Technologies and assays based on fluorescence give us the possibility to investigate the systems ... more Technologies and assays based on fluorescence give us the possibility to investigate the systems of interest from different aspects with unprecedented precision and high sampling rate. This has in turn led to the adoption of a "systems" approach in our research with an increasing trend towards study of entire GPCR signaling pathways: ligand binding, receptor interaction with G-protein, G-protein dissociation and the role of individual subunits, properties of adenylatecyclase in conversion of ATP into cAMP, cAMP production and its role in protein expression, etc. However, this approach also led to an explosive increase of raw data. To integrate data from multiple measurable signals into meaningful actionable knowledge that can yield real insight into studied processes, we use multivariable global data analysis combining various fluorescence readouts (intensity and anisotropy based).
A set of novel apomorphine derivatives were synthesized with diversely functionalized side chains... more A set of novel apomorphine derivatives were synthesized with diversely functionalized side chains in the proximity of position 2 of the aporphine skeleton. Amino and/or carboxylic functions were introduced to this region of the backbone to test their pharmacological effects. During the synthesis of 2-(S-3-mercaptopropionic acid)-derivative a heteroring-fused congener was also isolated. The structural elucidation confirmed that the formation of this product was in accordance with our previous observations on the reaction of thebaine (2) with thiosalycilic acid. All the novel apomorphine congeners 4a–g were neuropharmacologically characterized to discover their dopaminergic profiles. Two derivatives were identified as D2 full agonists equipotent with apomorphine (1) having significantly increased D2/D1 selectivity ratios.► Synthesis of novel apomorphine derivatives with diversely functionalized aminoalkyl side chains in the proximity of position 2 of the aporphine skeleton. ► A thiopyrano-fused byproduct was also isolated and characterized. ► Neuropharmacologically characterization revealed that two derivatives have equipotent D2 full agonists with apomorphine (1) having significantly increased D2/D1 selectivity ratio.
We have characterized the modulation of adenylyl cyclase (AC) activity by ligands of dopaminergic... more We have characterized the modulation of adenylyl cyclase (AC) activity by ligands of dopaminergic receptors in rat striatal homogenate and compared the results with receptor-ligand binding affinities. Despite the fact that rat striatum contains high level of both dopamine D(1) and D(2) receptors, only the D(1)-specific AC activation by agonists could be determined. All D(1)-receptor agonists (dopamine, dihydrexidine, and A 77636) used were able to increase cAMP accumulation in a concentration-dependent manner, while D(1)-receptor antagonists (SCH23390, SKF83566, and butaclamol) blocked the effects induced by the aforementioned agonists. At the same time, the D(2)-receptor agonist quinpirole and antagonist sulpiride had no effect on cAMP accumulation in striatal homogenate neither on the basal level nor on the activated level of AC, while inhibited [(3)H]raclopride binding to these membranes. Comparing the ligands of the D(1) receptor in modulating the activity of AC and displacing D(1)-receptor-specific radioligand [(3)H]SCH23390 binding revealed that the ligands modulate both of these processes with similar affinities. It indicates that under given experimental conditions, only dopamine D(1)-receptor-mediated stimulation of AC activity can be measured in membrane homogenate of rat striatum, while dopamine D(2)-receptor effects remain fully hidden.
A set of novel apomorphine derivatives were synthesized with diversely functionalized side chains... more A set of novel apomorphine derivatives were synthesized with diversely functionalized side chains in the proximity of position 2 of the aporphine skeleton. Amino and/or carboxylic functions were introduced to this region of the backbone to test their pharmacological effects. During the synthesis of 2-(S-3-mercaptopropionic acid)-derivative a heteroring-fused congener was also isolated. The structural elucidation confirmed that the formation of this product was in accordance with our previous observations on the reaction of thebaine (2) with thiosalycilic acid. All the novel apomorphine congeners 4a-g were neuropharmacologically characterized to discover their dopaminergic profiles. Two derivatives were identified as D(2) full agonists equipotent with apomorphine (1) having significantly increased D(2)/D(1) selectivity ratios.
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.
