Reactivity of rat abdominal aorta to U46619 following whole-body gamma irradiation

Rats exposed to 20 Gy whole-body irradiation demonstrated a depressed aortic responsiveness to the thromboxane mimic, U46619, 48 h postirradiation. The mechanism for this observed response was investigated. Shielding the abdominal aorta attenuated this altered vascular reactivity. Since this suggests that radiation exposure induces local changes in the aorta, vascular smooth muscle function was assessed with cumulative concentrations of KCl. Radiation-induced smooth muscle damage was insufficient to account for the decreased reactivity to U46619. Next, calcium availability for vascular smooth muscle function was evaluated and found not to be responsible for the radiation-induced depression in aortic responsiveness. Finally, the role that cyclooxygenase products play in the depressed contractile response was investigated. Indomethacin treatment prior to and for 48 h after irradiation attenuated the altered vascular reactivity to U46619. These data suggest that a radiation-induced increase in cyclooxygenase products may play a role in the decreased aortic reactivity to the thromboxane mimic.     

Conformational changes in human integrin alphaIIbbeta3 after platelet activation, monitored by FRET

Integrin alpha(IIb)beta(3), an abundant heterodimeric receptor at the surface of blood platelets, binds adhesive proteins after platelet activation and plays a primary role in haemostasis. In solution, it has been observed mainly in two conformations: the bent and the extended forms. Based on X-ray crystallography, electron microscopy and immunochemical observations of full-length integrin ectodomains and intact integrins, it has been agreed that unactivated integrins are in the bent conformation, both isolated in solution and in living cells. However, consensus is yet to emerge on the bent or extended conformation of activated integrins and on their mechanism of activation (the switchblade, the deadbolt and the S-S reduction models), which require further experimental tests at the cell level to become established facts. Here, we tested the proposed structural rearrangements undergone by integrin alpha(IIb)beta(3) after cell activation, by using F?rster-type fluorescence resonance energy transfer (FRET) and attached fluorescent labels to Fab fragments of monoclonal antibodies directed to the betaA domain of the beta(3) subunit (donor, Alexa488-P97 Fab) and to the Calf-2 domain of the alpha(IIb) subunit (acceptor, Cy3-M3 Fab or Cy3-M10 Fab). The FRET efficiencies observed after ADP or TRAP platelet activation changed less than 20% from the resting values, showing that the distance between the labeled Fab fragments changes only modestly after platelet activation by physiological agonists. This observation is consistent with a conformational model of the activated integrin in the cell less extended than in the switchblade model.     

Endothelin-1 and U46619 potentiate selectively the venous responses to nerve stimulation within the perfused superior mesenteric vascular bed of the rat

The effects were examined of endothelin-1 and U46619 on the responses to perivascular nerve stimulation of the simultaneously perfused arterial and venous vessels of the superior mesenteric arterial bed of the rat. Stimulation of the nerves at 4-16 Hz for 30 s caused frequency dependent constrictions of both the arterial and venous vessels similar to those produced by bolus doses of exogenous noradrenaline (0.1-10 nmol). Infusion of either endothelin-1 (0.1 nM) or U46619 (1-3 nM) caused small (less than or equal to 5 mmHg) increases in arterial and venous perfusion pressures and selectively potentiated the venous, but not arterial, responses to nerve stimulation. Conversely, endothelin-1 and U46619 potentiated the responses of both the arterial and venous vessels to exogenous noradrenaline. Thus, as reported previously for the arterial vessels of the rat mesentery, the isolated venous vessels constrict to perivascular nerve stimulation in a frequency dependent manner. In addition, endothelin-1 and U46619 potentiate selectively the effects of nerve stimulation on the veins.     

WR2721 ameliorates the radiation-induced depression in reactivity of rat abdominal aorta to U46619

Previous studies showed that 20 Gy whole-body gamma irradiation results in a decreased response of the abdominal aorta to the stable thromboxane A2 (TXA2) mimic, U46619. The present study evaluated the effect of WR2721 on this radiation-induced decrease in vascular responsiveness. Rats receiving WR2721 (200 mg/kg, i.p.) 20 min before irradiation showed no depression in vascular reactivity to U46619 compared to control. The abolition of the radiation-induced decrease in vascular responsiveness was not caused by a direct vasoconstrictor action of WR2721 or its metabolites. The vascular response of rat abdominal aortic rings to KCl was unchanged after in vivo exposure to ionizing radiation. WR2721 did not alter the vascular response to KCl. These studies confirm that exposure to whole-body ionizing radiation decreased abdominal aortic vascular responsiveness to U46619. This depressed vascular reactivity can be abolished by pretreatment with the radioprotectant, WR2721. These observations may provide a rapid initial screening method for evaluating the in vivo efficacy of radioprotectant drugs.     

