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Circulation Research. 1995;77:64-72

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(Circulation Research. 1995;77:64-72.)
© 1995 American Heart Association, Inc.

Articles

Localization of Atrial Natriuretic Factor Receptors in the Mesenteric Arterial Bed

Comparison With Angiotensin II and Endothelin Receptors

Héctor de León, Marie-Chantal Bonhomme, Gaétan Thibault, Raul Garcia

From the Laboratory of Experimental Hypertension and Vasoactive Peptides, Clinical Research Institute of Montreal (Canada), Montreal University.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract Although receptors for atrial natriuretic factor (ANF) and angiotensin II (Ang II) have been reported in rat mesenteric arteries, both peptides induce weak biological responses. Endothelin-1 (ET-1) evokes a potent vasoconstriction in the mesenteric artery. To identify the tissue localization of ANF, Ang II, and ET-1 receptors, radioligand binding experiments with 125I-ANF, 125I–[Sar1,Ile8]Ang II, and 125I–ET-1 were performed in defatted mesenteric arteries and in the surrounding adipose tissue. 125I-ANF binding assays in adipose tissue showed a single class of high-affinity binding sites (Bmax, 420±16 fmol/mg protein; Kd, 343±16 pmol/L). In vascular membranes, most 125I-ANF binding was nonspecific. The majority of receptors present in adipose tissue recognized ANF, C-type natriuretic peptide (CNP), and des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANF-(4-23) (C-ANF) with close affinities, with C-ANF competing for >98% of the binding sites. In adipocytes, ANF and CNP stimulated cGMP generation. cGMP production by mesenteric arteries was stimulated by sodium nitroprusside but not by ANF or CNP. Autoradiographic localization of 125I-ANF and 125I–ET-1 showed that in the case of ANF, most specific binding occurred in adipocytes, whereas for ET-1, specific binding was present in both adipose tissue and mesenteric arteries. Cross-linking of 125I-ANF followed by SDS-PAGE revealed two receptor species of 130 and 70 kD in adipose membranes and none in vascular tissue. Both were completely displaced by ANF, CNP, and C-ANF. 125I–[Sar1,Ile8]Ang II binding assays in adipose tissue exhibited a single class of binding sites (Bmax, 211±4 fmol/mg protein; Kd, 520±10 pmol/L). In mesenteric arteries, 125I–[Sar1,Ile8]Ang II saturation assays showed little specific binding. The profile of ligands competing for 125I–[Sar1,Ile8]Ang II was consistent with an AT1 receptor subtype. 125I–ET-1 binding assays demonstrated high-affinity binding sites in both mesenteric arteries and adipose tissue (Bmax, 179±21 and 312±7 fmol/mg protein, respectively; Kd, 215±45 and 180±111 pmol/L, respectively), the majority corresponding to BQ-123–sensitive sites. Thus, ANF and Ang II binding sites were either absent or barely expressed in the mesenteric artery. Binding sites for ET-1 were abundantly expressed in the mesenteric artery. Adipose tissue expressed high-affinity binding sites for ANF, Ang II, and ET-1, but their biological role, if any, remains to be defined.


Key Words: mesenteric artery • adipose tissue • natriuretic peptides • angiotensin II • endothelin-1


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Atrial natriuretic factor (ANF) evokes a variety a physiological responses affecting cardiovascular homeostasis. Two other members of the natriuretic peptide family, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), have also been identified. ANF, BNP, and CNP have high-sequence homology.1 2 3 ANF receptors belong to different subtypes, namely, guanylate cyclase–containing and non-guanylate cyclase–containing receptors. Guanylate cyclase–coupled receptors can be distinguished as ANP-A (atrial natriuretic peptide-A or guanylyl cyclaseA) and ANP-B (atrial natriuretic peptide-B or guanylyl cyclase-B) receptors. Non-guanylate cyclase–coupled receptors known as C receptors or ANP-C (atrial natriuretic peptide-C) have been postulated to be clearance receptors4 and to inhibit adenylate cyclase.5 All three receptors have been cloned.6 7 8 ANP-A and ANP-B receptors have conservative structural requirements. They will not bind several truncated ANF analogues such as des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANF-(4-23) (C-ANF),4 9 10 which binds with high affinity to ANP-C receptors. Recently, CNP has been reported to be the specific ligand for ANP-B receptors but also binds ANP-C receptors with high affinity.11

Angiotensin II (Ang II) is one of the major factors involved in blood pressure and volume regulation. With the use of specific nonpeptide Ang II antagonists typified by losartan (DuP753) and PD123319,12 13 Ang II receptor subtypes have been classified as AT1 and AT2. AT1 is inhibited by losartan and AT2, by PD123319.14 Most of the known physiological effects of Ang II are mediated by AT1 receptors. Recently, AT1 was cloned from the rat aorta and kidney15 16 and from bovine adrenal glands.17

Endothelin (ET), a potent vasoconstrictor vascular endothelium–derived factor, was also recently described18 and purified.19 It represents a family of three 21–amino acid peptides with high homology: ET-1, ET-2, and ET-3.20 ET-1 is the only peptide expressed in significant amounts in tissues. The biological effects of ET are mediated by specific membrane receptors. Two ET receptors have been cloned.21 22 ETA has a higher affinity for ET-1 than for ET-3; ETB has a similar affinity for ET-1 and ET-3.

