scholarly journals Pro-Resolving Mediator Annexin A1 Regulates Intracellular Ca2+ and Mucin Secretion in Cultured Goblet Cells Suggesting a New Use in Inflammatory Conjunctival Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Anne V. Lyngstadaas ◽  
Markus V. Olsen ◽  
Jeffrey A. Bair ◽  
Robin R. Hodges ◽  
Tor P. Utheim ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Ocular Surface ◽  
Tear Film ◽  
Goblet Cells ◽  
Optimal Level ◽  
Annexin A1 ◽  
Mucin Secretion ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Conjunctival Goblet Cells

The amount of mucin secreted by conjunctival goblet cells is regulated to ensure the optimal level for protection of the ocular surface. Under physiological conditions lipid specialized pro-resolving mediators (SPM) are essential for maintaining tissue homeostasis including the conjunctiva. The protein Annexin A1 (AnxA1) can act as an SPM. We used cultured rat conjunctival goblet cells to determine if AnxA1 stimulates an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion and to identify the signaling pathways. The increase in [Ca2+]i was determined using fura2/AM and mucin secretion was measured using an enzyme-linked lectin assay. AnxA1 stimulated an increase in [Ca2+]i and mucin secretion that was blocked by the cell-permeant Ca2+ chelator BAPTA/AM and the ALX/FPR2 receptor inhibitor BOC2. AnxA1 increased [Ca2+]i to a similar extent as the SPMs lipoxin A4 and Resolvin (Rv) D1 and histamine. The AnxA1 increase in [Ca2+]i and mucin secretion were inhibited by blocking the phospholipase C (PLC) pathway including PLC, the IP3 receptor, the Ca2+/ATPase that causes the intracellular Ca2+ stores to empty, and blockade of Ca2+ influx. Inhibition of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase also decreased the AnxA1-stimulated increase in [Ca2+]i and mucin secretion. In contrast inhibitors of ERK 1/2, phospholipase A2 (PLA2), and phospholipase D (PLD) did not alter AnxA1-stimulated increase in [Ca2+]i, but did inhibit mucin secretion. Activation of protein kinase A did not decrease either the AnxA1-stimulated rise in [Ca2+]i or secretion. We conclude that in health, AnxA1 contributes to the mucin layer of the tear film and ocular surface homeostasis by activating the PLC signaling pathway to increase [Ca2+]i and stimulate mucin secretion and ERK1/2, PLA2, and PLD to stimulate mucin secretion from conjunctival goblet cells.

scholarly journals Role of ion channels in the functional response of conjunctival goblet cells to dry eye

2018 ◽  
Vol 315 (2) ◽  
pp. C236-C246 ◽  
Author(s):  
Donald G. Puro
Keyword(s):  
Ion Channels ◽  
Dry Eye ◽  
Ocular Surface ◽  
Adaptive Response ◽  
Tear Film ◽  
Goblet Cells ◽  
Cation Conductance ◽  
Conjunctival Goblet Cells

Optimal vision requires an ocular surface with a stable tear film whose many critical tasks include providing >70% of the eye’s refractive power. However, for millions, tear film instability produces uncomfortable sight-impairing dry eye. Despite the multitude of etiologies for dry eye, a universal hallmark is hyperosmolarity of the tear film. Presently, knowledge of how the ocular surface responds to hyperosmolarity remains incomplete with little understood about the role of ion channels. This bioelectric analysis focused on conjunctival goblet cells whose release of tear-stabilizing mucin is a key adaptive response to dry eye. In freshly excised rat conjunctiva, perforated-patch recordings demonstrated that a ≥10% rise in osmolarity triggers goblet cells to rapidly generate a ~15-mV hyperpolarization due to the oxidant-dependent activation of ATP-sensitive K+ (KATP) channels. High-resolution membrane capacitance measurements used to monitor exocytosis revealed that this hyperpolarization results in an approximately fourfold boost in exocytotic activity evoked by cholinergic input, which in vivo occurs via a neural reflex and depends chiefly on calcium influxing down its electro-gradient. We discovered that this adaptive response is transient. During 30–80 min of hyperosmolarity, development of a depolarizing nonspecific cation conductance fully counterbalances the KATP-driven hyperpolarization and thereby eliminates the exocytotic boost. We conclude that hyperosmotic-induced hyperpolarization is a previously unappreciated mechanism by which goblet cells respond to transient ocular dryness. Loss of this voltage increase during long-term dryness/hyperosmolarity may account for the clinical conundrum that goblet cells in chronically dry eyes can remain filled with mucin even though the tear film is hyperosmotic and mucin-deficient.

scholarly journals Characterisation of Gel-Forming Mucins Produced In Vivo and In Ex Vivo Conjunctival Explant Cultures

