The collecting duct of the distal nephron marks the final location where adjustments to Na+ excretion can be made, therefore determining the final concentration of Na+ conserved in the extracellular fluid which plays a role in governing overall blood volume and pressure. This transport of Na+ is subject to hormonal regulation but the signalling pathways underpinning this regulation however, are not fully understood. In this thesis the signalling pathways allowing both basal and insulin-stimulated Na+ absorption were explored in the murine collecting duct cell line, mpkCCDcl4. The effects of two insulin-sensitizing drugs, TZDs, on ENaC-mediated Na+ transport were investigated and the signalling pathways underlying two other hormonal regulators of ENaC, dexamethasone and vasopressin, were also examined. Unstimulated monolayers of mpkCCDcl4 cells generated spontaneous Na+ absorption which was quantified by measuring equivalent short circuit current (Ieq). Selective inhibition of PI3-kinase, mTORC2 and SGK1 left ~80 % of the current intact, indicating these signalling molecules are not required for basal Na+ transport. Acute addition of insulin stimulated Ieq and this occurred with a concomitant increase in mTORC2, SGK1 and Akt activity. Inhibition of PI3-kinase abolished the insulin-stimulated response as well as phosphorylation of downstream substrates, indicating a crucial role of PI3-kinase. Inhibition of mTORC1 with rapamycin did not alter basal or insulin-stimulated Na+ transport. The mTOR inhibitors TORIN1 and PP242 could therefore be used to evaluate the role of mTORC2. These inhibitors greatly reduced insulin-stimulated ENaC-mediated Na+ transport and also abolished SGK1 and mTORC2 activity, indicating a novel role of mTORC2. An inhibitor of SGK1, GSK650394A abolished insulin-stimulated Na+ transport and specifically inhibited SGK1 acitivty demonstrating the importance of SGK1 in insulin signalling. The inhibitor Akti-1/2 also abolished insulin-mediated Na+ transport but this compound inhibited both Akt and SGK1 activity. The TZDs pioglitazone and rosiglitazone did not alter basal or insulin-stimulated Na+ transport and had no effect on SGK1 activity indicating these drugs do not alter Na+ absorption in this cell line. Dexamethasone stimulated ENaC-mediated Na+ transport in a similar manner to insulin and this could be blocked with rapamycin. This drug did not alter phosphorylation of NDRG1 indicating that dexamethasone stimulates Na+ transport in an mTORC1-dependent manner but without altering SGK1 activity. Arginine vasopressin also stimulated Ieq but did so by reducing Rt with an associated depolarisation of Vt. Ieq could be blocked with amiloride and vasopressin-stimulated Ieq was insensitive to TORIN1 and PP242. Vasopressin suppressed SGK1 phosphorylation of NDRG1 but did stimulate protein kinase A (PKA) activity. Therefore vasopressin stimulates Ieq via a PKA-dependent but mTOR- and SGK1-independent pathway.

Respiratory distress syndrome and pulmonary oedema often result in poor health and in the worst case scenario, death. Several studies have proposed that the eventual resolution of these dangerous conditions is due to active sodium reabsorption through the epithelial sodium channel (ENaC), which is crucial for lung fluid clearance. Although clinical prognosis can be improved by using glucocorticoid hormones to augment the ENaC-dependent removal of liquid from the lungs, we still require a better understanding of the underlying mechanism in order to improve treatments in the future. This thesis, therefore explores the role of serum / glucocorticoid-inducible protein kinase 1 (SGK1) and protein kinase A (PKA) in the responses of hormone-stimulated H441 human airway cells. Dexamethasone, a synthetic glucocorticoid hormone, is thought to evoke expression of the gene encoding SGK1 and, to become catalytically active, this gene product must then be phosphorylated via TORC2 and PDK1, protein kinases activated via the P13-kinase pathway. Once activated, SGK1 appears to exert control over the surface abundance of ENaC subunits by phosphorylation, and thus inactivating, a ubiquitin ligase (Nedd4-2), that normally mediate the withdrawal of ENaC subunits from the plasma membrane. Protein kinase A (PKA) may contribute to this control mechanism by also phosphorylating Nedd4-2. In order to clarify the way in which these pathways contribute to glucocorticoid-induced lung liquid clearance, the present thesis has explored the effects of dexamethasone and / or PKA activation upon the overall / surface expression of ENaC subunits, the activities of SGK1 and PKA and the phosphorylation status of physiologically-important residues within Nedd4-2 itself.

