Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, December 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.) - University of Queensland, 2006. / Includes bibliography.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

Thesis (Ph.D.)--Kent State University, 2007. / Title from PDF t.p. (viewed Jan. 16, 2007) Advisor: Chet C. Sherwood. Includes bibliographical references (p. 165-198)

Proefschrift Rijksuniversiteit Limburg, Maastricht. / Auteursnaam op omslag: Wim Riedel. Met samenvatting in het Nederlands. - Met bibliogr., lit. opg.

nÃo hà / O etanol tem influÃncia sobre diversos sistemas de neurotransmissores. VÃrios estudos tÃm relatado a interaÃÃo entre a via dopaminÃrgica e colinÃrgica e a interaÃÃo delas isoladamente com os efeitos do etanol. O bloqueador colinÃrgico atropina, que à um alcalÃide muito utilizado no caso de intoxicaÃÃes colinÃrgica e comprometimento cardÃaco e o haloperidol, à neurolÃptico, antagonista das vias dopaminÃrgicas, principalmente pelo bloqueio do receptor D2, utilizado no tratamento da esquizofrenia, tanto na fase aguda como crÃnica, psicoses e episÃdios manÃacos psicÃticos sÃo fÃrmacos utilizados para o estudo desses sistemas. Este trabalho objetivou estudar os efeitos comportamentais e neuroquÃmicas produzidos em corpo estriado induzidos pelo tratamento subcrÃnico com etanol na presenÃa e ausÃncia de atropina ou haloperidol. Foram utilizados camundongos Swiss, fÃmeas, com peso variando entre 25 â 30g. Os animais foram tratados com Ãgua destilada (controle), etanol (1 ou 3g/kg, v.o) ou prà tratados com haloperidol (0,5mg/kg, i.p) ou atropina (0,5mg/kg, i.p), essa aplicaÃÃo foi feita trinta minutos antes da administraÃÃo do etanol ou Ãgua destilada diariamente durante sete dias.Trinta minutos apÃs a Ãltima administraÃÃo das drogas por via intraperitoneal ou sessenta minutos apÃs a Ãltima administraÃÃo por via oral, os animais foram submetidos aos testes comportamentais de campo aberto e rota rod (atividade locomotora), placa perfurada e plus maze (atividade de ansiedade) e, posteriormente, sacrificados com dissecaÃÃo do corpo estriado para determinaÃÃo dos nÃveis de monoaminas (norepinefrina, dopamina e seu metabÃlico Ãcido 3, 4-dihidroxifenilacÃtico) e a atividade da acetilcolinesterase. O etanol apresentou efeito estimulante na menor dose e depressor na maior dose, principalmente nos testes de atividade locomotora. Quando associado à maior dose, o haloperidol apresentou, na maioria das vezes o efeito potencializador do etanol, jà a atropina apenas em alguns testes mostrou uma tendÃncia de reversÃo desse efeito apenas associado ao etanol na menor dose, embora nÃo observado em todos os paramentos avaliados. AlÃm disso, o efeito produziu um discreto aumento nos nÃveis de dopamina no corpo estriado, porÃm quando associado aos bloqueadores, houve reduÃÃo desses nÃveis, sendo mais acentuada quando essa associaÃÃo foi feita com o etanol na maior dose. Na avaliaÃÃo da atividade da enzima acetilcolinesterase, todas as substÃncias utilizadas associadas ou nÃo, provocaram a reduÃÃo de mais de 60% na atividade desta enzima, sendo mais evidente na administraÃÃo isolada de atropina, ou seja, as administraÃÃes que apresentaram reduÃÃo significativa da concentraÃÃo de DA, apresentaram tambÃm a atividade da AchE reduzida, evidenciando a interaÃÃo desses dois sistemas, sugerindo-os como possÃveis alvos de aÃÃo no desenvolvimento de medicamento que coadjuvem no tratamento do alcoolismo / Ethanol is an agent with nonspecific action, which may interfere in various neurotransmitter systems. Atropine is an alkaloid with blocking action of the cholinergic system, used in cholinergic intoxication and other with cardiac involvement. The neuroleptic, haloperidol, is an antagonist of dopaminergic pathways, mainly by blocking the D2 receptor, used in the treatment of schizophrenia in both acute and chronic psychoses and maniac psychotic episodes. Several studies have reported the interaction between the dopaminergic and cholinergic pathway and their interaction separately with the ethanol. This study investigated the behavioral and neurochemical effects produced in the striatum induced by subchronic treatment with ethanol in presence and absence of atropine or haloperidol. Swiss mice were used, females, weighing between 25 - 30g. The animals were treated with distilled water (control), ethanol (1 or 3g/kg, po) or pre-treated with haloperidol (0.5 mg / kg, ip) or atropine (0.5 mg / kg, ip), this application was thirty minutes before administration of ethanol or distilled water daily for seven days. Thirty minutes after the last drug administration intraperitoneally or sixty minutes after the last oral administration, the animals were subjected to behavioral tests of open field and track rod (locomotors activity), hole board and plus maze (anxiety activity) and then sacrificed with dissection of the striatum for determination of monoamines (norepinephrine, dopamine and its metabolic acid 3, 4-dihydroxyphenylacetic) or the concentration of acetylcholinesterase. Ethanol showed a stimulating effect in depressing the lower dose and higher dose, especially in tests of locomotors activity. When associated with a higher dose, haloperidol showed, in most cases the potentiating effect of ethanol, since atropine in some tests only showed a tendency to reverse this effect only associated with ethanol at the lowest dose, although not observed in all parameters of evaluated. However, the effect produced a modest increase in the concentrations of dopamine in the striatum, but when associated with blockers, a reduction of these levels was more pronounced when the association was made with ethanol at the highest dose. In the evaluation of the enzyme acetylcholinesterase, all substances used, combined or not, reduced more than 60% levels this enzyme, being more evident in the administration of atropine alone, or the authorities which showed a significant reduction in the concentration of AD, also showed reduced AChE, showing the interaction of these two systems, suggesting them as possible targets of action of drug development which contribute to the treatment of alcoholism

