Physiology of saliva

DENT 5302

Topics in Dental BiochemistryDr. Joel Rudney

Foundation knowledge

DENT 5315 Oral Histology Dr. Koutlas’ salivary gland lectures

Ten Cate’s Oral Histology Chapter on Salivary Glands

NSCI 6110 Neuroscience for Dental Students Material on neurotransmitters, signal transduction

PHSL 6051 Physiology for Dental Students Material on water transport, signal transduction Future foundation for gastrointestinal and kidney

Innervation of stimulation

Dual autonomic innervation of salivary glands Parasympathetic - secretion of water and ions Sympathetic - protein secretion Both act simultaneously and synergistically

Mediated by G-protein coupled receptors Parasympathetic - M3 muscarinic receptors

Minor players - neuropeptide; nucleotide receptors• VIP, Substance P, nucleotides, etc.

Sympathetic - 2 adrenergic receptorsMinor players - adrenergic receptors

Two different signal transduction pathways

Muscarinic messages

http://www.liv.ac.uk/~petesmif/teaching/1bds_mb/p4/15.gif

The Phospholipase C - IP3 pathway sends the message

Intracellular (and extracellular) Ca2+ flux is a major effector

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)

Water/electrolyte secretion Water secretion is driven by osmotic changes

Mediated by ionic fluxesFrom basolateral surfaces to the apex (lumen)

Involves ion pumps and channels Basolateral

Na+-K+-ATPase Ca2+ activated K+ channelNa+-K+-2Cl--cotransporter (NKCCl)Na+-H+ exchangerCl-- HCO3

- exchanger, plus Carbonic anhydrase

LumenalCa2+ activated Cl- channelHCO3

- channel (Ca2+ activated?) , plus Carbonic anhydrase

Alternative mechanisms

Na+-K+-ATPase

Ca2+ activated K+ channel

Na+-K+-2Cl--cotransporter

Ca2+ activated Cl- channel

Na+-H+ exchanger

Cl-- HCO3- exchanger

Carbonic anhydrase

Na+-H+ exchanger

HCO3- channel

Carbonic anhydrase

Adapted from Turner and Sugiya, Oral Dis. 2:3-11, 2002

Newly-discovered components

Channels for extracellular Ca2+ in basolateral membrane Initiate Ca2+ flux that activates other ion channels hTrp1, others?

How does water cross the apical membrane? Aquaporin family of water channels

Found in many organ systemsSalivary aquaporin is Aqp5

Ca2+ activated, open to let water outIonic flux pulls the water out - during stimulation

Low level of activation in resting state??

Validation of the model

http://wwwdir.nidcr.nih.gov/dirweb/common/sps.jpgGresz et al. Am. J. Phsiol. 287: G151-G161, 2004

Apical Aquaporin 5

Basolateral Na+-K+-ATPase

Ductal reabsorption

Saliva entering the lumen is isotonic Saliva entering the mouth is hypotonic Reabsorption of Na+ and Cl- by striated duct cells

Similar to distal tubules of kidneys Ion pumps and channels

LumenalNa+-H+ exchangerCl-- HCO3

- exchangerHCO3

- channelNa+-K+ exchangerNa+-Cl--cotransporter

BasolateralNa+-K+-ATPaseCl- channel

Striated duct cell

Cl-

3 Na+

2 K+ATP

Cl-Na+

Na+

Na+

K+

H+

Cl-

HCO3-

HCO3-

Lumen Interstitium

Nucleus

MitochondriaBasolateral membrane folds

Carbonic anhydrase

Clinical significance

Many points for drugs to interfere with water secretion Receptors, signal transduction, ion pumps/channels May explain why xerostomia is a widespread side effect

The M3 receptor is a key point Autoantibodies to M3 occur in some Sjogren’s patients

Sjogren’s etiology and pathogenesis is very complex Agonists can be useful in profound xerostomia treatment

Pilocarpine and CevimilineRequires some remaining functional tissue

Anti-cholinergicsMost likely to induce xerostomia as a side effect

Research “in the pipeline”

Can we repair damaged salivary glands? Gene therapy approach

Use viruses to transfect genes into host cells Infusion into ducts

Ducts are best preserved in Sjogren’s/radiation Transfect aquaporin into rat duct cells

Not normally present in duct cellsTransfection increased salivary flowShort-term effect, and only replaces waterWould need to replace many genes for full repair

Tissue engineering

May have more potential in the long run Step 1: Create a biocompatible scaffold

Must have a duct-like structure Step 2: Seed with cells

Engineer cells to function like secretory/duct cells OR Use stem cells and induce differentiation

Step 3: Implant into a patient Must induce vascularization and innervation Must suppress rejection or use compatible cells Will it make saliva??

Protein secretion

A parallel process to water/ion secretion Both occur side by side in the same secretory cell There is complex cross-talk between pathways

Classic exocytosis pathway Endoplasmic reticulum - translation, glycosylation Golgi - more extensive glycosylation Condensing vacuole - packaging, condensation Immature granule - sorting, major branching point Secretory granule - protein storage -adrenergic stimulation

Docking, membrane fusion, exocytosis

Classic exocytosis

Immediate response to NA:Docking and fusion of preformed granulesRelease of contents

Long-term response to NA:TranscriptionTranslationGlycosylationNew granuleshttp://www.liv.ac.uk/~petesmif/teaching/1bds_mb/p4/14.gif

(Noradrenaline)

Secretory granules

Complex internal structure Multiple types of proteins, compacted and folded

Membrane proteins that mediate docking and fusion V(esicle)-SNARES on granule membranes T(arget)-SNARES on inner side of cell apical membrane A Ca2+ -dependent process

Example of cross talk between pathways

The other protein pathways Constitutive-like pathway

Branches off from immature granules Proteins carried in vesicles to apex - fuse and open Always active - no stimulation required

Minor regulated pathway Branches off from immature granules Proteins carried in vesicles to apex - fuse and open Triggered by low levels of M3 cholinergic agonists Vesicle membranes contain t-SNARES for granules

Both are sources of proteins in basal and resting secretions Vesicle contents are different from granule contents Explains different protein composition after stimulation

Cross-talk is essential

Castle, A. M. et al. J Cell Sci 2002;115:2963-2973

Cholinergic agonist - very low doseConstitutive-like and Minor Regulated only

Cholinergic agonist - low doseConstitutive-like and Minor Regulated with occasional granule docking

Adrenergic agonist - standard doseConstitutive-like and Minor Regulated plus Classic Exocytosis

Adrenergic agonist - standard doseCholinergic agonist - low dose Constitutive-like and Minor Regulated plus synergistic Classic Exocytosis

Supplemental Reading

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