Cyperus esculentus L. and Tetracarpidium conophorum Müll. Arg. Supplemented Diet Improved Testosterone Levels, Modulated Ectonucleotidases and Adenosine Deaminase Activities in Platelets from L-NAME-Stressed Rats.

In hypertensive individuals, platelet morphology and function have been discovered to be altered, and this has been linked to the development of vascular disease, including erectile dysfunction (ED). The impact of nutritional supplementation with Cyperus esculentus (tiger nut, TN) and Tetracarpidium conophorum (walnut, WN) on androgen levels, ectonucleotidases, and adenosine deaminase (ADA) activities in platelets from L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) challenged rats were investigated. We hypothesized that these nuts may show a protective effect on platelets aggregation and possibly enhance the sex hormones, thereby reverting vasoconstriction. Wistar rats (male; 250-300 g; n = 10) were grouped into seven groups as follows: basal diet control group (I); basal diet/L-NAME/Viagra (5 mg/kg/day) as positive control group (II); ED-induced group (basal diet/L-NAME) (III); diet supplemented processed TN (20%)/L-NAME (IV); diet supplemented raw TN (20%)/L-NAME (V); diet supplemented processed WN (20%)/L-NAME (VI); and diet supplemented raw WN (20%)/L-NAME (VII). The rats were given their regular diet for 2 weeks prior to actually receiving L-NAME (40 mg/kg/day) for ten days to induce hypertension. Platelet androgen levels, ectonucleotidases, and ADA were all measured. L-NAME considerably lowers testosterone levels (54.5 ± 2.2; p < 0.05). Supplementing the TN and WN diets revealed improved testosterone levels as compared to the control (306.7 ± 5.7), but luteinizing hormone levels remained unchanged. Compared to control groups, the L-NAME-treated group showed a rise in ATP (127.5%) hydrolysis and ADA (116.7%) activity, and also a decrease in ADP (76%) and AMP (45%) hydrolysis. Both TN and WN supplemented diets resulted in substantial (p < 0.05) reversal effects. Enhanced testosterone levels and modulation of the purinergic system in platelets by TN and WN could be one of the mechanisms by which they aid in vasoconstriction control.

Purinergic Dysfunction in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by increased pulmonary arterial pressure and pulmonary vascular resistance, which result in an increase in afterload imposed onto the right ventricle, leading to right heart failure. Current therapies are incapable of reversing the disease progression. Thus, the identification of novel and potential therapeutic targets is urgently needed. An alteration of nucleotide- and nucleoside-activated purinergic signaling has been proposed as a potential contributor in the pathogenesis of PAH. Adenosine-mediated purinergic 1 receptor activation, particularly A2AR activation, reduces pulmonary vascular resistance and attenuates pulmonary vascular remodeling and right ventricle hypertrophy, thereby exerting a protective effect. Conversely, A2BR activation induces pulmonary vascular remodeling, and is therefore deleterious. ATP-mediated P2X7R activation and ADP-mediated activation of P2Y1R and P2Y12R play a role in pulmonary vascular tone, vascular remodeling, and inflammation in PAH. Recent studies have revealed a role of ectonucleotidase nucleoside triphosphate diphosphohydrolase, that degrades ATP/ADP, in regulation of pulmonary vascular remodeling. Interestingly, existing evidence that adenosine activates erythrocyte A2BR signaling, counteracting hypoxia-induced pulmonary injury, and that ATP release is impaired in erythrocyte in PAH implies erythrocyte dysfunction as an important trigger to affect purinergic signaling for pathogenesis of PAH. The present review focuses on current knowledge on alteration of nucleot(s)ide-mediated purinergic signaling as a potential disease mechanism underlying the development of PAH.

Mitochondria-derived ATP participates in the formation of neutrophil extracellular traps induced by platelet-activating factor through purinergic signaling in cows.

