Myotropic affinities of angiotensin I1 and des-Asp'-angiotensinII in rabbit aorta and femoral artery by lllicroassay IL ILL HA^^ H. WAUCHAND THEODORE E. BALES Departmenfs of ,Wedicitae ar~dPhysiology, Eosr Caroli~liaUtliverss'fySchool of iWcdicirte, Grcetaville, NC, U.S.A. 27834

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Received December 4, 1978 FVaerc~n,FV. II., and BALES,T. E. 1979. Myotropic afxmitier of angiotensin 11 and des-Asp1angiotensin 61 in rabbit aorta and femoral artery by n~icroassay.Can. 9. Physiol. Pharmacol. 57, 1254-1 266. Dose-dependent isornetr~ccontractions to [Asp1,11e6]1-angiotensinHI (AB [ ) and des-Asp'-[I leb]ang~otensinI1 (AIII) were obtained with 3-mm-wide rings of rabbit thoracic aorta and fenmoral artery in rnicrobaths. A period of 2.5-3 h wab required to obtain reproducible contractile responses of increased sensitivity. Contractioras developed faster and they were much more forceful but less sustained in femoral arterial rings than in aortic rings. Noncumulative doseresponse curves with A11 and AIIH were parallel and reached the 5arne maximum. Peak contractile responses were linearly proportiotaal to the receptor \timulation predicted from the mass action equation and the concept of intrinsic activity relating bath dosage of agonist to the number of myotropic receptors occupied by A11 and by AIIH. These fi~sdingsvalidated measurement of the myotropic afinities of both tissues for A11 and A111 by the obtained EDLO values. In 0.6-mL baths, developed with the use of a meshed screen for reoxygenation, the apparent aflinities of aortic muscle for A11 and A111 averaged 0.149 and 0.81030 nM, respectavely. The mean affinities were much greater at 0.594 and 0.236 nhf, respectively, in fenaoral arterial muscle. The inyotropic affinity for AIIH relative to that for A11 averaged 2.24(;, in the aorta but 40.8' in the femoral artery. The apparent aflinities were reduced anti contractions less forceful in 0.24-mL baths without regassing. The results suggest that A11 and ABII may stimulate the same angiotensin receptors in aorta and femoral artery and that the receptors may be different in structure or immediate enbiroiarnent in these two vascular tissues. WAUGII,W. H., et BALES,T. E. 1979. Myotropic affinities of angiotensin I1 and des-Asp1angiotensin 11 in rabbit aorta and femoral artery by microassay. Can. J. Physiol. Pharmacol. 57, 1256-1266. On a obtenu des contractions [email protected] sections d'aorte thoracique et d'artkre fernorale de lapin, de 3 mm d'dpaisseur plackes dans un microbain. Ces contractions soimt [email protected] de la [Aspi, Iley-angiotensine I I (Ail) et de la des-Asp1-[Ileq-angiotensine11 (AIII) et dkpenclent de la dose. Une pCriode de 2.5-3 h est recluise afin ci'oktenir des reponses contractiles reproductibles de sensibiilitt! croissante. Les contractions ont lieu plus rapidement et solmt plus puissantes inais de moindre dur6e avec les anneaux d'artkre fkmorale comparativenlent aux sections d'aorte. L-es courbcs reliant les reponses aux doses de AlI et de All1 de f a ~ o nnoncumulative, sont parallkles et passent par le m2me rnaxin~unm.Les valeurs rriaxirnales dcs reponses contractiles sont linkaire~nentproportionnelles B la stimulation des rkcepteurs, tel que predit par l'kquation d'action de masse et par le concept d'activitk inatrins6yue qui relie le dosage en agoniste de la solution ciu bain au nombre de rkcepteurs myotropiques coiacernds par AII et AIII. Ces rhultats valident la mestire des affinitks myotropiques ales deux tissus (? A % I et All1 par les valeurs de ED50 qui sont obtenues. Lorsqu'on travaille avec des bains de 0.6 rnL dans lesquels la reoxygknation se fait (? l'aide d'un tissu B mailles, les [email protected] du muscle B AH1 et %. A111 valent en moyenne respectivement 0.149 et 0.0030 11.44. Les affinites moyennes sont beaucoup plus importantes avec 16 muscle d'artkre fernorale et valent respectivement 0.594 et 0.246 nM. L'afinit6 myotropique & ATTI relativement celle Li AII est de 2.26(,; pour I'aorte et de 40.8'; pour 19artkre f6morale. Les affanit6s apparentes dilnin~lentet les contractions sont moins fortcs lorsqu'on utilise des brains de 0.24 rn12sans rioxygknation. Ces rksultats suggkrent que All et AIII pourraient stimuler les memes rkcepteurs B l'angiotensine dans l'aorte et Z9artkrefernorale et que ces rkcepteurs pourraient difT6rer par leur structure et par leur environne~nentimrnidiat B 13int6rieurdes deux tissus vasculaires. [Traduit par le journal]

Introduction The predominant angiotensin peptide of the renin-angiolensin system in circulating arterial u

L2

A s a ~ a v ~ ~ ~AII, r o ~IAsp1,ll~"-an%iotensin s: 11; AIII, dcsAsp1-[Tley-armgiotensin 11; PSS, physiological salt solution; gf, gram-force (lkilogram-force = 9.806 65 N ) ; EDSF, mean effective dose.

plasma is BIT in man and dog, and BIII in the rat (caravapgi et a]. 1976; Semple et al. 19'76; Sempk and Morton 1976). The A111 is about 20-38% as potent as A11 when assayed by pressor activity irk in the rat ( B et I~961 ; Campboll ~ et ~ al. 1977) and rabbit (Steele ct al. 19'76). However, the relative potencies of B I I and its heptapeptide homo-

0008-4212/'79jIll256-]t 1 $01.00/0

(a1979 National Research Council of CanadajConseil national de recherches du Canada

