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Endocrinology Vol. 141, No. 1 406-411
Copyright © 2000 by The Endocrine Society

ARTICLES

Coexpression of Receptors for Adrenomedullin, Calcitonin Gene-Related Peptide, and Amylin in Pancreatic ß-Cells1

Alfredo Martínez, Supriya Kapas, Mae-Jean Miller, Yvona Ward and Frank Cuttitta

Department of Cell and Cancer Biology, National Cancer Institute, National Institutes of Health (A.M., M.J.M., Y.W., F.C.), Bethesda, Maryland 20892; and Molecular Signaling Group, Clinical Sciences Research Center, St. Bartholomew’s and the Royal London School of Medicine and Dentistry (S.K.), London, United Kingdom E1 2AT

Address all correspondence and requests for reprints to: Dr. Alfredo Martínez, Department of Cell and Cancer Biology, National Cancer Institute, National Institutes of Health, Building 10, Room 13N262, 9000 Rockville Pike, Bethesda, Maryland 20892. E-mail: martineza{at}bprb.nci.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Three receptors have been characterized by their ability to bind adrenomedullin (AM): L1, RDC1, and CRLR. Immunohistochemical analysis and RT-PCR showed that all three receptors are expressed by the insulin-producing cells of the islets of Langerhans. RDC1 and CRLR in the presence of particular modifying proteins can also bind calcitonin gene-related peptide (CGRP). Such data suggest that the inhibitory effect caused by both AM and CGRP on insulin secretion is mediated by a direct interaction with the ß-cell. We also identified receptors for amylin, the third member of the AM peptide family, in mouse insulin-secreting cells. The ß-cells located closer to the periphery of the islets had a stronger immunoreactivity for the AM/CGRP receptors. This observation could be related to a paracrine mechanism, given the proximity of AM- and CGRP-secreting cells (F and {delta}-cells, respectively), which are located at the periphery of the islets. Interestingly, the smooth muscle cells in the pancreatic vasculature expressed only RDC1, which is in agreement with physiological data showing that AM functions in the cardiovascular system are mainly mediated through a CGRP1 receptor. These data further implicate AM and the other components of its peptide family as important regulators of insulin release.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADRENOMEDULLIN (AM), a recently characterized 52-amino acid bioactive peptide (1, 2, 3, 4), together with calcitonin gene-related peptide (CGRP) and amylin is part of a peptide family characterized by some common biological activities and the presence of a six-amino acid ring structure formed by an intramolecular disulfide bond. Calcitonin shares a lower degree of homology with the other three peptides and is sometimes also included in the same peptide superfamily (5).

In the pancreas, AM is located in the islet cells, with predominant expression in the F cells, which also contain pancreatic polypeptide (6). The expression of AM in endocrine pancreatic cells seems to be a highly conserved feature from both the phylogenetic (7) and the ontogenetic (8) perspective. The presence of AM in the pancreas is justified by its function as a regulator of ß-cell physiology (6, 9). AM acts as a tonic inhibitor of insulin release, which has been shown in both isolated islets and through glucose tolerance tests in vivo (6). Recently, AM has been implicated in the inhibition of amylase secretion by pancreatic acini (10), thereby establishing AM as a multifunctional pancreatic hormone.

