Role of nitric oxide on purinergic signalling in the cochlea

In the inner ear, there is considerable evidence that extracellular adenosine 5′-triphosphate (ATP) plays an important role in auditory neurotransmission as a neurotransmitter or a neuromodulator, although the potential role of adenosine signalling in the modulation of auditory neurotransmission has also been reported. The activation of ligand-gated ionotropic P2X receptors and G protein-coupled metabotropic P2Y receptors has been reported to induce an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons (SGNs), and supporting cells in the cochlea. ATP may participate in auditory neurotransmission by modulating [Ca²⁺]_i in the cochlear cells. Recent studies showed that extracellular ATP induced nitric oxide (NO) production in IHCs, OHCs, and SGNs, which affects the ATP-induced Ca²⁺ response via the NO-cGMP-PKG pathway in those cells by a feedback mechanism. A cross-talk between NO and ATP may therefore exist in the auditory signal transduction. In the present article, I review the role of NO on the ATP-induced Ca²⁺ signalling in IHCs and OHCs. I also consider the possible role of NO in the ATP-induced Ca²⁺ signalling in SGNs and supporting cells.     

ATP released by astrocytes mediates glutamatergic activity-dependent heterosynaptic suppression

Extracellular ATP released from axons is known to assist activity-dependent signaling between neurons and Schwann cells in the peripheral nervous system. Here we report that ATP released from astrocytes as a result of neuronal activity can also modulate central synaptic transmission. In cultures of hippocampal neurons, endogenously released ATP tonically suppresses glutamatergic synapses via presynaptic P2Y receptors, an effect that depends on the presence of cocultured astrocytes. Glutamate release accompanying neuronal activity also activates non-NMDA receptors of nearby astrocytes and triggers ATP release from these cells, which in turn causes homo- and heterosynaptic suppression. In CA1 pyramidal neurons of hippocampal slices, a similar synaptic suppression was also produced by adenosine, an immediate degradation product of ATP released by glial cells. Thus, neuron-glia crosstalk may participate in activity-dependent synaptic modulation.     

P2Y purine receptor responses and expression in the pulmonary circulation of juvenile rabbits

The purine nucleotide ATP mediates pulmonary vasodilation at birth by stimulation of P2Y purine receptors in the pulmonary circulation. The specific P2Y receptors in the pulmonary circulation and the segmental distribution of their responses remain unknown. We investigated the effects of purine nucleotides, ATP, ADP, and AMP, and pyrimidine nucleotides, UTP, UDP, and UMP, in juvenile rabbit pulmonary arteries for functional characterization of P2Y receptors. We also studied the expression of P2Y receptor subtypes in pulmonary arteries and the role of nitric oxide (NO), prostaglandins, and cytochrome P-450 metabolites in the response to ATP. In conduit size arteries, ATP, ADP, and AMP caused greater relaxation responses than UTP, UDP, and UMP. In resistance vessels, ATP and UTP caused comparable vasodilation. The response to ATP was attenuated by the P2Y antagonist cibacron blue, the NO synthase antagonist N(omega)-nitro-l-arginine methyl ester (l-NAME), and the cytochrome P-450 inhibitor 17-octadecynoic acid but not by the P2X antagonist alpha,beta-methylene ATP or the cyclooxygenase inhibitor indomethacin in conduit arteries. In the resistance vessels, l-NAME caused a more complete inhibition of the responses to ATP and UTP. Responses to AMP and UMP were NO and endothelium dependent, whereas responses to ADP and UDP were NO and endothelium independent in the conduit arteries. RT-PCR showed expression of P2Y(1), P2Y(2), and P2Y(4) receptors, but not P2Y(6) receptors, in lung parenchyma, pulmonary arteries, and pulmonary artery endothelial cells. These data suggest that distinct P2Y receptors mediate the vasodilator responses to purine and pyrimidine nucleotides in the juvenile rabbit pulmonary circulation. ATP appears to cause NO-mediated vasodilation predominantly through P2Y2 receptors on endothelium.     

Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex

Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca²⁺-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca²⁺-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using “sniff-cell” approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca²⁺ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca²⁺ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca²⁺-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes in the neocortex.     

