0013-7227/78/1034-1289$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society

Vol. 103, No. 4

Printed in U.S.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* BRIAN J. MORRISt AND IAN A. REID^ Department of Physiology, University of California, San Francisco, California 94143 ABSTRACT. The proposal that brain "rerun-like" activity may be due not to renin but to an action of brain cathepsin D on angiotensinogen during assay at low pH (ca. 5) was tested using pure bovine cathepsin D with dog and human angiotensinogen from cerebrospinal fluid (CSF) and nephrectomized plasma. Cathepsin D generated angiotensin I from CSF and plasma at a rate directly proportional to the concentration of enzyme added. The rate of hydrolysis of angiotensin I from angiotensinogen was estimated as 0.028% min~'/ig cathepsin D"1 for dog CSF, 0.001 for human CSF, 0.00035 for nephrectomized dog plasma, and 0.00001 for nephrectomized human plasma. Thus, cathepsin D hydrolyzes dog angiotensinogen about 30 times faster than it hydrolyzes human angiotensinogen. The rate of reaction in plasma is about l/100th that in CSF. The subcellular distribution of renin-like activity in the mid-

brain of dogs was compared with that of cathepsin D and acid phosphatase, both lysosomal enzymes, by differential and isopycnic gradient centrifugation. The distribution of each was similar, with the distributions of renin-like activity and cathepsin D bearing the greatest similarity. Taken together with previous data from this laboratory concerning similarities in enzymatic and physical properties of the latter protease activities, the present results add further support to the proposal that the renin-like activity that has been reported in brain is actually a reaction of brain cathepsin D with angiotensinogen during assay at low pH in vitro to form angiotensin I, the brain being devoid of genuine renin. The present work also provides the first direct evidence for a renin-like hydrolysis of the Leulo-Leu" bond of angiotensinogen by cathepsin D. (Endocrinology 103: 1289, 1978)

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HE CONCEPT of a renin-angiotensin system autonomous within the central nervous system has been advocated over the past few years (1-5). Certainly, injection of renin, angiotensin I (AI), or angiotensin II (All) into the cerebral ventricles elicits responses such as increased systemic arterial pressure, drinking, and increased secretion of vasopressin and corticotropin (3, 5, 6-8). Although there is convincing evidence for the existence of the protein substrate of renin, angiotensinogen, in CSF (9) and of angiotensin-converting enzyme (EC 3.4.15.1) (10) and angiotensin receptors (11) on the plasma membranes, the evidence for renin (EC 3.4.99.19) itself has recently been challenged.

Day and Reid (12) showed that the "reninlike" activity that had previously been measured in brain extracts (1, 2) corresponded in position to cathepsin D (EC 3.4.23.5) activity in samples during gel filtration and isoelectric focusing. T h e pH activity profile of brain renin-like activity also resembled that of cathepsin D, but differed markedly from that of renal renin. In particular, the enzymatic activity of the brain enzyme fell to zero above p H 6, whereas renin is quite active at the "physiological" pH of 7.4. Day and Reid suggested that the renin-like activity that had previously been measured in brain extracts using an assay pH of ca. 5 may have measured the hydrolysis by cathepsin D of AI from angiotensinogen during the course of nonspecific degradation Received December 7, 1977. of proteins by this intracellular carboxyl pro* This work was supported by the Skaggs Foundation. tease. This is possible because cathepsin D t Recipient of a C. J. Martin Research Fellowship from the National Health and Medical Research Council of has a substrate specificity similar to pepsin Australia and subsequently of an Advanced Fellowship and it has long been known that when pepsin from the Northern California Heart Association. To is incubated with plasma, AI is formed (13, whom requests for reprints should be addressed. $ Recipient of Research Career Development Award 14). The present study tested the hypothesis directly by incubating virtually pure cathepsin HL-00104. 1289

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MORRIS AND REID

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D with angiotensinogen present in cerebrospinal fluid (CSF) and in plasma of dog and man. To provide additional support for an identity of brain renin-like activity as cathepsin D, the subcellular distribution of each in dog midbrain was examined and compared. Materials and Methods Materials Cathepsin D from bovine spleen was purchased in powder form from Sigma Chemical Co., St. Louis, MO (lot 26C-8100); the purification method u§ed (15) gave virtually pure enzyme, as judged by electrophoresis. Samples of CSF were collected from the cisterna magna of four dogs anesthetized with sodium pentobarbital (30 mg/kg). Samples of ventricular CSF were obtained from four human patients. Reaction of cathepsin D with angiotensinogen Samples of CSF from four dogs and four humans were dialysed in 1 liter 50 mM sodium citrate, sodium phosphate buffer, pH 5, at 4 C overnight. Portions (1 ml) were then incubated at 37 C with cathepsin D (0,4, and 40 /Jg/ml, final concentration) added in 50 /xl pH 5 buffer. The reaction mixture also contained 15 mM EDTA, 1.6 mM 2,3-dimercapto-1-propanol, and 3.4 mM 8-quinolinol to inhibit angiotensinase activity. At 0, 0.5, and 1 h, the incubation mixture was diluted 1:1 (vol/vol) with cold distilled water, mixed, and placed in boiling water for 3 min. The tubes were centrifuged at 1000 X g for 15 min and the amount of AI present in two 0.01-ml and two 0.1-ml aliquots was measured by RIA (16). In a further experiment, dialyzed samples of plasma from a nephrectomized dog and a nephrectomized human were incubated, as above, with cathepsin D (400 /ig/ml) for 0, 0.5, 1, and 2 h, and AI was measured in a similar fashion. The nephrectomized plasma contained low residual renin activity (dog,

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.

0013-7227/78/1034-1289$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society Vol. 103, No. 4 Printed in U.S.A. A "Renin-Like" Enzymatic A...
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