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.

Enzymatically degraded low density lipoproteins are more potent inducers of egr-1 mRNA than oxidized or native low density lipoproteins.

OBJECTIVES: The transcription factor early growth response gene-1 (Egr-1) may contribute to atherosclerosis by inducing genes that mediate inflammation and thrombosis. Egr-1 mRNA is highly expressed in human atherosclerotic lesions. Enzymatic modification transforms LDL into atherogenic molecules (E-LDL) which are also present in atherosclerotic lesions. We have investigated whether E-LDL induces egr-1 mRNA in human monocytes. DESIGN AND METHODS: Mono-Mac-6 cells were incubated with E-LDL, oxidized (Ox-LDL) and native LDL (N-LDL). Egr-1 mRNA expression was followed by quantitative RT-PCR. RESULTS: E-LDL (25 microg cholesterol/mL) induced egr-1 mRNA maximally within 1 h and were 2.3 and 3.6 fold (p < 0.05) more effective than Ox-LDL or N-LDL. At a concentration of 10 microg/mL cholesterol, E-LDL were twofold less effective. CONCLUSIONS: These results show that E-LDL are potent inducers of egr-1 mRNA and may therefore represent a link between lipoproteins trapped in the subendothelium and enhanced expression of egr-1 in human atherosclerotic lesions.

In vitro evaluation of the effect of dimethyl sulfoxide on equine articular cartilage matrix metabolism.

OBJECTIVE: To evaluate the effects of dimethyl sulfoxide (DMSO) on equine articular cartilage matrix metabolism. STUDY DESIGN: Using a cartilage explant culture system, proteoglycan (PG) synthesis, PG release, lactate metabolism, chondrocyte viability, and metabolism recovery were determined after cartilage exposure to DMSO. SAMPLE POPULATION: Cartilage harvested from metacarpophalangeal and metatarsophalangeal joints of 12 horses (age range, 1 to 10 years). METHODS: Explants were exposed to concentrations of DMSO (1% to 20%) for variable times (3 to 72 hours). PG synthesis and release were determined by a radiolabel incorporation assay and dimethylmethylene blue (DMMB) dye assay, respectively. Lactate released into culture media was measured, and chondrocyte viability was assessed using the Formizan Conversion Assay and a paravital staining protocol. Metabolism recovery was assessed in explants that were allowed to recover in maintenance media after exposure to DMSO. RESULTS: PG synthesis and lactate metabolism were inhibited in a dose- and time-dependent manner after exposure to DMSO concentrations > or = 5%; there was no significant alteration in PG release. No change in chondrocyte viability was detected after incubation with DMSO. PG synthesis and lactate metabolism returned to baseline rates when allowed a recovery period after exposure to DMSO. CONCLUSIONS: DMSO concentrations > or = 5% suppress equine articular cartilage matrix metabolism. Suppression of PG synthesis and lactate metabolism is reversible and does not appear to be the result of chondrocyte death. CLINICAL RELEVANCE: Equine clinicians adding DMSO to intraarticular lavage solutions should be aware that DMSO may have deleterious effects on equine articular cartilage matrix metabolism.

A gravity-independent ergometer to be used for resistance training in space.

An ergometer, to be used for resistance training in space, has been developed and validated. It is designed to activate the extensor muscles of the knee and ankle joints while performing the leg press exercise. Resistance is provided independent of gravity by using the inertial focus of a flywheel. Eleven men performed two series of consecutive maximal voluntary concentric and eccentric muscle actions. Force, power, work and electromyographic (EMG) activity, measured during exercise on this ergometer and a traditional leg press resistive apparatus were similar. This mechanical ergometer seems to meet the operational and technical requirements of equipment that can be flown and used in space. Also, the physiological responses to acute exercise suggest that adaptations similar to those achieved by traditional weight training can be produced. Exercise using the inertia ergometer would, therefore, probably also be effective in combating the muscle atrophy and loss of strength that occur in microgravity.

A novel eubacterial phylum: comparative nucleotide sequence analysis of a tuf-gene of Flexistipes sinusarabici.

A tuf-gene of Flexistipes sinusarabici has been cloned and sequenced. The primary structure of the predicted elongation factor protein was compared with available sequences of homologous genes and elongation factors Tu or 1 alpha of eubacteria, archaebacteria and eukaryotes. Based on elongation factor Tu data Flexistipes sinusarabici belongs to the eubacterial kingdom but no specific relationship to any phylum was detected. The amino acid of the elongation factor Tu of F. sinusarabici exhibits no striking pecularities. Among the highly conserved positions only two are different. Sites of known or postulated functions are conserved.

Cloning and sequencing of the fus-gene encoding elongation factor 2 in the archaebacterium Thermoplasma acidophilum.

We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences.

Cloning and sequencing of the gene coding for the elongation factor 1 alpha from the archaebacterium Thermoplasma acidophilum.

The gene which encodes the elongation factor 1 alpha (EF-1 alpha) of the archaebacterium Thermoplasma acidophilum (tuf-gene) has been cloned and sequenced. The gene coding for elongation factor EF-2 was found downstream from the 3' end of the tuf-gene. Comparison of the predicted amino acid sequence of Thermoplasma EF-1 alpha with EF-1 alpha sequences of other organisms showed that the highest similarity values were found between T. acidophilum and Methanococcus vannielii.

Prevention of supraventricular tachyarrhythmias post coronary artery bypass surgery.

In a randomized prospective study 32 patients received either alinidine or a placebo for the first five postoperative days after coronary bypass surgery. The purpose of the study was to investigate the prophylactic antiarrhythmic properties of alinidine on supraventricular tachyarrhythmias (svt), which occur with incidence after open heart surgery. There was no significant difference in pretherapeutical parameters between the two groups. Eleven out of sixteen control patients (69%) and none of the patients treated with alinidine had svt. All arrhythmias occurred in the first three postoperative days and required medical treatment. Even after alinidine was stopped, patients in this group did not experience arrhythmias. The mean systolic blood pressure in the treatment group was 113 +/- 13 mmHg, in the control group it was 119 +/- 16 mmHg. The mean heart rate tended to be lower in the alinidine group (82 +/- 12 beats min-1 91 +/- 21 beats min-1. In 1/16 patients the alinidine treatment was stopped due to marked hypotension (less than 90 mmHg) and bradycardia (less than beats min-1). Two other patients in this group had short periods of mild bradycardia (less than 60 beats min-1) which was tolerated well. Additional medical treatment was not needed. In this study prophylactic treatment with alinidine proved to be highly effective in preventing postoperative arrhythmias following myocardial revascularisation.