Negative Regulation of CFTR Activity by Extracellular ATP Involves P2Y2 Receptors in CFTR-expressing CHO Cells

Extracellular nucleotides exert autocrine/paracrine effects on ion transport by activating P2 receptors. We studied the effects of extracellular ATP and UTP on the cystic fibrosis transmembrane conductance regulator (CFTR) channel stably expressed in Chinese Hamster Ovary cells (CHO-BQ1 cells). CFTR activity was measured using the (¹²⁵I) iodide efflux technique and whole-cell patch-clamp recording in response to either forskolin or xanthine derivatives. Using RT-PCR and intracellular calcium concentration ([Ca²⁺]_i) measurement, we showed that CHO-BQ1 cells express P2Y2 but not P2Y4 receptors. While ATP and UTP induced similar increases in [Ca²⁺]_i, pre-addition by one of these two agonists desensitized the response for the other, suggesting that ATP- and UTP-induced [Ca²⁺]_i increases were mediated by a common receptor, which was identified as the P2Y2 subtype. CFTR activity was reduced by ATP and UTP but not by ADP or adenosine applications. This inhibitory effect of ATP on CFTR activity was not due to a change in cAMP level. Furthermore, CFTR activation by forskolin or IBMX failed to promote [Ca²⁺]_i increase, suggesting that CFTR activation did not generate an ATP release large enough to stimulate P2Y2 receptors. Taken together, our results show that endogenous P2Y2 receptor activation downregulates CFTR activity in a cAMP-independent manner in CHO cells. B. Marcet and V. Chappe contributed equally to this work.     

Coexpression of several types of metabotropic nucleotide receptors in single cerebellar astrocytes

We have examined the expression of mRNA for several P2Y nucleotide receptors by northern blot analysis in purified type 1 cerebellar astrocyte cultures. These results suggest that different P2Y subtypes could be responsible for ATP metabotropic calcium responses in single type 1 astrocytes. To identify these subtypes we have studied the pharmacological profile of ATP calcium responses using fura-2 microfluorimetry. All tested astrocytes responded to ATP and UTP stimulations evoking similar calcium transients. Most astrocytes also responded to 2-methylthioATP and ADP challenges. The agonist potency order was 2-methylthioATP > ADP > ATP = UTP. Cross-desensitization experiments carried out with ATP, UTP, and 2-methylthioATP showed that 2-methylthioATP and UTP interact with different receptors, P2Y(1) and P2Y(2) or P2Y(4). In a subpopulation of type 1 astrocytes, ATP prestimulation did not block UTP responses, and UDP elicited clear intracellular Ca(2+) concentration responses at very low concentrations. 2-MethylthioATP and UTP calcium responses exhibited different sensitivity to pertussis toxin and different inhibition patterns in response to P2 antagonists. The P2Y(1)-specific antagonist N:(6)-methyl-2'-deoxyadenosine 3', 5'-bisphosphate (MRS 2179) specifically blocked the 2-methylthio-ATP responses. We can conclude that all single astrocytes coexpressed at least two types of P2Y metabotropic receptors: P2Y(1) and either P2Y(2) or P2Y(4) receptors. Moreover, 30-40% of astrocytes also coexpressed specific pyrimidine receptors of the P2Y(6) subtype, highly selective for UDP coupled to pertussis-toxin insensitive G protein.     

Possible Involvement of Extracellular ATP-P2Y Purinoceptor Signaling in Ischemia-induced Tolerance of Astrocytes in Culture

Extracellular adenosine 5′-triphosphate (ATP) activates specific G protein-coupled purinoceptors (P2Y), and ATP-P2Y signaling pathways induces intracellular Ca²⁺ mobilization resulting in changes in the gene expression of a variety of proteins in astrocytes. This study investigated whether the exposure of cultured astrocytes to sublethal ischemia produced resistance to subsequent lethal ischemic stress, and if so, whether the extracellular ATP-P2Y signaling pathways were responsible for the tolerance. Ischemia-like insults, sublethal oxygen-glucose deprivation (sOGD), produced tolerance to subsequent lethal OGD stress in cultured astrocytes. Early during reperfusion after sOGD, the amount of extracellular ATP and the expression of both P2Y₁ and P2Y₂ receptors were increased, leading to enhanced activation of the extracellular ATP-P2Y signaling pathways. The occurrence of intracellular spontaneous Ca²⁺ oscillations was also increased. In addition, sOGD treatment enhanced the expression of the phosphorylated form of extracellular signal-regulated protein kinases 1 and 2 (p-ERK 1/2), and treatment with an inhibitor of ERK significantly attenuated the sOGD-induced ischemic tolerance of astrocytes.     

