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Effects of prostaglandins and prostaglandin precursors on the arterial pressure of the bullfrog, Rana catesbiana
Comp. Biochem. Physiol., Vol. 66C, pp. 199 to 202
0306-4492/80/0701-0199502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
EFFECTS OF PROSTAGLANDINS AND PROSTAGLANDIN PRECURSORS ON THE ARTERIAL PRESSURE OF THE BULLFROG, R A N A CA TESBIANA CHARLES W. LEFFLER, ROBERT C. HANSON* and EDWARD G. SCHNEIDER Department of Physiology and Biophysics, University of Tennessee Center for the Health Sciences, Memphis, TN 38163, U.S.A. (Received 15 October 1979) Abstract--Effects of prostaglandin precursors, prostaglandins, and cyclooxygenase inhibitors upon arterial pressures of unanesthetized bullfrogs were determined. 1. The mean arterial pressure of the bullfrogs was 32 4- 2 mm Hg and was not affected by indomethatin. 2. Intravenous arachidonic acid decreased arterial pressure, producing a 40% decrease at 3 mg/kg per min for 3 min. Arachidonic acid responses were attenuated by indomethacin or ETYA. 3. Intranvenous PGE2, PGI2, and PGF2~ all decreased arterial pressure. PGE2 and PGI2 were similar in potency, with PGF2~ being less potent. All three were more potent than arachidonic acid. 4. Endogenous arterial PG concentrations were less than 0.04, 0.28, and 13 ng/ml of PGE2, PGF2,, and PGI2, respectively.
INTRODUCTION In mammals, prostaglandins are highly vasoactive, and can have pronounced effects upon arterial pressure in special circumstances (Bergstrom et al., 1968; von Euler, 1937; Fletcher & Ramwell, 1977; Leffler & Passmore, 1977; Tyler et al., 1976). In mammals, the prominent action of prostaglandins appears to be vasodilator, as exogenous arachidonic acid produces systemic hypotension, an effect that is blocked by fatty acid cyclo-oxygenase inhibitors (Rose et al., 1974; Tyler et al., 1976). Amphibians, likewise, are capable of synthesizing prostaglandins (Zusman et al., 1977a,b). Prostaglandins have been shown to have pronounced effects upon salt and water movement in amphibian bladders and intestines (Gerencser et al., 1978; Zusman et al., 1977a,b). Circulatory effects of prostaglandins in amphibians have not been reported. This investigation was designed to: (1) determine effects of exogenous arachidonic acid and prostaglandins upon arterial pressures of bullfrogs; and (2) determine if endogenous prostaglandin production is an important determinant of frog arterial pressure. METHODS Six bullfrogs, Rana catesbiana (200-600 g), were anesthetized with MS 222 (1-1.5 hr in 0.2% bath). The left common iliac artery and the abdominal vein were cannulated with heparinized and saline-filled cannulae. Arterial pressure was monitored via the arterial cannula. The frogs were placed in dechlorinated, aerated, freshwater and allowed to recover for 4-5 hr. All measurements were made in dechlorinated, aerated, fresh water at 22°C on unanesthetized frogs. * The present address of Dr Robert C. Hanson is: Department of Biologic Research, Mead Johnson, Evansville, Indiana, U.S.A. 199
Arachidonic acid (Sigma)was stored at -8°C under Nv 10 mg of arachidonic acid were dissolved in 0.1 ml ethanol, diluted with anoxic, 0.6?/o saline to 5 mi (2 nag arachidonic acid/ml), and buffered to pH 7.4-7.6 with NaHCO3. Fresh solution was prepared for each experiment, and was stored at 4°C between infusions. No loss of activity was observed over 3 hr. Indomethacin was dissolved with equimolar sodium carbonate to a concentration of 1.8 mg/ml and injected i.v. (4 mg/kg) through the same cannula that was used for arachidonic acid and prostaglandin infusions. 20 rain following treatment with indomethacin, post-indomethacin arachidonic acid infusions were performed, 5,831,14-eicosatetraynoic acid (ETYA) solution was prepared in a manner identical to arachidonic acid solution and infused alone or simultaneously with an equal concentration of arachidonic acid. Prostaglandins Fz~, E2, and E t were dissolved in ethanol (7.5 mg/ml) and stored at -8°C. These solutions were diluted to 150/~g/ml with 0.6% saline for infusion. PGI2 was dissolved in Tris buffer (pH = 10) to a concentration of 1 mg/ml and stored at -8°C. Immediately before infusion, the stock solution was allowed to melt, and a small aliquot was diluted with 0.6% saline to a concentration of 150/lg/ml. All prostaglandins were infused through the venous catheter. Arterial blood samples (20 ml) were taken from two large frogs into 60 ml of ethanol for determination of endogenous blood levels of prostaglandin-like compounds (Green & Samuelsson, 1964; McGiff et al., 1970). The precipitated elements were removed by filtration, washed twice with 50 ml ethanol, and the washes added to the filtrate. The filtrate was evaporated to the aqueous phase, which was acidified (pH 3) and extracted with ethyl acetate, then phosphate buffer (pH 8), then chloroform at pH 3. The acidic lipids so obtained were further purified by thin layer chromatography on silica gel (0.50 ram) using chloroform: methanol:acetic acid:water (90:9:1:0.65) as the solvent system. This procedure clearly separates authentic PGF2, from authentic PGE2 and 6-keto-PGF~ (the nonenzymatic reduction product of PGI2 at physiological pH). The zone corresponding to PGE2 + 6-keto-PGF~, was rechromatographed (silica gel, 0.25 ram) using the organic phase
200
CHARLES W.
