P2 receptor-mediated modulation of neurotransmitter release -- an update Presynaptic nerve terminals are equipped with a number of presynaptic auto - and heteroreceptors , including ionotropic P2X and metabotropic P2Y receptors . P2 receptors serve as modulation sites of transmitter release by ATP and other nucleotides released by neuronal activity and pathological signals . A wide variety of P2X and P2Y receptors expressed at pre - and postsynaptic sites as well as in glial cells are involved directly or indirectly in the modulation of neurotransmitter release . Nucleotides are released from synaptic and nonsynaptic sites throughout the nervous system and might reach concentrations high enough to activate these receptors . By providing a fine-tuning mechanism these receptors also offer attractive sites for pharmacotherapy in nervous system diseases . Here we review the rapidly emerging data on the modulation of transmitter release by facilitatory and inhibitory P2 receptors and the receptor subtypes involved in these interactions . Introduction Ionotropic P2X receptors and metabotropic P2Y receptors are the principal cell surface proteins , conveying the action of extracellular ATP , the ubiquitous signaling substance . P2X receptors are ligand-gated cation channels , composed of three individual subunits , whereas P2Y receptors belong to the superfamily of G protein-coupled receptors -LRB- GPCRs -RRB- , with seven transmembrane domains . Various subtypes of P2X and P2Y receptor families are expressed throughout the brain and involved in a wide array of functions from fast synaptic transmission to long-term plasticity and trophic changes important for development , neuron-glia interactions , and neuroimmunomodulation . In addition , ATP modulates synaptic transmission pre - and postsynaptically , both in a positive and negative direction via activation of P2X and P2Y receptors , respectively . The presynaptic nerve terminal is an important regulatory site , whereby the efficacy of synaptic transmission could be locally and efficiently controlled . Accordingly , axon terminals in the central nervous system and in the periphery are equipped with a wide variety of auto - and heteroreceptors -LSB- 1 -- 4 -RSB- . Whereas presynaptic metabotropic receptors convey negative feedback regulation of transmitter release , presynaptic ionotropic receptors could amplify synaptic transmission . Moreover the activation of ligand-gated cation channels with high Ca2 + permeability could directly elicit transmitter release triggered by the Ca2 + influx through the receptor-ion channel complex -LSB- 2 , 4 -RSB- . Coactivation of different presynaptic receptors provides a fine-tuning mechanism whereby different neurotransmitters and modulators can mutually influence the activity of each other . Presynaptic and extrasynaptic receptors controlling transmitter release also offer attractive target sites for existing and future pharmacotherapy , as they may modify the normal and pathological synaptic information processing without all-or-none actions -LSB- 3 -RSB- . Since ATP and its related nucleotides are ubiquitous signaling molecules , it is not surprising that their receptors , i.e. , ionotropic P2X and metabotropic P2Y receptors , participate both in the negative and positive feedback modulation of neurotransmitter release . Although the principal function proposed for ATP-sensitive P2 receptors was that they mediate the fast transmitter action of extracellular ATP in neuro-neuronal and neuro-effector synapses in the nervous system , it was already recognized in the early 1990s that they are also involved in the regulation of transmitter release -LSB- 5 , 6 -RSB- . It was subsequently revealed that the release of the major neurotransmitters of the brain and the peripheral neurons -LSB- acetylcholine -LRB- ACh -RRB- , noradrenaline -LRB- NA -RRB- , dopamine -LRB- DA -RRB- , serotonin , glutamate , γ-aminobutyric acid -LRB- GABA -RRB- -RSB- are modulated by P2X and/or P2Y receptors . In 2000 , Cunha and Ribeiro reviewed the literature on the presynaptic modulator role of ATP and suggested that there is a mismatch between the abundance of P2 receptor expression , the robust release of ATP in almost all parts of the central nervous system -LRB- CNS -RRB- and peripheral nervous system -LRB- PNS -RRB- , and the relative paucity of identified P2 receptor-mediated synapses , which implicates the major role of ATP as a neuromodulator , rather than a classic transmitter -LSB- 7 -RSB- . The focus of this mini-review is the facilitatory and inhibitory modulation of neurotransmitter release by different subtypes of P2X and P2Y receptors , irrespective of their localization , i.e. , whether they are pre - , post - , or extrasynaptic . Therefore , in addition to a brief summary of the determining factors of ATP availability in synapses , the structure , pharmacology , signal transduction , and distribution of P2X and P2Y receptors in the nervous system , available information on the release-modulating P2 receptors , and the receptor subtypes involved in these interactions will be detailed and updated . Determining factors of ATP availability in synapses The participation of ATP and related nucleotides in the regulation of neurotransmitter release presumes their accumulation in the extracellular space upon ongoing neuronal activity . Extracellular purine availability in the nervous system is basically determined by the balance of release and removal by enzymatic degradation and uptake . Sources and stimuli that trigger ATP release Since ATP is ubiquitous , all metabolically active cells of the nervous system provide a potential pool for its release . Therefore , besides the nerve terminals themselves , the cellular source of released purines participating in the modulation of neurotransmitter release could be any cell type located in contact with nerve terminals , i.e. , astrocytes , microglia , and endothelia . A wide variety of stimuli are known to release ATP to the extracellular space , which could lead to purine levels sufficiently high to activate nucleotide receptors expressed on the surface of pre - and postsynaptic membranes -LSB- 8 , 9 -RSB- . Although the stimulation-dependent release of ATP upon conventional -LSB- 10 , 11 -RSB- and high-frequency -LRB- e.g. , -LSB- 12 -RSB- -RRB- neuronal activity is well documented , these stimuli probably result in a spatially restricted , localized increase in extracellular purine levels , which serve the fast synaptic transmission and its modulation within the synaptic cleft . Furthermore , ATP-metabolizing ectoenzymes , present on the nerve terminal membrane , and glial cells -LSB- 13 -RSB- , such as ectoNTPDases , and the CD39/ecto -5 ′ nucleotidase -LSB- 14 -RSB- , may strongly limit nucleotide availability under these conditions . On the other hand , pathological events are known also to stimulate purine release . These signals include mechanical -LSB- 15 -- 17 -RSB- , chemical -LSB- 18 -RSB- , and hypotonic stimuli -LSB- 19 -RSB- , hypoxia/hypoglycemia/ischemia and consequent energy deprivation -LSB- 20 -- 25 -RSB- , inflammatory signals , such as bacterial lipopolysaccharide -LRB- LPS -RRB- -LSB- 26 , 27 -RSB- , interleukin-1β -LRB- IL-1β -RRB- -LSB- 28 -RSB- , and cellular injury . The pathological ATP release might result in a purine-rich extracellular milieu leading to a more widespread activation of receptors reaching also the extrasynaptic receptors on the neighboring nerve terminals or distant cells such as astrocytes . Therefore , P2 receptors could play a role in the modulation of not only neuronal but also astrocytic transmitter release . Finally , nucleotides and nucleosides may promote further release of purines , by a homo - or heteroexchange mechanism , if they reach a relatively high concentration in the extracellular space -LSB- 29 -RSB- . Mechanisms of ATP release Since ATP is a highly polarized molecule , which can not pass freely the cell membrane being released to the synaptic cleft , it could enter the extracellular space by the following mechanisms : -LRB- 1 -RRB- vesicular exocytosis , -LRB- 2 -RRB- carrier-mediated release , -LRB- 3 -RRB- release through channels and membrane pores , and -LRB- 4 -RRB- cytolytic release . -LRB- 1 -RRB- Vesicular exocytosis is a prototype mechanism for neurotransmitters and neuromodulators to enter the extracellular space , which is expected to be a -LSB- Ca2 + -RSB- o-dependent process . Indeed , ATP is taken up and stored in synaptic vesicles of nerve terminals -LSB- 8 -RSB- and astrocytes -LSB- 15 -RSB- and -LSB- Ca2 + -RSB- o-dependent ATP release in response to neuronal stimulation appears in many areas of the central and peripheral nervous system -LRB- for further references see -LSB- 8 , 9 , 24 , 30 , 