Effects of free and macromolecular-bound TXA2 receptor antagonist BM 1..505 on U46619-induced platelet aggregation

The goal of this study was to synthesize a macromolecular probe of the TXA₂ receptor antagonist BM13.505 which is unable to penetrate the platelet membrane for localization and characterization of the TXA₂ receptor. The active NHS-ester of BM13.505 was synthesized and purified. It was used for covalent coupling of BM13.505 to bovine serum albumin, a macromolecular carrier. Inhibitory effects of free and macromolecular bound BM13.505 on aggregatory properties of U46619-stimulated platelets were measured and compared to TXA₂ generation in platelets, as determined by TXB₂ radioimmuno assay. No inhibitory effects of free and macromolecular-bound BM13.505 on ADP- or thrombin-induced platelet aggregation were observed. Equimolar concentrations of free or macromolecular bound BM13.505 inhibited U46619-induced platelet aggregation and TXA₂ generation with equal potency. IC₅₀-values for platelet aggregation inhibition by free and macromolecular bound BM13.505 were 64 nM and 96 nM respectively. It appears that the TXA₂ receptor ligand binding site is located close to the outer membrane surface of platelets. Interaction of macromolecular bound BM13.505 with the platelet thromboxane receptor does not depend on the availability of the free carboxyl residue in BM13.505. The method for coupling a TXA₂ receptor antagonist to a macromolecule will aid in constructing probes for the localization and characterization of the TXA₂ receptor.     

Characteristics of an ADP receptor mediating platelet activation

Summary Adenosine diphosphate (ADP) is known to induce platelet shape change, aggregation and fibrinogen binding, followed by secretion. These processes are mediated by the binding of ADP to an externally oriented protein of the platelet plasma membrane. An affinity analog of ATP, a competitive inhibitor of the action of ADP, has been utilized to probe the structure and function of this receptor. FSBA (5-p-fluorosulfonylbenzoyl adenosine) covalently modifies a single protein in intact platelets with M_r = 100 000 and concomitantly inhibits platelet shape change, aggregation and fibrinogen binding. Studies on platelet membranes demonstrate non-covalent association of ADP-binding protein with actin which is also labeled by FSBA but only in isolated membranes. This finding suggests a structural and functional coupling of the receptor to the contractile process. The putative ADP receptor covalently modified with FSBA is cleaved by chymotrypsin, a process that reverses the inability of the platelets to bind fibrinogen. Thus, the M_r = 100 000 polypeptide may be involved in the proteolytic exposure of fibrinogen binding sites on the platelet surface. The ability of FSBA to inhibit platelet aggregation and fibrinogen binding by prostaglandin H₂ derivatives and epinephrine suggest that ADP is involved in these processes. However, the interaction is not at the receptor level since shape change, stimulated by PGH₂ derivatives and yohimbine (epinephrine antagonist) binding are unaffected by FSBA. Finally, the action of ADP to inhibit PGE₁- or PGI₂-stimulated adenylate cyclase appears to be mediated by a receptor distinct for the protein modified by FSBA.Abbreviation 5FSBA 5-p-fluorosulfonylbenzoyl adenosine     

Effects of prostaglandins, cAMP, and changes in cytosolic calcium on platelet aggregation induced by a thromboxane A2 mimic (U46619).

