Release Of Endogenous Adenosine Research Articles

The impact of Müller cells on development and resolution of macular edema

AbstractMüller cells support physiological neuronal activity and the integrity of the blood‐retinal barrier. A major function of Müller cells is the fluid absorption from the retinal tissue, which is mediated by transcellular water transport coupled to currents through potassium channels. In various disorders as retinal ischemia, ocular inflammation and /or retinal detachment, it was shown that Müller cells decrease the expression of the potassium channel (Kir4.1) which leads to an impairment of the water transport across Müller cell membranes, and induces the cellular swelling. Osmotic swelling of Müller cells is also induced by oxidative stress and by inflammatory mediators. The data suggest that a disturbed fluid transport through Müller cells is (in addition to vascular leakage) a pathogenic factor contributing to the development of retinal edema. Pharmacological re‐activation of the retinal water clearance by Müller cells may be used for treatment of macular edema. Intravitreal steroids as triamcinolone acetonide prevent osmotic swelling of Müller cells and induce the release of endogenous adenosine and subsequent A1 receptor activation which results in the opening of ion channels. Apparently, steroids resolve the edema by both inhibition of vascular leakage and stimulation of retinal fluid clearance by Müller cells.

Inhibition of Adenosine Pathway Alters Atrial Electrophysiology and Prevents Atrial Fibrillation.

BackgroundAdenosine leads to atrial action potential (AP) shortening through activation of adenosine 1 receptors (A1-R) and subsequent opening of G-protein-coupled inwardly rectifying K+ channels. Extracellular production of adenosine is drastically increased during stress and ischemia.ObjectiveThe aim of this study was to address whether the pharmacological blockade of endogenous production of adenosine and of its signaling prevents atrial fibrillation (AF).MethodsThe role of A1-R activation on atrial action potential duration, refractoriness, and AF vulnerability was investigated in rat isolated beating heart preparations (Langendorff) with an A1-R agonist [2-chloro-N6-cyclopentyladenosine (CCPA), 50 nM] and antagonist [1-butyl-3-(3-hydroxypropyl)-8-(3-noradamantyl)xanthine (PSB36), 40 nM]. Furthermore, to interfere with the endogenous adenosine release, the ecto-5′-nucleotidase (CD73) inhibitor was applied [5′-(α,β-methylene) diphosphate sodium salt (AMPCP), 500 μM]. Isolated trabeculae from human right atrial appendages (hRAAs) were used for comparison.ResultsAs expected, CCPA shortened AP duration at 90% of repolarization (APD90) and effective refractory period (ERP) in rat atria. PSB36 prolonged APD90 and ERP in rat atria, and CD73 inhibition with AMPCP prolonged ERP in rats, confirming that endogenously produced amount of adenosine is sufficiently high to alter atrial electrophysiology. In human atrial appendages, CCPA shortened APD90, while PSB36 prolonged it. Rat hearts treated with CCPA are prone to AF. In contrast, PSB36 and AMPCP prevented AF events and reduced AF duration (vehicle, 11.5 ± 2.6 s; CCPA, 40.6 ± 16.1 s; PSB36, 6.5 ± 3.7 s; AMPCP, 3.0 ± 1.4 s; P < 0.0001).ConclusionA1-R activation by intrinsic adenosine release alters atrial electrophysiology and promotes AF. Inhibition of adenosine pathway protects atria from arrhythmic events.

