Angiotensin II-induced Hypertension in the Rat

EFFECTS ON THE PLASMA CONCENTRATION,RENAL EXCRETION,

AND TISSUE RELEASE OF PROSTAGLANDINS

DEBRAI. Diz, PHILIP G. BAER, and ALBERTONASJLETTI, with the technicalassistance of VICTOR FOLARIN and CAROLYNMATTHEWS,Department ofPharmacology, University of Tennessee Center for the Health Sciences,Memphis, Tennessee 38163

A B S T R A C T Weexamined in rats the effects of in-traperitoneal angiotensin II (All) infusion for 12 d onurinary excretion, plasma concentration, and in vitrorelease of prostaglandin (PG) E2 and 6-keto-PGFI,,, aPGI2 metabolite. AII at 200 ng/min increased systolicblood pressure (SBP) progressively from 125±3 to170±9 mmHg(P < 0.01) and elevated fluid intake andurine volume. Urinary 6-keto-PGF,a excretion in-creased from 38±6 to 55±5 and 51±7 ng/d (P < 0.05)on days 8 and 11, respectively, of All infusion, buturinary PGE2 excretion did not change. Relative to acontrol value of 129±12 pg/ml in vehicle-infused (V)rats, arterial plasma 6-keto-PGFi,a concentration in-creased by 133% (P < 0.01) with All infusion. Aorticrings from AII-infused rats released more 6-keto-PGF1, (68±7 ng/mg) during 15-min incubation inKrebs solution than did rings from V rats (40±3 ng/mg); release of PGE2, which was <1% of that of 6-keto-PGFI,a, was also increased. Slices of inner renalmedulla from AII-infused rats released more 6-keto-PGFia (14±1 ng/mg) during incubation than did slicesfrom V rats (8±1 ng/mg, P < 0.05), but PGE2 releasewas not altered. In contrast, AII infusion did not alterrelease of 6-keto-PGFI,, or PGE2 from inferior venacava segments or from renal cortex slices. Infusion ofAII at 125 ng/min also increased SBP, plasma 6-keto-PGFia concentration, and in vitro release of 6-keto-PGFia from rings of aorta and renal inner medullaslices; at 75 ng/min AII had no effect. SBP on AIIinfusion day 11 correlated positively with both 6-keto-PGFia plasma concentration (r = 0.54) and net aortic

Address all correspondence to Dr. Alberto Nasjletti. Dr.Diz' current address is Building 10, Room 3D-48, NationalInstitutes of Mental Health, Bethesda, MD20205.

Received for publication I June 1982 and in revised form6 April 1983.

ring release (r = 0.70) when data from all rats werecombined. We conclude that augmentation of PGI2production is a feature of AII-induced hypertension.The enhancement of PGI2 production may be anexpression of nonspecific alteration in vascular struc-ture and metabolic functions during AII-induced hy-pertension, as well as the result of a specific effect ofthe peptide on the arachidonate-prostaglandin system.

INTRODUCTION

The concept that an interplay between prostaglandins(PG)' and angiotensin II contributes to circulatory ho-meostasis was first advanced in 1970 by McGiff et al.(1), who reported release of a PG-like substance intorenal venous blood in response to arterial angiotensinII infusion. Subsequently, this notion gained strengthwith reports that the vascular effects of angiotensin IIare augmented by prostaglandin synthetase inhibitors(2-4) and attenuated by both PGE2 and PGI2 (5, 6)and that, in conditions featuring activation of therenin-angiotensin system, the administration of pros-taglandin synthetase inhibitors causes renal vasocon-striction that is correlated positively with the level ofplasma renin activity (7). It is now also established thatangiotensin II can promote release of PG from a va-riety of organs, including kidneys (1, 2, 8), lungs (9,10), heart (3, 11), and blood vessels (12, 13), and thatthis action is due to stimulation of PGbiosynthesis (11).However, much of the information on the effects ofangiotensin II on PGbiosynthesis is derived from short-term studies of isolated perfused organs or of anes-thetized animals subjected to surgical stress and maynot, consequently, be applicable to more physiologicalsettings. For example, it is conceivable that the PG

' Abbreviations used in this paper: PG, prostaglandin(s).

466 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738/83/08/0466/12 $1.00Volume 72 August 1983 466-477

response of a tissue or organ to a brief and localizedincrease in angiotensin II varies from the response toa prolonged and systemic increase; in the latter casethe response may vary with time, and be affected bythe many functional and metabolic alterations that areassociated with chronic increases in angiotensin II lev-els. If so, any further consideration of an angiotensinII-PG interplay in long-term regulation of physiolog-ical events requires information on the consequencesof prolonged elevation of angiotensin II levels on thePG system. The present study in conscious rats was,therefore, designed to assess the effects of chronic an-giotensin II administration on the urinary excretion,plasma concentration, and in vitro tissue release ofPGE2 and 6-keto-PGFIa; the latter arises from non-enzymatic hydrolysis of PGI2 (11), and is viewed asan expression of PGI2 levels and production.

METHODSExperiments were performed on 51 male Wistar rats (HarlanSprague Dawley, Inc., Indianapolis, IN) weighing 231-290g; 27 of the rats were housed in individual metabolism cages,and the remainder were maintained in group cages withthree to five rats per cage. The rats were kept in a temper-ature (24°C)- and humidity (50%)-controlled room that wasilluminated between 6:00 a.m. and 6:00 p.m., had free accessto tap water, and were fed ad lib. a standard chow (PurinaNo. 5001, Ralston Purina Co., St. Louis, MO) containingsodium (174 meq/kg) and potassium (282 meq/kg).

Protocol. Studies were performed after a 7-d period ofacclimatization of the rats to the housing, feeding, and drink-ing conditions. Following a subsequent 4-d control period,each animal was anesthetized with ether and an Alzet os-motic minipump (model 2002, Alza Corp., Palo Alto, CA)filled with angiotensin II solution or vehicle was placed inthe abdominal cavity through a 1-cm midline incision, whichwas then closed with autoclips. Isoleucine5-angiotensin II(Sigma Chemical Co., St. Louis, MO) was dissolved to dif-ferent concentrations in 0.01 N acetic acid. Rats serving astime controls received only 0.01 N acetic acid. Assumingthat the angiotensin II did not degrade during the study, andthat the pumps dispensed fluid at the specified rate of 0.49;J/h, the calculated infusion rates of angiotensin II were 75,125, and 200 ng/min, in 9, 4, and 19 rats, respectively.Aguilera et al. (14) reported that intraperitoneal angiotensinII infusion at doses ranging from 50 to 250 ng/min elevatedthe concentration of angiotensin II in blood by about three-to 40-fold after 36 h of infusion.