~

WAUCH AND BALES

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logare, ,4111, in peripheral vascular beds are not known. It is also not known whether AT1 and A111 act on a comIalon angiotensin receptor in vascular smooth ~nuscleor whether they act on two vascular receptors, with the two angiotensin peptides working in tandem in the biological response of contraction (Peach 1 977; Ackerly, Moore et al. 1977). Functional heterogeneity may exist among vascular angiotensin receptors, since AIII and its amide dcrivative, [Asp1, Val5]-angiotensin 11, constrict the renal vasculature equally, while the latter peptide is more potent in the femoral bed of the dog (Taub et al. 1977). TIne characterizations c ~ the f direct interactions between the awgiotensins and their inyotropic receptors must be determined in isolated tissue systems rather than in in vivo systems (Furchgott 1955; Ariens et al. 1964n; Regoli et al. 1974; Devynck and hfeyer 1976). In vitrc~studies c ~ f~~oncu~nulative dose-response curves in helical strips of rabbit thoracic aorta have indicated that A11 acts directly on specific rnyotropic receptors, that A11 and ATTI have the same intrinsic activity, and that AIIII, on a molar basis, has a mean affinity or potency of oi~lyabout 1.6 or 5.7% of that of A11 (Wioux et al. 1973; Regoli ct al. 1974; iMoore and Khairallah 1976). In this investigation, the intrinsic activities and rnyotropic affinities of AII and A111 have been determined in both the isolated thoracic aorta and the isolated femoral artery of the rabbit. Vascular rings (circular segments) rather than helical strips were used because of the advantages shown by Hooker et al. (1977) with the use of vascular rings. The femoral artery was chosen for study since it has a greater sensitivity to awgiotcnsin ([Asnl, Val5]-angiotensin 11) than the thoracic aorta and since both rabbit aorta and fernoral artery are relatively resistant to the developnaent of angiotensin tachyphylaxis (Aiken and Vane 1970). In addition, a type of micro tissue bath, specifically developed for the bioassay of small amounts of angiotensin or other agonist in relatively srnalI volumes, was incorporated into the study. Methods P r ~ p u r a t t o nand Apparatus Male New Zc:aland White rabbits, 2.0-3.6 kg in weight, were anesthetized lightly with ether and sacrificed by intracardiac injection of a 1 Ikf KC1 solution (1 rnl_/kg). In some rabbits, the dercending thoracic aorta was excised and placeci pron~ptlyin a Petri dish containing PSS (for its composition see the section on solutions rind drugs), where the aorta was ive In cleaned of excess fat and surrounding c o ~ i ~ ~ e c t tissue. the other rabbits, the right and (or) left femoral arteries

1257

were cleaned of perivascular tissue in sitrr, then excised and placed in a Petri dish containing PSS. From the distal twothirds of the femoral artery o r from the middle part of the descending thoracic aorta, adjacent circ~alarsegn-nents (rings). each appreximately 3 mrn in width, were excised by cutting in par:illel. The vascular rings vcerc premptly suspended in individual 0.6-naI, tissue baths containing PSS, oxygenated with 100% 0.!(humidified) at a Wow of a few b~abblesper sncond. Bath PSS was kept at 37°C: by immersion of the lower two-thirds of the tissue bath assen-nblies (see Fig. 1 ) in a heated water bath. Femoral arterial rings and aortic rings were kept at resting tensions approximating 1 and 2 gf, re4pectively. while the bath fluids were replaced by new PSS, flowing at 0.19 or 0.38 rnL/min. Subdued room lighting of constant I~rrninositywas maintained during the experiments (F~rrchgott1960). Initial equilibration periods of 3 h were used before performing variable dose-response studies. Aerated PSS was infused into the tissue baths by dual iwfusiolz pumps (Harvard Apparatus Co.) through polyvinyl plastic tubing (Tygon S-53-Hl, microbore tubing of 0.76 nam internal diameter, Norton Co., Akron, Ohio). A 7-cm length of ',@gauge stainless steel tubing, interposed in each of the plastic pathways and immersed in the water bath, effected heating of the PSS frona room temperature to water bath temperature before the PSS entered each bath chamber. Also interposed in each plastic conduit was a three-wal tubing connector of 20-gauge stainless steel (Small Parts Tnc., Miami, Florida). When the inflow of PSS was stopped and the plabtic tubing clamped upstream, each three-way connector was wed to empty the bath of PSS by aspiration just before topwise addition of angiotensin (AIB or AIIT) contained in PSS to refill each tissue bath. Aliquots of PSS containing the AII or ABIB were prewarmed by imrnea-sion within containers in the water bath for 1-2 min just before their addition to the bath chambers. The chamber of each tissue bath consisted of a 1.9-cmlong hole (diameter, 6.4 rnria) in a Plexiglas rod (3.0 cm In length, 12.7 mm in diameter). As shown in Fig. 1 , the 0:! inlet of the bath was positioned near the bottom of the chamber. This inlet (23-gauge stainless steel tubing) was attached to pIastic tubing which conducted the 0. supply to each bath. A rectangular area ( 4 riarn in width) of 40-mesh st~~inless steel wire cloth (Small Parts Hnc.. Miami, Florida) extended from near the bottom to the top of each bath chamber. This wire c h t h was fixed close to the O2 inlet by prior in~ertionof the wire cloth through a heat-exchange bar. positionecl across the bath (see Fig. 1 ). The wire cloth, serving as a screen, directed the inflowing 8 2 bubbles upwards at the side of the bath channber and effected gentle oxygenation and stirring of the PSS. The heat-exchange bar (20-gauge stainless steel tubing with indwelling 24-gauge copper wire) served to maintain the contents of each tissue bath at a temperature close to the vvater bath temperature during periods of stopped PSS inflow. A removable support wire s f 28-gauge stainless steel, placed within two small holes, fixed each vascular ring about 1 lam above the heatexchange bar. (For convenience, only one of the holes for the support wire opened externally. The opening was made "liquid-tight" by external applicatioai of silicone elastomer adhesive paste shortly before immersion of each bath assenll3ly into the water bath.) The inside of each bath chamber, the wire screen, heat-exchange bar, and support wire were coated with silicone filnn (Siliclad, Clay-Adams Inc.. New York, New York). A 28-gauge stainless steel wire, with its lower part bent as a horizontal hook (3.5 rnm in longest axis), fixed each vascular ring to a Grass model

