共查询到20条相似文献,搜索用时 46 毫秒
1.
Kazuhide Inoue Kazuyuki Nakajima Takako Morimoto Yoshiaki Kikuchi Schuichi Koizumi Peter Illes Shinichi Kohsaka 《British journal of pharmacology》1998,123(7):1304-1310
- ATP (10–100 μM), but not glutamate (100 μM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 μM) nor glutamate (100 μM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 μM), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 μM). The Ca2+ ionophore induced plasminogen release in a concentration-dependent manner (0.3 μ A23187M to 10 μM).
- ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 μM) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 μM) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells).
- The ATP-evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2-Methylthio ATP was active (7 out of 7 cells), but α,β-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 μM) was shown not to inhibit the ATP-evoked increase in [Ca2+]i (20 out of 20 cells). 2′- and 3′-O-(4-Benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long-lasting increase in [Ca2+]i even at 1 μM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5′-triphosphate-2′, 3′-dialdehyde (oxidized ATP, 100 μM), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 μM).
- These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.
2.
C Jane Dixon Wayne B Bowler Catherine A Walsh James A Gallagher 《British journal of pharmacology》1997,120(5):777-780
- Human osteoblasts responded to the application of extracellular nucleotides, acting at P2-receptors, with increases in cytosolic free calcium concentration ([Ca2+]i).
- In populations of human osteoblasts, adenosine 5′-diphosphate (ADP) evoked a rise in [Ca2+]i with less than 40% of the amplitude of that induced by adenosine 5′-triphosphate (ATP).
- ATP and uridine 5′-triphosphate (UTP) were applied to single human osteoblasts and induced [Ca2+]i rises of comparable amplitude in every cell tested.
- However, from the results of single cell studies with ADP (and 2-methylthioATP (2-meSATP)) two groups of cells were delineated: one group responded to ADP (or 2-meSATP) with a rise in [Ca2+]i indistinguishable from that evoked by ATP; whereas the second group failed completely to respond to ADP (or 2-meSATP).
- Therefore heterogeneity of receptor expression exists within this population of human osteoblasts. The limited distribution of the ADP-responsive receptor underlies the small response to ADP, compared with ATP, recorded in populations of human osteoblasts. This heterogeneity may reflect differences in the differentiation status of individual cells.
3.
Caroline E Gargett Jean E Cornish James S Wiley 《British journal of pharmacology》1997,122(5):911-917
- Although extracellular adenosine 5′-triphosphate (ATP) is the natural ligand for the P2Z receptor of human lymphocytes it is less potent than 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) in opening the associated ion channel, which conducts a range of permeants including Ba2+ and ethidium+. We have quantified the influx of ethidium+ into lymphocytes produced by BzATP, ATP, 2-methylthio-ATP (2MeSATP) and ATPγS, studied competition between ATP and BzATP and investigated the effects of KN-62, a new and potent inhibitor of the P2Z receptor.
- BzATP and ATP stimulated ethidium+ influx with EC50 values of 15.4±1.4 μM (n=5) and 85.6±8.8 μM (n=5), respectively. The maximal response to ATP was only 69.8±1.9% of that for BzATP. Hill analysis gave nH of 3.17±0.24 (n=3) and 2.09±0.45 (n=4) for BzATP and ATP, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z receptor-operated ion channel.
- A rank order of agonist potency of BzATP>ATP=2MeSATP>ATPγS was observed for agonist-stimulated ethidium+ influx, while maximal influxes followed a rank order of BzATP>ATP>2MeSATP>ATPγS.
- Preincubation with 30–50 μM oxidized ATP (ox-ATP), an irreversible P2Z inhibitor, reduced the maximal response but did not change the steepness of the Ba2+ influx-response curve produced by BzATP (nH 3.2 and 2.9 for 30 and 50 μM ox-ATP, respectively (n=2)).
- ATP (300–1000 μM) added simultaneously with 30 μM BzATP (EC90) inhibited both ethidium+ and Ba2+ fluxes to a maximum of 30–40% relative to the values observed with BzATP alone. Moreover, ATP (300 μM) shifted the concentration-response curve to the right for BzATP-stimulated Ba2+ influx, confirming competition between ATP and BzATP.
- KN-62, a new and powerful inhibitor of the lymphocyte P2Z receptor, showed less potency in antagonizing BzATP-mediated fluxes than ATP-induced fluxes when maximal concentrations of both agonists (BzATP, 50 μM; ATP, 500 μM) were used.
- These data suggest that the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a more efficacious agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes.
4.
Carlo Centemeri Chiara Bolego Maria P Abbracchio Flaminio Cattabeni Lina Puglisi Geoffrey Burnstock Simonetta Nicosia 《British journal of pharmacology》1997,121(8):1700-1706
- This study was aimed at characterizing ATP-induced rises in cytosolic free calcium ion, [Ca2+]i, in a population of rat striatal astrocytes loaded with the fluorescent Ca2+ probe Fura2, by means of fluorescence spectrometry.
