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Vascular Angiotensin II Receptor and Calcium Signaling in Toadfish
General and Comparative Endocrinology 115, 122–131 (1999) Article ID gcen.1999.7297, available online at http://www.idealibrary.com on
Vascular Angiotensin II Receptor and Calcium Signaling in Toadfish Ze-lian Qin,1 Hong-Q. Yan,2 and Hiroko Nishimura3 Department of Physiology and Biophysics, University of Tennessee, Memphis, Tennessee 38163 Accepted March 28, 1999
membrane fractions was displaced completely by [Asn1, Val5]ANG II and [Sar1, Ile8]ANG II. Losartan, but not PD 123319, partly displaced ANG II binding at 10ⴚ10–10ⴚ6 M. Furthermore, ANG II (10ⴚ7 or 10ⴚ8 M) caused a rapid, transient increase in the cytosolic Ca2ⴙ signal (fluorescence ratio (FR) of 340/380 nm) of isolated VSM tissues measured by fura-2 and a dual wavelength fluorospectrometer, whereas extracellular Kⴙ induced sustained, dose-dependent (P F 0.01) increases in FR. The results indicate that toadfish VSM tissues possess a rather nonselective ANG receptor; partial inhibition of ANG II binding by losartan and stimulation of cytosolic Ca2ⴙ signaling by ANG II suggest that the receptor has some resemblance to AT1 homologous receptors. r 1999 Academic Press Key Words: vascular angiotensin receptor; calcium signaling; toadfish; angiotensin binding; angiotensin antagonist; angiotensin receptor subtypes; losartan
The renin–angiotensin system evolved during the early evolution of vertebrates and regulates blood pressure/ blood volume homeostasis in nonmammalian and mammalian vertebrates. Properties of vascular angiotensin (ANG) receptors and signal pathways in primitive animals are, however, not well understood. We aimed to determine whether vascular ANG II receptors in the toadfish, Opsanus tau, an aglomerular teleost, pharmacologically resemble either the ANG subtype 1 receptor (AT1) or the subtype 2 receptor (AT2) by examining (i) the effects of selective ANG receptor antagonists on ANG II-induced vasopressor action and binding and (ii) ANG II’s effect on cytosolic Ca2ⴙ signaling. [Asn1, Val5]ANG II (native teleost ANG II) dose-dependently increased the mean arterial pressure of conscious toadfish. ANG IIinduced pressor responses (100–500 ng/kg) were inhibited substantially (79–83%) by [Sar1, Ile8]ANG II (5 g · kgⴚ1 ⴙ 5 g · kgⴚ1 · minⴚ1) and moderately (34–53%) by losartan (AT1 antagonist, 10 mg/kg ⴙ 20 mg · kgⴚ1 · hⴚ1) and by PD 123319 (AT2 antagonist, 10 mg/kg ⴙ 20 mg · kgⴚ1 · hⴚ1) (36–60%). Likewise, the [Asp1, Val5, His9]ANG I-induced pressor effect was completely eliminated by an ANG I-converting enzyme inhibitor, SQ 14,225. Specific 125I-ANG II binding to vascular smooth muscle (VSM)
The renin–angiotensin system has been identified in elasmobranchs (Takei et al., 1993), holocephalians and primitive bony fishes (Nishimura et al., 1973), and teleosts and tetrapods (Sokabe et al., 1969) and appears to play important roles in cardiovascular homeostasis and osmoregulation (Nishimura, 1987; Henderson et al., 1993; Kobayashi and Takei, 1996). The structure of the angiotensin (ANG) II molecule has been maintained throughout the vertebrate phylogeny with variations in the amino acid in positions 1 (asparagine or aspartic acid) and 5 (valine or isoleucine) (for review see Henderson et al., 1993; Nishimura and Walker, 1994). Dogfish ANG II is unique in that the position 3
1 Present address: Department of Plastic Surgery, The Third School of Clinical Medicine, Beijing Medical University, Beijing 100083, People’s Republic of China. 2 Present address: University of Pittsburgh, Safar Center for Resuscitation Research, Pittsburgh, PA. 3 To whom reprint requests should be addressed at Department of Physiology and Biophysics, University of Tennessee–Memphis, 894 Union Avenue, Memphis, TN 38163.
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0016-6480/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
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amino acid is proline instead of valine (Takei et al., 1993). Although the amino acid sequence of toadfish ANG I is not known, the native ANG I of another aglomerular teleost, Lophius litulon, is [Asn1, Val5, His9]ANG I (Hayashi et al., 1978), and toadfish ANG I binds to antibody raised against [Asp1, Ile5, His9]ANG I (Nishimura et al., 1977). ANG receptors are also widely found in nonmammalian and mammalian animals (Nishimura et al., 1997). The recent development of novel nonpeptide ANG receptor antagonists has led to the discovery in mammals of type 1 (AT1) and type 2 (AT2) receptor subtypes (Chiu et al., 1989; Dudley et al., 1990). The molecular and pharmacological properties of the ANG receptors that show 60–75% amino acid sequence homology have been identified in Xenopus laevis (Ji et al., 1993; Bergsma et al., 1993), turkeys (Murphy et al., 1993), and chickens (Kempf et al., 1996). The structure of an ANG receptor in the trout kidney has also been partially identified (Parkyn et al., 1997). Radioligand binding studies, however, suggest the presence of more than one receptor subtype in nonmammalian tissues (Marsigliante et al., 1994; Nishimura et al., 1994; Tierney et al., 1997). In the present study, we therefore aimed to determine whether vascular ANG receptors in an aglomerular teleost, the toadfish, Opsanus tau, resemble the AT1 or AT2 receptor subtypes by examining (i) the effects of selective antagonists on ANG II-induced vasopressor actions and specific ANG II binding and (ii) ANG II’s effect on cytosolic calcium signaling.
MATERIALS AND METHODS Fishes and Maintenance Adult toadfish, O. tau, of both sexes, weighing 300–400 g, were purchased from the Whitney Marine Laboratory, University of Florida (St. Augustine, FL), and shipped by air to Memphis, Tennessee. Toadfish were kept in temperature-regulated aquaria (15°) (Living Stream, 130 gal, Frigid Unit; Toledo, OH) containing 50% seawater (Instant Ocean, synthetic seasalts, Aquarium Systems, Mentor, OH). We used 50% seawater because (i) the toadfish were collected from a brackish-water bay where the salinity is lower than full-strength, and (ii) in laboratory aquaria, toadfish
eat better and remain more active in 50% than in full-strength seawater. Toadfish maintain similar plasma osmolality and Na levels in 50 and 100% seawater by hypo-osmoregulation (Lahlou et al., 1969). The seawater was continuously circulated and aerated through a charcoal filter. The seawater at full strength contained 450 mM Na and 550 mM Cl, and its osmolality was about 1 osmol/kg water. Fish were fed fresh clams and shrimp twice a week.
Drugs and Reagents [Asn1, Val5]ANG II (native teleost fish ANG II), [Sar1, Ile8]ANG II (nonselective ANG receptor antagonist), and [Asp1, Val5, His9]ANG I were purchased from Peptide International (Louisville, KY). The ANG stock solutions (10⫺3 M in 50 mM Tris–HCl buffer solution, pH 7.2) were kept at ⫺70°. Losartan (DuP 753, AT1 antagonist, DuPont Merck Pharmaceuticals, Wilmington, DE), PD 123319 (AT2 antagonist, Parke-Davis Pharmaceutical Research Division, Ann Arbor, MI), and SQ 14,225 (ANG-converting enzyme inhibitor, Captopril, E. R. Squibb and Sons, Princeton, NJ) were kind gifts from the respective companies. The following drugs and reagents were commercially obtained: Bay K 8644 and calcium ionophore (Br-A23187) from Calbiochem Corporation (10⫺3 M in DMSO, San Diego, CA); Fura-2 AM (acetoxymethyl ester, 1 mM in dry DMSO and Pluronic TM F-127, Molecular Probes, Inc., Eugene, OR); bovine serum albumin (BSA) from ICN Biomedicals (Costa Mesa, CA); and Trizma base (Tris), EGTA, 1,10-phenanthroline, and bacitracin from Sigma Chemical Co. (St. Louis, MO). Pluronic F-127 was dissolved (20%) in DMSO and stocked at room temperature. The toadfish Ringer solution contained (in mM) 114.0 NaCl, 10.0 sodium acetate, 5.0 KCl, 2.5 CaCl2, 0.5 MgCl2, 0.2 NaH2PO4, 0.8 Na2HPO4, 25.0 NaHCO3, 11.1 dextrose, and 0.11 ascorbic acid (pH 7.4, 315 mosmol/kg H2O). Minimum essential medium with Hanks’ balanced salt solution (MEM–Hank) was purchased from Gibco BRL, Life Technologies, Inc. (Grand Island, NY).