Exogenous adenosine facilitates neuroprotection and functional recovery following cerebral ischemia in rats

Introduction & ObjectiveCerebral ischemia causes physiological and biochemical cellular changes that ultimately result in structural and functional damage to hippocampal neurons. Ischemia also raises endogenous adenosine release that in turn has neuroprotective effects. The purpose of this study was to evaluate the effect of exogenous adenosine on mitigating neuronal lesions to the CA1 region of hippocampus and A2A protein expression following cerebral I/R in rats. MethodsMale Wistar rats were randomly assigned to three experimental groups (sham, ischemia + control, and ischemia + adenosine). A daily dose of adenosine (0.1 mg/ml/kg, i.p.) was administered starting 24 h post-ischemia for 7 days. Ischemia was induced by occlusion of both common carotid arteries for 45 min. Cresyl violet and Hematoxylin Eosin staining were used to assess lesion extent and location. To investigate the expression and protein levels, immunohistochemistry and enzyme-linked immunosorbent assay method was used. ResultsThe cerebral ischemia caused neuronal loss in the CA1 region and reduced sensorimotor functions in lesion animals. Injection of adenosine significantly diminished cell death and improved sensorimotor functional recovery. Moreover, the expression and concentration of A2A protein was significantly greater in the adenosine group compared to the ischemia group. ConclusionThis study showed that the administration of exogenous adenosine promotes protection against cell death and supports functional recovery following ischemic injury.

Regulation of human brown adipose tissue by adenosine and A2A receptors \u2013 studies with [15O]H2O and [11C]TMSX PET/CT

PurposeBrown adipose tissue (BAT) has emerged as a potential target to combat obesity and diabetes, but novel strategies to activate BAT are needed. Adenosine and A2A receptor (A2AR) agonism activate BAT in rodents, and endogenous adenosine is released locally in BAT as a by-product of noradrenaline, but physiological data from humans is lacking. The purpose of this pilot study was to investigate the effects of exogenous adenosine on human BAT perfusion, and to determine the density of A2ARs in human BAT in vivo for the first time, using PET/CT imaging.MethodsHealthy, lean men (n = 10) participated in PET/CT imaging with two radioligands. Perfusion of BAT, white adipose tissue (WAT) and muscle was quantified with [15O]H2O at baseline, during cold exposure and during intravenous administration of adenosine. A2AR density of the tissues was quantified with [11C]TMSX at baseline and during cold exposure.ResultsAdenosine increased the perfusion of BAT even more than cold exposure (baseline 8.3 ± 4.5, cold 19.6 ± 9.3, adenosine 28.6 ± 7.9 ml/100 g/min, p < 0.01). Distribution volume of [11C]TMSX in BAT was significantly lower during cold exposure compared to baseline. In cold, low [11C]TMSX binding coincided with high concentrations of noradrenaline.ConclusionsAdenosine administration caused a maximal perfusion effect in human supraclavicular BAT, indicating increased oxidative metabolism. Cold exposure increased noradrenaline concentrations and decreased the density of A2AR available for radioligand binding in BAT, suggesting augmented release of endogenous adenosine. Our results show that adenosine and A2AR are relevant for activation of human BAT, and A2AR provides a future target for enhancing BAT metabolism.

Adenosine hypersensitivity and atrioventricular block.

Adenosine is aubiquitous substance that is released under several physiological and pathological conditions and has cardiovascular effects including cardioinhibition and vasodilation. It has been shown to be animportant modulator implicated in several forms of syncope. In patients with chronic low plasma levels of adenosine, atransient release of endogenous adenosine can be sufficient to block conduction in the atrioventricular node and induce prolonged asystole; conversely, when plasma adenosine levels are chronically high, adenosine release is responsible for vasodepression. Distinct purinergic profiles in patients presenting with syncope have recently been correlated with the clinical presentation: "low-adenosine patients," prone to asystole, may present with idiopathic atrioventricular block, carotid sinus syndrome, or syncope with no or very brief prodromes and normal heart; "high-adenosine patients," prone to vasodilation, experience vasovagal syncope. This pathophysiological classification may have therapeutic implications.

Mechanisms involved in adenosine pharmacological preconditioning-induced cardioprotection.

