal (GI) toxicity. 2. Freund's adjuvant (FA) arthritis was induced in rats by a single intraplantar injection into the right hindpaw of 100 microl of mycobacterium butirricum (6 mg ml(-1)). The effect of equimolar doses of naproxen (1, 3 and 10 mg kg(-1)) and NO-naproxen (1.5, 4.5 and 16 mg kg(-1)) was evaluated using two dosage regimen protocols: (i) preventive, starting oral administration of the drugs at the time of induction of arthritis and for the following 21 days (day 1 - 21); (ii) therapeutic, starting oral administration of the drugs 7 days after adjuvant injection and for the following 14 days (day 7 - 21). 3. Hindpaw swelling (days 3, 7, 11, 14, 17, 21) and nociception (days 15 and 21) were measured. On day 22 rats were sacrificed, draining lymph nodes were removed and T cells isolated. In vitro proliferation of T cells following stimulation with concanavalin A (0.5 - 5 microg ml(-1)) was measured using a tritiated thymidine incorporation assay. IL-2 receptor expression on T cells was measured by FACS analysis. 4. Naproxen and NO-naproxen showed similar activity in reducing oedema formation in the non-injected (controlateral) hindpaw. Both drugs showed anti-nociceptive effect. NO-naproxen was anti-nociceptive at a dose of 4.5 mg kg(-1) while naproxen showed the same extent of inhibition only at a dose of 10 mg kg(-1). 5. T cells were isolated and characterized by FACS analysis. Stimulation of isolated T cells with concanavallin A in vitro caused a significant increase in thymidine uptake. NO-naproxen at a dose of 4.5 mg kg(-1) inhibited T cell proliferation to the same extent as 10 mg kg(-1) of naproxen. 6. Inhibition of T cell proliferation was well correlated with reduced IL-2 receptor expression on T cells. In addition, NO-naproxen reduced both IL-1beta and TNFalpha plasma levels whilst naproxen reduced IL-1beta levels only. 7. In conclusion, both naproxen and NO-naproxen reduce inflammation and nociception associated with arthritis. In addition NO-naproxen interferes to a larger extent with cellular mechanism involved in T cell activation in rat adjuvant arthritis indicating that introduction of the NO moiety in the naproxen structure increases the effect at the level of the immune system.

Animals↗
PubMed2000

Geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) mediated signal transduction has anti-inflammatory effects and interacts with glucocorticoid receptor in vivo.

Histamine, vascular endothelial growth factor, acetylcholine, oestrogen as well as fluid shear stress activates a mechanism that recruits heat shock protein 90 to the endothelial nitric oxide synthase. The interaction between Hsp90 and eNOS enhances the activation of the enzyme in cells and in intact blood vessels leading to NO production. Intraplantar administration of carrageenan (50 microl paw(-1)) to mice causes an oedema lasting 72 h. Geldanamycin (0.1, 0.3, 1 mg kg(-1)), a specific inhibitor of Hsp-90, that inhibits endothelium-dependent relaxations of the rat aorta, mesentery and middle artery inhibits carrageenan-induced mouse paw oedema in a dose dependent manner. Co-administration to mice of dexamethasone (1 mg kg(-1)) with geldanamycin (0.3 mg kg(-1)) at anti-inflammatory dose causes a loss of the total anti-inflammatory effect of each agent alone. RU 486 (10 mg kg(-1)), a well known glucocorticoid receptorial antagonist, does not inhibit oedema formation but prevents the anti-inflammatory action of dexamethasone (1 mg kg(-1)). Similarly, RU 486 prevents the anti-inflammatory action of geldanamycin (0.3 mg kg(-1)). In conclusion we have described for the first time that geldanamycin, an inhibitor of Hsp90 dependent signal transduction, is anti-inflammatory in vivo implying that Hsp90 is critical for pathways involved in carrageenan-induced paw oedema. In addition the ability of GA to block NO release and reduce oedema formation suggests a therapeutic rationale for specific inhibitors of Hsp90 as potential anti-inflammatory drugs.

