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Using two newly synthesized inhibitors, Ro 31-8220 and CGP 41,251, of protein kinase C (PKC), we analysed: (1) how distinct PMN functions (shape changes, locomotion, pinocytosis) are regulated, and (2) the role of protein phosphorylation and PKC in this process. We were able to transform: (1) resting PMNs into locomoting cells using fNLPNTL, (2) locomoting cells into non-locomoting highly pinocytic cells using PMA, and (3) PMA-stimulated cells showing marked pinocytosis into locomoting or into resting cells using Ro 31-8220. It is thus possible to selectively manipulate PMN function (resting state, locomotion, marked pinocytosis), indicating that there are different regulatory pathways. It was not possible to induce locomotion and marked pinocytosis simultaneously, indicating crosstalk between pathways. Ro 31-8220 inhibited PMA-induced shape changes (nonpolar cells) and pinocytosis, but not fNLPNTL-induced shape changes (polarity) and pinocytosis. At higher concentrations, Ro 31-8220 alone elicited cell polarity and chemokinesis, indicating that a constitutively active protein kinase is involved in maintaining the spherical shape of resting PMNs. Functional effects of another PKC inhibitor, CGP 41,251, on neutrophil function were strikingly different. CGP 41,251 selectively inhibited fNLPNTL-induced polarity and locomotion (but not colchicine or Ro 31-8220-induced polarity), and it failed to inhibit PMA-induced, stimulated pinocytosis and shape changes. Although the effects of Ro 31-8220 vs. CGP 41,251 on PMN function were strikingly different, the inhibition of profiles for constitutive and for fNLPNTL- or PMA-induced protein phosphorylation in intact PMNs showed only small differences, which could not yet be conclusively related to cell function.

Brain microvessel endothelial cell monolayers have been shown to be a suitable blood-brain barrier in vitro system to study adrenergic regulation of permeability. We tested adrenergic drugs on bovine brain microvessel endothelial cell monolayer permeability to a biomembrane impermeant molecule, sodium fluorescein. Endogenous catecholamines noradrenaline and adrenaline were tested as well as the alpha-adrenoceptor agonist phenylephrine, the beta-adrenoceptor agonist clenbuterol and the alpha-adrenoceptor antagonist prazosin. Results showed an alpha-adrenoceptor mediated increase and a beta-adrenoceptor mediated decrease in monolayer permeability. Both alpha- and beta-adrenoceptor mediated changes in permeability were abolished by inhibiting fluid-phase pinocytosis, either by vincristine or by avoiding bovine brain microvessel endothelial cell's energy utilization. The reverse transport (i.e., from brain to blood side) was also influenced by adrenergic drugs; alpha- or beta-adrenoceptor stimulation induced a permeability-reducing effect. We conclude that alpha-adrenoceptor stimulation increases bovine brain microvessel endothelial cell monolayer permeability and that beta-adrenoceptor stimulation has the opposite effect. Reverse transport results obtained with beta-adrenoceptor stimulation seem controversial and deserve further study. These results also support in vivo findings that demonstrated adrenergic influences on blood brain barrier permeability.

Clathrin-coated vesicle endocytosis and macropinocytosis are distinct endocytic pathways demonstrable in several cell types including human epidermoid A431 cells (West, M.A., M.S. Bretscher, and C. Watts. 1989. J. Cell Biol. 109:2731-2739). Here we analyze the extent of mixing of macropinocytic endosome (macropinosome) content with that of conventional endosomes served by coated vesicle endocytosis. Using laser scanning confocal fluorescence microscopy we detected very little delivery of macropinosome content to either early or late endosomes-lysosomes as defined by labeling with transferrin or with LDL. Mixing of the contents of the macropinosomes and conventional endosomes was not induced by the addition of brefeldin A. Moreover, the morphology of macropinosomes was not grossly altered in the presence of brefeldin A, whilst in the same cells there were dramatic tubulation effects on conventional endosomes as reported by others. Although refractory to fusion with conventional endosomes, macropinosomes were nonetheless dynamic structures which sometimes exhibited vesiculo-tubular morphology in living cells and were capable of fusing with each other. We suggest that different endocytic mechanisms can give rise to distinct endosome populations.

Pinocytic activity is greatly stimulated in gamma-BHC (gamma isomer of benzene hexachloride) treated, vegetative cells of Dictyostelium discoideum as measured by 14C sucrose or FITC-dextran uptake. Transmission electron microscopic studies also reveal the presence of a greater number of pinosomal vesicles in the pesticide-treated Dictyostelium amoebae. The enhanced pinocytic activity has been discussed in relation to lipophilic interactions of gamma-BHC with the hydrophobic cell surface and the observed changes in the cytoskeletal proteins of the treated cells.

