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M. Fukumoto, E. Ishizaki, D. G. Puro; Retinal Microvascular KATP Channels: Redox Regulation, Spermine Modulation and Diabetic Alteration. Invest. Ophthalmol. Vis. Sci. 2009;50(13):18. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We assessed the interrelationships of microvascular redox status, spermine-mediated oxidation and diabetes on the function of KATP channels whose activation in retinal microvessels mediates the hyperpolarizing effects of the vasoactive signals, adenosine and dopamine.
Ionic currents were monitored via perforated-patch pipettes sealed onto mural cells located on microvascular complexes freshly isolated from the adult rat retina. Intracellular oxidants were monitored with the dye, carboxy-H2DCFDA. The KATP activator, pinacidil (5µM), was used to induce the KATP current. Rats were made diabetic by streptozotocin.
In a series of 27 experiments, the pinacidil-induced conductance detected in retinal microvessels was enhanced (p < 0.01) by the chemical oxidant, 5,5'-dithionitrobenzoate (DTNB), and diminished (p < 0.01) by dithiothreitol (DTT), a reductant. Other experiments tested the hypothesis that spermine, a polyamine whose catabolism generates H2O2 and other oxidants, plays a role in regulating KATP channel activity in retinal microvessels. Use of an oxidation-sensitive dye demonstrated that exposure to 5mM spermine increased (p < 0.001, n = 17) the concentration of oxidants in microvascular cells. In other experiments, we observed that spermine increased (p < 0.001, n = 7) the KATP conductance by a mechanism sensitive to the reductant, DTT. Conversely, inhibition of spermine synthesis by DFMO decreased (p < 0.001, n= 5) the KATP current. Because diabetes upregulates spermine in retinal microvessels, we conducted 63 experiments using microvessels from diabetic rats. We found that after 6.6± 0.4 weeks of hyperglycemia, the KATP conductance was increased (p < 0.001) by a mechanism sensitive to DTT (p < 0.001) and DFMO (p < 0.001).
We conclude that the function of KATP channels in retinal microvessels is potently regulated by a redox mechanism driven by the polyamine, spermine. In addition to the identification of spermine-dependent oxidation as a physiological mechanism regulating the activity of microvascular KATP channels, our results also support the possibility that spermine-dependent oxidation plays a role in causing functional changes in the microvasculature of the diabetic retina.
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