John M. Dickenson

Human adenosine A1 receptor and P2Y2‐purinoceptor‐mediated activation of the mitogen‐activated protein kinase cascade in transfected CHO cells

1 The mitogen‐activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric Gi/Go protein‐coupled and Gq/G11 protein‐coupled receptors. The aims of the current study were: (i) to investigate whether the Gi/Go protein‐coupled adenosine A1 receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO‐A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor. 2 The selective adenosine A1 receptor agonist N6‐cyclopentyladenosine (CPA) stimulated time and concentration‐dependent increases in MAP kinase activity in CHO‐A1 cells (EC50 7.1±0.4 nm). CPA‐mediated increases in MAP kinase activity were blocked by PD 98059 (50 μm; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre‐treating cells with pertussis toxin (to block Gi/Go‐dependent pathways). 3 Adenosine A1 receptor‐mediated activation of MAP kinase was abolished by pre‐treatment with the protein tyrosine inhibitor, genistein (100 μm; 6±10% of control). In contrast, daidzein (100 μm), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μm) were also sensitive to the phosphatidylinositol 3‐kinase inhibitors wortmannin (100 nm; 55±8% inhibition) and LY 294002 (30 μm; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31‐8220 (10 μm). 4 Activation of the endogenous P2Y2 purinoceptor with UTP also stimulated time and concentration‐dependent increases in MAP kinase activity in CHO‐A1 cells (EC50 = 1.6±0.3 μm). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31‐8220 (10 μm; 45±5% inhibition), indicating the possible involvement of both Gi/Go protein and Gq protein‐dependent pathways in the overall response to UTP. 5 CPA and UTP stimulated concentration‐dependent increases in the phosphorylation state of the 42 kDa and 44 kDa forms of MAP kinase as demonstrated by Western blotting. 6 Co‐activation of CHO‐A1 cells with CPA (10 nm) and UTP (1 μm) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31‐8220 (10 μm). 7 Adenosine A1 and P2Y2 purinoceptor activation increased the expression of luciferase in CHO cells transfected with a luciferase reporter gene containing the c‐fos promoter. However, co‐activating these two receptors produced only additive increases in luciferase expression. 8 In conclusion, our studies have shown that the transfected adenosine A1 receptor and the endogenous P2Y2 purinoceptor couple to the MAP kinase signalling pathway in CHO‐A1 cells. Furthermore, co‐stimulation of the adenosine A1 receptor and the P2Y2 purinoceptor produced synergistic increases in MAP kinase activity but not c‐fos mediated luciferase expression.

Functional expression of the P2Y14 receptor in murine T-lymphocytes

Quantitative reverse transcriptase polymerase chain reaction (RT–PCR) analysis has previously shown that the P2Y14 receptor is expressed in peripheral immune cells including lymphocytes. Although in transfected cells the P2Y14 receptor couples to pertussis toxin‐sensitive Gi/o protein, the functional coupling of endogenously expressed P2Y14 receptors to the inhibition of adenylyl cyclase activity has not been reported. Therefore, the primary aim of this study was to determine whether the P2Y14 receptor is functionally expressed in murine spleen‐derived T‐ and B‐lymphocyte‐enriched populations. RT–PCR analysis detected the expression of P2Y14 receptor mRNA in whole spleen and isolated T‐ and B‐lymphocytes. In T cells, UDP‐glucose (EC50=335u2003nM) induced a small but significant inhibition (circa 20%) of forskolin‐stimulated cAMP accumulation, suggesting functional coupling of endogenously expressed P2Y14 receptors to the inhibition of adenylyl cyclase activity. In contrast, the other putative P2Y14 receptor agonists UDP‐galactose, UDP‐glucuronic acid and UDP‐N‐acetylglucosamine had no significant effect alone but behaved as partial agonists by blocking UDP‐glucose responses. In B cells, UDP‐glucose (100u2003μM) had no significant effect on forskolin‐stimulated cAMP accumulation. Treatment of T cells with pertussis toxin (Gi/o blocker) abolished the inhibitory effects of UDP‐glucose on forskolin‐stimulated cAMP accumulation. T‐cell proliferation in response to anti‐CD3 monoclonal antibody (1u2003μgu2003ml−1) was significantly inhibited by UDP‐glucose (59% inhibition; p[IC50]=5.9±0.3), UDP‐N‐acetylglucosamine (37%; 6.1±0.3), UDP‐galactose (56%; 8.2±0.2) and UDP‐glucuronic acid (49%; 6.3±0.2). Interleukin‐2‐ (5u2003ngu2003ml−1) induced T‐cell proliferation was also significantly inhibited by all four agonists. In summary, we have shown that the P2Y14 receptor appears to be functionally expressed in murine spleen‐derived T‐lymphocytes. These observations suggest that UDP‐glucose and related sugar nucleotides presumably via the P2Y14 receptor may play an important role in modulating immune function.

