Renin Inhibitory Peptides Research Articles

Renin inhibitory peptides. A .beta.-aspartyl residue as a replacement for the histidyl residue at the P-2 site

In an effort to decrease the size and to increase the hydrophilicity of the previously prepared renin inhibitory peptides, it was postulated that one might be able to take advantage of the polar Thr-84 on the flap region of the enzyme renin by potential hydrogen bonding to polar functionality on the inhibitory peptide at the P-2 site. A beta-aspartyl residue with a carboxylic acid group was proposed to be a possible replacement for the histidyl residue at the P-2 site. A series of renin inhibitory peptides were prepared with the beta-aspartyl residue to probe the structure-activity relationship of the resulting peptides. Potent inhibitory peptides could be realized with activity in the subnanomolar range. Molecular modeling was also undertaken to investigate the interactions between the enzyme active site and the new inhibitors and to suggest a possible mode of binding of these ligands to the enzyme. From this modeling study, the role of Ser-229 at the active site in the bound conformer of the inhibitors was suggested. It was further noted in the analogue study that a malic acid residue, which is the oxygen analogue of the beta-aspartic acid residue, could lead to further enhancement of inhibitory potency of congeneric peptides. Small renin inhibitors, such as compound XII with molecular weight 535 and with no alpha-amino acid residue, could be prepared and exhibited renin inhibitory activity in the nanomolar range.

A primate bioassay for the determination of renin inhibitory peptides in serum

The study of renin inhibitory peptides (RIPs) in rodents and primates requires the establishment of a simple, high volume method for determining the concentration of RIPs in serum after intravenous or oral dosing. The human renin inhibition assay useful for rodents is not directly applicable to primates due to inherent production of angiotensin I from the primate serum angiotensinogen and added recombinant human renin. Therefore, a novel approach to analyze the serum concentrations of RIPs in primates is described based on in vitro studies with monkey serum. The procedure involves the inactivation of monkey angiotensinogen and monkey renin by thermal denaturation prior to analysis. Application of this assay was demonstrated by analyzing serum samples from an in vivo study in monkeys using ditekiren (U-71,038), a renin inhibitory peptide, and by validation of the assay and results using a tritium-based radioimmunoassay (RIA) for ditekiren. The minimum detectable limit of ditekiren for both the RIA and the bioassay for primates was 10 ng/ml serum. The reported bioassay should be of value for monitoring serum levels of thermostable RIPs from pharmacokinetic, bioavailability, and pharmacodynamic studies in primates as well as in humans.

Acute hypotensive responses to peptide inhibitors of renin in conscious monkeys: an effect on blood pressure independent of plasma renin inhibition

In order to investigate the hypotensive mechanisms of action of peptide renin inhibitors, blood pressure responses to five renin inhibitors were compared with those to the angiotensin converting enzyme inhibitor, enalaprilat, in conscious African green and rhesus monkeys. (3S-4S)-4-amino-5-cyclohexyl-3-hydroxy pentanoic acid (ACHPA)-containing renin inhibitory peptide (ACRIP) and enalaprilat both decreased blood pressure in euvolemic and volume-depleted African green monkeys. However, while a maximum dose of enalaprilat reduced blood pressure to 80 +/- 4 and 56 +/- 4 mmHg in the euvolemic and volume-depleted monkeys, respectively, ACRIP lowered pressure to life-threatening levels (less than 40 mmHg) under both conditions. The relative potencies of ACRIP and four other renin inhibitors for inhibiting in vitro plasma renin activity (PRA; IC50) were compared with their potencies in reducing blood pressure by 15 mmHg (ED15 mmHg) and lowering blood pressure more than enalaprilat in volume-depleted rhesus monkeys. All renin inhibitors lowered blood pressure significantly beyond the maximal response to enalaprilat. Despite a significant correlation (r = 0.99, P less than 0.05) between the in vitro PRA inhibitory potency and the in vivo ED15 mmHg, doses which lowered blood pressure beyond the maximal responses to enalaprilat were not significantly correlated (r = 0.53, P greater than 0.05) with the in vitro PRA IC50 values. Furthermore, the profound depressor responses to renin inhibitors in rhesus monkeys were accompanied by increases in the heart rate and decreases in pulse pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a sensitive activity assay for high-volume evaluation of human renin inhibitory peptides in rat serum: results with U-71,038.

