The cardiovascular pharmacology of prostacyclin (PGI2) in the rat

PROSTAGLANDINS

THE CARDIOVASCULAR PHARMACOLOGY OF PROSTACYCLIN (PGI,)

IN THE RAT James R. Weeks and Linda D. Compton Experimental Biology Research, The Upjohn Company, Kalamazoo, Michigan 49001, USA ABSTRACT Physiological roles have been suggested for prostacyclin in the cardiovascular system. Prostacyclin was administered by intravenous infusion to unanesthetized rats. Over a 24 hr period, 0.32 mg/kg/day caused only flushing of the ears. Larger doses (0.56 and 1 mg/kg/day) caused hypothermia, behavioral depression, and swelling of the paws. Cumulative dose-response curves for its depressor action were determined in both unanesthetized and anesthetized, vagotomized, ganglion-blocked rats. In unanesthetized rats, the threshold dose was about 0.1 ug/kg/ min. Respiratory depression precluded doses larger than 1 ug/kg/min. In anesthetized rats, the threshold dose was about 0.001 ug/kg/min, and the maximally effective dose was about 0.1 pg/kg/min. At 0.032 pg/kg/min, blood pressure first fell and then rose slightly. This compensatory rise did not occur in nephrectomized rats, suggesting renin release as the mechanism. Intravenous infusion of 0.1 but not 0.01 pg/kg/min in unanesthetized rats doubled plasma renin activity. In saline-loaded unanesthetized rats, urine volume and urinary sodium excretion were decreased by 0.1 pg/kg/min of prostacyclin. INTRODUCTION Prostacyclin (PGI2) was discovered as a metabolite of the cyclic endoperoxide prostaglandin HZ (PGH2) in blood vessels (l-3). It is a powerful inhibitor of platelet aggregation, and, since the blood platelets have the ability to form both PGH and the aggregatory thromboxane A , a physiological role in the form o a a balance between these two compounds has been proposed (4). Prostacyclin is also generated by the lungs and released into the arterial circulation (5,6). This circulating prostacyclin can affect platelet aggregation and a homeostatic role has been proposed. Prostacyclin has also been identified as the coronary vasodilator principle formed from arachidonic acid by the heart and coronary vessels (7,8). Prostacyclin is a depressor agent with vasodilator activity on the peripheral, coronary and pulmonary 1A preliminary account of some of this work was presented at the 2nd International Symposium on Prostaglandins and the Cardiovascular System, Halle (Saale), GDR, September 19-21, 1977 and the First Soviet Union Conference, Prostaglandins in Experimental and Clinical Medicine, Moscow, USSR, April 18-20, 1978.

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vasculature (9-12). Because prostacyclin, in contrast to PGE,, is not inactivated during passage through the lungs (g-14), prostacy clin should be considered as a factor controlling blood pressure (12,13,15). Finally, aortic tissue from spontaneous hypertensive rats has an increased capacity to form prostacyclin compared to normotensive rats, which was proposed as a protective or compensatory reaction against the increased pressure (13). If the pharmacological effect of a substance is to have relevance as a possible circulating mediator, such an effect should be produced by doses which would be tolerated by a normal, unanesthetized animal without overt toxicity or other gross pharmacological actions. Prostacyclin is unstable under physiological conditions, having a half-life of 3 min both in blood and in pH 7.5 buffer at 37" (14). Although not inactivated appreciably by the lungs (g-14), its activity disappears as rapidly as PGEz and PGF2 in a single passage through the hind quarters and liver of the dog (157. Therefore, if prostacyclin does play a physiological role, it is likely that it would be formed on a more or less continuous basis. Administration by infusion would more likely mimic the physiological situation, and, since there is no degradation in the lungs, intravenous infusion would not entail losses as with PGE2 and PGF2,. We report here studies on the cardiovascular action of prostacyclin in both anesthetized and unanesthetized rats, together with some comparisons with prostaglandin Es. In all studies, compounds were given by intravenous infusion. To assess relevance of these actions to possible physiological roles for prostacyclin, the maximum tolerated dose, given by prolonged intravenous infusion to unanesthetized rats, was studied. METHODS General. Rats were Upjohn Sprague-Dawley origin, specific pathogen free, females. Rats for renal function studies weighed 150-170 g, other unanesthetized rats weighed 338-416 g, and anesthetized rats weighed 256-312 g. Unanesthetized rats were prepared with chronic indwelling venous cannulas (16). For measurement of blood pressure from unanesthetized rats, chronic indwelling aortic cannulas, modified extensively from those originally described by Weeks and Jones (17), were used.l Prostacyclin sodium salt was dissolved in 0.05 M tromethamine (TRIS) buffer, pH 9.4, made isotonic with 7.42 g/L of sodium chloride. Stock solutions were 1 mg/ml, stored no longer than 4 days at -60". Each stock sample was thawed once only. Dilutions were prepared fresh daily and kept on ice. 'All doses are as the sodium salt. PGE, dilutions were prepared fresh daily in isotonic saline from a 10 mg/ml stock solution in absolute ethanol. lFurther details may be obtained from J. R. Weeks.