LEFFLER, ROBERT C.
HANSON
of ethyl acetate:isooctane:acetic acid:water (11:5:2:10) as a solvent to separate PGE from 6-keto-PGFt=. The eluants from the thin layer chromatographic plates were evaporated and reconstituted for bioassay. The prostaglandin-like compounds were quantified by bracket assay utilizing rat stomach strip, rat uterus, and rat colon superfused with Krebs solution containing indomethacin, scopolamine, and diphenhydramine hydrochioride. The minimum concentrations in blood that could be detected (assuming 100% recovery) were: 8ng 6-ketoPGFt,/ml; 0.03 ng PGE2/ml; and 0.2 ng PGF2dml. Recovery of added authentic prostaglandins processed through extraction and thin layer chromatography average 60, 70, and 72% for 6-keto-PGF~,, PGE2, and PGF2=, respectively.
and
EDWARD
G. SCHNEIDER
I untreated o] indomethacin. . . . . . '~ ETYA 50
•
|
o
o
40
SAP (%
decreose )
o 30
o o
o
o oD
o 20
. ~,..g-"
I0
I00
500
2000
ARACHIDONIC
(IJg.kg.min)
RESULTS The mean arterial pressure of the six bullfrogs before arachidonic acid infusions was 32 + 2* mm Hg (39 4- 2 systolic/28 4- 2 diastolic). Treatment with indomethacin caused no significant change in arterial pressure. Intravenous infusion of arachidonic acid decreased the arterial pressures of bullfrogs in a dosedependent manner (Fig. 1). The minimum infusion rate administered for 3 min that produced a detectable decrease in pressure was about 0.3 mg/kg per min. A 3-min infusion at 3 mg/kg per min decreased systemic arterial pressure approx 409/0. Within 10 min after the infusion, arterial pressures had returned toward controls. In 809/0 of the infusions between 1.5-3 mg/kg per min of arachidonic acid, the arterial pressure 10 min post-infusion was within 6% of the preinfusion pressure. Hypotensive effects of exogenous arachidonic acid were greatly attenuated by indomethacin and ETYA (Fig. 1). Thus, a 3-min infusion of 1 mg arachidonic acid/kg per min decreased systemic arterial pressure approx 259/0 prior to treatment with indomethacin, but only about 8% following indomethacin treatment. Equimolar ETYA infused simultaneously with arachidonic acid produced a similar attenuation of the response to arachidonic acid (Fig. 1). Intravenous PGE2 infusions produced dosedependent decreases in the arterial pressures of the bullfrogs (Fig. 2). Also, longer infusions (3 min) caused greater decreases than did shorter (2 min) infusions. The dosage of PGE2 necessary to produce a given decrease in pressure was approx 100 times less than the arachidonic acid dose. Also, the response to PGE2 was somewhat more prolonged than was the arachidonic acid response. In 88% (7 of 8) of the PGE2 infusions between 10-50/~g/kg per min for 2 min, the arterial pressure 10 min post-infusion was more than 10% less than the pre-infusion pressure (in the other, the arterial pressure was 9.7% less). The responses to PGE2 were not affected by treatment with indomethacin. Responses to PGI2 (3-30#g/kg per re_in for 2 min, 7 infusions) were quantitatively similar to responses to PGE2 (PGE2 and PGI2 regressions were not different at 95% confidence). Thus, a 2-min PGI2 infusion at a dose of 3 #g/kg per min produced an average decrease in arterial pressure of 16% while 10#g/kg per rain produced an average decrease of 27%. Within 10m in, the pressure had returned to within 10% of the before infusion pressure in 67% of *
Mean 4- SEM.
•
ACID
Fig. 1. Effects of 3 rain intravenous infusions of arachidonic acid upon the systemic arterial pressures (SAP) of bullfrogs before and after treatment with indomethacin (4 mg/kg) or ETYA (simultaneous, equimolar infusion). (Regressions were determined by method of least squares. Untreated and indomethacin treated regression lines are significantly different at P < 0.05). the infusions. PGEt is likewise a hypotensive agent, but we did not infuse PGEt into enough frogs for meaningful quantification of responses. In the bullfrog, PGF2= was a hypotensive agent. Two- and 3-min intravenous infusions of PGF2= between 4 and 500 #K/kg per rnin produced dosedependent decreases in arterial pressure (Fig. 3). Recovery was rapid, approaching before infusion pressures within 7 min after all infusions (up to 500 #g/kg per min). Responses to PGF2= were considerably less, at equal doses, than were responses to PGE2 and PGI2, but were greater than responses to arachidonic acid. We also observed the effects of PGE t and PGF2= on a single leopard frog (Rana pipiens). In this animal, PGE~ was, as in the bullfrog, a hypotensive agent; but PGF2= (12-500 #g/kg per min for 1-min periods) caused increases in arterial pressure. Prostaglandin-like activity was measured in three 20 ml arterial blood samples. In none of these samples was the prostaglandin concentration high enough for positive identification. We conclude, therefore, that arterial prostaglandin concentrations are less than
3 min i n f u s i o n - - 2 min infus on . . . . .
