piperidines and himbacine

Topics: Alkaloids; Animals; Anticoagulants; Atherosclerosis; Coronary Restenosis; Fibrinolytic Agents; Furans; Humans; Naphthalenes; Peptides; Piperidines; Platelet Aggregation Inhibitors; Receptor, PAR-1

Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling pathways. One is sensitive to pertussis toxin and acts rapidly by a membrane-delimited pathway on the channels. The other is not sensitive to pertussis toxin and acts more slowly through an unknown second messenger. These pathways are shared with several other agonists.

Topics: Alkaloids; Animals; Calcium Channels; Furans; GTP-Binding Proteins; Muscarinic Agonists; Muscarinic Antagonists; Naphthalenes; Neurons; Oxotremorine; Patch-Clamp Techniques; Pertussis Toxin; Piperidines; Pirenzepine; Rabbits; Rats; Receptors, Muscarinic; Superior Cervical Ganglion; Virulence Factors, Bordetella

Albeit time-consuming, the most rigorous method of classification of receptor subtypes is the determination of pA2 values. However, this methodology requires the knowledge of absolute concentrations of both agonists and antagonists--something that may not necessarily be calculable under certain experimental conditions. Wolfgang Kromer offers a different method for classification using antagonists which is not dependent on absolute affinities, and which has the additional advantage of speed.

Topics: Alkaloids; Binding, Competitive; Furans; Muscarinic Antagonists; Naphthalenes; Piperidines; Pirenzepine; Receptors, Muscarinic

The structures of five new natural products (GB 27-GB 31,

Topics: Alkaloids; Furans; Magnoliopsida; Molecular Structure; Naphthalenes; Papua New Guinea; Phytochemicals; Piperidines; Rainforest; Trees

The enantiomers of (4R/S)-4-hydroxy-N, N-diphenyl-2-pentynamide are key chiral synthons for the synthesis of thrombin receptor antagonists such as vorapaxar. In this paper, we report the enzymatic preparation of enantiomerically enriched (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide using lipase A from Burkholderia cepacia ATCC 25416 as the catalyst. First, the lipase gene (lipA) and its chaperone gene (lipB) was cloned and expressed in Escherichia coli system. After purification, lipase A activation was performed with the assistance of foldase lipase B. Enzyme assay revealed that the activated lipase A showed the optimal catalytic activity at 60 ºC and pH 7. The effects of various metals on the activity were investigated and results demonstrated that most of the metals inhibited the activity. To further improve the catalytic outcome, two-phase reaction was studied, and n-hexane proved to be a good organic solvent for the combination system. Using the optimize conditions, (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide with 94.5% ee value and 48.93% conversion ratio was achieved. Our investigation on this lipase reveals lipase A as a promising biocatalyst for producing chiral propargyl alcohol for preparation of novel himbacine analogs.

Topics: Alkaloids; Burkholderia cepacia; Catalysis; Escherichia coli; Furans; Gene Expression; Naphthalenes; Piperidines; Stereoisomerism; Sterol Esterase

One-pot sequential reactions using the acyl moieties installed by enzymatic dynamic kinetic resolution of alcohols have been little investigated. In this work, the acryloyl moiety installed via the lipase/oxovanadium combo-catalyzed dynamic kinetic resolution of a racemic dienol [4-(cyclohex-1-en-1-yl)but-3-en-2-ol or 1-(cyclohex-1-en-1-yl)but-2-en-1-ol] with a (Z)-3-(phenylsulfonyl)acrylate underwent an intramolecular Diels-Alder reaction in a one-pot procedure to produce an optically active naphtho[2,3-c]furan-1(3H)-one derivative (98% ee). This method was successfully applied to the asymmetric total synthesis of (-)-himbacine.

Topics: Alkaloids; Biocatalysis; Cycloaddition Reaction; Furans; Kinetics; Lipase; Molecular Structure; Naphthalenes; Piperidines; Stereoisomerism

Myopia is a huge public health problem worldwide, reaching the highest incidence in Asia. Identification of susceptible genes is crucial for understanding the biological basis of myopia. In this paper, we have identified and characterized a functional myopia-associated gene using a specific mouse-knockout model. Mice lacking the muscarinic cholinergic receptor gene (M2; also known as Chrm2) were less susceptible to lens-induced myopia compared with wild-type mice, which showed significantly increased axial length and vitreous chamber depth when undergoing experimental induction of myopia. The key findings of this present study are that the sclera of M2 mutant mice has higher expression of collagen type I and lower expression of collagen type V than do wild-type mice and mice that are mutant for other muscarinic subtypes, and, therefore, M2 mutant mice were resistant to the development of experimental myopia. Pharmacological blockade of M2 muscarinic receptor proteins retarded myopia progression in the mouse. These results suggest for the first time a role of M2 in growth-related changes in extracellular matrix genes during myopia development in a mammalian model. M2 receptor antagonists might thus provide a targeted therapeutic approach to the management of this refractive error.

Topics: Adult; Aged; Alkaloids; Animals; Cell Proliferation; Collagen; Disease Progression; Extracellular Matrix; Fibroblasts; Furans; Gene Knockdown Techniques; Humans; Mice; Mice, Knockout; Middle Aged; Myopia; Naphthalenes; Piperidines; Pirenzepine; Receptor, Muscarinic M2; RNA, Small Interfering; Sclera; Up-Regulation

Discovery of a novel nor-seco himbacine analog as potent thrombin receptor (PAR-1) antagonist is described. Despite low plasma level, these new analogs showed excellent ex vivo efficacy in the monkey platelet aggregation assay. A potent hydroxy metabolite generated in vivo was identified as the agent responsible for the ex vivo efficacy. Following this discovery, the metabolite series was optimized to obtain analogs that showed very good ex vivo efficacy along with excellent pharmacokinetic profile in c. monkey.

Topics: Administration, Oral; Alkaloids; Animals; Biological Availability; Blood Platelets; Drug Discovery; Furans; Humans; Macaca fascicularis; Naphthalenes; Piperidines; Platelet Aggregation; Platelet Aggregation Inhibitors; Protein Binding; Rats; Receptor, PAR-1; Structure-Activity Relationship; Thrombin

Cholinergic modulation of spontaneous GABAergic currents (mIPSC) was studied using whole-cell patch methods in mouse accessory olfactory bulb slices. Carbachol (above 100 microM) administration produced an increase in the mIPSC frequency in mitral cells, but did not affect the responses of mitral cells to GABA. The carbachol effect persisted in the presence of combined ionotropic and metabotropic glutamatergic receptor antagonists. The carbachol effect was reduced by the muscarinic receptor type-1 and -4 (M1 and M4) antagonist pirenzepine (10 microM), but not by the M2 and M4 antagonist himbacine (10 microM). The KCNQ/Kv7 potassium channel openers retigabine (80 microM) and diclofenac (300 microM) blocked the carbachol action, while the KCNQ potassium channel blocker XE-911 (20 microM) increased the mIPSC frequency. XE-911's action persisted in the presence of glutamate receptor blockers. In the presence of carbachol, mIPSCs were abolished by Ni (200 microM), while being insensitive to the calcium channel blocker nimodipine (30 microM), suggesting a role for R-type calcium channels in the GABA release. These results suggest that carbachol closed KCNQ channels by stimulating M1 receptors on granule cell dendrites, and the resulting depolarized and unstable membrane promoted calcium influx, thus increasing the GABA release. The possible role of acetylcholine in facilitating formation of a pheromone memory in mice is also discussed.

Topics: Alkaloids; Animals; Anticonvulsants; Calcium; Calcium Channel Blockers; Calcium Channels, R-Type; Carbachol; Carbamates; Cholinergic Agonists; Dendrites; Diclofenac; Excitatory Amino Acid Antagonists; Furans; GABA Antagonists; gamma-Aminobutyric Acid; In Vitro Techniques; Ion Channel Gating; KCNQ Potassium Channels; Mice; Mice, Inbred BALB C; Muscarinic Antagonists; Naphthalenes; Nimodipine; Olfactory Bulb; Patch-Clamp Techniques; Phenylenediamines; Piperidines; Pirenzepine; Potassium Channel Blockers; Receptor, Muscarinic M1; Receptors, Glutamate; Synapses

Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M(2), M(3), and M(4)) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M(3)-single KO and M(1)/M(3) double-KO mice. In addition, the M(3) antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M(5)-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M(2)/M(4) double-KO mice, but not M(2)- or M(4)-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M(2)/M(4) antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M(2) and M(4) receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M(5) is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord.

Topics: Alkaloids; Alkenes; Animals; Furans; Glutamic Acid; Mice; Mice, Knockout; Muscarinic Agonists; Naphthalenes; Oxotremorine; Parasympatholytics; Piperidines; Posterior Horn Cells; Receptors, Muscarinic; Spinal Cord; Synapses; Synaptic Transmission

Hawthorn (Crataegus spp.) plant extract is used as a herbal alternative medicine for the prevention and treatment of various cardiovascular diseases. Recently, it was shown that hawthorn extract preparations caused negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay, independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the effect of hawthorn extract to decrease the contraction rate of cultured cardiomyocytes. To test the hypothesis that hawthorn is acting via muscarinic receptors, the effect of hawthorn extract on atrial versus ventricular cardiomyocytes in culture was evaluated. As would be expected for activation of muscarinic receptors, hawthorn extract had a greater effect in atrial cells. Atrial and/or ventricular cardiomyocytes were then treated with hawthorn extract in the presence of atropine or himbacine. Changes in the contraction rate of cultured cardiomyocytes revealed that both muscarinic antagonists significantly attenuated the negative chronotropic activity of hawthorn extract. Using quinuclidinyl benzilate, L-[benzylic-4,4'-(3)H] ([(3)H]-QNB) as a radioligand antagonist, the effect of a partially purified hawthorn extract fraction to inhibit muscarinic receptor binding was quantified. Hawthorn extract fraction 3 dose-dependently inhibited [(3)H]-QNB binding to mouse heart membranes. Taken together, these findings suggest that decreased contraction frequency by hawthorn extracts in neonatal murine cardiomyocytes may be mediated via muscarinic receptor activation.