In Vivo Vasopressor Assay Three days prior to surgery, toadfish were adapted to a plastic chamber (13.5 liters) through which aerated and filtered 50% seawater (15°) was circulated by a ministaltic pump (Manostat, New York, NY) from a
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6-liter reservoir. Toadfish were anesthetized by immersion in a solution of 0.03% tricaine methanesulfonate (Finquel, Ayerst Laboratories; New York, NY) and 0.015% NaHCO3 for 15 min. Two clear vinyl expanded catheters (i.d. 0.45–0.75 mm, Dural Plastics & Engineering, Dural, Australia) were inserted (i) into a gastric or splenic branch of the celiac artery (the tip of the catheter floated in the celiac artery) for recording mean arterial pressure (BP) and agonist ANG II injections, and (ii) into the intestinal branch of the hepatomesenteric artery for ANG antagonist infusion/injection. The distal ends of catheters were exteriorized outside the chamber. Both celiac and hepatomesenteric arteries originate in the dorsal aorta, and there was no difference in BP measured in these two arteries. The experiment was performed after the fish had recovered from anesthesia and surgical stress (36–48 h). Mean dorsal aortic pressure was determined with a strain gauge pressure transducer (Statham P23 DC, Grass Instrument Co., Quincy, MA, or HewlettPackard 1280C, venous–arterial type, Andover, MA), using the heart’s position as a zero reference and recorded on a polygraph (Grass Model 7.8P–24.5 or Hewlett-Packard Model 7702B) (Nishimura et al., 1978, 1979). The vasopressor actions of ANG II and the effects of inhibitors on the vasopressor dose–response studies were determined as follows: [Asn1, Val5]ANG II (synthetic teleost ANG II) was injected into unanesthetized toadfish at doses of 10, 20, 50, 100, 200, and 500 ng/kg in a volume of 0.5 ml/kg. The return of BP to basal stable levels was ensured between injections. The prime (injection) and maintenance (infusion) doses of the vehicle (0.9% NaCl, 1 ml/kg ⫹ 7 ml · kg⫺1 · h⫺1), [Sar1, Ile8]ANG II (5 µg/kg ⫹ 300 µg · kg⫺1 · h⫺1), losartan (10 mg/kg ⫹ 10 to 20 mg · kg⫺1 · h⫺1), or PD 123319 (10 mg/kg ⫹ 10 to 20 mg · kg⫺1 · h⫺1) were administered; [Asn1, Val5]ANG II dose–response studies were repeated during antagonist infusion. Losartan often caused a transient decrease in BP (2–4 mm Hg), and an ANG dose–response study was conducted after the BP had returned toward the basal level. The same fish was used for different drugs after a 24to 48-h interval. The order of antagonists (or vehicle) was altered randomly. The effect of an ANG-converting enzyme inhibitor, SQ 14,225 (1 mg/kg), was examined on the last day since the effects of this drug usually remain more than 24 h. For this study, [Asp1, Val5, His9]ANG I (50–1000 ng/kg) was used as an agonist.
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Qin, Yan, and Nishimura
Measurement of Cytosolic Calcium Signaling The dorsal aorta was isolated under a dissecting microscope, excised, cut into 1-cm segments (usually two segments per fish), and cleared of surrounding adipose and connective tissues in MEM–Hank supplemented with an additional 10 mM NaCl to simulate the NaCl concentration of toadfish serum. Isolated dorsal aortae (final, 3–4 ⫻ 8 mm) were cut open and placed on a glass coverslip, filling the third quarter of space from the top (which includes the entire beam path), and were glued at the corners with a minimum amount of Super Glue (Super Glue Co., Hollis, NY). The aortic tissue specimens were equilibrated in toadfish Ringer solution (pH 7.4) at room temperature (⬃25°) for at least 30 min after being mounted on the coverslip and then incubated for 60 min at 25° with a fluorescent indicator, fura-2 AM (4 µM containing 0.02% Pluronic F-127). The isolated aortic segments mounted on glass coverslips were placed in a flowthrough fluorescent cuvette (approximately 1.8 ml) and superfused with prewarmed aerated toadfish Ringer solution by a peristaltic pump (3 ml/min); the cuvette was placed in a water-jacketed holder maintained at 25°. Autofluorescence was measured prior to fura-2 loading in each tissue, and excitation spectra before and after fura-2 loading were superimposed to confirm sufficient uptake of the fluorescent indicator (approximately two- to threefold increase at 340 nm fluorescence) by the aortic tissue. The excitation wavelength was alternated at 8-s intervals between 340 and 380 nm, while fluorescence at 510 nm was continuously recorded (dual-wavelength spectrofluorophotometer, Shimadzu, RF-5000) (Wang et al., 1992; Qin and Nishimura, 1998). After a stable baseline of fura-2 fluorescence ratio at 340/380 nm (referred to as FR) was obtained, the aortic smooth muscle (SM) tissue preparations were superfused first with Ringer solution for another 5 to 10 min to obtain control levels and then with Ringer containing a test drug. To ensure that the acetoxymethyl ester form of fura-2 was hydrolyzed in the cells by cellular enzymes to bind to cytosolic Ca2⫹, we released loaded fura-2 from the aortic tissue preparation by cell membrane lysis with digitonin (10–40 µg/ml). Digitonin increased fluorescence emission, whereas addition of EGTA (20 mM) to chelate Ca2⫹ decreased fluorescence and shifted the peak toward 380 nm, indicating that the changes in fluorescence are likely to reflect changes in cytosolic Ca2⫹ levels.
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We used two protocols: (i) The effects of [Asn1, Val5]ANG II were examined by superfusing the aortic segment with (in the following order): Ringer (for 10 min), [Asn1, Val5]ANG II (10⫺7 M) (12 min), Ringer (10 min), [Asn1, Val5]ANG II (10⫺7 M or 10⫺6 M) (12 min), Ringer (10 min), and calcium ionophore (Br-A23187, 10⫺6 M). (ii) Calcium signal responses to extracellular K⫹ concentration were tested by superfusing the tissues with Ringer (for 10 min), 10, 20, 30, and 50 mM K⫹-containing Ringer solutions, and 50 mM K⫹ and Bay K 8644 (10⫺6 M), each for 8 min in a cumulative fashion.
Vascular Membrane Fractions and Angiotensin II Binding Vascular smooth muscle (VSM) membrane fractions were prepared as reported previously (Takei et al., 1988; Nishimura et al., 1994). Briefly, ventral and dorsal aortae, brachial arteries, and the celiac artery were quickly excised and cleaned of adventitial connective tissues under a dissecting microscope in chilled phosphate-buffered saline (pH 7.4). Vascular tissues were then homogenized in 8 vol of chilled 50 mM Tris–HCl buffer containing 0.25 mM sucrose and 0.1 mM EDTA2Na (pH 7.2) with a Brinkman polytron (setting 8 for 10 s, four times) in an ice bath. The homogenate was centrifuged twice at 3000g for 15 min at 4°, and the supernatant was further centrifuged at 57,000g (4°) for 30 min. The obtained fraction contained crude cell membrane fractions devoid of mitochondria and lysosomal vesicles (Takei et al., 1988). The pellet was resuspended in 0.4 to 0.8 ml of 50 mM Tris–HCl buffer containing 10 mM MgCl2 (pH 7.2), and the protein concentration of the suspension was determined by the method of Lowry et al. (1951) using BSA as the standard. ANG II binding to the membrane fractions was examined in the same day. Radiolabeled ANG II binding to the membrane receptors was examined as reported (Takei et al., 1988; Nishimura et al., 1994). We examined the displacement of radiolabeled ANG II by ANG antagonists by incubating 125I-[Ile5]ANG II (Du Pont–New England Nuclear, Boston, MA) (0.5 nM) and the membrane fraction (50–80 µg) with 10⫺10 to 10⫺4 M of [Asn1, Val5]ANG II, [Sar1, Ile8]ANG II, losartan, or PD 123319 in assay buffer (50 mM Tris–HCl, 10 mM MgCl2, and 0.2% BSA, pH 7.2) containing 1 mM EGTA, 0.1 mM phenanthroline, and 0.01 mM bacitracin (metabolism inhibitors)
(total incubation mixture, 100 µl) at 12°C for 90 min. The incubation was terminated by adding 5 ml of ice-cold Tris–HCl buffer (50 mM, pH 7.2) to the test tubes and by filtering the mixture through a glass fiber filter (Schleicher and Schuell, No. 30, presoaked in 50 mM Tris–HCl containing 0.2% BSA) under negative pressure. The filters were rinsed twice with 5 ml of chilled Tris–HCl buffer and dried. The radioactivities trapped on the filters were counted in an automated well-type gamma counter (Packard Cobra, Packard, Downers Grove, IL). Total (triplicate) and nonspecific bindings (duplicate; with excess unlabeled ANG II) were measured to calculate specific binding.
Data Analysis All data were expressed as means ⫾ SE. In the vasopressor response study, the differences in dose– response curves before and after antagonist treatment were evaluated by a two-factor analysis of variance (ANOVA) followed by, where applicable, the Newman– Keuls multiple comparison with repeated measures. In the cytosolic Ca2⫹ signaling measurements, the autofluorescence at 340 and 380 nm was subtracted from the respective fura-2 fluorescence levels prior to the calculation of the fluorescence ratios (FRs) at 340/380 nm. ANOVA followed by the Newman–Keul’s multiple comparison test was used for statistical analysis of Ca signal responses.
