Adenosine A(2A) receptor facilitation of hippocampal synaptic transmission is dependent on tonic A(1) receptor inhibition

Adenosine tonically inhibits synaptic transmission through actions at A(1) receptors. It also facilitates synaptic transmission, but it is unclear if this facilitation results from pre- and/or postsynaptic A(2A) receptor activation or from indirect control of inhibitory GABAergic transmission. The A(2A) receptor agonist, CGS 21680 (10 nM), facilitated synaptic transmission in the CA1 area of rat hippocampal slices (by 14%), independent of whether or not GABAergic transmission was blocked by the GABA(A) and GABA(B) receptor antagonists, picrotoxin (50 microM) and CGP 55845 (1 microM), respectively. CGS 21680 (10 nM) also inhibited paired-pulse facilitation by 12%, an effect prevented by the A(2A) receptor antagonist, ZM 241385 (20 nM). These effects of CGS 21680 (10 nM) were occluded by adenosine deaminase (2 U/ml) and were made to reappear upon direct activation of A(1) receptors with N(6)-cyclopentyladenosine (CPA, 6 nM). CGS 21680 (10 nM) only facilitated (by 17%) the K(+)-evoked release of glutamate from superfused hippocampal synaptosomes in the presence of 100 nM CPA. This effect of CGS 21680 (10 nM), in contrast to the isoproterenol (30 microM) facilitation of glutamate release, was prevented by the protein kinase C inhibitors, chelerythrine (6 microM) and bisindolylmaleimide (1 microM), but not by the protein kinase A inhibitor, H-89 (1 microM). Isoproterenol (30 microM), but not CGS 21680 (10-300 nM), enhanced synaptosomal cAMP levels, indicating that the CGS 21680-induced facilitation of glutamate release involves a cAMP-independent protein kinase C activation. To discard any direct effect of CGS 21680 on adenosine A(1) receptor, we also show that in autoradiography experiments CGS 21680 only displaced the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyladenosine ([(3)H]DPCPX, 0.5 nM) with an EC(50) of 1 microM in all brain areas studied and CGS 21680 (30 nM) failed to change the ability of CPA to displace DPCPX (1 nM) binding to CHO cells stably transfected with A(1) receptors.Our results suggest that A(2A) receptor agonists facilitate hippocampal synaptic transmission by attenuating the tonic effect of inhibitory presynaptic A(1) receptors located in glutamatergic nerve terminals. This might be a fine-tuning role for adenosine A(2A) receptors to allow frequency-dependent plasticity phenomena without compromising the A(1) receptor-mediated neuroprotective role of adenosine.     

Cross talk between A(1) and A(2A) adenosine receptors in the hippocampus and cortex of young adult and old rats

Adenosine modulates synaptic transmission by acting on inhibitory A(1) and facilitatory A(2A) receptors, the densities of which are modified in aged animals. We investigated how A(2A) receptor activation influences A(1) receptor function and whether this interaction is modified in aged rats. In hippocampal and cortical nerve terminals from young adult (6 wk), but not old rats (24 mo), the A(2A) receptor agonist, 2-[4-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680; 30 nM) decreased the binding affinity of a selective A(1) receptor agonist, cyclopentyladenosine (CPA), an effect prevented by the A(2A) antagonist, (4-(2-[7-amino-2-(2-furyl (1,2,4)-triazolo(2,3-a (1,3,5)triazin-5-yl-aminoethyl)phenol (ZM 241385, 20 nM). This effect of CGS 21680 required intact nerve terminals and was also observed in the absence of Ca(2+). This A(2A)-induced "desensitization" of A(1) receptors was prevented by the protein kinase C inhibitor, chelerythrine (6 microM), and was not detected in the presence of the protein kinase C activator, phorbol-12,13-didecanoate (250 nM), which itself caused a reduction in binding affinity for CPA. The protein kinase A inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (10 microM), and the protein kinase A activator, 8-Br-cAMP (1 mM), had no effects on the A(2A)-induced A(1) receptor desensitization. This A(2A)-induced A(1) receptor desensitization had a functional correlation because CGS 21680 (10 nM) attenuated by 40% the inhibition caused by CPA (10 nM) on CA1 area population spike amplitude in hippocampal slices. This A(2A)/A(1) interaction may explain the attenuation by adenosine deaminase (2 U/ml), which removes tonic A(1) inhibition, of the facilitatory effect of CGS 21680 on synaptic transmission. The requirement of tonic A(1) receptor activation for CGS 21680 to induce facilitation of synaptic transmission was reinforced by the observation that the A(1) receptor antagonist, 1, 3-dipropyl-8-cyclopentylxanthine (20 nM) prevented CGS 21680 (10 nM) facilitation of population spike amplitude. The present results show the ability of A(2A) receptors to control A(1) receptor function in a manner mediated by protein kinase C, but not protein kinase A, in young adult but not in aged rats.     

