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Melanophores are pigment cells found in the skin of lower vertebrates. The brownish-black pigment melanin is stored in organelles called melanosomes. In response to different stimuli, the cells can redistribute the melanosomes, and thereby change colour. During melanosome aggregation, a height increase has been observed in fish and frog melanophores across the cell centre. The mechanism by which the cell increases its height is unknown. Changes in cell shape can alter the electrical properties of the cell, and thereby be detected in impedance measurements. We have in earlier studies of Xenopus laevis melanophores shown that pigment aggregation can be revealed as impedance changes, and therefore we were interested in investigating the height changes associated with pigment aggregation further. Accordingly, we quantified the changes in cell height by performing vertical sectioning with confocal microscopy. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that the elevation of plasma membrane is caused by local swelling due to influx of water through HgC12-sensitive aquaporins. We also measured the height of the microtubule structures to assess whether they are involved in the height increase. Our results show that pigment aggregation in X. laevis melanophores resulted in a significant height increase, which was substantially larger when aggregation was induced by latrunculin than with melatonin. Moreover, the elevation of the plasma membrane did not correlate with influx of water through aquaporins or formation of new microtubules, Rather, the accumulation of granules seemed to drive the change in cell height.

We show here, that activation of protein kinase C by the phorbol ester PMA improves barrier function in colon carcinoma (HT 29) cells. By contrast, in canine kidney (MDCK I) cells it caused increased permeability and opening of tight junctions; the latter has also been noticed in other studies. Thus, with PMA confluent HT 29 cells responded with a reduced passage of 330 kDa sodium fluorescein, increased transepithelial electrical resistance, and a change in the cell shape of the HT 29 cells from an irregular to a regular, hexagonal form. Confocal imaging revealed parallel distinct changes in the staining of occludin and caludin-1, viz. a translocation from cytoplasmic clusters to apical cell–cell contacts. Interestingly, in both cell lines protein kinase A activation caused a decreased in the threonine phosphorylation of occludin that correlated with tight junction assembly in HT 29 cells and tight junction disassembly in MDCK I cells. We conclude that protein kinase C regulation of the epithelial barrier involves specific molecular mechanisms and achieves distinct effects at different developmental stages.

The effects of the N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) on the lateral mobility of the complement receptor type 1 (CR1/CD35) in glass-adherent human neutrophils were investigated, using fluorescence recovery after photobleaching (FRAP) and confocal microscopy (CSLM). It was found that addition of 0.1–1 μM fMLF increased the diffusion constant (D) of CR1/CD35 to 167–278% of controls. No effect was observed on the receptor distribution or the mobile fraction of receptors. The effect of fMLF on the lateral diffusion of CR1/CD35 could be totally inhibited by addition of pertussis toxin (PT, 250 ng/ml) or of the free radical scavenger enzymes superoxide dismutase (SOD, 2000 U/ml) and catalase (CAT, 200 U/ml), added together the results show that oxidative metabolites produced by neutrophils in response to fMLF can modulate CR1/CD35 diffusion, and indicate a regulatory role for oxygen radicals in phagocytosis.

In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2–1.2 μM) and higher concentrations (1.5 μM and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain I (MDCK-I) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 μg/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure.

Phagocytosis by neutrophils, macrophages, and other professional phagocytes requires rapid remodeling of actin. Early phagosomes are surrounded by a rim of F-actin that is disassembled during phagosomoal maturation. Breakdown of periphagosomal F-actin and phagolysosome fusion are calcium dependent processes in neutrophils interacting with serum-opsonized prey, but appears to be calcium independent in macrophages interacting with serum- or IgG-opsonized prey. In the present study, we found that calcium was necessary for phagocytosis, breakdown of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey. We also observed that lipophosphoglycan (LPG) from Leishmania donovani promastigotes required calcium to exert its inhibitory effect on macrophage phagocytosis and periphagosomal F-actin breakdown. We conclude that calcium is essential for phagocytosis, depolymerization of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey, as well as for proper functioning of LPG.

When the receptors for platelet-derived growth factor (PDGF) are activatedthey aggregate, become tyrosine-phosphorylated and elicit a cascade ofdown-stream signals, including mobilization of Ca2+ from intra- andextracellular stores. Receptor mobility in the plane of the membrane isa prerequisite for receptor aggregation and further signalling. Using humanforeskin fibroblasts (AG 1523) and fluorescence recovery afterphotobleaching (FRAP), we therefore assessed the lateral mobilitycharacteristics of PDGF-β2 receptors by their diffusioncoefficient (D), and fraction of mobile receptors (R). This was done oncells stimulated with either normal human serum (NHS) or PDGF underdifferent Ca2+-conditions. The results suggest that both intra- and extracellular free Ca2+influence the mobility characteristics of the PDGF-β2receptor. Interestingly, the extracellular Ca2+ seems to imposegeneral restrictions on the mobility of receptors, since R increased whenextracellular Ca2+ was quenched with EGTA, whereas intracellularclamping of Ca2+ transients with MABTAM (BAPT/AM) primarily affectedD. When both intra- and extracellular Ca2+ were quenced, D remainedlow and R high, further supporting the proposition that they achievedistinct effects. Inhibition of tyrosine phosphorylation with Erbstatin,partly inhibited the NHS effects and released PDGF-induced receptorimmobilization. Ratio imaging with Fura-2 displayed that both NHS and PDGFinduced changes in intracellular free [Ca2+]. In view of the presentdata it might have important effects on the state of the receptor in themembrane, for instance by regulating its lateral mobility, communicationwith other receptors and signalling functions in the membrane.