RESULTS Effects of ANG Antagonists on ANG II-Induced Vasopressor Responses [Asn1, Val5]angiotensin (ANG) II significantly increased (P ⬍ 0.01) mean arterial pressure (BP) in conscious toadfish in a dose-related manner (Fig. 1). The ANG II-induced dose–pressor curve was not altered by vehicle (0.9% NaCl, Fig. 1A). [Sar1, Ile8]ANG II (Fig. 1B) showed a mild agonist effect and raised the basal BP by 7.3 ⫾ 1.5 mm Hg; it completely suppressed the ANG II-induced dose–pressor responses (P ⬍ 0.01, ANOVA). Losartan (AT1 antagonist, Fig. 1C) partly but significantly (P ⬍ 0.01, ANOVA) reduced the ANG II-induced vasopressor responses. The percentage inhibitions were larger at lower doses (20 ng/kg,
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FIG. 1. Effects of angiotensin II (ANG II) antagonists on [Asn1, Val5]ANG II-induced vasopressor responses in conscious toadfish. (A) Effects of vehicle (0.9% NaCl); (B) effects of [Sar1, Ile8]ANG II; (C) effects of Dup 753; (D) effects of PD123319. All test drugs were diluted in saline before using. Prior to treatments, ANG II caused significant dose-dependent increases in vasopressor actions (P ⬍ 0.01). The values between lines indicate significance levels (P) before and after antagonist treatment by ANOVA. *P ⬍ 0.05, **P ⬍ 0.01 before and after antagonist treatment by Newman–Keuls multiple comparison at each dose. NS, nonsignificant. Baseline blood pressure for each group: (A) 21.4 ⫾ 1.4; (B) 20.3 ⫾ 1.5; (C) 23.7 ⫾ 2.0; (D) 23.6 ⫾ 1.9.
80.2 ⫾ 9.7%; 50 ng/kg, 66.6 ⫾ 6.9%) than at higher doses (200 ng/kg, 40.7 ⫾ 9.4%; 500 ng/kg, 34.0 ⫾ 5.2%). PD 123319 (AT2 antagonist, Fig. 1D) partly suppressed ANG II-induced vasopressor responses (0.01 ⬍ P ⬍ 0.05, ANOVA). [Asp1, Val5, His9]ANG I also increased toadfish BP in a dose-related fashion (Figs. 2A and 2B). The vasopressor responses were, however, lower than those of ANG II and were completely eliminated by an ANGconverting enzyme inhibitor, SQ 14,225 (1 mg/kg, P ⬍ 0.01, ANOVA) (Fig. 2B). The mean body weight (g) of toadfish used for the vasopressor studies was 490 ⫾ 42 g (n ⫽ 8).
Effects of Extracellular Kⴙ, Bay K 8644, and ANG II on Cytosolic Calcium Signaling To see whether treatments that induce Ca2⫹ influx also increase the fura-2 fluorescence ratio in toadfish
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vascular smooth muscle tissues, we examined the effects of 10 to 50 mM K⫹ (which presumably stimulates the voltage-gated Ca2⫹ channel by membrane depolarization) on changes in the fura-2 fluorescence 340/380 nm ratio. K⫹ (20–50 mM) induced significant increases in fura-2 fluorescence ratios (Fig. 3). The addition of Bay K 8644, a voltage-gated calcium channel agonist, to 50 mM K⫹-containing Ringer solution further increased cytosolic Ca2⫹ signals (P ⬍ 0.01). [Asn1, Val5]ANG II (10⫺7 M) evoked a rapid and transient increase in Ca2⫹ signaling in toadfish vascular tissues (Fig. 4). The second ANG II application, however, caused a much smaller increase in Ca2⫹ signaling, whereas Ca2⫹ ionophore (10⫺6 M, Br-A23187) induced a large increase in Ca2⫹ signaling in the same vascular preparation (Figs. 4 and 5). The mean body weight of toadfish used for studies on Ca2⫹ signaling
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FIG. 2. (A) A representative recording of the effect of SQ 14,225 on [Asp1, Val5, His9]ANG I-induced pressor responses. (B) The dose–response study (mean ⫾ SE) done in conscious toadfish. ANG II-induced pressor effects were significantly reduced after SQ 14,225 (P ⬍ 0.01, ANOVA). *P ⬍ 0.05, **P ⬍ 0.01 before and after treatment with SQ 14,225 by Newman–Keuls multiple comparison at each dose. The basal blood pressure for the control was 19.6 ⫾ 3.1 mm Hg; after the SQ 14,225 study, it was 19.5 ⫾ 3.3 mm Hg.
for ANG II was 399 ⫾ 51 g (n ⫽ 11), and that for K⫹ and Bay K 8644 studies was 426 ⫾ 24 g (n ⫽ 9).
Effects of ANG Antagonists on Angiotensin II Binding to Vascular Membrane ANG II binding to VSM membrane fractions was dose-dependently displaced by unlabeled [Asn1, Val5]ANG II (ID50 ⫽ 1 ⫻ 10⫺9 M, pooled tissue from 3 fish) and by [Sar1, Ile8]ANG II (ID50 ⫽ 1.58 ⫻ 10⫺8 M, pooled tissues from three to four fish per experiment, repeated twice) with complete displacement at 10⫺6 M
(Fig. 6). Losartan partially (30–50%) displaced ANG II at 10⫺10–10⫺6 M) (pooled tissues from three to four fish per experiment, repeated twice), whereas no displacement was seen after PD 123319 at 10⫺10–10⫺6 M (pooled tissue from three fish per experiment, repeated twice). The very high doses of PD 123319 (10⫺5 and 10⫺4 M), however, substantially displaced [Asn1, Val5]ANG II (43–70%). Preliminary study indicates that specific 125I-[Ile5]ANG II binding to VSM membrane fractions increased with incubation time (data not shown). The mean body weight of toadfish used for the ANG binding study was 423 ⫾ 21 g (n ⫽ 28).
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FIG. 3. Increases in cytosolic Ca2⫹ signaling evoked in toadfish dorsal aortic smooth muscle tissues by increasing doses of K⫹ in the bath and K⫹ plus Bay K 8644, expressed by fura-2 fluorescence ratio at 340/380 nm. **P ⬍ 0.01 by Newman–Keuls multiple comparison from control. The baseline fluorescence ratio is 0.70 ⫾ 0.06 (n ⫽ 9).
FIG. 5. Summary (mean ⫾ SE, n ⫽ 11) of the effects of [Asn1, Val5]ANG II on cytosolic calcium signals. *P ⬍ 0.05, **P ⬍ 0.01 from pretreatment level. The basal fluorescence ratio is 0.95 ⫾ 0.01. Autofluorescence was subtracted before calculating the 340/380 ratio.
DISCUSSION
pounds, represented by DuP 753 (losartan) (Chiu et al., 1989); and those for AT2 are tetrahydroimidazopyridines, represented by PD 123177 and PD 123319 (Parke-Davis) (Dudley et al., 1990). A markedly modified ANG II analog, CGP 42112 A (Ciba-Geigy), is also
In mammals, both AT1 and AT2 receptors have seven-transmembrane domains and are coupled with G-protein (Chiu et al., 1994). The prototypical antagonists for the AT1 receptor are biphenylimidazole com-
Ca2⫹
FIG. 4. A representative recording of cytosolic response to [Asn1, Val5]ANG II (10⫺7 M) in VSM tissues of toadfish. The Ca2⫹ signal is expressed by the fluorescence ratio of 340/380 nm (emission, 510 nm). The second application of ANG II at a higher dose (10⫺6 M) did not induce a cytosolic Ca2⫹ signal, whereas the same tissue responded to bromo (Br)-A23287 (10⫺6 M).
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FIG. 6. Displacement of 125I-[Ile5]ANG II binding to toadfish vascular tissue membrane fractions with angiotensin antagonists. Each point in the displacement study is the mean of two binding experiments (pooled tissue from three to four fish and each assay in duplicate or triplicate). B and B0 are the specific (total minus nonspecific) binding in the presence and absence, respectively, of various doses of the test compound.
