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Evidence that the arginine esteropeptidase (gamma) subunit of nerve growth factor can activate inactive renin
Neuroscience Letters, 24 (1981) 87-92
87
Elsevier/North-Holland Scientific Publishers Ltd.
EVIDENCE THAT THE ARGININE ESTEROPEPTIDASE (GAMMA) SUBUNIT OF NERVE GROWTH FACTOR CAN ACTIVATE INACTIVE RENIN
BRIAN J. MORRIS, DANIEL F. CATANZAROand RITA T. DE ZWART Department of Physiology, The University of Sydney, New South Wales, 2006 (Australia)
(Received February 5th, 1981; Revised version received March 12th, 1981; Accepted March 23rd, 1981)
The beta and gamma subunits of nerve growth factor were isolated and tested for inactive-reninactivating activity using human amniotic fluid as a convenient source of inactive renin. Only the gamma subunit, which is an arginine esteropeptidase, could activate inactive renin. The role of this enzyme in sites containing both it and inactive renin warrants investigation.
Extracts o f brain contain a molecule that is recognized by antisera to pure renal renin and that can hydrolyze angiotensin I (ANG I) from angiotensinogen at pH 7.5 [7, 8, 10], suggesting that renin is present in the central nervous system. Immunocytochemical studies have demonstrated a wide distribution of renin-like material in various regions of the brain [5, 26]. Most recently the existence, in addition, of an inactive, prorenin-like molecule, has been demonstrated in brain extracts [8]. Activation of this molecule may be the primary event leading to intracerebral generation of angiotensin II, which is known to increase blood pressure, stimulate drinking and release vasopressin by central actions [2, 24, 25] and may play a role in the maintenance of hypertension in spontaneously hypertensive rats [9, 24]. However, the enzyme responsible for activation of inactive renin in neural tissue is not known. Many proteinases can activate inactive renin in biological fluids and kidney [12, 15-19, 21]. However, most of these are not present in the brain. One which is present in brain and can activate inactive renin in human amniotic fluid is cathepsin D [15], activity of which was responsible for earlier reports of renin-like activity in extracts of brain [4, 20]. Such a reaction would be restricted to intracellular compartments having a favourable pH in the acid range. A proteinase that is active at physiological pH and is present in a variety of tissue is the arginine esteropeptidase (gamma) subunit of nerve growth factor (NGF). -~NGF is part of the 75 NGF complex and functions to convert pro-~-NGF to/~NGF, the subunit responsible for activity in promoting nerve growth. The brain contains low levels of ~NGF [27] and 3,NGF might be expected to be present as well. In a recent preliminary communication a crude preparation of NGF was reported to cause activation o f inactive renin in human amniotic fluid [16]. However, the fact that 03404-3940/81/0000-0000/$ 02.50 ©Elsevier/North-Holland Scientific Publishers Ltd.
88 renin activity could be detected in the preparation suggested that other contaminating enzymes might have been responsible for the activation. Therefore, in the present experiment purification procedures were conducted in order to isolate the beta and gamma subunits of NGF for evaluation of possible inactive-reninactivating activity. H u m a n amniotic fluid, the richest most readily available source of inactive renin was used in the studies. Subunits of NGF were isolated from the submandibular glands of adult, male Quackenbush mice, the richest source of this substance. The t3 subunit was isolated as 2.5S NGF by the method of Mobley et al. [14], as described by Jeng and Bradshaw [11]. The final preparation, eluted from a CM52-Cellulose column by 5 mM Tris-HC1, pH 9, 0.5 M NaC1, ran as a single band of Mr 13,000 on SDS gel electrophoresis and was active in promoting nerve growth (as tested in the laboratory of Dr. M.R. Bennett by conventional techniques [13]). The ) subunit was isolated from glands, as described by Jeng and Bradshaw [11]. An assay based on the hydrolysis of benzoyl-arginine-para-nitroaniline was used to localize arginine esterase activity in fractions. Particularly good resolution was obtained during elution of the CM-Cellulose column with a parabolic 0-1 M NaC1 gradient. The final preparation ran as a Mr 25,000 band on SDS gel electrophoresis. A second preparation of ~,NGF (kindly supplied by Dr. E.M. Shooter, Stanford University) was