Competitive mode and site of interaction of ticagrelor at the human platelet P2Y12 -receptor.

BACKGROUND: The G-protein-coupled P2Y12 -receptor plays a crucial role in platelet aggregation. Recently, ticagrelor was licensed as the first perorally active and reversible P2Y12 -receptor antagonist. OBJECTIVE: The present study investigated the site and the antagonistic mode of action of ticagrelor at wild-type or mutant human P2Y12 -receptors. METHODS: Recombinant wild-type or mutant human P2Y12 -receptors were stably expressed in Chinese hamster ovary Flp-In cells. Receptor function was assessed by quantification of ADP- and 2-methylthio-ADP-mediated inhibition of forskolin-induced cellular cAMP production either using a [(3) H]cAMP-radioaffinity assay or a cAMP response element-driven luciferase reporter gene assay. RESULTS: The natural agonist ADP inhibited forskolin-induced cAMP formation at the wild-type P2Y12 -receptor with a lower potency (EC50 209 nm) than the synthetic agonist 2-methylthio-ADP (EC50 1.0 nm). Ticagrelor shifted the concentration-response curves of both agonists in a parallel and surmountable manner to the right. Increasing concentrations of ticagrelor caused increasing shifts. Schild-plot analysis revealed pA2 values of 8.85 for ticagrelor against ADP, and 8.69 against 2-methylthio-ADP, and slopes of the regression lines not different from unity. In cells expressing a recombinant C194A(5.43) -mutant P2Y12 -receptor construct, ticagrelor lost antagonistic potency when tested against ADP or 2-methylthio-ADP. CONCLUSIONS: The experiments reveal a surmountable and competitive mode of antagonism of ticagrelor at P2Y12 -receptors activated by either the natural agonist ADP or the synthetic agonist 2-methylthio-ADP. Cys194(5.43) is likely to be involved in the interaction of ticagrelor with ADP and 2-methylthio-ADP. The data give new insights into the site and mode of action of ticagrelor at the human P2Y12 -receptor.

Dual regulation of ß2-adrenoceptor messenger RNA expression in human lung fibroblasts by ß2-cAMP signaling; delayed upregulated inhibitors oppose a rapid in onset, direct stimulation of gene expression.

Based on their bronchodilatory effect, ß2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and COPD. As treatment with ß2-adrenoceptor agonists has been associated with worsening of airway hyper-reactivity, possibly because of loss of ß-adrenoceptor function, molecular mechanism of the regulation of ß2-adrenoceptor expression were studied. MRC-5 human lung fibroblasts were cultured in absence or presence of test substances followed by ß2-adrenoceptor messenger RNA (mRNA) determination by qPCR. After inhibition of mRNA synthesis by actinomycin D, ß2-adrenoceptor mRNA decreased with a half-life of 23 min, whereas inhibition of protein synthesis by cycloheximide caused an about 5- and 6-fold increase within 1.5 and 4 h, respectively. ß2-Adrenoceptor mRNA was increased by about 100 % after 1 h exposure to formoterol or olodaterol but decreased by about 60 % after 4 h agonist exposure. Both effects of ß2-adrenoceptor agonists were mimicked by forskolin, a direct activator of adenylyl cyclase and cholera toxin, which stimulates adenylyl cyclase by permanent activation of Gs. ß2-Adrenoceptor agonist-induced upregulation of ß2-adrenoceptor mRNA was blocked by the ß2-adrenoceptor antagonist ICI 118551 and prevented by actinomycin D, but not by cycloheximide. Moreover, in presence of cycloheximide, ß2-adrenoceptor agonist-induced reduction in ß2-adrenoceptor mRNA was converted into stimulation, resulting in a more than 10-fold increase. In conclusion, expression of ß2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. The ß2-adrenoceptor gene is under strong inhibitory control of short-living suppressor proteins. ß2-Adrenoceptor activation induces via adenylyl cyclase - cyclic adenosine monophosphate (cAMP) signaling a rapid in onset direct stimulation of the ß2-adrenoceptor gene transcription, an effect opposed by a delayed upregulation of inhibitory factors.