Characterization of ERK1/2 signalling pathways induced by adenosine receptor subtypes in newborn rat cardiomyocytes

Adenosine A1, A2A, and A3 receptors (ARs) and extracellular signal‐regulated kinase 1/2 (ERK1/2) play a major role in myocardium protection from ischaemic injury. In this study, we have characterized the adenosine receptor subtypes involved in ERK1/2 activation in newborn rat cardiomyocytes. Adenosine (nonselective agonist), CPA (A1), CGS 21680 (A2A) or Cl‐IB‐MECA (A3), all increased ERK1/2 phosphorylation in a time‐ and dose‐dependent manner. The combined maximal response of the selective agonists was similar to adenosine alone. Theophylline (nonselective antagonist) inhibited completely adenosine‐mediated ERK1/2 activation, whereas a partial inhibition was obtained with DPCPX (A1), ZM 241385 (A2A), and MRS 1220 (A3). PD 98059 (MEK1; ERK kinase inhibitor) abolished all agonist‐mediated ERK1/2 phosphorylation. Pertussis toxin (PTX, Gi/o blocker) inhibited completely CPA‐ and partially adenosine‐ and Cl‐IB‐MECA‐induced ERK1/2 activation. Genistein (tyrosine kinase inhibitor) and Ro 318220 (protein kinase C, PKC inhibitor) partially reduced adenosine, CPA and Cl‐IB‐MECA responses, without any effect on CGS 21680‐induced ERK1/2 phosphorylation. H89 (protein kinase A, PKA inhibitor) abolished completely CGS 21680 and partially adenosine and Cl‐IB‐MECA responses, without any effect on CPA response. Cl‐IB‐MECA‐mediated increases in cAMP accumulation suggest that A3AR‐induced ERK1/2 phosphorylation involves adenylyl cyclase activation via phospholipase C (PLC) and PKC stimulation. In summary, we have shown that ERK1/2 activation by adenosine in cardiomyocytes results from an additive stimulation of A1, A2A, and A3ARs, which involves Gi/o proteins, PKC, and tyrosine kinase for A1 and A3ARs, and Gs and PKA for A2AARs. Moreover, the A3AR response also involves a cAMP/PKA pathway via PKC activation.

Regulation of p42/p44 MAPK and p38 MAPK by the adenosine A1 receptor in DDT1MF-2 cells

