Stable ATP Analogue Research Articles

Extracellular ATP inhibits excitatory synaptic input on parvalbumin positive interneurons and attenuates gamma oscillations via P2X4 receptors.

P2X4 receptors (P2X4R) are ligand gated cation channels that are activated by extracellular ATP released by neurons and glia. The receptors are widely expressed in the brain and have fractional calcium currents comparable with NMDA receptors. Although P2X4Rs have been reported to modulate synaptic transmission and plasticity, their involvement in shaping neuronal network activity remains to be elucidated. We investigated the effects of P2X receptors at network and synaptic level using local field potential electrophysiology, whole cell patch clamp recordings and calcium imaging in fast spiking parvalbumin positive interneurons (PVINs) in rat and mouse hippocampal slices. The stable ATP analogue ATPγS, selective antagonists and P2X4R knockout mice were used. The P2XR agonist ATPγS reversibly decreased the power of gamma oscillations. This inhibition could be antagonized by the selective P2X4R antagonist PSB-12062 and was not observed in P2X4-/- mice. The phasic excitatory inputs of CA3 PVINs were one of the main regulators of the gamma power. Associational fibre compound excitatory postsynaptic currents (cEPSCs) in CA3 PVINs were inhibited by P2X4R activation. This effect was reversible, dependent on intracellular calcium and dynamin-dependent internalization of AMPA receptors. The results indicate that P2X4Rs are an important source of dendritic calcium in CA3 PVINs, thereby regulating excitatory synaptic inputs onto the cells and presumably the state of gamma oscillations in the hippocampus. P2X4Rs represent an effective target to modulate hippocampal network activity in pathophysiological conditions such as Alzheimer's disease and schizophrenia.

Functional atropine sensitive purinergic responses in the healthy rat bladder

While acetylcholine is regarded to be the main directly contractile transmitter substance in the urinary bladder, interactions with other transmitters likely occur. Presently, the interplay between purinergic and cholinergic signalling was investigated to unravel the involvement of the urothelium and efferent neurons in the functionally important purinergically evoked release of acetylcholine in vitro. Functional characterization of receptor subtypes involved in this interplay was also performed. In vitro organ bath experiments with electrical field stimulation (EFS) or administration of agonist were performed in the absence and presence of the neurotoxin tetrodotoxin (TTX; 5×10-7M) and/or receptor antagonists, in intact and urothelium-denuded full thickness rat bladder strip preparations. Interestingly, functional contractions to ATP (10-6-10-3M) remained unaffected by TTX, but were significantly lowered in the presence of the muscarinic antagonist atropine (10-6M). However, in urothelium-denuded strip preparations, this latter phenomenon was not present and the ATP response remained unaltered. To rule out purinergic interference caused by break-down of ATP, experiments were performed in which the stable ATP-analogue αβMeATP (10-7-10-5M) gave rise to functional atropine-sensitive contractions. Furthermore, contractions to ATP were not affected by P2Y6 purinoceptor blockade (by MRS2578; 10-7, 10-5M), nor were relaxatory responses to ATP sensitive to atropine, PPADS (3×10-5M) or αβMeATP. Lastly, relaxations to ADP (10-6-10-3M) or NECA (10-8-10-5M) were unaltered by the presence of atropine. To conclude, purinergic functional contractile, but not relaxatory, responses are supported by the cholinergic transmitter system in vitro, through non-neuronal mechanisms in the urothelium. Involved purinoceptors are of the P2X-subtype, most likely P2X1 and/or P2X3.

Shedding of Microvesicles from Microglia Contributes to the Effects Induced by Metabotropic Glutamate Receptor 5 Activation on Neuronal Death.

