Simonetta Nicosia

Production of leukotrienes in a model of focal cerebral ischaemia in the rat

The aim of this work was to evaluate the role of leukotrienes in brain damage in vivo in a model of focal cerebral ischaemia in the rat, obtained by permanent occlusion of middle cerebral artery. A significant (P<0.01) elevation of LTC4, LTD4 and LTE4 (cysteinyl‐leukotrienes) levels occurred 4 h after ischaemia induction in the ipsilateral cortices of ischaemic compared to sham‐operated animals (3998±475 and 897±170 fmol g−1 tissue, respectively, P<0.01). The NMDA receptor antagonist MK‐801 and the adenosine A2A receptor antagonist SCH 58261 were administered in vivo at doses known to reduce infarct size and compared with the leukotriene biosynthesis inhibitor MK‐886. MK‐886 (0.3 and 2 mg kg−1 i.v.) and MK‐801 (3 mg kg−1 i.p.) decreased cysteinyl‐leukotriene levels (−78%, P<0.05; −100%, P<0.01; −92%, P<0.01, respectively) 4 h after permanent occlusion of the middle cerebral artery, whereas SCH 58261 (0.01 mg kg−1 i.v.) had no significant effects. MK‐886 (2 mg kg−1 i.v.) was also able to significantly reduce the cortical infarct size by 30% (P<0.05). We conclude that cysteinyl‐leukotriene formation is associated with NMDA receptor activation, and that it represents a neurotoxic event, the inhibition of which is able to reduce brain infarct area in a focal ischaemic event.

Characterization of the Ca2+ responses evoked by ATP and other nucleotides in mammalian brain astrocytes

1 This study was aimed at characterizing ATP‐induced rises in cytosolic free calcium ion, [Ca2+]i, in a population of rat striatal astrocytes loaded with the fluorescent Ca2+ probe Fura2, by means of fluorescence spectrometry. 2 ATP triggered a fast and transient elevation of [Ca2+]i in a concentration‐dependent manner. The responses of the purine analogues 2‐methylthio‐ATP (2‐meSATP), adenosine‐5′‐O‐(2‐thiodiphosphate) (ADPβS), as well as uridine‐5′‐triphosphate (UTP) resembled that of ATP, while α,β‐methylene‐ATP (α,β‐meATP) and β,γ‐methylene‐ATP (β,γ‐meATP) were totally ineffective. 3 Suramin (50 μM) had only a minor effect on the ATP response, whereas pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (5 μM) significantly depressed the maximum response. 4 Extracellular Ca2+ did not contribute to the observed [Ca2+]i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its influx by means of either Ni2+ (1 mM) or Mn2+ (1 mM) did not modify the nucleotide responses. 5 Furthermore, after preincubation with 10 μM thapsigargin, the nucleotide‐evoked [Ca2+]i increments were completely abolished. In contrast, 10 mM caffeine did not affect the responses, suggesting that thapsigargin‐, but not caffeine/ryanodine‐sensitive stores are involved. 6 Both application of the G‐protein blocker guanosine‐5′‐O‐(2‐thiodiphosphate) (GDPβS) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml−1) partially inhibited the nucleotide‐mediated responses. Moreover, the phospholipase C (PLC) inhibitor U‐73122, but not its inactive stereoisomer U‐73343 (5 μM), significantly reduced the ATP‐evoked [Ca2+]i rise. 7 In conclusion, our results suggest that, in rat striatal astrocytes, ATP‐elicited elevation of [Ca2+]i is due solely to release from intracellular stores and is mediated by a G‐protein‐linked P2Y receptor, partially sensitive to PTx and coupled to PLC.

Vasoactive intestinal polypeptide (VIP) stimulates adenylate cyclase in selected areas of rat brain

Abstract VIP stimulated adenylate cyclase activity in homogenates of some areas of rat brain that are rich in this peptide, e.g., cerebral cortex, hypothalamus and hippocampus, as well as in cerebellar cortex, where VIP content is low. No stimulation occurred in caudate nucleus or brainstem. The enzyme stimulation was inhibited by Ca 2+ , but unaffected by guanine nucleotides. Synthetic fragments of VIP (VIP 6−28 & VIP 14–28 ) neither stimulated cyclase activity nor inhibited VIP-induced stimulation.