31 -RSB- -RRB- . Moreover , recent findings indicate that vesicular ATP could be released not only from nerve terminals but also from neuronal somata -LSB- 32 -RSB- and astrocytes -LSB- 15 -RSB- . -LRB- 2 -RRB- Although specific transporters capable of transmembrane movement of ATP are yet to be molecularly identified in the nervous system , ABC -LRB- ATP binding cassette -RRB- proteins have been implicated as ATP transporters -LSB- 19 , 33 , 34 -RSB- in non-neuronal cells . These transporters are also expressed in glial cells -LSB- 35 -RSB- and mediate ATP release upon hypo-osmotic challenge -LSB- 36 , 37 -RSB- . -LRB- 3 -RRB- Channels and pores , such as connexin hemichannels -LSB- 38 -RSB- , are also potential candidates to drive the transmembrane movement of ATP . They have been identified to mediate ATP release from astrocytes and other non-neuronal cells in response to mechanical stress -LSB- 39 , 40 -RSB- and other stimuli -LSB- 31 , 41 -RSB- . -LRB- 4 -RRB- Although only scarcely supported by direct experimental proof -LSB- 42 -RSB- , the general assumption is that any kind of cellular injury could result in high local ATP concentrations in the extracellular space . In this case the millimolar cytoplasmic ATP is expected to leak out of the cell through the membrane damage . Metabolism of ATP in the extracellular space Several enzyme families are responsible for the extracellular degradation of ATP in the nervous system . The first step of the inactivation of ATP is mediated by the family of ectonucleoside triphosphate diphosphohydrolases -LRB- ENTPDases , EC 3.6.1.5 , also known as ectoATPase or apyrase -RRB- , which are able to hydrolyze ATP and adenosine diphosphate -LRB- ADP -RRB- to AMP -LSB- 14 -RSB- . Among these enzymes ENTPDase 1 , 2 , 3 , and 8 are present in the brain -LSB- 43 , 44 -RSB- , having low micromolar Km for ATP and ADP giving rise to rapid and highly effective hydrolysis of ATP in almost all neuronal tissues . In addition to the ENTPDase family , ATP and other nucleotides could also be dephosphorylated by ectonucleotide pyrophosphatases -LRB- ENPPs -RRB- and by alkaline phosphatases , both having broader substrate specificity , but also widespread tissue distribution -LSB- 14 -RSB- . The final step of extracellular inactivation is the hydrolysis of AMP by the ecto-5 ′ - nucleotidase -LRB- EC 3.1.3.5 -RRB- enzyme , which is the rate-limiting step giving rise to the formation of adenosine that acts on P1 receptors , which include A1 , A2A , A2B , and A3 receptor subtypes . Thus , endogenous ATP is converted to adenosine to activate A1 adenosine receptors within a second in the hippocampus -LSB- 45 , 46 -RSB- , whereas the hydrolysis of ATP seems slower in other brain regions , such as the cerebral cortex -LSB- 47 -RSB- . Ectoenzymes therefore have an important role in the substrate delivery to different subtypes of P2X and P2Y receptors . In spite of its short half-life , effective concentrations of nucleotides can be reached in the synapse for the activation of ionotropic P2X receptors and metabotropic P2Y receptors . Structure , pharmacology , and signal transduction of P2X receptors Ionotropic P2X receptors are nonselective cation channels consisting of at least three subunits . P2X receptor subunits are 379-595 amino acid long polypeptide chains , having two transmembrane domains -LRB- TM1 and TM2 -RRB- and a large extracellular loop -LSB- 48 , 49 -RSB- . Until now seven members of this receptor family have been identified molecularly , which are numbered from P2X1 to P2X7 , and have individual kinetics and pharmacological phenotype -LSB- 50 -RSB- . These receptor proteins coassemble into various homo - or heterooligomeric assemblies to form functional receptors . Among possible combinations so far 16 variations have been proved to be functional -LSB- 51 -RSB- . These are all of the homooligomeric receptors , except P2X6 , which does not function in homooligomeric form , and the rest are heterooligomers , formed from P2X1-P2X6 subunits . However , recently it has been reported that by N-glycosylation even the homomeric P2X6 receptor could be rendered functional -LSB- 52 -RSB- . On the other hand , the P2X7 receptor functions only in homooligomeric form and does not coassemble with other known P2X receptor subunits . P2X receptors are permeable to both monovalent -LRB- Na + , K + -RRB- and divalent -LRB- Ca2 + -RRB- cations and the activation of the receptor generates an inward current leading to the local depolarization of the cell membrane ; in addition , the Ca2 + influx through the receptor-ion channel complex could directly trigger transmitter release . Moreover , upon prolonged or repetitive agonist application certain P2X receptors , especially the P2X7 receptor , display pore dilation which makes the channel permeable to high molecular weight cations up to 800 Da . Basically P2X receptors are sensitive to ATP and to its various synthetic analogues but not to AMP and adenosine and the ligand binding profiles of homomeric P2X receptors are well established -LRB- for further information , see -LSB- 50 , 53 -RSB- -RRB- . On the other hand , less is known about the pharmacology of heteromeric receptors ; among them , the pharmacological profile of P2X2/3 , P2X2/6 , P2X1/2 , P2X1/4 , P2X1/5 , and P2X4/6 are described -LSB- 54 -- 59 -RSB- . However , the expression pattern and the ligand binding profile of individual assemblies of P2X receptors are highly overlapping , often creating difficulties in the identification of the P2X receptor subunit composition of receptors expressed in native tissues . Moreover P2X receptor assemblies also share common ligand binding properties with certain members of the P2Y receptor family . Therefore , transgenic mice genetically deficient in individual P2X receptor subtypes are being increasingly used for P2X receptor identification . The distribution of P2X receptors in neuronal structures In situ hybridization studies with specific riboprobes , and immunocytochemical studies using antibodies raised against individual P2X receptor subunits , revealed that all seven P2X receptors are widely expressed in the nervous system . However , the expression of different receptor subunits show species - , region - , and cell type-specific distinct distribution -LSB- 60 -RSB- . Among the P2X receptors , P2X2 , P2X4 , and P2X6 seem to be most abundantly expressed in the brain , whereas other subunits show more restricted localization -LSB- 60 -- 63 -RSB- . The typical localization of the P2X2 receptor is on nerve terminals of the brain and the periphery -LSB- 61 , 64 , 65 -RSB- , although it also appears postsynaptically -LSB- 62 -RSB- . P2X1 receptors had initially been suggested to be exclusively expressed on smooth muscle membrane consistent with its role in mediating fast synaptic transmission at the autonomic neuroeffector junction -LSB- 60 -RSB- . However , recent studies with more sensitive probes revealed that its expression is more widespread , i.e. , it is also present on central and peripheral neurons -LSB- 63 , 66 -RSB- . The same holds true for P2X3 receptors , which are primarily associated with sensory pathways , but functional studies indicate that they are also expressed in other brain regions and autonomic pathways -LSB- 67 -- 69 -RSB- . The P2X4 receptor shows substantial expression in several brain areas such as the cerebral cortex , hippocampus , thalamus , and brainstem -LSB- 70 -RSB- and is associated with postsynaptic specialization of synaptic contacts -LSB- 62 -RSB- . P2X5 subunits have the most restricted localization in the brain , although it shows strong representation in certain areas , e.g. , the nucleus tractus solitarii -LRB- NTS -RRB- -LSB- 71 -RSB- . Finally , P2X7 receptors are also expressed in the brain , especially in reactive microglia and astroglia -LSB- 72 -RSB- and immunoelectron microscopic studies revealed a widespread presynaptic expression of P2X7 receptor immunoreactivity in a number of different brain areas , including the brainstem , hippocampus , cortex , spinal cord , and the skeletal neuromuscular junction -LSB- 73 -- 75 -RSB- . However , two studies , using the same antibodies , demonstrated that P2X7 receptor immunoreactivity is still observable in the brain of P2X7 receptor knockout animals and thereby raised doubts on the validity of previous immunocytochemical observations -LSB- 76 , 77 -RSB- . Therefore , available P2X7 receptor antibodies either recognize a site , which is not the P2X7 receptor , or , as a recent study indicates -LSB- 78 -RSB- , a brain analogue of the P2X7 receptor , which shares its antibody binding domain with the cloned P2X7 receptor and partially retains its functionality in P2X7 receptor knockout animals . Structure , pharmacology , and signal