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Endogenous renin inhibitor in neuroblastoma cells
Brain Research, 250 (1982) 373-377 Elsevier Biomedical Press
373
Endogenous renin inhibitor in neuroblastoma cells TAISUKE INAGAKI, TOMIO OKAMURA and TADASHI INAGAMI Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232 (U.S.A.)
(Accepted July 6th, 1982) Key words: neuroblastoma cell -- renin - - renin inhibitor - - intracellular regulation -- intracellular function
Cloned neuroblastoma cells (Neuro-2a) in culture were found to contain a renin inhibitory substance. The inhibitor in the extract of cloned neuroblastoma cells was separated from renin activity by anti-renin IgG-Sepharose and selectivelyconcentrated by adsorption to renin-agarose gel. The present study demonstrated the coexistence of renin and its inhibitor in the same cell and suggested a possible regulatory mechanism of intracellular renin activity by an endogenous renin inhibitor in neuronal cells. Recent findings of coexistence in cultured neuroblastoma cells of renin, angiotensin I and IIa, le, angiotensin converting enzyme 12 and, by implication, angiotensinogen, suggests a possible intracellular mechanism of the formation of angiotensin II in these cells in contrast to the well known extracellular mechanism in plasma. The coexistence of the enzymes and substrate within the same cells also suggests an intracellular control mechanism by compartmentalization of these components or by a specific inhibitor. The presence of possible renin inhibitor was suggested earlier in conjunction with activatable high molecular weight renins isolated from kidney homogenates 1,s,1°. However, these activatable renins have been considered as an intermediate of the activation of inactive, single polypeptide renin zymogen 9. Renin binding protein (RBP) binds to renin in the presence of sulfhydryl blocking reagentsa, 15 and forms a high molecular weight renin 6. However, the binding was reported to have little effect on the enzyme activity. Recently, an inhibitor of renin was found in the kidney homogenates~a,~5 and its possible identity with RBP was suggested 15. While renin is known to be localized in renin granules ofjuxtaglomerular cells in the cortex of the kidney, the inhibitor was shown to be localized in cytosol fractions of kidney homogenatela,15. Furthermore, RBP was shown to be localized in tubular cells 16 rather than in juxtaglomerular cells. The finding that the inhibi0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
tion was demonstrable only at a very high concentration casts doubt on the physiological significance of RBP in the control of renin. Thus, much remains to be clarified about the regulatory mechanism of the intracellular system. To obtain insight in the control mechanism of the intracellular angiotensin generating system, we have investigated the possible existence of renin inhibitor in a mouse neuroblastoma cell line (Neuro-2a). The study was extended to include partial purification of the inhibitor by a new affinity chromatographic method. Renin-inhibitory activity was determined by the decrease in renin activity caused by the addition of cell extract fractions to renin assay mixture. Renin activity was determined by radioimmunoassay of angiotensin 15 generated at 37 °C in a mixture consisting of 25 #1 of partially purified hog angiotensinogen (3 #M), 50/A of neuroblastoma renin solution (prepared as below), 50 #1 of inhibitor solution (or water for control experiments) and 100 tzl of 0.1 M Na-phosphate buffer, pH 7.0, containing 5 mM EDTA, 1 mM diisopropyffiuorophosphate (DFP), 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 #g/ ml soybean trypsin inhibitor (STI), 5/~g/ml trasylol, 6/~g/ml leupeptin and 2/~M (1-5-carboxy-3-phenylpropyl)-L-l-lysyl-L-proline (L-154, an angiotensin converting enzyme inhibitor). These inhibitors were used to eliminate artifact arising from destruction of angiotensin I or renin. Renin and renin inhibitor were prepared from
