The role of prostaglandins in the renin, catecholamine, and blood pressure responses to hemorrhage and captopril in conscious rabbits

Prostaglandins Leukotrienes and Medicine 9:

445-457, 1982

THE ROLE OF PROSTAGLANDINS IN TRR RRNIN, CATECHOLAMINR, AND BLOOD PRESSURE RRSPONSES TO RRMORRRAGE MD CONSCIWS

CAPTOPRIL IN

RABBITS

Michael S. Golub, Morris E. Serger,NicolasD. Vlachakist,Peter Eggens, and Mohinder P. Sambhi,HypertensionDivision, Sepulveda VeteransAdministrationMedical Center, UCLA-SanFernandoProgram, UCLA School of Medicine,Sepulveda,California,and tUSC !Mool of Medicine,Los Angeles, California. [Reprintrequaatsto MSG at SepulvedaVAMC, 16111 Plumer St., Sepulveda,California 913431

ABSTkACT The plasma renin activity(Pl?A)increasesand blood pressure changes seen followingmild (6 ml/kg) hemorrhageand angiotensinconverting enzyme inhibition(captopril,1 mg/kg bolus followedby 0.6 mg/kg/hr infueion)were studied in conscious rabbits. Two protocols were followedto evaluate the role of fatty acid cyclooxygenaseproducts and beta-adreuergicreceptors. In the first, the effect of indomethatin (I) (n-8) (4mg/kg/din drinkingwater for five days and 10 xg/kg intravenousbolus followedby 2 mg/kg/h infusionon day of study)was compared to control (C) (n-8) receivingI dilueat. In the second protocol, the combinationof propranolol(P) (5 mg/kg/d in drinkingwater for five days and 0.5 mg/kg bolus followedby 2.4 mg/kg/hr infusion) plus I (n-7) was comparedto P alone (n-7). After oral I, irunoassayableprostaglandinE (PGE) excretionwas 46% less than coutrol (p(O.05). Animals receivingP plus I had 68% (pp
445

administration of I (-7 .O !: 3.1 mm Hg). Thus, in the conscious rabbit, it is suggested that: (1) Renin release induced by hemorrhage has a cyclooxygenase dependent and a cyclooxygenase independent (betaadrenergic) component, (2) the PRA response to captopril is independent of the prostaglandin or beta-adrenergic systems, (3) inhibition of the cyclooxygenase enzyme does not influence plasma catecholamine responses, and (4) the hypotensive response to angiotensin converting enzyme inhibition has a prostaglandin related component.

INTRODUCTION The role of fatty acid cyclooxygenase products in the regulation of renin release has been intensively investigated in recent years. Although it is clear that inhibition of this enzyme system with nonsteroidal anti-inflammatory drugs frequently reduces renin activity, the site of this interaction and its mechanism have not been clarified. The baroreceptor and macula densa sensors have been linked to the prostaglandin system (l-3), but the possibility that sympatheticallymediated renin responses may have a prostaglandin-independent pathway has been suggested by experiments in the dog (4) and in man (6). In the rat (6) and cat (7) renin release induced by sympathetic stimuli Angiotensin II are prevented by prostaglandin synthetase inhibitors. suppresses renin release and inhibitors of its formation or antagonists of its action are associated with renin release. Pros taglandin synthesis inhibitors have decreased renin release associated with this mechanism in the rat (8) and in man (9,lO) but not in the rabbit (11) or dog In considering the influence of the prostaglandin system on (12,13). the possible influence of prostaglandin syntherenin release in vivo, sis inhibitors on the release of norepinephrine and epinephrine should also be considered (14). Accordingly, we have evaluated the effect of prostaglandin synthesis inhibition with indomethacin on the release of renin, epinephrine, and norepinephrine in conscious rabbits in response to mild hemorrhage To study and angiotensin converting enzyme inhibition with captopril. the contribution of a beta-adrenergic stimulus the experiments were performed with and without propranolol treatment. The results indicate that hemorrhage increases renin secretion by prostaglandin dependent inhibition does not and independent mechanisms, that prostaglandin substantially alter the catecholamine response to hemorrhage or converting enzyme inhibition, and that the renin response to captopril is not mediated by the beta-adrenergic or prostaglandin systems in this As indomethacin blunted the blood pressure response to capspecies. a role for cyclooxygenase products in the hypotensive mechanism topril, of converting enzyme inhibition is suggested.

