The Relationship of the Renal Vascular Activity of Angiotensin II to the Autonomic Nervous System * JOHN C

Journal of Clinical Investigation Vol. 44, No. 11, 1965

The Relationship of the Renal Vascular Activity of Angiotensin II to the Autonomic Nervous System * JOHN C. MCGIFF t AND THOMAS M. FASY (From the Departments of Pharmacology and Medicine, School of Medicine, University of Pennsylvania, and the Edward B. Robinette Foundation, Medical Clinic, Hospital of the University of Pennsylvania, Philadelphia, Pa.)

The vasoconstrictor activity of angiotensin II vasoconstrictor activity of angiotensin II, similar was originally considered to be due to a direct ef- to that produced by renal ischemia (9). The fect on vascular smooth muscle (1, 2). Subse- present study reports on the role of the nervous quently, it has been suggested that the sympathetic system in determining the renal vascular reactivity nervous system participates in the vascular re- to angiotensin II. The renal vascular bed was sponse to angiotensin II (3-5). After acute selected for this study because it is the most sensi- denervation of the limb, the reactivity of the limb tive of the regional beds tested to the vasoconstric- vasculature to angiotensin II is reduced in the rat tor effect of angiotensin II (10). Blockade by (3) and dog (4). Bickerton and Buckley (5) autonomic drugs of the renal vasoconstriction pro- have described two elements in the pressor reduced by angiotensin II was demonstrated. Au- sponse to angiotensin II: a centrally mediated ef- tonomic blocking agents selected were representa- fect, due to stimulation of central autonomic nerv- tive of several classes: ganglionic blockade (hexa- ous structures, and a peripheral action on the vas- methonium), alteration of the release of the neuro- cular smooth muscle. transmitter by either central (hydralazine) or Angiotensin II is not only influenced in its vas- peripheral activity (guanethidine and bretylium), cular activity by the autonomic nervous system, depletion of the transmitter (reserpine), and re- but itself will release or enhance the response to ceptor blockade (phentolamine). The capacity of stimuli releasing catecholamines. Thus, angio- autonomic blocking agents to oppose the effects of tensin II has been reported to enhance the pressor angiotensin II was related to the activity of the response to tyramine (6), to release catechola- drug at the neuroeffector site. Those blocking mines from the adrenal medulla (7), and to lower agents that reduced the release or depleted the the threshold to sympathetic nerve stimulation neurotransmitter were demonstrated to oppose the (8). renal vascular activity of angiotensin II. Renal ischemia was observed in this laboratory to alter the renal vascular response to angiotensin Methods II. While examining the role of the nervous sys- Dogs weighing 21 to 32 kg (mean, 24 kg) were anes- tem in the production of this effect, renal denerva- thetized with morphine sulfate (2 mg per kg, subcutane- tion was observed to produce a loss of the renal ously) and chloralose (70 mg per kg, intravenously). Heparin 1 (200 to 400 International U per kg) was ad- * Submitted for publication February 25, 1965; accepted ministered intravenously as the anticoagulant. The lungs August 5, 1965. were ventilated by a Starling Ideal pump through a Abstracts of this paper appeared in Circulation 1964, 30, Y-shaped glass tube inserted in the trachea. 124, and Clin. Res. 1965, 13, 214. Presented in part before A Sanborn multichannel direct writer recorded mean the American Heart Association, October 1964. arterial pressure and the venous outflow of one kidney. Supported by grants HE-08805 and HE-5239, general In one experiment the two renal blood flows were meas- research support grant 1S01-FR05115-01, and training ured simultaneously. The renal vein was cannulated with grant T5-GM-4-07 from the U.S. Public Health Service. a plastic cannula (4 mm, inside diameter). The time re- t Established Investigator, American Heart Associa- quired for cannulation, during which the arterial supply tion (July 1, 1964). was interrupted by a clamp, was less than 2 minutes. Address requests for reprints to Dr. John C. McGiff, The effluent was passed through a Shipley-Wilson ro- Dept. of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19104. 1 Panheprin, Abbott Laboratories, North Chicago, Ill. 1911 1912 JOHN C. McGIFF AND THOMAS M. FASY tameter (200 ml) that was placed below the cannulated nephrine and levarterenol (2 to 5 ug per kg per minute) vein. A common reservoir received the renal blood flow, were administered. The doses of hexamethonium, tyra- which was returned to the femoral vein by a Sigmamotor mine, and hydralazine are given in terms of the salt; for pump, automatically activated when 40 ml of blood was the other drugs, doses are expressed as the base. Intra- emptied into the reservoir. The amount of blood in the venous injections were made rapidly through a cannulated extracorporeal recording circuit was constant. The dead femoral vein in a fluid volume of less than 2 ml. In the space of the recording system was 120 ml; an equal volume experiments in which the kidney was denervated, renal of plasma expander (6% gelatin solution) was used to intra-arterial injections were made possible; 1 ml of the prime this system. drug in appropriate concentration was administered into In five experiments, acute denervation of the kidney was the cannula and tubing conducting the blood to the per- accomplished by transection of the renal artery. The fused kidney. Several schedules of reserpine 3 adminis- denervated kidney was then perfused with blood from tration were elected, for a usual dose of reserpine (e.g., the carotid artery through a cannula inserted into the 0.1 mg per kg per day intramuscularly for 2 days) proved distal end of the sectioned renal artery. insufficient in two experiments to alter the renal vascu- In four experiments the first cervical vertebra was ex- lar activity of angiotensin II. The dose of reserpine that posed and a laminectomy was performed. A 2-cm seg- effectively modified the renal vascular response to angio- ment of spinal cord was uncovered by removing the over- tensin II in four dogs was 0.1 mg per kg intramuscularly, lying dura mater. After the renal vascular responses to daily for 4 days, then 1 mg per kg intravenously, 24 hours the drugs were observed, the cord was transected. and 12 hours before the experiment. The following drugs were administered intravenously: To exclude tachyphylaxis or a time trend as a cause angiotensin II 2 (0.1 to 0.5 gg per kg), tyramine hydro- of the modified renal vascular response to angiotensin II, chloride (50 to 500 /Ag per kg), levarterenol bitartrate the following precautions were observed. a) In the con- (0.25 to 2.0 yg per kg), epinephrine bitartrate (0.4 to trol periods, i.e., before renal denervation, angiotensin II 2.0 /Ag per kg), phentolamine methanesulfonate (0.1 mg was administered only once. The only exception was that per kg), guanethidine sulfate (5 to 10 mg per kg), before spinal cord transection there were two injections bretylium tosylate (5 mg per kg), hexamethonium bro- of angiotensin II separated by an interval of 5 minutes. mide (5 to 10 mg per kg), nicotinic acid tartrate (30 to b) Successive injections of angiotensin II were separated 100 /Ag per kg), and hydralazine hydrochloride (2 mg per by an interval of at least 3 minutes, which was deter- kg). In several experiments intravenous infusions of epi- 3 Serpasil phosphate, Ciba Pharmaceutical Co., Summit, 2 Hypertensin, Ciba Pharmaceutical Co., Summit, N. J. N. J.