Activation of adenosine A3 receptor suppresses lipopolysaccharide-induced TNF-α production through inhibition of PI 3-kinase/Akt and NF-κB activation in murine BV2 microglial cells

Adenosine is an endogenous nucleoside that regulates many processes, including inflammatory responses, through activation of its receptors. Adenosine receptors have been reported to be expressed in microglia, which are major immune cells of brain, yet little is known about the role of adenosine receptors in microglial cytokine production. Thus, we investigated the effect of adenosine and adenosine A₃ receptor ligands on LPS-induced tumor necrosis factor (TNF-α) production and its molecular mechanism in mouse BV2 microglial cells. Adenosine and Cl-IB-MECA, a specific adenosine A₃ receptor agonist, suppressed LPS-induced TNF-α protein and mRNA levels. Moreover, MRS1523, a selective A₃ receptor antagonist, blocked suppressive effects of both adenosine and Cl-IB-MECA on TNF-α. We further examined the effect of adenosine on signaling molecules, such as PI 3-kinase, Akt, p38, ERK1/2, and NF-κB, which are involved in the regulation of inflammatory responses. Adenosine inhibited LPS-induced phosphatidylinositol (PI) 3-kinase activation and Akt phosphorylation, whereas it had no effect on the phosphorylation of p38 and ERK1/2. We also found that adenosine as well as Cl-IB-MECA inhibited LPS-induced NF-κB DNA binding and luciferase reporter activity. Taken together, these results suggest that adenosine A₃ receptor activation suppresses TNF-α production by inhibiting PI 3-kinase/Akt and NF-κB activation in LPS-treated BV2 microglial cells.     

Cardioprotection induced by hydrogen sulfide preconditioning involves activation of ERK and PI3K/Akt pathways

We previously reported that hydrogen sulfide (H₂S) preconditioning (SP) produces cardioprotective effects against ischemia in rat cardiac myocytes. The present study aims to elucidate the signaling mechanisms involved in SP-induced cardioprotection by investigating the role of extracellular signal regulated kinase (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt. We found that preconditioning with NaHS (a H₂S donor) for three cycles significantly decreased myocardial infarct size and improved heart contractile function in the isolated rat hearts. NaHS (1–100 μM) concentration-dependently increased cell viability and percentage of rod-shaped cardiac myocytes. Blockade of ERK1/2 with PD 98059 or PI3K/Akt with LY-294002 or Akt inhibitor III during either preconditioning or ischemia periods significantly attenuated the cardioprotection of SP, suggesting that both ERK1/2 and PI3K/Akt triggered and mediated the cardioprotection of SP. Moreover, SP induced ERK1/2 and Akt phosphorylation in isolated hearts. The phosphorylation of ERK1/2 induced by SP was attenuated by either glibenclamide, an ATP-sensitive K⁺ channel (K_ATP) blocker, or chelerythrine, a specific protein kinase C (PKC) blocker. In addition, ischemic-preconditioning-induced ERK1/2 activation was reversed by inhibiting endogenous H₂S production, suggesting that ERK1/2 activation induced by ischemic preconditioning was, at least partly, mediated by endogenous H₂S. In conclusion, K_ATP/PKC/ERK1/2 and PI3K/Akt pathways contributed to SP-induced cardioprotection.     

