Sodium butyrate-induced structural and functional modifications in proteins of cultured rabbit articular chondrocytes.

The effects of sodium butyrate (NaB), a potent growth inhibitory agent, on actin distribution, alkaline phosphatase (AP) activity and protein content were studied in rabbit articular chondrocytes in monolayer culture. When growth of randomly proliferating cells was arrested with NaB, actin stress fibers appeared; at the same time, vimentin-containing intermediate filaments and tubulin-containing microtubules were dispersed. Concomitantly, membrane AP activity and protein content were increased. Such effects support the hypothesis that NaB affects the expression of many proteins by modification of gene expression, probably at the transcriptional level.

Effects of retinoic acid on the growth of cultured rabbit articular chondrocytes: Relation with alkaline phosphatase activity and beta receptor.

The effects of retinoic acid (RA) on rabbit articular cartilage cells were studied for concentrations ranging from 5.10(-5) M to 10(-7) M; the treatment with RA over three days resulted in dose dependent inhibition of chondrocyte proliferation between 5.10(-5) and 10(-5) M with persistence of the inhibitory effect until 10(-6) M. RA until 10(-7) M induced a slight, but significant, enhancement of cell proliferation. This growth stimulating effect seems to be related to the Beta receptor system because Beta blockers, such as sotalol and DL propranolol, were able to suppress the stimulating action of agonist type isoprenaline. The activity of alkaline phosphatase (AP) was also determined. The highest dose of RA (5.10(-5) M) induced an increase (x 3) of AP activity, and 10(-7) M RA decreased it (x 0.4).

G2 arrest, binucleation, and single-parameter DNA flow cytometric analysis.

One important facet of flow cytometry involves the effects of pharmacological agents on cell cycle progression. Comparative G2 fraction perturbations were examined: effects of sodium butyrate on articular chondrocytes, effects of an antineoplastic agent (SOAZ) and an antirheumatic drug (D-penicillamine) on HeLa cells. Even though DNA flow cytometric analysis detects preferentially an induction of G2 arrest, the mode of action of these agents on the cell cycle is different. Sodium butyrate and D-penicillamine lead to an increase of binucleate cells due to cytokinesis perturbation. Because of similar fluorescence intensity, distinguishing G2 from binucleate GO/1 cells is not easily possible using DNA content measurement and reflects a failure of flow cytometry in the detection of binucleate cells. Rapid cell cycle analysis of single cells should contribute greatly to the study of pharmacological interactions, but DNA flow cytometric measurements obtained from cultured cells exposed to certain agents must be cautiously interpreted because those may interact on cytokinesis and induce artefacts in histogram interpretation.

Age-related changes in rabbit articular chondrocytes.

Cell culture techniques have been used extensively in the study of the aging process at the cellular level. The "senescent" articular chondrocyte seems to be a good model to examine the responses to aging in osteoarthritis, one of the most frequent diseases of old age. Thus in vitro chondrocyte "senescence", established by weekly subculture was characterized by a declining proliferation rate during late passages, from a rapid growth rate in early subculture to a complete loss of the proliferation capacity after 8 +/- 1 passages. Flow cytometric analysis show a time course decrease in the fraction S and G2 + M during the subculture, and a concomitant enhancement in protein content related to the increase of cell size. The immunocytochemistry assays revealed an appearance of a rigid cytoarchitecture with an increase in the number, and organization, of three cytoskeletal components: actin, tubulin and vimentin. The cultured chondrocytes therefore undergo in vitro aging analogous to that described for diploid fibroblasts, and could constitute a cellular model for pharmacological and toxicological assays.

Sodium butyrate-induced changes in cultured rabbit articular chondrocyte transmembrane electrical potentials. Relationship between these changes and the proliferative response.

Sodium butyrate at 5mM reversibly induced a significant increase of transmembrane potentials (Em) in normal chondrocytes (24 hours after seeding) and arrested their proliferation. This increase in Em levels, which could be temporarily abolished by Tetra-ethyl Ammonium (TEA 5mM), was related to an increase in membrane permeability to K+. This hyperpolarization was correlated with the reversible inhibition of growth in G1 induced by the sodium butyrate.

Effects of sodium butyrate on growth and cell-cycle kinetics of cultured rabbit articular chondrocytes.

The sodium salt of n-butyric acid was found to inhibit the growth of asynchronous cultures of rabbit articular chondrocytes. This inhibitory effect was dose-dependent between 1 mM and 5 mM, reversible, and accompanied by volume enhancement and modification of cellular morphology. Flow-cytometric analysis showed that drug exposure led to a slowing-down of the cell-cycle progression; after 1 day's exposure, cells accumulated in G1, and after 2 or 3 days' treatment, in G2, without a blockage in M; the increase of cells in G2 was in fact due to an enhancement of binculeated cells. The treated cells had an increased RNA content. Articular chondrocytes seem to be target cells for sodium butyrate and therefore it represents a valuable biological tool for studying the mechanisms of their growth regulation.

