Hypoxia Alters Caveolar Architecture and Adenylyl Cyclase 6 Regulation in Pulmonary Arterial Smooth Muscle
Bibliographic record
Abstract
Abstract Rationale Persistent pulmonary hypertension of the newborn (PPHN) represents a catastrophic failure of normal pulmonary vascular relaxation after birth. Non-responsiveness to nitric oxide therapy in 25-30% of cases suggests an underlying disorder of smooth muscle relaxation. Neonatal hypoxia diminishes cardiac inotropic responses and pulmonary artery relaxation due to reduced adenylyl cyclase (AC) activity. Among nine transmembrane AC isoforms, neonatal pulmonary artery myocytes express mainly AC isoform 6, followed by 3, 7 and 9; we have shown AC6 is inhibited by hypoxia. The plasma membrane of pulmonary arterial myocytes contains caveolae, lipid-rich invaginations important for protein scaffolding and signal transduction. Smooth muscle relaxation pathways, including AC signaling, are largely localized to caveolar lipid rafts. AC6 colocalizes with prostacyclin receptors in caveolae to mediate vascular relaxation. This study aimed to determine how hypoxia influences caveolar organization in pulmonary arterial smooth muscle cells, focusing on AC6 localization and coupling with G protein activators or inhibitors. We hypothesized that hypoxia disrupts the interaction of caveolar AC6 with activator Gαs, decreasing cAMP generation. Methods3rd-6th generation pulmonary arterial smooth muscle cells (PASMCs) were microdissected from <24hr old piglets and cultured; normoxic cells in 21% O2, hypoxic cells exposed to 10% O2 for 72 hours. Caveolar organization was analyzed by transmission electron microscopy (TEM; CCEM, McMaster University); caveolar fractions were isolated by sucrose gradient ultracentrifugation, to quantify AC6 and interacting proteins Gαi and Gαs by Western blot and co-immunoprecipitation in caveolar fractions and whole cell lysates. Gαs-based cAMP generation was measured in normoxic and hypoxic myocytes using an HTRF-based cAMP kit (CisBio), following AC6 knockdown using siRNA (Dharmacon). Results By blinded analysis of TEM images, we observed a significant decrease in caveolae density with increased width (but not depth) of caveolae under hypoxic conditions, indicating altered caveolar architecture. Abundance of AC6 increased in whole PASMCs, without change in Gαs or Gαi abundance. However, both AC6 and Gαi proteins increased significantly in caveolar fractions of hypoxic PASMCs. Co-immunoprecipitation confirmed that hypoxia enhances AC6-Gαi association. Hypoxic PASMC showed decreased cAMP generation to forskolin stimulation compared to normoxic PASMC; but after siRNA selective knockdown of AC6, we found no difference in cAMP generation between normoxic and hypoxic PASMC, indicating caveolar AC6 is responsible for the hypoxia sensitivity of PASMC cAMP generation. Conclusions Hypoxia alters PASMC caveolar organization and enriches caveolar abundance of hypoxia-inhibited AC6 as well as its inhibitor Gαi. This interaction decreases AC6 functionality in hypoxic pulmonary artery.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".