- Email: [email protected]
Angiotensin II-induced tachyphylaxis in aortas of normo- and hypertensive rats: changes in receptor affinity
European Journal of Pharmacology, 232 (1993) 173-180
173
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$0600
EJP 52943
Angiotensin II-induced tachyphylaxis in aortas of normoand hypertensive rats: changes in receptor affinity S.C. K u t t a n and M.K. Sire Department of Pharmacology, Faculty of Medicine, National Universityof Singapore, Singapore 0511, Singapore Received 10 August 1992, revised MS received 24 November 1992, accepted 8 December 1992
Angiotensin II-induced tachyphylaxiswas found to be associated with changes in agonist affinity (K a) and ECs0 values, as assessed by using Furchgott's equation derived for the determination of full agonist affinity. The diminished affinity during tachyphylaxis was observed in aorta ring preparations from both Wistar-Kyoto and spontaneously hypertensive rats. Noradrenaline (10 -9 M) reduced the increase in the K a value during tachyphylaxis in both strains. The results suggest that tachyphylaxis occurs at the level of the receptor, resulting in changes in the affinity of the ligand for the receptor and in the coupling efficiency of the receptor system. The results also support the probable role of modulators acting on allosteric receptor sites. Angiotensin II; Receptor affinity; Smooth muscle (vascular)
1. Introduction
We have observed that, in the rat aorta, tachyphylaxis to angiotensin II is characterised by a depressed maximum response and a significant change in the ECs0 value, and that these changes are attenuated by noradrenaline and prostaglandin F2~ (PGF2~ (Sim and Kuttan, 1992). The change in the ECs0 value suggests that there may be an alteration in the affinity of the receptor for the ligand or that the receptor coupling mechanism is compromised. Furthermore, others have suggested that the mechanism responsible for mediating tachyphylaxis is at the level of the receptor (Aboulafia et al., 1989; Abdellatif et al., 1991). In order to investigate whether angiotensin II-induced tachyphylaxis is associated with changes in receptor affinity, we made the assumption that angiotensin II-induced tachyphylaxis involves the irreversible deactivation of a homologous receptor population under in vitro conditions. The irreversibility is based on the observation that tachyphylaxis remains the same after 30, 60, 90 and 180 min of reequilibration following the first exposure. Furthermore, it has, been shown that there are no spare angiotensin II receptors for phospholipase C-mediated inositol tris-
Correspondence to: M.K. Sim, Department of Pharmacology, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore.
phosphate (IP3) formation in vascular smooth muscle cells (Ullian and Linas, 1990). On the basis of these assumptions, we used the equation which describes receptor deactivation by chemical alkylation (Furchgott, 1966) to evaluate the affinity constant of angiotensin II from the first and second concentration-response curves for angiotensin II. The relationship for determining receptor affinity for full agonists after the receptors have been irreversibly deactivated can be described as a double reciprocal equation, 1/[A] = 1/[A']. 1/q + 1 / K a • (1/q 1), where [A] and [A'] are equiactive concentrations of the agonist before and after deactivation, and q is the fraction (%) of receptors available for a ligand interaction. It follows that the amount of receptors involved in the response is given by 100 - q, assuming that deactivation results from a ligand-receptor interaction and response. The straight line from the double reciprocal plot of 1/[A] vs. 1/[A'] is then used to evaluate the K a value given by: Ka = ( s l o p e - 1)/intercept. On the basis of data obtained from three consecutive concentration-response curves for angiotensin II in rat aorta rings from both normo- and hypertensive adult rats, we determined the K a and q values of the receptors. If tachyphylaxis does not involve a change in receptor affinity, then the K a derived for the first and second cumulative concentration-response curves should not be significantly different from each other. The effects of noradrenaline on these K~ and q values were also investigated.
174 2. Materials and methods
2.1. Preparation of aorta rings Adult (20-24 weeks) Wistar-Kyoto rats (WKY; 269 + 7.0 g) and age-matched spontaneously hypertensive rats (SHR; 274 + 4.4 g) were obtained from the Animal Resource Centre, Western Australia. The animals were stunned by a blow to the base of the neck and immediately killed by cervical dislocation. The aorta was then removed and placed in Krebs buffer (composition in mM: NaCI 118, KCI 4.8, CaCI 2 2.5, MgSO 4 1.2, KH2PO 4 1.2, N a H C O 3 24, glucose 11, E D T A 0.03) maintained at 37°C. Aorta rings were prepared as described previously (Sim and Singh, 1987) with modifications. Briefly, the excised aorta was placed in Krebs solution and aerated with a mixture of 95% 0 2 and 5% CO 2. The aorta was allowed to equilibrate with the Krebs solution for at least 45 min with wash steps every 15 min to remove all biological fluids trapped within the aorta. Fat and connective tissue were gently removed from the aorta and rings of about 2 mm long were prepared. One end of the aorta ring was connected via a silk thread to an isotonic transducer with a 300 V per mm displacement of the lever arm, measured across 10 000 /2 load (Ugo Basile, Model 7006), giving an amplification factor of 300 from a pen deflection 0.3 mm per /zm of ring shortening when coupled to a Grass Polygraph with the preamplifier sensitivity set at 0.5 m V / c m (Model 7D). The other end was attached to a thin steel wire hook fixed onto the gas inlet tubing. The preparation was immersed in a 10-ml organ bath containing Krebs solution and constantly aerated. After 60 min of equilibration with a load of 0.5 g, the protocol described below was started.
2.2. Protocol The experimental protocol used t o study the development of angiotensin II-induced tachyphylaxis in the aorta rings of adult normo- and hypertensive rats was based on the detailed method worked out previously (Sim and Kuttan, 1992), but with slight modifications. Each ring was exposed to five 10-min preincubations with 10 -9 M noradrenaline. The second to fifth preincubations were preceded by a 30-rain interval during which each ring was washed with Krebs solution devoid of drugs. Ten minutes after the third preincubation, the ring was exposed to increasing cumulative concentrations of angiotensin II (10 -1° to 10 -6 M) to obtain a cumulative concentration-response curve. Each cumulative concentration-response curve for angiotensin II was obtained as described earlier for the rat aorta (Shibata et al., 1990) with slight modifications. Each cumulative concentration of angiotensin II (a 10-fold
increase) was added after the response to the preceding concentration had reached a plateau. To ensure that the maximum contraction had been attained, an additional dose was administered until no further response was elicited. The second and third concentration-response curves were obtained, under similar conditions as the first, after the fourth and fifth preincubations with noradrenaline, respectively. In the controls where noradrenaline was not used, rings were exposed to cumulative concentrations of angiotensin II until the maximum response was attained. The second and third cumulative concentration-response curves were made after 30 min of reequilibration and washing. At the end of the experiment and after two washes, the ring was treated with 10 -8 M noradrenaline followed by 10 -6 M acetylcholine to evaluate its contractility and endothelial integrity, respectively. Data from rings that attained maximum contractions of less than 90% of control rings or that did not relax to acetylcholine were not included in the analyses. Parallel control concentration-response curves for angiotensin II were obtained in aorta rings that were not preincubated with noradrenaline. The protocol of using three 10-min exposures of noradrenaline was adopted so as to (i) separate rings that contracted to the three exposures of the spasmogen from those that did not, and (ii) determine the magnitude of contraction and extent of desensitisation of the rings to the spasmogen.
2.3. Calculations Concentration-response curves were subjected to Probit transformation and regression analysis to yield a straight line. The intercept and gradient were then used to calculate equiactive concentrations between the two concentration-response curves. The K a and q values were evaluated by substituting these values into the double reciprocal plot. The ECs0 values were also calculated from the regression lines. The K a and q values for the first response were derived from the second response, which is expressed as a percentage of the maximum value of the first response, i.e. [A] from the first response and [A'] from the second response. Data for the second response were similarly derived from the third response, which is expressed as a percentage of the maximum value of the second response, i.e. [A] from the second response and [A'] from the third response. These K a values were then used to determine the fractional receptor occupancy with the following formula (Schwietert et al., 1991): % occupancy = 100 X [A]/([A] + Ka). Log [occupancy] was then plotted against percent maximum response of the first and second concentration-response curves. The pEOs0, defined as the logarithm of the occupancy that 'gives 50% response, was calculated and used to determine the significance of the shifts of the plots.