Systolic blood pressure and body weight were determinedin all rats during the control period and at intervals afterimplantation of the minipumps: in rats housed in individualmetabolism cages, 24-h food and electrolyte consumption,water intake, urine volume, and urinary excretion of sodium,potassium, PGE2, and 6-keto-PGFI. were also determined.Urine was collected for 24-h periods at room temperatureexcept during measurement of PG excretion, in which casethe urine was received in plastic bottles surrounded by dryice to keep the specimens frozen throughout the collectionperiod.

On day 4 or 12 of the infusion period, the rats were anes-thetized with ether and the abdomen was opened with amidline incision. The abdominal aorta was punctured and

a 4-6-ml blood sample was collected into a syringe contain-ing 15% EDTA (100 ju) and indomethacin (100 ug in 10Al ethanol): after centrifugation the plasma was frozen forlater determination of PGconcentration. The thoracic aorta,the supradiaphragmatic inferior vena cava, and the left kid-ney were then excised in some of the rats for estimation ofin vitro release of PG.

PG release studies. The aorta, vena cava, and kidneywere placed in ice-cold Krebs solution containing (mM) NaCl118, KCI 4.7, CaCl2 2.5, KH2PO41.2, MgSO41.17, dextrose8.4, and NaHCO325.0, and rinsed free of visible blood. Thethoracic aorta was freed of adventitial tissue and cut into 2-mmrings; the inferior vena cava was cut into 4-5-mm seg-ments. The kidney was decapsulated and bisected longitu-dinally, the inner medulla was excised and segmented intoslices with a blade, and the cortex was sliced (0.5 mm) witha Stadie-Riggs microtome. The vascular and renal tissueswere placed into 25-ml flasks containing 2 ml of Krebs so-lution and were incubated for 15 min at 370C under anatmosphere of 95% 02-5% CO2, with 100 cycle/min agi-tation. At the termination of the incubation the medium wasfrozen for later PG radioimmunoassay. Tissue specimens ineach incubation flask were dried to constant weight, aver-aging 1.31 mg for the aorta, 2.68 mg for the vena cava, 13.27mg for the renal cortex, and 3.06 mg for the renal innermedulla. Results are expressed as nanograms of immuno-reactive PG released during the 15-min incubation periodper milligram of dry tissue.

Radioimmunoassay of PGE2 and 6-keto-PGFia. Testsamples and radioimmunoassay reagents were diluted with0.1 M phosphate buffer, pH 7.4, containing 0.15 M sodiumchloride, 0.1% sodium azide, and 0.1% gelatin. Reactionmixtures consisting of equal volumes (100 Ml) of tritium-la-beled PGE2 or 6-keto-PGFI. (8,000 dpm; New England Nu-clear, Boston, MA), an appropriate antibody diluted to bind40% of the tracer in the absence of unlabeled ligand, andthe unknown sample or the corresponding PG standard(Upjohn Co., Kalamazoo, MI) were incubated at 4°C for 16h; after addition of dextran-coated charcoal the suspensionwas centrifuged and the radioactivity of the supernate wasdetermined. The PGE2 antibody (Institute Pasteur) had<0.5% cross-reactivity with PGF2a, 6-keto-PGFia, PGD2,and PGA2. The 6-keto-PGFIa antibody was a gift from Dr.Lawrence Levine (Department of Biochemistry, BrandeisUniversity, Waltham, MA) and cross-reacted <0.5% withPGE2, PGF2a, PGD2, and 6-keto-PGEIa. The amount of PGstandard required to produce 50% displacement of radio-labeled PG from antibody-binding sites was 14 pg for PGE2,and 56 pg for 6-keto-PGFIa. PGE2 and 6-keto-PGF1,, in theincubation media from the PG release studies were measuredin unextracted samples diluted with the radioimmunoassaybuffer. Alkali treatment (pH 12 at 100°C for 5 min) of in-cubation media samples did not affect the estimates of 6-keto-PGFIa, but resulted in complete disappearance of thePGE2immunoreactive material. The PGcontent of the urineand plasma was determined after extraction. The displace-ment of the tritium-labeled PGE2 and 6-keto-PGFia fromtheir corresponding antibodies by serially diluted samplesof incubation media or of lipids extracted from plasma orurine paralleled the displacement caused by varying amountsof the appropriate PG standard.

To extract PG from urine the specimen (2 ml) was acid-ified to pH 3.0 with formic acid and passed through a columnof octadecylsilyl silica (Sep Pak C18 cartridges, Waters As-sociates, Milipore Corp., Milford, MA) prewashed withmethanol (2 ml) and then with water (20 ml); the columnwas then eluted successively with water (20 ml), ethanol/

Angiotensin II Effects on Prostaglandins 467

water (15:85, vol/vol, 20 ml), petroleum ether (20 ml), andmethyl formate (10 ml) (15). The methyl formate fractioncontaining the prostanoids was evaporated to dryness undera stream of nitrogen and the dry residue was reconstitutedin toluene/ethyl acetate/methanol (60:40:1, vol/vol) andapplied to a silica column (Sep Pak silica cartridges, WatersAssociates) that had been prewashed with 2 ml of toluene/ethyl acetate/methanol/formic acid (60:40:20:1, vol/vol)and equilibrated with 20 ml of toluene/ethyl acetate (60:40,vol/vol). The column was eluted first with 10 ml of toluene/ethyl acetate (60:40, vol/vol) and then with 12 ml of toluene/ethyl acetate/methanol/formic acid (60:40:5:1, vol/vol); thelatter fraction, containing the prostanoids, was dried undera stream of nitrogen and was reconstituted in the radioim-munoassay buffer. The recovery of 6-keto-[3H]PGFIa addedto each urine sample before purification was 85.4±2.6% (n= 108): the individual 6-keto-PGF1,, recovery values servedto correct the estimates of both 6-keto-PGFIa and PGE2 forlosses incurred during purification, as in preliminary studiesthe recoveries of these PGadded separately to urine did notdiffer from each other. The urinary excretion of PGE2 andof 6-keto-PGFIa was calculated as the product of 24-h urinevolume and urinary PG concentration, and is expressed asnanograms per day. The coefficient of variation of PGassaysin the same urine sample on seven different days was 12.9%for PGE2 and 8.2% for 6-keto-PGFia.