Animals↗
PubMed2000

Effect of spironolactone and its metabolites on contractile property of isolated rat aorta rings.

Spironolactone and its active metabolites canrenone and potassium canrenoate are normally used as antihypertensive drugs. Although they are classified as antagonists of aldosterone, their mechanism of action cannot be ascribed solely to the regulation of ion transport in the distal tubule of nephrons. Here we have evaluated the effects of spironolactone, canrenone, and potassium canrenoate on contractile properties of isolated rat aorta rings. Spironolactone (1-300 microM), canrenone (1-300 microM), and potassium canrenoate (0.01-10 mM), in a concentration-dependent manner, relaxed rat aorta rings precontracted with phenylephrine (1 microM) or KCl (40 mM). These relaxant effects were not affected by prior treatment with either aldosterone (100 microM), glibenclamide (10 microM), or tetraethylammonium (10 mM), excluding the possibility that these drugs can be involved in either the nongenomic effect of aldosterone or on activation of potassium channels. Spironolactone and canrenone at concentrations of 30 and 100 microM, but not at 10 microM, and potassium canrenoate at concentrations of 0.3 and 1 mM, but not at 0.1 mM, significantly inhibited the phenylephrine (0.001-3 microM) concentration-response curve. Conversely, all tested concentrations of spironolactone (10, 30, and 100 microM), canrenone (10, 30, and 100 microM), and potassium canrenoate (0.1, 0.3, and 1 mM) significantly inhibited the concentration-response curve induced by cumulative concentrations of KCI (10-80 mM). Because both phenylephrine- and KCl-induced contractions imply an intracellular Ca2+ influx, we suggest that these drugs could act through an inhibition of voltage-dependent Ca2+ channels.

Animals↗
PubMed2000

Antihypertensive properties of a nitric oxide-releasing naproxen derivative in two-kidney, one-clip rats.

Nonsteroidal anti-inflammatory drugs have been reported to exacerbate hypertension. In this study, we tested the hypothesis that a nitric oxide-releasing derivative of naproxen would ameliorate hypertension in the rat. Hypertension was induced by partially occluding one renal artery (the "2K,1C" model), and 2 wk later the rats started receiving naproxen, the nitric oxide-releasing derivative HCT-3012, or vehicle each day for 2 wk. Naproxen significantly exacerbated the hypertension. HCT-3012 significantly reduced blood pressure relative to both the naproxen- and vehicle-treated groups. Both naproxen and HCT-3012 markedly suppressed whole blood thromboxane B(2) synthesis. In studies of anesthetized rats, naproxen significantly enhanced the late hypertensive response to endothelin-1 and significantly blunted the early hypotensive response. In contrast, HCT-3102 did not affect either response to endothelin-1. In vitro, HCT-3012 significantly reduced the responsiveness of aortic rings to the contractile effects of phenylephrine. These studies suggest that HCT-3012 reduces blood pressure in hypertensive rats, not simply through the vasodilatory actions of the nitric oxide it releases, but through alterations in the responsiveness of the vasculature to endogenous pressor agents.

Animals↗
PubMed1999

Protease-activated receptor-2 involvement in hypotension in normal and endotoxemic rats in vivo.