Topics: Alkaloids; Animals; Animals, Newborn; Cells, Cultured; Chromatography, Gel; Crataegus; Furans; Mice; Myocytes, Cardiac; Naphthalenes; Piperidines; Plant Extracts; Receptors, Muscarinic

The discovery of an exceptionally potent series of thrombin receptor (PAR-1) antagonists based on the natural product himbacine is described. Optimization of this series has led to the discovery of 4 (SCH 530348), a potent, oral antiplatelet agent that is currently undergoing Phase-III clinical trials for acute coronary syndrome (unstable angina/non-ST segment elevation myocardial infarction) and secondary prevention of cardiovascular events in high-risk patients.

Topics: Administration, Oral; Alkaloids; Animals; Furans; Humans; In Vitro Techniques; Lactones; Macaca fascicularis; Naphthalenes; Piperidines; Platelet Aggregation Inhibitors; Pyridines; Rats; Receptors, Thrombin; Structure-Activity Relationship

The inotropic response to muscarinic receptor stimulation of isolated chick ventricular myocardium was examined at various developmental stages, and the receptor subtype involved was pharmacologically characterized. In embryonic chick ventricles, carbachol (CCh) produced positive inotropy at micromolar concentrations. In hatched chick ventricles, CCh produced negative inotropy at nanomolar concentrations. Neither positive nor negative inotropy was observed in the 19 - 21-day-old embryos. Both positive and negative inotropy were also observed with acetylcholine and oxotremoline-M. The CCh-induced positive inotropy in 7 - 9-day-old embryonic ventricles and the negative inotropy in 1 - 3-day-old hatched chick ventricles were antagonized by muscarinic receptor antagonists; pA(2) values for the positive and negative responses of pirenzepine were 7.5 and 7.2, those of AF-DX116 (11-[(2-[(diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepine-6-one) were 6.8 and 6.9, those of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were 9.0 and 8.5, and those of himbacine were 7.0 and 8.0, respectively. CCh had no effect on action potential configuration. In conclusion, the positive inotropy is most likely mediated by muscarinic M(1) receptors and the negative inotropy is mostly likely mediated by muscarinic M(4) receptors.

Topics: Action Potentials; Alkaloids; Animals; Carbachol; Chick Embryo; Dose-Response Relationship, Drug; Furans; Heart Ventricles; Myocardial Contraction; Naphthalenes; Piperidines; Pirenzepine; Receptors, Muscarinic

The synthesis and biological activity of a novel series of thrombin receptor antagonists is described. This series of compounds showed excellent in vitro and in vivo potency. The most potent compound 40 had an IC(50) of 7.6 nM and showed robust inhibition of platelet aggregation in a cynomolgus monkey model after oral administration.

Topics: Administration, Oral; Alkaloids; Animals; Area Under Curve; Chemistry, Pharmaceutical; Furans; Inhibitory Concentration 50; Ligands; Macaca fascicularis; Models, Chemical; Naphthalenes; Parasympatholytics; Piperidines; Platelet Aggregation; Protein Binding; Rats; Receptors, Thrombin; Thrombin

The structure-activity relationship (SAR) of the vinyl pyridine region of himbacine derived thrombin receptor (PAR-1) antagonists is described. A 2-vinylpyridyl ring substituted with an aryl or a heteroaryl group at the 5-position showed the best overall PAR-1 affinity and pharmacokinetic properties. One of the newly discovered analogs bearing a 5-(3-pyridyl) substituent showed excellent PAR-1 affinity (Ki = 22 nM) and oral activity with reduced ClogP and improved off-target selectivity compared to an earlier development candidate.

Topics: Alkaloids; Animals; Anticoagulants; Furans; Molecular Structure; Naphthalenes; Piperidines; Platelet Aggregation Inhibitors; Rats; Receptor, PAR-1; Structure-Activity Relationship

Pursuing our earlier efforts in the himbacine-based thrombin receptor antagonist area, we have synthesized a series of compounds that incorporate heteroatoms in the C-ring of the tricyclic motif. This effort has resulted in the identification of several potent heterocyclic analogs with excellent affinity for the thrombin receptor. Several of these compounds demonstrated robust inhibition of platelet aggregation in an ex vivo model in cynomolgus monkeys following oral administration. A detailed profile of 28b, a benchmark compound in this series, with a Ki of 4.3 nM, is presented.

Topics: Administration, Oral; Alkaloids; Animals; Biological Availability; Blood Platelets; Furans; Heterocyclic Compounds, 3-Ring; Humans; In Vitro Techniques; Isoquinolines; Macaca fascicularis; Mice; Microsomes, Liver; Naphthalenes; Piperidines; Platelet Aggregation Inhibitors; Pyridines; Rats; Receptor, PAR-1; Stereoisomerism; Structure-Activity Relationship

Muscarinic antagonists, particularly atropine, can inhibit myopia development in several animal models and also in children. However, the biochemical basis of the inhibition of axial eye growth remains obscure, and there are doubts whether muscarinic receptors are involved at all. Experiments in chickens and monkeys have shown that the synthesis of the transcription factor ZENK, also named Egr-1, in retinal glucagon amacrine cells is strongly associated with inhibition of axial eye growth (assumed to create a STOP signal). We have tested whether the muscarinic antagonists atropine, pirenzepine, oxyphenonium, gallamine, MT-3, himbacine, and 4-DAMP can stimulate ZENK expression so that the drugs' inhibitory effect on myopia development could be explained by an enhanced STOP signal. Because it is known that intravitreal quisqualic acid (QA) eliminates most cholinergic neurons in the retina within 6 or 7 days, in a second set of experiments, we tested whether these antagonists could still stimulate ZENK production, 6 days after QA was applied. Muscarinic antagonists, injected intravitreally at various concentrations, affected ZENK synthesis in various and unpredictable ways. Pirenzepine, oxyphenonium, and MT-3 increased the proportion of glucagon cells that were ZENK-immunoreactive, whereas himbacine decreased that proportion, and gallamine and 4-DAMP had no significant effect. Atropine caused an upregulation of ZENK only if all positive amacrine and bipolar cells were counted and therefore appeared to affect primarily cells other than glucagon amacrines. The pattern of results remained unchanged after ablation of most cholinergic neurons by QA. Our results suggest that at least some muscarinic antagonists do not activate cells that synthesize ZENK when they inhibit axial eye growth. Therefore, in line with other studies they also cast doubt on the assumption that muscarinic transmission is crucial, and they suggest that muscarinic antagonists may inhibit myopia through extraretinal target sites or through non-cholinergic retinal actions.

Topics: Alkaloids; Animals; Atropine; Cell Count; Chickens; Excitatory Amino Acid Agonists; Furans; Gallamine Triethiodide; Glucagon; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Muscarinic Antagonists; Myopia; Naphthalenes; Peptides; Piperidines; Quisqualic Acid; Receptors, Cholinergic; Retina; Retinal Ganglion Cells

Spinal muscarinic acetylcholine receptors (mAChRs) play an important role in the regulation of nociception. To determine the role of individual mAChR subtypes in control of synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in lamina II neurons using whole-cell recordings in spinal cord slices of wild-type and mAChR subtype knockout (KO) mice. The mAChR agonist oxotremorine-M (3-10 microM) dose-dependently decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. However, in the presence of the M2 and M4 subtype-preferring antagonist himbacine, oxotremorine-M caused a large increase in the sIPSC frequency. In M3 KO and M1/M3 double-KO mice, oxotremorine-M produced a consistent decrease in the frequency of sIPSCs, and this effect was abolished by himbacine. We were surprised to find that in M2/M4 double-KO mice, oxotremorine-M consistently increased the frequency of sIPSCs and mIPSCs in all neurons tested, and this effect was completely abolished by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 subtype-preferring antagonist. In M2 or M4 single-KO mice, oxotremorine-M produced a variable effect on sIPSCs; it increased the frequency of sIPSCs in some cells but decreased the sIPSC frequency in other neurons. Taken together, these data strongly suggest that activation of the M3 subtype increases synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M2 and M4 subtypes predominantly attenuates GABAergic inputs to dorsal horn neurons in mice, an action that is opposite to the role of M2 and M4 subtypes in the spinal cord of rats.

