Magnetic "fishing" assay to screen small-molecule mixtures for modulators of protein-protein interactions.

Protein-protein interactions are an intricate part of biological pathways and have become important targets for drug discovery. Here we present a two-stage magnetic bead assay to functionally screen small-molecule mixtures for modulators of protein-based interactions, with simultaneous affinity-based isolation of active compounds and identification by mass spectrometry. Proteins of interest interact in solution prior to the addition of Ni(II)-functionalized magnetic beads to recover an intact protein-protein complex through affinity capture of a polyhistidine-tagged primary target ("protein-complex fishing"). Protein-complex fishing, utilizing His(6)-tagged calmodulin (CaM) as the primary (bait) protein and melittin (Mel) as the target, was used to screen a mass-encoded library of 1000 bioactive compounds (50 mixtures, 20 compounds each) and successfully identified three known antagonists, three naturally occurring phenolic compounds previously reported to disrupt CaM-activated phosphodiesterase activity, and two newly identified modulators of the CaM-Mel interaction, methylbenzethonium and pempidine tartrate. The ability to produce quantitative inhibition data is also shown through the development of dose-dependent response curves and the determination of inhibition constants (K(I)) for the novel compound methylbenzethonium (K(I) = 14-49 nM) and two known antagonists, calmidazolium (K(I) = 1.7-7.5 nM) and trifluoperazine (K(I) = 1.2-3.0 µM), with the latter two values being in close agreement with literature values.

Corticosterone implants to the amygdala and type 1 CRH receptor regulation: effects on behavior and colonic sensitivity.

Corticosterone (CORT) micropellets were stereotaxically placed bilaterally at the dorsal margin of the central nucleus of the amygdala (CeA). Both behavioral and physiological responses were recorded (plus maze and colonic discomfort) at 7 days post-implantation. Corticosterone reduced the exploration of the plus maze and increased colonic distress. The ability of a CRH type 1 receptor antagonist, antalarmin, to block behavioral and colonic effects of central placement of CORT was also examined. The diminished exploration in the plus maze and colon distress observed in response to CORT placement at the CeA were averted by the administration of antalarmin. These results provide further evidence for the role of the CRH type 1 receptor to ameliorate both behavioral and physiological functions.

Comparison of Outcomes between Burch Colposuspension with and without Concomitant Abdominal Hysterectomy

A total abdominal hysterectomy may cause a postoperative vesicourethral dysfunction due to an injury to the pelvic nerves. However, many incontinent women with benign diseases of the uterus and its adnexae have undergone a Burch colposuspension with a concomitant abdominal hysterectomy. This study was undertaken to compare the outcomes of a Burch colposuspension performed alone with that of a Burch with a concomitant abdominal hysterectomy. This study included 132 women, who, were treated for primary urinary incontinence from February 1999 to February 2002 and were diagnosed with stress urinary incontinence by means of the urodynamic test at the Department of Obstetrics and Gynecology at Yonsei University Hospital. Forty-two women underwent a Burch colposuspension alone (Burch group) and 90 women underwent a Burch colposuspension with a concomitant abdominal hysterectomy (hysterectomy group). Between the Burch and hysterectomy groups, the mean age, parity, menopausal rate, Hormone Replacement Therapy (HRT) rate, 1 year follow-up outcomes and postoperative complications were compared using the subjective and objective stress tests according to the retrospective chart review. The mean age (54.6 +/- 0.5 vs 58.6 +/- 9.2 years, p=0.382), parity (3.3 +/- 1.2 vs 3.6 +/- 1.7), menopausal rate (71.4 vs 77.7%), or HRT rate (23.3 vs 11.2%) of the two groups were similar. Complications related to surgery were encountered in 5 patients (11.9%) in the Burch group and in 7 patients (7.8%) in the hysterectomy group (p=0.842). One year follow-up subjective symptoms were encounterd in 2 patients in the Burch group and in 4 patients in the hysterectomy group (p=1.00). The stress test was positive in only one patient in the hysterectomy (p=1.00). No significant difference was observed in the 1 year follow-up outcomes, which were 91.4% (32/35 patients) in the Burch and 91.2% (73/80) in the hysterectomy groups. The results showed that there were no adverse effects on the 1 year follow-up outcomes or complications in patients who underwent a Burch colposuspension with an abdominal hysterectomy.

