Journal of Orthopaedic Research 1587.93 The Journal of Bone and Joint Surgery, Inc 0 1997 Orthopaedic Research Society

Nitric Oxide and G Proteins Mediate the Response of Bovine Articular Chondrocytes to Fluid-Induced Shear

*P. Das, *D. J. Schurman, and *tR. Lane Smith

*Orthopaedic Research Laboratory, Division of Orthopaedic Surgery, Department of Functional Restoration, Stanford, and fRehabilitation Research and Development Center, Department of Veterans Affairs Palo Alto Health Care System,

Palo Alto, California, U S A .

Summary: Mechanical loading alters the metabolism of articular cartilage, possibly due to effects of shear stress on chondrocytes. In cultured chondrocytes, glycosaminoglycan synthesis increases in response to fluid- induced shear. This study tested the hypothesis that shear stress increases nitric oxide production in chondro- cytes, and nitric oxide then influences glycosaminoglycan metabolism. Inhibitors of nitric oxide synthase, G proteins, phospholipase C, potassium channels, and calcium channels were also analyzed for effects on nitric oxide release and glycosaminoglycan synthesis. Fluid-induced shear was applied to primary high-density monolayer cultures of adult bovine articular chondrocytes using a cone viscometer. Nitric oxide release in chondrocytes increased in response to the duration and the magnitude of the fluid-induced shear. Shear- induced nitric oxide production was reduced in the presence of nitric oxide synthase inhibitors but was unaffected by pertussis toxin, neomycin, tetraethyl ammonium chloride, or verapamil. The increase in glycos- aminoglycan synthesis in response to shear stress was blocked by nitric oxide synthase inhibitors, pertussis toxin, and neomycin but not by tetraethyl ammonium chloride or verapamil. The phospholipase C inhibitor, neomycin, also decreased glycosaminoglycan synthesis in the absence of flow-induced shear. As studied here, shear stress increased nitric oxide production by chondrocytes, and the shear-induced change in matrix mac- romolecule metabolism was influenced by nitric oxide synthesis, G protein activation, and phospholipase C activation.

In diarthrodial joints, articular cartilage experi- ences a variety of mechanical conditions including stresses, strains, and pressures that are generated dur- ing normal daily activity (32) . Mechanical loads are distributed within the joint through a specialized ex- tracellular matrix that derives tensile strength from type-I1 collagen fibrils (28) and compressive resilience from aggregating proteoglycans and water (25). Long- term stability of the cartilage matrix depends in part on the response of chondrocytes to mechanical load- ing (13,14,40,48).

Mechanical forces play an important role in nor- mal cartilage homeostasis and in the development of disease (5,33,44). Insufficient or excessive loads lead to patterns of cartilage degradation that resemble human disease (43,4935). Carter et al. have hypothe- sized that degeneration of articular cartilage is ac-

Received September 8,1995; accepted October 9,1996. Address correspondence and reprint requests to R. L. Smith

at Orthopaedic Research Laboratory, R144, Rte3, Stanford Uni- versity Medical Center, Stanford, CA 94305-5341, U.S.A. E-mail: mu.rls@forsythe.stanford.edu

Presented in part at the 41st Annual Meeting of the Ortho- paedic Research Society, Orlando, Florida, U.S.A., February 13- 16,1995.

celerated by intermittent shear stresses and inhibited by intermittent hydrostatic pressure (5) . In human os- teoarthritis and in animal models of osteoarthritis, changes in cartilage that are characteristic of early stages of the disease include increased glycosamino- glycan synthesis, proteoglycan size, and expression of novel proteoglycan epitopes (6,41,47).

We have shown in an in vitro fluid-flow model that shear stress alters chondrocyte cell shape and align- ment, increases glycosaminoglycan synthesis, and enlarges proteoglycans and glycosaminoglycan side chains (51). Applying shear stress to chondrocytes also increases prostaglandin E2 release and mRNA levels of tissue inhibitor of metalloproteinase (51). Hu- man articular chondrocytes exposed to flow-induced shear exhibit increased expression of interleukin-6 (IL- 6) mRNA and protein (30). Chondrocytes that are freshly isolated from osteoarthritic cartilage also show increased levels of IL-6 expression (30).