To extract PG from plasma the specimen (2-3 ml) wasacidified to pH 3.0 with formic acid and applied to an oc-tadecylsilyl silica column that was eluted as described abovefor urine samples. The methyl formate fraction containingthe prostanoids was evaporated to dryness under nitrogenand the lipid residue was dissolved in methanol, applied asa band to a 0.25-mm thick silica gel G thin-layer chroma-tography plate (Redi-Plate, 20 X 20 cm, Fisher ScientificCo., Pittsburgh, PA), and chromatographed concurrentlywith authentic prostanoid standards, using as a solvent sys-tem the organic phase of ethyl acetate/isooctane/aceticacid/water (11:5:2:10, vol/vol). Authentic PG were locatedby exposing the plate to iodine vapor. Zones of the platecorresponding to the position of PGE2 and 6-keto-PGFI,,,were scraped off and eluted with methanol. The eluates weredried in nitrogen and the lipid residues were reconstitutedin radioimmunoassay buffer and assayed for PGE2 and 6-keto-PGFIa, respectively. The recovery of [3H]PGE2 and6-keto-[3H]PGFIa added to each plasma sample before pu-rification was 68.3±2.5 and 55.9±3.1%, respectively. Theindividual recovery values served to correct the estimatesof plasma PGE2and 6-keto-PGF1,, for losses incurred duringpurification. The plasma levels of PGE2 and 6-keto-PGF1,,are expressed as picograms per milliliter. The coefficient ofvariation of PGassays on 7 different days in the same sampleof plasma was 14.2% for PGE2and 13.0% for 6-keto-PGFIa.

Radioimmunoassay of 13,14-dihydro-15-keto-PGE2. Theconcentration of this metabolite of PGE2 in plasma wasmeasured in unextracted specimens with an antiserum do-nated by Dr. Lawrence Levine (16); the plasma levels areexpressed in picograms per milliliter. The intraassay coef-ficient of variation was 7.6%.

Systolic blood pressure determination. Systolic bloodpressure was determined by tail sphygmography after warm-ing the rats at 37°C for 10-15 min.

Urinary electrolytes and osmolality determinations. So-dium and potassium concentration in urine was determinedby flame photometry with lithium as the internal standard,and urine osmolality was determined by freezing pointdepression.

Statistical analyses. Results are expressed throughout the

text, tables and figures as mean±SEM. Data were analyzedinitially by one- or two-way analysis of variance, followedby unpaired Student's t test or Newman-Keul's a posterioritest to evaluate differences. The null hypothesis was rejectedwhen the P value was <0.05.

RESULTS

The effect of intraperitoneal infusion of angiotensinII on systolic blood pressure and body weight is shownin Fig. 1. Blood pressure increased progressively withthe administration of angiotensin II at 125 or 200 ng/min, reaching values '50 mmHgabove the prein-fusion level by the 11th d; blood pressure also increasedon the 2nd d of infusion of 75 ng/min, but fell onsubsequent days to values not different from those invehicle-infused rats. Body weight gain was not af-fected by administration of angiotensin II for 11 d at75 ng/min (+50±2 g), but it was reduced in rats re-ceiving 125 ng/min (+13±5 g, P < 0.01) and arrestedin rats receiving 200 ng/min (-12±8 g, P < 0.01),relative to values in vehicle-infused rats (+55±4 g).Failure of the rats infused with angiotensin II at 200ng/min to grow was associated with a reduction in the11-d cumulative food intake (171±11 g, P < 0.01) com-pared with food intake by the vehicle-infused rats(244±5 g); infusion of angiotensin II at 75 ng/d didnot affect cumulative food intake (244±6 g).

Shown in Table I are fluid intake and urine volume,osmolality, and electrolyte values. Coincident with theincrease in blood pressure, animals receiving angio-tensin II at 200 ng/min exhibited a two- to threefoldelevation in urine volume that was associated with asubstantial increase of water intake, and a loweringof urinary osmolality to about one-third of the prein-fusion value. The daily dietary intake of sodium andpotassium was reduced throughout the period of ad-ministration of angiotensin II at 200 ng/min, and theurinary excretion of these electrolytes was either un-changed or decreased relative to values in vehicle-in-fused rats. However, for both sodium and potassiumthe difference between dietary intake and urinary ex-cretion tended to fall in the rats infused with angio-tensin II at 200 ng/min, suggesting reduced retention.Infusion of angiotensin II at 75 ng/min had no effecton any of the above discussed variables.

Fig. 2 depicts the urinary excretion of immuno-reactive PGE2 and 6-keto-PGF1a before and duringadministration of angiotensin II. Relative to values invehicle-infused rats, urinary 6-keto-PGFIa was signif-icantly increased on days 8 and 11 of angiotensin IIinfusion at 200 ng/min. In contrast, the administrationof angiotensin II at this rate did not change the urinaryoutput of PGE2; at an infusion rate of 75 ng/min,angiotensin II did not affect the rates of urinary ex-cretion of either PGE2 or 6-keto-PGFIa.

468 D. I. Diz, P. G. Baer, and A. Nasjletti

200-

LU

Cl)

$ M

cLE0 Eo,~00-im

175-

150'

125-

100

330

I-30 P

a0CX

290-

250

I CONTROL! ANGIOTENSIN II ORVEHICLE INFUSION I*

-4 -2 0 4DAYS

12

DAYS

FIGURE 1 Systolic blood pressure and body weight before and during intraperitoneal infusionof angiotensin II (All) in rats. Results are expressed as means±SE, n = number of rats. Asterisksindicate P < 0.05 relative to corresponding values in vehicle-infused rats. 0: vehicle, n = 14;0: AII 75 ng/min, n = 9; A: AII 125 ng/min, n = 4; *: All 200 ng/min, n = 14.