2021 ◽  
Vol 22 (19) ◽  
pp. 10528
Author(s):  
Sara I. Van Acker ◽  
Bert Van den Bogerd ◽  
Zoë P. Van Acker ◽  
Agnė Vailionytė ◽  
Michel Haagdorens ◽  
...  
Keyword(s):  
Ocular Surface ◽  
Ex Vivo ◽  
Tear Film ◽  
Goblet Cells ◽  
Film Stability ◽  
Explant Cultures ◽  
Current Paradigm ◽  
Conjunctival Goblet Cells

One key element to the health of the ocular surface encompasses the presence of gel-forming mucins in the pre-ocular tear film. Conjunctival goblet cells are specialized epithelial cells that secrete mucins necessary for tear film stability and general homeostasis. Their dysfunction can be linked to a range of ocular surface inflammation disorders and chronic injuries. To obtain new perspectives and angles to tackle mucin deficiency, the need for an accurate evaluation of their presence and corresponding mucin secretion in ex vivo conjunctival cultures has become a requisite. In vitro, goblet cells show a significant decrease in the production and secretion of gel-forming mucins, accompanied by signs of dedifferentiation or transdifferentiation. Explant cultures on laminin-treated CLP-PEG hydrogels can, however, support the production of gel-forming mucins. Together, we challenge the current paradigm to evaluate the presence of cultured goblet cells solely based on their general mucin (MUC) content through imaging analyses, showing the need for additional techniques to assess the functionality of goblet cells. In addition, we broadened the gel-forming mucin profile of in vivo goblet cells with MUC5B and MUC6, while MUC2 and MUC6 is added to the profile of cultured goblet cells.

Modulation of K channels by coexpressed human alpha1C-adrenoceptor in Xenopus oocytes

1997 ◽  
Vol 272 (3) ◽  
pp. H1275-H1286
Author(s):  
G. N. Tseng ◽  
J. A. Yao ◽  
J. Tseng-Crank
Keyword(s):  
Protein Kinase ◽  
Model System ◽  
K Channel ◽  
K Channels ◽  
Channel Modulation ◽  
Channel Function ◽  
Adrenergic Modulation ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Multiple Receptor

alpha1-Adrenoceptors participate in the regulation of inotropy and chronotropy in the heart. Modulation of cardiac K-channel function plays an important role in these alpha1-adrenergic functions. Studies of the mechanisms of K-channel modulation by alpha1-adrenoceptors are hampered by the coexistence of multiple receptor and channel subtypes in the heart. We therefore used a model system of coexpressing a specific receptor (human alpha1c-adrenoceptor) and a K-channel clone (hIsK, rKv1.2, or rKv1.4) in oocytes. alpha1c-Adrenoceptor stimulation caused a rapid upregulation of hIsK by elevating the intracellular Ca concentration. At least part of this effect was due to an activation of calmodulin and Ca/calmodulin-dependent protein kinase II. On the other hand, alpha1c-adrenoceptor stimulation caused a slow downregulation of rKv1.2 and rKvl.4 by activating protein kinase C. The differential modulation of K channels by alpha1c-adrenoceptors demonstrated in our experiments corroborates the complexity of alpha1-adrenergic functions in the heart. Our results indicate that the oocyte model system can be a useful approach in studying alpha1-adrenergic modulation of ion-channel function and signal transduction.

Ca2+ and protein kinase C activation of mucin granule exocytosis in permeabilized SPOC1 cells

1998 ◽  
Vol 275 (1) ◽  
pp. C285-C292 ◽  
Author(s):  
C. E. Scott ◽  
Lubna H. Abdullah ◽  
C. William Davis
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Fold Increase ◽  
Mucin Secretion ◽  
Granule Exocytosis ◽  
Kinase C ◽  
Streptolysin O ◽  
Intracellular Ca ◽  
Mucin Release ◽  
Protein Kinase C Activation

Mucin secretion by airway goblet cells is under the control of apical P2Y2, phospholipase C-coupled purinergic receptors. In SPOC1 cells, the mobilization of intracellular Ca2+ by ionomycin or the activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) stimulates mucin secretion in a fully additive fashion [L. H. Abdullah, J. D. Conway, J. A. Cohn, and C. W. Davis. Am. J. Physiol. 273 ( Lung Cell. Mol. Physiol. 17): L201–L210, 1997]. This apparent independence between PKC and Ca2+ in the stimulation of mucin secretion was tested in streptolysin O-permeabilized SPOC1 cells. These cells were fully competent to secrete mucin when Ca2+ was elevated from 100 nM to 3.1 μM for 2 min following permeabilization; the Ca2+EC50 was 2.29 ± 0.07 μM. Permeabilized SPOC1 cells were exposed to PMA or 4α-phorbol at Ca2+ activities ranging from 10 nM to 10 μM. PMA, but not 4α-phorbol, increased mucin release at all Ca2+ activities tested: at 10 nM Ca2+ mucin release was 2.1-fold greater than control and at 4.7 μM Ca2+ mucin release was maximal (3.6-fold increase). PMA stimulated 27% more mucin release at 4.7 μM than at 10 nM Ca2+. Hence, SPOC1 cells possess Ca2+-insensitive, PKC-dependent, and Ca2+-dependent PKC-potentiated pathways for mucin granule exocytosis.