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Maximum tolerated dose studies. Prostacyclin was infused while rats were relatively unrestrained using the apparatus described separately (18). Volume was 4.1 ml/day using a Harvard 975 Infusion Pump with 5 ml syringes. Rectal temperature was measured with a Yellow Springs Instrument Co. Telethermometer thermistor probe. Behavioral depression was evaluated by the leg withdrawal and righting reflexes. A hind paw was gently extended and the leg reflex considered absent if it were not pulled back within 3 sec. The righting reflex was considered absent if the fore quarters were not righted within 5 set after placing the rat on its side. Pinkness of the pinnae were evaluated subjectively on a scale of 0 to 3. Hind paw volume was estimated by marking a point on the ankle with an indelible pen, and immersing the paw into a tared cup of water and noting the apparent increase in weight of the cup. The rat was anesthetized lightly with methoxyflurane. Blood pressure studies. Blood pressure was recorded on a Grass polygraph as the electronically damped mean, and each reading was the average pressure, estimated by eye, over a l-2 min period. Infusions of prostacyclin or PGE2 were continued until there was no apparent further change in pressure, about 12 (range 5-18) min for unanesthetized rats and about 7 (range 3-14) min for anesthetized rats. Continuous registration of blood pressure was assured by infusion of 2-3 pl/min of saline into a T-connector (TC-20/3, Small Parts, 6901 NE 3rd Ave., Miami, Florida 33138) on the pressure transducer (Statham P23Gc). This slow flow does not affect the pressure reading. Flow was controlled by a fluid resistance adapted in principle from Ardill and Fentem (19).l As a control for spontaneous changes in blood pressure, rats were prepared in the same manner as for the experiments described below (6 unanesthetized, 6 intact anesthetized and 7 nephrectomized anesthetized) and given only vehicle infusion. After stabilization, blood pressures were recorded about every 10 min for 60 min, an interval which corresponded roughly to the average infusion time for each dose of prostacyclin or PGE2. At no time did the average mean pressures differ significantly from the initial value. Intravenous infusions for blood pressure studies were at a rate of 61 pl/min for unanesthetized rats and 18 ul/min for anesthetized rats. Two infusion pumps were used for each rat so infusion solutions could be changed quickly. If prostacyclin infusions were interrupted only for a few seconds, blood pressure would rise. Unanesthetized rats were placed in glass battery jars. Connections were made from the cannulas to infusion pumps and transducers by polyethylene tubing (PE 20) suspended over the jars. IFurther details may be obtained from J. R. Weeks.

