Clinical and Experimental Pharmacology & Physiology (1978) 5,439-448.
CONVERSION OF ANGIOTENSIN I TO ANGIOTENSIN I1 IN SHEEP R.T. Femley, J . P.Coghlan, E.J. Cran, T.W. Fei, B. A. Scoggins and A. McCregor Howard Florey Institute of Experimental Physiology and Medicine, University ofMelbourne, Parkville, Victoria, Australia
(Received 24 Februav 1977;revision received 9 June 1977)
SUMMARY
1. The methodology for measurement of angiotensin Iin whole blood is described. 2. Angiotensin I was measured in arterial and venous blood samples from sodiumloaded, sodium-replete and sodium-depleted sheep. Venous blood concentrations were higher than arterial angiotensin I concentrations. Arterial angiotensin I concentrations increased with increasing sodium deficiency. 3. Angiotensin I1 was infused into six sodium-replete sheep, the blood concentrations of angiotensin I1 measured and the metabolic clearance rates calculated. The average value was 121 21 l/h. 4. Angiotensin I was then infused into these sheep and arterial and venous blood concentrations of angiotensins I and I1 determined. From this data and the metabolic clearance rate of angiotensin 11, the degree of conversion of angiotensin I to I1 was calculated. The values vaned in different sheep from 54 2% to 105 ?r 6% ( n = 6).
*
*
Key words: angiotensin, metabolic clearance rate, percentage conversion, radioimmunoassay. INTRODUCTION It has been shown by bioassay (Ng & Vane, 1968; Biron & Huggins, 1968), by radioimmunoassay (Fanburg & Glazier, 1973) and by chromatographic assay (Oparil, Sanders & Haber, 1970) that the lung is a major site of conversion of the inactive decapeptide, angiotensin I, to the active octapeptide, angiotensin 11. This does not mean, however, that the lung is the major source of endogenous angiotensin 11, and several papers have described the production of angiotensin I1 in tissues where the hormone has its site of action. Examples are in the kidney (Di Salvo et al., 1971; Franklin, Peach & Gilmore, 1970), in the mesenteric arteries (Malik & Nasjletti, 1976; Di Salvo & Montefusco, 1971) and in peripheral tissue in general (Kreye & Gross, 197 1 ;Collier & Robinson, 1974). Whether blood-borne or locally generated angiotensin I1 is more important physiologically has yet to be determined. Correspondence: Dr J . P. Coghlan, Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia. 0305-1 870/78/09004439$02.00 0 1 9 7 8 Blackwell Scientific Publications
439
440
R. T. Fernley et al.
This paper describes an assay for angiotensin I in blood which was used to measure the arterial and venous concentrations of the peptide in various physiological states in the sheep. With the angiotensin I1 assay it was used to determine the degree of conversion of exogenous angiotensin I to I1 in the normal conscious sheep. METHODS
Iod ination of' angiotensins Both angiotensins I and I1 were labelled with '?'I or 1 3 1 1 by the chloramine-T method (Hunter & Greenwood, 1962). Purification was achieved by passage through columns (1 .O X 6.0 cm) of the ionexchange resin Amberlite CG400 equilibrated in 0.05 mol/l sodium acetate buffer (pH 5.0), followed by gel filtration on Sephadex G-15 (angiotensin 11) or Sephadex G-25 (angiotensin I). These columns were 1.2 x 90cm and the gel equilibrated in 0.2mol/l acetic acid. The peak tubes of radioactivity were tested with the appropriate antibody for immunoreactivity, aliquoted and stored at - 20°C. Antibodies Antibodies were raised against 1-Asp-5-Ile-angiotensin I by coupling the peptide to haemocyanin by the carbodiimide method (Good friend, Levine & Fasman, 1964) and injecting the complex mixed with Freund's complete adjuvant into rabbits. Each rabbit received the equivalent of 1.O mg angiotensin I per injection at monthly intervals. Monthly bleeds were taken from the ear vein and tested for antibodies. If these were of sufficiently high titre, the antibodies were tested for sensitivity and cross-reactivity with related peptides. The angiotensin I1 antibodies were the same as those used by Cain, Coghlan & Catt (1972). Extraction The procedure used was essentially that of Cain et al. (1972), except that '251-labelled angiotensin I was used as recovery tracer in the samples. Blood samples (20 ml) were collected directly from catheters inserted in the artery or vein into 3.0 vols of ethanol, mixed and stored at - 20°C until assayed. Radioimmunoassay The radioimmunoassay for angiotensin I was essentially that for angiotensin I1 as described by Cain et al. (1972) except that angiotensin I antibodies were used and 1311-labelled angiotensin 1 used as tracer. Aliquots of the '251-labelled angiotensin I recovery tracer were included in the assay to measure the mass of tracer in the samples. Arterial and venous blood samples were obtained from sheep under various physiological conditions and these were used to assess the angiotensin I assay. Sheep were made sodiumdeficient by uncompensated loss of saliva from a parotid fistula, the degree of deficiency being measured, and sheep were sodium-loaded by including 10 g sodium chloride per day in the food offered to the sheep over a 10 day period. Infusions In a series of six experiments on conscious sodium-replete sheep, either angiotensin I or angiotensin I1 dissolved in normal saline was infused into the jugular vein and blood samples withdrawn from the carotid artery and the contralateral jugular vein. Blood pressure and cardiac rate were measured just before each bleed was taken. Angiotensin I was infused
Conversion of angiotensin I to angiotensin 11
441
at three rates: 0.29, 18.5 and 37 nmol/h for 1 h each, and bleeds were taken 1 5 , 3 0 and 60 min after the beginning of each rate of infusion. When the infusion was completed, the animal was allowed to rest for 2 h, then angiotensin I1 was infused at 24 and 48 nmol/h for 30 min each. Bleeds were taken at 15 and 30 min. RESULTS
Purification of tracer angiotensin I Separation of unlabelled angiotensin I from the 1251or 1311-labelledpeptide was achieved by gel filtration on Sephadex G-25. A specific activity of 800 pCi/pg was found for typical tracer preparations compared to a theoretical maximum of 1230 pCi/pg for monoiodoangiotensin I. Radioimmunoassay The sensitivity of the radioimmunoassay for angiotensin I was found to be 40 fmol giving, with a 50% recovery in the extraction and measuring the sample in duplicate, an assay sensitivity of 8.0 fmol/ml blood using a standard 20ml blood sample. The antibodies used showed only 1% cross-reactivity with angiotensin 11,and generally less than 1% cross-reactivity with sheep renin substrate. Extraction The average recovery of '251-labelled angiotensin I added to four water samples was 78 f 8%, while recovery from six blood samples was 51 k 3%. The recovery of 1.0 pmol angiotensin I added to blood from sodium-loaded sheep, after correction for losses, was 98 k 4.5% (n = 6). The water blank was found to vary considerably with each extraction and two water samples were included in each extraction to monitor the blank. Angiotensin I blood concentrations with valying sodium stants The angiotensin I concentration in arterial blood from salt-loaded sheep was 8.2 k 5.7 fmol/ml ( n = 16). Using pooled arterial blood from salt-loaded sheep and extracting six samples together, a value of 6.5 fmol/ml angiotensin I with an intra-assay variation of
Table 1. Comparison of arterial angiotensin I concentrations in sheep with varying sodium status Angiotensin I concentration (fmol/ml) Sodium status
Mean
s.d.