5o
o
#
4o SAP (% d e c r e o s e )
30 20
s
o
o
o
io ~) I
t I00
t 1000
PGE= 0 4 g ' k g ' m i n )
Fig. 2. Effects of intravenous infusion of PGEz upon: the systemic arterial pressures (SAP) of bullfrogs. (Regressions were determined by method of least squares.)
Effects of prostaglandins on frog blood pressure
~
min i n f u s i o n
*
rnin n f u s o n . . . . .
.
50 40
(% d e c r e a s e
l
30 o
ZO I0
o
o
o o . - "
o
o.~-" o
o I
I0
I00
IO00
PGFza{ ~g.kcJ.min)
Fig. 3. Effects of intravenous infusions of PGF2, upon the systemic arterial pressure (SAP) of bullfrogs. (Regressions were determined by method of least squares.) 0.04, 0.28, and t3 ng/ml of PGE, PGF, and PGI 2, respectively. DISCUSSION
Indomethacin had no effect upon the arterial pressure of unanesthetized, normotensive bullfrogs. Further, attempted detection of prostaglandin-like compounds in frog plasma indicated that high levels of circulating prostaglandins were not present. Thus, it appears that the role of prostaglandins in the regulation of blood pressure in the normotensive unanesthetized bullfrog is minimal. This finding is not surprising since indomethacin has only small effects upon systemic arterial pressures of untraumatized mammals (Leffler & Passmore, 1977; Leffler et al., 1978; Nowak & Wennmalm, 1978; Terraguo et al., 1977). In mammals, prostaglandins become significant contributors to blood pressure determination and blood flow distribution during hemorrhagic, septic, or traumatic shock (Fletcher & Ramwell, 1977; Green & Samuelsson, 1964; Leffler et al., 1978; Tyler et al., 1976). Whether endogenous prostaglandin production can have a role in amphibian cardiovascular regulation in specific circumstances remains to be determined. Exogenous arachidonic acid infused intravenously into bullfrogs produced dose-dependent arterial pressure decreases. This hypotensive effect was greatly attenuated following treatment with indomethacin or during simultaneous infusion of 5,8,11,14-eicosatetraynoic acid (ETYA). Indomethacin and ETYA both inhibit conversion of arachidonic acid into prostaglandin cyclic endoperoxide precursors to the prostaglandins ( A h e r n & Downing, 1970; Flower, 1974). Thus, the hypotensive activity of arachidonic acid in the bullfrog would appear to be the result of conversion into prostaglandins, rather than a direct effect of arachidonic acid itself. Likewise, in mammals, arachidonic acid has very little, direct cardiovascular effect (Tyler et al., 1978). The three metabolites of arachidonic acid examined (PGE2, PGI2, and PGF2~) all decreased the arterial pressures of bullfrogs. PGE2 and PGI2 are systemic vasodilators in mammals as well (Armstrong et al., 1978a; Armstrong et al., 1978b; Leffler et al., 1978; Leffler & Hessler, 1979). PGF2=, however, has a
201
hypertensive effect upon most mammals (Anderson et al., 1972; Leffier et al., 1979; Nakano & Cole, 1969). We do not know whether or not the hypotensive activity of PGF2= upon the bullfrog results from direct action of PGF2~ or from conversion into another compound, possibly PGE2. The greatly reduced activity of PGF2~ as compared to E2 could suggest such conversion. It is interesting to note that in another species within the same genus (Rana pipiens) PGF2, appears to increase arterial pressure. PGE2 was not infused into the leopard frog; but PGE1 was, as in the bullfrog a hypotensive agent. The hypotensive effects of PGE2 are more prolonged in the bullfrog than in normotensive mammals (Cassin et al., 1979). In mammals, PGEz is rapidly metabolized upon passage through the pulmonary circulation, thus precluding its action as a circulating hormone in the healthy mammal. The entire frog cardiac output does not pass through the pulmonary circulation. The parallel nature of the frog circulation could possibly contribute to the prolonged activity of PGE2 in the bullfrog vasculature. PGI2, which is not rapidly deactivated by mammalian lung, is reduced at physiological pH to a much less active compound, 6-keto-PGFl~. In frog, as in the mammal, the activity of PGI2 is short-lived. Prostaglandins, whether synthesized from arachidonic acid within the frog or infused directly into the frog, are vasoactive in bullfrogs. PGFz,, PGE2, PGE1, and PGI2 decrease the arterial pressure of the bullfrog. Endogenous prostaglandins do not appear to have significant influence upon the arterial pressure of the normotensive, unanesthetized bullfrog. Whether or not prostaglandins become more important determinants of arterial pressure following hemorrhage, trauma, or following administration of endotoxin, as apparently occurs in mammals, remains to be determined. SUMMARY
The effects of intravenous infusion of arachidonic acid upon the arterial pressures of unanesthetized bullfrogs, Rana catesbiana, were determined before and after treatment with indomethacin (4 mg/kg, i.v.) and during infusion of equimolar 5,8,11,14-eicosatetraynoic acid (ETYA). Effects of intravenous infusions of prostaglandin E2, F2=, and 12 upon arterial pressure were also determined. The mean arterial pressure of the bullfrogs was 32 + 2mmHg. Treatment with indomethacin had no effect upon this pressure. Intravenous arachidonic acid (0.3-3 mg/kg per min for 3 min) decreased arterial pressure in a dose-dependent manner, producing a 40% decrease at 3 mg/kg per min. The responses to arachidonic acid were greatly attenuated by indomethacin or ETYA. Intravenous PGE2, PGI2, and PGF2~ all decreased arterial pressure. PGE2 and PGI2 were similar in potency, with PGF2~ being more than 10 times less potent. All 3 prostaglandins, at equal dosage, had far more pronounced hypotensive effects than did arachidonic acid. High plasma levels of endogenous prostaglandins were not found when blood samples were analyzed by chemical extraction of acidic lipids, thin layer chromatography, and tissue cascade bioassay (arterial PG concentrations were less than 0.04, 0.28,
202
CHARLESW. LEFFLER,ROBERTC. HANSONand EDWARDG. SCHNEIDER
and 13 ng/ml of PGE2, PGF2~, and PGI2, respectively). Endogenous prostaglandin synthesis does not appear to have significant influence upon the arterial pressure of the normotensive, unanesthetized bullfrog, although prostaglandins, when present, can have profound cardiovascular effects upon the frog. Acknowledgements--We acknowledge the excellent assistance of S. D. Gleason and C. E. Wakelyn. Prostaglandins were a gift from Dr J. E. Pike, The Upjohn Co. Indomethacin was a gift from Dr C. A. Stone, Merck, Sharpe and Dohme. 5,8,11,14-eicosatetraynoic acid was a gift from Dr K. U. Malik, Department of Pharmacology, University of Tennessee Center for the Health Sciences. This study was supported, in part, by a General Research Support Grant from the University of Tennessee Center for the Health Sciences (from USPHS), the Tennessee Heart Association, and USPHS HL 16658.
