Modelagem celular in vitro da Doença de Huntington com ácido 3-nitropropiônico e ácido quinolínico & Avaliação das funções neuroprotetoras da adenosina / In vitro cell modeling of Huntington's Disease with 3-nitropropionic acid and quinolinic acid and evaluation of the neuroprotective functions of adenosine

A Doença de Huntington (Huntington's disease - HD) trata-se de uma patologia neurodegenerativa hereditária caracteriza por meio da expressão das proteínas huntingtinas mutantes (mHtt), das mortes dos neurônios espinhais médios (medium spiny neurons MSNs) GABAérgicos D2-positivos do striatum e da hipercinesia. Uma hipótese se refere à função das mHtts de potencializarem os efeitos excitotóxicos das estimulações dos receptores de NMDA (NMDAR) por meio da inibição da succinato desidrogenase, resultando em desequilibrio das [Ca2+]i, estresse oxidativo e apoptose. A adenosina agonista dos receptores purinérgicos P1 tem sido descrita por conta das suas funções neuroprotetoras e neuromodulatórias. Assim, estabelecemos dois modelos in vitro da HD fundamentados nas neurodiferenciações das linhagens murinas de célula-tronco embrionárias E14-TG2a e progenitoras neurais do hipocampo HT-22; seguidas pelos tratamentos com ácido quinolínico (QA) agonista seletivo dos NMDARs , na ausência e na presença do ácido 3-nitropropiônico (3-NP) inibidor irreversível da succinato desidrogenase. Estes modelos foram utilizados nas avaliações das funções neuroprotetoras da adenosina. Os neurônios pós-mitóticos das culturas de E14-TG2a diferenciadas foram caracterizados conforme os MSNs GABAérgicos do striatum; enquanto os neurônios HT-22 diferenciados foram caracterizados de modo inespecífico. Metodologia: imunofluorescência (microscopia e citometria); PCR em tempo real; análise das variações dos potenciais das membranas plasmáticas e das variações transientes das [Ca2+]i por microfluorimetria; e quantificações das reduções do AlamarBlue® (% de sobrevida celular) e das atividades extracelulares de LDH (U/L) (necrose) por espectrometria. Avaliamos a capacidade do 3-NP de potencializar os efeitos excitotóxicos do QA comparando dois grupos de neurônios HT-22 diferenciados: QA 8mM (EC50) (controle); e 3-NP 5mM/QA 8mM. Avaliarmos o potencial neuroprotetor da adenosina comparando quatro grupos de neurônios HT-22 diferenciados: QA 8mM; adenosina 250µM/QA 8mM; 3-NP 5mM/QA 8mM; 3-NP 5mM/adenosina 250µM/QA 8mM. Os neurônios pós-mitóticos derivados das E14TG2a foram classificados como MSNsGABAérgicos do striatum integrantes de uma cultura neuronal heterogênea semelhante às conexões nigroestriatais, corticoestriatais, striatonigral e striatopallidal. Os neurônios HT-22 diferenciados perfaziam uma cultura neuronal heterogênea, não totalmente madura, composta por neurônios glutamatérgicos, dopaminérgicos, colinérgicos e GABAérgicos. Os neurônios HT-22 diferenciados 3-NP 5mM apresentaram menores % de sobrevida celular após os tratamentos com QA 8mM por 24h (p<0.05); e maiores amplitudes das variações das [Ca2+]i dependentes do QA 8mM (p<0.05) (cinética 6 minutos). Por outro lado, os neurônios HT-22 diferenciados pré- tratados com 3-NP 5mM apresentaram menores atividades extracelulares de LDH após o tratamento com QA 8mM por 24h menor proporção de necrose. Os pré-tratamentos com adenosina 250µM indicaram uma tendência dos efeitos neuroprotetores (p>0.05) maiores % de sobrevida celular; menores atividades extracelulares de LDH; e menores amplitudes das variações transientes das [Ca2+]i. Em conjunto, nossos resultados indicam que a inibição da succinato desidrogenase potencializa os efeitos excitotóxicos dos NMDARs por meio da alteração das [Ca2+]i e, provavelmente, dos mecanismos de morte celular; enquanto a adenosina apenas tendeu à neuroproteção

