The purinergic receptor P2Y2 receptor mediates chemotaxis of dendritic cells and eosinophils in allergic lung inflammation.

BACKGROUND: Extracellular ATP contributes to the pathogenesis of asthma via signalling at purinergic receptors. However, the precise purinergic receptors subtypes mediating the pro-asthmatic effects of ATP have not been identified, yet. METHODS: In vivo studies were performed using the OVA-alum model. Functional expression of the P2Y(2) purinergic receptor subtype on human monocyte-derived dendritic cells and eosinophils was investigated using real-time PCR, migration assays, and production of reactive oxygen species. RESULTS: Compared to wild-type animals P2Y(2) -/- mice showed reduced allergic airway inflammation which can be explained by defective migration of blood myeloid DCs towards ATP in vitro and in vivo, whereas the influence of ATP on maturation and cytokine production was not changed. Additionally, ATP failed to induce migration of bone marrow-derived eosinophils from P2Y(2) R-deficient animals. The relevance of our findings for humans was confirmed in functional studies with human monocyte-derived DCs and eosinophils. Interestingly, stimulation of human DCs derived from allergic individuals with house dust mite allergen induced functional up-regulation of the P2Y(2) R subtype. Furthermore, eosinophils isolated from asthmatic individuals expressed higher levels of P2Y(2) R compared to healthy controls. This was of functional relevance as these eosinophils were more sensitive to ATP-induced migration and production of reactive oxygen metabolites. CONCLUSIONS: In summary, P2Y(2) R appears to be involved in asthmatic airway inflammation by mediating ATP-triggered migration of mDCs and eosinophils, as well as reactive oxygen species production. Together our data suggest that targeting P2Y(2) R might be a therapeutic option for the treatment of asthma.

[Reimbursement and rational prescription of laboratory tests]. / Financement de la biologie clinique et bonne prescription des analyses.

Following the important growth of the Belgian laboratory medicine budget in the eighties, the mechanisms of reimbursement by the social security system have become more and more complex in the last 20 years. The current system is a dual one, with a lump sum complemented by an amount per test. The rules differ for hospitalized and non-hospitalized patients. New recently launched measures ("reference amounts") intend to decrease the prescription of laboratory tests in hospitals, while others targeting non-hospital practice are being considered. Beside these purely financial initiatives targeting the laboratories, another approach involves fostering a rational prescription of tests according to the results of interventional trials or international guidelines consistent with evidence-based medicine. The recent report of the KCE on laboratory tests prescription by general practitioners is consistent with this strategy.

Determination of urinary iodine by inductively coupled plasma mass spectrometry.

BACKGROUND: Mild iodine deficiency is endemic in many countries of Europe including Belgium. Fast, accurate and specific methods for quantification of urinary iodine are needed. We describe in this report a specific ICP-MS method for the quantification of urinary iodine. METHOD: Samples and iodate calibrators were diluted 20 times into aqueous solution containing triton X-100, 1.5% HCl and (103)Rh as an internal standard. Prior digestion or oxidation was not necessary. Results were compared with those obtained by Sandell-Kolthoff (S-K) spectrophotometric method. RESULTS: Comparison of both methods showed good agreement. The Passing-Bablok regression between both methods was ICP-MS=0.986 (S-K)-7.51. The Bland-Altman difference plot showed a small but significant mean difference of -13.3 microg/L for ICP-MS. The between-day coefficient of variation (CV) was 13% at 89 microg/L. Limit of detection was 4 microg/L and limit of quantification was 20 microg/L. No carryover effect has been observed on series containing up to 50 samples. CONCLUSION: The ICP-MS method described here is fast, accurate and specific for the quantification of urinary iodine. Compared to the S-K method the urinary iodine concentrations measured by the ICP-MS method were slightly, but significantly lower. Consequently, the results of studies using S-K method should be compared with caution with those using the ICP-MS method.

Prostacyclin production by the deendothelialized rabbit aorta.

