Calcium Signaling and Exocytosis Monitored in Surgically Excised Human Minor Salivary Glands
The primary purpose of salivary glands is to produce saliva; a fluid consisting of water, electrolytes and proteins which is essential to the well being and proper functioning of the oral cavity. Fluid flow is severely diminished in the autoimmune disease, Sjogrens syndrome (SS) resulting in a significant decrease in the quality of life of patients. The levels of various cytokines including lymphotoxin α (LT) and interferon γ (IFN) are elevated in SS and are thought to contribute to the etiology of the disease. The aims of this study were two-fold. First, to assess if surgically resected minor salivary glands could be used to study the signal transduction pathways leading to fluid and protein secretion and secondly, to assess if cytokine acute exposure influenced these processes.
Methods:
Minor salivary glands were harvested during closing of the maxillary circumvestibular incisions made to perform Lefort I osteotomy / orthognathic surgery. These glands, directly in the incision line, are routinely removed and discarded in order to prevent any potential pathology. The isolated labial glands were incubated overnight (16hrs) with the indicated concentrations of cytokine in complete RPMI. Intracellular [Ca2+] was measured in fluo-2 loaded isolated lobules by multi-photon (MP) microscopy using an Olympus Fluoview1000MP microscope. Exocytosis was measured using MP microscopy by monitoring the endocytosis of sulphorrhodamine B included in the extracellular medium.
Results:
Human minor salivary glands maintained overnight retained polarized morphology. Specifically, nuclei stained with Hoechst dye predominately localized to the basal region, whereas in contrast the intracellular calcium release channel, Inositol 1,4,5-trisphosphate receptor was localized immediately below the apical plasma membrane. Fluo-2 loaded glands responded to the muscarinic agonist carbachol (CCh) to result in a robust, concentration-dependent increase in intracellular [Ca2+]. Exocytosis was also markedly stimulated by incubation of glands with CCh, or the cAMP raising agent forskolin. Incubation of glands with 200 ng/ml LT significantly reduced the Ca2+signals stimulated by CCh, whereas incubation with IFN did not significantly alter the signals. In contrast, incubation with LT or IFN alone did not reduce exocytosis, but co-incubation with both agents significantly reduced exocytosis.
Conclusions
These data establish that surgically resected glands are a useful tool to study the molecular mechanisms underlying the physiology of human salivary glands. Further this system may be a useful model to study the effects of cytokines on the molecular pathways which result in fluid and protein secretion from human salivary glands.