Peptides,Vol. 13, pp. 499-508, 1992

0196-9781/92 $5.00 + .00 Copyright© 1992 PergamonPressLtd.

Printed in the USA.

Molecular Characterization of Angiotensin II Type II Receptors in Rat Pheochromocytoma Cells

RANDY

M A R I A L. W E B B , *l E D D I E C.-K. LIU,* R O B E R T B. C O H E N , * A N D E R S H E D B E R G , t E L I Z A B E T H A. B O G O S I A N , * H O S S A I N M O N S H I Z A D E G A N , * C H R I S M O L L O Y , * S E R A F I N O , t S U Z A N N E M O R E L A N D , t T. J. M U R P H Y ~ A N D K E N N E T H E. J. D I C K I N S O N *

Departments of*Biochemistry and "pPharmacology, Bristol-Myers Squibb, Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543-4000 and ~cDepartment of Cardiology, Emory University, Atlanta, GA 30322 R e c e i v e d 23 D e c e m b e r 1991 WEBB, M. L., E. C.-K. LIU, R. B. COHEN, A. HEDBERG, E. A. BOGOSIAN, H. MONSHIZADEGAN, C. MOLLOY, R. SERAFINO, S. MORELAND, T. J. MURPHY AND K. E. J. DICKINSON. Molecularcharacterization ofangiotensin II type II receptors in rat pheochromocytoma cells. PEPTIDES 13(3) 499-508, 1992.--The binding sites and biochemical effects of angiotensin (A) II were investigated in rat pheochrnmocytoma (PC I2W) cells. Sareosine I, [12~I]-tyrosine4, isoleucinea-AI1([~25I]SI-AII) bound to a saturable population of sites on membranes with an equilibrium dissociation constant (Ka) of 0.4 nM and a binding site maximum of 254 fmol/mg protein. Competitive displacement of [~2~I]-SI-AIIby agonists and antagonists elucidated a rank order of potency ofAIII ~ AII> PD 123177 > AI > [des-Phe]AIl [All(l-7)] >> DuP 753. The stable guanine nucleotide analog 5'-guanylyl imidodiphosphate did not alter the binding affinity or slope of the inhibition curves for AI, AII, AIII, or AII( 17). Treatment of PCI2W cells with AII or AIII did not affect the free intracellular calcium concentration, phosphoinositide metabolism, arachidonate release, cyclic GMP, or cyclic AMP concentrations. [~25I]-AIIbinding sites remained on the cell surface and were not internalized after 2 h at 37°C. Angiotensin II did not stimulate tyrosine, serine, or threonine phosphorylation. Northern analysis of PCI2W mRNA with an AT~ receptor gene probe failed to produce an RNA:DNA hybrid at low stringency. These data indicate that PC 12W cells express a homogeneous population of AT2 binding sites which differ significantly from AT j receptors in signal transduction and molecular structure. AT2 sites may act via potentially novel, biochemical pathways or, alternatively, be vestigial receptors. Angiotensin II

AT2 sites

Signal transduction

Radioligand binding

TWO types of angiotensin (A) II receptors have been identified based on their pharmacological and biochemical properties. Type I (AT0 and type II (AT2) (6) receptors bind AII with high affinity but differentially bind the nonpeptidic antagonists DuP 753 and PD 123177. AT~ receptors bind DuP 753 with high affinity and PD 123177 with low affinity, while AT2 receptors bind DuP 753 with low affinity and PD 123177 with high affinity (11,12,33). ATt and AT2 receptors exhibit differential sensitivity to thiol reducing agents. Thus AT2 binding rites are resistant to reduction by dithiothreitol (DTT), while binding to AT~ receptors declines in the presence of DTT (11,19,37). The different pharmacological and biochemical properties of AT~ and AT2 receptors are also apparent in the physiological function of AII receptors. Whereas the ATI receptor mediates the AII-induced alterations in pressor responses, catecholamine and aldosterone secretion, and drinking behavior (38), and has recently been cloned (27,31), the function and structure of the AT2 binding site is unknown. I Requests for reprints should be addressed to Dr. Mafia L. Webb. 499

Elucidation of the role of AT2 binding rites is critical as part of an understanding of the actions of AII. Several observations with selective antagonists suggest that AII decreases the cyclic G M P levels in neural cells from the brain of l-day-old rats. The AII-induced decline in cyclic G M P was inhibited by PD 123177 but not by DuP 753 (34). This finding suggests that AT2 sites are associated with modulation of cyclic G M P levels. AT2 receptors have also been implicated in the [des-Phe]AII [AII(17)]-induced stimulation of prostaglandin release from human astrocytes (22,35). Recently, Swiss 3T3 fibroblasts (15) and ovarian granulosa (28) cells have been shown to express homogeneous populations of AT2 receptors. Although the effect of AII on numerous signal transduction pathways was thoroughly examined in these cells, the role of the AT2 receptor, if any, remains obscure. Additional cell lines and tissues, particularly of neural origin, that express AT2 receptors may aid in elucidation of the function of AII at these receptors.

500

WEBB ET AL.

