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To elucidate the direct effect of selective protein kinase Cβ inhibitor LY333531 on diabetic sensory neurons, we examined intracellular Ca2+ concentration in isolated rat dorsal root ganglion neurons using the fluorescent Ca2+ indicator fura-2. The duration of calcium transients induced by high (50 mM) extracellular K+ in small diabetic dorsal root ganglion neurons was significantly prolonged compared with that in control neurons. This prolonged intracellular Ca2+ concentration elevation in diabetic neurons was normalized rapidly and reversibly by LY333531 in a dose-dependent manner, and the effect of LY333531 was completely abolished by pretreating the neurons with mitochondrial calcium uniporter inhibitor, Ruthenium 360. These results suggest that LY333531 has an ameliorating effect on calcium homeostasis of diabetic sensory neurons via mitochondrial calcium buffering.