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 determine rate and degree of cooling
for the superficial digital flexor tendon (SDFT) during a
standard cryotherapy application in horses and evaluate
in vitro effects of cooling on survival of tendon
Sample Population—6 limbs of 5 adult horses and
cultured cells obtained from SDFT of 3 adult horses
Procedure—In vivo data were acquired by use of a
thermocouple temperature probe inserted into the
SDFT of a forelimb of each standing sedated horse.
After baseline temperatures were recorded, a commercial
compression splint with circulating coolant
was placed on each selected limb, which was then
exposed to cold treatment for 60 minutes.
Temperatures were recorded at 30-second intervals.
Mean minimum core temperature was calculated and
used to design a protocol for in vitro cold treatment of
cells. Specimens were obtained from the SDFT of
horses during necropsy; tendon cells were cultured in
suspension and exposed to 1-hour of cold treatment
that mimicked the in vivo procedure. Viability of cells
after cold treatment was compared with viability of
cells maintained at body temperature.
Results—After 1 hour of cold treatment, SDFT core
temperature was reduced by a mean of 21.8°C, reaching
a mean minimum temperature of 10oC. Viability
did not differ significantly between cold-treated and
Conclusion and Clinical Relevance—Results indicated
that topical application of cryotherapy significantly
reduced core SDFT temperature in standing
sedated horses. Temperatures achieved in vivo during
cold treatment were not detrimental to the in vitro
viability of tendon cells. (Am J Vet Res 2003;64:835–844)
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)