BACKGROUND: The protease-activated receptor-2 (PAR-2) is expressed by vascular endothelial cells and upregulated by lipopolysaccharide (LPS) in vitro. PAR-2 is activated by a tethered ligand created after proteolytic cleavage by trypsin or experimentally by a synthetic agonist peptide (PAR-2AP) corresponding to the new amino terminus of the tethered ligand. METHODS AND RESULTS: Intravenous administration of PAR-2AP (0.1, 0.3, and 1 mg/kg) to rats caused a dose-dependent hypotension. A scrambled peptide was without effect. A specific trypsin inhibitor, biotin-SGKR-chloromethylketone, inhibited trypsin-induced hypotension but not that stimulated by PAR-2AP. In animals treated with LPS 20 hours earlier, we found an increased sensitivity to trypsin and PAR-2AP in the hypotensive response. In particular, PAR-2AP caused hypotension at a low concentration of 30 ng/kg. Moreover, PAR-2 was immunolocalized to endothelial and smooth muscle cells in aorta and jugular vein in LPS-treated rats, and increased levels of PAR-2 mRNA were shown by reverse transcription-polymerase chain reaction analysis. CONCLUSIONS: Our findings suggest that PAR-2 is important in the regulation of blood pressure in vivo. A functional upregulation of PAR-2 by LPS was demonstrated by the activity of concentrations of PAR-2AP that were inactive in normal animals. We conclude that PAR-2 may play an important role in the hypotension associated with endotoxic shock and may represent a new therapeutic target.

Animals↗
PubMed1999

Antiphospholipid antibodies inhibit prostaglandin release by decidual cells of early pregnancy: possible involvement of extracellular secretory phospholipase A2.

OBJECTIVE: To investigate the effect of antiphospholipid antibodies on eicosanoid production by human decidual cells and the in vitro interaction between antiphospholipid antibodies and secretory phospholipase A2. DESIGN: Cultures of human decidual cells from early pregnancy. SETTING: All decidual specimens were obtained from the Obstetrics and Gynecology Department of the Catholic University, Rome, Italy. PATIENT(S): Patients were undergoing operative laparoscopy for extrauterine pregnancy, with a period of amenorrhea ranging from 6 to 9 weeks. INTERVENTION(S): Decidual samples were collected at laparoscopy by routine uterine curettage. MAIN OUTCOME MEASURE(S): Decidual cells were incubated with antiphospholipid antibodies, and eicosanoids (prostaglandin [PG] E2, PGF2alpha, and thromboxane B2) were assayed by RIA after 24 hours of culture. In vitro interactions between antiphospholipid antibodies and secretory phospholipase A2 were investigated with use of a modified ELISA for phospholipase A2. RESULT(S): Antiphospholipid antibodies reduced eicosanoid release from decidual cells in a dose-dependent fashion. In vitro assays showed that antiphospholipid antibodies bound secretory phospholipase A2 and that a competition occurred between antiphospholipid antibodies and secretory phospholipase A2 for the common substrate cardiolipin. CONCLUSION(S): In light of the critical role played by eicosanoids in decidual function, we suggest that an interaction between antiphospholipid antibodies and secretory phospholipase A2 occurring in vivo might impair important cellular communications at the decidual level in the antiphospholipid antibody syndrome.

Antibodies, Anticardiolipin↗
PubMed1999

Beneficial effects of ACE-inhibition with zofenopril on plaque formation and low-density lipoprotein oxidation in watanabe heritable hyperlipidemic rabbits.

