PKCβ and reactive oxygen species mediate enhanced pulmonary vasoconstrictor reactivity following chronic hypoxia in neonatal rats

Abstract

Reactive oxygen species (ROS), mitochondrial dysfunction, and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. On the basis of evidence that PKCβ and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCβ and mitochondrial ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCβ inhibitor LY-333,531, the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and the mitochondrial antioxidants mitoquinone mesylate (MitoQ) and (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) on vasoconstrictor responses in saline-perfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12-day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F_2α (U-46619). LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared with control rats, which was inhibited by both LY-333,531 and mitochondria-targeted antioxidants. Additional experiments assessing mitoROS generation with the mitochondria-targeted ROS indicator MitoSOX revealed that a PKCβ-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrially localized PKCβ in pulmonary arteries as assessed by Western blotting of subcellular fractions. We conclude that PKCβ activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure.NEW & NOTEWORTHY This research demonstrates a novel contribution of PKCβ and mitochondrial reactive oxygen species signaling to increased pulmonary vasoconstrictor reactivity in chronically hypoxic neonates. The results provide a potential mechanism by which chronic hypoxia increases both basal and agonist-induced pulmonary arterial smooth muscle tone, which may contribute to neonatal pulmonary hypertension.

Keywords: PKCβ; mitochondria; oxidative stress; pulmonary hypertension; vascular disease.

Fig. 1.

Chronic hypoxia (CH) increases baseline pulmonary vascular resistance and basal tone through reactive oxygen species signaling. A–C: total (R_t; A), arterial (R_a; B), and venous (R_v; C) baseline vascular resistance (mmHg·mL⁻¹·kg·min) in lungs (in situ) from control and CH neonatal rats in the presence or absence of the superoxide dismutase mimetic 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL, 1 mM). D–F: the contribution of basal tone to total (D), arterial (E), and venous (F) resistance is expressed as the change in resistance (ΔR) to 1,3-propanediamine, N-{4-[1-(3-aminopropyl)-2-hydroxy-2-nitro-sohydrazino]butyl} (spermine NONOate, 100 μM) in lungs (in situ) from control and CH neonates. All experiments were conducted in the presence of N^ω-nitro-l-arginine (300 μM). Values are means ± SE; n = 4–10 rats/group (indicated in bars). *P < 0.05 vs. control, ^#P < 0.05 vs. vehicle, analyzed by 2-way ANOVA followed by Student-Newman-Keuls post hoc comparison.

Fig. 2.

PKC contributes to augmented baseline pulmonary vascular resistance and basal tone in lungs from neonatal rats exposed to chronic hypoxia (CH). A–C: total (R_t; A), arterial (R_a; B), and venous (R_v; C) baseline vascular resistance (mmHg·mL⁻¹·kg·min) in lungs from control and CH neonatal rats in the presence or absence of the PKC inhibitor Ro 31-8220 (10 μM). D–F: the contribution of basal tone to total (D), arterial (E), and venous (F) resistance is expressed as the change in resistance (ΔR) to 1,3-propanediamine, N-{4-[1-(3-aminopropyl)-2-hydroxy-2-nitro-sohydrazino]butyl} (spermine NONOate, 100 μM) in lungs (in situ) from control and CH neonates. Experiments were conducted in the presence of N^ω-nitro-l-arginine (300 μM). Values are means ± SE; n = 6–13 rats/group (indicated in bars). *P < 0.05 vs. control, ^#P < 0.05 vs. vehicle, analyzed by 2-way ANOVA followed by Student-Newman-Keuls post hoc comparison.

Fig. 3.

Chronic hypoxia (CH) increases baseline pulmonary vascular resistance and basal tone through PKCβ signaling. A–C: total (R_t; A), arterial (R_a; B), and venous (R_v: C) baseline vascular resistance (mmHg·mL⁻¹·kg·min) in lungs (in situ) from control and CH neonatal rats in the presence or absence of the PKCβ inhibitor LY-333,531 (LY, 10 nM). D–F: the contribution of basal tone to total (D), arterial (E), and venous (F) resistance is expressed as the change in resistance (ΔR) to 1,3-propanediamine, N-{4-[1-(3-aminopropyl)-2-hydroxy-2-nitro-sohydrazino]butyl} (spermine NONOate, 100 μM) in lungs from control and CH neonates. Experiments were conducted in the presence of N^ω-nitro-l-arginine (300 μM). Values are means ± SE; n = 4–11 rats/group (indicated in bars). *P < 0.05 vs. control, ^#P < 0.05 vs. vehicle, analyzed by 2-way ANOVA followed by Student-Newman-Keuls post hoc comparison.

Fig. 4.

Reactive oxygen species (ROS) mediate enhanced vasoconstrictor sensitivity in lungs from chronic hypoxia (CH) neonates: changes in total (A), arterial (B), and venous (C) resistance (mmHg·mL⁻¹·kg·min) to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F_2α (U-46619) in lungs from control and CH neonates. Inset in C uses a smaller y-axis scale to allow better visualization of differences between groups. Experiments were conducted in the presence of the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL, 1 mM) or vehicle. All experiments were conducted in the presence of N^ω-nitro-l-arginine (300 μM). Values are means ± SE; n = 6 rats for control vehicle, n = 5 rats for CH vehicle, n = 6 rats for control + TEMPOL, n = 4 rats for CH + TEMPOL. *P < 0.05 vs. control, ^#P < 0.05 vs. vehicle, analyzed by 2-way ANOVA at each U-46619 concentration ([U-46619]) with a Student-Newman-Keuls post hoc comparison.

