Peptides,Vol. 13, pp. 291-296, 1992
0196-9781/92 $5.00 + .00 1992 PergamonPressLtd.
Printed in the USA.
Characterization of Angiotensin II Receptor Subtypes in the Rat Spleen KEISUKE TSUTSUMI, CHRISTER
STROMBERG
A N D J U A N M. S A A V E D R A
Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, 9000 Rockville Pike, Building 10, Room 2D-45, Bethesda, MD 20892 R e c e i v e d 22 J u l y 1991 TSUTSUMI, K., C. STROMBERG AND J. M. SAAVEDRA. Characterization of angiotensin H receptorsubtypes in the rat spleen. PEPTIDES 13(2) 291-296, 1992.--Quantitative autoradiography was used to determine the subtype of ANG receptors in the red pulp of the rat spleen. The AT1 antagonist DuP 753 competed for ANG binding with high atfinity; binding was abolished by dithiothreitol. The AT2 competitor CGP 42112 A showed lower affinity, and the AT2 competitor PD 123177 did not affect binding at 10-5 M. These data indicated the presence of only AT1 receptors. AT~ receptor number was similar in immature (2 weeks old) and adult (8 weeks old) rats. Binding was sensitive to guanine nucleotides, suggesting an association with G-proteins. Angiotensin II, at a dose ofl0 -7 M, stimulated inositol phosphate formation 33% over control values in spleen from 8-week-old rats. This effect was significantly blocked by 10-s M DuP 753. We suggest a possible role of AT~ receptors in the regulation of splenic volume, blood flow, and lymphocyte function. Renin angiotensin system Angiotensin receptors Dithiothreitol ATI receptors
Immune system
IN addition to its role in the regulation of blood pressure and fluid homeostasis (8), angiotensin II (ANG) has been shown to have effects on several components of the immune system (29). ANG releases prolactin from the anterior pituitary (8) and prolactin is involved in the regulation of the immune system (16). Binding sites for ANG have been found on human peripheral lymphocytes (18), on murine granuloma macrophages (10,28), and in the red pulp of the rat and mouse spleen (3). In addition, ANG suppresses human mononuclear cell reactivity by stimulating suppressor T-lymphocytes (19,20). These data indicate that ANG may play a role in the regulation of immune response and inflammation. With the use of selective ANG receptor antagonists, two ANG receptor subtypes have been recently characterized in peripheral tissues (4,23,26,30) and in the brain (24,25,27). ATI receptors are selectively displaced by DuP 753, and AT2 receptors by CGP 42112 A and PD 123177 (4,23-27,30). DuP 753 antagonizes the vasoconstriction produced by ANG, and reduces blood pressure in experimental and human hypertension (23). Because of the use of selective AT~ receptor blockers in clinical situations, it was of interest to characterize A N G receptor subtypes in ANG target organs.
DuP 753
CGP 42112 A
PD 123177
decapitation, between 0900 and 1000 h, and the spleens were removed immediately.
Quantitative A utoradiography For binding studies, the spleens were frozen immediately after removal in isopentane at - 3 0 ° C and stored at - 7 0 ° C for less than one week. Tissue sections (16 t~m thick) were cut in a cryostar at - 15°C, thaw-mounted on gelatin-coated glass slides, and dried overnight in a desiccator at 4°C. Consecutive sections were stained with toluidine blue or incubated as described below. Sections were labeled in vitro with [125i][SarI]ANG (Peninsula Laboratories, Belmont, CA, iodinated by DuPont New England Nuclear Laboratories, Wilmington, DE; spec.act. 2200 Ci/mmol) using experimentally determined incubation parameters previously described (13). Briefly, sections were preincubated for 15 min at 22°C in 10 mM Na phosphate buffer, pH 7.4, containing 120 mM NaC1, 5 mM Na2EDTA, 0.005% bacitracin (Sigma Chemical Co., St. Louis, MO), and 0.2% proteinase-free bovine serum albumin (Sigma), followed by incubation for 120 min in fresh buffer containing the appropriate ligand. After incubation, slides were rinsed 4 consecutive times, 1 minute each, in fresh ice-cold 50 mM Tris-HC1 buffer, pH 7.6, followed by a dip in ice-cold distilled water, and the sections were dried under air. To characterize AT receptor subtypes, consecutive sections were incubated with 5 × 10-1° M [t25I][Sart]ANG, a concentration close to the Ko value (13) in the presence of increasing concentrations (10 -~° to 10-4 M) of the selective AT~ competitor DuP 753 {2-n-butyl-4-chloro-5-hydroxymethyl-1-[2'-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole, from DuPont, Wilmington, DE} (4) or 10-l° to 10-4 M of the selective AT2 corn-
METHOD Groups of five 10-day-old (with lactating females) and 7-weekold male Sprague-Dawley rats were purchased from Zivic Miller (Alliston Park, PA) and were provided with standard rat food and water ad lib, with a 12:12 h light-dark cycle with lights on at 0600 h. Rats were killed when 14 days old or 8 weeks old, by
291
292
TSUTSUMI, STROMBERG AND SAAVEDRA
.n,
B
12
D
q
E 1ram
ANGIOTENSIN RECEPTOR SUBTYPES IN RAT SPLEEN
293
TABLE 1 ANGIOTENSIN RECEPTOR SUBTYPES 1N THE RAT SPLEEN
Age
Specific Binding
Dup 753 10-5 M
CGP 42112 A 10 7 M
PD 123177 10 5 M
DTT 5 × 10-3 M
2 weeks old 8 weeks old
16.8 _+ 1.2 17.3 _+0.5
ND ND
16.3 _+ 1.5 18.3 +_0.9
17.2 _+ 1.8 16.8 ___0.9
ND ND
Data are fmol/mg protein, means +_S.E.M., for groups for 5 rats, measured individually. [~25I][Sarl]ANGconcentration was 5 x 10-1° M. ND: not significantlydifferent from nonspecificbinding (p > 0.05).
