P2 Receptor Antagonist Research Articles

Myogenic tone in mouse mesenteric arteries: evidence for P2Y receptor-mediated, Na(+), K (+), 2Cl (-) cotransport-dependent signaling.

This study examines the action of agonists and antagonists of P2 receptors on mouse mesenteric artery contractions and the possible involvement of these signaling pathways in myogenic tone (MT) evoked by elevated intraluminal pressure. Both ATP and its non-hydrolyzed analog alpha,beta-ATP triggered transient contractions that were sharply decreased in the presence of NF023, a potent antagonist of P2X(1) receptors. In contrast, UTP and UDP elicited sustained contractions which were suppressed by MRS2567, a selective antagonist of P2Y(6) receptors. Inhibition of Na(+), K(+), 2Cl(-) cotransport (NKCC) with bumetanide led to attenuation of contractions in UTP- but not ATP-treated arteries. Both UTP-induced contractions and MT were suppressed by MRS2567 and bumetanide but were insensitive to NF023. These data implicate a P2Y(6)-mediated, NKCC-dependent mechanism in MT of mesenteric arteries. The action of heightened intraluminal pressure on UTP release from mesenteric arteries and its role in the triggering of P2Y(6)-mediated signaling should be examined further.

Evidence for the Involvment of Endothelial A2A Receptors in Adenosine Induced KATP Channel‐dependent Arteriolar Vasodilation

Adenosine (ADO) has an important role in many vascular processes including vasodilation, inflammation, permeability and injury protection. We have shown previously that endothelium is required for ADO mediated arteriolar dilation (Duza et al, AJP 285:H26, 2003). In the present study we investigated the receptor subtype and signaling cascade that mediate the ADO induced vasodilation in mouse cremaster arterioles. We examined which receptor subtype is implicated in the ADO induced vasodilation using specific P1 receptor antagonists; DPCPX, SCH442416 and PSB603 for A1, A2A and A2B receptors respectively. Control ADO dilation of 16.3±1.3 ?m (n=19) was decreased to 3.4±1.2 ?m (n=4, P<0.05) with SCH442416 (A2A antagonist). DPCPX (A1‐ antagonist) and PSB603 (A2B antagonist) had no effect on the ADO induced dilation: 14.8±3.3 ?m (n=5, P>0.05) and 16±4.1 ?m (n=5, P>0.05) respectively. Additionally we used the KATP channel blocker Glibenclamide to test the involvement of KATP channels in A2A receptor mediated dilation using the A2A specific agonist CGS21680. Control dilation was reduced from 10.4±1.8 ?m to 2.3±1.1µm (n=6, P<0.05) with Glibenclamide.Immunohistochemistry revealed that both A2A and A2B receptors are located on the endothelium. Thus, our results suggest that the primary pathway for this endothelium‐dependent ADO vasodilation is through A2A receptors acting via KATP channels. (Supported by HL76414)

Differentially Expressed MicroRNAs in Allergic Asthma Target Genes underlying Airway Hyper-Responsiveness and Goblet Cell Hyperplasia (136.22)

Abstract Asthma is an allergic disease that results in the obstruction of airways as a result of goblet cell hyperplasia and airway hyper-reactivity (AHR). Broad-spectrum P2-receptor antagonists and glucocorticoids are commonly used therapeutic agents for this disease. Niflumic acid (NA), an inhibitor of calcium-activated chloride channels is used to overcome corticosteroid-resistant asthma. Using Next Generation Sequencing, we dissected the asthma-related small RNAome in the lung before and after (FC) allergen challenge. We find a significant induction of microRNAs in FC from 40% to 74% suggesting an important role for miRNAs in asthma. Also, we uncovered 460 miRNA-up:mRNA-down and 17 miRNA-down:mRNA-up pairs that are significantly correlated in naïve and FC lung. The latter category contains a number of key genes induced during asthma and include miR-106b:P2X, miR-205:P2Y, miR-145:CLCA3 and miR-133a:GM-CSF. We also uncovered two novel miRNAs Asth-miR-1 and Asth-miR-2. Asth-miR-1 is predicted to target Toll-like receptor (TLR) adaptor TIRAP and IRAK which associates to activate NF-Kb, AP-1 and IRFs, and under some conditions, induce allergic lung disease. Asth-miR-2 is predicted to target ryanodine receptor 2 (RyR) that mediates Ca2+ release that induces airway smooth muscle contraction and bronchoconstriction. The integration of miR-106b, 205, 145 a, 133a and Asth-miR-1 and Asth-miR-1 2 with current therapies can potentially significantly enhance the efficacy and specificity of drugs used to combat asthma.