Dronedarone for prevention of atrial fibrillation: a dose-ranging study.

AIMS: Dronedarone, a benzofurane derivative without iodine substituents, shares the electrophysiologic properties of amiodarone. This study was designed to determine the most appropriate dose of dronedarone for prevention of atrial fibrillation (AF) after cardioversion. METHODS AND RESULTS: Patients with persistent AF were randomly allocated to 800, 1200, 1600 mg daily doses of dronedarone or placebo. The main analysis was conducted on 199/270 patients, who entered the maintenance phase following pharmacological cardioversion or, if unsuccessful, DC cardioversion. Within 6-month follow-up, the time to AF relapse increased on dronedarone 800 mg, with a median of 60 days vs 5.3 days in the placebo group (relative risk reduction 55% [95% CI, 28 to 72%] P=0.001). No significant effect was seen at higher doses. Spontaneous conversion to sinus rhythm on dronedarone occurred in 5.8 to 14.8% of patients (P=0.026). There were no proarrhythmic reactions. Drug-induced QT prolongation was only noticed in the 1600 mg group. Premature drug discontinuations affected 22.6% of subjects given 1600 mg dronedarone versus 3.9% on 800 mg and were mainly due to gastrointestinal side effects. No evidence of thyroid, ocular or pulmonary toxicity was found. CONCLUSION: Dronedarone, at a 800 mg daily dose, appears to be effective and safe for the prevention of AF relapses after cardioversion. The absence of thyroid side effects and of proarrhythmia are important features of the drug. Further studies are needed to better delineate the antiarrhythmic profile of the drug.

Understanding channel blocking in the nicotinic acetylcholine receptor.

Ion-channel blockers are molecules that obstruct the path used by ions to cross the membrane through a protein channel. Many of these are local anesthetics, toxins or drugs of abuse, and the knowledge of their mechanism of action at the atomic level is an important step towards the development of new compounds on a structural basis. A molecular model of the transmembrane region of the nicotinic acetylcholine receptor, an important brain and muscle fast signaling protein, was used as a target for docking several channel blockers by means of an automatic docking method. The combination of the independent docking method and molecular models (of the receptor and blockers) reproduced or explained quite accurately experimental data (photoaffinity labeling, site-directed mutagenesis, binding assays). This represents a strong support for the validity of the predictions made for those molecules for which no experimental data is available and also for the models and methods on which are based.

[A comparative study of the central H-cholinergic-blocking and NMDA-blocking actions of MK-801, memantin, amantadine, pyrilen and IEM-1754 in experiments on intact rats]. / Sravnitel'noe issledovanie tsentral'nogo H-kholinoblokiruiushcheo o NMDA-blokiruiushchego deistviia MK-801, memantina, amantadina, pirilena i IEM-1754 v opytakh na intaktnykh krys.

Pyrilene acts as a central H-cholinoblocker upon intramuscular injection at a dose of 0.02-0.08 mg/kg, and as an NMDA-blocker, when the dose is increased to 0.2-0.8 mg/kg. Similarly, amantadine exhibits the properties of H-cholinoblocker and NMDA-blocker in the dose intervals 10-15 mg/kg and 120-180 mg/kg, respectively. The activity of MK-801 markedly exceeds that of memantine, although close doses of both NMDA-blockers inhibit the NMDA and corazole effects, as well as the central effects (analgesia and seizure) of nicotine, thus showing no significant selectivity with respect to NMDA. IEM-1754 tested on intact animals exhibited a selective action upon the NMDA receptors, preventing the NMDA-induced analgesia and lethality and the corazole-induced convulsions at doses 10-100 times lower as compared to those preventing nicotine-induced seizure and analgesia.

