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.

     

Characterization of the P2 receptors in rabbit pulmonary artery

1. We have identified the P₂ receptors mediating vasomotor responses in the rabbit pulmonary artery.
2. Neither ATP nor UTP contracted intact or endothelium-denuded rings. However, both relaxed intact rings of rabbit pulmonary artery that had been preconstricted with phenylephrine (pD₂ 5.2 and 5.6, respectively).
3. The vasodilator effect of UTP was endothelium-dependent and abolished by the nitric oxide synthase inhibitor N^G-nitro-L-arginine (L-NOARG).
4. The vasodilator effect of ATP was only partially inhibited by removal of endothelium or addition of L-NOARG, suggesting an additional direct effect on vascular smooth muscle.
5. The endothelium-dependent vasodilator responses to UTP and ATP were competitively antagonized by suramin.
6. Preconstricted, endothelium-denuded rings were also relaxed by 2-methylthio ATP (pD₂ 6.6), a P_2Y receptor agonist.
7. Ca²⁺-mobilizing P_2U receptors were identified on smooth muscle cells on the basis of single cell responses to ATP (pD₂ 7.8) and UTP (pD₂ 7.9; 6.7 in the presence of 100 μM suramin).
8. There was no evidence of a Ca²⁺-mobilizing P_2Y receptor in these cultured cells.
9. The data suggest the presence of (i) a suramin-sensitive P_2U receptor on endothelial cells that induces vasorelaxation through NO release, (ii) a suramin-sensitive P_2U receptor on cultured smooth muscle cells that mobilizes Ca²⁺ but is not coupled to vasomotor responses and (iii) a putative P_2Y receptor on vascular smooth muscle cells that induces relaxation via a Ca²⁺-independent signal transduction pathway.

     

Enhancement of the response to purinergic agonists in P2Y1 transfected 1321N1 cells by antagonists suramin and PPADS

1. We have previously shown that both suramin and pyridoxal-phosphate-6-azophenyl-2′, 4′ disulphonic acid (PPADS) act as antagonists at transfected P2Y₁ receptors. Here we show that under certain experimental conditions these two P2 antagonists can enhance the response to agonists acting at these receptors.
2. The expression of either P2Y₁ or P2Y₂ receptors in 1321N1 human astrocytoma cells results, on a change of medium, in an elevation of basal (no added agonist) accumulation of [³H]-inositol(poly)phosphates([³H]-InsP_x) compared to cells not expressing these receptors. This elevation is much greater in P2Y₁ transfectants than in P2Y₂ transfectants.
3. Both PPADS and suramin reduced this basal level of [³H]-InsP_x accumulation in the P2Y₁ expressing cells.
4. When a protocol was used which required changing the culture medium, antagonists were added at a concentration which reduced the basal accumulation by about 50%, there was a significant stimulation in response to increasing concentrations of 2-methylthioadenosine 5′-triphosphate (2MeSATP), in the absence of antagonists there was no significant effect of the agonist.
5. However, when 2MeSATP was added in the absence of a change of medium and with no antagonist present, there was a several fold increase in [³H]-InsP_x accumulation. These results show that a release of endogenous agonist activity (possibly ATP/ADP) from the P2Y₁ expressing cells can create conditions in which a response to an agonist such as 2MeSATP can only be seen in the presence of a competitive antagonist.

     

A Ca²⁺-dependent chloride current and Ca²⁺ influx via Ca(v)1.2 ion channels play major roles in P2Y receptor-mediated pulmonary vasoconstriction

BACKGROUND AND PURPOSE

ATP, UTP and UDP act at smooth muscle P2X and P2Y receptors to constrict rat intrapulmonary arteries, but the underlying signalling pathways are poorly understood. Here, we determined the roles of the Ca²⁺-dependent chloride ion current (I_Cl,Ca), Ca_v1.2 ion channels and Ca²⁺ influx.