REFERENCES AHERN P. G. & DOWNING D. T. (1970) Inhibition of prostaglandin biosynthesis by eicosa-5,8,11,14-tetraynoic acid. Biochim. biophys. Acta 210, 456--461. ANDERSONF. L., KRALISSA. C., ISAGARIST. J. d~ KINDA H. (1972) Effects of prostaglandins F2, and E2 on the bovine circulation. Proc. Soc. exp. Biol. Med. 140, 1049-1053. ARMSTRONGJ. M., DUSTINGG. S., MONCADAS. & VANE J. R. (1978a) Cardiovascular actions of prostacyclin (PGI2), a metabolite of arachidonic acid which is synthesized by blood vessels. Circ. Res. Suppl. I, 43, Ii12-II19. ARMSTRONGJ. i . , LATTIMERN., MONCADAS. & VANEJ. R. (1978b) Comparison of the vasodepressor effects of prostacyclin and 6-oxo-prostaglandin Ft~ with those of prostaglandin E 2 in rats and rabbits. Br. d. Pharmac. 62, 125-130. BERGSTROMS., CARLSONL. A. & WEEKS J. R. (1968) The prostaglandins: a family of biologically active lipids. Pharmac. Rev. 20, 1-48. CASSIN S., TYLER T., LEFFLERC. & WALLISR. (1979) Pulmonary and systemic vascular responses of perinatal goats to prostaglandins E1 and E2. Am. J. Physiol. 236, H828-H832. EULER U. S. VON (1937) On the specific vasodilating and plain muscle stimulating substances from accessory genital glands in man and certain animals (prostaglandin and vesiglandin). J. Physiol., Lond. 88, 213-234. FLETCHERJ. S. & RAMWELLP. W. (1977) Modification, by aspirin and indomethacin, of haemodynamic and prosta-
glandin releasing effects of E. coil endotoxin in the dog. Br. J. Pharmac. 61, 175-181. FLOWER R. J. (1974) Drugs which inhibit prostaglandin biosynthesis. Pharmac. Rev. 26, 33-67. GERENCSERG. A., TYLER T. & CmSiN S. (1978) Sodium transport by isolated bullfrog small intestine: effect of prostaglandin Et. Biochim. biophys. Acta 509, 159-169. GREEN K. & SAMUELSSONB. (1964) Prostaglandins and related factors: XIX. Thin layer chromatography of prostaglandins. J. Lipid Res. 5, 117-120. LEFFLER C. W. & HESSLER J. R. (1979) Pulmonary and systemic vascular effects of exogenous prostaglandin I2 in fetal lambs. Eur. J. Pharmac. 54, 37--42. LEFFLER C. W., TYLER T. L. & CASSINS. (1978) Effects of indomethacin on cardiovascular hemodynamies of goats in hemorrhagic shock. Circ. Shock 5, 299--310. LEFFLER C. W. & PASSMOREJ. C. (1977) Effects of indomethacin on hemodynamics of dogs in refractory hemorrhagic shock. J. surg. Res. 23, 392-399. LEFFLERC. W., TYLERT. L. & CASSlNS. (1979) Pulmonary and systemic circulatory responses of perinatal goats to prostaglandin F2v Can. J. Physiol. 57, 167-173. McGIFF J. C., CROWSHAWK., TERRAGNON. A. & LONIGROW A. J. (1970) Release of a prostaglandin-like substance into renal venous blood in response to angiotensin--II. Circ. Res. 26, 27 (Suppl. 1) 1121-1130. NAKANOJ. ~; COLE B. (1969) Effects of PGEI and F2, on systemic, pulmonary, and splanchnic circulation in dogs. Am. J. Physiol. 217, 222-226. NOWAL J. & WENNMALMA. (1978) Influence of indomethatin and of prostaglandin E~ on total and regional blood flow in man. Acta. physiol, scand. 102, 484--492. ROSE J. C., JOhnSON M., RAMWELLP. W. & KOT P. A. (1974) Effects of arachidonic acid on systemic arterial pressure, myocardial contractility and platlets in the dog. Proc. Soc. exp. Biol. Med. 147, 652-655. TERRAGNON. A., TERRAGNOD. A. & McGIFF J. C. (1977) Contributions of prostaglandins to the renal circulation in conscious, anesthetized, and laparatomized dogs. Circ. Res. 40, 590-595. TYLERT., LEFFLERC. & CASSINS. (1976) Use of indomethacin to reverse neonatal hypotension. Experientia 32, 61. TYLERT. L., LEFFLERC. W. & CASSINS. (1978) Circulatory responses of perinatal goats to prostaglandin precursors. Prostagland. & Med. 1, 213-220. ZUSMAN R. M., KEISER H. R. & HANDLER J. S. (1977a) Vasopressin-stimulated prostaglandin E biosynthesis in the toad urinary bladder. J. clin. Invest. 60, 1339-1347. ZUSMAN R. M., KEISER H. R. & HANDLER J. S. (1977b) Inhibition of vasopressin stimulated prostaglandin E biosynthesis by chlorpropamide in the toad urinary bladder. J. clin. Invest. 60, 1348-1353.
