June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
The lack of a direct effect of bicarbonate on photoreceptor guanylyl cyclase
Author Affiliations & Notes
  • Igor V Peshenko
    Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania, United States
  • Elena V Olshevskaya
    Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania, United States
  • Alexander M Dizhoor
    Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Igor Peshenko, None; Elena Olshevskaya, None; Alexander Dizhoor, None
  • Footnotes
    Support  NIH grant EY11522; Pennsylvania Department of Health CURE Formula Grant
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 602. doi:
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      Igor V Peshenko, Elena V Olshevskaya, Alexander M Dizhoor; The lack of a direct effect of bicarbonate on photoreceptor guanylyl cyclase. Invest. Ophthalmol. Vis. Sci. 2017;58(8):602.

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

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Abstract

Purpose : Retinal membrane guanylyl cyclase 1 (RetGC1) plays important role in rod and cone physiology. Two types of regulatory proteins control enzymatic activity of RetGC1: Ca2+-sensitive guanylyl cyclase activating proteins (GCAPs) and retinal degeneration 3 (RD3) protein, which inhibits RetGC1 activity. Recently, bicarbonate was proposed as a novel phototransduction-linked RetGC regulator [1,2 ], contradicting other studies on the same subject [3,4]. The goal of this study was to independently verify the effects of bicarbonate on the basal and GCAP-regulated RetGC activity

Methods : The enzymatic activity of a recombinant RetGC1 expressed in HEK293 cells and the native RetGC in wild type and transgenic mouse retinas was assayed in the presence of sodium bicarbonate and GCAPs using a direct method monitoring conversion of 32P-GTP into 32P-cGMP by thin-layer chromatography

Results : No measurable effect of bicarbonate on basal activity of recombinant RetGC1 was detected in the presence of 0-100 mM bicarbonate. While GCAP1 activated recombinant RetGC1 expressed in HEK293 cells >100-fold, there was no increase in GCAP1-stimulated RetGC1 activity in the presence of bicarbonate. We also assayed the effects of bicarbonate on the activity of the native guanylyl cyclase in the retinas present in double-gene knockout mouse retinas lacking GCAP1 and GCAP2 (GCAPs-/-) and of the RetGC1 isozyme present in RetGC2-/-GCAPs-/- triple-knockout retinas. Bicarbonate did not stimulate basal guanylyl cyclase activity, while GCAP1 and GCAP2 produced 18- and 11-fold stimulation, respectively. There was no additional stimulation of the cyclase by bicarbonate in the presence of GCAPs. Instead, there was a small decrease in GCAP-stimulated guanylyl cyclase activity in the presence of 50 mM bicarbonate, which could be attributed to a non-specific effect of high salt concentrations in the assay

Conclusions : Our results, together with the earlier studies [3,4], do not confirm with the reports that bicarbonate can directly activate photoreceptor guanylyl cyclase [1,2]. Our data also argue that the effects of bicarbonate on rod physiology reported in [2] cannot be explained by its direct regulation of guanylyl cyclase activity.
References: [1] Duda et al. (2016) Front. Mol. Neurosci. 9:5; [2] Duda et al. (2015) J. Biol. Chem. 290, 11052-60; [3] Guo et al. (2009) Biochemistry 48, 4417-22; [4] Sun et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 2041-46

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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