n
Level o f significance* (degrees o f freedom)
Na+ replete Na+ depleted Na+ loaded
14 41 8.2
10
14 5 16
P < 0.005 (17) P < 0.05 (28)
8.0 5.7
* Determined by Student's t-test.
-
442
R. T. Fernley et al.
3.1 fmollml was found. Similarly, using pooled arterial blood from sodium-replete sheep and extracting samples in a number of separate assays carried out on different days, a value of 22 fmol/ml was obtained with an inter-assay variation o f f 6.0 fmol/ml. The arterial angiotensin I concentration in sodium-replete sheep was measured as 1 4 f 10 fmol/ml (n = 14) while the venous concentration was 19 f 18 fmollml (n = 6). The angiotensin I concentration in arterial blood of sodium-depleted sheep showed a proportional relationship to the degree of sodium depletion, the coefficient of correlation being 0.95 (n = 9). The concentration rose to as high as 95 fmol/ml in a sheep with severe deficiency. The mean arterial angiotensin I concentration in five sheep with a deficiency of 400 to 500 mequiv. of sodium was 41 f 8.0 fmol/ml. These values are summarized in Table 1, and they are shown to be significantly different from one another. Previous studies from this group +_
Table 2. Comparison of angiotensin I and angiotensin 11 levels in both arterial and venous blood of three mildly sodiumdepleted sheep Angiotensin I (fmol/ml) Animal name Ronil Oda Vida
Angiotensin I1 (fmol/ml)
Arterial
Venous
AN
Arterial
Venous
A/V
32 38 65
48 43 81
0.67 0.88 0.80
61
68 100 145
0.99 0.81 0.96
3001 200
81 139
I
-
50
f 0-1 I
I
1-
3 5
6
Time ( h )
Fig. 1. A typical angiotensin 1 and angiotensin I1 infusion experiment with experimental animal Fess. The lower part of the graph shows the rate of infusion of the peptides vs time, while the graphs above this show the resultant blood angiotensin I and angiotensin I1 concentrations and the blood pressure response. For infusion rate: (UB) angiotensin I; (€I) angiotensin 11.
Conversion of angiotensin I to angiotensin II
443
(Cain et al., 1970) have shown that the arterial angiotensin I1 levels in sodium-replete sheep are 14 f 11 fmol/ml and also in sodium deficiency there is a positive correlation between arterial angiotensin I1 concentration and the degree of sodium deficiency (r = 0.71, n = 22). Table 2 compares the concentration of angiotensins I and I1 in venous and arterial blood of three mildly sodiumdepleted sheep. The average arterial/venous ratio for angiotensin I is 0.78, showing loss of angiotensin I across the lung, probably by conversion to angiotensin 11. Angiotensin infusions
The results of a typical infusion experiment are shown with angiotensin I and I1 concentrations and blood pressure responses (Fig. 1). The arterial blood concentrations of angiotensin I1 in the six experimental animals at two levels of angiotensin I1 infusion are shown in Table 3. From these data the blood clearance rate of angiotensin I1 has been calculated for each animal (Tait et al., (1962). The clearance rate varies from 100 l/h to 157 l/h with a mean of 121 l/h (+ 21, s.d.). From the clearance rate for angiotensin I1 and the concentration of angiotensin I1 in the blood resulting from the infusion of angiotensin I, Table 3. Mean blood clearance rates for angiotensin I1 in six conscious sodium-replete sheep
Infusion rate of angiotensin I1 (nmol/h)
Arterial blood angiotensin I1 concentration (fmol/ml)
Clearance rate angiotensin I1 (Uh)
24 24 48 48
252 203 337 282
95 118 142 170
24 24 48 48
210 130 320 27 0
113 187 152 178
Hugo
48 48
450 510
106 94
Rigo 1
24 24 48 48
220 190 420 340
110 127 114 143
24 24 48 48
240 220 450 490
99 107 107 97
24 24 48 48
200 180 490 480
119 136 98 100
Animal name Talon I
Fess
Talon I1
Rigo I1
Overall clearance rate for angiotensin II = 121 f 21 l/h (mean
Clearance rate (l/h) Mean
s.d.
