- Email: [email protected]
[125I]CGP 42112 binding reveals differences between rat brain and adrenal AT2 receptor binding sites
Regulatory Peptides, 44 (1993) 189-197 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00
189
REGPEP 01283
[125I]CGP 42112 binding reveals differences between rat brain and adrenal AT 2 receptor binding sites Robert C. Speth Department of Veterinary and Comparative Anatomy, Pharmacologyand Physiology, Washington State University,Pullman, WA (USA) (Received 6 October 1992; accepted 11 December 1992)
Key words: Angiotensin II receptor; Ligand CGP42112; fl-Mercaptoethanol; Binding affinity
Summary The AT 2 angiotensin II receptor selective ligand CGP42112 was radioiodinated and used to study AT 2 receptor binding sites in the rat brain (combined olfactory bulb, septum, thalamus and midbrain) and whole adrenal. The [125I]CGP 42112 binding was of high affinity, saturable and specific in both tissues. Competition studies with nonselective and angiotensin II receptor subtype selective ligands, and evaluation of the effects of the sulfhydryl reducing agent ~-mercaptoethanol, confirmed that [125I]CGP 42112 bound selectively to the AT 2 angiotensin II receptor subtype. [125I]CGP 42112 bound with higher affinity in the brain than in the adrenal. fl-Mercaptoethanol enhanced [125I]CGP 42112 binding in the brain, but did not alter its binding in the adrenal. A similar difference in binding affinity for [125I]sarcosine1,isoleucine8 angiotensin II and enhancement of binding affinity by fl-mercaptoethanol was observed in the rat brain and adrenal. These observations suggest that the AT 2 angiotensin II receptor subtypes in the brain and adrenal may differ.
Introduction Discrimination of the two major AII receptor subtypes, AT1 and AT 2 can be traced back to observations of the differential effects of the sulfhydryl reducing agent, dithiothreitol, on AII receptor binding. Correspondence to: R.C. Speth, Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164-6520, USA.
Studies by Glossman et al. [9] and Bennett and Snyder [ 1] who used dithiothreitol as a peptidase inhibitor suggested that dithiothreitol enhanced AII receptor binding in the adrenal and brain, while studies by Gunther et al. [ 10,11 ] demonstrated that dithiothreitol inhibited All receptor binding in the vasculature. Several recent studies have used peptidic and nonpeptidic ligands selective for AII receptor subtypes, as well as sulfhydryl reducing agents to evaluate AII receptor binding in various tissues [6,7,17,21,
190
22,24,28]. These studies show that the predominant AII receptor subtype in the tissues in which binding is decreased by sulfhydryl reducing agents is AT 1 (vasculature and liver) and the predominant receptor subtype in tissue in which binding is enhanced by sulfhydryl reducing agents is AT 2 (bovine cerebellum, and to a lesser extent, the adrenal). Molecular biological studies of the genes encoding AII receptors have described the AT 1 receptor as a member of the G-protein linked receptor superfamily [16,20]. Subsequent studies have demonstrated the existence of two or more different transcripts similar to the AT 1 receptor in the rat which have been designated as AT1A and AT m or AT 3 [8,12,13,19]. The AT 2 receptor gene has yet to be identified so its structure and other characteristics are less well known. Arriving at a description of a functional response mediated by the AT 2 receptor has also been difficult and controversial [2,25,27]. Thus, radioligand binding studies of AII receptors remain the primary means of describing AT 2 receptors. CGP 42112 A has been shown to bind with high affinity to the AT2 subtype and has low affinity for the AT 1 subtype [28]. Whitebread et al. [29] radiolabeled CGP 42112 and characterized its binding to AT 2 receptors in human uterus and rat adrenal. This report describes the further application of radiolabeled CGP 42112 to characterize AT 2 receptors and provides results suggestive of the presence of multiple subtypes of AT 2 receptor binding sites.
Methods
Radioligands [125I]CGP 42112 was prepared using a modification of the procedure of Whitebread et al. [29]. CGP 42112 B (B indicates trifluoroacetate salt), generously provided by Dr. Marc de Gasparo of CibaGeigy, Basle, was reacted with Na125I (NEZ-033H, NEN Research Products, Boston, MA) in the presence of 1 mM chloramine T and 370 mM sodium
phosphate (pH 7.4) for 20 s. The reaction was stopped with sodium metabisulfate, 0.5 mg/ml final concentration. Purified mono[125I]CGP 42112 was isolated by H P L C using a C18 reverse phase column eluted with a mobile phase of 23 ~o acetonitrile/77 ~o triethylamine phosphate (83 mM phosphoric acid adjusted to pH 3.0 with triethylamine). Baseline separation of uniodinated, monoiodinated and diiodinated CGP 42112 was obtained. [ 125I] S arcosinel,isoleucine8 angiotensin II ([125I]SI AII) was prepared in the same manner as [125I]CGP 42112, and the mono[125I]peptide was also purified by HPLC as described above except that the eluting buffer contained only 16Yo acetonitrile. The fractions containing mono[ ~25I]CGP 42112 and mono[125I]SI All were stored up to 8 weeks at -20°C in the presence of 2 mg/ml bovine albumin.
Tissue preparation Freshly dissected brains (olfactory bulbs, thalamus, septum and midbrain of two rats were pooled together) and whole adrenal glands (pooled from two rats) were obtained from male Sprague-Dawley rats (Laboratory Animal Resource Center, Washington State University, ages 14-18 weeks) killed by decapitation. The brain regions were chosen based on previous studies indicating that they contained an abundance of AT 2 receptors. The tissues were weighed, placed in 20 ml of ice-cold hypotonic buffer; 20 mM sodium phosphate, pH 7.1-7.2 with 0.1 mM bacitracin, and homogenized (Tissuemizer, Tek-Mar, Cincinnati, OH). The homogenized tissue was centrifuged at 48,000 g for 20 min at 0-4°C. The supernatant was decanted and the pelleted tissue was resuspended in 20 ml of assay buffer: 50 mM sodium phosphate, pH 7.1-7.2, with 150 mM NaCI, 1 mM disodium EDTA and 0.1 mM bacitracin. The rehomogenized tissue was recentrifuged as before. The supernatant was again decanted and the pelleted tissue was resuspended in 7 ml of assay buffer. Protein concentration in the homogenates was determined by the method of Lowry et al. [ 15].
191 Radioligand binding assays Final tissue homogenate (0.05 ml) was incubated for 120-150 min at 22°C with [12SI]CGP or [125I]SI All in a volume of 0.1 ml of assay buffer. To obtain saturation isotherms, the tissues were incubated with 8 different concentrations ofradioligand ranging from 0.03 to 2.0 nM, in duplicate ([125I]SI AII) or triplicate ([125I]CGP 42112). For competition analyses an additional 0.01 ml of assay buffer containing either AII, CGP 42112A, SI AII or losartan (DuP 753), to obtain final concentrations of 1, 1, 1 and 10 /~M, respectively, was added to triplicate tubes containing a single (0.3-0.6 nM) concentration of radioligand. To determine the effect of a sulfhydryl reducing agent on radioligand binding, fl-mercaptoethanol (Sigma Chemical Co., St. Louis, MO) was added to an aliquot of tissue homogenate to attain a concentration of 30 mM. Bound radioligand was separated from free radioligand on glass fiber filters, (#32, Schleicher and Schuell, Keene, NH). The filters were imediately rinsed three times with 3 ml of 50 mM sodiumpotassium phosphate buffer (pH 7.4), 22°C and assayed by sodium iodide crystal gamma scintillation counting. The saturation isotherm data was evaluated by a weighted (1/y2) nonlinear regression analysis (Inplot, Graphpad Software, Inc., San Diego, CA) using the equation: Y= A ' X / ( B + X) + C ' X
where Y is bound radioligand, A is Bma x o r the amount of receptor, X is the radioligand concentration, B is the dissociation constant (KD) and C is the ratio of nonspecific binding. To verify its accuracy, the computer derived value for nonspecific binding was compared to the actual value for nonspecific binding determined in the presence of 1 # M A I I at a single radioligand concentration of 0.3-0.6 nM. In addition, the linearity of nonspecific binding of [125I]CGP 42112 at concentrations ranging from 75 to 800 pM was established in a preliminary assay of rat brain and adrenal. Nonspecific binding of[ 125I]SI
AII was also demonstrated to be linear over the range of 50-700 pM. Generally there was no disparity between empirical and computer generated nonspecific binding values, however in two instances there was a disparity and the empirical value was placed into the equation as a constant. Statistical analyses A two-way ( + fl-mercaptoethanol and radioligand) randomized block ANOVA was used to compare K D and Brnax values in the brain and in the adrenal. A two-way ( + fl-mercaptoethanol and tissue) randomized block ANOVA was also used to evaluate differences in radioligand affinity between tissues. A two-way ( + fl-mercaptoethanol and competing ligand) randomized block ANOVA was also used to evaluate the effects of a saturating concentration of selective and nonselective AII ligands on radioligand binding. Mean values were compared using Duncan's multiple comparison test [14].