The teratogenicity of chloroquine (CQ) has been hypothesized to result from its effects on lysosomal function, specifically the ability of the visceral yolk sac (VYS) to capture and degrade external macromolecules. Using the rat whole embryo culture system, we evaluated the ability of CQ to accumulate in conceptual tissues and its effects on aspects of VYS function known to be important in the uptake and processing of nutrients. When CQ was added directly to the culture medium, it was found to accumulate rapidly in conceptual tissues, particularly the VYS. Tissue concentrations of CQ in the embryo proper reached approximately 10-fold those in the medium, whereas concentrations in the VYS exceeded by 100-fold the medium concentration within a 4-hr exposure on gestational day (GD) 10. Embryotoxic concentrations of CQ (10-30 microM) enhanced the activity of lysosomal cysteine proteinases measured in vitro under optimum pH conditions in both embryonic and VYS homogenates after a 26-hr treatment from GD 10-11. A different pattern of response in enzyme activity was observed between embryos and VYSs that could be attributed to the preferential accumulation of CQ in the VYS. Nonembryotoxic concentrations of CQ (1-7.5 microM) induced a concentration-dependent increase in VYS enzyme activity that peaked in conceptuses exposed to 20 microM CQ (an intermediate embryotoxic concentration). The enhanced cysteine proteinase activity was time dependent and appeared to increase gradually in conceptuses exposed to 10-20 microM CQ during the 26-hr culture period. This was in contrast to the rapid accumulation of CQ in conceptual tissues seen on gestational day 10. Protein content in the VYS was increased significantly after a 9-hr exposure of whole conceptuses to CQ (20 microM), indicating an inhibition of VYS proteolytic activity in situ. After 24 hr of exposure to 20 microM CQ, VYS protein content was not significantly different from control, but embryonic protein was reduced significantly by 20%. These observations are consistent with a model of reversible inhibition of VYS proteolysis by CQ followed by a compensatory increase in lysosomal proteinase activity. VYS fluid-phase pinocytosis was also assessed after CQ exposure and found to be inhibited only in the highest CQ concentration tested (30 microM). Lower concentrations of CQ that were still embryotoxic (10-20 microM) did not affect VYS fluid-phase pinocytosis, suggesting that inhibition of this activity is not primarily responsible for CQ embryotoxicity.

Cell differentiation and proliferation are mutually exclusive processes in many cases. The transition of starving Dictyostelium cells from growth to differentiation phase has been shown to occur at a particular position (putative shift point; PS-point) in the cell cycle of D. discoideum Ax-2. The significance of phosphorylation states of proteins such as 101 kDa, 90 kDa, and 32 kDa phosphoproteins has been argued, particularly around the PS-point. In this study we examined effects of the protein kinase inhibitors and activators on the transition of Ax-2 cells from growth to differentiation. K252a, a potent inhibitor of protein kinases, inhibited growth possibly through the blockage of pinocytotic activity of cells, and promoted the progress of development after starvation when applied to Ax-2 cells at the growth phase. Such a K252a-effect was most pronouncedly exhibited on the cells located near the PS-point. Unexpectedly, however, the development of starved cells was found to be considerably delayed by staurosporine bearing a structural and functional resemblance to K252a when it was applied during the growth phase. Pulse-labelings of growing Ax-2 cells with inorganic 32P (32Pi) showed that K252a induces the disappearance of a 48 kDa phosphoprotein and the appearance of a 50 kDa phosphoprotein, specifically in the cells located around the PS-point. Phosphorylation of 32 kDa and 24 kDa proteins was also inhibited by K252a, but this inhibition was not necessarily specific to the K252a-treatment and occurred independently of the cell-cycle phases. The possible significance of these results is discussed in relation to a breakaway of cells from proliferation to differentiation at the PS-point.

