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Adrenomedullin receptors in rat cerebral microvessels
Molecular Brain Research 81 (2000) 1–6 www.elsevier.com / locate / bres
Research report
Adrenomedullin receptors in rat cerebral microvessels Hideyuki Kobayashi a , *, Shin-ichi Minami a , Ryuichi Yamamoto a , Keizo Masumoto a , Toshihiko Yanagita a , Yasuhito Uezono a , Kimiyuki Tsuchiya b , Motohiko Mohri c , Kazuo Kitamura d , Tanenao Eto d , Akihiko Wada a a
Department of Pharmacology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889 -1692, Japan Experimental Animal Center, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889 -1692, Japan c Marine Ecosystems Research Department, Japan Marine Science and Technology Center, 2 -15 Natsushima, Yokosuka 237 -0061, Japan d Department of Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889 -1692, Japan b
Accepted 9 May 2000
Abstract To characterize the sites of action of adrenomedullin (AM) in the cerebral microvasculature, we studied the effect of AM on cyclic AMP (cAMP) level as well as expression of AM and its receptor in the rat cerebral microvessels. The microvessels were prepared from rat cerebral cortex by albumin flotation and glass bead filtration technique. AM and calcitonin gene-related peptide (CGRP) increased cAMP level in the microvessels in a concentration-dependent manner. The effect of AM was more than 100 times more potent than that of CGRP. The accumulation of cAMP by AM was inhibited by AM[22–52], an AM receptor antagonist, but not by CGRP[8–37], a CGRP receptor antagonist, suggesting that AM increased cAMP accumulation by acting on receptors specific to AM. [ 125 I]AM binding to the microvessels was displaced by AM and less potently by AM[22–52]. The displacing potencies of CGRP and CGRP[8–37] were very weak. mRNAs for AM as well as calcitonin-receptor-like receptor and receptor-activity-modifying protein 2 which form a receptor specific to AM, were highly expressed in the microvessels. These results provide biochemical and pharmacological evidence that AM is produced in and acts on the cerebral microvessels in an autocrine / paracrine manner and is involved in regulation of cerebral microcirculation. 2000 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Blood–brain barrier Keywords: Adrenomedullin; Cerebral microvessel; cAMP; CGRP, Calcitonin-receptor-like receptor; Receptor-activity-modifying protein
1. Introduction Adrenomedullin (AM) has been discovered as a hypotensive peptide from pheochromocytoma by monitoring its action on cyclic AMP (cAMP) in platelet [12]. AM consists of 52 amino acids, and its intramolecular ring structure and amidated carboxy terminal are homologous to those of calcitonin gene-related peptide (CGRP), a potent vasodilating peptide [12]. AM and its mRNA are expressed not only in pheochromocytoma but also many tissues such as adrenal medulla, lung and heart [21]. The binding sites for AM are also present in various tissues *Corresponding author. Tel.: 181-985-851-786; fax: 181-985-842776. E-mail address: [email protected] (H. Kobayashi).
including heart, lung and kidney suggesting that AM is involved in regulation of various physiological functions [20]. In the brain, AM expression dramatically increased in neurons after focal cerebral ischemia. In addition, AM dilates pial and basilar arteries, and AM administration modified focal ischemic injury, suggesting AM plays a role in regulation of cerebral circulation [2,23]. Although the presence of its binding sites in peripheral vasculature was reported [9,22], it is not known whether AM acts directly on the cerebral microvessels or not. The signal transduction pathway of AM has not been clarified completely but in many cell types its biological activity is produced through the production of cAMP [4,9]. The functional receptor specific for AM is reported to be expressed by calcitonin-receptor-like receptor (CRLR) with receptor-activity-modifying protein 2 (RAMP2),
0169-328X / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00148-0
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H. Kobayashi et al. / Molecular Brain Research 81 (2000) 1 – 6
while combination of CRLR with RAMP1 produced a receptor for CGRP [17]. In the present study, to know whether the sites of action of AM are present in the cerebral microvessels, we studied the effect of AM on cAMP level, binding sites for AM as well as the expression of AM receptor in the cerebral microvessels.
fonylfluoride, 0.2 mg / ml bacitracin, 100 KIU / ml aprotinin and 1 mg / ml leupeptin at 48C for 120 min in a microtube. The [ 125 I]AM bound to the microvessels was separated by centrifugation through silicon oil and dinonyl phthalate (1:1) at 12,000 rev. / min for 5 min, and counted by a gamma-counter. The specific binding was defined as the total binding minus non-specific binding which was determined in the presence of an excess concentration of 1 mM AM, and was 70–80% of total binding.
2. Materials and methods
2.1. Materials cAMP radioimmunoassay kit was obtained from Yamasa (Chiba, Japan). TRIzolE, DNase I, SuperScript II reverse transcriptase and RNase H were from Life Technologies (Tokyo, Japan). Ex Taq polymerase was from Takara (Otsu, Japan). Rat AM, rat CGRP, human AM[22–52] and human CGRP[8–37] were from Peptide Institute (Osaka, Japan). Human AM (Peptide Institute) was iodinated by lactoperoxidase method and monoiodinated [ 125 I]AM was purified by reverse phase HPLC.
