Neben den cAMP- und cGMP-abhängigen Proteinkinasen (PKA bzw. PKG) sind als zyklonukleotid-regulierte Effektorproteine die Ionenkanäle CNG1-4, der "guanine nucleotide exhange factor“ Epac sowie die zyklonukleotid-spaltende Familie der Phosphodiesterasen (PDEs) von Bedeutung. Industriell synthetisierte cGMP- und cAMP-Analoga besitzen zwar meist eine hohe Affinität für ihr Zielprotein, über ihre Hydrolysestabilität gegenüber PDEs in der Zelle ist jedoch wenig bekannt. In dieser Arbeit wurden die kinetischen Konstanten von elf der am häufigsten genutzten cAMP-und cGMP-Analoga an verschiedenen Vertretern der PDE-Familien mittels Mikrokaloriemetrie bestimmt. Zudem konnte in den Messungen der inhibitorisch Effekt hydrolysestabiler Derivate auf die PDEs qualitativ und quantitativ ermittelt werden kann. Die Ergebnisse zeigen, dass Phosphodiesterasen in der Lage sind, auch chemisch modifizierte Analogsubstanzen der Cyclonukleotide cAMP und cGMP zu hydrolysieren. Hydrolysestabile Derivate dagegen entwickeln häufig inhibitorische Wirkung auf die PDEs und verursachen dadurch Veränderungen der intrazellulären cAMP und cGMP Konzentrationen. So vermag z. B. die Epac-spezifische Substanz Sp-8-pCPT-2’-O-Me-cAMPS in den in vitro Experimenten die PDEs mit ki-Werten im einstelligen mikromolaren Bereich zu inhibieren. In mit Sp-8-pCPT-2’-O-Me-cAMPS stimulierten Thrombozyten steigt als Folge dieser PDE-Hemmung die cGMP Konzentration in der Zelle an und man beobachtet eine als Folge eine PKG-vermittelte Phosphorylierung des Substratproteins VASP – eine unerwünschte Nebenreaktion. Die erhobenen Daten lassen außerdem Rückschlüsse auf den Inhibitionsmechanismus zu. Einige Analoga inhibieren die cGMP-bindenden GAF-Domänen in den PDEs 2A, 5A, 6cone und 10A sowie die PDE 4D3 nach dem linear-mixed-Typ und beeinflussen daher, neben der katalytischen Aktivität, vermutlich auch regulatorische Zentren dieser Enzyme. Zusammenfassend erleichtern die erhobenen Daten Wissenschaftlern die Auswahl des für ihre Fragestellung am besten geeigneten Derivates. / cAMP and cGMP are critical second messengers that regulate multiple targets including different cAMP/cGMP-dependent protein kinases (PKA/PKGs), exchange proteins directly activated by cAMP (Epacs), phosphodiesterases (PDEs) and cyclic nucleotide-gated ion channels (CNGs). Second and third generation cyclic nucleotide analogs are widely used to elucidate specificity of cellular signaling, mediated by these target proteins. However, the selectivity and stability of these analogs need to be fully understood in order to properly interpret results and rigorously assess the mechanisms by which these analogs work in the cell. To better understand the selectivity and cross-reactivity of these analogs I measured the activation or inhibitory activity of 13 commonly-used cyclic nucleotide analogs 8 different PDEs. To measure their stability to hydrolysis I utilized isothermal microcalorimetry, a method that allows to evaluate whether or not an analog can function as a substrate or inhibitor for PDEs. I demonstrate that indeed some of these analogs can be hydrolyzed by multiple PDEs and others are competitive inhibitors. Herein I provide Ki data for all of the non-hydrolyzable analogs and Km and Vmax values for all of the hydrolyzable analogs. Each of these values, as well as their mode of inhibition can be determined in a single experiment. The data strongly implied that several of these analogs might, in addition to their primary effects, also cause elevation of cAMP or cGMP indirectly by inhibiting PDEs in the cell. Such an effect could of course cloud interpretation of the use of these analogs. Similarly, those that are PDE substrates also might have their duration of action substantially reduced. To illustrate this point we show that Sp-8-pCPT-2’-O-Me-cAMPS, a highly specific non-hydrolyzable Epac activator in vitro, can under certain conditions enhance cGMP/PKG and cAMP/PKA signaling pathways in intact platelets. Specifically we found enhanced VASP phosphorylation at both PKA and PKG phosphorylation sites after the addition of Sp-8-pCPT-2’-O-Me-cAMPS. These data indicate that this “selective Epac activator” is able to indirectly activate the cAMP/PKA and cGMP/PKG signalling pathways presumably through inhibition of platelet PDE5 and/or PDE3. The data together allow to provide recommendations for how best to probe the different cyclic nucleotide signalling pathways using cyclic nucleotide analogs. In summary, the data provide evidence that most cAMP and cGMP analogs have multiple targets. Therefore, interpretation of any effects these analogs have in cells should take into consideration their possible cross-target reactivities.

Os pacientes que usam lítio (Li) para tratamento do transtorno bipolar freqüentemente apresentam poliúria e deficiência de concentração urinária, sintomas do Diabetes Insipidus nefrogênico (DIN). Animais tratados com Li apresentam baixos níveis de produção de adenosina monofosfato cíclico (AMPc) em resposta ao hormônio antidiurético (HAD). O Sildenafil (Sil), um inibidor da fosfodiesterase 5 (PDE5), eleva os níveis intracelulares de guanosina monofosfato cíclico (GMPc), levando a inserção de aquaporina 2 (AQP2) na membrana plasmática das células do ducto coletor. Portanto, inibidores de PDE podem promover a inserção de AQP2 na membrana plasmática mesmo sem a ativação do receptor de HAD, indicando a participação de uma via alternativa mediada pelo GMPc. Nós investigamos o efeito do Sil na expressão renal das proteínas de membrana AQP2, UT-A1, NKCC2, NHE3, P-ENaC em ratos com DIN induzido pelo Li. Ratos Wistar foram divididos nos seguintes grupos: grupo controle, recebendo dieta alimentar normal durante quatro semanas; grupo Li, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas; grupo Li + Sil, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas e 200 mg por quilo de dieta de Sil a partir da segunda semana; grupo Sil, recebendo dieta alimentar normal durante a primeira semana e a partir da segunda semana recebendo dieta normal com 200 mg de Sil por quilo de dieta. Os animais do grupo Li desenvolveram poliúria, diminuição da osmolalidade urinária e diminuição da expressão da AQP2. No grupo Li+Sil, o Sil foi capaz de reverter parcialmente a poliúria, diminuir o clearance de água livre, aumentar a osmolalidade urinária e aumentar a expressão da AQP2. A expressão de UTA1 foi completamente normalizada com o tratamento com Sil. A expressão das proteínas NKCC2 e NHE3 apresentaram-se aumentadas no grupo tratado com Li, e o Sil não foi capaz de reverter tal alteração. Além disso, o tratamento com Sil reverteu completamente o aumento da resistência vascular renal. Assim, concluímos que o tratamento com Sil em ratos com DIN melhora a poliúria, aumenta a smolalidade urinária e diminui o clearance de água livre pelo aumento da expressão de AQP2 e UT-A1. O tratamento com Sil pode ser benéfico para pacientes que sofrem com DIN induzida pelo Li. / Patients taking lithium to treat bipolar disorder often present polyuria and urinary concentrating defect. In addition, lithium-treated animals present lower cyclic adenosine monophosphate production in response to vasopressin. Sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, elevates intracellular cyclic guanosine monophosphate (cGMP) levels, leading to plasma membrane accumulation of aquaporin 2 (AQP2). Therefore, PDE inhibitors might induce AQP2 membrane insertion even without vasopressin receptor activation by activating a parallel cGMP-mediated signal transduction pathway. We investigated the effect of sildenafil on renal expression of AQP2, UT-A1, sodium/hydrogen exchanger (NHE3), type 1 bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), and the epithelial sodium channel alpha subunit (P-ENaC). Wistar rats received lithium (40 mmol/kg food) or not for 4 weeks (Li or control), some rats also receiving sildenafil (200 mg/kg food) in weeks 2-4, with or without lithium (Li+Sil or Sil). In Li+Sil rats, urine output was markedly lower, as was water free clearance, whereas urine osmolality was higher. Semiquantitative immunoblotting revealed the following: AQP2 expression was partially normalized; UT-A1 expression was completely normalized; expression of NKCC2 and NHE3 was significantly higher in Li rats (although not significantly different between Li+Sil rats and Li rats); and P-ENaC protein expression was unaltered in all groups. Sildenafil treatment completely reversed the lithium-induced increase in renal vascular resistance. In conclusion, sildenafil treatment of lithium-induced nephrogenic diabetes insipidus (NDI) improves polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1. Sildenafil treatment could be beneficial in patients with lithium-induced NDI.

