Rheumatoid arthritis (RA) is a chronic inflammatory disease mediated, in part, by pro-inflammatory cytokines such a sI L- I P, TNFa andI L-6. Many factors may contribute to cytokine imbalances in this disease, for example, biochemical modulation of PBMCsa ndt heir membranes A. key membrane proteini s the Na/KATPase( sodium pump) responsible for ionic homeostasis Sodiump ump activity on rheumatoid PBMCsw as found to be markedly depressed when compared with healthy control cells possibly through an oxidative mechanism. Inhibition of the sodium pump by a cardiac glycoside inhibitor, ouabain, transiently upregulated[N a'ji levels and rapidly induced IL-10 and TNFa mRNA and protein in human PBMCs. In contrast, IL-6 production was significantly depressed. The sodium ionophore, monensin, caused a similar Na-dependent cytokine response to that of ouabain. This cytokine profile however, was reversed when studying rheumatoids ynovial fibroblasts where ouabain induced I L-6; IL- I and TNFa, on the other hand, were not expressed. An elevation in intracellulars odiumc an causea secondary rise in intracellular calcium levels through the action of a Na/Ca2+ exchanger. In studies using the calcium ionophore, A23187, it was observed that an elevation in [Ca 2+]i brought aboutt he induction of IL- IP and TNF(xi n PBMCs with a corresponding repression of IL-6 production. The data obtained in this study suggest that impaired N a/K-ATPase activity in rheumatoid cells, through elevations in intracellular cation levels, might help promote over-production of IL- IP and TNF(x by monocytes and IL-6 by synovial fibroblasts. This pattern of cytokine production conforms to that observed in rheumatoid synovial tissue in situ, thus supporting a role for this biochemical defect in contributing to the perpetuation of the chronic inflammatory state.

Digoxin has been known as a treatment for chronic heart failure for over 200 years. Its effect on the heart itself has been extensively studied and its inotropic effect well established. The inotropic effect of digoxin is the result of its inhibition of the membrane sodium pump or Na+/K+-ATPase, which plays an important role in maintaining the resting membrane potential across the plasma membrane through constantly pumping Na+ and K+ across the plasma membrane. Na+/K+-ATPase is not found exclusively in heart muscle. It is also found extensively throughout the brain. As digoxin is the drug of choice for pregnant woman with chronic heart failure, this study aimed to examine how digoxin affects brain development and neurons in culture. The well established chicken embryo animal model was used in this study. To probe for deviations from normal brain development, chicken embryos were exposed in ovo. Brains were examined using both transmission and scanning electron microscopy. Microscopy indicated significant damage to the neurons, specifically membranes and mitochondria, as well as cellular death by means of aponecrosis. An unexpected result was premature myelinogenesis in the brain. Chick embryo neurons (CEN) were exposed to digoxin in vitro and cell viability was assessed by performing crystal violet (CV) assays. Results showed that cell number increased over time. This is however, impossible as CEN are non-dividing cells and results were therefore interpreted as an increase in protein synthesis over time, correlating with the myelinogenesis results seen with electron microscopy. To assess membrane integrity, fluorescence microscopy was performed using propidium iodide as stain. Results from this experiment showed a sharp increase in propidium iodide uptake in exposed cells indicative of the membrane damage caused by digoxin. These results also correlated with the aponecrosis seen with electron microscopy, as the nuclei indicated apoptosis while propidium iodide is normally only absorbed by cells undergoing necrosis. Finally, a literature search was conducted to shed some light on the role that digoxin plays in serotonin production and levels in the brain. From the literature it seems that digoxin could increase serotonin production and elevate serotonin levels in the brain, which may influence normal brain development and may therefore play a role in myelinogenesis in the brain. / Dissertation (MSc (Anatomy))--University of Pretoria, 2008. / Anatomy / unrestricted