Huntington's disease (HD) is a hereditary neurodegenerative pathology characterized by mutant huntingtin proteins (mHtt) expression, striatum D2-positive GABAergic medium spiny neurons (MSNs) cell death and hyperkinetic motor symptoms development. One hypothesis refers to the principle that mHtt potentiates the excitotoxic effects of NMDA receptor (NMDAR) stimulation by the inhibition of mitochondrial succinate dehydrogenase, resulting in [Ca2+]i imbalance, oxidative stress and apoptosis. Adenosine P1 purinergic receptor agonist is related to neuroprotective and neuromodulatory functions. Thus, we established two in vitro HD models based on the neurodifferentiation of murine embryonic stem cell lines E14-TG2a and hippocampal neuroprogenitor cell line HT-22 followed by treatment with quinolinic acid (QA) selective agonist of NMDARs , in the absence and in the presence of 3-nitropropionic acid (3-NP) irreversible inhibitor of succinate dehydrogenase. These models were used to assess the neuroprotective functions of adenosine. Post-mitotic neurons from differentiated E14-TG2a cultures were characterized according to striatum's GABAergic MSNs; while the differentiated HT-22 neurons were characterized in a non-specific way. Methodology included immunofluorescence (microscopy and cytometry); real-time PCR; analysis of variations in the plasma membrane potentials and of transient variations in the [Ca2+]i by microfluorimetry; and quantification of AlamarBlue® reductions (% cell survival) and of extracellular LDH activity (U/L) (necrosis) by spectrometry. We evaluated the ability of 3-NP to potentiate the excitotoxic effects of QA by comparing two groups of differentiated HT-22 neurons: 8mM QA (control); and 5mM 3-NP/8mM QA. We evaluated the neuroprotective potential of adenosine comparing four groups of differentiated HT-22 neurons: QA 8mM; 250µM adenosine/8mM QA; 5mM 3-NP/8mM QA; 5mM 3-NP/250µM adenosine/8mM QA. Postmitotic neurons derived from E14TG2a were classified as striatums GABAergic MSNs that are part of a heterogeneous neuronal culture similar to nigrostriatal, corticostriatal, striatonigral, and striatopallidal connections. Differentiated HT-22 neurons consisted of a heterogeneous neuronal culture and not fully mature glutamatergic,dopaminergic, cholinergic and GABAergic neurons. Differentiated HT-22 neurons following 5mM 3-NP treatment showed lower % cell survival after treatments with 8mM QA for 24h (p<0.05); and higher amplitudes of the variations of [Ca2+]i induced by 8mM QA (p<0.05) (kinetics 6 minutes). On the other hand, differentiated HT-22 neurons 5mM 3-NP showed lower extracellular LDH activities after treatment with 8mM QA for 24h indicating a lower proportion of necrotic cells. Pretreatments with 250µM adenosine indicated a trend towards neuroprotective effects, such as higher percentages of cell survival; lower extracellular LDH activities; and lower amplitudes of transient variations of [Ca2+]i. Taken together, our results indicate that succinate dehydrogenase inhibition potentiated the excitotoxic effects of NMDARs by altering [Ca2+]i and, probably, cell death mechanisms, while adenosine only to neuroprotection

Critical View on the Usage of Ribavirin in Already Existing Psychostimulant-Use Disorder.

Substance-use disorder represents a frequently hidden non-communicable chronic disease. Patients with intravenous drug addiction are at high risk of direct exposure to a variety of viral infections and are considered to be the largest subpopulation infected with the hepatitis C virus. Ribavirin is a synthetic nucleoside analog that has been used as an integral component of hepatitis C therapy. However, ribavirin medication is quite often associated with pronounced psychiatric adverse effects. It is not well understood to what extent ribavirin per se contributes to changes in drug-related neurobehavioral disturbances, especially in the case of psychostimulant drugs, such as amphetamine. It is now well-known that repeated amphetamine usage produces psychosis in humans and behavioral sensitization in animals. On the other hand, ribavirin has an affinity for adenosine A1 receptors that antagonistically modulate the activity of dopamine D1 receptors, which play a critical role in the development of behavioral sensitization. This review will focus on the current knowledge of neurochemical/ neurobiological changes that exist in the psychostimulant drug-addicted brain itself and the antipsychotic-like efficiency of adenosine agonists. Particular attention will be paid to the potential side effects of ribavirin therapy, and the opportunities and challenges related to its application in already existing psychostimulant-use disorder.

