The long-term goals of this proposal are to study angiotensin II (AII) receptor regulation in primary neuronal enriched (PNC) and vascular smooth muscle cell (VSMC) cultures from SHR, WKY and Sprague-Dawley (SD) rats, to examine the effects of captopril and other converting enzyme inhibitors (CEI) on AII receptor regulation in these rats and to determine whether the anti-hypertensive effect of CEI in SHR is related to a down-regulation of brain AII receptors. We will utilize PNC and VSMC cultures from neonates to compare AII receptor regulation in SHR vs normotensive rats and to determine the effects of CEI on central and peripheral AII receptors. This project is divided into two specific aims.
Specific Aim 1 : To test the hypothesis that brain AII receptors are biochemically and functionally different in SHR vs normotensive rat strains and that brain AII receptors in SHR are regulated differently from those of WKY and SD rats. We will utilize radioligand binding techniques to characterize AII receptor number and affinity in PNC (hypothalamus/brain stem co-cultures) from SHR, WKY and SD pups and compare these to AII receptors in VSMC cultured from the same pups. Binding studies will be performed in intact cells and in membranes prepared from these cells. We will incubate cells with agents known to stimulate or decrease AII binding and determine whether there are differences in the responses of AII receptors in SHR vs WKY and in PNC vs VSMC cultures. We will functionally assess AII receptors in PNC and VSMC from SHR as compared to WKY and SD by monitoring 45 Ca2+ efflux, cytosolic [Ca2+], phosphoinositide turnover and AII stimulated release of vasopressin (VP). We propose that a major signal pathway mediating AII action in neuronal tissue is the inositol phosphate/Ca2+ pathway.
Specific Aim 2 : To test the hypothesis that administration of CEI to SHR is associated with down regulation of brain AII receptors. We will a) compare AII receptor number and affinity in PNC and VSMC from SHR, WKY and SD pups subjected to in utero treatment with CEI, b) we will determine the effects of short-term incubation of PNC and VSMC cultures with CEI of different chemical structures on AII binding kinetics. We will study the effects of these agents on the Kd and Bmax and the association and dissociation constants. We will also study the effect of these agents on AII receptor internalization and the coupling of the AII receptor to its second messenger. In order to determine the specificity of the effect of CEI on the AII receptor we will simultaneously monitor the effect of CEI on norepinephrine (NE) and VP binding. Results from the proposed study should increase our understanding of brain AII receptors and the regulation and how these receptors are altered in a genetic model of hypertension.
|Zhang, L; Edwards, D G; Berecek, K H (1996) Effects of early captopril treatment and its removal on plasma angiotensin converting enzyme (ACE) activity and arginine vasopressin in hypertensive rats (SHR) and normotensive rats (WKY). Clin Exp Hypertens 18:201-26|
|Berecek, K H; Zhang, L (1995) Biochemistry and cell biology of angiotensin-converting enzyme and converting enzyme inhibitors. Adv Exp Med Biol 377:141-68|
|Wu, J N; Edwards, D; Berecek, K H (1994) Changes in renal angiotensin II receptors in spontaneously hypertensive rats by early treatment with the angiotensin-converting enzyme inhibitor captopril. Hypertension 23:819-22|
|Wu, J N; Berecek, K H (1993) Prevention of genetic hypertension by early treatment of spontaneously hypertensive rats with the angiotensin converting enzyme inhibitor captopril. Hypertension 22:139-46|