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / No presente trabalho, foi avaliado a interaÃÃo entre os sistemas dopaminÃrgico e colinÃrgico atravÃs do estudo dos efeitos comportamentais (campo aberto e catalepsia) e neuroquÃmicos (densidade de receptores dopaminÃrgicos (D1 e D2-sÃmile) e muscarÃnicos (M1+M2-sÃmile) em corpo estriado de rato. As seguintes drogas foram utilizadas: mazindol (agonista dopaminÃrgico indireto), apomorfina (agonista dopaminÃrgico D1-sÃmile e D2-sÃmile), pimozida (antagonista dopaminÃrgico D2-sÃmile), SCH 23390 (antagonista dopaminÃrgico D1-sÃmile), pilocarpina (agonista muscarÃnico M1-sÃmile), carbacol (agonista muscarÃnico M2-sÃmile), pirenzepina (antagonista muscarÃnico M1-sÃmile), atropina (antagonista muscarÃnico M1 e M2 nÃo seletivo), clozapina (neurolÃptico atÃpico). Os resultados mostraram que a pimozida e o carbacol, sozinhos ou associados, causaram um aumento da resposta catalÃptica e uma diminuiÃÃo da atividade locomotora. O mazindol tambÃm aumentou a atividade locomotora. Carbacol, nas menores doses, e o mazindol aumentaram a densidade de receptores D1-sÃmile. A pimozida e a atropina, isoladamente, aumentaram a densidade de receptores D1-sÃmile no corpo estriado enquanto que a atropina causou uma diminuiÃÃo dos receptores D2-sÃmile e uma upregulation dos receptores muscarÃnicos. O mazindol aumentou o binding de 3H-NMS no corpo estriado. O presente trabalho sugere, de maneira geral, que existe uma relaÃÃo entre os receptores muscarÃnicos M1 e M2 com os receptores dopaminÃrgicos D1 e D2, sendo que esta relaÃÃo pode ocorrer de maneira positiva ou negativa, dependendo da seletividade e da dose das drogas utilizadas / In the study, the interaction between the dopaminergic and cholinergic systems through the study of behavioral (open field and catalepsy) and neurochemical (density of dopaminergic receptor (D1 and D2-like) and muscarinic (M1+M2-like)) effect in striatum rat was evaluated. The following drugs were used: mazindol (indirect dopaminergic agonist), apomorphine (D1-like and D2-like dopaminergic agonist), pilocarpine (M1-like muscarnic aginist), carbachol (M-2like agonist muscarinc), pirenzepine (M1-like antagonist muscarinic), atropine (non-selective M1 and M2 antagonist muscarinic), clozapina(non-typical neuroleptic). The results showed that the pimozida and carbachol, alone or associated, caused increase in the cataleptic response and reduction in the motor activity. The mazindol also increased the motor activity. In small dosage, the carbachol and mazindol increased the density of D1-like receptors. Isolated, the pimozida and atropine increased the density of the D1-like receptors in striatum whereas the atropine caused a reduction of D2-like receptors and upregulation of muscarinic receptors. This work suggests a relationship, between muscarinc receptors M1 and M2 and dopaminergic receptors D1 and D2, and that this relationship can occur in a positive and negative manner, depending on the selectivity and the dose of the used drug