Plasma PG levels measured in arterial blood on the12th d of infusion are displayed in Fig. 3. The levelof immunoreactive 6-keto-PGFIa increased by -'55%(P < 0.05) and 133% (P < 0.01) with the administrationof angiotensin II for 12 d at 125 and 200 ng/min,respectively, relative to a control value of 129±12 pg/ml in vehicle-infused rats. The plasma level of 6-keto-PGF1, also was higher (P = 0.05) in rats receivingangiotensin II for 4 d at 200 ng/min (152±19 pg/ml;n = 5) than in animals receiving vehicle only (98±14pg/ml; n = 5). Plasma PGE2 concentration was notaffected by angiotensin II infused at 75 or 125 ng/minfor 12 d, but tended to increase the rats infused at 200ng/min (0.01 < P < 0.1). The plasma concentrationof 13,14-dihydro-15-keto-PGE2 in rats infused withangiotensin II for 12 d at 200 ng/min, but not at 75ng/min, was higher (P < 0.05) than the concentrationin animals receiving vehicle only. Linear regressionanalysis of combined data from the vehicle-infusedrats and all three groups of rats infused with angio-tensin II for 12 d revealed a positive correlation be-

tween plasma 6-keto-PGF1, concentration and systolicblood pressure (r = 0.54; P < 0.01; y = -91.7 + 1.92x).

Net release of PG from vascular tissues during in-cubation in Krebs solution for 15 min is illustrated inFig. 4. Release of PGE2 from the aorta and the venacava represented <0.4 and 4.2% of the net release of6-keto-PGFia, respectively. The net release of PG byvena cava segments taken from animals infused withangiotensin II for 12 d did not differ significantly fromthe release by vena cava segments taken from vehicle-inf used controls. In contrast, rings of aorta from ratsinfused with angiotensin II for 12 d at 125 or 200 ng/min released more 6-keto-PGFia and PGE2 than didrings from vehicle-infused rats. However, the releaseof 6-keto-PGFI. by rings of aorta from rats receivingangiotensin II at 200 ng/min for 4 d only (49.4±8.4ng/mg; n = 5) was not statistically different from therelease by rings taken from animals injected with ve-hicle (45.9±6.1 ng/mg; n = 5); also the release of PGE2was not different. When data from all the animals in-fused with angiotensin II or vehicle for 12 d were com-

Angiotensin II Effects on Prostaglandins 469

9I aI 9 II I 5I I

CO -4 OM I- ~DCe - cO - CD16- +l - + CO +1

cq o 0 - 10 t-- +1 - +1 CO +1

0 C6 C1 to t-- +1 - +1 CO +1

a - Go - o tc o i1 cli t- in- +1 - +1 CO +1

+1 - +1 C +1

N CD t'-- C 0OSCoC__ _

+1 +1 CO +1

- +1 - +- CO +1(m Go ;o o

CO5 0i C eq eq t-- +1 - +1 CO +1v oo Co oo ;o u

- +1 - +1 cq +l

0 10 CO 00 CO- +1 - +I ci +1

CDv r - eq 0> 1-

+1 +I- +I

k0 t-V - k0 0

- +1 -4 +1 -4 +1

ooCO - Os - q-c

- +l - +I -i +l

- +l - +l - +l

. .

> <<i-c

O O t- 0 go _CO I- t- t-b 00CD +l 10 +l 1 +1

_ _4

m eq d4 10- o o eqo000t- +1 " - Qc +14 4 +1

sr 001 0n C CO00 CD 00 eq 0 00CD +1 t - sc +l4 4 +1

00N

ozz

oo 0 o0 g t-oo000 0 - I-

t- +IC +I Z +Io - - -

1010._

> < <_) *_4 *_-

e £ £S. O

0-

04

0300)

L 00 t Lo 0

+1 +1 +1

CD t~- co ~v Ceq - de O- eO

+1 +1 +1oo

-o e -o Co a

+1 +1 +1

ol -o 0- 1 0C-- C- Co

+1 +1 +1

CD 1O -00 - 00 eqCD oCD o Co

+1 +1 +1

0o~- C- )0o Oo -om 04

+1 +1 +1

0 00 IV I- eq1 t-

M N CQ - oo

CD0eq00 0

+1 +1 +1

CO eq 101000

L-4 00 q10 -

Co o o1 -C<) CiCOOC O eqO

+1 +1 +1

0

oc 0 eq 00 CO-o4 to C- o

COO0 CO) C'104+1 +1 +1

E-00 0N 00 0010neq 1o0-_ co

+1 +1 +1

CO 00 CO 1 09M 0 00

+1 +1 +1

co t- 000 o 0

4 o 0)C)o- -o

+1 +1 +1

0 0 0S - 0 -

+1 +1 +1

.to .9

- o OOeo _

470 D. I. Diz, P. G. Baer, and A. Nasjletti

I", U0CO +1 C

- eq t-O

l t-

~CC4

t- --00C +1 CO

v 0 L-

C +1 CO

CO +1 o

0 - 0

C +1 co

00 0CO +1 '"

CO +1 e

09 eqCDeq - oCO +1 co

C10C_CO) LO o

06 - ci

C +1 co

eC +1 eq

CO +1 CO

+1 1 +1

eor~+1 C +1

+1 1 +1

CD C o

+l 1 +1

+q10o6t e~

10 +1

+1 10 +1

+1 1 +1

+1 10 +1

+1 10+1

09o 0

+1 o +1+l e~ +l

0- t- eq_1C +1

+l CI +l

._

0

r.

boco

c

0

Lo

0

000

0

0

.

0

0

0

00

.0

0

0.a.

0-

0

0

to

10

CO)

+1

10

+1

kO 0 0CO +1 C +1

CI M o. C4C6 o 'r -4CO +1 CO +1

. *_.110 _

,-, 0e

.CSN

-b

.5c)

0.)

0

IV NeI CO 0_co eq 10 eq --

Cq o Ci o c o+1 +1 +1

10 0000100Z eq-- 00 -U - Ce

ci+° ci +°lc6i +°+1 +1 +1

- IoCO ZDOo _L 00 e

+1 +1 +1

t 1 00 t- 00 0

C4 O Cq O Ci O6+1 +1 +1

010 - 10 CO CON - Co - Co Ce

C4 C6 C- C CO e

+1 +1 +1

CO ~ 10 I Co 10

t--0-10-

Ci O3Ci Oi (6

+1 +1 +1

eq t- 10 eI t- lCo - O - CO eq

+1 +1 +1

eqt- v 00 0 eq

Ci O 0 eq eq4

CO) eq

+1 +1 +1

CO 0 0 -_+1 +1 +1

CD _ 0- _

-0 eq- - eq

+1 +1 +10 0 0

_ O _10 CO- --

+1 +1 +1

0 C t- -_ C

4ci +° 4+° ci +°l<C- - 0 - CO -

+1 +1 +1

CO t-+1 +1 +1

1£C- *_ b

-

eq-

0)

0)

c"

r.

