Search

We have previously shown that phenyl saligenin phosphate (PSP), an organophosphorus compound which is classed as a weak inhibitor of acetylcholinesterase, triggered cytotoxicity in mitotic and differentiated H9c2 cardiomyoblasts. The aim of this study was to assess whether sublethal concentrations of PSP could disrupt the morphology of differentiating rat H9c2 cardiomyoblasts and human-induced pluripotent stem-cell-derived cardiomyocyte progenitor cells (hiPSC-CMs) and to assess the underlying cytoskeletal changes. PSP-induced changes in protein expression were monitored via Western blotting, immunocytochemistry, and proteomic analysis. PSP-mediated cytotoxicity was determined by measuring MTT reduction, LDH release, and caspase-3 activity. Sublethal exposure to PSP (3 μM) induced morphological changes in differentiating H9c2 cells (7, 9, and 13 days), reflected by reduced numbers of spindle-shaped cells. Moreover, this treatment (7 days) attenuated the expression of the cytoskeletal proteins cardiac troponin I, tropomyosin-1, and α-actin. Further proteomic analysis identified nine proteins (e.g., heat shock protein 90-β and calumenin) which were down-regulated by PSP exposure in H9c2 cells. To assess the cytotoxic effects of organophosphorus compounds in a human cell model, we determined their effects on human-induced pluripotent stem-cell-derived cardiomyocyte progenitor cells. Chlorpyrifos and diazinon-induced cytotoxicity (48 h) was evident only at concentrations >100 μM. By contrast, PSP exhibited cytotoxicity in hiPSC-CMs at a concentration of 25 μM following 48 h exposure. Finally, sublethal exposure to PSP (3 μM; 7 days) induced morphological changes and decreased the expression of cardiac troponin I, tropomyosin-1, and α-actin in hiPSC-CMs. In summary, our data suggest cardiomyocyte morphology is disrupted in both cell models by sublethal concentrations of PSP via modulation of cytoskeletal protein expression.

Adequate invasion and complete remodelling of the maternal spiral arteries by the invading extravillous trophoblasts are the major determinants of a successful pregnancy. Increase in oxidative stress during pregnancy has been linked to the reduction in trophoblast invasion and incomplete conversion of the maternal spiral arteries, resulting in pregnancy complications such as pre-eclampsia, intrauterine growth restriction, and spontaneous miscarriages resulting in foetal/maternal mortality. The use of antioxidant therapy (vitamin C and E) and other preventative treatments (such as low dose aspirin) have been ineffective in preventing pre-eclampsia. Also, as the majority of antihypertensive drugs pose side effects, choosing an appropriate treatment would depend upon the efficacy and safety of mother/foetus. Since pre-eclampsia is mainly linked to placental oxidative stress, new diet-based antioxidants can be of use to prevent this condition. The antioxidant properties of flavonoids (naturally occurring phenolic compounds which are ubiquitously distributed in fruits and vegetables) have been well documented in non-trophoblast cells. Therefore, this study aimed to investigate the effects of flavonoids (quercetin, hesperidin) and their metabolites (Quercetin 3-O-β-glucuronide and hesperetin), either alone or in combination, on first trimester trophoblast cell line HTR-8/SVneo during oxidative stress. The data obtained from this study indicate that selected flavonoids, their respective metabolites significantly reduced the levels of reduced glutathione (p < 0.0001) during HR-induced oxidative stress. These flavonoids also inhibited the activation of pro-apoptotic kinases (p38 MAPK and c-Jun N-terminal kinase) during HR-induced phosphorylation. In addition, they enhanced spheroid stem-like cell generation from HTR8/SVneo cells, aiding their invasion. Our data suggest that dietary intake of food rich in quercetin or hesperidin during early pregnancy can significantly improve trophoblast (placenta) health and function against oxidative stress., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study aimed to investigate the cytoprotective effects of flavonoids, their metabolites alone or in combination against hypoxia/reoxygenation induced oxidative stress in the transformed human first trimester trophoblast cell line (HTR-8/SVneo). Oxidative stress was achieved with hypoxia followed by reoxygenation and the following assays were performed: MTT, CellTox™ Green Cytotoxicity, CellTiter-Glo®, NADP/NADPH-Glo™, ROS-Glo™/H 2 O 2 , GSH/GSSG-Glo™ and Caspase-Glo® 3/7 assays. HTR-8/SVneo cells, pre-treated for 24 h with flavonoids or their metabolites were protected significantly from oxidative stress. Flavonoids were associated with ROS modulation, reducing the generation of superoxide/hydrogen peroxide. The activities of caspases 3/7 were also significantly reduced significantly in HTR-8/SVneo cells pre-treated with flavonoids. This study has shown for the first time that 24 h pre-treatment with flavonoids, their metabolites alone or in combination, protected against HR-induced oxidative stress in the trophoblast cell line. These data indicate that dietary flavonoids may be beneficial to placental health and invasion during early gestation., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

NGF (nerve growth factor) and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 transamidase activity by NGF in retinoic acid-induced differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. The role of TG2 in NGF-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with NGF in N2a and SH-SY5Y cells. NGF mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON (Z-ZON-Val-Pro-Leu-OMe; Benzyloxycarbonyl-(6-Diazo-5-oxonorleucinyl)-l-valinyl-l-prolinyl-l-leucinmethylester) and R283 (1,3,dimethyl-2[2-oxo-propyl]thio)imidazole chloride) and by pharmacological inhibition of extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB) and protein kinase C (PKC), and removal of extracellular Ca 2+ . Fluorescence microscopy demonstrated NGF induced in situ TG2 activity. TG2 inhibition blocked NGF-induced attenuation of hypoxia-induced cell death and neurite outgrowth in both cell lines. Together, these results demonstrate that NGF stimulates TG2 transamidase activity via a ERK1/2, PKB and PKC-dependent pathway in differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. Furthermore, NGF-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results suggest a novel and important role of TG2 in the cellular functions of NGF., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Pharmacologically-induced pre- and post-conditioning represent attractive therapeutic strategies to reduce ischaemia/reperfusion injury during cardiac surgery and following myocardial infarction. We have previously reported that transglutaminase 2 (TG2) activity is modulated by the A 1 adenosine receptor and β 2 -adrenoceptor in H9c2 cardiomyoblasts. The primary aim of this study was to determine the role of TG2 in A 1 adenosine receptor and β 2 -adrenoceptor-induced pharmacological pre- and post-conditioning in the H9c2 cells. H9c2 cells were exposed to 8h hypoxia (1% O 2 ) followed by 18h reoxygenation, after which cell viability was assessed by monitoring mitochondrial reduction of MTT, lactate dehydrogenase release and caspase-3 activation. N 6 -cyclopentyladenosine (CPA; A 1 adenosine receptor agonist), formoterol (β 2 -adrenoceptor agonist) or isoprenaline (non-selective β-adrenoceptor agonist) were added before hypoxia/reoxygenation (pre-conditioning) or at the start of reoxygenation following hypoxia (post-conditioning). Pharmacological pre- and post-conditioning with CPA and isoprenaline significantly reduced hypoxia/reoxygenation-induced cell death. In contrast, formoterol did not elicit protection. Pre-treatment with pertussis toxin (G i/o -protein inhibitor), DPCPX (A 1 adenosine receptor antagonist) or TG2 inhibitors (Z-DON and R283) attenuated the A 1 adenosine receptor-induced pharmacological pre- and post-conditioning. Similarly, pertussis toxin, ICI 118,551 (β 2 -adrenoceptor antagonist) or TG2 inhibition attenuated the isoprenaline-induced cell survival. Knockdown of TG2 using small interfering RNA (siRNA) attenuated CPA and isoprenaline-induced pharmacological pre- and post-conditioning. Finally, proteomic analysis following isoprenaline treatment identified known (e.g. protein S100-A6) and novel (e.g. adenine phosphoribosyltransferase) protein substrates for TG2. These results have shown that A 1 adenosine receptor and β 2 -adrenoceptor-induced protection against simulated hypoxia/reoxygenation occurs in a TG2 and G i/o -protein dependent manner in H9c2 cardiomyoblasts., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: Ensuring reliable administration and reporting of cognitive screening tests are fundamental in establishing good clinical practice and research. This study captured the rate and type of errors in clinical practice, using the Addenbrooke's Cognitive Examination-III (ACE-III), and then the reduction in error rate using a computerized alternative, the ACEmobile app., Methods: In study 1, we evaluated ACE-III assessments completed in National Health Service (NHS) clinics ( n  = 87) for administrator error. In study 2, ACEmobile and ACE-III were then evaluated for their ability to capture accurate measurement., Results: In study 1, 78% of clinically administered ACE-IIIs were either scored incorrectly or had arithmetical errors. In study 2, error rates seen in the ACE-III were reduced by 85%-93% using ACEmobile., Discussion: Error rates are ubiquitous in routine clinical use of cognitive screening tests and the ACE-III. ACEmobile provides a framework for supporting reduced administration, scoring, and arithmetical error during cognitive screening.