Introdução: Vários são os fatores que alteram a atividade sexual de homens e mulheres. Com o envelhecimento observa-se aumento das queixas de desejo hipoativo feminino e de disfunção erétil. Visto que o homem e sua parceira constituem um sistema dinâmico, antes do advento dos inibidores da fosfodiesterase eles se adaptavam às condições disfuncionais do casal. A eficácia aliada a poucos efeitos colaterais e à facilidade de administração da sildenafila e, posteriormente, da vardenafila e tadalafila, revolucionou o tratamento da disfunção erétil. Por outro lado, até a presente data, a terapêutica medicamentosa com testosterona para o desejo sexual hipoativo de mulheres, ainda gera controvérsias. Objetivo: Avaliar o uso de androgênio, utilizado para tratamento das queixas de desejo sexual hipoativo em mulheres, comparando dois períodos, ou seja, pré e após o aparecimento iPDE 5. Os efeitos colaterais e as divergências em relação a este tratamento também serão analisados. Método: Foram selecionados estudos em inglês, português e espanhol, publicados entre 1988 e os dias atuais, ou seja, na década pré-advento dos inibidores da fosfodiesterase 5 e após. A busca dos artigos foi feita em periódicos indexados nas bases Lilacs, Cochrane, Embase e Medline/PubMed, utilizando-se os seguintes descritores e suas combinações: sexualidade (sexuality), desejo sexual hipoativo em mulheres (female hypoactive sexual desire disorder), testosterona (testosterone), terapia androgênica em mulheres (androgen therapy in women). Discussão: Embora haja evidência sobre a efetividade do tratamento com testosterona para desejo hipoativo em mulheres, este uso ainda gera muitas controvérsias. Resultados: O número de estudos randomizados sobre uso de testosterona para tratamento de DSH feminino aumentou de 10%, comparando o período compreendido entre 1988 e 1998, para 90% entre 1999 e 2012. Todos os estudos randomizados analisados demonstraram benefícios sobre a resposta sexual, melhorando a libido, a excitação e/ou o orgasmo. Porém, como tiveram seguimento por um período máximo de 24 semanas, os riscos dessa administração não foram esclarecidos. Conclusão: A partir de 1988, ou seja, após a liberação comercial dos inibidores da fosfodiesterase do tipo 5, para tratamento da disfunção erétil, houve um aumento significativo do número de pesquisas com a finalidade de avaliar o uso de testosterona em mulheres com desejo hipoativo. Porém, ainda são necessários estudos de longo prazo para que os riscos e os benefícios, desta administração, sejam esclarecidos / Introduction: There are several factors that alter the sexual activity of both men and women. With age, an increase in complaints about hypoactive feminine desire and erectile dysfunction can be observed. Since the man and his partner constitute a dynamic system, before the advent of phosphodiesterase inhibitors, they would adapt to the dysfunctional conditions of the couple. The efficacy with few collateral effects and the easy administration of sildenafil and later, vardenafil and tadalafil, has revolutionized the treatment of erectile dysfunction. On the other hand, until now, the drug therapy with testosterone for hypoactive sexual desire of women still generates controversies. Objective: Assess the use of androgen, used for treating complaints of hypoactive sexual desire in women, comparing two periods, that is, before and after the emergence of iPDE 5. Side effects and divergences regarding this treatment are also analyzed. Method: Studies in English, Portuguese and Spanish were selected, published between 1988 and the present, that is, in the decade before the advent of phosphodieterase 5 inhibitors and after this fact. The search for papers was made in indexed journals on Lilacs, Cochrane, Embase and Medline/PubMed data bases, using the following descriptors and their combinations: sexuality, female hypoactive sexual desire disorder, testosterone or androgen therapy in women. Discussion: Although there is evidence on the effectiveness of treatment with testosterone for hypoactive desire in women, this use still generates many controversies. Results: The number of randomized studies on the use of testosterone for the treatment of female HSD has increased 10%, comparing the period between 1988 and 1998, to 90% between 1999 and 2012. All randomized studies analyzed showed benefits on the sexual response, improving libido, excitation and/or orgasm. However, since they were followed for a maximum of 24 weeks, the risks of this dosage were not clarified. Conclusion: From 1988, that is, after the commercial release of phosphodiesterase type 5 inhibitors, for the treatment of erectile dysfunction, there was a significant increase in the number of papers with the purpose of evaluating the use of testosterone in women with hypoactive desire. However, there is the need of long-term studies in order to clarify the risks and benefits of this use

Submitted by Eunice Novais (enovais@uepg.br) on 2018-09-06T16:39:14Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Nubia Nascimento.pdf: 1296891 bytes, checksum: 1fb7f4ddd43a4591eeb9f1b8e95bb14f (MD5) / Made available in DSpace on 2018-09-06T16:39:14Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Nubia Nascimento.pdf: 1296891 bytes, checksum: 1fb7f4ddd43a4591eeb9f1b8e95bb14f (MD5) Previous issue date: 2018-05-28 / O cilostazol é um inibidor seletivo da fosfodiesterase III que atua como antiagregante plaquetário e vasodilatador. Faz parte do tratamento clínico da doença aterosclerótica carotídea extracraniana,apresentando benefícios para o adequado fluxo sanguíneo local.Devido à suacaracterística lipofílica,é um excelente fármaco para o estudo do desenvolvimento de sistemas de liberação modificada, que objetivam melhorar a disponibilidade biológica. A aplicação de tecnologias em formulações, como nanosuspensões, caracteriza uma estratégia atrativa para melhorar a ação de compostos pouco solúveis em água como o Cilostazol. Este trabalho avalioua ação do Cilostazolnanoencapsulado na túnica média da artéria carótida esquerda e no perfil lipídico de ratos Wistar. Os animais foram divididos em 4 grupos, que receberam soluções de nanocápsulas contendo cilostazol, nanocápsulas sem cilostazol, solução de propilenoglicol e cilostazol pesado.Foi observada diferença entre a espessura da carótida em micrômetros, entre os grupos que receberam o cilostazolnanoencapsulado e o grupo que recebeu a solução de propilenoglicol [70.22(9.2±47-81)IC95% 62-72] (P 0,019), sendo a maior espessura observada no grupo que recebeu o cilostazolnanoencapsulado. Não houve diferença em relação à celularidade em números absolutos, entre os grupos. Em relação ao perfil lipídico, o