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Mahmoud M. El-mas - One of the best experts on this subject based on the ideXlab platform.
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Oestrogen compromises the facilitatory effect of chronic nicotine on Adenosine A2B Receptor-K(+) channel-mediated renal vasodilation.
Clinical and experimental pharmacology & physiology, 2014Co-Authors: Mahmoud M. El-mas, Lamia K. Elsalakawy, Sahar M. El-gowilly, Hanan M. El-gowelliAbstract:We have shown previously that the renal vasodilatory action of the Adenosine analogue 5'-N-ethylcarboxamidoAdenosine (NECA) in female rats is mediated via preferential activation of Adenosine A2B Receptor (A2B R)-K(+) channel signalling. In the present study, we tested the hypothesis that the renal vasodilatory effect of NECA and its A2B R/K(+) channel specificities are altered by chronic nicotine administration. The oestrogenic modulation of the nicotine-NECA renovascular interaction was also evaluated by determining the effect of ovariectomy (OVX) and oestrogen replacement (OVXE2) on the evoked responses. In isolated phenylephrine-preconstricted perfused kidneys obtained from sham-operated rats, vasodilation in response to cumulative bolus injections of NECA (1.6-50 nmol) or papaverine (1-243 nmol) were not affected by nicotine (1-8 mg/kg per day, i.p., 2 weeks). However, vasodilator responses to NECA, but not papaverine, were reduced in kidneys of OVX rats and restored to near-sham values after E2 replacement. Further, nicotine increased NECA-induced vasodilation in perfused kidneys from OVX rats, but failed to do so in OVXE2 preparations. The enhanced NECA responsiveness in nicotine-treated OVX preparations was abolished after infusion (into isolated kidneys) of 10 μmol/L alloxazine (A2B R antagonist) or BaCl2 plus glibenclamide (blockers of inward rectifier and ATP-sensitive K(+) channels, respectively). Vasodilator responses to 0.05-1.6 μmol minoxidil (a K(+) channel opener) were increased by nicotine in OVX, but not OVXE2, preparations and this increase was abolished after infusion of BaCl2 + glibenclamide. Together, the data suggest that chronic nicotine enhances A2B R/K(+) channel-mediated renal vasodilation in oestrogen-depleted rats.
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Nitric oxide synthase/K+ channel cascade triggers the Adenosine A2B Receptor-sensitive renal vasodilation in female rats
European Journal of Pharmacology, 2013Co-Authors: Hanan M. El-gowelli, Sahar M. El-gowilly, Lamia K. Elsalakawy, Mahmoud M. El-masAbstract:Abstract Adenosine A2B-Receptors mediate the Adenosine-evoked renal vasodilations in male rats. Here, we tested whether this finding could be replicated in female renal vasculature and whether K+ hyperpolarization induced by nitric oxide synthase (NOS) and/or heme oxygenase (HO) accounts for Adenosine A2B Receptor-sensitive renal vasodilations. In phenylephrine-preconstricted perfused kidneys, vasodilations caused by the Adenosine analog 5′-N-ethylcarboxamidoAdenosine (NECA, 1.6–50 nmol) were attenuated after blockade of Adenosine A2B (alloxazine) but not A2A [8-(3-Chlorostyryl) caffeine, CSC] or A3 Receptors (N-(2-methoxyphenyl)-N′-[2-(3-pyridinyl)-4-quinazolinyl]-urea, VUF 5574), confirming the preferential involvement of A2B Receptors in NECA responses. NOS activation mediated the A2B Receptor-mediated NECA response because: (i) NOS inhibition (Nω-nitro- l -arginine-methyl ester, l -NAME) attenuated NECA vasodilations, (ii) concurrent l -NAME/alloxazine exposure caused more inhibition of NECA responses, and (iii) inhibition of NECA responses by alloxazine disappeared in l -arginine-supplemented preparations. Although HO inhibition (zinc protoporphyrin) failed to modify NECA responses, the attenuation of these responses by alloxazine disappeared in hemin (HO inducer)-treated preparations. NECA vasodilations were also attenuated after exposure to BaCl2, glibenclamide but not tetraethylammonium (blockers of inward rectifier, ATP-sensitive, and Ca2+-dependent K+-channels, respectively). The combined alloxazine/BaCl2/glibenclamide infusion caused no additional attenuation of NECA vasodilations. Vasodilations caused by minoxidil (K+-channel opener) were reduced by l -NAME or BaCl2/glibenclamide, supporting the importance of NOS signaling in K+ hyperpolarization. NECA or minoxidil vasodilations were attenuated by ouabain, Na+/K+-ATPase inhibitor, and in KCl-preconstricted preparations. Overall, facilitation of Adenosine A2B Receptor/NOS/K+ channel/Na+/K+-ATPase cascade underlies NECA vasodilations in female rats. Enhancing HO activity, albeit not causally related to NECA vasodilations, improves the pharmacologically compromised (alloxazine) NECA response.
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Nitric oxide synthase/K+ channel cascade triggers the Adenosine A(2B) Receptor-sensitive renal vasodilation in female rats.