The effects of U46619, a thromboxane mimic, on cytosolic Ca2+ concentration and platelet aggregation were determined in human platelets. Cytosolic Ca2+ concentration was determined by Quin-2 fluorescence and platelet aggregation quantitated with an aggregometer. Addition of U46619 (1 x 10(-7) M) to the platelet suspension produced a rapid increase in cytosolic Ca2+ and platelet aggregation. Pretreatment of platelets with EGTA (3 x 10(-3) M), verapamil (5 x 10(-4) M), a calcium entry blocker, or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (1 x 10(-3) M), an inhibitor of intracellular Ca2+ release, either blunted or markedly delayed the rate, but not the magnitude, of increase in cytosolic Ca2+ and prevented platelet aggregation by U46619. Pretreatment of platelets with prostaglandin I2 (PGI2) (5 x 10(-8) M), PGD2 (5 x 10(-8) M), PGE1 (5 x 10(-8) M), PGF2 alpha (1 x 10(-5) M), dibutyryl cAMP (5 x 10(-3) M), or forskolin (1 x 10(-6) M) prevented both the increase in cytosolic Ca2+ and the associated platelet aggregation induced by U46619. These data suggest that U46619 may induce platelet aggregation through an increase in cytosolic Ca2+, and that both Ca2+ entry and its release from intracellular storage sites probably contribute to the increase in cytosolic Ca2+. Furthermore, the rate of the increase in cytosolic Ca2+ concentration, as well as the magnitude of the increase, appear to be critical for platelet aggregation induced by U46619. Our data are consistent with the hypothesis that PGs inhibit U46619-induced platelet aggregation by preventing the increase in cytosolic Ca2+, and that these effects may be mediated via an increase in cAMP, since they were induced by PGs and cAMP.     

Human platelet activation by an alkylacetyl analogue of phosphatidylcholine

The present study has evaluated the influence of semi-synthetic platelet-aggregating factor, (PAF) i.e., alkylacetylglycerophosphocholine, on human platelet morphology, biochemistry and function in order to determine if PAF serves as the corrective factor restoring sensitivity to refractory platelets after treatment with epinephrine. Threshold concentrations of PAF caused irreversible platelet aggregation which could be blocked by agents elevating endogenous levels or cyclic AMP or inhibited by antagonists of platelet prostaglandin synthesis and secretion. PAF did not stimulate platelets through α-adrenergic receptors or receptors for arachidonate, endoperoxides or thromboxanes. 24 h after aspirin ingestion, platelets could be aggregated irreversibly by high concentrations, but not by threshold amounts of PAF, even though they were still insensitive to arachidonate. Another less potent PAF derivative, alkenylacetylglycerophosphocholine, blocked aggregation of 24-h aspirin platelets by PAF, but did not inhibit restoration of arachidonate sensitivity and irreversible aggregation when the samples were treated first with epinephrine. Our findings indicate that threshold amounts of PAF activate human platelets in a physiologic manner and cause irreversible aggregation which is dependent on prostaglandin synthesis and the release reaction. The results do not support the concept that PAF is the mediator of the mechanism of membrane modulation through which epinephrine induces correction of the refractory state in prostaglandin I₂-treated or dissociated platelets, or cells obtained from individuals following aspirin ingestion. Thus, the mechanism of platelet membrane modulation is capable of securing irreversible aggregation of secretion, prostaglandin synthesis or PAF formation.     

Forskolin and prostacyclin inhibit fluoride induced platelet activation and protein kinase C dependent responses

Platelet activation (cytosolic [Ca2+] increase, aggregation and ATP secretion) was induced with A1F-4. This agent presumably interacts with a G protein which appears to mediate the coupling of the receptors for Ca mobilizing hormones and phospholipase C. All the A1F-4 evoked responses were inhibited by treatment with forskolin or prostacyclin, agents known to increase cellular cAMP. Thus the G protein-phospholipase C system appears to be the site of cAMP inhibition. Unexpectedly forskolin and prostacyclin also inhibited secretion and aggregation induced by the activators of protein kinase C, diglyceride and phorbol ester, suggesting that cAMP can also inhibit directly the protein kinase C dependent responses.     

Cardiac responses in relation to heart size

There is a correlation between heart size and the propensity to develop and maintain fibrillation. Atrial and ventricular fibrillation is more easily induced and sustained in large than in small hearts. But other factors are at work as well, such as the ability to hibernate. The hibernator's heart has an adrenergic innervation which is different in distribution than that in nonhibernators with adrenergic nerves in the ventricles exclusively accompanying the blood vessels, thus leaving the myocardium proper without adrenergic innervation. This indicates that the risk of inhomogeneity of the electrophysiological parameters of the myocardial cells at a high sympathetic tone is less in the heart of a hibernator than in that of a nonhibernator.     

