Frederick Ehlert - Profile on Academia.edu (original) (raw)
Papers by Frederick Ehlert
The FASEB Journal, Apr 1, 2015
Comparison of agonist occupancy of M2 muscarinic receptor‐G protein complexes in myocardial homogenates with functional estimates of active receptor‐state affinity in isolated atria
The FASEB Journal
We have recently developed methods for estimating the active (Kact) and inactive (Kinact) recepto... more We have recently developed methods for estimating the active (Kact) and inactive (Kinact) receptor‐state affinity constants of ligands in functional studies on G protein‐coupled receptors. Our approach is valid for in vitro responses measured downstream from receptor activation, such as second messenger signaling in cell lines and responses in isolated tissues. The data required for our analysis include agonist‐mediated responses measured in the absence and presence of reduced receptor expression and modulation with an allosteric agonist (J Pharmacol Toxicol Methods 69:253–279, 2014). We also described an analogous technique that employs a constitutively active receptor mutant (J Pharmacol Toxicol Methods 83:94–106, 2017) instead of an allosteric agonist. In this study, we sought to validate our functional determination of Kact by using an independent biophysical method for estimating Kact. It is well known that muscarinic agonists exhibit high affinity for a subset of M2 muscarinic...
Functional role for the M2 muscarinic receptor in smooth muscle of guinea pig ileum
PubMed, Jul 1, 1993
A functional role for the M2 muscarinic receptor in smooth muscle contraction was investigated in... more A functional role for the M2 muscarinic receptor in smooth muscle contraction was investigated in isolated guinea pig ileum. Contractile responses to the muscarinic agonist oxotremorine-M (oxo-M) were measured in isolated ilea that had been pretreated with histamine (0.32 microM) and isoproterenol (0.64 microM) to achieve conditions of elevated cAMP. The resulting concentration-effect curve was biphasic, consisting of high (0-50 nM) and low (> 50 nM) potency components. The reversible M2-selective antagonist AF-DX 116 ([[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one) (1 and 10 microM) shifted this curve in a manner that was inconsistent with competitive antagonism at a single receptor site; the high affinity component was significantly blocked, whereas there was little effect on the low affinity portion of the curve. To inactivate the M3 muscarinic receptors selectively, ilea were incubated with the irreversible M1/M3-selective muscarinic antagonist 4-DAMP mustard [N-(2-chloroethyl)-4-piperidinyldiphenylacetate] (40 nM) for 1 hr in the presence of AF-DX 116 (1 microM) and were then washed extensively. Under these conditions, the contractile responses to oxo-M, in the presence of histamine and isoproterenol or forskolin, were antagonized by AF-DX 116 (1 microM) in a manner consistent with that mediated by an M2 receptor. AF-DX 116 caused 6.6- and 11-fold increases in the EC50 value for oxo-M for ilea pretreated with isoproterenol and forskolin, respectively, and a significant increase in the Hill coefficient in both cases. Under basal conditions, AF-DX 116 caused only a 1.34-fold increase in the EC50 value and no change in the Hill coefficient. In addition, under basal conditions 4-DAMP mustard treatment shifted the oxo-M contractile response curve to the right approximately 20-fold. However, when histamine was present in combination with isoproterenol or forskolin 4-DAMP mustard treatment shifted the concentration-effect curves for oxo-M to the right only about 3.5-fold. Oxo-M produced an M3-mediated stimulation of phosphoinositide hydrolysis in the longitudinal muscle of rat ileum with an EC50 value of 30 microM. 4-DAMP mustard (10 nM; 1 hr) prevented this response, resulting in a 6.6-fold increase in the EC50 value with a 65% reduction of the maximal response. In contrast, this treatment blocked M2-mediated inhibition of isoproterenol-stimulated adenylate cyclase with only a 2-fold increase in EC50, without affecting maximum inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)
