Biomedical subjects

I M Shapiro

Publications and source records attributed to I M Shapiro.

At least 55 records · Page 3Linked to original sources

PubMed1997

Extracellular ATP modulates [Ca2+]i in retinoic acid-treated embryonic chondrocytes.

When treated with low doses of retinoic acid (RA), cephalic chondrocytes of the chick embryonic sternum mature and express phenotypic characteristics of postmitotic hypertrophic cells. In concert with these maturation-dependent changes, cells release adenine nucleotides into the culture medium. To ascertain if these compounds modulate chondrocyte function, we challenged chondrocytes with nucleotides and measured one determinant of the signal transduction pathway, intracellular Ca2+ concentration ([Ca2+]i). In the presence of micromolar concentrations of ATP, there was a dose-dependent elevation in chondrocyte [Ca2+]i; ADP caused a small but significant rise in the peak [Ca2+]i response. We found that the change in the [Ca2+]i response is linked to retinoid-dependent maturation of chondrocytes. Thus the [Ca2+]i rise was dependent on the RA concentration and treatment time. Immature caudal chondrocytes, cells that were not affected by RA, were used as control cells for this study. When treated with ATP, these cells did not exhibit a [Ca2+]i response. Although the purinergic subtype receptor was not characterized, the observation that cells responded to ATP and ADP but were refractory to AMP and adenosine suggested that P2 purinoceptors were expressed by chondrocytes. Because, during the same culture period, chondrocytes exhibited many of the unique characteristics of the terminally differentiated cell, the acquisition of purinergic receptors represents a new feature associated with expression of the mature phenotype. Finally, to ascertain if the ATP-dependent response was due to release of Ca2+ from intracellular stores, cells were treated with thapsigargin. Since this compound significantly reduced the [Ca2+]i signal, we concluded that the ATP response is mediated by release of cation, from the endoplasmic reticulum.

Adenosine Diphosphate↗

PubMed1997

Further observations on programmed cell death in the epiphyseal growth plate: comparison of normal and dyschondroplastic epiphyses.

The objective of the investigation was to provide information on apoptosis in the normal epiphysis and to assess apoptosis in the plate of the dyschondroplastic chick. Apoptosis was evaluated using two terminal deoxynucleotide transferase end-labeling procedures, DNA fragmentation and nuclear morphology. We found that there was a minimal level of apoptosis in the dyschondroplastic cartilage. In the tibial dyschondroplastic (TD) lesion itself, only about 3% of cells are positive in the articular and proliferative regions; 11% of prehypertrophic chondrocytes are stained by the end-labeling procedure, and most of the cells are localized around vascular channels at the calcifying front. This finding suggests that dyschondroplasia is linked to impairment of apoptosis, and as a result the tissue contains immature cells that have outlived their normal life span. In contrast, in the normal plate, we noted that when the proliferative period was complete, the cells became terminal transferase positive; in addition, chondrocytes in the normal plate exhibited DNA fragmentation. Semiquantitative analysis of stained chondrocytes in the growth plate indicate that in the proliferative zone 15.5% of cells are terminal deoxynucleotidyl transferase (TUNEL) positive; in contrast, 44% of postmitotic chondrocytes are stained by the TUNEL procedure. The presence of a sharp border between the pre- and postmitotic zones suggests that the stimulus for apoptosis is maturation dependent and reflects local metabolic control. We also examined apoptosis in metaphyseal osteoblasts. We found that adjacent to the epiphysis, many osteoblasts were undergoing apoptosis. In more mature sites in the metaphysis, there was less cell death, indicating that osteoblast apoptosis was delayed and cells were completing their normal life cycle. Although terminal transferase end-labeled cells were not seen in articular cartilage, we noted that fibroblasts, in the perichondrial ligament surrounding the articular as well as the epiphyseal regions of the plate, were undergoing apoptosis. Apoptosis at this site may be related to lateral expansion of the cartilages, reflect a high cell turnover rate at the junction between the tissues, and result from paracrine signals received from the underlying cartilage.

