<p> Asphyxia (hypoxia, hypercapnia, and acidosis) sensing by neonatal adrenal chromaffin cells (AMC) plays a key role in the adaptation of the neonate to extrauterine environment. During birth, exposure of the neonate to episodes of acute asphyxia results in secretion of catecholamines from AMC, independent of neural inputs. This catecholamine (CAT) secretion promotes re-absorption of fluid and surfactant production in the lung, improved cardiac conductance, and assists in neonatal arousal. The asphyxia-sensing responses in AMC are lost postnatally during a time course that parallels splanchnic innervation. </p> </p> <p> The primary goal of this thesis was to uncover the mechanisms of asphyxia sensing in neonatal rat AMC, using primary and immortalized chromaffin cells, and to elucidate the potential role of innervation in the postnatal loss of these mechanisms. The experimental approaches relied on patch clamp techniques to record ionic currents and membrane potential, carbon fiber amperometry to monitor CAT secretion, chemiluminescence to measure reactive oxygen species (ROS) production and ATP secretion, and molecular techniques to examine protein and gene expression. </p> <p> In primary neonatal, but not juvenile AMC, hypoxia (PO₂ = 15 mmHg) caused a suppression of outward K⁺ current, membrane depolarization, and increased secretion. These effects were associated with a decrease in mitochondrial ROS production, were reversed by exogenous H₂O₂, and where mimicked by antioxidants. Of several mitochondrial electron transport chain (ETC) inhibitors tested, only rotenone, a complex I blocker, mimicked and occulded the effects of hypoxia. The immortalized chromaffin cell line (MAH cells) behaved similarly, and became hypoxia-insensitive when depleted of functional mitochondria (ρ0 MAH). Both neonatal AMC and immortalized MAH cells also responded to increased CO₂ (hypercapnia) with K⁺ current inhibition, membrane depolarized, increased intracellular Ca²⁺, and CAT secretion. However, these responses were independent of functional mitochondria and were associated with the expression of carbonic anhydrase II (CAII). </p> <p> Since the splanchnic nerve supplies both cholinergic and opioid peptidergic innervation to AMC, I tested the hypothesis that postsynaptic activation of the corresponding receptors might underlie the postnatal loss of asphyxia-sensing. First, to determine whether nicotinic acetylcholine receptor stimulation was involved, pregnant rats were exposed to nicotine (or saline) during gestation and AMC were examined in the offspring, soon after birth. Control AMC isolated from pups born to saline-treated dams displayed typical responses to hypoxia and hypercapnia, including inhibition of outward K⁺ current, membrane depolarization, increased cytosolic calcium, and CAT secretion. In contrast, AMC from pups born to nicotine-treated dams showed a dramatic loss of hypoxic sensitivity, though hypercapnic sensitivity and the expression of CO₂ markers (i.e. carbonic anhydrase I and II) appeared normal. This effect of chronic nicotine could be reproduced in cultured chromaffin cells in vitro and was mediated via activation of α7-nicotinic acetylcholine receptors (nAChR), leading to upregulation of ATP-dependent (K_ATP) K⁺ channels, which open during hypoxia and cause membrane hyperpolarization. The upregulation of K_ATP channels involved expression ofthe hypoxia inducible transcription factor (HIF 2α). Second, to determine whether opioid receptor stimulation was also involved, cultured chromaffin cells were chronically exposed to mu, delta, or kappa opioid agonists in vitro. Treatment with both mu and delta, but not kappa, agonists resulted in the loss of both hypoxia and hypercapnia sensing and a decrease in CAII expression. </p> <p> Taken together, these studies suggest that neonatal chromaffin cells sense hypoxia via a reduction in ROS generation located at or upstream of mitochondrial complex I. Chronic stimulation of nicotinic α7 ACh receptors leads to a selective loss of hypoxia sensing and this appears to be mediated via a HIF 2α-dependent upregulation of K_ATP channels. These channels open during hypoxia causing membrane hyperpolarization and reduced excitability. The result of this upregulation of K_ATP channels results in cells deficient in the ability to respond to hypoxia. </p> / Thesis / Doctor of Philosophy (PhD)

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Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-17T11:03:10Z No. of bitstreams: 1 lilianesenapinheiro.pdf: 8108520 bytes, checksum: b03200480798ddb2cf88a9276e4c9d8d (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T13:09:54Z (GMT) No. of bitstreams: 1 lilianesenapinheiro.pdf: 8108520 bytes, checksum: b03200480798ddb2cf88a9276e4c9d8d (MD5) / Made available in DSpace on 2016-10-22T13:09:54Z (GMT). No. of bitstreams: 1 lilianesenapinheiro.pdf: 8108520 bytes, checksum: b03200480798ddb2cf88a9276e4c9d8d (MD5) Previous issue date: 2008-06-25 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O diabetes mellitus (DM) reduz a secreção de catecolaminas (CAs) das células cromafins adrenais, sendo esse um evento patofisiológico crítico por favorecer a ocorrência de episódios de hipoglicemia grave decorrentes do próprio tratamento da doença. Vários trabalhos relatam a participação de proteínas quinase C (PKCs) nas vias de síntese e secreção de CAs nas células cromafins. Os