grupo que recebeu propilenogicol apresentou valores maiores de HDL comparativamente ao grupo que recebeu a solução de cilostazolnanoencapsulado [56.85(10.5±37.7-83.7)IC95% 52.56-61.38] (P0,01). O nível de triglicerídeos em mg/dL foi maior no grupo que recebeu o cilostazolpesado em relação ao grupo que recebeu a solução de nanocápsulas [116,92(56.37±45-266)IC95% 74.15-132.33] (P0,01). O cilostazol pesado apresentou melhores resultados em relação ao perfil lipídico. A apresentação do cilostazolnanoecapsulado não demonstrou ser adequada para o tratamento da doença carotídea extracraniana devido ao fato de aumentar o perfil lipídico dos animais estudados. A estrutura da nanocápsula, por conter lipídios em seu interior, piorou o perfil lipídico. Os achados laboratoriais foram condizentes com os achados histopatológicos. / Cilostazol is a selective inhibitor of phosphodiesterase III that acts as a platelet antiaggregant and vasodilator. It is part of the clinical treatment of extracranial atherosclerotic carotid disease, presenting benefits for adequate local blood flow. Due to its lipophilic characteristics, it is an excellent drug for the study of the development of modified release systems, which aim to improve biological availability. The application of technologies in formulations, such as nanosuspensions, characterizes an attractive strategy to improve the action of compounds little soluble in water like Cilostazol. This study evaluated the effect of nanoencapsulated Cilostazol on the left carotid artery layers and in the lipid profile of Wistar rats. The animals were divided into 4 groups, which received solutions of nanocapsules containing cilostazol, nanocapsules without cilostazol, solution of propylene glycol and heavy cilostazol. Differences were observed between carotid thickness in micrometers between groups receiving nanoencapsulated cilostazol and the group receiving the propylene glycol solution (70.22 (9.2 ± 47-81) 95% CI 62-72) (P <0.019), with the highest thickness observed in the group receiving nanoencapsulated cilostazol. There was no difference in cellularity in absolute numbers between the groups. Regarding the lipid profile, the group receiving propylene glycol had higher values of HDL in mg/dL, compared to the group that received the nanoencapsulated cilostazol solution [56.85 (10.5 ± 37.7-83.7) 95% CI 52.56-61.38] (P 0.01). The level of triglycerides in mg/dL was higher in the group that received the heavy cilostazol compared to the group that received the nanocapsule solution [116 (56.37 ± 45-116.92) CI95% 74.15-132.33] (P 0.01). The heavy cilostazol presented better results in relation to the lipid profile. The presentation of nanoecapsulated cilostazol has not been shown to be adequate for the treatment of extracranial carotid disease due to the fact that it increases the lipid profile of the animals studied. The structure of the nanocapsule, as it contained lipids, worsened the lipid profile. Laboratory findings were consistent with histopathological findings.

Introdução: A hipertensão arterial sistêmica (HA) pode estar associada à diminuição na produção e liberação do óxido nítrico derivado do endotélio (NO). O uso do sildenafil leva ao aumento de monofosfato de guanosina cíclica (GMPc), um importante mediador de NO. Contudo, pouco se sabe sobre os efeitos da inibição da fosfodiesterase tipo 5 (PDE5) na monitorização da pressão arterial 24-h (MAPA), pressão arterial durante exercício, noraepinefrina (Nor) e capacidade ao exercício, principalmente após transplante de coração (TX). Métodos: Nós estudamos 22 pacientes pós TX, os quais foram randomizados, tomando dose única de sildenafil (50mg) ou placebo (50mg), aproximadamente uma hora antes de iniciar o protocolo. No dia 1, os pacientes realizaram avaliação clínica, teste cardiopulmonar de caminhada de seis minutos (TES) seguido de teste de esforço cardiopulmonar (TE), Após o término dos testes em esteira, foi colocado o MAPA. Determinamos em repouso (rep), último minuto do TES (6) e pico do TE (Ex): FC (bpm) PAS e PAD (mmHg), VO2(ml/kg/min), Slope VE/VCO2, tempo de exercício (TE, min), distância (TES, Km) e Nor (pg/ml). No dia 2 o protocolo foi repetido, realizando-se o cross-over. Dezessete pacientes apresentavam HA. Resultados: (Pl e Sil respectivamente), Sil reduziu (p<0.05): PAS-rep(138±7 vs 122±18); PAD-rep(83±12 vs 78±12); PAS-6(156± 20 vs 137± 22); PAD-6(82±13 vs 77±14); PAS-Ex(155± 27vs 124±36); PAD-Ex(79±16 vs 66± 16); PAS 24-h(121±10 vs 114±9), PAD 24-h(80±6 vs 76±5), PAS vigília(122±11 vs 115±9), PAD vigília(81± 6 vs 76±5) e PAS noturna(119±12 vs 112±10), PAD noturna(78±7 vs 73±8); e aumentou Nor-repouso(483±165 vs 622±211). Sil não alterou rep, 6 e EX: FC, VO2 e Slope. Conclusão: O ciclo NO-cGMP parece desempenhar papel importante no controle da pressão arterial em TX. Sendo que, a inibição da PDE5 parece apresentar efeitos benéficos no controle da hipertensão arterial em TX, podendo ser utilizada concomitantemente a terapia anti-hipertensiva usual. / Background: Systemic hypertension (SH) can be associated with a decrease in endothelium-dependent nitric oxide (NO). Sildenafil determines increment in cyclic guanosine monophosphate (cGMP) that a mediator of NO. However, little is known about the effects of PDE5 inhibition on 24-hour ambulatory (ABP) and exercise blood pressure, noreprinephrine (Nor) and exercise capacity, specially after heart transplantation (HT). Methods: We studied 22 HT pts that on the 1st day underwent a cardiopulmonary (CP) self-controlled treadmill 6walk test(6) and, after, an ECG monitored CP treadmill maximal exercise test(Ex) within 60 and 90 min after oral Sildenafil (Sil,50mg) or placebo(Pl) given at random, and ABP. We determined at basal position(b), last min of 6 and the peak Ex the HR(bpm), SBP and DBP (mmHg), VO2(ml/kg/min), Slope VE/VCO2, exercise time(ET, min), distance(D, Km) and Nor(pg/ml). Also, after CP tests 24-h SBP and DBP were monitored. It was repeated on the 2nd day when the cross-over was done. Seventeen pts had SH. Results: (Pl and Sil respectively), Sil reduced (p<0.05): b- SBP(138±7 vs 122±18); b-DBP(83±12 vs 78±12); 6-SBP(156± 20 vs 137± 22); 6-DBP(82±13 vs 77±14); Ex-SBP(155± 27vs 124±36); Ex-DBP(79±16 vs 66± 16); 24-h SBP(121±10 vs 114±9) and DBP(80±6 vs 76±5), daytime SBP(122±11 vs 115±9) and DBP(81± 6 vs 76±5) and nighttime SBP(119±12 vs 112±10) and DBP(78±7 vs 73±8); and increase b-Nor(483±165 vs 622±211). Sil did not change in b, 6 and EX; HR, Nor, VO2 and Slope. Conclusion: NO-cGMP pathway seems to play a role in blood pressure control in HT. The PDE5 inhibition could have potential beneficial effects on hypertensive HT in addition to antihypertensive therapy.

