Biochirnica et Biophysica Acta. 1074(1991) 151-158 © 1991 ElsevierSciencePublishersB.V.0304-4165/91/$03.50 ADONIS 030441659100169S
151
BBAGEN 23532
Evidence for the presence of P2-purinoceptors at the surface
of human articular chondrocytes in monolayer culture A l i s o n M . C a s w e l l ~, W e n g S. L e o n g ~ a n d R. G r a h a m G . R u s s e l l 2 i Department of Biochemist~, and Molecular Bi~!,~'. Unieer~iO, ,,f Leeds, Leeds (U.K.) and 2 Department of Human Metabolism and Clinical Biochemisto.. Unwersity of Sheffield Medical School. Sheffield ( U.K.)
(Received24 October1990)
Key words: Cartilage;Chondrocyte;ExtracellularATP; Purinoceptor Extraeellular purines can act at purinoceptors to influence metabolic processes. Nucleotide-metabolizing ectoenzymes may modulate such purinergie effects, and their occurrence in a tissue may suggest the presence of purinoceptors. Thus, following the identification of ecto-nucleoside triphosphate pyrophosphatase in cultured human articular ehondrecytes, we have studied whether these cells express Pz-type puriueceptors. Release of prostaglandin E (PGE) was monitored, since articular ehondrocytes synthesize and secrete PGE, and activation of P~-purinoceptors frequently results in enhanced prostaglandin production. Extracellular ATP and ADP stimulated PGE production, whereas AMP and adenosine had only limited effects. ATP concentrations as low as 5 / z M were effective, and maximal responses were achieved at 50-100 p M ATP. GTP, UTP and ITP also elicited reponses, but tended to be less effective than ATP at equivalent concentrations. Of the analogues of ATP that were tested, only adenosine 5'-(fl,¥-methyleue)triphosphate stimulated PGE production. The response to extracellular ATP was virtually abolished by indomethucin. Treatment of the cells with the Pt-purinoceptor antagonist, 8-phenyltheophylline, or with pertussis toxin reduced both basal and ATP-stimulated PGE production, but did net substantially decrease the ratio of ATP-stimulated to basal PGE production. These results indicate the presence of P2-purinoceptors in cultured human articular chondrocytes, and suggest that extracellular ATP may have physiological and pathological effects in human articular cartilage.
Introduction Cell surface receptors for adenine nucleotides and adenosine (purinoceptors) have been identified in many tissues, and extracellular purines may have important physiological and pathological actions [1,2]. Purinoceptors were originally classified by Burnstock [3]. P~purinoceptors are most responsive to adenosine, whereas P2-purinoceptors are most responsive to ATP [3]. Further studies using analogues of ATP led to the definition of two subclasses of Pz-purinoceptor [4]. At Pzx" purinoceptors analogues of ATP which have a methylene group substituted in the polyphosphate chain, e.g., adenosine 5'-(a,B-methylene)triphosphate (pp[CHz]pA) and adenosine 5'-(/L~,-methylene)triphosphate (p[CH2] ppA), are more potent than those which are substituted at position 2 of the adenine ring, e.g., 2-methylthioadenosine 5"-triphosphate (MeSATP), whereas at Pzy-
Correspondence: A.M. Casw¢ll, IX~partmentof Biochemistry and Molecular Biology,Universityof Lecds, Leeds. LS2 9JT, U.K.
purinoceptors this order of potency is reversed [4]. More recent work has confirmed that Pz-purinoceptors in many tissues can be subclassified on this basis [5], but there are probably other types of Pz-purinoceptor [1,5]. Activation of P2-purinoceptors can cause various cellular responses, and each subtype of receptor may be coupled to different effector systems. Elevation of the cytosolic concentration of free calcium frequently occurs, but may result either from the mobilization of calcium from intracellular stores [6-8], or from the opening of plasma membrane ion channels [9-11]. It has recently been suggested that the former results from the activation of P2y-purinoceptors, and the lattei- from the activation of P2x-purinoceptors [5]. Enhanced production of prostaglandins is also frequently associated with the activation of P2-purinoceptors [12-16], and may be secondary to increases in the cytosolic concentration of free calcium. Activation of Pl-purinoceptors does not usually result in this response [1,3], and hence, stimulation of the production of prostaglandins by extracellular ATP normally indicates the presence of P2-purinoceptors.
152 Coupling of P2-purinoceptors to their effector systems via G-proteins has been demonstrated in some types of cell, e.g., humar, neutrophils [8], rat aortic smooth muscle cells [17], rat renal mesangial cells [18] and chick myotubes [19]. However, responses to P2purinoceptor activation were insensitive to pertussis toxin in chick myotubes, whereas they were substantially inhibited by this toxin in the other three types of cell. This suggests the involvement of different G-proteins. Nucleotide-metabolizing enzymes are also present at the surface of many types of cell, and it has been suggested that one function of such enzymes is to modulate purinergic responses [20]. Thus, the identification of nucleotide-metabolizing ectoenzymes in a tissue raises the question of whether one or both types of purinoceptor are present. We have observed that human articular chondrocytes in monolayer culture express nucleoside triphosphate (NTP) pyrophosphatase at their surface. This ectoenzyme catalyses the conversion of nucleoside triphosphates to nucleoside monophosphates and inorganic pyrophosphate (PPi), and appears to serve in the degradation of extracellular ATP in h u m a n articular cartilage in vivo [21,22]. The identification of this nucleotide-metabolizing ectoenzyme prompted us to explore whether human articular chondrocytes also express purinoceptors of the P2-subclass. H u m a n articular chondrocytes in monolayer culture actively synthesize and secrete prostaglandin E (POE) [23], and we have therefore examined the effect of extracellular adenine nucleotides and adenosine on the production of PGE by these cells. Our initial studies indicated that human articular chondrocytes in monolayer culture express P2-purinoceptors, and these receptors have been further characterized with respect to agonist potency. In particular, we have studied the response of the cells to other nucleoside triphosphates, and to three analogues of ATP (pp[CH2lpA, p[CH2lppA and MeSATP) to determine whether the receptors could be subclassified as P2x or P,y. We have also used the cyclooxygenase inhibitor, indomethacin, to verify that extracellular ATP was stimulating de novo synthesis of PGE, and have used a potent Pl-purinoceptor antagonist, 8-phenyltheophylline [24], in an attempt to demonstrate that ATP was not acting at a P:pudnoceptor, following its metabolism to adenosine. Finally, we have studied the effect of pertussis toxin on the response to extracellular ATP. Some of this work has already appeared in abstract form [251. Experimental procedures
Culture techniques. Adult h u m a n articular cartilage was removed from knee joints obtained after surgical
amputation for lower limb ischaemia, or from femoral heads obtained after surgery for femoral neck fracture. Chondrocytes were isolated and cultured as described previously [22,23,26]. For experimental use, confluent primary cultures were dispersed with 0.05% w / v trypsin/0.02% w / v EDTA, and subcultured into 1.6 cm diameter wells (24-well plates) or 1.1 cm diameter wells (48-well plates) at a density of approx. 