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A (Na+,K+)-ATPase é uma proteína integral da membrana plasmática que está sujeita a uma complexa regulação. Na fauna dos manguezais, dentre os crustáceos se destaca o caranguejo Cardisoma guanhumi (Latreille, 1825), um crustáceo decápode que desempenha um papel significativo na dinâmica deste ecossistema, considerado relevante recurso pesqueiro. Este estudo fornece o efeito das poliaminas, das enzimas do estresse oxidativo, da toxidade do amônio, e também investiga atividade K+-fosfatase e atividade (Na+,K+)-ATPase por estimulação sinérgica de K+/ NH4+ e NH4+/K+ na fração microsomal de brânquias do guaiamum. A atividade K+-fosfatase e a atividade (Na+,K+)-ATPase foram determinadas continuamente, a 25°C, em um espectrofotômetro Shimadzu U1800 equipado com células termostatizadas. Todos os experimentos foram feitos em duplicata utilizando-se pelo menos três preparações diferentes (N 3). A atividade PNFFase insensível à ouabaína representa 40% da atividade PNFFase total, e valor do KI foi de 370,0 18,5mol L-1. A atividade específica máxima estimada foi de 29,30 ± 1,46 nmol Pi min-1 mg-1 e o KM = 2,90 ± 0,14 mmol L-1. Por outro lado, a utilização do substrato fisiológico (ATP) permitiu a determinação de parâmetros cinéticos da atividade (Na+,K+)-ATPase em relação aos moduladores ATP, potássio, sódio, magnésio, amônio e, ouabaína. A atividade ATPase total na fração microsomal do tecido branquial de C. guanhumi recém-capturado (16 S) foi aproximadamente 166 nmol Pi min-1 mg-1 e uma atividade ATPase insensível à ouabaína de 26,55 nmol Pi min-1 mg-1, enquanto que aclimatado a 22 S a atividade ATPase total foi de 303,28 ± 15,16 nmol Pi min-1 mg-1 e a atividade insensível à ouabaína de 68,60 ± 3,43 nmol Pi min-1 mg-1. A (Na+,K+)-ATPase presente nessas duas preparações, não apresentam uma estimulação sinergística por K+ e NH4+. Houve alterações na afinidade da enzima para o ATP nas três diferentes concentrações de NH4Cl (120 mg/L; 240 mg/L; 360 mg/L) em comparação com o controle sem NH4Cl (KM= 0,1 ± 0,005 mmol L-1).Não foram observados efeitos significativos utilizando aminas biogênicas. Nossas análises mostraram também que as enzimas do estresse oxidativo estão atuando nestas diferentes preparações para combater os oxirradicais. Análise por Western blotting com anticorpo monoclonal revelou a presença de uma banda correspondente a subunidade da (Na+,K+)-ATPase com massa molecular 110 kDa. A imunolocalização mostrou que a subunidade da (Na+,K+)-ATPase encontra-se predominantemente distribuída por todo o citoplasma das células pilares branquiais, incluindo a região apical abaixo da cutícula. Identificamos o gene constitutivo da sequência parcial de nucleotídeos do cDNA da proteína ribosomal L10 (PRL10) das brânquias deCardisoma guanhumi. O estudo demonstrou que a (Na+,K+)-ATPase constitui um importante regulador da osmorregulação nesta espécie, contribuindo para um melhor entendimento dos papéis exercidos por essa enzima nos processos de osmorregulação e excreção de amônia nos crustáceos. / The (Na+,K+)-ATPase is an integral plasma membrane protein that is subject to complex regulation. In the mangrove fauna, the crustaceans include Cardisoma guanhumi crab (Latreille, 1825), a decapod crustacean that plays a significant role in the dynamics of this ecosystem, considered a relevant fishing resource. This study provides the effect of polyamines, oxidative stress enzymes, ammonium toxicity, and also investigates K+-phosphatase activity and (Na+,K+)-ATPase activity by synergistic K+/NH4+ and NH4+/ K+ stimulation in the microsomal fraction