Under physiological conditions, calcium (Ca2+) regulates essential functions of polarized secretory cells by the stimulation of specific Ca2+ signaling mechanisms, such as increases in intracellular Ca2+ concentration ([Ca2+]i) via the store-operated Ca2+ entry (SOCE) and the receptor-operated Ca2+ entry (ROCE). Homer proteins are scaffold proteins that interact with G protein-coupled receptors, inositol 1,4,5-triphosphate (IP3) receptors, Orai1-stromal interaction molecule 1, and transient receptor potential canonical (TRPC) channels. However, their role in the Ca2+ signaling in exocrine cells remains unknown. In this study, we investigated the role of Homer2 in the Ca2+ signaling and regulatory channels to mediate SOCE and ROCE in pancreatic acinar cells. Deletion of Homer2 (Homer2–/–) markedly increased the expression of TRPC3, TRPC6, and Orai1 in pancreatic acinar cells, whereas these expressions showed no difference in whole brains of wild-type and Homer2–/– mice. Furthermore, the response of Ca2+ entry by carbachol also showed significant changes to the patterns regulated by specific blockers of SOCE and ROCE in pancreatic acinar cells of Homer2–/– mice. Thus, these results suggest that Homer2 plays a critical role in the regulatory action of the [Ca2+]i via SOCE and ROCE in mouse pancreatic acinar cells.

The salivary glands secrete saliva, which plays a role in the maintenance of a healthy oral environment. Under physiological conditions, saliva secretion within the acinar cells of the gland is regulated by stimulation of specific calcium (Ca2+) signaling mechanisms such as increases in the intracellular Ca2+ concentration ([Ca2+]i) via storeoperated Ca2+ entry, which involves components such as Orai1, transient receptor potential (TRP) canonical 1, stromal interaction molecules, and inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs). Homer proteins are scaffold proteins that bind to G protein-coupled receptors, IP3Rs, ryanodine receptors, and TRP channels. However, their exact role in Ca2+ signaling in the salivary glands remains unknown. In the present study, we investigated the role of Homer2 in Ca2+ signaling and saliva secretion in parotid gland acinar cells under physiological conditions. Deletion of Homer2 (Homer2−/−) markedly decreased the amplitude of [Ca2+]i oscillations via the stimulation of carbachol, which is physiologically concentrated in parotid acinar cells, whereas the frequency of [Ca2+]i oscillations showed no difference between wild-type and Homer2−/− mice. Homer2−/− mice also showed a significant decrease in amylase release by carbachol in the parotid gland in a dose-dependent manner. These results suggest that Homer2 plays a critical role in maintaining [Ca2+]i concentration and secretion of saliva in mouse parotid gland acinar cells.

The role of Cl channels in regulatory volume decrease (RVD) in human salivary gland acinar cells was examined using a whole-cell patch clamp technique. Human tissues were obtained from healthy volunteers or from patients with oromaxillofacial tumors. During the measurements, K+-free solutions were employed to eliminate contamination of whole-cell conductance by K+ currents. When the cells were exposed to a 70% hypotonic solution, outward-rectifying currents, which were not observed in the resting state, were found to have significantly increased both in human labial and parotid gland acinar cells. The amplitudes of the currents were reduced in a low CI bath solution. Furthermore, the addition of 100μM 5-Nitro-2- (3-phenyl propylamino) benzoic acid (NPPB) or 100μM 4,4'-diisothio cyanatostilbene-2,2'-disulphonic acid (DIDS), known to partially block Cl channels, significantly inhibited these currents. Its outward-rectifying current profile, shift in reversal potential in a low Cl bath solution and pharmacological properties suggest that this is a Cα2+ independent, volume activated Cl current. We conclude therefore that volume activated Cl channels play a putative role in RVD in human salivary gland acinar cells.