PC12W cells, a substrain of the pheochromocytoma PC12 cell line, have been shown to contain AT2 receptors by virtue of enhanced binding affinity in the presence of sulfhydryl reducing agents, and low affinity binding of DuP 753 [ICso > 100 #M (33)]. However, the biochemical signal pathways and structure of the PC12W A T 2 receptor have not been investigated. The purpose of the present investigation was to further characterize angiotensin II binding sites in PC12W cells and examine the biochemical mechanism(s) and structure of ATE receptors in these cells. METHOD Sarcosine l, [125I]-tyrosine4, isoleucineS-AII ([12sI]-SI-AII) (2200 Ci/mmol), [12sI]-tyrosine4-AII ([125I]-AII)(2200 Ci/mmol), ['r32p]-ATP (3000 Ci/mmol), and [14C]-arachidonate (55 mCi/ mmol) were obtained from NEN Research Products (Boston, MA); [3H]-myoinositol (18-94 Ci/mmol) was purchased from Amersham (Arlington Heights, IL); angiotensin peptides were from Peninsula Labs (Belmont, CA); and cell culture reagents were from Gibco (Rockville, MD). DuP 753 and PD 123177 were prepared at Bristol-Myers Squibb. All other chemicals were obtained from Fisher, Sigma, or Mallinckrodt and were reagent grade. RNA size markers were from BRL (Bethesda, MD) and PCR primers from Genosys.

5% CO2. Cells were passaged once every 7 days after gentle physical dispersion and disaggregation. The growth medium was changed once every 3 days. The RASM cells between passages 3 and 24 were used in these experiments and, unless noted, were used in experiments in suspension, Radioligand Binding Assays

Membrane proteins (10-30 #g) were incubated with 0.2-0.4 nM [t25I]-SI-AII for 1 to 2 h at 25°C in 50 mM Tris (pH 7.4), 5 mM MgC12, 0.24 unit/ml aprotinin, 10 #g/ml 1,10-phenanthroline, 1 mM EDTA, and 1 mg/ml BSA. These conditions minimized the effect of metabolism (data not shown) in PCI2W cells. Saturation binding was conducted in the absence or presence of 1 #M angiotensin II, over increasing concentrations of radioligand. The receptor-ligand complex was filtered through Filtermat B ~ (Pharmacia LKB) pretreated with 0.3% polyethyleneimine in 50 mM Tris-HC1, pH 7.4, using a T o m t e c ~ multiwell cell harvester (Orange, CT). For competition analyses of AI, AII, and AIII, 50 el of [125I]-SI-AII (0.4 nM) and 50 #l of competing compound with or without 100 #M of the guanine nucleotide analogue 5'-guanylyl imidodiphosphate (GppNHp) was mixed with 100 #1 of PC 12W membranes and incubated as above. Saturation and competition binding data were analyzed according to Scatchard (32) and Cheng and Prusoff (10), respectively.

PC12 W Cell Culture and Membrane Preparation

Measurement o f Intracellular Calcium

PC12W cells were generously provided by Drs. Kwan Hee Kim and Robert Speth (Washington State University, Pullman, WA). PC12W cells are a substrain of PCI2 cells and grow as loosely attached monolayer cultures. Cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2.5% delipidated fetal calf serum (Cocalico Biologieals Inc., Reamstown, PA), 6.25 #g/ml transferrin, 6.25 ng/ml selenium, 1.25 mg/ml bovine serum albumin (BSA), and 5.35 #g/ml linoleic acid (Collaborative Research, Bedford, MA) in a humidified atmosphere containing 5% CO2 as previously described (33). Cells were passaged once every 7 days after gentle physical dispersion and dk~ap%~.'gation. The growth medium was changed once every 3 days. PC 12W cells between passages 3 and 24 were used in these experiments. Membranes were prepared at 4°C from near confluent cultures by harvesting cells into cold 10 mM HEPES (pH 7.4/22°C), 250 mM sucrose, and 1 mM EGTA. Cells were washed once and resuspended in 20 volumes of ice-cold homogenization buffer (50 mM Tris, pH 7.4, 10 mM MgCI2, 1 mM EGTA, 0.24 unit/ml aprotinin and 10 #g/ml of 1,10-phenanthroline). Cells were homogenized with a polytron with 3 bursts of 6 s at setting 7. The homogenate was filtered through two layers of cheesecloth and centrifuged at 40,000 × g for 20 rain. The membrane pellet was washed three times in homogenization buffer, and the final washed membrane pellet was brought up in homogenization buffer and used fresh for guanine nucleotide experiments or stored in aliquots at - 8 0 ° C until use. Aliquots were not used more than once to prevent alterations due to freezing and thawing. Protein concentrations were determined using the BCAa~ assay (Pierce, Rockford, IL).

Confluent PC12W cell monolayers in 150 cm 2 culture flasks were washed with calcium and magnesium-free phosphate-buffered saline (PBS) containing 2 mM EDTA and 2 mg/ml BSA and were incubated in 20 ml of fresh PBS for 2-3 min at 37°C in order to detach the cells. Cell suspensions were collected by gentle trituration through a wide-bore pipette and centrifuged (150 × g for 3 min) at room temperature. The cell pellet was resuspended in a HEPES-buffered saline solution (HBS, pH 7.4 at 37°C) containing l mg/ml BSA such that cell number of the suspension was 6 × l06 cells/ml. A 1 ml aliquot of the cell suspension was diluted with 2 ml of riBS and autofluorescence was determined in a SPEX spectrofluorometer. Excitation wavelengths were set at 340 and 380 nm, while emission was monitored at 505 nanometer (nm). Excitation slits were set at 1.0 millimeter (mm) and emission slits were set at 1.0 and 0.5 ram. The cell suspension was incubated with 2 #M fura-2 acetoxymethyl ester (Molecular Probes Inc., Eugene, OR) for 30 min at 37°C. The fura-2-1oaded cells were washed once by centrifugation, then resuspended in HBS at 370C and incubated for 30 min. The suspension was centrifuged and the cell pellet was suspended in fresh HBS at room temperature. Fluorescence experiments were carried out in the SPEX speetrofluorometer at 37°C in a quartz cuvette. The [Ca2÷]i was calculated using data analysis software developed by SPEX Industries, Inc., based on the following formula:

Rat Aortic Smooth Muscle Cells

Rat aortic smooth muscle cells (RASM cells) were generously provided by Dr. Marschall S. Runge (Emory University, Atlanta, GA). The RASM cells grow as a tightly attached monolayer culture. Cultures were obtained as primary cultures and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS in a humidified atmosphere containing

[Ca2+]i = Kd (R - Rmi,/Rm,x - R) (S:2/Sb2) where R is the ratio of the fluorescence of the sample at 340 nm and 380 nm; Rm~ and Rmin represent the ratios for fura-2 free acid at the same wavelength in the presence of saturating calcium and zero calcium, respectively; S n / S ~ is the ratio of ~ - 2 at 380 nm in zero and saturating calcium; and Ka is the dissociation constant of fura-2 for calcium, which was assumed to be 224 nM at 37°C (18). Measurement o f Phosphoinositide Turnover

PCI2W cells were cultured in 35-ram wells, and labeled to isotopic equilibrium with [3H]-myoinositol (2 ~Ci/mt) in inositol-

ANGIOTENSIN II RECEPTORS

501

free DMEM for 48 h. Cell monolayers (approximately 90% confluent) were washed and incubated for 15 rain at 37°C in medium containing 10 mM LiC1 in the absence or presence of AII antagonist. The cells were stimulated with agonist for 30 min, the media removed, and 2 mM EDTA at 100°C was added to the cell monolayer to disrupt cell integrity and release soluble inositol phosphates (IP). The cells and supernatant were removed, reboiled, and centrifuged. The supernatant was applied to a Dowex AG-1X8 anion exchange column, the labeled inositol, glycerophosphoinositol, and inositol 1-, 2-, and 3-phosphates fractionated essentially as described (3), and aliquots were counted in a liquid scintillation counter using Packard Ultima Gold XR scintillation fluid (Packard Inst. Co., Meriden, CT).

Measurement of [t4C]-Arachidonic Acid Metabolism PCI2W cells were grown to 90% confluence, washed with DMEM, and incubated for 3-4 h at 37°C with 1 ~Ci/ml [~4C]arachidonic acid in a total volume of 5 ml. Cells were washed with DMEM containing 0.1% fatty acid-free BSA, and incubated for 30 rain at 37°C in this medium containing the appropriate stimuli. Aliquots of the supernatant were removed, centrifuged at 14,000 × gfor 3 min to remove cells, 0.5 ml of the supernatants added to 5 ml of Optifluor scintillation fluid (Packard Inst. Co., Meriden, CT), and counted in a scintillation counter.

Determination of Cyclic GMP and AMP PC12W cells were obtained from washed monolayers and resuspended at 3-4 X 106 cells/ml in DMEM. The cell suspension was incubated for 30 min at 22°C with 15 ~M zaprinast (cyclic GMP), or 15/zM 3-isobutylmethylxanthine (cyclic AMP) to in-

hibit phosphodiesterases. The cell suspension (0.35 ml) was added to 1.5 ml microcentrifuge tubes, which contained the stimuli (atrial natriure~ic factor, sodium nitroprusside, or forskolin), and angiotensin peptides, in a total volume of 0.5 ml, In some cases the cells were preincubated for 15 min at 37°C with the AII peptides prior to the addition of stimuli. The incubation proceeded at 37°C for 30 rain, the cells were cooled in an ice bath, and 0.9 ml of ethanol added to disrupt cellular integrity. The mixture was centrifuged at 13,000 × g for 5 min; the supernatant was removed and dried in a speed vac drier. The dried pellet was reconstituted in 0.1 ml 50 mM sodium acetate and total cellular (intra- and extracellular) cyclic GMP and cyclic AMP was determined in aliquots (10/zl, cyclic GMP, 20 #1 cyclic AMP) using a cyclic GMP or cyclic AMP radioimmunoassay kit (New England Nuclear, Boston, MA). Samples were acetylated for determination of cyclic GMP. Cyclic nucleotide determinations were performed in triplicate samples of cells from at least three different passage numbers.

Determination of Cell-Surface [1251]-AIIBinding Cells were incubated with ['25I]-AII (0.2 nM) in a total volume of 0.5 ml for 2 or 3 h at 37°C or 4°C, respectively, centrifuged (3,000 X g, 3 min), and the supernatant removed. The cells were resuspended in low pH buffer (150 mM NaCl, 50 mM glycine, pH 3.0), or PBS, pH 7.4, for l0 rain at 4°C. The cells were recentrifuged (6000 × g, 3 min), washed twice in PBS at 4°C, and the cell associated radioactivity was determined by gamma spectroscopy.

Analysis of Cellular Protein Tyrosine Phosphorylation Cells were incubated in DMEM in the absence of serum for 48-72 h prior to treatment. The cells were then either left un-

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FIG. I. Saturation binding (A) and Scatehard transformation (B) of [I~I]-SI-AII to PCI2W cell membranes. Binding was conducted under equilibrium binding conditions (25°C, 2 h). Specific binding (solid circles) was calculated as the difference between total (squares) and nonspecific (open circles) binding and the data were analyzed by nonlinear (in panel A) or linear (in panel B) regression least square curve fitting. The data are representative of three separate experiments. Abbreviations: RL, radioligand ([I2~I]-SI-AII);B, bound; F, free.