TABLE I The renal vascular activity of angiotensin II as modified by acute denervation of the kidney (summary offive experiments)

Mean change of renal blood flow from con- Blood pressure* Renal blood flow* trolt h SE of 4 SE of mean d SE of mean mean difference mm Hg mil/min ml/min Predenervation Controlt 116 :1 14t 175 ± 18: Angiotensin, iv, 0.1 ug/kg 165 ± 12§ 135 i 201 -42±10§ Control 126 4t 20 170 ± 17 Levarterenol, iv, 1.0 jug/kg 171 i1= 1911 94 i 23|| -76415 Postdenervation Controlt 95 ± 71: 123 ± 24t Angiotensin, iv, 0.1 ttg/kg 144 ± 11§ 143 ± 23§ +20 4 2§ Control 91±12 145± 8 Levarterenol, iv, 1.0 Mg/kg 136 ± 23 62 i 181 -80±15 Control 95 ± 11 115 ± 20 Angiotensin, renal intra-arterial, 0.05 M~g/kg 97 ± 9 58 ± 22§ -57± 9 * The mean values of the renal blood flows and blood pressures resulting from administration of angiotensin II and levarterenol were compared with the mean of the control values. Differences were statistically evaluated; unless otherwise indicated p > 0.05. t The mean changes of renal blood flow from control resulting from intravenous administration of angiotensin II and levarterenol were compared before and after denervation of the kidney. Differences were statistically evaluated; unless otherwise indicated p > 0.05. t The mean values for the angiotensin control periods, pre- and postdenervation, did not differ significantly. §p <0.01. II p <0.05. ANGIOTENSIN AND THE AUTONOMIC NERVOUS SYSTEM 1913

E0 _9 c R Mo0 E oi -- N A L on D z E N E R V A 0- T 0 N

AENGONSIN ANGIOTENSIN ANGIOTENSIN ANGIOTENSIN O.Ipg/kg iv QOIug/ kg ia 0.2,ug/kg ivj 0.5kpg/kg iv ANGIOTENSIN O.O1ug/ kg ia 24 kg DOG 8-28-63 IImin I FIG. 1. EFFECT OF RENAL DENERVATION ON THE RENAL VASCULAR RESPONSE TO ANGIOTENSIN II IN A DOG UNDER MORPHINE-CHLORALOSE. Acute renal denervation abolished the constriction of the renal vasculature produced by angiotensin II intravenously and reduced the response to intra-arterial (ia) angiotensin II. Ten minutes elapsed between, the left and right panels. BP = blood pressure. mined by earlier work as an appropriate interval (9). of angiotensin II before denervation always re- c) The observations were confined to the first 90 min- sulted in a reduction of renal blood flow (- 42 ml utes after having set up the preparation, during which no appreciable time trend was observed (9, 11). d) The per minute) (Table I). After denervation, even renal vascular response to angiotensin II when altered a fivefold increase in the amount of intravenous was then compared to levarterenol or epinephrine to ex- angiotensin II failed to constrict the renal blood clude the development of vascular refractoriness. vessels (Figure 1). The mean change of renal The results were expressed as the maximal change in blood flow elicited by intravenous levarterenol (1 renal blood flow, mean aortic blood pressure, and calcu- before and after renal of lated vascular resistance elicited by the stimulus. Vascu- ,Mg per kg) denervation, lar resistances for the experimental period were deter- - 76 and - 80 ml per minute, respectively, did mined by dividing the mean aortic blood pressure by the not differ significantly (Table I). value expressed in milliliters per minute, representing the In all experiments in which the kidney was maximal change in renal blood flow produced by the denervated, angiotensin II (Figure 1) adminis- stimulus. Statistical analyses were made on paired tered into the renal artery (0.01 ug per kg) con- analyses of control and experimental values of the maximal change in renal blood flow and simultaneous blood tinued to elicit renal vasoconstriction (Table I and pressure after application of the stimulus (12). Figure 1). In Figure 2 the effect of angiotensin II upon the renal blood flows measured simultane- Results ously is shown after denervation of one kidney. Under these conditions, angiotensin II elicited a Acute renal denervation differential effect on renal blood flows; the blood In five experiments, acute renal denervation re- flow to the denervated kidney increased in resulted in a loss of the renal vasoconstrictor activity sponse to intravenous angiotensin II, whereas vaso- of angiotensin II (0.1 ug per kg) administered constriction occurred simultaneously in the inner- intravenously (Table I and Figure 1). After vated kidney. acute renal denervation, intravenous administration of 0.1 ,ug per kg of angiotensin II increased Spinal cord section renal blood flow in all cases (20 ml per minute, In four experiments, nervous activity was modi- mean of five experiments), whereas the same dose fied by interrupting the autonomic nerve supply at 1914 JOHN C. McGIFF AND THOMAS M. FASY

