Inorganic pyrophosphate release by rabbit articular chondrocytes in vitro

Abstract

Release of inorganic pyrophosphate (PPI) by rabbit articular chondrocytes in vitro was measured by a newly developed assay which utilizes radioactive orthophosphate (32Pi) labeling and anion exchange high performance liquid chromatography. Chondrocytes in monolayer and high density culture failed to release PPi. Explants (cartilage fragments), however, released newly formed PPi into the culture medium. Trypsin treatment of cartilage fragments almost completely blocked the PPi extrusion. Collagenase treatment had no effect on PPi extrusion. There was no clear correlation between proteoglycan synthesis, measured by 35S04 incorporation, and PPi release. Suppression of proteoglycan synthesis with tunicamycin did not influence the PPi release of the explants.Calcification of articular cartilage above the tidemark (chondrocalcinosis), either by calcium pyrophosphate dihydrate (CPPD) crystals or by hydroxyapatite (HA) crystals, is a relatively frequent pathologic finding. Both CPPD crystal deposition and HA crystal deposition are associated with osteoarthritis (OA) (1,2). These calcifications may be locally elaborated. CPPD crystal deposition has been found in humans following meniscectomy (3) and as a postarthritis phenomenon (4), and in animals after trauma From the Jan van Breemen Institute,

Interleukin 1 beta suppresses transforming growth factor-induced inorganic pyrophosphate (PPi) production and expression of the PPi-generating enzyme PC-1 in human chondrocytes.

“…Because PPi does not freely diffuse across membranes, it was hypothesized that the PPi might be released through an anion channel (23) or cosecreted with basic proteins such as collagen (24). Chondrocyte PPi elaboration is not dependent on de novo synthesis of glycosaminoglycans (25,26) but appears to be partially mediated by de novo protein synthesis (24). In this regard, TGF3 treatment of porcine chondrocytes up-regulated membrane and extracellular NT-PPPH activity by up to 36% (22).…”

Cartilage inorganic pyrophosphate elaboration is independent of sulfated glycosaminoglycan synthesis

“…Uronate elaboration in culture may reflect released degradation products rather than newly synthesized uronate. And, results of more recent work by Prins and coworkers, using rabbit cartilage, dispute the suggested tie between PP, and GAG extrusion (10).…”

“…More recently, studies of rabbit articular cartilage in organ culture failed to support this notion (10). No correlation of PP, elaboration and sulfation of macromolecules was observed.…”

“…Among all of the articular tissues that have been studied (cartilage, subchondral bone, synovium, synovial fluid), only hyaline or fibrocartilage elaborates extracellular PP, (8)(9)(10). Since PP, is commonly considered a by-product of synthetic reactions (1 I), we reasoned that biosyntheses prominent in or peculiar to chondrocytes might produce PP,, which could be exported from the cell together with the synthetic coproduct and participate in articular crystal formation.…”

“…No correlation of PP, elaboration and sulfation of macromolecules was observed. Moreover, those investigators found that suppression of proteoglycan synthesis by tunicamycin failed to suppress PP, elaboration (10).…”