Astrocytes mediate in vivo cholinergic-induced synaptic plasticity

Long-term potentiation (LTP) of synaptic transmission represents the cellular basis of learning and memory. Astrocytes have been shown to regulate synaptic transmission and plasticity. However, their involvement in specific physiological processes that induce LTP in vivo remains unknown. Here we show that in vivo cholinergic activity evoked by sensory stimulation or electrical stimulation of the septal nucleus increases Ca²⁺ in hippocampal astrocytes and induces LTP of CA3-CA1 synapses, which requires cholinergic muscarinic (mAChR) and metabotropic glutamate receptor (mGluR) activation. Stimulation of cholinergic pathways in hippocampal slices evokes astrocyte Ca²⁺ elevations, postsynaptic depolarizations of CA1 pyramidal neurons, and LTP of transmitter release at single CA3-CA1 synapses. Like in vivo, these effects are mediated by mAChRs, and this cholinergic-induced LTP (c-LTP) also involves mGluR activation. Astrocyte Ca²⁺ elevations and LTP are absent in IP₃R2 knock-out mice. Downregulating astrocyte Ca²⁺ signal by loading astrocytes with BAPTA or GDPβS also prevents LTP, which is restored by simultaneous astrocyte Ca²⁺ uncaging and postsynaptic depolarization. Therefore, cholinergic-induced LTP requires astrocyte Ca²⁺ elevations, which stimulate astrocyte glutamate release that activates mGluRs. The cholinergic-induced LTP results from the temporal coincidence of the postsynaptic activity and the astrocyte Ca²⁺ signal simultaneously evoked by cholinergic activity. Therefore, the astrocyte Ca²⁺ signal is necessary for cholinergic-induced synaptic plasticity, indicating that astrocytes are directly involved in brain storage information.     

UTP is not a biased agonist at human P2Y11 receptors

Biased agonism describes a multistate model of G protein-coupled receptor activation in which each ligand induces a unique structural conformation of the receptor, such that the receptor couples differentially to G proteins and other intracellular proteins. P2Y receptors are G protein-coupled receptors that are activated by endogenous nucleotides, such as adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP). A previous report suggested that UTP may be a biased agonist at the human P2Y₁₁ receptor, as it increased cytosolic [Ca²⁺], but did not induce accumulation of inositol phosphates, whereas ATP did both. The mechanism of action of UTP was unclear, so the aim of this study was to characterise the interaction of UTP with the P2Y₁₁ receptor in greater detail. Intracellular Ca²⁺ was monitored in 1321N1 cells stably expressing human P2Y₁₁ receptors using the Ca²⁺-sensitive fluorescent indicator, fluo-4. ATP evoked a rapid, concentration-dependent rise in intracellular Ca²⁺, but surprisingly, even high concentrations of UTP were ineffective. In contrast, UTP was slightly, but significantly more potent than ATP in evoking a rise in intracellular Ca²⁺ in 1321N1 cells stably expressing the human P2Y₂ receptor, with no difference in the maximum response. Thus, the lack of response to UTP at hP2Y₁₁ receptors was not due to a problem with the UTP solution. Furthermore, coapplying a high concentration of UTP with ATP did not inhibit the response to ATP. Thus, contrary to a previous report, we find no evidence for an agonist action of UTP at the human P2Y₁₁ receptor, nor does UTP act as an antagonist.     

