B Kull

Karolinska Institutet, Solna, Stockholm, Sweden

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Publications (25)74.38 Total impact

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    ABSTRACT: Adenosine and adenosine analogues have been reported to act as agonists or partial agonists at the growth hormone secretagogue receptor 1a (GHSR1a). We have re-examined this question. A concentration-dependent increase in intracellular calcium concentration ([Ca(2+)](i)) was observed in GHSR1a transfected HEK 293-EBNA cells stimulated with adenosine (EC50: 0.2 microM) or 2-chloroadenosine (EC50: 1.1 microM) but also in untransfected HEK 293-EBNA cells stimulated with 2-chloroadenosine (EC50: 0.67 microM) or 5'-N-ethylcarboxamidoadenosine (NECA) (EC50: 0.045 microM). These findings support endogenous expression of adenosine receptors, presumably A(2B) receptors in HEK 293-EBNA cells. In GHSR1a transfected CHO cells, lacking adenosine receptors, the GHSR1a agonist hGhrelin (EC50: 2.4 nM) increased [Ca(2+)](i), but no effects of adenosine, 2-chloroadenosine or NECA were detected. An inverse agonist of GHSR1a, [d-Arg-1, d-Phe-5, d-Trp-7, 9, Leu-11] substance P, reduced hGhrelin effects but adenosine, 2-chloroadenosine or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not. NECA increased the [Ca(2+)](i) in co-transfected (GHSR1a and A(2B) receptor) CHO cells (EC50: 0.053 microM), but no additive or synergistic effects on [Ca(2+)](i) or cAMP formation were observed after stimulation with NECA in the absence or in the presence of hGhrelin. In binding studies on GHSR1a transfected CHO cell membranes, [(125)I]-hGhrelin binding could be displaced by the GHSR1a agonist MK-0677 (IC50: 0.34 nM), hGhrelin (IC50: 1.5 nM), and the substance P analogue (IC50: 0.64 microM) but not by adenosine or 2-chloroadenosine. We conclude that adenosine and analogues do not act as agonists or partial agonists at the GHSR1a and that cross-talk between the GHSR1a and A(2B) receptors is limited.
    Biochemical Pharmacology 09/2005; 70(4):598-605. · 4.58 Impact Factor
  • Peter Salmi, Ruben Isacson, Björn Kull
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    ABSTRACT: The functional role of dopamine D(1) receptors is still controversial. One reason for this controversy is that for a long time the only available agonists for in vivo characterization of dopamine D(1) receptors were benzazepines. Among them was the prototype dopamine D(1) receptor partial agonist, SKF 38393. The lack of a selective and fully efficacious dopamine D(1) receptor agonist hampered basic research on dopamine D(1) receptors and left the potential clinical utility of dopamine D(1) receptor agonists elusive. The research situation improved when the first potent full dopamine D(1) receptor agonist dihydrexidine, a phenanthridine, was introduced in the late 1980s. In contrast to SKF 38393, dihydrexidine was shown to stimulate cyclic AMP synthesis just as well or better than dopamine, and potently displaced [(3)H]SCH 23390 from rat and monkey striatal membranes. Also, dihydrexidine was the first dopamine D(1) receptor agonist that had potent antiparkinsonian activity in a primate model of Parkinson's disease. This finding suggested clinical utility for dopamine D(1) receptor agonists in Parkinson's disease and that this utility might be critically dependent on the intrinsic efficacy of the drug. Clinical utility for dopamine D(1) receptor agonists in other central nervous disorders might also be dependent on the intrinsic efficacy of the drug. However, even though studies with dihydrexidine as a pharmacological tool have pointed to the clinical use for dopamine D(1) receptor agonists, dihydrexidine's unfavorable pharmacokinetic profile and various adverse effects are likely to restrict or even preclude its use in humans. This review article provides an updated overview of the pharmacology of dihydrexidine and discusses possible clinical utility of dopamine D(1) receptor agonists in various central nervous system disorders.
