Studies on the Role of Angiotensin in Experimental

Renovascular Hypertension: an Immunologic Approach

A. R. CHEISTLIEB, T. U. L. BiBER, and R. B. HIcKER

From the Hypertension Unit and the Department of Medicine, Peter BentBrigham Hospital, and Harvard Medical School,Boston, Massachusetts 02115

A B S T R A C T The role of angiotensin in three formsof experimental hypertension was assessed in rats. First,the acute blood pressure response to injected angiotensinamide and angiotensin acid was determined. Rats madehypertensive with deoxycorticosterone and salineshowed exaggerated responses; rats made hypertensiveby clipping one renal artery showed depressed responses;and rats made hypertensive by clipping one renal arteryand contralateral nephrectomy showed normal respon-sivity to angiotensin amide but depressed responsivityto angiotensin acid. These findings suggested that dif-ferent mechanisms may be involved in the three typesof hypertension studied.

To assess the role of angiotensin in these hypertensiverats the blood pressure response, the presence of anti-bodies determined by radioimmune techniques, and thedegree of refractoriness to injected angiotensin after im-munization with angiotensin were studied. None of sixrats made hypertensive by deoxycorticosterone and sa-line, and none of five mock immunized rats with renalhypertension of both types had a fall in blood pressure.By contrast, of the 20 rats with both types of renalhypertension in which antibody determinations weremade, 11 had developed a significant antibody titer, ofwhich seven showed a significant reduction in bloodpressure at the time of antibody determination, and threeof the remaining four had a significant blood pressurereduction earlier in their course. None of the nine renalhypertensive rats without demonstrable antibodies hada reduced blood pressure at the time of antibody de-termination, and only one had an earlier reduction in

This work was presented in part at the 60th Annual Meet-ing of the American Society for Clinical Investigation, At-lantic City, May 1968 and abstracted in 1968 J. Clin. Invest.49: 19a, and in 1967 Clin. Res. 15: 198.

Dr. R. B. Hickler is an Investigator of the HowardHughes Medical Institute.

Received for publication 31 December 1968 and in revisedform 28 March 1969.

blood pressure. The renal hypertensive rats were all re-fractory to injected angiotensin after immunization.

These results suggest a primary role for angiotensinin both forms of renal hypertension.

INTRODUCTIONEvidence for an association between the renin-angio-tensin system and the hypertension seen after constric-tion of a renal artery has been accumulating since 1898(1-7). The most direct evidence for this associationhas been the production of antirenin, which results inthe neutralization of the pressor effect of injected reninin the assay dog and which is associated with a lower-ing of the arterial pressure in renal hypertensive dogs,rats, and monkeys (8-13). However, nonspecificity ofthis response for renal hypertension is suggested bystudies showing a reduction in the blood pressure ofspontaneously hypertensive and pyelonephritic hyper-tensive dogs with the production of antirenin (14).

The production of hypertension and hypertensive vas-cular disease after injections of homologous crude renininto uninephrectomized rats provides further evidencethat the renin-angiotensin system may be instrumentalin producing renal hypertension (15-16). Recently, theblood pressure of rats with acute and chronic renalhypertension has been reduced by a renin-inhibitingphospholipid isolated from kidneys (17).

Although angiotensin has been thought to be the ulti-mate mediator of renovascular hypertension, definiteevidence for its role in this disease has not been estab-lished. With the advent of conjugates of angiotensin ca-pable of producing angiotensin antibodies (18-23) whichare biologically effective (24), an immunologic ap-proach to the role of angiotensin in the pathogenesisof renovascular hypertension seemed feasible.

This work is divided into two parts. Part I assessesendogenous angiotensin production in experimentally

1506 The Journal of Clinical Investigation Volume 48 1969

Rat Albumin 5 mg Asparginyl V0. 25 ml dist. H20 Angiotensin ]

(1) (2)

falineno 1-Cyclohexyl- 3-II 10 mg Carbodiimide 75 mg

0. 125 ml dist. H20

(3)

40 minutes room temp

Dialyze against distilled water at 5'C for 14 hours

Mix with equal volume Freund's Adjuvant

FIGURE 1 Preparation of the angiotensin antigen.

hypertensive rats indirectly by recording the bloodpressure sensitivity to injected angiotensin, and Part IIassesses the acute and chronic blood pressure responseto active immunization against angiotensin in these rats.

METHODSPart I. Four groups of male albino Sprague-Dawley rats

weighing 250-350 g were studied. Group I consisted ofeight normal rats; Group II of seven rats made hyper-tensive by unilateral nephrectomy, deoxycorticosteroneacetate (DOCA) 5 mg intramuscularly three times/wk,and fluid intake as 0.8%o saline throughout the period ofstudy (DOCA rats); Group III of seven rats made hyper-tensive by placing a constricting silver clip on the left renalartery together with contralateral nephrectomy (GN rats);and Group IV of 12 rats made hypertensive by placing aconstricting silver clip on the left renal artery (G rats).After blood pressures of 170-260 were recorded in GroupsII, III, and IV by the indirect caudal method (CaudalPlethysmograph System, Model 320-1, Decker Corp., Bala-Cynwyd, Pa.), each rat was anesthetized with sodium pen-tobarbital 30 mg/kg intraperitoneally which lowered themean blood pressure to 40-120 mmHg. The right carotidartery was canulated with a polyethylene catheter for di-rect recording of the blood pressure on a Bird Kymograph(Catalogue #70-060, Phipps & Bird, Richmond, Va.) fittedwith a Palmer mercury manometer (Palmer Instruments,Inc., Cincinnati, Ohio). The left jugular vein was similarlycatheterized for pharmacologic injections using Micro Syringe-Burettes (California Laboratory Equipment Co., Berkeley,Calif.) capable of delivering accurately as little as 0.0001ml.