374 extract o f c u l t m e d neuro-2a cells by immunoaffinity chromatography and by affinity chromatography on renin-Affi-Gel 10, respectively. The anti-renin lgG gel was prepared by the method of Yokosawa et al. 17 using rabbit anti-mouse submaxillary gland renin antibody n. Renin-Affi-Gel 10 was prepared by coupling 2 mg of purified mouse renin 2 to 2 ml of Affi-Gel 10 (Bio Rad) in 0.1 M phosphate buffer, pH 7.0, for 48 h at 4 °C with gentle agitation followed by treatment (3 h) with ethanolamine HCI by adding 0.2 ml of 1 M solution to the mixture. The gel was successively washed with 10 m M phosphate buffer, pH 7.0, the same buffer containing 1 M NaCI, 0.1 M acetic acid, again with 10 mM phosphate buffer pH 7.0 and stored at 4 °C. Neuro-2a cells 7 were grown in Dulbecco modified Eagle's medium containing 10% fetal calf serum, transferred to the serum-free medium for 24 h prior to harvest, detached from culture flasks with 1 m M EDTA and washed twice with the serumfree medium by the published method 12. Cells were then homogenized with polytron (Brinkman) in an ice bath for 15 s 4 times in 3 ml of water containing
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1 mM PMSF, 5 m M EDTA, 6 ffg/ml leupeptin, 1.25 ffM L-154 and 0.1% Triton X-100. The homogenate was centrifuged at 700 g for 15 rain and supernatant was recentrifuged at 32,000 g for 30 min. The supernatant designated as 'crude extract' was used as the source of renin and renin inhibitor. Renin was isolated by one step of immunoaffinity chromatography. The mixture of 2.5 m l of the crude extract, 2.5 ml of 20 mM Na-phosphate buffer, pH 7.0, containing 1 M NaCI and 5 ml of wet gel ofantirenin lgG-Sepharose previously equilibrated with 10 m M phosphate buffer, pH 7.0, containing 0.5 M NaCI were agitated for 24 h at 4 °C, and poured into a column (1.5 x 2.8 cm). The column was washed with the same buffer then eluted with 0.2 M acetic acid containing 0.5 M NaC1. The p H of the eluate was quickly adjusted to neutrality with 1 M TrisHCI buffer, pH 7.8. Renin activity was recovered at a greater than 40% yield (Fig. 1A). Renin inhibitor was found in the non-retained fractions which passed through the IgG-Sephaiose column (Fig. 1B). Thus, renin inhibitory activity was separated from renin. The mixture of 3 ml of the
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Fig. 1. Chromatography of crude extract of Neuro-2a cells on anti-renin IgG-Sepharose column (A) and inhibitory effect of the crude inhibitor from A on Neuro-2a renin activity (B). Crude extract was obtained from 275 x tOe cells of neuroblastoma. Total renin activity of crude extract was 27 ng angiotensin I/h and the renin recovered was 11.5 ng angiotensin I/h. The protein concentration in the fraction containing renin was too low to be determined. At the arrow, 0.2 M acetic acid containing 0.5 M NaC1 was applied to wash the column. The volume of each fraction was 0.5 ml. Horizontal bars indicate the fractions containing inhibitor (I), and fractions containing renin (II), respectively. In B, aliquots of crude inhibitor from A were incubated with 20/4 of Neuro-2a renin (capable of generating 135 pg of angiotensin 1/20 h) at 37 °C for 20 h. Renin-inhibitory activity was determined as described in text. The renin activity in the absence of the inhibitor was set equal to 100%.
