Involvement of P2Y1, P2Y6, A1 and A2A Receptors in the Purinergic Inhibition of NMDA-Evoked Noradrenaline Release in the Rat Brain Cortex.

In the cerebral cortex, glutamate activates NMDA receptors (NMDARs), localized in noradrenergic neurons, inducing noradrenaline release that may have a permissive effect on glutamatergic transmission, and therefore, on the modulation of long-term plasticity. ATP is co-released with noradrenaline, and with its metabolites (ADP and adenosine) is involved in the purinergic modulation of electrically-evoked noradrenaline release. However, it is not known if noradrenaline release evoked by activation of NMDARs is also under purinergic modulation. The present study aimed to investigate and to characterize the purinergic modulation of noradrenaline release evoked by NMDARs. Stimulation of rat cortical slices with 30 µM NMDA increased noradrenaline release, which was inhibited by ATP upon metabolization into ADP and adenosine and by the selective agonists of A1 and A2A receptors, CPA and CGS2680, respectively. It was also inhibited by UTP and UDP, which are mainly released under pathophysiological situations. Characterization of the effects mediated by these compounds indicated the involvement of P2Y1, P2Y6, A1 and A2A receptors. It is concluded that, in the rat brain cortex, NMDA-evoked noradrenaline release is modulated by several purinergic receptors that may represent a relevant mechanism to regulate the permissive effect of noradrenaline on NMDA-induced neuroplasticity.

Microglia P2Y13 Receptors Prevent Astrocyte Proliferation Mediated by P2Y1 Receptors.

Cerebral inflammation is a common feature of several neurodegenerative diseases that requires a fine interplay between astrocytes and microglia to acquire appropriate phenotypes for an efficient response to neuronal damage. During brain inflammation, ATP is massively released into the extracellular medium and converted into ADP. Both nucleotides acting on P2 receptors, modulate astrogliosis through mechanisms involving microglia-astrocytes communication. In previous studies, primary cultures of astrocytes and co-cultures of astrocytes and microglia were used to investigate the influence of microglia on astroglial proliferation induced by ADPßS, a stable ADP analog. In astrocyte cultures, ADPßS increased cell proliferation through activation of P2Y1 and P2Y12 receptors, an effect abolished in co-cultures (of astrocytes with ∼12.5% microglia). The possibility that the loss of the ADPßS-mediated effect could have been caused by a microglia-induced degradation of ADPßS or by a preferential microglial localization of P2Y1 or P2Y12 receptors was excluded. Since ADPßS also activates P2Y13 receptors, the contribution of microglial P2Y13 receptors to prevent the proliferative effect of ADPßS in co-cultures was investigated. The results obtained indicate that P2Y13 receptors are low expressed in astrocytes and mainly expressed in microglia. Furthermore, in co-cultures, ADPßS induced astroglial proliferation in the presence of the selective P2Y13 antagonist MRS 2211 (3 µM) and of the selective P2Y12 antagonist AR-C66096 (0.1 µM), suggesting that activation of microglial P2Y12 and P2Y13 receptors may induce the release of messengers that inhibit astroglial proliferation mediated by P2Y1,12 receptors. In this microglia-astrocyte paracrine communication, P2Y12 receptors exert opposite effects in astroglial proliferation as a result of its cellular localization: cooperating in astrocytes with P2Y1 receptors to directly stimulate proliferation and in microglia with P2Y13 receptors to prevent proliferation. IL-1ß also attenuated the proliferative effect of ADPßS in astrocyte cultures. However, in co-cultures, the anti-IL-1ß antibody was unable to recover the ADPßS-proliferative effect, an effect that was achieved by the anti-IL-1α and anti-TNF-α antibodies. It is concluded that microglia control the P2Y1,12 receptor-mediated astroglial proliferation through a P2Y12,13 receptor-mediated mechanism alternative to the IL-1ß suppressive pathway that may involve the contribution of the cytokines IL-1α and TNF-α.

Reactivation of wild-type and mutant p53 by tryptophanolderived oxazoloisoindolinone SLMP53-1, a novel anticancer small-molecule.

