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Comparison of angiotensin receptors in isolated smooth muscle tissues by photoaffinity labelling
European Journal of Pharmacology, 115 (1985) 53-58
53
Elsevier
C O M P A R I S O N O F A N G I O T E N S I N R E C E P T O R S IN I S O L A T E D S M O O T H M U S C L E T I S S U E S BY P H O T O A F F I N I T Y L A B E L L I N G YAN G. KWOK
*
and GRAHAM
J. M O O R E
÷
Dept. of Medical Biochemistry, University of Calgary, Calgary T2N 4NI, Canada Received 20 February 1985, revised MS received 29 May 1985, accepted 12 June 1985
Y.C. KWOK and G.J. MOORE, Comparison of angiotensin receptors in isolated smooth muscle tissues by photoaffinity labelling, European J. Pharmacol. 115 (1985) 53-58. The isolated rat uterus and rabbit aorta were photolabelled with [Azidobenzoic acid 1, isoleucineS]angiotensin II ([AB', IleS]ANG II) and dose-response curves to angiotensins II and III were determined before and after the irreversible labelling proce.dure. The data obtained were used to calculate affinities, efficacies and 'spare' receptors for angiotensins II and III. In the rat uterus angiotension II was the higher affinity ligand, whereas in the rabbit aorta angiotensins II and III had similar affinities. 'Spare' receptors were present for both ligands in both tissues. In the rat uterus ANG II and ANG III had similar efficacies, whereas in the rabbit aorta ANG II was the more efficacious ligand. Angiotensin receptors in the rabbit aorta, rat uterus, and rat portal vein appear to be substantially different from one another, and point to the possibility of different functional roles for angiotensins II and III in different smooth muscle tissues. For the vascular tissues, tachyphylaxis to angiotensin was more marked after irrevesible labelling than before, suggesting that the mechanism of angiotensin tachyphylaxis is receptor deactivation. Isolated rat uterus and rabbit aorta Photolabelling of smooth muscle
Efficacies and 'spare' receptors Angiotensins II and III
I. Introduction Angiotensin II ( A N G II, Asp-Arg-Val-Tyr-IleHis-Pro-Phe) acts at receptors in target tissues to produce contractile and secretory responses. The contractile action of A N G II in vascular smooth muscle is generally believed to be of physiological importance in blood pressure regulation and the etiology of certain forms of hypertension. The des-aspartyl heptapeptide A N G III, on the other hand, has a less well-defined physiological role, being less potent than A N G II in mediating the contraction of most smooth muscle tissues (Freeman et al., 1977). In the present report we have compared affinities, efficacies and 'spare' recep-
Affinities
tors for A N G II and III in three isolated smooth muscle tissues using a specific photolabelling technique. The data illustrate distinct differences in the recognition of A N G II and A N G III by individual smooth muscle tissues.
2. Materials and methods Peptides used in this study were synthesized and purified as described previously (Kwok and Moore, 1980). All of the reagents were of the highest grade available from a variety of commercial sources.
2.1. Rat uterus * Present address: Banting and Best, Dept. of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada. ÷ To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Experimental animals were killed by cervical dislocation and defatted uterine horns from diethylstibesterol-primed virgin Sprague-Dawley rats
54 (150-250 g), cut into three equal sections and suspended under 1 g of tension in a solution of 150 mM NaC1, 5.6 m M KC1, 0.18 m M CaCI 2, 1.8 m M N a H C O 3 and 1.4 m M glucose, p H 7.0, gassed with oxygen at 30°C. The tissue baths were 4 ml spectrophotometric cells which were permeable to UV light, and the lower end of the tissue was tied to the bent needle of a syringe, which also served to direct oxygen flow (Kwok and Moore, 1980). The upper end of the tissue was attached to a Statham force-displacement transducer which was connected to a Beckman Dynograph. Washing steps were carried out by withdrawing the solution in the cell with a syringe and replacing it with fresh oxygenated bathing solution at 30°C. Noncumulative dose-dependent responses to A N G II, A N G III and oxytocin were first obtained for each tissue. The tissue was challenged at 12 min intervals with increasing doses of each agonist and was washed out immediately after the tissue had contracted completely to each dose. Using this protocol, tachyphylaxis to angiotensin was avoided. The tissues were then subjected to the photolysis procedure. Photolysis was carried out with two 275 W General Electric Sunlamps placed on opposite sides of the tissue at a distance of 15 cm from the spectrophotometric cell. Tissues were incubated with 2.5 × 10 -6 M [AB 1, lie s] A N G II for 15 min in the dark, exposed to UV light for 3 min, and then washed out. The incubation-photolysiswashout procedure was repeated 5 times. The tissues were washed out periodically for another hour before dose-dependent responses were redetermined. The total time to complete the experiment was 5 - 6 h. 2.2. Rabbit aorta The upper part of the descending aorta was removed from New Zealand rabbits (2-4 kg), cleaned of adherent tissue, cut spirally into 6 equal sections and suspended under 1 g of tension at 37°C in a solution of 118 m M NaC1, 1.18 m M MgSO4, 1.18 m M K H 2 P O 4, 25 m M N a H C O 3, 2.5 m M CaC12, 4.7 m M KCI and 5.5 m M glucose, gassed with oxygen. Non-cumulative dose-response curves to A N G II and A N G I I I or noradrenaline were obtained. The tissue was chal-
lenged with A N G II or A N G III at 45 min intervals and was washed out immediately after the tissue had contracted completely to each dose. Noradrenaline doses were given between angiotensin challenges. For photoaffinity labelling the rabbit aorta was equilibrated with 2.5 × 10 -6 M [AB 1, Ile8]ANG II for 15 min in the dark and exposed to UV light for 1 min. The tissue was washed out and the protocol repeated once. Doseresponse curves were determined 1 h later. Tachyphylaxis was observed to maximum doses of A N G II and A N G III but not to noradrenaline after photolabelling. There were no statistically significant differences between the maximum responses of the rabbit aorta to noradrenaline and A N G II or A N G III before labelling, and therefore all angiotensin responses were calculated relative to the noradrenaline maximum. In both bioassays used, neither exposure to irradiation with UV light alone nor incubation with [AW, IleS]ANG II in the dark alone affected subsequent tissue responsiveness. Straiglit line fits to data points were obtained by the method of least squares analysis.
