Autoregulation in PC12 cells via P2Y receptors : Evidence for non-exocytotic nucleotide release from neuroendocrine cells Nucleotides are released not only from neurons , but also from various other types of cells including fibroblasts , epithelial , endothelial and glial cells . While ATP release from non-neural cells is frequently Ca2 + independent and mostly non-vesicular , neuronal ATP release is generally believed to occur via exocytosis . To evaluate whether nucleotide release from neuroendocrine cells might involve a non-vesicular component , the autocrine/paracrine activation of P2Y12 receptors was used as a biosensor for nucleotide release from PC12 cells . Expression of a plasmid coding for the botulinum toxin C1 light chain led to a decrease in syntaxin 1 detected in immunoblots of PC12 membranes . In parallel , spontaneous as well as depolarization-evoked release of previously incorporated -LSB- 3H -RSB- noradrenaline from transfected cells was significantly reduced in comparison with the release from untransfected cells , thus indicating that exocytosis was impaired . In PC12 cells expressing the botulinum toxin C1 light chain , ADP reduced cyclic AMP synthesis to the same extent as in non-transfected cells . Likewise , the enhancement of cyclic AMP synthesis either due to the blockade of P2Y12 receptors or due to the degradation of extracellular neucleotides by apyrase was not different between non-transfected and botulinum toxin C1 light chain expressing cells . However , the inhibition of cyclic AMP synthesis caused by depolarization-evoked release of endogenous nucleotides was either abolished or greatly reduced in cells expressing the botulinum toxin C1 light chain . Together , these results show that spontaneous nucleotide release from neuroendocrine cells may occur independently of vesicle exocytosis , whereas depolarization-evoked nucleotide release relies predominantly on exocytotic mechanisms . Introduction Adenine and uridine nucleotides are present in and released from all types of cells -LSB- 2 -RSB- and exert effects via P2X and/or P2Y receptors : P2X receptors are ATP-gated cation channels -LSB- 25 -RSB- ; P2Y receptors are G protein-coupled receptors , and eight different types have been identified -LRB- P2Y1 , 2 , 4 , 6 , 11 , 12 , 13 , 14 ; -LSB- 1 -RSB- -RRB- . P2Y1 , 11 , 12 and 13 are activated by adenine nucleotides , whereas P2Y6 is activated by uridine nucleotides . P2Y2 and 4 are sensitive to adenine and uridine nucleotides , and P2Y14 is activated by UDP-glucose . In a large number of non-neural tissues , e.g. liver , kidney , bones and blood vessels , released nucleotides subserve autocrine/paracrine functions by activating certain P2Y receptors -LSB- 32 , 16 , 38 , 12 , 30 -RSB- . In a given cell , this autocrine/paracrine regulation depends on the release of nucleotides , the presence of P2Y receptors and occasionally on the enzymatic activities of nucleotidases : in hepatoma cells -LSB- 16 -RSB- , for instance , released ATP is degraded to ADP to stimulate P2Y1 receptors ; in epithelial -LSB- 32 -RSB- and osteoblastic -LSB- 38 -RSB- cells , in contrast , ATP itself activates P2Y11 and P2Y2 receptors , respectively . Although ATP has first been described to be released from neurons -LSB- 15 -RSB- and to act as a neurotransmitter -LSB- 6 -RSB- , available information about neuronal autoregulation via released nucleotides and P2Y receptors is limited . Early examples were the feedback inhibition of sympathetic transmitter release via presynaptic P2Y receptors -LRB- see -LSB- 4 -RSB- -RRB- and the ATP-mediated autocrine inhibition of voltage-activated Ca2 + channels in bovine chromaffin cells -LSB- 9 -RSB- . More recently , autoregulation via P2Y receptors has been described for the phaeochromocytoma cell line PC12 -LSB- 24 -RSB- . There , endogenous P2Y12 receptors were activated by spontaneous nucleotide release to achieve half-maximal inhibition of cyclic AMP -LRB- cAMP -RRB- synthesis . Depolarization of PC12 cells by 100 mM K + further enhanced nucleotide release and thereby led to full inhibition of cAMP generation . This latter effect was prevented when voltage-activated Ca2 + channels were blocked by Cd2 + or when extracellular nucleotides were degraded by apyrase . Thus , the inhibition of cAMP synthesis in PC12 cells via P2Y receptors can be viewed as a biosensor for nucleotide release -LSB- 24 -RSB- . Nucleotides are released from various cells including fibroblast-like , epithelial , endothelial , glial and neuronal cells -LSB- 36 , 28 , 21 , 26 , 35 -RSB- . ATP release from non-neural cells can be elicited by different stimuli , such as hypotonic solutions -LSB- 36 -RSB- , mechanical stimulation -LSB- 28 -RSB- or exchange of culture media -LSB- 21 , 26 -RSB- and is frequently Ca2 + independent and therefore most probably non-vesicular . The mechanisms underlying this non-vesicular ATP release remained largely elusive , but ATP binding cassette transporters , stretch-activated cation channels as well as connexin hemichannels have been implicated -LSB- 22 -RSB- . In addition , evidence has been presented that indicates that nucleotide release from non-neural cells may to some extent be vesicular -LRB- e.g. -LSB- 17 , 8 -RSB- -RRB- . In neurons and neuroendocrine cells , ATP is stored in vesicles either alone or together with classic neurotransmitters and is thus released by exocytosis -LSB- 27 -RSB- . Vesicle exocytosis occurs spontaneously at a slow rate and is largely accelerated when Ca2 + entry is triggered by depolarization-induced opening of voltage-activated Ca2 + channels -LSB- 29 -RSB- . Accordingly , depolarization-evoked ATP release from neurons and neuroendocrine cells is entirely Ca2 + dependent -LSB- 35 , 13 -RSB- , but it remained unknown whether all of the spontaneous nucleotide release from neuronal sources is also vesicular . Vesicle exocytosis in neurons and neuroendocrine cells involves the SNARE proteins syntaxin , SNAP-25 and VAMP/synaptobrevin . These proteins are cleaved by various serotypes of clostridial neurotoxins which thereby prevent exocytosis . In particular , botulinum neurotoxin C1 cleaves syntaxin , and in some cells also SNAP-25 , and thereby impedes spontaneous as well as Ca2 + - evoked vesicle exocytosis -LSB- 5 -RSB- . Transfection of PC12 cells with a plasmid coding for the botulinum toxin C1 light chain was reported to largely reduce spontaneous as well as depolarization-evoked release of co-transfected human growth hormone -LSB- 10 -RSB- . Here , we used an analogous method to investigate whether in PC12 cells the activation of P2Y12 receptors by endogenously released nucleotides relies on vesicle exocytosis . Materials and methods Materials -LSB- 2,8 − 3H -RSB- adenine -LRB- specific activity 32 Ci mmol − 1 -RRB- and levo - -LSB- ring-2,5,6-3H -RSB- noradrenaline -LRB- specific activity 37 Ci mmol − 1 -RRB- were obtained from NEN -LRB- Vienna , Austria -RRB- . Na-ADP , 4 - -LRB- 3-butoxy-4-methoxybenzyl -RRB- imidazoline-2-one -LRB- RO 20-1724 -RRB- , 3 ′ ,5 ′ - cyclic AMP , apyrase -LRB- grade VII , with an approximately 1:1 ratio in ATPase and ADPase activity -RRB- , 2-methylthio-AMP , and 2-p - -LRB- 2-carboxyethyl -RRB- phenethylamino-5 ′ - N-ethylcarboxamido-adenosine -LRB- CGS 21680 -RRB- were purchased from Sigma -LRB- Vienna , Austria -RRB- . Cell culture and transfection PC12 cells were maintained and subcultured as previously described in detail -LSB- 24 -RSB- . The cells were plated onto collagen-coated -LRB- Biomedical Technologies Inc. , Stoughton , MA , USA -RRB- culture dishes -LRB- NUNC , Roskilde , Denmark -RRB- and were kept in OptiMEM -LRB- InVitrogen , Vienna , Austria -RRB- supplemented with 0.2 mM L-glutamine -LRB- HyClone , Aalst , Belgium -RRB- , 25,000 IU l − 1 penicillin and 25 mg l − 1 streptomycin -LRB- Sigma , Vienna , Austria -RRB- , 5 % fetal calf serum and 10 % horse serum -LRB- both InVitrogen , Vienna , Austria -RRB- . Once per week , cell cultures were split ; the medium was exchanged twice per week . To investigate the outflow of previously incorporated -LSB- 3H -RSB- noradrenaline under continuous superfusion , PC12 cells were plated onto 5-mm discs , for assays of cAMP accumulation they were plated onto 6-well culture dishes and for membrane preparations for Western blots they were plated onto 100-mm culture dishes . All this tissue culture plastic was coated