Melatonin and the time window for the expression of the alpha 8 nicotinic acetylcholine receptor in the membrane of chick retinal cells in culture

We have previously shown that melatonin influences the development of alpha 8 nicotinic acetylcholine receptor (nAChR) by measurement of the acetylcholine-induced increase in the extracellular acidification rate (ECAR) in chick retinal cell cultures. Cellular differentiation that takes place between DIV (days in vitro) 4 and DIV 5 yields cells expressing alpha 8 nAChR and results in a significant increase in the ECAR acetylcholine-induced. Blocking melatonin receptors with luzindole for 48 h suppresses the development of functional alpha 8 nAChR. Here we investigated the time window for the effect of melatonin on retinal cell development in culture, and whether this effect was dependent on an increase in the expression of alpha 8 nAChR. First, we confirmed that luzindole was inhibiting the effects of endogenous melatonin, since it increases 2-[(125)I] iodomelatonin (23 pM) binding sites density in a time-dependent manner. Then we observed that acute (15, 60 min, or 12 h) luzindole treatment did not impair acetylcholine-induced increase in the ECAR mediated by activation of alpha 8 nAChR at DIV 5, while chronic treatment (from DIV 3 or DIV 4 till DIV 5, or DIV 3.5 till DIV 4.5) led to a time-dependent reduction of the increase in the acetylcholine-induced ECAR. The binding parameters for [(125)I]-alpha-bungarotoxin (10 nM) sites in membrane were unaffected by melatonin suppression that started at DIV 3. Thus, melatonin surges in the time window that occurs at the final stages of chick retinal cell differentiation in culture is essential for development of the cells expressing alpha 8 nAChR subtype in full functional form. (C) 2010 ISDN. Published by Elsevier Ltd. All rights reserved.

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+](i)) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G alpha(q/11)-coupled M-1, M-3 and M-5 receptors and intracellular calcium stores, whereas G alpha(i/o)-protein coupled M-2 receptor activity mediated neuronal differentiation. (C) 2008 Elsevier Inc. All rights reserved.

Inhibition Mechanism of Rat alpha(3)beta(4) Nicotinic Acetylcholine Receptor by the Alzheimer Therapeutic Tacrine

Nicotinic acetylcholine receptors (nAChRs) were studied in detail in the past regarding their interaction with therapeutic and drug addiction related compounds. Using fast kinetic whole-cell recording, we have now studied effects of tacrine, an agent used clinically to treat Alzheimer`s disease, on currents elicited by activation of rat alpha(3)beta(4) nAChR heterologously expressed in KX alpha(3)beta(4)R2 cells. Characterization of receptor activation by nicotine used as agonist revealed a K(d) of 23 +/- 0.2 mu M and 4.3 +/- 1.3 for the channel opening equilibrium constant, Phi(-1). Experiments were performed to investigate whether tacrine is able to activate the alpha(3)beta(4) nAChR. Tacrine did not activate whole-cell currents in KX alpha(3)beta(4)R2 cells but inhibited receptor activity at submicromolar concentration. Dose response curves obtained with increasing agonist or inhibitor concentration revealed competitive inhibition of nAChRs by tacrine, with an apparent inhibition constant, K(I), of 0.8 mu M. The increase of Phi(-1) in the presence of tacrine suggests that the drug stabilizes a nonconducting open channel form of the receptor. Binding studies with TCP and MK-801 ruled out tacrine binding to common allosteric sites of the receptor. Our study suggests a novel mechanism for action of tacrine on nAChRs besides inhibition of acetylcholine esterase.

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha 7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca(2+) influx. However, the study of recombinant alpha 7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha 3, alpha 5, alpha 7, beta 2 and beta 4 subunits was present during the course of differentiation, while mRNAs coding for alpha 2, alpha 4 and beta 3 subunits were not expressed in PC12 cells. alpha 7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha 7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha 7 agonist choline. Increased alpha 7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

Central muscarinic receptors signal pilocarpine-induced salivation

Although cholinergic agonists such as pilocarpine injected peripherally can act directly on salivary glands to induce salivation, it is possible that their action in the brain may contribute to salivation. To investigate if the action in the brain is important to salivation, we injected pilocarpine intraperitoneally after blockade of central cholinergic receptors with atropine methyl bromide (atropine-mb). In male Holtzman rats with stainless steel cannulas implanted into the lateral ventricle and anesthetized with ketamine, atropine-mb (8 and 16 nmol) intracerebroventricularly reduced the salivation induced by pilocarpine (4 mumol/kg) intraperitoneally (133 +/- 42 and 108 +/- 22 mg/7 min, respectively, vs. saline, 463 +/- 26 mg/7 min), but did not modify peripheral cardiovascular responses to intravenous acetylcholine. Similar doses of atropine-mb intraperitoneally also reduced pilocarpine-induced salivation. Therefore, systemically injected pilocarpine also enters the brain and acts on central muscarinic receptors, activating autonomic efferent fibers to induce salivation.