3. Results Dose-response curves to A N G II, A N G III and oxytocin before and after photoaffinity labelling of the rat isolated uterus are shown in fig. 1A. Parallel displacement of the dose-response curve to A N G II and A N G III, with retention of the maximum response, were observed after the photoaffinity labelling procedure. In contrast, the dose-response curve to oxytocin was unaffected by photolabelling of angiotensin receptors, demonstrating the specificity of the labelling procedure. The data in fig. 1A were used to construct a double reciprocal plot of equiactive concentrations of A N G II or A N G III before and after photoaffinity labelling (fig. 1B). Dose-response curves to A N G II, A N G III and noradrenaline before and after photoaffinity labelling of the rabbit aorta are shown in fig. 2A. A parallel shift in the dose-response curve to A N G II after photolabelling was observed, whereas the m a x i m u m response to A N G III appeared to be
55
A
100
80
1.0
60
/ /-7
4°I 20
i
ii!!ljZ.o :o., =
~ I
10l-9
It
10-8
,
10-10
i
10-7
10-9 I
101-6
0.05
I
0.10
1 1 x 10 "9~) [ANG]'* ('M"
MOLAR CONCENTRATION OF PEPTIDE
Fig. 1. Responses of the rat isolated uterus to angiotensins II and III before and after photoaffinity labelling. (A) Dose-response curves to ANG II, ANG III and oxytocin before and after irreversible labelling of the tissues. Before labelling: (©) ANG lI, (A) ANG III, (m) oxytocin (inset). After labelling: (e) ANG II, (A) ANG III, (I) oxytocin (inset). Points are represented by mean + S.E.M. (N = 4-6). (B) Double reciprocal plots of matchin responses before and after irreversible labelling of the tissues. * indicates photolabelled tissues. (e) ANG II, (A) ANG III. d e p r e s s e d after the p h o t o l a b e l l i n g procedure. A t t e m p t s to o b t a i n m a x i m u m responses to A N G II ~ a n d A N G I I I after p h o t o l a b e l l i n g gave results which could not be utilized; r e p e a t e d challenges with m a x i m a l doses of the agonists gave progressively d i m i n i s h i n g responses i n d i c a t i n g t a c h y p h y l axis to m a x i m a l dose of angiotensin after p h o t o l a 100
belling. This was n o t o b s e r v e d with s u b - m a x i m a l doses. Similar p r o b l e m s have been e n c o u n t e r e d p r e v i o u s l y with the rat p o r t a l vein ( K w o k a n d M o o r e , 1984). T h e d a t a in fig. 2A were used to c o n s t r u c t fig. 2B. A c c o r d i n g to a m e t h o d d e v e l o p e d b y F u r c h g o t t a n d Bursztyn (1967), it is possible to calculate the
A
t =,, ~o Z O Q. t/)
8O 1.0 8o
.o
60
,,=,
40
~40
L
x
I-Z 0: a.
2°I
0.5
2O
i 10-8
I 10 -7
I 10 -6
MOLAR CONCENTRATION OF PEPTIDE
10-8
i
10-;' 0
I 0.05
I 0.10
jAnGle1 (.~_1 x 10 "e)
Fig. 2. Responses of the rabbit isolated aorta to angiotensins I] and III before and after photoaffinity labeling. See fig. ] for details; squares represent noradrenaline (inset).