The mitogen-activated protein kinase (MAPK) family consists of the p42/p44 MAPKs and the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 MAPK. We have previously reported that the human adenosine A(1) receptor stimulates p42/p44 MAPK in transfected Chinese hamster ovary cells. In this study, we have investigated whether the endogenous adenosine A(1) receptor in the smooth muscle cell line, DDT(1)MF-2 activates p42/p44 MAPK, JNK and p38 MAPK. The adenosine A(1) receptor agonist N(6)-cyclopentyladenosine stimulated time and concentration-dependent increases in p42/p44 MAPK and p38 MAPK phosphorylation in DDT(1)MF-2 cells. No increases in JNK phosphorylation were observed following adenosine A(1) receptor activation. N(6)-cyclopentyladenosine-mediated increases in p42/p44 MAPK and p38 MAPK phosphorylation were blocked by the selective adenosine A(1) receptor antagonist 1,3-dipropylcyclopentylxanthine and following pretreatment of cells with pertussis toxin. Furthermore, adenosine A(1) receptor-mediated increases in p42/p44 MAPK were sensitive to the MAPK kinase 1 inhibitor PD 98059 (2-amino-3-methoxyflavone), whereas p38 MAPK responses were blocked by the p38 MAPK inhibitor SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole). The broad range protein tyrosine kinase inhibitors genistein and tyrphostin A47 (alpha-cyano-(3,4-dihydroxy)thiocinnamide) did not block adenosine A(1) receptor stimulation of p42/p44 MAPK. For comparison, insulin-mediated increases in p42/p44 MAPK were blocked by genistein and tyrphostin A47. The Src tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and the epidermal growth factor receptor tyrosine kinase inhibitor AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline) also had no effect on adenosine A(1) receptor stimulation of p42/p44 MAPK. Furthermore, the protein kinase C inhibitors Ro 31-8220 (3-[1-[3-(2-isothioureido) propyl]indol-3-yl]-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione), chelerythrine and GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) were without effect on adenosine A(1) receptor-induced p42/p44 MAPK phosphorylation. In contrast, wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), inhibitors of phosphatidylinositol 3-kinase, attenuated adenosine A(1) receptor stimulation of p42/p44 MAPK phosphorylation. In conclusion, the adenosine A(1) receptor stimulates p42/p44 MAPK through a pathway which appears to be independent of tyrosine kinase activation but involves phosphatidylinositol 3-kinase. Finally, adenosine A(1) receptor stimulation in DDT(1)MF-2 cells also activated p38 MAPK but not JNK via a pertussis toxin-sensitive pathway.

Functional expression of adenosine A2A and A3 receptors in the mouse dendritic cell line XS-106.

There is increasing evidence to suggest that adenosine receptors can modulate the function of cells involved in the immune system. For example, human dendritic cells derived from blood monocytes have recently been described to express functional adenosine A1, A2A and A3 receptors. Therefore, in the present study, we have investigated whether the recently established murine dendritic cell line XS-106 expresses functional adenosine receptors. The selective adenosine A3 receptor agonist 1-[2-chloro-6[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide (2-Cl-IB-MECA) inhibited forskolin-mediated [3H]cyclic AMP accumulation and stimulated concentration-dependent increases in p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. The selective adenosine A2A receptor agonist 4-[2-[[-6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene-propanoic acid (CGS 21680) stimulated a robust increase in [3H]cyclic AMP accumulation and p42/p44 MAPK phosphorylation. In contrast, the selective adenosine A1 receptor agonist CPA (N6-cyclopentyladenosine) did not inhibit forskolin-mediated [3H]cyclic AMP accumulation or stimulate increases in p42/p44 MAPK phosphorylation. These observations suggest that XS-106 cells express functional adenosine A2A and A3 receptors. The non-selective adenosine receptor agonist 5-N-ethylcarboxamidoadenosine (NECA) inhibited lipopolysaccharide-induced tumour necrosis factor-alpha (TNF-alpha) release from XS-106 cells in a concentration-dependent fashion. Furthermore, treatment with Cl-IB-MECA (1 microM) or CGS 21680 (1 microM) alone produced a partial inhibition of lipopolysaccharide-induced TNF-alpha release (when compared to NECA), whereas a combination of both agonists resulted in the inhibition of TNF-alpha release comparable to that observed with NECA alone. Treatment of cells with the adenosine A2A receptor selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol (ZM 241385; 100 nM) and 5-amino-2-(2-furyl)-7-phenylethyl-pyrazolo[4,3-e]-1,2,4-triazolo[1,5c]pyrimidine (SCH 58261; 100 nM) and the adenosine A3 receptor selective antagonist N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS 1220; 100 nM) partially blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. Combined addition of MRS 1220 and SCH 58261 completely blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. In conclusion, we have shown that the mouse dendritic cell line XS-106 expresses functional adenosine A2A and A3 receptors, which are capable of modulating TNF-alpha release.

Regulation of p42/p44 mitogen-activated protein kinase by the human adenosine A3 receptor in transfected CHO cells.

