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Systematic synthesis of prostaglandin I2 following sustained infusion of angiotensin II in conscious dogs
European Journal of Pharmacology, 127 (1986) 9-16 Elsevier
9
S Y S T E M I C S Y N T H E S I S OF P R O S T A G L A N D I N 12 F O L L O W I N G S U S T A I N E D I N F U S I O N O F A N G I O T E N S I N II IN C O N S C I O U S D O G S MICHAEL L. WATSON *, ROBERT J. WORKMAN, WILLIAM HERZER, ROBERT A. BRANCH, JOHN A. OATES and ALAN R. BRASH
Department of Clinical Pharmacology, Vanderbilt University, Nashville, TN 37232, U.S.A. Received 28 April 1986, accepted 6 May 1986
M.L. WATSON, R.J. WORKMAN, W. HERZER, R.A. BRANCH, J.A. OATES and A.R. BRASH, Systemic synthesis of prostaglandin 12 following sustained infusion of angiotensin II in conscious dogs, European J. Pharmacol. 127 (1986) 9-16. Acute infusion of pharmacological doses of angiotensin II stimulates the release of prostaglandin 12 (PGI 2), which may modulate the vasoconstrictor response. It is uncertain whether sustained small increases in the plasma concentration of angiotensin II has the same effect. To investigate this further, low doses of angiotensin II were infused into conscious sodium replete dogs for 3 h. PGI 2 synthesis was assessed by measurement of a major metabolite of PGI 2, 2,3-dinor-6-keto PGFI~, in urine and plasma, using gas chromatography mass spectrometry. Angiotensin II infusion (15 ng/min per kg body weight) resulted in a 3-fold increase in plasma angiotensin II (50.8 ___5.4 to 149 ___11.2 pg/ml, P < 0.01). Mean blood pressure increased (84.8 + 4.3 to 108 + 4.7 mm Hg, P < 0.02) and renal blood flow decreased (201 + 46 to 127 + 13 ml/min, P < 0.01) throughout the infusion. However there was no change in either the plasma concentration (11.3 + 2.5 to 9.1 + 1.0 pg/ml) or rate of urinary excretion of dinor-6-keto PGFu~ (1.75 + 0.28 to 1.85 + 0.41 ng/30 min) during the angiotensin II infusion. The results suggest that small sustained elevations of the plasma concentration of angiotensin II such as are likely to occur in conscious animals, do not persistently stimulate release of PGI 2 in the systemic circulation. Angiotensin II
Blood pressure
Prostaglandin 12
1. Introduction The hypothesis that vasodilator prostaglandins modulate the vasoconstrictor activity of angiotensin II has been suggested by a number of observations. Renal release of P G E is increased in both animals and man during infusion of angiotensin II (McGiff et al., 1970; Frolich et al., 1975) and the renal vasoconstrictor activity,of angiotensin II is enhanced in anaesthetised animals by pretreatment with the prostaglandin synthetase inhibitor indomethacin (Aiken and Vane, 1973). Angiotensin II has also been reported to promote release of
* To whom all correspondenceshould be addressed at present address: Department of Medicine, Medical Renal Unit, Royal Infirmary, Edinburgh EH3 9YW, U.K. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Renal haemodynamics
PGI 2 from the renal (Shebuski and Aiken, 1980) mesenteric (Dusting et al., 1980) and pulmonary vascular beds (Voelkel et al., 1981). Injection of a large bolus of angiotensin II into both anaesthetised cats and conscious rabbits also increased the plasma concentration of 6-keto PGFI~ (Machleidt et al., 1981; Rowe and Nasjletti, 1983). However these studies do not establish to what extent PGI 2 modulates the vasoconstrictor effects of the sustained small increases in plasma concentration of angiotensin II that might be expected to occur under normal conditions in conscious animals and man. The objective of the present study was to determine whether sustained infusion of angiotensin II into conscious dogs in a dose sufficient to cause a modest increase in systemic blood pressure and renal vascular resistance also resulted in a sus-
10 tained increased release of PGI 2 into the systemic circulation. The rate of entry of PGI 2 into the circulation was assessed by measurement of the plasma concentration and rate of urinary excretion of a major metabolite of PGI2, 2,3-dinor-6keto PGFI~ using gas chromatography mass spectrometry techniques (Fitzgerald et al., 1981).
2. Materials and methods
2.1. Surgical preparation Six female mongrel dogs (17-24 kg) were surgically prepared by insertion of indwelling polyethylene catheters in the inferior vena cava and thoracic aorta via the femoral vessels. The catheters were exteriorised in the left flank. An electromagnetic flow probe (Statham, 3 ram) was placed around the left renal artery and the connecting lead exteriorised in the flank. The catheters were protected by a harness on the animals and at least one week was allowed after surgery before experiments were performed. Patency of catheters during this period was ensured by flushing with 0.9% sodium chloride solution on alternate days and filling them with heparinised sodium chloride solution (1000 I.U./ml heparin solution) in the intervening period.
2.2. Protocol Animals were maintained on a diet containing approximately 40 mmol sodium for two days before a study. On the day of a study a catheter (Foley 5F) was inserted into the bladder for collection of urine. The animals then stood quietly in a restraining sling. An initial intravenous (i.v.) infusion of 500 ml dextrose (50 g/l) was given over 30 min via the catheter in the inferior cava followed by continuous infusion of dextrose (50 g/l) at 3 ml/min and sodium chloride solution (150 mmol/1) at 0.6 ml/min, using a Harvard infusion pump. After a 60 min control period, either the sodium chloride infusion was continued (vehicle) or a freshly prepared solution containing angiotensin II amide (Hypertensin®, Ciba) in sodium chloride solution, was substituted. The
concentration of angiotensin II solution was adjusted for each dog such that the final rate of infusion was 15 ng/min per kg body weight. After a further 180 min sodium chloride solution was substituted for the angiotensin II and the infusion continued for a further 60 min recovery period. To permit subsequent calculation of glomerular filtration rate 99Tc diethylene triaminepentaacetic acid (DTPA) in sodium chloride solution was also continuously infused via the same catheter at a rate of 0.18 ml/min (Harvard pump), after an appropriate loading dose (Chervu et al., 1977). After an initial period of 90 min, 30 min collections of urine were made throughout the study. Complete emptying of the bladder at the end of each urine collection period was facilitated by both manual palpation and injection of air. Blood samples were collected every 30 min for estimation of DTPA activity while samples for estimation of plasma angiotensin II, dinor-6-keto PGFI~ and hematocrit were collected every hour. Each of the six animals underwent two studies during which either angiotensin II or vehicle was infused.
