AJH
1992;
5:495-501
ORIGINAL CONTRIBUTIONS
Inhibition of Human Renin by Rat Plasma
Rat Angiotensinogen Is a Competitive Inhibitor of the Human Renin-Substrate Interaction
Fuad Gahnem, Jean E. Sealey, Steven A Atlas, and John H. Laragh
Human renin can cleave rat angiotensinogen, yet infusion of human renin into rats causes only a modest increase in blood pressure. We therefore investigated whether there is a factor in rat plasma which inhibits human renin activity. The addition of 20% normal rat plasma to human plasma had a slight, but not significant, inhibitory effect on the rate of angiotensin formation, while nephrectomized rat plasma, which had a seven-fold higher concentration of angiotensinogen, caused a dose dependent inhibition (20 to 70%). The rat plasma inhibitor copurified with angiotensinogen. Analysis of the kinetics of the human renin-human substrate reaction and of the human renin-rat substrate reaction revealed that the rate of angiotensin I production in the presence of both substrates could be entirely accounted for by assuming that rat and
R
enin is an aspartyl protease whose major source is the kidney and angiotensinogen is its only known protein substrate. Although the plasma angiotensinogen concentration is close to 1000-fold higher than the hourly rate of angiotensin I (Ang I) generation, the concentration of angiotensinogen is normally rate-limiting in both human and animal p l a s m a . The demonstration of a positive relationship between levels of plasma renin activity and 1-3
human angiotensinogens are competitive inhibitors of each other. These results show that human renin can cleave rat substrate but the reaction rate is extremely slow relative to the cleavage of human angiotensinogen. They also indicate that rat angiotensinogen is an effective competitive inhibitor of the human reninsubstrate reaction. These results may be relevant to the development of renin inhibitors and to transfection studies involving heterologous renin or substrate genes. Am J Hypertens 1992;5:495-501
KEY WORDS: Human renin, rat angiotensinogen, human angiotensinogen, renin substrate reaction rate, Michaelis-Menten constant, angiotensin generation rate.
heart attacks in hypertensive patients, makes especially relevant the issue of how human renin can be inhibited. Renin activity is not inhibited by calcium chelators, serum protease inhibitors, reducing agents, heavy metals, or sulphydryl binders. Several different approaches have been taken to inhibit the effect of renin in vivo. Renin secretion is inhibited by ^-adrenergic antagonists. Angiotensin converting enzyme (ACE) inhibitors and angiotensin II (Ang II) receptor antagonists block Ang II production or its effect, respectively. ' Another approach has been to interfere with the reaction of renin with its substrate and to prevent Ang I formation. The development of renin inhibitors has focused in part around the structure-function relationship of angiotensinogen. Angiotensinogen exhibits species specificity due to amino acid substitutions at the cleavage site. Valine and isoleucine at positions 11,12 of 4,5
1
6
7 8
Received March 11, 1992. Accepted May 28, 1992. From the Cardiovascular Center, Cornell University Medical College, New York, New York. This study was supported by research grants from the National Institutes of Health, Bethesda, Maryland (HL 18323 SCR, HL 40152) and from the Mellon Foundation. Address correspondence and reprint requests to Jean E. Sealey, DSc, Cardiovascular Center, New York Hospital—Cornell University Medical College, 525 East 68th Street, New York, NY 10021.
© 1992 by the American Journal of Hypertension, Inc.
940
11
0895-7061/92/'$5.00
496
GAHNEM ET AL
AJH-AUGUST
human angiotensinogen are substituted by leucine and valine in other species. Human angiotensinogen can be cleaved only by primate renins whereas human renin can cleave angiotensinogen from most species. Never theless, infusion of human renin in rats causes only a slight increase in blood pressure ' and the addition of rat plasma to human plasma reduces the rate of angio tensin formation even though the concentration of an giotensinogen is increased. In this study we examined if this impairment in response is the result of an inhibitor of human renin in rat plasma. 1
12
13
METHODS Source of Rat Angiotensinogen Male Sprague-Dawley rats (300 g body weight) were bilaterally nephrecto mized. The rats were decapitated 24 h later and blood was collected in K3-EDTA vacutainers (final concentra tion 3 mmol/L EDTA) and placed immediately on ice. Plasma was separated by centrifugation at 2000 g for 20 min at 4°C, and stored at - 4 0 ° C . Purification of Rat Angiotensinogen Rat angioten sinogen was purified at 4°C as described by Bouhnik et a l . Nephrectomized rat plasma (64 mL, containing 300 //g Ang 1/7.3 g protein) was diluted ( 1 : 1 ) with 50 mmol/L Tris-HCI, pH 8.0, containing 0.1 mol/L NaCI and 3 mmol/L Na -EDTA (Tris buffer #1). An giotensinogen was precipitated between 30 and 6 0 % ammonium sulfate, dialyzed against Tris buffer #1 and 14
2
ο ο
FIGURE 1. Highly purified human renal renin (0.1 mGU/mU was incubated with human angiotensinogen (1400 ngAng I/mL) in the presence of either normal, Nx rat plasma, or rat angiotensinogen at two differ ent stages of purification (see Table 1). The data are represented as percent of control (12.5 ng/mL/h). Ν = 3 per each angioten sinogen concentration, a = Ρ < .05 com pared to the next lowest concentration of rat angiotensinogen.