The cellular mechanisms underlying the response of articular chondrocytes to shear stress are unknown. In endothelial cells, shear stress stimulates nitric oxide synthesis (3,29,45,46), and the nitric oxide then modu- lates cell metabolism. Induction of nitric oxide release in the endothelial cell involves either activation of a

87

P: DAS ET AL.

50-

l), tumor necrosis factor-a (TNF-a), endotoxin (39, 0 Control 52,54) and Staph factor (50). In chondrocytes, nitric

Shear T oxide activates neutral metalloproteinase activity (34) and inhibits IL-lP induction of prostaglandin E2 and IL-6 (16) and synthesis of IL-1 receptor antagonist (42). The inducible calcium-independent nitric oxide synthase is expressed in human articular chondrocytes (7,23), and its expression increases in osteoarthritic chondrocytes (9).

This study tested the hypothesis that articular chon- drocytes would release nitric oxide as a function of the

*

0 2 4 8 1 6 2 4

Time (hr)

FIG, 1. Time-dependent release of nitric oxide from articular chon- drocytes in response to shear. Shear stress was applied to the cells at an angular velocity of 200 rpm for varying intervals of time. Control cells were not exposed to shear. The concentration of nitrite in the culture medium was measured as an indicator of nitric oxide release using the Griess reaction, as described in the Meth- ods section. The figure represents means and SEM of the nitrite concentrations. (N = 10: *p < 0.01 relative to unsheared controls.) Each experiment was performed three times, with three or four culture plates for each trial.

pertussis toxin refractory G protein and phospholipase C (20) or potassium efflux and calcium influx (38) leading to activation of nitric oxide synthase. However, endothelial nitric oxide production in response to sus- tained shear does not require calcium/calmodulin or G protein activation (19).

In a variety of other cell types, including chon- drocytes, nitric oxide functions as an autocrine or paracrine regulatory signal (31,36,37). Chondrocytes release nitric oxide in response to interleukin-1 (IL-

. T

0 5 0 100 200

Cone Angular Velocity (rprn)

FIG. 2. Effects of magnitude of shear stress on nitric oxide release. Shear stress was applied to the cells at angular velocities of 0 (unsheared controls), 50,100, or 200 rpm for 24 hours. The concen- tration of nitrite in the culture medium was measured as described in the Methods section. The figure represents means and SEM of the nitrite concentrations. N = 6 for 0 and 200 rpm, n = 3 for 50 and 100 rpm; *p < 0.05 relative to unsheared controls.

duration and magnitude of shear stress. In addition, the effects of inhibitors of nitric oxide synthase, G proteins, phospholipase C, and potassium and calcium channels on the induction of nitric oxide release and on the shear stress-induced increase in glycosamino- glycan synthesis were characterized.

MATERIALS AND METHODS Cell Culture

Articular cartilage was removed under sterile conditions from the radiocarpal joints of 5 to 7-year-old bovines and dissociated overnight at 37OC in 20 ml of Dulbecco’s modified Eagle medium (GIBCO, Grand Island, NY, U.S.A.) with 50 &ml gentamicin and 0.6 mg/ml each Class 2 and Class 4 bacterial collagenase (Wor- thington Chemical, Freehold, NJ, U.S.A.). Subsequently, dissoci- ated cells were diluted 2.7-fold in Dulbecco’s phosphate buffered saline without Mg2+ and CaZ+ and were collected by centrifugation at 450 g for 17 minutes. Cells were washed twice in Dulbecco’s phosphate buffered saline without Mg2’ and CaZ’, washed once in Dulbecco’s modified Eagle medium, resuspended in 10 ml of Dul- becco’s modified Eagle medium, and then filtered through Nitex (Tetko, Monterey Park, CA, USA). The cells were then plated at a concentration of lo6 cells per plate on 100 mm dishes and were cultivated in Dulbecco’s modified Eagle medium-Ham’s F12 (1:l) medium (GIBCO) with 25 pg/ml gentamicin (Sigma Chemical, St. Louis, MO, U.S.A.) and 10% fetal bovine serum (GIBCO). Under these plating conditions, the chondrocytes typically reached con- fluence between days 5 and 7, with an average of 5 X 106 cells per plate. After 7 days, the medium was changed to serum-free Dul- becco’s modified Eagle medium-Ham’s F12 (1:l) containing 25 pg/ml gentamicin, supplemented with selenium and liposomes. Af- ter 48 hours in serum-free medium, the cells were exposed to fluid-induced shear.