2.3. Collection of data and handling of samples Systemic blood pressure and renal blood flow were recorded on a Hewlett Packard chart recorder throughout the study. Measurements of renal blood flow were calibrated with reference to an electrical zero (Statham Instruments) and the zero position was confirmed at the end of the experiment by a bolus i.v. injection of 2 #g angiotensin II, which transiently reduced renal blood flow to zero. Samples for angiotensin II were added to tubes containing EDTA, pepstatin and nonapeptide converting enzyme inhibitor (Beckman Instruments Inc., Palo Alto, CA) in final concentrations of 0.015, 10 -5 and 10 -6 M, respectively. A further 10 ml aliquot of blood was also collected into tubes containing indomethacin (25 #g) and 0.6 ml citrate buffer solution (3.8 g sodium citrate in 100 ml water). All samples were kept in ice and centrifuged at 2 000 × g for 20 min soon after collection. Plasma samples were stored at - 2 0 ° C . Further blood samples were collected into heparin and centrifuged at room temperature. A 1 ml aliquot of plasma was then separated and
11 D T P A measured in a gamma counter (Packard Instrument Co., Douners Grove, IL). Urine was collected into plastic containers. A 1 ml aliquot was removed for estimation of DTPA activity whilst the rest was stored at - 2 0 ° C.
2.4. Analytical methods Plasma angiotensin II was estimated by radioimmunoassay (IgG Corp., Nashville, TN). Dinor6-keto PGFI~ was measured using a modification of a method already described (Falardeau et al., 1981). In brief 10 ng of a tetradeuterated internal standard was added to 10-20 ml of urine or 5 ml plasma, which was purified by separation on a Clin-Elut column followed by a series of organic solvent extractions. A pentafiuorobenzyl ester methoxime derivative was then synthesised, and samples were further purified by thin layer chromatography. After elution from the silicic acid, the trimethyl silyl ether derivative was formed and samples were analysed by negative ion chemical ionization gas chromatography mass spectrometry. A 3 foot SP2100 gas chromatography column was used with methane as carrier gas, which was led directly into the ion source of an HP5982 mass spectrometer. Ions of the protonated and deuterated compound were monitored at 586 and 590 m / z units, and the initial quantity of endogenous material was calculated from the D O / D 4 ratio observed in the mass spectrometer. Plasma and urine sodium and potassium concentration were estimated by flame photometry.
2.5. Calculation of results Glomerular filtration rate (GFR) was determined from the expression: GFR=(Udtpa × V)//Pcltpa, where Udtpa and Patpa are the urinary and plasma activity of DTPA ( d p m / m l ) and V is the rate of urine flow (ml/min). The values of successive pairs of 30 min urine collections have been combined and expressed in the figures as the mean results at hourly intervals. Results are expressed as means + S.E.M. and comparisons have been made using a two-tailed Student's t-test. A significant difference between parameters was assumed when P < 0.05.
3. Results
3.1. Renal and systemic haemodynamics There were no significant differences in systemic blood pressure, glomerular filtration rate and renal blood flow between the two groups during the pre-infusion control period. On starting infusion of angiotensin II there was a prompt increase in systemic blood pressure and decrease in renal plasma flow which was sustained at the same level throughout the angiotensin II infusion (fig. 1). There was no change in these parameters during vehicle infusion and both returned to control values after completion of the angiotensin II infusion.
3.2. Humoral factors Plasma angiotensin II increased by 3-fold within 1 h of starting the infusion, (P < 0.01) (fig. 1), remained at the same level during the second hour, and then increased further during the third hour before returning to control values. The rate of urinary excretion of dinor-6-keto PGFI~ was unchanged during infusion of angiotensin II (fig. 2). Since there were significant changes in glomerular filtration rate during the angiotensin II infusion the results have also been expressed as a fraction of the glomerular filtration rate. The plasma concentration of dinor-6-keto PGFI~ was measured in four of the animals before and after infusion of angiotensin II and in five during the vehicle infusion. There was no change during infusion of either angiotensin II (11.3 ___2.5 pre and 9.1 + 1.0 p g / m l post) or vehicle (14.6_ 5.8 pre and 8.7 + 1.2 p g / m l post), although in one animal the plasma concentration was substantially higher than the others before infusion of vehicle. The urinary exretion rate of dinor-6-keto PGF1, was also high in this animal, which in part explained the apparent higher excretion rate in the vehicle infusion group.
3.3. Renal excretory function Urine flow rate, sodium and potassium excretion are shown in table 1. Angiotensin II tended to
12 200
150 ..
~
x
too js
~.
I/s
5
so
I
i 3o~ '~
200
,..1 I00
~
tt
r~ / ! 90 ,..1
I
m ~
@
r
7o
50
~.
I
I
I
I
I
C
60
120
180
240
TIME (mins)
Fig. 1. Plasma concentration of angiotensin II, renal blood flow and systemic blood pressure during infusion of angiotensin II ( . . . . . . ) or sodium chloride ( ). * P < 0.05, ** P < 0.01 for difference between groups.
TABLE 1 Urine flow sodium and potassium excretion before, during and after infusion of angiotensin II or sodium chloride solution. Time (min)
Urine flow (ml/min) Sodium excretion (~M/mio) Potassium excretion (/zM/min)
Before
During
0-60
60-120
120-180
180-240
After
Control A II Control AII
1.25 ± 0.39 a 2.85 4. 0.39 23.3 4- 8.0 46.2 ±11.8
2.18 ± 0.57 1.62±0.30 b 23.0 4-8.0 13.5 ±4.7 b
2.19 ± 0.44 2.20±0.41 17.9 ±6.3 5.9 4-1.7 b
2.13 ± 0.34 2.114-0.37 b 14.9 4-4.6 5.8 4-1.5 b
Control A II
15.0 + 4.5 29.9 4- 7.9
21.2 +6.0 9.6 +1.8 b
24.4 +4.3 a 8.2 4-3.1d
26.0 +3.5 ¢ 7.9 +2.0 b
240-300 1.97 ± 0.34 c 4.384-0.57 b 6.3 -I-1.2 b 7.7 4-2.5 b 27.2 +7.0 26.4 +8.2
a Between group differences; b differences from control in same group; a,b p < 0.05; c.d p < 0.01. A II, angiotensin II.
