Roles and mechanism of protein kinase D in vasoconstriction

Abstract

Protein kinase D (PKD) is known to be implicated in a variety of cellular processes and physiological functions, including cell motility, cardiac contraction, angiogenesis and smooth muscle contraction through regulation of multiple molecules. Although these regulations are presumed to be closely associated with vasomotor activity, role of PKD in vascular function is unknown. Then, we investigated the role of PKD in systemic circulation and contraction of aorta in in rats. We also investigated precise role s of PKD isoforms using human aortic smooth muscle cells (HASMCs) with RNA interference.Material and methodsSystemic blood pressure (SBP) through femoral artery, cardiac output (CO) and systemic vascular resistances (SVR) were measured in Sprague‐Dawley rats. The hemodynamic changes in response to intravenous injections of PKD inhibitor, CRT0066101 were recorded. Isometric tension of aorta ring samples was measured to examine inhibitory effect of CRT0066101 on norepinephrine (NE)‐induced contraction. To explore mechanisms of PKD in vasomotor activity, phosphorylations of PKD, myosin targeting subunit‐1 (MYPT1), CPI‐17, regulatory myosin light chain (RLC) and heat shock protein 27 (HSP27) stimulated with NE were measured as an index of activation of the pathways in aorta rings. In HASMCs, simultaneous measurements of intracellular calcium concentrations and cell shortening with NE stimulation were performed. PKD1, 2, and 3 were knocked down by their specific siRNA respectively in HASMCs for measurements of phosphorylation of PKD, MYPT1, CPI‐17 and RLC and actin polymerization.ResultsInjection of CRT0066101 decreased SBP and SVR. CRT0066101 also inhibited NE‐induced contraction in aorta rings dose‐dependently. In the rings, NE increased phosphorylations of PKD, MYPT1, RLC and CPI‐17, which were attenuated by pretreatment of CRT0066101 except CPI‐17. NE did not increase HSP27 phosphorylation in the rings. In HASMCs, CRT0066101 inhibited NE‐induced cell shortening without affecting increased calcium concentrations. In HASMCs, increased phosphorylations of MYPT1 and RLC with NE were significantly inhibited by PKD1 but not PKD2, 3 knock down. NE did not affect actin polymerization in the cells. Collectively, attenuation of NE‐induced contraction of aorta rings with CRT0066101 was associated with decreased phosphorylations of MYPT1 and RLC, but not CPI‐17 and HSP27. In HASMCs, PKD1 knocked down or PKD inhibition attenuated NE‐induced phosphorylation of MYPT1 and RLC without affecting actin polymerization and intracellular calcium regulation.ConclusionsPKD1 may play roles in contraction of aorta and systemic circulation. The regulation of vasomotor activity by PKD1 may be associated with phosphorylation of MYPT1. Understanding of PKD may lead to novel regulation of circulation.Support or Funding InformationThis work was supported by Grant‐in‐Aid for Scientific Research (C) 17K11058 from Japan Society for the Promotion of ScienceThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.