INTRODUÇÃO: A hipertensão arterial pulmonar associada a cardiopatias congênitas (HAP-CCg) é um tipo específico de doença vascular pulmonar com várias complicações. Os pacientes com HAP-CCg avançada são considerados inoperáveis e podem apresentar hipoxemia crônica e eritrocitose secundária devido ao desvio de sangue da direita para a esquerda através das comunicações cardíacas. O presente estudo teve como objetivo analisar a resposta a dois inibidores de fosfodiesterase 5 (iPDE 5), a saber, sildenafila e tadalafila, administrados por seis meses. Os fármacos haviam sido previamente aprovados para uso na HAP em geral. No entanto, a literatura é escassa no que diz respeito à sua utilização especificamente na HAP-CCg. Propusemo-nos a investigar potenciais efeitos destes fármacos além de suas ações como vasodilatadores. MÉTODOS: Foram incluídos 31 pacientes com HAP-CCg avançada (idade entre 10 e 54 anos). A maioria apresentava redução na saturação sistêmica de oxigênio, com hematócrito elevado. A sildenafila e a tadalafila foram administradas por via oral (respectivamente, 20 mg três vezes ao dia e dose única diária de 40 mg). A capacidade física foi avaliada pela distância percorrida em 6 minutos (teste de caminhada de 6 minutos, protocolo American Thoracic Society). A qualidade de vida relacionada à saúde foi avaliada utilizando-se o questionário genérico SF-36. Níveis circulantes de marcadores de disfunção microvascular foram analisados por ensaios imunoenzimáticos. Todas as medidas foram realizadas na condição basal, e aos três e seis meses de tratamento com iPDE 5. Utilizando casuística ampliada com análise de dados tanto de forma retrospectiva como prospectiva, investigamos variáveis com possível valor prognóstico. RESULTADOS: Os tratamentos com ambos os fármacos foram associados à melhora da capacidade física (classe funcional e distância percorrida em 6 minutos, p < 0,05), hematócrito e nível de hemoglobina (p < 0,05), e qualidade de vida relacionada à saúde (componentes físico e mental, p < 0,05). Comparados ao grupo controle, na condição basal, os pacientes apresentaram níveis plasmáticos elevados de beta-tromboglobulina (beta-TG, p=0,002), selectina P (p=0,018), ativador do plasminogênio tipo tecidual (t-PA, p=0,005) e do antígeno do fator de von Willebrand (VWF:Ag, p=0,005); a trombomodulina mostrou-se significantemente reduzida (TM, p < 0,001). A administração de tadalafila foi seguida de melhora no nível de beta-TG (p=0,004), t-PA (p=0,003) e TM (p=0,046), enquanto a selectina P modificou-se apenas com a sildenafila (p=0,034). O VWF:Ag mostrou-se com melhora significante, porém transitória, no grupo tratado com sildenafila (p=0,019). Usando casuística ampliada que incluiu pacientes de estudos anteriores de nosso grupo (coorte estendida de 75 indivíduos), analisamos dados coletados no início do seguimento para todos eles, assim como outros relacionados ao tratamento. Uma série de variáveis clínicas e laboratoriais foram testadas quanto à sua possível correlação com a sobrevida livre de evento (seguimento de nove anos). Nesta parte do estudo, os pacientes receberam não apenas iPDE 5 como tratamento para HAP. Alguns deles receberam antagonistas dos receptores de endotelina isoladamente, enquanto outros receberam estes medicamentos (bosentana, ambrisentana) em combinação com iPDE 5. Após realização de análises univariada, bivariada e multivariada foi possível a identificação de quatro preditores de prognóstico desfavorável: 1, menor tempo de exposição a fármacos específicos para HAP (p < 0,001); 2, classe funcional avançada na admissão (p=0,010); 3, função sistólica ventricular direita deprimida (p=0,010); e 4, níveis circulantes elevados de VWF:Ag (p=0,013). Menor tempo de exposição aos medicamentos específicos para HAP foi caracterizado como preditor independente de desfechos. CONCLUSÕES E COMENTÁRIOS: Com base nestas observações, concluímos que os iPDE 5 são importantes no manejo da HAP-CCg avançada. Os efeitos foram observados não apenas na capacidade física e na qualidade de vida, mas também em marcadores biológicos de disfunção microvascular, potencialmente relacionados à progressão e/ou complicações da doença. Além disso, as observações em longo prazo sugeriram que maior tempo de exposição aos fármacos para HAP (incluindo iPDE 5) foi benéfico, enfatizando a necessidade de instituição precoce das terapias. O fato de o estudo ter sido realizado apenas com fármacos de administração oral torna os resultados relevantes no que diz respeito a regiões onde o acesso a tratamentos específicos para HAP é limitado a tais modalidades / INTRODUCTION: Pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH) is a particular form of pulmonary vascular disease with a number of complications. Patients with advanced CHD-PAH are deemed inoperable, and may present with chronic hypoxemia and secondary erythrocytosis due to right-to-left shunting across the cardiac communications. The present study was aimed at analyzing the response to 2 phosphodiesterase type 5 inhibitors (PDE5is), namely sildenafil and tadalafil, which were administered for 6 months. The drugs had been previously approved for management of PAH in general. However, there has been scarce literature on their use in the specific setting of CHD-PAH. We wished to investigate potential effects beyond their actions as vasodilators. METHODS: Thirty-one patients with advanced CHD-PAH were enrolled (age 10 to 54 years). Most of them had systemic oxygen desaturation with elevated hematocrit. Sildenafil and tadalafil were administered orally (respectively, 20 mg t.i.d and single daily dose of 40 mg). The physical capacity was assessed by registering the 6-minute walked distance (6-minute walk test, American Thoracic Society protocol). Health-related quality of life was assessed using the SF-36 generic questionnaire. Circulating levels of microvascular dysfunction markers were analyzed by enzyme-linked immunoassays. All measurements were performed at baseline, 3 months and 6 months of PDE5i administration. Using a larger patient population with retrospective as well as prospective data analysis, we looked for potential predictors of prognosis. RESULTS: Both therapies were associated with improvement of the physical capacity (functional class and 6-minute walked distance, p < 0.05), hematocrit and hemoglobin level (p < 0.05), and health-related quality of life (physical and mental components, p < 0.05). Compared to controls, patients had elevated baseline plasma levels of beta-thromboglobulin (beta-TG, p=0.002), P-selectin (p=0.018), tissue-type plasminogen activator (t-PA, p=0.005) and von Willebrand factor antigen (VWF:Ag, p=0.005), while thrombomodulin was importantly reduced (TM, p < 0.001). Tadalafil administration was followed by improvement of beta-TG (p=0.004), t-PA (p=0.003) and TM level (p=0.046), while P-selectin was improved by sildenafil only (p=0.034). VWF:Ag improved significantly, but only transiently in the sildenafil group (p=0.019). Using an extended population which included additional patients from previous studies of ours (the extended cohort consisted of 75 individuals), we analyzed data collected at the beginning of follow-up for all subjects, and treatment-related data as well. A number of clinical and laboratory parameters were tested for their possible correlation with event-free survival (9-year survival analysis). In this part of the study, patients were on monotherapy or combination therapy with PDE5is (sildenafil or tadalafil) and endothelin receptor antagonists (bosentan or ambrisentan). Univariate, bivariate and multivariate analyses were used to identify four predictors of a more worrisome prognosis: 1, a shorter time of exposure to specific PAH therapies (p < 0.001); 2, an advanced functional class at entry (p=0.010); 3, a depressed right ventricular systolic function (p=0.010); and 4, high circulating levels of VWF:Ag (p=0.013). A shorter exposure to PAH therapies was characterized as an independent predictor of poor outcome. CONCLUSIONS AND COMMENTS: Our observations point towards beneficial effects of PDE5is in advanced CHD-PAH. Importantly, the observed effects were not only on the physical capacity, and quality of life, but also biological markers of microvascular injury potentially related to disease progression and/or complications. Furthermore, long-term patient observation showed that a longer exposure to PAH drugs (including PDE5is) was beneficial, thus emphasizing the need for early initiation of therapies. Because only oral drugs were used, results may be relevant for countries and regions with limited access to PAH-specific medications