European journal of pharmacology, 2013Co-Authors: Hanan M. El-gowelli, Sahar M. El-gowilly, Lamia K. Elsalakawy, Mahmoud M. El-masAbstract:Adenosine A2B-Receptors mediate the Adenosine-evoked renal vasodilations in male rats. Here, we tested whether this finding could be replicated in female renal vasculature and whether K(+) hyperpolarization induced by nitric oxide synthase (NOS) and/or heme oxygenase (HO) accounts for Adenosine A2B Receptor-sensitive renal vasodilations. In phenylephrine-preconstricted perfused kidneys, vasodilations caused by the Adenosine analog 5'-N-ethylcarboxamidoAdenosine (NECA, 1.6-50 nmol) were attenuated after blockade of Adenosine A2B (alloxazine) but not A2A [8-(3-Chlorostyryl) caffeine, CSC] or A3 Receptors (N-(2-methoxyphenyl)-N'-[2-(3-pyridinyl)-4-quinazolinyl]-urea, VUF 5574), confirming the preferential involvement of A2B Receptors in NECA responses. NOS activation mediated the A2B Receptor-mediated NECA response because: (i) NOS inhibition (N(ω)-nitro-L-arginine-methyl ester, L-NAME) attenuated NECA vasodilations, (ii) concurrent L-NAME/alloxazine exposure caused more inhibition of NECA responses, and (iii) inhibition of NECA responses by alloxazine disappeared in L-arginine-supplemented preparations. Although HO inhibition (zinc protoporphyrin) failed to modify NECA responses, the attenuation of these responses by alloxazine disappeared in hemin (HO inducer)-treated preparations. NECA vasodilations were also attenuated after exposure to BaCl2, glibenclamide but not tetraethylammonium (blockers of inward rectifier, ATP-sensitive, and Ca(2+)-dependent K(+)-channels, respectively). The combined alloxazine/BaCl2/glibenclamide infusion caused no additional attenuation of NECA vasodilations. Vasodilations caused by minoxidil (K(+)-channel opener) were reduced by L-NAME or BaCl2/glibenclamide, supporting the importance of NOS signaling in K(+) hyperpolarization. NECA or minoxidil vasodilations were attenuated by ouabain, Na(+)/K(+)-ATPase inhibitor, and in KCl-preconstricted preparations. Overall, facilitation of Adenosine A2B Receptor/NOS/K(+) channel/Na(+)/K(+)-ATPase cascade underlies NECA vasodilations in female rats. Enhancing HO activity, albeit not causally related to NECA vasodilations, improves the pharmacologically compromised (alloxazine) NECA response.
Hanan M. El-gowelli - One of the best experts on this subject based on the ideXlab platform.
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Oestrogen compromises the facilitatory effect of chronic nicotine on Adenosine A2B Receptor-K(+) channel-mediated renal vasodilation.
Clinical and experimental pharmacology & physiology, 2014Co-Authors: Mahmoud M. El-mas, Lamia K. Elsalakawy, Sahar M. El-gowilly, Hanan M. El-gowelliAbstract:We have shown previously that the renal vasodilatory action of the Adenosine analogue 5'-N-ethylcarboxamidoAdenosine (NECA) in female rats is mediated via preferential activation of Adenosine A2B Receptor (A2B R)-K(+) channel signalling. In the present study, we tested the hypothesis that the renal vasodilatory effect of NECA and its A2B R/K(+) channel specificities are altered by chronic nicotine administration. The oestrogenic modulation of the nicotine-NECA renovascular interaction was also evaluated by determining the effect of ovariectomy (OVX) and oestrogen replacement (OVXE2) on the evoked responses. In isolated phenylephrine-preconstricted perfused kidneys obtained from sham-operated rats, vasodilation in response to cumulative bolus injections of NECA (1.6-50 nmol) or papaverine (1-243 nmol) were not affected by nicotine (1-8 mg/kg per day, i.p., 2 weeks). However, vasodilator responses to NECA, but not papaverine, were reduced in kidneys of OVX rats and restored to near-sham values after E2 replacement. Further, nicotine increased NECA-induced vasodilation in perfused kidneys from OVX rats, but failed to do so in OVXE2 preparations. The enhanced NECA responsiveness in nicotine-treated OVX preparations was abolished after infusion (into isolated kidneys) of 10 μmol/L alloxazine (A2B R antagonist) or BaCl2 plus glibenclamide (blockers of inward rectifier and ATP-sensitive K(+) channels, respectively). Vasodilator responses to 0.05-1.6 μmol minoxidil (a K(+) channel opener) were increased by nicotine in OVX, but not OVXE2, preparations and this increase was abolished after infusion of BaCl2 + glibenclamide. Together, the data suggest that chronic nicotine enhances A2B R/K(+) channel-mediated renal vasodilation in oestrogen-depleted rats.
