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Carbachol-induced in vitro secretion of certain human submandibular proteins investigated by mass-spectrometry
archives of oral biology 53 (2008) 1077–1083
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/arob
Short communication
Carbachol-induced in vitro secretion of certain human submandibular proteins investigated by mass-spectrometry Tiziana Cabras a, Massimo Castagnola b,c, Rosanna Inzitari b, Jo¨rgen Ekstro¨m d, Michela Isola e, Alessandro Riva e, Irene Messana a,* a
Department of Applied Sciences in Biosystems, University of Cagliari, I-09042 Monserrato, Italy Institute of Biochemistry and Clinical Biochemistry, Faculty of Medicine, Catholic University, Rome, Italy c Institute of Chemistry of Molecular Recognition, CNR, Rome, Italy d Institute of Neuroscience and Physiology, The Sahlgrenska Academy, Go¨teborg University, Go¨teborg, Sweden e Department of Cytomorphology, University of Cagliari, Monserrato, Italy b
article info
abstract
Article history:
Objective: To investigate protein content of saliva produced in vitro by samples of human
Accepted 4 June 2008
submandibular gland following stimulation with the muscarinic agent carbachol. Design: Tissue samples, obtained at surgery from seven patients and showing normal
Keywords:
morphological appearance, were tested for 30 min: in absence of carbachol and atropine;
Human submandibular gland
in presence of carbachol (10 mM); in presence of carbachol (10 mM) and atropine (20 mM); or in
Carbachol
presence of just atropine (20 mM). Medium was analysed by high-performance liquid
HPLC–MS
chromatography–mass-spectrometry. Neither before nor during surgery were the patients
Acidic proline-rich proteins
exposed to drug treatments that were likely to influence the in vitro secretion.
Peptide PC
Results: Proline-rich proteins (PRP)-1 and -3, peptide PC and PB, statherin, cystatins SN, S1
Peptide PB
and S2 were invariably found in control gland tissue medium. Mean concentrations of these
Statherin
proteins/peptides in the medium were non-proportionally elevated following carbachol
Cystatins
exposure to the gland tissues. Difference between basal release and carbachol-induced secretion achieved statistical significance as to all the proteins/peptides under study but for statherin. Atropine alone or atropine plus carbachol caused no significant changes compared to the basal release of proteins/peptides. Conclusions: In vitro studies on salivary glands make it possible to study protein secretion from individual glands and thus, to reveal the contribution of the various types of gland to protein/ peptide content of whole saliva. The disproportional responses to carbachol may imply that the proteins/peptides are not confined to the same cells or to the same intracellular locations and are therefore not secreted as packages at parasympathetic cholinergic activity. # 2008 Elsevier Ltd. All rights reserved.
1.
Introduction
The oral fluid is a mixture of saliva from major and minor salivary glands, fluid from the gingival pockets, cell debris and
bacteria. Depending on type and intensity of the reflex stimulus different types of salivary glands are mobilised to various extent to create the most purposeful salivary response as to volume and composition under physiological condi-
* Corresponding author. Tel.: +39 070 6574520; fax: +39 070 6754523. E-mail addresses: [email protected], [email protected] (I. Messana). 0003–9969/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2008.06.001
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tions.1 In animals, pure saliva is obtained for experimental purposes from duct-cannulated major salivary glands. In humans, studies are usually focused ‘‘on whole saliva’’, i.e. the oral fluid. Collection of saliva from a single gland is obtained by a device placed over the gland duct (or ducts).2 However, usually no direct cannulation is performed in humans. Thus, it is difficult to avoid contamination of the saliva samples by the oral fluid. Whereas in animal studies, secretion is often evoked by stimulation of the parasympathetic and sympathetic innervations or by injecting selective receptor agonists into the blood stream, human secretion is usually evoked by taste or chewing, procedures most likely engaging a number of transmission mechanisms.1,3 In vitro observations allow the contribution of various salivary gland types to the composition of whole saliva to be studied. Furthermore, pieces of gland tissues can be exposed to selectively acting drugs, e.g. mimicking nerve-induced actions, without having to dissect nerves or to consider any systemic drug effects in the body. Whereas secretory in vitro studies on salivary gland tissues from animals are not uncommon, see e.g. Refs. [4–7], such studies on human salivary gland tissues are, on the whole, few.8–11 Yet, the ultimate vindication for in vitro studies on salivary glands would be those using operational specimens from humans as pointed out by Garrett.12 Hence, in the present study, the effect of the muscarinic receptor agonist carbachol on the in vitro secretion into the medium of certain proteins/peptides from pieces of human submandibular glands was analysed by applying high-performance liquid chromatography (HPLC) coupled to mass-spectrometry in order to investigate submandibular contribution to the protein content of mixed saliva in response to parasympathetic cholinergic activity.
2.
Materials and methods
2.1.
Reagents and instruments
All common chemicals and reagents were of analytical grade and were purchased from Merck (Darmstadt, Germany) and Sigma–Aldrich (St. Louis, MO, USA). The HPLC apparatus was a Thermo Finnigan (San Jose, CA, USA) Surveyor HPLC connected by a T-splitter to a diode-array UV–vis detector and to an LCQ Deca XP Plus mass-spectrometer. The mass-spectrometer was equipped with an electrospray ion (ESI) source. The reversed-phase chromatographic column was a Vydac (Hesperia, CA, USA) C8 middle-bore column, with 5 mm particle diameter (column dimensions 150 mm 2.1 mm).
2.2.
Collection and treatment of the glandular tissue
Samples of human submandibular glands were obtained at tumour surgery from seven patients, six males and one female, aged 48–75 years. The glands had not been exposed to radiation. Furthermore, prior to surgery or during surgery the patients were not treated with drugs that were likely to interfere with the in vitro secretion under present experimental set-up. Informed consent was obtained in each case and permission was granted by the local ethical committee (A.S.L. 8, Cagliari). Immediately after the resection, gland
tissue was washed twice with physiological saline solution and subdivided into pieces of about 5 mm long, 2 mm wide, 2 mm high. Incubations were performed as previously described.13 Briefly, glandular tissue was incubated at 37 8C for 30 min in 10 mL of an oxygenated medium (6 mM Tris–HCl, 123 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4 and 5.5 mM glucose, pH 7.4) without or with carbachol chloride (10 mM), when appropriate, combined with atropine (20 mM) in 25 mL sealed Erlenmeyer flasks, placed in a water-bath and continuously shaken. At the end of incubation, the glandular tissue was removed from the medium, weighed and subjected to cytomorphological and ultrastructural analysis.10 This examination of semithin tissue sections, allowed further assessment of the normality of the gland pieces incubated. Only incubation media from specimens showing a normal histological feature based on cytomorphological and ultrastructural analysis were used for biochemical characterization of their content. The mean wet weight of normally looking tissue, subdivided into small pieces, incubated in each Erlenmeyer flask was 53 14 mg (n = 22). Medium was centrifuged (6000 g, for 10 min) to remove eventual gross material, lyophilized and stored at 80 8C, awaiting the following HPLC– MS analysis.
2.3.
RP-HPLC–ESI-MS analysis
Lyophilized powder was dissolved in 150 mL of 0.2% aqueous trifluoroacetic acid (TFA), centrifuged at 10,000 g (10 min) and concentration of proteins in the supernatant determined by the bicinchoninic assay (Pierce, Rockford, IL, USA) corresponded to 0.15 0.05 mg/(mL mg) of tissue under basal conditions and to 1.3 0.3 mg/(mL mg) of tissue in stimulated experiments. 100 mL aliquots of the solution were directly injected into the HPLC–MS apparatus. The following solutions were utilized for the reversed-phase chromatography: (eluent A) 0.056% aqueous TFA and (eluent B) 0.050% TFA in acetonitrile–water 80/20 (v/v). The gradient applied was linear from 0 to 55% in 40 min, at a flow-rate of 0.30 mL/min. The Tsplitter addressed a flow-rate of about 0.20 mL/min towards the diode-array detector and a flow-rate of about 0.10 mL/min towards the electrospray ionization source. The diode-array detector was set at a wavelength of 214 and 276 nm. The eluent was not directed towards the electrospray source for the first 5 min of separation, in order to avoid damage to the ion trapmass-spectrometer (IT-MS), due to the elevated concentration of electrolytes (salts, small polar molecules). The ESI source spray voltage was 4.50 kV and the capillary temperature 220 8C with sheath gas flow-rate of 70 arbitrary unities. The ion trap apparatus operated in positive mode in the 300–2000 m/z range and mass spectra were collected every 3 ms in the positive ion mode.
2.4.
Characterization of peptides and quantification
Proteins detected in incubation media corresponded in terms of both chromatographic elution time and ESI spectrum to salivary peptides characterized in human saliva in previous studies.14–17 Molecular mass values were determined by deconvolution of ESI spectra, performed either by the software provided with the Deca-XP instrument (Bioworks Browser) or
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Table 1 – Muticharged ions (m/z) utilized to extract the ion current (XIC) peak of proteins/peptides secreted in the incubation medium Protein/peptide
Mass/charge (charge)
PB peptide Statherin Histatin1 Histatin 5 PRP-1
828.7 (+7), 966.7 (+6), 1159.5 (+5), 1449.1 (+4), 1931.7 (+3) 1345.7 (+4), 1794.1 (+3) 986.9 (+5), 1233.3 (+4), 1643.9 (+3) 760.3 (+4), 1013.3 (+3) 913.6 (+17), 970.7 (+16), 1035.3 (+15), 1109.2 (+14), 1194.5 (+13), 1293.9 (+12), 1411.5 (+11), 1552.5 (+10), 1724.9 (+9), 1940.4 (+8) 859.6 (+13), 931.1 (+12), 1015.7 (+11), 1117.2 (+10), 1241.2 (+9), 1396.2 (+8), 1595.5 (+7), 1861.3 (+6) 875.4 (+5), 1094.0 (+6), 1458.3 (+7) 955.2 (+15), 1023.4 (+14), 1102.0 (+13), 1193.8 (+12), 1302.2 (+11), 1432.3 (+10), 1591.3 (+9), 1790.1 (+8) 952.0 (+15), 1019.9 (+14), 1098.3 (+13), 1189.7 (+12), 1297.8 (+11), 1427.5 (+10), 1586.0 (+9), 1784.1 (+8) 957.3 (+15), 1025.6 (+14), 1104.4 (+13), 1196.4 (+12), 1305.1 (+11), 1435.5 (+10), 1594.9 (+9), 1794.1 (+8)
PRP-3 PC peptide Cystatin SN Cystatin S1 Cystatin S2
by MagTran 1.0.18 Experimental average molecular mass values were compared to those reported upon the Swiss-Prot (http://us.expasy.org/tools) and EMBL (http://www.embl-heidelberg.de) data banks. For quantification we utilized the extracted ion current (XIC) peak areas. XIC peaks were revealed by searching appropriate multi-charged ions reported in Table 1. Area under the ion current peaks was normalized with respect to the weight of the glandular tissue and expressed in relative units per mg of gland tissue. The present results were derived from seven non-carbachol exposed control specimens (in case of more than one control tissue from the same gland a mean value was calculated for each gland), three carbachol exposed specimens, three carbachol and atropine exposed specimens and five specimens just exposed to atropine.
2.5.
Statistics
Student’s t-test for unpaired comparisons was based on log values. Probabilities of less than 5% were considered significant. Values are mean S.E.M.
3.
Results
A basal release of a number of proteins/peptides occurred invariably: the acidic proline-rich proteins PRP-1 and -3, the cystatins SN, S1 and S2, statherin, peptide PB and PC as previously reported.13 Histatin 1 and histatin 5, occurred sporadically, as also observed previously.13 The total ion current (TIC) RP-HPLC profile of the products released from a specimen under control condition is shown in Fig. 1 (panel a). The list of the released proteins/peptides, their Swiss-Prot code number, the theoretical average molecular mass value and the experimental mass value are reported in Table 2. In order to reveal low protein/peptide concentrations of the medium of control tissue, we applied the XIC strategy. The concentration of histatins of the medium was lower than the concentrations of the other proteins/peptides under study. The lack of histatins in some samples was probably due to concentrations below the limit of detection under our experimental condition. The XIC method is based on the contemporaneous search of all the multi-charged ions expected for a given protein along the TIC
Table 2 – Experimental masses (Da) detected in the incubation medium by RP-HPLC–ESI-MS compared to the theoretical values reported in international data banks Peptide/protein
Swiss-Prot code
Theoretical average masses
Experimental masses
Acidic PRPs PRP-1, Pif-s (PRP-2) Diphos PRP-3, Pif-f (PRP-4) Diphos. PC (a-PRP fr. 107–150)
P02810 P02810 P02810
15515 (15514) 11162 (11161) 4371
Basic PRPs PB
P02814
5793
5792 1
Histatins Histatin 1 Histatin 5
P15515 P15516
4928 3036
4928 1 3036 1
Statherin
P02808
5379
5380 1
Cystatins Cystatin SN Cystatin S1 Cystatin S2
P01037 P01036 –
14312 14265 14345
14313 2 14266 1 14346 1
15515 2 11161 1 4371 1
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archives of oral biology 53 (2008) 1077–1083
Fig. 1 – RP-HPLC TIC (total ion current) profiles of the incubation medium of not stimulated (a) and 10 mM carbachol stimulated (b) submandibular gland tissue. The elution ranges of acidic PRPs, histatins, cystatins, PB peptide and statherin are evidenced. Panels (c) and (d) show the XIC (extracted ion current) peak of cystatin SN.
chromatographic profile. For example the panels c and d of Fig. 1 show the peak of cystatin SN recorded by using this strategy. A carbachol-induced secretion of proteins/peptides was demonstrated. From a qualitative point of view, no additional proteins/peptides appeared as compared to the basal release, as evident from Fig. 1 (panel b). As was the case under basal condition, the presence of histatins was not a consistent finding in the medium of those three samples exposed to carbachol. Therefore, this type of peptides was not included in the comparisons. For each protein/peptide consistently found, the figures for the mean response to carbachol were severalfold higher than those obtained under basal condition (Fig. 2a and b). However, the magnitude of the response of the individual proteins/ peptides (related to the control value in the absence of carbachol) was not uniform. The difference achieved statistical significance as to all proteins/peptides subjected to comparison but for statherin. In the presence of atropine (20 mM) and carbachol (10 mM), the concentration of proteins/peptides was not statistically significantly different from that of the medium of control tissue (Fig. 2a and b). However, the figures for mean values following exposure to both carbachol and atropine were usually higher than those in the absence of the combination. Exposure to just atropine (20 mM), showed no tendency for elevated mean values of proteins/peptides (Fig. 2a and b).
4.
Discussion
In vivo, it is particularly difficult to collect submandibular saliva not contaminated by sublingual saliva (see Ref. [19]). The present in vitro investigation allows the submandibular gland secretion to be studied separately and to show variations in the protein secretion following stimulation with the muscarinic agent carbachol. A basal release of a number of proteins from the incubated gland tissue was demonstrated, in agreement with previous observations.13 However, we were not able to show some major secretory products of submandibular glands, such as mucins,20–22 lysozyme,23 and amylase.24 Lack of mucin detection is related to the acidic treatment of the incubation medium in preparation for the mass-spectrometry analysis, which causes high molecular weight proteins to precipitate, whereas amylase and lysozyme were probably not revealed due to concentrations below the detection limit of our method. Likewise, lack of histatins in some samples was probably also due to levels below the detection limit. As would be expected only PB of the basic PRPs appeared in the media.25 As judged from the present findings, the submandibular gland is one contributor (not necessarily excluding the sublingual gland) to the concentrations of histatin 1, cystatin S and SN reported in mixed submandibular–sublingual saliva.11 The present findings of proline-rich proteins and histatins in the medium are in agreement with their
archives of oral biology 53 (2008) 1077–1083
Fig. 2 – Ion current peak area in relative units per mg of gland tissue of secreted proteins. (a) Proline-rich protein-1 (PRP-1), proline-rich protein-3 (PRP-3), PC peptide (PC), cystatin SN (Cyst SN), cystatin S1 (Cyst 1) and cystatin S2 (Cyst S2) and (b) PB peptide (PB) and statherin; note the difference in scale. Columns represent mean values and vertical bars + S.E.M. From left to right: (1) open columns in the absence of carbachol and atropine (controls, n = 7), (2) filled columns in the presence of carbachol (n = 3); columns with horizontal lines in the presence of carbachol and atropine (n = 3); and partly filled columns in the presence of atropine (n = 5). In some cases, the S.E.M. values were too small to show up in the graph. *P < 0.05 and **P = 0.01 as compared to control responses.
immunohistochemical demonstration in human submandibular glands.26 The basal release may reflect on-going constitutive secretion.27 Though the morphological appearance of the tissue seemed normal even at the ultrastructural level, it cannot be excluded that a certain fraction of the basal release
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was due to proteins/peptides that leaked from cells damaged as a consequence of the preparation. The human submandibular gland is innervated by parasympathetic and sympathetic nerves, and both cholinesterase-positive nerve fibres and catecholamine-fluorescence nerve fibres reach acinar as well as duct cells.28 Furthermore, the secretory elements are innervated by peptide-containing nerve fibres.29 Thus, reflex stimulation is likely to involve not only the activity of both divisions of the autonomic nervous system but also the action of conventional (acetylcholine and noradrenaline) and non-conventional (e.g. neuropeptides) transmitters to various degrees. In the present study, the muscarinic agonist carbachol most likely exerted its effect by the stimulation of muscarinic receptors of submandibular gland tissue to cause severalfold increases in the output of PRP-1 and -3, the cystatins SN, S1 and S2, peptide PB and PC. Support for this idea was gained from the finding that the levels of proteins/peptides in the medium were not significantly elevated, when the tissues were exposed to the combined action of carbachol and atropine. However, there was a clear tendency for higher figures for mean values following the combination of carbachol and atropine. This was probably a reflection of too a low concentration of atropine. Control experiments showed that atropine, in itself, caused no increases in the output of the proteins/peptides under study. Hence, a parasympathetic cholinergic transmission mechanism is probably involved in the secretion of these proteins/ peptides. Taking into account the relatively small number of observations and the large variations in the output of the individual proteins, conclusions should be drawn with caution. Nevertheless, the secretory pattern seemed to suggest that the proteins under study are not responding uniformly, exemplified by differences in the magnitude of response (in relation to basal value) within the cystatin family, and the lack of significant increase as to statherin. The disproportion may indicate that the proteins are not confined to the same cells or to the same intracellular locations and will therefore not be secreted as packages in response to parasympathetic nerve activity. In this connection, it may be recalled that in man the submandibular acinar cells are of two types, serous (90%) and mucous (10%) and that cystatingene transcripts are localized to the cytoplasm of only serous acinar cells of submandibular glands.30 Moreover, electron microscopic immunocytochemistry investigation of acinar cells in human submandibular glands shows some proteins to be differentially distributed within the granular content (amylase and agglutinins), while others (proline-rich proteins and histatins) are distributed uniformly or randomly throughout the granular content, suggesting that different mechanisms may be involved in the packaging of individual proteins within the same granule.26 The present demonstration of a carbachol-induced secretion of proteins may be combined with earlier cytomorphological findings on human submandibular gland tissue.31 Under the same experimental conditions and with the same concentration of carbachol as presently, a number of ultrastructural events takes place that indicates secretory activity; besides enlargement of lumina and appearance of V-shaped profiles and vacuoles, the density of the microvilli decreases, whereas that of the microbuds increases.10 Thus, the present
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experimental set-up maybe suitable for exploration of both ultrastructural changes and outputs of proteins and peptides of human salivary glands in response to a number of putative regulatory agonists of secretory activities such as neuropeptides and gastrointestinal hormones32,33; using the present preparation, unpublished observations of ours reveal a secretory effect of pentagastrin in human glands (Loy F, Diana M, Ekstro¨m J, Riva A). In conclusion, carbachol-induced the in vitro secretion of a number of proteins/peptides from human submandibular gland tissue specimens, thus indicating a parasympathetic cholinergic transmission mechanism involved in their secretion under normal conditions. In relative terms, the proteins/ peptides were not uniformly affected, suggesting that they may be stored at different locations and therefore not secreted as packages.
Acknowledgements We acknowledge the financial support of Cagliari University, Catholic University in Rome, MUR, Italian National Research Council (CNR-programs of scientific research promotion and diffusion), The Swedish Science Council (project no. 05927), The LUA/ALF agreement (project no. ALFGBG-11907) and the ‘‘INTERLINK’’-agreement (project no. II04C00H8M).
references
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10. Testa Riva F, Puxeddu R, Loy F, Conti G, Riva A. Cytomorphological study on human submandibular gland following treatment with secretagogue drugs. Cell Tissue Res 2006;324:347–52. 11. Nakamoto T, Srivastava A, Romanenko VG, Ovitt CE, PerezCornejo P, Arreola J, et al. Functional and molecular characterization of the fluid secretion mechanism in human parotid acinar cells. Am J Physiol-Reg I 2007;292:R2380–90. 12. Garrett JR. Innervation of salivary glands: neurohistological and functional aspects. In: Sreebny LM, editor. The salivary system. Boca Raton, FL, USA: CRC Press; 1988. p. 69–93. 13. Messana I, Loffredo F, Inzitari R, Cabras T, Giardina B, Onnis G, et al. The coupling of RP-HPLC and ESI-MS in the study of small peptides and proteins secreted in vitro by human salivary glands that are soluble in acidic solution. Eur J Morphol 2003;41:103–6. 14. Lupi A, Messana I, Denotti G, Schinina` ME, Gambarini G, Fadda MB, et al. Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry. Proteomics 2003;3:461–7. 15. Castagnola M, Inzitari R, Rossetti DV, Olmi C, Cabras T, Piras V, et al. A cascade of 24 histatins (histatin 3 fragments) in human saliva. Suggestions for a pre-secretory sequential cleavage pathway. J Biol Chem 2004;279:41436–43. 16. Inzitari R, Cabras T, Onnis G, Olmi C, Mastinu A, Sanna MT, et al. Different isoforms and post-translational modifications of human salivary acidic proline-rich proteins. Proteomics 2005;5:805–15. 17. Inzitari R, Cabras T, Rossetti DV, Fanali C, Vitali A, Pellegrini M, et al. Detection in human saliva of different statherin and P–B fragments and derivatives. Proteomics 2006;6:6370–9. 18. Zhang Z, Marshall AG. A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra. J Am Soc Mass Spectrom 1998;9:225–33. 19. Walz A, Stu¨hler K, Wattenberg A, Hawranke E, Meyer HE, Schmalz G, et al. Proteome analysis of glandular parotid and submandibular–sublingual saliva in comparison to whole human saliva by two dimensional gel electrophoresis. Proteomics 2006;6:1631–9. 20. Ramasubbu N, Reddy MS, Bergey EJ, Haraszthy GG, Soni SD, Levine MJ. Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular–sublingual saliva. Biochem J 1991;280:341–52. 21. Becerra L, Soares RV, Bruno LS, Siqueira CC, Oppenhaim FG, Offner GD, et al. Patterns of secretion of mucins and nonmucin glycoproteins in human submandibular/sublingual secretion. Arch Oral Biol 2003;48:147–54. 22. Rathman WM, van den Keybus PA, Van Zeyl MJ, Do¨pp EA, Veerman EC, New Amerongen AV. Characterization of monoclonal antibodies to human salivary (glyco) proteins. Cellular localization of mucins, cystatin-like 14 kD protein and 20 kD glycoprotein in the human submandibular gland. J Biol Buccale 1990;18:19–27. 23. MacKay BJ, Goodman H, Cox D, Grossbard BL, Iacono VJ, Pollock JJ. Development of an enzyme-linked immunosorbent assay for determination of lysozyme in human parotid and submandibular–sublingual salivas. J Clin Microbiol 1984;19:844–8. 24. Kauffman DL, Watanabe S, Evans JR, Keller PJ. The existence of glycosylated and non-glycosylated forms of human submandibular amylase. Arch Oral Biol 1973;18:1105–11. 25. Isemura S, Saitoh E. Molecular cloning and sequence analysis of cDNA coding for the precursor of the human salivary proline-rich peptide P–B. J Biochem (Tokyo) 1994;115:1101–6. 26. Takano K, Malamud D, Bennick A, Oppenheim F, Hand AR. Localization of salivary proteins in granules of human
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available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/arob
Short communication
Carbachol-induced in vitro secretion of certain human submandibular proteins investigated by mass-spectrometry Tiziana Cabras a, Massimo Castagnola b,c, Rosanna Inzitari b, Jo¨rgen Ekstro¨m d, Michela Isola e, Alessandro Riva e, Irene Messana a,* a
Department of Applied Sciences in Biosystems, University of Cagliari, I-09042 Monserrato, Italy Institute of Biochemistry and Clinical Biochemistry, Faculty of Medicine, Catholic University, Rome, Italy c Institute of Chemistry of Molecular Recognition, CNR, Rome, Italy d Institute of Neuroscience and Physiology, The Sahlgrenska Academy, Go¨teborg University, Go¨teborg, Sweden e Department of Cytomorphology, University of Cagliari, Monserrato, Italy b
article info
abstract
Article history:
Objective: To investigate protein content of saliva produced in vitro by samples of human
Accepted 4 June 2008
submandibular gland following stimulation with the muscarinic agent carbachol. Design: Tissue samples, obtained at surgery from seven patients and showing normal
Keywords:
morphological appearance, were tested for 30 min: in absence of carbachol and atropine;
Human submandibular gland
in presence of carbachol (10 mM); in presence of carbachol (10 mM) and atropine (20 mM); or in
Carbachol
presence of just atropine (20 mM). Medium was analysed by high-performance liquid
HPLC–MS
chromatography–mass-spectrometry. Neither before nor during surgery were the patients
Acidic proline-rich proteins
exposed to drug treatments that were likely to influence the in vitro secretion.
Peptide PC
Results: Proline-rich proteins (PRP)-1 and -3, peptide PC and PB, statherin, cystatins SN, S1
Peptide PB
and S2 were invariably found in control gland tissue medium. Mean concentrations of these
Statherin
proteins/peptides in the medium were non-proportionally elevated following carbachol
Cystatins
exposure to the gland tissues. Difference between basal release and carbachol-induced secretion achieved statistical significance as to all the proteins/peptides under study but for statherin. Atropine alone or atropine plus carbachol caused no significant changes compared to the basal release of proteins/peptides. Conclusions: In vitro studies on salivary glands make it possible to study protein secretion from individual glands and thus, to reveal the contribution of the various types of gland to protein/ peptide content of whole saliva. The disproportional responses to carbachol may imply that the proteins/peptides are not confined to the same cells or to the same intracellular locations and are therefore not secreted as packages at parasympathetic cholinergic activity. # 2008 Elsevier Ltd. All rights reserved.
1.
Introduction
The oral fluid is a mixture of saliva from major and minor salivary glands, fluid from the gingival pockets, cell debris and
bacteria. Depending on type and intensity of the reflex stimulus different types of salivary glands are mobilised to various extent to create the most purposeful salivary response as to volume and composition under physiological condi-
* Corresponding author. Tel.: +39 070 6574520; fax: +39 070 6754523. E-mail addresses: [email protected], [email protected] (I. Messana). 0003–9969/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2008.06.001
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tions.1 In animals, pure saliva is obtained for experimental purposes from duct-cannulated major salivary glands. In humans, studies are usually focused ‘‘on whole saliva’’, i.e. the oral fluid. Collection of saliva from a single gland is obtained by a device placed over the gland duct (or ducts).2 However, usually no direct cannulation is performed in humans. Thus, it is difficult to avoid contamination of the saliva samples by the oral fluid. Whereas in animal studies, secretion is often evoked by stimulation of the parasympathetic and sympathetic innervations or by injecting selective receptor agonists into the blood stream, human secretion is usually evoked by taste or chewing, procedures most likely engaging a number of transmission mechanisms.1,3 In vitro observations allow the contribution of various salivary gland types to the composition of whole saliva to be studied. Furthermore, pieces of gland tissues can be exposed to selectively acting drugs, e.g. mimicking nerve-induced actions, without having to dissect nerves or to consider any systemic drug effects in the body. Whereas secretory in vitro studies on salivary gland tissues from animals are not uncommon, see e.g. Refs. [4–7], such studies on human salivary gland tissues are, on the whole, few.8–11 Yet, the ultimate vindication for in vitro studies on salivary glands would be those using operational specimens from humans as pointed out by Garrett.12 Hence, in the present study, the effect of the muscarinic receptor agonist carbachol on the in vitro secretion into the medium of certain proteins/peptides from pieces of human submandibular glands was analysed by applying high-performance liquid chromatography (HPLC) coupled to mass-spectrometry in order to investigate submandibular contribution to the protein content of mixed saliva in response to parasympathetic cholinergic activity.
2.
Materials and methods
2.1.
Reagents and instruments
All common chemicals and reagents were of analytical grade and were purchased from Merck (Darmstadt, Germany) and Sigma–Aldrich (St. Louis, MO, USA). The HPLC apparatus was a Thermo Finnigan (San Jose, CA, USA) Surveyor HPLC connected by a T-splitter to a diode-array UV–vis detector and to an LCQ Deca XP Plus mass-spectrometer. The mass-spectrometer was equipped with an electrospray ion (ESI) source. The reversed-phase chromatographic column was a Vydac (Hesperia, CA, USA) C8 middle-bore column, with 5 mm particle diameter (column dimensions 150 mm 2.1 mm).
2.2.
Collection and treatment of the glandular tissue
Samples of human submandibular glands were obtained at tumour surgery from seven patients, six males and one female, aged 48–75 years. The glands had not been exposed to radiation. Furthermore, prior to surgery or during surgery the patients were not treated with drugs that were likely to interfere with the in vitro secretion under present experimental set-up. Informed consent was obtained in each case and permission was granted by the local ethical committee (A.S.L. 8, Cagliari). Immediately after the resection, gland
tissue was washed twice with physiological saline solution and subdivided into pieces of about 5 mm long, 2 mm wide, 2 mm high. Incubations were performed as previously described.13 Briefly, glandular tissue was incubated at 37 8C for 30 min in 10 mL of an oxygenated medium (6 mM Tris–HCl, 123 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4 and 5.5 mM glucose, pH 7.4) without or with carbachol chloride (10 mM), when appropriate, combined with atropine (20 mM) in 25 mL sealed Erlenmeyer flasks, placed in a water-bath and continuously shaken. At the end of incubation, the glandular tissue was removed from the medium, weighed and subjected to cytomorphological and ultrastructural analysis.10 This examination of semithin tissue sections, allowed further assessment of the normality of the gland pieces incubated. Only incubation media from specimens showing a normal histological feature based on cytomorphological and ultrastructural analysis were used for biochemical characterization of their content. The mean wet weight of normally looking tissue, subdivided into small pieces, incubated in each Erlenmeyer flask was 53 14 mg (n = 22). Medium was centrifuged (6000 g, for 10 min) to remove eventual gross material, lyophilized and stored at 80 8C, awaiting the following HPLC– MS analysis.
2.3.
RP-HPLC–ESI-MS analysis
Lyophilized powder was dissolved in 150 mL of 0.2% aqueous trifluoroacetic acid (TFA), centrifuged at 10,000 g (10 min) and concentration of proteins in the supernatant determined by the bicinchoninic assay (Pierce, Rockford, IL, USA) corresponded to 0.15 0.05 mg/(mL mg) of tissue under basal conditions and to 1.3 0.3 mg/(mL mg) of tissue in stimulated experiments. 100 mL aliquots of the solution were directly injected into the HPLC–MS apparatus. The following solutions were utilized for the reversed-phase chromatography: (eluent A) 0.056% aqueous TFA and (eluent B) 0.050% TFA in acetonitrile–water 80/20 (v/v). The gradient applied was linear from 0 to 55% in 40 min, at a flow-rate of 0.30 mL/min. The Tsplitter addressed a flow-rate of about 0.20 mL/min towards the diode-array detector and a flow-rate of about 0.10 mL/min towards the electrospray ionization source. The diode-array detector was set at a wavelength of 214 and 276 nm. The eluent was not directed towards the electrospray source for the first 5 min of separation, in order to avoid damage to the ion trapmass-spectrometer (IT-MS), due to the elevated concentration of electrolytes (salts, small polar molecules). The ESI source spray voltage was 4.50 kV and the capillary temperature 220 8C with sheath gas flow-rate of 70 arbitrary unities. The ion trap apparatus operated in positive mode in the 300–2000 m/z range and mass spectra were collected every 3 ms in the positive ion mode.
2.4.
Characterization of peptides and quantification
Proteins detected in incubation media corresponded in terms of both chromatographic elution time and ESI spectrum to salivary peptides characterized in human saliva in previous studies.14–17 Molecular mass values were determined by deconvolution of ESI spectra, performed either by the software provided with the Deca-XP instrument (Bioworks Browser) or
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Table 1 – Muticharged ions (m/z) utilized to extract the ion current (XIC) peak of proteins/peptides secreted in the incubation medium Protein/peptide
Mass/charge (charge)
PB peptide Statherin Histatin1 Histatin 5 PRP-1
828.7 (+7), 966.7 (+6), 1159.5 (+5), 1449.1 (+4), 1931.7 (+3) 1345.7 (+4), 1794.1 (+3) 986.9 (+5), 1233.3 (+4), 1643.9 (+3) 760.3 (+4), 1013.3 (+3) 913.6 (+17), 970.7 (+16), 1035.3 (+15), 1109.2 (+14), 1194.5 (+13), 1293.9 (+12), 1411.5 (+11), 1552.5 (+10), 1724.9 (+9), 1940.4 (+8) 859.6 (+13), 931.1 (+12), 1015.7 (+11), 1117.2 (+10), 1241.2 (+9), 1396.2 (+8), 1595.5 (+7), 1861.3 (+6) 875.4 (+5), 1094.0 (+6), 1458.3 (+7) 955.2 (+15), 1023.4 (+14), 1102.0 (+13), 1193.8 (+12), 1302.2 (+11), 1432.3 (+10), 1591.3 (+9), 1790.1 (+8) 952.0 (+15), 1019.9 (+14), 1098.3 (+13), 1189.7 (+12), 1297.8 (+11), 1427.5 (+10), 1586.0 (+9), 1784.1 (+8) 957.3 (+15), 1025.6 (+14), 1104.4 (+13), 1196.4 (+12), 1305.1 (+11), 1435.5 (+10), 1594.9 (+9), 1794.1 (+8)
PRP-3 PC peptide Cystatin SN Cystatin S1 Cystatin S2
by MagTran 1.0.18 Experimental average molecular mass values were compared to those reported upon the Swiss-Prot (http://us.expasy.org/tools) and EMBL (http://www.embl-heidelberg.de) data banks. For quantification we utilized the extracted ion current (XIC) peak areas. XIC peaks were revealed by searching appropriate multi-charged ions reported in Table 1. Area under the ion current peaks was normalized with respect to the weight of the glandular tissue and expressed in relative units per mg of gland tissue. The present results were derived from seven non-carbachol exposed control specimens (in case of more than one control tissue from the same gland a mean value was calculated for each gland), three carbachol exposed specimens, three carbachol and atropine exposed specimens and five specimens just exposed to atropine.
2.5.
Statistics
Student’s t-test for unpaired comparisons was based on log values. Probabilities of less than 5% were considered significant. Values are mean S.E.M.
3.
Results
A basal release of a number of proteins/peptides occurred invariably: the acidic proline-rich proteins PRP-1 and -3, the cystatins SN, S1 and S2, statherin, peptide PB and PC as previously reported.13 Histatin 1 and histatin 5, occurred sporadically, as also observed previously.13 The total ion current (TIC) RP-HPLC profile of the products released from a specimen under control condition is shown in Fig. 1 (panel a). The list of the released proteins/peptides, their Swiss-Prot code number, the theoretical average molecular mass value and the experimental mass value are reported in Table 2. In order to reveal low protein/peptide concentrations of the medium of control tissue, we applied the XIC strategy. The concentration of histatins of the medium was lower than the concentrations of the other proteins/peptides under study. The lack of histatins in some samples was probably due to concentrations below the limit of detection under our experimental condition. The XIC method is based on the contemporaneous search of all the multi-charged ions expected for a given protein along the TIC
Table 2 – Experimental masses (Da) detected in the incubation medium by RP-HPLC–ESI-MS compared to the theoretical values reported in international data banks Peptide/protein
Swiss-Prot code
Theoretical average masses
Experimental masses
Acidic PRPs PRP-1, Pif-s (PRP-2) Diphos PRP-3, Pif-f (PRP-4) Diphos. PC (a-PRP fr. 107–150)
P02810 P02810 P02810
15515 (15514) 11162 (11161) 4371
Basic PRPs PB
P02814
5793
5792 1
Histatins Histatin 1 Histatin 5
P15515 P15516
4928 3036
4928 1 3036 1
Statherin
P02808
5379
5380 1
Cystatins Cystatin SN Cystatin S1 Cystatin S2
P01037 P01036 –
14312 14265 14345
14313 2 14266 1 14346 1
15515 2 11161 1 4371 1
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Fig. 1 – RP-HPLC TIC (total ion current) profiles of the incubation medium of not stimulated (a) and 10 mM carbachol stimulated (b) submandibular gland tissue. The elution ranges of acidic PRPs, histatins, cystatins, PB peptide and statherin are evidenced. Panels (c) and (d) show the XIC (extracted ion current) peak of cystatin SN.
chromatographic profile. For example the panels c and d of Fig. 1 show the peak of cystatin SN recorded by using this strategy. A carbachol-induced secretion of proteins/peptides was demonstrated. From a qualitative point of view, no additional proteins/peptides appeared as compared to the basal release, as evident from Fig. 1 (panel b). As was the case under basal condition, the presence of histatins was not a consistent finding in the medium of those three samples exposed to carbachol. Therefore, this type of peptides was not included in the comparisons. For each protein/peptide consistently found, the figures for the mean response to carbachol were severalfold higher than those obtained under basal condition (Fig. 2a and b). However, the magnitude of the response of the individual proteins/ peptides (related to the control value in the absence of carbachol) was not uniform. The difference achieved statistical significance as to all proteins/peptides subjected to comparison but for statherin. In the presence of atropine (20 mM) and carbachol (10 mM), the concentration of proteins/peptides was not statistically significantly different from that of the medium of control tissue (Fig. 2a and b). However, the figures for mean values following exposure to both carbachol and atropine were usually higher than those in the absence of the combination. Exposure to just atropine (20 mM), showed no tendency for elevated mean values of proteins/peptides (Fig. 2a and b).
4.
Discussion
In vivo, it is particularly difficult to collect submandibular saliva not contaminated by sublingual saliva (see Ref. [19]). The present in vitro investigation allows the submandibular gland secretion to be studied separately and to show variations in the protein secretion following stimulation with the muscarinic agent carbachol. A basal release of a number of proteins from the incubated gland tissue was demonstrated, in agreement with previous observations.13 However, we were not able to show some major secretory products of submandibular glands, such as mucins,20–22 lysozyme,23 and amylase.24 Lack of mucin detection is related to the acidic treatment of the incubation medium in preparation for the mass-spectrometry analysis, which causes high molecular weight proteins to precipitate, whereas amylase and lysozyme were probably not revealed due to concentrations below the detection limit of our method. Likewise, lack of histatins in some samples was probably also due to levels below the detection limit. As would be expected only PB of the basic PRPs appeared in the media.25 As judged from the present findings, the submandibular gland is one contributor (not necessarily excluding the sublingual gland) to the concentrations of histatin 1, cystatin S and SN reported in mixed submandibular–sublingual saliva.11 The present findings of proline-rich proteins and histatins in the medium are in agreement with their
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Fig. 2 – Ion current peak area in relative units per mg of gland tissue of secreted proteins. (a) Proline-rich protein-1 (PRP-1), proline-rich protein-3 (PRP-3), PC peptide (PC), cystatin SN (Cyst SN), cystatin S1 (Cyst 1) and cystatin S2 (Cyst S2) and (b) PB peptide (PB) and statherin; note the difference in scale. Columns represent mean values and vertical bars + S.E.M. From left to right: (1) open columns in the absence of carbachol and atropine (controls, n = 7), (2) filled columns in the presence of carbachol (n = 3); columns with horizontal lines in the presence of carbachol and atropine (n = 3); and partly filled columns in the presence of atropine (n = 5). In some cases, the S.E.M. values were too small to show up in the graph. *P < 0.05 and **P = 0.01 as compared to control responses.
immunohistochemical demonstration in human submandibular glands.26 The basal release may reflect on-going constitutive secretion.27 Though the morphological appearance of the tissue seemed normal even at the ultrastructural level, it cannot be excluded that a certain fraction of the basal release
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was due to proteins/peptides that leaked from cells damaged as a consequence of the preparation. The human submandibular gland is innervated by parasympathetic and sympathetic nerves, and both cholinesterase-positive nerve fibres and catecholamine-fluorescence nerve fibres reach acinar as well as duct cells.28 Furthermore, the secretory elements are innervated by peptide-containing nerve fibres.29 Thus, reflex stimulation is likely to involve not only the activity of both divisions of the autonomic nervous system but also the action of conventional (acetylcholine and noradrenaline) and non-conventional (e.g. neuropeptides) transmitters to various degrees. In the present study, the muscarinic agonist carbachol most likely exerted its effect by the stimulation of muscarinic receptors of submandibular gland tissue to cause severalfold increases in the output of PRP-1 and -3, the cystatins SN, S1 and S2, peptide PB and PC. Support for this idea was gained from the finding that the levels of proteins/peptides in the medium were not significantly elevated, when the tissues were exposed to the combined action of carbachol and atropine. However, there was a clear tendency for higher figures for mean values following the combination of carbachol and atropine. This was probably a reflection of too a low concentration of atropine. Control experiments showed that atropine, in itself, caused no increases in the output of the proteins/peptides under study. Hence, a parasympathetic cholinergic transmission mechanism is probably involved in the secretion of these proteins/ peptides. Taking into account the relatively small number of observations and the large variations in the output of the individual proteins, conclusions should be drawn with caution. Nevertheless, the secretory pattern seemed to suggest that the proteins under study are not responding uniformly, exemplified by differences in the magnitude of response (in relation to basal value) within the cystatin family, and the lack of significant increase as to statherin. The disproportion may indicate that the proteins are not confined to the same cells or to the same intracellular locations and will therefore not be secreted as packages in response to parasympathetic nerve activity. In this connection, it may be recalled that in man the submandibular acinar cells are of two types, serous (90%) and mucous (10%) and that cystatingene transcripts are localized to the cytoplasm of only serous acinar cells of submandibular glands.30 Moreover, electron microscopic immunocytochemistry investigation of acinar cells in human submandibular glands shows some proteins to be differentially distributed within the granular content (amylase and agglutinins), while others (proline-rich proteins and histatins) are distributed uniformly or randomly throughout the granular content, suggesting that different mechanisms may be involved in the packaging of individual proteins within the same granule.26 The present demonstration of a carbachol-induced secretion of proteins may be combined with earlier cytomorphological findings on human submandibular gland tissue.31 Under the same experimental conditions and with the same concentration of carbachol as presently, a number of ultrastructural events takes place that indicates secretory activity; besides enlargement of lumina and appearance of V-shaped profiles and vacuoles, the density of the microvilli decreases, whereas that of the microbuds increases.10 Thus, the present
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experimental set-up maybe suitable for exploration of both ultrastructural changes and outputs of proteins and peptides of human salivary glands in response to a number of putative regulatory agonists of secretory activities such as neuropeptides and gastrointestinal hormones32,33; using the present preparation, unpublished observations of ours reveal a secretory effect of pentagastrin in human glands (Loy F, Diana M, Ekstro¨m J, Riva A). In conclusion, carbachol-induced the in vitro secretion of a number of proteins/peptides from human submandibular gland tissue specimens, thus indicating a parasympathetic cholinergic transmission mechanism involved in their secretion under normal conditions. In relative terms, the proteins/ peptides were not uniformly affected, suggesting that they may be stored at different locations and therefore not secreted as packages.
Acknowledgements We acknowledge the financial support of Cagliari University, Catholic University in Rome, MUR, Italian National Research Council (CNR-programs of scientific research promotion and diffusion), The Swedish Science Council (project no. 05927), The LUA/ALF agreement (project no. ALFGBG-11907) and the ‘‘INTERLINK’’-agreement (project no. II04C00H8M).
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