Restoration of the p53 pathway, namely by reactivation of mutant (mut) p53, represents a valuable anticancer strategy. Herein, we report the identification of the enantiopure tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a novel reactivator of wild-type (wt) and mut p53, using a yeast-based screening strategy. SLMP53-1 has a p53-dependent anti-proliferative activity in human wt and mut p53R280K-expressing tumor cells. Additionally, SLMP53-1 enhances p53 transcriptional activity and restores wt-like DNA binding ability to mut p53R280K. In wt/mut p53-expressing tumor cells, SLMP53-1 triggers p53 transcription-dependent and mitochondrial apoptotic pathways involving BAX, and wt/mut p53 mitochondrial translocation. SLMP53-1 inhibits the migration of wt/mut p53-expressing tumor cells, and it shows promising p53-dependent synergistic effects with conventional chemotherapeutics. In xenograft mice models, SLMP53-1 inhibits the growth of wt/mut p53-expressing tumors, but not of p53-null tumors, without apparent toxicity. Collectively, besides the potential use of SLMP53-1 as anticancer drug, the tryptophanol-derived oxazoloisoindolinone scaffold represents a promissing starting point for the development of effective p53-reactivating drugs.

Enhanced cytotoxicity of prenylated chalcone against tumour cells via disruption of the p53-MDM2 interaction.

AIM: Chalcones are naturally occurring compounds with recognized anticancer activity. It was recently shown that the O-prenyl derivative (2) of 2'-hydroxy-3,4,4',5,6'-pentamethoxychalcone (1) had a remarkably increased cytotoxicity against human tumour cells compared to its precursor. With this study, we aimed to investigate the molecular mechanism underlying the improved tumour cytotoxicity of prenylchalcone 2. MAIN METHODS: The impact of chalcones 1 and 2 on p53-MDM2 interaction was investigated using yeast growth-inhibitory and p53 transactivation assays. Their tumour growth-inhibitory effects were assessed on human colon adenocarcinoma HCT116 cell lines with wild-type p53 and its p53-null derivative, followed by analysis of cell cycle and apoptosis. In tumour cells, the activation of a mitochondrial pathway was checked by analysis of reactive oxygen species generation, Bax mitochondrial translocation and cytochrome c release. Additionally, the up-regulation of p53 transcriptional activity was investigated through Western blot analysis of p53 target expression levels, and the disruption of the p53-MDM2 interaction was confirmed by co-immunoprecipitation. KEY FINDINGS: The potent cell tumour growth-inhibitory activity of prenylchalcone 2 was associated with the activation of a p53 pathway involving cell cycle arrest and a mitochondria-dependent apoptosis. Furthermore, a correlation between the distinct cytotoxicity of chalcones 1 and 2 and their ability to disrupt the p53-MDM2 interaction was established. SIGNIFICANCE: This work shows that prenylation is a determinant factor for the enhancement of chalcones tumour cytotoxicity by improving their ability to disrupt the p53-MDM2 interaction. Prenylchalcone 2 represents a starting basis for the design of new p53-MDM2 interaction inhibitors with improved antitumor properties.

A tryptophanol-derived oxazolopiperidone lactam is cytotoxic against tumors via inhibition of p53 interaction with murine double minute proteins.

Inactivation of the p53 tumor suppressor protein by interaction with murine double minute (MDM) proteins, MDM2 and MDMX, is a common event in human tumors expressing wild-type p53. In these tumors, the simultaneous inhibition of these interactions with MDMs, for a full p53 reactivation, represents a promising anticancer strategy. Herein, we report the identification of a dual inhibitor of the p53 interaction with MDM2 and MDMX, the (S)-tryptophanol derivative OXAZ-1, from the screening of a small library of enantiopure tryptophanol-derived oxazolopiperidone lactams, using a yeast-based assay. With human colon adenocarcinoma HCT116 cell lines expressing wild-type p53 (HCT116 p53(+/+)) and its p53-null isogenic derivative (HCT116 p53(-/-)), it was shown that OXAZ-1 induced a p53-dependent tumor growth-inhibitory effect. In fact, OXAZ-1 induced p53 stabilization, up-regulated p53 transcription targets, such as MDM2, MDMX, p21, Puma and Bax, and led to PARP cleavage, in p53(+/+), but not in p53(-/-), HCT116 cells. In addition, similar tumor cytotoxic effects were observed for OXAZ-1 against MDMX-overexpressing breast adenocarcinoma MCF-7 tumor cells, commonly described as highly resistant to MDM2-only inhibitors. In HCT116 p53(+/+) cells, the disruption of the p53 interaction with MDMs by OXAZ-1 was further confirmed by co-immunoprecipitation. It was also shown that OXAZ-1 potently triggered a p53-dependent mitochondria-mediated apoptosis, characterized by reactive oxygen species generation, mitochondrial membrane potential dissipation, Bax translocation to mitochondria, and cytochrome c release, and exhibited a p53-dependent synergistic effect with conventional chemotherapeutic drugs. Collectively, in this work, a novel selective activator of the p53 pathway is reported with promising antitumor properties to be explored either alone or combined with conventional chemotherapeutic drugs. Moreover, OXAZ-1 may represent a promising starting scaffold to search for new dual inhibitors of the p53-MDMs interaction.

Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53-MDM2 interaction.

One of the most appealing targets for anticancer treatment is the p53 tumor suppressor protein. In half of human cancers, this protein is inactivated due to endogenous negative regulators such as MDM2. Actually, restoring the p53 activity, particularly through the inhibition of its interaction with MDM2, is considered a valuable therapeutic strategy against cancers with a wild-type p53 status. In this work, we report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams and the evaluation of their biological effects as p53-MDM2 interaction inhibitors. Using a yeast-based screening assay, two oxazoloisoindolinones, compounds 1b and 3a, were identified as potential p53-MDM2 interaction inhibitors. The molecular mechanism of oxazoloisoindolinone 3a was further validated in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53(+/+)) and in its isogenic derivative without p53 (HCT116 p53(-/-)). Indeed, using these cells, we demonstrated that oxazoloisoindolinone 3a exhibited a p53-dependent in vitro antitumor activity through induction of G0/G1-phase cell cycle arrest and apoptosis. The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported by the occurrence of PARP cleavage only in p53-expressing HCT116 cells. Moreover, oxazoloisoindolinone 3a led to p53 protein stabilization and to the up-regulation of p53 transcriptional activity with increased expression levels of several p53 target genes, as p21(WAF1/CIP1), MDM2, BAX and PUMA, in p53(+/+) but not in p53(-/-) HCT116 cells. Additionally, the ability of oxazoloisoindolinone 3a to block the p53-MDM2 interaction in HCT116 p53(+/+) cells was confirmed by co-immunoprecipitation. Finally, the molecular docking analysis of the interactions between the synthesized compounds and MDM2 revealed that oxazoloisoindolinone 3a binds to MDM2. Altogether, this work adds, for the first time, the oxazoloisoindolinone scaffold to the list of chemotypes activators of a wild-type p53-pathway with promising antitumor activity. Moreover, it may open the way to the development of a new class of p53-MDM2 interaction inhibitors.

Microglia P2Y6 receptors mediate nitric oxide release and astrocyte apoptosis.

BACKGROUND: During cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive phenotype. Chronically activated microglia acquire an anti-inflammatory phenotype that favors neuronal differentiation, but the impact of these microglia on astrogliosis is unknown. We investigated the contribution of pyrimidine receptors to microglia-astrocyte signaling in a chronic model of inflammation and its impact on astrogliosis. METHODS: Co-cultures of astrocytes and microglia were chronically treated with lipopolysaccharide (LPS) and incubated with uracil nucleotides for 48 h. The effect of nucleotides was evaluated in methyl-[3H]-thymidine incorporation. Western blot and immunofluorescence was performed to detect the expression of P2Y6 receptors and the inducible form of nitric oxide synthase (iNOS). Nitric oxide (NO) release was quantified through Griess reaction. Cell death was also investigated by the LDH assay and by the TUNEL assay or Hoechst 33258 staining. RESULTS: UTP, UDP (0.001 to 1 mM) or PSB 0474 (0.01 to 10 µM) inhibited cell proliferation up to 43 ± 2% (n = 10, P <0.05), an effect prevented by the selective P2Y6 receptor antagonist MRS 2578 (1 µM). UTP was rapidly metabolized into UDP, which had a longer half-life. The inhibitory effect of UDP (1 mM) was abolished by phospholipase C (PLC), protein kinase C (PKC) and nitric oxide synthase (NOS) inhibitors. Both UDP (1 mM) and PSB 0474 (10 µM) increased NO release up to 199 ± 20% (n = 4, P <0.05), an effect dependent on P2Y6 receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y6 receptors were expressed in the cultures being mainly localized in microglia. Moreover, the expression of iNOS was mainly observed in microglia and was upregulated by UDP (1 mM) or PSB 0474 (10 µM). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia. CONCLUSIONS: In LPS treated co-cultures of astrocytes and microglia, UTP is rapidly converted into UDP, which activates P2Y6 receptors inducing the release of NO by microglia that causes astrocyte apoptosis, thus controlling their rate of proliferation and preventing an excessive astrogliosis.