We previously reported that S-(N-methylcarbamoyl)glutathione (SMG), a conjugate formed by the reversible reaction between methyl isocyanate and glutathione, inhibited the development of mouse embryos in culture. The present study was done to determine whether SMG produced embryotoxicity by inhibiting yolk sac functions. For this purpose we determined the effects of an embryotoxic concentration of SMG on mouse yolk sac uptake mechanisms and lysosomal proteolysis as well as on the incorporation of [3H]leucine in mouse embryonic and limb-bud proteins. After 5 h of culture, SMG inhibited the uptake of [14C]sucrose and 125I-labelled bovine serum albumin in isolated day 15 yolk sacs to 62 and 77% of control, respectively. Lysosomal proteolysis was not inhibited, as judged by the release of trichloroacetic acid soluble radioactivity into the culture media. Uptake and incorporation of [3H]leucine from free [3H]leucine or from [3H]leucine-labelled protein in SMG-treated day 9 embryos were inhibited, respectively, to 61 and 25% of the control uptake during a 16-h labelling period. SMG also inhibited the incorporation of free [3H]leucine into limb-bud proteins. SMG-induced inhibition of 125I-labelled bovine serum albumin uptake by yolk sacs was partially prevented by the thiol donors N-acetylcysteine and glutathione but not by acivicin (gamma-glutamyl transpeptidase inhibitor) and aminooxyacetic acid (cysteine conjugate beta-lyase inhibitor). These data suggest that SMG suppresses embryonic growth primarily by an inhibition of nutrient uptake by the yolk sac. We postulate that this inhibition is due to tissue carbamoylation by methyl isocyanate released from SMG.

Adsorptive endocytosis of bovine beta-glucuronidase by human fibroblasts is mediated by two different membrane receptors: one recognizes phosphomannosyl residues on the enzyme, the other is yet a undefined recognition marker (A. González-Noriega, R. Coutiño, V. M. Saavedra, and R. Barrera (1989) Arch. Biochem. Biophys. 268, 649-658). We have purified a bovine liver inhibitor for the endocytosis of the bovine beta-glucuronidase mediated by the recently proposed recognition marker. The inhibitor is partially susceptible to periodate oxidation, can be released from a peptide backbone by mild alkali treatment, can be reduced by sodium borohydride, and can be adsorbed to anionic but not to cationic resins. Although the chemical structure of the isolated marker has not been determined, results indicate a 122-Da molecule which may contain amino alcohol groups and may be found in a 1800-Da glycosidic chain.

Fluid-phase uptake of macromolecules by cultured bovine aortic endothelial cells was measured using FITC-dextran (70,000 Da). Low doses of hydrogen peroxide added extracellularly stimulated the uptake of macromolecules by the endothelial cells. There was no general increase in the passive permeation or the transport across the cell layer. Moreover, when endothelial cells were stimulated with phorbol myristate acetate (PMA), macromolecules uptake was also enhanced. The PMA effect was blocked with superoxide dismutase (SOD) and catalase, suggesting a pivotal role of oxygen metabolites in fluid phase uptake of macromolecules by endothelial cells.

The PKC-inhibitor Ro 31-8220 inhibits stimulated fluid pinocytosis of human PMNs induced by the PKC-activators phorbol myristate acetate (PMA, IC50 = 1.35 x 10(-6) M) or diacylglycerols (OAG, diC8) by 95%, whereas Ro 31-8220 has no effect on D2O- or fNLPNTL-induced pinocytosis and enhances cytochalasin D- induced pinocytosis. Also formation of F-actin induced by PMA or diacylglycerols is selectively inhibited. The results indicate that a Ro 31-8220 -sensitive PKC is involved in signal transduction for enhanced pinocytosis and F-actin formation in response to one class of stimuli (classical activators of PKC) but not to others.

Fluid-phase endocytosis (pinocytosis) is highly active in amoebae of the cellular slime mould Dictyostelium discoideum as it provides an efficient entry of nutrients in axenic strains. Detailed kinetic analyses were conducted using fluorescein-labeled dextran (FITC-dextran) as fluid-phase marker and pH probe. Cells were first pulsed with FITC-dextran during a short period then chased by suspension in probe-free medium. Chase kinetics were characterized by a lag phase of about 40 min before pseudo-first order FITC-dextran efflux and thus reflected the progression of the probe cohort through the various endosomal compartments along the endosomal pathway. Temporal evolution of endo-lysosomal pH showed a rapid acidification (T1/2 approximately 10 min) to pH 5.0 followed by an increase up to pH 6.2 to 6.3. The effects of cycloheximide and caffeine, two inhibitors of endocytosis in Dictyostelium amoebae, on the evolution of endosomal pH during fluid-phase endocytosis, have been investigated. Cycloheximide fully blocked the cellular transit of FITC-dextran but acidification of endo-lysosomal compartments still took place. Caffeine increased endo-lysosomal pH, probably as a consequence of an elevation of cytosolic [Ca2+]. Furthermore, it allowed the functional identification of a caffeine-insensitive terminal segment of the endocytic pathway. It corresponded to a recycling, postlysosomal compartment at pH 6.2 to 6.3 with an apparent volume of 160 microns 3/amoebae.