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Anesthetized rats were given sodium pentobarbital, 30 mg/kg i.p. and placed on a temperature-controlled plate (MK4, Narco Biosystems, Houston, Texas). Rectal temperature was maintained between 36.8" and 38.4". The trachea was cannulated, vagi sectioned, and blood pressure recorded from the left carotid artery. A femoral vein was cannulated for intravenous infusions. For the first experiments rats were given pentolinium tartrate 2 mg/kg i.v., but in some rats ganglionic blockade did not persist for the duration of the experiment. Thereafter all rats were given three 1 mg/kg doses at intervals during surgical preparation. Nephrectomy was through flank incisions immediately after induction of anesthesia. Renin studies. Rats were handled in the same manner as for unanesthetized rats in blood Pressure studies. Rats were left undisturbed for at least 45 minutes, then vehicle infusion and initial blood pressure measurement started. Treatment consisted of either 10 min infusion of prostacyclin or vehicle. Then the pressure transducer was disconnected, about 0.1 ml of blood drawn from the aortic cannula (to rinse the connecting tubing) and discarded. A plastic 1 ml syringe was rinsed with a saturated solution of disodium edathamil (EDTA) and all air expelled. A 1 ml sample (or as much as possible) of blood was drawn into the syringe and imnediately transferred to a Wintrobe tube in ice, centrifuged at 0' for 5 min at 1500 g. Three 100 ~1 samples of plasma were taken and frozen. Plasma renin activity was determined by radioimmunoassay of generated angiotensin I by a slightly modified method of Haber -et al. (20) using the New England Nuclear kit. Renal function studies. Rats were in modified metabolism cages. The leash between the flow-through swivel and rat was l/8 in OD x l/16 in ID vinyl tubing. A 25 mn length of 16 ga hypodermic tubing was bent at a 45" angle and soldered at the center of a 25 x 30 mn piece of 0.13 mm thick stainless steel spring shim. This metal piece was cemented directly to the rat's back, without clipping the hair, using silicone rubber adhesive. Food was withheld overnight, and water removed one hour before the test. Rats were given 25 ml/kg of 0.9 percent sodium chloride orally and infusion of either prostacyclin or vehicle started. After 5 hr, urine was collected and analyzed for sodium and potassium using a Technicon Autoanalyzer 1. Statistical evaluation. Mean values are expressed f standard error, and significance of differences evaluated by the Student t test. RESULTS Maximum tolerated dose. Preliminary studies showed that the overt effects of high doses of prostacyclin were behavioral depression, hypothermia, flushing ("pinkness") of the pinnae, and swelling of the hind paws. Prostacyclin was infused intravenously and the effects evaluated after 4 and 24 hours. Infusions were a vehicle control and prostacyclin at 0.32, 0.56 and 1.0 mg/kg/day, or 0.22, 0.4 and 0.7 pg/kg/min respec-

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tively. Eight rats were used, of which seven received every treatment. Four days were allowed for recovery following each prostacyclin infusion. All parameters remained remarkably constant during the vehicle infusion. The only noticeable effect at 0.32 mg/kg/day was a mild flushing of the pinnae, present both at 4 and 24 hours. At 0.56 mg/ kg/day, pinnae coloring was more intense, body temperature fell slightly in every rat at 4 hours (mean change from 37.5' + 0.09 to 36.2' + 0.28), but recovered by 24 hours. At 1 mg/kg/day there was overt behavioral depression, the hind limb withdrawal reflex lost in 5 of 8 rats at 4 hours only, but the righting reflex was always present. The overt appearance seemed out of proportion to the minimal effect on reflexes. Pinnae were intensely flushed. Body temperature was profoundly lowered after 4 hours from 37.6" f 0.08 to 32.8" + 0.10, with only partial recovery to 34.1' * 0.70 after 24 hours. Paw volume increased progressively in all rats, being 7.6 f 1.3 percent greater after 4 hours and 19.8 ?:2.0 percent greater after 24 hours. Blood pressure,studies: unanesthetized rats. Dose-effect curves were determined for prostacyclin and PGEz by intravenous infusion. Vehicle was infused until blood pressure stabilized, and then compounds infused at dose rates increasing in 0.25 log increments. Results are sumnarized in Fig. 1. The threshold dose for prostacyclin was between 0.1 and 0.18 Pg/kg/min. At mid-range doses rats became less active and immobile, at higher doses they were prostrate with slow, labored respiration which was so severe that doses higher than 1.0 ug/kg/min were not given. In contrast, PGE:,caused only equivocal effects on blood pressure even at 5.6 Ftg/kg/minwhen a similar appearing depression was

L

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0.32

0.56

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Fig. 1. Prostacyclin and PGE, infusion on the blood pressure of unanesthetized, unrestrained rats. Initial blood pressures were 110.7 f 2.6 mm Hg for prostacyclin and 107.4 + 3.7 mm Hg for PGE2.

