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PKC β Signals Through Mitochondrial Reactive Oxygen Species Production in Pulmonary Arterial Smooth Muscle Cells of Neonatal Rats
Author(s) -
Sheak Joshua R.,
Jernigan Nikki L.,
Walker Benjimen R.,
Resta Thomas C.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1073.7
Subject(s) - mitochondrial ros , hypoxic pulmonary vasoconstriction , protein kinase c , reactive oxygen species , oxidative stress , microbiology and biotechnology , mitochondrion , medicine , stimulation , endocrinology , chemistry , vasoconstriction , biology , signal transduction
Chronic hypoxia (CH)‐induced vasoconstriction has been implicated in the pathogenesis of pulmonary hypertension in infants with chronic cardiorespiratory disorders. Although mechanisms of pulmonary vasoconstriction resulting from neonatal CH are not well understood, preliminary studies from our group support a role for both PKC β and reactive oxygen species (ROS) in this response. However, neither the signaling relationship between PKC β and ROS nor the cellular source of ROS involved in this response has been previously addressed. Considering the importance of PKC β in mitochondrial dysfunction and oxidative stress in neurodegenerative diseases, we hypothesized that PKC β signals to induce mitochondrial ROS generation in neonatal pulmonary arterial smooth muscle cells. To test this hypothesis, we transiently cultured primary pulmonary arterial smooth muscle cells (PASMC) collected from neonatal normoxic control rats. The production of mitochondrial ROS was assessed using the mitochondrial selective fluorescent O 2 − indicator, mitoSOX (5 μM). We treated PASMC with PMA (10 μM), a phorbol ester known to activate PKC isoforms, including PKC β , and observed PMA‐dependent increases in mitoSOX fluorescence that were prevented by selective PKC β inhibition with LY‐333,531 (10 nM, p<0.05 vs. PMA alone, n=6 rats). Selectivity of mitoSOX for mitochondrial‐derived O 2 − was verified using the mitochondrial‐targeted O 2 − scavenger, mitoTEMPO (200 μM), which prevented PMA‐induced increases in mitoSOX fluorescence. Western blot analysis confirmed the expression of PKC βI and PKC βII splice variants in homogenates of isolated pulmonary arteries from neonatal normoxic control rats. We conclude that PKC β stimulation leads to mitoROS production in PASMC from neonatal rats. Future studies are planned to evaluate the contribution of mitochondrial ROS to enhanced vasoconstrictor reactivity and associated pulmonary hypertension resulting from neonatal CH, and the cellular signaling mechanisms involved. Support or Funding Information Awarded to Thomas C. Resta, PhD: NIH R01 HL132883 and AHA 16GRNT27700010. Awarded to Joshua R. Sheak: NIH F31 HL131334.