Adenosine is a naturally occurring breakdown product of adenosine triphosphate and plays an important role in different physiological and pathological conditions. Adenosine also serves as an important trigger in ischemic and remote preconditioning and its release may impart cardioprotection. Exogenous administration of adenosine in the form of adenosine preconditioning may also protect heart from ischemia-reperfusion injury. Endogenous release of adenosine during ischemic/remote preconditioning or exogenous adenosine during pharmacological preconditioning activates adenosine receptors to activate plethora of mechanisms, which either independently or in association with one another may confer cardioprotection during ischemia-reperfusion injury. These mechanisms include activation of KATP channels, an increase in the levels of antioxidant enzymes, functional interaction with opioid receptors; increase in nitric oxide production; decrease in inflammation; activation of transient receptor potential vanilloid (TRPV) channels; activation of kinases such as protein kinase B (Akt), protein kinase C, tyrosine kinase, mitogen activated protein (MAP) kinases such as ERK 1/2, p38 MAP kinases and MAP kinase kinase (MEK 1) MMP. The present review discusses the role and mechanisms involved in adenosine preconditioning-induced cardioprotection.

Relaxation effect of abacavir on rat basilar arteries.

BackgroundThe use of abacavir has been linked with increased cardiovascular risk in patients with human immunodeficiency virus infection; however, the mechanism involved remains unclear. We hypothesize that abacavir may impair endothelial function. In addition, based on the structural similarity between abacavir and adenosine, we propose that abacavir may affect vascular contractility through endogenous adenosine release or adenosine receptors in blood vessels.MethodsThe relaxation effect of abacavir on rat basilar arteries was studied using the myograph technique. Cyclic GMP and AMP levels were measured by immunoassay. The effects of abacavir on nucleoside transporters were studied using radiolabeled nucleoside uptake experiments. Ecto-5′ nucleotidase activity was determined by measuring the generation of inorganic phosphate using adenosine monophosphate as the substrate.ResultsAbacavir induced the relaxation of rat basilar arteries in a concentration-dependent manner. This relaxation was abolished when endothelium was removed. In addition, the relaxation was diminished by the nitric oxide synthase inhibitor, L-NAME, the guanylyl cyclase inhibitor, ODQ, and the protein kinase G inhibitor, KT5820. Abacavir also increased the cGMP level in rat basilar arteries. Abacavir-induced relaxation was also abolished by adenosine A2 receptor blockers. However, abacavir had no effect on ecto-5’ nucleotidase and nucleoside transporters. Short-term and long-term treatment of abacavir did not affect acetylcholine-induced relaxation in rat basilar arteries.ConclusionAbacavir induces acute endothelium-dependent relaxation of rat basilar arteries, probably through the activation of adenosine A2 receptors in endothelial cells, which subsequently leads to the release of nitric oxide, resulting in activation of the cyclic guanosine monophosphate/protein kinase G-dependent pathway in vascular smooth muscle cells. It is speculated that abacavir-induced cardiovascular risk may not be related to endothelial dysfunction as abacavir does not impair relaxation of blood vessels. The most likely explanation of increased cardiovascular risk may be increased platelet aggregation as suggested by other studies.

Endogenous adenosine release is involved in the control of heart rate in rats.

Intravenous (i.v.) injections of adenosine exert marked effects on heart rate (HR) and arterial blood pressure (BP), but the role of an endogenous adenosine release by vagal stimulation has not been evaluated. In anaesthetized rats, we examined HR and BP changes induced by 1 min electrical vagal stimulation in the control condition, and then after i.v. injections of (i) atropine, (ii) propranolol, (iii) caffeine, (iv) 8 cyclopentyl-1,3-dipropylxanthine (DPCPX), or (v) dipyridamole to increase the plasma concentration of adenosine (APC). APC was measured by chromatography in the arterial blood before and at the end of vagal stimulation. The decrease in HR in the controls during vagal stimulation was markedly attenuated, but persisted after i.v. injections of atropine and propranolol. When first administered, DPCPX modestly but significantly reduced the HR response to vagal stimulation, but this disappeared after i.v. caffeine administration. Both the HR and BP responses were significantly accentuated after i.v. injection of dipyridamole. Vagal stimulation induced a significant increase in APC, proportional to the magnitude of HR decrease. Our data suggest that the inhibitory effects of electrical vagal stimulations on HR and BP were partly mediated through the activation of A1 and A2 receptors by an endogenous adenosine release. Our experimental data could help to understand the effects of ischemic preconditioning, which are partially mediated by adenosine.

