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A number of situations that result in abnormal permeability pathways in human red blood cells (RBCs) have been investigated. In sickle cell disease (SCD), RBCs contain HbS, rather than the normal HbA. When deoxygenated, an abnormal conductance pathway, termed P(sickle), is activated, which contributes to cell dehydration, largely through allowing Ca(2+) entry and subsequent activation of the Gardos channel. Whole-cell patch-clamp recordings from sickle RBCs show a deoxygenated-induced conductance, absent from normal RBCs, which shares some of the properties of P(sickle): equivalent Na(+) and K(+) permeability, significant Ca(2+) conductance, partial inhibition by DIDS and also Zn(2+). Gd(3+) markedly attenuates conductance in both normal and sickle RBCs. In addition, deoxygenated sickle cells, but not oxygenated ones or normal RBCs regardless of the oxygen tension, undergo haemolysis in isosmotic non-electrolyte solutions. Non-electrolyte entry was confirmed radioisotopically whilst haemolysis was inhibited by DIDS. These findings suggest that under certain circumstances P(sickle) may also be permeable to non-electrolytes. Finally, RBCs from certain patients with hereditary stomatocytosis have a mutated band 3, which appears able to act as a conductance pathway for univalent cations. These results extend our understanding of the abnormal permeability pathways of RBCs.

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Chondrocytes in cartilage are embedded in a matrix containing a high concentration of proteoglycans and hence of fixed negative charges. Their extracellular ionic environment is thus different from that of most cells, with extracellular Na+ being 250-350 mM and extracellular osmolality 350-450 mOsm. When chondrocytes are isolated from the matrix and incubated in standard culture medium (DMEM; osmolality 250-280 mOsm), their extracellular environment changes sharply. We incubated isolated bovine articular chondrocytes and cartilage slices in DMEM whose osmolality was altered over the range 250-450 mOsm by Na+ or sucrose addition. 35S-sulphate and 3H-proline incorporation rates were at a maximum when the extracellular osmolality was 350-400 mOsm for both freshly isolated chondrocytes and for chondrocytes in cartilage. The incorporation rate per cell of isolated chondrocytes was only 10% that of chondrocytes in situ both 4 and 24 hours after isolation. For freshly isolated chondrocytes, the rate increased 30-50% in DMEM to which NaCl or sucrose had been added to increase osmolality. In chondrocytes incubated overnight in DMEM, the rate was greatest in DMEM of normal osmolality and fell from the maximum in proportion to the change in osmolality. The effects of sucrose addition on incorporation rates were similar but not identical to those of Na+ addition. Changes in cell volume might be linked to changes in synthesis rates since the cell volume of chondrocytes (measured by Coulter-counter) increased 30-40% when the cells were removed from their in situ environment into DMEM. Synthesis rates can thus be partly regulated by changes in extracellular osmolality, which in cartilage is controlled by proteoglycan concentration. This provides a mechanism by which the chondrocytes can rapidly respond to changes in extracellular matrix composition.

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The direct effects of hydrostatic pressure on matrix synthesis in articular cartilage can be studied independently of the other factors that change during loading. We have found that the influence of hydrostatic pressure on incorporation rates of 35SO4 and [3H]proline into adult bovine articular cartilage slices in vitro depends on the pressure level and on the time at pressure. Pressures in the "physiological" range (5-15 MPa) applied for 20 s or for 5 min could stimulate tracer incorporation (30-130%) during the following 2 h, but higher pressures (20-50 MPa) had no effect on incorporation rates. The degree of stimulation in cartilage obtained from different animals was found to vary; in some animals none was seen. Stimulation also varied with position along the joint. Physiological pressures (5-10 MPa) applied continuously for the 2-h incubation period also stimulated incorporation rates, but pressures greater than 20 MPa always produced a decrease that was related to the applied pressure and that was reversible. These results suggests that the hydrostatic pressure that occurs during loading is a signal that can stimulate matrix synthesis rates in articular cartilage.

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The overall internal pH of the acid-tolerant green alga, Chlorella saccharophila, was determined in the light and in the dark by the distribution of 5,5-dimethyl-2-[(14)C]oxazolidine-2,4-dione ([(14)C]DMO) or [(14)C]benzoic acid ([(14)C]BA) between the cells and the surrounding medium. [(14)C]DMO was used at external pH of 5.0 to 7.5 while [(14)C]BA was used in the range pH 3.0 to pH 5.5. Neither compound was metabolized by the algal cells and intracellular binding was minimal. The internal pH of the algae obtained with the two compounds at external pH values of 5.0 and 5.5 were in good agreement. The internal pH of C. saccharophila remained relatively constant at pH 7.3 over the external pH range of pH 5.0 to 7.5. Below pH 5.0, however, there was a gradual decrease in the internal pH to 6.4 at an external pH of 3.0. The maintenance of a constant internal pH requires energy and the downward drift of internal pH with a drop in external pH may be a mechanism to conserve energy and allow growth at acid pH.