502

WEBB ET AL. TABLE 1 PHARMACOLOGICALCHARACTERISTICSOF [~2~I]-SI-AIIBINDING SITES ON PCI2W CELL MEMBRANES AND EFFECT OF GppNHp ON THE BINDING OF AGONISTS Control

+ 100 uM GppNHp

Ligand

Kd(nM)

Slope

Kd(nM)

Slope

AI AII AIII AII~I_7) PD 123177 DuP 753

276 + 37 0.7 + 0.03 0.4 _+0.1 974 + 216" 29 + 13 69,673 + 14,000

1.0 + 0.02 0.9 + 0.03 0.9 _ 0.1 0.9 + 0.02* 0.9 _+0.1 1.8 + 0.5f

297 _+ 57 0.7 _+0.1 0.6 _+0.1 1148 + 242* ---

1.1 + 0.1 1.0 + 0.03 1.1 _+0.1 1.0 + 0.03* ---

Competition binding of [ t25I]-SI-AII (0.4 nM) to PC12W membranes with or without 100/~M of the guanine nucteotide analog 5'-guanylyl imidodiphosphate (GppNHp). PC I2W membranes, competing compound, and radioligand were incubated 2 h at 25°C in the absence or presence of GppNHp as described in the Method section. Data (means + SEM or SD*; n = 2*-6) were analyzed according to Cheng and Prusoff(10). f Denotes a significant difference from 1 (p < 0.05).

treated or stimulated with EGF (100 nedml) and/or All for 15 min. Cultures were rinsed twice in ice-cold PBS containing 1 mM Na3VO4, and lysed on ice in P-TYR lysis buffer (50 mM HEPES, pH 7.5, 1% Triton X-100, 50 mM NaCI, 50 mM NaF, 10 mM sodium pyrophosphate, 5 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, plus 10 mg/ml ofaprotinin and leupeptin). Lysates were then subjected to ultrasonic disruption for 10 s followed by centrifugation at 14,000 × g at 4°C for 10 min. Equal amounts of protein (100/~g) were resolved by SDS polyacrylamide electrophoresis and immunoblotting as described (14). For antiphosphotyrosine immunoblotting, a 1:1000 dilution of monoclonal antiphosphotyrosine antibody (Upstate Biotechnology Inc., Lake Placid, NY) was used. Immunoreactive bands were visualized using [t25I]-protein A followed by autoradiography or phosphoimage analysis (Molecular Dynamics, Sunnyvale, CA), and radioactivity quantitated by phosphoimage analysis.

In Vitro Kinase Assay Cells were harvested in cold PBS containing 1 mM Na3VO4, washed once, and the cell pellet lysed in 50 m M HEPES (pH 7.5 at 220C), 50 mM NaCI, 1% Triton X-100, 5 mM EDTA, 1 mM Na3VO4, 0.24 unit/ml aprotinin, and 10/~g/ml 1,10-phenanthroline. Lysates were clarified by centrifugation at 14,000 × g at 4°C for 15 rain. Proteins (100 ug) were incubated with 10 /~Ci [-y32p]-ATP and 100 uM ATP in 50 m M HEPES (pH 7.5 at 22°C), 60 mM KCI for 30 min at 300C. Proteins were electrophoresed on 7.5 or 10% denaturing gel according to Laemmli (23). Gels were dried and radioactivity quantitated by phosphoimage analysis.

Northern Analysis Polyadenylated RNA from PC 12W cells and rat kidney were prepared according to Maniatis (26). Poly(A+) RNA was electrophoresed through a 0.67% formaldehyde, 1% agarose gel, transferred to nylon falters (Hybond N, Amenham, Arlington Heights, IL), and prehybridized for 2 h at 42"C. Hybridization proceeded for 16 h at 420C with a random primed (T7-polymerase, [a-32p]-dCTP-labeled) 2.2 kb Hindlll to Notl fragment of the rat vascular smooth muscle AT1 receptor eDNA. The hybridization buffer was 50 m M Tris-HC1, 5 × Denhardt's solution (0.1% BSA, 0.1% Ficoll, 0.1% polyvinylpyrrolidone), 1 M NaCI, 50% deionized f o r m a m i ~ , 0.5% sodium dodeeyl sulfate (SDS), and 10/~g/ml denatured mlmon ~ DNA. Filters were washed in 3 M sodium chloride, 0.3 M citrate (SSC) buffer con-

taining 0.1% SDS for 1 h at 50°C and exposed to Kodak XAR film.

PCR Analysis Primers of 40 bp in length corresponding to the amino and carboxy termini of the AT~ receptor were used to amplify singlestranded eDNA made from PC I2W cell mRNA. Thirty to 60 cycles were performed at 94°C for 1 min, 37°C for 2 min, and 72°C for 2 min. The presence of PCR products was evaluated on 1% agarose gels stained with ethidium bromide. RESULTS

Binding of [m l]-SI-AH in PC12 W Membranes Equilibrium binding of [12sI]-SI-AII in PCI2W membranes was attained after 60 min at 25°C and was maintained for approximately 60 min thereafter (data not shown); Saturation binding of [J25I]-SI-AII in PC12W membranes was studied in the absence and presence of an excess of unlabeled AII, Specific binding was saturable (Bm~ = 254 + 29 fmol/mg) and represented 60-95% of the total binding over a range of radioligand concentrations (0. !-5 nM) (Fig. 1A). The apparent Kd for [t25I]SI-AII was 0.4 nM. Scatchard analyses of saturation binding isotherms were linear and hence consistent with the presence of a single class of receptor binding sites (Fig. 1B). Specific binding of [t25I]-SI-AII to PCI2W membranes was completely inhibited in a concentration-dependent manner by AII receptor agonists and antagonists. The rank order of potency was AIII (0.4 nM) >_ AII (0.7 nM) > PD 123177 (29 nM) > AI (276 nM) > AII(1-7) (974 nM) > > DuP 753 (69,673 riM) (Table 1). Slope factors of the competition curves did not differ significantly from unity with the exception of that for DuP 753 (1.8; p < 0.05), for which a similarly steep slope was previously reported (33).

