- Email: [email protected]
A renin-like enzyme in luteal tissue
Molecular and Cellular Endocrinology, Elsevier Scientific Publishers Ireland,
269
41 (1986) 269-273 Ltd.
MCE 01531
Short Communication
A renin-like R.R. Cabrera, Luhoratorio de Reproduccibn y Lmtnncia
isorenin;
D.C. Guardia
and E. De Vito **
(LA RLA C), Consejo National de Investigaciones Casilla de Correo 855, Mendora (Argentina)
(Received
Kqy words: renin;
enzyme in luteal tissue *
renin of extrarenal
14 February
origin:
non-renin
1986; accepted
proteases;
Cientificas y Tknicas
(CONICET),
17 June 1986)
angiotensin:
corpus
luteum.
Summary The aim of this study was to identify immunologically and biologically a renin-like enzyme (RLE) in rat corpora lutea (CL). The biological activity of partially purified extracts of CL was tested in vivo by injection into anesthetized pentolinium-treated rats, obtaining a pressor response similar to renal renin. The enzyme activity in vitro was inhibited to about 50% by pretreatment with a specific antibody against renal renin. When the extracts were incubated with angiotensinogen, the product was inhibited mainly by angiotensin I antibody. The fact that there was no change in RLE content in 24 or 48 h nephrectomized rats, suggested the idea of a local production rather than an active blood renin sequestration.
Introduction The formation of angiotensin I (Ang I) from angiotensinogen hydrolysis has been detected in extracts of several mammalian tissues (Eskildsen, 1973; Ganten et al., 1976) suggesting that the occurrence of either renin or renin-like enzymes (RLE) is not restricted to the kidney. However, it is difficult to determine if the Ang I-generating activity is primarily due to an RLE or to non-renin proteases. Enzymes similar to kidney renin have been reported repeatedly in the female reproductive tract (Ganten et al., 1976). They have also been found in gonadotropin-producing cells, both in
* Preliminary
results of these studies were presented at The Fifth Scientific Meeting of the Inter-American Society of Hypertension, Guaruja, SZo Paulo, Brazil, March 1983. ** Members of Consejo National de Investigaciones Cientificas y Tknicas (CONICET) of Argentina. Reprint requests to Dr. Eduardo De Vito.
0303-7207/86/$03.50
0 1986 Elsevier Scientific
Publishers
Ireland,
pituitary and placenta, suggesting that they may play a role in the control of reproduction (Naruse et al., 1981; Poisner et al., 1981). The possibility of a contribution to plasma renin concentration has also been postulated (Fowler et al., 1981). However, the biological role of this extrarenal Ang I forming enzymes is still far from clear. In the present paper we present evidence on the existence of an enzyme capable to generate Ang I from angiotensinogen in luteal tissue from rats. Materials and methods Collection of CL and preparation of extracts Luteal tissue from adult female rats of a locally bred strain (originally Wistar stock) was used in this study. They were housed under conditions of standardized light (lights on from 06.00 to 20.00 h) and temperature (24 f 2°C) with free access to a balanced diet and water. Unless otherwise indicated they were sacrificed at 18:00 h. When nephrectomized rats were used, bilateral nephrecLtd.
