Training in metabolomics research. I. Designing the experiment, collecting and extracting samples and generating metabolomics data.

Metabolomics is perhaps the most challenging of the -omics fields, given the complexity of an organism's metabolome and the rapid rate at which it changes. When one sets out to study metabolism there are numerous dynamic variables that can influence metabolism that must be considered. Recognizing the experimental challenges confronting researchers who undertake metabolism studies, workshops like the one at University of Alabama at Birmingham have been established to offer instructional guidance. A summary of the UAB course training materials is being published as a two-part Special Feature Tutorial. In this month's Part I the authors discuss details of good experimental design and sample collection and handling. In an upcoming Part II, the authors discuss in detail the various aspects of data analysis.

XCMS2: processing tandem mass spectrometry data for metabolite identification and structural characterization.

Mass spectrometry based metabolomics represents a new area for bioinformatics technology development. While the computational tools currently available such as XCMS statistically assess and rank LC-MS features, they do not provide information about their structural identity. XCMS(2) is an open source software package which has been developed to automatically search tandem mass spectrometry (MS/MS) data against high quality experimental MS/MS data from known metabolites contained in a reference library (METLIN). Scoring of hits is based on a "shared peak count" method that identifies masses of fragment ions shared between the analytical and reference MS/MS spectra. Another functional component of XCMS(2) is the capability of providing structural information for unknown metabolites, which are not in the METLIN database. This "similarity search" algorithm has been developed to detect possible structural motifs in the unknown metabolite which may produce characteristic fragment ions and neutral losses to related reference compounds contained in METLIN, even if the precursor masses are not the same.

Endogenously produced adenosine regulates articular cartilage matrix homeostasis: enzymatic depletion of adenosine stimulates matrix degradation.

OBJECTIVE: Enhanced extracellular levels of adenosine have been shown to inhibit experimentally induced cartilage degradation. The objective of this study was to investigate the role of adenosine and A(2)adenosine receptors in regulating cartilage homeostasis in the absence of inflammatory stimuli. METHODS: Cartilage explants were exposed to adenosine deaminase (ADA) to deplete extracellular adenosine, and conditioned medium was collected for evaluation of glycosaminoglycan (GAG), prostaglandin E(2)(PGE(2)), nitric oxide (NO), and matrix metalloproteinases-3 and -13 (MMP-3, MMP-13) levels. In a second set of experiments, cartilage incubated with ADA was simultaneously exposed to the adenosine kinase inhibitor 5'-iodotubercidin (ITU) to inhibit adenosine breakdown, or to the A(2A)adenosine receptor agonist N(6)-[2-(3,5-dimethoxyphenyl)-ethyl]adenosine (DPMA). Finally, explants were incubated with the adenosine receptor antagonists ZM241385, CGS15943, theophylline or caffeine to block normal receptor activation by endogenous adenosine. RESULTS: Exposure to ADA induced a concentration-dependent increase in GAG release and production of total MMP-3, MMP-13, PGE(2), and NO. Both ITU and DPMA inhibited the ADA-mediated increases in GAG release and PGE(2), and NO production, but only ITU inhibited MMP-13 release. Exposure to ZM 241385 increased GAG, MMP-3 and MMP-13 release. Additionally, CGS 15943 increased MMP-3 production while theophylline increased GAG, PGE(2), and NO release. CONCLUSIONS: Endogenous adenosine levels appear to regulate cartilage matrix homeostasis even in the absence of inflammation. Regulation occurs, at least in part, through activation of cell surface receptors. This study suggests that autocrine and paracrine responses to adenosine release are important for maintenance of healthy articular cartilage.

Chondrocytes respond to adenosine via A(2)receptors and activity is potentiated by an adenosine deaminase inhibitor and a phosphodiesterase inhibitor.