Metabotropic glutamate (mGlu) receptor 5 is involved in neuroinflammation and has been shown to mediate reduced inflammation and neurotoxicity and to modify microglia polarization. On the other hand, blockade of mGlu5 receptor results in inhibition of microglia activation. To dissect this controversy, we investigated whether microvesicles (MVs) released from microglia BV2 cells could contribute to the communication between microglia and neurons and whether this interaction was modulated by mGlu5 receptor. Activation of purinergic ionotropic P2X7 receptor with the stable ATP analog benzoyl-ATP (100 μM) caused rapid MVs shedding from BV2 cells. Ionic currents through P2X7 receptor increased in BV2 cells pretreated for 24 h with the mGlu5 receptor agonist CHPG (200 μM) as by patch-clamp recording. This increase was blunted when microglia cells were activated by exposure to lipopolysaccharide (LPS; 0.1 μg/ml for 6 h). Accordingly, a greater amount of MVs formed after CHPG treatment, an effect prevented by the mGlu5 receptor antagonist MTEP (100 μM), as measured by expression of flotillin, a membrane protein enriched in MVs. Transferred MVs were internalized by SH-SY5Y neurons where they did not modify neuronal death induced by a low concentration of rotenone (0.1 μM for 24 h), but significantly increased rotenone neurotoxicity when shed from CHPG-treated BV2 cells. miR146a was increased in CHPG-treated MVs, an effect concealed in MVs from LPS-activated BV2 cells that showed per se an increase in miRNA146a levels. The present data support a role for microglia-shed MVs in mGlu5-mediated modulation of neuronal death and identify miRNAs as potential critical mediators of this interaction.

ATP-sensitive muscle afferents activate spinal trigeminal neurons with meningeal afferent input in rat - pathophysiological implications for tension-type headache.

BackgroundTension-type headache and other primary headaches may be triggered or aggravated by disorders of pericranial muscles, which is possibly due to convergent or collateral afferent input from meningeal and muscular receptive areas. In rodent models high extracellular concentrations of ATP caused muscle nociception and central sensitization of second order neurons. In a rat model of meningeal nociception we asked if spinal trigeminal activity induced by ATP can be modulated by local anaesthesia of distinct muscles.MethodsOngoing activity was recorded from spinal trigeminal neurons with afferent input from the cranial dura mater, the temporal muscle and neck muscles. The stable ATP analogue α,β-methylene adenosine 5′-triphosphate (α,β-meATP, 10 mM) was injected into the ipsilateral temporal muscle, 30 min later followed by injection of local anaesthetics (lidocaine, 2 %) into the ipsilateral neck muscles and/or the temporal muscle.ResultsInjection of α,β-meATP into the temporal muscle caused progressive increase in ongoing activity of most of the spinal trigeminal neurons within 30 min. Injection of lidocaine into the neck muscles and/or the temporal muscle reduced this activation to previous levels within 10 min.ConclusionsDistinct spinal trigeminal neurons processing meningeal nociceptive information are under the control of convergent afferent input from several pericranial muscles. Blockade of at least one of these inputs can normalize central trigeminal activity. This may explain why therapeutic manipulations of head muscles can be beneficial in primary headaches.

Smoking enhances the proinflammatory effects of nucleotides on cytokine release from human lung.

Nucleotides have effects on immune cells which are complex but generally proinflammatory, and have been suggested to play a role in smoking-related lung diseases. However, there have been no studies directly measuring functional responses to nucleotides in human lungs taken from smokers. We used fragments of post mortem human lung from smokers and non-smokers, incubated them with a range of nucleotides (4–1000 µM) in the presence of lipopolysaccharide (LPS; 10 µg/ml) for 24 hours and measured cytokines (IL-1β, IFNγ, IL-17, TNFα, IL-6, IL-8, IL-2 and IL-10) in the supernatants using multiplex immunoassays. Although the basal cytokine levels in the smokers were generally higher in the smokers than the non-smokers, there were no significant differences in either the basal release or the LPS-stimulated release of any of the cytokines when lungs from smokers and non-smokers were compared. There were no significant effects of ATP, ADP, AMP, UTP, α,β-methylene-ATP, P1, P4-diATP, 2-methylthio-ATP or Bz-ATP on the release of cytokines from the lungs. However, the stable ATP analogue ATPγS increased the release of IL-1β and IFNγ, and the effect was greatly increased in lungs from smokers. In non-smokers but not in smokers ATPγS increased the release of IL-17. Overall these results clearly demonstrate for the first time that in normal human lung a stable ATP analogue can enhance LPS-induced pro-inflammatory cytokine release, and that these effects are greatly altered by a prior history of smoking. This provides strong support for the suggestion that nucleotides are involved in the pathogenesis of smoking-related diseases.