Effect of GppNHp on [1251]-SI-AHBinding in PC12W Cell Membranes Guanine nucloatide binding proteins (G-proteins)regulate agonist binding at AT, txx~ptors in several tissues (24). Guanine nucleotide has been shown to decrease the affinity of A l l for A T~ receptors and increase the slope factor of the inhibition curve (13). To inv(mtigate the role of G-la'ot~.ns in attpatemin peptide binding to AT2 sites, a g t m i s t . ~ i n h k . ~ of [t2~I]SI-AII binding to PC12W membranes was examined for All-

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mation of IP- 1 was determined in cells preincubated with LiCI (10 mM). There was clear stimulation of IP-l by bradykinin (5.5-fold), whereas AII was unable to generate significant amounts of IP-l above basal levels (Fig. 3). A series ofAII peptides were tested for their ability to stimulate the release of [t4C]-arachidonate from PCI2W cells. Incubation of PCI2W cells with [~4C]-arachidonic acid resulted in rapid uptake, with 80-90% of the label associated with cells after 3 h at 37°C. Basal release of [14C]-arachidonate and [|4C]-labeled products into the medium was linear for 100 min. The ability of maximal concentrations (calculated from binding data) of AII (l #M), AIII (1 pM), or AII(l-7) (100 #M) to stimulate the release of [~4C]-arachidonic acid from PC 12W cells was examined following incubation for 30 min at 37°C. These peptides were unable to cause a significant stimulation of [|4C]-arachidonate release (Fig. 4). Lower concentrations of AII were also examined in view of reported sigmoidal concentration-response curves for nerve growth factor-stimulated [~4C]-arachidonate release in PCI2 cells (16). [m4C]-Arachidonate release stimulated by l, 10, and 100 nM AII was 93 + 14, 104 + 12, and 93 + 8 % (n = 24) of control values, respectively (data not shown). These values were not significantly different from control (p > 0.05). Bradykinin ( 1 #M) produced a small (29%) but significant stimulation of [~4C]-arachidonate release from PCI2W cells, and the Ca 2+ ionophore provoked a much larger [t4C]-arachidonate release (208% of control).

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"rime ( s e c ] FIG. 2. Effect of angiotensin II (AII) on intracellular calcium concentration ([Ca2+]i).PC 12W cells were treated with 10 nM bradykinin (BK), 1 #M arginine vasopressin (VSP), or 300 nM All. The All treatment was applied to unstimulated cells in the upper tracing and to VSP-treated cells during the maintenance phase of [Ca2+]idecline in the lower tracing. related peptides in the presence of 100 #M of the stable guanine nucleotide GppNHp. Inhibition of [~25I]-SI-AII binding by AI, All, AIII, or AII(1-7) was unchanged by the inclusion of GppNHp. Neither the inhibition constants nor slope factors of the competition curves were significantly altered (Table 1).

Effect of AH on lntracellular Calcium Concentration, Phosphoinositide Metabolism, and Arachidonate Release The effects of All on the concentration of intracellular calcium [Ca2+]i were examined in fura-2-1oaded PCI2W cells. Basal [Ca2+]i averaged 126 +_ 4 nM (n = 3) in unstimulated PCI2W cells. Calcium mobilization in PC 12W cells was stimulated in a dose-dependent fashion by the addition of bradykinin, with 10 nM, 100 nM, and 1 pM bradykinin increasing [Ca2+]i 90, 110, and 203%, respectively. These cells also responded to 1 #M vasopressin and 80 mM KCI with 60% and 100% increases in [Ca2+]i over basal levels, respectively. However, addition of 1 izM angiotensin II had no effect on [Ca2+]i (Fig. 2). The effect of AIII on [Ca2+]i was also examined, because AIII was a potent inhibitor of [12~I]-SI-AII binding (0.4 nM). However, 1 #M, 10 t~M, and 30 #M angiotensin III had no effect on [Ca2+]i levels (data not shown). PC12W cells were labeled with [3H]-myoinositol and stimulated for 30 min with All (1 #M) or bradykinin (1 uM). For-

Effect of AH on Cyclic GMP and AMP Total levels ofintra- and extracellular cGMP were measured in PC12W cells stimulated with All or AIII for 30 min in the presence of the cGMP phosphodiesterase inhibitor zaprinast (Table 2). Neither peptide (at 1 pM) altered basal cGMP levels. Atrial natriuretic peptide (ANP) produced a 15-fold increase in cGMP, and the stimulated cGMP levels were not significantly influenced by All or AIII. The soluble guanylate cyclase activator sodium nitroprusside stimulated total cGMP levels by 6-fold,

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FIG. 3. Effect of angiotensin II (All) on phosphoinositide hydrolysis. [3H]-Myoinositol-labeled PC12W cells were preincubated for 15 min with LiCI (10 raM) and stimulated with bradykinin (BK) (1 pM) or All (1 pM) for 30 rain at 37°C. Inositol phosphates (IP) were extracted and the IP-I fraction separated and quantitated. IP-I formation is expressed as the percent of basal IP-I. Results show the mean _+ SEM of three experiments. AII stimulated IP-I levels were not significantly different from basal IP-I levels (p > 0.05).