Nicotinic antagonists (piperidines and quinuclidines) reduce the susceptibility of early sea urchin embryos to agents evoking calcium shock.

1. Some nicotinic antagonists (piperidine and quinuclidine derivatives and bis-quaternary compounds) protect early embryos of the sea urchin Lytechinus pictus against a calcium shock evoked by ionomycin or a mixture of phorbol myristate acetate and nicotine. 2. Maximal protective potency was found for drugs that did not penetrate the plasma membrane. 3. Early sea urchin embryos have nicotinic acetylcholine receptors (nAChR) or nAChR-like structures localized on the cell surface that, apparently, take part in the control of Ca2+ influx.

[3H]benzylpempidine, a new radioligand for probing a putative channel site on nicotinic cholinergic receptors.

A study was undertaken to assess the receptor binding characteristics of [3H]4-benzylpempidine to an allosteric site on calf brain membranes associated with nicotinic cholinergic receptors and to compare the binding affinity of novel arylpempidine analogs with their ability to antagonize the behavioral effects of nicotine in mice. Scatchard analysis of the binding yielded a K(d) of 20 nM and a B(max) of 330 fmols/mg membrane protein. [3H]4-benzylpempidine appears to be a more satisfactory ligand than [3H]mecamylamine, since it possessed a 50-fold greater affinity and its binding was far less sensitive to inorganic ions and Tris. Among the arylpempidine analogs 4-m-chlorobenzylidenepempidine and 4-benzylidenepempidine had the lowest K(i) values (1.4 nM and 5.0 nM, respectively) and were the most potent in antagonizing nicotine-induced seizures in mice. Although the K(i) values for pempidine and mecamylamine were 1-2 orders of magnitude greater than any of the arylpempidines, the dose required to antagonize nicotine-induced seizures in mice was comparable to the arylpempidines. One explanation for this apparent discrepancy in the correlation of binding affinity and nicotine antagonism is the lower brain penetration of arylpempidines compared to mecamylamine, following their systemic administration to mice.

[Effects of drugs on re-excitability of synapses of isolated frog ganglia]. / Farmakonok hatása a béka izolált ganglion szinapszisának újraingerelhetóségére.

Effects of different pharmacons on the non-excitable period (NEP) and on the relative excitable period (REP) was studied in the sympathetic ganglionic synapses of the frog (Rana esculenta). Using paired stimulation we demonstrated that hexamethonium, magnesium, pempidine, tetraethylammonium (TEA) and d-tubocurarine chlorides significantly prolonged both NEP and REP at ganglion blocking threshold concentrations. Their maximum effect occurred within 30-60 min after the start of the exposition. Hemicholine and neostigmine prolonged only NEP but not REP while lidocaine influenced neither period applying continuous repeated stimulation at low frequencies (0.1-12.5 Hz). TEA showed no effect, however, at higher frequencies (14.3-20.0 Hz) it exerted a frequency-dependent depressant effect on the amplitude of the compound action potential.

Pharmacological characterization of a nicotinic autoreceptor in rat hippocampal synaptosomes.