Topics: Alkaloids; Animals; Electrophysiology; Furans; gamma-Aminobutyric Acid; Mice; Mice, Knockout; Muscarinic Agonists; Muscarinic Antagonists; Naphthalenes; Neurons; Oxotremorine; Piperidines; Posterior Horn Cells; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Spinal Cord

In rat striatal slices labelled with [(3)H]-adenine and in the presence of 1 mM 3-isobutyl-1-methylxantine (IBMX), cyclic [(3)H]-AMP ([(3)H]-cAMP) accumulation induced by the dopamine D(1) receptor agonist SKF-81297 (1 microM; 177 +/- 13% of basal) was inhibited by the general muscarinic agonist carbachol (maximum inhibition 72 +/- 3%, IC(50) 0.30 +/- 0.06 microM). The muscarinic toxin 7 (MT-7), a selective antagonist at muscarinic M(1) receptors, reduced the effect of SKF-81297 by 40+/-7% (IC(50) 251+/- 57 pM) and enhanced the inhibitory action of a submaximal (1 microM) concentration of carbachol (69 +/- 4% vs. 40 +/- 7% inhibition, IC(50) 386 +/- 105 pM). The toxin MT-1, agonist at M(1) receptors, stimulated [(3)H]-cAMP accumulation in a modest but significant manner (137 +/- 11% of basal at 400 nM), an action additive to that of D(1) receptor activation and blocked by MT-7 (10 nM). The effects of MT-7 on D(1) receptor-induced [(3)H]-cAMP accumulation and the carbachol inhibition were mimicked by the PKC inhibitors Ro-318220 (200 nM) and Gö-6976 (200 nM). Taken together our results indicate that in addition to the inhibitory role of M(4) receptors, in rat striatum acetylcholine stimulates cAMP formation through the activation of M(1 )receptors and PKC stimulation.

Topics: Alkaloids; Animals; Benzazepines; Carbachol; Cholinergic Agonists; Corpus Striatum; Cyclic AMP; Dopamine Agonists; Furans; Male; Muscarinic Antagonists; N-Methylscopolamine; Naphthalenes; Parasympatholytics; Piperidines; Pirenzepine; Protein Kinase C; Radioligand Assay; Rats; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M4; Receptors, Dopamine D1

The structure-activity relationship (SAR) of the lactone ring of himbacine derived thrombin receptor (PAR-1) antagonists (e.g., 2-5) is described. The effect of the lactone carbonyl group on binding to PAR-1 is dependent on the substitution pattern of the pyridine ring. A stereoselective intramolecular Michael addition reaction to the vinyl pyridine group was observed for these pyridine analogs of himbacine in basic conditions at elevated temperature.

Topics: Alkaloids; Furans; Inhibitory Concentration 50; Lactones; Molecular Structure; Naphthalenes; Piperidines; Receptor, PAR-1; Structure-Activity Relationship

Resting superior cervical ganglion (SCG) neurones are phasic cells that switch to a tonic mode of firing upon muscarinic receptor stimulation. This effect is partially due to the muscarinic inhibition of the M-current. Because delayed rectifier K+ channels are essential to sustain tonic firing in central neurones, we asked whether the delayed rectifier current IKV in SCG neurones was modulated by the muscarinic receptors expressed in these cells.. Whole-cell patch-clamp records of M-current and IKV were done in cultured or acutely dissociated rat SCG neurones. To characterize the receptor that regulates IKV, cells were bathed with muscarinic agonists and antagonists, relatively specific for receptor subtypes.. The muscarinic agonist oxotremorine-M (Oxo-M) enhanced IKV by approximately 46% relative to its basal value. This effect remained unaltered when M-current was suppressed by linopirdine or Ba2+. Enhancement of IKV was insensitive to the M1-antagonist pirenzepine, whereas it was inhibited (approximately 60%) by the M2/4-antagonist himbacine. Further, the relatively specific M2-agonist bethanechol was as potent as Oxo-M in enhancing IKV. The modulation of IKV was insensitive to pertussis toxin (PTX), but was severely attenuated when internal ATP was replaced by its non-hydrolysable analogue AMP-PNP.. These results suggest that an M2-like muscarinic receptor couples to a PTX-insensitive G-protein and to an ATP-dependent pathway to enhance IKV. Modulation of IKV must be taken into consideration in order to understand more precisely how muscarinic receptors acting on different ion channels regulate sympathetic excitability.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Bethanechol; Cells, Cultured; Delayed Rectifier Potassium Channels; Furans; Membrane Potentials; Muscarinic Agonists; Muscarinic Antagonists; Naphthalenes; Neurons; Oxotremorine; Patch-Clamp Techniques; Piperidines; Rats; Rats, Wistar; Receptor, Muscarinic M2; Superior Cervical Ganglion

On treatment with trifluoroacetic acid butenolide 14 undergoes N-Boc deprotection and condensation followed by an iminium ion activated intramolecular Diels-Alder cycloaddition to give the (+)-himbacine precursor 11 on reductive work up. Compound 11 was converted into (+)-himbacine in four synthetic steps. [reaction: see text]

Topics: 4-Butyrolactone; Aldehydes; Alkaloids; Alkenes; Furans; Models, Molecular; Naphthalenes; Piperidines; Stereoisomerism; Trifluoroacetic Acid

Structurally novel thrombin receptor (protease activated receptor 1, PAR-1) antagonists based on the natural product himbacine are described. The prototypical PAR-1 antagonist 55 showed a Ki of 2.7 nM in the binding assay, making it the most potent PAR-1 antagonist reported. 55 was highly active in several functional assays, showed excellent oral bioavailability in rat and monkey models, and showed complete inhibition of agonist-induced ex vivo platelet aggregation in cynomolgus monkeys after oral administration.

Topics: Administration, Oral; Alkaloids; Animals; Biological Availability; Blood Platelets; Chromatography, High Pressure Liquid; Fibrinolytic Agents; Furans; Heterocyclic Compounds, 3-Ring; Humans; In Vitro Techniques; Macaca fascicularis; Naphthalenes; Piperidines; Platelet Aggregation Inhibitors; Pyridines; Radioligand Assay; Rats; Receptor, PAR-1; Stereoisomerism; Structure-Activity Relationship

A number of analogs of the natural product himbacine were synthesized employing variations at the heterocyclic unit and the tether that links the heterocyclic unit to the tricyclic motif. Several of these analogs had M(2) affinity and M(1)/M(2) selectivity comparable to those of himbacine. The structural and stereochemical requirements of the heterocyclic unit for muscarinic binding are discussed in the light of these data.

Topics: Alkaloids; Cloning, Molecular; Furans; Heterocyclic Compounds; Humans; Molecular Structure; Muscarinic Antagonists; Naphthalenes; Parasympatholytics; Piperidines; Receptors, Muscarinic; Recombinant Proteins; Structure-Activity Relationship

With an aim to disclose the convergency and flexibility of our previously explored synthetic route to natural himbacine 1, and moreover, to clarify some novel aspects of the structure-activity relationships of 1, we prepared various structural types of novel himbacine congeners, 3-demethylhimbacine (3-norhimbacine) 2 and 4-epi-3-demethylhimbacine (4-epi-3-norhimbacine) 4-epi-2 and their enantiomers (ent-2 and ent-4-epi-2), 11-methylhimbacine 3, and 3-epihimbacine 4 in optically pure forms by employing our methodology. All of the synthesized congeners correspond to the compounds modified at the C-3 position of gamma-lactone moiety involved in 1. Among these congeners, 3-demethylhimbacine (3-norhimbacine) 2 was found to exhibit more potent muscarinic M(2) receptor binding affinity than natural 1.

Topics: Alkaloids; Animals; Binding, Competitive; Brain Stem; Cerebral Cortex; Furans; In Vitro Techniques; Indicators and Reagents; Kinetics; Lactones; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Muscarinic Antagonists; Naphthalenes; Piperidines; Quinuclidinyl Benzilate; Rats; Receptor, Muscarinic M2; Receptors, Muscarinic; Stereoisomerism

[reaction: see text] A formal total synthesis of the selective muscarinic receptor antagonist himbacine is presented. Key C-C bond-forming steps include an intramolecular Diels-Alder reaction, Stille coupling reactions, and a 6-exo-trig acyl radical cyclization to a conjugated enyne. An unexpected secondary alcohol to chloride conversion is witnessed during attempted thionocarbonate formation.

Topics: Alkaloids; Alkynes; Cyclization; Free Radicals; Furans; Naphthalenes; Piperidines; Plants, Medicinal; Trees

A series of himbacine (1)-related analogues has been prepared featuring three different isomeric configurations with respect to the B-ring (a, b and natural c) and three different interconnecting two-carbon unsaturated units [natural (E)-ene, (Z)-ene, and yne]. The study of the binding affinities of the nine resulting compounds, including synthetic (+)-himbacine (3c), towards the M(1)-M(4) muscarine receptor subtypes revealed that analogues 3a and 5c display a promising 10-fold selectivity for the M(2) receptor as compared to the M(1) receptor.

Topics: Alkaloids; Animals; Binding, Competitive; CHO Cells; Cricetinae; Furans; Humans; Muscarinic Antagonists; Naphthalenes; Piperidines; Protein Binding; Receptors, Muscarinic; Stereoisomerism; Structure-Activity Relationship; Substrate Specificity

The title three compounds ent-1, 6, and ent-6 were synthesized by coupling the chiral sulfone 4 or ent-4 with the chiral piperidinaldehyde 5 or ent-5, which were readily prepared following the synthetic routes previously established by the novel total synthesis of natural himbacine 1. Their muscarinic M2 subtype binding affinity was evaluated in comparison to that of 1, disclosing that the stereochemistry of both the tricyclic moiety and the piperidine part of 1 plays crucial roles in its potent activity.