The effects of angiotensin-converting enzyme (ACE)-inhibition with zofenopril on the development of atherosclerosis and low-density lipoprotein (LDL) oxidation were determined in Watanabe Heritable Hyperlipidemic (WHHL) rabbits. Rabbits received either placebo (n = 6) or 0.5 mg/kg/day of zofenopril (n = 6). After 6 weeks of treatment, the computer-assisted analysis revealed that zofenopril reduced the aortic and common carotid corrected cumulative lesion area by 34% and 39%, respectively (p < 0.05 vs placebo-treated group). The intimal/medial ratio of the largest fatty streaks was 0.426+/-0.158 in the zofenopril-treated group and 0.875+/-0.238 in the placebo-treated group (p < 0.05). Furthermore, we found in the zofenopril-treated group smaller lesions with an intimal/medial ratio of zofenopril also reduced plasmatic LDL oxidation, as shown by significant reduction of malondialdehyde content (p < 0.01) and relative agarose gel mobility (p < 0.05), as well as by the prolongation of the lag-time (p < 0.05). Compared to zofenopril-treated rabbits, arterial sections of the placebo-group had significant increase in the intimal presence of macrophages-derived foam cells (p < 0.05), ox-LDL (p < 0.01), and native LDL (p < 0.01) detected by immunocytochemistry with RAM-11, MDA2 and NP1533975 monoclonal antibodies, respectively. To investigate the amount of platelet accumulation in the atherosclerotic plaque we also measured platelet-associated radioactivity. Autologous platelets were labeled with 111Indiumoxine and injected intravenously. After 2 hours, WHHL were sacrificed and arterial sections were counted for platelet-associated radioactivity. In the placebo-treated group, platelet radioactivity was 0.52+/-0.12 equivalent of radioactivity per mg of tissue in the common carotid and 0.25+/-0.18 in the abdominal aorta; in contrast, rabbits treated by zofenopril had 0.20+/-0.12 in the common carotid and 0.06+/-0.01 in the abdominal aorta. These data indicate that ACE-inhibition with zofenopril has antiatherosclerotic and antioxidant effects in WHHL-rabbits. Our results also shows that these effects could be linked to a reduced wall-associated platelet deposition at the site of atherosclerotic lesions.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

Bronchoconstrictor effect of thrombin and thrombin receptor activating peptide in guinea-pigs in vivo.

1. Several thrombin cellular effects are dependent upon stimulation of proteinase activated receptor-1 (PAR-1) localized over the cellular surface. Following activation by thrombin, a new N-terminus peptide is unmasked on PAR-1 receptor, which functions as a tethered ligand for the receptor itself. Synthetic peptides called thrombin receptor activating peptides (TRAPs), corresponding to the N-terminus residue unmasked, reproduce several thrombin cellular effects, but are devoid of catalytic activity. We have evaluated the bronchial response to intravenous administration of human alpha-thrombin or a thrombin receptor activating peptide (TRAP-9) in anaesthetized, artificially ventilated guinea-pigs. 2. Intravenous injection of thrombin (100 microkg(-1)) caused bronchoconstriction that was recapitulated by injection of TRAP-9 (1 mg kg(-1)). Animal pretreatment with the thrombin inhibitor Hirulog (10 mg kg(-1) i.v.) prevented thrombin-induced bronchoconstriction, but did not affect bronchoconstriction induced by TRAP-9. Both agents did not induce bronchoconstriction when injected intravenously to rats. 3. The bronchoconstrictor effect of thrombin and TRAP-9 was subjected to tolerance; however, in animals desensitized to thrombin effect, TRAP-9 was still capable of inducing bronchoconstriction, but not vice versa. 4. Depleting animals of circulating platelets prevented bronchoconstriction induced by both thrombin and TRAP-9. 5. Bronchoconstriction was paralleled by a biphasic change in arterial blood pressure, characterized by a hypotensive phase followed by a hypertensive phase. Thrombin-induced hypotension was not subject to tolerance and was inhibited by Hirulog; conversely, hypertension was subject to tolerance and was not inhibited by Hirulog. Hypotension and hypertension induced by TRAP-9 were neither subject to tolerance nor inhibited by Hirulog. 6. Our results indicate that thrombin causes bronchoconstriction in guinea-pigs through a mechanism that requires proteolytic activation of its receptor and the exposure of the tethered ligand peptide. Platelet activation might be triggered by the thrombin effect.

Animals↗
PubMed1999

Nitric oxide-releasing NSAIDs inhibit interleukin-1beta converting enzyme-like cysteine proteases and protect endothelial cells from apoptosis induced by TNFalpha.