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Nitric oxide synthase/K+ channel cascade triggers the Adenosine A2B Receptor-sensitive renal vasodilation in female rats
European Journal of Pharmacology, 2013Co-Authors: Hanan M. El-gowelli, Sahar M. El-gowilly, Lamia K. Elsalakawy, Mahmoud M. El-masAbstract:Abstract Adenosine A2B-Receptors mediate the Adenosine-evoked renal vasodilations in male rats. Here, we tested whether this finding could be replicated in female renal vasculature and whether K+ hyperpolarization induced by nitric oxide synthase (NOS) and/or heme oxygenase (HO) accounts for Adenosine A2B Receptor-sensitive renal vasodilations. In phenylephrine-preconstricted perfused kidneys, vasodilations caused by the Adenosine analog 5′-N-ethylcarboxamidoAdenosine (NECA, 1.6–50 nmol) were attenuated after blockade of Adenosine A2B (alloxazine) but not A2A [8-(3-Chlorostyryl) caffeine, CSC] or A3 Receptors (N-(2-methoxyphenyl)-N′-[2-(3-pyridinyl)-4-quinazolinyl]-urea, VUF 5574), confirming the preferential involvement of A2B Receptors in NECA responses. NOS activation mediated the A2B Receptor-mediated NECA response because: (i) NOS inhibition (Nω-nitro- l -arginine-methyl ester, l -NAME) attenuated NECA vasodilations, (ii) concurrent l -NAME/alloxazine exposure caused more inhibition of NECA responses, and (iii) inhibition of NECA responses by alloxazine disappeared in l -arginine-supplemented preparations. Although HO inhibition (zinc protoporphyrin) failed to modify NECA responses, the attenuation of these responses by alloxazine disappeared in hemin (HO inducer)-treated preparations. NECA vasodilations were also attenuated after exposure to BaCl2, glibenclamide but not tetraethylammonium (blockers of inward rectifier, ATP-sensitive, and Ca2+-dependent K+-channels, respectively). The combined alloxazine/BaCl2/glibenclamide infusion caused no additional attenuation of NECA vasodilations. Vasodilations caused by minoxidil (K+-channel opener) were reduced by l -NAME or BaCl2/glibenclamide, supporting the importance of NOS signaling in K+ hyperpolarization. NECA or minoxidil vasodilations were attenuated by ouabain, Na+/K+-ATPase inhibitor, and in KCl-preconstricted preparations. Overall, facilitation of Adenosine A2B Receptor/NOS/K+ channel/Na+/K+-ATPase cascade underlies NECA vasodilations in female rats. Enhancing HO activity, albeit not causally related to NECA vasodilations, improves the pharmacologically compromised (alloxazine) NECA response.
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Nitric oxide synthase/K+ channel cascade triggers the Adenosine A(2B) Receptor-sensitive renal vasodilation in female rats.
European journal of pharmacology, 2013Co-Authors: Hanan M. El-gowelli, Sahar M. El-gowilly, Lamia K. Elsalakawy, Mahmoud M. El-masAbstract:Adenosine A2B-Receptors mediate the Adenosine-evoked renal vasodilations in male rats. Here, we tested whether this finding could be replicated in female renal vasculature and whether K(+) hyperpolarization induced by nitric oxide synthase (NOS) and/or heme oxygenase (HO) accounts for Adenosine A2B Receptor-sensitive renal vasodilations. In phenylephrine-preconstricted perfused kidneys, vasodilations caused by the Adenosine analog 5'-N-ethylcarboxamidoAdenosine (NECA, 1.6-50 nmol) were attenuated after blockade of Adenosine A2B (alloxazine) but not A2A [8-(3-Chlorostyryl) caffeine, CSC] or A3 Receptors (N-(2-methoxyphenyl)-N'-[2-(3-pyridinyl)-4-quinazolinyl]-urea, VUF 5574), confirming the preferential involvement of A2B Receptors in NECA responses. NOS activation mediated the A2B Receptor-mediated NECA response because: (i) NOS inhibition (N(ω)-nitro-L-arginine-methyl ester, L-NAME) attenuated NECA vasodilations, (ii) concurrent L-NAME/alloxazine exposure caused more inhibition of NECA responses, and (iii) inhibition of NECA responses by alloxazine disappeared in L-arginine-supplemented preparations. Although HO inhibition (zinc protoporphyrin) failed to modify NECA responses, the attenuation of these responses by alloxazine disappeared in hemin (HO inducer)-treated preparations. NECA vasodilations were also attenuated after exposure to BaCl2, glibenclamide but not tetraethylammonium (blockers of inward rectifier, ATP-sensitive, and Ca(2+)-dependent K(+)-channels, respectively). The combined alloxazine/BaCl2/glibenclamide infusion caused no additional attenuation of NECA vasodilations. Vasodilations caused by minoxidil (K(+)-channel opener) were reduced by L-NAME or BaCl2/glibenclamide, supporting the importance of NOS signaling in K(+) hyperpolarization. NECA or minoxidil vasodilations were attenuated by ouabain, Na(+)/K(+)-ATPase inhibitor, and in KCl-preconstricted preparations. Overall, facilitation of Adenosine A2B Receptor/NOS/K(+) channel/Na(+)/K(+)-ATPase cascade underlies NECA vasodilations in female rats. Enhancing HO activity, albeit not causally related to NECA vasodilations, improves the pharmacologically compromised (alloxazine) NECA response.
Christa E. Müller - One of the best experts on this subject based on the ideXlab platform.
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PSB 603 - a known selective Adenosine A2B Receptor antagonist - has anti-inflammatory activity in mice.