Halotolerant fungus, A. repens, showed a considerable difference in its growth rate, morphology, ultrastructural and molecular composition under NaCl stress as compared to control i.e. non-stressed condition. Light microscopic observations revealed significant differences in their mycelial thickness, their branching and septa. Transmission electron microscopic observations of both the conditions depicted significant differences in the qualitative and quantitative changes in mitochondria. Frequent pinocytotic vesiculation (vacuoles) of plasma membrane was observed in fungus under stress but no such vesiculation in control. The multivesiculate structures observed under stress with their origin from the cell membranes and subsequent release into vacuoles have not been reported in fungi under normal physiological conditions. The observations on pinocytosis are discussed in relation to ion compartmentation and salt tolerance in A. repens.

Treatment with hepatocyte growth factor/scatter factor (HGF/SF) rapidly induced the formation of conspicuous circular ruffles on the apical surfaces of two kidney cell lines, MDCK and PtK2. The ruffles were found to contain significant amounts of F-actin and myosin as judged by immunofluorescence microscopy. Time-lapse photomicroscopy demonstrated that the ruffles constrict, closing over, and were followed by the formation of phase bright structures. That these structures were macropinocytotic vesicles was confirmed using fluorescein isothiocyanate (FITC)-dextran as a marker for fluid uptake. It is hypothesized that the constriction of the ruffles followed by membrane fusion causes the vesicles to form. Treatment with suramin blocked both circular ruffle formation and scattering, suggesting that ligand binding was the causal agent for ruffle formation. The drugs amiloride and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) also completely inhibited ruffle formation, suggesting that ion transport was an early consequence of HGF/SF binding and that these transport effects had a major role in the cytoskeletal changes leading to circular ruffle formation. The appearance of macropinocytotic vesicles was also blocked by amiloride treatment. Surprisingly though, subsequent scattering was not blocked by amiloride treatment, although suramin and SITS both entirely inhibited scattering.

We previously reported that polymyxin B (PMB) enhances cellular catabolism of low density lipoproteins (LDLs) through a non-LDL receptor-mediated endocytotic pathway. These data were obtained mainly by using Hep G2 cells, a well differentiated human hepatoma cell line. In the current study, we explore the mechanisms of PMB-mediated endocytotic catabolism of LDL. We found that PMB enhanced LDL catabolism also in homozygous familial hypercholesterolemia fibroblasts, thereby establishing that PMB-mediated cellular catabolism of LDL does not involve LDL receptors. By using [14C]sucrose, and ligands for the asialoglycoprotein (ASGP) receptors, possibilities were excluded that PMB enhances cellular endocytosis of LDL, by inducing a general increase of cellular pinocytic activity or by causing endocytosis of LDL via the ASGP receptors in Hep G2 cells. We further show, by using polymyxin B coupled Sepharose 4B (PMB-Sepharose 4B) beads, that PMB binds to LDL to form a complex. This binding was tight, and changes in pH and salt concentrations had no significant effect on the binding, but unlabelled LDL competed with 125I-LDL to bind to PMB-Sepharose 4B. Urea and endotoxins decreased this binding, suggesting that PMB binds to LDL at least partially through hydrophobic interactions. Agarose gel electrophoresis of PMB-LDL indicates that PMB cationizes LDL. In conclusion, PMB binds to LDL to form a PMB-LDL complex presumably through interactions between lipid groups. This endows LDL with positive charges, which enhances LDL binding to negatively charged cell membranes, and such bound LDL is rapidly internalized through absorptive endocytosis.

Protein phosphatases 1 and 2A are important in regulating cellular functions by controlling the phosphorylation state of their substrates. In human monocytes, the inhibitors of these phosphatases, okadaic acid and calyculin A, were found to increase the mRNA accumulation and cytokine production of interleukin-1 beta and interleukin-1 alpha. The increased mRNA accumulation was found to be primarily because of the increase in the transcription rate of the interleukin-1 genes. Stimulation of interleukin-1 gene transcription may be caused by the stimulation of transcription factor activities, including those of AP-1, by these protein phosphatase inhibitors. Okadaic acid increased the synthesis of the interleukin-1 beta precursor and mature forms and their secretion. This increased processing and secretion correlated with the stimulation of IL-1 beta convertase mRNA accumulation. The stimulation of interleukin-1 alpha production by okadaic acid was more modest than that of interleukin-1 beta. However, the phosphorylation of the precursor interleukin-1 alpha cytokine was increased. These results show that protein phosphatase 1 and 2A inhibitors exert multiple effects on cytokine production in human monocytes and suggest that these two phosphatases play important roles in regulating interleukin-1 production.