OBJECTIVE: To test the mechanisms by which adenosine and adenosine analogues stimulate adenylate cyclase and suppress lipopolysaccharide (LPS)-induced production of nitric oxide (NO) by chondrocytes. METHODS: Primary chondrocytes isolated from equine articular cartilage were plated in monolayer. Intracellular cyclic-AMP (cAMP) accumulation was measured following exposure to medium containing adenosine, the non-hydrolyzable adenosine analogue N(6)-methyladenosine, the A(2A)specific agonist N(6)-(dimethoxyphenyl)-ethyl]adenosine (DPMA), the adenosine deaminase inhibitor erythro-9-(2-Hydroxy-3-nonyl)adenine hydrochloride (EHNA), or forskolin, a potent stimulator of adenylate cyclase. Regulation of NO production by LPS-stimulated chondrocytes, as determined by nitrite concentration, was assessed in the presence of adenosine, N(6)-methyladenosine, DPMA, the broad agonist 5'-N-ethylcarboxamidoadenosine (NECA), or forskolin. Alternatively, LPS-stimulated chondrocytes were exposed to EHNA or the phosphodiesterase inhibitor rolipram in the presence or absence of supplemental adenosine. RESULTS: Adenosine, N(6)-methyladenosine, DPMA, and forskolin each increased intracellular cAMP accumulation in a concentration-dependent manner and suppressed NO production by LPS-stimulated chondrocytes. NECA also decreased NO production by chondrocytes stimulated with LPS. Incubation with EHNA, to protect endogenously produced adenosine, or rolipram, which prevents the degradation of cAMP, similarly suppressed LPS-stimulated NO production. The addition of exogenous adenosine with EHNA or rolipram further suppressed NO production. CONCLUSIONS: This study documents functional responses to adenosine by articular chondrocytes. These responses are mimicked by the A(2A)receptor agonist, DPMA. Effects were enhanced by protecting adenosine using an adenosine deaminase inhibitor or by potentiating the cAMP response with rolipram. These experiments suggest that adenosine may play a physiological role in regulation of chondrocytes and that adenosine pathways could represent a novel target for therapeutic intervention.

Two-photon excitation laser scanning microscopy of human, porcine, and rabbit nasal septal cartilage.

Two-photon excitation laser scanning microscopy (TPM) was used to image human, porcine, and rabbit nasal septal cartilage. TPM provides optical sections of thick tissue specimens in situ without the use of exogenous dyes or need for tissue fixation. The cartilage tissue was imaged using near-infrared light generated by a mode-locked titanium/sapphire laser that was raster-scanned and coupled to an inverted microscope. Absorption of two photons by endogenous molecules and subsequent fluorescence was filtered to specific spectral bandwidths and detected with photomultiplier tubes. Two-photon stimulated fluorescence was detected with photomultiplier tubes optimized to specific spectral bandwidths. Signal intensity corresponds to the concentration of fluorophores, principally NADH, NADPH, and flavoproteins hence providing a means of redox imaging the cellular metabolic state. Specimens were scanned from the surface to a depth of about 150 microm. Image size was 50 x 50 microm with a diffraction limited pixel size of 0.4 microm. Cell membranes, nuclei, and matrix structures were identified in human, pig, and rabbit tissues. TPM provides a means to study three dimensional chondrocyte structure and matrix organization in situ at substantial depths, and permits longitudinal examination of cultured tissue explants without the need for exogenous dyes, tissue preparation, or fixation.

The roles of interleukin-6 and interleukin-10 in B cell hyperactivity in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease most prevalent in women between the ages of twenty and sixty. Successful treatment remains challenging due to a lack of understanding of the underlying mechanisms and multiple symptoms ranging from skin rashes to glomerulonephritis. The pathogenesis of SLE has been linked to a B-cell hyperproliferation unique to afflicted patients. These B-cells generate large quantities of IgG autoantibodies, ultimately capable of leading to lupus nephritis and renal failure. The significance of cytokines in SLE and in murine lupus, a related disease in mice, has been debated, particularly with respect to B-cell activity. Potential roles of auto-regulatory and inflammatory cytokines have been investigated. In particular, IL-6 and IL-10 have been shown to be key factors in regulating autoantibody-secreting B-cell activity in lupus. Here, we will provide a critical overview of our current knowledge of the regulatory roles of these two cytokines in SLE.

Expression of both P1 and P2 purine receptor genes by human articular chondrocytes and profile of ligand-mediated prostaglandin E2 release.