THE ROLE OF HISTAMINE Hr AND H2‐RECEPTORS IN THE GENERATION OF THROMBOXANE A2 IN PERFUSED GUINEA‐PIG LUNGS

1 When isolated perfused lungs from normal and ovalbumin sensitized guinea‐pigs were challenged with histamine and 2‐methylhistamine (agonists for Hj‐receptor), a release of thromboxane A2‐like substance was observed. The effect of histamine on production of thromboxane A2 (TXA2) in sensitized lungs, was more pronounced than in normal lungs (P < 0.01). 2 Specific activation of histamine H2‐receptors in normal lungs with large doses (100 jag) of dimaprit and 4‐methylhistamine, does not produce thromboxane‐like or prostaglandin‐like substances. 3 Perfusion of the lungs with pyrilamine (10 μg/ml) inhibited the release of arachidonate metabolites induced by histamine H1‐receptor stimulation, whereas cimetidine (5 μg/ml) was ineffective. 4 It is concluded that only the stimulation of histamine H1‐receptors appears to be responsible for generation of thromboxane A2 and other prostaglandin‐like substances in normal guinea‐pig lungs. In sensitized lungs, an increased ability of histamine to release TXA2 could be due to a possible interconversion of H2 into Hj‐receptors.

myc-immortalized microglial cells express a functional platelet-activating factor receptor.

Abstract: The autacoid platelet‐activating factor (PAF) takes part in a complex network of interactions regarding the cellular components of nervous tissues. Efforts aimed at characterizing the effects of PAF in the brain have been recently focalized on neurons because PAF exerts pleiotropic effects on these cells. Less attention has instead been paid to the glial component of the brain. We have used microglial cell lines immortalized from 13‐day‐old mouse embryo brains by a myc‐transducing retrovirus. When exposed to physiological doses of PAF, immortalized microglial cells showed increases in intracellular free calcium concentrations due to release of calcium from internal stores, as well as to extracellular calcium influxes. These profiles of reactivity were independent from the immortalizing process, being observable in primary microglial cultures and in immortalized clones showing different proliferative rates. PAF was also able to induce transient expression of the c‐fos protooncogene in serum‐starved cultures and induced a strong chemotactic response in microglial cells. In contrast with control macrophage cultures, PAF did not promote prostaglandin or leukotriene synthesis in immortalized cells. This was most likely due to the low amount of total arachidonic acid found in immortal microglia, with respect to that observed in freshly isolated cells. Our data suggest that several of the effects observed after PAF stimulation might be independent from PAF‐induced arachidonic acid metabolism. The availability of an in vitro microglial model might now help in studying the proinflammatory effects of PAF, both direct or microglia mediated, in the neural environment.

Thromboxane prostanoid receptor in human airway smooth muscle cells: a relevant role in proliferation

Thromboxane A(2) has been implicated as a mediator of bronchial hyperresponsiveness in asthma. Modulating agents are currently marketed in Japan and under clinical evaluation in the US, but full characterization of the thromboxane A(2) receptor and the signaling pathways that link it to the proliferative events taking place during airways structural remodeling has not been achieved. Here, we report that the presence of mRNA for both alpha and beta isoforms of the thromboxane A(2) receptor in smooth muscle cells from human bronchi correlates with protein expression evaluated by radioligand binding of the antagonist, SQ29,548 ([1S-[1alpha,2alpha(Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic-acid) (K(d)=3.4 nM+/-44%CV, coefficient of variation, B(max)=41 fmol/mg prot+/-38%CV). The receptor is functional, as the agonist, U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic-acid), induced a concentration-dependent Ca(2+) transient (EC(50)=0.12 microM+/-27%CV). Furthermore, U46619 concentration dependently increased DNA synthesis and markedly potentiated the epidermal growth factor mitogenic effect. Both events were specifically inhibited by SQ29,548, independently from transactivation of the epidermal growth factor receptor and partially sensitive to pertussis toxin.

Identification of specific binding sites for leukotriene C4 in membranes from human lung