Neutrophil extracellular trap (NET) formation eliminates/prevents the spread of infectious agents. Platelet activating factor (PAF) is involved in infectious diseases of cattle because it recruits and activates neutrophils. However, its ability to induce NET release and the role of metabolism in this process is not known. We investigated if inhibition of glycolysis, mitochondrial-derived adenosine triphosphate (ATP) synthesis and purinergic signaling though P2X1 purinoceptors interfered with NET formation induced by PAF. We inhibited bovine neutrophils with 2-deoxy-d-glucose, rotenone, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and NF449 to evaluate PAF-mediated NET extrusion. PAF induced mitochondrial hyperpolarization and triggered extracellular ATP release via pannexin-1. Inhibition of mitochondrial metabolism prevented extracellular ATP release. Inhibition of glycolysis, complex-I activity and oxidative phosphorylation prevented NET formation induced by PAF. Inhibition of P2X1 purinergic receptors inhibited mitochondrial hyperpolarization and NET formation. We concluded that PAF-induced NET release is dependent upon glycolysis, mitochondrial ATP synthesis and purinergic signaling.

Small Molecules Enhance Scaffold-Based Bone Grafts via Purinergic Receptor Signaling in Stem Cells.

The need for bone grafts is high, due to age-related diseases, such as tumor resections, but also accidents, risky sports, and military conflicts. The gold standard for bone grafting is the use of autografts from the iliac crest, but the limited amount of accessible material demands new sources of bone replacement. The use of mesenchymal stem cells or their descendant cells, namely osteoblast, the bone-building cells and endothelial cells for angiogenesis, combined with artificial scaffolds, is a new approach. Mesenchymal stem cells (MSCs) can be obtained from the patient themselves, or from donors, as they barely cause an immune response in the recipient. However, MSCs never fully differentiate in vitro which might lead to unwanted effects in vivo. Interestingly, purinergic receptors can positively influence the differentiation of both osteoblasts and endothelial cells, using specific artificial ligands. An overview is given on purinergic receptor signaling in the most-needed cell types involved in bone metabolism-namely osteoblasts, osteoclasts, and endothelial cells. Furthermore, different types of scaffolds and their production methods will be elucidated. Finally, recent patents on scaffold materials, as wells as purinergic receptor-influencing molecules which might impact bone grafting, are discussed.

A central role for P2X7 receptors in human microglia.

BACKGROUND: The ATP-gated ionotropic P2X7 receptor (P2X7R) has the unusual ability to function as a small cation channel and a trigger for permeabilization of plasmalemmal membranes. In murine microglia, P2X7R-mediated permeabilization is fundamental to microglial activation, proliferation, and IL-1ß release. However, the role of the P2X7R in primary adult human microglia is poorly understood. METHODS: We used patch-clamp electrophysiology to record ATP-gated current in cultured primary human microglia; confocal microscopy to measure membrane blebbing; fluorescence microscopy to demonstrate membrane permeabilization, caspase-1 activation, phosphatidylserine translocation, and phagocytosis; and kit-based assays to measure cytokine levels. RESULTS: We found that ATP-gated inward currents facilitated with repetitive applications of ATP as expected for current through P2X7Rs and that P2X7R antagonists inhibited these currents. P2X7R antagonists also prevented the ATP-induced uptake of large cationic fluorescent dyes whereas drugs that target pannexin-1 channels had no effect. In contrast, ATP did not induce uptake of anionic dyes. The uptake of cationic dyes was blocked by drugs that target Cl- channels. Finally, we found that ATP activates caspase-1 and inhibits phagocytosis, and these effects are blocked by both P2X7R and Cl- channel antagonists. CONCLUSIONS: Our results demonstrate that primary human microglia in culture express functional P2X7Rs that stimulate both ATP-gated cationic currents and uptake of large molecular weight cationic dyes. Importantly, our data demonstrate that hypotheses drawn from work on murine immune cells accurately predict the essential role of P2X7Rs in a number of human innate immune functions such as phagocytosis and caspase-1 activation. Therefore, the P2X7R represents an attractive target for therapeutic intervention in human neuroinflammatory disorders.