Animals↗

PubMed1996

Retinoic acid modulation of glutathione and cysteine metabolism in chondrocytes.

The major objective of this investigation was to determine the thiol status of chondrocytes and to relate changes in the level of glutathione and cysteine to maturation of the cells as they undergo terminal differentiation. Chondrocytes were isolated from the cephalic portion of chick embryo sterna and treated with all-trans retinoic acid for one week. We found that the addition of 100 nM retinoic acid to the cultures decreased the intracellular levels of glutathione and cysteine from 6.1 to 1.6 and 0.07 to 0.01 nmol/microgram DNA respectively; retinoic acid also caused a decrease in the extracellular concentration of cysteine. The decrease in chondrocyte thiols was dose and time dependent. To characterize other antioxidant systems of the sternal cell culture, the activities of catalase, glutathione reductase and superoxide dismutase were determined. Activities of all of those enzymes were high in the retinoic acid-treated cells; the conditioned medium also contained these enzymes and the cytosolic isoenzyme of superoxide dismutase. We probed the specificity of the thiol response by using immature caudal chondrocytes. Unlike the cephalic cells, retinoic acid did not change intracellular glutathione and extracellular cysteine levels, although the retinoid caused a reduction in the intracellular cysteine concentration. Finally, we explored the effect of medium components on chondrocyte thiol status. We noted that while ascorbate alone did not change cell thiol levels, it did cause a 4-fold decrease in the extracellular cysteine concentration. When retinoic acid and ascorbic acid were both present in the medium, there was a marked decrease in the level of glutathione. In contrast, the phosphate concentration of the culture medium served as a powerful modulator of both glutathione and cysteine. Results of the study clearly showed that there is a profound decrease in intracellular levels of both cysteine and glutathione and that thiol levels are responsive to ascorbic acid and the medium phosphate concentration. These findings point to a critical role for thiols in modulating events linked to chondrocyte maturation and cartilage matrix synthesis and mineralization.

Animals↗

PubMed1996

Adaptation of chondrocytes to low oxygen tension: relationship between hypoxia and cellular metabolism.

In endochondral bone, the growth cartilage is the site of rapid growth. Since the vascular supply to the cartilage is limited, it is widely assumed that cells of the cartilage are hypoxic and that limitations in the oxygen supply regulate the energetic state of the maturing cells. In this report, we evaluate the effects of oxygen tension on chondrocyte energy metabolism, thiol status, and expression of transcription elements, HIF and AP-1. Imposition of an hypoxic environment on cultured chondrocytes caused a proportional increase in glucose utilization and elevated levels of lactate synthesis. Although we observed a statistical increase in the activities of phosphofructokinase, pyruvate kinase, lactate dehydrogenase, and creatine kinase after exposure to lowered oxygen concentrations, the effect was small. The cultured cells exhibited a decreased utilization of glutamine, possibly due to down regulation of mitochondrial function and inhibition of oxidative deamination. With respect to total energy generation, we noted that these cells are quite capable of maintaining the energy charge of the cell at low oxygen tensions. Indeed, no changes in the absolute quantity of adenine nucleotides or the energy charge ratio was observed. Hypoxia caused a decrease in the glutathione content of cultured chondrocytes and a concomitant rise in cell and medium cysteine levels. It is likely that the fall in cell glutathione level is due to decreased synthesis of the tripeptide under reduced oxygen stress and the limited supply of glutamate. The observed rise in cellular and medium cysteine levels probably reflects an increase in the rate of degradation of glutathione and a decrease in synthesis of the peptide. To explore how cells transduce these metabolic effects, gel retardation assays were used to study chondrocyte HIF and AP-1 binding activities. Chondrocyte nuclear preparations bound an HIF-oligonucleotide; however, at low oxygen tensions, no increase in HIF binding was observed. In addition, we found that AP-1 binding activities in chondrocytes exposed to low oxygen tensions was elevated, although the response was lower than that exhibited by fibroblasts exposed to the same range of oxygen concentrations. We compared these results to HIF and AP-1 binding by cells in the growth plate. There was strong HIF and AP-1 binding throughout the plate, but no evidence of selective binding to any one zone. The results of the study lend strong support to the view that chondrocytes are very well adapted to low oxygen tensions; thus, under hypoxic conditions, there is a high level of expression of both HIF and AP-1, and energy conservation appears to be near-maximum.

