FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors.

Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M(1)-, M(3)-, and M(5)-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M(1)- and M(5)-acetylcholine receptors and the amplitude of these signals was larger at the M(1)-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M(1)-, M(3)- and M(5)-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation.

Aequorin functional assay for characterization of G-protein-coupled receptors: implementation with cryopreserved transiently transfected cells.

Assay technologies that measure intracellular Ca(2+) release are among the predominant methods for evaluation of GPCR function. These measurements have historically been performed using cell-permeable fluorescent dyes, although the use of the recombinant photoprotein aequorin (AEQ) as a Ca(2+) sensor has gained popularity with recent advances in instrumentation. The requirement of the AEQ system for cells expressing both the photoprotein and the GPCR target of interest has necessitated the labor-intensive development of cell lines stably expressing both proteins. With the goal of streamlining this process, transient transfections were used to either (1) introduce AEQ into cells stably expressing the GPCR of interest or (2) introduce the GPCR into cells stably expressing the AEQ protein, employing the human muscarinic M(1) receptor as a model system. Robust results were obtained from cryopreserved cells prepared by both strategies, yielding agonist and antagonist pharmacology in good agreement with literature values. Good reproducibility was observed between multiple transient transfection events. These results indicate that transient transfection is a viable and efficient method for production of cellular reagents for use in AEQ assays.

Exploring the mechanism of agonist efficacy: a relationship between efficacy and agonist dissociation rate at the muscarinic M3 receptor.

Although there are several empirical approaches that enable the comparison of relative agonist efficacy, the molecular basis that underlies differences in the ability of G protein-coupled receptor agonists to elicit a response is still largely unexplained. Several models have been described that incorporate the kinetics of receptor-mediated initiation of the G protein cycle, but these have not directly addressed the influence of agonist-binding kinetics. To test this, we investigated the relationship between the efficacy of seven M(3) muscarinic receptor agonists and their rate of dissociation (k(off)) from the M(3) receptor. The association and dissociation rate constants of the agonists were determined using a l-[N-methyl]-[(3)H]scopolamine methyl chloride competition binding assay in the presence of GTP. The agonists displayed a range of association and dissociation rates. Relative agonist efficacy was measured at two points after M(3) receptor activation: the stimulation of guanosine 5'-O-(3-[(35)S]thio)triphosphate binding to G alpha subunits, and the subsequent increase in intracellular calcium levels. These experiments revealed a range of intrinsic efficacy, from the low-efficacy pilocarpine and oxotremorine to high-efficacy acetylcholine. There was no relationship between agonist efficacy and the equilibrium binding affinity of each agonist (K(d)). When efficacy was compared with the dissociation rate constant, however, the two were highly correlated, suggesting a relationship between the duration of agonist binding at the receptor and the intrinsic efficacy. These data suggest that kinetic models incorporating the mean lifetime of specific complexes will be required to fully explain the nature of agonist efficacy.

L- and N-current but not M-current inhibition by M1 muscarinic receptors requires DAG lipase activity.

Stimulation of postsynaptic M(1) muscarinic receptors (M(1)Rs) increases firing rates of both sympathetic and central neurons that underlie increases in vasomotor tone, heart rate, and cognitive memory functioning. At the cellular level, M(1)R stimulation modulates currents through various voltage-gated ion channels, including KCNQ K+ channels (M-current) and both L- and N-type Ca2+ channels (L- and N-current) by a pertussis toxin-insensitive, slow signaling pathway. Depletion of phosphatidylinositol-4,5-bisphosphate (PIP2) during M(1)R stimulation suffices to inhibit M-current. We found previously that following PIP2 hydrolysis by phospholipase C, activation of phospholipase A2 and liberation of a lipid metabolite, most likely arachidonic acid (AA) are necessary for L- and N-current modulation. Here we examined the involvement of a third lipase, diacylglycerol lipase (DAGL), in the slow pathway. We documented the presence of DAGL in superior cervical ganglion neurons, and then tested the highly selective DAGL inhibitor, RHC-80267, for its capacity to antagonize M(1)R-mediated modulation of whole-cell Ca2+ currents. RHC-80267 significantly reduced L- and N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M) but did not affect their inhibition by exogenous AA. Moreover, voltage-dependent inhibition of N-current by Oxo-M remained in the presence of RHC-80267, indicating selective action on the slow pathway. RHC also blocked inhibition of recombinant N-current. In contrast, RHC-80267 had no effect on native M-current inhibition. These data are consistent with a role for DAGL in mediating L- and N-current inhibition. These results extend our previous findings that the signaling pathway mediating L- and N-current inhibition diverges from the pathway initiating M-current inhibition.