375 solution with 30 ml of phosphate buffer, p H 7.0, was concentrated to 2.5 mi by ultratiltration over a PM 10 filter membrane. The concentrate was used to titrate the renin-inhibitory activity as shown in Fig. lB. Although the extent of inhibition of renin was progressively increased with increasing amount of inhibitor solution, the inhibitory effect leveled off without reaching complete inhibition. The renin inhibitor was purified further by making use of its affinity to renin. Two ml of the crude inhibitor solution was mixed with 2 ml of renin-Affi-Gel 10 previously equilibrated with 10 mM phosphate buffer, pH 7.0, and gently agitated for 3 h at 22 °C in the presence of protease inhibitors and L-154 added to concentrations as described above for the preparation of the crude extract. The mixture was then poured into a column (0.5 x 2.5 cm). After a wash with the same buffer, the column was eluted with the same buffer containing 0.5 M NaC1 then with 0.01 M acetic acid. As shown in Fig. 2A, the inhibitory substance was found to bind to the renin column and is released by 0.5 M NaC1. i
1-
(A)
However, the non-retained fractions, which passed through the renin gel column, were found to contain more than half of the inhibitory activity in spite o f 3 h of shaking of the mixture of the crude inhibitor and the renin gel. This incomplete binding seems to reflect a weak affinity between the inhibitor and submaxillary gland renin. The inhibitory substance released from the renin-Affi-Gel 10 column was not capable of inhibiting renin completely even with an increasing concentration of the inhibitor (Fig. 2B). In order to exclude the possibility that the apparent inhibition was due to destruction of angiotensin I by angiotensinase or destruction of renin by proteases in 'crude extract', the following control experiments were carried out. Synthetic angiotensin I (25 #1 of 5 ng/ml) was incubated at 37 °C with 25 #1 of the crude extract and 100 #1 of 0.1 M Na-phosphate buffer, pH 7.0, containing the mixture of the protease inhibitors and L-154 at concentrations identical to those used in the inhibition experiments. The reaction was terminated by heating in a boiling water bath for 3 rain and the remaining angiotensin
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Fig, 2. Renin-Affi-Gel10 affinitychromatography of the crude inhibitor from Fig. 1A (A) and effect of concentration of the isolated inhibitor on Neuro-2a renin activity (B). In A, an aliquot (100/4) of each fraction was incubated with 50 pl of Neuro-2a renin and the inhibitory activity was determined. The volume of fractions 1 through 14 was 0.6 ml and that of fractions 15 through 35 was 0.3 ml. The arrows represent the time at which the buffer change was made: arrow a, 10 rnM phosphate buffer, pH 7.0, containing 0.5 M NaCi; arrow b, 0.01 M acetic acid. The dotted line represents a level of renin activity pre-existing in each assay system. Horizontal bar is the inhibitor fractions pooled, In B, 50 pl of the isolated inhibitor from (A) was used for each assay.
376
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Fig. 3. Recovery of angiotensin I (A) and time course of Neuro-2a renin in crude extract (B) in the present assay system. In A, after incubation of standard angiotensin I (125 pg) with the crude extract in the presence of protease inhibitors and L-154 at 37 °C, remaining Angiotensin I was determined by radioimmunoassay. In B, 10 ffl of the crude extract was incubated at 37 °C in the presence of protease inhibitors and L-154.
I was determined. Recovery of angiotensin I added to the crude extract was practically quantitative over a period of 20 h as shown in Fig. 3A. Renin catalyzed reaction in the crude extract proceeded linearly for 24 h in the presence of the protease inhibitors and L-154, as shown in Fig. 3B, indicating that destruction of renin did not occur under the assay conditions employed in these studies. In the present study, we have devised methods for the separation of renin and its inhibitor, and demonstrated the presence of renin inhibitor in a cloned and cultured neuroblastoma cell line. It was shown that the inhibition is not the artifact of destruction of renin or angiotensin I. Earlier, we have demonstrated intracellular renin-angiotensin system in neuroblastoma cells and postulated an intracellular mechanism for the formation of angiotensin lI in neuronal cells. The demonstration of renin and renin inhibitor in the present studies indicates the endogenous inhibitor may play a role in the regulation of renin in the intracellular renin-angiotensin system in neuroblastoma cells and presumably in
neuronal cells. The renin inhibitor in neuroblastoma cells shows a distinct difference from renin binding protein in the kidney. While very high concentrations of renin (ffg/ml) and renal binding proteins were needed to detect the inhibition in the kidney extract 15, the renin inhibitor in the neurobtastoma cells is able to inhibit renin at a far lower concentration range of the enzyme (ng/ml), whose activity can be detected only after prolonged reaction ( > 12 h) with its substrate. Renal extract used at the high concentration contains a strong angiotensinase activity which may not be suppressed under the condition employed by Ueno et al. 15. This may account for the differences in the apparent discrepancy between the kidney and neuroblastoma studies. It seems that renin inhibitor in neuronal cells is different from renin binding protein. This work was supported by Research Grants HL-24112 and HL-14192 from National Institutes of Health. The able assistance of Ms. Trinita Fitzgerald is gratefully acknowledged.