175
Angiotensin II (acetate salt) was purchased from Sigma (St. Louis, U.S.A) and noradrenaline (tartrate salt) was purchased from Fluka Biochemika (Buchs, Switzerland). Angiotensin II was prepared fresh in Krebs solution. Noradrenaline was dissolved in 1% ascorbic acid, and acetylcholine was prepared as a stock solution of 1 M in 0.5 M sodium dihydrogen phosphate solution. Drugs concentrations reflect the concentrations of their salts. All other reagents used were of analytical grade.
2.4. Statistics The data are expressed as mean values + S.E.M. Student's t-test was used in the statistical analyses of the data when only two sets of independent variables were compared. A paired t-test was used to analyse data with dependent variables, e.g. tachyphylaxis. In all cases, P values less than 0.05 were considered significant.
3.2. Angiotensin II-induced tachyphylaxis The results shown in fig. 1 indicate that the maximum response to the second exposure to angiotensin II (see table 1), was significantly (P < 0.05, paired t-test) depressed in the WKY (lst: 129 + 10 Izm; 2nd: 52 + 4 /xm) and the age-matched SHR (lst: 121 + 7/zm; 2nd: 55 _+3 tzm). The maximum shortening induced in both strains during the second response was not significantly different from each other (P < 0.05, Student's t-test). Comparison of the values for the first and second responses shows that the increase in the ECs0 value of both WKY (lst: 4.48 + 0.45 nM; 2nd: 6.00 + 0.58 nM) and SHR (lst: 4.55 + 0.42 nM; 2nd: 18.4 + 0.98 nM) was significantly different from each other (P < 0.05, Student's t-test). This was also reflected in a diminished affinity, with t h e tachyphylactic response being characterised by a significant increase in the Ka values in the WKY (lst: 76 +_3 nM; 2nd: 517 Jr 10 nM) and SHR (lst: 8.8 + 1.9 nM; 2nd: 170 + 17nM).
3.3. Effect of noradrenaline on responses elicited by angiotensin H in SHR and WKY rats
3. Results
3.1. Angiotensin II-induced response in SHR and WKY In the case of the first response in the absence of noradrenaline, table 1 shows that the K a value of the SHR (8.8 + 1.9 nM) was significantly (P < 0.05, Student's t-test) lower than that of the WKY (76 + 3 nM). However, the ECs0 (WKY: 4.48 + 0.5 nM; SHR: 4.55 + 0.4 nM) and maximum response (WKY: 129 + 10 /~m; SHR: 121 + 7.0/.~m) values were not significantly different. The q value derived from the equation indicates that a higher proportion of receptors in the SHR (91.2%) were not deactivated, whereas only 3.8% in the WKY were not deactivated.
The maximum response of WKY aorta rings to angiotensin II in the presence of noradrenaline was significantly depressed (P < 0.05, Student's t-test) from 129 + 10 Ixm (control) to 91 + 6 /~m (table 1). Although there was a significant reduction (P < 0.05, Student's t-test) in the ECs0 value (control: 4.48 + 0.45 nM; noradrenaline: 1.47 +0.15 riM) for the WKY, there was no corresponding reduction in the K a value (control: 76 + 3 nM; noradrenaline: 84 + 9 nM). The maximum response of SHR aorta rings to angiotensin II in the presence of noradrenaline was also depressed (control: 121 + 7 gM; noradrenaline: 85 + 7 /xM) but there was no significant (P < 0.05, Student's t-test) change in either the ECs0 value (control: 4.55 +
TABLE 1 Constants for angiotensin II concentration-response curves derived from the double reciprocal equation. Parameter
Ka(nM) ECso(nM) Max. resp.(/~m) q (%)
Response
1st 2nd 1st 2nd 1st 2nd 2nd 3rd
WKY
SHR
- noradrenaline (n - 12)
+ noradrenaline (n = 12)
- noradrenaline (n = 19)
+ noradrenaline (n = 13)
76 + 3 517 -I-10 b 4.48+ 0.5 6.00+ 0.6 b 129 ±10 52 ± 4b 3.8 2.3
84 +9 17.8 ± 1 b 1.47-/-0.2 ¢ 6.86±0.7 b 91.0 ±6.0 c 64.6 ±4.2 39.3 9.4
8.8 + 1.9 a 170 ±17 b 4.55± 0.4 18.4 ± 1.0 b 121 ± 7.0 55 ± 3.2 b 91.2 39.5
2.9 +2.3 10.2 ±0.3 b 4.64+0.4 12.6 ±0.8 b 85.0 ±7.0 c 31.6 +2.5 41.9 35.2
a Significantly different from the corresponding value of the WKY (Student's t-test), b Significantly different from the corresponding value in the first response (paired t-test), c Significantly different from the corresponding value in the absence of noradrenaline (Student's t-test).
176
la
lb
Wlstar-Kyoto Rats m R
m R
i
100
'100
i il ~d Me
Lof,Io[AII
"
"
LoItlO[AI!
M!
lc
MI
ld
Spontaneously Hypertensive Rats N
Ill W
i •"-
:.
' -10
-9'
4
:7
-6'
L%o[~al M!
k-
' -I1
-10
-g
-8'
-7'
-6'
L . ~ o I A n M!
Fig. 1. Three consecutive concentration-response curves for angiotensin II ( II, first; o, second; A, third) in aorta rings of the WKY (a and b) and SHR (c and d), in the absence (a and c) and presence (b and d) of 10 - 9 M noradrenaline. Number of rings used in (a), (b), (c) and (d) were 12, 12, 19 and 13, respectively.
0.4 nM; noradrenaline: 4.64 + 0.4 nM) or the K~ value (control: 8.8 5- 1.9 nM; noradrenaline: 2.9 + 2.3 nM). However, the q value indicated that there was an increase in receptor activation (control: 8.8%; noradrenaline: 58.1%) i.e. a decrease in receptors available for interaction (control: 91.3%; noradrenaline: 41.9%).
3.4. Effect of noradrenaline on angiotensin II-induced tachyphylaxis In the WKY, comparison of the first and second responses in the presence of noradrenaline, showed
that there was a significant decrease (P < 0.05, paired t-test) in the K a value (lst: 84 + 9 nM; 2nd: 17.8 + 1 nM). However, there was a significant increase (P < 0.05, paired t-test) in the ECs0 value (lst: 1.47 + 0.15 nM; 2nd: 6.86 + 0.70 nM). In the SHR, comparison of the first and second responses showed that there was a smaller increase in the K a value in the presence of noradrenaline (lst: 2.9 + 2.3 nM; 2nd: 10.2 + 0.3 nM) than in the control (lst: 8.8 + 1.9 nM; 2nd: 170 + 17 nM). T h e r e was also a •similar trend with respect to the ECs0 value in the presence of noradrenaline (lst: 4.64 5-0.4 nM; 2nd:
177 12.6 + 0.8 nM) and in the controls (lst: 4.55 + 0.4 nM; 2nd: 18.4 + 1.0 nM). The q value indicated that in the presence of noradrenaline a smaller proportion of receptors was being recruited to elicit the response, as seen in the difference between the q values obtained for the second and third response (control: 51.7%; noradrenaline: 6.7%).