Effects of thromboxane analog U46619 on endothelial damaged canine coronary arteries in vivo

We studied the effects of the thromboxane analog, U46619, infused into the left anterior descending (LAD) artery of intact dogs before and after producing endothelial denudation of the mid portion of the LAD. Proximal artery cross-sectional area (CSA) decreased by 47% with 0.1 microgram/min infusion of U46619 with intact and denuded endothelium, while resting CSA reduced spontaneously following denudation. Coronary resistance vessels demonstrated a marked constrictor response to U46619 with a rise in resistance and a fall in flow and myocardial O2 consumption. U46619 produces significant narrowing of proximal epicardial coronary arteries as well as resistance coronary vessels. This effect could cause ischemia in patients with moderate coronary atherosclerosis.     

Acute inhibition of superoxide formation and Rac1 activation by nitric oxide and iloprost in human vascular smooth muscle cells in response to the thromboxane A2 analogue, U46619

BACKGROUND: The over-production of superoxide (O(2)(-)) derived from NADPH oxidase (NOX) plays a central role in cardiovascular diseases. By contrast, nitric oxide (NO) and prostacyclin (PGI(2)) are vasculoprotective. The effect of the NO donor, NONOate and iloprost on O(2)(-) formation, p47(phox) and Rac(1) activation in human vascular smooth muscle cells (hVSMCs) was investigated. METHODS: hVSMCs were incubated with 10nM thromboxane A(2) analogue, U46619 for 16h, and then with apocynin (a NOX inhibitor), NONOate or iloprost for 1h and O(2)(-) measured spectrophometrically. The role of cyclic AMP and cyclic GMP was examined by co-incubation of drugs with protein kinase (PK) A and G inhibitors listed above. Rac(1) was studied using pull-down assays. RESULTS: NONOate and iloprost inhibited O(2)(-) formation, acutely, effects blocked by inhibition of PKG and PKA, respectively. Rac(1) and p47(phox) activation and translocation to the plasma membrane was completely inhibited by NONOate and iloprost, effects again reversed by co-incubation with PKG or PKA inhibitors. CONCLUSIONS: NO and PGI(2) block the acute activity of NOX in hVSMCs via the cGMP-PKG axis (for NO) and by the cAMP-PKA axis (for iloprost) through inhibition of Rac(1) and p47(phox) translocation. These findings have implications in the pathophysiology and treatment of CVD.     

Antagonism by ethanol of endotoxin-induced tissue factor activation in relation to the depressed endotoxin binding to monocyte-like U937 cells

Our previous study has reported that ethanol (ETOH) partially inhibited the endotoxin (LPS)-induced tissue factor (TF)-activation in monocytes including blood peripheral monocytes as well as cultured leukemic U937 and THP-1 cells. The present study shows a strong correlation (r=0·92; p<0·01) between TF-activation and depression in LPS binding blocked by ETOH in U937 cells. The antagonism by ETOH of LPS binding was not due to a direct extracellular blockade, since ETOH did not affect the affinity of fluorescein isothiocyanate (FITC)-LPS or -anti CD14 mAb on U937 cells. After U937 cells were treated with 2 per cent (v/v) ETOH for 3 h, LPS binding was however drastically inhibited as shown by immunostaining with FITC-LPS which was viewed on a confocal laser scanning microscope. The results imply that cellular events of the ETOH effect mediate this inhibition of LPS binding. Anti-CD14 mAb (UCHM-1) inhibited LPS binding in a dose-dependent fashion, revealing a competitive specific binding to the LPS receptor. The results suggest that CD14 plays an important role in the recognition of LPS. FITC-UCHM-1 binding was significantly reduced in the cells pretreated with 2 per cent (v/v) ETOH for 3 h, indicating that ETOH modulates the ability to express CD14. CD14 expression was upregulated by priming with LPS which was offset by ETOH. Acetaldehyde, a possible metabolite of ETOH, was tested with no effect on CD14 expression. Taken together, our results show that ETOH downregulates the recognition of LPS, and suggest that the inhibitory action is likely to be mediated by the depression in CD14 expression which was also accompanied by a significantly altered membrane fluidity. Thus, the antagonism by ETOH of the binding of LPS results in a depression in the LPS-induced TF-activation. © 1997 John Wiley & Sons, Ltd.     