The pressor response to synthetic a-L-asparaginyl1-valyl'angiotensin II (angiotensin amide) and to norepinephrinewere compared in each of the 34 rats studied. In 30 of theserats, the pressor response to a-L-aspartyl 1-valyl 6 angiotensinII (angiotensin acid) was also compared with that to nor-epinephrine. The dose of norepinephrine that would give apressor response of 16 mmHg or less (the comparativelylinear range of the dose response curve) was determined in

each rat. After the blood pressure had returned to baseline levels, the dose of angiotensin (amide or acid) thatwould give an identical rise in arterial pressure was found.Repeated responses were determined with each drug to as-sure reproductibility of the results. From these measure-ments the dosage ratio norepinephrine/angiotensin givingequipressor responses was calculated as an index of the rela-tive responsivity to these agents. The dosage range for nor-epinephrine was 10-20 mug, for angiotensin amide 2-4 mpg,and for angiotensin acid 0.8-10.5 mujg. The maximum fluidvolume delivered was 0.04 ml.

26 of the rats were sacrificed after this procedure. Afterligating the right carotid artery and the left jugular vein, theremaining 10 rats were saved for the studies described inPart II.

Part II. Four groups of rats indentical with those inPart I were studied. Ten normal rats, six DOCArats,eight GN rats, and fourteen G rats received immunizationinjections with angiotensin chemically linked to a carrierprotein. Two DOCArats, three GN rats, and two G ratsreceived mock immunization injections. The antigen wasprepared according to the method of Goodfriend, Levine, andFasman (20) as modified by Oken and Biber (24) (Fig. 1).To 5 mg of rat albumin (Pentex, Inc., Kankakee, Ill.) in0.25 ml of distilled water were dissolved 10 mg of syntheticasparginyl valine5 angiotensin II (Ciba Pharmaceutical Co.,Summit, N. J.). 75 mg of 1-cyclohexyl-3 (2 morpholino-ethyl) carbodiimide (Aldrich Chemical Co., Inc., Milwaukee,Wis.) were dissolved in 0.125 ml of distilled water. Aftermixing the two solutions and allowing them to stand atroom temperature for 40 min to permit conjugation of thereactants to albumin, the mixture was dialyzed against 800ml distilled water at 50C for 14 hr. This antigen was thenmixed thoroughly with an equal volume of Freund's adju-vant. The injectant for the mock immunized rats was pre-pared identically except that no angiotensin was added.

Each rat was injected three to nine times with 0.03 ml ofthe mixture into the footpad at intervals of 7 days or more.In 16 rats a series of three or four immunizations were fol-lowed by a period of 30-100 days during which time noinjections were given. Reimmunization was then undertaken

Angiotensin in Experimental Hypertension, an Immunologic Approach

- - cf

1507

to study possible booster responses. Blood pressures (BP)were followed throughout the period of study. Each indi-vidual recorded BP represents the average value of fourreadings obtained with duplicate recordings from each oftwo occluding tail cuffs of different size.

Rats were followed for up to 269 days after the onset of astable hypertension with mean values of 170-230 mmHgrecorded for at least 8 days before any immunization. Dur-ing this control period, blood pressures were recorded threeto seven times (average 4i). From 10 to 27 days after thefinal immunization series, the rats were prepared for directB P recording as described in Part I and the pressor re-sponses to angiotensin amide and to angiotensin acid werecompared with that to norepinephrine. The results were ex-pressed as norepinephrine/angiotensin equipressor dose ra-tios. In 10 rats, this pressor response was studied bothbefore and after the period of immunization or mock im-munization.

The plasmas from 20 rats were assayed for antibodyagainst angiotensin according to the radioimmune assaymethod of Vallotton, Page, and Haber (25) by Dr. Lot B.Page. 12"I angiotensin purified with rabbit antiserum raisedagainst poly-L-lysine-angiotensin copolymers was used. 501d of rat serum was incubated with approximately 0.02 m/Ag"I angiotensin standard for 18 hr at 40C. Bound angiotensinwas precipitated from free angiotensin by ammonium sulfate.The results are expressed as bound to free (B/F) ratiosof angiotensin. A B/F ratio of greater than 0.2 is consideredto represent the presence of antibody against angiotensinwhereas a B/F ratio of less than 0.2 represents nonspecificprotein binding. Serial antibody determinations were notperformed because of the mortality risk involved in thislong-term study.

At the termination of the study, the kidneys of 23 ot therenal hypertensive rats were assayed for renin accordingto the method of Boucher, Menard, and Genest (26). Theindividual kidneys were stripped of their capsule, weighed,homogenized, and the pH of the homogenate adjusted to 5.5.After filtration of the homogenate, the filtrate was diluted1: 200 with normal saline and neomycin. One-tenth and 0.2ml of the diluted filtrate was incubated at 370C for 1 hrin the presence of Dowex resin with 1 ml of plasma (pHadjusted to 5.5) from rats nephrectomized 24 hr previously.Elution and bioassay was completed according to the methodof Boucher et al. as modified by Blaufox, Birbari, Hickler,and Merrill (27). Results are expressed as millimicrogramsof angiotensin per gram of kidney per min of incubation.

Statistical analysis. Student's Mtest was used in the sta-tistical evaluation of the data. Means are shown with ±+1 SD.

