Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 102-118).

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [114]-130).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves [106]-128).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 160-188).

Cyclic AMP and cyclic GMP (cAMP and cGMP) are important second messengers in key signal-transduction pathways that mediate various physiological functions in bacteria and eukaryotes. Adenylyl Cyclases (ACs) and Guanylyl Cyclases (GCs) cyclize ATP and GTP to produce cAMP and cGMP, respectively. Though most nucleotide cyclises show exquisite specificity for their substrates, there are instances where ACs were observed to have low GC activity as well, and vice versa. To understand structural basis of substrate (ATP or GTP) recognition, discrimination and binding by an adenylyl cyclase, we have taken up Ma1120, an AC from Mycobacterium avium, for our studies. Work presented in the thesis includes crystal structures of Ma1120 in the presence of substrate (ATP or GTP), by-product pyrophosphate and ATP analogue 2′,5′-dideoxyd-3′-adenosine triphosphate (2′,5′-dd-3′-ATP). A triple mutant of Ma1120 (K101→E, D157→G, A167→Y) was generated to increase specificity of Ma1120 towards GTP by mutation in the substrate specifying residues, but the enzyme showed equal specificity for ATP as well as for GTP. Ma1120 exists as a monomer in solution and crystallized as a monomer in the absence of substrate or inhibitor. The substrate specifying lysine residue plays a dual role of interacting with the substrate and stabilizing the dimer. The dimerization loop region harbouring the second substrate specifying residue, an aspartate, shows significant differences in conformation and position between the monomeric and dimeric structures. Thus, this study has not only revealed that significant structural transitions are required for the interconversion of the inactive and the active forms of the enzyme, but also provided precise nature of these transitions. ATP bound to Ma-Cat has two different conformations, one with C2′-endo and the other with C3′-endo puckering for the ribose. C3′-endo conformation is favourable for catalysis as it brings 3′-OH group of ribose and free oxygen of α-phosphate closer to each other. The crystal structure of GTP bound to Ma-Cat showed a novel mode of GTP binding to AC. This is the first report of GTP bound to AC. ATP bound to Ma-Cat-KDA→EGY forms non-cognate substrate complex and ATP is stabilized by stacking of adenines over each other with Tyr167 flanking on both sides of adenines. Ma-Cat-KDA→EGY+GTP complex is the first report of GTP bound to a guanylyl cyclase. GTP is bound in reverse orientation when compared to ATP bound to AC. Reverse orientation of GTP is attained to stabilize the guanine in highly electronegative guanine binding pocket. Also, O3' of GTP is placed in opposite orientation as compared to ATP bound to Ma-Cat. Therefore, during cyclization reaction guanine and ribose changes their orientation to bring O3' atom of ribose closer to α-phosphate, after cleavage of the bond between α- and β-phosphates. Thus, this study has revealed novel modes of binding of ATP and GTP to catalytic domains of Ma1120 and its triple mutant, mechanism of substrate discrimination and residual activity for the non-cognate substrate.

Orientador: Edson Antunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T06:23:28Z (GMT). No. of bitstreams: 1 Bau_FernandoRicardo_M.pdf: 739046 bytes, checksum: e73f0e5383c7082e68c3cbf81bdfce5f (MD5) Previous issue date: 2009 / Resumo: A síndrome da bexiga hiperativa atinge grande parte da população mundial, e gera sintomas que prejudicam a qualidade de vida dos portadores. Está associada com a hiperatividade do detrusor que se dá por um aumento das contrações espontâneas. Alguns estudos têm mostrado que a deficiência de NO é um dos fatores responsáveis por gerar estas contrações espontâneas. É sabido que o mecanismo de sinalização do NO envolve a ativação da guanilil ciclase solúvel e produção de GMPc. Atualmente, algumas drogas têm sido sintetizadas para mimetizar o efeito exercido pelo NO, tal como o BAY 41-2272, um potente estimulador da guanilil ciclase solúvel independente de NO. Vários trabalhos mostraram que o BAY 41-2272 causa relaxamento de vários tipos de musculatura lisa, podendo ser um composto com grande potencial terapêutico em doenças onde a via do NO/GMPc está prejudicada. O objetivo deste trabalho é investigar a capacidade do BAY 41-2272 de relaxar detrusor isolado