2.2. Preparation of cerebral microvessels Cerebral microvessels were prepared from male Sprague–Dawley rats using the albumin flotation and glass bead filtration techniques. The purity of the microvessels was checked by phase contrast microscopy and they were free of neural and glial elements. In addition, activity of g-glutamyl transpeptidase, a marker enzyme of cerebral capillaries, was about 30 times higher than that in the cerebral cortex as described previously [13,14].
2.3. Measurement of cAMP The microvessels were preincubated with 1 mM isobutylmethylxanthine at 378C for 5 min in Ringer’s solution (KRH) composed of 137 mM NaCl, 5 mM KCl, 1.2 mM MgSO 4 , 2.2 mM CaCl 2 , 12 mM NaHCO 3 , 15 mM HEPES and 5 mM glucose (pH 7.4) containing 0.2% bovine serum albumin (BSA). They were further incubated with peptides for 5 min, and the incubation was terminated by the addition of 0.5 M HCl to make a final concentration of 0.1 M and heated at 958C for 3 min. After centrifugation at 10,0003g for 10 min, cAMP in the supernatant was measured by radioimmunoassay. Aliquots of microvessels were washed with phosphate buffered saline, solubilized by 1 M NaOH, and the protein concentration was measured by the method of Lowry et al. [16].
2.4. [ 125 I] AM binding assay Microvessels were incubated with 160 pM [ 125 I]AM in the presence of various concentrations of peptide in KRH containing 0.2% BSA, 0.5 mM phenylmethylsul-
2.5. Reverse transcription-polymerase chain reaction ( RT-PCR) RNA of the cerebral microvessels and cerebral cortex was isolated using TRIzolE, treated with DNase I and reverse transcribed to cDNA with SuperScript II using oligo(dT) 12 – 18 . After treatment with RNase H, cDNA (derived from 2-200 ng of total RNA) was amplified with 1.25 units of Ex Taq, 0.5 mM of each primer, 200 mM of each dNTP at 2 mM of Mg 21 concentration in a total volume of 50 ml. The amplification reaction consisted of 30 (RAMPs, CRLR, AM) or 25 (GAPDH) cycles of denaturation (948C, 1 min), annealing (668C for AM, 608C for CRLR and GAPDH, 558C for RAMPs, 1 min), and extension (728C, 1 min). Primers and sequence numbers are as follows: CRLR, 59 primer, 768–792, 39 primer, 1310–1286 of L27487; PAMP1, 59 primer, 189–208, 39 primer, 451–435 of AJ001014; RAMP2, 59 primer 385– 402, 39 primer, 517–498 of AJ001015; RAMP3, 59 primer, 189–208, 39 primer, 347–328 of AJ001016; AM, 59 primer, 145–175, 39 primer, 733–704 of D15069; GAPDH, 59 primer, 207–236, 39 primer, 893–864 of M17701. PCR products were subjected to electrophoresis on 2 or 3% agarose gel. The gels were stained with ethidium bromide and photographed. The density of band was quantified by Gel Doc 2000 (Bio Rad, Richmond, CA).
3. Results
3.1. Increase in cAMP by AM in cerebral microvessels AM increased cAMP level of the cerebral microvessels in a concentration-dependent manner. AM at concentration as low as 10 29 M increased cAMP level significantly. cAMP level was raised to 2.4-, 4.7-, 5.5- and 6.2-fold of control by 10 29 , 10 28 , 10 27 and 10 26 M, respectively (Fig. 1). CGRP also increased cAMP level of the cerebral microvessels in a concentration-dependent manner, but the effect was much weaker than that of AM. The response by both peptides did not reach a plateau level at concentration of 10 26 M. To get the same increase in cAMP, CGRP required more than 100 times higher concentration than AM. Thus, cAMP production in the cerebral microvessels is preferentially stimulated by AM (Fig. 1, upper panel).
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Fig. 2. Displacement of [ 125 I]AM binding to the cerebral microvessels by AM-related peptides. Microvessels were incubated with [ 125 I]AM in the presence of various concentrations of AM (s), AM[22–52] (n), CGRP (d) or CGRP[8–37] (h), and [ 125 I]AM bound to the microvessels was counted. Data are mean6S.E.M. of triplicate determinations of a representative of three experiments with similar results.
AM[22–52] (IC 50 : 7.6310 29 M) and much less potently by CGRP or CGRP[8–37] (IC 50 : .10 27 M) (Fig. 2). The potency of AM to displace [ 125 I]AM binding to the microvessels was three orders of magnitude more potent than CGRP or CGRP[8–37], suggesting [ 125 I]AM binding sites in the cerebral microvessels are highly specific to AM. Fig. 1. Increase in cAMP in cerebral microvessels by AM and CGRP. Upper panel: microvessels were preincubated with 1 mM isobutylmethylxanthine for 5 min and further incubated with AM (s) or CGRP (d) at 378C for 5 min, and cAMP was measured by radioimmunoassay. Each value is mean6S.E.M. of four experiments. Lower panel: microvessels preincubated with 1 mM AM[22–52] (n) or 1 mM CGRP[8–37] (h) in the presence of 1 mM isobutylmethylxanthine for 5 min were incubated with various concentrations of AM at 378C for 5 min, and cAMP was measured by radioimmunoassay. Each value is mean6S.E.M. of four experiments. *P,0.05 significantly different from stimulated by AM (s) at corresponding concentration by two-way ANOVA with Scheffe F-test.