3-104 cells per cm 2. When 48-well plates were used, variations between different positions on the plate were minimized by only placing cells in the central 24 wells. Incubation procedure. The incubation medium was serum-free Hepes-buffered minimal essential medium (Eagle) supplemented with Earle's salts, 100 U / m l penicillin and 100 p,g/ml streptomycin. At 5 - 9 days after subculturing, confluent monolayers of cells were washed three times with incubation medium. Cells were then incubated for 4 h at 37°C in incubation medium (0.5 ml/well for 1.6 cm diameter wells or 0.25 ml/well for 1.1 cm diameter wells) together with agonists as indicated. At the end of the incubation, media were removed and stored at - 2 0 ° C . Cell monolayers were rinsed with phosphate-buffered saline (Dulbecco's, without calcium and magnesium) to remove residual traces of medium. The plates were then stored at - 2 0 ° C , until the protein content of the wells was measured. Determination of prostaglandin E (PGE). PGE was measured in aliquots of incubation medium by radioimmunoassay methods in which free antigen was selectively adsorbed onto dextran-coated charcoal. Two different antisera were used for these studies, since one antibody (supplied by Steranti Research) ceased to be commercially available. However, both methods gave similar results. When using the antiserum supplied by Steranti Research, the assay buffer was 10 m M Tris-HCl (pH 7.4) containing 140 m M NaCI, 0.1% w / v gelatin and 0.02% w / v sodium azide, and 1 vial of lyophilized antiserum was reconstituted in 100 mi buffer. The protocol was as follows: Sample or standard (0.1 ml) was incubated overnight at 4°C with 0.1 ml [3HIPGE (248 Bq) and 0.1 ml antiserum. U n b o u n d PGE was then removed by the addition of 0.2 ml ice-cold assay buffer containing 0.6~ w / v Norit A charcoal and 0.125% w / v dextran. Following incubation at 4°C for 10 min, charcoal was removed by centrifugation for 15 min at 4°C at 2000 × g (ray = 24.5 cm), and 0.2 ml of the supernatant was counted in 4 ml Emulsifier Safe scintillation fluid. When using the antiserum supplied by Sigma Chemicals, the assay buffer was 10 m M sodium phosphate (pH 7.4) containing 150 m M NaCI, 0.1% w / v bovine serum albumin and 0.1% w / v sodium azide, and 1 vial of lyophilized antiserum was reconstituted in 150 ml buffer. The protocol was as follows: Sample or standard (0.1 ml) was incubated for 30 min at 4 ° C with 0.5 ml
153 antiserum. [3H]PGE was then added (0.1 ml, 148 Bq), and the samples incubated overnight at 4°C. Unbound PGE was removed by the addition of 0.2 ml ice-cold assay buffer containing 1% w / v activated charcoal and 0.170 w / v dextran. Following incubation at 4°C for 10 min, charcoal was removed by centrifugation for 15 min at 4°C at 2000 x g (ray = 24.5 cm), and 0.7 ml of the supernatant was counted in 4 ml Emulsifier Safe scintillation fluid. For both assays, results were calculated from standard curves, range. 5-2000 pg PGE2 per tube. Determination of protein. Cell monolayers were solubilized in 1 M NaOH and protein was measured by a modification of the Lowry method, using bovine serum albumin (range 4-20 /~g) as standard [27]. All results were corrected for the protein content of the wells. Activation ofpertussis toxin. Immediately prior to use, 1 #g pertussis toxin was incubated for 30 min at 37°C in 0.5 ml 0.1 M sodium phosphate (pH 7.0), containing 0.5 M NaCI and 20 mM dithiothreitol. Experimental design. Within experiments, each condition was tested in quadruplicate and the results expressed as the mean _+ standard error (S.E.). There was some variation in basal production of PGE between muitiwell plates. Hence control wells (n = 4) were included in each plate, and only results obtained from the same multiwell plate were compared statistically. The significance of differences between mean values was determined using the Student t-test. Experiments were performed at least three times, using cells from different donors, and qualitatively similar results were obtained on each occasion. Data from representative experiments are presented here.
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Materials Tissue culture media, enzymes and antibiotics were purchased from Gibco BRL. Life Technologies. Paisley. Strathclyde. U.K. Foetal calf serum was obtained from Northumbrian Biologicals, Cramlington, Northumberland, U.K. [5,6,8,11.12,14,15(n)-3H]Prostaglandin E 2 was supplied by Amersbam International, Aylesbury, U.K. Norit A charcoal was purchased from Aldrich Chemicals. Gillingham. U.K. Activated charcoal (untreated powder. 250-350 mesh), pp[CH2lpA (lithium salt), p[CH,lppA, ADP, AMP, anti-POE, ATP, bovine serum albumin (fraction V, for protein assay), bovine serum albumin (essentially fatty acid free, for radioimmunoassay of PGE), dextran (approximate average molecular weight, 70000), DL-dithiothreitol, forskolin, GTP, indomethacin, ITP, pertussis toxin, 8-phenyhheophylline, PGE2 and UTP were all supplied by Sigma Chemicals, Poole, U.K. MeSATP (tetrasodium salt) was supplied on behalf of Research Biochemicals, by Semal Technical, St. A1bans, U.K. Anti-PGE was purchased from Steranti Research, St AIbans, U.K. All other chemicals were obtained from BDH Chemicals, Poole, U.K., and were of AR grade or of the highest purity available.
Results In preliminary experiments, it was noted that extracellular ATP stimulated production of PGE, and that PGE accumulated steadily in the medium for 4 h when the concentration of ATP was 100 p.M (data not shown).
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Cont ATP ADP AMP Ado Cont ATP AOP AMP Ado Cont ATP ADP AMP Ado
Fig. 1. The effect of adenine nucleotidesand adenosineon the production of PGE by humanarticular chondrocytesin monolayerculture. Rinsed cell monolayerswereincubated for 4 h with adenine nucleotidesor adenosine(Ado). as indicated. Resultsare presented as the mean_+S.E. of four determinations. Valuesof P refer to a comparisonof results for each agonistwithcontrol(Cont). * P < 0.05; " * P < 0.01; * * * P < 0.00l.
154 ATP
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NUCLEOTIDE CONCENTRATION (JJM)
Fig. 2. Concentration dependence of the responses to ATP and ADP. Rinsed cell monolayers were incubated for 4 h with varying concentrations of ATP or ADP. as indicated. ATP and ADP were tested on different multiwell plates. Results are presented as the mean+S.E, of four determinations.
Thus, in subsequent experiments, P G E p r o d u c t i o n was measured at the end of a 4 h incubation period. Comparison of ATP with other adenine nucleotides and adenosine at concentrations of 2 0 , 1 0 0 and 500 .aM, revealed that A T P and A D P elicited similar responses at each concentration, whereas A M P and adenosine were much less effective (Fig. 1). The response to A T P was always greater than the corresponding response to adenosine ( P < 0 . 0 0 1 ) . Responses to A T P and A D P were concentration-dependent, and were m a x i m a l at 50-100 .aM (Fig. 2). There was some variation between chondrocyte cultures, probably reflecting different rates of metabolism of A T P and A D P at the cell surface. UTP and ITP stimulated p r o d u c t i o n of P G E at concentrations of 20, 100 and 500 #M, and G T P elicited responses at the two higher concentrations. However, CTP was ineffective under these conditions (Fig. 3). The
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observed responses were lower than the responses to equivalent c o n c e n t r a t i o n s of A T P ( P < 0.01). M a x t m a l responses were achieved at 5 0 - 1 0 0 .aM UTP, a n d ai 100-250 /~M ITP, whereas the response to G T P increased steadily up to a concentration of 500 .aM (Fig. 4). Again, there was some variation between chondrocyte cultures. A t low c o n c e n t r a t i o n s (1, 5 and 20 /~M), neither M e S A T P nor p p [ C H 2 ] p A elicited any respo':se, b u t at all three concentrations, p [ C H 2 ] p p A induced some s t i m u l a t i o n of P G E p r o d u c t i o n (Fig. 5). W h e n the concentration of agonist was 1 .aM, responses to p [ C H 2 ] p p A a n d A T P were similar, whereas when it was 20 .aM, the response to A T P was greater ( P < 0.05). I n d o m e t h a c i n slightly decreased basal p r o d u c t i o n of P G E and s u b s t a n t i a l l y decreased A T P - s t i m u l a t e d production of P G E (Table I), suggesting that extraceilular
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Fig. 3. The etfect of nuc!eoside triphosphates c'~n the production of PGE by human articular chondrocytes in monolayer culture. Rinsed cell monolaycrs w:re il;cubated for 4 h with nucleoside triphosphates, as indicated. Results are presented as the mean+S.E, of four determinations. values of P refer to a comparison of results for each agonist with control (Cont). * P < 0.05 * * P < 0.0l ; * * * P < 0.001.
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Fig. 4. Concentration dependence of the responses to GTP, ITP and UTP. Rinsed cell monolayers were incubated for 4 h with varying concentrations of GTP, ITP and UTP, as indicated. Each agonist was tested on a different muhiwell plate. Results are presented as the mean_+ S.E. of four determinations.