de camundongo, coelho e rato in vitro e os mecanismos farmacológicos envolvidos na resposta relaxante. Camundongos C57b6 machos (30-40 g), coelhos New Zealand machos (2-3 kg) e ratos Wistar machos (250-300 g) foram anestesiados e mortos. As bexigas foram removidas e fragmentos de detrusor foram montados em banho para órgãos isolados contendo 10 ml de solução de Krebs. Curvas concentração-resposta ao BAY 41-2272 (10-9 - 10-4 M) foram construídas em tecidos précontraídos com carbacol (10 µM) ou KCl (80 mM), na ausência ou na presença de LNAME (inibidor da óxido nítrico sintase; 100 µM), ODQ (inibidor da guanilato ciclase solúvel; 100 µM), Sildenafil (inibidor da fosfodiesterase tipo-5; 10 µM), ou inibidores de canais de potássio (0,1 µM charibdotoxina + 1 µM apamina; 1µM tetraetilamônio; ou 10 µM glibenclamida). Curvas concentração-resposta ao nitroprussiato de sódio (SNP; 10-8 - 10-4 M), gliceril trinitrato (GTN; 10-8 - 10-4 M) e 8Br-GMPc (10-8 - 10-4 M) foram também construídas. Contrações induzidas por CaCl2 extracelular foram avaliadas na presença do BAY 41-2272, bem como o efeito no influxo de cálcio em plaquetas isoladas de coelho. Níveis de GMPc e AMPc foram avaliados após a estimulação do detrusor com BAY 41- 2272 (10 e 100 µM) e SNP (100 µM) na ausência ou na presença de ODQ (100 µM), através de imunoensaio enzimático (ELISA). O BAY 41-2272 produziu relaxamento de detrusor isolado de camundongos, ratos e coelhos de maneira concentração-dependente, com valores de resposta máxima de 61,3 ± 6,6%, 91,7 ± 5,9% e 95,1 ± 9,9%, respectivamente. Detrusor de coelhos foram selecionados para os experimentos subseqüentes. Os doadores de NO, SNP e GTN, bem com o 8Br-GMPc produziram um discreto relaxamento comparado ao BAY 41-2272. O tratamento dos tecidos com L-NAME (100 µM) ou sildenafil (10 µM) não afetou de maneira significativa o relaxamento induzido pelo BAY 41-2272. Entretanto, o ODQ (100 µM), reduziu significativamente a resposta ao BAY 41-2272. Os bloqueadores de canais de K+ (apamin + charibdotoxina, glibenclamida ou tetraetilamônio) também não afetaram a resposta relaxante do BAY 41-2272. O BAY 41-2272 (10 e 100 µM) elevou os níveis de GMPc em cerca de 14 e 20 vezes respectivamente, sem afetar os níveis de AMPc. Na menor concentração do BAY 41-2272 (10 µM), o ODQ aboliu a elevação dos níveis de GMPc, ao passo que na maior concentração do BAY 41-2272 (100 µM), o ODQ inibiu parcialmente a elevação dos níveis de GMPc. A adição de CaCl2 (0,01-30 mM) extracelular em detrusor isolado de coelhos causou contração de maneira concentração-dependente que foi significativamente reduzida pelo tratamento prévio com BAY 41-2272 (1 e 10 µM), sendo que este efeito não foi prevenido pelo ODQ. O BAY 41-2272 reduziu significativamente o aumento dos níveis intracelulares de cálcio em plaquetas de coelho induzido por trombina. Em resumo, o BAY 41-2272 produz relaxamento em detrusor isolado de camundongos, coelhos e ratos através da produção de GMPc e da inibição do influxo de cálcio que independe de GMPc / Abstract: Overactive bladder (OAB) is a highly prevalent condition that affects millions of people worldwide with a profound effect on quality of life. The bladder overactivity is related to spontaneous contractions of the detrusor smooth muscle causing an increase in the intravesical pressure and consequently stimulation of the micturirion reflex. Evidences suggest that impairment of nitric oxide (NO) signaling pathway may account for OAB. It is well established that NO signaling pathways involves soluble guanylate cyclase (sGC) stimulation and cyclic GMP production. Recently, pharmacological agents capable of directly stimulating soluble guanylate cyclase independenly of NO, such as BAY 41-2272 has been reported to produce relaxation of different types of smooth muscle, showing great therapeutic potential in disturbs which NO pathway is impaired. The present study aimed to evaluate the capacity of BAY 41-2272 to relax isolated mouse, rat and rabbit DSM and the mechanism underlying these response. C57b6 male mice, Wistar male rats and New Zealand male rabbits were anesthetized, and urinary bladder removed. DSM was transferred to 10-mL organ baths containing oxygenated and warmed Krebs-Henseleit solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. Concentration-response curves to BAY 41-2272 (10-9 - 10-4M) were constructed, in previously contracted tissues with carbachol (10 µM) or KCl (80 mM), in the absence and in the presence of L-NAME (Nitric Oxide Synthase inhibitor; 100 