Circuit Robustness to Temperature Perturbation Is Altered by Neuromodulators.

In the ocean, the crab Cancer borealis is subject to daily and seasonal temperature changes. Previous work, done in the presence of descending modulatory inputs, had shown that the pyloric rhythm of the crab increases in frequency as temperature increases but maintains its characteristic phase relationships until it "crashes" at extremely high temperatures. To study the interaction between neuromodulators and temperature perturbations, we studied the effects of temperature on preparations from which the descending modulatory inputs were removed. Under these conditions, the pyloric rhythm was destabilized. We then studied the effects of temperature on preparations in the presence of oxotremorine, proctolin, and serotonin. Oxotremorine and proctolin enhanced the robustness of the pyloric rhythm, whereas serotonin made the rhythm less robust. These experiments reveal considerable animal-to-animal diversity in their crash stability, consistent with the interpretation that cryptic differences in many cell and network parameters are revealed by extreme perturbations.

Impaired cardiac muscarinic receptor function in dogs with heart failure.

Prior physiological studies have suggested that parasympathetic control is altered in heart failure. The goal of our studies was to investigate the influence of heart failure on the muscarinic receptor, and its coupling to adenylate cyclase. Ligand binding studies using [3H]quinuclidinyl benzilate and enriched left ventricular (LV) sarcolemma, demonstrated that muscarinic receptor density in heart failure declined 36% from a control of 5.6 +/- 0.6 pmol/mg, with no change in antagonist affinity. However, agonist competition studies with both carbachol and oxotremorine showed that it was a loss of high affinity agonist binding sites in the sarcolemma from failing LV that accounted for this difference. The functional efficacy of the muscarinic receptor was also examined. When 1 microM methacholine was added to 0.1 mM GTP and 0.1 mM isoproterenol, adenylate cyclase stimulated activity was inhibited by 15% in normal LV but only 5% in LV sarcolemma from animals with heart failure even when the reduced adenylate cyclase in these heart failure animals was taken into account. Even at 100-fold greater concentrations of methacholine, significantly less inhibition of adenylate cyclase activity was observed in LV failure as compared with normal LV sarcolemma. Levels of the GTP-inhibitory protein known to couple the muscarinic receptor to adenylate cyclase, as measured with pertussis toxin labeling, were not depressed in LV failure. Thus, the inhibitory pathway regulating LV adenylate cyclase activity is defective in heart failure. The decrease in muscarinic receptor density, and in particular the specific loss of the high affinity agonist binding component of this receptor population, appears to be the major factor underlying this abnormality.

Comparative in vivo effects of parathion on striatal acetylcholine accumulation in adult and aged rats.