    CNS Drug Reviews 02/2004; 10(3):230-42. · 4.92 Impact Factor
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    ABSTRACT: The behavioral and biochemical effects of the full dopamine D(1/5) receptor agonists, dihydrexidine and (1R,3S)-1-aminomethyl-5,6-dihydroxy-3-phenylisochroman HCl (A 68930), were examined in rats. Both A 68930 (0-4.6 mg kg(-1), s.c.) and dihydrexidine (0-8.0 mg kg(-1), s.c.) caused a dose-dependent suppression of locomotor activity, as assessed in an open-field. This locomotor suppression was dose-dependently antagonized by the selective dopamine D(1/5) receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine HCl (SCH 23390; 0-5.0 microg kg(-1), s.c.), but not by the selective dopamine D(2/3) receptor antagonist raclopride (0-25.0 microg kg(-1), s.c.). Furthermore, A 68930 and dihydrexidine did not cause any locomotor activity in habituated rats that displayed a very low base-line activity. Neither did A 68930 nor dihydrexidine produce any excessive stereotypies that could possibly interfere with and mask ambulatory activity. In fact, both A 68930 and dihydrexidine potently blocked hyperactivity produced by d-amphetamine (0-4.0 mg kg(-1), s.c.). Such findings traditionally would be interpreted as a sign of potential antipsychotic properties of A 68930 and dihydrexidine. Examination of neuronal activation, as indexed by the immediate early gene c-fos, showed that A 68930 and dihydrexidine caused a highly significant expression of c-fos in the medial prefrontal cortex. This c-fos expression was sensitive to treatment with SCH 23390, but not with raclopride. The effects of A 68930 and dihydrexidine on c-fos expression in caudate putamen or nucleus accumbens were less marked, or undetectable. The results indicate that stimulation of dopamine D(1/5) receptors, possibly in the medial prefrontal cortex, is associated with inhibitory actions on locomotor activity and d-amphetamine-induced hyperactivity. Assuming an important role of prefrontal dopamine D(1/5) receptors in schizophrenia, such inhibitory actions of dopamine D(1/5) receptor stimulation on psychomotor activation may have interesting clinical implications in the treatment of schizophrenia.
    Neuroscience 02/2004; 124(1):33-42. · 3.12 Impact Factor
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    ABSTRACT: The intrastriatal infusions of 'naked' small interfering RNA (siRNA) targeted to dopamine D1 receptors (1.0-10.0 nmol over 3 days) did not reduce dopamine D1 receptor messenger RNA levels or receptor protein, assessed by [125I] SCH 23982 binding in intact rats. This was in contrast to results in vitro where a 76% reduction in dopamine D1 receptor ligand binding could be observed. CONCLUSION: The results suggest that synthetic siRNA, when applied directly to rat brain, is not capable of inducing RNA interference.
    Acta Physiologica Scandinavica 11/2003; 179(2):173-7. · 2.55 Impact Factor
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    ABSTRACT: Undifferentiated and NGF-treated PC12 cells were subjected to anoxia for up to 24 h. The adenosine A(2A) receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidine (SCH 58261) decreased viability of undifferentiated, but not NGF-treated PC12 cells after 6 h of anoxia. Anoxia also transiently enhanced cAMP responses induced via activation of adenosine A(2A) receptors in undifferentiated PC12 cells (20-fold decrease in the EC(50) value for the agonist 2-[ p-(2-carbonylethyl) phenylethylamino]-5'- N-ethylcarboxamidoadenosine, CGS 21680). In NGF-treated PC12 cells, by contrast, anoxia decreased both the maximal response to and the potency of CGS 21680. In undifferentiated PC12 cells subjected to anoxia a very modest increase of A(2A) receptor mRNA was detected in cells by Northern blotting, but no changes in the amount of the receptor protein could be seen by Western blotting. However, surface biotinylation of the cells followed by avidin pull-down showed that the A(2A) receptor at the cell membrane was increased after anoxia. This was supported by immunolabelling of the A(2A) receptors: much of the receptor protein was present in the cytoplasm of normoxic cells, but in cells subjected to anoxia the A(2A) receptor immunolabelling at the cell membrane was more pronounced, indicating redistribution of the receptors from intracellular pools to the cell membrane during anoxia.