Purinergic modulation of norepinephrine release and uptake in rat brain cortex: contribution of glial cells.

The pathogenesis of psychiatric and neurodegenerative diseases is often associated with a deregulation of noradrenergic transmission. Considering the potential involvement of purinergic signaling in the modulation of noradrenergic transmission in the brain cortex, this study aimed to identify the P2Y receptor subtypes involved in the modulation of neuronal release and neuronal/glial uptake of norepinephrine. Electrical stimulation (100 pulses at 5 Hz) of rat cortical slices induced norepinephrine release that was inhibited by ATP and ADP (0.01-1 mM), adenosine 5'-O-(2-thiodiphosphate) (ADPßS, 0.03-0.3 mM), and UDP (0.1-1 mM). The effect of ADPßS was mediated by P2Y1 receptors and possibly by A1/P2Y1 heterodimers since it was attenuated by the A1 receptor antagonist DPCPX and by the P2Y1 receptor antagonist MRS 2500 but was resistant to the effect of adenosine deaminase (ADA). UDP inhibited norepinephrine release through activation of P2Y6 receptors, an effect that was abolished by the P2Y6 receptor antagonist MRS 2578 and by DPCPX, indicating that it depends on the formation and/or release of adenosine and activation of A1 receptors. Supporting this hypothesis, the inhibitory effect of UDP was also prevented by inhibition of ectonucleotidases, by ADA and was attenuated by the inhibitor of nucleoside transporter 6-[(4-nitrobenzyl)thio]-9-ß-d-ribofuranosylpurine (NBTI). Additionally, the inhibitory effect of UDP was attenuated when norepinephrine uptake 1 or 2 was inhibited. In astroglial cultures, ADPßS and UDP increased norepinephrine uptake mainly by activation of P2Y1 and P2Y6 receptors, respectively. The results indicate that neuronal and glial P2Y1 and P2Y6 receptors may represent new targets of intervention to regulate noradrenergic transmission in CNS diseases.

P2Y receptors on astrocytes and microglia mediate opposite effects in astroglial proliferation.

Nucleotides released upon brain injury signal to astrocytes and microglia playing an important role in astrogliosis, but the participation of microglia in the purinergic modulation of astrogliosis is still unclear. Highly enriched astroglial cultures and co-cultures of astrocytes and microglia were used to investigate the influence of microglia in the modulation of astroglial proliferation mediated by nucleotides. In highly enriched astroglial cultures, adenosine-5'-triphosphate (ATP), adenosine 5'-O-(3-thio)-triphosphate (ATPγS), adenosine 5'-O-(3-thio)-diphosphate (ADPßS; 0.01-1 mM), and adenosine-5'-diphosphate (ADP; 0.1-1 mM) increased proliferation up to 382%, an effect abolished in co-cultures containing 8% of microglia. The loss of ATP proliferative effect in co-cultures is supported by its fast metabolism and reduced ADP accumulation, an agonist of P2Y(1,12) receptors that mediate astroglial proliferation. No differences in ADPßS and ATPγS metabolism or P2Y(1,12) receptors expression were found in co-cultures that could explain the loss of their proliferative effect. However, conditioned medium from microglia cultures or co-cultures treated with ADPßS, when tested in highly enriched astroglial cultures, also prevented ADPßS proliferative effect. None of the uracil nucleotides tested had any effect in proliferation of highly enriched astroglial cultures, but uridine-5'-triphosphate (UTP; 0.1-1 mM) inhibited proliferation up to 66% in co-cultures, an effect that was dependent on uridine-5'-diphosphate (UDP) accumulation, coincident with a co-localization of P2Y(6) receptors in microglia and due to cell apoptosis. The results indicate that microglia control astroglial proliferation by preventing the proliferative response to adenine nucleotides and favouring an inhibitory effect of UTP/UDP. Several microglial P2Y receptors may be involved by inducing the release of messengers that restrain astrogliosis, a beneficial effect for neuronal repair mechanisms following brain injury.

