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-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, 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-alpha. These results suggest that, in ADF cells, TNF-alpha 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.     

Local stimulation of the adenosine A2B receptors induces an increased release of IL-6 in mouse striatum: an in vivo microdialysis study

Both adenosine and interleukin-6 (IL-6) have been implicated in the pathophysiology of, e.g., epileptic seizures, traumatic brain injury, and affective disorders. Stimulation of adenosine A_2B receptors on astrocytes in vitro leads to the increased synthesis and secretion of IL-6. We investigated whether or not activation of adenosine receptors evokes an increase of IL-6 release also in vivo . 5'- N -ethylcarboxamidoadenosine, a non-specific adenosine-agonist or vehicle was administered into the striatum of freely moving mice by reverse microdialysis. A statistical significant increase of the IL-6 concentration in the perfusate was detected already 60 min after 5'- N -ethylcarboxamidoadenosine administration. IL-6 increased progressively and reached a maximum after 240 min. This effect appears to be mediated through adenosine A_2B receptors since it was counteracted by the specific A_2B receptor antagonist MRS1706 but not by the specific A₁ receptor antagonist DPCPX. We conclude that adenosine via activation of A_2B receptors evokes IL-6 release also in vivo .     

Mechanism of isoproterenol-induced RGS2 up-regulation in astrocytes

Regulators of G protein signaling (RGSs) are inducibly expressed in response to various stimuli and the up-regulation of RGSs leads to significant decreases in GPCR responsiveness. Isoproterenol, an adrenergic receptor agonist, stimulated RGS2 mRNA in C6 rat astrocytoma cells. The up-regulation of RGS2 mRNA was abrogated by genistein, a protein tyrosine kinase inhibitor (PTK), and by broad-spectrum protein kinase C (PKC) inhibitors (staurosporine and GF109203X). alpha-Adrenergic antagonist (prazocin), beta-adrenergic antagonist (prazocin), and pertussis toxin only partially blocked the RGS2 up-regulation, suggesting that the RGS2 up-regulation is concomitantly mediated by Galphai, Galphas, and Galphaq. It is interesting to note that SB203580, a potent p38 mitogen-activated protein kinase (MAPK) inhibitor, completely inhibited the isoproterenol-mediated RGS2 expression. In addition, isoproterenol also markedly stimulated RGS2 mRNA in rat primary astrocytes, which were sensitive to SB203580 and staurosporine. Therefore, our data suggest that adrenergic receptor-mediated signaling (induced by isoproterenol) may be involved in the regulation of RGS2 expression in astrocytes via activating PTK, PKC, and p38 MAPK.     

Second messengers regulate RGS2 expression which is targeted to the nucleus.

Regulators of G-protein Signaling (RGS) proteins attenuate signaling activities of G proteins, and modulation of expression appears to be a primary mechanism for regulating RGS proteins. In human astrocytoma 1321N1 cells RGS2 expression was increased by activation of muscarinic receptors coupled to phosphoinositide signaling with carbachol, or by increased cyclic AMP production, demonstrating that both signaling systems can increase the expression of a RGS family member in a single cell type. Carbachol-stimulated increases in endogenous RGS2 protein levels appeared by immunocytochemical analysis to be largely confined to the nucleus, and this localization was confirmed by Western blot analysis which showed increased nuclear, but not cytosolic, RGS2 after carbachol treatment. Additionally, transiently expressed green fluorescent protein (GFP)-tagged, 6xHis-tagged, or unmodified RGS2 resulted in a predominant nuclear localization, as well as a distinct accumulation of RGS2 along the plasma membrane. The intranuclear localization of GFP-RGS2 was confirmed with confocal microscopy. Thus, RGS2 expression is rapidly and transiently increased by phosphoinositide signaling and by cyclic AMP, and endogenous and transfected RGS2 is largely, although not entirely, localized in the nucleus.     