OBJECTIVE: To assess the expression and function of purine receptors in articular chondrocytes. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to screen human chondrocyte RNA for expression of P1 and P2 purine receptor subtypes. Purine-stimulated prostaglandin E2 (PGE2) release from chondrocytes, untreated or treated with recombinant human interleukin-1alpha (rHuIL-1alpha), was assessed by radioimmunoassay. RESULTS: RT-PCR demonstrated that human articular chondrocytes transcribe messenger RNA for the P1 receptor subtypes A2a and A2b and the P2 receptor subtype P2Y2, but not for the P1 receptor subtypes A1 and A3. The P1 receptor agonists adenosine and 5'-N-ethylcarboxamidoadenosine did not change PGE2 release from chondrocytes. The P2Y2 agonists ATP and UTP stimulated a small release of PGE2 that was potentiated after pretreatment with rHuIL-1alpha. PGE2 release in response to ATP and UTP cotreatment was not additive, but release in response to coaddition of ATP and bradykinin (BK) or UTP and BK was additive, consistent with ATP and UTP competition for the same receptor site. The potentiation of PGE2 release in response to ATP and UTP after rHuIL-1alpha pretreatment was mimicked by phorbol myristate acetate. CONCLUSION: Human chondrocytes express both P1 and P2 purine receptor subtypes. The function of the P1 receptor subtype is not yet known, but stimulation of the P2Y2 receptor increases IL-1-mediated PGE2 release.

Adenosine 5'-triphosphate, uridine 5'-triphosphate, bradykinin, and lysophosphatidic acid induce different patterns of calcium responses by human articular chondrocytes.

Small calcium-mobilizing inflammatory mediators have been implicated in joint pathology. Here we demonstrate that bradykinin, adenosine 5'-triphosphate, uridine 5'-triphosphate, and lysophosphatidic acid raise the intracellular calcium concentration ([Ca2+]i) in human articular chondrocytes. Heterologous cross-desensitization experiments showed that the uridine 5'-triphosphate response was abolished by prior treatment with adenosine 5'-triphosphate and, conversely, that the adenosine 5'-triphosphate response was abolished by prior treatment with uridine 5'-triphosphate; this indicated competition for the same receptor site, whereas bradykinin and lysophosphatidic acid did not compete with other ligands. Pretreatment with thapsigargin abolished ligand-mediated Ca2+ responses but not vice versa; this confirmed that Ca2+ release occurred from intracellular stores. Single-cell analysis of Fura-2 acetoxymethyl ester loaded chondrocytes showed mediator-dependent patterns of oscillatory Ca2+ changes in a subset of cells when challenged with submaximal concentrations of bradykinin, adenosine 5'-triphosphate, or uridine 5'-triphosphate in the presence of extracellular Ca2+. However, no oscillatory responses were seen after a challenge with lysophosphatidic acid. Therefore, although a number of different Ca2+-mobilizing ligands activate chondrocytes, the differences that occur in the temporal patterning of Ca2+ responses may result in unique mediator-dependent changes in cellular activity.

ATP and UTP activate calcium-mobilizing P2U-like receptors and act synergistically with interleukin-1 to stimulate prostaglandin E2 release from human rheumatoid synovial cells.

OBJECTIVE: To pharmacologically and functionally characterize calcium-mobilizing purine receptors on adherent human rheumatoid synovial cells. METHODS: Fura-2-loaded synovial cells were screened for changes in cytosolic calcium concentration after the addition of purine receptor agonists. Release of interleukin-1 (IL-1) and prostaglandin E2 (PGE2) was assessed by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. The effect of IL-1 prestimulation on purine-mediated PGE2 release was determined. RESULTS: ATP (1-100 microM) and UTP (1-100 microM), but not 2-methylthio-ATP or adenosine, stimulated mobilization of calcium from intracellular stores in synovial cells. ATP and UTP stimulated a small, but significant, increase in PG release from resting synoviocytes and a dramatic increase in PG release from synoviocytes prestimulated with recombinant human IL-1alpha. Neither ATP nor UTP stimulated synoviocyte release of IL-1 as measured by specific ELISA. The effects of ATP and UTP on PG secretion were mimicked by phorbol 12-myristate 13-acetate and thapsigargin, and blocked by BAPTA buffering of cytosolic calcium. CONCLUSION: Adherent human rheumatoid synovial cells mobilize intracellular calcium via a P2U-like purine receptor. P2U receptor agonists stimulate PGE2 release from synoviocytes, an effect that is greatly enhanced by IL-1alpha prestimulation and blocked by intracellular calcium buffering.

Calcium-mobilizing purine receptors on the surface of mammalian articular chondrocytes.