transduction of P2Y receptors P2Y receptors all belong to G protein-coupled receptors , having seven hydrophobic transmembrane domains , and possess their ATP binding site on the external side of TM3 and TM7 domains -LSB- 79 -- 82 -RSB- . The P2Y receptor family has eight individual members , numbered P2Y1 , P2Y2 , P2Y4 , P2Y6 , P2Y11 , P2Y12 , P2Y13 , and P2Y14 . P2Y receptors are basically activated by adenine and uridine nucleotides , such as ATP , ADP , uridine diphosphate -LRB- UDP -RRB- , and uridine triphosphate -LRB- UTP -RRB- , but not by nucleosides and they are classified according to their sensitivity to purines and/or pyrimidines : P2Y1 , 12 and P2Y13 are adenine nucleotide-preferring receptors ; P2Y6 is preferred by uridine nucleotides ; P2Y2 , 4 and P2Y11 are receptors with mixed selectivity ; whereas P2Y14 is activated by UDP-glucose , UDP-galactose , UDP-N-acetylglucosamine , and UDP-glucuronic acid -LSB- 83 -RSB- . Although a minority of P2Y receptor subtypes are incompletely characterized and the pharmacological profiles of individual P2Y receptors are partially overlapping , ligands are available which display some selectivity to certain subtypes of the P2Y receptor family -LSB- for further information see 84 , 85 -RSB- . Nevertheless , identification of individual P2Y receptors requires careful pharmacological analysis and the use of receptor knockout animals , if available , and exclusion of the involvement of adenosine receptors in the effect of nucleotides used as P2Y agonists , because they may be metabolized to adenosine -LRB- see , e.g. , -LSB- 45 , 47 -RSB- -RRB- . In addition , the potential heteromerization of P2Y receptor subtypes with each other and with A1 adenosine receptors -LSB- 86 -RSB- should also be taken into account when individual P2Y receptors are identified . Whereas P2X receptors convey rapid changes in the neuronal excitability on the millisecond timescale , P2Y receptors act on a longer , second timescale , appropriate for the fine-tuning of synaptic transmission . As for the signal transduction pathways activated by various subtypes of P2Y receptors , P2Y1 , P2Y2 , P2Y4 , P2Y6 , and P2Y11 receptors are coupled via Gq/11 proteins to stimulate phospholipase C , followed by increases in inositol phosphates and mobilization of Ca2 + from intracellular stores ; in addition , the P2Y11 receptor mediates an increase in adenyl cyclase activity -LSB- 84 -RSB- . On the other hand , P2Y12 , P2Y13 , and P2Y14 receptors are coupled via Gi/o proteins to inhibit adenyl cyclase activity followed by a decrease in intracellular cAMP levels -LSB- 84 -RSB- . The activation of Gi protein-coupled P2Y receptors leads to the voltage-dependent inhibition of N-type voltage-sensitive Ca2 + channels directly or indirectly and subsequent inhibition of neurotransmitter release -LSB- 85 -RSB- . The inhibition of voltage-sensitive Ca2 + currents has also been demonstrated for those P2Y receptors , which are coupled to the Gq/11 proteins -LSB- 85 -RSB- ; however , this inhibition is voltage independent -LSB- 85 -RSB- . In addition , the Gi/o-coupled P2Y receptors are known to activate voltage-sensitive GIRK K + channels via direct interaction of Kir3 channel protein -LSB- 85 -RSB- , which hyperpolarizes the neuronal membrane . The distribution of P2Y receptors in neuronal structures mRNA encoding all known P2Y receptors , i.e. , P2Y1 , P2Y2 , P2Y4 , P2Y6 , P2Y12 , P2Y13 , and P2Y14 are present in the brain -LSB- 81 , 87 -- 89 -RSB- . Although our knowledge of their cell-specific localization at the protein level is still incomplete , it appears that a number of them , such as P2Y1 , P2Y2 , and P2Y6 receptors , are expressed both on neurons and astrocytes -LSB- 90 -- 96 -RSB- , whereas others are not exclusively , but predominantly localized to astrocytes -LRB- P2Y13 : -LSB- 97 -RSB- P2Y14 : -LSB- 98 -RSB- -RRB- , oligodendrocytes -LRB- P2Y12 : -LSB- 99 -RSB- -RSB- , or microglia -LRB- P2Y12 : -LSB- 100 -RSB- -RRB- . P2Y receptor mRNA and protein can be detected in a number of different structures , including sympathetic and parasympathetic and sensory nerve terminals , basal ganglia , brainstem , cerebellum , cortex , hypothalamus , and hippocampus -LRB- for further references , see -LSB- 85 -RSB- -RRB- . However , immunocytochemical data should be handled with caution due to the lack of verification of the specificity of many of the available antibodies . For detailed information on the distribution of individual P2Y receptor mRNAs and proteins , we refer to recent reviews on this particular topic -LSB- 85 , 101 -RSB- . Modulation of neurotransmitter release by facilitatory P2 receptors ACh PNS P2X receptors Since P2X receptors have relatively high Ca2 + permeability -LSB- 102 , 103 -RSB- , this property makes them capable of initiating neurotransmitter release by Ca2 + influx through the receptor-ion channel complex or facilitating Ca2 + - dependent neurotransmitter release , provided that they are located nearby the release sites -LRB- Table 1 -RRB- . The first report suggesting that P2 receptors are involved in the facilitatory modulation of neurotransmitter release stems from 1991 when we found that opposite to the well-known inhibitory action of adenosine , α,β-methylene ATP , a metabolically stable analogue of ATP , enhanced electrically evoked acetylcholine release from the myenteric plexus of guinea pig and facilitated the related contractile response -LSB- 6 -RSB- . This effect was not blocked by the antagonists of adenosine receptors , and therefore was proposed to be mediated by ATP-sensitive P2 receptors -LSB- 6 -RSB- . In the same time Fu and Poo observed that ATP potentiates the spontaneous secretion of acetylcholine from developing neuromuscular synapses in Xenopus cell culture by promoting Ca2 + influx through the plasma membrane . However , this effect was not recognized to be the result of P2 receptor activation -LSB- 104 -RSB- . Later on , P2 receptor-mediated facilitation of acetylcholine release was confirmed by electrophysiological recordings in chicken ciliary ganglion -LSB- 105 -RSB- and mouse motor nerve terminals -LSB- 106 -RSB- . Presynaptic P2X receptors , involved in the facilitation of acetylcholine release , have also been identified in developing and adult neuromuscular synapses of Xenopus -LSB- 107 -RSB- and rat -LSB- 108 -RSB- . Homomeric P2X7 receptors are inserted into the membrane of mouse motor nerve terminals and their activation elicits vesicular exocytosis -LSB- 74 , 109 -RSB- . However , there is no report about the presence of other subunit compositions of P2X receptors at the neuromuscular junction and it is also unclear whether such facilitatory receptors also exist on the terminals of central cholinergic neurons . Table 1Facilitatory modulation of neurotransmitter release in the PNS and CNSPreparationMeasured effectProposed receptorReferenceAcetylcholine Guinea pig ileumNT quantificationP2 -LSB- 6 -RSB- Chicken ciliary ganglionPresynaptic currentP2X -LSB- 105 -RSB- Xenopus NMJsEPP/mEPPP2 -LSB- 104 , 107 -RSB- Mouse NMJEPP/mEPPP2 -LSB- 106 -RSB- Mouse NMJVesicular destaining , EPSCP2X7-like -LSB- 74 , 109 -RSB- Rat NMJNT quantificationP2X -LSB- 108 -RSB- Noradrenaline Rabbit ear arteryEJPP2 -LSB- 110 -RSB- PC12 cellsNT quantificationP2 -LSB- 111 -RSB- Guinea pig ileumNT quantificationP2 -LSB- 6 -RSB- Rat vas deferensNT quantificationP2X1 , P2X3 , P2X2/3 -LSB- 113 -RSB- Guinea pig atriumNT quantificationP2X3 , P2X2/3 -LSB- 114 , 115 , 118 -RSB- Human and porcine heartNT quantificationP2X -LSB- 119 -RSB- Sympathetic neuronsNT quantificationP2X2 -LSB- 116 -RSB- LC neuronsAP dischargeP2X -LSB- 120 -RSB- Rat hippocampusNT quantificationP2X1 , P2X3 -LSB- 68 -RSB- Serotonin Rat hippocampusNT quantificationP2 -LSB- 121 -RSB- Dopamine Rat striatumNT quantificationP2Y -LSB- 122 , 123 -RSB- Rat nucleus accumbensNT quantificationP2 -LSB- 124 -- 128 -RSB- Glutamate Rat brainstemmEPSC/NT quantificationP2X1 -LSB- 131 , 157 -RSB- Rat NTSEPSCP2X3 , P2X2/3 -LSB- 129 , 130 , 132 -RSB- Rat hippocampusNT quantificationP2X1 , P2X3 , P2X2/3 -LSB- 69 -RSB- Rat hippocampusNT quantificationP2X7 -LSB- 75 -RSB- Mouse hippocampusNT quantificationP2X7 -LSB- 137 -RSB- Rat hippocampusEPSCP2X2 -LSB- 133 -RSB- Rat hippocampusEPSCP2X7 -LSB- 134 -RSB- Rat hippocampal neuronsEPSCP2 -LSB- 135 -RSB- Rat cortical synaptosomesNT quantificationP2X7 -LSB- 136 -RSB- Cultured astrocytesNT quantificationP2X7 -LSB- 142 -RSB- Retinal Müller glial cellsNT uptakeP2X7 -LSB- 143 -RSB- Rat spinal cordEPSC/mEPSCP2X3 , P2X1/5 , P2X4/6 -LSB- 144 -- 148 -RSB- Rat nucleus accumbensNT quantificationP2 -LSB- 149 -RSB- Rat medial habenulaEPSCP2Y4 -LSB- 150 -RSB- Cultured astrocytesNT quantificationP2Y1 -LSB- 151 -RSB- Cultured Schwann cellsNT quantificationP2 -LSB- 152 -RSB- GABA Midbrain