Topics: Alkaloids; Brain Chemistry; Furans; Muscarinic Antagonists; Naphthalenes; Piperidines; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptors, Muscarinic; Stereoisomerism; Structure-Activity Relationship

In the course of our studies of the structure-activity relationships of himbacine 1, a potent antagonist of the M(2) subtype of muscarinic receptor, the four title compounds, 2, ent-2, 3, and ent-3, were synthesized with a highly stereoselective intermolecular Diels-Alder reaction of tetrahydroisobenzofuran 4 with achiral furan-2(5H)-one 5 as a key step, followed by simultaneous optical resolution and epimer separation of the racemic intermediates. Among these compounds, 3-demethylhimbacine (3-norhimbacine) 2, bearing an absolute configuration corresponding to that of 1, was found to show more potent muscarinic M(2) subtype receptor binding activity than natural 1.

Topics: Alkaloids; Alzheimer Disease; Animals; Brain Stem; Cerebral Cortex; Furans; Muscarinic Antagonists; Naphthalenes; Parasympatholytics; Piperidines; Radioligand Assay; Rats; Receptor, Muscarinic M2; Receptors, Muscarinic; Stereoisomerism; Structure-Activity Relationship

[reaction: see text]. The IMDA reaction of 9 leads with good stereoselectivity to exo-adduct 10b. The functionalized ABC-ring core in 10 is well suited for the convergent synthesis of analogues of himbacine, a naturally occurring M2 selective muscarine receptor antagonist, as illustrated with the further synthesis of the dehydro-derivative 5.

Topics: Alkaloids; Australia; Crystallography, X-Ray; Furans; Indicators and Reagents; Models, Molecular; Muscarinic Antagonists; Naphthalenes; New Guinea; Piperidines; Plants, Medicinal; Stereoisomerism

[reaction: see text] The syntheses of 4,4a-didehydrohimbacine and 4,4a-didehydrohimandravine are presented. Key steps include an intramolecular Diels-Alder reaction of a bromopentadienyl acrylate and Suzuki-Miyaura and Stille coupling reactions.

Topics: Acrylates; Alkaloids; Furans; Indicators and Reagents; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Naphthalenes; Parasympatholytics; Piperidines; Plants, Medicinal

1. Muscarinic cholinoceptors (MChR) in freshly dispersed rat salivary gland (RSG) cells were characterized using microphysiometry to measure changes in acidification rates. Several non-selective and selective muscarinic antagonists were used to elucidate the nature of the subtypes mediating the response to carbachol. 2. The effects of carbachol (pEC(50) = 5.74 +/- 0.02 s.e.mean; n = 53) were highly reproducible and most antagonists acted in a surmountable, reversible fashion. The following antagonist rank order, with apparent affinity constants in parentheses, was noted: 4-DAMP (8.9)= atropine (8.9) > tolterodine (8.5) > oxybutynin (7.9) > S-secoverine (7.2) > pirenzepine (6.9) > himbacine (6.8) > AQ-RA 741 (6.6) > methoctramine (5.9). 3. These studies validate the use of primary isolated RSG cells in microphysiometry for pharmacological analysis. These data are consistent with, and extend, previous studies using alternative functional methods, which reported a lack of differential receptor pharmacology between bladder and salivary gland tissue. 4. The antagonist affinity profile significantly correlated with the profile at human recombinant muscarinic M(3) and M(5) receptors. Given a lack of antagonists that discriminate between M(3) and M(5), definitive conclusion of which subtype(s) is present within RSG cells cannot be determined.

Topics: Alkaloids; Animals; Atropine; Benzhydryl Compounds; Benzodiazepinones; Binding, Competitive; Biosensing Techniques; Carbachol; Cholinergic Agonists; Cresols; Diamines; Dose-Response Relationship, Drug; Furans; Male; Mandelic Acids; Muscarinic Antagonists; Naphthalenes; Phenethylamines; Phenylpropanolamine; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Submandibular Gland; Tolterodine Tartrate

The success of the M(1)-selective muscarinic antagonist pirenzepine in preventing myopia development in animal models implicates a role for the M(1) receptor. However, the relatively high dose of pirenzepine required may indicate that the drug acts through another receptor subtype. This study examined whether the M(4)-selective antagonist, himbacine, could also prevent myopia. Daily intravitreal injections of himbacine inhibited the inducement of myopia in chick eyes in a dose- dependent manner. Doses < or = 200 microg caused no significant inhibition of induced myopia compared to controls (-13.7 +/- 2.3 vs -16.2 +/- 0.9D, ANOVA p = 0.37), whilst a dose of 800 microg almost completely inhibited the induced myopia (-2.4 +/- 2.0, p < 0.01). Findings demonstrate himbacine is effective at preventing the development of myopia in chick and implicates a role for the M4 receptor.

Topics: Age Factors; Alkaloids; Animals; Chickens; Disease Models, Animal; Dose-Response Relationship, Drug; Furans; Muscarinic Antagonists; Myopia; Naphthalenes; Parasympatholytics; Piperidines; Pirenzepine; Receptor, Muscarinic M4; Receptors, Muscarinic; Refraction, Ocular; Retina; Sensory Deprivation; Vision, Monocular; Vitreous Body

Activation of the parasympathetic ciliary ganglion input to the choroid causes increases in choroidal blood flow. We examined the role and the type of muscarinic receptors within the choroid that are involved in these increases in choroidal blood flow, using electrical stimulation of the nucleus of Edinger-Westphal (EW) to activate the ciliary ganglion input to choroid in ketamine anesthetized pigeons. Baseline choroidal blood flow and its EW-evoked increases measured as peak and total (area under the curve) responses were determined using laser Doppler flowmetry. The EW-evoked responses were reduced dose-dependently after administration of 4-diphenyl-acetoxy-N-methylpiperedine (4-DAMP), a relatively selective antagonist of M3 type muscarinic receptors, with a maximal mean decrease of 86% (peak response) and 93% (total response) at a dose of 10 microg kg(-1)i.v. without a significant effect on baseline choroidal blood flow, heart rate or systemic arterial blood pressure. Atropine, a non-selective antagonist of muscarinic receptors, decreased the EW-evoked responses to a lesser extent than 4-DAMP after intravenous administration of 1 mg kg(-1)(by 67% for peak response and by 53% for total response) or topical administration of a 5% solution (by 41% for peak response and by 62% for total response), both of which increased heart rate and systemic arterial blood pressure without a consistent effect on baseline choroidal blood flow. In contrast, himbacine (i.p. 10 microg kg(-1)), a relatively selective antagonist of M2 type muscarinic receptors, increased the EW-evoked parasympathetic cholinergic vasodilation (by 93% for the peak response and by 142% for the total response) without a significant effect on heart rate, systemic arterial blood pressure or baseline choroidal blood flow. The results of our study suggest a major role of M3 type muscarinic receptors in the EW-evoked increases in choroidal blood flow. Based on findings that the ciliary ganglion input to choroid does not synthesize nitric oxide but inhibitors of NO production do block EW-evoked choroidal vasodilation, it seems likely that the M3 receptors acted on by 4-DAMP are present on choroidal endothelial cells and mediate choroidal vasodilation via stimulation of endothelial release of nitric oxide. In contrast, M2 muscarinic receptors may play a presynaptic role in downregulating EW-evoked parasympathetic cholinergic vasodilation in avian choroid.

Topics: Alkaloids; Animals; Atropine; Blood Pressure; Choroid; Columbidae; Electric Stimulation; Furans; Heart Rate; Muscarinic Antagonists; Naphthalenes; Parasympatholytics; Piperidines; Receptors, Muscarinic; Regional Blood Flow; Vasodilation

Acetylcholine outflow can be modulated through inhibitory presynaptic muscarinic autoreceptors. This study was to identify which subtype is involved in mouse cortex. Five muscarinic antagonists and their ability to elevate stimulation-induced (S-I) acetylcholine outflow were tested in the presence of neostigmine, which decreased S-I outflow. The potency of each antagonist was determined, expressed as a ratio of the potency of each other antagonist and compared with the potency ratios of the antagonists for each of the defined muscarinic receptors (M(1)-M(4)), as recorded in the literature. Linear regression analysis revealed that the data fitted the M(2) (r(2)>0.97) and M(4) (r(2)>0.85) subtypes best, with no correlation for the M(1) and M(3) subtypes.

Topics: Acetylcholine; Alkaloids; Animals; Atropine; Autoreceptors; Binding, Competitive; Cerebral Cortex; Furans; Male; Mice; Muscarinic Antagonists; Naphthalenes; Neostigmine; Parasympatholytics; Parasympathomimetics; Piperidines; Pirenzepine; Receptor, Muscarinic M2; Receptor, Muscarinic M4; Receptors, Muscarinic

Structure activity studies on [4-(phenylsulfonyl)phenyl]methylpiperazine led to the discovery of 4-cyclohexyl-alpha-[4-[[4-methoxyphenyl(S)-sufinyl]phenyl]-1-pi perazineacetonitrile, 1, an M2 selective muscarinic antagonist. Affinity at the cloned human M2 receptor was 2.7 nM; the M1/M2 selectivity is 40-fold.

