O peptídeo natriurético tipo C (CNP) é descrito como agonista vasodilatador do músculo liso vascular de artérias e veias, podendo ser produzido e liberado do endotélio vascular frente a diversos estímulos. Este promove o relaxamento vascular através da interação com seu receptor associado à enzima guanilil ciclase particulada (GCp) de membrana, levando ao aumento dos níveis de GMPc ou pelo receptor NPR-C associado à inibição de enzima adenilil ciclase e ativação de enzima fosfolipase C e canais para K+, via proteína Gi. Esta vasodilatação é descrita envolver parcialmente a via NO-GCs-GMPc, estando relacionada com a mobilização intracelular de cálcio. O modelo de hipertensão 2R-1C está associada ao consumo de NO, no ambiente celular, via reação com espécies reativas de oxigênio (EROs), especialmente ânion superóxido (O2-), caracterizando um prejuízo na vasodilatação dependente do endotélio. Desta forma, considerando as alterações descritas no modelo de hipertensão renovascular 2R-1C quanto ao estresse oxidativo e a importância do CNP como agonista vasodilatador, a hipótese do presente trabalho foi de que na aorta torácica isolada de ratos hipertensos 2R-1C, o relaxamento vascular induzido pelo CNP estaria prejudicado devido o estresse oxidativo. Neste contexto, o objetivo do presente trabalho foi de elucidar os mecanismos celulares envolvidos nas respostas vasculares induzidas pelo CNP. Células endoteliais isoladas de aorta de ratos 2R-1C apresentaram menor [NO]c comparadas as de 2R, sugerindo disfunção endotelial. A exposição das células endoteliais à agente sequestrador de O2- (Tiron) demonstrou a participação deste ânion em 2R-1C, mas não em 2R. CNP promoveu relaxamento de anéis de aorta de ratos 2R e 2R-1C, o qual foi mais pronunciado em preparações desprovidas de camada endotelial em 2R-1C. Hidroxicobalamina (sequestrador de NO0), mas não L-NAME (inibidor de NOS), causou redução da potência do CNP em 2R-1C. ODQ (inibidor de GCs) reduziu a potência em 2R E+, atenuando isoladamente o efeito máximo e o número de Hill em preparações E- de 2R-1C ao nível controle. Em presença do inibidor seletivo de GK (Rp-8-Br-PET-cGMPS) ocorreu atenuação da potência do CNP em todos os grupos experimentais, porém o efeito máximo do CNP somente foi reduzido daquelas preparações E-. A inibição da SERCA reduziu da potência do CNP em todos os grupos experimentais, porém o efeito máximo em 2R-1C foi reduzido ao nível controle. Sobre pré-contração induzida pela EC50 de solução de cloreto de potássio (KCl) ocorreu atenuação de potência e efeito máximo do CNP em todos os grupos experimentais. Contudo, com a utilização de inibidores seletivos de canais para K+, apenas observou-se a participação de BKCa e SKCa em 2R e 2R-1C e de KV em 2R-1C no relaxamento induzido pelo CNP. A inibição de NAD(P)H-oxidase (Apocinina) foi capaz de normalizar o efeito máximo em preparações E- de 2R-1C. A inibição de junções gap mioendoteliais (ácido 18--glicirretínico) promoveu redução da potência do CNP em 2R E+. Por fim, a inibição não-seletiva de COX (Indometacina) apenas reduziu a potência do CNP em preparações E+ de 2R. A partir da análise do conjunto de dados obtidos em estudos realizados em células endoteliais isoladas e funcionais pode-se concluir a participação do sistema de peptídeos natriuréticos sobre a modulação do tônus vascular em preparações isoladas de ratos 2R e 2R-1C. Assim, o relaxamento vascular induzido pelo CNP não se encontra prejudicado em aorta torácica isolada de 2R-1C. O endotélio vascular modula negativamente o relaxamento induzido pelo CNP em aorta de ratos 2R e 2R-1C. Embora haja disfunção endotelial em 2R-1C, esta não é suficiente para alterar a mobilização de cálcio induzida pelo CNP. O relaxamento induzido pelo peptídeo envolve a participação de metabólitos de NOS e de espécie NO0, assim como as enzimas NAD(P)H oxidase, GCs, GK e SERCA parecem estar relacionadas com o efeito vasodilatador induzido pelo CNP mais pronunciado em 2R-1C. Assim também, a sinalização desencadeada pelo CNP leva à ativação de canais