synaptosomesNT quantificationP2X3 , dinucleotide R -LSB- 153 -RSB- Cultured dorsal horn neuronsIPSCP2X -LSB- 154 -RSB- Cultured hippocampal cellsIPSCP2 -LSB- 156 -RSB- Cultured cortical cellsNT quantificationP2X7 -LSB- 155 -RSB- Rat brainstemIPSCP2X1 -LSB- 157 -RSB- Rat , mouse , and guinea pig hippocampusNT quantification/IPSCP2X7 -LRB- indirect -RRB- -LSB- 75 , 137 , 158 -RSB- Cultured astrocytesNT quantificationP2X7 -LSB- 159 -RSB- Rat hippocampusIPSCP2Y1 -LSB- 163 , 206 -RSB- Glycine Rat dorsal hornIPSCP2X -LSB- 161 -RSB- Rat trigeminal nucleussIPSCP2X -LSB- 162 -RSB- AP action potential , EJP excitatory junction potential , EPP end plate potential , EPSC excitatory postsynaptic current , IPSC inhibitory postsynaptic current , mEPP miniature EPP , mEPSC miniature EPSC , NMJ neuromuscular junction , NT neurotransmitter , sEPP spontaneous EPP , sIPSC spontaneous IPSC Monoamines -LRB- NA , serotonin , DA -RRB- PNS P2X receptors The presynaptic facilitatory action of ATP on noradrenergic transmission was described for the first time by Miyahara and Suzuki in rabbit ear artery -LSB- 110 -RSB- . It was followed by the demonstration of the facilitatory effect of ATP and its metabolically stable analogue α,β-methylene ATP on -LSB- 3H -RSB- noradrenaline efflux in PC12 cells -LSB- 111 -RSB- and in the guinea pig ileum -LSB- 6 -RSB- . However , the knowledge on P2 receptors at that time did not allow the identification of P2 receptor subtypes involved in these effects . The issue has been reinvestigated and it was found that sympathetic nerve terminals are equipped with ionotropic P2X receptors , activation of which directly elicits or facilitates noradrenaline release elicited by nerve stimulation -LSB- 1 , 112 -- 115 -RSB- via a direct Ca2 + influx through the receptor-ion channel complex . The pharmacological phenotype of these receptors varies between species , between transmission sites of the sympathetic nervous system , and even between the somata and nerve terminals of an individual neuron . Thus , in cultured sympathetic neurons of the rat an α,β-methylene ATP-insensitive P2X2-like receptor was identified -LSB- 116 -RSB- , whereas in the guinea pig right atrium we found that α,β-methylene ATP stimulates noradrenaline outflow and the pharmacological profile of the underlying receptor was similar to that of P2X3 or P2X2/P2X3 receptors , consistent with the expression of their mRNA in the sympathetic ganglia -LSB- 115 -RSB- . In another study the facilitatory P2X receptors involved in the modulation of noradrenaline outflow in the rat vas deferens were identified as P2X1 , P2X3 , or P2X2/P2X3 receptors -LSB- 113 -RSB- . In contrast , cultured mouse sympathetic nerve terminals do not seem to express facilitatory nucleotide-sensitive receptors -LSB- 117 -RSB- . Importantly , these receptors seem to be endogenously activated by ATP released in response to ongoing neuronal activity -LSB- 115 , 116 -RSB- and by myocardial ischemia in the guinea pig -LSB- 118 -RSB- , porcine , and human heart -LSB- 119 -RSB- and could contribute to ischemia-induced arrhythmia and ischemic heart dysfunction . CNS P2X receptors In the central nervous system , locus coeruleus -LRB- LC -RRB- neurons of the rat are equipped with ATP-sensitive P2X-like receptors , which facilitate the discharge of spontaneous action potentials -LSB- 120 -RSB- . Facilitatory P2X receptors have also been described in the noradrenergic axon terminals innervating the hippocampus , and the homomeric P2X1 and P2X3 receptors were identified as the most likely subunits responsible for this action -LSB- 68 -RSB- . P2Y receptors P2 receptors enhance the release of serotonin from the hippocampus -LSB- 121 -RSB- and that of dopamine from the striatum -LSB- 122 , 123 -RSB- , and the latter effects are thought to be mediated by P2Y receptors . However , the pre - or postsynaptic localization of receptors responsible for these effects were not clarified in these studies . The P2 receptor agonist 2-methyl-thio ATP releases dopamine from the nucleus accumbens through direct and indirect mechanisms -LSB- 124 -- 126 -RSB- in vivo . Interestingly , P2 receptor activation-evoked dopamine release seems to play a role in the modulation of feeding behavior as P2 receptor antagonists inhibit feeding-induced dopamine release and concomitant behavioral changes after food deprivation -LSB- 127 , 128 -RSB- . Excitatory amino acids -LRB- glutamate , aspartate -RRB- CNS P2X receptors In addition to ACh and monoamines , the release of excitatory amino acid transmitters is also modulated by presynaptic P2X receptors in the CNS , as demonstrated partly by neurochemical and partly by electrophysiological methods.Activation of P2X receptors elicits glutamate release in the brainstem -LSB- 129 -- 132 -RSB- , hippocampus -LSB- 69 , 75 , 133 -- 135 -RSB- , and cortical synaptosomes -LSB- 136 -RSB- . As for the underlying receptor subunits involved in these effects , P2X1 -LSB- 69 -RSB- , P2X2 -LSB- 133 -RSB- , P2X3 , and P2X2/3 receptors -LSB- 69 , 130 -RSB- as well as P2X7 -LSB- 75 , 132 , 134 , 136 , 137 -RSB- were identified . The involvement of P2X2 receptors -LSB- 133 -RSB- and P2X7 receptors -LSB- 137 -RSB- has been confirmed by the use of transgenic mice deficient in P2X2 and P2X7 receptors , respectively . Moreover the activation of P2X7 receptors not only elicits glutamate release but also permits the activation of other ligand-gated ion channels on the nerve terminals , such as α7 nicotinic receptors , as demonstrated recently in rat cortical synaptosomes -LSB- 136 -RSB- . The activation of a P2X7-like receptor promotes Ca2 + influx in cortical synaptosomes -LSB- 138 -RSB- and in isolated midbrain synaptic terminals -LSB- 139 -RSB- and activates p38MAP kinase enzyme in the hippocampus -LSB- 140 -RSB- . This latter effect seems to participate in the effect of ATP to elicit glutamate release as it was sensitive to the inhibition by the specific p38MAP kinase inhibitor , SB203580 -LSB- 140 -RSB- . Nevertheless , the exact mechanism whereby the P2X7 receptor and subsequent activation of p38MAP kinase enzyme leads to increased glutamate release awaits further investigation . In addition , it has been reported that the P2X7 receptor agonist BzATP depresses synaptic transmission at the mossy fiber-CA3 synapse -LSB- 141 -RSB- in a p38MAPK-dependent way . However , more recently the participation of P2X7 receptors in this latter effect has been disproved -LSB- 76 , 77 -RSB- . In addition to nerve terminals , P2X7 receptor activation also elicits glutamate release from cultured astrocytes -LSB- 142 -RSB- and inhibits the uptake of glutamate in Müller glial cells of the retina -LSB- 143 -RSB- . The activation of P2X receptors facilitates excitatory transmission in the spinal cord , releasing glutamate from primary afferent fibers terminating in lamina II -LSB- 144 -- 148 -RSB- and lamina V -LSB- 144 -RSB- ; these actions are mediated by P2X3 , P2X1/5 , and P2X4/6 receptors , respectively . Finally , the ability of the P2 receptor ligand 2-methyl-thio ATP to release glutamate has also been demonstrated in vivo in the dopamine-depleted nucleus accumbens -LSB- 149 -RSB- , although the underlying receptor subtype was not identified in this study . P2Y receptors Interestingly , the activation of P2Y receptors is also implicated to elicit and potentiate glutamate release in the central nervous system . In the medial habenula nucleus UDP and UTP increase presynaptic release probability and elicit a non-Hebbian-type long-term potentiation of excitatory transmission , an effect probably mediated by P2Y4 receptors -LSB- 150 -RSB- . In addition , the activation of P2Y1 receptors elicits vesicular glutamate release from astrocytes -LSB- 151 -RSB- and from cultured Schwann cells -LSB- 152 -RSB- . Inhibitory amino acids -LRB- GABA , glycine -RRB- CNS P2X receptors ATP or P1,P5-di -LRB- adenosine-5 ′ -RRB- pentaphosphate -LRB- Ap5A -RRB- elicits an increase in the intrasynaptosomal calcium and induces subsequent GABA release in midbrain GABAergic synaptosomes via activation of P2X3 and a dinucleotide receptor -LSB- 153 -RSB- . The regulation of GABA release by P2X receptors has also been reported in the spinal cord -LSB- 154 -RSB- , cultured cortical -LSB- 155 -RSB- and hippocampal -LSB- 156 -RSB- cells , and the brainstem , where the excitatory and inhibitory synaptic transmission is facilitated via P2X3 and P2X1 receptors , respectively -LSB- 157 -RSB- . In addition to direct modulation of glutamate release , P2X7 receptor activation also releases GABA from the hippocampus through the activation on non-NMDA-type glutamate receptors -LSB- 75 -RSB- . This effect is absent in mice genetically deficient in P2X7 receptors -LSB- 137 -RSB- and mediated by the sodium-dependent reversal