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 2020Co-Authors: Magdalena Kotańska, Małgorzata Szafarz, Kamil Mika, Anna Dziubina, Marek Bednarski, Christa E. Müller, Jacek Sapa, Katarzyna Kieć-kononowiczAbstract:Abstract A2B Adenosine Receptors are present in a wide spectrum of tissues, especially on cells of the immune system. Since these particular Receptors have the lowest, of all Adenosine Receptor subtypes, affinity for Adenosine they are believed to play a special role in immunological processes associated with elevated Adenosine levels such as inflammation. The aim of this preliminary study was to determine the potential anti-inflammatory properties of compound PSB-603, a potent and selective Adenosine A2B Receptor antagonist, in two different experimental models of local and systemic inflammation. In a model of inflammation induced by local carrageenan administration paw edema was measured using a pletysmometer. Additionally, levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and reactive oxygen species (ROS) were determined in the inflamed paw. Using the mouse model of peripheral inflammation induced by intraperitoneal (ip) administration of zymosan A, the influence of the A2B antagonist on the infiltration of neutrophils into the peritoneum and its effect on the plasma levels of CRP, TNF-α, and IL-6 were investigated. The results showed that PSB-603 administered at a dose of 5 mg/kg b.w. ip significantly reduced inflammation in both tested models. Particularly, it significantly decreased levels of the inflammatory cytokines IL-6, TNF-α and of ROS in the inflamed paw and reduced inflammation of the peritoneum by significantly decreasing the infiltration of leukocytes. Additionally, in the latter model, no statistically significant difference was observed in the CRP level between the control group without inflammation and the group which has been treated with the PSB-603 compound. Thus, the results may indicate the anti-inflammatory activity of Adenosine A2B Receptor antagonists in two different models of inflammation.
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fluorescent labeled selective Adenosine A2B Receptor antagonist enables competition binding assay by flow cytometry
Journal of Medicinal Chemistry, 2018Co-Authors: Meryem Kose, Sabrina Gollos, Tadeusz Karcz, Amelie Fiene, Fabian Heisig, Andrea Behrenswerth, Vigneshwaran Namasivayam, Katarzyna Kieckononowicz, Christa E. MüllerAbstract:Fluorescent ligands represent powerful tools for biological studies and are considered attractive alternatives to radioligands. In this study, we developed fluorescent antagonists for A2B Adenosine Receptors (A2BARs), which are targeted by antiasthmatic xanthines and were proposed as novel targets in immuno-oncology. Our approach was to merge a small borondipyrromethene (BODIPY) derivative with the pharmacophore of 8-substituted xanthine derivatives. On the basis of the design, synthesis, and evaluation of model compounds, several fluorescent ligands were synthesized. Compound 29 (PSB-12105), which displayed high affinity for human, rat, and mouse A2BARs (Ki = 0.2–2 nM) and high selectivity for this AR subtype, was selected for further studies. A homology model of the human A2BAR was generated, and docking studies were performed. Moreover, 29 allowed us to establish a homogeneous Receptor–ligand binding assay using flow cytometry. These compounds constitute the first potent, selective fluorescent A2BAR li...
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Domains for activation and inactivation in G protein-coupled Receptors--a mutational analysis of constitutive activity of the Adenosine A2B Receptor.
Biochemical pharmacology, 2014Co-Authors: Miriam C. Peeters, Christa E. Müller, Rachel J. J. Elands, Gerard J. P. Van Westen, Eelke B. Lenselink, Adriaan P. IjzermanAbstract:G protein-coupled Receptors (GPCRs) are a major drug target and can be activated by a range of stimuli, from photons to proteins. Most, if not all, GPCRs also display a basal level of biological response in the absence of such a stimulus. This level of so-called constitutive activity results from a delicate energy equilibrium that exists between the active and the inactive state of the Receptor and is the first determinant in the GPCR activation mechanism. Here we describe new insights in specific regions of the Adenosine A2B Receptor that are essential in activation and inactivation. We developed a new screening method using the MMY24 S. Cerevisiae strain by which we were able to screen for constitutively inactive mutants Receptors (CIMs). We applied this screening method on a mutagenic library of the Adenosine A2B Receptor, where random mutations were introduced in transmembrane domains four and five (TM4 and TM5) linked by extracellular loop 2 (EL2). The screen resulted in the identification of 22 single and double mutant Receptors, all showing a decrease in constitutive activity as well as in agonist potency. By comparing these results with a previous screen of the same mutagenic library for constitutively active mutant Receptors (CAMs), we discovered specific regions in this G protein-coupled Receptor involved in either inactivation or activation or both. The results suggest the activation mechanism of GPCRs to be much less restricted to sites of high conservation or direct interaction with the ligand or G protein and illustrate how dynamic the activation process of GPCRs is.