Adenosine A(3) receptor-induced proliferation of primary human coronary smooth muscle cells involving the induction of early growth response genes.

In human coronary smooth muscle cells adenosine A(2B) receptors mediate the inhibition of platelet-derived growth factor (PDGF)-induced proliferation via induction of the transcription factor nuclear receptor subfamily 4, group A, member 1 (NR4A1). In the absence of PDGF, adenosine analogues increased proliferation. In the present study we characterised the adenosine receptor mediating the increase in proliferation of these cells and identified involved transcription factors. Cultured human coronary smooth muscle cells were treated with selective A(3) receptor ligands. Effects on proliferation were determined by counting cells and measuring changes in impedance. The induction of transcription factors was assessed by qPCR. The A(3) receptor agonist 2-chloro-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide) enhanced the number of human coronary smooth muscle cells with a half-maximal concentration of only 1 nM. 2-chloro-IB-MECA also increased the expression of the transcription factors early growth response protein (EGR)2 and EGR3, but not of EGR1, NR4A1, NR4A2 and NR4A3. The responses to 2-chloro-IB-MECA were blocked by two A(3) receptor antagonists, MRS1523 (3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate; 10-300 µM) and VUF 5574 (N-(2-methoxyphenyl)-N'-[2-(3-pyridinyl)-4-quinazolinyl]-urea; 1-100 nM, as well as by the phospholipase C-inhibitor U73343 (0.2 µM). Small interfering RNA directed against EGR2 and EGR3 abolished the increases in proliferation induced by 2-chloro-IB-MECA. In summary, this is the first report demonstrating a coupling of smooth muscle adenosine A(3) receptors to increases in proliferation of human coronary smooth cells by the activation of phospholipase C and an induction of the transcriptions factors EGR2 and EGR3. The results facilitate the understanding of the role of adenosine A(3) receptors in the cardiovascular system.

Interaction of the active metabolite of prasugrel, R-138727, with cysteine 97 and cysteine 175 of the human P2Y12 receptor.

BACKGROUND: The P2Y(12) receptor plays a crucial role in platelet aggregation and is the target of platelet aggregation inhibitors, including the thienopyridine compound prasugrel. OBJECTIVE: The present study analyzed the effects of R-138727 (2-[1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid), the active metabolite of prasugrel, on recombinant wild-type and mutant human P2Y(12) receptors in order to identify the molecular site of action of R-138727. METHODS: The function of wild-type and mutant P2Y(12) receptors stably expressed in Chinese hamster ovary cells was assessed by measuring the 2-methylthio-ADP-mediated inhibition of forskolin-stimulated cellular cAMP production. RESULTS: In cells expressing wild-type receptors, R-138727 potently inhibited receptor function with a half-maximal concentration below 1 microm. The mode of action was irreversible. The same effect of R-138727 was observed in cells expressing Cys17Ala/Cys270Ala constructs. In contrast, in cells expressing either a Cys97Ala construct or a Cys175Ala construct, R-138727 failed to inhibit the response to the agonist. When cells expressing wild-type receptors were pretreated with the P2 receptor antagonists ATP or suramin, no effect of R-138727 was observed. Similar experiments with N-acetylcysteine 10 microm showed no interference of N-acetylcysteine with R-138727. CONCLUSIONS: The experiments demonstrate a potent and irreversible action of R-138727 at the recombinant human P2Y(12) receptor. The data suggest that R-138727 interacts with cysteine 97 (upper portion of the predicted third transmembrane region) and cysteine 175 (second extracellular loop) of the receptor, which are likely to form a disulfide bridge in native receptors. Moreover, the data also suggest that this site of action of R-138727 is close to the ligand-binding site of the receptor.

