Localization of angiotensin converting enzyme (kininase II). II. Immunocytochemistry and immunofluorescence

Abstract

The cellular and subcellular sites of angiotensin converting enzyme (kininase II) in lung tissue and endothelial cells in culture were examined by immunocytochemical and immunofluorescence techniques. Converting enzyme is capable of inactivating bradykinin and of converting angiotensin I to its potent lower homolog, angiotensin II. Immunocytochemistry at the electron microscope level used goat anti-(pig lung angiotensin converting enzyme) coupled to 11-MP (11-microperoxidase) via glutaraldehyde or to 8-MP (8-microperoxidase) via a bifunctional active ester, bissuccinyl succinate. The latter conjugate, which does not contain complex polymers, has been characterized in detail in terms of immunoreactivity and peroxidatic activity Both conjugates yield similar results: angiotensin converting enzyme appears to be localized along the luminal surface of pulmonary endothelial cells. Endothelial cells of large and small vessels react with the antibody conjugates, but reactions are most prominent at the level of the capillaries and venules. The conjugates are also reactive with pig lung and aortic endothelial cells in monolayer culture. These cell lines were shown to possess converting enzyme activity using a functional assay [hydrolysis of ( 125I)Tyr 8-bradykinin to yield ( 125I)Tyr-Arg]. Cells identified as smooth muscle of pulmonary artery did not possess converting enzyme activity and did not react with the antibody-conjugates. Converting enzyme was also shown to be associated with pulmonary endothelial cells in culture by direct and indirect immunofluorescence. The direct studies used specific antibody coupled to MDPF. The indirect studies used cells incubated with specific antibody and then with FITC-labelled rabbit anti-(goat globulins). Our results support the conclusion that circulating bradykinin can be inactivated and angiotensin can be activated by an enzyme situated on the luminal surface of pulmonary endothelial cells. The strategic position of the lungs in the general circulation and the strategic position of the enzyme within the lungs suggest that the enzyme may help regulate the quantities of bradykinin and angiotensin II allowed to enter the systemic arterial circulation. The ability of pulmonary converting enzyme to inactivate a hypotensive substance, bradykinin, and to form a hypertensive substance, angiotensin II, may bespeak a role of the enzyme in blood pressure homeostasis.