0

a),C.)

0)

._0CU

2

COe -4 X O

-0 0- 1o CO"+1 +1 +1

ci ci c

-q - q-°°eqcs_ Ci -ci

+1 +1 +1

00 0 0s t-I-s - oCOeq

+1 +1 +1

r)- 10t - 10 -COIf- 1

-10 C*,

+l +1 +1

0100 CID 00 10

+1 +1 +1

v 10o v CI t- 0

ci ci ci~- - ~- q ~o e

+1 +1 +1

00 CO 00 t- t- coeq eq eq - o eq

0 ci 6 c]

+1 +1 +1

CO 0 C10 CO

c ci 6tc

+1 +1 +1

to Co co t- -00

o - 0 C-

-4 6 6o. ci

+1 +1 +1

0 0 o00 00000 Ce 00 - Cq

+1 +1 +1

- 010 Cq C C0CO - - +q - +°l

+1 +1 +1

0 O 00 0o0 10C'O 0 -4 -10 -4

+1 +1 +H-eq o_-__ - _Co- o

+1 +1 +1

. .

> <0 <C

-

eqs

0-

0)

CU._X2r._

CU

0o

0s Ib 10 0x 1tF Co b e 00 CDCS6 Oi CD6 O t O

+1 +1 +1

eq Co C - Co eqeo~eq C00 CO 0 IC-

0'-46 064+1 +1 +1

cq CO oo CD q) t-O - _0 -eq CO C4

+1 +1 +1

C) - CO eq C>- kko CO 00 CIe 0D C

+1 +1 +1

001 eq ~ 0

+1 +1 +1

eq

t 0 e100 CoO+1 +1 +1

q 10 0t- r-

6_6c i4r

+1 +1 +10 CO 0 COCZO

Co -D t- eq eq t-

+1 +1 +l00 C-D V 0 r

- eq O-iO C CO

C- -° cc -° t- -°

+1 +1 +1

0 CO eq : CD

Co eqCOv -

O6 CikCi _C66

+1 +1 +1O I" NC140

OCD 05 CD CDC O 1 0CN O

+l +l +l

10 CDC

. .q. C+l +l +lCD +° C+l CD +°

_ C6 cU CD C

+l +l +l

t- tw §

-C

S

04

2'

o0

4._

CL)S._

UL)

co

+l +l +l

00 o0- eq 0101o U)-

U0 eq eq - eq

Co 0CO 0 CO0+1 +1 +1

001li0 C o 10

6+° 6+°l ci °

- ceq eq eq eCD0 Co) N- 0C13 Oi C O C6 O

+1 +1 +1

0010 - eq-o

0 - eq 00 eqO4Ci

cl0:1C+H +1 +1

c:s 0s sr t- _00C _ COD -_ _

+1 +1 +1

cO eq Ci O

+1 +1 +1

Co0 e q0

C6Cs O O

C6CO

+1 +1 +1+1 +1 +1

0 00 CO-0 CD

C--000eq6Co-6+1 +1 +1

10> 00 0 0

0 -q - eq -

C6 +°l C6 +° i +°l

+1 +1 +1

0 00CD t O o0 Cq 00 C 0 _

Ci Oi cl O C4 O

+l +1 +1

O 0 1¢C ~ C)coOCOO >O

+l +l +l

. .

10 -gN -00 Oe _

0 C6cDC Co Oi

+l +l +l

t?O ) Dq

0-

0)

--~

0)Co

e

0L)c0

".0)I..

L)U.-

r.0)CU

CD

0.b

m I" 0 ) co0- C C cOC r COei .i _;..

+1 +1 +1

+1 +1 +1

10 CO eq 10 COD

eqCO- Co1 Co

c-1 sr cs t c

+1 +1 +1eq _ q0 -0

v o CD-o oo

+1 +1 +1

kO 010C,1 04Ci C- -i eq+1 +1 +1

CD 0 eko e

IV _- U: i e CiC. .4

+1 +1 +1

i+lc-i6 +° - °

10 0 000m

cq CO-04 F c

eC4 -eqCOeq+1 +1 4-I

C-i 0-C-O _

+l +l +l

CS O C4 kO _ O

+1 +1 +1Ci CO co Ci Cqeq

+1 +1 +1eq COe - b

co-0 -00 0

C4 C4_i Ci

+1 +1 +1

o- Co Co co 0-C i -O COCO

+1 +1 +l

C- 000Co C-- O-

CX 0CQ V0

+1+1 +1

-°- °l °q +°

>i +° <° °

Angiotensin II Effects on Prostaglandins 471

coCo

0)

cu

cU

co.e

co

6

CU

cis

0

S.

9

V

Cl

b0)

5

0.

Q

E

0

"0

._

G)o

>)0)

CU

"0

0)

U) U

CUZ

CU

2~0

-

0-

ol

0.0IL)

xC.)

0)'S

," 60

1 IV

P CM 30c

150]

100

gcs_ _ _

50-

-4

* *

1 2 DaysCONTRALIITENSIN RVEHICLEINFUSI N

FIGURE 2 Urinary excretion of immunoreactive 6-keto-PGFIa and PGE2 before and duringintraperitoneal infusion of angiotensin II (AII) in rats. Results are expressed as means±SE, n= number of rats. Asterisks indicate P < 0.05 relative to corresponding values in vehicle-infusedrats. l: vehicle, n = 9; 0: AII 75 ng/min, n = 9; *: AII 200 ng/min, n = 9.