P2X1 receptor-mediated pressor responses in the anesthetized mouse

P2X1 receptors and adrenoceptors are mainly responsible for vasoconstriction in a variety of blood vessels. However, previous studies have shown that α,β-methylene adenosine 5′-triphosphate (α,β-MeATP), a stable analogue of ATP, can induce both pressor and depressor responses in laboratory animals. In this study, the effects of increasing intravenous doses of α,β-MeATP and noradrenaline (NA) (0–30nmol/kg) administered at 20min intervals on systolic (SBP), diastolic (DBP) and mean (MBP) blood pressure in groups of anesthetized mice (n=6) were compared. Both α,β-MeATP and NA caused transient, dose-dependent increases in SBP, DBP and MBP but the effect of α,β-MeATP was more rapid and significantly larger at doses of 10 and 30nmol/kg (P<0.01). At the dose of 30nmol/kg, α,β-MeATP increased SBP, DBP and MBP by 65.8±7.0, 65.7±5.0 and 65.7±5.5mmHg, respectively, compared to increases of 36.8±4.6, 33.3±4.9 and 34.5±4.7mmHg, respectively, produced by NA. These results indicate P2X1 receptors play an important role in BP regulation although purinergic vasoconstriction alone may not explain the more potent pressor response to α,β-MeATP in the anesthetized mouse.

Oxytocin is expressed in epidermal keratinocytes and released upon stimulation with adenosine 5′‐[γ‐thio]triphosphate in vitro

Oxytocin is a neuropeptide produced primarily in the hypothalamus and is best known for its roles in parturition and lactation. It also influences behaviour, memory and mental state. Recent studies have suggested a variety of roles for oxytocin in peripheral tissues, including skin. Here we show that oxytocin is expressed in human skin. Immunohistochemical studies showed that oxytocin and its carrier protein, neurophysin I, are predominantly localized in epidermis. RT-PCR confirmed the expression of oxytocin in both skin and cultured epidermal keratinocytes. We also show that oxytocin is released from keratinocytes after application of adenosine 5'-[γ-thio]triphosphate (ATPγS, a stable analogue of ATP) in a dose-dependent manner. The ATPγS-induced oxytocin release was inhibited by removal of extracellular calcium, or by the P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). These results suggest that oxytocin is produced in human epidermal keratinocytes and is released in response to calcium influx via P2X receptors.

Extracellular ATP and P2Y Receptor Activation Induce a Proinflammatory Host Response in the Human Urinary Tract

Extracellular ATP can be released by many cell types under conditions of cellular stress and signals through activation of purinergic receptors. Bladder uroepithelial cells grown in vitro have previously been shown to release ATP in response to stretch. In the present study, we investigated ATP release from uroepithelial cells infected with bacteria and the effect of ATP on the host cell proinflammatory interleukin 8 (IL-8) response. The human kidney epithelial cell line A498 and the human uroepithelial cell line UROtsa were grown in culture and stimulated by the uropathogenic Escherichia coli (UPEC) IA2 strain or the stable ATP analogue ATP-gamma-S. ATP and IL-8 levels were measured in cell culture medium with a luciferin-luciferase assay and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that UPEC infection of uroepithelial cells for 1 h significantly increased (P < 0.01) the extracellular ATP levels. ATP-gamma-S (10 and 100 microM) stimulated release of IL-8 from UROtsa and A498 cells after 6 and 24 h. Experiments with different purinoceptor agonists suggested that P2Y receptors, and not P2X receptors, were responsible for the ATP-gamma-S-induced IL-8 release. The potency profile further suggested involvement of P2Y(1), P2Y(2), and/or P2Y(11) receptors, and reverse transcription-PCR (RT-PCR) studies confirmed that the cells expressed these receptors. The amount of IL-8 released increased 12-fold in UPEC-infected cells, and apyrase, an enzyme that degrades ATP, reduced this increase by approximately 50%. The present study suggests that enhanced ATP release and P2Y receptor activation during urinary tract infection may represent a novel, non-TLR4-mediated mechanism for production of proinflammatory IL-8 in human urinary tract epithelial cells.