Several receptors with different affinities for AM have been cloned and sequenced. All of them belong to the seven-transmembrane domain G protein-coupled receptor superfamily and were previously characterized as orphan receptors. The first molecule to be identified for its ability to bind AM was L1, a 395-amino acid polypeptide isolated from rat lung that was able to elevate cAMP in COS-7 cells after exposure to AM, with a Kd of 8.2 x 10-9 M (11). Another molecule able to bind AM is RDC1, a CGRP1 receptor initially isolated from a dog thyroid library and later shown to bind both CGRP and AM, although with different affinities (3 x 10-9 and 1 x 10-7 M, respectively) (12). Another player in this complex physiology is the calcitonin receptor-like receptor (CRLR), which was cloned in 1993 (13) and characterized as a CGRP receptor 3 yr later (14). Interestingly, this receptor requires the presence of modulating proteins with a single transmembrane domain known as receptor activity-modifying proteins (RAMPs). When CRLR and RAMP1 were cotransfected into Xenopus oocytes, a CGRP1 pharmacological response was acquired (CGRP > AM). On the other hand, cotransfection of CRLR and RAMP2 or RAMP3 resulted in a typical AM receptor pharmacology (AM >> CGRP) (15). Recently, a fourth putative receptor for AM has been isolated from human tissue and shown to be expressed by hemopoietic cells (16). This receptor presents a high sequence homology to the rat L1, but, unfortunately, when it was expressed in COS-7 cells it was not functional (17). None of these receptors has a measurable affinity for amylin, the third member of this peptide family, but recently Muff et al. (18) observed that cotransfection of human calcitonin receptor isotype 2 with either RAMP1 or RAMP3 evoked selective binding of amylin in rabbit endothelial cells. There are still several discrepancies between the pharmacological properties observed in many organs and the characteristics of the identified receptors. Given these controversies, many researchers in the field believe that new receptor molecules will be found in the near future. In fact, we recently reported the existence of an AM binding protein in the bloodstream of numerous species that might be a soluble receptor (19).

To better understand the physiological actions of the AM peptide family in the pancreas, we developed antibodies and molecular probes for the three best characterized AM receptors and some related proteins and studied their presence and distribution in the pancreas.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues
Eight Sprague Dawley rats were killed with CO2, and the pancreas was removed, fixed in Bouin’s fluid (Sigma, St. Louis, MO), and embedded in paraffin.

Antibodies
Three peptides were selected from the extracellular regions of L1, RDC1, and CRLR (Table 1Go) and synthesized. They were coupled to keyhole limpet hemocyanin (Calbiochem-Behring, La Jolla, CA) via glutaraldehyde cross-linkage, and the conjugate was used to hyperimmunize New Zealand White rabbits as previously described (20).


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  		Table 1. Synthetic peptides selected from the sequence of the AM receptors and used to generate the antibodies employed in this study

 
Monoclonal antibodies against somatostatin, glucagon, and pancreatic polypeptide (21) were provided by Ms. H. C. Wong/Dr. John Walsh (Digestive Diseases Research Center, UCLA, Los Angeles, CA). A guinea pig antibovine insulin antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Immunocytochemistry
The avidin-biotin-peroxidase complex method was performed in paraffin sections as previously described (6). Optimal concentrations for the primary antibodies were 1:500 (anti-RDC1), 1:1000 (anti-L1), and 1:5000 (anti-CRLR). Negative controls included substitution of the primary antibody by preimmune rabbit serum and preabsorption of the antibody with 10 nmol/ml homologous or heterologous synthetic peptides.

Confocal microscopy
To better estimate putative hormone colocalizations, triple labeling followed by confocal micoscopy were performed as previously described (7). Paraffin sections were dewaxed, rehydrated, and exposed to donkey normal serum (Jackson ImmunoResearch Laboratories, Inc.; 1:30) in PBS. Then, sections were exposed to a mixture of three primary antisera obtained in different species, overnight at 4 C. The mixture consisted of guinea pig antibovine insulin (1:2,000; Jackson ImmunoResearch Laboratories, Inc.), a rabbit antibody against one of the receptors (in-house), and one monoclonal antibody at a time. Optimal concentrations for the receptor antibodies were previously described for immunocytochemistry. Monoclonal antibodies used in this study include antisomatostatin (1:10,000; UC 102L, CURE, UCLA), antiglucagon (1:1,000; CURE, UCLA; with permission from Prof. Gregor, Department of Internal Medicine, Eberhard-Karls University, Tubingen, Germany), and antirat pancreatic polypeptide (1:500; CURE, UCLA) (22). The next day, the sections were incubated for 1 h in the second layer, which was a mixture of Cy5-donkey antiguinea pig (Jackson ImmunoResearch Laboratories, Inc.), Bodipy goat antirabbit (Molecular Probes, Inc., Eugene, OR), and biotinylated goat antimouse (Dakopatts, Glostrup, Denmark), all of them at a final concentration of 1:200. A third layer was added for an additional hour, containing lissamine rhodamine streptavidin (1:200; Jackson ImmunoResearch Laboratories, Inc.). After thorough washes, the slides were mounted in SlowFade solution (Molecular Probes, Inc.) and observed with a Carl Zeiss Laser Scanning Microscope 510 (New York, NY) equipped with four lasers.