56 TABLE 1 Comparison of bioactivity determinants for angiotensins II and III in isolated tissues by photoaffinity labelling. Values are expressed as means +- S.E.M. for 4 - 6 tissues. Angiotensin II
Rat uterus Rabbit aorta Rat portal vein c
Angiotensin III
EDs0 (M)
K(M)
Ra (%) a
Rs (%)
8 + 3 x 1 0 -1° 8+-3x10 -9 1.5 +-0.4x 10 - s
3+2x10 -s 5+3x10 -7 3+-2×10 -7
13+_7 15+-8 30+-10
86 90 58
b
EDs0 (M)
K(M)
Ra (%) a
Rs (%) b
8+-3x10 -9 2 4 - 1 × 1 0 -7 8 + - 6 x 1 0 -8
3+1×10 -7 7+-3×10 -7 6 + 2 × 1 0 -8
20+9 14+-7 20±7
87 32 4
Ra is the percent available (active) receptors after irrevesible labelling, b Rs is the percent %pare' receptors, c Data calculated from Kwok and Moore (1984).
a
fraction of receptors unoccupied by the irreversible label and the affinity of a given agonist for its receptors from the dose-response curves before and after labelling. The following conditions must apply: (i) all the receptors have the same affinity and efficacy with a given agonist, (ii) steady-state kinetics governed by the law of mass action apply, (iii) the affinity and efficacy of the agonist is unaltered by irreversible labelling of a fraction of the receptors, and (iv) the agonist does not cause desensitization. Double reciprocal plots for equiactive concentrations of the agonist before and after inactivation of a portion of the receptors (figs. 1B and 2B) should yield a straight line from which a dissociation constant, K (slope divided by ordinate intercept), and the fraction of receptors unoccupied by the irreversible label, q (1/slope), can be calculated (Furchgott and Bursztyn, 1967). Once the value of K has been determined for given agonist, it is possible to obtain information on the efficacy of an agonist from the law of mass action:
[RA]max [R,]
[A]max K + [A]max
where [A]max is the concentration of agonist required to produce the maximum response, [RA] max is the concentration of the. receptor-agonist complex required to produce the maximum response, and [ R t ] is the total concentration of receptors. The value of [A]max for each agonist can be determined from the dose-response curves before labelling. The relative efficacies of two agonist are obtained from the ratio of the values of [ R A ] m a x / [ R t ] for each agonist. In practice, a more accurate estimate of relative efficacies can be ob-
tained using half-maximal values. The validity of the calculated ratio of efficacies for the two agonists is dependent on all the criteria listed above. In addition, both agonists must act at the same receptors with the same mechanism, and their rates of uptake and metabolism must be the same. The data can be quantified in percentage format: R m = 100 X [ R k ] m a x / [ R t ] = % receptors which must be occupied in order to produce the maximum response, R S = 100 - R m = % 'spare' receptors, Rtm/R 2 = ratio of efficacies of two agonists, R a = 100 x q = % receptors unoccupied by the irreversible label (% active receptors), R i = 1 0 0 - R a = % receptors inactivated by the irreversible label. The data in figs. 1 and 2 have been used to calculate, by the methods described above, the various determinats of the biological activity of A N G II and A N G III in the rat uterus and rabbit aorta (table 1). For comparison, previously published data on the rat portal vein (Kwok and Moore, 1984) have been included. The relative
TABLE 2 Relative affinities and efficacies of angiotensins II and Ill in isolated tissues determined by photoaffinity labelling.
Rat uterus Rabbit aorta Rat portal vein
Affinity ratio
Efficacy ratio a
A N G I I / A N G III
A N G I I / A N G III
10 1 0.2
0.9 14 11
a Calculated from the [ A ] / ( K + [ A ] ) ratio at half-maximal response.
57 affinities and efficacies of A N G II and A N G III in each of the three tissues are given in table 2.
4. Discussion The data in figs. 1A and 2A demonstrate the feasibility of irreversible labelling of angiotensin receptors in isolated smooth muscle tissues with the photolabile angiotensin antagonist. The photoaffinity labelling procedure appears to be specific since the dose-response curves for the alternative agonists, oxytocin and noradrenaline, used in the rat uterus and rabbit aorta assays respectively, were not significantly affected by the photolabelling. For the rat uterus, evidence has been obtained previously which suggests that irreversible labelling of the receptors can be protected by large doses of angiotensin (Kwok and Moore, 1980). This type of investigation on the specificity of receptor labelling could not be carried out with the isolated rat portal vein (Kwok and Moore, 1984) because of the tachyphylaxis induced by large doses of angiotensin. Similarly, the present studies illustrate that supermaximal doses of angiotensin also cause densensitization of the isolated rabbit aorta and, since the validity of the irreversible labelling technique requires that this phenomenon not be present, large doses of angiotensin have been avoided throughout the experimental procedures with the rabbit aorta. Evidence has been obtained previously with the rat portal Vein which illustrates that the affinity constant does not change after different degrees of irreversible labelling (Kwok and Moore, 1984), suggesting the presence of a uniform population of receptors in this tissue, and possibly other smooth muscle tissues. Experiments in this laboratory indicate that A N G II and A N G III act at the same high affinity receptors in both the rat uterus (Kwok, 1982) and the rat portal vein (Scanlon and Moore, unpublished), and it is presumed that this is also the case for the rabbit aorta. For comparing the bioactivity determinants of angiotensins II and III, it has been assumed that the rates of uptake and metabolism of these two agonists by the isolated tissues are the same, and that the rate of conversion of A N G II to A N G III in the tissue bath is