petitor CGP 42112 A [nicotinic acid-Tyr-(NZ-benzyloxycarbonyl-Arg)Lys-His-Pro-Ile-OH, from CIBA-Geigy, Basle, Switzerland] (30). In another experiment, consecutive sections were incubated with 5 × 10-~° M [~2sI][Sar~]ANG and single concentrations of DuP 753 (10 -5 M), CGP 42112 A (10 -7 M), or the selective AT2 competitor PD 123177 {1-(4-amino-3methylphenyl)methyl- 5-diphenylacety1-4,5,6,7-tetrahydro- 1H imidazo[4,5-C]pyridine-6-carboxylic acid-2HC1, from Parke Davis, Ann Arbor, MI} (10 -5 M), or the reducing agent dithiothreitol (5 × 10 3 M). At these concentrations, DuP 753 totally competed for AT~ receptors (4,23,25,27), CGP 42112 A and PD 123177 totally competed for AT2 receptors (26,27), and dithiothreitol totally inhibited ATt binding (24). Nonspecific binding was determined in the presence of 5 × 10_6 M unlabeled ANG (Peninsula Laboratories, Belmont, CA). Specific ANG binding was the difference between total binding in the abscence of competitors and nonspecific binding. Total binding minus binding in the presence of DuP 753 represented AT~ receptors. Total binding minus binding in the presence of CGP 42112 A or PD 123177 represented AT2 receptors. We studied the possible involvement of guanine nucleotide binding proteins in the regulation of AT binding. Spleen sections were incubated in the absence or presence of increasing concentrations (10 -8 to 10-3 M) of the metabolically stable analogue of GTP: guanosine-5'-O-(3-thiotriphosphate) (GTP3,S) (Sigma) (26). The incubation conditions were similar to those used for characterization of AT receptor subtypes, with the exception that the preincubation and incubation buffers consisted of 50 mM Na phosphate buffer, pH 7.4, containing 120 mM NaC1, 10 mM MgCi2, 0.005% bacitracin (Sigma), and 0.2% proteinasefree bovine serum albumin (Sigma) (26). The dry labeled sections, together with a set of [~25I]standards (Amersham Co., Arlington Heights, IL), were apposed against [SH]Hyperfilm (Amersham) in X-ray cassettes (CGR Medical Corp., Baltimore, MD). Films were developed with D 19 Kodak developer for 4 min at 4°C. Optical densities of the autoradiograms were determined by computerized microdensitometry and the results expressed in fmol/mg protein after comparison with the [~25I] standard curve (15). ICs0 values were calculated with the GraphPad Inplot program (GraphPad, San Diego, CA). Results were expressed as means +_ S.E.M. and were analyzed by unpaired t-test and one-way ANOVA with post hoc Bonferroni test.
Determination of Phosphoinositide Hydrolysis The phosphoinositide hydrolysis was assessed as the production of inositol phosphate (IP0 in the presence of l0 mM LiCl (14). Briefly, whole spleens from 8-week-old rats were crosschopped with a McIlwain tissue chopper (Brinkmann Instruments, Inc., Westbury, NY) to yield 350 × 350-#m slices. The slices were washed and prelabeled with [3H]myoinositol in Krebs Ringer buffer (KRB) at 37°C for 2 hours and continuously gassed with 95% 02-5% CO2. After prelabeling, the slices were washed and dispensed in aliquots of 50 #1 into individual tubes containing 310 ul freshly gassed KRB with l0 mM LiCl and incubated for 10 min at 37°C. The method was validated by incubating spleen slices in the presence of l0 -5 M norepinephrine (Sigma). To test the effects of ANG, the slices were stimulated with 10-8 or l0 -7 M of ANG. To show a functional correlate to the visualized ATt receptors, slices were incubated in the presence of l0 -s M DuP 753 and l 0 -7 M ANG. The AT2 antagonist was not used, because no ATE receptors were seen by autoradiography. All drugs were added in 40 #l Li÷-KRB and the incubation was continued for a further 45 min. In control samples 40 #l saline was added. To standardize variation due to unequal sample size, IPI accumulation was expressed for individual samples as a percentage of the radioactivity associated with membranes. RESULTS
A utoradiography of ANG Receptors A high density of ANG receptors was localized throughout the red pulp of the rat spleen (Fig. IA and B). Binding was totally displaced by incubation with 10-5 M DuP 753 (Fig. 1C, Table 1), was completely inhibited by 5 × 10-3 M dithiothreitol (Table 1), was insensitive to incubation with 10-7 M CGP 42112 A (Fig. 1D, Table 1) or PD 123177 (Table 1), and was totally displaced by unlabeled ANG (Fig. 1E). Analysis of competition curves indicated that DuP 753 competed for ANG binding with an affinity about 6 times higher than CGP 42112 A (Fig. 2). In 8-week-old rats, the ICso for DuP 753 was 1.03 _+ 0.08 × 10 -7 M , and the ICso for CGP 42112 A was 6.58 + 3.93 × 10-6 M, for groups of 5 rats, measured individually.
FIG. 1. Autoradiography of angiotensin II receptor subtypes in the rat spleen. The figure represents consecutive sections of spleen from an 8-weekold rat. (A) Stained with toluidine blue. (B) Total binding, incubated with 5 × l0 lOM [t2sI][Sarl]ANG. 1: white pulp. 2: red pulp. (C) Incubated as in B, with addition of l0 -5 M DuP 753. (D) Incubated as in B, with addition ofl0 -7 M CGP 42112 A. (E) Incubated as in B, with addition of 5 × 10 -6 M unlabeled ANG.
294
TSUTSUMI, STROMBERG AND SAAVEDRA
,2°t 80
60
40
'11 .
.
.
.
.
.
.
oo -I0 -9 -8 -7 -6 -5 -4 log [M] FIG. 2. Displacement of [~2Sl][Sart]ANGin rat spleen. Displacement curves obtained from consecutive sections from spleen of 8-week-old rats, incubated in the presence of 5 × 10 J0 M [~251][SarqANG and increasingconcentrationsof competitors. Results are expressed as means + S.E.M. of groups of 5 rats, measured individually.Closed circles: DuP 753. Open circles: CGP 42112 A.