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CAN. J. PHYSHBL. PHARMACBL. VOL. 57. 1979

WIRE

C L O T H , 48

W 1 RE

H O O K , 28

S U P P O R T

MESH

gauge

W I R E , 28

gauge

O2 INLET, 2 3 gauge Cu 20

WBRE B N S i D E

gauge

TUBiNG

F L U I D I N L E T , 20

gauge

FIG. 1. Diagram, drawn to approximate scale, of the 0.6-mE-capacity tissue bath assembly. The upper end of the wire hook is connected to a transducer. The tissue bath is partly itnmersed in a heated water bath.

femoral arterial rings for 4-10 min. The devel~pedpeak isometric tensions were measured. The solutions were then aspirated and inflows of PSS lchumed at 0.38 or 0.76 mk; min. In order to avoid tachyphylaxis, the intervals between each application of AIH 2nd ABII were set at 20-30 nain far small doses of angiotensin (to 5 nLWABI in both tissues and to 20 nA1 A111 in femoral artery and 100 wM AIII in aorta) and at 40-45 rnin for larger doses. The intervals between applications were generally 5 min longer in aortic rings than ian femoral arterial rings. Only one noncumulative doscresponse series to AH1 and to AIII was measured in each Solutions a i d Drrrgs ring. Paired dose-response curves to AIT and to A111 were The PSS was of the following composition: NaC1, obtained by using adjacent11 cut rings o l aorta or of fen~oral 135 anh4: KCI. 4.0 m1W: CaCi,, 1.5 miW; MgSO*. 0.7 rnit1; artery. The ED," values obtained from each dose-response Na2HPOk, 1.3 mA4; creatine, 0.2 mM; gla~cose, 5.5 m.44; curve of A l I and of AIBI wcre averaged to calculate the calciian~ disodiinm ethylenediaminetetraa~ctate~ 0.026 m1W; overall mean 9 §EM values of the ED.;,, for AT1 and AIHI neomycin sulfate, 5 mg/dk; 3,-(N-2-hydroxyethylpiperazin- ~ I Ieach series of vascular rings studied. The values of the N'-yl ) ethanesulfo~aic acid, 10 mL?4 (HEPES buffer agent, reciprocal of the AT[ ED,,, value divided by the reciprocal pK 7.3 at 37°C (Good ct al. 1966) ) ; with pH adjusted to 7.3 of the AHHI ED5,,value from each paired experinaent were at 3'7"c' by addition of NaOH. Synthetic All (Asp-Arg-Val- averaged to calculate the mean -t. %EMpaired values of the 'lTry-J1e-His-Pr(7-Phe) and AHIT (Arg-Viil-Try-Ile-His-Pro- reIative myolropic afinity (potency) of AIIT. Statistical l'he ) , obtained froin IJ.S, Biochem. Corp., Cleveland, Ohio, significances were determined by use o f an appropriate and of chemical homogeneity by thin-layer chronmatography, Student's t-test (Sokal and Kohlf 1969 ) . were dissolved in distilled water containing weomycin sulfate, 80 rng/dL. to stock angiotensin concentrations of 4.0 rng/dL. Each angiotensin solution was stored frozen in polyethylene Results containers and only used several times in preparing working solutions of A11 and AIJI. Working solutions were made by Drug Ser-~sitbr~a'by with Time and R e s p o ~ ?Repro-.~~ diluting the stock standards with PSS, prepared on the day ducibdkity of use, to the desired concentrations sf AII and ATII. These The co~atractiIeresponses of the femoral arterial angiotensin solutions were kept within plastic contairners at rings to low test doses of AII (2 IIM) i~~crcaseci proice-cold temperature. FT.03 force-displacement transducer, adjusted to a nominal comnpliance of 5 pm/'gf (Grass lnstrunnent Cs., Quincy, Massachusetts). 'Phe transducers connected, in turn, to a model DIi8 recorder (Electronics for Medicine Iwc.. Pleasantvillc. New York) for ~ncasurenmcnt of rna~sclecomtractions. FOB.smooth drainage during conctant inflow of PSS. a wet cotton thread (size 8 ) was positioned at the top of each tissue bath for siphonage, or else a side hole (3.6 Inmn in diameter) positioned just below the top of the tissue bath was employed as a liquid exit port.

iWeuszerrrnc~tof Drug Responses AnEiolensin added to

bath chambers, were

Ieft in contact with the aortic sings for 7-14 rnin and the

gressively and reached a maximurn wit11 2.5-3 h of incubatioi~in the baths of oxygenated HEPES- buffered I%S (Fig. 2 ) . Once maxi~nalsensitivity had

1259

WAUGH AND BALES H I G H E S T

TABLE1. Development times and plateau times of peak isometric tensions of arterial rings in response to A11 and AIII

TENSION

Tissue

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Aorta ( n

=

7)

Fernoral artery ( n = 8)

Peptide dose, n!%P

Development time, s

Plateau time, s

AII, 100 AIII, 5000

243 5 15* 171 _6 14*

65k 12

ATH, 10 AIH, 108 AIIE, 10 AIII, 1000

260k 14 71 -b3* 245 f 32 58 +6*

64-f-13 20kIt 48 5 10 15 + 2 t

*Difference between means in some tissue, P tDifTeerence between means in samt: tissue, P 1

3

4

INCUBATION

TIME,

2

5

6

< 0.01. < 0.05.