- ATP triggered a fast and transient elevation of [Ca2+]i in a concentration-dependent manner. The responses of the purine analogues 2-methylthio-ATP (2-meSATP), adenosine-5′-O-(2-thiodiphosphate) (ADPβS), as well as uridine-5′-triphosphate (UTP) resembled that of ATP, while α,β-methylene-ATP (α,β-meATP) and β,γ-methylene-ATP (β,γ-meATP) were totally ineffective.
- Suramin (50 μM) had only a minor effect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (5 μM) significantly depressed the maximum response.
- Extracellular Ca2+ did not contribute to the observed [Ca2+]i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its influx by means of either Ni2+ (1 mM) or Mn2+ (1 mM) did not modify the nucleotide responses.
- Furthermore, after preincubation with 10 μM thapsigargin, the nucleotide-evoked [Ca2+]i increments were completely abolished. In contrast, 10 mM caffeine did not affect the responses, suggesting that thapsigargin-, but not caffeine/ryanodine-sensitive stores are involved.
- Both application of the G-protein blocker guanosine-5′-O-(2-thiodiphosphate) (GDPβS) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml−1) partially inhibited the nucleotide-mediated responses. Moreover, the phospholipase C (PLC) inhibitor U-73122, but not its inactive stereoisomer U-73343 (5 μM), significantly reduced the ATP-evoked [Ca2+]i rise.
- In conclusion, our results suggest that, in rat striatal astrocytes, ATP-elicited elevation of [Ca2+]i is due solely to release from intracellular stores and is mediated by a G-protein-linked P2Y receptor, partially sensitive to PTx and coupled to PLC.
5.
- The ionotropic purinoceptors in isolated Deiters'' cells of guinea-pig cochlea were characterized by use of the whole-cell variant of the patch-clamp technique.
- Extracellular application of adenosine 5′-triphosphate (ATP) induced a dose-dependent inward current when the cells were voltage-clamped at −80 mV. The ATP-induced current showed desensitization and had a reversal potential around −4 mV.
- Increasing intracellular free Ca2+ by decreasing the concentration of EGTA in the pipette solution reduced the amplitude of the ATP-gated current.
- The order of agonist potency was: 2-methylthioATP (2-meSATP)>ATP>benzoylbenzoyl-ATP (BzATP)>α,β-methyleneATP (α,β,meATP>adenosine 5′-diphosphate (ADP)>uridine 5′-triphosphate (UTP)>adenosine 5′-monophosphate (AMP)=adenosine (Ad).
- Pretreatment with forskolin (10 μM), 8-bromoadenosine-3′,5′-cyclophosphate (8-Br-cyclic AMP, 1 mM), 3-isobutyl-1-methylxanthine (IBMX, 1 mM) or phorbol-12-myristate-13-acetate (PMA, 1 μM) reversibly reduced the ATP-induced peak current.
- The results are consistent with molecular biological data which indicate that P2X2 purinoceptors are present in Deiters'' cells. In addition, the reduction of the ATP-gated current by activators of protein kinase A and protein kinase C indicates that these P2X2 purinoceptors can be functionally modulated by receptor phosphorylation.
6.
Alan R Conant Michael J Fisher Alexander G McLennan Alec W M Simpson 《British journal of pharmacology》1998,125(2):357-364
- To characterize the P2 receptors present on the human umbilical vein endothelial-derived cell line, ECV304, cytosolic Ca2+, ([Ca2+]c), responses were recorded in single cells and in cell suspensions to a series of nucleotides and nucleotide agonists.
- Concentration response curves were obtained in fura-2-loaded ECV304 cell suspensions, with EC50 values of 4.2 μM for ATP, 2.5 μM for UTP and 14 μM for adenosine-5′-O-(3-thio)triphosphate (ATPγS). EC50 values for 2-methylthioATP, ADP, adenosine-5′-O-(2-thio)diphosphate (ADPβS) and AMP were 0.5 μM, 3.5 μM, 15 μM and 4.7 μM respectively, but maximal [Ca2+]c responses were less than those produced by a maximal addition of ATP/UTP. ECV304 cells were unresponsive to UDP and β,γ,methyleneATP.
- Cross-desensitization studies on ECV304 cells suggested that ATP and UTP recognized the same receptor. However, ADP recognized a receptor distinct from the UTP-sensitive receptor and AMP recognized a third distinct receptor.
- ECV304 [Ca2+]c responses to 2-methylthioATP were inhibited in the presence of 30 μM pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), whereas [Ca2+]c responses to UTP were unaffected by this treatment.
