alteredgolgi apparatus articular · altered golgi apparatus in hydrostatically loaded articular...

Annals of the Rheumatic Diseases 1993; 52: 192-198

Altered Golgi apparatus in hydrostatically loadedarticular cartilage chondrocytes

Jyrki J Parkkinen, Mikko J Lammi, Alpo Pelttari, Heikki J Helminen, Markku Tammi,Ismo Virtanen

AbstractObjectives Articular cartilage proteo-glycan content is controlled by jointloading. This study aimed to elucidate therole of hydrostatic pressure in thisregulation.Methods Primary cultures of chondro-cytes from bovine articular cartilage,grown on coverslips, were subjected to 5,15, or 30 MPa hydrostatic pressure,applied continuously or cyclically at 0-125or 0O05 Hz. The Golgi apparatus wasvisualised either by a fluorochromecoupled wheat germ agglutinin or bytransmission electron microscopy.Proteoglycan synthesis was studied by theincorporation of sulphur-35 labelledsulphate.Results After 30 MPa continuoushydrostatic pressure, the Golgi apparatuswas observed in a compact form with aconcomitant decrease in proteoglycansynthesis. The normal stacked appearanceofthe Golgi apparatus was no more visiblein the electron microscopy preparation ofthe pressurised chondrocytes. This effectwas reversible and was also noticed after15 MPa continuous load, though to aminozr extent. Cyclic pressures (5-30 MPa)caused no apparent change in the Golgiapparatus. The shape of some cellschanged to a more retracted form after 30MPa continuous pressure. Nocodazole,which causes disassembly of themicrotubules, blocked the compactinginfluence of pressurisation on the Golgiapparatus, and reduced proteoglycansynthesis to about half ofthe control level.Conclusions The packing of the Golgiapparatus is dependent on microtubulesand may contribute to the inhibition ofproteoglycan synthesis observed inarticular cartilage subjected to highhydrostatic pressure.

(Ann Rheum Dis 1993; 52: 192-198)

Mechanical forces play an important part inthe regulation of articular cartilage matrix.'Moderate running exercise increases theglycosaminoglycan content of articularcartilage,2 3whereas strenuous exercise has anopposite influence with proteoglycans reduced,particularly from the superficial zone.4 Lack ofload bearing results in a reduced proteoglycancontent in cartilage.' 6 The changes in

proteoglycan content lead to alterations in thebiomechanical properties of cartilage.7 Theatrophy following reduced loading and the lossof superficial zone proteoglycans followingstrenuous exercise may put articular cartilageat risk of permanent injury.During joint loading chondrocytes in

articular cartilage are first exposed to ahydrostatic pressure gradient which is followedby deformation and fluid flow. The latterevents are associated with streaming currentsand physicochemical changes in theextracellular tissue matrix. Of these factorsonly hydrostatic pressure can be investigatedindependently, because it can be applied oncells and tissues without simultaneous fluidflow or tissue deformation. When appliedexperimentally, its action is uniform in all partsof the tissue. The most striking effects ofhydrostatic pressure are exerted on sites ofphase changes such as the water/lipidinterphase in membranes.8 Hydrostaticpressure has been suggested to control proteinand proteoglycan synthesis in articularcartilage.9 10

Hydrostatic pressure has multiple effects oncellular processes.8 Most studies, however,have been performed on single cell organismsand few data are available on the effects ofhydrostatic pressure on mammalian cells.Relatively small pressures (5 MPa) stimulateNa-K ATPase activity in erythrocytes," buthigher pressures have inhibitory effects, e.g. onexocytosis'2 and protein synthesis.'3 Malignantcells may be more resistant to hydrostaticpressure than normal cells.'4The Golgi apparatus of the mammalian cell

is an organelle which consists of aninterconnected network of stacks of flattenedcistemae and tubular structures.'5 The Golgiapparatus is localised in a juxtanuclear positiontogether with the microtubule organisingcentre. Intact interphase microtubules areessential in maintaining the structural integrityand location of the Golgi apparatus as shownexperimentally by the aid of microtubuledisrupting agents, such as colchicine,nocodazole, and vinca alkaloids.'5 16 In cellstreated with these agents, the Golgi apparatusis dissociated into small cytoplasmic vesicles.Funtionally, the Golgi apparatus affects theconstruction, processing, and intracellulartargeting of synthetic products.