Functional crosstalk of prejunctional receptors on the modulation of noradrenaline release in mesenteric vessels: A differential study of artery and vein.

The role of angiotensin II receptors, bradykinin receptors and ß-adrenoceptors in the modulation of noradrenaline release and the influence of α(2)-autoinhibition in these effects was investigated in the mesenteric artery and vein. Rings of mesenteric vessels of male Wistar rats were labelled with [(3)H]-noradrenaline and the effects of modulators on tritium overflow evoked by 100 pulses at 2Hz (marked α(2)-autoinhibition) and by 20 pulses at 50Hz or 100 pulses at 2Hz plus yohimbine (1µM; reduced α(2)-autoinhibition) were evaluated. Angiotensin II and bradykinin enhanced noradrenaline release evoked by 100 pulses at 2Hz, in a concentration-dependent manner, in both vessels. These effects were attenuated under conditions of reduced α(2)-autoinhibition. The attenuation was partially reversed by activation of adenosine A(1) receptors in both vessels and by activation of P2Y receptors in the vein. Isoprenaline and the selective ß(2)-adrenoceptor agonist formoterol enhanced tritium overflow independently of α(2)-autoinhibition in the vein. In the artery, the enhancement by formoterol was only observed under reduced α(2)-autoinhibition. Pharmacological characterization of the ß-adrenoceptors indicated that in the artery the effect of isoprenaline was mediated by the ß(1)-subtype under marked α(2)-autoinhibition and by the ß(2)-subtype under reduced α(2)-autoinhibition whereas in the vein the effect was independent of α(2)-autoinhibition. The results indicate that α(2)-autoinhibition is a key determinant of the magnitude of facilitation caused by angiotensin II and bradykinin in both types of mesenteric vessels and regulates the effects mediated by ß(1)-and ß(2)-adrenoceptors which co-exist in the artery.

Ethnopharmacological notes about ancient uses of medicinal plants in Trás-os-Montes (northern of Portugal).

AIM OF THE STUDY: In order to preserve the ancestral knowledge, an ethnopharmacological study has been carried out in two councils belonging to Trás-os-Montes region a small area located in the northern of Portugal. In that area, medicinal plants, most of the species wild, are still in use among farmers, shepherds and other people who live far from villages and built-up areas. MATERIALS AND METHODS: Among the 46 people that were interviewed (mean age of 66 years old), 88 species belonging to 42 families of vascular plants were identified for treatment of various human ailments. An ethnopharmacological report is made consisting of species names, vernacular names, popular uses of the plants and their pharmacological properties. RESULTS AND CONCLUSION: The most dominant family is Lamiaceae (18%) and the most frequently part of the plant used for the treatment of diseases are leaves (37.9%). The largest number of taxa is used to treat gastrointestinal disorders (73.9%).

The P2Y(1) and P2Y(12) receptors mediate autoinhibition of transmitter release in sympathetic innervated tissues.

In the sympathetic nervous system, ATP is a co-transmitter and modulator of transmitter release, inhibiting noradrenaline release by acting on P2Y autoreceptors, but in peripheral tissues the subtypes involved have only scarcely been identified. We investigated the identity of the noradrenaline release-inhibiting P2Y subtypes in the epididymal portion of vas deferens and tail artery of the rat. The subtypes operating as autoreceptors, the signalling mechanism and cross-talk with alpha(2)-autoreceptors, was also investigated in the epididymal portion. In both tissues, the nucleotides 2-methylthioATP, 2-methylthioADP, ADP and ATP inhibited noradrenaline release up to 68%, with the following order of potency: 2-methylthioADP=2-methylthioATP>ADP=ATP in the epididymal portion and 2-methylthioADP=2-methylthioATP=ADP>ATP in the tail artery. The selective P2Y(1) antagonist 2'-deoxy-N(6)-methyladenosine 3',5'-bisphosphate (30microM) and the P2Y(12) antagonist 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl ester (30microM) increased noradrenaline release per se by 25+/-8% and 18+/-3%, respectively, in the epididymal portion but not in tail artery. Both antagonists attenuated the effect of nucleotides in the epididymal portion whereas in tail artery only the P2Y(1) antagonist was effective. The agonist of P2Y(1) and P2Y(12) receptors, 2-methylthioADP, caused an inhibition of noradrenaline release that was not prevented by inhibition of phospholipase C or protein kinase C but was abolished by pertussis toxin. 2-methylthioADP and the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine were less potent at inhibiting noradrenaline release under marked influence of alpha(2)-autoinhibition. In both tissues, nucleotides modulate noradrenaline release by activation of inhibitory P2Y(1) receptors but in the epididymal portion P2Y(12) receptors also participate. P2Y(1) and P2Y(12) receptors are coupled to G(i/o)-proteins and operate as autoreceptors in the vas deferens where they interact with alpha(2)-adrenoceptors on the modulation of noradrenaline release.