- ECV304 cells responded to the diadenosine polyphosphate Ap3A with rises in [Ca2+]c. Apparent responses to Ap4A, Ap5A and Ap6A, were shown to be due to a minor nucleotide contaminant that could be removed by pre-treatment of the diadenosine samples with either alkaline phosphatase or apyrase.
- ECV304 cells display a pharmacology consistent with the presence of at least two P2 receptors; a P2Y2 receptor insensitive to the diadenosine polyphosphates and a P2Y1 receptor sensitive to Ap3A. In addition, ECV304 cells respond to AMP with increases in [Ca2+]c via an as yet uncharacterized receptor.
7.
Jennifer L Albert John P Boyle Jonathan A Roberts R A John Challiss Sharon E Gubby Michael R Boarder 《British journal of pharmacology》1997,122(5):935-941
- The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca2+ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK).
- Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca2+ had little effect on the peak Ca2+-response, but resulted in a more rapid decline to basal. There was no response to α,β-MethylATP (α,βMeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage.
- ATP (log EC50 −5.1±0.2) also caused an increase in the total [3H]-inositol (poly)phosphates ([3H]-InsPx) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and α,βMeATP giving no detectable response.
- Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P3.
- None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPγS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 μM forskolin.
- Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with activation at P2Y2 receptors. 2MeSATP gave a much smaller response with a lower potency than UTP.
- These results are consistent with brain endothelial regulation by P2Y2 receptors coupled to phospholipase C, Ca2+ and MAPK; and by P2Y1-like (2MeSATP-sensitive) receptors which are linked to Ca2+ mobilization by a mechanism apparently independent of agonist stimulated Ins (1,4,5)P3 levels. A further response to ATP, acting at an undefined receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The differential MAPK coupling of these receptors suggests that they exert fundamentally distinct influences over brain endothelial function.
8.
Katsuichi Matsuo Takeshi Katsuragi Sono Fujiki Chiemi Sato Tatsuo Furukawa 《British journal of pharmacology》1997,121(8):1744-1748
- The present study was addressed to clarify the subtypes of P2-purinoceptor involved in ATP release and contraction evoked by α,β-methylene ATP (α,β-mATP) and other P2-agonists in guinea-pig ileum.
- α,β-mATP 100 μM produced a transient and steep contraction followed by ATP release from tissue segments. These maximum responses appeared with different time-courses and their ED50 values were 5 and 25 μM, respectively. The maximum release of ATP by α,β-mATP was markedly reduced by 250 μM suramin, 30 μM pyridoxal-phosphate-6-azophenyl-2′,5′-disulphonic acid (PPADS) and 30 μM reactive blue 2 (RB-2), P2-receptor antagonists. However, the contractile response was inhibited by suramin, tetrodotoxin and atropine, but not by PPADS and RB-2.
- Although the contraction caused by α,β-mATP was strongly diminished by Ca2+-removal and nifedipine, and also by tetrodotoxin and atropine at 0.3 μM, the release of ATP was virtually unaffected by these procedures.
- UTP, β,γ-methylene ATP (β,γ-mATP) and ADP at 100 μM elicited a moderate release of ATP. The release caused by UTP was virtually unaffected by RB-2. However, these P2-agonists failed to elicit a contraction of the segment.
- The potency order of all the agonists tested for the release of ATP was α,β-mATP>UTP>β,γ-mATP>ADP.
- In superfusion experiments with cultured smooth muscle cells from the ileum, α,β-mATP (100 μM) enhanced the release of ATP 5 fold above the basal value. This evoked release was inhibited by RB-2.
- These findings suggest that ATP release and contraction induced by P2-agonists such as α,β-mATP in the guinea-pig ileum result mainly from stimulation of different P2-purinoceptors, P2Y-like purinoceptors on the smooth muscles and, probably, P2X-purinoceptors on cholinergic nerve terminals, respectively. However, the ATP release may also be mediated, in part, by P2U-receptors, because UTP caused RB-2-insensitive ATP release.
9.
- The effects of exogenous adenosine 5′-triphosphate (ATP) and α,β-methylene ATP (α,βmeATP) on C6BU-1 cells transfected with P2X2 and P2X3 subtypes, separately or together (P2X2+3), were investigated using fura-2 fluorescence recording and whole-cell patch clamp recording methods.
- Untransfected C6BU-1 cells showed no intracellular Ca2+ ([Ca2+]i) increase in response to depolarizing stimulation with high K+ or stimulation with ATP. There was no current induced by ATP under voltage clamp conditions in untransfected C6BU-1 cells. ATP caused Ca2+ influx only from extracellular sources in C6BU-1 cells transfected with the P2X subtypes, suggesting that the C6BU-1 cell line is suitable for the characterization of Ca2+ influx through the P2X subtypes.
