Regulatory volume response of erythrocytes exposed to a gradual and slow decrease in medium osmolality

 A sudden decrease in external medium osmolality (90 mosmol/kg) causes an immediate swelling of trout erythrocytes, followed by a regulatory volume decrease (RVD) due to activation of both a KCl cotransporter and a taurine transport pathway. Here, we determined how trout red cells respond when they are exposed to a gradual and slow decrease in medium osmolality (80 mosmol/kg at a rate of 0.7 mosmol/kg per min). Erythrocytes were unable to regulate their volume efficiently when swollen gradually and it increased continuously throughout the experimental period (120 min). As long as volume was increased slowly by 15–25%, regulatory pathways remained essentially inactivated, erythrocytes losing no significant amount of intracellular osmotically active solutes. Above this swelling threshold, a response was triggered but the quantity of solutes lost via the regulatory pathways was still not sufficient to counterbalance the continuous entry of water due to the slow and gradual decrease in medium tonicity. Received: 18 January 1999 / Received after revision: 10 February 1999 / Accepted: 11 February 1999     

Flow-mediated cell stress induction in adherent leukocytes is accompanied by modulation of morphology and phagocytic function

Leukocytes adherent to the surfaces of both vascular biomaterials and normal blood vessels experience blood flow induced shear stress. The goal of the reported studies was to investigate the effect of fluid flow on the morphology, phagocytic function and stress response induction in adherent immune cells. Shear approximating arterial, venous and intermediate levels were applied onto glass-adherent IC21 macrophages in a temperature-controlled parallel plate flow system. The results indicate that fluid flow induces a shear-dependent physiological stress response in adherent macrophages and that significant morphological changes accompany macrophage responses to shear stress. In addition, arterial flow conditions induce not only significant cell polarisation, but also enhanced phagocytic ingestion in glass-adherent IC21 macrophages. These findings suggest that blood flow induced shear stress may not only be consequent to adherent leukocyte activation, but may also be integral to the regulation of adherent leukocyte behaviour in vivo.     

Cellular dynamics of corneal wound re-epithelialization in the rat. I. Fate of ocular surface epithelial cells synthesizing DNA prior to wounding

The fate of ocular surface epithelial cells in response to injury of the cornea was examined. Corneal epithelial cells were labeled during DNA synthesis with [3H]thymidine 1 h prior to wounding. A 3-mm diameter epithelial defect was made in the center of the rat cornea, with the basement membrane remaining intact. Within 12 h of abrasion, labeled cells were detected in the regenerating surface. At 18 h, there was a 2.7- and 17-fold increase of labeled basal and suprabasal cells, respectively, in the epithelium adjacent to the wound, and at 24 and 30 h there was an excessive number of cell layers (up to 7) at the margin of the abrasion. Re-epithelialization progressed as a gradient of cell layers that became diminished towards the center of the wound. Completion of layers 1, 2, 3, and 4 were recorded at 24, 30, 36, and 72 h, respectively. No changes in the labeling index of the limbus or conjunctiva were noted. These results suggest that processes of centripetal and vertical migration, as well as events related to cell division, in the uninjured corneal surface are not impeded by wounding of the corneal epithelium. However, wound healing appears to require cells with a basal phenotype, presumably because of this cell type's migratory capability.     

Intracellular ATP depletion inhibits swelling-induced -[H]aspartate release from primary astrocyte cultures

Volume expansion-sensing outward rectifier (VSOR) anion channel, also referred to as volume-sensitive organic osmolyte-anion channel (VSOAC), appears to be responsible for cell swelling-induced amino acid release in a variety of cells. One prominent feature of the VSOR/VSOAC is that non-hydrolyzed intracellular ATP binding to the channel or an accessory protein is required for its activation. In this study, the effect of intracellular ATP depletion on the swelling-induced release of -[³H]aspartate from rat primary astrocyte cultures due to exposure to either high K⁺ or hypotonic media was studied. When the cells were pretreated for 10 min with a combination of the metabolic inhibitors 2-deoxyglucose and rotenone, 100 mM K⁺ media- or hypotonic media-induced -[³H]aspartate release was completely suppressed. Added separately, each inhibitor showed only partial or no inhibition of -[³H]aspartate release, which correlated with its relative effectiveness in decreasing intracellular ATP levels. These data are consistent with the view that during high [K⁺]_o or hypotonic media-induced swelling of primary astrocyte cultures an ATP-dependent swelling-activated VSOAC channel is responsible for -[³H]aspartate release and close to normal ATP is required for full channel activation.     

