Objective—To determine whether adenosine influences
the in vitro release of nitric oxide (NO) from differentiated
primary equine articular chondrocytes.
Sample Population—Articular cartilage harvested
from the metacarpophalangeal and metatarsophalangeal
joints of 11 horses (3 to 11 years old) without
history or clinical signs of joint disease.
Procedure—Chondrocytes were isolated, plated at a
high density (105 cells/well), and treated with adenosine,
the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine
(NECA), bradykinin, or other
agents that modify secondary messenger pathways
alone or in combination with bacterial lipopolysaccharide
(LPS) or recombinant human interleukin-1α (rhIL-1α). Nitric oxide release was measured indirectly by
use of the Griess reaction and was expressed as
µmol of nitrite in the supernatant/µg of protein in the
cell layer. Inducible nitric oxide synthase (iNOS) activity
was determined by measuring the conversion of
radiolabeled arginine to radiolabeled citrulline.
Results—Treatment of chondrocytes with adenosine
alone had no significant effect on NO release.
However, adenosine and NECA inhibited LPS- and
rhIL-1α-induced NO release. This response was mimicked
by forskolin, which acts to increase adenylate
cyclase activity, but not by the calcium ionophore
A23187. Treatment of chondrocytes with phorbol
myristate acetate, which acts to increase protein
kinase C activity, potentiated LPS-induced NO
release. Adenosine treatment also significantly inhibited
the LPS-induced increase in iNOS activity.
Conclusions and Clinical Relevance—Adenosine
and the nonspecific adenosine receptor agonist
NECA inhibited inflammatory mediator-induced
release of NO from equine articular chondrocytes.
Modulation of adenosine receptor-mediated pathways
may offer novel methods for treatment of
inflammation in horses with joint disease. (Am J Vet Res 2002;63:204–210)
Objective—To investigate the activities of
hyaluronidases in equine sera and synovial fluid samples
and sera from fetal and adult bovids and evaluate
the extent to which the degradation of hyaluronan is
influenced by chondrocytes.
Sample Population—Commercial and noncommercial
samples of equine (n = 6) and bovine (6) sera and
16 synovial fluid samples from horses.
Procedure—Hyaluronidase activities in sera and synovial
fluid samples were assessed via enzyme
zymography (performed at pH 4, 5, 6, or 7).
Chondrocytes were isolated from equine cartilage
and cultured with or without hyaluronan (1 mg/mL);
the degradation of hyaluronan was assessed via
agarose gel electrophoresis.
Results—Hyaluronidase activity was detected in
equine sera and synovial fluid samples at pH 4, but not
at pH 7, and in bovine sera at both pH values. In all
samples at pH 4, a major band of activity (molecular
weight, approx 60 kd) and some additional higher molecular
weight bands were detected; high- and low-molecular-weight activities were detected in bovine
sera at pH 7. Hyaluronan in tissue culture medium with
or without fetal calf serum was degraded in the presence,
but not the absence, of equine chondrocytes.
Conclusions and Clinical Relevance—Hyaluronidase
activity was detected in equine sera and synovial fluid
at pH 4 and in bovine sera at pH 4 and 7. Primary chondrocytes
in monolayer culture can degrade exogenous
hyaluronan. Modulating native hyaluronidase activity
may offer a new approach to improve the quantity and
quality of hyaluronan in articular joints. ( Am J Vet Res 2005;66:984–990)
Objective—To investigate accumulation of extracellular
adenosine (ADO) by equine articular chondrocytes
and to compare effects of adenosine kinase inhibition
and adenosine deaminase inhibition on the amount of
nitric oxide (NO) produced by lipopolysaccharide
Sample Population—Articular cartilage from
metacarpophalangeal and metatarsophalangeal joints
of 14 horses.
Procedure—Chondrocytes were cultured as monolayers,
and cells were incubated with LPS, the adenosine
kinase inhibitor 5'-iodotubercidin (ITU), or the
adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-
nonyl)adenine hydrochloride (EHNA). Concentrations
of ADO in cell supernatants were measured by use of
reverse-phase high-performance liquid chromatography.
Effect of inhibition of enzymatic metabolism of
ADO on induced NO production was evaluated by
exposing cells to a combination of LPS and ITU or LPS
Results—Articular chondrocytes accumulated extracellular
ADO when exposed to LPS or ITU.
Chondrocytes exposed to ITU accumulated ADO in a
time-dependent manner. Unstimulated chondrocytes
did not accumulate ADO. Similarly, EHNA alone did
not produce detectable ADO concentrations; however,
addition of EHNA and ITU resulted in a synergistic
effect on accumulation of ADO. Lipopolysaccharideinduced
NO production was more effectively suppressed
by exposure to ITU than to EHNA
Conclusions and Clinical Relevance—Equine articular
chondrocytes release ADO in response to the
proinflammatory stimulus of bacterial LPS. Inhibition
of the metabolism of ADO increases accumulation of
extracellular ADO. Autocrine release of ADO from
chondrocytes may play a role in the cellular response
to tissue damage in arthritic conditions, and pharmacologic
modulation of these pathways in joints of
arthritic horses could be a potential method of therapy.
(Am J Vet Res 2002;63:1512–1519)