Since the splanchnic circulation is thought to make a major contribution to total peripheral resistance, rat mesenteric arteries have been used extensively as a model of resistance-sized vessels for ANF and Ang II receptor characterization and regulation.23 24 However, in vitro and in vivo experiments on mesenteric artery preparations have demonstrated that these peptides are either devoid of any biological activity (ANF)25 26 27 or are weak vasoconstrictors (Ang II).28 On the other hand, ET-1 is reported to have a potent vasoconstrictor effect on the isolated perfused mesenteric artery.29 Mesenteric arteries and arterioles are distinctively encircled by a thick layer of adipose tissue, which is routinely removed, at least in part, when pharmacological (rings and strip preparations) as well as radioligand binding experiments (particulate fractions) are performed.23 24 25 On the other hand, when the whole vascular bed is studied in perfusion experiments, no attempt is made to remove the adipose tissue.25 26 28 There is evidence suggesting that perivascular adipose tissue may not only influence vascular responsiveness to vasoactive peptides30 but that it may also be a local site of angiotensinogen generation.31

The reported presence of ANF and Ang II receptors in the mesenteric artery23 24 in the absence of a direct potent biological effect,25 26 together with the powerful vasoconstrictor action of ET31 via pharmacologically defined ET receptors32 in the same vascular territory, prompted us to hypothesize that ANF and Ang II receptors in the rat mesentery are localized in tissues other than the arterial bed. Studies were therefore performed in completely defatted mesenteric arteries on the one hand and in surrounding adipose tissue on the other to assess the precise localization of ANF and Ang II receptors by radioligand binding.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Preparation of Vascular and Fatty Membranes
Sprague-Dawley rats (300 to 325 g) were decapitated, and their mesenteric arcades were removed by blunt dissection and placed in ice-cold saline. A large piece of fat overlying the superior mesenteric artery was eliminated, because preliminary experiments showed it to have three to four times lower 125I-ANF binding than that found in adipose tissue around the mesenteric arteries and arterioles. The superior mesenteric vein and branching veins were eliminated. The vascular preparation consisted of the mesenteric arterial bed, including the superior mesenteric artery, ileal and jejunal arteries, and all branching arterioles. The smallest outer diameter of the mesenteric vessels in the preparation was 90 to 100 µm. Adipose tissue surrounding the mesenteric arteries and arterioles was detached by stripping them with forceps. Vascular and fatty tissues were homogenized in 0.25 mol/L fresh ice-cold sucrose solution with a Polytron homogenizer (setting 8, four times for 10 seconds) and centrifuged at 1075g for 10 minutes at 4°C. The supernatant was kept on ice, and the process was repeated for the pellet. The supernatants of both centrifugations were pooled, filtered through 20-µm nylon mesh, and centrifuged at 100 000g for 60 minutes at 4°C. The pellet was resuspended in 0.05 mol/L Tris-HCl buffer, pH 7.4. Protein concentration was assessed by the Coomassie blue technique described by Spector.33

Binding Assay
The binding assay was performed as described previously.34 35 Aliquots of fatty and vascular membranes (30 to 40 µg of protein) were incubated in duplicate for 90 minutes at 22°C in the presence of increasing concentrations of unlabeled peptides (1 pmol/L to 1 µmol/L). In competition experiments, membranes were incubated with 90 to 105 pmol/L 125I-ANF (750 Ci/mmol), 70 to 80 pmol/L 125I–[Sar1,Ile8]Ang II (1550 Ci/mmol), or 125I–ET-1 (1470 Ci/mmol) in a final volume of 0.25 mL. The reaction was stopped by dilution with 3.5 mL assay buffer and rapid filtration through Whatman GF/C filters, which were then rinsed three times with 3 mL of 0.05 mol/L Tris-HCl at pH 7.4. The assay buffer contained 0.05 mol/L Tris-HCl (pH 7.4), 1 µmol/L aprotinin, 0.1% bacitracin, 5 mmol/L MgCl2, 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), 0.4 µmol/L phosphoramidon, and 0.5% bovine serum albumin (BSA). Filtration and rinsing were performed with a semiautomatic harvester system (Brandel), which processes 30 tubes simultaneously. The filters were counted in an LKB gamma counter with 70% efficiency. 125I-ANF, 125I–[Sar1,Ile8]Ang II, and 125I–ET-1 were prepared by the lactoperoxidase method.36 Nonspecific binding was defined as binding remaining in the presence of 1 µmol/L unlabeled peptides. Three saturation experiments and three to four competitive inhibition assays for each displacing agent were undertaken on three different membrane preparations for each tissue. Ten to 12 rats were used in each experiment, and all binding assays were performed in duplicate the same day the tissues were isolated.