13
6
~ 0
~
2
,; 6
,0 ~ 2
0
i........... 1
C
+__--
I
60
f
120 TIME (mins)
I
180
]
240
Fig. 2. Dinor-6-keto PGFI~ excretion with and without corrections for changes in glomerular filtration rate during angiotensin II (15 n g / k g per min) ( . . . . . . ) or sodium chloride ( ) infusion.
decrease sodium and potassium excretion, although differences were not significant during all collection periods. 4. D i s c u s s i o n
Angiotensin II is a powerful vasoconstrictor. Studies in anaesthetised animals have demonstrated that the renal vasoconstriction caused by infusion of angiotensin II into the kidney is partially opposed by the release of vasodilator prostaglandins (McGiff et al., 1970; Aiken and Vane, 1973). Two more recent studies particularly implicate PGI 2 as the vasodilator released by angiotensin II in both the renal and mesenteric vasculature (Shebuski and Aiken, 1973; Dusting et al., 1980). In both studies very large doses of angiotensin II were infused and the surgical trauma inherent in such studies stimulates renal prostaglandin synthesis (Terragno et al., 1977). Evidence that vasodilator prostaglandins modulate
the vasoconstrictor activity of angiotensin II in the systemic circulation is less clear. Aiken and Vane were unable to demonstrate any effect of indomethacin on angiotensin II induced vasoconstriction in the isolated hind limb of dogs (Aiken and Vane, 1973). However, inhibitors of prostaglandin synthetase have been reported to increase the pressor response to infused angiotensin II in man (Negus et al., 1976; Vierhapper et al., 1981). Measurement of changes in PGE 2 synthesis in anaesthetised dogs (Dunn et al., 1978) and PGI 2 synthesis in conscious rabbits (Rowe and Nasjletti, 1983) suggest that increased synthesis of both prostaglandins in response to large doses of angiotensin II is transient. It is unclear to what extent these observations can be extended to conscious unstressed animals in which the circulating concentration of angiotensin II is low. Moreover the sustained increases in concentration of angiotensin II that occur under normal conditions may not be associated with the increases in
14 PGI 2 synthesis reported in response to short-term changes. In order to clarify the role of PGI 2 as a systemic modulator of angiotensin II activity low doses of angiotensin II were infused into conscious unstressed animals and changes in plasma concentration and urinary excretion of a major metabolite of PGI 2 (2,3-dinor-6-keto PGFa~ ) were monitored. This metabolite constitutes only a small percentage of the total number of PGI 2 metabolites in urine (Brash et al., 1983) and plasma (Taylor et al., 1983). However after a systemic infusion of PGI 2 the size and time course of the increase in plasma concentration of dinor-6-keto PGFI~ was similar to that for another metabolite of PGI 2, dinor-6,15-diketo PGFI~ (Taylor et al., 1983). The rate of urinary excretion of either of these metabolites has been shown to correlate well with the rate of entry of PGI 2 into the systemic circulation (Fitzgerald et al., 1981) which in man is depressed by aspirin therapy (Fitzgerald et al., 1983b) and increased within 6 h of administration of a thromboxane synthetase inhibitor (Fitzgerald et al., 1983a). A large proportion of PGI 2 synthesised in the kidney appears in urine as 6-keto PGFI~ (Wilson et al., 1982) and therefore urinary dinor-6-keto PGFa~ most likely reflects total body PGI 2 synthesis minus a proportion of renal synthesis. In the present study, despite infusion of a dose of angiotensin II sufficient to cause a mean increase in blood pressure of 24 mm Hg and decrease in renal blood flow of 31% for 3 h, there was no change in excretion of the metabolite of PGI 2. Moreover it is highly unlikely that changes in glomerular filtration rate altered excretion of the metabolite since there was also no change in excretion rate when expressed as a fraction of glomerular filtration rate. These findings contrast with previous observations that angiotensin II increases the concentration of 6-keto PGFI~ and dinor-6,15-diketo PGFI~ in plasma (Machleidt et al., 1981). Since it has already been established that dinor-6-keto PGFI~ and dinor-6,15-diketo P G F ~ increase in plasma in a similar fashion after PGI z infusion (Taylor et al., 1983), an increase in plasma or urinary dinor6-keto PGF1~ would have been expected after angiotensin II infusion. The reported increase in concentration of dinor-6,15-diketo PGFa~ after
angiotensin II was however only transient (Machleidt et al., 1981). In this study dinor-6-keto PGF1, was only measured in plasma 60 min after the angiotensin II infusion started. A transient release of PGI 2 into the circulation immediately on starting the infusion of angiotensin II may therefore not have been detectable 60 min later. The increase may have been so small and transient as to be insufficient to result in any detectable change in the rate of urinary excretion of dinor-6-keto PGFI~ during the first 30 min collection period. On this basis it could be postulated that the immediate effects of a sudden change in plasma concentration of angiotensin II, such as after a change in posture (Brown et al., 1966), might be modulated by a small increase in systemic PGI 2 release. However the pressor response to angiotensin II in the present study reached its maximum within 2 min of starting the infusion and was sustained throughout, suggesting that the initial pressor effect was not significantly modulated by release of a vasodepressor compound. The lack of change in plasma concentration and urinary excretion of dinor-6-keto PGFI~ over 3 h indicates that even if such an effect did occur it is not sustained. The systemic vascular response to infused angiotensin II is greatly diminished during activation of the renin angiotensin system by sodium depletion (Reid and Laragh, 1965). Perhaps it is only under such conditions of apparent tachyphalaxis to angiotensin II that systemic PGI 2 synthesis is increased. However, other results in man suggest that this is not the case, since the rate of excretion of dinor-6-keto PGFI~ is actually decreased when the renin angiotensin system is activated either by removal of aldosterone secreting adenomas, or during sodium depletion (Watson et al., 1984). Although angiotensin II may stimulate the synthesis of some other prostaglandins, the apparent resistance to the vasopressor effects of angiotensin II can best be accounted for by changes in receptor occupany (Thurston and Laragh, 1975). If dose response curves are constructed for the effects of angiotensin II on blood pressure as a function of the plasma concentration of angiotensin II, then changes in response can be entirely accounted for by changes in position of
15
the dose response curve, and the associated release of a vasodilator need not be postulated (Bean et al., 1979). Such an explanation might also explain the apparently increased response of normal subjects to angiotensin II during treatment with indomethacin (Negus and Tannen, 1976; Vierhapper et al., 1981; Speckhart et al., 1977). In two of these studies (Vierhapper et al., 1981; Speckhart et al., 1977), plasma renin activity decreased while the subjects were on indomethacin, although the decrease only reached significance in the former study, implying that basal synthesis of angiotensin II was also decreased. The resulting decreased receptor occupancy by endogenous angiotensin II would then result in an increased response to the infused compound. The present results do not exclude the possible stimulation by angiotensin II of PGI 2 synthesis within the renal vasculature. Such PGI 2 synthesis may be measured better by monitoring urinary excretion of 6-keto PGF1,, the stable hydrolysis product of PGI 2 (Rosenkranz et al., 1981; Patrono et al., 1982; Wilson et al., 1982). In summary, despite obvious effects of sustained infusion of angiotensin II on the circulation, there was no detectable change in systemic synthesis of PGI 2. The results, therefore, do not support the hypothesis that release of PGI 2 from the systemic circulation modulates the sustained pressor effects of angiotensin II during sodium replete conditions.