Molecular basis for the antiproliferative effect of agmatine in tumor cells of colonic, hepatic, and neuronal origin.

The aim of the present study was to challenge potential mechanisms of action underlying the inhibition of tumor cell proliferation by agmatine. Agmatine inhibited proliferation of the human hepatoma cells HepG2, the human adenocarcinoma cells HT29, the rat hepatoma cells McRH7777, and the rat pheochromocytoma cells PC-12. Inhibition of proliferation of HepG2 cells was associated with an abolition of expression of ornithine decarboxylase (ODC) protein and a doubling of mRNA content encoding ODC. In HepG2 cells, silencing of ODC-antizyme-1, but not of antizyme inhibitor, by RNA interference resulted in an increase of agmatine's antiproliferative effect. Thus, the distinct decrease in intracellular polyamine content by agmatine was due to a reduced translation of the synthesizing protein ODC but was not essentially mediated by induction of ODC-antizyme or blockade of antizyme inhibitor. In interaction experiments 1 mM L-arginine, 1 mM D-arginine, 1 mM citrulline, 100 microM N(omega)-nitro-L-arginine methyl ester, 1 and 10 microM sodium nitroprusside, and 1 microM N1-guanyl-1,7-diaminoheptane failed to alter agmatine's antiproliferative effect. Hence, the antiproliferative effect of agmatine in HT29 and HepG2 cells is due to an interaction with neither the NO synthases, the hypusination of eIF5A, nor an agmatine-induced reduction in availability of intracellular L-arginine. L-Arginine and citrulline, but not d-arginine, inhibited tumor cell proliferation by themselves. Their inhibitory effect was abolished after silencing of arginine decarboxylase (ADC) expression by RNA interference indicating the conversion to agmatine by ADC. Finally, in the four cell lines under study, agmatine-induced inhibition of cell proliferation was paralleled by an increase in intracellular caspase-3 activity, indicating a promotion of apoptosis.

M-type K+ currents in rat cultured thoracolumbar sympathetic neurones and their role in uracil nucleotide-evoked noradrenaline release.

Cultured sympathetic neurones are depolarized and release noradrenaline in response to extracellular ATP, UDP and UTP. We examined the possibility that, in neurones cultured from rat thoracolumbar sympathetic ganglia, inhibition of the M-type potassium current might underlie the effects of UDP and UTP. Reverse transcriptase-polymerase chain reaction indicated that the cultured cells contained mRNA for P2Y(2)-, P2Y(4)- and P2Y(6)-receptors as well as for the KCNQ2- and KCNQ3-subunits which have been suggested to assemble into M-channels. In cultures of neurones taken from newborn as well as from 10 day-old rats, oxotremorine, the M-channel blocker Ba(2+) and UDP all released previously stored [(3)H]-noradrenaline. The neurones possessed M-currents, the kinetic properties of which were similar in neurones from newborn and 9 - 12 day-old rats. UDP, UTP and ATP had no effect on M-currents in neurones prepared from newborn rats. Oxotremorine and Ba(2+) substantially inhibited the current. ATP also had no effect on the M-current in neurones prepared from 9 - 12 day-old rats. Oxotremorine and Ba(2+) again caused marked inhibition. In contrast to cultures from newborn animals, UDP and UTP attenuated the M-current in neurones from 9 - 12 day-old rats; however, the maximal inhibition was less than 30%. The results indicate that inhibition of the M-current is not involved in uracil nucleotide-induced transmitter release from rat cultured sympathetic neurones during early development. M-current inhibition may contribute to release at later stages, but only to a minor extent. The mechanism leading to noradrenaline release by UDP and UTP remains unknown.

Role of action potentials and calcium influx in ATP- and UDP-induced noradrenaline release from rat cultured sympathetic neurones.