para K+ em ambos os grupos, envolvendo a mediação através de junções gap mioendoteliais em 2R, mas não em 2R-1C. / C-type natriuretic peptide (CNP) acts as a vasodilator agonist that relaxes vascular smooth muscle cells from arteries and veins, and it can be produced and released from the vascular endothelium by several stimuli. This peptide induces vascular relaxation through interaction with its receptor which is associated with particulate guanylyl cylase (pGC), leading to the increase in cGMP levels or it can also can interacts with NPR-C receptor associated with the inhibition of adenylyl cyclase and the activation of phospholipase C and K+ channels, via Gi protein. This vasodilation is related to partially involves NO-sGC-cGMP and intracellular calcium mobilization. 2K-1C hypertension is related to NO consumption, in the cellular environment through reaction with reactive oxygen species (ROS), mainly superoxide anion (O2-), characterizing impaired endothelium-dependent relaxation. So, in view of the changes in renovascular hypertension 2K-1C through oxidative stress and the importance of CNP like a vasodilator agonist, the hypothesis of the present work was on thoracic aorta isolated from renal hypertensive rats (2K-1C) the vascular relaxation induced by CNP would be impaired due to oxidative stress. In this context, the study aimed to demonstrate the cellular mechanisms involved on vascular responses induced by CNP. Endothelial cells isolated from 2K-1C rat aortas showed decreased [NO]c compared to 2K, suggesting endothelial dysfunction. In the presence of O2- scavenger (Tiron) there was the participation of this anion on endothelial cells from 2K-1C, but not on 2K ones. CNP caused relaxation of aortic rings from 2K and 2K-1C rats which was enhanced on denuded rings from 2K-1C. Hydroxicobalamine (NO0 scavenger) but not L-NAME (NOS inhibitor) decreased the potency of CNP on intact aortic rings from 2K-1C. ODQ (sGC inhibitor) decreased potency in intact vascular rings from 2K, just reducing maximum effect and Hill number on denuded aortic rings from 2K-1C to a control level. In the presence of selective GK inhibitor (Rp-8-Br-PET-cGMPS) occurred an attenuation of CNP potency in all experimental groups, but the maximum effect of CNP was reduced only in aortic rings without endothelial layer. SERCA inhibition decreased CNP potency of all experimental groups, but maximum effect was reduced on 2K-1C to control levels. Under pre-contraction elicited by EC50 of potassium chloride (KCl) solution there was a decreased CNP potency and maximum effect in all experimental groups. However, the presence of selective inhibitor of K+ channels showed the contribution of BKCa and SKCa on 2K and 2K-1C and KV on 2K-1C in the CNP induced relaxation. NAD(P)H-oxidase inhibition (Apocinin) was able to normalize maximum effect of CNP on denuded vessels from 2K-1C. Myoendothelial gap junctions inhibition (18--glicirrhetinic acid) reduced CNP potency on intact aortic rings from 2K. Finally, the non-selective COX inhibition (Indometacin) induced a lower CNP potency on intact aortic rings from 2K. The analysis of the data obtained from isolated endothelial cells and functional studies allow us to conclude the participation of natriuretic peptide system in the modulation of vascular tone on aortic rings isolated from 2K and 2K-1C rats. Thus, the vascular relaxation induced by CNP is not impaired on aortic rings isolated from 2K-1C. Vascular endothelium negatively modulates the relaxation induced by CNP on 2K and 2K-1C rat aortas. Although 2K-1C exhibits endothelial dysfunction, it is not sufficient to impair calcium mobilization induced by CNP. CNP induced relaxation involves NOS metabolites and NO0 specie, as well as the activity of NAD(P)H oxidase, sGC, GK and SERCA is related to the enhanced vasodilator effect induced by CNP on 2K-1C. Similarly, CNP pathway leads to K+ channels activation in both experimental groups and the signal mediation through myoendothelial gap junctions on 2K aortas, but not on 2K-1C ones.