    Archiv für Experimentelle Pathologie und Pharmakologie 03/2002; 365(2):150-7. · 2.15 Impact Factor
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    ABSTRACT: Adenosine tonically inhibits synaptic transmission through actions at A(1) receptors. It also facilitates synaptic transmission, but it is unclear if this facilitation results from pre- and/or postsynaptic A(2A) receptor activation or from indirect control of inhibitory GABAergic transmission. The A(2A) receptor agonist, CGS 21680 (10 nM), facilitated synaptic transmission in the CA1 area of rat hippocampal slices (by 14%), independent of whether or not GABAergic transmission was blocked by the GABA(A) and GABA(B) receptor antagonists, picrotoxin (50 microM) and CGP 55845 (1 microM), respectively. CGS 21680 (10 nM) also inhibited paired-pulse facilitation by 12%, an effect prevented by the A(2A) receptor antagonist, ZM 241385 (20 nM). These effects of CGS 21680 (10 nM) were occluded by adenosine deaminase (2 U/ml) and were made to reappear upon direct activation of A(1) receptors with N(6)-cyclopentyladenosine (CPA, 6 nM). CGS 21680 (10 nM) only facilitated (by 17%) the K(+)-evoked release of glutamate from superfused hippocampal synaptosomes in the presence of 100 nM CPA. This effect of CGS 21680 (10 nM), in contrast to the isoproterenol (30 microM) facilitation of glutamate release, was prevented by the protein kinase C inhibitors, chelerythrine (6 microM) and bisindolylmaleimide (1 microM), but not by the protein kinase A inhibitor, H-89 (1 microM). Isoproterenol (30 microM), but not CGS 21680 (10-300 nM), enhanced synaptosomal cAMP levels, indicating that the CGS 21680-induced facilitation of glutamate release involves a cAMP-independent protein kinase C activation. To discard any direct effect of CGS 21680 on adenosine A(1) receptor, we also show that in autoradiography experiments CGS 21680 only displaced the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyladenosine ([(3)H]DPCPX, 0.5 nM) with an EC(50) of 1 microM in all brain areas studied and CGS 21680 (30 nM) failed to change the ability of CPA to displace DPCPX (1 nM) binding to CHO cells stably transfected with A(1) receptors. Our results suggest that A(2A) receptor agonists facilitate hippocampal synaptic transmission by attenuating the tonic effect of inhibitory presynaptic A(1) receptors located in glutamatergic nerve terminals. This might be a fine-tuning role for adenosine A(2A) receptors to allow frequency-dependent plasticity phenomena without compromising the A(1) receptor-mediated neuroprotective role of adenosine.
    Neuroscience 02/2002; 112(2):319-29. · 3.12 Impact Factor
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    ABSTRACT: This overview will focus on recent data from our laboratory. The four cloned human adenosine receptors stably transfected into Chinese hamster ovary cells were studied with respect to signaling via cyclic AMP and mitogen-activated protein (MAP) kinases. Adenosine acted as a full agonist at all the adenosine receptors when increases (A2A or A2B receptors) or decreases (A1 and A3 receptors) in cyclic AMP accumulation were studied. Adenosine was approximately equipotent at A1, A2A, and A3 receptors, but approximately 50 times higher concentrations were needed at A2B receptors. The potency of adenosine was such that levels encountered under basal physiological conditions (30–300 nM) were sufficient to activate all but the A2B receptors. Inosine was a low-efficacy agonist at A1 and A3 receptors but was inactive at A2A and A2B receptors. Thus, adenosine is the most important agonist and the only one at A2A and A2B receptors. When MAP kinase signaling was examined, adenosine was equipotent at all the four receptors, and significant activation was noted at 100 nM adenosine. The potency of adenosine is dependent not only on the type of receptor but also on receptor number. Thus, high potency of adenosine is seen only where the receptor is expressed abundantly. These and other results are reviewed and discussed in relation to agonist and antagonist effects in vivo. In particular, we summarize recent data that show that adenosine tonically activates A2A receptors in the basal ganglia. Some aspects of the use of adenosine A2A receptor antagonists in the treatment of Parkinson disease also are highlighted. Drug Dev. Res. 52:274–282, 2001. © 2001 Wiley-Liss, Inc.