of guaiamum gills. The K+-phosphatase activity and (Na+,K+)-ATPase activity were determined continuously at 25°C on a Shimadzu U1800 spectrophotometer equipped with thermostated cells. All experiments were done in duplicate using at least three different preparations (N 3). The PNFFase activity insensitive to ouabain represents 40% of the total PNFFase activity, and KI value was 370,0 18,5mol L-1. The maximum specific activity estimated was 29.30 ± 1.46 nmol Pi min-1 mg-1 and KM = 2.90 ± 0.14 mmol L-1. On the other hand, the use of the physiological substrate (ATP) allowed the determination of kinetic parameters of the activity (Na+,K+)-ATPase in relation to the modulators ATP, potassium, sodium, magnesium, ammonium and ouabain.The total ATPase activity in the microsomal fraction of freshly caught C. guanhumi (16 S) gill tissue was approximately 166 nmol Pi min-1 mg-1 and a 26.55 nmol Pi min-1 mg-1 ouabain ATPase activity, while acclimated at 22 S the total ATPase activity was 303.28 ± 15.16 nmol Pi min-1 mg-1 and the ouabain insensitive activity of 68.60 ± 3.43 nmol Pi min-1 mg-1. The (Na+,K+)-ATPase present in these two preparations, do not present a synergistic stimulation by K+ and NH4+. There were changes in the enzyme affinity for ATP at the three different concentrations of NH4Cl (120 mg / L, 240 mg / L, 360 mg / L) compared to the control without NH4Cl (KM = 0.1 ± 0.005 mmol L-1). No significant effects were observed using biogenic amines. No significant effects were observed using biogenic amines. Our analyzes have also shown that oxidative stress enzymes are acting in these different preparations to combat oxirradicals. Analysis by Western blotting with monoclonal antibody revealed the presence of a band corresponding to sub subunit of (Na+,K+)-ATPase with molecular mass 110 kDa. Immunolocalization showed that the (Na+,K+)-ATPase sub subunit is predominantly distributed throughout the cytoplasm of the gill pillars, including the apical region below the cuticle. We identified the constitutive gene of the nucleotide partial sequence of the cDNA of ribosomal protein L10 (PRL10) of the gills of Cardisoma guanhumi. The study demonstrated that (Na+,K+)-ATPase is an important regulator of osmoregulation in this species, contributing to a better understanding of the roles played by this enzyme in the processes of osmoregulation and excretion of ammonia in crustaceans.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Toledo, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Zi-Jian Xie. Includes abstract. Document formatted into pages: iii, 156 p. Title from title page of PDF document. Bibliography: pages 64-67, 97-100, 116-117, 125-155.

Made available in DSpace on 2014-06-12T15:49:36Z (GMT). No. of bitstreams: 2 arquivo1526_1.pdf: 3081334 bytes, checksum: 9e2891701f044d8665f61bd792f09dae (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Uma dieta equilibrada deve fornecer os nutrientes necessários, inclusive os ácidos graxos essenciais (AGE) ao animal, durante toda a vida. O presente trabalho objetivou estudar aspectos funcionais, estruturais e moleculares renais em ratos no crescimento e adultos, em dieta carente em AGE (CAGE) desde o desmame. Os estudos foram realizados em gaiolas metabólicas. Os resultados, expressos como médias ± DP, foram corrigidos por 100g de peso e considerados significativos quando p0,05. Apesar de ingestão de sólido similar, os ratos CAGE pesaram menos desde a 6ª semana até adultos. A ingestão de água e a diurese foram similares entre os grupos em todas as semanas, exceto pela maior diurese dos CAGE na 7ª semana. As excreções de