I00
eo
6o
E "~
40
u
-~.~-io-i.~-;.o-4.~-~.o-;.~
4. D i s c u s s i o n
o'.o
Log [Occupancy]
The maximum contraction elicited by the first exposure of the WKY and S H R to angiotensin II was of comparable magnitude although the SHR showed a higher receptor affinity for the ligand compared to the WKY. As a greater proportion of receptors were required to induce the same response in the WKY (96.2%) than in the SHR (8.8%), this suggests that the angiotensin II-receptor interaction can induce a greater response in the SHR than in the WKY. This suggestion is supported by the log [occupancy] vs. response plots (see fig. 2), where the S H R plots are on the left of those of the WKY. From fig. 1, the extent of angiotensin II-induced tachyphylaxis, in terms of a depressed maximum response, was similar in the adult W K Y and age-matched SHR, i.e. 60 and 55%, respectively. Furthermore, in both strains the second (tachyphylactic) response to angiotensin II was comparable. In terms of K a and ECs0 values, there was a greater increase in S H R (Ka: 19.3 × ; E C s 0 : 4 . 0 × ) than in the WKY (Ka: 7.1 × ; E C s 0 : 1 . 3 × ), implying the occurrence of a greater degree of tachyphylaxis in the SHR. This discrepancy may be explained by the balance achieved between the counteracting influences of reduced receptor reserve
~D
100
~
so
E
so
x
4O
o
~
o -6
-5
-4
-3
-2
-1
0
Log [Occupancy] Fig. 2. Log [occupany]-responseplots for the first cumulative concentration-response curve for angiotensin II in SHR ( • ) and WKY (•); and second cumulative concentration-response curve for angiotensin II in SHR (zx) and WKY (1:3), in the absence of 10 - 9 M noradrenaline. The pEOso of the first (1.546+0.11, n = 19) and second (1.199+0.062, n = 19) concentration-response curves for SHR was significantly different (P < 0.05, paired t-test). The pEOso of the first (1.54:[:0.08, n = 12) and second (3.549+_0.013, n = 12) concentrationresponse curves for WKY was significantlydifferent (P < 0.05, paired t-test).
Fig. 3, Log [occupancy]-responseplots for the first cumulative concentration-response curve for angiotensin II in WKY, in the absence ([:3) and presence ( • ) of 10 - 9 M noradrenaline; and SHR, in the absence (,x) and presence ( • ) of the catecholamine. The pEOs0 of WKY in the absence (1.562+0.085, n = 12) and presence (1.900+ 0.053, n = 12) of 10 - 9 M noradrenaline was significantly different (P <0.05, Student's t-test). The pEOs0 of SHR in the absence (1.546_+0.11, n = 19) and presence (0.544+0.06, n = 13) of 10 - 9 M noradrenaline was significantly different (P < 0.05, Student's t-test). and increased coupling efficiency. However, the K a value for the tachyphylactic response in the SHR was almost 3 times lower than that in the WKY (SHR: 170 + 17 nM; WKY: 517 + 10 nM), whereas the ECs0 value (SHR: 18.4 + 0.98 nM; WKY: 6.00 + 0.58 nM) was almost three times higher. Since the ECs0 value is a parameter of the observed response, this implies that in the S H R the coupling efficiency had been compromised by tachyphylaxis. This is reflected in the log [occupancy] vs. response plot of the S H R where the second response is on the right of the first response. In contrasts in the WKY, the coupling efficiency was improved under conditions in which there was a reduced number of available receptors, with the log [occupancy] vs. response plot appearing on the left of the first response. This can also be inferred from the q values, which indicate that more receptors were recruited in the S H R than in the WKY (SHR: 51.7%, WKY: 1.5%; values derived from the difference in q values obtained from the second and third exposure to angiotensin II in the absence of noradrenaline). There seems to have been a marginal increase in the coupling efficiency of the angiotensin II-receptor interaction in the presence of noradrenaline in WKY aorta rings (see fig. 3). Although there was an approximately 30% decrease in the maximum response, partly as a result of a 37% decrease in the percentage of receptors activated (control: 96.2%; noradrenaline: 60.7%), there was a 3-fold decrease in the ECs0 value, implying that the efficiency of the ligand-receptor response coupling had increased. In the case of the SHR, the insignificant changes in ECs0 and K a values coupled with the decreased maximum response (30%) but increased receptor activation (6.6 × ) suggest that noradrenaline compromises the responses of the S H R to angiotensin II. This decrease in coupling efficiency is seen in fig. 3,
178 100 c o D.
8O
E
60
==
the observed attenuation of angiotensin II-induced tachyphylaxis. Assuming that deactivation of receptors occurs when ligand binding results in a response, the q values suggest that the response in the SHR is governed by the concept of 'spare-receptors' as only a small number were required for a maximum response, i.e. q = 91.2%, whereas in the WKY almost all receptors were bound during the maximum response, i.e. q = 3.8%. During tachyphylaxis, there is an increase in the influence of spare-receptors in the WKY tissue, with a reduction in the SHR. This apparent switch cannot be explained at the moment; however, it suggests that the receptor system may not be bound by any particular mechanism of ligand-receptor interaction. In protein allosterism, proteins are considered malleable structures with a spectrum of conformations, as opposed to rigid matrices. Cooperative protein effects are not specifically accounted for in the occupation, rate and inactivation receptor theories (Kenakin, 1987). If one drug molecule binds to one receptor to generate a response, the binding reaction can be described by a simple hyperbola according to the Langmuir adsorption isotherm. However, angiotensin II concentrationresponse curves are sigmoidal rather than hyperbolic, thereby denoting cooperative binding during the receptor-ligand interaction. This cooperativity may be similar to that described recently i.e. heterotropic and homotropic (Scalon et al., 1990). Cooperativity may involve allosteric sites (Timmermans et al., 1991) which may accommodate natural inhibitors (Moore et al., 1989) or modulators (Kuttan and Sim, 1991a). The response of the aorta rings to angiotensin II is determined by the K a value and the coupling efficiency of the activated receptors. In the SHR this coupling efficiency was greater in the absence of noradrenaline and decreased in its presence. In vivo, this may manifest itself as a reduced feedback signal in the regulation of angiotensin II response, thus prompting a greater response to the octapeptide. In the WKY, the presence of noradrenaline had an opposite effect by increasing the coupling efficiency. In vivo this may be manifested as an enhanced regulatory feedback signal and hence a limited response to the octapeptide. Although we believe that tachyphylaxis may be an in vitro phenomenon only (Sim and Kuttan, 1992), the existence of the phenomenon in vitro can be used to give insight into receptor function in normal and pathological conditions. From the present work, there seems to be a balance between the receptor affinity, the number of activated receptors and the coupling efficiency. The counteracting influences of these three parameters ultimately influence the observed ECs0 and maximum response values. There seems to be an overall reduction in the coupling efficiency in the SHR compared to the WKY in the tachyphylactic state. This -
4o ¢: 20 o.. 0
~ -6
, -5
-4 Log
-3
-2
-1
0
[Occupancy]