377 1 Boyd, G. W.. A protein bound form of porcine renal renin, Circulat. Res., 35 (1974) 426--438. 2 Cohen, S., Taylor, J. M., Murakami, K., Michelakis, A. M. and Inagami, T., Isolation and characterization of renin-like enzymes from mouse submaxillary glands, Biochemistry, 23 (1972) 4286-4293. 3 Fishman, M. C., Zimmerman, E. A. and Slater, E. E., Renin and anglotensins: The complete system within the neuroblastoma × glioma cell, Science, 214 1.1981) 921923. 4 Funakawa, S., Funae, Y. and Yamamoto, K., Conversion between renin and high-molecular weight renin in the dog, Biochem. J., 176 (1978) 977-981. 5 Haber, E., Koerner, T., Page, L. B,, Kliman, B. and Purnode, A., Application of a radioimmunoassay for anglotensin I to the physiologic measurements of plasma renin activity in normal human subjects, J. Clin. Endocrinol., 29 (1969) 1349-1355. 6 lnagarni, T. and Murakami, K., Purification of high molecular weight forms of renin from hog kidney, Circulat. Res., 41 (1977) II 11-II 16. 7 Klebe, R. J. and Ruddle, F. H., Neuroblastorrm: cell culture analysis of a differentiating stem cell system, J. Cell BioL, 43 (1969) 69A. 8 Leckie, B. J. and McConnell, A., A renin inhibitor from rabbit kidney: conversion of a larger inactive renin to a smaller active enzyme, Circulat. Res., 36 (1975) 513-519. 9 Leckie, B. J., McConnell, A. and Jordan, J., Inactive renin - - A renin proenzyme? In J. Tang (Ed.), Acidproteases, Advances in Exp. Med. BioL, VoL 95, Plenum Press, New York, 1977, pp. 249-270. 10 Levine, M., Lentz, K. E., Kahn, J. R., Dorer, F. E. and
Skeggs, L. T., Studies on high molecular weight renin from hog kidney, Cireulat. Res., 42 (1978) 368-375. 11 Michelakis, A. M., Yoshida, H., Memzie, J., Murakami, K. and Inagami, T., A radioimmunoassay for the direct measurement of renin in mice and its application to submaxillary gland and kidney studies, Endocrinology, 94 (1974) 1101-1105. 12 Okamura, T., Clemens, D. L. and Inagami, T., Renin, angiotensins, and angiotensin-converting enzyme in neuroblastoma cells: evidence for intracellular formation of angiotensins, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 6940~943. 13 Sagnella, G. A., Caldwell, P. R. B. and Peart, W. S., Subcellular distribution of low- and high-molecularweight renin and its relation to a renin inhibitor in pig renal cortex, Clin. Sci., 59 (1980) 337-345. 14 Sagnella, G. A. and Peart, W. S., Properties of a renin inhibitor isolated from the pig kidney cortex, Clin. Sci., 60 (1981) 639-651. 15 Ueno, N., Miyazaki, H., Hirose, S. and Murakami, K., A 56,000-dalton renin-bindingprotein in hog kidney is an endogenous renin inhibitor, J. Biol. Chem., 256 (1981) 12023-12027. 16 Yamamoto, K., Ikemoto, F., Takaori, K. and Iwao, H., Nature of the renin binding substance, Proceedings of Satellite Symposium of 8th Interr,ational Congress of Pharmacology, (1981) in press. 17 Yokosawa, H., Yokosawa, N. and Inagami, T., Specific antibody to human renal renin and ;ts cross-reactivity with inactive human plasma prorenin (40897), Proc. Soc. Exp. Biol. Med., 164 (1980) 466-470.
373
Endogenous renin inhibitor in neuroblastoma cells TAISUKE INAGAKI, TOMIO OKAMURA and TADASHI INAGAMI Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232 (U.S.A.)