With the advent of large scale genome sequencing efforts along with more sophisticated methods of genetic mapping, a number of loci have been identified that are associated with human diseases. Intriguingly, many genes identified in these loci remain uncharacterized. Although current annotation can provide a prediction of putative function of some of these proteins at a biochemical level, understanding their cellular roles require analysis at a single gene level. Bioinformatic analysis carried out in the laboratory during studies on cyclic nucleotide metabolism in mycobacteria identified putative Class III cyclic nucleotide phosphodiesterases (Class III cNMP PDEs) from the non-redundant database of proteins. One of the proteins identified was the Rv0805 gene product from Mycobacterium tuberculosis. Detailed biochemical characterization of this protein revealed that Rv0805 is indeed a phosphodiesterase (PDE) and could hydrolyze 3’, 5’-cyclic adenosine monophosphate (cAMP) as well as 3’, 5’-cyclic guanosine monophosphate (cGMP). Structural analysis of Rv0805 revealed a metallophosphoesterase (MPE) like fold and presence of two metal atoms at the binuclear metal centre of the protein. Moreover, overexpression of Rv0805 in E. coli and M. smegmatis reduced intracellular cAMP levels indicating that it possesses cAMP PDE activity in vivo. The majority of proteins identified in this bioinformatic analysis were of bacterial or archaebacterial in origin but it was interesting to find some mammalian proteins, since, till date, no Class III cNMP PDE has been found in higher eukaryotes. Interestingly, two genes were identified in the human genome. These genes, 239FB and 239AB, are expressed in the fetal brain and adult brain, respectively and have been annotated as metallophosphoesterases but there has been no biochemical or functional characterization of these proteins. The 239FB gene is present between the FSHB and PAX6 genes on chromosome 11. This gene locus is present within a deletion interval (11p13-14) that is associated with the mental retardation phenotype of WAGR syndrome (Wilms’ tumor, aniridia, genitourinary anomalies, mental retardation). Inspection of available sequenced mammalian genomes indicated a shared synteny of the genes in the WAGR locus, highlighting it’s evolutionary conservation. Most interestingly, nucleotide sequences within the WAGR locus (which include the 5 genes WT1, PAX6, RCN1, ELP4 and 239FB) are amongst the 481 ultra conserved regions of the human genome. Moreover, 239FB is one of only 24 instances where an ortholog of an ultra-conserved element could be partially traced back by sequence similarity in lower eukaryotes such as Ciona intestinalis, Drosophila melanogaster, or Caenorhabditis elegans. Although the function of the 239FB protein is unknown so far, the distinctive expression of the gene in the fetal brain and the presence of an “ancient conserved region” in this gene suggest that this gene may be vital for the development of the nervous system. The work carried out in this thesis has attempted to understand the physiological functions of the 239FB/AB gene family. Amino acid sequence comparison revealed two amino acids changes between the human and rat proteins indicating the extra-ordinary sequence conservation of these proteins. Therefore, to characterize the biochemical properties of 239FB and 239AB proteins, rat proteins were used as model enzymes. Reverse transcription-PCR analysis of RNA prepared from the fetal and adult rat brains as well as Western blot analysis on cytosolic fractions of rat brains from various developmental stages indicated that 239FB is predominantly expressed in fetal brain. Detailed biochemical analyses of the rat 239FB and 239AB proteins were performed which showed that they possess metallophosphodiesterase activity. 239FB showed activity only in the presence of Mn2+ and Co2+ as the added metal cofactors. Surprisingly, the Km for Mn2+ of 239FB was found to be 1.5 mM, which is nearly 60-fold higher than that of its mycobacterial ortholog, Rv0805. A systematic mutational analysis was performed to characterize the residues that are involved in binding either one or both the metals found in the catalytic site of 239FB. Although 239FB shares some of the residues that have been shown to be essential for metal binding and catalytic activity with other MPEs including Rv0805, there are some differences as well. One histidine residue that has been conserved in other MPEs and has been shown to be important for metal binding is replaced by glycine (Gly-252) in 239FB. To study the consequence of replacing the glycine with a histidine in 239FB, a 239FBGly252His mutant protein was generated and characterized. Interestingly, the single mutation of Gly-252 to a histidine residue not only increased the affinity of the protein for metals but increased catalytic activity as well with various phosphodiesters. Moreover, 239FBGly252His mutant protein showed significant activity with cAMP and cGMP which were not hydrolysed by wild type 239FB. Interestingly, in the 239AB protein, histidine 284 is present at a position equivalent to Gly-252 in the 239FB protein. Biochemical characterization of 239AB showed 2’, 3’-cAMP hydrolyzing activity similar to 239FBGly252His mutant protein. A rat 239FB protein with a mutation (His67Arg) corresponding to a single nucleotide polymorphism seen in human 239FB, led to complete inactivation of the protein. The occurrence of this SNP at a very low frequency and only as a heterozygous condition suggests that a complete loss-of-function mutation of 239FB in human populations cannot be tolerated. To gain insights into the function of 239FB in its physiological milieu, yeast two-hybrid screening was performed with 239FB using human fetal brain cDNA library. Dipeptidyl peptidase III, a zinc dependent metallopeptidase, was found as an interacting partner of 239FB in this analysis and the functional consequences of this interaction would be an interesting area of study in future. While a number of metallophosphoesterases have been characterized biochemically and structurally, their biological role(s) and in vivo substrate(s) remain elusive. In order to elucidate the physiological role of 239FB/AB family, the ortholog of 239FB/AB in D. melanogaster was characterized. Sequence comparison of Drosophila ortholog with both the mammalian proteins indicated that it may be an ortholog of 239AB and hence, it was named as d239AB. Enhancer-promoter analysis with a putative promoter region of the d239AB indicated the expression of d239AB in the mushroom bodies in brain and in enterocytes in mid gut. Characterization of a Drosophila line, BS#16242, with a piggybac element inserted in the intron of d239AB showed disruption of d239AB expression. This suggested that BS#16242 line can serve as a d239AB knockout line and hence, was selected for further phenotypic characterization to unravel the physiological roles of d239AB. Though, BS#16242 flies did not show any developmental defects, a