Adenine and uracil nucleotides release noradrenaline from rat postganglionic sympathetic neurones by activation of P2X-receptors and distinct receptors for uracil nucleotides, respectively. The present study on cultured neurones of rat thoracolumbal paravertebral ganglia has analysed the involvement of action potentials and calcium influx in the nucleotide-induced transmitter release. ATP and UDP (100 microM each) caused a marked release of previously incorporated [3H]noradrenaline. The P2-receptor antagonists suramin (300 microM) and cibacron blue 3GA (3 microM) decreased the ATP-induced but not the UDP-induced release. The response to ATP was decreased by the sodium channel blocker tetrodotoxin (0.5 microM; by 47%), by the N-type calcium channel blocker omega-conotoxin GVIA (100 nM; by 35%), and by the alpha2-adrenoceptor agonist UK-14,304 (1 microM; by 45%); it was not changed by the potassium channel blocker tetraethylammonium (10 mM). The response to UDP was abolished by tetrodotoxin, greatly decreased by omega-conotoxin (by 78%), also abolished by UK-14,304, and increased by tetraethylammonium (by 410%). ATP (100 microM) caused a marked increase in intra-axonal free calcium as measured by fura-2 microfluorimetry. Withdrawal of extracellular calcium diminished the calcium response to ATP by 85%, and tetrodotoxin and omega-conotoxin diminished it by about 40%. As studied with the amphotericin B-perforated patch method, ATP (100 microM) induced a large depolarisation and action potential firing. UDP (100 microM) induced only a small depolarisation but it also elicited action potentials. UDP increased the excitability of the neurones. The results indicate that the ATP-induced release of noradrenaline depends on influx of calcium from the extracellular space. Calcium passes through two structures: voltage-gated channels and - probably - the P2X-receptor itself. Only that component of ATP-induced transmitter release which is triggered by opening of voltage-gated calcium channels is sensitive to modulation by alpha2-adrenoceptors. In contrast to ATP, the UDP-induced release of noradrenaline is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels. All of the UDP-induced release is therefore sensitive to alpha2-adrenoceptor modulation.

A study of the mechanism of the release of ATP from rat cortical astroglial cells evoked by activation of glutamate receptors.

Glutamate and the selective agonists at ionotropic glutamate receptors N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate release ATP from superfused primary cultures of rat cortical astrocytes. The mechanism of this release was investigated. The release of ATP elicited by N-methyl-D-aspartate and kainate was abolished or greatly reduced in the absence of external calcium as well as in the presence of cadmium (1 mM) and nicardipine (10 microM). The release of ATP elicited by AMPA, in contrast, was not changed by these interventions. The calcium ionophore ionomycin (5 microM) released ATP in the presence but not in the absence of external calcium. No release was obtained with alpha-latrotoxin. Of several compounds tested as potential blockers of ATP transporters or channels only glibenclamide (100 microM) and diphenylamine-2-carboxylate (500 microM), which block the cystic fibrosis transmembrane conductance regulator, caused any change: both reduced the effect of AMPA without changing the effects of N-methyl-D-aspartate and (only glibenclamide tested) kainate. Lithium (1 mM) abolished the release of ATP evoked by glutamate and AMPA and significantly reduced the release evoked by N-methyl-D-aspartate and kainate. The three glutamate receptor agonists did not increase the release of lactate dehydrogenase. The results confirm the previous observation that activation of N-methyl-D-aspartate, AMPA and kainate receptors induces release of ATP from astrocytes in culture. Two different mechanisms seem to be involved. The N-methyl-D-aspartate- and kainate-induced release of ATP requires an influx of calcium, is not due to neuron-like exocytosis, is not mediated by cystic fibrosis transmembrane conductance regulator or a mechanism regulated by cystic fibrosis transmembrane conductance regulator, and is reduced (by an unknown mechanism) but not abolished by lithium. The AMPA-induced release does not require extracellular calcium, may be mediated by cystic fibrosis transmembrane conductance regulator or a mechanism regulated by cystic fibrosis transmembrane conductance regulator, and is abolished (by an unknown mechanism) by lithium. The ability of astrocytes to both release ATP and respond to ATP suggests that ATP may act as an autocrine or paracrine messenger between these glial cells.

