Neuroscience Scholars Summer Program Project

Kirsten R. GambrellSummer 2005Mentors: Dr. Elise Gomez-SanchezDr. Celso Gomez-Sanchez

The Gomez-Sanchez Lab –General Questions about Hypertension How do steroids of the adrenal cortex

induce hypertension?

Particularly, how does the mineralocorticoid aldosterone alter the central control of the blood pressure?

How is the synthesis of steroids of the adrenal cortex (aldosterone and the glucocorticoids, cortisol and corticosterone) regulated?

Mineralocorticoid Receptor (MR)

MR transcription factor Promotes transcription of different

genes depending on the cell type and binding

responsible for sodium and water transport in the kidney & other transport epithelia

The MR is activated by binding to an agonist

Adrenal Cortex (low salt diet)

Zona

glom

erulos

a

Zona

inter

med

ia

Zona

retic

ularis

Zona

fasc

iculat

a

adrenocorticotropic hormone (ACTH)adrenocorticotropic hormone (ACTH)

Aldosterone synthaseAldosterone synthase

11-11-ββ Hydroxylase Hydroxylase (corticosterone/cortisol)(corticosterone/cortisol)

Ang II, low salt, high KAng II, low salt, high K

Aldosterone synthase & 11Aldosterone synthase & 11ββ-Hydroxylase -Hydroxylase expression in rat adrenal expression in rat adrenal

LOW SODIUM DIET HIGH SODIUM DIETLOW SODIUM DIET HIGH SODIUM DIET

Aldosterone Aldosterone synthasesynthase

1111ββ -Hydroxylase-Hydroxylase

Brain MR and its link to hypertension

The affinity of the MR for its primary ligands is tissue dependent

11β-hydroxysteroid dehydrogenase provides extrinsic specificity for aldo in the kidney.

Cortisol & corticosterone hippocampus

Aldosterone Hippocampus Paraventricular n (hypothal) Amygdala Cortex cerebellum

The mechanism for ligand preference in the brain is not known

The circumventricular organs & amygdala are involved with blood pressure and fluid and electrolyte homeostasis.

deKloet Front Neuroendocrinology 1991, 2:95-164AV3V

Excessive & Inappropriate activation of the Excessive & Inappropriate activation of the MR in circumventricular nuclei & amygdalaMR in circumventricular nuclei & amygdala

central sympathetic drive to kidneys, heart & vsmcentral sympathetic drive to kidneys, heart & vsm

release of argenine Vasopressin (aVP)release of argenine Vasopressin (aVP)

baroreceptor sensitivitybaroreceptor sensitivityJaniak, Lewis, Brody AJP 259:R1025-R1034, 1990

sodium appetitesodium appetitesakai et al AJP 251:R762-R768 1986

proinflammatory cytokines in plasma & heartproinflammatory cytokines in plasma & heart

Felder et al AJP 284:R259-76 2003

Specific Questions for NSSP

Do oxytocin and vasopressin have a direct relationship with MR? Do oxytocin and vasopressin receptors co-

localize with those of MR?

Does RACK1 protein interact with the MR and give it ligand specificity?

MR Slides

MR 1-18 6G1, 488, 20x, SON MR 365 4D6, 594, 20x, PVN

Oxytocin and Vasopressin

Synthesized in the PVN and SON of the hypothalamus and released into the general circulation from the pituitary

Nine-aa, peptide neurotransmitters similar in aa sequence but different in function

Oxytocin: uterine contraction, milk let-down, and blood pressure control, counteracting the affects of vasopressin

Vasopressin: vasoconstriction and renal fluid absorption

http://www.driesen.com/hypothalamic_control_of_the_pituitary_gland.htm

Protocol

Neuroanatomy Selective

expression for vasopressin, oxytocin and MR

Rat brain – hypothalamus, specifically PVN and SON

IHC Bathing brain

sections in the primary Ab (MR, oxytocin, vasopressin) then secondary Ab

Staining both with DAB and immuno-fluorescence

Vasopressin Stained Slides

Vaso 647, MR365 546, DAPI in the PVN

Vaso 647, MR 1-18 546, DAPI in the PVN

Vaso 546, 20x, in the PVN

Oxytocin 647, MR365 546, DAPI, 20x, SON

Oxytocin Double Stained Slides

Results Based on double stained slides, both

vasopressin and oxytocin are expressed in the same cell.

Sharp 3D images of the slides using either a confocal microscope or de-convolution software with a Z-motor will confirm these results.

This suggests that vasopressin and oxytocin may interact directly with MR

Conclusion What does this mean?

In the normal animal model, we found that there is a relationship between vasopressin and MR, as well as oxytocin and MR.

What’s next? These relationships will be studied in animal

models, both genetic and pharmacologic, of human hypertension, including salt-sensitive hypertension, that are responsive to MR antagonists.

RACK1

RACK1 is a scaffolding protein that co-precipitates in a yeast-2-hybrid assay with the MR.

The hypothesis is that RACK1 alters the configuration of MR, thus increasing its specificity for aldosterone

Special Thanks to …Dr. Elise Gomez-SanchezDr. Celso Gomez-SanchezAngela F de RodriguezMiriam Gomez-SanchezDr. Ian PaulDetra ScottAnn DautenhahnGomez-Sanchez LabNeuroscience Summer Scholars