Cell culture
Two well-characterized cell lines originated from pancreatic ß cells: CRL 1777 (hamster), and CRL 2055 (mouse) were obtained from the American Type Culture Collection (Rockville, MD). They were maintained in F12K medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with either 10% horse serum plus 2.5% FCS (CRL 1777) or 10% FCS (CRL 2055), at 37 C in a 5% CO2 atmosphere.

RT-PCR and Southern blot
The MicroFast Track kit (Invitrogen, San Diego, CA) was used to extract messenger RNA (mRNA) from the cell lines. RT, PCR, and Southern blot were carried out as previously described (20). Primers specific for the three AM/CGRP receptors, the human calcitonin receptor, and the RAMP proteins were designed and are shown in Table 2Go. mRNA from mouse liver and the human tumor cell line NCI-H209 were used as controls.


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  		Table 2. Oligonucleotides used for the amplification of the AM receptors, the calcitonin receptor (Calc. R.), and related proteins

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of AM receptors in the pancreas
Immunohistochemical methods detected the presence of the three AM receptors studied in the islets of Langerhans (Fig. 1Go). Most of the cells in the islets were labeled, with stronger staining in the peripheral cells (Fig. 1CGo). In addition to the endocrine components, the antibody against RDC1 stained the smooth muscle cells in the arteries and arterioles of the pancreatic parenchyma (Fig. 1BGo). Interestingly, no immunoreactivity was observed for any of the three receptors in the exocrine acini or the ductal system.



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  		Figure 1. Paraffin section of rat pancreas stained with antibodies against L1 (A), RDC1 (B), and CRLR (C). Note the staining in the vascular smooth muscle cells in B. Magnification, x370.

 
Specificity controls demonstrated that absorption of the antibodies with their homologous synthetic peptides resulted in complete quenching of the immunostaining (Fig. 2Go, A and B), whereas preabsorption with any of the other heterologous peptides did not modify staining intensity (Fig. 2Go, C and D).



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  		Figure 2. Consecutive sections of rat pancreas stained with anti-CRLR (A), anti-CRLR preabsorbed with CRLR synthetic peptide (B), anti-L1 (C), and anti-L1 preabsorbed with RDC1 peptide (D). Note the complete quenching of the staining produced by the homologous peptide and the unchanged reaction when a heterologous peptide was used. Magnification, x370.

 
Colocalization pattern
Triple immunofluorescence followed by confocal microscopy were applied to investigate the colocalization of the AM receptors with the classical pancreatic hormones and thereby identify the cell type in which these epitopes are expressed. In all cases, complete colocalization with insulin was observed for the three receptors, whereas receptor immunoreactivity was never in the same cells as glucagon, somatostatin, or pancreatic polypeptide (Fig. 3Go). The strong immunoreactivity observed in the periphery of the islets by immunohistochemistry was also seen by immunofluorescence, but it labeled a subset of insulin-positive cells located near the border of the islets (Fig. 3Go).



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  		Figure 3. Triple immunofluorescence followed by confocal microscopy showing complete colocalization of insulin (purple, first column) with the three receptors (green, second column). The receptors did not colocalize with any of the other pancreatic hormones (red, third column). The fourth column represents a composite of the previous three images. The colocalization of insulin and the receptors renders a white color. Magnification, x370.

 
Molecular analysis
To further characterize the presence of the three AM receptors and related proteins in the ß-cells of the pancreas, two well characterized cell lines that secrete insulin in culture were studied by RT-PCR, followed by Southern blot. Both cells expressed the three AM/CGRP receptors (Fig. 4Go). In addition, the mouse ß-cell line CRL 2055 was positive for the calcitonin receptor, RAMP1, and RAMP3. In contrast, the hamster ß-cell line CRL 1777 expressed high amounts of RAMP3, but was negative for calcitonin receptor and the other two RAMPs. Surprisingly, neither pancreatic cell line had a positive signal for RAMP2, even though the control human cell line NCI-H209 had a high expression of this message.