In chondrocytes, proteoglycan synthesisrequires a well coordinated assembly of theprotein and carbohydrate components. In ratchondrosarcoma cells, proteoglycan synthesis

Department ofAnatomy, UniversityofKuopio, PO Box1627, SF-70211 Kuopio,FinlandJ J ParkkinenM J LammiH J HelminenM TammiLaboratory of ElectronMicroscopy,University ofKuopio,PO Box 1627, SF-70211Kuopio, FinlandA PelttariDepartment ofAnatomy, Universityof Helsinki,Siltavuorenpenger 20,00170 Helsinki, FinlandI VirtanenCorrespondence to:Dr Parkkinen.

Accepted for publication28 October 1992

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Altered Golgi apparatus in hydrostatically loaded articular cartilage chondrocytes

from translation to secretion is accomplished in70-90 minutes, of which glycosaminoglycanchain elongation and sulphation in the Golgiapparatus takes about 15 minutes.'7 We haveearlier found that in short term loadingexperiments dynamic pressure appears tomodulate the post-translational events ofproteoglycan synthesis in articular cartilageexplants.10 In this study we have investigatedthe effects of hydrostatic pressure on thestructural organisation of the Golgi apparatusin cultured articular chondrocytes.

Materials and methodsCELL CULTURE

Primary bovine chondrocyte cultures wereestablished from 1 to 2 year old animals(Itikka-Lihapolar, Kuopio, Finland). Articularcartilage from the patellar surface of the femurwas minced into 1-2 mm3 pieces andsequentially digested by trypsin and crudecollagenase (Sigma, St. Louis, MO, USA).Cells (106) were plated on 35 mm Petri dishesand cultured in Basal Medium Eagle (BME)with 25 mM Hepes and Hanks's salts (GibcoLtd, Paisley, United Kingdom) supplementedwith 10% fetal calf serum (Gibco), 3 mMglutamine (Gibco), antibiotics (100 U/mlpenicillin and 100 ,ug/ml streptomycin, 2-5gg/ml amphotericin B) (Flow Laboratories,Irvine, United Kingdom) at 37°C. Forimmunostaining and electron microscopy,glass (diameter 13 mm) and plastic coverslips(Thermanox, Nunc Inc, Naperville, IL, USA)were used respectively. The medium waschanged every second day.

COVERING METHOD AND THE LOADINGAPPARATUS

After the cells reached confluence the disheswere filled by medium and sealed by a coveringof Surlyn 180 1-Bynel CXA 3048 bilayermembrane (thickness 90 ,um; Du Pont,Wilmington, DE, USA) after excluding all air.The membrane was attached to the rim of thePetri dish with Jet Melt 3764 adhesive (3MUnited Kingdom plc, Bracknell, UnitedKingdom).The loading apparatus is described in detail

elsewhere.'8 Briefly, the apparatus consists oftwo water filled cylindrical chambers one ofwhich serves as the control, non-pressurisedreference chamber and the other as thepressure chamber. During the experiments thedishes were immersed in the prewarmed(37°C) distilled water. The pressure developedin the test chamber could be selected between4 and 32 MPa. The loading and unloadingperiods in the cyclic pressure mode were freelyselectable.

LOADING PROCEDURES

The chondrocytes on glass coverslips forimmunostaining were exposed to 5, 15, or 30MPa hydrostatic pressure with three cyclealternatives. In the first, the cells were exposedto continuous pressure during the whole

experiment- that is, for two hours. The otherloading procedures were cyclic: four secondspressure on, followed by four seconds nopressure (0-125 Hz), or one second pressure onthen 19 seconds no pressure (0-05 Hz) for atwo hour period.To study proteoglycan synthesis, dishes

without the coverslips were filled by mediumand covered by the membrane as describedearlier. The chondrocyte cultures were labelledwith 5 gCi/ml sulphur-35 labelled sulphate(carrier free, Amersham International, LittleChalfont, United Kingdom) injected throughthe membrane. The membrane was patched bya small piece of tape and parallel dishes wereimmediately incubated at 37°C in the pressureand reference chambers. The cultures (seven ineach chamber) were pressurised continuouslyat 30 MPa for two hours. After loading thedishes were put on ice and the incorporatedsulphur-35 radioactivity was isolated byPD-10 columns (Sephadex G-25, Pharmacia,Sweden) and measured by liquid scintillationcounter (LKB, Bromma, Sweden). The DNAcontent of each dish was determined by themethod of Kim et al.'9 For every dish thesulphate incorporation was calculated as pmol/ig DNA/h. The results were finally expressedas values related to the control level(pressurised/control ratio).