also used in some experiments. Human amniotic fluid, which contained - 1800 pmol ANG I . h l/ml of acid activatable renin together with low concentrations of proteinase inhibitors, was dialyzed overnight at 4°C in 15 vol. of 100 mM sodium phosphate buffer, pH 7.4, containing 1 mM EDTA and 75 mM NaC1. Portions of 200/~1 were mixed with 10 izl aliquots of NGF subunit to give 0, 0.4, 2 and 10 izg/ml. The mixtures were incubated at 22°C for 0, 0.2, 0.5, 1, 2, 3, 4 and 5 h and renin assay performed using 10 #1 sample and 200 #1 plasma from a nephrectomized sheep (1 h, 37°C, pH 7.4) [22]. The reaction was stopped by dilution and boiling before ANG I radioimmunoassay. Inhibition of arginine esteropeptidase (3,NGF) activity in the experiments was achieved by addition of a mixture of 5 mM (final concentration) phenylmethanesulphonylfluoride (PMSF), 0.3 mg/ml lima bean trypsin inhibitor (LBTI) and 0.3 mg/ml soya bean trypsin inhibitor (SBTI). Electrophoretically pure t3NGF is commonly found to be contaminated with renin [3, 6] and our preparation was no exception. Renin activity was 404 + 21 pmol ANG I-h l/#g (mean +_ S.D. of 8 replicates). All of the renin activity in mixtures of amniotic fluid and/~NGF could be accounted for entirely by the summation of the pre-existing renin activities in each. The mixture of PMSF, LBTI and SBTI does not inhibit renin activity so that addition of this to the mixtures had no effect on renin activities observed. At final concentrations of 0, 0.4, 2 and 10 /~g ~ N G F / m l amniotic fluid, renin activities were 261 + 21 and 276 + 16 with PMSF, LBTI, SBTI, 3 0 5 + 4 0 and 328+__27, 833___59 and 8 8 5 + 4 2 , and 4 2 6 2 + 5 5 5 and 4 3 3 8 + 6 8 9 pmol ANG I . h - l / m l ( m e a n ± S . D . of 8 replicates). When the
89
/3NGF/amniotic fluid mixtures were incubated at 22°C over 5 h there was no increase in renin activity above the levels seen at zero time. The absence of any increase in renin during incubation of amniotic fluid with /3NGF indicates that /3NGF has no inactive-renin-activating activity. Experiments were then performed with 3,NGF. In a preliminary experiment the importance of inhibiting TNGF activity before the assay of renin was demonstrated. In the absence of PMSF/LBTI/SBTI there was increased apparent formation of ANG I (Table I). However, this was not due to renin because if 3,NGF activity was inhibited by PMSF/LBTI/SBTI (which do not inhibit renin) before samples were assayed there was a 96°70 reduction in the generation of ANG I-like immunoreactive material in the renin assay. The activity might therefore have been due to generation by 3,NGF from sheep plasma of material that cross-reacted with the ANG I antiserum. The necessity of adding PMSF/LBTI/SBTI before renin assay was vital for renin measurements to be valid. The results of these experiments also indicate that the "rNGF used had little, if any, contamination with renin. In order to test for inactive-renin-activating activity of 3,NGF, amniotic fluid was initially acidified briefly since this has been found to increase the sensitivity of inactive renin to activation by arginine esteropeptidases such a trypsin (J.G. McGirr and B.J. Morris, unpublished results). Therefore the pH of amniotic fluid was adjusted to 3.3 at 4°C by dropwise addition of conc. H2SO4 and after 30 min was readjusted to pH 7.4 with 10 M NaOH. After adding 1/20 vol. of 1 M sodium phosphate buffer, pH 7.4, portions of 200 #1 were incubated at 22°C with 10 ~1 3,NGF, as before. At the end of each period of incubation 5 /d of solutions of PMSF, LBTI and SBTI were added to give final concentrations of 5 mM, 0.3 mg/ml
TABLE 1 INTERFERENCE RENIN ASSAY
BY T H E G A M M A SUBUN1T O F N E R V E G R O W T H F A C T O R 1N T H E
R e n i n (pmol A N G I • h - I / m l a m n i o t i c fluid):
Aa +Inhibitorsb Ba Alone +lnhibitors
c o n c e n t r a t i o n o f ",/NGF 0zg/ml): 0 0.4
2
10
231 _+ 42 243 _+ 15
365 + 47 238 + 18
798 +_ 75 261 _+ 23
8740 +_ 548 588 +_ 52
253 _+ 7 229 _+ 18
276 _+ 28 246 _+ 23
501 +_ 59 258 _+ 19
2830 _+ 522 338 __+ 33
a T w o d i f f e r e n t p r e p a r a t i o n s o f - r N G F : A = ours: B = S h o o t e r ' s (The lower activity of the latter m a y be due to losses d u r i n g s h i p m e n t o f the l y o p h i l i z e d p r e p a r a t i o n . ) b The "),NGF i n h i b i t o r s - P M S F , L B T I a n d SBTI - were a d d e d after 2 2 ° C i n c u b a t i o n o f "~NGF with a m n i o t i c fluid, i.e. b e f o r e renin assay.
