Stimulation by intragastrically administered E2 prostaglandins of human gastric mucus output

Abstract. Prostaglandin E₂ and 15(R)15 methyl prostaglandin E₂ were instilled intragastrically to study gastric mucus output in healthy male subjects during an infusion of pentagastrin. Mucus was measured by determining total, free, and bound N-acetyl neuraminic acid (NANA) in gastric recoveries. NANA is a sialic acid located at the end terminals on the carbohydrate chains of mucus glycoprotein. It contributes to viscosity and prevents enzymatic degradation of mucus.
The methyl analogue of prostaglandin E₂ increased the gastric output of NANA and inhibited gastric acid secretion in a dose-dependent fashion. NANA produced in response to the analogue was bound to mucus glycoprotein. Prostaglandin E₂ increased the output of NANA without affecting the gastric acid secretion. Both prostaglandin E₂ and its methyl analogue increased volumes, pH, and NANA content of gastric aspirates withdrawn prior to start of the pentagastrin infusion, indicating a stimulation of the alkaline gastric secretion.
The results show that oral E₂ prostaglandins have dual effects on gastric secretion, and that their ability to stimulate mucus production is not secondary to gastric acid inhibition. Further studies are needed to examine whether their effect is mainly to increase the incorporation of NANA into mucus glycoproteins, or to stimulate the release and/or production of gastric mucus, and also to quantitate the gastric nonparietal secretion.
The stimulatory properties of E₂ prostaglandins on gastric mucus and alkaline secretion may be one mechanism by which they protect the gastric mucosa against experimentally induced damage.     

Activation of GPVI by collagen is regulated by α2β1 and secondary mediators

Summary.  We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin α₂β₁ in the activation of washed platelets by collagen in the presence of the α_IIbβ₃ antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca²⁺ elevation and dense granule secretion are more severely suppressed by the above inhibitors. α₂β₁ blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca²⁺ that is delayed in the presence of the above inhibitors and which is accompanied by α-granule secretion. These results demonstrate that secondary mediators and α₂β₁ modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of α₂β₁ is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of α₂β₁.     

Functionally inactive apolipoprotein E3 in a type III hyperlipoproteinaemic patient

Abstract. Type III hyperlipoproteinaemia (HLP) is, amongst others, characterized by the E_2/2 phenotype as determined by isoelectric focusing of apolipoprotein E. However, one of our clinically symptomatic type III HLP patients showed a E_3/3 phenotype.
After complexation with phospholipid vesicles, apo E from this patient was, in contrast with apo E from a type IV HLP patient (E_3/4 phenotype), unable to compete with low density lipoprotein (LDL) for binding to the specific LDL receptors on cultured human fibroblasts. This defect in binding to the LDL receptor was not due to an impaired lipid binding capability. The clinical symptomatic type III hyperlipoproteinaemia of our patient is probably due to a functionally inactive apo E₃.     

Prostaglandin E2 (PGE2) induces headache in healthy subjects

The role of prostanoids in nociception is well established. The headache-eliciting effects of prostaglandin E₂ (PGE₂) and its possible mechanisms have previously not been systematically studied in man. We hypothesized that infusion of PGE₂ might induce headache and vasodilation of cranial vessels. PGE₂ (0.40 µg kg⁻¹ min⁻¹) or saline was infused for 25 min into 11 healthy subjects in a cross-over, double-blind study. Headache intensity was scored on a verbal rating scale from 0 to 10. In addition, we recorded mean flow in the middle cerebral artery (V_MCA) by transcranial Doppler and diameter of the superficial temporal artery (STA) by high-resolution ultrasonography. All 11 subjects reported headache on the PGE₂ day and no subjects reported headache on the placebo day ( P  = 0.001). During the immediate phase (0–30 min) ( P  = 0.005) and the postinfusion phase (30–90 min) ( P  = 0.005), the area under the curve for headache score was significantly larger on the PGE₂ day compared with the placebo day. PGE₂ caused dilatation of the STA (23.5%; 95% CI 14.0, 37.8) and the MCA (8.3%; 95% CI 4.0, 12.6). We suggest that PGE₂ induces headache by activation and sensitization of cranial perivascular sensory afferents.     