RESULTS

Part I. Response to angiotensin in experimentalhypertensionThe dosage of norepinephrine required to give a

blood pressure rise of 9-16 mmHg varied between rats.To obviate this the pressor variation ratios of norepi-nephrine/angiotensin amide, and of norepinephrine/angiotensin acid required to give an equal blood pressurerise were determined. A high ratio results from in-creased sensitivity to angiotensin and a low ratio re-sults from increased resistance to angiotensin. The groupmean intrinsic vascular reactivity of the three groups of

hypertensive rats under anesthesia was equivalent asmeasured by the amount of norepinephrine required togive the same blood pressure rise (Table I). Therefore,the difference in the response to angiotensin cannot beattributed to varying states of cardiovascular reactivity.

Table I compares the norepinephrine/angiotensin ra-tios for each group of rats. In normal and GN rats themean norepinephrine/angiotensin amide ratios were simi-lar whereas the DOCArats had a significantly highermean ratio (P < 0.001), which showed increased sensi-tivity to angiotensin amide; the G rats had a significantlylower ratio (P < 0.001), showing marked resistance toangiotensin amide.

Although the group differences with angiotensin acidare similar to those seen with angiotensin amide, thereis a greater spread of the individual points. The GNratswere more resistant to angiotensin acid than were nor-mal rats (P = 0.005) and less resistant than were Grats (P = 0.05). On comparison with normal rats, theDOCArats did not have a significantly greater sensi-tivity to angiotensin acid whereas the G rats were highlyresistant (P < 0.001). DOCA rats were more sensi-tive than either GN rats (P < 0.005) or G rats (P <0.001).

Part II. Immunization of rats with a conjugateof angiotensin

A fall in blood pressure of 30 mmHg or more(> 2 SD of our observed spontaneous mean pressurevariation in renovascular hypertensive rats) from av-erage control levels and lasting for 8 or more days wasconsidered significant and occurred after at least oneseries of immunizations in 8 of 14 G rats (57%) andthree of eight GNrats (38%). Four rats had significantbooster responses after a subsequent series of immuniza-tions, and three rats had blood pressures which fell sig-nificantly and never returned to control levels.

Significant blood pressure reduction occurred on theaverage of 42 days after the first injection in the firstseries of immunizations; in one rat the blood pressurefell significantly 38 days after a single immunization in-jection. Booster responses occurred on the average of15 days after the first injection of the booster series.The average duration of a given blood pressure reduc-tion was longer after the first series of immunizations(54 days) than after subsequent series (12 days).

Table II presents the following data: the total periodeach rat was followed; the average blood pressures be-fore any immunization; the average blood pressures forthe 10 days preceding termination of each study (timeplasma taken for radioimmune assay); the change inblood pressure from the control period to the 10 daysbefore completion of the study; the interval between thelast series of immunization and death; and the presence

1508 A. R. Christlieb, T. U. L. Biber, and R. B. Hickler

of angiotensin antibody expressed as the bound/free rats had a significant blood pressure fall from controlratios. blood pressure levels during the period of observation.

None of the immunized DOCAhypertensive rats and In fact, there was, in general, an increase in blood pres-none of the mock immunized renovascular hypertensive sure during the period of observation in these groups,

TABLE IPressor Response of Normal Rats and Rats with Experimental Hypertension to Injections of Norepinephrine (NE),

a-L-asparaginyll-valine5 angiotensin II (AA), and a-L-aspartyll-valine5 angsotensin II (AAc)

mig giving equal pressorAve BP response Ratios

before BP duringGroup* Rat No. study anesthesia NE AA AAc BP rise NE/amide NE/acid

Normal 12456789

Mean

mmHg mmHg604850

- 60- 65

5550

- 75

57

DOCA DI 215 80D3 220 40D4 215 50D6 185 80D7 200 90D12 200 60D14 210 65

Mean 206 66

GN GN2 190 85GN3 180 65GNII 195 100GN13 190 85GN16 195 95GN18 190 90GNT 200 70

Mean 191 84

G GIG3G4G7G8G9GIlG13G14G18G19G23

175 90170 80170 65195 80175 80180 90230 120190 50200 110240 75260 100220 75

Mean 200 85

20 10.0 2.220 11.0 3.015 5.5 1.820 9.4 2.320 11 2.320 10 2.320 7.8 1.920 6.8 -

19 8.9 2.3

10 3.4 1.520 7.1 1.820 5 2.910 2.1 0.810 2.0 0.910 2.9 0.910 2.6 0.9

12.9 3.6 1.4

10 3.6 2.020 11 2.710 8.9 2.120 7.5 2.520 13.5 3.610 4.3 1.520 10

15.7 8.4 2.4

20 2020 30 510 6.9 2.120 2210 6.7 1.2510 20 510 7.5 1.2520 41 10.510 10 220 42 12.510 37.5 5.920 33 8.0

13.5 23 5.4

mmHg

12.510.51116131212.5

9.5

12.1

159.5

141510.5

912

12.1

1311.5121213.511.513

12.3

1515121013121511.51212.5109

12.2

2.0 9.01.8 6.72.7 8.32.1 8.71.8 8.72.0 8.72.6 10.52.9

2.2 0441 8.6 ±1.14I

3.02.84.04.85.03.53.8

3.8 ±0.9+

2.81.81.22.71.52.32.0

2.0 1:0.64

1.00.71.40.91.50.51.30.51.00.40.30.6

0.8 :10.4:

6.711.1

7.012.511.111.111.1

10.1 ±2.34

5.07.44.88.05.66.7

6.3 ±1.31

44.8

8.02.08.01.95.01.61.72.5

4.0 ±2.54

* See text for description of hypertensive rats.I Mean ±1 SD.