    Drug Development Research 05/2001; 52(1‐2):274 - 282. · 0.87 Impact Factor
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    ABSTRACT: The potency of adenosine and inosine as agonists at human adenosine receptors was examined in a functional assay using changes in cyclic AMP (cAMP) formation in intact Chinese hamster ovary (CHO) cells stably transfected with the human A1, A2A, A2B, and A3 receptors. Adenosine increased cAMP formation in cells expressing the A2A (ec50: 0.7 μM) and A2B (ec50: 24 μM) receptors and inhibited forskolin (0.3–3 μM)-stimulated cAMP formation in cells expressing the A1 (ec50: 0.31 μM) and A3 receptors (ec50: 0.29 μM). The potency of adenosine at the A2A and A2B receptors was not altered by the presence of the uptake inhibitor nitrobenzylthioinosine (NBMPR), whereas it was increased about 6-fold by NBMPR at the A1 and A3 receptors. In the presence of NBMPR, inosine was a potent agonist (ec50: 7 and 0.08 μM at the A1 and A3 receptors, respectively), but with low efficacy especially at the A3 receptors. No effect of inosine was seen at the A2 receptors. Caffeine, theophylline, and paraxanthine shifted the dose–response curve for adenosine at the A1, A2A, and A2B receptors. These results indicate that adenosine is the endogenous agonist at all human adenosine receptors and that physiological levels of this nucleoside can activate A1, A2A, and A3 receptors on cells where they are abundantly expressed, whereas pathophysiological conditions are required to stimulate A2B receptors to produce cyclic AMP.
    Biochemical Pharmacology 03/2001; · 4.58 Impact Factor
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    ABSTRACT: The potency of adenosine and inosine as agonists at human adenosine receptors was examined in a functional assay using changes in cyclic AMP (cAMP) formation in intact Chinese hamster ovary (CHO) cells stably transfected with the human A1, A2A, A2B, and A3 receptors. Adenosine increased cAMP formation in cells expressing the A2A (ec50: 0.7 μM) and A2B (ec50: 24 μM) receptors and inhibited forskolin (0.3–3 μM)-stimulated cAMP formation in cells expressing the A1 (ec50: 0.31 μM) and A3 receptors (ec50: 0.29 μM). The potency of adenosine at the A2A and A2B receptors was not altered by the presence of the uptake inhibitor nitrobenzylthioinosine (NBMPR), whereas it was increased about 6-fold by NBMPR at the A1 and A3 receptors. In the presence of NBMPR, inosine was a potent agonist (ec50: 7 and 0.08 μM at the A1 and A3 receptors, respectively), but with low efficacy especially at the A3 receptors. No effect of inosine was seen at the A2 receptors. Caffeine, theophylline, and paraxanthine shifted the dose–response curve for adenosine at the A1, A2A, and A2B receptors. These results indicate that adenosine is the endogenous agonist at all human adenosine receptors and that physiological levels of this nucleoside can activate A1, A2A, and A3 receptors on cells where they are abundantly expressed, whereas pathophysiological conditions are required to stimulate A2B receptors to produce cyclic AMP.
    Biochemical Pharmacology - BIOCHEM PHARMACOL. 01/2001; 61(4):443-448.
