Test – The morphology and function of salivary glands (answers)(answers)

1. Fundamental importance for embryonic salivary development:

A. proliferation B. apoptosis C. gastrulation D. all three E. none of them

2. Location of secretory protein (export protein) storage in salivary glands A. Golgi vesicle

B. zymogen granule C. Golgi cysternae D. cell nucleus

3. Which one of these transmitters stimulates both salivary secretion, gastric acid secretion and pancreatic enzyme secretion?

A. adrenaline B. acetylcholine C. histamine D. somatostatin

13. 1.13. Salivary gland electrolyte, water and protein secretion – Gabor Varga

The most abundant and important component of saliva is water, about 98 % of the secreted quantity. The rest is made up of electrolytes, proteins, and a few minor components, lipids, glucose and urea. The water and electrolyte secretion is an energy consuming, active two-stage process. First the acini produce isotonic primary saliva. This fluid is then modified in the ductal system by electrolyte reabsorption to form a hypotonic secretion.

Figure 1.138. Figure 1. – Salivation – two-stage hypothesis

Figure 1.139. Figure 2. – Flow rate curves of saliva and the two-stage hypothesis 1

Figure 1.140. Figure 3. – Flow rate curves of saliva and the two-stage hypothesis 2

Primary saliva formation by acini

In various secretory epithelia fluid can either be transported through the cells by transcellular transport or between the cells through junction complexes by paracellular transport. The transcellular transport pathway in

the salivary glands primarily requires aquaporin 5 (AQP5) water channels, localized to the luminal surface of acinar cells. The lack of AQP5 decreases transport not only through the plasma membrane but also through the tight junction complex by decreasing the expression of tight junction proteins such as claudins and occluding.

The basolateral-to-apical water movement by acini is rather due to osmotic gradient initiated by electrolyte transport and apical discharge. Therefore, passive water movement is a consequence of preceeding acinar secretion, resulting an isotonic primary secretory fluid.

Figure 1.141. Figure 4. – Main transporters-channels-pumps

Acinar water secretion is primarily driven by vectorial, transcellular Cl^– transport. The energy for Cl^– movement across the cell is provided from a highly active pump, the Na+/K+ ATPase that uses ATP to extrude 3 Na⁺ and to allow entry of 2 K⁺ into the cell. A consequence, Na⁺ gradient between the intracellular and the extracellular space becomes much higher, than that in K⁺ concentration difference. At the expense of the Na⁺ concentration gradient, the Na⁺ K⁺ 2 Cl^– cotransporter (NKCC1) brings 3 Na⁺, 3 K⁺ and 6 Cl^– ions into the acinar cell from the interstitium, so concentration of K+ and Cl^– in the intracellular space well above their equilibrium potential.

Figure 1.142. Figure 5. – Acinar cell transporters

In response to muscarinic receptor stimulation intracellular Ca²⁺ becomes elevated, Cl ions are released into the acinar lumen by Ca²⁺-activated chloride channels at the apical membrane and Ca²⁺-activated potassium channels open to release K⁺ into the interstitium at the basolateral membrane. Additionally, Na+ is driven through tight junctions from the interstitium into acinar luminal to compensate the developing electrochemical gradient.

The participating K⁺ and Cl^– channels have been recently identified. Two different K⁺ channels with somewhat different characteristics are involved. One of these is the named IK1 or SK4, a Ca²⁺-activated K⁺ channel of intermediate single channel conductance. The other one called maxi K or Slo is both Ca²⁺- and voltage-activated with a large single channel conductance. Fluid secretion is severely impaired when both IK1 and Slo channels are missing. The recycling of K⁺ ions into the extracellular space and hyperpolarizes the membrane and increases the drive for Cl^– to exit on the opposite side. The osmotic gradient, due to luminal NaCl accumulation then draws water through the acinar cells.

Figure 1.143. Figure 6. - Acini

There are two alternative mechanisms accounting about for about 30 % of isotonic primary saliva secretion. The alternative Cl^– is achieved the joint activities of basolateral Na⁺/H⁺ and Cl^–/HCO3– exchangers. CO2 diffuses through the basolateral membrane into the cell. In the cytosol carbonic anhydrase enzyme activity catalyzes H⁺ and HCO3– production from H2O and CO2. The newly synthetized HCO3– is exchanged to Cl^– by the Cl^–/HCO3–

exchanger while intracellular extra H⁺ is extruded by the Na⁺/H⁺ exchanger. Accumulated intracellular Cl^– then secreted apically. The secretory process can also be achieved to a certain degree even under Cl^–-free conditions, since an apical anion conductance may also use HCO3^– through the channels instead of Cl^– at somewhat lower level.

Secondary saliva modifications by ductal cells

Among the ductal cells in salivary glands intercalated and striated ducts are intralobular, and excretory ducts are primarily extralobular. NaCl reabsorption happens both in intralobular and extralobular ducts. Ducts are impermeable for water, thus, ductal electrolyte reabsorption directly lead to hypotonic saliva formation. Final salivary electrolyte concentration highly depends on salivary flow rate. As basal flow rate is low, secretion goes through the ductal system at low speed. Thus, reabsorption is almost complete leading to extremely hypotonic solution. Food intake stimulated secretion is accelerated, so salivary fluid flushes through the ductal system at

high speed. In such conditions ductal reabsorption is incomplete, and the ionic strength of saliva reaching the mouth is very similar to the primary isotonic secretion of acini.

Figure 1.144. Figure 7. – Salivary gland transporters

The energy for reabsorbing electrolytes comes from the highly active Na⁺/K⁺ ATPase producing extremely low Na⁺ level in the cell. ENaC, the epithelial Na⁺ channel, expressed in the apical ductal membrane clearly plays a crucial role in ductal Na⁺ reabsorption. Amiloride block of ENaC function severely impairs Na⁺ reabsorption, while two Na⁺/H⁺ exchangers, NHE2 and NHE3 play a secondary, minor role in the process.

Cl^– is also reabsorbed by ducts. Apical Cl^– channels, namely the cAMP-dependent Cl^– channel CFTR and Cl^– /HCO3– exchangers are responsible for this process.

Figure 1.145. Figure 8. – Ductal reabsorption mechanisms

Salivary K⁺ concentration is higher than that found in blood plasma. K⁺ accumulation happens at the level of the intra- and extralobular salivary gland ducts. Apical K⁺/H⁺ exchangers and K⁺-HCO3– cotransporters may also be involved in this process.

Mechanism of protein secretion

Acinar salivary cells continuously synthesize the export proteins. Synthesized nascent amino acid chains undergo post-translational modifications in the Golgi apparatus, then stored in zymogen granules. Zymogen granules are transported and fused with the apical cell membrane in a process called exocytosis in response to appropriate stimulation. Various salivary glands produce proteins in different composition. The secretory products of each salivary gland are unique. The two types of acinar cells are serous and mucinous. Both serous and mucinous acinar cells form secretory acini. Serous acini discharge a watery fluid rich in α-amylase and additional enzymes, anti-viral and anti-microbial-bacterial proteins. α-amylase is the most abundant protein in serous saliva. About 70 % of amylase is parotid product. Mucous acini secrete a thick, mucin-rich fluid. Sero-mucous acini produce both mucins and serous proteins. The parotid is made up mostly of serous acinar cells.

The sublingual gland and most minor glands are mostly mucinous, while the submandibular gland is a mixed by serous and mucinous acini. Besides proteins, all salivary glands produce electrolytes and water for the protection of the oral soft tissues and teeth against acidic and demineralizing conditions.

13.1. Test – Salivary gland electrolyte, water and protein