A novel pharmacological approach to treating cardiac ischemia. Binary conjugates of A1 and A3 adenosine receptor agonists

Adenosine released during cardiac ischemia exerts a potent, protective effect in the heart via activation of A(1) or A(3) receptors. However, the interaction between the two cardioprotective adenosine receptors and the question of which receptor is the more important anti-ischemic receptor remain largely unexplored. The objective of this study was to test the hypothesis that activation of both receptors exerted a cardioprotective effect that was significantly greater than activation of either receptor individually. This was accomplished by using a novel design in which new binary conjugates of adenosine A(1) and A(3) receptor agonists were synthesized and tested in a novel cardiac myocyte model of adenosine-elicited cardioprotection. Binary drugs having mixed selectivity for both A(1) and A(3) receptors were created through the covalent linking of functionalized congeners of adenosine agonists, each being selective for either the A(1) or A(3) receptor subtype. MRS 1740 and MRS 1741, thiourea-linked, regioisomers of a binary conjugate, were highly potent and selective in radioligand binding assays for A(1) and A(3) receptors (K(i) values of 0.7-3.5 nm) versus A(2A) receptors. The myocyte models utilized cultured chick embryo cells, either ventricular cells expressing native adenosine A(1) and A(3) receptors, or engineered atrial cells, in which either human A(3) receptors alone or both human A(1) and A(3) receptors were expressed. The binary agonist MRS 1741 coactivated A(1) and A(3) receptors simultaneously, with full cardioprotection (EC(50) approximately 0.1 nm) dependent on expression of both receptors. Thus, co-activation of both adenosine A(1) and A(3) receptors by the binary A(1)/A(3) agonists represents a novel general cardioprotective approach for the treatment of myocardial ischemia.     

Differential capacities of the RGS1, RGS16 and RGS-GAIP regulators of G protein signaling to enhance alpha2A-adrenoreceptor agonist-stimulated GTPase activity of G(o1)alpha

Recombinant RGS1, RGS16 and RGS-GAIP, but not RGS2, were able to substantially further stimulate the maximal GTPase activity of G(o1)alpha promoted by agonists at the alpha2A-adrenoreceptor in a concentration-dependent manner. Kinetic analysis of the regulation of an alpha2A-adrenoreceptor-G(o1)alpha fusion protein by all three RGS proteins revealed that they had similar affinities for the receptor-G protein fusion. However, their maximal effects on GTP hydrolysis varied over threefold with RGS16 > RGS1 > RGS-GAIP. Both RGS1 and RGS16 reduced the potency of the alpha2A-adrenoreceptor agonist adrenaline by some 10-fold. A lower potency shift was observed for the partial agonist UK14304 and the effect was absent for the weak partial agonist oxymetazoline. Each of these RGS proteins altered the intrinsic activity of both UK14304 and oxymetazoline relative to adrenaline. Such results require the RGS interaction with G(o1)alpha to alter the conformation of the alpha2A-adrenoreceptor and are thus consistent with models invoking direct interactions between RGS proteins and receptors. These studies demonstrate that RGS1, RGS16 and RGS-GAIP show a high degree of selectivity to regulate alpha2A-adrenoreceptor-activated G(o1)alpha rather than G(i1)alpha, G(i2)alpha or G(i3)alpha and different capacities to inactivate this G protein.     

Adenosine inhibits the release of interleukin-1beta in activated human peripheral mononuclear cells

The effects of adenosine and subtype-specific activators of adenosine receptors (A1, A2A, A2B and A3) were studied on the release of interleukin-1beta (IL-1beta) from peripheral mononuclear cells, monocytes and lymphocytes. In the cells activated by the protein kinase C specific phorbol ester (phorbol 12-myristate 13-acetate) and Ca(2+) ionophore (A23187) both adenosine and the subtype-specific receptor agonists, CPA (A1), CGS 21680 (A2A) and IB-MECA (A3) induced a concentration-dependent inhibition of IL-1beta release. The rank order of potency in the inhibition of IL-1beta release was CPA=CGS 21680>IB-MECA>adenosine>NECA (in the presence of A1, A2A and A3 receptor inhibitors). The inhibitory actions of CPA, CGS 21680 or IB-MECA were significantly reduced in the presence of DPCPX, ZM 243185 or MRS 1191 as subtype-specific antagonists on A1, A2A and A3 adenosine receptors, respectively. It can be concluded that adenosine inhibits the release of IL-1beta from the activated human peripheral mononuclear cells. In this process A1, A2A and A3 receptors are involved.     