COLD TREATMENTAfter coverage with the Surlyn membrane,Petri dishes with coverslips were cooled on icefor 30 minutes. One dish was exposed to 30MPa continuous load for two hoursimmediately after the cold treatment and theother served as a control in the referencechamber. To confirm disruption of themicrotubules in cold treatment, one part of thesamples was fixed immediately after treatmentwith methanol at -20°C for five minutes. Themicrotubules were visualised by using theindirect immunofluorescence technique. Theprimary antibody was against (x tubulin andthe secondary antibody was fluoresceinisothiocyanate (FITC) conjugated antimouseIgG.

NOCODAZOLE TREATMENTS

Chondrocytes on coverslips were firstincubated with 10 iM nocodazole (Sigma)dissolved in dimethyl sulphoxide (DMSO) for90 minutes at 37°C. Some of the dishes werethen exposed to 30 MPa continuous load fortwo hours, whereas some served as controlswithout pressurisation.A series of dishes (10 in each group) treated

with nocodazole and controls with a similaramount ofDMSO (vehicle) were labelled with185 kBq/ml 35SO4 for 20 hours to show theinfluence of nocodazole on proteoglycansynthesis. After labelling, the cultures werehandled as described earlier.

In the third nocodazole experiment, 10dishes were covered by membrane as describedearlier and 10 ,uM nocodazole in DMSO wasinjected through the membrane. In nine

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control dishes the same amount ofDMSO wasadded. After 90 minutes of treatment, 185kBq/ml 35SO4 was injected and the dishes wereexposed to 5 MPa load with a 0 5 Hz cycle for20 hours. The membranes were patched by asmall piece of tape.

STAINING OF THE GOLGI APPARATUS

The chondrocytes on coverslips were fixedwith 3.5% paraformaldehyde for five minutesat room temperature. The coverslips weretreated with 0.1% Triton X-100 in phosphatebuffered saline for five minutes. Thechondrocytes were allowed to react withtetramethylrhodamine isothiocyanate con-jugated wheat germ agglutinin (TRITC-WGA; Vector Lab. Inc., Burlingame, CA,USA) to visualise the Golgi apparatus.20 ANikon Microphot-FXA (Nikon, Tokyo, Japan)microscope equipped with epi-illuminator andfilters for TRITC was used for photography onKodak T-MAX 400 film.

ELECTRON MICROSCOPYCells on plastic coverslips were fixed with 2%glutaraldehyde in 0-1 M sodium cacodylate(pH 7A4) at room temperature for one hour.Postfixation was performed in a mixture (1:1)of 2% glutaraldehyde in 0-I M cacodylatebuffer, pH 7 4, and 1% osmium tetroxide ins-collidine buffer, pH 7-3, for one hour at+4°C. After staining with 05% uranyl acetatefor 40 minutes, the chondrocytes were rinsedwith sodium cacodylate buffer supplementedwith 0-22 M sucrose for one hour, dehydratedin a graded ethanol series and embedded inLX-1 12 (Ladd Research Industries,Burlington, CA, USA) as small fragments lyingtangentially to the sectioning plane. Silvercoloured thin sections were cut withultramicrotome and stained with uranyl acetateand lead citrate. The sections were examinedand photographed in a JEOL JEM 1200EXtransmission electron microscope (JEOL Ltd,Tokyo, Japan).

ResultsIn these experiments the Golgi apparatus wasvisualised with TRITC-WGA, whichspecifically binds to sialic acid andN-acetylglucosamine residues, mainly presentin the Golgi apparatus after fixation anddetergent permeabilisation.20 In TRITC-WGA-stained chondrocytes a typical, bright,reticular, juxtanuclear staining pattern waspresent after paraformaldehyde fixation andTriton X-100 extraction. The Golgi apparatusformed a stacked structure polarised to onepole of the chondrocyte, leaving the other polelargely unstained (fig 1).The chondrocytes were subjected to various

loading procedures after reaching confluence.After continuous 30 MPa pressure the Golgiapparatus was packed into a clump butremained in the above-mentioned region of thecell (fig 2). The morphology of some cells wasalso altered to a more retracted form. Under

Figure 1 Chondrocytes from bovine articular cartilagecultured on coverslips, fixed with paraformaldehyde, andpermeabilised with Triton X-100. The Golgi apparatus wasstained with TRITC-WGA. The Golgi apparatus shows atypical reticular staining pattern polarising to one pole of thecell. Bar=5,um.