Adenosine binds predominantly to adenosine receptor A1 subtype in astrocytes and mediates an immunosuppressive effect.

The four kinds of adenosine receptor subtypes (ARs), named as ARA1, ARA2A, ARA2B and ARA3, have multiple biological functions. ARs are differently distributed across the body and have distinguished ability of binding adenosine. We try to figure out how these ARs were expressed in astrocytes and which one has the first priority of utilizing adenosine. Firstly, mRNA expressions and membrane localization of all ARs were evaluated by qPCR and western blot. After the membrane localization of all ARs in astrocytes was being confirmed their individual adenosine binding ability was determined by radio-active ligand binding assay respectively. It was revealed that ARA1 had much superior adenosine binding ability than other AR subtypes. Functional study demonstrated that ARA1 potentially mediated an immune suppressive effect in astrocytes. The activation of ARA1 signaling lead to decreased IL-12 and IL-23 production, and decreased chemokine production, including CCL2, CXCL8 and IP-10. When interacted with CD4 cells ARA1 agonist pre-treated astrocytes showed hindered ability of stimulating CD4 cells to secret IL-17 and IFN-γ and inducing CD4 cells' chemo taxi. Finally, in vivo experiment confirmed that local administration of ARA1agonist ameliorated EAE in wild type B6 recipients, but not Ara1-/- recipients. As a conclusion, this paper suggested that adenosine receptor A1 subtype predominantly binds adenosine in astrocytes and mediates an immunosuppressive effect.

Adenosine Signaling through A1 Receptors Inhibits Chemosensitive Neurons in the Retrotrapezoid Nucleus.

A subset of neurons in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors by regulating depth and frequency of breathing in response to changes in tissue CO2/H+. The activity of chemosensitive RTN neurons is also subject to modulation by CO2/H+-dependent purinergic signaling. However, mechanisms contributing to purinergic regulation of RTN chemoreceptors are not entirely clear. Recent evidence suggests adenosine inhibits RTN chemoreception in vivo by activation of A1 receptors. The goal of this study was to characterize effects of adenosine on chemosensitive RTN neurons and identify intrinsic and synaptic mechanisms underlying this response. Cell-attached recordings from RTN chemoreceptors in slices from rat or wild-type mouse pups (mixed sex) show that exposure to adenosine (1 µM) inhibits chemoreceptor activity by an A1 receptor-dependent mechanism. However, exposure to a selective A1 receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine, DPCPX; 30 nM) alone did not potentiate CO2/H+-stimulated activity, suggesting activation of A1 receptors does not limit chemoreceptor activity under these reduced conditions. Whole-cell voltage-clamp from chemosensitive RTN neurons shows that exposure to adenosine activated an inward rectifying K+ conductance, and at the network level, adenosine preferentially decreased frequency of EPSCs but not IPSCs. These results show that adenosine activation of A1 receptors inhibits chemosensitive RTN neurons by direct activation of a G-protein-regulated inward-rectifier K+ (GIRK)-like conductance, and presynaptically, by suppression of excitatory synaptic input to chemoreceptors.

Purinergic signalling links mechanical breath profile and alveolar mechanics with the pro-inflammatory innate immune response causing ventilation-induced lung injury.