Aged rats are more sensitive to the acute toxicity of the prototype organophosphate insecticide, parathion. We compared the acute effects of parathion on diaphragm and brain regional cholinesterase activity, muscarinic receptor binding and striatal acetylcholine levels in 3- and 18-month-old male Sprague-Dawley rats. Adult and aged rats were surgically implanted with a microdialysis cannula into the right striatum 5-7 days prior to parathion treatment. Rats were given either vehicle (peanut oil, 2 ml/kg) or one of a range of dosages of parathion (adult: 1.8, 3.4, 6.0, 9.0, 18 and 27 mg/kg, s.c.; aged: 1.8, 3.4, 6 and 9 mg/kg, s.c.) and body weight, functional signs of toxicity, and nocturnal motor activity were recorded for seven days. Three and seven days after parathion treatment, microdialysis samples were collected and rats were subsequently sacrificed for biochemical measurements. Higher dosages of parathion led to significant time-dependent reductions in body weight in both age groups. Rats in both age groups treated with lower dosages showed few overt signs of cholinergic toxicity while equitoxic high dosages (adult, 27 mg/kg; aged, 9 mg/kg) elicited marked signs of cholinergic toxicity (involuntary movements and SLUD [i.e., acronym for Salivation, Lacrimation, Urination and Defecation] signs) with peak effects being noted 3-4 days after treatment. Nocturnal activity (ambulation and rearing) was reduced in both age groups following parathion dosing, with more prominent effects in adults and rearing being more consistently affected. Dose- and time-dependent inhibition of cholinesterase activity was noted in both diaphragm and striatum. Total muscarinic receptor ([(3)H]quinuclidinyl benzilate, QNB) binding was significantly lower in aged rats, and both total binding and muscarinic agonist ([(3)H]oxotremorine methiodide] binding was significantly reduced in both age-groups treated with the highest dosages of parathion (adult, 27 mg/kg; aged, 9 mg/kg). In contrast to relatively similar levels of cholinesterase inhibition, striatal extracellular acetylcholine levels were significantly lower (2.2- to 2.9-fold) in aged rats at both 3 and 7 day time-points compared to adult rats treated with equitoxic dosages (i.e., 9 and 27 mg/kg, respectively). No age-related differences in in vitro striatal acetylcholine synthesis or in vivo acetylcholine accumulation following direct infusion of the cholinesterase inhibitor neostigmine (1 microM) were noted. While aged rats are more sensitive than adults to the acute toxicity of parathion, lesser acetylcholine accumulation was noted in the striatum of aged rats exhibiting similar levels of cholinesterase inhibition. These findings suggest that lesser acetylcholine accumulation may be required to elicit cholinergic signs in the aged rat, possibly based on aging-associated changes in muscarinic receptor density.

Muscarinic receptor coupling to intracellular calcium release in rat pancreatic acinar carcinoma.

Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cholinergic receptor protein affinity labeled with the muscarinic antagonist [3H]propylbenzilylcholine mustard revealed a major polypeptide with molecular weight of 80,000-83,000 in both acinar carcinoma and normal acinar cells of rat pancreas. Muscarinic receptor protein is therefore conserved in pancreatic acinar carcinoma. A small but significant difference was detected in the affinity of carcinoma cell receptors (Kd approximately 0.6 nM) and normal cell receptors (Kd approximately 0.3 nM) for reversible binding of the muscarinic antagonist drug, N-methylscopolamine. In addition, carcinoma cell muscarinic receptors displayed homogeneous binding of the agonist drugs carbamylcholine (Kd approximately 31 microM) and oxotremorine (Kd approximately 4 microM), whereas normal cell receptors demonstrated heterogeneous binding, with a minor receptor population showing high affinity binding for carbamylcholine (Kd approximately 3 microM) and oxotremorine (Kd approximately 160 nM), and a major population showing low affinity binding for carbamylcholine (Kd approximately 110 microM) and oxotremorine (Kd approximately 18 microM). Both carcinoma and normal cells exhibited concentration-dependent carbamylcholine-stimulated increases in cytosolic free Ca2+, as measured by 45Ca2+ outflux assay and intracellular quin 2 fluorescence. However, carcinoma cells were observed to be more sensitive to Ca2+ mobilizing actions of submaximal carbamylcholine concentrations, demonstrating 50% maximal stimulation of intracellular Ca2+ release at a carbamylcholine concentration (approximately 0.4 microM) approximately one order of magnitude below that seen for normal cells. These results indicate altered muscarinic receptor coupling to intracellular Ca2+ release in acinar carcinoma cells, which manifests as a single activated receptor state for agonist binding, and increased sensitivity of Ca2+ release in response to muscarinic receptor stimulation.

Agonist specific effects of guanine nucleotides on muscarinic cholinergic receptors in rat anterior pituitary membranes.