Technologies and assays based on fluorescence give us the possibility to investigate the systems ... more Technologies and assays based on fluorescence give us the possibility to investigate the systems of interest from different aspects with unprecedented precision and high sampling rate. This has in turn led to the adoption of a "systems" approach in our research with an increasing trend towards study of entire GPCR signaling pathways: ligand binding, receptor interaction with G-protein, G-protein dissociation and the role of individual subunits, properties of adenylatecyclase in conversion of ATP into cAMP, cAMP production and its role in protein expression, etc. However, this approach also led to an explosive increase of raw data. To integrate data from multiple measurable signals into meaningful actionable knowledge that can yield real insight into studied processes, we use multivariable global data analysis combining various fluorescence readouts (intensity and anisotropy based).
A set of novel apomorphine derivatives were synthesized with diversely functionalized side chains... more A set of novel apomorphine derivatives were synthesized with diversely functionalized side chains in the proximity of position 2 of the aporphine skeleton. Amino and/or carboxylic functions were introduced to this region of the backbone to test their pharmacological effects. During the synthesis of 2-(S-3-mercaptopropionic acid)-derivative a heteroring-fused congener was also isolated. The structural elucidation confirmed that the formation of this product was in accordance with our previous observations on the reaction of thebaine (2) with thiosalycilic acid. All the novel apomorphine congeners 4a–g were neuropharmacologically characterized to discover their dopaminergic profiles. Two derivatives were identified as D2 full agonists equipotent with apomorphine (1) having significantly increased D2/D1 selectivity ratios.► Synthesis of novel apomorphine derivatives with diversely functionalized aminoalkyl side chains in the proximity of position 2 of the aporphine skeleton. ► A thiopyrano-fused byproduct was also isolated and characterized. ► Neuropharmacologically characterization revealed that two derivatives have equipotent D2 full agonists with apomorphine (1) having significantly increased D2/D1 selectivity ratio.
We have characterized the modulation of adenylyl cyclase (AC) activity by ligands of dopaminergic... more We have characterized the modulation of adenylyl cyclase (AC) activity by ligands of dopaminergic receptors in rat striatal homogenate and compared the results with receptor-ligand binding affinities. Despite the fact that rat striatum contains high level of both dopamine D(1) and D(2) receptors, only the D(1)-specific AC activation by agonists could be determined. All D(1)-receptor agonists (dopamine, dihydrexidine, and A 77636) used were able to increase cAMP accumulation in a concentration-dependent manner, while D(1)-receptor antagonists (SCH23390, SKF83566, and butaclamol) blocked the effects induced by the aforementioned agonists. At the same time, the D(2)-receptor agonist quinpirole and antagonist sulpiride had no effect on cAMP accumulation in striatal homogenate neither on the basal level nor on the activated level of AC, while inhibited [(3)H]raclopride binding to these membranes. Comparing the ligands of the D(1) receptor in modulating the activity of AC and displacing D(1)-receptor-specific radioligand [(3)H]SCH23390 binding revealed that the ligands modulate both of these processes with similar affinities. It indicates that under given experimental conditions, only dopamine D(1)-receptor-mediated stimulation of AC activity can be measured in membrane homogenate of rat striatum, while dopamine D(2)-receptor effects remain fully hidden.
A set of novel apomorphine derivatives were synthesized with diversely functionalized side chains... more A set of novel apomorphine derivatives were synthesized with diversely functionalized side chains in the proximity of position 2 of the aporphine skeleton. Amino and/or carboxylic functions were introduced to this region of the backbone to test their pharmacological effects. During the synthesis of 2-(S-3-mercaptopropionic acid)-derivative a heteroring-fused congener was also isolated. The structural elucidation confirmed that the formation of this product was in accordance with our previous observations on the reaction of thebaine (2) with thiosalycilic acid. All the novel apomorphine congeners 4a-g were neuropharmacologically characterized to discover their dopaminergic profiles. Two derivatives were identified as D(2) full agonists equipotent with apomorphine (1) having significantly increased D(2)/D(1) selectivity ratios.
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.
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