Adenosine mediates nitric-oxide-independent renal vasodilation by activation of A2A receptors.

OBJECTIVE: Adenosine dilates rabbit renal arteries by an endothelium-dependent, nitric oxide (NO)- and prostaglandin-independent mechanism. The aim was to identify the responsible P1-purinoceptor subtype and to investigate the involvement of K+-channels. METHODS: Rabbit renal arteries were perfused with medium containing indomethacin (10 micromol/l). After preconstriction with noradrenaline (0.4 micromol/l), changes in vessel diameter by P1-purinoceptor agonists were measured with a photoelectric device. The P1-receptor subtype was characterised by selective antagonists. RESULTS: Adenosine caused concentration-dependent dilation (EC50 approximately 7 micromol/l). The mRNA for A1, A2A and A3 receptors were demonstrated by reverse transcription-polymerase chain reaction from total RNA of renal arteries. The agonists CPCA (A2) and CGS21680 (A2A) dilated renal arteries (EC50 approximately 0.1 micromol/l), and CPA (A1) was ineffective. As demonstrated by experiments using two arteries in sequence, CPCA induced release of an endothelium-derived relaxing factor. NO synthase inhibition by NG-nitro-L-arginine methyl ester (L-NAME) had no effect on CPCA-induced dilation. The concentration-response curves of adenosine, CPCA and CGS21680 were shifted to the right by the A2A antagonist ZM241385 (1 micromol/l), but not by the A1 and A3 antagonists DPCPX (1 micromol/l) and MRS1220 (1 micromol/l). Iberiotoxin (0.1 micromol/l), a blocker of Ca2+-activated K+-channels, slightly shifted the dose- response curve of CPCA. Arteries preconstricted by KCl showed dilation to CPCA, but not to acetylcholine chloride (ACh). CONCLUSION: Adenosine induces dilation of rabbit renal arteries through activation of A2A receptors. This effect depends on the release of an endothelium-derived relaxing factor, which is not NO. Dilation by ACh in the presence of L-NAME is likely to be mediated by K+ as an endothelium-derived relaxing factor. However, in the A2A-receptor-induced dilation of rabbit renal arteries, K+ does not play this role, suggesting the involvement of a further soluble factor in the receptor-induced dilatory function of the endothelium.

Purinoceptors mediate renal vasodilation by nitric oxide dependent and independent mechanisms.

BACKGROUND: Adenosine triphosphate (ATP) and its metabolites including adenosine modulate renal vascular tone under physiological and pathophysiological conditions. Their effects are brought about by activation of membrane bound P1- and P2-purinoceptors located on smooth muscle and endothelial cells. In this study we analyzed the purinoceptor mediated dilation of rabbit and human renal arteries, and evaluated the possible involvement of endothelium-derived relaxing factors. METHODS: Segments of rabbit and human renal arteries were incubated and perfused with medium containing indomethacin. After preconstriction, drug induced changes in the vessel diameters were measured by a photoelectric device. RESULTS: ATP (EC50 = 1 mumol/liter), added intraluminally, caused maximal vasodilation of 80 to 100% of the preconstriction response in both species. This effect was inhibited by the P1-purinoceptor antagonist 8-p-(sulphophenyl)theophylline (100 mumol/liter), suggesting that it was in part due to breakdown of ATP to adenosine. The nature of purinoceptor mediated renal vasodilation was studied further in rabbit renal arteries. Adenosine (EC50 = 1 mumol/liter) as well as the P2Y-receptor agonists ADP beta S (EC50 = 0.4 mumol/liter) and 2-MeSATP (EC50 = 0.2 mumol/liter) dilated the arteries by 80 to 100%. The effects of 2-MeSATP, which were to a much lesser extent that of ADP beta S but not that of adenosine, were attenuated by the P2Y-antagonist reactive blue 2 (3 mumol/liter). Removal of the endothelium almost abolished the vasodilation induced by adenosine and ATP. In contrast, these dilator response were only slightly attenuated by the nitric oxide synthase blockers NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine (300 mumol/liter each), whereas acetylcholine and 2-MeSATP induced dilation was markedly reduced by NG-nitro-L-arginine methyl ester. CONCLUSIONS: P1-purinoceptors activated by adenosine dilate rabbit renal arteries by an endothelium-derived relaxing factor that appears to be distinct from nitric oxide. In contrast, P2Y-purinoceptor induced renal dilation is mediated by nitric oxide. ATP, the physiological activator of P2Y-purinoceptors, is rapidly broken down to adenosine in rabbit and human renal arteries. Therefore, in rabbit and human renal arteries the vasodilatory effect of exogenous ATP mainly results from P1-purinoceptor activation probably through its breakdown product, adenosine.