The effects of guanine nucleotides on the binding affinity of muscarinic cholinergic receptors for muscarinic agents were studied in rat anterior pituitary membranes using direct ligand binding methods with [3H]quinuclidinyl benzylate, GTP and Gpp(NH)p at a concentration of 0.1 mM markedly decreased the binding affinity of the agonist, oxotremorine, for the receptors but had no effect on the binding of the antagonist, atropine. Mg2+ (1 mM) on the other had markedly increased the binding affinity of oxotremorine but not that of atropine. Thus, it is conceivable that the release of the growth hormone or the inhibition of prolactin release by acetylcholine, which we and others have previously shown, is modulated by the opposite actions of guanine nucleotides and divalent metal ions such as Mg2+.

Arachidonic acid release in cell lines transfected with muscarinic receptors: a simple functional assay to determine response of agonists.

Muscarinic agonists stimulated arachidonic acid release from 10- to 32-fold in Chinese hamster ovary (CHO) cells transfected with muscarinic M1, M3 and M5 receptor subtypes. Muscarinic agonists liberated arachidonic acid from the cAMP-coupled M2 and M4 cells only in the presence of ATP. Partial agonists were less efficacious at liberating arachidonic acid than full agonists. The ability of muscarinic agonists to liberate arachidonic acid and stimulate phosphoinositide hydrolysis in the same CHO M1, M3 and M5 cells was well correlated; however, partial agonists were more efficacious at stimulating phosphoinositide hydrolysis than arachidonic acid release. The efficacy and potency of 13 muscarinic agonists to liberate arachidonic acid was characterised. Influx of external calcium was required for arachidonic acid release even after initiation of agonist-induced release. It is concluded that arachidonic acid release is a simple assay suitable for evaluation of muscarinic agonists, antagonists and the flux of external calcium into cells.

Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging.

The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of [3H]-antagonist as well as of the labeled natural neurotransmitter, [3H]-acetylcholine [( 3H]-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of [3H]-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity [3H]-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with [3H]-antagonist represent a loss of low-affinity agonist binding sites. In contrast, [3H]-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms.

Redox modulation of Gi protein expression and adenylyl cyclase signaling: role of nitric oxide.

Nitric oxide (NO) has been shown to regulate a variety of physiological functions, including vascular tone. The inhibition of NO synthase by N(omega)-nitro-L-arginine methyl ester (L-NAME) has been reported to increase arterial blood pressure. The present studies were undertaken to investigate if the increased blood pressure by L-NAME is associated with enhanced expression of Gi proteins, implicated in the pathogenesis of hypertension. L-NAME was administered orally into Sprague-Dawley rats for a period of 4 weeks. Control rats were given plain tap water only. The systolic blood pressure was enhanced in L-NAME-treated rats as compared with control rats; however, the heart-to-body weight ratio was not different in the two groups. The levels of Gialpha-2 and Gialpha-3 proteins and their mRNA as determined by western and northern blotting, respectively, were significantly augmented in hearts from L-NAME-treated rats, whereas the levels of Gsalpha and Gbeta were unaltered. In addition, the effect of low concentrations of GTPgammaS on forskolin-stimulated adenylyl cyclase activity (receptor-independent functions of Gialpha) was significantly enhanced, whereas the receptor-dependent inhibitions of adenylyl cyclase were completely attenuated in L-NAME-treated rats. Whereas cholera toxin-mediated stimulation of adenylyl cyclase was unaltered in both group of rats, the stimulatory effects of some agonists on adenylyl cyclase activity were diminished in L-NAME-treated rats. These results suggest the implication of NO in the modulation of Gi protein expression and associated adenylyl cyclase signaling.

Limbic thalamus in rabbit: architecture, projections to cingulate cortex and distribution of muscarinic acetylcholine, GABAA, and opioid receptors.