Signaling of the human P2Y<Subscript>1</Subscript> receptor measured by a yeast growth assay with comparisons to assays of phospholipase C and calcium mobilization in 1321N1 human astrocytoma cells

The human P2Y₁ receptor was expressed in the yeast Saccharomyces cerevisiae strain MPY578q5, which is engineered to couple to mammalian G protein-coupled receptors (GPCRs) and requires agonist-induced activation for growth. A range of known P2Y₁ receptor agonists were examined with the yeast growth assay system, and the results were validated by comparing with potencies in the transfected 1321N1 astrocytoma cell line, in which calcium mobilization was measured with a FLIPR (fluorometric-imaging plate reader). The data were also compared with those from phospholipase C activation and radioligand binding with the use of a newly available radioligand [³H]MRS2279 (2-chloro-N ⁶-methyl-(N)-methanocarba-2’-deoxyadenosine-3’,5’bisphosphate). In the yeast growth assay, the rank order of potency of 2-MeSADP (2-methylthioadenosine 5’-diphosphate), ADP (adenosine 5’-diphosphate), and ATP (adenosine 5’-triphosphate) is the same as those in other assay systems, i.e., 2-MeSADP>ADP>ATP. The P2Y₁-selective antagonist MRS2179 (N ⁶-methyl-2-deoxyadenosine-3’,5’-bisphosphate) was shown to act as an antagonist with similar potency in all systems. The results suggest that the yeast expression system is suitable for screening P2Y₁ receptor ligands, both agonists and antagonists. The yeast system should be useful for random mutagenesis of GPCRs to identify mutants with certain properties, such as selective potency enhancement for small synthetic molecules and constitutive activity.     

Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 ⁴²⁰AKKAA⁴²⁴ mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gα_q association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.     

RGS-7 completes a receptor-independent heterotrimeric G protein cycle to asymmetrically regulate mitotic spindle positioning in C. elegans

Heterotrimeric G proteins promote microtubule forces that position mitotic spindles during asymmetric cell division in C. elegans embryos. While all previously studied G protein functions require activation by seven-transmembrane receptors, this function appears to be receptor independent. We found that mutating a regulator of G protein signaling, RGS-7, resulted in hyperasymmetric spindle movements due to decreased force on one spindle pole. RGS-7 is localized at the cell cortex, and its effects require two redundant Galphao-related G proteins and their nonreceptor activators RIC-8 and GPR-1/2. Using recombinant proteins, we found that RIC-8 stimulates GTP binding by Galphao and that the RGS domain of RGS-7 stimulates GTP hydrolysis by Galphao, demonstrating that Galphao passes through the GTP bound state during its activity cycle. While GTPase activators typically inactivate G proteins, RGS-7 instead appears to promote G protein function asymmetrically in the cell, perhaps acting as a G protein effector.     