with collagen -LRB- as above -RRB- . For the generation of PC12 cell clones stably expressing the botulinum toxin C1 light chain -LRB- BoNT/C1 -RRB- , 15 μg of a pcDNA3 vector harbouring the botulinum toxin C1 light chain -LRB- pBoNT/C1 ; kindly provided by R.D. Burgoyne , Liverpool , UK -RRB- -LSB- 10 -RSB- were mixed with 50 μl of the TransFast transfection reagent -LRB- Promega , Mannheim , Germany -RRB- and added to semiconfluent PC12 cell cultures in serum-free medium . After a 1 h incubation at 37 °C , two additional volumes of serum-free medium and the appropriate amount of serum -LRB- as above -RRB- were added . This medium was exchanged 48 h after transfection for a medium supplemented with 500 μg/ml neomycin -LRB- G418 -RRB- to allow for selection of drug resistance . This selection medium was replaced every 3 -- 4 days until distinct islands of surviving cells were visible . Individual clones of antibiotic-resistant cells were transferred to 24-well plates and grown in medium containing 200 μg/ml G418 . Immunoblotting of SNARE proteins Preparation of protein extracts of PC12 membranes was performed as described previously -LSB- 18 -RSB- . Materials and methodology used for production and analysis of ECL-immunoblots were also essentially as described therein , with the following modifications : protein extracts in SDS sample buffer were incubated at 95 °C for 5 min and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis -LRB- SDS-PAGE -RRB- on 5 % -LRB- stacking -RRB- and 10 % -LRB- separating -RRB- gels . After electrophoretic transfer of proteins to nitrocellulose , membranes were cut horizontally along appropriate molecular weight markers in order to separate regions harbouring proteins with apparent molecular weights of 10 -- 20 kD , 21 -- 29 kD and 30 -- 50 kD . These membrane strips were incubated in one of the following monoclonal SNARE antibodies : syntaxin 1 antibody clone 78.3 , synaptobrevin 2 antibody clone 69.1 -LRB- both obtained from Synaptic Systems , Göttingen , Germany -RRB- and SNAP-25 antibody MAB331 -LRB- purchased from Chemicon , Temecula , CA , USA -RRB- . Measurement of -LSB- 3H -RSB- noradrenaline release -LSB- 3H -RSB- noradrenaline uptake in and superfusion of PC12 cell cultures were performed as described previously -LSB- 23 -RSB- . Culture discs with PC12 cells were incubated in 0.1 μM -LSB- 3H -RSB- noradrenaline in culture medium supplemented with 1 mM ascorbic acid at 36 °C for 1 h . After labelling , culture discs were transferred to small chambers and superfused with a buffer containing -LRB- mM -RRB- NaCl -LRB- 120 -RRB- , KCl -LRB- 3.0 -RRB- , CaCl2 -LRB- 2.0 -RRB- , MgCl2 -LRB- 2.0 -RRB- , glucose -LRB- 20 -RRB- , hydroxyethylpiperazine ethanesulphonic acid -LRB- HEPES -RRB- -LRB- 10 -RRB- , fumaric acid -LRB- 0.5 -RRB- , Na-pyruvate -LRB- 5.0 -RRB- , ascorbic acid -LRB- 0.57 -RRB- and desipramine -LRB- 0.001 -RRB- , adjusted to pH 7.4 with NaOH . Superfusion was performed at 25 °C at a rate of about 1.0 ml/min . Collection of 4-min superfusate fractions was started after a 60-min washout period to remove excess radioactivity . Depolarization-dependent overflow of previously incorporated -LSB- 3H -RSB- noradrenaline was triggered by inclusion of 30 or 100 mM KCl in the superfusion buffer during two 30-s periods 20 min apart -LRB- Fig. 2a -RRB- . At the end of the experiments , the radioactivity remaining in the cells was extracted by immersion of the discs in 2 % -LRB- v/v -RRB- perchloric acid followed by sonication . Radioactivity in extracts and collected fractions was determined by liquid scintillation counting -LRB- Packard Tri-Carb 2100 TR -RRB- . The spontaneous -LRB- unstimulated -RRB- rate of -LSB- 3H -RSB- outflow was obtained by expressing the radioactivity of a collected fraction as percentage of the total radioactivity in the cultures at the beginning of the corresponding collection period . Values obtained during the 4-min period directly preceding the application of 30 mM K + were used for the comparison of spontaneous -LRB- basal -RRB- tritium outflow in wild-type and pBoNT/C1 expressing PC12 cells . K + - evoked tritium overflow was calculated as the difference between the total -LSB- 3H -RSB- outflow during and after stimulation and the estimated basal outflow which was assumed to decline linearly throughout the experiments . Therefore , basal outflow during periods of stimulation was assumed to equate the arithmetic mean of the samples preceding and those following stimulation , respectively . The difference between the total and the estimated basal outflow was expressed as a percentage of the total radioactivity in the cultures at the beginning of the respective stimulation -LRB- % of total activity -RRB- . Determination of cyclic AMP The accumulation of cAMP in PC12 cell cultures was determined as described before -LSB- 33 -RSB- . After labelling of cellular purines with tritiated adenine -LRB- 2.5 μCi/ml for 12 h -RRB- , the medium was replaced by a buffer -LRB- 120 mM NaCl , 3 mM KCl , 2 mM MgCl2 , 2 mM CaCl2 , 20 mM glucose , 10 mM HEPES , 10 mM LiCl , adjusted to pH 7.4 with NaOH -RRB- supplemented with 100 μM of the phosphodiesterase inhibitor Ro-20-1724 -LSB- 4 - -LRB- 3-butoxy-4-methoxybenzyl -RRB- imidazolidin-2-one -RSB- and 1 U/ml adenosine deaminase . Dishes were then kept at room temperature for 105 min . During the last 15 min of this incubation period , the adenosine A2A receptor agonist 2-p - -LRB- 2-carboxyethyl -RRB- phenethylamino-5 ′ - N-ethylcarboxamido-adenosine -LRB- CGS 21680 -RRB- , ADP -LRB- 10 μM -RRB- , apyrase -LRB- 1 U/ml -RRB- or 100 mM KCl -LRB- NaCl was reduced accordingly -RRB- were also included in the medium . When appropriate , the P2Y12 receptor antagonist 2MesAMP -LRB- 100 μM ; -LSB- 14 -RSB- -RRB- was present for the last 25 min . The incubation was terminated by exchanging the buffer for 1 ml of 2.5 % perchloric acid containing 100 μM non-labelled cAMP followed by a 20-min incubation at 4 °C . Subsequently , cAMP was separated from the other purines by a chromatographic procedure described before -LSB- 33 -RSB- . One tenth of each sample obtained as described above was used for the determination of the total radioactivity . The remaining 900 μl were neutralised by addition of 100 μl 4.2 M KOH and applied to Dowex 50 columns -LRB- AG 50W-X4 ; Bio-Rad , Vienna , Austria -RRB- , which were then rinsed with 3 ml H2O . The eluate obtained by the subsequent application of 8 ml H2O was directly poured onto alumina columns -LRB- Bio-Rad , Vienna , Austria -RRB- , which were then washed with 6 ml H2O . Finally , cAMP was eluted with 4 ml imidazole buffer -LRB- 20 mM imidazole in 0.2 M NaCl ; pH 7.45 -RRB- . Radioactivity within the samples obtained was determined by liquid scintillation counting . The radioactivity in the fraction of cAMP was expressed as percentage of the total radioactivity incorporated in the cells . Stimulation of PC12 cells , whether they were clonal cell lines or wild-type cells , with the adenosine A2A receptor agonist CGS 21680 caused reproducible increases in the values of cAMP . However , the extent of basal and stimulated cAMP synthesis may vary between different preparations -LSB- 33 -RSB- . Hence , to be able to directly compare changes in CGS 21680-induced cAMP synthesis in different cell clones , values obtained in the presence of ADP , apyrase , 2MesAMP or KCl were expressed as percentage of the values obtained in the very same preparation , but in the absence of these agents -LRB- % of control -RRB- . Statistics All data represent arithmetic means ± SEM ; n represents numbers of culture dishes . Statistical significance of differences between single data points was evaluated by the non-parametric Mann-Whitney test , and p values < 0.05 were accepted as indicators of significance . Results To assess the role of vesicle exocytosis in the release of endogenous nucleotides from neuroendocrine cells , this study employed the autocrine/paracrine control of cAMP synthesis in PC12 cells via P2Y12 receptors . To prevent vesicle exocytosis , we generated cell clones stably expressing pBoNT/C1 . Depending on the cell types being investigated , this toxin cleaves one or more SNARE proteins and thereby prevents vesicular transmitter release or hormone secretion -LSB- 5 -RSB- . Cleavage of SNARE proteins by the expression of the BoNT/C1 light chain The major target