In this study we have investigated whether the human adenosine A3 receptor activates p42/p44 mitogen-activated protein kinase (MAPK) in transfected Chinese hamster ovary (CHO) cells (designated CHO-A3). The high affinity adenosine A3 receptor agonist IB-MECA (1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-beta-D-ribofuranuronamide) stimulated time (peak activation occurring after 5 min) and concentration-dependent (pEC50=9.0+/-0.2) increases in p42/p44 MAPK in CHO-A3 cells. Adenosine A3 receptor-mediated increases in p42/p44 MAPK were sensitive to pertussis toxin and the MAPK kinase 1 inhibitor PD 98059 (2-amino-3-methoxyflavone). The broad range protein tyrosine kinase inhibitor genistein and the phosphatidylinositol 3-kinase inhibitors wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) also blocked adenosine A3 receptor stimulation of p42/p44 MAPK. In contrast, inhibition of protein kinase C had no significant effect on adenosine A3 receptor-induced p42/p44 MAPK activation. IB-MECA (pEC50=10.1+/-0.2) also increased the expression of luciferase in CHO-A3 cells transiently transfected with a luciferase reporter gene containing the c-fos promoter. Furthermore, IB-MECA-induced increases in luciferase gene expression were sensitive to pertussis toxin, PD 98059, genistein, wortmannin and LY 294002. In conclusion, we have shown that the human adenosine A3 receptor stimulates p42/p44 MAPK and c-fos-mediated luciferase gene expression in transfected CHO cells.

Characterization of P2Y receptor subtypes functionally expressed on neonatal rat cardiac myofibroblasts

Background and purpose:u2002 Little is known about P2Y receptors in cardiac fibroblasts, which represent the predominant cell type in the heart and differentiate into myofibroblasts under certain conditions. Therefore, we have characterized the phenotype of the cells and the different P2Y receptors at the expression and functional levels in neonatal rat non‐cardiomyocytes.

Activation of the p38 and p42/p44 mitogen‐activated protein kinase families by the histamine H1 receptor in DDT1MF‐2 cells

The mitogen‐activated protein kinases (MAPKs) consist of the p42/p44 MAPKs and the stress‐activated protein kinases, c‐Jun N‐terminal kinase (JNK) and p38 MAPK. In this study we have examined the effect of histamine H1 receptor activation on MAPK pathway activation in the smooth muscle cell line DDT1MF‐2. Histamine stimulated time and concentration‐dependent increases in p42/p44 MAPK activation in DDT1MF‐2 cells. Responses to histamine were inhibited by the histamine H1 receptor antagonist mepyramine (KD 3.5u2003nM) and following pre‐treatment with pertussis toxin (PTX; 57% inhibition). Histamine‐induced increases in p42/p44 MAPK activation were blocked by inhibitors of MAPK kinase 1 (PD 98059), tyrosine kinase (genistein and tyrphostin A47), phosphatidylinositol 3‐kinase (wortmannin and LY 294002) and protein kinase C (Ro 31‐8220; 10u2003μM; 41% inhibition). Inhibitors of Src tyrosine kinase (PP2) and the epidermal growth factor tyrosine kinase (AG1478) were without effect. Removal of extracellular Ca2+, chelation of intracellular Ca2+ with BAPTA and inhibition of focal adhesion assembly (cytochalasin D) had no significant effect on histamine‐induced p42/p44 MAPK activation. Histamine stimulated time and concentration‐dependent increases in p38 MAPK activation in DDT1MF‐2 cells but had no effect on JNK activation. Histamine‐induced p38 MAPK activation was inhibited by pertussis toxin (74% inhibition) and the p38 MAPK inhibitor SB 203580 (95% inhibition). In summary, we have shown the histamine H1 receptor activates p42/p44 MAPK and p38 MAPK signalling pathways in DDT1MF‐2 smooth muscle cells. Interestingly, signalling to both pathways appears to involve histamine H1 receptor coupling to Gi/Go‐proteins.