A sensitive activity assay for high volume evaluation of human renin inhibitory peptides (RIPs) in rat sera (range 2-80 ng/ml) was developed based on the low affinity of RIPs to rat renin and their high affinity to human renin. The utility of this activity assay was tested by measuring concentrations of a human RIP, U-71,038 (BOC-Pro-Phe-N-MeHis-Leu psi [CHOHCH2]Val-Ile-Amp), in rat sera, determined by the activity assay, by a sensitive radioimmunoassay (RIA), and by tracking tritiated drug. Rats were given radiolabeled drug as an intravenous bolus, and blood samples were collected at various times after dosing. The serum level of U-71,038 equivalents was determined by the three techniques. Whole blood was also counted for total radioactivity to evaluate the potential for U-71,038 incorporation into red blood cells. Results from the three serum assays indicate good agreement between the calculated U-71,038 equivalents for the 30 min and 1 hr collection times. The 2 and 4 hr collection times show excellent agreement for the activity assay and RIA; [3H]-U-71,038 determinations gave substantially higher values. Serum levels for U-71,038 determined 30 min after dosing averaged less than 300 ng equivalents/ml suggesting that less than 1% of the administered dose was in the systemic circulation at that time. Thus, U-71,038 was rapidly cleared. At the 4 hr collection time, the level of U-71,038 equivalents, as determined by activity assay and RIA, was ten times the in vitro IC50 for the renin inhibitory activity of U-71,038.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of renin inhibition in the conscious primate Macaca fascicularis.

Pro-His-Pro-Phe-His-Statine-Ile-Phe-NH2 (R-Pep-27), a potent renin inhibitory peptide, was infused into the conscious, sodium-depleted Macaca fascicularis at doses of 0, 0.1, 1, 4, 16, and 32 micrograms/kg/min for 10 minutes. At all doses greater than 0.1 microgram/kg/min, there was a parallel decrease in mean arterial pressure (MAP), plasma renin activity, and plasma angiotensin II (Ang II) concentration. On the other hand, assays with monoclonal antibodies specific for total renin and active renin demonstrated that the peptide's inhibition of circulating active renin stimulated the release of both. The maximal effective R-Pep-27 dose was approximately 16 micrograms/kg/min, which reduced MAP by an average of 15.8 +/- 1.4 mm Hg (n = 14) and plasma renin activity and plasma Ang II concentration to 3% (n = 9) and 15% (n = 5), respectively, of the pretreatment values. At 0.1 microgram/kg/min, there was no significant decrease in MAP; however, measurement of plasma renin activity showed an average decrease in activity of 42% (n = 3). No significant change in the heart rate was observed at all the doses studied. For comparison, intravenous captopril (400 micrograms/kg bolus) was administered after the MAP of the monkeys had recovered from the peptide experiments, and it reduced MAP by 25.1 +/- 2.4 mm Hg (n = 10) without significantly changing plasma renin activity. As anticipated, injection of angiotensin I (80-160 ng/kg bolus) into sodium-depleted monkeys during peptide infusion caused a transient rise in MAP of 14.8 +/- 5.4 mm Hg (n = 4) above the mean pretreatment value.(ABSTRACT TRUNCATED AT 250 WORDS)

Renin inhibitory peptides: a study of structural modifications in the peptide backbone.

We studied the peptide backbone modifications that improve the metabolic stability of the resulting peptides and yet retain high inhibitory activity against human plasma renin. A systematic investigation of N-alpha-methyl and C-alpha-methyl modifications at the P2 and P3 sites of renin-inhibitory peptides that contain part of the human angiotensinogen sequence led to the discovery of N-alpha-methyl amino acids at the P2 site as a useful structural modification. U-71,038 (11) inhibited human plasma renin with an in vitro potency (IC50) of 2.6 x 10(-10) mol/l. It is highly selective for renin and, as anticipated, resistant to proteolytic degradation. Additional study based on molecular graphic modelling has led us to propose a gamma-lactam conformational constraint at the P2-P3 site. This pseudo-dipeptide has proved useful in the preparation of active renin inhibitors. Compound 18a inhibited human plasma renin with an in vitro potency (IC50) of 2.1 x 10(-9) mol/l. This class of compounds also offers structural features for the study of enzyme-bound conformers.

ChemInform Abstract: Structure‐Conformation‐Activity Relationships of Renin Inhibitory Peptides Having P1‐P1′ Xaaψ(CH2NH)Yaa Substitutions: Molecular Modeling and Crystallography Studies.