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    ABSTRACT: In situ hybridization with cRNA probes showed A(2A) receptor and G(olf) mRNAs to be abundantly expressed in caudate putamen, nucleus accumbens, and olfactory tubercle, whereas G(s) mRNA shows a comparatively low expression in regions expressing A(2A) receptors. In caudate putamen, 49% of the medium-sized neuron-like cells exhibited a strong signal for adenosine A(2A) receptor mRNA, and 98% showed a strong signal for G(olf) mRNA. In contrast, G(s) mRNA was found in only 12% of the medium-sized neuron-like cells in caudate putamen. The coexpression of adenosine A(2A) receptor mRNA with that of G(olf) or G(s) mRNAs was studied with double in situ hybridization. A large majority (91-95%) of the neurons in caudate-putamen that contained adenosine A(2A) receptor mRNA also expressed G(olf) mRNA, whereas only 3 to 5% of the neurons with adenosine A(2A) receptor mRNA coexpressed G(s) mRNA. The A(2A) receptor agonist CGS 21680 [2-[p-(2-carbonylethyl)phenylethylamino-5'-N-ethylcarboxa midoadenosin e] dose dependently activated G(olf) subunits in striatal membranes as shown by photolabeling with [alpha-(32)P]m-acetylanilido-GTP followed by immunoprecipitation with a specific antibody against G(olf). Transfection of G(olf) cDNA into Chinese hamster ovary cells, which stably express human adenosine A(2A) receptors, led to an increased efficacy of CGS 21680, as evidenced by a stronger cAMP response, indicating that activation of G(olf) by A(2A) receptors leads to a biological signal. In conclusion, these results provide anatomical and biochemical evidence that adenosine A(2A) receptors stimulate G(olf) rather than G(s) in striatum.
    Molecular Pharmacology 11/2000; 58(4):771-7. · 4.41 Impact Factor
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    ABSTRACT: The effect of guanosine triphosphate (GTP) on the interaction of antagonists with human adenosine A1 and A2A receptors was studied using whole-hemisphere sections from human brain and membranes from Chinese hamster ovary (CHO) cells expressing human A1 and A2A receptors. Adenosine A1 receptors, studied using 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) as radioligand, showed the expected regional distribution in human brain. Addition of 500 μM GTP significantly increased (23–55%) DPCPX binding in all regions measured. In CHO cells transfected with human adenosine A1 receptor cDNA, the number of receptors, Bmax, increased from 401 (359–442) to 667 (592–743) fmol/mg protein upon addition of GTP. 5-Amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]-pyrimidine (SCH 58261), a selective adenosine A2A receptor ligand, showed saturable binding to membranes from CHO cells transfected with adenosine A2A receptor cDNA and was localized to striatum and globus pallidus in human brain sections. Addition of GTP did not significantly change SCH 58261 binding to brain sections or CHO cell membranes. These results indicate that human A1 and A2A receptors are not substantially different from those of the rat as regards regulation by GTP and interactions with endogenous adenosine in binding experiments. However, the relative abundance of the receptors differs between species, and this may be related to the differences observed in the potency of the endogenous agonist.
    Neuropharmacology 11/2000; 39(12):2374–2380. · 4.11 Impact Factor
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    ABSTRACT: The effect of guanosine triphosphate (GTP) on the interaction of antagonists with human adenosine A(1) and A(2A) receptors was studied using whole-hemisphere sections from human brain and membranes from Chinese hamster ovary (CHO) cells expressing human A(1) and A(2A) receptors. Adenosine A(1) receptors, studied using [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) as radioligand, showed the expected regional distribution in human brain. Addition of 500 microM GTP significantly increased (23-55%) [3H]DPCPX binding in all regions measured. In CHO cells transfected with human adenosine A(1) receptor cDNA, the number of receptors, B(max), increased from 401 (359-442) to 667 (592-743) fmol/mg protein upon addition of GTP. [3H]5-Amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo-[4,3-e]-1,2, 4-triazolo-[1,5-c]-pyrimidine ([3H]SCH 58261), a selective adenosine A(2A) receptor ligand, showed saturable binding to membranes from CHO cells transfected with adenosine A(2A) receptor cDNA and was localized to striatum and globus pallidus in human brain sections. Addition of GTP did not significantly change [3H]SCH 58261 binding to brain sections or CHO cell membranes. These results indicate that human A(1) and A(2A) receptors are not substantially different from those of the rat as regards regulation by GTP and interactions with endogenous adenosine in binding experiments. However, the relative abundance of the receptors differs between species, and this may be related to the differences observed in the potency of the endogenous agonist.