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D +/- standard error of the mean, SEM) of 4.2 +/- 0.2 x 10(-10) cm2/s, and 1.8 +/- 0.2 x 10(-10) cm2/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3 +/- 0.3 x 10(-10) cm2/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1 +/- 0.8 x 10(-10) cm2/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.

When platelet-derived growth factor (PDGF) binds to its receptors a number of biochemical reactions are elicited in the cell. Several models have been presented for the effects of ligand-induced receptor conformation and aggregation on signal transduction but little is known about the direct effects on receptor diffusion. This study concerns the lateral mobility of PDGF receptors in fibroblasts. It was assessed with fluorescence recovery after photobleaching (FRAP), using rhodaminated receptor antibodies or Fab-fragments of the antibody as ligands. The aims of the investigation were: (a) to compare the lateral mobility of membrane receptors of human fibroblasts labelled with either antibodies against the PDGF receptor or Fab-fragments of the same antibodies, and (b) to study the effects of serum or PDGF on the mobility of the receptors. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard or under serum-free conditions yielding “normal” and “starved” cells, respectively. Two parameters of the diffusion were evaluated; the diffusion coefficient (D) and the mobile fraction (R) of the receptors. We found that normal fibroblasts had a smaller diffusion coefficient and a lower mobile fraction compared to starved cells using antibodies for receptor labelling. The addition of PDGF, just before the measurement, increased the D and R for normal cells, while starved cells, showing higher initial values, displayed slightly reduced values of D and R. After the addition of serum, D increased and R remained low for normal cells, whereas for starved cells both D and R increased to upper limits of 11.0×10−10 cm2s−1 and >90% respectively. In general, the D and R values, both in normal and starved cells, were higher for cells labelled with Fab-fragments than for antibody-labelled cells. The results are discussed in relation to the natural complexity of the receptor, and how PDGF, serum, antibodies and Fab-fragments might interfere with receptor structure, aggregation state and membrane diffusion characteristics.

Protein kinase C (PK-C) and casein kinase II (CK-II) activities were studied in two human colon carcinoma cell lines (HT-29 and CaCO-2) undergoing differentiation in vitro resulting, in small-intestine-like cells. CaCo-2 cells, when grown under standard conditions, appear to undergo spontaneous differentiation. In these cells PK-C and CK-II activities were determined on day 5, 10 and 15. No significant differences in activities were seen either in PK-C or CK-II activity. HT-29 cells, when grown in glucose-free medium can be stimulated to undergo differentiation which is completed within 20 days. PK-C and CK-II activities were determined after 5, 10, 15, 20 and 25 days, respectively. PK-C activity rose from 7.9 +/- 3.5 pmole 32P/mg protein/min at day 5 to 37.5 +/- 14.8 pmole 32P/mg protein/min at day 20. After 25 days the activity was reduced to 20.0 +/- 7.8 pmole 32P/mg protein/min. CK-II activity did not change significantly during day 5 to 20, but on day 25 there was a significant decrease in CK-II activity from 94.9 +/- 6.4 pmole 32P/mg protein/min (day 20) to 62.6 +/- 3.9 pmole 32P/mg protein/min (day 25) p = 0.003. The results in this study indicate a role for PK-C and CK-II in cell growth and differentiation.

Rat peritoneal mast cells in vitro released [3H]-serotonin in the presence of halide (I-), myeloperoxidase (MPO), and a H2O2-generating system. The degranulation was partly inhibited with low concentrations of bovine serum albumin (BSA; 0.1–l% w/v), or polyethyleneglycols (PEG 600 or PEG 6000, 0.1% w/v). Furthermore, PEG 600 and PEG 6000 enhanced the stabilization achieved with 0.1% BSA. The results may provide a new aspect on antigenicity of PEG-modified allergens, and on mast-cell response at an inflammatory site.

The aims of the present investigation were (a) to compare the lateral mobility of membrane receptors of human fibroblasts and polymorphonuclear leukocytes (PMNL) labelled with either platelet-derived growth factor (PDGF), or the lectin wheat germ agglutinin (WGA), and (b) to study effects of serum or PDGF on the mobility of these receptor molecules in human fibroblasts. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard (10%) or under serum-free conditions yielding "normal" and "starved" cells, respectively. The receptor mobility was studied in response to exposure to PDGF, or serum, in short time or prolonged incubations. Human polymorphonuclear leukocytes (PMNL) were adhered to microscope slides by clotting drops of blood. They were stained with rhodaminated PDGF or fluoresceinated WGA. The diffusion of labelled receptors was assessed with fluorescence recovery after photobleaching (FRAP). It was found that (a) fibroblasts grown at normal serum concentration had a lower diffusion coefficient (D = 3 x 10(-10) cm2 s-1) for the PDGF-receptor and a slightly lower mobile fraction (R = 60%) than starved cells (D = 5 x 10(-10) cm2 s-1 and R = 73%), (b) addition of serum to starved cells increased both D and R for the PDGF receptor to 12 x 10(-10) cm2 s-1 and 96%, respectively, (c) a similar pattern was obtained for WGA-labelled glycoconjugates indicating general membrane effects of serum-induced cell stimulation, and (d) in PMNL the PDGF receptor displayed motility characteristics (D = 3 - 4 x 10(-10) cm2 s-1 and R = 59%) similar to those in fibroblasts, possibly suggesting equivalent anchorage mechanisms in the membrane.