Presynaptic adenosine and P2Y receptors.

Adenine-based purines, such as adenosine and ATP, are ubiquitous molecules that, in addition to their roles in metabolism, act as modulators of neurotransmitter release through activation of presynaptic P1 purinoceptors or adenosine receptors (activated by adenosine) and P2 receptors (activated by nucleotides). Of the latter, the P2Y receptors are G protein-coupled, whereas the P2X receptors are ligand-gated ion channels and not covered in this review.

Involvement of G-protein ßγ subunits on the influence of inhibitory α2-autoreceptors on the angiotensin AT1-receptor modulation of noradrenaline release in the rat vas deferens.

The influence of alpha2-autoreceptors on the facilitation of [3H]-noradrenaline release mediated by angiotensin II was studied in prostatic portions of rat vas deferens preincubated with [3H]-noradrenaline. Angiotensin II enhanced tritium overflow evoked by trains of 100 pulses at 8 Hz, an effect that was attenuated by the AT1-receptor antagonist losartan (0.3-1 microM), at concentrations suggesting the involvement of the AT1B subtype. The effect of angiotensin II was also attenuated by inhibition of phospholipase C (PLC) and protein kinase C (PKC) indicating that prejunctional AT1-receptors are coupled to the PLC-PKC pathway. Angiotensin II (0.3-100 nM) enhanced tritium overflow more markedly, up to 64%, under conditions that favor alpha2-autoinhibition, observed when stimulation consisted of 100 pulses at 8 Hz, than under poor alpha2-autoinhibition conditions, only up to 14%, observed when alpha2-adrenoceptors were blocked with yohimbine (1 microM) or when stimulation consisted of 20 pulses at 50 Hz. Activation of PKC with 12-myristate 13-acetate (PMA, 0.1-3 microM) also enhanced tritium overflow more markedly under strong alpha2-autoinhibition conditions. Inhibition of Gi/o-proteins with pertussis toxin (8 microg/ml) or blockade of Gbetagamma subunits with the anti-betagamma peptide MPS-Phos (30 microM) attenuated the effects of angiotensin II and PMA. The results indicate that activation of AT1-receptors coupled to the PLC-PKC pathway enhances noradrenaline release, an effect that is markedly favoured by an ongoing activation of alpha2-autoreceptors. Interaction between alpha2-adrenoceptors and AT1-receptors seems to involve the betagamma subunits released from the Gi/o-proteins coupled to alpha2-adrenoceptors and protein kinase C activated by AT1-receptors.

Interaction between adenosine A 2B-receptors and alpha2-adrenoceptors on the modulation of noradrenaline release in the rat vas deferens: possible involvement of a group 2 adenylyl cyclase isoform.