Leukotriene C4 (LTC4), one of the major components of the slow-reacting substance of anaphylaxis (SRS-A), is a potent constrictor of bronchial smooth muscle in many species including humans. Here we report the identification and characterization of specific binding sites for LTC4 in membranes from human lung parenchyma. At 4 degrees, 3H-LTC4 binding is specific, saturable (Bmax = 32-41 pmoles/mg prot.), rapid (equilibrium being attained within 15 min), reversible and of high affinity (Kd = 3.6-7 X 10(-8) M). The binding sites are sensitive to heat and probably possess a protein moiety, being inactivated upon trypsinization. CaCl2 affects both the association and the dissociation rate and dose-dependently enhances the binding of 3H-LTC4 at equilibrium; maximal enhancement (4-fold) occurred at 10(-2)M CaCl2. Unlabelled LTC4 is able to complete with 3H-LTC4 for its binding sites with an IC50 of 7.8 X 10(-8) M. The addition of 10(-2) M CaCl2 increases the potency of LTC4 in inhibiting the binding (2.2-fold); both the competition curves are monophasic, indicating the existence of a homogeneous class of binding sites. In the presence of CaCl2, LTD4, LTE4 and the SRS-A antagonist FPL 55712 can inhibit 3H-LTC4 specific binding, being, however, less potent than LTC4 (IC50 S = 2.2 X 10(-6), 2.4 X 10(-5) M, for LTD4, LTE4 and FPL 55712, respectively). FPL 55712 displayed a competitive mechanism; its affinity, however, was lower if absorption to glass was not prevented. The present studies indicate that specific binding sites for 3H-LTC4 exist in human lung parenchyma, and that a receptor-mediated process might be involved in the bronchoconstriction induced by LTC4.

(5Z)-carbacyclin discriminates between prostacyclin-receptors coupled to adenylate cyclase in vascular smooth muscle and platelets.

1 (5E)‐ and (5Z)‐carbacyclin are prostacyclin (PGI2) analogues endowed with antiaggregating and vasodilator properties, which stimulate adenylate cyclase activity in membranes from human platelets and cultured myocytes from rabbit mesenteric artery. 2 In platelets they display the same efficacy as prostaglandin E1 (PGE1), and hence PGI2, both as activators of adenylate cyclase and as inhibitors of aggregation. 3 In contrast, in vascular smooth muscle cells (5Z)‐carbacyclin fails to produce the same degree of stimulation of the enzyme as PGI2, (5E)‐carbacyclin and PGE, nor does it induce the maximal relaxation of the mesenteric artery as do the other prostaglandins. 4 (5Z)‐carbacyclin is also able to antagonize the activation of adenylate cyclase and the relaxation elicited by PGE1 or PGI2 in the mesenteric artery, and therefore it displays partial agonist properties in these cells. 5 We conclude that the receptors for PGI2 coupled to adenylate cyclase in platelets and vascular smooth muscle cells are different from each other, because (5Z)‐carbacyclin can discriminate between them, being a partial agonist at myocyte but not at platelet level.

Effects of loratadine on cytosolic Ca2+ levels and leukotriene release: novel mechanisms of action independent of the anti-histamine activity.

Loratadine, a non-sedating anti-histamine drug, displays in vitro potential anti-allergic properties not related to its interaction with the histamine H1 receptor. In a search for the mechanisms of these actions, we have found that loratadine induces an elevation of cytosolic calcium ion, [Ca2+]i, in rat peritoneal macrophages or human platelets. The mechanism of this elevation resides in the ability of loratadine to discharge intracellular Ca2+ stores, similarly to thapsigargin. This in turn brings about the inhibition of [Ca2+]i rise induced by physiological activators (platelet activating factor and ADP), as well as by thapsigargin. One of the active metabolites of loratadine, descarbo-ethoxy-loratadine, and another anti-histamine, namely terfenadine, exhibit the same effects. In addition, loratadine partially inhibits antigen-induced leukotriene release from human bronchi, but is unable to inhibit the concomitant contraction. We conclude that loratadine can interfere with the mechanisms controlling Ca2+ release, thus inhibiting the cell activation elicited by various agonists through [Ca2+]i elevation. This might be the mechanism underlying its anti-allergic actions in vitro. Furthermore, loratadine might represent an interesting tool in the study of Ca2+ homeostasis.

Effects of PGI2 on platelet aggregation and adenylate cyclase activity in human type IIa hypercholesterolemia

The sensitivity to PGI2 of platelets of 20 selected type IIa hypercholesterolemic patients was studied and compared to that of platelets of 14 normocholesterolemic subjects. Type IIa subjects required higher concentrations of PGI2 to inhibit platelet aggregation elicited by 1 microM ADP, 1 microgram/ml collagen and 1.4 microM epinephrine. Adenylate cyclase activity was also measured in washed platelet membranes from the two groups of subjects. Adenylate cyclase activity, both basal and PGI2-stimulated, was not statistically different in the two groups examined. Therefore changes at the level of PGI2 receptors coupled to adenylate cyclase are not likely to be responsible for the different platelet sensitivity to prostacyclin.