Roles of purines in synaptic modulation evoked by hypercapnia in isolated spinal cord of neonatal rat in vitro

The purine compounds, adenosine 5'-triphosphate (ATP) and adenosine, are known to accumulate in the extracellular space and to elicit various cellular responses during hypoxia/ischemia, whereas the roles of purines during hypercapnia are poorly understood. In this study, we examined the effects of various drugs affecting purine turnover on the responses to hypercapnia in the spinal cord. Electrically evoked reflex potentials were measured in an in vitro preparation of the isolated spinal cord of the neonatal rat by extracellular recording. Extracellular adenosine concentrations were assayed by high performance liquid chromatography (HPLC) methods. Hypercapnia (20% CO2) depressed the reflex potentials, which were partially reversed by an adenosine A1 receptor antagonist, 8-cyclopentyl theophylline, but not by a P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid. Exogenous adenosine and ATP also depressed the reflex potentials via adenosine A1 receptors. The hypercapnia-evoked depression was not reversed by inhibitors of gap junction hemichannels, anion channels, P2X7 receptors or equilibrative nucleoside transporters, all of which might be involved in purine efflux pathways. The adenosine accumulation evoked by hypercapnia was not inhibited by tetrodotoxin, ethylene glycol-bis(beta-amino ethyl ether) tetraacetic acid (EGTA) or an ecto-ATPase inhibitor, ARL 67156. Homocysteine thiolactone, used to trap intracellular adenosine, significantly reduced extracellular adenosine accumulation during hypercapnia. These results suggest that hypercapnia released adenosine itself from intracellular sources, using pathways different from the conventional exocytotic mechanism, and that this adenosine depressed spinal synaptic transmission via adenosine A1 receptors.

Modulation of firing activity by ATP in dopamine neurons of the rat substantia nigra pars compacta

ATP acts as a neurotransmitter or co-neurotransmitter in many areas of the CNS and peripheral nervous systems; however, little is known about the expression and functional role of purinoceptors (P2) in midbrain dopaminergic neurons. Therefore, we investigated P2X receptor expression and regulation of spontaneous firing activity in dopaminergic neurons of the substantia nigra pars compacta (SNc) in rats using patch-clamp and Ca2+-imaging techniques. In most neurons, application of ATP (1 μM–1 mM) increased firing rate dose-dependently (EC50=1.26±0.26 μM, n=45). When the P2-receptor agonists such as 2-methylthio-adenosine 5′-triphosphate (2-MeSATP) or ATPγS were applied or pressure-applied to the neuron, the firing activity increased together with a rise in cytosolic Ca2+ concentration ([Ca2+]c), but application of beta,gamma-methylene ATP (P2X1, 3 agonist) or methylthio-adenosine 5′-diphosphate (P2Y1 agonist) had no effect. In many neurons, the effect of ATP was abolished by the application of the P2-receptor antagonists, suramin or pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). When ATP was applied in a Ca2+-free solution, there was no detectable change in [Ca2+]c, suggesting that ATP does not release Ca2+ from intracellular stores. In the single-cell reverse transcription polymerase chain reaction (RT-PCR), we found that 65% of dopaminergic neurons expressed mRNAs for P2X receptors; positive amplifications of P2X6 (57.1%), P2X2/6 (25.0%), and P2X4 mRNA (17.9%), respectively. From the above results, we could conclude that ATP modulates firing activities in the rat SNc dopaminergic neurons, possibly via P2X2, P2X2/6, and/or P2X4 receptors.