IlMINI elimination of the renal vasoconstrictor effect of 0 0-N, angiotensin II administered intravenously was 0 noted. 0 After cord section, angiotensin II (0.1 jug per E J0 0- - kg) administered intravenously increased the re- A_ 0 'n- nal blood flow by 29 ml per minute (mean of four 0-. experiments), whereas -J before cord section a re- U. 0 duction of 88 ml per minute (mean of four ex- a 8- E 0 _ N periments) was recorded (p < 0.01; Table II). 0 °_0 E R The decreased reactivity of the renal vasculature _~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~V I I.Sl 0- A to angiotensin II produced by cord section was not <- 0 T z 0 observed for catecholamines. An epinephrine in- w a:- 0- fusion (4 Mg per kg per minute) after cord section N resulted in cessation of the renal blood flow. oI o- Levarterenol by intravenous injection (1 pg per .0 kg) as well as by infusion a. A--\ i (5 Mag per kg per min- m0 I- /K\~ A - ute) increased renal vascular resistance by 220% U 1I 0 and 604% of control values, respectively, after cord section. OE41E 0- t t ANGIOTENSIN ANGIOTENSIN Autonomic blocking agents O.Ipg/kg iv 0.2 jig/kg iv 25 kg DOG The effect of acute renal denervation and cord 8-30-63 section on the renal vascular response to angio- FIG. 2. DIFFERENTIAL REACTION OF THE RENAL BLOOD tensin II prompted the use of drugs that modify FLOWS TO ANGIOTENSIN II IN A DOG UNDER MORPHINE- autonomic nervous activity. The selection of au- CHLORALOSE. Between the left and right panels, the right tonomic blocking agents was determined by their kidney was denervated. Fifteen minutes separates the two panels. possessing major blocking activity at different sites, either prejunctionally, along the autonomic the level of the spinal cord (Table II). The cervi- nerve and its central connections, or at the sympa- cal cord was sectioned immediately after recording thetic receptor (a) that mediates renal vasocon- control observations of the response of the renal striction. Phentolamine was used to block the a vasculature to angiotensin II. After cord section, receptors. The following drugs were used to in-

TABLE II Renal vascular effects of angiotensin II before and after spinal cord transection (summary offour experiments)

Mean change of renal blood flow Blood pressure* Renal blood flow* from controlt + SE Procedure i SE of mean h SE of mean of mean di 'erence mm Hg mi/min ml/misn Before cord section Control 135 i 13 176 :1: 11 Angiotensin, iv, 0.1 pg/kg 180 :4= 17t 89 i- 15§ -88 + 17t After cord section Control 84 6 106 A1: 8 Angiotensin, iv, 0.1 pg/kg 111 1: 7t 135 4t 12 +29 :1: 10t * The mean values of the blood pressures and renal blood flows produced by angiotensin II were compared with the mean values of the control blood pressures and renal blood flows. Differences were statistically evaluated. t The mean changes of renal blood flow from control resulting from administration of angiotensin II, before and after cord transection, were significantly different from each other. t p < 0.01. § p <0.05. ANGIOTENSIN AND THE AUTONOMIC NERVOUS SYSTEM 1915

TABLE III The effect of guanethidine upon the renal vascular activity of angiotensin II

Renal Renal Blood blood vascular pressure flow resistance

mm Hg Ml!mi% Dog 1 Control 95 190 Angiotensin, iv, 0.1 pug/kg 155 100 +210 Guanethidine, iv, 10 mg/kg Control 150 200 Angiotensin, iv, 0.1 ug/kg 165 200 +11 Dog 2 Control 80 195 Angiotensin, iv, 0.2 Ag/kg 110 160 +68 Guanethidine, iv, 10 mg/kg Control 90 210 Angiotensin, iv, 0.2 ,&g/kg 110 210 +21 Dog 3 Control 140 170 Angiotensin, iv, 0.1 Mg/kg 185 110 +105 Guanethidine, iv, 10 mg/kg Control 115 130 Angiotensin, iv, 0.1 pg/kg 145 145 +14 Dog 4 Hexamethonium, iv, 10 mg/kg (administered 20 minutes previously) Control 45 150 Angiotensin, iv, 0.1 Ag/kg 85 55 +413 Guanethidine, iv, 8 mg/kg Control 110 125 Angiotensin, iv, 0.1 M&g/kg 140 140 +14 terrupt autonomic nervous activity: hydralazine angiotensin II administered intravenously in- (central effect), hexamethonium (ganglionic creased renal vascular resistance by 117% and blockade), guanethidine and bretylium (modi- 105%o; after phentolamine a 160%o and 81% in- fication of the release of the autonomic neurotrans- crease, respectively, in renal vascular resistance mitter), and reserpine (depletion of the neuro- was produced by angiotensin II. The differences transmitter). In most cases the renal vascular (Figure 3) of + 37%o and - 23%o between the effects of angiotensin II and levarterenol were com- per cent increase of renal vascular resistance angio- pared before and after administration of the block- tensin II produced before and after drug inter- ing agent. Because of the duration of activity of vention were not significant. To be significant the autonomic blocking drugs, a single set of ob- (p < 0.05) a greater than 62%o reduction in the servations for one drug was made per experiment. renal vasoconstrictor effect of angiotensin II or No animal received more than one blocking agent levarterenol, after the administration of the block- except for the use of guanethidine after hexa- ing drug, was required (Symbols falling below methonium (Table III) in one experiment. the broken line in Figure 3 represent significant Phentolamine and hexamethonium. Phentola- reductions by the blocking drug of the renal vaso- mine (0.1 mg per kg, intravenously) and hexa- constriction produced by the pressor agent). As methonium (5 to 10 mg per kg, intravenously) expected, phentolamine (0.1 mg per kg, intrave- failed to block the renal vasoconstriction elicited nously) in two experiments did block the renal by angiotensin II (Figures 3 and 4, Table IV). vasoconstriction produced by levarterenol (Fig- In Figure 3, the vasoconstrictor activity of angio- ure 3), whereas the renal vascular effects of levar- tensin II and levarterenol after drug intervention terenol were not reduced by hexamethonium. In is expressed in terms of their per cent change four dogs that had received hexamethonium (Ta- from control values. Thus before phentolamine, ble IV), 0.1 ug per kg of angiotensin II intrave- 1916 JOHN C. McGIFF AND THOMAS M. FASY