The phosphatase inhibitors okadaic acid and calyculin A were found to elicit or to modify several neutrophil responses, suggesting that dephosphorylation plays a regulatory role. The concentrations of okadaic acid (> or = 1 mumol/L) that were effective on neutrophil functions (shape changes and marginal stimulation of pinocytosis) were shown to stimulate the incorporation of 32PO4 into many neutrophil proteins several-fold. Calyculin A was effective at 50-fold lower concentrations. In the presence of the inhibitors, the cells exhibited a nonpolar shape and the polarization response induced by chemotactic peptide was inhibited. Both phosphatase inhibitors also induced the association of F-actin with the cell membrane. A steady-state phosphatase activity is thus involved in maintaining shape and F-actin localization of resting cells. Inhibitors alone had no significant effect on the amount of cytoskeleton-associated actin. The increase in cytoskeletal actin observed at 30 minutes of stimulation with phorbol ester or 5 to 30 minutes of stimulation with chemotactic peptide, however, was abolished by okadaic acid or calyculin A, suggesting an important role of a phosphatase. In contrast, the early increase in cytoskeleton-associated actin observed at 1 minute of stimulation with peptide was not affected. This finding indicates that the increased association of actin with the cytoskeleton in the early and the later stages of neutrophil activation may be mediated by different signalling pathways.

During craniofacial development in the mouse embryo (days 9-12 of gestation; plug day = day 1), transient expression of serotonin (5-HT) uptake in epithelial structures of this region correlates with critical morphogenetic events (Lauder et al., '88; Shuey, '91; Shuey et al., '89, '92). The purpose of the present investigation was to assess the possible functional significance of these uptake sites by examination of patterns of dysmorphology following exposure of embryos to selective 5-HT uptake inhibitors. Exposure of mouse embryos in whole embryo culture to sertraline, at a concentration (10 microM) which produced no evidence of general embryotoxicity, caused craniofacial malformations consistent with direct action at 5-HT uptake sites. Two other 5-HT uptake inhibitors, fluoxetine and amitriptyline, produced similar defects. The critical period of sertraline exposure occurred on days 10-11. The observed craniofacial defects were associated with decreased proliferation and extensive cell death in mesenchyme located 5-6 cell layers deep from the overlying epithelium. In contrast, the subepithelial mesenchymal layers showed normal or elevated levels of proliferation. From these results it appears that inhibition of 5-HT uptake into craniofacial epithelia may produce developmental defects by interference with serotonergic regulation of epithelial-mesenchymal interactions important for normal craniofacial morphogenesis.

Lucifer yellow (LY) accumulation was used to measure macrophage pinocytosis. The hematopoietic growth factors, macrophage colony-stimulating factor (CSF-1), granulocyte-macrophage CSF (GM-CSF), and interleukin 3, and the macrophage activators, lipopolysaccharide and zymosan, all stimulated LY uptake in both murine bone marrow-derived macrophages (BMMs) and resident peritoneal macrophages (RPMs) without affecting LY efflux. The stimulation of pinocytosis in the poorly cycling RPMs and in BMMs by nonmitogens dissociates stimulation of pinocytosis from subsequent DNA synthesis. Regulation of pinocytosis in BMMs appears to be independent of that of urokinase-type plasminogen activator expression. The increases in CSF-mediated BMM pinocytosis were not inhibited by pertussis toxin, by elevations in intracellular cAMP, or by glucocorticoids and were only partially inhibited by inhibitors of Na+/H+ antiport and Na+/K(+)-ATPase activities. Protein kinase C activation could be involved in regulating BMM pinocytosis because phorbol myristate acetate, oleoylacyglycerol, and exogenously added phospholipase C can all stimulate it. Ca2+ ionophores were inactive, whereas the Na+/H+ ionophore monensin potently inhibited BMM pinocytosis.

The experiments on the rabbits and dogs have revealed that in the initial period of endotoxin shock the number of secretory granules in the atrial cardiomyocytes decreases and vascular permeability increases due to transendothelial micropinocytosis. In the intermediate period of endotoxin shock the ultrastructural alterations progress. The development of ultrastructural manifestations of endotoxemia greatly depends on the condition of endocrine heart function.