504

WEBB ET AL.

and this increase was unaffected by the presence of AII or AIII. Similar results were obtained for cells preincubated with angiotensin peptides for 15 min prior to addition of stimuli (data not shown). The AT2 binding site-selective ligand PD 123,177 had no effect on basal cGMP levels, or cGMP levels determined in the presence of stimuli + angiotensin peptides (data not shown). The effect of angiotensin II on PC12W cell total (intra- and extracellular) cyclic AMP levels is shown in Table 2. Angiotensin II (1 pM) had no significant effect on basal cyclic AMP levels. The adenylate cyclase activator forskolin (1 ~tM) stimulated cyclic AMP levels 2-fold, whereas its inactive analogue 1,9-dideoxyforskolin was without effect. Angiotensin II had no inhibitory effect on forskolin-stimulated cAMP levels. PDI23,177 did not alter basal cyclic AMP levels, or cyclic AMP levels stimulated by forskolin in the presence of All (data not shown). Similar results were obtained using AIII as the AT2 site stimulant (data not shown).

Internalization of [1251]-AII AT2 binding site-mediated internalization of [125I]-AII in PC 12W cells was compared to that occurring via the AT t receptor on rat aortic smooth muscle (RASM) cells. Incubation of PC12W cells with [t25I]-AII for 2 h at 37°C, or 3 h at 4°C, resulted in specific binding, which was totally extractable with an acid wash, indicative of cell surface binding at both temperatures (Fig. 5). By contrast, [t25I]-AII incubated with RASM cells at 37°C was uniformly distributed between cell surface and intracellular sites. When binding was conducted at 4°C, a temperature that inhibits internalization, all binding to RASM cell membranes was acid extractable. The presence of the lysomotropic agent NH4C1 (10 raM) markedly enhanced (86%) the binding of [12Sl]-AIl to RASM cells at 37°C, but produced no significant augmentation of binding to PCI2W cells.

Effect of AH on the Phosphorylation of Cellular Protein In order to determine if All could stimulate protein tyrosine phosphorylation in PC I2W cells, lysates from control and All-

TABLE 2 EFFECT OF ANGIOTENSINPEPTIDES ON BASALAND STIMULATED CYCLIC GMP AND CYCLIC AMP LEVELS Agent None (basal) All (1 pM) AIII (1 pM) ANP (1 pM) ANP + AII ANP + AIII Nitroprusside (10 pM) NP + All NP + AIII DDF ( 1 pM) Forskolin (I pM) Forskolin + All

CyclicGMP (fmol/10~cells)

CyclicAMP (fmol/106cells)

264 __+200 276 + 174 270 _+ 180 4,190 ___700 4,260 + 1,050 4,396 _+ 1,050 1,750 __+680 1,810 + 770 1,643 + 600

27.7 + 5.0 25.7 _+ 4.0

27.1 + 6.4 63.3 + 10.6 59.9 -+ 15.6

PCI2W cells were stimulated for 30 rain with the agents indicated and total extra- and intracellular cyclic GMP or cyclic AMP measured. Results show mean values _+SEM of experiments performed three times in triplicate. Abbreviations: ANP, atrial natrietic peptide; NP, sodium nitroprusside, DDF, 1,9-dideoxyforskolin.

stimulated cells were analyzed by immunoblotting with antiphosphotyrosine antibodies (Fig. 6A). In these experiments, cells were also challenged with epidermal growth factor (EGF) as a positive control, since binding of this hormone to specific surface receptors causes rapid autophosphorylation on tyrosine residues. Unstimulated PCI2W lysates contained several tyrosine phosphorylated proteins in the range of 30-300 kDa (Fig. 6A, lane 1). Angiotensin II stimulation of P C I 2 W cells did not induce any detectable increase or decrease in the overall pattern of protein tyrosine phosphorytation (Fig. 6A, lane 2). In contrast, EGF induced rapid tyrosine phosphorylation of the 175 kDa EGF receptor, as well as a few minor bands (Fig, 6A, lane 3). Treat-

200 A

..I

O E lZ

0

All (1-7) AIII All BK ~ I N

0

100 0

ql'

Q tu

Z

TREATMENT FIG. 4. Release of ['*C]-arachidonic acid and products from PC12W cells, PC12W ceils were labeled with [~(C]-arachidonic acid and stimulated with agonim: 100 ~M All(l-7), 1 ;(M AlII, 1 tiM All, I ~tM bradykinin (BK), and 1 pM ionomycin for 30 min at 37°C. The media was transferred to tubes, centrifuged to remove cells, and aliquots of the supernatant were used to determine total media [~4C]. Results are expressed as the percent change from the unstimulated control, and they represent the mean + SEM of three to nine experiments.