446

MRTEODS Materials: Indomethacin (Merck, Sharpe 6:Dohme Laboratories, Rahwap, N.J.) was freshly prepared each day (25 mg/ml) in equimolar sodium carbonate with maintenance of the pE between 8 and 9 by gradual addition of the buffer. Captopril (Squibb Institute for Medical Research, Princeton, N.J.) and propranolol (Ayerst Laboratories, New York, N.Y.) were reconstituted from powder in sterile water on the day of administration. Measurements: Mean arterial pressure (MAP) and pulse rates were recorded continuously from the ear artery on a multichannel recorder (RSllA Beckman Instruments, Schiller Park, 111) with a pressure transducer (P231D, Statham Instruments Inc., Oxnard, CA). Plasma renln activity (PRA) was measured by radloiuzzunoassayof generated angiotensin I in the presence of anglotensinase inhibitors (15). Urinary prostaglandin E (PGE) was measured by the radioimmunoassay method of Zia et al. (16) on unextracted urine (17). The antibody, generated in rabbits to a PGE2 bovine serum albumin complex (la), has 28% cross reactivity to PGEl and is reported as PGR2 equivalents. The sensitivity of the assay is 100 pgfml of urine and the interassay coefficient of variability is 13%. Plasma epinephrine (II)and norepinephrlne (NE) concentrations were determined in reduced glutathione - EGTA treated plasma samples. The blood was spun in a refrigerated centrifuge within two hours of collection and then frozen. Samples were assayed within two months using the radioenzymatic msthod of Vlachakis and Mendlowitz (19). This method has a sensitivity of 20 pgfml for both noreplnephrine and epinephrine and an interassay coefficient of variation of 2.3X;and 3.2X, respectively. Protocol: Thirty female New Zealand rabbits, 2.5 to 3.5 kg body weight, were maintained on ad libitum water and rabbit chow. Each rabbit was housed singly In a metabolic cage for five days and urine was collected for the last 48 hours of this period. Experiment 1. In this study, rabbits receiving the prostaglandin synthetase inhibitor indomethacin (1) were compared with control (C) animals who received I diluent (titrated to pH 7.0 with glacial acetic acid). Indomethacin (or an equal volume of diluent) was added to the animals drinking water to provide a dose of 4 mg/kg/d for five days. On the sixth morning, the animals were removed from the metabolic cages and placed in adjustable restraining boxes. The animals' ears were taped to a lucite platform. Both central ear arteries and marginal veins were catheterized percutaneously with 22 gauge Teflon catheters following lidocaine nerve blocks. The animals were allowed to acclimate to the restraint and when stable pulse and blood pressure measurements were obtained (approximately l/2 hour) a dose of 10 mg/kg I (or diluent) in 1.0 - 1.5 ml was administered intravenously followed by a sustaining infusion of 2 mgfkgjh (1.0 ml/h). Thirty minutes following the start of the infusion measurements of blood pressure and pulse rate were made and three ml of arterial blood was drawn for PRA, NZ and E determinations. The animals were then hemorrhaged from the ear artery

447

(6 ml/kg) with a withdrawal pump at a rate of 2.3 ml per minute. Thirty minutes following hemorrhage MAP and pulse rate were measured and blood samples for PRA, NE and E determinations were drawn (post hemorrhage). Captopril was then administered as an intravenous bolus dose of 1 mg/kg in 0.75 ml followed by a continuous infusion of 0.6 mg/kg/h (1.0 ml/h). This dose nearly eliminates the pressor response to angiotensin I in our laboratory. Forty-five minutes following the start of captopril (post captopril) the measurements were repeated and the experiment terminated. Experiment 2. In this experiment, the effect of cyclooxygenase inhibition with I was tested in animals receiving the beta-adrenergic blocking drug propranolol (P) . The control group received P in the drinking water at a dose of 5 mg/kg/d. The experimental group received P (5 mg/kg/d) plus I (4 mg/kg/d) for the same period. Urine was collected on the last two days of drug administration. On the sixth day, the animals were studied as in experiment 1 except that each animal also received P as a bolus dose of 0.5 mg/kg in 1 ml followed by a sustaining infusion of 2.4 mg/kg/hr (1:0 ml/h). This dose of P has been shown to markedly block beta-adrenergic responses in the rabbit (20)

??

The results were expressed as the mean +- SE and Statistics. values of P less than 0.05 were considered significant. Comparisons between the two groups in experiments 1 and 2 were performed with Student’s t test. Paired analysis was performed for all comparisons within a group. When comparisons of all four groups were made, analysis of variance was performed and between group comparisons utilized Tukey’s tables (21). RESULTS The urinary excretion of PGE was &creased by oral I (1.00 2 0.23 pg/d) compared to control (1.84 + 0.51 ug/d) by 46% (p
448

Table 1. Plasma CatecholamineResponses Norepinephrlne(pg/ml) Baseline

Post Hemorrhage Post Captopril

Control (n-8)

271 + 29

521 f 81 **

568 + 97

Indomethacin(n=8)

225 + 31

387 +-80 *

679 ?:158

Propranolol(n=7)

390 f 95

492 2 77

642 +_144

Propranolol+ Indomethacin (n=7)

300 + 58

491 + 85

689 + 218

Total (n=30)

293 +-29

472 + 40 **

645 + 77 **

Epinephrine(pg/ml) Control

52 k 10

99 + 38

68 + 26

Indomethacin

63 + 12

79 + 23

170 + 83

Propranolol

44 +_11

67 + 18

273 ? 109

Propranolol+ Indomethacin

66 f 33

75 ? 18

308 ?:177

Total

63 f 11

76 2 11

198 It58 *

* Pp
449

1

24222018161412-

I

IO864-

i-

2-

c Fig.