270
tomy was performed under ether anesthesia either 24 or 48 h before sacrifice. Extract preparation. Luteal tissue was separated from the rest of the ovary by careful dissection under a stereoscopic microscope. It was later homogenized at 4°C in 0.154 M NaCl (100 mg of tissue per ml) in an Ultra-Turrax homogenizer and centrifuged at 24000 x g for 30 min. The clear supernatant was kept at -20°C if not immediately assayed. In a pool of luteal extracts, a primary purification of the enzyme was performed according to Procedure A of the method described by Haas et al. (1966). The fraction having renin-like activity will be referred to as ‘fraction II’ (FII). Protein concentration in the homogenates and FII preparations was determined by the method of Lowry et al. (1951). Assay of renin-like activity Renin-like activity was assayed by incubating the luteal extracts with an angiotensinogen preparation that was obtained from 48 h nephrectomized male rats or dogs, as was previously described for bovine serum angiotensinogen (De Vito et al., 1970). 25 ~1 of each luteal extract were added to tubes containing 0.875 ml of the angiotensinogen solution, pH 6.8 (2.5 pg of Ang I). The mixture was incubated at 37°C in the presence of 1.4 mM phenylmethylsulfonyl fluoride, 3.4 mM 8-hydroxyquinoline, 10 mM EDTA disodium salt, 3.3 mM l,lO-phenanthroline and 1.1 mM neomycin sulfate. After 16 h of incubation the reaction was stopped by adding 0.1 ml of 0.1 M HCl and placing the tubes in a boiling water bath for 5 min. The precipitate was removed and the clear supernatant was brought to pH 7 with 5 N NaOH. The substance generated during incubation (Ang I) was measured by either a modification of the radioimmunoassay of Haber et al. (1969) or by a biological assay on the basis of its pressor activity in anesthetized, pentolinium-treated rats (De Vito and Fasciolo, 1965). Renin-like activity in the extracts was expressed as ng of Ang I formed per mg of fresh tissue and per hour of incubation (ng Ang I/mg/h). Angiotensinase activity was controlled in every assay. Blanks for the angiotensinogen solution and for each extract were also run.
Assay of non-renin proteases A modified version of the method of Anson (1937) was used. 10 ~1 samples of the different extracts were incubated at 37°C with 500 ~1 of a 2% hemoglobin. Most of the incubations were run at pH 3.5 and pH 5.6 for either 30 min or 16 h. A few were done at pH 7 for 16 h. The reaction was stopped by addition of 1.0 ml 0.3 M trichloroacetic acid. Light absorption at 280 nm was measured in the clear supernatant. Protease activity was expressed as prnol of generated tyrosine per mg of tissue and per hour incubation (pm01 tyr/mg/h). Characterization of the renin-like enzyme Immunological studies. Rat renin was obtained from rat kidney cortex treated according to Procedure A of Haas et al. (1966) followed by an additional purification using an ion-exchange chromatograhy with DEAE-Sephadex (in batch), chromatography on Sephadex G-100 and after dialysis and concentration, the active fraction was applied to a DEAE-cellulose column. The fraction containing renin was dialyzed against 0.154 M NaCl and stored frozen at - 30°C. A specific antibody against rat kidney renin, generous gift from Dr. Tadashi Inagami, was used. The ability of this antibody to neutralize rat renin was examined by measuring inhibition of the enzymatic activity of renal renin (30 ng Ang I/h incubation at 37°C) by serial dilutions. This antibody resulted in 50% inhibition of renin activity when diluted 1 : 3200 and a complete inhibition when diluted 1 : 500. Experiments on luteal tissue renin were performed by incubating 25 ~1 of tissue extracts (100 mg/ml) with the antibody in a final dilution of 1 : 500 for 24 h at 4°C. FII was also incubated with diluted antibody, so that 25 ~1 yielded approximately the same amount of Ang I as the tissue extracts. Right after, those samples were incubated with angiotensinogen at 37°C as previously described. Characterization of the enzyme-substrate reaction product In some experiments, to further characterize the product of the enzyme-substrate reaction, it was incubated overnight at 4°C with several concentrations of antisera against Ang I and Ang II.