severe reduction in the fecundity of these files was observed. Further analysis revealed defective ovulation as a probable reason for reduced fecundity of these flies. In addition to compromised fecundity, BS#16242 flies showed a significant reduction in the life span of male as well as female flies. Moreover, these flies showed less resistance to thermal stress and desiccation. Most interestingly, all these phenotypes were rescued upon neuronal expression of the d239AB transgene in BS#16242 flies indicating that neuronal function of d239AB is important for diverse physiological processes. The phenotypes observed in BS#16242 flies mimic the physiological state under increased insulin signaling, such as decrease in life span, and susceptibility to various stress conditions suggesting that d239AB could play a role in the insulin signaling pathway. Interestingly, overexpression of d239AB transgene in neurons reduced cAMP levels in the brains of Drosophila, indicating that the protein may have cAMP phosphodiesterase activity in vivo. This is the first analysis of the presence of a Class III phosphodiesterase in eukaryotes. Thus, d239AB mediated regulation of cAMP levels in a particular subsets of cells, such as neurons, could also be one of the molecular mechanisms responsible for reduced fecundity and longevity of BS#16242 flies. Interacting partners of d239AB were inspected in the Drosophila interactome (built on protein-protein interactions identified using a yeast two-hybrid approach). Strikingly, most of the d239AB interacting proteins were involved either in transcriptional or translational regulation indicating that d239AB could be involved in the regulation of expression of genes involved in diverse physiological processes. This could explain why disruption of d239AB led to various physiological defects such as reduced fecundity, decreased life span and compromised fitness. In summary, studies described in this thesis suggest that 239FB and 239AB proteins are the first Class III cyclic nucleotide phosphodiesterases reported in eukaryotes. Results shown here suggest the critical role of their ortholog in the physiology of Drosophila. Further genetic manipulation in D. melanogaster and other organisms which harbor orthologs of the 239FB/AB gene could throw light on the diverse biological roles of these enzymes in humans.

Insulin and glucagon are released in pulses from pancreatic β- and α-cells, respectively. Both cell types are electrically excitable, and elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) due to depolarization with voltage-dependent entry of the cation is the main trigger of hormone secretion. Store-operated Ca2+ entry  (SOCE) also contributes to the [Ca2+]i elevation and this process has been suggested to be particularly important for glucagon secretion. cAMP is another important messenger that amplifies Ca2+-triggered secretion of both hormones, but little is known about cAMP dynamics in islet cells. In type-2 diabetes, there is deteriorated β-cell function associated with elevated concentrations of fatty acids, but the underlying mechanisms are largely unknown. To clarify the processes that regulate insulin and glucagon secretion, cAMP signalling and the store-operated pathway were investigated in β- and α-cells, primarily within their natural environment in intact mouse and human islets of Langerhans. Fluorescent biosensors and total internal reflection microscopy were used to investigate signalling specifically at the plasma membrane (PM). Adrenaline increased and decreased the sub-PM cAMP concentration ([cAMP]pm) in immuno-identified α-cells and β-cells, respectively, which facilitated cell identification. Glucagon elicited [cAMP]pm oscillations in α- and β-cells, demonstrating both auto- and paracrine effects of the hormone. Whereas glucagon-like peptide 1 (GLP-1) consistently elevated [cAMP]pm in β-cells, only few α-cells responded, indicating that GLP-1 regulates glucagon secretion without changes of α-cell [cAMP]pm. Both α- and β-cells responded to glucose with pronounced oscillations of [cAMP]pm that were partially Ca2+-dependent and synchronized among islet β-cells. The glucose-induced cAMP formation was mediated by plasma membrane-bound adenylyl cyclases. Several phosphodiesterases (PDEs), including the PDE1, -3, -4, and -8 families, were required for shaping the [cAMP]pm signals and pulsatile insulin secretion. Prolonged exposure of islets to the fatty acid palmitate deteriorated glucose-stimulated insulin secretion with loss of pulsatility. This defect was associated with impaired cAMP generation, while [Ca2+]i signalling was essentially unaffected. Stromal interacting molecule 1 (STIM1) is critical for activation of SOCE by sensing the Ca2+ concentration in the endoplasmic reticulum (ER). ER Ca2+ depletion caused STIM1 aggregation, co-clustering with the PM Ca2+ channel protein Orai1 and SOCE activation. Glucose, which inhibits SOCE by filling the ER with Ca2+, reversed the PM association of STIM1. Consistent with a role of the store-operated pathway in glucagon secretion, this effect was maximal at the low glucose concentrations that inhibit glucagon release, whereas considerably higher concentrations were required in β-cells. Adrenaline induced STIM1 translocation to the PM in α-cells and the reverse process in β-cells, partially reflecting the opposite effects of adrenaline on cAMP in the two cell types. However, cAMP-induced STIM1 aggregates did not co-cluster with Orai1 or activate SOCE, indicating that STIM1 translocation can occur independently of Orai1 clustering and SOCE.

Hypothyroidism is a common endocrine disorder affecting women of reproductive age. On a global level, iodine deficiency is still the most common cause of hypothyroidism. Also genetic variations, in particular SNP rs4704397 in the PDE8B gene, are responsible for a significant proportion of TSH variations.  Untreated hypothyroidism has significant adverse effects on pregnancy and fetal outcome. Most international guidelines suggest targeted thyroid testing in pregnant women with risk factors for thyroid disturbances. In a case-control study, an association between homozygous A/A as well as homozygous G/G carriers of SNP rs 4704397 in PDE8B and recurrent miscarriage was found. The explanation for this association is unknown. In a nationwide survey, all guidelines for thyroid testing and management of hypothyroidism during pregnancy in Sweden were collected and compared with international guidelines. The local guidelines were variable and poorly compliant with the international guidelines. In a follow-up in one district, 5,254 pregnant women were included for subsequent review of their medical reports. We found a targeted thyroid testing rate of 20.1% in clinical practice, with an overall frequency of women with trimester-specific elevated TSH of 18.5%. More disturbingly, half of the women who were on levothyroxine treatment at the time of conception had an elevated TSH level at thyroid testing. In a subsequent cohort study of the 5,254 women, we found the prevalence of trimester-specific elevated TSH and overt hypothyroidism to be equal in targeted thyroid tested and untested women. In a cross-sectional study, a median urinary iodine concentration (UIC) of 98 μg/l was found in the study population. According to WHO/UNICEF/IGN criteria, the population-based median UIC during pregnancy should be 150-249 μg/l. In conclusion, genetic variations may contribute to adverse pregnancy outcomes. In clinical practice, thyroid testing and the management of hypothyroidism during pregnancy is unsatisfactory, regarding the whole chain from development of local guidelines to their implementation and to targeted thyroid testing. Moreover, our results indicate insufficient iodine status in the pregnant population of Sweden.