Angiotensin in Experimental Hypertension, an Immunologic Approach 1509

H"

IMMUNIZEDAB Bound/Free >0.2

40-

20-

A BP

0+-mmHg

20-

40-

60-

"IMMUNIZED"AB Boundc/Free <0.2

MOCKIMMUNIZED

mm

- - -

mGoldblatt, non-nephrex

IGoldblatt, uni -nephrex

FIGuRE 2 Change in blood pressure between control levels and levelsrecorded at time of antibody determination in Goldblatt rats. AB (anti-body) bound/free > 0.2 represents presence of antibodies whereas AB(antibody) bound/free < 02 represents nonspecific binding. Nonnephrex= nonnephrectomized; uninephrex = uninephrectomized.

indicating progression of the hypertensive process; therewas an average increase of 21 mmHg in the immunizedDOCArats and 24 mmHg in the mock immunized Gand GN rats. By contrast, of the 20 immunized reno-vascular hypertensive rats in whom antibodies were de-termined, 7 of the 11 in which a B/F ratio > 0.2 de-veloped did show a significant reduction in blood pres-

240 NON-NI

200

sure. This included one GN and six G rats. The re-maining nine (four GNand five G) had a B/F ratio of< 0.2, and none had a significant BP reduction. In fact,seven of the nine showed a BP increase, similar to theimmunized DOCArats and mock immunized renovascu-lar hypertensive rats. Fig. 2 summarizes these changes.Additionally, of the four rats which did not show a sig-

EPHREX

I K * *

Mock Immunized

1601240 -

aImmunizedw Bound/Free 0.06200 _

160 __ ____ ------------------- ___________________- ___ t t t~ t t t tmmHg

1 2 3t Angio. Immunization

FIGURE 3 Blood pressure response and times of immunizations in each ofthree immunized or mock immunized rats rendered hypertensive by clippingone renal artery (G-rats). A bound/free ratio of > 0.2 indicates the pres-ence of angiotensin antibodies. A blood pressure fall of 30 mmHg or moreand lasting for 8 days or more is considered significant. Nonnephrex =

nonnephrectomized.

1510 A. R. Christlieb, T. U. L. Biber, and R. B. Hickler

G + GNrats§ witha B/F ratio$ > 0.2

G + GNrats§ witha B/F ratiot < 0.2

GNrats§ without ABdetermination*

G + GNratsi, mockimmunized

DOCArats§ withoutAB determination:

DOCAratsin mockimmunized

TABLE I IRelationship Between Antibody and Blood Pressure Change from Control Period to the

10 day Period before Antibody Determination

Timebetween

finalinocu- BP 10 days before

lations Control BP before antibody determina- Anti-Total + AB inoculation tion or death body

Duration inocu- determi- BP B/FRat studied* lations nation Mean High Low Mean High Low change Ratiot

or deathl

days days mmHg mmHg mmHg

G14G24G30G25G26G27G28G20G31GN2SGN24

Mean

GilGIG7G8G33GN6GN18GN22GN2

Mean

GN17GN13

Mean

G13GN3GN16GN11G4

Mean

D12D14D6D4DSDIS

Mean

D7Dl

Mean

102 5 12 193 195 185269 8 12 185 200 180238 7 19 193 200 185240 8 14 203 220 180242 7 24 187 205 170244 8 25 196 210 185246 8 27 230 245 210225 8 26 195 210 180216 8 26 208 230 195203 7 14 193 205 180226 8 25 202 215 190

223 7 20 199 212 185

104 6 13 230 240 225128 7 13 172 175 170124 6 10 195 210 180190 10 22 178 180 175152 6 26 210 220 200191 9 22 203 220 185129 6 14 195 205 180236 8 25 183 215 170122 6 18 188 200 170

153 7 18 195 207 184

218106

162

6 27 193 200 1806 17 188 200 175

6 22 191 200 178

117 5 16 188 200 17562 3 1S 190 215 170

135 6 20 218 220 180138 6 25 202 215 190131 5 30 170 180 160

117 5 2 1 193 206 175

78 4 19 210 220 20082 3 1 1 220 225 215

102 5 18 188 195 180107 5 3 180 190 17056 3 12 183 185 18039 3 3 220 - -

77 4 1 1 200 206 194

94 5 14 187 200 16054 3 6 220 225 215

74 4 10 204 213 188

125151200157140163170183230140203

169

2401118822021320011231205173225

210

145 105165 145200 200170 145145 135170 150170 170200 160235 225145 130210 200

177 160

190 185225 215235 200

250 220210 200185 145230 220

218 203

265 28022511 -

245 253

212 220 205225 230 220238 250 225232 245 220197 220 185

217 233 211

201 220 185220 225 21523011 - -223 225 210230 230 230220 220 220

221 225 215

225 230 22023011 - -

228 230 225

-68 0.20-34 0.36+7 0.57

-46 0.40-47 0.84-33 0.34-60 1.47-12 0.25+22 0.47-53 0.27+1 0.92

-30 0.55

+10+16+25+35-10+28+10-10+37

+15

00.06

00.080.130.11

00.06

0

0.04

240 +72- +37

233 +55

+24+35+20+30+27

+27

-90

+42+43+47

0

+21

+38+10

+24

* Total study period after onset of hypertension.t AB determination = antibody expressed as B/F (bound/free) ratio.5 See text for description of hypertensive rats.11 Solitary pressure determination during 10 days before termination of study. Previous pressures equal or higher.

