Abstract 152: Unleashed intracellular Ca2+ Releases From Ryanodine Receptor-2 Plays An Important Role In Diabetic Cerebral Vasoconstriction

Abstract

Many devastating cerebral vascular diseases occur due to abnormally increased [Ca 2+ ] i and associated contraction in cerebral artery smooth muscle cells (CASMCs); however, the underlying molecular mechanisms are largely unknown. In this study, we assessed the novel hypothesis that FK506 binding protein 12.6 (FKBP12.6) is dissociated from ryanodine receptor-2 (RyR2), leading to unleashed intracellular Ca 2+ release in diabetic CASMCs and thus cerebral vasoconstriction. Using Förster resonance energy transfer and other biochemical assays, we have found that FKBP12.6 was tightly associated with RyR2 in mouse CASMCs. The association of these two Ca 2+ signaling molecules was disrupted in diabetic cells. Investigations using a laser scanning confocal microscope have revealed that targeted gene knockout (KO) of FKBP12.6 significantly increased local intracellular Ca 2+ release (Ca 2+ spark). A similar change in local intracellular Ca 2+ release was observed in diabetic cells. FKBP12.6 KO and diabetes did not produce additive effects. Global (whole-cell) intracellular Ca 2+ release following activation of RyRs with caffeine was increased to a similar extent in both FKBP12.6 KO and diabetic CASMCs. The increased caffeine-induced intracellular Ca 2+ release was not further enhanced in diabetic/FKBP12.6 KO cells. Stimulation of -adrenergic receptors with norepinephrine could result in a large increase in local and global intracellular Ca 2+ release with the same degree in FKBP12.6 KO, diabetic and diabetic/FKBP12.6 KO. FKBP12.6 KO, diabetic and diabetic/FKBP12.6 KO mice were all hypertensive, and their hypertensive levels were similar. In conclusion, we have, for the first time, provided compelling evidence that FKBP12.6 is dissociated from RyR2, which leads to unleashed intracellular Ca 2+ release through RyR2 in CASMCs and associated cerebral vasoconstriction, thereby playing an essential role in the development of diabetic and other cerebral vascular diseases.