Acknowledgements We are grateful to Ciba Geigy Ltd. for supplies of angiotensin If amide and Upjohn Ltd. for prostagiandin standards. Dr. M.L. Watson was supported by U.S. Public health Service International Research Fellowship F05 TWO 3114-02. The study was supported by a Specialized Center of Research Grant in Hypertension (HL14192).
References Aiken, J.W. and J.R. Vane, 1973, Intrarenal prostaglandin release attenuates the renal vasoconstrictor activity of angiotensin, J. Pharmacol. Exp. Ther. 184, 678. Bean, B.L., J.J. Brown, J. Casals-Stenzel, R. Fraser, A.F. Lever,
J.A. Millar, J.J. Morton, B. Petch, A.J.G. Riegger, J.I.S. Robertson and M. Tree, 1979, The relation of arterial pressure and plasma angiotensin II concentration, Circ. Res. 44, 452. Brash, A.R., E.K. Jackson, C. Saggese, J.A. Lawson, J.A. Oates and G.A. Fitzgerald, 1983, The metabolic disposition of prostacyclin in man, J. Pharmacol. Exp. Ther. 226, 78. Brown, J.J., D.L. Davies, A.F. Lever, D. McPherson and J.I.S. Robertson, 1966, Plasma renin concentration in relation to changes in posture, Clin. Sci. 30, 279. Chervu, L.R., H.B. Lee, Q. Goyal and M.D. Blaufox, 1977, Use of 99mTc-Cu-DTPA complex as a renal function agent, J. Nucl. Med. 18, 62. Dunn, M.J., J.F. Liard and F. Dray, 1978, Basal and stimulated rates of renal secretion and excretion of prostaglandins E2, F2~ and 13,14-dihydro 15-keto PGFI~ in the dog, Kidney Int. 13, 136. Dusting, G.J., E.M. Mullins and A.E. Doyle, 1980, Angiotensin-induced prostacyclin release may contribute to the hypotensive action of converting enzyme inhibitors, Adv. Prostagl. Throm. Res. 7, 815. Falardeau, P., J.A. Oates and A.R. Brash, 1981, Quantitative analysis of two dinor urinary metabolites of prostaglandin 12, Anal. Biochem. 115, 359. Fitzgerald, G.A., A.R. Brash, P. Falardeau and J.A. Oates, 1981, Estimated rate of prostacyclin secretion into the circulation of normal man, J. Clin. Invest. 68, 1272. Fitzgerald, G.A., A.R. Brash, J.A. Oates and A.K. Pedersen, 1983a, Endogenous prostacyclin biosynthesis and platelet function during selective inhibition of thromboxane synthase in man, J. Clin. Invest. 71, 1336. Fitzgerald, G.A., J.A. Oates, J. Hawiger, R.L. Maas, L.J. Roberts, J.A. Lawson and A.R. Brash, 1983b, Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man, J. Clin. Invest. 71,676. Frolich, J.C., T.W. Wilson, B.J. Sweetman, M. Smigel, A.S. Nies, K. Carr, J.T. Watson and J.A. Oates, 1975, Urinary protaglandins; identification and origin, J. Clin. Invest. 55, 763. Machleidt, C., U. Forstermann, H. Anhut and G. Hertting, 1981~ Formation and elimination of prostacyclin metabolites in the cat in vivo as determined by radioimmunoassay of unextracted plasma, European J. Pharmacol. 74, 19. McGiff, J.C., K. Crowshaw, N.A. Terragno and A.J. Lonigro, 1970, Release of a prostaglandin-like substance into renal venous blood in response to angiotensin II, Circ. Res. 27 (Suppl. 1), 121. Negus, P., R.L. Tannen and M.J. Dunn, 1976, Indomethacin potentiates the vasoconstrictor actions of angiotensin II in normal man, Prostaglandins 12, 175. Patrono, C., F. Pugliese, G. Ciabattoni, P. Patrignani, A. Maseri, S. Chierchia, B.A. Peskar, G.A. Cinotti, B.M. Simonetti and A. Pierucci, 1982, Evidence for direct stimulatory effect of prostacyclin on renin release in man, J. Clin. Invest. 69, 231. Reid, W.D. and J.H. Laragh, 1965, Sodium and potassium
16 intake, blood pressure and pressor response to angiotensin, Proc. Soc. Exp. Biol. Med. 120, 26. Rosenkranz, B., W. Kitajima and J.C. Frolich, 1981, Relevance of urinary 6-keto prostaglandin Flu determinaton, Kidney Int. 19, 755. Rowe, B.P. and A. Nasjletti, 1983, Biphasic blood pressure response to angiotensin II in the conscious rabbit: Relation to prostaglandins, J. Pharmacol. Exp. Ther. 225, 559. Shebuski, R.J. and J.W. Aiken, 1980, Angiotensin II stimulation of renal prostaglandin synthesis elevates circulating prostacyclin in the dog, J. Cardiovasc. Pharmacol. 2, 667. Speckhart, P., P. Zia, R. Zipser and R. Horton, 1977, The effect of sodium restriction and prostaglandin inhibition on the renin angiotensin system in man, J. Clin. Endocrinol. Metab. 44, 832. "~ Taylor, B.M., R.J. Shebuski and F.F. Sun, 1983, Circulating prostacyclin metabolites in the dog, J. Pharmacol. Exp. Ther. 224, 692. Terragno, N.A., D.A. Terragno and J.C. McGiff, 1977, Contribution of prostaglandins to the renal circulation in conscious anesthetised and laparotomized dogs, Circ. Res. 40, 590.
Thurston, H. and J.H. Laragh, 1975, Prior receptor occupancy as a determinant of the pressor activity of infused angiotensin II in the rat, Circ. Res. 36, 113. Vierhapper, H., L.W. Waldhaus and P. Nowotay, 1981, Effect of indomethacin upon angiotensin-induced changes in blood pressure and plasma aldosterone in normal man, European J. Clin. Invest. 11, 85. Voelkel, N.F., J.G. Gerber, I.F. McMurty, A.S. Nies and J.T. Reeves, 1981, Release of vasodilator prostaglandin PGI 2 from isolated rat lung during vasoconstriction, Circ. Res. 48, 207. Watson, M.L., R.P. Goodman, J.R. Gill, R.A. Branch, A.R. Brash and G.A. Fitzgerald, 1984, Endogenous prostacyclin synthesis is decreased during activation of the renin-angiotensin system in man, J. Clin. Endocrinol. Metab. 58, 304. Wilson, T.W., C.B. Loadholt, P.J. Privitera and P.V. Halushka, 1982, Frusemide increases urine 6-keto PGFI~. Relation to natriuresis vasodilatation and renin release, Hypertension 4, 634.