Fig. 5.

Greater vasoconstrictor sensitivity in lungs from chronic hypoxia (CH) vs. control neonates is PKC dependent: changes in total (A), arterial (B), and venous (C) resistance (mmHg·mL⁻¹·kg·min) to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F_2α (U-46619) in lungs from control and CH neonates. Inset in C uses a smaller y-axis scale to allow better visualization of differences between groups. Experiments were conducted in the continued presence of N^ω-nitro-l-arginine (300 μM) with or without the PKC inhibitor Ro 31-8220 (10 μM). Values are means ± SE; n = 5 rats for control vehicle, n = 6 rats for CH vehicle, n = 5 rats for control + Ro 31-8220, n = 4 rats for CH + Ro 31-8220. *P < 0.05 vs. control, ^#P < 0.05 vs. vehicle, analyzed by 2-way ANOVA at each U-46619 concentration ([U-46619]) with a Student-Newman-Keuls post hoc test.

Fig. 6.

PKCβ mediates greater vasoconstrictor sensitivity in lungs from chronic hypoxia (CH) neonates: changes in total (A), arterial (B), and venous (C) resistance (mmHg·mL⁻¹·kg·min) to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F_2α (U-46619) in lungs from control and CH neonates. Inset in C uses a smaller y-axis scale to allow better visualization of differences between groups. Experiments were conducted in the continued presence of N^ω-nitro-l-arginine (300 μM) with or without the PKCβ inhibitor LY-333,531 (LY, 10 nM). Values are means ± SE; n = 6 rats for control vehicle, n = 5 rats for CH vehicle, n = 5 rats for control + LY, n = 5 rats for CH + LY. *P < 0.05 vs. control, ^#P < 0.05 vs. vehicle, analyzed by 2-way ANOVA at each U-46619 concentration ([U-46619]) with a Student-Newman-Keuls post hoc test.

Fig. 7.

PKCβ and mitochondrial reactive oxygen species facilitate enhanced basal tone in isolated pulmonary arteries from chronic hypoxia (CH) neonates. Experiments were conducted in the presence of the mitochondria-targeted $O_2^{-}$ scavenger (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO, 200 μM; A), the coenzyme Q10 analog mitoquinone mesylate (MitoQ, 1 μM; B), the PKCβ inhibitor LY-333,531 (LY, 10 nM; C), or vehicle. Basal tone (% vasoconstriction) is expressed as % maximally dilated (Ca²⁺ free) inner diameter. All experiments were conducted in the presence of N^ω-nitro-l-arginine (300 μM). Values are means ± SE; n = 5–10 rats/group (indicated in bars). *P < 0.05 vs. control, ^#P < 0.05 vs. vehicle, analyzed by 2-way ANOVA followed by Student-Newman-Keuls post hoc test.

Fig. 8.

PKCβ does not contribute to elevated basal tone in isolated pulmonary arteries from chronic hypoxia (CH) adult rats. Experiments were conducted in the presence of the PKCβ inhibitor LY-333,531 (LY, 10 nM) or vehicle. Basal tone (% vasoconstriction) is expressed as % maximally dilated (Ca²⁺ free) inner diameter. All experiments were conducted in the presence of N^ω-nitro-l-arginine (300 μM). Values are means ± SE; n = 8 or 9 rats/group (indicated in bars). *P < 0.05 vs. control, analyzed by 2-way ANOVA followed by Student-Newman-Keuls post hoc test.

Fig. 9.

PKCβ stimulation leads to mitochondrial reactive oxygen species production in pulmonary arterial smooth muscle cells (PASMCs) from control neonates. A: representative photomicrographs of MitoSOX fluorescence from transiently cultured PASMCs collected from pulmonary arteries of control neonates. B: summary data of MitoSOX mean fluorescence intensity (MFI) under vehicle conditions or in response to the PKC agonist PMA (10 μM) in the presence of each treatment condition: PKCβ inhibition with LY-333,531 (LY, 10 nM), oxidant scavenging with the superoxide dismutase mimetics 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL, 1 mM) and Tiron (10 mM), and the mitochondria-targeted $O_2^{-}$ scavenger (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO, 200 μM). Values are means ± SE; n = 6 rats/group (indicated in bars). *P < 0.05 vs. vehicle, ^#P < 0.05 vs. PMA vehicle, analyzed by 2-way ANOVA followed by Student-Newman-Keuls post hoc test.

Fig. 10.

Pulmonary arterial PKCβ protein expression is lower in chronic hypoxia (CH) compared with control neonates. A: representative Western blot for PKCβ. B: summary data for PKCβ levels in pulmonary arterial homogenates from control and CH neonatal rats. PKCβ bands were normalized to those of β-actin. Values are means ± SE; n = 7 rats/group (indicated in bars). *P < 0.05 vs. control, analyzed by unpaired t-test.

Fig. 11.

Mitochondrially localized PKCβ is greater in intrapulmonary arteries from neonatal chronic hypoxia (CH) vs. control rats. A: representative Western blot for PKCβ and cytochrome-c oxidase (Cyt. C Ox.) in mitochondria-enriched pellet (P) and extramitochondrial supernatant (S) fractions. B: summary data for the P-to-S PKCβ ratio in fractionated pulmonary arterial homogenates collected from control and CH neonatal rats. Values are means ± SE; n = 8 rats/group (indicated in bars). *P < 0.05 vs. control, analyzed by unpaired t-test.