An hypothesis for the interpretation of the contractile response of vascular smooth muscle at the cellular level.

The long preservation and recovery of functional (contractile) properties in cultured aortic smooth muscle cells, even after replating or deep-frozen storage and the measurement of their responses are now technically settled issues. We could thus study extensively the responses of single cultured cells from rat thoracic aorta. Responses were elicited by the addition of KCl 40 mmol/L without or with a calcium blocker PN 200-100 (10(-6) mol/L); angiotensin II (10(-11)-10(-6) mol/L) without or with antagonist (losartan 10(-5) mol/L); or serotonin (10(-9)-10(-4) mol/L) without or with antagonist (naftidrofuryl 10(-5) mol/L). Results thus obtained enabled us to propose a new hypothesis for the interpretation of the contractile responses of an elastic vascular smooth muscle. The different maximal effects of different agonists result mainly from the different proportions of cells they can mobilize; the agonist concentration-contraction relationship is mainly due to the increase of the proportion of cells involved up to a maximal value typical of the agonist used. An antagonist primarily reduce the proportion of cells an agonist can mobilize. Some of the consequences of this hypothesis are briefly outlined.

Use of a renal tubule cell line (LLC-PK1) to study the nephrotoxic potential of a kappa-type Bence-Jones protein.

The cytotoxicity of a Bence-Jones protein was assessed using a porcine renal tubule cell line (LLC-PK1), with the aim of developing a model for studying the potential nephrotoxicity of these proteins. The effects of a kappa Bence-Jones protein on cell viability were studied by means of biochemical methods (supravital dye uptake and measurement of cellular enzyme activities) and morphological electron microscopy. After a 24-h-treatment with Bence-Jones protein, a moderate cytotoxicity (about 15%) was noted but only a minor difference compared to treatment with bovine albumin in the same conditions. The morphological study showed a few cells in the process of lysis, but their numbers were insufficient for the demonstration of a clear cytotoxic effect. Immunocytochemical studies showed Bence-Jones protein fixation on some cells, especially on the outer membrane. Labeling of the hyaloplasm and basal pole of a few cells pointed to internalization of protein by LLC-PK1 cells. Although the cytotoxicity of the Bence-Jones protein tested here was only moderate, the use of this model enabled its cytotoxic effect to be distinguished from that of beta-lactoglobulin. This isolate could serve as a "moderate control" for a later study with a BJP having caused acute renal failure.

Mitochondrial biogenesis in rabbit articular chondrocytes transferred to culture.

The effect of the switch to aerobic growth conditions was examined in rabbit articular chondrocytes transferred to culture. Spectroscopic analysis of the cytochromes of the respiratory chain shows that only cytochrome b is present in chondrocytes from cartilage, cytochromes c, c1, and a.a3 being undetectable as compared with the typical spectrum found in a primary cell culture on day 4. Steady state levels of RNA transcripts of nuclear (cytochrome c) and mitochondrial genes (cytochrome b and cytochrome oxidase subunits II and III) involved in the oxidative metabolism were determined relative to the RNA transcripts of the nuclear gene for glyceraldehyde phosphate dehydrogenase involved in the glycolytic pathway and to mitochondrial ribosomal RNAs. Chondrocytes transferred to culture showed a general increase in the levels of all transcripts, but the effect on mitochondrial transcripts was much greater (x 20) than the effect on nuclear transcripts (x 3-4). These results show the absence of a coordinate regulation of the expression of mitochondrial and nuclear genes coding for components of the respiratory chain. The increase in mitochondrial DNA triggered by culture conditions does not appear to be sufficient to account for the enhanced transcription. Concomitant with these mitochondrial changes, the level of transcripts for the collagen II gene involved in the differentiation function decreases dramatically (3% of the control on day 3).

Compared flow cytometric analysis of mitochondria using 10-n-nonyl acridine orange and rhodamine 123.

