Neuroscience Scholars Summer Program Project

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Neuroscience Scholars Summer Program Project

  1. 1. Neuroscience Scholars Summer Program Project Kirsten R. Gambrell Summer 2005 Mentors: Dr. Elise Gomez-Sanchez Dr. Celso Gomez-Sanchez
  2. 2. 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?
  3. 3. 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
  4. 4. Adrenal Cortex (low salt diet) Zona glom erulosa Zona interm edia Zona reticularis Zona fasciculata adrenocorticotropic hormone (ACTH)adrenocorticotropic hormone (ACTH) Aldosterone synthaseAldosterone synthase 11-11-ββ HydroxylaseHydroxylase (corticosterone/cortisol)(corticosterone/cortisol) Ang II, low salt, high KAng II, low salt, high K
  5. 5. Aldosterone synthase & 11Aldosterone synthase & 11ββ-Hydroxylase-Hydroxylase expression in rat adrenalexpression in rat adrenal LOW SODIUM DIET HIGH SODIUM DIETLOW SODIUM DIET HIGH SODIUM DIET AldosteroneAldosterone synthasesynthase 1111ββ-Hydroxylase-Hydroxylase
  6. 6. 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, 2:95-164 AV3V
  7. 7. Excessive & Inappropriate activation of theExcessive & 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 sensitivity Janiak, Lewis, Brody AJP 259:R1025-R1034, 1990 sodium appetitesodium appetite sakai et al AJP 251:R762-R768 1986 proinflammatory cytokines in plasma & heartproinflammatory cytokines in plasma & heart Felder et al AJP 284:R259-76 2003
  8. 8. 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?
  9. 9. MR Slides MR 1-18 6G1, 488, 20x, SON MR 365 4D6, 594, 20x, PVN
  10. 10. 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
  11. 11. http://www.driesen.com/hypotha lamic_control_of_the_pituitary_g land.htm
  12. 12. 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
  13. 13. 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
  14. 14. Oxytocin 647, MR365 546, DAPI, 20x, SON Oxytocin Double Stained Slides
  15. 15. 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
  16. 16. 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.
  17. 17. 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
  18. 18. Special Thanks to … Dr. Elise Gomez-Sanchez Dr. Celso Gomez-Sanchez Angela F de Rodriguez Miriam Gomez-Sanchez Dr. Ian Paul Detra Scott Ann Dautenhahn Gomez-Sanchez Lab Neuroscience Summer Scholars

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