of the GABA transporter -LSB- 75 -RSB- . P2X receptor-mediated , TTX-sensitive GABA release has been implicated in the accelerated recovery of guinea pig hippocampal slices from a hypoxic/hypoglycemic insult -LSB- 158 -RSB- . The activation of P2X7 receptors also releases GABA from cultured RBA astrocytes , however , with a different mechanism , by participation of the HCO3 - / Cl - exchanger -LSB- 159 -RSB- . On the other hand , no evidence was found for a direct facilitation of GABA release by P2 receptors in the hippocampal nerve terminal preparation -LSB- 160 -RSB- . Nevertheless , the release of another inhibitory transmitter , glycine , is augmented by P2X receptor activation in the dorsal horn -LSB- 161 -RSB- and in the brainstem trigeminal nucleus -LSB- 162 -RSB- . P2Y receptors In addition to P2X receptors , activation of P2Y1 receptors leads to an increase of the inhibitory postsynaptic current -LRB- IPSC -RRB- frequency in an acute hippocampal slice in a manner dependent on action potential generation , indicating that this effect is related to the activation of receptors present on the somata/dendrites of hippocampal interneurons -LSB- 163 -RSB- . Modulation of neurotransmitter release by inhibitory P2 receptors In addition to facilitatory modulation , P2 receptors are also involved in the inhibitory modulation of the release of various transmitters and the metabotropic P2Y receptors are thought to play a major role in these actions -LRB- Table 2 -RRB- . Table 2Inhibitory modulation of neurotransmitter release in the PNS and CNSPreparationMeasured effectProposed receptorReferenceAcetylcholine Frog ganglionEPSCP2 -LSB- 165 -RSB- Guinea pig ileumNT quantificationP2 -LSB- 164 , 166 , 167 -RSB- Ileal synaptosomesNT quantificationP1 -LSB- 168 -RSB- Guinea pig submucosal neuronsEJPP3 -LSB- 169 -RSB- Frog NMJEPPP2 -LSB- 170 -RSB- Rat submandibular ganglionEPSCP2 -LSB- 171 -RSB- Rabbit retinaNT quantificationP2 -LSB- 172 -RSB- Mouse NMJsEPPP2Y -LSB- 173 -RSB- Noradrenaline Mouse vas deferensNT quantificationP2Y-like -LSB- 5 , 174 , 181 , 182 -RSB- Rat vas deferensNT quantificationP3 -LSB- 178 -RSB- Rat vas deferensNT quantificationP2Y -LSB- 181 -RSB- Rat vas deferensNT quantificationP2Y12 , P2Y13 -LSB- 113 -RSB- Guinea pig vas deferensNT quantificationP2Y-like -LSB- 179 -RSB- Guinea pig saphenous arteryEJPP2 -LSB- 175 -RSB- Rat caudal arteryNT quantificationP3 , P2Y -LSB- 176 , 177 , 180 -RSB- Rat atriumNT quantificationP2Y-like183 Rat irisNT quantificationP2Y-like -LSB- 184 -RSB- Rat kidneyNT quantificationP2 -LSB- 185 -RSB- Rat pancreasNT quantificationP2 -LSB- 186 -RSB- Chick sympathetic neuronsNT quantificationP2 , P2Y12 -LSB- 187 , 188 , 189 -RSB- Bovine chromaffin cellsNT quantificationP2Y12 -LSB- 190 , 194 -RSB- Rat cortexNT quantificationP2Y -LSB- 195 -RSB- Rat hippocampusNT quantificationP2Y -LSB- 196 -RSB- Serotonin Rat cortexNT quantificationP2 -LSB- 197 -RSB- Dopamine Rat striatumNT quantificationP2 -LSB- 198 -RSB- Glutamate Rat cortexEPSCP2 -LSB- 199 -RSB- Rat hippocampusEPSCP2Y -LSB- 200 , but see 201 -RSB- Rat spinal cordPolysynaptic EPSPP2Y -LSB- 204 -RSB- Hippocampal slice cultureEPSCP2Y -LSB- 205 -RSB- EJP excitatory junction potential , EPP end plate potential , EPSC excitatory postsynaptic current , EPSP excitatory postsynaptic potential , NMJ neuromuscular junction , NT neurotransmitter , SEPP spontaneous EPP ACh PNS It has been known for a long time that ATP is involved in the inhibitory presynaptic modulation of cholinergic transmission -LSB- 164 -RSB- . However , it has been the subject of a long-standing debate whether ATP itself is responsible for this effect or its degradation product adenosine -LSB- 165 -- 167 -RSB- , whereas an alternative was that ATP itself acts on adenosine receptors -LSB- 168 -RSB- or activates a putative P3 receptor bearing pharmacological features of both P1 and P2 receptors -LSB- 169 -RSB- . A more definitive proof for the involvement of P2 receptors in the inhibition of acetylcholine release was obtained later in the frog neuromuscular junction -LSB- 170 -RSB- , rat submandibular ganglia -LSB- 171 -RSB- , and rabbit retina -LSB- 172 -RSB- . Presynaptic P2Y receptors responsible for the inhibition of spontaneous acetylcholine release were recently identified at the mouse neuromuscular junction -LSB- 173 -RSB- . In this study the underlying subcellular mechanism of the inhibition of acetylcholine release was also explored : the activation of P2Y receptors is coupled to Gi/o proteins and modulates presynaptic Ca2 + channels related to tonic secretion of acetylcholine -LSB- 173 -RSB- . CNS Inhibitory P2 receptors involved in the modulation of ACh release have been demonstrated in rat cerebral cortex -LSB- 47 -RSB- . By contrast , in the hippocampus , ATP primarily inhibits acetylcholine release through its breakdown to adenosine and subsequent action on A1 adenosine receptors -LSB- 47 -RSB- . Monoamines -LRB- NA , serotonin , DA -RRB- PNS The presence of nucleotide-sensitive inhibitory P2 receptors on postganglionic sympathetic neurons was recognized relatively early -LSB- 5 , 174 -- 179 -RSB- , although initially these receptors were qualified as P2Y-like -LSB- 5 -RSB- or as putative P3 receptors , which are `` hybrid '' receptors between P1 and P2 receptors and sensitive to adenine nucleotides but also to theophylline derivatives -LSB- 176 -- 178 -RSB- . These receptors have been described and characterized in sympathetic nerves innervating the rat caudal artery -LSB- 176 , 177 , 180 -RSB- , guinea pig saphenous artery -LSB- 175 -RSB- , vas deferens -LSB- 5 , 178 , 179 , 181 , 182 -RSB- , atrium -LSB- 183 -RSB- , iris -LSB- 184 -RSB- , kidney -LSB- 185 -RSB- , and pancreas -LSB- 186 -RSB- as well as in cultured sympathetic neurons -LSB- 187 , 188 -RSB- . As for subtype-specific identification , Queiroz et al. -LSB- 113 -RSB- identified presynaptic inhibitory nucleotide receptors on the noradrenergic axon terminals of the rat vas deferens as P2Y12 and/or P2Y13 receptors , whereas on cultured sympathetic neurons -LSB- 189 -RSB- and bovine adrenal chromaffin cells -LSB- 190 -RSB- only P2Y12 receptors have been identified . Interestingly , it appears that mouse sympathetic neurons -LSB- 191 -RSB- and noradrenergic nerves innervating the rat adrenal cortex -LSB- 192 -RSB- do not express an inhibitory P2 receptor . The mechanism of P2Y receptor-mediated inhibition of noradrenaline release has also been explored in several studies : the activation of P2Y receptors inhibits voltage-dependent Ca2 + influx and thereby limits the Ca2 + - dependent vesicular exocytosis and subsequent efflux of noradrenaline to the extracellular space -LSB- 189 , 193 , 194 -RSB- . CNS Similar inhibitory P2Y receptors have also been reported in the CNS in the rat brain cortex -LSB- 195 -RSB- and hippocampus -LSB- 196 -RSB- ; however P2Y receptor subtypes involved were not identified in these early studies . In the CNS , ATP inhibits the release of serotonin -LSB- 197 -RSB- and dopamine -LSB- 198 -RSB- via activation of metabotropic P2 receptors . Excitatory amino acids -LRB- glutamate , aspartate -RRB- CNS ATP and its metabolically stable analogue ATP-γ-S inhibits depolarization-evoked glutamate release from rat brain cortex slices -LSB- 199 -RSB- and inhibits glutamatergic EPSPs in hippocampal CA1 synapses -LSB- 200 -RSB- . Although the underlying receptor was sensitive to theophylline derivatives , the authors proposed that ATP acted through a putative pertussis toxin-sensitive P2Y receptor . However , this hypothesis was challenged by showing the rapid and highly effective hydrolysis of ATP in the hippocampal slices -LSB- 45 , 46 -RSB- and by the demonstration of the complete absence of nucleotide-mediated modulation of excitatory synaptic transmission in the hippocampi of A1 receptor - / - mice -LSB- 201 -RSB- . In a recent study Rodrigues et al. demonstrated that single hippocampal pyramidal neurons do express P2Y1 , P2Y2 , and P2Y4 receptors , and the release of glutamate , measured by a neurochemical technique , is inhibited by these receptors -LSB- 69 -RSB- . The discrepancy between the observations obtained in electrophysiological and neurochemical studies might be explained by the fact that in the former , individual synapses , whereas in the latter , glutamate release from all synapses of the hippocampal slice were simultaneously investigated . Nevertheless , the exact conditions under which the activation of P2Y receptors by endogenous ligands gain significance remain to be identified . Functional data suggest that the release of glutamate in the spinal cord is modulated by inhibitory P2Y receptors . The activation of P2Y receptors causes blockade of the N-type calcium channels in dorsal root ganglion -LRB- DRG -RRB- cells -LSB- 202 -RSB- , and this