Figure 2 Articular cartilage chondrocytes as infig 1, butexposed to 30 MPa continuous pressure for two hours. TheGolgi apparatus was packed into a clump. Bar=S,um.

electron microscopic examination controlchondrocytes displayed the Golgi apparatus asa set of flattened cisternae arranged in a stack(fig 3A), but in pressurised cells the Golgiapparatus was disorganised and no stacks werefound (fig 3B). Sulphate incorporation, whichreflects the proteoglycan synthesis, wasdecreased by 18 (12%) (mean (SE)) from thecontrol values. If the 30 MPa continuous loadwas extended up to 20 hours, the synthesis ratedecreased by 36% (Lammi et al, unpublisheddata).The reversibility of the altered structure of

the Golgi apparatus was studied by fixation ofcoverslips 15, 30, 60, and 120 minutes afterwithdrawing the pressure. After 15 minutes norecovery was seen, but after 60 minutes theorganisation of the Golgi apparatus was fullyre-established (data not shown).

If the chondrocytes were exposed to cyclicloading with 30 MPa using 0 125 Hz (fig 4) or0 05 Hz loading regime for two hours, nostructural change in the Golgi apparatus wasobserved.

After a continuous 15 MPa pressure thepacking of the Golgi apparatus was also visible,

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Figure 3 Transmission electron microscopy vicw of the perinuclear area of chondrocytes in

the control culture (A) and after two hours continuous loading under 30 MPa pressure (B).T'he cells were fixed in 2% glutaraldehyde, posffixed in a mixture ofglutaraldehyde and

osmium tetroxide, and embedded in LX-1 12 resin. The sections were stained with uranylacetate and lead citrate. In control chondrocytes (A) the Golgi apparatus (arrows) was

organised in stacks near the centriole but in the pressurised cells numerous vesicles (arrows)

of various sizes were visible in the same region and no stacks of cisternae were visible (B).Bar=1 pim.

but not as striking and not in every cell as after30 MPa (fig 5). These morphologicalalterations did not appear when 15 MPa cyclicpressure (0'125 Hz, 0 05 Hz) was used. Thestructure of the Golgi apparatus showed nogross alterations after 5 MPa continuouspressure (fig 6).The microtubules in chondrocytes were

disrupted after half an hour of cold treatment(fig 7A). The typical radial staining pattern ofthe microtubules seen in control cells (data notshown) was absent following cold treatment.The morphology of the Golgi apparatusremained intact through this treatment (fig7B), however, and during the subsequentrearrangement period of the microtubules at37°C (fig 7C). Pressurisation at 30 MPaimmediately after cold treatment led toclumping of the Golgi apparatus into theperinuclear area as seen without cold treatment(fig 7D).Nocodazole, which reversibly and highly

specifically disrupts the microtubules, led tofragmentation and dispersion of the Golgiapparatus over the cytoplasm (fig 8A). Afterpressurisation of the nocodazole treatedchondrocytes, the Golgi apparatus remained

Figure 5 Chondrocytes as infig 1 but exposed to 15 MPacontinuous pressurefor two hours. Cell morphology issimilar to control cultures but the Golgi apparatus iscondensed toform a packed structure. Bar=5 pm.

Figure 4 Chondrocytes exposed to 30MPa cyclic Figure 6 Chondrocytes exposed to 5 MPa continuous(0-125 Hz) pressurefor two hours. No alterations in the hydrostatic pressure for two hours. The Golgi apparatusmorphology of the cell or the Golgi apparatus compared represents the normal staining pattern in culturedwithfig 1. Bar=5 m. chondrocytes as in fig 1. Bar=S,um.

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Figure 7 Chondrocytes kept at 0°Cfor 30 minutes before pressurisation. The cold treatment leads to disruption ofmicrotubules (A), but the Golgi apparatus is locatedjuxtanuclearly with the typical staining pattern. There were noalterations in the morphology of the cells (B). After cold treatment the cells were placed to 37°Cfor two hours (C). Thestaining pattern of the Golgi apparatus remains as in fig 7B. In continuously pressurised chondrocytes after 30 MPapressure for two hours at 37°C the Golgi apparatus is clumped into the perinuclear area (D) as without cold treatment(fig 2). Bar=5,um.