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The four cysteine residues in the second extracellular loop of the human Adenosine A2B Receptor: Role in ligand binding and Receptor function
Biochemical pharmacology, 2011Co-Authors: Anke C. Schiedel, Sonja Hinz, Farag F. Sherbiny, Astrid Maaß, Dominik Thimm, Thomas Borrmann, Christa E. MüllerAbstract:The Adenosine A2B Receptor is of considerable interest as a new drug target for the treatment of asthma, inflammatory diseases, pain, and cancer. In the present study we investigated the role of the cysteine residues in the extracellular loop 2 (ECL2) of the Receptor, which is particularly cysteine-rich, by a combination of mutagenesis, molecular modeling, chemical and pharmacological experiments. Pretreatment of CHO cells recombinantly expressing the human A2B Receptor with dithiothreitol led to a 74-fold increase in the EC50 value of the agonist NECA in cyclic AMP accumulation. In the C783.25S and the C17145.50S mutant high-affinity binding of the A2B antagonist radioligand [3H]PSB-603 was abolished and agonists were virtually inactive in cAMP assays. This indicates that the C3.25–C45.50 disulfide bond, which is highly conserved in GPCRs, is also important for binding and function of A2B Receptors. In contrast, the C16645.45S and the C16745.46S mutant as well as the C16645.45S–C16745.46S double mutant behaved like the wild-type Receptor, while in the C15445.33S mutant significant, although more subtle effects on cAMP accumulation were observed – decrease (BAY60-6583) or increase (NECA) – depending on the structure of the investigated agonist. In contrast to the X-ray structure of the closely related A2A Receptor, which showed four disulfide bonds, the present data indicate that in the A2B Receptor only the C3.25–C45.50 disulfide bond is essential for ligand binding and Receptor activation. Thus, the cysteine residues in the ECL2 of the A2B Receptor not involved in stabilization of the Receptor structure may have other functions.
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gpcr structure and activation an essential role for the first extracellular loop in activating the Adenosine A2B Receptor
The FASEB Journal, 2011Co-Authors: Miriam C. Peeters, Margot W. Beukers, Christa E. Müller, Gerard J. P. Van Westen, Dong Guo, Lisanne E Wisse, Adriaan P. IjzermanAbstract:The highly variable extracellular loops in G protein-coupled Receptors (GPCRs) have been implicated in Receptor activation, the mechanism of which is poorly understood. In a random mutagenesis screen on the human Adenosine A(2B) Receptor (A(2B)R) using the MMY24 Saccharomyces cerevisiae strain as a read-out system, we found that two residues in the first extracellular loop, a phenylalanine and an aspartic acid at positions 71 and 74, respectively, are involved in Receptor activation. We subsequently performed further site-directed and site-saturation mutagenesis. These experiments revealed that the introduction of mutations at either of the identified positions results in a wide variety of Receptor activation profiles, with changes in agonist potency, constitutive activity, and intrinsic activity. Radioligand binding studies showed that the changes in activation were not due to changes in Receptor expression. We interpret these data in the light of the recently revealed structure of the Adenosine A(2A)R, the closest homologue of the A(2B)R. The two residues are suggested to be vital in maintaining the tertiary structure of a β sheet in the extracellular domain of the A(2B)R. We hypothesize that deterioration of structure in the extracellular domains of GPCRs compromises overall Receptor structure with profound consequences for Receptor activation and constitutive activity.
Adriaan P. Ijzerman - One of the best experts on this subject based on the ideXlab platform.
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Domains for activation and inactivation in G protein-coupled Receptors--a mutational analysis of constitutive activity of the Adenosine A2B Receptor.
Biochemical pharmacology, 2014Co-Authors: Miriam C. Peeters, Christa E. Müller, Rachel J. J. Elands, Gerard J. P. Van Westen, Eelke B. Lenselink, Adriaan P. IjzermanAbstract:G protein-coupled Receptors (GPCRs) are a major drug target and can be activated by a range of stimuli, from photons to proteins. Most, if not all, GPCRs also display a basal level of biological response in the absence of such a stimulus. This level of so-called constitutive activity results from a delicate energy equilibrium that exists between the active and the inactive state of the Receptor and is the first determinant in the GPCR activation mechanism. Here we describe new insights in specific regions of the Adenosine A2B Receptor that are essential in activation and inactivation. We developed a new screening method using the MMY24 S. Cerevisiae strain by which we were able to screen for constitutively inactive mutants Receptors (CIMs). We applied this screening method on a mutagenic library of the Adenosine A2B Receptor, where random mutations were introduced in transmembrane domains four and five (TM4 and TM5) linked by extracellular loop 2 (EL2). The screen resulted in the identification of 22 single and double mutant Receptors, all showing a decrease in constitutive activity as well as in agonist potency. By comparing these results with a previous screen of the same mutagenic library for constitutively active mutant Receptors (CAMs), we discovered specific regions in this G protein-coupled Receptor involved in either inactivation or activation or both. The results suggest the activation mechanism of GPCRs to be much less restricted to sites of high conservation or direct interaction with the ligand or G protein and illustrate how dynamic the activation process of GPCRs is.
