NOTES 92 1

direct effect of the drug on A-V transmission, the rest being secondary to the pressor response of the drug. The direct effect is seen when bleeding prevents the pressor response, and is approximately one-half that observed when the tdood pressure is allowed to rise.

lrlcreases in blood pressure are normally compensated for by an increased discharge of the cardiac vagus nerves and decreased sympathetic discharge, both of ~vfiich would slow A-V transmission. The vagi were cut in the present experiments but the syrrlpathetic nerves were left intact. I t is therefore possible that a part of the effect of the pressor response was due to decreased sympathetic discharge. Ho~vevcr, it is unlikely that this was of major importance because the effects of increases in blood pressure which we observed, either on injection of the drug or on meclnanical occlusion of the aorta, were quantitatively very similar to those reported by Kohli et nl. (6), who measured FRP in spinal vagotor~lized animals.

1. K. I . ~IELVIH,H,E and F. C. 1,er. Arch. Intern. I'harmacodyra. 92, 109 (1952). 2. A. BIAXCIII, A. F. Dl< ~ S C H A E P D R ~ ~ E K , G. R. DE VLEESCFIITOUWER, P. PKEZIBSI, and HI.

STORMORKEK. Arch. Intern. Pharmacodyn. 120,459 (1959). 3. P. PKEZIOSI, G. K. rm VLEESCHIIOUWEIQ, A. 1;. DE SCWABPDKYVEK, arid A. RIANCWH. Arch.

Iiatern. Pharmacodyn. 121, 506 (1959). 4. j. L. GILBEKT, 6. ~,AX(;E, E. POLEVOY, and 6. H ~ s o ~ c s . J. Pharmacol. Exptl. Tkerap.

123,9 (1958). 5. B). EWEIJ and C. M E ~ E Z . Arch. Intern. E'kysid. 72,44 (1964). 6, J. D. KOHLI, R. ?'uT'I'I.E, P. E. DRESEL, and I. K. INNES. J. Pharnaacol. Exptl. Therap. 153,

505 (1966). 7. 0. KKAYEK, J . J. ~ I A N D O K I , and C. -MENDF:Z. J. Pkarmacol. Exptl. Therap. 103,412 (1951j.

NOTE ON THE PREPARATION OF RENIN SUBSTRATE'

A new ra~icrolmethod for tlle ~neasureilaelmt of plasma rerain activity and renal re~niim content of rats has L~een establisl~ed by Bolmcller et al. (Ij . Th i s ~netliod is based on the yririciple that reniim contained in 0.1 1x11 of plasn~a when incubated over a 12-hour period witla excess substrate (obtained , according

I\Vork supported through grants from the Medical Research Co~ancil of Canada, the 1,ife lrlsurarlce Xledical I<es=rch Fund, Kose~nont, Pa., the Quebec Heart Fouladation, and the Ciba Company, Montreal,

2I;eiilow of the Nedical Research Council of Canada. jXTedica1 ~Zssociate of the Rledieal Research Council of Canada, and Associate Professor of

Riledicime, University of Montreal.

Canadian Journal of Physiology and Phar~nacology. Volurne 45, 921 (1989)

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922 CANAHPIAN JCBURPu'Ak OF PBIYSHOLOGY AND PHAKMACOLOGU. VOL. 45, 1967

to lIaas' procedtare (%), from rats bilaterally riephrectol~lized 24 tloul-s pre- viously) will form detectable arnourats of a~igiotensin. The latter is protected from proteolytic degradation by the addition of Dowex resin 50LV-X2 (NM,+) to the iancubation nledium and is n~easured by a rat pressor assay. 'Tllis pro- cedure is 11 iglily reprodalcible, sensitive, and specific, and permits the repeated and sequential measurement of reni11 activity in rats.

,- I lie application of this nlicromethod to dogs is Interesting and imysortar~t. YBrevious findings of renin a-aaer-tsurerments in this species were h s e d naostly on the vasopressor activity of crude plasma and often j~ielded conflicti~~g results. In applying Bouclner's rraicrolnethod to dogs, important difliculties were en- countered, related to the great variations in substrate concent rations ohta iiie(B from plasma of different nelshrectoinized dogs. Althoargh it is known that this species has a low conceiitration of ren in sul~strate, \2'17 ic17 is increased only %fold after nephrectomy (31, as conipared with 4- to 15-fold in rats (-I-G), it was thorrgl~ t that the variations of suhstra te concentration calcountercd ill dogs deep1 y anesthetized ~vitli pen tobarbital and to a lesser extent n-itll cl~loralose, coultf be explained on the insis of the effect of anestlaesia on the release of renin. I t has Exen previous8~- sllo~vn it1 our laboratory by S t r o ~ ~ g and 'Fre~nl~lay (ua~pul~lisl~ed data) tllxt deep perltobarhital anesthesia ill dogs frecguea~tly indeices a 2.5- to 3-fold rise in blood reriin :ictivity. Sin-nilar increases \vetre found in anesthetized 111nmans by the sctme workers.