    Neuropharmacology 10/2000; 39(12):2374-80. · 4.11 Impact Factor
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    ABSTRACT: ATP-induced Ca2+ transients were examined in individual PC12 cells of a well defined clone, before and after treatment with nerve growth factor (NGF) to induce a neurone-like phenotype. Using reverse transcriptase PCR these cells were found to express mRNA for several P2 receptors. In undifferentiated cells the ATP-induced Ca2+ response was entirely dependent on Ca2+ influx, could not be mimicked by UTP, alpha,beta-methylene ATP or dibenzoyl ATP or be blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). ATP had no significant effect on levels of cyclic AMP or inositol 1,4,5-trisphosphate (InsP3). These results suggest that in undifferentiated PC12 cells ATP mainly acts on a P2X receptor, possibly the P2X4 subtype. After treatment with NGF for 7 days the ATP response was increased and partially sensitive to PPADS. A component of the ATP-induced Ca2+ increase was due to mobilisation of intracellular Ca2+ stores and another to capacitative Ca2+ entry. UTP caused an increase in intracellular Ca2+, and InsP3 formation could be stimulated by ATP and UTP. ATP also caused a small increase in cyclic AMP, but this was abolished in the presence of indomethacin. Thus, after NGF treatment ATP acts partially via a P2Y receptor, possibly the P2Y2 subtype.
    Neuropharmacology 02/2000; 39(3):482-96. · 4.11 Impact Factor
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    ABSTRACT: Four adenosine receptors have been cloned from many mammalian and some non-mammalian species. In each case the translated part of the receptor is encoded by two separate exons. Two separate promoters regulate the A1 receptor expression, and a similar situation may pertain also for the other receptors. The receptors are expressed in a cell and tissue specific manner, even though A1 and A2B receptors are found in many different cell types. Emerging data indicate that the receptor protein is targeted to specific parts of the cell. A1 and A3 receptors activate the Gi family of G proteins, whereas A2A and A2B receptors activate the Gs family. However, other G proteins can also be activated even though the physiological significance of this is unknown. Following the activation of G proteins several cellular effector pathways can be affected. Signaling via adenosine receptors is also known to interact in functionally important ways with signaling initiated via other receptors.
    Archiv für Experimentelle Pathologie und Pharmakologie 01/2000; 362(4):364-374. · 2.15 Impact Factor
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    ABSTRACT: Human adenosine A2A and rat dopamine D2 receptors (A2A and D2 receptors) were co-transfected in Chinese hamster ovary (CHO) cells to study the interactions between two receptors that are co-localized in striatopallidal γ-aminobutyric acid-(GABA)ergic neurons. Membranes from transfected cells showed a high density of D2 (3.6 pmol per mg protein) and A2A receptors (0.56 pmol per mg protein). The D2 receptors were functional: an agonist, quinpirole, could stimulate GTPγS binding and reduce stimulated adenylyl cyclase activity. The A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680) decreased high-affinity binding of the agonist dopamine at D2 receptors. Activation of adenosine A2A receptors shifted the dose–response curve for quinpirole on adenosine 3′,5′-cyclic monophosphate (cAMP) to the right. However, CGS 21680 did not affect dopamine D2 receptor-induced GTPγS binding, but did cause a concentration-dependent increase in cAMP accumulation. The maximal cAMP response was decreased by the D2 agonist quinpirole in a concentration-dependent manner, but there was no change in ec50 and no effect in cells transfected only with adenosine A2A receptors. A2A receptor activation also increased phosphorylation of cAMP response element-binding protein and expression of c-fos mRNA. These effects were also strongly counteracted by quinpirole. These results show that the antagonistic actions between adenosine A2A and dopamine D2 receptors noted previously in vivo can also be observed in CHO cells where the two receptors are co-transfected. Thus, no brain cell-specific factors are required for such interactions. Furthermore, the interaction at the second messenger level and beyond may be quantitatively more important than A2A receptor-mediated inhibition of high affinity D2 agonist binding to the receptor.