EXPERIMENTAL APPROACH

Isometric tension was recorded from endothelium-denuded rat intrapulmonary artery rings (i.d. 200–500 µm) mounted on a wire myograph.

KEY RESULTS

The I_Cl,Ca blockers, niflumic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and the Ca_v1.2 channel blocker, nifedipine, reduced peak amplitude of contractions evoked by UTP and UDP by ∼45–50% and in a non-additive manner. Ca²⁺-free buffer inhibited responses by ∼70%. Niflumic acid and nifedipine similarly depressed contractions to ATP, but Ca²⁺-free buffer almost abolished the response. After peaking, contractions to UTP and UDP decayed slowly by 50–70% to a sustained plateau, which was rapidly inhibited by niflumic acid and nifedipine. Contractions to ATP, however, reversed rapidly and fully. Tannic acid contracted tissues per se and potentiated nucleotide-evoked contractions.

CONCLUSIONS AND IMPLICATIONS

I_Cl,Ca and Ca²⁺ influx via Ca_v1.2 ion channels contribute substantially and equally to contractions of rat intrapulmonary arteries evoked by UTP and UDP, via P2Y receptors. ATP also activates these mechanisms via P2Y receptors, but the greater dependence on extracellular Ca²⁺ most likely reflects additional influx through the P2X1 receptor pore. The lack of a sustained response to ATP is probably due to it acting at P2 receptor subtypes that desensitize rapidly. Thus multiple signalling mechanisms contribute to pulmonary artery vasoconstriction mediated by P2 receptors.     

UTP induces vascular responses in the isolated and perfused canine epicardial coronary artery via UTP-preferring P2Y receptors

1. Vasoconstrictor responses of the isolated and perfused canine epicardial coronary artery to uridine 5′-triphosphate (UTP) were analysed pharmacologically.
2. At basal perfusion pressure, UTP induced vasoconstriction in a dose-related manner and the vasoconstriction was sometimes followed by a slight vasodilatation at large doses (more than 10 nmol). The rank order of potency for vasoconstriction was UTP=UDP>ATP>TTP⩾ITP>> UMP. At raised perfusion pressure by 20 mM KCl, the vasoconstriction was not changed and a small vasodilatation was induced at large doses. The rank order of potency for vasodilatation was induced at large doses. The rank order of potency for vasodilatation was ATP>>ITP⩾UDP>UTP⩾TTP. The maximal vasodilator response to UTP was much less than that to ATP. UMP did not induce vasodilatation.
3. The P2X receptor agonist and desensitizing agent α,β-methylene ATP (1 μM) and the P2 receptor antagonist suramin (100 μM) inhibited the vasoconstrictor responses to ATP but not those to UTP and UDP. The P2 receptor antagonist reactive blue 2 (30 μM) did not inhibit the vascular responses to UTP.
4. UTP (200 μM) desensitized the vasoconstrictor responses to UTP, but not either the vasodilator responses to UTP or the vasoconstrictor responses to ATP and UDP. UDP (200 μM) did not desensitize the vascular responses to UTP.
5. Preincubating the UDP stock solution and arterial preparation with hexokinase (10 and 1 uml⁻¹, respectively) did not change the vasoconstrictor responses to UDP.
6. The Ca channel blocker diltiazem (1 μM) inhibited the vasoconstrictor responses to UTP but not those to ATP and UDP. Incubation in a Ca²⁺-free solution containing 1 mM EGTA inhibited the vascular responses to ATP, UTP and UDP.
7. Removal of the endothelium by an intraluminal injection of saponin (1 mg) inhibited the vasodilator responses to UTP. Indomethacin, a cyclo-oxygenase inhibitor (1 μM), inhibited the vasodilator responses to UTP, but N^G-nitro-L-arginine, a nitric oxide synthase inhibitor (300 μM), did not have an inhibitory effect.
8. The results suggest that (1) UTP induces vasoconstriction via UTP-preferring P2Y receptors on the smooth muscle and vasodilatation via receptors different from those mediating the vasoconstriction induced by UTP and mediating the vasodilatation by ATP on the endothelium, through mainly the release of prostacyclin in the canine epicardial coronary artery; (2) UDP induces vasoconstriction via UDP-preferring P2Y receptors; and (3) L-type Ca ion channels are involved in the vasoconstriction induced by UTP, but not in that induced by UDP.