64 _+ 7

+

Wors: Reoxygenated 0.6-rnL baths were used. Time values are means SEM; n is the number of paired rings in which each response was ineasured

once.

h

Frc. 2. Change in rnyotreplc sensitivity of femoral arterial rings with serial doses of 2 nA4 AT1 as a function of time at 37°C in oxygenated WEPES-bufiered PSS. Responses are expressed as the percentage of the highest tension response to 2 n,WABI observed in each ring during the entire incubation period. Points indicate mean responses and vertical bars are or,e SEM of four rings. Highest tension reynnses were 3.99 & 0.60 gf (mean & §EM). A 20-min interval of drug &ashslat and recovery followed each 10-nlin application of AHT.

been attained, the variation of the femoral arterial response to a low test dose of AH1 was small for the next 3-4 h when 20 rnin elapsed between initial washout of the agonist and the next application of the AIH (Fig. 2 ) . The initial equilibration time of 2.5-3 h for increased sensitivity and then an essentially constant response to A11 was similar to that found for helical strips of rabbit aorta in oxygeiaated Krebs-bicarbonate solution in response to adrenaline at l t ~ wdosage (Furehgott and Bhadrakonl 1953) and to [A~nl,Val~~]-angiotensii~ I%and other agorlists at maximal dosages (Altura and Altura 1970). The mean cocffacier~tof variation of the tension responses to 2 nlV AII was 5.596 (range 2,6 to 1 1.4% ) in four femoral arterial rings from the 3rd to the 6th h of irlcuhation with 20-min dnag-washout recovery intervals between each of seven serial applications of the drug. The coeficient of variation of the responses to 2 nM AHT averaged 13.6% (range 7.5 to 1 7.9 96 ) in another four femoral arterial rings over the same time period with 12-mi11 washout recovery inatervals betureen each of 10 serial drug applications. Pattern o f Responses in Aorta and Femoral Artery The tension responses in aorta and femoral artery began within a few seconds of application of AIT and AIPI. The time required for attainment of peak

developed tension (development time) and the period ol stable, maintained peak tension response (plateau time) varied inversely with the dosage of AH1 and AIII. At low concentrations (2 and 3 n M ) of AIT, the development time was about 11-1 4 min in aortic rings and about 8-1 0 rnin in femoral arterial rings. At the maximal myotropic dosage of AII (100 nM), the development time was about 4 min iia aortic rings and about 1-1.5 rnin in femoral arterial rings. The development times and plateau times are summarized in Table 1 for maximal doses of ATI and AIII in aortic rings and for submaximal and maximal doses of the peptides in femoral arterial rings. Plateau times of the peak responses to maximal doses of AT1 and A111 averaged less than 2 min irn the aorta and less than 0.5 min in the fcnloral artery. The durations of the peak isometric contractions were apparently not tern~iisatedby degradation of peptide to smaller concentl-ations in the small bath volumes since the plateau tinres varied iisversely with peptide dosage, as shown for the femoral artery in Table 1. Also, the durations of the peak contractions in the aortic rings were similar to the short plateaux described for isometric contractions in rabbit aortic strips when the responses were induced by submaxiinal and maximal doses of A11 superfused colatinuously at a constant rate or when the resporases were induced by maximal doses of AII, ATII, and a nondegradable analogue of AT1 applied to aortic strips in baths of much larger volume (10 m k ) (St-Louis et al. 1977). At equiactive doses (which required greater bath concentrations of AHIH), development times were significantly shorter for A111 than for AII in both tissues. However, at submaximal doses of the same concentration (e.g., I 0 nAf), the mean development times and the mean plateau times of the femoral ar-

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1260

CAN. J. PHYSIQL. PHARMACOL. VQL. 57, 1979

FIG.3. Typical tracings of rnyotropic efTects of submaxirnal doses of equal rnolarity (10 nM) and of maximal doses s f AIH and AIHI in a pair of femoral arterial rings in reoxygenated 0.6-mL baths. Ordinates, tension in gram-force; abscissas, time. Arrows indicate peptide applications. Drug washouts are not shown.

terial responses to All' and AIII were not different significantly (Table 8 ) . The typical patterns of the contractile effects in two paired rinags of fenaoral artery in response to the same dose sf both peptides (10 nM) and to maximal effective doses of the peptides (1 00 nM API, 1008 11'44 AIII) are illustrated in Fig. 3. Maximal Developed Teazsions Tension responses to ~naximaldoses of both AII and AIII were measured in the same vascular rings at the end of each response study that involved progressively increasing doses of one of the two peptides. The intervals between application of the two drugs to the same rings were identical (40-45 min). Pn seven pairs of aortic rings, the responses to maximal closes of AT1 ( 100 nlW) and A111 (5080 laM) averaged 2.60 zk 1.21 and 2.60 -t- 1.29 gf (mean & SD, n = 14) , respectively. In eight pairs of femoral arterial rings, the responses to maximal doses of AIH ( 108 nM) and APII ( 1008 IM) averaged 9.17 -t- 1.50 and 8.88 1.27 gf, respectively. Thus, A11 and A111 produced the same maximal tension responses both in aorta and femoral artery. However, the maximal contractions were much greater in femoral artery than in the aorta.

*

Dose-Response Relationships The dose-response relationships in rings of aorta and femoral artery are summarized in Fig. 4. The responses are expressed as percentages of the maximal tensions induced by the most effective dose of AII or AIIH. Plotted semilogarithmically, the doseresponse curves of A11 and AIII were parallel and

reached the same maximnkzrn in both aorta and femoral artery. The aorta and fe~noralartery exhibited greater sensitivity to A11 than to AIII, as shown by the more left-sided locations of the d o s e response curves of A11 in Fig. 4. Tn addition, the differences in sensitivity between the two peptides were very much less in the femoral artery than the aorta. Also, the senasitivity of the femoral artery to A111 exceeded the sensitivity of the aorta to API. The bath concentrations inducing a half-maximal contraction (ED,,,) averaged 8.46 -t- 2.52 and 364 -t44 nahf (mean -t- SEM, n = 7 ) for AIP and AIII, respectively, in the aorta. In fc~noralarterial rings, the EDsoaveraged 2.78 zk 0.1 9 and 4.50 ?I 0.44 nM (re == 8 ) for AZZ and AIII, respectively. In application of the mass actiorn e q u a t i ~ nof Clark at equilibrium to the action of stimulating agonist, the effect (response) is related to bath concentration of agonist, the receptor stimulus is linearly proportional to the quantity of drug-receptor cornplex, and the effect is linearly proportional to the receptor stimulus (Ariens et al. 1964~1,1964b). The linear relationships may be represented by where a is a proportionality constant termed the intrinsic activity, expressed as the ratio between the naaximal effect of the drug under study and E,,, that of a reference dmg able to produce a rnaxirnal obtainable effect, KL4is the apparent dissociation constant of the drug-receptor complex, A is the free drug corlcentration in the bath, SA is the evoked receptor stimrilus or activation as a percentage of the maximal obtainable stimulus (S,),and Eg is the

