Hypoxia-induced hyaluronan synthesis by articular chondrocytes: the role of nitric oxide.
OBJECTIVE
Articular cartilage is an avascular tissue in which chondrocytes are exposed to hypoxic conditions. We previously demonstrated that reactive oxygen species (ROS) induced apoptosis of chondrocytes. We also demonstrated that nitric oxide (NO) was induced when chondrocytes were exposed to hypoxia and that NO inhibited the ROS-induced apoptosis. Hyaluronan (HA) is a high molecular weight glycosaminoglycan whose antioxidative effects have been reported. The purpose of the present study was to determine whether HA synthesis was induced in chondrocytes exposed to hypoxia, and, if so, whether the hypoxia-induced HA synthesis is regulated by NO.


METHODS
Bovine articular chondrocytes were used in this study. Levels of HA were determined by the sandwich enzyme-binding assay. Expression of HA synthase (HAS) was determined with reverse transcription-polymerase chain reaction. The production of NO was examined using the Griess reaction. We also determined inducible nitric oxide synthase (iNOS) enzyme synthesis using the histochemistry and Western blot analysis.


RESULTS
Chondrocytes cultured under hypoxic conditions exhibited enhanced HA synthesis. When the NO inhibitors, L-NMMA and L-NAME, were added, the hypoxia-enhanced HA levels in the culture medium were significantly inhibited.


CONCLUSIONS
Endogenous NO synthesis plays an important role in hypoxia-enhanced HA synthesis.
Hashimoto
K
K
Department of Orthopaedic Surgery, Kinki University School of Medicine, 377-2 Ohno-higashi, Osaka-sayama, 589-8511, Osaka, Japan.
Fukuda
K
K
Yamazaki
K
K
Yamamoto
N
N
Matsushita
T
T
Hayakawa
S
S
Munakata
H
H
Hamanishi
C
C
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology* / Q000378:metabolism; D002417:Cattle; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism; D006820:Hyaluronic Acid / Q000096:biosynthesis* / Q000737:chemistry; D000860:Hypoxia; D007375:Interleukin-1 / Q000378:metabolism; D008970:Molecular Weight; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
D007375:Interleukin-1; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D006820:Hyaluronic Acid; D052247:Nitric Oxide Synthase Type II; D014453:Glucuronosyltransferase; D010100:Oxygen