(Accepted July 6th, 1982) Key words: neuroblastoma cell -- renin - - renin inhibitor - - intracellular regulation -- intracellular function
Cloned neuroblastoma cells (Neuro-2a) in culture were found to contain a renin inhibitory substance. The inhibitor in the extract of cloned neuroblastoma cells was separated from renin activity by anti-renin IgG-Sepharose and selectivelyconcentrated by adsorption to renin-agarose gel. The present study demonstrated the coexistence of renin and its inhibitor in the same cell and suggested a possible regulatory mechanism of intracellular renin activity by an endogenous renin inhibitor in neuronal cells. Recent findings of coexistence in cultured neuroblastoma cells of renin, angiotensin I and IIa, le, angiotensin converting enzyme 12 and, by implication, angiotensinogen, suggests a possible intracellular mechanism of the formation of angiotensin II in these cells in contrast to the well known extracellular mechanism in plasma. The coexistence of the enzymes and substrate within the same cells also suggests an intracellular control mechanism by compartmentalization of these components or by a specific inhibitor. The presence of possible renin inhibitor was suggested earlier in conjunction with activatable high molecular weight renins isolated from kidney homogenates 1,s,1°. However, these activatable renins have been considered as an intermediate of the activation of inactive, single polypeptide renin zymogen 9. Renin binding protein (RBP) binds to renin in the presence of sulfhydryl blocking reagentsa, 15 and forms a high molecular weight renin 6. However, the binding was reported to have little effect on the enzyme activity. Recently, an inhibitor of renin was found in the kidney homogenates~a,~5 and its possible identity with RBP was suggested 15. While renin is known to be localized in renin granules ofjuxtaglomerular cells in the cortex of the kidney, the inhibitor was shown to be localized in cytosol fractions of kidney homogenatela,15. Furthermore, RBP was shown to be localized in tubular cells 16 rather than in juxtaglomerular cells. The finding that the inhibi0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
tion was demonstrable only at a very high concentration casts doubt on the physiological significance of RBP in the control of renin. Thus, much remains to be clarified about the regulatory mechanism of the intracellular system. To obtain insight in the control mechanism of the intracellular angiotensin generating system, we have investigated the possible existence of renin inhibitor in a mouse neuroblastoma cell line (Neuro-2a). The study was extended to include partial purification of the inhibitor by a new affinity chromatographic method. Renin-inhibitory activity was determined by the decrease in renin activity caused by the addition of cell extract fractions to renin assay mixture. Renin activity was determined by radioimmunoassay of angiotensin 15 generated at 37 °C in a mixture consisting of 25 #1 of partially purified hog angiotensinogen (3 #M), 50/A of neuroblastoma renin solution (prepared as below), 50 #1 of inhibitor solution (or water for control experiments) and 100 tzl of 0.1 M Na-phosphate buffer, pH 7.0, containing 5 mM EDTA, 1 mM diisopropyffiuorophosphate (DFP), 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 #g/ ml soybean trypsin inhibitor (STI), 5/~g/ml trasylol, 6/~g/ml leupeptin and 2/~M (1-5-carboxy-3-phenylpropyl)-L-l-lysyl-L-proline (L-154, an angiotensin converting enzyme inhibitor). These inhibitors were used to eliminate artifact arising from destruction of angiotensin I or renin. Renin and renin inhibitor were prepared from