WAUGH AND BALES

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-

%

M A X I M A L

TENSION

FIG. 4. Dose-response curves obtained with AII and AIIT in seven pairs of aortic rings and eight pairs of femoral arterial rings in reoxjgenated 0.6-mL tissue baths. Solid circles and asterisks are mean data points for A11 acting on fenaoral artery and aorta, se5pectively. Sc~lidsquares and open circles are mean data points for AIIL acting on femoral artery and aorta, respectively. Vertical bars represent one SEM. Maximal tension responses to AIIf were of the same magnitude as thosc to AIL in both aorta and femoral artery (see text).

observed effect as a fraction of the maximal obtainable effect (Ariens et al. 1964a, 1 9 6 4 ) . Each of the four mean ED,,, values was used to test the above assumptions, since each mean ED5o value would then represent the mean apparent dissociation constai~t(K,\) of the drug-receptor complex, which is equal to the reciprocal of the apparent affinity of the drug for the rnyotropic receptors (Ariens et al. 1964a. 1964b). For both drugs, an equal intrinsic activity of unity was used. The plots of the observed EA/E,,,values as a function of the calculated S,l/S,,, values in aorta and femoral artery are shown in Figs. 5 and 6, respectively. Inspection of the plots and the ill~astratedstraight line of unity slope for the theoretical equation suggest that the predicted dmg stimulus is approxin~atelyproportional to the peak tension response for the various bath concentrations of A11 and A111 applied both to aorta and femoral artery. The best-fit linear regression equations for the specific data points for A11 and A111 in the aorta (Fig. 5 ) were y 1 . 1 5 ~- 4.3 and y 1 . 0 3 ~ 0.6, respectively. The regression equations for tlle data points for A11 and AITI in the femoral artery (Fig. 1 . 0 3 ~- 8.7, 6 ) were y = 1 . 1 5 ~- 7.7 a12d y respectively. The standard errors of the mean y values at extrapolated zero values for x in these four equatio~ls were k 2 . 4 , 2 2 . 1 , 2 9 . 6 , and t 8 . 6 , respectively. Thc standard errors of the regression coefficieilts (slopes) were t 0.042, t 0 . 0 3 8 , t0.13, and to.13, respectively. None of the four equations differed significantly from the equation for the

-

-

-

theoretical Bine of unity with respect to passing through the zero origin o r with respect to slope ( P values > 0.1 ), except for the slope of the equation for the relationship of A11 in the aorta (P value < 0.05 and > 0.02). Mre have no sure explanation

+

40

sAbs,,

60

80

100

'A

FIG. 5. Relationship between calculated receptor stimulus and observed effect (isometric contraction) of AII and AIII in seven pairs of aortic rings in the 0.6-mId baths. Closed circles are mean data points for AII; open circles are mean data points for AIII. The theoretical line of unity for the direct proportionality is also shown.

CAN. J. PHYSIOIL. PP3ARMACOL. VOL. 57, 1979

TABLE2. Mean eRective concentrations and apparent affinities of AT1 and A111 in rabbit aorta and femoral artery

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Peptide

Tissue

A1H ATII

Aortic Aortic

AI H AIHI

Femoral Femoral

ED60, nh4

Affinity, nil1

$.46+2.52 364 1t 44 1.78 kO.19 4.50k0.44

0.149 LO .021 0.0O30+0.0003 0.594k0.047 0.236 *0.020

Relative affinity, 100 2.26k0.37 100 40.8 - t 3 -42

+

NOTE:Reoxygenated 0.6-n?L baths were used. Values are means SEM in seven pairs of aortic rings and eight pairs of femoral artery rings. The ED50 values are bath concentrations of peptide producing 50% of m a x i m i isometric values. response. ..ZEnities Rere salcuiated as equal t o the recipl~ocalsof the individual EDSO

for the modest difference in slope sf 15 zk 4.2% between the nacan relationship for A11 in the aorta and the theoretical unity line. Linear plots of unity slope for receptor stimeaIation of rabbit aortic strips by A11 have been documented (Rioux et al. 1973). The approxinaately linear relationships shown in Figs. 5 and 6 and the approximations of the four regression equations to the equation for the theoretical unity relationships provide the following: ( a ) indication of the absence of a reserve of receptors for A11 and for AIIH in aorta and femoral artery, (b) exclusion of a threshold phenomenon for action of AII and AIHI in aorta and femoral artery, and (c.) validation of the use of the EDzovalues as close

0

40

60

80

loo

sA/sm, 040 Frc;. 6. Relationship between calculated receptor stimulus and observed effect (isometric contraction) of AII and AIII in eight pairs of femoral arterial rings in the 0.6-mL baths. Closed circles and open circles are the mean data points for AII and AIII, respectively. The theoretical line of unity for the direct proportionality is also shown.