Developmental changes of purinergic control of intestinal motor activity during metamorphosis in the African clawed frog,Xenopus laevis

Little is known about the purinergic regulation of intestinal motor activity in amphibians. Purinergic control of intestinal motility is subject to changes during development in mammals. The aim of this study was to investigate purinergic control of intestinal smooth muscle in the amphibian Xenopus laevis and explore possible changes in this system during the developmental phase of metamorphosis. Effects of purinergic compounds on mean force and contraction frequency in intestinal circular muscle strips from prometamorphic, metamorphic, and juvenile animals were investigated. Before metamorphosis, low concentrations of ATP reduced motor activity, whereas the effects were reversed at higher concentrations. ATP-induced relaxation was not inhibited by the P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) but was blocked by the ecto-nucleotidase inhibitor 6-N,N-diethyl-d-beta,gamma-dibromomethylene ATP (ARL67256), indicating that an ATP-derived metabolite mediated the relaxation response at this stage. Adenosine induced relaxation before, during, and after metamorphosis, which was blocked by the A(1)-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The stable ATP-analog adenosine 5'-[gamma-thio]-triphosphate (ATPgammaS) and 2-methylthioATP (2-MeSATP) elicited contractions in the circular muscle strips in prometamorphic tadpoles. However, in juvenile froglets, 2-MeSATP caused relaxation, as did ATPgammaS at low concentrations. The P2Y(11)/P2X(1)-receptor antagonist NF157 antagonized the ATPgammaS-induced relaxation. The P2X-preferring agonist alpha-beta-methyleneadenosine 5'-triphosphate (alpha-beta-MeATP) evoked PPADS-sensitive increases in mean force at all stages investigated. This study demonstrates the existence of an adenosine A(1)-like receptor mediating relaxation and a P2X-like receptor mediating contraction in the X. laevis gut before, during, and after metamorphosis. Furthermore, the development of a P2Y(11)-like receptor-mediated relaxation during metamorphosis is shown.

Novel Functions of Small Conductance Ca2+-activated K+ Channel in Enhanced Cell Proliferation by ATP in Brain Endothelial Cells

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB), which is essential for maintaining homeostasis of the brain. Net cellular turnover, which results from the balance between cell death and proliferation, is important in maintaining BBB homeostasis. Here we report a novel mechanism that underlies ATP-induced cell proliferation in t-BBEC 117, a cell line derived from bovine brain endothelial cells. Application of 0.1-30 mum ATP to t-BBEC 117 concentration-dependently increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in two phases: an initial transient phase and a later and smaller sustained one. These two phases of [Ca(2+)](i) rise were mainly due to Ca(2+) release and sustained Ca(2+) influx, respectively. The pretreatment with apamin, a selective blocker of small conductance Ca(2+)-activated K(+) channels (SK), significantly reduced both the [Ca(2+)](i) increase and K(+) current induced by ATP. Transcripts corresponding to P2Yx, SK2, and transient receptor potential channels were detected in t-BBEC 117. Knock down of SK2 protein, which was the predominant Ca(2+)-activated K(+) channel expressed in t-BBEC 117, by siRNA significantly reduced both the sustained phase of the [Ca(2+)](i) rise and the K(+) current induced by ATP. Cell proliferation was increased significantly by the presence of the stable ATP analogue ATPgammaS. This effect was blunted by UCL1684, a synthesized SK blocker. In conclusion, in brain endothelial cells ATP-induced [Ca(2+)](i) rise activates SK2 current, and the subsequent membrane hyperpolarization enhances Ca(2+) entry presumably through transient receptor potential channels. This positive feedback mechanism can account for the augmented cell proliferation by ATP.

