Immunocytochemical and Biochemical Characterization of Angiotensin I and II in Cultured Neuronal and Glial Cells from Rat Brain

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Abstract
Neuronal and glial cells cultured from neonatal rat brains showed staining for both angiotensin I and II using the peroxidase-antiperoxidase method. In glial cell extracts of normotensive Wistar-Kyoto rats, the concentrations of angiotensin I and II were 12.47 ± 2.71 (n = 4) and 66.73 ± 13.28 fmol/mg protein (n = 4). Angiotensin I and II found in neuronal cell extracts of normotensive Wistar-Kyoto rats were 11.29 ± 2.99 (n = 4) and 60.25 ± 12.77 fmol/mg protein (n = 4). No significant difference was found in the concentration of angiotensin I and II in both cell types from the same rat strain. Angiotensin I concentrations of 16.83 ± 3.43 fmol/mg protein (n = 5) determined in neuronal cell extracts derived from spontaneously hypertensive rats did not differ significantly from those found in neuronal cell extracts of Wistar-Kyoto rats. However, neuronal cell extracts from spontaneously hypertensive rats revealed values of 25.19 ± 4.31 fmol angiotensin Π/mg protein (n = 4). This was significantly different (p < 0.05) and represented a 58% reduction in the angiotensin II levels in neuronal cells from spontaneously hypertensive rats compared to Wistar-Kyoto rat cultures. Angiotensin I and II measured in the growth medium containing 10% plasma-derived horse serum was below the detection limit of both radioimmunoassays. No difference in the angiotensin I and II levels was found in cells kept in serum-free medium. The angiotensin I and II immunoreactive material determined in the cell extracts could be characterized on reversed-phase high pressure liquid chromatography as (Ile5)-angiotensin I and II. (Ile5)-angiotensin III was not detectable.

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