O peptídeo natriurético tipo C (CNP) é descrito como agonista vasodilatador do músculo liso vascular de artérias e veias, podendo ser produzido e liberado do endotélio vascular frente a diversos estímulos. Este promove o relaxamento vascular através da interação com seu receptor associado à enzima guanilil ciclase particulada (GCp) de membrana, levando ao aumento dos níveis de GMPc ou pelo receptor NPR-C associado à inibição de enzima adenilil ciclase e ativação de enzima fosfolipase C e canais para K+, via proteína Gi. Esta vasodilatação é descrita envolver parcialmente a via NO-GCs-GMPc, estando relacionada com a mobilização intracelular de cálcio. O modelo de hipertensão 2R-1C está associada ao consumo de NO, no ambiente celular, via reação com espécies reativas de oxigênio (EROs), especialmente ânion superóxido (O2-), caracterizando um prejuízo na vasodilatação dependente do endotélio. Desta forma, considerando as alterações descritas no modelo de hipertensão renovascular 2R-1C quanto ao estresse oxidativo e a importância do CNP como agonista vasodilatador, a hipótese do presente trabalho foi de que na aorta torácica isolada de ratos hipertensos 2R-1C, o relaxamento vascular induzido pelo CNP estaria prejudicado devido o estresse oxidativo. Neste contexto, o objetivo do presente trabalho foi de elucidar os mecanismos celulares envolvidos nas respostas vasculares induzidas pelo CNP. Células endoteliais isoladas de aorta de ratos 2R-1C apresentaram menor [NO]c comparadas as de 2R, sugerindo disfunção endotelial. A exposição das células endoteliais à agente sequestrador de O2- (Tiron) demonstrou a participação deste ânion em 2R-1C, mas não em 2R. CNP promoveu relaxamento de anéis de aorta de ratos 2R e 2R-1C, o qual foi mais pronunciado em preparações desprovidas de camada endotelial em 2R-1C. Hidroxicobalamina (sequestrador de NO0), mas não L-NAME (inibidor de NOS), causou redução da potência do CNP em 2R-1C. ODQ (inibidor de GCs) reduziu a potência em 2R E+, atenuando isoladamente o efeito máximo e o número de Hill em preparações E- de 2R-1C ao nível controle. Em presença do inibidor seletivo de GK (Rp-8-Br-PET-cGMPS) ocorreu atenuação da potência do CNP em todos os grupos experimentais, porém o efeito máximo do CNP somente foi reduzido daquelas preparações E-. A inibição da SERCA reduziu da potência do CNP em todos os grupos experimentais, porém o efeito máximo em 2R-1C foi reduzido ao nível controle. Sobre pré-contração induzida pela EC50 de solução de cloreto de potássio (KCl) ocorreu atenuação de potência e efeito máximo do CNP em todos os grupos experimentais. Contudo, com a utilização de inibidores seletivos de canais para K+, apenas observou-se a participação de BKCa e SKCa em 2R e 2R-1C e de KV em 2R-1C no relaxamento induzido pelo CNP. A inibição de NAD(P)H-oxidase (Apocinina) foi capaz de normalizar o efeito máximo em preparações E- de 2R-1C. A inibição de junções gap mioendoteliais (ácido 18--glicirretínico) promoveu redução da potência do CNP em 2R E+. Por fim, a inibição não-seletiva de COX (Indometacina) apenas reduziu a potência do CNP em preparações E+ de 2R. A partir da análise do conjunto de dados obtidos em estudos realizados em células endoteliais isoladas e funcionais pode-se concluir a participação do sistema de peptídeos natriuréticos sobre a modulação do tônus vascular em preparações isoladas de ratos 2R e 2R-1C. Assim, o relaxamento vascular induzido pelo CNP não se encontra prejudicado em aorta torácica isolada de 2R-1C. O endotélio vascular modula negativamente o relaxamento induzido pelo CNP em aorta de ratos 2R e 2R-1C. Embora haja disfunção endotelial em 2R-1C, esta não é suficiente para alterar a mobilização de cálcio induzida pelo CNP. O relaxamento induzido pelo peptídeo envolve a participação de metabólitos de NOS e de espécie NO0, assim como as enzimas NAD(P)H oxidase, GCs, GK e SERCA parecem estar relacionadas com o efeito vasodilatador induzido pelo CNP mais pronunciado em 2R-1C. Assim também, a sinalização desencadeada pelo CNP leva à ativação de canais