131
32
157
33
100
122
102
113
f
s.d.).
15
5.0
18
100
9.25 18.5 37
9.25
Hugo
Rigo I
113
37
Rig0 I1
18% (mean
* s.d.1.
-
-
76 72 104
56 53 53
59 93
50
97.5 107 109
Percentage
t Each angiotensin I1 concentration is the mean of three determinations.
* As determined by angiotensin I1 infusion as shown in Table 3.
i
21 7
235
58 109 316
52 98.5 197
30 69.5 219
69 151 308
Angiotensin I1 concentration (fmol/ml)t
Overall percentage conversion = 73.5
102
37
37
Talon I1
122
157
9.25 18.5 37
Fess
18.5
131
Mean angiotensin I1 clearance rate (l/h)*
9.25 18.5 37
Angiotensin I infusion rate (nmol/h)
Talon 1
Animal name
66
65
84
54
67
105
Mean percentage
Conversion
Table 4. Percentage conversions of infused angiotensin I to angiotensin I1 in sodium-replete sheep
17
2
23
6
s.d.
>
%
m a
-G23
.r
.a
Conversion o f angiotensin I to angiotensin II
445
Table 5. Arterial and venous levels of angiotensin I from sodium-replete sheep infused with angiotensin I (37 nmol/h) Animal name
Hugo
Rigo I
Talon I1
Rig0 I1
Mean s.d.
Arterial angiotensin I (fmol/ml)
Venous angiotensin I (fmol/ml)
55 68 39
36 32
32 25 49
16 22
37 47 52
23
53 80 89
22 35
52 19
26.5 1.7
These bleeds were taken when the steady state in blood angiotensin I concentration was achieved.
under steady state conditions, the rate of formation of angiotensin I1 can be calculated. The degree of conversion is then the ratio of the production rate of angiotensin I1 from the infused angiotensin I to the infusion rate of angiotensin I: percentage conversion = PAU -x 100 = IAI
cAU
[BIAU
IAI
Where PA,, = production rate of angiotensin II;-IAI = infusion rate of angiotensin I; C,, = blood clearance rate of angiotensin 11; and [B] = blood concentration of angiotensin 11. These values are listed in Table 4 and they vary from 54 f 2% to 105 f 6% conversion (mean = 73.5% 18%, n = 6 ) . Table 5 compares the steady state venous angiotensin I concentrations with the corresponding arterial angiotensin I concentrations when the sheep were infused with angiotensin I at 37 nmol/h. The venous concentration was 26.5 f 7.7 fmol/ml while the average arterial angiotensin I concentration was twice this at 52 f 19 fmol/ml.
*
DISCUSSION
The assay for angiotensin I in blood has been established and fundamental parameters such as blank, sensitivity, interference by angiotensin I1 and renin substrate, and precision, have been measured. The assay has given consistent results using blood samples obtained under various physiological conditions and during infusion of the peptide. Sodium loading is
446
R. T. Fernley et al.
known to suppress renin secretion and hence lower circulating levels of the angiotensins, and this was shown in the analyses of blood from sodium-loaded sheep. Conversely, sodium depletion leads to higher angiotensin I levels and this was also found in the analyses of blood samples from sodium-depleted sheep. The assay is not dissimilar to that described by Waite (1973), although it is a somewhat shorter procedure. His values for normal man ranged from 8.5 to 68 fmol/ml (mean 39 fmol/ml) in venous blood and from 9.0 t o 59 (mean 23) fmol/ml in arterial blood. The differences in angiotensin I concentrations between venous and arterial blood shown in the mildly sodiumdepleted sheep indicates conversion of endogenous angiotensin I to I1 across the pulmonary circulation. The angiotensin I1 concentration in arterial blood did not rise correspondingly; however, this may be explained by the presence of a larger percentage of immunoreactive fragments of angiotensin I1 in venous blood than in arterial blood (Cain, Catt & Coghlan, 1969). This would require that the fragments be cleared in the lung, but there is as yet no direct evidence for this. Angiotensin I1 is perhaps formed as well as cleared in peripheral tissue, which would also increase the venous concentration to an extent not expected from the calculated clearance rate. This problem has not yet been resolved. During infusion of angiotensin I into the jugular vein, the angiotensin I concentration in the blood from the contralateral jugular vein is about half the carotid artery angiotensin I concentration. This suggests that the clearance rate of angiotensin I across the peripheral vascular beds is about the same as that of angiotensin 11: that is, about 120 l/h. The high degree of conversion of angiotensin I to I1 indicates a highly efficient system of activation of the peptide for presentation to the target tissues via the arterial blood. This reinforces the concept that the lung is the major site of physiological activation of the hormone. However, it does not rule out the possibility of local generation and action of angiotensin at target tissues. An example is the local production of angiotensin I1 in the kidney, where conflicting evidence has been obtained. Abe et al. (1975) infused renin with or without an angiotensin antagonist into the renal artery of anaesthetized dogs. By observing the effects on renal blood flow, plasma renin activity and renal venous plasma concentrations of angiotensin I and 11, they concluded that local production played at the most a minor role in the control of renal haemodynamics. Itskovitz, Herbert & McGiff (1973) measured renal blood flow in isolated blood-perfused dog kidneys and observed changes in renal blood flow patterns when the endogenous renin substrate was depleted. The original blood flow pattern was restored by infusion of tetradecapeptide renin substrate but not by angiotensin 11, suggesting that local production and not circulating angiotensin I1 mediated