Hypoxia induces differential changes of dopamine metabolism in mature and immature mesencephalic and diencephalic cell cultures

Summary. Perinatal hypoxia is known as a high risk factor for the development of long-lasting abnormalities in dopaminergic system. The early developmental alterations of dopamine (DA) metabolism induced by hypoxia could contribute to these abnormalities. To understand the hypoxia-induced changes of intra- and extracellular dopamine levels and its main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in immature dopaminergic neurons, we compared these changes in rat mesencephalic and diencephalic cell cultures on day in vitro (DIV) 2 (immature cells), DIV 8 and DIV 13 (mature cells). Cell cultures were exposed to an oxygen-free gas mixture in a Billups chamber for 2–4 hours. Mature cell cultures responded to hypoxia with an increase of DA levels in the cells and in the medium during the first 45 min (by an average of 57 and 114% respectively). Thereafter, DA levels decreased, and returned to the baseline within the next 30 min. The cellular DA levels continued to decrease up to 15% of the baseline during 255 min hypoxia whereas the extracellular DA content stabilized at the prehypoxic levels. Immature cell cultures (DIV 2) in contrast to mature ones, were unable to maintain normal extracellular DA levels during hypoxia and showed a decrease of the cellular and extracellular levels to 50% of the prehypoxic levels. DOPAC and HVA changes mimick, however, at a lower level, the pattern of DA changes during the exposure to hypoxia. In principle, in the diencephalic cell culture similar effects of hypoxia exposure on the investigated parameters were found (studied during 0–120 min). The present study demonstrates that mature and immature dopaminergic cells differ in the regulation of the extra- and intracellular DA levels during hypoxia. In immature cells the low synthetic capacity of tyrosine hydroxylase and the deficient capacities of the transport and storage processes result in decreased extracellular DA levels. This could be an important factor for the long-term modulation of the expression of tyrosine hydroxylase and subsequent long-term behavioral and/or neurological abnormalities induced by perinatal hypoxia. Received June 8, 1998; accepted July 21, 1998     

Preservation of the non-rectangular cuticular plate/cell axis angle of outer hair cells

Motile properties of outer hair cells (OHCs) may contribute to sharp tuning and amplification in the mammalian cochlea. Shape changes of isolated OHCs in response to various physical and chemical influences have been investigated intensively. However, determinations of shape may have been influenced by unanticipated effects of preparation and preservation of the OHCs investigated. Thus, in a first step, lengths of freshly isolated OHCs from the guinea pig cochlea were determined using a video-enhancing magnification system. The cuticular plate/cell axis angle (CP/CA angle) was then measured in native cells and under the influence of potassium chloride and potassium gluconate incubation. To show the influence of glutaraldehyde (GA) fixation on the isolated OHCs, fixative dependent changes on cell length and CP/CA angle were recorded in native and preincubated OHCs. In these experiments, the cell length of vital isolated OHCs was between 41.5 m, in the basal turn, and 103.7 m, in the apical turn. The average CP/CA angle was 106° ± 4.2° (n = 324 cells, turns 1–4) with no statistically significant differences for the four turns. Under the influence of potassium chloride, cell length was reduced by 8.1%. Potassium gluconate incubation led to a shortening of cell length, followed by a 5.3% increase after 5 min. The CP/CA angle under potassium chloride was decreased (97.0°) and was then increased under the influence of potassium gluconate (110.7°) as a result of cuticular plate tilting. Cell shrinkage after fixation depended on the fixative's osmolarity and on the GA concentration. Increased GA levels amplified cell shrinkage from 34% for hypo-osmolar solutions to 15% in iso-osmolar and 29% in hyperosmolar solutions. The CP/CA angle of native and incubated OHCs was not different from those fixed with GA. The present data provide a rational basis for isolated OHC shape parameters. Moreover, functionally induced changes can be better interpreted when OHCs are influenced by fixatives, as shown in the GA experiments.     

Correlation of freeze-fracture and scanning electron microscopy of epiphyseal chondrocytes

Summary Chondrocytes in epiphyseal cartilage were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) using freeze-fracture techniques. Freeze-fracture replicas showed large numbers of fingerlike, 0.11–0.15 m diameter, projections from the chondrocyte surface, with numerous 95–180 Å diameter intramembranous particles associated with both the cell membrane surface and these projections. With SEM, these cytoplasmic projections were also obvious, but appeared collapsed into clusters of globular-shaped projections on the surface of the chondrocytes. With freeze-fracture techniques, in which shrinkage artifacts were essentially eliminated, the cytoplasmic projections were often seen in intimate contact with the extracapsular matrix. However, with chondrocytes prepared by both SEM and conventional TEM, there was evidence of shrinkage, the cytoplasmic projections having little contact with the extracapsular matrix. These findings show that the cytoplasmic processes are not artifacts of tissue processing and provide morphological evidence in support of the hypothesis that matrix vesicles are of cellular origin.Correlation of Freeze-Fracture and Scanning Electron Microscopy of Epiphyseal Chondrocytes     