Labeled peptide binding to vascular and fatty membranes was studied over a range of protein concentrations from 5 to 50 µg. All experiments were performed with 20 to 40 µg protein. Total, specific, and nonspecific binding is depicted in all saturation figures. In competition experiments, only specific binding is shown. Specific binding was obtained by subtracting nonspecific from total binding.

Covalent Cross-linking of Adipose and Vascular Tissue ANF Receptors
Aliquots (100 µg) of vascular and fatty membranes were incubated in binding assay buffer for 60 minutes at 22°C, with 200 pmol/L 125I-ANF in the absence and presence of 1 µmol/L unlabeled ANF, CNP, or C-ANF. Disuccinimidyl suberate (DSS) in dimethyl sulfoxide (DMSO) was added to a final concentration of 0.5 mmol/L and incubated for 15 minutes at 22°C. The reaction was quenched by the addition of ammonium acetate to a final concentration of 50 mmol/L. Sample buffer (100 µL) containing 1% SDS (wt/vol), 100 mmol/L Tris-HCl (pH 6.8), 20% glycerol, 2% ß-mercaptoethanol, and traces of bromophenol blue was then added. The samples were boiled for 5 minutes before being analyzed by electrophoresis.

SDS-PAGE of Adipose and Vascular Tissue ANF Receptors
Electrophoresis was performed with a modification of the method of Schägger and von Jagow37 in 8% unidimensional slab gel in the presence of 0.1% SDS. The gels were stained with Coomassie brilliant blue R-250, destained, and dried. Molecular weights were identified by reference standards. The dried gels were exposed to Kodak Omat XRP6 film for 4 to 6 days at -70°C. The relative density of high- and low-molecular-weight ANF receptor subtypes in the autoradiograms was assessed by densitometry.

Autoradiographic Localization of 125I-ANF
125I-ANF (24 pmol) in sodium phosphate buffer containing 0.1 g/dL BSA was injected in a volume of 300 µL into the superior mesenteric artery of male Sprague-Dawley rats (100 g body weight) under pentobarbital anesthesia (n=3). In parallel experiments, unlabeled ANF (7 nmol) was mixed with 125I-ANF and injected into rats under the same conditions (n=3). Two minutes after injection, the mesenteric vascular bed was perfused with 200 mL of Bouin's solution for 10 minutes. The animals were then killed, and the entire mesenteric vascular bed was removed, fixed for 24 hours in the same fixative, and embedded in paraffin. Sections (5 µm thick) were cut and stained with hematoxylin and lithium carbonate before being dipped into Ilford K5 emulsion. All sections were then exposed for 4 weeks, developed in D19 solution, and fixed with sodium thiosulfate for 4 minutes.

Autoradiographic Localization of 125ET-1
Sprague-Dawley rats (300 to 325 g body weight) were decapitated, and their mesenteric arcades were removed and placed in saline. The superior mesenteric vein and branching veins as well as a large piece of fat overlying the superior mesenteric vein were removed and eliminated. The preparation, corresponding to the mesenteric arterial bed and surrounding adipose tissue, was snapped frozen in isopentane at -40°C. Frozen sections were cut in a cryostat at -30°C. The sections were thaw-mounted onto gelatin-coated slides and kept at -70°C until used. Before the assay, the slides were dried in a dessicator for 4 hours at 4°C. They were then preincubated at room temperature for 15 minutes in 50 mmol/L Tris-HCl buffer (pH 7.4) containing 0.5% BSA and subsequently incubated for 60 minutes in fresh buffer supplemented with 150 pmol/L of 125I–ET-1 as well as 5 µmol/L MgCl2, 100 mmol/L NaCl, 1 µmol/L phosphoramidon, 1 µmol/L aprotinin, 1 µmol/L EDTA, 1 µmol/L PMSF, and 0.05% bacitracin. Nonspecific binding was determined in the presence of an excess of unlabeled ligand (1 µmol/L). After incubation, the sections were transferred through four successive 1-minute washes of ice-cold 50 mmol/L Tris-HCl buffer (pH 7.4) containing 0.5% BSA, fixed (30 minutes) in 2% glutaraldehyde (pH 7.4, 4°C), washed in distilled water, dehydrated in ethanol, and dried overnight at 40°C. Subsequently, the slides were dipped in Ilford K5 clear emulsion, exposed for 5 to 6 days at 4°C, developed in Kodak D19, and stained with hematoxylin and eosin.

Histological Preparation
Male Sprague-Dawley rats (300 to 350 g) were decapitated, their mesenteric artery arcades were excised, and their intestines were removed. The entire mesenteric artery arcades or defatted mesenteric vascular beds, prepared as above, were fixed for 24 hours in Bouin's solution and embedded in paraffin. Sections (5 µm) were cut and stained with hematoxylin, lithium carbonate, and eosin.