measures of the apparent dissociation constants (MA)of the receptor complexes of AII and AIII in the muscle cells of the aorta and femoral artery in thcse experiments (Ariens et al. 1964a, 1964b). Accordingly, use of the reciprocals of the observed ED,,, values as the apparent afinities of these muscle cells for A11 and A111 is validated. The ratios of these reciprocals are measures of the relative myotropic affinities and "true" relative potencies of these two drugs in the described experiments (Arieias et al. 1964a, 1964b). The values of the affinities and relative affinities of the muscle cells of the aorta and femoral artery for A11 aiad AIII are summarized in Table 2. The affinities for A111 relative to AII averaged 2.26 t 0.37% (n = 7 ) in the aorta and 40.8 t 3.42% ( n = 8 ) in the femoral artery. Dsse-Response Relationships without Reoxygeaaation Similar experiments were performed with the use of femoral arterial rings in tissue baths of smaller capacity (0.24 mL) . The chambers were 4.8 mm in internal diameter and 14.5 mm in depth. Bath construction was similar to the 0.6-mL baths except that the 0.24-mL baths contained neither inlet for reoxygenation nor a wire cloth screen. Application of maximal doses of A11 ( 180 nM) and A111 (1000 nM) in aerated PSS volumes of 0.24 mL resulted in equal peak tension responses which averaged only 60-62% of the mean maximal responses to A11 and A111 of femoral arterial rings in the 0.6-mL baths. In paired rings, the maximal responses to A11 and AIII were 5.48 t 2.06 and 5.52 t 1.99 gf (mean t SD, n 20), respectively. Plateau times of tlme peak tension responses to maximal doses of A11 and AIII measured 13 zk 1 and 10 1 s (mean SEM, n = l o ) , respectively. ~h~~~values were significantly shorter than the mean plateau times in with doses of A11 and A111 in the reoxygenated baths

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%

MAXIMAL

PENSION

FIG. '7. Dose-response curves obtained with AT1 and AIIT on 80 pairs of femoral artery rings in nonreoxygenated tissue baths of 0.24 mE capacity. Solid circles and solid squares are mean data points for A11 and ATII, respectively. Vertical bars represent one %EM.Maximal tension responses to A11 and A111 were equal (see text).

( P < 0.01 and P < 0.05, respectively). Thus, fading this mean value and the mearm relative affinity for of the maximal contractile responses to both peptides AIII (40.8 76 ) in the experiments using the reoxywas accentuated by use of the smaller baths which genated baths was not significant (P > 0.1 ) (by use featured neither regassing nor stirring during the re- of a t-test appropriate for samples with unequal varisponse periods. These sensitivities of the arterial rings ances). to AH1 and AIII were also reduced in the 0.24-mE Discussion baths. The mean dose-response curves of A11 and T o detect small amounts of drugs by their effects AIll in 10 pairs OF femoral arterial rings within these baths are shown in Fig. 7 . The ED,, values for A11 on isolated smooth muscle, small baths are useful in and A111 were 6.48 L- 1.00 and 1'7.7 t 4.53 nM order to increase the concentration produced by a given total dose of drug or to reduce the total sample (mean t SEM), respectively. The predicted stimulus - observed effect relation- volume required. With the usual type of tissue bath ships for AIH and AIII, calculated with use of the it is generally not practical to diminish the chamber mean ED,, values, were plotted, Results showed volume below about 2 mL (Gaddurn and Stephenthat the data points for both peptides approximated son 1958). Serious physical csmplications of rethe theoretical line of unity for a direct propor- gassing in bath chambers less than about 12-15 mm tionality between receptor stimulation by A11 and in internal diameter are excess mechanical agitation AH11 arad the peak contractions evoked in these ex- of the tissue and loss of bath solution with the escapperiments (Ariens et al. 1964a, 1964b). The linear ing gas bubbles. The method developed, using a regression equatims for the data points of A11 and laterally placed 40-mesh screen to confine and direct AH11 did not differ significantly from the linear equa- upwardly the entering gas bubbles, obviates these tion of unity slope with respect to zero origin of y complications in small chambers only 6.4 mm in values ( P values > 0.8 ) or with respect to slope ( P internal diameter. This regassing method provides values > 0.05). These findings justified the use of concurrent mixing by continuous stirring of the bath the EDjo values to determine the myotropic a f i i - liquid. Without irrigation, the temperature of the ties. small volume of bath solution is kept stable by the The apparent rnyotropic afinities for A11 and metallic heat-exchange bar when the Plexiglas bath AT11 were 0.8 84 t 0.822 and 0.095 t 0,019 nM is immersed in a water bath containing a thermostat, (rnsan + SEM, n 1G ) , respectively. The relative heater, and stirrer. In similar Baath chambers sf aEinity for AIIB averaged 64.2 t % 6,896 (mean 2 7.9 mrn internal diameter, 28 m m depth, and 1.4 mL SEM) by paired analysis, The difference between capacity, eumu2ative dose-response studies (van

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Rossurn 1963) may be conveniently and satisfactorily performed with the use of only microlitre a~olurrmes of agonist and a I -0-mE bath solution (Waugh 1979). In 0.24-mL baths without rcgassing, the femoral arterial responses to A%Iand AIII were much impaired when compared with the tension responses in the reoxygenated 0.4-mE baths. However, no evidence was found to suggest that AYI and AIIY acted on different vascular receptors. The impairment was likely related to a diffusion-limitcd supply of oxygen i~ the static bath volunle of 0.24 mL when the contracting muscle cells presumably lowered surroundi~agoxygetl tension ( P o , ) . Oxygen consumption increases during isometric coiltractions of vascular snlosth muscle caused by angiotensin or other agoalist (Coburra et al. 1979) and critically reduced bath POLpromptly reduces the contractile responses (Detar and Bohr 1968; Coburn et al. 1979). Our results suggest that it is not ideal ger~erallyto use small baths to study the contractile behavior of isolated smootI1 muscle near 37°C in the absence of reoxygcnration or continuous mixing of solution containing air or oxygen. This reservation should apply to isormetric contractions of vascular segments measured by manometry in microbaths (Waugh 1978) and perhaps to contractile responses measured in designed microbaths of less than 50-pL capacity (Gaddum and Stephenson 195 8 ) . The employed PSS contained neornycin sulfate in an antibacterial concentration of 5 mg/dE (0.055 mllg). This concentration of neomycin did not depress the contractile responses to AII and AIII. Neomycin coa~centrationsof 0.7 mM or higher do depress the responses of arterial muscle to various agonists (Cioodman et al. 1974; Adarns and Goodman 1975). Tlle parallel dose-response curves of A l l and A111 with the same maxiinum levels suggest that the myotropic receptors are the same for A11 and AIII in the aorta and in the femoral artery. It does not seem likely that there exist two diffcrent myotropic receptors for AII and AHIT in each of these two tissues which act in tandem in producing the same response of cqual tension, as suggested by Peach (1977). This follows from the findings that ( I ) ,the apparent affinity for AIPP in the femoral artery was about 80 times greater than that for AIII in the aorta, while the apparent affinity for AII in the femoral artery was only fourfold greater than that of A11 in the aorta (Table 2), (2) the affinities were greater for A11 in both tissues, and (3) the maximal tensions induced by A11 and AIII were equal in each tissue.