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AT2-selective (de Gasparo et al., 1994). Interestingly, all the major known biological effects of ANG II are mediated via the AT1 receptor by stimulation of the phosphatidylinositol hydrolysis/protein kinase C pathway or by reduction of cAMP (Chiu et al., 1994). Furthermore, ANG II promotes VSM cell growth by stimulating tyrosine kinases (Berk and Corson, 1997) via the AT1 receptor. In contrast, the AT2 receptor appears to play a role during development and cell proliferation in pathological conditions such as injuryinduced vascular neointima formation and atherosclerosis (Dzau et al., 1991; Nakajima et al., 1995). Signal transduction via protein tyrosine phosphatase coupled to guanylate cyclase inhibition has been suggested but not established (de Gasparo et al., 1994). The present study indicates that [Asn1, Val5]ANG II induced dose-dependent pressor actions that were inhibited completely by [Sar1, Ile8]ANG II, a peptide antagonist, and partially by losartan and PD 123319. The partial inhibition of the ANG II-induced vasopressor effect by both losartan and PD 123319 suggests that the toadfish vascular ANG II receptor has low selectivity and is not readily distinguishable as either the AT1 or the AT2 subtype. Likewise, the specific binding to labeled ANG II was displaced completely by [Asn1, Val5]ANG II and [Sar1, Ile8]ANG II and partially displaced by losartan, but not by PD 123319, except at very high doses (10⫺5 and 10⫺4 M). The partial displacement by losartan suggests that both losartan-sensitive and -insensitive binding sites are present in the toadfish vascular ANG receptor. The displacement of ANG binding by PD 123319 only at high doses may presumably be nonspecific, since in mammalian ANG receptors high doses of losartan bind to the AT2 receptor, whereas high doses of EXP 655 (PD 123177), pharmacologically very similar to PD 123319, bind to the AT1 receptor (Chiu et al., 1989). Likewise, high doses of losartan and PD 123319 displaced ANG II binding sites in fowl VSM receptors, which functionally differ from both AT1 and AT2 receptor subtypes (Nishimura et al., 1994; Walker et al., 1993). ANG II evoked a rapid increase in cytosolic Ca2⫹ signaling in VSM tissues of toadfish dorsal aortae, measured by the fluorescent indicator fura-2. It is not clear from the present study, however, whether the increase in cytosolic Ca signaling is due to stimulation of Ca2⫹ release from a cellular store such as the
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endoplasmic reticulum or to the stimulation of Ca influx. Taken together, the present findings suggest that the toadfish vascular ANG receptor is rather nonselective, although it appears to have some resemblance to the AT1-homologous receptor. The present study also indicates that, similar to mammalian VSM cells, toadfish vascular tissues possess a voltage-gated calcium channel that is activated by K⫹-induced membrane depolarization and a calcium channel agonist, Bay K 8644. Our previous studies indicate that toadfish have renin substrate, renin, converting enzyme, and ANGs I and II (Nishimura et al., 1977, 1979). Renin secretion in toadfish is markedly stimulated by hemorrhage and hypotensive drugs (Nishimura et al., 1979) and mediated by renal arterial baroreceptors via cytosolic Ca2⫹ in renin secretory cells (Nishimura and Madey, 1989) but not by stimulation of the adrenergic nervous system (Nakamura et al., 1992) or cAMP (Nishimura and Madey, 1989, and unpublished). Thus, toadfish possess the functioning renin–angiotensin cascade and ANG receptor, although they may differ biochemically from those of more advanced vertebrates. It has been shown that ANG receptors are present in the cardiovascular system (Nishimura et al., 1978; Olson et al., 1994), liver and intestines (Marsigliante et al., 1994), and kidney (Cobb and Brown 1993; 1994) of various teleosts. Cobb and Brown (1993, 1994) reported that renal glomeruli isolated from rainbow trout show a specific ANG II binding site that has considerable affinity to losartan. The Kd for [Asn1, Val5]ANG II is 0.38 nM, whereas the IC50 for losartan is approximately 12.5 nM (Cobb and Brown, 1993, 1994). ANG II binding was also weakly displaced by [Sar1, Ala8]ANG II but not by PD 123319. Furthermore, ANG II induced a transient rise in Ca2⫹ signaling (Cobb et al., 1999), and ANG II and ANG III induced antidiuresis in the perfused rainbow trout, perhaps via the same ANG receptor; losartan also decreased urine flow and glomerular filtration rate (Brown et al., 1997). Recently, Parkyn and co-workers (1997) constructed primers based on the conserved amino acid residues and partially sequenced the ANG receptor from rainbow trout liver genomic DNA using polymerase chain reaction (PCR). The deduced peptide sequence of the trout receptor shows 47% identity with the mammalian AT1 and 41% identity with the AT2 receptor
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(Parkyn et al., 1997). This agrees with our present finding that the toadfish vascular ANG receptor is rather nonselective. Myocardial ANG receptors (Ji et al., 1993; Bergsma et al., 1993) cloned from X. laevis have 60% amino acid identity and 65% nucleotide homology with the coding region of the mammalian AT1 receptor; the Xenopus receptor transfected in Xenopus oocytes mobilized intracellular Ca2⫹ (Ji et al., 1993) and produced a Ca2⫹-dependent Cl current (Bergsma et al., 1993). Furthermore, Murphy et al. (1993) and, more recently, Kempf and co-workers (1996) cloned ANG receptors in the turkey and chicken adrenal cortex, respectively, that show 75% amino acid identity to the AT1 receptor and mediate the phosphatidylinositol/Ca2⫹ pathway. The above Xenopus cardiac and avian adrenal receptors are coupled to G-protein and show seven-transmembrane structures. Therefore, from a phylogenetical point of view, it appears that ANG receptors that show some homology to the AT1 receptor evolved early in the vertebrate phylogeny. These nonmammalian ANG receptors (Ji et al., 1993; Bergsma et al., 1993; Murphy et al., 1993; Nishimura et al., 1994; Kempf et al., 1996) show high affinity to peptide antagonist but show no selectivity to losartan/PD 123319, although some species have affinity to CGP 41221A (AT2-selective). Pharmacological differences in nonmammalian ANG receptors, such as the absence of affinity to selective AT1/ AT2 antagonists, may suggest that the amino acid sequence important for selective antagonist binding may be different in nonmammalian AT1 homologous receptors (Sandberg, 1994). In nonmammalian tissues, however, one or more unidentified ANG receptors appear to exist. For example, the dogfish, Triakis scyllia, and Scyliorhinus canicula have high-affinity ANG receptors in the gills, rectal gland, and/or interrenal gland that bind to CGP 41221A and losartan with nearly equal potencies (Tierney et al., 1997; Hazon et al., 1997). Likewise, ANG receptors in fowl VSM membranes do not show affinity to either losartan or PD 123319 and do not stimulate cytosolic Ca2⫹ (Walker et al., 1993; Nishimura et al., 1994). Whether this unidentified receptor has some molecular similarity to the AT2 receptor subtype or belongs to an entirely new group of ANG receptors remains to be determined.
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Qin, Yan, and Nishimura
ACKNOWLEDGMENTS We thank Ms. Xiaoying He and Ms. Hongying Liu for their excellent technical assistance. This study was supported by National Heart, Lung, and Blood Institute Grant HL-52881 and American Heart Association Grant-in-Aid 92012400. A preliminary study was presented at the Experimental Biology 1995 meeting, Atlanta, GA.
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Vascular Smooth Muscle Cell Growth mechanisms of vascular renin-angiotensin system in myointimal hyperplasia. Hypertension 18(Suppl II), 100–105. Hayashi, T., Nakayama, T., Nakajima, T., and Sokabe, H. (1978). Comparative studies on angiotensins. V. Structure of angiotensin formed by the kidney of Japanese goosefish and its identification by Dansyl method. Chem. Pharm. Bull. 26, 215. Hazon, N., Cerra, M. C., Tierney, M. L., Tota, B., and Takei, Y. (1997). Elasmobranch renin angiotensin system and the angiotensin receptor. In ‘‘Advances in Comparative Endocrinology, Proceedings of the XIII International Congress of Comparative Endocrinology,’’ pp. 1307–1312. Yokohama, Japan. Henderson, I. W., Brown, J. A., and Balment, R. J. (1993). The renin–angiotensin system and volume homeostasis. In ‘‘New Insights in Vertebrate Kidney Function’’ (J. A. Brown, R. J. Balment, and J. C. Rankin, Eds.), Society for Experimental Biology Seminar Series 52, pp. 311–350. Cambridge Univ. Press, UK. Ji, H., Sandberg, K., Zhang, Y., and Catt, K. J. (1993). Molecular cloning, sequencing and functional expression of an amphibian angiotensin II receptor. Biochem. Biophys. Res. Commun. 194, 756– 762. Kempf, H., le Moullec, J.-M., Corvol, P., and Gasc, J.-M. (1996). Molecular cloning, expression and tissue distribution of a chicken angiotensin II receptor. FEBS Lett. 399, 198–202. Kobayashi, H., and Takei, Y. (1996). The renin angiotensin system, comparative aspects. Zoophysiology 35. Lahlou, B., Henderson, I. W., and Sawyer, W. H. (1969). Renal adaptations by Opsanus tau, a euryhaline aglomerular teleost, to dilute media. Am. J. Physiol. 216, 1266–1272. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Marsigliante, S., Verri, T., Barker, S., Jimenez, E., Vinson, G. P., and Storelli, C. (1994). Angiotensin II receptor subtypes in eel (Anguilla anguilla). J. Mol. Endocrinol. 12, 61–69. Murphy, T. J., Nakamura, Y., Takeuchi, K., and Alexander, W. (1993). Accelerated Communication: A cloned angiotensin receptor isoform from the turkey adrenal gland is pharmacologically distinct from mammalian angiotensin receptors. Mol. Pharmacol. 44, 1–7. Nakajima, M., Hutchinson, H. G., Fujinaga, M., Hayashida, W., Morishita, R., Zhang, L., Horiuchi, M., Pratt, R. E., and Dzau, V. J. (1995). The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: Gain-of-function study using gene transfer. Proc. Natl. Acad. Sci. USA 92, 10663–10667. Nakamura, Y., Madey, M. A., Nishimura, H., Quach, D., and Barajas, L. (1992). Lack of control of renin release by adrenergic nervous system in the aglomerular toadfish. Gen. Comp. Endocrinol. 88, 62–75. Nishimura, H. (1987). Role of the renin–angiotensin system in osmoregulation. In ‘‘Vertebrate Endocrinology: Fundamentals and Biomedical Implications’’ (H. Nishimura, Ed.), pp. 157–187. Academic Press, New York. Nishimura, H., Crofton, J. T., Norton, V. M., and Share, L. (1977). Angiotensin generation in teleost fish determined by radioimmunoassay and bioassay. Gen. Comp. Endocrinol. 32, 236–247.
