Occludin is required for TRPV1-modulated paracellular permeability in the submandibular gland

Abstract

SummaryOccludin plays an important role in maintaining tight junction barrier function in many types of epithelia. We previously reported that activation of transient receptor potential vanilloid subtype 1 (TRPV1) in rabbit submandibular gland promoted salivary secretion, partly by an increase in paracellular permeability. We have now explored the role of occludin in TRPV1-modulated paracellular permeability in a rat submandibular gland cell line SMG-C6. Both TRPV1 and occludin were expressed in SMG-C6 cells, and capsaicin induced redistribution of occludin, but not claudin-3, claudin-4 or E-cadherin, from the cell membrane into the cytoplasm. Capsaicin also decreased transepithelial electrical resistance (TER) and increased the Trypan Blue and FITC-dextran flux. Capsazepine (CPZ), a TRPV1 antagonist, inhibited the capsaicin-induced occludin redistribution and TER decrease. Moreover, occludin knockdown by shRNA suppressed, whereas occludin re-expression restored, the TER response to capsaicin. Mechanistically, TRPV1 activation increased ERK1/2 and MLC2 phosphorylation. PD98059, an ERK1/2 kinase inhibitor, abolished the capsaicin-induced MLC2 phosphorylation, whereas ML-7, an MLC2 kinase inhibitor, did not affect ERK1/2 phosphorylation, suggesting that ERK1/2 is the upstream signaling molecule of MLC2. Capsaicin also induced F-actin reorganization, which was abolished by CPZ, PD98059 and ML-7, indicating that TRPV1 activation altered F-actin organization in an ERK1/2-and MLC2-dependent manner. Furthermore, either PD98059 or ML-7 could abolish the capsaicin-induced TER response and occludin redistribution, whereas knockdown of ERK1/2 further confirmed that the TRPV1-modulated paracellular permeability was ERK1/2 dependent. Taken together, these results identified a crucial role of occludin in submandibular epithelial cells, and more importantly, demonstrated that occludin was required to mediate TRPV1-modulated paracellular permeability.

Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells

“…The extracellular loops of claudins interact with each other to seal the cellular sheet and regulate paracellular transport between luminal and basolateral spaces (Lal-Nag and Morin, 2009). Claudin-4 has been detected in diverse epithelia, such as salivary, renal, lung, intestinal and epidermal cells (Akazawa et al, 2013;Cong et al, 2013;Gong et al, 2014;Kage et al, 2014;Shrestha et al, 2014). In rat submandibular SMIE cells, overexpression of claudin-4 increases TER and decreases the epithelial permeability of 70 kDa dextran (Michikawa et al, 2008).…”

“…Total RNA extraction and reverse transcription were performed as previously reported (Cong et al, 2013). The primer sequences for rat tight junction components are shown in supplementary material Table S1.…”

“…Immunofluorescence staining was performed as previously reported (Cong et al, 2013). Fluorescence images were captured on a confocal microscope (Leica TCS SP5, Wetzlar, Germany).…”

“…The immunoprecipitation assay was performed as previously reported (Cong et al, 2013). For statistical analysis, the amounts of the same proteins in the input fraction (shown as 'cell lysate') or the reprobed membranes with the immunoprecipitated antibodies were detected as controls.…”

The timing of acid-induced increase in saliva secretion in transplanted submandibular glands

“…In addition to autonomic nerves, secretion-related receptors on the cell membrane, such as TRPV1, and tight junctions also play an important role in regulating secretion from normal and transplanted SMGs. [17][18][19][20][21][22][23] Therefore, further study on sympathetic reinnervation and secretion-related receptors is required.…”