Rapid Regulation Of Aldosterone On Epithelia Sodium Channel

Object: Aldosterone play an important role in regulation of extracellular volume, electrolyteshomeostasis and blood pressure control. These physical function depend on the limitedregulation of sodium reabsorption by epithelial sodium channel (ENaC) in cortical connectingduct, the mechanisms of ENaC regulation by Aldo is compose of genomic and nongenomicaction in present study. The mechanism of genomic is clear: the Aldo enter the principle cellof cortical connecting duct (CCD) and binding with MR in intracytoplasm, and Aldo-MRcomplex transport into intranuclear and interact with the specific DNA bingding site whichfinally regulate specific mRNA transcripte and synthesis several aldosterone induced protein(AIP). And then AIP increase the ENaC activity hence promote the sodium reabsorption byENaC. An other mechanism is nongenomic ation,the character of nogenomic of Aldo is rapidand non-MR-dependence, but the underlying mechanism is still unclear,. For furtherunderstand the physiology and pathology meaning of ENaC regulation by aldosterone, wewill explore the rapid regulation of aldosterone on ENaC and its possible mechanism.On theother hand,it could help to promote the development of new drugs like aldosteronicantagonist.Content:1Establishment and assessment of the mammalian epithelial cell model that have ENaCactivity.2Research the rapid regulation of ENaC activity by aldosterone.3Explore the mechanism of the rapid regulation by aldosterone.Method:1. Establishment of cell modelWe estabish mammalian renal epithelia cell mode that have ENaC activity. MDCKand mpkCCD were cultured to integrate monolayer which have high resistance and sensitiveto amiloride. Finally, the cell model were assessed by morphologic observation, TEERmeasurement and sigle channel recording and the better one were selected for furtherresearch.2. Rapid regulation by aldosteroneMR blocker-aldactone were added to Verify the non-MR-depended rapid regulation(＜ 3hours).after addition of10-6M/L aldosterone on mpkCCD monolayer, toppgraphic imagewere observed, amiloride-sensitive△TEER were measured,single channel current wererecorded and intracellular calcium concentration were detected.3. The mechanism of rapid ENaC regulation by aldosterone(1) The specific PI3-K blocker were used to identify the function of PI3-K pathway inrapid regulation on ENaC by aldosterone.(2) Cacium loader A23187were used to increase the concentration of intracellularcalcium and explore the effect of intracellular calcium change on rapid regulation on ENaCby aldosterone.(3) Cyt D were used to disrupt the F-acin of cystoskeleton and discuss the relationship ofcell morphology and EaNC activity.4. Main index and measurement(1) Morphology observation: SICM(2) TEER: EVOM2(3) Single channel recording: patch clamp cell-attached configurationResult:1. Establish mpkCCD mammalian epithelia cell model that have EaNC activity.2. After addition of aldosteron, cell transversal contract and ongitudinal stretching, EaNCactivity increase both in monolayer and single channel and display amiloride-sensitive△TEER and channel open probability improve.3. LY294002block the increase of aldosterone induced EaNC activity and recover thetopographic change,decrese the miloride-sensitive△TEER and channel open probability.4. A23187rapid increase the intracellular calcium concentration and increaseamiloride-sensitive△TEER and channel open probability.5. Cyt D make the cell transversal contract and longitudinal stretching and increaseamiloride-sensitive△TEER and channel open probability.Conclusion:1. Aldosterone rapid increase the EaNC activity.2. PI3-K pathway and cacium play an important role in aldosterone rapid increase EaNCactivity. 3. Morphological change (transversal contract and longitudinal stretching) increase EaNCactivity reveal that morphology and EaNC activity close relative.