The modulation of [3H]ACh release by nicotinic compounds was studied in superfused rat hippocampal synaptosomes loaded with [3H]choline, (-)-Nicotine (0.1-10 microM) evoked a dose-dependent increase in [3H]ACh release; higher concentrations were less effective. Nicotine-evoked release was Ca(2+)-dependent, and blocked by the nicotinic antagonists dihydro-beta-erythroidine, mecamylamine, and pempidine. The alpha 7-selective antagonist methyllycaconitine did not inhibit nicotine-evoked release when tested at 1 microM, although at 10 microM some attenuation of the response was observed. Six agonists tested were equally efficacious in stimulating [3H]ACh release, as judged by the maximum responses, and gave the following EC50 values: (+/-)-epibatidine 0.12 microM; (+)-anatoxin-a 0.14 microM; (-)-nicotine 0.99 microM; (-)-cytisine 1.06 microM; ABT-418 2.6 microM; isoarecolone 43 microM. Each agonist generated a "bell-shaped" dose response curve, suggesting desensitisation at higher concentrations. This is supported by analysis of repetitive stimulation with (-)-nicotine and (-)-cytisine: S2/S1 ratios declined sharply with increasing concentration, whereas subsequent KC1-evoked release remained constant. These results are discussed in terms of possible nicotinic receptor subtypes that might be present on hippocampal nerve terminals.

Distinct presynaptic control of dopamine release in striosomal- and matrix-enriched areas of the rat striatum by selective agonists of NK1, NK2, and NK3 tachykinin receptors.

Using a sensitive in vitro microperfusion method, the effects of selective and potent agonists of NK1, NK2, and NK3 tachykinin receptors ([Pro9]SP, ([Lys5,MeLeu9,Nle10]NKA-(4-10), and [Pro7]NKB, respectively) on the presynaptic control of dopamine release were investigated in striosomal-enriched (area rich in [3H]naloxone binding sites) and matrix-enriched areas of the rat striatum. Marked differences could be demonstrated as follows: (i) when used at 0.1 microM, the NK1 agonist stimulated the release of [3H]dopamine continuously synthesized from [3H]tyrosine in both compartments, while the NK2 and NK3 agonists enhanced the release of [3H]dopamine only in the matrix; (ii) the stimulatory effect of the NK3 agonist was less pronounced than those of the NK1 and NK2 agonists; (iii) the NK1 agonist-evoked responses were tetrodotoxin (1 microM) sensitive, while those of the NK2 and NK3 agonists were, respectively, partially and totally tetrodotoxin resistant; (iv) specific receptors are involved in these responses since the stimulatory effects of the NK1 and NK2 agonists were, respectively, blocked by potent antagonists of NK1 (RP-67580; 1 microM) and NK2 (SR-48968; 1 microM) receptors, while these antagonists did not affect the NK3 agonist-evoked response; (v) the indirect stimulatory effect of the NK1 agonist was partially reduced under local blockade of cholinergic transmission in the matrix but not in the striosomal-enriched area. Interestingly, this study also revealed mismatches between autoradiographic data and receptor-mediated responses, since NK2 binding sites could not be observed in the striatum while NK3 but not NK1 binding sites were visualized in the striosomal-enriched area.

Functional heterogeneity of the matrix compartment in the cat caudate nucleus as demonstrated by the cholinergic presynaptic regulation of dopamine release.

Previously, using a new in vitro microsuperfusion procedure, we have demonstrated marked differences in the cholinergic presynaptic regulation of the release of [3H]dopamine continuously synthesized from [3H]tyrosine in two close striosomal- and matrix-enriched areas of the cat caudate nucleus. A tetrodotoxin-resistant stimulatory effect of acetylcholine mediated by muscarinic receptors was observed in both compartments. However, in addition, two opposing types of tetrodotoxin-sensitive acetylcholine-evoked regulation of [3H]dopamine release were only seen in the matrix: one facilitatory, involving nicotinic receptors located on as yet unidentified neurons, and the other inhibitory, mediated by muscarinic receptors located on dynorphin-containing neurons. In the present study, using the same approach, a functional heterogeneity was demonstrated in the matrix. Indeed, in various conditions the effects of acetylcholine (50 microM) on the release of [3H]dopamine were different in a matrix-enriched area (matrix 2) distinct from that previously investigated (matrix 1); these areas being characterized by the presence or absence of islands of striatonigral cells, respectively. As in matrix 1, acetylcholine induced a short-lasting stimulation of [3H]dopamine release in matrix 2 but, in contrast to that observed in matrix 1, the acetylcholine-evoked response in matrix 2 was not modified in the presence of tetrodotoxin (1 microM). Experiments made in the presence of the tetrodotoxin and atropine (1 microM) indicated that both muscarinic and nicotinic receptors are located on dopaminergic nerve terminals in matrix 2 while muscarinic receptors are only present in matrix 1. In the absence of tetrodotoxin, the short-lasting stimulation of [3H]dopamine release was transformed into a long-lasting response in the presence of pempidine (50 microM), in matrix 2 but not in matrix 1 while prolonged responses were seen in both matrix areas in the presence of atropine. Finally, the acetylcholine short stimulatory effect on [3H]dopamine release was transformed into a long stimulatory response in the presence of bicuculline (50 microM) but not naloxone (1 microM) in matrix 2 while the reverse was observed in matrix 1. By providing further evidence for a functional heterogeneity of the matrix, our results suggest that depending on the matrix area investigated, dynorphin- or GABA-containing neurons are involved in the indirect cholinergic inhibitory control of dopamine release.