Results
Binding of [125I]CGP 42112 was saturable and displayed high affinity and specificity in both the brain (olfactory bulb, septum, thalamus, midbrain) and whole adrenal gland. Fig. 1 describes a saturation isotherm for [ 125I]CGP 42112 binding to brain membranes in the presence of fl-mercaptoethanol. The values for specific and nonspecific binding derived from the regression analysis are also displayed. The computer generated value for nonspecific binding was consistent with that determined empirically at 1 /~M AII at a single concentration of [125I]CGP 42112. Fig. 2 describes the binding of [125I]CGP 42112 and [125I]SI AII to an adrenal membrane preparation in the presence and absence of 30 mM fl-mercaptoethanol. Figs. 3 and 4 describe Rosenthal (Scatchard) plots for specific [ 125I]CGP and [lZSI]SI AII binding in the brain and adrenal, respectively, derived from the data shown in Figs. 1 and 2. The average values for the dissociation constant
192 125I-CGP 42112 Binding to Rat Brain Olfactory Bulb, Septum, Thalamus, Midbrain • Total Binding
11
u M All * 1 u M C G P 42112 oi0 u M h o s a r t a n _ .... •
o
1
'4-, V
..m"
.
.
%"
......
Angiotensin II Receptor Binding in Rat Adrenal 8 • • 30 m M 8-mercaptoethanol [] • I251-CGP 42112 ! o , 12SI-Sl All o 6¸
,
4
" "
r~
g m
; ~ 0 0
~ T
d
~ N ° n s p e cific
S p e c i f i c Bindinc
Bindlng
500 1251-CGP 42112 (pN)
i
1000
Fig. 1. Saturation isotherm of total [125I]CGP 42112 binding to rat brain (olfactory bulb, septum, thalamus and midbrain) in the presence of 30 m M fl-mercaptoethanol. The amount of membrane protein present in each tube, in 0.1 ml total assay volume, was 286 #g. This was equivalent to an initial wet weight of 3.0 mg. Samples were run in triplicate. Nonspecific binding in the presence of 1 #M AII was determined at a single, 510 pM, concentration of [ 125I]CGP 42112. ['25I]CGP 42112 binding in the presence of 1 #M CGP42112 and 10 #M losartan was also determined at 510 pM [IzsI]CGP 42112. The losartan data points are barely distinquishable because of the superimposition of the total binding data points and their placement is indicated with an arrow. The curves for total and specific binding and the line for nonspecific binding were generated from the equation: Y= Bmax'X/(g D + X ) + C ' X where Y= total bound, X = [125I]CGP 42112 concentration, and C is the ratio of nonspecific binding. The derived values for this example were 1.93 fmol for B . . . . 66 pM for KD, and 0.00255 for the nonspecific binding ratio. A Rosenthal (Scatchard) plot of these data is presented in Fig. 4.
i
k
500 i000 1500 1251-Radioligand (pM)
2000
Fig. 2. Saturation isotherm of total [125I]CGP 42112 and [msI]SI AII binding in rat adrenal in the absence or presence of fl-mercaptoethanol. Curves drawn are derived from nonlinear regression analysis for specific plus nonspecific binding as described in Materials and Methods and legend to Fig. 1. The amount of membrane protein present in each tube, in 0.1 ml total assay volume, was 28 #g. This was equivalent to an initial wet weight of 0.5 rag.
(KD) and receptor density (nmax) from four such experiments are presented in Table I. Analysis of variance revealed that 30 mM /3-mercaptoethanol caused a significant (P< 0.01) reduction in the Bmax for [nSI]SI All binding in both the brain (33 ~o) and the adrenal (52~o), but did not significantly alter that for [ 1251]CGP 42112. Analysis of variance also demonstrated 30 mM fl-mercaptoethanol significantly
TABLEI Comparison of [~25I]GCP 42112 and [nsI]SI AII binding in rat brain and adrenal Tissue
Ligand
- ME
Brain Adrenal
CGP SI AII CGP SI AII
Bmax (fmol/mg protein)
K D (pM)
150 + 275 + 186 + 430 +
+ ME
16 59 80 192
63 128 200 429
+ 18"* + 9** + 135 + 130
- ME
+ ME
6.2 10.4 125 371
6.1 + 1.0 7.1 + 0.8* 107 + 10 176 + 87*
+ 0.7 + 0.4 + 38 + 131
Values are mean, n = 4, +_S.D. - ME is no fl-mercaptoethanol, + ME is in the presence of 30 mM fl-mercaptothanol *P < 0.01 versus - ME; **P< 0.02 versus - ME.
193 A n g i o t e n s i n II Receptor B i n d i n g in Rat Adrenal 125I-CGP 42112 v e r s u s 1 2 5 I - S a r l , I l e S AII 10 • •
•
30
mM 8-mercaptoethanol
o v ¢-
"~II
o=
o
nn
0 0
o.01
0,02
003
o,04
o.05
Bound/Free (ml) Fig. 3. Rosenthal (Scatchard) plot of specific []25I]CGP 42112 and [ ~25I]SI A l l binding to rat adrenal membranes derived from data shown in Fig. 2. Specific binding was determined as described in Materials and Methods.
enhanced the binding affinity (decreased the Ko) for both [ 1251]SI All and [ 125I]CGP 42112 in the brain, but did not alter the binding affinity for either radiA n g i o t e n s i n II Receptor Binding in Rat Brain Olfactory bulb - Thalamus - Septum - Midbrain 3Ù • •
2.5 ~ "~
\
2.0""
D
I-..
3 0 mM 8 - m e r c a p t o e t h a n o l o • 1251-CGp 42112 o • leSI-sI AII
-i--. i............. l. "'"'O
....... l"
0.5. 0.(3
"'"~'"',
o005
Q010 0.015 Bound/Free (ml)
o020
Fig.4. Rosenthal (Scatchard) plot of specific[t2~I]CGP 42112 and []251]SIAll binding to rat brain membranes obtained from the olfactorybulb,septum, thalamus and midbrain. Specificbinding was determined as described in Materials and Methods. The amount of membrane protein present in each tube, in 0.1 ml total assay volume, was 286 #g. This was equivalent to an initial wet weight of 3.0 mg.