Altered neurogenic and mechanical responses to acetylcholine, ATP and substance P in detrusor from rat with outlet obstruction

The well-known side effects of anticholinergic compounds used to treat urinary incontinence caused by detrusor overactivity have addressed the interest on other pharmacological intervention. The purpose of the present work was to investigate the possible changes in purinergic and cholinergic components of parasympathetic neurotransmission in obstructed rat bladders with detrusor overactivity, and to examine the effect of the association of suramin, atropine and indomethacin on nerve-mediated responses to electrical field stimulation (EFS). Mechanical responses to exogenous acetylcholine, ATP and substance P were also evaluated. Altered sensitivities to acetylcholine and to the sensory neurotransmitter substance P, but unchanged sensitivity to the stable ATP analogue α,β-methyleneATP were observed in bladders from obstructed rats. Suramin and atropine inhibited purinergic and cholinergic components of the neurogenic responses evoked by EFS in detrusor strips from control and obstructed rats. Interestingly, suramin enhanced the antagonistic effect of atropine on neurogenic responses of detrusor strips at all frequencies of stimulation tested. Our results suggest that the association between an antimuscarinic drug and an antagonist of P2X purinoceptors such as suramin might be helpful to reduce the therapeutic dosage of the antimuscarinic drug, along with its side effects. This approach may be of interest in the therapy of patients with bladder incontinence caused by detrusor overactivity, which do not even respond to a maximal dosage of antimuscarinic drug.

Role of Purinoceptors in Cardiac Function in Rats during Ontogeny

Intravenous injection of exogenous ATP (10(-4) M) to rats aging 21, 56, and 100 days increased the heart rate by the 15th sec postinjection. Stable ATP analogue alpha,beta-methylene-ATP in an equimolar concentration increased the heart rate in rats aging 56 and 100 days (by the 15th second after treatment), but had no effect on 21-day-old animals. alpha,beta-Methylene-ATP in a concentration of 10(-7) M increased the heart rate in 21-day-old rat pups, which attests to high sensitivity of P2 purinoceptors. Administration of ATP and alpha,beta-methylene-ATP after treatment with nonselective purinoceptor antagonist suramin did not increase the heart rate in rats of different age groups. Infusion of ATP and its stable analogue after administration of selective P2Y receptor antagonist reactive blue 2 increased the heart rate in rats of different age groups. These changes reflect activation of P2X receptors in the heart.

Renovascular effects of sympathetic cotransmitters ATP and NPY are age-dependent in spontaneoulsy hypertensive rats

Hypertension is characterized by sympathetic overactivity. Neuropeptide Y (NPY) and ATP are cotransmitters of norepinephrine (NE) and regulate renovascular resistance. The present study analyzes sympathetic nonadrenergic neurotransmission in hypertensive (SH-SP) and normotensive (WKY) rats. In addition, adult and young hypertensive rats were compared to investigate the role of aging on sympathetic nonadrenergic cotransmission in hypertensive disease. Pressor responses to renal nerve stimulations (RNS) and drugs were measured on isolated perfused kidneys of young (8-10 weeks) and adult (18-24 weeks) WKY, and SH-SP rats. RNS evoked contractions at 1 Hz were resistant to blockade by the alpha-adrenoceptor antagonist phentolamine (1 microM) but abolished by the P2 receptor blocker suramin (100 microM). Compared to adult WKY, RNS-induced pressor responses were unchanged in adult SH-SP and young WKY, but significantly greater in young SH-SP rats. The NPY-Y1 receptor antagonist BIBP3226 (1 microM) reduced phentolamine-resistant pressor responses in adult and young WKY, young SH-SP, but not in adult SH-SP rats. In contrast to WKY and young SH-SP rats, exogenously perfused NPY (0.1 microM) was unable to potentiate RNS-induced, phentolamine-resistant pressor responses in adult SH-SP rats. NE and the stable ATP analogue alpha,beta-mATP increased the perfusion pressor response more potently in adult SH-SP than in WKY rats. Neuronally released NPY plays a major role in potentiating RNS-induced nonadrenergic pressor responses in kidneys of WKY and young SH-SP rats. In adult SH-SP rats NPY fails to enhance these responses. In this hypertensive model ageing seems to be associated with a loss of a modulatory role of renal NPY Y1 receptors. Since pressor responses to NE and ATP are higher in SH-SP animals, functional NPY-Y1 receptor downregulation might be an adaptive mechanism.