Adenine Nucleotides↗

PubMed1995

Bioactive material template for in vitro synthesis of bone.

We describe the synthesis of a new, porous, modified bioactive glass for use as a template for bone formation in vitro. The porosity of the glass was 36.4%; the pore size ranged from 10-160 mm, and there was no incipient devitrification. Prior to seeding the glass with cells, it was necessary to condition the disks. Optimum conditioning was achieved by immersing the templates in a tris buffer at pH 6.8 for 48 h and then treating the glass with tissue culture medium for 1 h at 37 degrees C. The conditioned glass disks were seeded with 10(6) neonatal rat calvaria osteoblast-like cells; cells on the substrate were maintained in culture for 3-7 days. To prevent pH shifts due to corrosion of the conditioned glass, the medium:glass ratio was maintained at 90 ml/g. We found that the templates were rapidly invaded by cells which maintained the osteoblast phenotype; thus, they exhibited high alkaline phosphatase activity and synthesized type I collagen and osteocalcin. SEM-EDAX showed that the cells elaborated substantial amounts of extracellular matrix and a bonelike tissue was present throughout the entire template thickness. FTIR analysis of material formed in the glass indicated that the mineral phase was a biologic hydroxyapatite. Controls (cells without substrate and substrate without cells) exhibited none of these features. Results of the study suggest that this porous glass can function as a template for generating bone in vitro.

Animals↗

PubMed1995

The phosphatidylinositol-glycolipid anchor on alkaline phosphatase facilitates mineralization initiation in vitro.

Alkaline phosphatase (AP) is required for the proper mineralization of cartilage and bone. The enzyme is localized to the outer surface of cells through a phosphatidylinositol-glycolipid anchor, which is covalently attached to the carboxyl terminus of the protein. In calcifying cartilage, AP-rich matrix vesicles (MVs) are released into the matrix from chondrocytes, and apatite formation is initiated within and around these particles. In this paper we examine the role of the AP glycolipid anchor using an in vitro mineralization assay system. AP was purified to homogeneity, and the purified enzyme was used to drive mineral formation in vitro with and without the anchor. Mineral formation was initiated through phosphate release from beta-glycerol phosphate (beta-GP). The amount of PO4(-3) released was similar whether the anchor was present or absent. However, SEM and X-ray microanalysis revealed that the mineral produced by anchored AP was indistinguishable from that produced by MVs and that both of those minerals were more apatite-like than mineral formed by soluble AP or through spontaneous precipitation. Taken together, the data suggest that in addition to providing PO4(-3) to drive mineralization, AP influences the nature of the mineral formed. Further, AP containing its glycolipid anchor produces mineral comparable with that formed by tissue-derived MVs. Thus, in the absence of extracellular matrix, MV mineralization in vitro can be emulated by glycolipid-anchor containing AP.

Alkaline Phosphatase↗

PubMed1995

End labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosis in terminally differentiated chondrocytes.

The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTag and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate for apoptosis. Dual parameteric flow cytometric contour plots of Hoechst and 7-amino-actinomycin D fluorescence showed that abut 8% of cells in the plate were apoptotic. Most of these cells were in hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate the number of cells undergoing this event, the data lend credence to the view that cells are removed from the epiphysis through apoptosis. If this is the case, then chondrocytes probably enter the terminal phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death.

Animals↗

PubMed1995

Adenine nucleotide metabolism by chondrocytes in vitro: role of ATP in chondrocyte maturation and matrix mineralization.