not a significant factor. We have not observed any difference in the rates of onset or duration of the responses to A N G II and A N G III which, to a first approximation, suggests there may be no gross difference in rates of uptake and metabolism of these two ligands. Recent experiments in this laboratory also suggest that the presence of inhibitors of angiotensinase A, the enzyme that converts A N G II to A N G III, does not affect the dose-response curves to A N G II (Scanlon and Moore, unpublished observations). Furthermore, the photoaffinity label should also inactivate angiotensindegrading enzymes in these tissues. Therefore the comparison of relative affinities and efficacies for A N G II and A N G III in these isolated tissues may not be unreasonable. Based on the data in table 2, it appears that A N G II is the higher affinity ligand in the rat uterus and that both A N G II and A N G III have similar efficacies in this tissue. Thus the biological potencies of these two ligands are dependent only on the affinity in the rat uterus. In contrast, A N G II and A N G III appear to have the same affinities for angiotensin receptors in the rabbit aorta, and the higher potency of A N G II is determined solely by its increased efficacy. For the rat portal vein, A N G III is the higher affinity ligand but has the lower efficacy of the two agonists, suggesting that A N G III, under certain conditions, could perhaps reduce the response to a given concentration of A N G II (Kwok and Moore, 1984). The relative efficacies of A N G II and A N G III in each tissue are reflected in their spare receptor populations, with greater efficacy being concomitant with greater receptor reserve. An appreciable receptor reserve is present for both ligands in all tissues, except for the rat portal vein where there are no spare receptors for A N G III. The significantly different relative affinities and efficacies of A N G II and A N G III in the three isolated smooth muscle tissues studied suggest that angiotensin receptors in all of these tissues are kinetically different from one another. For vascular smooth muscle tissues, subtle differences between selected vascular beds could have relevant physiological consequences in the regulation of blood flow and blood pressure. Although the two vascular smooth muscle tissues investigated in this
58 study derived from different species, the possibility should be considered that arterial and venous tissues may display substantially different reactivities towards A N G II and A N G III. The calcium and magnesium concentration utilized for assays of the rat uterus and rat portal vein were sub-physiological, whereas the divalent cation concentrations utilized in rabbit aorta assays approached physiological concentrations. It is possible that the relative affinities and efficacies of A N G II and A N G III are dependent on divalent cation concentrations. A difference in the susceptibility of uterine tissue, c o m p a r e d to vascular tissue, to photoaffinitiy labelling was observed. In order to produce 70-90% blockade of angiotensin receptors in all three tissues, the rat uterus required five photoaffinity labellings with a flash time of 3 min in each case, whereas the rat portal vein and rabbit aorta required only two photoaffinity labellings with a flash time of 1 min. These findings suggest that angiotensin receptors in the rat uterus are much less accessible to photoaffinity labelling than are receptors in either of the vascular smooth muscle tissue investigated. For the rat uterus, there is a possible indication here that the endometrial cells, which are located on the inside of the tubular tissue m o u n t e d for the bioassay, m a y be the primary cellular determinants of angiotensin activity. Alternatively, the relative efficiency of photolabelling m a y be a direct consequence of the thickness of the tissue as it relates to accessibility of the photolabel and U V light to the total smooth muscle population. Finally, these photoaffinity labelling experiments have raised an issue concerning the mechanism of angiotensin tachyphylaxis. For both the rat portal vein (Kwok and Moore, 1984) and the rabbit aorta, it was observed that desensitization to maximal doses of angiotensin was more marked after photolabelling than before. Apparently susceptibility to angiotensin tachyphylaxis is dependent on the n u m b e r of active receptors present.
This suggests that angiotensin itself m a y be able to deactivate a fraction of the available receptors, such that: (i) there is no notable induction of tachyphylaxis when there is a large receptor reserve present before labelling; and (ii) desensitization occurs when the size of the spare receptor pool is decreased after labelling. This appears to indicate that the mechanism of angiotensin tachyphylaxis is a ligand-induced receptor ' d o w n regulation' phenomenon, resulting from a decrease in receptor reactivity or possibly internalization of angiotensin receptors (Robertson and Khairallah, 1971; Re et al., 1981).
Acknowledgments This work was supported by the Alberta Heart Foundation, the Alberta Heritage Foundation for Medical Research and the Medical Research Council of Canada. We thank Joanne Ward for typing the manuscript and Martin Scanlon for helpful discussion.