Activation of protein kinase B by the A1‐adenosine receptor in DDT1MF‐2 cells

In this study the effect of insulin and A1‐adenosine receptor stimulation on protein kinase B (PKB) activation has been investigated in the hamster vas deferens smooth muscle cell line DDT1MF‐2. Increases in PKB phosphorylation were determined by Western blotting using an antibody that detects PKB phosphorylation at Ser473. Insulin, a recognized activator of PKB, stimulated a concentration‐dependent increase in PKB phosphorylation in DDT1MF‐2 cells (EC50 5±1u2003pM). The selective A1‐adenosine receptor agonist N6‐cyclopentyladenosine (CPA) stimulated time and concentration‐dependent increases in PKB phosphorylation in DDT1MF‐2 cells (EC50 1.3±0.5u2003nM). CPA‐mediated increases in PKB phosphorylation were antagonized by the A1‐adenosine receptor selective antagonist 1,3‐dipropylcyclopentylxanthine (DPCPX) yielding an apparent KD value of 2.3u2003nM. Pre‐treatment of DDT1MF‐2 cells with pertussis toxin (PTX, 100u2003ngu2003ml−1 for 16u2003h), to block Gi/Go‐dependent pathways, abolished CPA (1u2003μM) induced phosphorylation of PKB. In contrast, responses to insulin (100u2003nM) were resistant to PTX pre‐treatment. The phosphatidylinositol 3‐kinase (PI‐3K) inhibitors wortmannin (IC50 10.3±0.6u2003nM) and LY 294002 (IC50 10.3±1.2u2003μM) attenuated the phosphorylation of PKB elicited by CPA (1u2003μM) in a concentration‐dependent manner. Wortmannin (30u2003nM) and LY 294002 (30u2003μM) also blocked responses to insulin (100u2003nM). Removal of extracellular Ca2+ and chelation of intracellular Ca2+ with BAPTA had no significant effect on CPA‐induced PKB phosphorylation. Similarly, pretreatment (30u2003min) with inhibitors of protein kinase C (Ro 31‐8220; 10u2003μM), tyrosine kinase (genistein; 100u2003μM), mitogen‐activated protein (MAP) kinase kinase (PD 98059; 50u2003μM) and p38 MAPK (SB 203580; 20u2003μM) had no significant effect on CPA‐induced PKB phosphorylation. In conclusion, these data demonstrate that A1‐adenosine receptor stimulation in DDT1MF‐2 cells increases PKB phosphorylation through a PTX and PI‐3K‐sensitive pathway.

Stimulation of protein kinase B and p70 S6 kinase by the histamine H1 receptor in DDT1MF-2 smooth muscle cells

Previous studies have shown that the histamine H1 receptor activates p42/p44 mitogen‐activated protein kinases (MAPK) in DDT1MF‐2 smooth muscle cells via a phosphatidylinositol 3‐kinase (PI‐3K)‐dependent pathway. In this study the effect of histamine H1 receptor stimulation on protein kinase B (PKB) and p70 S6 kinase, both of which are downstream targets of PI‐3K, has been investigated. Increases in PKB and p70 S6 kinase activation were monitored by Western blotting using phospho‐specific PKB (Ser473) and p70 S6 kinase (Thr421/Ser424) antibodies. Histamine stimulated time and concentration‐dependent increases in the phosphorylation of PKB and p70 S6 kinase in DDT1MF‐2 cells. Both responses were completely inhibited by the histamine H1 receptor antagonist mepyramine and following pre‐treatment with pertussis toxin, to block Gi/Go protein dependent pathways. The PI‐3K inhibitors wortmannin (IC50 5.9±0.5u2003nM) and LY 294002 (IC50 6.9±0.8u2003μM) attenuated the increase in PKB phosphorylation induced by histamine (100u2003μM) in a concentration‐dependent manner. Histamine‐induced increases in p70 S6 kinase phosphorylation were partially sensitive to rapamycin (20u2003nM; 68% inhibition) but completely blocked by wortmannin (100u2003nM), LY 294002 (30u2003μM) and the MAPK kinase inhibitor PD 98059 (50 μM). In summary, these data demonstrate that the histamine H1 receptor stimulates PKB and p70 S6 kinase phosphorylation in DDT1MF‐2 smooth muscle cells. However, functional studies revealed that histamine does not stimulate DDT1MF‐2 cell proliferation or attenuate staurosporine‐induced caspase‐3 activity. The challenge for future research will be to link the stimulation of these kinase pathways with the physiological and pathophysiological roles of the histamine H1 receptor.