Topics: Acetylcholine; Administration, Oral; Alkaloids; Animals; Benzene Derivatives; Corpus Striatum; Dibenzazepines; Drug Design; Furans; Humans; Muscarinic Antagonists; Naphthalenes; Piperidines; Pyridines; Rats; Receptor, Muscarinic M2; Receptors, Muscarinic; Recombinant Proteins; Structure-Activity Relationship

The purpose of this study was to evaluate the effects of solubilizing excipients on Caco-2 transport parameters of poorly water-soluble NCEs (new chemical entities), and determine their permeability class under the BCS guidance (Biopharmaceutics Classification System). The effect of solubilizing excipients on soluble donor concentration of Sch 56592, Sch-X and Sch-Y was estimated. The transport of reference compounds and NCEs was studied across Caco-2 monolayers in absence or presence of solubilizing agents. The Caco-2 permeability of reference compounds showed good correlation with their extent of human oral absorption data. Sch 56592, Sch-X and Sch-Y exhibited high baseline Caco-2 permeability (>10(-5) cm/s). Povidone (1%) improved soluble donor concentration and flux of Sch 56592 by 40%. Other solubilizing excipients predominantly improved Sch 56592 soluble donor concentration, with either no change or a decrease in flux. With Sch-X, 1% povidone, pluronic F68, gelucir 44/14, and 3:2 propylene glycol/Tween-80 markedly improved soluble donor concentration, while increasing Sch-X flux by 40-65%. The soluble donor concentration of Sch-Y was also enhanced by excipients; however, only 1% pluronic F68 and PEG 300 increased Sch-Y flux by 35-50%. Sch 56592, Sch-X and Sch-Y are low solubility-high permeability compounds under the BCS guidance. For such poorly water-soluble NCEs, solubilizing excipients should be carefully screened based on their effects on solubility profiles and membrane transport.

Topics: Alkaloids; Biological Transport; Caco-2 Cells; Cell Membrane Permeability; Cimetidine; Diltiazem; Excipients; Furans; Humans; Hydrogen-Ion Concentration; Imidazoles; Intestinal Absorption; Naphthalenes; Phenytoin; Piperidines; Poloxamer; Polyethylene Glycols; Povidone; Propranolol; Solubility; Triazoles

Epibatidine, a neurotoxin isolated from the skin of Epipedobates tricolor, is an efficacious antinociceptive agent with a potency 200 times that of morphine. The toxicity of epibatidine, because of its nonspecificity for both peripheral and central nicotinic receptors, precludes its development as an analgesic. During the synthesis of epibatidine analogs we developed potent antinociceptive agents, typified by CMI-936 and CMI-1145, whose antinociception, unlike that of epibatidine, is mediated via muscarinic receptors. Subsequently, we used specific muscarinic toxins and antagonists to delineate the muscarinic receptor subtype involved in the antinociception evoked by these agents. Thus, the antinociception produced by CMI-936 and CMI-1145 is inhibited substantially by 1) intrathecal injection of the specific muscarinic M4 toxin, muscarinic toxin-3; 2) intrathecally administered pertussis toxin, which inhibits the G proteins coupled to M2 and M4 receptors; and 3) s.c. injection of the M2/M4 muscarinic antagonist himbacine. These results demonstrate that the antinociception elicited by these epibatidine analogs is mediated via muscarinic M4 receptors located in the spinal cord. Compounds that specifically target the M4 receptor therefore may be of substantial value as alternative analgesics to the opiates.

Topics: Alkaloids; Analgesics, Non-Narcotic; Animals; Body Temperature; Bridged Bicyclo Compounds, Heterocyclic; Female; Furans; Mice; Muscarinic Agonists; Naphthalenes; Oxadiazoles; Piperidines; Pyridines; Receptor, Muscarinic M4; Receptors, Muscarinic; Salivation

A parallel synthesis of racemic himbacine analogs was carried out by N-alkylation of various commercially available cyclic amine derivatives with the alkylating agent 4 which bears the tricyclic unit of himbacine. Several of these analogs have potency comparable to that of himbacine, albeit lacking the desired selectivity. Structure-activity relationship studies support the existence of a hydrophobic pocket in the receptor where the piperidine ring of dihydrohimbacine binds.

Topics: Alkaloids; Furans; Kinetics; Models, Chemical; Models, Molecular; Muscarinic Antagonists; Naphthalenes; Piperidines; Structure-Activity Relationship

Striatal m4 muscarinic receptors are important because their blockade controls movement, and they are preferentially located on striatal neurons that project to the internal globus pallidus. The following studies were performed in vitro to provide a basis for using antimuscarinic toxins to study the effects of selective m4 blockade on movement in vivo. Because m4-toxin has limited selectivity alone (102-fold higher affinity for m4 than m1 receptors), m1-toxin was used first to occlude m1 receptors selectively, fully and irreversibly. It blocked 42% of the sites for 1.0 nM 3H-N-methylscopolamine in rat striatal membranes and 43% in sections of cat striatum. m4-Toxin (>500-fold higher affinity for m4 than m2, m3 or m5 receptors) blocked 88% of the residual, non-m1 sites in membranes, showing 64 pmol m4 receptors/g tissue. In comparison, AFDX-116, biperiden, clozapine, gallamine, hexahydrodifenidol, himbacine, R(+)hyoscyamine, methoctramine, pirenzepine, silahexocyclium, trihexyphenidyl and tripitramine did not distinguish m4 from other non-m1 receptors. 3H-Pirenzepine dissociated twice as rapidly from non-m1 as m1 receptors. Autoradiography was used to test the idea that m4 receptors are localized preferentially in the striosomes of the cat striatum. Non-m1 receptors were distributed equally in striosomes and matrix, indicating that striatal neurons with m4 receptors are in both compartments. Thus m1-toxin facilitates studies of m4 receptors by occluding m1 receptors, and m4-toxin is a selective antagonist for residual m4 receptors.

Topics: Alkaloids; Animals; Benzodiazepines; Binding, Competitive; Cats; CHO Cells; Corpus Striatum; Cricetinae; Elapid Venoms; Furans; Gallamine Triethiodide; Humans; Male; Muscarinic Antagonists; N-Methylscopolamine; Naphthalenes; Neurotoxins; Piperidines; Pirenzepine; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M1; Receptor, Muscarinic M4; Receptors, Muscarinic; Recombinant Proteins

The green mamba (Dendroaspis angusticeps) "muscarinic toxin', MT1, was radioiodinated by the chloramine T method. 125I-MT1 labelled the muscarinic M1 receptor subtype with a very good selectivity in rat brain. It had no preference for the receptor states with high or low affinity for agonists, and was not affected by Gpp(NH)p addition to the incubation medium. The 125I-MT1 binding was reversible, with a half life of 45 min at 25 degrees C. The effect of competitive and allosteric muscarinic antagonists on 125I-MT1 binding and dissociation can be rationalized by assuming that the radioiodinated toxin is able to label the muscarinic (acetylcholine) binding site.

Topics: Alkaloids; Animals; Atropine; Binding, Competitive; Brain; Carbachol; Elapid Venoms; Furans; Gallamine Triethiodide; In Vitro Techniques; Iodine Radioisotopes; Isotope Labeling; Kinetics; Muscarinic Agonists; Muscarinic Antagonists; Naphthalenes; Oxotremorine; Parasympatholytics; Pilocarpine; Piperidines; Pirenzepine; Protein Binding; Rats; Receptor, Muscarinic M1; Receptors, Muscarinic; Synaptic Membranes; Tubocurarine

1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Alkaloids; Animals; Arecoline; Bethanechol; Carbachol; Diamines; Dose-Response Relationship, Drug; Furans; Ganglia, Parasympathetic; Ganglionectomy; Iris; Male; Methacholine Chloride; Muscarinic Agonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Naphthalenes; Parasympatholytics; Pilocarpine; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic

1. The effects of pertussis toxin (PTX) on contraction and/or relaxation induced by agonists or transmural nerve stimulation (TNS) were examined in the rat iris dilator and sphincter muscles. 2. TNS in the presence of phentolamine induced an atropine-sensitive biphasic response: initial contraction followed by relaxation in dilator muscles. Exogenously applied acetylcholine (ACh) elicited a large relaxation at low doses (3 microM or less) and a concentration at high doses. 3. Only the ACh-induced relaxation was affected by injection of PTX (10 ng) into the anterior eye chamber. Relaxation was decreased 12 h after injection and had completely disappeared after 24 h. Relaxation recovered in part 3 weeks and almost completely 8 weeks after PTX treatment. A gradual decrease in muscarinic relaxation in a dilator muscle was also observed in vitro after addition of PTX to the bathing solution. 4. The pA2 values of muscarinic blockers, pirenzepine, AF-DX 116, 4-DAMP, and himbacine for competitive antagonism to ACh-induced contraction were 7.14, 6.53, 9.03, and 6.80, respectively, in PTX-pretreated dilator muscles. These values are comparable to those obtained in parasympathectomized dilator muscles and may indicate involvement of M3 or M3-like receptors in muscle contraction. 5. Pretreatment with PTX did not significantly affect contraction induced by noradrenaline or 5-hydroxytryptamine or the relaxation induced by isoprenaline in dilator muscles. 6. In conclusion, among several agonist-induced responses in the rat iris dilator and sphincter muscles, only muscarinic relaxation in dilator muscle occurs via activation of PTX-sensitive GTP binding proteins.