Altered release of endothelin-1,2 and thromboxane B2 from trophoblastic cells in pre-eclampsia

The aim of the study was to investigate whether pre-eclampsia is associated with an altered release of vasoactive substances from trophoblastic cells in vitro . Trophoblastic cells from 15 uncomplicated control pregnancies and 18 pre-eclamptic pregnancies at preterm (weeks 31–36; n  = 12) and term (weeks 37–40; n  = 21) were cultured for 5 days. The concentrations of angiotensin II (AII), endothelin-1,2 (ET-1,2), thromboxane B₂ (TXB₂), 6-keto-prostaglandin F_1α (6-keto-PGF1_α) and leukotriene B₄ (LTB₄) were measured daily in culture media for 5 days by radioimmunoassay. In pre-eclampsia, concentrations of ET-1,2 were decreased ( P < 0.01) at both preterm and term, TXB₂ concentrations were increased P < 0.05) only at preterm and the TXB₂–6-keto-PGF_1α ratio was increased at both preterm and term ( P < 0.01) as compared with the controls. Concentrations of AII, 6-keto-PGF_1α and LTB₄ were similar to the controls. The data suggest that pre-eclampsia is associated with a decreased release of ET-1 and an increased release of TXB₂ from trophoblastic cells in vitro     

Effects of cyclooxygenase inhibitors, prostaglandins F2α and I2, on isolated coeliac and basilar arteries of alloxan-diabetic dogs

Abstract. The effects of prostaglandin synthetase inhibitors PGF_2α and PGI₂ on the tone of isolated basilar and coeliac arteries were studied in healthy and alloxan-diabetic dogs. PGF_2α (1 μmol l^-1) produced a significantly higher tone in diabetic basilar arteries (1·15 ± 0·16 mN) than in normal cerebral vessels (0·7 + 0·10 mN). By contrast, the contractile responses of normal and diabetic coeliac arteries to PGF_2α did not differ. The cyclooxygenase inhibitors indomethacin (3 μmol l^-1) and suprofen (0·58 μmol l^-1) potentiated the PGF_2α-evoked contractions in all of the vessels studied. The percent potentiation was greater (50–60%) in the basilar arteries from alloxan-treated dogs than in normal basilar vessels (22–30%). There was not such a difference between diabetic and normal coeliac arteries. Prostacyclin produced a concentration-related relaxation in the presence of indomethacin or indomethacin + PGF_2α. The relaxant potencies of PGI₂ were similar in the vessels from metabolically healthy and diabetic dogs. The IC₅₀ values for PGI₂ were 11·6 ± 1·3 and 11·8 ± 1·8 nmol l^-1 in normal and diabetic basilar arteries, respectively; they were 25·4 ± 3·2 and 26·2 ± 3·9 nmol l^-1 in control and alloxan-treated coeliac vessels. These results indicate that normal and diabetic vessels may have differential reactivity to cyclooxygenase inhibitors, this difference being dependent on the vascular region.     

Cytoplasmic regions of the β3 subunit of integrin αIIbβ3 involved in platelet adhesion on fibrinogen under flow conditions