1. Retinae of crabs, Leptograpsus variegatus, held on a 12:12 h light-dark cycle were prepared for culture in vitro shortly before light-off. After an hour in darkness to permit the assembly of "night" rhabdoms, retinae were exposed to various combinations of drugs: 1 microM okadaic acid (OKA); 60 microM SC-9; 10 microM phorbol, 12,13-diacetate (PDA). 2. The effects of the specific protein phosphatase inhibitor, OKA, are confirmed as light-dependent. Rhabdom sizes were not compromised by OKA, nor by either of the two protein kinase activators, SC-9 or PDA when each was deployed alone in darkness. 3. In combination with OKA, PDA induced demolition of rhabdoms by abnormal macropinocytosis of microvillar membranes. 4. Combined with OKA, SC-9 induced a transient reduction of rhabdoms, followed by overgrowth to abnormal sizes. Overgrowth was blocked by the transcription inhibitor actinomycin D. 5. Disparate consequences of combining OKA with SC-9 or PDA imply that more than one protein kinase C may be involved.

Coumarins, which are thought to stimulate macrophage proteinase activity, have been advocated for the treatment of high protein oedemas, such as obstructive lymphoedema. In experiments with cultured murine macrophages, coumarin and 7-hydroxycoumarin (10(-3) or 10(-4) M) had no significant effect on plasminogen-activator activity, plasminogen-independent fibrinolytic activity or pinocytosis. Although no in vitro effect on macrophage proteinase activity was found, it is possible that coumarins activate other cell types in vivo and thus effectively treat lymphoedema.

The cytotoxic effects of gelonin on various phagocytic and nonphagocytic cells were studied. Peritoneal exudate cells (PEC) are found to be more sensitive to gelonin compared to P388D1 and J774A.1 cells, nonphagocytic cells being the least sensitive. While chloroquine markedly enhances the cytotoxicity of gelonin in macrophages (greater than 100-fold) ammonium chloride confers protection. A higher rate of uptake of 125I-gelonin in PEC (7 times the rate observed in other cells) is probably mediated by an interaction of terminal mannose residues of gelonin with mannose receptors on PEC plasma membrane as inferred from a pronounced inhibitory effect of mannan. In contrast to a pronounced inhibitory effect of mannan on the uptake of gelonin in PEC (7-fold), the cytotoxicity is reduced only by 2-fold. On the other hand, mannan has little or no effect on enhancement of the toxicity of gelonin by chloroquine. The studies have suggested that the internalization of gelonin in PEC may involve two pathways: (a) mannose receptor-mediated endocytosis, which plays a minor role in the intoxication process, and (b) nonspecific fluid phase pinocytosis, which is susceptible to enhancement by chloroquine and has a major role to play in the manifestation of the toxic effect of gelonin. In macrophage-like cells only the latter pathway operates.

When Concanavalin A, 1-20 micrograms ml-1 binds to the surface of Amoeba proteus the cell's response to Ca2+ and to cationic inducers of pinocytosis is strikingly altered. Separately, Concanavalin A and Ca2+ are weak inducers but their combined effects are intense pinocytosis and suppression of the normal inhibitory effect of Ca2+ on cation-induced pinocytosis. At high concentrations (greater than 25 micrograms ml-1) the lectin increases cellular uptake and binding of 45Ca in the cell surface but the ionic permeability and the potential of the cell membrane are little changed by treatment with Concanavalin A. Subsequent addition of Ca2+ starts the pinocytic cycle and causes a fivefold increase of the membrane resistance without depolarizing the cell. Neither a rise of free intracellular calcium nor an influx of 45Ca precedes formation of pinocytotic channels when Con A is applied to cells in the presence of calcium. All effects of Concanavalin A are blocked by alpha-methylmannoside while succinyl-Concanavalin A is without effects on pinocytosis. These findings suggest that crosslinking of cell surface carbohydrates reverses calcium inhibition of pinocytosis and increases the cell's pinocytotic response to cations without modifying either the bioelectrical effects or the intracellular free concentration of calcium.