Addition of increasing concentrations of GTP3,S resulted in a progressive inhibition (up to about 60%) of ANG binding, with an ICs0 of 2.52 + 0.37 × 10 s M (Fig. 3).
cleotides. Addition of a metabolically stable analogue of GTP, GTP3,S, decreased agonist binding affinity. Inhibition of binding in the presence of guanine nucleotides has been reported earlier for ANG (7). The present results are consistent with recent studies demonstrating that binding to fetal AT, receptors in liver and lung parenchyma (26), as well as to rat vascular ANG receptors (31) and to vascular smooth muscle cells, which have been recently classified as AT~ receptors (23), is also sensitive to guanine nucleotides. The inhibition of ANG binding to its AT~ receptors by guanine nucleotides indicates that spleen AT, receptors, like other peripheral AT, receptors, may be coupled functionally to effectors by G-proteins, and that they belong to the superfamily of G-protein-coupled receptors (1). In the rat spleen, ANG stimulated phosphoinositide hydrolysis, and this effect was blocked by the selective AT, receptor antagonist DuP 753. These results indicate that AT, receptors in the spleen, as in other tissues (11,21,26), are associated with this intracellular second messenger system as one of their signal transduction mechanisms. This is the first report demonstrating an AT, receptor-linked second messenger response in an immune organ of the rat. The immunological activity of the spleen is located in the white pulp (2,12,17), which is devoid of ANG receptors [present results and (3)]. This observation, and the absence of ANG binding in another immune system organ, the thymus (3), questions a direct role of ANG in the regulation of the immune response. However, ANG receptors are present in human peripheral blood lymphocytes (19) and murine granuloma macrophages (22,28), and ANG stimulates the suppressor T-cells in humans (20). In the rat, the small numbers of ANG receptors detectable in isolated spleen cells (3) may be located on lymphocytes or macrophages, or in mature T-cells, but not in the maturing lymphocytes located in the thymus. Alternatively, the effects of ANG described in humans may be absent in rodents.
Phosphoinositide Hydrolysis Stimulation with 10 -7 M ANG resulted in a 33 + 6% increase in inositol-l-phosphate accumulation in the spleen from 8-weekold rats (p < 0.001 vs. control). The effect of 1 0 -7 M ANG was significantly antagonized by 10-5 M DuP 753 (p < 0.05) (Fig. 4). In the same experiment, 10-5 M norepinephrine increased inositol-l-phosphate accumulation by 58 + 6% (p < 0.001 vs. control).
I00 A
8O c
8 DISCUSSION
The localization and characterization of high affinity ANG receptors in the red pulp of the rat spleen (3) and the recent discovery of ANG receptor subtypes (4,23,30) raised the question of the relative role of each subtype in the control of the spleen functions and immune response. We report that previously described ANG receptors in the red pulp of the rat spleen (3) belong to the AT, subtype, as determined by the selective competition by DuP 753, the sensitivity of the binding to the reducing agent dithiothreitol, and the low affinity of AT2 competitors PD 123177 and CGP 42112 A (4,23-27,30). We have examined spleen ANG receptors in adult and immature rats, since ANG receptor expression varies with age (25). Both the number and the subtype of ANG receptors are similar at 2 weeks and 8 weeks of age, indicating that ANG could play a role in the spleen through stimulation of AT1 receptors not only in adulthood but also early in postnatal life. Our present results indicate that binding of the ANG agonist [12sI][Sar']ANG to all AT, receptors is sensitive to guanine nu-
6O .,,,~
40 ,,,,I,-
2O
'
oo
II
.
-8
.
.
-7
.
.
.
-6 -5 lo 9 [M]
-4
-3
FIG. 3. Effects of GTP~S on ANG binding. Consecutive sections from spleen of 8-week-oldrats were incubatedwith 5 × 10-1° M [~25I][Sar~]ANG and increasing concentrations of GTP-yS. Results are expressed as means _+S.E.M. of groups of 5 rats, measured individually.
A N G I O T E N S I N R E C E P T O R SUBTYPES IN R A T SPLEEN
40
g.
30
co o (_
g
20
b
.fi
I0
ANG ANG ANG 10-SM lO-7M I0-7M +
DuP IO-~M FIG. 4. Stimulation of inositol-1-phosphate accumulation by ANG and the effect of DuP 753 on ANG-induced stimulation in rat spleen. Each bar represents the mean + S.E.M. (n = 5). *p < 0.001 vs. control, and p < 0.05 vs, ANG 10-7 M + DuP 753 l0 -5 M; paired t-test.
295
The physiological role of AT~ receptors in the red pulp of the spleen has not yet been clarified. In the spleen, the red pulp is an important storage of intact red blood cells, and has metabolic activity (12). Smooth muscle cells in the capsule and trabeculae of the stroma of the red pulp, probably together with reticular cells, which have been shown to have contractile properties (2), receive noradrenergic innervation (9) and regulate the volume of the spleen (2). Smooth muscle cells in the splenic stroma are also responsive to ANG, which causes a marked vasoconstriction and a reduction in the volume of the spleen (5,6). This action is not caused indirectly by catecholamine release, because A N G effects cannot be abolished by either denervation or pharmacological blockade ofcatecholamine receptors (5). On the other hand, A N G may interact with adrenergic innervation, since the blockade of adrenergic receptors increases the effects of A N G on the spleen (5). The identification of the cell type(s) expressing these receptors in the red pulp was not possible with quantitative autoradiography because of the inherent limitation in the resolution of the technique. However, it is possible that most of the AT~ receptors are located in the red pulp stroma, because red blood cells do not bind A N G and the concentration of A N G receptors in isolated rat spleen cells was reported to be one order of magnitude less than that in the rat spleen sections (3). AT~ receptors in the splenic stroma might mediate the actions of A N G on the blood flow and volume regulation of the spleen (2,5), which is consistent with a physiological role for circulating ANG, and for AT~ receptors, in the regulation of blood volume and pressure (8). Since hemorrhage and hypovolemia increase plasma A N G (8), stimulation of spleen AT~ receptors by the circulating hormone, which results in contraction of the spleen and release of blood to the general circulation, could play a role in the maintenance of blood volume under those conditions.