Involvement of central cholinergic mechanisms on sodium intake induced by gabaergic activation of the lateral parabrachial nucleus

Bilateral injections of the GABAA agonist muscimol into the lateral parabrachial nucleus (LPBN) disrupt satiety and induce strong ingestion of water and 0.3M NaCl in fluid-replete rats by mechanisms not completely clear. In the present study, we investigated the effects of the blockade of central muscarinic cholinergic receptors with atropine injected intracerebroventricularly (i.c.v.) on 0.3M NaCl and water intake induced by muscimol injections into the LPBN in fluid-replete rats. Male Holtzman rats with stainless steel cannulas implanted bilaterally into the LPBN and unilaterally into the lateral ventricle (LV) were used. Bilateral injections of muscimol (0.5nmol/0.2μL) into the LPBN induced 0.3M NaCl (32.2±9.9mL/4h, vs. saline: 0.4±0.2mL/4h) and water intake (11.4±4.4mL/4h, vs. saline: 0.8±0.4mL/4h) in fluid-replete rats previously treated with i.c.v. injection of saline. The previous i.c.v. injection of atropine (20nmol/1μL) reduced the effects of LPBN-muscimol on 0.3M NaCl (13.5±5.0mL/4h) and water intake (2.9±1.6mL/4h). The i.c.v. injection of atropine did not affect 0.3M NaCl (26.8±6.2mL/2h, vs. saline i.c.v.: 36.5±9.8mL/2h) or water intake (14.4±2.5mL/2h, vs. saline i.c.v.: 15.6±4.8mL/2h) in rats treated with furosemide+captopril subcutaneously combined with bilateral injections of moxonidine (α2-adrenoceptor/imidazoline agonist, 0.5nmol/0.2μL) into the LPBN, suggesting that the effect of atropine was not due to non-specific inhibition of ingestive behaviors. The results show that active central cholinergic mechanisms are necessary for the hypertonic NaCl and water intake induced by the blockade of the inhibitory mechanisms with injections of muscimol into the LPBN in fluid-replete rats. The suggestion is that in fluid-replete rats the action of LPBN mechanisms inhibits facilitatory signals produced by the activity of central cholinergic mechanisms to maintain satiety. © 2012 Elsevier B.V.

Erythrina mulungu Alkaloids Are Potent Inhibitors of Neuronal Nicotinic Receptor Currents in Mammalian Cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Immunohistochemical localization of nicotinic acetylcholine-receptor subunits in the mesencephalon and diencephalon of the chick (Gallus-gallus)

Monoclonal antibodies against two alpha-bungarotoxin-binding subunits (alpha-7 and alpha-8) of the nicotinic acetylcholine receptors (nAChRs) were used as immunohistochemical probes to map their distribution in the chick diencephalon and mesencephalon. The distribution of the alpha-7 and alpha-8 nAChR subunits was compared to the distribution of immunoreactivity produced by a monoclonal antibody against the beta-2 structural subunit of the nAChRs.Structures that contained high numbers of alpha-7-like immunoreactive (LI) somata included the intergeniculate leaflet, nucleus intercalatus thalami, nucleus ovoidalis, organum paraventricularis, nucleus rotundus, isthmic nuclei, nucleus trochlearis, oculomotor complex, nucleus interstitio-pretecto-subpretectalis, stratum griseum centrale of the optic tectum, and nucleus semilunaris. Neuropil staining for alpha-7-LI was intense in the nucleus dorsomedialis hypothalami, nucleus geniculatus lateralis ventralis, griseum tecti, isthmic nuclei, nucleus lentiformis mesencephali, nucleus of the basal optic root, and stratum griseum et fibrosum superficiale of the tectum. High numbers of alpha-8-LI somata were found in the stratum griseum et fibrosum superficiale of the tectum and the nucleus interstitio-pretecto-subpretectalis, and intense neuropil staining for alpha-8-LI was found in the dorsal thalamus, nucleus geniculatus lateralis ventralis, lateral hypothalamus, griseum tecti, nucleus lentiformis mesencephali, nucleus interpeduncularis, and stratum griseum et fibrosum superficiale of the tectum. High numbers of beta-2-LI somata were found only in the nucleus spiriformis lateralis, whereas neuropil staining for beta-2-LI was intense in the nucleus geniculatus lateralis ventralis, nucleus suprachiasmaticus, nucleus lateralis anterior, nucleus habenularis lateralis, area pretectalis, griseum tecti, nucleus lentiformis mesencephali, nucleus externus, and nucleus interpeduncularis, and in the stratum griseum centrale, stratum griseum et fibrosum superficiale, and stratum opticum of the tectum.These results indicate that there are major disparities in the localization of the alpha-bungarotoxin-binding alpha-7 and alpha-8 nAChR subunits and the beta-2 structural nAChR subunit in the chick diencephalon and mesencephalon. These nAChR subunits appear, however, to coexist in several regions of the chick brain.