Radially expanding transglial calcium waves in the intact cerebellum

Multicellular glial calcium waves may locally regulate neural activity or brain energetics. Here, we report a diffusion-driven astrocytic signal in the normal, intact brain that spans many astrocytic processes in a confined volume without fully encompassing any one cell. By using 2-photon microscopy in rodent cerebellar cortex labeled with fluorescent indicator dyes or the calcium-sensor protein G-CaMP2, we discovered spontaneous calcium waves that filled approximately ellipsoidal domains of Bergmann glia processes. Waves spread in 3 dimensions at a speed of 4-11 microm/s to a diameter of approximately 50 microm, slowed during expansion, and were reversibly blocked by P2 receptor antagonists. Consistent with the hypothesis that ATP acts as a diffusible trigger of calcium release waves, local ejection of ATP triggered P2 receptor-mediated waves that were refractory to repeated activation. Transglial waves represent a means for purinergic signals to act with local specificity to modulate activity or energetics in local neural circuits.

Interaction of hydrocortisone with ATP and adenosine on nerve-mediated contractions of frog skeletal muscle

The inhibitory effects of ATP and adenosine on the nerve-mediated contractile responses of isolated sartorius muscle of the frog, Rana ridibunda, evoked by electrical field stimulation (EFS) were studied using pharmacological organ-bath technique. The effects of hydrocortisone applied in vitro and in vivo on contractility of sartorius muscle were also examined. ATP (100 μM) significantly reduced the amplitude of contraction to EFS of sartorius muscle, while pyridoxalphosphate-6-azonphenyl-2′,4′-disulfonic acid (PPADS; 10 μM), a P2 receptor antagonist, abolished inhibitory effect of ATP. A similar inhibitory effect of adenosine (100 μM) was fully antagonized by 8-( p-sulfophenyl)-theophylline (8-SPT, 100 μM), a P1 receptor antagonist. Incubation of the tissue with hydrocortisone (10 μM) caused a slight, but significant, decrease of muscle contractions. After incubation of muscle preparations with both hydrocortisone and ATP, no inhibition of muscle contractility was registered. A single injection of hydrocortisone (100 mg/kg) 12 h prior to experiments to frogs did not significantly change the nerve-mediated contractility of isolated sartorius muscle; however, it abolished the inhibitory action of ATP without changing inhibitory activity of adenosine. After treatment of frogs with hydrocortisone for 14 days (100 mg/kg/day), both ATP and adenosine retained their inhibitory action on EFS-induced contractions of the muscle, and their effects were antagonized by PPADS and 8-SPT, respectively. It is concluded that hydrocortisone has antagonistic actions against the inhibitory effects of ATP at the frog neuromuscular junction, although this effect is lost following long-term treatment with hydrocortisone.

MECHANICALLY INDUCED REACTIVE GLIOSIS CAUSES ATP-MEDIATED ALTERATIONS IN ASTROCYTE STIFFNESS

Reactive gliosis is a process triggered in astrocytes after traumatic injury, yet the exact consequences of gliosis on cellular survival and neural regenerative processes in the injured brain remain only partly understood. One recently discovered feature influencing neuronal growth and differentiation is the physical stiffness of the environment surrounding pioneering neurites. In this study, the mechanical properties of cultured cortical astrocytes are measured following a mechanical stretch injury that induces reactive gliosis. In mechanically injured cultures, there was a significant increase in glial fibrillary acidic protein (GFAP) immunoreactivity 24 h following a rapid, transient 15% strain. In these same cultures, astrocytes in the surrounding region--the "mechanical penumbra"--also exhibited increased GFAP immunoreactivity compared to naive cultures. Correlated with these changes in GFAP was a general softening of the non-nuclear regions of the astrocytes, both in the injured and penumbra cells, as measured by atomic force microscopy (AFM). The elastic modulus in naive cultures was observed to be 57.7+/-5.8 kPa in non-nuclear regions of naive cultures, while 24 h after injury the modulus was observed to be 26.4+/-4.9 kPa in the same region of injured cells. In the penumbra of injured cultures, the modulus was 23.7+/-3.6 kPa. Alterations in astrocyte stiffness in the area of injury and mechanical penumbra were ameliorated by pretreating cultures with a nonselective P2 receptor antagonist (PPADS). Since neuronal cells generally prefer softer substrates for growth and neurite extension, these findings may indicate that the mechanical characteristics of reactive astrocytes are favorable for neuronal recovery after traumatic brain injury.

