Background - The role of protein phosphatases (PPs) during ischemic preconditioning in the rabbit heart was examined. Methods and Results - Fostriecin, a potent inhibitor of PP2A, was administered to isolated rabbit hearts starting either 15 minutes before or 10 minutes after the onset of a 30-minute period of regional ischemia and continuing until the onset of reperfusion. After 2 hours of reperfusion, infarct size was measured with triphenyltetrazolium chloride. In a second study with isolated rabbit cardiomyocytes, the effect of fostriecin pretreatment was assessed by measuring changes in cell osmotic fragility during simulated ischemia. PP1 and PP2A activities of isolated control and ischemically preconditioned cells were also measured. In a third series of experiments, left ventricular biopsies of isolated rabbit hearts were obtained before and at selected times during 60 minutes of global ischemia, and the tissue was assayed for PP1 and PP2A activities. In isolated hearts pretreated with fostriecin, only 8% of the ischemic zone infarcted, significantly less than that in untreated control hearts (33%; P<0.001) but comparable to that in ischemically preconditioned hearts (9%; P<0.001 versus control). Significant protection was also observed in the hearts treated only after the onset of ischemia (18% infarction; P<0.05 versus control). In isolated myocytes, fostriecin also provided protection comparable to that produced by metabolic preconditioning. Preconditioning had no apparent effect on the activity of either PP1 or PP2A in isolated ventricular myocytes or ventricular tissue obtained from heart biopsies. Conclusions - Fostriecin, a potent inhibitor of PP2A, can protect the rabbit heart from infarction even when administered after the onset of ischemia. But inhibition of either PP1 or PP2A does not appear to be the mechanism of protection from ischemic preconditioning.

Orientador: Ronaldo Aloise Pilli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T20:06:35Z (GMT). No. of bitstreams: 1 Sant'Ana_CarolinaMartinsAvilade_D.pdf: 25003165 bytes, checksum: deb5835cc499985cdc35e9f74c89025a (MD5) Previous issue date: 2014 / Resumo: No capítulo 1 estudou-se uma rota sintética inédita para a obtenção da fostriecina e seus análogos, a qual possui como etapa chave a adição aldólica 1,4-anti, mediada por enolato de boro, para instalar o centro estereogênico em C11. Neste contexto, foi avaliada a influência do substituinte em ? nas reações aldólicas envolvendo enolatos de boro de metilcetonas quirais, que contêm em sua estrutura um centro estereogênico quaternário em C-8. Explorou-se seu escopo reacional, utilizando aldeídos que diferem entre si no que se refere aos aspectos estéreo e eletrônico. Obteve-se adutos com excelente diastereoseletividade (rd 1,4-anti versus 1,4-syn > 90:10). Esta metodologia foi aplicada na síntese do fragmento C5-C13 da fostriecina sendo obtido em 10 etapas e 8,4% de rendimento global a partir do (2E)-2-metil-2-buten-1-ol, disponível comercialmente. Este fragmento foi utilizado por G. A. O¿Doherty e colaboradores na síntese total mais recente da fostriecina, sendo preparado em 15 etapas a partir do (2E)-pent-2-en-4-in-1-ol (Org. Lett., 12, 3752, 2010). No capítulo 2, foi desenvolvida uma rota sintética flexível utilizando-se uma abordagem modular que permitiu a preparação de quatro estereoisômeros do produto natural coibacina A a fim de determinar a configuração absoluta do produto natural. Esta rota sintética possui como etapas chave olefinações de Wittig e Julia e ciclopropanação assimétrica de Charette. Após comparação por HPLC, utilizando coluna quiral, dos tempos de retenção destes quatro estereoisômeros com uma amostra da coibacina A de origem natural, foi possível estabelecer inequivocamente a configuração absoluta deste policetídeo como sendo 5R,16S,18S. A estereoquímica do centro estereogênico em C-5 da lactona ?