oligand in the adrenal. The ANOVA comparing radioligand binding affinity in the brain and adrenal indicated that the brain had a significantly higher affinity for [125I]CGP 42112 (P<0.05) and [125I]SI All (P<0.01) than the adrenal. However, it did not reveal the significant effect of fl-mercaptoethanol on binding affinity nor did it show a significant interaction. Table II describes the specificity of [125I]CGP 42112 and [125I]SI AII binding for AII receptor binding sites. Fig. 1 also describes the specificity of [125I]CGP 42112 binding in the brain. At a concentration averaging 440 pM, 54~o of the [125I]CGP 42112 binding in the brain was specific for All receptors as defined by displacement with 1 #M All. A similar amount of [125I]CGP 42112 binding was displaced by 1/aM unlabeled CGP 42112. Losartan, the AT 1 selective antagonist, at 10/aM, did not displace any [125I]CGP 42112 binding in the brain. In the presence of 30 mM fl-mercaptoethanol there was a significant (P< 0.05) increase in the proportion of 1 #M All and CGP 42112 displaceable [125I]CGP 42112 binding. This increase is attributable to enhanced binding affinity for this radioligand. Specific 1 # M A I I displaceable [125I]CGP 42112 binding in the adrenal was 79~ of total binding. 1 /aM CGP 42112 displaceable [ 125I]CGP 42112 binding was 83~o, while 10 #M losartan displaceable binding was only 3 ~o of total binding. Addition of 30 mM fl-mercaptoethanol did not significantly alter the proportion of 1 #M All, 1 /aM CGP 42112 and 10 #M losartan displaceable [125I]CGP 42112 binding. Specific 1/aM AII displaceable [125I]SI AII binding was 69~o of total binding in the brain. 1/aM CGP 42112 and 10/aM losartan displaceable [ |25I]SI AII binding was 48 ~o and 26 ~'o, respectively. The sum of the 1 /aM CGP 42112 (putative AT2) and 10 /aM losartan (putative AT 1) displaceable binding is 76 ~o. This is slightly greater than 69~o and may reflect some overlap of CGP 42112 to inhibit a small proportion of AT 1 binding and/or losartan inhibition of a small proportion of AT 2 binding. In the presence
194 TABLE II Competition for [125I]GGP 42112 and [125I]SI AII binding by All, CGP 42112, losartan and SI AII in rat brain and adrenal Tissue
Brain
Adrenal
Radioligand*
[125I]CGPa ( + ME) a [ 125I]SI All c ( + ME) d [ 1251]CGP a
( + ME) a [125I]SI AIIf ( + ME) g
Competing ligand (~o of total binding remaining) All
CGP 42112
losartan
SI All
46.0 _+8.4 38.9 + 8.1 b 31.0 + 7.4 25.4 + 5.2 20.9 + 8.6 17.8 + 5.2 6.4 + 1.8 12.3 + 3.9
45.7 _+9.0 34.1 _+5.6b 52.0 + 2.1 35.0 + 4.9 e 16.8 + 5.6 15.1 + 3.6 56.6 + 5.7 47.4 + 5.9
101.2 + 13.5 99.0 _+6.2 74.0 + 14.3 79.3 + 15.0 97.1 + 2.5 96.0 + 6.5 33.3 + 10.1 56.7 + 7.2
ND ND 28.2 + 9.0 22.1 +_3.2 ND ND 12.3 + 3.9 8.5 _+2.0
Values are mean + S.D. (n = 4) except SI AII (n = 3). The percent of specific binding is: 100 - the values shown. Lower values indicate a higher percent of specific binding to All receptors or individual All receptor subtypes. ND is not determined. * The average radioligand concentration was 440 pM for [125I]CGP 42112 and 540 pM for [12sI]SI All. Concentrations of competing ligands were 1 #M for All, CGP 42112 and SI All, and 10 #M for losartan. a P<0.01, losartan>AII-=CGP 42112. b p < 0.05, combined AI1 and CGP 42112 < no fl-mercaptoethanol combined AII and CGP 42112. c p < 0.05, losartan > CGP 42112 > All ----SI All. d P<0.05, losartan>CGP 42112~AII-=-SI All. ° P<0.05,losartan>AII=SI AII. g P<0.05, losartan>CGP 42112>AII=SI AII.
o f 30 m M fl-mercaptoethanol, specific 1 # M A I I displaceable [ 125I] SI A I I binding was 75 ~o. O n e # M C G P 42112 d i s p l a c e a b l e [125I]SI A l l binding increased to 65 ~o while 10/~M l o s a r t a n d i s p l a c e a b l e binding d e c r e a s e d to 21 ~o. Specific 1 # M A l l d i s p l a c e a b l e [125I]SI A l l binding in the whole adrenal was 94~o. O n e # M C G P 42112 a n d 10 ~tM l o s a r t a n d i s p l a c e a b l e [125I] SI A I I binding were 43~o and 67~o, respectively. In the presence of 30 m M fl-mercaptoethanol, specific 1 /~M A l l displaceable [125I]SI A I I binding was 88~o. O n e / ~ M C G P 42112 d i s p l a c e a b l e [125I]SI A I I binding increased to 53 ~o while 10 # M l o s a r t a n displaceable binding d e c r e a s e d to 43 ~o.
Discussion A s previously r e p o r t e d by W h i t e b r e a d et al. [29] in h u m a n myometrium, [125I]CGP 42112 b i n d s sat-
urably a n d with high a t ~ i t y in the rat brain a n d adrenal. The K D values for [125I]CGP 42112 in this study are in excellent agreement with those found in h u m a n m y o m e t r i u m [29]. Nonspecific binding for [125I]CGP 42112 defined either by 1 # M A I I a n d 1 /~M C G P 42112B was nearly identical, in b o t h the brain a n d adrenal, suggesting that the A T 2 r e c e p t o r is the only site at which [12SI]CGP 42112 binds with high affinity. The A T 1 selective ligand losartan, at 10 /~M, did n o t significantly inhibit [125I]CGP 42112 binding, confirming that r a d i o i o d i n a t i o n o f C G P 42112 d i d not alter its A l l receptor subtype selectivity. C o m p a r i s o n o f specific [125I]CGP 42112 binding with specific [125I]SI A l l binding allows estimates o f the p r o p o r t i o n o f A T 1 and A T 2 subtypes in the rat brain ( c o m b i n e d olfactory bulb, septum, thalamus, m i d b r a i n ) a n d whole a d r e n a l to be m a d e . B a s e d on Bma x values for the two radioligands in the a b s e n c e
195 of fl-mercaptoethanol, 40~o of the brain All receptors were AT 1 and 60~o were AT 2. This estimate is the same as that reported by Chang et al., [5] for similar rat brain regions. From the reduction in the Bmax for [125I]SI AII in the presence of 30 mM fl-mercaptoethanol, an estimate of 32~ AT 1 and 68~o AT2 in brain could be made. However, in the presence of 30 mM fl-mercaptoethanol, 10/~M losartan (a concentration more than 10-fold below its IC5o for the AT 2 subtype) still retained the ability to displace 20~o of the [ a25I]SI All binding in the brain. This suggests that 30 mM fl-mercaptoethanol does not fully inactivate brain AT 1 receptors or that AT 2 receptors become more sensitive to losartan in the presence of fl-mercaptoethanol. A third estimate of the proportion of subtypes, based on the amount of specific [ 125I]SI AII binding displaced by 1 #M CGP 42112 and 10 ktM losartan in the absence of fl-mercaptoethanol, is 37~o AT1 and 63~o AT 2. This estimate has the limitation of assuming equal affinity of [125I]SI AII for each of the AII receptor subtypes, and that the concentration of each competing ligand was selective for only one of the subtypes. Neither assumption can be met based on previous studies by Rowe et al. [18] and Whitebread et al. [29]. Thus, the estimate using [125I]CGP 42112 remains the most reliable. An estimate of 66~o ATa/34~o AT2 in the whole adrenal can be made from comparing Bmax values for [tzsI]CGP 42112 versus [125I]SI AII. This estimate differs moderately from the 50~ ratio reported by Chang and Lotti [4]. Two observations from this study are suggestive of differences in AT 2 receptors in the brain and adrenal. One is the differential effects of fl-mercaptoethanol on [125I]CGP 42112 binding affinity in the brain and adrenal. In the brain, 30 mM fl-mercaptoethanol clearly increased binding affinity of [125I]CGP 42112 for AT 2 receptors. By contrast, fl-mercaptoethanol did not alter [125I]CGP 42112 binding affinity for whole adrenal AT 2 receptors. An identical pattern of enhanced binding affinityfor [ 1251]SI AlI in the brain, and no change in binding affinity in the adrenal, was
observed in the presence of 30 mM fl-mercaptoethanol. With [125I]SI All however, it is not possible to attribute this change entirely to AT 2 receptors, since [125I]SI AII binds to AT 1 receptors as well. The second observation is the higher binding affinity of both [ 125I]CGP 42112 and [ 125I]SI AII for the brain compared to the adrenal. But again, the [125I]SI AII effect may not be limited to AT 2 receptors. While these data are consistent with the existence of two AT 2 receptor subtypes, other explanations of the data should be acknowledged. It is possible that the adrenal may be less sensitive to the effects of the sulfhydryl reducing agent, e.g., the adrenal may have more oxidizable substrates, or be more capable of oxidizing fl-mercaptoethanol, but the centrifugal washing and substantial dilution of the tissue to < 0.3 mg protein/ml make this unlikely. It is still uncertain as to whether CGP 42112 is an agonist or antagonist at the AT 2 receptor. Thus, it is possible that [125I]CGP 42112 is recognizing different agonist affinity states of the AT 2 receptor. However the likelihood of such an effect being tissue dependent is small. Finally, the ANOVA comparing K D values did not reveal a significant interaction between fl-mercaptoethanol and tissue as expected. However, this may result from the large variability in the adrenal binding data compared to the brain binding data. Tsutsumi and Saavedra [26] have reported the existence of two AT 2 receptor subtypes. The subtype they call AT2A is sensitive to guanine nucleotides and pertussis toxin in a manner similar to several G-protein linked receptors. The subtype they call AT2B is insensitive to guanine nucleotides and pertussis toxin and conforms more to the criteria others have established for the AT 2 receptor [3,6,7,23,28]. Whether the two putative receptor subtypes we see in the brain and the adrenal fit within the AT2A and ATzB framework of Tsutsumi and Saavedra [26] for brain AT 2 receptor subtypes remains to be determined. The differential effects of sulfhydryl reducing agents on AII receptor binding was the first solid
196
evidence for the AII receptor subtypes that were later confirmed pharmacologically. Therefore, it is possible that sulfhydryl reducing agents are once again indicating additional AII receptor subtypes that will ultimately be confirmed with new generations of All receptor subtype selective agents. [125I]CGP will be useful for further studies of AT 2 receptor subtypes since the extraneous binding to AT 1 receptors seen with other angiotensinergic radioligands will not be present or need to be eliminated. Preliminary evaluation of rat brain sections incubated with [ ~25I]CGP 42112 (Speth and Grove, unpublished observations) indicates that this radioligand is also suitable for in vitro receptor autoradiographic analysis of AT 2 receptors, showing no specific binding in brain areas containing exclusively the AT 1 receptor subtype.