proteína e creatinina não diferiram entre os grupos, no crescimento e adultos. Os animais CAGE apresentaram caliurese aumentada na 7ª (616,5±135,2 vs 414,4±140,5 μmol/100g/24h) e na 8ª semana (554,7±135,1 vs 367,1±93,2 μmol/100g/24h), nesta acompanhada de maior natriurese (351,1±90,7 vs 277,4± 96,1 μmol/100g/24h), que ocorreu, também, na 13ª semana (256,8±54 vs 203,4±52,1 μmol/100g/24h) quando houve maior consumo alimentar. Na restrição hídrica de 12h, tanto os animais controles como os CAGE concentraram urina, embora estes com natriurese (223,6±16 vs 166,2±37,7 μmol/100g/12h) e caliurese (169,9±16,7 vs 126,2±22 μmol/100g/12h). Nos animais adultos, a CAGE não alterou a filtração glomerular (342,8±121,8 vs 382,9±113,1 μl/min/100g), a função tubular proximal (40±20 vs 42,5±20,9 μl/min/100g), o manuseio tubular de Na+ e a estrutura renal, esta tanto à histologia como à morfometria. Entretanto, a CAGE aumentou a atividade da Na+, K+-ATPAse e diminuiu a da Na+-ATPase, que não foi estimulada pela angiotensina II nos animais adultos. A ingestão da CAGE desde o desmame retardou o crescimento, aumentou episodicamente a excreção de Na+ e K+, sem afetar a estrutura e a função no adulto, embora tenham ocorrido alterações da atividade ATPásica no túbulo proximal renal

A (Na+,K+)-ATPase é uma proteína integral da membrana plasmática que está sujeita a uma complexa regulação. Na fauna dos manguezais, dentre os crustáceos se destaca o caranguejo Cardisoma guanhumi (Latreille, 1825), um crustáceo decápode que desempenha um papel significativo na dinâmica deste ecossistema, considerado relevante recurso pesqueiro. Este estudo fornece o efeito das poliaminas, das enzimas do estresse oxidativo, da toxidade do amônio, e também investiga atividade K+-fosfatase e atividade (Na+,K+)-ATPase por estimulação sinérgica de K+/ NH4+ e NH4+/K+ na fração microsomal de brânquias do guaiamum. A atividade K+-fosfatase e a atividade (Na+,K+)-ATPase foram determinadas continuamente, a 25°C, em um espectrofotômetro Shimadzu U1800 equipado com células termostatizadas. Todos os experimentos foram feitos em duplicata utilizando-se pelo menos três preparações diferentes (N 3). A atividade PNFFase insensível à ouabaína representa 40% da atividade PNFFase total, e valor do KI foi de 370,0 18,5mol L-1. A atividade específica máxima estimada foi de 29,30 ± 1,46 nmol Pi min-1 mg-1 e o KM = 2,90 ± 0,14 mmol L-1. Por outro lado, a utilização do substrato fisiológico (ATP) permitiu a determinação de parâmetros cinéticos da atividade (Na+,K+)-ATPase em relação aos moduladores ATP, potássio, sódio, magnésio, amônio e, ouabaína. A atividade ATPase total na fração microsomal do tecido branquial de C. guanhumi recém-capturado (16 S) foi aproximadamente 166 nmol Pi min-1 mg-1 e uma atividade ATPase insensível à ouabaína de 26,55 nmol Pi min-1 mg-1, enquanto que aclimatado a 22 S a atividade ATPase total foi de 303,28 ± 15,16 nmol Pi min-1 mg-1 e a atividade insensível à ouabaína de 68,60 ± 3,43 nmol Pi min-1 mg-1. A (Na+,K+)-ATPase presente nessas duas preparações, não apresentam uma estimulação sinergística por K+ e NH4+. Houve alterações na afinidade da enzima para o ATP nas três diferentes concentrações de NH4Cl (120 mg/L; 240 mg/L; 360 mg/L) em comparação com o controle sem NH4Cl (KM= 0,1 ± 0,005 mmol L-1).Não foram observados efeitos significativos utilizando aminas biogênicas. Nossas análises mostraram também que as enzimas do estresse oxidativo estão atuando nestas diferentes preparações para combater os oxirradicais. Análise por Western blotting com anticorpo monoclonal revelou a presença de uma banda correspondente a