Summary.  Platelet adhesion to surface-bound fibrinogen depends on integrin α_IIbβ₃. In the present study, we investigated the role of the regions ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T of the β₃ cytoplasmic tail in the regulation of platelet adhesion under flow conditions, by introducing peptide mimetics in platelets. Introduction of peptide EATSTFTN (E–N) increased surface coverage by 35%, an effect caused by 25% more adhesion. In contrast, peptide TNITYRGT (T–T) decreased surface coverage by 16%, as a result of 25% less adhesion. An S→P substitution in the E–N peptide, thereby mimicking a mutation in Glanzmann's thrombasthenia, abolished the effect of E–N. A suboptimal concentration of cytochalasin D is known to enhance ligand binding to α_IIbβ₃ in platelet suspensions. Under flow, cytochalasin D (1 µmol L⁻¹) induced 50% more platelet adhesion, with a strong reduction in platelet spreading. Both peptides opposed the increase in adhesion by cytochalasin D and partly (E–N) and completely (T–T) restored platelet spreading. Thus, the ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T regions of β₃ contribute to the regulation of αIIbβ3 anchorage to the cytoskeleton and platelet spreading to an adhesive surface.     

Effects of prostaglandin-E1 on ST segment elevation and regional myocardial blood flow during experimental myocardial ischaemia in dogs

Abstract. The effects of prostaglandin E₁ (PGE₁) on myocardial ischaemia (as measured by epicardial ST segment changes), myocardial flow and substrate exchange has been studied in dogs. Myocardial ischaemia was induced by intermittent external clipping of a branch of the left anterior descending coronary artery.
During occlusion with a continuous intravenous infusion of isoprenaline, elevated epicardial ST segments (∑ST), were raised to 46 ± 6 mV (mean ± SEM). Pretreatment with PGE₁ reduced ∑ST to 34 ± 6 mV ( P <0.001), but had no effect on mean aortic blood pressure (AP¯) or on regional myocardial blood flow. Isoprenaline infusion increased plasma free fatty acids (FFA) to 1925 ± 150 μmol/l and this was reduced to 1320 ± 220 μmol/l by PGE₁ ( P <0.005).
During occlusion without isoprenaline, PGE₁ did not effect ∑ST or plasma FFA when infused intravenously or into the left atrium. Mean aortic blood pressure decreased from 131 ± 7 to 108 ± 10 (PGE₁ i.v.) or 109 ± 8 mmHg (PGE₁ i.a.) ( P <0.001). This was associated with a decrease in regional myocardial blood flow, both in the ischaemic and non-ischaemic myocardium. However, when blood pressure was maintained constant, intravenous PGE₁ tended to decrease occlusion-induced ST segment elevation.
These results suggest that PGE₁ can reduce isoprenaline-stimulated myocardial ischaemia through its antilipolytic action but in the absence of catecholamine stimulation the main effect is to reduce blood pressure thereby counterbalancing any potentially beneficial effects on the ischaemic myocardium.     

The Effect of Ergotamine on Prostaglandin Formation in Guinea Pig''s Ear Slices

SYNOPSIS
We have studied the effect of ergotamine tartrate (10⁻⁷ and 10⁻⁵ M) on prostaglandin (PG) formation in guinea pig's ear slices because of the suggested role of PGs in the pathophysiology of the migraine attack. The ear slices were incubated with arachidonic acid (AA) and the amount of different PGs (6-keto-F_1a, F_2a, E₂, D₂ and A₂) in the incubation medium was determined. The results indicate that ergotamine is an inhibitor of PG synthesis and that it probably affects either the phospholipase A₂ or the cyclo-oxygenase activity.     

Radioimmunoassay of prostaglandins Falpha, E1 and E2 in human plasma.

