The quest for mineralocorticoid receptors
Download
1 / 18

The quest for - PowerPoint PPT Presentation


  • 285 Views
  • Updated On :

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'The quest for ' - Angelica


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
The quest for mineralocorticoid receptors l.jpg

The quest for Mineralocorticoid Receptors

Sodium

MR

Yutong Liu

Gomez-Sanchez Lab

Aldosterone


Slide2 l.jpg

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


Slide3 l.jpg

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


Slide4 l.jpg

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


Slide5 l.jpg

Developing tools to study MR & Aldo action in the brain

Molecular Biology

Biochemistry

Behavior

APPROACHES


Slide6 l.jpg

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


Slide7 l.jpg

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.


Slide8 l.jpg

  • 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


Slide9 l.jpg

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


Slide10 l.jpg

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


Slide11 l.jpg

Molecular weight Protocol for MRcombined 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


Slide12 l.jpg

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


Slide13 l.jpg

F3; AS4433 +/+ male, normal salt diet, observe in one organism, if it’s significant to life itself, will likely be 

13.5 mo old,

Congestive Heart Failure & glomerulosclerosis

Sprague Dawley male

normal salt diet

12 mo old

control


Syn1 as 4433 transgenic rat mean blood pressure l.jpg
Syn1/AS observe in one organism, if it’s significant to life itself, will likely be 4433 Transgenic Rat: Mean Blood Pressure

3 months old;

normal salt diet


Slide15 l.jpg

Experimental Setup of Behavioral Study: observe in one organism, if it’s significant to life itself, will likely be 

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



Slide17 l.jpg

Conclusion to Saline

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.


Slide18 l.jpg

Acknowledgements to Saline

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


ad