Cyclic Nucleotide Measurement
cGMP production was measured as described previously.34 Briefly, mesenteric perivascular adipose tissue and arteries were prepared as in the binding assay, but instead of 0.9% NaCl solution, Hanks' balanced salt solution (HBSS containing [mmol/L] NaCl 137, KCl 5.4, KH2PO4 0.44, Na2HPO4 0.33, MgSO4 0.04, MgCl2 0.50, CaCl2 1.25, and NaHCO3 4.0, at pH 7.4) was used with neither PMSF nor bacitracin. The tissues were resuspended in final HBSS assay buffer containing 0.2 g/dL BSA, 5.5 mmol/L glucose, and 25 mmol/L HEPES. Fatty and vascular samples were preincubated in assay buffer at 37°C for 15 minutes before isobutylmethylxanthine (IBMX) was added at a final concentration of 0.5 mmol/L. Three minutes later, ANF, CNP, C-ANF, or sodium nitroprusside (SNP) was added to achieve a range of concentrations varying from 0.1 nmol/L to 1 µmol/L in a final assay buffer incubation volume of 500 µL. After incubation for 5 minutes, the reaction was stopped by the addition of 500 µL of ice-cold 10% trichloroacetic acid (TCA). The samples were centrifuged at 1075g at 4°C for 15 minutes, the supernatant was retained, and the pellet was digested with 1 mol/L NaOH for protein determination.33 TCA was removed by extracting the supernatant four times with 2 mL of water-saturated ether, and the remaining ether was evaporated at room temperature. The aqueous phase was dried with a Speed Vac concentrator (Savant Instruments, Inc). Each sample was resuspended in 300 µL of 50 mmol/L sodium acetate buffer, pH 6.2. A 100-µL aliquot was acetylated, and cGMP concentration was assessed by radioimmunoassay. The fatty and vascular samples were preincubated for 5 minutes with IBMX. Three fresh preparations were obtained for each tissue (five to seven rats for each preparation), and two separate determinations were conducted for each preparation. All experiments were performed the same day the tissues were isolated.

Mesenteric Artery Perfusion
Vasoconstrictor responses to norepinephrine (NE), vasopressin (AVP), ET-1, and Ang II were determined in mesenteric preparations in which the surrounding adipose tissue was either completely removed or left intact. Male Sprague-Dawley rats (300 to 350 g) were used in these experiments. They were anesthetized with pentobarbital sodium (60 mg/kg body wt IP). The superior mesenteric artery was cannulated with PE-90 tubing, flushed with 20 mL Krebs' solution, and dissected free from the intestine. The whole vascular bed (with or without surrounding fat) was placed in a water-jacketed chamber maintained at 37°C and perfused with Krebs' solution (mmol/L: NaCl 112, KCl 5.0, NaH2PO4 1.0, MgSO4 1.2, CaCl2 2.5, NaHCO3 2.5, and glucose 11.2), which was kept at 37°C and aerated with a mixture of 5% CO2/95% O2 to obtain a pH of 7.4. Intact mesenteric preparations and defatted arteries were perfused at 7.5±0.5 and 15±2 mL/min, respectively, with a peristaltic pump to achieve a constant pressure of {approx}20 mm Hg. Perfusion pressure was recorded with a pressure transducer (Statham P231D) connected to a polygraph (Grass 7D). The preparations were allowed to equilibrate for 1 hour before a dose-response curve to each agent was established.

Chemicals
All materials were of the highest reagent grade available. Tricine, Coomassie brilliant blue, lactoperoxidase, bacitracin, PMSF, pepstatin, phosphoramidon, polyethylene glycol, BSA, and gamma globulin were from Sigma Chemical Co; DSS, from Pierce; 2-mercaptoethanol, from J.T. Baker Inc; DMSO and glycerol, from Anachemia; aprotinin, from Miles Laboratories; molecular weight standards, from Pharmacia Fine Chemicals; SDS, acrylamide, bisacrylamide, and tetramethylethylendiamine, from Bio-Rad; 125I-sodium, from Amersham Canada Limited; ANF-(99-126), CNP, C-ANF, Ang II, [Sar1,Ile8]Ang II, and ET-1, from Bachem; BQ-123, from the Peptide Institute Inc; and Omat XRP6 film, from Eastman Kodak. Losartan potassium and PD123319 were generous gifts from DuPont Merck Pharmaceuticals Co and Parke-Davis, respectively.

Analysis of Data
Whenever appropriate, the results are expressed as mean±SEM. Binding data were analyzed by processing raw data with the computer-based EBDA program (Elsevier, Biosoft). The binding capacity (Bmax) and apparent affinity (Kd) of binding sites were then determined by the computer-based LIGAND program.38

When the Hofstee plot and line of best fit displayed by the LIGAND program with an assumed one-site model were inadequate, suggesting departure from linearity, a two-site model investigated the same data; these procedures were followed by testing of whether the second model provided a better statistical fit than the first model.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Light Microscopy
Fig 1Down is a low-power magnification (x1.5) of two mesenteric preparations before (top panel) and after (bottom panel) the surrounding adipose tissue and mesenteric veins were removed. Two light micrographs of untouched (top panel) and completely defatted (bottom panel) mesenteric arteries are shown in the insets. The walls of both arteries present undamaged intimal, medial, and adventitial layers.