these effects. Thurston & Swales (1974) used acute and chronic two kidney Goldblatt hypertensive rats with infusions of angiotensin I1 antibody or angiotensin I1 antagonist to investigate the significance of local generation of angiotensin 11. For rats with short term hypertension, injection of sufficient angiotensin I1 antiserum to substantially block an administered dose of 50 ng of angiotensin I1 failed to reduce their elevated blood pressure. However, injection of the angiotensin I1 antagonist, 1-Sar-8-Ala-angiotensin 11, again in sufficient doses to block the effect of exogenous angiotensin, did lower the blood pressure substantially. Similar observations were made on chronic hypertensive rats, although the drop in blood pressure was less than that in the first group. This evidence supports the idea of formation of angiotensin I1 at the site of its action, which is accessible to the small antagonist molecule but not to the much larger antibody molecule. Similar results using a different approach have been found by the same workers (Swales & Thurston, 1973). However, Bing & Poulsen (1970) did report a transient fall in blood pressure in normal
Conversion o f angiotensin I to angiotensin 11
447
rats on injection of anti-angiotensin I1 antibody, an effect not found in nephrectomized rats. This argues for a role for circulating angiotensin I1 in the control of blood pressure, but the question of local production of physiologically active angiotensin I1 remains, as yet, unresolved. ACKNOWLEDGMENTS
This work was supported in part by Grants in Aid from the National Heart Foundation of Australia and the National Health and Medical Research Council of Australia. REFERENCES Abe, Y., Omosu, M., Iwao, H., Okahara, T., Kishimoto, T. & Yamamoto, K. (1975) Intrarenal roles of renin-angiotensin system. IV. Effects of homologous renin on renal function during angiotensin I1 antagonist infusion in dogs. Japanese Journal of Pharmacology, 25, Suppl. 1,41-42. Bing, J. & Poulsen, K. (1970) Effect of anti-angiotensin I1 on blood pressure and sensitivity to angiotensin and renin. Acta pathologica et microbiologica scandinavica, 78a, 6-1 8. Biron, P. & Huggins, C.G. (1968) Pulmonary activation of synthetic angiotensin. I. Life Sciences, 7, 9 6 5-9 70. Chin, M.D., Catt, KJ., Coghlan, J.P. & Blair-West, J.R. (1970) Evaluation of angiotensin I1 metabolism in sheep by radioimmunoassay. Endocrinology, 86,955-964. Cain, M.D., Catt, K J . & Coghlan, J.P. (1969) Effect of circulating fragments of angiotensin I1 on radioirnmunoassay in arterial and venous blood. Journal of Clinical Endocrinology and Metabolism, 29,1639-1643. Cain, M.D., Coghlan, J.P. & Catt, K J . (1972) Measurement of angiotensin I1 in blood by radioimmunoassay. Clinica chimica acta, 39,21-34. Collier, J.G. & Robinson, B.F. (1974) Comparison of effects of locally infused angiotensin I and I1 on hand veins and forearm arteries in man: evidence for converting enzyme activity in limb vessels. Clinical Science and Molecular Medicine, 47, 189-1 92. Di Salvo, J. & Montefusco, C.B. (1971) Conversion of angiotensin I to angiotensin I1 in the canine mesenteric circulation. American Journal of Physiology, 221, 1576-1579. Di Salvo, J., Peterson, A., Montefusco, C. & Menta, M. (1971) Intrarenal conversion of angiotensin I to angiotensin I1 in the dog. Circulation Research, 29, 398-406. Fanburg, B.L. & Glazier, J.B. (1973) Conversion of angiotensin I to angiotensin I1 in the isolated perfused dog lung. Journal ofApplied Physiology, 35, 325-331. Franklin, W.G., Peach, M J . & Gilmore, J.P. (1970) Evidence for the renal conversion of angiotensin I in the dog. Circulation Research, 27, 321-324. Goodfriend, T.L., Levine, L. & Fasman, G.D. (1964) Antibodies to bradykinin and angiotensin: a use of carbodiimides in immunology. Science, 144, 1344-1 346. Hunter, W.M. & Greenwood, F.C. (1962) Preparation of iodine-1 31-labelled human growth hormone of high specific activity. Nature, 194,495496. Itskovitz, H.D., Herbert, L.A. & McGiff, J.C. (1973) Angiotensin as a possible intrarenal hormone in isolated dog kidneys. Circulation Research, 32,550-555. Kreye,V.A.W. & Gross, F. (1971) Conversion of angiotensin I to angiotensin I1 in peripheral vascular beds of the rat. American Journal ofPhysiology, 220, 1294-1296. Malik, K.U. & Nasjletti, A. (1976) Fascilitation of adrenergic transmission by locally generated angiotensin I1 in rat mesenteric arteries. Circulation Research, 38,26-30. Ng, K.K.F. & Vane, J.R. (1968) Fate of angiotensin I in the circulation. Nature, 218, 144-150. Oparil, S., Sanders, G.A. & Haber, E. (1970) In vivo and in vitro conversion of angiotensin I to I1 in dog blood. Circulation Research, 26,591-599. Swales, J.D. & Thurston, H. (1973) Generation of angiotensin 11 at peripheral vascular level: studies using angiotensin I1 antisera. Clinical Science and Molecular Medicine, 45,69 1-700. Tait, J.F., Little, B., Tait, S.A.S. & Flood, C. (1962) The metabolic clearance rate of aldosterone in pregnant and non-pregnant subjects estimated by both single injection and constant infusion methods. Journal of Clinical Investigation, 41, 2093-2100.
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Thurston, H. & Swales, J.D. (1974) Comparison of angiotensin 11 antagonist and antiserum infusion with nephrectomy in the rat with two-kidney Goldblatt hypertension. Circularion Research, 35, 3 25-3 29. Waite, M.A. (1973) Measurement of concentrations of angiotensin I in human blood by radioimmunoassay. Clinical Science and Molecular Medicine, 45, 5 1-64.