Light entering the eye is transformed by the retina into electrical signals. Extensive processing takes place in the retina before these signals are transmitted to the brain. Beginning in the outer retina, light-evoked electrical signals are distributed into parallel pathways specialized for different visual tasks, such as the detection of dark vs. bright ambient light, the onset or offset of light, and the direction of stimulus motion. Pathway diversity is a consequence of cell type diversity, differential cell connectivity, synapse organization, receptor expression, or any combination thereof. Cell connectivity itself can be accomplished through excitatory or inhibitory chemical synapses, or electrical coupling via gap junctions. Gap junctions are further specialized based on the expression of different connexin subunit isoforms. In aggregate, this diversity gives rise to ganglion cells with highly specialized functions, including ON and/or OFF responses, contrast-tuning and direction-selectivity (DS). The directionally-selective circuit, a circuit specialized for the encoding of stimulus motion, makes use of many of these circuit specializations. Bipolar cells, in response to glutamate release from cone photoreceptors, provide highly-sensitive glutamatergic input to amacrine cells and DS ganglion cells (DSGCs) in this circuit, while amacrine cells provide cholinergic and directionally-tuned GABAergic input to DSGCs. One population of DSGCs also transmit signals laterally to one another via gap junctions. Thus numerous specializations in bipolar cells, amacrine cells and ganglion cells endow DSGCs with their unique encoding abilities. In Chapters 2 and 3 of this dissertation I focus on synchronized firing between gap junction-coupled DSGCs. sDSGCs exhibit fine-scale correlations, with action potentials in an sDSGC more likely within ~2ms of action potential firing in a coupled neighbour. I first characterize electrical coupling of DSGCs through the identification of the molecular composition of DSGC gap junctions (Chapter 2). Physiological and immunohistochemical methods allowed me to demonstrate an important role for connexin 36 subunits in DSGC electrical coupling. Next (Chapter 3) I investigate the sub-cellular mechanisms underlying neuronal correlations between electrically coupled DSGCs. Using paired recordings, I show that chemical input (from bipolar cells and amacrine cells), electrical input (from gap junctions), and Na+ channel activity in DSGC dendrites underlie the generation of correlated spiking activity. While a common feature of electrically coupled networks, the mechanisms underlying correlations were previously unclear. In Chapter 4 I focus on the mechanisms within the DS circuit that endow these neurons with impressive sensitivity to stimulus contrast. Using physiological and pharmacological methods I first assess the relative contrast sensitivity of ganglion cells and starburst amacrine cells (SACs) in the DS circuit. The sensitivity of DSGC and SAC excitatory currents to antagonists of Na+ channels suggests an important role for these channels in amplifying low contrast responses and other weak inputs to the circuit. This role is later attributed to the differential expression of voltage-gated Na+ channels in specific bipolar cell populations. In aggregate, this dissertation describes several novel circuit mechanisms within the well-studied DS circuit. I also provide specific roles for such specializations in visual coding. / Graduate

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24485号 / 医博第4927号 / 新制||医||1063(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 江藤 浩之, 教授 湊谷 謙司, 教授 大鶴 繁 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

Ion channels are fundamental molecules in the nervous system that catalyze the flux of ions across the cell membrane. There are mounting evidences suggesting that the kinetic properties of ion channels undergo activity-dependent changes in various pathophysiological conditions. Here such activity-dependent changes were studied in case of two different ion channels; the rat brain derived voltage-gated Na+ channel, rNav1.2 and the human background leak K+ channel, hTREK1 using the single channel patch-clamp technique. Our results on the voltage-gated Na+ channel (Chapter III) illustrated that sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, induced a defined non-linear variation in the unitary conductance, activation, inactivation and recovery kinetic properties of the channel. Signal processing tools attributed a pseudo-oscillatory nature to the non-linearity observed in the channel properties. Prolonged membrane depolarization also induced a “molecular memory” phenomenon, characterized by clustering of dwell time events and strong autocorrelation in the dwell time series. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Similar plastic changes were observed in case of the hTREK1 channel in presence of saturating concentrations of agonist, trichloroethanol (TCE) (Chapter IV). TREK1 channel behaves similar to single enzyme molecules with a single binding site for the substrate K+ ion whereas TCE acts as an allosteric activator of the channel. We observed that with increasing concentration of TCE (10 M to 10 mM) the catalytic turnover rate exhibited progressive departure from monoexponential to multi-exponential distribution suggesting the presence of ‘dynamic disorder’ analogous to single enzyme molecules. In addition, we observed the induction of strong correlation in successive waiting times and flux intensities, exemplified by distinct mode switching between high and low flux activity, which implied the induction of memory in single ion channel. Our observation of such molecular memory in two different ion channels in different experimental conditions highlights the importance and generality of the phenomenon which is normally hidden under the ensemble behaviour of ion channels. In the final part of the work (chapter V) we observed strong negative cooperativity and half-of-sites saturation kinetics in the interaction of local anesthetic, lidocaine with hTREK1 channel. We also mapped the specific anesthetic binding site in the c-terminal domain of the channel. Further, single channel analysis and the heterodimer studies enabled us to propose a model for this interaction and provide a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

No description available.