Severe pulmonary infection or vigorous cyclic deformation of the alveolar epithelial type I (AT I) cells by mechanical ventilation leads to massive extracellular ATP release. High levels of extracellular ATP saturate the ATP hydrolysis enzymes CD39 and CD73 resulting in persistent high ATP levels despite the conversion to adenosine. Above a certain level, extracellular ATP molecules act as danger-associated molecular patterns (DAMPs) and activate the pro-inflammatory response of the innate immunity through purinergic receptors on the surface of the immune cells. This results in lung tissue inflammation, capillary leakage, interstitial and alveolar oedema and lung injury reducing the production of surfactant by the damaged AT II cells and deactivating the surfactant function by the concomitant extravasated serum proteins through capillary leakage followed by a substantial increase in alveolar surface tension and alveolar collapse. The resulting inhomogeneous ventilation of the lungs is an important mechanism in the development of ventilation-induced lung injury. The high levels of extracellular ATP and the upregulation of ecto-enzymes and soluble enzymes that hydrolyse ATP to adenosine (CD39 and CD73) increase the extracellular adenosine levels that inhibit the innate and adaptive immune responses rendering the host susceptible to infection by invading microorganisms. Moreover, high levels of extracellular adenosine increase the expression, the production and the activation of pro-fibrotic proteins (such as TGF-ß, α-SMA, etc.) followed by the establishment of lung fibrosis.

Neonatal Maternal Deprivation Enhances Presynaptic P2X7 Receptor Transmission in Insular Cortex in an Adult Rat Model of Visceral Hypersensitivity.

AIMS: Insular cortex (IC) is involved in processing the information of pain. The aim of this study was to investigate roles and mechanisms of P2X7 receptors (P2X7Rs) in IC in development of visceral hypersensitivity of adult rats with neonatal maternal deprivation (NMD). METHODS: Visceral hypersensitivity was quantified by abdominal withdrawal reflex threshold to colorectal distension (CRD). Expression of P2X7Rs was determined by qPCR and Western blot. Synaptic transmission in IC was recorded by patch-clamp recording. RESULTS: The expression of P2X7Rs and glutamatergic neurotransmission in IC was significantly increased in NMD rats when compared with age-matched controls. Application of BzATP (P2X7R agonist) enhanced the frequency of spontaneous excitatory postsynaptic currents (sEPSC) and miniature excitatory postsynaptic currents (mEPSC) in IC slices of control rats. Application of BBG (P2X7R antagonist) suppressed the frequencies of sEPSC and mEPSC in IC slices of NMD rats. Microinjection of BzATP into right IC significantly decreased CRD threshold in control rats while microinjection of BBG or A438079 into right IC greatly increased CRD threshold in NMD rats. CONCLUSION: Data suggested that the enhanced activities of P2X7Rs in IC, likely through a presynaptic mechanism, contributed to visceral hypersensitivity of adult rats with NMD.

Adenosine-dependent phrenic motor facilitation is inflammation resistant.

Phrenic motor facilitation (pMF), a form of respiratory plasticity, can be elicited by acute intermittent hypoxia (i.e., phrenic long-term facilitation, pLTF) or direct application of drugs to the cervical spinal cord. Moderate acute intermittent hypoxia (mAIH; 3 × 5-min episodes of 35-50 mmHg arterial Po2, 5-min normoxic intervals) induces pLTF by a serotonin-dependent mechanism; mAIH-induced pLTF is abolished by mild systemic inflammation induced by a low dose of lipopolysaccharide (LPS; 100 µg/kg ip). In contrast, severe acute intermittent hypoxia (sAIH; 3 × 5-min episodes of 25-30 mmHg arterial Po2, 5-min normoxic intervals) elicits pLTF by a distinct, adenosine-dependent mechanism. Since it is not known if systemic LPS blocks the mechanism giving rise to sAIH-induced pLTF, we tested the hypothesis that sAIH-induced pLTF and adenosine 2A (A2A) receptor-induced pMF are insensitive to mild systemic inflammation elicited by the same low dose of LPS. In agreement with our hypothesis, neither sAIH-induced pLTF nor cervical intrathecal A2A receptor agonist (CGS-21680; 200 µM, 10 µl × 3)-induced pMF were affected 24 h post-LPS. Pretreatment with intrathecal A2A receptor antagonist injections (MSX-3; 10 µM, 12 µl) blocked sAIH-induced pLTF 24 h post LPS, confirming that pLTF was adenosine dependent. Our results give insights concerning the differential impact of systemic inflammation and the functional significance of multiple cascades capable of giving rise to phrenic motor plasticity. The relative resistance of adenosine-dependent pMF to inflammation suggests that it provides a "backup" system in animals lacking serotonin-dependent pMF due to ongoing inflammation associated with systemic infections and/or neural injury.NEW & NOTEWORTHY This study gives novel insights concerning how a mild systemic inflammation impacts phrenic motor plasticity (pMF), particularly adenosine-dependent pMF. We suggest that since this adenosine-dependent pathway is insensitive to systemic inflammation, it represents an alternative or "backup" mechanism of pMF when other mechanisms are suppressed.

