In contrast to lacritin, the mechanism for secretion by Cch has been well studied, and occurs by a calcium-dependent pathway.
10,22,23 In agreement with these previous findings, Cch increased intracellular Ca
2+ in our cultured monkey lacrimal acinar cells (
Fig. 3A). On the other hand, increased intracellular Ca
2+ was not detected in lacritin-treated cells, although protein secretion was stimulated. Additional lacritin added to the Cch treatment showed an additional effect on protein secretion without further increasing Ca
2+ (
Fig. 3B). These results were different from those of a previous report indicating that lacritin increased intracellular Ca
2+ and mitosis in human salivary ductal cells.
18 Therefore, we performed Ca
2+ measurements using the calciumsensitive fluorophore Calcium 5 (Molecular Devices) with a microscopic imaging technique (SP5 AOBS spectral confocal system; Leica Microsystems, Wetzlar, Germany), similar to the previous report,
18 resulting in a significant increase in calcium by Cch, but not by lacritin (data not shown). The previous report also showed that activation of PKCα occurred upstream of elevated Ca
2+ and that an inhibitor of PKCα inhibited increased Ca
2+. In our monkey lacrimal acinar cells, a PKCα inhibitor did not inhibit lacritin-induced protein secretion (data not shown), although Cch-induced secretion was inhibited as previously reported.
10 In addition, lacritin-induced protein secretion and even monkey lacrimal acinar cells were stimulated in Ca
2+-free medium (
Fig. 3C), suggesting that lacritin may not require Ca
2+ influx from extracellular. All these suggest a different mechanism for lacritin-induced protein secretion in monkey lacrimal acinar cells. Minor or very transient increases in Ca
2+ might not have been detected in our monkey cells, but Ca
2+ signaling was not a major mechanism for lacritin as it is for Cch-induced secretion. Similarly, Ca
2+-independent secretion of insulin was observed in pancreatic islet cells cultured with EGTA.
24 Further studies are needed to clarify the exact mechanism for lacritin-induced protein secretion.