5, NO. 8
centrifuged at 10,000 g for 30 min. The supernatant was applied to a DEAE-Sepharose (Pharmacia, Uppsala, Sweden) column equilibrated with the same buffer and eluted with a NaCI gradient (0.1 to 0.4mol/L). Fractions that contained the renin substrate were pooled, dialyzed against Tris buffer # 1 , concentrated 15-fold to 12.4 mL (Amicon filter, Beverly, MA), and applied to a Sephacryl S-200 (Pharmacia) column that was preequilibrated in the same buffer. Fractions containing the angiotensino gen were pooled, concentrated to the original loading volume, and stored at — 40°C. Purification of Human Angiotensinogen Blood from normal human volunteers was collected into K3-EDTA vacutainers. Plasma was separated by centrifugation at 2000 g and 25 °C for 20 min and stored at - 4 0 ° C Human angiotensinogen was purified as previously de scribed. The final concentration of renin substrate was 20 //g Ang I/mL. It was stored at - 4 0 ° C . 15
Purification of Human Renal Renin Human renal renin was purified as previously described. An addi tional antihuman renin immunoaffinity chromatogra phy step was used. The antibodies (F-15) were a gift from Drs. Pierre Corvol, Bernard Pau, and Joel Menard. The renin preparation gave a single band on polyacrylamide gel electrophoresis. The concentration was 1.5 Goldblatt units/mL (GU/mL). Incubation with 16
17
Sll
100
1992-VOL
8 ill I
1 3 ι >
5 1
Ξ
so μ
ι
Ο
< 25
μ
ζ
1992;
5:495-501
ORIGINAL CONTRIBUTIONS
Inhibition of Human Renin by Rat Plasma
Rat Angiotensinogen Is a Competitive Inhibitor of the Human Renin-Substrate Interaction
Fuad Gahnem, Jean E. Sealey, Steven A Atlas, and John H. Laragh
Human renin can cleave rat angiotensinogen, yet infusion of human renin into rats causes only a modest increase in blood pressure. We therefore investigated whether there is a factor in rat plasma which inhibits human renin activity. The addition of 20% normal rat plasma to human plasma had a slight, but not significant, inhibitory effect on the rate of angiotensin formation, while nephrectomized rat plasma, which had a seven-fold higher concentration of angiotensinogen, caused a dose dependent inhibition (20 to 70%). The rat plasma inhibitor copurified with angiotensinogen. Analysis of the kinetics of the human renin-human substrate reaction and of the human renin-rat substrate reaction revealed that the rate of angiotensin I production in the presence of both substrates could be entirely accounted for by assuming that rat and
R
enin is an aspartyl protease whose major source is the kidney and angiotensinogen is its only known protein substrate. Although the plasma angiotensinogen concentration is close to 1000-fold higher than the hourly rate of angiotensin I (Ang I) generation, the concentration of angiotensinogen is normally rate-limiting in both human and animal p l a s m a . The demonstration of a positive relationship between levels of plasma renin activity and 1-3
human angiotensinogens are competitive inhibitors of each other. These results show that human renin can cleave rat substrate but the reaction rate is extremely slow relative to the cleavage of human angiotensinogen. They also indicate that rat angiotensinogen is an effective competitive inhibitor of the human reninsubstrate reaction. These results may be relevant to the development of renin inhibitors and to transfection studies involving heterologous renin or substrate genes. Am J Hypertens 1992;5:495-501
KEY WORDS: Human renin, rat angiotensinogen, human angiotensinogen, renin substrate reaction rate, Michaelis-Menten constant, angiotensin generation rate.