Competitive Interactions between Orthostheric Ligands at Ligand-Gated Ion Channels
Affinity and Efficacy, 2014
Effect of streptozotocin on neurogenic M 2 and M 3 muscarinic receptor‐mediated contractions in mouse urinary bladder
The FASEB Journal, 2010
The M2 muscarinic receptor inhibits the development of streptozotocin-induced neuropathy in mouse... more The M2 muscarinic receptor inhibits the development of streptozotocin-induced neuropathy in mouse urinary bladder
Estimating Affinity and Efficacy by Reverse-Engineering and Response-Clamp Analysis
Affinity and Efficacy, 2014
Complex binding properties of the benzodiazepine receptor
Analysis of Agonism and Inverse Agonism in Signaling Pathways that Exhibit Constitutive Activity
Affinity and Efficacy, 2014
Correlation between pharmacological activity and the influence of GABA on the binding of flunitrazepam and the specific benzodiazepine antagonist, Ro 15-1788
Psychopharmacology Bulletin, 1982
Cardiac calcium antagonist channels labelled by [3H]nitrendepine: Characterization, drug and ionic specificity
Clinical research, 1982
The GABA shift: A predictor of anxiolytic efficacy
Guanine nucleotide and sodium ion regulation of agonist muscarinic cholinergic receptor binding
Cardiac muscarinic receptors: Regulation of the agonist binding using the ligand [3H]-cismethyldioxolane (CD)
Circulation, 1980
Bnezodiazepine Benzodiazepine binding using [3H]propyl β-carboline-3-carboxylate
IUPHAR/BPS Guide to Pharmacology CITE, 2021
Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee... more Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [50]) are activated by the endogenous agonist acetylcholine. All five (M1-M5) mAChRs are ubiquitously expressed in the human body and are therefore attractive targets for many disorders. Functionally, M1, M3, and M5 mAChRs preferentially couple to Gq/11 proteins, whilst M2 and M4 mAChRs predominantly couple to Gi/o proteins. Both agonists and antagonists of mAChRs are clinically approved drugs, including pilocarpine for the treatment of elevated intra-ocular pressure and glaucoma, and atropine for the treatment of bradycardia and poisoning by muscarinic agents such as organophosphates.
IUPHAR/BPS Guide to Pharmacology CITE, 2019
Muscarinic acetylcholine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Musca... more Muscarinic acetylcholine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [45]) are GPCRs of the Class A, rhodopsin-like family where the endogenous agonist is acetylcholine. In addition to the agents listed in the table, AC-42, its structural analogues AC-260584 and 77-LH-28-1, N-desmethylclozapine, TBPB and LuAE51090 have been described as functionally selective agonists of the M1 receptor subtype via binding in a mode distinct from that utilized by non-selective agonists [243, 242, 253, 155, 154, 181, 137, 11, 230]. There are two pharmacologically characterised allosteric sites on muscarinic receptors, one defined by it binding gallamine, strychnine and brucine, and the other defined by the binding of KT 5720, WIN 62,577, WIN 51,708 and staurosporine [161, 162].
Regulation of Muscarinic Receptor Binding
Advances in Behavioral Biology, 1981
The specific binding of [3H] antagonists to muscarinic receptors from neuronal tissue has been de... more The specific binding of [3H] antagonists to muscarinic receptors from neuronal tissue has been demonstrated by several investigators (6,19,24,28,32,37). In each case, the binding of [3H] antagonists is consistent with the law of mass action for a single class of independent receptors. Similarly, the results of antagonist/ [3H] antagonist competition experiments are also consistent with the law of mass action (23). Perhaps the most convincing evidence demonstrating that the binding of [3H] antagonists represents a specific interaction with the muscarinic receptor is the excellent quantitative agreement between the values of dissociation constants determined by binding experiments and by antagonism of agonist induced contractions of the guinea pig ileum (8,10).