- In C6BU-1 cells transfected with the P2X2 subtype, ATP (more than 10 μM) but not α,βmeATP (up to 100 μM) evoked a rise in [Ca2+]i.
- In the cells transfected with the P2X3 subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 μM of α,βmeATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca2+]i rise and the current response was seen only with the P2X3 subtype. In addition, the agonist-induced rise in [Ca2+]i was observed only after the first application because of desensitization of this subtype.
- In C6BU-1 cells co-transfected with P2X2 and P2X3, ATP at 1 μM evoked a [Ca2+]i rise. This responsiveness was higher than that of the other subtype combinations tested. The efficiency of expression was improved by co-transfection with P2X2 and P2X3, when compared to transfection with the P2X3 subtype alone. The desensitization of the P2X2+3 was apparently slower than that of the P2X3 subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca2+]i rise.
- These results suggest that the P2X2 and P2X3 subtypes assemble a heteromultimer and that this heterogeneous expression acquires more effective Ca2+ dynamics than that by homogenously expressed P2X2 or P2X3
10.
- Recently, 4-chloro-3-ethyl phenol (CEP) has been shown to cause the release of internally stored Ca2+, apparently through ryanodine-sensitive Ca2+ channels, in fractionated skeletal muscle terminal cisternae and in a variety of non-excitable cell types. Its action on smooth muscle is unknown. In this study, we characterized the actions of CEP on vascular contraction in endothelium-denuded dog mesenteric artery. We also determined its ability to release Ca2+, by use of Ca2+ imaging techniques, on dog isolated mesenteric artery smooth muscle cells and on bovine cultured pulmonary artery endothelial cells.
- After phenylephrine-(PE, 10 μM) sensitive Ca2+ stores were depleted by maximal PE stimulation in Ca2+-free medium, the action of CEP on refilling of the emptied PE stores was tested, by first pre-incubating the endothelium-denuded artery in CEP for 15 min before Ca2+ was restored for a 30 min refilling period. At the end of this period, Ca2+ and CEP were removed, and the arterial ring was tested again with PE to assess the degree of refilling of the internal Ca2+ store.
- In a concentration-dependent manner (30, 100 and 300 μM), CEP significantly reduced the size of the post-refilling PE contraction (49.4, 28.9 and 5.7% of control, respectively) in Ca2+-free media. This suggests that Ca2+ levels are reduced in the internal stores by CEP treatment. CEP alone did not cause any contraction either in Ca2+-containing or Ca2+-free Krebs solution.
- Restoring Ca2+ in the presence of PE caused a large contraction, which reflects PE-induced influx of extracellular Ca2+. The contraction of tissues pretreated with 300 μM CEP was significantly less compared with controls. However, tissues pretreated with 30 and 100 μM CEP were unaffected. Washout of CEP over 30 min produced complete recovery of responses to PE in Ca2+-free and Ca2+-containing medium suggesting a rapid reversal of CEP effects.
- Concentration-response curves were constructed for PE, 5-hydroxytryptamine (5-HT) and K+ in the absence of and after 30 min pre-incubation with 30, 100 and 300 μM CEP. In all cases, CEP caused a concentration-dependent depression of the maximum response to PE (84.8, 43.4 and 11.6% of control), 5-HT (65.4, 25.7 and 6.9% of control) and K+ (77.6, 41.1 and 10.8% of control).
- Some arterial rings were pre-incubated with ryanodine (30 μM) for 30 min before the construction of PE concentration-response curves. In Ca2+-free Krebs solution, ryanodine alone did not cause any contraction. However, 58% (11 out of 19) of the tissues tested with ryanodine developed contraction (6.9±1.2% of 100 mM K+ contraction, n=11) in the presence of external Ca2+. EC50 values for PE in ryanodine-treated tissues (1.7±0.25 μM, n=16) were not significantly different from controls (2.5±0.41 μM, n=22). Maximum contractions to PE (118.5±4.4% of 100 mM K+ contraction, n=16) were also unaffected by ryanodine when compared to controls (129±4.2%, n=23).
- When fura-2 loaded smooth muscle cells (n=13) and endothelial cells (n=27) were imaged for Ca2+ distribution, it was observed that 100 and 300 μM CEP in Ca2+-free medium caused Ca2+ release in both cell types. Smooth muscle cells showed a small decrease in cell length. Addition of EGTA (5 mM) reversed the effect of CEP on intracellular Ca2+ to control values.
- These data show, for the first time in vascular smooth muscle and endothelial cells, that CEP releases Ca2+ more rapidly than ryanodine. Unlike ryanodine, CEP caused no basal contraction but depressed contractions to PE, 5-HT and K+. The lack of basal contraction may result from altered responsiveness of the contractile system to intracellular Ca2+ elevation.
11.