The use of the supravital mitochondrial-specific dye Rhodamine 123 (Rh 123) in combination with flow cytometry permits the monitoring of the changes in the mitochondrial transmembrane potential, reflecting the overall mitochondrial activity of the living cell. While this probe appears to be a potent tool for these studies, it also exhibits an important limit in the interpretation of the results: it cannot distinguish between an increase in mitochondrial activity without biogenesis and a modification of mitochondrial content. 10-n-Nonyl Acridine Orange chloride (NAO) constitutes another mitochondrial specific fluorochrome. In contrast with Rh 123, NAO accumulation in the cell does not seem to be driven by the proton-motrice force but does seem to be related to specific interactions with mitochondrial membrane proteins and/or lipids. In this work, the cytotoxicity of NAO, the kinetics of cellular uptake and the release of the dye have been determined using flow cytometry. The use of several ionophores or mitochondrial inhibitors has confirmed the independence of NAO uptake regarding mitochondrial transmembrane potential. NAO was also used to examine the changes in the mitochondrial compartment during the transfer of articular chondrocytes from cartilage to the culture conditions, where Rh 123 evidenced changes in mitochondrial activity and/or biogenesis, in order to know whether the use of probes with different specificity allows one to distinguish between mitochondrial activity and biogenesis.

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).

Subculture of rabbit articular chondrocytes within a collagen gel: growth and analysis of differentiation.

Primary cultures of rabbit articular chondrocytes have been subcultured within three-dimensional (3D) collagen gels. Under these conditions, the cells remained viable and divided, but with a lower proliferation rate than that observed in control monolayer cultures. Flow cytometric analysis of progression of the cells into the cell cycle has confirmed and extended these findings. Also the cellular volume was decreased in 3D-culture, being in the same range as thein vivo size of cartilage cells. Specific staining for proteoglycans and type II collagen immunolocalization on sections of gels showed the expression of differentiated phenotypes and revealed the accumulation of these matrix components in the immediate surroundings of the cells. The use of Ultroser G (a serum substitute) improved the conditions for 3D- culture of rabbit articular chondrocytes.

Rhodamine 123 uptake and mitochondrial DNA content in rabbit articular chondrocytes evolve differently upon transfer from cartilage to culture conditions.

Mitochondrial DNA (mtDNA) represents 0.15% of the total cell DNA (at least an order of magnitude less than in liver or heart) of rabbit articular chondrocytes. Besides the already well-documented low respiratory activity, chondrocyte differentiation thus involves a specific control of mitochondrial biogenesis. When transferred to in vitro conditions, chondrocytes increase their stock of mtDNA at the same time they resume growth, even more efficiently (8 times) than they do for cell volume (4.4 times). On the contrary, overall mitochondrial activity, estimated as the uptake of rhodamine 123, does not follow the same trend (2.5 times increase). Chondrocytes apparently keep these functional characteristics for some generations in culture.

Mitochondrial uptake of rhodamine 123 by rabbit articular chondrocytes.

Rhodamine 123 was used to stain and analyze by flow cytometry the mitochondria of rabbit articular chondrocytes. Stationary primary cultures and exponentially growing subcultures were compared to enzymatically released chondrocytes from cartilage. The increase in mitochondrial fluorescence, when chondrocytes are transferred from cartilage to culture environment, is suggestive of some change in chondrocyte adaptation and/or differentiation in these conditions.

Mitochondrial analysis in living cells: the use of rhodamine 123 and flow cytometry.

Flow cytometry combines the advantages of microscopy and biochemical analysis in a single highly sensitive technique for a rapid examination of numerous individual living cells. It has become a potent and essential tool in the studies of the physiology of the whole cell and its organelles. Rhodamine 123 is a vital fluorescent dye used in flow cytometry. As it is specifically concentrated in mitochondria because of the transmembrane potential that these organelles maintain in living cells, rhodamine 123 is thus a useful probe for monitoring the abundance and activity of mitochondria. A critical survey of the routine use of rhodamine 123 together with flow cytometry in mitochondrial research is presented.