[Modification of the growth of chondrocytes by the action of concanavalin A]. / Modulation de la croissance des chondrocytes sous l'influence de la concanavaline A.

The effects of Concanavalin A have been assessed on the growth of articular cultured chondrocytes. A differential action in relation with cellular density has been observed. Concanavalin A (0.02, 0.2 and 2 micrograms/ml) exerted a stimulating effect on low density cultures, whereas it depressed the cell growth curve with high cell density cultures, especially at the concentration of 2 micrograms/ml. Such a discrepancy in the behaviour of cells related to the density does not seem to have been previously observed with chondrocytes. It could be explained by a modification of the accessibility of Concanavalin A receptors.

[Aggregation of macrophages by a human lymphokine (MAgF). Role of the eicosanoid system]. / Agrégation des macrophages par une lymphokine humaine (MAgF). Rôle du système des eicosanoides.

Peritoneal exudate cells aggregate when exposed to the lymphokine Macrophage Aggregating Factor (MAgF). The role of prostaglandins in the aggregation of these cells has been investigated with a quantitative assay. Results are discussed in the context of the mode of action of the migration inhibiting factor (MIF) and of the other non immune aggregating stimuli such as C5a and fMLP. Prostaglandins E1, E2, and F2 alpha alone did not cause aggregation of macrophages but partially inhibited the MAgF aggregation of macrophages and this effect was not different from the one obtained with MAgF. MAgF, calcium ionophore A23187 and arachidonic acid induced aggregation were blocked by 5, 8, 11, 14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism, by indomethacin (10(-4), 10(-6) M) by corticosteroids (dexamethazone, methylprednisolone, hydrocortisone) but not by aspirin (10(-2), 10(-4) M) nor by phenylbutazone (10(-4), 10(-5) M). These results suggest a causal relationship between MAgF induced macrophage aggregation and that due to other stimuli with respect to the derivatives of arachidonic acid. The lipoxygenase pathways metabolites stimulate aggregation whereas the cyclo-oxygenase pathways metabolites inhibit it.

Lymphokine-induced macrophage aggregation: involvement of cyclic-GMP and microtubules.

Human lymphokine (LK) is known to induce guinea pig macrophage aggregation. This effect can be quantitatively measured with a Born modified platelet aggregometer. This method has been well correlated with the state of delayed hypersensitivity. Previous findings about the mechanism of action of the aggregation indicated that this aggregation is not due to an increase in the cellular level of c-AMP. It is doubtful whether c-AMP is a messenger of the LK action. Using a radio-immunoassay, small but significant increases were found in c-GMP levels of LK-aggregated macrophages. In addition, exogenous dibutyryl c-GMP and carbamylcholine induced a macrophage aggregation, as did the divalent cation ionophore A-23187. These data, together with the fact that LK-induced macrophage aggregation is inhibited by colchicine and at 0 degree C, suggest that microtubule polymerization is involved in this process.

Lymphokine-induced Macrophage aggregation: the possible role of cyclic nucleotides.

The possible role of cyclic nucleotides in guinea pig macrophage aggregation, induced by human lymphokine (LK) has been investigated. Small increases were found in guanosine 3'5'-cyclic monophosphate (cGMP), but not adenosine 3'5'-cyclic monophosphate (cAMP), levels of lymphokine aggregated macrophages. Addition of exogenous dibutyryl (DB) cAMP, L-isoproterenol, or theophylline did not induce macrophage aggregation. By contrast, both exogenous DBcGMP and carbamyl-choline induced a macrophage aggregation; although DBcGMP was more effective. In addition, both L-isoproterenol and DBcAMP in the presence of theophylline decreased LK-induced macrophage aggregation, whereas D-isoproterenol and DBcGMP had no effect. The results obtained here are discussed in the context of the previously reported effects of cyclic nucleotides on migration inhibitory factor (MIF) activity and the possible role of these agents in the mechanism of action of MIF.

Anti-beta adrenoreceptor blockade activity of plasma ultrafiltrate in two uraemic patients: effect of parathyroidectomy.

A possible interaction between d-1 propranolol and hyperparathyroid plasma ultrafiltrate on guinea pig auricles has been studied in "in vitro" experiments. Plasma ultrafiltrates have been samples in two patients on chronci haemodialysis before (pre-PTx) and after (post-PTx) parathyroidectomy. A significant inhibition of propranolol depressant activity on cardiac contractile strength has been observed in the presence of pre-PTx plasma ultrafiltrates. On the contrary, no such inhibition was noted in the presence of post-PTx plasma ultrafiltrates.