A brief challenge of rat astrocytes with either a,b-methyleneATP (a,b-meATP) or basic ®broblast growth factor (bFGF) resulted, three days later, in morphological di erentiation of cells, as shown by marked elongation of astrocytic processes. The P2 receptor antagonist suramin prevented a,b-meATPbut not bFGF-induced astrocytic elongation. Similar e ects on astrocytic elongation were also observed with ATP and other P2 receptor agonists (b,gmeATP, ADPbS, 2meSATP and, to a lesser extent, UTP). 2 Pertussis toxin completely abolished a,b-meATP-but not bFGF-induced e ects. No e ects were exerted by a,b-meATP on cyclic AMP production; similarly, neomycin had no e ects on elongation of processes induced by the purine analogue, suggesting that adenylyl cyclase and phospholipase C are probably not involved in a,b-meATP-induced e ects (see also the accompanying paper by Centemeri et al., 1997). The tyrosine-kinase inhibitor genistein greatly reduced bFGF-but not a,b-meATP-induced astrocytic elongation. 3 Challenge of cultures with a,b-meATP rapidly and concentration-dependently increased [ 3 H]-arachidonic acid (AA) release from cells, suggesting that activation of phospholipase A 2 (PLA 2 ) may be involved in the long-term functional e ects evoked by purine analogues. Consistently, exogenously added AA markedly elongated astrocytic processes. Moreover, various PLA 2 inhibitors (e.g. mepacrine and dexamethasone) prevented both the early a,b-meATP-induced [ 3 H]-AA release and/or the associated long-term morphological changes, without a ecting the astrocytic elongation induced by bFGF. Finally, the protein kinase C (PKC) inhibitor H7 fully abolished a,b-meATP-but not bFGF-induced e ects. 4 Both a,b-meATP and bFGF rapidly and transiently induced the nuclear accumulation of Fos and Jun. Both c-fos and c-jun induction by the purine analogue could be fully prevented by pretreatment with suramin. In contrast, the e ects of bFGF were una ected by this P2 receptor antagonist. 5 It was concluded that a,b-meATP-and bFGF-morphological di erentiation of astrocytes occurs via independent transductional pathways. For the purine analogue, signalling involves a G i /G o proteincoupled P2Y-receptor which may be linked to activation of PLA 2 (involvement of an arachidonatesensitive PKC is speculated); for bFGF, a tyrosine kinase receptor is involved. Both pathways merge on some common intracellular target, as suggested by induction of primary response genes, which in turn may regulate late response genes mediating long-term phenotypic changes of astroglial cells. 6 These ®ndings implicate P2 receptors as novel targets for the pharmacological regulation of reactive astrogliosis, which has intriguing implications in nervous system diseases characterized by degenerative events.

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1 This study was aimed at characterizing ATP-induced rises in cytosolic free calcium ion, [Ca 2+ ] i , in a population of rat striatal astrocytes loaded with the¯uorescent Ca 2+ probe Fura2, by means of uorescence spectrometry. 2 ATP triggered a fast and transient elevation of [Ca 2+ ] i in a concentration-dependent manner. The responses of the purine analogues 2-methylthio-ATP (2-meSATP), adenosine-5'-O-(2-thiodiphosphate) (ADPbS), as well as uridine-5'-triphosphate (UTP) resembled that of ATP, while a,b-methylene-ATP (a,b-meATP) and b,g-methylene-ATP (b,g-meATP) were totally ine ective. 3 Suramin (50 mM) had only a minor e ect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (5 mM) signi®cantly depressed the maximum response. 4 Extracellular Ca 2+ did not contribute to the observed [Ca 2+ ] i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its in¯ux by means of either Ni 2+ (1 mM) or Mn 2+ (1 mM) did not modify the nucleotide responses. 5 Furthermore, after preincubation with 10 mM thapsigargin, the nucleotide-evoked [Ca 2+ ] i increments were completely abolished. In contrast, 10 mM ca eine did not a ect the responses, suggesting that thapsigargin-, but not ca eine/ryanodine-sensitive stores are involved. 6 Both application of the G-protein blocker guanosine-5'-O-(2-thiodiphosphate) (GDPbS) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml 71 ) partially inhibited the nucleotide-mediated responses. Moreover, the phospholipase C (PLC) inhibitor U-73122, but not its inactive stereoisomer U-73343 (5 mM), signi®cantly reduced the ATP-evoked [Ca 2+ ] i rise. 7 In conclusion, our results suggest that, in rat striatal astrocytes, ATP-elicited elevation of [Ca 2+ ] 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.