Pharmacological profile of adenosine A2 receptor in PC12 cells

The PC12 cell line, a clone isolated from a pheochromocytoma tumor of rat adrenal medulla, was shown to exclusively contain stimulatory adenosine (A2) receptors linked to adenylate cyclase (AC). AC was stimulated 6-7 fold by several agonists with a rank order of potency of 5'-N-Ethyl carboxamidoadenosine (NECA) greater than 2-Chloroadenosine (2-CADO) greater than (R)-N-Phenylisopropyladenosine (R-(-)-PIA) greater than N6-Cyclopentyladenosine (CPA) greater than N6-Cyclohexyladenosine (CHA) greater than S-(+)-PIA. AC activity was antagonized by a variety of adenosine receptor antagonists with a potency order of 1,3,-Dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) greater than 1,3,-Diethyl-8-phenylxanthine (DPX) greater than 8-Phenyltheophylline greater than 3-Isobutyl-1-methylxanthine (IBMX) greater than 8-(p-sulfophenyl)theophylline (PST) greater than 7-(beta-chloroethyl)theophylline greater than theophylline = enprofylline = caffeine. Under conditions known to favour receptor-mediated Ni-coupled inhibition of AC, R-(-)-PIA failed to inhibit both basal and forskolin stimulated AC activity in PC12 cells, confirming the absence of an A1 mediated response. On the other hand, adenosine agonists inhibited AC activity in rat cortical membranes with a rank order of potency of CPA greater than R-(-)-PIA greater than CHA greater than NECA greater than S-(+)-PIA greater than 2-CADO. These findings suggest that PC12 cells are functionally deficient in an A1 receptor linked AC response but are efficiently coupled to A2 stimulatory receptors. The cells should prove useful for further study of A2 adenosine receptors and to establish selectivity profiles of compounds acting at both A1 and A2 receptors.     

Nucleotide coronary vasodilation in guinea pig hearts

The role of P1 receptors and P2Y1 receptors in coronary vasodilator responses to adenine nucleotides was examined in the isolated guinea pig heart. Bolus arterial injections of nucleotides were made in hearts perfused at constant pressure. Peak increase in flow was measured before and after addition of purinoceptor antagonists. Both the P1 receptor antagonist 8-(p-sulfophenyl)theophylline and adenosine deaminase inhibited adenosine vasodilation. AMP-induced vasodilation was inhibited by P1 receptor blockade but not by adenosine deaminase or by the selective P2Y1 antagonist N6-methyl-2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179). ADP-induced vasodilation was moderately inhibited by P1 receptor blockade and greatly inhibited by combined P1 and P2Y1 blockade. ATP-induced vasodilation was antagonized by P1 blockade but not by adenosine deaminase. Addition of P2Y1 blockade to P1 blockade shifted the ATP dose-response curve further rightward. It is concluded that in this preparation ATP-induced vasodilation results primarily from AMP stimulation of P1 receptors, with a smaller component from ATP or ADP acting on P2Y1 receptors. ADP-induced vasodilation is largely due to P2Y1 receptors, with a smaller contribution by AMP or adenosine acting via P1 receptors. AMP responses are mediated solely by P1 receptors. Adenosine contributes very little to vasodilation resulting from bolus intracoronary injections of ATP, ADP, or AMP.     

Regulator of G-protein signalling 3 redirects prototypical Gi-coupled receptors from Rac1 to RhoA activation

The small GTPases, Rac1 and RhoA, are pivotal regulators of several essential, but distinct cellular processes. Numerous G-protein-coupled receptors signal to these GTPases, but with different specificities. Specifically, Gi-coupled receptors (GiPCRs) are generally believed to activate Rac1, but not RhoA, a process involving Gbetagamma-dimers and phosphatidylinositol 3-kinase (PI3K). Here we show that, depending on the expression level of the 519 amino acid isoform of regulator of G-protein signalling 3 (RGS3L), prototypical GiPCRs, like M2 muscarinic, A1 adenosine, and alpha2-adrenergic receptors, activate either Rac1 or RhoA in human embryonic kidney cells and neonatal rat cardiomyocyte-derived H10 cells. The switch from Rac1 to RhoA activation in H10 cells was controlled by fibroblast growth factor-2 (FGF-2), lowering the expression of RGS3L. Activation of both, Rac1 and RhoA, seen at low and high expression levels of RGS3L, respectively, was sensitive to pertussis toxin and the PI3K inhibitor LY294002 and mediated by Gbetagamma-dimers. We conclude that RGS3L functions as a molecular switch, redirecting GiPCRs via Gbetagamma-dimers and PI3K from Rac1 to RhoA activation. Considering the essential roles of Rac1 and RhoA in many signalling pathways, this additional function of RGS3L indicates a specific role of this protein in cellular signalling networks.     