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One rat was almost in extremis at 3.2 Pg/kg/min, and was not given the higher dose. Whenprostacyclin infusions were discontinued, blood pressure started rising within a few seconds and within a few minutes exceeded initial pressure. Behavioral recovery was likewise prompt.

severe.

Blood pressure studies: anesthetized rats. Dose-effect curves were next determined using pentobarbital anesthetized, vagotomized, pentolinium-treated rats. Dose rates increased in 0.5 log increments. The threshold dose for prostacyclin was between 0.001 and 0.0032 Pg/kg/min. For doses up to and including 0.01 pg/kg/min, blood pressure fell promptly to a new steady state with each increase in infusion rate. At higher doses, for most rats, pressure would fall promptly but after 1 to 2 min would partially recover before attaining a steady state (Fig. 2). The maximally effective dose was between 0.1 and 0.32 Pg/kg/min.

mmHg

INTACT

NEPHRECTOMIZED

80 60 40

L

Fig. 2. Blood pressure response of an intact and nephrectomized rat to an intravenous infusion of prostacyclin. At the arrow, infusion rate was increased from 0.032 to 0.1 Pg/kg/min. Since compensatory mechanisms through the autonomic nervous system should have been eliminated by the vagotomv and ganglionic blockade, the experiment was repeated using nephrectomized rats to eliminate a possible renal compensatory mechanism. Results for both experiments are sunarized in Fig. 3. In the nephrectomized rats there was no partial recovery after increasing the infusion rate at any dose (Fig. 2). The maximum fall in intact rats nearly equalled the steady state in nephrectomized rats. The results of a similar experiment with PGE, are summarized in Fig. 4. The depressor effect is now revealed in these anesthetized rats. The threshold dose was between 0.01 and 0.032 Pg/kg/min, about ten-fold greater than for prostacyclin. Linear regressions for IIXTI Hg fall vs. log dose were calculated for PGE, and prostacyclin in nephrectomized rats, limiting consideration to the four mid-range doses. The slope for PGE, was 17.1 mm Hg, much less than the 31.4 nnn Hg for prostacyclin. Based upon doses calculated to give a 25 mn Hg fall, prostacyclin is 37 times more potent than PGE,.

506

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0

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Fig. 3. Prostacyclin infusion on the blood pressure of anesthetized intact and nephrectomized rats. Initial blood pressures were 114.2+ 5.4 mn Hg for intact and 99.226.8 nm Hg for nephrectomized rats. The asterisk means the maximum effect differs significantly (PcO.05) from the steady state in intact rats. 0

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Fig. 4. Prostaglandin E2 infusion on the blood pressure of anesthetized intact and nephrectomized rats. Initial blood pressures were 102.6k5.6 mm Hg for intact and 91.4f6.7 mn Hg for nephrectomized rats. The asterisk means the maximum effect differs significantly (P~0.05) from the steady state effect in intact rats.