Ultrastructural changes of crop-sac and lactotrophs after systemic or intraventricular administration of drugs enhancing cholinergic transmission in pigeons.

The ultrastructural effects of drugs enhancing, by different mechanisms, cholinergic transmission in the crop-sac (the target for prolactin secretion in birds) and the anterior pituitary lactotrophs, were studied in pigeons (Columba livia). The systemic or intraventricular administration of physostigmine, carbachol and muscarine produced maximal crop-sac stimulation with milk-like secretion, as demonstrated by the observation of ultrastructural changes in the lactiferous areas through scanning and transmission electron microscopy of the crop-sac mucosa. A marked activation was also observed in anterior pituitary lactotrophs. Crop-sac and anterior pituitary lactotrophs stimulatory effects were prevented by an atropine pretreatment, but not by mecamylamine and pempidine pretreatments. The present results suggest that muscarinic receptors at the hypothalamic and/or anterior pituitary level are involved in avian species in the control of prolactin secretion.

Pharmacological evaluation of the antagonism of nicotine's central effects by mecamylamine and pempidine.

The nature of mecamylamine's and pempidine's antagonism of nicotine in the central nervous system has not been defined clearly. Although these compounds are thought to be noncompetitive antagonists in the brain due to the fact that they do not compete effectively for agonist binding to brain tissue in vitro, pharmacological evidence is lacking. The alteration of nicotine's dose-response curves for depression of spontaneous activity and antinociception was determined in the presence of increasing concentrations of pempidine. Pempidine was found to increase the ED50 of nicotine (0.73 mg/kg) for depression of spontaneous activity in a dose-related manner. At a dose of 3 mg/kg, pempidine increased nicotine's ED50 4.7-fold. The maximum effect of nicotine was achieved in the presence of the highest dose of pempidine, suggesting competitive antagonism. However, pempidine did decrease the maximum effect of nicotine in producing antinociception at doses that increased the ED50 13.7-fold which suggests a noncompetitive action. The structural requirements for mecamylamine's antagonism of these nicotine effects was also determined in order to address the question of whether the antagonists are interacting at a receptor site. The structure-activity relationships of the mecamylamine analogs revealed that the N-, 2- and 3-methyl groups were important for optimal potency. Optical isomerism was found to have little effect on potency. Addition of pyridinyl groups to the nitrogen abolished the activity of these compounds. The structural requirements for the agonists and antagonists therefore appear to be quite different. The alterations produced similar results for antagonism of both effects of nicotine. Mecamylamine and pempidine therefore appear to exhibit both competitive and noncompetitive properties in antagonizing the central effects of nicotine.

Distinct presynaptic control of dopamine release in striosomal and matrix areas of the cat caudate nucleus.

