The renin substrate, angiotensinogen, was localized by immunocytochemistry in liver and kidney of normal rats by the use of an antiserum directed against pure rat angiotensinogen. This substrate was also examined in rats after bilateral nephrectomy, which is known to increase plasma angiotensinogen, and in rats treated with colchicine, which inhibits serum protein secretion. In normal rat liver, light microscopy showed the presence of immunoreactive material in a very few cells. The number of stained hepatocytes rose in rats treated with colchicine or after bilateral nephrectomy. Immuno-staining increased further when rats were both nephrectomized and colchicine treated. In the kidney, angiotensinogen was specifically located as granular formations in nephrocytes of the proximal tubule but never in the granular cells of the juxtaglomerular apparatus. The localization of these granular formations under the brush border suggests that angiotensinogen is reabsorbed from the glomerular ultrafiltrate rather than synthesized in the kidney.

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Angiotensin modifies a number of functions of the central nervous system (CNS) including blood pressure regulation, thirst, and the secretion of the antidiuretic and adrenocorticotropic hormones. Whereas peripheral administration of angiotensin produces these effects, accumulating evidence implicates a brain angiotensin system which operates independently of the circulating renin-angiotensin system (reviewed in refs 1-3). All components of the renin-angiotensin system have been identified in the brain, but there is considerable controversy over whether these components are synthesized in the brain or derived from plasma and the extent to which these components interact to generate angiotensin in the brain in vivo. Whereas many different enzymes are able to cleave angiotensinogen (renin substrate) to release angiotensin I (AI) or angiotensin II (AII), angiotensinogen is the only known precursor of AI and AII. Definitive evidence for an independent brain angiotensin system requires the demonstration that the brain synthesizes an angiotensin precursor. Here we report the identification of angiotensinogen mRNA in rat brain by cell-free translation and show that the brain angiotensinogen precursors are identical to those previously identified for liver, the source of plasma angiotensinogen.

SUMMARY The role of angiotensinogen ID blood pressure control was assessed in nonnotensive rate by observing the changes resulting from inhibition by specific rat angiotensinogen antiserum. The antiserura decreased blood pressure in rats on normal sodium as well as sodium-free diets (respectively ABP = -3 0 ± 6 mm Hg and -42 ± 8 mm Hg). In binephrectomlzed sodium-replete rats, administration of antiserum did not reduce blood pressure, whereas in sodium-depleted animals it slightly decreased blood pressure by 11 ± 3 mm Hg. These results suggest that angiotensinogen participates in the regulation of blood pressure in normotensive rats, even in the sodium-replete state. (Hypertension 4: 185-189, 1982) KEY WORDS • angiotensinogen antibodies * blood pressure • normotensive rats • sodium status T HE renin-angiotensin system plays an important role in regulating blood pressure; 1 * * its role in normal homeostasis, hypertensive diseases, and edematous states continues to be defined by study of the use of effective blocking agents.The initial step of the renin-angiotensin system, the action of renin on angiotensinogen, can be inhibited by specific antibodies, pepstatin and its derivatives, phospholipids, and substrate analogs.1 Angiotensinogen concentration is known to be a rate-limiting factor for angiotensin generation in human 4 or rat* plasma. Treatment with estrogen** 7 or glucocorticoids 8 causes a rise in angiotensinogen levels, which probably contributes to the increased incidence of high blood pressure observed in women receiving oral contraceptives* and the hypertension associated with glucocorticoid excess. 10In the present study, specific angiotensinogen antibodies obtained by immunizing rabbits against highly purified rat angiotensinogen have been used to inactivate the renin-angiotensin system in conscious rats on normal and sodium-free diets. This method of blockade has not been investigated previously and provides a useful comparison with other blocking methods. Methods Production of AntiserumRenin substrate (angiotensinogen) in binephrectomized rat plasma was purified in six successive steps: ammonium sulfate precipitation, followed by chromatography on blue dextran sepharose, DEAEsephacel, hydroxylapatite, agarose-acrylamide, and then preparative isoelectric focusing. Purity was checked by sodium dodecyl sulfate (SDS)-gel electrophoresis and amino acid analysis, which demonstrated that the first 10 amino acids were exactly those of angiotensin I."Antibodies to rat angiotensinogen were obtained in rabbits. They were injected intradermally with 50 fig of pure angiotensinogen mixed with complete Freund's adjuvant, and then after 8 weeks a booster injection of SO ng of angiotensinogen mixed with incomplete Freund's adjuvant was administered at multiple intradermal sites. High titer antisera were obtained 3 weeks after the third injection. Binding of pure iodinated angiotensinogen was used to follow the successive titers of antibodies during the immunization procedure.18 Antisera selected for the experi...

The influence of thyroidectomy on the renin-angiotensin system was studied in the rat. From 1-6 weeks after thyroidectomy, PRA and plasma renin substrate (PRS) decreased, but the plasma renin concentration remained unchanged, and the renal renin content increased. T3 injection corrected the changes in the plasma renin-angiotensin system of thyroidectomized rats within 20-40 h. After ethinylestradiol treatment, the PRS in thyroidectomized rats rose in the same proportion as that in normal rats, but remained below the normal level. After binephrectomy, on the other hand, the PRS was high, and PRS levels in normal and thyroidectomized animals were similar. Isoproterenol increased PRA and plasma renin concentration in control animals but had no effect on thyroidectomized rats. From the above results it may be concluded that angiotensinogen production is dependent on thyroid hormones and that renin release depends on beta-adrenergic receptor sensitivity to catecholamines, which is reduced by thyroidectomy. (Endocrinology 108: 647, 1981)