Antibodies against prostaglandins (PG)F2alpha, E1 and E2 were obtained inrabbits immunized with respectively PG F2alpha, PG E1 and PG E2 conjugated to bovine serum albumin by carbodiimide. A radioimmunoassay capable of measuring 7 pg of PG Falpha, 2 pg of PG E2 and 14 pg of PG E1 in human peripheral plasma is described. Plasma samples (pH 3, citric acid) are extracted with cyclohexane: ethyl acetate, 1:1 and then chromatographed on silicic acid columns to separate the prostaglandins into three fractions: fraction I, PG A, PG B and some unknown immunoreactive compounds; fraction II, PG E and fraction III, PG Falpha. The recovery is 80+ +/- 6.2. Mean plasma levels in adults of PG Falpha and PG E, expressed in pg/ml: -PG Falpha 12 +/- 2.8 (n- 25 men), 8 +/- 2.3 (n= 18 women, follicular phase), 7 +/- 1.4 (n= 18 women, luteal phase). -PG E1 40.5 +/- 7.6 (n= 13 men), 38 +/- 17.1 (n= 10 women). -PG E2 4.5 +/- 1 (n= 12 adult subjects). The major characteristics of the method described herein are the following: - a large volume of plasma has to be processed (10 ml or more for PG Falpha and PG E1, 5 ml or more ofr PG E2). - a chromatographic step is necessary to separate the different prostaglandins which makes it possible to circumvent probelms of immunological cross reactivity and interference with unknown immunoreactive compounds. - great care has been taken in collection of blood samples, especially to insure complete removal of blood cells namely platelets.     

Endothelin-1 does not modulate O2· release and [Ca2+]i variations in resting or differentiated HL-60 cells

Summary— Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing superoxide (O₂*) generation in human resting or DMSO-differentiated neutrophil-like HL-60 cells. ET-1 (0.01 – 100 nM) was not able to modulate O₂* generation stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, EC₅₀ = 4.24 ± 1.63 nM in the absence and 3.16 ± 1.95 nM in the presence of ET-1). Neither did ET-1 (0.01 – 100 nM) promote the mobilization of intracellular calcium ions or modulate fMLP-induced [Ca²⁺]_i increase in this model of human neutrophils. Phosphoramidon, a neutral endopeptidase inhibitor, was not able to reveal any biological (O₂*) or biochemical ([Ca²⁺]_i) response to ET-1 in the absence or in the presence of fMLP in these cells. These results indicate that DMSO-differentiated neutrophil-like HL-60 cells are not sensitive to ET-1 in terms of O₂* generation or [Ca²⁺]_i variations.     

CIB1 deficiency results in impaired thrombosis: the potential role of CIB1 in outside-in signaling through integrin αIIbβ3

Summary.  Background: Agonist-induced inside-out signaling activates platelet integrin α_IIbβ₃, rendering it to bind plasma fibrinogen (Fg). Fg binding induces outside-in signaling that culminates in platelet aggregation, leading to physiological hemostasis and pathological thrombosis. How outside-in signaling through α_IIbβ₃ regulates hemostasis and thrombosis is not well understood. We have previously shown that CIB1 is involved in regulating α_IIbβ₃ function. Objective: To determine the in vivo role of CIB1 in the process of hemostasis and thrombosis. Methods and Results: Genetic ablation of Cib1 significantly increased mouse tail bleeding time. Greater than 50% of the Cib1 null mice showed a rebleeding phenotype. Time taken for complete occlusion of carotid artery upon 10% FeCl₃-induced injury was significantly delayed in the absence of Cib1. This was also associated with unstable thrombus formation. The inside-out signaling appears normal as ADP-, collagen- and PAR4 peptide-induced aggregation and fibrinogen binding was unaffected. The absence of Cib1 also affected the ability of platelets to spread on immobilized Fg, but not filopodia formation. Spreading could be restored in Cib1 null platelets by the addition of exogenous ADP. Outside-in signaling-dependent tyrosine phosphorylation of the integrin β₃ subunit was significantly reduced in the absence of Cib1 as determined by Western blot analysis. Conclusion: Using gene knockout mice, we show for the first time that lack of Cib1 results in impaired thrombosis. CIB1 regulates these processes by affecting platelet spreading, but not platelet filopodia formation. These in vivo and in vitro results clearly show that CIB1 is a key regulator of thrombosis.     