ChemInformVolume 19, Issue 23 Natural Products ChemInform Abstract: Structure-Conformation-Activity Relationships of Renin Inhibitory Peptides Having P1-P1′ Xaaψ(CH2NH)Yaa Substitutions: Molecular Modeling and Crystallography Studies. T. K. SAWYER, T. K. SAWYER Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorD. T. PALS, D. T. PALS Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorB. MAO, B. MAO Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorL. L. MAGGIORA, L. L. MAGGIORA Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorD. J. STAPLES, D. J. STAPLES Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorA. E. DEVAUX, A. E. DEVAUX Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorH. J. SCHOSTAREZ, H. J. SCHOSTAREZ Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorJ. H. KINNER, J. H. KINNER Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorC. W. SMITH, C. W. SMITH Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this author T. K. SAWYER, T. K. SAWYER Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorD. T. PALS, D. T. PALS Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorB. MAO, B. MAO Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorL. L. MAGGIORA, L. L. MAGGIORA Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorD. J. STAPLES, D. J. STAPLES Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorA. E. DEVAUX, A. E. DEVAUX Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorH. J. SCHOSTAREZ, H. J. SCHOSTAREZ Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorJ. H. KINNER, J. H. KINNER Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this authorC. W. SMITH, C. W. SMITH Biopolym. Chem. Regul. Pept. Res., Upjohn Co., Kalamazoo, MI 49001, USASearch for more papers by this author First published: June 7, 1988 https://doi.org/10.1002/chin.198823313Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume19, Issue23June 7, 1988 RelatedInformation

Renin inhibitors containing psi[CH2O] pseudopeptide inserts.

Renin inhibitors 2-4 with the D-Lys renin inhibitory peptide (RIP) sequence, but containing Leu psi[CH2O]Ala (2), Leu psi[CH2O]Val (3), and Leu psi[CH2O]Leu (4) at the P1-P1' site, were of a comparable potency to RIP. N-Terminal Boc-protected inhibitors containing Pro psi[CH2O]Phe in positions P4-P3 were potent inhibitors of renin, with Boc-Phe-Pro psi[CH2O]Phe-His-Leu psi[CH(OH)CH2]Val-Ile-(2-aminomethyl) pyridine (17) having an IC50 of 1.6 X 10(-9) M.

Structure-conformation-activity relationships of renin inhibitory peptides having P 1-P 1' Xaaψ[CH 2NH]Yaa substitutions: molecular modeling and crystallography studies 1

Structure-activity studies of angiotensinogen (ANG)-based renin inhibitors having carboxy-terminal pseudodipeptidyl P 1-P 1' Xaaψ(CH 2NH]Yaa-NH 2 and related transition state synthetic mimetics were performed and integrated with molecular modeling using a computer simulated human renin active site model, and compared to a known crystallographic structure of a P 1-P 1' PheΨ[CH 2NH]Phe substituted renin inhibitor bound to rhizopuspepsin. Renin is an aspartic acid protease (EC 3.4.99.19) which catalyzes the first and rate-limiting step of the enzyme cascade that exists for the biosynthesis of angiotensin II (ANG II) 2–4. Renin inhibition may be therapeutically important in the development of useful antihypertensive agents 5–9 based on the role of the renin-angiotensin converting enzyme cascade in the physiological and, perhaps, pathophysiological control of blood pressure and electrolyte homeostasis. In retrospect, the development of ANG-based inhibitors of human renin has essentially focused on systematic structure-activity studies 10–20 related to p 1-p 1' pseudodipeptidyl substitutions of the Leu-Val cleavage site (see below) of various substrate fragments in which the scissile amide (CONH) has been replaced by CH(OH)CH 2 or CH 2NH type transition state mimics of the proposed aminol (C(OH) 2NH) that is believed to be generated during enzyme-catalyzed hydrolysis of ANG at the renin active site.

Structure--activity relationship of fluorine-containing renin inhibitory peptides based upon the tertiary structure of human renin.

The structure-activity relationship of acyl-His-trifluorinated leucinol derivatives as inhibitors of human renal renin is discussed based upon the tertiary structure of human renin, which was deduced from the crystal structure of penicillopepsin by assuming structural similarity. The structural requirements for the inhibitors and possible interactions at the renin binding site are discussed.

New human renin inhibitory peptides: angiotensinogen transition-state analogues containing novel Leu-Val replacement and a retro-inverso amide bond.

New human renin inhibitors were designed from transition-state analogues of angiotensinogen, synthesized and evaluated. The peptide derovative, which contained 1-naphthylmethylsuccinylamide residue (P3) with a retro-inverso amide bond and a norstatine isoamylamide residue (P1-P1'), was stable to proteases and had potent human renin inhibitory activity. This compact inhibitor exhibited hypotension when administered orally to a monkey.