The increase in cAMP by AM was suppressed by an AM receptor antagonist, AM[22–52] but not by a CGRP receptor antagonist, CGRP[8–37], indicating that the cerebral microvessels express receptors highly specific to AM (Fig. 1, lower panel). The concentration–response curve for AM was shifted to the right by AM[22–52] indicating that AM[22–52] antagonizes AM receptor in a competitive manner (Fig. 1, lower panel). The IC 50 value of AM[22– 52] for the increase in cAMP caused by 10 28 M AM was 1.860.6 mM (n53), whereas that of CGRP[8–37] was greater than 10 mM (data not shown).
3.2. Displacement of [ 125 I] AM binding to cerebral microvessels by AM-related peptides Binding of [ 125 I]AM to the cerebral microvessels was displaced by AM (IC 50 : 3.0310 210 M), less potently by
3.3. RT-PCR analysis of mRNA for AM receptor and AM To examine the gene expression of AM receptors in the cerebral microvessels, mRNAs encoding CRLR and RAMPs were analyzed by RT-PCR; 543 base pairs of 768 base–1310 base of rat CRLR cDNA were amplified by PCR. When the reverse transcription was omitted, no amplification was observed, suggesting the PCR product was amplified from cDNA reverse transcribed from RNA but not from genomic DNA of the cells. PCR product increased in a concentration-dependent manner to cDNA in the reaction mixture, and was abundant both in the cerebral cortex and microvessels (Fig. 3). Expression of RAMP2 mRNA was much higher, whereas those for RAMP1 and RAMP3 were lower in the microvessels than in the cerebral cortex, suggesting that receptors specific to AM in the microvessels were formed by a high expression of RAMP2 with CRLR. Expression of AM was also high in the cerebral microvessels (Fig. 3e), suggesting that AM is produced and secreted from the microvessels and may act as an autocrine / paracrine regulator of cerebral microcirculation. The specificity of RT-PCR was confirmed by cleavage of the products by restriction enzymes (Fig. 3b, d and f). Since mRNA for GAPDH was more abundant in the cerebral cortex than in the cerebral microvessels, the expression of CRLR, RAMP2 and AM corrected with
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Fig. 3. Expression of CRLR, RAMPs, AM and GAPDH mRNA in cerebral cortex and cerebral microvessels. RT-PCR products of CRLR (a), RAMPs (c), AM (e) and GAPDH (g) mRNA (2 ng, lanes 2, 6; 20 ng, lanes 3, 7; 200 ng, lanes 1, 4, 5, 8) of the cerebral cortex (1–4) and cerebral microvessels (5–8) were separated by agarose gel electrophoresis. Reverse transcription was omitted in lanes 1, 5. PCR reaction was also carried out with H 2 O as a negative control (lane 9). The DNA size marker was a 50 bp ladder with intense bands of 50, 350 and 800 bp as shown on the left. The sizes of RT-PCR product of CRLR (543 bp), RAMP1 (263 bp), RAMP2 (133 bp), RAMP3 (159 bp), AM (589 bp), and GAPDH (687 bp) are shown on the right. (b) Cleavage of 543 bp CRLR PCR product of the cerebral cortex (1–5) and microvessels (6–10) by BarI (2, 7) to 506, 21 and 16 bp fragments, by BamHI (3, 8) to 73 and 470 bp, by RsaI (4, 9) to 170 and 373 bp, and by TaqI (5, 10) to 473 and 70 bp, respectively. (d) Cleavage of PCR products of RAMP1 (1–4), RAMP2 (5–8) and RAMP3 (9–14) of the cerebral cortex (1, 2, 5, 6, 9–11) and microvessels (3, 4, 7, 8, 12–14). PCR product of RAMP1 (263 bp) was cleaved by SacI to 50 and 213 bp fragments (2, 4), RAMP2 (133 bp) by EcoT14I to 25, 54 and 54 bp (6, 8), or RAMP3 (159 bp) by RsaI to 34 and 125 bp (10, 13) or by HindIII to 41 and 118 bp fragments (11, 14), respectively. (f) Cleavage of 589 bp AM PCR product of the cerebral cortex (1–3) and microvessels (4–6) by NarI (2, 5) to 447 and 142 bp fragments, and by TaqI (3, 6) to 80, 196 and 313 bp fragments, respectively. PCR products of RAMP1 slightly larger than 263 bp and of AM slightly smaller than 589 bp were also detected, but these bands are not likely to be a specific PCR product from each mRNA since they were not cut by restriction enzymes specific to each sequence. Each photograph is a representative of three experiments of different preparations of the microvessels.
H. Kobayashi et al. / Molecular Brain Research 81 (2000) 1 – 6
GAPDH was higher in the cerebral microvessel than in the cerebral cortex. The ratio of PCR product density of RAMP2 / GAPDH as well as AM / GAPDH in the cerebral microvessels was more than 10 times greater than that in the cerebral cortex.