Thromboxane A2 and related prostaglandins in airways

Summary— Asthma is now thought to be a chronic inflammatory disease of the airways. The roles of prostanoids, thromboxane A₂ (TXA₂) and the prostaglandins (PGs) in the pathogenesis and pathophysiology of asthma have fostered a wealth of studies but remain controversial. TXA₂ and the bronchoconstrictor PGs, PGD₂ and PGF_2α, are generated in greater amounts in asthmatic than in normal subjects. TXA₂ is a potent constrictor of airway smooth muscle, an inducer of acetylcholine release and of airway microvascular leakage. It may participate in the thickening and the remodeling of the airway wall which may contribute to the airway hyperresponsiveness, a typical feature of asthma. Strategies for inhibition of TXA₂ effects include antagonism of the TXA₂ receptor (TP receptor) and inhibition of the thromboxane synthase. TP receptor antagonists could block the effects of all the bronchoconstrictor prostanoids because TXA₂ as well as the bronchoconstrictor PGs act through activation of lung TP receptor. The recent development of specific and potent TP receptor antagonists and inhibitors of thromboxane synthase has provided tools to assess the role of TXA₂ and bronchoconstrictor PGs in the pathophysiology of asthma.     

The in vitro pharmacological profile of KR31080, a nonpeptide AT1 receptor antagonist

Summary— KR31080 (2-butyl-5-methyl-6-(1-oxopyridin-2-yl)-3-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl]methyl]-3H-imidazo[4,5-b] pyridine) is a potent inhibitor of angiotensin type 1 (AT₁) receptors in rabbit aorta and human recombinant AT₁ receptors. In the isolated rabbit thoracic aorta, KR31080 caused a nonparallel shift to the right of the concentration-response curves to angiotensin II (All) with decreased maximal response (pD'₂ = 10.1 ± 0.1), but had no effect on the contractile response induced by norepinephrine. KR31080 inhibited specific [¹²⁵I]AII binding to rabbit aortic membranes (AT, receptors) and [¹²⁵I][Sar¹, Ile⁸]AII binding to human recombinant AT₁ receptors in a concentration-dependent manner with IC₅₀ values of 0.84 ± 0.08 nM and 1.92 ± 0.15 nM, respectively, but did not inhibit specific [¹²⁵I)AII binding to bovine cerebellum membranes (ÀT₂ receptors). In the Scatchard analysis, KR31080 interacted with rabbit aortic AT₁ receptors in a competitive manner, similar to losartan. These results demonstrate that KR31080 is a potent and AT₁ selective angiotensin receptor antagonist which exerts a competitive antagonism in the [¹²⁵I]AII binding assay and insurmountable AT₁ receptor antagonism in the functional study.     

Prostaglandin - E 2 Levels in the Saliva of Common Migrainous Women

SYNOPSIS
Prostaglandin E₂ (PG-E₂) levels were measured in the saliva of 6 women suffering from common migraine, during an attack, and in the interval between attacks; the results obtained were compared to the levels found in a matched control group of healthy women. There was a significant increase in the levels of PG-E₂ during migraine attack P<0.05. These results may suggest that PG-E₂ takes an important role in the mechanism of migraine.     

Cisplatin-induced renal effects and thromboxane A2 receptor blockade

The aim of this study was to evaluate the renal protective effect of linotroban, a thromboxane A₂ receptor antagonist, in 25 patients with malignant tumours scheduled for cisplatin therapy. Cisplatin was administered 1 h after the start of a 24-h continuous infusion of linotroban or placebo. Glomerular filtration rate and effective renal plasma flow were measured. Infusions of cisplatin decreased glomerular filtration rate by 17 ± 25 mL min⁻¹ ( P  = 0.049 vs. baseline) and effective renal plasma flow by 94 ± 150 mL min⁻¹ ( P  = 0.049 vs. baseline) in the placebo group. In the linotroban group a decrease in glomerular filtration rate by 11 ± 18 mL min⁻¹ ( P  = 0.050 vs. baseline) and in effective renal plasma flow by 26 ± 63 mL min⁻¹ ( P  = 0.2 vs. baseline) was noted. However, no difference was noted between groups in response to treatment. Our findings indicate that linotroban may not be useful for prevention of cisplatin's acute nephrotoxic effects.     