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PROSTAGLANDINS The maximally effective dose of PGEz was probably not achieved, but higher doses were not given considering the profound depression observed at comparable doses in unanesthetized rats. A secondary rise in pressure was noted only at the highest dose, and, as with prostacyclin, only in intact rats. Renin studies. To test the hypothesis that the compensatory rise in pressure following prostacyclin infusion was mediated by renin release, we measured plasma renin activity after intravenous infusion of prostacyclin. Unanesthetized, unrestrained rats were used since anesthesia can release renin (21) and handling can in crease plasma catecholamine concentrations (22), which would be expected to stimulate renin release (23). Prostacyclin 0.01 and 0.1 pg/kg/min or vehicle were infused for 10 min. The low dose was clearly depressor in anesthetized rats, but without indirect evidence of renin release, whereas the higher dose showed clear indirect evidence of renin release. In unanesthetized rats, even the high dose had only an equivocal effect on blood pressure. Eight rats were used, each rat received each treatment, and the order of testing was balanced insofar as possible. To minimize the influence of hemorrhage from blood sampling, at least 3 days elapsed between each test. Result are given in Table 1. There was no effect on blood pressure. With succeeding tests, there was a progressive slight fall in hematocrit (47.4 + 0.98 to 44.0 f 0.89 percent), which was significant (PcO.05) only between the first and third tests. Plasma renin activity after the low dose did not differ from vehicle infusion, but was more than doubled after 0.1 ug/kg/min. Table 1 Effect of Intravenous Infusion of Prostacyclin on Blood Pressure and Plasma Renin Activity in Unanesthetized Rats Treatment

Blood Pressure (mn Hg) Initial Change

Plasma Renin Activity ng/ml/hr AT I

Vehicle

108 f 2.1

-2.4 + 2.37

3.83 + 0.69

PG12 Na 0.01 pg/kg/min

104 f 2.2

-0.2 + 1.63

4.13 * 0.75

PG12 Na 0.1 ug/kg/min

106 f 2.0

0.9 f 1.80

8.86 f l.lla

aP<0.05 from vehicle Renal function studies. We studied only the effect of infusion of prostacyclin, 0.1 ug/kg/min, on the excretion of a sodium

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chloride load in unanesthetized rats. Nine rats received the prostacyclin and 9 other rats an equal volume of vehicle. Results are surrmarizedin Table 2. Prostacyclin infusion significantly decreased both urinary volume and sodium excretion compared to vehicle-treated controls. Potassium excretion was highly variable in both treated and control rats, and the difference was not significant. Table 2 Effect of Intravenous Infusion of Prostacyclin on Renal Function in Unanesthetized Rats

Treatment

uv ml/5 hr

INa+

UK+

ueql5 hr

veql5 hr

Vehicle

2.6 ?r0.33

259 f 33.1

141 * 22.0

PGIz Na 0.1 Pg/kg/min

1.8 f 0.16a

158 + 25.4"

112 + 15.2

aP<0.05 from vehicle

DISCUSSION The threshold for overt intolerance to prostacyclin in the anesthetized rat is about 0.4 Pg/kg/min (0.56 mg/kg/day), and was manifested by a short-lasting hypothermia and flushing of the pinnae. Other than some flushing of the pinnae, rats tolerated 0.22 pg/kg/min (0.32 mg/kg/day). Unless normal production of prostacyclin is close to the maximum tolerated level, any pharmacological effect which is proposed to have possible physiological relevance as a humoral substance should be achieved by doses below 0.4 Pg/kg/min at the most and preferably below 0.2 ug/kg/min. The reddening of the pinnae during infusion of prostacyclin in unanesthetized rats suggests cutaneous vasodilatation. Further experiments would be required to determine whether such dilatation could promote heat loss sufficient to explain the hypothermia. Since the behavioral depression noticed at 1 mg/kg/day seemed out of proportion to the minimal effects on reflexes, the depression may have been only secondary to the profound hypothermia. We have no ready explanation for the swelling of the hind paws. A similar effect followed PGE, infusion, but a larger dose over a longer time was required (24). The ability of normal, unanesthetized rats to compensate for cardiovascular disturbances is dramatically illustrated by the 30- to