+250 9C) ANGIOTENSIN I iv; io +100 LEVARTERENOL o iv; * io

z z I0- +50[ w o-

a:-oCu-)

4 0- 0 0 E -i - _ CL-W-

'Ii 0 A 0-. 4

0 -501- ANGIOTENSIN A HEXAMETHONUMtAGOTENSN 0.1,pg/ kg iv 5 mg/kg iv A ~~A O.I.ig/kg iv A -100 A BRETYLIUM HYDRALAZINE PHENTOLAMINE 11-7-63 limin I 32 kg DOG 5 mg/kg iv 2 mg/kg iv 0.1 mg/kg iv FIG. 4. THE EFFECT OF HEXAMETHONIUM ON THE RE- FIG. 3. EFFECT OF AUTONOMIC BLOCKING DRUGS ON THE NAL VASCULAR RESPONSE TO ANGIOTENSIN II IN A DOG RENAL VASCULAR RESPONSE TO ANGIOTENSIN II AND UNDER MORPHINE-CHLORALOSE. LEVARTERENOL. The circles (levarterenol) and triangles (angiotensin II) represent the difference expressed as per cent change (%o A) between two observations: the nously produced a mean increase in renal vascular per cent increase in renal vascular resistance produced by resistance of 194%, which did not differ signifi- the pressor agent before and after drug intervention. cantly (p > 0.05) from the mean change in renal Each triangle represents a single experiment. Of the vascular resistance that angiotensin II elicited be- eight experiments depicted here, levarterenol was adminis- fore hexamethonium, + 172%o. The control mean tered also in six. Angiotensin was used in 0.1 pug per kg doses iv and 0.05 ug per kg ia (renal artery), levar- blood pressure of 74 mm Hg after hexamethonium, terenol 1.0 pag per kg iv and 0.5 pAg per kg ia. Significant when contrasted to that before hexamethonium of reduction of the renal vascular response to angiotensin II 140 mm Hg (Table IV), testifies to the adequacy or levarterenol (p < 0.05) by the blocking agent occurred of the dosage of hexamethonium. when the symbol falls below the broken line. Thus bretylium and hydralazine altered significantly the renal Guanethidine, bretylium, and hydralazine. vascular response to angiotensin II, whereas phentolamine Guanethidine (5 to 10 mg per kg) administered modified that to levarterenol. intravenously abolished or reduced markedly the

TABLE IV The effect of hexamethonium upon the renal vascular response to angiotensin II (summary offour experiments)

Renal vascular Blood pressure* Renal blood flow* resistancet lSE of mean tSE of mean tSE of mean mm Hg mi/min % change Control 140 i 7 201 ± 14 Angiotensin, iv, 0.1 p&g/kg 180 i 6t 118 i 26§ +172 ± 79t Hexamethonium, iv, 5-10 mg/kg Control 74 i 10 184 i 13 Angiotensin, iv, 0.1 pg/kg 121 4t 13t 121 i 22§ +194 ± 75t * The mean values of the renal blood flows and blood pressures resulting from intravenous administration of angiotensin II were compared with the mean of the control values. Differences were statistically evaluated. t The mean values for the per cent change in renal vascular resistance elicited by angiotensin II before and after hexamethonium were compared. The differences were not significantly different, p > 0.05. t p < 0.01. § p <0.05. ANGIOTENSIN AND THE AUTONOMIC NERVOUS SYSTEM 1917

|I MINI 0 0 -4 0- LL o a 0 o_a0 0 9- m-4 JIc 0 N Zc1 1|.- 0- T H O I W- D a. - - -a a.) _-In N 0' E 0o to oE 0- ANGIOTENSIN4 ANGIOTENSIN EPINEPHRINE ANGIOTENSIN O. I pg/kg i v 0.1 pg/kg iv O.4pg/kg iv O.Ijg/kg iv 27 kg DOG 7-12-63

FIG. 5. GUANETHIDINE BLOCKADE OF THE RENAL VASOCONSTRICTOR ACTIVITY OF ANGIOTENSIN II IN A DOG UNDER MORPHINE-CHLORALOSE. The two panels are separated by 5 minutes, during which guanethidine, 10 mg per kg, was administered intravenously.

renal vasoconstrictor activity of intravenous angio- vascular resistance produced by angiotensin II was tensin II in four dogs (Table III and Figure 5), in each case greater than 62%o of control (p < whereas the renal vascular effects of catechola- 0.05). The renal vascular effect of levarterenol mines were unimpaired. This effect was rapid in (1 ug per kg) was not reduced significantly by onset (within 20 seconds of guanethidine adminis- either hydralazine or bretylium (Figure 3). tration, intravenously), was reversed by tyramine, Tyramine and renal vascular reactivity. The and was more effective than acute renal denerva- ability of guanethidine or bretylium to abolish the tion. Thus acute renal denervation did not abol- response to angiotensin II administered intra-ar- ish the renal vasoconstriction elicited by angioten- terially was compromised by previous administra- sin II administered into the renal artery (Table tion of tyramine. The administration of tyramine I), whereas guanethidine (5 mg intra-arterially (100 to 500 ,g per kg) restored the renal vascular or 5 to 10 mg per kg intravenously) eliminated or activity of angiotensin II after that activity had markedly reduced the renal vascular activity of been blocked by guanethidine (Figure 6). After angiotensin II administered intra-arterially as well spinal cord section, the vasoconstrictor effects of (Figure 6). The blockade of the vascular activity angiotensin II were abolished; however, after ad- of angiotensin II by guanethidine was unrelated to ministration of tyramine (50 to 200 lig per kg), the degree of pre-existing vascular tone, for it was partial restoration of the renal vascular effect of seen at control blood pressures as disparate as 45 angiotensin II was possible. It should be noted, mm Hg (posthexamethonium) and 150 mm Hg however, that there are forms of altered renal vas- (Table III). The pressor effect of angiotensin II cular reactivity to angiotensin II that tyramine will administered intravenously was not eliminated by not restore, e.g., that produced by reserpine. guanethidine. Reserpine. The results thus far suggest a re- In three animals each, bretylium (5 mg per kg) lationship between autonomic nervous activity and and hydralazine (2 mg per kg) also opposed the the vascular action of angiotensin II. Reserpine renal vascular activity of angiotensin IT (Figure was administered in six dogs in an attempt to 3), for the drug-induced reduction in the renal modify the renal vascular response to angiotensin 1918 JOHN C. McGIFF AND THOMAS M. FASY