Topics: Acetylcholine; Alkaloids; Animals; Atropine; Diamines; Dose-Response Relationship, Drug; Furans; GTP-Binding Proteins; In Vitro Techniques; Iris; Male; Microinjections; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Naphthalenes; Parasympatholytics; Pertussis Toxin; Phentolamine; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Virulence Factors, Bordetella

1. The effects of seven muscarinic receptor antagonists were used to characterize the receptors which mediate carbachol-evoked contractions of intertaenial circular and taenial longitudinal muscle in human isolated colon. The effects of these antagonists were studied upon colon contractions induced by cumulatively added carbachol which had mean EC50 values of 11.7 +/- 2.3 microM (n = 8) and 12.6 +/- 2.3 microM (n = 8) respectively upon circular and longitudinal smooth muscle. 2. All antagonists displaced concentration-response curves to carbachol to the right in a parallel manner. The maximum concentration of each antagonist added (30 nM-10 microM) did not significantly suppress the maximum response. 3. In circular muscle, the M3 muscarinic receptor antagonists, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), hexahydrosiladiphenidol (HHSiD) and para-fluoro-hexahydrosiladiphenidol (p-F-HHSiD) inhibited responses with pA2 values of 9.41 +/- 0.23, 7.17 +/- 0.07, 6.94 +/- 0.18 respectively. The M2 muscarinic receptor antagonist, AF-DX 116, the M2/M4 muscarinic receptor antagonist, himbacine, and the M1 muscarinic receptor antagonist, pirenzepine, yielded pA2 values of 7.36 +/- 0.43, 7.47 +/- 0.14 and 7.23 +/- 0.48 respectively. The non-selective antagonist, atropine, had a pA2 of 8.72 +/- 0.28. 4. In longitudinal muscle 4-DAMP, HHSiD, p-F-HHSiD, AF-DX 116, himbacine and pirenzepine gave pA2 values of 9.09 +/- 0.16, 7.45 +/- 0.43, 7.44 +/- 0.21, 6.44 +/- 0.1, 7.54 +/- 0.40, 6.87 +/- 0.38 respectively. Atropine yielded a pA2 value of 8.60 +/- 0.08. 5. The pharmacological profile of antagonist affinities at the muscarinic receptor population responding to muscarinic agonist-evoked contraction is similar to that widely accepted as characterizing the activation of an M3 muscarinic receptor subtype, although pA2 values of some antagonists are lower than that seen in other investigations.

Topics: Alkaloids; Atropine; Carbachol; Carcinoma; Colon; Colonic Neoplasms; Dose-Response Relationship, Drug; Furans; Humans; In Vitro Techniques; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Naphthalenes; Piperidines; Pirenzepine

This study describes the antagonistic properties of himbacine, in comparison with those of pirenzepine, at muscarinic receptors mediating the depolarization of rat superior cervical ganglion, the inhibition of electrically-induced twitch contractions of rabbit vas deferens and the contraction of dog saphenous vein, currently classified as putative muscarinic M1 sites. The affinity of himbacine for the vas deferens site (pA2 8.08) was nearly ten times higher than those for the M1 receptors of rat ganglion and dog saphenous vein (pA2 7.14 and 7.16, respectively); affinity estimates for pirenzepine were similar throughout the different preparations. The present data are consistent with the allocation of ganglion and saphenous vein receptors into the M1 subclass; the profile of the vas deferens site, conversely, appears to be different, and possibly more closely related to that reported for the M4/m4 receptor.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Alkaloids; Animals; Binding, Competitive; Dogs; Female; Furans; Male; Muscarine; Naphthalenes; Parasympatholytics; Parasympathomimetics; Piperidines; Pirenzepine; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Saphenous Vein; Superior Cervical Ganglion; Vas Deferens

The aim of this study was to characterise the muscarinic receptor present in the uterus of the virgin rat. Homogenate binding studies were undertaken using [3H]quinuclidinyl benzilate as the radioligand and atropine (10 microM) to determine non-specific binding. [3H]Quinuclidinyl benzilate binding was saturable with a Kd of 63 pM and a Bmax of 3 fmol/mg protein. The pKi values obtained using antagonists with high affinity for differing muscarinic receptor subtypes were pirenzepine, 6.2; hexahydrosiladifenidol, 6.9; AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]5,11-dihydro-6H - pyrido[2,3-b][1,4]benzodiazepine-6-one), 7.0; and himbacine, 7.8. These findings suggest that muscarinic M2 receptors are present in rat uterus.

Topics: Alkaloids; Animals; Atropine; Binding, Competitive; Computer Simulation; Female; Furans; In Vitro Techniques; Ligands; Muscarinic Antagonists; Naphthalenes; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Uterus

Muscarinic toxin 3 (MT3) (65 amino acids, four disulphides, M(r) 7379) was isolated from the venom of the African snake Dendroaspis angusticeps (green mamba) and its amino acid sequence determined. Its ability to inhibit the binding of [3H]N-methylscopolamine ([3H]NMS) to Chinese hamster ovary cells stably expressing subtypes of muscarinic receptors was studied. MT3 displayed high affinity for the m4 receptor (pKi = 8.7 +/- 0.06), 40-fold lower affinity at ml receptors (pKi = 7.11 +/- 0.17) whereas no inhibition of [3H]NMS binding to m2, m3 and m5 receptors was observed at concentrations up to 1 microM. This makes MT3 the most selective m4 receptor ligand known to date.

Topics: Alkaloids; Amino Acid Sequence; Amino Acids; Animals; Elapid Venoms; Elapidae; Furans; Intercellular Signaling Peptides and Proteins; Kinetics; Molecular Sequence Data; Naphthalenes; Peptides; Piperidines; Pirenzepine; Receptors, Muscarinic; Sequence Analysis

Synaptosomes prepared from fresh specimens of human cerebral cortex were labeled with [3H]glycine ([3H]Gly) and distributed in parallel superfusion chambers. Exposure to 15 mM KCl evoked a tritium overflow which was largely prevented by 10 mM Mg++, suggesting a consistent component of Ca(++)-dependent [3H]Gly release. Acetylcholine (ACh; 1-100 microM), added during K(+)-depolarization, increased the release of tritium in a concentration-dependent manner (maximal effect, 60%; EC50 = 7 microM). Oxotremorine (1-100 microM) mimicked ACh. The effect of 10 microM ACh was insensitive to the nicotinic antagonist mecamylamine (100 microM), but it was blocked by the muscarinic antagonist atropine (0.1 microM). Three muscarinic receptor antagonists, pirenzepine, AF-DX 116 (11-[12-[diethylamino-methyl]-1-piperidinyl]acetyl-5-11-dihydro -6H-pyrido-[2-3-b][1,4]benzodiazepine-6-one) and himbacine, endowed with relative selectivity for various muscarinic receptor subtypes, prevented with differential affinities the effect of 10 microM ACh. Himbacine was the most potent antagonist of ACh, its pA2 (8.34) being 20- or 50-fold higher than that of pirenzepine (7.27) or AF-DX 116 (6.65). It is concluded that: 1) ACh can increase the release of Gly in human cerebral cortex; 2) the interaction occurs through muscarinic receptors which resemble most the M4 subtype; and 3) considering that Gly is required to activate the N-methyl-D-aspartate glutamate receptor, the ACh-evoked Gly release may represent a linkage between cholinergic and glutamatergic transmission, two systems strongly implicated in cognitive processes.

Topics: Acetylcholine; Alkaloids; Cerebral Cortex; Cholinergic Fibers; Drug Synergism; Female; Furans; Glycine; Humans; Kinetics; Male; Muscarinic Antagonists; Naphthalenes; Parasympatholytics; Piperidines; Pirenzepine; Potassium Chloride; Receptors, Muscarinic; Stimulation, Chemical; Synaptic Transmission; Synaptosomes; Tritium

Carbachol, a full muscarinic receptor agonist, stimulated [3H]inositol phosphate accumulation in both the ventral and dorsal hippocampus, but its efficacy and affinity were higher in the former area. The partial agonist oxotremorine had a weak stimulatory effect in both regions. The affinity profiles of pirenzepine and AF-DX 116 in antagonizing carbachol-stimulated [3H]inositol phosphate accumulation indicated that M1 and M3 receptors contributed equally to the response in either region. On the other hand, there were no differences in the receptor density, or in the distribution of muscarinic receptor subtypes between the two regions of the hippocampus which could account for the effect as determined in binding experiments with selective antagonists. Analysis of carbachol binding curves did, instead, indicate a difference in the way the agonist interacted with the receptors within the hippocampus, i.e., carbachol recognized three agonist affinity states (superhigh, high and low) in the ventral hippocampus, and only two (high and low) in the dorsal part. The findings thus suggested that the regional diversity in the efficacy of carbachol in stimulating phosphoinositide turnover was related to the complexity with which it bound to muscarinic receptors. Transduction processes that intervene between changes in the muscarinic receptors' conformation and activation of phospholipase C might be relevant to these differences.

Topics: Alkaloids; Animals; Carbachol; Furans; Guanylyl Imidodiphosphate; Hippocampus; Male; Membranes; Mitochondria; N-Methylscopolamine; Naphthalenes; Oxotremorine; Parasympatholytics; Parasympathomimetics; Phosphatidylinositols; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine Derivatives; Synaptosomes; Type C Phospholipases

1. Carbachol produced a relaxation of dilator muscle at a concentration lower than 1 microM and a contraction at a concentration higher than 1 microM. 2. We studied the effects of the M1-selective antagonist, pirenzepine, the M2-selective antagonist, himbacine, the M3-selective antagonist, 4-diphenyl-acetoxy-N-methylpiperidine methiodide (4-DAMP) and the non-selective antagonist, atropine, on carbachol-induced relaxation and contraction of the rat iris dilator smooth muscle. All the antagonists competitively inhibited both the responses to carbachol. 3. In relaxation and contraction, the low affinity of pirenzepine and himbacine suggest that the rat iris dilator smooth muscle receptors are not of the M1 and M2 subtypes. In contrast, 4-DAMP potently inhibited the carbachol-induced relaxation and contraction with affinities similar to those reported for the M3 subtype. 4. Carbachol-induced relaxation and contraction of the rat iris dilator appears to be mediated through a homogeneous population of M3 subtype.