4. Discussion In this paper, we found that AM increased cAMP level by reacting with receptors of high affinity and specific to AM in the cerebral microvessels. In addition, mRNAs for AM, RAMP2 and CRLR were highly expressed in the cerebral microvessels suggesting that AM is produced in the microvessels and act on themselves in an autocrine / paracrine manner and is involved in the regulation of cerebral microcirculation. Receptors for AM may be subdivided into two subtypes. In the rat mesenteric vasculature contracted with methoxiamine, AM decreased perfusion pressure which is suppressed by CGRP[8–37] [19]. In addition, in cultured vascular smooth muscle cells of rat, AM increased cAMP, and the effect was suppressed by CGRP[8–37]. Therefore, the receptors present in the cells such as vascular smooth muscle react not only with AM but also with CGRP [4]. On the other hand, AM increased cAMP level in the cultured endothelial cells of human [3] and bovine [22], Rat-2 fibroblast cells [1] and mouse Swiss 3T3 fibroblast cells [24], but the effect was not suppressed by CGRP[8– 37] indicating that receptors in these cells are specific to AM. The receptor for AM in the cerebral microvessel was of this type highly specific to AM. Because the reactivity of cells may change during culture, our results show, for the first time, that native endothelial cells express receptors specific to and high affinity to AM. The high specificity of receptors to AM in the cerebral microvessel is much different from the reactivity of the cerebral arterioles where CGRP dilates at lower concentrations than did AM, and the vasodilatatory effect of AM is strongly suppressed by CGRP[8–37] [18]. Thus, the difference of specificity to the peptides between the cerebral microvessels and arterioles suggests a distinct functional role of the receptors depending on the size of vessels. An orphan G-protein coupled receptor (L1) was previously reported as an AM receptor [8]. It has been shown to mediate an increase of cAMP response to AM and to bind with [ 125 I]AM with high affinity (KD : 8.2 nM). Its expression pattern was similar to the distribution of [ 125 I]AM binding sites in rat tissues. However, cAMP formation by AM and AM binding to the COS-7 cells expressing L1 receptor has not been confirmed [11], suggesting that L1 did not represent a real receptor for AM. On the other hand, a receptor cloned as CRLR behaved as an AM receptor when it was co-expressed with RAMP2 [17]. In fact, a functional AM receptor composed of CRLR with
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RAMP2 was expressed in human endothelial and vascular smooth muscle, and the reactivity to the peptides was confirmed in the COS-7 cells expressing in these clones [7]. Therefore, we examined the expression of CRLR and RAMPs in the cerebral microvessels, and found a very high expression of RAMP2. The high expression of RAMP2 with CRLR would form a specific receptor for AM, which is in good agreement with the increase in cAMP and the displacement of [ 125 I]AM binding caused specifically by AM. AM receptors in the cerebral microvessels may play a role in regulation of production of biologically active peptides such as endothelin. Endothelin, a potent basoconstrictor, is produced and secreted from the cerebral microvessels. The secretion of endothelin is suppressed by cAMP [25], whereas endothelin inhibits adenylate cyclase in the endothelial cells of brain microvessels [15]. Thus, AM and endothelin may regulate cerebral microcirculation by interacting the action of each peptide. Although cAMP has been reported to have the function of regulation of macromolecular transport of proteins such as albumin [5], its precise role in regulation of permeability of the cerebral microvessels is still unclear [6]. On the other hand, since forskolin, an activator of adenylate cyclase, decreased incorporation of thymidine in cultured cerebral microvessel endothelial cells [10], AM might be involved in regulation of cell growth. AM may activate signal transduction pathways other than cAMP pathway such as Ca / calmodulin and mitogen activated protein kinase pathways but only at higher concentration in other cell types [1,22]. In addition to the demonstration of an autocrine / paracrine regulator of AM in the cerebral microvessels of the present study, the elucidation of transduction pathways of AM would serve for better understanding for regulatory mechanisms of the cerebral microcirculation.
Acknowledgements This work is supported in part by research grants from the Ministry of Education, Science and Culture of Japan, Miyazaki Medical College (grant for Special Research Project) and Japan Marine Science and Technology Center.
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[15] A. Ladoux, C. Frelin, Endothelins inhibit adenylate cyclase in brain capillary endothelial cells, Biochem. Biophys. Res. Commun. 180 (1991) 169–173. [16] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [17] L.M. McLatchie, N.J. Fraser, M.J. Main, A. Wise, J. Brown, N. Thompson, R. Solari, M.G. Lee, S.M. Foord, RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor, Nature 393 (1998) 333–339. [18] Y. Mori, M. Takayasu, Y. Suzuki, M. Shibuya, J. Yoshida, H. Hidaka, Effects of adrenomedullin on rat cerebral arterioles, Eur. J. Pharmacol. 330 (1997) 195–198. [19] C. Nuki, H. Kawasaki, K. Kitamura, M. Takenaga, K. Kangawa, T. Eto, A. Wada, Vasodilator effect of adrenomedullin and calcitonin gene-related peptide receptors in rat mesenteric vascular beds, Biochem. Biophys. Res. Commun. 196 (1993) 245–251. [20] A.A. Owji, D.M. Smith, H.A. Coppock, D.G. Morgan, R. Bhogal, M.A. Ghatei, S.R. Bloom, An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat, Endocrinology 136 (1995) 2127–2134. [21] J. Sakata, T. Shimokubo, K. Kitamura, M. Nishizono, Y. Iehiki, K. Kangawa, H. Matsuo, T. Eto, Distribution and characterization of immunoreactive rat adrenomedullin in tissue and plasma, FEBS Lett. 352 (1994) 105–108. [22] Y. Shimekake, K. Nagata, S. Ohta, Y. Kambayashi, H. Teraoka, K. Kitamura, T. Eto, K. Kangawa, H. Matsuo, Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca 21 mobilization, in bovine aortic endothelial cells, J. Biol. Chem. 270 (1995) 4412–4417. [23] X. Wang, T.L. Yue, F.C. Barone, R.F. White, R.K. Clark, R.N. Willette, A.C. Sulpizio, N.V. Aiyar, R.R. Ruffolo Jr., G.Z. Feuerstein, Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage, Proc. Natl. Acad. Sci. USA 92 (1995) 11480–11484. [24] D.J. Withers, H.A. Coppock, T. Seufferlein, D.M. Smith, S.R. Bloom, E. Rozengurt, Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells, FEBS Lett. 378 (1996) 83–87. [25] M.A. Yakubu, C.W. Leffler, Regulation of ET-1 biosynthesis in cerebral microvascular endothelial cells by vasoactive agents and PKC, Am. J. Physiol. 276 (1999) C300–305.