REFERENCES 1. Birnbaumer, L. Transduction of receptor signal into modulation of 10. Foils, G.; Deso, B.; Medgyesi, G. A.; Fust, (3. Effect of angiotensin effector activity by G proteins: The first 20 years or so. FASEB J. 4: II on macrophage functions. Immunology 48:529-535; 1983. 3068-3078; 1990. 11. Garcla-S~nz, J.; Macias-Silva, M. Angiotensin II stimulates phosphoinositide turnover and phosphorylase through AII-1 receptors 2. Blue, J.; Weiss, L. Electron microscopy of the red pulp of the dog in isolated rat hepatocytes. Biochem. Biophys. Res. Commun. 172: spleen including vascular arrangements, periarterial macrophage 780-785; 1990. sheats (ellipsoids), and the contractile, innervated reticular meshwork. 12. Hartwig, H.; Hartwig, H. G. Structural characteristics of the mamAm. J. Anat. 161:189-218; 1981. malian spleen indicatingstorage and release of red blood cells. Aspects 3. Castrtn, E.; Kurihara, M.; Saavedra, J. M. Autoradiographic localof evolutionary and environmental demands. Experientia 41:159ization and characterization of angiotensin II binding sites in the 163; 1985. spleen of rats and mice. Peptides 8:737-742; 1987. 13. Israel, A.; Correa, F. M. A.; Niwa, M.; Saavedra, J. M. Quantitative 4. Chiu, A. T.; Herblin, W. F.; McCall, D. E.; Ardecky, R. J.; Cailni, determination ofangiotensin lI bindingsites in rat brain and pituitary D. J.; Duncia, J. V.; Pease, L. J.; Wong, P. C.; Wexler, R. R.; Johnson, gland by radioautography. Brain Res. 322:341-345; 1984. A. L.; Timmermans, P. B. M. W. M. Identification of angiotensin 14. Laitinen, J. T.; Torda, T.; Saavedra, J. M. Cholinergic stimulation II receptor subtypes. Biochem. Biophys. Res. Commun. 165:196of phosphoinositide hydrolysis in the rat pineal gland. Eur. J. Phar203; 1989. macol. 161:237-240; 1989. 5. Davies, B. N.; Withilngton, P. G. The responses of the isolated, 15. Nazarali, A. J.; Gutkind, J. S.; Saavedra, J. M. Calibration of[nsI]blood-perfused spleen of the dog to angiotensin, oxytocin and vapolymer standards with [t25I]-brain paste standards for use in quansopressin. Br. J. Pharmacol. 54:205-212; 1975. titative receptor autoradiography. J. Neurosci. Methods 30:247-253; 6. Davies, B. N.; Withrington, P. G. The actions of drugs on the smooth 1989. muscle of the capsule and blood vessels of the spleen. Pharmacol. 16. Nicoll, C. S. Physiological actions of prolactin. In: Greep, R. O.; Rev. 25:373-413; 1973. Astwood, E. B., eds. Handbook of physiology, sect. 7, vol. 4, pt. 2, 7. De l_6an, A.; Ong, H.; Gutkowska, J.; Schiller, P. W.; McNicoll, N. chap. 32. Washington, DC: American Physiological Society; 1974: Evidence for agonist-induced interaction ofangiotensin receptor with 253-292. a guanine nucleotide-binding protein in bovine adrenal zona glo17. Reilly, F. D. Innervation and vascular pharmacodynamics of the merulosa. Mol. Pharmacol. 26:498-508; 1984. mammalian spleen. Experientia 4 l: 187-192; 1985. 8. Dzau, V. J.; Pratt, R. E. Renin-angiotensin system: Biology, phys18. Shimada, K,; Yazaki, Y. Binding sites for angiotensin II in human iology, and pharmacology. In: Fozzard, H. A., ed. The heart and mononuclear leucocytes. J. Biochem. 84:1013-1015; 1978. cardiovascular system. New York: Raven Press; 1986:1631-1662. 19. Simon, M. R.; Engel, D. E.; Weinstock, J. V.; Roi, L. D. The effect 9. Felten, D. L.; Felten, S. Y.; Carlson, S. L.; Oscfiowka, J. A.; Livnat, of angiotensin II on human mononuclear cell reactivity: Suppression S. Noradrenergic and peptidergic innervation of lymphoid tissue. J. of PHA-P-induced thymidine incorporation. Immunol. Invest. 14: Immunol. 135:755S-765S; 1985. 389-400; 1985.
296 20. Simon, M. R.; Engel, D. E.; Weinstock, J. V. Angiotensin II suppression of human mononuclear cell reactivity is associated with enhanced OKT8 + lymphocyte tymidine incorporation. J. Immunopharmacol. 8:289-297; t986. 21. Strrmberg, C.; Tsutsumi, K.; Viswanathan, M.; Saavedra, J. M. Angiotensin 11 AT~ receptors in rat superior cervical ganglia: Characterization and stimulation of phosphoinositide hydrolysis. Eur. J. Pharmacol. Mol. Pharmacol. 208:331-336; 1991. 22. Thomas, D. W.; Hoffman, M. D. Identification of macrophage receptors for angiotensins: A potential role in antigen uptake for T lymphocyte responses? J. Immunol. 132:2807-2812; 1984. 23. Timmermans, P. B. M. W. M.; Wong, P. C.; Chiu, A. T.; Herblin, W. F. Nonpeptide angiotensin II receptor antagonists. Trends Pharmacol. Sci. 12:55-62; 1991. 24. Tsutsumi, K.; Saavedra, J. M. Increased dithiothreitol-insensitive, type 2 angiotensin II receptors in selected brain areas of young rats. Cell. Mol. Neurobiol. 11:295-299; 1991. 25. Tsutsumi, K.; Saavedra, J. M. Characterization and development of angiotensin II receptor subtypes (ATj and AT2) in rat brain. Am. J. Physiol. 261:R209-R216; 1991.
TSUTSUMI, S T R O M B E R G A N D SAAVEDRA 26. Tsutsumi, K.; Strrmberg, C.; Viswanathan, M.; Saavedra, J. M. Angiotensin II receptor subtypes in fetal tissues of the rat: Autoradiography, guanine nucleotide sensitivity and association with phosphoinositide hydrolysis. Endocrinology 129:1075-1082; 1991. 27. Tsutsumi, K.; Saavedra, J. M. Differential development of angiotensin II receptor subtypes in the rat brain. Endocrinology 128:630632; 1991. 28. Weinstock, J. V.; Kassab, J. T. Functional angiotensin II receptors on macrophages from isolated liver granulomas of murine schistosoma mansoni. J. Immunol. 132: 2598-2602; 1984. 29. Werner, G. H.; Floc'h, F.; Migliore-Samour, D.; Jolles, P. Immunomodulating peptides. Experientia 42:521-531; 1986. 30. Whitebread, S.; Mele, M.; Kamber, B.; de Gasparo, M. Preliminary biochemical characterization of two angiotensin II receptor subtypes. Biochem. Biophys. Res. Commun. 163:284-291; 1989. 31. Wright, G. B.; Alexander, R. W.; Ekstein, L. S.; Gimbrone, M. A., Jr. Sodium, divalent cations, and guanine nucleotides regulate the affinity of the rat mesenteric artery angiotensin II receptor. Circ. Res. 50:462-469; 1982.