P2-receptor-mediated inhibition of noradrenaline release in the rat pancreas.

The aim of the study was to find out whether, and if so through which receptors, nucleotides modulate the release of noradrenaline in the rat pancreas. Segments of the pancreas were preincubated with [3H]-noradrenaline, superfused with medium containing desipramine (1 microM) and yohimbine (1 microM), and stimulated electrically, in most experiments by 60 pulses/l Hz. The adenosine A1-receptor agonist N6-cyclopentyl-adenosine (CPA; EC50 32 nM), the non-subtype-selective adenosine receptor agonists adenosine (EC50 15 microM) and 5'-N-ethylcarboxamidoadenosine (NECA; EC50 135 nM), and the nucleotides ATP (EC50 13 microM), adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS; EC50 19 microM) and adenosine-5'-O-(2-thiodiphosphate) (ADPbetaS; EC50 16 microM) decreased the evoked overflow of tritium. The adenosine A2A-agonist 2-p-(2-carboxyethyl)-phenethylamino-5 '-N-ethylcarboxamido-adenosine (CGS 21680) caused no change. The concentration-response curve of CPA was shifted to the right by the A -antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 10 nM; pKd 9.1) but, like the concentration-response curve of adenosine, hardly affected by the P2-receptor antagonist cibacron blue 3GA (30 microM). Combined administration of a high concentration of DPCPX (1 microM) and 8-phenyltheophylline (10 microM) abolished the effects of CPA and NECA. The concentration-response curves of ATP and ADPbetaS were shifted to the right by both DPCPX (10 nM; pKd 8.7 and 8.9, respectively) and cibacron blue 3GA (30 microM; pKd 5.0 and 5.2, respectively). The antagonist effects of DPCPX (10 nM) and cibacron blue 3GA (30 microM) against ATP were additive in a manner compatible with the blockade of two separate receptors for ATP. In the presence of the high concentration of DPCPX (1 microM) and 8-phenyltheophylline (10 microM), ATP and ADPbetaS still decreased evoked tritium overflow, and this decrease was attenuated by additional administration of cibacron blue 3GA (30 microM). The P2-antagonists cibacron blue 3GA, reactive blue 2, reactive red 2, and to a limited extent also suramin and 8-(3,5-dinitro-phenylenecarbonylimino)- 1,3,5-naphthalenetrisulphonate (XAMR0721), increased the evoked overflow of tritium by up to 114%. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS) caused no change. The results indicate that the postganglionic sympathetic axons of the rat pancreas possess A1-adenosine and P2-receptors. Both receptors mediate an inhibition of noradrenaline release. The presynaptic P2-receptors are activated by an endogenous ligand, presumably ATP, during appropriate trains of action potentials. This is the first demonstration of presynaptic P2-receptors at postganglionic sympathetic neurons that are located in prevertebral ganglia.