The acute effect of in vitro deendothelialization on the production of prostacyclin (PGI2) by the rabbit aorta has been investigated. The effectiveness of removing endothelium by rubbing it against filter paper or scraping it with a scalpel was demonstrated by scanning electron microscopy and en face examination after silver staining. Endothelium removal produced an immediate stimulation of PGI2 release, resulting in 408% of the control after rubbing and 367% of the control after scraping, during the first 30-min period of incubation. This increased production of PGI2 gradually declined over time to reach values similar to the control after 2h. At that time, the deendothelialized aorta was totally unresponsive to the stimuli that increase PGI2 release in the intact aorta (acetylcholine, ADP, ionophore A23187, and arachidonic acid). The enhanced production of PGI2 in the deendothelialized aorta was associated with an increased release of free arachidonic acid (353% of the control): in contrast with PGI2, this stimulation was maintained for at least 150 min. A transient exposure of the deendothelialized aorta to ibuprofen (250 microM) was followed by a rebound of PGI2 production, which was also prolonged by BW-755C (3-10 microM). In conclusion, removal of the endothelium triggered an immediate and sustained mobilization of free arachidonic acid in the rabbit aorta: the resulting increase of PGI2 production was short-lived, probably as a consequence of cyclooxygenase self-inactivation. Our results indicate that the subendothelium has a significant capacity to produce PGI2, but that this capacity is expressed only briefly.

Novel transformations of arachidonic acid by the rat thyroid in vitro.

The transformation of arachidonic acid by the rat thyroid in vitro has been investigated. At least two metabolites have been partially characterized: they differed from known metabolites of arachidonic acid in terms of retention volume in liquid chromatography, ultraviolet spectrophotometry and pharmacology (formation not inhibited by indomethacin and enhanced by eicosatetraynoic acid). The analysis by chemical ionization mass spectrometry suggested that these metabolites might be diketo-monohydroxy- and monoketo-dihydroxy-compounds. The conversion of arachidonic acid into these compounds was stimulated by ionophore A23187, decreased by the peroxidase inhibitor methimazole and potentiated by iodide, suggesting that this pathway is under the control of Ca2+ and of a peroxidase product.

Metabolic disposition of prostaglandin E1 in man.

Metabolism of [17, 18-3H]prostaglandin E1 was investigated in three healthy male volunteers during intravenous infusion. The infusion rate was 5.0 ng/kg per min. Blood samples were obtained before the end of the infusion as well as 5, 10, 20, 40, 90 and 180 min afterwards; urine and feces were collected until 96 and 72 h, respectively, after the experiment. All samples were analyzed for radioactivity. Urine was further chromatographed, including by high-pressure liquid chromatography, and subsequently analyzed by gas chromatography-mass spectrometry. Radioactivity in plasma rapidly declined during the first 10 min after termination of the infusion, and then was eliminated exponentially with a mean half-life of 181 min, probably reflecting slow excretion of one or more metabolite. 12% of the administered radioactivity could be recovered from feces and 88% from urine. From the radioactive material obtained from urine the following metabolites could be identified (each number represents data of one volunteer): 7 alpha-hydroxy-5,11-diketotetranor-prostane-1,16-dioic acid (10.4, 20.4 and 30.1%), 7 alpha-hydroxy-5,11-diketotetranor-prostanoic acid (8.2, 6.9 and 9.3%), 5 alpha, 7 alpha-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid and its delta-lactone (together accounting for 4.1, 2.1 and 3.8%).

Effects of prostaglandins F alpha on dog thyroid cyclic AMP level and function.

Prostaglandins F1 alpha and F2 alpha, at high concentrations (greater than or equal to 28 microM) enhanced cyclic AMP accumulation in dog thyroid slices. At lower concentrations, they inhibited the cyclic AMP accumulation induced by thyrotropin (TSH), prostaglandin E1, and cholera toxin. This effect was rapid in onset and of short duration, calcium-dependent and suppressed by methylxanthines. Prostaglandin F alpha also inhibited TSH-induced secretion and activated iodide binding to proteins. These characteristics are similar to those of carbamylcholine action, except that prostaglandins F did not enhance cyclic GMP accumulation. The effect of prostaglandin F alpha was not inhibited by atropine, phentolamine and adenosine deaminase and can therefore not be ascribed to an induced secretion of acetylcholine, norepinephrine or adenosine. It is suggested that prostaglandins F act by increasing influx of extracellular Ca2+. Arachidonic acid also inhibited the TSH-induced cyclic AMP accumulation. However this effect was specific for TSH, it was enhanced in the absence of calcium and was not inhibited by methylxanthines or by indomethacin at concentrations which completely block its conversion to prostaglandin F alpha. Arachidonic acid action is sustained. This suggests that arachidonic acid inhibits thyroid adenylate cyclase at the level of its TSH receptor and that this effect is not mediated by prostaglandin F alpha or any other cyclooxygenase product.

