Pig pancreatic kallikrein liberates kallidin from kininogen, whereas trypsin releases bradykinin. Recently, both kallikrein and trypsin have been reported to convert inactive plasma renin to active renin. However, we found that at pH 6.0, trypsin generated an angiotensin II-like pressor substance from human plasma protein in the absence of converting enzyme. This has been isolated and found to have the same amino acid composition as angiotensin II. Thus, in vitro trypsin can directly liberate both the depressor, bradykinin, in weak alkaline conditions, and the pressor, angiotensin II, at weakly acidic pH, from the appropriate substrates. We have now investigated whether kallikrein--a serine protease like trypsin--also generates a pressor substance at weakly acidic pH. Our results demonstrate that it does. We therefore suggest that kallikrein may be involved in a direct link between the pressor and depressor systems and we propose the term 'kinin-tensin system' for this sort of one-enzyme system capable of generating both depressor and pressor substances.

A nerve-sparing procedure during robot-assisted radical prostatectomy has been considered one of the most important techniques for preserving postoperative genitourinary function. The reason is that adequate nerve-sparing procedures could preserve both erectile function and lower urinary tract function after surgery. When a nerve-sparing procedure is carried out, the cavernous nerves themselves cannot be visualized, despite the magnified viewing field during robot-assisted radical prostatectomy. Thus, nerve-sparing procedures have been considered challenging operations, even now. However, because not all surgeons have carried out a sufficient number of nerve-sparing procedures, the development of new nerve-sparing procedures or new methods for mapping the cavernous nerves is required. Recently, various new operative techniques, for example, Retzius-sparing robot-assisted radical prostatectomy, transvesical robot-assisted radical prostatectomy and retrograde release of neurovascular bundle technique during robot-assisted radical prostatectomy, have been developed. In addition, new surgical devices, for example, biological/bioengineering solutions for cavernous nerve protection and devices for identifying the cavernous nerves during radical prostatectomy, have developed to preserve the cavernous nerves. In contrast, limitations or problems in preserving cavernous nerves and postoperative erectile function have become apparent. In particular, the recovery rate of erectile function, the positive surgical margin rate at the site of nerve-sparing and the indications for nerve sparing have become obvious with the accumulation of much evidence. Furthermore, predictive factors for postoperative erectile function after nerve-sparing procedures have also been clarified. In this article, the importance of a comprehensive approach for early recovery of erectile function in the robot-assisted radical prostatectomy era is discussed.

This study was undertaken to confirm our previous preliminary observation that hog pancreas kallikrein (EC 3.4.21.35) directly liberated an angiotensin-like substance from human plasma protein Cohn fraction IV-4 at an acidic pH of 4.0-5.0. First, the possibility of proangiotensin or des-Asp1-angiotensin being the pressor substance was ruled out by t.l.c. Secondly, the pressor substance was purified by Sephadex G-25 and Bio-Gel P-2 gel filtration, and finally by high-performance liquid chromatography. The amino acid composition of the isolated pressor substance (residues/mol) was: Asp, 1.03; Val, 1.03; Ile, 1.00; Tyr, 0.69; Phe, 1.04; His, 0.91; Arg, 0.86; Pro, 0.86. This composition was identical with that of angiotensin. Since the reaction mixture was not contaminated with common proteolytic enzymes, such as trypsin, chymotrypsin, renin, cathepsin D and proangiotensin-converting enzyme, and other enzymes activated by kallikrein, it is clear that hog kallikrein directly produces angiotensin in vitro.

An in vivo study was done to determine whether angiotensin II is directly formed by kallikrein or kallikrein-like proteases. Dogs were bilaterally nephrectomized 24 hours before ligation of the coronary artery. This acute coronary artery occlusion led to a regionally increased acidic state in the ischemic tissue and resulted in an elevation of immunoreactive angiotensin II (IR-Angiotensin II) levels in the coronary sinus blood, but not in systemic aortic blood. Elevation of the IR-Angiotensin II level was specifically inhibited by aprotinin, a kallikrein inhibitor. It was not affected by either captopril, a potent angiotensin converting enzyme inhibitor, or by pepstatin, a renin inhibitor. The concentrations of immunoreactive angiotensin I (IR-Angiotensin I), plasma renin activity and angiotensin converting enzyme activity remained unaltered in the presence of coronary artery occlusion. These results suggest that IR-Angiotensin II in the ischemic heart may be generated directly by kallikrein or kallikrein-like proteases, independently of the systemic renin angiotensin system.

SUMMARY Enzymatic activity of tonin-o^-macroglobulin complex was studied in vitro and in vivo, using an immunoimmobilization technique. Tonin-ai-macroglobulin complex, which was immunologically immobilized by anti-a r macroglobulin antibody covalently coupled to agarose gels, could quantitatively hydrolyze angiotensin I and synthetic tridecapeptide renin substrate to form angiotensin II. However, the solid-phase antibody-bound tonin-a,-macroglobulin complex could not hydrolyze the plasma protein renin substrate. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, inhibited both free tonin and the solid-phase antibody-bound tonin-a|-macroglobulin complex. The hydrolytic activity of the solid-phase antibody-bound tonin-«,-macroglobulin complex against angiotensin I was not inhibited by soybean trypsin inhibitor (molecular weight, 23,000), a potent inhibitor of free tonin. Taken together, these results suggest that tonin bound to c^-macroglobulin keeps the active site intact and that inhibition of the enzyme activity is due to a steric hindrance. When 500 /xl of tonin was administered intravenously to rats, the immunoimmobilization method was used to show that the tonin-a r -macroglobulin complex in the plasma formed angiotensin II. Thus, the tonin-a r macroglobulin complex in the plasma may be linked to some forms of hypertension through angiotensin II formation. (Hypertension 11: 63-70, 1988) KEY WORDS • tonin immunoimmobilization angiotensin I • angiotensin II • a r -macroglobulin