P2X1 Receptor-Mediated Ca2+ Influx Triggered by DA-9801 Potentiates Nerve Growth Factor-Induced Neurite Outgrowth.

Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. An increase in intracellular calcium ion level was induced by DA-9801, and chelation of extracellular calcium ions with ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

The microglial ATP-gated ion channel P2X7 as a CNS drug target.

Based on promising preclinical evidence, microglial P2X7 has increasingly being recognized as a target for therapeutic intervention in neurological and psychiatric diseases. However, despite this knowledge no P2X7-related drug has yet entered clinical trials with respect to CNS diseases. We here discuss the current literature on P2X7 being a drug target and identify unsolved issues and still open questions that have hampered the development of P2X7 dependent therapeutic approaches for CNS diseases. It is concluded here that the lack of brain penetrating P2X7 antagonists is a major obstacle in the field and that central P2X7 is a yet untested clinical drug target. In the CNS, microglial P2X7 activation causes neuroinflammation, which in turn plays a role in various CNS disorders. This has resulted in a surge of brain penetrant P2X7 antagonists. P2X7 is a viable, clinically untested CNS drug target. GLIA 2016;64:1772-1787.

Adenosine A1 receptor-mediated suppression of carbamazepine-resistant seizure-like events in human neocortical slices.

OBJECTIVE: The need for alternative pharmacologic strategies in treatment of epilepsies is pressing for about 30% of patients with epilepsy who do not experience satisfactory seizure control with present treatments. In temporal lobe epilepsy (TLE) even up to 80% of patients are pharmacoresistant, and surgical resection of the ictogenic tissue is only possible for a minority of TLE patients. In this study we investigate purinergic modulation of drug-resistant seizure-like events (SLEs) in human temporal cortex slices. METHODS: Layer V/VI field potentials from a total of 77 neocortical slices from 17 pharmacoresistant patients were recorded to monitor SLEs induced by application of 8 mM [K(+) ] and 50 µm bicuculline. RESULTS: Activating A1 receptors with a specific agonist completely suppressed SLEs in 73% of human temporal cortex slices. In the remaining slices, incidence of SLEs was markedly reduced. Because a subportion of slices can be pharmacosensitive, we tested effects of an A1 agonist, in slices insensitive to a high dose of carbamazepine (50 µm). Also in these cases the A1 agonist was equally efficient. Moreover, ATP and adenosine blocked or modulated SLEs, an effect mediated not by P2 receptors but rather by adenosine A1 receptors. SIGNIFICANCE: Selective activation of A1 receptors mediates a strong anticonvulsant action in human neocortical slices from pharmacoresistant patients. We propose that our human slice model of seizure-like activity is a feasible option for future studies investigating new antiepileptic drug (AED) candidates.

Purinergic and Cholinergic Drugs Mediate Hyperventilation in Zebrafish: Evidence from a Novel Chemical Screen.