Fig. 4. Log [occupancy]-responseplots for the second cumulative concentration-response curve for angiotensin II in the WKY in the absence ([]) and presence ( • ) of 10-9 M noradrenaline; and SHR, in the absence (0) and presence (e) of 10 - 9 M noradrenaline. The pEOs0 of WKY in the absence (3.549+0.013, n = 12) and presence (0.954+0.007, n =12) of 10 - 9 M noradrenaline was significantly different (P < 0.05, Student's t-test). The pEOs0 of SHR in the absence (1.199+ 0.062, n = 19) and presence (1.4635:0.008, n = 13)of 10 - 9 M noradrenaline was significantlydifferent (P < 0.05, Student's t-test). where the log [occupancy] vs. response plot of the SHR is on the right of the control. During tachyphylaxis and in the presence of noradrenaline the affinity of the angiotensin II receptor was increased 4.7-fold increase in the normotensive animals. However, there was also a 4.7-fold increase in the ECs0 value, which was greater than that seen in the absence of the catecholamine (control: 1.3 X; noradrenaline: 4.7 Z ). This seems to suggest a reduced ligand-receptor response coupling efficiency, which is reflected in fig. 4. Thus under these conditions the observed attenuation of angiotensin II-induced tachyphylaxis in normotensive animals by noradrenaline may be due to the greater number of activated receptors. This is reflected in the difference between q values obtained from the second and third concentration-response curves for angiotensin II in the WKY (control: 1.5%; noradrenaline: 29.9%). Thus, tachyphylaxis is apparently associated with an increase in the K a value, i.e. decrease in affinity, which is more pronounced in the SHR than the WKY. Although on the second exposure to angiotensin II there was a similar degree of depression of the maximum response in both strains, there was a greater increase in the ECs0 value in the SHR than the WKY. In SHR aorta rings, noradrenaline affected its response to angiotensin II by decreasing the ligand receptor response coupling efficiency, whereas noradrenaline increased this coupling efficiency in the WKY. This effect was reversed, in both cases, in the second tachyphylactic response (see fig. 4). Although there was a decreased coupling efficiency in the WKY, the increase in the proportion of receptors recruited and the decrease in the K a value result in an increased maximum response
179
again may cause a weak regulatory negative feedback signal, leading to a greater recruitment of receptors in the SHR. The presence of noradrenaline seems to favour the SHR rather than the WKY in terms of coupling efficiency during the tachyphylactic response. However, because of the low numbers of receptors activated in the SHR, this is not manifested in the response of the aorta rings to angiotensin II. Tachyphylaxis is associated with changes at the receptor level, i.e. change in affinity and coupling efficiency. Factors which influence these parameters can be derived from the endothelium or the smooth muscle cell. The present work provides us with some insight into changes in certain parameters associated with ligand-receptor interaction and helps us reformulate our ideas about the receptor theory. The variable affinity hypothesis of receptors has been proposed earlier, albeit with reference to o~-adrenoceptors (Oriowo et al., 1991). Oriowo et al. proposed that factors in the microenvironment of the receptor could alter the affinity of the receptor. The possible involvement of such factors is also suggested from the observation that the endothelium may be a source of an endothelium-derived modulating factor which influences the affinity of the angiotensin receptor (Kuttan and Sim, 1991b). The recent demonstration of an allosteric site modulating the affinity of angiotensin II for its AT 1 receptor (Boulay et al., 1992) suggests the probable existence of ligands that could function as allosteric modulators of receptor affinity. In the in vitro environment, tachyphylaxis may therefore involve the displacement of the factor and uncoupling of the receptor-signal transduction system, resulting in a reduction in receptor affinity and in the tissue response. Thus analogues of angiotensin that bind to the receptor and do not initiate the displacement of the modulator will be seen to be nontachyphylactic (Oshiro et al., 1989). Furthermore, auxilIary agonists may reactivate the receptor through intracellular biochemical processes (i.e. phosphorylation of intracellular domains of the receptor), which may explain the partial reversal or attenuation of in vitro tachyphylaxis that occurs in the presence of other spasmogens, e.g. noradrenaline and PGF2,,. It has also been shown that vasoconstrictors, e.g. angiotensin II, vasopressin, serotonin and noradrenaline, stimulate tyrosine-protein phosphorylation of the same set of proteins in vascular smooth muscle cells (Tsuda et al., 1991) and may augment each others response in vivo. With the recent discovery of angiotensin peptides in the blood (Lawrence et al., 1990), one wonders whether multiple receptor subtypes or multiple receptor affinity states are the key which allows angiotensin receptors to discern one ligand from another. The theory of a modulating factor changing the affinity state of the receptor is relevant as improved technology reveals
that physiological systems have the ability to respond to much lower levels of ligands than previously thought. The possibility that other positive and negative allosteric modulating factors exist for other receptor systems cannot be discounted either.
References Abdellatif, M.M., C.F. Neubauer, W.J. Lederer and T.B. Rogers, 1991, Angiotensin-induced desensitisation of the phosphoinositide pathway in cardiac cell occurs at the level of the receptor, Circ. Res. 69, 800. Aboulafia, J., M.E.M. Oshiro, T. Feres, S.I. Shimuta and A.C.M. Paiva, 1989, Angiotensin II desensitisation and Ca 2+ and Na + fluxes in vascular smooth muscle, Pfliigers Arch. 415, 230. Boulay, G., G. Servant, T.T. Luong, E. Escher and G. Guillemette, 1992, Modulation of angiotensin II binding affinity by allosteric interaction of polyvinyl sulfate with an intracellular domain of the DuP753 sensitive angiotensin II receptor of bovine adrenal glomerulosa, Mol. Pharmacol. 41, 809. Furchgott, R.F., 1966, The use of beta-haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist complexes, in: Advances in Drug Research, eds. N.J. Harper and A.B. Simmonds (Academic Press. New York) p. 21. Kenakin, T.P., 1987, Pharmacologic Analysis of Drug-Receptor Interaction (Raven Press). Kuttan, S.C. and M.K. Sim, 1991a, Endothelium-dependent response of rabbit aorta to femtomolar concentrations of angiotensin II, J. Cardiovasc. Pharmacol. 17, 929. Kuttan, S.C. and M.K. Sim, 1991b, Isolation and purification of the putative EDMF from the rabbit aortic endothelium (Abstract), Pharmacologist 33, P470. Lawrence, A.C., G. Evin, A. Kladis and D.S. Campbell, 1990, An alternative strategy for the radioimmunoassay of angiotensin peptides using amino-terminal directed antisera: measurement of eight angiotensin peptides in human plasma, J. Hypert. 8, 715. Moore, G.J., R.C. Ganter and K.J. Franklin, 1989, Angiotensin 'antipeptides': ( - ) messenger RNA complimentary to human All ( + ) messenger RNA encodes an angiotensin receptor antagonist, Biochem. Biophys. Res. Commun. 160, 1387. Oriowo, M.A., R.D. Bevan and J.A. Bevan, 1991, Variable receptor affinity and tissue sensitivity, Blood Vessels 28, 115. Oshiro, M.E.M., S.I. Shimuta, T.B. Paiva and A.C.M. Paiva, 1989, Evidence for a regulatory site in angiotensin II receptor of smooth muscle, Eur. J. Pharmacol. 166, 411. Scalon, M.N., P. Koniaz and G.J. Moore, 1990, The relationship between homotropic and heterotropic cooperativity for angiotensin receptors in smooth muscle, Gen. Pharmacol. 21, 59. Schwietert, H.R., M.A.M. Gouw, D. Wilhelm, B. Wiffert and P.A. Van Zwieten, 1991, The role of al-adrenoceptor subtypes in the phasic and tonic responses to phenylephrine in the longitudinal smooth muscle of the rat portal vein, Naunyn-Schmiedeb. Arch. Pharmacol. 343, 463. Shibata, R., S. Morita, K. Nagai, S. Miyata and T. lwasaki, 1990, Effects of H7 (protein kinase inhibitor) and phorbol ester on aortic strips from spontaneously hypertensive rats, Eur. J. Pharmacol. 175, 161. Sim, M.K. and S.C. Kuttan, 1992, Effects of noradrenaline and prostaglandin F2, on angiotensin-induced contraction and tachyphylaxis in rat aortic rings, Pharmacol. Toxicol. 70, 60. Sim, M.K. and M. Singh, 1987, Decreased responsiveness of the aortae of hypertensive rats to acetylcholine, histamine and noradrenaline, Br. J. Pharmacol. 90, 147.
180 Timmermans, P.B.M.W.M., P.C. Wong, A.T. Chiu and W.F. Herblin, 1991, Non-peptide angiotensin II receptor antagonist, Trends Pharmacol. 12, 55. Tsuda, T., Y. Kawahara, H. Shii, M. Koide, Y. Ishida and M. Yokogama, 1991, Vasoconstrictor-induced protein-tyrosine phos-
phorylation in cultured vascular smooth muscle cells, FEBS Lett. 285, 444. Ullian, M.E. and S.L. Linas, 1990, Angiotensin II surface receptor coupling to inositol trisphosphate formation in vascular smooth muscle cells, J. Biol. Chem. 265, 195.