3-19-64 1lminI 25 kg DOG

0 ..---;.-.

U.0 ......

.-- -T-R- m - - l : :--- : --- 1 - j ...... +...i ...... +......

F ..

ANGIOTE:NSIN G;UANETHIDCNE UAiNETHIDCIlNE LEVARTEREFNOL ANG. e,,, ia (ANG) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..5 mg ic | Smg io 05 pg/ko io 0.05 pgkg 0.Ipg./kg i v o~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~AdG. AING. AlG. TYtRAMINE 0.05 pg/kg io 0.5 Pg/kg ia 0.05 pg/kg ia 5 mg ia

FIG. 6. BLOCKADE AND RESTORATION OF THE RENAL VASCULAR RESPONSE TO ANGIO- TENSIN II IN A DOG UNDER MORPHINE-CHLORALOSE. Thirty minutes previously the kidney was denervated acutely. The renal vascular response to angiotensin II in- jected into the renal artery was blocked by guanethidine and restored by tyramine.

II. In four animals receiving reserpine, the re- jug per kg, intravenously) increased renal vascular sults of which are presented in Table V, angioten- resistance by 303%o, 250%o, 577%, and 330%o of sin II (0.1 ug per kg, intravenously) produced control, which were not significantly different changes in renal vascular resistance of + 14%o (p > 0.05) from the increase in renal vascular re- (mean of four experiments) that were significantly sistance produced by levarterenol in dogs that did different (p < 0.05) from the mean values not receive reserpine (368 95%). (+ 247) produced by angiotensin II in the four To evaluate the completeness of depletion of animals not receiving reserpine. In two dogs re- catecholamines, intravenous nicotine (30 to 100 ceiving smaller doses of reserpine (see Methods) ,ug per kg) was used. Nicotine has been demon- there was no reduction in the renal vasoconstrictor strated in this preparation (13) to be one of the effect of angiotensin II. In the four dogs given most potent releasers of catecholamines; e.g., 50 reserpine that demonstrated a reduced renal vas- /Ag per kg of nicotine administered intravenously cular response to angiotensin II, levarterenol (1 increased renal vascular resistance by 373% and

TABLE V The effect of reserpine upon the renal vascular activity of angiotensin II (summary of eight experiments)

Renal vascular Blood pressure* Renal blood flow* resistance + SE of mean + SE of mean =1: SEof mean mm Hg mil/min % change Without reserpine (4 dogs) Control 144 i 13 153 ± 8 Angiotensin, iv, 0.1 pg/kg 201 i 9* 73 + 20* +247 z4 63t Reserpine (4 dogs) Control 62 ± 2 119 ± 14 Angiotensin, iv, 0.1 pg/kg 81 :1: 7 136 4- 11 + 14 ± 3t * The mean values of the blood pressures and renal blood flows produced by angiotensin II in dogs with and without reserpine were compared with the mean values of the control blood pressures and renal blood flows. Differences were statistically evaluated; p < 0.05. t The per cent change of control in renal vascular resistances produced by angiotensin II in dogs with and without reserpine was statistically evaluated for significant differences; p < 0.05. ANGIOTENSIN AND THE AUTONOMIC NERVOUS SYSTEM 1919 396%2o of control. A parallel effect on the renal tensin II, was reduced by 22%, 12%o, 13%o, and vascular bed was observed for nicotihne and angio- 35%o of control by intravenous angiotensin II (0.1 tensin II. When the renal vascularr response to Mg per kg) after levarterenol administration in the nicotine was eliminated or reduced, a similar re- reserpine treated dogs. Simultaneously, levar- sponse to angiotensin II was observed. terenol restored partially the renal vascular re- The relationship between the renaI vascular ef- sponse to nicotine (50 ug per kg, intravenously). fects of nicotine and angiotensin II vvas expressed in several ways. The effect of angi(otensin II on Discussion the renal vasculature was usually pre31didtfl-d:UULV- herTuY th0-LIM The renal vascular activity of angiotensin II has response of the renal vascular bed to A been demonstrated in the present relationship between nicotine and ang>.nicotine. experiments to Iotensin be related closely to the autonomic nervous system. terms of an available tissue store of coatecholamines the vascular action of II is op- was suggested further by a reduce'Itchoamineof Thus, angiotensin triction when posed by those agents whose primary effect is on angiotensin II to elicit renal vasocons the fiber administered shortly after nicotine (Figure 7) sympathetic postganglionic (bretylium or ted dog, ex- and guanethidine) (14) by agents that alter Furthermore, in the reserpine treat sympathetic activity by a haustion by repeated intravenous iedjtio, ef central action (hydrala- nicotine (50 per kg) of an alreadyinjections do zine) (15). Thoseautonomicblockingagentsthat fug do not interfere with release of the sympathetic pleted sympathetic neurotransmitter E tpartiallyede- stretresulted neurotransmitter, e.g., hexamethonium (16), or in a loss of the renal vasoconstrictc ativity coincident with the have a primary effect at the sympathetic a recep- angiotensin II, lc tor site nal vascular effect of nicotine. (phentolamine) (17) will not block the renal vascular of more restoration activity angiotensin II. A In the animals receiving reserpin( Xgiotensin precise definition of this relationship may be made of the renal vascular response to a!rigiotensin II only conditionally. For example, the vascular ac- was not accomplished by tyramine (100 to 300 ~Lg tivity of angiotensin II may have a cholinergic de- per kg). In these reserpine treated animals, ad- terminant. Thus, the stimulation by angiotensin ministration of as small an amount of levarterenol II of the isolated intestines has been reported to as 0.25 ug per kg resulted in a partia i restoration depend on the release of acetylcholine, since the of the renal vascular reactivity to ax grotenlsbl Id response is abolished by depleting actetylcholine administered intravenously. Thus, th and is potentiated by anticholinesterases (18, 19). flow, which previously was unchang4ed by angio_ A cholinergic link for sympathetic nervous activity has been proposed (20, 21). According to Eo 1lmini Burn and Rand, acetylcholine liberated by the adrenergic nerve impulse releases catecholamines. Whether or not a cholinergic mechanism is in- IL- volved in the vascular activity of angiotensin II Z o cannot be stated from the evidence provided by the a 0- present experiments.