The selective A2 adenosine receptor agonist CGS 21680 enhances excitatory transmitter amino acid release from the ischemic rat cerebral cortex.

The effects of the selective adenosine A2 receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride (CGS 21680) on aspartate and glutamate release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four vessel occlusion. Pretreatment with CGS 21680 failed to alter basal excitatory amino acid levels, however, CGS 21680 at 10(-6) M significantly enhanced the ischemia-evoked release. Thus, aspartate and glutamate release during ischemia can be stimulated via the activation of A2 receptors, in addition to the suppression of excitatory amino acid release mediated by selective A1 receptor agonists.     

Mechanism of phosphatidylinositol 3-kinase-dependent increases in BAC1.2F5 macrophage-like cell density in response to M-CSF: Phosphatidylinositol 3-kinase inhibitors increase the rate of apoptosis rather than inhibit DNA synthesis

OBJECTIVE AND DESIGN: To determine the role of phosphatidylinositol 3-kinase (PI 3-kinase) in macrophagecolony stimulating factor (M-CSF)-induced macrophage proliferation. MATERIALS: The M-CSF-dependent BAC1.2F5 murine macrophage cell line was used. METHODS: PI 3-kinase activity, Protein kinase B activation, increased cell numbers, induction of DNA synthesis and apoptosis were measured in response to serum, M-CSF and PI 3-kinase inhibitors. RESULTS: Wortmannin or LY294002 inhibited M-CSF-stimulated increases in BAC1.2F5 cell density. Further analysis showed that inhibition of PI 3-kinase had an insignificant effect on DNA synthesis, but significantly induced apoptosis. Other co-factors in serum mediated cell survival and prevented programmed cell death, in a PI 3-kinase-dependent manner. Stimulation of BAC1.2F5 macrophages with M-CSF induced phosphorylation of PKB/Akt as detected by activation-specific antibodies. Activation of PKB/Akt correlated with PI 3-kinase activation, suggesting that the protection from apoptosis in these cells is mediated by PKB/Akt. CONCLUSIONS: These results indicate that the lack of increase in cell numbers when cells are stimulated with M-CSF in the presence of PI 3-kinase inhibitors is due to a preferential PI 3-kinase requirement for protection against apoptosis, rather than a requirement for PI 3-kinase activation during the proliferation signal.     

Role of adenosine receptors in neuroprotective effect during global cerebral ischemia

Selective A₁ adenosine receptor agonists produced a considerable neuroprotective effect during global cerebral ischemia. The neuroprotective effect decreased in the order: A₁ agonists-NECA-adenosine-A_2A agonist CGS 21680, while selective A₃ adenosine receptor agonist was ineffective. Inhibitory analysis showed that A₁ adenosine receptors mediate the neuroprotective effect of CPA, are involved in the effects of NECA and adenosine (but not CGS 21680), and participate in natural resistance to cerebral ischemia. The role of A_2B adenosine receptors in the realization of neuroprotective effects was also demonstrated.     

Human skeletal muscle cell differentiation is associated with changes in myogenic markers and enhanced insulin-mediated MAPK and PKB phosphorylation

AIM: We hypothesized that myogenic differentiation of HSMC would yield a more insulin responsive phenotype. METHODS: We assessed expression of several proteins involved in insulin action or myogenesis during differentiation of primary human skeletal muscle cultures (HSMC). RESULTS: Differentiation increased creatine kinase activity and expression of desmin and myocyte enhancer factor (MEF)2C. No change in expression was observed for big mitogen-activated protein kinase (BMK1/ERK5), MEF2A, insulin receptor (IR), hexokinase II, and IR substrates 1 and 2, while expression of glycogen synthase, extracellular signal-regulated kinase 1 and 2 (ERK1/2 MAP kinase) and the insulin responsive aminopeptidase increased after differentiation. In contrast to protein kinase B (PKB)a, expression of (PKB)b increased, with differentiation. Both basal and insulin-stimulated PI 3-kinase activity increased with differentiation. Insulin-mediated phosphorylation of PKB and ERK1/2 MAP kinase increased after differentiation. CONCLUSION: Components of the insulin-signalling machinery are expressed in myoblast and myotube HSMC; however, insulin responsiveness to PKB and ERK MAP kinase phosphorylation increases with differentiation.     