Application of Flow-Induced Shear Stress Shear stress was applied to the cells by exposure to continuous

laminar fluid flow in a cone viscometer modeled after the device described by Bussolari et al. (4). Briefly, a 95 mm diameter cone having a cone angle of 0.5” was rotated in the culture medium, and the rate of rotation was maintained and monitored with an elec- tronic motor controller. The tip of the cone was fixed 1 mm above the bottom of the culture plate. Mechanical restraints prevented plate movement. As described by Bussolari et al. (4), this system applies a nearly uniform level of shear stress across the plate. At the angular velocities used for this study, the culture medium ex- hibits a predominantly azimuthal or concentric flow, accompanied by a radial secondary flow with little or no turbulence.

The levels of shear stress produced by the 95 mm diameter cone rotating at different speeds were calculated as described pre- viously. When the cone is rotated at 200 rpm, the flow-induced shear ranges from 1.64 Pa near the center of the cone to 1.93 Pa at the edge of the cone. Similarly when the cone is rotated at 50

3 Orthop Res, Vol. 15, No. I , 1997

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SHEAR-INDUCED NtTRtC OXtDE RELEASE BY CHONDROCYTES 89

T 0 Control

N o n e L-NYA

lnh lb l tora

FIG. 3. Inhibition of shear-induced nitric oxide release by nitric oxide synthase inhibitors. Shear stress was applied to the cells at 200 rpm for 24 hours. Both sheared and control cultures were treated without (n = 5) and with 1 mM NG-methyl-L-arginine (L- NMA) (n = 7). The concentration of nitrite in the culture medium was measured as described in the Methods section. The figure represents means and SEM, expressed as percentages of the ni- trite level for cells sheared without inhibitors (**p < 0.05 relative to cells sheared without inhibitors). One millimolar ~-N~-( l - imino- ethy1)ornithine hydrochloride gave results similar to those of 1 mM L-NMA, except that baseline levels of nitric oxide release were observed (2-3.5 pM with and without exposure to fluid- induced shear).

and 100 rpm, the flow-induced shear ranges from 0.41 to 0.42 Pa and from 0.82 to 0.87 Pa, respectively. Rotation rates higher than 200 rpm were not used since these levels dislodged the cells and caused turbulence and loss of culture medium. No change in tem- perature was observed for the sheared cultures.

The cells were exposed to flow-induced shear for varying lengths of time, ranging from 2 to 24 hours. Control cells were not exposed to shear but were maintained under identical culture conditions. In some experiments, both control and experimental cells were treated with one of the following inhibitors: the potas- sium-channel blocker tetraethyl ammonium chloride (3 mM), the calcium-channel blocker verapamil hydrochloride (10 mM), the G protein inhibitor pertussis toxin (100 ng/ml), the phospholipase C inhibitor neomycin sulfate (5 mM, buffered with 30 mM HEPES), or the nitric oxide synthase inhibitors NG-methyl-L-arginine (L- NMA, 1 m M ) and l-N5-(~-iminoethyl)ornithine hydrochloride (1 mM) (all Sigma Chemical).

Quantification of Nitric Oxide Release The concentration of nitrite, the stable end-product of nitric

oxide oxidation, was used as an indicator of nitric oxide release. Nitrite concentration in the culture medium was measured spec- trophotometrically using the Griess reaction with sodium nitrite as the standard (10). Three hundred microliters of culture medium was removed and replaced with an equal volume of fresh medium in the culture dishes at designated times. Collected samples were incubated with 150 pl of 1% sulfanilamide and 150 p1 of 0.1% N-1-naphthyl-ethylenediamine dihydrochIoride (Sigma Chemical) for 10 minutes for measurement of absorbance at 550 nm.