0196-9781/92 $5.00 + .00 1992 PergamonPressLtd.
Printed in the USA.
Characterization of Angiotensin II Receptor Subtypes in the Rat Spleen KEISUKE TSUTSUMI, CHRISTER
STROMBERG
A N D J U A N M. S A A V E D R A
Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, 9000 Rockville Pike, Building 10, Room 2D-45, Bethesda, MD 20892 R e c e i v e d 22 J u l y 1991 TSUTSUMI, K., C. STROMBERG AND J. M. SAAVEDRA. Characterization of angiotensin H receptorsubtypes in the rat spleen. PEPTIDES 13(2) 291-296, 1992.--Quantitative autoradiography was used to determine the subtype of ANG receptors in the red pulp of the rat spleen. The AT1 antagonist DuP 753 competed for ANG binding with high atfinity; binding was abolished by dithiothreitol. The AT2 competitor CGP 42112 A showed lower affinity, and the AT2 competitor PD 123177 did not affect binding at 10-5 M. These data indicated the presence of only AT1 receptors. AT~ receptor number was similar in immature (2 weeks old) and adult (8 weeks old) rats. Binding was sensitive to guanine nucleotides, suggesting an association with G-proteins. Angiotensin II, at a dose ofl0 -7 M, stimulated inositol phosphate formation 33% over control values in spleen from 8-week-old rats. This effect was significantly blocked by 10-s M DuP 753. We suggest a possible role of AT~ receptors in the regulation of splenic volume, blood flow, and lymphocyte function. Renin angiotensin system Angiotensin receptors Dithiothreitol ATI receptors
Immune system
IN addition to its role in the regulation of blood pressure and fluid homeostasis (8), angiotensin II (ANG) has been shown to have effects on several components of the immune system (29). ANG releases prolactin from the anterior pituitary (8) and prolactin is involved in the regulation of the immune system (16). Binding sites for ANG have been found on human peripheral lymphocytes (18), on murine granuloma macrophages (10,28), and in the red pulp of the rat and mouse spleen (3). In addition, ANG suppresses human mononuclear cell reactivity by stimulating suppressor T-lymphocytes (19,20). These data indicate that ANG may play a role in the regulation of immune response and inflammation. With the use of selective ANG receptor antagonists, two ANG receptor subtypes have been recently characterized in peripheral tissues (4,23,26,30) and in the brain (24,25,27). ATI receptors are selectively displaced by DuP 753, and AT2 receptors by CGP 42112 A and PD 123177 (4,23-27,30). DuP 753 antagonizes the vasoconstriction produced by ANG, and reduces blood pressure in experimental and human hypertension (23). Because of the use of selective AT~ receptor blockers in clinical situations, it was of interest to characterize A N G receptor subtypes in ANG target organs.
DuP 753
CGP 42112 A
PD 123177
decapitation, between 0900 and 1000 h, and the spleens were removed immediately.
Quantitative A utoradiography For binding studies, the spleens were frozen immediately after removal in isopentane at - 3 0 ° C and stored at - 7 0 ° C for less than one week. Tissue sections (16 t~m thick) were cut in a cryostar at - 15°C, thaw-mounted on gelatin-coated glass slides, and dried overnight in a desiccator at 4°C. Consecutive sections were stained with toluidine blue or incubated as described below. Sections were labeled in vitro with [125i][SarI]ANG (Peninsula Laboratories, Belmont, CA, iodinated by DuPont New England Nuclear Laboratories, Wilmington, DE; spec.act. 2200 Ci/mmol) using experimentally determined incubation parameters previously described (13). Briefly, sections were preincubated for 15 min at 22°C in 10 mM Na phosphate buffer, pH 7.4, containing 120 mM NaC1, 5 mM Na2EDTA, 0.005% bacitracin (Sigma Chemical Co., St. Louis, MO), and 0.2% proteinase-free bovine serum albumin (Sigma), followed by incubation for 120 min in fresh buffer containing the appropriate ligand. After incubation, slides were rinsed 4 consecutive times, 1 minute each, in fresh ice-cold 50 mM Tris-HC1 buffer, pH 7.6, followed by a dip in ice-cold distilled water, and the sections were dried under air. To characterize AT receptor subtypes, consecutive sections were incubated with 5 × 10-1° M [t25I][Sart]ANG, a concentration close to the Ko value (13) in the presence of increasing concentrations (10 -~° to 10-4 M) of the selective AT~ competitor DuP 753 {2-n-butyl-4-chloro-5-hydroxymethyl-1-[2'-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole, from DuPont, Wilmington, DE} (4) or 10-l° to 10-4 M of the selective AT2 corn-
METHOD Groups of five 10-day-old (with lactating females) and 7-weekold male Sprague-Dawley rats were purchased from Zivic Miller (Alliston Park, PA) and were provided with standard rat food and water ad lib, with a 12:12 h light-dark cycle with lights on at 0600 h. Rats were killed when 14 days old or 8 weeks old, by
291
292
TSUTSUMI, STROMBERG AND SAAVEDRA
.n,
B
12
D
q
E 1ram
ANGIOTENSIN RECEPTOR SUBTYPES IN RAT SPLEEN
293
TABLE 1 ANGIOTENSIN RECEPTOR SUBTYPES 1N THE RAT SPLEEN
Age
Specific Binding
Dup 753 10-5 M
CGP 42112 A 10 7 M
PD 123177 10 5 M
DTT 5 × 10-3 M
2 weeks old 8 weeks old
16.8 _+ 1.2 17.3 _+0.5
ND ND
16.3 _+ 1.5 18.3 +_0.9
17.2 _+ 1.8 16.8 ___0.9
ND ND
Data are fmol/mg protein, means +_S.E.M., for groups for 5 rats, measured individually. [~25I][Sarl]ANGconcentration was 5 x 10-1° M. ND: not significantlydifferent from nonspecificbinding (p > 0.05).