     

Molecular cloning and characterization of rat P2Y4 nucleotide receptor

An intronless open reading frame encoding a protein (361aa in length) was isolated from a rat genomic library probed with a DNA fragment from rat heart. This protein showed 83% sequence identity with the human P2Y₄ (hP2Y₄) receptor and represents a homologue of the human pyrimidinoceptor. However, the rP2Y₄ receptor is not selective for uridine nucleotides and, instead, shows an agonist potency order of ITP=ATP=ADP(pure)=UTP=ATPγS=2-MeSATP=Ap₄A>UDP(pure). ADP, ATPγS, 2-MeSATP and UDP are partial agonists. Thus, in terms of agonist profile, rP2Y₄ is more like the P2U receptor subtype. The rP2Y₄ receptor was reversibly antagonized by Reactive blue 2 but not by suramin which, otherwise, inhibits the hP2Y₂ receptor (a known P2U receptor). Thus, rP2Y₄ and the P2Y₂ subtype appear to be structurally distinct forms of the P2U receptor (where ATP and UTP are equi-active) but can be distinguished as suramin-insensitive and suramin-sensitive P2U receptors, respectively.     

Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

BACKGROUND AND PURPOSE

Despite the abundant expression of the UDP-sensitive P2Y₆ receptor in urothelial cells and sub-urothelial myofibroblasts its role in the control of bladder function is not well understood.

EXPERIMENTAL APPROACH

We compared the effects of UDP and of the selective P2Y₆ receptor agonist, PSB0474, on bladder urodynamics in anaesthetized rats; the voided fluid was tested for ATP bioluminescence. The isolated urinary bladder was used for in vitro myographic recordings and [³H]-ACh overflow experiments.

KEY RESULTS

Instillation of UDP or PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions; an effect blocked by the P2Y₆ receptor antagonist, MRS2578. Effects mediated by urothelial P2Y₆ receptors required extrinsic neuronal circuitry as they were not detected in the isolated bladder. UDP-induced bladder hyperactvity was also prevented by blocking P2X3 and P2Y₁ receptors, respectively, with A317491 and MRS2179 applied i.v.. UDP decreased [³H]-ACh release from stimulated bladder strips with urothelium, but not in its absence. Inhibitory effects of UDP were converted into facilitation by the P2Y₁ receptor antagonist, MRS2179. The P2Y₆ receptor agonist increased threefold ATP levels in the voided fluid.

CONCLUSIONS AND IMPLICATIONS

Activation of P2Y₆ receptors increased the voiding frequency indirectly by releasing ATP from the urothelium and activation of P2X3 receptors on sub-urothelial nerve afferents. Bladder hyperactivity may be partly reversed following ATP hydrolysis to ADP by E-NTPDases, thereby decreasing ACh release from cholinergic nerves expressing P2Y₁ receptors.     

Characterization of P2 receptors for purine and pyrimidine nucleotides in human placental cotyledons