Innate immunity signaling: cytosolic Ca2+ elevation is linked to downstream nitric oxide generation through the action of calmodulin or a calmodulin-like protein

Ca²⁺ rise and nitric oxide (NO) generation are essential early steps in plant innate immunity and initiate the hypersensitive response (HR) to avirulent pathogens. Previous work from this laboratory has demonstrated that a loss-of-function mutation of an Arabidopsis (Arabidopsis thaliana) plasma membrane Ca²⁺-permeable inwardly conducting ion channel impairs HR and that this phenotype could be rescued by the application of a NO donor. At present, the mechanism linking cytosolic Ca²⁺ rise to NO generation during pathogen response signaling in plants is still unclear. Animal nitric oxide synthase (NOS) activation is Ca²⁺/calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants: a pathogen-induced Ca²⁺ signal leads to CaM and/or a CaM-like protein (CML) activation of NOS. In wild-type Arabidopsis plants, the use of a CaM antagonist prevents NO generation and the HR. Application of a CaM antagonist does not prevent pathogen-induced cytosolic Ca²⁺ elevation, excluding the possibility of CaM acting upstream from Ca²⁺. The CaM antagonist and Ca²⁺ chelation abolish NO generation in wild-type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca²⁺/CaM dependent in vitro. The CaM-like protein CML24 has been previously associated with NO-related phenotypes in Arabidopsis. Here, we find that innate immune response phenotypes (HR and [avirulent] pathogen-induced NO elevation in leaves) are inhibited in loss-of-function cml24-4 mutant plants. Pathogen-associated molecular pattern-mediated NO generation in cells of cml24-4 mutants is impaired as well. Our work suggests that the initial pathogen recognition signal of Ca²⁺ influx into the cytosol activates CaM and/or a CML, which then acts to induce downstream NO synthesis as intermediary steps in a pathogen perception signaling cascade, leading to innate immune responses, including the HR.     

β-Amyloid activates nitric oxide synthesis and causes neuronal death in hippocampal astrocytes

Digital fluorescence imaging was used to study the effects of the amyloid-beta peptide βA (full-length peptide, βA 1–42) and its neurotoxic fragment (βA 25–35) on nitric oxide (NO) synthesis and cell viability in mixed cultures of rat hippocampal neurons and astrocytes. It was found that both βA 1–42 and βA 25–35 stimulated NO synthesis in astrocytes, but not in neurons. L-NAME, an inhibitor of the inducible NO synthase, blocked the effect of βA on NO production almost completely, reduced βA-induced mitochondrial depolarization in astrocytes, and partly prevented neuronal death. The rate of NO synthesis was decreased in Ca²⁺-free medium, increased in the presence of antioxidants and the NADPH oxidase inhibitor, and decreased in the presence of the SH-reagent thimerosal.     

Cold storage of rabbit thoracic aorta in University of Wisconsin solution attenuates P2Y(2) purine receptors

Post-transplantation thrombosis may occur in donor segments of iliac arteries and livers following surgical removal and storage in University of Wisconsin (UW) solution for transplantation. We have previously suggested that purine receptors are vulnerable to denaturation after UW storage. The aims of the present study were to determine what particular subtypes of purine P2Y receptors in rabbit thoracic aorta deteriorate after 8 days of UW storage by studying vascular reactivity to acetylcholine, ATP, 2MeSATP and UTP. Ring segments of aortae from male New Zealand White rabbits were mounted upon fine-wire myographs and vasodilatation to the above agents tested on fresh tissue, and after 8 days of UW storage. Vasodilatation to ATP was attenuated by 100 microM L-NAME in fresh tissue suggesting that the relaxant response was, in part, due to nitric oxide (NO). P2Y-mediated relaxation to ATP was significantly attenuated by UW storage and cholinergic responses were not. This attenuated relaxation to ATP was not further attenuated by L-NAME, suggesting a loss of the NO-dependent mechanism. De-endothelialisation indicated that UTP-mediated vasorelaxation, via P2Y(2) receptors, was endothelium-dependent. Any residual endothelium-independent relaxation to UTP was abolished by UW storage and endothelium-dependent UTP relaxation was reduced to the same level as that seen in fresh, de-endothelialised tissue. In contrast responses to 2MeSATP, via P2Y(1) receptors, were predominantly endothelium-independent and were only partially attenuated by UW storage. Responses to pyridoxalphosphate-6-azophenyl-2('),4(')-disulphonic acid (PPADS) and L-NAME suggested that vasorelaxation to 2MeSATP and UTP was mediated by P2Y(1) and P2Y(2) receptors, respectively. It is therefore concluded that UW storage predominantly decreases P2Y(2) receptor-mediated vascular reactivity.     

