Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase.

Abstract

BACKGROUND:

Hypoxia-induced pulmonary hypertension is a major cause of morbidity and mortality. Hypoxia induces pulmonary vasoconstriction, in part, by decreasing endothelial nitric oxide synthase (eNOS) expression. The mechanism by which hypoxia decreases eNOS expression is not known but may involve Rho-kinase-induced actin cytoskeletal changes in vascular endothelial cells.

METHODS AND RESULTS:

To determine whether hypoxia regulates eNOS expression through Rho-kinase, we exposed human saphenous and pulmonary artery endothelial cells to hypoxia (3% O2) with and without a Rho-kinase inhibitor, hydroxyfasudil (0.1 to 100 micromol/L), for various durations (0 to 48 hours). Hypoxia increased Rho-kinase expression and activity by 50% and 74%, decreased eNOS mRNA and protein expression by 66+/-3% and 57+/-5%, and inhibited eNOS activity by 48+/-9%. All of these effects of hypoxia on eNOS were reversed by cotreatment with hydroxyfasudil. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase or Rho-kinase by overexpression of dominant-negative Rho-kinase reversed hypoxia-induced decrease in eNOS expression. Indeed, disruption of the actin cytoskeleton, the downstream target of Rho-kinase, by cytochalasin D also upregulated eNOS expression. Hypoxia reduced eNOS mRNA half-life from 22+/-2 to 13+/-2 hours, which was reversed by cotreatment with hydroxyfasudil. However, neither hypoxia nor hydroxyfasudil had any effects on eNOS gene transcription.

CONCLUSIONS:

These results indicate that hypoxia-induced decrease in eNOS expression is mediated by Rho-kinase and suggest that Rho-kinase inhibitors may have therapeutic benefits in patients with hypoxia-induced pulmonary hypertension.

• Authors: Masao Takemoto, Jianxin Sun, Junko Hiroki, Hiroaki Shimokawa, James K Liao
• Journal: Circulation (Circulation) Vol. 106 Issue 1 Pg. 57-62 (Jul 02 2002) ISSN: 1524-4539 [Electronic] United States
• PMID: 12093770 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Enzyme Inhibitors
• Intracellular Signaling Peptides and Proteins
• RNA, Messenger
• hydroxyfasudil
• 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
• NOS3 protein, human
• Nitric Oxide Synthase
• Nitric Oxide Synthase Type III
• Protein Serine-Threonine Kinases
• rho-Associated Kinases

Topics

• 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine (analogs & derivatives, pharmacology)
• Cell Hypoxia
• Cells, Cultured
• Down-Regulation
• Endothelium, Vascular (drug effects, enzymology)
• Enzyme Inhibitors (pharmacology)
• Gene Expression Regulation
• Humans
• Intracellular Signaling Peptides and Proteins
• Nitric Oxide Synthase (genetics, metabolism)
• Nitric Oxide Synthase Type III
• Protein Serine-Threonine Kinases (antagonists & inhibitors, metabolism)
• Pulmonary Artery (cytology, enzymology)
• RNA Stability (drug effects)
• RNA, Messenger (biosynthesis)
• Transcription, Genetic (drug effects)
• rho-Associated Kinases