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Biomedical subjects

F J Ehlert

Publications and source records attributed to F J Ehlert.

At least 19 recordsLinked to original sources
PubMed1992

Heparin, dextran and trypan blue allosterically modulate M2 muscarinic receptor binding properties and interfere with receptor-mediated inhibition of adenylate cyclase.

The influences of heparin, dextran and trypan blue on muscarinic receptor binding properties and inhibition of adenylate cyclase were investigated in homogenates of the rat heart. These compounds caused a concentration-dependent enhancement in the specific binding of the muscarinic antagonist [3H]N-methylscopolamine ([3H]NMS) when measured at a radioligand concentration of approximately 0.5 nM in magnesium-containing, low ionic strength buffer. The maximal enhancements of [3H]NMS binding were 2.89-, 1.68- and 1.43-fold increases for heparin, dextran and trypan blue, respectively; the EC50 values for this effect were 0.12, 0.033 and 4.6 microM, respectively. The effects of heparin, dextran and trypan blue on [3H]NMS binding were attributed mainly to an increase in the overall affinity of muscarinic receptors for [3H]NMS, and were greatly attenuated by 100 mM NaCl. These effects were qualitatively similar to those produced by GTP. Heparin, dextran and trypan blue also affected the binding of the muscarinic agonist oxotremorine-M in a manner similar to that of GTP; that is, in the presence of these compounds, agonist affinity was decreased. Our experiments also showed that heparin and dextran attenuate the inhibition of adenylate cyclase activity caused by oxotremorine-M in myocardial homogenates without influencing basal adenylate cyclase activity. We conclude that heparin and dextran interfere with the muscarinic receptor-G protein coupling in the rat heart.

Adenylyl Cyclase Inhibitors↗
PubMed1992

Specific inhibition of isoproterenol-stimulated cyclic AMP accumulation by M2 muscarinic receptors in rat intestinal smooth muscle.

The ability of oxotremorine-M to inhibit cyclic AMP accumulation in the presence of a variety of adenylate cyclase activators was studied in slices from the longitudinal muscle of the rat ileum. Oxotremorine-M was found to inhibit forskolin- and isoproterenol-stimulated cyclic AMP accumulation maximally by 17 and 32%, respectively, but not the stimulation due to other activators of adenylate cyclase. Inhibition of cyclic AMP accumulation by oxotremorine-M was unaffected by tetrodotoxin and was completely reversed by atropine. AF-DX 116 (11[[2-[(diethylamino)methyl]-1- piperidynyl]acetyl]-5,11-dihydro-6H-pyrido[2,3- b][1,4]benzodiazepine-6-one) an M2-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant (KB) of 0.20 microM, consistent with the dissociation constants for binding at the M2 muscarinic receptor site (KD = 0.092 microM) and inhibition of adenylate cyclase activity (KB = 0.13 microM). Hexahydrosiladifenidol, an M3-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant of 0.67 microM, again consistent with the dissociation constant for binding at the M2 site (KD = 0.83 microM). The agreement between the estimates of the dissociation constants of muscarinic antagonists for binding and for inhibition of cyclic AMP accumulation suggest that oxotremorine-M inhibition of isoproterenol-stimulated cyclic AMP accumulation in slices of rat intestinal smooth muscle is mediated by the M2 receptor.

Adenylyl Cyclases↗
PubMed1992

Conversion of N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) to an aziridinium ion and its interaction with muscarinic receptors in various tissues.

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 significant change in the dissociation constants. The affinity of 4-DAMP mustard and its transformation products for muscarinic receptors was determined in competitive binding experiments with N-[3H] methylscopolamine, and the results show that the aziridinium ion of 4-DAMP mustard was the most potent form, compared with the parent 2-chloroethylamine (4-DAMP mustard) and the alcoholic hydrolysis product. The rates of receptor alkylation by 4-DAMP mustard were measured in the rat heart and gland. Virtually no alkylation (less than 1%) occurred in the heart at a 4-DAMP mustard concentration of 1.6 nM, after 30 min, whereas almost 50% alkylation was observed in the gland under the same conditions. Almost complete alkylation of receptors in the gland could be achieved at a 4-DAMP mustard concentration of 200 nM, after 1 hr. Treatment of the isolated rat ileum with 4-DAMP mustard caused an irreversible blockade of contractions elicited by the muscarinic agonist oxotremorine-M, and this blockade persisted after extensive washing. The results presented here show that 4-DAMP mustard forms an aziridinium ion that binds irreversibly to muscarinic receptors and exhibits selectivity for M3, compared with M2 muscarinic receptors.

