Background: Ethanol is a cardiotoxic substance that damages the heart by increasing apoptosis, free radical formation and calcium overloading. Consequently, there is an increase in cell death leaving fewer functioning myocytes leading to heart failure. Sildenafil is a phosphodiesterase type-5 (PDE-5) inhibitor approved for treatment of erectile dysfunction. Studies from our lab have demonstrated that PDE-5 inhibition reduces myocardial infarct size and attenuates post-ischemic cardiac dysfunction in both ischemia-reperfusion and permanent coronary artery ligation models. Therefore, in the present study, we hypothesized that treatment with sildenafil will prevent cardiotoxicity associated with acute alcohol exposure by reducing myocyte apoptosis and preserving cardiac function through PKG signaling. Methods and Results: Adult cardiomyocytes were isolated and treated with 100 mM of 100% ethanol ± 10 µM sildenafil. At 24 hours necrosis was assessed via trypan blue exclusion assay, JC-1 staining assessed mitochondrial membrane potential and ROS production was measured by DCF fluorescence. At 48 hours apoptosis was assessed by TUNEL assay. Ethanol increased the rate of necrotic and apoptotic cell death. This was attenuated by co-treatment with sildenafil. Ethanol disrupted the mitochondrial membrane potential and increased ROS production. Sildenafil preserved mitochondrial membrane potential and attenuated ROS production. Treatment of myocytes with 5-HD, a mitochondrial K+atp channel antagonist, blocked the protective effect of sildenafil. Knockdown of PKG using adenoviral siRNA blocked the protective effect of sildenafil, while overexpression of PKG1α conferred protection against ethanol cytotoxicity. To further demonstrate the effect of sildenafil ethanol-cardiotoxicity in vivo, mice were treated with ethanol (3 g/kg/day) with or without sildenafil (0.7 mg/kg) by i.p. injection for three consecutive days. After treatment, the animals were sacrificed and the hearts removed and perfused on a Langendorff system to measure function. After functional analysis, apoptosis and PKG activity was measured in the heart samples. Ethanol decreased the rate-force product and increased myocardial apoptosis. Sildenafil preserved cardiac function and significantly reduced apoptosis. Sildenafil treated myocardium also showed an increase in PKG activity. Conclusion: Sildenafil attenuates the toxic effect of ethanol by reducing apoptosis and maintaining the mitochondrial integrity in cardiomyocytes. Sildenafil also preserved cardiac function in ethanol-treated mice. Protein kinase G-dependent signaling plays a critical role in attenuating cardiotoxic effect of ethanol.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Spermidine (SPD) is an endogenous polyamine with polycationic structure present in the central nervous system of mammals. SPD regulates biological processes, such as Ca2+ influx by glutamatergic N-methyl-D-aspartate receptor (NMDA receptor), which has been associated with nitric oxide synthase (NOS) and cGMP/PKG pathway activation and a decrease of Na+,K+-ATPase activity in rats cerebral córtex synaptossomes. Decreased Na+,K+-ATPase activity, as well as decreased enzyme expression, directly impairs neurotransmitter signaling with deleterious consequences on learning and memory. The enzyme Na+,K+-ATPase establishes Na+ and K+ gradients across membranes of excitable cells and by this means maintains membrane potential and controls intracellular pH and volume. However, it has not been defined whether SPD modulates Na+,K+-ATPase activity in the hippocampus. In this study we investigated whether SPD alters Na+,K+-ATPase activity in slices of hippocampus from rats, and possible underlying mechanisms. Hippocampal slices and homogenates were incubated with SPD (0.05-10 μM) for 30 minutes. SPD (0.5 and 1 μM) decreased Na+,K+-ATPase activity in slices, but not in homogenates. MK-801 (100 μM), a non-competitive antagonist of NMDA receptor, arcaine (0.5 μM), an antagonist of the polyamine binding site at the NMDA receptor, and L-NAME (100 μM), a nitric oxide synthase inhibitor, prevented the inhibitory effect of SPD (0.5 μM). ODQ (10 μM), a guanylate cyclase inhibitor, and KT5823 (2 μM), a protein kinase G inhibitor, also prevented the inhibitory effect of SPD on Na+,K+-ATPase activity. SPD (0.5 and 1.0 μM) increased NO2 plus NO3 (NOx) levels in slices, MK-801 (100 μM) and arcaine (0.5 μM) prevented the effect of SPD (0.5 μM) on the NOx content. These results suggest that SPD-induced decrease of Na+,K+-ATPase activity involves NMDA/NOS/cGMP/PKG pathway. / A espermidina (SPD) é uma poliamina endógena com estrutura policatiônica presente no sistema nervoso central (SNC) de mamíferos. A SPD regula muitos processos biológicos, como o influxo de cálcio através dos receptores glutamatérgicos N-metil-D-aspartato (NMDA), o qual tem sido associado com a ativação da enzima óxido nítrico sintase (NOS) e da via de sinalização da guanosina mono fosfato cíclica/proteína quinase dependente de GMPc (GMPc/PKG). Sabe-se que uma diminuição da atividade da Na+,K+-ATPase, bem como a sua expressão, prejudica diretamente a sinalização de neurotransmissores, comprometendo tanto aprendizado e a memória. A enzima Na+,K+-ATPase estabelece os gradientes de Na+ e K+ através da membrana de células excitáveis e desta forma mantém o potencial de membrana e contribui para o controle do volume e do pH celular. No entanto, não está bem definido se a SPD modula a atividade da Na+,K+-ATPase no hipocampo de ratos. Neste estudo foi investigado se SPD altera a atividade da Na+,K+-ATPase em fatias de hipocampo de ratos, e o possível mecanismo envolvido neste processo. As fatias e o homogeneizado de hipocampo foram incubados com SPD (0,05-10 M) por 30 minutos. SPD (0,5 e 1 M) diminuíram a atividade da Na+,K+-ATPase em fatias, mas não no homogeneizado de hipocampo. MK-801 (100 μM), um antagonista não competitivo do receptor NMDA, arcaína (0,5 M), um antagonista do sítio de ligação das poliaminas no receptor NMDA, e L-NAME (100 μM), um inibidor da NOS, preveniram o efeito inibitório da SPD (0,5 M). ODQ (10 μM), um inibidor da enzima guanilato ciclase, e KT5823 (2 μM), um inibidor da proteína quinase dependente de GMPc, também preveniram o efeito inibitório da SPD sobre a atividade da Na+,K+-ATPase. SPD (0,5 e 1,0 μM) aumentaram os níveis de NO2 plus NO3 (NOx) nas fatias de hipocampo. MK-801 (100 μM) e arcaína (0,5 μM) preveniram o efeito da SPD (0,5 μM) sobre conteúdo de NOx. Estes resultados sugerem que a diminuição da atividade da Na+,K+-ATPase induzida pela SPD envolve a via de sinalização NMDA/NOS/GMPc/PKG.