271
These antisera were raised in rabbits according to the method of Menard and Catt (1972). Both antibodies were specific, inhibiting only the pressor effect of the corresponding angiotensin. Statistics Data are expressed as means + standard of the mean. Significances of differences calculated by Student’s t-test.
error were
Results Presence of renin-like activity in luteal extracts and ‘fraction II’ preparations All luteal tissue specimens examined has measurable activity after incubation with both rat or dog angiotensinogen. However, extracts of nonluteal ovarian tissues were unable to generate Ang I when incubated with angiotensinogen. The same was true when luteal extracts were treated with trypsin or boiled before incubation (Fig. 1A). Ang I recoveries ranged from 81 to 115%: indicating
kw AI 1 2.5ng
2
3
A
4 AI 5 AI 2.6ng 25ng
8
AI 123 2bng
4
S
that the angiotensinase activity was either low or absent. No Ang I was detected in any of the blanks after incubation. Enzymatic activity in luteal extracts from 30 randomly cycling intact virgin rats incubated with the dog angiotensinogen was 1.08 * 0.17 ng Ang I/mg/h (mean + SE). Bilateral nephrectomy, performed 24 or 48 h before sacrifice (six animals each), did not cause any significant change in luteal renin activity when compared with shamoperated animals (1.09 f 0.17 ng Ang I/mg/h and 1.11 k 0.18 ng Ang/I/mg/h, respectively). After dialysis, the FII fraction (non-incubated with angiotensinogen) was tested by intravenous injection in the rat bioassay. The result was a pressor response similar to those produced by renal renin (Fig. 2). Characteristics of the luteal enzyme To perform immunological studies, 12 different luteal tissue extracts were examined, as well as FII fraction. The total RLE activity and the percent inhibition by antibody pretreatment are shown in Table 1. Inhibition of RLE activity was found in all the rat samples examined after antibody pretreatment. Note that inhibition was between 31 and 62% for the luteal extracts suggesting that specific renin activity is about 50% of the total RLE activity. The pressor activity generated by incubation of different luteal tissue extracts with dog angiotensinogen was tested by treating with antisera against Ang I and Ang II. When the antiserum against Ang I was used, more than 94% of the pressor activity was inhibited. Less than 20% of the initial
AI
l.Sng
Fig. 1. Blood pressure response in the rat biological assay. Panel A: Injection of Ang I standard and 50 pl of the incubation product of three different rat CL extracts (1, 2 and 3). Extracts 4 and 5: response to 100 81 of the incubation product from extracts 1 and 2 previously boiled and treated with trypsin respectively prior to angiotensinogen incubation. Panel E: Inhibition of the blood pressure response to pressor material generated by incubation of CL extracts with dog angiotensinogen (1) treated with antibody against Ang I (2) and Ang II (4) and regeneration of pressor effect by antibody destruction after boiling (3-5).
AI 2.Sng
Fll 20~81
Al 2.5ng
FlI@ 20~11
FII SO_ul
Fig. 2. Blood pressure response to fraction II without angiotensinogen incubation. Blood pressure response to fraction II (FII) obtained according to Procedure A of Haas et al. (1966). No response was observed after sample boiling (FII 5).
272
TABLE
1
EFFECT ON ANG I PRODUCTION AGAINST RAT RENIN
OF
ANTIBODIES
Samples were incubated with dog angiotensinogen after pretreatment with antibody against renin, as described in the text. The production of Ang I was compared with the sample run without antibody pretreatment, Mean * SE (CL extract of rats R l-10) first column =1.07kO.O9 ng Ang I/mg/h. Second column = 0.55 k 0.06 ng Ang I/mg/h. The values differ significantly: P c 0.001. Sample
Not pretreated with antibody (ng Ang I/ml/h)
‘Fraction 11’ 1.67 CLofRl 1.17 CLofR2 1.27 CLofR3 1.33 CLofR4 1.18 CLofR5 0.76 CLofR6 1.22 CLofR7 1.46 CLofR8 0.78 CLofR9 0.82 CL of R 10 0.72
Pretreated with antibody (ng Ang I/ml/h)
% Inhibition
0.47 0.49 0.77 0.64 0.81 0.29 0.50 0.80 0.49 0.43 0.30
