CHARACTERIZATION OF LACTIC ACID TRANSPORT IN BOVINE ARTICULAR CHONDROCYTES
+*Wilkins, R (A-Arthritis Research Campaign); *McClure, B; *Browning, J (A-Martin Wronker Trust)
+*University of Oxford, Oxford, UK. University Laboratory of Physiology, Parks Road, Oxford, OX1 3PT, UK, +44-1865-282164, Fax: +44-1865-272488,
[email protected]
have been used here to assess the properties of a membrane transport system
which would be expected to operate as an efflux pathway in vivo.
Addition of increasing concentrations of lactic acid elicited a saturable
intracellular acidification, indicative of a carrier-mediated process. This effect
was inhibited by the well-characterised inhibitor of MCT, α-CHC, confirming
that H+-lactate- cotransport mediated by this transporter is responsible for the
acidification observed. The kinetic parameters of the MCT-mediated
acidification are those of a high-capacity system (high Vmax) with low affinity
(high Km) for lactate-. Such properties are consistent with the MCT4 isoform,
previously described in other glycolytic tissues [3], and are well-suited to the
large quantities of lactic acid which must efflux from the anaerobic
chondrocyte. The significant acidification of steady-state pHi which was
observed upon addition of α-CHC indicates that there is a high basal level of
lactic acid extrusion from these cells under resting conditions and is consistent
with the high rates of acid production which have been reported by other
workers [1]. This study is a first step towards the characterisation of a
membrane transport process contributing to matrix pH, the activity of which
will therefore dictate in part cartilage turnover.
Figure 1 (a)
7.4
pH
7.2
untreated
7.0
10mM α-CHC
1mM α-CHC
control
6.8
0
50
100
150
200
Time (s)
(b)
-1
Acid Flux (mM min )
15
10
5
0
0
5
10
15
20
7.5
10.0
[lactic acid] (mM)
Figure 2
10.0
-1
Acid Flux (mM min )
INTRODUCTION
The avascular environment of the articular chondrocyte dictates that
metabolism is largely by anaerobic pathways, with the consequent production
of large quantities of lactic acid. The lactic acid produced by chondrocytes
must diffuse from the cells, through the extracellular matrix to the synovial
fluid and thereafter to capillaries and the systemic circulation. There are
reports of high, graded concentrations of lactic acid within the cartilage matrix
[1], and we have previously described inhibition of matrix turnover by
extracellular lactic acid [2].
At physiological pH values, lactic acid exists predominantly as the ionic
species H+ and lactate-. These charged lipophobic species will not readily
cross the plasma membrane of cells, and their exchange between the
cytoplasm and extracellular solution is commonly mediated by a family of
membrane-bound H+-lactate- cotransport carrier proteins, called the
monocarboxylate transporters, or MCT. To date, seven MCT isoforms have
been reported, with tissue-specific distribution. For highly glycolytic tissues,
such as 'white' skeletal muscle fibres and leukocytes, MCT-4 is the
predominant isoform. This protein mediates H+-lactate- efflux from the cell,
and demonstrates a low affinity for lactate- and a high transport capacity,
properties appropriate for cells producing large quantities of the substrate [3].
In the present study, the lactic acid transport properties of articular
chondrocytes have been characterised. The efflux of lactic acid - along with
fixed charged density and the operation of other H+ extrusion proteins - will
be an important determinant of matrix pH, and hence macromolecular
turnover.
METHODS
Chondrocytes were isolated from bovine metacarpophalangeal cartilage using
collagenase (0.8mg ml-1, 18h) in DMEM. Intracellular pH (pHi) was measured
in cells loaded with the fluorescent indicator BCECF (BCECF-AM, 10µM,
37°C, 30min) using a spectrofluorimeter (EX = 490nm/439nm, EM = 535nm).
The ratio 490nm/439nm was calibrated using nigericin (3µM) in a high-K+
solution [4]. Cells (1 × 106 ml-1) were suspended in a HEPES-buffered saline
(pH 7.4) and pHi recorded at steady-state, or following the addition of lactic
acid (1-20mM) to the extracellular solution. The effects of treatment with the
MCT inhibitor α-cyanohydroxycinnamic acid (α-CHC, 1-10mM) on steady-state
pHi and on the response to lactic acid addition were also investigated. In
experiments using α-CHC, the fluorescent signal was corrected for the
concentration-dependent influence of the inhibitor on the fluorescent properties of
BCECF. pHi changes were converted to acid equivalent fluxes (JH, mM min-1, ±
SEM, n ≥ 3) using the equation JH = ∆pHi/∆t × buffering power (βi), where βi = 43.3pHi + 337 [5].
RESULTS
Chondrocyte steady-state pHi was 7.1 - 7.2, consistent with previous reports
for this cell type [4]. Addition of lactic acid (1-20mM) produced a rapid
intracellular acidification (Figure 1a) which increased in magnitude with
increasing concentrations of lactic acid, and saturated at the highest doses
tested (Figure 1b). These data were used to determine Vmax and Km values for
the influx, which were 19.2 ± 2 mmol (l cells)-1 min-1 and 10.5 ± 2.05mM
respectively.
For cells at steady-state pHi, treatment with α-CHC (1-10mM) produced an
acidification, which was dependent on the concentration of α-CHC used
(Figure 2). Treatment with α-CHC reduced the magnitude of acidification
induced by addition of 10mM lactic acid (Figure 1b), with increasing
concentrations of the inhibitor producing increasingly large effects.
DISCUSSION
For a predominantly anaerobic cell type, there must be an effective efflux
from the cell of the large quantities of lactic acid produced if pHi is to be
maintained. Given that the pKa of lactic acid means that it exists almost
entirely as H+ and lactate- ions at physiological pH, a carrier-mediated
transport of these lipophobic species must be responsible for the efflux of the
acid [3]. In this study, the pHi changes arising upon addition of lactic acid or
of the MCT inhibitor α-CHC have been characterised in articular
chondrocytes using an H+-sensitive dye. Hence, the influx of H+ and lactate-
7.5
5.0
2.5
0.0
0.0
2.5
5.0
[α
α -CHC] (mM)
REFERENCES
[1] Lee, R.B. & Urban, J.P.G. (1997) Biochem. J. 321:95-102; [2] Wilkins,
R.J. & Hall, A.C. (1995) J. Cell. Physiol. 164:474-481; [3] Price, N.T.,
Jackson, V.N. & Halestrap, A.P. (1998) Biochem. J. 329:321-328; [4]
Wilkins, R.J. & Hall A.C. (1992) Exp. Physiol. 77:521-524; [5] Browning,
J.A. & Wilkins, R.J. (1998) J. Physiol. 506P:129.
ACKNOWLEDGMENTS
Work funded by the Arthritis Research Campaign and Royal Society, UK.
Poster Session - Cartilage Cell Biology - VALENCIA D
46th Annual Meeting, Orthopaedic Research Society, March 12-15, 2000, Orlando, Florida
0925