ANGIOTENSIN II RECEPTORS

505

1251]AII BINDING TO PC12-W CELLS

i 100 =13 ¢¢ 131 ¢¢ ~m

BINDINGAT 37°C

•

Total bound • Non specific • Acid stripped [ ] NH4Cl(10raM) t~,d ql~

o

BINDINGAT 4°C

f

1251]AII

~'~

BINDING

TO

RASM

[ ] Totalbound • Non specific [ ] Acid stripped

CELLS

200

it

m'U c

loo

,_o

FIG. 5. ['25I]-AIIbinding to PCI2W (upper panel) and RASM (lower panel) ceils. Cells were incubated with [t25I]-AII(0.2 nM) for 2 or 3 h at 37°C or 4°C, respectively. Cell-associated radioactivity was determined in cells incubated in PBS (pH 7.4) or acidified saline (pH 3.0) (acid stripped), and to cells incubated in the presence of NH4CI (l0 raM). Binding was normalized to percent of total binding values. Results show the mean _+SEM of three experiments. Asterisk denotes significant difference from nonspecific binding value (p < 0.05).

ment of cells with AII prior to stimulation with EGF did not affect the subsequent tyrosine phosphorylation of EGF receptors (Fig. 6A, lane 4). Taken together, these results suggest that the AT2 binding site in PCI2W cells is not coupled to intracellular pathways involving protein tyrosine kinases. The effects of AII on protein phosphorylation were also evaluated using in vitro kinase assays. Lysates prepared from PC12W cells incubated with AII for 30 min displayed the same pattern of phosphorylation as lysates from untreated cells (Fig. 6B). In addition, AII did not alter the pattern of EGF-stimulated phosphorylation, suggesting that AII does not stimulate the activity of a phosphatase involved in EGF signalling (data not shown).

Northern Analysis of PC12 W mRNA With the A I"1 Receptor eDNA To investigate the possibility of sequence similarity between AT2 and ATt nucleic acids, PC12W poly(A+) mRNA was examined by low stringency Northern hybridization analysis using an AT~ receptor eDNA probe. Messenger RNA from PCI2W cells did not hybridize to a 2.2 kb probe from the AT~ receptor eDNA. In the same experiment, rat kidney mRNA was run as

a positive control. Kidney mRNA showed strong hybridization signals at 2.2 and 3.5 kb with the AT~ probe (Fig. 7).

Amplification of AT1 eDNA in PC12W Cells Primers to the ATt receptor eDNA, which corresponded to the amino and carboxyl termini of the AT~ receptor, were used in PCR reactions to amplify primer-related sequences in PC12W cell eDNA. Thirty amplification cycles from several preparations of PC 12W mRNA and the corresponding eDNA failed to produce any products. In contrast, amplification of RASM eDNA resulted in a DNA product of I kb in length (data not shown), which corresponded to the size and sequence of the ATt receptor eDNA [data not shown, (27)]. DISCUSSION This investigation demonstrates that, in addition to the pharmacological differences between the ATt and AT2 binding sites, based predominantly upon binding of selective ligands (6,11,12), numerous biochemical and molecular properties also distinguish these proteins. While most functions of AII are mediated via the AT1 receptor and are antagonized by DuP 753, the biological

506

WEBB ET AL.

(A)

1

2

3

4

(B)

1

2

200--

200--

93--

97.4--.

69--

6 9 ¸- 46-46--

All

-

-t-

-

+

EGF

-

-

+

+

30--

FIG. 6. Effectof AII of on phosphorylation. (A) Tyrosine phosphorylation of cell proteins in response to AII and EGF. Antiphosphotyrosine immunoblot analysis of whole cell lysates from unstirnulated cells (lane 1) or cells treated for 15 rain with AII (1 pM, lane 2), EGF (100 ng/ml, lane 3), or All plus EGF (lane 4). Tyrosine-phosphorylated bands, including the activated EGF receptor, are indicated by arrows. Positions of molecular weight marker standards are shown on the left. (B) Effect of AIl as measured by in vitro ldnase assay. Lysates from control PCI2W cells (lane 1) or cells incubated with AII (1 #M) for 30 rain at 37"C (lane 2) were used to stimulate in vitro phosphorylation of lysate proteins. functions and biochemical events distal to All binding at the AT2 sites are unknown. A recent report showed that neural cells from brains of 1-day-old rats contain A'I"2receptors that respond to All with a decrease in cyclic GMP (34). The observed cyclic GMP response to All in the neudtes contrasts that observed in the ovarian granulosa cell (28). Thus it was of interest to investigate the biochemical effects of All and related peptides in the neuronal PC12W pheochromocytoma cell. The radioligand binding data are consistent with the presence of a homogeneous population of AII binding sites in PC 12W cells with a Bm~ of 254 fmol/mg protein, a Kd for [~25I]-SI-AII of 0.4 nM, and a rank order of potency (ICso) of AIII > A I I > PD 123177 > AI > AII(I-7) > > DuP 753. The original pharmacological characterization of AII binding sites in PCI2W cells by Speth and Kim (33) reported IC~o values for inhibition of [~25I]-SI-AII binding of 2.1 nM, 271 tzM, and 12.2 nM for AII, DuP 753, and para-aminophenylalanine-AII, an AT2-selective agonist, respectively, a B ~ of 326 fmol/mg protein, and a / G of 0.4 riM. Thus the data reported here confirm and extend the original de'~ription of AT2 sites in PC 12W cells. Studies with [t2q]-AH revealed similar pharmacology in saturation and competition analyses, indicating that these two radioligands bind at the same site (data not shown). In addition to PC12W cells, homogeneous populations of AT2 sites have been described in Swiss 3T3 fibroblasts (15) and ovarian granulosa cells (28). The AT2 site is also found in the rat adrenal (8,11), ovary (28), and