The objective of the investigation was to explore the notion that chondrocytes in the growth plate secrete nucleotides and that these compounds are used to regulate cell maturation and matrix mineralization. Chondrocytes were isolated from the cephalic region of chick embryo sterna and maintained in culture until confluent. To promote expression of the mature phenotype, cultures were then treated with retinoic acid. During the culture period, medium was removed and analyzed for nucleotides using a modified reverse-phase high-performance liquid chromatography (HPLC) procedure. We found that culture medium, conditioned by the chondrocytes, contained significant quantities of nucleotides. Moreover, the nucleotide concentrations were similar in magnitude to levels reported for media conditioned by other cell types. In terms of species, adenosine diphosphate (ADP) was the major nucleotide present in the conditioned medium; adenosine monophosphate (AMP) was present, but at a lower concentration than ADP. To examine the possibility that adenosine triphosphate (ATP) was released by the cultured chondrocytes, but was rapidly degraded into ADP and AMP, we examined the kinetics of ATP breakdown by chondrocytes. We found that chondrocytes degraded over 70% of exogenous ATP within 15 minutes. Similar experiments performed with ADP and AMP indicated that these nucleotides were also degraded by the cells, but at a slower rate than ATP. To determine whether the extracellular nucleotides modulate cartilage development, we examined the effect of exogenous ATP on four major determinants of chondrocyte function: alkaline phosphatase activity, cell proliferation rate, anaerobic metabolism, and mineral deposition. We found that ATP caused only minimum alterations in cell number and alkaline phosphatase activity; however, it increased the lactate content of the medium probably by stimulating anaerobic glycolysis. We noted that ATP had a significant effect on the amount and type of mineral deposited into chondrocyte cultures. Compared with untreated controls, ATP stimulated formation of a small amount of poorly crystallized calcium phosphate. The results of the study show for the first time that chondrocytes release nucleotides into the extracellular milieu. Although they are rapidly degraded, they serve to regulate both mineral formation and energy metabolism.

Adenosine Triphosphate↗

PubMed1995

A rapid and ultrasensitive method for measurement of DNA, calcium and protein content, and alkaline phosphatase activity of chondrocyte cultures.

Most investigators are cognizant of the problems inherent in counting cells embedded in a complex and abundant extracellular matrix. To overcome these obstacles, we developed a new method of isolating nucleic acids from chondrocytes which facilitates measurement of cell number by DNA analysis. Chondrocytes were isolated from chick embryo sterna and grown continuously without subculturing for 2-3 weeks in monolayer. The cells were treated with triton X-100 and the nucleic acid content of the extract was determined by measuring DNA fluorescence in the presence of Hoechst dye 33258. To minimize background fluorescence due to the triton, we precipitated the DNA with alcohol and then solubilized the nucleic acids in EDTA. This simple procedure removed the detergent and substantially increased the sensitivity of the method. Thus, we could measure with high precision and high recovery, the DNA content of cultures of 10,000-50,000 cells. In a single well containing 0.5-1.0 million cells, sufficient material remained for subsequent measurements of alkaline phosphatase activity and protein and calcium content. As the mineral present in the triton-treated samples was soluble in EDTA, we experienced no problems in measuring the calcium content of the culture. In addition, as triton X-100 is a nonionic detergent, we were able to measure cell and matrix proteins; moreover, the presence of the triton maintained the catalytic state of alkaline phosphatase. We conclude that this procedure provides a simple and rapid approach to measuring major indicators of chondrocyte maturation and function.

Alkaline Phosphatase↗

PubMed1995

Growth plate pathology in feline mucopolysaccharidosis VI.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases that result from the accumulation of partially catabolized glycosaminoglycans (GAGs) within lysosomes. A characteristic of most affected individuals is radiographic evidence of symmetrical epiphyseal dysplasia, with short stature and degenerative joint disease. Although there is evidence of epiphyseal dysfunction, little is known of the changes that occur at the morphological level. The growth plate of the femoral head was studied by light and electron microscopy in five cats with MPS VI (Maroteaux-Lamy syndrome, arylsulfatase B deficiency) and 12 normal cats. Compared with the normals, the MPS VI cat growth plates exhibited poorly organized proliferative zones, an almost total loss of column formation in the hypertrophic zone, an uneven chondro-osseous junction, a disorganized calcifying cartilage zone, and abnormal or reduced numbers of osteoclasts. By electron microscopy, the cytoplasm of affected cat chondrocytes was filled with membrane-bound vacuoles. Together these findings indicate that the MPS diseases cause major changes in growth plate structure and function.

