Thesis (M.S.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thesis (M.S.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [128]-151).

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 110-125). Also available online via the World Wide Web.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 68-82).

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2007" Includes bibliographical references.

Made available in DSpace on 2015-05-14T12:59:43Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1379485 bytes, checksum: ae907e91268da44815445844aa82dc49 (MD5) Previous issue date: 2008-03-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / 8(17),12e,14-labdatrien-18 oic acid (labdane-302), is a diterpene isolated from the stem bark of Xylopia langsdorfiana A. St.-Hil. & Tul. In a preliminary study, Ribeiro (2003) demonstrated that labdane-302 inhibited the phasic contractions induced by carbachol (CCh) or histamine (IC50 = 1.7  0.5 and 0.9  0.2 x 10-5 M, respectively) on guinea-pig ileum. The aim of the present study was to investigate the spasmolytic action mechanism of labdane-302 in that organ. Isometric and isotonic contractions were monitored, and the parameters of relative potency and efficacy were determined from cumulative concentration-response curves. Labdane-302 inhibited (pD´2 = 4.9  0.2; r2 = 0.8  0.1) the cumulative concentration-response curves to histamine, and these were shifted to the right, in a non-parallel manner (Schild plot slope = 4.2  1.4), with depression of the maximal effect (Emax), suggesting a noncompetitive antagonism. Labdane-302 relaxed, in an equipotent manner, the ileum pre-contracted with KCl, CCh or histamine, suggesting that this diterpene could be acting on Cav. This assumption was confirmed by observation that labdane-302 antagonized the CaCl2 induced contractions in the depolarizing medium without Ca2+ (pD´2 = 5.3  0.1; r2 = 0.5  0.05 ), with shift of the concentration-response curve to the right, in a non-parallel manner (Schild plot slope = 2.6  0.5), with depression of Emax. As in the guinea-pig ileum the major CaV subtype is the Cav 1, we decided to investigated its role in the action mechanism of labdane-302, and was verified that the diterpene relaxed (EC50 = 3.6  0.8 x 10-5 M) the ileum pre-contracted with S-(-)-Bay K8644, a selective agonist of the Cav1. As the potent relaxing of labdane-302 was no different when ileum pre-contracted with KCl or S-(-)-Bay K8644, it is suggestive of indirect blockade of the Cav1. Since K+ channels play a major role in the regulation of membrane potential and modulation of CaV, we decided to investigate the participation of K+ channels in the spasmolytic action of labdane-302. The relaxant potency of labdane-302 (EC50 = 1.5  0.3 x 10-5 M) was decreased on 2.4 times in the presence of CsCl, a non-selective K+channels blocker (CE50 = 3,5  0,6 x 10-5 M), suggesting a possible involvement of the K+ channels in the spasmolytic effect caused by labdane-302. In order to verify which subtypes of K+ channels could be involved we used selectives blockers of these channels. The observation that 4-aminopyridine, a selective blocker of Kv, and that TEA+ 1 mM, a selective blocker of the BKca did not change the relaxant effect of labdane-302 suggests that KV and BKca are not involved in its action mechanism. On the other hand, the log concentration-response curve induced by labdane-302 was shifted to the right in the presence of apamine, selective blocker of the SKCa, (EC50 = 3.3  0.4 x 10-5 M) or glibenclamide, selective blocker of the KATP, (EC50 = 3.2  0.3 x 10-5 M), suggesting the involvement of SKCa and KATP in the spasmolytic action mechanism induced by labdane-302 on guinea-pig ileum. In the presence of aminophylline the potency of labdane-302 was increased (EC50 = 0.4 ± 0.03 x 10-5 M), which indicate participation of cyclic nucleotides. These results suggest that the relaxant effect of labdane-302 on guinea-pig ileum, involves the activation of the SKCa and KATP with the consequent blocking of Cav-L and involvement of the cyclic nucleotides. / O ácido 8(17),12e,14-labdatrieno-18-óico (labdano-302) é um diterpeno isolado das cascas do caule de Xylopia langsdorfiana A. St.-Hil. & Tul. Em estudos anteriores Ribeiro (2003) demonstrou que o labdano-302 inibiu as contrações fásicas induzidas por carbacol (CCh) e histamina (CI50 = 1,7  0,5 e 0,9  0,2 x 10-5 M, respectivamente) em íleo de cobaia. O objetivo desse estudo foi investigar o mecanismo de ação espasmolítica do labdano-302 neste órgão. As contrações isométricas e isotônicas foram monitoradas e os parâmetros de potência e eficácia relativas foram determinados a partir de curvas de concentrações-resposta cumulativas. O labdano-302 inibiu (pD´2 = 4,9  0,2; r2 = 0,8  0,1) as curvas cumulativas à histamina e estas foram desviadas para direita, de forma não paralela ( slope de Schild = 4,2  1,4) e com redução do Emax, sugerindo um antagonismo não competitivo. O labdano-302 relaxou, de maneira equipotente, o íleo pré-contraído com KCl, CCh ou histamina sugerindo que este diterpeno deve estar agindo Cav. Essa sugestão foi confirmada pelo fato do labdano-302 ter antagonizado as contrações induzidas por CaCl2 em meio despolarizante nominalmente sem Ca2+ (pD´2 = 5,3  0,1; r2 = 0,5  0,05 ), com desvio da curva concentrações-resposta para direita, de forma não paralela ( slope de Schild = 2,6  0,5) e com redução do Emax. Como em íleo de cobaia o CaV mais expresso é o Cav 1, decidiu-se investigar a sua participação no mecanismo de ação do labdano-302 e foi verificado que o diterpeno relaxou (CE50 = 3,6  0,8 x 10-5 M) o íleo pré-contraído com S-(-)-Bay K8644, um ativador seletivo dos Cav1. Como a potência relaxante do labdano-302 não foi diferente quando o órgão era pré-contraído com KCl ou S-(-)-Bay K8644, isto é sugestivo de bloqueio indireto dos Cav1. Uma vez que os canais de K+ desempenham um papel chave na regulação do potencial de membrana e modulação dos CaV, decidiu-se investigar a participação dos canais de K+ na ação espasmolítica do labdano-302. A potência relaxante de labdano-302 (CE50 = 1,5  0,3 x 10-5 M) foi reduzida em aproximadamente 2,4 vezes na presença de CsCl, bloqueador não seletivo dos canais de K+ (CE50 = 3,5  0,6 x 10-5 M), sugerindo a participação de canais de K+ no efeito do labdano-302. Para verificar qual(is) canal(is) de K+ estariam envolvidos usou-se bloqueadores seletivos desses canais. O fato da 4-aminopiridina, bloqueador seletivo dos Kv, e do tetraetilamônio 1 mM, bloqueador seletivo dos BKca não alterar o efeito relaxante do labdano-302 indica que os KV e os BKca não estão envolvidos em seu mecanismo de ação. Entretanto, a curva de relaxamento induzida pelo labdano-302 foi desviada para direita na presença de apamina, um bloqueador seletivo dos SKCa (CE50 = 3,3  0,4 x 10-5 M) ou de glibenclamida, um bloqueador seletivo dos KATP, (CE50 = 3,2  0,3 x 10-5 M), sugerindo o envolvimento dos SKCa e dos KATP no mecanismo de ação espasmolítica do labdano-302 em íleo isolado de cobaia. Na presença de aminofilina, a potência do labdano-302 foi aumentada (CE50 = 0.4 ± 0.03 x 10-5 M), o que indica a participação de nucleotídeos cíclicos. Esses resultados sugerem que o efeito relaxante do labdano-302 em íleo de cobaia, envolve a ativação dos SKCa e KATP com o conseqüente bloqueio de Cav-L e o envolvimento de nucleotídeos cíclicos.