Quantification of Glycosaminoglycan Synthesis Five microcuries per milliliter [35S]04 as sulfuric acid (pur-

chased as 185 MBq/ml; New England Nuclear, Wilmington, DE, U.S.A.) was added to the control and experimental cultures at the

beginning of each test. After 24 hours of exposure to shear, gly- cosaminoglycan synthesis was determined by measuring the in- corporation of [35S]04 into cetylpyridinium chloride-precipitable material (8). Aliquots of culture medium (0.2 ml) were added to 0.2 ml of a solution of 0.5% cetylpyridinium chloride and 1.6 ml of a solution of 0.031 M Na,SO, and 25 pg/ml chondroitin sulfate. The mixture was then vortexed and incubated at 37°C for 1 hour. The precipitates were collected on glass fiber filters (2.5 cm GFlA filters; Whatman, Maidstone, England) and washed with 10 ml of an ice-cold solution of 0.025 M Na2S0,, 0.005% cetylpyridinium chloride, and 5 ml of ice-cold distilled water. The filters were placed in vials and the precipitates were solubilized with 0.5 ml of distilled water. Six milliliters of Opti-Fluor (Packard Instruments, Meriden, CT, U.S.A.) was added, and the radioactivity was quan- tified using a Beckman LS-7500 liquid scintillation counter (Beck- man Instruments, Fullerton, CA, U.S.A.).

In experiments that used neomycin sulfate, the ratio of labelled sulfate to total sulfate in the culture medium decreased, and con- sequently [35S]04 incorporation into glycosaminoglycan also de- creased. To avoid this interference, in some experiments 5 p 3 m l of [3H]glucosamine (purchased as 1.2 TBq/mmole; New England Nuclear) was added to the control and experimental cultures at the beginning of the test period, and the incorporation of [3H]gluco~- amine into glycosaminoglycan was measured as described above.

Statistical Analysis One-way analysis of variance (ANOVA) and Student's two-

sample t test (two-tailed) were used for statistical comparisons with p < 0.05 considered to be significant.

RESULTS Articular chondrocytes showed an increase in nitric

oxide release with the duration of fluid-induced shear (Fig. 1). A 5-fold increase in nitric oxide release in the

- L -

El Sheared T

" N o n e TEA Ver PTX Neo

l n h l b l t o r a FIG. 4. Shear-induced nitric oxide synthesis in the presence of tetraethyl ammonium chloride (TEA), verapamil hydrochloride (Ver), pertussis toxin (PTx) or neomycin sulfate (Neo). Shear stress was applied to the cells at 200 rpm for 24 hours. Both sheared and control cultures were treated with either no inhibitor (n = 15) or with 3 mM tetraethyl ammonium chloride (n = 4). 10 pM verap- amil hydrochloride (n = 7), 100 ng/ml pertussis toxin (n = 4), or 5 mM neomycin sulfate (n = 8). The concentration of nitrite in the culture medium was measured as described in the Methods sec- tion. The figure represents means and SEM, expressed as percen- tages of the nitrite level for cells sheared without inhibitors (*p < 0.05 relative to unsheared controls).

J Orthop Res, Vol. 15, No. 1, 1997

90 P. DAS ET AL.

2 2 5

T

None TEA

t

T

Ver

0 Control

Sheared

T T

PTx Neo L-NMA

Inhibiton

FIG. 5. Effects of shear and signal transduction inhibitors on gly- cosaminoglycan (GAG) synthesis. Shear stress was applied to the cells at 200 rpm for 24 hours. Both sheared and control cultures were treated with either no inhibitor (n = 10) or with 3 mM tetraethyl ammonium chloride (TEA) (n = 4), 10 F M verapamil hydrochloride (Ver) (n = 7). 100 ng/ml pertussis toxin (PTx) (n = 4), 1 mM NG-methyl-L-arginine (L-NMA) (n = 7), or 5 mM neo- mycin sulfate (Neo) (n = 5). Glycosaminoglycan synthesis was quantified by measuring 35S incorporation into material precipi- tated by cetylpyridinium chloride as described in the Methods section. The figure represents means and SEM of 35S incorpora- tion into glycosaminoglycan, expressed as percentages of that in unsheared controls (*p < 0.05 relative to unsheared controls). Sim- ilar results were obtained with 1 mM ~-N~-(l-iminoethyl)orni- thine hydrochloride, except that baseline levels of cetylpyridinium chloride-precipitable material were observed (unsheared con- trols + L-N~-[ 1-iminoethyl]ornithine hydrochloride, 4,709 2 1,503 dpm compared with shear-exposed + ~-N~-[l-iminoethyl]ornithine hydrochloride, 4,569 5 2,622 dpm). Only neomycin influenced baseline levels of glycosaminoglycan synthesis in control cultures.