Alkylation↗
PubMed1991

Heterogeneity of muscarinic receptor subtypes in cerebral blood vessels.

The identity and distribution of muscarinic cholinergic receptor subtypes and associated signal transduction mechanisms was characterized for the cerebral circulation using correlated functional and biochemical investigations. Subtypes were distinguished by the relative affinities of a panel of muscarinic antagonists, pirenzepine, AF-DX 116 [11-2-[[2-[diethylaminomethyl]- 1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one], hexahydrosiladifenidol, methoctramine, 4-diphenylacetoxy-N-methylpiperidine methobromide, dicyclomine, para-fluoro-hexahydrosiladifenidol and atropine. Muscarinic receptors characterized by inhibition of [3H]quinuclidinylbenzilate binding in membranes of bovine pial arteries were of the M2 subtype. In contrast pharmacological analysis of [3H]-quinuclidinylbenzilate binding in bovine intracerebral microvessels suggests the presence of an M4 subtype. Receptors mediating endothelium-dependent vasodilation in rabbit pial arteries were of the M3 subtype, whereas muscarinic receptors stimulating endothelium-independent phosphoinositide hydrolysis in bovine pial arteries were of the M1 subtype. These findings suggest that characteristics of muscarinic receptors in cerebral blood vessels vary depending on the type of vessel, cellular location and function mediated.

Animals↗
PubMed1991

N-[3H]methylscopolamine labeling of non-M1, non-M2 muscarinic receptor binding sites in rat brain.

Radioligand binding and quantitative autoradiographic techniques were used to characterize the pharmacological profile and anatomical distribution of N-[3H]methylscopolamine [( 3H] NMS)-labeled sites under assay conditions in which radioligand binding to M1 and M2 muscarinic receptors was blocked by addition of pirenzepine and AF-DX 116 (11[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b] [1,4]benzodiazepine-6-one) to the incubation buffer. Nonlinear regression analysis of saturation data demonstrated that a large proportion of atropine-displaceable [3H]NMS binding persisted in the presence of saturating concentrations of M1 and M2 blockers. The residual [3H]NMS [( 3H]NMSb) sites were widely distributed throughout rat brain and represented the predominant muscarinic receptor population. The autoradiographic distribution of [3H]NMSb sites did not correspond to that of [3H]pirenzepine or [3H]AF-DX 116, indicating that [3H]NMSb labeled non-M1, non-M2 muscarinic sites. Moreover, the pharmacological profile of [3H]NMSb differed from that of [3H]pirenzepine at M1 sites, and was inconsistent with that of M2 receptor binding sites. Although we were unable to pharmacologically distinguish subpopulations of non-M1, non-M2 binding sites, the anatomical distribution of [3H]NMSb sites corresponded to that of the combined mRNA distributions for m3 and m4.

Animals↗
PubMed1990

Muscarinic M1 receptors stimulate phosphoinositide hydrolysis in bovine cerebral arteries.

The muscarinic agonist oxotremorine-M produced a concentration-dependent increase in phosphoinositide hydrolysis in bovine pial arteries. The maximal effect was 5.9 +/- 0.89 fold over basal levels, and the EC50 for oxotremorine-M was 8.9 x 10(-6) M. The phosphoinositide response in arteries with the luminal endothelium removed was similar to the response in intact arteries. The specific muscarinic antagonists pirenzepine, 4-DAMP and methoctramine produced parallel shifts of the concentration-response curve to oxotremorine-M, with the following order of potency (pKB): 4-DAMP (8.59 +/- 0.10) greater than pirenzepine (8.12 +/- 0.11) greater than methoctramine (6.77 +/- 0.20). These results indicate that muscarinic stimulation activates phosphoinositide hydrolysis in cerebral arteries, and that the muscarinic receptors mediating this increase are similar to the M1 subtype.

Animals↗
PubMed1990

Tertiary 2-haloethylamine derivatives of the muscarinic agent McN-A-343, [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride.

4-[(2-Chloroethyl)methylamino]-2-butynyl N-(3-chlorophenyl)carbamate (2) and 4-[(2-bromoethyl)methylamino]-2-butynyl N-(3-chlorophenyl)carbamate (3) were synthesized. Compounds 2 and 3 cyclized at neutral pH to an aziridinium ion (4). The rate constants for the cyclization of 2 and 3 at 37 degrees C were about 0.01 and 0.4 min-1, respectively, as measured by titrimetric analysis and by 1H NMR spectroscopy. The aziridinium ion had 1/4 the potency of McN-A-343 (1) as a ganglionic muscarinic stimulant in the anesthetized, pentolinium-treated rat but showed no muscarinic effects on the isolated guinea pig ileum. It caused alkylation of muscarinic receptors in homogenates of the rat cerebral cortex. An irreversible blockade of central muscarinic receptors was also observed after intravenous administration of 3 to mice. Because of its selectivity, irreversible actions, and ability to pass into the central nervous system, 3 should become a valuable tool in studies of muscarinic receptors.