Effect of A2B adenosine receptor gene ablation on proinflammatory adenosine signaling in mast cells

Pharmacological studies suggest that A(2B) adenosine receptors mediate proinflammatory effects of adenosine in human mast cells in part by up-regulating production of Th2 cytokines and angiogenic factors. This concept has been recently challenged by the finding that mast cells cultured from bone marrow-derived mast cells (BMMCs) of A(2B) knockout mice display an enhanced degranulation in response to FcepsilonRI stimulation. This finding was interpreted as evidence of anti-inflammatory functions of A(2B) receptors and it was suggested that antagonists with inverse agonist activity could promote activation of mast cells. In this report, we demonstrate that genetic ablation of the A(2B) receptor protein has two distinct effects on BMMCs, one is the previously reported enhancement of Ag-induced degranulation, which is unrelated to adenosine signaling; the other is the loss of adenosine signaling via this receptor subtype that up-regulates IL-13 and vascular endothelial growth factor secretion. Genetic ablation of A(2B) receptors had no effect on A(3) adenosine receptor-dependent potentiation of Ag-induced degranulation in mouse BMMCs, but abrogated A(2B) adenosine receptor-dependent stimulation of IL-13 and vascular endothelial growth factor secretion. Adenosine receptor antagonists MRS1706 and DPCPX with known inverse agonist activity at the A(2B) subtype inhibited IL-13 secretion induced by the adenosine analog NECA, but did not mimic the enhanced Ag-induced degranulation observed in A(2B) knockout BMMCs. Thus, our study confirmed the proinflammatory role of adenosine signaling via A(2B) receptors and the anti-inflammatory actions of A(2B) antagonists in mouse BMMCs.     

Endogenous RGS proteins attenuate Galpha(i)-mediated lysophosphatidic acid signaling pathways in ovarian cancer cells

Lysophosphatidic acid is a bioactive phospholipid that is produced by and stimulates ovarian cancer cells, promoting proliferation, migration, invasion, and survival. Effects of LPA are mediated by cell surface G-protein coupled receptors (GPCRs) that activate multiple heterotrimeric G-proteins. G-proteins are deactivated by Regulator of G-protein Signaling (RGS) proteins. This led us to hypothesize that RGS proteins may regulate G-protein signaling pathways initiated by LPA in ovarian cancer cells. To determine the effect of endogenous RGS proteins on LPA signaling in ovarian cancer cells, we compared LPA activity in SKOV-3 ovarian cancer cells expressing G(i) subunit constructs that are either insensitive to RGS protein regulation (RGSi) or their RGS wild-type (RGSwt) counterparts. Both forms of the G-protein contained a point mutation rendering them insensitive to inhibition with pertussis toxin, and cells were treated with pertussis toxin prior to experiments to eliminate endogenous G(i/o) signaling. The potency and efficacy of LPA-mediated inhibition of forskolin-stimulated adenylyl cyclase activity was enhanced in cells expressing RGSi G(i) proteins as compared to RGSwt G(i). We further showed that LPA signaling that is subject to RGS regulation terminates much faster than signaling thru RGS insensitive G-proteins. Finally, LPA-stimulated SKOV-3 cell migration, as measured in a wound-induced migration assay, was enhanced in cells expressing Galpha(i2) RGSi as compared to cells expressing Galpha(i2) RGSwt, suggesting that endogenous RGS proteins in ovarian cancer cells normally attenuate this LPA effect. These data establish RGS proteins as novel regulators of LPA signaling in ovarian cancer cells.     