6-KETO-PGFia

r-

I Iz

*

PGE2 13, 14-DIHYDRO-1 5-KETO-PGE2

11z

*m

I I

z

I Iz

I Iz

L-0

0111z

0

ANGIOTENSIN II (ng/min)

FIGURE 3 Immunoreactive 6-keto-PGFIa, PGE2, and 13,14-dihydro-15-keto-PGE2 concentra-tions in arterial plasma on the 12th d of angiotensin II intraperitoneal infusion. Results areexpressed as means±SE, n = number of rats. Asterisks indicate P < 0.05 relative to values invehicle-infused rats (open columns).

472 D. I. Diz, P. G. Baer, and A. Nasjletti

400 -

Eco

0.z

z

4I-000.4

Cl

200-

O-

I

VENACAVA

801

40a 0,~ E0 -

D-I

II11z

*

*0.80

lLl Eg -- 0.40

tx

c

0

ANGIOTENSIN 11(ng/min)

FIGURE 4 Net release of immunoreactive 6-keto-PGF1. and PGE2 from segments of thoracicaorta and inferior vena cava during incubation in Krebs solution for 15 min at 37°C. Resultsare expressed as means±SE, n = number of rats. Asterisks indicate P < 0.05 relative to valuesin vehicle-infused rats (open columns).

bined, as shown in Fig. 5, the net release of 6-keto-PGF1, from rings of aorta correlated positively withblood pressure; PGE2 release and blood pressure were

similarly correlated. However, the release of PG fromrings of aorta did not correlate significantly with bloodpressure in rats receiving angiotensin II at 200 ng/min,or vehicle, for 4 d only (for 6-keto-PGF,a r = 0.21; forPGE2 r = 0.24).

Shown in Table II is the net release of PG fromrenal tissues during incubation in Krebs solution for15 min. Slices of inner medulla from rats infused withangiotensin II at 125 or 200 ng/min for 12 d releasedmore 6-keto-PGFia than did slices from vehicle-in-fused rats. In contrast, the net release of PGE2 frominner medulla slices was not affected. Similarly, neitherPGE2nor 6-keto-PGFia release from renal cortex slicesobtained from angiotensin II-inf used rats differed sig-nificantly from corresponding values for slices fromvehicle-infused rats.

DISCUSSION

PGE2 in the urine arises within the kidney (17) andits rate of excretion is a function of renal PGE2 syn-

thesis (18). In the present study a chronic infusion ofangiotensin II did not affect either urinary PGE2 ex-

cretion or net release of PGE2 from renal tissue invitro, suggesting that the renal production of PGE2didnot increase. These findings appear to be at variancewith reports that angiotensin II promotes renal PGE2synthesis and release. However, all such prior reportshave been restricted to the effects on PG synthesis ofshort-term angiotensin II administration and the re-

sults, therefore, can not be considered to conflict di-rectly with those of our present chronic infusion study.Further, data contained in two early reports (1, 8) ofthe effect of close renal arterial infusion of angiotensinII on renal PGshow that renal PGE2 release was aug-

mented initially, but that the effect was not sustained.Rather, the angiotensin II infusion was shown to in-duce prompt rises in urinary PGE2excretion and renalvenous blood PGE2concentration, and both tended toreturn to or toward control levels after several minutesof continued infusion.

Contrasting with the lack of effect of chronic an-

giotensin II infusion on urinary PGE2 excretion, theurinary output of 6-keto-PGFia increased during an-

Angiotensin II Effects on Prostaglandins 473

II

z

*

SnIIz

II11z

If)11z

*

0

AORTA

150-

_ X

30O- E

~e -

100-

50-

A a

0

00

00

* 0

0

100

1.5

1.0-

EC.d

0.5-

0

100

0 00

00

00

0

0

a*A

0 A*

r=0.70p<O.01y =-24.9 + 0.50X

150 200BLOODPRESSURE(mmHg)

250

-AO

A

00

0 0

0g*088oo-

a a.

0

* 0r=0.64p<O.O1y =-0.71 + 0.007X

A A

150 200BLOODPRESSURE(mmHg)

250

FIGURE 5 Relationship between the systolic blood pressure measured on the 11th d of angio-tensin II (All) or vehicle infusion and the arterial plasma concentrations of immunoreactive6-keto-PGFIs,, and PGE2 measured on the 12th d of infusion. 0: vehicle; 0: All 75 ng/min;A: AII 125 ng/min; *: All 200 ng/min.

giotensin II administration. Urinary 6-keto-PGFia mayarise both from glomerular ultrafiltration of plasma(19) and from several renal cell types (20, 21). There-fore, the augmentation of urinary 6-keto-PGF,a ex-cretion may have resulted from both stimulation ofrenal PGI2 synthesis and elevation of the plasma con-centration of PGI2 and/or 6-keto-PGFia. In this regardwe found that slices of inner medulla from rats infusedwith angiotensin II for 12 d released more 6-keto-

PGFI,a than did slices from vehicle-infused rats. Wealso found that the arterial plasma concentration of6-keto-PGFia increased with administration of angio-tensin II.

That chronic angiotensin II infusion raised theplasma level of 6-keto-PGF1,a in rats is consistent withreports that the blood concentration of a PGI2-like sub-stance increased during short-term angiotensin II in-fusion in dogs (10, 22, 23); the increase in blood PGI2

474 D. I. Diz, P. G. Baer, and A. Nasjletti

9 5-

* w

A

A

A

A

TABLE IINet Release of 6-Keto-PGFI,, and PGE2from Slices of Renal Cortex and Inner

Medulla Incubated in Krebs Solution for 15 min

6-Keto-PGFI. PGE,

Cortex Medulla Cortex Medulla

ng/mg

Vehicle (n = 5) 0.05±0.01 7.92±1.09 0.17±0.02 91.97±7.08AII, 125 ng/min (n = 4) 0.06±0.01 18.36±2.49' 0.14±0.02 67.52±13.29AII, 200 ng/min (n = 5) 0.06±0.01 13.53±1.46" 0.23±0.02 76.59±15.93

Values are means±SE, n = number of observations, the asterisk denotes significantdifference from value used in vehicle-infused rats (P < 0.05); angiotensin II (All) orvehicle were infused for 12 d before the study.

in dogs was attributed to stimulation of PGI2 synthesisin the lungs (10, 22) and kidneys (22, 23). The plasmalevel of 13,14-dihydro-15-keto-PGE2 increased withadministration of angiotensin II suggesting stimulationof PGE2 tissue production. Metabolism of PGE2 to13,14-dihydro-15-keto-PGE2, which occurs readily inthe lung and in other tissues, may have prevented thearterial plasma concentration of PGE2from rising dur-ing angiotensin infusion (24).