Cultured glomerular mesangial cells (MC) respond to low concentrations of bacterial endotoxin (ET) by secreting prostaglandins and interleukin-1. To evaluate further the nature of ET-induced mesangial cell activation, the authors evaluated the effects of this agent on MC morphology and cytoskeletal organization. Bacterial ET, in concentrations as low as I ng/ml, induced reversible membrane ruffling, cellular rounding, and extension of many filopodia and lamellopodia. Augmented fluid-phase pinocytosis occurred in parallel, as determined by transmission electron microscopy and tritiated sucrose uptake. These cellular morphologic and functional changes were associated with an extensive, but reversible, depolymerization of actin microfilaments. Actin gene expression was also modified by ET. At 4 to 6 hours after ET exposure, Northern blot analysis showed a twofold to fourfold increase in actin mRNA levels. In situ hybridizations of ET-stimulated cells at the light and electron microscopic levels demonstrated a markedly asymmetric distribution of actin mRNA, which was localized in the cellular periphery at filopodial and lamellopodial extensions, presumably sites of new actin protein synthesis. It is concluded that ET effects on MC are distinct from the nonspecific lytic or 'toxic' actions described for other cell types. Endotoxin induces a global activation of this cell type associated with major changes in membrane structure, cytoskeletal organization, and gene expression, which resemble in many respects the responses to peptide mitogens.

We examined the effects of isoproterenol and carbachol on fluid-phase endocytosis by Chinese hamster ovary (CHO) cells transfected with beta-adrenergic, M1, or M3 cholinergic receptors. Isoproterenol increased cAMP production and carbachol increased intracellular Ca, indicating successful expression of the receptor genes and coupling to typical signal transduction pathways. Carbachol inhibited the uptake of horseradish peroxidase (HRP) or Lucifer yellow (markers of fluid-phase endocytosis) in both M1- and M3-containing cells but not in wild-type cells, whereas isoproterenol did not affect pinocytosis in cells transfected with beta-adrenergic receptors. Carbachol inhibited the transit of HRP from an exchangeable pool to a nonexchangeable pool by a latent process requiring minimally 5 min of incubation. During the latent period, only one peak of low-density HRP-containing vesicles was found on Percoll gradients; after 5 min, HRP appeared in both high- and low-density vesicles. Carbachol-treated cells contained less HRP in the high-density fraction enriched in lysosomal markers. Early endosomes from CHO cells labeled for 5 min with HRP underwent fusion to make a more dense population of vesicles in the presence of ATP and KCl at 37 degrees C but not at 4 degrees C. The fused material contained increased levels of G proteins as detected either by ADP ribosylation with appropriate toxins or by immunoblotting with specific antibodies. These findings suggest that GTP binding proteins are internalized in endocytic vesicles and enter into a complex trafficking process involving fusion with other vesicular compartments. Trafficking of endosomes to these compartments is inhibited by activated M1 and M3 muscarinic receptors in CHO cells.

The central adrenergic innervation of the cerebral microvessels may play a role in the control of blood-brain barrier permeability. To pursue the study of this hypothesis we investigated the effect of noradrenaline on both the permeability of the blood-brain barrier to sodium fluorescein and on the pinocytotic activity of cerebral endothelial cells in the rat. Noradrenaline, stereotactically injected in the right lateral cerebral ventricle, significantly increased the cerebral extraction ratio of sodium fluorescein in a dose-dependent way. The same effect was induced by phenylephrine. Prostaglandin F2 alpha had no significant effect on the passage of sodium fluorescein through the blood-brain barrier. The effect of noradrenaline (150 micrograms) on the cerebral extraction ratio of sodium fluorescein was totally blocked by phenoxybenzamine (25 mg/kg i.p., 24 h before noradrenaline). Noradrenaline (150 micrograms) significantly increased the pinocytotic activity of cerebral endothelial cells. Phenoxybenzamine (as above) reduced the effect of noradrenaline on pinocytosis. It is concluded that noradrenaline increases the blood-brain barrier's permeability to sodium fluorescein, most probably through an effect on alpha adrenoceptors. The increase induced in the blood-brain barrier's permeability by noradrenaline seems to be due, at least in part, to an increase in the pinocytotic activity of endothelial cells.