1.

-

P

P+I

PRA increase over baseline following hemorrhage (6 ml/kg). C = Control, I = Indomethacin, P = Propranolol, P + I = Propranolol plus Indomethacin *p
Table II.

PRA Responses (ng/ml/hr) Post Captopril

Baseline

Post-Hemorrhage

11.9 + 3.4

35.1 f 6.2 *

281.4 + 68.9**

Indomethacin (n=8)

8.2 f 3.6

16.4 + 5.7 t

229.8 ?164.4*

Propranolol (n-7)

16.6 + 6.3

26.8 + 4.9

359.5 + 76.7*

Propranolol + Indomethacin (n=7)

12.0 f 5.6

12.1 + 4.0 tt

350.7 + 118.4*

Control (n=8)

*p
tp<0.05 vs Control ttp
450

Blood pressure did not differ signlf icantly among the four groups at the start of the protocol (Table III). The control animals did not show a significant change in MAP (-2.0 ? 1.1 mm Hg) between the baseline and post-hemorrhage measurements. However, the I-treated rabbits had a modest (-8.1 2 2.4 mm Hg) decrease in blood pressure and the difference between the two groups was significant. In experiment 2, both the P and P + I groups had a fall in MAP (-6.9 + 2.4 and -10.0 2 2.9 mm which were not significantly different from each Hg, respectively) other. The MAP fall induced by captoprll was blunted by I (-7.0 +- 3.1 vs. -16.6 k 3.3 mm Hg, p(O.05) in experiment 1. A smaller and insignificant ef feet of I was seen when it was added to P (-12.5 ?: 2.7 vs. -17.7 + 3.5) (Pig. 2). The two groups receiving I (I and P + I) had a significantly (p
Table III.

MAP Responses

Baseline Control

(n=8)

(mm Hg)

Post Heraorrhage

Post Captopril

80.3 + 1.8

78.3 +- 1.7

61.6 t 3.4 **

Indomethacin (n-8)

84.8 + 1.7

76.6 + 2.7 *

69.6 +-3.7 *

Propranolol (n-7)

79.1 + 2.3

72.3 +-3.1 *

54.6 f 5.2 **

Propranolol + Indomethacin (n=7)

82.3 +- 4.3

72.3 + 2.9 *

61.2 + 2.7

*P
value value

451

0

-C

I

P -

P+I -

I-

- 2- 4- 6-8-

1+

-IO-12 -

I

-14 -I 6-18 -

l-

I

Fig.

2.

MAP changes following captopril (difference hemorrhage and post captopril pressures). In Figure 1. *p
of post Abbreviation5

as

DISCUSSION Renin release from the juxtaglomerulat apparatus is affected by several distinct mechanisms including renal baroreceptors, renal nerves , the macula densa, circulating catecholaminea, and feedback inhfbition by anglotensin II. Several studies have shown that drug5 which inhibit the prostaglandin cyclooxygenaee eneyme can inhibit or prevent renin release related to these mechanisms, but the result5 are not consistent between epeciea or between -in vitro and -In vivo systems. In the non-filtering, denervated dog kidney lntrarenal indomethatin did not block renin release induced by suprarenal aort ic constriction or by administration of isoproterenol (1). Data et al. (2) prepared their dogs with propranolol and adrenalectomy prior to decreasing renal perfusion pressure in the non-filtering denetvated kidney. Under these circumstance5 indomethacin significantly decreased renin release. Thus, it has been suggested that beta-adrenergic stimuli may act independently of the cyclooxygenaee system and that the effect5 of the beta-adrenerglc receptor must be eliminated in order to evaluate the contribution of the proetaglandin syetem. A similar conclusion was reached by Iienrich et al. (3) who studied renin release