We, therefore, favor the view that A11 and A111 act on the same angiotensin receptors both in aortic muscle and femoral arterial muscle. With this premise, a different stereochemical structure of the angiotermsin receptor in femoral artery nauscle or a difference in the immediate receptor environment in these two tissues appears to be responsible for the much greater difference between the A111 affinities than that between the AII afiinities in the two muscles. However, these differences may be interprcted to support the two difFerent receptor Baypothesis if one asstunes a much different ratio of the rlumber of thc two receptors in the two muscles. The hypothesized ratio of A11 :ATIP receptors would approximate 2.4: 1 in the femoral arterial muscle and 44: 1 in aortic muscle if calculated from the mean affinity data. The relative mean affinity value for A111 of 2.3% cabserved in the aortic rings with the use of the reoxygenated baths is not substantially different from the mean relative affinity values for AIII of 5.7 and 1.6% previously found ila strips of rabbit aorta, with the use of noncunnulative dosc-response studies (RIOUXet al. 1974; Moore and Khairallah 1976). Furthermore, the absolute mean ED,,, values of aortic muscle for AII and AIII' found in our study are not significantly different from the mean EDno values found by Moore and Khairallah ( 1976) with the use of 10-mL tissue baths. The potency of AIIT relative to A11 has been reported higher, at 1 7 % , in curnt~latia~e dose-response studies of A l l and ,4111in strips of rabbit aorta (Ackerly. Tsai et al. 1977). Perhaps the greater relative potency of A111 found by Ackerly, Tsai et al. ( 1977) may be at least partly due to rncasurement of responses before establishment of the plateau equilibrium phase of each contraction. This seems evident from inspection of typical cumulative dose-response rccords employed in the same laboratory (Ackerly, Moore et al. 1977). We tentatively conclude from our study that the rabbit femoral artery is generally snore desirable as a test object for study of test drugs acting on isolated arterial muscle than the rabbit thoracic aorta, as championed by Furchgott (Furchgott and Bhadrakom 1953; Furchgott 1960) and employed by others for the assay of angiotensin peptides on vascular sn~oc~th muscle. Rabbit femoral arteries are more readily cleared of perivascular tissue with less trauma than the thoracic aorta. The femoral arteries arc substantially thinner than the aorta; this greater thinness permits more rapid diffusion of drugs and oxygen fron-n the surrcpunding medium into the deeper layers of the tissue. (The unstretched wall thicknesses of the