Studying the collective motions of the adenosine A2A receptor as a result of ligand binding using principal component analysis.

Adenosine receptors (ARs) belong to family A of GPCRs that are involved in many diseases, including cerebral and cardiac ischemic diseases, immune and inflammatory disorders, etc. Thus, they represent important therapeutic targets to treat these conditions. Computational techniques such as molecular dynamics (MD) simulations permit researchers to obtain structural information about these proteins, and principal component analysis (PCA) allows for the identification of collective motions. There are available structures for the active form (3QAK) and the inactive form (3EML) of A2AR which permit us to gain insight about their activation/inactivation mechanism. In this work, we have proposed an inverse strategy using MD simulations where the active form was coupled to the antagonist caffeine and the inactive form was coupled to adenosine agonist. Moreover, we have included four reported thermostabilizing mutations in the inactive form to study A2AR structural differences under different conditions. Some observations stand out from the PCA studies. For instance, the apo structures showed remarkable similarities, and the principal components (PCs) were rearranged in a ligand-dependent manner. Additionally, the active conformation was less stable compared to the inactive one. Some PCs inverted their direction in the presence of a ligand, and comparison of the PCs between 3EML and 3EML_ADN showed that adenosine induced major changes in the structure of A2AR. Rearrangement of PCs precedes and drives conformational changes that occur after ligand binding. Knowledge about these conformational changes provides important insights about the activity of A2AR.

Glaucoma Drugs in the Pipeline.

Glaucoma is a chronic disease that can be challenging to treat for both patients and physicians. Most patients will require more than 1 medication over time to maintain their intraocular pressure (IOP) at a physiologically benign level. Patients may become refractory to existing compounds and many struggle with adherence to multiple topical drop regimens. The field of glaucoma therapeutics has been advancing rapidly with an emphasis on compounds comprising multiple molecules/mechanisms of action that offer additivity and are complementary to current therapeutics. Several new topical drop compounds directly targeting the trabecular meshwork (TM)/Schlemm canal/conventional outflow pathway to reduce outflow resistance have obtained US Food and Drug Administration approval in the past year. These include rho kinase inhibitors and nitric oxide donating compounds. Alternative therapies that offer long-term IOP lowering while removing the patient from the drug delivery system are moving forward in development. These include gene therapy and stem cell strategies, which could ease or eliminate the burden of topical drop self-administration for several years. Additionally, a variety of novel formulations and devices are in development that aim for controlled, steady state delivery of therapeutics over periods of months. The future of glaucoma therapy is focusing on an increase in specificity for the individual patient: their type of glaucoma; underlying mechanisms; genetic make-up; comorbid conditions; and rate of progression. Maintaining functional vision and improving patient outcomes remains the goal in glaucoma therapeutics. The current collection of novel therapeutics offers an expanded set of tools to achieve that goal.

The Psychopharmacology of Effort-Related Decision Making: Dopamine, Adenosine, and Insights into the Neurochemistry of Motivation.

Effort-based decision making is studied using tasks that offer choices between high-effort options leading to more highly valued reinforcers versus low-effort/low-reward options. These tasks have been used to study the involvement of neural systems, including mesolimbic dopamine and related circuits, in effort-related aspects of motivation. Moreover, such tasks are useful as animal models of some of the motivational symptoms that are seen in people with depression, schizophrenia, Parkinson's disease, and other disorders. The present review will discuss the pharmacology of effort-related decision making and will focus on the use of these tasks for the development of drug treatments for motivational dysfunction. Research has identified pharmacological conditions that can alter effort-based choice and serve as models for depression-related symptoms (e.g., the vesicular monoamine transport-2 inhibitor tetrabenazine and proinflammatory cytokines). Furthermore, tests of effort-based choice have identified compounds that are particularly useful for stimulating high-effort work output and reversing the deficits induced by tetrabenazine and cytokines. These studies indicate that drugs that act by facilitating dopamine transmission, as well as adenosine A2A antagonists, are relatively effective at reversing effort-related impairments. Studies of effort-based choice may lead to the identification of drug targets that could be useful for treating motivational treatments that are resistant to commonly used antidepressants such as serotonin transport inhibitors.

Pharmacological targeting of adenosine receptor signaling.