µM), ODQ (sGC inhibitor; 100 µM), Sildenafil (phosphodiesterase type-5 inhibitor; 10 µM), or potassium channel blockers (0.1 µM charybdotoxin + 1 µM apamin; 1 µM tetraethylammonium; or 10 µM glybenclamide). Concentration-response curves to sodium nitroprusside (SNP; 10-8 - 10-4 M), glyceryl trinitrate (GTN; 10-8 - 10-4 M) and 8Br-cGMP (10-8 - 10-4 M) were also constructed. CaCl2-induced contractions in DSM and calcium influx in rabbit isolated platelets were evaluated in the presence of BAY 41-2272. Levels of cAMP and cGMP in DSM strips were determined after treatment with BAY 41-2272 (10 and 100 µM), SNP (100 µM) in the absence or in the presence of ODQ (100 µM) using specific EIA kit. BAY 41-2272 (0.001-100 µM) produced concentration-dependent DSM relaxations in mouse, rat and rabbit with maximal responses of 61.3 ± 6.6%, 95.1 ± 9.9% and 91.7 ± 5.9%, respectively. The NO-donors sodium nitroprusside and glyceryl trinitrate, as well as 8-bromo-cGMP also produced concentration-dependent rabbit DSM relaxations, but to a lesser extent than BAY 41-2272. Pretreatment with L-NAME (NO synthesis inhibitor) or sildenafil (phosphodiesterase-5 inhibitor) had no effect in BAY 41-2272- induced responses. However, the soluble guanylyl cyclase inhibitor ODQ significantly reduced BAY 41-2272-induced relaxantions. BAY 41-2272 (10 and 100 µM) increased the bladder cGMP levels by about of 14- and 20-fold, respectively, without affecting the cAMP levels. The cGMP increases in response to BAY 41-2272 and SNP were markedly reduced by ODQ. CaCl2 caused a concentration-dependent contraction in DSM strips and BAY 41- 2272 significantly reduced the contractile responses to extracellular Ca2+ in an ODQinsensitive manner. BAY 41-2272 also significantly reduced the increase of intracellular calcium levels induced by thrombin. This inhibitory effect was completely reverted after the treatment with ODQ. BAY 41-2272 relaxes DSM of the three animal species studied. BAY 41-2272-induced DSM relaxation involves mainly cGMP production, but an additional mechanism involving Ca2+ influx blockade independently of cGMP production appears to be involved / Mestrado / Farmacologia / Mestre em Farmacologia

Orientador: Gilberto de Nucci / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T20:24:02Z (GMT). No. of bitstreams: 1 Toque_HaroldoAlfredoFlores_D.pdf: 1734292 bytes, checksum: a664da49d6a032202e54c1490b0647dc (MD5) Previous issue date: 2008 / Resumo: O sildenafil, inibidor específico de PDE5, é utilizado no tratamento da disfunção erétil. Na procura de um inibidor mais potente e seletivo de PDE5, testamos vinte novos análogos do sildenafil (6a-v), caracterizados pela presença de um grupo sulfonil ou de um substituinte n'n'etilendiamina na posição do grupo metil da piperazina. Os objetivos deste trabalho foram: 1) Avaliar a atividade inibitória da PDE5 de plaquetas humanas; 2) Avaliar o efeito relaxante dos análogos do sildenafil em corpo cavernoso de coelho; 3) Avaliar o efeito relaxante dos análogos do sildenafil em anéis de aorta de coelho. Foi utilizado sangue de voluntários sadios para a atividade inibitória da PDE5 e coelhos New Zealand machos (2-3 kg) procedentes do CEMIB-UNICAMP para os estudos funcionais. Os animais foram anestesiados com uretana, o corpo cavernoso e a artéria aorta foram rapidamente removidos. O tecido cavernoso e anéis de aorta foram montados em banho para órgão isolado contendo solução de Krebs (37oC, 95% O2 / 5% CO2). Os tecidos foram ligados a transdutores isométricos conectados a um sistema PowerLab® de aquisição de dados. Nossos resultados mostraram que a atividade inibitória da PDE5 induzida pelos análogos do sildenafil em plaquetas humanas foi de maneira dependente da concentração. Os compostos 6m, 6n e 6q mostraram valores elevados de IC50 para inibir a PDE5 plaquetária, enquanto que os compostos 6a, 6b, 6d, 6g e 6p produziram inibição inferior a 50%. A potência inibitória (IC50) dos compostos 6c, 6e, 6f, 6h, 6i, 6l e 6o foi similar ao sildenafil (IC50: 0,05 µM) com valores variando entre 0,05 e 0,15 µM. Os análogos que derivaram da molécula n'n'etilendiamina (6r, 6s, 6t e 6v) mostraram uma boa atividade inibitória com valores entre 0,20-0,51 µM. Interessantemente, o composto 6u mostrou uma potência de 0,04 µM, o qual representou o menor valor obtido dos análogos do sildenafil. Nos estudos funcionais, todos os análogos do sildenafil, à exceção do análogo 6m, relaxaram preparações de corpo cavernoso de coelho de maneira dependente da concentração. Particularmente, o análogo 