1. The aim of this study was to characterize P2 receptors in the arterial vascular bed of human perfused placental cotyledons. Vasoconstrictor responses to bolus injections of purine and pyrimidine nucleotides were tested at basal tone, and vasodilator responses in preparations with tone raised by perfusion with prostaglandin F_2α (PGF_2α; 10–50 nM).
2. At basal tone, bolus injections of the P2X-selective agonist α,β-methylene ATP (α,β-meATP; 0.5–500 nmol) elicited dose-dependent vasoconstriction. ATP (0.005–5 μmol) also elicited dose-dependent vasoconstriction, but was less potent than α,β-meATP. Vasoconstriction was also elicited by other nucleotides, but only at the highest dose tested (5 μmol): UTP > CTP=ITP (n=6). GTP and TTP did not cause vasoconstriction.
3. Constrictor responses to bolus injections of α,β-meATP were resistant to desensitization and were not significantly affected when carried out in the presence of 1 μM α,β-meATP added to the perfusate. However, responses to bolus injections of α,β-meATP were partially blocked by perfusion with 10 μM α,β-meATP. In contrast, responses to ATP and UTP were unaffected by 10 μM α,β-meATP. The P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10 and 100 μM) had no significant effect on vasoconstriction mediated by α,β-meATP and ATP.
4. Removal of the endothelium had no significant effect on constrictor responses to α,β-meATP, ATP and UTP. Inhibition of nitric oxide (NO) synthesis with N^G-nitro-L-arginine methyl ester (L-NAME; 100 μM) had no significant effect on vasoconstriction to ATP and α,β-meATP.
5. In preparations with tone raised with PGF_2α (10–50 nM) vasodilatation was elicited by nucleotides with the following order of potency: 2MeSATP=ADP >> ATP > UTP > CTP=GTP=ITP=TTP. pD₂ values were: 2MeSATP, 10.03±0.26 (n=7); ADP, 9.97±0.40 (n=5); ATP, 8.89±0.18 (n=7); UTP, 7.79±0.35 (n=7). Maximal responses to 2MeSATP and ADP were similar and were approximately 40% greater than maximal responses to ATP and UTP.
6. Vasodilator responses to nucleotides were abolished by L-NAME (100 μM) and by removal of the endothelium.
7. In conclusion, contractile responses mediated by α,β-meATP and ATP in human placental smooth muscle are resistant to desensitization and insensitive to PPADS and, thus, show a dissimilar pharmacological profile to the classic smooth muscle P2X₁ receptor. There may be two subtypes of smooth muscle P2 receptor based on differential antagonism of α,β-meATP and ATP with α,β-meATP. A smooth muscle P2 receptor mediates vasoconstriction to UTP, and may indicate a further subtype. Endothelium-dependent, NO-dependent, vasodilatation to 2MeSATP and ADP may be mediated by P2Y₁ receptors, while endothelial P2Y₂ receptors are likely to mediate NO-dependent relaxation to ATP and UTP.

     

UDP induces intestinal epithelial migration via the P2Y6 receptor

BACKGROUND AND PURPOSE

Extracellular nucleotides are released at high concentrations from damaged cells and function through P2 receptor activation. Intestinal epithelial restitution, which is defined as cell migration independent of cell proliferation, is an important initial step in the process of wound healing. In this study, we investigated the role of extracellular nucleotides in intestinal epithelial migratory responses.

EXPERIMENTAL APPROACH

Wound-healing and trans-well migration assays were performed with a rat intestinal epithelial cell line (IEC-6). The concentrations of extracellular nucleotides released from injured IEC-6 cells were measured by HPLC. TGF-β expression was assessed by RT-PCR and elisa.

KEY RESULTS

Scratching the monolayer of IEC-6 cells induced cell migration. Pretreatment with apyrase or MRS2578, a selective P2Y₆ antagonist, inhibited the wound-induced cell migration. Among the cellular nucleotides, only ATP and uridine 5''-diphosphate (UDP) were detected in the culture medium after cell wounding. Exogenously applied UDP dose-dependently enhanced the migration more effectively than ATP but did not induce proliferation. In addition, cell wounding and UDP increased the expression of TGF-β, and both the wound-induced and UDP-enhanced migration were inhibited by MRS2578 or ALK5Inhibitor (ALK5i), a TGF-β receptor blocker. Furthermore, cell wounding and UDP stimulation up-regulated the expression of P2Y₆ receptor mRNA, and this effect was suppressed by MRS2578 or ALK5i.

CONCLUSION AND IMPLICATIONS

Wound-induced UDP evokes intestinal epithelial restitution by activation of P2Y₆ receptors, which mediates de novo synthesis of TGF-β. In addition, the expression of P2Y₆ receptors is increased by cell wounding and UDP, which constitutes a positive-feedback loop for mucosal repair.     

