Biomedical subjects

F Vittur

Publications and source records attributed to F Vittur.

At least 37 records · Page 2Linked to original sources

PubMed1996

Investigation of laminar appearance of articular cartilage by means of magnetic resonance microscopy.

Magnetic resonance (MR) images and relaxation and diffusion maps of articular cartilage were obtained to explain discrepancies in its MR appearance. Porcine specimens were studied only by MR microscopy. For human specimens a combination of MR microscopy and large-scale MR imaging was used. Common features in the laminar structures of human and porcine samples are described. It was found that the decay of transverse magnetization was nonexponential with a rapidly decaying component which prevented construction of reliable proton-density maps. Dependence of T2 values on the orientation of specimens in the magnetic field as well as magnetization transfer experiments supported the previous suggestions about a significant role of dipolar interaction with protons of collagen in the laminar appearance of articular cartilage. The loss of the laminar structure induced by rotation of the human cartilage specimen around the axis normal to its surface demonstrated nonuniform angular distribution of the collagen fibers within the layer.

Animals↗

PubMed1996

Gap junctions mediate intercellular calcium signalling in cultured articular chondrocytes.

Gap junction-mediated intercellular communication has been implicated in a variety of cellular functions. Among these, signal transduction can be coordinated among several cells due to gap junctional permeability to intracellular second messengers. Chondrocytes from articular cartilage in primary culture respond to extracellular ATP by rhythmically increasing their cytosolic Ca2+ concentration. Digital imaging fluorescence microscopy of Fura-2 loaded cells was used to monitor Ca2+ in confluent and semi-confluent cell layers. Under these conditions, Ca2+ spikes propagate from cell to cell giving rise to intercellular Ca2+ waves. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in scrape-loading experiments. Intercellular dye transfer was blocked by the gap junction inhibitor 18 alpha-glycyrrhetinic acid. In imaging experiments, the inhibitor caused the loss of synchrony of ATP-induced Ca2+ oscillations, and blocked the intercellular Ca2+ propagation induced by mechanical stimulation of a single cell in a monolayer. It is concluded that gap junctions mediate intercellular signal transduction in cartilage cells and may provide a mechanism for co-ordinating their metabolic activity.

Adenosine Triphosphate↗

PubMed1995

Spatial and temporal Ca2+ signalling in articular chondrocytes.

Stimulation of pig articular chondrocytes with either bradykinin, fetal calf serum or the Ca(2+)-ATPase inhibitor thapsigargin induced increases of the cytosolic Ca2+ concentration. By computerized videoimaging, the spatial and temporal aspects of the Ca2+ signal were revealed at single cell level. The cell response depended on Ca2+ release from intracellular stores without significant contribution of Ca2+ influx. A great heterogeneity in the cell population was found with respect to the Ca2+ storage ability. The Ca2+ response initiated in a discrete subcellular region and then spread in a nondecremental fashion to involve the whole cytosol. Such a behaviour was independent of the stimulus applied, thus suggesting a functional heterogeneity of the intracellular Ca2+ stores involved. In the region from which the response started, local Ca2+ spikes were recorded, revealing a spatially restricted pulsatile activity.

Animals↗

PubMed1995

Calcium, sulfur, and zinc distribution in normal and arthritic articular equine cartilage: a synchrotron radiation-induced X-ray emission (SRIXE) study.

Calcium, sulfur, and zinc content in normal and arthritic equine cartilage have been studied by synchrotron radiation-induced X-ray emission (SRIXE). Ranging from the superficial to the columnar zone of the normal tissue, calcium and zinc concentrations are increasingly higher, whereas sulfur is at its highest concentration in the transitional zone. In the arthritic tissue, calcium concentration is at its maximum in the transitional zone, whereas zinc and sulfur distributions are relatively homogeneous. Sulfur concentration in arthritic cartilage is reduced to about one-third with respect to that in normal tissue. The possibility that zinc concentration reflects the distribution of the zinc-containing enzyme alkaline phosphatase is presented.

Animals↗

PubMed1995

Detection and quantitation of phosphorus metabolites in crude tissue extracts by 1H and 31P NMR: use of gradient assisted 1H-31P HMQC experiments, with selective pulses, for the assignment of less abundant metabolites.

