Abstract
PurposeGap junction intercellular communication (GJIC) plays a critical role in the maintenance of corneal endothelium homeostasis. We determined if benzalkonium chloride (BAK) alters GJIC activity in the rabbit corneal endothelium since it is commonly used as a drug preservative in ocular eyedrop preparations even though it can have cytotoxic effects.MethodsThirty-six adult New Zealand albino rabbits were randomly divided into three groups. BAK at 0.01%, 0.05%, and 0.1% was applied twice daily to one eye of each of the rabbits in one of the three groups for seven days. The contralateral untreated eyes were used as controls. Corneal endothelial morphological features were observed by in vivo confocal microscopy (IVCM). Immunofluorescent staining resolved changes in gap junction integrity and localization. Western blot analysis and RT-PCR evaluated changes in levels of connexin43 (Cx43) and tight junction zonula occludens-1 (ZO-1) gene and protein expression, respectively. Cx43 and ZO-1 physical interaction was detected by immunoprecipitation (IP). Primary rabbit corneal endothelial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing BAK for 24 hours. The scrape-loading dye transfer technique (SLDT) was used to assess GJIC activity.ResultsTopical administration of BAK (0.05%, 0.1%) dose dependently disrupted corneal endothelial cell morphology, altered Cx43 and ZO-1 distribution and reduced Cx43 expression. BAK also markedly induced increases in Cx43 phosphorylation status concomitant with decreases in the Cx43-ZO-1 protein-protein interaction. These changes were associated with marked declines in GJIC activity.ConclusionsThe dose dependent declines in rabbit corneal endothelial GJIC activity induced by BAK are associated with less Cx43-ZO-1 interaction possibly arising from increases in Cx43 phosphorylation and declines in its protein expression. These novel changes provide additional evidence that BAK containing eyedrop preparations should be used with caution to avoid declines in corneal transparency resulting from losses in GJIC activity and endothelial function.
Highlights
Corneal endothelial functional activity and homeostasis are essential for this tissue layer to counter the natural tendency of the stroma to imbibe fluid and lose its transparency. [1] The maintenance of corneal deturgescence can be compromised by a variety of stresses that disrupt endothelial integrity
Associated with the progressive declines in punctate Cx43 staining, western blot analysis shown in Fig. 2B further indicates that gap junctional (1% Triton X-100 insoluble) Cx43 fell
[33] In the present study, we examined the effect of benzalkonium chloride (BAK) on gap junctional communication (GJIC) activity and its physical interaction with a tight junctional barrier protein zonula occludens-1 (ZO-1) in rabbit corneal endothelial cells
Summary
Corneal endothelial functional activity and homeostasis are essential for this tissue layer to counter the natural tendency of the stroma to imbibe fluid and lose its transparency. [1] The maintenance of corneal deturgescence can be compromised by a variety of stresses that disrupt endothelial integrity. [7] Losses in endothelial integrity caused by cell loss are instead compensated for through cell enlargement and stretching at sites proximal to the defect Given this inability to undergo proliferation, it is essential to characterize specific stress-induced cellular changes since such insight can help design strategies to hasten and improve restoration of transparency caused by an injury. Gap junctions are formed by two hemichannels or connexons which consist of connexins in either homomeric or heteromeric configurations They are needed for the transfer between cells of small molecules, ions, phosphorylated nucleotides, nutrients, and second messengers (,1KDa) such as cAMP, IP3, and Ca2+ exchange. [9,10] Gap junction connectivity is required for numerous cellular processes that underlie the maintenance of tissue homeostasis They include proliferation, differentiation, and embryonic development. They include proliferation, differentiation, and embryonic development. [11] Disruption of gap junction complexes can change cell-cell communication and is associated with certain diseases such as cardio-cerebrovascular disease, [12,13] tumor, [14,15] a variety of skin diseases, [16] and congenital cataract. [17] There are approximately 21 human connexin genes and 20 mouse connexin genes which have been identified. [18] Connexin (Cx43) is widely expressed and ubiquitously present in a variety of cell types including macrophages. [19] It is the most common connexin subtype expressed in the corneal endothelium of rat [20], rabbit [21] and human. [22] It has been shown that Cx43 gene knockdown accelerated rabbit corneal epithelial [23] and endothelial [24] wound healing. [25] The fact that losses in gap junctional communication (GJIC) has disparate effects on maintenance of tissue layer homeostasis and wound healing indicates the importance of delineating the effects of specific stresses on GJIC and tissue homeostasis
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