Enhancement of rapid eye movement sleep in the rat by actions at A1 and A2a adenosine receptor subtypes with a differential sensitivity to atropine

The adenosine agonist cyclohexaladenosine injected into the medial pontine reticular formation of the rat induces a long-lasting increase in rapid eye movement sleep. To investigate the adenosine receptor-subtype(s) mediating this effect, the dose-response relationships for increasing rapid eye movement sleep by two highly selective adenosine receptor agonists were compared. Rats were surgically prepared for chronic sleep recording and bilateral guide cannulae were aimed at medial sites in the caudal, oral pontine reticular formation. Injections were made unilaterally in 60 nl volumes within 1 h after lights-on. The adenosine agonists used were A1-selective cyclohexaladenosine (10(-6)-10(-4) M) and A2a-selective CGS 21680 (10(-7)-10(-3) M). Each animal also received a series of three, paired-consecutive injections of the muscarinic receptor antagonist atropine (4x10(-3) M) followed by the lowest effective dose of each agonist or saline as control. The A2a receptor agonist, CGS 21680, was one order of magnitude more potent than the A1 receptor agonist, cyclohexaladenosine, in inducing rapid eye movement sleep increases. Preinjection of atropine at a dose that did not itself affect rapid eye movement sleep resulted in antagonism of CGS 21680, but not cyclohexaladenosine-induced rapid eye movement sleep.The differential sensitivity of these ligands to antagonism by atropine supports the conclusion that both A1 and A2a adenosine receptor subtypes in the reticular formation subserve agonist-induced rapid eye movement sleep and that they do so by independent mechanisms. The A2a mechanism requires the cholinergic system and may act through the increased release of acetylcholine. The A1 mechanism operates at a different locus possibly through an inhibition of GABA neurotransmission.     

Triggering of BDNF facilitatory action on neuromuscular transmission by adenosine A2A receptors

Motor nerve terminals possess adenosine A(2A) receptors and brain derived neurotrophic factor (BDNF) TrkB receptors. In the present work we evaluated how BDNF actions on neuromuscular transmission would be influenced by adenosine A(2A) receptors activation. BDNF (20-100 ng/ml) on its own was devoid of effect on evoked endplate potentials (EPPs) recorded intracellularly from rat innervated diaphragms paralysed with tubocurarine. However, when BDNF was applied 45 min after a brief (2 min) depolarizing KCl (10 mM) pulse or when the adenosine A(2A) receptors were activated with CGS 21680 (10 nM), BDNF (20 ng/ml) increased EPPs amplitude without influencing the resting membrane potential of the muscle fibre. The action of BDNF was prevented by the adenosine A(2A) receptor antagonist, ZM 241385 (50 nM) as well as by the TrkB receptor phosphorylation inhibitor, K252a (200 nM). The PKA inhibitor, H-89 (1 microM), prevented the excitatory effect of CGS 21680 (10 nM) on EPPs as well as prevented its ability to trigger a BDNF effect. The PLCgamma inhibitor, U73122 (5 microM), did not prevent the excitatory action of CGS 21680 (10 nM) on neuromuscular transmission, but abolished the action of BDNF in the presence of the A(2A) receptor agonist. The results suggest the following sequence of events in what concerns cooperativity between A(2A) receptors and TrkB receptors at the neuromuscular junction: A(2A) receptor activates the PKA pathway, which promotes the action of BDNF through TrkB receptors coupled to PLCgamma, leading to enhancement of neuromuscular transmission.     

