Joseph A. Browning

Surviving in a matrix: membrane transport in articular chondrocytes.

The separation of the extracellular milieu from the cytosol is especially important for articular chondrocytes, given the atypical and arguably challenging environment that these cells inhabit [77, 110, 117]. Chondrocytes, like all other cells, must possess effective membrane transport systems to minimize changes of cellular composition in the face of fluctuating surroundings [53, 82, 108]. They must also scavenge adequate precursor molecules for matrix macromolecule synthesis, a task hindered by their remoteness from the vasculature [62, 110]. The realization in the last decade that characterization of the cellular physiology of chondrocytes will further our understanding of cartilage pathologies such as osteoarthritis has led to increasing interest in the field of chondrocyte membrane transport [2, 51, 53, 55, 82, 132]. In this short, and necessarily selective, review we will consider the challenges faced by chondrocytes in articular cartilage, the ways in which these cells control their intracellular composition, and the role of membrane transporters in maintaining matrix integrity.

Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease.

1 This study was aimed at identifying the signalling pathways involved in the activation of volume‐regulatory mechanisms of human cervical cancer cells. 2 Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. 3 The activation of swelling‐activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling‐activated Cl− channel. 4 Cell swelling triggered mitogen‐activated protein (MAP) kinase cascades leading to the activation of extracellular signal‐regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume‐responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl− channels, and taurine transport. However, the volume‐regulatory mechanism was independent of the activation of p38 MAP kinase. 5 The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up‐regulated by protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate (PMA) and down‐regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen‐activated/ERK‐activating kinase (MEK) activity. 6 Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume‐regulatory mechanism of human cervical cancer cells.

Variations of Intracellular pH in Human Erythrocytes via K+(Na+)/H+ Exchange Under Low Ionic Strength Conditions

Abstract. The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K+ efflux and Cl− loss. When human erythrocytes were suspended in a physiological NaCl solution (pHo= 7.4), the measured pHi was 7.19 ± 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pHi to 7.70 ± 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl− content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H+ influx with the measured unidirectional K+ efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H+ influx and a K+ efflux in LIS solution of 108.2 ± 20.4 mmol (lcells hr)−1 and 98.7 ± 19.3 mmol (lcells hr)−1, respectively. For bovine and porcine erythrocytes, in LIS media, H+ influx and K+ efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K+(Na+)/H+ exchanger, inhibited the K+ efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K+(Na+)/H+ exchanger in the human erythrocyte membrane.

Deoxygenation-Induced Non-Electrolyte Pathway in Red Cells from Sickle Cell Patients

Red cells from patients with sickle cell disease contain HbS rather than the normal HbA (here termed HbS cells). On deoxygenation, HbS cells exhibit a distinctive solute permeability pathway, Psickle, activated stochastically, and partially inhibited by DIDS and dipyridamole. It is often referred to as a cation channel although its permeability characteristics remain vague and its molecular identity is unknown. We show that, in contrast to normal red cells, a proportion of HbS cells underwent haemolysis when deoxygenated in isosmotic non-electrolyte solutions. Haemolysis was stochastic: cells unlysed after an initial deoxygenation pulse showed lysis when harvested, reoxygenated and subsequently exposed to a second period of deoxygenation. O2 dependence of haemolysis was similar to that of Psickle activation. Haemolysis was accompanied by high rates of sucrose influx, and both haemolysis and sucrose influx were inhibited by DIDS and dipyridamole. Sucrose influx was only detected as ionic strength was reduced below 80 mM. These findings are consistent with the postulate that deoxygenation of HbS cells, under certain conditions, activates a novel non-electrolyte pathway. Their significance lies in understanding the nature of the deoxygenation-induced permeability in HbS cells, together with its relationship with novel pathways induced by a variety of manipulations in normal red cells.

Modulation of H+ transport mechanisms by interleukin-1 in isolated bovine articular chondrocytes.