ATP was promoting de novo synthesis of PGE. 8-Phenyitheophylline moderately decr..~ased both basal and ATP-stimulated production of PGE (Table I). However, the presence of 8-phenyltheophylline did not decrease the ratio of ATP-stimulated to basal production of PGE, s n ~ e s t i n g that this Pl-purinoceptor antagonist was not directly inhibiting the ATP-stimulated component of the- response. Interestingly, production of PGE by rabbit articular chondrocytes was decreased by treating the cells with forskolin to elevate the intracellular concentration of cAMP [28]. Hence, since 8-phenyltheophyUine could have increased the intracellular concentration of cAMP in h u m a n articular chondrocytes, we investigated the effect of forskolin (10 btM) on both basal and ATP-stimulated production of POE. In the presence and absence of forskolin, respectively, basal
60
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production of PGE was 26.0 +_ 1.64 and 25.9 +__3.49 pg/,ug protein per 4 h, and ATP-stimulated production of PGE (100 ttM ATP) was 89.5 + 11.1 and 95.3 _+ 10.5 p g / ~ g protein per 4 h. Finally, pretreatment of the cells with pertussis toxin also decreased both ATP-stimulated and basal production of PGE (Table II), and although the ratio of ATP-stimulated to basal production of PGE tended to fall, the decrease was not ahvays statistically significant. Discussion These findings indicate the presence of P:-purinoceptors at the surface of h u m a n articular chondrocytes in monolayer culture. Extracellular ATP evoked a response that is normally associated with the activation of
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Fig. 5. The effect of ATP and analogues of ATP on the production of PGE by human articular chon¢' ~cytes in monolayer culture. Rinsed cell monolayers were incubated for 4 h with ATP or analogues of ATP, as indicated. Some control wells were treated with lithium (Li) at 2.5-times the concentration of the agonist, because the lithium salt of adenosine 5'-(adi-methylene)triphosphate was used. AMPCPP, adenosine 5'-(a,/i'-methyl-
ene)triphosphate;AMPPCP, adenosine5'-(/],y-methylene)triphosphate.Results are presented as the mean+ S.E. of four determinations.Valuesof P refer to a comparisonof results for each agonistwith control (Cnnt). * P < 0 . 0 5 ; * * P < 0 . 0 l , * * * P < 0 . 0 0 l .
156 TABLE 1 Effect of indomethacin and 8-phenyltheoph_vlline on ATP-stimulated production of PGE
lndomethacin was dissolved in dimethylsulphoxide to give a concentration of 14 mM, and the solution was diluted with incubation medium to give a final concentration of 1.4 .aM. 8-Phenyltheophylline (8-PT) was dissolved in 80% v/v methanol containing 0.2 M NaOH to give a concentration of 10 raM. and the solution was diluted with incubation medium to give a final concentration of 10 .aM. Rinsed cell monolayers were incubated for 4 h with ATP, as indicated, in the presence or absence of indomethacin or 8-phenyltheophylline. Control wells contained 0.0l~ v/v dimetbylsulphoxide for the study of the effect of indomethacin, and 0.08% v/v methanol/0.2 mM NaOH for the study of the effect of 8-phenyhheophylline. Results are presented as the mean _+S.E. of four determinations. PGE (pg/~g protein per 4 h) NoATP Control Indomethacin Control 8-PT
20 #M ATP
8.48 4-1.94 57.6 4-7.82 5.64+1.53 13.1+2.23"* 22.8 +2.55 93.8+9.88 12.4 4-2.48 * 54.14-2.95 **
100/tM ATP 86.6 + 10.2 12.1+ 1.18"** 144 ±14.6 111 + 8.63
Values of P refer to a comparison of concentrations of PGE in the presence and absence of indomethacin or S-phenyltheophylline. as appropliate. * P < 0.05. ** P < 0.01. *** P < 0.001.
P2-purinoceptors, a n d responses to A T P w e r e m u c h g r e a t e r t h a n responses to equivalent c o n c e n t r a t i o n s o f adenosine. However, since A T P c a n b e m e t a b o l i z e d a t the surface of these cells to A M P a n d a d e n o s i n e [21|, the involvement o f activation o f P~-purinoceptors in m e d i a t i n g p a r t o f this response c a n not b e entirely ruled out. T h e results with 8 - p h e n y l t h e o p h y l l i n e are s o m e w h a t curious, b u t d o n o t a p p e a r to r e f u t e the c o n t e n t i o n t h a t A T P was a c t i n g directly at P2-purinoceptors. T h e r e a r e several m e c h a n i s m s w h e r e b y 8 - p h e n y l t h e o p h y l l i n e c o u l d influence b o t h b a s a l a n d A T P - s t i m u l a t e d p r o d u c t i o n o f
P G E . Firstly, 8 - p h e n y l t h e o p h y l l i n e m i g h t have inhibited production of PGE by human articular chondrocytes by raising the i n t r a c e l l u l a r c o n c e n t r a t i o n o f c A M P via inh i b i t i o n o f the activity o f c A M P phosphodiesterase(s). H o w e v e r , this possibility w o u l d a p p e a r to be ruled o u t b y the o b s e r v a t i o n t h a t forskolin d i d not affect either b a s a l o r A T P - s t i m u l a t e d p r o d u c t i o n o f P G E b y hz.man a r t i c u l a r c h o n d r o c y t e s in m o n o l a y e r culture. Secondly, it h a s b e e n o b s e r v e d t h a t 3 - i s o b u t y l - l - m e t h y l x a n t h i n e at c o n c e n t r a t i o n s in excess o f 0.1 m M inhibits the activity o f c y c l o o x y g e n a s e [29], b u t it is n o t k n o w n w h e t h e r 8 - p h e n y l t h e o p h y l l i n e a t the c o n c e n t r a t i o n u s e d in this s t u d y (10 ILM), also inhibits the enzyme. T h i r d l y , the i n h i b i t o r y effect o f 8 - p h e n y l t h e o p h y l l i n e c o u l d indicate that both basal and ATP-stimulated production of P G E are e n h a n c e d b y a c t i v a t i o n o f P~-purinoceptors b y e n d o g e n o u s l y p r o d u c e d a d e n o s i n e . T h e p r e s e n c e o f Ptp u r i n o c e p t o r s in h u m a n a r t i c u l a r c h o n d r o c y t e s d o e s n o t a p p e a r to h a v e b e e n investigated. H o w e v e r , it has b e e n r e p o r t e d t h a t a c t i v a t i o n o f Pt-like p u r i n o c e p t o r s in the r a t t h y r o i d cell line, F R T L - 5 , permissively e n h a n c e s r e s p o n s e s to a c t i v a t i o n o f P2-purinoeeptors [30]. Clearly, possible i n t e r a c t i o n s b e t w e e n P~-purinoceptors a n d P2p u r i n o c e p t o r s in h u m a n a r t i c u l a r c h o n d r o c y t e s s h o u l d be explored further. Pertussis t o x i n a l s o a p p e a r e d to a c t p r i m a r i l y o n basal production of PGE by human articular chondrocytes in m o n o l a y e r culture, a n d it w a s n o t entirely c l e a r w h e t h e r it h a d a n a d d i t i o n a l effect o n the A T P - m e d i a ted c o m p o n e n t o f t h e response. Interestingly, pertussis toxin inhibited both basal and ATP-stimulated production o f P G D b y p r i m a r y c u l t u r e s o f r a t a s t r o e y t e s [31]. A g a i n , o n e e x p l a n a t i o n f o r o u r f i n d i n g s is t h a t a n o t h e r signalling p a t h w a y , w h o s e activity d e t e r m i n e s b o t h b a s a l and ATP-stimulatod production of PGE, contains a pertussis toxin-sensitive O - p r o t e i n . It is n o t e w o r t h y t h a t in F R T L - 5 cells, a pertussis toxin-sensitive G - p r o t e i n w a s involved in the t r a n s d u c t i o n o f the signal f r o m the Pt-like p u r i n o c e p t o r s , b u t n o t f r o m the P 2 - p u r i n o c e p t o r s [30]. T h e i d e n t i t y o f this p u t a t i v e s e c o n d signalling
TABLE 11 Effect of pretreatment with pertussis toxin on A TP-stimulated production of PGE
Cells were treated overnight with 20 ng/ml dithiothreitol-activated pertussis toxin (PT), and control wells were preincubated with an equivalent concentration of dithiothreitol (0.2 mM). Rinsed cell monolayers were then incubated for 4 h with ATP, as indicated. Each concentration of ATP was tested on a different multiwell plate. Results are presented as the mean 4-S.E. of four determinations. PGE (pg/p.g protein per 4 h) Untreated. no ATP Untreated, 20 .aM ATP PT-treated. no ATP PT-treated, 20 b~M ATP
18.1 + 0.83 42.1 4-2.32 16.1 + 0.20 28.1 + 1.06 * *
Untreated, no ATP Untreated, 100 .aM ATP PT-treated, no ATP PT-treated, 100 pM ATP
27.3 + 1.98 61.24- 2.44 18.7 + 1.79 * 37.1 :t: 1.15 * * *
Untreated, no ATP Untreated, 500 .aM ATP PT-treated, no ATP PT-treated, 500/~ M ATP
Values of P refer to a comparison of results for pertussis toxin-treated cells with those for the corresponding untreated cells. * P < 0.05. ** P < 0.01. P < 0.001.