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Objective: Reductions in sodium pump (SP) abundance can give rise to increases in contractile force in uterine and vascular smooth muscle as well as an increased secretion in secretory cells, including potentially those of the placenta. To determine whether the mouse might serve as a model for human pregnancy in terms of the SP and to determine whether changes in SP abundance anticipate or follow labor, we studied pregnant mice over the final trimester of their pregnancy. Study Design: C57Bl6 dams (n=46) were bred and studied during their pregnancy. Animals (n=4) were sacrificed at specific gestational time points. Other mice had labor induced with LPS on Gestational day 15 and were then studied at specific time points after induction. Specimens were studied for mRNA abundance as well as protein abundance using methods such as Real time RT-PCR and Western blot analysis. Data were analyzed by ANOVA with post hoc Duncan's pair-wise comparisons. Results: Levels of uterine SP α3 isoform mRNA were most abundant on day 14 near the beginning of the third trimester. There was a significant fall in SP &alpha3 mRNA abundance by day 18 with a slightly lower level on the day of birth but an increased SP α3 mRNA abundance by one day post partum. Contrary to the uterus, SP α3 mRNA levels in the placenta increased over the last trimester, from day 14 to the day of birth. Western blot analysis on the two tissues demonstrated a somewhat similar pattern. In the LPS studies of uterus and placenta, the SP α3 isoform protein abundance appeared to fall when compared to the 2 hour time point. Those animals which were injected with a vehicle control showed very little change in SP α3 abundance after injection. While protein levels were reduced, there was no significant reduction in mRNA for all specimens. Conclusion: Uterine SP α3 isoform protein expression fell late in mouse pregnancy but prior to labor and appeared to be mediated by reductions in its mRNA. These reductions paralleled changes observed in term pregnant women. Such reductions would increase the sensitivity of the uterus to agents causing contraction but may directly increase the force, duration and frequency of contractions. Placental SP α3 isoform protein expression had no significant change over the final trimester. However, unlike uterine protein, the placental protein may not be mediated by its mRNA. Reductions in SP α3 protein abundance were also seen in preterm labor produced by LPS induction. These changes may not be mediated by mRNA. Taken together, changes in the SP α3 isoform may represent a fundamental mechanism in the initiation and/ or progression of term labor and in preterm in mouse and potentially in human.

The sodium pump (SP or Na+,K+-ATPase) is a membrane embedded protein complex that pumps 3 sodium ions out and 2 potassium ions into the cell per cycle and in so doing creates a cell membrane electrochemical potential. The membrane potential is critical for any functional cell. In the vasculature, reduction in the voltage potential causes vascular smooth muscle contraction and a narrowing of blood vessels (vasoconstriction) which can lead to increased blood pressure (hypertension). Substantial research over the past several decades has provided a vast amount of research on SP inhibitors, sometimes called endogenous digitalis-like factors (EDLF). Increased levels of these factors have been implicated in many hypertensive disorders including preeclampsia (PE), a life-threatening complication of pregnancy. It has been demonstrated that EDLF might be a causative factor in the pathophysiology of hypertension in PE. In order to elucidate EDLF production and regulation in PE, We developed a radioimmunoassay (RIA) measuring EDLF that could be applied to serum from pregnant women, placental homogenate and placental tissue culture. This assay employs Digibind, a commercially available Fab fragment derived from polyclonal antidigoxin antibodies that