CONVERSION OF ANGIOTENSIN I TO ANGIOTENSIN I1 IN SHEEP R.T. Femley, J . P.Coghlan, E.J. Cran, T.W. Fei, B. A. Scoggins and A. McCregor Howard Florey Institute of Experimental Physiology and Medicine, University ofMelbourne, Parkville, Victoria, Australia
(Received 24 Februav 1977;revision received 9 June 1977)
SUMMARY
1. The methodology for measurement of angiotensin Iin whole blood is described. 2. Angiotensin I was measured in arterial and venous blood samples from sodiumloaded, sodium-replete and sodium-depleted sheep. Venous blood concentrations were higher than arterial angiotensin I concentrations. Arterial angiotensin I concentrations increased with increasing sodium deficiency. 3. Angiotensin I1 was infused into six sodium-replete sheep, the blood concentrations of angiotensin I1 measured and the metabolic clearance rates calculated. The average value was 121 21 l/h. 4. Angiotensin I was then infused into these sheep and arterial and venous blood concentrations of angiotensins I and I1 determined. From this data and the metabolic clearance rate of angiotensin 11, the degree of conversion of angiotensin I to I1 was calculated. The values vaned in different sheep from 54 2% to 105 ?r 6% ( n = 6).
*
*
Key words: angiotensin, metabolic clearance rate, percentage conversion, radioimmunoassay. INTRODUCTION It has been shown by bioassay (Ng & Vane, 1968; Biron & Huggins, 1968), by radioimmunoassay (Fanburg & Glazier, 1973) and by chromatographic assay (Oparil, Sanders & Haber, 1970) that the lung is a major site of conversion of the inactive decapeptide, angiotensin I, to the active octapeptide, angiotensin 11. This does not mean, however, that the lung is the major source of endogenous angiotensin 11, and several papers have described the production of angiotensin I1 in tissues where the hormone has its site of action. Examples are in the kidney (Di Salvo et al., 1971; Franklin, Peach & Gilmore, 1970), in the mesenteric arteries (Malik & Nasjletti, 1976; Di Salvo & Montefusco, 1971) and in peripheral tissue in general (Kreye & Gross, 197 1 ;Collier & Robinson, 1974). Whether blood-borne or locally generated angiotensin I1 is more important physiologically has yet to be determined. Correspondence: Dr J . P. Coghlan, Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia. 0305-1 870/78/09004439$02.00 0 1 9 7 8 Blackwell Scientific Publications
439
440
R. T. Fernley et al.
This paper describes an assay for angiotensin I in blood which was used to measure the arterial and venous concentrations of the peptide in various physiological states in the sheep. With the angiotensin I1 assay it was used to determine the degree of conversion of exogenous angiotensin I to I1 in the normal conscious sheep. METHODS
Iod ination of' angiotensins Both angiotensins I and I1 were labelled with '?'I or 1 3 1 1 by the chloramine-T method (Hunter & Greenwood, 1962). Purification was achieved by passage through columns (1 .O X 6.0 cm) of the ionexchange resin Amberlite CG400 equilibrated in 0.05 mol/l sodium acetate buffer (pH 5.0), followed by gel filtration on Sephadex G-15 (angiotensin 11) or Sephadex G-25 (angiotensin I). These columns were 1.2 x 90cm and the gel equilibrated in 0.2mol/l acetic acid. The peak tubes of radioactivity were tested with the appropriate antibody for immunoreactivity, aliquoted and stored at - 20°C. Antibodies Antibodies were raised against 1-Asp-5-Ile-angiotensin I by coupling the peptide to haemocyanin by the carbodiimide method (Good friend, Levine & Fasman, 1964) and injecting the complex mixed with Freund's complete adjuvant into rabbits. Each rabbit received the equivalent of 1.O mg angiotensin I per injection at monthly intervals. Monthly bleeds were taken from the ear vein and tested for antibodies. If these were of sufficiently high titre, the antibodies were tested for sensitivity and cross-reactivity with related peptides. The angiotensin I1 antibodies were the same as those used by Cain, Coghlan & Catt (1972). Extraction The procedure used was essentially that of Cain et al. (1972), except that '251-labelled angiotensin I was used as recovery tracer in the samples. Blood samples (20 ml) were collected directly from catheters inserted in the artery or vein into 3.0 vols of ethanol, mixed and stored at - 20°C until assayed. Radioimmunoassay The radioimmunoassay for angiotensin I was essentially that for angiotensin I1 as described by Cain et al. (1972) except that angiotensin I antibodies were used and 1311-labelled angiotensin 1 used as tracer. Aliquots of the '251-labelled angiotensin I recovery tracer were included in the assay to measure the mass of tracer in the samples. Arterial and venous blood samples were obtained from sheep under various physiological conditions and these were used to assess the angiotensin I assay. Sheep were made sodiumdeficient by uncompensated loss of saliva from a parotid fistula, the degree of deficiency being measured, and sheep were sodium-loaded by including 10 g sodium chloride per day in the food offered to the sheep over a 10 day period. Infusions In a series of six experiments on conscious sodium-replete sheep, either angiotensin I or angiotensin I1 dissolved in normal saline was infused into the jugular vein and blood samples withdrawn from the carotid artery and the contralateral jugular vein. Blood pressure and cardiac rate were measured just before each bleed was taken. Angiotensin I was infused
Conversion of angiotensin I to angiotensin 11
441
at three rates: 0.29, 18.5 and 37 nmol/h for 1 h each, and bleeds were taken 1 5 , 3 0 and 60 min after the beginning of each rate of infusion. When the infusion was completed, the animal was allowed to rest for 2 h, then angiotensin I1 was infused at 24 and 48 nmol/h for 30 min each. Bleeds were taken at 15 and 30 min. RESULTS
Purification of tracer angiotensin I Separation of unlabelled angiotensin I from the 1251or 1311-labelledpeptide was achieved by gel filtration on Sephadex G-25. A specific activity of 800 pCi/pg was found for typical tracer preparations compared to a theoretical maximum of 1230 pCi/pg for monoiodoangiotensin I. Radioimmunoassay The sensitivity of the radioimmunoassay for angiotensin I was found to be 40 fmol giving, with a 50% recovery in the extraction and measuring the sample in duplicate, an assay sensitivity of 8.0 fmol/ml blood using a standard 20ml blood sample. The antibodies used showed only 1% cross-reactivity with angiotensin 11,and generally less than 1% cross-reactivity with sheep renin substrate. Extraction The average recovery of '251-labelled angiotensin I added to four water samples was 78 f 8%, while recovery from six blood samples was 51 k 3%. The recovery of 1.0 pmol angiotensin I added to blood from sodium-loaded sheep, after correction for losses, was 98 k 4.5% (n = 6). The water blank was found to vary considerably with each extraction and two water samples were included in each extraction to monitor the blank. Angiotensin I blood concentrations with valying sodium stants The angiotensin I concentration in arterial blood from salt-loaded sheep was 8.2 k 5.7 fmol/ml ( n = 16). Using pooled arterial blood from salt-loaded sheep and extracting six samples together, a value of 6.5 fmol/ml angiotensin I with an intra-assay variation of
Table 1. Comparison of arterial angiotensin I concentrations in sheep with varying sodium status Angiotensin I concentration (fmol/ml) Sodium status
Mean
s.d.
