The Conversion of Angiotensin I to Angiotensin Ii by Rabbit Glomeruli

Abstract

Experiments were designed to test the possibility that glomeruli can convert angiotensin I (AI) to angiotensin II (AII) (Thurau, Dahlheim & Granger, 1969). In six experiments, 10\g=m\g(1-asp-5-ile) AI in 1\m=.\1 ml 0\m=.\15m-phosphate\p=m-\salinebuffer, pH 5\m=.\7, were incubated in stoppered neoprene tubes with 20-25 glomeruli dissected as previously described (Brown, Davies, Lever, Parker & Robertson, 1965) from kidneys of New Zealand White rabbits. The glomeruli were stored frozen, then thawed and incubated. In 2 control experiments AI was incubated with the paper fragments used to transfer glomeruli to the incubation tube, in 1 control experiment with 25 boiled glomeruli, and in 4 control experiments with rabbit blood (0\m=.\1\g=m\l )equal in volume to 25 glomeruli (Vimtrup, 1928). In another control experiment glomeruli were incubated without angiotensin. Ten \g=m\g AI were incubated with buffer alone with each set of experiments.An incubation pH of 5\m=.\7was chosen because single glomeruli show little angiotensinase activity at this pH (Brown et al. 1965). Microbial contamination (Vane, 1971) was prevented by making buffers from boiled salt solutions and sterile distilled water. Tubes, stoppers and dishes for microdissection were boiled for 20 min or heated in an oven overnight before use. The incubation buffer contained neomycin (0-2%).The tubes were incubated at 37°C for 48 h. Any AH produced was separated from AI by a modification of the method of Walaszek, Bunag & Huggins (1962). 0-5 ml incubation mixture was diluted with 1 ml boiled, cooled, distilled water, applied to a 0-9x12 cm column of CM Sephadex equilibrated with 0-05M-sodium phosphate buffer, pH 5-7, and eluted with a gradient of 50 ml 0-05M-sodium phosphate, pH 5-7, in the mixing chamber and 50 ml 0-1 M-sodium phosphate, pH 7-0, in the reservoir. Fractions (1 ml) were collected and assayed by their pressor effect on the rat blood pressure preparation (Plate). 0-1 ml of fractions containing AI or All were pooled, bioassayed against the respective AI or AH standard, and also tested by radio¬ immunoassay for All (Dusterdieck & McElwee, 1972). The total amounts of angio¬ tensin in each peak and the percentage recovery of angiotensin from the column could then be found.Complete separation of standard l-asp-5-ile AI and l-asp-5-ile AH was obtained (Plate). The mean recovery of standard AI in four experiments was 76 % and that of AH was 82%. The concentration of All found by radioimmunoassay of an All peak (77-4 ng/ml) agreed with that found by bioassay (60-80 ng/ml). Material from the AI fraction contained 300-400 ng angiotensin/ml by bioassay, but only 5-4 ng/ml

Conversion of Angiotensin I into Angiotensin II in the Isolated Perfused Rat Kidney

“…This finding is in agreement with observations in the dog (di Salvo, Peterson, Montefusco & Menta, 1971 ;Aiken & Vane, 1972). For isolated glomeruli of rabbit kidney a mean conversion rate of 6.8% was found (Leckie, Gavras, McGregor & McElwee, 1972). However, it is possible that in the isolated rat kidney perfused with a cell-free medium, RPF is much higher than in vivo, and that the rapid passage of A1 through the kidney might limit its rate of conversion.…”

Angiotensin II immunoreactivity coexists with renin in the juxtaglomerular granular cells of the kidney.

“…Although circulating All has mainly been considered responsible for the effects in the kidneys, it also has been suggested that the intrarenal regulatory functions are performed by a second, tissuebound intrarenal renin-angiotensin system localized in the region of the juxtaglomerular apparatus (5). This hypothesis was supported by the fact that the enzymes involved in the metabolism of All are all located in this area (9)(10)(11). However, the microtopographical details ofthe localization ofthe various components of the renin-angiotensin system, particularly the exact site of generation of All, remained unclear.…”

Renin substrate in granules from rat kidney cortex

“…Renal converting enzyme may be located in the cell membrane, as it is in the lung, and Oshima et al (1974) suggest that it may be physically separated from the renal circulation to serve a local intrarenal function. Further, since isolated glomeruli have been shown to convert angiotensin I into angiotensin II (Leckie et al, 1972) and converting-enzyme activity has been measured in the juxtaglomerular apparatus (Granger et al, 1972) it is likely that the juxtaglomerular cells may contain converting enzyme in the cell membrane. Conversion of angiotensin I to angiotensin II may occur here or in more distal vessel walls, e.g.…”