Bradykinin (BK) acts through eNOS to reduce MAPK-mediated feedback inhibition of insulin signalling. Preliminary data suggest that the sGC-cGMP-PKG pathway, a prominent NO target, is involved. Our present study aimed to support the role of this pathway with atrial natriuretic peptide (ANP), which uses a receptor associated GC (NPR-A) to generate cGMP. We found that treating adipocytes with ANP mimicked BK effects on insulin-stimulated glucose uptake, Tyr-IRS-1 and Akt/PKB phosphorylation, as well as JNK and ERK1/2 inhibition. These outcomes depended on GC-cGMP-PKG signalling since A71915 (NPR-A antagonist), and KT-5823 (PKG inhibitor), completely abrogated them, while zaprinast (phosphodiesterase inhibitor), prolonged ANP actions. Furthermore, decreased MAPK phosphorylation was independent of upstream kinase activity, suggesting that MAPK phosphatases may be involved. These data indicate that BK and ANP act through the GC-cGMP-PKG pathway to potentiate insulin signalling via attenuated feedback inhibition. Stimulating the GC-cGMP-PKG pathway may, therefore, be a promising therapy for T2DM.

The role of cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG) in necrotic and apoptotic pathways of many cell types is well established; however its role in the ischemic preconditioning (IPC) of cardiomyocytes is not clearly defined. In the current study, we assessed the hypothesis that PKG protects against cell death following ischemidreperfusion injury in myocytes subjected to IPC. Freshly isolated adult rat ventricular myocytes were subjected to IPC by incubating in ischemic buffer for 30 minutes (min) followed by incubation in normal medium for 30 min. Prolonged simulated ischemia (SI) was created by incubating myocytes in the ischemic buffer for 90 min and reoxygenation (RO) for 120 min in the normal medium. Necrosis was determined by trypan blue exclusion and apoptosis was assessed by TUNEL assay. IPC reduced necrosis as shown by significant decrease in trypan blue positive cells as compared to virgin non-preconditioned myocytes subjected to SI and RO alone (p<.01). Similarly, the number of TUNEL positive myocytes following SI and 18 hrs of RO were significantly reduced in the IPC group. Treatment with PKG inhibitor, KT5832 (2pM) completely abolished the protection against necrosis by IPC. However, KT5832 failed to abolish the protective affect of IPC against apoptosis. Furthermore, myocytes infected with an adenoviral construct of PKG-la (1 x 1 o4 particles/cell) significantly reduced the number of trypan blue and TUNEL positive cells. These results suggest that the PKG signaling pathway plays an essential role in the preconditioning of myocytes against necrosis following SI / RO injury. Furthermore, while the overexpression of PKG protects myocytes against necrosis, as well as apoptosis, IPC may not induce a sufficient level of PKG during 18 hours of RO to induce protection against apoptosis.