The analysis of crude tissue extracts by NMR has proven to be of use in the study of metabolism due to the non-destructive and non-selective character of the technique. Lists of 1H and 31P NMR assignments of phosphorus metabolites in water solution at specified pH and ionic composition are of large general value but their usefulness may be limited when analysing complex mixtures of metabolites at low concentrations. In this work we report on the use of gradient-assisted proton detected multiple quantum 1H and 31P coherence experiments with selective pulses for the rapid and unambiguous assignments of some crowded regions in 1H and 31P spectra of crude extracts from rat liver. The amplitudes of the gradient episodes were calibrated to optimize the coherence transfer pathway between proton and phosphorus, and the delay for the evolution of the long-range coupling was calculated from values of 3JPH and 4JPH ranging from 1.4 to 7.5 Hz. Moreover, a selective 90 degrees Gaussian pulse on the 31P channel was introduced to increase the resolution in the F1-domain and make the method even faster. The procedure was then applied to unambiguously assign the ID 31P and 1H spectra of perchloric acid extracts of rat livers that had been stimulated with phenylephrine, dBcAMP and glucagon and thus detect changes in the concentration of less abundant metabolites such as phosphoenolpyruvate, UDP-glucose and AMP. The fact that the quantification of these metabolites by either 31P and 1H methods lead to different results is discussed, and the use of 1H NMR spectroscopy for the quantification of phosphorus metabolites whose signal are too weak or poorly resolved in a 31P spectrum is proposed.

Adenosine Monophosphate↗

PubMed1995

Oxygen-derived free radical (ODFR) action on hyaluronan (HA), on two HA ester derivatives, and on the metabolism of articular chondrocytes.

Oxygen-derived free radicals (ODFR) appear to be involved in the pathogenesis of arthritic disorders. In order to gain new insight on their role in the phenomenon and as a basis for a therapeutic approach, the effect of ODFR (produced by the xanthine oxidase-hypoxantine system) on hyaluronic acid, on two HA ester derivatives, and on pig articular chondrocytes was investigated. High M(r) HA (1.1 x 10(6)) and low M(r) HA (16 x 10(4)) were depolymerized by ODFR but the methyl and hydrocortisone esters of HA (HYAFF 2P50 and HYC13) turned out to be nearly unaffected. When articular chondrocytes were treated with ODFR, a rapid nucleoside triphosphate (NTP) depletion, a transient appearance of pyrophosphate (PPi), and an increase of phosphomonoester and diphosphodiester concentrations have been observed. The NTP depletion and the DPDE increase are related to the concentration of free radicals. Glyceraldehyde-3-phosphate accumulation during ODFR treatment suggests that ATP depletion can occur as a consequence of the blockage of glycolysis at the level of glyceraldehyde-3-P dehydrogenase. The hypothesis is presented that PPi can be produced from the pathway of the FAD-NAD (DPDE) biosynthesis and then either hydrolyzed by endogenous pyrophosphatases or precipitated in the form of insoluble calcium salts. Long-term treatment (16 h) with ODFR causes a loss of chondrocyte membrane integrity which can be revealed both by an increased free LDH activity and by the characteristic signal of free phospholipids in the 31P-NMR spectra. While high M(r) HA shows a significant protective activity for chondrocytes against ODFR action, low M(r) HA and ester derivatives do not. It is suggested that the therapeutic activity of HA ester derivatives can be ascribed to their in vivo hydrolysis products.

Adenosine Triphosphate↗

PubMed1994

Potassium channels of pig articular chondrocytes are blocked by propofol.

The effect of propofol on the voltage-activated potassium channels in pig articular chondrocytes was investigated. Propofol was found to reversibly block the potassium channels in a dose-dependent manner. The blocking effect was voltage-independent and the Hill coefficient was 1.85 +/- 0.18. No changes either in the slope conductance or in the single channel kinetics were observed. The half-blocking concentration (Ec50) was 6.0 +/- 0.49 microM which is much lower than the concentrations used to observe the scavenging effect of the drug in an artificial synovial fluid. Interestingly, Ec50 found in our experiments is also smaller than the blood concentration of propofol used in anaesthesia. These results show that propofol may strongly affect the potassium channels in some non-excitable cells.

Animals↗

PubMed1994

Energy metabolism, replicative ability, intracellular calcium concentration, and ionic channels of horse articular chondrocytes.