n
Level o f significance* (degrees o f freedom)
Na+ replete Na+ depleted Na+ loaded
14 41 8.2
10
14 5 16
P < 0.005 (17) P < 0.05 (28)
8.0 5.7
* Determined by Student's t-test.
-
442
R. T. Fernley et al.
3.1 fmollml was found. Similarly, using pooled arterial blood from sodium-replete sheep and extracting samples in a number of separate assays carried out on different days, a value of 22 fmol/ml was obtained with an inter-assay variation o f f 6.0 fmol/ml. The arterial angiotensin I concentration in sodium-replete sheep was measured as 1 4 f 10 fmol/ml (n = 14) while the venous concentration was 19 f 18 fmollml (n = 6). The angiotensin I concentration in arterial blood of sodium-depleted sheep showed a proportional relationship to the degree of sodium depletion, the coefficient of correlation being 0.95 (n = 9). The concentration rose to as high as 95 fmol/ml in a sheep with severe deficiency. The mean arterial angiotensin I concentration in five sheep with a deficiency of 400 to 500 mequiv. of sodium was 41 f 8.0 fmol/ml. These values are summarized in Table 1, and they are shown to be significantly different from one another. Previous studies from this group +_
Table 2. Comparison of angiotensin I and angiotensin 11 levels in both arterial and venous blood of three mildly sodiumdepleted sheep Angiotensin I (fmol/ml) Animal name Ronil Oda Vida
Angiotensin I1 (fmol/ml)
Arterial
Venous
AN
Arterial
Venous
A/V
32 38 65
48 43 81
0.67 0.88 0.80
61
68 100 145
0.99 0.81 0.96
3001 200
81 139
I
-
50
f 0-1 I
I
1-
3 5
6
Time ( h )
Fig. 1. A typical angiotensin 1 and angiotensin I1 infusion experiment with experimental animal Fess. The lower part of the graph shows the rate of infusion of the peptides vs time, while the graphs above this show the resultant blood angiotensin I and angiotensin I1 concentrations and the blood pressure response. For infusion rate: (UB) angiotensin I; (€I) angiotensin 11.
Conversion of angiotensin I to angiotensin II
443
(Cain et al., 1970) have shown that the arterial angiotensin I1 levels in sodium-replete sheep are 14 f 11 fmol/ml and also in sodium deficiency there is a positive correlation between arterial angiotensin I1 concentration and the degree of sodium deficiency (r = 0.71, n = 22). Table 2 compares the concentration of angiotensins I and I1 in venous and arterial blood of three mildly sodiumdepleted sheep. The average arterial/venous ratio for angiotensin I is 0.78, showing loss of angiotensin I across the lung, probably by conversion to angiotensin 11. Angiotensin infusions
The results of a typical infusion experiment are shown with angiotensin I and I1 concentrations and blood pressure responses (Fig. 1). The arterial blood concentrations of angiotensin I1 in the six experimental animals at two levels of angiotensin I1 infusion are shown in Table 3. From these data the blood clearance rate of angiotensin I1 has been calculated for each animal (Tait et al., (1962). The clearance rate varies from 100 l/h to 157 l/h with a mean of 121 l/h (+ 21, s.d.). From the clearance rate for angiotensin I1 and the concentration of angiotensin I1 in the blood resulting from the infusion of angiotensin I, Table 3. Mean blood clearance rates for angiotensin I1 in six conscious sodium-replete sheep
Infusion rate of angiotensin I1 (nmol/h)
Arterial blood angiotensin I1 concentration (fmol/ml)
Clearance rate angiotensin I1 (Uh)
24 24 48 48
252 203 337 282
95 118 142 170
24 24 48 48
210 130 320 27 0
113 187 152 178
Hugo
48 48
450 510
106 94
Rigo 1
24 24 48 48
220 190 420 340
110 127 114 143
24 24 48 48
240 220 450 490
99 107 107 97
24 24 48 48
200 180 490 480
119 136 98 100
Animal name Talon I
Fess
Talon I1
Rigo I1
Overall clearance rate for angiotensin II = 121 f 21 l/h (mean
Clearance rate (l/h) Mean
s.d.