References Freeman, R.J., Davis, J.O., Lohmeier, T.A. and Speilman, W.S., 1977, [Des-Aspl]angiotensin II: Mediator of the angiotensin system? Fed. Proc. 36, 1766-1770. Furchgott, R. and Bursztyn, P., 1967, Comparison of dissociation constants and of relative efficacies of selected agonists acting on parasympathetic nerves, Ann. N.Y. Acad. Sci. 144, 882-898. Kwok, Y.C., 1982, Angiotensin receptors in smooth muscle, Ph.D. Thesis, University of Calgary. Kwok, Y.C. and Moore G.J., 1980, Affinity labelling of angiotensin receptors in the isolated rat uterus with a photolabile antagonist, Mol. Pharmacol. 18, 210-214. Kwok, Y.C. and Moore G.J., 1984, Photoaffinity labelling of the rat isolated portal vein: determination of affinity constants and 'spare' receptors for angiotensins II and III, J. Pharm. Exp. Ther. 231, 137-140. Re, R.N., Macphee, A.A. and Fallon J.T., 1981, Specificnuclear binding of angiotensin II by rat liver and spleen nuclei, Clin. Sci. 61, 245s-247s. Robertson, A.L. and Khairallah, P.A., 1971, Angiotensin II: rapid location in nuclei of smooth and cardiac muscle, Science 171, 1138-1140.
53
Elsevier
C O M P A R I S O N O F A N G I O T E N S I N R E C E P T O R S IN I S O L A T E D S M O O T H M U S C L E T I S S U E S BY P H O T O A F F I N I T Y L A B E L L I N G YAN G. KWOK
*
and GRAHAM
J. M O O R E
÷
Dept. of Medical Biochemistry, University of Calgary, Calgary T2N 4NI, Canada Received 20 February 1985, revised MS received 29 May 1985, accepted 12 June 1985
Y.C. KWOK and G.J. MOORE, Comparison of angiotensin receptors in isolated smooth muscle tissues by photoaffinity labelling, European J. Pharmacol. 115 (1985) 53-58. The isolated rat uterus and rabbit aorta were photolabelled with [Azidobenzoic acid 1, isoleucineS]angiotensin II ([AB', IleS]ANG II) and dose-response curves to angiotensins II and III were determined before and after the irreversible labelling proce.dure. The data obtained were used to calculate affinities, efficacies and 'spare' receptors for angiotensins II and III. In the rat uterus angiotension II was the higher affinity ligand, whereas in the rabbit aorta angiotensins II and III had similar affinities. 'Spare' receptors were present for both ligands in both tissues. In the rat uterus ANG II and ANG III had similar efficacies, whereas in the rabbit aorta ANG II was the more efficacious ligand. Angiotensin receptors in the rabbit aorta, rat uterus, and rat portal vein appear to be substantially different from one another, and point to the possibility of different functional roles for angiotensins II and III in different smooth muscle tissues. For the vascular tissues, tachyphylaxis to angiotensin was more marked after irrevesible labelling than before, suggesting that the mechanism of angiotensin tachyphylaxis is receptor deactivation. Isolated rat uterus and rabbit aorta Photolabelling of smooth muscle
Efficacies and 'spare' receptors Angiotensins II and III
I. Introduction Angiotensin II ( A N G II, Asp-Arg-Val-Tyr-IleHis-Pro-Phe) acts at receptors in target tissues to produce contractile and secretory responses. The contractile action of A N G II in vascular smooth muscle is generally believed to be of physiological importance in blood pressure regulation and the etiology of certain forms of hypertension. The des-aspartyl heptapeptide A N G III, on the other hand, has a less well-defined physiological role, being less potent than A N G II in mediating the contraction of most smooth muscle tissues (Freeman et al., 1977). In the present report we have compared affinities, efficacies and 'spare' recep-
Affinities
tors for A N G II and III in three isolated smooth muscle tissues using a specific photolabelling technique. The data illustrate distinct differences in the recognition of A N G II and A N G III by individual smooth muscle tissues.
2. Materials and methods Peptides used in this study were synthesized and purified as described previously (Kwok and Moore, 1980). All of the reagents were of the highest grade available from a variety of commercial sources.