Some aspects of the physiology of chondrocytes from horse articular cartilage were studied, since this animal model can be helpful in understanding arthritic processes. The replicative ability of articular chondrocytes, measured by the incorporation of [3H]thymidine, and their capacity of proteoglycan production, evaluated from the incorporation of [35S] sulfate, are very low. In addition, these cells do not differentiate in vitro as shown by the constant specific activity of alkaline phosphatase measured at different times in culture. Two types of potassium channels were identified by patch clamp experiments in the cell-attached configuration, one characterized by a conductance of 40 pS and the other of 100 pS. No active K+ channels were found at Vpip = 0. It was shown by Fura-2 experiments that the low replicative ability is paralleled by a modest variation of the intracellular calcium concentration after a mitogenic stimulus. 31P NMR experiments, both on slices of whole articular cartilage and on isolated cells, demonstrate that chondrocytes derive their energy mainly from the glycolytic pathway.

Animals↗

PubMed1993

Culture and differentiation of chondrocytes entrapped in alginate gels.

We studied the response to culture conditions and the differentiative ability in suspension culture in alginate gels of resting chondrocytes from the preosseous cartilage of adult pig scapula. It was found that the maximum rate of chondrocyte duplication is reached at the fourth day in culture whereas the rate of proteoglycan synthesis and alkaline phosphatase expression do not gain a maximum value before the seventh day. During the culture time, the chondrocytes undergo differentiation as it is demonstrated by the alkaline phosphatase specific activity increase and by morphological criteria (hypertrophy, increase of the number of mitochondria per cell, increased endoplasmic reticulum, matrix vesicle production). The alginate gels can be easily dissolved to obtain cell populations in which the variation of cytosolic calcium concentration following a proliferative stimulus can be conveniently observed using the conventional procedure of Fura 2.

Alginates↗

PubMed1992

Elemental analysis of growth plate cartilage by synchrotron-radiation-induced X-ray emission (SRIXE).

The elemental composition of growth plate cartilage from calf scapula has been studied by means of SRIXE. X-ray emission spectra were obtained from the resting, hypertrophic and calcified regions of cartilage; then, each element was mapped with a lateral definition of about 10 microns x 10 microns. Evidence was found for a homogeneous distribution of the elements in resting cartilage compared to changes in local concentration of some atoms in the hypertrophic-calcified tissue. In this zone Ca, Sr, Ni, Zn, S, reach the maximal concentration at the calcification front while Cu shows a uniform distribution. A Zn distribution similar to that of the Zn-containing enzyme alkaline phosphatase, the key enzyme of calcification, is found.

Animals↗

PubMed1992

Calcium-activated potassium channels in chondrocytes.

The presence of calcium-activated potassium channels in chondrocytes of growing cartilage was tested. Results obtained with fura-2 on cultured resting chondrocytes indicate that the cells respond to an elevation of extracellular calcium concentration ([Ca2+]o) from 0.1 to 2 mM increasing the intracellular concentration of the ion ([Ca2+]i) from 117 to 187 nM. This increment may be blocked by 3 microM La3+. Patch clamp experiments in cell-attached configuration showed that, when [Ca2+]i rises, the open probability (Po) of the K+ channels increases. Increments in both Po and unitary currents of the K+ channels can be obtained after applying 2.5 microM A23187 with 2 mM [Ca2+]o. Hence, the results demonstrate that, in chondrocytes, a class of Ca(2+)-activated K+ channels is present and their activity is related to an increase of [Ca2+]i.

Animals↗

PubMed1992

31P NMR studies of resting zone cartilage from growth plate.

31P NMR of superfused resting cartilage demonstrated the presence of phosphocreatine in chondrocytes. Changes in pH and in the NTP level were followed during carbon source starvation. From 31P spectra of perchloric acid extracts, phosphoethanolamine, phosphocholine, and the corresponding glycerol diesters were identified as the major phosphomonoester and phosphodiester components.

Animals↗

PubMed1991

Energy state of chondrocytes assessed by 31P-NMR studies of preosseous cartilage.

The energy state of resting and hypertrophic chondrocytes from growth plate was studied by 31P-NMR spectroscopy of superfused cartilage slices. The presence of phosphocreatine was demonstrated in both cell types, using a repetition time of 3 s. By comparing the decline in the nucleoside triphosphate level after adding blockers of the glycolysis or of the mitochondrial respiration, it was deduced that resting and hypertrophic chondrocytes use both metabolic pathways for energy production, but the glycolysis dominates. Hypertrophic cells rely more on the mitochondrial respiration than the resting cells.

Animals↗

PubMed1990

A potassium channel in cultured chondrocytes.

