Physiology of saliva

DENT 5302

Topics in Dental Biochemistry

Dr. Joel Rudney

Foundation knowledge
DENT 5315 Oral Histology
Dr. Koutlas salivary gland lectures
Ten Cates 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 receptors
Minor players - adrenergic receptors
Two different signal transduction pathways

Muscarinic messages
The Phospholipase
C - IP3 pathway
sends the message

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

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

Adrenergic messages
(Noradrenaline)

The adenylate cyclase

- cAMP pathway
sends the message
Effectors are activated by a
phophorylation cascade

http://www.liv.ac.uk/~petesmif/teaching/1bds_mb/p4/16.gif

Water/electrolyte secretion
Water secretion is driven by osmotic changes
Mediated by ionic fluxes
From basolateral surfaces to the apex (lumen)
Involves ion pumps and channels
Basolateral
Na+-K+-ATPase
Ca2+ activated K+ channel
Na+-K+-2Cl--cotransporter (NKCCl)
Na+-H+ exchanger
Cl-- HCO3- exchanger, plus Carbonic anhydrase
Lumenal
Ca2+ activated Cl- channel
HCO3- channel (Ca2+ activated?) , plus Carbonic anhydrase

Alternative mechanisms
Na+-H+ exchanger
Cl-- HCO3- exchanger
Carbonic anhydrase
Na+-H+ exchanger
HCO3- channel
Carbonic anhydrase
Na+-K+-ATPase
Ca2+ activated K+ channel
Na+-K+-2Cl--cotransporter
Ca2+ activated Cl- channel
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 systems
Salivary aquaporin is Aqp5
Ca2+ activated, open to let water out
Ionic flux pulls the water out - during stimulation
Low level of activation in resting state??

Validation of the model

Gresz et al. Am. J. Phsiol. 287: G151-G161, 2004

http://wwwdir.nidcr.nih.gov/dirweb/common/sps.jpg

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
Lumenal
Na+-H+ exchanger
Cl-- HCO3- exchanger
HCO3- channel
Na+-K+ exchanger
Na+-Cl--cotransporter
Basolateral
Na+-K+-ATPase
Cl- channel

Striated duct cell

Lumen

Interstitium
Na+

H+
HCO3HCO3-

Nucleus

ClCarbonic
anhydrase

2K

ATP

Na+

3 Na+

Cl-

K+
Na+
ClMitochondria

Basolateral
membrane
folds

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 Sjogrens patients
Sjogrens etiology and pathogenesis is very complex
Agonists can be useful in profound xerostomia treatment
Pilocarpine and Cevimiline
Requires some remaining functional tissue
Anti-cholinergics
Most 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 Sjogrens/radiation
Transfect aquaporin into rat duct cells
Not normally present in duct cells
Transfection increased salivary flow
Short-term effect, and only replaces water
Would 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
(Noradrenaline)
Immediate response to NA:
Docking and fusion of
preformed granules
Release of contents

http://www.liv.ac.uk/~petesmif/teaching/1bds_mb/p4/14.gif

Long-term response to NA:

Transcription
Translation
Glycosylation
New granules

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

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