Sleep-Wake Sensitive Mechanisms of Adenosine Release in the Basal Forebrain of Rodents: An In Vitro Study

Adenosine acting in the basal forebrain is a key mediator of sleep homeostasis. Extracellular adenosine concentrations increase during wakefulness, especially during prolonged wakefulness and lead to increased sleep pressure and subsequent rebound sleep. The release of endogenous adenosine during the sleep-wake cycle has mainly been studied in vivo with microdialysis techniques. The biochemical changes that accompany sleep-wake status may be preserved in vitro. We have therefore used adenosine-sensitive biosensors in slices of the basal forebrain (BFB) to study both depolarization-evoked adenosine release and the steady state adenosine tone in rats, mice and hamsters. Adenosine release was evoked by high K+, AMPA, NMDA and mGlu receptor agonists, but not by other transmitters associated with wakefulness such as orexin, histamine or neurotensin. Evoked and basal adenosine release in the BFB in vitro exhibited three key features: the magnitude of each varied systematically with the diurnal time at which the animal was sacrificed; sleep deprivation prior to sacrifice greatly increased both evoked adenosine release and the basal tone; and the enhancement of evoked adenosine release and basal tone resulting from sleep deprivation was reversed by the inducible nitric oxide synthase (iNOS) inhibitor, 1400 W. These data indicate that characteristics of adenosine release recorded in the BFB in vitro reflect those that have been linked in vivo to the homeostatic control of sleep. Our results provide methodologically independent support for a key role for induction of iNOS as a trigger for enhanced adenosine release following sleep deprivation and suggest that this induction may constitute a biochemical memory of this state.

Neurabin scaffolding of adenosine receptor and RGS4 regulates anti‐seizure effect of endogenous adenosine

An increase in adenosine receptor responsiveness to endogenous adenosine would enhance neuroprotection while avoiding the confounding effects of exogenous ligands. Here we report novel regulation of adenosine‐evoked responses by a neural tissue‐specific protein, neurabin. Neurabin attenuated adenosine A1 receptor (A1R) signaling by assembling a complex between the A1R and the regulator of G protein signaling 4 (RGS4). Inactivation of the neurabin gene enhanced A1R signaling and promoted the protective effect of adenosine against excitotoxic seizure and neuronal death in mice. Furthermore, administration of a small molecule inhibitor of RGS4 significantly attenuated seizure severity in mice. The enhanced anti‐seizure and neuroprotective effect achieved by disruption of the A1R/neurabin/RGS4 complex is elicited by the on‐site and on‐demand release of endogenous adenosine, and does not require administration of A1R ligands. These data identify neurabin‐RGS4 as a novel tissue‐selective regulatory mechanism for fine‐tuning adenosine receptor function in the nervous system. Moreover, these findings implicate the A1R/neurabin/RGS4 complex as a valid therapeutic target for specifically manipulating the neuroprotective effects of endogenous adenosine. This research is supported by UAB faculty development grant (QW) and NIH grants MH081917 (QW) and DA10044 (PG).