37.4.4-1.81 65.8 + 2.18 26.6 + 1.43 * * 36.6 + 5.35 * *
157 pathway in human articular chondrocytes is not known, but coupling of Pl-purinoceptors of the Al-subclass (inhibitory) to pertussis toxin-sensitive G-proteins has been widely documented [32]. An alternative explanation for our findings is that phospholipase A 2 is itself coupled via a pertussis toxin-sensitive G-protein in human articular chondrocytes, since it has been demonstrated that transducin is directly involved in the activation of this enzyme in rod outer segments of bovine retina [331. As human articular chondrocytes in monolayer culture metabolize ATP at their surface, it is difficult to assess the sensitivity of the receptor. However, in three experiments using low concentrations of ATP, production of POE was stimulated significantly on each occasion by 5 .ttM ATP, and on one occasion by 1 iaM ATP. This suggests that the receptor could be sensitive to very low concentrations of ATP in vivo. It is also difficult to define the specificity of the receptor because of the metabolism of agonists. However, the limited effect of p[CH2]ppA, and the lack of effect of MeSATP and pp[CH2]pA, suggest that the receptor can not be designated a s P2x or P2y. This is not entirely unexpected, since it was noted in the introduction that not all P2-purinoceptors can be classified on this basis. The ability of GTP, ITP and UTP to elicit responses indicates that the receptor is not specific for the adenine base or for pudnes, or alternatively, that there is more than one type of receptor for nucleoside triphosphates present at the surface of human articular chondrocytes. Interestingly, evidence has recently been presented for the presence of separate receptors for ATP and UTP at the s u r f a c e of human HL60 cells [34]. It is not possible, at present, to define the role of P2-purinoceptors in human articular cartilage, but it is now recognized that articular chondrocytes can respond to many systemically and locally produced agents. For example, recent studies have demonstrated the presence of receptors for serotonin, dopamine and histamine in human articular chondrocytes [35,36], and for bradykinin in pig articular chondrocytes [37]. The extraceUular concentration of ATP in human articular cartilage is not known. However, it is probably considerably higher than concentrations in plasma and synovial fluid, which a r e of the order of 50 nM [38,39], because ATP is metabolized at the surface of human articular chondrocytes [21] and of many other types of cell [20]. The sources of extracellular ATP in human articular cartilage are also not known, but since this tissue is not innervated [40], release of ATP from nerve endings is ruled out. Articular chondrocytes synthesize and secrete the components of the cartilage extracellular matrix [41], and small amounts of ATP could be released in association with these macromolecules. In addition, human articular chondrocytes might specifically secrete ATP in response to certain stimuli, since there are
mechanis/ns for the release of ATP from some types of cell, e.g., porcine and human vascular endothelial cells [42.43], and rat heart cells [44,45]. There could be enhanced release of ATP from human articular chondrocytes in pathological situations, and the concentration of ATP in synovial fluid from subjects with chondrocalcinosis is elevated [39]. Release of ATP may be increased when human articular chondrocytes are promoted to repair the matrix following physical injury or enzymic destruction, if ATP is secreted in association with macromolecules. Physical injury to the cells themselves could also result in release of ATP (along with other intracellular components). There is circumstantial evidence that this latter process occurs, because chondrocalcinosis is sometimes associated with previous tissue injury [46,47], and the PPi which is deposited may have arisen from the action of ecto-NTP pyrophosphatase on ATP released from damaged cells. in conclusion, we have provided evidence that human articular chondrocytes in monolayer culture, express P.,-purinoceptors at their surface, which are sensitive to low concentrations of ATP. Such receptors could therefore be activated in vivo, One consequence of our findings is a possible mechanism for the observed association between chondrocalcinosis and osteoarthritis [47,48], since ATP released from articular chondrocytes, can both induce alterations in cellular metabolism through its action at P,-purinoceptors, and serve as a source of PPi. Furthermore, we have recently observed that extracellular ATP stimulates resorption of proteoglycans from bovine nasal cartilage [49], which suggests that activation of P2-purinoceptors in chondrocytes may have wide-ranging biological consequences, Acknowledgement We are grateful to the Wellcome Trust, U.K. for financial support for this work. References
t Gordon,J.L (1986) Biochem.J. 233,309-319. 2 Williams, M. (1987) Annu. Rev. Pharmacol.Toxicol. 27, 315-345. 3 Burnstock,G. (1978) in Cell Membrane Receptors for Drugs and Hormones(Straub, R.W. and Bolis, L., eds.), pp. 107-118. Raven Press, New York. 4 Burnstock G. and Kennedy, C. (1985) Gen. Pharmacol. 16, 433440. 5 Kennedy,C. (1990) Arch. Int. Pharmacodyn.Ther. 303, 30-50. 6 Charest, R., Blaekmore, P.F. and Exton.J.H. (1985)J. Biol. Chem. 260, 15789-15794. 7 Hallam. T.J. and Pearson, J.D. (1986) FEBS Lett. 207, 95-99. 8 Cockcruft. S. and Stutchfield, J. (1989) FEBS Len. 245, 25-29. 9 Benham,C.D. and Tsien, R.W. (1987) Nature 328, 275-278. l0 EI-Moatassim,C., Domand, J. and Mani, J.-C. (1987) Biochim. Biophys. Acta 927, 437-444. It Danziger, R.S., Raffaeli, S., Moreno-Sanchez, R., Sakai, M.. Capogrossi, M.C., S~argeon, H.A, Hansford, R.G. and Lakana. E.G. (1988) Cell Calcium 9, 193-199.
158 12 Needleman, P., Minkes, M.S. and Douglas. J.R. (1974) Circ. Res. 34. 455-460. 13 Brown. C.M. and Burnstock, G. (1981) Eur. J. Pharmacol. 69. 81-86. 14 Pearson. J.D.. Slakey, L.L. and Gordon. J.L. (1983) Biochem. J. 214. 273-276. 15 Forsberg, E.J., Feuerstein, G., Shohami, E. and Pollard, H.B. (1987l Proc. Natl. Acad. Sci. U.S.A. 84, 5630-5634. 16 Pfeilschifter. J.. Thiiring. B. and Festa. F. (1989) Eur. J. Biochem. 186. 509-513. 17 Tsuda, T., Kawahara, Y.. Fukumoto, Y., Takai, Y. and Fukuzaki, H. (1988) Jpn. Circ. J. 52. 570-579. 18 Pfeilschifter. J. (1990) Cell. Signal. 2. 129-138. 19 Haggblad, J, and Heilbronn, E. (1988) FEBS Lett. 235, 133-136. 20 Pearson, J.D. (1987) in Mammalian Ectoenzymes (Kenny, A.J. and Turner. A.J., eds.), pp. 139-167, Elsevier, Amsterdam. 21 Caswell. ±,.M. and Russell, R.G,G. (1985) Biochim. Biophys. Aeta 847, 40-47. 22 Caswell, A.M. and Russell, R.G.G. (1988) Biochim. Biophys. Acta 966, 310-317. 23 Meats, J.E.. McGuire, M.B. and Russell, R.G.G. (1980) Nature 286, 891-892. 24 Griffith. S.G., Meghji. P., Moody, C.J. and Burnstock, G. (1981) Eur. J. Pharmacol. 75, 61-64. 25 Caswell, A.M. and Russell, R.G.G. (1988) Br. J. RheumatoL 27, Suppl. A2, 125. 26 Srivastava, V.M.L., Malemud. C.J. and Sokoloff, L. (1974) Connect. Tissue Res. 2, 127-136. 27 Lowry, O.H., Rosebrough, N.J., Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 28 Malemud. C.J., Mills, T.M. and Papay, R.S. (1986) Prostaglandins 32. 495-501. 29 Whorton, A.R,, Collawn, J.B., Montgomery, M.E., Young, S.L. and Kent. R.S, (1985) Biochem. Pharmacol. 34,119-123.