of BoNT/C1 is syntaxin 1 , which is cleaved by this toxin in all types of preparations -LSB- 5 -RSB- . Therefore , several pBoNT/C1 expressing clones were initially tested in immunoblots of harvested membranes for a lack of full-length syntaxin . Two of these pBoNT/C1 expressing clones -LRB- BT2 and BT7 -RRB- were selected for further analysis . In bovine chromaffin cells , BoNT/C1 was reported to cleave not only syntaxin 1 , but also SNAP-25 , even though the toxin failed to cleave purified and recombinant SNAP-25 -LSB- 11 -RSB- . To evaluate the efficiency of SNARE protein cleavage by the BoNT/C1 light chain expressed in PC12 cells , the immunostaining of syntaxin and SNAP-25 in Western blots was compared with that of synaptobrevin , which is undoubtedly insensitive to BoNT/C1 -LSB- 11 , 5 -RSB- . As shown in Fig. 1 , the stable expression of pBoNT/C1 markedly reduced or even abolished the staining of a protein with an apparent molecular weight of 37 kD by an anti-syntaxin antibody . In contrast , there were no clear-cut reductions in the staining of a 25-kD band by an anti-SNAP-25 antibody , and occasionally the immunostaining of this protein appeared to be even enhanced in pBoNT/C1 expressing clones -LRB- Fig. 1a -RRB- . To quantify these changes in SNARE protein expression , the immunoblots were quantified by densitometry and the values obtained for the anti-syntaxin and anti-SNAP-25 immunoreactive bands were set in relation to those of the anti-synaptobrevin immunoreactive bands -LRB- Fig. 1b -RRB- . This procedure revealed a significant reduction of syntaxin in the pBoNT/C1 expressing clones when compared with wild-type PC12 cells . SNAP-25 immunoreactivity , in contrast , was not significantly altered in these cell clones . Fig. 1Identification of SNARE proteins in membrane extracts from either wild-type -LRB- WT , untransfected -RRB- PC12 cells or from two PC12 cell clones stably expressing pBoNT/C1 -LRB- BT2 and BT7 -RRB- . SDS-membrane extracts were separated on SDS-PAGE and transferred to nitrocellulose membranes . The membranes were cut horizontally to separate regions harbouring proteins with apparent molecular weights of 1 -- 20 kD , 21 -- 29 kD and 30 -- 50 kD and immunostained with anti-syntaxin 1 antibody clone 78.3 -LRB- Stx -RRB- , anti-SNAP-25 antibody MAB331 -LRB- S25 -RRB- and anti-synaptobrevin 2 antibody clone 69.1 -LRB- Sb -RRB- , respectively . a Representative blot obtained as described above . b The relation between the anti-syntaxin and the anti-synaptobrevin immunoreactivities -LRB- Stx/Sb -RRB- as determined by densitometric analysis -LRB- n = 3 -RRB- . c The relation between the anti-SNAP-25 and the anti-synaptobrevin immunoreactivities -LRB- Stx/Sb -RRB- as determined by densitometric analysis -LRB- n = 3 -RRB- . * Significant differences versus the results obtained with membranes from wild-type cells at p < 0.05 Inhibition of transmitter release by the expression of the BoNT/C1 light chain Previously , it has been shown that the transient expression of pBoNT/C1 reduced stimulated vesicular catecholamine release from PC12 cells -LSB- 10 -RSB- . To evaluate whether catecholamine release was reduced in the pBoNT/C1 expressing clones , clonal and wild-type PC12 cells were labelled with -LSB- 3H -RSB- noradrenaline and the basal and depolarization-dependent outflow of radioactivity was determined . After washout of excess radioactivity during a 1-h washout period , all PC12 cell cultures steadily released small amounts of radioactivity in the superfusion buffer -LRB- Fig. 2a -RRB- . However , this spontaneous outflow of radioactivity was significantly less in the two pBoNT/C1 expressing clones than in untransfected PC12 cells -LRB- Fig. 2b -RRB- . Fig. 2Basal and depolarization-evoked -LSB- 3H -RSB- noradrenaline release from either wild-type -LRB- WT , untransfected -RRB- PC12 cells or from two PC12 cell clones stably expressing pBoNT/C1 -LRB- BT2 and BT7 -RRB- . PC12 cell cultures were labelled with -LSB- 3H -RSB- noradrenaline and superfused . Subsequent to a 60-min washout period , 4-min fractions of superfusate were collected . Tritium overflow was stimulated twice -LRB- S1 after 72 min and S2 after 92 min of superfusion -RRB- by the presence of KCl -LRB- 30 or 100 mM , each for 30 s -RRB- . a Comparison of the time courses of fractional -LSB- 3H -RSB- outflow from the three different kinds of cell cultures -LRB- n = 5 to 6 -RRB- . b The amount of basal -LSB- 3H -RSB- outflow from the three PC12 cell cultures -LRB- n = 8 to 9 -RRB- . c The amount of -LSB- 3H -RSB- overflow from the three PC12 cell cultures triggered by 30 mM KCl -LRB- n = 8 to 9 -RRB- . d The amount of -LSB- 3H -RSB- overflow from the three PC12 cell cultures triggered by 100 mM KCl -LRB- n = 8 to 9 -RRB- . * , ** , *** Significant differences versus the results obtained with wild-type cells at p < 0.05 , p < 0.01 and p < 0.001 , respectively In wild-type cells , the presence of 30 mM K + -LRB- Na + was reduced accordingly -RRB- for 30 s caused a reproducible increase in the outflow of radioactivity -LRB- Fig. 2a -RRB- , but such an effect was hardly observed in the pBoNT/C1 expressing clones . Accordingly , the overflow of radioactivity triggered by 30 mM K + was significantly reduced in the pBoNT/C1 expressing clones in comparison with wild-type cells -LRB- Fig. 2c -RRB- . This confirms that the expression of the BoNT/C1 light chain reduced spontaneous noradrenaline release and almost abolished release due to mild depolarizations . Large intracellular Ca2 + increases have been reported to rescue transmitter release from cells poisoned with BoNT/C1 -LSB- 7 -RSB- . Therefore , PC12 cultures were also exposed to 100 mM K + -LRB- Na + was again reduced -RRB- for 30 s. Although this stimulation paradigm caused unequivocal increases in the outflow of radioactivity even from pBoNT/C1 expressing cells -LRB- Fig. 2a -RRB- , the amount of tritium overflow was still reduced when compared with the values obtained in untransfected PC12 cells -LRB- Fig. 2d -RRB- . Thus , the expression of the BoNT/C1 light chain efficiently reduced spontaneous as well as depolarization-evoked vesicular transmitter release . Changes in the regulation of cAMP synthesis via P2Y receptors due to the expression of the BoNT/C1 light chain In PC12 cells , activation of endogenous P2Y12 receptors by the exposure to nucleotides causes an inhibition of adenylyl cyclase -LSB- 33 -RSB- . Furthermore , spontaneously released nucleotides are also sufficient to partially activate these receptors , and depolarization of the cells fully activates the receptors through enhanced nucleotide release to maximally suppress cAMP synthesis -LSB- 24 -RSB- . In accordance with these previous data , exposure of wild-type PC12 cells to 10 μM ADP significantly reduced the amount of cAMP generated due to the activation of A2A adenosine receptor by 1 μM CGS 21680 present for 15 min . In the two pBoNT/C1 expressing clones , the inhibition of cAMP accumulation was as pronounced as in the wild-type cells -LRB- Fig. 3a -RRB- . Thus , the adenylyl cyclase inhibiting P2Y receptors were operating equally well in transfected as well as untransfected PC12 cells . Fig. 3Changes in cAMP synthesis caused by exogenous ADP or endogenously released nucleotides in either wild-type -LRB- WT , untransfected -RRB- PC12 cells or in two PC12 cell clones stably expressing pBoNT/C1 -LRB- BT2 and BT7 -RRB- . After loading with -LSB- 3H -RSB- adenine , PC12 cells were incubated in RO 20 -- 1724 -LRB- 100 μM -RRB- for 105 min . During the last 15 min of this incubation period , 1 μM CGS 21680 was present either alone or together with 10 μM ADP -LRB- a -RRB- , 1 U/ml apyrase -LRB- b -RRB- , 100 μM MeSAMP -LRB- c -RRB- or 100 mM KCl -LRB- d -RRB- . The amount of radioactivity retrieved within the fraction of cyclic AMP -LRB- cAMP -RRB- was calculated as percentage of the total radioactivity extracted from the cell cultures . In order to compare the results of different cell cultures , values obtained in the presence of ADP , apyrase , 2MesAMP or KCl were expressed as percentage of the values obtained in the very same preparation , but in the absence of these agents -LRB- % of control -RRB- . a Results obtained in the presence of 10 μM ADP are expressed as percentage of the results obtained in its absence -LRB- % of control ; n = 6 to 10 -RRB- . b Results obtained in the