If we are to fully understand mechanisms of cartilage homeostasis, it is essential that we know the full catalogue of receptors present on the surface of a chondrocyte and the pathways regulated by ligands that bind to these receptors. In this study, we describe chondrocyte responses to adenosine 5'-triphosphate and related molecules. Adenosine 5'-triphosphate stimulated a statistically significant, dose-dependent, transient rise in the concentration of calcium ions in Fura 2-loaded, differentiated, primary chondrocytes. The increase occurred in the absence of extracellular calcium, indicating a mobilization from intracellular stores. The increase in concentration of cytoplasmic calcium ions induced by adenosine 5'-triphosphate was mimicked by uridine 5'-triphosphate but not by 2-methylthioadenosine 5'-triphosphate, cytidine 5'-triphosphate, or adenosine. Heterologous desensitization experiments demonstrated that chondrocytes showed no subsequent response to uridine 5'-triphosphate after initial stimulation with adenosine 5'-triphosphate nor did they respond to adenosine 5'-triphosphate in inverse conditions, thereby indicating competition for the same receptor site. Together, these results are consistent with the presence of a P2U receptor on the cell surface of chondrocytes. Purine-induced calcium mobilization in passaged chondrocytes showed the same pharmacological profile with respect to agonist sensitivity, but responses were of greater magnitude than responses in primary differentiated chondrocytes, suggesting upregulation of the receptor with time in culture. Adenosine 5'-triphosphate and uridine 5'-triphosphate (1-100 microM) did not alter cartilage matrix synthesis as measured by rate of incorporation of [35S]sulfate into glycosaminoglycan by cartilage explants or primary chondrocytes. Matrix degradation, measured by release of glycosaminoglycan from cartilage explants, was also unaltered by adenosine 5'-triphosphate or uridine 5'-triphosphate (1-100 microM). Production of prostaglandin E2 was upregulated by incubation with either adenosine 5'-triphosphate or uridine 5'-triphosphate. These data demonstrate the presence of a functional P2U-like purine receptor on the surface of primary articular chondrocytes and support the hypothesis that altered concentrations of extracellular purines may influence chondrocyte metabolism.

Effect of carprofen on sulfated glycosaminoglycan metabolism, protein synthesis, and prostaglandin release by cultured osteoarthritic canine chondrocytes.

OBJECTIVE: To determine whether the nonsteroidal anti-inflammatory drug carprofen directly influences canine chondrocyte metabolism. ANIMALS: Cartilage from the femoral heads of 13 dogs undergoing total hip replacement. PROCEDURE: Rates of glycosaminoglycan (GAG) synthesis and degradation, protein synthesis, cell viability, and prostaglandin release were determined in canine explant cartilage or monolayer canine chondrocyte cultures in the presence of 0 to 100 micrograms of carprofen/ml. Rate of GAG synthesis was assessed as incorporation of [35S]sulfate into cartilage matrix during a 3-hour pulse label. Degradation of cartilage GAG was assessed as rate of release of [35S]sulfate from prelabeled explant cultures. Rates of total protein synthesis were assessed as incorporation of [35S]methionine into trichloracetic acid precipitable material during a 3-hour pulse label. Radiolabeled chondrocyte proteins were separated by polyacrylamide gel electrophoresis and visualized by fluorography. Rates of prostaglandin E2 release were assessed by radioimmunoassay. RESULTS: Carprofen stimulated a significant increase in the rate of GAG synthesis at concentrations of 1 and 10 micrograms/ml, with no change in total protein synthesis, pattern of new protein synthesis, or cell viability. At concentration > or = 20 micrograms/ml, inhibition of GAG synthesis and total protein synthesis was observed. There was no significant change in rate of release of GAG from cartilage explants, but potent inhibition of prostaglandin release was observed. CONCLUSIONS: Carprofen has a direct influence on chondrocyte activity, resulting in changes in rate of production of cartilage matrix. CLINICAL RELEVANCE: In determining the optimal therapeutic dose of carprofen for arthritic conditions in dogs, it is important to consider potential influences on cartilage, as well as anti-inflammatory actions.

Effect of activated equine neutrophils on sulfated proteoglycan metabolism in equine cartilage explant cultures.

OBJECTIVE: To determine the influence of activated equine neutrophils on sulfated glycosaminoglycan metabolism of equine articular cartilage in vitro. ANIMALS: Articular cartilage explants harvested from the metacarpophalangeal joints of 7 horses. PROCEDURE: Proteoglycan degradation and synthesis were measured by release of glycosaminoglycan from the explants, and incorporation of [35S]sulfate into newly synthesized glycosaminoglycan. RESULTS: Activated equine neutrophils significantly increased the release of glycosaminoglycan from explant matrix and the magnitude of that response was influenced by duration of exposure. This response varied significantly between horses, but was detected as early as 3 hours after co-cultures were initiated. In addition to enhancing degradation, incubation of explants with activated neutrophils for 3 days caused significant inhibition of glycosaminoglycan synthesis during a subsequent 3-hour pulse-labeling period. This response varied significantly between individual animals, but age was not a predictive factor. CONCLUSION: Neutrophils may have a critical role in the process of cartilage degradation during equine inflammatory joint disease.