Research report
Adrenomedullin receptors in rat cerebral microvessels Hideyuki Kobayashi a , *, Shin-ichi Minami a , Ryuichi Yamamoto a , Keizo Masumoto a , Toshihiko Yanagita a , Yasuhito Uezono a , Kimiyuki Tsuchiya b , Motohiko Mohri c , Kazuo Kitamura d , Tanenao Eto d , Akihiko Wada a a
Department of Pharmacology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889 -1692, Japan Experimental Animal Center, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889 -1692, Japan c Marine Ecosystems Research Department, Japan Marine Science and Technology Center, 2 -15 Natsushima, Yokosuka 237 -0061, Japan d Department of Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889 -1692, Japan b
Accepted 9 May 2000
Abstract To characterize the sites of action of adrenomedullin (AM) in the cerebral microvasculature, we studied the effect of AM on cyclic AMP (cAMP) level as well as expression of AM and its receptor in the rat cerebral microvessels. The microvessels were prepared from rat cerebral cortex by albumin flotation and glass bead filtration technique. AM and calcitonin gene-related peptide (CGRP) increased cAMP level in the microvessels in a concentration-dependent manner. The effect of AM was more than 100 times more potent than that of CGRP. The accumulation of cAMP by AM was inhibited by AM[22–52], an AM receptor antagonist, but not by CGRP[8–37], a CGRP receptor antagonist, suggesting that AM increased cAMP accumulation by acting on receptors specific to AM. [ 125 I]AM binding to the microvessels was displaced by AM and less potently by AM[22–52]. The displacing potencies of CGRP and CGRP[8–37] were very weak. mRNAs for AM as well as calcitonin-receptor-like receptor and receptor-activity-modifying protein 2 which form a receptor specific to AM, were highly expressed in the microvessels. These results provide biochemical and pharmacological evidence that AM is produced in and acts on the cerebral microvessels in an autocrine / paracrine manner and is involved in regulation of cerebral microcirculation. 2000 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Blood–brain barrier Keywords: Adrenomedullin; Cerebral microvessel; cAMP; CGRP, Calcitonin-receptor-like receptor; Receptor-activity-modifying protein
1. Introduction Adrenomedullin (AM) has been discovered as a hypotensive peptide from pheochromocytoma by monitoring its action on cyclic AMP (cAMP) in platelet [12]. AM consists of 52 amino acids, and its intramolecular ring structure and amidated carboxy terminal are homologous to those of calcitonin gene-related peptide (CGRP), a potent vasodilating peptide [12]. AM and its mRNA are expressed not only in pheochromocytoma but also many tissues such as adrenal medulla, lung and heart [21]. The binding sites for AM are also present in various tissues *Corresponding author. Tel.: 181-985-851-786; fax: 181-985-842776. E-mail address: [email protected] (H. Kobayashi).
including heart, lung and kidney suggesting that AM is involved in regulation of various physiological functions [20]. In the brain, AM expression dramatically increased in neurons after focal cerebral ischemia. In addition, AM dilates pial and basilar arteries, and AM administration modified focal ischemic injury, suggesting AM plays a role in regulation of cerebral circulation [2,23]. Although the presence of its binding sites in peripheral vasculature was reported [9,22], it is not known whether AM acts directly on the cerebral microvessels or not. The signal transduction pathway of AM has not been clarified completely but in many cell types its biological activity is produced through the production of cAMP [4,9]. The functional receptor specific for AM is reported to be expressed by calcitonin-receptor-like receptor (CRLR) with receptor-activity-modifying protein 2 (RAMP2),
0169-328X / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00148-0
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H. Kobayashi et al. / Molecular Brain Research 81 (2000) 1 – 6
while combination of CRLR with RAMP1 produced a receptor for CGRP [17]. In the present study, to know whether the sites of action of AM are present in the cerebral microvessels, we studied the effect of AM on cAMP level, binding sites for AM as well as the expression of AM receptor in the cerebral microvessels.
fonylfluoride, 0.2 mg / ml bacitracin, 100 KIU / ml aprotinin and 1 mg / ml leupeptin at 48C for 120 min in a microtube. The [ 125 I]AM bound to the microvessels was separated by centrifugation through silicon oil and dinonyl phthalate (1:1) at 12,000 rev. / min for 5 min, and counted by a gamma-counter. The specific binding was defined as the total binding minus non-specific binding which was determined in the presence of an excess concentration of 1 mM AM, and was 70–80% of total binding.
