Endothelial Cell Autophagy Maintains Shear Stress-Induced Nitric Oxide Generation via Glycolysis-Dependent Purinergic Signaling to Endothelial Nitric Oxide Synthase

Leena P Bharath; Jae Min Cho; Seul-Ki Park; Ting Ruan; Youyou Li; Robert Mueller; Tyler Bean; Van Reese; Russel S Richardson; Jinjin Cai; Ashot Sargsyan; Karla Pires; Pon Velayutham Anandh Babu; Sihem Boudina; Timothy E Graham; J David Symons

doi:10.1161/ATVBAHA.117.309510

Endothelial Cell Autophagy Maintains Shear Stress-Induced Nitric Oxide Generation via Glycolysis-Dependent Purinergic Signaling to Endothelial Nitric Oxide Synthase

Arterioscler Thromb Vasc Biol. 2017 Sep;37(9):1646-1656. doi: 10.1161/ATVBAHA.117.309510. Epub 2017 Jul 6.

Authors

Affiliations

¹ From the Department of Nutrition and Integrative Physiology, College of Health (L.P.B., J.M.C., S.-K.P., T.R., Y.L., R.M., T.B., R.S.R., K.P., V.A.B., S.B., T.E.G., J.D.S.) and Molecular Medicine Program (J.C., A.S., S.B., T.E.G., J.D.S.), University of Utah, Salt Lake City; Division of Endocrinology, Metabolism, and Diabetes, University of Utah School of Medicine, Salt Lake City (L.P.B., J.M.C., S.-K.P., T.R., Y.L., R.M., T.B., J.C., A.S., K.P., S.B., T.E.G., J.D.S.); and University of Utah Geriatric Research, Education, and Clinical Center, George E. Whalen VA Medical Center, Salt Lake City (V.R., R.S.R.).
² From the Department of Nutrition and Integrative Physiology, College of Health (L.P.B., J.M.C., S.-K.P., T.R., Y.L., R.M., T.B., R.S.R., K.P., V.A.B., S.B., T.E.G., J.D.S.) and Molecular Medicine Program (J.C., A.S., S.B., T.E.G., J.D.S.), University of Utah, Salt Lake City; Division of Endocrinology, Metabolism, and Diabetes, University of Utah School of Medicine, Salt Lake City (L.P.B., J.M.C., S.-K.P., T.R., Y.L., R.M., T.B., J.C., A.S., K.P., S.B., T.E.G., J.D.S.); and University of Utah Geriatric Research, Education, and Clinical Center, George E. Whalen VA Medical Center, Salt Lake City (V.R., R.S.R.). j.david.symons@hsc.utah.edu.

Abstract

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-eNOS^S1177) 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-eNOS^S1177 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-eNOS^S1177 and NO generation in ECs with intact autophagy. Supporting a central role for PKCδ^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 PKCδ^T505 is negated by inhibiting autophagy, (2) shear-induced p-eNOS^S1177 and NO generation are restored in autophagy-impaired ECs via pharmacological (eg, bryostatin) or genetic (eg, constitutively active PKCδ) activation of PKCδ^T505, and (3) pharmacological (eg, rottlerin) and genetic (eg, PKCδ small interfering RNA) PKCδ inhibition prevents shear-induced p-eNOS^S1177 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 PKCδ has strategic potential to restore compromised NO generation in pathologies associated with suppressed EC autophagy.

Keywords: autophagy; cell physiological phenomena; endothelial cells; nitric oxide; reactive oxygen species.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Adenosine Triphosphate / metabolism*
Animals
Autophagy* / drug effects
Autophagy-Related Proteins / deficiency
Autophagy-Related Proteins / genetics
Cattle
Cells, Cultured
Endothelial Cells / drug effects
Endothelial Cells / enzymology*
Endothelial Cells / pathology
Glucose Transporter Type 1 / genetics
Glucose Transporter Type 1 / metabolism
Glycolysis* / drug effects
Humans
Mechanotransduction, Cellular* / drug effects
Mice, Inbred C57BL
Mice, Knockout
Nitric Oxide / metabolism*
Nitric Oxide Synthase Type III / metabolism*
Phosphorylation
Protein Kinase C-delta / antagonists & inhibitors
Protein Kinase C-delta / genetics
Protein Kinase C-delta / metabolism
Protein Kinase Inhibitors / pharmacology
Purinergic P2Y Receptor Antagonists / pharmacology
RNA Interference
Reactive Oxygen Species / metabolism
Receptors, Purinergic P2Y1 / drug effects
Receptors, Purinergic P2Y1 / genetics
Receptors, Purinergic P2Y1 / metabolism*
Serine
Stress, Mechanical
Transfection
Ubiquitin-Conjugating Enzymes / deficiency
Ubiquitin-Conjugating Enzymes / genetics

Substances

Autophagy-Related Proteins
Glucose Transporter Type 1
Protein Kinase Inhibitors
Purinergic P2Y Receptor Antagonists
Reactive Oxygen Species
Receptors, Purinergic P2Y1
Nitric Oxide
Serine
Adenosine Triphosphate
Nitric Oxide Synthase Type III
Ubiquitin-Conjugating Enzymes
Protein Kinase C-delta
Atg3 protein, mouse

Abstract

Publication types

MeSH terms

Substances

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