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No. of bitstreams: 1 arcarofilho_ca_dr_arafcf_int.pdf: 7378512 bytes, checksum: cb295c066a7be41be3508e2daf64d24f (MD5) Previous issue date: 2018-04-27 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Considerando os avanços no conhecimento acerca dos mecanismos que controlam o metabolismo de proteínas na musculatura esquelética que permitiram a busca por novas opções para o tratamento das atrofias musculares, o presente estudo teve como objetivo a compreensão do potencial antiproteolítico de inibidores de fosfodiesterase, PDE (pentoxifilina, inibidor não-seletivo de PDE; rolipram, inibidor seletivo de PDE4) em músculos esqueléticos de ratos submetidos à atrofia muscular devido à insuficiência insulínica (diabetes mellitus experimental), com ênfase na elucidação da participação de componentes da sinalização do AMP cíclico (AMPc) nesta resposta. Ratos normais e diabéticos (60 mg/kg de estreptozotocina, administração intravenosa) foram tratados com salina (NS e DS) ou com 2 mg/kg de rolipram (NROL e DROL), ou com 25 mg/kg de pentoxifilina (NPTX e DPTX) durante 3 dias, por via intraperitoneal. Após três dias de tratamento, músculos soleus e extensor digitorum longus (EDL) foram removidos, pesados, congelados e processados para diversas análises: (i) conteúdo de AMPc (ensaio imunoenzimático); (ii) atividades das proteases proteassoma, calpaínas e caspase-3 (uso de substratos específicos fluorigênicos); (iii) níveis proteicos e/ou níveis de fosforilação de componentes das vias proteolíticas, efetores intracelulares sinalizatórios e fatores de transcrição (Western blotting); (iv) determinação dos níveis séricos de insulina e citocinas pró-inflamatórias. Foram realizados experimentos ex-vivo, para verificar a ação direta dos fármacos no controle da proteólise muscular e ativação de efetores intracelulares, via incubações dos músculos na presença de rolipram ou de agonistas de EPAC (Exchange protein directly activated by cAMP) e de PKA (proteína quinase dependente de AMPc), proteínas efetoras ativadas pelo AMPc. Também foram realizados experimentos no Laboratório do Prof. Dr. Marco Sandri, no Venetian Institute of Molecular Medicine, Padova, Itália, para a avaliação do papel de PDE4D no controle do processo autofágico-lisossomal em músculos esqueléticos de camundongos jejuados. Os tratamentos de animais diabéticos com rolipram (DROL) ou com pentoxifilina (DPTX) promoveram uma redução nas atividades do proteassoma e calpaínas em soleus e EDL, bem como nos níveis de componentes-chave do sistema proteolítico ubiquitina-proteassoma (MuRF-1, atrogin-1, conjugados poliubiquitinados), e aumento nos níveis de calpastatina (inibidor das calpaínas). Interesante ressaltar que o grupo DROL apresentou redução na atividade e níveis proteicos de caspase-3, em ambos os músculos, enquanto que o grupo DPTX apenas em músculos EDL. Contribuindo com a redução observada na atividade de caspase-3, houve uma redução nos níveis de Bax (proteína pró-apoptótica) e aumento nos níveis de Bcl-2 (proteína anti-apoptótica) em ambos os músculos de animais DROL. Animais diabéticos tratados com salina (DS) apresentaram aumento nas atividades das três proteases, bem como nos níveis de componentes participantes destes processos proteolíticos. Animais normais e diabéticos tratados com salina (NS e DS) apresentaram níveis de AMPc basais e semelhantes entre si, tanto em soleus quanto em EDL, enquanto que os tratamentos de ratos normais e diabéticos com pentoxifilina (NPTX e DPTX) ou rolipram (NROL e DROL) promoveram aumentos de AMPc, em ambos os músculos. Um dos mecanismos que podem estar envolvidos na inibição da proteólise muscular após aumentos nas concentrações de AMPc envolve a proteína EPAC, responsável por integrar a sinalização do AMPc e a sinalização insulínica via ativação da quinase AKT. Animais diabéticos tratados com pentoxifilina ou com rolipram apresentaram aumento nos níveis proteicos de EPAC 1 e na fosforilação de AKT, quando comparados ao grupo DS. Observamos também um aumento na fosforilação inibitória de fatores de transcrição FoxO 1 e 3a em ambos os músculos de animais DROL. Podemos sugerir que parte das ações de rolipram que culminaram em ativação de AKT e inibição de FoxO na musculatura esquelética possam estar associadas aos aumentos observados nos níveis circulantes de insulina em animais DROL. Investigamos, apenas nos animais tratados com rolipram, a possibilidade de participação da proteína PKA no controle da proteólise muscular. Em animais DROL houve ativação da PKA, verificada tanto pelo aumento na fosforilação de substratos de PKA, bem como do fator de transcrição CREB, em soleus e EDL. Vale destacar que animais DS apresentaram níveis reduzidos de p-CREB e de substratos fosforilados por PKA em soleus e EDL. Animais diabéticos tratados com os inibidores de PDE apresentaram uma diminuição de citocinas pró-inflamatórias séricas (TNF-, PTX e ROL; IL-1, ROL) e aumento nos níveis de insulina sérica (ROL) em relação aos animais DS. Nos estudos ex vivo, as incubações de músculos soleus e EDL com rolipram levaram a uma redução da proteólise total, bem como aumento na fosforilação de substratos de PKA e de AKT. Músculos soleus e EDL incubados com agonistas de EPAC apresentaram aumento na fosforilação de AKT, enquanto que a incubação com agonista de PKA promoveu aumento na fosforilação dos substratos de PKA (em ambos os músculos) e aumento na fosforilação de AKT (apenas em EDL), quando comparados aos músculos incubados na ausência do fármaco. Nos estudos para compreensão do papel de PDE4D no controle do processo autofágico-lisossomal, observou-se que o silenciamento gênico da PDE4D em músculos tibialis anterior promoveu uma preservação da massa muscular e da área da fibra em animais jejuados, quando comparados ao músculo controle. Músculos flexor digitorium brevis, silenciados para PDE4D, apresentaram diminuição na expressão de proteínas-chave do processo autofágico-lisossomal, tais como LC3 e p62. Estes resultados evidenciam os mecanismos que podem estar envolvidos na ação direta de inibidores de PDE no controle do metabolismo proteico muscular esquelético, via ativação de duas vias dependentes de AMPc: (i) a via PKA/CREB, que pode participar do controle da transcrição de Bcl-2 e calpastatina, bem como na inativação direta de caspases, inibindo assim os processos proteolíticos dependentes de caspase-3 e calpaínas, (ii) a via EPAC/AKT, via fosforilação e inibição de FoxO 1 e 3A, regulando a expressão dos atrogenes (MuRF-1 e atrogin-1) e promovendo uma diminuição na atividade do sistema ubiquitina-proteassoma. Além disso, o tratamento com inibidores de PDE diminuem o processo inflamatório e aumentam os níveis circulantes de insulina, ações que podem contribuir para os efeitos antiproteolíticos. Evidências iniciais também sugerem que PDE4D participa no controle do sistema autofágico-lisossomal na musculatura esquelética. Todos estes resultados indicam que PDE participam no controle