Adenine triphosphate nucleotides are antagonists at the P2Y12 receptor

The aim of the present study was to characterize the pharmacological profile of the P2Y(12) receptor for several adenine triphosphate nucleotides in view of their possible roles as partial agonists or true antagonists. Two distinct cellular systems were used: P2Y(1) receptor deficient mouse platelets ( platelets) previously shown to express a native and functional P2Y(12) receptor and 1321 N1 astrocytoma cells stably expressing the human P2Y(12) receptor (1321 N1 P2Y(12)). ADP and its structural analogues inhibited cAMP accumulation in a dose-dependent manner in both platelets and 1321 N1 P2Y(12) cells with a similar rank order of potency, 2 methylthio-ADP (2MeSADP) >>ADP - Adenosine 5'-(betathio) diphosphate (AlphaDPbetaS). Commercial ATP, 2 chloro; ATP (2ClATP) and 2 methylthio-ATP (2MeSATP) also inhibited cAMP accumulation in both cell systems. In contrast, after creatine phosphate (CP)/creatine phosphokinase (CPK) regeneration, adenine triphosphate nucleotides lost their agonistic effect on platelets and behaved as antagonists of ADP (0.5 microm)-induced adenylyl cyclase inhibition with IC(50) of 13.5 +/- 4.8, 838 +/- 610, 1280 +/- 1246 microm for 2MeSATP, ATP and 2ClATP, respectively. In 1321 N1 P2Y(12) cells, CP/CPK regenerated ATP and 2ClATP lost their agonistic effect only when CP/CPK was maintained during the cAMP assay. The stable ATP analogue ATPgammaS antagonized ADPbetaS-induced inhibition of cAMP accumulation in both platelets and 1321 N1 P2Y(12) cells. Thus, ATP and its triphosphate analogues are not agonists but rather antagonists at the P2Y(12) receptor expressed in platelets or transfected cells, provided care is taken to remove diphosphate contaminants and to prevent the generation of diphosphate nucleotide derivatives by cell ectonucleotidases.

ATP Potentiates Interleukin-1β-induced MMP-9 Expression in Mesangial Cells via Recruitment of the ELAV Protein HuR

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.

Regional differences in the adenosine A 2 receptor-mediated modulation of contractions in rat vas deferens

Adenosine receptors involved in modulation of contractions were characterized in the bisected rat vas deferens by combining pharmacological and immunohistochemical approaches. In both portions, noradrenaline-elicited contractions were enhanced by the adenosine A 1 receptor agonist N 6-cyclopentyladenosine (CPA), and inhibited by the non-selective adenosine receptor agonist 5′- N-ethylcarboxamidoadenosine (NECA) in the presence of the adenosine A 1 receptor antagonist 1,3-dipropyl-8-cyclopentyl-l,3-dipropylxanthine (DPCPX). The adenosine A 2A receptor agonist 2- p-(2-carboxyethyl)phenethyl-amino-5′- N-ethylcarboxamidoadenosine (CGS 21680) also inhibited noradrenaline-elicited contractions but only in the prostatic portion. Contractions elicited by the stable ATP analogue α,β-methyleneATP (α,β-MeATP) were inhibited only by NECA in the presence of DPCPX and only in the prostatic portion. This study provides functional evidence for the presence, in both portions of the rat vas deferens, of an adenosine A 1 receptor-mediated enhancement and of an adenosine A 2 receptor-mediated inhibition of contractions. The latter effect is mediated by both A 2A and A 2B subtypes in the prostatic portion but only by the A 2B subtype in the epididymal portion. This regional variation is supported by the immunohistochemical results that revealed an adenosine A 2A receptor immunoreactivity not co-localized with nerve fibres more abundant in the prostatic than in the epididymal portion.