0306-4492/80/0701-0199502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
EFFECTS OF PROSTAGLANDINS AND PROSTAGLANDIN PRECURSORS ON THE ARTERIAL PRESSURE OF THE BULLFROG, R A N A CA TESBIANA CHARLES W. LEFFLER, ROBERT C. HANSON* and EDWARD G. SCHNEIDER Department of Physiology and Biophysics, University of Tennessee Center for the Health Sciences, Memphis, TN 38163, U.S.A. (Received 15 October 1979) Abstract--Effects of prostaglandin precursors, prostaglandins, and cyclooxygenase inhibitors upon arterial pressures of unanesthetized bullfrogs were determined. 1. The mean arterial pressure of the bullfrogs was 32 4- 2 mm Hg and was not affected by indomethatin. 2. Intravenous arachidonic acid decreased arterial pressure, producing a 40% decrease at 3 mg/kg per min for 3 min. Arachidonic acid responses were attenuated by indomethacin or ETYA. 3. Intranvenous PGE2, PGI2, and PGF2~ all decreased arterial pressure. PGE2 and PGI2 were similar in potency, with PGF2~ being less potent. All three were more potent than arachidonic acid. 4. Endogenous arterial PG concentrations were less than 0.04, 0.28, and 13 ng/ml of PGE2, PGF2,, and PGI2, respectively.
INTRODUCTION In mammals, prostaglandins are highly vasoactive, and can have pronounced effects upon arterial pressure in special circumstances (Bergstrom et al., 1968; von Euler, 1937; Fletcher & Ramwell, 1977; Leffler & Passmore, 1977; Tyler et al., 1976). In mammals, the prominent action of prostaglandins appears to be vasodilator, as exogenous arachidonic acid produces systemic hypotension, an effect that is blocked by fatty acid cyclo-oxygenase inhibitors (Rose et al., 1974; Tyler et al., 1976). Amphibians, likewise, are capable of synthesizing prostaglandins (Zusman et al., 1977a,b). Prostaglandins have been shown to have pronounced effects upon salt and water movement in amphibian bladders and intestines (Gerencser et al., 1978; Zusman et al., 1977a,b). Circulatory effects of prostaglandins in amphibians have not been reported. This investigation was designed to: (1) determine effects of exogenous arachidonic acid and prostaglandins upon arterial pressures of bullfrogs; and (2) determine if endogenous prostaglandin production is an important determinant of frog arterial pressure. METHODS Six bullfrogs, Rana catesbiana (200-600 g), were anesthetized with MS 222 (1-1.5 hr in 0.2% bath). The left common iliac artery and the abdominal vein were cannulated with heparinized and saline-filled cannulae. Arterial pressure was monitored via the arterial cannula. The frogs were placed in dechlorinated, aerated, freshwater and allowed to recover for 4-5 hr. All measurements were made in dechlorinated, aerated, fresh water at 22°C on unanesthetized frogs. * The present address of Dr Robert C. Hanson is: Department of Biologic Research, Mead Johnson, Evansville, Indiana, U.S.A. 199
Arachidonic acid (Sigma)was stored at -8°C under Nv 10 mg of arachidonic acid were dissolved in 0.1 ml ethanol, diluted with anoxic, 0.6?/o saline to 5 mi (2 nag arachidonic acid/ml), and buffered to pH 7.4-7.6 with NaHCO3. Fresh solution was prepared for each experiment, and was stored at 4°C between infusions. No loss of activity was observed over 3 hr. Indomethacin was dissolved with equimolar sodium carbonate to a concentration of 1.8 mg/ml and injected i.v. (4 mg/kg) through the same cannula that was used for arachidonic acid and prostaglandin infusions. 20 rain following treatment with indomethacin, post-indomethacin arachidonic acid infusions were performed, 5,831,14-eicosatetraynoic acid (ETYA) solution was prepared in a manner identical to arachidonic acid solution and infused alone or simultaneously with an equal concentration of arachidonic acid. Prostaglandins Fz~, E2, and E t were dissolved in ethanol (7.5 mg/ml) and stored at -8°C. These solutions were diluted to 150/~g/ml with 0.6% saline for infusion. PGI2 was dissolved in Tris buffer (pH = 10) to a concentration of 1 mg/ml and stored at -8°C. Immediately before infusion, the stock solution was allowed to melt, and a small aliquot was diluted with 0.6% saline to a concentration of 150/lg/ml. All prostaglandins were infused through the venous catheter. Arterial blood samples (20 ml) were taken from two large frogs into 60 ml of ethanol for determination of endogenous blood levels of prostaglandin-like compounds (Green & Samuelsson, 1964; McGiff et al., 1970). The precipitated elements were removed by filtration, washed twice with 50 ml ethanol, and the washes added to the filtrate. The filtrate was evaporated to the aqueous phase, which was acidified (pH 3) and extracted with ethyl acetate, then phosphate buffer (pH 8), then chloroform at pH 3. The acidic lipids so obtained were further purified by thin layer chromatography on silica gel (0.50 ram) using chloroform: methanol:acetic acid:water (90:9:1:0.65) as the solvent system. This procedure clearly separates authentic PGF2, from authentic PGE2 and 6-keto-PGF~ (the nonenzymatic reduction product of PGI2 at physiological pH). The zone corresponding to PGE2 + 6-keto-PGF~, was rechromatographed (silica gel, 0.25 ram) using the organic phase
200
CHARLES W.