Cyclic loading opens hemichannels to release ATP as part of a chondrocyte mechanotransduction pathway

The process of chondrocyte mechanotransduction is poorly understood. However, recent studies suggest the involvement of a purinergic calcium signaling pathway although the mechanism of ATP release has not been identified. The present study tests the hypothesis that cyclic compression opens hemichannels thereby triggering the release of ATP into the extracellular milieu activating P2 receptors. The well-established chondrocyte-agarose model was utilized enabling chondrocytes to be subjected to a 40-min period of cyclic compression at 0-15% strain and 1 Hz. The opening of hemichannels was determined using Lucifer yellow (LY) incorporation and fluorescence microscopy, whereas the release of ATP into the surrounding media was quantified using the luciferin-luciferase assay. Results indicated that cyclic compression activated hemichannels such that the percentage of cells showing LY incorporation increased from 50 to 70%. This was associated with a sevenfold increase in the release of ATP. Both LY incorporation and ATP release in response to mechanical loading were blocked by the hemichannel inhibitor, flufenamic acid. Treatment with apyrase or P2 receptor antagonists, suramin or oxidated-ATP, did not prevent the mechanically induced response. In conclusion, mechanical loading triggers release of ATP via hemichannels. Hence, this study provides the first evidence of hemichannel involvement in chondrocyte mechanobiology.

Effects of ATP on the Intracellular Calcium Level in the Osteoblastic TBR31-2 Cell Line

We investigated the effects of extracellular ATP on TBR31-2 cells established from the bone marrow of transgenic mice harboring the temperature-sensitive simian virus (SV) 40 T-antigen gene. These cells showed the capacity to differentiate toward osteoblasts and could be enhanced by bone morphogenetic protein (BMP)-2, an inducer of osteoblasts. The intracellular calcium ion level ([Ca(2+)](i)) in differentiating TBR31-2 cells was measured by fluorescence confocal microscopic imaging using the Ca(2+)-sensitive probe, Calcium Green 1/AM. P2 receptor agonists, such as ATP (1 microM), uridine 5'-triphosphate (1 microM), and ADP (1 microM), significantly increased the [Ca(2+)](i) of TBR31-2 cells in 2-d and 5-d cultures, but a potent P2X receptor agonist, alpha,beta-methylene ATP (10 microM), did not increase [Ca(2+)](i). The increase in [Ca(2+)](i) induced by ATP in the 2-d culture tended to be higher than in the 5-d culture. The increase in [Ca(2+)](i) of both cultures was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, a P2 receptor antagonist. However, in an external Ca(2+)-free condition ATP-induced increase in [Ca(2+)](i) was unchanged at either stage. U73122, phospholipase C inhibitor and Thapsigargin, a calcium-pump inhibitor, significantly inhibited the increase in [Ca(2+)](i) at both stages. Reverse transcription-polymerase chain reaction analysis showed that the expression of P2Y receptor mRNA was higher in the 2-d culture than in the 5-d culture. These results indicate that ATP induces the increase in [Ca(2+)](i) from the calcium store through activating P2Y receptors in TBR31-2 cells and that the 2-d culture can respond to ATP more than the 5-d culture due to the higher expression of P2Y receptors. This suggests that the physiological role of ATP in osteoblasts is altered during differentiation.

Translocation of neuronal nitric oxide synthase to the plasma membrane by ATP is mediated by P2X and P2Y receptors.

