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J Dent Res 83(4): 333-337, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Expression of Adrenomedullin and its Receptors in Human Salivary Tissue

S. Kapas1, K. Pahal1, A.T. Cruchley1, E. Hagi-Pavli1, and J.P. Hinson2,*

1 Molecular Signalling Group, Clinical Sciences Research Centre, and
2 Department of Endocrinology, Barts & the London, Queen Mary’s School of Medicine & Dentistry, Suite 12, Dominion House, Bartholomew Close, London EC1A 7BE, UK;

* corresponding author, j.p.hinson{at}qmul.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adrenomedullin is a multifunctional peptide produced by a wide range of different cells and tissues. This study was designed to investigate whether adrenomedullin is present in human saliva and in salivary glands. It was expected that saliva may contain high concentrations of adrenomedullin, which has antimicrobial activity in vitro, which may have functional implications in the oral cavity. Saliva from the submandibular and parotid glands contained higher concentrations of adrenomedullin than did the circulation, but lower concentrations than in whole saliva. This suggests that oral epithelium may contribute the majority of the adrenomedullin peptide found in saliva. Specific adrenomedullin receptors were found in cell lines from the submandibular (HSG) and parotid (HSY) salivary glands. These findings suggest a paracrine/autocrine role for adrenomedullin in these tissues; however, the concentration of adrenomedullin in saliva was insufficient to suggest a significant antimicrobial action in the healthy oral cavity.

KEY WORDS: adrenomedullin • saliva • salivary gland • oral mucosa


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adrenomedullin is a multifunctional peptide (Hinson et al., 2000) which has physiological significance, since recent studies in transgenic mice demonstrate that disruption of the adrenomedullin gene results in a lethal knockout (Caron and Smithies, 2001, 2002). Studies from our laboratory, and others, have shown that adrenomedullin is expressed in key epithelial surfaces and is emerging as an important effector molecule in host defense (Cameron and Fleming, 1998; Allaker et al., 1999; Kapas et al., 2001a; Marutsuka et al., 2001; Welsch et al., 2002). Adrenomedullin is produced in epithelial surfaces such as the skin, lung, gut, and oral cavity (Martínez et al., 1995, 1997; Allaker et al., 1999; Kapas et al., 2001b). Adrenomedullin has been demonstrated to accumulate in the apical regions of normal human bronchial epithelium, in human skin, and in the skin of the Xenopus laevis toad (Martínez et al., 1995, 1997; Hinson et al., 2000; Kapas et al., 2001b). Cameron and Fleming (1998) localized adrenomedullin mRNA in epithelial cells lining the uterus, bronchioles, and gastrointestinal tract in mice and rats. The epithelium lining these mucosal surfaces provides a first line of defense against potentially pathogenic micro-organisms. Analysis of previous data (Walsh et al., 1998; Allaker et al., 1999; Kapas et al., 2001a; Marutsuka et al., 2001) provides evidence that adrenomedullin also has antimicrobial properties against both Gram-positive and -negative bacteria isolated from skin, oral cavity, respiratory tract, and the gut. The concentration of adrenomedullin required to kill/inhibit bacterial growth is higher than those levels found in the circulation, but may be compatible with an effect of locally produced peptide. However, in certain circumstances, such as sepsis, elevated plasma adrenomedullin levels may contribute to the response to bacterial challenge.

Until recently, the mucosal lining of the mouth was regarded as a simple physical barrier, preventing bacterial invasion and the escape of body fluids. Considering the hostile environment of the mouth, being awash with food particles and commensal micro-organisms, there is surprisingly little incidence of bacterial infection in the oral cavity under normal circumstances. It is clear that the saliva contains a range of antimicrobial substances. We hypothesize that adrenomedullin may be an additional antimicrobial factor in saliva.