Receptors responsive to extracellular pyrimidine nucleotides.

Extracellular adenine nucleotides have long been known to mediate pharmacological responses via several subtypes of P2 purinoceptors. More recently, however, similar responses to uracil nucleotides have been demonstrated. Here, Didier Communi and Jean-Marie Boeynaems discuss the evidence to suggest that distinct pyrimidinoceptors do indeed exist, although they do not appear to constitute a separate receptor family of their own.

P2 purinoceptors on vascular endothelial cells: physiological significance and transduction mechanisms.

The characterization of P2 gamma purinoceptors on vascular endothelial cells has progressed rapidly since their existence was first demonstrated in 1983. They transduce the actions of extracellular ATP and ADP--endothelium-dependent relaxation, prostacyclin synthesis, endothelial cell mitogenesis--which play a vital role in the interaction between platelets (a rich source of extracellular adenine nucleotides) and the vessel wall. Release of prostacyclin limits the extent of intravascular platelet aggregation following vascular damage and platelet stimulation, while the mitogenic effect may accelerate the repair of a lesion. P2 gamma receptors on endothelial cells are coupled to a phospholipase C by a GTP-binding protein. Jean-Marie Boeynaems and Jeremy Pearson explain how the increases in cytoplasmic Ca2+ and diacylglycerol resulting from this initial event mediate several further effects. In particular, activation of a Ca2(+)-sensitive phospholipase A2 explains the increased synthesis of prostacyclin, while the phosphorylation of several proteins by calmodulin-dependent kinases modulates other endothelial cell functions.

Advances in signalling by extracellular nucleotides. the role and transduction mechanisms of P2Y receptors.

Nucleotides are ubiquitous intercellular messengers whose actions are mediated by specific receptors. Since the first clonings in 1993, it is known that nucleotide receptors belong to two families: the ionotropic P2X receptors and the metabotropic P2Y receptors. Five human P2Y receptor subtypes have been cloned so far and a sixth one must still be isolated. In this review we will show that they differ by their preference for adenine versus uracil nucleotides and triphospho versus diphospho nucleotides, as well as by their transduction mechanisms and cell expression.

Involvement of multiple receptors in the actions of extracellular ATP: the example of vascular endothelial cells.

The role of ATP and ADP as intercellular mediators is now well established. The presence of the nucleotides in extracellular fluids can result from several mechanisms: cell lysis, selective permeabilization of the plasma membrane and exocytosis of secretory vesicles, such as platelet dense bodies. Extracellular adenine nucleotides are rapidly degraded by ectonucleotidases expressed inter alia on the surface of endothelial cells. They act on cells via the family of P2 receptors which encompasses more than 5 subtypes, some of which have been cloned recently. The P2T, P2U and P2Y receptors belong to the superfamily of receptors coupled to G proteins, whereas the P2X receptor is a cation channel and the P2Z receptor a non-selective pore. ATP and ADP stimulate the endothelial production of prostacyclin (PGI2) and nitric oxide (NO), two vasodilators and inhibitors of platelet aggregation, via an increase in cytosolic Ca2+. This action of adenine nucleotides is believed to limit the extent of intravascular platelet aggregation and to help localize thrombus formation to areas of endothelial damage. The endothelial response to nucleotides is mediated by at least two distinct subtypes of P2 receptors, P2Y and P2U, both coupled to phospholipase C.

Cholinergic and alpha-adrenergic stimulation of prostaglandin release by dog thyroid in vitro.

Dog thyroid slices released in their incubation medium prostaglandins E2 (PGE2), F2 alpha (PGF2 alpha), 15-keto-13,14-dihydro-F2 alpha, and thromboxane B2 (TxB2), as measured by direct RIA. This release could be inhibited by indomethacin and naproxen, indicating that it corresponded to a neosynthesis. Carbamylcholine (2--100 microns) stimulated the release of PGE2, PGF2 alpha, and TxB2, whereas epinephrine (20 microns to 1 nM) enhanced the release of PGE2 and PGF2 alpha but had no effect on TxB2. These stimulations were inhibited by atropine and dihydroergocryptine, respectively, suggesting that a muscarinic and an alpha-adrenergic receptor were involved. The ionophore A23187 reproduced these stimulations, and the stimulatory effects of carbamylcholine and epinephrine were inhibited in the absence of exogenous Ca++ and after EGTA depletion. Ca++ thus appears as a major regulator of PG production in the thyroid. TSH (0.06--10 MU/ml) and dibutyryl cAMP had no effect on the release of PGE2, PGF2 alpha, or TxB2 by dog thyroid in vitro.