Modulation of hippocampal glutamatergic transmission by ATP is dependent on adenosine a(1) receptors.

Excitatory glutamatergic synapses in the hippocampal CA1 region of rats are potently inhibited by purines, including adenosine, ATP, and ATP analogs. Adenosine A(1) receptors are known to mediate at least part of the response to adenine nucleotides, either because adenine nucleotides activate A(1) receptors directly, or activate them secondarily upon the nucleotides' conversion to adenosine. In the present studies, the inhibitory effects of adenosine, ATP, the purportedly stable ATP analog adenosine-5'-O-(3-thio)triphosphate (ATPgammaS), and cyclic AMP were examined in mice with a null mutation in the adenosine A(1) receptor gene. ATPgammaS displaced the binding of A(1)-selective ligands to intact brain sections and brain homogenates from adenosine A(1) receptor wild-type animals. In homogenates, but not in intact brain sections, this displacement was abolished by adenosine deaminase. In hippocampal slices from wild-type mice, purines abolished synaptic responses, but slices from mice lacking functional A(1) receptors showed no synaptic modulation by adenosine, ATP, cAMP, or ATPgammaS. In slices from heterozygous mice the dose-response curve for both adenosine and ATP was shifted to the right. In all cases, inhibition of synaptic responses by purines could be blocked by prior treatment with the competitive adenosine A(1) receptor antagonist 8-cyclopentyltheophylline. Taken together, these results show that even supposedly stable adenine nucleotides are rapidly converted to adenosine at sites close to the A(1) receptor, and that inhibition of synaptic transmission by purine nucleotides is mediated exclusively by A(1) receptors.

Extracellular ATP and UTP activate the protein kinase B/Akt cascade via the P2Y(2) purinoceptor in renal mesangial cells.

Extracellular nucleotides can activate a common purinoceptor mediating various cell responses. In this study we report that stimulation of rat mesangial cells with ATP and UTP leads to a rapid activation of the protein kinase B/Akt (PKB) pathway. Time-course studies reveal a rapid and transient phosphorylation of both Ser(473) and Thr(308) of PKB with a maximal effect after 5 min of stimulation. The response is concentration-dependent with a maximal effect at 30 microM of ATP and UTP. Western blot analysis of mesangial cells reveals the expression of the isoenzymes PKB-alpha and PKB-gamma, but not the PKB-beta. ATP and UTP also activate the upstream located PI 3-kinase-dependent kinase. Furthermore, the ATP- and UTP-induced PKB phosphorylation is abolished by two inhibitors of the PI 3-kinase. In addition, suramin, a putative P2Y(2) receptor antagonist, and pertussis toxin, an inhibitor of G(i)/G(o) activation, markedly block ATP- and UTP-induced PKB phosphorylation. A series of ATP and UTP analogues were tested for their ability to stimulate PKB phosphorylation. UTP, ATP and gamma-thio-ATP are the only compounds capable of activating PKB. Stress-induced apoptosis of mesangial cells is reduced by the stable ATP analogue, gamma-thio-ATP, and this inhibitory effect is reversed in the presence of LY 294002. In summary, these results demonstrate that extracellular nucleotides are able to activate the PI 3-kinase/PDK/PKB cascade via the P2Y(2)-receptor and a pertussis toxin-sensitive G(i) protein. Moreover, in mesangial cells this cascade may have an important role in the antiapoptotic response but not in the mitogenic or inflammatory response produced by extracellular nucleotides.

Involvement of purinergic signalling in central mechanisms of body temperature regulation in rats.

