Roles of protein kinase D in vasoconstriction and hemodynamics
Authors: Sugawara Yoh , Mizuno Yusuke , Kawakami Hiromasa , Goto Takahisa 
Published: November 2023

Protein kinase D (PKD), formerly classified as a member of PKC family, is known to be implicated in a variety of cellular processes and physiological functions, including cell motility, cardiac contraction, angiogenesis. Recently, PKD has been noted to be involved in various pathological conditions such as heart failure, cardiac hypertrophy. PKD thus, is thought to be a novel target for the treatments. However, little is known about a role of PKD in vasomotor activity and pulmonary circulation. Then, we investigated the role of PKD in pulmonary vasoconstriction and the circulation in normal and monocrotaline (MCT)‐induced pulmonary hypertension (PH) in rats.Material and methodsEight‐week‐old male rats were given either 60 mg/kg (MCT group) or saline (control group) 4 weeks before experiments. Measurements of right ventricular systolic pressure (RVSP), systemic blood pressure (SBP), cardiac output (CO) and systemic and pulmonary vascular resistances were performed by the thermodilution technique. Hemodynamic changes in response to intravenous bolus injections of PKD inhibitor, CRT0066101 were recorded. Isometric tension was measured using ring samples of pulmonary arteries and aortas to examine the effect of CRT0066101 on norepinephrine (NE)‐induced contraction. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1), CPI‐17, regulatory myosin light chain (RLC), heat shock protein 27 (HSP27) and PKD in the ring arteries was measured. Phosphorylations of PKD and MYPT1 are known to be indexes of activity of PKD and Rho kinase respectively. Simultaneous measurements of intracellular calcium concentrations with Fura‐2 and cell shortening were performed using human pulmonary artery smooth muscle cells (HPSMC).ResultsThe injection of CRT0066101 decreased RVSP in MCT group with small increase in CO but did not in control. CRT0066101 also decreased SBP in both groups similarly. NE‐induced contraction in pulmonary artery rings was inhibited almost totaly with CTR0066101 to a greater extent in MCT group than in control. On the other hand, NE‐induced contraction in aortas was inhibited with CRT0066101 in both groups similarly. NE induced phosphorylations of PKD, MYPT1 and RLC, but not HSP27, CPI‐17 in pulmonary arteries in both groups. The increased phosphorylations were all attenuated by pretreatment of CRT0066101. Protein expression of Rho kinase in pulmonary arteries was increased in MCT group compared to in control. In HPSMC, CRT0066101 inhibited NE‐induced cell shortening without affecting increase in calcium concentrations. Therefore, PKD was activated with NE and involved in contraction of pulmonary arteries and aortas. Attenuation of the vascular contraction with CRT0066101 was associated with decreased phosphorylations of MYPT1 and RLC, but not affecting intracellular calcium. In addition, inhibition of PKD appeared to be beneficial for RVSP and circulation in pulmonary hypertension.ConclusionsPKD was involved in contraction of aorta and pulmonary arteries. and might play a role in pulmonary and systemic circulations. The mechanism of PKD in vasoconstriction appeared to be associated with MYPT1 phosphorylation but not intracellular calcium regulation.Support or Funding InformationThis work was supported by Grant‐in‐Aid for Scientific Research (C) 26462342 from Japan Society for the Promotion of Science