Primary culture of vital marginal cells from cochlear explants of the stria vascularis

Summary Explants of the stria vascularis and spiral ligament were dissected from guinea pig cochleae and were successfully cultivated for several weeks. After 2 days, fibroblast-like cells of the spiral ligament covered the bottom of the cell culture dish around the explant. Marginal cells of the stria vascularis proliferated and grew on the luminal surface towards the border of the explant at a rate of 15 m/day. At day 6 in culture the proliferating marginal cells reached the border of the explant and then advanced to the bottom of the cell-culture dish. There the marginal cells replaced fibroblast-like cells and built an epithelial hexagonal-shaped monolayer. Light microscopic and transmission electron microscopic investigations revealed that the cultured cells were viable and that typical morphological characteristics of marginal cells were preserved. Cultivation of these cells provides a unique model for studies of physiological properties of marginal cells of the stria vascularis.     

Oxalate transport in cultured porcine renal epithelial cells

Summary Oxalate-containing kidney stones are the most common type (75%) of renal stones. In order to control oxalate excretion in the urine, a basic understanding of the cellular transport of oxalate is imperative. We have utilized the technique of continuous cell culture to establish and characterize a model system to study renal epithelial cell (LLCPK1) oxalate transport. Our data demonstrate that oxalate uptake in these cells is dependent on time, concentration and energy. TheK _m for oxalate uptake was 200 m. Oxalate uptake was decreased at lower temperatures and elevated in an acidic extracellular environment. Both anion exchange inhibitors DIDS and SITS inhibited oxalate oxalate uptake. Sulfate, chloride, and bicarbonate decreased oxalate uptake, as did the diuretics bumetanide and furosemide. There was no evidence for the co-transport of oxalate with sodium. Our data show that monolayers of cultured kidney epithelial cells are a valuable model system for study of the basic cellular mechanisms of oxalate transport.     

Adrenergic stimulation of isolated rat gastric mucosal cells

Summary Adrenergic stimulation of the adenylate cyclase (AC)-cAMP-system and ¹⁴C-aminopyrine accumulation, an indirect measure of parietal cell H⁺-production, was studied in different preparations of gastric mucosal cells.The ₂-adrenoceptor agonist hexoprenaline activated AC of crude homogenates from the gastric corpus of mouse, rat, guinea-pig, hog, dog and man. In isolated rat gastric cells (20% parietal cells), treated by low power sonication, 10^–8 to 10^–3 mol/l adrenaline and hexoprenaline activated AC equally potently and efficaciously by maximally 170%. Isoprenaline proved to be less effective activating up to 80%. 5·10^–5 mol/l GMP-PNP augmented basal activity 8.5 times and reduced the maximal efficacy. Adrenaline and hexoprenaline activated AC by maximally 120%, isoprenaline by 40%. The potency of adrenaline was 4 times lower, that of hexoprenaline 2 and that of isoprenaline 4 times higher in the presence of GMP-PNP. Adrenergic stimulation was inhibited by the -adrenoceptor antagonist propranolol, the effect of -adrenoceptor-blockade by phenoxybenzamine was less pronounced. In fractions with 7–80% of parietal cells, prepared by isopycnic centrifugation with Percoll, adrenaline and hexoprenaline activated AC or hexoprenaline enhanced the cellular level of cAMP in parietal cell poor and rich fractions. The degree of activation in response to histamine correlated with the number of parietal cells. ¹⁴C-Aminopyrine uptake was increasingly stimulated through 10^–8 to 10^–5 mol/l hexoprenaline, maximally by doubling the basal accumulation. 10^–4 mol/l histamine was 8 times more effective. 3·10^–7 mol/l propranolol inhibited the effect of 10^–5 mol/l hexoprenaline by 80%.The data suggest the localization of -adrenoceptors (likely -adrenoceptor) on parietal and other nonidentified gastric cells. At the parietal cell, adrenaline and hexoprenaline initiate activation of AC and hexoprenaline leads to H⁺-production. The responses are small compared to the effect of histamine. Thus, -adrenoceptor agonists exert intrinsic activity in relation to H⁺-production. Their influence on stimulated secretion of isolated cells remains to be elucidated.