In this present study we found that rings of aortafrom rats infused with angiotensin II for 12 d releasedmore 6-keto-PGFia than did rings from vehicle-in-fused rats, suggesting augmentation of arterial vas-cular PGI2 synthesis in rats receiving angiotensin II.However, PGI2 synthesis by the aorta need not be rep-resentative of that by other vascular segments, in par-ticular the microvasculature. Although rings of aortafrom angiotensin II-infused rats also released morePGE2 than did rings from vehicle-infused rats, itshould be noted that the amount of PGE2released was<1% of the amount of 6-keto-PGFia released.

Overall, our data on the urinary excretion, plasmaconcentration, and in vitro tissue release of 6-keto-PGF1,a suggest that augmentation of renal and extra-renal PGI2 production and, presumably, plasma PGI2concentration is a feature of the response of the intactrat to chronic infusion of angiotensin II. Informationderived from acute studies indicates that the action ofangiotensin II to increase PG synthesis relates to stim-ulation of phospholipid deacylation (25), which in-creases the free arachidonic acid available to cycloox-ygenase and, consequently, the formation of endo-peroxide intermediates and PG. Induction of PGsynthesis by angiotensin is characterized by rapidityof onset and reversibility after removal of the stimulus(25). Therefore, it is intriguing that in the presentstudy angiotensin II did not increase the urinary ex-cretion of 6-keto-PGFsa or the release of 6-keto-PGFs,from rings of aorta until after a lag period of several

days. Furthermore, under in vitro conditions that didnot preserve a critical feature of the in vivo environ-ment, i.e., high angiotensin II levels, rings of aorta andrenal medullary tissue obtained from rats infused withangiotensin II for 12 d still released more 6-keto-PGFsathan did corresponding tissues from vehicle-infusedrats. These observations require consideration of thepossibility that during chronic angiotensin II infusionthe enhancement of urinary excretion and in vitro tis-sue release of 6-keto-PGFia relates to one or moreslowly developing and persistent alterations in func-tion and/or tissue structure rather than to a direct,immediate, and rapidly reversible action of angioten-sin II on the phospholipid-arachidonate-prostaglandinsystem. In this respect, we found that angiotensin IIcaused a progressive elevation of blood pressure, thefinal level of which correlated positively on the 12thbut not on the 4th d of infusion with the net releaseof 6-keto-PGFIa from aortic rings in vitro. Severalother investigators have also noted an association be-tween high blood pressure and increased vascular PGsynthesis (26, 27). Similarly, various features of renalPG metabolism have been found to be abnormal inother animal models of hypertension (28-30). In ourstudy, angiotensin II also produced substantial alter-ations in renal function, caloric intake, and electrolytebalance that conceivably could have contributed to theoverall effect of the peptide on PG production. Forexample, there are reports that the renal synthesis ofPGE2 is affected by changes in the intake of sodium(31) and potassium (32), and that the vascular synthesisof PGI2 depends on extracellular potassium concen-tration (33).

That chronic infusion of angiotensin II in the ratresulted in sustained hypertension confirms a recentreport (34). The tendency for the difference betweensodium intake and urinary sodium excretion to fallduring angiotensin administration at 200 ng/min sug-gests that renal sodium retention was reduced; this,

Angiotensin II Effects on Prostaglandins 475

and the associated rise in urine volume, may be relatedto pressure-induced diuresis and natriuresis.

This study was not designed to assess the functionalsignificance of the increase in PG associated with an-giotensin II-induced hypertension. However, it is wellrecognized that several PG are capable of affectingvascular and renal mechanisms involved in blood pres-sure regulation (35). For example, both PGI2 and PGE2dilate resistance blood vessels and attenuate vascularreactivity to angiotensin II and other pressor stimuli(6). It follows, then, that increased PGproduction dur-ing chronic angiotensin II infusion may be part of ahomeostatic response that acts to minimize the angio-tensin II-induced blood pressure elevation.

In conclusion, our study shows that chronic angio-tensin II infusion in rats produces a progressive in-crease in blood pressure associated with augmentationof plasma concentration, urinary excretion, and net invitro release of 6-keto-PGFia from rings of aorta andslices of renal inner medulla. We found no evidenceof increased renal PGE2production during angiotensinII infusion. On the basis of these findings, we concludethat augmentation of PGI2 production is a feature ofthe response to angiotensin II-induced hypertension.

ACKNOWLEDGMENTSThis work was supported by U.S. Public Health Servicegrants HL-23548 and HL-18579. Dr. Philip G. Baer is therecipient of National Institutes of Health Career ResearchDevelopment Award 1 K04 HL-00806.

REFERENCES1. McGiff, J. C., K. Crowshaw, N. A. Terragno, and A. J.

Lonigro. 1970. Release of a prostaglandin-like substanceinto renal venous blood in response to angiotensin II.Circ. Res. 26-27(Suppl. 1):I-121-I-130.

2. Aiken, J. W., and J. R. Vane. 1973. Intrarenal prosta-glandin release attenuates the renal vasoconstrictor ac-tivity of angiotensin II. J. Pharmacol. Exp. Ther.184:678-687.

3. Gunther, S., and P. J. Cannon. 1980. Modulation of an-giotensin II coronary vasoconstriction by cardiac pros-taglandin synthesis. Am. J. Physiol. 238:895-901.

4. Negus, P., R. L. Tannen, and M. J. Dunn. 1976. Indo-methacin potentiates the vasoconstrictor actions of an-giotensin II in the normal man. Prostaglandins. 12:175-180.

5. Lonigro, A. J., N. A. Terragno, K. U. Malik. and J. C.McGiff. 1973. Differential inhibition by prostaglandinsof the renal actions of pressor stimuli. Prostaglandins.3:595-606.

6. Susic, H., and K. U. Malik. 1981. Attenuation by ara-chidonic acid of the effect of vasoconstrictor stimuli inthe canine kidney. J. Pharmacol. Exp. Ther. 219:377-382.

7. Blasingham, M. C., and A. Nasjletti. 1980. Differentialrenal effects of cyclooxygenase inhibition in sodium-re-

plete and sodium-deprived dog. Am. J. Physiol.239:F360-F365.

