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

Abstract

Low-affinity A 2B adenosine receptors (A 2B 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 A 2B 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, A 2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-a) treatment (24-h) increased A 2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-a markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A 2B ARs desensitization. In the presence of TNF-a, A 2B 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 A 2B AR antagonist MRS 1706 and required the presence of TNF-a. These results suggest that, in ADF cells, TNF-a selectively modulates A 2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A 2B AR in response to brain damage. Keywords: A 2B adenosine receptors, cerebral damage, cytokines, functional cross-talk, human astrocytoma cells, reactive astrogliosis. Astrocytes are the most important source of extracellular adenine-based purines in brain (Ciccarelli et al. 1999) and also express specific adenosine receptors (ARs) including the A 1 , A 2A, A 2B and A 3 subtypes (Peakman and Hill 1996;Trincavelli et al. 2002a). Recent data suggest a role for the adenosine system in the autocrine and heterocrine regulation of these cells under both physiological and pathological conditions, such as hypoxia or ischaemia, when astrocytes turn into an activated form called 'reactive astrogliosis ' (Ciccarelli et al. 1994;Brambilla and Abbracchio 2001;Brambilla et al. 2003). There is still debate as to the functional significance of this reaction. Although it is accepted that activated astrocytes contribute to isolate damaged brain areas from surrounding healthy cells and participate in neuronal recovery synthesizing neurotrophins and pleiotrophins, these cells also release several potentially toxic compounds (e.g. nitric oxide and cytokines; Neary et al. 1996). There is a suggestion that excessive and prolonged astrogliosis, such as that found in acute and chronic neurodegenerative diseases, may contribute to brain damage; hence, the characterization of the endogenous Address correspondence and reprint requests to Claudia Martini, Department Psychiatry, Neurobiology, Pharmacology and Biotechnology. Via Bonanno, 6. 56126 Pisa, Abbreviations used: ARs, adenosine receptors; ADF, hum...

Tumor Necrosis Factor-α Prevents Desensitization of Gαs-Coupled Receptors by Regulating GRK2 Association with the Plasma Membrane

“…Together, these data suggest that TNF-␣ and probably some other inflammatory cytokines promote A 2A R signaling and function through multiple effects, not only enhancing the receptor number but also regulating mediators of receptor signaling and desensitization. Our demonstration that TNF-␣ inhibits A 2A R desensitization is also consistent with the previous report by Trincavelli et al (2004) that TNF-␣ inhibits desensitization of A 2B Rs and enhances the functions of these receptors in human astroglial cells. Moreover, the current study provides further mechanistic insight into TNF-␣-mediated inhibition of receptor desensitization.…”

“…Although GRK2 and other GRKs are regulated by both endogenous and exogenous factors (Penela et al, 2003), there has been some evidence that expression and activity of GRKs are regulated by inflammation and inflammatory cytokines in certain cells or tissues (Lombardi et al, 1999(Lombardi et al, , 2001). More recent studies by Trincavelli et al (2004) demonstrated that TNF-␣ reduced agonist-dependent receptor phosphorylation and attenuated agonist-mediated desensitization of A 2B Rs in human astroglial cells. Precise mechanisms of TNF-␣ regulation of adenosine receptor desensitization and the role of this cytokine in regulation of GPCR activity in general, however, remain unknown.…”

Astrocytes in the damaged brain: Molecular and cellular insights into their reactive response and healing potential

“…These transcription factors drive GFAP upregulation and cell proliferation [22,78,79,80]. The PKB/Akt system 2) [61]. A similar convergence on STAT transducers is operated by the Janus family kinases (JAK) in response to extracellular GFs and cytokines including the astrogliosis triggers IL6, CNTF, EGF, and TGFα (Fig.…”

“…Indeed, a regulatory connection between inflammatory cytokines,Ado and its receptors has been demonstrated in astrocytes grown in vitro. For example, exposure to the astrogliosis-promoting agent TNF reduced the internalization and down-regulation of the A2B receptor subtype [61].…”

Targeting tumour necrosis factor-α in hypoxia and synaptic signalling

“…TNF-α has been shown to interact with adenosine in its effects during hypoxia. Very little is known about this interaction [49,50]. Hypoxia induces oxidative stress in the brain [51,52] and is associated with increased levels of proinflammatory cytokines, such as TNF-α in pig [53], in humans [54] and in mice [55].…”