131 Nishimura, H., Lunde, L. G., and Zucker, A. (1979). Renin response to hemorrhage and hypotension in the aglomerular toadfish Opsanus tau. Am. J. Physiol. 237, H105–H111. Nishimura, H., and Madey, M. A. (1989). Signals controlling renin release in aglomerular toadfish. Fish Endocrinol. 7, 323–329. Nishimura, N., Norton, V. M., and Bumpus, F. M. (1978). Lack of specific inhibition of angiotensin II in eels by angiotensin antagonists. Am. J. Physiol. 235, H95–H103. Nishimura, H., Ogawa, M., and Sawyer, W. H. (1973). Renin– angiotensin system in primitive bony fishes and a holocephalian. Am. J. Physiol. 224, 950–956. Nishimura, H., Qin, Z., and Shimada, T. (1997). Evolution of angiotensin receptors and signaling. In ‘‘Advances in Comparative Endocrinology, Proceedings of the XIII International Congress of Comparative Endocrinology,’’ pp. 1299–1305. Yokohama, Japan. Nishimura, H., and Walker, O. E. (1994). Angiotensin and vascular cell interaction in birds. In ‘‘Perspectives in Comparative Endocrinology’’ (K. G. Davey, R. E. Peter, and D. D. Tobe, Eds.), pp. 166–176. National Research Council of Canada, Ottawa. Nishimura, H., Walker, O., Patton, C. M., Madison, A. B., Chiu, A. T., and Keiser, J. (1994). Novel angiotensin receptor subtypes in fowl. Am. J. Physiol. 267, R1174–R1181. Olson, K. R., Chavez, A., Conklin, D. J., Cousins, K. L., Farrell, A. P., Ferlic, R., Keen, J. E., Kne, T., Kowalski, K. A., and Veldman, T. (1994). Localization of angiotensin II responses in the trout cardiovascular system. J. Exp. Biol. 194, 117–138. Parkyn, G., Aves, S. J., and Brown, J. A. (1997). Cloning and characterisation of the rainbow trout angiotensin receptor. In ‘‘Advances in Comparative Endocrinology, Proceedings of the XIII International Congress of Comparative Endocrinology,’’ pp. 1329– 1332. Yokohama, Japan. Qin, Z.-L., and Nishimura, H. (1998). Ca2⫹ signaling in fowl aortic smooth muscle increases during maturation but is impaired in neointimal plaques. J. Exp. Biol. 201, 1695–1705. Sandberg, K. (1994). Structural analysis and regulation of angiotensin II receptors. Trends Endocrinol. Metab. 5, 28–35. Sokabe, H., Ogawa, M., Oguri, M., and Nishimura, H. (1969). Evolution of the juxtaglomerular apparatus in the vertebrate kidneys. Texas Rept. Biol. Med. 27, 867–885. Takei, Y., Hasegawa, Y., Watanabe, Y. X., Nakajima, K., and Hazon, N. (1993). A novel angiotensin I isolated from an elasmobranch fish. J. Endocrinol. 139, 281–285. Takei, Y., Stallone, J. N., Nishimura, H., and Campanile, P. (1988). Angiotensin II receptors in the fowl aorta. Gen. Comp. Endocrinol. 69, 205–216. Tierney, M., Takei, Y., and Hazon, N. (1997). The presence of angiotensin II receptors in elasmobranchs. Gen. Comp. Endocrinol. 105, 9–17. Walker, O. E., Nishimura, H., Taylor, A., and Lester, B. (1993). Novel angiotensin receptor subtypes in fowl. FASEB J. 7, A406. Wang, W., Hayama, N., Robinson, T. V., Kramer, R. E., and Schneider, E. G. (1992). Effect of osmolality on cytosolic free calcium and aldosterone secretion. Am. J. Physiol. 262, E68–E75.
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Vascular Angiotensin II Receptor and Calcium Signaling in Toadfish Ze-lian Qin,1 Hong-Q. Yan,2 and Hiroko Nishimura3 Department of Physiology and Biophysics, University of Tennessee, Memphis, Tennessee 38163 Accepted March 28, 1999
membrane fractions was displaced completely by [Asn1, Val5]ANG II and [Sar1, Ile8]ANG II. Losartan, but not PD 123319, partly displaced ANG II binding at 10ⴚ10–10ⴚ6 M. Furthermore, ANG II (10ⴚ7 or 10ⴚ8 M) caused a rapid, transient increase in the cytosolic Ca2ⴙ signal (fluorescence ratio (FR) of 340/380 nm) of isolated VSM tissues measured by fura-2 and a dual wavelength fluorospectrometer, whereas extracellular Kⴙ induced sustained, dose-dependent (P F 0.01) increases in FR. The results indicate that toadfish VSM tissues possess a rather nonselective ANG receptor; partial inhibition of ANG II binding by losartan and stimulation of cytosolic Ca2ⴙ signaling by ANG II suggest that the receptor has some resemblance to AT1 homologous receptors. r 1999 Academic Press Key Words: vascular angiotensin receptor; calcium signaling; toadfish; angiotensin binding; angiotensin antagonist; angiotensin receptor subtypes; losartan
The renin–angiotensin system evolved during the early evolution of vertebrates and regulates blood pressure/ blood volume homeostasis in nonmammalian and mammalian vertebrates. Properties of vascular angiotensin (ANG) receptors and signal pathways in primitive animals are, however, not well understood. We aimed to determine whether vascular ANG II receptors in the toadfish, Opsanus tau, an aglomerular teleost, pharmacologically resemble either the ANG subtype 1 receptor (AT1) or the subtype 2 receptor (AT2) by examining (i) the effects of selective ANG receptor antagonists on ANG II-induced vasopressor action and binding and (ii) ANG II’s effect on cytosolic Ca2ⴙ signaling. [Asn1, Val5]ANG II (native teleost ANG II) dose-dependently increased the mean arterial pressure of conscious toadfish. ANG IIinduced pressor responses (100–500 ng/kg) were inhibited substantially (79–83%) by [Sar1, Ile8]ANG II (5 g · kgⴚ1 ⴙ 5 g · kgⴚ1 · minⴚ1) and moderately (34–53%) by losartan (AT1 antagonist, 10 mg/kg ⴙ 20 mg · kgⴚ1 · hⴚ1) and by PD 123319 (AT2 antagonist, 10 mg/kg ⴙ 20 mg · kgⴚ1 · hⴚ1) (36–60%). Likewise, the [Asp1, Val5, His9]ANG I-induced pressor effect was completely eliminated by an ANG I-converting enzyme inhibitor, SQ 14,225. Specific 125I-ANG II binding to vascular smooth muscle (VSM)
The renin–angiotensin system has been identified in elasmobranchs (Takei et al., 1993), holocephalians and primitive bony fishes (Nishimura et al., 1973), and teleosts and tetrapods (Sokabe et al., 1969) and appears to play important roles in cardiovascular homeostasis and osmoregulation (Nishimura, 1987; Henderson et al., 1993; Kobayashi and Takei, 1996). The structure of the angiotensin (ANG) II molecule has been maintained throughout the vertebrate phylogeny with variations in the amino acid in positions 1 (asparagine or aspartic acid) and 5 (valine or isoleucine) (for review see Henderson et al., 1993; Nishimura and Walker, 1994). Dogfish ANG II is unique in that the position 3
1 Present address: Department of Plastic Surgery, The Third School of Clinical Medicine, Beijing Medical University, Beijing 100083, People’s Republic of China. 2 Present address: University of Pittsburgh, Safar Center for Resuscitation Research, Pittsburgh, PA. 3 To whom reprint requests should be addressed at Department of Physiology and Biophysics, University of Tennessee–Memphis, 894 Union Avenue, Memphis, TN 38163.
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amino acid is proline instead of valine (Takei et al., 1993). Although the amino acid sequence of toadfish ANG I is not known, the native ANG I of another aglomerular teleost, Lophius litulon, is [Asn1, Val5, His9]ANG I (Hayashi et al., 1978), and toadfish ANG I binds to antibody raised against [Asp1, Ile5, His9]ANG I (Nishimura et al., 1977). ANG receptors are also widely found in nonmammalian and mammalian animals (Nishimura et al., 1997). The recent development of novel nonpeptide ANG receptor antagonists has led to the discovery in mammals of type 1 (AT1) and type 2 (AT2) receptor subtypes (Chiu et al., 1989; Dudley et al., 1990). The molecular and pharmacological properties of the ANG receptors that show 60–75% amino acid sequence homology have been identified in Xenopus laevis (Ji et al., 1993; Bergsma et al., 1993), turkeys (Murphy et al., 1993), and chickens (Kempf et al., 1996). The structure of an ANG receptor in the trout kidney has also been partially identified (Parkyn et al., 1997). Radioligand binding studies, however, suggest the presence of more than one receptor subtype in nonmammalian tissues (Marsigliante et al., 1994; Nishimura et al., 1994; Tierney et al., 1997). In the present study, we therefore aimed to determine whether vascular ANG receptors in an aglomerular teleost, the toadfish, Opsanus tau, resemble the AT1 or AT2 receptor subtypes by examining (i) the effects of selective antagonists on ANG II-induced vasopressor actions and specific ANG II binding and (ii) ANG II’s effect on cytosolic calcium signaling.