Angiotensin in Experimental Hypertension, an Immunologic Approach 1511

UNI-NEPHREX

Mock Immunized

' + 4 44 41601 -- -------J-

240~~~~~~~~~~~~~~t "Immunized"240 -

Bound/Free 0

200 -

160 ----- __----_________________________--________________mm t t t t t tHg

Immunized Bound/Free 0.27

200 [

1620 [2 3

t Angio. Immunization4 5 6 7 Months 8

FIGURE 4 Blood pressure response and times of immunizations in each ofthree immunized or mock immunized rats rendered hypertensive by clippingone renal artery and contralateral nephrectomy (GN rats). A bound/freeratio of > 0.2 indicates the presence of angiotensin antibodies. A blood pres-

sure fall of 30 mmHg or more lasting for 8 days or more is considered sig-nificant. Uninephrex = uninephrectomized.

nificant BP fall between control levels and the timeof antibody determination despite a B/F ratio of > 0.2,3 (Nos. G20, G31, and GN24, Table II) had shown a

significant BP reduction earlier in the course. Of the ninerats with a B/F ratio of < 0.2, only one rat (No. GN22,Table II) had shown a significant blood pressure re-

duction earlier in its course. The immunized DOCArats lost weight and died before pressor responses toangiotensin and norepinephrine were determined andwithout plasma radioimmune assay for antibodies. RatNos. GN17 and GN13 (Table II) also died withoutfinal studies.

Fig. 3 details the blood pressure response and thetimes of immunization or mock immunization in each ofthree representative G rats. The course of rat No. G13,which was mock immunized and studied for 106 days, isseen in the upper plot. No significant blood pressure falloccurred. The course of rat No. G1, which received im-munization injections but had no significant antibodies(B/F of 0.06) demonstrable at the end of the study, isshown in the center plot. This rat was followed for 128days. Again, no significant blood pressure reductionsoccurred; as shown, the blood pressure actually increasedduring the period of observation. In the lower plot isseen the course of rat No. G26, which was followed for187 days after which time a significant antibody B/F

ratio of 0.84 was found. A significant blood pressure

fall occurred after the first series of immunizations.This rat retained a significantly lowered blood pressure

throughout the period of study.Similar results for three GN rats can be seen in

Fig. 4. The upper plot shows the course of rat No. GN11,which was mock immunized and followed for 104 days.A persistent increase in blood pressure was observedover the period of observation. The center plot followsrat No. GN2 for a period of 188 days after which timeno significant antibodies were measured (B/F ratio of0). There was no significant fall in blood pressure afterany of the immunizations. The course of rat No. GN25,which was followed for 193 days is seen in the lowerplot. A significant reduction in blood pressure occurredafter the first immunization series. Further falls in BP("booster responses") are seen after the second and thirdseries of immunizations. At the end of the study at whichtime the blood pressure was 53 mmHg below controllevels, a significant B/F ratio of 0.27 was measured.

Pressor responsiveness of inoculated rats. After im-munization of normal rats, the mean ratios fell from2.2 to 1.0 with angiotensin amide (P < 0.001) (TablesI and III). These changes are consistent with thosepreviously described (21) and reveal a highly signifi-cant increase in refractoriness to angiotensin amide as

1512 A. R. Christlieb, T. U. L. Biber, and R. B. Hickler

240

200

TABLE IIIPressor Response of Normal Rats and Rats with Experimental Hypertension to Inject ofions Norepinephrine (NE),

a-L-asparaginyll-valine5 Angiotensin II (AA), and a-L-aspartyll-valine5 Angiotensin II (AAc) after Inoculationwith an Angiotensin Conjugate or Mock Immunization

Anti- BP mpg giving equal pressor RatiosTotal body during responseinocu- B/F anesthe-

Group* Rat No. lations ratiot sia NE AA AAc BP rise NE/AA NE/AAc

mmHg mmHgInoculation with angiotensin conjugate

Normal 1 42 7 -3 65 76 47 48 5

10 611 712 650 6

Mean 6

GN GN25 7 0.27GN24 8 0.92GN6 9 0.11GN18 6 0GN22 8 0.06GN2 6 0GN17 6GN13 6

Mean 7

G G14 5 0.20G24 8 0.36G30 7 0.57G25 8 0.40G26 7 0.84G27 8 0.34G28 8 1.47G20 8 0.25G31 8 0.47Gil 6 0GI 7 0.06G7 6 0G8 10 0.08G33 6 0.13

Mean 7

Mock immunizedGN GN11 6

GN16 6 -

Mean 6

13 54 5

Mean 5

6540

1156050757070506075

66

509090

8585

80

556085505050908075

12070

10070

73

60100

80

6580

73

20 7.520 2730 4920 5020 6010 7.520 2020 11.840 5120 3020 24

21.8 30.7

40 4620 3120 15

20 810 200

22 60

20 6420 1820 1520 10030 4920 1020 2020 720 3110 2020 2010 1620 17

19.2 29.8

2115

511692313

22.4

2237.2

3.9

3.8150

43.4

14.57.36.4

4915

5.97.43.8

126.85.52.93.4

10.8

20 40 1020 13 3.6

20 26.5 6.810 28.5 5.310 8.3 1.9

10 18.4 3.6

1289

11.512121010

8.51011

10.4

10.5138

11.510

10.6

16898.59

11.51

98.5

1088

12

9.9

811

9.5

89.5

8.8

2.70.70.60.40.31.3\1.01.70.80.70.8

1.0 -0.7§0.90.61.3

2.50

1.1 409.§

0.31.11.30.20.62.01.02.80.70.51.00.61.2

1.41.3

4.01.80.60.91.5

1.6 41.1§1.80.55.1

5.30.1

2.6 42.5§1.42.73.10.42.03.42.75.31.71.53.63.54.5

1.0 ±0.7§ 2.8 41.35§

0.5 2.01.5 5.6

1.0 3.80.4 1.91.2 5.2

0.8 3.6

* See text for description of hypertensive rats.$ Antibody expressed as B/F (bound/free) ratio where determined.§ Mean 41 SD.