ChemInform Abstract: α‐Methylproline‐Containing Renin Inhibitory Peptides: In vivo Evaluation in an Anesthetized, Ganglion‐Blocked, Hog Renin Infused Rat Model.

ChemInformVolume 18, Issue 31 Other Subjects ChemInform Abstract: α-Methylproline-Containing Renin Inhibitory Peptides: In vivo Evaluation in an Anesthetized, Ganglion-Blocked, Hog Renin Infused Rat Model. S. THAISRIVONGS, S. THAISRIVONGS Royal Danish School Pharm., Dep. Pharm. Chem. AD, 2100 Copenhagen, DenmarkSearch for more papers by this authorD. T. PALS, D. T. PALS Royal Danish School Pharm., Dep. Pharm. Chem. AD, 2100 Copenhagen, DenmarkSearch for more papers by this authorJ. A. LAWSON, J. A. LAWSON Royal Danish School Pharm., Dep. Pharm. Chem. AD, 2100 Copenhagen, DenmarkSearch for more papers by this authorS. R. TURNER, S. R. TURNER Royal Danish School Pharm., Dep. Pharm. Chem. AD, 2100 Copenhagen, DenmarkSearch for more papers by this authorD. W. HARRIS, D. W. HARRIS Royal Danish School Pharm., Dep. Pharm. Chem. AD, 2100 Copenhagen, DenmarkSearch for more papers by this author S. THAISRIVONGS, S. THAISRIVONGS Royal Danish School Pharm., Dep. Pharm. Chem. AD, 2100 Copenhagen, DenmarkSearch for more papers by this authorD. T. PALS, D. T. PALS Royal Danish School Pharm., Dep. Pharm. Chem. AD, 2100 Copenhagen, DenmarkSearch for more papers by this authorJ. A. LAWSON, J. A. LAWSON Royal Danish School Pharm., Dep. Pharm. Chem. AD, 2100 Copenhagen, DenmarkSearch for more papers by this authorS. R. TURNER, S. R. TURNER Royal Danish School Pharm., Dep. Pharm. Chem. AD, 2100 Copenhagen, DenmarkSearch for more papers by this authorD. W. HARRIS, D. W. HARRIS Royal Danish School Pharm., Dep. Pharm. Chem. AD, 2100 Copenhagen, DenmarkSearch for more papers by this author First published: August 4, 1987 https://doi.org/10.1002/chin.198731341AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLThe site is currently under maintenance and this functionality is not available right now. Please try later. Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume18, Issue31August 4, 1987 RelatedInformation

Renin inhibitors. Design of angiotensinogen transition-state analogues containing novel. (2R,3R,4R,5S)-5-amino-3,4-dihydroxy-2-isopropyl-7-methyloctanoic acid.

A highly stereoselective synthesis of 2(R)-[5(R)-[1(S)-[(tert-butyloxycarbonyl)amino]-3-methylbutyl]-2,2- dimethyl-4(R)-dioxolanyl]-3-methylbutanoic acid is described. This is a suitably protected carboxylic acid useful as an intermediate for the preparation of renin inhibitory peptides. Angiotensinogen analogues such as peptides IX and X that contain the dipeptide isostere (2R,3R,4R,5S)-5-amino-3,4-dihydroxy-2-isopropyl-7-methyloctanoic acid residue at the scissile site are shown to be potent inhibitors of human plasma renin. The glycol moiety in this novel acid, dihydroxyethylene isostere, is suggested to act as a transition-state analogue and mimics the tetrahedral intermediate formed during the enzyme-catalyzed hydrolysis of the peptidic bond.

Alpha-Methylproline-containing renin inhibitory peptides: in vivo evaluation in an anesthetized, ganglion-blocked, hog renin infused rat model.

A structure-activity analysis of peptides containing backbone C alpha-methyl modification at the P4 site of the angiotensinogen sequence led to the discovery of potent renin inhibitors with apparent in vitro metabolic stability. Boc-alpha-MePro-Phe-His-Leu psi[CHOHCH2]Val-Ile-Amp dicitrate (Va) is a potent inhibitor of human plasma renin with an IC50 value of 1.8 nM. This peptide was shown not to be degraded in vitro by chymotrypsin, elastase, pepsin, and a rat liver homogenate preparation. It is also a potent inhibitor of hog renin with an IC50 value of 1.6 nM and was shown to elicit in vivo activity and cause dose-dependent hypotensive responses when given intravenously to anesthetized ganglion-blocked, hog renin infused rats.