BACKGROUND: Nitric oxide (NO)-releasing NSAIDs are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. Although it has been demonstrated that NO-NSAIDs spare gastric mucosal blood flow, molecular determinants involved in this effect are unknown. AIM: To investigate the effect of aspirin, naproxen and flurbiprofen, and their NO-derivatives, on gastric apoptosis and endothelial cell damage induced by tumour necrosis factor-alpha (TNFalpha). In other systems, TNFalpha-induced apoptosis is mediated by caspases, a growing family of cysteine proteases similar to the IL-1beta converting enzyme (ICE), and so we have investigated whether NO-NSAIDs modulate ICE-like endopeptidases. METHODS: Rats were treated orally with aspirin, naproxen and flurbiprofen, or their NO-releasing derivatives in equimolar doses, and were killed 3 h later to assess mucosal damage and caspase activity. Endothelial cells (HUVECs) were obtained from human umbilical cord by enzymatic digestion. Caspase 1 and 3 activities were measured by a fluorimetric assay using selective peptides as substrates and inhibitors. Apoptosis was quantified by ELISA specific for histone-associated DNA fragments and by the terminal transferase nick-end translation method (TUNEL). RESULTS: In vivo NSAID administration caused a time-dependent increase in gastric mucosal damage and caspase activity. NCX-4016, NO-naproxen and NO-flurbiprofen did not cause any mucosal damage and prevented cysteine protease activation. NSAIDs and NO-NSAIDs stimulated TNFalpha release. Exposure to TNFalpha resulted in a time- and concentration-dependent HUVEC apoptosis, an effect that was prevented by pretreating the cells with NCX-4016, NO-naproxen, NO-flurbiprofen, SNP or Z-VAD.FMK, a pan-caspase inhibitor. The activation of ICE-like cysteine proteases was required to mediate TNFalpha-induced apoptosis of HUVECs. Exogenous NO donors inhibited TNFalpha-induced cysteine protease activation. Inhibition of caspase activity was due to S-nitrosylation of ICE/CPP32-like proteases. NO-NSAIDs prevented IL-1beta release from endotoxin-stimulated macrophages. CONCLUSIONS: NO-releasing NSAIDs are a new class of non-peptide caspase inhibitors. Inhibition of ICE-like cysteine proteases prevents endothelial cell damage induced by pro-inflammatory agents and might contribute to the gastro-protective effects of NO-NSAIDs.

Animals↗
PubMed1999

Nitric oxide as a signaling molecule in the vascular system: an overview.

In retrospect, basic research in the fields of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) during the past two decades appears to have followed a logical course, beginning with the findings that NO and cGMP are vascular smooth muscle relaxants, that nitroglycerin relaxes smooth muscle by metabolism to NO, progressing to the discovery that mammalian cells synthesize NO, and finally the revelation that NO is a neurotransmitter mediating vasodilation in specialized vascular beds. A great deal of basic and clinical research on the physiologic and pathophysiologic roles of NO in cardiovascular function has been conducted since the discovery that endothelium-derived relaxing factor (EDRF) is NO. The new knowledge on NO should enable investigators in this field to develop novel and more effective therapeutic strategies for the prevention, diagnosis, and treatment of numerous cardiovascular disorders. The goal of this review was to highlight the early research that led to our current understanding of the pathophysiologic role of NO in cardiovascular medicine. Furthermore, we discussed the possible mechanism of some drugs interfering with NO signaling cascade.

Animals↗
PubMed1999

Mechanisms of prostaglandin E2 release by intact cells expressing cyclooxygenase-2: evidence for a 'two-component' model.