Inhibition by iodide of the cholinergic stimulation of prostaglandin synthesis in dog thyroid.

Iodide is shown to inhibit the cholinergic stimulation of prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) synthesis in dog thyroid slices. The inibitory effect of iodide was already detectable at 1 micron and reached its maximal level at 10 microns. At this concentration, iodide inhibited the carbamylcholine-stimulated release of PGE2 and PGF2 alpha by 59% and 73%, respectively (eight experiments). The effect of iodide was neither immediate nor rapidly reversed after a change of incubation medium. The unstimulated release of PGE2 and PGF2 alpha and the stimulation by epinephrine and ionophore A23187 were not modified by iodide in concentrations up to 0.1 mM. The effect of iodide was suppressed in the presence of methimazole (0.1 mM) but not NaClO4 (2 mM). Iodid (0.1 mM) did not inhibit the stimulation by carbamylcholine of PGF2 alpha and PGE2 release by rat pancreas in vitro. These data demonstrate a new action of iodide on the thyroid and establish a link among iodide metabolism, PG synthesis, and cholinergic action in the dog thyroid.

Kinetics of dog thyroid secretion in vitro.

The time sequence of radioiodine sequestration and secretion (BE131I) have been compared in dog thyroid slices prelabeled with 131I in vivo and incubated in vitro with or without TSH. Sequestration has been taken to be the amount of radioiodine present in phagocytic vacuoles or colloid droplets; the TSH or (Bu)2cAMP stimulation of the basal values was suppressed by endocytosis blocking drugs. TSH induced a sequestrated radioactivity (S) after 5 min and a stimulated secretion after 20 min. The secretion rate was constant: 1%/h (mean +/- SD = 1.0 +/- 0.4; n = 7) of the total radioactivity of the slices. At equilibrium, S was constant and equal to less than 1% of the total radioactivity. The half-life of S, assuming a disappearance rate proportional to S, was 26 min (26 +/- 4; n = 5); assuming a disappearance rate independent of S, the lifetime was 44 min (44 +/- 7; n = 6). At the steady state, the limiting step of maximally stimulated secretion was the hydrolysis of the sequestrated radioactivity and endocytosis rate was equal to secretion rate. Without TSH, a constant BEI release (0.23% +/- 0.07%/h; n = 7), insensitive to cytochalasin B, was observed, which corresponded to basal secretion.

Regulation of cyclic nucleotide and prostaglandin formation in normal human thyroid tissue and in autonomous nodules.

We have investigated the regulation of the human throid gland based on controls discovered in the dog thyroid gland. TSH and thyroid-stimulating immunoglobulin enhanced cAMP accumulation, which supports the validity of the Sutherland model for the action of TSH on the human thyroid. Iodide inhibited TSH- and thyroid-stimulating immunoglobulin-activated cAMP accumulation and this effect was reduced by methimazole, showing that, in this tissue, iodide, through an oxidized derivative, depresses the TSH-cAMP system. Contrary to the hypothesis of a short feedback loop of thyroid hormone, no thyroid effect of T3 or T4 was found. Adrenergic agents (norepinephrine and isoproterenol) enhanced cAMP accumulation; this effect was inhibited by dl-propranolol but not by d-propranolol or phentolamine. This suggests a positive control of the thyroid cAMP system by beta-adrenergic receptors. Histamine also increased cAMP accumulation. However, the role of these controls is unknown. Acetylcholine, by a muscarinic type effect, enhanced cGMP accumulation and prostaglandin E2 and prostaglandin F2 alpha release. These effects were mimicked by ionophore A23187 and abolished in a calcium-deprived medium, which suggests that they are secondary to a raised Ca++ influx. The results are summarized in a general working model of human thyroid regulation. These biochemical controls have been compared in normal tissue and autonomous nodules. No evidence of increased sensitivity to TSH of the nodular tissue was found. On the other hand, this tissue was less sensitive to acetylcholine (cGMP accumulation) and more sensitive to norepinephrine (cAMP accumulation).

Lack of prostaglandin involvement in the mitogenic effect of TSH on canine thyroid cells in primary culture.