72 58 39 52 31 62 59 45 37 48 58
pressor activity was inhibited by the antiserum against Ang II. The inhibitory activity of both antisera was destroyed by boiling them before the incubation (Fig. 1 B). Non-renin proteolytic actiuity The activity of non-renin proteases was measured in several luteal specimens. A high concentration of acid proteases (acting at pH 3.5) was found in most cases (0.36 = 0.02 pmol tyr/mg/h, mean + SD). The activity of proteases was very low, but not absent, at pH 5.6, after 16 h of incubation (0.05 k 0,004 pmol tyr/mg/h, mean + SE). The activity of non-renin proteases was not detected when FII was incubated during 16 h at pH 7. Discussion This study describes the presence of an Ang I-releasing enzyme in the corpora lutea of rats. One key question is whether or not this enzyme found in the CL is a renin-like enzyme (isorenin). The finding that a specific antibody against renin
neutralized about 50% of the total Ang I-releasing activity demonstrates the presence of specific immunoreactive renin in rat luteal tissue. On the other hand, these data support the concept that renin-like activity in luteal tissue is a mixture of immunoreactive renin and other Ang I-releasing enzymes. This fact is usual for all tissue isorenins hitherto studied. Another important observation was the finding of a pressor response similar to that produced by renin, when the FII preparations (primary purification) from CL extracts were tested directly in the rat bioassay, indicating an in vivo effect produced by a biologically active enzyme under physiological conditions. Non-specific neutral proteases seem not to be involved in this response, since no activity of these proteases was detected in the in vitro assay at pH 7. On the other hand, the product of the enzymesubstrate reaction is primarily Ang I since it was inhibited mainly by the specific antiserum against Ang I. The antibodies we used showed a noncrossreaction when they were tested against synthetic Ang I and II. A partial inhibition by antibody against Ang II was, however, observed when the product of the enzyme-substrate reaction was tested. A similar result was found by Carretero et al. (1972) with an enzyme extracted from the dog uterus. They could not, and neither could we, explain satisfactorily this fact. But it is clear that the pressor substance released is primarily Ang I. According to the present results, an important question is whether the tissue renin found in the CL has been synthesized in this tissue. In spite of the results obtained using nephrectomized rats, cellular storage of renal renin in luteal tissue cannot be excluded. However, the lack of correlation between luteal activity and plasma renin activity or concentration (unpublished observations) may favor the idea of a local production. On the other hand, only through an active storage process, as far as we know never before described, it would be possible to explain the amount of enzyme found in the luteal tissue, taking into account the considerably limited amount of blood which could remain in the CL. Finally, the results obtained with an antibody capable to distinguish specific renin from enzymes with non-specific Ang I-generating properties,
273
demonstrate the presence of renin in luteal tissue. As far as we know, we presented the first evidence on the existence of a renin-like enzyme in luteal tissue. Little is known about the physiological significance of renin-like enzymes of extrarenal origin and further studies are required for the elucidation of its biological role in the corpus luteum. Acknowledgements The generous gift of specific antibody against rat renin from Dr. Tadashi Inagami of the Vanderbilt University, Nashville, TN, U.S.A., is greatly appreciated. The authors express their thanks to Dr. R.P. Deis for several fruitful discussions during the course of this research, and to Dr. A. Castro Vazquez for the critical reading of this manuscript. This work was supported by a grant from the BID-CONICET Program.
