OSMOTIC STRESS INITIATES INTRACELLULAR CALCIUM WAVES IN CHONDROCYTES THROUGH
EXTRACELLULAR INFLUX, THE INOSITOL PHOSPHATE, AND A G-PROTEIN MEDIATED PATHWAY
*Erickson, G (A-NIH); +*Guilak, F (A-NIH)
+*Duke University Medical Center, Box 3093/Durham, NC 27710. Box 3093/Durham, NC 27710, 919-684-2521, Fax: 919-681-8490, [email protected]
Introduction: The biophysical environment of chondrocytes plays an
important role in the health, turnover, and homeostasis of articular cartilage.
Under normal physiologic loading, chondrocytes are exposed to a complex
and diverse array of biophysical signals, including mechanical and osmotic
stresses, fluid flow and pressures [4]. Due to the charged and hydrated nature
of the extracellular matrix, mechanical compression causes exudation of
interstitial fluid in cartilage, which alters the osmotic environment of the
chondrocytes. Confocal microscopy studies have shown that chondrocytes
lose or gain volume in response to tissue compression [4] or changes in
extracellular osmolarity [3]. The active process of volume recovery
subsequent to osmotic shock has been shown to initiate intracellular signaling
cascades [2], which may in turn alter cellular metabolism [6]. Although the
mechanisms of intracellular signaling in response to osmotic stress are not
fully understood, it has been hypothesized that intracellular transients and
oscillations of calcium ion (Ca2+) are involved. The objective of this study was
to examine the hypothesis that osmotic stress initiates a transient increase in
the concentration of intracellular calcium ion ([Ca2+]i), and to determine the
mechanisms of Ca2+ mobilization in isolated chondrocytes exposed to hypoand hyper-osmotic stress.
Materials and Methods: Cell Culture and Isolation: Articular chondrocytes
were isolated from knee cartilage of skeletally mature pigs (N=7) immediately
after sacrifice. Cells were isolated with sequential pronase and collagenase
digestion, seeded on glass and cultured in DMEM medium with 10% fetal
bovine serum overnight in a humidified atmosphere at 37oC, 5% CO2.
Osmotic Stress: All tests were performed within 12 hrs of isolation. Solutions
of varying osmolalities (150, 250, 450, and 550 mOsm) were prepared by
adding sucrose or deionized water to the iso-osmotic solution (DMEM, 310
mOsm). All experiments were performed at 37oC. Calcium Imaging: Cells
were incubated for 20 min. prior to testing with Fluo-3-AM (15µM) and FuraRed-AM (20µM). Fluorescence ratio imaging was performed using dualwavelength laser scanning confocal microscope (LSM 510, Zeiss Inc.) [5]. A
series of fluorescence and differential interference contrast (DIC) images were
recorded at a scan rate of 0.33 Hz for 10 min. to determine [Ca2+]i and cell
volume, respectively. Inhibitors of Ca2+ Mobilization: In order to determine
the source of Ca2+ mobilization in response to osmotic stress, the following
inhibitors were used: gadolinium (10µM, which blocks mechano-sensitive
channels), Ca2+-free media with EGTA (10mM, a Ca2+ chelator), U73122
(10µM, a phospholipase C (PLC) inhibitor), thapsigargin (3µM, inhibits CaATPases on endoplasmic reticulum), heparin (blocks inositol trisphosphate
(IP3) mediated calcium release), and pertussis toxin (PTX, 1µg/ml, ADP
ribosylates G-proteins). Cells were exposed to inhibitors for 30 min. prior to
dye loading, except for EGTA, which was introduced for 15 min. prior to
testing and PTX which was introduce 6 hrs prior to testing. Heparin was coelectroporated into cells with similarly sized cascade blue dextran 30 min.
prior to testing and confirmed via UV fluorescent microscopy.
Results: Chondrocyte Response to Osmotic Stress: Control experiments
demonstrated that replacement of the iso-osmotic medium with an identical
solution resulted in no change of [Ca2+]i or cell volume. Replacement of the
iso-osmotic medium by hypo- or hyper-osmotic medium caused significant
changes in cell volume as well as transient increases of [Ca2+]i. A typical Ca2+
transient that occurred following osmotic stimulation consisted of an initial
peak of [Ca2+]i which was often followed by several oscillations. A significant
increase in [Ca2+]i (10% above baseline) was observed at all levels of osmotic
stress. Over 95% of cells showed an increase in [Ca2+]i at 150, 250, or 450
mOsm, while at 550 mOsm, approximately 67% of cells responded with a
significant increase over baseline (Table 1). Pretreatment with gadolinium
blocked the increase of [Ca2+]i with hyper-osmotic stress, but had no effect
with hypo-osmotic stress. Ca2+-free medium eliminated [Ca2+]i increases in all
cases. Pretreatment with U73122, thapsigargin, heparin, and PTX significantly
decreased the percentage of cells responding to osmotic stress.