subunidade da (Na+,K+)-ATPase com massa molecular 110 kDa. A imunolocalização mostrou que a subunidade da (Na+,K+)-ATPase encontra-se predominantemente distribuída por todo o citoplasma das células pilares branquiais, incluindo a região apical abaixo da cutícula. Identificamos o gene constitutivo da sequência parcial de nucleotídeos do cDNA da proteína ribosomal L10 (PRL10) das brânquias deCardisoma guanhumi. O estudo demonstrou que a (Na+,K+)-ATPase constitui um importante regulador da osmorregulação nesta espécie, contribuindo para um melhor entendimento dos papéis exercidos por essa enzima nos processos de osmorregulação e excreção de amônia nos crustáceos. / The (Na+,K+)-ATPase is an integral plasma membrane protein that is subject to complex regulation. In the mangrove fauna, the crustaceans include Cardisoma guanhumi crab (Latreille, 1825), a decapod crustacean that plays a significant role in the dynamics of this ecosystem, considered a relevant fishing resource. This study provides the effect of polyamines, oxidative stress enzymes, ammonium toxicity, and also investigates K+-phosphatase activity and (Na+,K+)-ATPase activity by synergistic K+/NH4+ and NH4+/ K+ stimulation in the microsomal fraction of guaiamum gills. The K+-phosphatase activity and (Na+,K+)-ATPase activity were determined continuously at 25°C on a Shimadzu U1800 spectrophotometer equipped with thermostated cells. All experiments were done in duplicate using at least three different preparations (N 3). The PNFFase activity insensitive to ouabain represents 40% of the total PNFFase activity, and KI value was 370,0 18,5mol L-1. The maximum specific activity estimated was 29.30 ± 1.46 nmol Pi min-1 mg-1 and KM = 2.90 ± 0.14 mmol L-1. On the other hand, the use of the physiological substrate (ATP) allowed the determination of kinetic parameters of the activity (Na+,K+)-ATPase in relation to the modulators ATP, potassium, sodium, magnesium, ammonium and ouabain.The total ATPase activity in the microsomal fraction of freshly caught C. guanhumi (16 S) gill tissue was approximately 166 nmol Pi min-1 mg-1 and a 26.55 nmol Pi min-1 mg-1 ouabain ATPase activity, while acclimated at 22 S the total ATPase activity was 303.28 ± 15.16 nmol Pi min-1 mg-1 and the ouabain insensitive activity of 68.60 ± 3.43 nmol Pi min-1 mg-1. The (Na+,K+)-ATPase present in these two preparations, do not present a synergistic stimulation by K+ and NH4+. There were changes in the enzyme affinity for ATP at the three different concentrations of NH4Cl (120 mg / L, 240 mg / L, 360 mg / L) compared to the control without NH4Cl (KM = 0.1 ± 0.005 mmol L-1). No significant effects were observed using biogenic amines. No significant effects were observed using biogenic amines. Our analyzes have also shown that oxidative stress enzymes are acting in these different preparations to combat oxirradicals. Analysis by Western blotting with monoclonal antibody revealed the presence of a band corresponding to sub subunit of (Na+,K+)-ATPase with molecular mass 110 kDa. Immunolocalization showed that the (Na+,K+)-ATPase sub subunit is predominantly distributed throughout the cytoplasm of the gill pillars, including the apical region below the cuticle. We identified the constitutive gene of the nucleotide partial sequence of the cDNA of ribosomal protein L10 (PRL10) of the gills of Cardisoma guanhumi. The study demonstrated that (Na+,K+)-ATPase is an important regulator of osmoregulation in this species, contributing to a better understanding of the roles played by this enzyme in the processes of osmoregulation and excretion of ammonia in crustaceans.