Animals↗

PubMed1995

Effects of deferoximine on chondrocyte alkaline phosphatase activity: proxidant role of deferoximine in thalassemia.

The homozygous form of beta-thalassemia, the most common single gene disorder, is treated by red cell transfusion therapy. Following transfusion, the chelator, deferoximine, is administered to patients to remove excess iron. However, when this drug is given to young children, metaphyseal dysplasia and abnormalities of linear growth are frequently observed. To explore the notion that deferoximine interferes with endochondral growth by chelating zinc, we examined the effect of the drug on chondrocytes maintained in long-term culture. We found that deferoximine caused a dose-dependent inhibition of a wide range of functions including cell proliferation, protein synthesis (and possibly under-hydroxylation of type X collagen), and mineral deposition. Directly relevant to the mineralization process was the observation that the drug dramatically lowered the activity of alkaline phosphatase, a zinc-requiring enzyme. To test the hypothesis that enzyme inhibition was due to chelation of zinc by deferoximine, the cell culture medium was supplemented with excess zinc. However, this treatment did not overcome the deferoximine-dependent change in enzyme activity. We next examined the possibility that deferoximine, in the presence of ascorbate, could form a free radical system that would serve to inactivate the enzyme. Using alkaline phosphatase extracted from chick cartilage, we noted that the activity of the phosphatase was markedly reduced in the presence of deferoximine and ascorbate. These effects were consistant with the notion that deferoximine and ascorbate can act as a prooxidant couple. This conclusion was confirmed when we measured the oxidative activities of the system using nitrobule tetrazolium and cytochrome c. Indeed, we noted that deferoximine markedly activates the autocatalytic oxidation of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1994

Retinoic acid is a major regulator of chondrocyte maturation and matrix mineralization.

During the process of endochondral bone formation, chondrocytes undergo a series of complex maturational changes. Our recent studies indicate that this maturational process is influenced by the vitamin A derivative retinoic acid (RA). To learn how this agent regulates chondrocyte development, we characterized matrix gene expression during maturation of cartilage cells in chick sternum. RNAs were isolated from the cephalic portion of day 13, 14, 16, 18, and 20 chick embryo sternum and analyzed via northern blots. Type II collagen RNA levels remained fairly constant during this developmental period. In contrast, expression of type X collagen and alkaline phosphatase (APase) genes was first detected at day 16, followed by that of osteonectin (ON) and osteopontin (OP). To explore the mechanisms triggering these changes, chondrocytes were isolated from the cephalic portion of day 17-18 sternum (US cells) and grown in monolayer in standard serum-containing medium. After 3 weeks in culture, most of the cells enlarged and became type X collagen-positive, but they exhibited low APase activity and contained only trace amounts of ON and OP mRNAs. Treatment of parallel 3-week-old cultures with RA (10-100 nM) rapidly increased expression of the APase, ON, and OP genes severalfold. In concert with a significant increase in APase activity, there was abundant calcium accumulation in the RA-treated cultures. Electron microscopy confirmed the formation of large matrix-associated mineral crystals and the presence of numerous matrix vesicles. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum (LS cells).(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1994

Retinoic acid induces a shift in the energetic state of hypertrophic chondrocytes.