By use of a sensitive in vitro microsuperfusion method, the cholinergic prsynaptic control of dopamine release was investigated in a prominent striosome (areas poor in acetylcholinesterase activity) located within the core of cat caudate nucleus and also in adjacent matrix area. The spontaneous release of [3H]dopamine continuously synthesized from [3H]tyrosine in the matrix area was found to be twice that in the striosomal area; the spontaneous and potassium-evoked releases of [3H]dopamine were calcium-dependent in both compartments. With 10(-6) M tetrodotoxin, 5 x 10(-5) M acetylcholine stimulated [3H]dopamine release in both striosomal and matrix areas, effects completely antagonized by atropine (10(-6) M), thus showing the involvement of muscarinic receptors located on dopaminergic nerve terminals. Experiments without tetrodotoxin revealed a more complex regulation of dopamine release in the matrix: (i) In contrast to results seen in the striosome, acetylcholine induced only a transient stimulatory effect on matrix dopamine release. (ii) Although 10(-6) M atropine completely abolished the cholinergic stimulatory effect on [3H]dopamine release in striosomal area, delayed and prolonged stimulation of [3H]dopamine release was seen with atropine in the matrix. The latter effect was completely abolished by the nicotinic antagonist pempidine (10(-5) M). Therefore, in the matrix, in addition to its direct (tetrodotoxin-insensitive) facilitatory action on [3H]dopamine release, acetylcholine exerts two indirect (tetrodotoxin-sensitive) opposing effects: an inhibition and a stimulation of [3H]dopamine release mediated by muscarinic and nicotinic receptors, respectively.

Antagonism of the nicotine-induced changes of the striatal dopamine metabolism in mice by mecamylamine and pempidine.

The ability of nicotinic receptor blockers, mecamylamine and pempidine, to antagonize the changes in striatal dopamine (DA) metabolism induced by repeated nicotine administration was studied. The contents of DA and its metabolites 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured. Mice kept at 20-22 degrees C were given nicotine, 3 mg/kg, s.c., four times, at 30 min intervals, and sacrificed 20 min after the last dose. Hexamethonium, 10 mg/kg, i.p., was administered at 30 min before the first nicotine dose in order to prevent the peripheral effects of nicotine. Mecamylamine, 0.6 or 10 mg/kg, i.p., and pempidine, 0.6 or 20 mg/kg, i.p., were given at 60 min before sacrifice. Mecamylamine and pempidine decreased clearly the striatal 3-MT content, which suggests that the nigrostriatal dopaminergic neurons are physiologically controlled by a stimulatory nicotinic mechanism. The repeatedly administered nicotine caused deep hypothermia, and increased the striatal DOPAC content but decreased the 3-MT and HVA contents. The small dose of mecamylamine, which was the only dose found to effectively antagonize the nicotine-induced hypothermia, antagonized the decrease of HVA content. The large but not the small doses of mecamylamine and pempidine antagonized the nicotine-induced increase of DOPAC content but none of the doses studied antagonized the decrease of 3-MT content. Thus it seems that nicotine decreases the 3-MT content by a mechanism distinct from the mechanism mediating the increase of the DOPAC content. The decreased 3-MT content most probably results from desensitization of nicotinic cholinergic receptors (nAChR) and following decrease of cholinergic regulation of nigrostriatal dopaminergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of naturally occurring cell death in the sympathetic and parasympathetic ganglia of the chicken embryo following blockade of ganglionic transmission.

Both presynaptic and postsynaptic blockade of ganglionic transmission during the period of naturally occurring ganglion cell death reduced the number of surviving neurons in the sympathetic ganglia (SG) and ciliary ganglion (CG). The CG was chosen for analysis because there was a temporal separation between cell proliferation and death in the CG but not in the SG. Ganglion cell proliferation and migration were unaffected by ganglionic blockade. The increased ganglion cell loss that followed ganglionic blockade was accompanied by an increased number of degenerating cells. These results indicate that the decreased number of healthy ganglion cells that followed ganglionic blockade was the result of enhanced naturally occurring cell death.