Use of adverse conditions to stimulate a cellular stress response by equine articular chondrocytes.

OBJECTIVE: To determine the response of equine articular cartilage cells to heat and calcium stresses. DESIGN: Analysis of newly synthesized, [35S]methionine-labeled proteins after treatment of isolated primary equine chondrocytes. PROCEDURE: Primary cultures of equine articular chondrocytes were incubated at temperatures ranging from 37 to 42 C for heat stress experiments or incubated in the presence or absence of the intracellular calcium pump inhibitor, thapsigargin, for calcium stress experiments. Patterns of new protein synthesis were determined by incubating with [35S]methionine followed by separation of proteins by use of one- or two-dimensional gel electrophoresis and visualization of labeled proteins by use of fluorography. RESULTS: Equine chondrocytes cultured at temperature of 42 C had increased synthesis of specific proteins, compared with the profile of protein synthesis in control chondrocytes cultured at 37 C. These changes were characteristic of the heat shock stress response described in a number of other mammalian cell-types. Equine chondrocytes cultured in the presence of thapsigargin also had increased synthesis of specific proteins. Two-dimensional gel electrophoresis of these newly synthesized proteins revealed the changes to be consistent with the induction of the glucose-regulated protein family of stress proteins. CONCLUSIONS: Changes in the pattern of new protein synthesis can be induced in differentiated equine articular chondrocytes by heat shock or calcium stress. These responses are characteristic of a widely described mammalian stress response that has been postulated to be involved in cellular protective mechanisms. The ability of equine chondrocytes to mount a robust stress response may be important in the processes of tissue damage and recovery in articular joints of horses.

Regulation of glycosaminoglycan metabolism by bone morphogenetic protein-2 in equine cartilage explant cultures.

OBJECTIVE: To investigate whether recombinant human bone morphogenetic protein-2 (rhBMP-2) regulates glycosaminoglycan (GAG) synthesis and release from equine articular cartilage explant cultures. DESIGN: Equine articular cartilage explants were maintained in vitro for 7 days in the presence of 0 (control), 1, 10, or 100 ng of rhBMP-2/ml. Synthesis and release of GAG were assessed as measures of production and degradation of the extracellular matrix, respectively. ANIMALS: 6 horses (age range, 2 to 25 years old) without clinically detectable musculoskeletal abnormalities. PROCEDURE: Rate of synthesis of GAG was assessed by incorporation of [36S]sulfate during the final 24 hours of the 7-day incubation period. Release of GAG was assessed on days 3, 6, and 7, using 1,9-dimethylmethylene blue. RESULTS: Explants from all 6 horses had a significant (P = 0.05) increase in release of GAG in response to incubation with 100 ng of rhBMP-2/ml. There was a significant (P = 0.05) decrease in GAG synthesis in explants from only 2 of the 6 horses at the same concentration of rhBMP-2. There was no significant age correlation between responsive and nonresponsive horses. CONCLUSIONS: A concentration of 100 ng of rhBMP-2/ml stimulates GAG release from explant cultures of equine articular cartilage. The data suggest that bone morphogenetic proteins may be potential regulators of equine cartilage degradation and repair. CLINICAL RELEVANCE: Surgical procedures that damage subchondral bone may stimulate generation of improved cartilage-like tissue. It is, therefore, crucial to understand how bone-derived factors may influence cartilage metabolism in horses.

Influence of alginate polysaccharide composition and culture conditions on chondrocytes in three-dimensional culture.

Alginate-embedded chondrocytes have been used for experimental analysis of cartilage matrix metabolism and as a potential bioartificial system for repairing cartilage defects. Alginates are linear copolymers composed of 1,4-linked beta-n-mannuronic acid and 1,4 linked alpha-L-guluronic acid, which occur as regions made up exclusively of one unit or the other, or as regions in which the monomers approximate an alternating sequence. Data presented here demonstrate that the mannuronic to guluronic acid (M/G) ratio and molecular weight of the alginate utilized effects the tissue-culture handling properties of the resultant gel and the activity of embedded chondrocytes. In experiments comparing chondrocyte survival and matrix synthesis, optimal conditions were obtained with an alginate of both high mannuronic acid content and high molecular weight. Chondrocytes survived and proliferated when maintained in unsupplemented media, in media supplemented with fetal calf serum, and in media supplemented with the defined serum replacement ITS+ (6.25 microg/ml insulin, 6.25 microg/ml transferrin, 6.25 ng/ml selenous acid, 1.25 mg/ml bovine serum albumin, 5.35 microg/ml linoleic acid). High cell survival rate and increase in cell number was obtained in the absence of serum. In contrast, long-term matrix synthesis and deposition required media supplementation as indicated by uptake of [(35)S]sulfate into glycosaminoglycans and by immunofluorescence using antibodies specific for cartilage matrix molecules.