90 a n d 0.3 m g / m l , respectively, in o r d e r to stop the activity o f . y N G F in activating inactive r e n i n a n d in f o r m i n g i m m u n o r e a c t i v e m a t e r i a l d u r i n g the r e n i n assay. Renin activity in a m n i o t i c fluid i n c r e a s e d with time o f i n c u b a t i o n at 2 2 ° C with 3,NGF a n d was d e p e n d e n t on t h e c o n c e n t r a t i o n o f 3,NGF a d d e d ( T a b l e II).
TABLE II ACTIVATION OF INACTIVE RENIN BY THE GAMMA SUBUNIT OF NERVE GROWTH FACTOR Renin (pmol ANG I. h - t/ml amniotic fluid)a Time of incubation of ",/NGF with amniotic fluid (h) Concentration of ",/NGF 0zg/ml) 0 0.4 2 10
0
0.2
0.5
1
2
3
4
5
260 260 290 390
250 260 350 500
290 300 400 610
290 300 470 960
240 310 500 1450
280 320 500 1540
290 320 550 1850
260 330 590 2100
a The ",/NGF inhibitors - PMSF, LBTI and SBTI - were added after 22°C incubation of 3,NGF with amniotic fluid, i.e. before renin assay.
T h e r e f o r e the p r e s e n t s t u d y has e l i m i n a t e d p o t e n t i a l a r t e f a c t s due to c o n t a m i n a t i o n by renin a n d interference in the renin a s s a y a n d has d e m o n s t r a t e d t h a t the g a m m a s u b u n i t o f nerve g r o w t h f a c t o r can a c t i v a t e inactive renin. T h e rate o f a c t i v a t i o n in the present s t u d y was c o n s i d e r a b l y less t h a n that r e p o r t e d earlier [16], suggesting that the earlier results were due in p a r t , to c o n t a m i n a t i o n o f the c r u d e p r e p a r a t i o n o f N G F by o t h e r enzymes c a p a b l e o f a c t i v a t i n g inactive renin, p o s s i b l y k a l l i k r e i n [17]. T h e arginine e s t e r o p e p t i d a s e , 3,NGF, c o n v e r t s pro-13NGF to /3NGF [1] a n d can also activate p l a s m i n o g e n [23]. T h e results o f the present e x p e r i m e n t s indicate t h a t this enzyme s u b u n i t o f nerve g r o w t h f a c t o r can activate inactive ( ' p r o - ' ) renin. W h e t h e r such a r e a c t i o n has p h y s i o l o g i c a l relevance in or a d j a c e n t to neurones o r o t h e r cells which c o u l d p o s s i b l y c o n t a i n b o t h s u b s t a n c e s will require m u c h m o r e extensive investigation. H o w e v e r , the present results show the p o t e n t i a l for such a m e c h a n i s m . This s t u d y was s u p p o r t e d by grants f r o m the N a t i o n a l H e a l t h a n d M e d i c a l R e s e a r c h C o u n c i l o f A u s t r a l i a a n d the C h i l d r e n ' s A s s i s t a n c e F u n d .
