Abstract
Purpose :
Keratocyte signaling has previously been studied following major disruptions to the different cell types of the cornea or the cornea itself. Transient plasma membrane disruption (TPMD) is a common form of injury to individual cells that can be created by mechanical loading. The purpose of this study was to determine if TPMD in a single keratocyte can trigger calcium signaling events in neighboring keratocytes.
Methods :
Corneal stromal cells were cultured from human (HCSC) and mouse corneas (MCSC). A single TPMD was produced using a multiphoton microscope in Cal-520-AM dye-loaded cells. TPMD-induced cytoplasmic calcium (Ca++i) increases were measured in neighboring cells in control and Ca++-free (K-SFM) solutions containing the Ca++ATPase inhibitor thapsigargin (TGN), gap junction inhibitor 18-α-glycyrrhetinic acid (18a-GA), apyrase (catalyzes ATP hydrolysis), or the TRPV1 inhibitor BCTC. Fluorescence intensity was recorded and the number of neighboring cells responding to the single cell TPMD was counted, as was the area under the intensity vs time curve (fluorescence area). The maximum distance of neighboring cell Ca++ TPMD responses within ex vivo human corneas was also measured.
Results :
TPMD resulted in transient Ca++i increases in the targeted cell and in non-wounded neighboring cells. The number of neighbor cells showing elevated TPMD-induced Ca++i was reduced in K-SFM, TGN, K-SFM + TGN, and apyrase groups; fluorescence area was reduced in K-SFM, K-SFM + TGN, and apyrase groups. The number of MCSC neighboring cells showing TPMD-induced Ca++i increases was reduced in TGN and K-SFM + TGN groups; MCSC fluorescence area was reduced in K-SFM, 18a-GA, and K-SFM + TGN groups. In human corneal tissue, the distance of TPMD induced calcium signal transmission was reduced in K-SFM, K-SFM + TGN, and 18a-GA groups.
Conclusions :
Single keratocyte TPMD results in a transient Ca++i increase in neighboring HCSC, MCSC, and keratocytes within ex vivo human corneas. The source of increased Ca++i is both intra-and extra-cellular calcium. The signal can be mediated by extracellular ATP and/or gap junctions, and is species dependent. The TRPV1 channel is not involved in the response. Because TPMD can result from physiological mechanical loading, common occurrences such as eye rubbing and corneal trauma could lead to keratocyte TPMD, which would likely be a normal aspect of corneal physiology and pathophysiology.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.