Effect of bacterial lipopolysaccharides on sulfated glycosaminoglycan metabolism and prostaglandin E2 synthesis in equine cartilage explant cultures.

The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (LPS) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of LPS from 0 (control) to 100 micrograms/ml. Glycosaminoglycan (GAG) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-dimethylmethylene blue. Newly synthesized GAG content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (PGE2) synthesis was quantified, using a [3H]PGE2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to LPS, and explants from 1 horse were used to compare responses to stimulation with LPS derived from 2 bacterial sources. Equine explants cultured with bacterial LPS had a dose-dependent decrease in synthesis and increase in release of GAG, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of PGE2 released into the culture medium in response to incubation with LPS. Comparison of data for GAG synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to LPS between the 2 species. Equine explants tended to have a greater suppression of GAG synthesis in response to incubation with increasing concentrations of LPS than did age-corrected bovine samples. However, similar analysis of data on GAG release did not indicate any difference in sensitivity between the 2 species for this response. There was no evidence that the presence or absence of serum supplementation or the use of LPS derived from different bacterial sources made a significant difference in the response of explants to incubation with LPS.

The intracellular Ca(2+)-pump inhibitors thapsigargin and cyclopiazonic acid induce stress proteins in mammalian chondrocytes.

Primary cultures of mammalian articular chondrocytes respond to treatment with the intracellular Ca(2+)-pump inhibitors thapsigargin (TG) and cyclopiazonic acid by specific changes in protein synthesis consistent with a stress response. Two-dimensional gel electrophoresis of newly synthesized proteins confirmed that the response was consistent with the induction of glucose-regulated proteins. The effects of low-dose TG (10 nM), measured by changes in [35S]methionine labelling of newly synthesized proteins, can first be observed by 10 h and are maximal by 24 h. The pattern of changes induced by TG is shared with cyclopiazonic acid, but effects of both perturbants differ significantly from changes induced by heat shock. Upon removal of TG, normal protein synthesis is restored by 48 h. Immunoblots showed increased concentrations of the stress proteins HSP90, HSP72/73 and HSP60 in chondrocytes treated with TG, but induction of newly synthesized heat-shock proteins by TG was not apparent on [35S]methionine-labelled gels. The alterations in protein synthesis induced by Ca(2+)-pump inhibitors were unaffected by BAPTA-AM loading, which clamped cytosolic Ca2+ at resting levels. We conclude that inhibition of intracellular Ca(2+)-pump activity can elicit a stress response, which has important implications for the interpretation of chronic use of Ca(2+)-pump inhibitors. In particular, the activation of the cellular shock response should be considered in interpreting the regulation of protein synthesis and cell survival by Ca(2+)-pump inhibitors such as TG.

Regulation of matrix metabolism in equine cartilage explant cultures by interleukin 1.

Explant cultures were set up, using articular cartilage obtained from metatarsophalangeal joints of 11 horses. Explants from 2 horses were used to determine culture conditions appropriate for tissue viability. The cartilage explants maintained steady-state metabolism of proteoglycans during a 13-day evaluation period. The metabolic response of equine articular cartilage to incubation with recombinant human interleukin 1 (0.01 to 100 ng/ml) was studied, using cartilage obtained from the remaining 9 horses, age of which ranged from 3 months to 20 years. Interleukin 1 induced a dose-dependent release of glycosaminoglycan from the matrix during a 3-day incubation period. It also caused dose-dependent inhibition of glycosaminoglycan synthesis during a 3-hour pulse-labeling period. Explants obtained from older horses were significantly (P < 0.05) less responsive to interleukin 1, with respect to synthesis and release of glycosaminoglycan.

Cytokines and their receptors.

Cytokines act via receptor-mediated pathways to influence the regulation of both immune and non-immune cells. This review will discuss some of the most important developments over the past year which have contributed to the elucidation of the mechanisms of cell activation by these molecules.