Adenosine receptor signaling plays important roles in normal physiology, but is also known to modulate the development or progression of several different diseases. The design of new, efficient, and safe pharmacological approaches to target the adenosine system may have considerable therapeutic potential, but is also associated with many challenges. This review summarizes the main challenges of adenosine receptor targeted treatment including tolerance, disease stage, cell type-specific effects, caffeine intake, adenosine level assessment and receptor distribution in vivo. Moreover, we discuss several potential ways to overcome these obstacles (i.e., the use of partial agonists, indirect receptor targeting, allosteric enhancers, prodrugs, non-receptor-mediated effects, neoreceptors, conditional knockouts). It is important to address these concerns during development of new and successful therapeutic approaches targeting the adenosine system.

Dopamine D2 Receptor-Mediated Regulation of Pancreatic ß Cell Mass.

Understanding the molecular mechanisms that regulate ß cell mass and proliferation is important for the treatment of diabetes. Here, we identified domperidone (DPD), a dopamine D2 receptor (DRD2) antagonist that enhances ß cell mass. Over time, islet ß cell loss occurs in dissociation cultures, and this was inhibited by DPD. DPD increased proliferation and decreased apoptosis of ß cells through increasing intracellular cAMP. DPD prevented ß cell dedifferentiation, which together highly contributed to the increased ß cell mass. DRD2 knockdown phenocopied the effects of domperidone and increased the number of ß cells. Drd2 overexpression sensitized the dopamine responsiveness of ß cells and increased apoptosis. Further analysis revealed that the adenosine agonist 5'-N-ethylcarboxamidoadenosine, a previously identified promoter of ß cell proliferation, acted with DPD to increase the number of ß cells. In humans, dopamine also modulates ß cell mass through DRD2 and exerts an inhibitory effect on adenosine signaling.

Beneficial and detrimental role of adenosine signaling in diseases and therapy.

Adenosine is a major signaling nucleoside that orchestrates cellular and tissue adaptation under energy depletion and ischemic/hypoxic conditions by activation of four G protein-coupled receptors (GPCR). The regulation and generation of extracellular adenosine in response to stress are critical in tissue protection. Both mouse and human studies reported that extracellular adenosine signaling plays a beneficial role during acute states. However, prolonged excess extracellular adenosine is detrimental and contributes to the development and progression of various chronic diseases. In recent years, substantial progress has been made to understand the role of adenosine signaling in different conditions and to clarify its significance during the course of disease progression in various organs. These efforts have and will identify potential therapeutic possibilities for protection of tissue injury at acute stage by upregulation of adenosine signaling or attenuation of chronic disease progression by downregulation of adenosine signaling. This review is to summarize current progress and the importance of adenosine signaling in different disease stages and its potential therapeutic effects.

[The receptorial responsiveness method (RRM): a new possibility to estimate the concentration of pharmacologic agonists at their receptors]. / Uj lehetoség farmakológiai agonisták receptorközeli koncentrációjának becslésére: a receptoriális válaszkészség módszer (RRM).

Cardiovascular disease is the biggest challenge in terms of life expectancy in developed countries. Adenosine contributes to the adaptation of the heart to ischemia and hypoxia, because adenosine, in addition to its metabolite role in the nucleic acid metabolism, is the endogenous agonist of the ubiquitous adenosine receptor family. Adenosine receptor activation is beneficial in most cases, it improves the balance between energy supply and consumption, reduces injury caused by stressors and inhibits the unfavorable tissue remodeling. Pharmacological manipulation of cardioprotective effects evoked by adenosine is an important, although to date not sufficiently utilized endeavor that may have therapeutic and preventive implications in cardiovascular diseases. As the ligand binding site of adenosine receptors is accessible from the extracellular space, it is especially important to know the adenosine concentration of the interstitial fluid ([Ado](ISF)). However, in the functioning heart, [Ado](ISF) values range in an extremely wide interval, spanning from nano- to micromolar concentrations, as estimated by the commonly used methods. Our recently developed procedure, the receptorial responsiveness method (RRM), may resolve this problem in certain cases. RRM enables quantification of an acute increase in the concentration of a pharmacological agonist, uniquely in the microenvironment of the receptors of the given agonist. As a limitation, concentration of agonists with short half-life (just like adenosine) at their receptors can only be quantified with the equieffective concentration of a stable agonist exerting the same action. In a previous study using RRM, inhibition of the transmembrane nucleoside transport in the euthyroid guinea pig atrium produced an increase in [Ado](ISF) that was equieffective with 18.8 +/- 3 nM CPA (N6-cyclopentyladenosine, a stable, selective A1 adenosine receptor agonist). This finding is consistent with observations of others, i.e., in the normoxic heart, adenosine flow is directed into the cell interior, and thus transport blockade elevates the extracellular adenosine level. In turn, nucleoside transport inhibition in the hyperthyroid guinea pig atrium caused a rise in [Ado](ISF) equieffective with 46.5 +/- 13.7 nM CPA. In sum, our work team was the first to demonstrate that adenosine transport in the hyperthyroid atrium has the same direction but is more intense as/than that in the euthyroid one.