MATERIALS AND METHODS Fishes and Maintenance Adult toadfish, O. tau, of both sexes, weighing 300–400 g, were purchased from the Whitney Marine Laboratory, University of Florida (St. Augustine, FL), and shipped by air to Memphis, Tennessee. Toadfish were kept in temperature-regulated aquaria (15°) (Living Stream, 130 gal, Frigid Unit; Toledo, OH) containing 50% seawater (Instant Ocean, synthetic seasalts, Aquarium Systems, Mentor, OH). We used 50% seawater because (i) the toadfish were collected from a brackish-water bay where the salinity is lower than full-strength, and (ii) in laboratory aquaria, toadfish
eat better and remain more active in 50% than in full-strength seawater. Toadfish maintain similar plasma osmolality and Na levels in 50 and 100% seawater by hypo-osmoregulation (Lahlou et al., 1969). The seawater was continuously circulated and aerated through a charcoal filter. The seawater at full strength contained 450 mM Na and 550 mM Cl, and its osmolality was about 1 osmol/kg water. Fish were fed fresh clams and shrimp twice a week.
Drugs and Reagents [Asn1, Val5]ANG II (native teleost fish ANG II), [Sar1, Ile8]ANG II (nonselective ANG receptor antagonist), and [Asp1, Val5, His9]ANG I were purchased from Peptide International (Louisville, KY). The ANG stock solutions (10⫺3 M in 50 mM Tris–HCl buffer solution, pH 7.2) were kept at ⫺70°. Losartan (DuP 753, AT1 antagonist, DuPont Merck Pharmaceuticals, Wilmington, DE), PD 123319 (AT2 antagonist, Parke-Davis Pharmaceutical Research Division, Ann Arbor, MI), and SQ 14,225 (ANG-converting enzyme inhibitor, Captopril, E. R. Squibb and Sons, Princeton, NJ) were kind gifts from the respective companies. The following drugs and reagents were commercially obtained: Bay K 8644 and calcium ionophore (Br-A23187) from Calbiochem Corporation (10⫺3 M in DMSO, San Diego, CA); Fura-2 AM (acetoxymethyl ester, 1 mM in dry DMSO and Pluronic TM F-127, Molecular Probes, Inc., Eugene, OR); bovine serum albumin (BSA) from ICN Biomedicals (Costa Mesa, CA); and Trizma base (Tris), EGTA, 1,10-phenanthroline, and bacitracin from Sigma Chemical Co. (St. Louis, MO). Pluronic F-127 was dissolved (20%) in DMSO and stocked at room temperature. The toadfish Ringer solution contained (in mM) 114.0 NaCl, 10.0 sodium acetate, 5.0 KCl, 2.5 CaCl2, 0.5 MgCl2, 0.2 NaH2PO4, 0.8 Na2HPO4, 25.0 NaHCO3, 11.1 dextrose, and 0.11 ascorbic acid (pH 7.4, 315 mosmol/kg H2O). Minimum essential medium with Hanks’ balanced salt solution (MEM–Hank) was purchased from Gibco BRL, Life Technologies, Inc. (Grand Island, NY).
In Vivo Vasopressor Assay Three days prior to surgery, toadfish were adapted to a plastic chamber (13.5 liters) through which aerated and filtered 50% seawater (15°) was circulated by a ministaltic pump (Manostat, New York, NY) from a
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6-liter reservoir. Toadfish were anesthetized by immersion in a solution of 0.03% tricaine methanesulfonate (Finquel, Ayerst Laboratories; New York, NY) and 0.015% NaHCO3 for 15 min. Two clear vinyl expanded catheters (i.d. 0.45–0.75 mm, Dural Plastics & Engineering, Dural, Australia) were inserted (i) into a gastric or splenic branch of the celiac artery (the tip of the catheter floated in the celiac artery) for recording mean arterial pressure (BP) and agonist ANG II injections, and (ii) into the intestinal branch of the hepatomesenteric artery for ANG antagonist infusion/injection. The distal ends of catheters were exteriorized outside the chamber. Both celiac and hepatomesenteric arteries originate in the dorsal aorta, and there was no difference in BP measured in these two arteries. The experiment was performed after the fish had recovered from anesthesia and surgical stress (36–48 h). Mean dorsal aortic pressure was determined with a strain gauge pressure transducer (Statham P23 DC, Grass Instrument Co., Quincy, MA, or HewlettPackard 1280C, venous–arterial type, Andover, MA), using the heart’s position as a zero reference and recorded on a polygraph (Grass Model 7.8P–24.5 or Hewlett-Packard Model 7702B) (Nishimura et al., 1978, 1979). The vasopressor actions of ANG II and the effects of inhibitors on the vasopressor dose–response studies were determined as follows: [Asn1, Val5]ANG II (synthetic teleost ANG II) was injected into unanesthetized toadfish at doses of 10, 20, 50, 100, 200, and 500 ng/kg in a volume of 0.5 ml/kg. The return of BP to basal stable levels was ensured between injections. The prime (injection) and maintenance (infusion) doses of the vehicle (0.9% NaCl, 1 ml/kg ⫹ 7 ml · kg⫺1 · h⫺1), [Sar1, Ile8]ANG II (5 µg/kg ⫹ 300 µg · kg⫺1 · h⫺1), losartan (10 mg/kg ⫹ 10 to 20 mg · kg⫺1 · h⫺1), or PD 123319 (10 mg/kg ⫹ 10 to 20 mg · kg⫺1 · h⫺1) were administered; [Asn1, Val5]ANG II dose–response studies were repeated during antagonist infusion. Losartan often caused a transient decrease in BP (2–4 mm Hg), and an ANG dose–response study was conducted after the BP had returned toward the basal level. The same fish was used for different drugs after a 24to 48-h interval. The order of antagonists (or vehicle) was altered randomly. The effect of an ANG-converting enzyme inhibitor, SQ 14,225 (1 mg/kg), was examined on the last day since the effects of this drug usually remain more than 24 h. For this study, [Asp1, Val5, His9]ANG I (50–1000 ng/kg) was used as an agonist.
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Qin, Yan, and Nishimura
Measurement of Cytosolic Calcium Signaling The dorsal aorta was isolated under a dissecting microscope, excised, cut into 1-cm segments (usually two segments per fish), and cleared of surrounding adipose and connective tissues in MEM–Hank supplemented with an additional 10 mM NaCl to simulate the NaCl concentration of toadfish serum. Isolated dorsal aortae (final, 3–4 ⫻ 8 mm) were cut open and placed on a glass coverslip, filling the third quarter of space from the top (which includes the entire beam path), and were glued at the corners with a minimum amount of Super Glue (Super Glue Co., Hollis, NY). The aortic tissue specimens were equilibrated in toadfish Ringer solution (pH 7.4) at room temperature (⬃25°) for at least 30 min after being mounted on the coverslip and then incubated for 60 min at 25° with a fluorescent indicator, fura-2 AM (4 µM containing 0.02% Pluronic F-127). The isolated aortic segments mounted on glass coverslips were placed in a flowthrough fluorescent cuvette (approximately 1.8 ml) and superfused with prewarmed aerated toadfish Ringer solution by a peristaltic pump (3 ml/min); the cuvette was placed in a water-jacketed holder maintained at 25°. Autofluorescence was measured prior to fura-2 loading in each tissue, and excitation spectra before and after fura-2 loading were superimposed to confirm sufficient uptake of the fluorescent indicator (approximately two- to threefold increase at 340 nm fluorescence) by the aortic tissue. The excitation wavelength was alternated at 8-s intervals between 340 and 380 nm, while fluorescence at 510 nm was continuously recorded (dual-wavelength spectrofluorophotometer, Shimadzu, RF-5000) (Wang et al., 1992; Qin and Nishimura, 1998). After a stable baseline of fura-2 fluorescence ratio at 340/380 nm (referred to as FR) was obtained, the aortic smooth muscle (SM) tissue preparations were superfused first with Ringer solution for another 5 to 10 min to obtain control levels and then with Ringer containing a test drug. To ensure that the acetoxymethyl ester form of fura-2 was hydrolyzed in the cells by cellular enzymes to bind to cytosolic Ca2⫹, we released loaded fura-2 from the aortic tissue preparation by cell membrane lysis with digitonin (10–40 µg/ml). Digitonin increased fluorescence emission, whereas addition of EGTA (20 mM) to chelate Ca2⫹ decreased fluorescence and shifted the peak toward 380 nm, indicating that the changes in fluorescence are likely to reflect changes in cytosolic Ca2⫹ levels.
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We used two protocols: (i) The effects of [Asn1, Val5]ANG II were examined by superfusing the aortic segment with (in the following order): Ringer (for 10 min), [Asn1, Val5]ANG II (10⫺7 M) (12 min), Ringer (10 min), [Asn1, Val5]ANG II (10⫺7 M or 10⫺6 M) (12 min), Ringer (10 min), and calcium ionophore (Br-A23187, 10⫺6 M). (ii) Calcium signal responses to extracellular K⫹ concentration were tested by superfusing the tissues with Ringer (for 10 min), 10, 20, 30, and 50 mM K⫹-containing Ringer solutions, and 50 mM K⫹ and Bay K 8644 (10⫺6 M), each for 8 min in a cumulative fashion.