Tissue transglutaminase 2 (TG2) is modulated by protein kinase A (PKA) mediated phosphorylation: however, the precise mechanism(s) of its modulation by G-protein coupled receptors coupled to PKA activation are not fully understood. In the current study we investigated the potential regulation of TG2 activity by the β 2 -adrenoceptor in rat H9c2 cardiomyoblasts. Transglutaminase transamidation activity was assessed using amine-incorporating and protein cross-linking assays. TG2 phosphorylation was determined via immunoprecipitation and Western blotting. The long acting β 2 -adrenoceptor agonist formoterol induced time- and concentration-dependent increases in TG2 transamidation. Increases in TG2 activity were reduced by the TG2 inhibitors Z-DON (Benzyloxycarbonyl-(6-Diazo-5-oxonorleucinyl)-L-valinyl-L-prolinyl-L-leucinmethylester) and R283 ((1,3,dimethyl-2[2-oxo-propyl]thio)imidazole chloride). Responses to formoterol were blocked by pharmacological inhibition of PKA, extracellular signal-regulated kinase 1 and 2 (ERK1/2), or phosphatidylinositol 3-kinase (PI-3K) signalling. Furthermore, the removal of extracellular Ca 2+ also attenuated formoterol-induced TG2 activation. Fluorescence microscopy demonstrated TG2-induced biotin-X-cadaverine incorporation into proteins. Formoterol increased the levels of TG2-associated phosphoserine and phosphothreonine, which were blocked by inhibition of PKA, ERK1/2 or PI-3K signalling. Subsequent proteomic analysis identified known (e.g. lactate dehydrogenase A chain) and novel (e.g. Protein S100-A6) protein substrates for TG2. Taken together, the data obtained suggest that β 2 -adrenoceptor-induced modulation of TG2 represents a novel paradigm in β 2 -adrenoceptor cell signalling, expanding the repertoire of cellular functions responsive to catecholamine stimulation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The PAC 1 receptor and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 activity by the PAC 1 receptor in retinoic acid-induced differentiating N2a neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. The role of TG2 in PAC 1 receptor-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27). PACAP-27 mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON and R283 and by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), protein kinase C (Ro 31-8220), MEK1/2 (PD 98059), and removal of extracellular Ca 2+ . Fluorescence microscopy demonstrated PACAP-27 induced in situ TG2 activity. TG2 inhibition blocked PACAP-27 induced attenuation of hypoxia-induced cell death and outgrowth of axon-like processes. TG2 activation and cytoprotection were also observed in human SH-SY5Y cells. Together, these results demonstrate that TG2 activity was stimulated downstream of the PAC 1 receptor via a multi protein kinase dependent pathway. Furthermore, PAC 1 receptor-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results highlight the importance of TG2 in the cellular functions of the PAC 1 receptor., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection., (Copyright © 2016 Elsevier Inc. All rights reserved.)

At present, little is known about the effect(s) of organophosphorous compounds (OPs) on cardiomyocytes. In this study, we have investigated the effects of phenyl saligenin phosphate (PSP), two organophosphorothioate insecticides (diazinon and chlorpyrifos), and their acutely toxic metabolites (diazoxon and chlorpyrifos oxon) on mitotic and differentiated H9c2 cardiomyoblasts. OP-induced cytotoxicity was assessed by monitoring MTT reduction, LDH release, and caspase-3 activity. Cytotoxicity was not observed with diazinon, diazoxon, or chlorpyrifos oxon (48 h exposure; 200 μM). Chlorpyrifos-induced cytotoxicity was only evident at concentrations >100 μM. In marked contrast, PSP displayed pronounced cytotoxicity toward mitotic and differentiated H9c2 cells. PSP triggered the activation of JNK1/2 but not ERK1/2, p38 MAPK, or PKB, suggesting a role for this pro-apoptotic protein kinase in PSP-induced cell death. The JNK1/2 inhibitor SP 600125 attenuated PSP-induced caspase-3 and JNK1/2 activation, confirming the role of JNK1/2 in PSP-induced cytotoxicity. Fluorescently labeled PSP (dansylated PSP) was used to identify novel PSP binding proteins. Dansylated PSP displayed cytotoxicity toward differentiated H9c2 cells. 2D-gel electrophoresis profiles of cells treated with dansylated PSP (25 μM) were used to identify proteins fluorescently labeled with dansylated PSP. Proteomic analysis identified tropomyosin, heat shock protein β-1, and nucleolar protein 58 as novel protein targets for PSP. In summary, PSP triggers cytotoxicity in differentiated H9c2 cardiomyoblasts via JNK1/2-mediated activation of caspase-3. Further studies are required to investigate whether the identified novel protein targets of PSP play a role in the cytotoxicity of this OP, which is usually associated with the development of OP-induced delayed neuropathy.

Whilst mitotic rat embryonic cardiomyoblast-derived H9c2 cells have been widely used as a model system to study the protective mechanisms associated with flavonoids, they are not fully differentiated cardiac cells. Hence, the aim of this study was to investigate the cardioprotective and cardiotoxic actions of quercetin and two of its major in vivo metabolites, quercetin 3-glucuronide and 3'-O-methyl quercetin, using differentiated H9c2 cells. The differentiated cardiomyocyte-like phenotype was confirmed by monitoring expression of cardiac troponin 1 after 7 days of culture in reduced serum medium containing 10 nM all-trans retinoic acid. Quercetin-induced cardiotoxicity was assessed by monitoring MTT reduction, lactate dehydrogenase (LDH) release, caspase 3 activity and reactive oxygen species production after prolonged flavonoid exposure (72 hr). Cardiotoxicity was observed with quercetin and 3'-O-methyl quercetin, but not quercetin 3-glucuronide. Cardioprotection was assessed by pre-treating differentiated H9c2 cells with quercetin or its metabolites for 24 hr prior to 2-hr exposure to 600 μM H2 O2, after which oxidative stress-induced cell damage was assessed by measuring MTT reduction and LDH release. Cardioprotection was observed with quercetin and 3'-O-methyl quercetin, but not with quercetin 3-glucuronide. Quercetin attenuated H2 O2 -induced activation of ERK1/2, PKB, p38 MAPK and JNK, but inhibitors of these kinases did not modulate quercetin-induced protection or H2 O2 -induced cell death. In summary, quercetin triggers cardioprotection against oxidative stress-induced cell death and cardiotoxicity after prolonged exposure. Further studies are required to investigate the complex interplay between the numerous signalling pathways that are modulated by quercetin and which may contribute to the cardioprotective and cardiotoxic effects of this important flavonoid., (© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Background and Purpose: Tissue transglutaminase (TG2) has been shown to mediate cell survival in many cell types. In this study, we investigated whether the role of TG2 in cytoprotection was mediated by the activation of PKA and PKC in cardiomyocyte-like H9c2 cells., Experimental Approach: H9c2 cells were extracted following stimulation with phorbol-12-myristate-13-acetate (PMA) and forskolin. Transglutaminase activity was determined using an amine incorporating and a protein crosslinking assay. The presence of TG isoforms (TG1, 2, 3) was determined using Western blot analysis. The role of TG2 in PMA- and forskolin-induced cytoprotection was investigated by monitoring H₂O₂-induced oxidative stress in H9c2 cells., Key Results: Western blotting showed TG2 >> TG1 protein expression but no detectable TG3. The amine incorporating activity of TG2 in H9c2 cells increased in a time and concentration-dependent manner following stimulation with PMA and forskolin. PMA and forskolin-induced TG2 activity was blocked by PKC (Ro 31-8220) and PKA (KT 5720 and Rp-8-Cl-cAMPS) inhibitors respectively. The PMA- and forskolin-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Immunocytochemistry revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (β-tubulin) and novel (α-actinin) protein substrates for TG2. Pretreatment with PMA and forskolin reversed H2 O2 -induced decrease in MTT reduction and release of LDH. TG2 inhibitors R283 and Z-DON blocked PMA- and forskolin-induced cytoprotection., Conclusions and Implications: TG2 activity was stimulated via PKA- and PKC-dependent signalling pathways in H9c2 cells These results suggest a role for TG2 in cytoprotection induced by these kinases., (© 2014 The British Pharmacological Society.)

As the hardware of FLIM technique becomes mature, the most important criterion for FLIM application is the correct interpretation of its data. In this research, first of all, a more orthogonal phasor approach, called as Modified Phasor Approach (MPA), is put forward. It is a way to calculate the lifetime of the complex fluorescent process, and a rule to measure how much the fluorescence process deviates from single exponential decay. Secondly, MPA is used to analysis the time-resolved fluorescence processes of the transfected CHO-K1 Cell lines expressing adenosine receptor A1R tagged by CYP and YFP, measured in the channel of the acceptor. The image of the fluorescence lifetime and the multiplication of the fluorescence lifetime and deviation from single exponential decay reveal the details of the Homo-FRET. In one word, MPA provides the physical meaning in its whole modified phasor space, and broadens the way for the application of the fluorescence lifetime imaging.

