Pre- and Post-synaptic Effects of Botulinum Toxin A on Submandibular Glands.

Intraglandular injection of botulinum toxin type A (BoNT/A) is an effective treatment for sialorrhea. Despite numerous experimental and clinical studies on inhibition of saliva section by BoNT/A, the proteolysis of synaptosomal-associated protein 25 (SNAP-25) following BoNT/A treatment has not yet been confirmed in the salivary gland after injection of BoNT/A. More important, it is not known whether BoNT/A exerts a direct effect in acinar cells. Here, we show that injection of BoNT/A into the rat submandibular gland (SMG) decreased salivary flow in a dose-dependent manner; the inhibitory effect lasted at least 4 wk, and salivary flow recovered to normal levels by 12 wk. During the inhibitory period, SMG neurons and synapses expressed lower levels of full-length SNAP-25, and cleavage of SNAP-25 was observed, as indicated by detection of reduced molecular weight SNAP-25 using Western blotting. In addition, the water channel aquaporin 5 (AQP5) was downregulated and abnormally distributed in rat SMG after injection of BoNT/A. The direct effects of BoNT/A on AQP5 expression and distribution were assessed in vitro to exclude the influence of BoNT/A-induced inhibitory neurotransmission. In stable GFP-AQP5-transfected SMG-C6 cells, treatment with BoNT/A reduced the cell surface protein level of AQP5 in a dose- and time-dependent manner without affecting total AQP5 protein expression. Cell surface biotinylation and immunofluorescence demonstrated translocation of AQP5 from the membrane to the cytoplasm, which was confirmed by decreased levels of AQP5 protein in the membrane fraction and increased levels in the cytoplasmic fraction, suggestive of AQP5 redistribution. Taken together, these results indicated that BoNT/A reversibly decreased saliva secretion in rat SMGs through not only the presynaptic SNAP-25 cleavage but also the postsynaptic AQP5 redistribution. These data provide the first evidence for a direct effect of BoNT/A on the salivary gland.