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.

Surgical and pharmacological reductions in sympathetic nerve activity increase the neuropeptide Y-immunoreactivity content of the rat iris but not the vas deferens.

Decentralization of the superior cervical ganglion (S.C.G.) of the rat elevated the neuropeptide-Y immunoreactivity (NPY-ir) content of the ganglion on day 1 (+43%) but not on day 3 post-surgery. The content of NPY-ir in the iris was increased by decentralization (+40%) 3 days post-surgery, and treatment with clonidine (+43%), and pempidine (+82%). The levels of NPY-ir in the rat vas deferens were not affected by either surgical or pharmacological treatment. These results suggest NPY is released from sympathetic nerves in the iris but not vas deferens during normal sympathetic activity.

Neurotransmitter modulation of corticotropin releasing factor secretion into the hypophysial-portal circulation.

Secretion of corticotropin releasing factor (CRF) from the hypothalamic paraventricular nuclei into the hypophysial-portal circulation is modulated by a variety of neuronal afferents. Effects of intracerebroventricular acetylcholine (ACH), gamma-aminobutyric acid (GABA) and epinephrine (EPI) on immunoreactive (ir) CRF concentration in portal plasma were directly evaluated in urethanized male rats. ACH (0.1-10 nmole) administration was associated with a dose-dependent elevation of portal irCRF which was attenuated by pretreatment with either muscarinic or nicotinic receptor antagonists. GABA (0.1-10 nmole) also caused inhibition of irCRF concentration in the portal plasma which was prevented by bicuculline pretreatment. Finally, EPI (0.1-10 nmole) facilitated irCRF secretion via alpha 1- and beta-adrenergic mechanisms. These observations provide the first direct information on possible neurotransmitter actions on hypothalamic CRF secretion.

Inhibition by neosurugatoxin of nicotine-induced antinociception.

We have found a selective inhibition of nicotine-induced antinociception in mice by neosurugatoxin (NSTX, 0.4-3.8 nmol/kg), a neurotoxin with a high affinity for ganglionic nicotinic receptors (ED50 = 0.65 nmol/kg). The toxin also reduced specific [3H]nicotine binding in mouse brain membranes (IC50 = 95 nM). The anti-nicotinic activity of NSTX was markedly greater than that of mecamylamine and pempidine. These data indicate that NSTX may block functional nicotinic cholinoceptors possibly in the central nervous system.

Mediation of passive avoidance learning by nicotinic hippocampo-entorhinal components in young rats.

Young rats, 11, 16, and 20 days of age, received bilateral injections of three antinicotinic agents into the posteroventral hippocampo-subiculo-entorhinal area, and were trained to learn a cool-draft-stimulus, passive-avoidance task shortly after (17 min). Gallamine triethiodide had no action at low doses and provoked convulsions at higher concentrations. Pempidine tartrate produced age- and dose-dependent impairments of the passive avoidance, and was much more effective in younger groups (11 and 16 days) than at 20 days. alpha-bungarotoxin also induced dose-dependent deficits. These results, together with the mecamylamine-induced deficits already reported, suggest that nicotinic cholinergic synapses located in the posteroventral part of the hippocampal complex play a role in passive-avoidance learning in the young rat as soon as this type of conditioning is possible, but become relatively less important at older ages, when muscarinic mechanisms also become involved.

Detection of the messenger RNAs coding for the opioid peptide precursors in pituitary and adrenal by "in situ' hybridization: study in several mammal species.

The messenger RNAs coding for opioid peptide precursors have been detected and mapped in histological sections by "in situ' hybridization using specific DNA probes labelled with 32P. Using bovine preproenkephalin A (PPA) cDNA, PPA mRNA was detected in adrenal medulla of bull, hamster and guinea pig. No signal was detected in adrenal of man, rat and cat. The pro-opiomelanocortin (POMC) mRNA was detected in pituitary of man, bull, cat, rat and pig, in all cells of the intermediate lobe as well as in scattered cells of the anterior lobe producing POMC. Adequate controls demonstrated the specificity of the labelling. These results provide evidence of the expression of the gene coding for PPA in the adrenal and for POMC in the pituitary. They show cross-hybridization of one DNA probe with mRNAs of various mammals and then provide evidence that one single probe can be used to analyze expression of a given gene in tissues of several animal species by "in situ' hybridization.

Lack of stereoselectivity in ability of nicotine to release dopamine from rat synaptosomal preparations.

Both the naturally occurring (-)-isomer and the synthetic (+)-isomer of nicotine caused release of 3H from a crude synaptosomal fraction of rat brain preincubated with [3H]dopamine. The isomers were equipotent in producing this response, which was concentration-dependent, a significant effect on the fractional release of dopamine being observed at 10(-4) M nicotine. The effect did not appear to be the result of synaptosomal damage, as levels of the intrasynaptosomal marker lactate dehydrogenase did not increase in the supernatant. Nicotine-induced release was inhibited by removal of external Ca2+ and by the presence in vitro of pempidine (230 microM). Neither hexamethonium (500 microM) in vitro nor the chronic administration of (-)-nicotine in vivo had any effect on the nicotine-induced release of [3H]dopamine. It is concluded that nicotine exerts this effect via a presynaptic nicotinic receptor of the "ganglionic" type, but that this receptor differs from that in the periphery by showing a relative lack of stereospecificity. There is no evidence of a functional "down regulation" in this receptor on chronic exposure to nicotine in vivo.