Phospholemman-Dependent Regulation of Na/K-Atpase Modulates Constriction and Relaxation in Aortic Smooth Muscle

Abstract

The Na/K ATPase (NKA) plays a role in modulating vascular tone through both NO-dependent and independent pathways. Phospholemman (PLM) is a muscle-specific regulator of Na/K ATPase and, in cardiac muscle, links PKA, PKC, and NO-signaling pathways to NKA stimulation. PLM is expressed in vascular smooth muscle (VSM) and we hypothesized that its phosphorylation may modulate the vascular responses to agonists. We tested this using wild-type (WT), PLM knock-out (KO), and mutant PLM knock-in mice (3SA) in which the PKC and PKA phosphorylation sites (S63, S68, S69) are mutated to alanines. Agonist-induced constriction and relaxation were measured in aortic rings in an isometric wire myograph. Vascular NKA activity was assessed by K-induced relaxation (a surrogate measure of Na/K ATPase activation) and PLM phosphorylation by immunoblotting. In WT aortae, PLM phosphorylation (S63 and S68) was significantly increased in response to phenylephrine (PE) and K-induced relaxation was significantly higher in WT than KO mice (85±7% vs 58±4% of PE-induced pre-constriction; p<0.01), suggesting PLM regulates NKA activity in VSM. The dose-response curve for PE-induced constriction was profoundly shifted up and to the left in 3SA aortae compared to WT suggesting PLM phosphorylation normally limits constriction. Ouabain (300μM) completely abolished this difference. Pretreatment with L-NAME (300μM) also potentiated constrictor responses to PE to a greater extent in WT than 3SA vessels. Relaxation induced by the NO donor spermine NONOate was blunted in vessels from 3SA mice: 67±6% vs 84±2% (U46619-induced constriction; p<0.01 cf WT). In summary, isolated aortic rings from mice expressing unphosphorylatable PLM showed markedly elevated constriction in response to PE and attenuated relaxation in response to an NO donor. Thus, PLM phosphorylation regulates the activity of vascular NKA and this contributes to modulation of aortic constriction and relaxation.