Acknowledgements Dr. Marc de Gasparo, Ciba-Geigy, Basle, Switzerland, donated the CGP 42112B used for these studies and Dr. Ronald Smith of DuPont Merck Pharmaceuticals, Wilmington, DE, donated the losartan used for these studies. This work was supported by USPHS, NIH grant NS-21305. Frances Yang, who provided technical assistance was supported by NIH, RR 03368, Minority High School Student Research Apprentice Program. Jeanne Jensen, Kevin Grove and Dr. Joseph Harding graciously reviewed the manuscript, providing valuable suggestions. References 1 Bennett, J.P. Jr. and Snyder, S.H., Angiotensin II binding to mammalian brain membranes, J. Biol. Chem., 251 (1976) 7423-7430. 2 Bottari, S., King, I.N., Reichlin, S., Dahlstroem, I., Lydon, N. and De Gasparo, M., The angiotensin AT 2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase, Biochem. Biophys. Res. Commun., 183 (1992) 206-211. 3 Bottari, S.P., Taylor, V., King, I.N., Bogdal, Y., Whitebread, S. and De Gasparo, M., Angiotensin II AT2 receptors do not
interact with guanine nucleotide binding proteins, Eur. J. Pharmacol., 207 (1991) 157-163. 4 Chang, R.S.L. and Lotti, V.J., Two distinct angiotensin II receptor binding sites in rat adrenal revealed by new selective nonpeptide ligands, Mol. Pharmacol., 29 (1990) 347-351. 5 Chang, R.S.L., Lotti, V.J., Chen, T.B. and Faust, K.A., Two angiotensin II binding sites in rat brain revealed using [125I]Sarl,IleS-angiotensin II and selective nonpeptide antagonists, Biochem. Biophys. Res. Commun., 171 (1990) 813817. 6 Chiu, A.T., Herblin, W.F., McCall, D.E., Ardecky, R.J., Carini, D.J., Duncia, J.V., Pease, L,J., Wong, P.C., Wexler, R.R., Johnson, A.L. and Timmermans, P.B.M.W.M., Identification of angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun., 165 (1989) 196-203. 7 Chiu, A.T., McCall, D.E., Nguyen, T.T., Carini, D.J., Duncia, J.V., Herblin, W.F., Uyeda, R.T., Wong, P.C., Wexler, R.R., Johnson, A.L. and Timmermans, P.B.M.W.M., Discrimination of angiotensin II receptor subtypes by dithiothreitol, Eur. J. Pharmacol., 170 (1989) 117-118. 8 Elton, T.S., Stephan, C.C., Taylor, G.R., Kimball, M.G., Martin, M.M., Durand, J.N. and Oparil, S., Isolation of two distinct type 1 angiotensin II receptor genes, Biochem. Biophys. Res. Commun., 184 (1992) 1067-1073. 9 Glossmann, H., Baukal, A.J. and Catt, K.J., Properties of angiotensin II receptors in the bovine and rat adrenal cortex, J. Biol. Chem., 249 (1974) 825-834. 10 Gunther, S., Gimbrone, M.A. Jr. and Alexander, R.W., Regulation by angiotensin II of its receptors in resistance blood vessels, Nature, 287(5779) (1980) 230-232. 11 Gunther, S., Gimbrone, M.A. Jr. and Alexander, R.W., Identification and characterization of the high affinity vascular angiotensin II receptor in rat mesenteric artery, Circ. Res., 47 (1980) 278-286. 12 Iwai, N. and Inagami, T., Identification of two subtypes in the rat type I angiotensin II receptor, FEBS. Lett., 298 (1992) 257-260. 13 Kakar, S.S., Sellers, J.C., Devor, D.C., Musgrove, L.C. and Neill, J.D., Angiotensin II type-1 receptor subtype cDNAs: differential tissue expression and hormonal regulation, Biochem. Biophys. Res. Commun., 183 (1992) 1090-1096. 14 Kirk, R.E., Multiple comparison tests. In Experimental Design: Procedures for the Behavioral Sciences, Brooks/Cole, Belmont, 1982, pp. 106. 15 Lowry, O.H., Rosebrough, N.J., Fan', A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 16 Murphy, T.J., Alexander, R.W., Griendling, K.K., Runge, M.S. and Bernstein, K.E., Isolation of a eDNA encoding the vascular type-1 angiotensin II receptor, Nature, 351 (1991) 233-236.
197 17 Rowe, B.P., Grove, K.L., Saylor, D.L. and Speth, R.C., Angiotensin II receptor subtypes in the rat brain, Eur. J. Pharmacol., 186 (1990) 339-342. 18 Rowe, B.P., Saylor, D.L. and Speth, R.C., Analysis of angiotensin II receptor subtypes in individual rat brain nuclei, Neuroendocrinology, 55 (1992) 563-573. 19 Sandberg, K., Ji, H., Clark, A.J., Shapira, H. and Catt, K.J., Cloning and expression of a novel angiotensin II receptor subtype, J. Biol. Chem., 267 (1992) 9455-9458. 20 Sasaki, K., Yamano, Y., Bardhan, S., Iwai, N., Murray, J.J., Hasegawa, M., Matsuda, Y. and Inagami, T., Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-I receptor, Nature, 351 (1991) 230233. 21 Song, K., Allen, A.M., Paxinos, G. and Mendelsohn, F.A.O., Mapping of angiotensin II receptor subtype heterogeneity in rat brain, J. Comp. Neurol., 316 (1992) 467-484. 22 Speth, R.C., Angiotensin II receptor subtypes in the bovine cerebellum, FASEB J., 5 (1991) A870 (Abstract). 23 Speth, R.C. and Kim, K.H., Discrimination of two angiotensin II receptor subtypes with a selective analogue of angiotensin II, p-aminophenylalanine 6 angiotensin II, Biochem. Biophys. Res. Commun., 169 (1990) 997-1006. 24 Speth, R.C., Rowe, B.P., Grove, K.L., Carter, M.R. and Say-
25
26
27
28
29
lor, D.L., Sulfhydryl reducing agents distinguish two subtypes of angiotensin II receptors in the rat brain, Brain Res., 548 (1991) 1-8. Sumners, C., Tang, W., Zelezna, B. and Raizada, M.K., Angiotensin II receptor subtypes are coupled with distinct signaltransduction mechanisms in neurons and astrocytes from rat brain, Proc. Natl. Acad. Sci. USA, 88 (1991) 7567-7571. Tsutsumi, K. and Saavedra, J.M., Heterogeneity of angiotensin II AT 2 receptors in the rat brain, Mol. Pharmacol., 41 (1992) 290-297. Webb, M.L., Liu, E.C.-K., Cohen, R.B., Hedberg, A., Bogosian, E.A., Monshizadegan, H., Molloy, C., Serafino, R., Moreland, S., Murphy, T.J. and Dickinson, K.E.J., Molecular characterization of angiotensin II type II receptors in rat pheochromocytoma cells, Peptides, 13 (1992) 499-508. Whitebread, S., Mele, M., Kamber, B. and De Gasparo, M., Preliminary biochemical characterization of two angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun., 163 (1989) 284-291. Whitebread, S.E., Taylor, V., Bottari, S.P., Kamber, B. and De Gasparo, M., Radioiodinated CGP 42112A: a novel high affinity and highly selective ligand for the characterization of angiotensin AT2 receptors, Biochem. Biophys. Res. Commun., 181 (1991) 1365-1371.
