Coordinated Regulation of Shear Stress-Induced Nitric Oxide Production by TRPC Channel-Derived Calcium Fluxes and Protein Kinase C Modulation in Endothelial Cells
Authors:   , Muzorewa Tenderano , Barbee Kenneth A 
Published: November 2022

Endothelial dysfunction, characterised by impaired nitric oxide (NO) bioavailability, arises in response to a variety of cardiovascular risk factors and precedes atherosclerosis. NO is produced by tight regulation of endothelial nitric oxide synthase (eNOS) activity in response to vasodilatory stimuli. This regulation of eNOS is mediated in part by store-operated calcium entry (SOCE) and Protein Kinase C (PKC), a promiscuous serine/threonine kinase. Calcium-dependent PKCbeta (PKC[beta]) and calcium-independent PKCeta (PKC[eta]) have both been implicated in the regulation and dysfunction of endothelial responses to shear stress and agonists. This signaling pathway is thought to be compartmentalized in caveolae. This study seeks to quantify the contribution of PKC[eta], PKC[beta], Orai1 channels and transient receptor potential canonical (TRPC) channels in regulating calcium signaling and endothelial nitric oxide synthase (eNOS) activation after exposure of endothelial cells to ATP or shear stress. The role of Cav1 as a scaffold for the proteins that regulate this pathway was also investigated. Bovine and human aortic endothelial cells were stimulated in vitro under pharmacological inhibition of target PKCs, and calcium channels as well as following knockdown of Cav1. The participation of these proteins in calcium flux, eNOS phosphorylation and NO synthesis was assessed following stimulation with agonist or shear stress. PKC[eta] proved to be a robust regulator of agonist- and shear stress-induced eNOS activation, modulating calcium fluxes and tuning eNOS activity by multi-site phosphorylation. PKC[beta] showed modest influence in this pathway, promoting eNOS activation basally and in response to shear stress. Both PKC isozymes contributed to the constitutive and induced phosphorylation of eNOS. The observed PKC signaling architecture shows gossamer complexity, recruiting Src to mediate a portion of PKC[eta]'s control on calcium entry and eNOS phosphorylation. Elucidation of the importance of PKC[eta] in this pathway was tempered by evidence of a single stimulus producing concurrent phosphorylation at ser1179 and thr497 which are antagonistic to eNOS activity. The peak and sustained ATP-induced calcium signal and the resulting eNOS activation were attenuated by inhibition of TRPC3, which was store operated. TRPC4 blockade reduced the transient peak in calcium concentration after ATP stimulation but did not significantly reduce eNOS activity. Simultaneous TRPC3 & 4 inhibition reduced flow-induced NO production via alterations in phosphorylation-mediated eNOS activity. Inhibition of TRPC1/6 or Orai1 failed to lower ATP-induced calcium entry or eNOS activation. Cav1 silencing reduced ATP-initiated eNOS activation. For the first time, in a single species in vitro, shear stress- and ATP-stimulated NO production are shown to be differentially regulated by classical and novel PKCs. TRPC3 is shown to be store-operated in BAECs and HAECs and is the key regulator of ATP-induced eNOS activation, whereas flow stimulation also recruits TRPC4 into the pathway for the synthesis of NO in BAECs. This study furthers our understanding of the PKC and TRPC isozyme interplay that optimizes NO production and the importance of Cav1 localization of this pathway. These considerations will inform the ongoing design of drugs for the treatment of PKC-sensitive cardiovascular pathologies.