374 extract o f c u l t m e d neuro-2a cells by immunoaffinity chromatography and by affinity chromatography on renin-Affi-Gel 10, respectively. The anti-renin lgG gel was prepared by the method of Yokosawa et al. 17 using rabbit anti-mouse submaxillary gland renin antibody n. Renin-Affi-Gel 10 was prepared by coupling 2 mg of purified mouse renin 2 to 2 ml of Affi-Gel 10 (Bio Rad) in 0.1 M phosphate buffer, pH 7.0, for 48 h at 4 °C with gentle agitation followed by treatment (3 h) with ethanolamine HCI by adding 0.2 ml of 1 M solution to the mixture. The gel was successively washed with 10 m M phosphate buffer, pH 7.0, the same buffer containing 1 M NaCI, 0.1 M acetic acid, again with 10 mM phosphate buffer pH 7.0 and stored at 4 °C. Neuro-2a cells 7 were grown in Dulbecco modified Eagle's medium containing 10% fetal calf serum, transferred to the serum-free medium for 24 h prior to harvest, detached from culture flasks with 1 m M EDTA and washed twice with the serumfree medium by the published method 12. Cells were then homogenized with polytron (Brinkman) in an ice bath for 15 s 4 times in 3 ml of water containing
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1 mM PMSF, 5 m M EDTA, 6 ffg/ml leupeptin, 1.25 ffM L-154 and 0.1% Triton X-100. The homogenate was centrifuged at 700 g for 15 rain and supernatant was recentrifuged at 32,000 g for 30 min. The supernatant designated as 'crude extract' was used as the source of renin and renin inhibitor. Renin was isolated by one step of immunoaffinity chromatography. The mixture of 2.5 m l of the crude extract, 2.5 ml of 20 mM Na-phosphate buffer, pH 7.0, containing 1 M NaCI and 5 ml of wet gel ofantirenin lgG-Sepharose previously equilibrated with 10 m M phosphate buffer, pH 7.0, containing 0.5 M NaCI were agitated for 24 h at 4 °C, and poured into a column (1.5 x 2.8 cm). The column was washed with the same buffer then eluted with 0.2 M acetic acid containing 0.5 M NaC1. The p H of the eluate was quickly adjusted to neutrality with 1 M TrisHCI buffer, pH 7.8. Renin activity was recovered at a greater than 40% yield (Fig. 1A). Renin inhibitor was found in the non-retained fractions which passed through the IgG-Sephaiose column (Fig. 1B). Thus, renin inhibitory activity was separated from renin. The mixture of 3 ml of the
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Fig. 1. Chromatography of crude extract of Neuro-2a cells on anti-renin IgG-Sepharose column (A) and inhibitory effect of the crude inhibitor from A on Neuro-2a renin activity (B). Crude extract was obtained from 275 x tOe cells of neuroblastoma. Total renin activity of crude extract was 27 ng angiotensin I/h and the renin recovered was 11.5 ng angiotensin I/h. The protein concentration in the fraction containing renin was too low to be determined. At the arrow, 0.2 M acetic acid containing 0.5 M NaC1 was applied to wash the column. The volume of each fraction was 0.5 ml. Horizontal bars indicate the fractions containing inhibitor (I), and fractions containing renin (II), respectively. In B, aliquots of crude inhibitor from A were incubated with 20/4 of Neuro-2a renin (capable of generating 135 pg of angiotensin 1/20 h) at 37 °C for 20 h. Renin-inhibitory activity was determined as described in text. The renin activity in the absence of the inhibitor was set equal to 100%.