Chondrocytes, obtained from preosseous cartilage, were studied by patch clamp technique in cell-attached recording configuration, and single potassium channels were characterized at different stages of culture. After 3 days, outward currents were present, with an open probability increasing with depolarization, and the K+ channels showing a mean slope conductance of 82 pS in asymmetric and 168 pS in symmetric potassium solution. Tetraethylammonium (TEA) and quinidine blocked the channels. Cells at confluence showed similar channel activity, with conductances of 121 and 252 pS, respectively. We suggest that culture time and/or conditions may modify K+ channels or induce the expression of a new type of channels.

Action Potentials↗

PubMed1990

Serum-induced cytosolic calcium movements and mitogenesis in cultured preosseous chondrocytes.

The differentiation of preosseous chondrocytes begins with the proliferation of resting cells and results in the expression of the hypertrophic phenotype. The effect of fetal calf serum on chondrocyte mitogenesis and intracellular Ca2+ concentration was studied in resting and hypertrophic cells in primary culture. Resting chondrocytes respond to the growth stimulus with immediate release of Ca2+ from intracellular stores and with opening of the plasma membrane Ca2+ channels. These events may be related to the elevated [3H]thymidine incorporation observed after serum exposure. In contrast, in hypertrophic chondrocytes the lower rate of DNA synthesis seems to be coupled with a lower activity of the Ca2+ signaling mechanism and, probably, with reduced intracellular calcium stores. It is proposed that expression of the Ca2+ signaling mechanism may be modulated during the differentiation of preosseous chondrocytes.

Animals↗

PubMed1990

Modification of plasma membrane of differentiating preosseous chondrocytes: evidence for a degradative process in the mechanism of matrix vesicle formation.

Chondrocytes of the growth plate are differentiating cells. Their evolution leads to matrix vesicle formation and to cartilage mineralization. This is an in vitro study of the plasma membrane of chondrocytes at two differentiation stages. Differences in protein and glycoprotein components, increased membrane fluidity, and responsiveness to PTH indicate that hypertrophic ("ossifying") chondrocytes possess a plasma membrane widely different from that of resting chondrocytes. Their plasma membrane is particularly enriched in alkaline phosphatase (Mr 70K). Purified matrix vesicles contain the 70K form of alkaline phosphatase, but a 50K species is also detectable, a signal of degradative process. In fact, proteins and glycoproteins of matrix vesicles are less numerous than those of cell plasma membranes. It is suggested that, in vivo, matrix vesicle formation may be mediated by Ca2(+)-activated neutral proteases.

Alkaline Phosphatase↗

PubMed1986

A possible role for polyamines in cartilage in the mechanism of calcification.

The role of polyamines in cartilage is not known: they may be somehow related to the mechanism of calcification. In epiphyseal cartilage from calf scapulas, they are more concentrated in the ossifying area, where calcification takes place, than in the resting region. Spermidine is present in greater amounts than spermine and putrescine. Since ornithine decarboxylase (EC 4.1.1.17) is measurable only in the resting region of the tissue, it is in this area that polyamine biosynthesis occurs, while they accumulate in the ossifying area. Immunohistochemical evidence is obtained that only in the ossifying zone is spermidine extracellular. It is at this level that the matrix is rearranged to become calcified, and proteoglycans are dissociated and partially removed. The effect of polyamines on solutions of proteoglycan subunits has been studied in vitro by following variations of turbidity and viscosity. While in the presence of putrescine the specific viscosity decreases to asymptotic values, in the presence of either 30 mM spermidine or 2.5-10 mM spermine, the decrement is more marked. At the same concentrations, increase of the turbidity of proteoglycan subunit solutions was observed. Only spermidine showed the capacity of displacing proteoglycan subunits from a column of Sepharose 4B-type II collagen: at 15 mM concentration, about 90% of proteoglycans were removed from the column. Alkaline phosphatase activity, which plays an important role in calcification, is enhanced by spermidine and spermine. These results obtained in vitro support the hypothesis that polyamines may be related to calcification of preosseous cartilage.

Alkaline Phosphatase↗

PubMed1984

Alkaline phosphatase binds to collagen; a hypothesis on the mechanism of extravesicular mineralization in epiphyseal cartilage.

Affinity chromatography on Sepharose 4B-collagen gels was used to test the affinity of alkaline phosphatase for collagen. Results indicate that alkaline phosphatase of preosseous cartilage binds to collagen probably by electrostatic interactions, this interaction is inhibited by proteoglycan subunits. These results suggest that, in vivo, the formation of a collagen-alkaline phosphatase complex may be a step of the process leading to cartilage calcification.

Alkaline Phosphatase↗