[Adenosine alleviates hypoxia-induced rat right ventricular hypertrophy through the NHE-1/CaN signal pathway].

OBJECTIVE: To investigate the effect of adenosine and its agonist on hypoxia-induced right ventricular hypertrophy (RVH) and explore the underlying mechanism. METHODS: Fifty-six rats were randomly divided into normoxia group, hypoxia group, and treated hypoxia groups (with different treatments with adenosine, A1 receptor agonist CPA, A2 receptor agonist NECA, CPA plus A1 receptor inhibitor DPCPX, or NECA plus A2B receptor inhibitor MRS1754). The rats except for those in normoxia group were exposed to normobaric chronic hypoxia (9.5%-10.5% oxygen) for 21 days, and the corresponding treatments were administered since the 7th day of hypoxia till day 21 via implantable capsule with a pressure pump. After the treatments, the right ventricles were then removed and weighed for evaluation of hypertrophy, and the expressions of NHE-1 and CnAß mRNA in the myocardial tissue were detected using RT-PCR. RESULTS: After a 21-day hypoxia, the rats showed significantly increased RV/(LV+S) ratio (0.369∓0.033) and RV/BW ratio (0.75∓0.095) compared to those in normoxia group (0.271∓0.010 and 0.59∓0.039, respectively; P<0.001), adenosine treatment group (0.281∓0.022 and 0.65∓0.077, respectively; P<0.001, P=0.025), hypoxia with CPA group (0.313∓0.021 and 0.66∓0.067, respectively P<0.001), and hypoxia with NECA group(0.333∓0.019, and 0.68∓0.074, respectively P<0.001). The NHE-1 and CnAß mRNA levels in hypoxia group were significantly higher than those in normoxia group, adenosine treatment group, hypoxia with CPA group, and hypoxia with NECA group(P<0.001). CONCLUSION: Adenosine and its agonist can inhibit hypoxia-induced RVH in rats through the NHE-1/CaN signal pathway.

Current antiplatelet options for NSTE-ACS patients.

Non-ST elevation (NSTE) myocardial infarction and unstable angina are the most common clinical presentations of acute coronary syndrome (ACS). Platelet activation is central to the pathogenesis of NSTE-ACS and consensus guidelines that advocate early revascularization supported by intensive antiplatelet therapy. This review examines the drugs used concurrently with aspirin as dual antiplatelet therapy in the NSTE-ACS setting. Clopidogrel represented an important therapeutic advance. However, variations in platelet response and a relatively slow onset of action compromise outcomes with clopidogrel. Evidence reviewed in this article shows that in NSTE-ACS patients, ticagrelor and prasugrel are more effective than clopidogrel and are relatively well tolerated, with an acceptable and manageable bleeding risk. The literature suggests several differences between ticagrelor and prasugrel that should allow clinicians to better tailor treatment to the patient. Head-to-head comparisons are now needed to compare directly the risks and benefits of ticagrelor and prasugrel in NSTE-ACS. Further studies also need to address other outstanding issues such as the benefits and risks of prasugrel pre-treatment and to stratify efficacy and tolerability according to diabetes mellitus (DM) and other co-morbidities. In the meantime, the issues discussed in this review should enhance clinicians' ability to optimize and individualize NSTE-ACS treatment, thereby further reducing the morbidity and mortality associated with this common cardiovascular condition.

The effect of adenosine on pro-inflammatory cytokine production by porcine T cells.