U46619 and carbocyclic thromboxane A2-induced increases in tracheal mucous gel layer thickness

U46619 or carbocyclic thromboxane A2 (CTA2) administered intravenously (IV) to rats produced dose-related increases of tracheal mucous gel layer thickness. Significant gel thickening was observed at doses ranging from 3.5 pg to 35 ng and from 500 pg to 50 ng for U46619 and CTA2, respectively. Intravenous treatment with pinane thromboxane A2 (PTA2), a thromboxane antagonist, prior to injection of U46619 or CTA2 attenuated the mucous gel layer response. The effect of PTA2 on U46619 and CTA2 was dose-dependent over the dosage range of PTA2 tested (1.0-31.6 micrograms/kg). PTA2 was equiactive against U46619 and CTA2 stimulated increases in gel thickness (ED50's 6.64 and 6.43 micrograms/kg respectively) suggesting a similar site of action for U46619 and CTA2. Slow reacting substance (SRS) injected IV into rats stimulated mucous gel layer thickening that was also inhibited by pretreatment with PTA2. These findings lend further support for the role of thromboxane in pathophysiologic conditions in which bronchorrhea contributes to the symptomatology.     

Grazing responses in herbs in relation to herbivore selectivity and plant traits in an alpine ecosystem

Herbivores shape plant communities through selective foraging. However, both herbivore selectivity and the plant’s ability to tolerate or resist herbivory may depend on the density of herbivores. In an alpine ecosystem with a long history of grazing, plants are expected to respond to both enhanced and reduced grazing pressures, and the interaction between plant traits and changes in species abundance are expected to differ between the two types of alteration of grazing regime. To understand the mechanisms behind species response, we investigated the relationship between sheep selectivity (measured in situ), plant traits and experimentally derived measures of change in species abundance as a response to the enhancement (from low to high density) or cessation (from low to zero density) of sheep grazing pressure over a six-year time period for 22 abundant herb species in an alpine habitat in south Norway. Sheep selected large, late-flowering herbs with a low leaf C/N ratio. Species that increased in abundance in response to enhanced grazing pressure were generally small and had high root/shoot ratios, thus exhibiting traits that reflect both resistance (through avoidance) and tolerance (through regrowth capacity) strategies. The abundance of selected species remained stable during the study period, and also under the enhanced grazing pressure treatment. There was, however, a tendency for selected species to respond positively to cessation of grazing, although overall responses to cessation of grazing were much less pronounced than responses to enhanced grazing. Avoidance through short stature (probably associated with increased light availability through the removal of tall competitors) as well as a certain amount of regrowth capacity appear to be the main mechanisms behind a positive response to enhanced grazing pressure in this study. The plant trait perspective clearly improves our insight into the mechanisms behind observed changes in species abundance when the disturbance regime is altered. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

G13 is an essential mediator of platelet activation in hemostasis and thrombosis

Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A(2) (TXA(2)) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through G(q), G(i) and G(z) (refs. 4-6). However, the role and relative importance of G(12) and G(13), which are activated by various platelet stimuli, are unclear. Here we show that lack of Galpha(13), but not Galpha(12), severely reduced the potency of thrombin, TXA(2) and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Galpha(13) deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G(13)-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.     

Conformational changes and complement activation induced upon antigen binding to antibodies

The interaction between antibodies specific for the loop region of lysozyme and this monovalent antigenic determinant was compared to their interaction with either the dimeric derivative of the loop (bis-loop) or the polyvalent antigen loop-A--L. This comparison was based on complement fixation capacity, and on spectroscopic changes in the circular polarized fluorescence (CPL) of the antibodies. The binding of the loop to its antibodies causes spectroscopic changes, assigned to conformational changes in the Fc region, which are not accompanied by complement activation. However, the binding of the bisloop led to distinctly different CPL changes and concomitantly to complement fixation comparable to that induced upon binding of the loop-A--L. Complement fixation and the CPL changes can be induced in the intact antibodies only; reduction of the interchain disulfide bridges, or cleavage by papain or pepsin digestion abolishes both activities.