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Activation of Stretch-Activated Channels and Maxi-K⁺ Channels by Membrane Stress of Human Lamina Cribrosa Cells

¹From the Molecular Medicine Laboratories, RCSI Education and Research Centre, Beaumont Hospital, Dublin, Ireland; ³Ophthalmology, Mater Misericordiae University Hospital and Conway Institute, University College Dublin, Dublin, Ireland; and ⁴Glaucoma Research, Alcon Research, Ltd., Forth Worth, Texas.

PURPOSE. The lamina cribrosa (LC) region of the optic nerve head is considered the primary site of damage in glaucomatous optic neuropathy. Resident LC cells have a profibrotic potential when exposed to cyclical stretch. However, the mechanosensitive mechanisms of these cells remain unknown. Here the authors investigated the effects of membrane stretch on cell volume change and ion channel activity and examined the associated changes in intracellular calcium ([Ca²⁺]_i).

METHODS. The authors used primary LC cells obtained from normal human donor eyes. Confocal microscopy was used to investigate the effect of hypotonic cell membrane stretch on cell volume changes. Whole-cell patch-clamp and calcium imaging techniques were used to investigate the effect of hypotonicity on ion channel(s) activity and [Ca²⁺]_i changes, respectively. RT-PCR was used to examine for the maxi-K⁺ signature in LC cells.

RESULTS. In this study, LC cells showed significant volume changes in response to hypotonic cell swelling. The authors characterized a large conductance K⁺ channel (maxi-K⁺) in LC cells and demonstrated its increased activity during cell membrane hypotonic stretch. RT-PCR revealed the presence of maxi-K⁺ signature in LC cells. The authors showed the [Ca²⁺]_i and maxi-K⁺ channels to be dependent on extracellular Ca²⁺ and inhibited by gadolinium, which blocks stretch-activated channels (SACs). Pretreatment with thapsigargin, which blocks the release of Ca²⁺ from endoplasmic reticulum stores, showed no significant difference in [Ca²⁺]_i concentration on hypotonic swelling.

CONCLUSIONS. The results show that hypotonic stress of human LC cells activates SAC and Ca²⁺-dependent maxi-K⁺ channels and that the increase in [Ca²⁺]_i during cell swelling was predominantly from extracellular sources (or intracellular stores other than the endoplasmic reticulum). These findings improve the understanding of how LC cells respond to cell membrane stretch. Further experiments in this area may reveal future targets for novel therapeutic intervention in the management of glaucoma.