30 Okajlma, F., Sato, K., Nazarea, M., Sho, K. and Kondo, Y. (1989) J. Biol. Chem. 264, 13029-13037. 31 Gelficke-Haerter, P.J., Wurster, S., Schobert, A. and Hertting, G. (1938) Arch. PharmacoL 338, 704-707. 32 Stiles, G L. (1990) Clin. Res. 38, 10-18. 33 Jelsema, C.L. (1987) J. Biol. Chem. 262,163-168. 34 Stutchfield, J. and Cockcroft, S. (1990) FEBS Lett. 262, 256-258. 35 Taylor, D.J. and Woolley, D.E. (1987) Ann. Rheum. Dis. 46, 431-435. 36 Vignon, E., Broquet, P., Mathieu, P., Louisot. P. and Richard, M. (1990) Biochem. Int. 20, 251-255. 37 Benton, H.P., Jackson, T.R. and Hanley, M.R. (1989) Biochem. J. 258, 861-867. 38 Parkinson, P.i, (1973) J. Physiol. 234, 72P-74P. 39 Ryan, L.M., Rachow, J.W. and McCarty, D.J. (1987) Arthritis Rheum. 30, $131. 40 Meaehim, G. and Stockwell, R.A. (1979) in Adult Articular Cartilage (Freeman, M.A.R., ed.), pp. 1-67, Pitman Medical, Tunbridge Wells. 41 Stockwell, R.A. and Meachim, G. (1979) in Adult Articular Cartilage (Freeman, M.A.R., ed.), pp. 69-144, Pitman Medical. Tunbridge Wells. 42 Pearson, J.D. and Gordon, J.L. (1979) Nature 281. 384-386. 43 Lollar, P. and Owen, W.G. (1981) Ann. NY Acad. Sci. 370, 51-56. 44 Forrester, T. and Williams, C.A. (1977) J. Physiol. 268, 371-390. 45 Clemens, M.G, and Forrester, T. (1980) J. Physiol. 312. 143-158. 46 Doherty, M.. Watt, I. and Dieppe, P.A. (1982) Lancet i, 1207-1210. 47 Dieppe, P.A., Alexander, G.J.M., Jones, H.E., Doherty, M., Scott, D.G.I.. Manhire, A. and Watt, I. (1982) Ann. Rheum. Dis. 41, 371-376. 48 Felson, D.T.. Anderson, J.J., Naimark, A., Kannel, W. and Meenan, R.F. (1989) J. Rheumatol. 16,1241-1245. 49 Leong, W.S., Russell, R.G.G. and Caswell, A.M. (1990) Biochem. Soc. Trans. 18, 951-952.
151
BBAGEN 23532
Evidence for the presence of P2-purinoceptors at the surface
of human articular chondrocytes in monolayer culture A l i s o n M . C a s w e l l ~, W e n g S. L e o n g ~ a n d R. G r a h a m G . R u s s e l l 2 i Department of Biochemist~, and Molecular Bi~!,~'. Unieer~iO, ,,f Leeds, Leeds (U.K.) and 2 Department of Human Metabolism and Clinical Biochemisto.. Unwersity of Sheffield Medical School. Sheffield ( U.K.)
(Received24 October1990)
Key words: Cartilage;Chondrocyte;ExtracellularATP; Purinoceptor Extraeellular purines can act at purinoceptors to influence metabolic processes. Nucleotide-metabolizing ectoenzymes may modulate such purinergie effects, and their occurrence in a tissue may suggest the presence of purinoceptors. Thus, following the identification of ecto-nucleoside triphosphate pyrophosphatase in cultured human articular ehondrecytes, we have studied whether these cells express Pz-type puriueceptors. Release of prostaglandin E (PGE) was monitored, since articular ehondrocytes synthesize and secrete PGE, and activation of P~-purinoceptors frequently results in enhanced prostaglandin production. Extracellular ATP and ADP stimulated PGE production, whereas AMP and adenosine had only limited effects. ATP concentrations as low as 5 / z M were effective, and maximal responses were achieved at 50-100 p M ATP. GTP, UTP and ITP also elicited reponses, but tended to be less effective than ATP at equivalent concentrations. Of the analogues of ATP that were tested, only adenosine 5'-(fl,¥-methyleue)triphosphate stimulated PGE production. The response to extracellular ATP was virtually abolished by indomethucin. Treatment of the cells with the Pt-purinoceptor antagonist, 8-phenyltheophylline, or with pertussis toxin reduced both basal and ATP-stimulated PGE production, but did net substantially decrease the ratio of ATP-stimulated to basal PGE production. These results indicate the presence of P2-purinoceptors in cultured human articular chondrocytes, and suggest that extracellular ATP may have physiological and pathological effects in human articular cartilage.
Introduction Cell surface receptors for adenine nucleotides and adenosine (purinoceptors) have been identified in many tissues, and extracellular purines may have important physiological and pathological actions [1,2]. Purinoceptors were originally classified by Burnstock [3]. P~purinoceptors are most responsive to adenosine, whereas P2-purinoceptors are most responsive to ATP [3]. Further studies using analogues of ATP led to the definition of two subclasses of Pz-purinoceptor [4]. At Pzx" purinoceptors analogues of ATP which have a methylene group substituted in the polyphosphate chain, e.g., adenosine 5'-(a,B-methylene)triphosphate (pp[CHz]pA) and adenosine 5'-(/L~,-methylene)triphosphate (p[CH2] ppA), are more potent than those which are substituted at position 2 of the adenine ring, e.g., 2-methylthioadenosine 5"-triphosphate (MeSATP), whereas at Pzy-
Correspondence: A.M. Casw¢ll, IX~partmentof Biochemistry and Molecular Biology,Universityof Lecds, Leeds. LS2 9JT, U.K.
purinoceptors this order of potency is reversed [4]. More recent work has confirmed that Pz-purinoceptors in many tissues can be subclassified on this basis [5], but there are probably other types of Pz-purinoceptor [1,5]. Activation of P2-purinoceptors can cause various cellular responses, and each subtype of receptor may be coupled to different effector systems. Elevation of the cytosolic concentration of free calcium frequently occurs, but may result either from the mobilization of calcium from intracellular stores [6-8], or from the opening of plasma membrane ion channels [9-11]. It has recently been suggested that the former results from the activation of P2y-purinoceptors, and the lattei- from the activation of P2x-purinoceptors [5]. Enhanced production of prostaglandins is also frequently associated with the activation of P2-purinoceptors [12-16], and may be secondary to increases in the cytosolic concentration of free calcium. Activation of Pl-purinoceptors does not usually result in this response [1,3], and hence, stimulation of the production of prostaglandins by extracellular ATP normally indicates the presence of P2-purinoceptors.