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gpcr structure and activation an essential role for the first extracellular loop in activating the Adenosine A2B Receptor
The FASEB Journal, 2011Co-Authors: Miriam C. Peeters, Margot W. Beukers, Christa E. Müller, Gerard J. P. Van Westen, Dong Guo, Lisanne E Wisse, Adriaan P. IjzermanAbstract:The highly variable extracellular loops in G protein-coupled Receptors (GPCRs) have been implicated in Receptor activation, the mechanism of which is poorly understood. In a random mutagenesis screen on the human Adenosine A(2B) Receptor (A(2B)R) using the MMY24 Saccharomyces cerevisiae strain as a read-out system, we found that two residues in the first extracellular loop, a phenylalanine and an aspartic acid at positions 71 and 74, respectively, are involved in Receptor activation. We subsequently performed further site-directed and site-saturation mutagenesis. These experiments revealed that the introduction of mutations at either of the identified positions results in a wide variety of Receptor activation profiles, with changes in agonist potency, constitutive activity, and intrinsic activity. Radioligand binding studies showed that the changes in activation were not due to changes in Receptor expression. We interpret these data in the light of the recently revealed structure of the Adenosine A(2A)R, the closest homologue of the A(2B)R. The two residues are suggested to be vital in maintaining the tertiary structure of a β sheet in the extracellular domain of the A(2B)R. We hypothesize that deterioration of structure in the extracellular domains of GPCRs compromises overall Receptor structure with profound consequences for Receptor activation and constitutive activity.
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Structure-affinity relationships of Adenosine A2B Receptor ligands.
Medicinal research reviews, 2006Co-Authors: Margot W. Beukers, Illiana Meurs, Adriaan P. IjzermanAbstract:Many selective and high affinity agonists and antagonists have been developed for the Adenosine A1, A2A, and A3 Receptors. Very recently such compounds have been identified for the Adenosine A2B Receptors. This review presents an overview of the structure-affinity relationships of antagonists and agonists for this Receptor subtype as published in the scientific and patent literature. To date the most selective >370-fold, high affinity Adenosine A2B Receptor antagonist is the xanthine analog, compound 16 (8-(1-(3-phenyl-1,2,4-oxadiazol-5-yl)methyl)-1H-pyrazol-4-yl)-1,3-dipropyl-1H-purine-2,6(3H,7H)-dione). The pyrrolopyrimidine analog OSIP339391 (73) is slightly less selective, 70-fold, but has a higher affinity 0.41 nM compared to 1 nM for compound 16. Other promising classes of compounds with selectivities ranging from 10- to 160-fold and affinities ranging from 3 to 112 nM include triazolo, aminothiazole, quinazoline, and pyrimidin-2-amine analogs. Progress has also been achieved concerning the development of selective high affinity agonists for the Adenosine A2B Receptor. For years the most potent, albeit non-selective Adenosine A2B Receptor agonist was (S)PHPNECA (88). Last year, a new class of non-ribose ligands was reported. Several compounds displayed selectivity with respect to Adenosine A2A and A3 Receptors. In addition, full and partial agonists for the Adenosine A2B Receptor were identified with EC50 values of 10 nM (LUF5835, 103) and 9 nM (LUF5845, 105), respectively. © 2006 Wiley Periodicals, Inc. Med Res Rev, 26, No. 5, 667–698, 2006
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Random mutagenesis of the human Adenosine A2B Receptor followed by growth selection in yeast. Identification of constitutively active and gain of function mutations
Molecular pharmacology, 2004Co-Authors: Margot W. Beukers, Clara C. Blad, Hans Den Dulk, Jaap Brouwer, Joris Van Oppenraaij, Patrick P. W. Van Der Hoorn, Adriaan P. IjzermanAbstract:To gain insight in spontaneous as well as agonist-induced activation of the human Adenosine A2B Receptor, we applied a random mutagenesis approach in yeast to create a large number of Receptor mutants and selected mutants of interest with a robust screening assay based on growth. The amino acid sequence of 14 mutated Receptors was determined. All these mutated Receptors displayed constitutive activity. In particular, single-point mutations at T42A, V54L, and F84S and a triple-point mutation at N36S, T42A, and T66A resulted in high constitutive activity. In addition, a C-terminally truncated (after Lys269) mutant, Q214L I230N V240M V250M N254Y T257S K269stop, was highly constitutively active. The T42A, V54L, and F84S mutants showed a considerable decrease, 4.9- to 6.9-fold, in the EC50 value of 5′- N -ethylcarboxamidoAdenosine (NECA), an Adenosine analog. Combined mutation of I242T, K269R, V284A, and H302Q, as well as F84L together with S95G, resulted in an even greater potency of NECA of 10- and 18-fold, respectively. In fact, all constitutively active mutants had an increased potency for NECA. This suggests that the wild-type (wt) human A2B Receptor itself is rather silent, which may explain the low affinity of agonists for this Receptor. To verify the ability of the mutant Receptors to activate mammalian second messenger systems, cAMP experiments were performed in CHO cells stably expressing the wt and T42A Receptors. These experiments confirmed the increased sensitivity of T42A for NECA, because the EC50 values of T42A and the wt Receptor were 0.15 ± 0.04 and 1.3 ± 0.4 μM, respectively.
