June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
TRPV1-dependent NKCC1 activation in mouse lens involves integrin and the tubulin cytoskeleton
Author Affiliations & Notes
  • Mohammad Shahidullah
    Physiology, The University of Arizona, Tucson, Arizona, United States
    Ophthalmology and Vision Science, The University of Arizona, Tucson, Arizona, United States
  • Amritlal Mandal
    Physiology, The University of Arizona, Tucson, Arizona, United States
  • Nicholas A Delamere
    Physiology, The University of Arizona, Tucson, Arizona, United States
    Ophthalmology and Vision Science, The University of Arizona, Tucson, Arizona, United States
  • Footnotes
    Commercial Relationships   Mohammad Shahidullah, None; Amritlal Mandal, None; Nicholas Delamere, None
  • Footnotes
    Support  This research was supported by a grant from the National Institute of Health, Grant number: NIH EY009532.
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 2058. doi:
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      Mohammad Shahidullah, Amritlal Mandal, Nicholas A Delamere; TRPV1-dependent NKCC1 activation in mouse lens involves integrin and the tubulin cytoskeleton. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2058.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : In earlier studies we showed the response of the mouse lens to hyperosmotic solution involves TRPV1-dependent ERK1/2 and NKCC1 activation. In studies on different tissues, other investigators have suggested the cytoskeleton plays an important role in responses to osmotic stress. In the present study, we examine whether integrins and tubulins are involved in the activation of NKCC1 in mouse lens epithelium.

Methods : Lenses were obtained from Wild-type (WT) and TRPV1 knockout (KO) mice. Rubidium (Rb) uptake by the intact lens was quantified by atomic absorption spectrophotometry. Previous studies used bumetanide-sensitivity to validate Rb uptake as a measure of lens NKCC activity. ERK1/2 phosphorylation was detected and quantified by Western blot analysis. Cytoplasmic calcium was measured in cultured lens epithelium loaded with Fura-2 using a ratiometric imaging technique.

Results : In WT mice, an integrin agonist leukadherin 1 (LA-1, 25 μM) increased Rb uptake by intact lenses from a control value of 3.9±0.4 to 5.2±0.4 (n=7, P<0.05). LA-1 also increased the cytoplasmic calcium concentration in cultured WT lens epithelium from 197±18 to 341±25 nM in (n=5, P<0.001). In TRPV1 KO lenses, LA-1 failed to produce a significant change of Rb uptake. Moreover, LA-1 failed to increase cytoplasmic calcium concentration in cultured lens epithelium from TRPV1 KO mice. Importantly, the rise of Rb uptake caused by LA-1 in WT lenses was prevented by the putative tubulin stabilizer, paclitaxel (100 nm). The Rb uptake response to LA-1 was abolished by a TRPV1 antagonist, A889425 (1.0 μM). LA-1 was observed to cause ERK1/2 activation in WT cultured lens epithelium. The TRPV1 agonist capsaicin and hyperosmotic solution (350 mOsm) were shown to cause a similar pattern of ERK1/2 activation in WT cultured lens epithelium. The ERK1/2 responses were transient and displayed a peak at ~5 min. Paclitaxel as well as A889425 markedly reduced the magnitude of the ERK1/2 activation responses to hyperosmotic stress and LA-1.

Conclusions : The findings suggest a functional link between integrins, the tubulin cytoskeleton, and the TRPV1-dependent increase of NKCC1 that occurs when the lens is subjected to hyperosmotic stress. It is noteworthy that the integrin agonist LA-1 as well as hyperosmotic stress both were found to activate ERK1/2, an important step in the signaling associated with activation of NKCC1 in the lens.

This is a 2021 ARVO Annual Meeting abstract.

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