cGMP production is coupled to Ca-dependent nitric oxide generation in rabbit parotid acinar cells

We investigated the mechanism of guanosine 3′,5′-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a β-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca²⁺ mobilization, because intracellular Ca ²⁺ elevating reagents, such as thapsigargin and the Ca²⁺ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca²⁺ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by N^G-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca²⁺. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca²⁺ mobilization.     

Purinergic mechanisms in the control of gastrointestinal motility

For many years, ATP and adenosine have been implicated in movement regulation of the gastrointestinal tract. They act through three major receptor subtypes: adenosine or P1 receptors, P2X receptors and P2Y receptors. Each of these major receptor types can be subdivided into several different classes and is widely distributed amongst various neurons, muscle types, glia and interstitial cells that regulate intestinal functions. Several key roles for the different receptors and their endogenous ligands have been identified in physiological and pharmacological studies. For example, adenosine acting at A(1) receptors appears to inhibit intestinal motility in various pathological conditions. Similarly, ATP acting at P2Y receptors is an important component of inhibitory neuromuscular transmission, acting as a cotransmitter with nitric oxide. ATP acting at P2X and P2Y(1) receptors is important for synaptic transmission in simple descending excitatory and inhibitory reflex pathways. Some P2Y receptor subtypes prefer uridine nucleotides over purine nucleotides. Thus, roles for UTP and UDP as enteric transmitters in place of ATP cannot be excluded. ATP also appears to be important for sensory transduction, especially in chemosensitive pathways that initiate local inhibitory reflexes. Despite this evidence, data are lacking about the roles of either adenosine or ATP in more complex motility patterns such as segmentation or the interdigestive migrating motor complex. Clarification of roles for purinergic transmission in these common, but understudied, motility patterns will depend on the use of subtype-specific antagonists that in some cases have not yet been developed.     

Reactive oxygen species mediate the potentiating effects of ATP on GABAergic synaptic transmission in the immature hippocampus

Reactive oxygen species (ROS) constitute important signaling molecules in the central nervous system. They regulate a number of different functions both under physiological conditions and under pathological conditions. Here we tested the hypothesis that in the immature hippocampus ATP, the most diffuse neurotransmitter in the brain, modulates synaptic transmission via ROS. We show that ATP, acting on metabotropic P2Y1 receptors, increased the frequency of GABA(A)-mediated spontaneous postsynaptic currents (SPSCs) in CA3 principal cells, an effect that was prevented by the antioxidant N-acetyl-cysteine or by catalase, an enzyme that breaks down H2O2. The effect of ATP on SPSCs was mimicked by H2O2 or by the pro-oxidant, Fe2+, which, through the Fentol reaction, catalyzes the conversion of H2O2 into highly reactive hydroxyl radicals. MRS-2179, a P2Y1 receptor antagonist, removed the facilitatory action of Fe2+ on SPSCs, suggesting that endogenous ATP acting on P2Y1 receptors is involved in Fe2+-induced modulation of synaptic transmission. Imaging ROS with the H2O2-sensitive dye DCF revealed that ATP induces generation of peroxide in astrocytes via activation of P2Y1 receptors coupled to intracellular calcium rise. Neither N-acetyl-cysteine nor catalase prevented Ca2+ transients induced by ATP in astrocytes. Since a single hippocampal astrocyte can contact many neurons, ATP-induced ROS signaling may control thousands of synapses. This may be crucial for information processing in the immature brain when GABAergic activity is essential for the proper wiring of the hippocampal network.     