2.1. Rat uterus * Present address: Banting and Best, Dept. of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada. ÷ To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Experimental animals were killed by cervical dislocation and defatted uterine horns from diethylstibesterol-primed virgin Sprague-Dawley rats
54 (150-250 g), cut into three equal sections and suspended under 1 g of tension in a solution of 150 mM NaC1, 5.6 m M KC1, 0.18 m M CaCI 2, 1.8 m M N a H C O 3 and 1.4 m M glucose, p H 7.0, gassed with oxygen at 30°C. The tissue baths were 4 ml spectrophotometric cells which were permeable to UV light, and the lower end of the tissue was tied to the bent needle of a syringe, which also served to direct oxygen flow (Kwok and Moore, 1980). The upper end of the tissue was attached to a Statham force-displacement transducer which was connected to a Beckman Dynograph. Washing steps were carried out by withdrawing the solution in the cell with a syringe and replacing it with fresh oxygenated bathing solution at 30°C. Noncumulative dose-dependent responses to A N G II, A N G III and oxytocin were first obtained for each tissue. The tissue was challenged at 12 min intervals with increasing doses of each agonist and was washed out immediately after the tissue had contracted completely to each dose. Using this protocol, tachyphylaxis to angiotensin was avoided. The tissues were then subjected to the photolysis procedure. Photolysis was carried out with two 275 W General Electric Sunlamps placed on opposite sides of the tissue at a distance of 15 cm from the spectrophotometric cell. Tissues were incubated with 2.5 × 10 -6 M [AB 1, lie s] A N G II for 15 min in the dark, exposed to UV light for 3 min, and then washed out. The incubation-photolysiswashout procedure was repeated 5 times. The tissues were washed out periodically for another hour before dose-dependent responses were redetermined. The total time to complete the experiment was 5 - 6 h. 2.2. Rabbit aorta The upper part of the descending aorta was removed from New Zealand rabbits (2-4 kg), cleaned of adherent tissue, cut spirally into 6 equal sections and suspended under 1 g of tension at 37°C in a solution of 118 m M NaC1, 1.18 m M MgSO4, 1.18 m M K H 2 P O 4, 25 m M N a H C O 3, 2.5 m M CaC12, 4.7 m M KCI and 5.5 m M glucose, gassed with oxygen. Non-cumulative dose-response curves to A N G II and A N G I I I or noradrenaline were obtained. The tissue was chal-
lenged with A N G II or A N G III at 45 min intervals and was washed out immediately after the tissue had contracted completely to each dose. Noradrenaline doses were given between angiotensin challenges. For photoaffinity labelling the rabbit aorta was equilibrated with 2.5 × 10 -6 M [AB 1, Ile8]ANG II for 15 min in the dark and exposed to UV light for 1 min. The tissue was washed out and the protocol repeated once. Doseresponse curves were determined 1 h later. Tachyphylaxis was observed to maximum doses of A N G II and A N G III but not to noradrenaline after photolabelling. There were no statistically significant differences between the maximum responses of the rabbit aorta to noradrenaline and A N G II or A N G III before labelling, and therefore all angiotensin responses were calculated relative to the noradrenaline maximum. In both bioassays used, neither exposure to irradiation with UV light alone nor incubation with [AW, IleS]ANG II in the dark alone affected subsequent tissue responsiveness. Straiglit line fits to data points were obtained by the method of least squares analysis.
3. Results Dose-response curves to A N G II, A N G III and oxytocin before and after photoaffinity labelling of the rat isolated uterus are shown in fig. 1A. Parallel displacement of the dose-response curve to A N G II and A N G III, with retention of the maximum response, were observed after the photoaffinity labelling procedure. In contrast, the dose-response curve to oxytocin was unaffected by photolabelling of angiotensin receptors, demonstrating the specificity of the labelling procedure. The data in fig. 1A were used to construct a double reciprocal plot of equiactive concentrations of A N G II or A N G III before and after photoaffinity labelling (fig. 1B). Dose-response curves to A N G II, A N G III and noradrenaline before and after photoaffinity labelling of the rabbit aorta are shown in fig. 2A. A parallel shift in the dose-response curve to A N G II after photolabelling was observed, whereas the m a x i m u m response to A N G III appeared to be
55
A
100
80
1.0
60
/ /-7
4°I 20
i
ii!!ljZ.o :o., =
~ I
10l-9
It
10-8
,
10-10
i
10-7
10-9 I
101-6
0.05
I
0.10
1 1 x 10 "9~) [ANG]'* ('M"
MOLAR CONCENTRATION OF PEPTIDE
Fig. 1. Responses of the rat isolated uterus to angiotensins II and III before and after photoaffinity labelling. (A) Dose-response curves to ANG II, ANG III and oxytocin before and after irreversible labelling of the tissues. Before labelling: (©) ANG lI, (A) ANG III, (m) oxytocin (inset). After labelling: (e) ANG II, (A) ANG III, (I) oxytocin (inset). Points are represented by mean + S.E.M. (N = 4-6). (B) Double reciprocal plots of matchin responses before and after irreversible labelling of the tissues. * indicates photolabelled tissues. (e) ANG II, (A) ANG III. d e p r e s s e d after the p h o t o l a b e l l i n g procedure. A t t e m p t s to o b t a i n m a x i m u m responses to A N G II ~ a n d A N G I I I after p h o t o l a b e l l i n g gave results which could not be utilized; r e p e a t e d challenges with m a x i m a l doses of the agonists gave progressively d i m i n i s h i n g responses i n d i c a t i n g t a c h y p h y l axis to m a x i m a l dose of angiotensin after p h o t o l a 100
belling. This was n o t o b s e r v e d with s u b - m a x i m a l doses. Similar p r o b l e m s have been e n c o u n t e r e d p r e v i o u s l y with the rat p o r t a l vein ( K w o k a n d M o o r e , 1984). T h e d a t a in fig. 2A were used to c o n s t r u c t fig. 2B. A c c o r d i n g to a m e t h o d d e v e l o p e d b y F u r c h g o t t a n d Bursztyn (1967), it is possible to calculate the
A
t =,, ~o Z O Q. t/)
8O 1.0 8o
.o
60
,,=,
40
~40
L
x
I-Z 0: a.
2°I
0.5
2O
i 10-8
I 10 -7
I 10 -6
MOLAR CONCENTRATION OF PEPTIDE
10-8
i
10-;' 0
I 0.05
I 0.10
jAnGle1 (.~_1 x 10 "e)
Fig. 2. Responses of the rabbit isolated aorta to angiotensins I] and III before and after photoaffinity labeling. See fig. ] for details; squares represent noradrenaline (inset).
