Statement of the problem: The epithelial sodium channels (ENaC) play an important role in regulation of blood pressure (BP). Although the genes are identical in Dahl salt sensitive (S) and Dahl salt resistant (R) rats, expression of ENaC subunits is increased in kidneys of S rats on high salt diet. Intracerebroventricular (icv) infusion of ENaC blocker benzamil prevents Na+ induced hypertension. It was not known whether ENaC subunits are expressed in the brain and whether or not brain ENaC plays a role in regulation of [Na+] in CNS. Hypothesis: 1. Epithelial sodium channels are expressed in the brain. 2. Expression of ENaC is increased in the kidneys and brain of Dahl S rats on high salt diet. 3. ENaC in the brain contributes to regulation of [Na+] in the CSF and brain interstitium. Methods of investigation: We studied expression and distribution of the ENaC subunits and assessed the effects of icv infusion of Na+-rich aCSF in Wistar rats or high salt diet in Dahl S rats in different areas of the brain. Function of ENaC in the choroid plexus was evaluated by studying the effects of benzamil and ouabain on Na+ transport. Major findings: In Wistar rats, both mRNA and protein of all three ENaC subunits are expressed in brain epithelia and magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nucleus. ENaC abundance is higher on the apical versus basolateral membrane of choroid cells. Benzamil decreases Na+ influx into choroid cells by 20-30% and increases CSF [Na+] by ~8 mmol/L. Na+ rich aCSF increases apical membrane expression of βENaC in the choroid cells and of α and βENaC in basolateral membrane of ependymal cells, but has no effect on neuronal ENaC. Expression of ENaC is higher in choroid cells and SON of Dahl S versus R rats and the higher expression persists on a high salt diet. High salt attenuates the ouabain blockable efflux of Na+ from choroid cells and has no effect on CSF [Na+] in Dahl R rats. In contrast, high salt does not attenuate ouabain blockable efflux of 22Na+ and CSF [Na+] increases in Dahl S. Main Conclusion: ENaC in the brain contributes to Na+ transport into the choroid cells and appear to be involved in reabsorption of Na+ from the CSF. Aberrant regulation of Na+ transport and of Na+K+ATPase activity, might contribute to increases in CSF [Na+] in Dahl S rats on high-salt diet. ENaC in magnocellular neurons may contribute to enhanced secretion of mediators such as ‘ouabain’ leading to sympathetic hyperactivity in Dahl S rats.

Statement of the problem: The epithelial sodium channels (ENaC) play an important role in regulation of blood pressure (BP). Although the genes are identical in Dahl salt sensitive (S) and Dahl salt resistant (R) rats, expression of ENaC subunits is increased in kidneys of S rats on high salt diet. Intracerebroventricular (icv) infusion of ENaC blocker benzamil prevents Na+ induced hypertension. It was not known whether ENaC subunits are expressed in the brain and whether or not brain ENaC plays a role in regulation of [Na+] in CNS. Hypothesis: 1. Epithelial sodium channels are expressed in the brain. 2. Expression of ENaC is increased in the kidneys and brain of Dahl S rats on high salt diet. 3. ENaC in the brain contributes to regulation of [Na+] in the CSF and brain interstitium. Methods of investigation: We studied expression and distribution of the ENaC subunits and assessed the effects of icv infusion of Na+-rich aCSF in Wistar rats or high salt diet in Dahl S rats in different areas of the brain. Function of ENaC in the choroid plexus was evaluated by studying the effects of benzamil and ouabain on Na+ transport. Major findings: In Wistar rats, both mRNA and protein of all three ENaC subunits are expressed in brain epithelia and magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nucleus. ENaC abundance is higher on the apical versus basolateral membrane of choroid cells. Benzamil decreases Na+ influx into choroid cells by 20-30% and increases CSF [Na+] by ~8 mmol/L. Na+ rich aCSF increases apical membrane expression of βENaC in the choroid cells and of α and βENaC in basolateral membrane of ependymal cells, but has no effect on neuronal ENaC. Expression of ENaC is higher in choroid cells and SON of Dahl S versus R rats and the higher expression persists on a high salt diet. High salt attenuates the ouabain blockable efflux of Na+ from choroid cells and has no effect on CSF [Na+] in Dahl R rats. In contrast, high salt does not attenuate ouabain blockable efflux of 22Na+ and CSF [Na+] increases in Dahl S. Main Conclusion: ENaC in the brain contributes to Na+ transport into the choroid cells and appear to be involved in reabsorption of Na+ from the CSF. Aberrant regulation of Na+ transport and of Na+K+ATPase activity, might contribute to increases in CSF [Na+] in Dahl S rats on high-salt diet. ENaC in magnocellular neurons may contribute to enhanced secretion of mediators such as ‘ouabain’ leading to sympathetic hyperactivity in Dahl S rats.