91 1 Berger, E.A. and Shooter, E.M., Evidence for pro-3-nerve growth factor, a biosynthetic precursor to
3-nerve growth factor, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3647-3651. 2 Brooks, V.L. and Malvin, R.L., An intracerebral, physiological role for angiotensin: effects of central blockade, Fed. Proc., 38 (1979) 2272-2275. 3 Cozzari, C., Angeletti, P.U., Lazar, J., Orth, H. and Gross, F., Separation of isorenin activity from nerve growth factor (NGF) activity in mouse submaxillary gland extracts, Biochem. Pharmacol., 22 (1973) 1321-1327. 4 Day, R.P. and Reid, I.A., Renin activity in dog brain: enzymological similarity to cathepsin D., Endocrinology, 99 (1976) 93-100. 5 Fuxe, K., Ganten, D., HOkfelt, T., Locatelli, V., Poulsen, K., Stock, G., Rix, E. and Taugner, R., Renin-like immunocytochemical activity in the rat and mouse brain, Neurosci. Lett., 18 (1980) 245 -250. 6 Guroff, G., Montgomery, P., Tolson, N., Lewis, M.E. and End, D., Induction of ornithine decarboxylase by renin-free nerve growth factor, Proc. nat. Acad. Sci. (Wash.), 77 (1980) 4607 -4609. 7 Hirose, S., Yokosawa, H. and Inagami, T., Immunochemical identification of renin in rat brain and distinction from acid proteases, Nature (Lond.), 274 (1978) 392-393. 8 Hirose, S., Yokosawa, H., Inagami, T. and Workman, R.J., Renin and prorenin in hog brain: ubiquitous distribution and high concentration in the pituitary and pineal, Brain Res., 191 (1980) 489-499. 9 Hutchinson, J.S., Mendelsohn, F.A.O. and Doyle, A.E., Blood pressure responses of conscious normotensive and spontaneously hypertensive rats to intracerebroventricular and peripheral administration of captopril, Hypertension, 2 (1980) 546-550. 10 lnagami, T., Yokosawa, H. and Hirose, S., Definitive evidence for renin in rat brain by affinity chromatographic separation from protease, Clin. Sci. mol. Med., 55 (1978) 121s-123s. 11 Jeng, I. and Bradshaw, R.A., The preparation of nerve growth factor. In N. Marks and R. Rodnight (Eds.), Research Methods in Neurochemistry, Vol. 4, Plenum Press, New York, 1978, pp. 265-288. 12 Lawrence, C.H. and Morris, B.J., Mechanism of activation of inactive renin in human plasma by puff adder venom, Biochim. biophys. Acta (Amst.)657 (1981) 13-25. 13 Levi-Montalcini, R., Meyer, H. and Hamburger, V., In vitro experiments on the effects of mouse sarcoma 180 and 37 on the spinal and sympathetic ganglia of the chick embryo, Cancer Res., 14 (1954) 49-57. 14 Mobley, W.C., Schenker, A. and Shooter, E.M., Characterization and isolation of proteolytically modified nerve growth factor, Biochemistry, 15 (1976) 5543-5552. 15 Morris, B.J., Activation of human inactive ('pro-') renin by cathepsin D and pepsin, J. clin. endocr. Metab., 46 (1978) 153-157. 16 Morris, B.J. and Catanzaro, D.F., Activation of inactive renin by nerve growth factor, IRCS Med. Sci., 8 (1980) 433. 17 Morris, B.J. and Day, R.P., Activation of inactive renin by kallikrein, IRCS Med. Sci., 6 (1978) 348. 18 Morris, B.J. and Day, R.P., Activation of inactive renin by fibrinolysin, IRCS Med. Sci., 6 (1978) 348. 19 Morris, B.J. and Lumbers, E.R., The activation of renin in human amniotic fluid by proteolytic enzymes, Biochim. biophys. Acta (Amst.), 289 (1972) 385-391. 20 Morris, B.J. and Reid, I.A., A 'renin-like' enzymatic action of cathepsin D and the similarity in subcellular distributions of 'renin-like' activity and cathepsin D in the midbrain of dogs, Endocrinology, 103 (1978) 1289-1296. 21 Morris, B.J., de Zwart, R.T. and Brown, C.R., Activation of inactive renin by acrosin, 1RCS Med. Sci. 8 (1980) 721. 22 Morris, B.J., de Zwart, R.T. and Young, J.A., Renin in mouse but not in rat submandibular glands, Experientia (Basel), 36 (1980) 1333-1334.
