Objective Mitochondrial depolarization following ATP-sensitive potassium (mitoKATP) channel activation has been shown to induce cerebral vasodilation by the generation of ‘calcium sparks’ in smooth muscle. It is unclear, however, if mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitoKATP channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. Methods and Results Diameter studies in isolated rat cerebral arteries showed BMS and diazoxide induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitoKATP channels, phosphoinositide-3 kinase (PI3K) or nitric oxide synthase (NOS). Scavenging of reactive oxygen species (ROS), however, diminished vasodilation induced by diazoxide but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells (CMVECs) showed that BMS elicited mitochondrial depolarization, and enhanced nitric oxide (NO) production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial ROS production. Measurements of intracellular calcium ([Ca2+]i) in CMVECs and arteries showed that both diazoxide and BMS increased endothelial [Ca2+]i. Western blot analyses revealed increased phosphorylation of Akt and endothelial NOS (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by PI3K inhibition. Electron spin resonance spectroscopy confirmed vascular NO generation in response to diazoxide and BMS. Conclusions Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca2+]i as well as by PI3K-Akt-induced eNOS phosphorylation. Both mitochondrial ROS-dependent and –independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.

Our objective was to investigate the mitochondrial dynamics following oxygen-glucose deprivation (OGD) in cultured rat cortical neurons. We documented changes in morphology, protein expression, and DNA levels in mitochondria following OGD and examined the roles of mitochondrial fission [dynamin-related protein 1 (Drp1), fission protein-1 (Fis1)] and fusion [mitofusin-1 (Mfn1), mitofusin-2 (Mfn2), and optic atrophy-1 protein (OPA1)] proteins on mitochondrial biogenesis and morphogenesis. We tested the effects of two Drp1 blockers [15-deoxy-Δ12,14-Prostaglandin J2 (PGJ2) and Mitochondrial Division Inhibitor (Mdivi-1)] on mitochondrial dynamics and cell survival. One hour of OGD had minimal effects on neuronal viability but mitochondria appeared condensed. Three hours of OGD caused a 60% decrease in neuronal viability accompanied by a transition from primarily normal/tubular and lesser number of rounded mitochondria during normoxia to either poorly labeled or small and large rounded mitochondria. The percentage of rounded mitochondria remained the same. The mitochondrial voltage-dependent anion channel, Complex V, and mitoDNA levels increased after OGD associated with a dramatic reduction in Drp1 expression, less reduction in Mfn2 expression, an increase in Mfn1 expression, with no changes in either OPA1 or Fis1. Although PGJ2 increased polymerization of Drp1, it did not reduce cell death or alter mitochondrial morphology following OGD and Mdivi-1 did not protect neurons against OGD. In summary, mitochondrial biogenesis and maintained fusion occurred in neurons along with mitochondrial fission following OGD; thus Mfn1 but not Drp1 may be a major regulator of these processes.

Transforming growth factors-beta1 (TGF-beta1), -2, and -3 form a small group of related proteins involved in the regulation of proliferation, differentiation, and survival of various cell types. Recently, TGF-betas were also demonstrated to be neuroprotective. In the present study, we investigated their distribution in the rat brain as well as their expression following middle cerebral artery occlusion. Probes were produced for all types of TGF-betas, and in situ hybridization was performed. We demonstrated high TGF-beta1 expression in cerebral cortex, hippocampus, central amygdaloid nucleus, medial preoptic area, hypothalamic paraventricular nucleus, substantia nigra, brainstem reticular formation and motoneurons, and area postrema. In contrast, TGF-beta2 was abundantly expressed in deep cortical layers, dentate gyrus, midline thalamic nuclei, posterior hypothalamic area and mamillary body, superior olive, areas of monoaminergic neurons, spinal trigeminal nucleus, dorsal vagal complex, cerebellum, and choroid plexus, and a high level of TGF-beta3 mRNA was found in cerebral cortex, hippocampus, basal amygdaloid nuclei, lateral septal nucleus, several thalamic nuclei, arcuate and supramamillary nuclei, superior colliculus, superior olive, brainstem reticular formation and motoneurons, area postrema, and inferior olive. Focal brain ischemia induced TGF-betas with markedly different expression patterns. TGF-beta1 was induced in the penumbral region of cortex and striatum, whereas TGF-beta2 and -beta3 were induced in different layers of the ipsilateral cortex. The expression of the subtypes of TGF-betas in different brain regions suggests that they are involved in the regulation of different neurons and bind to different latent TGF-beta binding proteins. Furthermore, they might have subtype-specific functions following ischemic attack.