Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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).
Conclusions: :
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.
Keywords: retina • ion channels • diabetes