In the epiphyseal growth plate, chondrocyte maturation is accompanied by dramatic alterations in energy metabolism. To explore the relationship between these two events, we used retinoic acid (RA) to promote chondrocyte maturation in culture. The specific question that was addressed was, does RA treatment of cultured chondrocytes in vitro induce a change in energy status similar to that seen in hypertrophic chondrocytes in vivo. Maturing chondrocytes isolated from the cephalic region of day 18 chick embryo sterna were allowed to grow for 7-14 days in monolayer until confluent and then treated with 10-300 nM RA. Immature chondrocytes from the caudal region of sternum were grown in parallel and served as control cells for the study. We found that in maturing cephalic cell cultures, RA had a rapid and profound effect on oxidative metabolism. The retinoid caused a reduction in the energy charge ratio (ECR) and the ATP/ADP ratio and a sharp decrease in cell ATP levels. Maximum inhibition was observed when the RA concentration was 10-35 nM. Compared with the adenine nucleotides, creatine phosphate levels were decreased to a lesser extent by RA, although there was substantial inhibition of creatine kinase activity. We expected to find a compensatory elevation in glycolytic activities; however, the lactate levels in the medium of the treated cells indicated that anaerobic glycolysis was depressed. In contrast to the cephalic chondrocytes, when caudal cell cultures were treated with RA, lactate formation was stimulated and there were minimal effects on oxidative metabolism. To determine the mechanism of inhibition of glycolysis, we measured the activity of pyruvate kinase in RA-treated cephalic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1993

Retinoic acid induces rapid mineralization and expression of mineralization-related genes in chondrocytes.

Numerous studies of experimental hypo- and hypervitaminosis A have long suggested that retinoic acid (RA) is involved in chondrocyte maturation during endochondral ossification and skeletogenesis. However, the specific and direct roles of RA in these complex processes remain unclear. Based on recent studies from our laboratories, we tested the hypothesis that RA induces the expression of genes associated with the terminal mineralization phase of chondrocyte maturation and promotes apatite deposition in the extracellular matrix. Cell populations containing chondrocytes at advanced stages of maturation were isolated from the upper portion of Day 18 chick embryo sterna and grown for 2 weeks in monolayer until confluent. The cells were then treated with low doses (10-100 nM) of RA for up to 6 days in the presence of a phosphate donor (beta-glycerophosphate) but in the absence of ascorbic acid. Within 4 days of treatment, RA dramatically induced expression of the alkaline phosphatase (APase), osteonectin, and osteopontin genes, caused a several-fold increase in APase activity, and provoked massive mineral formation while it left type X collagen gene expression largely unchanged. The mineral had a mean Ca/Pi molar ratio of 1.5; Fourier transform infrared spectra confirmed that it represented hydroxyapatite. Mineralization was completely abolished by treatment with parathyroid hormone; this profound effect confirmed that RA induced cell-mediated mineralization and not nonspecific precipitation. When cultures were treated with both RA and ascorbic acid, there was a slight further increase in APase activity and increased calcium accumulation. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum; however, RA only had minimal effects on mineralization and gene expression in these cells. Thus, RA appears to be a rapid, potent, maturation-dependent, ascorbate-independent promoter of terminal maturation and matrix calcification in chondrocytes.

Alkaline Phosphatase↗

PubMed1993

Computer modeling of the oxygen supply and demand of cells of the avian growth cartilage.

We have used computer modeling studies to investigate the oxygen supply to the prehypertrophic and hypertrophic regions of avian growth plate. We measured experimentally the characteristics of the oxygen consumption of chondrocytes at different oxygen tensions. The oxygen consumption decreases at low oxygen tensions. This relation between oxygen tension and oxygen consumption serves as a protective mechanism that prevents the cells in the prehypertrophic zone from becoming anoxic in the regions farthest from the blood vessels. The results of the calculations, when combined with redox measurements of the cells in the growth plate, indicate that the metabolism of the chondrocytes is not controlled simply by the available oxygen supply.

Animals↗

PubMed1993

Measurement of metabolic events in the avian epiphyseal growth cartilage using a bioluminescence technique.