I O- Bretylium and guanethidine, which have been 0NE)E ,,, reported to block sympathetic nervous activity, op- Q.a. CU__ posed the renal vascular activity of angiotensin II Cr00 (Figures 3 and 5). It would be premature, how- 0 0- ever, to adduce this as evidence of the dependency T T NICOTINE ANGIOTENSIN ANGIOTENSIN of the vascular activity of angiotensin II upon the I mg iv 0.2 pg/kg iv 0.1 pg/kg iv sympathetic neurotransmitter, for bretylium and 7-10-64 22 kg DOG guanethidine may have a primary anticholinergic FIG. 7. THE ALTERATION OF THE RENAL VASCULAR AC- effect. Thus, Bhagat showed that the activity of TIVITY OF ANGIOTENSIN II BY NICOTINE IN A DOG UNDER guanethidine upon catecholamine stores of the rat MORPHINE-CHLORALOSE. ventricle was prevented by hemicholinium, which 1920 JOHN C. McGIFF AND THOMAS M. FASY inhibits acetylcholine synthesis (22). Bhagat lease of levarterenol-3H from the rat heart, which further demonstrated that administration of cho- release is an index of transmission of impulses in line chloride, which promotes acetylcholine syn- postganglionic fibers (16). In the same prepara- thesis, will restore the capacity of guanethidine to tion, on the other hand, bretylium reduced the re- deplete catecholamines from subcellular fractions. lease of levarterenol-3H, which is consistent with Bretylium has been suggested by Burn and Froede the presumed effect of bretylium on sympathetic to have anticholinergic activity (23). With these nerve transmission and the renal vascular response qualifications in mind, a tentative construction of to angiotensin II. In addition, Robertson and the relationship between angiotensin II and the Rubin (18), using an isolated system, showed that autonomic nervous system will be attempted. hexamethonium did not block the contractile re- The effects of acute renal denervation upon the sponse of the guinea pig ileum to angiotensin II. renal vascular activity of angiotensin II suggested The present results suggest that the renal vas- at least two elements in the renal vascular re- cular effect of angiotensin II depends in part at sponse. After renal denervation, the renal vascu- least on the presence of catecholamines, for reser- lar effect of angiotensin II administered intrave- pine will reduce or abolish the reactivity of the re- nously was eliminated, whereas renal intra-arterial nal vasculature to angiotensin II. A reduction of injection of angiotensin II continued to elicit renal the vascular activity of angiotensin II in the hind vasoconstriction. These observations agree with limb of the dog has been reported after reserpine those of Laverty (3) and Zimmerman (4). (25). In two of the present experiments, a Laverty demonstrated that the vasoconstriction of schedule of reserpine administration that might be the rat hind limb elicited by intravenous angio- expected to reduce catecholamines to less than tensin II was immediately abolished by denerva- 10% of the control levels (26) failed to reduce tion of the limb. Zimmerman showed in the dog the renal vasoconstrictor activity of angiotensin that after acute sympathectomy of the hindquarter, II. In all of the experiments in which reserpine the vasoconstrictor response to intra-arterial ad- was used, tyramine failed to elicit renal vasocon- ministration of angiotensin II was reduced. A striction. This observation confirms the near de- bimodal mechanism of the action of angiotensin II, pletion of catecholamines effected by reserpine, for one nerve mediated and the other direct, has been Crout, Muskus, and Trendelenburg have reported postulated by Benelli, Della Bella, and Gandini that in isolated guinea pig atria pretreated with (8). In cross-perfusion experiments in dogs, reserpine, only 2% of the normal tissue catechola- Bickerton and Buckley (5) described a central mines was required for tyramine to produce a re- hypertensive effect of angiotensin II that was sponse (27). Nicotine, the renal vasoconstrictor blocked peripherally by an adrenergic blocking activity of which depends on the presence of local agent, piperoxan. In the present experiments, the catecholamines, had effects similar to angiotensin effects of cervical cord transection upon the renal II. In the four experiments in which the renal vascular activity of angiotensin II administered vascular effect of angiotensin II was abolished or intravenously suggest the importance of central reduced by reserpine, the renal vascular activity of nervous activity in the fully developed renal vas- nicotine was similarly attenuated. Furthermore, cular response to angiotensin II. administration of angiotensin II directly after The failure of hexamethonium to alter the renal nicotine, after the return of renal blood flow to vascular response to angiotensin II would appear control levels, resulted in reduced vasoconstriction to exclude a central nervous mechanism in the re- elicited by angiotensin II (Figure 7). These renal vascular response to angiotensin II adminis- sults suggest that nicotine and angiotensin II re- tered intravenously. However, hexamethonium quire an identical or similar store of the sympa- has been reported to be ineffective in the blockade thetic neurotransmitter for their activity. This of the pressor response to some sympathetic gang- store is either affected by these agents directly or lionic stimulants, whereas atropine will block this through a common intermediary of catecholamine pressor response (24). Hertting, Potter, and