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A Functional Screening of Adenosine Analogues at the Adenosine A2B Receptor: A Search for Potent Agonists
Nucleosides & nucleotides, 1998Co-Authors: De Zwart M, R. Link, Von Frijtag Drabbe Künzel Jk, Gloria Cristalli, Kenneth A. Jacobson, Andrea Townsend-nicholson, Adriaan P. IjzermanAbstract:Various Adenosine analogues were tested at the Adenosine A2B Receptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A2B Receptors. 5'-N-Substituted carboxamidoAdenosines were most potent. 5'-N-EthylcarboxamidoAdenosine (NECA) was most active with an EC50 value of 3.1 microM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N6) of the purine ring system. The most active N6-substituted derivative N6-methyl-NECA was 5 fold less potent than NECA. C8- and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3- and 7-deazaanalogues were not active.
Yang Xia - One of the best experts on this subject based on the ideXlab platform.
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Adenosine A2B Receptor a pathogenic factor and a therapeutic target for sensorineural hearing loss
The FASEB Journal, 2020Co-Authors: Jeanne M Manalo, Hong Liu, Hong Sun, Rodney E. Kellems, Dalian Ding, John Hicks, Richard Salvi, Fred A Pereira, Yang XiaAbstract:Over 466 million people worldwide are diagnosed with hearing loss (HL). About 90% of HL cases are sensorineural HL (SNHL) with treatments limited to hearing aids and cochlear implants with no FDA-approved drugs. Intriguingly, ADA-deficient patients have been reported to have bilateral SNHL, however, its underlying cellular and molecular basis remain unknown. We report that Ada-/- mice, phenocopying ADA-deficient humans, displayed SNHL. Ada-/- mice cochlea with elevated Adenosine caused substantial nerve fiber demyelination and mild hair cell loss. ADA enzyme therapy in these mice normalized cochlear Adenosine levels, attenuated SNHL, and prevented demyelination. Additionally, ADA enzyme therapy rescued SNHL by restoring nerve fiber structure in Ada-/- mice post two-week drug withdrawal. Moreover, elevated cochlear Adenosine in untreated mice was associated with enhanced AdorA2B gene expression. Preclinically, ADORA2B-specific antagonist treatment in Ada-/- mice significantly improved HL, nerve fiber density, and myelin compaction. We also provided genetic evidence that ADORA2B is detrimental for age-related SNHL by impairing cochlear myelination in WT aged mice. Overall, understanding purinergic molecular signaling in SNHL in Ada-/- mice allows us to further discover that ADORA2B is also a pathogenic factor underlying aged-related SNHL by impairing cochlear myelination and lowering cochlear Adenosine levels or blocking ADORA2B signaling are effective therapies for SNHL.
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abstract 17080 Adenosine A2B Receptor mediated erythrocyte oxygen release counteracts renal hypoxia and its progression
Circulation, 2017Co-Authors: Zhangzhe Peng, Weiru Zhang, Rodney E. Kellems, Renna Luo, Lijian Tao, Yang XiaAbstract:Introduction: Insufficient tissue oxygen (O2) availability (hypoxia) is commonly seen in patients with cardiovascular diseases (CVD) ,chronic kidney diseases (CKD), respiratory diseases and certain cancers. Although erythrocyte is the only cell type responsible for delivering and sensoring O2, its function in pathological tissue hypoxia remains largely unknown. Hypothesis: Erythrocyte A2B Adenosine Receptor (ADORA2B) plays a key protective role in Ang II-induced tissue damage by increasing 2,3-biphosphoglycerate(2, 3-BPG) and oxygen release to counteract tissue hypoxia. Methods: We infused angiotensin II (Ang II, 140ng/kg/min) to mice with specific deletion of ADORA2B in erythrocytes (ADORA2B f/f /EpoR-Cre + ) for 14 days and measured the levels of 2,3-BPG,P50,p-AMPK in erythrocytes of mild and severe CKD patients. Results: We found that 2,3-BPG, an erythrocyte-specific metabolite enhancing O2 delivery, was significantly induced in the erythrocytes of mice infused with Ang II. Mouse genetic studies demonstrated that Ang II induced local tissue accumulation of Adenosine and that elevated Adenosine-mediated erythrocyte ADORA2B activation was beneficial by inducing 2,3-BPG production, triggering O2 release to counteract tissue hypoxia, heart and kidney damage including proteinuria along with the mRNA levels of collagen I, fibronectin, prepro-ET-1 and endothelin Receptor type A, which are known tissue damage associated genes. Mechanistically, we revealed that AMPK is an intracellular signaling molecular functioning downstream of ADORA2B underlying elevated 2,3-BPG production by inducing BPG mutase activity and protects tissue hypoxia, kidney dysfunction and renal fibrosis. Finally, we translated our mouse study to human and confirmed that the levels of 2,3-BPG, P50 and p-AMPK were elevated in erythrocytes of hypertensive CKD patients compared to healthy individuals and correlated to disease severity. Conclusions: We demonstrate that erythrocyte ADORA2B-mediated AMPK activation plays a key protective role in Ang II-induced tissue damage by increasing 2, 3-BPG and oxygen release to counteract tissue hypoxia and immediately suggest novel therapies for hypertension and CKD.
