Biological activity of an angiotensin II-ferritin conjugate on rabbit aortic smooth muscle cells

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Abstract
Specific binding sites for [Asp1,Ile5]angiotensin II (angiotensin) were demonstrated in homogenates and subcellular fractions of aortic medial smooth muscle cells, but the localization of the angiotensin receptor responsible for contraction was not determined. To establish the location of this receptor, a membrane-impermeable analog of angiotensin was prepared by acylating its N-terminal amino group with the N-hydroxysuccinimide ester of succinylated ferritin. This angiotensin-ferritin conjugate possessed the same intrinsic activity as angiotensin but was .apprx. 200 times less potent in inducing contraction in rabbit aortic strips. The stability of the conjugate was investigated, and .apprx. 5% of the contractile activity of the angiotensin-ferritin conjugate was attributable to low MW components that were present before or after exposure to aortic strips. The time required for aortic strips to reach a plateau of contraction in response to angiotensin-ferritin was significantly longer than that required by free angiotensin to produce the same level of contraction. With enzymatically dispersed aortic smooth muscle cells, the time taken to produce contractions by both angiotensin and angiotensin-ferritin was indistinguishable. [Sar1,-Ala8]angiotensin II, a competitive inhibitor of angiotensin, completely suppressed contractions induced by angiotensin or angiotensin-ferritin in aortic strips or dispersed aortic smooth muscle cells. Angiotensin apparently need not directly penetrate the plasma membrane to cause contraction and the angiotensin receptor responsible for initiating contraction of aortic smooth muscle cells is located on the plasma membrane.