Aerobic training attenuates nicotinic acetylcholine receptor changes in the diaphragm muscle during heart failure

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Role of cholinergic and adrenergic pathways of the medial septal area in the control of water intake and renal excretion in rats

In this study, we investigated an interaction between noradrenergic and cholinergic pathways of the medial septal area (MSA) on the control of water intake and urinary electrolyte excretion by means of injection of their respective agonists. Noradrenaline (a nonspecific α-adrenergic agonist) and clonidine (an α2-adrenergic agonist), but not phenylephrine (an α1-adrenergic agonist), induced natriuresis and kaliuresis. α-Adrenergic activation had no effect on the natriuresis and kaliuresis induced by carbachol (a cholinergic agonist) and it inhibited the antinatriuresis and antikaliuresis induced by isoproterenol (a ß-adrenergic agonist). Interactions related to volume excretion are complex. α-Adrenergic activation induced a mild diuresis and inhibited the antidiuresis induced by isoproterenol, but phenylephrine combined with carbachol induced antidiuresis. The water intake induced by carbachol was inhibited by clonidine and noradrenaline, but not phenylephrine. These results show an asymmetry in the interaction between α-adrenergic and cholinergic receptors concerning water intake and electrolyte excretion. © 1992.

Aerobic training attenuates nicotinic acethylcholine receptor changes in the diaphragm muscle during heart failure

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Lipophilicity as a determinant of binding of procaine analogs to rat alpha(3)beta(4) nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (nAChRs) have been studied in detail with regard to their interaction with therapeutic and drug addiction-related compounds. Using a structureactivity approach, we have examined the relationship among the molecular features of a set of eight para-R-substituted N,N-[(dimethylamino)ethyl] benzoate hydrochlorides, structurally related to procaine and their affinity for the a3 beta 4 nAChR heterologously expressed in KXa3 beta 4R2 cells. Affinity values (log[1/IC50]) of these compounds for the a3 beta 4 nAChR were determined by their competition with [3H]TCP binding. Log(1/IC50) values were analyzed considering different hydrophobic and electronic parameters and those related to molar refractivity. These have been experimentally determined or were taken from published literature. In accordance with literature observations, the generated cross-validated quantitative structureactivity relationship (QSAR) equations indicated a significant contribution of hydrophobic term to binding affinity of procaine analogs to the receptor and predicted affinity values for several local anesthetics (LAs) sets taken from the literature. The predicted values by using the QSAR model correlated well with the published values both for neuronal and for electroplaque nAChRs. Our work also reveals the general structure features of LAs that are important for interaction with nAChRs as well as the structural modifications that could be made to enhance binding affinity. (c) 2012 Wiley Periodicals, Inc.

From Folke Medicine to Medicine Chemistry: Study, Using in Vitro and Cellular Assays, of Receptors Mechanism Involved in the Activities of Natural Compounds