56 TABLE 1 Comparison of bioactivity determinants for angiotensins II and III in isolated tissues by photoaffinity labelling. Values are expressed as means +- S.E.M. for 4 - 6 tissues. Angiotensin II
Rat uterus Rabbit aorta Rat portal vein c
Angiotensin III
EDs0 (M)
K(M)
Ra (%) a
Rs (%)
8 + 3 x 1 0 -1° 8+-3x10 -9 1.5 +-0.4x 10 - s
3+2x10 -s 5+3x10 -7 3+-2×10 -7
13+_7 15+-8 30+-10
86 90 58
b
EDs0 (M)
K(M)
Ra (%) a
Rs (%) b
8+-3x10 -9 2 4 - 1 × 1 0 -7 8 + - 6 x 1 0 -8
3+1×10 -7 7+-3×10 -7 6 + 2 × 1 0 -8
20+9 14+-7 20±7
87 32 4
Ra is the percent available (active) receptors after irrevesible labelling, b Rs is the percent %pare' receptors, c Data calculated from Kwok and Moore (1984).
a
fraction of receptors unoccupied by the irreversible label and the affinity of a given agonist for its receptors from the dose-response curves before and after labelling. The following conditions must apply: (i) all the receptors have the same affinity and efficacy with a given agonist, (ii) steady-state kinetics governed by the law of mass action apply, (iii) the affinity and efficacy of the agonist is unaltered by irreversible labelling of a fraction of the receptors, and (iv) the agonist does not cause desensitization. Double reciprocal plots for equiactive concentrations of the agonist before and after inactivation of a portion of the receptors (figs. 1B and 2B) should yield a straight line from which a dissociation constant, K (slope divided by ordinate intercept), and the fraction of receptors unoccupied by the irreversible label, q (1/slope), can be calculated (Furchgott and Bursztyn, 1967). Once the value of K has been determined for given agonist, it is possible to obtain information on the efficacy of an agonist from the law of mass action:
[RA]max [R,]
[A]max K + [A]max
where [A]max is the concentration of agonist required to produce the maximum response, [RA] max is the concentration of the. receptor-agonist complex required to produce the maximum response, and [ R t ] is the total concentration of receptors. The value of [A]max for each agonist can be determined from the dose-response curves before labelling. The relative efficacies of two agonist are obtained from the ratio of the values of [ R A ] m a x / [ R t ] for each agonist. In practice, a more accurate estimate of relative efficacies can be ob-
tained using half-maximal values. The validity of the calculated ratio of efficacies for the two agonists is dependent on all the criteria listed above. In addition, both agonists must act at the same receptors with the same mechanism, and their rates of uptake and metabolism must be the same. The data can be quantified in percentage format: R m = 100 X [ R k ] m a x / [ R t ] = % receptors which must be occupied in order to produce the maximum response, R S = 100 - R m = % 'spare' receptors, Rtm/R 2 = ratio of efficacies of two agonists, R a = 100 x q = % receptors unoccupied by the irreversible label (% active receptors), R i = 1 0 0 - R a = % receptors inactivated by the irreversible label. The data in figs. 1 and 2 have been used to calculate, by the methods described above, the various determinats of the biological activity of A N G II and A N G III in the rat uterus and rabbit aorta (table 1). For comparison, previously published data on the rat portal vein (Kwok and Moore, 1984) have been included. The relative
TABLE 2 Relative affinities and efficacies of angiotensins II and Ill in isolated tissues determined by photoaffinity labelling.
Rat uterus Rabbit aorta Rat portal vein
Affinity ratio
Efficacy ratio a
A N G I I / A N G III
A N G I I / A N G III
10 1 0.2
0.9 14 11
a Calculated from the [ A ] / ( K + [ A ] ) ratio at half-maximal response.
57 affinities and efficacies of A N G II and A N G III in each of the three tissues are given in table 2.