No description available.

O mosquito Aedes (Stegomyia) aegypti Linnaeus, 1762 é o principal vetor do vírus do dengue sorotipo 1-4 (DEN 1-4) e da febre amarela. Por não haver vacina disponível, a redução da transmissão da dengue só pode ser alcançada mediante o controle do vetor. Entre as medidas de controle, os órgãos responsáveis utilizam-se de compostos químicos, principalmente organofosforados. Além disso, devido ao grande incomodo causado pelo Aedes, a população faz uso de inseticidas domésticos, a base de piretróides, na tentativa de eliminar e ou repelir o mosquito. As repetidas aplicações destes inseticidas e seu uso contínuo possibilitam o desenvolvimento de resistência em populações de mosquitos, processo resultante do efeito seletivo de exposição a dosagens que matam os indivíduos suscetíveis, sobrevivendo os resistentes, que transferem essa capacidade a seus descendentes. Dentre os mecanismos de resistência, a redução da sensibilidade do sítio alvo é dada por mutações pontuais no sitio de ação dos inseticidas. Tais mutações podem levar a uma substituição de aminoácidos na molécula alvo e a uma diminuição da afinidade do inseticida com essa molécula. No caso dos piretróides a mudança estrutural na molécula formadora do canal de sódio (Nav), sítio alvo deste inseticida, é a causa da resistência tipo knockdown resistence (kdr), um mecanismo de resistência bastante conhecido. Em Aedes aegypti, a associação entre a presença da mutação V1016I e o fenótipo de resistência a piretróide já foi verificada em algumas regiões Brasileiras, utilizando primers alelo-específicos. A mutação I1011M também está associada à resistência a piretróides e já foi descrita no Brasil. O objetivo deste estudo foi determinar a frequência destas mutações que levam às substituições V1016I e I1011M no AaNav de populações de Aedes aegypti no estado de São Paulo e avaliar a evolução desta frequência no período de dez anos. Para isso, indivíduos coletados em 2001 e 2011 tiveram o DNA extraído e primers alelo específicos foram utilizados para a realização de PCR a fim de verificar a presença da mutação. Houve um aumento significativo do alelo 1016 Ile nas populações estudadas, comparando-se os anos 2001 e 2011. Entretanto para o alelo 1011 Met, somente a população de Santos apresentou essa diferença significativa. Este aumento na frequência destas mutações pode ter sido ocasionado pela utilização de inseticidas domésticos à base de piretróides, uma vez que os órgãos de controle interromperam a utilização desses compostos / The mosquito Aedes (Stegomyia) aegypti Linnaeus, 1762 is the main vector of the virus dengue serotypes 1-4 (DEN 1-4) and the yellow fever virus. As there is not vaccine available, the reduction of the transmission of dengue can only be achieved by controlling the vector. Among the control measures, the responsible agencies use chemical compounds, organophosphate mainly. Furthermore, due to the big nuisance caused by Aedes, the population makes use of domestic insecticide, pyrethroids based, trying to eliminate or repel the mosquito. The repeated applications of these insecticides and the continuous use of them enable the resistance development in the mosquitoes population. This process is the result of selective effect of exposure to dosages that can kill the susceptible individuals, and then the surviving individuals transfer these characteristics to their descendents. Among the mechanism of resistance, the reduction of sensibility of the target site is given by punctual mutation (SNIP) in the action site of the insecticides. This mutation can lead to a substitution of amino acids in the target molecule and a reduction of affinity of the insecticide with this molecule. In the case of pyrethroids, the structural change in the forming molecule of the sodium channel (Nav), target site of this insecticide, is the cause of knockdown resistence (kdr) type. In Aedes aegypti, the association between the presence of the mutation V1016I and the pyrethroid resistance phenotype has been previously verified in many Brazilian regions, using Allele Specific primers The mutation I1011M is also associated with the pyrethroids resistance and it has been described in Brazil. The objective of the this study was to determine the frequency of the mutations that leads to the substitutions V1016I e I1011M in AaNav of the Aedes aegypti population in the state of Sao Paulo and to evaluate the evolution of this frequency in the period of ten years. Therefore, collected individuals in 2001 and 2011 had their DNA extracted and specific primers were used for PCR, in order to verify the presence of the mutation. There was a significant raise of allele 1016 Ile in the studied population, comparing the years 2001 and 2011. Nevertheless for allele 1011 Met, only the population of Santos showed this significant difference. This raise in the frequency of these mutations may have been caused by the utilization of domestic insecticides pyrethroid based, whereas the control agencies interrupted the utilization of this compost

No description available.