BackgroundThe translocation of neuronal nitric oxide synthase (nNOS) from the cytosol to the membrane is functionally coupled to the activation of N-methyl-D-aspartate (NMDA) receptors at synapses. Whereas there is abundant evidence indicating that ATP and nitric oxide are involved in nociceptive transmission, whether nNOS is activated by ATP remains unknown. We recently established a fluorescence imaging system for examining nNOS translocation in PC12 cells expressing a yellow fluorescence protein-tagged nNOS N-terminal mutant, nNOSNT-YFP, and examined the effect of ATP on nNOS translocation using the system.ResultsThe translocation of nNOS was induced by ATP in the presence of NMDA and forskolin, an adenylate cyclase activator. The purinergic P2X receptor agonist 2-MeSATP and the P2Y agonist UTP significantly enhanced nNOS translocation; and simultaneous stimulation with 2-MeSATP and UTP exhibited the same concentration-response curve for the translocation as obtained with ATP. ATP, 2-MeSATP, and UTP increased the intracellular Ca2+ concentration ([Ca2+]i) in PC12 cells. Conversely, whereas the P2X receptor antagonist PPADS and the P2Y antagonist reactive blue-2 partially inhibited increases in the translocation of nNOS and [Ca2+]i by ATP, the non-selective P2 receptor antagonist suramin completely blocked them. In addition, the increase in the nNOS translocation by ATP was blocked by NMDA receptor antagonists and inhibitors of protein kinase A, protein kinase C, and Src kinase. Consistent with the expression of P2X and P2Y receptors in the spinal cord, ATP and UTP increased the [Ca2+]i in primary cultured spinal neurons. ATP potentiated and prolonged the [Ca2+]i increase produced by NMDA in the dorsal horn of the spinal cord. Furthermore, the selective P2X3/P2X2/3 antagonist A-317491 inhibited nNOS activation assessed by NO formation in spinal slices prepared from neuropathic pain model mice.ConclusionATP is involved in nNOS translocation mediated by protein kinase C via activation of P2X and P2Y receptors and nNOS translocation may be an action mechanism of ATP in nocieptive processing in the spinal cord.

Mysteries of Renal Autoregulation

Autoregulation is an important renal regulatory mechanism that provides an important protective role in glomerular hemodynamics.1 The phenomenon of autoregulatory behavior has been recognized for many decades, but a clear understanding of the underlying mechanisms involved in mediating resistance adjustments remains to be established. As illustrated in Figure 1, pressure-mediated autoregulatory behavior operates by recognizing a change in renal perfusion pressure, or renal vascular transmural pressure, and initiating induction of autoregulatory resistance changes intended to stabilize renal blood flow and/or glomerular filtration rate. The net result is that renal blood flow and glomerular filtration rate remain relatively stable over a wide range of renal perfusion pressures. Autoregulatory behavior is exquisitely sensitive in healthy normal kidneys but significantly less efficient in pathological settings, such as diabetes mellitus and some forms of hypertension. Complete loss of autoregulatory control results in renal blood flow that behaves more passively, thus increasing variability and rendering flow more directly determined by renal perfusion pressure.1,2 Figure 1. Outline of the pressure-induced signaling scheme postulated for autoregulatory adjustments in the afferent arteriolar diameter. Whole kidney autoregulation reflects regulatory input from the myogenic and tubuloglomerular feedback mechanisms at the very least.3–7 Recently, contributions of a third and possibly a fourth mechanism have been suggested.6,7 Consequently, with continued study, the mechanisms underlying renal autoregulatory resistance adjustments are slowly becoming clearer. In addition to the P1 receptor hypothesis put forth to explain signal transduction for tubuloglomerular feedback-dependent resistance changes, we have explored the hypothesis that pressure-mediated autoregulatory responses are mediated by extracellular ATP and activation of P2X1 receptors.3,8,9 Conclusive identification of the signaling molecules that mediate autoregulation remains a point of debate, but it appears clear that the release of a purine-based moiety is required for autoregulatory adjustments in afferent arteriolar resistance.3–5,8–10 This …

Acute lung inflammation and ventilator-induced lung injury caused by ATP via the P2Y receptors: an experimental study