cross reacts with EDLF, as the primary antibody. Using Digibind RIA, we demonstrated that placenta is a source of EDLF production and regulation. Moreover, the identification of an inhibitor, ketoconazole and a substrate, 17-hydroxyprogesterone of the synthetic pathway of EDLF in placenta proved that this pathway shares steps with the steroid synthetic pathway. Some potential regulatory agents which have elevated levels in PE or be associated in PE and thus are thought to mediate PE, such as hydrogen peroxide, tumor necrosis factor-α (TNF-α) and hypoxia have also been demonstrated to be stimuli of EDLF production in placenta. These findings are helpful to the further study on EDLF synthesis and regulation in placenta. Once we elucidate the mechanisms, it could be easier to provide deeper insights into the pathogenesis of PE and subsequently develop earlier diagnosis and effective prevention of or therapeutic approaches to PE.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Na+,K+-ATPase is ubiquitously expressed in the plasma membrane of all animal cells where serves as the principal regulator of intracellular ion homeostasis. Na+,K+-ATPase activity is activated by Na+ and K+ and current evidence indicates that total Na+,K+-ATPase activity is, in general, inhibited by anions. However, the effect of pharmacologically-induced Cl- flux on α1- and α2/3-subunit containing Na+,K+-ATPase activity is not established. In this study we investigated the effect of diazepam, a GABAA receptor positive allosteric modulator, on α1- and α2/3-subunit containing Na+,K+-ATPase activity. Hippocampal and cortical slices were incubated with diazepam (0, 0.05, 0.15 or 0.5 μM) and/or flumazenil (0, 0.005, 0.015, 0.05, 0.15, 0.5 or 1.5 μM) for 10 minutes. After incubation the slices were homogenized and α1 and α2/3 Na+,K+-ATPase activity were assayed using ouabain 3 μM (that inhibits α2/3-subunit containing Na+,K+-ATPase) and 4 mM (that inhibits both isoforms). Diazepam caused a 50% decrease of α2/3-subunit containing Na+,K+-ATPase activity in the hippocampus, but did not alter enzyme activity in the entorhinal cortex. The effect of diazepam was prevented by flumazenil, indicating that the decrease of Na+,K+-ATPase was involved GABAA receptors. Furthermore, a low chloride medium abolished the diazepam-induced decrease of Na+,K+-ATPase activity. Our data suggests that Na+,K+-ATPase in the hippocampus is sensitive to the pharmacological effects of a benzodiazepine by GABAA receptor-mediated mechanisms. Keywords: sodium pump. GABAA receptor. diazepam. flumazenil. chloride ion. hippocampus. entorhinal córtex. / A enzima Na+,K+-ATPase, ou bomba de sódio, é expressa na membrana plasmática de células eucarióticas, onde atua como principal regulador da homeostase iônica intracelular. A enzima Na+,K+-ATPase é ativada pelos íons Na+ and K+ e evidências indicam que a atividade total da enzima Na+,K+-ATPase é inibida por ânions. Entretanto, o efeito do fluxo de cloreto induzido farmacologicamente sobre a atividade das subunidades α1 e α2/3 da enzima Na+,K+-ATPase ainda não foi investigado. Neste estudo, nós investigamos o efeito do diazepam, um modulador alostérico positivo do receptor GABAA na atividade específica das subunidades α1 e α2/3 da Na+,K+-ATPase. Fatias de hipocampo e de córtex entorrinal foram incubadas com diazepam (0; 0,05; 0,15 ou 0,5 μM) e/ou flumazenil (0; 0,005, 0,015; 0,05; 0,15; 0,5 ou 1,5 μM) por 10 minutos. Após a incubação, as fatias foram homogeneizadas e a atividade das subunidades α1 e α2/3 da enzima Na+,K+-ATPase foi determinada. Diazepam diminuiu 50% a atividade da subunidade α2/3 da Na+,K+-ATPase no hipocampo, mas não alterou a atividade da enzima em córtex entorrinal. O efeito do diazepam foi prevenido por flumazenil, indicando que a diminuição da atividade da Na+,K+-ATPase envolveu a ativação dos receptores GABAA. Além disso, a baixa concentração de cloreto no meio de incubação aboliu a diminuição da atividade enzimática induzida por diazepam. Nossos dados sugerem que a enzima Na+,K+-ATPase no hipocampo é sensível a efeitos farmacológicos dos benzodiazepínicos por meio de mecanismos ativados por receptores GABAérgicos.