British Journal of Pharmacology, 2010
Background and purpose: We investigated how McN-A-343 inhibited the alkylation of the M1 muscarin... more Background and purpose: We investigated how McN-A-343 inhibited the alkylation of the M1 muscarinic receptor by its nitrogen mustard derivative and that of ACh to identify whether it interacts allosterically or orthosterically. Experimental approach: We incubated the M1 muscarinic receptor expressed in Chinese hamster ovary cells with ACh mustard for various periods of time in the presence of McN-A-343 or known allosteric and orthosteric ligands. After stopping the reaction and removing unreacted ligands, unalkylated receptors were measured using [ 3 H]N-methylscopolamine. Analogous experiments were done using a nitrogen mustard analog of McN-A-343. Affinity constants, cooperativity values for allosteric interactions and rate constants for receptor alkylation were estimated using a mathematical model. Key results: The kinetics of receptor alkylation by the nitrogen mustard derivatives of ACh and McN-A-343 were consistent with a two-step model in which the aziridinium ion rapidly forms a reversible receptor complex, which converts to a covalent complex at a slower rate. The inhibition of receptor alkylation by acetycholine, N-methylscopolamine and McN-A-343 was consistent with competitive inhibition, whereas that caused by gallamine was consistent with allosterism. Affinity constants estimated from alkylation kinetics agreed with those measured by displacement of [ 3 H]N-methylscopolamine binding. Conclusions and implications: Our results suggest that McN-A-343 and its nitrogen mustard derivative interact competitively with ACh and N-methylscopolamine at the orthosteric site on the M1 muscarinic receptor. Measuring how drugs modulate the kinetics of receptor alkylation by an irreversible ligand is a powerful approach for distinguishing between negative allosteric modulators and competitive inhibitors.
Conversion of N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) to an aziridinium ion and its interaction with muscarinic receptors in various tissues
Molecular pharmacology, 1992
A 2-chloroethylamine derivative [N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard)... more A 2-chloroethylamine derivative [N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard)] of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) was synthesized, and its conversion to an aziridinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4 and 37 degrees, 4-DAMP mustard released an equivalent amount of chloride. The release of chloride was consistent with a first-order process having a half-time of 5.7 min. The aziridinium ion reached a peak concentration at 32 min, corresponding to 75% of the initial concentration of 4-DAMP mustard. When homogenates of rat brain, heart, and submaxillary gland were incubated with 4-DAMP mustard (9 nM) for 1 hr, washed extensively, and then assayed for muscarinic receptor binding properties, a 56% decrease in the binding capacity of N-[3H]methylscopolamine in the heart and brain and a 71% decrease in the gland were observed, without a sign...
The FASEB Journal, Apr 1, 2015
Comparison of agonist occupancy of M2 muscarinic receptor‐G protein complexes in myocardial homogenates with functional estimates of active receptor‐state affinity in isolated atria
The FASEB Journal
We have recently developed methods for estimating the active (Kact) and inactive (Kinact) recepto... more We have recently developed methods for estimating the active (Kact) and inactive (Kinact) receptor‐state affinity constants of ligands in functional studies on G protein‐coupled receptors. Our approach is valid for in vitro responses measured downstream from receptor activation, such as second messenger signaling in cell lines and responses in isolated tissues. The data required for our analysis include agonist‐mediated responses measured in the absence and presence of reduced receptor expression and modulation with an allosteric agonist (J Pharmacol Toxicol Methods 69:253–279, 2014). We also described an analogous technique that employs a constitutively active receptor mutant (J Pharmacol Toxicol Methods 83:94–106, 2017) instead of an allosteric agonist. In this study, we sought to validate our functional determination of Kact by using an independent biophysical method for estimating Kact. It is well known that muscarinic agonists exhibit high affinity for a subset of M2 muscarinic...