Figure 8 Chondrocytes cultured in the presence of microtubule disrupting agent (O,uM nocodazolefor 90 minutes), whichled to thefragmentation and dispersion of the Golgi apparatus over the cytoplasm (A), and after pressurisation in 30 MPacontinuous load (B). There is no packing of the Golgi apparatus in the absence of microtubules. Bar=5 pm.

dispersed in the cytoplasm and loading had noeffect on its vesicular appearance (fig 8B). Themorphology of the chondrocytes remainedunchanged during nocodazole treatment.Nocodazole had a drastic influence onproteoglycan synthesis, which decreased to50(2)% of the control values. Nocodazoletreatment also blocked the effect of a 5 MPa,0-5 Hz loading regimen, which was shown tostimulate proteoglycan synthesis withoutnocodazole. 18

DiscussionThe Golgi apparatus plays an important partin the processing and sorting of secretoryproteins.15 It is particularly essential in cells

such as chondrocytes, the main function ofwhich is to synthesise and exportmacromolecules for the maintenance of theextracellular matrix. In chondrocytes theglycosylation and sulphation of proteoglycanstake place in the Golgi apparatus, wherespecific enzymes sequentially add more than1000 sugar residues into the chondroitinsulphate and keratan sulphate chains of a singlelarge cartilage proteoglycan molecule(aggrecan). In this study we have shown thathydrostatic pressure altered the structure of theGolgi apparatus, which may account for thepreviously observed decrease in proteoglycansynthesis.2"

Sialic acid, the terminal residue of severaloligosaccharides including cartilage keratan

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sulphate, is attached to its substrates by theGolgi apparatus sialyl transferases. ThereforeWGA, which binds to sialic acid residues, canbe used for staining the Golgi apparatus.TRITC-WGA binding sites are localised in themedial and trans cisternae of the Golgiapparatus.22 In TRITC-WGA stained cells theGolgi apparatus was clumped juxtanuclearlyafter continuous 30 MPa pressure. Electronmicroscopy after this loading procedurerevealed that the clumping of the Golgiapparatus resulted from the disorganisation ofthe Golgi stacks. The Golgi apparatus wasprobably fragmented and condensed into thejuxtanuclear cytoplasm and wads visualised asa clumped structure by light microscopy.The stresses acting in the bovine knee joint

have not been measured, but it has beenestimated that the static compressive stress inthe knee joints of cows is 08 MPa.23 Thestresses on the cartilage surface in variousspecies of different sizes, including humans,are relatively constant. During the walkingcycle, 0-8-6-3 MPa pressures can be found inthe human knee joint,24 but in the human hipjoint peak pressures exceeding 18 MPa havebeen measured.25 Therefore, 5 MPa appears torepresent an ordinary physiological pressurelevel, 15 MPa a high physiological pressure,whereas 30 MPa is possibly beyond thephysiological pressure range.Thus far little is known of the mechanism

by which the pressure influences thechondrocytes. We have suggested thathydrostatic pressure is one of the maincomponents controlling proteoglycan synthesisrates in articular cartilage."' The appliedhydrostatic pressures have been small in manyprevious experiments compared withphysiological pressure levels in joints.26 Ourresults with 30 MPa agree, however, with thefindings of Hall and Urban2' that highcontinuous hydrostatic pressure (two hours, 50MPa) inhibits sulphate incorporation. Thecited study also indicated that the generalprotein synthesis in chondrocytes, measured asproline incorporation, decreased due to thecontinuous high hydrostatic pressure. With 5MPa we found no changes in the structure ofthe Golgi apparatus. A cyclic hydrostaticpressure of 5 MPa, however, influencesproteoglycan synthesis,"8 suggesting thathydrostatic pressure can modulateproteoglycan synthesis without changes in thegross organisation of the Golgi apparatus.The polygonal morphology of the cells,

maintained by the actin filaments and themicrotubules, is typical of culturedchondrocytes.2" The general morphology ofthechondrocytes was not affected bydepolymerisation of the microtubules withnocodazole, indicating that the microtubuleshad no direct effect on the cell shape.28 29 Afterpressurisation, however, the shape of somecells was altered to a retracted form, possiblydue to disruption of the stress fibres.28 Thesefindings suggest that the disruption of cell tocell and cell to substrate adhesions wasresponsible for the alterations of the generalcell morphology.28 Our results agree with the