Adenosine A2A receptors interact with GABAergic pathways to modulate respiration in neonatal piglets

GABA and adenosine contribute to respiratory inhibition in early postnatal life. In this study the adenosine A2A receptor agonist CGS21680 was used to evaluate adenosine receptor specificity and the interrelation of adenosine and GABA in the inhibition of inspiratory drive. In neonatal piglets (n = 10), CGS21680 was injected into the fourth ventricle resulting in apnea and/or decreased burst area and frequency of phrenic discharge. Phrenic burst area decreased to 58.9 +/- 8.6% (S.E.M.) after CGS21680 injection (control = 91.8 +/- 1.0%). Expiratory time increased 261.0 +/- 59.9% after CGS21680 from control (87.7 +/- 2.7%). When bicuculline was injected locally within the rostral ventrolateral medulla (n = 5), or into the fourth ventricle (n = 5), the CGS21680 induced inhibition of phrenic was abolished. To define expression of A2A receptor at the message level (mRNA), we employed in situ hybridization with a digoxigenin-coupled oligonucleotide. Adenosine A2A receptor mRNA was expressed in regions of the medulla oblongata known to contain GABAergic neurons. We conclude that GABAergic inputs affecting respiratory timing and inspiratory drive are modulated by activation of A2A receptors. These findings offer new insight into the mechanism whereby xanthine therapy diminishes apnea of prematurity.     

Adrenaline potentiates insulin-stimulated PKB activation in the rat fast-twitch epitrochlearis muscle without affecting IRS-1-associated PI 3-kinase activity

We have previously shown in the rat slow-twitch soleus muscle that adrenaline greatly potentiates insulin-stimulated protein kinase B (PKB) phosphorylation without having an effect alone. However, insulin signalling capacity through the PKB pathway is higher in soleus than in fast-twitch muscles, whereas adrenaline activates phosphorylase more strongly in epitrochlearis. Therefore, the aim of the present study was to investigate the interaction between adrenaline and insulin signalling in the fast-twitch epitrochlearis muscle. Insulin increased insulin receptor substrate-1 (IRS-1)-associated phosphoinositide (PI) 3-kinase activity threefold, and adrenaline did not influence basal or insulin-stimulated PI 3-kinase activity. Insulin but not adrenaline increased PKB activity and phosphorylation of Ser(473) and Thr(308). It is interesting to note that adrenaline potentiated insulin-stimulated PKB activity and PKB Ser(473) and Thr(308) phosphorylation. These effects were mimicked by dibutyryl-cyclic adenosine monophosphate (db-cAMP). Adrenaline and db-cAMP increased glycogen synthase kinase (GSK)-3beta Ser(9) phosphorylation independently of PKB activation and enhanced insulin-stimulated GSK-3beta Ser(9) phosphorylation. Although adrenaline increased GSK-3 phosphorylation (inhibiting activity), phosphorylation of its target sites on glycogen synthase was increased, and adrenaline blocked insulin-stimulated glycogen synthase dephosphorylation of Ser(641) and Ser(645,649,653,657), glycogen synthase activation and glycogen synthesis. Insulin-stimulated glucose transport was not influenced by adrenaline despite the increased PKB activation. In conclusion, as in the slow-twitch soleus muscle, adrenaline potentiates insulin-stimulated PKB activation in the fast-twitch glycolytic epitrochlearis muscle without increasing IRS-1-associated PI 3-kinase activity. Furthermore, adrenaline induces phosphorylation of a pool of GSK-3 that is not involved in the regulation of glycogen metabolism. These results indicate that the combination of adrenaline and insulin may activate novel signalling molecules rather than just summing up their effects on linear pathways.     

Ligation of the T cell co-stimulatory receptor CD28 activates the serine-threonine protein kinase protein kinase B

The intracellular signaling pathways activated upon ligation of the co-stimulatory receptor CD28 remain relatively ill-defined, although CD28 ligation does result in the strong association with, and activation of, phosphatidylinositol (PI) 3-kinase. The downstream effector targets of the CD28-activated PI 3-kinase-dependent signaling pathway remain poorly defined, but recent evidence from other systems has shown that Akt/protein kinase B (PKB) is a major target of PI 3-kinase and have indicated that a major function of PKB is the regulation of cell survival events. Given the strong coupling of CD28 to PI 3-kinase and the known protective effects of both CD28 and PI 3-kinase against apoptosis in different cell models, we investigated the effects of CD28 on PKB activation. We demonstrate that ligation of CD28 by either anti-CD28 monoclonal antibodies or the natural ligand B7.1, results in the marked activation of PKB in both the leukemic T cell line Jurkat and freshly isolated human peripheral blood-derived normal T lymphocytes. Our data suggest therefore, that PKB may be an important intracellular signal involved in CD28 signal transduction and demonstrate CD28 coupling to downstream elements of a signaling cascade known to promote cell survival.     