173
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$0600
EJP 52943
Angiotensin II-induced tachyphylaxis in aortas of normoand hypertensive rats: changes in receptor affinity S.C. K u t t a n and M.K. Sire Department of Pharmacology, Faculty of Medicine, National Universityof Singapore, Singapore 0511, Singapore Received 10 August 1992, revised MS received 24 November 1992, accepted 8 December 1992
Angiotensin II-induced tachyphylaxiswas found to be associated with changes in agonist affinity (K a) and ECs0 values, as assessed by using Furchgott's equation derived for the determination of full agonist affinity. The diminished affinity during tachyphylaxis was observed in aorta ring preparations from both Wistar-Kyoto and spontaneously hypertensive rats. Noradrenaline (10 -9 M) reduced the increase in the K a value during tachyphylaxis in both strains. The results suggest that tachyphylaxis occurs at the level of the receptor, resulting in changes in the affinity of the ligand for the receptor and in the coupling efficiency of the receptor system. The results also support the probable role of modulators acting on allosteric receptor sites. Angiotensin II; Receptor affinity; Smooth muscle (vascular)
1. Introduction
We have observed that, in the rat aorta, tachyphylaxis to angiotensin II is characterised by a depressed maximum response and a significant change in the ECs0 value, and that these changes are attenuated by noradrenaline and prostaglandin F2~ (PGF2~ (Sim and Kuttan, 1992). The change in the ECs0 value suggests that there may be an alteration in the affinity of the receptor for the ligand or that the receptor coupling mechanism is compromised. Furthermore, others have suggested that the mechanism responsible for mediating tachyphylaxis is at the level of the receptor (Aboulafia et al., 1989; Abdellatif et al., 1991). In order to investigate whether angiotensin II-induced tachyphylaxis is associated with changes in receptor affinity, we made the assumption that angiotensin II-induced tachyphylaxis involves the irreversible deactivation of a homologous receptor population under in vitro conditions. The irreversibility is based on the observation that tachyphylaxis remains the same after 30, 60, 90 and 180 min of reequilibration following the first exposure. Furthermore, it has, been shown that there are no spare angiotensin II receptors for phospholipase C-mediated inositol tris-
Correspondence to: M.K. Sim, Department of Pharmacology, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore.
phosphate (IP3) formation in vascular smooth muscle cells (Ullian and Linas, 1990). On the basis of these assumptions, we used the equation which describes receptor deactivation by chemical alkylation (Furchgott, 1966) to evaluate the affinity constant of angiotensin II from the first and second concentration-response curves for angiotensin II. The relationship for determining receptor affinity for full agonists after the receptors have been irreversibly deactivated can be described as a double reciprocal equation, 1/[A] = 1/[A']. 1/q + 1 / K a • (1/q 1), where [A] and [A'] are equiactive concentrations of the agonist before and after deactivation, and q is the fraction (%) of receptors available for a ligand interaction. It follows that the amount of receptors involved in the response is given by 100 - q, assuming that deactivation results from a ligand-receptor interaction and response. The straight line from the double reciprocal plot of 1/[A] vs. 1/[A'] is then used to evaluate the K a value given by: Ka = ( s l o p e - 1)/intercept. On the basis of data obtained from three consecutive concentration-response curves for angiotensin II in rat aorta rings from both normo- and hypertensive adult rats, we determined the K a and q values of the receptors. If tachyphylaxis does not involve a change in receptor affinity, then the K a derived for the first and second cumulative concentration-response curves should not be significantly different from each other. The effects of noradrenaline on these K~ and q values were also investigated.
174 2. Materials and methods
2.1. Preparation of aorta rings Adult (20-24 weeks) Wistar-Kyoto rats (WKY; 269 + 7.0 g) and age-matched spontaneously hypertensive rats (SHR; 274 + 4.4 g) were obtained from the Animal Resource Centre, Western Australia. The animals were stunned by a blow to the base of the neck and immediately killed by cervical dislocation. The aorta was then removed and placed in Krebs buffer (composition in mM: NaCI 118, KCI 4.8, CaCI 2 2.5, MgSO 4 1.2, KH2PO 4 1.2, N a H C O 3 24, glucose 11, E D T A 0.03) maintained at 37°C. Aorta rings were prepared as described previously (Sim and Singh, 1987) with modifications. Briefly, the excised aorta was placed in Krebs solution and aerated with a mixture of 95% 0 2 and 5% CO 2. The aorta was allowed to equilibrate with the Krebs solution for at least 45 min with wash steps every 15 min to remove all biological fluids trapped within the aorta. Fat and connective tissue were gently removed from the aorta and rings of about 2 mm long were prepared. One end of the aorta ring was connected via a silk thread to an isotonic transducer with a 300 V per mm displacement of the lever arm, measured across 10 000 /2 load (Ugo Basile, Model 7006), giving an amplification factor of 300 from a pen deflection 0.3 mm per /zm of ring shortening when coupled to a Grass Polygraph with the preamplifier sensitivity set at 0.5 m V / c m (Model 7D). The other end was attached to a thin steel wire hook fixed onto the gas inlet tubing. The preparation was immersed in a 10-ml organ bath containing Krebs solution and constantly aerated. After 60 min of equilibration with a load of 0.5 g, the protocol described below was started.
2.2. Protocol The experimental protocol used t o study the development of angiotensin II-induced tachyphylaxis in the aorta rings of adult normo- and hypertensive rats was based on the detailed method worked out previously (Sim and Kuttan, 1992), but with slight modifications. Each ring was exposed to five 10-min preincubations with 10 -9 M noradrenaline. The second to fifth preincubations were preceded by a 30-rain interval during which each ring was washed with Krebs solution devoid of drugs. Ten minutes after the third preincubation, the ring was exposed to increasing cumulative concentrations of angiotensin II (10 -1° to 10 -6 M) to obtain a cumulative concentration-response curve. Each cumulative concentration-response curve for angiotensin II was obtained as described earlier for the rat aorta (Shibata et al., 1990) with slight modifications. Each cumulative concentration of angiotensin II (a 10-fold
increase) was added after the response to the preceding concentration had reached a plateau. To ensure that the maximum contraction had been attained, an additional dose was administered until no further response was elicited. The second and third concentration-response curves were obtained, under similar conditions as the first, after the fourth and fifth preincubations with noradrenaline, respectively. In the controls where noradrenaline was not used, rings were exposed to cumulative concentrations of angiotensin II until the maximum response was attained. The second and third cumulative concentration-response curves were made after 30 min of reequilibration and washing. At the end of the experiment and after two washes, the ring was treated with 10 -8 M noradrenaline followed by 10 -6 M acetylcholine to evaluate its contractility and endothelial integrity, respectively. Data from rings that attained maximum contractions of less than 90% of control rings or that did not relax to acetylcholine were not included in the analyses. Parallel control concentration-response curves for angiotensin II were obtained in aorta rings that were not preincubated with noradrenaline. The protocol of using three 10-min exposures of noradrenaline was adopted so as to (i) separate rings that contracted to the three exposures of the spasmogen from those that did not, and (ii) determine the magnitude of contraction and extent of desensitisation of the rings to the spasmogen.
2.3. Calculations Concentration-response curves were subjected to Probit transformation and regression analysis to yield a straight line. The intercept and gradient were then used to calculate equiactive concentrations between the two concentration-response curves. The K a and q values were evaluated by substituting these values into the double reciprocal plot. The ECs0 values were also calculated from the regression lines. The K a and q values for the first response were derived from the second response, which is expressed as a percentage of the maximum value of the first response, i.e. [A] from the first response and [A'] from the second response. Data for the second response were similarly derived from the third response, which is expressed as a percentage of the maximum value of the second response, i.e. [A] from the second response and [A'] from the third response. These K a values were then used to determine the fractional receptor occupancy with the following formula (Schwietert et al., 1991): % occupancy = 100 X [A]/([A] + Ka). Log [occupancy] was then plotted against percent maximum response of the first and second concentration-response curves. The pEOs0, defined as the logarithm of the occupancy that 'gives 50% response, was calculated and used to determine the significance of the shifts of the plots.