References Anson, M.L. (1937) J. Gen. Physiol. 20, 561-569. Carretero, O.A., Bujak, B. and Houle, J.A. (1972) Am. J. Physiol. 220, 1468-1472 De Vito, E. and Fasciolo, J.C. (1965) Acta Physiol. Pharmacol. Latinoam. 15, 129-137. De Vito, E., Gordon, S.B., Cabrera, R.R. and Fasciolo, J.C. (1970) Am. J. Physiol. 219, 1036-1041. Eskildsen, P.C. (1973) Acta Pathol. Microbial. Stand. 81, 263-268. Fowler, Jr., W.L., Johnson, J.A., Kurz, K.D., Kilfoil, J., Love, S. and Payne, C.G. (1981) Endocrinology 109, 290-295. Ganten, D., Schelling, P., Vecsei, P. and Ganten, U. (1976) Am. J. Med. 60, 760-772. Haas, E., Goldblatt, H., Gipson, E.C. and Lewis, L. (1966) Circ. Res. 19, 739-749. Haber, E., Koemer, D., Page, L.B., Kliman, B. and Purnode, A. (1969) J. Clin. Endocrinol. Metab. 29, 1349-1355. Lowry, D.M., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Menard, J. and Catt, K.J. (1972) Endocrinology 90, 422-430. Naruse, K., Takii, Y. and Inagami, T. (1981) Proc. Natl. Acad. Sci. 78, 7579-7583. Poisner, A.M., Wood, G.W., Poisner, R. and Inagami, T. (1981) Endocrinology 109, 1150-1155. Skeggs, L.T., Lentz, K.E., Hochstrasser, H. and Kahn, J.R. (1963) J. Exp. Med. 118, 73-98.
269
41 (1986) 269-273 Ltd.
MCE 01531
Short Communication
A renin-like R.R. Cabrera, Luhoratorio de Reproduccibn y Lmtnncia
isorenin;
D.C. Guardia
and E. De Vito **
(LA RLA C), Consejo National de Investigaciones Casilla de Correo 855, Mendora (Argentina)
(Received
Kqy words: renin;
enzyme in luteal tissue *
renin of extrarenal
14 February
origin:
non-renin
1986; accepted
proteases;
Cientificas y Tknicas
(CONICET),
17 June 1986)
angiotensin:
corpus
luteum.
Summary The aim of this study was to identify immunologically and biologically a renin-like enzyme (RLE) in rat corpora lutea (CL). The biological activity of partially purified extracts of CL was tested in vivo by injection into anesthetized pentolinium-treated rats, obtaining a pressor response similar to renal renin. The enzyme activity in vitro was inhibited to about 50% by pretreatment with a specific antibody against renal renin. When the extracts were incubated with angiotensinogen, the product was inhibited mainly by angiotensin I antibody. The fact that there was no change in RLE content in 24 or 48 h nephrectomized rats, suggested the idea of a local production rather than an active blood renin sequestration.
Introduction The formation of angiotensin I (Ang I) from angiotensinogen hydrolysis has been detected in extracts of several mammalian tissues (Eskildsen, 1973; Ganten et al., 1976) suggesting that the occurrence of either renin or renin-like enzymes (RLE) is not restricted to the kidney. However, it is difficult to determine if the Ang I-generating activity is primarily due to an RLE or to non-renin proteases. Enzymes similar to kidney renin have been reported repeatedly in the female reproductive tract (Ganten et al., 1976). They have also been found in gonadotropin-producing cells, both in
* Preliminary
results of these studies were presented at The Fifth Scientific Meeting of the Inter-American Society of Hypertension, Guaruja, SZo Paulo, Brazil, March 1983. ** Members of Consejo National de Investigaciones Cientificas y Tknicas (CONICET) of Argentina. Reprint requests to Dr. Eduardo De Vito.
0303-7207/86/$03.50
0 1986 Elsevier Scientific
Publishers
Ireland,
pituitary and placenta, suggesting that they may play a role in the control of reproduction (Naruse et al., 1981; Poisner et al., 1981). The possibility of a contribution to plasma renin concentration has also been postulated (Fowler et al., 1981). However, the biological role of this extrarenal Ang I forming enzymes is still far from clear. In the present paper we present evidence on the existence of an enzyme capable to generate Ang I from angiotensinogen in luteal tissue from rats. Materials and methods Collection of CL and preparation of extracts Luteal tissue from adult female rats of a locally bred strain (originally Wistar stock) was used in this study. They were housed under conditions of standardized light (lights on from 06.00 to 20.00 h) and temperature (24 f 2°C) with free access to a balanced diet and water. Unless otherwise indicated they were sacrificed at 18:00 h. When nephrectomized rats were used, bilateral nephrecLtd.