452

during hemorrhage in the dog. Rowever, in the rat (6) and cat (7) sympathetic stimuli of renin release can be completely prevented by suggested an cyclooxygenase enzyme inhibitors. --In vivo the8e results effect on cyclic AMP’s ability to stimulate renin release (6) but in (22)7 vitro studies point to a locus prior to cyclic AMP generation In the rabbit, Romero et al. were able to completely suppress the renin response to hemorrhage with prostaglandln synthetase inhibitors The current study reinvestigated the effect of proetaglandin (23) synthesis inhibition with and without beta-adrenergic blockade. The reduction in imunoassayable PGE attests to the effectiveness of the oral I in inhibiting the cyclooxygenase enzyme. Other studies bave shown a similar degree of reduction of urinary prostaglandln E excretion following gastrointestinal administration of lndomethacin (24,25). Thirty minutes after the intravenous administration of indomethacln at 1.5 mg/Rg, renal tissue prostaglandin levels were reduced by 95% (26). Romero et al. (23) reported a greater than 90% reduction in PGE levels in renal tissue six hours after a 9 ng/Rg dose. Thus, our protocol of oral medication followed by an intravenous bolus (10 mg/Rg and a sustaining infusion should have markedly reduced renal prostaglandin synthesis. This, of course, does not ensure that all renal sites were inhibited equally. Actions of the drug other than prostaglandin synthesis inhibition also might have contributed to the observed results. ??

The effect of lndomethacin on hemorrhage-induced increases in plasma renin levels in both our experiments suggests that the prostaglandin-dependent component of this renin release is separable from a beta-adrenergic mechanism. The fact that the combination of indomethacin and propranolol totally prevented a renin response is compatible with the thesis that hemorrhage-induced renin release in the conscious rabbit is composed of two elements, one prostaglandin dependent and one prostaglandin independent (beta-adrenergic). It Is also possible that one of the drugs may have potentlated the action of the other. With regard to the effect of converting enzyme inhibition on renin release, species differences have also been reported. In the rat, the renin activity increases after saralasin, an angiotensin II antagonist, are prevented by prostaglandin synthesis inhibition (8) and indomethatin decreases renin activity after captopril in man (9,lO). In the salt depleted (12) and hemorrhaged (13) dog, no significant effect was seen. The present results show that the vigorous response seen in the rabbit &es not appear to be mediated through either beta-adrenergic or prostaglandln mechanisms. Blackshear et al. (27) showed that reduction of renal blood below cutoregulatory limits will raise renin activity by a prostaglandin-independent mechanism. Rowever, our previous Studies with the same volume of hemorrhage and dose of captopril did not show a decrease in ef feet ive renal blood flow ( 11). Thus, the Pechaniam of the prostaglandin and beta-adrenergic-independent Increase in renin release remain8 speculative.

453

PGE has been shown to impair norepinephrine release from nerve terminals (14). Therefore, inhibition of prostaglandin synthesis might be expected to increase sympathetic activity. However, in man, no effect (28) or decreased circulating norepinephrine levels (29) have been reported following indomethacin treatment. For these reasons, we measured the circulating levels of E and NE during these studies to evaluate their possible contribution to renin activity changes. The results are in accord with those of Henrich et al. (30) who found no effect of I on circulating catecholamines on hemorrhaged dogs. Additionally, Oliver et al. (31) found no effect of I or meclofenamate on renal venous NE after converting enzyme inhibition hypotension or interruption in vena caval blood flow. The changes in renin release induced by I cannot, therefore, be attributed to changes in sympathoadrenal activity. The cardiovascular effects of I are of interest. The first measured pulse rate was significantly lower in the indomethacin treated and the propranolol treated groups. The lowering of the pulse rate with beta-adrenergic blockade is an expected pharmacologic effect but the decrease due to I is unexplained. We noted that such an effect previously (11) and Seymour and Zehr (1) also reported a lower pulse It is possible that oral I resulted in salt and water rate in the dog. retention although the blood pressure was not affected. The similarity of the catecholamine levels in the subgroups does not suggest an effect The fall in blood pressure after mild on sympathetic discharge. anihemorrhage in this group, similar to that seen in the propranolol synthesis inhibition might limit carmals, suggests that prostaglandin In the absence of cardiac output determinations, this diac responses. possibility cannot be directly assessed. Of significant import was the ef feet of I on the captopril-induced that a fall in MAP. The data gives support to the possibility prostaglandin related mechanism underlies part of the hypotensive In the second experiment the effect of I on the action of this drug. blood pressure response to captopril did not reach statistical significance . This may represent variability of this effect or possible interactions of the three medications. A recent report in man showed that I impaired the hypotenslve response to captoprll and simultaneously reduced a circulating proataglandin metabolite (10). In our study, the renin activity levels were somewhat lower in the I-treated animals so there may have been less potential for the angiotenain II Overall, however, the PRA levels at the reducing action of the drug. time of captopril administration were a poor and insignificant predlctor of the blood pressure response. This suggests that mechanisms other than blockade of angiotensin formation, such aa increased proataglandin formation, may be important in the action of angiotensin converting enzyme inhibitors.

454

The authors thank Mr. Keith Rough and Mrs. Susan Negnone for their help. We are also grateful to Mrs. Susanne Fogel for preparing the manuscript. The research was supported by Veterans Administration research funds.

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2.

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3.

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4.

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8.

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9.

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