175
Angiotensin II (acetate salt) was purchased from Sigma (St. Louis, U.S.A) and noradrenaline (tartrate salt) was purchased from Fluka Biochemika (Buchs, Switzerland). Angiotensin II was prepared fresh in Krebs solution. Noradrenaline was dissolved in 1% ascorbic acid, and acetylcholine was prepared as a stock solution of 1 M in 0.5 M sodium dihydrogen phosphate solution. Drugs concentrations reflect the concentrations of their salts. All other reagents used were of analytical grade.
2.4. Statistics The data are expressed as mean values + S.E.M. Student's t-test was used in the statistical analyses of the data when only two sets of independent variables were compared. A paired t-test was used to analyse data with dependent variables, e.g. tachyphylaxis. In all cases, P values less than 0.05 were considered significant.
3.2. Angiotensin II-induced tachyphylaxis The results shown in fig. 1 indicate that the maximum response to the second exposure to angiotensin II (see table 1), was significantly (P < 0.05, paired t-test) depressed in the WKY (lst: 129 + 10 Izm; 2nd: 52 + 4 /xm) and the age-matched SHR (lst: 121 + 7/zm; 2nd: 55 _+3 tzm). The maximum shortening induced in both strains during the second response was not significantly different from each other (P < 0.05, Student's t-test). Comparison of the values for the first and second responses shows that the increase in the ECs0 value of both WKY (lst: 4.48 + 0.45 nM; 2nd: 6.00 + 0.58 nM) and SHR (lst: 4.55 + 0.42 nM; 2nd: 18.4 + 0.98 nM) was significantly different from each other (P < 0.05, Student's t-test). This was also reflected in a diminished affinity, with t h e tachyphylactic response being characterised by a significant increase in the Ka values in the WKY (lst: 76 +_3 nM; 2nd: 517 Jr 10 nM) and SHR (lst: 8.8 + 1.9 nM; 2nd: 170 + 17nM).
3.3. Effect of noradrenaline on responses elicited by angiotensin H in SHR and WKY rats
3. Results
3.1. Angiotensin II-induced response in SHR and WKY In the case of the first response in the absence of noradrenaline, table 1 shows that the K a value of the SHR (8.8 + 1.9 nM) was significantly (P < 0.05, Student's t-test) lower than that of the WKY (76 + 3 nM). However, the ECs0 (WKY: 4.48 + 0.5 nM; SHR: 4.55 + 0.4 nM) and maximum response (WKY: 129 + 10 /~m; SHR: 121 + 7.0/.~m) values were not significantly different. The q value derived from the equation indicates that a higher proportion of receptors in the SHR (91.2%) were not deactivated, whereas only 3.8% in the WKY were not deactivated.
The maximum response of WKY aorta rings to angiotensin II in the presence of noradrenaline was significantly depressed (P < 0.05, Student's t-test) from 129 + 10 Ixm (control) to 91 + 6 /~m (table 1). Although there was a significant reduction (P < 0.05, Student's t-test) in the ECs0 value (control: 4.48 + 0.45 nM; noradrenaline: 1.47 +0.15 riM) for the WKY, there was no corresponding reduction in the K a value (control: 76 + 3 nM; noradrenaline: 84 + 9 nM). The maximum response of SHR aorta rings to angiotensin II in the presence of noradrenaline was also depressed (control: 121 + 7 gM; noradrenaline: 85 + 7 /xM) but there was no significant (P < 0.05, Student's t-test) change in either the ECs0 value (control: 4.55 +
TABLE 1 Constants for angiotensin II concentration-response curves derived from the double reciprocal equation. Parameter
Ka(nM) ECso(nM) Max. resp.(/~m) q (%)
Response
1st 2nd 1st 2nd 1st 2nd 2nd 3rd
WKY
SHR
- noradrenaline (n - 12)
+ noradrenaline (n = 12)
- noradrenaline (n = 19)
+ noradrenaline (n = 13)
76 + 3 517 -I-10 b 4.48+ 0.5 6.00+ 0.6 b 129 ±10 52 ± 4b 3.8 2.3
84 +9 17.8 ± 1 b 1.47-/-0.2 ¢ 6.86±0.7 b 91.0 ±6.0 c 64.6 ±4.2 39.3 9.4
8.8 + 1.9 a 170 ±17 b 4.55± 0.4 18.4 ± 1.0 b 121 ± 7.0 55 ± 3.2 b 91.2 39.5
2.9 +2.3 10.2 ±0.3 b 4.64+0.4 12.6 ±0.8 b 85.0 ±7.0 c 31.6 +2.5 41.9 35.2
a Significantly different from the corresponding value of the WKY (Student's t-test), b Significantly different from the corresponding value in the first response (paired t-test), c Significantly different from the corresponding value in the absence of noradrenaline (Student's t-test).
176
la
lb
Wlstar-Kyoto Rats m R
m R
i
100
'100
i il ~d Me
Lof,Io[AII
"
"
LoItlO[AI!
M!
lc
MI
ld
Spontaneously Hypertensive Rats N
Ill W
i •"-
:.
' -10
-9'
4
:7
-6'
L%o[~al M!
k-
' -I1
-10
-g
-8'
-7'
-6'
L . ~ o I A n M!
Fig. 1. Three consecutive concentration-response curves for angiotensin II ( II, first; o, second; A, third) in aorta rings of the WKY (a and b) and SHR (c and d), in the absence (a and c) and presence (b and d) of 10 - 9 M noradrenaline. Number of rings used in (a), (b), (c) and (d) were 12, 12, 19 and 13, respectively.
0.4 nM; noradrenaline: 4.64 + 0.4 nM) or the K~ value (control: 8.8 5- 1.9 nM; noradrenaline: 2.9 + 2.3 nM). However, the q value indicated that there was an increase in receptor activation (control: 8.8%; noradrenaline: 58.1%) i.e. a decrease in receptors available for interaction (control: 91.3%; noradrenaline: 41.9%).
3.4. Effect of noradrenaline on angiotensin II-induced tachyphylaxis In the WKY, comparison of the first and second responses in the presence of noradrenaline, showed
that there was a significant decrease (P < 0.05, paired t-test) in the K a value (lst: 84 + 9 nM; 2nd: 17.8 + 1 nM). However, there was a significant increase (P < 0.05, paired t-test) in the ECs0 value (lst: 1.47 + 0.15 nM; 2nd: 6.86 + 0.70 nM). In the SHR, comparison of the first and second responses showed that there was a smaller increase in the K a value in the presence of noradrenaline (lst: 2.9 + 2.3 nM; 2nd: 10.2 + 0.3 nM) than in the control (lst: 8.8 + 1.9 nM; 2nd: 170 + 17 nM). T h e r e was also a •similar trend with respect to the ECs0 value in the presence of noradrenaline (lst: 4.64 5-0.4 nM; 2nd:
177 12.6 + 0.8 nM) and in the controls (lst: 4.55 + 0.4 nM; 2nd: 18.4 + 1.0 nM). The q value indicated that in the presence of noradrenaline a smaller proportion of receptors was being recruited to elicit the response, as seen in the difference between the q values obtained for the second and third response (control: 51.7%; noradrenaline: 6.7%).