    Biochemical Pharmacology 10/1999; · 4.58 Impact Factor
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    ABSTRACT: To examine possible species differences in pharmacology, rat adenosine A2A receptors were studied in PC12 (pheochromocytoma) cells, and human receptors in Chinese hamster ovary (CHO) cells transfected with the cloned human A2A receptor cDNA. Using [3H]-5-amino-7(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine ([3H]-SCH 58261) as radioligand, the estimated Bmax (maximal binding) was 538 and 2085 fmol/mg in CHO and PC12 cells, respectively. The Kd (dissociation constant) values for [3H]-SCH 58261 were 1.05 and 5.6 nM in the two cell types, respectively. The order of potency of antagonists and most agonists was the same in both cell types, but 2-phenylaminoadenosine and 2-chloroadenosine were relatively less potent in PC12 cells than in CHO cells. In the functional assay, using cyclic AMP accumulation, all agonists tested were more potent in CHO than in PC12 cells, but this could not be readily explained by differences in adenylyl cyclase or in the expression of G proteins. As in the case of binding, the relative agonist potencies were similar for most compounds, but 2-phenylaminoadenosine and 2-chloroadenosine were more potent at human A2A receptors in CHO cells than predicted from the data obtained on rat A2A receptors in PC12 cells. Antagonists were approximately equipotent in the two cells. These results show that, despite only small differences in amino acid sequences and no difference in antagonist pharmacology, the relative order of potency of receptor agonists can differ between species homologues of the adenosine A2A receptor.
    Biochemical Pharmacology 02/1999; 57(1):65-75. · 4.58 Impact Factor
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    ABSTRACT: To examine possible species differences in pharmacology, rat adenosine A2A receptors were studied in PC12 (pheochromocytoma) cells, and human receptors in Chinese hamster ovary (CHO) cells transfected with the cloned human A2A receptor cDNA. Using [3H]-5-amino-7(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([3H]-SCH 58261) as radioligand, the estimated Bmax (maximal binding) was 538 and 2085 fmol/mg in CHO and PC12 cells, respectively. The Kd (dissociation constant) values for [3H]-SCH 58261 were 1.05 and 5.6 nM in the two cell types, respectively. The order of potency of antagonists and most agonists was the same in both cell types, but 2-phenylaminoadenosine and 2-chloroadenosine were relatively less potent in PC12 cells than in CHO cells. In the functional assay, using cyclic AMP accumulation, all agonists tested were more potent in CHO than in PC12 cells, but this could not be readily explained by differences in adenylyl cyclase or in the expression of G proteins. As in the case of binding, the relative agonist potencies were similar for most compounds, but 2-phenylaminoadenosine and 2-chloroadenosine were more potent at human A2A receptors in CHO cells than predicted from the data obtained on rat A2A receptors in PC12 cells. Antagonists were approximately equipotent in the two cells. These results show that, despite only small differences in amino acid sequences and no difference in antagonist pharmacology, the relative order of potency of receptor agonists can differ between species homologues of the adenosine A2A receptor.
    Biochemical Pharmacology - BIOCHEM PHARMACOL. 01/1999; 57(1):65-75.