In the prostatic portion of rat vas deferens, activation of adenosine A 2B-receptors, beta2-adrenoceptors, adenylyl cyclase or protein kinase A caused a facilitation of noradrenaline release. Blockade of alpha2-adrenoceptors with yohimbine (1 microM) attenuated the facilitation mediated by adenosine A 2B-receptors and by direct activation of adenylyl cyclase with forskolin but not that mediated by beta2-adrenoceptors or by direct activation of protein kinase A with 8-bromoadenosine-3',5'-cyclicAMP. The adenosine A 2B- and the beta2-adrenoceptor-mediated facilitation was prevented by the adenylyl cyclase inhibitors, 2',5'-dideoxy-adenosine (3 microM) and 9-cyclopentyladenine (100 microM), at concentrations that also attenuated the release enhancing effect of forskolin, but were not changed by the phospholipase C inhibitor 1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122, 1 microM). Facilitation of noradrenaline release mediated by adenosine A 2B-receptors was also attenuated by activation of protein kinase C with the phorbol ester 12-myristate 13-acetate (1 microM) and by inhibition of Gbetagamma subunits with an anti-betagamma peptide; facilitation mediated by beta2-adrenoceptors was mainly attenuated by the calmodulin inhibitor calmidazolium (10 microM) and by the calmodulin kinase II inhibitor (N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzene-sulfonamide phosphate (KN-93, 5 microM). The results suggest that adenosine A 2B- but not beta2-adrenoceptor-mediated facilitation of noradrenaline release is enhanced by an ongoing activation of alpha2-adrenoceptors. They further suggest that adenosine A 2B-receptors and beta2-adrenoceptors are coupled to distinct adenylyl cyclase isoforms what may explain the different influence of alpha2-adrenoceptor signalling pathway on the facilitatory effects mediated by the two adenylyl cyclase coupled receptors.

Coupling to protein kinases A and C of adenosine A2B receptors involved in the facilitation of noradrenaline release in the prostatic portion of rat vas deferens.

In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1-30 microM), but not the A(2A) agonist CGS 21680 (0.001-10 microM), caused a facilitation of electrically evoked noradrenaline release (up to 43 +/- 4%), when inhibitory adenosine A(1) receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A(2B) receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of alpha(1)-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A(2B) receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase-protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A(2B) receptor-mediated facilitation of noradrenaline release.

ATP modulates noradrenaline release by activation of inhibitory P2Y receptors and facilitatory P2X receptors in the rat vas deferens.

The role of ATP on the modulation of noradrenaline release elicited by electrical stimulation (100 pulses/8 Hz) was studied in the prostatic portion of rat vas deferens preincubated with [3H]noradrenaline. In the presence of P1 antagonists, the nucleotides 2-methylthioadenosine-5'-triphosphate (2-MeSATP), 2-methylthioadenosine 5'-diphosphate (2-MeSADP), ADP, and ATP decreased electrically evoked tritium overflow up to 44%, with the following order of potency: 2-MeSATP > 2-MeSADP > ADP > or = ATP. The P2Y antagonists reactive blue 2 (RB2) and 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) increased, whereas the P2X antagonist pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) decreased evoked tritium overflow. The inhibitory effect of 2-MeSATP was antagonized by RB2 (10 microM) and by 2-MeSAMP (10 microM) but not by the selective P2Y1 receptor antagonist 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179; 10 microM). When, besides P1 receptors, inhibitory P2Y receptors were blocked with RB2, alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-meATP), beta,gamma-imidoadenosine 5'-triphosphate (beta,gamma-imidoATP), beta,gamma-methyleneadenosine 5'-triphosphate (beta,gamma-meATP), 2-MeSATP, and ATP enhanced tritium overflow up to 140%, with the following order of potency: alpha,beta-meATP > 2-MeSATP = ATP = beta,gamma-meATP > or = beta,gamma-imidoATP. The facilitatory effects of alpha,beta-MeATP and beta,gamma-imidoATP were prevented by PPNDS. Under the same conditions, apyrase attenuated, whereas the ectonucleotidase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethylene 5'-triphosphate enhanced tritium overflow, an effect that was prevented by PPNDS. In the prostatic portion of the rat vas deferens, endogenous ATP exerts a dual and opposite modulation of noradrenaline release: an inhibition through activation of P2Y receptors with a pharmacological profile similar to that of the P2Y12 and P2Y13 receptors and a facilitation through activation of P2X receptors with a pharmacological profile similar to that of P2X1 and P2X3, or PX2/P2X3 receptors.

Adenosine A2A receptor-mediated facilitation of noradrenaline release involves protein kinase C activation and attenuation of presynaptic inhibitory receptor-mediated effects in the rat vas deferens.