objetivos desse trabalho foram analisar o efeito do DM sobre a expressão das isoformas α, ε e ζ de PKC em células cromafins de ratos e avaliar se o controle glicêmico reverte os efeitos da doença. Foram utilizados ratos Wistar com DM induzido por estreptozotocina. Foram estabelecidos três grupos experimentais, ratos controles (C), diabéticos tratados com salina (DTS) ou com insulina (DTI). As análises foram feitas 15 dias após a indução. Utilizamos as técnicas de imunohistoquímica e Western Blot. A insulinoterapia foi estabelecida após estudos do comportamento alimentar e da variação dos níveis glicêmicos de ratos controles e doentes durante 24h consecutivas. Foi testada a eficácia de diferentes esquemas de tratamento com insulina. O tratamento estabelecido consistiu em injeções de insulina NPH, sendo 1U aplicada às 13h e 4U às 19h. Após os 15 dias de tratamento, o ganho médio de massa corporal dos ratos C (+37±3g) e DTI (+43±3g) foram similares enquanto os DTS emagreceram (-9±6g). A média da glicemia de jejum dos ratos C (74±1mg/dl) e dos DTI (93±6mg/dl) foram similares e dentro dos níveis normais, enquanto que a dos ratos DTS foi elevada (471±23mg/dl). A insulinoterapia restabeleceu os níveis plasmáticos do colesterol total, c-LDL e c-VLDL nos ratos DTI. O DM não alterou os níveis de c-HDL, triglicerídos e frutosamina. As análises da expressão de PKCs mostraram que a PKCα é a mais expressada seguida de ζ e depois de ε. O DM reduziu em 39,5% a expressão da PKCα, enquanto a de ζ foi aumentada em 74,2%. A expressão da PKCε não foi afetada pelo DM. O tratamento com insulina reverteu o efeito do DM sobre a expressão de PKCα, a expressão da PKCε continuou inalterada e a expressão da PKCζ permaneceu elevada (+32,6%) quando comparada aos ratos C. Concluímos que em células cromafins adrenais, o diabetes afeta a expressão de isoformas de PKCs de maneira diferenciada. Trabalhos realizados em nosso laboratório mostraram que o DM reduz o conteúdo total (21,1%), a secreção basal (-24,3%) e a estimulada por carbacol (-28,9%) e K+ (42,2%) de CAs. Como observado para PKCα, a insulinoterapia reverteu o efeito do DM sobre o conteúdo total. Já foi demonstrado que PKCα participa de uma via de sinalização que estimula a atividade de tirosina hidroxilase. Por outro lado, o tratamento não restabeleceu os processos secretórios, sugerindo que PKCζ possa estar envolvida nessa alteração. Há fortes evidências de que PKCζ regula canais de K+ retificadores, o que pode explicar o efeito da doença sobre o processo de secreção via despolarização da membrana. / The diabetes mellitus (DM) reduces the catecholamine (CAs) secretion of adrenal chromaffin cells, a critical pathophysiologic event that promotes the occurrence of serious hypoglycemia episodes, consequence of the disease treatment. Several papers report the participation of protein kinase C (PKC) on catecholamine synthesis signal pathways of adrenal chromaffin cells. The objectives of this work were to study the effect of DM on expression of PKC isoforms α, ε and ζ in rat chromaffin cells and to evaluate if the glicemic control revert the effect of the illness. Male Wistar rats with diabetes induced by streptozotocin were used. Three experimental groups were determined: Control (C), diabetic rats receiving saline solution (DS) and diabetic rats receiving insulin (DI). The analyses were made after 15 days of DM induction. Immunohistochemistry and western blotting techniques were done. The insulin therapy protocol was established after studying the feeding behavior and glycemic level variations during the whole 24h. The information made possible to establish the time of insulin applications. Several schemes of insulin treatments were tested to keep the diabetic rat as close as possible to normoglycemia path. The best results were found by using 1U at 1:00 PM and 4U at 7:00 PM of NPH insulin. After 15 days of treatment the acquired body weight was similar between C and DI rats, 37±3g and 43±3g, respectively. The DS rats emaciated 9±6g. The fasting glycemic levels were 74±1mg/dl, 93±6mg/dl and 471±23mg/dl to C, DI and DS rats, respectively. The insulin therapy reestablishes the plasmic levels of total cholesterol, c-LDL and c-VLDL on DI rats. The DM did not change the levels of c-HDL, triglycerides and frutosamine. The PKCα is the more expressed isoform in adrenal chromaffin cells, followed by ζ and ε. The DM reduced 39,5% the PKCα expression and, unlike, increased 74,2% the expression of PKCζ. The expression of PKCε was not affected by DM. The insulin treatment reverted the effect of DM on PKCα, the expression of PKCε remained unchanged and the expression of PKCζ remained higher than the control group (+32,6%). Studies of our laboratory show that the DM causes reduction on adrenal catecholamine content (21,1%), basal secretion (-24,3%) and catecholamine secretion stimulated by carbachol (-28,9%) and high K+ (-42,2%). The insulin therapy, in like manner as observed on PKCα, reverted the DM effect on adrenal catecholamine content. It was shown that PKCα participates on signal transduction pathway that stimulates the activity of tyrosine hydroxylase. Otherwise, the insulin treatment did not restore the secretory processes, suggesting that PKCζ could be involved in this process. There are strong evidences showing that PKCζ regulates the voltage-dependent delayed rectifier K (Kv) and its expression was not normalized by insulin therapy.