LEFFLER, ROBERT C.
HANSON
of ethyl acetate:isooctane:acetic acid:water (11:5:2:10) as a solvent to separate PGE from 6-keto-PGFt=. The eluants from the thin layer chromatographic plates were evaporated and reconstituted for bioassay. The prostaglandin-like compounds were quantified by bracket assay utilizing rat stomach strip, rat uterus, and rat colon superfused with Krebs solution containing indomethacin, scopolamine, and diphenhydramine hydrochioride. The minimum concentrations in blood that could be detected (assuming 100% recovery) were: 8ng 6-ketoPGFt,/ml; 0.03 ng PGE2/ml; and 0.2 ng PGF2dml. Recovery of added authentic prostaglandins processed through extraction and thin layer chromatography average 60, 70, and 72% for 6-keto-PGF~,, PGE2, and PGF2=, respectively.
and
EDWARD
G. SCHNEIDER
I untreated o] indomethacin. . . . . . '~ ETYA 50
•
|
o
o
40
SAP (%
decreose )
o 30
o o
o
o oD
o 20
. ~,..g-"
I0
I00
500
2000
ARACHIDONIC
(IJg.kg.min)
RESULTS The mean arterial pressure of the six bullfrogs before arachidonic acid infusions was 32 + 2* mm Hg (39 4- 2 systolic/28 4- 2 diastolic). Treatment with indomethacin caused no significant change in arterial pressure. Intravenous infusion of arachidonic acid decreased the arterial pressures of bullfrogs in a dosedependent manner (Fig. 1). The minimum infusion rate administered for 3 min that produced a detectable decrease in pressure was about 0.3 mg/kg per min. A 3-min infusion at 3 mg/kg per min decreased systemic arterial pressure approx 409/0. Within 10 min after the infusion, arterial pressures had returned toward controls. In 809/0 of the infusions between 1.5-3 mg/kg per min of arachidonic acid, the arterial pressure 10 min post-infusion was within 6% of the preinfusion pressure. Hypotensive effects of exogenous arachidonic acid were greatly attenuated by indomethacin and ETYA (Fig. 1). Thus, a 3-min infusion of 1 mg arachidonic acid/kg per min decreased systemic arterial pressure approx 259/0 prior to treatment with indomethacin, but only about 8% following indomethacin treatment. Equimolar ETYA infused simultaneously with arachidonic acid produced a similar attenuation of the response to arachidonic acid (Fig. 1). Intravenous PGE2 infusions produced dosedependent decreases in the arterial pressures of the bullfrogs (Fig. 2). Also, longer infusions (3 min) caused greater decreases than did shorter (2 min) infusions. The dosage of PGE2 necessary to produce a given decrease in pressure was approx 100 times less than the arachidonic acid dose. Also, the response to PGE2 was somewhat more prolonged than was the arachidonic acid response. In 88% (7 of 8) of the PGE2 infusions between 10-50/~g/kg per min for 2 min, the arterial pressure 10 min post-infusion was more than 10% less than the pre-infusion pressure (in the other, the arterial pressure was 9.7% less). The responses to PGE2 were not affected by treatment with indomethacin. Responses to PGI2 (3-30#g/kg per re_in for 2 min, 7 infusions) were quantitatively similar to responses to PGE2 (PGE2 and PGI2 regressions were not different at 95% confidence). Thus, a 2-min PGI2 infusion at a dose of 3 #g/kg per min produced an average decrease in arterial pressure of 16% while 10#g/kg per rain produced an average decrease of 27%. Within 10m in, the pressure had returned to within 10% of the before infusion pressure in 67% of *
Mean 4- SEM.
