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. 2014 Feb;171(3):676-87.
doi: 10.1111/bph.12487.

Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324

K T Santhosh  1 , A S Sikarwar, M Hinton, P Chelikani, S Dakshinamurti
Affiliations

Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324

K T Santhosh et al. Br J Pharmacol. 2014 Feb.

Abstract

Background and purpose: Dysregulation of the thromboxane A₂ (TP) receptor, resulting in agonist hypersensitivity and hyper-responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA-accessible serine residues in determining TP receptor affinity, using site-directed mutational analysis.

Experimental approach: Vasoconstriction to a thromboxane mimetic and phosphorylation of TP receptor serine was examined in pulmonary arteries from neonatal swine with persistent pulmonary hypertension and controls. Effects of hypoxia were determined in porcine and human TP receptors. Human TPα serines at positions 324, 329 and 331 (C-terminal tail) were mutated to alanine and transiently expressed in HEK293T cells. Saturation binding and displacement kinetics of a TP antagonist and agonist were determined in porcine TP, wild-type human TPα and all TP mutants. Agonist-elicited calcium mobilization was determined for each TP mutant, in the presence of a PKA activator or inhibitor, and in hypoxic and normoxic conditions.

Key results: The Ser324A mutant was insensitive to PKA activation and hypoxia, had a high affinity for agonist and increased agonist-induced calcium mobilization. Ser329A was no different from wild-type TP receptors. Ser331A was insensitive to hypoxia and PKA with a decreased agonist-mediated response.

Conclusions and implications: In hypoxic pulmonary hypertension, loss of site-specific phosphorylation of the TP receptor causes agonist hyper-responsiveness. Ser324 is the primary residue phosphorylated by PKA, which regulates TP receptor-agonist interactions. Ser331 mutation confers loss of TP receptor-agonist interaction, regardless of PKA activity.

Keywords: PKA; hypoxia; pulmonary hypertension; thromboxane.

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Figures

Figure 1
Figure 1
TP receptor phosphorylation is decreased in pulmonary artery myocytes from PPHN animals. (A) Pulmonary arterial rings from newborn (D0), 3-day-old normoxic (D3 Control) and hypoxic pulmonary hypertensive animals (D3 PPHN) were subjected to isometric myography (set at optimal length by length–tension curve; force measurements normalized to maximal KCl-induced force). n = 4 animals per group. Contraction force to TP agonist U46619 declines over the first 3 days of life in normal animals. Hypoxic PPHN increases TP receptor-mediated contraction, **P < 0.001. (B) Representative blot and histogram showing phosphorylation of TP receptors in contractile pulmonary arterial myocytes primary cultured from 3 day hypoxic PPHN swine and age-matched normoxic controls. Cell lysates were subjected to immunoprecipitation using polyclonal antibody (rabbit) to thromboxane receptor, and TP serine phosphorylation quantified in immunoprecipitates by Western blot (mouse monoclonal antibody to phospho-serine; *P < 0.05, n = 8). Total TP receptor protein content was unchanged (not shown).
Figure 2
Figure 2
TP receptor-specific GTPase activity is increased in hypoxic pulmonary artery myocytes. Neonatal pulmonary artery myocytes grown in hypoxia or normoxia, challenged for 2 min with TP receptor agonist 10−6 M U46619, with or without TP receptor blockade by 10−5 M SQ29546, then flash-frozen. GTPase activity quantified in membrane fractions. Organic Pi liberated by membrane GTPase from substrate GTP in response to U46619 was calculated from a standard curve as [Pi] min−1·mg−1 protein, after subtraction of baseline (unstimulated) Pi release. Measured GTPase activity represents Pi release specifically due to TP-G-protein complex GTPase activity. This activity is sensitive to TP receptor blockade (+SQ) (data from three separate experiments in triplicate). *P < 0.05.
Figure 3
Figure 3
Human and porcine TP receptors expressed in HEK293T cells are hyper-responsive after 72 h of hypoxia. HEK293T clones stably expressing wild-type porcine TP (A) or human TPα (B) were exposed to hypoxic or normoxic culture conditions for 72 h, loaded with calciometric dye fluo-4 and challenged with serial concentrations of the thromboxane mimetic U46619. Ca2+ mobilization response to U46619 challenge under normoxic or hypoxic conditions quantified as relative fluorescence units following baseline subtraction; *P < 0.001, n = 4.
Figure 4
Figure 4
PKA, but not PKC, activity in HEK 293T cells is reduced by hypoxia. After 72 h of hypoxic or normoxic conditions,HEK293T cells were incubated with 10−6 M forskolin (activates PKA), 10−6 M H8 (inhibits PKA), 10−8 M milrinone (PDE-3 inhibitor), PMA (activates PKC), 10−6 M Go6983 (inhibits PKC) or diluent for 30 min. PKA (A) or PKC (C) activity measured in cell lysates, expressed as fold-change from normoxic control. TP-mediated Ca2+ mobilization in the presence of PKA activation or inhibition (B) or PKC activation or inhibition (D) determined using fluo-4, quantified as relative fluorescence units after baseline subtraction, normalized to maximal Ca2+ response. n = 4, **P < 0.001 compared with normoxic control.
Figure 5
Figure 5
PKA sensitivity of wild-type TPα and amino acid substitution mutants. HEK293T cells transfected with human wild-type TPα, or serine-to-alanine substitution mutants Ser239A, Ser324A, Ser329A or Ser331A were incubated with 10−6 M forskolin (activates PKA), 10−6 M H8 (inhibits PKA) or diluent, then challenged with serial concentrations of TP agonist U46619. (A) Alanine substitution of Ser239 did not alter PKA sensitivity of the TP agonist concentration-response curve relative to the wild-type TP receptor. (B) Substitution of Ser324 sensitized the receptor to the agonist and abolished PKA responsiveness. (C) The Ser329A mutant had PKA sensitivity comparable to wild-type TP receptors. (D) Ser331 substitution reduced the sensitivity of the receptor to the TP receptor agonist, regardless of PKA activation state. Corresponding EC50 values are given in Table 2.
Figure 6
Figure 6
Hypoxia increases competitive binding of agonist to TPα; effect of C-terminal amino acid substitution mutation. HEK293T cells transfected with (A) human wild-type TPα, or serine-to-alanine mutants (B) Ser324A, (C) Ser329A or (D) Ser331A, following 48 h of hypoxic or normoxic exposure, were incubated with a saturating concentration of [3H]-SQ29548, before antagonist displacement with serial concentrations of cold agonist U46619. Data are from three experiments.
Figure 7
Figure 7
Hypoxia increases agonist-induced Ca2+ mobilization by TPα; effect of C-terminal amino acid substitution mutation. HEK293T cells transfected with human wild-type TPα, or serine-to-alanine substitution mutants Ser324A, Ser329A or Ser331A were incubated for 48 h in hypoxic or normoxic conditions, followed by challenge with serial concentrations of agonist U46619. Absolute Ca2+ mobilization curves showing (A) differing effects of Ser324 substitution on agonist sensitivity in normoxic versus hypoxic conditions, relative to wild-type TP receptors; (B) effect of Ser329 substitution on hypoxia sensitivity of TP receptor-mediated Ca2+ response; and (C) effect of Ser331 substitution on hypoxic and normoxic TP agonist responses.
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