para K+ em ambos os grupos, envolvendo a mediação através de junções gap mioendoteliais em 2R, mas não em 2R-1C. / C-type natriuretic peptide (CNP) acts as a vasodilator agonist that relaxes vascular smooth muscle cells from arteries and veins, and it can be produced and released from the vascular endothelium by several stimuli. This peptide induces vascular relaxation through interaction with its receptor which is associated with particulate guanylyl cylase (pGC), leading to the increase in cGMP levels or it can also can interacts with NPR-C receptor associated with the inhibition of adenylyl cyclase and the activation of phospholipase C and K+ channels, via Gi protein. This vasodilation is related to partially involves NO-sGC-cGMP and intracellular calcium mobilization. 2K-1C hypertension is related to NO consumption, in the cellular environment through reaction with reactive oxygen species (ROS), mainly superoxide anion (O2-), characterizing impaired endothelium-dependent relaxation. So, in view of the changes in renovascular hypertension 2K-1C through oxidative stress and the importance of CNP like a vasodilator agonist, the hypothesis of the present work was on thoracic aorta isolated from renal hypertensive rats (2K-1C) the vascular relaxation induced by CNP would be impaired due to oxidative stress. In this context, the study aimed to demonstrate the cellular mechanisms involved on vascular responses induced by CNP. Endothelial cells isolated from 2K-1C rat aortas showed decreased [NO]c compared to 2K, suggesting endothelial dysfunction. In the presence of O2- scavenger (Tiron) there was the participation of this anion on endothelial cells from 2K-1C, but not on 2K ones. CNP caused relaxation of aortic rings from 2K and 2K-1C rats which was enhanced on denuded rings from 2K-1C. Hydroxicobalamine (NO0 scavenger) but not L-NAME (NOS inhibitor) decreased the potency of CNP on intact aortic rings from 2K-1C. ODQ (sGC inhibitor) decreased potency in intact vascular rings from 2K, just reducing maximum effect and Hill number on denuded aortic rings from 2K-1C to a control level. In the presence of selective GK inhibitor (Rp-8-Br-PET-cGMPS) occurred an attenuation of CNP potency in all experimental groups, but the maximum effect of CNP was reduced only in aortic rings without endothelial layer. SERCA inhibition decreased CNP potency of all experimental groups, but maximum effect was reduced on 2K-1C to control levels. Under pre-contraction elicited by EC50 of potassium chloride (KCl) solution there was a decreased CNP potency and maximum effect in all experimental groups. However, the presence of selective inhibitor of K+ channels showed the contribution of BKCa and SKCa on 2K and 2K-1C and KV on 2K-1C in the CNP induced relaxation. NAD(P)H-oxidase inhibition (Apocinin) was able to normalize maximum effect of CNP on denuded vessels from 2K-1C. Myoendothelial gap junctions inhibition (18--glicirrhetinic acid) reduced CNP potency on intact aortic rings from 2K. Finally, the non-selective COX inhibition (Indometacin) induced a lower CNP potency on intact aortic rings from 2K. The analysis of the data obtained from isolated endothelial cells and functional studies allow us to conclude the participation of natriuretic peptide system in the modulation of vascular tone on aortic rings isolated from 2K and 2K-1C rats. Thus, the vascular relaxation induced by CNP is not impaired on aortic rings isolated from 2K-1C. Vascular endothelium negatively modulates the relaxation induced by CNP on 2K and 2K-1C rat aortas. Although 2K-1C exhibits endothelial dysfunction, it is not sufficient to impair calcium mobilization induced by CNP. CNP induced relaxation involves NOS metabolites and NO0 specie, as well as the activity of NAD(P)H oxidase, sGC, GK and SERCA is related to the enhanced vasodilator effect induced by CNP on 2K-1C. Similarly, CNP pathway leads to K+ channels activation in both experimental groups and the signal mediation through myoendothelial gap junctions on 2K aortas, but not on 2K-1C ones.