Masahiro Naramatsu Toshikazu Yamashita Shinichiro Kokubun 《British journal of pharmacology》1997,122(3):558-562
- The signalling pathway which causes contractions to adenosine 5′-O-2-thiodiphosphate (ADPβS) and α,β-methylene adenosine 5′-diphosphate (α,β-Me ADP) was investigated in rat urinary bladder smooth muscle by measuring isotonic tension.
- The responses to 10 μM α,β-methylene adenosine 5′-triphosphate (α,β-Me ATP) in 0 and 3.6 mM Ca2+ were 5.9±1.3 (n=10) and 122.2±6.4 (n=8) % respectively of those obtained in 1.8 mM Ca2+, whereas those to 100 μM ADPβS were 34.6±3.3 (n=8) and 96.8±7.2 (n=8) %, in 0 and 3.6 mM Ca2+, respectively. In both experimental conditions, the responses to the two agonists expressed as % of the control responses were significantly different (P<0.01).
- Indomethacin at high concentrations (>1 μM) decreased the responses to α,β-Me ATP (10 μM), ADPβS (100 μM) and α,β-Me ADP (100 μM). However, no significant difference was obtained between the responses to all the agonists at 30 μM indomethacin.
- 2-Nitro-4-carboxphenyl n,n-diphenylcarbamate (NCDC) at concentrations between 1 μM and 100 μM concentration-dependently decreased the responses to ADPβS (100 μM) and α,β-Me ADP (100 μM) and almost completely inhibited them at 100 μM. Although the responses to α,β-Me ATP (10 μM) were also inhibited by the drug, at 50 and 100 μM NCDC the responses to α,β-Me ATP were significantly larger than those to ADPβS and α,β-Me ADP (P<0.01).
- NCDC 100 μM significantly inhibited the KCl-induced contraction to 65.9±4.9% (n=6) of the control (P<0.01).
- It is suggested that the contraction via ADPβS-sensitive receptors in the rat urinary bladder smooth muscle mainly depends on Ca2+ ions liberated from intracellular Ca2+ stores, though the contribution of Ca2+ ions from the extracellular space cannot be neglected. The release of Ca2+ ions from stores is mainly mediated by the production of inositol trisphosphate (IP3) via the activation of phospholipase C.
12.
- The electrophysiological actions of several agonists which may differentiate between P2X1- and P2X3-receptors were studied under concentration and voltage-clamp conditions in dissociated neurones of 1–4 day old rat dorsal root ganglia.
- β,γ-Methylene-D-ATP (β,γ-me-D-ATP) (1–300 μM), diadenosine 5′,5′′′-P1,P5-pentaphosphate (AP5A) (100 nM–300 μM), diadenosine 5′,5′′′-P1,P4-tetraphosphate (AP4A) (300 nM–300 μM) and uridine 5′-triphosphate (UTP) (1 μM–1 mM) all activated concentration-dependent inward currents with a latency to onset of a few ms.
- The concentration-response curves for β,γ-me-D-ATP and AP5A and ATP had similar maximum values, while that for AP4A had a lower maximum. The concentration-response curve to UTP was shallow and did not reach a maximum. β,γ-Methylene-L-ATP was virtually inactive. The rank order of agonist potency was ATP>AP5A≈amp;AP4A>β,γ-me-D-ATP>UTP>>β,γ-methylene-L-ATP.
- The inward currents were inhibited by the P2-receptor antagonists suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (10 μM). PPADS also inhibited responses to ATP (800 nM) and α,β-methylene ATP (2 μM) in a concentration-dependent manner.
- This study shows that β,γ-me-D-ATP, AP5A, AP4A and UTP all act via a suramin- and PPADS-sensitive P2X-receptor to evoke rapid, transient inward currents in dissociated neurones of rat dorsal root ganglia. The very low activity of β,γ-methylene-L-ATP suggests that the agonists were acting at the P2X3-subtype to produce these effects.
13.
14.
M S Fagura I A Dainty G D McKay I P Kirk R G Humphries M J Robertson I G Dougall P Leff 《British journal of pharmacology》1998,124(1):157-164
- In the present study we have classified the receptor(s) mediating increases in intracellular calcium concentration ([Ca2+]i) in human washed platelets and compared the pharmacological profile obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y1-receptor.
- The P2Y1-receptor antagonist, adenosine-3′-phosphate-5′-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar affinities (pKB of 5.8 and 6.0, respectively).
- The selective P2YADP antagonist, AR-, exhibited partial agonism in the Jurkat cells with an affinity (pKA) of 4.9. This value is consistent with its known P2Y1-receptor activity. In platelets, AR- C66096 at a concentration (0.1 μ C66096M) approximately 100 fold greater than its known P2YADP receptor affinity, had no effect on ADP-induced increases in [Ca2+]i.
