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Astrocyte-induced cortical vasodilation is mediated by D-serine and endothelial nitric oxide synthase.

ABSTRACT: Astrocytes play a critical role in neurovascular coupling by providing a physical linkage from synapses to arterioles and releasing vaso-active gliotransmitters. We identified a gliotransmitter pathway by which astrocytes influence arteriole lumen diameter. Astrocytes synthesize and release NMDA receptor coagonist, D-serine, in response to neurotransmitter input. Mouse cortical slice astrocyte activation by metabotropic glutamate receptors or photolysis of caged Ca(2+) produced dilation of penetrating arterioles in a manner attenuated by scavenging D-serine with D-amino acid oxidase, deleting the enzyme responsible for D-serine synthesis (serine racemase) or blocking NMDA receptor glycine coagonist sites with 5,7-dichlorokynurenic acid. We also found that dilatory responses were dramatically reduced by inhibition or elimination of endothelial nitric oxide synthase and that the vasodilatory effect of endothelial nitric oxide synthase is likely mediated by suppressing levels of the vasoconstrictor arachidonic acid metabolite, 20-hydroxy arachidonic acid. Our results provide evidence that D-serine coactivation of NMDA receptors and endothelial nitric oxide synthase is involved in astrocyte-mediated neurovascular coupling.

SUBMITTER: Stobart JL

PROVIDER: S-EPMC3581926 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation.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 mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca(2+)]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca(2+)]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS.Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca(2+)]i as well as by phosphoinositide-3 kinase-protein kinase B-induced eNOS phosphorylation. Both mitochondrial reactive oxygen species-dependent and -independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.

Project description:OBJECTIVE:Impaired endothelial cell (EC) autophagy compromises shear stress-induced nitric oxide (NO) generation. We determined the responsible mechanism. APPROACH AND RESULTS:On autophagy compromise in bovine aortic ECs exposed to shear stress, a decrease in glucose uptake and EC glycolysis attenuated ATP production. We hypothesized that decreased glycolysis-dependent purinergic signaling via P2Y1 (P2Y purinoceptor 1) receptors, secondary to impaired autophagy in ECs, prevents shear-induced phosphorylation of eNOS (endothelial nitric oxide synthase) at its positive regulatory site S1117 (p-eNOSS1177) and NO generation. Maneuvers that restore glucose transport and glycolysis (eg, overexpression of GLUT1 [glucose transporter 1]) or purinergic signaling (eg, addition of exogenous ADP) rescue shear-induced p-eNOSS1177 and NO production in ECs with impaired autophagy. Conversely, inhibiting glucose transport via GLUT1 small interfering RNA, blocking purinergic signaling via ectonucleotidase-mediated ATP/ADP degradation (eg, apyrase), or inhibiting P2Y1 receptors using pharmacological (eg, MRS2179 [2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetrasodium salt]) or genetic (eg, P2Y1-receptor small interfering RNA) procedures inhibit shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Supporting a central role for PKC?T505 (protein kinase C delta T505) in relaying the autophagy-dependent purinergic-mediated signal to eNOS, we find that (1) shear stress-induced activating phosphorylation of PKC?T505 is negated by inhibiting autophagy, (2) shear-induced p-eNOSS1177 and NO generation are restored in autophagy-impaired ECs via pharmacological (eg, bryostatin) or genetic (eg, constitutively active PKC?) activation of PKC?T505, and (3) pharmacological (eg, rottlerin) and genetic (eg, PKC? small interfering RNA) PKC? inhibition prevents shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Key nodes of dysregulation in this pathway on autophagy compromise were revealed in human arterial ECs. CONCLUSIONS:Targeted reactivation of purinergic signaling and PKC? has strategic potential to restore compromised NO generation in pathologies associated with suppressed EC autophagy.

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Astrocyte-induced cortical vasodilation is mediated by D-serine and endothelial nitric oxide synthase.

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