exposed cultures occurred after 4 hours of shear com- pared with unsheared controls (p < 0.01). After 24 hours of exposure to shear, nitric oxide release was 18-fold greater than controls. Nitric oxide release also increased with the magnitude of fluid-induced shear stress (Fig. 2). At an angular velocity of 50 rpm (0.41- 0.42 Pa of shear stress), chondrocytes exposed to shear exhibited a 2-fold increase in the release of nitric ox- ide compared with unsheared controls after 24 hours (p < 0.01). At 100 rpm (0.82-0.87 Pa) and 200 rpm (1.64-1.93 Pa), a 6-fold (p < 0.05) and 17-fold (p < 0.05) increase in nitric oxide release occurred, respec- tively, when compared with the control cultures. The levels of nitrite released per 106 cells in response to exposure to fluid-induced shear at 200 rpm were in the range of 4.5-5.5 pg, whereas control levels were in the vicinity of 0.3 pg.

The competitive nitric oxide synthase antagonist L- NMA inhibited the shear-induced nitric oxide release (p < 0.05) (Fig. 3). Similar results were obtained with the irreversible inhibitor, L-NS-(1-iminoethy1)orni- thine hydrochloride, except that baseline levels of nitric oxide release were observed (2-3.5 pM). No sig-

nificant change in shear-induced nitric oxide release occurred in the presence of tetraethyl ammonium chlo- ride, verapamil, pertussis toxin, or neomycin (Fig. 4).

Fluid-induced shear increased glycosaminoglycan synthesis by 1.7-fold (p < 0.05) (Fig. 5). Neither tet- raethyl ammonium chloride nor verapamil altered the rate of glycosaminoglycan synthesis. In contrast, pertussis toxin, neomycin, L-NMA and L-NS-( l-imino- ethy1)ornithine hydrochloride inhibited the shear- induced glycosaminoglycan synthesis. The irreversible inhibitor, ~-N~-(l-iminoethyl)ornithine hydrochloride, reduced levels of cetylpyridinium chloride-precip- itable material to baseline (unsheared controls + L-N~- [1-iminoethyllornithine hydrochloride, 4,709 2 1,503 dpm compared with shear-exposed + L-N~-[I -imino- ethyllornithine hydrochloride, 4,569 2 2,622 dpm). In the presence of neomycin sulfate, the basal rate of 35S incorporation into glycosaminoglycan decreased by 15-fold. When 5 mM neomycin sulfate was added to the culture medium, the ratio of labeled sulfate to total sulfate decreased significantly, which interfered with the incorporation of labeled sulfate into glycos- aminoglycan. To rule out this interference and to ex- amine whether neomycin sulfate actually affects the basal rate of glycosaminoglycan synthesis, we also looked at the incorporation of [3H]glucosamine into glycosaminoglycan (Fig. 6). In the absence of any in- hibitors, shear stress induced an increase in PHIglu- cosamine incorporation into glycosaminoglycan (p < 0.05). The addition of neomycin reduced the [3H]glu- cosamine incorporation into glycosaminoglycan in the

T 0 Control

0 Sheared

None Neo inhibitors

FIG. 6. Effect of neomycin (Neo) on glycosaminoglycan (GAG) synthesis in the presence and absence of shear. Shear stress was applied to the cells at 200 rpm for 24 hours. Both sheared and control cultures were treated either with no inhibitor or with 5 mM neomycin sulfate. Glycosaminoglycan synthesis was quanti- fied by measuring (3H]glucosamine incorporation into material precipitated by cetylpyridinium chloride as described in the Meth- ods section. The figure represents means and SEM of [3H]glu- cosamine incorporation into glycosaminoglycan. N = 4; *p < 0.05 relative to unsheared controls and #p < 0.05 relative to cultures without neomycin.

