There are several evidences that some functions of D1 dopamine receptors can be modulated by colo... more There are several evidences that some functions of D1 dopamine receptors can be modulated by colocalized adenosine A1 receptors. To elucidate the role of particular components of the receptor complex in the ligand binding and second messenger activation level we have used Sf9 cell expression system. The expression of D1 and A1 receptors was confirmed by proper binding of specific radioligands [3H]SCH23390 (Kd=1.1+/-0.1 nM, Bmax=2.2+/-0.1 pmol/mg protein) and [3H]DPCPX (Kd=2.1+/-0.8nM, Bmax=2.9+/-0.4 pmol/mg protein), respectively. The kinetics of [3H]SCH23390 binding corresponded to the simplest reversible bimolecular binding reaction of complex formation, with k(on)=0.20+/-0.02 min(-1)nM(-1) and k(off)=0.13+/-0.01 min(-1). Dopaminergic agonists increased the accumulation of cAMP in the transfected cells in concentration-dependent manner, indicating a correct coupling of receptor to second messenger system. The coupling of the A1 receptor to Gi proteins was confirmed by both GTPgammaS dependent agonist binding and inhibition of cAMP accumulation by N-cyclopentyladenosine (NCPA). Activation of the A1 receptor by NCPA had no significant influence on neither affinities of dopaminergic ligands nor the radioligand binding kinetics to the co-exprssed D1 receptors in Sf9 cell membranes. On the other hand, the activation of the A1 receptors inhibited the D1 receptor-specific accumulation of cAMP, but only in cells where Gi proteins were expressed with the receptors.
We have characterized the binding of [2-(3)H]-4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-... more We have characterized the binding of [2-(3)H]-4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-[1,3,5]-triazin-5-ylamino]ethyl)phenol ([(3)H]ZM241385) to adenosine A(2A) receptors in membranes of rat striatum and transfected CHO cells. Saturation experiments showed that [(3)H]ZM241385 binds to a single class of binding sites with high affinity (K(d) = 0.23 nM and 0.14 nM in CHO cell and striatal membranes, respectively). The membranes of CHO cells required pretreatment with adenosine deaminase (ADA) to achieve high-affinity binding, while ADA had no influence on the ligand binding properties in striatal membranes. The binding of [(3)H]ZM241385 was fast and reversible, achieving equilibrium within 20 minutes at all radioligand concentrations. The kinetic analysis of the [(3)H]ZM241385 interaction with A(2A) receptors indicated that the reaction had at least two subsequent steps. The first step corresponds to a fast equilibrium, which also determines the antagonist potency to competitively inhibit CGS21680-induced accumulation of cAMP (first equilibrium constant K(A) = 6.6 nM). The second step corresponds to a slow process of conformational isomerization (equilibrium constant K(i) = 0.03). The combination of the two steps gives the dissociation constant K(d) = 0.20 nM based on the kinetic data, which is in good agreement with the directly measured value. The data obtained shed light on the mechanism of the [(3)H]ZM241385 interaction with adenosine A(2A) receptors from different sources in vitro. The isomerization step of the A(2A) antagonist radioligand binding has to be taken into account for the interpretation of the binding parameters obtained from the various competition assays and explain the discrepancy between antagonist affinity in saturation experiments versus its potency in functional assays.
A novel set of 1-substituted apomorphines as dopaminergic agonists were synthesized according to ... more A novel set of 1-substituted apomorphines as dopaminergic agonists were synthesized according to our new strategy employing the acid-catalyzed rearrangement of diversely functionalized 5β-substituted-6-demethoxythebaines. The activities of new compounds for dopamine receptors subtypes were evaluated using HEK293 based stable cell lines expressing D1, D2L or D3 receptor subtypes. All studied compounds had affinities in nanomolar range for D2L and D3 receptors and the change of the nature of substituent in position 1 had only moderate effect. D1 receptors were sensitive to the introduction of the 4-OH-benzyl function resulting in an increased affinity. The small hydrophilic group (hydroxymethyl) highly reduced the agonist affinity and potency thereby increasing subtype selectivity. This strategy for selective modulation of affinities and potencies of 1-substituted apomorphines gives essential hints for future design of subtype selective dopaminergic ligands.