270
tomy was performed under ether anesthesia either 24 or 48 h before sacrifice. Extract preparation. Luteal tissue was separated from the rest of the ovary by careful dissection under a stereoscopic microscope. It was later homogenized at 4°C in 0.154 M NaCl (100 mg of tissue per ml) in an Ultra-Turrax homogenizer and centrifuged at 24000 x g for 30 min. The clear supernatant was kept at -20°C if not immediately assayed. In a pool of luteal extracts, a primary purification of the enzyme was performed according to Procedure A of the method described by Haas et al. (1966). The fraction having renin-like activity will be referred to as ‘fraction II’ (FII). Protein concentration in the homogenates and FII preparations was determined by the method of Lowry et al. (1951). Assay of renin-like activity Renin-like activity was assayed by incubating the luteal extracts with an angiotensinogen preparation that was obtained from 48 h nephrectomized male rats or dogs, as was previously described for bovine serum angiotensinogen (De Vito et al., 1970). 25 ~1 of each luteal extract were added to tubes containing 0.875 ml of the angiotensinogen solution, pH 6.8 (2.5 pg of Ang I). The mixture was incubated at 37°C in the presence of 1.4 mM phenylmethylsulfonyl fluoride, 3.4 mM 8-hydroxyquinoline, 10 mM EDTA disodium salt, 3.3 mM l,lO-phenanthroline and 1.1 mM neomycin sulfate. After 16 h of incubation the reaction was stopped by adding 0.1 ml of 0.1 M HCl and placing the tubes in a boiling water bath for 5 min. The precipitate was removed and the clear supernatant was brought to pH 7 with 5 N NaOH. The substance generated during incubation (Ang I) was measured by either a modification of the radioimmunoassay of Haber et al. (1969) or by a biological assay on the basis of its pressor activity in anesthetized, pentolinium-treated rats (De Vito and Fasciolo, 1965). Renin-like activity in the extracts was expressed as ng of Ang I formed per mg of fresh tissue and per hour of incubation (ng Ang I/mg/h). Angiotensinase activity was controlled in every assay. Blanks for the angiotensinogen solution and for each extract were also run.
Assay of non-renin proteases A modified version of the method of Anson (1937) was used. 10 ~1 samples of the different extracts were incubated at 37°C with 500 ~1 of a 2% hemoglobin. Most of the incubations were run at pH 3.5 and pH 5.6 for either 30 min or 16 h. A few were done at pH 7 for 16 h. The reaction was stopped by addition of 1.0 ml 0.3 M trichloroacetic acid. Light absorption at 280 nm was measured in the clear supernatant. Protease activity was expressed as prnol of generated tyrosine per mg of tissue and per hour incubation (pm01 tyr/mg/h). Characterization of the renin-like enzyme Immunological studies. Rat renin was obtained from rat kidney cortex treated according to Procedure A of Haas et al. (1966) followed by an additional purification using an ion-exchange chromatograhy with DEAE-Sephadex (in batch), chromatography on Sephadex G-100 and after dialysis and concentration, the active fraction was applied to a DEAE-cellulose column. The fraction containing renin was dialyzed against 0.154 M NaCl and stored frozen at - 30°C. A specific antibody against rat kidney renin, generous gift from Dr. Tadashi Inagami, was used. The ability of this antibody to neutralize rat renin was examined by measuring inhibition of the enzymatic activity of renal renin (30 ng Ang I/h incubation at 37°C) by serial dilutions. This antibody resulted in 50% inhibition of renin activity when diluted 1 : 3200 and a complete inhibition when diluted 1 : 500. Experiments on luteal tissue renin were performed by incubating 25 ~1 of tissue extracts (100 mg/ml) with the antibody in a final dilution of 1 : 500 for 24 h at 4°C. FII was also incubated with diluted antibody, so that 25 ~1 yielded approximately the same amount of Ang I as the tissue extracts. Right after, those samples were incubated with angiotensinogen at 37°C as previously described. Characterization of the enzyme-substrate reaction product In some experiments, to further characterize the product of the enzyme-substrate reaction, it was incubated overnight at 4°C with several concentrations of antisera against Ang I and Ang II.