Transient activation and delayed inhibition of Na+,K+,Cl- cotransport in ATP-treated C11-MDCK cells involve distinct P2Y receptor subtypes and signaling mechanisms

In C11-MDCK cells, which resemble intercalated cells from collecting ducts of the canine kidney, P2Y agonists promote transient activation of the Na+,K+,Cl- cotransporter (NKCC), followed by its sustained inhibition. We designed this study to identify P2Y receptor subtypes involved in dual regulation of this carrier. Real time polymerase chain reaction analysis demonstrated that C11-MDCK cells express abundant P2Y1 and P2Y2 mRNA compared with that of other P2Y receptor subtypes. The rank order of potency of agents (ATP approximately UTP > 2-(methylthio)-ATP (2MeSATP); adenosine 5'-[beta-thio]diphosphate (ADPbetaS) inactive) indicated that P2Y2 rather than P2Y1 receptors mediate a 3-4-fold activation of NKCC within the first 5-10 min of nucleotide addition. NKCC activation in ATP-treated cells was abolished by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin (CaM) antagonists trifluoroperazine and W-7, and KN-62, an inhibitor of Ca2+/CaM-dependent protein kinase II. By contrast with the transient activation, 30-min incubation with nucleotides produced up to 4-5-fold inhibition of NKCC, and this inhibition exhibited a rank order of potency (2MeSATP > ADPbetaS > ATP > UTP) typical of P2Y1 receptors. Unlike the early response, delayed inhibition of NKCC occurred in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded cells and was completely abolished by the P2Y1 antagonists MRS2179 and MRS2500. Transient activation and delayed inhibition of NKCC in C11 cell monolayers were observed after the addition of ATP to mucosal and serosal solutions, respectively. NKCC inhibition triggered by basolateral application of ADPbetaS was abolished by MRS2500. Our results thus show that transient activation and delayed inhibition of NKCC in ATP-treated C11-MDCK cells is mediated by Ca2+/CaM-dependent protein kinase II- and Ca2+-independent signaling triggered by apical P2Y2 and basolateral P2Y1 receptors, respectively.     

Adenosine A2A receptor-mediated facilitation of noradrenaline release involves protein kinase C activation and attenuation of presynaptic inhibitory receptor-mediated effects in the rat vas deferens

In the epididymal portion of rat vas deferens, facilitation of noradrenaline release mediated by adenosine A2A receptors, but not that mediated by beta2-adrenoceptors or by direct activation of adenylyl cyclase, was attenuated by blockade of alpha2-adrenoceptors and abolished by simultaneous blockade of alpha2-adrenoceptors, adenosine A1 and P2Y receptors. The adenosine A2A receptor-mediated facilitation was not changed by inhibitors of protein kinase A, protein kinase G or calmodulin kinase II but was prevented by inhibition of protein kinase C with chelerythrine or bisindolylmaleimide XI. Activation of protein kinase C with phorbol 12-myristate 13-acetate caused a facilitation of noradrenaline release that was abolished by bisindolylmaleimide XI and reduced by antagonists of alpha2-adrenoceptors, adenosine A1 and P2Y receptors. Activation of adenosine A2A receptors attenuated the inhibition of noradrenaline release mediated by the presynaptic inhibitory receptors. This effect was mimicked by phorbol 12-myristate 13-acetate and prevented by bisindolylmaleimide XI. It is concluded that adenosine A2A receptors facilitate noradrenaline release by a mechanism that involves a protein kinase C-mediated attenuation of effects mediated by presynaptic inhibitory receptors, namely alpha2-adrenoceptors, adenosine A1 and P2Y receptors.     

On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells

Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation.     

Adenosine modulates cell growth in human epidermoid carcinoma (A431) cells.

Adenosine mediates many physiological functions via activation of extracellular receptors. The modulation of cell growth by adenosine was found to be receptor-mediated. In A431 cells adenosine evoked a biphasic response in which a low concentration (approximately 10 microM) produced inhibition of colony formation but at higher concentrations (up to 100 microM) this inhibition was progressively reversed. Evidence for the involvement of A1 (inhibitory) and A2 (stimulatory) adenosine receptors in regulating cell growth of these tumor cells was obtained through plating efficiency studies based on the relative potency of adenosine agonists and antagonists. When both A1 and A2 receptors were blocked, colony formation or growth was not inhibited at low concentrations of adenosine but was inhibited at high adenosine concentrations.