Daniela Tuscano

Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells

Low‐affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real‐time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF‐α) treatment (24‐ h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF‐α markedly reduced agonist‐dependent receptor phosphorylation on threonine residues and attenuated agonist‐mediated A2B ARs desensitization. In the presence of TNF‐α, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF‐α. These results suggest that, in ADF cells, TNF‐α selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.

Agonist‐Induced Internalization and Recycling of the Human A3 Adenosine Receptors

Abstract: A3 adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen‐dependent nature of the therapeutic effects probably related to receptor desensitization and down‐regulation. Here we studied the agonist‐induced internalization of human A3 adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N6‐(4‐amino‐3‐[125I]iodobenzyl)adenosine‐5′‐N‐methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A3 adenosine receptor showed a profile typical of these receptors in other cell lines (KD = 1.3 ± 0.08 nM; Bmax = 400 ± 28 fmol/mg of proteins). The iodinated agonist, bound at 4°C to whole transfected cells, was internalized by increasing the temperature to 37°C with a rate constant of 0.04 ± 0.034 min‐1. Agonist‐induced internalization of A3 adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short‐term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02 ± 0.0017 min‐1. Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.

Serotonin-mediated phosphorylation of extracellular regulated kinases in platelets of patients with panic disorder versus controls

Phosphorylation of extracellular signal-regulated kinases (ERK 1/2) represents a converging intracellular signalling pathway which is involved in the modulation of gene transcription and may contribute to the feed-back regulation of neurotransmitter receptor functioning. The purpose of the current study was to investigate the serotonin-mediated phosphorylation of ERK 1/2 in platelets from patients (n = 17) with panic disorder, with respect to healthy volunteers (n = 17). Patients presented a severe symptomatology as assessed by the self-report rating scales for panic-agoraphobic (PAS-SR) and mood (MOOD-SR) spectrum, and by Clinical Global Impression Severity Scale (CGI-S). In platelets from healthy volunteers, serotonin induced a rapid increase of ERK 1/2 phosphorylation with a transient monophasic kinetic. The dose-response curves showed this effect was concentration dependent with an average of the EC(50) value of 22.8 +/- 2.4 microM. Platelet pre-incubation with 5HT(1A) and 5HT(2A) antagonists, pindobind and ritanserin, significantly inhibited serotonin-mediated kinase activation with an EC(50) of 3.2 +/- 0.2 and 1.99 +/- 0.08 nM, respectively, suggesting an involvement of these specific receptor subtypes in serotonin-mediated response. Furthermore, the 5HT(1A) and 5HT(2A) agonists, 8-hydroxy-N,N-dipropyl-aminotetralin (8OH-DPAT) and 1-(2,5-dimethoxy)-4-iodophenyl-2-aminopropane (DOI), were able to modulate ERK 1/2 phosphorylation in a concentration-dependent manner with an EC(50) value of 3.1 +/- 0.2 and 76 +/- 4.5 nM, respectively. ERK 1/2 phosphorylation was not observed after serotonin treatment of platelets from drug-free panic disorder patients, suggesting an alteration in intracellular phosphorylative pathways. Since ERK 1/2 responsiveness to other stimulus, such as collagen and thrombin, was comparable in platelets from healthy volunteers and patients, our results suggested that a specific alteration of serotonergic system occurred in panic disorder. Further studies to investigate 5HT(1A) and 5HT(2A) receptor expression and threonine phosphorylation levels showed that, nevertheless no significant differences in the receptor expression levels were detected, an increase of both 5HT receptor phosphorylation, on threonine residues, occurred in platelet from panic patients with respect to controls, suggesting that a reduction of serotonin receptor functioning was involved in the loss of serotonin responsiveness in panic.

7-Nitrobenzofurazan (NBD)-Derivatives Of 5'-N-Ethylcarboxamidoadenosine (NECA) As New Fluorescent Probes For Human A3 Adenosine Receptors

New fluorescent ligands for adenosine receptors (ARs), obtained by the insertion, in the N(6) position of NECA, of NBD-moieties with linear alkyl spacers of increasing length, proved to possess a high affinity and selectivity for the A(3) subtype expressed in CHO cells. In fluorescence microscopy assays, compound 2d, the most active and selective for human A(3)-AR, permitted visualization and localization of this human receptor subtype, showing its potential suitability for internalization and trafficking studies in living cells.

Serotonin-Mediated Cyclic AMP Inhibitory Pathway in Platelets of Patients Affected by Panic Disorder

Cyclic adenosine monophosphate (cAMP) pathway abnormalities have been suggested to be involved in anxiety disorders including panic (PD). The present study sought at investigating the downstream inhibitory adenylyl cyclase (AC) pathway activated by 5-HT in platelets obtained from 22 patients with a diagnosis of PD versus 22 healthy volunteers. In PD patients, a significant impairment of 5-HT potency to inhibit AC was observed. One month of treatment with paroxetine induced a significant increase of 5-HT potency in T1 patients close to the control values. [35S]GTPγS binding studies showed that in PD patients, a reduction of 5-HT receptor-G protein coupling occurred without any significant changes in G protein levels. These findings demonstrated that (1) a reduction of the inhibitory AC pathway activated by 5-HT occurred in platelets from PD patients; (2) the reduced 5-HT responsiveness in PD was related to an impairment of 5-HT receptor-G protein coupling, and (3) after 1 month of treatment with paroxetine, such a dysfunction significantly reversed together with a significant improvement of clinical symptoms.

4-[6-(Dansylamino)hexylamino]-7-methyl-2-phenyl-1,8-naphthyridine as a new potential fluorescent probe for studying A1-adenosine receptor.

A new fluorescent ligand for adenosine receptors, obtained by the insertion of a dansylamino-moiety with a linear hexyl spacer in the N4 position of a 1,8-naphthyridine adenosine receptor ligand, proved to possess a high affinity and selectivity for the A1 receptor subtype.

Do Acetylcholinesterase Inhibitors Have a Role in Improving Cognitive Impairment in Patients with Schizophrenia

At present, there are no really efficacious tools available to counteract cognitive deficits in patients with schizophrenia: even though new atypical antipsychotic drugs represent an advance compared with typical antipsychotic drugs, the results obtained with this class of drugs are actually partial. Acetylcholinesterase inhibitors (AChEIs) that have been proven to be effective on psychiatric symptoms, behavioural abnormalities and cognitive dysfunction of patients with dementia may be effective on cognitive deficit in patients with schizophrenia, and may also improve their psychopathology and behaviour.In the present paper we review the use of AChEIs in the treatment of schizophrenia. Although these AChEIs have different action mechanisms (donepezil only inhibits acetylcholinesterase; rivastigmine also inhibits butyryl-cholinesterase; galantamine also interacts with nicotinic acetylcholine receptors), they have similar clinical effects.We have observed no or mild effects on cognitive deficits and symptoms in double-blind studies, a dramatic effect on a patient’s subjective well-being and ability to cope and subjective judgement of psychiatrists in the case reports and open studies.The question remains as to how we can accurately measure a patient’s capacity to feel, to cope and his/her desire to live with other people — aspects very different from intelligence and cognitive function. Further double-blind placebo studies are required to determine the role of AChEIs in the improvement of quality of life for patients affected by schizophrenia.