I00
eo
6o
E "~
40
u
-~.~-io-i.~-;.o-4.~-~.o-;.~
4. D i s c u s s i o n
o'.o
Log [Occupancy]
The maximum contraction elicited by the first exposure of the WKY and S H R to angiotensin II was of comparable magnitude although the SHR showed a higher receptor affinity for the ligand compared to the WKY. As a greater proportion of receptors were required to induce the same response in the WKY (96.2%) than in the SHR (8.8%), this suggests that the angiotensin II-receptor interaction can induce a greater response in the SHR than in the WKY. This suggestion is supported by the log [occupancy] vs. response plots (see fig. 2), where the S H R plots are on the left of those of the WKY. From fig. 1, the extent of angiotensin II-induced tachyphylaxis, in terms of a depressed maximum response, was similar in the adult W K Y and age-matched SHR, i.e. 60 and 55%, respectively. Furthermore, in both strains the second (tachyphylactic) response to angiotensin II was comparable. In terms of K a and ECs0 values, there was a greater increase in S H R (Ka: 19.3 × ; E C s 0 : 4 . 0 × ) than in the WKY (Ka: 7.1 × ; E C s 0 : 1 . 3 × ), implying the occurrence of a greater degree of tachyphylaxis in the SHR. This discrepancy may be explained by the balance achieved between the counteracting influences of reduced receptor reserve
~D
100
~
so
E
so
x
4O
o
~
o -6
-5
-4
-3
-2
-1
0
Log [Occupancy] Fig. 2. Log [occupany]-responseplots for the first cumulative concentration-response curve for angiotensin II in SHR ( • ) and WKY (•); and second cumulative concentration-response curve for angiotensin II in SHR (zx) and WKY (1:3), in the absence of 10 - 9 M noradrenaline. The pEOso of the first (1.546+0.11, n = 19) and second (1.199+0.062, n = 19) concentration-response curves for SHR was significantly different (P < 0.05, paired t-test). The pEOso of the first (1.54:[:0.08, n = 12) and second (3.549+_0.013, n = 12) concentrationresponse curves for WKY was significantlydifferent (P < 0.05, paired t-test).
Fig. 3, Log [occupancy]-responseplots for the first cumulative concentration-response curve for angiotensin II in WKY, in the absence ([:3) and presence ( • ) of 10 - 9 M noradrenaline; and SHR, in the absence (,x) and presence ( • ) of the catecholamine. The pEOs0 of WKY in the absence (1.562+0.085, n = 12) and presence (1.900+ 0.053, n = 12) of 10 - 9 M noradrenaline was significantly different (P <0.05, Student's t-test). The pEOs0 of SHR in the absence (1.546_+0.11, n = 19) and presence (0.544+0.06, n = 13) of 10 - 9 M noradrenaline was significantly different (P < 0.05, Student's t-test). and increased coupling efficiency. However, the K a value for the tachyphylactic response in the SHR was almost 3 times lower than that in the WKY (SHR: 170 + 17 nM; WKY: 517 + 10 nM), whereas the ECs0 value (SHR: 18.4 + 0.98 nM; WKY: 6.00 + 0.58 nM) was almost three times higher. Since the ECs0 value is a parameter of the observed response, this implies that in the S H R the coupling efficiency had been compromised by tachyphylaxis. This is reflected in the log [occupancy] vs. response plot of the S H R where the second response is on the right of the first response. In contrasts in the WKY, the coupling efficiency was improved under conditions in which there was a reduced number of available receptors, with the log [occupancy] vs. response plot appearing on the left of the first response. This can also be inferred from the q values, which indicate that more receptors were recruited in the S H R than in the WKY (SHR: 51.7%, WKY: 1.5%; values derived from the difference in q values obtained from the second and third exposure to angiotensin II in the absence of noradrenaline). There seems to have been a marginal increase in the coupling efficiency of the angiotensin II-receptor interaction in the presence of noradrenaline in WKY aorta rings (see fig. 3). Although there was an approximately 30% decrease in the maximum response, partly as a result of a 37% decrease in the percentage of receptors activated (control: 96.2%; noradrenaline: 60.7%), there was a 3-fold decrease in the ECs0 value, implying that the efficiency of the ligand-receptor response coupling had increased. In the case of the SHR, the insignificant changes in ECs0 and K a values coupled with the decreased maximum response (30%) but increased receptor activation (6.6 × ) suggest that noradrenaline compromises the responses of the S H R to angiotensin II. This decrease in coupling efficiency is seen in fig. 3,
178 100 c o D.
8O
E
60
==
the observed attenuation of angiotensin II-induced tachyphylaxis. Assuming that deactivation of receptors occurs when ligand binding results in a response, the q values suggest that the response in the SHR is governed by the concept of 'spare-receptors' as only a small number were required for a maximum response, i.e. q = 91.2%, whereas in the WKY almost all receptors were bound during the maximum response, i.e. q = 3.8%. During tachyphylaxis, there is an increase in the influence of spare-receptors in the WKY tissue, with a reduction in the SHR. This apparent switch cannot be explained at the moment; however, it suggests that the receptor system may not be bound by any particular mechanism of ligand-receptor interaction. In protein allosterism, proteins are considered malleable structures with a spectrum of conformations, as opposed to rigid matrices. Cooperative protein effects are not specifically accounted for in the occupation, rate and inactivation receptor theories (Kenakin, 1987). If one drug molecule binds to one receptor to generate a response, the binding reaction can be described by a simple hyperbola according to the Langmuir adsorption isotherm. However, angiotensin II concentrationresponse curves are sigmoidal rather than hyperbolic, thereby denoting cooperative binding during the receptor-ligand interaction. This cooperativity may be similar to that described recently i.e. heterotropic and homotropic (Scalon et al., 1990). Cooperativity may involve allosteric sites (Timmermans et al., 1991) which may accommodate natural inhibitors (Moore et al., 1989) or modulators (Kuttan and Sim, 1991a). The response of the aorta rings to angiotensin II is determined by the K a value and the coupling efficiency of the activated receptors. In the SHR this coupling efficiency was greater in the absence of noradrenaline and decreased in its presence. In vivo, this may manifest itself as a reduced feedback signal in the regulation of angiotensin II response, thus prompting a greater response to the octapeptide. In the WKY, the presence of noradrenaline had an opposite effect by increasing the coupling efficiency. In vivo this may be manifested as an enhanced regulatory feedback signal and hence a limited response to the octapeptide. Although we believe that tachyphylaxis may be an in vitro phenomenon only (Sim and Kuttan, 1992), the existence of the phenomenon in vitro can be used to give insight into receptor function in normal and pathological conditions. From the present work, there seems to be a balance between the receptor affinity, the number of activated receptors and the coupling efficiency. The counteracting influences of these three parameters ultimately influence the observed ECs0 and maximum response values. There seems to be an overall reduction in the coupling efficiency in the SHR compared to the WKY in the tachyphylactic state. This -
4o ¢: 20 o.. 0
~ -6
, -5
-4 Log
-3
-2
-1
0
[Occupancy]