9
S Y S T E M I C S Y N T H E S I S OF P R O S T A G L A N D I N 12 F O L L O W I N G S U S T A I N E D I N F U S I O N O F A N G I O T E N S I N II IN C O N S C I O U S D O G S MICHAEL L. WATSON *, ROBERT J. WORKMAN, WILLIAM HERZER, ROBERT A. BRANCH, JOHN A. OATES and ALAN R. BRASH
Department of Clinical Pharmacology, Vanderbilt University, Nashville, TN 37232, U.S.A. Received 28 April 1986, accepted 6 May 1986
M.L. WATSON, R.J. WORKMAN, W. HERZER, R.A. BRANCH, J.A. OATES and A.R. BRASH, Systemic synthesis of prostaglandin 12 following sustained infusion of angiotensin II in conscious dogs, European J. Pharmacol. 127 (1986) 9-16. Acute infusion of pharmacological doses of angiotensin II stimulates the release of prostaglandin 12 (PGI 2), which may modulate the vasoconstrictor response. It is uncertain whether sustained small increases in the plasma concentration of angiotensin II has the same effect. To investigate this further, low doses of angiotensin II were infused into conscious sodium replete dogs for 3 h. PGI 2 synthesis was assessed by measurement of a major metabolite of PGI 2, 2,3-dinor-6-keto PGFI~, in urine and plasma, using gas chromatography mass spectrometry. Angiotensin II infusion (15 ng/min per kg body weight) resulted in a 3-fold increase in plasma angiotensin II (50.8 ___5.4 to 149 ___11.2 pg/ml, P < 0.01). Mean blood pressure increased (84.8 + 4.3 to 108 + 4.7 mm Hg, P < 0.02) and renal blood flow decreased (201 + 46 to 127 + 13 ml/min, P < 0.01) throughout the infusion. However there was no change in either the plasma concentration (11.3 + 2.5 to 9.1 + 1.0 pg/ml) or rate of urinary excretion of dinor-6-keto PGFu~ (1.75 + 0.28 to 1.85 + 0.41 ng/30 min) during the angiotensin II infusion. The results suggest that small sustained elevations of the plasma concentration of angiotensin II such as are likely to occur in conscious animals, do not persistently stimulate release of PGI 2 in the systemic circulation. Angiotensin II
Blood pressure
Prostaglandin 12
1. Introduction The hypothesis that vasodilator prostaglandins modulate the vasoconstrictor activity of angiotensin II has been suggested by a number of observations. Renal release of P G E is increased in both animals and man during infusion of angiotensin II (McGiff et al., 1970; Frolich et al., 1975) and the renal vasoconstrictor activity,of angiotensin II is enhanced in anaesthetised animals by pretreatment with the prostaglandin synthetase inhibitor indomethacin (Aiken and Vane, 1973). Angiotensin II has also been reported to promote release of
* To whom all correspondenceshould be addressed at present address: Department of Medicine, Medical Renal Unit, Royal Infirmary, Edinburgh EH3 9YW, U.K. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Renal haemodynamics
PGI 2 from the renal (Shebuski and Aiken, 1980) mesenteric (Dusting et al., 1980) and pulmonary vascular beds (Voelkel et al., 1981). Injection of a large bolus of angiotensin II into both anaesthetised cats and conscious rabbits also increased the plasma concentration of 6-keto PGFI~ (Machleidt et al., 1981; Rowe and Nasjletti, 1983). However these studies do not establish to what extent PGI 2 modulates the vasoconstrictor effects of the sustained small increases in plasma concentration of angiotensin II that might be expected to occur under normal conditions in conscious animals and man. The objective of the present study was to determine whether sustained infusion of angiotensin II into conscious dogs in a dose sufficient to cause a modest increase in systemic blood pressure and renal vascular resistance also resulted in a sus-
10 tained increased release of PGI 2 into the systemic circulation. The rate of entry of PGI 2 into the circulation was assessed by measurement of the plasma concentration and rate of urinary excretion of a major metabolite of PGI2, 2,3-dinor-6keto PGFI~ using gas chromatography mass spectrometry techniques (Fitzgerald et al., 1981).
2. Materials and methods
2.1. Surgical preparation Six female mongrel dogs (17-24 kg) were surgically prepared by insertion of indwelling polyethylene catheters in the inferior vena cava and thoracic aorta via the femoral vessels. The catheters were exteriorised in the left flank. An electromagnetic flow probe (Statham, 3 ram) was placed around the left renal artery and the connecting lead exteriorised in the flank. The catheters were protected by a harness on the animals and at least one week was allowed after surgery before experiments were performed. Patency of catheters during this period was ensured by flushing with 0.9% sodium chloride solution on alternate days and filling them with heparinised sodium chloride solution (1000 I.U./ml heparin solution) in the intervening period.
2.2. Protocol Animals were maintained on a diet containing approximately 40 mmol sodium for two days before a study. On the day of a study a catheter (Foley 5F) was inserted into the bladder for collection of urine. The animals then stood quietly in a restraining sling. An initial intravenous (i.v.) infusion of 500 ml dextrose (50 g/l) was given over 30 min via the catheter in the inferior cava followed by continuous infusion of dextrose (50 g/l) at 3 ml/min and sodium chloride solution (150 mmol/1) at 0.6 ml/min, using a Harvard infusion pump. After a 60 min control period, either the sodium chloride infusion was continued (vehicle) or a freshly prepared solution containing angiotensin II amide (Hypertensin®, Ciba) in sodium chloride solution, was substituted. The
concentration of angiotensin II solution was adjusted for each dog such that the final rate of infusion was 15 ng/min per kg body weight. After a further 180 min sodium chloride solution was substituted for the angiotensin II and the infusion continued for a further 60 min recovery period. To permit subsequent calculation of glomerular filtration rate 99Tc diethylene triaminepentaacetic acid (DTPA) in sodium chloride solution was also continuously infused via the same catheter at a rate of 0.18 ml/min (Harvard pump), after an appropriate loading dose (Chervu et al., 1977). After an initial period of 90 min, 30 min collections of urine were made throughout the study. Complete emptying of the bladder at the end of each urine collection period was facilitated by both manual palpation and injection of air. Blood samples were collected every 30 min for estimation of DTPA activity while samples for estimation of plasma angiotensin II, dinor-6-keto PGFI~ and hematocrit were collected every hour. Each of the six animals underwent two studies during which either angiotensin II or vehicle was infused.