Angiotensin II responses of vascular smooth muscle cells from hypertensive rats: enhancement at the level of p42 and p44 mitogen activated protein kinase

1. Stimulation of the AT₁ receptor by angiotensin II (AII) gives a larger mitogenic response in vascular smooth muscle cells from spontaneously hypertensive rats (SHR) compared to those from normotensive (WKY) controls. Here we investigated whether the p42 and p44 mitogen activated protein kinase (MAPK) pathway is differentially regulated in these cells by AT₁ receptors.
2. We showed that there is a similar level of p42 and p44 MAPK immunoreactivity in the SHR and WKY derived cells.
3. However, by use of an antiserum specific for the tyrosine phosphorylated form of MAPK, and an assay with a nonapeptide MAPK substrate, we showed that AII (100 nM)-stimulated phosphorylation and activation of p42^mapk and p44^mapk are enhanced in the SHR derived cells.
4. This increased MAPK activity in SHR derived cells was also seen on protein kinase C activation with 100 nM phorbol myristate acetate (PMA). The size and time course of the response to PMA was the same as that to AII in each cell type.
5. The protein kinase C inhibitor Ro 31-8220 attenuated the early (2 min) phase of AII stimulation of MAPK activity and the entire stimulation caused by PMA. At longer times of AII stimulation both p42^mapk and p44^mapk were activated by an Ro 31-8220-insensitive mechanism.
6. Agonist or PMA stimulation of MAPK activity was inhibited by the tyrosine kinase inhibitor genistein. AII stimulated tyrosine protein phosphorylation to a greater degree in SHR than WKY cells.
7. These results show that the MAPK response of SHR derived cells is increased over that of WKY cells by mechanisms independent of the enhanced stimulation of phospholipase C; amplification at the level of sequential protein kinase C and tyrosine kinase steps leads to the enhanced responsiveness of MAPK in the SHR derived cells.

     

Human adenosine A1 receptor and P2Y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells

1. The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric G_i/G_o protein-coupled and G_q/G₁₁ protein-coupled receptors. The aims of the current study were: (i) to investigate whether the G_i/G_o protein-coupled adenosine A₁ receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor.
2. The selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1±0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 μM; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block G_i/G_o-dependent pathways).
3. Adenosine A₁ receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 μM; 6±10% of control). In contrast, daidzein (100 μM), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55±8% inhibition) and LY 294002 (30 μM; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 μM).
4. Activation of the endogenous P2Y₂ purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC₅₀=1.6±0.3 μM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 μM; 45±5% inhibition), indicating the possible involvement of both G_i/G_o protein and G_q protein-dependent pathways in the overall response to UTP.
5. CPA and UTP stimulated concentration-dependent increases in the phosphorylation state of the 42 kDa and 44 kDa forms of MAP kinase as demonstrated by Western blotting.
6. Co-activation of CHO-A1 cells with CPA (10 nM) and UTP (1 μM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 μM).
7. Adenosine A1 and P2Y2 purinoceptor activation increased the expression of luciferase in CHO cells transfected with a luciferase reporter gene containing the c-fos promoter. However, co-activating these two receptors produced only additive increases in luciferase expression.
8. In conclusion, our studies have shown that the transfected adenosine A₁ receptor and the endogenous P2Y₂ purinoceptor couple to the MAP kinase signalling pathway in CHO-A1 cells. Furthermore, co-stimulation of the adenosine A1 receptor and the P2Y₂ purinoceptor produced synergistic increases in MAP kinase activity but not c-fos mediated luciferase expression.

     

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.

     

Novel vasocontractile role of the P2Y14 receptor: characterization of its signalling in porcine isolated pancreatic arteries

Background and Purpose

The P2Y₁₄ receptor is the newest member of the P2Y receptor family; it is G_i/o protein-coupled and is activated by UDP and selectively by UDP-glucose and MRS2690 (2-thiouridine-5′-diphosphoglucose) (7–10-fold more potent than UDP-glucose). This study investigated whether P2Y₁₄ receptors were functionally expressed in porcine isolated pancreatic arteries.

Experimental Approach

Pancreatic arteries were prepared for isometric tension recording and UDP-glucose, UDP and MRS2690 were applied cumulatively after preconstriction with U46619, a TxA₂ mimetic. Levels of phosphorylated myosin light chain 2 (MLC2) were assessed with Western blotting. cAMP concentrations were assessed using a competitive enzyme immunoassay kit.

Key Results

Concentration-dependent contractions with a rank order of potency of MRS2690 (10-fold) > UDP-glucose ≥ UDP were recorded. These contractions were reduced by PPTN {4-[4-(piperidin-4-yl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthoic acid}, a selective antagonist of P2Y₁₄ receptors, which did not affect responses to UTP. Contraction to UDP-glucose was not affected by MRS2578, a P2Y₆ receptor selective antagonist. Raising cAMP levels and forskolin, in the presence of U46619, enhanced contractions to UDP-glucose. In addition, UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP levels. Removal of the endothelium and inhibition of endothelium-derived contractile agents (TxA₂, PGF_2α and endothelin-1) inhibited contractions to UDP glucose. Y-27632, nifedipine and thapsigargin also reduced contractions to the agonists. UDP-glucose and MRS2690 increased MLC2 phosphorylation, which was blocked by PPTN.

Conclusions and Implications

P2Y₁₄ receptors play a novel vasocontractile role in porcine pancreatic arteries, mediating contraction via cAMP-dependent mechanisms, elevation of intracellular Ca²⁺ levels, activation of RhoA/ROCK signalling and MLC2, along with release of TxA₂, PGF_2α and endothelin-1.     

P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice

Background and purpose:

P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis.

Experimental approach:

mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE^–/–) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE^–/– mice.

Key results:

P2Y₆ receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y₂ receptor expression remained unchanged. Expression of P2Y₁ or P2Y₄ receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y₆ mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y₆-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y₆ receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y₆-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100–300 µM) or pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS, 10–30 µM). Finally, 4-week treatment of cholesterol-fed apoE^–/– mice with suramin or PPADS (50 and 25 mg·kg⁻¹·day⁻¹ respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication.

Conclusions and implications:

These results suggest involvement of nucleotide receptors, particularly P2Y₆ receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y₆ receptor-deficient mice.     

P2Y1 receptors mediate inhibitory neuromuscular transmission in the rat colon

Background and purpose:

Inhibitory junction potentials (IJP) are responsible for smooth muscle relaxation in the gastrointestinal tract. The aim of this study was to pharmacologically characterize the neurotransmitters [nitric oxide (NO) and adenosine triphosphate (ATP)] and receptors involved at the inhibitory neuromuscular junctions in the rat colon using newly available P2Y₁ antagonists.

Experimental approach:

Organ bath and microelectrode recordings were used to evaluate the effect of drugs on spontaneous mechanical activity and resting membrane potential. IJP and mechanical relaxation were studied using electrical field stimulation (EFS).

Key results:

N^ω-nitro-L-arginine (L-NNA) inhibited the slow component of the IJP and partially inhibited the mechanical relaxation induced by EFS. MRS2179, MRS2500 and MRS2279, all selective P2Y₁ receptor antagonists, inhibited the fast component of the IJP without having a major effect on the relaxation induced by EFS. The combination of both L-NNA and P2Y₁ antagonists inhibited the fast and the slow components of the IJP and completely blocked the mechanical relaxation induced by EFS. Sodium nitroprusside caused smooth muscle hyperpolarization and cessation of spontaneous motility that was prevented by oxadiazolo[4,3-α]quinoxalin-1-one. Adenosine 5′-O-2-thiodiphosphate, a preferential P2Y agonist, hyperpolarized smooth muscle cells and decreased spontaneous motility. This effect was inhibited by P2Y₁ antagonists.