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  		Figure 4. Southern blot analysis of the cell lines CRL 2055 and CRL 1777. Both cell lines express mRNA for the three AM receptors and some of the RAMPs. Water controls and total RNA from mouse liver and from the cell line NCI-H209 were used as negative and positive controls of the assay.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data clearly demonstrate that the three receptors known to have AM binding capabilities (L1, RDC1, and CRLR) are expressed by the ß-cells in the pancreas. This finding was confirmed by immunohistochemistry, immunofluorescence, and molecular analysis. The specificity of the immunoreaction was demonstrated by a successful competition with the synthetic peptides used to generate the antibodies and by the lack of cross-reactivity with the heterogeneous peptides. In addition, PCR bands for the calcitonin receptor, RAMP1, and RAMP3 were found in an insulin-producing mouse cell line. The specificity of the PCR products was confirmed by Southern blot with internal probes.

It became clear that the other pancreatic endocrine cell types do not express any of the AM receptors. Therefore these results indicate that the inhibitory effect of AM in insulin secretion (6) is caused by a direct action on the ß-cell, rather than through an indirect mechanism involving the secretion of secondary mediators. This is in agreement with our previous study on rat isolated islets in which we did not observe any change in the secretion of glucagon, somatostatin, or pancreatic polypeptide after exposure of the islets to AM, but we saw a clear dose-dependent inhibition of insulin secretion (6). In the same study we observed an increase in cAMP after stimulation with AM (6). cAMP is the second messenger for the three AM receptors (12, 13, 14, 15), and its elevation indicates the presence of physiologically active AM receptors in the rat islets.

The coexpression of calcitonin receptor and RAMP1 and/or RAMP3 has been shown to generate an amylin receptor physiology in endothelial cells (18). In the mouse cell line CRL 2055 we have demonstrated the presence of these three molecules, providing molecular evidence for the existence of the amylin receptor in ß-cells. Amylin is produced by the ß-cells of the pancreas and also influences insulin secretion (23, 24, 25), indicating that a receptor for this peptide must exist in the islets. In consequence, the action of amylin in the pancreas may be mediated through an autocrine mechanism. Nevertheless, physiological experiments must be conducted to ascertain this. The lack of calcitonin receptor, RAMP1, and RAMP2 signal in the cell line CRL 1777 may be due to the absence of these molecules from the hamster ß-cells or to a low homology of the hamster’s genes with the human sequences used to generate the probes.

The presence of the receptors for the three components of this peptide family: AM (L1, RDC1, and CRLR plus RAMP3), CGRP (RDC1 and CRLR plus RAMP1), and amylin (calcitonin receptor plus RAMP3) in the same cell type suggests the existence of a redundant mechanism for the reception of the three peptide signals. This seems to be the rule rather than the exception in ß-cells, which are known for the implementation of several similar fail-safe mechanisms (26). Although L1 and CRLR in the presence of RAMP2 or RAMP3 are able to bind AM at low concentrations (~10-9 M), RDC1 has a lower affinity (~10-7 M). The expression of all of these molecules in the same cell may implicate a variety of responses depending on the local concentration of AM. On the other hand, RDC1 and CRLR coexpressed with RAMP1 are excellent CGRP1 receptors. CGRP has been shown to inhibit insulin secretion as well and to be present in the somatostatin-producing cells (22, 23).

The observation of two populations of ß-cells, the one toward the periphery with higher immunoreactivity for AM/CGRP receptors and the other closer to the center with lower positivity, may relate to the expression patterns for AM and CGRP. AM is produced by F cells, which are always in the periphery of the islets and also express pancreatic polypeptide (6), whereas CGRP is released from {delta}-cells, which are also at the periphery (22, 23). If we assume a paracrine secretion for these hormones, it makes sense that the ß-cells closer to the source of AM and CGRP should contain a higher concentration of the receptors.

