James Burton

A substrate analog inhibitor of renin that is effective in vivo

Summary A potent inhibitor of primate renin effective in vivo has been synthesized. The peptide, Pro-His-Pro-Phe-His-Phe-Phe-Val-Tyr-Lys, is an analog of the amino acid sequence found between residues 6 and 13 of renin natural substrate (angiotensinogen). The inhibitor is eight times more soluble at physiologic pH and is cleared from circulation about two-orders of magnitude more slowly than earlier substrate analog inhibitors which were not active in vivo. Infusion of the inhibitor into the monkey (M. fascicularis) blocks the rise in mean arterial pressure caused by exogenous human renin, but not by angiotensin I or II. When injected into a salt-depleted monkey, the peptide lowers blood pressure in a dose-dependent manner about as effectively as the converting enzyme inhibitor, teprotide.

On the specificity of human renin. Studies with peptide inhibitors

The amino acid sequence around the renin substrate site is known to be identical to the N-terminal tetradecapeptide: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser. Renin (EC 3.4.99.19) from both primates and non-primates cleaves this peptide at the leucylleucine bond. Several analogs of the octapeptide segment: His-Pro-Phe-His-Leu-Leu-Val-Tyr of this tetradecapeptide act as competitive inhibitors for human renin with inhibition constants down to 1 muM. The same peptides were shown, however, to have no or only slight affinity for non-primate renin. The substrate site has been preserved throughout evolution whereas the enzyme site for human renin is different from that of non-primate renins. The findings suggest that species-specific peptides must be developed for both studies of renin inhibition and for renin purification.

Purification of hog renin by affinity chromatography using the synthetic competitive inhibitor (D-Leu6)octapeptide.

The renin substrate analog His-Pro-Phe-His-Leu-D-Leu-Val-Tyr ([D-Leu6]-octapeptide) acts as a potent inhibitor of renin because of the D-amino acid substitution at the cleavage site. This inhibitor was coupled to CNBr-activated Sepharose 4B to yield a support for affinity chromatography. Hog renin with a specific activity of 1.2 Goldblatt units/mg was in one step purified 195-fold to a final specific activity of 234 Goldblatt units/mg. Application of a pH gradient from 5.0 to 7.5 to the support was found to be the most successful elution program, probably because the [D-Leu6]-octapeptide is not an inhibitor for renin at neutral pH.

Acute hypertension in a nonhuman primate: humoral and hemodynamic mechanisms.

The present study assessed the contribution of the renin-angiotensln system (RAS), dietary sodium, and cardiac output (CO) to the genesis of primate hypertension in a one-kidney model which was developed to test species-specific renin inhibitors. Reduction of renal perfusion pressure increased mean arterial pressure (MAP) from 105 ± 4 to 127 ± 3 mm Hg (p < 0.0005), associated with increased plasma renin activity (PRA) from 4.9 ± 0.7 to 13.8 db 1.1 ng.ml−1hr−1 (p < 0.0005). Correlation of MAP with PRA yielded an r value of 0.662 (p < 0.0005). Significant blood pressure elevation was obtained with both regular (R) and low sodium (LS) diet (p < 0.0005), although the MAP change was greater with LS. With both R and LS diet, hypertension was associated with increased PRA (p < 0.0005), and normotensive pressures were achieved with converting enzyme inhibitor (teprodde). The hemodynamic change with hypertension was an increase of systemic vascular resistance (SVR) from 0.89 ± 0.12 to 1.17 ± 0.09 units {p < 0.05); cardiac output (CO) and central blood volume did not change significantly. Thus, acute hypertension, mediated by the RAS, was developed in a one-kidney primate model. The hemodynamic correlate of hypertension was increased SVR; CO and volume redistribution were not Initiating factors. (Hypertension 4: 219–225, 1982)

ANTISERA TO HUMAN RENIN NORMALIZE RENIN-DEPENDENT HYPERTENSION IN THE MONKEY: THERAPEUTIC IMPLICATIONS

Polyclonal antisera raised against pure human renin normalize renin-dependent blood pressure elevation in the monkey (M. fascicularis). In vitro, comparable inhibition of either human or monkey plasma renin by the antisera was demonstrated. In vivo, intravenous infusion of 2 ml of antisera did not change mean arterial pressure of salt-repleted monkeys, however, its administration to salt-depleted monkeys with elevated plasma renin activity lowered mean arterial pressure 10 Torr. A 25 Torr rise in mean arterial pressure and increase in plasma renin activity occurred promptly after inflation of a suprarenal aortic cuff in conscious uninephrectomized monkeys. Administration of 2 ml of antisera to these monkeys normalized mean pressure, which was reduced by an additional 10 Torr if the animals were previously salt-depleted. Maximal hypotension occurred within 1 hour and was sustained for approximately 10 hours. Because of the differing specificities of polyclonal antisera, sera raised in two laboratories against human renin purified from different sources were employed. Identical results were obtained. This is the first demonstration of the use of antisera to inhibit endogenous renin activity in primates and predicts the in vivo efficacy of renin antisera as experimental, diagnostic and therapeutic agents.

Hypotensive Effects of the Renin Inhibitor (RI-78) and the Converting Enzyme Inhibitor (Teprotide) in Conscious Monkeys

Effects of the pentapeptide renin inhibitor (RI-78; Phe(4Cl)-Phe-Val-Tyr-Lys-NH2) and the angiotensin converting enzyme (ACE) inhibitor (teprotide) on mean arterial pressure (MAP) were examined in conscious monkeys (M. fascicularis). In salt depleted normotensive monkeys with a MAP of 95 +/- 4 mmHg and plasma renin activity (PRA) of 15.9 +/- 2.7 ngAI/ml/h, a bolus injection of a dose of 375 micrograms/kg of RI-78 caused a prompt hypotensive effect. Maximal hypotensive action was seen within 1 min, and MAP returned to the basal level within 15 min. With this dose, MAP was reduced by 20 +/- 6 mmHg. Teprotide (1 mg/kg) decreased MAP and reached a nadir after 13 min. There was no significant difference between maximal hypotensive responses seen with RI-78 (375 micrograms/kg) and with teprotide (1 mg/kg). Hypotensive effects of RI-78 and teprotide were also examined in acute renal hypertensive monkeys with a MAP of 125 +/- 5 mmHg and a PRA of 27.1 +/- 5.7 ngAI/ml/h. Again, similar hypotensive effects were observed. We conclude that antihypertensive effect of RI-78 is comparable to that seen with teprotide.