- The ability of adenine nucleotide analogues to elevate [Ca2+]i in the Jurkat cells was also determined. The rank order of agonist potency (p[A]50) was: 2-MeSADP (8.3)>2-ClATP (7.8)>ADP (7.5)=2-MeSATP (7.4)>ATPγS (6.5)>ATP (6.2), with ATP appearing to be a partial agonist.
- The same rank order of potency was observed when similar experiments were performed in platelets. However, the absolute potencies of all the agonists and the intrinsic activities of both ATPγS and ATP were lower in platelets.
- The operational model of agonism was used to test whether the agonist concentration-effect profiles obtained in these two cell types could be explained on the basis of differences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y1-receptor system.
- The hypothesis that the observed partial agonist behaviour of ATP was the result of receptor activation by contaminating ADP with concomitant receptor blockade by ATP, was tested in the platelet system. This hypothesis was supported by a theoretical analysis, which yielded an affinity value for ATP similar to that obtained previously at P2Y1-receptors.
- In summary, the results of this study indicate that human washed platelets contain P2Y1-receptors which mediate increases in [Ca2+]i and that this receptor population is pharmacologically distinct from P2YADP-receptors.
15.
- The mechanism underlying 5-hydroxytryptamine (5-HT) and/or dopamine release induced by (+)-amphetamine ((+)-Amph), 3,4-methylendioxymethamphetamine (MDMA), p-chloroamphetamine (pCA) and (+)-fenfluramine ((+)-Fen) was investigated in rat brain superfused synaptosomes preloaded with the 3H neurotransmitters.
- Their rank order of potency for [3H]-5-HT-releasing activity was the same as for inhibition of 5-HT uptake (pCA⩾MDMA⩾(+)-Fen>>(+)-Amph). Similarly, their rank order as [3H]-dopamine releasers and dopamine uptake inhibitors was the same ((+)-Amph>>pCA=MDMA>>(+)-Fen). We also confirmed that the release induced by these compounds was prevented by selective transporter inhibitors (indalpine or nomifensine).
- [3H]-5-HT and/or [3H]-dopamine release induced by all these compounds was partially (31–80%), but significantly Ca2+-dependent. Lack of extracellular Ca2+ did not alter uptake mechanisms nor did it modify the carrier-dependent dopamine-induced [3H]-dopamine release. (+)-Amph-induced [3H]-dopamine release and pCA- and MDMA-induced [3H]-5-HT release were significantly inhibited by ω-agatoxin-IVA, a specific blocker of P-type voltage-operated Ca2+-channels, similar to the previous results on (+)-Fen-induced [3H]-5-HT release.
- Methiothepin inhibited the Ca2+-dependent component of (+)-Amph-induced [3H]-dopamine release with high potency (70 nM), as previously found with (+)-Fen-induced [3H]-5-HT release. The inhibitory effect of methiothepin was not due to its effects as a transporter inhibitor or Ca2+-channel blocker and is unlikely to be due to its antagonist properties on 5-HT1/2, dopamine or any other extracellular receptor.
- These results indicate that the release induced by these compounds is both ‘carrier-mediated'' and Ca2+-dependent (possibly exocytotic-like), with the specific carrier allowing the amphetamines to enter the synaptosome. The Ca2+-dependent release is mediated by Ca2+-influx (mainly through P-type Ca2+-channels), possibly triggered by the drug interacting with an unknown intracellular target, affected by methiothepin, common to both 5-HT and dopamine synaptosomes.
16.
Medardo Hernández Gillian E Knight Scott SP Wildman Geoffrey Burnstock 《British journal of pharmacology》2009,157(8):1463-1473
Background and purpose:
As adenosine 5′-triphosphate (ATP) is one of the inhibitory mediators of the bladder outflow region, this study investigates the possible release of ATP or related purines in response to electrical field stimulation (EFS) and the purinoceptor(s) involved in nerve-mediated relaxations of the pig urinary bladder neck.Experimental approach:
Urothelium-denuded and intact phenylephrine-precontracted strips were mounted in organ baths containing physiological saline solution at 37°C and gassed with 95% O2 and 5% CO2 for isometric force recordings.Key results:
EFS, in the presence of atropine, guanethidine and NG-nitro-L-arginine, and exogenous purines, produced frequency- and concentration-dependent relaxations respectively. Adenosine 5′-diphosphate (ADP) and adenosine were more potent than ATP in producing relaxation, while uridine 5′-triphosphate, uridine 5′-diphosphate and α,β-methylene ATP were less effective. The non-selective P2 antagonist suramin, and the P2Y1 and P1 receptor blockers 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium and 8-(p-sulphophenyl)theophylline, respectively, inhibited the responses to EFS and ATP. The P1 agonist''s potency was: 5′-N-ethylcarboxamidoadenosine (NECA)>4-2[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene propanoic acid hydrochloride>2-chloro-N6-cyclopentyladenosine>-2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide = adenosine. 4-(-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol, an A2A antagonist, reduced the relaxations to EFS, adenosine and NECA. In urothelium-intact samples, relaxations to EFS and purines were smaller than in urothelium-denuded preparations. Neuronal voltage-gated Na+ channels blockade failed to modify ATP relaxations. At basal tension, EFS- and ATP-induced contractions were resistant to desensitization or blockade of P2X1 and P2X3 receptors.Conclusions and implications:
ATP is involved in the non-adrenergic, non-cholinergic, non-nitrergic inhibitory neurotransmission in the pig bladder neck, producing relaxation largely through muscle A2A receptors after breakdown to adenosine, and P2Y1 receptors after breakdown to ADP. Antagonists of these receptors may be useful for urinary incontinence treatment produced by intrinsic sphincteric deficiency. 相似文献17.