2. Materials and methods
2.1. Materials cAMP radioimmunoassay kit was obtained from Yamasa (Chiba, Japan). TRIzolE, DNase I, SuperScript II reverse transcriptase and RNase H were from Life Technologies (Tokyo, Japan). Ex Taq polymerase was from Takara (Otsu, Japan). Rat AM, rat CGRP, human AM[22–52] and human CGRP[8–37] were from Peptide Institute (Osaka, Japan). Human AM (Peptide Institute) was iodinated by lactoperoxidase method and monoiodinated [ 125 I]AM was purified by reverse phase HPLC.
2.2. Preparation of cerebral microvessels Cerebral microvessels were prepared from male Sprague–Dawley rats using the albumin flotation and glass bead filtration techniques. The purity of the microvessels was checked by phase contrast microscopy and they were free of neural and glial elements. In addition, activity of g-glutamyl transpeptidase, a marker enzyme of cerebral capillaries, was about 30 times higher than that in the cerebral cortex as described previously [13,14].
2.3. Measurement of cAMP The microvessels were preincubated with 1 mM isobutylmethylxanthine at 378C for 5 min in Ringer’s solution (KRH) composed of 137 mM NaCl, 5 mM KCl, 1.2 mM MgSO 4 , 2.2 mM CaCl 2 , 12 mM NaHCO 3 , 15 mM HEPES and 5 mM glucose (pH 7.4) containing 0.2% bovine serum albumin (BSA). They were further incubated with peptides for 5 min, and the incubation was terminated by the addition of 0.5 M HCl to make a final concentration of 0.1 M and heated at 958C for 3 min. After centrifugation at 10,0003g for 10 min, cAMP in the supernatant was measured by radioimmunoassay. Aliquots of microvessels were washed with phosphate buffered saline, solubilized by 1 M NaOH, and the protein concentration was measured by the method of Lowry et al. [16].
2.4. [ 125 I] AM binding assay Microvessels were incubated with 160 pM [ 125 I]AM in the presence of various concentrations of peptide in KRH containing 0.2% BSA, 0.5 mM phenylmethylsul-
2.5. Reverse transcription-polymerase chain reaction ( RT-PCR) RNA of the cerebral microvessels and cerebral cortex was isolated using TRIzolE, treated with DNase I and reverse transcribed to cDNA with SuperScript II using oligo(dT) 12 – 18 . After treatment with RNase H, cDNA (derived from 2-200 ng of total RNA) was amplified with 1.25 units of Ex Taq, 0.5 mM of each primer, 200 mM of each dNTP at 2 mM of Mg 21 concentration in a total volume of 50 ml. The amplification reaction consisted of 30 (RAMPs, CRLR, AM) or 25 (GAPDH) cycles of denaturation (948C, 1 min), annealing (668C for AM, 608C for CRLR and GAPDH, 558C for RAMPs, 1 min), and extension (728C, 1 min). Primers and sequence numbers are as follows: CRLR, 59 primer, 768–792, 39 primer, 1310–1286 of L27487; PAMP1, 59 primer, 189–208, 39 primer, 451–435 of AJ001014; RAMP2, 59 primer 385– 402, 39 primer, 517–498 of AJ001015; RAMP3, 59 primer, 189–208, 39 primer, 347–328 of AJ001016; AM, 59 primer, 145–175, 39 primer, 733–704 of D15069; GAPDH, 59 primer, 207–236, 39 primer, 893–864 of M17701. PCR products were subjected to electrophoresis on 2 or 3% agarose gel. The gels were stained with ethidium bromide and photographed. The density of band was quantified by Gel Doc 2000 (Bio Rad, Richmond, CA).
3. Results
3.1. Increase in cAMP by AM in cerebral microvessels AM increased cAMP level of the cerebral microvessels in a concentration-dependent manner. AM at concentration as low as 10 29 M increased cAMP level significantly. cAMP level was raised to 2.4-, 4.7-, 5.5- and 6.2-fold of control by 10 29 , 10 28 , 10 27 and 10 26 M, respectively (Fig. 1). CGRP also increased cAMP level of the cerebral microvessels in a concentration-dependent manner, but the effect was much weaker than that of AM. The response by both peptides did not reach a plateau level at concentration of 10 26 M. To get the same increase in cAMP, CGRP required more than 100 times higher concentration than AM. Thus, cAMP production in the cerebral microvessels is preferentially stimulated by AM (Fig. 1, upper panel).
H. Kobayashi et al. / Molecular Brain Research 81 (2000) 1 – 6
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Fig. 2. Displacement of [ 125 I]AM binding to the cerebral microvessels by AM-related peptides. Microvessels were incubated with [ 125 I]AM in the presence of various concentrations of AM (s), AM[22–52] (n), CGRP (d) or CGRP[8–37] (h), and [ 125 I]AM bound to the microvessels was counted. Data are mean6S.E.M. of triplicate determinations of a representative of three experiments with similar results.