The production of prostaglandin E2 (PGE2) by cultured dog thyroid cells was high in a serum-containing medium and low in a serum-free, completely defined medium. Thyrotropin (TSH) and epidermal growth factor (EGF), two mitogenic factors for these cells, did not stimulate PGE2 release. Indomethacin, at a concentration which completely inhibited PGE2 production, had no effect on thyroid cell multiplication and DNA synthesis stimulated by TSH and EGF. It is concluded that cyclooxygenase products are not involved in the proliferation of canine thyroid cells and its control by TSH.

Rapid up-regulation of P2Y messengers during granulocytic differentiation of HL-60 cells.

HL-60 cells are human promyelocytic cells expressing two ATP receptors: the P2Y(2) and P2Y(11) subtypes. Our Northern blotting experiments have shown that P2Y(2) and P2Y(11) messengers were up-regulated in these cells, rapidly and independently of protein synthesis, following treatment with granulocytic differentiating agents such as retinoic acid, dimethylsulfoxide, granulocyte-colony stimulating factor, dibutyryl cyclic AMP and ATP. AR-C67085 and adenosine 5'-O-(3-thiotriphosphate), two potent agonists of the recombinant P2Y(11) receptor, increased intracellular cAMP concentration in HL-60 cells more potently than ATP itself. These observations support the conclusion that the effect of ATP on HL-60 cell differentiation is mediated by the P2Y(11) receptor.

Rapid phosphorylation of a 27 kDa protein induced by tumor necrosis factor.

Tumor necrosis factor (TNF) has been shown to induce the phosphorylation of a 27 kDa protein in a time- and concentration-dependent manner in HeLa D98/AH2, ME 180 and bovine aortic endothelial cells. This phosphorylation could be reproduced by the calcium ionophore, A23187. However, this phosphorylation was not observed in L929 cells, for which TNF is highly cytotoxic, suggesting that it might play a role in actions of TNF other than the induction of cell death.

Plasma homocysteine concentration in a Belgian school-age population.

BACKGROUND: Total plasma homocysteine (tHcy) is an independent risk factor for cardiovascular disease in adults. Data for children and adolescents are lacking. OBJECTIVE: The aim of this study was to provide a reference range for tHcy and to explore the relation between tHcy and nutritional indexes in a Belgian pediatric population. DESIGN: tHcy, folate, and vitamin B-12 were measured in 647 healthy children (353 girls and 294 boys) aged 5-19 y. RESULTS: The tHcy distribution was, as in adults, skewed to the right [geometric mean (-1 SD, +1 SD): 7.41 micromol/L (5.51, 9.96)]. Concentrations were lowest in younger children and increased with age. After the tHcy distribution was examined according to age, 3 age ranges were distinguished: 5-9 y [6.21 micromol/L (5.14, 7.50)], 10-14 y [7.09 micromol/L (5.69, 8.84)], and 15-19 y [8.84 micromol/L (6.36, 12.29)]. We observed no significant differences in tHcy values between girls and boys in children aged < 15 y; in postpubertal children, however, concentrations were higher in boys than in girls. In the 3 age groups, folate was inversely correlated with tHcy; the negative relation between tHcy and vitamin B-12 was less strong. Familial cardiovascular disease was more frequent in children who had hyperhomocysteinemia. CONCLUSIONS: These observations suggest that in children, as in adults, genetic, nutritional, and endocrine factors are determinants of the metabolism of homocysteine. The significance of tHcy values in childhood and young adulthood in terms of predicting cardiovascular risk in adulthood should be investigated.

Metabolism of arachidonic acid in rabbit iris and retina.

Both the iris and the retina of the rabbit released prostaglandins (PG) E2, F2 alpha, 6-keto-F1 alpha and thromboxane (Tx) B2, when incubated in vitro. PGE2 was the major cyclooxygenase product formed by each tissue. The kinetics of PGE2 release by the iris and the retina were similar: high initial output followed by a decline to a steady-state value. The production of PGE2 was inhibited by indomethacin and stimulated by ionophore A23187. The iris and the retina converted exogenous arachidonic acid into 12- and 15-hydroxy-eicosatetraenoic acid (HETE): inhibition by eicosatetraynoic acid (ETYA) indicated the involvement of lipoxygenase enzymes. This lipoxygenase activity was important relatively to cyclooxygenase in the retina, but was only a minor pathway in the iris. Leukotriene (LT) B4 was released by the iris and the retina in amounts smaller than PGE2, but compatible with a biological activity: ionophore A23187 stimulated LTB4 production in both tissues. Our data support the hypothesis that PGE2 and LTB4 could play a role in the initiation of ocular inflammation.