presence of 1 U/ml apyrase are expressed as percentage of the results obtained in its absence -LRB- % of control ; n = 9 to 10 -RRB- . c Results obtained in the presence of 100 μM MeSAMP are expressed as percentage of the results obtained in its absence -LRB- n = 6 to 9 -RRB- . d Results obtained in the presence of 100 mM KCl are expressed as percentage of the results obtained in its absence -LRB- n = 9 ; *** p < 0.001 versus the results obtained in wild-type cells ; # , ### p < 0.05 and p < 0.001 versus the results obtained in the absence of KCl -RRB- In the presence of apyrase -LRB- 1 U/ml -RRB- to degrade extracellular nucleotides , the cAMP accumulation triggered by CGS 21680 was almost doubled . Again , this effect was not different between wild-type and pBoNT/C1 expressing cells -LRB- Fig. 3b -RRB- . In accordance with these latter results , the blockade of the receptors by the P2Y12 receptor antagonist 2-MeSAMP -LRB- 100 μM ; -LSB- 14 -RSB- -RRB- also enhanced the stimulation of cAMP about twofold and there were no significant differences between the three types of PC12 cultures -LRB- Fig. 3b -RRB- . Thus , the activation of the P2Y receptors negatively linked to adenylyl cyclase by spontaneously released nucleotides was the same whether pBoNT/C1 was expressed or not . When wild-type PC12 cells were depolarized by 100 mM K + , the CGS 21680-induced cAMP accumulation was reduced to about the same extent as by 10 μM ADP . However , in one -LRB- BT2 -RRB- of the two pBoNT/C1 expressing clones , the depolarization of PC12 cells failed to cause any alteration in the CGS 21680-induced cAMP accumulation ; and in the other clone -LRB- BT7 -RRB- , this inhibition was significantly weaker when compared with the values obtained in wild-type PC12 cells -LRB- Fig. 3d -RRB- . Thus , depolarization-dependent inhibition of the cAMP synthesis via P2Y12 receptors was greatly impaired in the pBoNT/C1 expressing cells . Discussion In non-neural cells , release of nucleotides is mostly non-vesicular and involves , for instance , ATP binding cassette transporters , stretch-activated cation channels or connexin hemichannels -LSB- 22 -RSB- . In neurons , however , nucleotide release is generally believed to rely on vesicle exocytosis -LSB- 27 -RSB- . Here , we employed the autocrine/paracrine activation of P2Y12 receptors in PC12 cells to investigate whether nucleotide release from neuroendocrine cells might involve a non-vesicular component . Vesicle exocytosis involves the SNARE proteins syntaxin , SNAP-25 and synaptobrevin , and cleavage of one or more of these proteins by clostridial neurotoxins reduces or even abolishes exocytotic transmitter release -LSB- 5 -RSB- . The primary target of BoNT/C1 is syntaxin , and its inhibitory effect on transmitter release has been correlated with the cleavage of this protein -LSB- 3 -RSB- . However , in cultures of bovine chromaffin cells -LSB- 11 -RSB- , spinal cord -LSB- 37 -RSB- or hippocampal neurons -LSB- 7 -RSB- , BoNT/C1 was reported to cleave not only syntaxin , but also SNAP-25 . In contrast , in sensory neurons the toxin was reported to act exclusively on syntaxin -LSB- 31 -RSB- . Furthermore , in cell-free assays BoNT/C1 was found to leave either native SNAP-25 after subcellular fractionation or recombinant SNAP-25 unaltered -LSB- 11 , 37 -RSB- . Thus , the effects of BoNT/C1 on SNARE proteins appear to vary between different preparations . In this study , expression of the BoNT/C1 light chain reduced syntaxin , but not SNAP-25 immunoreactivity detected in PC12 cells . Likewise , in the insulin secreting β-cell line HIT-T15 expression of the BoNT/C1 light chain only affected syntaxin , even though application of recombinant toxin to permeabilized cells did lead to a reduction in SNAP-25 -LSB- 20 -RSB- . Hence , heterologous expression of the toxin 's light chain in neuroendocrine cells does not entirely reproduce the effects of toxin application to these cells . Syntaxin was reported to be more sensitive to BoNT/C1 light chain than SNAP-25 -LSB- 20 -RSB- and a 1000-fold higher light chain concentration of BoNT/C was required for SNAP-25 cleavage when compared with the other SNAP-25-targeting serotypes BoNT/A or BoNT/E -LSB- 34 -RSB- . Thus , the resistance of SNAP-25 to heterologously expressed BoNT/C1 light chain is likely due to the relatively low toxin affinity and insufficient expression levels of the peptide . Nevertheless , in functional experiments , expression of the BoNT/C1 light chain significantly reduced spontaneous as well as depolarization-evoked release of -LSB- 3H -RSB- noradrenaline . On the one hand , this confirms that the cleavage of syntaxin alone is sufficient to reduce transmitter release -LSB- 3 -RSB- . On the other hand , this result corroborates that catecholamines are released from PC12 cells through vesicular mechanisms and these are largely impaired by the expression of the BoNT/C1 light chain -LSB- 10 -RSB- . Considering that the heterologous expression of the BoNT/C1 light chain did efficiently reduce vesicle exocytosis , the question arises as to how this manipulation altered the release of endogenous nucleotides . In PC12 cells , endogenously released nucleotides activate P2Y12 receptors , which mediate a decrease in the synthesis of cAMP triggered by the activation of A2A adenosine receptors -LSB- 24 -RSB- . In accordance with these previous results , exposure of non-transfected PC12 cells to the P2Y12 receptor antagonist 2-MesAMP enhanced the cAMP synthesis . An equivalent effect was achieved when the cells were incubated in the nucleotide degrading enzyme apyrase , thus indicating that spontaneously released nucleotides continuously activated the P2Y12 receptors . To be able to evaluate whether the generation of PC12 clones stably expressing the BoNT/C1 light chain might interfere with the above autocrine/paracrine signalling loop only by affecting exocytosis , we had to establish that the function of the P2Y12 receptor was not compromised by the toxin 's light chain or by the clonal selection procedure . Accordingly , 10 μM ADP , a saturating concentration of the nucleotide in the inhibition of cAMP synthesis in PC12 cells -LSB- 33 -RSB- , were applied to non-transfected cells and to the two chosen cell clones -LRB- BT2 and BT7 -RRB- : the inhibition of cAMP accumulation was not different in the three cultures . Thus , the control of cAMP synthesis via the P2Y12 receptor was not altered by pBoNT/C1 expression or by clonal selection . Previously , we had found that the control of voltage-gated Ca2 + channels via this receptor subtype was the same in a number of PC12 cell clones expressing various types of membrane receptors -LSB- 19 -RSB- . Knowing that the P2Y12 receptors operated independently of the BoNT/C1 light chain , the autocrine/paracrine activation of the receptors was assessed . In the toxin expressing clones , the degradation of extracellular nucleotides by apyrase and the blockade of P2Y12 receptors by 2-MesAMP had identical effects on cAMP accumulation as in non-transfected cells . This demonstrates that the spontaneous release of nucleotides was not significantly affected by the BoNT/C1 light chain . Enhancement of nucleotide release by depolarization of PC12 cells fully activates P2Y12 receptors and thereby reduces cAMP synthesis to about the same extent as 10 μM ADP . This phenomenon is based on Ca2 + - dependent vesicle exocytosis as it is suppressed when Ca2 + influx is prevented by the blockade of voltage-gated Ca2 + channels or when extracellular nucleotides are degraded by apyrase -LSB- 24 -RSB- . In support of this conclusion , the inhibition of cAMP synthesis through a depolarization of PC12 cells by 100 mM K + was either significantly attenuated or entirely abolished in the pBoNT/C1 expressing clones . Hence , the K + - evoked and thus Ca2 + - dependent release of nucleotides was largely reduced by the BoNT/C1 light chain . Taken together , the present results show that the expression of the BoNT/C1 light chain impairs vesicular transmitter release from PC12 cells , but does not alter the autocrine/paracrine activation of P2Y receptors by spontaneously released nucleotides . Thus , the spontaneous nucleotide release from neuroendocrine cells may occur independently of vesicle exocytosis . The depolarization-evoked nucleotide release , in contrast , relies predominantly on exocytotic mechanisms .