189
REGPEP 01283
[125I]CGP 42112 binding reveals differences between rat brain and adrenal AT 2 receptor binding sites Robert C. Speth Department of Veterinary and Comparative Anatomy, Pharmacologyand Physiology, Washington State University,Pullman, WA (USA) (Received 6 October 1992; accepted 11 December 1992)
Key words: Angiotensin II receptor; Ligand CGP42112; fl-Mercaptoethanol; Binding affinity
Summary The AT 2 angiotensin II receptor selective ligand CGP42112 was radioiodinated and used to study AT 2 receptor binding sites in the rat brain (combined olfactory bulb, septum, thalamus and midbrain) and whole adrenal. The [125I]CGP 42112 binding was of high affinity, saturable and specific in both tissues. Competition studies with nonselective and angiotensin II receptor subtype selective ligands, and evaluation of the effects of the sulfhydryl reducing agent ~-mercaptoethanol, confirmed that [125I]CGP 42112 bound selectively to the AT 2 angiotensin II receptor subtype. [125I]CGP 42112 bound with higher affinity in the brain than in the adrenal. fl-Mercaptoethanol enhanced [125I]CGP 42112 binding in the brain, but did not alter its binding in the adrenal. A similar difference in binding affinity for [125I]sarcosine1,isoleucine8 angiotensin II and enhancement of binding affinity by fl-mercaptoethanol was observed in the rat brain and adrenal. These observations suggest that the AT 2 angiotensin II receptor subtypes in the brain and adrenal may differ.
Introduction Discrimination of the two major AII receptor subtypes, AT1 and AT 2 can be traced back to observations of the differential effects of the sulfhydryl reducing agent, dithiothreitol, on AII receptor binding. Correspondence to: R.C. Speth, Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164-6520, USA.
Studies by Glossman et al. [9] and Bennett and Snyder [ 1] who used dithiothreitol as a peptidase inhibitor suggested that dithiothreitol enhanced AII receptor binding in the adrenal and brain, while studies by Gunther et al. [ 10,11 ] demonstrated that dithiothreitol inhibited All receptor binding in the vasculature. Several recent studies have used peptidic and nonpeptidic ligands selective for AII receptor subtypes, as well as sulfhydryl reducing agents to evaluate AII receptor binding in various tissues [6,7,17,21,
190
22,24,28]. These studies show that the predominant AII receptor subtype in the tissues in which binding is decreased by sulfhydryl reducing agents is AT 1 (vasculature and liver) and the predominant receptor subtype in tissue in which binding is enhanced by sulfhydryl reducing agents is AT 2 (bovine cerebellum, and to a lesser extent, the adrenal). Molecular biological studies of the genes encoding AII receptors have described the AT 1 receptor as a member of the G-protein linked receptor superfamily [16,20]. Subsequent studies have demonstrated the existence of two or more different transcripts similar to the AT 1 receptor in the rat which have been designated as AT1A and AT m or AT 3 [8,12,13,19]. The AT 2 receptor gene has yet to be identified so its structure and other characteristics are less well known. Arriving at a description of a functional response mediated by the AT 2 receptor has also been difficult and controversial [2,25,27]. Thus, radioligand binding studies of AII receptors remain the primary means of describing AT 2 receptors. CGP 42112 A has been shown to bind with high affinity to the AT2 subtype and has low affinity for the AT 1 subtype [28]. Whitebread et al. [29] radiolabeled CGP 42112 and characterized its binding to AT 2 receptors in human uterus and rat adrenal. This report describes the further application of radiolabeled CGP 42112 to characterize AT 2 receptors and provides results suggestive of the presence of multiple subtypes of AT 2 receptor binding sites.
Methods
Radioligands [125I]CGP 42112 was prepared using a modification of the procedure of Whitebread et al. [29]. CGP 42112 B (B indicates trifluoroacetate salt), generously provided by Dr. Marc de Gasparo of CibaGeigy, Basle, was reacted with Na125I (NEZ-033H, NEN Research Products, Boston, MA) in the presence of 1 mM chloramine T and 370 mM sodium
phosphate (pH 7.4) for 20 s. The reaction was stopped with sodium metabisulfate, 0.5 mg/ml final concentration. Purified mono[125I]CGP 42112 was isolated by H P L C using a C18 reverse phase column eluted with a mobile phase of 23 ~o acetonitrile/77 ~o triethylamine phosphate (83 mM phosphoric acid adjusted to pH 3.0 with triethylamine). Baseline separation of uniodinated, monoiodinated and diiodinated CGP 42112 was obtained. [ 125I] S arcosinel,isoleucine8 angiotensin II ([125I]SI AII) was prepared in the same manner as [125I]CGP 42112, and the mono[125I]peptide was also purified by HPLC as described above except that the eluting buffer contained only 16Yo acetonitrile. The fractions containing mono[ ~25I]CGP 42112 and mono[125I]SI All were stored up to 8 weeks at -20°C in the presence of 2 mg/ml bovine albumin.
Tissue preparation Freshly dissected brains (olfactory bulbs, thalamus, septum and midbrain of two rats were pooled together) and whole adrenal glands (pooled from two rats) were obtained from male Sprague-Dawley rats (Laboratory Animal Resource Center, Washington State University, ages 14-18 weeks) killed by decapitation. The brain regions were chosen based on previous studies indicating that they contained an abundance of AT 2 receptors. The tissues were weighed, placed in 20 ml of ice-cold hypotonic buffer; 20 mM sodium phosphate, pH 7.1-7.2 with 0.1 mM bacitracin, and homogenized (Tissuemizer, Tek-Mar, Cincinnati, OH). The homogenized tissue was centrifuged at 48,000 g for 20 min at 0-4°C. The supernatant was decanted and the pelleted tissue was resuspended in 20 ml of assay buffer: 50 mM sodium phosphate, pH 7.1-7.2, with 150 mM NaCI, 1 mM disodium EDTA and 0.1 mM bacitracin. The rehomogenized tissue was recentrifuged as before. The supernatant was again decanted and the pelleted tissue was resuspended in 7 ml of assay buffer. Protein concentration in the homogenates was determined by the method of Lowry et al. [ 15].
191 Radioligand binding assays Final tissue homogenate (0.05 ml) was incubated for 120-150 min at 22°C with [12SI]CGP or [125I]SI All in a volume of 0.1 ml of assay buffer. To obtain saturation isotherms, the tissues were incubated with 8 different concentrations ofradioligand ranging from 0.03 to 2.0 nM, in duplicate ([125I]SI AII) or triplicate ([125I]CGP 42112). For competition analyses an additional 0.01 ml of assay buffer containing either AII, CGP 42112A, SI AII or losartan (DuP 753), to obtain final concentrations of 1, 1, 1 and 10 /~M, respectively, was added to triplicate tubes containing a single (0.3-0.6 nM) concentration of radioligand. To determine the effect of a sulfhydryl reducing agent on radioligand binding, fl-mercaptoethanol (Sigma Chemical Co., St. Louis, MO) was added to an aliquot of tissue homogenate to attain a concentration of 30 mM. Bound radioligand was separated from free radioligand on glass fiber filters, (#32, Schleicher and Schuell, Keene, NH). The filters were imediately rinsed three times with 3 ml of 50 mM sodiumpotassium phosphate buffer (pH 7.4), 22°C and assayed by sodium iodide crystal gamma scintillation counting. The saturation isotherm data was evaluated by a weighted (1/y2) nonlinear regression analysis (Inplot, Graphpad Software, Inc., San Diego, CA) using the equation: Y= A ' X / ( B + X) + C ' X
where Y is bound radioligand, A is Bma x o r the amount of receptor, X is the radioligand concentration, B is the dissociation constant (KD) and C is the ratio of nonspecific binding. To verify its accuracy, the computer derived value for nonspecific binding was compared to the actual value for nonspecific binding determined in the presence of 1 # M A I I at a single radioligand concentration of 0.3-0.6 nM. In addition, the linearity of nonspecific binding of [125I]CGP 42112 at concentrations ranging from 75 to 800 pM was established in a preliminary assay of rat brain and adrenal. Nonspecific binding of[ 125I]SI
AII was also demonstrated to be linear over the range of 50-700 pM. Generally there was no disparity between empirical and computer generated nonspecific binding values, however in two instances there was a disparity and the empirical value was placed into the equation as a constant. Statistical analyses A two-way ( + fl-mercaptoethanol and radioligand) randomized block ANOVA was used to compare K D and Brnax values in the brain and in the adrenal. A two-way ( + fl-mercaptoethanol and tissue) randomized block ANOVA was also used to evaluate differences in radioligand affinity between tissues. A two-way ( + fl-mercaptoethanol and competing ligand) randomized block ANOVA was also used to evaluate the effects of a saturating concentration of selective and nonselective AII ligands on radioligand binding. Mean values were compared using Duncan's multiple comparison test [14].