271
These antisera were raised in rabbits according to the method of Menard and Catt (1972). Both antibodies were specific, inhibiting only the pressor effect of the corresponding angiotensin. Statistics Data are expressed as means + standard of the mean. Significances of differences calculated by Student’s t-test.
error were
Results Presence of renin-like activity in luteal extracts and ‘fraction II’ preparations All luteal tissue specimens examined has measurable activity after incubation with both rat or dog angiotensinogen. However, extracts of nonluteal ovarian tissues were unable to generate Ang I when incubated with angiotensinogen. The same was true when luteal extracts were treated with trypsin or boiled before incubation (Fig. 1A). Ang I recoveries ranged from 81 to 115%: indicating
kw AI 1 2.5ng
2
3
A
4 AI 5 AI 2.6ng 25ng
8
AI 123 2bng
4
S
that the angiotensinase activity was either low or absent. No Ang I was detected in any of the blanks after incubation. Enzymatic activity in luteal extracts from 30 randomly cycling intact virgin rats incubated with the dog angiotensinogen was 1.08 * 0.17 ng Ang I/mg/h (mean + SE). Bilateral nephrectomy, performed 24 or 48 h before sacrifice (six animals each), did not cause any significant change in luteal renin activity when compared with shamoperated animals (1.09 f 0.17 ng Ang I/mg/h and 1.11 k 0.18 ng Ang/I/mg/h, respectively). After dialysis, the FII fraction (non-incubated with angiotensinogen) was tested by intravenous injection in the rat bioassay. The result was a pressor response similar to those produced by renal renin (Fig. 2). Characteristics of the luteal enzyme To perform immunological studies, 12 different luteal tissue extracts were examined, as well as FII fraction. The total RLE activity and the percent inhibition by antibody pretreatment are shown in Table 1. Inhibition of RLE activity was found in all the rat samples examined after antibody pretreatment. Note that inhibition was between 31 and 62% for the luteal extracts suggesting that specific renin activity is about 50% of the total RLE activity. The pressor activity generated by incubation of different luteal tissue extracts with dog angiotensinogen was tested by treating with antisera against Ang I and Ang II. When the antiserum against Ang I was used, more than 94% of the pressor activity was inhibited. Less than 20% of the initial
AI
l.Sng
Fig. 1. Blood pressure response in the rat biological assay. Panel A: Injection of Ang I standard and 50 pl of the incubation product of three different rat CL extracts (1, 2 and 3). Extracts 4 and 5: response to 100 81 of the incubation product from extracts 1 and 2 previously boiled and treated with trypsin respectively prior to angiotensinogen incubation. Panel E: Inhibition of the blood pressure response to pressor material generated by incubation of CL extracts with dog angiotensinogen (1) treated with antibody against Ang I (2) and Ang II (4) and regeneration of pressor effect by antibody destruction after boiling (3-5).
AI 2.Sng
Fll 20~81
Al 2.5ng
FlI@ 20~11
FII SO_ul
Fig. 2. Blood pressure response to fraction II without angiotensinogen incubation. Blood pressure response to fraction II (FII) obtained according to Procedure A of Haas et al. (1966). No response was observed after sample boiling (FII 5).