Adenosine is a well described anti-inflammatory modulator of immune responses. The aim of the present study was to describe the role of common adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) in cytokine production by main porcine T cell subpopulations. TNF-α, IFN-γ, IL-2 and IL-10 were detected by multicolor flow cytometry together with cell surface markers CD3, CD4 and CD8. It was found that NECA inhibits (in a dose-dependent manner) production of pro-inflammatory TNF-α and Th1-associated cytokines IFN-γ, IL-2 in all concanavalin A-stimulated T cell subpopulations. Moreover, production of IL-10 was potentiated in all T cell subpopulations tested. These corresponded well with the fact that all T cell subsets expressed mRNA for adenosine receptor (AR) subtypes to comparable extents. Contrary to concanavalin A-stimulated cells, NECA had a moderate effect on PMA-stimulated T cells, suggesting that AR in pigs acts via signaling pathways not associated with protein-kinase C. Non-selective antagonist CGS15943 as well as allosteric modulator SCH202676 failed to reverse the effect of NECA in pigs. In conclusion, NECA has an anti-inflammatory effect on porcine T cell subpopulations.

Acute down-regulation of adenosine A(1) receptor activity in status epilepticus.

PURPOSE: Status epilepticus (SE) remains a potentially devastating condition that quickly becomes refractory to antiepileptic drug treatment and arises as a result of a failure of the brain's endogenous antiepileptic mechanisms. Understanding these mechanisms and how they are disrupted in SE is necessary in order to identify novel therapeutic approaches. Adenosine is considered an endogenous anticonvulsant. Extracellular concentrations increase coinciding with seizure termination; activation of A(1) receptors (A(1)Rs) reduces seizure-induced damage and epileptiform activity. The present study examines the effectiveness of focal drug delivery in a model of limbic SE that closely resembles the human condition and describes, for the first time, alterations in A(1)R signaling during prolonged seizures that may contribute to the progression from self-terminating seizures to self-sustaining SE (SSSE). METHODS: We developed a rat perforant path stimulation model in which 50% of rats develop SSSE and tested whether modulation of A(1)Rs influenced SSSE development when drugs were infused to the dentate gyrus. We further determined the ability of A(1)Rs to modulate perforant path to granule cell transmission in hippocampal slices taken from sham-operated control and post-SE animals. KEY FINDINGS: Adenosine (3 µM) and the A(1)R-selective agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 10 µM) reduced the severity of SSSE as measured by spike count, electroencephalography power and behavioral seizure score. In addition, CCPA suppressed the progression to SSSE. Surprisingly, the A(1)R-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 µm) had no effect on the severity of or progression to SSSE, suggesting a lack of intrinsic A(1)R activation. Immunohistochemistry revealed no alterations in total A(1)R expression. However, we observed a marked down-regulation of A(1)R modulation of neurotransmission in vitro, indicating acute A(1)R desensitization. SIGNIFICANCE: These findings indicate that A(1)R activation can prevent the progression to SE and suggest that reduced A(1)R signaling promotes the transition of seizures to SSSE.

Differential AMPK phosphorylation sites associated with phenylephrine vs. antihypertrophic effects of adenosine agonists in neonatal rat ventricular myocytes.

Stimulation of cardiac AMP-activated protein kinase (AMPK) has been demonstrated in both prohypertrophic and antihypertrophic settings, although the reasons for such discrepant results are not well understood. We determined how AMPK is regulated in response to phenylephrine-induced cardiomyocyte hypertrophy and assessed whether AMPK activity may be a factor underlying the antihypertrophic effect of adenosine receptor agonists. The role of AMPK in hypertrophic responses was determined by assessing the effect of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside on three hypertrophic indexes, including protein synthesis, cell surface area, and fetal gene expression. The changes in phosphorylation of the catalytic alpha-subunit of AMPK at two different sites, Thr(172) and Ser(485/491), in response to phenylephrine and adenosine receptor agonists were also examined. 5-Aminoimidazole-4-carboxyamide ribonucleoside completely abolished phenylephrine-induced increases in protein synthesis, cell surface area, and fetal gene expression. AMPK phosphorylation time course studies revealed that phenylephrine induced a time-dependent activation at site Ser(485/491), in contrast to adenosine receptor agonists, which demonstrated rapid AMPK phosphorylation at Thr(172). Furthermore, the phosphorylation at Ser(485/491) by phenylephrine was not affected by the addition of adenosine receptor agonists, although, conversely, phosphorylation of AMPK at Thr(172) by adenosine receptor agonists was abrogated by the addition of phenylephrine. We propose from these results that cardiomyocyte hypertrophic and antihypertrophic responses, at least with respect to inhibition of phenylephrine-induced hypertrophy by adenosine receptor agonists, are mediated by multisite AMPK regulation. The latter are reflected by increased phosphorylation at Ser(485/491) and at Thr(172), associated with prohypertrophic and antihypertrophic responses, respectively.