4. Discussion The data in figs. 1A and 2A demonstrate the feasibility of irreversible labelling of angiotensin receptors in isolated smooth muscle tissues with the photolabile angiotensin antagonist. The photoaffinity labelling procedure appears to be specific since the dose-response curves for the alternative agonists, oxytocin and noradrenaline, used in the rat uterus and rabbit aorta assays respectively, were not significantly affected by the photolabelling. For the rat uterus, evidence has been obtained previously which suggests that irreversible labelling of the receptors can be protected by large doses of angiotensin (Kwok and Moore, 1980). This type of investigation on the specificity of receptor labelling could not be carried out with the isolated rat portal vein (Kwok and Moore, 1984) because of the tachyphylaxis induced by large doses of angiotensin. Similarly, the present studies illustrate that supermaximal doses of angiotensin also cause densensitization of the isolated rabbit aorta and, since the validity of the irreversible labelling technique requires that this phenomenon not be present, large doses of angiotensin have been avoided throughout the experimental procedures with the rabbit aorta. Evidence has been obtained previously with the rat portal Vein which illustrates that the affinity constant does not change after different degrees of irreversible labelling (Kwok and Moore, 1984), suggesting the presence of a uniform population of receptors in this tissue, and possibly other smooth muscle tissues. Experiments in this laboratory indicate that A N G II and A N G III act at the same high affinity receptors in both the rat uterus (Kwok, 1982) and the rat portal vein (Scanlon and Moore, unpublished), and it is presumed that this is also the case for the rabbit aorta. For comparing the bioactivity determinants of angiotensins II and III, it has been assumed that the rates of uptake and metabolism of these two agonists by the isolated tissues are the same, and that the rate of conversion of A N G II to A N G III in the tissue bath is
not a significant factor. We have not observed any difference in the rates of onset or duration of the responses to A N G II and A N G III which, to a first approximation, suggests there may be no gross difference in rates of uptake and metabolism of these two ligands. Recent experiments in this laboratory also suggest that the presence of inhibitors of angiotensinase A, the enzyme that converts A N G II to A N G III, does not affect the dose-response curves to A N G II (Scanlon and Moore, unpublished observations). Furthermore, the photoaffinity label should also inactivate angiotensindegrading enzymes in these tissues. Therefore the comparison of relative affinities and efficacies for A N G II and A N G III in these isolated tissues may not be unreasonable. Based on the data in table 2, it appears that A N G II is the higher affinity ligand in the rat uterus and that both A N G II and A N G III have similar efficacies in this tissue. Thus the biological potencies of these two ligands are dependent only on the affinity in the rat uterus. In contrast, A N G II and A N G III appear to have the same affinities for angiotensin receptors in the rabbit aorta, and the higher potency of A N G II is determined solely by its increased efficacy. For the rat portal vein, A N G III is the higher affinity ligand but has the lower efficacy of the two agonists, suggesting that A N G III, under certain conditions, could perhaps reduce the response to a given concentration of A N G II (Kwok and Moore, 1984). The relative efficacies of A N G II and A N G III in each tissue are reflected in their spare receptor populations, with greater efficacy being concomitant with greater receptor reserve. An appreciable receptor reserve is present for both ligands in all tissues, except for the rat portal vein where there are no spare receptors for A N G III. The significantly different relative affinities and efficacies of A N G II and A N G III in the three isolated smooth muscle tissues studied suggest that angiotensin receptors in all of these tissues are kinetically different from one another. For vascular smooth muscle tissues, subtle differences between selected vascular beds could have relevant physiological consequences in the regulation of blood flow and blood pressure. Although the two vascular smooth muscle tissues investigated in this
58 study derived from different species, the possibility should be considered that arterial and venous tissues may display substantially different reactivities towards A N G II and A N G III. The calcium and magnesium concentration utilized for assays of the rat uterus and rat portal vein were sub-physiological, whereas the divalent cation concentrations utilized in rabbit aorta assays approached physiological concentrations. It is possible that the relative affinities and efficacies of A N G II and A N G III are dependent on divalent cation concentrations. A difference in the susceptibility of uterine tissue, c o m p a r e d to vascular tissue, to photoaffinitiy labelling was observed. In order to produce 70-90% blockade of angiotensin receptors in all three tissues, the rat uterus required five photoaffinity labellings with a flash time of 3 min in each case, whereas the rat portal vein and rabbit aorta required only two photoaffinity labellings with a flash time of 1 min. These findings suggest that angiotensin receptors in the rat uterus are much less accessible to photoaffinity labelling than are receptors in either of the vascular smooth muscle tissue investigated. For the rat uterus, there is a possible indication here that the endometrial cells, which are located on the inside of the tubular tissue m o u n t e d for the bioassay, m a y be the primary cellular determinants of angiotensin activity. Alternatively, the relative efficiency of photolabelling m a y be a direct consequence of the thickness of the tissue as it relates to accessibility of the photolabel and U V light to the total smooth muscle population. Finally, these photoaffinity labelling experiments have raised an issue concerning the mechanism of angiotensin tachyphylaxis. For both the rat portal vein (Kwok and Moore, 1984) and the rabbit aorta, it was observed that desensitization to maximal doses of angiotensin was more marked after photolabelling than before. Apparently susceptibility to angiotensin tachyphylaxis is dependent on the n u m b e r of active receptors present.
This suggests that angiotensin itself m a y be able to deactivate a fraction of the available receptors, such that: (i) there is no notable induction of tachyphylaxis when there is a large receptor reserve present before labelling; and (ii) desensitization occurs when the size of the spare receptor pool is decreased after labelling. This appears to indicate that the mechanism of angiotensin tachyphylaxis is a ligand-induced receptor ' d o w n regulation' phenomenon, resulting from a decrease in receptor reactivity or possibly internalization of angiotensin receptors (Robertson and Khairallah, 1971; Re et al., 1981).
Acknowledgments This work was supported by the Alberta Heart Foundation, the Alberta Heritage Foundation for Medical Research and the Medical Research Council of Canada. We thank Joanne Ward for typing the manuscript and Martin Scanlon for helpful discussion.
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