131
32
157
33
100
122
102
113
f
s.d.).
15
5.0
18
100
9.25 18.5 37
9.25
Hugo
Rigo I
113
37
Rig0 I1
18% (mean
* s.d.1.
-
-
76 72 104
56 53 53
59 93
50
97.5 107 109
Percentage
t Each angiotensin I1 concentration is the mean of three determinations.
* As determined by angiotensin I1 infusion as shown in Table 3.
i
21 7
235
58 109 316
52 98.5 197
30 69.5 219
69 151 308
Angiotensin I1 concentration (fmol/ml)t
Overall percentage conversion = 73.5
102
37
37
Talon I1
122
157
9.25 18.5 37
Fess
18.5
131
Mean angiotensin I1 clearance rate (l/h)*
9.25 18.5 37
Angiotensin I infusion rate (nmol/h)
Talon 1
Animal name
66
65
84
54
67
105
Mean percentage
Conversion
Table 4. Percentage conversions of infused angiotensin I to angiotensin I1 in sodium-replete sheep
17
2
23
6
s.d.
>
%
m a
-G23
.r
.a
Conversion o f angiotensin I to angiotensin II
445
Table 5. Arterial and venous levels of angiotensin I from sodium-replete sheep infused with angiotensin I (37 nmol/h) Animal name
Hugo
Rigo I
Talon I1
Rig0 I1
Mean s.d.
Arterial angiotensin I (fmol/ml)
Venous angiotensin I (fmol/ml)
55 68 39
36 32
32 25 49
16 22
37 47 52
23
53 80 89
22 35
52 19
26.5 1.7
These bleeds were taken when the steady state in blood angiotensin I concentration was achieved.
under steady state conditions, the rate of formation of angiotensin I1 can be calculated. The degree of conversion is then the ratio of the production rate of angiotensin I1 from the infused angiotensin I to the infusion rate of angiotensin I: percentage conversion = PAU -x 100 = IAI
cAU
[BIAU
IAI
Where PA,, = production rate of angiotensin II;-IAI = infusion rate of angiotensin I; C,, = blood clearance rate of angiotensin 11; and [B] = blood concentration of angiotensin 11. These values are listed in Table 4 and they vary from 54 f 2% to 105 f 6% conversion (mean = 73.5% 18%, n = 6 ) . Table 5 compares the steady state venous angiotensin I concentrations with the corresponding arterial angiotensin I concentrations when the sheep were infused with angiotensin I at 37 nmol/h. The venous concentration was 26.5 f 7.7 fmol/ml while the average arterial angiotensin I concentration was twice this at 52 f 19 fmol/ml.
*
DISCUSSION
The assay for angiotensin I in blood has been established and fundamental parameters such as blank, sensitivity, interference by angiotensin I1 and renin substrate, and precision, have been measured. The assay has given consistent results using blood samples obtained under various physiological conditions and during infusion of the peptide. Sodium loading is
446
R. T. Fernley et al.
known to suppress renin secretion and hence lower circulating levels of the angiotensins, and this was shown in the analyses of blood from sodium-loaded sheep. Conversely, sodium depletion leads to higher angiotensin I levels and this was also found in the analyses of blood samples from sodium-depleted sheep. The assay is not dissimilar to that described by Waite (1973), although it is a somewhat shorter procedure. His values for normal man ranged from 8.5 to 68 fmol/ml (mean 39 fmol/ml) in venous blood and from 9.0 t o 59 (mean 23) fmol/ml in arterial blood. The differences in angiotensin I concentrations between venous and arterial blood shown in the mildly sodiumdepleted sheep indicates conversion of endogenous angiotensin I to I1 across the pulmonary circulation. The angiotensin I1 concentration in arterial blood did not rise correspondingly; however, this may be explained by the presence of a larger percentage of immunoreactive fragments of angiotensin I1 in venous blood than in arterial blood (Cain, Catt & Coghlan, 1969). This would require that the fragments be cleared in the lung, but there is as yet no direct evidence for this. Angiotensin I1 is perhaps formed as well as cleared in peripheral tissue, which would also increase the venous concentration to an extent not expected from the calculated clearance rate. This problem has not yet been resolved. During infusion of angiotensin I into the jugular vein, the angiotensin I concentration in the blood from the contralateral jugular vein is about half the carotid artery angiotensin I concentration. This suggests that the clearance rate of angiotensin I across the peripheral vascular beds is about the same as that of angiotensin 11: that is, about 120 l/h. The high degree of conversion of angiotensin I to I1 indicates a highly efficient system of activation of the peptide for presentation to the target tissues via the arterial blood. This reinforces the concept that the lung is the major site of physiological activation of the hormone. However, it does not rule out the possibility of local generation and action of angiotensin at target tissues. An example is the local production of angiotensin I1 in the kidney, where conflicting evidence has been obtained. Abe et al. (1975) infused renin with or without an angiotensin antagonist into the renal artery of anaesthetized dogs. By observing the effects on renal blood flow, plasma renin activity and renal venous plasma concentrations of angiotensin I and 11, they concluded that local production played at the most a minor role in the control of renal haemodynamics. Itskovitz, Herbert & McGiff (1973) measured renal blood flow in isolated blood-perfused dog kidneys and observed changes in renal blood flow patterns when the endogenous renin substrate was depleted. The original blood flow pattern was restored by infusion of tetradecapeptide renin substrate but not by angiotensin 11, suggesting that local production and not circulating angiotensin I1 mediated