Neurabin Scaffolding of Adenosine Receptor and RGS4 Regulates Anti-Seizure Effect of Endogenous Adenosine

Endogenous adenosine is an essential protective agent against neural damage by various insults to the brain. However, the therapeutic potential of adenosine receptor-directed ligands for neuroprotection is offset by side effects in peripheral tissues and organs. An increase in adenosine receptor responsiveness to endogenous adenosine would enhance neuroprotection while avoiding the confounding effects of exogenous ligands. Here we report novel regulation of adenosine-evoked responses by a neural tissue-specific protein, neurabin. Neurabin attenuated adenosine A1receptor (A1R) signaling by assembling a complex between the A1R and the regulator of G-protein signaling 4 (RGS4), a protein known to turn off G-protein signaling. Inactivation of the neurabin gene enhanced A1R signaling and promoted the protective effect of adenosine against excitotoxic seizure and neuronal death in mice. Furthermore, administration of a small molecule inhibitor of RGS4 significantly attenuated seizure severity in mice. Notably, the dose of kainate capable of inducing an ∼50% rate of death in wild-type (WT) mice did not affect neurabin-null mice or WT mice cotreated with an RGS4 inhibitor. The enhanced anti-seizure and neuroprotective effect achieved by disruption of the A1R/neurabin/RGS4 complex is elicited by the on-site and on-demand release of endogenous adenosine, and does not require administration of A1R ligands. These data identify neurabin-RGS4 as a novel tissue-selective regulatory mechanism for fine-tuning adenosine receptor function in the nervous system. Moreover, these findings implicate the A1R/neurabin/RGS4 complex as a valid therapeutic target for specifically manipulating the neuroprotective effects of endogenous adenosine.

Novel Trends in the Treatment of Cardiovascular Disorders: Site- and Event- Selective Adenosinergic Drugs

This review focuses on the potential role of site- and event-selective adenosinergic drugs in the treatment of cardiovascular diseases. Adenosine is released from the myocardium and vessels in response to various forms of stress and acts on four receptor subtypes (A1, A2A, A2B and A3). Adenosine is an important endogenous substance with important homeostatic activity in the regulation of cardiac function and circulation. Adenosine receptors are also involved in the modulation of various cellular events playing crucial role in physiological and pathological processes of the cardiovascular system. These actions are associated to activation of distinct adenosine receptor subtypes, therefore drugs targeting specific adenosine receptors might be promising therapeutic tools in treatment of several disorders including various forms of cardiac arrhythmia, myocardial ischemia-reperfusion injury, angina pectoris, chronic heart failure, etc. Recently, in addition to subtype-specific adenosine receptor agonists and antagonists, a number of substances that enhance adenosine receptor activation locally at the site where the release of endogenous adenosine is the most intensive have been developed. Thus global actions of adenosine receptor agonists and antagonists, as well as desensitization or down-regulation following chronic administration of these orthosteric compounds can possibly be avoided. We discuss the chemical, pharmacological and clinical features of these compounds: (1) inhibitors of membrane adenosine transporters (NBTI, dipyridamole), (2) inhibitors of adenosine deaminase (coformycin, EHNA), (3) inhibitors of adenosine kinase (tubercidin, aristeromycin), (4) inhibitors of AMP deaminase (GP3269), (5) activators of 5'-nucleotidase (methotrexate), (6) adenosine regulators (acadesine) and (7) allosteric adenosine receptor modulators (PD81723, LUF6000). The development of this type of substances might offer a novel therapeutic approach for treating cardiovascular diseases in the near future.

Endogenous Activation of adenosine A 1 receptors promotes post-ischemic electrocortical burst suppression