release as acetylcholine. A parallel reduction of Axelrod showed that hexamethonium failed to re- the effect of nicotine and angiotensin II on the duce, or only slightly reduced, the spontaneous re- contraction of the isolated ileum of the guinea pig ANGIOTENSIN AND THE AUTONOMIC NERVOUS SYSTEM 192 t and rabbit after depletion of acetylcholine has been shown potentiation by angiotensin II of the con- observed by Robertson and Rubin (18). tractions of the vas deferens and spleen produced Further evidence to support the participation of by sympathetic nerve stimulation (8). catecholamines in the renal vascular response to Autonomic blocking agents have been considered angiotensin II is provided by the effects of tyra- to be ineffective in blocking the vasoconstrictor ef- mine. After blockade of the renal vascular effects fect of angiotensin II (4, 31). The pressor re- of angiotensin II by guanethidine or section of the sponse in normal human subjects to angiotensin II spinal cord, tyramine restored the reactivity of the was shown by Laurence and Nagle to be unmodi- renal vasculature to angiotensin II. Tyramine will fied by bretylium or guanethidine (32). An not restore the renal vascular activity of angio- awareness that the pressor effect of angiotensin II tensin II after depletion of catecholamines by re- may be related to several actions of angiotensin serpine. Several lines of evidence relating to the II, such as release of catecholamines from the effects of tyramine (or other indirect acting sym- adrenal medulla (7) and a positive inotropic ef- pathomimetic amines) and guanethidine converge fect (33), should invalidate any conclusions drawn on the autonomic nervous mechanism, which re- from assuming a direct relationship between the leases the sympathetic neurotransmitter. Gua- pressor response and vasoconstrictor properties nethidine has been reported to block the pressor of angiotensin II. Guanethidine has been reported effect and the development of tension in isolated to be effective in treating the hypertension of oc- atria of rats induced by tyramine, in the absence clusive renal arterial disease (34) in which angio- of any significant reduction of tissue catechola- tensin II probably initiates and may well sustain, mines (28). Bhagat has suggested that guanethi- in concert with other factors, the hypertension dine and tyramine act on the same store of levar- (31). The present investigation suggests that the terenol; a competitive antagonism between gua- efficacy of autonomic blocking agents in reducing nethidine and tyramine was considered probable the blood pressure of nephrogenic hypertension is (22, 29). Confirmation of this view is afforded predictable. Further exploration of the relation- by Matsumoto and Horita (30), who demon- ship between autonomic blocking agents and al- strated that other indirect acting sympathomimetic terations of the release of the neurotransmitter is amines (dexamphetamine and methamphetamine) necessary before the present formulation of the of which tyramine is the prototype reduce the up- relationship between autonomic blockade and take of guanethidine by rabbit hearts and fulfill the angiotensin II may be considered more than conditions of competitive antagonism. Competi- tentative. tive antagonism between tyramine and guanethi- Summary dine posits overlapping activities varying in strength. Thus, guanethidine has weak sympatho- The effect of angiotensin II upon the renal vas- mimetic actions and strong sympathetic nerve culature is dependent upon two elements: 1) in- blocking activity, whereas the indirect acting sym- tactness of autonomic innervation of the kidney, pathomimetic amines have strong sympathomi- and 2) a local effect that may have an autonomic metic actions and weak sympathetic nerve blocking nervous determinant either in terms of a critical activity. store of sympathetic neurotransmitter or cholinergic Additional confirmation of the close relationship involvement in the vascular effect of angiotensin between sympathetic nerve activity and angioten- II. Interruption of autonomic nervous activity by sin II is provided by observations on the aug- renal denervation and cervical cord section will re- mentation by angiotensin II of the effects of sym- duce or abolish the renal vascular reactivity to pathetic nerve stimulation and sympathomimetic angiotensin II. The effect of autonomic blocking agents. Thus, angiotensin II has been demon- agents on renal vascular reactivity to angiotensin strated by McCubbin and Page to augment the II was predicted according to their activity at the pressor effect produced by sympathomimetic neuroeffector site. Those agents, guanethidine, agents (tyramine and ephedrine) and those pro- bretylium, and hydralazine, which influence sym- cedures that release levarterenol from sympathetic pathetic nervous activity by reducing the release of nerve endings (6). Benelli and associates have the neurotransmitter, block the renal vascular ac- 1922 JOHN C. McGIFF AND THOMAS M. FASY tivity of angiotensin II, whereas those agents that 11. Zimmerman, B. G. Influence of several autonomic primarily block receptor sites (phentolamine) or blocking agents on segmental vascular reactivity. influence ganglionic transmission (hexametho- J. Pharmacol. exp. Ther. 1964, 146, 200. 