earlier findings that a certain pressure levelmust be exceeded to achieve a change in cellmorphology.28The alterations of the Golgi apparatus

depended on the pressure level and the cycleapplied. Within the range of physiological pres-sures (15 MPa or less) some packing occurredin the Golgi apparatus but no changes wereobserved in the general cell morphology. Thissuggests that the change in the Golgi apparatusis one of the first structural alterations follow-ing an increase in hydrostatic pressure and notdue to the general impact of pressure on thecell shape.The morphology of the Golgi apparatus is

closely associated with the integrity of themicrotubules,30 possibly through microtubuleassociated proteins.3' The role of the micro-tubules in the packing of the Golgi apparatuswas tested by exposing the chondrocytes to 30MPa pressure after depolymerising the micro-tubules. This was accomplished by nocoda-zole, which binds tubulin heterodimers andinhibits their polymerisation, resulting in pro-gressive depolymerisation of the microtubules.The treatment led to fragmentation of theGolgi apparatus into numerous vesicles scat-tered throughout the cytoplasm. High hydro-static pressure did not induce clumping of theGolgi apparatus in these cells, suggesting thatintact microtubules were essential for the pack-ing of the Golgi apparatus on increased hydro-static pressure. Proteoclycan synthesis wasreduced to half of the control level due to thedepolymerisation of the microtubules andpacking of the Golgi apparatus. As proteogly-can secretion still proceeded, though at areduced rate, it seems that there must be routesindependent of microtubules to transport new-ly synthesised proteoglycans to plasma mem-brane. The microtubules are possibly essentialin gathering the Golgi cisternae into a singlestack.'5 The role of the microtubules in thesecretion of proteoglycans is then more facili-tatory than obligatory. The individual Golgicisternae or vesicles may operate independent-ly but not as efficiently as the whole Golgiapparatus.32 33

After depolymerisation of the microtubules,hydrostatic pressure had no effect on sulphateincorporation with the stimulatory loadingregime.'8 Therefore, without microtubules, thechondrocytes could not respond to the hydro-static pressure or, alternatively, the capacity ofeven slightly dispersed stacks was insufficientfor the stimulatory response.The situation after the cold treatment was

different. The microtubules are depolymerisedat low temperatures34 35 but there was no frag-mentation or dispersion ofthe Golgi apparatus.This was probably due to lowered metabolismin the cold,33 and may also indicate that thestructural change in the Golgi apparatus isdependent on a continuous supply of energy.When the cold treated cells were placed at37°C and immediately pressurised the Golgiapparatus changed into the packed form.

This is the first report of the influence ofhydrostatic pressure on the Golgi apparatusorganisation in eukaryotic cells. These results

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suggest that in chondrocytes the hydrostaticpressure modifies the structure of the Golgiapparatus. The gross alterations of the Golgiapparatus occur at high pressure levels, but stillwithin the range measured in cartilage invivo.25 Cartilage proteoglycan synthesis is alsoinhibited by the high hydrostatic pressure9(Lammi et al, unpublished data). Although thecomparison of the in vivo and in vitro experi-ments is complicated, the pressure response

can be induced in the chondrocytes and thecartilage explants. The matrix of cartilageprobably modulates the response to loading.Cultured chondrocytes can be used, however,to study the mechanisms by which pressure

effects are mediated in the cells. Although no

changes in the Golgi apparatus were seen withlow pressures, it is still possible that low pres-

sures influence the Golgi apparatus withoutchanges in the gross appearance of the organ-

elle. These data thus indicate that high hydro-static pressure causes structural changes in theGolgi apparatus which may affect thedecreased processing rate of proteoglycans inthe chondrocytes.

This work was supported by grants from The North Savo Fundof the Finnish Cultural Foundation, the Research and ScienceFoundation of Farmos, the Paulo Foundation, the Academy ofFinland and the Finnish Research Council for Physical Educa-tion and Sports, Ministry of Education. The authors give theircompliments to Mrs Eija Rahunen, Mrs Elma Sorsa, Mrs EijaVoutilainen, and Ms Eija Antikainen for their skilful technicalassistance.

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