Angiotensin in Experimental Hypertension, an Immunologic Approach 1513

G GG.

a result of immunization. Previously (21) it was shownthat no significant increase in angiotensin refractorinessoccurred after mock immunization. Nonimmunized G ratshad low mean ratios with angiotensin amide similar tothe low mean ratios after "immunization" regardless ofwhether or not a B/F ratio of > 0.2 was found. Afterimmunization of GNrats, the mean ratios with angioten-sin amide fell from 2.0 to 1.1 (P < 0.05) with no sig-nificant difference between those which developed anti-bodies and those which did not.

After immunization of normal rats, the mean ratioswith angiotensin acid fell from 8.6 to 1.6 (P < 0.001).In G rats the mean ratio fell from 4 to 2.8 (statisticallynot significant); the response was the same in thosewith and those without demonstrable antibodies. Thenonimmunized GN rats had a mean ratio of 6.3 whichfell to 2.6 after immunization (P < 0.01); the two ratswith demonstrable antibodies had a mean ratio of 1.1compared with a mean ratio of 3.7 in the three ratswithout demonstrable antibodies.

The pressor responsiveness as expressed by norepi-nephrine/angiotensin ratios was also studied both be-fore and after immunization and mock immunization ineach of 10 rats. Figs. 5 and 6 summarize these paired ra-tios for each rat. The mean norepinephrine/angiotensinamide ratio for all of the immunized rats so studied fellfrom 1.4 ±0.7 to 0.6 ±0.5 (P < 0.025). For the G ratsonly, the mean ratio fell from 1.1 ±0.3 to 0.7 ±0.4 (P <0.05). There was no significant difference between themean norepinephrine/angiotensin amide ratios before andafter mock immunization (1.1 ±0.4 and 0.9 ±0.5 re-spectively). In three of these four rats, the ratio re-mained essentially unchanged after mock immunization.The mean norepinephrine/angiotensin acid ratio fellfrom 6.5 ±1.7 to 1.9 ±1.9 (P < 0.01) in inoculated ratsand was not dependent on the presence of demonstrable

4rRatio:

Equt- PressorResponse

Norepi /Amide

21

Immunized Mock Immunized

FIGuRE 5 Pressor responsivity expressed as norepineph-rine/angiotensin amide ratios before and after immunizationor mock immunization in G and GN rats. A low ratio re-sults from increased refractoriness to angiotensin.

Ratio:Equi- Pressor

ResponseNorepi/Acid

4-

2

01Immunized Mock Immunized

FIGURE 6 Pressor responsivity expressed as norepinephrine/angiotensin acid ratios before and after immunization ormock immunization in G and GN rats. A low ratio resultsfrom increased refractoriness to angiotensin.

antibodies. With mock immunization, the norepinephrine/angiotensin acid ratios did not change significantly (4.3-+-1.6 and 3.7 ±2.0 respectively).

Renal renin activity. The results of the renal reninactivity determined in the kidneys of immunized ratsare shown in Table IV. In the G rats the renal reninactivity in the clipped kidneys was consistently higherthan in the unclipped kidney and was comparable in thosewith and those without demonstrable antibodies. By con-trast, in rats with antibodies the mean renin activity inthe unclipped kidneys was three times greater than thatof the unclipped kidney in rats without antibodies. In thegroup with antibodies, the ratio of mean renal reninactivity in the clipped to the unclipped kidney was 6; inthe group without antibodies it was 15. In the GN rats,the mean renal renin activity in those without anti-bodies was slightly higher than the mean renal reninactivity of normal rats, and lower than that of theclipped kidney of G rats.

Plasma potassium. The plasma potassium and bodyweight of all rats with demonstrable antibodies and ofsix rats without demonstrable antibodies were deter-mined. The group mean potassium was 3.2 mEq/literand 3.4 mEq/liter respectively (group mean of five nor-mal rats was 3.2 mEq/liter). Mean body weights weresimilar, 453 g and 503 g respectively.

1514 A. R. Christlieb, T. U. L. Biber, and R. B. Hickler

I-

TABLE IVRenal Renin Activity (RRA)* in Kidneys of Normal Rats and Renal Hypertensive Rats

Inoculated with an Angiotensin Conjugate

G ratst

B/F > 0.21 B/F < 0.2§ GNrats*

Normal rats Unclipped Clipped Unclipped Clipped B/F > 0.2§ B/F < 0.2§kidney kidney kidney kidney

Rat No. RRA Rat No. RRA RRA Rat No. RRA RRA Rat No. RRA Rat No. RRA

1 4150 G14 1970 - G11 350 3850 GN26 5311 GN6 43952 2790 G24 270 2945 GI 390 GN18 85803 3460 G30 1139 15430 G7 250 4600 GN22 26354 3855 G25 565 4495 G8 130 5035 GN2 20105 4270 G26 385 3465 G33 508 57206 2525 G27 2610 60307 2890 G28 617 89908 4730 G20 442 6094

G31 1582 5978

Mean 3583 1064 6678 326 4801 5311 4405

* RRAexpressed as millimicrograms angiotensin per gram kidney per minute of incubation.t See text for description of hypertensive rats.§ Antibody expressed as B/F (bound/free) ratio.