P2Y nucleotide receptor interaction with alpha integrin mediates astrocyte migration

Astrocytes become activated in response to brain injury, as characterized by increased expression of glial fibrillary acidic protein (GFAP) and increased rates of cell migration and proliferation. Damage to brain cells causes the release of cytoplasmic nucleotides, such as ATP and uridine 5'-triphosphate (UTP), ligands for P2 nucleotide receptors. Results in this study with primary rat astrocytes indicate that activation of a G protein-coupled P2Y(2) receptor for ATP and UTP increases GFAP expression and both chemotactic and chemokinetic cell migration. UTP-induced astrocyte migration was inhibited by silencing of P2Y(2) nucleotide receptor (P2Y(2)R) expression with siRNA of P2Y(2)R (P2Y(2)R siRNA). UTP also increased the expression in astrocytes of alpha(V)beta(3/5) integrins that are known to interact directly with the P2Y(2)R to modulate its function. Anti-alpha(V) integrin antibodies prevented UTP-stimulated astrocyte migration, suggesting that P2Y(2)R/alpha(V) interactions mediate the activation of astrocytes by UTP. P2Y(2)R-mediated astrocyte migration required the activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (Akt) and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways, responses that also were inhibited by anti-alpha(V) integrin antibody. These results suggest that P2Y(2)Rs and their associated signaling pathways may be important factors regulating astrogliosis in brain disorders.     

P2Y purinoceptors induce changes in intracellular calcium in acinar cells of rat lacrimal glands

Adenosine 5′-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca²⁺]_i in acinar cells. The removal of extracellular Ca²⁺ or the use of Ca²⁺ channel blockers partially inhibited the ATP-induced [Ca²⁺]_i increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP₃ receptors) did not completely inhibit the ATP-induced [Ca²⁺]_i increase. P1 purinoceptor agonists did not induce any changes in [Ca²⁺]_i, whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca²⁺]_i. A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca²⁺]_i, although UTP (a P2Y_2,4,6 receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP ⋙ UTP) suggested that P2Y₁ played a significant role in the cellular response to ATP. BzATP (a P2X₇ receptor agonist) induced a small increase in [Ca²⁺]_i, but α,β-meATP (a P2X_1,3 receptor agonist) had no effect. RT-PCR indicated that P2X_2,3,4,5,6,7 and P2Y_1,2,4,12,14 are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y₁) as well as by P2X purinoceptors.     

Comparative analysis of optogenetic actuators in cultured astrocytes

Astrocytes modulate synaptic transmission via release of gliotransmitters such as ATP, glutamate, d-serine and l-lactate. One of the main problems when studying the role of astrocytes in vitro and in vivo is the lack of suitable tools for their selective activation. Optogenetic actuators can be used to manipulate astrocytic activity by expression of variants of channelrhodopsin-2 (ChR2) or other optogenetic actuators with the aim to initiate intracellular events such as intracellular Ca²⁺ ([Ca²⁺]_i) and/or cAMP increases. We have developed an array of adenoviral vectors (AVV) with ChR2-like actuators, including an enhanced ChR2 mutant (H134R), and a mutant with improved Ca²⁺ permeability (Ca²⁺ translocating channelrhodopsin, CatCh). We show here that [Ca²⁺]_i elevations evoked by ChR2(H134R) and CatCh in astrocytes are largely due to release of Ca²⁺ from the intracellular stores. The autocrine action of ATP which is released under these conditions and acts on the P2Y receptors also contributes to the [Ca²⁺]_i elevations. We also studied effects evoked using light-sensitive G-protein coupled receptors (opto-adrenoceptors). Activation of optoα1AR (Gq-coupled) and optoβ2AR (G_s-coupled) resulted in astrocytic [Ca²⁺]_i increases which were suppressed by blocking the corresponding intracellular signalling cascade (phospholipase C and adenylate cyclase, respectively). Interestingly, the bulk of [Ca²⁺]_i responses evoked using either optoAR was blocked by an ATP degrading enzyme, apyrase, or a P2Y1 receptor blocker, MRS 2179, indicating that they are to a large extent triggered by the autocrine action of ATP. We conclude that, whilst optimal tools for control of astrocytes are yet to be generated, the currently available optogenetic actuators successfully initiate biologically relevant signalling events in astrocytes.     