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  		Figure 1. Low-power magnifications (original magnifications x1.5) of mesenteric arterial bed consisting of superior mesenteric, jejunal, and ileal arteries. A, Intact arcade. B, Defatted preparation (adipose tissue and veins removed). Insets are light micrographs of intact (A) and defatted (B) preparations (original magnifications x63).

Saturation and Competitive Inhibition of 125I-ANF, 125I–[Sar1,Ile8]Ang II, and 125I–ET-1 Binding to Fatty and Vascular Membranes
125I-ANF saturation binding assays in adipose tissue membranes revealed a single class of high-affinity binding sites with a Bmax of 420±16 fmol/mg protein and a Kd of 343±16 pmol/L (n=3) (Fig 2aDown and 2cDown). In contrast, 125I-ANF saturation assays in vascular membranes indicated that most of the 125I-ANF binding was nonspecific (Fig 2bDown). The scatter of data on poor specific binding was extremely high, rendering Scatchard transformation meaningless and suggesting that ANF receptor expression in the arterial mesenteric vasculature was either extremely low or absent. The pharmacological profile of ligands competing for 125I-ANF (Fig 2dDown) indicated that the majority of receptors present in adipose tissue did not discriminate between the different natriuretic peptides, because they recognized ANF, CNP, and C-ANF with close affinities (424±85, 930±87, and 770±48 pmol/L, respectively). C-ANF, a specific ligand for the ANP-C receptor, potently competed for >98% of 125I-ANF binding sites, suggesting that ANP-C receptors were predominant in mesenteric adipocytes.



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  		Figure 2. Pharmacological characterization of atrial natriuretic factor (ANF) receptors in mesenteric adipose and vascular tissues. a and b, Representative 125I-ANF saturation binding curves in membranes prepared from mesenteric adipose tissue (a) and arteries (b). {circ} indicates total binding; {triangledown}, nonspecific binding; and {bullet}, specific binding. c, Scatchard transformation of specific binding data in mesenteric adipose tissue (Bmax, 404 fmol/mg protein; Kd, 333 pmol/L) and vascular membranes ({circ}). d, Representative competitive inhibition curves of 125I-ANF binding to mesenteric adipose tissue. {bullet} indicates ANF; {square}, C-type natriuretic peptide; and {circ}, des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANF-(4-23).

125I–[Sar1,Ile8]Ang II saturation binding curves in adipose tissue (Fig 3aDown and 3cDown) exhibited a single class of high-affinity binding sites with a Bmax of 211±4 fmol/mg protein and a Kd of 520±10 pmol/L (n=3). In mesenteric artery membranes, 125I–[Sar1,Ile8]Ang II saturation binding assays showed little specific binding, with a Bmax 10 times lower than that observed in mesenteric fat (20±5 fmol/mg protein) and a similar Kd (620±40 pmol/L, n=3, Fig 3bDown and 3cDown). The pharmacological profile of the ligand competing for 125I–[Sar1,Ile8]Ang II binding was consistent with an AT1 receptor subtype, with the following rank order of potency: [Sar1,Ile8]Ang II>Ang II>losartan>PD123319 (Fig 3dDown). Losartan, a specific AT1 antagonist, inhibited 100% of 125I–[Sar1,Ile8]Ang II binding, whereas PD123319, a nonpeptide AT2 antagonist, did not compete for radioligand binding at concentrations of up to 10 µmol/L.



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  		Figure 3. Pharmacological characterization of angiotensin II (Ang II) receptors in mesenteric adipose and vascular tissues. a and b, Representative 125I–[Sar1,Ile8]Ang II saturation binding curves in membranes prepared from mesenteric fat (a) and arteries (b). {circ} indicates total binding; {triangledown}, nonspecific binding; and {bullet}, specific binding. c, Scatchard transformation of the specific binding data. {bullet} indicates fatty membranes (Bmax, 210 fmol/mg protein; Kd, 500 pmol/L); {circ}, vascular membranes (Bmax, 15 fmol/mg protein; Kd, 580 pmol/L). d, Representative competitive inhibition curves of 125I–[Sar1,Ile8]Ang II binding to mesenteric adipose tissue. {bullet} indicates [Sar1,Ile8]Ang II; {circ}, Ang II; {triangledown}, losartan; and {square}, PD123319.

125I–ET-1 saturation binding experiments in adipose tissue showed a single class of binding sites. Unlike 125I–[Sar1,Ile8]Ang II and 125I-ANF, 125I–ET-1 binding to mesenteric arteries was a saturable process, with saturation being reached between 1 and 2 nmol/L (Fig 4aDown and 4bDown). Scatchard analysis demonstrated a single class of high-affinity binding sites. The binding parameters derived from competitive inhibition with ET-1 showed a Bmax of 179±21 and 312±7 fmol/L mg protein and a Kd of 215±45 and 580±111 pmol/L for mesenteric arteries and adipose tissue, respectively. BQ-123–sensitive binding was 79% and 64% for mesenteric arteries and adipose tissue, respectively (Fig 4cDown and 4dDown and TableDown).