petitor CGP 42112 A [nicotinic acid-Tyr-(NZ-benzyloxycarbonyl-Arg)Lys-His-Pro-Ile-OH, from CIBA-Geigy, Basle, Switzerland] (30). In another experiment, consecutive sections were incubated with 5 × 10-~° M [~2sI][Sar~]ANG and single concentrations of DuP 753 (10 -5 M), CGP 42112 A (10 -7 M), or the selective AT2 competitor PD 123177 {1-(4-amino-3methylphenyl)methyl- 5-diphenylacety1-4,5,6,7-tetrahydro- 1H imidazo[4,5-C]pyridine-6-carboxylic acid-2HC1, from Parke Davis, Ann Arbor, MI} (10 -5 M), or the reducing agent dithiothreitol (5 × 10 3 M). At these concentrations, DuP 753 totally competed for AT~ receptors (4,23,25,27), CGP 42112 A and PD 123177 totally competed for AT2 receptors (26,27), and dithiothreitol totally inhibited ATt binding (24). Nonspecific binding was determined in the presence of 5 × 10_6 M unlabeled ANG (Peninsula Laboratories, Belmont, CA). Specific ANG binding was the difference between total binding in the abscence of competitors and nonspecific binding. Total binding minus binding in the presence of DuP 753 represented AT~ receptors. Total binding minus binding in the presence of CGP 42112 A or PD 123177 represented AT2 receptors. We studied the possible involvement of guanine nucleotide binding proteins in the regulation of AT binding. Spleen sections were incubated in the absence or presence of increasing concentrations (10 -8 to 10-3 M) of the metabolically stable analogue of GTP: guanosine-5'-O-(3-thiotriphosphate) (GTP3,S) (Sigma) (26). The incubation conditions were similar to those used for characterization of AT receptor subtypes, with the exception that the preincubation and incubation buffers consisted of 50 mM Na phosphate buffer, pH 7.4, containing 120 mM NaC1, 10 mM MgCi2, 0.005% bacitracin (Sigma), and 0.2% proteinasefree bovine serum albumin (Sigma) (26). The dry labeled sections, together with a set of [~25I]standards (Amersham Co., Arlington Heights, IL), were apposed against [SH]Hyperfilm (Amersham) in X-ray cassettes (CGR Medical Corp., Baltimore, MD). Films were developed with D 19 Kodak developer for 4 min at 4°C. Optical densities of the autoradiograms were determined by computerized microdensitometry and the results expressed in fmol/mg protein after comparison with the [~25I] standard curve (15). ICs0 values were calculated with the GraphPad Inplot program (GraphPad, San Diego, CA). Results were expressed as means +_ S.E.M. and were analyzed by unpaired t-test and one-way ANOVA with post hoc Bonferroni test.
Determination of Phosphoinositide Hydrolysis The phosphoinositide hydrolysis was assessed as the production of inositol phosphate (IP0 in the presence of l0 mM LiCl (14). Briefly, whole spleens from 8-week-old rats were crosschopped with a McIlwain tissue chopper (Brinkmann Instruments, Inc., Westbury, NY) to yield 350 × 350-#m slices. The slices were washed and prelabeled with [3H]myoinositol in Krebs Ringer buffer (KRB) at 37°C for 2 hours and continuously gassed with 95% 02-5% CO2. After prelabeling, the slices were washed and dispensed in aliquots of 50 #1 into individual tubes containing 310 ul freshly gassed KRB with l0 mM LiCl and incubated for 10 min at 37°C. The method was validated by incubating spleen slices in the presence of l0 -5 M norepinephrine (Sigma). To test the effects of ANG, the slices were stimulated with 10-8 or l0 -7 M of ANG. To show a functional correlate to the visualized ATt receptors, slices were incubated in the presence of l0 -s M DuP 753 and l 0 -7 M ANG. The AT2 antagonist was not used, because no ATE receptors were seen by autoradiography. All drugs were added in 40 #l Li÷-KRB and the incubation was continued for a further 45 min. In control samples 40 #l saline was added. To standardize variation due to unequal sample size, IPI accumulation was expressed for individual samples as a percentage of the radioactivity associated with membranes. RESULTS
A utoradiography of ANG Receptors A high density of ANG receptors was localized throughout the red pulp of the rat spleen (Fig. IA and B). Binding was totally displaced by incubation with 10-5 M DuP 753 (Fig. 1C, Table 1), was completely inhibited by 5 × 10-3 M dithiothreitol (Table 1), was insensitive to incubation with 10-7 M CGP 42112 A (Fig. 1D, Table 1) or PD 123177 (Table 1), and was totally displaced by unlabeled ANG (Fig. 1E). Analysis of competition curves indicated that DuP 753 competed for ANG binding with an affinity about 6 times higher than CGP 42112 A (Fig. 2). In 8-week-old rats, the ICso for DuP 753 was 1.03 _+ 0.08 × 10 -7 M , and the ICso for CGP 42112 A was 6.58 + 3.93 × 10-6 M, for groups of 5 rats, measured individually.
FIG. 1. Autoradiography of angiotensin II receptor subtypes in the rat spleen. The figure represents consecutive sections of spleen from an 8-weekold rat. (A) Stained with toluidine blue. (B) Total binding, incubated with 5 × l0 lOM [t2sI][Sarl]ANG. 1: white pulp. 2: red pulp. (C) Incubated as in B, with addition of l0 -5 M DuP 753. (D) Incubated as in B, with addition ofl0 -7 M CGP 42112 A. (E) Incubated as in B, with addition of 5 × 10 -6 M unlabeled ANG.
294
TSUTSUMI, STROMBERG AND SAAVEDRA
,2°t 80
60
40
'11 .
.
.
.
.
.
.
oo -I0 -9 -8 -7 -6 -5 -4 log [M] FIG. 2. Displacement of [~2Sl][Sart]ANGin rat spleen. Displacement curves obtained from consecutive sections from spleen of 8-week-old rats, incubated in the presence of 5 × 10 J0 M [~251][SarqANG and increasingconcentrationsof competitors. Results are expressed as means + S.E.M. of groups of 5 rats, measured individually.Closed circles: DuP 753. Open circles: CGP 42112 A.
