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-N(6)-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.