152 Coupling of P2-purinoceptors to their effector systems via G-proteins has been demonstrated in some types of cell, e.g., humar, neutrophils [8], rat aortic smooth muscle cells [17], rat renal mesangial cells [18] and chick myotubes [19]. However, responses to P2purinoceptor activation were insensitive to pertussis toxin in chick myotubes, whereas they were substantially inhibited by this toxin in the other three types of cell. This suggests the involvement of different G-proteins. Nucleotide-metabolizing enzymes are also present at the surface of many types of cell, and it has been suggested that one function of such enzymes is to modulate purinergic responses [20]. Thus, the identification of nucleotide-metabolizing ectoenzymes in a tissue raises the question of whether one or both types of purinoceptor are present. We have observed that human articular chondrocytes in monolayer culture express nucleoside triphosphate (NTP) pyrophosphatase at their surface. This ectoenzyme catalyses the conversion of nucleoside triphosphates to nucleoside monophosphates and inorganic pyrophosphate (PPi), and appears to serve in the degradation of extracellular ATP in h u m a n articular cartilage in vivo [21,22]. The identification of this nucleotide-metabolizing ectoenzyme prompted us to explore whether human articular chondrocytes also express purinoceptors of the P2-subclass. H u m a n articular chondrocytes in monolayer culture actively synthesize and secrete prostaglandin E (POE) [23], and we have therefore examined the effect of extracellular adenine nucleotides and adenosine on the production of PGE by these cells. Our initial studies indicated that human articular chondrocytes in monolayer culture express P2-purinoceptors, and these receptors have been further characterized with respect to agonist potency. In particular, we have studied the response of the cells to other nucleoside triphosphates, and to three analogues of ATP (pp[CH2lpA, p[CH2lppA and MeSATP) to determine whether the receptors could be subclassified as P2x or P,y. We have also used the cyclooxygenase inhibitor, indomethacin, to verify that extracellular ATP was stimulating de novo synthesis of PGE, and have used a potent Pl-purinoceptor antagonist, 8-phenyltheophylline [24], in an attempt to demonstrate that ATP was not acting at a P:pudnoceptor, following its metabolism to adenosine. Finally, we have studied the effect of pertussis toxin on the response to extracellular ATP. Some of this work has already appeared in abstract form [251. Experimental procedures
Culture techniques. Adult h u m a n articular cartilage was removed from knee joints obtained after surgical
amputation for lower limb ischaemia, or from femoral heads obtained after surgery for femoral neck fracture. Chondrocytes were isolated and cultured as described previously [22,23,26]. For experimental use, confluent primary cultures were dispersed with 0.05% w / v trypsin/0.02% w / v EDTA, and subcultured into 1.6 cm diameter wells (24-well plates) or 1.1 cm diameter wells (48-well plates) at a density of approx. 3-104 cells per cm 2. When 48-well plates were used, variations between different positions on the plate were minimized by only placing cells in the central 24 wells. Incubation procedure. The incubation medium was serum-free Hepes-buffered minimal essential medium (Eagle) supplemented with Earle's salts, 100 U / m l penicillin and 100 p,g/ml streptomycin. At 5 - 9 days after subculturing, confluent monolayers of cells were washed three times with incubation medium. Cells were then incubated for 4 h at 37°C in incubation medium (0.5 ml/well for 1.6 cm diameter wells or 0.25 ml/well for 1.1 cm diameter wells) together with agonists as indicated. At the end of the incubation, media were removed and stored at - 2 0 ° C . Cell monolayers were rinsed with phosphate-buffered saline (Dulbecco's, without calcium and magnesium) to remove residual traces of medium. The plates were then stored at - 2 0 ° C , until the protein content of the wells was measured. Determination of prostaglandin E (PGE). PGE was measured in aliquots of incubation medium by radioimmunoassay methods in which free antigen was selectively adsorbed onto dextran-coated charcoal. Two different antisera were used for these studies, since one antibody (supplied by Steranti Research) ceased to be commercially available. However, both methods gave similar results. When using the antiserum supplied by Steranti Research, the assay buffer was 10 m M Tris-HCl (pH 7.4) containing 140 m M NaCI, 0.1% w / v gelatin and 0.02% w / v sodium azide, and 1 vial of lyophilized antiserum was reconstituted in 100 mi buffer. The protocol was as follows: Sample or standard (0.1 ml) was incubated overnight at 4°C with 0.1 ml [3HIPGE (248 Bq) and 0.1 ml antiserum. U n b o u n d PGE was then removed by the addition of 0.2 ml ice-cold assay buffer containing 0.6~ w / v Norit A charcoal and 0.125% w / v dextran. Following incubation at 4°C for 10 min, charcoal was removed by centrifugation for 15 min at 4°C at 2000 × g (ray = 24.5 cm), and 0.2 ml of the supernatant was counted in 4 ml Emulsifier Safe scintillation fluid. When using the antiserum supplied by Sigma Chemicals, the assay buffer was 10 m M sodium phosphate (pH 7.4) containing 150 m M NaCI, 0.1% w / v bovine serum albumin and 0.1% w / v sodium azide, and 1 vial of lyophilized antiserum was reconstituted in 150 ml buffer. The protocol was as follows: Sample or standard (0.1 ml) was incubated for 30 min at 4 ° C with 0.5 ml
153 antiserum. [3H]PGE was then added (0.1 ml, 148 Bq), and the samples incubated overnight at 4°C. Unbound PGE was removed by the addition of 0.2 ml ice-cold assay buffer containing 1% w / v activated charcoal and 0.170 w / v dextran. Following incubation at 4°C for 10 min, charcoal was removed by centrifugation for 15 min at 4°C at 2000 x g (ray = 24.5 cm), and 0.7 ml of the supernatant was counted in 4 ml Emulsifier Safe scintillation fluid. For both assays, results were calculated from standard curves, range. 5-2000 pg PGE2 per tube. Determination of protein. Cell monolayers were solubilized in 1 M NaOH and protein was measured by a modification of the Lowry method, using bovine serum albumin (range 4-20 /~g) as standard [27]. All results were corrected for the protein content of the wells. Activation ofpertussis toxin. Immediately prior to use, 1 #g pertussis toxin was incubated for 30 min at 37°C in 0.5 ml 0.1 M sodium phosphate (pH 7.0), containing 0.5 M NaCI and 20 mM dithiothreitol. Experimental design. Within experiments, each condition was tested in quadruplicate and the results expressed as the mean _+ standard error (S.E.). There was some variation in basal production of PGE between muitiwell plates. Hence control wells (n = 4) were included in each plate, and only results obtained from the same multiwell plate were compared statistically. The significance of differences between mean values was determined using the Student t-test. Experiments were performed at least three times, using cells from different donors, and qualitatively similar results were obtained on each occasion. Data from representative experiments are presented here.
20yM
Materials Tissue culture media, enzymes and antibiotics were purchased from Gibco BRL. Life Technologies. Paisley. Strathclyde. U.K. Foetal calf serum was obtained from Northumbrian Biologicals, Cramlington, Northumberland, U.K. [5,6,8,11.12,14,15(n)-3H]Prostaglandin E 2 was supplied by Amersbam International, Aylesbury, U.K. Norit A charcoal was purchased from Aldrich Chemicals. Gillingham. U.K. Activated charcoal (untreated powder. 250-350 mesh), pp[CH2lpA (lithium salt), p[CH,lppA, ADP, AMP, anti-POE, ATP, bovine serum albumin (fraction V, for protein assay), bovine serum albumin (essentially fatty acid free, for radioimmunoassay of PGE), dextran (approximate average molecular weight, 70000), DL-dithiothreitol, forskolin, GTP, indomethacin, ITP, pertussis toxin, 8-phenyhheophylline, PGE2 and UTP were all supplied by Sigma Chemicals, Poole, U.K. MeSATP (tetrasodium salt) was supplied on behalf of Research Biochemicals, by Semal Technical, St. A1bans, U.K. Anti-PGE was purchased from Steranti Research, St AIbans, U.K. All other chemicals were obtained from BDH Chemicals, Poole, U.K., and were of AR grade or of the highest purity available.
Results In preliminary experiments, it was noted that extracellular ATP stimulated production of PGE, and that PGE accumulated steadily in the medium for 4 h when the concentration of ATP was 100 p.M (data not shown).
t0OpM
""
S00pM
i
Cont ATP ADP AMP Ado Cont ATP AOP AMP Ado Cont ATP ADP AMP Ado
Fig. 1. The effect of adenine nucleotidesand adenosineon the production of PGE by humanarticular chondrocytesin monolayerculture. Rinsed cell monolayerswereincubated for 4 h with adenine nucleotidesor adenosine(Ado). as indicated. Resultsare presented as the mean_+S.E. of four determinations. Valuesof P refer to a comparisonof results for each agonistwithcontrol(Cont). * P < 0.05; " * P < 0.01; * * * P < 0.00l.
154 ATP
== o
so
0
20
50
25O
100
I
500
0
20
50
100
250
500
NUCLEOTIDE CONCENTRATION (JJM)
Fig. 2. Concentration dependence of the responses to ATP and ADP. Rinsed cell monolayers were incubated for 4 h with varying concentrations of ATP or ADP. as indicated. ATP and ADP were tested on different multiwell plates. Results are presented as the mean+S.E, of four determinations.