Results
Binding of [125I]CGP 42112 was saturable and displayed high affinity and specificity in both the brain (olfactory bulb, septum, thalamus, midbrain) and whole adrenal gland. Fig. 1 describes a saturation isotherm for [ 125I]CGP 42112 binding to brain membranes in the presence of fl-mercaptoethanol. The values for specific and nonspecific binding derived from the regression analysis are also displayed. The computer generated value for nonspecific binding was consistent with that determined empirically at 1 /~M AII at a single concentration of [125I]CGP 42112. Fig. 2 describes the binding of [125I]CGP 42112 and [125I]SI AII to an adrenal membrane preparation in the presence and absence of 30 mM fl-mercaptoethanol. Figs. 3 and 4 describe Rosenthal (Scatchard) plots for specific [ 125I]CGP and [lZSI]SI AII binding in the brain and adrenal, respectively, derived from the data shown in Figs. 1 and 2. The average values for the dissociation constant
192 125I-CGP 42112 Binding to Rat Brain Olfactory Bulb, Septum, Thalamus, Midbrain • Total Binding
11
u M All * 1 u M C G P 42112 oi0 u M h o s a r t a n _ .... •
o
1
'4-, V
..m"
.
.
%"
......
Angiotensin II Receptor Binding in Rat Adrenal 8 • • 30 m M 8-mercaptoethanol [] • I251-CGP 42112 ! o , 12SI-Sl All o 6¸
,
4
" "
r~
g m
; ~ 0 0
~ T
d
~ N ° n s p e cific
S p e c i f i c Bindinc
Bindlng
500 1251-CGP 42112 (pN)
i
1000
Fig. 1. Saturation isotherm of total [125I]CGP 42112 binding to rat brain (olfactory bulb, septum, thalamus and midbrain) in the presence of 30 m M fl-mercaptoethanol. The amount of membrane protein present in each tube, in 0.1 ml total assay volume, was 286 #g. This was equivalent to an initial wet weight of 3.0 mg. Samples were run in triplicate. Nonspecific binding in the presence of 1 #M AII was determined at a single, 510 pM, concentration of [ 125I]CGP 42112. ['25I]CGP 42112 binding in the presence of 1 #M CGP42112 and 10 #M losartan was also determined at 510 pM [IzsI]CGP 42112. The losartan data points are barely distinquishable because of the superimposition of the total binding data points and their placement is indicated with an arrow. The curves for total and specific binding and the line for nonspecific binding were generated from the equation: Y= Bmax'X/(g D + X ) + C ' X where Y= total bound, X = [125I]CGP 42112 concentration, and C is the ratio of nonspecific binding. The derived values for this example were 1.93 fmol for B . . . . 66 pM for KD, and 0.00255 for the nonspecific binding ratio. A Rosenthal (Scatchard) plot of these data is presented in Fig. 4.
i
k
500 i000 1500 1251-Radioligand (pM)
2000
Fig. 2. Saturation isotherm of total [125I]CGP 42112 and [msI]SI AII binding in rat adrenal in the absence or presence of fl-mercaptoethanol. Curves drawn are derived from nonlinear regression analysis for specific plus nonspecific binding as described in Materials and Methods and legend to Fig. 1. The amount of membrane protein present in each tube, in 0.1 ml total assay volume, was 28 #g. This was equivalent to an initial wet weight of 0.5 rag.
(KD) and receptor density (nmax) from four such experiments are presented in Table I. Analysis of variance revealed that 30 mM /3-mercaptoethanol caused a significant (P< 0.01) reduction in the Bmax for [nSI]SI All binding in both the brain (33 ~o) and the adrenal (52~o), but did not significantly alter that for [ 1251]CGP 42112. Analysis of variance also demonstrated 30 mM fl-mercaptoethanol significantly
TABLEI Comparison of [~25I]GCP 42112 and [nsI]SI AII binding in rat brain and adrenal Tissue
Ligand
- ME
Brain Adrenal
CGP SI AII CGP SI AII
Bmax (fmol/mg protein)
K D (pM)
150 + 275 + 186 + 430 +
+ ME
16 59 80 192
63 128 200 429
+ 18"* + 9** + 135 + 130
- ME
+ ME
6.2 10.4 125 371
6.1 + 1.0 7.1 + 0.8* 107 + 10 176 + 87*
+ 0.7 + 0.4 + 38 + 131
Values are mean, n = 4, +_S.D. - ME is no fl-mercaptoethanol, + ME is in the presence of 30 mM fl-mercaptothanol *P < 0.01 versus - ME; **P< 0.02 versus - ME.
193 A n g i o t e n s i n II Receptor B i n d i n g in Rat Adrenal 125I-CGP 42112 v e r s u s 1 2 5 I - S a r l , I l e S AII 10 • •
•
30
mM 8-mercaptoethanol
o v ¢-
"~II
o=
o
nn
0 0
o.01
0,02
003
o,04
o.05
Bound/Free (ml) Fig. 3. Rosenthal (Scatchard) plot of specific []25I]CGP 42112 and [ ~25I]SI A l l binding to rat adrenal membranes derived from data shown in Fig. 2. Specific binding was determined as described in Materials and Methods.
enhanced the binding affinity (decreased the Ko) for both [ 1251]SI All and [ 125I]CGP 42112 in the brain, but did not alter the binding affinity for either radiA n g i o t e n s i n II Receptor Binding in Rat Brain Olfactory bulb - Thalamus - Septum - Midbrain 3Ù • •
2.5 ~ "~
\
2.0""
D
I-..
3 0 mM 8 - m e r c a p t o e t h a n o l o • 1251-CGp 42112 o • leSI-sI AII
-i--. i............. l. "'"'O
....... l"
0.5. 0.(3
"'"~'"',
o005
Q010 0.015 Bound/Free (ml)
o020
Fig.4. Rosenthal (Scatchard) plot of specific[t2~I]CGP 42112 and []251]SIAll binding to rat brain membranes obtained from the olfactorybulb,septum, thalamus and midbrain. Specificbinding was determined as described in Materials and Methods. The amount of membrane protein present in each tube, in 0.1 ml total assay volume, was 286 #g. This was equivalent to an initial wet weight of 3.0 mg.
