THE CONTROL OF INTRACELLULAR PH IN CULTURED AVIAN CHONDROCYTES

1. Mechanical loading of cartilaginous tissue generates an increase in the concentration of cations in the extracellular matrix. This includes a decrease of the extracellular pH (pH(o)), which is known to affect the intracellular pH (pH(i)), thereby modifying the intracellular metabolism. Thus, the regulation of pH(i) is essential for the physiological function of cartilage. The fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF AM) was employed in order to assess the mechanisms responsible for control of the pH(i) in an embryonic avian chondrocyte cell suspension. 2. Steady-state pH(i) in the absence of physiological HCO3- was 7.15+/-0.01 pH units as compared to a pH(i) of 6.94+/-0.02 pH units in its presence (P < 0.01). The intrinsic buffering power of chondrocytes (beta(i)) was 38-9 mm/pH unit and the total buffering capacity (beta(T)) was 65.8 mm/pH unit. 3. Cells maintained in a Hepes-buffered solution were exposed to an intracellular acid load by the NH4+ prepulse technique (20 mm NH4Cl). The initial rate of pH(i) recovery was 0.106 pH units/min (n = 18). Amiloride (0-33 mm), an inhibitor of the Na+-H+ exchanger, or replacement of external sodium [Na+]o with choline induced a 60 % inhibition of the recovery rate, indicating a predominant involvement of this antiporter in the response to intracellular acidification. 4. H+-ATPase inhibitors (oligomycin 20 mug/ml; NN;-dieyelohexylcarbodiimide (DCC), 0.5 mm; N-ethylmaleimide (NEM), 0.25 mm) and iodomycin (2 mM), a metabolic cell suppressor, reduced acid extrusion by 25 % as measured by the NH4Cl prepulse in Hepes-bathed cells. 5. Chondrocytes transferred from a Hepes-buffered solution to a 5 % CO2 - 25 mm HCO3- medium (HCO3- solution) underwent a pH(i) decrease of almost-equal-to 0.20 pH units, followed by a regulatory alkalinizing response of 0.118 pH units/min. The Na+-H+ exchanger was responsible for only 15 % of this alkalinization (amiloride, 0.33 mm), in contrast to its primary role in HCO3--free solution. 6. The activity of a Na+-dependent Cl--HCO3- exchanger in physiological HCO3- solution was estimated by addition of the inhibitors 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS; 0.5 mm) or diisothiocyanatostilbene 2.2'-disulphonic acid (DIDS: 100 mum) and by the suspensions of chondrocytes in a Na+-free solution. Acidification performed under these conditions resulted in a 45% inhibition of the recovery rate as compared to control rates. Since SITS addition to cells kept in gluconate medium and depleted of intracellular chloride was without effect. it is concluded that a Na+ HCO3- co-transporter does not operate under the experimental conditions. 7. H+-ATPase in physiological HCO3- solution accounts for 25% of the acid extrusion (oligomycin 20 pg/ml: DCC 0.5 mm). About 15% of the extrusion rate is due to other unidentified processes. 8. Exposure of chondrocytes to a Cl--free HCO3- solution caused an alkalinization response inhibitable by stilbene derivatives. Comparably, pH(i) alkalinization induced by 20 mm NH4Cl resulted in a regulatory acidifying response independent of [Na+]. and inhibitable by stilbene derivatives. The results indicate that a Na+-independent Cl--HCO3- exchanger is the main chondrocytes acid loader. 9. Cells were maintained in a HCO3- solution and the pH(o) was varied by acid addition (pH. values of 7.05 and 6.55). After an initial acidification, the pH(i) returned within 5.15 min to its initial value of about 6.90. We conclude that in avian chondrocytes a pH(o) decrease is opposed by effective acid extrusion mechanisms in order to reduce pH(i) variations. A hypothesis of proteoglycans metabolism control through mechanical stress-induced pH. alterations and an ensuing pH(i) regulation is suggested.