Nuclei of the thalamus that project to cingulate cortex have been implicated in responses to noxious stimuli, cholinergic and motor functions. The rabbit limbic thalamus may play an important role in these functions, but has not been studied extensively in terms of its cytoarchitecture, the topographical organization of its cortical projections, and differential transmitter regulation of its subnuclei. Therefore, the architecture, projections to cingulate cortex, and radioligand binding were investigated in the anterior, ventral, lateral, and midline nuclei of rabbit thalamus. The anterior nuclei are highly differentiated because both the dorsal and ventral nuclei have parvicellular and magnocellular divisions. Fluorescent dyes were injected into cingulate cortex to evaluate limbic thalamocortical connections. The anterior medial, submedial, and parafascicular nuclei project primarily to anterior cingulate cortex, while they have small or no projections to posterior areas. The ventral anterior and ventral lateral nuclei have a significant projection to dorsal cingulate cortex, including areas 24b and 29d. Projections of the anterior ventral nucleus are topographically organized, since medial parts of the parvicellular division project to rostral area 29, and lateral parts project to caudal area 29. The lateral nuclei and the parvicellular and magnocellular divisions of the anterior dorsal nucleus project with progressively higher densities in the rostrocaudal plane of area 29. Finally, the magnocellular division of the anterior ventral nucleus projects almost exclusively to caudal and ventral area 29, i.e., granular retrosplenial cortex. Ligand binding studies employed coverslip autoradiography and single grain counting techniques. Muscarinic receptor binding was moderate for both pirenzepine and oxotremorine-M in the parvicellular anterior ventral nucleus, while in other nuclei, there was an inverse relationship in the binding for these ligands. Most notably, the anterior dorsal nucleus, which receives no cholinergic input, had very high oxotremorine-M and low pirenzepine binding, while the anterior medial nucleus, which receives a moderate cholinergic input, had the highest pirenzepine binding and very low oxotremorine-M binding. Muscimol binding to GABAA receptors was highest in the anterior ventral nucleus, while it was at moderate levels in the anterior dorsal and lateral nuclei. The binding of Tyr-D-Ala-Gly-MePhe-Gly-ol to mu opioid receptors and 2-D-penicillamine-5-D-penicillamine-enkephalin to delta opioid receptors were both high in the parvicellular and low in the magnocellular divisions of the anterior dorsal nucleus. The magnocellular division of the anterior ventral, the lateral dorsal, and the parafascicular nuclei had high mu opioid binding, while the lateral dorsal and lateral magnocellular nuclei had low levels of delta opioid binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoamine neurotransmitter receptors: ligand-receptor models.

Recent medicinal chemistry studies have led to the idea that muscarinic ligands of differing efficacy employ distinctly different pharmacophores when interacting with their specific receptor. Although the primary recognition element, the cationic head group, remains constant for all ligands, two H-bonding interactions are used selectively to stabilize agonist binding. In contrast, antagonists rely predominantly upon hydrophobic binding in the vicinity of the acetylmethyl group present in the endogenous transmitter. These ideas have been evaluated in the context of a three-dimensional model of the muscarinic receptor derived originally from bacteriorhodopsin. Together these studies have allowed speculations to be made regarding the sequence of events which lead to coupling to G-protein. Although an Asp residue on helix III of the seven-helical model is known to be involved as the primary recognition site for all ligands, a second, more deeply buried Asp on helix II is suggested to represent the ultimate agonist-binding site from which receptor activation is triggered. Similar arguments are also applied to homologous receptor systems, in particular the beta-adrenergic receptor.

Subtypes of muscarinic receptors in cultured explants of the hippocampus of the rat.

Using the tritiated, muscarinic antagonist quinuclidinyl benzilate ([3H]QNB) as a ligand, muscarinic receptors have been identified and characterized in intact, cultured explants of the hippocampus of the rat. Competition studies with scopolamine and oxotremorine indicated a certain heterogeneity in the population of muscarinic receptors, whereas atropine and pirenzepine competed with [3H]QNB in a manner consistent with only one binding site for these substances. Thus, the observed heterogenity does not fit in with the M1/M2 receptor concept. Extended studies, with the aim of determining to what extent these putative subtypes of receptors are functional, would be of interest.

Effect of irreversible loss of muscarinic receptors on (3H)-acetylcholine release from the hippocampus.