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lOO-fold smaller doses needed to lower blood pressure in anesthetized rats. The intrinsic activity of both prostacyclin and PGE2 was revealed only when compensatory mechanisms were blocked. Pinelis et al. (10) studied prostacyclin in rats by intravenous infusion and found depressor activity comparable to that reported here. Armstrong et al. (12) and Pace-Asciak et al. (15) also reported effects of prostacyclin on blood pressure of anesthetized rats. Meaningful comparisons of their results cannot be made, since not only were the anesthetics different but also they used bolus injections of prostacyclin rather than infusions. Noteworthy is that the intrinsic depressor activity of prostacyclin, as revealed in the anesthetized rat (0.01 to 0.032 ug/kg/min), is at the most about one-tenth of the dose which induced overt pharmacological effects in unanesthetized rats. These studies do not argue against proposals that prostacyclin may function as a factor in controlling blood pressure (12,13,15). Armstrong et al. (12) published intravenous dose-response curves for PGE;!and prostacyclin. In their experiments, compounds were given by bolus injections. Dose-response curves were parallel and prostacyclin was 4 to 8 times more potent than PGE,. At least part of the greater potency of prostacyclin is because PGE, is partially inactivated in the lungs. In our experiments, the dose-response curves were not parallel, with the slope for PGE, less than that of prostacyclin. At a 25 mm Hg fall in pressure, prostacyclin was 37 times more potent than The discrepancy between our results and those of Armstrong et al. PGE (lilf'maybe a consequence of bolus injections. Bolus injections of PGE, might give blood concentrations in the lungs which greatly exceed the maximum rate of metabolism (or transport into metabolizing cells), and a considerable excess would reach the systemic circulation. By infusion, the lower blood levels would permit a more efficient metabolism of PGE,, and thus it would be relatively less effective. Furthermore, as the bolus dose increases, relatively more PGE, would escape destruction and contribute to an apparently steeper slope of the dose-response curve. The rise in plasma renin activity is consistent with the hypothesis that the compensatory secondary rise in pressure following infusion of higher doses of prostacyclin was mediated by release of renin. This hypothesis is supported by reports that prostacyclin releases renin from kidney slices of rabbits (25) and rats (26), from isolated perfused rat kidneys (26), and into the renal vein during infusion into the renal artery of anesthetized dogs (27). The release we observed could not be secondary to lowered renal perfusion pressure, since arterial pressure remained unchanged. However, the 0.1 ug/kg/min dose of prostacyclin was nearly maximally vasodilator in anesthetized rats, so there may have been considerable compensatory activity through the sympathetic nervous system. Catecholamines so released could themselves release renin (23).

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At the higher doses of prostacyclin, the maximum fall in blood pressure was approximately the same in intact as in nephrectomized rats. If a compensatory pressor effect were continually operating in the intact rats, one would have expected the maximum fall to be less. Such a difference may be obscured not only by the variability of these responses but also because the initial pressures of the nephrectomized rats were about 15 mm Hg lower than in the intact rats. Whether PGEz can release renin is controversial (28). At 3.2 pg/kg/min of PGEz, the difference between the maximum effect and steady state blood pressures was significant (PcO.02). For PGI,, the differences were significant at 0.032 pg/kg/min and higher. In the unanesthetized rat, even 0.2 ug/kg/min of prostacyclin was well tolerated, but 3.2 pg/kg/min of PGEz caused severe depression. Accordingly, any physiological role for PGEz as a mediator for renin release would most likely be limited to a locally-mediated effect. Human and rabbit renal cortical microsomes have the ability to form prostacyclin from PGGz (29). Prostacyclin must also be considered as a possible intra-renal as well as circulating mediator of the release of renin by the kidney. In anesthetized dogs, Hill and Moncada (30) reported that intravenous infusion of prostacyclin in sub-hypotensive doses increased renal blood flow but decreased urinary excretion of sodium and chloride without affecting urinary volume. Hypotensive doses reduced urinary volume as well. In these experiments, since the prostacyclin dose was actually a compensated hypotensive dose, increased sympathetic nervous system activity may have decreased renal blood flow and caused the observed reductions in excretion of sodium and urine volume.

In summary, these studies show that prostacyclin has depressor activity in doses far below that causing overt evidence of intolerance, and therefore does not argue against a possible physiological role as a mediator or hormone. Furthermore, evidence is consistent with a reninreleasing effect of prostacyclin also in well-tolerated doses. ACKNOWLEDGEMENTS

We are indebted to James H. Ludens and Catherine J. Taylor for electrolyte assays, to Donald T. Pals and E. R. Micalizzi for plasma renin activity determinations, and to Philip I. Good for statistical advice and services. Dorothy M. Sutter provided technical assistance. Prostacyclin sodium salt was supplied by Frank H. Lincoln of The Upjohn Company. REFERENCES 1.