Prostaglandin (PG) release in cells expressing constitutive cyclooxygenase-1 is known to be regulated by liberation of arachidonic acid by phospholipase A2 followed by metabolism by cyclooxygenase. However, the relative contribution of phospholipase A2 to the release of PGs in cells expressing cyclooxygenase-2 is not clear. We addressed this question by using radioimmunoassay to measure PGE2 release by human cells (A549) induced to express cyclooxygenase-2 (measured by Western blot analysis) by interleukin-1beta. Cells were either unstimulated or stimulated with agents known to activate phospholipase A2 (bradykinin, Des-Arg10-kallidin, or the calcium ionophore A23187) or treated with exogenous arachidonic acid. When cells were treated to express cyclooxygenase-2, the levels of PGE2 released over 15 min were undetectable; however, in the same cells stimulated with bradykinin, A23187, or arachidonic acid, large amounts of prostanoid were produced. Using selective inhibitors/antagonists, we found that the effects of bradykinin were mediated by B2 receptor activation and that prostanoid release was due to cyclooxygenase-2, and not cyclooxygenase-1, activity. In addition, we show that the release of PGE2 stimulated by either bradykinin, A23187, or arachidonic acid was inhibited by the phospholipase A2 inhibitor arachidonate trifluoromethyl ketone. Hence, we have demonstrated that PGE2 is released by two components: induction of cyclooxygenase-2 and supply of substrate, probably via activation of phospholipase A2. This is illustrated in A549 cells by a clear synergy between the cytokine interleukin-1beta and the kinin bradykinin.

Adrenergic beta-Agonists↗
PubMed1999

Nitric oxide-releasing NSAIDs: GI-safe antithrombotics.

Aspirin is increasingly being used for long-term prophylaxis of myocardial infarction and stroke, but its use is limited by toxicity in the gastrointestinal tract. Even very low doses of aspirin can markedly increase the risk of gastrointestinal bleeding and ulceration. While proven effective in prophylaxis of stroke and myocardial infarction, the efficacy of aspirin is limited. Addition of a nitric oxide-releasing moiety to several non-steroidal anti-inflammatory drugs results in a profound reduction in their toxicity in the gastrointestinal tract and kidney. A similar derivatization of aspirin has recently been shown to result in a more potent, gastrointestinal-sparing antithrombotic drug. Two such compounds (NCX-4215 and NCX-4016; NicOx SA) have undergone detailed evaluation thus far. In each case, the NO-aspirin has shown improved anti-aggregatory activity while not inducing detectable gastric damage. The compounds have also been shown to exert protective effects in the gastrointestinal tract exposed to other injurious agents. The NO-aspirin derivatives significantly inhibit leukocyte adherence to the vascular endothelium, which may contribute to their anti-thrombotic activity. NO-releasing derivatives of aspirin and naproxen also exhibit beneficial effects in experimental hypertension, which would also contribute to improved anti-thrombotic activity. NO-releasing derivatives of NSAIDs offer great potential as gastrointestinal-sparing anti-thrombotic drugs.

Journal Article↗
PubMed1998

Dynamic activation of endothelial nitric oxide synthase by Hsp90.

Heat-shock protein 90 (Hsp90) coordinates the trafficking and regulation of diverse signalling proteins, but its precise role in regulating specific cellular targets is not known. Here we show that Hsp90 associates with endothelial nitric oxide synthase (eNOS) and is rapidly recruited to the eNOS complex by agonists that stimulate production of nitric oxide, namely vascular endothelial growth factor, histamine and fluid shear stress. Moreover, the binding of Hsp90 to eNOS enhances the activation of eNOS. Inhibition of signalling through Hsp90 attenuates both agonist-stimulated production of nitric oxide and endothelium-dependent relaxation of isolated blood vessels. Our results indicate that Hsp90 facilitates signalling mediated by growth-factor, G-protein and mechanotransduction pathways that lead to the activation of eNOS. These observations indicate that in addition to its role as a molecular chaperone involved in protein folding and maturation, Hsp90 may also be recruited to cellular targets depending on the activation state of the cell.

Animals↗
PubMed1998

Hypotension and inflammatory cytokine gene expression triggered by factor Xa-nitric oxide signaling.