Mikrobielle Rhodopsine sind Membranproteine, die Lichtsensitivität mit sensorischer, enzymatischer oder ionenleitender Funktion in einem Protein vereinen. Ihre strukturelle Kompaktheit macht lichtgetriebene Ionenpumpen und lichtgesteuerte Kanalrhodopsine (ChRs) zu weit verbreiteten optogenetischen Werkzeugen in der biologischen Forschung. Die Entdeckung und Entwicklung weiterer Ionenselektivitäten eröffnet neue Möglichkeiten der optogenetischen Manipulation des Membranpotentials elektrogener Zellen wie Neuronen. Seit langem wird an lichtgesteuerten K+-selektiven Ionenkanälen geforscht, um biologisch kompatible inhibierende Proteine zu kreieren. Eine Punktmutation im Rhodopsin KR2, einer lichtgesteuerten Na+-Pumpe aus dem Meeresbakterium Dokdonia eikasta, induzierte K+-selektive Leckströme. In dieser Studie wurden die Limitationen dieser KR2-R109Q-Mutante mithilfe elektrophysiologischer Methoden experimentell charakterisiert, begleitet von computergestützten pKa-Vorhersagen und MD-Simulationen. Eine Mutationsstudie offenbarte die molekularen Ursachen für die nachteilige pH-Abhängigkeit und die verbleibende Na+-Pumpaktivität. Durch Kombination von Schlüsselmutationen im Extrazellularraum des Proteins konnten diese Einschränkungen reduziert werden und größere K+-Leitfähigkeit unter physiologischen Bedingungen erzielt werden. Währenddessen wurden die ersten K+-selektiven ChRs unter physiologischen Bedingungen entdeckt, die KCRs. HcKCR1 aus der stramenopilen Alge Hyphochytrium catenoides und Mutanten der Ionenleitpore wurden elektrisch charakterisiert, unterstützt durch strukturelle Vorhersagen. Ein neuartiger hydrophober Selektivitätsfilter wurde identifiziert und seine Konservierung in verwandten Stramenopilen-ChRs nachgewiesen. WiChR aus Wobblia lunata zeigte hierbei eine beispiellose K+-Permeabilität und erreichte in Herzmuskelzellen und Neuronen hohe Eignung in Ein- und Zweiphotoneninhibition bei niedriger Lichtintensität und geringer Gewebeerwärmung. / Microbial rhodopsins are light-sensitive membrane proteins found in all domains of life. They combine photosensitivity with sensory, enzymatic or ion-translocating functions. Their structural simplicity makes light-driven ion pumps and light-gated channelrhodopsins (ChRs) valuable optogenetic tools for controlling cellular activity with light. Discovering and engineering new forms of ion selectivity expands possibilities for manipulating the membrane potential of electrogenic cells like neurons. Light-sensitive K+-selective ChRs have been highly anticipated as inhibitory optogenetic tools. A point mutation in the central gate of KR2, a light-driven Na+-pump rhodopsin from the marine bacterium Dokdonia eikasta, resulted in K+-selective leak photocurrents. This study experimentally characterized the main limitations of this KR2-R109Q variant using two-electrode and whole-cell voltage-clamp methods, supported by computational pKa prediction and classical MD simulations. An extensive mutational study revealed the molecular cause for the detrimental pH-sensitivity and residual Na+-pumping activity. Combining key mutations in the extracellular part of the protein reduced these limitations, yielding larger K+-selective photocurrents under physiological conditions. During this study, a novel ChR family with superior properties, Kalium ChRs (KCRs) was discovered, representing the first K+-selective ChRs under physiological conditions. HcKCR1 from the stramenopile alga Hyphochytrium catenoides and mutants of its putative ion conduction pore were electrically characterized in WCVC experiments, supported by structural predictions. A novel type of hydrophobic selectivity filter was identified and found to be conserved in related stramenopile ChRs. Among them, WiChR from Wobblia lunata exhibited an unmatched preference for K+ over Na+ and favorable performance in cardiac myocytes and neurons, allowing single- and two-photon inhibition at low irradiance and reduced tissue heating.

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