BackgroundExtracellular adenosine 5'-triphosphate (ATP) is an endogenous signaling molecule involved in multiple biological phenomena, including inflammation. The effects of extracellular ATP in the lung have not been fully clarified. This study examined 1) the biological roles of extracellular ATP in the pathogenesis of lung inflammation and 2) the possibility of involvement of extracellular ATP in mechanical ventilation-induced lung injury.MethodsThe effects of intratracheal ATP on lung permeability, edema or lung inflammation were assessed by measurements of the lung wet-to-dry weight ratio and lung permeability index, immunohistochemistry and expression of key cytokines by real-time polymerase chain reaction. The ATP concentration in broncho-alveolar lavage (BAL) fluid from mice mechanically ventilated was measured by luciferin-luciferase assay. The suppressive effects of a P2 receptor antagonist on ventilator-induced lung inflammation were also examined.ResultsATP induced inflammatory reactions in the lung mainly via the ATP-P2Y receptor system. These reactions were alleviated by the co-administration of a specific P2 receptor antagonist. Mechanical ventilation with a large tidal volume caused lung inflammation and increased the ATP concentration in BAL fluid. P2 receptor antagonism partially mitigated the inflammatory effects of large tidal volume ventilation.ConclusionOur observations suggest that the ATP-P2Y receptor system is partially involved in the pathogenesis of ventilator-induced lung injury.

Extracellular ATP and zinc are co-secreted with insulin and activate multiple P2X purinergic receptor channels expressed by islet beta-cells to potentiate insulin secretion

It is well established that ATP is co-secreted with insulin and zinc from pancreatic beta-cells (beta-cells) in response to elevations in extracellular glucose concentration. Despite this knowledge, the physiological roles of extracellular secreted ATP and zinc are ill-defined. We hypothesized that secreted ATP and zinc are autocrine purinergic signaling molecules that activate P2X purinergic receptor (P2XR) channels expressed by beta-cells to enhance glucose-stimulated insulin secretion (GSIS). To test this postulate, we performed ELISA assays for secreted insulin at fixed time points within a "real-time" assay and confirmed that the physiological insulin secretagogue glucose stimulates secretion of ATP and zinc into the extracellular milieu along with insulin from primary rat islets. Exogenous ATP and zinc alone or together also induced insulin secretion in this model system. Most importantly, the presence of an extracellular ATP scavenger, a zinc chelator, and P2 receptor antagonists attenuated GSIS. Furthermore, mRNA and protein were expressed in immortalized beta-cells and primary islets for a unique subset of P2XR channel subtypes, P2X(2), P2X(3), P2X(4), and P2X(6), which are each gated by extracellular ATP and modulated positively by extracellular zinc. On the basis of these results, we propose that, within endocrine pancreatic islets, secreted ATP and zinc have profound autocrine regulatory influence on insulin secretion via ATP-gated and zinc-modulated P2XR channels.

Regulation of smooth muscle contractility by the epithelium in rat vas deferens: role of ATP‐induced release of PGE2

Recent studies suggest that the epithelium might modulate the contractility of smooth muscle. However, the mechanisms underlying this regulation are unknown. The present study investigated the regulation of smooth muscle contraction by the epithelium in rat vas deferens and the possible factor(s) involved. Exogenously applied ATP inhibited electrical field stimulation (EFS)-evoked smooth muscle contraction in an epithelium-dependent manner. As the effects of ATP on smooth muscle contractility were abrogated by inhibitors of prostaglandin synthesis, but not by those of nitric oxide synthesis, prostaglandins might mediate the effects of ATP. Consistent with this idea, PGE(2) inhibited EFS-evoked smooth muscle contraction independent of the epithelium, while ATP and UTP induced the release of PGE(2) from cultured rat vas deferens epithelial cells, but not smooth muscle cells. The ATP-induced PGE(2) release from vas deferens epithelial cells was abolished by U73122, an inhibitor of phospholipase C (PLC) and BAPTA AM, a Ca(2+) chelator. ATP also transiently increased [Ca(2+)](i) in vas deferens epithelial cells. This effect of ATP on [Ca(2+)](i) was independent of extracellular Ca(2+), but abolished by the P2 receptor antagonist RB2 and U73122. In membrane potential measurements using a voltage-sensitive dye, PGE(2), but not ATP, hyperpolarized vas deferens smooth muscle cells and this effect of PGE(2) was blocked by MDL12330A, an adenylate cyclase inhibitor, and the chromanol 293B, a blocker of cAMP-dependent K(+) channels. Taken together, our results suggest that ATP inhibition of vas deferens smooth muscle contraction is epithelium dependent. The data also suggest that ATP activates P2Y receptor-coupled Ca(2+) mobilization leading to the release of PGE(2) from epithelial cells, which in turn activates cAMP-dependent K(+) channels in smooth muscle cells leading to the hyperpolarization of membrane voltage and the inhibition of vas deferens contraction. Thus, the present findings suggest a novel regulatory mechanism by which the epithelium regulates the contractility of smooth muscle.