cGMP-dependent protein kinases (PKG) are key players in intracellular second messenger signaling within many cellular systems throughout the body. Most notably PKG is known for its role in smooth muscle relaxation (Pfeiffer et.al, 1999). The Iα PKG isozyme has been identified as the primary effector of the nitric oxide pathway (and serves to be a novel drug target). To date the overall knowledge of structure and function of PKG is lacking in terms of the mechanisms of activation and the structural orientations that coordinate them. Recently, our laboratory has solved the crystal structure of the regulatory domain of PKG Iα, which revealed a previously unknown α-helical domain dubbed the Switch Helix (SW) (Osborne et.al, 2011). The SW domain was found to be a site of interprotomer communication via hydrophobic interactions between its C-terminus and hydrophobic residues, named the nest located on the opposing protomer. Synthetic peptides derived from the SW domain, named S-tides, dosedependently activate PKG Iα (Moon et.al, 2015). In addition, the amino acid residues of the nest are in proximity to the cGMP binding site B. It was hypothesized that the binding site for S-tides (nest) and the cGMP binding site B interact and are co-dependent of one another. The hypothesis of this thesis is the binding site for the S-tides (nest) and the cGMP binding site B interact and are co-dependent of one another. To test this hypothesis two aims were constructed: Aim 1: To develop an S1.5 analog that utilizes both the nest and the B-site to increase S-tide activity, Aim 2: To explore the intricacies of these modes of activation and how they interact with each other to obtain a better understanding of the interplay between these two sites. First, based on the most potent S-tide S1.5 (YEDAEAKAKYEAEAAFFANLKLSD, Ka=6 μM), two analogs were synthesized. The peptide S2.5 which lacked the amino acids LSD at the C-terminus showed a three-fold lower activation constant (Ka= 15 μM), although the molecule retained its helicity as demonstrated by circular dichroism. The second analog, S3.5 contained unnatural amino acid components from a molecular modeling approach in an effort to further increase the affinity by interacting with the adjacent cGMP binding site B. However, S3.5 showed further reduction in activity with an activation constant of 70 μM. These findings led us to conclude that the failure of the SAR approach indicates a different mode of S-tide activation as had been previously thought. Next, we investigated the role of the cGMP binding site B in the mechanism of S-tide mediated PKG Iα activation. Co-activation assays with cGMP and S1.5 demonstrated that cGMP activation is not altered in the presence of S1.5. Furthermore, S1.5 mediated activation is negatively affected in the presence of cGMP. These results suggest that the B-site of cGMP does not positively enforce the S1.5 activation kinetics. Next, we employed the PKG Iα mutant E292A, which cannot bind cGMP to the B-site (Moon et.al., 2018). Interestingly, this mutant retains the activation kinetics of PKG Iα WT when activated via S1.5 and cGMP. Thus, the cGMP binding site B is not crucial in the activation mechanisms of activating PKG Iα with cGMP. Likewise, the cGMP binding site B is not crucial in the activation mechanisms of activating PKG Iα with S1.5. To further support these findings, the PKG Iα mutant C42A, which showed crippled cGMP activation kinetics could be activated with S1.5 with a potency similar to wild type. Taken together, the results in this thesis demonstrate that in contrast to the initial hypothesis the binding sites for S-tides and cGMP, although in proximity, show no experimental support of a positive interaction. These findings are significant as they reveal that S1.5 mediated activation of PKG is truly independent of cGMP, thereby providing a molecular platform for the therapeutic development of these unique peptides.

Foraging behaviour in Drosophila melanogaster larvae is influenced by natural allelic variation in the foraging (for) gene that encodes a cyclic GMP – dependent protein Kinase (PKG), such that rovers (forR) traverse greater distances while foraging than sitters (fors). Foraging behaviour is also influenced by natural allelic variation in the wings up A (wupA) gene that encodes the Troponin-I protein (TnI). Specifically, wupAlow allele suppresses the dominance of the forR allele, turning rovers into sitters. The dominance of the natural wupA alleles and their interactions with allelic combinations in for has not been characterized. I conducted various crosses and found that wupA alleles exhibit incomplete dominance. More importantly, I found that allelic combinations of wupA and for gave rise to a range in larval foraging behaviour. In this study, I propose that this gradient effect in foraging behaviour is due to variation in levels of PKG activity and TnI phosphorylation potential.