Moncada, S., R. J. Gryglewski, S. Bunting, and J. R. Vane. An Enzyme Isolated from Arteries Transforms Prostaglandin

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PROSTAGLANDINS Endoperoxides to an Unstable Substance that Inhibits Platelet Aggregation. Nature 263:663, 1976. 2. Johnson, R. A., D. R. Mor%i, J. H. Kinner, R. R. Gorman, J. C. McGuire, F. F. Sun, N. Whittaker, S. Bunting, J. Salmon, S. Moncada, and J. R. Vane. The Chemical Structure of Prostaglandin X (Prostacyclin). Prostaglandins 12:915, 1976. 3. Moncada, S., E. A. Higgs, and J. R. Vane. Human Arterial and Venous Tissues Generate Prostacyclin (Prostaglandin X) a Potent Inhibitor of Platelet Aggregation. Lancet l_:18, 1977. 4. Moncada, S., R. J. Gryglewski, S. Bunting, and J. R. Vane. A Lipid Peroxide Inhibits the Enzyme in Blood Vessel Microsomes that Generate from Prostaglandin Endoperoxides the Substance (Prostaglandin X) which Prevents Platelet Aggregation. Prostaglandins 12:715, 1976. 5. Gryglewski, R. r, R.,Korbut, A. Ocetkiewicz, J. Sp?awiiiski, B. Wojtaszek, and J. Swies. Lungs as a Generator of Prostacyclin -- Hypothesis on Physiological Significance. NaunynSchmiedeberg's Arch. Pharmacol. 304:45, 1978. 6. Moncada, S., R. Korbut, S. Bunting, and J. R. Vane. Prostacyclin is a Circulating Hormone. Nature 273~767, 1978. 7. de Deckere, E. A. M., D. H. Nugteren, F. % Hoor. Prostacyclin is the Major Prostaglandin Released from the Isolated Perfused Rabbit and Rat Heart. Nature 268:160, 1977. 8. Dusting, G. J., S. Moncada, and J. R. Vane. Prostacyclin (PGX) is the Endogenous Metabolite Responsible for Relaxation of Coronary Arteries Induced by Arachidonic Acid. Prostaglandins 13:3, 1977. 9. smstrong, J. M., D. Chapple, G. J. Dusting, R. Hughes, S. Moncada, and J. R. Vane. Cardiovascular Actions of Prostacyclin (PG12) in Chloralose Anesthetized Dogs. Br. J. Pharmacol. 61:136P, 1.977. 10. Enelis, V. G., Ch.M. Markov, Sh.1. Ismailov, W. Fb'rster.The Influence of Prostacyclin and Prostaglandin El on Haemodynamics in Rats. Abstracts, First Soviet Union Conference, Prostaglandins in Experimental and Clinical Medicine, Moscow, April 18-20, 1978, p. 40. 11. DuCharme, D. W., G. L. DeGraaf, S. J. Humphrey, and M. G. Wendling. In: Prostaglandins in Cardiovascular and Renal Function. (A. Scriabine, A. N. Lefer and S. A. Kuehl, eds.). Spectrum, New York, 1978, in press. 12. Armstrong, J. M., N. Lattimer, S. Moncada, and J. R. Vane. Comparison of the Vasodepressor Effects of Prostacyclin and 6-oxo-prostaglandin F,, with Those of Prostaglandin E, in Rats and Rabbits. Br. J. Pharmacol. 62:125, 1978. 13. Pace-Asciak, C. R., H. C. Carrara, G.Rangaraj, and K. C. Nicolaou. Enhanced Formation of PGI,, a Potent Hypotensive Substance, by Aortic Rings and Homogenates of the Spontaneous 14. Dusting, G. J., S. Moncada, and J. R. Vane. Disappearance of Prostacyclin (PGI,) in the Circulation of the Dog. Br. J.

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15.

16. 17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28. 29.

30.

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