effect may decrease the release of glutamate from DRG terminals in the spinal cord and thereby partly counterbalance the algogenic effect of ATP -LSB- 203 , 204 -RSB- . This assumption is supported by the findings that the P2Y1/12/13 receptor agonist ADP-β-S inhibits polysynaptic , but not monosynaptic excitatory postsynaptic potentials in the hemisected spinal cord and exhibits antinociceptive potential in the tail flick test -LSB- 204 -RSB- . Recent studies revealed that modulators released from glial cells also regulate neurotransmitter release from nearby nerve terminals by the activation of P2 receptors . Hence mechanical stimulation of astrocytes in hippocampal cell culture leads to the generation of Ca2 + waves in astrocytes , which spread by the release of ATP and subsequent activation of P2 receptors and lead to the depression of excitatory synaptic transmission between neurons -LSB- 205 -RSB- . This glia-driven synaptic depression is partly mediated by ATP itself acting on P2Y receptors and partly by adenosine acting on A1 adenosine receptors -LSB- 205 -RSB- . A similar mechanism has also been demonstrated in intact hippocampal slices , where ATP released from neurons and astrocytes acts on P2Y1 receptors to excite interneurons , resulting in increased synaptic inhibition within intact hippocampal circuits -LSB- 206 -RSB- . On the other hand , to our knowledge there is no information regarding whether the release of GABA and other inhibitory amino acids is subject to modulation via inhibitory P2 receptors . Potential therapeutic utilization of P2 receptors involved in the regulation of neurotransmitter release P2X and P2Y receptors involved in the regulation of neurotransmitter release offer attractive , although not yet utilized sites for pharmacotherapy in nervous system diseases . For instance , facilitatory P2X receptors present on axon terminals could be activated not only during normal neuronal activity , but also during pathological situations , when cellular damage provides an ATP-rich extracellular milieu nearby the receptors . Thus , P2X receptors present on the sympathetic nerve terminals supplying the heart seem to be endogenously activated by ATP by myocardial ischemia -LSB- 118 , 119 -RSB- and could contribute to ischemia-induced arrhythmia and ischemic heart dysfunction . Therefore , inhibition of these facilitatory P2X receptors might have therapeutic relevance in ischemic heart disease . The pathological activation of CNS P2X receptors , regulating the release of glutamate during ischemic-like conditions , was also recently described -LSB- 207 -RSB- . Increased activation of P2X receptors could contribute to ischemia-evoked glutamate release and thereby to glutamatergic excitotoxicity and resultant neuronal death ; therefore , inhibition of these receptors could be a promising approach to treat ischemia-related neurodegenerative diseases . An analogous mechanism could play a role in the spinal cord during the sensitization process leading to various forms of sensory neuropathy ; therefore , attenuation of increased glutamate release from the central terminals of primary sensory neurons by the inhibition of P2X receptors is a potential pathway which could be utilized in neuropathic pain . Since inhibitory P2Y receptors are frequently coexpressed on nerve terminals that are equipped with P2X receptors , activation of P2Y receptors could have a similar effect as the inhibition of P2X receptors . Therefore P2Y receptor agonists might also have therapeutic value in the areas described above . However , one should bear in mind that different subtypes of P2X and P2Y receptors affect various other aspects of physiological and pathological neuronal functions , which could also modify their potential . Conclusion In conclusion , substantial advances have been obtained in the identification and characterization of neurotransmitter release modulating P2 receptors in recent years . It appears that almost all major neurotransmitters of the nervous system are subject to neuromodulation by nucleotide-sensitive P2 receptors . Although there are exceptions to this rule , in general the release of different transmitters is subject to a dual modulation similar to modulation of other transmitters of the CNS and PNS : facilitatory modulation is conveyed by ionotropic P2X receptors , whereas inhibitory modulation is mediated by G protein-coupled metabotropic P2Y receptors . Amongst P2X receptors , P2X1 , P2X2 , P2X3 , P2X2/3 , P2X1/5 , P2X4/6 , and P2X7 receptors were identified to be responsible for facilitatory modulation in different areas of the CNS and PNS . In addition , P2Y receptors -LRB- P2Y1 , P2Y4 -RRB- could also mediate facilitation of transmitter release in certain areas . Inhibitory modulation of neurotransmitter release is mediated by P2Y12 and P2Y13 receptors ; however , individual P2Y receptor subtypes involved in these interactions are far from fully explored yet . It appears that not only neuronal , but also glia-derived ATP play a role in the modulation of neurotransmitter release . The intensity of P2 receptor-mediated modulation , the balance between the facilitatory and inhibitory modulation and the participating individual receptor subtypes , however , varies between individual transmission sites , depending on the expression pattern of P2 receptors and the factors determining the nucleotide levels in the vicinity of release of modulatory P2 receptors . Therefore , further progress is necessary in order to obtain a precise mapping of P2 receptor-mediated modulation of neurotransmitter release . The in vivo relevance of most of the in vitro observations on presynaptic P2 receptors awaits further investigation . Finally , physiological and pathological situations where presynaptic P2 receptors become endogenously activated by released nucleotides need to be identified .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O

neuromodulation
presynaptic
p2x receptor
p2y receptors
release
transmitter


Time sequence of the damage to the acceptor and donor sides of photosystem II by UV-B radiation as evaluated by chlorophyll a fluorescence The effects of ultraviolet-B -LRB- UV-B -RRB- radiation on photosystem II -LRB- PS II -RRB- were studied in leaves of Chenopodium album . After the treatment with UV-B the damage was estimated using chlorophyll a fluorescence techniques . Measurements of modulated fluorescence using a pulse amplitude modulated fluorometer revealed that the efficiency of photosystem II decreased both with increasing time of UV-B radiation and with increasing intensity of the UV-B . Fluorescence induction rise curves were analyzed using a mechanistic model of energy trapping . It appears that the damage by UV-B radiation occurs first at the acceptor side of photosystem II , and only later at the donor side . Introduction The solar spectrum that reaches the earth 's surface includes wavelengths in the visible or photosynthetically active range -LRB- PAR , 400 -- 700 nm -RRB- , in the ultraviolet-A -LRB- UV-A , 320 -- 400 nm -RRB- , ultraviolet-B -LRB- UV-B , 280 -- 320 nm -RRB- and in the ultraviolet-C -LRB- UV-C , 200 -- 280 nm -RRB- ranges . While UV-C is totally absorbed by the atmosphere , ozone is the principal atmospheric attenuator of UV-B radiation -LRB- Cockell and Horneck 2001 -RRB- . The depletion of ozone by the emission into the atmosphere of man-made chlorine - and bromine-containing compounds has been correlated with an increase in the background level of UV-B radiation . UV-B radiation can affect a multitude of physiological and morphological plant processes that ultimately can lead to inhibition of growth and reproduction -LRB- Jansen et al. 1998 -RRB- . Potential molecular targets for direct UV-B damage via photomodification or photosensitization include nucleotides , proteins , lipids and pigments -LRB- Jordan 2002 -RRB- . Besides direct macromolecular damage , UV-B can also induce specific signal transduction pathways and changes in gene expression that stimulates acclimation and repair processes -LRB- Hollósy 2002 ; Brosché and Strid 2003 -RRB- . The photosynthetic process can be affected by UV-B radiation at different levels , including alterations in plant and leaf morphology that decreases light interception -LRB- Jansen 2002 -RRB- , changes in stomatal function that limit the availability of CO2 , changes in photosynthetic pigments -LRB- Strid and Porra 1992 -RRB- , on the expression of photosynthetic genes and on enzymes of the carbon fixation pathway . However , it is the effects of UV-B radiation on light harvesting and primary photochemical reactions of photosynthetic membranes , particularly on the Photosystem II -LRB- PS II -RRB- reaction center , which has attracted much attention and study -LRB- Vass 1997 ; Vass