Foraging behaviour in Drosophila melanogaster larvae is influenced by natural allelic variation in the foraging (for) gene that encodes a cyclic GMP – dependent protein Kinase (PKG), such that rovers (forR) traverse greater distances while foraging than sitters (fors). Foraging behaviour is also influenced by natural allelic variation in the wings up A (wupA) gene that encodes the Troponin-I protein (TnI). Specifically, wupAlow allele suppresses the dominance of the forR allele, turning rovers into sitters. The dominance of the natural wupA alleles and their interactions with allelic combinations in for has not been characterized. I conducted various crosses and found that wupA alleles exhibit incomplete dominance. More importantly, I found that allelic combinations of wupA and for gave rise to a range in larval foraging behaviour. In this study, I propose that this gradient effect in foraging behaviour is due to variation in levels of PKG activity and TnI phosphorylation potential.

It is now well accepted, through the use of mutational studies, that genes influence behavioural variation. However, we have little knowledge of the cellular and neuronal mechanisms underlying the effects of specific genes. This thesis broadens our understanding of the neurogenetic underpinnings of naturally occurring differences in behaviour using the genetically tractable model organism Drosophila melanogaster. The thesis focuses on allelic variation at the foraging (for) gene which influences both larval and adult behaviour. In particular, for’s cellular/neural contributions to food-related behaviours and learning and memory is investigated. In the first study, we map FOR protein distribution patterns in the adult brain and use this knowledge to demonstrate a neural-specific function for the for gene in adult food-related behaviour. In the second study we demonstrate a novel role for for in the regulation of naturally existing differences in fly learning and memory in the mushroom bodies of the fly brain. In the third study, I explore FOR distribution patterns in larval tissues. I show that FOR is expressed both in neural and non-neural tissues suggesting a distributed function for FOR in food-related behaviours in the larva. In the last study, I describe naturally existing differences in fat metabolism in the Drosophila larva fat storage tissue. FOR is expressed in the fat storage tissue and may regulate lipid packaging, a trait linked to foraging. In general, my thesis is a cellular and neurogenetic analysis of natural variation in behavioural and physiological traits of D. melanogaster. The functions of FOR in food-related behaviours, nutrient physiology and cognition are conserved across taxa. The findings of this thesis should provide a framework to understand these phenomena in a wide range of organisms.

It is now well accepted, through the use of mutational studies, that genes influence behavioural variation. However, we have little knowledge of the cellular and neuronal mechanisms underlying the effects of specific genes. This thesis broadens our understanding of the neurogenetic underpinnings of naturally occurring differences in behaviour using the genetically tractable model organism Drosophila melanogaster. The thesis focuses on allelic variation at the foraging (for) gene which influences both larval and adult behaviour. In particular, for’s cellular/neural contributions to food-related behaviours and learning and memory is investigated. In the first study, we map FOR protein distribution patterns in the adult brain and use this knowledge to demonstrate a neural-specific function for the for gene in adult food-related behaviour. In the second study we demonstrate a novel role for for in the regulation of naturally existing differences in fly learning and memory in the mushroom bodies of the fly brain. In the third study, I explore FOR distribution patterns in larval tissues. I show that FOR is expressed both in neural and non-neural tissues suggesting a distributed function for FOR in food-related behaviours in the larva. In the last study, I describe naturally existing differences in fat metabolism in the Drosophila larva fat storage tissue. FOR is expressed in the fat storage tissue and may regulate lipid packaging, a trait linked to foraging. In general, my thesis is a cellular and neurogenetic analysis of natural variation in behavioural and physiological traits of D. melanogaster. The functions of FOR in food-related behaviours, nutrient physiology and cognition are conserved across taxa. The findings of this thesis should provide a framework to understand these phenomena in a wide range of organisms.

Bradykinin (BK) acts through eNOS to reduce MAPK-mediated feedback inhibition of insulin signalling. Preliminary data suggest that the sGC-cGMP-PKG pathway, a prominent NO target, is involved. Our present study aimed to support the role of this pathway with atrial natriuretic peptide (ANP), which uses a receptor associated GC (NPR-A) to generate cGMP. We found that treating adipocytes with ANP mimicked BK effects on insulin-stimulated glucose uptake, Tyr-IRS-1 and Akt/PKB phosphorylation, as well as JNK and ERK1/2 inhibition. These outcomes depended on GC-cGMP-PKG signalling since A71915 (NPR-A antagonist), and KT-5823 (PKG inhibitor), completely abrogated them, while zaprinast (phosphodiesterase inhibitor), prolonged ANP actions. Furthermore, decreased MAPK phosphorylation was independent of upstream kinase activity, suggesting that MAPK phosphatases may be involved. These data indicate that BK and ANP act through the GC-cGMP-PKG pathway to potentiate insulin signalling via attenuated feedback inhibition. Stimulating the GC-cGMP-PKG pathway may, therefore, be a promising therapy for T2DM.