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  		Figure 4. Pharmacological characterization of endothelin-1 (ET-1) receptors in mesenteric adipose and vascular tissues. a, Representative 125I–ET-1 saturation specific-binding curves in membranes prepared from mesenteric adipose tissue ({bullet}) and arteries ({circ}). b, Scatchard transformation of specific binding data in mesenteric adipose tissue ({bullet}; Bmax, 390 fmol/mg protein; Kd, 600 pmol/L) and mesenteric arteries ({circ}; Bmax, 205 fmol/mg protein; Kd, 200 pmol/L). c and d, Representative competitive inhibition curves of 125I–ET-1 binding to mesenteric arteries (c) and mesenteric adipose tissue (d). {circ} indicates ET-1; {bullet}, BQ-123.


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  		Table 1. Binding Capacities and Affinities Derived From Competitive Inhibition Experiments on 125I–Endothelin-1 Binding

cGMP Measurements
ANF and CNP stimulated cGMP generation by adipose tissue in a dose-dependent manner (Fig 5aDown). The threshold concentration was 1 to 10 nmol/L, with maximal stimulation occurring in the presence of 1 µmol/L of either ANF or CNP as 50- and 22-fold increases, respectively, over baseline values. Mesenteric arteries did not respond to either ANF or CNP concentrations of 1 µmol/L (Fig 5bDown), indicating the absence of guanylate cyclase–coupled receptors. C-ANF (1 µmol/L) did not evoke any cGMP stimulation by either tissue (Fig 5aDown). SNP stimulated cGMP production by the mesenteric arteries in a dose-dependent manner (Fig 5bDown).



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  		Figure 5. Graphs showing cGMP stimulation by atrial natriuretic factor (ANF), C-type natriuretic peptide (CNP), and des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANF-(4-23) (C-ANF) in mesenteric adipose tissue (a) and by ANF, CNP, C-ANF, and sodium nitroprusside (SNP) in mesenteric arteries (b).

Affinity Cross-linking Studies
To further characterize ANF receptor subtypes, affinity cross-linking studies were performed on fatty and vascular membranes. DSS cross-linking of 125I-ANF revealed two receptor species of 130 and 70 kD on adipose tissue (Fig 6Down, lane 1). The proportion of high- and low-molecular-weight bands assessed by densitometry was {approx}3% and 97%, respectively. ANF, C-ANF, or CNP (1 µmol/L each) (Fig 6Down, lanes 2, 3, and 4, respectively) completely inhibited 125I-ANF binding in both high- and low-molecular-weight bands. Vascular tissue did not exhibit any chemical cross-linking of 125I-ANF (Fig 6Down, lanes 5 and 6).



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  		Figure 6. Autoradiography by SDS-PAGE of cross-linked atrial natriuretic factor (ANF) receptors in adipose (lanes 1 through 4) and vascular (lanes 5 and 6) tissues in the absence (lanes 1 and 5) and presence (lanes 2 and 6) of 10-6 mol/L ANF, des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANF-(4-23) (lane 3), and C-type natriuretic peptide (lane 4).

Autoradiographic Localization of 125I-ANF
To further substantiate our findings in vascular and fatty particulate fractions, we undertook in situ autoradiographic localization of 125I-ANF binding sites in the mesenteric vascular bed. Two minutes after injecting 125I-ANF, most adipocytes surrounding the mesenteric arteries were labeled, as indicated by dense deposits of overlaid silver grains that followed the contour of the adipocyte plasma membrane (Fig 7aDown). Mesenteric arterioles also exhibited a significant accumulation of silver grains. Injection of an excess of unlabeled ANF together with 125I-ANF resulted in a significant decrease in the number of silver grains over adipocytes (Fig 7bDown). Reduced radioactivity was also observed in the mesenteric arteries but to a lesser extent, indicating its nonspecific nature.



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  		Figure 7. In situ autoradiographic localization of 125I–atrial natriuretic factor binding sites in the mesentery in the absence (a) and presence (b) of unlabeled atrial natriuretic factor (original magnification x160).

Autoradiographic Localization of 125I–ET-1
Fig 8Down depicts in vitro autoradiographic localization of 125I–ET-1 binding sites in mesenteric artery and surrounding adipose tissue. Dense deposits of silver grains labeled the arteriole wall (Fig 8aDown) and the contour of adipocyte plasma membranes (Fig 8cDown). Excess of unlabeled ET-1 together with 125I–ET-1 resulted in the almost complete disappearance of silver grains in both arteriole (Fig 8bDown) and adipose tissue (Fig 8dDown), indicating a specific binding.



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  		Figure 8. In vitro autoradiographic localization of 125I–endothelin-1 binding sites in mesenteric arterioles (a and b) and surrounding adipose tissue (c and d) in the absence (a and c) and presence (b and d) of unlabeled endothelin-1 (original magnification x400).

Mesenteric Artery Perfusion
In isolated rat mesenteric arteries perfused with Krebs' solution, mean basal perfusion pressure in intact and defatted preparations was 21±2 and 19±1 mm Hg, respectively. NE, AVP, ET-1, and Ang II were administered in the perfusate of both intact and defatted preparations in amounts varying from 10 to 100 µg. Ang II up to 100 µg did not evoke a significant pressor response in intact (not shown) or defatted mesenteric arteries (Fig 9Down). NE, AVP, and ET-1 induced a dose-dependent pressor effect in both intact (not shown) and defatted preparations. NE exerted a transient pressor effect, whereas ET-1 elicited a long-lasting vasopressor response (Fig 9Down).



