1. Physiology of saliva DENT 5302 Topics in Dental Biochemistry Dr. Joel Rudney

2. Foundation knowledge zDENT 5315 Oral Histology yDr. Koutlas’ salivary gland lectures zTen Cate’s Oral Histology yChapter on Salivary Glands zNSCI 6110 Neuroscience for Dental Students yMaterial on neurotransmitters, signal transduction zPHSL 6051 Physiology for Dental Students yMaterial on water transport, signal transduction yFuture foundation for gastrointestinal and kidney

3. Innervation of stimulation zDual autonomic innervation of salivary glands yParasympathetic - secretion of water and ions ySympathetic - protein secretion yBoth act simultaneously and synergistically zMediated by G-protein coupled receptors yParasympathetic - M3 muscarinic receptors xMinor players - neuropeptide; nucleotide receptors VIP, Substance P, nucleotides, etc. ySympathetic -  2 adrenergic receptors xMinor players -  adrenergic receptors yTwo different signal transduction pathways

4. Muscarinic messages http://www.liv.ac.uk/~petesmif/teaching/1bds_mb/p4/15.gif The Phospholipase C - IP 3 pathway sends the message Intracellular (and extracellular) Ca 2+ flux is a major effector

5. Adrenergic messages http://www.liv.ac.uk/~petesmif/teaching/1bds_mb/p4/16.gif The adenylate cyclase - cAMP pathway sends the message Effectors are activated by a phophorylation cascade (Noradrenaline)

6. Water/electrolyte secretion zWater secretion is driven by osmotic changes yMediated by ionic fluxes xFrom basolateral surfaces to the apex (lumen) zInvolves ion pumps and channels yBasolateral xNa + -K + -ATPase xCa 2+ activated K + channel xNa + -K + -2Cl - -cotransporter (NKCCl) xNa + -H + exchanger xCl - - HCO 3 - exchanger, plus Carbonic anhydrase yLumenal xCa 2+ activated Cl - channel xHCO 3 - channel (Ca 2+ activated?), plus Carbonic anhydrase

7. Alternative mechanisms Na + -K + -ATPase Ca 2+ activated K + channel Na + -K + -2Cl - -cotransporter Ca 2+ activated Cl - channel Na + -H + exchanger Cl - - HCO 3 - exchanger Carbonic anhydrase Na + -H + exchanger HCO 3 - channel Carbonic anhydrase Adapted from Turner and Sugiya, Oral Dis. 2:3-11, 2002

8. Newly-discovered components zChannels for extracellular Ca 2+ in basolateral membrane yInitiate Ca 2+ flux that activates other ion channels yhTrp1, others? zHow does water cross the apical membrane? yAquaporin family of water channels xFound in many organ systems xSalivary aquaporin is Aqp5 yCa 2+ activated, open to let water out xIonic flux pulls the water out - during stimulation yLow level of activation in resting state??

9. Validation of the model http://wwwdir.nidcr.nih.gov/dirweb/common/sps.jpg Gresz et al. Am. J. Phsiol. 287: G151-G161, 2004 Apical Aquaporin 5 Basolateral Na + -K + -ATPase

10. Ductal reabsorption zSaliva entering the lumen is isotonic zSaliva entering the mouth is hypotonic zReabsorption of Na + and Cl - by striated duct cells ySimilar to distal tubules of kidneys zIon pumps and channels yLumenal xNa + -H + exchanger xCl - - HCO 3 - exchanger xHCO 3 - channel xNa + -K + exchanger xNa + -Cl - -cotransporter yBasolateral xNa + -K + -ATPase xCl - channel

11. Striated duct cell Cl - 3 Na + 2 K + ATP Cl - Na + K+K+ H+H+ Cl - HCO 3 - Lumen Interstitium Nucleus Mitochondria Basolateral membrane folds Carbonic anhydrase

12. Clinical significance zMany points for drugs to interfere with water secretion yReceptors, signal transduction, ion pumps/channels yMay explain why xerostomia is a widespread side effect zThe M3 receptor is a key point yAutoantibodies to M3 occur in some Sjogren’s patients xSjogren’s etiology and pathogenesis is very complex yAgonists can be useful in profound xerostomia treatment xPilocarpine and Cevimiline xRequires some remaining functional tissue yAnti-cholinergics xMost likely to induce xerostomia as a side effect

13. Research “in the pipeline” zCan we repair damaged salivary glands? zGene therapy approach yUse viruses to transfect genes into host cells yInfusion into ducts xDucts are best preserved in Sjogren’s/radiation yTransfect aquaporin into rat duct cells xNot normally present in duct cells xTransfection increased salivary flow xShort-term effect, and only replaces water xWould need to replace many genes for full repair

14. Tissue engineering zMay have more potential in the long run zStep 1: Create a biocompatible scaffold yMust have a duct-like structure zStep 2: Seed with cells yEngineer cells to function like secretory/duct cells OR yUse stem cells and induce differentiation zStep 3: Implant into a patient yMust induce vascularization and innervation yMust suppress rejection or use compatible cells yWill it make saliva??

15. Protein secretion zA parallel process to water/ion secretion yBoth occur side by side in the same secretory cell yThere is complex cross-talk between pathways zClassic exocytosis pathway yEndoplasmic reticulum - translation, glycosylation yGolgi - more extensive glycosylation yCondensing vacuole - packaging, condensation yImmature granule - sorting, major branching point ySecretory granule - protein storage y  -adrenergic stimulation xDocking, membrane fusion, exocytosis

16. Classic exocytosis Immediate response to NA: Docking and fusion of preformed granules Release of contents Long-term response to NA: Transcription Translation Glycosylation New granules http://www.liv.ac.uk/~petesmif/teaching/1bds_mb/p4/14.gif (Noradrenaline)

17. Secretory granules zComplex internal structure yMultiple types of proteins, compacted and folded zMembrane proteins that mediate docking and fusion yV(esicle)-SNARES on granule membranes yT(arget)-SNARES on inner side of cell apical membrane yA Ca 2+ -dependent process xExample of cross talk between pathways

18. The other protein pathways zConstitutive-like pathway yBranches off from immature granules yProteins carried in vesicles to apex - fuse and open yAlways active - no stimulation required zMinor regulated pathway yBranches off from immature granules yProteins carried in vesicles to apex - fuse and open yTriggered by low levels of M3 cholinergic agonists yVesicle membranes contain t-SNARES for granules zBoth are sources of proteins in basal and resting secretions yVesicle contents are different from granule contents yExplains different protein composition after stimulation

19. Cross-talk is essential Castle, A. M. et al. J Cell Sci 2002;115:2963-2973 Cholinergic agonist - very low dose Constitutive-like and Minor Regulated only Cholinergic agonist - low dose Constitutive-like and Minor Regulated with occasional granule docking Adrenergic agonist - standard dose Constitutive-like and Minor Regulated plus Classic Exocytosis Adrenergic agonist - standard dose Cholinergic agonist - low dose Constitutive-like and Minor Regulated plus synergistic Classic Exocytosis

20. Supplemental Reading Turner RJ, Sugiya H (2002). Understanding salivary fluid and protein secretion. Oral Diseases 8:3-11.