ATP stimulation of Ca2+-dependent plasminogen release from cultured microglia

1. 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 {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 induced plasminogen release in a concentration-dependent manner (0.3 μM to 10 μM).
2. 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).
3. 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).
4. 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.

     

Effects of extracellular nucleotides on single cells and populations of human osteoblasts: contribution of cell heterogeneity to relative potencies

1. Human osteoblasts responded to the application of extracellular nucleotides, acting at P₂-receptors, with increases in cytosolic free calcium concentration ([Ca²⁺]_i).
2. 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).
3. ATP and uridine 5′-triphosphate (UTP) were applied to single human osteoblasts and induced [Ca²⁺]_i rises of comparable amplitude in every cell tested.
4. 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).
5. 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.

     

ATP,a partial agonist for the P2Z receptor of human lymphocytes

1. 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.
2. 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 n_H 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.
3. 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.
4. 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 (n_H 3.2 and 2.9 for 30 and 50 μM ox-ATP, respectively (n=2)).
5. 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.
6. 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.
7. 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.

     

Characterization of the Ca2+ responses evoked by ATP and other nucleotides in mammalian brain astrocytes

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.

     

P2X receptors in cochlear Deiters' cells

1. 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.
2. 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.
3. Increasing intracellular free Ca²⁺ by decreasing the concentration of EGTA in the pipette solution reduced the amplitude of the ATP-gated current.
4. 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).
5. 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.
6. 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.

     

Characterization of the P2 receptors on the human umbilical vein endothelial cell line ECV304

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.

     

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase

1. 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).
2. 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.
3. ATP (log EC₅₀ −5.1±0.2) also caused an increase in the total [³H]-inositol (poly)phosphates ([³H]-InsP_x) 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.
4. 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.
5. 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.
6. 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.
7. 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.

     

ATP release and contraction mediated by different P2-receptor subtypes in guinea-pig ileal smooth muscle

1. 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.
2. 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.
3. 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.
4. 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.
5. The potency order of all the agonists tested for the release of ATP was α,β-mATP>UTP>β,γ-mATP>ADP.
6. 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.
7. 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.

     

Characterization of Ca2+ influx through recombinant P2X receptor in C6BU-1 cells

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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

     

Actions of 4-chloro-3-ethyl phenol on internal Ca2+ stores in vascular smooth muscle and endothelial cells

1. 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.
2. 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.
3. 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.
4. 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.
5. 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).
6. 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).
7. 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.
8. 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.

     

The signalling pathway which causes contraction via P2-purinoceptors in rat urinary bladder smooth muscle

1. 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.
2. 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).
3. 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.
4. 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).
5. NCDC 100 μM significantly inhibited the KCl-induced contraction to 65.9±4.9% (n=6) of the control (P<0.01).
6. 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.

     

Pharmacological properties of P2X3-receptors present in neurones of the rat dorsal root ganglia

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.

     

P2Y1-receptors in human platelets which are pharmacologically distinct from P2YADP-receptors

1. 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.
2. The P2Y₁-receptor antagonist, adenosine-3′-phosphate-5′-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar affinities (pK_B of 5.8 and 6.0, respectively).
3. The selective P2Y_ADP antagonist, AR-{"type":"entrez-nucleotide","attrs":{"text":"C66096","term_id":"2424801","term_text":"C66096"}}C66096, exhibited partial agonism in the Jurkat cells with an affinity (pK_A) of 4.9. This value is consistent with its known P2Y₁-receptor activity. In platelets, AR-{"type":"entrez-nucleotide","attrs":{"text":"C66096","term_id":"2424801","term_text":"C66096"}}C66096 at a concentration (0.1 μM) approximately 100 fold greater than its known P2Y_ADP receptor affinity, had no effect on ADP-induced increases in [Ca²⁺]_i.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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 P2Y_ADP-receptors.

     

Carrier-dependent and Ca2+-dependent 5-HT and dopamine release induced by (+)-amphetamine, 3,4-methylendioxy-methamphetamine,p-chloroamphetamine and (+)-fenfluramine

1. 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.
2. 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).
3. [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.
4. 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-HT_1/2, dopamine or any other extracellular receptor.
5. 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.

     

Role of ATP and related purines in inhibitory neurotransmission to the pig urinary bladder neck

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 N^G-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 A_2A 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 A_2A 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.     

Purinergic facilitation of ATP-sensitive potassium current in rat ventricular myocytes

1. The effects of different purinergic agonists on the cardiac adenosine 5′-triphosphate (ATP)-sensitive potassium current (I_K(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.
2. The extracellular application of ATP in the micromolar range induced, besides known inward currents through cationic and chloride channels, the facilitation of I_K(ATP) once I_K(ATP) had already been partially activated during the low-ATP dialysis.
3. Analogues of ATP, α,β-methyleneadenosine 5′-triphosphate (α,βmeATP), 2-methylthioadenosine triphosphate (2MeSATP), adenosine 5′-O-3-thiotriphosphate (ATPγS) similarly facilitated I_K(ATP). UTP and ADP were very weak agonists while AMP and adenosine had no detectable effect.
4. 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.
5. Suramin, a nonselective P₂-purinoceptor antagonist, altered I_K(ATP) at the relatively high concentration required to inhibit purinoceptors. Pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), a supposedly predominantly P_2X-purinoceptor antagonist, at micromolar concentration inhibited the transient inward current but did not block the facilitation of I_K(ATP).
6. Our results demonstrate that ATP and its analogues facilitate I_K(ATP) in rat ventricular myocytes by stimulation of non-P₁-, non-P_2X-purinoceptors.

     

Pharmacological profile of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 leukemia cells

1. Extracellular ATP (EC₅₀=146±57 μM) and various ATP analogues activated cyclic AMP production in undifferentiated HL-60 cells.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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).
7. Taken together, the data indicate the presence of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 cells.

     

Ca2+ mobilization in the aortic endothelium in streptozotocin-induced diabetic and cholesterol-fed mice

1. 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.
2. 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.
3. The weaker [Ca²⁺]_i response in Ca²⁺-containing medium in STZ-induced diabetic and cholesterol-fed mice was normalized by chronic administration of cholestyramine.
4. 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.
5. Lysophosphatidylcholine (LPC) decreased the [Ca²⁺]_i responses to ACh in the aortic endothelium from normal mice.
6. 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.

     

Effects of levcromakalim and nucleoside diphosphates on glibenclamide-sensitive K+ channels in pig urethral myocytes

1. 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).
2. 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.
3. 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.
4. 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.
5. 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.
6. 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 (K_i) of approximately 520 nM.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.