these effects. Thurston & Swales (1974) used acute and chronic two kidney Goldblatt hypertensive rats with infusions of angiotensin I1 antibody or angiotensin I1 antagonist to investigate the significance of local generation of angiotensin 11. For rats with short term hypertension, injection of sufficient angiotensin I1 antiserum to substantially block an administered dose of 50 ng of angiotensin I1 failed to reduce their elevated blood pressure. However, injection of the angiotensin I1 antagonist, 1-Sar-8-Ala-angiotensin 11, again in sufficient doses to block the effect of exogenous angiotensin, did lower the blood pressure substantially. Similar observations were made on chronic hypertensive rats, although the drop in blood pressure was less than that in the first group. This evidence supports the idea of formation of angiotensin I1 at the site of its action, which is accessible to the small antagonist molecule but not to the much larger antibody molecule. Similar results using a different approach have been found by the same workers (Swales & Thurston, 1973). However, Bing & Poulsen (1970) did report a transient fall in blood pressure in normal
Conversion o f angiotensin I to angiotensin 11
447
rats on injection of anti-angiotensin I1 antibody, an effect not found in nephrectomized rats. This argues for a role for circulating angiotensin I1 in the control of blood pressure, but the question of local production of physiologically active angiotensin I1 remains, as yet, unresolved. ACKNOWLEDGMENTS
This work was supported in part by Grants in Aid from the National Heart Foundation of Australia and the National Health and Medical Research Council of Australia. REFERENCES Abe, Y., Omosu, M., Iwao, H., Okahara, T., Kishimoto, T. & Yamamoto, K. (1975) Intrarenal roles of renin-angiotensin system. IV. Effects of homologous renin on renal function during angiotensin I1 antagonist infusion in dogs. Japanese Journal of Pharmacology, 25, Suppl. 1,41-42. Bing, J. & Poulsen, K. (1970) Effect of anti-angiotensin I1 on blood pressure and sensitivity to angiotensin and renin. Acta pathologica et microbiologica scandinavica, 78a, 6-1 8. Biron, P. & Huggins, C.G. (1968) Pulmonary activation of synthetic angiotensin. I. Life Sciences, 7, 9 6 5-9 70. Chin, M.D., Catt, KJ., Coghlan, J.P. & Blair-West, J.R. (1970) Evaluation of angiotensin I1 metabolism in sheep by radioimmunoassay. Endocrinology, 86,955-964. Cain, M.D., Catt, K J . & Coghlan, J.P. (1969) Effect of circulating fragments of angiotensin I1 on radioirnmunoassay in arterial and venous blood. Journal of Clinical Endocrinology and Metabolism, 29,1639-1643. Cain, M.D., Coghlan, J.P. & Catt, K J . (1972) Measurement of angiotensin I1 in blood by radioimmunoassay. Clinica chimica acta, 39,21-34. Collier, J.G. & Robinson, B.F. (1974) Comparison of effects of locally infused angiotensin I and I1 on hand veins and forearm arteries in man: evidence for converting enzyme activity in limb vessels. Clinical Science and Molecular Medicine, 47, 189-1 92. Di Salvo, J. & Montefusco, C.B. (1971) Conversion of angiotensin I to angiotensin I1 in the canine mesenteric circulation. American Journal of Physiology, 221, 1576-1579. Di Salvo, J., Peterson, A., Montefusco, C. & Menta, M. (1971) Intrarenal conversion of angiotensin I to angiotensin I1 in the dog. Circulation Research, 29, 398-406. Fanburg, B.L. & Glazier, J.B. (1973) Conversion of angiotensin I to angiotensin I1 in the isolated perfused dog lung. Journal ofApplied Physiology, 35, 325-331. Franklin, W.G., Peach, M J . & Gilmore, J.P. (1970) Evidence for the renal conversion of angiotensin I in the dog. Circulation Research, 27, 321-324. Goodfriend, T.L., Levine, L. & Fasman, G.D. (1964) Antibodies to bradykinin and angiotensin: a use of carbodiimides in immunology. Science, 144, 1344-1 346. Hunter, W.M. & Greenwood, F.C. (1962) Preparation of iodine-1 31-labelled human growth hormone of high specific activity. Nature, 194,495496. Itskovitz, H.D., Herbert, L.A. & McGiff, J.C. (1973) Angiotensin as a possible intrarenal hormone in isolated dog kidneys. Circulation Research, 32,550-555. Kreye,V.A.W. & Gross, F. (1971) Conversion of angiotensin I to angiotensin I1 in peripheral vascular beds of the rat. American Journal ofPhysiology, 220, 1294-1296. Malik, K.U. & Nasjletti, A. (1976) Fascilitation of adrenergic transmission by locally generated angiotensin I1 in rat mesenteric arteries. Circulation Research, 38,26-30. Ng, K.K.F. & Vane, J.R. (1968) Fate of angiotensin I in the circulation. Nature, 218, 144-150. Oparil, S., Sanders, G.A. & Haber, E. (1970) In vivo and in vitro conversion of angiotensin I to I1 in dog blood. Circulation Research, 26,591-599. Swales, J.D. & Thurston, H. (1973) Generation of angiotensin 11 at peripheral vascular level: studies using angiotensin I1 antisera. Clinical Science and Molecular Medicine, 45,69 1-700. Tait, J.F., Little, B., Tait, S.A.S. & Flood, C. (1962) The metabolic clearance rate of aldosterone in pregnant and non-pregnant subjects estimated by both single injection and constant infusion methods. Journal of Clinical Investigation, 41, 2093-2100.
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Thurston, H. & Swales, J.D. (1974) Comparison of angiotensin 11 antagonist and antiserum infusion with nephrectomy in the rat with two-kidney Goldblatt hypertension. Circularion Research, 35, 3 25-3 29. Waite, M.A. (1973) Measurement of concentrations of angiotensin I in human blood by radioimmunoassay. Clinical Science and Molecular Medicine, 45, 5 1-64.
