Little is known about the relationship between stimulation of lacrimal gland (LG) tear protein secretion by parasympathetic versus sympathetic nerves, particularly whether the spectrum of tear proteins evoked through each innervation pathway varies. We have previously shown that activity and abundance of cathepsin S (CTSS), a cysteine protease, is greatly increased in tears of Sjögren's syndrome (SS) patients and in tears from the male NOD mouse of autoimmune dacryoadenitis that recapitulates SS-associated dry eye disease. Beyond the increased synthesis of CTSS detected in the diseased NOD mouse LG, increased tear CTSS secretion in NOD mouse tears was recently linked to increased exocytosis from a novel endolysosomal secretory pathway. Here, we have compared secretion and trafficking of CTSS in healthy mouse LG acinar cells stimulated with either the parasympathetic acetylcholine receptor agonist, carbachol (CCh), or the sympathetic α₁-adrenergic agonist, phenylephrine (PE). In situ secretion studies show that PE significantly increases CTSS activity and protein in tears relative to CCh stimulation by 1.2-fold (***, p = 0.0009) and ∼5-fold (*, p-0.0319), respectively. A similar significant increase in CTSS activity with PE relative to CCh is observed when cultured LGAC are stimulated in vitro. CCh stimulation significantly elevates intracellular [Ca²⁺], an effect associated with increases in the size of Rab3D-enriched vesicles consistent with compound fusion, and subsequently decreases in their intensity of labeling consistent with their exocytosis. PE stimulation induces a lower [Ca²⁺] response and has minimal effects on Rab3D-enriched SV diameter or the intensity of Rab3D-enriched SV labeling. LG deficient in Rab3D exhibit a higher sensitivity to PE stimulation, and secrete more CTSS activity. Significant increases in the colocalization of endolysosomal vesicle markers (Lamp1, Lamp2, Rab7) with the subapical actin suggestive of fusion of endolysosomal vesicles at the apical membrane occur both with CCh and PE stimulation, but PE demonstrates increased colocalization. In conclusion, the α₁-adrenergic agonist, PE, increases CTSS secretion into tears through a pathway independent of the exocytosis of Rab3D-enriched mature SV, possibly representing an alternative endolysosomal secretory pathway.

关于副交感神经和交感神经刺激泪腺 (LG) 泪液蛋白分泌之间的关系知之甚少，特别是通过每个神经支配途径诱发的泪液蛋白谱是否不同。我们之前已经表明，在干燥综合征 (SS) 患者的泪液和重现 SS 相关干眼病的自身免疫性泪腺炎的雄性 NOD 小鼠的泪液中，组织蛋白酶 S (CTSS)（一种半胱氨酸蛋白酶）的活性和丰度大大增加. 除了在患病的 NOD 小鼠 LG 中检测到 CTSS 合成增加外，NOD 小鼠泪液中 CTSS 分泌增加最近与新的内溶酶体分泌途径的胞吐作用增加有关。这里，_1-肾上腺素能激动剂，去氧肾上腺素（PE）。原位分泌研究表明，相对于 CCh 刺激，PE 显着增加眼泪中的 CTSS 活性和蛋白质，分别增加 1.2 倍（***，p = 0.0009）和 5 倍（*，p-0.0319）。当体外刺激培养的 LGAC 时，观察到 PE 相对于 CCh 的 CTSS 活性有类似的显着增加。CCh 刺激显着提高了细胞内 [Ca ²⁺ ]，这是一种与富含 Rab3D 的囊泡大小增加相关的效应，与复合融合一致，并随后降低其标记强度与其胞吐作用一致。PE 刺激诱导较低的 [Ca ²⁺] 响应并对富含 Rab3D 的 SV 直径或富含 Rab3D 的 SV 标记的强度的影响最小。缺乏 Rab3D 的 LG 对 PE 刺激表现出更高的敏感性，并分泌更多的 CTSS 活性。内溶酶体囊泡标志物（Lamp1、Lamp2、Rab7）与顶端膜下肌动蛋白的共定位显着增加，表明内溶酶体囊泡在顶端膜上的融合发生在 CCh 和 PE 刺激下，但 PE 表明共定位增加。总之，α ₁ -肾上腺素能激动剂 PE 通过与富含 Rab3D 的成熟 SV 的胞吐作用无关的途径增加 CTSS 分泌到泪液中，这可能代表了另一种内溶酶体分泌途径。