Calcium is involved in nitric oxide- and auxin-induced lateral root formation in rice

In the present study, the role of nitric oxide (NO) in the regulation of lateral root (LR) formation in rice was examined. Application of sodium nitroprusside (SNP; a NO donor) and indole-3-butyric acid (IBA; a naturally occurring auxin) to rice seedlings induced LR formation. The effect is specific for NO because the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (cPTIO) blocked the action of SNP and IBA. Endogenous NO was detected by the specific fluorescence probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. SNP- and IBA-induced NO fluorescence was specifically suppressed by cPTIO. Nitrate reductase (NR) inhibitor sodium tungstate completely inhibited IBA-induced LR formation and NO fluorescence. However, nitric oxide synthase inhibitor N ^G-nitro-l-arginine methyl ester hydrochloride slightly reduced IBA-induced LR formation and NO generation. It appears that NO generation that occurs in response to IBA might primarily involve NR activity. Moreover, NO production caused by SNP and IBA was localized in root area corresponding to LR emergence. The effects of Ca²⁺ chelators, Ca²⁺-channel inhibitors, and calmodulin antagonists on LR formation induced by SNP and IBA were also examined. All these inhibitors were effective in reducing the action of SNP and IBA. However, Ca²⁺ chelators and Ca²⁺-channel inhibitors had no effect on SNP- and IBA-induced NO generation. It is concluded that cytosolic levels of Ca²⁺ may regulate SNP and IBA action through calmodulin-dependent mechanism.     

Multiple P2Y receptors couple to calcium-dependent,chloride channels in smooth muscle cells of the rat pulmonary artery

Background

Uridine 5''-triphosphate (UTP) and uridine 5''-diphosphate (UDP) act via P2Y receptors to evoke contraction of rat pulmonary arteries, whilst adenosine 5''-triphosphate (ATP) acts via P2X and P2Y receptors. Pharmacological characterisation of these receptors in intact arteries is complicated by release and extracellular metabolism of nucleotides, so the aim of this study was to characterise the P2Y receptors under conditions that minimise these problems.

Methods

The perforated-patch clamp technique was used to record the Ca²⁺-dependent, Cl^- current (I_Cl,Ca) activated by P2Y receptor agonists in acutely dissociated smooth muscle cells of rat small (SPA) and large (LPA) intrapulmonary arteries, held at -50 mV. Contractions to ATP were measured in isolated muscle rings. Data were compared by Student''s t test or one way ANOVA.

Results

ATP, UTP and UDP (10^-4M) evoked oscillating, inward currents (peak = 13–727 pA) in 71–93% of cells. The first current was usually the largest and in the SPA the response to ATP was significantly greater than those to UTP or UDP (P < 0.05). Subsequent currents tended to decrease in amplitude, with a variable time-course, to a level that was significantly smaller for ATP (P < 0.05), UTP (P < 0.001) and UDP (P < 0.05) in the SPA. The frequency of oscillations was similar for each agonist (mean≈6–11.min^-1) and changed little during agonist application. The non-selective P2 receptor antagonist suramin (10^-4M) abolished currents evoked by ATP in SPA (n = 4) and LPA (n = 4), but pyridoxalphosphate-6-azophenyl-2'',4''-disulphonic acid (PPADS) (10^-4M), also a non-selective P2 antagonist, had no effect (n = 4, 5 respectively). Currents elicited by UTP (n = 37) or UDP (n = 14) were unaffected by either antagonist. Contractions of SPA evoked by ATP were partially inhibited by PPADS (n = 4) and abolished by suramin (n = 5). Both antagonists abolished the contractions in LPA.

Conclusion

At least two P2Y subtypes couple to I_Cl,Ca in smooth muscle cells of rat SPA and LPA, with no apparent regional variation in their distribution. The suramin-sensitive, PPADS-resistant site activated by ATP most resembles the P2Y₁₁ receptor. However, the suramin- and PPADS-insensitive receptor activated by UTP and UDP does not correspond to any of the known P2Y subtypes. These receptors likely play a significant role in nucleotide-induced vasoconstriction.