Statement of the problem: The epithelial sodium channels (ENaC) play an important role in regulation of blood pressure (BP). Although the genes are identical in Dahl salt sensitive (S) and Dahl salt resistant (R) rats, expression of ENaC subunits is increased in kidneys of S rats on high salt diet. Intracerebroventricular (icv) infusion of ENaC blocker benzamil prevents Na+ induced hypertension. It was not known whether ENaC subunits are expressed in the brain and whether or not brain ENaC plays a role in regulation of [Na+] in CNS. Hypothesis: 1. Epithelial sodium channels are expressed in the brain. 2. Expression of ENaC is increased in the kidneys and brain of Dahl S rats on high salt diet. 3. ENaC in the brain contributes to regulation of [Na+] in the CSF and brain interstitium. Methods of investigation: We studied expression and distribution of the ENaC subunits and assessed the effects of icv infusion of Na+-rich aCSF in Wistar rats or high salt diet in Dahl S rats in different areas of the brain. Function of ENaC in the choroid plexus was evaluated by studying the effects of benzamil and ouabain on Na+ transport. Major findings: In Wistar rats, both mRNA and protein of all three ENaC subunits are expressed in brain epithelia and magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nucleus. ENaC abundance is higher on the apical versus basolateral membrane of choroid cells. Benzamil decreases Na+ influx into choroid cells by 20-30% and increases CSF [Na+] by ~8 mmol/L. Na+ rich aCSF increases apical membrane expression of βENaC in the choroid cells and of α and βENaC in basolateral membrane of ependymal cells, but has no effect on neuronal ENaC. Expression of ENaC is higher in choroid cells and SON of Dahl S versus R rats and the higher expression persists on a high salt diet. High salt attenuates the ouabain blockable efflux of Na+ from choroid cells and has no effect on CSF [Na+] in Dahl R rats. In contrast, high salt does not attenuate ouabain blockable efflux of 22Na+ and CSF [Na+] increases in Dahl S. Main Conclusion: ENaC in the brain contributes to Na+ transport into the choroid cells and appear to be involved in reabsorption of Na+ from the CSF. Aberrant regulation of Na+ transport and of Na+K+ATPase activity, might contribute to increases in CSF [Na+] in Dahl S rats on high-salt diet. ENaC in magnocellular neurons may contribute to enhanced secretion of mediators such as ‘ouabain’ leading to sympathetic hyperactivity in Dahl S rats.

Prostaglandin E2 (PGE2), a metabolite of arachidonic acid, plays a role in water and sodium reabsorption in the collecting duct of the kidney. The collecting duct is responsible for the fine tuning of water and electrolytes. Only a small fraction of the filtered water and sodium is reabsorbed in the collecting duct, a fraction crucial to the regulation of water and electrolyte balance. This current study addresses the role of EP1, one of four PGE2 receptors, in the collecting duct. It is well documented that PGE2 inhibits sodium and water reabsorption in the collecting duct, however the exact mechanism is still debated. To determine whether the EP1 receptor mitigates AngII renal effects, an in vivo study was performed with EP1-/- mice. Global EP1-/- knockout mice were crossed with a renin overexpressing mouse line (herein denoted as “Ren”) and subjected to a high salt (HS) and low salt (LS) diet. Ren mice displayed an 11mmHg increase in systolic blood pressure (BP) on a HS diet and a decrease in BP of 14mmHg on a LS diet compared to the normal salt (NS) diet. Ren EP1-/- mice did not display a significant increase or decrease in BP on a HS or LS diet. On a LS diet, Ren EP1-/- displayed a drop in urine osmolarity (1641 mOsm/ kgH2O) vs. wild type (WT) mice (2107 mOsm/ kgH2O), consistent with increased sodium reabsorption. Narrowing in on the collecting duct, Ren EP1-/- mice had enhanced αENaC levels compared to Ren mice. In ex vivo microperfusion experiments, EP1-/- tubules show no response to PGE2 in the presence of AVP, whereas PGE2 inhibits AVP induced water reabsorption in WT mice. An increase in αENaC membrane accumulation due to EP1 gene ablation results in increased sodium reabsorption subsequently leading to a rise in BP. This contributes to the lack of salt sensitivity in EP1-/- mice. Overall, the EP1 receptor in the collecting duct represents a potential therapeutic target for the treatment of hypertension.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23423号 / 医博第4768号 / 新制||医||1053(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 湊谷 謙司, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / The  cardiac  voltage-­gated  sodium  channels  (Nav1.5)  play  a  specific  and   critical  role  in  regulating  cardiac  electrical  activity  by  initiating  and  propagating   action  potentials  in  the  heart.  The  association  between  Nav1.5  dysfunctions  and   generation  of  various  types  of  cardiac  arrhythmia  disease,  including  long-­QT3   and  Brugada  syndrome,  is  well  established.  