The peripheral and highly lipid-exposed M4 α-helix, although distant from the agonist binding site, channel gate, and other important gating structures, is involved in modulating function of the nicotinic acetylcholine receptor. M4 "senses" changes in the surrounding lipid environment and may consequently affect receptor function by altering specific interactions between the M4 C-terminus and the Cys-loop. An example of this lipid sensing ability is demonstrated by a lipid-facing Cys418 to Trp substitution on αM4 (αM4 C418W) of the muscle-type receptor, which subtly alters protein-lipid interactions and potentiates channel function 16-fold, leading to a slow-channel congenital myasthenic syndrome. Through the use of mutational studies and mutant cycle analysis, I determine that, contrary to previous studies, M4–Cys-loop interactions are not critical to wild-type muscle-type receptor function, nor are they involved in C418W-induced potentiation. Instead, C418W potentiates channel activity by enhancing local M4-M1 interactions mediated by three polar side-chains, which are absolutely critical to potentiation. I show that altered M4-M1 interactions are ultimately translated to two important gating structures, which work in tandem to stabilize the open conformation of the receptor. These studies highlight how altered protein-lipid interactions can affect channel function and contribute to our understanding of the underlying gating mechanism of the muscle-type receptor.

One of the major physiological responses to insulin secretion is the activation of lipogenesis in target tissues (principally fat and liver). As acetyl-CoA carboxylase (ACC) is the rate limiting enzyme in fatty acid synthesis, the mechanisms involved in the short term regulation of this enzyme represent a pertinent model system for determining elements involved in amplification of the signals produced in response to stimulation of cells with lipogenic and counter regulatory hormones. The regulation of mammalian ACC by hormones is a complex phenomenon involving interplay between allosteric and covalent mechanisms. While the effects of adrenaline and glucagon are well characterised, the mechanism of regulation by insulin has still to be defined and formed the focus for the work presented in this thesis. To study the role of phosphorylation in the response of ACC to insulin, the site-specific phosphorylation of the enzyme observed following exposure of intact cells to insulin has been reproduced in vitro. These studies for the first time describe the conditions required to achieve distribution of [³²P] in vitro among sites of acetyl-CoA carboxylase, very similar to that seen after hormone treatment of intact cells and employing endogenous polyamine-sensitive kinase(s). No corresponding increase in catalytic activity was detected following phosphorylation, in vitro, of insulin directed phosphorylation sites on purified rat liver acetyl-CoA carboxylase in these studies. Subsequently, ACC was phosphorylated by an exogenous protein kinase from maturation activated sea-star oocytes which led to high stoichiometric incorporation of ³²P into the unique site (I-site) phosphorylated in intact cells in response to insulin (0.3 mol phosphate / mol 240,000 kD subunit). Again no change in ACC activity was observed following I-site phosphorylation. The peptide containing the I-site was separated from other tryptic phosphopeptides by reverse phase HPLC and then sequenced. Phosphorylation of serine 1186 was determined to be the major phosphorylation site of ACC in response to insulin. The amino acid sequence corresponding to the peptide containing Ser 1186 is located in the putative "hinge" region of ACQ which is some 300 amino acids towards the C-terminal of the biotin binding site (Lys-784). Subsequent re-evaluation of the kinetic properties of acetyl-CoA carboxylase during purification has led to the identification of a fraction containing low Mr inhibitor(s) and an apparently novel protein activator present in rat liver. Affinity purified rat liver acetyl-CoA carboxylase can be activated 2-3 fold at physiological citrate concentrations (0.1-0.5mM) by the addition of the heat and pro tease-sensitive cytosolic protein. The ACC activator has been extensively purified (though not yet to homogeneity) from a 100,000 g supernatant fractions from rat liver extract, by a combination of ammonium sulphate precipitation, ion-exchange chromatography and gel filtration. From these results we concluded that the activator is a protein and the native molecular weight in solution is estimated to be approximately 75 kDaltons. A popular hypothesis regarding the short term regulation of ACC involves a phosphorylation-dephosphorylation mechanism resulting in inhibition and activation respectively. A number of experiments have been carried out in order to test the hypothesis that the activator preparation may contain protein phosphatase activity directed towards ACC. The results strongly suggest that under the assay conditions described for the expression of activation of catalytic activity of ACC, there is little or no apparent dephosphorylation. Indeed, the most purified preparations of ACC activator do not contain any detectable phosphatase activity towards the model substrates histone III-S and casein. The activation of ACC occurs rapidly, in a time dependent manner (within 20 min at 37°C) and involves protein-protein interaction which is antagonized by avidin. The interactions between ACC, avidin and activator protein suggest that the activator not only induces conformational change at the active site of ACC but may also bind in such a way as to be displaced (perhaps directly) by avidin. From the data presented it is concluded that this acetyl-CoA carboxylase activator protein represents a novel factor which may be involved in the short term regulation of ACC activity. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