1. P2 purinoreceptors are present in hypothalamic and brainstem nuclei that are involved in the regulation of body temperature (T(b)). The role of ATP acting on these P2 receptors in thermoregulation was investigated by studying the effects of the stable ATP analogue alpha,beta-methyleneATP (alpha,beta-meATP) and P2 receptor antagonists suramin and pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on T(b) when injected intracerebroventricularly (i.c.v.) via a pre-implanted cannula in conscious rats at various ambient temperatures and during lipopolysaccharide (LPS)-induced fever. 2. Depending on ambient temperature, alpha,beta-meATP (0.2 micromol, i.c.v.) induced a fall in T(b) (-3.3 degrees C, P<0.05), no changes in T(b) when compared to pre-injection levels, or an increase in T(b) ( approximately 1.0 degrees C, P<0.05) in rats maintained at 10 degrees C, 25 degrees C and 30 degrees C ambient temperature, respectively. 3. Suramin (7 nmol, i.c.v.) induced a lasting (up to 6 h) increase in T(b) (on average 1.2 degrees C, P<0.05) in rats kept at 25 degrees C or 30 degrees C, but failed to induce any rise in T(b) in rats at 10 degrees C ambient temperature. An increase in T(b) was also observed in rats (25 degrees C ambient temperature) treated with PPADS (0.2 micromol, i.c.v.). 4. alpha,beta-meATP (0.2 micromol) injected i.c.v. or directly into the anterior hypothalamus caused a profound fall in T(b) (by 0.9 degrees C and 1.0 degrees C, respectively; P<0.05) during LPS (E.coli; 50 microg kg(-1))-induced fever in rats at 25 degrees C ambient temperature. Fever was initiated more rapidly in rats treated with suramin (7 nmol) or PPADS (70 nmol), however its late phase was unaffected. Suramin (7 nmol) and PPADS (70 nmol) injected at the time when fever was already developed (2.5 h after LPS injections) did not alter febrile T(b). 5. These data indicate that purinergic signalling may play a significant role in central mechanisms of T(b) regulation at various ambient temperatures and during fever.

Somatostatin inhibits exocytosis in rat pancreatic α‐cells by Gi2‐dependent activation of calcineurin and depriming of secretory granules

1. Measurements of cell capacitance were used to investigate the molecular mechanisms by which somatostatin inhibits Ca(2+)-induced exocytosis in single rat glucagon-secreting pancreatic alpha-cells. 2. Somatostatin decreased the exocytotic responses elicited by voltage-clamp depolarisations by 80 % in the presence of cyclic AMP-elevating agents such as isoprenaline and forskolin. Inhibition was time dependent and half-maximal within 22 s. 3. The inhibitory action of somatostatin was concentration dependent with an IC(50) of 68 nM and prevented by pretreatment of the cells with pertussis toxin. The latter effect was mimicked by intracellular dialysis with specific antibodies to G(i1/2) and by antisense oligonucleotides against G proteins of the subtype G(i2). 4. Somatostatin lacked inhibitory action when applied in the absence of forskolin or in the presence of the L-type Ca(2+) channel blocker nifedipine. The size of the omega-conotoxin-sensitive and forskolin-independent component of exocytosis was limited to 60 fF. By contrast, somatostatin abolished L-type Ca(2+) channel-dependent exocytosis in alpha-cells exposed to forskolin. The magnitude of the latter pool amounted to 230 fF. 5. The inhibitory effect of somatostatin on exocytosis was mediated by activation of the serine/threonine protein phosphatase calcineurin and was prevented by pretreatment with cyclosporin A and deltamethrin or intracellularly applied calcineurin autoinhibitory peptide. Experiments using the stable ATP analogue AMP-PCP indicate that somatostatin acts by depriming of granules. 6. We propose that somatostatin receptors associate with L-type Ca(2+) channels and couple to G(i2) proteins leading to a localised activation of calcineurin and depriming of secretory granules situated close to the L-type Ca(2+) channels.