Experimental Data Prelirninary experiillents were done to adapt the metllod to dogs by stud>-ing

t l ~ e kinetics of the enzymatic reaction. Renin substrate was prepared according to ETar~s' procedure. In the first series of experiralents, plasma was c:ollecteci from nine mongrel dogs, l~ilaterally riephrectornized 24 (-11, 48 (21, and 7% ljours (3) previorasly . I-'entol-jarbital was arsed as the ancstkretic agent dtaa-iug t11 e rlephrecton~ y procede~re. Sut~strate c o n c e ~ ~ tra tion was studied in each anisaial Ily incubating the sul~strate preparation in increasiing amounts (50 to 500 ~ n g of freeze-dried sul~strate) in the presence of a cor~stant cl~t;tiltity of slog kidney extract. I n these experi~nents, the rate of angiotensin prczdalction n ;is forand to be depcnde~at on sulsstrnte concerntration. In five instances, the substrate preparatiori was iacubatetl for 24 hours, without added renir~. Three of tllese five preparations gerieratetd vasopressor su bstances, the highest yicf d being obtained with ttae sutsstrate preparation fro~m dogs ~lephrectornized 23 1-1ours previous1 y.

In a second series of experiments, tlae substrate obtained from six nlorlgrel dogs pretreated with angiotensiai before and during tlse anestl7esi;t ~ ~ r o c c d urc for nepl~rectonly, \vas used. Tl, is was cfone by a constant intravenous infusion of vr-tline-5 angiotensin I l , aspartic p-amide (El y~aerterisia~, ('iba) a t 200 ng,'kg per min. The infusion was l)egu11 half an hour before cliloralose anestlaesia (20 n~l,/kg of a saturated solution in 0.9y0 saline) and continued until the sccoild kidney was reniovcd. The total duration of angioiensi~l irafuslon airas

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NOTES

SUBSTRATE (mg)

I 1 Effect on dog'$ rcilin s~tbstratc, of ai lgiutensi~~ iilfusiotl (200 ng-/kg pcr nlinj before and during chioralose ancsthesi,~ rmtl ncphrectonly. Dogs anesthctiscd with c.hlorc~iose and infu4cd ( I ) with angiotensin and (2) with control isotonic: saline. The substrate obtcrincct from thc two dogs was iilcubctted with the same amount of dog kidney extract, with two cotltrol incubations v;ithout renil1 extract.

a t least 80 rnin. These dogs were bled 4s hours after nephrectonlj-, a11rH t11c p1asn1;-1 was processed in tlae same Inanrler as in the first group, lor substrate prcpr-tratior~. Kinetics of the renin sul ,strate reaction in arigioterlsir~-treated dogs show that arngiotensin formation reaches a plateau \vitIa 150 rng of freeze- dried substrate preparation (Fig. 1). Irm corltrast, su1,stratc frornl four control dogs that received tlle stkrne dose of c.hloralose ancl a control infusion of isotonic saline forlned angiotensi~a i11 a~noun ts parallel to the sn bstra te concentrc~tion (up to 300 mg), 11-helm incubated with dog kidney extract.

Substrate obtained frorrl three dogs pretreated \I-it11 arlgioterlsin failed, n-lien irlcubateti for 24 hours ~v i t l~o~a t added renin, to generate vasopressor saabstarlces in t ~ v o cases and lil~err-ated only traces in the third one. On the o t l~er hand, substrate ol~ttair~ed from four dogs anestlletizcd with cllloralosc formed, I\-lleri incubated alone for 24 hcsurs, detectable amounts of arlgioter~sin in t\\-o instan(-es.

Discussion Because of the effects of deep a~sestl~esla on renil1 litm-ation, it was thought

tlmt the variable amounts of renin released in sol-rle dogs at the time of r-tncs- tllesia 11-ere prolsaljly responsible for the decrease ira saal~strate level after nepl~rectomy. This coulci \\-ell explain the I-raarlted variations itn sul>strate coracentration in control dogs.

'Tlre second series of experinlents \?-as tllercfore designed to test this possi- Ijilit>-, and tile release of re~nin I V ~ S prcven tcci by thc infnsiorl of nngiotensin 30 mi11 hefore and during the anesthesia procedure until complete removal of both kidneys, since it 1x1s 1)eerl dernonstrated hot11 in llumans (9) and iri clogs (3) il~;tt angiotensin suppresses re~ain release.

rI'l~is new step permits the o1)telltion of a reprodncit,le suk~strate preparl~tion

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924 Crlh'ADIAZN J(PURN.lL OF PEIYSIBI,BGY AND PIIL\KMX@0LOGY. VOL. 45, 1967

from one aninla1 to the other and has greatly facilitated the adaptation t ( ~ dogs of Boucber's micromethod for the measurement of renin activity. Iden tical restnlts have been obtained in rats and have confir~ned these data (1).

1. I<. Roucar~ra, J. M ~ N A R D , and J. GENEST. Can. J. PhysiaH. Pharmacol. This issue. 2. E. IIAAS, 11. GOL~BT~ATT, E. C . G~PSON, and Id. L~L\VIS. Gircialatian Res. 19, 73'3 (1066). 3. D. COLLINS and C. D. I IARAK~~T~. Circ~~lation Res. 2, 196 (1954). 4. IP. I~EAQIIIER. Am. J. I'hysio1. 208, 1083 (196.5). 5. TIs REGOLI, Pi. B R ~ N N B R , and F. G~zoss. Helv. Physiol. I'harrnacol. Acta, 19, ClOl (1961). 6. J . BIXG. Acta H'athol. Microbiol. Scand, 60, 311 (1964). 7 . J . GENEST, J. DE CHAMPLAIN, et al. Pruceedings of the Council for High Blood Preesnre

Research, Cleveland. Kov. 13 and 14, 1964. American lIeart Associatiurn. p. 97. S. -4. J. I'ANDEK. Proceedings of the Coancil for Iiigh Blood E'ressure I<esearch, Cleveland.

Nov. 13 and 14, 1964. Anxericail Heart Association. p. 126.

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