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  		Figure 9. Tracings showing representative responses of isolated defatted, perfused mesenteric arteries to several agents. NE indicates norepinephrine; ET-1, endothelin-1; AVP, vasopressin; and ANG II, angiotensin II.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
One of the facts that has been perplexing us during the last few years has been the lack of correlation between the absence of a biological effect of ANF in some resistance vessels, like the mesenteric arterial bed, and the reported presence of high-affinity ANF receptors in these same tissues. Thus, we have reported previously25 that ANF did not modify vascular resistance of the NE-perfused isolated rat mesenteric artery. This finding was later confirmed and extended to other resistance vascular beds.26 Moreover, Shen et al27 observed that the only vascular territory whose resistance was modified by ANF infusion into the dog was the kidney. It was evident that their findings contrasted with the description and characterization of high-affinity ANF receptors in the rat mesenteric artery bed.24 39

We now present evidence that the mesenteric artery in the rat does not contain ANF receptors, providing a valid and coherent explanation for the controversial reports on ANF's lack of biological activity and the presence of ANF receptors in the mesenteric artery. As shown in Fig 2bUp, no ANF binding could be discerned in mesenteric vascular membranes when the mesenteric arteries were completely defatted. Moreover, when the arteries were incubated in the presence of either ANF or CNP, no cGMP production was detected. The functional and histological integrity of the defatted mesenteric arteries was demonstrated by light microscopy, showing undamaged intimal, medial, and adventitial layers in the walls of all vessels examined (Fig 1bUp, inset), and cGMP production by these vessels was stimulated with SNP. Together, these results indicate that defatted mesenteric vessels are anatomically intact and metabolically active. Further evidence of the absence of ANF receptors in the mesenteric artery was provided by in situ autoradiography, 125I-ANF cross-linking, and ANF-stimulated cGMP production. In agreement with our binding experiments, an excess of unlabeled ANF failed to fully displace 125I-ANF from mesenteric arterioles (Fig 7Up). Furthermore, no band was observed during cross-linking experiments with mesenteric vascular membranes (Fig 6Up, lanes 5 and 6), and no ANF-stimulated cGMP production by mesenteric vessels could be detected (Fig 5bUp).

ANF receptors have recently been characterized in isolated rat renal preglomerular vessels,40 supporting previous physiological data demonstrating that ANF has a direct effect in the preglomerular vasculature.41 These findings and the present work underline the biological relevance of regional specificity and the differential distribution of receptors within the entire vascular tree. Moreover, our results emphasize the highly specific effect of ANF on the renal vasculature, which remains the only resistance vascular bed where both biological actions and ANF receptors are present. As internal controls for our ANF experiments, we chose two peptides known to be either weak (Ang II) or powerful (ET-1) vasoconstrictors of the arterial mesenteric bed. Although canine mesenteric arteries are sensitive to Ang II,42 vascular Ang II receptors have been identified and characterized mainly in rat mesenteric arteries,23 43 which are less responsive to Ang II than to either NE or AVP.28 In our experiments on isolated perfused mesenteric vascular beds, where the surrounding adipose tissue was completely removed or left intact, bolus administration of up to 100 µg Ang II did not modify perfusion pressure of either clean or intact preparations (Fig 9Up). These negative results correlate well with our Ang II binding studies in mesenteric vascular membranes, where very low Ang II receptor density was detected. In contrast, ET1 elicited strong vasoconstriction (Fig 9Up), and in agreement with its biological effect, 125I–ET-1 saturation binding experiments on clean defatted mesenteric preparations revealed the presence of high-affinity and high-capacity binding sites. By exposing isolated perfused mesenteric arteries to BQ-123, a highly specific ETA antagonist, it has been suggested32 that ET-1 vasoconstricts mesenteric arteries by activation of its ETA receptors. Moreover, our in vitro autoradiography of 125I–ET-1 clearly demonstrates specific ET binding sites in both arterial vascular bed and adipose tissue. We believe that our experiments on mesenteric vascular membranes and our radioligand studies are the first to offer direct evidence that in the rat mesenteric artery the predominant ET-1 receptor is indeed ETA. The presence of ETA receptors in defatted mesenteric arteries serves as a positive control, providing further proof that the absence or scarcity of ANF and Ang II receptors in these vessels is not due to tissue damage.

Unpublished results from our laboratory (R. Garcia, C. Crilley, M.-C. Bonhomme, 1994) have demonstrated that when adipocytes from the mesenteric territory are isolated by the collagenase method,44 their binding characteristics to Ang II, ANF, and ET-1 are conserved, suggesting that receptors for those peptides are truly present on adipose membranes.