Addition of increasing concentrations of GTP3,S resulted in a progressive inhibition (up to about 60%) of ANG binding, with an ICs0 of 2.52 + 0.37 × 10 s M (Fig. 3).
cleotides. Addition of a metabolically stable analogue of GTP, GTP3,S, decreased agonist binding affinity. Inhibition of binding in the presence of guanine nucleotides has been reported earlier for ANG (7). The present results are consistent with recent studies demonstrating that binding to fetal AT, receptors in liver and lung parenchyma (26), as well as to rat vascular ANG receptors (31) and to vascular smooth muscle cells, which have been recently classified as AT~ receptors (23), is also sensitive to guanine nucleotides. The inhibition of ANG binding to its AT~ receptors by guanine nucleotides indicates that spleen AT, receptors, like other peripheral AT, receptors, may be coupled functionally to effectors by G-proteins, and that they belong to the superfamily of G-protein-coupled receptors (1). In the rat spleen, ANG stimulated phosphoinositide hydrolysis, and this effect was blocked by the selective AT, receptor antagonist DuP 753. These results indicate that AT, receptors in the spleen, as in other tissues (11,21,26), are associated with this intracellular second messenger system as one of their signal transduction mechanisms. This is the first report demonstrating an AT, receptor-linked second messenger response in an immune organ of the rat. The immunological activity of the spleen is located in the white pulp (2,12,17), which is devoid of ANG receptors [present results and (3)]. This observation, and the absence of ANG binding in another immune system organ, the thymus (3), questions a direct role of ANG in the regulation of the immune response. However, ANG receptors are present in human peripheral blood lymphocytes (19) and murine granuloma macrophages (22,28), and ANG stimulates the suppressor T-cells in humans (20). In the rat, the small numbers of ANG receptors detectable in isolated spleen cells (3) may be located on lymphocytes or macrophages, or in mature T-cells, but not in the maturing lymphocytes located in the thymus. Alternatively, the effects of ANG described in humans may be absent in rodents.
Phosphoinositide Hydrolysis Stimulation with 10 -7 M ANG resulted in a 33 + 6% increase in inositol-l-phosphate accumulation in the spleen from 8-weekold rats (p < 0.001 vs. control). The effect of 1 0 -7 M ANG was significantly antagonized by 10-5 M DuP 753 (p < 0.05) (Fig. 4). In the same experiment, 10-5 M norepinephrine increased inositol-l-phosphate accumulation by 58 + 6% (p < 0.001 vs. control).
I00 A
8O c
8 DISCUSSION
The localization and characterization of high affinity ANG receptors in the red pulp of the rat spleen (3) and the recent discovery of ANG receptor subtypes (4,23,30) raised the question of the relative role of each subtype in the control of the spleen functions and immune response. We report that previously described ANG receptors in the red pulp of the rat spleen (3) belong to the AT, subtype, as determined by the selective competition by DuP 753, the sensitivity of the binding to the reducing agent dithiothreitol, and the low affinity of AT2 competitors PD 123177 and CGP 42112 A (4,23-27,30). We have examined spleen ANG receptors in adult and immature rats, since ANG receptor expression varies with age (25). Both the number and the subtype of ANG receptors are similar at 2 weeks and 8 weeks of age, indicating that ANG could play a role in the spleen through stimulation of AT1 receptors not only in adulthood but also early in postnatal life. Our present results indicate that binding of the ANG agonist [12sI][Sar']ANG to all AT, receptors is sensitive to guanine nu-
6O .,,,~
40 ,,,,I,-
2O
'
oo
II
.
-8
.
.
-7
.
.
.
-6 -5 lo 9 [M]
-4
-3
FIG. 3. Effects of GTP~S on ANG binding. Consecutive sections from spleen of 8-week-oldrats were incubatedwith 5 × 10-1° M [~25I][Sar~]ANG and increasing concentrations of GTP-yS. Results are expressed as means _+S.E.M. of groups of 5 rats, measured individually.
A N G I O T E N S I N R E C E P T O R SUBTYPES IN R A T SPLEEN
40
g.
30
co o (_
g
20
b
.fi
I0
ANG ANG ANG 10-SM lO-7M I0-7M +
DuP IO-~M FIG. 4. Stimulation of inositol-1-phosphate accumulation by ANG and the effect of DuP 753 on ANG-induced stimulation in rat spleen. Each bar represents the mean + S.E.M. (n = 5). *p < 0.001 vs. control, and p < 0.05 vs, ANG 10-7 M + DuP 753 l0 -5 M; paired t-test.
295
The physiological role of AT~ receptors in the red pulp of the spleen has not yet been clarified. In the spleen, the red pulp is an important storage of intact red blood cells, and has metabolic activity (12). Smooth muscle cells in the capsule and trabeculae of the stroma of the red pulp, probably together with reticular cells, which have been shown to have contractile properties (2), receive noradrenergic innervation (9) and regulate the volume of the spleen (2). Smooth muscle cells in the splenic stroma are also responsive to ANG, which causes a marked vasoconstriction and a reduction in the volume of the spleen (5,6). This action is not caused indirectly by catecholamine release, because A N G effects cannot be abolished by either denervation or pharmacological blockade ofcatecholamine receptors (5). On the other hand, A N G may interact with adrenergic innervation, since the blockade of adrenergic receptors increases the effects of A N G on the spleen (5). The identification of the cell type(s) expressing these receptors in the red pulp was not possible with quantitative autoradiography because of the inherent limitation in the resolution of the technique. However, it is possible that most of the AT~ receptors are located in the red pulp stroma, because red blood cells do not bind A N G and the concentration of A N G receptors in isolated rat spleen cells was reported to be one order of magnitude less than that in the rat spleen sections (3). AT~ receptors in the splenic stroma might mediate the actions of A N G on the blood flow and volume regulation of the spleen (2,5), which is consistent with a physiological role for circulating ANG, and for AT~ receptors, in the regulation of blood volume and pressure (8). Since hemorrhage and hypovolemia increase plasma A N G (8), stimulation of spleen AT~ receptors by the circulating hormone, which results in contraction of the spleen and release of blood to the general circulation, could play a role in the maintenance of blood volume under those conditions.