DISCUSSIONPathophysiologic studies in renal hypertension. The

three types of experimental hypertension studied wereselected because each type is reported to have a differentlevel of both renal and circulating renin activity. In ratsmade hypertensive with salt loading and deoxycorticos-terone (DOCA rats), the renal and peripheral reninactivities are low (28, 29). Mechanisms to account forthis type of hypertension have been reviewed recently(30). In experimental renal hypertension produced byclipping one renal artery and contralateral nephrec-tomy (GN rats), the renal and circulating renin activi-ties are normal (28, 29, 31). In experimental renal hy-pertension produced by clipping one renal artery withthe contralateral kidney intact (G rats), there is elevatedrenin activity in the clipped kidney and in the circula-tion but depressed renin activity in the untouched kid-ney (28, 29, 31). Recent studies have shown a goodcorrelation between changes in plasma renin activityand the concentration of angiotensin in the blood (32,33).

An inverse relationship has been noted between themagnitude of the blood pressure response to exogenousangiotensin and renin activity (28). The results in PartI of the current study are generally in accord with this.DOCArats were highly sensitive to both angiotensinamide and acid presumably reflecting the suppression ofthe renin-angiotensin system in this form of hyperten-sion. G rats were highly resistant to both peptides, pre-sumably due to high levels of circulating angiotensin.

GN rats however, which are reported to have normalrenal renin activity (31) had normal pressor responsive-ness to injected angiotensin amide, but had significantlydecreased pressor responsiveness to angiotensin acid.It is probable that the natural angiotensin in rats isaspartyl angiotensin, and these results suggest that inGN rats the circulating angiotensin level may be higherthan in normal rats but lower than in G rats.

The results of Part II indicate that the hypertensionin the two forms of experimental renal hypertension canbe ameliorated after successful in vivo production ofantibodies against angiotensin, strongly suggesting thatangiotensin is directly involved in the mechanism of thehypertension.

In this study immunization was started during the 1stmonth of hypertension in most of the rats. However,booster inoculations resulted in blood pressure falls aslong as 8 months after the onset of stable hypertension.It appears, therefore, that immunization against angio-tensin can result in blood pressure reduction in both theacute and the chronic phase of experimental renalhypertension.

Injections of angiotensin coupled to albumin throughcarbodiimide cross-linkage reversed the hypertensiveprocess in 50% of the rats with experimental renal hy-pertension. Further, there was excellent correlation be-tween the presence of antibody, expressed as bound/freeratios using the radioimmune assay, and the differencebetween the average control blood pressures taken be-fore any immunization injections and the average blood

Angiotensin in Experimental Hypertension, an Immunologic Approach 1515

pressures recorded for 10 days preceding antibody de-termination. 7 of 11 rats (64%) with a bound/free ra-tio of > 0.2 had a significant blood pressure fall betweenthese periods. Although four of the rats with a bound/free ratio of > 0.2 did not have a similar blood pressurefall between these periods, three of the four did not havethe progressive increase in blood pressure seen in therats with a bound/free ratio of < 0.2 and in the mockimmunized rats. In addition, three of these four rats hadshown a significant blood pressure reduction earlier intheir course. Booster injections with the antigen afterthe blood pressure had returned to control hypertensivelevels resulted in repeated significant blood pressure fallsin four rats.

It is conceivable that the blood pressure reduction ob-served in the rats with demonstrable antibodies was anonspecific consequence of successful immunization.However, it is unlikely that the blood pressure reduc-tion resulted from a nonspecific effect of the immuniza-tion procedure itself since none of the nine rats withexperimental renal hypertension which had a bound/free ratio of < 0.2 and none of the five mock immunizedrats with experimental renal hypertension had a signifi-cant fall in blood pressure between average controllevels and the average levels for the 10 days precedingantibody determination. Most of these rats had a gradualincrease in blood pressure over the period of study, con-sistent with the natural progression of renal hyper-tension. That none of the six rats with hypertension pro-duced with saline and deoxycorticosterone acetate had asignificant reduction in blood pressure after the attemptto actively immunize them against angiotensin lends fur-ther evidence for the specificity of angiotensin antibodiesin effecting the blood pressure fall seen in the renal hy-pertensive rats. Although no antibody determinationswere obtained in these rats because of their prematuredeath, it is possible that none developed antibodies. Thisappears unlikely, however, because more than 50% ofthe Goldblatt rats had a bound/free antibody ratio of> 0.2.

The results of these studies suggest that angiotensinprobably is the specific mediator in the hypertension as-sociated with renal artery stenosis. Although aldosteronehas been thought to play only a permissive role in theetiology of renovascular hypertension, the possibilityexists that the reduction in blood pressure seen afterimmunization occurred as a consequence of a suppressionof aldosterone secretion secondary to a decrease in circu-lating angiotensin. The normal plasma potassium in thegroup with and without demonstrable antibodies, andthe similar body weights between the two groups sug-gest that mineralocorticoid activity was present. Inaddition, several studies have suggested that aldosteronesecretion is not abolished following the removal of an-

giotensin from the circulation and that aldosterone isnot the mediator of renovascular hypertension (16, 34-38).