AM[22–52] (IC 50 : 7.6310 29 M) and much less potently by CGRP or CGRP[8–37] (IC 50 : .10 27 M) (Fig. 2). The potency of AM to displace [ 125 I]AM binding to the microvessels was three orders of magnitude more potent than CGRP or CGRP[8–37], suggesting [ 125 I]AM binding sites in the cerebral microvessels are highly specific to AM. Fig. 1. Increase in cAMP in cerebral microvessels by AM and CGRP. Upper panel: microvessels were preincubated with 1 mM isobutylmethylxanthine for 5 min and further incubated with AM (s) or CGRP (d) at 378C for 5 min, and cAMP was measured by radioimmunoassay. Each value is mean6S.E.M. of four experiments. Lower panel: microvessels preincubated with 1 mM AM[22–52] (n) or 1 mM CGRP[8–37] (h) in the presence of 1 mM isobutylmethylxanthine for 5 min were incubated with various concentrations of AM at 378C for 5 min, and cAMP was measured by radioimmunoassay. Each value is mean6S.E.M. of four experiments. *P,0.05 significantly different from stimulated by AM (s) at corresponding concentration by two-way ANOVA with Scheffe F-test.
The increase in cAMP by AM was suppressed by an AM receptor antagonist, AM[22–52] but not by a CGRP receptor antagonist, CGRP[8–37], indicating that the cerebral microvessels express receptors highly specific to AM (Fig. 1, lower panel). The concentration–response curve for AM was shifted to the right by AM[22–52] indicating that AM[22–52] antagonizes AM receptor in a competitive manner (Fig. 1, lower panel). The IC 50 value of AM[22– 52] for the increase in cAMP caused by 10 28 M AM was 1.860.6 mM (n53), whereas that of CGRP[8–37] was greater than 10 mM (data not shown).
3.2. Displacement of [ 125 I] AM binding to cerebral microvessels by AM-related peptides Binding of [ 125 I]AM to the cerebral microvessels was displaced by AM (IC 50 : 3.0310 210 M), less potently by
3.3. RT-PCR analysis of mRNA for AM receptor and AM To examine the gene expression of AM receptors in the cerebral microvessels, mRNAs encoding CRLR and RAMPs were analyzed by RT-PCR; 543 base pairs of 768 base–1310 base of rat CRLR cDNA were amplified by PCR. When the reverse transcription was omitted, no amplification was observed, suggesting the PCR product was amplified from cDNA reverse transcribed from RNA but not from genomic DNA of the cells. PCR product increased in a concentration-dependent manner to cDNA in the reaction mixture, and was abundant both in the cerebral cortex and microvessels (Fig. 3). Expression of RAMP2 mRNA was much higher, whereas those for RAMP1 and RAMP3 were lower in the microvessels than in the cerebral cortex, suggesting that receptors specific to AM in the microvessels were formed by a high expression of RAMP2 with CRLR. Expression of AM was also high in the cerebral microvessels (Fig. 3e), suggesting that AM is produced and secreted from the microvessels and may act as an autocrine / paracrine regulator of cerebral microcirculation. The specificity of RT-PCR was confirmed by cleavage of the products by restriction enzymes (Fig. 3b, d and f). Since mRNA for GAPDH was more abundant in the cerebral cortex than in the cerebral microvessels, the expression of CRLR, RAMP2 and AM corrected with
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H. Kobayashi et al. / Molecular Brain Research 81 (2000) 1 – 6
Fig. 3. Expression of CRLR, RAMPs, AM and GAPDH mRNA in cerebral cortex and cerebral microvessels. RT-PCR products of CRLR (a), RAMPs (c), AM (e) and GAPDH (g) mRNA (2 ng, lanes 2, 6; 20 ng, lanes 3, 7; 200 ng, lanes 1, 4, 5, 8) of the cerebral cortex (1–4) and cerebral microvessels (5–8) were separated by agarose gel electrophoresis. Reverse transcription was omitted in lanes 1, 5. PCR reaction was also carried out with H 2 O as a negative control (lane 9). The DNA size marker was a 50 bp ladder with intense bands of 50, 350 and 800 bp as shown on the left. The sizes of RT-PCR product of CRLR (543 bp), RAMP1 (263 bp), RAMP2 (133 bp), RAMP3 (159 bp), AM (589 bp), and GAPDH (687 bp) are shown on the right. (b) Cleavage of 543 bp CRLR PCR product of the cerebral cortex (1–5) and microvessels (6–10) by BarI (2, 7) to 506, 21 and 16 bp fragments, by BamHI (3, 8) to 73 and 470 bp, by RsaI (4, 9) to 170 and 373 bp, and by TaqI (5, 10) to 473 and 70 bp, respectively. (d) Cleavage of PCR products of RAMP1 (1–4), RAMP2 (5–8) and RAMP3 (9–14) of the cerebral cortex (1, 2, 5, 6, 9–11) and microvessels (3, 4, 7, 8, 12–14). PCR product of RAMP1 (263 bp) was cleaved by SacI to 50 and 213 bp fragments (2, 4), RAMP2 (133 bp) by EcoT14I to 25, 54 and 54 bp (6, 8), or RAMP3 (159 bp) by RsaI to 34 and 125 bp (10, 13) or by HindIII to 41 and 118 bp fragments (11, 14), respectively. (f) Cleavage of 589 bp AM PCR product of the cerebral cortex (1–3) and microvessels (4–6) by NarI (2, 5) to 447 and 142 bp fragments, and by TaqI (3, 6) to 80, 196 and 313 bp fragments, respectively. PCR products of RAMP1 slightly larger than 263 bp and of AM slightly smaller than 589 bp were also detected, but these bands are not likely to be a specific PCR product from each mRNA since they were not cut by restriction enzymes specific to each sequence. Each photograph is a representative of three experiments of different preparations of the microvessels.