Many  types  of  post-­translational   modifications  have  been  shown  to  regulate  Nav1.5  biophysical  properties,   including  phosphorylation,  glycosylation  and  ubiquitination.  However,  our   understanding  about  how  post-­translational  lipid  modification  affects  sodium   channel  function  and  cellular  excitability,  is  still  lacking.  The  goal  of  this   dissertation  is  to  characterize  Nav1.5  palmitoylation,  one  of  the  most  common   post-­translational  lipid  modification  and  its  role  in  regulating  Nav1.5  function  and   cardiac  excitability.     In  our  studies,  three  lines  of  biochemistry  evidence  were  shown  to  confirm   Nav1.5  palmitoylation  in  both  native  expression  background  and  heterologous   expression  system.  Moreover,  palmitoylation  of  Nav1.5  can  be  bidirectionally   regulated  using  2-­Br-­palmitate  and  palmitic  acid.  Our  results  also  demonstrated   that  enhanced  palmitoylation  in  both  cardiomyocytes  and  HEK293  cells   increases  sodium  channel  availability  and  late  sodium  current  activity,  leading  to   enhanced  cardiac  excitability  and  prolonged  action  potential  duration.  In  contrast,   blocking  palmitoylation  by  2-­Br-­palmitiate  increases  closed-­state  channel inactivation  and  reduces  myocyte  excitability.  Our  computer  simulation  results   confirmed  that  the  observed  modification  in  Nav1.5  gating  properties  by  protein   palmitoylation  are  adequate  for  the  alterations  in  cardiac  excitability.  Mutations  of   potential  palmitoylation  sites  predicted  by  CSS-­Palm  bioinformatics  tool  were   introduced  into  wild-­type  Nav1.5  constructs  using  site-­directed  mutagenesis.   Further  studies  revealed  four  cysteines  (C981,  C1176,  C1178,  C1179)  as   possible  Nav1.5  palmitoylation  sites.  In  particular,  a  mutation  of  one  of  these   sites(C981)  is  associated  with  cardiac  arrhythmia  disease.  Cysteine  to   phenylalanine  mutation  at  this  site  largely  enhances  of  channel  closed-­state   inactivation  and  ablates  sensitivity  to  depalmitoylation.  Therefore,  C981  might  be   the  most  important  site  that  regulates  Nav1.5  palmitoylation.  In  summary,  this   dissertation  research  identified  novel  post-­translational  modification  on  Nav1.5   and  revealed  important  details  behind  this  process.  Our  data  provides  new   insights  on  how  post-­translational  lipid  modification  alters  cardiomyocyte   excitability  and  its  potential  role  in  arrhythmogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / The voltage-gated sodium channels (VGSCs) are critical determinants of neuronal excitability. They set the threshold for action potential generation. The VGSC isoform Nav1.6 participates in various physiological processes and contributes to distinct pathological conditions, but how Nav1.6 function is differentially regulated in different cell types and subcellular locations is not clearly understood. Some VGSC isoforms are subject to S-palmitoylation and exhibit altered functional properties in different S-palmitoylation states. This dissertation investigates the role of S-palmitoylation in Nav1.6 regulation and explores the consequences of S-palmitoylation in modulating neuronal excitability in physiological and pathological conditions. The aims of this dissertation were to 1) provide biochemical and electrophysiological evidence of Nav1.6 regulation by S-palmitoylation and identify specific S-palmitoylation sites in Nav1.6 that are important for excitability modulation, 2) determine the biophysical consequences of epilepsy-associated mutations in Nav1.6 and employ computational models for excitability prediction and 3) test the modulating effects of S-palmitoylation on aberrant Nav1.6 activity incurred by epilepsy mutations. To address these aims, an acyl-biotin exchange assay was used for Spalmitoylation detection and whole-cell electrophysiology was used for channel characterization and excitability examination. The results demonstrate that 1) Nav1.6 is biochemically modified and functionally regulated by S-palmitoylation in an isoform- and site-specific manner and altered S-palmitoylation status of the channel results in substantial changes of neuronal excitability, 2) epilepsy associated Nav1.6 mutations affect different aspects of channel function, but may all converge to gain-of-function alterations with enhanced resurgent currents and increased neuronal excitability and 3) S-palmitoylation can target specific Nav1.6 properties and could possibly be used to rescue abnormal channel function in diseased conditions. Overall, this dissertation reveals S-palmitoylation as a new mechanism for Nav1.6 regulation. This knowledge is critical for understanding the potential role of S-palmitoylation in isoform-specific regulation for VGSCs and providing potential targets for the modulation of excitability disorders. / 2022-05-06