et al. 2005 -RRB- . PS II is one of the reaction centers that , together with the cytochrome b6f complex , the photosystem I -LRB- PS I -RRB- reaction center , and the ATP-synthase , forms the electron transport chain that drives energy transduction in the thylakoid membranes of oxygenic eukaryotes . The photochemical core of PS II is formed by the D1/D2 heterodimer , where the redox electron carriers and cofactors of electron transport are bound . Light absorbed by the antenna system of PS II induces the excitation of a special reaction center chlorophyll -LRB- P680 -RRB- , which is photo-oxidized on the first electron transfer reaction of PS II , with a pheophytin molecule -LRB- Pheo -RRB- acting as the primary electron acceptor and the formation of a radical pair state -LRB- P680 + Pheo − -RRB- . This radical pair state is `` stabilized '' at the reducing -LRB- acceptor -RRB- side of PS II by the electron transfer from Pheo − to a primary D2-bound quinone electron acceptor -LRB- QA -RRB- and then to the secondary D1-bound quinone acceptor -LRB- QB -RRB- , which , upon accumulation of two reducing equivalents , and becoming protonated to plastoquinol , dissociates from the reaction center . At the oxidizing -LRB- donor -RRB- side of PS II , P680 + is reduced by a redox-active tyrosine residue of D1 -LRB- Yz -RRB- which acts as an electron transfer intermediate between P680 + and the oxygen-evolving complex -LRB- OEC -RRB- , the metalloenzyme system composed of a cluster of four Mn ions and inorganic cofactors -LRB- Ca2 + and Cl − -RRB- that oxidizes water into molecular oxygen . Accordingly , the consecutive photochemical formation of the primary radical pair P680 + Pheo − by light absorption and the reduction of P680 + forming the radical YZ + drives the sequential four-step oxidation of the OEC inducing the S-state transitions and ultimately the splitting of the water molecule . For detailed reviews on oxygen evolution and PS II structure and function see , Diner and Babcock -LRB- 1996 -RRB- , Xiong et al. -LRB- 1998 -RRB- , and Van Rensen and Curwiel -LRB- 2000 -RRB- . From the above brief description of the PS II components and the potential molecular targets of UV-B radiation , it is clear that several components of PS II could be directly affected , as actually has been observed -LRB- Vass et al. 1996 -RRB- . Several studies , most of them in vitro , have shown that UV-B can inhibit PS II electron transport by damage to the quinone electron acceptors ' redox function -LRB- Rodrigues et al. 2006 -RRB- , to Yz function , to the OEC , and to the D1 protein . It should be noted that the time-sequence of events that ultimately leads to inactivation of PS II function is not completely clear , as it might involve a direct destruction or impairment of the absorbing molecule , modification of the protein environment to which these redox components are bound as well as a process of energy transfer by the sensitizer species to the damaged site -LRB- Vass 1997 -RRB- . The time sequence of damage to acceptor and donor sides of PS II , respectively has never been measured in a single experiment . In this work , we have studied the effect of UV-B radiation on the function of PS II in leaves of Chenopodium album using both steady-state -LRB- light-adapted -RRB- fluorescence and the time-resolved fast Chl-a fluorescence induction curve . For a review on Chl-a fluorescence , see Govindjee -LRB- 1995 -RRB- . The results were analyzed based on a mechanistic model of energy trapping and electron transport of PS II -LRB- Vredenberg 2000 -RRB- . It appears that damage to the acceptor side of PS II occurs first , and that only later the donor side becomes affected . Materials and methods Plant growth Plants of Chenopodium album L. were grown as described earlier -LRB- Rodrigues et al. 2006 -RRB- . After around 28 days the plants were transferred to a growth cabinet with a constant temperature of 20 °C , 70 % relative humidity and a PAR level of 60 μmol m − 2 s − 1 . The plants were watered daily and after 10 -- 14 days the sampling of leaves for the UV exposure treatments started . Several different batches of plants were used and no significant difference could be detected when comparing results from experiments replicated with plants with similar ages but from different batches . UV-B radiation treatments The plant material used for the exposure to UV light was either fully developed detached leaves or leaf discs -LRB- 0.6 cm2 -RRB- floating on petri-dishes filled with demineralized water with the abaxial leaf surface exposed to the radiation . The control samples were either kept in the darkness or exposed to white light -LRB- PAR -RRB- . In some experiments leaves were vacuum infiltrated with 3 - -LRB- 3,4-dichlorophenyl -RRB- -1,1-dimethylurea -LRB- DCMU -RRB- in 0.5 % ethanol before being exposed to UV-B , with the controls infiltrated with a 0.5 % ethanol solution . UV-B irradiations were performed using a Vilber Lourmat lamp with filter -LRB- VL-115M , Marne-la-Vallée , France -RRB- . This lamp has a peak emission at 312 nm . UV-B irradiance was measured with an optometer -LRB- United Detector Technology Inc , Baltimore , USA -RRB- equipped with a probe specific for UV radiation . Chlorophyll a fluorescence measurements Measurements of Chl-a fluorescence were performed at room temperature on leaf samples that had been dark adapted for 1 h after the exposure to UV-B radiation , unless otherwise stated . Steady-state Chl-a fluorescence was measured with a pulse-amplitude fluorometer -LRB- PAM 101 -- 103 , Walz Inc. , Effeltrich , Germany -RRB- . A weak modulated measuring light -LRB- 1.6 kHz , 650 nm -RRB- was used for the determination of the minimal fluorescence level Fo , followed by a 1 s pulse of saturating light -LRB- 5,500 μmol m − 2 s − 1 -RRB- for the measurement of the maximum fluorescence level Fm . The leaf was then irradiated with a fixed or increasing level of actinic PAR and , at each level , we measured the steady state light-adapted fluorescence -LRB- Fo ′ -RRB- , the maximum fluorescence of a light-adapted state -LRB- Fm ′ -RRB- induced by a saturating light pulse superimposed on the actinic light , and after removing the actinic light and the application during 10 s of far-red light -LRB- 6 μmol m − 2 s − 1 , 720 nm -RRB- , the minimum fluorescence of a light-adapted state -LRB- Fo ′ -RRB- . During the saturating pulses the modulation of the measuring light was automatically changed to 100 kHz . Several fluorescence parameters were calculated from the measured fluorescence levels -LRB- Schreiber et al. 1986 ; Genty et al. 1989 ; Van Kooten and Snel 1990 -RRB- : the potential photochemical efficiency -LRB- quantum yield -RRB- of PS II , expressed as the ratio Fv/Fm with Fv = Fm − Fo ; the coefficient of photochemical quenching , qP , given by -LRB- Fm ′ − Fs -RRB- / Fv ′ with Fv ′ = Fm ′ − Fo ′ ; the efficiency of excitation energy capture by open PS II reaction centers , Fv ′ / Fm ′ ; and non-photochemical quenching , qN , given by 1 − -LRB- Fm ′ − Fo ′ -RRB- / -LRB- Fm − Fo -RRB- -LRB- Walters and Horton 1991 -RRB- . The fast Chl-a fluorescence rise was measured with the Plant Efficiency Analyser -LRB- PEA , Hansatech Ltd , Norfolk , UK -RRB- . Leaf samples were excited with a saturating pulse of red light -LRB- peak at 650 nm , 3,500 μmol m − 2 s − 1 -RRB- and the fluorescence signal recorded at an acquisition interval of 10 μs in the first 2 ms of excitation -LRB- for more details on this instrument see Strasser et al. -LRB- 1995 , 2000 -RRB- : a model describing discrete steps of excitation energy and electron transfer associated with PS II was published by Zhu et al. -LRB- 2005 -RRB- . Due to the limiting response time of the fluorescence detector in the time region below 50 μs , the Fo level was taken as the model-calculated -LRB- see below -RRB- fluorescence level at 10 μs . This procedure gave similar results to the method of extrapolation as proposed by Vredenberg -LRB- 2000 -RRB- . The fast Chl-a fluorescence rise is plotted in a log-time scale either as the absolute measured values or as the relative variable fluorescence -LRB- rFv -RRB- , calculated as -LRB- Fm − Ft -RRB- / -LRB- Fm − Fo -RRB- , with Ft as the fluorescence level at any time . Modeling and interpretation of fast Chlorophyll a fluorescence rise The fast Chl-a fluorescence rise curve was modeled according to Vredenberg et al. -LRB- 2001 -RRB- . The mathematical formulation is based on a three-state model of energy trapping and fluorescence -LRB- Vredenberg 2000 -RRB- and equates the polyphasic fluorescence rise upon a saturating pulse of light to a multi-state transfer pattern of the PS II reaction center . Results and discussion Exposure of the leaves to UV-B radiation caused an increase of Fo and