Topics: Alkaloids; Animals; Atropine; Carbachol; Furans; In Vitro Techniques; Iris; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Naphthalenes; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic

The binding potencies for the putative M2-selective antagonist himbacine were determined in radioligand binding and in functional response assays in neuronal tissue and Chinese hamster ovary cells containing transfected muscarinic receptors. Himbacine was shown to bind to all five cloned muscarinic receptor subtypes in the order of potencies: hM2 = hM4 > hM3 > hM1 > hM5 (Kd values were 4, 7, 59, 83 and 296 nM, respectively). Himbacine was shown to bind to M2 receptors in rat heart and brain stem with Kd values of 6.9 and 4.6 nM, respectively. In rat brain tissues with complex mixtures of muscarinic receptors, and using the radioligand [3H] +/- -5,11-dihydro-11-([(2-(2-[(dipropylamino)methyl]-1- peperidinyl)ethyl)amino]carbonyl)-6H-pyrido(2,3-b)(1,4)benzodiazep ine-6-one to demarcate M2 and M4 receptors, himbacine was shown to bind to 80% of cortical or striatal receptors with Kd values of 4.5 and 3.8 nM, respectively, consistent with the involvement of M2 and/or M4 receptors in both these brain regions. Himbacine was a potent blocker of oxotremorine-M-mediated cyclic AMP inhibition in rat striatum (4.4 nM) and in N1E-115 neuroblastoma cells (10.6 nM), responses mediated by M4 receptors. Himbacine also reversed oxotremorine-M-mediated inhibition of evoked acetylcholine release from hippocampal tissue with a Kd value of 8.6 nM, a value consistent with the involvement of M2 or M4 receptors. At the cortical postsynaptic muscarinic receptors involved with phosphoinositide turnover (putative M1 and M3 receptors), himbacine was 21-fold less potent. Himbacine appears to be a potent muscarinic antagonist that displays selectivity for M2 or M4 receptors, as compared to M1 or M3 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Alkaloids; Animals; Binding, Competitive; Brain Chemistry; CHO Cells; Cricetinae; Furans; Humans; Male; Myocardium; Naphthalenes; Parasympatholytics; Phosphatidylinositols; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Recombinant Proteins; Synapses; Tumor Cells, Cultured

The subtype of propylbenzilylcholine mustard (PrBCM)-sensitive and -resistant muscarinic cholinoceptors in guinea pig ileal muscle was examined using four selective muscarinic antagonists, pirenzepine, AF-DX 116, himbacine and 4-DAMP. The pA2 values of the four antagonists against pilocarpine were not different from their values against carbachol after the treatment with PrBCM and was identified with the values for the m3 subtype. These results suggest that the subtype of PrBCM-sensitive and -resistant muscarinic cholinoceptors in guinea pig ileal muscle is the m3 subtype only and not other subtypes.

Topics: 4-Aminopyridine; Alkaloids; Animals; Drug Resistance; Furans; Guinea Pigs; Ileum; In Vitro Techniques; Kinetics; Male; Muscarinic Antagonists; Muscle, Smooth; Naphthalenes; Parasympatholytics; Piperidines; Pirenzepine; Propylbenzilylcholine Mustard; Receptors, Muscarinic; Sensitivity and Specificity

The five muscarinic receptors (m1-m5), although structurally closely related, can be distinguished pharmacologically by the use of subtype-selective ligands. Various tricyclic muscarinic antagonists, including the AF-DX derivative AQ-RA 741 and the alkaloid himbacine, for example, have been shown to display up to 200-fold higher affinities for m2 and m4 than for m5 receptors. On the other hand, antagonists such as sila-hexocyclium and the pirenzepine derivative UH-AH 37 exhibit lower affinities for m2 than for m5 and all other muscarinic receptors. To identify receptor epitopes that contribute to the subtype selectivities of these antagonists, we prepared a series of chimeric m2/m5 muscarinic receptors in which regions of the m5 receptor were systematically replaced with the homologous regions of the m2 receptor. AQ-RA 741, himbacine, and sila-hexocyclium bound to the various chimeric receptors, expressed in COS-7 cells, with affinity profiles indicative of multiple receptor domains contributing to the subtype selectivities of these antagonists. On the other hand, the higher affinity of UH-AH 37 for m5 than for m2 receptors appears to be largely dependent on a short stretch of 31 amino acids comprising most of transmembrane region VI and the third extracellular loop, a region that does not contribute to the subtype selectivity of AQ-RA 741 and himbacine. Our data indicate that different receptor epitopes are involved in conferring subtype selectivity on structurally different muscarinic antagonists.

Topics: Alkaloids; Amino Acid Sequence; Benzodiazepinones; Chimera; Dibenzazepines; Furans; Humans; Molecular Sequence Data; Muscarinic Antagonists; N-Methylscopolamine; Naphthalenes; Parasympatholytics; Piperazines; Piperidines; Receptors, Muscarinic; Scopolamine Derivatives; Sequence Homology, Nucleic Acid

1. The muscarine receptor mediating electrically-stimulated acid secretion in the mouse isolated stomach was characterized using a variety of muscarine receptor antagonists confirming the M1 nature of the antagonist effect of telenzepine. 2. Field stimulation (7 V, 10 Hz, 0.5 ms) resulted in a plateau acid secretion over at least 90 min which was completely blocked by either 1 mumol/l TTX or H2 receptor antagonists (100 mumol/l cimetidine or 10 mumol/l lupitidine). Ranitidine, which is known to potently inhibit mucosal acetylcholine esterase, was ineffective. Compound 48/80 at 100 mumol/l, which depletes mucosal histamine stores, initially mimicked electrical stimulation but subsequently prevented it from inducing acid secretion. 3. 10 muscarine receptor antagonists with differing relative affinities for M1, M2 and M3 receptors were introduced at 1 mumol/l to inhibit electrically-stimulated acid secretion. The percentages inhibition were plotted against binding affinities of the antagonists at either M1, M2 or M3 binding sites. A statistically significant correlation between functional and binding data was detected only when based on M1 affinities. 4. It is concluded that field stimulation, which probably mimicks vagal drive, results in muscarinic M1 receptor activation on paracrine cells to release histamine. Histamine then stimulates parietal cells to secrete acid. Hence, according to the present and our previous data, telenzepine inhibits acid secretion under these conditions by blocking M1 receptors at least partially located on histamine-releasing paracrine cells.

Topics: Acetylcholine; Alkaloids; Animals; Anti-Ulcer Agents; Electric Stimulation; Furans; Gastric Acid; Gastric Mucosa; Histamine; Mice; Muscarinic Antagonists; Naphthalenes; p-Methoxy-N-methylphenethylamine; Parasympatholytics; Piperidines; Pirenzepine; Stomach; Thiazepines; Vagus Nerve

A selective amplification of the coding sequence of the rat M2 muscarinic receptor gene was achieved by the polymerase chain reaction. The error rate of this amplification system under conditions specified was 1 nucleotide substitution in 841 base pairs. In vitro expression of this gene in murine fibroblasts (B82) via the eukaryotic expression vector, pH beta APr-1-neo, resulted in high level expression of specific [3H] (-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of [3H] (-)MQNB binding with a Kd value of 265 pM and a Bmax value of 411 +/- 50 fmol/10(6) cells. Cardiac selective muscarinic antagonists such as himbacine and AF-DX 116 show high affinities for this binding site in the M2LKB2-2 cells. The rank order of potency of several antagonists in inhibiting [3H] (-)MQNB binding in these cells conformed to the characteristics of an M2 type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a Ki value of 2.0 microM. This receptor appeared to be inversely coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. Carbachol also had a slight stimulatory effect on the hydrolysis of inositol lipids. The polymerase chain reaction proves highly effective in cloning genes from genomic material, as demonstrated by the first in vitro functional expression of the rat M2 type muscarinic receptor.

Topics: Alkaloids; Amino Acid Sequence; Animals; Base Sequence; Carbachol; Cell Line; Cloning, Molecular; Cyclic AMP; Fibroblasts; Furans; Gene Amplification; Gene Expression; Molecular Sequence Data; Muscarinic Antagonists; Naphthalenes; Phosphatidylinositols; Piperidines; Pirenzepine; Plasmids; Polymerase Chain Reaction; Rats; Receptors, Muscarinic; Transfection

The possibility of an allosteric interaction by himbacine, a cardioselective antagonist, with rat cardiac muscarinic receptors was studied. Himbacine allosterically decelerated the dissociation of bound [3H]N-methylscopolamine [( 3H]NMS) in a concentration-dependent manner with an IC50 value of 103.7 microM. When compared to the IC50 values of other cardioselective antagonists, the rank order of potencies was: methoctramine greater than gallamine greater than himbacine greater than AF-DX 116. In contrast, the potencies of these compounds to displace [3H]NMS binding were: himbacine greater than methoctramine greater than AF-DX 116 greater than gallamine. The allosteric potencies were found not to be correlated with binding potencies (correlation coefficient = -0.15). A striking common feature of the cardioselective antagonists is their ability to bind to an allosteric site on cardiac muscarinic receptors.