de processos proteolíticos, portanto inibidores de PDE emergem como uma opção interessante na ativação da sinalização do AMPc na musculatura esquelética, com vistas à utilização futura no tratamento de quadros de perda de massa muscular durante situações de atrofia. / Considering the advances in the knowledge of the mechanisms controlling the protein metabolism in skeletal muscles that allowed the discover of new options for the treatment of muscle atrophies, the present study aimed to understand the antiproteolytic potential of phosphodiesterase (PDE) inhibitors (pentoxifylline, a non-selective PDE inhibitor; rolipram, a selective PDE 4 inhibitor), in skeletal muscles of rats submitted to muscle atrophy due to insulin insufficiency (experimental diabetes mellitus), with emphasis on the elucidation of the participation of cyclic AMP (cAMP) signaling components. Normal and diabetic rats (60 mg/kg streptozotocin, intravenous administration) were treated intraperitoneally with saline (NS and DS) or with 2 mg/kg rolipram (NROL and DROL) or with 25 mg/kg pentoxifylline (NPTX and DPTX) for 3 days. After three days of treatments, soleus and extensor digitorum longus (EDL) muscles were removed, weighed, frozen and processed for several analyzes: (i) cAMP content; (ii) activities of proteasome, calpain and caspase-3 (use of specific fluorigenic substrates); (iii) protein levels and/or phosphorylation levels of components of proteolytic pathways, intracellular signaling effectors and transcription factors (Western blotting); (iv) determination of serum insulin and proinflammatory cytokines levels. Ex vivo experiments were performed to verify the direct action of the drugs in the control of muscle proteolysis and activation of intracellular effectors, via muscle incubations in the presence of rolipram or agonists of EPAC (Exchange protein directly activated by cAMP) and PKA (cAMP-dependent protein kinase), intracellular effectors activated by cAMP. Experiments were also carried out in the Laboratory of Prof. Dr. Marco Sandri at the Venetian Institute of Molecular Medicine, Padova, Italy, for the evaluation of the role of PDE4D in controlling the autophagic-lysosomal process in skeletal muscles of starved mice. Treatments of diabetic animals with rolipram (DROL) or pentoxifylline (DPTX) promoted a reduction in the activities of proteasome and calpain in soleus and EDL, as well as reduced the levels of key components of the ubiquitin-proteasome system (MuRF-1, atrogin-1, polyubiquitinated conjugates), and increased the levels of calpastatin (calpain inhibitor). Interestingly, DROL rats showed a reduction in the activity and in the protein levels of caspase-3 in both muscles, whereas DPTX rat had reductions only in EDL muscles. Contributing to the reduction in caspase-3 activity, it was observed a reduction in the content of Bax (pro-apoptotic protein) and an increase of Bcl-2 (anti-apoptotic protein) in both muscles of DROL rats. Diabetic animals treated with saline (DS) showed an increase in the activities of the three proteases, as well as increases in the levels of components belonging to these proteolytic processes. Normal and diabetic animals treated with saline (NS and DS) had basal and similar levels of cAMP in both soleus and EDL, whereas the treatments of normal and diabetic rats with pentoxifylline (NPTX and DPTX) or with rolipram (NROL and DROL) promoted increases in cAMP in both muscles. One of the mechanisms that may be involved in the muscle proteolysis inhibition after increases in cAMP involves the EPAC protein, responsible for integrating the cAMP and the insulin signaling pathways via AKT activation. Diabetic animals treated with pentoxifylline or with rolipram showed an increase in the protein levels of EPAC 1 and in the phosphorylation of AKT, when compared with the DS group. We also observed an increase in the phosphorylation (inhibitory) of FoxO 1 and 3a in both muscles of DROL rats. It can be suggested that part of the rolipram actions causing AKT activation and FoxO inhibition in skeletal muscles may be associated with the increases in the circulating levels of insulin observed in DROL animals. It was investigated, only in animals treated with rolipram, the possible involvement of PKA in the control of muscle proteolysis. DROL rats had activation of PKA, verified both by the increase in the phosphorylation of PKA substrates, as well as in the phophorylation of the transcription factor CREB, in soleus and EDL. DS rats had decreased levels of p-CREB and of the PKA substrates, in soleus and EDL. Diabetic animals treated with PDE inhibitors showed a decrease in serum proinflammatory cytokines (TNF-, PTX and ROL; IL-1, ROL) when compared with DS. In ex vivo studies, incubations of soleus and EDL with rolipram caused a reduction of the total proteolysis as well as an increase in the phosphorylation of PKA substrates and and of AKT. Soleus and EDL muscles incubated with EPAC agonist showed increased in the AKT phosphorylation, whereas incubation with PKA agonist promoted an increase in the phosphorylation of PKA substrates (in both muscles) and and increase in the AKT phosphorylation (EDL), when compared with muscles incubated in the absence of the drugs. In studies to understand the role of PDE4D in the control of the autophagic-lysosomal process, it was observed that the PDE4D gene silencing in anterior tibialis muscles caused a preservation of the muscle mass and fiber area in fasted animals when compared with control muscle. Flexor digitorium brevis muscles, silenced for PDE4D, showed a decreased expression of key proteins of the autophagic-lysosomal process, such as LC3 and p62. These results suggested the mechanisms that may be involved in the direct action of PDE inhibitors in the control of skeletal muscle protein metabolism, through activation of two cAMP-dependent pathways: (i) the PKA/CREB pathway, which may participate in transcriptional control of Bcl-2 and calpastatin, as well as causing direct inactivation of caspases, thus inhibiting the proteolytic processes dependent on caspase-3 and calpains, (ii) the EPAC/AKT pathway, via phosphorylation and inhibition of FoxO 1 and 3a factors, regulating the expression of atrogenes (MuRF-1 and atrogin-1) and promoting a decrease in activity of ubiquitin-proteasome system. Treatments with PDE inhibitors also decreased the inflammatory process and increased the circulating linsulin levels, which may be contributing to the antiproteolytic responses. Initial evidence also suggests that PDE4D participates in the control of the autophagy-lysosomal system in skeletal muscles. All these results indicate that PDE participate in the control of proteolytic processes, therefore PDE inhibitors emerge as an interesting option to activate the cAMP signaling in the skeletal muscles, which may be used in the future in treatments muscle mass loss during atrophy situations. / FAPESP: Processo 2013/18861-2 / FAPESP: Processo 2014/12202-0 / FAPESP: Processo 2017/02348-5