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Mohammad Shahidullah, Amritlal Mandal, David Krizaj, Sarah Redmon, Nicholas A Delamere; TRPV1-dependent activation of NKCC1 in mouse lens epithelium. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3177. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In porcine lenses the response to a hyperosmotic stimulus involves an increase in the activity of an ion cotransporter NKCC1. Recent studies with agonists and antagonists pointed to a mechanism that appears to depend on activation of TRPV1 ion channels. Here we examine responses in the mouse, using a knockout strategy to examine lenses from mice that do not express TRPV1.
Lenses were obtained from TRPV1 KO and WT C57/BL6 mice. Studies were carried out in primary cultured lens epithelium obtained from WT and TRPV1 KO mouse. TRPV1 and NKCC1 expression and phosphorylation was studied by RT-PCR and western blot analysis. Rb uptake was measured by atomic absorption spectrometry.
RT-PCR confirmed TRPV1 expression in the WT but not in the KO mouse lens. Capsaicin (1 µM) and moderate hyperosmotic solution (350 mOsm) elicited rapid (2 min) phosphorylation of NKCC1 in epithelial cells derived from WT but not in cells derived from TRPV1 KO mice. In the wild type cells, phosphorylated NKCC1 band density was increased from a normalized value of 1 to a value of 1.5±0.05 (P<0.001, n=3, capsaicin) and 2.0±0.1 (P<0.001, n=3, hyperosmotic solution). Rb uptake was measured as an index of NKCC1 stimulation. Capsaicin as well as hyperosmotic solution caused significant increase in Rb uptake in WT cells but not in TRPV1 KO cells. The increase in Rb uptake was from 333±3 (control) to 401±6 (P<0.001), n=6, capsaicin) and from 309±6 (control) to 379±5 (P<0.001, n=6, hyperosmotic) nmoles/mg protein/10 min. The capsaicin and hyperosmotic solution-induced increases in Rb uptake were abolished by the NKCC inhibitor, bumetanide (10 nM). Furthermore, hyperosmotic solution-induced NKCC1 phosphorylation and Rb uptake increases both were abolished by a TRPV1 antagonist A889415 (1.0µM).
A variety of responses to capsaicin and hyperosmotic solution point to a functional role for TRPV1 channels in mouse lens. Lack of NKCC1 phosphorylation and Rb uptake responses in TRPV1 KO mouse epithelium reinforces the notion that a hyperosmotic challenge causes TRPV1-dependent NKCC1 activation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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