The signaling pathway initiated by factor Xa on vascular endothelial cells was investigated. Factor Xa stimulated a 5- to 10-fold increased release of nitric oxide (NO) in a dose-dependent reaction (0.1-2.5 microG/ml) unaffected by the thrombin inhibitor hirudin but abolished by active site inhibitors, tick anticoagulant peptide, or Glu-Gly-Arg-chloromethyl ketone. In contrast, the homologous clotting protease factor IXa or another endothelial cell ligand, fibrinogen, was ineffective. A factor Xa inter-epidermal growth factor synthetic peptide L (83)FTRKL(88) (G) blocking ligand binding to effector cell protease receptor-1 inhibited NO release by factor Xa in a dose-dependent manner, whereas a control scrambled peptide KFTGRLL was ineffective. Catalytically active factor Xa induced hypotension in rats and vasorelaxation in the isolated rat mesentery, which was blocked by the NO synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) but not by D-NAME. Factor Xa/NO signaling also produced a dose-dependent endothelial cell release of interleukin 6 (range 0.55-3.1 ng/ml) in a reaction inhibited by L-NAME and by the inter-epidermal growth factor peptide Leu(83)-Leu(88) but unaffected by hirudin. Maximal induction of interleukin 6 mRNA required a brief, 30-min stimulation with factor Xa, unaffected by subsequent addition of tissue factor pathway inhibitor. These data suggest that factor Xa-induced NO release modulates endothelial cell-dependent vasorelaxation and cytokine gene expression. This pathway requiring factor Xa binding to effector cell protease receptor-1 and a secondary step of ligand-dependent proteolysis may preserve an anti-thrombotic phenotype of endothelium but also trigger acute phase responses during activation of coagulation in vivo.

Animals↗
PubMed1998

Mast cell degranulation induced by two phospholipase A2 homologues: dissociation between enzymatic and biological activities.

Bothropstoxin-I and bothropstoxin-II are phospholipase A2 homologues isolated from Bothrops jararacussu snake venom. The former is devoid of phospholipase A2 activity whereas the latter has very low enzymatic activity. In this study, we have investigated the in vivo (rat paw and skin oedema) and in vitro (mast cell degranulation) inflammatory effects caused by bothropstoxin-I and bothropstoxin-II. Bothropstoxin-I (25-100 microg/paw) and bothropstoxin-II (12.5-50 microg/paw) caused dose-dependent rat paw oedema. The intradermal injection of bothropstoxin-I (0.125-5 microg/site) and bothropstoxin-II (0.125-5 microg/site) into rat skin also resulted in dose-dependent oedema formation. These oedematogenic activities were largely reduced in animals pretreated with the histamine/5-hydroxytryptamine (5-HT) receptor antagonist cyproheptadine (2 mg/kg, i.p. 0.5 h before). Similarly, p-bromophenacyl bromide, a compound known to inhibit phospholipase A2 activity, significantly inhibited rat paw and skin oedema induced by both phospholipase A2 homologues. The polyanion heparin (5 IU/site) significantly reduced the rat skin oedema induced by either bothropstoxin-I or bothropstoxin-II as well as the paw oedema (50 IU/site) induced by the former. When assayed in the rat peritoneal mast cells in vitro, both bothropstoxin-I (10 and 100 microg/ml) and bothropstoxin-II (3 and 10 microg/ml) significantly caused [14C]5-HT release. The [14C]5-HT release caused by these phospholipase A2 homologues were reduced by p-bromophenacyl bromide and heparin (50 IU/ml). Our results indicate that oedema formation induced by bothropstoxin-I and bothropstoxin-II is mostly dependent on in vivo mast cell degranulation. Since heparin greatly reduced the oedematogenic activity of these phospholipase A2 homologues, it is likely that the cationic charge of these substances plays a major role in the mast cell activation. Our results also indicate that p-bromophenacyl bromide may not be a suitable pharmacological tool to investigate the correlation between enzymatic activity and the inflammatory effects of phospholipases A2.

Animals↗