Following transient global cerebral ischemia (GCI), spontaneous electrocortical activity resumes from the isoelectric line through a sequence of “bursts” of activity alternating with periods of electrical “suppression,” commonly referred to as the post-ischemic burst suppression (BS) pattern. Several lines of evidence suggest that BS reflects an impairment of neocortical connectivity. Here we tested in vivo whether synaptic depression by adenosine A 1 receptor (A 1R) activation contributes to BS patterns following GCI. Male Wistar rats were subjected to 1, 5 or 10 min of GCI using a “four-vessel occlusion” model under chloral hydrate anesthesia. Quantification of BS recovery was carried out using BS ratio. During GCI full electrocortical suppression was attained (BS ratio reached 100%). During the following reperfusion the BS ratio returned to 0. The time course of the decay was exponential after 1 and 5-min GCI and bi-exponential after 10-min GCI. The BS recovery was progressively delayed with the duration of ischemia. Administration of the A 1R antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 1.25 mg/kg i.p.) accelerated the post-ischemic BS recovery for all GCI durations. Following the 10-min GCI the effect of DPCPX was only apparent on the initial fast decay of the BS ratio. These data suggest that endogenous adenosine release promotes BS patterns during reperfusion following transient cerebral ischemia. Furthermore, the endogenous A 1R activation may be the primary underlying cause of post-ischemic BS patterns following brief ischemic episodes. It is likely that synaptic depression by post-ischemic A 1R activation functionally disrupts the connectivity within the cortical networks to an extent that promotes BS patterns.

Advance in molecular signal mechanism of myocardial ischemia/reperfusion injury

Ischemia/reperfusion injury is a common physiopathologic change. Lots of researches on mechanism of ischemia/ reperfusion injury have been carried out and new progress in molecular signal mechanism has been obtained. Adenosine binds to adenosine 2A receptor (A2AAR) and activates the Gs protein-cAMP signaling axis, leading to attenuation of reperfusion injury via anti-in- flammatory mechanism. Activation of opioid receptors alleviates reperfusion injury by interaction with anti-apoptotic pro-survival kinase signaling pathways. Ischemic post-conditioning seems to involve in enhanced release of endogenous adenosine. Key words: reperfusion injury ; adenosine ; opioids ; postconditioning ; myocardial ; ischemia/reperfusion ;

Purinergic receptor activation inhibits osmotic glial cell swelling in the diabetic rat retina

The anti-inflammatory glucocorticoid, triamcinolone acetonide, is used clinically for the rapid resolution of diabetic macular edema. Osmotic swelling of glial cells may contribute to the development of retinal edema. Triamcinolone inhibits the swelling of retinal glial cells of diabetic rats. Here, we determined whether the effect of triamcinolone is mediated by a receptor-dependent mechanism. Hyperglycemia was induced in rats with streptozotocin injection. After 6–10 months, the swelling properties of glial cells in retinal slices upon hypotonic challenge were determined. Nucleotide-degrading ecto-enzymes were immunostained in retinal slices and glial cells. Hypotonic challenge did not change the size of glial cell bodies from control retinas but induced swelling of cells from diabetic animals. Triamcinolone inhibited glial cell swelling; this effect was prevented by a selective antagonist of adenosine A1 receptors, an inhibitor of nucleoside transporters, inhibitors of adenylyl cyclase and protein kinase A activation, and inhibitors of potassium and chloride channels. In diabetic (but not control) retinas, the effect of triamcinolone apparently involves extracellular nucleotide degradation. Glial cells from diabetic retinas displayed immunolabeling against nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) which was not observed in control retinas. The mRNA expression for NTPDase1 was significantly increased in the retina of diabetic rats. It is suggested that triamcinolone induces the release and formation of endogenous adenosine that subsequently activates A1 receptors resulting in ion efflux through potassium and chloride channels and prevention of osmotic swelling. Whereas adenosine is liberated via facilitated transport in control retinas, an extracellular formation of adenosine contributes to the effect of triamcinolone in diabetic retinas.