2.3. Collection of data and handling of samples Systemic blood pressure and renal blood flow were recorded on a Hewlett Packard chart recorder throughout the study. Measurements of renal blood flow were calibrated with reference to an electrical zero (Statham Instruments) and the zero position was confirmed at the end of the experiment by a bolus i.v. injection of 2 #g angiotensin II, which transiently reduced renal blood flow to zero. Samples for angiotensin II were added to tubes containing EDTA, pepstatin and nonapeptide converting enzyme inhibitor (Beckman Instruments Inc., Palo Alto, CA) in final concentrations of 0.015, 10 -5 and 10 -6 M, respectively. A further 10 ml aliquot of blood was also collected into tubes containing indomethacin (25 #g) and 0.6 ml citrate buffer solution (3.8 g sodium citrate in 100 ml water). All samples were kept in ice and centrifuged at 2 000 × g for 20 min soon after collection. Plasma samples were stored at - 2 0 ° C . Further blood samples were collected into heparin and centrifuged at room temperature. A 1 ml aliquot of plasma was then separated and
11 D T P A measured in a gamma counter (Packard Instrument Co., Douners Grove, IL). Urine was collected into plastic containers. A 1 ml aliquot was removed for estimation of DTPA activity whilst the rest was stored at - 2 0 ° C.
2.4. Analytical methods Plasma angiotensin II was estimated by radioimmunoassay (IgG Corp., Nashville, TN). Dinor6-keto PGFI~ was measured using a modification of a method already described (Falardeau et al., 1981). In brief 10 ng of a tetradeuterated internal standard was added to 10-20 ml of urine or 5 ml plasma, which was purified by separation on a Clin-Elut column followed by a series of organic solvent extractions. A pentafiuorobenzyl ester methoxime derivative was then synthesised, and samples were further purified by thin layer chromatography. After elution from the silicic acid, the trimethyl silyl ether derivative was formed and samples were analysed by negative ion chemical ionization gas chromatography mass spectrometry. A 3 foot SP2100 gas chromatography column was used with methane as carrier gas, which was led directly into the ion source of an HP5982 mass spectrometer. Ions of the protonated and deuterated compound were monitored at 586 and 590 m / z units, and the initial quantity of endogenous material was calculated from the D O / D 4 ratio observed in the mass spectrometer. Plasma and urine sodium and potassium concentration were estimated by flame photometry.
2.5. Calculation of results Glomerular filtration rate (GFR) was determined from the expression: GFR=(Udtpa × V)//Pcltpa, where Udtpa and Patpa are the urinary and plasma activity of DTPA ( d p m / m l ) and V is the rate of urine flow (ml/min). The values of successive pairs of 30 min urine collections have been combined and expressed in the figures as the mean results at hourly intervals. Results are expressed as means + S.E.M. and comparisons have been made using a two-tailed Student's t-test. A significant difference between parameters was assumed when P < 0.05.
3. Results
3.1. Renal and systemic haemodynamics There were no significant differences in systemic blood pressure, glomerular filtration rate and renal blood flow between the two groups during the pre-infusion control period. On starting infusion of angiotensin II there was a prompt increase in systemic blood pressure and decrease in renal plasma flow which was sustained at the same level throughout the angiotensin II infusion (fig. 1). There was no change in these parameters during vehicle infusion and both returned to control values after completion of the angiotensin II infusion.
3.2. Humoral factors Plasma angiotensin II increased by 3-fold within 1 h of starting the infusion, (P < 0.01) (fig. 1), remained at the same level during the second hour, and then increased further during the third hour before returning to control values. The rate of urinary excretion of dinor-6-keto PGFI~ was unchanged during infusion of angiotensin II (fig. 2). Since there were significant changes in glomerular filtration rate during the angiotensin II infusion the results have also been expressed as a fraction of the glomerular filtration rate. The plasma concentration of dinor-6-keto PGFI~ was measured in four of the animals before and after infusion of angiotensin II and in five during the vehicle infusion. There was no change during infusion of either angiotensin II (11.3 ___2.5 pre and 9.1 + 1.0 p g / m l post) or vehicle (14.6_ 5.8 pre and 8.7 + 1.2 p g / m l post), although in one animal the plasma concentration was substantially higher than the others before infusion of vehicle. The urinary exretion rate of dinor-6-keto PGF1, was also high in this animal, which in part explained the apparent higher excretion rate in the vehicle infusion group.
3.3. Renal excretory function Urine flow rate, sodium and potassium excretion are shown in table 1. Angiotensin II tended to
12 200
150 ..
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60
120
180
240
TIME (mins)
Fig. 1. Plasma concentration of angiotensin II, renal blood flow and systemic blood pressure during infusion of angiotensin II ( . . . . . . ) or sodium chloride ( ). * P < 0.05, ** P < 0.01 for difference between groups.
TABLE 1 Urine flow sodium and potassium excretion before, during and after infusion of angiotensin II or sodium chloride solution. Time (min)
Urine flow (ml/min) Sodium excretion (~M/mio) Potassium excretion (/zM/min)
Before
During
0-60
60-120
120-180
180-240
After
Control A II Control AII
1.25 ± 0.39 a 2.85 4. 0.39 23.3 4- 8.0 46.2 ±11.8
2.18 ± 0.57 1.62±0.30 b 23.0 4-8.0 13.5 ±4.7 b
2.19 ± 0.44 2.20±0.41 17.9 ±6.3 5.9 4-1.7 b
2.13 ± 0.34 2.114-0.37 b 14.9 4-4.6 5.8 4-1.5 b
Control A II
15.0 + 4.5 29.9 4- 7.9
21.2 +6.0 9.6 +1.8 b
24.4 +4.3 a 8.2 4-3.1d
26.0 +3.5 ¢ 7.9 +2.0 b
240-300 1.97 ± 0.34 c 4.384-0.57 b 6.3 -I-1.2 b 7.7 4-2.5 b 27.2 +7.0 26.4 +8.2
a Between group differences; b differences from control in same group; a,b p < 0.05; c.d p < 0.01. A II, angiotensin II.