•
ACID
Fig. 1. Effects of 3 rain intravenous infusions of arachidonic acid upon the systemic arterial pressures (SAP) of bullfrogs before and after treatment with indomethacin (4 mg/kg) or ETYA (simultaneous, equimolar infusion). (Regressions were determined by method of least squares. Untreated and indomethacin treated regression lines are significantly different at P < 0.05). the infusions. PGEt is likewise a hypotensive agent, but we did not infuse PGEt into enough frogs for meaningful quantification of responses. In the bullfrog, PGF2= was a hypotensive agent. Two- and 3-min intravenous infusions of PGF2= between 4 and 500 #K/kg per rnin produced dosedependent decreases in arterial pressure (Fig. 3). Recovery was rapid, approaching before infusion pressures within 7 min after all infusions (up to 500 #g/kg per min). Responses to PGF2= were considerably less, at equal doses, than were responses to PGE2 and PGI2, but were greater than responses to arachidonic acid. We also observed the effects of PGE t and PGF2= on a single leopard frog (Rana pipiens). In this animal, PGE~ was, as in the bullfrog, a hypotensive agent; but PGF2= (12-500 #g/kg per min for 1-min periods) caused increases in arterial pressure. Prostaglandin-like activity was measured in three 20 ml arterial blood samples. In none of these samples was the prostaglandin concentration high enough for positive identification. We conclude, therefore, that arterial prostaglandin concentrations are less than
3 min i n f u s i o n - - 2 min infus on . . . . .
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Fig. 2. Effects of intravenous infusion of PGEz upon: the systemic arterial pressures (SAP) of bullfrogs. (Regressions were determined by method of least squares.)
Effects of prostaglandins on frog blood pressure
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Fig. 3. Effects of intravenous infusions of PGF2, upon the systemic arterial pressure (SAP) of bullfrogs. (Regressions were determined by method of least squares.) 0.04, 0.28, and t3 ng/ml of PGE, PGF, and PGI 2, respectively. DISCUSSION
Indomethacin had no effect upon the arterial pressure of unanesthetized, normotensive bullfrogs. Further, attempted detection of prostaglandin-like compounds in frog plasma indicated that high levels of circulating prostaglandins were not present. Thus, it appears that the role of prostaglandins in the regulation of blood pressure in the normotensive unanesthetized bullfrog is minimal. This finding is not surprising since indomethacin has only small effects upon systemic arterial pressures of untraumatized mammals (Leffler & Passmore, 1977; Leffler et al., 1978; Nowak & Wennmalm, 1978; Terraguo et al., 1977). In mammals, prostaglandins become significant contributors to blood pressure determination and blood flow distribution during hemorrhagic, septic, or traumatic shock (Fletcher & Ramwell, 1977; Green & Samuelsson, 1964; Leffler et al., 1978; Tyler et al., 1976). Whether endogenous prostaglandin production can have a role in amphibian cardiovascular regulation in specific circumstances remains to be determined. Exogenous arachidonic acid infused intravenously into bullfrogs produced dose-dependent arterial pressure decreases. This hypotensive effect was greatly attenuated following treatment with indomethacin or during simultaneous infusion of 5,8,11,14-eicosatetraynoic acid (ETYA). Indomethacin and ETYA both inhibit conversion of arachidonic acid into prostaglandin cyclic endoperoxide precursors to the prostaglandins ( A h e r n & Downing, 1970; Flower, 1974). Thus, the hypotensive activity of arachidonic acid in the bullfrog would appear to be the result of conversion into prostaglandins, rather than a direct effect of arachidonic acid itself. Likewise, in mammals, arachidonic acid has very little, direct cardiovascular effect (Tyler et al., 1978). The three metabolites of arachidonic acid examined (PGE2, PGI2, and PGF2~) all decreased the arterial pressures of bullfrogs. PGE2 and PGI2 are systemic vasodilators in mammals as well (Armstrong et al., 1978a; Armstrong et al., 1978b; Leffler et al., 1978; Leffler & Hessler, 1979). PGF2=, however, has a
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hypertensive effect upon most mammals (Anderson et al., 1972; Leffier et al., 1979; Nakano & Cole, 1969). We do not know whether or not the hypotensive activity of PGF2= upon the bullfrog results from direct action of PGF2~ or from conversion into another compound, possibly PGE2. The greatly reduced activity of PGF2~ as compared to E2 could suggest such conversion. It is interesting to note that in another species within the same genus (Rana pipiens) PGF2, appears to increase arterial pressure. PGE2 was not infused into the leopard frog; but PGE1 was, as in the bullfrog a hypotensive agent. The hypotensive effects of PGE2 are more prolonged in the bullfrog than in normotensive mammals (Cassin et al., 1979). In mammals, PGEz is rapidly metabolized upon passage through the pulmonary circulation, thus precluding its action as a circulating hormone in the healthy mammal. The entire frog cardiac output does not pass through the pulmonary circulation. The parallel nature of the frog circulation could possibly contribute to the prolonged activity of PGE2 in the bullfrog vasculature. PGI2, which is not rapidly deactivated by mammalian lung, is reduced at physiological pH to a much less active compound, 6-keto-PGFl~. In frog, as in the mammal, the activity of PGI2 is short-lived. Prostaglandins, whether synthesized from arachidonic acid within the frog or infused directly into the frog, are vasoactive in bullfrogs. PGFz,, PGE2, PGE1, and PGI2 decrease the arterial pressure of the bullfrog. Endogenous prostaglandins do not appear to have significant influence upon the arterial pressure of the normotensive, unanesthetized bullfrog. Whether or not prostaglandins become more important determinants of arterial pressure following hemorrhage, trauma, or following administration of endotoxin, as apparently occurs in mammals, remains to be determined. SUMMARY
