Improved Models for Pharmacological Null Experiments: Calculation of Drug Efficacy at Recombinant D1A Dopamine Receptors Stably Expressed in Clonal Cell Lines

Mak, Chun; Avalos, Melva; Randall, Patrick K.; Kwan, Sau-Wah; Abell, Creed W.; Neumeyer, John L.; Whisennand, Richard; Wilcox, Richard E.

doi:10.1016/0028-3908(96)84625-9

Cited by 12 publications

(7 citation statements)

References 32 publications

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“…The null model for receptor occlusion was used to estimate the equilibrium dissociation constant (K A ) of the full agonist DAMGO (31)(32)(33). In this model, the occluded curve obtained for DAMGO after receptor inactivation with ␤-CNA is represented as a right-shifted version of the control curve.…”

Section: Methodsmentioning

confidence: 99%

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Opioid Agonists Have Different Efficacy Profiles for G Protein Activation, Rapid Desensitization, and Endocytosis of Mu-opioid Receptors

Borgland

Connor

Osborne

et al. 2003

Journal of Biological Chemistry

144

179

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The differential ability of various -opioid receptor (MOP) agonists to induce rapid receptor desensitization and endocytosis of MOP could arise simply from differences in their efficacy to activate G proteins or, alternatively, be due to differential capacity for activation of other signaling processes. We used AtT20 cells stably expressing a low density of FLAG-tagged MOP to compare the efficacies of a range of agonists to 1) activate G proteins using inhibition of calcium channel currents (I Ca ) as a reporter before and after inactivation of a fraction of receptors by ␤-chlornaltrexamine, 2) produce rapid, homologous desensitization of I Ca inhibition, and 3) internalize receptors. Relative efficacies determined for G protein coupling were [Tyr-D-Ala-Gly-MePhe-Glyol]enkephalin (DAMGO) (1) > methadone (0.98) > morphine (0.58) > pentazocine (0.15). The same rank order of efficacies for rapid desensitization of MOP was observed, but greater concentrations of agonist were required than for G protein activation. By contrast, relative efficacies for promoting endocytosis of MOP were DAMGO (1) > methadone (0.59) > > morphine (0.07) > pentazocine (0.03). These results indicate that the efficacy of opioids to produce activation of G proteins and rapid desensitization is distinct from their capacity to internalize -opioid receptors but that, contrary to some previous reports, morphine can produce rapid, homologous desensitization of MOP.Tolerance to the analgesic and other effects of opioid drugs, such as morphine, undermines their use in long term treatment (1). Although analgesic tolerance is likely to be a complex phenomenon, an understanding of how -opioid (MOP) 1 receptors are regulated by opioid agonists will lead to important insights into this phenomenon. MOPs are similar to many other G protein-coupled receptors (GPCR) in that they undergo desensitization within several minutes of stimulation by agonists (2-4), and continued agonist exposure results in removal of receptors from the cell surface (5-7). MOP phosphorylation is increased as a consequence of agonist exposure, leading to a commonly held assumption that receptor phosphorylation is essential for MOP desensitization and internalization (5, 8 -10). The mechanism that may be responsible for uncoupling MOP from G protein activation and then promoting receptor sequestration is as follows: phosphorylation of the agonist occupied MOP by a G protein receptor kinase (GRK), subsequent binding of ␤-arrestins to the phosphorylated receptor, and removal of MOP from the cell surface via a clathrin-dependent process (11-15). The enhanced analgesic potency of morphine and diminished analgesic tolerance in ␤-arrestin2 knockout mice suggest that the capacity of opioid agonists to cause receptor desensitization and endocytosis is one of the key cellular mechanisms underlying tolerance (16). Phosphorylation of MOP by protein kinase C (PKC) or calmodulin-dependent kinase II can also reduce the capacity of MOP to activate G proteins (8,17), also contributing to opioid tole...

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Section: Methodsmentioning

confidence: 99%

“…A null model was also used to estimate the equilibrium dissociation constant of the partial agonists (K B ) and their relative efficacy to DAMGO (33). The relationship between equally effective concentrations of a partial agonist and DAMGO can be described by,…”

Section: Equation 2 Is Equivalent To Ln([aј]) ϫ Ln(shift) Which Can mentioning

confidence: 99%

Opioid Agonists Have Different Efficacy Profiles for G Protein Activation, Rapid Desensitization, and Endocytosis of Mu-opioid Receptors

Borgland

Connor

Osborne

et al. 2003

Journal of Biological Chemistry

144

179

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“…The increase of NNC 01-0012 intrinsic activity at the D1B!D1CcT chimera was not strictly limited to this compound and is apparent with other benzazepine agents that act as partial agonists at Dl-!ike receptors. Thus, SKF-38393 can behave as a partial agonist at cloned DI-like receptors, depending on a variety of conditions (Pedersen et al, 1994;Jensen et al, 1995;Watts et al, 1995;Mak et al, 1996). As depicted in Fig.…”

Section: Effects Of D1b Carboxyl-terminal Tail Substitution On Other mentioning

confidence: 98%

Dopamine D1B Receptor Chimeras Reveal Modulation of Partial Agonist Activity by Carboxyl‐Terminal Tail Sequences