Vascular Membrane Fractions and Angiotensin II Binding Vascular smooth muscle (VSM) membrane fractions were prepared as reported previously (Takei et al., 1988; Nishimura et al., 1994). Briefly, ventral and dorsal aortae, brachial arteries, and the celiac artery were quickly excised and cleaned of adventitial connective tissues under a dissecting microscope in chilled phosphate-buffered saline (pH 7.4). Vascular tissues were then homogenized in 8 vol of chilled 50 mM Tris–HCl buffer containing 0.25 mM sucrose and 0.1 mM EDTA2Na (pH 7.2) with a Brinkman polytron (setting 8 for 10 s, four times) in an ice bath. The homogenate was centrifuged twice at 3000g for 15 min at 4°, and the supernatant was further centrifuged at 57,000g (4°) for 30 min. The obtained fraction contained crude cell membrane fractions devoid of mitochondria and lysosomal vesicles (Takei et al., 1988). The pellet was resuspended in 0.4 to 0.8 ml of 50 mM Tris–HCl buffer containing 10 mM MgCl2 (pH 7.2), and the protein concentration of the suspension was determined by the method of Lowry et al. (1951) using BSA as the standard. ANG II binding to the membrane fractions was examined in the same day. Radiolabeled ANG II binding to the membrane receptors was examined as reported (Takei et al., 1988; Nishimura et al., 1994). We examined the displacement of radiolabeled ANG II by ANG antagonists by incubating 125I-[Ile5]ANG II (Du Pont–New England Nuclear, Boston, MA) (0.5 nM) and the membrane fraction (50–80 µg) with 10⫺10 to 10⫺4 M of [Asn1, Val5]ANG II, [Sar1, Ile8]ANG II, losartan, or PD 123319 in assay buffer (50 mM Tris–HCl, 10 mM MgCl2, and 0.2% BSA, pH 7.2) containing 1 mM EGTA, 0.1 mM phenanthroline, and 0.01 mM bacitracin (metabolism inhibitors)
(total incubation mixture, 100 µl) at 12°C for 90 min. The incubation was terminated by adding 5 ml of ice-cold Tris–HCl buffer (50 mM, pH 7.2) to the test tubes and by filtering the mixture through a glass fiber filter (Schleicher and Schuell, No. 30, presoaked in 50 mM Tris–HCl containing 0.2% BSA) under negative pressure. The filters were rinsed twice with 5 ml of chilled Tris–HCl buffer and dried. The radioactivities trapped on the filters were counted in an automated well-type gamma counter (Packard Cobra, Packard, Downers Grove, IL). Total (triplicate) and nonspecific bindings (duplicate; with excess unlabeled ANG II) were measured to calculate specific binding.
Data Analysis All data were expressed as means ⫾ SE. In the vasopressor response study, the differences in dose– response curves before and after antagonist treatment were evaluated by a two-factor analysis of variance (ANOVA) followed by, where applicable, the Newman– Keuls multiple comparison with repeated measures. In the cytosolic Ca2⫹ signaling measurements, the autofluorescence at 340 and 380 nm was subtracted from the respective fura-2 fluorescence levels prior to the calculation of the fluorescence ratios (FRs) at 340/380 nm. ANOVA followed by the Newman–Keul’s multiple comparison test was used for statistical analysis of Ca signal responses.
RESULTS Effects of ANG Antagonists on ANG II-Induced Vasopressor Responses [Asn1, Val5]angiotensin (ANG) II significantly increased (P ⬍ 0.01) mean arterial pressure (BP) in conscious toadfish in a dose-related manner (Fig. 1). The ANG II-induced dose–pressor curve was not altered by vehicle (0.9% NaCl, Fig. 1A). [Sar1, Ile8]ANG II (Fig. 1B) showed a mild agonist effect and raised the basal BP by 7.3 ⫾ 1.5 mm Hg; it completely suppressed the ANG II-induced dose–pressor responses (P ⬍ 0.01, ANOVA). Losartan (AT1 antagonist, Fig. 1C) partly but significantly (P ⬍ 0.01, ANOVA) reduced the ANG II-induced vasopressor responses. The percentage inhibitions were larger at lower doses (20 ng/kg,
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FIG. 1. Effects of angiotensin II (ANG II) antagonists on [Asn1, Val5]ANG II-induced vasopressor responses in conscious toadfish. (A) Effects of vehicle (0.9% NaCl); (B) effects of [Sar1, Ile8]ANG II; (C) effects of Dup 753; (D) effects of PD123319. All test drugs were diluted in saline before using. Prior to treatments, ANG II caused significant dose-dependent increases in vasopressor actions (P ⬍ 0.01). The values between lines indicate significance levels (P) before and after antagonist treatment by ANOVA. *P ⬍ 0.05, **P ⬍ 0.01 before and after antagonist treatment by Newman–Keuls multiple comparison at each dose. NS, nonsignificant. Baseline blood pressure for each group: (A) 21.4 ⫾ 1.4; (B) 20.3 ⫾ 1.5; (C) 23.7 ⫾ 2.0; (D) 23.6 ⫾ 1.9.
80.2 ⫾ 9.7%; 50 ng/kg, 66.6 ⫾ 6.9%) than at higher doses (200 ng/kg, 40.7 ⫾ 9.4%; 500 ng/kg, 34.0 ⫾ 5.2%). PD 123319 (AT2 antagonist, Fig. 1D) partly suppressed ANG II-induced vasopressor responses (0.01 ⬍ P ⬍ 0.05, ANOVA). [Asp1, Val5, His9]ANG I also increased toadfish BP in a dose-related fashion (Figs. 2A and 2B). The vasopressor responses were, however, lower than those of ANG II and were completely eliminated by an ANGconverting enzyme inhibitor, SQ 14,225 (1 mg/kg, P ⬍ 0.01, ANOVA) (Fig. 2B). The mean body weight (g) of toadfish used for the vasopressor studies was 490 ⫾ 42 g (n ⫽ 8).
Effects of Extracellular Kⴙ, Bay K 8644, and ANG II on Cytosolic Calcium Signaling To see whether treatments that induce Ca2⫹ influx also increase the fura-2 fluorescence ratio in toadfish
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vascular smooth muscle tissues, we examined the effects of 10 to 50 mM K⫹ (which presumably stimulates the voltage-gated Ca2⫹ channel by membrane depolarization) on changes in the fura-2 fluorescence 340/380 nm ratio. K⫹ (20–50 mM) induced significant increases in fura-2 fluorescence ratios (Fig. 3). The addition of Bay K 8644, a voltage-gated calcium channel agonist, to 50 mM K⫹-containing Ringer solution further increased cytosolic Ca2⫹ signals (P ⬍ 0.01). [Asn1, Val5]ANG II (10⫺7 M) evoked a rapid and transient increase in Ca2⫹ signaling in toadfish vascular tissues (Fig. 4). The second ANG II application, however, caused a much smaller increase in Ca2⫹ signaling, whereas Ca2⫹ ionophore (10⫺6 M, Br-A23187) induced a large increase in Ca2⫹ signaling in the same vascular preparation (Figs. 4 and 5). The mean body weight of toadfish used for studies on Ca2⫹ signaling
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FIG. 2. (A) A representative recording of the effect of SQ 14,225 on [Asp1, Val5, His9]ANG I-induced pressor responses. (B) The dose–response study (mean ⫾ SE) done in conscious toadfish. ANG II-induced pressor effects were significantly reduced after SQ 14,225 (P ⬍ 0.01, ANOVA). *P ⬍ 0.05, **P ⬍ 0.01 before and after treatment with SQ 14,225 by Newman–Keuls multiple comparison at each dose. The basal blood pressure for the control was 19.6 ⫾ 3.1 mm Hg; after the SQ 14,225 study, it was 19.5 ⫾ 3.3 mm Hg.
for ANG II was 399 ⫾ 51 g (n ⫽ 11), and that for K⫹ and Bay K 8644 studies was 426 ⫾ 24 g (n ⫽ 9).
Effects of ANG Antagonists on Angiotensin II Binding to Vascular Membrane ANG II binding to VSM membrane fractions was dose-dependently displaced by unlabeled [Asn1, Val5]ANG II (ID50 ⫽ 1 ⫻ 10⫺9 M, pooled tissue from 3 fish) and by [Sar1, Ile8]ANG II (ID50 ⫽ 1.58 ⫻ 10⫺8 M, pooled tissues from three to four fish per experiment, repeated twice) with complete displacement at 10⫺6 M
(Fig. 6). Losartan partially (30–50%) displaced ANG II at 10⫺10–10⫺6 M) (pooled tissues from three to four fish per experiment, repeated twice), whereas no displacement was seen after PD 123319 at 10⫺10–10⫺6 M (pooled tissue from three fish per experiment, repeated twice). The very high doses of PD 123319 (10⫺5 and 10⫺4 M), however, substantially displaced [Asn1, Val5]ANG II (43–70%). Preliminary study indicates that specific 125I-[Ile5]ANG II binding to VSM membrane fractions increased with incubation time (data not shown). The mean body weight of toadfish used for the ANG binding study was 423 ⫾ 21 g (n ⫽ 28).
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FIG. 3. Increases in cytosolic Ca2⫹ signaling evoked in toadfish dorsal aortic smooth muscle tissues by increasing doses of K⫹ in the bath and K⫹ plus Bay K 8644, expressed by fura-2 fluorescence ratio at 340/380 nm. **P ⬍ 0.01 by Newman–Keuls multiple comparison from control. The baseline fluorescence ratio is 0.70 ⫾ 0.06 (n ⫽ 9).