6f mostrou o melhor perfil farmacológico no relaxamento, com potência similar ao sildenafil, e pode servir de base para o desenvolvimento de novos inibidores de PDE5 para o tratamento da disfunção erétil. Além disso, nossos resultados mostraram que os análogos do sildenafil produzem relaxamento dependente da concentração em anéis de aorta de coelho com endotélio íntegro ou removido. Somente o análogo 6b e 6m apresentaram valores de potência inferiores quando comparados ao sildenafil em tecidos com endotélio íntegro. A remoção do endotélio ou a adição do LNAME ou do ODQ (inibidor da NO sintase e da guanilil ciclase solúvel, respectivamente) em tecidos com endotélio íntegro, provocou deslocamento à direita para o sildenafil e seus análogos, à exceção do 6r e 6u que não apresentaram diminuição da potência decorrente da inibição do NO, seja pela remoção do endotélio, pela inibição da NO sintase ou pela inibição da guanilil ciclase solúvel. Nossos dados também mostram que o 6r e 6u, em combinação com o BAY 41-2272, aumentam a potência evocada por estes análogos tanto em anéis de aorta com endotélio íntegro ou removido. O relaxamento evocado pelo sildenafil, 6r e 6u não envolve a participação de canais de potássio e de cálcio, nem envolve a formação de prostanóides. As respostas relaxantes destes dois análogos não foram alteradas em tecidos desprovidos de endotélio, mostrando-se independentes da via NO/GMPc. Isto sugere que estes dois análogos podem ser de particular interesse em patologias decorrentes de disfunção endotelial. / Abstract: Sildenafil, a cGMP-specific phosphodiesterase-5 (PDE5) inhibitor, is used for the treatment of erectile dysfunction. In the search for a potent and selective PDE5 inhibitor, new sildenafil analogues (6a-v), characterized by the presence on the sulphonyl group in the 5' position of novel N-4-substituted piperazines or ethylenediamine moiety, were synthesized. The aim of this work was 1) To evaluate the PDE5 inhibitory activity in human platelets; 2) To evaluate the relaxing effect of sildenafil analogues in rabbit corpus cavernosum; 3) To evaluate the relaxing effect of sildenafil analogues in rabbit isolated aorta. Blood from human volunteers were collected and used for PDE5 inhibitory activity and Male New Zealand rabbit (2-3 kg) for functional studies. The rabbits were anaesthetized with urethane and sacrificed. The cavernosal tissue and aortic rings were mounted in organ bath containing Krebs solution (37oC, 95% O2 / 5% CO2). Each tissue was connected to an isometric transducer which was connected to a data acquisition system Powerlab®. Our results showed that sildenafil and its analogues concentration-dependently inhibited PDE5 activity in human platelets. Compounds 6m, 6n and 6q showed higher values of IC50 to inhibit PDE5 of platelets, whereas compounds 6a, 6b, 6d, 6g and 6p did not reach 50% of inhibition. The inhibitory potency of PDE5 for 6c, 6e, 6f, 6h, 6i, 6l and 6o were similar with sildenafil (IC50: 0,05 µM) with values between 0,05-0,15 µM. Derived analogues from n'n' substitution showed great PDE5 inhibitory activity. Interesting, compound 6u exhibited greater IC50 value (0,04 µM). In functional studies, all sildenafil analogues with exception of 6m, relaxed concentration-dependently rabbit corpus cavernosum. Compound 6f exhibited great pEC50 value in corpus cavernosum and could be used as base for developing new PDE5 inhibitors. Moreover, our results showed that sildenafil analogues concentration-dependently relaxed both endothelium-intact and - denuded aortic rings with similar potency values of sildenafil. Compounds 6b and 6m showed lower values of potency when compared to sildenafil in endothelium intact. Endothelium denudation or addition of L-NAME or ODQ (NOS and sGC inhibitors, respectively) caused marked rightward shifts in the curve to sildenafil and its analogues, whereas the relaxation curves for 6r and 6u were not altered after endothelium removal or either by the NO synthase or sGC inhibition. Moreover, our data also suggest that compound 6r and 6u increased the potency values in combination with BAY 41-2272 in both intact and denuded endothelium. The relaxation evoked by sildenafil, 6r and 6u does not involve either calcium or potassium channels or prostanoids formation. The relaxing responses by these compounds were independent of NO/cGMP pathway, suggesting that these compounds may be used in several diseases involving endothelium dysfunction. / Doutorado / Doutor em Farmacologia

No description available.

No / Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex, and may contribute to the pathophysiology of stroke and migraine. Previous studies demonstrated that nitric oxide (NO) formation promotes the repolarisation phase of CSD, and this effect may be cyclic GMP (cGMP)-mediated. Here, we have examined how phosphodiesterase (PDE) inhibition, either alone or superimposed on NO synthase (NOS) inhibition, alters CSD and the associated changes in extracellular cGMP. Microdialysis probes incorporating an electrode were implanted into the frontoparietal cortex of anaesthetised rats for quantitative recording of CSD, pharmacological manipulations, and dialysate sampling for cGMP measurements. CSD was induced by cathodal electrical stimulation in the region under study by microdialysis. Extracellular cGMP increased, but only slightly, during CSD. Perfusion of either zaprinast or sildenafil through the microdialysis probe, at concentrations that inhibited both PDE5 and PDE9 (and possibly other PDE), increased significantly extracellular cGMP. Unexpectedly, these levels remained high when NOS was subsequently inhibited with N¿-nitro- -arginine methyl ester hydrochloride ( -NAME, 1 mM). The most interesting pharmacological effect on CSD was obtained with sildenafil. This drug altered neither CSD nor the subsequent characteristic effect of NOS inhibition, i.e. a marked widening of CSD. The fact that NOS inhibition still widened CSD in the presence of the high extracellular levels of cGMP associated with PDE inhibition, suggests that NO may promote CSD recovery, independently of cGMP formation.

Cyclic 3',5'-guanosine monophosphate (cGMP) is a key signaling molecule involved in a myriad of physiological processes, including vascular smooth muscle (VSM) tone, water- and electrolyte homeostasis, platelet aggregation, airway smooth muscle tone, smooth muscle proliferation and bone formation. Increased occurrence of vascular disorders including erectile dysfunction, hypertension, stroke and coronary artery disease, have made it increasingly important to study the dynamic interplay between cGMP synthesis and hydrolysis in VSM cells. This dissertation examines the spatial distribution of intracellular cGMP, [cGMP]i, in response to NO and atrial natriuretic peptide (ANP) in VSM cells. To investigate the spatial patterning of [cGMP]i, we have developed a new generation of non-FRET (fluorescence resonance energy transfer) cGMP biosensors that are suitable to monitor [cGMP]i in response to physiological (low-nanomolar) NO and ANP concentrations and that qualify for real-time, confocal imaging techniques. We have termed these indicators FlincGs, for green fluorescent indicators of cGMP. For the development of FlincGs, we made use of the specific cGMP binding characteristics of PKG. We utilized site-specific mutagenesis, kinetic cGMP binding, dissociation and kinase assays, as well as crystallography, in order to investigate PKG activation and cGMP binding dynamics in greater detail. Based on these studies, our novel, non-FRET cGMP biosensors were designed by attaching cGMP binding fragments of PKG to the N-terminus of circular permutated green fluorescent protein. We applied FlincGs in cultured VSM cells as well as in intact tissue to determine whether two spatially distinct populations of guanlylyl cyclase (cytosolic versus membrane bound) underlie the generation of spatiotemporally-specific patterns of [cGMP]i formation.