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1977. (Bes-Aspl)Angiotensin J : A study of its pressor and steroic!ogenic activities in conscious rats. Endocrinology, POO,46-5 1. CARAVICGI, A. M., BIANCHI, G., BROWN,J. J., LEVER,A. F., J. D., MOBFRISON, MORTON,J. J., POWELI.-JACKSON, J. I. S., and S I M P I F , P. h;. 1976. Blood pressure kind plaqma angiotenhin 11 cnncentratio~l after renal artery constriclio~land angiotensin infusion in the dog. Circ. Res. 38, 335-321. CouerRN, K.F., GRUBB, B., and ARONSON, R. D. 1979. Effect of cyanide on oxygen tension-dependent mechanical tension in rabbit aorta. Circ. Res. 44, 368-378. DE~I'AK. K., and BOIIR,D. F. 3968. Oxygen and vascular smooth muscle contraction. Am. J . Physiol. 214, 241-250. DEVYNCK, M.-A., and ME-YER, 22. 1076. Angiotensin receptors in vascular tissue. Am. J. Med. 61, 758-767. Fu19c~c;orr., R. F. 1955. The pharmacology of vascular smooth muscle. Pharmacol. Rev. 7, 183-265. 1960. Spiral-cut strip of rabbit aorta for in vitro studie\ of responses of arterial snaooth muscle. In Methods in medical research. Vol. 8. EdirvJ by H. D. Bruner. Year Book Medical Publishers Inc., Chicago. pp. 177-186. F U R C I I ( ~ OK. ~ TF., , and B H ~ D K A K OS.M1953. , Reactions of strips of rabbit aorta to epinephrine, isopropylanterenol, sodium nitrite and other drugs. J. Pharmacol. Exp. Ther. 108, 129-143. GAIIDIIM, J. H., and STEPHI-NSON, K. P. 1958. A microbath. Br. J. Ph:umacol. 13, 493-497. Gooah N . E., \Vrxc;r,-r. G . D., \VINTER,W., CONNOLLY, T. N., I Z ~ W AS., , and SINGH,R. M. M. 1966. Hydrogen ion buffers for biological research. Biochemistry, 5, 467-477. GOOD~.IAN, F. R., WFISS,G. R., and ADAMS,H. R. 1974. A C K E R I , ~J., L\., MOORE,A. F., and PEACH,M. J. 1977. Alterations by neomycin of "Ca movements and contracDesnonstraticzn of diaferent contractile mechanisms for tile responses in vascailar smooth muscle. J. Pkamacol. angiotensin I1 and des-asp1-angiotensin I1 in rabbit aortic Exp. Ther. 188,472-480. strips. Proc. Nati. Acad. Sci. U.S.A. 74. 5725-5728. HOOKER, C. S., CALKINS, P. A., and FLEISCH,J. H. 1977. On ACKFRLY, J. A., TSAI.5.-S., and P E ~ C HM. , J . 1977. Role of the measurement of vascular and respiratory smooth converting enzyme in the receptors of rabbit atria, aortas, muscie responses in vitro. Blood Veshels, 14, 1-1 1. and adrenal zona glomeruiosa to (de5-al;pl) angiotensin I. MOORF,A., and KIIAIKALI*AK. P. A. 1976. Further studies on Circ. Res. 41, 23 1-238. angiotellsin tachyphylaxis. J. Pharmacol. Exp. Ther. 197, A D ~ M SPI., R.. and G ~ O I ~ M A F.NR, . 1975. Difrerential in575-58 1 . hibitory efiect of neonlpcin on contractile responses of various canine arteries. J. Pharmacol. Exp. Ther. 193, Pr A C H , M.J. 1977. Renin-angiotensin system: biochemistry and mechanisms of action. Physiol. Rev. 57, 313-370. 393-102. Kr.cor~,I)., PARK,W.K., and Krocx, F . 1974. PharmacolAIKEN,J, W., and VANE,J. R. 1970, The renin-angiotensin ogy of angiotensin. Pharmacol. Rev. 26, 69-123. system: inhibition of converting enzyme in isolated tissues. Rroux, F., PARK,W. I;.,and RECOLI,D. 1973. Application Nature (London), 228, 30-34. of drug-receptor theories to angiotensin. Can. 3. Physiol. ALTVRA,5. M.. and ALTURA.5 . T, 1970. Calcium content I'harmacoi. 51, 665-672. and force of drug-induced contractions of arterial muscle during recovery in vitro. Proc. SOC.Exp. Biol. Med. 135, R o s s u ~J., M. VAN. 1963. Cumulative dose-response curves. 11. Technique for the making of dose-response curves in 739-744. isolated organs and the evaluation of drug parameters. ARILNS,E. J., SIMONIS,A. hfva., and ROssuh4, J. M. VAN. Arch. Int. Pharmacodyn. Tker. 143, 299-330. 1964n. The relation between stimulus and effect. I n J.,~and , PARK,W. K. 1977. Molecular pharmacology. Vol. I. Edited h y E. J. Ariens. ST-I,orrrs, J., KEGOLI,B., B A K ~ I B Myotropic actions of angiotensin and i~oradrenalinein Academic Press Inc., New York. pp. 394-466. strips of rabbit aortae. Can. J. Physiol. Pharmacol. 55, 1964b. Drug-receptor interaction : interaction of one 1056-1069. or more drugs with one receptor system. In Molecular F, P. F., BOYL),A. S., DAWFS. P. h4., and MORTON, pharmacology. Vol. I. Edited / ~ yE. J. Ariens. Academic SEMPL Press Inc., New York. pp. 119-286. J. J. 1976. Angiotensin 11 and its heptapeptide (2-a), BUMPUS,F. M., KHAIRALI-AH, P. A., ARAKAWA, K., PAGE.. hexapeptide (3-8 ) and pentapeptide (4-8 ) metabolites I. H., and SMEBY,K. R. 1961. The relationships of strucin arterial and venous blood of man. Circ. Kes. 39, 671ture to pressor and oxytocic actions of isoleucineocta678. peptide and \arious analogues. Biochim. Biophys. Acta, SEMPLE,P. F., and MORTON,J. J. 1976. Angiotensin I1 and 46, 38-44. angiotensin 111 in rat blood. Circ. Res. 38 (Suppl. XI), CAMPBEI-L.'1Y. B., SCHMITZ,J. M., and ITSKOVTTZ, H. D. 122-1 26.

femoral arterial rings and aortic rings were usually betwee11 0.15 and 8.28 Inm and 0.32 and 0.40 mm, respectively.) In additic~n,in the femoral artery, the sensitivities to A11 arad A111 were greater, tlae responses developed faster, the contractions were muc%lmore forceful, and the tinaes for relaxation and recoklery after washing werc shorter. Rioux ct al. (1973) demonstrated a direct proportioilality between the number of myotropic receptors for 411 and the mechallical response in rabbit aortic strips undergoing isotonic ccjntractions. Our findings indicate a similar direct proportionality for isonletric contractile responses to A11 and AT11 in rabbit aor~icnaelscle and femoral arterial muscle. The direct proportionality has been used as a criterion in the selection of rabbit aorta as the model target tissue for biochemical studies on the binding of ,411 to its specific receptor in vascular smooth muscle (Devynck and Meyer 1976). The rabbit femoral artery also fulfils this criterion and the angiotensin receptor - contractile characteristics of femoral artery should be expected to resemble, more closely than the thoracic aorta, those of many smaller peripheral arteries.

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SOKAL, R. R.. and ROHLF,F. J. 1969. Biometry. W. H. Freeman & Company, San Francisco. STEI:I-E,J. M., JR., NEUSY,A.-J., and LOW~NSTEIN, J. 1976. The effects of des-asp1-angiotensin II on blood pressure, plasma aldostersne concentration, and plasma renin activity in the rabbit. Circ. Res. 38 (Suppl. 111, 113-1 16. TAUB,K. J., CALDICOTT, W. J. H., and WOLLENBERG, N.K. 1977. Angiotensin antagonists with increased specificity

for the renal v a s c ~ l a t u r eJ.~ Clin. Invest. 59, 528-535. WAUGH,W. H. 1978. Manometric microbath assay of vasoactive agents: potencies of angiotensin IT and angiotensin If1 in rabbit f e r n ~ r a larteries. Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 822. (Abstr.) 1979. Femoral artery responses to norepinephrine in small tissue baths with regassing. Fed. Broc. Fed. Am. Soc. Exp. Biol. 38, 1244. (Abstr.)

Myotropic affinities of angiotensin II and des-AspI-angiotensin II in rabbit aorta and femoral artery by microassay.

Myotropic affinities of angiotensin I1 and des-Asp'-angiotensinII in rabbit aorta and femoral artery by lllicroassay IL ILL HA^^ H. WAUCHAND THEODORE...
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