Alkylation↗
PubMed1990

Differential coupling of subtypes of the muscarinic receptor to adenylate cyclase and phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum.

The binding affinities of selective muscarinic antagonists were compared with their ability to block receptor-mediated inhibition of adenylate cyclase and stimulation of phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum. When measured by competitive inhibition of the binding of N-[3H]methylscopolamine, the binding properties of selective muscarinic antagonists were consistent with a two-site model. Approximately 84% of the binding sites (major sites) had high affinity for the M2-selective antagonists methoctramine and AF-DX 116 (11[[2-[(diethylamino)methyl]-1- piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one), whereas the remainder of the sites (minor sites) had high affinity for hexahydrosiladifenidol and its para-fluoro derivative. There was good agreement between the estimates of the dissociation constants of muscarinic antagonists for the major binding site and those measured by antagonism of the adenylate cyclase response. There was also good agreement between the dissociation constants of muscarinic antagonists for the minor binding site and those measured by antagonism of the phosphoinositide response and the contractile response. Our data indicate that there are at least two types of muscarinic receptors in the longitudinal muscle of the ileum, the more abundant being an M2 receptor, which mediates an inhibition of adenylate cyclase activity, and the less abundant being an M3 receptor, which triggers contraction and phosphoinositide hydrolysis.

Adenylyl Cyclases↗
PubMed1990

Regional distribution of M1, M2 and non-M1, non-M2 subtypes of muscarinic binding sites in rat brain.

The distribution of subtypes of the muscarinic receptor in homogenates of the rat brain was investigated by measuring the competitive inhibition of the binding [3H]N-methylscopolamine by pirenzepine and AF-DX 116 (11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one). In most brain regions, the competitive binding curves for AF-DX 116 and pirenzepine were consistent with a two-site model. The dissociation constant of pirenzepine for its high-affinity site (M1 receptor) was approximately 10(-8) M, whereas the dissociation constant of AF-DX 116 for its high affinity site (M2 receptor) was approximately 10(-7) M. In many regions, particularly those in the forebrain, the sum of the densities of the M1 and M2 binding sites was substantially less than 100% of the total sites, indicating the existence of a third population of sites lacking high affinity for both pirenzepine and AF-DX 116. We have designated these latter sites as non-M1, non-M2 muscarinic receptors. In general, the densities of the M1 and non-M1, non-M2 binding sites were highest in cerebral cortex, corpus striatum and hippocampus, intermediate in thalamus and hypothalamus, and lowest in midbrain, medulla-pons and cerebellum, whereas the M2 binding site had a relatively low, uniform density throughout the brain. The binding capacity of [3H]N-methylquinuclidinyl benzilate was estimated to be 20 to 30% lower than that of [3H]quinuclidinyl benzilate in various regions of the forebrain, but not in more caudal regions of the brain where the two radioligands had approximately the same binding capacities. Treatment of homogenates of the cerebral cortex with benzilylcholine mustard caused a selective loss of the majority of the [3H]N-methylscopolamine binding sites but spared 25% of the sites labeled by [3H]quinuclidinyl benzilate The results of pirenzepine/[3H]quinuclinyl benzilate competitive binding experiments on cerebral cortex treated with benzilylcholine mustard showed that the residual binding sites for [3H] quinuclidinyl benzilate were enriched in M1 muscarinic receptors.

Animals↗
PubMed1990

The interaction of amitriptyline, doxepin, imipramine and their N-methyl quaternary ammonium derivatives with subtypes of muscarinic receptors in brain and heart.

The interaction of amitriptyline, doxepin, imipramine and their N-methyl quaternary derivatives with muscarinic receptors was investigated in the brain and heart. The potency of the tricyclic derivatives for inhibiting the binding of 11[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b] [1,4] benzodiazepine-6-one to M2 muscarinic receptors in cerebral cortex was similar to that measured in competitive binding experiments with the nonselective muscarinic antagonist [3H]N-methylscopolamine in the corpus striatum and heart. Moreover, the tricyclic derivatives antagonized muscarinic receptor-mediated inhibition of adenylate cyclase activity with similar potency in the corpus striatum and heart, and there was good agreement between the affinities of the tricyclic derivatives when measured by radioligand binding and by antagonism of the adenylate cyclase response. Our results show that amitriptyline, doxepin and imipramine lack selectivity for subtypes of the muscarinic receptor.