92 23 Orenstein, N.S., Dvorak, H.F., Blanchard, M . H . and Young, M., Nerve growth factor: a proteasc that can activate plasminogen, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 5497-5500. 24 Phillips, M.I., Weyhenmeyer, J., Felix, D., Ganten, D. and H o f f m a n , W.E., Evidence for an endogenous brain renin-angiotensin system, Fed. Proc., 38 (1979) 2260 2266. 25 Reid, 1.A., The brain renin-angiotensin system: a critical analysis, Fed. Proc., 38 (1979) 2255-2259. 26 Slater, E.E., Defendini, R. and Z i m m e r m a n , E.A., Wide distribution of immunoreactive renin in nerve cells of h u m a n brain, Proc. nat. Acad. Sci. (Wash.), 77 (1980) 5458-5460. 27 Vinores, S. and Guroff, G., Nerve growth factor: mechanism of action, Ann. Rev. Biophys. Bioengng, 9 (1980) 223-257.
87
Elsevier/North-Holland Scientific Publishers Ltd.
EVIDENCE THAT THE ARGININE ESTEROPEPTIDASE (GAMMA) SUBUNIT OF NERVE GROWTH FACTOR CAN ACTIVATE INACTIVE RENIN
BRIAN J. MORRIS, DANIEL F. CATANZAROand RITA T. DE ZWART Department of Physiology, The University of Sydney, New South Wales, 2006 (Australia)
(Received February 5th, 1981; Revised version received March 12th, 1981; Accepted March 23rd, 1981)
The beta and gamma subunits of nerve growth factor were isolated and tested for inactive-reninactivating activity using human amniotic fluid as a convenient source of inactive renin. Only the gamma subunit, which is an arginine esteropeptidase, could activate inactive renin. The role of this enzyme in sites containing both it and inactive renin warrants investigation.
Extracts o f brain contain a molecule that is recognized by antisera to pure renal renin and that can hydrolyze angiotensin I (ANG I) from angiotensinogen at pH 7.5 [7, 8, 10], suggesting that renin is present in the central nervous system. Immunocytochemical studies have demonstrated a wide distribution of renin-like material in various regions of the brain [5, 26]. Most recently the existence, in addition, of an inactive, prorenin-like molecule, has been demonstrated in brain extracts [8]. Activation of this molecule may be the primary event leading to intracerebral generation of angiotensin II, which is known to increase blood pressure, stimulate drinking and release vasopressin by central actions [2, 24, 25] and may play a role in the maintenance of hypertension in spontaneously hypertensive rats [9, 24]. However, the enzyme responsible for activation of inactive renin in neural tissue is not known. Many proteinases can activate inactive renin in biological fluids and kidney [12, 15-19, 21]. However, most of these are not present in the brain. One which is present in brain and can activate inactive renin in human amniotic fluid is cathepsin D [15], activity of which was responsible for earlier reports of renin-like activity in extracts of brain [4, 20]. Such a reaction would be restricted to intracellular compartments having a favourable pH in the acid range. A proteinase that is active at physiological pH and is present in a variety of tissue is the arginine esteropeptidase (gamma) subunit of nerve growth factor (NGF). -~NGF is part of the 75 NGF complex and functions to convert pro-~-NGF to/~NGF, the subunit responsible for activity in promoting nerve growth. The brain contains low levels of ~NGF [27] and 3,NGF might be expected to be present as well. In a recent preliminary communication a crude preparation of NGF was reported to cause activation o f inactive renin in human amniotic fluid [16]. However, the fact that 03404-3940/81/0000-0000/$ 02.50 ©Elsevier/North-Holland Scientific Publishers Ltd.