13
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60
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120 TIME (mins)
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240
Fig. 2. Dinor-6-keto PGFI~ excretion with and without corrections for changes in glomerular filtration rate during angiotensin II (15 n g / k g per min) ( . . . . . . ) or sodium chloride ( ) infusion.
decrease sodium and potassium excretion, although differences were not significant during all collection periods. 4. D i s c u s s i o n
Angiotensin II is a powerful vasoconstrictor. Studies in anaesthetised animals have demonstrated that the renal vasoconstriction caused by infusion of angiotensin II into the kidney is partially opposed by the release of vasodilator prostaglandins (McGiff et al., 1970; Aiken and Vane, 1973). Two more recent studies particularly implicate PGI 2 as the vasodilator released by angiotensin II in both the renal and mesenteric vasculature (Shebuski and Aiken, 1973; Dusting et al., 1980). In both studies very large doses of angiotensin II were infused and the surgical trauma inherent in such studies stimulates renal prostaglandin synthesis (Terragno et al., 1977). Evidence that vasodilator prostaglandins modulate
the vasoconstrictor activity of angiotensin II in the systemic circulation is less clear. Aiken and Vane were unable to demonstrate any effect of indomethacin on angiotensin II induced vasoconstriction in the isolated hind limb of dogs (Aiken and Vane, 1973). However, inhibitors of prostaglandin synthetase have been reported to increase the pressor response to infused angiotensin II in man (Negus et al., 1976; Vierhapper et al., 1981). Measurement of changes in PGE 2 synthesis in anaesthetised dogs (Dunn et al., 1978) and PGI 2 synthesis in conscious rabbits (Rowe and Nasjletti, 1983) suggest that increased synthesis of both prostaglandins in response to large doses of angiotensin II is transient. It is unclear to what extent these observations can be extended to conscious unstressed animals in which the circulating concentration of angiotensin II is low. Moreover the sustained increases in concentration of angiotensin II that occur under normal conditions may not be associated with the increases in
14 PGI 2 synthesis reported in response to short-term changes. In order to clarify the role of PGI 2 as a systemic modulator of angiotensin II activity low doses of angiotensin II were infused into conscious unstressed animals and changes in plasma concentration and urinary excretion of a major metabolite of PGI 2 (2,3-dinor-6-keto PGFa~ ) were monitored. This metabolite constitutes only a small percentage of the total number of PGI 2 metabolites in urine (Brash et al., 1983) and plasma (Taylor et al., 1983). However after a systemic infusion of PGI 2 the size and time course of the increase in plasma concentration of dinor-6-keto PGFI~ was similar to that for another metabolite of PGI 2, dinor-6,15-diketo PGFI~ (Taylor et al., 1983). The rate of urinary excretion of either of these metabolites has been shown to correlate well with the rate of entry of PGI 2 into the systemic circulation (Fitzgerald et al., 1981) which in man is depressed by aspirin therapy (Fitzgerald et al., 1983b) and increased within 6 h of administration of a thromboxane synthetase inhibitor (Fitzgerald et al., 1983a). A large proportion of PGI 2 synthesised in the kidney appears in urine as 6-keto PGFI~ (Wilson et al., 1982) and therefore urinary dinor-6-keto PGFa~ most likely reflects total body PGI 2 synthesis minus a proportion of renal synthesis. In the present study, despite infusion of a dose of angiotensin II sufficient to cause a mean increase in blood pressure of 24 mm Hg and decrease in renal blood flow of 31% for 3 h, there was no change in excretion of the metabolite of PGI 2. Moreover it is highly unlikely that changes in glomerular filtration rate altered excretion of the metabolite since there was also no change in excretion rate when expressed as a fraction of glomerular filtration rate. These findings contrast with previous observations that angiotensin II increases the concentration of 6-keto PGFI~ and dinor-6,15-diketo PGFI~ in plasma (Machleidt et al., 1981). Since it has already been established that dinor-6-keto PGFI~ and dinor-6,15-diketo P G F ~ increase in plasma in a similar fashion after PGI z infusion (Taylor et al., 1983), an increase in plasma or urinary dinor6-keto PGF1~ would have been expected after angiotensin II infusion. The reported increase in concentration of dinor-6,15-diketo PGFa~ after
angiotensin II was however only transient (Machleidt et al., 1981). In this study dinor-6-keto PGF1, was only measured in plasma 60 min after the angiotensin II infusion started. A transient release of PGI 2 into the circulation immediately on starting the infusion of angiotensin II may therefore not have been detectable 60 min later. The increase may have been so small and transient as to be insufficient to result in any detectable change in the rate of urinary excretion of dinor-6-keto PGFI~ during the first 30 min collection period. On this basis it could be postulated that the immediate effects of a sudden change in plasma concentration of angiotensin II, such as after a change in posture (Brown et al., 1966), might be modulated by a small increase in systemic PGI 2 release. However the pressor response to angiotensin II in the present study reached its maximum within 2 min of starting the infusion and was sustained throughout, suggesting that the initial pressor effect was not significantly modulated by release of a vasodepressor compound. The lack of change in plasma concentration and urinary excretion of dinor-6-keto PGFI~ over 3 h indicates that even if such an effect did occur it is not sustained. The systemic vascular response to infused angiotensin II is greatly diminished during activation of the renin angiotensin system by sodium depletion (Reid and Laragh, 1965). Perhaps it is only under such conditions of apparent tachyphalaxis to angiotensin II that systemic PGI 2 synthesis is increased. However, other results in man suggest that this is not the case, since the rate of excretion of dinor-6-keto PGFI~ is actually decreased when the renin angiotensin system is activated either by removal of aldosterone secreting adenomas, or during sodium depletion (Watson et al., 1984). Although angiotensin II may stimulate the synthesis of some other prostaglandins, the apparent resistance to the vasopressor effects of angiotensin II can best be accounted for by changes in receptor occupany (Thurston and Laragh, 1975). If dose response curves are constructed for the effects of angiotensin II on blood pressure as a function of the plasma concentration of angiotensin II, then changes in response can be entirely accounted for by changes in position of
15
the dose response curve, and the associated release of a vasodilator need not be postulated (Bean et al., 1979). Such an explanation might also explain the apparently increased response of normal subjects to angiotensin II during treatment with indomethacin (Negus and Tannen, 1976; Vierhapper et al., 1981; Speckhart et al., 1977). In two of these studies (Vierhapper et al., 1981; Speckhart et al., 1977), plasma renin activity decreased while the subjects were on indomethacin, although the decrease only reached significance in the former study, implying that basal synthesis of angiotensin II was also decreased. The resulting decreased receptor occupancy by endogenous angiotensin II would then result in an increased response to the infused compound. The present results do not exclude the possible stimulation by angiotensin II of PGI 2 synthesis within the renal vasculature. Such PGI 2 synthesis may be measured better by monitoring urinary excretion of 6-keto PGF1,, the stable hydrolysis product of PGI 2 (Rosenkranz et al., 1981; Patrono et al., 1982; Wilson et al., 1982). In summary, despite obvious effects of sustained infusion of angiotensin II on the circulation, there was no detectable change in systemic synthesis of PGI 2. The results, therefore, do not support the hypothesis that release of PGI 2 from the systemic circulation modulates the sustained pressor effects of angiotensin II during sodium replete conditions.