A rapid test to identify drugs that affect autonomic responses to hypoxia holds therapeutic and ecologic value. The zebrafish (Danio rerio) is a convenient animal model for investigating peripheral O2 chemoreceptors and respiratory reflexes in vertebrates; however, the neurotransmitters and receptors involved in this process are not adequately defined. The goals of the present study were to demonstrate purinergic and cholinergic control of the hyperventilatory response to hypoxia in zebrafish, and to develop a procedure for screening of neurochemicals that affect respiration. Zebrafish larvae were screened in multi-well plates for sensitivity to the cholinergic receptor agonist, nicotine, and antagonist, atropine; and to the purinergic receptor antagonists, suramin and A-317491. Nicotine increased ventilation frequency (fV) maximally at 100 µM (EC50 = 24.5 µM). Hypoxia elevated fV from 93.8 to 145.3 breaths min-1. Atropine reduced the hypoxic response only at 100 µM. Suramin and A-317491 maximally reduced fV at 50 µM (EC50 = 30.4 and 10.8 µM) and abolished the hyperventilatory response to hypoxia. Purinergic P2X3 receptors were identified in neurons and O2-chemosensory neuroepithelial cells of the gills using immunohistochemistry and confocal microscopy. These studies suggest a role for purinergic and nicotinic receptors in O2 sensing in fish and implicate ATP and acetylcholine in excitatory neurotransmission, as in the mammalian carotid body. We demonstrate a rapid approach for screening neuroactive chemicals in zebrafish with implications for respiratory medicine and carotid body disease in humans; as well as for preservation of aquatic ecosystems.

Presynaptic Adenosine Receptor-Mediated Regulation of Diverse Thalamocortical Short-Term Plasticity in the Mouse Whisker Pathway.

Short-term synaptic plasticity (STP) sets the sensitivity of a synapse to incoming activity and determines the temporal patterns that it best transmits. In "driver" thalamocortical (TC) synaptic populations, STP is dominated by depression during stimulation from rest. However, during ongoing stimulation, lemniscal TC connections onto layer 4 neurons in mouse barrel cortex express variable STP. Each synapse responds to input trains with a distinct pattern of depression or facilitation around its mean steady-state response. As a result, in common with other synaptic populations, lemniscal TC synapses express diverse rather than uniform dynamics, allowing for a rich representation of temporally varying stimuli. Here, we show that this STP diversity is regulated presynaptically. Presynaptic adenosine receptors of the A1R type, but not kainate receptors (KARs), modulate STP behavior. Blocking the receptors does not eliminate diversity, indicating that diversity is related to heterogeneous expression of multiple mechanisms in the pathway from presynaptic calcium influx to neurotransmitter release.

Effect of Ginger and Turmeric Rhizomes on Inflammatory Cytokines Levels and Enzyme Activities of Cholinergic and Purinergic Systems in Hypertensive Rats.

Inflammation exerts a crucial pathogenic role in the development of hypertension. Hence, the aim of the present study was to investigate the effects of ginger (Zingiber officinale) and turmeric (Curcuma longa) on enzyme activities of purinergic and cholinergic systems as well as inflammatory cytokine levels in Nω-nitro-L-arginine methyl ester hydrochloride-induced hypertensive rats. The rats were divided into seven groups (n = 10); groups 1-3 included normotensive control rats, hypertensive (Nω-nitro-L-arginine methyl ester hydrochloride) rats, and hypertensive control rats treated with atenolol (an antihypertensive drug), while groups 4 and 5 included normotensive and hypertensive (Nω-nitro-L-arginine methyl ester hydrochloride) rats treated with 4 % supplementation of turmeric, respectively, and groups 6 and 7 included normotensive and hypertensive rats treated with 4 % supplementation of ginger, respectively. The animals were induced with hypertension by oral administration of Nω-nitro-L-arginine methyl ester hydrochloride, 40 mg/kg body weight. The results revealed a significant increase in ATP and ADP hydrolysis, adenosine deaminase, and acetylcholinesterase activities in lymphocytes from Nω-nitro-L-arginine methyl ester hydrochloride hypertensive rats when compared with the control rats. In addition, an increase in serum butyrylcholinesterase activity and proinflammatory cytokines (interleukin-1 and - 6, interferon-γ, and tumor necrosis factor-α) with a concomitant decrease in anti-inflammatory cytokines (interleukin-10) was observed in Nω-nitro-L-arginine methyl ester hydrochloride hypertensive rats. However, dietary supplementation of both rhizomes was efficient in preventing these alterations in hypertensive rats by decreasing ATP hydrolysis, acetylcholinesterase, and butyrylcholinesterase activities and proinflammatory cytokines in hypertensive rats. Thus, these activities could suggest a possible insight about the protective mechanisms of the rhizomes against hypertension-related inflammation.