O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O B I O O O O O O O O O O

cyclic amp
nucleotide release
pc12 cells
vesicle exocytosis


Mapping P2X and P2Y receptor proteins in striatum and substantia nigra : An immunohistological study Our work aimed to provide a topographical analysis of all known ionotropic P2X1 -- 7 and metabotropic P2Y1 ,2,4,6,11 -- 14 receptors that are present in vivo at the protein level in the basal ganglia nuclei and particularly in rat brain slices from striatum and substantia nigra . By immunohistochemistry-confocal and Western blotting techniques , we show that , with the exception of P2Y11 ,13 receptors , all other subtypes are specifically expressed in these areas in different amounts , with ratings of low -LRB- P2X5 ,6 and P2Y1 ,6,14 in striatum -RRB- , medium -LRB- P2X3 in striatum and substantia nigra , P2X6 ,7 and P2Y1 in substantia nigra -RRB- and high . Moreover , we describe that P2 receptors are localized on neurons -LRB- colocalizing with neurofilament light , medium and heavy chains -RRB- with features that are either dopaminergic -LRB- colocalizing with tyrosine hydroxylase -RRB- or GABAergic -LRB- colocalizing with parvalbumin and calbindin -RRB- , and they are also present on astrocytes -LRB- P2Y2 ,4 , colocalizing with glial fibrillary acidic protein -RRB- . In addition , we aimed to investigate the expression of P2 receptors after dopamine denervation , obtained by using unilateral injection of 6-hydroxydopamine as an animal model of Parkinson 's disease . This generates a rearrangement of P2 proteins : most P2X and P2Y receptors are decreased on GABAergic and dopaminergic neurons , in the lesioned striatum and substantia nigra , respectively , as a consequence of dopaminergic denervation and/or neuronal degeneration . Conversely , P2X1 ,3,4,6 on GABAergic neurons and P2Y4 on astrocytes augment their expression exclusively in the lesioned substantia nigra reticulata , probably as a compensatory reaction to dopamine shortage . These results disclose the presence of P2 receptors in the normal and lesioned nigro-striatal circuit , and suggest their potential participation in the mechanisms of Parkinson 's disease . Introduction It is now well established that the arrangement of ionotropic P2X and metabotropic P2Y receptors -LSB- 1 , 2 -RSB- on a cell membrane is a very dynamic process , often related to developmental or physiopathological conditions . Moreover , it is common knowledge that multiple P2 proteins are simultaneously recruited on a cell membrane for triggering biological functions . As a consequence , P2 receptors are rightly considered more than the sum of their single entities and must be therefore regarded as a complex network of cooperating receptors . Under this perspective , a numerical model was also introduced , the combinatorial receptor web model , which explains the biological efficacy of combining an assorted array of different P2 proteins on a given cell , in order to integrate , upgrade , guarantee and optimize specific receptor-dependent functions -LSB- 3 -RSB- . This trend of course applies to the central nervous system -LRB- CNS -RRB- as well , where in situ hybridization of P2 mRNA subtypes and immunohistochemistry of P2 proteins shows , for instance , wide but heterogeneous simultaneous distribution of both P2X -LSB- 4 -- 11 -RSB- and P2Y -LSB- 12 -- 15 -RSB- classes of receptors . In particular , P2X2 ,4,6 and P2Y1 subtypes are abundant and widespread approximately in the entire brain , while P2X1 protein is enriched in the cerebellum , P2X3 in the brain stem , and P2X7 is largely prejunctional . The hippocampus concurrently expresses all P2X and , moreover , P2Y1 ,2,4,6,12 receptor subtypes . Particularly in the basal ganglia -LRB- BG -RRB- , neostriatal medium-spiny neurons and cholinergic interneurons highly express P2X2 and P2Y1 receptors , but it appears that they become functional only under certain , as yet unknown , conditions -LSB- 16 -RSB- . Moreover , P2X2 receptor protein was described in substantia nigra pars compacta -LRB- SNC -RRB- -LSB- 17 -RSB- , whereas both protein and mRNA were described in SNC and striatum -LSB- 18 -RSB- . Finally , only very low levels of P2X4 ,6 mRNAs were detected in substantia nigra -LRB- SN -RRB- and striatum -LSB- 19 -RSB- . By functional analysis , ATP release was demonstrated from cultured embryonic neostriatal neurons -LSB- 20 -RSB- , and ATP-evoked potassium currents in rat striatal neurons were shown to be mediated by P2 receptors -LSB- 21 -RSB- . ATP was also proved to increase extracellular dopamine levels in rat striatum through stimulation of P2Y subtypes -LSB- 22 -RSB- , although it was claimed to inhibit dopamine release in the neostriatum -LSB- 23 -RSB- . Extracellular ATP via P2 receptors was finally reported to induce neurotoxicity in vitro -LSB- 24 -RSB- and in vivo -LSB- 25 -RSB- in the striatum . Besides P2 receptors on neurons , in BG there is also evidence of P2 receptors on , and release of ATP from , glial cells . P2Y12 subtype is present , for instance , on oligodendrocytes in striatum and SN -LSB- 26 -RSB- , and P2X7 receptor is upregulated on microglia in striatum after middle cerebral artery occlusion -LSB- 27 -RSB- . In spite of these results , there is a general paucity of studies addressing the cellular distribution of all P2 receptor proteins in BG . Our work thus aimed to provide the complete topographical analysis of known P2X and P2Y subtypes that are present in rat striatum and SN in vivo , and to investigate the dynamic presence of P2 proteins after the induction of experimental parkinsonism by dopamine-denervation achieved by using the unilateral 6-hydroxydopamine -LRB- 6-OHDA -RRB- rat model . By upgrading the current map of P2 receptors expressed in the brain , our study discloses the potential impact of these receptors in the normal and lesioned nigro-striatal circuit . Materials and methods Histological procedures Wistar rats -LRB- Harlan , Udine , Italy -RRB- were anesthetized by i.p. injections of sodium pentobarbital -LRB- 60 mg/kg -RRB- , and transcardially perfused with saline -LRB- 0.9 % NaCl -RRB- followed by 4 % paraformaldehyde , in phosphate buffer -LRB- PB , 0.1 M pH 7.4 -RRB- . Each brain was immediately removed , post-fixed in the same fixative for 2 h , and then transferred to 30 % sucrose in PB at 4 °C , until it sank . The experimental protocol used in this study was approved by the Italian Ministry of Health and was in agreement with the guidelines of the European Communities Council Directive of November 24 , 1986 -LRB- 86/609/EEC -RRB- for the care and use of laboratory animals . All efforts were made to minimize the number of animals used and their suffering . Double immunofluorescence Transverse sections -LRB- 40-μm thick -RRB- were cut on a freezing microtome and were processed for double immunofluorescence studies . Non-specific binding sites were blocked with 10 % normal donkey serum in 0.3 % Triton X-100 , in phosphate buffered saline -LRB- PBS -RRB- for 30 min at room temperature . The sections were incubated in a mixture of primary antisera for 24 -- 48 h in 0.3 % Triton X-100 in PBS . Rabbit anti-P2r -LRB- 1:300 , Alomone , Jerusalem , Israel -RRB- was used in combination with either mouse anti-calbindin-D-28K -LRB- 1:200 , Sigma , Mi , Italy -RRB- , mouse anti-tyrosine hydroxylase -LRB- TH , 1:500 , Sigma -RRB- , mouse anti-parvalbumin -LRB- 1:200 , Chemicon International , Temecula , CA , USA -RRB- , mouse anti-glial fibrillary acidic protein -LRB- GFAP -RRB- -LRB- 1:400 , Sigma -RRB- , mouse anti-myelin basic protein -LRB- MBP , 1:200 , Chemicon International -RRB- , mouse anti-neurofilament H non-phosphorylated -LRB- SMI 32 , 1:500 , Sternberger Monoclonals , Lutherville , MD , USA -RRB- , mouse anti-neurofilament H and M non-phosphorylated -LRB- SMI 33 , 1:500 , Sternberger Monoclonals -RRB- , mouse anti-neurofilament 160 -LRB- NF160 , 1:500 , Sigma -RRB- or goat anti-neurofilament-L protein -LRB- NF-L , 1:100 , Santa Cruz , Mi , Italy -RRB- . The secondary antibodies used for double labeling were Cy3-conjugated donkey anti-rabbit IgG -LRB- 1:100 , red immunofluorescence , Jackson Immunoresearch , West Baltimore Pike , PA , USA -RRB- , Cy2-conjugated donkey anti-mouse IgG -LRB- 1:100 , green immunofluorescence , Jackson Immunoresearch -RRB- or Cy2-conjugated donkey anti-goat IgG -LRB- 1:100 , green immunofluorescence , Jackson Immunoresearch -RRB- . The sections were washed in PBS three times for 5 min each , and then incubated for 3 h in a solution containing a mixture of the secondary antibodies in 1 % normal donkey serum in PBS . After rinsing , the sections were mounted on slide glasses , allowed to air dry and coverslipped with gel/mount anti-fading medium -LRB- Biomeda , Foster City , CA , USA -RRB- . Confocal microscopy Double - or triple-label immunofluorescence was analyzed by means of a confocal laser scanning microscope -LRB- CLSM -RRB- -LRB- LSM 510 , Zeiss , Arese , Mi , Italy -RRB- equipped with argon laser emitting at 488 nm , helium/neon laser emitting at 543 nm , and helium/neon laser emitting at 633 nm . Specificity of the antibodies was positively proved by performing confocal analysis in the absence of the primary antibodies , but in the presence of either anti-rabbit or anti-mouse secondary antibodies . Specificity was further confirmed for the P2r antiserum by performing immunoreactions in the simultaneous presence of the P2r neutralizing immunogenic peptides . Isolation of cerebral areas and protein extraction Wistar rats were anesthetized by i.p. injections of sodium pentobarbital -LRB- 60 mg/kg -RRB- and , after decapitation , brains were removed . Each brain was transversally cut on a vibratome -LRB- 300 μm -RRB- . The specific cerebral areas were isolated with the aid of a dissection microscope and homogenized in RIPA buffer -LRB- 1 % Nonidet P-40 , 0.5 % sodium deoxycholate , 0.1 % SDS in PBS containing protease inhibitors -RRB- . After short sonication , the homogenates were incubated on ice for 1 h and centrifuged at 14,000 r.p.m. for 10 min at 4 °C . Protein quantification was performed in the supernatants by Bradford colorimetric assay -LRB- Biorad , Milan , Italy -RRB- . Western blot analysis Equal amounts of cell lysate -LRB- 20 -- 30 μg of protein from each cerebral area -RRB- were separated by electrophoresis on 10 -- 12 % SDS-PAGE and transferred to nitrocellulose membranes Hybond-C extra -LRB- Amersham Biosciences , Cologno Monzese , Italy -RRB- . The filters were pre-wetted in 5 % non-fat milk in TBS-T -LRB- 10 mM Tris pH 8 , 150 mM NaCl , 0.1 % Tween 20 -RRB- and hybridized overnight with P2X1 ,2,4 antisera -LRB- Alomone , 1:500 -RRB- , P2X5 and P2Y4/14 -LRB- 1:200 -RRB- , P2Y6 -LRB- 1:300 -RRB- or P2Y2 -LRB- 1:400 -RRB- . The antisera were immunodetected with an anti-rabbit HRP-conjugated antibody -LRB- 1:5,000 -RRB- and developed by ECL chemiluminescence -LRB- Amersham Biosciences -RRB- , using Kodak Image Station -LRB- KDS IS440CF -RRB- . Anti-P2r specificity The polyclonal P2r antisera used in this study were raised against P2r highly purified peptides -LRB- identity confirmed by mass spectrography and amino acid analysis , as indicated in the certificate of analysis provided by the manufacturer -RRB- , corresponding to specific epitopes not present in any other known protein . The specificity of the P2r signals was moreover assessed by incubating Western blots either in the absence of the primary antiserum , or in the presence of the primary antiserum together with the neutralizing P2r immunogenic peptides -LRB- μg protein ratio 1:1 between peptide and antiserum -RRB- . 6-OHDA lesion and Nissl staining Deeply anesthetized rats -LRB- 45 days old , about 150 g body weight -RRB- were injected with 8 μg/4 μl 6-OHDA in saline 0.1 % ascorbic acid in the medial forebrain bundle -LRB- stereotaxic coordinates ap = − 4.4 ; l = +1.2 ; vd = − 7.8 , see also Paxinos et al. -LSB- 28 -RSB- -RRB- at a rate of 0.38 μl/min . Fifteen days later , the lesioned rats were tested with 0.05 mg/kg s.c. of the D1/D2 dopamine agonist apomorphine , in order to verify the efficacy of the 6-OHDA lesion , and contralateral turns to the lesion were counted for 40 min . Only those rats that made at least 200 contralateral turns were used for the study . It has been previously demonstrated that rats meeting this screening criterion have greater than 95 % depletion of striatal dopamine -LSB- 29 -RSB- . At 1.5 months after the 6-OHDA lesion , rats were used for immunohistological experiments -LRB- n = 3 -RRB- . In order to evaluate cell damage , 40-μm rat brain sections were mounted onto gelatinized slides . They were dehydrated through alcohols , and then rehydrated and stained in 2 % cresyl violet for 45 min . Following deionized water rinses , the slides were dehydrated in a standard alcohol series , cleared in xylene , and coverslipped . Results P2X and P2Y receptor proteins in rat striatum We describe in this work the cellular and subcellular in vivo distribution of P2X and P2Y receptors in transverse sections of adult rat striatum , showing by double immunofluorescence confocal analysis that the various P2 subtype proteins are distinguished by different degrees of expression and are not uniformly distributed throughout the entire tissue -LRB- Fig. 1 -RRB- . Fig. 1P2X and P2Y receptor proteins in rat striatum . Transverse sections through the striatum of adult rats were processed for double immunofluorescence studies . Rabbit polyclonal antisera against P2X1 ,2,4 and P2Y4 receptors -LRB- red Cy3 immunofluorescence -RRB- were used in combination with antibodies against neuronal or glial markers -LRB- green Cy2 immunofluorescence -RRB- . Panel A P2X1 : confocal images illustrate clear colocalization of P2X1 receptor with neurofilament-light protein -LRB- NF-L -RRB- . The merged field of inset a shows absence of colocalization between the neuronal GABAergic marker calbindin -LRB- green -RRB- -LRB- a calcium-binding protein expressed mainly in medium spiny neurons of the striatum -RRB- and P2X1 receptor -LRB- red -RRB- . The merged field of inset b shows the merged field of P2X1 -LRB- red -RRB- and MBP -LRB- green -RRB- overlapping immunoreactivities at higher magnification . Panel B P2X2 : double immunofluorescence demonstrates that P2X2 receptor immunoreactivity -LRB- red -RRB- colocalizes with calbindin protein -LRB- green -RRB- . The insets e and f show colocalization with the neuronal GABAergic marker parvalbumin -LRB- green -RRB- -LRB- a calcium-binding protein that is expressed in interneurons of the striatum -RRB- . Panel C P2X4 : red immunofluorescence for P2X4 protein merges with the green signals of the three types of neurofilament proteins : NF-L -LRB- inset g , merged field -RRB- , NF160 -LRB- inset i , merged field -RRB- and SMI 32 and , moreover , with parvalbumin -LRB- inset h , merged field -RRB- and calbindin -LRB- inset i , merged field -RRB- . Panel D P2Y4 : red P2Y4 immunoreactivity is present on calbindin-positive neurons -LRB- green -RRB- , on parvalbumin-positive neurons -LRB- green -RRB- -LRB- insets n -- q -RRB- , and on GFAP-positive astrocytes -LRB- inset o , merged field -RRB- . Western blot analysis also confirms the presence of receptor proteins P2X1 ,2,4 -LRB- insets c in panel A , d in panel B , m in panel C , respectively -RRB- and P2Y4 -LRB- inset r in panel D -RRB- in striatum . Specificity of the P2 receptor signals was assessed by incubations of the primary antisera with the corresponding neutralizing immunogenic peptides -LRB- μg protein ratio 1:1 between peptide and antiserum -RRB- . Scale bars are 10 μm in A ; 100 μm in inset a ; 2 μm in inset b ; 50 μm in B and in insets e and f ; 20 μm in C ; 10 μm in insets h , i , l ; 5 μm in inset g ; 50 μm in D ; 5 μm in inset n ; and 20 μm in inset o. Similar results were obtained in at least four independent experiments In particular , a strong P2X1 receptor immunoreactivity -LRB- red -RRB- confers a patchy appearance to the striatum , being localized mainly in white matter , while sparing the projecting calbindin-positive GABAergic neurons that are highly enriched in gray matter -LRB- Fig. 1A , inset a , green -RRB- . Moreover , P2X1 protein immunofluorescence is present on NF-L positive , transversally oriented neuronal fibers , although the merged field provides only partial colocalization between the two signals -LRB- Fig. 1A -RRB- . In addition , the high magnification analysis -LRB- Fig. 1A , inset b -RRB- of P2X1 -LRB- red -RRB- and MBP -LRB- green -RRB- immunoreactive signals shows that P2X1 receptor is surrounded by MBP , proving the presence of P2X1 protein on myelinated fibers . Due to the close vicinity of the two signals , overlapping yellow immunofluorescence is also observed . Finally , P2X1 receptor in striatum is recognized by Western blot analysis as a single protein band of 60 -- 65 kDa , additionally abolished in the presence of the P2X1 receptor -- neutralizing immunogenic peptide -LRB- Fig. 1A , inset c -RRB- . Conversely , an abundant P2X2 receptor immunoreactivity -LRB- red -RRB- is found in gray matter of striatum -LRB- Fig. 1B -RRB- , while sparing the bundles of white matter . Specific receptor immunolabeling is present not only on the highly expressed calbindin-positive projecting GABAergic neurons , but also on the fewer parvalbumin-positive GABAergic interneurons -LRB- Fig. 1B , insets e , f -RRB- . By Western blot analysis , we show that P2X2 receptor is present in striatum under two isoforms of about 45 and 32 kDa , furthermore , it is abolished in the presence of the P2X2 receptor -- neutralizing immunogenic peptide -LRB- Fig. 1B , inset d -RRB- . P2X3 receptor immunostaining in striatum is of medium intensity -LRB- Table 1 -RRB- , and mainly localizes on GABAergic neurons of gray matter -LRB- data not shown -RRB- . Table 1Map of P2 receptor proteins in striatum and substantia nigra StriatumSubstantia nigraP2X1 + + + + + + P2X2 + + + + + + P2X3 + + + + P2X4 + + + + + + P2X5 + + + + P2X6 + + + P2X7 + + + + P2X1 + + + P2X2 + + + + + + P2X4 + + + + + + P2X6 + + + + P2X11 -- -- P2X12 + + + + + + P2X13 -- -- P2X14 -- + + + Relative abundance of all P2X and P2Y receptor proteins was analyzed by confocal immunofluorescence microscopy , as described in `` Materials and methods '' . The intensity of the specific immunostaining was scored as follow : -- = not detected ; + = just sufficient to evaluate presence and outline of positive cells ; + + = adequate to assess morphological features of cell bodies and/or cellular processes ; + + + = very bright P2X4 receptor signal is instead very copious in white matter , although present on a few fibers of gray matter as well -LRB- Fig. 1C , red -RRB- . It partially colocalizes with all types of heavy - , light - and medium-chain neurofilament proteins -LRB- merged fields -RRB- : SMI 32 -LRB- green -RRB- , NF-L -LRB- inset g -RRB- , and NF160 -LRB- inset i -RRB- . Moreover , we find P2X4 protein also on GABAergic interneurons -LRB- inset h -RRB- and GABAergic spiny neurons -LRB- inset l -RRB- . By Western blot analysis , we demonstrate that P2X4 receptor is present in striatum as a single band of about 60 kDa , moreover , it is abolished in the presence of the P2X4 receptor -- neutralizing immunogenic peptide -LRB- Fig. 1C , inset m -RRB- . P2X5 ,6,7 and P2Y1 receptor immunoreactivities in striatum are very weak -LRB- Table 1 -RRB- in gray matter , although totally absent from white matter under our experimental conditions -LRB- data not shown -RRB- . The P2Y2 receptor is highly expressed in striatum on axons of white matter and astrocytes of gray matter -LRB- Table 1 -RRB- . Moreover , it is detected as a double protein band in the 55 -- 65 kDa range -LRB- data not shown -RRB- . A strong P2Y4 receptor immunoreactivity is present only in gray matter of striatum , localized on both types of GABAergic neurons : calbindin-positive -LRB- Fig. 1D -RRB- and parvalbumin-positive -LRB- insets n -- q -RRB- . Nevertheless , the receptors are also widespread throughout the striatum on astrocytes , as shown by colocalization with the GFAP marker -LRB- inset o -RRB- . By Western blot analysis , we prove that P2Y4 receptor is present in striatum as a double band of about 42 and 85 kDa -LRB- inset r -RRB- , likely corresponding to the monomeric and dimeric aggregation states of the receptor -LSB- 30 , 31 -RSB- . While P2Y6 receptor is barely detectable -LRB- Table 1 -RRB- on GABAergic neurons in striatum -LRB- data not shown -RRB- , P2Y11 ,13,14 receptor proteins were not identified by any means under our experimental conditions -LRB- Table 1 -RRB- . Finally , P2Y12 receptor in striatum -LRB- Table 1 -RRB- is abundantly expressed only on oligodendrocytes and myelin sheets , as previously shown -LSB- 26 -RSB- . P2X and P2Y receptor proteins in substantia nigra We conducted a parallel analysis on the cellular and subcellular in vivo distribution of P2X and P2Y receptors in transverse sections of adult rat SN . We showed by double immunofluorescence confocal analysis that the different P2 receptor proteins possess more comparable levels of expression with respect to the striatum , and are also more uniformly , although differently , distributed throughout the entire SNC and SNR -LRB- Fig. 2 -RRB- . In particular , strong signals for ionotropic P2X2 ,5 -LRB- red , Fig. 2A , B -RRB- , P2X1 ,4 -LRB- Table 1 -RRB- , metabotropic P2Y6 ,14 -LRB- red , Fig. 2C , D -RRB- and P2Y4 -LRB- Table 1 -RRB- , or moderate signals for P2X3 ,6 and P2Y1 receptors -LRB- Table 1 -RRB- are present on dopaminergic neurons -LRB- TH-positive -RRB- of SNC . Moreover , P2Y2 and P2Y12 receptors are abundantly expressed in SN -LRB- Table 1 -RRB- , but P2Y2 is expressed on axons and astrocytes , and P2Y12 only on oligodendrocytes and myelin sheets -LSB- 26 -RSB- . Conversely , in SNR , a weak P2X/Y receptor immunoreactivity is limited to sparse neuronal bodies , likely identified as GABAergic neurons by colocalization with parvalbumin -LRB- data not shown -RRB- . The presence at the tissue level in SN of ionotropic P2X2 ,5 -LRB- insets a in panel A , and b in panel B of Fig. 2 , respectively -RRB- and metabotropic P2Y6 ,14 -LRB- insets c in panel C , and d in panel D of Fig. 2 , respectively -RRB- proteins is confirmed by Western blot analysis performed in all cases in the presence of specific receptor -- neutralizing immunogenic peptides . Similarly to the striatum , immunoreactive signals for P2Y11 ,13 receptors were not identified by any means under our experimental conditions -LRB- Table 1 -RRB- . Fig. 2P2X and P2Y receptor proteins in rat substantia nigra . Double immunofluorescence visualized by confocal analysis was performed in transverse sections through the substantia nigra of adult rats . Strong signals for ionotropic P2X2 ,5 and metabotropic P2Y6 ,14 -LRB- red Cy3 immunofluorescence -RRB- are present on dopaminergic neurons -LRB- TH-positive , green Cy2 immunofluorescence -RRB- of substantia nigra pars compacta -LRB- SNC -RRB- , whereas in substantia nigra pars reticolata -LRB- SNR -RRB- P2X/Y immunoreactivity is limited to sparse neuronal bodies . Western blot analysis confirms the presence in substantia nigra of receptor proteins P2X2 ,5 -LRB- insets a in panel A , and b in panel B , respectively -RRB- and P2Y6 ,14 -LRB- insets c in panel C , and d in panel D , respectively -RRB- . Specificity of the P2 receptor signals was assessed by incubation of the primary antisera with the corresponding neutralizing immunogenic peptides -LRB- μg protein ratio 1:1 between peptide and antiserum -RRB- . Scale bars in all panels are 50 μm . Similar results were obtained in at least four independent experiments 6-Hydroxydopamine modulates the expression of selected P2 receptors in striatum and substantia nigra No contralateral rotation as a sign of motor deficit was reported in rats before being 6-OHDA-lesioned , but was instead detected after the lesion rotation -LRB- data not shown -RRB- , together with loss of dopaminergic TH-positive neurons only from the ipsilateral hemisphere of SNC -LRB- Fig. 3A and insets a , b -RRB- . Fig. 36-Hydroxydopamine modulates the expression of selected P2 receptor proteins in striatum and substantia nigra . Staining of rat substantia nigra after 6-hydroxydopamine treatment . Panel A Conventional microscopy images of Nissl staining shows several dopaminergic neurons -LRB- arrows -RRB- in the contralateral control hemisphere , which are lost -LRB- asterisks -RRB- in the ipsilateral lesioned hemisphere . Specific ipsilateral dopaminergic lesion of substantia nigra pars compacta -LRB- SNC -RRB- was also visualized by confocal TH-immunostaining -LRB- green -RRB- -LRB- insets a , b -RRB- . Panel B Confocal merged yellow images show upregulation of P2X1 receptor protein -LRB- red -RRB- in parvalbumin-positive GABAergic neurons -LRB- green -RRB- in the lesioned side of substantia nigra pars reticolata -LRB- SNR -RRB- of 6-hydroxydopamine-treated rats . Panel C Confocal merged yellow images show a drastic increase in GFAP-positive astrocytes -LRB- green -RRB- in the lesioned side of 6-hydroxydopamine-treated rats and , correspondingly , an augment of P2Y4 signal -LRB- red -RRB- -LRB- inset c -RRB- . Scale bars are 100 μm in A , B and in insets a , b ; 20 μm in C ; and 10 μm in inset c. Similar results were obtained in at least three independent experiments Concomitantly , we prove that dopamine denervation in the 6-OHDA-lesioned rat generates a significant and selective rearrangement of P2 receptor proteins . Whereas the expression pattern and immunofluorescence intensities of P2X1 ,4 , P2Y2 -LRB- colocalizing with all neurofilaments and present in white matter on fibers projecting from the cortex -RRB- , and P2Y12 -LRB- present on oligodendrocytes of white matter -RRB- remain constant in both ipsi - and contralateral hemispheres after 6-OHDA treatment -LRB- as well as in control animals -RRB- , all other P2X and P2Y receptors are decreased on parvalbumin - and calbindin-positive GABAergic neurons of deafferented ipsilateral striatum -LRB- but not contralateral and in control animals -RRB- , as measured by semiquantitative analysis -LRB- Table 2 -RRB- -LRB- n = 3 -RRB- . Table 2Map of P2 receptor modulation after dopamine denervation Ipsilateral Striatum Ipsilateral SNP2X1 = ↑ GABAP2X2 ↓ GABA ↓ THP2X3 ↓ GABA ↓ TH , ↑ GABAP2X4 ↓ GABA ↓ TH , ↑ GABAP2X5 = ↓ THP2X6 = ↓ TH , ↑ GABAP2X7 == P2X1 = ↓ THP2X2 == P2X4 ↓ GABA ↑ GFAPP2X6 = ↓ THP2X11 == P2X12 == P2X13 == P2X14 = ↓ THRelative increase -LRB- ↑ -RRB- or decrease -LRB- ↓ -RRB- in P2X and P2Y receptor proteins analyzed by confocal immunofluorascence microscopy in striatum and SN after treatment in rat in vivo with 6-hydroxydopamine -LRB- ipsilateral -RRB- , and in control -LRB- not lesioned -RRB- brain hemisphere -LRB- contralateral -RRB- . TH = presence in dopaminergic neurons , GABA = presence in GABAergic neurons , GFAP = presence in astrocytes Similarly , all P2X and P2Y receptors are lost in the lesioned -LRB- but not contralateral -RRB- substantia nigra pars compacta , consequent to the degeneration of the majority of TH-positive dopaminergic neurons -LRB- Table 2 -RRB- . Conversely , P2X1 -LRB- Fig. 3B -RRB- and P2X3 ,4,6 -LRB- Table 2 -RRB- receptors present on GABAergic neurons , and P2Y4 receptors on astrocytes augment their expression only in ipsilateral substantia nigra pars reticulata adjacent to the lesioned pars compacta . In this same area , a phenomenon of astrogliosis is also induced , as detected by more abundant expression of GFAP-positive astrocytes -LRB- Fig. 3C -RRB- . Discussion Because the roles of ATP in the CNS have received less attention until recently , often due to lack of appropriate research tools , our knowledge of the functional qualification of P2 receptors in the brain is limited , although rapidly improving . As a group of nuclei interconnected with cerebral cortex , thalamus and brainstem , and associated with a variety of functions , such as motor control , cognition , emotions and learning , the BG -LSB- 32 -RSB- is an area that deserves thorough analysis . Our work was aimed at mapping in vivo the presence of P2 receptor subtypes in the BG nuclei of striatum and SN by immunofluorescence-confocal and Western blotting techniques . The specificity of the highly sensitive molecular probes used for the detection of all known P2X and P2Y receptor proteins has been previously validated -LSB- 33 , 34 -RSB- . In addition , we undertook an analysis that excluded possible cross-reactivity for all antisera used . Our results not only establish that the majority of P2X -LRB- P2X1 -- 7 -RRB- and P2Y -LRB- P2Y1 ,2,4,6,11 -- 14 -RRB- receptors so far cloned from mammalian tissues are found in striatum and SN , but also prove their distinctive localization on neurons and/or glial cells . In detail we show that , with the exception of only P2Y11 and P2Y13 receptors -LRB- whose immunoreactivity was not identified by any means under our experimental conditions -RRB- , all other subtypes are specifically localized in striatum and SN -LRB- both pars compacta and reticulata -RRB- , although with different levels of expression , rated as low -LRB- P2X5 ,6 and P2Y1 ,6,14 in striatum -RRB- , medium -LRB- P2X3 in striatum and SN ; P2X6 ,7 and P2Y1 in SN -RRB- and high . Moreover , while we show a prevalence of P2 receptors on neurons -LRB- P2X1 ,4 and P2Y2 colocalizing with neurofilament light , medium and heavy chains -RRB- with features that are either dopaminergic -LRB- P2X2 -- 5 and P2Y1 ,4,6,14 colocalizing with TH , in SN -RRB- or GABAergic -LRB- P2X2 -- 4 and P2Y4 colocalizing with parvalbumin and calbindin , in striatum -RRB- , we also describe their expression on astrocytes -LRB- P2Y2 ,4 in striatum and SN , colocalizing with GFAP -RRB- , microglia -LRB- P2X7 , colocalizing with OX42 -RRB- -LSB- 27 -RSB- , and oligodendrocytes -LRB- P2Y12 , colocalizing with MBP and RIP -RRB- -LSB- 26 -RSB- . By confirming previous autoradiographic studies -LSB- 35 , 36 -RSB- , our results therefore prove the widespread but diversified P2-receptor protein distribution in striatum and SN , and extend to these nuclei the great level of biological complexity and molecular sophistication pertaining to P2 receptors -LSB- 3 -RSB- . Although the configuration of receptor subunits required for assembly into functional cation channels gated by extracellular ATP in different regions of the CNS comprising the BG is not known yet , colocalization of so many different P2X subtypes in striatum and SN is definitely compatible with heteromultimeric assembly of ionotropic subunits . Since a growing body of biochemical and biophysical evidence now indicates that the propensity to form homo - and especially hetero-multimers is frequent also for G protein-coupled receptors -LSB- 37 -RSB- comprising the P2Y subtypes -LSB- 30 , 31 -RSB- , the concurrent expression in striatum and SN of as many metabotropic receptors could explain once more a complex hetero-oligomeric architecture . Nevertheless , the biological phenomenon of redundancy could also justify the simultaneous presence of multiple P2 receptor subtypes in these nuclei , with the final outcome of increasing the structural and pharmacological heterogeneity of these brain regions . Finally , the composite architecture of P2 receptors that we depicted in striatum and SN might likely also signify a multipart mechanism of receptor cooperative behavior -LRB- Volonté et al. , personal communication -RRB- that sustains the concomitant level of complexity of this brain area in several tasks , such as planning and modulation of movement pathways , cognitive processes involving executive functions , reward and addiction . These possibilities are , of course , not mutually exclusive . Striatal neurons , including the most abundant medium spiny neurons , receive convergent synaptic modulation from nigral dopaminergic neurons and from cortical glutamatergic projections -LSB- 38 -RSB- . The present study showing that lesions of nigral dopaminergic neurons do not significantly affect purinergic receptors present on axons of striatum white matter , but do generate a significant overall decrease in P2X and P2Y receptor proteins from striatal spiny neurons and GABAergic interneurons , thus confirms and extends the involvement of P2 receptors and extracellular ATP to the cortex-basal ganglia circuit -LSB- 21 -RSB- . Since dopaminergic denervation affects not only the nigrostriatal dopaminergic pathway but , as a consequence , the corticostriatal glutamatergic pathway with an increase in glutamatergic transmission -LSB- 39 -- 41 -RSB- and extracellular glutamate levels in the striatum -LSB- 42 -RSB- , the reduced P2 receptor protein expression that we demonstrate in striatum gray matter could thus not only be a direct effect of the nigrostriatal inhibition , but also a cause of de-inhibitory mechanisms occurring in the corticostriatal circuit . In this regard , it is common knowledge that extracellular ATP participates in excitatory neurotransmission in the CNS -LSB- 43 -RSB- , that release of extracellular ATP occurs in CNS under both normal and pathological conditions -LSB- 44 -RSB- and , not least , that glutamate release is induced by extracellular ATP in CNS glutamatergic neurons -LSB- 45 -RSB- . Neurons of the pars compacta responsible for dopamine production in the brain , which we have shown here to completely lose their array of P2 receptors as a consequence of neurodegeneration induced by 6-OHDA treatment , receive inhibiting signals also from neurons of the pars reticulata that produce GABA -LSB- 46 -RSB- . Loss of dopamine neurons in the SNC , one of the main pathological features of Parkinson 's disease leading to a marked reduction in dopamine function in the brain , thus also impedes the inhibitory pathway of SNR , with a consequent overactivation of GABAergic neurons . Our findings that specific expression of both ionotropic P2X1 ,3,4,6 receptors on GABAergic neurons and metabotropic P2Y4 receptors on astrocytes is remarkably increased in SNR after dopamine denervation thus probably reflects a parallel compensatory overreaction of GABAergic neurons to dopamine shortage . One possible explanation is that purinergic mechanisms might thus play a crucial role in the fine-tuned regulation not only of dopaminergic and glutamatergic cross-talk in striatum , as it occurs in nucleus accumbens -LSB- 47 -RSB- , but also of GABAergic and dopaminergic interplay in SN , as it occurs in the mesolimbic neuronal circuit -LSB- 48 -RSB- . This is consistent with the overall versatile functions accomplished by P2 receptors in the CNS under both normal and pathological conditions -LSB- 43 , 44 , 49 -RSB- and , in particular , with the intermediary role in oligodendrocyte-to-neuron -LSB- 26 -RSB- , Bergmann glia-to-neuron , and neuron-to-neuron communication -LSB- 50 -RSB- proposed for P2 receptors in various brain regions . In summary , the importance of our work is twofold . We first provide the complete topographical analysis of all known P2X and P2Y receptor subtypes expressed in vivo at their protein levels in rat striatum and SN , which , when considered alongside functional studies , supports a key role for extracellular ATP as a cotransmitter/neuromodulator in these brain areas . Then , we prove that dopamine denervation in the 6-OHDA animal model of Parkinson 's disease generates a significant rearrangement of P2 receptor proteins in these nuclei , therefore disclosing the participation of P2 receptors in the lesioned nigro-striatal circuit . While requiring further investigation , our findings indicate a potential but noteworthy pharmacological and therapeutic novel outcome for Parkinson 's disease .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

purinergic receptors
rat brain
tyrosine hydroxylase

parkinson’s disease
γ-aminobutyric acid
6-hydroxydopamine