RISK信号通路在心肌预处理及后处理中的作用

促生存激酶磷脂酰肌醇-3激酶(phosphatidylinositol-3-OH kinase,PI3K)和细胞外信号调节蛋白激酶1/2(extra-cellular signal-regulated protein kinase 1/2,ERK1/2)均为丝氨酸/苏氨酸激酶.缺血预处理(ischemic preconditioning,IPC)是1986年Murry等发现的一种内源性心肌保护现象,即预先反复短暂缺血/再灌注可以提高心肌组织对随后持续缺血/再灌注损伤的耐受性.2003年,Zhao等[1]相对于IPC首次提出缺血后处理(ischemic postconditioning),方法是在持续再灌注开始前进行反复短暂缺血/再灌注,与IPC一样,可以缩小心肌梗死面积.此后许多研究均证实了缺血后处理的心脏保护作用[2-4],而且还发现,心肌缺血后处理和缺血预处理以及模拟上述两者的药物后处理和药物预处理都于再灌注早期阶段通过磷酸化激活PI3K和ERK1/2减轻心肌缺血/再灌注损伤.Hausenloy等[5]首次将PI3K和ERK1/2两者合称为再灌注损伤挽救激酶(reperfusion injury salvage kinase,RISK)信号通路,本文就RISK信号通路在心肌预处理及后处理中的作用进行综述.     

Adenosine affects the release of Ca2+ from the sarcoplasmic reticulum via A2A receptors in ferret skinned cardiac fibres

In this study, it was shown that adenosine potentiates caffeine-induced Ca2+ release. It was then proposed that the enhancement of the caffeine-induced Ca2+ release might occur by a direct effect on the ryanodine Ca2+ release channel or on other Ca2+ regulation mechanisms. Furthermore, A2A receptors may be functional on the ferret cardiac sarcoplasmic reticulum. Using chemically skinned fibres, experiments were conducted on ferret cardiac muscle to find out whether adenosine and the A1 and A2A adenosine receptor agonists (CCPA and CGS 21680) and antagonists (DPCPX and ZM 241385) affected caffeine-induced Ca2+ release and the Ca2+ sensitivity of contractile proteins. Changes in the caffeine-induced contracture brought about by adenosine and by adenosine-receptor agonists and antagonists were recorded in saponin-skinned fibres (50 microg ml(-1)). Tension-pCa relationships were then obtained by exposing Triton X-100-skinned fibres (1% v/v) sequentially to solutions of decreasing pCa. Adenosine (1-100 nm) and the specific A2A receptor agonist CGS 21680 (1-50 nm) produced a concentration-dependant potentiation of the caffeine-induced Ca2+ release from saponin-skinned fibres. The data plotted versus adenosine and CGS 21680 concentrations displayed sigmoid relationships (Hill relationship), with potentiation of Ca2+ release by 22.2 +/- 1.6 (n = 6) and 10.9 +/- 0.4% (n = 6), respectively. In addition, the potentiation of caffeine-induced Ca2+ release by adenosine (50 nm; 15.3 +/- 1.0%; n = 6) and by CGS 21680 (50 nm; 11.2 +/- 0.4%; n = 6) was reduced by the specific A2A receptor antagonist ZM 241385 (50 nm) to 8.0 +/- 1.4 (n = 4) and 5.4 +/- 1.2% (n = 4), respectively. The A1 receptor agonist CCPA (1-50 nm) and antagonist DPCPX (50 nm) had no significant effects on caffeine responses. In Triton X-100-skinned fibres, the maximal Ca(2+)-activated tension of the contractile proteins (41.3 +/- 4.1 mN mm(-2); n = 8), the Hill coefficient (nH = 2.2 +/- 0.1; n = 8) and the pCa50 (6.15 +/- 0.05; n = 8) were not significantly modified by adenosine (100 nm) or by CGS 21680 (50 nm).