<p>Insulin release from single islets of Langerhans is pulsatile. The secretory activities of the islets in the pancreas are coordinated resulting in plasma insulin oscillations. Nutrients amplitude-regulate the insulin pulses without influencing their frequency. Diabetic patients show an abnormal plasma insulin pattern, but the cause of the disturbance remains to be elucidated. Ithe present thesis the influence of the cytoplasmic calcium concentratio([Ca²⁺]_i) and cell metabolism on pulsatile insulin release was examined in single islets of Langerhans from <i>ob/ob</i>-mice. Glucose stimulation of insulin release involves closure of ATP-sensitive K⁺ channels (K_ATP channels), depolarization, and Ca²⁺ influx in β-cells. In the presence of 11 mM glucose, pulsatile insulin secretion occurs in synchrony with oscillations i[Ca²⁺]_i. When [Ca²⁺]_i is low and stable, e.g. under basal conditions, low amplitude insulin pulses are still observed. When [Ca²⁺]_i is elevated and non-oscillating, e.g. when the β-cells are depolarized by potassium, high amplitude insulin pulses are observed. The frequency of the insulin pulses under these conditions is similar to that observed when [Ca²⁺]_i oscillations are present. By permanently opening or closing the K_ATP channels with diazoxide or tolbutamide, respectively, it was investigated if glucose can modulate pulsatile insulin secretion when it does not influence the channel activity. Under these conditions, [Ca²⁺]_i remained stable whereas the amplitude of the insulin pulses increased with sugar stimulation without change in the frequency. Metabolic inhibition blunted but did not prevent the insulin pulses. The results indicate that oscillations in metabolism can generate pulsatile insulin release when [Ca²⁺]_i is stable. However, under physiological conditions, pulsatile secretion is driven by oscillations in metabolism and [Ca²⁺]_i, acting in synergy.</p>

Insulin release from single islets of Langerhans is pulsatile. The secretory activities of the islets in the pancreas are coordinated resulting in plasma insulin oscillations. Nutrients amplitude-regulate the insulin pulses without influencing their frequency. Diabetic patients show an abnormal plasma insulin pattern, but the cause of the disturbance remains to be elucidated. Ithe present thesis the influence of the cytoplasmic calcium concentratio([Ca2+]i) and cell metabolism on pulsatile insulin release was examined in single islets of Langerhans from ob/ob-mice. Glucose stimulation of insulin release involves closure of ATP-sensitive K+ channels (KATP channels), depolarization, and Ca2+ influx in β-cells. In the presence of 11 mM glucose, pulsatile insulin secretion occurs in synchrony with oscillations i[Ca2+]i. When [Ca2+]i is low and stable, e.g. under basal conditions, low amplitude insulin pulses are still observed. When [Ca2+]i is elevated and non-oscillating, e.g. when the β-cells are depolarized by potassium, high amplitude insulin pulses are observed. The frequency of the insulin pulses under these conditions is similar to that observed when [Ca2+]i oscillations are present. By permanently opening or closing the KATP channels with diazoxide or tolbutamide, respectively, it was investigated if glucose can modulate pulsatile insulin secretion when it does not influence the channel activity. Under these conditions, [Ca2+]i remained stable whereas the amplitude of the insulin pulses increased with sugar stimulation without change in the frequency. Metabolic inhibition blunted but did not prevent the insulin pulses. The results indicate that oscillations in metabolism can generate pulsatile insulin release when [Ca2+]i is stable. However, under physiological conditions, pulsatile secretion is driven by oscillations in metabolism and [Ca2+]i, acting in synergy.