8. Dunn, M. J., J. F. Liard, and F. Dray. 1978. Basal andstimulated rates of renal secretion and excretion of pros-taglandins E2, Fa, and 13,14-dyhydro-15-keto F, in thedog. Kidney Int. 13:136-143.

9. Gryglewski, R. J. 1980. Prostacyclin as a circulatory hor-mone. Biochem. Pharmacol. 28:3161-3166.

10. Dusting, G. J. 1981. Angiotensin-induced release of aprostacyclin-like substance from the lungs. J.Cardiovasc.Pharmacol. 3:197-206.

11. Needleman, P., S. D. Bronson, A. Wyche, M. Sivakoff,and K. C. Nicolaou. 1978. Cardiac and renal prosta-glandin 12. Biosynthesis and biological effects in isolatedperfused rabbit tissues. J. Clin. Invest. 61:839-849.

12. Blumberg, A. L., S. E. Denny, G. R. Marshall, and P.Needleman. 1977. Blood vessel-hormone interactions:angiotensin, bradykinin, and prostaglandins. Am. J. Phy-siol. 232:H306-H310.

13. Dusting, G. J., and E. M. Mullins. 1980. Stimulation byangiotensin of prostacyclin biosynthesis in rats and dogs.Clin. Exp. Pharmacol. Physiol. 7:545-550.

14. Aguilera, G., A. Schirar, A. Baukal, and K. J. Catt. 1980.Angiotensin II receptors. Properties and regulation inadrenal glomerulosa cells. Circ. Res. 46(Suppl. 1):I-118-I-127.

15. Powell, W. S. 1980. Rapid extraction of oxygenatedmetabolites of arachidonic acid from biological samplesusing octadecylsilyl silica. Prostaglandins. 20:947-957.

16. Levine, L. 1977. Levels of 13,14-dihydro-15-keto-PGE2in some biological fluids as measured by radioimmu-noassay. Prostaglandins. 14:1125-1139.

17. Williams, W. M., J. Frolich, A. S. Nies, and J. A. Oates.1977. Urinary prostaglandins: site of entry into renaltubular fluid. Kidney Int. 11:256-269.

18. Frolich, J. C., I. W. Wilxon, B. J. Sweetman, M. Smigel,A. S. Nies, K. Carr, J. T. Watson, and J. A. Oates. 1975.Urinary prostaglandins: identification and origin. J.Clin. Invest. 55:763-770.

19. Rosenkranz, B., W. Kitajima, and J. C. Frolich. 1981.Relevance of urinary 6-keto-prostaglandin F,, deter-mination. Kidney Int. 19:755-759.

20. Sraer, J., J. Foidart, D. Chansel, P. Mahieu, and R. Ar-daillou. 1980. Prostaglandin synthesis by rat isolatedglomeruli and glomerular cultures cells. Int. J. Biochem.12:203-207.

21. Grenier, F. C., and W. L. Smith. 1978. Formation of 6-keto-PGFIa,, by collecting tubule cells isolated from rab-bit renal papillae. Prostaglandins. 16:759-772.

22. Shebuski, R. J., and J. W. Aiken. 1980. Angiotensin IIstimulation of renal prostaglandin synthesis elevates cir-culating prostacyclin in the dog. J. Cardiovasc. Phar-macol. 2:667-677.

23. Mullane, K. M., and S. Moncada. 1980. Prostacyclin re-lease and the modulation of some vasoactive hormones.Prostaglandins. 20:25-49.

24. Anggard, E., C. Larsson, and B. Samuelsson. 1971. Thedistribution of 15-hydroxy prostaglandin dehydrogenaseand prostaglandin-13-reductase in tissues of the swine.Acta Physiol. Scand. 81:396-404.

25. Schwartzman, M., and A. Raz. 1980. Biochemical actionsof vasoactive peptide hormones. Biochem. J. 192:127-131.

26. Pace-Asciak, C. R., M. C. Carrara, G. Rangaraj, andK. C. Nicolaou. 1978. Enhanced formation of PGI2, apotent hypotensive substance, by aortic rings and ho-

476 D. I. Diz, P. C. Baer, and A. Nasjletti

mogenates of the spontaneously hypertensive rat. Pros-taglandins. 15:1005-1012.

27. Rioux, F., R. Quiron, and D. Regoli. 1977. The role ofprostaglandins in hypertension. I. The release of pros-taglandins by aorta strips of renal, DOCA-salt, and spon-taneously hypertensive rats. Can. J. Physiol. Pharmacol.55:1330-1338.

28. Dunn, M. J. 1976. Renal prostaglandin synthesis in thespontaneously hypertensive rat. J. Clin. Invest. 58:862-870.

29. Pugsley, D. J., L. J. Beilin, and R. Peto. 1975. Renalprostaglandin synthesis in the Goldblatt hypertensiverat. Circ. Res. 36(Suppl 1):I-81-I-88.

30. Limas, C., and C. J. Limas. 1979. Enhanced renome-dullary prostaglandin synthesis in spontaneously hyper-tensive rats: role of a phospholipase A2. Am. J. Physiol.236:H65-H72.

31. Chaumet-Riffaud, P., J. P. Oudinet, J. Sraer, C. Lajotte,

and R. Ardaillou. 1981. Altered PGE2 and PGF2a pro-duction by glomeruli and papilla of sodium-depleted andsodium-loaded rats. Am. J. Physiol. 241:F517-F524.

32. Beck, A., and J. 0. Shaw. 1981. Thromboxane B2 andprostaglandin E2 in the K+-depleted rat kidney. Am. J.Physiol. 240:F151-F157.

33. Dusing, R., R. Scherhag, R. Tippelmann, U. Budde, K.Glanzer, and H. J. Kramer. 1982. Arachidonic acid me-tabolism in isolated rat aorta. J. Biol. Chem. 257:1993-1996.

34. Textor, S. C., H. R. Brunner, and H. Gavras. 1981. Con-verting enzyme inhibition during chronic angiotensin IIinfusion in rats. Hypertension. 3:269-276.

35. Nasjletti, A., and K. U. Malik. 1982. Interrelations be-tween prostaglandins and vasoconstrictor hormones:contribution to blood pressure regulation. Fed Proc.41:2394-2399.

Angiotensin II Effects on Prostaglandins 477