Platelet Antistaphylococcal Responses Occur through P2X 1 and P2Y 12 Receptor-Induced Activation and Kinocidin Release

Platelets (PLTs) act in antimicrobial host defense by releasing PLT microbicidal proteins (PMPs) or PLT kinocidins (PKs). Receptors mediating staphylocidal efficacy and PMP or PK release versus isogenic PMP-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains were examined in vitro. Isolated PLTs were incubated with ISP479C or ISP479R (PLT/S. aureus ratio range, 1:1 to 10,000:1) in the presence or absence of a panel of PLT inhibitors, including P2X and P2Y receptor antagonists of increasingly narrow specificity, and PLT adhesion receptors (CD41, CD42b, and CD62P). PLT-to-S. aureus exposure ratios of > or = 10:1 yielded significant reductions in the viability of both strains. Results from reversed-phase high-performance liquid chromatography indicated that staphylocidal PLT releasates contained PMPs and PKs. At ratios below 10:1, the PLT antistaphylococcal efficacy relative to the intrinsic S. aureus PMP-susceptible or -resistant phenotype diminished. Apyrase (an agent of ADP degradation), suramin (a general P2 receptor antagonist), pyridoxal 5'-phosphonucleotide derivative (a specific P2X(1) antagonist), and cangrelor (a specific P2Y(12) antagonist) mitigated the PLT staphylocidal response against both strains, correlating with reduced levels of PMP and PK release. Specific inhibition occurred in the presence and absence of homologous plasma. The antagonism of the thromboxane A(2), cyclooxygenase-1/cyclooxygenase-2, or phospholipase C pathway or the hindrance of surface adhesion receptors failed to impede PLT anti-S. aureus responses. These results suggest a multifactorial PLT anti-S. aureus response mechanism involving (i) a PLT-to-S. aureus ratio sufficient for activation; (ii) the ensuing degranulation of PMPs, PKs, ADP, and/or ATP; (iii) the activation of P2X(1)/P2Y(12) receptors on adjacent PLTs; and (iv) the recursive amplification of PMP and PK release from these PLTs.

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

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

Release of ATP and glutamate in the nucleus tractus solitarii mediate pulmonary stretch receptor (Breuer–Hering) reflex pathway

The Breuer–Hering inflation reflex is initiated by activation of the slowly adapting pulmonary stretch receptor afferents (SARs), which monosynaptically activate second-order relay neurones in the dorsal medullary nucleus of the solitary tract (NTS). Here we demonstrate that during lung inflation SARs release both ATP and glutamate from their central terminals to activate these NTS neurones. In anaesthetized and artificially ventilated rats, ATP- and glutamate-selective microelectrode biosensors placed in the NTS detected rhythmic release of both transmitters phase-locked to lung inflation. This release of ATP and glutamate was independent of the centrally generated respiratory rhythm and could be reversibly abolished during the blockade of the afferent transmission in the vagus nerve by topical application of local anaesthetic. Microionophoretic application of ATP increased the activity of all tested NTS second-order relay neurones which receive monosynaptic inputs from the SARs. Unilateral microinjection of ATP into the NTS site where pulmonary stretch receptor afferents terminate produced central apnoea, mimicking the effect of lung inflation. Application of P2 and glutamate receptor antagonists (pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulphonic acid, suramin and kynurenic acid) significantly decreased baseline lung inflation-induced firing of the second-order relay neurones. These data demonstrate that ATP and glutamate are released in the NTS from the central terminals of the lung stretch receptor afferents, activate the second-order relay neurones and hence mediate the key respiratory reflex — the Breuer—Hering inflation reflex.