272
TABLE
1
EFFECT ON ANG I PRODUCTION AGAINST RAT RENIN
OF
ANTIBODIES
Samples were incubated with dog angiotensinogen after pretreatment with antibody against renin, as described in the text. The production of Ang I was compared with the sample run without antibody pretreatment, Mean * SE (CL extract of rats R l-10) first column =1.07kO.O9 ng Ang I/mg/h. Second column = 0.55 k 0.06 ng Ang I/mg/h. The values differ significantly: P c 0.001. Sample
Not pretreated with antibody (ng Ang I/ml/h)
‘Fraction 11’ 1.67 CLofRl 1.17 CLofR2 1.27 CLofR3 1.33 CLofR4 1.18 CLofR5 0.76 CLofR6 1.22 CLofR7 1.46 CLofR8 0.78 CLofR9 0.82 CL of R 10 0.72
Pretreated with antibody (ng Ang I/ml/h)
% Inhibition
0.47 0.49 0.77 0.64 0.81 0.29 0.50 0.80 0.49 0.43 0.30
72 58 39 52 31 62 59 45 37 48 58
pressor activity was inhibited by the antiserum against Ang II. The inhibitory activity of both antisera was destroyed by boiling them before the incubation (Fig. 1 B). Non-renin proteolytic actiuity The activity of non-renin proteases was measured in several luteal specimens. A high concentration of acid proteases (acting at pH 3.5) was found in most cases (0.36 = 0.02 pmol tyr/mg/h, mean + SD). The activity of proteases was very low, but not absent, at pH 5.6, after 16 h of incubation (0.05 k 0,004 pmol tyr/mg/h, mean + SE). The activity of non-renin proteases was not detected when FII was incubated during 16 h at pH 7. Discussion This study describes the presence of an Ang I-releasing enzyme in the corpora lutea of rats. One key question is whether or not this enzyme found in the CL is a renin-like enzyme (isorenin). The finding that a specific antibody against renin
neutralized about 50% of the total Ang I-releasing activity demonstrates the presence of specific immunoreactive renin in rat luteal tissue. On the other hand, these data support the concept that renin-like activity in luteal tissue is a mixture of immunoreactive renin and other Ang I-releasing enzymes. This fact is usual for all tissue isorenins hitherto studied. Another important observation was the finding of a pressor response similar to that produced by renin, when the FII preparations (primary purification) from CL extracts were tested directly in the rat bioassay, indicating an in vivo effect produced by a biologically active enzyme under physiological conditions. Non-specific neutral proteases seem not to be involved in this response, since no activity of these proteases was detected in the in vitro assay at pH 7. On the other hand, the product of the enzymesubstrate reaction is primarily Ang I since it was inhibited mainly by the specific antiserum against Ang I. The antibodies we used showed a noncrossreaction when they were tested against synthetic Ang I and II. A partial inhibition by antibody against Ang II was, however, observed when the product of the enzyme-substrate reaction was tested. A similar result was found by Carretero et al. (1972) with an enzyme extracted from the dog uterus. They could not, and neither could we, explain satisfactorily this fact. But it is clear that the pressor substance released is primarily Ang I. According to the present results, an important question is whether the tissue renin found in the CL has been synthesized in this tissue. In spite of the results obtained using nephrectomized rats, cellular storage of renal renin in luteal tissue cannot be excluded. However, the lack of correlation between luteal activity and plasma renin activity or concentration (unpublished observations) may favor the idea of a local production. On the other hand, only through an active storage process, as far as we know never before described, it would be possible to explain the amount of enzyme found in the luteal tissue, taking into account the considerably limited amount of blood which could remain in the CL. Finally, the results obtained with an antibody capable to distinguish specific renin from enzymes with non-specific Ang I-generating properties,
273
demonstrate the presence of renin in luteal tissue. As far as we know, we presented the first evidence on the existence of a renin-like enzyme in luteal tissue. Little is known about the physiological significance of renin-like enzymes of extrarenal origin and further studies are required for the elucidation of its biological role in the corpus luteum. Acknowledgements The generous gift of specific antibody against rat renin from Dr. Tadashi Inagami of the Vanderbilt University, Nashville, TN, U.S.A., is greatly appreciated. The authors express their thanks to Dr. R.P. Deis for several fruitful discussions during the course of this research, and to Dr. A. Castro Vazquez for the critical reading of this manuscript. This work was supported by a grant from the BID-CONICET Program.
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