a decrease of Fm -LRB- Fig. 1B -RRB- , resulting in a decrease of the Fv/Fm value -LRB- Fig. 1A -RRB- . Using a lower intensity of UV-B radiation on grape leaves , Pfündel -LRB- 2003 -RRB- also found a decrease of Fm . However , in his experiments , the Fo first decreased with time , and only increased after about 3 h . The increase of the Fo indicates an increase in the amount of QB-nonreducing centers ; the decrease of the Fv/Fm value indicates that UV-B has a strong damaging effect on the functioning of PS II . Fig. 1Effect of UV-B radiation of variable duration on Fo , Fm , and the Fv/Fm ratio in leaf discs . The UV-B radiation level was 720 -LRB- ■ -RRB- , or 150 -LRB- ◯ -RRB- μmol m − 2 s − 1 , respectively -LRB- PAR level was zero -RRB- ; control samples were kept in darkness . Each value is the mean of four measurements and the bars indicate ± S.D. ; bars not shown are within the symbols Illumination with white light during 6 h decreased the photochemical quenching qP from 1.0 at the lowest intensity to a value of 0.58 at 656 μmol m − 2 s − 1 -LRB- Fig. 2 -RRB- . Curwiel et al. -LRB- 1993 -RRB- , using the same plant material , reported a decrease to about 0.80 after illumination at about 500 μmol m − 2 s − 1 . Up to 100 μmol m − 2 s − 1 , irradiation with UV-B has a stronger effect on qP than white light ; at 200 μmol m − 2 s − 1 and higher , the effect of UV-B is smaller . The efficiency of excitation energy capture by open PS II reaction centers , Fv ′ / Fm ′ , was already decreased strongly after 6 h at 5.4 μmol m − 2 s − 1 of UV-B , while the effect of white light is much smaller ; comparable results were found for the quantum yield of PS II given by -LRB- Fm ′ − Fs -RRB- / Fm ′ . The non-photochemical qN increased from 0 at the lowest intensities to 0.51 -LRB- white light -RRB- and 0.41 -LRB- UV-B -RRB- at the highest irradiation levels . Curwiel et al. -LRB- 1993 -RRB- reported a value of about 0.6 after illumination with white light at about 550 μmol m − 2 s − 1 . Fig. 2Effects of 6 h irradiation , at various levels , of detached leaves on some fluorescence parameters . CO is control ; control is white light -LRB- PAR -RRB- ; UV-B is given by the Vilber lamp -LRB- zero PAR -RRB- . Each value is the average of 4 measurements and the bars indicate ± S.D. ; bars not shown are within the symbols Measurements of Chl-a fluorescence transients of intact leaves with the PEA fluorometer are presented in Fig. 3 . In Fig. 3A the effects of 0 , 1 , 3 , and 6 h irradiation with the Vilber UV-B lamp at 150 μmol m − 2 s − 1 are illustrated . Also in these measurements , the most obvious effects of UV-B are an increase of the Fo level and a decrease in Fm . After infiltration with water -LRB- plus 0.5 % ethanol -RRB- the effects of UV-B appear the same -LRB- compare in Fig. 3B , □ with ■ -RRB- . Compared with infiltration with water , infiltration with DCMU -LRB- ● -RRB- causes a much higher Fo level , and a faster increase to the J-P level ; irradiation with UV-B after DCMU-infiltration -LRB- ◯ -RRB- causes a still higher Fo level and a slower increase to the J-P level . Comparable results were found using another PS II-inhibiting herbicide , atrazine -LRB- not shown -RRB- . The higher Fo level indicates an increased amount of QB-nonreducing centers . Apart from the Fo level , the differences between the curves before and after UV-B treatment are much smaller in the presence of DCMU . This may be related with the finding by Jansen et al. -LRB- 1993 -RRB- that PS II-inhibitors like DCMU , which replace QB from its niche on the D1 protein , inhibit UV-B-driven D1 degradation . Fig. 3Fluorescence induction curves after various treatments of leaves . -LRB- A -RRB- Fluorescence transients after 0 , 60 , 180 , and 360 min irradiation with UV-B light at 150 μmol m − 2 s − 1 . -LRB- PAR is zero -RRB- . -LRB- B -RRB- ■ , After infiltration with 0.5 % ethanol -LRB- CO is control -RRB- , no UV-B ; ● , after infiltration with 10 − 5 M DCMU in 0.5 % ethanol , no UV-B ; □ , after infiltration with 0.5 % ethanol and 6 h irradiation with 150 μmol m − 2 s − 1 UV-B light ; ◯ , after infiltration with 10 − 5 M DCMU in 0.5 % ethanol and 6 h irradiation with UV-B . Each curve is the average of three measurements To study the kinetics of the various parts of the transients , induction curves were normalized -LRB- Fig. 4 -RRB- . While the level of the J transient increases a little after 60 min UV-B , it decreases clearly after 180 and 360 min UV-B ; the kinetics of the O-J increase become lower after UV-B irradiation . In all cases the level of the I transient decreases and the kinetics of the J - -LRB- I -RRB- P rise decreases . From curves like those in Fig. 4 , rate constants of reactions involved in the photochemical trapping in the reaction center of PS II were calculated using the Three-State Trapping Model of PS II -LRB- Vredenberg 2000 ; Vredenberg et al. 2001 -RRB- . The rate constants for QA − oxidation by QB -LRB- kAB1 -RRB- decreased very fast , both at 150 and 720 μmol m − 2 s − 1 ; it is down to about 50 % of control already after 15 min UV-B irradiation -LRB- Fig. 5A -RRB- . The rate constant for QA − oxidation by QB − -LRB- kAB2 -RRB- was down to 5 % of control , already after 15 min UV-B -LRB- not shown -RRB- . This indicates that UV-B irradiation has a fast action on the reducing side of PS II . The rate constants for Yz + reduction by the OEC in S1 and S2 -LRB- k1 and k2 in Fig. 5B and C , respectively -RRB- , were also affected , but much slower . From this we conclude that the damage by UV-B starts at the reducing side , and affects the donor side later . In Fig. 5D it is illustrated that kL -LRB- excitation rate -RRB- increases by UV-B irradiation . This indicates that UV-B radiation decreases the antenna size , or that a lower number of open reaction centers is available after UV-B treatment . Fig. 4Relative variable fluorescence measured after illumination of leaves during various times -LRB- T , in min -RRB- with UV-B light at 150 -LRB- A -RRB- or 720 μmol m − 2 s − 1 -LRB- B -RRB- ; PAR is zero . CO is control . Each curve is the average of three -LRB- A -RRB- or four -LRB- B -RRB- measurementsFig . 5Rate constants of some reactions involved in the photochemical trapping in the reaction center of PS II , calculated using the Three-State Trapping Model of PS II -LRB- Vredenberg 2000 -RRB- . Curves like those presented in Fig. 5 were taken as a source for the calculations ; these curves were measured after various times of irradiation with the Vilber lamp at 150 μmol m − 2 s − 1 -LRB- ○ -RRB- or 720 μmol m − 2 s − 1 -LRB- ■ -RRB- ; PAR was zero . Average of four measurements and the bars indicate ± S.D. ; bars not shown are within the symbols As may be concluded from the results presented in Fig. 5 , UV-B radiation affects rate constants of reactions at both the acceptor and donor sides of PS II . With respect to the acceptor side , Rodrigues et al. -LRB- 2006 -RRB- reported that QA − is a photosensitizer for UV-B radiation ; absorption of UV-B by this semiquinone radical initiates reactions leading to damage to PS II . The group of Vass , most often using isolated plant preparations , has reported several times on the effect of UV-B radiation on the donor side of PS II . In a recent paper -LRB- Szilard et al. 2006 -RRB- , they describe a model where UV-B-induced inhibition of water oxidation is caused either by direct absorption within the catalytic manganese cluster or by damaging intermediates of the water oxidation process . Vass et al. -LRB- 1996 -RRB- measured the effects of UV-B radiation on the OEC , the QAFe2 + complex , the redox-active tyrosines , and the D1 protein in isolated spinach PS II membrane particles . While all these functions were affected , the OEC appeared to be the most sensitive ; the authors concluded that the primary damage by UV-B occurs at the OEC , and that modification and/or inactivation of tyrosine and the quinone acceptor complex are subsequent events . Our work confirms that UV-B radiation affects both acceptor and donor sides of PS II . However , our analysis leads to the conclusion that the acceptor side of PS II is affected first and the donor side later . This different conclusion may be caused by the different material and methods of the two works . Vass et al. -LRB- 1996 -RRB- used PS II particle membranes , while in our work intact leaves were measured . Furthermore , Vass et al. -LRB- 1996 -RRB- measured the effect of UV-B separately on the various PS II functions , while we calculated the effects of UV-B from one single measurement .

O O O O O O O O O O O O B I O O O O O O B I I O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

chenopodium album
chlorophyll a fluorescence
photosystem ii

uv-b radiation
photodamage
photosynthesis