-? insaturada, previamente assinalada como S, foi corrigida para R e a estereoquímica dos centros em C-16 e C-18 foi estabelecida como sendo S e S, respectivamente. Dessa forma concluímos a primeira síntese total do policetídeo insaturado coibacina A em 13 etapas (etapa linear mais longa) e 3,4% de rendimento global a partir do (S)-glicidol, obtido comercialmente. Adicionalmente, sintetizamos o isômero 5R,14S,16S da coibacina B em 13 etapas (etapa linear mais longa) e 5,7% de rendimento global / Abstract: In chapter 1, a new synthetic route for fostriecin and analogues was studied with the 1,4-anti boron-mediated aldol reaction as the key step in the formation of the stereogenic center at C11. In this context, the influence of the ?-alkoxy substituent in the aldol reaction involving the boron enolate of the chiral methyl ketones with a quaternary stereogenic center at C-8 was evaluated. The reaction scope was explored by using aldehydes with different steric and electronic patterns. Products were obtained in excellent diastereoselectivity (rd 1,4-anti versus 1,4-syn > 90:10). This methodology was applied to the synthesis of C5-C13 fragment of fostriecin in 10 steps and 8.4% overall yield from commercially available (2E)-2-methyl-2-buten-1-ol. This intermediate was used by G. A. O¿Doherty and co-workers in the most recent total synthesis of fostriecin and it was prepared in 15 steps from (2E)-pent-2-en-4-yn-1-ol (Org. Lett., 12, 3752, 2010). In chapter 2, a flexible synthetic route for four stereoisomers of the natural product coibacin A was developed based on a modular approach in order to establish the absolute configuration of this natural product. This synthetic strategy possesses as key steps the Wittig and Julia olefination reactions and Charette asymmetric cyclopropanation reaction. After comparison by chiral HPLC chromatography of the retention time of the four synthetic stereoisomers with a sample of coibacin A obtained from natural sources, the absolute configuration of the natural product was unequivocally established as 5R,16S,18S. The stereochemistry of the stereogenic center at C-5 of the ?,?-unsaturated ?-lactone, that had been previously assigned as S, was corrected to R and the absolute configurations at C-16 and C-18 were assigned as S and S, respectively. Therefore, we accomplished the first total synthesis of the unsaturated polyketide coibacin A in 13 steps (longest linear sequence) and 3.4% overall yield from commercially available (S)-glycidol. Additionally, we synthesized the 5R,14S,16S isomer of coibacin B in 13 steps (longest linear sequence) and 5.7% overall yield / Doutorado / Quimica Organica / Doutor em Ciências

Calcium-tolerant rabbit cardiomyocytes were isolated using retrograde aortic perfusion with a nominally calcium-free, collagenase buffer. In vitro ischemic preconditioning was induced by a 10-min episode of ischemic pelleting, followed by a 15-min post-incubation and a prolonged period of ischemic pelleting. Injury was assessed by determination of cell contracture and trypan blue permeability following hypotonic swelling and correlated with metabolic assays of lactate and adenine nucleotides. The protein phosphatase PP1/2A inhibitor calyculin A and PP2A-selective fostriecin protected isolated rabbit cardiomyocytes from lethal injury after a 10-min pre-incubation and when added late into ischemic pellets after a delay of 75 min. At the time of late drug addition, cells were severely ATP-depleted and in rigor contracture. Protection with Calyculin A from 1 nM to 1 μM was dose-related. Cells pre-incubated with 10 nM to 10 μM fostriecin 10 min prior to ischemic pelleting were protected with an EC50 approximating 71 nM, implying protection at a PP2A-selective dose. The selective protein kinase C inhibitor, calphostin C, blocked ischemic preconditioning protection but not protection from 1 μM calyculin A. Protection of severely ischemic cardiomyocytes following protein phosphatase inhibition appears not to require PKC activity or ATP conservation. Pre-incubation of cells with calyculin A induced high levels of phosphorylation in p38 mitogen activated protein kinase (MAPK), as compared to the ischemia-induced phosphorylation observed in the untreated group only at 30 min of ischemia, providing evidence of protein phosphatase activity in cardiomyocytes. Pharmacological protection in late ischemia has been demonstrated, but the mechanism of protection is undetermined.