Background: Previous studies have revealed that the mitochondrial permeability transition pore (MPTP) plays a critical role in necrotic and apoptotic cell death. Given the opposed roles of the MPTP in cardioprotection (transient versus sustained opening) the primary aim of this study was to determine how two structurally different MPTP inhibitors (cyclosporine A and bongkrekic acid) administered for varying time regimes influenced ischemia/reperfusion (I/R)-induced injury in myocardial slices from rat left ventricle. A second objective was to explore how pharmacologic MPTP opening (using atractyloside) at different time points during I/R modulated myocardial injury., Materials and Methods: Myocardial slices from rat left ventricle were subjected to 90 min ischemia/120 min reoxygenation at 37°C. MPTP inhibitors and openers were added at various time points during the experimental regime. Tissue injury was assessed by creatine kinase (CK) released and determination of cell necrosis and apoptosis. Myocardial caspase 3 activity was also determined., Results: The results show that the status of MPTP can dramatically influence ischemic/reoxygenation induced injury and protection of the rat left ventricular myocardium. Importantly, the status of the MPTP during first 10 min of reoxygenation is of critical importance with both opening and closing of the pore being as protective as ischemic preconditioning., Conclusions: The present study has shown that both formation and inhibition of the MPTP can be exploited for therapeutic purposes and that there is a defined therapeutic window, with the first few minutes of reoxygenation being a crucial period to achieve cardioprotection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Ischaemic postconditioning is a phenomenon whereby short periods of ischaemia applied during the start of reperfusion protect the myocardium from the damaging consequences of reperfusion. As such, pharmacological-induced postconditioning represents an attractive therapeutic strategy for reducing reperfusion injury during cardiac surgery and following myocardial infarction. The primary aim of this study was to determine the role of large-conductance Ca²(+)-activated potassium channels (BK(Ca) channels) in adenosine A₁ receptor-induced pharmacological postconditioning in the rat embryonic cardiomyoblast-derived cell line H9c2. H9c2 cells were exposed to 6 h hypoxia (0.5% O₂) followed by 18 h reoxygenation (H/R) after which cell viability was assessed by monitoring lactate dehydrogenase (LDH) release and caspase-3 activation. The adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA; 100 nmol/L) or the BK(Ca) channel opener NS1619 (10 µmol/L) were added for 30 min at the start of reoxygenation following 6 h hypoxic exposure. Where appropriate, cells were treated (15 min) before pharmacological postconditioning with the BK(Ca) channel blockers paxilline (1 µmol/L) or iberiotoxin (100 nmol/L). Pharmacological postconditioning with CPA or NS1619 significantly reduced H/R-induced LDH release. Treatment with paxilline or iberiotoxin attenuated adenosine A₁ receptor and NS1619-induced pharmacological postconditioning. These results have shown for the first time that BK(Ca) channels are involved in adenosine A₁ receptor-induced pharmacological postconditioning in a cell model system.

Protein kinase B (PKB/Akt) plays a critical role in cell survival but the investigation of its involvement has been limited by the lack of specific pharmacological agents. In this study, using novel PKB inhibitors (VIII and XI), we investigated the role of PKB in cardioprotection of the rat and human myocardium, the location of PKB in relation to mitoK(ATP) channels and p38 mitogen-activated protein kinase (p38 MAPK), and whether the manipulation of PKB can overcome the unresponsiveness to protection of the diabetic myocardium. Myocardial slices from rat left ventricle and from the right atrial appendage of patients undergoing elective cardiac surgery were subjected to 90 min ischaemia/120 min reoxygenation at 37 degrees C. Tissue injury was assessed by creatine kinase (CK) released and determination of cell necrosis and apoptosis. The results showed that blockade of PKB activity caused significant reduction of CK release and cell death, a benefit that was as potent as ischaemic preconditioning and could be reproduced by blockade of phosphatidylinositol 3-kinase (PI-3K) with wortmannin and LY 294002. The protection was time dependent with maximal benefit seen when PKB and PI-3K were inhibited before ischaemia or during both ischaemia and reoxygenation. In addition, it was revealed that PKB is located downstream of mitoK(ATP) channels but upstream of p38 MAPK. PKB inhibition induced a similar degree of protection in the human and rat myocardium and, importantly, it reversed the unresponsiveness to protection of the diabetic myocardium. In conclusion, inhibition of PKB plays a critical role in protection of the mammalian myocardium and may represent a clinical target for the reduction of ischaemic injury.

Large-conductance Ca(2+)-activated potassium channels, located on the inner mitochondrial membrane, have recently been implicated in cytoprotection. Therefore, the primary aim of this study was to determine the role of large-conductance Ca(2+)-activated potassium channels in adenosine A(1) receptor-induced pharmacological preconditioning in the rat embryonic cardiomyoblast-derived cell line H9c2. For pharmacological preconditioning, H9c2 cells were exposed to the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (100 nM) or the Ca(2+)-activated potassium channel opener NS1619 (10 microM) for 30 min prior to 6 h hypoxia (0.5% O(2)) in glucose-free and serum-free media. Where appropriate cells were treated (15 min) before pharmacological preconditioning with the Ca(2+)-activated potassium channels blockers paxilline (1 microM) or iberiotoxin (100 nM). Cell viability following 6 h hypoxia was assessed by monitoring lactate dehydrogenase (LDH) release and caspase-3 activation. Ca(2+)-activated potassium channel subunit protein expression and cell survival protein kinase (ERK1/2 and PKB/Akt) activation were assessed by Western blotting. The results demonstrate that the adenosine A(1) receptor is functionally expressed in H9c2 cells and when activated protects against hypoxia-induced LDH release and caspase-3 activation. Treatment with paxilline or iberiotoxin attenuated adenosine A(1) receptor and NS1619-induced pharmacological preconditioning. Large-conductance Ca(2+)-activated potassium channel alpha and beta4 protein subunits were detected in mitochondrial fractions isolated from H9c2 cells. NS1619 (10 microM) induced no significant changes in ERK1/2 or PKB phosphorylation. These results have shown for the first time that large-conductance Ca(2+)-activated potassium channels are involved in adenosine A(1) receptor-induced pharmacological preconditioning in a cell model system.

Background and Purpose: 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., Experimental Approach: Non-cardiomyocyte phenotype was determined by confocal microscopy by using discoidin domain receptor 2, alpha-actin and desmin antibodies. P2Y receptor expression was investigated by reverse transcription-polymerase chain reaction and immunocytochemistry, and receptor function by cAMP and inositol phosphate (IP) accumulation induced by adenine or uracil nucleotides in the presence or absence of selective antagonists of P2Y(1) (MRS 2179, 2-deoxy-N(6)-methyl adenosine 3',5'-diphosphate diammonium salt), P2Y(6) (MRS 2578) and P2Y(11) (NF 157, 8,8'-[carbonylbis[imino-3,1-phenylenecarbonylimino(4-fluoro-3,1-phenylene)carbonylimino]]bis-1,3,5-naphthalene trisulphonic acid hexasodium salt) receptors. G(i/o) and G(q/11) pathways were evaluated by using Pertussis toxin and YM-254890 respectively., Key Results: The cells (>95%) were alpha-actin and discoidin domain receptor 2-positive and desmin-negative. P2Y(1), P2Y(2), P2Y(4), P2Y(6) were detected by reverse transcription-polymerase chain reaction and immunocytochemistry, and P2Y(11)-like receptors at protein level. All di- or tri-phosphate nucleotides stimulated IP production in an YM-254890-sensitive manner. AMP, ADPbetaS, ATP and ATPgammaS increased cAMP accumulation, whereas UDP and UTP inhibited cAMP response, which was abolished by Pertussis toxin. MRS 2179 and NF 157 inhibited ADPbetaS-induced IP production. MRS 2578 blocked UDP- and UTP-mediated IP responses., Conclusion and Implications: P2Y(1)-, P2Y(2)-, P2Y(4)-, P2Y(6)-, P2Y(11)-like receptors were co-expressed and induced function through G(q/11) protein coupling in myofibroblasts. Furthermore, P2Y(2) and P2Y(4) receptor subtypes were also coupled to G(i/o). The G(s) response to adenine nucleotides suggests a possible expression of a new P2Y receptor subtype.

We report the development of a system combining the capabilities of fluorescence imaging spectroscopy (x, lambda, I), fluorescence lifetime (tau) and static and dynamic fluorescence anisotropy (r), enabling the wide-field measurement of the spectroscopic parameters of fluorophores: (x, lambda, I, tau, r). The system employs a frequency domain data collection strategy with a modulated light emitting diode as the light source. A polarization rotator placed in the excitation path after a polarizer allows alternating parallel and perpendicular images to be collected without moving parts. A second polarizer on the emission side serves as the analyzer, leading to estimations of the wavelength-dependent dynamic anisotropies. The spectrograph has a nominal range of 365-920 nm; however, the light-emitting diodes and filter sets used in this study restricted the usable range from about 510 to 700 nm. The system was tested on rhodamine 6G (R6G) solutions containing 0, 15, 37, 45, 59, 74 and 91 glycerol. These experiments gave rotational diffusion results comparing favourably with literature values while also demonstrating a trend towards shorter measured lifetimes at high refractive index. The ability of the system to resolve mixtures was tested on mixtures of anti-human IgG-FITC (gamma-chain-specific) and R6G. These fluorophores have similar lifetimes but could be separated using anisotropy parameters. The imaging capabilities of the system were tested on mixtures of fluorescent beads with glycerol solutions of R6G.