- The effects of different purinergic agonists on the cardiac adenosine 5′-triphosphate (ATP)-sensitive potassium current (IK(ATP)), appearing during dialysis of rat isolated ventricular myocytes with a low-ATP (100 μM) internal solution under whole-cell patch-clamp conditions, were examined in the presence of a P1 purinoceptor antagonist.
- The extracellular application of ATP in the micromolar range induced, besides known inward currents through cationic and chloride channels, the facilitation of IK(ATP) once IK(ATP) had already been partially activated during the low-ATP dialysis.
- Analogues of ATP, α,β-methyleneadenosine 5′-triphosphate (α,βmeATP), 2-methylthioadenosine triphosphate (2MeSATP), adenosine 5′-O-3-thiotriphosphate (ATPγS) similarly facilitated IK(ATP). UTP and ADP were very weak agonists while AMP and adenosine had no detectable effect.
- The half-maximal stimulating concentration (C50) of α,βmeATP, an analogue that did not elicite the interfering inward cationic current was 1.5 μM. Similar apparent C50 (1–2 μM) were observed for ATP and analogues tested with somewhat less maximal effect of ATPγS.
- Suramin, a nonselective P2-purinoceptor antagonist, altered IK(ATP) at the relatively high concentration required to inhibit purinoceptors. Pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), a supposedly predominantly P2X-purinoceptor antagonist, at micromolar concentration inhibited the transient inward current but did not block the facilitation of IK(ATP).
- Our results demonstrate that ATP and its analogues facilitate IK(ATP) in rat ventricular myocytes by stimulation of non-P1-, non-P2X-purinoceptors.
18.
Arthur D Conigrave Joanne Y Lee Louise van der Weyden Lele Jiang Peter Ward Vit Tasevski Brian M Luttrell Michael B Morris 《British journal of pharmacology》1998,124(7):1580-1585
- Extracellular ATP (EC50=146±57 μM) and various ATP analogues activated cyclic AMP production in undifferentiated HL-60 cells.
- The order of agonist potency was: ATPγS (adenosine 5′-O-[3-thiotriphosphate])⩾BzATP (2′&3′O-(4-benzoylbenzoyl)-adenosine-5′-triphosphate)⩾dATP>ATP. The following agonists (in order of effectiveness at 1 mM) were all less effective than ATP at concentrations up to 1 mM: β,γ methylene ATP⩾2-methylthioATP>ADP⩾Ap4A (P1, P4-di(adenosine-5′) tetraphosphate)⩾Adenosine>UTP. The poor response to UTP indicates that P2Y2 receptors are not responsible for ATP-dependent activation of adenylyl cyclase.
- Several thiophosphorylated analogs of ATP were more potent activators of cyclic AMP production than ATP. Of these, ATPγS (EC50=30.4±6.9 μM) was a full agonist. However, adenosine 5′-O-[1-thiotriphosphate] (ATPαS; EC50=45±15 μM) and adenosine 5′-O-[2-thiodiphosphate] (ADPβS; EC50=33.3±5.0 μM) were partial agonists.
- ADPβS (IC50=146±32 μM) and adenosine 5′-O-thiomonophosphate (AMPS; IC50=343±142 μM) inhibited cyclic AMP production by a submaximal concentration of ATP (100 μM). Consistent with its partial agonist activity, ADPβS was estimated to maximally suppress ATP-induced cyclic AMP production by about 65%. AMPS has not been previously reported to inhibit P2 receptors.
- The broad spectrum P2 receptor antagonist, suramin (500 μM), abolished ATP-stimulated cyclic AMP production by HL-60 cells but the adenosine receptor antagonists xanthine amine congener (XAC; 20 μM) and 8-sulpho-phenyltheophylline (8-SPT; 100 μM) were without effect.
- Extracellular ATP also activated protein kinase A (PK-A) consistent with previous findings that PK-A activation is involved in ATP-induced differentiation of HL-60 cells (Jiang et al., 1997).