Thus, in subsequent experiments, P G E p r o d u c t i o n was measured at the end of a 4 h incubation period. Comparison of ATP with other adenine nucleotides and adenosine at concentrations of 2 0 , 1 0 0 and 500 .aM, revealed that A T P and A D P elicited similar responses at each concentration, whereas A M P and adenosine were much less effective (Fig. 1). The response to A T P was always greater than the corresponding response to adenosine ( P < 0 . 0 0 1 ) . Responses to A T P and A D P were concentration-dependent, and were m a x i m a l at 50-100 .aM (Fig. 2). There was some variation between chondrocyte cultures, probably reflecting different rates of metabolism of A T P and A D P at the cell surface. UTP and ITP stimulated p r o d u c t i o n of P G E at concentrations of 20, 100 and 500 #M, and G T P elicited responses at the two higher concentrations. However, CTP was ineffective under these conditions (Fig. 3). The
20JJM
i i i
observed responses were lower than the responses to equivalent c o n c e n t r a t i o n s of A T P ( P < 0.01). M a x t m a l responses were achieved at 5 0 - 1 0 0 .aM UTP, a n d ai 100-250 /~M ITP, whereas the response to G T P increased steadily up to a concentration of 500 .aM (Fig. 4). Again, there was some variation between chondrocyte cultures. A t low c o n c e n t r a t i o n s (1, 5 and 20 /~M), neither M e S A T P nor p p [ C H 2 ] p A elicited any respo':se, b u t at all three concentrations, p [ C H 2 ] p p A induced some s t i m u l a t i o n of P G E p r o d u c t i o n (Fig. 5). W h e n the concentration of agonist was 1 .aM, responses to p [ C H 2 ] p p A a n d A T P were similar, whereas when it was 20 .aM, the response to A T P was greater ( P < 0.05). I n d o m e t h a c i n slightly decreased basal p r o d u c t i o n of P G E and s u b s t a n t i a l l y decreased A T P - s t i m u l a t e d production of P G E (Table I), suggesting that extraceilular
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i i
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I
i °°
i
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e
Cent ATP GTP CTP UTP ITP Cont ATP GTP CTP UTP ITP Cont ATP GTP CTP UTP ITP
Fig. 3. The etfect of nuc!eoside triphosphates c'~n the production of PGE by human articular chondrocytes in monolayer culture. Rinsed cell monolaycrs w:re il;cubated for 4 h with nucleoside triphosphates, as indicated. Results are presented as the mean+S.E, of four determinations. values of P refer to a comparison of results for each agonist with control (Cont). * P < 0.05 * * P < 0.0l ; * * * P < 0.001.
155 loo ! I
~
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i
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il ~'
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20
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:
100 250 500
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100 250 500
NUCLEOTIDECONCENTRATIONl uM)
Fig. 4. Concentration dependence of the responses to GTP, ITP and UTP. Rinsed cell monolayers were incubated for 4 h with varying concentrations of GTP, ITP and UTP, as indicated. Each agonist was tested on a different muhiwell plate. Results are presented as the mean_+ S.E. of four determinations.
ATP was promoting de novo synthesis of PGE. 8-Phenyitheophylline moderately decr..~ased both basal and ATP-stimulated production of PGE (Table I). However, the presence of 8-phenyltheophylline did not decrease the ratio of ATP-stimulated to basal production of PGE, s n ~ e s t i n g that this Pl-purinoceptor antagonist was not directly inhibiting the ATP-stimulated component of the- response. Interestingly, production of PGE by rabbit articular chondrocytes was decreased by treating the cells with forskolin to elevate the intracellular concentration of cAMP [28]. Hence, since 8-phenyltheophyUine could have increased the intracellular concentration of cAMP in h u m a n articular chondrocytes, we investigated the effect of forskolin (10 btM) on both basal and ATP-stimulated production of POE. In the presence and absence of forskolin, respectively, basal
60
1 uM
0,
Cent Li
ATP MeS AMP AMP Cent U ATP CPP PCP
production of PGE was 26.0 +_ 1.64 and 25.9 +__3.49 pg/,ug protein per 4 h, and ATP-stimulated production of PGE (100 ttM ATP) was 89.5 + 11.1 and 95.3 _+ 10.5 p g / ~ g protein per 4 h. Finally, pretreatment of the cells with pertussis toxin also decreased both ATP-stimulated and basal production of PGE (Table II), and although the ratio of ATP-stimulated to basal production of PGE tended to fall, the decrease was not ahvays statistically significant. Discussion These findings indicate the presence of P:-purinoceptors at the surface of h u m a n articular chondrocytes in monolayer culture. Extracellular ATP evoked a response that is normally associated with the activation of
S VM
11
ATP MeS AMP AMP Con! U ATP CPP PCP
20pM ..
ATP MeS AMP AMP ATP CPP PCP
Fig. 5. The effect of ATP and analogues of ATP on the production of PGE by human articular chon¢' ~cytes in monolayer culture. Rinsed cell monolayers were incubated for 4 h with ATP or analogues of ATP, as indicated. Some control wells were treated with lithium (Li) at 2.5-times the concentration of the agonist, because the lithium salt of adenosine 5'-(adi-methylene)triphosphate was used. AMPCPP, adenosine 5'-(a,/i'-methyl-
ene)triphosphate;AMPPCP, adenosine5'-(/],y-methylene)triphosphate.Results are presented as the mean+ S.E. of four determinations.Valuesof P refer to a comparisonof results for each agonistwith control (Cnnt). * P < 0 . 0 5 ; * * P < 0 . 0 l , * * * P < 0 . 0 0 l .
156 TABLE 1 Effect of indomethacin and 8-phenyltheoph_vlline on ATP-stimulated production of PGE
lndomethacin was dissolved in dimethylsulphoxide to give a concentration of 14 mM, and the solution was diluted with incubation medium to give a final concentration of 1.4 .aM. 8-Phenyltheophylline (8-PT) was dissolved in 80% v/v methanol containing 0.2 M NaOH to give a concentration of 10 raM. and the solution was diluted with incubation medium to give a final concentration of 10 .aM. Rinsed cell monolayers were incubated for 4 h with ATP, as indicated, in the presence or absence of indomethacin or 8-phenyltheophylline. Control wells contained 0.0l~ v/v dimetbylsulphoxide for the study of the effect of indomethacin, and 0.08% v/v methanol/0.2 mM NaOH for the study of the effect of 8-phenyhheophylline. Results are presented as the mean _+S.E. of four determinations. PGE (pg/~g protein per 4 h) NoATP Control Indomethacin Control 8-PT
20 #M ATP
8.48 4-1.94 57.6 4-7.82 5.64+1.53 13.1+2.23"* 22.8 +2.55 93.8+9.88 12.4 4-2.48 * 54.14-2.95 **
100/tM ATP 86.6 + 10.2 12.1+ 1.18"** 144 ±14.6 111 + 8.63
Values of P refer to a comparison of concentrations of PGE in the presence and absence of indomethacin or S-phenyltheophylline. as appropliate. * P < 0.05. ** P < 0.01. *** P < 0.001.