Chronic hypoxia is a state of oxygen limitation that is common in many aquatic and terrestrial environments. Metabolic suppression is an essential strategy that is used by hypoxia-tolerant champions such as goldfish and naked mole-rats to cope with prolonged low oxygen. This thesis examines the physiological processes used by goldfish and naked mole-rats to survive in low oxygen environments. It proposes a novel mechanism - the remodeling of membrane lipids - to reduce ATP use and production. Temperature (homeoviscous adaptation), diet (natural doping in migrant birds) and body mass (membrane pacemaker of metabolism) have an impact on the lipid composition of membranes that, in turn, modulates metabolism. In chapters 2 and 3 of this thesis, I demonstrate that vertebrate champions of hypoxia tolerance undergo extensive changes in membrane lipid composition upon in vivo exposure to low oxygen. These changes and those observed in hibernating mammals can promote the downregulation of Na⁺/K⁺-ATPase (major ATP consumers), mitochondrial respiration capacity [OXPHOS (phosphorylating conditions), proton leak (non-phosphorylating conditions), cytochrome c oxidase], and energy metabolism (β-oxidation and glycolysis) as discussed in chapters 3 and 4. A common membrane signal regulating the joint inhibition of ion pumps and channels could be an exquisite way to preserve the balance between ATP supply and demand in hypometabolic states. In chapter 5, I show that the reduction in ATP turnover is also orchestrated by mechanisms that involve post-translational and post-transcriptional modifications and epigenetic changes. Membrane remodeling, together with these more traditional molecular mechanisms, could work in concert to cause metabolic suppression.

The Na/K pumps are essential for living system and widely expressed in all eukaryotic cell membranes. By actively transporting sodium ions out of and potassium ions into the plasma membrane, Na/K pumps creates both an electrical and a chemical gradient across the plasma membrane, which are crucial for maintaining membrane potential, cell volume, and secondary active transporting of other solutes, etc. Previously, oscillating electric field with a frequency close to the mean physiological turnover rate was used to synchronize and modulate the Na/K pump molecules. Results showed that the turnover rate of Na/K pumps can be accelerated by folds. However, this what we called first generation synchronization modulation (SM) technique can only synchronize sodium and potassium translocations into their corresponding half cycles. The detailed location of each sodium extrusion and potassium intrusion can not be determined. As a result, the synchronized pumps were uniformly distributed, generating steady-state macroscopic currents. Based on these studies, Dr.Chen developed a new generation synchronization modulation technique. The waveform of original SM by adding an overshoot pulse at the end of each half cycle. This overshoot pulse has a function of energy barrier which will force all of the Na/K pumps into the same state in the pumping cycle until the membrane polarity change. As a result, Na/K pump molecules are not only synchronized into half cycles of oscillating electric field, but individual steps of the pumping cycle. Accordingly, transient pump currents or so called 'pre-steady state' pump currents are generated, from which some detailed information abut the mechanism of Na/K pumps can be dissected. In this dissertation, we firstly characterized the synchronized pump currents by modified SM. The results showed that transient currents were induced at the beginning of each half cycle as expected. The ratio between positive and negative transient currents was close to 3:2, stoichiometric number of Na/K pump. Moreover, the transient currents were significantly reduced in the presence of ouabain in a time dependent manner. In addition, by gradually increasing the frequency of SM electric field in a step-wise fashion, the synchronized pump current can be modulated to the corresponding level. Next,we utilized this technique to study some detailed mechanisms of Na/K pump, including single channel configuration in transmembrane domain and extracellular D2O effect on the turnover rate. Lastly, we extended our study to applications of this new technique and found that the modified Synchronization Modulation technique can significantly hyperpolarize the membrane potential of skeletal muscle fiber in both physiological and high potasssium conditions. During intensive exercise, the interstitial potassium ions are accumulated and temporarily reach a high level, which will attenuate the contraction force and induce muscle fatigue. Na/K pumps are crucial in the maintenance of skeletal muscle excitability and contractility by restoring the Na and K concentration gradients. By accelerating the turnover rate of Na/K pumps, SM can efficiently re-establish the membrane potential and enhance skeletal muscle contractivity, which unleashes its potential in improving certain pathological conditions, such as exercise-induced hyperkalemia.

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