The present study was designed to investigate the adrenomedullin content of saliva and to determine the contributions made by different salivary glands. Adrenomedullin receptors in salivary cell lines were investigated by ligand-binding studies to establish whether there may be a case for adrenomedullin having a paracrine or autocrine role in these tissues.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All the human samples used in these studies were obtained with the person giving informed consent to protocols approved by the East London and City Research Ethics Committee (ref. t/02/043 November 2002 and P/03/122 November 2003).

Measurement of Adrenomedullin in Saliva and Serum by Enyme-linked Immune Assay
Saliva was obtained from adult donors (male, n = 8; female, n = 9; 21–60 yrs of age). Donors were free of periodontal inflammation and other oral pathologic conditions, as assessed by clinical examination, and had no medical abnormalities that affect the salivary glands. Stimulated whole saliva (5 mL; subject chewed on neutral-based gum) and stimulated parotid and submandibular/sublingual saliva (5 mL; subject sucked on lemon candy) were collected on ice. Whole saliva was centrifuged for removal of bacterial and cellular debris (10,000 rpm, 4°C for 5 min). Parotid saliva was secured by means of Curby cups placed over the parotid papilla (Sreebny, 1996). Submandibular/sublingual secretions (referred to as submandibular saliva) were obtained with collection devices placed in the anterior floor of the mouth. Saliva samples were stored at -70°C until required.

Blood samples were collected from healthy volunteers, the serum extracted, and kept at -70°C until analyzed. Samples were thawed on ice, mixed with an equal volume of 0.1% alkaline-treated casein (Martínez et al., 1997), and extracted through C-18 Sep-Pak 400 mg cartridges (Waters-Millipore, Milford, MA, USA). The proteins were eluted with 80% isopropanol. The eluate was then lyophilized and stored at -20°C until required.

On the day of assay, samples were reconstituted in 500 µL EIA buffer, separated into 50-µL aliquots, and assayed according to the manufacturer’s instructions (Phoenix Pharmaceuticals Inc., Belmont, CA, USA). The minimal amount of adrenomedullin detected in this assay was 0.2 ng/mL. The assay does not cross-react with human CGRP (calcitonin gene-related peptide), PAMP (pro-adrenomedullin N-terminal 20-peptide), amylin, or calcitonin (data supplied by manufacturer).

Immunoblotting of AM in Saliva
Lyophilized saliva samples were reconstituted in buffer. A 50-µg quantity of protein was heated to 99°C for 4 min, loaded into sample wells, resolved on a 10–20% tricine SDS-polyacrylamide gel (Novex, San Diego, CA, USA), and run at 120 V for 2 hrs. Transfer blotting was accomplished with the use of the same apparatus, and proteins were transferred to a PDVF membrane (Immobilin, Millipore) at 30 V for 4 hrs. Membranes were blocked overnight in a solution of 5% dried milk in PBS containing 0.1% Tween 20 at 4°C. Membranes were then washed and then incubated for 60 min at room temperature in a 1:500 dilution of rabbit anti-human AM antibody (Allaker and Kapas, 2003), washed 3 times in PBS, and incubated in 1:200 goat anti-rabbit biotinylated IgG (Vector Laboratories, Peterborough, UK) for 60 min at room temperature. Membranes were washed 3 times in PBS and the signal amplified/detected by means of ECL according to the manufacturer’s instructions (Amersham International plc, UK).

Immunohistochemical Staining of Adrenomedullin in Salivary Tissues
Adrenomedullin was identified in 5-µm-thick tissue sections of formalin-fixed submandibular and parotid glands as described previously (Taichman et al., 1998). Previously characterized rabbit antibodies to human adrenomedullin were used at a concentration of 1:1000 (Allaker and Kapas, 2003). We performed antigen retrieval by subjecting the tissue sections to microwaving (750 W, 20 min) while in 10 mM citrate buffer, pH 6.0. After this, the avidin-biotin complex (ABC) method was used as described previously (Taichman et al., 1998). Bound antibodies were visualized by Vector Red (Vector Labs, Peterborough, UK). Sections were lightly counterstained with hematoxylin. Pre-incubation of the antiserum with 10 nmol/mL synthetic antigen was used as a negative control.