We developed a technique to map the distribution of selected metabolites in the growth cartilage in situ using luciferase-NAD(P)H:FMN oxidoreductase. Chick tibial epiphyses were freeze-trapped, sectioned, and freeze-dried. For evaluating lactate, luciferase was suspended in a buffer containing polyvinylalcohol, gelatin, NAD, FMN, and lactic dehydrogenase (LDH). The buffer was frozen into a layer 800 microns thick and placed in contact with the tissue section. The temperature of the frozen reagent mixture was then allowed to increase; the emitted light was focused through a photographic lens and collected on film. We found that lactate was synthesized by cells in all regions of the growth plate. The highest concentration of the metabolite was observed in the calcified hypertrophic region. Substantial levels of lactate were also present in articular cartilage. By modifying the composition of the buffer solution, we were able to map the distribution of glucose and glucose-6-phosphate and the activity of LDH. Maximal levels of each of the three components were present in hypertrophic cartilage. Chemical analysis of the tissue section confirmed the luminographic studies and provided further evidence that there was reliance on glycolytic metabolism in terminally differentiated chondrocytes. Use of enzyme couples similar to those described above should permit the technique to be used to study most, if not all, of the major metabolic components of cartilage.

Animals↗

PubMed1993

Immunotoxic effects of mercuric compounds on human lymphocytes and monocytes. III. Alterations in B-cell function and viability.

The major goal of the study was to determine the effects of high and low levels of mercury on human B-cells. Following treatment of B-cells with HgCl2 (0-1000 ng) and MeHgCl2 (0-100 ng), their activation by mitogens was evaluated. Both forms of mercury caused a dose dependent reduction in B-cell proliferation in the presence or absence of monocytes. MeHgCl was approximately 10 times more potent than HgCl2. Mercury also inhibited the ability of these cells to synthesize IgM and IgG. Analysis of the expression of activation markers indicated that CD69, an early marker of cell activation, was not effected by mercury. In comparison, B-cell expression of the low affinity IgE receptor and the transferrin receptor were significantly reduced. Of particular interest, cells activated by mitogen for 48 hr became refractory to the immunotoxic effects of mercury. When exposed to high levels of HgCl2 (0.5-10 micrograms/ml) and MeHgCl (0.05-1 micrograms/ml), there was minimal reduction in B-cell viability at 1-4 hr, however, after exposure to mercury for 24 hr, cell death was apparent. MeHgCl was approximately 5-10 times more potent than HgCl2. Electron microscopic analysis revealed early nuclear alterations characterized by hyperchromaticity, nuclear fragmentation and condensation of nucleoplasm. Both forms of mercury caused a rapid and sustained elevation in the intracellular levels of Ca++. The results of this investigation clearly show that mercury-containing compounds are immunomodulatory; moreover, the decrease in B-cell function indicates that this metal is immunotoxic at very low exposure levels. Furthermore, the cytotoxic events are consistent with the notion that mercury initiates changes associated with programmed cell death.

B-Lymphocytes↗

PubMed1992

Developmental regulation of creatine kinase activity in cells of the epiphyseal growth cartilage.

During the process of endochondral bone formation, the maturing chondrocyte exhibits profound changes in energy metabolism. To explore the mechanism of energy conservation in cartilage we examined the expression of creatine kinase, an enzyme that catalyzes the formation of ATP in tissues under oxygen stress. Measurement of creatine kinase activity and cytochemical assessment of enzyme distribution clearly showed that the level of enzyme activity was related to chondrocyte maturation. Thus, as the cells hypertrophied, there was a progressive increase in creatine kinase activity. Similarly, an elevation in creatine kinase activity was noted in chondrocyte cultures as the cells assumed an hypertrophic state. When cartilage calcification was disturbed by rickets, there was a decrease in enzyme activity in the hypertrophic region. Studies were performed to examine the creatine kinase isozyme profile of cells of the epiphysis. In resting and proliferating cartilage, the isoform was MM. In hypertrophic cartilage, the predominant isoforms were MB and BB. In terms of the creatine phosphate content, the highest values were seen in the proliferative region; lower amounts were present in hypertrophic and resting cartilage; and no creatine phosphate was detected in calcified cartilage. These data suggest that turnover of creatine phosphate is greatest in the mineralized region of the epiphysis. The results of these investigations point to creatine kinase as being under developmental control. The activity of the enzyme in cartilage cells should serve as a marker of developmental events associated with chondrocyte proliferation, hypertrophy, and mineralization.

Animals↗