Energetic, oxidative and ionic exchange in rat brain and liver mitochondria at experimental audiogenic epilepsy (Krushinsky-Molodkina model).

The role of brain and liver mitochondria at epileptic seizure was studied on Krushinsky-Molodkina (KM) rats which respond to sound with an intensive epileptic seizure (audiogenic epilepsy). We didn't find significant changes in respiration rats of brain and liver mitochondria of KM and control rats; however the efficiency of АТР synthesis in the KM rat mitochondria was 10% lower. In rats with audiogenic epilepsy the concentration of oxidative stress marker malondialdehyde in mitochondria of the brain (but not liver) was 2-fold higher than that in the control rats. The rate of H2O2 generation in brain mitochondria of КМ rats was twofold higher than in the control animals when using NAD-dependent substrates. This difference was less pronounced in liver mitochondria. In KM rats, the activity of mitochondrial ATP-dependent potassium channel was lower than in liver mitochondria of control rats. The comparative study of the mitochondria ability to retain calcium ions revealed that in the case of using the complex I and complex II substrates, permeability transition pore is easier to trigger in brain and liver mitochondria of KM and КМs rats than in the control ones. The role of the changes in the energetic, oxidative, and ionic exchange in the mechanism of audiogenic epilepsy generation in rats and the possible correction of the epilepsy seizures are discussed.

Pathophysiology of osteoarthritis: evidence against the viscoelastic theory.

OBJECTIVES: Whether changes in the hyaluronan moiety of synovial fluid are associated with osteoarthritis (OA) is unresolved experimentally, notwithstanding frequent statements in the literature that the disease leads to degraded hyaluronan. We evaluated this hypothesis by comparing the molecular weight and concentration of hyaluronan in synovial fluid from patients with and without OA. METHODS: Synovial fluid was obtained by needle aspiration from patients with advanced OA (Kellgren-Lawrence Grade IV) and from patients with no radiological or arthroscopic evidence of OA. The distribution of the molecular weight of hyaluronan was measured using both gel electrophoresis and size-exclusion chromatography, and hyaluronan concentration was determined by immunosorbent assay and differential refractometry. RESULTS: The distributions of molecular weight were highly variable within each group of patients; the average distributions, however, were identical in the 2 groups. The average concentration of hyaluronan also did not differ statistically between the 2 groups. CONCLUSIONS: We found no evidence of hyaluronan degradation in synovial fluid of patients with OA. Commonly expressed opinion in the literature to the contrary may have resulted from a failure to adequately consider the limitations of previous experimental studies.

Hyaluronan-binding receptors: possible involvement in osteoarthritis.

Our objectives were to compare the expression of the hyaluronan receptors CD44 and RHAMM in knee synovial tissue of patients with and without advanced osteoarthritis (OA). Both receptors were detected immunohistochemically; the staining appeared more intense in the tissues from the patients with advanced OA. Expression of CD44 and RHAMM were each significantly increased (p < 0.05) in synovial tissue from patients with OA, as determined by means of Western-blot analysis. The findings suggested that changes in levels of the HA-binding proteins might be implicated in the development or progression of OA.

Hyaluronan suppresses IL-1beta-induced metalloproteinase activity from synovial tissue.

Intraarticular injection of hyaluronan (viscosupplementation) is commonly used to treat knee pain from osteoarthritis. The therapeutic benefit might derive from hyaluronan inhibition of the activity of the cytokine-regulated catabolic enzymes that attack joint cartilage (matrix metalloproteinases). We tested the hypothesis that hyaluronan inhibited interleukin-1beta-induced matrix metalloproteinase activity secreted by explants of synovial tissue from patients with osteoarthritis and investigated the mechanism of the effect. Hyaluronan with a molecular mass of 12.8 MDa (number average) antagonized induced metalloproteinase activity in proportion to hyaluronan concentration in the clinically relevant range of 2 to 8 mg/mL. The effect was not attributable solely to molecular mass because 1.2-MDa hyaluronan produced comparable inhibition. Based on measurements involving hyaluronans of different average molecular masses, polydispersity and viscosity were similarly ruled out as primary responsible factors. The effect of hyaluronan on induced metalloproteinase activity was mediated partially by CD44, the principal cell surface receptor for hyaluronan. Hyaluronan inhibited interleukin-1beta-induced metalloproteinase production from osteoarthritic synovial tissue by a process that was not solely dependent on hyaluronan molecular mass but that was partly mediated by hyaluronan binding to CD44. The efficacy of viscosupplementation could be explained if hyaluronan also blocked catabolic enzyme activity in the joint.

Glycoproteins bound to ion channels mediate detection of electric fields: a proposed mechanism and supporting evidence.