REFERENCES 1. Birnbaumer, L. Transduction of receptor signal into modulation of 10. Foils, G.; Deso, B.; Medgyesi, G. A.; Fust, (3. Effect of angiotensin effector activity by G proteins: The first 20 years or so. FASEB J. 4: II on macrophage functions. Immunology 48:529-535; 1983. 3068-3078; 1990. 11. Garcla-S~nz, J.; Macias-Silva, M. Angiotensin II stimulates phosphoinositide turnover and phosphorylase through AII-1 receptors 2. Blue, J.; Weiss, L. Electron microscopy of the red pulp of the dog in isolated rat hepatocytes. Biochem. Biophys. Res. Commun. 172: spleen including vascular arrangements, periarterial macrophage 780-785; 1990. sheats (ellipsoids), and the contractile, innervated reticular meshwork. 12. Hartwig, H.; Hartwig, H. G. Structural characteristics of the mamAm. J. Anat. 161:189-218; 1981. malian spleen indicatingstorage and release of red blood cells. Aspects 3. Castrtn, E.; Kurihara, M.; Saavedra, J. M. Autoradiographic localof evolutionary and environmental demands. Experientia 41:159ization and characterization of angiotensin II binding sites in the 163; 1985. spleen of rats and mice. Peptides 8:737-742; 1987. 13. Israel, A.; Correa, F. M. A.; Niwa, M.; Saavedra, J. M. Quantitative 4. Chiu, A. T.; Herblin, W. F.; McCall, D. E.; Ardecky, R. J.; Cailni, determination ofangiotensin lI bindingsites in rat brain and pituitary D. J.; Duncia, J. V.; Pease, L. J.; Wong, P. C.; Wexler, R. R.; Johnson, gland by radioautography. Brain Res. 322:341-345; 1984. A. L.; Timmermans, P. B. M. W. M. Identification of angiotensin 14. Laitinen, J. T.; Torda, T.; Saavedra, J. M. Cholinergic stimulation II receptor subtypes. Biochem. Biophys. Res. Commun. 165:196of phosphoinositide hydrolysis in the rat pineal gland. Eur. J. Phar203; 1989. macol. 161:237-240; 1989. 5. Davies, B. N.; Withilngton, P. G. The responses of the isolated, 15. Nazarali, A. J.; Gutkind, J. S.; Saavedra, J. M. Calibration of[nsI]blood-perfused spleen of the dog to angiotensin, oxytocin and vapolymer standards with [t25I]-brain paste standards for use in quansopressin. Br. J. Pharmacol. 54:205-212; 1975. titative receptor autoradiography. J. Neurosci. Methods 30:247-253; 6. Davies, B. N.; Withrington, P. G. The actions of drugs on the smooth 1989. muscle of the capsule and blood vessels of the spleen. Pharmacol. 16. Nicoll, C. S. Physiological actions of prolactin. In: Greep, R. O.; Rev. 25:373-413; 1973. Astwood, E. B., eds. Handbook of physiology, sect. 7, vol. 4, pt. 2, 7. De l_6an, A.; Ong, H.; Gutkowska, J.; Schiller, P. W.; McNicoll, N. chap. 32. Washington, DC: American Physiological Society; 1974: Evidence for agonist-induced interaction ofangiotensin receptor with 253-292. a guanine nucleotide-binding protein in bovine adrenal zona glo17. Reilly, F. D. Innervation and vascular pharmacodynamics of the merulosa. Mol. Pharmacol. 26:498-508; 1984. mammalian spleen. Experientia 4 l: 187-192; 1985. 8. Dzau, V. J.; Pratt, R. E. Renin-angiotensin system: Biology, phys18. Shimada, K,; Yazaki, Y. Binding sites for angiotensin II in human iology, and pharmacology. In: Fozzard, H. A., ed. The heart and mononuclear leucocytes. J. Biochem. 84:1013-1015; 1978. cardiovascular system. New York: Raven Press; 1986:1631-1662. 19. Simon, M. R.; Engel, D. E.; Weinstock, J. V.; Roi, L. D. The effect 9. Felten, D. L.; Felten, S. Y.; Carlson, S. L.; Oscfiowka, J. A.; Livnat, of angiotensin II on human mononuclear cell reactivity: Suppression S. Noradrenergic and peptidergic innervation of lymphoid tissue. J. of PHA-P-induced thymidine incorporation. Immunol. Invest. 14: Immunol. 135:755S-765S; 1985. 389-400; 1985.
296 20. Simon, M. R.; Engel, D. E.; Weinstock, J. V. Angiotensin II suppression of human mononuclear cell reactivity is associated with enhanced OKT8 + lymphocyte tymidine incorporation. J. Immunopharmacol. 8:289-297; t986. 21. Strrmberg, C.; Tsutsumi, K.; Viswanathan, M.; Saavedra, J. M. Angiotensin 11 AT~ receptors in rat superior cervical ganglia: Characterization and stimulation of phosphoinositide hydrolysis. Eur. J. Pharmacol. Mol. Pharmacol. 208:331-336; 1991. 22. Thomas, D. W.; Hoffman, M. D. Identification of macrophage receptors for angiotensins: A potential role in antigen uptake for T lymphocyte responses? J. Immunol. 132:2807-2812; 1984. 23. Timmermans, P. B. M. W. M.; Wong, P. C.; Chiu, A. T.; Herblin, W. F. Nonpeptide angiotensin II receptor antagonists. Trends Pharmacol. Sci. 12:55-62; 1991. 24. Tsutsumi, K.; Saavedra, J. M. Increased dithiothreitol-insensitive, type 2 angiotensin II receptors in selected brain areas of young rats. Cell. Mol. Neurobiol. 11:295-299; 1991. 25. Tsutsumi, K.; Saavedra, J. M. Characterization and development of angiotensin II receptor subtypes (ATj and AT2) in rat brain. Am. J. Physiol. 261:R209-R216; 1991.
TSUTSUMI, S T R O M B E R G A N D SAAVEDRA 26. Tsutsumi, K.; Strrmberg, C.; Viswanathan, M.; Saavedra, J. M. Angiotensin II receptor subtypes in fetal tissues of the rat: Autoradiography, guanine nucleotide sensitivity and association with phosphoinositide hydrolysis. Endocrinology 129:1075-1082; 1991. 27. Tsutsumi, K.; Saavedra, J. M. Differential development of angiotensin II receptor subtypes in the rat brain. Endocrinology 128:630632; 1991. 28. Weinstock, J. V.; Kassab, J. T. Functional angiotensin II receptors on macrophages from isolated liver granulomas of murine schistosoma mansoni. J. Immunol. 132: 2598-2602; 1984. 29. Werner, G. H.; Floc'h, F.; Migliore-Samour, D.; Jolles, P. Immunomodulating peptides. Experientia 42:521-531; 1986. 30. Whitebread, S.; Mele, M.; Kamber, B.; de Gasparo, M. Preliminary biochemical characterization of two angiotensin II receptor subtypes. Biochem. Biophys. Res. Commun. 163:284-291; 1989. 31. Wright, G. B.; Alexander, R. W.; Ekstein, L. S.; Gimbrone, M. A., Jr. Sodium, divalent cations, and guanine nucleotides regulate the affinity of the rat mesenteric artery angiotensin II receptor. Circ. Res. 50:462-469; 1982.