Previous studies using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis have shown that the P2Y(14) receptor is expressed at high levels in human neutrophils. Therefore the primary aim of this study was to determine whether the P2Y(14) receptor is functionally expressed in human neutrophils. In agreement with previous studies RT-PCR analysis detected the expression of P2Y(14) receptor mRNA in human neutrophils. UDP-glucose (IC(50)=1 microM) induced a small but significant inhibition (circa 30%) of forskolin-stimulated cAMP accumulation suggesting functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity in human neutrophils. In contrast, the other putative P2Y(14) receptor agonists UDP-galactose and UDP-glucuronic acid (at concentrations up to 100 microM) had no significant effect, whereas 100 microM UDP-N-acetylglucosamine-induced a small but significant inhibition of forskolin-stimulated cAMP accumulation (20% inhibition). UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine behaved as partial agonists by blocking UDP-glucose mediated inhibition of forskolin-induced cAMP accumulation. Treatment of neutrophils with pertussis toxin (G(i/o) blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. UDP-glucose (100 microM) also induced a modest increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, whereas the other sugar nucleotides had no effect on ERK1/2 activation. Finally, UDP-glucose and related sugar nucleotides had no significant effect on N-formyl-methionyl-leucyl-phenylalanine-induced elastase release from neutrophils. In summary, although we have shown that the P2Y(14) receptor is functionally expressed in human neutrophils (coupling to inhibition of forskolin-induced cAMP and ERK1/2 activation) it does not modulate neutrophil degranulation (assessed by monitoring elastase release). Clearly further studies are required in order to establish the functional role of the P2Y(14) receptor expressed in human neutrophils.

This study aimed to characterize beta(3)-adrenergic receptors (ARs) in rat neonatal cardiomyocytes using the noradrenaline (NOR) properties to modulate the expression and function of the three beta-ARs. We assessed the effect of NOR (physiological nonselective agonist), isoprenaline (ISO, beta-nonselective agonist), dobutamine (DOB, beta(1)-selective agonist), and procaterol (PROC, beta(2)-selective agonist) on cAMP accumulation using cardiomyocytes untreated or treated with 100 microM NOR for 24 h. The inhibition of forskolin-stimulated cAMP accumulation was determined using NOR, isoprenaline, and the beta(3)-selective agonists 4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid (BRL 37344) and 5-[-2-([-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243). The experiments were performed in the absence or presence of propranolol or 2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate (CGP 20712A) and/or 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118551) to inhibit beta(1)- and beta(2)-AR stimulation and 1-(2-ethylphenoxy)-3-[[1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino-(2S)-2-propanol hydrochloride (SR 59230A) (beta(3)-selective antagonist). In addition, the level of the three subtypes was determined by reverse transcription polymerase chain reaction and Western blotting. NOR pretreatment decreased the activation of cAMP induced by NOR, isoprenaline, and DOB, whereas PROC response was abolished. The inhibition of NOR response by CGP 20712A or ICI 118551 demonstrated that beta(1)- and beta(2)-ARs are down-regulated and that beta(2)-AR functional activity was also abolished in cardiomyocytes exposed to chronic stimulation. beta(3)-AR function was observed with NOR and ISO when beta(1)-/beta(2)-ARs were blocked and with both beta(3)-selective agonists in NOR-treated cells only. This response was completely inhibited by SR 59230A and involved G(i) protein. Furthermore, the results from functional studies agree well with those from expression experiments. In conclusion, these data provide strong evidence that beta(3)-ARs are functionally up-regulated and coupled to G(i) protein in rat neonatal cardiomyocytes following chronic exposure to NOR when beta(1)- and beta(2)-ARs are down-regulated.

Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis has previously shown that the P2Y(14) receptor is expressed in peripheral immune cells including lymphocytes. Although in transfected cells the P2Y(14) receptor couples to pertussis toxin-sensitive G(i/o) protein, the functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity has not been reported. Therefore, the primary aim of this study was to determine whether the P2Y(14) receptor is functionally expressed in murine spleen-derived T- and B-lymphocyte-enriched populations. RT-PCR analysis detected the expression of P2Y(14) receptor mRNA in whole spleen and isolated T- and B-lymphocytes. In T cells, UDP-glucose (EC(50) = 335 nM) induced a small but significant inhibition (circa 20%) of forskolin-stimulated cAMP accumulation, suggesting functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity. In contrast, the other putative P2Y(14) 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 (100 microM) had no significant effect on forskolin-stimulated cAMP accumulation. Treatment of T cells with pertussis toxin (G(i/o) blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. T-cell proliferation in response to anti-CD3 monoclonal antibody (1 microg ml(-1)) was significantly inhibited by UDP-glucose (59% inhibition; p[IC(50)] = 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- (5 ng ml(-1)) induced T-cell proliferation was also significantly inhibited by all four agonists. In summary, we have shown that the P2Y(14) 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 P2Y(14) receptor may play an important role in modulating immune function.

Three subtypes of adenosine receptors (A(1), A(2A) and A(3) ARs) are functionally expressed in cardiomyocytes. Adenosine released during ischemia and ischemia/reperfusion plays a major role in cardioprotection. Phosphatidylinositol 3-kinase (PI-3K)/protein kinase B (PKB) and MEK/ERK1/2 pathways are involved in cell survival. Since the role of these pathways in AR-mediated preconditioning is poorly understood, we have investigated whether PI-3K/PKB and/or MEK1/ERK1/2 pathways are involved in AR-induced cardioprotection in neonatal rat cardiomyocytes. Cells were pre-treated (15 min) with adenosine (non-selective), CPA (A(1)), CGS 21680 (A(2A)) or Cl-IB-MECA (A(3)) before 4 h hypoxia (0.5% O(2)) and 18 h reoxygenation (HX4/R). HX4/R-induced increase in LDH release was significantly reduced by adenosine (70%), CPA (59%) and Cl-IB-MECA (46%). The MEK1 inhibitor PD 98059 suppressed the effects of adenosine, CPA, and Cl-IB-MECA on LDH release, whereas the PI-3K inhibitor wortmannin did not reverse this cardioprotection. Western blotting of phosphorylated ERK1/2 and PKB during HX4/R supported the involvement of ERK1/2 and not PKB in A(1) and A(3) agonist-mediated cardioprotection. In addition, adenosine, CPA and Cl-IB-MECA inhibited HX4/R-induced caspase 3 activity by 75%, 70% and 59%, respectively, and this inhibition was abolished by PD 98059. Interestingly, wortmannin inhibited by 66% the anti-apoptotic response triggered by Cl-IB-MECA but had no effect on adenosine or CPA-induced inhibition of caspase 3. CGS 21680 did not modify cell survival or caspase 3 activity. In conclusion, these data show that the preconditioning effect of adenosine requires A(1) and A(3) but not A(2A) ARs and involves an anti-apoptotic effect via MEK1/ERK1/2 pathway in neonatal rat cardiomyocytes. In addition, A(3)AR-induced preconditioning also involves a PI-3K dependent pathway.

Mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase 1 and 2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (PKB; also known as Akt) are important antiapoptotic signalling pathways which have recently been implicated in cardioprotection. However, at present the involvement of ERK1/2 and PI3-kinase/PKB in adenosine receptor-mediated cardioprotection is poorly understood. In this study we used isolated rat right ventricular strips, contracted by electrical-field stimulation, in order to investigate the role of ERK1/2 and PI3-kinase/PKB in adenosine receptor-induced cardioprotection. Ventricle strips were pretreated for 2 min with the agonists adenosine (non-selective), CPA (A1 selective), CGS 21680 (A2A selective) and Cl-IB-MECA (A3 selective) before 30 min hypoxia followed by 30 min reoxygenation. Each agonist significantly improved posthypoxic percentage contraction recovery compared to control strips. Similarly hypoxic preconditioning (10 min hypoxia followed by 20 min reoxygenation) significantly improved posthypoxic percentage contraction recovery compared to non-preconditioned strips. The selective adenosine receptor antagonists DPCPX (A1), ZM 241385 (A2A) and MRS 1220 (A3) attenuated cardioprotection induced by CPA, CGS 21680 and Cl-IB-MECA, respectively. Pre-incubation (30 min) of ventricle strips with the MEK1 inhibitor PD 98059 (50 microM) or the PI3-kinase inhibitor wortmannin (100 nM) significantly reduced posthypoxic percentage contraction recovery induced by hypoxic preconditioning. In contrast, PD 98059 and wortmannin had no significant effect on cardioprotection induced by CPA, Cl-IB-MECA or CGS 21680. Overall these data indicate that although selective A1, A2A and A3 adenosine receptor agonists induce preconditioning in rat right ventricular strips the effects are independent of ERK1/2- and PI3-kinase-dependent pathways. In contrast ERK1/2 and PI3-kinase-dependent pathways do appear to be involved in early hypoxic preconditioning.

