An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat

Abstract

Rat adrenomedullin is a novel 50-amino acid peptide with structural similarities to the calcitonin family of peptides, calcitonin, calcitonin gene-related peptide (CGRP), and islet amyloid polypeptide (IAPP). Using rat [125I]adrenomedullin, specific binding sites were demonstrated in heart, lung, spleen, liver, soleus, diaphragm, gastrocnemius, and spinal cord membranes. The highest binding was present in heart and lung, which was further characterized. These sites exhibited saturation, dissociation, and competition. In rat lung, only rat (IC50 = 5.8 nM) and human (IC50 = 94 nM) adrenomedullin competed with [125I]adrenomedullin. However, in rat heart, rat (IC50 = 0.2 nM) and human (IC50 = 4.2 nM) adrenomedullin, IAPP (IC50 = 240 nM), and CGRP (IC50 = 1050 nM) all competed with [125I] adrenomedullin. Saturation analysis revealed binding capacities and dissociation constants of 2.8 +/- 0.3 pmol/mg protein and 1.3 +/- 0.3 nM, respectively, in lung and 0.47 +/- 0.11 pmol/mg protein and 0.41 +/- 0.14 nM in heart. Comparison with [125I]CGRP- and [125I]IAPP-binding sites in lung showed that rat adrenomedullin could potently inhibit at these sites (IC50 = 5 and 6 nM, respectively). Chemical cross-linking demonstrated a major band of 83,000 mol wt in lung, diaphragm, spleen, and liver and a band of 94,000 mol wt in heart, soleus, and gastrocnemius. Thus, [125I]adrenomedullin-binding sites in rat lung are abundant and can be differentiated from binding sites in rat heart, both pharmacologically and by mol wt.

Purification and characterization of PAMP‐12 (PAMP[9–20]) in porcine adrenal medulla as a major endogenous biologically active peptide

“…N-Tyr-PAMP [10][11][12][13][14][15][16][17][18][19][20], human PAMP[l-20] (PAMP-20), PAMP [9][10][11][12][13][14][15][16][17][18][19][20] (PAMP-12), [10][11][12][13][14][15][16][17][18][19][20], [13][14][15][16][17][18][19][20], and [16][17][18][19][20] were synthesized by the solid phase method with a peptide synthesizer (Model 431 A, Applied Biosystems, Foster, CA, USA), and purified by reversed-phase high performance liquid chromatography (RP-HPLC).…”

“…PAMP [10][11][12][13][14][15][16][17][18][19][20] (8 mg) and bovine thyroglobulin (10 mg) were conjugated by the carbodimide method as described [13]. The reaction mixture was dialyzed four times against one liter of 0.9% NaCl and twice against one liter of 0.05 M sodium phosphate buffer (pH 7.4) containing 0.08 M NaCl.…”

“…The RIA for PAMP [10][11][12][13][14][15][16][17][18][19][20] was carried out as follows. The RIA incubation buffer for PAMP was 0.05 M sodium phosphate buffer (pH 7.4) containing 0.5% bovine serum albumin, 0.5% Triton X-100, 0.025 M EDTA-2Na, 0.05% NaN 3 , and 500 KlU/ml of aprotinin.…”

“…The RIA incubation buffer for PAMP was 0.05 M sodium phosphate buffer (pH 7.4) containing 0.5% bovine serum albumin, 0.5% Triton X-100, 0.025 M EDTA-2Na, 0.05% NaN 3 , and 500 KlU/ml of aprotinin. The RIA incubation mixture was composed of 100 ul of the standard PAMP [10][11][12][13][14][15][16][17][18][19][20] or the unknown sample with 200 ul of an antiserum which contained 0.5% normal rabbit serum, which was designated PAMP Ab-M#6-3, at the dilution of 1:6650. After incubation for 12 h, 100 itl of 125 I-N-Tyr-PAMP [10][11][12][13][14][15][16][17][18][19][20]NH 2 (18000 cpm) was added and incubated for 24 h. One hundred ul of anti-rabbit IgG goat serum diluent was then added, and incubation was continued for 24 h. The tubes were centrifuged at 2000 Xg at 4°C for 30 min, and the radioactivity of the precipitate was measured with a gamma-counter (Aloka ARC-600, Tokyo).…”

“…PAMP The antiserum to PAMP [10][11][12][13][14][15][16][17][18][19][20], P A M P A b -M # 6 -3 , recognized synthetic P A M P [10][11][12][13][14][15][16][17][18][19][20] with high affinity at a final dilution of 1:13 300. As shown in Fig.…”