12. Snedecor, G. W. Statistical Methods Applied to Ex- nium) are without effect on the renal vascular periments in Agriculture and Biology. Ames, response to angiotensin II. The restoration by Iowa, Iowa State College Press, 1956. tyramine of the renal vascular response to angio- 13. McGiff, J. C. Regional flow response to cigarette tensin II suggests the participation of a particular smoking in the dog. Fed. Proc. 1963, 22, 509. store of sympathetic neurotransmitter in the vas- 14. Green, A. F., and R. D. Robson. Comparison of the cular activity of angiotensin II. effects of bretylium, guanethidine and bethanidine on smooth muscle responses to different rates of sympathetic nerve stimulation. Brit. J. Pharmacol. Acknowledgments 1964, 22, 349. We acknowledge gratefully the assistance of Dr. Solo- 15. Tangri, K. K., and K. P. Bhargava. The central mon D. Erulkar in performing the cervical cord sections. hypotensive action of l-hydrazinophthalazine We are indebted to Dr. Calvin F. Kay for support, ad- (C-5968). Arch. int. Pharmacodyn. 1960, 125, vice, and critical review of the work. Drs. George B. 331. Koelle, Francis C. Wood, and Robert L. Volle gave 16. Hertting, G., L. T. Potter, and J. Axelrod. Effect helpful advice in the preparation of the manuscript. Mr. of decentralization and ganglionic blocking agents Leon H. King rendered excellent technical assistance. on the spontaneous release of H'-norepinephrine. Dr. William E. Wagner of Ciba Pharmaceutical Co. J. Pharmacol. exp. Ther. 1962, 136, 289. generously supplied reserpine, phentolamine, guanethidine, 17. McGiff, J. C. The renal vascular response to and angiotensin II. hemorrhage. J. Pharmacol. exp. Ther. 1964, 145, 181. References 18. Robertson, P. A., and D. Rubin. Stimulation of in- testinal nervous elements by angiotensin. Brit. J. 1. Yonkman, F. F., R. Jeremias, and D. Stilwell. Angio- Pharmacol. 1962, 19, 5. tonin myotropism. Proc. Soc. exp. Biol. (N. Y.) 19. Khairallah, P. A., and I. H. Page. Mechanism of ac- 1943, 54, 204. tion of angiotensin and bradykinin on smooth 2. Furchgott, R. F. The pharmacology of vascular muscle in situ. Amer. J. Physiol. 1961, 200, 51. smooth muscle. Pharmacol. Rev. 1955, 7, 183. 20. Burn, J. H., and M. J. Rand. Sympathetic post- 3. Laverty, R. A nervously-mediated action of angio- ganglionic mechanism. Nature (Lond.) 1959, 184, tensin in anaesthetised rats. J. Pharm. Pharmacol. 163. 1963, 15, 63. Zimmerman, B. G. Effect of acute sympathectomy on 21. Jacobowitz, D., and G. B. Koelle. Histochemical 4. and responses to angiotensin and norepinephrine. Cir- correlations of acetylcholinesterase catecholamines in postganglionic autonomic nerves of the culat. Res. 1962, 11, 780. Pharmacol. 5. Bickerton, R. K., and J. P. Buckley. Evidence for a cat, rabbit, and guinea pig. J. exp. central mechanism in angiotensin induced hyper- Ther. 1965, 148, 225. tension. Proc. Soc. exp. Biol. (N. Y.) 1961, 22. Bhagat, B. Modification of the effects of guanethidine 106, 834. on cardiac catechol amines by various agents. 6. McCubbin, J. W., and I. H. Page. Renal pressor Brit. J. Pharmacol. 1964, 22, 238. system and neurogenic control of arterial pressure. 23. Burn, J. H., and H. Froede. The action of sub- Circulat. Res. 1963, 12, 553. stances which block sympathetic postganglionic 7. Feldberg, W., and G. P. Lewis. The action of pep- nervous transmission. Brit. J. Pharmacol. 1963, tides on the adrenal medulla. Release of adrenaline 20, 378. by bradykinin and angiotensin. J. Physiol. (Lond.) 24. Roszkowski, A. P. An unusual type of sympathetic 1964, 171, 98. ganglionic stimulant. J. Pharmacol. exp. Ther. 8. Benelli, G., D. Della Bella, and A. Gandini. Angio- 1961, 132, 156. tensin and peripheral sympathetic nerve activity. 25. Baum, T. Vascular reactivity of reserpine-pretreated Brit. J. Pharmacol. 1964, 22, 211. dogs. J. Pharmacol. exp. Ther. 1963, 141, 30. 9. McGiff, J. C., and H. D. Itskovitz. Loss of the 26. Kuntzman, R., and M. M. Jacobson. On the mecha- renal vasoconstrictor activity of angiotension II nism of heart norepinephrine depletion by tyramine, during renal ischemia. J. clin. Invest. 1964, 43, guanethidine and reserpine. J. Pharmacol. exp. 2359. Ther. 1964, 144, 399. 10. Mandel, M. J., and L. A. Sapirstein. Effect of angio- 27. Crout, J. R., A. J. Muskus, and U. Trendelenburg. tensin infusion on regional blood flow and regional Effect of tyramine on isolated guinea-pig atria in vascular resistance in the rat. Circulat. Res. 1962, relation to their noradrenaline stores. Brit. J. 10, 807. Pharmacol. 1962, 18, 600. ANGIOTENSIN AND THE AUTONOMIC NERVOUS SYSTEM 1923

28. Bhagat, B., and F. E. Shideman. Mechanism of the 32. Laurence, D. R., and R. E. Nagle. The effects of inhibitory action of guanethidine on cardiovascular bretylium and guanethidine on the pressor responses responses to tyramine and amphetamine. J. Phar- to noradrenaline and angiotensin. Brit. J. Pharma- macol. exp. Ther. 1963, 140, 317. col. 1963, 21, 403. 29. Day, M. D. Effect of sympathomimetic amines on 33. Fowler, N. O., and J. C. Holmes. Coronary and the blocking action of guanethidine, bretylium and myocardial actions of angiotensin. Circulat. Res. xylocholine. Brit. J. Pharmacol. 1962, 18, 421. 30. Matsumoto, C, and A. Horita. Studies of the 1964, 14, 191. antagonism of guanethidine by methamphetamine. 34. Dustan, H. P., I. H. Page, E. F. Poutasse, and L. Biochem. Pharmacol. 1963, 12, 295. Wilson. An evaluation of treatment of hyperten- 31. Page, I. H., and F. M. Bumpus. Angiotensin. sion associated with occlusive renal arterial disease. Physiol. Rev. 1961, 41, 331. Circulation 1963, 27, 1018.