In transfected cells, the P2Y14 receptor reportedly couples to pertussis toxin-sensitive G(i/o)-proteins. However, 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 investigate the effects of uridine 5'-diphosphoglucose (UDP-glucose) on forskolin-stimulated cyclic AMP (cAMP) accumulation in two cell lines that reportedly express P2Y14 receptor mRNA, namely human neuroblastoma SH-SY5Y cells and human astrocytoma U373 MG cells. In U373 MG cells, UDP-glucose inhibited forskolin-stimulated cAMP accumulation in a concentration-dependent manner (pEC50=4.5 +/- 0.3). Furthermore, treatment with pertussis toxin abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation in U373 MG cells. In SH-SY5Y cells, UDP-glucose had no significant effect on forskolin-stimulated cAMP accumulation. To confirm the expression of P2Y14 receptor mRNA in U373 MG and SH-SY5Y cells, we performed reverse transcriptase polymerase chain reaction (RT-PCR) analysis. However, RT-PCR did not detect the expression of P2Y14 receptor mRNA in SH-SY5Y cells or surprisingly in U373 MG cells. In conclusion, we have shown that although UDP-glucose inhibits forskolin-stimulated cAMP accumulation in human U373 MG astrocytoma cells, we did not detect P2Y14 receptor mRNA in these cells. These results would suggest that the effects of UDP-glucose in U373 MG cells are independent of P2Y14 receptor expression. Thus, results obtained with UDP-glucose should be interpreted with caution, since they clearly may not necessarily reflect the involvement of the P2Y14 receptor.

It is well established that adenosine receptors are involved in cardioprotection and that protein kinase B (PKB) is associated with cell survival. Therefore, in this study we have investigated whether adenosine receptors (A(1), A(2A) and A(3)) activate PKB by Western blotting and determined the involvement of phosphatidylinositol 3-kinase (PI-3K)/PKB in adenosine-induced preconditioning in cultured newborn rat cardiomyocytes. Adenosine (non-selective agonist), CPA (A(1) selective agonist) and Cl-IB-MECA (A(3) selective agonist) all increased PKB phosphorylation in a time- and concentration-dependent manner. The combined maximal response to CPA and Cl-IB-MECA was similar to the increase in PKB phosphorylation induced by adenosine alone. CGS 21680 (A(2A) selective agonist) did not stimulate an increase in PKB phosphorylation. Adenosine, CPA and Cl-IB-MECA-mediated PKB phosphorylation were inhibited by pertussis toxin (PTX blocks G(i)/G(o)-protein), genistein (tyrosine kinase inhibitor), PP2 (Src tyrosine kinase inhibitor) and by the epidermal growth factor (EGF) receptor tyrosine kinase inhibitor AG 1478. The PI-3K inhibitors wortmannin and LY 294002 blocked A(1) and A(3) receptor-mediated PKB phosphorylation. The role of PI-3K/PKB in adenosine-induced preconditioning was assessed by monitoring Caspase 3 activity and lactate dehydrogenase (LDH) release induced by exposure of cardiomyocytes to 4 h hypoxia (0.5% O(2)) followed by 18 h reoxygenation (HX4/R). Pre-treatment with wortmannin had no significant effect on the ability of adenosine-induced preconditioning to reduce the release of LDH or Caspase 3 activation following HX4/R. In conclusion, we have shown for the first time that adenosine A(1) and A(3) receptors trigger increases in PKB phosphorylation in rat cardiomyocytes via a G(i)/G(o)-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning.

Reactive oxygen species including H(2)O(2) activate an array of intracellular signalling cascades that are closely associated with cell death and cell survival pathways. The human neuroblastoma SH-SY5Y cell line is widely used as model cell system for studying neuronal cell death induced by oxidative stress. However, at present very little is known about the signalling pathways activated by H(2)O(2) in SH-SY5Y cells. Therefore, in this study we have investigated the effect of H(2)O(2) on extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (PKB) activation in undifferentiated and differentiated SH-SY5Y cells. H(2)O(2) stimulated time and concentration increases in ERK1/2, JNK and PKB phosphorylation in undifferentiated and differentiated SH-SY5Y cells. No increases in p38 MAPK phosphorylation were observed following H(2)O(2) treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 ((2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited H(2)O(2)-induced increases in ERK1/2 and PKB phosphorylation. Furthermore, H(2)O(2)-mediated increases in ERK1/2 activation were sensitive to the MAPK kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK responses were blocked by the JNK inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Treatment of SH-SY5Y cells with H(2)O(2) (1 mM; 16 h) significantly increased the release of lactate dehydrogenase (LDH) into the culture medium indicative of a decrease in cell viability. Pre-treatment with wortmannin, SP 600125 or SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; p38 MAPK inhibitor) had no effect on H(2)O(2)-induced LDH release from undifferentiated or differentiated SH-SY5Y cells. In contrast, PD 98059 and LY 294002 significantly decreased H(2)O(2)-induced cell death in both undifferentiated and differentiated SH-SY5Y cells. In conclusion, we have shown that H(2)O(2) stimulates robust increases in ERK1/2, JNK and PKB in undifferentiated and differentiated SH-SY5Y cells. Furthermore, the data presented clearly suggest that inhibition of the ERK1/2 pathway protects SH-SY5Y cells from H(2)O(2)-induced cell death.

1. Adenosine A(1), A(2A), and A(3) 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. 2. Adenosine (nonselective agonist), CPA (A(1)), CGS 21680 (A(2A)) or Cl-IB-MECA (A(3)), 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 (A(1)), ZM 241385 (A(2A)), and MRS 1220 (A(3)). 3. PD 98059 (MEK1; ERK kinase inhibitor) abolished all agonist-mediated ERK1/2 phosphorylation. Pertussis toxin (PTX, G(i/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. 4. Cl-IB-MECA-mediated increases in cAMP accumulation suggest that A(3)AR-induced ERK1/2 phosphorylation involves adenylyl cyclase activation via phospholipase C (PLC) and PKC stimulation. 5. In summary, we have shown that ERK1/2 activation by adenosine in cardiomyocytes results from an additive stimulation of A(1), A(2A), and A(3)ARs, which involves G(i/o) proteins, PKC, and tyrosine kinase for A(1) and A(3)ARs, and Gs and PKA for A(2A)ARs. Moreover, the A(3)AR response also involves a cAMP/PKA pathway via PKC activation.

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.

1. Previous studies have shown that the histamine H(1) receptor activates p42/p44 mitogen-activated protein kinases (MAPK) in DDT(1)MF-2 smooth muscle cells via a phosphatidylinositol 3-kinase (PI-3K)-dependent pathway. In this study the effect of histamine H(1) 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 (Ser(473)) and p70 S6 kinase (Thr(421)/Ser(424)) antibodies. 2. Histamine stimulated time and concentration-dependent increases in the phosphorylation of PKB and p70 S6 kinase in DDT(1)MF-2 cells. Both responses were completely inhibited by the histamine H(1) receptor antagonist mepyramine and following pre-treatment with pertussis toxin, to block G(i)/G(o) protein dependent pathways. 3. The PI-3K inhibitors wortmannin (IC(50) 5.9+/-0.5 nM) and LY 294002 (IC(50) 6.9+/-0.8 microM) attenuated the increase in PKB phosphorylation induced by histamine (100 microM) in a concentration-dependent manner. 4. Histamine-induced increases in p70 S6 kinase phosphorylation were partially sensitive to rapamycin (20 nM; 68% inhibition) but completely blocked by wortmannin (100 nM), LY 294002 (30 microM) and the MAPK kinase inhibitor PD 98059 (50 microM). 5. In summary, these data demonstrate that the histamine H(1) receptor stimulates PKB and p70 S6 kinase phosphorylation in DDT(1)MF-2 smooth muscle cells. However, functional studies revealed that histamine does not stimulate DDT(1)MF-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 H(1) receptor.

NF-kappaB is a transcription factor that plays a vital role in regulating inducible gene expression in immune and inflammatory responses. In view of the well documented effects of adenosine on immune and inflammatory responses, we have explored whether adenosine A1, A2B and A3 receptors regulate NF-kappaB activity in transfected Chinese hamster ovary (CHO) cells using a luciferase reporter gene construct. No increases in NF-kappaB activity were observed in CHO-A1, -A2B and -A3 cells stimulated with the non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine. Elevating intracellular cyclic AMP (cAMP) levels using forskolin (direct activator of adenylyl cyclase) and rolipram (type IV, cAMP-specific phosphodiesterase inhibitor), inhibited NF-kappaB activity in CHO cells. Adenosine A2B receptor stimulation also inhibited NF-kappaB activity, whereas adenosine A1 and A3 receptor activation had no effect. These data reflect the known coupling of adenosine A2B receptors to increases in cAMP. In conclusion, adenosine A1, A2B and A3 receptors do not directly activate NF-kappaB in CHO cells. However, adenosine A2B receptor activation significantly inhibited NF-kappaB activity. Inhibition of NF-kappaB activity by the adenosine A2B receptor may contribute to the anti-inflammatory effects of adenosine.