P2-purinoceptors, a n d responses to A T P w e r e m u c h g r e a t e r t h a n responses to equivalent c o n c e n t r a t i o n s o f adenosine. However, since A T P c a n b e m e t a b o l i z e d a t the surface of these cells to A M P a n d a d e n o s i n e [21|, the involvement o f activation o f P~-purinoceptors in m e d i a t i n g p a r t o f this response c a n not b e entirely ruled out. T h e results with 8 - p h e n y l t h e o p h y l l i n e are s o m e w h a t curious, b u t d o n o t a p p e a r to r e f u t e the c o n t e n t i o n t h a t A T P was a c t i n g directly at P2-purinoceptors. T h e r e a r e several m e c h a n i s m s w h e r e b y 8 - p h e n y l t h e o p h y l l i n e c o u l d influence b o t h b a s a l a n d A T P - s t i m u l a t e d p r o d u c t i o n o f
P G E . Firstly, 8 - p h e n y l t h e o p h y l l i n e m i g h t have inhibited production of PGE by human articular chondrocytes by raising the i n t r a c e l l u l a r c o n c e n t r a t i o n o f c A M P via inh i b i t i o n o f the activity o f c A M P phosphodiesterase(s). H o w e v e r , this possibility w o u l d a p p e a r to be ruled o u t b y the o b s e r v a t i o n t h a t forskolin d i d not affect either b a s a l o r A T P - s t i m u l a t e d p r o d u c t i o n o f P G E b y hz.man a r t i c u l a r c h o n d r o c y t e s in m o n o l a y e r culture. Secondly, it h a s b e e n o b s e r v e d t h a t 3 - i s o b u t y l - l - m e t h y l x a n t h i n e at c o n c e n t r a t i o n s in excess o f 0.1 m M inhibits the activity o f c y c l o o x y g e n a s e [29], b u t it is n o t k n o w n w h e t h e r 8 - p h e n y l t h e o p h y l l i n e a t the c o n c e n t r a t i o n u s e d in this s t u d y (10 ILM), also inhibits the enzyme. T h i r d l y , the i n h i b i t o r y effect o f 8 - p h e n y l t h e o p h y l l i n e c o u l d indicate that both basal and ATP-stimulated production of P G E are e n h a n c e d b y a c t i v a t i o n o f P~-purinoceptors b y e n d o g e n o u s l y p r o d u c e d a d e n o s i n e . T h e p r e s e n c e o f Ptp u r i n o c e p t o r s in h u m a n a r t i c u l a r c h o n d r o c y t e s d o e s n o t a p p e a r to h a v e b e e n investigated. H o w e v e r , it has b e e n r e p o r t e d t h a t a c t i v a t i o n o f Pt-like p u r i n o c e p t o r s in the r a t t h y r o i d cell line, F R T L - 5 , permissively e n h a n c e s r e s p o n s e s to a c t i v a t i o n o f P2-purinoeeptors [30]. Clearly, possible i n t e r a c t i o n s b e t w e e n P~-purinoceptors a n d P2p u r i n o c e p t o r s in h u m a n a r t i c u l a r c h o n d r o c y t e s s h o u l d be explored further. Pertussis t o x i n a l s o a p p e a r e d to a c t p r i m a r i l y o n basal production of PGE by human articular chondrocytes in m o n o l a y e r culture, a n d it w a s n o t entirely c l e a r w h e t h e r it h a d a n a d d i t i o n a l effect o n the A T P - m e d i a ted c o m p o n e n t o f t h e response. Interestingly, pertussis toxin inhibited both basal and ATP-stimulated production o f P G D b y p r i m a r y c u l t u r e s o f r a t a s t r o e y t e s [31]. A g a i n , o n e e x p l a n a t i o n f o r o u r f i n d i n g s is t h a t a n o t h e r signalling p a t h w a y , w h o s e activity d e t e r m i n e s b o t h b a s a l and ATP-stimulatod production of PGE, contains a pertussis toxin-sensitive O - p r o t e i n . It is n o t e w o r t h y t h a t in F R T L - 5 cells, a pertussis toxin-sensitive G - p r o t e i n w a s involved in the t r a n s d u c t i o n o f the signal f r o m the Pt-like p u r i n o c e p t o r s , b u t n o t f r o m the P 2 - p u r i n o c e p t o r s [30]. T h e i d e n t i t y o f this p u t a t i v e s e c o n d signalling
TABLE 11 Effect of pretreatment with pertussis toxin on A TP-stimulated production of PGE
Cells were treated overnight with 20 ng/ml dithiothreitol-activated pertussis toxin (PT), and control wells were preincubated with an equivalent concentration of dithiothreitol (0.2 mM). Rinsed cell monolayers were then incubated for 4 h with ATP, as indicated. Each concentration of ATP was tested on a different multiwell plate. Results are presented as the mean 4-S.E. of four determinations. PGE (pg/p.g protein per 4 h) Untreated. no ATP Untreated, 20 .aM ATP PT-treated. no ATP PT-treated, 20 b~M ATP
18.1 + 0.83 42.1 4-2.32 16.1 + 0.20 28.1 + 1.06 * *
Untreated, no ATP Untreated, 100 .aM ATP PT-treated, no ATP PT-treated, 100 pM ATP
27.3 + 1.98 61.24- 2.44 18.7 + 1.79 * 37.1 :t: 1.15 * * *
Untreated, no ATP Untreated, 500 .aM ATP PT-treated, no ATP PT-treated, 500/~ M ATP
Values of P refer to a comparison of results for pertussis toxin-treated cells with those for the corresponding untreated cells. * P < 0.05. ** P < 0.01. P < 0.001.
37.4.4-1.81 65.8 + 2.18 26.6 + 1.43 * * 36.6 + 5.35 * *
157 pathway in human articular chondrocytes is not known, but coupling of Pl-purinoceptors of the Al-subclass (inhibitory) to pertussis toxin-sensitive G-proteins has been widely documented [32]. An alternative explanation for our findings is that phospholipase A 2 is itself coupled via a pertussis toxin-sensitive G-protein in human articular chondrocytes, since it has been demonstrated that transducin is directly involved in the activation of this enzyme in rod outer segments of bovine retina [331. As human articular chondrocytes in monolayer culture metabolize ATP at their surface, it is difficult to assess the sensitivity of the receptor. However, in three experiments using low concentrations of ATP, production of POE was stimulated significantly on each occasion by 5 .ttM ATP, and on one occasion by 1 iaM ATP. This suggests that the receptor could be sensitive to very low concentrations of ATP in vivo. It is also difficult to define the specificity of the receptor because of the metabolism of agonists. However, the limited effect of p[CH2]ppA, and the lack of effect of MeSATP and pp[CH2]pA, suggest that the receptor can not be designated a s P2x or P2y. This is not entirely unexpected, since it was noted in the introduction that not all P2-purinoceptors can be classified on this basis. The ability of GTP, ITP and UTP to elicit responses indicates that the receptor is not specific for the adenine base or for pudnes, or alternatively, that there is more than one type of receptor for nucleoside triphosphates present at the surface of human articular chondrocytes. Interestingly, evidence has recently been presented for the presence of separate receptors for ATP and UTP at the s u r f a c e of human HL60 cells [34]. It is not possible, at present, to define the role of P2-purinoceptors in human articular cartilage, but it is now recognized that articular chondrocytes can respond to many systemically and locally produced agents. For example, recent studies have demonstrated the presence of receptors for serotonin, dopamine and histamine in human articular chondrocytes [35,36], and for bradykinin in pig articular chondrocytes [37]. The extraceUular concentration of ATP in human articular cartilage is not known. However, it is probably considerably higher than concentrations in plasma and synovial fluid, which a r e of the order of 50 nM [38,39], because ATP is metabolized at the surface of human articular chondrocytes [21] and of many other types of cell [20]. The sources of extracellular ATP in human articular cartilage are also not known, but since this tissue is not innervated [40], release of ATP from nerve endings is ruled out. Articular chondrocytes synthesize and secrete the components of the cartilage extracellular matrix [41], and small amounts of ATP could be released in association with these macromolecules. In addition, human articular chondrocytes might specifically secrete ATP in response to certain stimuli, since there are
mechanis/ns for the release of ATP from some types of cell, e.g., porcine and human vascular endothelial cells [42.43], and rat heart cells [44,45]. There could be enhanced release of ATP from human articular chondrocytes in pathological situations, and the concentration of ATP in synovial fluid from subjects with chondrocalcinosis is elevated [39]. Release of ATP may be increased when human articular chondrocytes are promoted to repair the matrix following physical injury or enzymic destruction, if ATP is secreted in association with macromolecules. Physical injury to the cells themselves could also result in release of ATP (along with other intracellular components). There is circumstantial evidence that this latter process occurs, because chondrocalcinosis is sometimes associated with previous tissue injury [46,47], and the PPi which is deposited may have arisen from the action of ecto-NTP pyrophosphatase on ATP released from damaged cells. in conclusion, we have provided evidence that human articular chondrocytes in monolayer culture, express P.,-purinoceptors at their surface, which are sensitive to low concentrations of ATP. Such receptors could therefore be activated in vivo, One consequence of our findings is a possible mechanism for the observed association between chondrocalcinosis and osteoarthritis [47,48], since ATP released from articular chondrocytes, can both induce alterations in cellular metabolism through its action at P,-purinoceptors, and serve as a source of PPi. Furthermore, we have recently observed that extracellular ATP stimulates resorption of proteoglycans from bovine nasal cartilage [49], which suggests that activation of P2-purinoceptors in chondrocytes may have wide-ranging biological consequences, Acknowledgement We are grateful to the Wellcome Trust, U.K. for financial support for this work. References
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