Major dorsoventral differences in the modulation of the local CA1 hippocampal network by NMDA, mGlu5, adenosine A2A and cannabinoid CB1 receptors.

Recent research points to diversification in the local neuronal circuitry between dorsal (DH) and ventral (VH) hippocampus that may be involved in the large-scale functional segregation along the long axis of the hippocampus. Here, using CA1 field recordings from rat hippocampal slices, we show that activation of N-methyl-d-aspartate receptors (NMDARs) reduced excitatory transmission more in VH than in DH, with an adenosine A1 receptor-independent mechanism, and reduced inhibition and enhanced postsynaptic excitability only in DH. Strikingly, co-activation of metabotropic glutamate receptor-5 (mGluR5) with NMDAR, by CHPG and NMDA respectively, strongly potentiated the effects of NMDAR in DH but had not any potentiating effect in VH. Furthermore, the synergistic actions in DH were occluded by blockade of adenosine A2A receptors (A2ARs) by their antagonist ZM 241385 demonstrating a tonic action of these receptors in DH. Exogenous activation of A2ARs by 4-[2-[[6-amino-9-(N-ethyl-ß-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS 21680) did not change the effects of mGluR5-NMDAR co-activation in either hippocampal pole. Importantly, blockade of cannabinoid CB1 receptors (CB1Rs) by their antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM 281) restricted the synergistic actions of mGluR5-NMDARs on excitatory synaptic transmission and postsynaptic excitability and abolished their effect on inhibition. Furthermore, AM 281 increased the excitatory transmission only in DH indicating that CB1Rs were tonically active in DH but not VH. Removing the magnesium ions from the perfusion medium neither stimulated the interaction between mGluR5 and NMDAR in VH nor augmented the synergy of the two receptors in DH. These findings show that the NMDAR-dependent modulation of fundamental parameters of the local neuronal network, by mGluR5, A2AR and CB1R, markedly differs between DH and VH. We propose that the higher modulatory role of A2AR and mGluR5, in combination with the role of CB1Rs, provide DH with higher functional flexibility of its NMDARs, compared with VH.

Medicinal chemistry of adenosine, P2Y and P2X receptors.

Pharmacological tool compounds are now available to define action at the adenosine (ARs), P2Y and P2X receptors. We present a selection of the most commonly used agents to study purines in the nervous system. Some of these compounds, including A1 and A3 AR agonists, P2Y1R and P2Y12R antagonists, and P2X3, P2X4 and P2X7 antagonists, are potentially of clinical use in treatment of disorders of the nervous system, such as chronic pain, neurodegeneration and brain injury. Agonists of the A2AAR and P2Y2R are already used clinically, P2Y12R antagonists are widely used antithrombotics and an antagonist of the A2AAR is approved in Japan for treating Parkinson's disease. The selectivity defined for some of the previously introduced compounds has been revised with updated pharmacological characterization, for example, various AR agonists and antagonists were deemed A1AR or A3AR selective based on human data, but species differences indicated a reduction in selectivity ratios in other species. Also, many of the P2R ligands still lack bioavailability due to charged groups or hydrolytic (either enzymatic or chemical) instability. X-ray crystallographic structures of AR and P2YRs have shifted the mode of ligand discovery to structure-based approaches rather than previous empirical approaches. The X-ray structures can be utilized either for in silico screening of chemically diverse libraries for the discovery of novel ligands or for enhancement of the properties of known ligands by chemical modification. Although X-ray structures of the zebrafish P2X4R have been reported, there is scant structural information about ligand recognition in these trimeric ion channels. In summary, there are definitive, selective agonists and antagonists for all of the ARs and some of the P2YRs; while the pharmacochemistry of P2XRs is still in nascent stages. The therapeutic potential of selectively modulating these receptors is continuing to gain interest in such fields as cancer, inflammation, pain, diabetes, ischemic protection and many other conditions. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.