There are several evidences that some functions of D1 dopamine receptors can be modulated by colo... more There are several evidences that some functions of D1 dopamine receptors can be modulated by colocalized adenosine A1 receptors. To elucidate the role of particular components of the receptor complex in the ligand binding and second messenger activation level we have used Sf9 cell expression system. The expression of D1 and A1 receptors was confirmed by proper binding of specific radioligands [3H]SCH23390 (Kd=1.1+/-0.1 nM, Bmax=2.2+/-0.1 pmol/mg protein) and [3H]DPCPX (Kd=2.1+/-0.8nM, Bmax=2.9+/-0.4 pmol/mg protein), respectively. The kinetics of [3H]SCH23390 binding corresponded to the simplest reversible bimolecular binding reaction of complex formation, with k(on)=0.20+/-0.02 min(-1)nM(-1) and k(off)=0.13+/-0.01 min(-1). Dopaminergic agonists increased the accumulation of cAMP in the transfected cells in concentration-dependent manner, indicating a correct coupling of receptor to second messenger system. The coupling of the A1 receptor to Gi proteins was confirmed by both GTPgammaS dependent agonist binding and inhibition of cAMP accumulation by N-cyclopentyladenosine (NCPA). Activation of the A1 receptor by NCPA had no significant influence on neither affinities of dopaminergic ligands nor the radioligand binding kinetics to the co-exprssed D1 receptors in Sf9 cell membranes. On the other hand, the activation of the A1 receptors inhibited the D1 receptor-specific accumulation of cAMP, but only in cells where Gi proteins were expressed with the receptors.
We have characterized the binding of [2-(3)H]-4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-... more We have characterized the binding of [2-(3)H]-4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-[1,3,5]-triazin-5-ylamino]ethyl)phenol ([(3)H]ZM241385) to adenosine A(2A) receptors in membranes of rat striatum and transfected CHO cells. Saturation experiments showed that [(3)H]ZM241385 binds to a single class of binding sites with high affinity (K(d) = 0.23 nM and 0.14 nM in CHO cell and striatal membranes, respectively). The membranes of CHO cells required pretreatment with adenosine deaminase (ADA) to achieve high-affinity binding, while ADA had no influence on the ligand binding properties in striatal membranes. The binding of [(3)H]ZM241385 was fast and reversible, achieving equilibrium within 20 minutes at all radioligand concentrations. The kinetic analysis of the [(3)H]ZM241385 interaction with A(2A) receptors indicated that the reaction had at least two subsequent steps. The first step corresponds to a fast equilibrium, which also determines the antagonist potency to competitively inhibit CGS21680-induced accumulation of cAMP (first equilibrium constant K(A) = 6.6 nM). The second step corresponds to a slow process of conformational isomerization (equilibrium constant K(i) = 0.03). The combination of the two steps gives the dissociation constant K(d) = 0.20 nM based on the kinetic data, which is in good agreement with the directly measured value. The data obtained shed light on the mechanism of the [(3)H]ZM241385 interaction with adenosine A(2A) receptors from different sources in vitro. The isomerization step of the A(2A) antagonist radioligand binding has to be taken into account for the interpretation of the binding parameters obtained from the various competition assays and explain the discrepancy between antagonist affinity in saturation experiments versus its potency in functional assays.
A novel set of 1-substituted apomorphines as dopaminergic agonists were synthesized according to ... more A novel set of 1-substituted apomorphines as dopaminergic agonists were synthesized according to our new strategy employing the acid-catalyzed rearrangement of diversely functionalized 5β-substituted-6-demethoxythebaines. The activities of new compounds for dopamine receptors subtypes were evaluated using HEK293 based stable cell lines expressing D1, D2L or D3 receptor subtypes. All studied compounds had affinities in nanomolar range for D2L and D3 receptors and the change of the nature of substituent in position 1 had only moderate effect. D1 receptors were sensitive to the introduction of the 4-OH-benzyl function resulting in an increased affinity. The small hydrophilic group (hydroxymethyl) highly reduced the agonist affinity and potency thereby increasing subtype selectivity. This strategy for selective modulation of affinities and potencies of 1-substituted apomorphines gives essential hints for future design of subtype selective dopaminergic ligands.
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