The proinflammatory cytokine interleukin-1 (IL-1) promotes the degradation of articular cartilage by inhibiting matrix synthesis and stimulating degradative enzyme activity. Generation of nitric oxide (NO) in response to IL-1 is implicated in these actions. The catabolic actions of IL-1 can be inhibited by manoeuvres which are predicted to dissipate H+ gradients across the chondrocyte plasma membrane. In the present study, the effects of IL-1 on H+ extrusion from bovine articular chondrocytes were investigated. pH was measured using the H+-sensitive fluorescent dye BCECF. Cells were acidified by ammonium rebound and the contribution of the Na+-H+ exchanger (NHE) and of the vacuolar H+-ATPase to acid extrusion was characterised by ion substitution and inhibitor studies. Overnight (18h) exposure to IL-1 stimulated acid extrusion in a dose-dependent fashion. This effect represented stimulation of both NHE and the ATPase. Characterisation of the timecourse of this response indicated that, while stimulation of acid extrusion was rapid, effects on the ATPase were only apparent after greater than 8h incubation with the cytokine. In keeping with this observation, the protein synthesis inhibitor cycloheximide abolished the stimulatory effect of IL-1 on ATPase-mediated extrusion. The upregulation of ATPase activity by IL-1 was inhibited by the NOS inhibitor L-NAME and by the NO scavenger PTIO. In cells which had not been exposed to IL-1, treatment with the NO donor SNAP also stimulated acid extrusion by the ATPase. In contrast, NHE activity was not altered by any of these compounds. Taken together, these results imply that IL-1 can stimulate acid extrusion in chondrocytes and that this reflects rapid upregulation of NHE with slower induction of H+-ATPase activity which requires elevated levels of NO. While ATPase induction involves protein synthesis, this process may not constitute synthesis of ATPase proteins per se, but rather of some associated regulatory process.

Functional and molecular determination of carbonic anhydrase levels in bovine and cultured human chondrocytes

In this study, bovine articular and human chondrocytes from the C-20/A4 cell line were tested for the functional activity and molecular presence of the enzyme carbonic anhydrase. This enzyme is classically considered to be important in the maintenance of high cellular buffering capacity by catalysing the slow attainment of equilibrium between CO(2) and HCO(3)(-). The first functional assay measured the rate of pH equilibration after administration of a fixed dose of CO(2) solution to cell lysates. Compared to positive controls (human erythrocytes, murine M1 cells and purified carbonic anhydrase), chondrocyte lysates attained equilibrium at a significantly slower rate, similar to the rate obtained with a negative control (Xenopus oocytes). A second functional assay studied CO(2) hydration kinetics in intact C-20/A4 cells, using a pH-sensitive fluorescent dye, as the CO(2) content of the extracellular solution was changed. It was shown that C-20/A4 cells accelerate hydration only to a small degree. Hydration kinetics were reduced to the spontaneous rate in the presence of acetazolamide. Western immunoblotting with isoform-nonspecific antibodies to carbonic anhydrase demonstrated weak staining in both bovine and human chondrocytes.

Novel permeability characteristics of red blood cells from sickle cell patients heterozygous for HbS and HbC (HbSC genotype).

Individuals heterozygous for HbS and HbC (HbSC) represent about 1/3(rd) of sickle cell disease (SCD) patients. Whilst HbSC disease is generally milder, there is considerable overlap in symptoms with HbSS disease. HbSC patients, as well as HbSS ones, present with the chronic anaemia and panoply of acute vaso-occlusive complications that characterize SCD. However, there are important clinical and haematological differences. Certain complications occur with greater frequency in HbSC patients (like proliferative retinopathy and osteonecrosis) whilst intravascular haemolysis is reduced. Patients with HbSC disease can be considered as a discrete subset of SCD cases. Although much work has been carried out on understanding the pathogenesis of SCD in HbSS homozygotes, including the contribution of altered red blood cell permeability, relatively little pertains directly to HbSC individuals. Results reported in the literature suggest that HbSC cells, and particularly certain subpopulations, present with similar permeability to HbSS cells but there are also important differences - these have not been well characterized. We hypothesise that their unique cell transport properties accounts for the different pattern of disease in HbSC patients and represents a potential chemotherapeutic target not shared in red blood cells from HbSS patients. The distinct pattern of clinical haematology in HbSC disease is emphasised here. We analyse some of the electrophysiological properties of single red blood cells from HbSC patients, comparing them with those from HbSS patients and normal HbAA individuals. We also use the isosmotic haemolysis technique to investigate the behaviour of total red blood cell populations. Whilst both HbSS and HbSC cells show increased monovalent and divalent (Ca(2+)) cation conductance further elevated upon deoxygenation, the distribution of current magnitudes differs, and outward rectification is greatest for HbSC cells. In addition, although Gd(3+) largely abolishes the cation conductance of both HbSS and HbSC cells, only in HbSS ones are currents inhibited by the aminoglycosides like streptomycin. This distinction is retained in isosmotic lysis experiments where both HbSS and HbSC cells undergo haemolysis in sucrose solutions but streptomycin significantly inhibits lysis only in HbSS cells. These findings emphasise similarities but also differences in the permeability properties of HbSS and HbSC cells, which may be important in pathogenesis.

Characterisation of inorganic phosphate transport in bovine articular chondrocytes.