375 solution with 30 ml of phosphate buffer, p H 7.0, was concentrated to 2.5 mi by ultratiltration over a PM 10 filter membrane. The concentrate was used to titrate the renin-inhibitory activity as shown in Fig. lB. Although the extent of inhibition of renin was progressively increased with increasing amount of inhibitor solution, the inhibitory effect leveled off without reaching complete inhibition. The renin inhibitor was purified further by making use of its affinity to renin. Two ml of the crude inhibitor solution was mixed with 2 ml of renin-Affi-Gel 10 previously equilibrated with 10 mM phosphate buffer, pH 7.0, and gently agitated for 3 h at 22 °C in the presence of protease inhibitors and L-154 added to concentrations as described above for the preparation of the crude extract. The mixture was then poured into a column (0.5 x 2.5 cm). After a wash with the same buffer, the column was eluted with the same buffer containing 0.5 M NaC1 then with 0.01 M acetic acid. As shown in Fig. 2A, the inhibitory substance was found to bind to the renin column and is released by 0.5 M NaC1. i
1-
(A)
However, the non-retained fractions, which passed through the renin gel column, were found to contain more than half of the inhibitory activity in spite o f 3 h of shaking of the mixture of the crude inhibitor and the renin gel. This incomplete binding seems to reflect a weak affinity between the inhibitor and submaxillary gland renin. The inhibitory substance released from the renin-Affi-Gel 10 column was not capable of inhibiting renin completely even with an increasing concentration of the inhibitor (Fig. 2B). In order to exclude the possibility that the apparent inhibition was due to destruction of angiotensin I by angiotensinase or destruction of renin by proteases in 'crude extract', the following control experiments were carried out. Synthetic angiotensin I (25 #1 of 5 ng/ml) was incubated at 37 °C with 25 #1 of the crude extract and 100 #1 of 0.1 M Na-phosphate buffer, pH 7.0, containing the mixture of the protease inhibitors and L-154 at concentrations identical to those used in the inhibition experiments. The reaction was terminated by heating in a boiling water bath for 3 rain and the remaining angiotensin
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Fig, 2. Renin-Affi-Gel10 affinitychromatography of the crude inhibitor from Fig. 1A (A) and effect of concentration of the isolated inhibitor on Neuro-2a renin activity (B). In A, an aliquot (100/4) of each fraction was incubated with 50 pl of Neuro-2a renin and the inhibitory activity was determined. The volume of fractions 1 through 14 was 0.6 ml and that of fractions 15 through 35 was 0.3 ml. The arrows represent the time at which the buffer change was made: arrow a, 10 rnM phosphate buffer, pH 7.0, containing 0.5 M NaCi; arrow b, 0.01 M acetic acid. The dotted line represents a level of renin activity pre-existing in each assay system. Horizontal bar is the inhibitor fractions pooled, In B, 50 pl of the isolated inhibitor from (A) was used for each assay.
376
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Fig. 3. Recovery of angiotensin I (A) and time course of Neuro-2a renin in crude extract (B) in the present assay system. In A, after incubation of standard angiotensin I (125 pg) with the crude extract in the presence of protease inhibitors and L-154 at 37 °C, remaining Angiotensin I was determined by radioimmunoassay. In B, 10 ffl of the crude extract was incubated at 37 °C in the presence of protease inhibitors and L-154.
I was determined. Recovery of angiotensin I added to the crude extract was practically quantitative over a period of 20 h as shown in Fig. 3A. Renin catalyzed reaction in the crude extract proceeded linearly for 24 h in the presence of the protease inhibitors and L-154, as shown in Fig. 3B, indicating that destruction of renin did not occur under the assay conditions employed in these studies. In the present study, we have devised methods for the separation of renin and its inhibitor, and demonstrated the presence of renin inhibitor in a cloned and cultured neuroblastoma cell line. It was shown that the inhibition is not the artifact of destruction of renin or angiotensin I. Earlier, we have demonstrated intracellular renin-angiotensin system in neuroblastoma cells and postulated an intracellular mechanism for the formation of angiotensin lI in neuronal cells. The demonstration of renin and renin inhibitor in the present studies indicates the endogenous inhibitor may play a role in the regulation of renin in the intracellular renin-angiotensin system in neuroblastoma cells and presumably in
neuronal cells. The renin inhibitor in neuroblastoma cells shows a distinct difference from renin binding protein in the kidney. While very high concentrations of renin (ffg/ml) and renal binding proteins were needed to detect the inhibition in the kidney extract 15, the renin inhibitor in the neurobtastoma cells is able to inhibit renin at a far lower concentration range of the enzyme (ng/ml), whose activity can be detected only after prolonged reaction ( > 12 h) with its substrate. Renal extract used at the high concentration contains a strong angiotensinase activity which may not be suppressed under the condition employed by Ueno et al. 15. This may account for the differences in the apparent discrepancy between the kidney and neuroblastoma studies. It seems that renin inhibitor in neuronal cells is different from renin binding protein. This work was supported by Research Grants HL-24112 and HL-14192 from National Institutes of Health. The able assistance of Ms. Trinita Fitzgerald is gratefully acknowledged.
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