Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that Ca2+ may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular Ca2+ concentration ([Ca2+]i) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced [Ca2+]i increase with nonspecific Ca2+ channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive [Ca2+]i elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a Ca2+ chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that Ca2+ may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream Ca2+ regulation of the WNK-OSR1 pathway in intact cells.

(1) Background: Botulinum toxin (BoNT) injection is an esthetically effective and safe treatment for contouring the lower face. This study aimed to evaluate the combined effects of BoNT and supplementary oral appliance (OA) therapy on lower facial contouring. (2) Methods: We conducted a prospective randomized controlled trial from January 2015 to June 2016 at the Yonsei University Dental Hospital. Volunteers aged 20–45 years with masseter hypertrophy were randomly assigned to one of two groups: the non-OA group and the OA group. The non-OA group received BoNT injections alone, whereas the OA group received an OA in addition to BoNT injections. Changes in the bulkiest height of the lower face were evaluated by three-dimensional laser scanning before and 4, 8, 12, and 24 weeks after injections in both groups. (3) Results: In both groups, the bulkiest height reductions decreased, with a significant interaction between group (p = 0.046) and time (p < 0.001), although the overall reduction was at a similar level at 24 weeks. (4) Conclusions: The pattern of the bulkiest height reduction of the lower face after BoNT injection differed between standalone treatment and OA therapy, implying a normalizing effect of OA on masseter muscle activity.