1. 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 H(1) receptor activation on MAPK pathway activation in the smooth muscle cell line DDT(1)MF-2. 2. Histamine stimulated time and concentration-dependent increases in p42/p44 MAPK activation in DDT(1)MF-2 cells. Responses to histamine were inhibited by the histamine H(1) receptor antagonist mepyramine (K(D) 3.5 nM) and following pre-treatment with pertussis toxin (PTX; 57% inhibition). 3. 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; 10 microM; 41% inhibition). Inhibitors of Src tyrosine kinase (PP2) and the epidermal growth factor tyrosine kinase (AG1478) were without effect. Removal of extracellular Ca(2+), chelation of intracellular Ca(2+) with BAPTA and inhibition of focal adhesion assembly (cytochalasin D) had no significant effect on histamine-induced p42/p44 MAPK activation. 4. Histamine stimulated time and concentration-dependent increases in p38 MAPK activation in DDT(1)MF-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). 5. In summary, we have shown the histamine H(1) receptor activates p42/p44 MAPK and p38 MAPK signalling pathways in DDT(1)MF-2 smooth muscle cells. Interestingly, signalling to both pathways appears to involve histamine H(1) receptor coupling to G(i)/G(o)-proteins.

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.

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.

Activation of A(1) adenosine receptors leads to the inhibition of cAMP accumulation and the stimulation of inositol phosphate accumulation via pertussis toxin-sensitive G-proteins. In this study we have investigated the signaling of the A(1) adenosine receptor in Chinese hamster ovary (CHO) cells, when expressed at approximately 203 fmol/mg (CHOA1L) and at approximately 3350 fmol/mg (CHOA1H). In CHOA1L cells, the agonists N(6)-cyclopentyladenosine (CPA), (R)-N(6)-(2-phenylisopropyl)adenosine, and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited cAMP production in a concentration-dependent manner. After pertussis toxin treatment, the agonist NECA produced a stimulation of cAMP production, whereas CPA and (R)-N(6)-(2-phenylisopropyl)adenosine were ineffective. In CHOAIH cells, however, all three agonists produced both an inhibition of adenylyl cyclase and a pertussis toxin-insensitive stimulation of adenylyl cyclase. All three agonists were more potent at inhibiting adenylyl cyclase in CHOA1H cells than in CHOA1L cells. In contrast, A(1) agonists (and particularly NECA) were less potent at stimulating inositol phosphate accumulation in CHOA1H cells than in CHOA1L cells. After pertussis toxin treatment, agonist-stimulated inositol phosphate accumulation was reduced in CHOA1H cells and abolished in CHOA1L cells. The relative intrinsic activity of NECA in stimulating inositol phosphate accumulation, compared to CPA (100%), was much greater in the presence of pertussis toxin (289.6%) than in the absence of pertussis toxin (155.2%). These data suggest that A(1) adenosine receptors can couple to both pertussis toxin-sensitive and -insensitive G-proteins in an expression level-dependent manner. These data also suggest that the ability of this receptor to activate different G-proteins is dependent on the agonist present.

In this study the effect of insulin and A(1)-adenosine receptor stimulation on protein kinase B (PKB) activation has been investigated in the hamster vas deferens smooth muscle cell line DDT(1)MF-2. Increases in PKB phosphorylation were determined by Western blotting using an antibody that detects PKB phosphorylation at Ser(473). Insulin, a recognized activator of PKB, stimulated a concentration-dependent increase in PKB phosphorylation in DDT(1)MF-2 cells (EC(50) 5+/-1 pM). The selective A(1)-adenosine receptor agonist N(6)-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in PKB phosphorylation in DDT(1)MF-2 cells (EC(50) 1.3+/-0.5 nM). CPA-mediated increases in PKB phosphorylation were antagonized by the A(1)-adenosine receptor selective antagonist 1,3-dipropylcyclopentylxanthine (DPCPX) yielding an apparent K(D) value of 2.3 nM. Pre-treatment of DDT(1)MF-2 cells with pertussis toxin (PTX, 100 ng ml(-1) for 16 h), to block G(i)/G(o)-dependent pathways, abolished CPA (1 microM) induced phosphorylation of PKB. In contrast, responses to insulin (100 nM) were resistant to PTX pre-treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin (IC(50) 10.3+/-0.6 nM) and LY 294002 (IC(50) 10.3+/-1.2 microM) attenuated the phosphorylation of PKB elicited by CPA (1 microM) in a concentration-dependent manner. Wortmannin (30 nM) and LY 294002 (30 microM) also blocked responses to insulin (100 nM). Removal of extracellular Ca(2+) and chelation of intracellular Ca(2+) with BAPTA had no significant effect on CPA-induced PKB phosphorylation. Similarly, pretreatment (30 min) with inhibitors of protein kinase C (Ro 31-8220; 10 microM), tyrosine kinase (genistein; 100 microM), mitogen-activated protein (MAP) kinase kinase (PD 98059; 50 microM) and p38 MAPK (SB 203580; 20 microM) had no significant effect on CPA-induced PKB phosphorylation. In conclusion, these data demonstrate that A(1)-adenosine receptor stimulation in DDT(1)MF-2 cells increases PKB phosphorylation through a PTX and PI-3K-sensitive pathway.

1. The effect of protein tyrosine kinase inhibitors on human adenosine A1 receptor-mediated [3H]-inositol phosphate ([3H]-IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO-A1) cells. 2. In agreement with our previous studies the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) stimulated the accumulation of [3H]-IPs in CHO-A1 cells. Pre-treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 microM; 30 min) potentiated the responses elicited by 1 microM (199+/-17% of control CPA response) and 10 microM CPA (234+/-15%). Similarly, tyrphostin A47 (100 microM) potentiated the accumulation of [3H]-IPs elicited by 1 microM CPA (280+/-32%). 3. Genistein (EC50 = 13.7+/-1.2 microM) and tyrphostin A47 (EC50 = 10.4+/-3.9 microM) potentiated the [3H]-IP response to 1 microM CPA in a concentration-dependent manner. 4. Pre-incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 microM; 30 min) and tyrphostin A1 (100 microM; 30 min), respectively, had no significant effect on the accumulation of [3H]-IPs elicited by 1 microM CPA. 5. Genistein (100 microM) had no significant effect on the accumulation of [3H]-IPs produced by the endogenous thrombin receptor (1 u ml(-1); 100+/-10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [3H]-IP response (148+/-13%). 6. Genistein (100 microM) had no effect on the [3H]-IP response produced by activation of the endogenous Gq-protein coupled CCK(A) receptor with the sulphated C-terminal octapeptide of cholecystokinin (1 microM CCK-8; 96+/-6% of control). In contrast, tyrphostin A47 (100 microM) caused a small but significant increase in the response to 1 microM CCK-8 (113+/-3% of control). 7. The phosphatidylinositol 3-kinase inhibitor LY 294002 (30 microM) and the MAP kinase kinase inhibitor PD 98059 (50 microM) had no significant effect on the [3H]-IP responses produced by 1 microM CPA and 1 microM CCK-8. 8. These observations suggest that a tyrosine kinase-dependent pathway may be involved in the regulation of human adenosine A1 receptor mediated [3H]-IP responses in CHO-A1 cells.

In transfected Chinese hamster ovary (CHO-A1) cells the human adenosine A1 receptor directly stimulates pertussis toxin-sensitive increases in inositol phosphate production and potentiates (synergistically) the inositol phosphate responses mediated by Gq-coupled P2Y2 purinoceptor and CCK(A) receptors. In the present study we have investigated the role of Gbetagamma subunits in mediating adenosine A1 receptor effects on phospholipase C activation (both direct and synergistic) by transiently transfecting CHO-A1 cells with a scavenger of Gbetagamma subunits: the C-terminus of beta-adrenoceptor kinase 1 (beta ark1 residues 495-689). [3H]inositol phosphate responses to the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA; 1 microM) were inhibited (41 +/- 1%) in CHO-A1 cells transiently transfected with the Gbetagamma scavenger, beta ark1 (495-689). Expression of beta ark1 (495-689) protein was confirmed by Western blotting. In contrast, adenosine A1 receptor-mediated inhibition of forskolin stimulated [3H]cyclic AMP accumulation was unaffected by transient expression of beta ark1 (495-689). Beta ark1 (495-689) expression had no significant effect on the [3H]inositol phosphate responses produced by activation of the endogenous P2Y2 purinoceptor (100 microM UTP; 92 +/- 0.8% of control). [3H]inositol phosphate accumulation in response to adenosine A receptor activation was also attenuated in CHO-K1 cells co-transfected with the beta ark1 (495-689) minigene (59 +/- 4% inhibition of control response to 1 microM CPA). Finally, transient expression of beta ark1 (495-689) in CHO-A1 cells inhibited the augmentation of [3H]inositol phosphate responses resulting from co-activation of adenosine A1 receptors and P2Y2 purinoceptors. These experiments indicate that Gbetagamma subunits are involved in the direct coupling the adenosine A1 receptor to phospholipase C and that they also participate in the augmentation of P2Y2 purinoceptor-mediated [3H]inositol phosphate responses by the adenosine A1 receptor.

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 microM; 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 microM; 6+/-10% of control). In contrast, daidzein (100 microM), the inactive analogue of genistein had no significant effect (96+/-12 of control). MAP kinase responses to CPA (1 microM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55+/-8% inhibition) and LY 294002 (30 microM; 40+/-5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 microM). 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 microM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67+/-3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 microm; 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 microM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 microM). 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.