The mechanism by which animals detect weak electric and magnetic fields has not yet been elucidated. We propose that transduction of an electric field (E) occurs at the apical membrane of a specialized cell as a consequence of an interaction between the field and glycoproteins bound to the gates of ion channels. According to the model, a glycoprotein mass (M) could control the gates of ion channels, where M > 1.4 x 10(-18)/E, resulting in a signal of sufficient strength to overcome thermal noise. Using the electroreceptor organ of Kryptopterus as a mathematical and experimental model, we showed that at the frequency of maximum sensitivity (10 Hz), fields as low as 2 microV/m could be detected, and that the observation could be explained if a glycoprotein mass of 0.7 x 10(-12) kg (a sphere 11 microm in diameter) were bound to channel gates. Antibodies against apical membrane structures in Kryptopterus blocked field transduction, which was consistent with the proposal that it occurred at the membrane surface. Although the target of the field was hypothesized to be an ion channel, the proposed mechanism can easily be extended to include other kinds of membrane proteins.

Assessment of immunologic mechanisms for flare reactions to Synvisc.

Intraarticular injection of Synvisc for treatment of knee pain sometimes results in an acute local reaction (flare). We tested the hypothesis that the flare was a Type-1 hypersensitivity reaction as manifested by the presence of Synvisc antibodies in the synovial fluid and serum and by an increase in the concentration of the mast-cell enzyme tryptase in the synovial fluid. Our second objective was to determine whether the ratio of CD4+ to CD8+ lymphocytes in the synovial fluid was increased, as would be expected in a Type-4 hypersensitivity reaction. The study population was a prospective, consecutive series of 16 patients who had a flare, and 20 control patients. We found no differences in product-specific antibodies in the synovial fluid or serum between patients with flares and patients without flares. The mean tryptase level in the synovial fluid of patients with flares, 3.8 +/- 0.8 microg/L, was not different from the corresponding level in the control patients. The CD4+/CD8+ ratio in the synovial fluid was more than eight times greater in patients with flares. Flares that sometimes occur after treatment with Synvisc are probably not Type-1 (antibody-mediated) hypersensitivity reactions, but may be Type-4 (cell-mediated) hypersensitivity reactions.

Increased intercellular communication through gap junctions may contribute to progression of osteoarthritis.

Our aim was to support the hypothesis of a specific association between gap junctions in synovial tissue and the presence of osteoarthritis, as evidenced by differences between osteoarthritis and non-osteoarthritis synovia in the number of gap junctions, the amount of gap-junction protein, and the amount of enzymatic activity produced through a pathway mediated by gap-junction intercellular communication. An average of 4.41 gap junctions were found per 100 cells counted in the osteoarthritis synovia, compared with 1.00 in the controls. The amount of the gap-junction protein connexin 43 in synovial lining cells was approximately 50% greater in patients with osteoarthritis. Synovial lining cells from patients with osteoarthritis produced matrix metalloproteinases constitutively and, at higher levels, in response to stimulation by interleukin-1 beta. In both cases, intercellular communication through gap junctions was shown to be critical to the ability of the cells to secrete matrix metalloproteinases. Overall, the results indicated that gap junctions between synovial lining cells were altered significantly in patients with osteoarthritis, as a consequence of the disease process or as part of the causal chain. In either case, gap junctions seem to be a rational therapeutic target.

Extracellular currents alter gap junction intercellular communication in synovial fibroblasts.

We studied the effect of extremely low frequency (ELF) currents on gap junction intercellular communication (GJIC) mediated by connexin43 protein. Confluent monolayers of synovial fibroblasts (HIG-82) and neuroblastoma cells (5Y) were exposed in bath solution to 0-75 mA/m(2) (0-56 mV/m), 60 Hz. Single channel conductance, cell membrane current-voltage (I-V) curves, and Ca(2+) influx were measured using the nystatin single and double patch methods. The conductances of the closed and open states of the gap junction channel in HIG-82 cells were each significantly reduced (by 0.76 and 0.39 pA, respectively) in cells exposed to 20 mA/m(2). Current densities as low as 10 mA/m(2) significantly increased Ca(2+) influx in HIG-82 cells. No effects were seen in 5Y cells. The I-V curves of the plasma membranes of both types of cells were independent of 60 Hz electric fields and current densities, 0-75 mA/m(2), indicating that the effect of the 60 Hz fields on GJIC in HIG-82 cells was not mediated by a change in membrane potential. We conclude that ELF electric fields can alter GJIC in synovial cells via a mechanism that does not depend on changes in membrane potential, but may depend on Ca(2+) influx. The results open the possibility that GJIC mediated responses in synovial cells, such as for example, their secretory responses to proinflammatory cytokines, could be antagonized by the application of ELF electric fields.

IL-1beta-induced production of metalloproteinases by synovial cells depends on gap junction conductance.

Synovial cells can form networks connected by gap junctions. The purpose of this study was to obtain evidence for a necessary role of gap junction intercellular communication in protein secretion by synovial cells. We developed a novel assay to measure the enzymatic activity of metalloproteinases (MMPs) produced by synovial cells in response to interleukin-1beta (IL-1beta) and employed the assay to explore the biological function of gap junctions. IL-1beta produced a dose-dependent increase in MMP activity that was blocked by exposure to the gap junction inhibitors 18alpha-glycyrrhetinic acid and octanol for as few as 50 min. The inhibitors produced an immediate and marked reduction in intercellular communication, as assessed by transient current analysis using the nystatin perforated-patch method. These observations suggest that communication through gap junctions early in IL-1beta signal transduction is critical to the process of cytokine-regulated secretion of MMPs by synovial cells.