Adenylyl Cyclase Inhibitors↗
PubMed1990

Influence of pH on the binding of scopolamine and N-methylscopolamine to muscarinic receptors in the corpus striatum and heart of rats.

The influence of pH on the binding of scopolamine and [3H]N-methylscopolamine to muscarinic receptors in the heart and corpus striatum was investigated. The specific binding of [3H]N-methylscopolamine in the heart and corpus striatum was relatively insensitive to pH over the range of 6 through 10 but decreased markedly below pH 6.0. This reduction in binding was attributed to a reversible decrease in the observed affinity without an effect on the binding capacity. The data are consistent with the postulate that [3H]N-methylscopolamine competes with hydrogen ions for an acidic group on the muscarinic receptor that has a pKA of approximately 5.5 in both the heart and corpus striatum. When measured by competitive inhibition of the binding of [3H]N-methylscopolamine, the affinity of scopolamine decreased relative to that of [3H]N-methylscopolamine as the pH increased from 6 to 10, confirming that it is primarily the protonated form of scopolamine that binds with muscarinic receptors.

Animals↗
PubMed1990

Signaling through the muscarinic receptor-adenylate cyclase system of the heart is buffered against GTP over a range of concentrations.

The influence of GTP on muscarinic receptor occupancy and inhibition of adenylate cyclase activity was investigated in well washed homogenates of the rat myocardium. In these homogenates, the highly efficacious muscarinic agonist oxotremorine-M was without effect on adenylate cyclase activity in the absence of exogenous GTP but caused a maximal 38% inhibition of the enzyme in the presence of 0.1 microM GTP. Increasing the concentration of GTP to 0.1 mM caused small to moderate increases in the maximal inhibition of adenylate cyclase elicited by oxotremorine-M and in the concentration of this agonist required for half-maximal inhibition of the enzyme. In contrast, the same change in the concentration of GTP (0.1 microM to 0.1 mM) caused a relatively large increase (46-fold) in the concentration of oxotremorine-M necessary for half-maximal receptor occupancy. Similar observations were made for the highly efficacious muscarinic agonist carbachol. Our results show that GTP increases receptor coupling efficiency and decreases agonist affinity and that these two effects oppose one another, so that the level of muscarinic agonist-mediated inhibition of adenylate cyclase activity remains relatively constant over a range of concentrations of GTP. We have also used a model to predict the influence of GTP on receptor binding properties and agonist-mediated inhibition of adenylate cyclase activity and have calculated theoretical results generally consistent with the experimental observations.

Adenylyl Cyclase Inhibitors↗
PubMed1989

Coupling of subtypes of the muscarinic receptor to adenylate cyclase in the corpus striatum and heart.

The binding properties of a series of muscarinic antagonists were compared with their ability to antagonize muscarinic receptor mediated inhibition of adenylate cyclase activity in homogenates of the corpus striatum and heart of rats. When measured by the competitive inhibition of the binding of the muscarinic antagonist N-[3H]methylscopolamine, the binding properties of selective muscarinic antagonists in the corpus stratum and cerebral cortex were consistent with a model incorporating a minimum of three populations of muscarinic receptors, a high affinity site for pirenzepine (M1), a high affinity site for AF-DX 116 [11] [2-[ (diethylamino)methyl]-1-piperidinyl] acetyl] -5, 11-dihydro-6H-pyrido [2,3-b] 1,4] benzodiazepine-6-one (M2) and a third population (non-Ml, non-M2 sites) displaying low affinity for the latter antagonists. The results of similar experiments on the heart showed that this tissue contained a uniform population of M2 muscarinic receptors. The binding properties of the M2 receptor in cerebral cortex and corpus stratum were also investigated directly in antagonist [3H] AF-DX 116 competition experiments and, although the high affinity AF-DX 116 site in brain (M2) exhibited selectivity for the cardioselective antagonists AF-DX 116 and gallamine, some differences were noted between M2 sites in brain and heart. The muscarinic adenylate cyclase response in the corpus striatum was relatively insensitive to the M2 selective antagonists AF-DX 116 and gallamine as well as the M1 selective antagonist pirenzepine, suggesting that non-M1, non-M2 sites inhibit adenylate cyclase activity in the corpus striatum. In contrast, the effects of muscarinic antagonists on the muscarinic adenylate cyclase response in the heart were consistent with the postulate that M2 receptors inhibit adenylate cyclase activity in this tissue.

Adenylyl Cyclase Inhibitors↗