Functional role for the M2 muscarinic receptor in smooth muscle of guinea pig ileum
PubMed, Jul 1, 1993
A functional role for the M2 muscarinic receptor in smooth muscle contraction was investigated in... more A functional role for the M2 muscarinic receptor in smooth muscle contraction was investigated in isolated guinea pig ileum. Contractile responses to the muscarinic agonist oxotremorine-M (oxo-M) were measured in isolated ilea that had been pretreated with histamine (0.32 microM) and isoproterenol (0.64 microM) to achieve conditions of elevated cAMP. The resulting concentration-effect curve was biphasic, consisting of high (0-50 nM) and low (> 50 nM) potency components. The reversible M2-selective antagonist AF-DX 116 ([[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one) (1 and 10 microM) shifted this curve in a manner that was inconsistent with competitive antagonism at a single receptor site; the high affinity component was significantly blocked, whereas there was little effect on the low affinity portion of the curve. To inactivate the M3 muscarinic receptors selectively, ilea were incubated with the irreversible M1/M3-selective muscarinic antagonist 4-DAMP mustard [N-(2-chloroethyl)-4-piperidinyldiphenylacetate] (40 nM) for 1 hr in the presence of AF-DX 116 (1 microM) and were then washed extensively. Under these conditions, the contractile responses to oxo-M, in the presence of histamine and isoproterenol or forskolin, were antagonized by AF-DX 116 (1 microM) in a manner consistent with that mediated by an M2 receptor. AF-DX 116 caused 6.6- and 11-fold increases in the EC50 value for oxo-M for ilea pretreated with isoproterenol and forskolin, respectively, and a significant increase in the Hill coefficient in both cases. Under basal conditions, AF-DX 116 caused only a 1.34-fold increase in the EC50 value and no change in the Hill coefficient. In addition, under basal conditions 4-DAMP mustard treatment shifted the oxo-M contractile response curve to the right approximately 20-fold. However, when histamine was present in combination with isoproterenol or forskolin 4-DAMP mustard treatment shifted the concentration-effect curves for oxo-M to the right only about 3.5-fold. Oxo-M produced an M3-mediated stimulation of phosphoinositide hydrolysis in the longitudinal muscle of rat ileum with an EC50 value of 30 microM. 4-DAMP mustard (10 nM; 1 hr) prevented this response, resulting in a 6.6-fold increase in the EC50 value with a 65% reduction of the maximal response. In contrast, this treatment blocked M2-mediated inhibition of isoproterenol-stimulated adenylate cyclase with only a 2-fold increase in EC50, without affecting maximum inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)
Competitive Interactions between Orthostheric Ligands at Ligand-Gated Ion Channels
Affinity and Efficacy, 2014
Effect of streptozotocin on neurogenic M 2 and M 3 muscarinic receptor‐mediated contractions in mouse urinary bladder
The FASEB Journal, 2010
The M2 muscarinic receptor inhibits the development of streptozotocin-induced neuropathy in mouse... more The M2 muscarinic receptor inhibits the development of streptozotocin-induced neuropathy in mouse urinary bladder
Estimating Affinity and Efficacy by Reverse-Engineering and Response-Clamp Analysis
Affinity and Efficacy, 2014
Complex binding properties of the benzodiazepine receptor
Analysis of Agonism and Inverse Agonism in Signaling Pathways that Exhibit Constitutive Activity
Affinity and Efficacy, 2014
Correlation between pharmacological activity and the influence of GABA on the binding of flunitrazepam and the specific benzodiazepine antagonist, Ro 15-1788
Psychopharmacology Bulletin, 1982
Cardiac calcium antagonist channels labelled by [3H]nitrendepine: Characterization, drug and ionic specificity
Clinical research, 1982
The GABA shift: A predictor of anxiolytic efficacy
Guanine nucleotide and sodium ion regulation of agonist muscarinic cholinergic receptor binding
Cardiac muscarinic receptors: Regulation of the agonist binding using the ligand [3H]-cismethyldioxolane (CD)
Circulation, 1980
Bnezodiazepine Benzodiazepine binding using [3H]propyl β-carboline-3-carboxylate
IUPHAR/BPS Guide to Pharmacology CITE, 2021
Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee... more Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [50]) are activated by the endogenous agonist acetylcholine. All five (M1-M5) mAChRs are ubiquitously expressed in the human body and are therefore attractive targets for many disorders. Functionally, M1, M3, and M5 mAChRs preferentially couple to Gq/11 proteins, whilst M2 and M4 mAChRs predominantly couple to Gi/o proteins. Both agonists and antagonists of mAChRs are clinically approved drugs, including pilocarpine for the treatment of elevated intra-ocular pressure and glaucoma, and atropine for the treatment of bradycardia and poisoning by muscarinic agents such as organophosphates.