88 renin activity could be detected in the preparation suggested that other contaminating enzymes might have been responsible for the activation. Therefore, in the present experiment purification procedures were conducted in order to isolate the beta and gamma subunits of NGF for evaluation of possible inactive-reninactivating activity. H u m a n amniotic fluid, the richest most readily available source of inactive renin was used in the studies. Subunits of NGF were isolated from the submandibular glands of adult, male Quackenbush mice, the richest source of this substance. The t3 subunit was isolated as 2.5S NGF by the method of Mobley et al. [14], as described by Jeng and Bradshaw [11]. The final preparation, eluted from a CM52-Cellulose column by 5 mM Tris-HC1, pH 9, 0.5 M NaC1, ran as a single band of Mr 13,000 on SDS gel electrophoresis and was active in promoting nerve growth (as tested in the laboratory of Dr. M.R. Bennett by conventional techniques [13]). The ) subunit was isolated from glands, as described by Jeng and Bradshaw [11]. An assay based on the hydrolysis of benzoyl-arginine-para-nitroaniline was used to localize arginine esterase activity in fractions. Particularly good resolution was obtained during elution of the CM-Cellulose column with a parabolic 0-1 M NaC1 gradient. The final preparation ran as a Mr 25,000 band on SDS gel electrophoresis. A second preparation of ~,NGF (kindly supplied by Dr. E.M. Shooter, Stanford University) was also used in some experiments. Human amniotic fluid, which contained - 1800 pmol ANG I . h l/ml of acid activatable renin together with low concentrations of proteinase inhibitors, was dialyzed overnight at 4°C in 15 vol. of 100 mM sodium phosphate buffer, pH 7.4, containing 1 mM EDTA and 75 mM NaC1. Portions of 200/~1 were mixed with 10 izl aliquots of NGF subunit to give 0, 0.4, 2 and 10 izg/ml. The mixtures were incubated at 22°C for 0, 0.2, 0.5, 1, 2, 3, 4 and 5 h and renin assay performed using 10 #1 sample and 200 #1 plasma from a nephrectomized sheep (1 h, 37°C, pH 7.4) [22]. The reaction was stopped by dilution and boiling before ANG I radioimmunoassay. Inhibition of arginine esteropeptidase (3,NGF) activity in the experiments was achieved by addition of a mixture of 5 mM (final concentration) phenylmethanesulphonylfluoride (PMSF), 0.3 mg/ml lima bean trypsin inhibitor (LBTI) and 0.3 mg/ml soya bean trypsin inhibitor (SBTI). Electrophoretically pure t3NGF is commonly found to be contaminated with renin [3, 6] and our preparation was no exception. Renin activity was 404 + 21 pmol ANG I-h l/#g (mean +_ S.D. of 8 replicates). All of the renin activity in mixtures of amniotic fluid and/~NGF could be accounted for entirely by the summation of the pre-existing renin activities in each. The mixture of PMSF, LBTI and SBTI does not inhibit renin activity so that addition of this to the mixtures had no effect on renin activities observed. At final concentrations of 0, 0.4, 2 and 10 /~g ~ N G F / m l amniotic fluid, renin activities were 261 + 21 and 276 + 16 with PMSF, LBTI, SBTI, 3 0 5 + 4 0 and 328+__27, 833___59 and 8 8 5 + 4 2 , and 4 2 6 2 + 5 5 5 and 4 3 3 8 + 6 8 9 pmol ANG I . h - l / m l ( m e a n ± S . D . of 8 replicates). When the
89
/3NGF/amniotic fluid mixtures were incubated at 22°C over 5 h there was no increase in renin activity above the levels seen at zero time. The absence of any increase in renin during incubation of amniotic fluid with /3NGF indicates that /3NGF has no inactive-renin-activating activity. Experiments were then performed with 3,NGF. In a preliminary experiment the importance of inhibiting TNGF activity before the assay of renin was demonstrated. In the absence of PMSF/LBTI/SBTI there was increased apparent formation of ANG I (Table I). However, this was not due to renin because if 3,NGF activity was inhibited by PMSF/LBTI/SBTI (which do not inhibit renin) before samples were assayed there was a 96°70 reduction in the generation of ANG I-like immunoreactive material in the renin assay. The activity might therefore have been due to generation by 3,NGF from sheep plasma of material that cross-reacted with the ANG I antiserum. The necessity of adding PMSF/LBTI/SBTI before renin assay was vital for renin measurements to be valid. The results of these experiments also indicate that the "rNGF used had little, if any, contamination with renin. In order to test for inactive-renin-activating activity of 3,NGF, amniotic fluid was initially acidified briefly since this has been found to increase the sensitivity of inactive renin to activation by arginine esteropeptidases such a trypsin (J.G. McGirr and B.J. Morris, unpublished results). Therefore the pH of amniotic fluid was adjusted to 3.3 at 4°C by dropwise addition of conc. H2SO4 and after 30 min was readjusted to pH 7.4 with 10 M NaOH. After adding 1/20 vol. of 1 M sodium phosphate buffer, pH 7.4, portions of 200 #1 were incubated at 22°C with 10 ~1 3,NGF, as before. At the end of each period of incubation 5 /d of solutions of PMSF, LBTI and SBTI were added to give final concentrations of 5 mM, 0.3 mg/ml
TABLE 1 INTERFERENCE RENIN ASSAY
BY T H E G A M M A SUBUN1T O F N E R V E G R O W T H F A C T O R 1N T H E
R e n i n (pmol A N G I • h - I / m l a m n i o t i c fluid):
Aa +Inhibitorsb Ba Alone +lnhibitors
c o n c e n t r a t i o n o f ",/NGF 0zg/ml): 0 0.4
2
10
231 _+ 42 243 _+ 15
365 + 47 238 + 18
798 +_ 75 261 _+ 23
8740 +_ 548 588 +_ 52
253 _+ 7 229 _+ 18
276 _+ 28 246 _+ 23
501 +_ 59 258 _+ 19
2830 _+ 522 338 __+ 33
a T w o d i f f e r e n t p r e p a r a t i o n s o f - r N G F : A = ours: B = S h o o t e r ' s (The lower activity of the latter m a y be due to losses d u r i n g s h i p m e n t o f the l y o p h i l i z e d p r e p a r a t i o n . ) b The "),NGF i n h i b i t o r s - P M S F , L B T I a n d SBTI - were a d d e d after 2 2 ° C i n c u b a t i o n o f "~NGF with a m n i o t i c fluid, i.e. b e f o r e renin assay.