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SHEAR-IND UCED NITRIC OXIDE RELEASE 3 Y CHOND RUCYTES 91

controls by 4-fold (p < 0.05) and prevented shear- induced increase of glycosaminoglycan synthesis.

DISCUSSION This study demonstrated that shear stress induced

the release of nitric oxide from primary monolayer cultures of bovine articular chondrocytes and that ni- tric oxide mediated the metabolic response of the cells to shear stress. The increased release of nitric oxide by the chondrocytes varied with the duration and the magnitude of shear stress. The nitric oxide synthase inhibitors, L-NMA and L-NS-( 1-iminoethy1)ornithine hydrochloride, inhibited shear-induced nitric oxide synthesis as well as increased glycosaminoglycan syn- thesis. The increase in glycosaminoglycan synthesis by fluid flow-induced shear observed in this study was comparable to that reported previously (51).

Chondrocytes release nitric oxide in response to a variety of stimuli such as IL-1, TNF-a, endotoxin (39,52,54), and Staph factor (50). Nitric oxide medi- ates IL-1p-induced inhibition of glycosaminoglycan synthesis (15,22) but not IL-1 or Staph factor-induced proteoglycan loss (22,50). In this study, increased glycosaminoglycan synthesis in response to shear- induced release of nitric oxide suggests that the effects of nitric oxide on chondrocytes may vary with the stimulus. The potential for pleiotropic effects of nitric oxide on chondrocytes is consistent with observations from other studies. Synthesis of gelatinase and pros- taglandin E2 is suppressed by L-NMA when chon- drocytes are activated by IL-1 alone, but they are enhanced when the cells are activated by a combina- tion of IL-1 and other cytokines (52). Nitric oxide production in response to IL-1 and transforming growth factor-f! also varies with the differentiation state of chondrocytes (l), and nitric oxide can cause apoptosis in chondrocytes (2).

In cartilage, nitric oxide may act as a localized or a widespread messenger depending on the nature and extent of the stimulus. As a small, neutral molecule in aqueous phase (21,24), nitric oxide can diffuse rapidly through the matrix. The distance over which nitric oxide functions depends on its diffusion rate and half- life; Lancaster proposed that nitric oxide acts primar- ily as a paracrine signal (21). In monolayer cultures of chondrocytes, the paracrine nature of the nitric oxide effects may be exacerbated due to rapid dissemination of the nitric oxide into the culture medium, enabling all cells to be exposed to the compound. In v i m , the extracellular matrix may influence distribution of ni- tric oxide to chondrocytes such that shear stress re- sults in locally high concentrations of nitric oxide and focal regions of altered cartilage metabolism.

This study investigated signal transduction events that may mediate shear-induced nitric oxide release. Our results show that nitric oxide release does not oc-

cur secondary to activation of a tetraethyl ammonium chloride-sensitive potassium channel, a verapamil- sensitive calcium channel, a pertussis toxin-sensitive G protein, or phospholipase C. These data suggest that a distinct pathway may underlie the shear-induced nitric oxide production by chondrocytes in v i m . This path- way may be similar to that for the sustained nitric oxide production by endothelial cells that does not re- quire calcium/calmodulin or G protein activation (19).

Other signal transduction events that may mediate shear-induced increases in glycosaminoglycan syn- thesis by chondrocytes were also investigated here. Inhibitors of tetraethyl ammonium chloride-sensitive potassium channels or verapamil-sensitive calcium channels had no effect on glycosaminoglycan synthe- sis, whereas pertussis toxin, an antagonist of G, and Go proteins (12,20), inhibited this response. Therefore, ac- tivation of Gi or Go proteins appears to mediate the change in glycosaminoglycan synthesis in response to shear. Since pertussis toxin did not inhibit nitric oxide release, G protein activation may occur secondary to nitric oxide release, or G protein activation and nitric oxide release may occur independently in response to shear. However, both events appeared necessary for increased glycosaminoglycan synthesis in response to shear.