The effects of intravenous infusion of arachidonic acid upon the arterial pressures of unanesthetized bullfrogs, Rana catesbiana, were determined before and after treatment with indomethacin (4 mg/kg, i.v.) and during infusion of equimolar 5,8,11,14-eicosatetraynoic acid (ETYA). Effects of intravenous infusions of prostaglandin E2, F2=, and 12 upon arterial pressure were also determined. The mean arterial pressure of the bullfrogs was 32 + 2mmHg. Treatment with indomethacin had no effect upon this pressure. Intravenous arachidonic acid (0.3-3 mg/kg per min for 3 min) decreased arterial pressure in a dose-dependent manner, producing a 40% decrease at 3 mg/kg per min. The responses to arachidonic acid were greatly attenuated by indomethacin or ETYA. Intravenous PGE2, PGI2, and PGF2~ all decreased arterial pressure. PGE2 and PGI2 were similar in potency, with PGF2~ being more than 10 times less potent. All 3 prostaglandins, at equal dosage, had far more pronounced hypotensive effects than did arachidonic acid. High plasma levels of endogenous prostaglandins were not found when blood samples were analyzed by chemical extraction of acidic lipids, thin layer chromatography, and tissue cascade bioassay (arterial PG concentrations were less than 0.04, 0.28,
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CHARLESW. LEFFLER,ROBERTC. HANSONand EDWARDG. SCHNEIDER
and 13 ng/ml of PGE2, PGF2~, and PGI2, respectively). Endogenous prostaglandin synthesis does not appear to have significant influence upon the arterial pressure of the normotensive, unanesthetized bullfrog, although prostaglandins, when present, can have profound cardiovascular effects upon the frog. Acknowledgements--We acknowledge the excellent assistance of S. D. Gleason and C. E. Wakelyn. Prostaglandins were a gift from Dr J. E. Pike, The Upjohn Co. Indomethacin was a gift from Dr C. A. Stone, Merck, Sharpe and Dohme. 5,8,11,14-eicosatetraynoic acid was a gift from Dr K. U. Malik, Department of Pharmacology, University of Tennessee Center for the Health Sciences. This study was supported, in part, by a General Research Support Grant from the University of Tennessee Center for the Health Sciences (from USPHS), the Tennessee Heart Association, and USPHS HL 16658.
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glandin releasing effects of E. coil endotoxin in the dog. Br. J. Pharmac. 61, 175-181. FLOWER R. J. (1974) Drugs which inhibit prostaglandin biosynthesis. Pharmac. Rev. 26, 33-67. GERENCSERG. A., TYLER T. & CmSiN S. (1978) Sodium transport by isolated bullfrog small intestine: effect of prostaglandin Et. Biochim. biophys. Acta 509, 159-169. GREEN K. & SAMUELSSONB. (1964) Prostaglandins and related factors: XIX. Thin layer chromatography of prostaglandins. J. Lipid Res. 5, 117-120. LEFFLER C. W. & HESSLER J. R. (1979) Pulmonary and systemic vascular effects of exogenous prostaglandin I2 in fetal lambs. Eur. J. Pharmac. 54, 37--42. LEFFLER C. W., TYLER T. L. & CASSINS. (1978) Effects of indomethacin on cardiovascular hemodynamies of goats in hemorrhagic shock. Circ. Shock 5, 299--310. LEFFLER C. W. & PASSMOREJ. C. (1977) Effects of indomethacin on hemodynamics of dogs in refractory hemorrhagic shock. J. surg. Res. 23, 392-399. LEFFLERC. W., TYLERT. L. & CASSlNS. (1979) Pulmonary and systemic circulatory responses of perinatal goats to prostaglandin F2v Can. J. Physiol. 57, 167-173. McGIFF J. C., CROWSHAWK., TERRAGNON. A. & LONIGROW A. J. (1970) Release of a prostaglandin-like substance into renal venous blood in response to angiotensin--II. Circ. Res. 26, 27 (Suppl. 1) 1121-1130. NAKANOJ. ~; COLE B. (1969) Effects of PGEI and F2, on systemic, pulmonary, and splanchnic circulation in dogs. Am. J. Physiol. 217, 222-226. NOWAL J. & WENNMALMA. (1978) Influence of indomethatin and of prostaglandin E~ on total and regional blood flow in man. Acta. physiol, scand. 102, 484--492. ROSE J. C., JOhnSON M., RAMWELLP. W. & KOT P. A. (1974) Effects of arachidonic acid on systemic arterial pressure, myocardial contractility and platlets in the dog. Proc. Soc. exp. Biol. Med. 147, 652-655. TERRAGNON. A., TERRAGNOD. A. & McGIFF J. C. (1977) Contributions of prostaglandins to the renal circulation in conscious, anesthetized, and laparatomized dogs. Circ. Res. 40, 590-595. TYLERT., LEFFLERC. & CASSINS. (1976) Use of indomethacin to reverse neonatal hypotension. Experientia 32, 61. TYLERT. L., LEFFLERC. W. & CASSINS. (1978) Circulatory responses of perinatal goats to prostaglandin precursors. Prostagland. & Med. 1, 213-220. ZUSMAN R. M., KEISER H. R. & HANDLER J. S. (1977a) Vasopressin-stimulated prostaglandin E biosynthesis in the toad urinary bladder. J. clin. Invest. 60, 1339-1347. ZUSMAN R. M., KEISER H. R. & HANDLER J. S. (1977b) Inhibition of vasopressin stimulated prostaglandin E biosynthesis by chlorpropamide in the toad urinary bladder. J. clin. Invest. 60, 1348-1353.