The role of muscarinic receptors in the regulation of (3H)-acetylcholine (3H-Ach) release from the hippocampus was investigated with the irreversible cholinergic agonist BM-123 (N-[4(2-chloroethylmethylamino)-2-butynyl]2-pyrrolidone). Pretreatment with BM-123 had no significant effect on spontaneous (3H)-Ach release at 12, 24, 72, and 144 hr. However, this treatment significantly reduced the inhibitory effect of oxotremorine (10 microM) on (3H)-Ach release at 12, 24, and 72 hr (p less than 0.02). At these time intervals, there was a significant loss in muscarinic receptors as determined by (3H)-oxotremorine-M binding. The maximum loss of both oxotremorine-induced inhibition of (3H)-Ach release and muscarinic receptors occurred at 12 hr. Both parameters returned to normal by 144 hr. There was a linear relationship between the recovery of (3H)-Ach release and (3H)-oxotremorine-M binding sites. These results indicate that muscarinic receptors play a significant role in the regulation of (3H)-Ach release in hippocampus, and that this receptor system may lack spare receptors.

Synthesis and muscarinic activities of 1,2,4-thiadiazoles.

A series of novel 1,2,4-thiadiazoles bearing a mono- or bicyclic amine at C5 were prepared. Quinuclidine and 1-azabicyclo[2.2.1]heptane derivatives were synthesized by reaction of the lithium enolate of the 3-methoxycarbonyl compounds followed by ester hydrolysis and decarboxylation. The receptor-binding affinity and efficacy of these compounds as muscarinic ligands was assessed by radioligand binding assays using [3H]-N-methylscopolamine and [3H]oxotremorine-M. Optimal agonist affinity was observed for 3'-methyl compounds. Smaller substituents (H) retained efficacy with reduced affinity while larger groups led to substantially lower efficacy. The observed binding affinity was influenced both by the conformational energy of rotation around the C3-C5' bond and the steric requirement of the mono- or bicyclic amine.

The synthesis and biochemical pharmacology of enantiomerically pure methylated oxotremorine derivatives.

Previous pharmacological studies of methylated oxotremorine derivatives bearing substituents at the 3-, 4-, and 5-positions of the pyrrolidinone ring have been conducted using racemic mixtures, and not with optically active compounds. The synthesis and radioligand binding data of optically active, methylated oxotremorine derivatives at the 3- and 4-positions are described. There are significant pharmacological differences between the 3- and 4-position derivatives. The 4-position enantiomers have weak, approximately equal affinity and antagonist-like profiles, whereas the 3-position enantiomers have significantly different affinities and partial agonist-like profiles.

Muscarinic analgesics with potent and selective effects on the gastrointestinal tract: potential application for the treatment of irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a pathopysiolocal condition characterized by abnormal bowel habits that are frequently accompanied by abdominal pain. Current therapy based on reducing high-amplitude GI contractions with nonselective muscarinic antagonists is limited in efficacy due to typical muscarinic side effects and provides no pain relief. We have previously found potent antinociceptive agents acting through muscarinic receptors. In the present work, new 1,2,5-thiadiazole-based structures with muscarinic activity have been evaluated both for activity as analgesics in the mouse withing assay and for activity in normalizing spontaneous cluster contractions in ferret jejunum as a model of IBS in humans. (5R,6R)-exo-6-[4-[(4,4,4-Trifluorobutyl)thio]-1,2,5-thiadiazol+ ++-3-yl] -1-azabicyclo[3.2.1]octane (35, LY316108/NNC11-2192) was found to offer an exceptional profile combining analgesic potency in mouse writhing (ED50 = 0.1 mg/kg) along with potency for normalization of GI motility (ED50 = 0.17 mg/kg). This combination of GI and analgesic potency suggests 35 as an excellent candidate for evaluation as a potential treatment of IBS.

2-Methyl-1,3-dioxaazaspiro[4.5]decanes as novel muscarinic cholinergic agonists.

Many nonquaternary ammonium muscarinic agonists have been developed over the last few years, but most of the existing compounds (e.g., arecoline, RS-86, AF-30) behave as weak partial agonists at cholinergic receptors in tissues of limited receptor reserve. The current paper describes the synthesis and biochemical assessment of analogues of AF-30 designed to have sufficient conformational freedom to allow greater receptor flexibility and hence activation. The new compounds and important standards were tested in a new biochemical assay designed to measure both receptor affinity and intrinsic activity of each compound and for their ability to stimulate phosphatidylinositol turnover in rat cerebral cortex. Two azaspirodecanes (5a and 5b) were shown to have far greater predicted efficacy than AF-30.