Acknowledgements We are grateful to Ciba Geigy Ltd. for supplies of angiotensin If amide and Upjohn Ltd. for prostagiandin standards. Dr. M.L. Watson was supported by U.S. Public health Service International Research Fellowship F05 TWO 3114-02. The study was supported by a Specialized Center of Research Grant in Hypertension (HL14192).
References Aiken, J.W. and J.R. Vane, 1973, Intrarenal prostaglandin release attenuates the renal vasoconstrictor activity of angiotensin, J. Pharmacol. Exp. Ther. 184, 678. Bean, B.L., J.J. Brown, J. Casals-Stenzel, R. Fraser, A.F. Lever,
J.A. Millar, J.J. Morton, B. Petch, A.J.G. Riegger, J.I.S. Robertson and M. Tree, 1979, The relation of arterial pressure and plasma angiotensin II concentration, Circ. Res. 44, 452. Brash, A.R., E.K. Jackson, C. Saggese, J.A. Lawson, J.A. Oates and G.A. Fitzgerald, 1983, The metabolic disposition of prostacyclin in man, J. Pharmacol. Exp. Ther. 226, 78. Brown, J.J., D.L. Davies, A.F. Lever, D. McPherson and J.I.S. Robertson, 1966, Plasma renin concentration in relation to changes in posture, Clin. Sci. 30, 279. Chervu, L.R., H.B. Lee, Q. Goyal and M.D. Blaufox, 1977, Use of 99mTc-Cu-DTPA complex as a renal function agent, J. Nucl. Med. 18, 62. Dunn, M.J., J.F. Liard and F. Dray, 1978, Basal and stimulated rates of renal secretion and excretion of prostaglandins E2, F2~ and 13,14-dihydro 15-keto PGFI~ in the dog, Kidney Int. 13, 136. Dusting, G.J., E.M. Mullins and A.E. Doyle, 1980, Angiotensin-induced prostacyclin release may contribute to the hypotensive action of converting enzyme inhibitors, Adv. Prostagl. Throm. Res. 7, 815. Falardeau, P., J.A. Oates and A.R. Brash, 1981, Quantitative analysis of two dinor urinary metabolites of prostaglandin 12, Anal. Biochem. 115, 359. Fitzgerald, G.A., A.R. Brash, P. Falardeau and J.A. Oates, 1981, Estimated rate of prostacyclin secretion into the circulation of normal man, J. Clin. Invest. 68, 1272. Fitzgerald, G.A., A.R. Brash, J.A. Oates and A.K. Pedersen, 1983a, Endogenous prostacyclin biosynthesis and platelet function during selective inhibition of thromboxane synthase in man, J. Clin. Invest. 71, 1336. Fitzgerald, G.A., J.A. Oates, J. Hawiger, R.L. Maas, L.J. Roberts, J.A. Lawson and A.R. Brash, 1983b, Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man, J. Clin. Invest. 71,676. Frolich, J.C., T.W. Wilson, B.J. Sweetman, M. Smigel, A.S. Nies, K. Carr, J.T. Watson and J.A. Oates, 1975, Urinary protaglandins; identification and origin, J. Clin. Invest. 55, 763. Machleidt, C., U. Forstermann, H. Anhut and G. Hertting, 1981~ Formation and elimination of prostacyclin metabolites in the cat in vivo as determined by radioimmunoassay of unextracted plasma, European J. Pharmacol. 74, 19. McGiff, J.C., K. Crowshaw, N.A. Terragno and A.J. Lonigro, 1970, Release of a prostaglandin-like substance into renal venous blood in response to angiotensin II, Circ. Res. 27 (Suppl. 1), 121. Negus, P., R.L. Tannen and M.J. Dunn, 1976, Indomethacin potentiates the vasoconstrictor actions of angiotensin II in normal man, Prostaglandins 12, 175. Patrono, C., F. Pugliese, G. Ciabattoni, P. Patrignani, A. Maseri, S. Chierchia, B.A. Peskar, G.A. Cinotti, B.M. Simonetti and A. Pierucci, 1982, Evidence for direct stimulatory effect of prostacyclin on renin release in man, J. Clin. Invest. 69, 231. Reid, W.D. and J.H. Laragh, 1965, Sodium and potassium
16 intake, blood pressure and pressor response to angiotensin, Proc. Soc. Exp. Biol. Med. 120, 26. Rosenkranz, B., W. Kitajima and J.C. Frolich, 1981, Relevance of urinary 6-keto prostaglandin Flu determinaton, Kidney Int. 19, 755. Rowe, B.P. and A. Nasjletti, 1983, Biphasic blood pressure response to angiotensin II in the conscious rabbit: Relation to prostaglandins, J. Pharmacol. Exp. Ther. 225, 559. Shebuski, R.J. and J.W. Aiken, 1980, Angiotensin II stimulation of renal prostaglandin synthesis elevates circulating prostacyclin in the dog, J. Cardiovasc. Pharmacol. 2, 667. Speckhart, P., P. Zia, R. Zipser and R. Horton, 1977, The effect of sodium restriction and prostaglandin inhibition on the renin angiotensin system in man, J. Clin. Endocrinol. Metab. 44, 832. "~ Taylor, B.M., R.J. Shebuski and F.F. Sun, 1983, Circulating prostacyclin metabolites in the dog, J. Pharmacol. Exp. Ther. 224, 692. Terragno, N.A., D.A. Terragno and J.C. McGiff, 1977, Contribution of prostaglandins to the renal circulation in conscious anesthetised and laparotomized dogs, Circ. Res. 40, 590.
Thurston, H. and J.H. Laragh, 1975, Prior receptor occupancy as a determinant of the pressor activity of infused angiotensin II in the rat, Circ. Res. 36, 113. Vierhapper, H., L.W. Waldhaus and P. Nowotay, 1981, Effect of indomethacin upon angiotensin-induced changes in blood pressure and plasma aldosterone in normal man, European J. Clin. Invest. 11, 85. Voelkel, N.F., J.G. Gerber, I.F. McMurty, A.S. Nies and J.T. Reeves, 1981, Release of vasodilator prostaglandin PGI 2 from isolated rat lung during vasoconstriction, Circ. Res. 48, 207. Watson, M.L., R.P. Goodman, J.R. Gill, R.A. Branch, A.R. Brash and G.A. Fitzgerald, 1984, Endogenous prostacyclin synthesis is decreased during activation of the renin-angiotensin system in man, J. Clin. Endocrinol. Metab. 58, 304. Wilson, T.W., C.B. Loadholt, P.J. Privitera and P.V. Halushka, 1982, Frusemide increases urine 6-keto PGFI~. Relation to natriuresis vasodilatation and renin release, Hypertension 4, 634.