Fig. 4. Log [occupancy]-responseplots for the second cumulative concentration-response curve for angiotensin II in the WKY in the absence ([]) and presence ( • ) of 10-9 M noradrenaline; and SHR, in the absence (0) and presence (e) of 10 - 9 M noradrenaline. The pEOs0 of WKY in the absence (3.549+0.013, n = 12) and presence (0.954+0.007, n =12) of 10 - 9 M noradrenaline was significantly different (P < 0.05, Student's t-test). The pEOs0 of SHR in the absence (1.199+ 0.062, n = 19) and presence (1.4635:0.008, n = 13)of 10 - 9 M noradrenaline was significantlydifferent (P < 0.05, Student's t-test). where the log [occupancy] vs. response plot of the SHR is on the right of the control. During tachyphylaxis and in the presence of noradrenaline the affinity of the angiotensin II receptor was increased 4.7-fold increase in the normotensive animals. However, there was also a 4.7-fold increase in the ECs0 value, which was greater than that seen in the absence of the catecholamine (control: 1.3 X; noradrenaline: 4.7 Z ). This seems to suggest a reduced ligand-receptor response coupling efficiency, which is reflected in fig. 4. Thus under these conditions the observed attenuation of angiotensin II-induced tachyphylaxis in normotensive animals by noradrenaline may be due to the greater number of activated receptors. This is reflected in the difference between q values obtained from the second and third concentration-response curves for angiotensin II in the WKY (control: 1.5%; noradrenaline: 29.9%). Thus, tachyphylaxis is apparently associated with an increase in the K a value, i.e. decrease in affinity, which is more pronounced in the SHR than the WKY. Although on the second exposure to angiotensin II there was a similar degree of depression of the maximum response in both strains, there was a greater increase in the ECs0 value in the SHR than the WKY. In SHR aorta rings, noradrenaline affected its response to angiotensin II by decreasing the ligand receptor response coupling efficiency, whereas noradrenaline increased this coupling efficiency in the WKY. This effect was reversed, in both cases, in the second tachyphylactic response (see fig. 4). Although there was a decreased coupling efficiency in the WKY, the increase in the proportion of receptors recruited and the decrease in the K a value result in an increased maximum response
179
again may cause a weak regulatory negative feedback signal, leading to a greater recruitment of receptors in the SHR. The presence of noradrenaline seems to favour the SHR rather than the WKY in terms of coupling efficiency during the tachyphylactic response. However, because of the low numbers of receptors activated in the SHR, this is not manifested in the response of the aorta rings to angiotensin II. Tachyphylaxis is associated with changes at the receptor level, i.e. change in affinity and coupling efficiency. Factors which influence these parameters can be derived from the endothelium or the smooth muscle cell. The present work provides us with some insight into changes in certain parameters associated with ligand-receptor interaction and helps us reformulate our ideas about the receptor theory. The variable affinity hypothesis of receptors has been proposed earlier, albeit with reference to o~-adrenoceptors (Oriowo et al., 1991). Oriowo et al. proposed that factors in the microenvironment of the receptor could alter the affinity of the receptor. The possible involvement of such factors is also suggested from the observation that the endothelium may be a source of an endothelium-derived modulating factor which influences the affinity of the angiotensin receptor (Kuttan and Sim, 1991b). The recent demonstration of an allosteric site modulating the affinity of angiotensin II for its AT 1 receptor (Boulay et al., 1992) suggests the probable existence of ligands that could function as allosteric modulators of receptor affinity. In the in vitro environment, tachyphylaxis may therefore involve the displacement of the factor and uncoupling of the receptor-signal transduction system, resulting in a reduction in receptor affinity and in the tissue response. Thus analogues of angiotensin that bind to the receptor and do not initiate the displacement of the modulator will be seen to be nontachyphylactic (Oshiro et al., 1989). Furthermore, auxilIary agonists may reactivate the receptor through intracellular biochemical processes (i.e. phosphorylation of intracellular domains of the receptor), which may explain the partial reversal or attenuation of in vitro tachyphylaxis that occurs in the presence of other spasmogens, e.g. noradrenaline and PGF2,,. It has also been shown that vasoconstrictors, e.g. angiotensin II, vasopressin, serotonin and noradrenaline, stimulate tyrosine-protein phosphorylation of the same set of proteins in vascular smooth muscle cells (Tsuda et al., 1991) and may augment each others response in vivo. With the recent discovery of angiotensin peptides in the blood (Lawrence et al., 1990), one wonders whether multiple receptor subtypes or multiple receptor affinity states are the key which allows angiotensin receptors to discern one ligand from another. The theory of a modulating factor changing the affinity state of the receptor is relevant as improved technology reveals
that physiological systems have the ability to respond to much lower levels of ligands than previously thought. The possibility that other positive and negative allosteric modulating factors exist for other receptor systems cannot be discounted either.
References Abdellatif, M.M., C.F. Neubauer, W.J. Lederer and T.B. Rogers, 1991, Angiotensin-induced desensitisation of the phosphoinositide pathway in cardiac cell occurs at the level of the receptor, Circ. Res. 69, 800. Aboulafia, J., M.E.M. Oshiro, T. Feres, S.I. Shimuta and A.C.M. Paiva, 1989, Angiotensin II desensitisation and Ca 2+ and Na + fluxes in vascular smooth muscle, Pfliigers Arch. 415, 230. Boulay, G., G. Servant, T.T. Luong, E. Escher and G. Guillemette, 1992, Modulation of angiotensin II binding affinity by allosteric interaction of polyvinyl sulfate with an intracellular domain of the DuP753 sensitive angiotensin II receptor of bovine adrenal glomerulosa, Mol. Pharmacol. 41, 809. Furchgott, R.F., 1966, The use of beta-haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist complexes, in: Advances in Drug Research, eds. N.J. Harper and A.B. Simmonds (Academic Press. New York) p. 21. Kenakin, T.P., 1987, Pharmacologic Analysis of Drug-Receptor Interaction (Raven Press). Kuttan, S.C. and M.K. Sim, 1991a, Endothelium-dependent response of rabbit aorta to femtomolar concentrations of angiotensin II, J. Cardiovasc. Pharmacol. 17, 929. Kuttan, S.C. and M.K. Sim, 1991b, Isolation and purification of the putative EDMF from the rabbit aortic endothelium (Abstract), Pharmacologist 33, P470. Lawrence, A.C., G. Evin, A. Kladis and D.S. Campbell, 1990, An alternative strategy for the radioimmunoassay of angiotensin peptides using amino-terminal directed antisera: measurement of eight angiotensin peptides in human plasma, J. Hypert. 8, 715. Moore, G.J., R.C. Ganter and K.J. Franklin, 1989, Angiotensin 'antipeptides': ( - ) messenger RNA complimentary to human All ( + ) messenger RNA encodes an angiotensin receptor antagonist, Biochem. Biophys. Res. Commun. 160, 1387. Oriowo, M.A., R.D. Bevan and J.A. Bevan, 1991, Variable receptor affinity and tissue sensitivity, Blood Vessels 28, 115. Oshiro, M.E.M., S.I. Shimuta, T.B. Paiva and A.C.M. Paiva, 1989, Evidence for a regulatory site in angiotensin II receptor of smooth muscle, Eur. J. Pharmacol. 166, 411. Scalon, M.N., P. Koniaz and G.J. Moore, 1990, The relationship between homotropic and heterotropic cooperativity for angiotensin receptors in smooth muscle, Gen. Pharmacol. 21, 59. Schwietert, H.R., M.A.M. Gouw, D. Wilhelm, B. Wiffert and P.A. Van Zwieten, 1991, The role of al-adrenoceptor subtypes in the phasic and tonic responses to phenylephrine in the longitudinal smooth muscle of the rat portal vein, Naunyn-Schmiedeb. Arch. Pharmacol. 343, 463. Shibata, R., S. Morita, K. Nagai, S. Miyata and T. lwasaki, 1990, Effects of H7 (protein kinase inhibitor) and phorbol ester on aortic strips from spontaneously hypertensive rats, Eur. J. Pharmacol. 175, 161. Sim, M.K. and S.C. Kuttan, 1992, Effects of noradrenaline and prostaglandin F2, on angiotensin-induced contraction and tachyphylaxis in rat aortic rings, Pharmacol. Toxicol. 70, 60. Sim, M.K. and M. Singh, 1987, Decreased responsiveness of the aortae of hypertensive rats to acetylcholine, histamine and noradrenaline, Br. J. Pharmacol. 90, 147.
180 Timmermans, P.B.M.W.M., P.C. Wong, A.T. Chiu and W.F. Herblin, 1991, Non-peptide angiotensin II receptor antagonist, Trends Pharmacol. 12, 55. Tsuda, T., Y. Kawahara, H. Shii, M. Koide, Y. Ishida and M. Yokogama, 1991, Vasoconstrictor-induced protein-tyrosine phos-
phorylation in cultured vascular smooth muscle cells, FEBS Lett. 285, 444. Ullian, M.E. and S.L. Linas, 1990, Angiotensin II surface receptor coupling to inositol trisphosphate formation in vascular smooth muscle cells, J. Biol. Chem. 265, 195.