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Influence of Dietary Sodium Restriction on Angiotensin II Receptors in Rat Adrenals¹

Department of Biochemistry (J.G.L., L.D.), and Department of Anatomy and Cell Biology (N.B., D.M.), Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4, and Department of Obstetrics-Gynecology (I.M.B.), University of Wisconsin, Madison, Wisconsin 53715

Address all correspondence and requests for reprints to: Jean-Guy LeHoux, Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. E-mail: j.lehoux{at}courrier.usherb.ca

We studied the distribution of angiotensin II (AII) receptors type 1 (AT₁) and type 2 (AT₂) and the effects of a low sodium intake on these two subtypes of receptors in male rat adrenals. Binding studies on adrenal slices, on cell membranes and on cell suspensions were performed using [¹²⁵I]AII and specific analogs for AT₁ (Losartan) and AT₂ (PD 123319) receptors. The distribution of AT₁ was also studied by immunofluorescence. Complementary approaches were necessary to reach our goal. Indeed, by autoradiography on adrenal slices, [¹²⁵I]AII was shown to bind to the zona glomerulosa (ZG) and to the medulla (M). When coincubated with [¹²⁵I]AII, PD 123319 displaced [¹²⁵I]AII from the medulla and from the ZG, indicating the presence of AT₂ receptors in both zones. Losartan partially displaced [¹²⁵I]AII from the ZG, indicating the presence of AT₁ receptors in that zone. Furthermore, the labeling intensity of the medulla (AT₂ receptors) was much stronger in adrenal sections from rats kept on a low sodium regimen than from controls. Immunofluorescence microscopy revealed that AT₁ receptors were located mainly in the ZG of control rats. After sodium restriction, AT₁ receptors appeared to be uniformly distributed within an enlarged ZG; furthermore AT₁ receptor-positive cells were found to a limited degree in the zona fasciculata and possibly in the zona reticularis, and a greater number of these positive cells appeared in these zones under sodium restriction. Cell suspensions from rats fed a low sodium diet showed a 2.7- and 2.1-fold increase in total AII receptors in adrenal ZG and ZFR + M cells when compared with controls. Based on Losartan displacement, we calculated that [¹²⁵I]AII bound to AT₁ and to AT₂ receptors was increased in both ZG and ZFR + M cell preparations under sodium restriction. Results of binding studies on cell membranes were also indicative of an increasing effect of sodium restriction on AT₁ and AT₂ receptors binding capacity. Furthermore, Northern blotting analysis revealed 3.0- and 2.5-fold increases in the level of AT₁ receptor mRNA in the ZG and the ZFR + M of rats fed a low sodium diet as compared with those fed a normal diet. The low sodium intake resulted in a weaker increase (1.5-fold) in the level of AT₂ receptor messenger RNA in the ZG, with no changes in the ZFR + M preparations. In conclusion, in this study complementary approaches were needed to determine the localization of AT₁ and AT₂ receptors in the rat adrenal, and to show the increasing effects of a low sodium regimen on the adrenal level of these receptors. Immunofluorescence studies revealed AT₁ receptors mainly in the ZG and also in some cells of the inner adrenal cortex zones; in adrenals of rats kept on a low sodium diet the ZG was markedly enlarged, and an increased number of immunoreactive cells with AT₁ receptors were observed throughout that zone; also more immunoreactive cells were present in the inner zones of the adrenal cortex. Furthermore in the adrenals of rats kept on a low sodium diet, we observed: 1) an increased number of AT₁ and AT₂ receptors in cell suspensions from the ZG, and in cell suspensions of the ZFR + M; 2) an increased level of AT₁ and AT₂ receptor mRNAs in the ZG; 3) an increased level of AT₁ receptor mRNA, with no changes in the AT₂ mRNA level in the ZFR + M. These results suggest a role for AT₁ as well as for AT₂ receptors in controlling adrenal function and differentiation under normal as well as under physiological stimulation of AII production following sodium restriction.

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