The phospholipase C antagonist, neomycin, inhib- ited the shear-induced increase in glycosaminoglycan synthesis and caused a 4-fold reduction in the rate of glycosaminoglycan synthesis in unsheared controls. Since neomycin reduced basal levels of glycosamino- glycan synthesis, the inhibition of the shear response by neomycin could be either because phospholipase C activation mediates the shear response, or because phospholipase C activity is necessary for glycosamino- glycan synthesis in chondrocytes. Neomycin did not affect nitric oxide release in response to flow-induced shear; this indicates that phospholipase C activation may be secondary to or independent of nitric oxide release. It is also possible that neomycin may have rendered the chondrocytes unresponsive to shear stress through a generalized inhibition of glycosamino- glycan metabolism.

Inhibition of receptor-mediated phospholipase C activation by pertussis toxin in some cell types (12) implies that certain members of the pertussis-sensitive Gi and Go protein families activate phospholipase C. Thus, stimulation of glycosaminoglycan synthesis by shear may involve activation of a Gi or Go protein, leading to phospholipase C activation. Phospholipase C activation generates diacylglycerol, which in turn can activate protein kinase C (35). Matsubara et al. reported that a protein kinase C activator increases glycosaminoglycan synthesis in cultured chondrocytes (26). Therefore, protein kinase C activation secondary to phospholipase C activity may mediate the shear-

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92 l? DAS ET AL.

induced increase in glycosaminoglycan synthesis. In this study, the levels of shear stress that induced

nitric oxide release ranged from 0.41 to 1.93 Pa. It is currently not possible to determine the precise levels of shear stress in the cartilage of a normal loaded joint (44). In normal joint loading, the cartilage extracellu- lar matrix ameliorates the shear stress imparted to chondrocytes (18). As studied in this in virro model, articular chondrocytes in the primary high-density monolayer cultures were exposed to fluid-induced shear stress without a fully developed, mature articu- lar cartilage matrix to protect the cells as might occur in a mechanically loaded joint. In vivo, the added pre- sence of the cartilage extracellular matrix may dictate that significantly higher forces be applied to generate the same level of distortion that the cells achieved in this model system (18).

It is important to emphasize that cells in monolayer culture may respond differently to testing than chon- drocytes localized in siru within the cartilage extracel- lular matrix. A number of factors may influence the in v i m behavior; these may be linked to organization of the cellular cytoskeleton and to the presence of focal adhesions that bind the cells to the tissue culture sub- stratum. In addition, the application of fluid-induced shear on a continuous basis may not adequately rep- resent an intermittent type of fluid-flow that might occur in vivo. Nevertheless, the system represents one model by which chondrocytes may be uniformly ex- posed to shear as a small population of cells.

Since mechanical forces, including shear stress, are hypothesized to contribute to joint diseases such as osteoarthritis (44), the regulatory mechanisms ob- served here, i.e., nitric oxide release and G protein activation, may influence cartilage degeneration. In- creased nitric oxide synthase expression in human os- teoarthritic chondrocytes (9) and elevated nitrite and nitrate levels in synovial fluid from arthritic joints (11,17) support this hypothesis. Furthermore, in ani- mal models, nitric oxide synthase inhibitors suppress development of inflammatory arthritis and adjuvant arthritis (27,53). As demonstrated here, shear stress stimulated chondrocytes to release nitric oxide that, in turn, regulated matrix metabolism; these observations suggest that shear stress acts in part through nitric oxide-dependent pathways to modulate articular car- tilage metabolism.

Acknowledgmenk We thank G. Kajiyama, B.S., for excellent technical assistance and J. P. Cooke, M.D., and D. R. Carter, Ph.D., for helpful suggestions. This work was supported in part by a Hughes Foundation Undergraduate Research Opportunities Grant to P.D., the Orthopaedic Research Fund, Stanford Univer- sity, and by the Department of Veterans Affairs (Merit Review Project A857RO) and the Rehabilitation Research and Develop- ment Center, Department of Veterans Affairs Palo Alto Health Care System, Palo Alto, California.

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