The sodium bicarbonate cotransporter (NBC) protein is functionally expressed in salivary glands. In this experiment, we examined the role of NBC in HCO₃-formation in human parotid gland acinar cells. Intracellular pH (pHi) was measured in 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded cells. Acetazolamide (0.1 mM) and 4,4'-diisothio cyanatostilbene-2,2'-disulphonic acid (DIDS, 0.5 mM) were used as specific inhibitors of carbonic anhydrase and NBC, respectively. The degree of inhibition was assessed by measuring the pHi recovery rate (△pHi/min) after cell acidification using an ammonium prepulse technique. In control experiments, △pHi/min was 1.40±0.06. Treatment of cells with 0.5 mM DIDS or 0.1 mM acetazolamide significantly reduced △pHi/min to 1.14±0.14 and 0.74±0.15, respectively. Simultaneous application of DIDS and acetazolamide further reduced △pHi/min to 0.47±0.10. Therefore, DIDS and acetazolamide reduced △pHi/min by 19% and 47%, respectively, while simultaneous application of both DIDS and acetazolamide caused a reduction in △pHi/min of 67%. These results suggest that in addition to carbonic anhydrase, NBC also partially contributes to HCO₃- formation in human parotid gland acinar cells.

H₂O₂, a member of reactive oxygen species (ROS), is known to be involved in the mediation of physiological functions in a variety of cell types. However, little has been known about the physiological role of H₂O₂in exocrine cells. Therefore, in the present study, the effect of H₂O₂on cholecystokinin (CCK)-evoked Cα²+ mobilization and amylase release was investigated in rat pancreatic acinar cells. Stimulation of the acinar cells with sulfated octapeptide form of CCK (CCK-8S) induced biphasic increase in amylase release. Addition of 30μM H₂O₂ enhanced amylase release caused by 10 pM CCK-8S, but inhibited the amylase release induced by CCK-8S at concentrations higher than 100 pM. An ROS scavenger, 10 μM Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, increased amylase release caused by CCK-8S at concentrations higher than 100 pM, although lower concentrations of CCK-8S-induced amylase release was not affected. To examine whether the effect of H₂O₂on CCK-8S-induced amylase release was exerted via modulation of intracellular Cα²+ signaling, we measured the changes in intracellular Cα²+ concentration ([Cα²+]i) in fura-2 loaded acinar cells. Although 30 μM H₂O₂did not induce any increase in([Cα²+]i by itself, it increased the frequency and amplitude of([Cα²+]i oscillations caused by 10 pM CCK-8S. However, 30μM H₂O₂had little effect on 1 nM CCK-8S-induced increase in [Cα²+]i. ROS scavenger, 1 mM N-acetylcysteine, did not affect [Cα²+]i changes induced by 10 pM or 1 nM CCK-8S. Therefore, it was concluded that 30 μM H₂O₂ enhanced low concentration of CCK-8S-induced amylase release probably by increasing [Cα²+]i oscillations while it inhibited high concentration of CCK-8S-induced amylase release.

Von Ebner's glands (vEG) are minor salivary glands associated with circumvallate and foliate papilla. The secretions of vEG consist of microenvironment of the taste buds in the circumvallate and foliate papillae, and thus saliva from vEG plays a role in the perception of taste. The Ca²+ signaling system in rat vEG acinar cell was examined using the Ca²+-sensitive fluorescent indicator Fura-2. Agonist-induced increase in intracellular Ca²+([Ca²+]i) was stimulated by carbachol (CCh) and substance P (SP), but not by norepinephrine (NE), and recovered to control levels by their receptor antagonists dose-dependently. The effects were also observed in Ca²+-free medium, suggesting mobilization from intracellular Ca²+ store. These results in the vEG acinar cell indicate that 1) [Ca²+]i is at least regulated by muscarinic and neurokininergic (NK1) receptors; 2) the increases in [Ca²+]i activated by CCh and SP are mainly mediated by discharge of cytosolic calcium pool.