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    ABSTRACT: PC12 cells are genetically labile and so-called wild-type cells comprise multiple subclones. We have examined the A2A adenosine receptor signal transduction pathways in four such clones (denoted clones 1, 19, 21 and 27) of PC12 cells. Adenosine A2A, A2B and A1 receptor mRNAs were detected in all four clones by RT-PCR, whereas no A3 receptor mRNA was found. A2A receptors were quantitated by radioligand binding using the antagonist radioligand [3H]SCH 58261 ([3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine). The B max was highest in clone 1 followed by clones 21, 19 and 27. Whereas the amount of Gi protein appeared similar in all four clones, the amount of Gs protein was higher in clones 21 and 27 than in the other two clones. Maximal responses to the non-selective adenosine analogue NECA (5’-N-ethylcarboxamidoadenosine) were similar to those observed with the selective adenosine A2A receptor agonist CGS 21680 (2-[p-(2-carbonylethyl) phenylethylamino]-5’-N-ethylcarboxamidoadenosine), and were approximately equal in clones 1 and 21, but lower in clone 19 and very low in clone 27. For both compounds EC50 was significantly higher in clone 27 than in clone 1. In both clones the response to NECA could be competitively antagonized by a selective adenosine A2A antagonist, SCH 58261. The present results show that different clones of PC12 cells differ widely in the cAMP increase induced by adenosine analogues and that this is due to differences in the amount of adenosine A2A receptor, G protein and effector. A large difference in receptor number resulted in differences in potency of an agonist.
    Archiv für Experimentelle Pathologie und Pharmakologie 12/1998; 359(1):28-32. · 2.15 Impact Factor
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    ABSTRACT: Four adenosine receptor subtypes of the family of G protein-coupled receptors, designated A1, A2A, A2B and A3 are currently known. In this study all human subtypes were stably transfected into Chinese hamster ovary (CHO) cells in order to be able to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). The A1 subtype showed the typical pharmacological profile with 2-chloro-N6-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N6-phenylisopropyladenosine (PIA) diastereomers. In competition with antagonist radioligand biphasic curves were observed for agonists. In the presence of GTP all receptors were converted to a single low affinity state indicating functional coupling to endogenous G proteins. For A2A adenosine receptors CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadeno sine) and N-ethylcarboxamidoadenosine (NECA) were found to be the most potent agonists followed by R- and S-PIA with minor stereoselectivity. The relative potencies of agonists for the A2B adenosine receptor could only be tested by measurement of receptor-stimulated adenylyl cyclase activity. NECA was the most potent agonist with an EC50-value of 2.3 microM whereas all other compounds tested were active at concentrations in the high micromolar range. Inhibition of NECA-stimulated adenylyl cyclase identified xanthine amino congener (XAC; 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine) as the most potent antagonist at this receptor subtype. The A3 receptor was characterized utilizing the nonselective agonist [3H]NECA. The N6-benzyl substituted derivatives of adenosine-5'-N-methyluronamide (MECA) turned out to be the most potent agonists. The notion of xanthine-insensitivity of the A3 receptor should be dropped at least for the human receptor as xanthines with submicromolar affinity were found. Overall, the pharmacological characteristics of the human receptors are similar to other species with some species-specific characteristics. In this study we present for the first time the comparative pharmacology of all known human adenosine receptor subtypes. The CHO cells with stably transfected adenosine receptors provide an identical cellular background for such a pharmacological characterization. These cells are valuable systems for further characterization of specific receptor subtypes and for the development of new ligands.
    Archiv für Experimentelle Pathologie und Pharmakologie 02/1998; 357(1):1-9. · 2.15 Impact Factor
  • 01/1998;

Publication Stats

1k Citations
74.38 Total Impact Points

Institutions

  • 1997–2004
    • Karolinska Institutet
      • Institutionen för fysiologi och farmakologi
      Solna, Stockholm, Sweden
    • San Raffaele Scientific Institute
      Milano, Lombardy, Italy
  • 1999
    • Lund University
      Lund, Skåne, Sweden