Effects of high-altitude chronic hypoxia on platelet α2-receptors in man

During chronic high-altitude (HA) exposure, basal and exercise-induced noradrenaline (NA) increases do not parallel blood pressure (BP) changes observed; unlike β-adrenergic receptors, to our knowledge no data are available on α-receptors. We studied platelet α₂- and leucocyte β-receptors and basal catecholamine levels in 11 trained climbers before and after they had spent a 15-day period at a height of over 4400 m. In six of the climbers we also evaluated catecholamines after maximal bicycle ergometer exercise. After chronic high-altitude exposure, a significant decrease was found in platelet α₂-receptor density and affinity [ B _max from 92.6 ± 6.7 to 54.6 ± 4.2 fmol mg⁻¹ protein ( P  < 0.001) and K _D from 1.271 ± 0.034 to 1.724 ± 0.077 nmol L⁻¹ ( P  < 0.05)], although no changes to β-receptors were observed. No changes were found in basal pre- and post-expedition NA and adrenaline (A), and there was only a slight decrease in post-expedition NA after maximal exercise. Our results suggest that prolonged exposure to hypoxia induces a down-regulation of α₂-receptors, which may be a contributory factor in the regulation of the physiological vascular response to acclimatization.     

Multiple ways to switch platelet integrins on and off

Summary.  In the classical concept of platelet integrin activation, it is considered that unidirectional conformational changes of α_IIbβ₃ and α₂β₁ regulate the adhesiveness of platelets for fibrin(ogen) and collagen, respectively. Here, we summarize recent evidence that these conformational changes: (i) can also occur in the reverse direction; and (ii) are not independent events. Platelet stimulation through the P2Y₁₂ receptors provokes only transient α_IIbβ₃ activation via signaling routes involving phosphoinositide 3-kinases and Rap1b. Furthermore, α_IIbβ₃ can be secondarily inactivated in platelets with prolonged high Ca²⁺ rises, which expose phosphatidylserine and bind coagulation factors. Thus, platelet stimulation with strong agonists (collagen and thrombin) also results in transient integrin activation. Integrin α₂β₁ is found to be activated by a mechanism that is directly linked to α_IIbβ₃ activation. Integrin α₂β₁ can adopt different activation states, depending on the trigger. Conclusively, reversibility and synchrony of platelet integrin activation are newly identified mechanisms to restrict thrombus growth and to allow optimal coagulation factor binding. Back-shifting of activated integrins towards their resting state may be a novel goal of antithrombotic medication.     

Potentiation of platelet activation through the stimulation of P2X1 receptors

Summary.  The platelet P2X₁ purinergic receptor is a ligand-gated ion channel that responds to ATP. The precise role of P2X₁ in platelet function is unknown, though stimulation with the P2X₁ agonist α,β-Me-ATP is known to result in platelet shape change through elevation of calcium levels. The aim of the present study was to examine further the effects of P2X₁ stimulation on platelet activation. Stimulation of P2X₁ with α,β-Me-ATP resulted in shape change and small aggregate formation in heparin-anticoagulated platelet preparations. Given the ability of heparin to potentiate platelet activation, subsequent experiments were performed in hirudin. In these platelet preparations, aggregate formation in response to α,β-Me-ATP alone was less than that observed in heparin; however, α,β-Me-ATP significantly potentiated platelet aggregate formation when added in conjunction with other weak platelet agonists [epinephrine or thrombopoietin (TPO)]. Platelet aggregate formation was confirmed by single platelet loss (microaggregate formation), microscopy, and light transmittance studies. Further, the P2X₁ antagonist MRS-2159 inhibited platelet shape change and aggregation responses induced by α,β-Me-ATP. Overall, this study demonstrates that P2X₁ stimulation can induce/potentiate platelet activation in combination with other platelet agonists. These results are the first demonstration of platelet aggregation mediated through direct P2X₁ stimulation, supporting a role for this receptor in regulating platelet activation.