We have previously reported that the transfected Gi/Go protein-coupled human adenosine A1 receptor (expressed at 200 fmol/mg of protein) and the endogenous 5-HT1B receptor (not detectable using radioligand binding) suppress forskolin-stimulated cyclic AMP accumulation and stimulate increases in [Ca2+]i in Chinese hamster ovary cells (CHO). In addition, co-activation of the adenosine A1 receptor (but not the 5-HT1B receptor) potentiates the hydrolysis of inositol phospholipids elicited by receptors coupled to Gq-proteins (Dickenson and Hill, 1996. Eur. J. Pharmacol. 320, 141-151). In order to establish whether this difference in ability to modulate Gq-coupled receptor responses is a consequence of low 5-HT1B receptor density, we have stably transfected CHO-KI cells with the human 5-HT1Dbeta cDNA (the human homologue of the rodent 5-HT1B receptor). We initially isolated a clonal cell line (designated CHO5-HT1B cells) displaying moderate specific [3H]5-HT binding (pKd of 8.17+/-0.07 and a Bmax of 140 fmol/mg protein). In CHO5-HT1B cells, the selective human 5-HT1B/1D receptor agonist sumatriptan produced a concentration-dependent inhibition of forskolin-stimulated cyclic AMP accumulation (pEC50=7.92+/-0.04). Sumatriptan also elicited a moderate and pertussis toxin-sensitive increase in [3H]inositol phosphate formation in CHO-5HT1B cells (pEC50=6.51+/-0.05). Finally, sumatriptan synergistically enhanced P2U purinoceptor stimulated [3H]inositol phosphate accumulation through a pertussis toxin-sensitive mechanism. These findings clearly show the significance of 5-HT1B receptor expression level in determining whether 5-HT1B receptor activation can modulate the accumulation of [3H]inositol phosphates elicited by a Gq-protein coupled receptor. The observation that 5-HT1B receptor activation can potentiate Gq-coupled receptor stimulated second messenger responses may have an important physiological role in the regulation of vascular smooth muscle contraction.

Neuropeptide Y (NPY) significantly potentiates the constrictor actions of noradrenaline and ATP on blood vessels via a pertussis toxin (PTX)-sensitive mechanism involving Gi/o (alpha beta gamma) protein subunits (Gi/o, GTP-binding proteins sensitive to PTX). In Chinese hamster ovary K1 (CHO K1) cells expressing specific receptors for these neurotransmitters, stimulation of Gi/o protein-coupled receptors for NPY and other neurotransmitters can augment the Gq/11-coupled (Gq/11, GTP-binding proteins insensitive to PTX) alpha 1B adrenoceptor- or ATP receptor-induced arachidonic acid (AA) release and inositol phosphate (IP) production (early events which may precede vasoconstriction). In this study, we have assessed the role of G beta gamma subunits in the synergistic interaction between Gi/o- (NPY Y1, 5-hydroxytryptamine 5-HT1B, adenosine A1) and Gq/11- [ATP P2Y2 (P2U)]-coupled receptors on AA release by using the specific abilities of regions of the beta-adrenergic receptor kinase (beta ARK1 residues 495-689) and the transducin alpha subunit to associate with G-protein beta gamma subunit dimers and to act as G beta gamma subunit scavengers. Transient expression of beta ARK1(495-689) in CHO K1 cells heterologously expressing NPY Y1 receptors had no significant effect on the PTX-insensitive ability of ATP to stimulate AA release. Stimulation of NPY Y1 receptors (as well as the endogenous 5-hydroxytryptamine 5-HT1B receptor and the transiently expressed human adenosine A1 receptor) resulted in a PTX-sensitive augmentation of ATP-stimulated AA release, which was inhibited by expression of both G beta gamma subunit scavengers. Expression of beta ARK1(495-689) similarly inhibited NPY Y1 receptor augmentation of ATP-stimulated IP production (a measure of phospholipase C activity), a step thought to precede the NPY Y1 receptor-augmented protein kinase C-dependent AA release previously observed in these cells. These experiments demonstrate that G beta gamma subunits, as inhibited by two different G beta gamma scavengers, significantly contribute to the synergistic interaction between NPY Y1 Gi/o- and Gq/11-coupled receptor activity, and are required for the augmentation of IP production and AA release observed in this model cell system.

Thrombin receptor activation in Chinese hamster ovary (CHO) cells stimulates the hydrolysis of inositol phospholipids and the release of arachidonic acid. Our previous studies have shown that activation of the human transfected adenosine A1 receptor in CHO cells (CHO-A1) potentiates the accumulation of inositol phosphates elicited by endogenous P2U purinoceptors and CCKA receptors. In this study we have investigated whether adenosine A1 receptor activation can modulate thrombin-stimulated arachidonic acid release and/or inositol phospholipid hydrolysis in CHO-A1 cells. Thrombin stimulated [3H]arachidonic acid release and total [3H]inositol phosphate accumulation in CHO-A1 cells. Both these responses to thrombin were were insensitive to pertussis toxin. The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), potentiated thrombin-stimulated [3H]arachidonic acid. In marked contrast, PMA inhibited thrombin-stimulated [3H]inositol phosphate accumulation. The selective protein kinase C inhibitor Ro 31-8220 (3-¿1-[3-(2-isothioureido)propyl] indol-3-yl¿-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione) had no effect on thrombin-stimulated [3H]arachidonic acid release but reversed the potentiation of thrombin-stimulated [3H]arachidonic acid release elicited by PMA. The selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) augmented the release of [3H]arachidonic acid produced by thrombin. Co-activation of the adenosine A1 receptor also potentiated thrombin-stimulated [3H]inositol phosphate accumulation. The synergistic interactions between the adenosine A1 receptor and thrombin were abolished in pertussis-toxin-treated cells. The potentiation of [3H]arachidonic acid release by CPA was blocked by the protein kinase C inhibitors Ro 31-8220 and GF 109203X (3-[1-[3-(dimethylamino)propyl]-1 H-indol-3-yl]-4-(1 H-indol-3-yl)- 1H-pyrrole-2,5-dione). In conclusion, thrombin receptor activation in CHO-A1 cells stimulates the accumulation of [3H]inositol phosphates and the release of [3H]arachidonic acid through pertussis-toxin-insensitive G-proteins. Experiments using PMA suggest that protein kinase C differentially regulates thrombin receptor activation of phospholipase C and phospholipase A2. Co-activation of the transfected human adenosine A1 receptor augments thrombin-stimulated phospholipase C and phospholipase A2 activity. Finally, the augmentation of phospholipase A2 activity by the adenosine A1 receptor is inhibited by selective protein kinase C inhibitors, suggesting the involvement of protein kinase C.

The effect of Gi coupled receptor activation (adenosine A1 and 5-HT1B receptors) on cholecystokinin receptor-stimulated inositol phosphate accumulation has been investigated in Chinese hamster ovary cells transfected with the human adenosine A1 receptor cDNA (CHO-A1). CHO cells constitutively express the 5-HT1B receptor [Berg, Clarke, Sailstad, Saltzman and Maayani (1994) Mol. Pharmacol. 46, 477-484]. Our previous studies using CHO-A1 cells have revealed that both the adenosine A1 and 5-HT1B receptor are negatively coupled to adenylyl cyclase activity and stimulate increases in [Ca2+]i, through a pertussis toxin-sensitive pathway. In the present study the selective adenosine A1 receptor agonist N6-cyclopentyladenosine stimulated a pertussis toxin-sensitive increase in total [3H]inositol phosphate accumulation. The sulphated C-terminal octapeptide of cholecystokinin (CCK-8) stimulated a robust and pertussis toxin-insensitive increase in [3H]inositol phosphate accumulation through the activation of CCKA receptors. Co-stimulation of CHO-A1 cells with N6-cyclopentyladenosine and CCK-8 produced a synergistic increase in [3H]inositol phosphate accumulation. The synergistic interaction between N6-cyclopentyladenosine and CCK-8 was abolished in pertussis toxin-treated cells. Synergy between N6-cyclopentyladenosine and CCK-8 still occurred in the absence of extracellular calcium. The 5-HT1B receptor agonist 5-carboxyamidotryptamine did not stimulate a measurable increase in [3H]inositol phosphate accumulation. Furthermore, 5-carboxyamidotryptamine had no significant effect on CCK-8 mediated [3H]inositol phosphate production. Activation of endogenous P2U receptors (Gq/Gll coupled) with ATP gamma S produced a significant increase in [3H]inositol phosphate accumulation. Co-stimulation of CHO-A1 cells with ATP gamma S and CCK-8 produced additive increases in [3H]inositol phosphate accumulation. These data indicate that CHO-A1 cells may prove a useful model system in which to investigate further the mechanisms underlying the intracellular 'cross-talk' between phospholipase C coupled receptors (Gq/Gll linked) and Gi/Go coupled receptors.