heart attacks in hypertensive patients, makes especially relevant the issue of how human renin can be inhibited. Renin activity is not inhibited by calcium chelators, serum protease inhibitors, reducing agents, heavy metals, or sulphydryl binders. Several different approaches have been taken to inhibit the effect of renin in vivo. Renin secretion is inhibited by ^-adrenergic antagonists. Angiotensin converting enzyme (ACE) inhibitors and angiotensin II (Ang II) receptor antagonists block Ang II production or its effect, respectively. ' Another approach has been to interfere with the reaction of renin with its substrate and to prevent Ang I formation. The development of renin inhibitors has focused in part around the structure-function relationship of angiotensinogen. Angiotensinogen exhibits species specificity due to amino acid substitutions at the cleavage site. Valine and isoleucine at positions 11,12 of 4,5
1
6
7 8
Received March 11, 1992. Accepted May 28, 1992. From the Cardiovascular Center, Cornell University Medical College, New York, New York. This study was supported by research grants from the National Institutes of Health, Bethesda, Maryland (HL 18323 SCR, HL 40152) and from the Mellon Foundation. Address correspondence and reprint requests to Jean E. Sealey, DSc, Cardiovascular Center, New York Hospital—Cornell University Medical College, 525 East 68th Street, New York, NY 10021.
© 1992 by the American Journal of Hypertension, Inc.
940
11
0895-7061/92/'$5.00
496
GAHNEM ET AL
AJH-AUGUST
human angiotensinogen are substituted by leucine and valine in other species. Human angiotensinogen can be cleaved only by primate renins whereas human renin can cleave angiotensinogen from most species. Never theless, infusion of human renin in rats causes only a slight increase in blood pressure ' and the addition of rat plasma to human plasma reduces the rate of angio tensin formation even though the concentration of an giotensinogen is increased. In this study we examined if this impairment in response is the result of an inhibitor of human renin in rat plasma. 1
12
13
METHODS Source of Rat Angiotensinogen Male Sprague-Dawley rats (300 g body weight) were bilaterally nephrecto mized. The rats were decapitated 24 h later and blood was collected in K3-EDTA vacutainers (final concentra tion 3 mmol/L EDTA) and placed immediately on ice. Plasma was separated by centrifugation at 2000 g for 20 min at 4°C, and stored at - 4 0 ° C . Purification of Rat Angiotensinogen Rat angioten sinogen was purified at 4°C as described by Bouhnik et a l . Nephrectomized rat plasma (64 mL, containing 300 //g Ang 1/7.3 g protein) was diluted ( 1 : 1 ) with 50 mmol/L Tris-HCI, pH 8.0, containing 0.1 mol/L NaCI and 3 mmol/L Na -EDTA (Tris buffer #1). An giotensinogen was precipitated between 30 and 6 0 % ammonium sulfate, dialyzed against Tris buffer #1 and 14
2
ο ο
FIGURE 1. Highly purified human renal renin (0.1 mGU/mU was incubated with human angiotensinogen (1400 ngAng I/mL) in the presence of either normal, Nx rat plasma, or rat angiotensinogen at two differ ent stages of purification (see Table 1). The data are represented as percent of control (12.5 ng/mL/h). Ν = 3 per each angioten sinogen concentration, a = Ρ < .05 com pared to the next lowest concentration of rat angiotensinogen.
5, NO. 8
centrifuged at 10,000 g for 30 min. The supernatant was applied to a DEAE-Sepharose (Pharmacia, Uppsala, Sweden) column equilibrated with the same buffer and eluted with a NaCI gradient (0.1 to 0.4mol/L). Fractions that contained the renin substrate were pooled, dialyzed against Tris buffer # 1 , concentrated 15-fold to 12.4 mL (Amicon filter, Beverly, MA), and applied to a Sephacryl S-200 (Pharmacia) column that was preequilibrated in the same buffer. Fractions containing the angiotensino gen were pooled, concentrated to the original loading volume, and stored at — 40°C. Purification of Human Angiotensinogen Blood from normal human volunteers was collected into K3-EDTA vacutainers. Plasma was separated by centrifugation at 2000 g and 25 °C for 20 min and stored at - 4 0 ° C Human angiotensinogen was purified as previously de scribed. The final concentration of renin substrate was 20 //g Ang I/mL. It was stored at - 4 0 ° C . 15
Purification of Human Renal Renin Human renal renin was purified as previously described. An addi tional antihuman renin immunoaffinity chromatogra phy step was used. The antibodies (F-15) were a gift from Drs. Pierre Corvol, Bernard Pau, and Joel Menard. The renin preparation gave a single band on polyacrylamide gel electrophoresis. The concentration was 1.5 Goldblatt units/mL (GU/mL). Incubation with 16
17
Sll
100
1992-VOL
8 ill I
1 3 ι >
5 1
Ξ
so μ
ι
Ο
< 25
μ
ζ