oligand in the adrenal. The ANOVA comparing radioligand binding affinity in the brain and adrenal indicated that the brain had a significantly higher affinity for [125I]CGP 42112 (P<0.05) and [125I]SI All (P<0.01) than the adrenal. However, it did not reveal the significant effect of fl-mercaptoethanol on binding affinity nor did it show a significant interaction. Table II describes the specificity of [125I]CGP 42112 and [125I]SI AII binding for AII receptor binding sites. Fig. 1 also describes the specificity of [125I]CGP 42112 binding in the brain. At a concentration averaging 440 pM, 54~o of the [125I]CGP 42112 binding in the brain was specific for All receptors as defined by displacement with 1 #M All. A similar amount of [125I]CGP 42112 binding was displaced by 1/aM unlabeled CGP 42112. Losartan, the AT 1 selective antagonist, at 10/aM, did not displace any [125I]CGP 42112 binding in the brain. In the presence of 30 mM fl-mercaptoethanol there was a significant (P< 0.05) increase in the proportion of 1 #M All and CGP 42112 displaceable [125I]CGP 42112 binding. This increase is attributable to enhanced binding affinity for this radioligand. Specific 1 # M A I I displaceable [125I]CGP 42112 binding in the adrenal was 79~ of total binding. 1 /aM CGP 42112 displaceable [ 125I]CGP 42112 binding was 83~o, while 10 #M losartan displaceable binding was only 3 ~o of total binding. Addition of 30 mM fl-mercaptoethanol did not significantly alter the proportion of 1 #M All, 1 /aM CGP 42112 and 10 #M losartan displaceable [125I]CGP 42112 binding. Specific 1/aM AII displaceable [125I]SI AII binding was 69~o of total binding in the brain. 1/aM CGP 42112 and 10/aM losartan displaceable [ |25I]SI AII binding was 48 ~o and 26 ~'o, respectively. The sum of the 1 /aM CGP 42112 (putative AT2) and 10 /aM losartan (putative AT 1) displaceable binding is 76 ~o. This is slightly greater than 69~o and may reflect some overlap of CGP 42112 to inhibit a small proportion of AT 1 binding and/or losartan inhibition of a small proportion of AT 2 binding. In the presence
194 TABLE II Competition for [125I]GGP 42112 and [125I]SI AII binding by All, CGP 42112, losartan and SI AII in rat brain and adrenal Tissue
Brain
Adrenal
Radioligand*
[125I]CGPa ( + ME) a [ 125I]SI All c ( + ME) d [ 1251]CGP a
( + ME) a [125I]SI AIIf ( + ME) g
Competing ligand (~o of total binding remaining) All
CGP 42112
losartan
SI All
46.0 _+8.4 38.9 + 8.1 b 31.0 + 7.4 25.4 + 5.2 20.9 + 8.6 17.8 + 5.2 6.4 + 1.8 12.3 + 3.9
45.7 _+9.0 34.1 _+5.6b 52.0 + 2.1 35.0 + 4.9 e 16.8 + 5.6 15.1 + 3.6 56.6 + 5.7 47.4 + 5.9
101.2 + 13.5 99.0 _+6.2 74.0 + 14.3 79.3 + 15.0 97.1 + 2.5 96.0 + 6.5 33.3 + 10.1 56.7 + 7.2
ND ND 28.2 + 9.0 22.1 +_3.2 ND ND 12.3 + 3.9 8.5 _+2.0
Values are mean + S.D. (n = 4) except SI AII (n = 3). The percent of specific binding is: 100 - the values shown. Lower values indicate a higher percent of specific binding to All receptors or individual All receptor subtypes. ND is not determined. * The average radioligand concentration was 440 pM for [125I]CGP 42112 and 540 pM for [12sI]SI All. Concentrations of competing ligands were 1 #M for All, CGP 42112 and SI All, and 10 #M for losartan. a P<0.01, losartan>AII-=CGP 42112. b p < 0.05, combined AI1 and CGP 42112 < no fl-mercaptoethanol combined AII and CGP 42112. c p < 0.05, losartan > CGP 42112 > All ----SI All. d P<0.05, losartan>CGP 42112~AII-=-SI All. ° P<0.05,
o f 30 m M fl-mercaptoethanol, specific 1 # M A I I displaceable [ 125I] SI A I I binding was 75 ~o. O n e # M C G P 42112 d i s p l a c e a b l e [125I]SI A l l binding increased to 65 ~o while 10/~M l o s a r t a n d i s p l a c e a b l e binding d e c r e a s e d to 21 ~o. Specific 1 # M A l l d i s p l a c e a b l e [125I]SI A l l binding in the whole adrenal was 94~o. O n e # M C G P 42112 a n d 10 ~tM l o s a r t a n d i s p l a c e a b l e [125I] SI A I I binding were 43~o and 67~o, respectively. In the presence of 30 m M fl-mercaptoethanol, specific 1 /~M A l l displaceable [125I]SI A I I binding was 88~o. O n e / ~ M C G P 42112 d i s p l a c e a b l e [125I]SI A I I binding increased to 53 ~o while 10 # M l o s a r t a n displaceable binding d e c r e a s e d to 43 ~o.
Discussion A s previously r e p o r t e d by W h i t e b r e a d et al. [29] in h u m a n myometrium, [125I]CGP 42112 b i n d s sat-
urably a n d with high a t ~ i t y in the rat brain a n d adrenal. The K D values for [125I]CGP 42112 in this study are in excellent agreement with those found in h u m a n m y o m e t r i u m [29]. Nonspecific binding for [125I]CGP 42112 defined either by 1 # M A I I a n d 1 /~M C G P 42112B was nearly identical, in b o t h the brain a n d adrenal, suggesting that the A T 2 r e c e p t o r is the only site at which [12SI]CGP 42112 binds with high affinity. The A T 1 selective ligand losartan, at 10 /~M, did n o t significantly inhibit [125I]CGP 42112 binding, confirming that r a d i o i o d i n a t i o n o f C G P 42112 d i d not alter its A l l receptor subtype selectivity. C o m p a r i s o n o f specific [125I]CGP 42112 binding with specific [125I]SI A l l binding allows estimates o f the p r o p o r t i o n o f A T 1 and A T 2 subtypes in the rat brain ( c o m b i n e d olfactory bulb, septum, thalamus, m i d b r a i n ) a n d whole a d r e n a l to be m a d e . B a s e d on Bma x values for the two radioligands in the a b s e n c e
195 of fl-mercaptoethanol, 40~o of the brain All receptors were AT 1 and 60~o were AT 2. This estimate is the same as that reported by Chang et al., [5] for similar rat brain regions. From the reduction in the Bmax for [125I]SI AII in the presence of 30 mM fl-mercaptoethanol, an estimate of 32~ AT 1 and 68~o AT2 in brain could be made. However, in the presence of 30 mM fl-mercaptoethanol, 10/~M losartan (a concentration more than 10-fold below its IC5o for the AT 2 subtype) still retained the ability to displace 20~o of the [ a25I]SI All binding in the brain. This suggests that 30 mM fl-mercaptoethanol does not fully inactivate brain AT 1 receptors or that AT 2 receptors become more sensitive to losartan in the presence of fl-mercaptoethanol. A third estimate of the proportion of subtypes, based on the amount of specific [ 125I]SI AII binding displaced by 1 #M CGP 42112 and 10 ktM losartan in the absence of fl-mercaptoethanol, is 37~o AT1 and 63~o AT 2. This estimate has the limitation of assuming equal affinity of [125I]SI AII for each of the AII receptor subtypes, and that the concentration of each competing ligand was selective for only one of the subtypes. Neither assumption can be met based on previous studies by Rowe et al. [18] and Whitebread et al. [29]. Thus, the estimate using [125I]CGP 42112 remains the most reliable. An estimate of 66~o ATa/34~o AT2 in the whole adrenal can be made from comparing Bmax values for [tzsI]CGP 42112 versus [125I]SI AII. This estimate differs moderately from the 50~ ratio reported by Chang and Lotti [4]. Two observations from this study are suggestive of differences in AT 2 receptors in the brain and adrenal. One is the differential effects of fl-mercaptoethanol on [125I]CGP 42112 binding affinity in the brain and adrenal. In the brain, 30 mM fl-mercaptoethanol clearly increased binding affinity of [125I]CGP 42112 for AT 2 receptors. By contrast, fl-mercaptoethanol did not alter [125I]CGP 42112 binding affinity for whole adrenal AT 2 receptors. An identical pattern of enhanced binding affinityfor [ 1251]SI AlI in the brain, and no change in binding affinity in the adrenal, was
observed in the presence of 30 mM fl-mercaptoethanol. With [125I]SI All however, it is not possible to attribute this change entirely to AT 2 receptors, since [125I]SI AII binds to AT 1 receptors as well. The second observation is the higher binding affinity of both [ 125I]CGP 42112 and [ 125I]SI AII for the brain compared to the adrenal. But again, the [125I]SI AII effect may not be limited to AT 2 receptors. While these data are consistent with the existence of two AT 2 receptor subtypes, other explanations of the data should be acknowledged. It is possible that the adrenal may be less sensitive to the effects of the sulfhydryl reducing agent, e.g., the adrenal may have more oxidizable substrates, or be more capable of oxidizing fl-mercaptoethanol, but the centrifugal washing and substantial dilution of the tissue to < 0.3 mg protein/ml make this unlikely. It is still uncertain as to whether CGP 42112 is an agonist or antagonist at the AT 2 receptor. Thus, it is possible that [125I]CGP 42112 is recognizing different agonist affinity states of the AT 2 receptor. However the likelihood of such an effect being tissue dependent is small. Finally, the ANOVA comparing K D values did not reveal a significant interaction between fl-mercaptoethanol and tissue as expected. However, this may result from the large variability in the adrenal binding data compared to the brain binding data. Tsutsumi and Saavedra [26] have reported the existence of two AT 2 receptor subtypes. The subtype they call AT2A is sensitive to guanine nucleotides and pertussis toxin in a manner similar to several G-protein linked receptors. The subtype they call AT2B is insensitive to guanine nucleotides and pertussis toxin and conforms more to the criteria others have established for the AT 2 receptor [3,6,7,23,28]. Whether the two putative receptor subtypes we see in the brain and the adrenal fit within the AT2A and ATzB framework of Tsutsumi and Saavedra [26] for brain AT 2 receptor subtypes remains to be determined. The differential effects of sulfhydryl reducing agents on AII receptor binding was the first solid
196
evidence for the AII receptor subtypes that were later confirmed pharmacologically. Therefore, it is possible that sulfhydryl reducing agents are once again indicating additional AII receptor subtypes that will ultimately be confirmed with new generations of All receptor subtype selective agents. [125I]CGP will be useful for further studies of AT 2 receptor subtypes since the extraneous binding to AT 1 receptors seen with other angiotensinergic radioligands will not be present or need to be eliminated. Preliminary evaluation of rat brain sections incubated with [ ~25I]CGP 42112 (Speth and Grove, unpublished observations) indicates that this radioligand is also suitable for in vitro receptor autoradiographic analysis of AT 2 receptors, showing no specific binding in brain areas containing exclusively the AT 1 receptor subtype.