Caffeine stimulates locomotor activity in the mammalian spinal cord via adenosine A1 receptor-dopamine D1 receptor interaction and PKA-dependent mechanisms.

Caffeine is a potent psychostimulant that can have significant and widely variable effects on the activity of multiple neuronal pathways. The most pronounced caffeine-induced behavioral effect seen in rodents is to increase locomotor activity which has been linked to a dose-dependent inhibition of A1 and A(2A) receptors. The effects of caffeine at the level of the lumbar spinal central pattern generator (CPG) network for hindlimb locomotion are lacking. We assessed the effects of caffeine to the locomotor function of the spinal CPG network via extracellular ventral root recordings using the isolated neonatal mouse spinal cord preparation. Addition of caffeine and of an A1 receptor antagonist significantly decreased the cycle period accelerating the ongoing locomotor rhythm, while decreasing burst duration reversibly in most preparations suggesting the role of A1 receptors as the primary target of caffeine. Caffeine and an A1 receptor antagonist failed to stimulate ongoing locomotor activity in the absence of dopamine or in the presence of a D1 receptor antagonist supporting A1/D1 receptor-dependent mechanism of action. The use of caffeine or an A1 receptor blocker failed to stimulate an ongoing locomotor rhythm in the presence of a blocker of the cAMP-dependent protein kinase (PKA) supporting the need of this intracellular pathway for the modulatory effects of caffeine to occur. These results support a stimulant effect of caffeine on the lumbar spinal network controlling hindlimb locomotion through the inhibition of A1 receptors and subsequent activation of D1 receptors via a PKA-dependent intracellular mechanism.

Electrical properties of purinergic transmission in smooth muscle of the guinea-pig prostate.

Prostatic smooth muscle develops spontaneous myogenic tone which is modulated by autonomic neuromuscular transmission. This study aimed to investigate the role of purinergic transmission in regulating electrical activity of prostate smooth muscle and whether its contribution may be altered with age. Intracellular recordings were simultaneously made with isometric tension recordings in smooth muscle preparations of the guinea-pig prostate. Immunostaining for P2X1 receptors on whole mount preparations was also performed. In prostate preparations which generated spontaneous slow waves, electrical field stimulation (EFS)-evoked excitatory junction potentials (EJPs) which were abolished by guanethidine (10 µM), α-ß-methylene ATP (10 µM) or pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (PPADS, 10 µM) but not phentolamine (1 µM). Consistently, immunostaining revealed the expression of P2X1 receptors on prostatic smooth muscle. EJPs themselves did not cause contractions, but EJPs could sum to trigger a slow wave and associated contraction. Yohimbine (1 µM) and 3,7-dimethyl-1-propargylxanthine (DMPX, 10 µM) but not propranolol (1 µM) potentiated EJPs. Although properties of EJPs were not different between young and aging guinea-pig prostates, ectoATPase inhibitor ARL 67156 (100 µM) augmented EJP amplitudes by 64.2 ± 29.6% in aging animals, compared to 22.1 ± 19.9% in young animals. These results suggest that ATP released from sympathetic nerves acts on P2X1 purinoceptors located on prostate smooth muscle to evoke EJPs, while pre-junctional α2-adrenergic and adenosine A2 receptors may play a role in preventing excessive transmitter release. Age-related up-regulation of enzymatic ATP breakdown may be a compensatory mechanism for the enhanced purinergic transmission which would cause hypercontractility arising from increased ATP release in older animals.