H. Kobayashi et al. / Molecular Brain Research 81 (2000) 1 – 6
GAPDH was higher in the cerebral microvessel than in the cerebral cortex. The ratio of PCR product density of RAMP2 / GAPDH as well as AM / GAPDH in the cerebral microvessels was more than 10 times greater than that in the cerebral cortex.
4. Discussion In this paper, we found that AM increased cAMP level by reacting with receptors of high affinity and specific to AM in the cerebral microvessels. In addition, mRNAs for AM, RAMP2 and CRLR were highly expressed in the cerebral microvessels suggesting that AM is produced in the microvessels and act on themselves in an autocrine / paracrine manner and is involved in the regulation of cerebral microcirculation. Receptors for AM may be subdivided into two subtypes. In the rat mesenteric vasculature contracted with methoxiamine, AM decreased perfusion pressure which is suppressed by CGRP[8–37] [19]. In addition, in cultured vascular smooth muscle cells of rat, AM increased cAMP, and the effect was suppressed by CGRP[8–37]. Therefore, the receptors present in the cells such as vascular smooth muscle react not only with AM but also with CGRP [4]. On the other hand, AM increased cAMP level in the cultured endothelial cells of human [3] and bovine [22], Rat-2 fibroblast cells [1] and mouse Swiss 3T3 fibroblast cells [24], but the effect was not suppressed by CGRP[8– 37] indicating that receptors in these cells are specific to AM. The receptor for AM in the cerebral microvessel was of this type highly specific to AM. Because the reactivity of cells may change during culture, our results show, for the first time, that native endothelial cells express receptors specific to and high affinity to AM. The high specificity of receptors to AM in the cerebral microvessel is much different from the reactivity of the cerebral arterioles where CGRP dilates at lower concentrations than did AM, and the vasodilatatory effect of AM is strongly suppressed by CGRP[8–37] [18]. Thus, the difference of specificity to the peptides between the cerebral microvessels and arterioles suggests a distinct functional role of the receptors depending on the size of vessels. An orphan G-protein coupled receptor (L1) was previously reported as an AM receptor [8]. It has been shown to mediate an increase of cAMP response to AM and to bind with [ 125 I]AM with high affinity (KD : 8.2 nM). Its expression pattern was similar to the distribution of [ 125 I]AM binding sites in rat tissues. However, cAMP formation by AM and AM binding to the COS-7 cells expressing L1 receptor has not been confirmed [11], suggesting that L1 did not represent a real receptor for AM. On the other hand, a receptor cloned as CRLR behaved as an AM receptor when it was co-expressed with RAMP2 [17]. In fact, a functional AM receptor composed of CRLR with
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RAMP2 was expressed in human endothelial and vascular smooth muscle, and the reactivity to the peptides was confirmed in the COS-7 cells expressing in these clones [7]. Therefore, we examined the expression of CRLR and RAMPs in the cerebral microvessels, and found a very high expression of RAMP2. The high expression of RAMP2 with CRLR would form a specific receptor for AM, which is in good agreement with the increase in cAMP and the displacement of [ 125 I]AM binding caused specifically by AM. AM receptors in the cerebral microvessels may play a role in regulation of production of biologically active peptides such as endothelin. Endothelin, a potent basoconstrictor, is produced and secreted from the cerebral microvessels. The secretion of endothelin is suppressed by cAMP [25], whereas endothelin inhibits adenylate cyclase in the endothelial cells of brain microvessels [15]. Thus, AM and endothelin may regulate cerebral microcirculation by interacting the action of each peptide. Although cAMP has been reported to have the function of regulation of macromolecular transport of proteins such as albumin [5], its precise role in regulation of permeability of the cerebral microvessels is still unclear [6]. On the other hand, since forskolin, an activator of adenylate cyclase, decreased incorporation of thymidine in cultured cerebral microvessel endothelial cells [10], AM might be involved in regulation of cell growth. AM may activate signal transduction pathways other than cAMP pathway such as Ca / calmodulin and mitogen activated protein kinase pathways but only at higher concentration in other cell types [1,22]. In addition to the demonstration of an autocrine / paracrine regulator of AM in the cerebral microvessels of the present study, the elucidation of transduction pathways of AM would serve for better understanding for regulatory mechanisms of the cerebral microcirculation.
Acknowledgements This work is supported in part by research grants from the Ministry of Education, Science and Culture of Japan, Miyazaki Medical College (grant for Special Research Project) and Japan Marine Science and Technology Center.
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