Comparable levels of hypertension resulted from eitherclipping one renal artery (high renin renal hyperten-sion) or clipping one renal artery coupled with contra-lateral nephrectomy (normal or intermediate renin hy-pertension). The quantitative difference in the sensitivityto injected angiotensin found between the two prepara-tions suggests a difference in the circulating level ofangiotensin. This may be explained by invoking theantihypertensive function which has been postulatedfor the contralateral kidney, requiring that the clippedkidney secrete more renin (angiotensin) to sustain acomparable level of hypertension when both kidneysare present. It would appear, then, that angiotensin maybe intimately involved in the initiation and maintenanceof both forms of experimental renal hypertension.

Immunologic considerations. The immunologic cir-cumstances in this study are unique in that active im-munization against an endogenously produced low mo-lecular weight peptide, angiotensin, has resulted notonly in the production of antibodies against angiotensin,but also in a physiological response (blood pressurereduction). Booster inoculations resulted in physio-logic responses which were not typical of anamnesticantibody responses in that the delay before the bloodpressure fall averaged 15 days and the blood pressurereduction was of short duration, averaging 12 days.In this immunologic situation where there is con-stant endogenous production of the hapten, antibodybinding must keep pace with production to block the bio-logical effects (blood pressure fall). Similarly, in vitrodetection of antibody would be limited to some degree bythe amount of endogenous angiotensin present and boundto the antibody.

Several studies involving haptens have demonstratedthat exposure to the hapten preceding the active im-munization may result in suppression of antibody pro-duction and that this immunologic unresponsiveness maybe due to persistence of the hapten during the period ofattempted immunization (39-42). Partial immunologicunresponsiveness might explain the failure to demon-strate antibodies in some of the inoculated rats and alsoexplain the delay in blood pressure fall after booster im-munization inoculations.

It is postulated, therefore, that to achieve a surplusof antibody would take several days during which timeangiotensin would have to be formed in greater amountsto maintain the hypertension. Similarly, once the bloodpressure has fallen, the stimulus for angiotensin releasewould be augmented, resulting in a further increase inangiotensin and saturation of the antibody. The excess

1516 A. R. Christlieb, T. U. L. Biber, and R. B. Hickler

angiotensin would thereby produce renewed hyperten-sion in a short period of time.

Evidence supporting this is found in the results of therenal renin activity determinations (Table IV) and theangiotensin injection tests (Table III). An elevatedrenal renin activity would suggest increased renin se-cretion and elevated angiotensin levels. An analysis ofthe renal renin activity levels in the clipped kidneys ofthe G rats fails to indicate any convincing difference be-tween the group which did as opposed to the groupwhich did not develop a bound to free antibody ratio of> 0.2. Because these clipped kidneys are constantlystimulated to produce renin, this similarity between thetwvo groups may be expected. By contrast, in the un-clipped kidneys of the G rats, the average renal reninactivity was three times as high in the group which didas compared with the group which did not developa bound to free ratio of > 0.2, suggesting an increasedlevel of circulating angiotensin in the group withdemonstrable antibodies. Such an increase in angio-tensin may be sufficient to bind available angiotensinantibody binding sites in a short period of time and ac-count for the short duration of the blood pressure fallsafter booster injections. This might also explain themodified blood pressure course seen in the rats with aB/F ratio of > 0.2 but which did not have a significantblood pressure fall at the time of antibody determination.Here it appears that although antibody binding sitessufficient to bind angiotensin were present earlier in thecourse when a blood pressure fall was recorded, at thetime of antibody determination, angiotensin productionwas stimulated with a resultant antibody neutralizationof only enough angiotensin to prevent progression ofthe hypertensive process.

With the exception of the rats with a clip on one re-nal artery (G rats), which were highly resistant to in-jected angiotensin before immunization, immunized ratsdeveloped significantly greater refractoriness to in-jected angiotensin amide and acid, whether or not anti-bodies were demonstrated, when compared with nonim-munized or mock immunized rats. When paired norepi-nephrine/angiotensin ratios were performed before andafter immunization or mock immunization in the samerats (Figs. 5 and 6), a significantly increased refrac-toriness to both angiotensin amide and angiotensin acidwas observed in the inoculated rats and was not de-pendent on the presence of demonstrable antibodies. Nosignificant change in refractoriness was observed in themock immunized rats studied similarly. There are twoexplanations which may account for these results.First, it is possible that a slow release of the angiotensinfrom the antigen complex may lead to angiotensin tachy-phvlaxis. The quantity of angiotensin in the injectedantigen however was less than half that causing refrac-

toriness to an acute angiotensin injection 1 hr after dis-continuation of a 12 day infusion in four of seven rats(43). In addition, there was no observed increase inblood pressure after inoculations suggesting that if an-giotensin were released from the antigen complex, itwas not released in pressor amounts. The second ex-planation is that the observed increase in refractorinessis consistent with antibody neutralization of angiotensinin all of the inoculated rats, including those in whichno blood pressure reduction was observed. The ratswithout demonstrable antibodies by radioimmune tech-niques could well have had an excess of endogenousangiotensin at the time of in vitro antibody studies.

ACKNOWLEDGMENTSWe express our appreciation to Dr. Lot B. Page for per-forming the radioimmune assays for angiotensin, to Dr.Charles B. Carpenter for his assistance in the interpretationof the immunological aspects of this study, and to Dr. Ben-jamin J. Murawski for his assistance in the statisticalanalysis of the data.

This work was supported by U. S. Public Health ServiceGrant HE 11306-02 and by The John A. Hartford Founda-tion, Inc.

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