Acknowledgements Dr. Marc de Gasparo, Ciba-Geigy, Basle, Switzerland, donated the CGP 42112B used for these studies and Dr. Ronald Smith of DuPont Merck Pharmaceuticals, Wilmington, DE, donated the losartan used for these studies. This work was supported by USPHS, NIH grant NS-21305. Frances Yang, who provided technical assistance was supported by NIH, RR 03368, Minority High School Student Research Apprentice Program. Jeanne Jensen, Kevin Grove and Dr. Joseph Harding graciously reviewed the manuscript, providing valuable suggestions. References 1 Bennett, J.P. Jr. and Snyder, S.H., Angiotensin II binding to mammalian brain membranes, J. Biol. Chem., 251 (1976) 7423-7430. 2 Bottari, S., King, I.N., Reichlin, S., Dahlstroem, I., Lydon, N. and De Gasparo, M., The angiotensin AT 2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase, Biochem. Biophys. Res. Commun., 183 (1992) 206-211. 3 Bottari, S.P., Taylor, V., King, I.N., Bogdal, Y., Whitebread, S. and De Gasparo, M., Angiotensin II AT2 receptors do not
interact with guanine nucleotide binding proteins, Eur. J. Pharmacol., 207 (1991) 157-163. 4 Chang, R.S.L. and Lotti, V.J., Two distinct angiotensin II receptor binding sites in rat adrenal revealed by new selective nonpeptide ligands, Mol. Pharmacol., 29 (1990) 347-351. 5 Chang, R.S.L., Lotti, V.J., Chen, T.B. and Faust, K.A., Two angiotensin II binding sites in rat brain revealed using [125I]Sarl,IleS-angiotensin II and selective nonpeptide antagonists, Biochem. Biophys. Res. Commun., 171 (1990) 813817. 6 Chiu, A.T., Herblin, W.F., McCall, D.E., Ardecky, R.J., Carini, D.J., Duncia, J.V., Pease, L,J., Wong, P.C., Wexler, R.R., Johnson, A.L. and Timmermans, P.B.M.W.M., Identification of angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun., 165 (1989) 196-203. 7 Chiu, A.T., McCall, D.E., Nguyen, T.T., Carini, D.J., Duncia, J.V., Herblin, W.F., Uyeda, R.T., Wong, P.C., Wexler, R.R., Johnson, A.L. and Timmermans, P.B.M.W.M., Discrimination of angiotensin II receptor subtypes by dithiothreitol, Eur. J. Pharmacol., 170 (1989) 117-118. 8 Elton, T.S., Stephan, C.C., Taylor, G.R., Kimball, M.G., Martin, M.M., Durand, J.N. and Oparil, S., Isolation of two distinct type 1 angiotensin II receptor genes, Biochem. Biophys. Res. Commun., 184 (1992) 1067-1073. 9 Glossmann, H., Baukal, A.J. and Catt, K.J., Properties of angiotensin II receptors in the bovine and rat adrenal cortex, J. Biol. Chem., 249 (1974) 825-834. 10 Gunther, S., Gimbrone, M.A. Jr. and Alexander, R.W., Regulation by angiotensin II of its receptors in resistance blood vessels, Nature, 287(5779) (1980) 230-232. 11 Gunther, S., Gimbrone, M.A. Jr. and Alexander, R.W., Identification and characterization of the high affinity vascular angiotensin II receptor in rat mesenteric artery, Circ. Res., 47 (1980) 278-286. 12 Iwai, N. and Inagami, T., Identification of two subtypes in the rat type I angiotensin II receptor, FEBS. Lett., 298 (1992) 257-260. 13 Kakar, S.S., Sellers, J.C., Devor, D.C., Musgrove, L.C. and Neill, J.D., Angiotensin II type-1 receptor subtype cDNAs: differential tissue expression and hormonal regulation, Biochem. Biophys. Res. Commun., 183 (1992) 1090-1096. 14 Kirk, R.E., Multiple comparison tests. In Experimental Design: Procedures for the Behavioral Sciences, Brooks/Cole, Belmont, 1982, pp. 106. 15 Lowry, O.H., Rosebrough, N.J., Fan', A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 16 Murphy, T.J., Alexander, R.W., Griendling, K.K., Runge, M.S. and Bernstein, K.E., Isolation of a eDNA encoding the vascular type-1 angiotensin II receptor, Nature, 351 (1991) 233-236.
197 17 Rowe, B.P., Grove, K.L., Saylor, D.L. and Speth, R.C., Angiotensin II receptor subtypes in the rat brain, Eur. J. Pharmacol., 186 (1990) 339-342. 18 Rowe, B.P., Saylor, D.L. and Speth, R.C., Analysis of angiotensin II receptor subtypes in individual rat brain nuclei, Neuroendocrinology, 55 (1992) 563-573. 19 Sandberg, K., Ji, H., Clark, A.J., Shapira, H. and Catt, K.J., Cloning and expression of a novel angiotensin II receptor subtype, J. Biol. Chem., 267 (1992) 9455-9458. 20 Sasaki, K., Yamano, Y., Bardhan, S., Iwai, N., Murray, J.J., Hasegawa, M., Matsuda, Y. and Inagami, T., Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-I receptor, Nature, 351 (1991) 230233. 21 Song, K., Allen, A.M., Paxinos, G. and Mendelsohn, F.A.O., Mapping of angiotensin II receptor subtype heterogeneity in rat brain, J. Comp. Neurol., 316 (1992) 467-484. 22 Speth, R.C., Angiotensin II receptor subtypes in the bovine cerebellum, FASEB J., 5 (1991) A870 (Abstract). 23 Speth, R.C. and Kim, K.H., Discrimination of two angiotensin II receptor subtypes with a selective analogue of angiotensin II, p-aminophenylalanine 6 angiotensin II, Biochem. Biophys. Res. Commun., 169 (1990) 997-1006. 24 Speth, R.C., Rowe, B.P., Grove, K.L., Carter, M.R. and Say-
25
26
27
28
29
lor, D.L., Sulfhydryl reducing agents distinguish two subtypes of angiotensin II receptors in the rat brain, Brain Res., 548 (1991) 1-8. Sumners, C., Tang, W., Zelezna, B. and Raizada, M.K., Angiotensin II receptor subtypes are coupled with distinct signaltransduction mechanisms in neurons and astrocytes from rat brain, Proc. Natl. Acad. Sci. USA, 88 (1991) 7567-7571. Tsutsumi, K. and Saavedra, J.M., Heterogeneity of angiotensin II AT 2 receptors in the rat brain, Mol. Pharmacol., 41 (1992) 290-297. Webb, M.L., Liu, E.C.-K., Cohen, R.B., Hedberg, A., Bogosian, E.A., Monshizadegan, H., Molloy, C., Serafino, R., Moreland, S., Murphy, T.J. and Dickinson, K.E.J., Molecular characterization of angiotensin II type II receptors in rat pheochromocytoma cells, Peptides, 13 (1992) 499-508. Whitebread, S., Mele, M., Kamber, B. and De Gasparo, M., Preliminary biochemical characterization of two angiotensin II receptor subtypes, Biochem. Biophys. Res. Commun., 163 (1989) 284-291. Whitebread, S.E., Taylor, V., Bottari, S.P., Kamber, B. and De Gasparo, M., Radioiodinated CGP 42112A: a novel high affinity and highly selective ligand for the characterization of angiotensin AT2 receptors, Biochem. Biophys. Res. Commun., 181 (1991) 1365-1371.