Previous reports of ANF receptor localization in interscapular, epididymal, and mammary gland adipose tissue45 46 and the present study suggest that ANF receptors have a general distribution in adipose tissue. We have now extended these results by characterizing the ANF receptor subtype present in mesenteric adipocytes. Competitive binding experiments with ANF, CNP, and C-ANF showed that all three peptides displaced 125I-ANF from adipocyte membranes with very close affinities, not allowing the discrimination of any particular ANF receptor subtype. On the other hand, as shown in Fig 5aUp, incubation of adipose tissue with increasing doses of ANF or CNP clearly induced cGMP production in a dose-dependent manner, evidencing the presence of guanylate cyclase–coupled ANF receptors. Since previous studies in cells exclusively expressing the ANP-A receptor have demonstrated that CNP does not stimulate cGMP production,11 we must conclude that guanylate cyclase–coupled ANP-A and ANP-B receptors are both expressed in mesenteric adipocytes. The presence of a guanylate cyclase–bound receptor was further substantiated by cross-linking experiments of 125I-ANF with adipocyte membranes, showing a clear high-molecular-weight band (Fig 6Up, lane 1) representing {approx}3% of total cross-linked 125I-ANF. This high-molecular-weight band, together with the low-molecular-weight band, was completely displaced by unlabeled ANF, CNP, and C-ANF (Fig 6Up, lanes 2 through 4). This unusual behavior for a high-molecular-weight ANF receptor suggests the presence of either a new ANF receptor subtype or an isoform of already known ANF receptors. It is also possible that for unknown reasons high-molecular-weight ANF receptors (ANP-A and ANP-B) have a different affinity for ANF, CNP, and C-ANF in adipocyte membranes than in other tissues.

During the preparation of this manuscript, Ang II receptors were identified and characterized in rat epididymal adipose tissue.47 We now report that rat mesenteric adipocytes contain high-affinity Ang II binding sites that, according to competitive binding experiments with a highly specific nonpeptide Ang II antagonist, correspond to AT1.

As far as we know, this is the first report that describes and characterizes ET receptors in mature rat adipocytes. The presence of ET binding sites in rat preadipocytes has been reported previously, without further characterization.48 As noted above for mesenteric arteries, mesenteric adipocytes present high-affinity ET binding sites, being 64% BQ-123 sensitive. This suggests that as for arteries, most ET receptors in mesenteric adipose tissue correspond to ETA.

Resistance-sized vessels from different vascular beds may respond to vasoactive peptides in a distinctive manner that may be the result of the tissue-specific expression of their receptors. Thus, rat renal resistance vessels express well-characterized ANF and Ang II receptors35 40 as well as a functional response.41 49 On the other hand, ANF and Ang II receptors are not expressed or are only scantly expressed in the mesenteric artery (present results), which is not responsive to these peptides.25 26 28 The absence of arterial receptors does not necessarily mean that Ang II may not play a role in blood flow distribution in the vascular mesenteric bed, since it does have a potent vasoconstrictor effect in the venous vascular side.50 Moreover, the influence of periaortic adipose tissue on rat aortic responsiveness to vasoactive agents,30 the presence of angiotensinogen mRNA in periaortic and mesenteric adipocytes,31 and our present results suggest that perivascular adipose tissue may play a role in blood flow distribution by sympathetic, autocrine, or paracrine mechanisms. On the other hand, contrary to ANF and Ang II, ET has both a potent vasoconstrictor effect29 32 and high-affinity receptors, suggesting that it may play a local role in blood flow regulation.

We have previously reported51 52 that ANF receptors in mesenteric arteries could be pathophysiologically regulated. However, failure to totally remove all adipose tissue surrounding the mesenteric vascular bed may account for our previous findings.

In view of the fact that ANF, Ang II, and ET receptors in adipose tissue have no apparent association with the known major biological actions of these peptides, the physiological relevance of our observations in adipocytes remains to be elucidated.

In conclusion, we have reported in the present study that in concordance with the lack of biological effects of ANF and Ang II, receptors for these peptides are absent or scantly expressed in the mesenteric arterial bed. On the other hand, ET-1 exerts powerful vasoconstriction in the mesenteric artery, where high-affinity specific ET binding sites are present. Adipose tissue expresses high-affinity binding sites for ANF, Ang II, and ET-1, but their physiological role remains to be defined. Last, a high-molecular-weight ANF receptor present in adipocytes behaves in a manner different from that described in other tissues.


*    Acknowledgments
 
This study was supported by grants to Dr Garcia from the Medical Research Council of Canada (MT-11558) and the Kidney Foundation of Canada. H. de León was supported in part by a studentship from the Consejo Nacional de Ciencia y Tecnologia (CONACYT), Mexico, DF, Mexico. The authors thank Suzanne Diebold, Christian Charbonneau, and Feli Faraco-Cantin for their excellent technical assistance, Angie Poliseno for her invaluable secretarial help, and Ovid Da Silva for his editorial input.


*    Footnotes
 
Reprint requests to Raul Garcia, MD, Clinical Research Institute of Montreal, 110 Pine Ave West, Montreal, Quebec H2W 1R7, Canada.

Received December 27, 1993; accepted March 13, 1995.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
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 up arrowDiscussion
 *References
 

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