P2-receptor-mediated inhibition of serotonin release in the rat brain cortex.

The possibility of a P2-receptor-mediated modulation of the release of serotonin in the rat brain cortex was investigated in occipito-parietal slices preincubated with [3H]serotonin and then superfused and stimulated electrically (10 pulses, 1 Hz). Adenosine receptor agonists decreased the stimulation-evoked overflow of tritium at best slightly; the selective A1 agonist N6-cyclopentyl-adenosine caused no change. Several nucleotides had more marked effects: ATP (3-1000 microM), adenosine-5'-O-(3-thiotriphosphate) (3-300 microM) and P1,P5-di(adenosine-5')-pentaphosphate (3-300 microM) decreased the evoked overflow by up to ca 35%. AMP, alpha,beta-methylene-ATP and UTP produced smaller decreases and 2-methylthio-ATP and UMP caused no change. The inhibition by ATP was attenuated both by the P1-receptor antagonist 8-(p-sulphophenyl)-theophylline (100 microM) and by the P2-receptor antagonist suramin (300 microM) but was not changed by indomethacin (10 microM) and NG-nitro-L-arginine (10 microM). We conclude that the release of serotonin in the rat brain cortex is inhibited through presynaptic P1-receptors (which are not A1) as well as P2-receptors. Inhibition of release via P2-receptors has been previously shown for noradrenaline (brain cortex and hippocampus) and dopamine (neostriatum) and, hence, may be widespread. Differences between transmitter systems exist, however, in the degree of their sensitivity to presynaptic P2-receptor-mediated modulation.

P2-receptor modulation of noradrenergic neurotransmission in rat kidney.

1. ATP has previously been shown to act as a sympathetic cotransmitter in the rat kidney. The present study analyses the question of whether postganglionic sympathetic nerve endings in the kidney possess P2-receptors which modulate noradrenaline release. Rat kidneys were perfused with Krebs-Henseleit solution containing the noradrenaline uptake blockers cocaine and corticosterone and the alpha2-adrenoceptor antagonist rauwolscine. The renal nerves were electrically stimulated, in most experiments by 30 pulses applied at 1 Hz. The outflow of endogenous noradrenaline (or, in some experiments, of ATP and lactate dehydrogenase) as well as the perfusion pressure were measured simultaneously. 2. The P2-receptor agonist adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS, 3-30 microM) reduced the renal nerve stimulation (RNS)-induced outflow of noradrenaline (estimated EC50 =8 microM). The P2-receptor antagonist cibacron blue 3GA (30 microM) shifted the concentration-inhibition curve for ATPgammaS to the right (apparent pKB value 4.7). 3. Cibacron blue 3GA (3-30 microM) and its isomer reactive blue 2 (3-30 microM) significantly increased RNS-induced outflow of noradrenaline in the presence of the P1-receptor antagonist 8-(p-sulphophenyl)theophylline (8-SPT, 100 microM) by about 70% and 90%, respectively. The P2-receptor antagonist suramin (30-300 microM) only tended to enhance RNS-induced outflow of noradrenaline. When the nerves were stimulated by short pulse trains consisting of 6 pulses applied at 100 Hz (conditions under which autoinhibition is inoperative), reactive blue 2 did not affect the RNS-induced outflow of noradrenaline. 4. RNS (120 pulses applied at 4 Hz) induced the outflow of ATP but not of the cytoplasmatic enzyme lactate dehydrogenase. 5. ATPgammaS (3-30 microM) concentration-dependently reduced pressor responses to RNS at 1 Hz. Cibacron blue 3GA, reactive blue 2 as well as suramin also reduced pressor responses to RNS (maximally by 50 to 70%). 6. This study in rat isolated kidney, in which the release of endogenous noradrenaline was measured, demonstrates that renal sympathetic nerves possess prejunctional P2-receptors that mediate inhibition of transmitter release. These prejunctional P2-receptors are activated by endogenous ligands, most likely ATP, released upon nerve activity. Both, P2-receptor agonists and P2-receptor antagonists reduced pressor responses to RNS either by inhibiting transmitter release or by blocking postjunctional vasoconstrictor P2-receptors.