IUPHAR/BPS Guide to Pharmacology CITE, 2019
Muscarinic acetylcholine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Musca... more Muscarinic acetylcholine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [45]) are GPCRs of the Class A, rhodopsin-like family where the endogenous agonist is acetylcholine. In addition to the agents listed in the table, AC-42, its structural analogues AC-260584 and 77-LH-28-1, N-desmethylclozapine, TBPB and LuAE51090 have been described as functionally selective agonists of the M1 receptor subtype via binding in a mode distinct from that utilized by non-selective agonists [243, 242, 253, 155, 154, 181, 137, 11, 230]. There are two pharmacologically characterised allosteric sites on muscarinic receptors, one defined by it binding gallamine, strychnine and brucine, and the other defined by the binding of KT 5720, WIN 62,577, WIN 51,708 and staurosporine [161, 162].
Regulation of Muscarinic Receptor Binding
Advances in Behavioral Biology, 1981
The specific binding of [3H] antagonists to muscarinic receptors from neuronal tissue has been de... more The specific binding of [3H] antagonists to muscarinic receptors from neuronal tissue has been demonstrated by several investigators (6,19,24,28,32,37). In each case, the binding of [3H] antagonists is consistent with the law of mass action for a single class of independent receptors. Similarly, the results of antagonist/ [3H] antagonist competition experiments are also consistent with the law of mass action (23). Perhaps the most convincing evidence demonstrating that the binding of [3H] antagonists represents a specific interaction with the muscarinic receptor is the excellent quantitative agreement between the values of dissociation constants determined by binding experiments and by antagonism of agonist induced contractions of the guinea pig ileum (8,10).
British Journal of Pharmacology, 2010
Background and purpose: We investigated how McN-A-343 inhibited the alkylation of the M1 muscarin... more Background and purpose: We investigated how McN-A-343 inhibited the alkylation of the M1 muscarinic receptor by its nitrogen mustard derivative and that of ACh to identify whether it interacts allosterically or orthosterically. Experimental approach: We incubated the M1 muscarinic receptor expressed in Chinese hamster ovary cells with ACh mustard for various periods of time in the presence of McN-A-343 or known allosteric and orthosteric ligands. After stopping the reaction and removing unreacted ligands, unalkylated receptors were measured using [ 3 H]N-methylscopolamine. Analogous experiments were done using a nitrogen mustard analog of McN-A-343. Affinity constants, cooperativity values for allosteric interactions and rate constants for receptor alkylation were estimated using a mathematical model. Key results: The kinetics of receptor alkylation by the nitrogen mustard derivatives of ACh and McN-A-343 were consistent with a two-step model in which the aziridinium ion rapidly forms a reversible receptor complex, which converts to a covalent complex at a slower rate. The inhibition of receptor alkylation by acetycholine, N-methylscopolamine and McN-A-343 was consistent with competitive inhibition, whereas that caused by gallamine was consistent with allosterism. Affinity constants estimated from alkylation kinetics agreed with those measured by displacement of [ 3 H]N-methylscopolamine binding. Conclusions and implications: Our results suggest that McN-A-343 and its nitrogen mustard derivative interact competitively with ACh and N-methylscopolamine at the orthosteric site on the M1 muscarinic receptor. Measuring how drugs modulate the kinetics of receptor alkylation by an irreversible ligand is a powerful approach for distinguishing between negative allosteric modulators and competitive inhibitors.
Conversion of N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) to an aziridinium ion and its interaction with muscarinic receptors in various tissues
Molecular pharmacology, 1992
A 2-chloroethylamine derivative [N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard)... more A 2-chloroethylamine derivative [N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard)] of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) was synthesized, and its conversion to an aziridinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4 and 37 degrees, 4-DAMP mustard released an equivalent amount of chloride. The release of chloride was consistent with a first-order process having a half-time of 5.7 min. The aziridinium ion reached a peak concentration at 32 min, corresponding to 75% of the initial concentration of 4-DAMP mustard. When homogenates of rat brain, heart, and submaxillary gland were incubated with 4-DAMP mustard (9 nM) for 1 hr, washed extensively, and then assayed for muscarinic receptor binding properties, a 56% decrease in the binding capacity of N-[3H]methylscopolamine in the heart and brain and a 71% decrease in the gland were observed, without a sign...