Pancreatic ß-cells are heterogeneous in their secretory responsiveness, glucose sensitivity and metabolic rate. A diminished and delayed first-phase insulin release is an early sign of failing ß-cells in diabetes. Mechanisms controlling functional characteristics, such as lag time for insulin release or magnitude of the response in each individual cell are unknown. To find out whether the heterogeneity represents a random phenomenon in ß-cell or is a manifestation of reproducible characteristics, we compared parameters of Ca2+ response in Fura-2 labelled ob/ob mouse ß-cells during two consecutive stimulations with glucose. Lag times, as well as peak heights and nadirs of initial lowering showed a strong correlation between the first and second stimulation. Thus, timing and magnitude of the early Ca2+ response were specific for each cell. ß-Cells from lean mice, diabetic db/db mice and rats also showed cell-specific responses characteristics. This indicates that a cell-specific Ca2+ response to glucose is common in rodent ß-cells, both normal and diabetic. Another question was whether aggregated ß-cells show cell-specific responses. Using the same protocol as for dispersed ß-cells, we analysed Ca2+ responses in clusters of different size and in intact islets from ob/ob and lean mice. Correlations were found between the first and second stimulation for timing and magnitude of [Ca2+]i rise, and for the initial lowering. Next, we tested if the ß-cell response is cell-specific, when induced at different steps of the stimulus-secretion coupling. The glycolytic intermediate glyceraldehyde, the mitochondrial substrate KIC, the KATP-channel blocker tolbutamide and arginine were used as tools. [Ca2+]i changes were studied in dispersed ß-cells from lean, ob/ob and db/db mice. NADH responses to glucose and KIC were analyzed as a measure of metabolic flux. The correlation between Ca2+ and insulin response from individual ß-cells was tested using Fluo-3 and Fluozin-3. Both timing and magnitude of calcium responses were cell-specific in lean mouse ß-cells with all tested secretagogues. ß-Cells from ob/ob and db/db mice showed cell-specific timing of Ca2+ responses to glyceraldehyde but not to KIC, tolbutamide or arginine. However, ob/ob mouse ß-cells within intact islets showed cell-specific timing of tolbutamide-induced response. NADH responses to glucose were cell-specific in all three mouse models, but the timing of NADH responses to KIC was cell-specific only in lean mice. Thus, a cell-specific response can be induced in normal ß-cells at several steps of stimulus-secretion coupling for nutrient-stimulated insulin release. Cell-specific properties of ß-cell ion channels and the mitochondrial metabolism are affected in db/db and ob/ob mice. The relation between mitochondrial mass and parameters of Ca2+ responses were investigated in Mitotracker Red and Fluo-3 labelled ß-cells using confocal microscopy. Data show that ß-cell mitochondrial state may play an important role in determining the timing of [Ca2+]i changes. In summary, the early Ca2+ response pattern in ß-cells, including the lag time, the nadir of initial lowering and the height of the first peak response is cell-specific. Isolated and functionally coupled ß-cells show cell-specific timing of Ca2+ responses when stimulated with metabolic and non-metabolic agents. This may be a robust mechanism of importance for the adequate function of ß-cells and a basis for the pacemaker function of some cells. A disturbed cell specificity of the mitochondrial metabolism and ion channel function appears to be a marker of ß-cell dysfunction in hyperglycemia and diabetes and may explain the delayed insulin release in ß-cells from diabetic subjects.