Topics: Alkaloids; Allosteric Regulation; Animals; Atropine; Binding, Competitive; Diamines; Dose-Response Relationship, Drug; Furans; Gallamine Triethiodide; Male; Myocardium; N-Methylscopolamine; Naphthalenes; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine Derivatives

Topics: Alkaloids; Animals; Atropine; Carbachol; Furans; Guinea Pigs; Heart Atria; Ileum; In Vitro Techniques; Naphthalenes; Parasympatholytics; Piperidines; Receptors, Cholinergic

To compare the proportions of four muscarinic receptors in different rat brain regions, we used competition curves with four selective antagonists, at 1-[N-methyl-3H]scopolamine methyl chloride [( 3H]NMS) binding equilibrium and after allowing [3H]NMS dissociation for 35 min. Himbacine and methoctramine were shown to discriminate two muscarinic receptor subtypes having a high affinity for 4-diphenylacetoxy-N-methylpiperidine methiodide and hexahydrosiladifenidol, intermediate affinity for pirenzepine, and low affinity for AF-DX 116. One M4 subtype had a high affinity for himbacine and methoctramine; it was found predominantly in homogenates from rat striatum (46% of total [3H]NMS receptors) and in lower proportion in cortex (33% of [3H]NMS receptors) and hippocampus (16% of [3H]NMS receptors). Its binding properties were identical to those of muscarinic receptors in the neuroblastoma x glioma NG 108-15 hybrid, suggesting that it was encoded by m4 mRNA. The M3 subtype (typically found in rat pancreas, a tissue expressing the m3 mRNA) had a low affinity for himbacine and methoctramine and represented about 10% of all [3H]NMS receptors in rat brain cortex, hippocampus, striatum, and cerebellum. M1 and M2 receptors were identified in rat brain by their high affinity for pirenzepine and AF-DX 116, respectively.

Topics: Alkaloids; Animals; Brain; Diamines; Furans; Humans; Hybrid Cells; N-Methylscopolamine; Naphthalenes; Parasympatholytics; Piperidines; Rats; Receptors, Muscarinic; Scopolamine Derivatives; Tumor Cells, Cultured

Himbacine, himbeline, N-methylhimandravine and himandravine together with their dihydro-derivatives were evaluated as antagonists of muscarinic receptors in guinea-pig ileal longitudinal muscle and electrically stimulated left atrium. Himbacine was the most potent compound and the 15-fold selectivity exhibited for the M2 muscarinic receptor was greater than that found with any of the other compounds examined. Reduction of the double bond linking the decalin ring system and the piperidine ring almost abolished selectivity in dihydrohimbacine. Removal of the N-methyl group in himbacine to form himbeline was associated with reduced selectivity. However the corresponding change in converting N-methylhimandravine to himandravine was not associated with any change in selectivity suggesting that orientation of the 2-methyl group in the piperidine ring may be important for selectivity.

Topics: Alkaloids; Animals; Carbachol; Electric Stimulation; Furans; Guinea Pigs; Ileum; In Vitro Techniques; Muscle Contraction; Muscle, Smooth, Vascular; Naphthalenes; Parasympatholytics; Piperidines; Plants, Medicinal; Structure-Activity Relationship

Vecuronium and himbacine are antimuscarinic compounds which in functional studies exhibited a ca 6- and 10-fold higher potency at cardiac muscarinic receptors than at ileal muscarinic receptors. However in binding studies both compounds failed to differentiate between [3H](-)-QNB binding sites in guinea-pig atrial and ileal muscle homogenates. In the latter experiments, the dissociation constants of vecuronium in atria and ileum and that of himbacine in ileum were lower than the values determined functionally. The basis for the lack of cardioselectivity in binding studies is not known. These compounds add to the list of functional cardioselective muscarinic receptor antagonists that failed to display selectivity in binding studies with [3H](-)-QNB.

Topics: Alkaloids; Animals; Binding, Competitive; Furans; Guinea Pigs; Heart; Heart Atria; Ileum; In Vitro Techniques; Naphthalenes; Parasympatholytics; Piperidines; Vecuronium Bromide

1. The action of methoctramine and himbacine at muscarinic receptors has been studied using guinea-pig isolated trachea, oesophageal muscularis mucosae, paced left atria, and rat aortic preparations. 2. Methoctramine (1 x 10(-6)-3.2 x 10(-4) M), but not himbacine, elicited positive inotropic responses. These responses were enhanced by pretreating the animals with reserpine. The responses in reserpine-treated animals were not antagonized by phentolamine (1 x 10(-6) M) but were antagonized by propranolol (1 x 10(-6) M). 3. Methoctramine, but not himbacine, exhibited allosteric inhibitory effects at cardiac muscarinic receptors, resulting in a curvilinear Schild plot. Deviations from competitive antagonism were also observed in combination dose-ratio experiments using atropine and methoctramine. At 1 x 10(-6) M, the pKB value for methoctramine was 7.88 +/- 0.15 (mean +/- s.e.mean, n = 5). The pA2 value for himbacine at cardiac muscarinic receptors was 8.52 +/- 0.06 (n = 3). 4. At tracheal and oesophageal muscularis mucosal smooth muscle receptors, the Schild plots for both antagonists were linear. The pA2 values for methoctramine at receptors in these two preparations were similar (6.08 +/- 0.05 and 6.03 +/- 0.09 respectively, n = 4) and were approximately 60 fold less than those values observed at atrial receptors. Himbacine, also exhibited similar values at muscarinic receptors in the trachea and oesophageal muscularis mucosae (7.61 +/- 0.05 and 7.57 +/- 0.04 respectively, n = 4). 5. Muscarinic receptors mediating relaxation of the rat aortic endothelium exhibited pA2 values for methoctramine (5.87 +/- 0.12, n = 6) which were similar to those observed in the smooth muscle, but not the atria. The pA2 values for himbacine at endothelial muscarinic receptors were approximately 0.5 pA2 units lower than those observed at muscarinic receptors in smooth muscle (6.92 + 0.80, n = 6). In addition, the Schild slopes for methoctramine and himbacine at these receptors were significantly (P < 0.05) less than unity. 6. Methoctramine, and to a lesser extent himbacine, are potent and selective antagonists for cardiac muscarinic receptors. However, caution should be used in interpretation of the data with methoctramine in view of the inhibitory allosteric properties and direct inotropic actions of this compound.

Topics: Alkaloids; Animals; Diamines; Drug Interactions; Endothelium; Furans; Guinea Pigs; Heart Atria; In Vitro Techniques; Male; Muscle, Smooth; Naphthalenes; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic

The in vitro receptor binding properties of a muscarinic antagonist himbacine have been studied in rat cerebral cortical, cardiac and ileal membranes. Himbacine displayed high affinity (KH = 2.94 nM) for 19%, and low affinity (KL = 71.2 nM) for the remaining muscarinic receptors in rat cerebral cortex. This high affinity of himbacine agrees with its affinity for the 62% of cerebral cortical [3H]AF-DX 116 binding sites (KH = 2.30 nM). The affinity of himbacine for cardiac receptors (Ki = 9.06 nM) and ileal receptors (Ki = 12.4 nM) was the same. Therefore, himbacine appears to be a high-affinity M2-selective ligand which recognizes a subtype of M2 receptors in the cerebral cortex.

Topics: Alkaloids; Animals; Binding, Competitive; Cell Membrane; Cerebral Cortex; Furans; Ileum; Kinetics; Male; Myocardium; Naphthalenes; Organ Specificity; Parasympatholytics; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic

The interaction of himbacine with carbachol was studied and compared with that of homatropine in guinea-pig atria and rat stomach fundus. Himbacine was a competitive antagonist of carbachol in both preparations. In atria, himbacine had similar affinity for the muscarinic receptors associated with force (pA2 = 8.33) and rate of contraction (pA2 = 8.42). In stomach, himbacine had a pA2 value of 7.29 which corresponds to a 10-fold less affinity than for atria. In contrast, homatropine had similar affinities for muscarinic receptors in stomach (pA2 = 7.13) and for those in atria mediating force (pA2 = 7.21) and rate (pA2 = 7.07) responses. These results indicate that himbacine distinguishes between cardiac and smooth muscle muscarinic receptors whereas it is unable to distinguish between possible subtypes of cardiac receptors associated with force and rate responses.

Topics: Alkaloids; Animals; Carbachol; Female; Furans; Guinea Pigs; Heart; Heart Rate; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Myocardial Contraction; Naphthalenes; Parasympatholytics; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Stomach

The antimuscarinic actions of himbacine were compared with those of atropine and/or homatropine on atria, ileum and trachea from guinea-pigs and rat uterus preparations. The antagonism of acetylcholine or carbachol by all the antagonists was competitive on the preparations studied. The pA2 values of himbacine in all smooth muscle preparations were similar (around 7.2) whereas in atria it exhibited about 10-fold higher affinity (pA2 = 8.2). In contrast, both atropine and homatropine had similar affinities for muscarine receptors (pA2 values around 9.1 and 7.2 respectively) in both atria and smooth muscle. It may be concluded from these results that cardiac and smooth muscle muscarine receptors are not homogeneous and that himbacine is a relatively potent and selective antagonist for cardiac receptors. The cardio-selectivity of himbacine supports the concept of heterogeneity of muscarine receptors.

Topics: Alkaloids; Animals; Atropine; Diethylstilbestrol; Female; Furans; Guinea Pigs; Heart; Ileum; In Vitro Techniques; Muscle, Smooth; Myocardial Contraction; Naphthalenes; Parasympatholytics; Piperidines; Rats; Trachea; Uterine Contraction