Sugamori

Scheideler

Vernier

et al. 1998

Journal of Neurochemistry

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NNC 01‐0012, a second‐generation benzazepine compound, pharmacologically differentiates multiple vertebrate D1 receptor subtypes (D1A, D1B, D1C, and D1D) and displays high selectivity and affinity for dopamine D1C receptors. Functionally, whereas NNC 01‐0012 acts as a full or poor antagonist at D1C and D1A receptor‐mediated cyclic AMP production, respectively, it exhibits partial agonist activity at the D1B receptor. To define some of the structural motifs that regulate the pharmacological and functional differentiation of vertebrate dopamine D1 receptors by NNC 01‐0012, a series of receptor chimeras were constructed in which the divergent carboxyl‐terminal (CT) receptor tails were replaced with the corresponding sequences of D1A, D1B, or D1C receptors. Substitution of the vertebrate D1B carboxyl‐terminal‐tail at position Tyr345 with carboxyl‐terminal‐tail sequences of the D1A receptor abolished the partial agonist activity of NNC 01‐0012 without affecting dopamine‐stimulated cyclic AMP accumulation. At vertebrate D1B/D1Cct‐tail receptor mutants, however, the intrinsic activity of the partial agonist NNC 01‐0012 (10 µM) was markedly enhanced (∼60% relative to 10 µM dopamine) with no concomitant alteration in the molecule's ligand binding affinity or constitutive activity of the chimeric receptor. Similar results were obtained with other benzazepines such as SKF‐38393 and SCH‐23390, which act as partial agonists at vertebrate D1B receptors. Substitution of D1A and D1C receptor carboxyl‐terminal tails with sequences encoded by the D1B receptor carboxyl‐terminal tail did not, however, produce receptors with functional characteristics significantly different from wild type. Taken together, these data clearly suggest that in addition to well‐characterized domains and amino acid residues in the third cytoplasmic loop, partial agonist activity at the D1B receptor is modulated by sequence‐specific motifs within the carboxyl‐terminal tail, a region that may underlie the possible structural basis for functionally divergent roles of multiple dopamine D1‐like receptors.

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“…According to standard methods for nonlinear analyses [Severson and Wilcox, 1988;Mak et al, 1996;Avalos et al, 2001] displacement assay data were first analyzed together within species to increase the precision and accuracy of binding parameter determinations. Displacement curves were analyzed using nonlinear regression (NLR) of logistic dose response curves using the NLR subroutine within SPSS [Norusis, 1990], which is similar to ALLFIT [DeLean et al, 1982].…”

Section: Discussionmentioning

confidence: 99%

Pharmacological Characterization of the D1- and D2-Like Dopamine Receptors from the Brain of the Leopard Frog, <i>Rana pipiens</i>

Chu

Wilczynski

Wilcox³

2001

Brain Behav Evol

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The pharmacological profiles of D1- and D2-like dopamine receptors were investigated for native brain receptors in the leopard frog, Rana pipiens, using direct binding assays, which characterize functional receptors rather than assess total receptor protein. We used homogenate assays of R. pipiens fore- and midbrains to determine, via saturation isotherms, that the dissociation constant, Kd, for ³H-SCH-23390 binding to the D1-like receptors was 0.29 nM, and the maximal receptor density, Bmax, was 40 fmoles/mg protein. This compares with the more than 10-fold higher density of D1 sites in rat striatum. Specific binding for the D2-like receptors was measurable using these methods with ³H-spiperone as the ligand. However, saturation of binding was not achieved. This contrasts with the > 400 fmoles/mg protein Bmax in rat striatum. Pharmacological profiles (rank order of potency of displacing drugs) for each receptor type were determined. We used non-radioactive SCH-23390, SKF-38393, sulpiride, and spiperone to displace ³H-SCH-23390 and ³H-spiperone at D1 and D2 receptors, respectively. Parallel displacement assays were performed with rat striatal controls. Results indicated that the relative rank order displacements in anuran dopamine receptors were characteristic of D1- and D2-like receptors. However, the rank orders were not identical to those in mammals. The rank order for affinity at D1-like receptors in both rats and frogs was SCH-23390 > SKF-38393 > spiperone > sulpiride. The rank order for affinity at D2-like receptors was spiperone > SCH-23390 > sulpiride > SKF-38393 in frogs, and spiperone > sulpiride > SCH-23390 > SKF-38393 in rats. SKF-38393 and spiperone had similar affinities for the ‘D1’ receptors in both species. SCH-23390 had a slightly lower affinity for the D1-like receptors in Rana, whereas sulpiride had a significantly lower affinity for Rana D1-like receptors compared to rat D1 receptors. In Rana D2-like receptors, spiperone and sulpiride were significantly less potent compared to rat. However, SCH-23390 and SKF-38393 were equally potent for the D2-like receptors in both species. The results indicate that amphibian brain dopamine receptors fall into two classes similar to the mammalian D1 and D2 subfamilies, but with binding characteristics slightly different from those typically described in mammals. This work represents the first pharmacological characterization of native brain dopaminergic receptors in an anuran amphibian. Because direct binding assays measure the initial aspect of the functional interaction between transmitter and receptor, these data provide an important complement to studies using cell expression systems.

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Improved Models for Pharmacological Null Experiments: Calculation of Drug Efficacy at Recombinant D1A Dopamine Receptors Stably Expressed in Clonal Cell Lines

Cited by 12 publications

References 32 publications

Opioid Agonists Have Different Efficacy Profiles for G Protein Activation, Rapid Desensitization, and Endocytosis of Mu-opioid Receptors

Opioid Agonists Have Different Efficacy Profiles for G Protein Activation, Rapid Desensitization, and Endocytosis of Mu-opioid Receptors

Dopamine D1B Receptor Chimeras Reveal Modulation of Partial Agonist Activity by Carboxyl‐Terminal Tail Sequences

Pharmacological Characterization of the D1- and D2-Like Dopamine Receptors from the Brain of the Leopard Frog, <i>Rana pipiens</i>

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