No description available.

KCNE β-subunits modulate KCNQ1 (Q1) voltage-gate K+channels providing the current diversity required for Q1 channels to function in a wide variety of cell types and tissues. In the present thesis, the stoichiometry of KCNE1 (E1) β-subunits in functioning Q1 channels is investigated, along with the formation of heteromeric channel complexes, complexes containing 2 different KCNE β-subunits. The chemical approaches used to answer these questions were then expanded to generate a novel labeling reagent. To determine the stoichiometry of the Q1/E1 complex, I devised an iterative subunit counting approach that relies on a chemically releasable K+channel blocking reagent. The extracellularly applied reagent irreversibly blocks charybdotoxin (CTX) sensitive Q1 channels by chemically modifying E1 peptides that contain an N-terminal cysteine residue. Chemical release of the inhibitor and subsequent iterative applications of the reagent reported that Q1 channels partner with two KCNE β-subunits. To determine whether heteromeric Q1-KCNE complexes form, I synthesized a similar, but non-cleavable, K+channel blocking reagent that detects specific KCNE peptides in functioning complexes by irreversible channel inhibition. Using this “KCNE sensor”, heteromeric Q1/E1/E3, Q1/E1/E4 and Q1/E3/E4 complexes were shown to form, traffic to the cell surface and function. Using mathematical subtraction to visualize the irreversibly blocked current, the currents and gating kinetics of the different heteromeric complexes were revealed and a hierarchy of KCNE subunit modulation of Q1 channels was determined: E3>E1>>E4. Building on this technology, a chemically releasable K+ channel blocking reagent was created to specifically label KCNE β-subunits with biotin. The reagent delivers biotin to CTX sensitive Q1 channels and labeling occurs through free thiols provided by either cysteine residues or thiol modified sugars. This preliminary data demonstrates a novel strategy for labeling endogenous K+ channels in native cells.

No description available.