Identification and regulation of renin in human cultured mesangial cells.

Renin activity was measured in the incubation medium, and the cellular extract of human mesangial cells, which had been cultured in the presence of renin-free human plasma (three kidneys; 4-7 passages). Active renin and total renin obtained after trypsin treatment was estimated by radioimmunoassay of angiotensin I using renin-free human plasma as a substrate. Mesangial cell renin had characteristics similar to those of standard human renin; optimum enzymatic activity at pH 5.8, marked inhibition in the presence of two (monoclonal and polyclonal) human renin-specific antibodies and of SR 42128, a new potent statine-containing renin inhibitory peptide. The synthetic capability of the mesangial cells varied markedly with the original kidney (1-49 and 0.3-0.9 ng X h-1 X mg-1 for total renin in the medium and the cellular extract respectively). Renin was secreted mainly as inactive renin. Prostaglandin E2 (PGE2) and carba-prostaglandin I2 (PGI2) (a stable analogue) produced a dose-dependent (0.1-1.10 microM) increase in renin activity in both the cellular extract and the culture medium. Isoproterenol (200 microM) increased renin activity only in the medium. The effects of these agonists were more marked on inactive than on active renin. These results demonstrate that cultured human mesangial cells synthesize and release renin in a stable manner over a long period of culture, thus providing a useful tool for the in vitro study of renin secretion and its control.

Haemodynamic responses to specific renin-angiotensin inhibitors in hypertension and congestive heart failure. A review.

The renin-angiotensin system is an important regulator of vascular resistance in many patients with hypertension and congestive heart failure. To quantitatively evaluate this contribution requires correlation of markers of the renin-angiotensin system with haemodynamic parameters, notably blood pressure, cardiac output, and calculated systemic vascular resistance. In addition, to determine ventricular loading properties, assessment of cardiac filling pressures is also required. The availability of specific pharmacological inhibitors of the renin-angiotensin system greatly enhances such correlation, as the haemodynamic consequence of blocking the renin-angiotensin system can then more fully identify its contribution. In the last decade, highly specific pharmacological inhibitors have become available to serve such a purpose. Renin inhibitory peptides, and renin-specific antibodies can block the rate-limiting step of the renin-angiotensin cascade: namely, the cleavage of 4 amino acids from the angiotensinogen substrate by renin. However, this method of blockade is still at the early stages of investigation. More readily available are converting enzyme inhibitors which block the formation of angiotensin II, the potent vasoconstrictor which mediates increased systemic vascular resistance, and angiotensin II analogues which compete with endogenous angiotensin II for vascular and adrenal receptors. Although hypertension and chronic congestive heart failure are clinically distinct entities in many respects, their common bond is the fact that both pathological mechanisms are mediated by an increase of systemic vascular resistance. The implications of blocking the resulting vasoconstriction in both entities are therefore quite similar. This review summarises our present knowledge of the contribution of the renin-angiotensin system to the vasoconstriction of hypertension and congestive heart failure, and also summarises the haemodynamic consequences of such inhibition. The implications of the response to these specific pharmacological probes, as well as their limitations, are discussed. Their importance rests not only in their therapeutic application, but also in their contribution as probes for pathophysiological mechanisms of vasoconstriction in cardiovascular disease.

Specific inhibition of renin by an angiotensinogen analog: studies in sodium depletion and renin-dependent hypertension.

The angiotensin substrate analog Pro-His-Pro-Phe-His-Phe-Phe-Val-Tyr-Lys has no significant effect on blood pressure in sodium-replete monkeys (Macaca fascicularis) but blocks the pressor response to infused human renin. Pressor responses to angiotensin I and angiotensin II are not attenuated. In five studies in sodium-depleted monkeys, an infusion of 2 mg of the peptide per kg of body weight resulted in a reduction of mean arterial pressure (MAP) from 105 +/- 4 to 79 +/- 3 mm Hg, which is not significantly different from the response to 1 mg of the angiotensin I-converting enzyme inhibitor teprotide per kg. In uninephrectomized monkeys, inflation of a suprarenal aortic cuff caused an increase in MAP from 107 +/- 3 to 131 +/- 3 mm Hg. Infusion of 0.6 mg of the renin-inhibitory peptide per kg was followed by a return of blood pressure to 107 +/- 4 mm Hg--a depressor response similar to that observed with teprotide. This specific in vivo inhibitor of renin can now be applied to a wide variety of physiologic studies.