In the epididymal portion of rat vas deferens, facilitation of noradrenaline release mediated by adenosine A2A receptors, but not that mediated by beta2-adrenoceptors or by direct activation of adenylyl cyclase, was attenuated by blockade of alpha2-adrenoceptors and abolished by simultaneous blockade of alpha2-adrenoceptors, adenosine A1 and P2Y receptors. The adenosine A2A receptor-mediated facilitation was not changed by inhibitors of protein kinase A, protein kinase G or calmodulin kinase II but was prevented by inhibition of protein kinase C with chelerythrine or bisindolylmaleimide XI. Activation of protein kinase C with phorbol 12-myristate 13-acetate caused a facilitation of noradrenaline release that was abolished by bisindolylmaleimide XI and reduced by antagonists of alpha2-adrenoceptors, adenosine A1 and P2Y receptors. Activation of adenosine A2A receptors attenuated the inhibition of noradrenaline release mediated by the presynaptic inhibitory receptors. This effect was mimicked by phorbol 12-myristate 13-acetate and prevented by bisindolylmaleimide XI. It is concluded that adenosine A2A receptors facilitate noradrenaline release by a mechanism that involves a protein kinase C-mediated attenuation of effects mediated by presynaptic inhibitory receptors, namely alpha2-adrenoceptors, adenosine A1 and P2Y receptors.

Facilitation of noradrenaline release by adenosine A(2A) receptors in the epididymal portion and adenosine A(2B) receptors in the prostatic portion of the rat vas deferens.

The adenosine-receptor modulation of noradrenaline release was compared in prostatic and epididymal portions of rat vas deferens. In both portions, tritium overflow elicited by electrical stimulation (100 pulses/8 Hz) was reduced by the adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine, and enhanced by the nonselective receptor agonist, 5'-N-ethylcarboxamidoadenosine, in the presence of the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 and 100 nM). The adenosine A(2A) receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine, increased tritium overflow, but only in the epididymal portion. The enhancement caused by NECA was prevented by the adenosine A(2A) receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 20 nM), in the epididymal and by the adenosine A(2B) receptor antagonist, alloxazine (1 microM), in the prostatic portion. Inhibition of adenosine uptake enhanced tritium overflow in both portions, an effect blocked by ZM 241385 in the epididymal and by alloxazine in the prostatic portion. The results indicate that adenosine exerts an adenosine A(1) receptor-mediated inhibition, in both portions, and facilitation mediated by adenosine A(2A) receptors in the epididymal and by A(2B) receptors in the prostatic portion.

Release inhibitory receptors activation favours the A2A-adenosine receptor-mediated facilitation of noradrenaline release in isolated rat tail artery.

1. Interactions between A(2A)-adenosine receptors and alpha(2)-, A(1)- and P2- release-inhibitory receptors, on the modulation of noradrenaline release were studied in isolated rat tail artery. Preparations were labelled with [(3)H]-noradrenaline, superfused with desipramine-containing medium, and stimulated electrically (100 pulses at 5 Hz or 20 pulses at 50 Hz). 2. Blockade of alpha(2)-autoreceptors with yohimbine (1 microM) increased tritium overflow elicited by 100 pulses at 5 Hz but not by 20 pulses at 50 Hz. 3. The selective A(2A)-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 1-100 nM) enhanced tritium overflow elicited by 100 pulses at 5 Hz. Yohimbine prevented the effect of CGS 21680, which was restored by the A(1)-receptor agonist N(6)-cyclopentyladenosine (CPA; 100 nM) or by the P2-receptor agonist 2-methylthioadenosine triphosphate (2-MeSATP; 80 microM). 4. CGS 21680 (100 nM) failed to increase tritium overflow elicited by 20 pulses at 50 Hz. The alpha(2)-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304; 30 nM), the A(1)-receptor agonist CPA (100 nM) or the P2-receptor agonist 2-MeSATP (80 microM) reduced tritium overflow. In the presence of these agonists CGS 21680 elicited a facilitation of tritium overflow. 5. Blockade of potassium channels with tetraethylammonium (TEA; 5 mM) increased tritium overflow elicited by 100 pulses at 5 Hz to values similar to those obtained in the presence of yohimbine but did not prevent the effect of CGS 21680 (100 nM) on tritium overflow. 6. It is concluded that, in isolated rat tail artery, the facilitation of noradrenaline release mediated by A(2A)-adenosine receptors is favoured by activation of release inhibitory receptors.