In mineralising tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV) possess membrane transporters that accumulate Ca²⁺ and inorganic phosphate (P_i), and initiate the formation of hydroxyapatite crystals. MV are also present in articular cartilage, and hydroxyapatite crystals are believed to promote cartilage degradation in osteoarthritic joints. In the present study, P_i transport pathways in isolated bovine articular chondrocytes have been characterised. P_i uptake was temperature-sensitive and could be resolved into Na⁺-dependent and Na⁺-independent components. The Na⁺-dependent component saturated at high concentrations of extracellular P_i, with a K_m for P_i of 0.17mM. In solutions lacking Na⁺, uptake did not fully saturate, implying that under these conditions carrier-mediated uptake is supplemented by a diffusive pathway. Both Na⁺-dependent and Na⁺-independent components were sensitive to the P_i transport inhibitors phosphonoacetate and arsenate, although a fraction of Na⁺-independent P_i uptake was resistant to these anions. Total P_i uptake was optimal at pH 7.4, and reduced as pH was made more acidic or more alkaline, an effect that represented reduced Na⁺-dependent influx. RT-PCR analysis confirmed that two members of the NaPi III family, Pit-1 and Pit-2, are expressed, but that NaPi II transporters are not.

Effect of Peroxynitrite on Passive K+ Transport in Human Red Blood Cells

Peroxynitrite is generated in vivo by the reaction between nitric oxide, from endothelial and other cells, and the superoxide anion. It is therefore pertinent to examine its effects on the membrane permeability of red blood cells. Treatment of human red blood cells with peroxynitrite (nominally 1 mM) markedly stimulated passive K⁺ permeability. The main effect was on a Cl^--independent K⁺ pathway, which remains unidentified. Although K⁺-Cl^- cotransport (KCC) was stimulated, this was dependent on saline composition, being inhibited by physiological levels of glucose (IC₅₀ 4 mM), and also by sucrose and MOPS. Effects on the Cl^--independent K⁺ pathway were less dependent on saline composition, and were not inhibited by amiloride, ethylisopropylamiloride, dimethylamiloride or gadolinium. Na⁺-K⁺-2Cl^- cotransporter was inhibited whilst there was little effect on the Gardos channel (Ca²⁺-activated K⁺ channel). Peroxynitrite was markedly more effective in oxygenated cells than deoxygenated ones. Treatment with peroxynitrite per se did not affect initial cell volume. Anisotonic swelling modestly increased the Cl_--independent K⁺ influx, but did not affect peroxynitrite-stimulated KCC. Decreasing extracellular pH from 7.4 to 7.2 or 7.0 increased KCC stimulation, whilst the Cl^--independent component of K⁺ transport was lowest at pH 7.2. Finally, protein phosphatase inhibition with calyculin A (100 nM) inhibited KCC, implying that, as with other KCC stimuli, peroxynitrite acts via decreased protein phosphorylation; pre-treatment with calyculin A also inhibited the Cl^--independent component of K⁺ transport. These findings are relevant to the actions of peroxynitrite in vivo.

The effect of intracellular alkalinisation on intracellular Ca(2+) homeostasis in a human chondrocyte cell line.

Abstract. Intracellular pH (pHi) is a well-established determinant of cartilage matrix metabolism. Changes to chondrocyte pHi, and therefore matrix turnover rates, arise following joint loading. It is not yet clear whether pH changes exert their effects on matrix metabolism directly, or by changing the concentration of another, as yet unidentified, intracellular factor. In this study the effect of intracellular alkalinisation on intracellular [Ca2+] has been examined using the human chondrocyte C-20/A4 cell line. pHi was manipulated by the addition of weak bases to suspensions of chondrocytes and fluorimetric techniques were employed to measure pHi and [Ca2+]i. The effect of pHi changes on intracellular inositol 1,4,5-trisphosphate (IP3) levels was also determined. The pH-sensitive properties of the Ca2+-sensitive fluoroprobe employed in this study, Fura-2, were investigated such that artefactual effects of pH changes upon the dye could be discounted. It was demonstrated that, for dye loaded into cells, alkalinisation resulted in a small increase in the affinity of the dye for Ca2+ ions. Intracellular alkalinisation elicited by treatment with either of the weak bases trimethylamine or ammonium chloride initiated a rise in [Ca2+]i. This effect was too large to be explicable by the effects of pH changes on Fura-2 and was not dependent on the presence of extracellular Ca2+ ions. Prior depletion of intracellular Ca2+ stores by treatment with thapsigargin inhibited alkalinisation-induced increases in [Ca2+]i and intracellular alkalinisation was also associated with increased levels of intracellular IP3. These results confirm that alkaline pHi changes associated with dynamic loading of cartilage also result in knock-on alterations to [Ca2+]i. Given the sensitivity of cartilage matrix metabolism to [Ca2+]i it is likely that this signalling cascade forms an important part of the mechanotransduction pathway that determines the response of chondrocytes to applied load.