Maintenance of Salivary Cell Lines
Two epithelial cell lines established from submandibular (HSG) and parotid (HSY) salivary glands (Myoken et al., 1996; Sato et al., 1996) were used in this study. HSY was established from an adenocarcinoma of the parotid gland. Based on its morphological and immunocytochemical properties, it is thought to originate from the intercalated duct or acinar region (Hayashi et al., 1987). HSG was established from the submandibular gland of a patient with squamous cell carcinoma. Based on its morphological and immunocytochemical characteristics, it is considered to originate from the intercalated duct (Shirasuna et al., 1981). Cells were maintained in T75 cm2 flasks in MEM (Invitrogen, Paisley, Scotland) supplemented with 10% FBS and routine antibiotics in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. Cells were allowed to grow to 80% confluence before being passaged in trypsin-EDTA solution. Twenty-four hours before experiments were carried out, 70% confluent T75 cm2 flasks of cells were rendered quiescent by being placed in serum-free MEM (sfMEM). On the day of experiments, cells were washed in sterile PBS, and a 3-mL quantity of fresh sfMEM was placed on the cells, after which the flasks were incubated overnight. The cell culture supernatant was harvested, lyophilized, and stored at 4°C until required for adrenomedullin assay. Adrenomedullin was assayed as described above, without the extraction step.

Adrenomedullin Receptor Binding Assays of Salivary Cells
Adrenomedullin receptors were measured with the use of [125I] adrenomedullin. Briefly, HSG or HSY cells were incubated at room temperature for 60 min with 0.1 nmol/L [125I] adrenomedullin and increasing concentrations of unlabeled human adrenomedullin, CGRP, PAMP, calcitonin, or amylin, in binding buffer (20 mmol/L HEPES, pH 7.4, 5 mmol/L MgCl2, 10 mmol/L NaCl, 4 mmol/L KCl, 1 mmol/L EDTA). Cells were solubilized in 0.1 mol/L NaOH, and tracer bound to the cells was determined by gamma spectroscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adrenomedullin Levels in Saliva, Salivary Cells, and Serum
Adrenomedullin was detected in whole saliva and various salivary ductal secretion of normal adults, and from the supernatant of salivary ductal cell lines (Fig. 1Go). The range of values obtained in whole saliva was 55 to 65 pg/mL. ANOVA indicated that adrenomedullin levels in whole saliva were significantly higher than those found in either submandibular or parotid ductal saliva (p < 0.001). All three sources of saliva contained significantly higher concentrations of adrenomedullin than was found in normal serum (p < 0.001). Conditioned media obtained from cells derived from human submandibular salivary gland (HSY) and from human parotid gland (HSG) was assayed for adrenomedullin content. Adrenomedullin was produced by both these cell lines, with the amounts secreted by cells derived from the submandibular gland (HSY 11.1 ± 5.6 pg/mL) higher than those secreted by cells derived from the parotid salivary gland (HSG 4.6 ± 3.2 pg/mL).



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  		Figure 1. Adrenomedullin concentrations (pmol/L) in normal human saliva and serum. Each value represents means ± SEM, n = 4. *P < 0.05 and ***P < 0.001 compared with levels in serum. {dagger} p < 0.001 compared with either parotid or submandibular alone (one-way ANOVA followed by a Dunnett’s test).

 
Western Blot and Immunocytochemical Analysis of Adrenomedullin in Saliva and Salivary Tissues
We measured adrenomedullin levels by Western blotting in whole saliva, submandibular, or parotid ductal saliva. Fig. 2Go illustrates that a single protein band was observed with an apparent molecular weight of 6 kDa. Saliva from the submandibular gland contained more adrenomedullin than did the parotid gland saliva. Synthetic human adrenomedullin was used as a reference control (1 µg).



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  		Figure 2. Western blotting of synthetic human adrenomedullin and saliva. Pooled fractions of submandibular, parotid, or whole saliva were subjected to immunoblotting. Only one band, of approximately 6 kDa, was observed in all samples. Experiments were carried out 3 times, with the same result.

 
Since the antibody appeared to detect only authentic adrenomedullin, we then used the same polyclonal antibody to detect adrenomedullin in normal human salivary glands. Intense cytoplasmic staining with adrenomedullin antibody was seen in the ductal epithelial cells of the minor salivary gland specimen (Fig. 3Go, lower panel). This immunostaining was lost when the antibody was pre-absorbed with a blocking peptide, which was the antigen sequence against which it was raised (Fig. 3Go, upper panel). Similar results were observed in major salivary glands (data not shown).



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  		Figure 3. Immunohistochemical localization of adrenomedullin in a human minor salivary gland. Note ductal epithelial cell staining (lower panel). Similar findings were observed in the parotid and submandibular salivary glands (not illustrated). Upper panel, negative staining control involving the primary rabbit adrenomedullin antibody absorbed with adrenomedullin antigen before application to the specimen. Scale bar = 50 µm. This image was chosen on the basis that the histology is clearly identifiable and the localization of adrenomedullin can be seen clearly.

 
Adrenomedullin Receptors in Salivary Cells
The binding characteristics of adrenomedullin receptors were studied in the salivary gland cell lines HSG and HSY. Binding of [125I] adrenomedullin to the HSY cells was shown to be saturable for the receptor and analysis of the data, by the curve-fitting program LIGAND, and Hill analysis predicted the probability of a single-site model vs. a two-site model (p < 0.05; Hill coefficient 0.891). Scatchard analysis revealed the presence of a single population of binding sites (Fig. 4aGo), and the calculated KD of the receptor was 0.4 nmol/L, with a Bmax of 630 fmol/mg protein. Fig. 4bGo shows the displacement of [125I] adrenomedullin by unlabeled adrenomedullin, CGRP, calcitonin, PAMP, or amylin, with the use of intact HSY cells. Displacement of [125I] adrenomedullin binding by unlabeled peptide showed that AM tracer was displaced in a dose-dependent manner, and up to 90% was displaced by 50 nmol/L adrenomedullin. Fig. 4bGo shows that CGRP, calcitonin, PAMP, or amylin did not significantly displace [125I] adrenomedullin binding in parallel cultures of HSY cells, even at concentrations up to 10-6 mol/L. Analysis of these data demonstrates that HSY cells express specific receptors for adrenomedullin.



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  		Figure 4. Scatchard analysis and displacement curves of human [125I] adrenomedullin. (A,B) Data from HSY cells. (C,D) Data from HSG cells. (A,C) Scatchard analysis of specific [125I] adrenomedullin binding; displacement with adrenomedullin. (B,D) Displacement of [125I] adrenomedullin binding by increasing concentrations of adrenomedullin (filled squares), CGRP (open squares), calcitonin (open circles), amylin (filled circles), or PAMP (filled triangles). Each point represents the mean of triplicate determinations.

 
Similar experiments were carried out with HSG cells. Scatchard analysis revealed the presence of a single population of binding sites (Fig. 4aGo), and the calculated KD of the receptor was 0.65 nmol/L, with a Bmax of 740 fmol/mg protein. Fig. 5b shows the displacement of [125I] adrenomedullin by unlabeled adrenomedullin, CGRP, calcitonin, PAMP, or amylin, with the use of intact HSG cells. Displacement of [125I] adrenomedullin binding by unlabeled peptide showed that adrenomedullin tracer was displaced in a dose-dependent manner, and up to 80% was displaced by 50 nmol/L adrenomedullin. Calcitonin, PAMP, or amylin did not significantly displace [125I] adrenomedullin binding; however, CGRP was able to displace up to 20% of radiolabeled adrenomedullin binding in HSY cells. Analysis of these data suggests that HSY cells express receptors specific for adrenomedullin, but also have a small population of CGRP receptors.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Adrenomedullin was detected in the whole saliva of healthy adults as well as in ductal secretions from the submandibular/sublingual and parotid glands (Figs. 1Go, 2Go). These levels of adrenomedullin production were higher than those found in the circulation (ca. 60 pmol/L vs. 10 pmol/L), and thus it appears that adrenomedullin is actively secreted into saliva from submandibular and parotid salivary glands. Cell lines derived from the submandibular and parotid glands secreted large amounts of adrenomedullin constitutively, and clearly there is a case for adding salivary cells to the long list of cell types that produce and secrete adrenomedullin (Hinson et al., 2000).

It is noteworthy, however, that the concentration of adrenomedullin in whole saliva was significantly greater than that found in submandibular or parotid saliva alone. It is very likely that other cell types in the oral cavity contribute to salivary adrenomedullin levels. Recently, we have shown that oral epithelial cells (keratinocytes), in vitro, secrete adrenomedullin, and that production is increased in response to a wide variety of agents such as cytokines and steroid hormones (Kapas et al., 2001b). It is probable that these cells contribute to salivary adrenomedullin concentrations.

It is not clear what the functions of adrenomedullin in saliva might be. Adrenomedullin is known to exert a wide range of effects in a wide range of tissues, including stimulation of angiogenesis (Zhao et al., 1998), influencing vascular permeability (Chu et al., 2001), bactericidal actions (Allaker et al., 1999), causing vasodilatation, and both increases and decreases in cell division (for a review, see Hinson et al., 2000). The concentration of adrenomedullin found in saliva, although higher than in plasma, is not high enough to exert a significant bactericidal effect on the main oral pathogens, such as Porphyromonas gingivalis. This organism is killed by concentrations of adrenomedullin above 500 pmol/L (Allaker et al., 1999), around eight-fold higher than the levels found in these studies of around 60 pmol/L. However, there is evidence that adrenomedullin expression in oral keratinocytes is up-regulated in response to challenge with live oral pathogens (Kapas et al., 2001a). Such a response may conceivably bring salivary adrenomedullin concentrations within the bactericidal range. It is also possible that adrenomedullin may act in concert with other salivary factors to enhance their antimicrobial effects, although this hypothesis remains to be tested. It is unlikely that salivary adrenomedullin activates adrenomedullin receptors in oral tissues, since the binding affinity of adrenomedullin receptors is in the nanomolar range (Hinson et al., 2000). It is likely that adrenomedullin is exerting very local effects in the oral cavity and salivary glands, as in other tissues.

Using the cell lines derived from the submandibular (HSG) and parotid (HSY) glands, we investigated the presence of specific receptors for adrenomedullin. The single binding site proposed by Scatchard analysis, and that fact that CGRP did not displace adrenomedullin binding to HSG cells, suggests that there are specific adrenomedullin receptors on these cells (Fig. 4Go). To a certain extent, this was also true of HSY cells, except that these cells also appear to have a small population of CGRP receptors, since CGRP displaced up to 20% of radiolabeled adrenomedullin (Fig. 5). One clear conclusion of these binding data is that circulating levels, or even salivary concentrations, of adrenomedullin are not high enough to activate these specific receptors. It is therefore most likely that locally produced adrenomedullin has an autocrine or paracrine effect on adrenomedullin receptors within the salivary gland. The nature of this effect has not been investigated.


   ACKNOWLEDGMENTS
 
We are grateful to Professors Norton Taichman and Daniel Malamud (Dept. of Oral Pathology and Biochemistry, School of Dental Medicine, University of Pennsylvania) for collecting ductal saliva samples. The authors are also grateful to the Nuffield Foundation, The Royal Society, and The Oral and Dental Research Trust for grant support.

Received May 1, 2003; Last revision January 27, 2004; Accepted January 30, 2004


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Allaker RP, Kapas S (2003). Adrenomedullin and mucosal defence: interaction between host and microorganism. Regul Pept 112:147–152.[ISI][Medline]

Allaker RP, Zihni C, Kapas S (1999). An investigation into the antimicrobial effects of adrenomedullin on members of the skin, oral, respiratory tract and gut microflora. FEMS Immunol Med Microbiol 23:289–293.[ISI][Medline]

Cameron VA, Fleming AM (1998). Novel sites of adrenomedullin gene expression in mouse and rat tissues. Endocrinology 139:2253–2264.[Abstract/Free Full Text]

Caron KM, Smithies O (2001). Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional adrenomedullin gene. Proc Natl Acad Sci USA 98:615–619.[Abstract/Free Full Text]

Caron KM, Smithies O (2002). Multiple roles of adrenomedullin revealed by animal models. Microsc Res Tech 57:55–59.[ISI][Medline]

Chu DQ, Smith DM, Brain SD (2001). Studies of the microvascular effects of adrenomedullin and related peptides. Peptides 22:1881–1886.[ISI][Medline]

Hinson JP, Kapas S, Smith DM (2000). Adrenomedullin, a multifunctional regulatory peptide. Endocr Rev 21:138–167.[Abstract/Free Full Text]

Hayashi Y, Yanagawa T, Yoshida H, Azuma M, Nishida T, Yura Y, et al. (1987). Expression of vasoactive intestinal polypeptide and amylase in a human parotid gland adenocarcinoma cell line in culture. J Natl Cancer Inst 79:1025–1037.

Kapas S, Bansal A, Bhargava V, Maher R, Malli R, Hagi-Pavli E, et al. (2001a). Adrenomedullin expression in pathogen challenged oral epithelial cells. Peptides 22:1485–1489.[ISI][Medline]

Kapas S, Tenchini ML, Farthing PM (2001b). Regulation of adrenomedullin secretion in cultured human skin and oral keratinocytes. J Invest Dermatol 171:353–359.

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]

Martínez A, Elsasser TH, Muro-Corcho C, Moody TW, Miller MJ, Macri CJ, et al. (1997). Expression of adrenomedullin and its receptor in normal and malignant human skin: a potential pluripotent role in the integument. Endocrinology 138:5597–5604.[Abstract/Free Full Text]

Marutsuka K, Nawa Y, Asada Y, Hara S, Kitamura K, Eto T, et al. (2001). Adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP) are present in human colonic epithelia and exert an antimicrobial effect. Exp Physiol 86:543–545.[Abstract]

Myoken Y, Myoken Y, Okamoto T, Kan M, McKeehan WL, Sato JD, et al. (1996). Expression of fibroblast growth factor-1 (FGF-1), FGF-2 and FGF receptor-1 in a human salivary-gland adenocarcinoma cell line: evidence of growth. Int J Cancer 65:650–657.[ISI][Medline]

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Sreebny LM (1996). Xerostomia: diagnosis, management and clinical complications. In: Saliva and oral health. 2nd ed. Edgar WM, O’Mullane DM, editors. London: British Dental Association, pp. 43–66.

Taichman NS, Cruchley AT, Fletcher LM, Hagi-Pavli EP, Paleolog EM, Abrams WR, et al. (1998). Vascular endothelial growth factor in normal human salivary glands and saliva: a possible role in the maintenance of mucosal homeostasis. Lab Invest 78:869–875.[ISI][Medline]

Welsch U, Unterberger P, Hofter E, Cuttitta F, Martinez A (2002). Adrenomedullin in mammalian and human skin glands including the mammary gland. Acta Histochem 104:65–72.[ISI][Medline]

Zhao Y, Hague S, Manek S, Zhang L, Bicknell R, Rees MC (1998). PCR display identifies tamoxifen induction of the novel angiogenic factor adrenomedullin by a nonestrogenic mechanism in the human endometrium. Oncogene 16:409–415.[ISI][Medline]




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