Ca2+
U73122 Thaps. Hep.
free
150
95.7 *
100
4.9 ** 48.7 **
79.2 ** 18.2**
250
100 *
100
2.7 ** 15 **
31.7 ** 0**
310
0
450
96.4 *
15 **
0 **
0 **
0 **
11.7**
550
66.7 *
15 **
0 **
0 **
0 **
0**
Table 1: Percentage of cells showing a significant increase in [Ca2+]i
*p<0.001 vs. 310mOsm control **p<0.005 vs. control group of same
osmolality, (n=30-40 per group, Chi-squared test).
Con.
Gadol.
PTX
57.7**
4.8**
0**
0**
Ca2+
U73122 Thaps. Hep.
PTX
free
150
56.8 *
85.4 ** 0 **
15 **
4.2 **
0**
38.5
250
96.9 *
96.4
0 **
10 **
2.4 **
0**
0**
310
0
450
96.4 *
12.5 ** 0 **
0 **
0 **
0**
0**
550
54.6 *
5 **
0 **
0 **
0 **
0**
0**
2+
Table 2: Percentage of cells exhibiting oscillations in [Ca ] *p<0.001 vs.
310mOsm control **p<0.005 vs. control group of same osmolality (n=30-40
per group, Chi-squared test).
Con.
Gadol.
Discussion: The results of this study indicate that both hypo- and hyperosmotic stress result in significant increases and oscillations in [Ca2+]i in
chondrocytes. These observations provide support for the hypothesis that
changes in the osmotic environment of the chondrocyte, secondary to
mechanical compression of the extracellular matrix, can potentially initiate
intracellular signaling pathways. The mechanisms inducing the initial peak
increase of [Ca2+]i differed significantly in response to hypo- or hyper-osmotic
stress. Treatment with gadolinium, a blocker of mechanosensitive ion
channels, abolished the response to hyper-osmotic stress but had no effect
with hypo-osmotic stress. The results suggest that mechano-sensitive ion
channels are not involved in the response to hypo-osmotic stress and are
consistent with the hypothesis that rapid swelling of the cell creates small
tears which allow entry of Ca2+ as well as other solutes [1]. This conclusion is
also supported by a more rapid time to onset of the first [Ca2+]i peak as
compared with hyper-osmotic stress (not shown). In all cases, the transient
increases and oscillations of [Ca2+]i were inhibited by removing extracellular
Ca2+ or by blocking Ca2+ release from intracellular stores, suggesting a
mechanism of a Ca2+-induced Ca2+ release consistent with the inositol
triphosphate (IP3) pathway. This finding was supported by the fact that
treatment with U73122 , thapsigargin, and heparin decreased or abolished
[Ca2+]i transients in all cases. Interestingly, our findings did not implicate a
role for IP3 in the initial peak of [Ca2+]i in response to hypo-osmotic stress. It
was also found that PTX significantly decreased the number of cells
responding to osmotic challenge implicating a possible role of G-proteins in
the induced calcium response. The findings of this study indicate that osmotic
stress may play an important role in intracellular signaling in response to
matrix compression by inducing transient increases of [Ca2+]i. Further study is
required to determine whether these intracellular signals are correlated to
reported changes chondrocyte metabolism [4].
References: [1] Baumann M and Grebe R Mol Mem Bio, 15(4):193-201,
1998. [2] Cohen DM Comp Biochem & Phys 117:291-9, 1996. [3] Deshayes
CMP et al. J. Physiol, 467:214P, 1993. [4] Guilak F et al. In:Basic
Orthopaedic Biomechanics, Ed. VC Mow and WC Hayes, Lippincott-Raven,
Phil., 1997. [5] Lipp P and Niggli E Cell Calcium 14:359-372, 1993. [6]
Urban et al. J Cell Phys 154:262-270, 1992.
Acknowledgments
Supported by NIH grants AR43876, AG15768, and GM08555.
Poster Session - Cartilage Cell Biology - VALENCIA D
46th Annual Meeting, Orthopaedic Research Society, March 12-15, 2000, Orlando, Florida
0923