This study examined the effects of epinephrine, norepinephrine, AVP and ACh on fluid movement by the lungs of the late-term guinea pig fetus. Catecholamines and AVP are secreted in high amounts by the fetus during delivery, and could be important with respect to fetal lung fluid removal; this event is vital at the time of birth. The lungs were supported in vitro for a duration of three hours, and production rates were measured using a dye-dilution technique. The average resting production rate in terms of ml/kg‧h declined with gestational age (54-67 days gestation; n=171). There was a lesser decline in the average resting production rate in terms of ml/h. The average production rate of untreated preparations in the first hour was 1.60 ± 0.26 ml/kg body weight per hour, and rates did not change significantly during the remaining two hours of experimentation (n=30). This rate is comparable to those reported from chronically catheterized fetal sheep. Treatment was administered during the second hour of experimentation, following an ABA design. Lungs (n=36) were transferred to fresh Krebs-Henseleit saline containing one of the following concentrations of epinephrine: (a) 10‾⁵ M; (b) 10‾⁶ M; (c) 10‾⁷ M; (d) 5 x 10‾⁸ M; (e) 10‾⁸ M; and (f) 10‾⁹ M. With the exception of the top dose, epinephrine treatment caused an immediate reduction in fluid secretion, or fluid reabsorption. Sodium followed the movement of water in all cases. The effect of epinephrine at 10‾⁷ M was maximal, and the threshold dose for epinephrine was calculated at 1.78 x 10‾¹¹ M. Phentolamine and propranolol had no effect in control preparations. However, phentolamine completely blocked the effect of epinephrine, whereas propranolol was ineffective. Isoproterenol had no effect on pulmonary fluid production. Alpha-adrenergic receptors apparently mediate the effect of epinephrine on pulmonary fluid movement in the fetal guinea pig lung. This conclusion is different from that obtained in fetal sheep, in which beta-adrenergic receptors are utilized. A possible synergism between epinephrine and AVP was examined. Lungs (n=12) were transferred to fresh Krebs-Henseleit saline containing either (a) 0.6 mU/ml AVP, or b) 0.6 mU/ml AVP combined with epinephrine at 10‾⁷ M. Treatment with AVP caused a slow, prolonged reduction in fluid production. Treatment with AVP together with epinephrine did not demonstrate synergism. The effect of norepinephrine (NE) was examined. Lungs (n=36) were transferred to fresh Krebs-Henseleit saline containing one of the following concentrations of NE: (a) 1.24 x 10‾⁵ M; (b) 1.24 x 10‾⁶ M; (c) 1.24 x 10‾⁷ M; (d) 5.24 x 10‾⁸ M; (e) 1.24 x 10‾⁸ M; and (f) 1.24 x 10‾⁹ M. In all preparations, treatment with NE resulted in an immediate reduction in fluid production, and reabsorptions were observed at the higher doses. Sodium followed the movement of water in every case. The threshold dose was calculated at 3.16 x 10‾¹⁰ M. Phentolamine blocked the effect of NE, reinforcing the importance of pulmonary alpha-adrenergic receptors in the fetal guinea pig. There was no relationship between age and degree of response with treatment of either epinephrine or NE, but fetuses under 78.0 g did not respond to NE. The effect of ACh was examined. Lungs (n=24) were transferred to fresh Krebs-Henseleit saline containing one of the following concentrations of ACh: (a) 10‾⁴ M; (b) 10‾⁵ M; (c) 10‾⁶ M; and (d) 10‾⁸ M. At the three top doses, immediate and powerful reabsorptions of pulmonary fluid were observed in older fetuses (60 days gestation and above); significant falls were observed in the younger fetuses. This result was unexpected, as it was hypothesized that ACh would stimulate fluid production. The threshold dose for ACh was between 10‾⁶ M and 10‾⁸ M. Phentolamine blocked the effect of ACh. This result suggested that reabsorption is a result of an indirect effect of ACh acting through pulmonary alpha receptors. The results in this study show that epinephrine, NE, AVP and ACh are all important promoters of fetal pulmonary fluid removal in the fetal guinea pig. Pulmonary alpha-adrenergic receptors mediate the effects of epinephrine, NE and ACh (indirectly). The conclusions drawn from this study emphasize the importance of species' comparison in fetal research. LIST OF ABBREVIATIONS AVP Arginine Vasopressin NE Norepinephrine DOPA dihydroxyphenylalanine PNMT Phenylethanolamine n-methyltransferase ACh Acetylcholine / Science, Faculty of / Zoology, Department of / Graduate