FIG. 5. Summary (mean ⫾ SE, n ⫽ 11) of the effects of [Asn1, Val5]ANG II on cytosolic calcium signals. *P ⬍ 0.05, **P ⬍ 0.01 from pretreatment level. The basal fluorescence ratio is 0.95 ⫾ 0.01. Autofluorescence was subtracted before calculating the 340/380 ratio.
DISCUSSION
pounds, represented by DuP 753 (losartan) (Chiu et al., 1989); and those for AT2 are tetrahydroimidazopyridines, represented by PD 123177 and PD 123319 (Parke-Davis) (Dudley et al., 1990). A markedly modified ANG II analog, CGP 42112 A (Ciba-Geigy), is also
In mammals, both AT1 and AT2 receptors have seven-transmembrane domains and are coupled with G-protein (Chiu et al., 1994). The prototypical antagonists for the AT1 receptor are biphenylimidazole com-
Ca2⫹
FIG. 4. A representative recording of cytosolic response to [Asn1, Val5]ANG II (10⫺7 M) in VSM tissues of toadfish. The Ca2⫹ signal is expressed by the fluorescence ratio of 340/380 nm (emission, 510 nm). The second application of ANG II at a higher dose (10⫺6 M) did not induce a cytosolic Ca2⫹ signal, whereas the same tissue responded to bromo (Br)-A23287 (10⫺6 M).
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FIG. 6. Displacement of 125I-[Ile5]ANG II binding to toadfish vascular tissue membrane fractions with angiotensin antagonists. Each point in the displacement study is the mean of two binding experiments (pooled tissue from three to four fish and each assay in duplicate or triplicate). B and B0 are the specific (total minus nonspecific) binding in the presence and absence, respectively, of various doses of the test compound.
Vascular Smooth Muscle Cell Growth
AT2-selective (de Gasparo et al., 1994). Interestingly, all the major known biological effects of ANG II are mediated via the AT1 receptor by stimulation of the phosphatidylinositol hydrolysis/protein kinase C pathway or by reduction of cAMP (Chiu et al., 1994). Furthermore, ANG II promotes VSM cell growth by stimulating tyrosine kinases (Berk and Corson, 1997) via the AT1 receptor. In contrast, the AT2 receptor appears to play a role during development and cell proliferation in pathological conditions such as injuryinduced vascular neointima formation and atherosclerosis (Dzau et al., 1991; Nakajima et al., 1995). Signal transduction via protein tyrosine phosphatase coupled to guanylate cyclase inhibition has been suggested but not established (de Gasparo et al., 1994). The present study indicates that [Asn1, Val5]ANG II induced dose-dependent pressor actions that were inhibited completely by [Sar1, Ile8]ANG II, a peptide antagonist, and partially by losartan and PD 123319. The partial inhibition of the ANG II-induced vasopressor effect by both losartan and PD 123319 suggests that the toadfish vascular ANG II receptor has low selectivity and is not readily distinguishable as either the AT1 or the AT2 subtype. Likewise, the specific binding to labeled ANG II was displaced completely by [Asn1, Val5]ANG II and [Sar1, Ile8]ANG II and partially displaced by losartan, but not by PD 123319, except at very high doses (10⫺5 and 10⫺4 M). The partial displacement by losartan suggests that both losartan-sensitive and -insensitive binding sites are present in the toadfish vascular ANG receptor. The displacement of ANG binding by PD 123319 only at high doses may presumably be nonspecific, since in mammalian ANG receptors high doses of losartan bind to the AT2 receptor, whereas high doses of EXP 655 (PD 123177), pharmacologically very similar to PD 123319, bind to the AT1 receptor (Chiu et al., 1989). Likewise, high doses of losartan and PD 123319 displaced ANG II binding sites in fowl VSM receptors, which functionally differ from both AT1 and AT2 receptor subtypes (Nishimura et al., 1994; Walker et al., 1993). ANG II evoked a rapid increase in cytosolic Ca2⫹ signaling in VSM tissues of toadfish dorsal aortae, measured by the fluorescent indicator fura-2. It is not clear from the present study, however, whether the increase in cytosolic Ca signaling is due to stimulation of Ca2⫹ release from a cellular store such as the
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endoplasmic reticulum or to the stimulation of Ca influx. Taken together, the present findings suggest that the toadfish vascular ANG receptor is rather nonselective, although it appears to have some resemblance to the AT1-homologous receptor. The present study also indicates that, similar to mammalian VSM cells, toadfish vascular tissues possess a voltage-gated calcium channel that is activated by K⫹-induced membrane depolarization and a calcium channel agonist, Bay K 8644. Our previous studies indicate that toadfish have renin substrate, renin, converting enzyme, and ANGs I and II (Nishimura et al., 1977, 1979). Renin secretion in toadfish is markedly stimulated by hemorrhage and hypotensive drugs (Nishimura et al., 1979) and mediated by renal arterial baroreceptors via cytosolic Ca2⫹ in renin secretory cells (Nishimura and Madey, 1989) but not by stimulation of the adrenergic nervous system (Nakamura et al., 1992) or cAMP (Nishimura and Madey, 1989, and unpublished). Thus, toadfish possess the functioning renin–angiotensin cascade and ANG receptor, although they may differ biochemically from those of more advanced vertebrates. It has been shown that ANG receptors are present in the cardiovascular system (Nishimura et al., 1978; Olson et al., 1994), liver and intestines (Marsigliante et al., 1994), and kidney (Cobb and Brown 1993; 1994) of various teleosts. Cobb and Brown (1993, 1994) reported that renal glomeruli isolated from rainbow trout show a specific ANG II binding site that has considerable affinity to losartan. The Kd for [Asn1, Val5]ANG II is 0.38 nM, whereas the IC50 for losartan is approximately 12.5 nM (Cobb and Brown, 1993, 1994). ANG II binding was also weakly displaced by [Sar1, Ala8]ANG II but not by PD 123319. Furthermore, ANG II induced a transient rise in Ca2⫹ signaling (Cobb et al., 1999), and ANG II and ANG III induced antidiuresis in the perfused rainbow trout, perhaps via the same ANG receptor; losartan also decreased urine flow and glomerular filtration rate (Brown et al., 1997). Recently, Parkyn and co-workers (1997) constructed primers based on the conserved amino acid residues and partially sequenced the ANG receptor from rainbow trout liver genomic DNA using polymerase chain reaction (PCR). The deduced peptide sequence of the trout receptor shows 47% identity with the mammalian AT1 and 41% identity with the AT2 receptor
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(Parkyn et al., 1997). This agrees with our present finding that the toadfish vascular ANG receptor is rather nonselective. Myocardial ANG receptors (Ji et al., 1993; Bergsma et al., 1993) cloned from X. laevis have 60% amino acid identity and 65% nucleotide homology with the coding region of the mammalian AT1 receptor; the Xenopus receptor transfected in Xenopus oocytes mobilized intracellular Ca2⫹ (Ji et al., 1993) and produced a Ca2⫹-dependent Cl current (Bergsma et al., 1993). Furthermore, Murphy et al. (1993) and, more recently, Kempf and co-workers (1996) cloned ANG receptors in the turkey and chicken adrenal cortex, respectively, that show 75% amino acid identity to the AT1 receptor and mediate the phosphatidylinositol/Ca2⫹ pathway. The above Xenopus cardiac and avian adrenal receptors are coupled to G-protein and show seven-transmembrane structures. Therefore, from a phylogenetical point of view, it appears that ANG receptors that show some homology to the AT1 receptor evolved early in the vertebrate phylogeny. These nonmammalian ANG receptors (Ji et al., 1993; Bergsma et al., 1993; Murphy et al., 1993; Nishimura et al., 1994; Kempf et al., 1996) show high affinity to peptide antagonist but show no selectivity to losartan/PD 123319, although some species have affinity to CGP 41221A (AT2-selective). Pharmacological differences in nonmammalian ANG receptors, such as the absence of affinity to selective AT1/ AT2 antagonists, may suggest that the amino acid sequence important for selective antagonist binding may be different in nonmammalian AT1 homologous receptors (Sandberg, 1994). In nonmammalian tissues, however, one or more unidentified ANG receptors appear to exist. For example, the dogfish, Triakis scyllia, and Scyliorhinus canicula have high-affinity ANG receptors in the gills, rectal gland, and/or interrenal gland that bind to CGP 41221A and losartan with nearly equal potencies (Tierney et al., 1997; Hazon et al., 1997). Likewise, ANG receptors in fowl VSM membranes do not show affinity to either losartan or PD 123319 and do not stimulate cytosolic Ca2⫹ (Walker et al., 1993; Nishimura et al., 1994). Whether this unidentified receptor has some molecular similarity to the AT2 receptor subtype or belongs to an entirely new group of ANG receptors remains to be determined.
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Qin, Yan, and Nishimura
ACKNOWLEDGMENTS We thank Ms. Xiaoying He and Ms. Hongying Liu for their excellent technical assistance. This study was supported by National Heart, Lung, and Blood Institute Grant HL-52881 and American Heart Association Grant-in-Aid 92012400. A preliminary study was presented at the Experimental Biology 1995 meeting, Atlanta, GA.
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