Estrogen is known to induce rapid vasodilatory response in isolated arteries. Because estrogen is a nonselective receptor agonist, the involvement of estrogen receptor (ER) subtypes in acute estrogenic responses has remained elusive. Acute administration of the selective ER␣ agonist 4,4Ј,4Љ-(4-propyl-[ 1 H]pyrazole-1,3,5-triyl) tris-phenol (PPT) to precontracted aortic rings from intact female rats dose-dependently induced an ER-dependent vascular relaxation fully overlapping to that induced by 17␤-estradiol. By contrast, the selective ER␤ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) had no acute effect on vasomotion. This short-term vasorelaxant action of PPT was abolished by the NO synthase inhibitor N -nitro-L-arginine methyl ester and by endothelium removal. In aortic tissues from ovariectomized (OVX) rats, however, neither 17␤-estradiol nor PPT induced acute vascular relaxation. The effect of PPT was restored in preparations from estrogen-replaced OVX rats, whereas DPN remained ineffective even after estrogen replacement. PPT acted through an ER-dependent mechanism, as shown by impaired response in the presence of the anti-estrogen ICI 182,780 (7␣,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol). Accordingly, isolated rat aortic endothelial cells expressed both ER␣ and ER␤. These data show that selective ER␣ but not ER␤ agonists reproduced the acute vasodilation of estrogen via a receptor-mediated pathway in the aorta from intact as well as 17␤-estradiolreplaced OVX rats. This beneficial effect was undetectable in tissues from OVX rats. Selective pharmacological targeting of ER subtypes may thus represent a novel and promising approach in the treatment of vascular disease.The vascular wall is clearly one of the target organs of estrogens. A number of studies have shown that estrogens modulate vasomotor responses after both acute application and in vivo short-or long-term treatment (for review, see Mendelsohn and Karas, 1999;Cignarella et al., 2001). Although different mechanisms have been reported (Shaw et al., 2001), such effects seem to be mediated by specific estrogen receptors (ERs) that are located on the plasma membrane as well as intracellularly. So far, two ER subtypes have been described: ER␣ and ER␤. Both subtypes are found in vascular smooth muscle (Register and Adams, 1998;Hodges et al., 2000;Maggi et al., 2003) and human endothelial cells (Caulin-Glaser et al., 1996). Although similar, the two ER isoforms are distinct gene products with nonoverlapping functions. They are coexpressed in most tissues but increasing evidence suggests that ER␣ and ER␤ mediate opposite effects in a kind of yin-yang manner (Gustafsson, 2003).The relative contribution of each ER subtype to vascular responses has been difficult to investigate because the physiological ER ligand 17␤-estradiol (E 2 ) has no binding affinity preference for ER␣ and ER␤. Selective ER agonists have now become available, the most widely used being 4,4Ј,4Љ-(4-propyl-[1 H]pyrazole-1,3,5-triyl) tris-phen...

Adenosine‐5′‐triphosphate (ATP) is known to exert a variety of biological effects via the activation of either ionotropic P2X‐ or G‐protein coupled P2Y‐purinoceptor subtypes. In this study the effects induced by ATP and ATP analogues on rat bladder strips were characterized at resting tone and in carbachol‐prestimulated tissues. ATP exerted a clear concentration‐dependent biphasic response, which was maximal at 1 mM concentration and was characterized by an immediate and transient contraction, followed by a slower sustained relaxation. The receptor mediating contraction was susceptible to desensitization by ATP and by the ATP analogue, α,β‐methyleneATP (α,β‐meATP) showing the typical features of the P2X‐purinoceptor; conversely, ATP‐evoked relaxation did not undergo tachyphylaxis following either ATP or α,β‐meATP. The slower and sustained relaxant phase seemed to be due to activation of P2Y‐purinoceptors, based on responses obtained with the P2Y agonist, 2‐methyl‐thioATP (2‐meSATP) and, more importantly, based on the clear involvement of the G‐proteins. In fact, the G‐protein activator, guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) significantly potentiated and the G‐protein blocking agent, guanosine 5′‐O‐(2‐thio‐diphosphate) (GDPβS) completely abolished the ATP‐induced relaxation. No effects were exerted by these two G‐protein modulators on the ATP‐induced contraction. The relaxant component of the ATP response of bladder tissue was not significantly influenced by nitro‐benzyl‐thioinosine (NBTI) or by 8‐phenyltheophylline (8‐PT), suggesting that the contribution of the ATP metabolite adenosine to this response was negligible. Moreover, relaxation evoked by ATP and by the adenosine analogue, 5′‐N‐ethylcarboxamidoadenosine (NECA) was additive. Suramin was unable to modify either the relaxant or the contractile responses of bladder strips to ATP. However, when tested on the concentration‐response curve to the slowly hydrolysable P2x‐agonist α,β‐meATP, a rightward shift was detected, suggesting that ATP contractile responses are mediated by suramine‐sensitive P2x‐purinoceptors. Uridine‐5′‐triphosphate (UTP) only induced a rapid and concentration‐dependent contraction of the rat bladder preparation, which was not desensitized by pre‐exposure to α,β‐meATP, suggesting that UTP responses were not mediated by the ‘classical’ P2X‐purinoceptor. It is therefore concluded that both P2X‐ and P2Y‐purinoceptors, which mediate ATP‐induced contraction and relaxation, respectively, are present in rat bladder. Moreover, removal of epithelium did not affect ATP‐elicited contraction, whereas ATP‐induced relaxation was significantly augmented. These data suggest that P2x‐ and P2y‐ purinoceptors are localized in smooth muscle cells and that the relaxant response is probably modulated by excitatory factor(s) released by epithelial cells.

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