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The Adenosine A2B Receptor promotes tumor progression of bladder urothelial carcinoma by enhancing MAPK signaling pathway
Oncotarget, 2017Co-Authors: Yihong Zhou, Xi Chu, Fei Deng, Liang Tong, Guoxiong Tong, Jianye Liu, Jin Tang, Yuxin Tang, Yang XiaAbstract:// Yihong Zhou 1 , Xi Chu 1 , Fei Deng 1 , Liang Tong 1 , Guoxiong Tong 1 , Ye Yi 1 , Jianye Liu 1 , Jin Tang 1 , Yuxin Tang 1 , Yang Xia 2 and Yingbo Dai 1 1 Department of Urology, The Third Xiangya Hospital of Central South University, Changsha, China 2 Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas, USA Correspondence to: Yingbo Dai, email: // Keywords : Adenosine A2B Receptor, bladder urothelial carcinoma, prognosis, MAPK signaling Received : March 14, 2017 Accepted : May 01, 2017 Published : May 12, 2017 Abstract The Adenosine A2B Receptor (A2BR) was considered to play an oncogenic role in many human malignancies. However, the expression and molecular function of A2BR in bladder urothelial carcinoma (BUC) have not been well elucidated. Herein, we found that the expression of A2BR was higher than other Adenosine Receptors in BUC tissues and cells, and it was upregulated in BUC tissues compared with matched normal bladder tissues. Furthermore, high expression of A2BR was associated with poor prognosis of patients with BUC. In addition, suppression of A2BR inhibited the proliferation, migration and invasion of BUC cells and arrested the cell cycle at the G1 phase. Finally, we demonstrated that downregulation of A2BR inhibited the proliferation, migration and invasion of BUC in part via the MAPK signaling pathway, increasing the levels of P21 but decreasing the levels of cyclin B1, D, E1, MMP-2 and MMP-9. Overexpression of MMP-2 could rescue BUC cells migration and invasion. Thus, the present study indicates that A2BR may play a potential oncogenic role in BUC progression and act as a potential biomarker to identify BUC patients with poor clinical outcomes.
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Adenosine A2B Receptor promotes erythrocyte oxygen release to counteract tissue hypoxia and injury
Blood, 2016Co-Authors: Yang Xia, Yujin Zhang, Hong Liu, Anren Song, Kaiqi Sun, Morayo G. Adebiyi, Zhangzhe Peng, Renna Luo, Rodney E. KellemsAbstract:Insufficient tissue oxygen (O 2 ) availability (hypoxia) is commonly seen in patients with cardiovascular diseases (CVD), chronic kidney diseases (CKD), respiratory diseases, hemolytic disorders and certain cancers. Without interference, pathological hypoxia is dangerous because it promotes disease progression, organ damage and failure. Although erythrocyte is the only cell type responsible for delivering O 2 , its function in pathological tissue hypoxia remains largely unknown. Here using unbiased high throughput metabolomic profiling, we found that 2,3-biphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite enhancing O2 delivery, was significantly induced in the erythrocytes of mice infused with angiotensin II (Ang II), a potent vasopeptide commonly increased in the circulation of CVD and CKD patients to induce tissue hypoxia and injury via vasoconstriction. Mouse genetic studies demonstrated that Ang II induced local tissue accumulation of Adenosine and that elevated Adenosine-mediated erythrocyte A2B Adenosine Receptor (ADORA2B) activation was beneficial by inducing 2,3-BPG production, triggering O 2 release to prevent multiple tissue hypoxia, inflammation and kidney damage. Mechanistically, we revealed that AMPK is an intracellular signaling molecular functions downstream of ADORA2B underlying elevated 2,3-BPG production by inducing BPG mutase activity. Finally, we translated our mouse study to human and confirmed that the levels of 2,3-BPG and P50 were elevated in erythrocytes of mild CKD patients compared to healthy individuals and further increased in severe CKD patients compared to mild CKD patients. Taken together, we demonstrate that erythrocyte ADORA2B plays a key protective role in Ang II-induced tissue damage by increasing 2, 3-BPG and oxygen release to counteract tissue hypoxia. Disclosures No relevant conflicts of interest to declare.
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Adenosine A2B Receptor and Hyaluronan Modulate Pulmonary Hypertension Associated with Chronic Obstructive Pulmonary Disease
American journal of respiratory cell and molecular biology, 2013Co-Authors: Harry Karmouty-quintana, Tingting Weng, Luis J. Garcia-morales, Ning Yuan Chen, Mesias Pedroza, Hongyan Zhong, Jose G. Molina, R.r. Bunge, Brian A. Bruckner, Yang XiaAbstract:Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. The development of pulmonary hypertension (PH) in patients with COPD is strongly associated with increased mortality. Chronic inflammation and changes to the lung extracellular matrix (ECM) have been implicated in the pathogenesis of COPD, yet the mechanisms that lead to PH secondary to COPD remain unknown. Our experiments using human lung tissue show increased expression levels of the Adenosine A2B Receptor (ADORA2B) and a heightened deposition of hyaluronan (HA; a component of the ECM) in remodeled vessels of patients with PH associated with COPD. We also demonstrate that the expression of HA synthase 2 correlates with mean pulmonary arterial pressures in patients with COPD, with and without a secondary diagnosis of PH. Using an animal model of airspace enlargement and PH, we show that the blockade of ADORA2B is able to attenuate the development of a PH phenotype that correlates with reduced levels of HA deposition in the vessels and the down-regulation of genes involved in the synthesis of HA.
