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Mohammad Shahidullah, Amritlal Mandal, Ting Wei, Nicholas A Delamere; A role for EGF signaling in the response of mouse lens epithelium to osmotic stress. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4636.
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© ARVO (1962-2015); The Authors (2016-present)
Previous data show TRPV1 activation by hyperosmotic solution leads to NKCC stimulation. Accordingly, TRPV1 knockout (KO) lenses do not appear sensitive to modest increases of bathing solution osmolarity. However, in order to be viable, TRPV1 KO lenses must have some capacity for osmoregulation. Here we examine the ability of TRPV1 KO epithelium to respond to hyperosmolar stimuli and the underlying mechanism.
Studies were done in cultured wild-type (WT) and TRPV1 KO mouse lens epithelial cells. Rubidium (Rb) uptake, measured by atomic absorption spectrophotometry, was used as an indicator of inwardly-directed potassium ion transport. Protein phosphorylation was detected and measured by quantitative western blot analysis.
Rb uptake was measured in TRPV1 KO cells that were exposed to a range of hyperosmolar (hyper) solutions (325 to 450 mOsm). Rb uptake was increased to 380 ± 3 and 440 ± 7 nmoles/mg protein/10 min at 400 and 450 mOsm, respectively, compared to a control value of 328 ± 8 (n=6, p<0.001). The response was prevented by the NKCC inhibitor bumetanide (10 nM) (Control 358 ± 14; Hyper 441 ± 6.4 (n=6, p<0.001); hyper+Bum 327 ± 11 nmoles/mg protein/10 min; (n=6, p=0.38). The Rb uptake response also was abolished by the EGF receptor antagonist CI-1033 (CI, 1 µM); Con 358 ± 4, Hyper 441 ± 6 (n=6, p<0.001) and CI+Hyper 355 ± 13; n=6, p< 0.3). To determine whether EGF effects on NKCC-dependent Rb uptake are a unique feature of TRPV1 KO cells, similar experiments were done on WT cells. Exposing WT cells to 400 mOsm solution caused an increase of bumetanide-sensitive Rb uptake that could be prevented by CI-1033 (Control 424 ± 9, Hyper 534 ± 9 and CI+Hyper 459 ± 14; n=6-12 p<0.001 and p=0.251 ). In both WT and KO cells, 400 mOsm solution or EGF (10 ng/ml) caused 20 fold increase in ERK1/2 phosphorylation that was inhibited by CI-1033. ERK activation is a required step in osmotic stress-induced NKCC activation.
The data show that under certain conditions EGF signaling plays a role in the NKCC response of lens epithelium to hyperosmotic stress. The EGF-dependent response was only detectable at high stress conditions. The mechanism permits a hyperosmotic stress response in KO cells that lack TRPV1-mediated form of NKCC stimulation. However, a similar mechanism was evident in WT cells.
This is a 2020 ARVO Annual Meeting abstract.
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