90 a n d 0.3 m g / m l , respectively, in o r d e r to stop the activity o f . y N G F in activating inactive r e n i n a n d in f o r m i n g i m m u n o r e a c t i v e m a t e r i a l d u r i n g the r e n i n assay. Renin activity in a m n i o t i c fluid i n c r e a s e d with time o f i n c u b a t i o n at 2 2 ° C with 3,NGF a n d was d e p e n d e n t on t h e c o n c e n t r a t i o n o f 3,NGF a d d e d ( T a b l e II).
TABLE II ACTIVATION OF INACTIVE RENIN BY THE GAMMA SUBUNIT OF NERVE GROWTH FACTOR Renin (pmol ANG I. h - t/ml amniotic fluid)a Time of incubation of ",/NGF with amniotic fluid (h) Concentration of ",/NGF 0zg/ml) 0 0.4 2 10
0
0.2
0.5
1
2
3
4
5
260 260 290 390
250 260 350 500
290 300 400 610
290 300 470 960
240 310 500 1450
280 320 500 1540
290 320 550 1850
260 330 590 2100
a The ",/NGF inhibitors - PMSF, LBTI and SBTI - were added after 22°C incubation of 3,NGF with amniotic fluid, i.e. before renin assay.
T h e r e f o r e the p r e s e n t s t u d y has e l i m i n a t e d p o t e n t i a l a r t e f a c t s due to c o n t a m i n a t i o n by renin a n d interference in the renin a s s a y a n d has d e m o n s t r a t e d t h a t the g a m m a s u b u n i t o f nerve g r o w t h f a c t o r can a c t i v a t e inactive renin. T h e rate o f a c t i v a t i o n in the present s t u d y was c o n s i d e r a b l y less t h a n that r e p o r t e d earlier [16], suggesting that the earlier results were due in p a r t , to c o n t a m i n a t i o n o f the c r u d e p r e p a r a t i o n o f N G F by o t h e r enzymes c a p a b l e o f a c t i v a t i n g inactive renin, p o s s i b l y k a l l i k r e i n [17]. T h e arginine e s t e r o p e p t i d a s e , 3,NGF, c o n v e r t s pro-13NGF to /3NGF [1] a n d can also activate p l a s m i n o g e n [23]. T h e results o f the present e x p e r i m e n t s indicate t h a t this enzyme s u b u n i t o f nerve g r o w t h f a c t o r can activate inactive ( ' p r o - ' ) renin. W h e t h e r such a r e a c t i o n has p h y s i o l o g i c a l relevance in or a d j a c e n t to neurones o r o t h e r cells which c o u l d p o s s i b l y c o n t a i n b o t h s u b s t a n c e s will require m u c h m o r e extensive investigation. H o w e v e r , the present results show the p o t e n t i a l for such a m e c h a n i s m . This s t u d y was s u p p o r t e d by grants f r o m the N a t i o n a l H e a l t h a n d M e d i c a l R e s e a r c h C o u n c i l o f A u s t r a l i a a n d the C h i l d r e n ' s A s s i s t a n c e F u n d .
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