Post-translational modifications of thromboxane receptor G-protein alpha q complex in hypoxic PPHN
| dc.contributor.author | Sikarwar, Anurag Singh | |
| dc.contributor.examiningcommittee | Chelikani, Prashen (Oral Biology) Halayko, Andrew (Physiology and Pathophysiology) Shen, Gary (Physiology and Pathophysiology) | en_US |
| dc.contributor.supervisor | Dakshinamurti, Shyamala (Pediatrics and Physiology) | en_US |
| dc.date.accessioned | 2016-09-09T18:16:47Z | |
| dc.date.available | 2016-09-09T18:16:47Z | |
| dc.date.issued | 2014-01 | en_US |
| dc.date.issued | 2014-01 | en_US |
| dc.degree.discipline | Physiology and Pathophysiology | en_US |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | en_US |
| dc.description.abstract | Introduction: Persistent pulmonary hypertension of the newborn (PPHN) is associated with an elevated thromboxane to prostacyclin ratio, pulmonary artery (PA) hyperreactivity and hypersensitivity. Thromboxane receptor (TP), coupling with G-protein Gαq causes pulmonary vasoconstriction; whereas prostacyclin receptor (IP), coupling with Gαs, causes vasodilation and TP phosphorylation via adenylyl cyclase (AC)-cAMP-protein kinase A (PKA), desensitizes TP. Both TP phosphorylation and Gαq palmitoylation play major roles in regulation of signaling through the TP-Gαq complex. We hypothesized that increased Gαq palmitoylation and decreased AC activity could cause hypoxic TP hyperresponsiveness. We studied the impact of hypoxia on selected post-translational modifications of the receptor-G-protein complex, determining TP vasoconstriction: Gαq palmitoylation, TP phosphorylation and upstream AC activity. Methods: Force responses to thromboxane mimetic U46619, palmitoylation inhibition by 2-bromopalmitate (2-BP) and AC activation (forskolin) were studied by myography in hypoxic PPHN and control newborn swine pulmonary artery. Ca2+ mobilization was studied by fluorescent calcium indicators fura-2AM in pulmonary myocytes (PASMC), and fluo-4NW in HEK293 cells. Effects of hypoxia on Gαq palmitoylation were studied by metabolic labeling. Gαq cysteines and TP serines were mutated to determine sites of post-translational modifications. Protein expression and receptor-G-protein coupling were studied by Western blot and co-immunoprecipitation. PKA activity was assayed; and AC activity quantified. Results: Hypoxia increases Gαq palmitoylation, without increasing total palmitate uptake. Palmitoylation inhibition decreases U46619-stimulated force generation as well as Ca2+ mobilization in PPHN PA rings and hypoxic PASMC. Mutation of palmitoylable cysteine and palmitoylation inhibition proportionately decrease U46619-mediated Ca2+ mobilization in HEK293 cells. TP serine phosphorylation is decreased by hypoxia due to decreased PKA activity; this causes TP hypersensitivity and hyper-reactivity. Serine 324 of TPα is the target of PKA-mediated desensitization. AC activator-induced relaxation is reduced in PPHN PA. Basal and receptor-stimulated AC activity are decreased in hypoxic PASMC. Decreased AC activity is not due to decreased AC expression, ATP availability nor increased Gαi activation. Conclusion: Increased Gαq palmitoylation plays a role in TPα hyper-responsiveness in hypoxic PPHN. Hypoxia also reduces responses to agents acting through AC, unleashing TP-mediated vasoconstriction. Reactivation of pulmonary AC might be useful therapeutically to promote vasodilation and TP desensitization. | en_US |
| dc.description.note | October 2016 | en_US |
| dc.identifier.citation | Sikarwar, A. S., et al. 2014 Palmitoylation of Galphaq determines its association with the thromboxane receptor in hypoxic pulmonary hypertension. Am J Respir Cell Mol Biol 50(1):135-43. | en_US |
| dc.identifier.citation | Santhosh, K. T., et al. 2014 Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324. Br J Pharmacol 171(3):676-87. | en_US |
| dc.identifier.uri | http://hdl.handle.net/1993/31664 | |
| dc.language.iso | eng | en_US |
| dc.publisher | American Thoracic Society | en_US |
| dc.publisher | John Wiley and Sons, Inc | en_US |
| dc.rights | open access | en_US |
| dc.subject | PPHN, post-translational modifications | en_US |
| dc.title | Post-translational modifications of thromboxane receptor G-protein alpha q complex in hypoxic PPHN | en_US |
| dc.type | doctoral thesis | en_US |