Changes induced by formalin pain in central alpha1-adrenoceptor density are modulated by adenosine receptor agonists.

We aimed to elucidate the role of alpha(1)-adrenoceptors in adenosine analgesia in the formalin test. Formalin was injected into the hind paw of male CD-1 mice after injection of adenosine A(1) or A(2a) receptor agonists, CPA, [N(6)-cyclopentyladenosine], and CGS21680 [2-p-(2-carboxyethyl)-phenylethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride]. In the behavioral experiment, alpha(1)-adrenoceptors were blocked by an alpha(1)-adrenoceptor antagonist prazosin, 0.01 mg/kg i.p., and the time mice spent paw licking was recorded for the early (0-15 min) and late (15-60 min) phase of formalin pain. In the neurochemical experiments, mice were killed 15 or 45 min after formalin injection. The density of alpha(1)-adrenoceptors was assessed in various brain areas and in the lumbar spinal cord by [(3)H]prazosin autoradiography. Adenosine agonists produced analgesia in both phases of formalin pain, while prazosin showed a tendency to pronociceptive action in the late phase, and antagonized the effect of CGS21680. After formalin injection, alpha(1)-adrenoceptor density was elevated in some brain areas, mainly in the late phase (some contralateral amygdaloid and ipsilateral thalamic nuclei) and depressed in others (early phase in the ipsilateral spinal cord and late phase in both ipsi- and contralateral sensorimotor cortex). Elevation of alpha(1)-adrenoceptor density, which may be interpreted as a defensive response, did not develop in several cases of CPA-pretreated mice. This suggests that the analgesic effect of adenosine A(1) receptor activation renders the defensive response unnecessary. The depression of alpha(1)-adrenoceptors may suggest development of hypersensitivity in a given structure, and this was antagonized by CGS21680, suggesting the role of A(2a) receptors in control of inflammatory formalin pain.

Estimation of endogenous adenosine activity at adenosine receptors in guinea-pig ileum using a new pharmacological method.

AIM: Adenosine modulates neurotransmission and in the intestine adenosine is continuously released both from nerves and from smooth muscle. The main effect is modulation of contractile activity by inhibition of neurotransmitter release and by direct smooth muscle relaxation. Estimation of adenosine concentration at the receptors is difficult due to metabolic inactivation. We hypothesized that endogenous adenosine concentrations can be calculated by using adenosine receptor antagonist and agonist and dose ratio (DR) equations. METHODS: Plexus-containing guinea-pig ileum longitudinal smooth muscle preparations were made to contract intermittently by electrical field stimulation in organ baths. Schild plot regressions were constructed with 2-chloroadenosine (agonist) and 8-(p-sulfophenyl)theophylline (8-PST; antagonist). In separate experiments the reversing or enhancing effect of 8-PST and the inhibiting effect of 2-chloroadenosine (CADO) were analysed in the absence or presence of an adenosine uptake inhibitor (dilazep), and nucleoside overflow was measured by HPLC. RESULTS: Using the obtained DR, baseline adenosine concentration was calculated to 28 nm expressed as CADO activity, which increased dose dependently after addition of 10(-6) m dilazep to 150 nm (P < 0.05). HPLC measurements yielded a lower fractional increment (80%) in adenosine during dilazep, than found in the pharmacological determination (440%). CONCLUSION: Endogenous adenosine is an important modulator of intestinal neuro-effector activity, operating in the linear part of the dose-response curve. Other adenosine-like agonists might contribute to neuromodulation and the derived formulas can be used to calculate endogenous agonist activity, which is markedly affected by nucleoside uptake inhibition. The method described should be suitable for other endogenous signalling molecules in many biological systems.

A2A adenosine receptor overexpression and functionality, as well as TNF-alpha levels, correlate with motor symptoms in Parkinson's disease.