Calcium signaling in human airway goblet cells following purinergic activation

2007 ◽  
Vol 292 (1) ◽  
pp. L92-L98 ◽  
Author(s):  
Andrea H. Rossi ◽  
Wendy C. Salmon ◽  
Michael Chua ◽  
C. William Davis
Keyword(s):  
Goblet Cell ◽  
Primary Cultures ◽  
Goblet Cells ◽  
Pulmonary Diseases ◽  
Mucin Secretion ◽  
Peak Response ◽  
General Importance ◽  
Intracellular Ca ◽  
High Doses ◽  
Purinergic Stimulation

Despite the general importance of Ca2+ signaling in signal transduction, and of goblet cell mucin hypersecretion in inflammatory pulmonary diseases, measurement of airway goblet cell intracellular Ca2+ (Ca[Formula: see text]) has not been reported. In this article, we describe the results of experiments measuring Ca[Formula: see text] in primary cultures of human bronchial goblet cells after stimulation with the purinergic agonist adenosine 5′- O-(3-thiotriphosphate) (ATPγS) and phorbol 12-myristate 13-acetate (PMA). Ca2+ signaling in human goblet cells after purinergic stimulation follows the classic paradigm of a Ca[Formula: see text] transient from a basal activity of 110 nM to a peak response of 260.1 ± 41.2 nM within 2 min, followed by a long superbasal plateau (155.3 ± 0.2 nM) between 10 and 15 min. The rise in Ca[Formula: see text] appears to result from a mobilization of intracellular stores, because the transient was nearly abolished by inhibition of PLC with the phosphatidylinositol-specific PLC inhibitor U-73122, and it was not affected significantly by removal of extracellular Ca2+. Loading goblet cells with BAPTA inhibited the ATPγS-induced Ca2+ transient by 86.0 ± 13.1%, relative to control. Finally, in contrast to the massive effects of high doses of PMA (300 nM) on mucin secretion from goblet cells, phorbol ester stimulated a small (27.1 ± 7% of the ATPγS control peak), brief rise in Ca[Formula: see text]. This diminutive signal likely denotes a local Ca2+ gradient, which may be associated with the mucin granule exocytotic process.

scholarly journals Stimulation of Basal Secretion in the Treatment of Dry Eye Syndrome

2019 ◽  
Vol 16 (1) ◽  
pp. 70-75
Author(s):  
L. I. Uchakova ◽  
S. V. Balalin ◽  
V. L. Zagrebin
Keyword(s):  
Dry Eye ◽  
Tear Film ◽  
Goblet Cells ◽  
Dry Eye Syndrome ◽  
Ophthalmological Examination ◽  
Basal Secretion ◽  
Tear Production ◽  
Before And After ◽  
The Stability ◽  
Conjunctival Goblet Cells

The aim is to evaluate the effectiveness of Stillavit in the treatment of dry eye syndrome (DES) and its effect on the density of conjunctival goblet cells and the functional parameters of tear production. Methods. There were analyzed treatment results of 18 patients (36 eyes) with DES at the age of 48 to 80 years (average 60.6 ± 1.3 years) including 17 women and 1 male before and after 1 and 3 months after Stillavit prescription. In all patients in addition to a standard ophthalmological examination the results of Schirmer I, Norn, as well as the LIPCOF test were evaluated and the density of conjunctival goblet cells was examined. Results. After 1 and 3 months there was a significant improvement in the results of the Norn test: from 5.17 ± 0.22 seconds to 7.18 ± 0.17 seconds which indicated an increase in the stability of the tear film. After 3 months there was a significant increase in the density of conjunctival goblet cells, improvement in the Schirmer sample: from 9.26 ± 0.89 to 11.7 ± 0.6 mm (t = 2.28, p < 0.05) and LIPCOF test (t = 2.18, p < 0.05) which indicated an increase in tear production and a significant decrease in the inflammatory process and edema of the conjunctiva. Conclusion. Treatment of DES with the use of Stillavit leads after 3 months to a significant increase in the density of conjunctival goblet cells, increases tear production and stability of the tear film.

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