brain (29). These receptors exhibit pharmacological characteristics similar to those for the PC12W AT2 receptor. Interestingly, in all cases the affinity of AIII for the AT2 site is greater than that of AII for this site. Given the diversity of cell types in which AT2 sites are found, it seems likely that AT2 rites may mediate some yet undescribed actions of the angiotemin ~ . Most of the physiological effects of AII are mediated by high affinity surface receptors that are coupled via G-proteins to adenylyl cyclase or phospholipase C (PLC) (7,13). This is based on the ability of guanine nueleotides to modiflatc agonist binding to these receptors. Previous investigators have reported that the presence of nonhydrolyzable guanine nucleotides does not alter the binding affinity of AII to AT2 sites (4,15,28,33). Because AIII has at least equivalent binding affinity as AH and is known to have potent biological potency, it was of interest to eomlmre the effect of GppNHp on All and AIII binding to AT2 sites. The presence of GppNHp did not alter the affinity, or the slopes of the inhibition curves, of AI1 or AIII for AT2 site, in PCI2W membranes. Additionally, binding of AI or All(l-7) was unaffected by the presence of GppNHp. We have also been unable to show an effect of guanine nucleotides on the ~ ' o n rate of [12sI]-AIl (data not shown), which is a more ~millve of guanine nueleotide regulation. These findialll m l l e ~ that the PCI2W cell AT2 sites, like those in Swiss 3T3 ~ and ovarian granulosa cells, are not coupled directly t o a ~ i n . G-proteins other than Gq play a role in mediation o f the diverse

ANGIOTENSIN II RECEPTORS

M

PC 12W

507

K

kb 7.5-4.4-2.4--

FIG. 7. Northern hybridization analysis of polyadenylated RNA from PCi2W cells and rat kidney. The positions and sizes of the RNA markers (M) are shown on the left, PCI2W RNA in the center, and kidney RNA (K) on the right. effects of AII. In the rat liver, AT~ receptors are coupled via Gi to adenylate cyclase as well as via Gq to PLC. Both the AIIinduced increase in inositol phosphate metabolism and [Ca2+]i, and the decrease in adenyl cyclase are attenuated by DuP 753 (2). The physiologic consequences of the All-induced decrease in adenyl cyclase are presently unknown and may represent species diversity within the AT) receptors (20,30). Investigation of second messenger systems in PCI2W cells demonstrated that All did not increase [Ca2+]i nor did it affect phosphoinositide metabolism, arachidonate metabolite release, cyclic GMP or cyclic AMP concentrations. These data indicate that the AT2 receptor on PC12W cells is not coupled to pathways of Ca 2+, PLC, or PLA2 activation. This is in direct contrast to the mechanism of AT) receptor action where AII increases the concentration of intracellular calcium (1) and the turnover of inositol phospholipids in rat aortic smooth muscle (RASM) cells (5,17). We have observed similar effects in RASM cells (data not shown). Additionally, AII did not affect protein phosphorylation of PC12W proteins as evaluated by tyrosine phosphorylation and in vitro kinase activities, indicating that the AT2 receptor is distinct from membrane-spanning receptor kinases such as growth factor receptors. Consideration was given to the possibility that the AT2 sites functioned as clearance proteins similar to the ANP clearance receptors (25). The data presented here indicate that All binds exclusively to cell surface sites on PC 12W cells and that these AT2 sites are not internalized at 4°C or 37°C. By contrast, [125I]-

All binding to RASM ceils at 37°C consisted of cell surface bound and internalized radioligand. The lysosomotropic agent NH4CI increased [12q]-AII binding to RASM cells, whereas it produced no change in binding to PC 12W cells. These findings suggest that AII was internalized in RASM cells by pathways involving lysosomal compartments. Conversely, AT2 sites on PCI2W cells, as in Swiss 3T3 cells (15), did not mediate agonist internalization, suggesting that they do not function as clearance receptors. The recent cloning of the AT~ receptor cDNA (27,31) revealed that this AII receptor is a member of the large seven membrane spanning domain receptor family. Data presented here and elsewhere suggest that AT2 binding sites are not coupled to GTP binding proteins and, as such, may not belong to the seven membrane spanning domain receptor family. This suggestion is consistent with two lines of evidence. Northern analysis of PCI2W RNA with AT~ receptor cDNA under low stringency conditions failed to produce a hybrid, suggesting that AT2 RNA sequence varies significantly from the AT~ cDNA sequence or that it is present at very low levels. The latter possibility can probably be excluded, since amplification of PC 12W RNA with oligonucleotide primers to the AT~ receptor did not yield AT~ cDNA product. The lack of PCR products generated from the PC I2W amplification reaction suggests the absence of stable ATt mRNA in PCI2W cells. Additionally, specific binding to AT2 sites was unaffected by concentrations of DTT (data not shown), which dramatically reduced binding to AT, receptors (6,37). Structural diversity among a receptor family is not uncommon, as exemplified by the glutamate receptor family comprised of ionotropic, glutamate-gated cation channels (36), and metabotropic G-protein-coupled proteins (21). In summary, this investigation demonstrates that the high affinity binding sites for AII on undifferentiated PCI2W cells are AT2 sites. Occupancy of these sites with AII, or in some cases All-related peptides, did not affect free intracellular calcium concentration, phosphoinositide hydrolysis, arachidonate release, the pattern of cellular protein phosphorylation, or induce internalization of ligand. Moreover, PC I2W mRNA failed to hybridize with AT~ cDNA under low stringency conditions. The nature and role of these sites in differentiated PC12W cells is presently unclear. Collectively, these data indicate that the AT2 sites differ significantly from ATt receptors. The possibility remains that this high affinity AII receptor stimulates an as yet unidentified or novel signal transduction pathway or, alternatively, is vestigial in PC 12W cells. ACKNOWLEDGEMENTS The authors thank Drs. Bud Weller and Denis Ryono for synthesis of angiotensin II antagonists.

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