The antagonistic interaction between adenosine and dopamine receptors could have important pathophysiological and therapeutic implications in Parkinson's disease (PD). The primary aim of this study was to investigate the expression, affinity, and density of A(1), A(2A), A(2B), and A(3) adenosine receptors (ARs) and D(2) dopamine receptors (D(2)Rs) in PD. An increase in A(2A)AR density in putamen was found. The presence and functionality of ARs in human lymphocyte and neutrophil membranes from patients with PD revealed a specific A(2A)AR alteration compared with healthy subjects. A statistically significant linear correlation among the A(2A)AR density, functionality, or tumor necrosis factor-alpha (TNF-alpha) levels and Unified Parkinson's Disease Rating Scale (UPDRS) motor score was reported. Adenosine concentration and TNF-alpha levels were increased in plasma of patients with PD. In rat adrenal pheochromocytoma (PC12) cells, a widely useful model, adenosine antagonists decreased dopamine uptake, and an opposite effect was mediated by A(2A) agonists. This is the first report showing the presence of an A(2A)AR alteration in putamen in PD that mirrors a similar up-regulation in human peripheral blood cells. Moreover, the correlation found between A(2A)AR density or A(2A) agonist potency and UPDRS motor score highlights the central role of A(2A)ARs in the pharmacological treatment of PD.

A highly conserved tryptophan residue in the fourth transmembrane domain of the A adenosine receptor is essential for ligand binding but not receptor homodimerization.

Dimerization between G protein-coupled receptors (GPCRs) is a clearly established phenomenon. However, limited information is currently available on the interface essential for this process. Based on structural comparisons and sequence homology between rhodopsin and A(1) adenosine receptor (A(1)R), we initially hypothesized that four residues in transmembrane (TM) 4 and TM5 are involved in A(1)R homodimerization. Accordingly, these residues were substituted with Ala by site-directed mutagenesis. Interestingly, the mutant protein displayed no significant decrease in homodimer formation compared with wild-type A(1)R, as evident from coimmunoprecipitation and BRET(2) analyses (improved bioluminescence resonance energy transfer system offered by Perkin-Elmer Life Sciences), but lost ligand binding activity almost completely. Further studies disclosed that this effect was derived from the mutation of one particular residue, Trp132, which is highly conserved among many GPCRs. Confocal immunofluorescence and cell-surface biotinylation studies revealed that the mutant receptors localized normally at transfected cell membranes, signifying that loss of ligand binding was not because of defective cellular trafficking. Molecular modeling of the A(1)R-ligand complex disclosed that Trp132 interacted with several residues located in TM3 and TM5 that stabilized agonist binding. Thus, loss of interactions of Trp with these residues may, in turn, disrupt binding to agonists. Our study provides strong evidence of the essential role of the highly conserved Trp132 in TM4 of adenosine receptors.

Regulation of somatostatin release by adenosine in the mouse stomach.

Adenosine inhibits gastric acid secretion, either directly by acting on acid-secreting parietal cells or indirectly by stimulating the release of the acid inhibitor, somatostatin. The present study examined the role of adenosine on somatostatin release in an isolated vascularly perfused mouse stomach model. Concentrations of exogenous adenosine >or= 1.0 microM stimulated gastric release of somatostatin-like immunoreactivity (SLI), and this effect was blocked by the A(2A) receptor antagonist ZM 241385 [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol]. The A(2A) receptor agonist CGS 21680 [2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride] augmented SLI release in a concentration-dependent manner, suggesting that A(2A) receptor activation is involved in the stimulatory effect of adenosine on SLI release. Conversely, SLI release was inhibited by the A(1) receptor agonists N(6)-cyclopentyladenosine and 2-chloro-N(6)-cyclopentyladenosine and lower concentration of adenosine (0.01 microM). The involvement of specific adenosine receptors in controlling the release of gastric SLI was also examined using A(2A) receptor knockout (A(2A)R-KO) mice. In these mice, adenosine (10 microM) inhibited SLI release, and the effect was abolished by the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, suggesting a link between the selective A(1) activation and inhibition of SLI release. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride augmented SLI release in wild-type controls but not in the presence of ZM 241385 or in A(2A)R-KO mice. We conclude that adenosine has dual actions on regulating mouse gastric SLI release: stimulatory at higher concentrations through the A(2A) receptor and inhibitory at lower concentrations through the A(1) receptor, whereas A(2B) and A(3) receptors have a minimal role.