Another interesting finding is the expression of RDC1, but not the other receptors, in the smooth muscle cells of the pancreatic vessels. It has been repeatedly reported that the vasodilatory action of AM is mediated through a CGRP1 receptor, as it can be inhibited by CGRP-(8–37), a specific peptide antagonist of such pharmacology (27, 28). Our results clearly agree with these reports and suggest that RDC1 may be the only AM receptor present in the vascular system, at least in the pancreas. This is in agreement with a recent report that found RDC1 to be the only AM receptor present in rat aortic vascular smooth muscle cells (29).

Surprisingly, none of the receptors studied was expressed by the exocrine acini, even though Tsuchida et al. (10) showed an inhibitory role for AM in the secretion of amylase. These observations might suggest the existence of a different AM receptor in the exocrine cells of the pancreas.

Many factors have been shown to modulate AM expression in different cell types (30). In bovine pancreas it has been demonstrated that a mild infection and/or lipopolysaccharide injection results in an impressive overexpression of inducible nitric oxide synthase and AM, which, in turn, reduces circulating insulin and elevates glucose (31). Therefore, any compound or physiological state that modulates AM expression may have a direct impact on insulin release and glucose homeostasis. In fact, it has been recently suggested that AM may be responsible for the onset of type 2 diabetes in some individuals (32).

In summary, the three AM receptors are restricted to ß-cells of the islets, constituting a complex system designed to regulate insulin secretion. Unraveling this physiological puzzle may contribute to a better understanding of the normal function of the endocrine pancreas as well as the diseases produced by dysregulation of insulin secretion.


   Acknowledgments
 
We thank Ms. H. C. Wong/Dr. John Walsh for providing the monoclonal antibodies against glucagon, somatostatin, and pancreatic polypeptide.


   Footnotes
 
1 This work was supported by CURE/UCLA/DDC Antibody/RIA Core, NIH Grant DK-41301. Back

Received August 11, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  3. Sakata J, Shimokubo T, Kitamura K, Nakamura S, Kangawa K, Matsuo H, Eto T 1993 Molecular cloning and biological activities of rat adrenomedullin, a hypotensive peptide. Biochem Biophys Res Commun 195:921–927[CrossRef][Medline]
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  11. Kapas S, Catt KJ, Clark JL 1995 Cloning and expression of cDNA encoding a rat adrenomedullin receptor. J Biol Chem 270:24344–25347
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  15. McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM 1998 RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393:333–339[CrossRef][Medline]
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  18. Muff R, Buhlmann N, Fischer JA, Born W 1999 An amylin receptor is revealed following co-transfection of a calcitonin receptor with receptor activity modifying proteins-1 or -3. Endocrinology 140:2924–2927[Abstract/Free Full Text]
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  20. Martínez A, Miller MJ, Unsworth EJ, Siegfried JM, Cuttitta F 1995 Expression of adrenomedullin in normal human lung and in pulmonary tumors. Endocrinology 136:4099–4105[Abstract]
  21. Wong HC, Andersen DK, Sternini C, Ruiz CL, Hull EC, Walsh JH, Brunicardi FC 1995 Production of a rat pancreatic polypeptide-specific monoclonal antibody and its influence on glucose homeostasis by in vivo immunoneutralization. Hybridoma 14:369–376[Medline]
  22. Fujimura M, Greeley GH, Hancock MB, Alwmark A, Santos A, Cooper CW, Reumont KJ, Ishizuka J, Thompson JC 1988 Colocalization of calcitonin gene-related peptide and somatostatin in pancreatic islet cells and inhibition of insulin secretion by calcitonin gene-related peptide in the rat. Pancreas 3:49–52[Medline]
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  28. Nishimura Y, Suzuki A 1997 Relaxant effects of vasodilator peptides on isolated basilar arteries from stroke-prone spontaneously hypertensive rats. Clin Exp Pharmacol Physiol 24:157–161[Medline]
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  31. Elsasser TH, Sartin JL, Martínez A, Kahl S, Montuenga L, Pío R, Fayer R, Miller MJ, Cuttitta F 1999 Underlying disease stress augments plasma and tissue adrenomedullin responses to endotoxin: co- localized increases of adrenomedullin and iNOS within pancreatic islets. Endocrinology 140:5402–5411[Abstract/Free Full Text]
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals