Physical exercise stimulates salivary secretion of cystatins

Autoři: Marcelo de Lima Sant’Anna aff001;  Leandro Teixeira Oliveira aff001;  Diego Viana Gomes aff002;  Sergio Tadeu Farinha Marques aff004;  D. William Provance, Jr aff005;  Martha Meriwether Sorenson aff001;  Verônica Pinto Salerno aff002
Působiště autorů: Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil aff001;  Department of Physical Activity Biosciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil aff002;  Almirante Sylvio de Carmargo Training Center, Brazilian Navy, Rio de Janeiro, Brazil aff003;  Naval Sports Commisson, Brazilian Navy, Rio de Janeiro, Brazil aff004;  Center for Technological Development in Health, Oswaldo Cruz Insitute, Rio de Janeiro, Brazil aff005
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


Physical exercise is known to activate the sympathetic nervous system, which influences the production of saliva from salivary glands. Our examination of saliva collected from highly trained athletes before and after a number of physical competititions showed an increase in the secretion of S-type cystatins and cystatin C as a subacute response to aerobic and anaerobic exercise. The elevation in salivary cystatins was transient and the recovery time course differed from that of amylase and other salivary proteins. An in vitro assay was developed based on a cell line from a human submandibular gland (HSG) that differentiated into acinus-like structures. Treatments with the β-adrenergic agonist isoproterenol caused a shift in the intracellular distribution of S-type cystatins and cystatin C, promoting their accumulation at the outer regions of the acinus prior to release and suggesting the activation of a directional transport involving co-migration of both molecules. In another treatment using non-differentiated HSG cells, it was evident that both expression and secretion of cystatin C increased upon addition of the β-adrenergic agonist, and these effects were essentially eliminated by the antagonist propranolol. The HSG cell line appears to have potential as a model for exploring the mechanism of cystatin secretion, particularly the S-type cystatins that originate primarily in the submandibular glands.

Klíčová slova:

Exercise – Running – Saliva – Salivary glands – Secretion – Sports – Submandibular glands – Isoproterenol


1. Papacosta E, Nassis GP. Saliva as a tool for monitoring steroid, peptide and immune markers in sport and exercise science. J Sci Med Sport. 2011;14: 424–434. doi: 10.1016/j.jsams.2011.03.004 21474377

2. de Sousa-Pereira P, Amado F, Abrantes J, Ferreira R, Esteves PJ, Vitorino R. An evolutionary perspective of mammal salivary peptide families: cystatins, histatins, statherin and PRPs. Arch Oral Biol. 2013;58(5): 45145–45148. doi: 10.1016/j.archoralbio.2012.12.011 23352445

3. Chicharro JL, Lucía A, Pérez M, Vaquero AF, Ureña R. Saliva composition and exercise. Sports Med. 1998;26(1): 17–27. doi: 10.2165/00007256-199826010-00002 9739538

4. Ekstrom J, Khosravani N, Castagnola M, Messana I. Saliva and control of its secretion. In: Olle Ekberg editor. Dysphagia: diagnosis and treatment. Berlin: Springer; 2012. pp. 19–47.

5. Proctor GB, Carpenter GH. Salivary secretion: mechanism and neural regulation. Monogr Oral Sci. 2014;24: 14–29. doi: 10.1159/000358781 24862591

6. Henskens YM, Veerman EC, Mantel MS, van der Velden U, Nieuw Amerongen AV. Cystatins S and C in human whole saliva and in glandular salivas in periodontal health and disease. J Dent Res. 1994,73(10): 1606–14. doi: 10.1177/00220345940730100501 7929975

7. Babad H, Ben-Zvi R, Bdolah A, Schramm M. The mechanism of enzyme secretion by the slices. Eur J Biochem. 1967, 1(1): 96–101. doi: 10.1111/j.1432-1033.1967.tb00049.x 6059351

8. Kjaer M, Secher NH, Galbo H. Physical stress and catecholamine release. Baillieres Clin Oral Dis. 2018 Nov;24(8):1477–1483. doi: 10.1111/odi.12920 Epub 2018 Jul 10.

9. Bocanegra OL, Diaz MM, Teixeira RR, Soares SS, Espindola FS. Determination of the lactate threshold by means of salivary biomarkers: chromogranin A as novel marker of exercise intensity. Eur J Appl Physiol. 2012;112(9): 3195–3203. doi: 10.1007/s00421-011-2294-4 22227853

10. Ventre G, Colonna C, Smith J, Alfano D, Moldow R. Salivary VIP concentrations are elevated in humans after acute stress. Peptides. 2013;49: 27–31. doi: 10.1016/j.peptides.2013.08.014 23994551

11. Aydin S, Aydin S, Kuloglu T, Yilmaz M, Kalayci M, Sahin I, et al. Alterations of irisin concentrations in saliva and serum of obese and normal-weight subjects, before and after 45 min of a Turkish bath or running. Peptides. 2013;50: 13–18. doi: 10.1016/j.peptides.2013.09.011 24096106

12. Ligtenberg AJ, Brand HS, van den Keijbus PA, Veerman EC. The effect of physical exercise on salivary secretion of MUC5B, amylase and lysozyme. Arch Oral Biol. 2015;60(11): 1639–1644. doi: 10.1016/j.archoralbio.2015.07.012 26351746

13. Henskens YM, van den Keijbus PA, Veerman EC, Van der Weijden GA, Timmerman MF, Snoek CM, et al. Protein composition of whole and parotid saliva in healthy and periodontitis subjects. Determination of cystatins, albumin, amylase and IgA. J Periodontal Res. 1996;31(1): 57–65. doi: 10.1111/j.1600-0765.1996.tb00464.x 8636877

14. CISM—Conseil International du Sport Militaire. Naval Pentathlon: Sports Regulation. Available from:, 2009 (accessed 01.10.2015).

15. Ricardo DR, de Almeida MB, Franklin BA, Araújo CG. Initial and final exercise heart rate transients: influence of gender, aerobic fitness, and clinical status. Chest. 2005;127(1): 318–27. doi: 10.1378/chest.127.1.318 15653999

16. Sant’Anna ML, Casimiro-Lopes G, Boaventura G, Marques STF, Sorenson MS, Salerno VP.Anaerobic exercise affects the saliva antioxidant/oxidant balance in high-performance pentathlon athetes. Humo. 2016;17(1): 50–55. doi: org/10.1515/humo-2016-0003

17. Zagatto AM, Beck WR, Gobatto CA. Validity of the running anaerobic sprint test for assessing anaerobic power and predicting short-distance performances. J Strength Cond Res. 2009;23(6):1820–7. doi: 10.1519/JSC.0b013e3181b3df32 19675478

18. Ljungberg G, Ericson T, Ekblom B, Birkhed D. Saliva and marathon running. Scand J Med Sci Sports. 1997;7: 214–219. 9241026

19. Dawes C. The effects of exercise on protein and electrolyte secretion in parotid saliva. J Physiol. doi: 10.1113/jphysiol.1981.sp013940 7320933

20. Bellagambi FG, Degano I, Ghimenti S, Lomonaco T, Dini V, Romanelli M, et al. Determination of salivary α-amylase and cortisol in psoriatic subjects undergoing the Trier Social Stress Test. Microchem J. 2018 Jan 136: 177–184.

21. Navazesh M. Methods for collecting saliva. Ann N Y Acad Sci. 1993;694: 72–7. doi: 10.1111/j.1749-6632.1993.tb18343.x 8215087

22. Gonçalves Lda R, Soares MR, Nogueira FC, Garcia C, Camisasca DR, Domont G. et al. Comparative proteomic analysis of whole saliva from chronic periodontitis patients. Proteomics. 2010;73(7): 1334–1341. doi: 10.1016/j.jprot.2010.02.018 20215060

23. Bradford MM. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72: 248–254. doi: 10.1006/abio.1976.9999 942051

24. Aguilar HN, Zielnik B, Tracey CN, Mitchell BF. Quantification of rapid Myosin regulatory light chain phosphorylation using high-throughput in-cell Western assays: comparison to Western immunoblots. PLoS One 2010;5(4):e9965. doi: 10.1371/journal.pone.0009965 20376358

25. Shirasuna K, Sato M, Miyazaki T. A neoplastic epithelial duct cell line established from an irradiated human salivary gland. Cancer (Phila.). 1981;48: 745–752.

26. Capes-Davis A, Theodosopoulos G, Atkin I, Drexler HG, Kohara A, MacLeod RA. Check your cultures! A list of cross-contaminated or misidentified cell lines. Int J Cancer. 2010 Jul 1;127(1):1–8. doi: 10.1002/ijc.25242 20143388

27. Lin LC, Elkashty O, Ramamoorthi M, Trinh N, Liu Y, Sunavala-Dossabhoy G. Cross-contamination of the human salivary gland HSG cell line with HeLa cells: A STR analysis study. Oral Dis. 2018;24(8):1477–1483. doi: 10.1111/odi.12920 29923277

28. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7): 676–682. doi: 10.1038/nmeth.2019 22743772

29. Kakanis MK, Peake J, Brenu EW, Simmonds M, Gray B, Hooper SL, et al. The open window of susceptibility to infection after acute exercise in healthy young male elite athletes. Exerc Immunol Rev. 2010;16: 119–137. 20839496

30. Mikulski T, Ziemba A, Nazar K. Influence of body carbohydrate store modification on catecholamine and lactate responses to graded exercise in sedentary and physically active subjects. J Physiol Pharmacol. 2008;59(3): 603–616 18953101

31. Scharhag-Rosenberger F, Carlsohn A, Lundby C, Schüler S, Mayer F, Scharhag J. Can more than one incremental cycling test be performed within one day? Eur J Sport. Sci. 2014;14(5): 459–467. doi: 10.1080/17461391.2013.853208 24168437

32. Fernandes AL, Lopes-Silva JP, Bertuzzi R, Casarini DE, Arita DY, Bishop DJ. Effect of time of day on performance, hormonal and metabolic response. during a 1000-M cycling time trial. PLoS ONE. 2014;9(10): e109954. doi: 10.1371/journal.pone.0109954 25289885

33. Soltoff S, Hedden LJ. Isoproterenol and cAMP block ERK phosphorylation and enhance [Ca2+]i Increases and oxygen consumption by muscarinic receptor stimulation in rat parotid and submandibular acinar cells. Biol Chem. 2010;285(18): 13337–13348. doi: 10.1074/jbc.M110.112094 20207737

34. Carpenter GH. The secretion, components, and properties of saliva. Annu Rev Food Sci Technol. 2013;4: 267–276. doi: 10.1146/annurev-food-030212-182700 23464573

35. Messenger SW, Falkowski MA, Groblewski GE. Ca2+-regulated secretory granule exocytosis in pancreatic and parotid acinar cells. Cell Calc. 2014;55(6): 369–375. doi: 10.1016/j.ceca.2014.03.003 24742357

36. Chopra DP, Xue-Hu IC. Secretion of alpha-amylase in human parotid gland epithelial cell culture. J Cell Physiol. 1993;155(2): 223–33. doi: 10.1002/jcp.1041550202 8097745

37. Vag J, Byrne EM, Hughes DH, Hoffman M, Ambudkar I, Maguire P, et al. Morphological and functional differentiation of HSG cells: role of extracellular matrix and trpc 1. J Cell Physiol. 2007;212(2): 416–23. doi: 10.1002/jcp.21035 17348017

38. Castle D, Castle A. Intracellular transport and secretion of salivary proteins. Crit Rev Oral Biol Med. 1998;9(1): 4–22. 9488245

39. Chatterton RT, Jr, Vogelsong KM, Lu YC, Ellman AB, Hudgens GA. Salivary α-amylase as a measure of endogenous adrenergic activity. Clin Physiol. 1996;16: 433–448. doi: 10.1111/j.1475-097x.1996.tb00731.x 8842578

40. Ghimenti S, Lomonaco T, Onor M, Murgia L, Paolicchi A, Fuoco R, et al. Measurement of warfarin in the oral fluid of patients undergoing anticoagulant oral therapy. PLoS One. 2011;6(12): e28182. doi: 10.1371/journal.pone.0028182 22164240

41. Lindh E, Brännström J, Jones P, Wermeling F, Hässler S, Betterle C, et al. Autoimmunity and cystatin SA1 deficiency behind chronic mucocutaneous candidiasis in autoimmune polyendocrine syndrome type 1. J Autoimmun. 2013;42: 1–6. doi: 10.1016/j.jaut.2012.10.001 23122533

42. Imoto Y, Tokunaga T, Matsumoto Y, Hamada Y, Ono M, Yamada T, et al. Cystatin SN upregulation in patients with seasonal allergic rhinitis. PLoS ONE. 2013;8(8): e67057. doi: 10.1371/journal.pone.0067057 23950865

43. Dickinson DP, Thiesse M, Hicks MJ. Expression of type 2 cystatin genes CST1-CST5 in adult human tissues and the developing submandibular gland. DNA Cell Biol. 2002;21(1): 47–65. doi: 10.1089/10445490252810311 11879580

44. Ganeshnarayan K, Velliyagounder K, Furgang D, Fine DH. Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans. J Periodont Res 2012; 47: 661–673. doi: 10.1111/j.1600-0765.2012.01481.x 22582873

45. Blankenvoorde MF, Henskens YM, van't Hof W, Veerman EC, Nieuw Amerongen AV. Inhibition of the growth and cysteine proteinase activity of Porphyromonas gingivalis by human salivary cystatin S and chicken cystatin. Biol Chem. 1996;377(12): 847–850. 8997496

46. Morgan-Bathke M, Lin HH, Chibly AM, Zhang W, Sun X, Chen CH, et al. Deletion of ATG5 shows a role of autophagy in salivary homeostatic control. J Dent Res 2013; 92(10):911–917. doi: 10.1177/0022034513499350 23884556

47. Watanabe S, Komine O, Endo F, Wakasugi K, Yamanaka K, et al. Intracerebroventricular administration of Cystatin C ameliorates disease in SOD1-linked amyotrophic lateral sclerosis mice. J Neurochem. 2018;145(1): 80–89. doi: 10.1111/jnc.14285 29282717

48. West NP, Pyne DB, Kyd JM, Renshaw GM, Fricker PA, Cripps AW. The effect of exercise on innate mucosal immunity. Br J Sports Med. 2010;44: 227–231. doi: 10.1136/bjsm.2008.046532 18499767

49. Davison G, Allgrove J, Gleeson M. Salivary antimicrobial peptides (LL-37 and alpha-defensins HNP1–3), antimicrobial and IgA responses to prolonged exercise. Eur J Appl Physiol. 2009;106: 277–284. doi: 10.1007/s00421-009-1020-y 19263072

50. Fábián TK, Hermann P, Beck A, Fejérdy P, Fábián G. Salivary defense proteins: their network and role in innate and acquired oral immunity. Int J Mol Sci. 2012;13(4): 4295–4320. doi: 10.3390/ijms13044295 22605979

51. Nieman DC. Exercise, Infection, and Immunity Int. J. Sports Med. 1994(15): S131—S141.

52. Nieman DC. Risk of upper respiratory tract infection in athletes: an epidemiologic and immunologic perspective. J Athl Train. 1997;32(4): 344–349. 16558471

53. Walsh NP, Gleeson M, Shephard RJ, Gleeson M, Woods JA, Bishop NC, et al. Position statement. Part one: Immune function and exercise. Exerc Immunol Rev. 2011;17: 6–63 21446352

54. Gleeson M, David BP, Lisa JE, Sharron TH, John RA, Christopher O, et al. Developing a multi-component immune model for evaluating the risk of respiratory illness in athletes Exerc Immunol Rev. 2017;23:52–64. 28230530

55. Campbell JP, Turner JE. Debunking the myth of exercise- induced immune suppression: redefining the impact of exercise on immunological health across the lifespan. Front. Immunol. 2018;9: 648. doi: 10.3389/fimmu.2018.00648 29713319

56. Jasim H, Olausson P, Hedenberg-Magnusson B, Ernberg M, Ghafouri B. The proteomic profile of whole and glandular saliva in healthy pain-free subjects Scientific Reports. 2016;6: 39073. doi: 10.1038/srep39073 27976689

57. Kunz H, Bishop NC, Spielmann G,· Pistillo M, Reed J, Ograjsek T, et al.·Fitness level impacts salivary antimicrobial protein responses to a single bout of cycling exercise. Eur J Appl Physiol. 2015;115: 1015–1027. doi: 10.1007/s00421-014-3082-8 25557386

58. Zouhal H, Jacob C, Delamarche P, Gratas-Delamarche A. Catecholamines and the effects of exercise, training and gender. Sports Med. 2008;38(5): 401–423. doi: 10.2165/00007256-200838050-00004 18416594

59. Ji X, Yao L, Wang M, Liu X, Peng S, Li K, et al. Cystatin C attenuates insulin signaling transduction by promoting endoplasmic reticulum stress in hepatocytes. FEBS Lett. 52015;589(24): 3938–3944. doi: 10.1016/j.febslet.2015.11.029 26592151

60. Takuma T, Shitara A, Arakawa T, Okayama M, Mizoguchi I, Tajima Y. Isoproterenol stimulates transient SNAP23-VAMP2 interaction in rat parotid glands. FEBS Lett. 2013;587(6): 583–9. doi: 10.1016/j.febslet.2013.01.039 23380067

61. Kim SK, Jones TP, Cuzzort LM. Protein synthesis and amylase messenger RNA content in rat parotid salivary glands after total or partial stimulation with isoproterenol. Arch Oral Biol. 1989;34(11): 895–901. doi: 10.1016/0003-9969(89)90147-7 2482020

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