Corneal Re-epithelialization Stimulated by Diadenosine Polyphosphates Recruits RhoA/ROCK and ERK1/2 Pathways

To investigate the role of ERK1/2 and RhoA/ROCK intracellular pathways in the modification of corneal re-epithelialization when stimulated by the diadenosine polyphosphates Ap(4)A and Ap(3)A. In wounded confluent SIRC (Statens Seruminstitut rabbit cornea) cell monolayers and in the presence or absence of Ap(4)A or Ap(3)A 100 microM, a battery of P2 receptor antagonists and inhibitors of tyrosin kinases, MAPK, and cytoskeleton pathways (AG1478 100 microM, U0126 100 microM, Y27632 100 nM, and (-)-blebbistatin 10 microM; n = 8 each) were assayed. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with Ap(4)A and Ap(3)A (100 microM), with or without suramin, RB-2, U0126, and Y27632. The intracellular distribution of pERK and ROCK-I was examined in the presence of Ap(4)A or Ap(3)A (100 microM) with U0126 and Y27632 (100 nM). In the presence of Ap(4)A, U0126, Y27632, AG1478, and (-)-blebbistatin, reduced the migration rate compared to the effect of Ap(4)A alone (P < 0.0001, P < 0.001, P < 0.01, and P < 0.1 versus Ap(4)A, respectively). In the presence of Ap(3)A 100 microM, U0126 and Y27632 accelerated the migration rate when compared with the effect of Ap(3)A alone, whereas AG1478 and (-)-blebbistatin (P < 0.0001 versus Ap(3)A) slowed the migration rate. Western blot assays demonstrated that both dinucleotides activated the ERK1/2 pathway but only Ap(4)A activated the ROCK-I pathway. The intracellular distribution of pERK1/2 and ROCK-I reflected cross-talk between these two pathways. The activation of the Ap(4)A/P2Y(2) receptor, accelerates corneal epithelial cell migration during wound healing with the activation of MAPK and cytoskeleton pathways, whereas activation of the Ap(3)A/P2Y(6) receptor signals only the MAPK pathway.

Purinergic P2X receptors facilitate inhibitory GABAergic and glycinergic neurotransmission to cardiac vagal neurons in the nucleus ambiguus

This study examined whether adenosine 5′-triphosphate (ATP) modulated inhibitory glycinergic and GABAergic neurotransmission to cardiac vagal neurons. Inhibitory activity to cardiac vagal neurons was isolated and examined using whole-cell patch-clamp recordings in an in vitro brain slice preparation in rats. ATP (100 μM) evoked increases in the frequency of glycinergic and GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in cardiac vagal neurons which were blocked by the broad P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (100 μM). Application of the P2Y agonists uridine triphosphate (15 μM) and adenosine 5′-0-( Z-thiodiphosphate) (60 μM) did not enhance inhibitory neurotransmission to cardiac vagal neurons however, application of the selective P2X` receptor agonist, α, β-methylene ATP (100 μM), increased glycinergic and GABAergic mIPSC neurotransmission to cardiac vagal neurons. The increase in inhibitory neurotransmission evoked by α, β-methylene ATP was abolished by the selective P2X receptor antagonist 2′,3′- O-(2,4,6-Trinitrophenyl) adenosine 5′-triphosphate (100 μM) indicating P2X receptors enhance the release of inhibitory neurotransmitters to cardiac neurons. The voltage-gated calcium channel blocker cadmium chloride did not alter the evoked increase in inhibitory mIPSCs. This work demonstrates that P2X receptor activation enhances inhibitory neurotransmission to parasympathetic cardiac vagal neurons and demonstrates an important functional role for ATP mediated purinergic signaling to cardiac vagal neurons.