P2-receptor-mediated inhibition of noradrenaline release in the rat hippocampus.

Experiments on hippocampal slices were carried out in order to find out whether the release of noradrenaline in the hippocampus can be modulated through P2-receptors. The slices were preincubated with [3H]-noradrenaline, superfused with medium containing desipramine (1 microM), and stimulated electrically, in most experiments by 4 pulses/100 Hz. The adenosine A1-receptor agonist N6-cyclopentyl-adenosine (CPA) and the nucleotides ATP, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) and adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) decreased the evoked overflow of tritium by up to 55%. The adenosine A2a-agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamido-adenosin e (CGS 21680; 0.003-0.3 microM) caused no change. The concentration-response curve of CPA was shifted to the right by the A1-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 3 nM) but not by the P2-receptor antagonists cibacron blue 3GA (30 microM) and reactive blue 2 (30 microM); the apparent pKB value of DPCPX against CPA was 9.0. In contrast, the concentration-response curve of ATP was shifted to the right by DPCPX (3 nM), apparent pKB 8.7, as well as by cibacron blue 3GA (30 microM), apparent pKB 5.2, and reactive blue 2 (30 microM), apparent pKB 5.6; the antagonist effects of DPCPX and cibacron blue 3GA were additive in a manner compatible with the blockade of two separate receptors for ATP. The same pattern was obtained with ATP gamma S: its concentration-response curve was shifted to the right by DPCPX as well as by cibacron blue 3GA and reactive blue 2. Suramin (300 microM) antagonized neither the effect of ATP nor that of ATP gamma S. The 5'-nucleotidase inhibitor alpha, beta-methylene-ADP (100 microM) did not change the effect of ATP. Only cibacron blue 3GA (30 microM) but not reactive blue 2 (30 microM), given alone, consistently caused a small increase of the evoked overflow of tritium. Hippocampal slices degraded exogenous ATP, and this degradation was reduced by cibacron blue 3GA (30 microM), reactive blue 2 (30 microM) and suramin (300 microM). The results indicate that the noradrenergic terminal axons of the rat hippocampus possess P2-receptors in addition to the known A1-adenosine receptors. The presynaptic P2-receptors mediate an inhibition of noradrenaline release, are activated by nucleotides but not nucleosides, and are blocked by cibacron blue 3GA and reactive blue 2. ATP and ATP gamma S act at both the A1- and the P2-receptors. An autoreceptor function of cerebral presynaptic P2-receptors remains doubtful.

Differences in the mode of stimulation of cultured rat sympathetic neurons between ATP and UDP.

Postganglionic sympathetic neurons possess at least two excitatory receptors for nucleotides: P2X-purinoceptors and separate receptors for uracil nucleotides. In cultured neurons from rat superior cervical ganglia (SCG), both receptors, when activated, induce release of noradrenaline. Here we describe marked differences between the responses of cultured neurons from rat thoracolumbal paravertebral ganglia to ATP and UDP. ATP elicited release of previously taken up [3H]noradrenaline, induced an inward current, and increased the intra-axonal free calcium level, over the same range of micromolar concentrations. UDP was more potent than ATP in releasing [3H]noradrenaline but induced an inward current only at a concentration of 1 mM and caused much smaller increases in intraaxonal free calcium. The mechanism of action of ATP presumably consists of P2X-purinoceptor activation followed by depolarization, calcium entry through voltage-sensitive channels and exocytosis. The mode of action of UDP is different. It probably activates a G-protein-coupled pyrimidinoceptor. The pyrimidinoceptor then possibly mediates mobilization of intracellular calcium close to the sites of transmitter release and in addition an increase in the calcium sensitivity of the exocytotic apparatus.