1. Chinese hamster ovary cells (CHO-K1) express an endogenous 5-hydroxytryptamine (5-HT)1B-like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)-sensitive mechanism. Furthermore, the human adenosine A1 receptor when expressed in CHO-K1 cells (CHO-A1) has been shown to mobilize intracellular Ca2+ through a PTX-sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5-HT1B-like receptor was able to stimulate increases in intracellular free [Ca2+] ([Ca2+]i) in CHO-A1 cells. 2. In agreement with previous studies using CHO cells, 5-hydroxytryptamine (5-HT) elicited a concentration-dependent inhibition of forskolin-stimulated [3H]-cyclic AMP production in CHO-A1 cells (p[EC50] = 7.73 +/- 0.13). 5-HT (1 microM) inhibited 47 +/- 5% of the [3H]-cyclic AMP accumulation induced by 3 microM forskolin. Forskolin stimulated [3H]-cyclic AMP accumulation was also inhibited by the 5-HT1 receptor agonists (p[EC50] values) 5-carboxyamidotryptamine (5-CT; 8.07 +/- 0.08), RU 24969 (8.12 +/- 0.33) and sumatriptan (5.80 +/- 0.31). 3. 5-HT elicited a concentration-dependent increase in [Ca2+]i in CHO-A1 cells (p[EC50] = 8.07 +/- 0.05). In the presence of 2 mM extracellular Ca2+, 5-HT (1 microM) increased [Ca2+]i from 174 +/- 17 nM to 376 +/- 22 nM. The 5-HT1 receptor agonists (p[EC50] values), 5-carboxyamidotryptamine (5-CT; 7.9 +/- 0.02), RU 24969 (8.1 +/- 0.07) and sumatriptan (5.9 +/- 0.11) all elicited concentration-dependent increases in [Ca2+]i. Similar maximal increases in [Ca2+]i were obtained with each agonist. The selective 5-HT1A receptor agonist, 8-OH-DPAT (10 microM) did not stimulate increases in [Ca2+]i. 5-HT (100 microM) and 5-CT (10 microM) did not stimulate a measurable increase in [3H]-inositol phosphate accumulation in CHO-A1 cells. 4. 5-HT (1 microM)-mediated increases in [Ca2+]i were insensitive to the 5-HT receptor antagonist, ritanserin (5-HT2; 100 nM), ketanserin (5-HT2; 100 nM), LY-278,584 (5-HT3; 1 microM) and WAY 100635 (5-HT1A; 1 microM). The response to 5-HT (100 nM) was antagonized by the non-selective 5-HT1 antagonist, methiothepin (pKb = 8.90 +/- 0.09) and the 5-HT1D antagonist GR 127935 (pKb = 10.44 +/- 0.06). 5. Pretreatment with PTX (200 ng ml-1 for 4 h) completely attenuated the Ca2+ response to 100 microM 5-HT. 6. In untransfected CHO-K1 cells, 5-HT (1 microM), RU 24969 (1 microM), and 5-CT (1 microM) elicited increases in [Ca2+]i similar to those observed in CHO-A1 cells. 7. These data demonstrate that in CHO-K1 cells the endogenously expressed 5-HT1B-like receptor couples to the phospholipase C/Ca2+ signalling pathway through a PTX-sensitive pathway, suggesting the involvement of Gi/Go protein(s).

1. The effect of adenosine A1-receptor and P2-purinoceptor agonists on [3H]-inositol phosphate accumulation has been investigated in CHO-K1 cells transfected with the human adenosine A1-receptor. 2. Adenosine receptor agonists stimulated [3H]-inositol phosphate accumulation in CHO-K1 cells with a rank potency order of N6-cyclopentyladenosine (CPA) > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine > N6-2-(4-aminophenyl) ethyladenosine (APNEA). The responses to both CPA and APNEA were antagonized by the A1 selective antagonist, 1,3-dipropylcyclopentylxanthine (DPCPX) yielding KD values of 1.2 nM and 4.3 nM respectively. 3. ATP, UTP and ATP gamma S were also able to stimulate [3H]-inositol phosphate accumulation in these cells with EC50 values of 1.9 microM, 1.3 microM and 5.0 microM respectively. 2-Methyl-thio-ATP was a weak agonist of this response (EC50 > 100 microM). 4. The [3H]-inositol phosphate response to CPA was completely attenuated by pertussis toxin treatment (24 h; 100 ng ml-1). In contrast, the responses to ATP, UTP and ATP gamma S were only reduced by circa 30% in pertussis toxin-treated cells. 5. The simultaneous addition of CPA and either ATP, UTP or ATP gamma S produced a large augmentation of [3H]-inositol phospholipid hydrolysis. This was due to an increase in the maximal response and was significantly greater than the predicted additive response for activation of these two receptor systems. The synergy was not observed in pertussis toxin-treated cells. 6. No synergy was observed between the [3H]-inositol phosphate responses to histamine and ATP in CHO-K1 cells transfected with the bovine histamine H1-receptor. In these cells the response to histamine was completely resistant to inhibition by pertussis toxin treatment. 7. This study provides a clear demonstration of a synergy between pertussis toxin-sensitive and insensitive receptor systems in a model cell system which is an ideal host for transfected cDNA sequences. This model system should provide a unique opportunity to unravel the mechanisms underlying this example of receptor cross-talk involving phospholipase C.

Keratan sulfate (KS) chains prepared from both bovine tracheal rings and bovine femoral head cartilage were digested with the enzyme keratanase from Pseudomonas species; large repeat-sequence and non-reducing terminal oligosaccharides were fractionated and purified using high-performance ion-exchange chromatography. The main beta-linked pentasulfated hexasaccharide repeat segment, [R6], GlcNAc(6S)1-1-3Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S)1-3Gal-ol and the asialo beta-linked capping pentasulfated heptasaccharide, [C7], Gal1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) 1-3Gal-ol have been completely characterized by high-field NMR spectroscopy using one- and two-dimensional methods. Partial 1H assignments are summarized for three homologous series of higher oligosaccharides: GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]2-5(1-3)Gal-0l [R8,R10,R12] Gal1-4GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]3-5(1-3)Gal-ol [C9,C11,C13] NeuAc alpha 2-3Gal1-4GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]2-4(1-3)Gal-ol [C8,C10,C12] obtained from keratan sulfate by keratanase cleavage. The first shows that the unsulfated galactose residues within the repeat sequence region of KS may be separated by fully sulfated segments which have a wide distribution of lengths. The others, viz. those with sialylated caps, and the related galactose capped asialo-segments (derived from a KS digestion in which the keratanase also exhibited sialidase activity) represent an homologous series of epitopes in which the first internal unsulfated galactose is located at a position which may be up to five or more fully sulfated N-acetyllactosamine disaccharide repeat units along from the non-reducing terminus of the KS polymer.

Skeletal keratan sulfates have been degraded by three independent techniques and the resultant, borohydride-reduced, disaccharides have been characterised by NMR spectroscopy. The 1H and 13C (where available) chemical shifts are reported for the following substances, where GalNAc-ol represents N-acetyl-galactosaminitol, GlcNAc-ol represents N-acetyl-glucosaminitol, GlcNAc(6S)-ol represents N-acetyl-glucosaminitol 6-O-sulfate and 2,5AnMan(6S)-ol represents 2,5-anhydro-D-mannitol 6-O-sulfate. (a) GlcNAc(6S)beta(1-3)Gal-ol, isolated after keratanase (from Pseudomonas sp.) digestion. (b) Gal beta(1-4)GlcNAc(6S)-ol and Gal(6S)beta(1-4) GlcNAc(6S)-ol, the 1H chemical shifts have been reported previously [Brown, G. M., Huckerby, T. N., Morris, H. G., Abram, B. L. & Nieduszynski, I. A. (1994) Biochemistry 33, 4836-4846; Brown, G. M., Huckerby, T. N. & Nieduszynski, I. A. (1994) Eur. J. Biochem. 224, 281-308], GlcNAc(6S)beta(1-6)GalNAc-ol, [formula: see text], [formula: see text], all isolated after keratanase II digestion. (c) Gal beta(1-4)2,5AnMan(6S)-ol and Gal(6S)beta(1-4)2,5AnMan(6S)-ol, isolated after hydrazinolysis and nitrous acid digestion. In addition, the model compounds Gal beta(1-4)GlcNAc-ol and Gal beta(1-6)GlcNAc-ol have also been examined by 1H and 13C NMR spectroscopy. The value of these data for microstructural analysis of keratan sulfate samples is discussed.

Adenosine A1 receptors directly stimulate inositol phospholipid hydrolysis and Ca2+ mobilization through a pertussis toxin sensitive mechanism in DDT1MF-2 cells. In the present study we have investigated whether G protein beta gamma subunits (G beta gamma) are capable of stimulating phospholipase C in DDT1MF-2 cell membrane preparations using lipid vesicles containing [3H]phosphatidylinositol 4,5-bisphosphate. DDT1MF-2 cell membrane and soluble fractions were found to contain phospholipase C activity which was stimulated by increases in free Ca2+ ion concentration. G beta gamma purified from bovine retinal transducin produced significant increases in phospholipase C activity in DDT1MF-2 cell membranes. G beta gamma-dependent activation of phospholipase C, while virtually absent in the presence of low Ca2+ ion concentrations, increased markedly with increasing free Ca2+ ion concentration. These data suggest that membrane bound phospholipase C in DDT1MF-2 cells is sensitive to Ca2+, and may be stimulated conditionally by G beta gamma subunits, i.e. G beta gamma subunits activate the enzyme only in the presence of Ca2+. G beta gamma subunits also stimulated soluble phospholipase C in DDT1MF-2 cells. These findings support the hypothesis that Gi beta gamma subunits are involved in adenosine A1 receptor stimulated phospholipase C/Ca2+ signaling in DDT1MF-2 cells.