Conclusions and implications:

The co-transmission process in the rat colon involves ATP and NO. P2Y₁ receptors mediate the fast IJP and NO the slow IJP. The rank order of potency of the P2Y₁ receptor antagonists is MRS2500 greater than MRS2279 greater than MRS2179. P2Y₁ receptors might be potential pharmacological targets for the regulation of gastrointestinal motility.     

ATP released via pannexin-1 hemichannels mediates bladder overactivity triggered by urothelial P2Y6 receptors

In contrast to the well-known signaling role of urothelial ATP to control bladder function, the hypothesis that uracil nucleotides (UTP and/or UDP) also exert autocrine/paracrine actions only recently gained experimental support. Urothelial cells express UDP-sensitive P2Y₆ receptors, yet their role in the control of bladder activity has been mostly neglected. This study was designed to investigate the ability of PSB0474, a stable UDP analogue which exhibits selectivity for P2Y₆ receptors, to modulate urodynamic responses in the anaesthetized rat in vivo. Instillation of PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions. PSB0474-induced bladder overactivity was prevented by the selective P2Y₆ antagonist, MRS2578. The increase in the VF produced by PSB0474 was also blocked by inhibitors of pannexin-1 hemichannels, ¹⁰Panx or carbenoxolone, when these drugs were applied inside the bladder lumen but not when they were administered intravenously. Reduction of hemichannels pore permeability with H1152 also prevented PSB0474-induced bladder overactivity, but the exocytosis inhibitor, Exo-1, was inactive. PSB0474 increased by 3-fold the urinary ATP content. Implication of hemichannels permeability on PSB0474-induced ATP release was demonstrated by real-time fluorescence video-microscopy measuring the uptake of propidium iodide by intact urothelial cells in the absence and in the presence of MRS2578 or carbenoxolone. Confocal microscopy studies confirmed the co-localization of pannexin-1 and P2Y₆ receptors in the rat urothelium. Data indicate that activation of P2Y₆ receptors causes bladder overactivity in the anaesthetized rat indirectly by releasing ATP from the urothelium via pannexin-1 hemichannels.     

Facilitation of transmitter release from rat sympathetic neurons via presynaptic P2Y(1) receptors

BACKGROUND AND PURPOSE

P2Y₁, P2Y₂, P2Y₄, P2Y₁₂ and P2Y₁₃ receptors for nucleotides have been reported to mediate presynaptic inhibition, but unequivocal evidence for facilitatory presynaptic P2Y receptors is not available. The search for such receptors was the purpose of this study.

EXPERIMENTAL APPROACH

In primary cultures of rat superior cervical ganglion neurons and in PC12 cell cultures, currents were recorded via the perforated patch clamp technique, and the release of [³H]-noradrenaline was determined.

KEY RESULTS

ADP, 2-methylthio-ATP and ATP enhanced stimulation-evoked ³H overflow from superior cervical ganglion neurons, treated with pertussis toxin to prevent the signalling of inhibitory G proteins. This effect was abolished by P2Y₁ antagonists and by inhibition of phospholipase C, but not by inhibition of protein kinase C or depletion of intracellular Ca²⁺ stores. ADP and a specific P2Y₁ agonist caused inhibition of Kv7 channels, and this was prevented by a respective antagonist. In neurons not treated with pertussis toxin, ³H overflow was also enhanced by a specific P2Y₁ agonist and by ADP, but only when the P2Y₁₂ receptors were blocked. ADP also enhanced K⁺-evoked ³H overflow from PC12 cells treated with pertussis toxin, but only in a clone expressing recombinant P2Y₁ receptors.

CONCLUSIONS AND IMPLICATIONS

These results demonstrate that presynaptic P2Y₁ receptors mediate facilitation of transmitter release from sympathetic neurons most likely through inhibition of Kv7 channels.