Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells

Cholinergic agonists are major stimuli for fluid secretion in parotid acinar cells. Saliva bicarbonate is essential for maintaining oral health. Electrogenic and electroneutral Na+-HCO3− cotransporters (NBCe1 and NBCn1) are abundant in parotid glands. We previously reported that angiotensin regulates NBCe1 by endocytosis in Xenopus oocytes. Here, we studied cholinergic regulation of NBCe1 and NBCn1 membrane trafficking by confocal fluorescent microscopy and surface biotinylation in parotid epithelial cells. NBCe1 and NBCn1 colocalized with E-cadherin monoclonal antibody at the basolateral membrane (BLM) in polarized ParC5 cells. Inhibition of constitutive recycling with the carboxylic ionophore monensin or the calmodulin antagonist W-13 caused NBCe1 to accumulate in early endosomes with a parallel loss from the BLM, suggesting that NBCe1 is constitutively endocytosed. Carbachol and PMA likewise caused redistribution of NBCe1 from BLM to early endosomes. The PKC inhibitor, GF-109203X, blocked this redistribution, indicating a role for PKC. In contrast, BLM NBCn1 was not downregulated in parotid acinar cells treated with constitutive recycling inhibitors, cholinergic stimulators, or PMA. We likewise demonstrate striking differences in regulation of membrane trafficking of NBCe1 vs. NBCn1 in resting and stimulated cells. We speculate that endocytosis of NBCe1, which coincides with the transition to a steady-state phase of stimulated fluid secretion, could be a part of acinar cell adjustment to a continuous secretory response. Stable association of NBCn1 at the membrane may facilitate constitutive uptake of HCO3− across the BLM, thus supporting HCO3− luminal secretion and/or maintaining acid-base homeostasis in stimulated cells

A secretagogue-siRNA conjugate confers resistance to cytotoxicity in a cell model of Sjögren\u27s syndrome

Objective—Sjögren\u27s syndrome (SjS) is characterized by xerophthalmia and xerostomia resulting from loss of secretory function due to immune cell infiltration in lacrimal and salivary glands. Current SjS therapeutic strategies employ secretagogues to induce secretion via muscarinic receptor stimulation. Based on our expertise on muscarinic type-3-receptor (M3R), we are utilizing ligands specific for muscarinic receptor to deliver siRNA into cells via receptor-mediated endocytosis, thereby altering epithelial cell responses to external cues such as pro-inflammatory or death signals while simultaneously stimulating secretion. Methods—Carbachol was synthesized with an active choline group and conjugated with siRNA targeting caspase-3, and a human salivary gland cell line (HSG) was used to test the efficacy of this conjugate. Results—Lipofectamine transfection of conjugate into cells resulted in 78%-reduction in caspase-3 gene expression, while external conjugate treatment of HSG cells resulted in similar intracellular calcium release and induction of endocytosis as carbachol stimulation indicating that the siRNA and carbachol portions of conjugate retained function after conjugation. HSG cells treated with conjugate (without Lipofectamine transfection) exhibited a 50% reduction in caspase-3 gene and protein expression indicating our conjugate is effective in delivering functional siRNA into cells via receptor-mediated endocytosis. Furthermore, TNF-α induced apoptosis was significantly reduced in conjugate treated cells. Conclusions—In conclusion, a secretagogue-siRNA conjugate prevented cytokine-induced apoptosis in salivary epithelial cells, which is critical to maintain fluid secretion and potentially reverse the clinical hallmark of SjS