CREB Activation and Ischaemic Preconditioning

Previous studies from our laboratory showed that activation of p38 MAPK is one of the triggers of ischaemic preconditioning. The signalling events downstream of p38 MAPK and their links to the putative final effectors of preconditioning are not clear. The cAMP responsive element-binding protein (CREB) is also phosphorylated by exposure to short episodes of ischaemia/reperfusion, suggesting a triggering action. The aim of this study was to systematically investigate (1) the signalling pathways leading to CREB phosphorylation during an ischaemic or beta-adrenergic preconditioning protocol (2) changes in CREB phosphorylation during sustained ischaemia and their significance in ischaemia/reperfusion injury. The isolated perfused working rat heart was preconditioned by 1 x 5 min global ischaemia or 3 x 5 min global ischaemia and freeze-clamped. Drugs to manipulate CREB activation were added 5 min before onset of ischaemia. Non-preconditioned and preconditioned hearts were subjected to 25 min global or 35 min regional ischaemia, followed by 30 min reperfusion. Infarct sizes were determined using tetrazolium staining. Phosphorylation of CREB was determined by Western blots. Exposure of hearts to 5 min global ischaemia followed by reperfusion, significantly increased CREB phosphorylation This is mediated by, amongst others, release of endogenous catecholamines and adenosine, as indicated by the use of receptor blockers. Events downstream of receptor stimulation were evaluated using inhibitors for PKA (H89), MSK-1 (H89, Ro318220), PKC (bisindolylmaleimide), p38 MAPK (SB203580) and ERK (PD98059). Activation of PKA, PKC, ERK and p38 MAPK is involved in preconditioning-induced CREB phosphorylation. Ischaemia-induced activation of iPLA(2) and cPLA(2) also contribute to CREB phosphorylation as indicated by the use of the inhibitors 4-bromo-enol-lactone (BEL) and AACOF(3,) respectively. Inhibition of CREB phosphorylation by either BEL or AACOF(3) during a preconditioning protocol partially attenuated cardioprotection. CREB phosphorylation was attenuated during sustained global ischaemia of both non-preconditioned and preconditioned hearts. These data suggest that CREB phosphorylation during an ischaemic preconditioning protocol may contribute to triggering preconditioning, while reduced phosphorylation during sustained ischaemia does not appear to be associated with cardioprotection.

Recent insights in the pharmacological actions of methotrexate in the treatment of rheumatoid arthritis

This review presents recent data supporting the methotrexate (MTX) mechanisms of action, which are likely to account for its anti-proliferative and immunosuppressive effects in rheumatoid arthritis (RA). The effects of MTX in vivo may be mediated by reducing cell proliferation, increasing the rate of apoptosis of T cells, increasing endogenous adenosine release, altering the expression of cellular adhesion molecules, influencing production of cytokines, humoral responses and bone formation. Several reports indicate that the effects of MTX are influenced by genetic variants, specific dynamic processes and micro-environmental elements such as nucleotide deprivation or glutathione levels. The challenge for the future will be linking biological and genetic markers relevant to the response to MTX in RA.

Activation of central adenosine A2Areceptors enhances superior laryngeal nerve stimulation-induced apnea in piglets via a GABAergic pathway

Activation of the laryngeal mucosa results in apnea that is mediated through, and can be elicited via electrical stimulation of, the superior laryngeal nerve (SLN). This potent inhibitory reflex has been suggested to play a role in the pathogenesis of apnea of prematurity and sudden infant death syndrome, and it is attenuated by theophylline and blockade of GABA(A) receptors. However, the interaction between GABA and adenosine in the production of SLN stimulation-induced apnea has not been previously examined. We hypothesized that activation of adenosine A(2A) receptors will enhance apnea induced by SLN stimulation while subsequent blockade of GABA(A) receptors will reverse the effect of A(2A) receptor activation. The phrenic nerve responses to increasing levels of SLN stimulation were measured before and after sequential intracisternal administration of the adenosine A(2A) receptor agonist CGS (n = 10) and GABA(A) receptor blocker bicuculline (n = 7) in ventilated, vagotomized, decerebrate, and paralyzed newborn piglets. Increasing levels of SLN stimulation caused progressive inhibition of phrenic activity and lead to apnea during higher levels of stimulation. CGS caused inhibition of baseline phrenic activity, hypotension, and enhancement of apnea induced by SLN stimulation. Subsequent bicuculline administration reversed the effects of CGS and prevented the production of apnea compared with control at higher SLN stimulation levels. We conclude that activation of adenosine A(2A) receptors enhances SLN stimulation-induced apnea probably via a GABAergic pathway. We speculate that SLN stimulation causes endogenous release of adenosine that activates A(2A) receptors on GABAergic neurons, resulting in the release of GABA at inspiratory neurons and subsequent respiratory inhibition.

