The quest for Mineralocorticoid Receptors. Sodium. MR. Yutong Liu Gomez-Sanchez Lab. Aldosterone. The Focus: Unraveling causes of hypertension. The Players:. Mineralocorticoid receptor (MR) Activation of MR in the amygdala increases salt appetite
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The quest for Mineralocorticoid Receptors Sodium MR Yutong Liu Gomez-Sanchez Lab Aldosterone
The Focus: Unraveling causes of hypertension The Players: • Mineralocorticoid receptor (MR) • Activation of MR in the amygdala increases salt appetite • 2. MR in circumventricular organs are involved in cardiovascular homeostasis & inflammatory process • 3. MR in hippocampal neurons are involved in learning and memory • Aldosterone (aldo) • Aldo is a steroid hormone of the mineralocorticoid family, which maintains fluid, electrolyte, and blood pressure homeostasis • 2. Synthesis- • a. Adrenal glands • b. synthesis tightly regulated by Na, K, & angiotensin II
The Focus: Unraveling causes of hypertension • Chronic excessive activation of MR • Hypertension • 2. Heart, vessel, and kidney inflammation and scarring • 3. Heart and Vessel hypertrophy
The Connections ? Mineralocorticoid Receptor Aldosterone --*#@!-- In kidney, heart, brain, & many other organs • HYPERTENSION • Effects 65 million Americans according to the American Heart Association • 2. Primary risk factor for heart attack, stroke, and kidney failure • 3. 7-12% hypertension is due to hyperaldosteronism Relevance
Developing tools to study MR & Aldo action in the brain Molecular Biology Biochemistry Behavior APPROACHES
Hypotheses … so many to tell, which one? • Small interfering (si) mRNA carried by a lentivirus vector can be used to eliminate MR expression 2. An ELISA can be used for a very sensitive MR detection 3. A transgenic rat over-expressing aldosynthase in neurons will have an increased appetite for sodium
The purpose of the Western Blot In general: To confirm the existence/absence of a protein of the same molecular weight & charge as MR in cells transfected with si mRNA for MR or scrambled si mRNA. In this case: To demonstrate a decrease in the expression of MR by the si mRNA method.
1. Sample Preparation • *apparatus: a) water bath, b) centrifuge • a. boil • b. centrifuge • 2. Electrophoresis • *apparatus: Hoefer Mighty Small II • a. load sample • b. load marker • c. run @ 150v for 1 hour • 3. Transfer • *apparatus: BioRad MiniProtean II • a. soak filter pads in transfer buffer • b. soak PVDF membrane in methanol (1min), then transfer buffer (30min) • c. soak gel in transfer buffer (5min) • d. Assemble transfer sandwich • e. fill apparatus w/ transfer buffer • f. run for 1.5 hours at 350 mA • 4. Immuno Detection • *apparatus: Belly Dancer • a. block w/ milk solution for 1 hr • b. incubate w/ primary antibody overnight in cold room • c. wash membrane w/ 1x PBS + .05% Tween • d. incubate w/ secondary antibody for 1 hr • e. wash membrane in 1x PBSTween • 5. Developing • ~location: a) 4° C cold room, b) dark room • make a 1:1 working solution of West Pico reagents • incubate membrane in West Pico solution in cold room (5 min) • in dark room, dab excess WP • wrap membrane in plastic wrap • expose film to membrane Western Blot Methods
Developing an Enzyme Linked ImmunoSorbent Assay (ELISA) Protocol for MR What the MR ELISA reveals: How much MR protein is in a particular solution More protocols in planning • Chicken anti-sheep • Sheep anti-mr • Mouse anti-mr • Primary AB (MR 365 & MRN2 2B7) • Donkey anti-mouse HRP Protocols we have tried • anti-sheep IgG • Sheep anti-mr • Mouse anti-mr • Primary AB • Goat anti-mouse
Developing an Enzyme Linked ImmunoSorbent Assay (ELISA) Protocol for MR Sandwich ELISA for MR: side view of one well in a 96 well plate Rabbit x-mouse Horseradish Peroxidase Primary Antibody: 365 and 2B7 Samples: Variable & Control Sheep x-MR Coating antibody: chicken x-sheep; donkey x-sheep
Molecular weight combined w/ antibody is more accurate The Good Antibody may not be completely specific The Bad Western Blot ELISA Can’t do many samples simultaneously; not very quantitative 1.Quantitative (can make a standard curve) 2. More sensitive: measure smaller amounts The Bad The Good
Biologists had a dogma that what you observe in one organism, if it’s significant to life itself, will likely be appropriate to other organisms. Gina Kolata
F3; AS4433 +/+ male, normal salt diet, 13.5 mo old, Congestive Heart Failure & glomerulosclerosis Sprague Dawley male normal salt diet 12 mo old control
Syn1/AS4433 Transgenic Rat: Mean Blood Pressure 3 months old; normal salt diet
Experimental Setup of Behavioral Study: Do Aldosynthase-over-expressing rats prefer saline over water? Aldosynthase rats (7 male, 7 female) Normal Sprague-Dawley rats (7 male, 7 female) Saline Water Saline Water
Comparison of Volume Imbibed per Rat Type upon 3rd Exposure to Saline 3rd Exposure: July 16-20th
Conclusion The siRNA and the ELISA for MR are still works in progress. The salt appetite study of transgenic rats revealed that both type of rats preferred saline over water. As to which type of rat preferred saline more, over the length of 3 exposures, a guesstimated conclusion would be the same.
Acknowledgements Dr. Ian Paul Anne Dautenhahn Catherine Kaime Neuroscience Summer Scholars Program Dr. Elise P. Gomez-Sanchez Dr. Celso E. Gomez-Sanchez Angela F. de Rodriguez Gomez-Sanchez lab