- Taken together, the data indicate the presence of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 cells.
19.
- Experiments were performed to compare Ca2+ mobilization in the aortic endothelium in streptozotocin (STZ)-induced diabetic and cholesterol-fed mice with that in age-matched controls.
- The intracellular free Ca2+ ([Ca2+]i) in the fura PE-3 loaded endothelium of aortic rings was dose-dependently increased by cumulative administration of acetylcholine (ACh). ACh caused a transient rise in [Ca2+]i in Ca2+-free medium. The ACh-induced increase in [Ca2+]i in normal or Ca2+-free medium was significantly weaker in both STZ-induced diabetic and cholesterol-fed mice.
- The weaker [Ca2+]i response in Ca2+-containing medium in STZ-induced diabetic and cholesterol-fed mice was normalized by chronic administration of cholestyramine.
- The increased low density lipoprotein (LDL) levels seen in both STZ-induced diabetic and cholesterol-fed mice were normalized by the same chronic administration of cholestyramine (300 mg kg−1, p.o. daily for 10 weeks). Chronic administration of cholestyramine had no effect on the plasma glucose level.
- Lysophosphatidylcholine (LPC) decreased the [Ca2+]i responses to ACh in the aortic endothelium from normal mice.
- These results suggest that ACh increases both Ca2+ influx and Ca2+ release from storage in the aortic endothelium. The weaker [Ca2+]i influx seen in the endothelium of aortae from both STZ-induced diabetic and cholesterol-fed mice was improved by the chronic administration of cholestyramine, and we suggest that this improvement is due, at least in part, to a lowering of the plasma LDL level. It is further suggested that LPC may have an important influence over Ca2+ mobilization in the endothelium.
20.
Noriyoshi Teramoto Gordon McMurray Alison F Brading 《British journal of pharmacology》1997,120(7):1229
- Effects of levcromakalim and nucleoside diphosphates (NDPs) on both membrane currents and unitary currents in pig proximal urethra were investigated by use of patch clamp techniques (conventional whole-cell configuration, nystatin perforated patch, cell-attached configuration and inside-out patches).
- Levcromakalim produced a concentration-dependent outward current at a holding potential of −50 mV. The peak current amplitude showed little variation when measured by either conventional whole-cell or nystatin perforated patch configurations.
- In conventional whole-cell configuration, the levcromakalim (100 μM)-induced outward current decayed by about 90% in 18 min. In contrast, with the nystatin perforated patch, approximately 86% of the levcromakalim-induced outward current still remained after 18 min.
- The peak amplitude of the levcromakalim (100 μM)-induced outward membrane current recorded by the conventional whole-cell configuration was greatly reduced by inclusion of 5 mM EDTA in the pipette. The much smaller but significant outward membrane current remaining was abolished by glibenclamide.
- In conventional whole-cell recordings, inclusion of an NDP in the pipette solution induced a small outward current which slowly reached a maximal amplitude (in 2 to 10 min) and was suppressed by glibenclamide. Addition of 100 μM levcromakalim after the NDP-induced current had peaked activated a further outward current which was larger than that recorded in the absence of NDPs. Approximately 50% of this current still remained at 18 min, even when conventional whole-cell configuration was used.
- In the cell-attached mode in symmetrical 140 mM K+ conditions, glibenclamide inhibited the 100 μM levcromakalim-activated 43 pS K+ channel in a concentration-dependent manner, showing an inhibitory dissociation constant (Ki) of approximately 520 nM.
- In inside-out patches in which the glibenclamide-sensitive K+ channel had run down after exposure to levcromakalim, both uridine 5′-diphosphate (UDP) and MgATP were capable of reactivating the channel. Further application of Mg2+ to the UDP-reactivated K+ channels enhanced the channel activity reversibly.
- In inside-out patches UDP was capable of activating the glibenclamide-sensitive K+ channel without levcromakalim, providing that there was free Mg2+ present (either UDP in 5 mM EGTA or UDP in 5 mM EDTA with Mg2+). Additional application of levcromakalim caused a further reversible activation of channel opening.
- In the presence of levcromakalim, application of adenosine 5′-triphosphate (ATP) to the inner surface of the membrane patch inhibited UDP-reactivated channel opening in a concentration-dependent manner.
- Addition of an untreated cytosolic extract of pig proximal urethra reactivated the glibenclamide-sensitive K+ channel in the presence of 100 μM levcromakalim in inside-out patches.
- These results demonstrate the presence in the pig proximal urethra of a glibenclamide-sensitive K+ channel that is blocked by intracellular ATP and can be activated by levcromakalim. Intracellular UDP can reactivate the channel after rundown. Additionally, intracellular Mg2+ may play an important role in regulating the channel activity.