Diquafosol tetrasodium elicits total cholesterol release from rabbit meibomian gland cells via P2Y2 purinergic receptor signalling

Ken-Ichi Endo,Koushi Fujisawa,Asuka Sakamoto

doi:10.1038/s41598-021-86433-6

Abstract

Diquafosol tetrasodium (DQS), a purinergic P2Y2 receptor agonist, stimulates secretion of both water and mucins from the conjunctiva into tears. Hence, DQS-containing eye drops have been approved as a therapeutic option for dry eye disease in some Asian countries, including Japan. Recent clinical reports state that instilling DQS-containing eye drops significantly increases the lipid layer thickness in tears. Therefore, we examined this compound’s direct actions on holocrine lipid-secreting meibomian gland cells and their function. Isolated meibomian gland cells (meibocytes) were procured from rabbits and cultivated in serum-free culture medium. Differentiated meibocytes with pioglitazone were used for the subsequent experiments. Intracellular Ca2+ signalling of the cells was dramatically elevated with DQS addition in a dose-dependent manner. This DQS-induced elevation was almost completely cancelled by the coexistence of the selective P2Y2 receptor antagonist AR-C118925XX. DQS treatment also facilitated total cholesterol (TC) release from cells into the medium. This effect of DQS on TC was suppressed significantly by the intracellular Ca2+ chelator BAPTA-AM as well as by AR-C118925XX. DNA fragmentation analysis revealed that DQS may have enhanced the apoptotic DNA fragmentation caused spontaneously by cells. Thus, DQS could stimulate meibocytes to release lipids through the P2Y2 receptor and possibly facilitate holocrine cell maturation.

Highlights

Diquafosol tetrasodium (DQS), a purinergic P2Y2 receptor agonist, stimulates secretion of both water and mucins from the conjunctiva into tears
The P2RY2 transcript was detected at a similar level (134% of just after harvesting [ΔCt = 9.7]) in meibocytes cultured with the peroxisome proliferator-activated receptor (PPAR) agonist
These results indicated that rabbit meibocytes, both in vivo and in our culture system, expressed a relative level of the P2RY2 gene transcript

Summary

Introduction

Diquafosol tetrasodium (DQS), a purinergic P2Y2 receptor agonist, stimulates secretion of both water and mucins from the conjunctiva into tears. Recent clinical reports state that instilling DQS-containing eye drops significantly increases the lipid layer thickness in tears We examined this compound’s direct actions on holocrine lipid-secreting meibomian gland cells and their function. P 2Y2 receptors are found at several anterior eye sites, such as the corneal and conjunctival epithelia and conjunctival goblet cells, and DQS stimulates the secretion of both water and mucins from cells into tears[5,6] Because of these pharmacological actions, ophthalmic solutions containing DQS have been approved as therapeutic options for dry eye disease in some Asian countries, including Japan, South Korea and China. Most lipids in tears, such as wax esters, triglycerides and total cholesterol (TC), i.e., the sum of cholesterol esters and free cholesterol, are supplied as Scientific Reports | (2021) 11:6989

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