Wissenschaftlicher Wirksamkeitsnachweis einer fixen Extraktkombination (Ze 91019) von Baldrian und Hopfen, welche traditionell als Einschlafhilfe genutzt wird

Valerian and hops are traditionally used as sleep-inducing aids. Alertness reduces gradually with the prolongation of wakefulness through the release of endogenous adenosine in the frontal basal cortex. Valerian has an adenosine-like action and supports the readiness to fall asleep. The control of the sleep-wake rhythm induces sleep when the time-related interaction is operating properly. The control is closely related to endogenous melatonin secretion. Hops act in a similar way to melatonin. Therefore, the efficacy of a valerian and hops combination in sleep disorder can scientifically be explained.

Reply

To the Editor: We thank Dr. Riksen and colleagues for their comments on our pharmacogenetics study. Although the precise mechanism of action of MTX in rheumatoid arthritis (RA) is unclear, we agree with Dr. Riksen that the effects are probably contributed by endogenous adenosine release and the interactions of MTX with enzymes involved in the folate pathway. Dr. Riksen is right that the mutant 34T allele of AMPD1 leads to reduced activity of AMPD. In skeletal and heart biopsy specimens, heterozygous (CT) individuals have ≥50% reduced enzyme activity, whereas homozygous (TT) individuals have ≤1% residual activity. Riksen et al also speculate that the mutant variant of AMPD reduces the interaction with AICAR. As a consequence of this assumption, it would be less likely that carriage of the 34T allele increases adenosine release. However, there are no data to support their assumption that the affinity of AMPD for AICAR is affected by the mutant 34T allele variant. In addition, studies involving other folate pathway enzymes, such as dihydrofolate reductase and folylpolyglutamate synthase, showed that the activity of the mutant enzyme was reduced, whereas the affinity of the mutant enzymes for their substrates, including antifolates, remained similar or was affected only for other substrates such as L-glutamate. Therefore, we think that the reduced activity of AMPD due to the mutant AMPD1 34T allele in the enzyme and the inhibition of AMPD via AICAR may act complementarily in enhancing adenosine release. Biochemical studies are urgently needed to investigate the effect of the AMPD1 34C >T variant in health and disease. The effect of the AMPD1 34C>T variant itself on plasma adenosine is hardly documented. The scarce evidence indicates that the T allele probably increases adenosine: during exercise (not during rest), venous adenosine levels increase in carriers of 34TT. Furthermore, the AMPD1 34C >T variant improves survival of patients with established congestive heart failure or patients with established coronary artery disease (9, 10), probably by promoting release of adenosine, a well-known cardioprotective substance (11). How AMPD1 34C >T interacts with MTX in RA (factors that influence purine enzyme kinetics) is purely speculative. Second, Dr. Riksen and colleagues raise concerns about confounding in our results due to the efficacy cutoff points used in our data analysis. Our approach increases the power of the study, because it categorizes all patients with a Disease Activity Score (DAS) into 2 groups instead of 3 groups. If we use the precise EULAR response criteria, patients are categorized into 66 responders, 41 nonresponders, and 79 moderate responders. Comparing the precise EULAR definition of nonresponse and good response with our definition of response and nonresponse with a DAS of ≤2.4 shows that only 4 patients were misclassified. The genotype analysis with the precise EULAR definition resulting in 66 responders and 41 nonresponders shows similar results. The association for the ATIC CC genotype remains significant, while the AMPD1 and ITPA genotypes are nonsignificant due to the reduced numbers of responders and nonresponders.