My Doctorate Research has been focused on the evaluation of the pharmacological activity of a natural extract of chestnut wood (ENC) towards the cardiovascular and gastrointestinal system and on the identification of the active compounds. The ENC has been shown to contain more than 10% (w/w) of phenolic compounds, of which tannins as Vescalgin and Castalgin are the more representative. ENC cardiovascular effects have been investigated in guinea pig cardiac preparations; furthermore its activity has been evalueted in guinea pig aorta strips. ENC induced transient negative chronotropic effect in isolated spontaneously beating right atria and simultaneously positive inotropic effect in left atria driven at 1 Hz. Cardiac cholinergic receptors are not involved in the negative chronotropic effect and positive inotropic effects are not related to adrenergic receptors. In vascular smooth muscle, natural extract of chestnut did not significantly change the contraction induced by potassium (80 mM) or that induced by noradrenaline (1μM). In guinea pig ileum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. In guinea pig ileum, the antispasmodic activity of ENC showed a significant antispasmodic activity against a variety of different spasmogenic agents including histamine, KCl, BaCl2. In guinea pig proximal colon, stomach and jejunum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. ENC contracted gallbladder guinea pig in a reversible and concentration-dependent manner. This effect does not involve cholinergic and cholecystokinin receptors and it is reduced by nifedipine. ENC relaxed Oddi sphincter smooth muscle. The cholecystokinetic and Oddi sphincter relaxing activities occurred also in guinea pigs fed a lithogenic diet. The cholecystokinetic occurred also in human gallbladder. The Fractionation of the extract led to the identification of the active fraction.

Haemonchus contortus acetylcholine receptors of the DEG-3 subfamily and their role in sensitivity to monepantel

Gastro-intestinal nematodes in ruminants, especially Haemonchus contortus, are a global threat to sheep and cattle farming. The emergence of drug resistance, and even multi-drug resistance to the currently available classes of broad spectrum anthelmintics, further stresses the need for new drugs active against gastro-intestinal nematodes. A novel chemical class of synthetic anthelmintics, the Amino-Acetonitrile Derivatives (AADs), was recently discovered and the drug candidate AAD-1566 (monepantel) was chosen for further development. Studies with Caenorhabditis elegans suggested that the AADs act via nicotinic acetylcholine receptors (nAChR) of the nematode-specific DEG-3 subfamily. Here we identify nAChR genes of the DEG-3 subfamily from H. contortus and investigate their role in AAD sensitivity. Using a novel in vitro selection procedure, mutant H. contortus populations of reduced sensitivity to AAD-1566 were obtained. Sequencing of full-length nAChR coding sequences from AAD-susceptible H. contortus and their AAD-1566-mutant progeny revealed 2 genes to be affected. In the gene monepantel-1 (Hco-mptl-1, formerly named Hc-acr-23H), a panel of mutations was observed exclusively in the AAD-mutant nematodes, including deletions at intron-exon boundaries that result in mis-spliced transcripts and premature stop codons. In the gene Hco-des-2H, the same 135 bp insertion in the 5' UTR created additional, out of frame start codons in 2 independent H. contortus AAD-mutants. Furthermore, the AAD mutants exhibited altered expression levels of the DEG-3 subfamily nAChR genes Hco-mptl-1, Hco-des-2H and Hco-deg-3H as quantified by real-time PCR. These results indicate that Hco-MPTL-1 and other nAChR subunits of the DEG-3 subfamily constitute a target for AAD action against H. contortus and that loss-of-function mutations in the corresponding genes may reduce the sensitivity to AADs.

Activation of nicotinic receptor-induced postsynaptic responses to luteinizing hormone-releasing hormone in bullfrog sympathetic ganglia via a Na+-dependent mechanism

Nicotine at very low doses (5–30 nM) induced large amounts of luteinizing hormone-releasing hormone (LHRH) release, which was monitored as slow membrane depolarizations in the ganglionic neurons of bullfrog sympathetic ganglia. A nicotinic antagonist, d-tubocurarine chloride, completely and reversibly blocked the nicotine-induced LHRH release, but it did not block the nerve-firing-evoked LHRH release. Thus, nicotine activated nicotinic acetylcholine receptors and produced LHRH release via a mechanism that is different from the mechanism for evoked release. Moreover, this release was not caused by Ca2+ influx through either the nicotinic receptors or the voltage-gated Ca2+ channels because the release was increased moderately when the extracellular solution was changed into a Ca2+-free solution that also contained Mg2+ (4 mM) and Cd2+ (200 μM). The release did not depend on Ca2+ release from the intraterminal Ca2+ stores either because fura-2 fluorimetry showed extremely low Ca2+ elevation (≈30 nM) in response to nicotine (30 nM). Moreover, nicotine evoked LHRH release when [Ca2+] elevation in the terminals was prevented by loading the terminals with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid and fura-2. Instead, the nicotine-induced release required extracellular Na+ because substitution of extracellular NaCl with N-methyl-d-glucamine chloride completely blocked the release. The Na+-dependent mechanism was not via Na+ influx through the voltage-gated Na+ channels because the release was not affected by tetrodotoxin (1–50 μM) plus Cd2+ (200 μM). Thus, nicotine at very low concentrations induced LHRH release via a Na+-dependent, Ca2+-independent mechanism.