Functional properties of ramified microglia were investigated in primary cultures of rat cerebral cortical cells. These microglia could be readily identified in both fixed and living cultures through previously established features. Based on their destruction by 5 mM L-leucine methyl ester, a high level of intrinsic endocytotic activity was established. When cultures were incubated with fluorescent latex beads to assess phagocytosis, little or no such activity was exhibited by ramified cells. However, when cultures were incubated with dyes or other soluble tracer compounds, these cells always exhibited labeling. This labeling was selective for ramified microglia in the cultures and was demonstrated using a variety of compounds, including trypan blue, lucifer yellow, horseradish peroxidase (HRP), and India ink. Intracellular label could be observed in vesicular structures; this localization corresponded to an active cellular process. Also, cellular labeling was inhibited by the presence of colchicine. These features supported the inference that the labeling was attributable to pinocytosis, and this process appeared to account for the vast majority of endocytotic activity in the ramified microglia. Possible physiological significance of this pinocytotic activity was indicated by the accumulation of various neurotransmitters/modulators: gamma-aminobutyric acid and vasoactive intestinal polypeptide (VIP). Ramified cells in these cultures have been previously noted to exhibit a constant and rapid pattern of motility, which was consistently observed here through time-lapse video recording; pinocytosis and rapid motility were shown to concur in individual cells. Based on their high intrinsic pinocytotic activity and pattern of cellular motility, the ramified microglia specifically are suggested to serve a constitutive function of fluid cleansing within the interstitial spaces of brain tissue.

A fraction from diabetic rat serum containing a low-molecular-weight (800-1000) somatomedin inhibitor (SI) alters growth and development in both neurulation and early limb bud staged mouse embryos in vitro. Previous studies suggested that an accumulation of serum proteins and morphological changes of the visceral yolk sac (VYS) were produced following exposure to the SI in early limb bud staged conceptuses. The morphological changes, characterized by the presence of large endosomes in the endodermal cells, suggested that the SI altered histiotrophic nutrition, whereby proteins are pinocytosed by the endodermal VYS cells and degraded to constituent amino acids. Therefore, the effects of the SI on pinocytosis and protein degradation by the VYS were evaluated using the whole embryo culture system. Results showed that the SI reduced fluid phase pinocytosis as determined by the uptake of [U-14C]sucrose, but that accumulation of [3H]leucine-labeled hemoglobin ([3H]Hb) by the VYS was greater following exposure to the SI than in controls. In contrast, the accumulation of 3H-labeled amino acids in the embryo (produced from the degradation of [3H]Hb by the VYS) was reduced by the SI. The extent of amino acid reduction in embryonic accumulation is dependent upon the concentration of SI in the culture medium and correlates with the incidence of malformations produced by the SI, i.e., high rates of malformations occur with large reductions in embryonic 3H-labeled amino acid accumulation. The apparent paradox of high [3H]Hb accumulation in the presence of decreased pinocytosis appears to be the result of altered processing of the [3H]Hb in the endodermal cells. The altered processing decreases the "elimination" of the proteins from the VYS and results in the decrease in 3H-labeled amino acid present in the embryo proper. Therefore, the SI appears to alter two processes of VYS histiotrophic function. (1) decreased pinocytosis and (2) altered protein processing, ultimately resulting in a decreased availability of substrates for the embryo. During the early stages of embryogenesis in the human, the trophoblast cells of the placenta are responsible for the transport of nutrients from the maternal to embryonic systems. Since these cells show high phagocytic and pinocytotic activities, the SI may also disrupt these processes in the chorioallantoic placenta and contribute to diabetes-induced embryopathies.

A fermentation broth of Streptomyces sp. SIPI-A4-0044 inhibited in vitro growth of src or ras oncogene-expressed (onc+) cells more strongly than that of oncogene-unexpressed (onc-) counterparts. The active components were isolated and identified as hygrolidin family antibiotics (HGL). In mixed cultures consisting of onc+ and onc- cells, at an appropriate ratio, HGL showed selective toxicity to focus like structures of onc+ cells, leaving monolayer areas of onc- cells little damaged. HGL rapidly inhibited pinocytosis, or the influx of neutral red into the cells, at concentrations partially inhibitory to the cell growth. In contrast, HGL only slightly inhibited the influx of 2-deoxyglucose, nucleosides and leucine and the syntheses of DNA, RNA and protein even at high concentrations. Upon prolonged exposure to sublethal concentrations of HGL, onc- cells but not onc+ cells recovered pinocytotic activity and resumed growth.

The high-affinity, type I Ig Fc receptor (Fc gamma RI) for human IgG1, human IgG3, murine IgG2a, and murine IgG3 is highly expressed on monocytes, neutrophils (PMN) in certain disease states, and phagocytes treated with interferon-gamma (IFN-gamma). We studied the activation of the human PMN oxidative burst and stimulated fluid pinocytosis induced by three monoclonal antibodies (MoAbs) directed against Fc gamma RI (CD64) to study the role of this receptor in Fc-mediated cellular activation. All three MoAbs were capable of triggering PMN activation from IFN-gamma-treated PMN when cross-linked with goat antimurine Ig reagents. However, MoAb 197 alone demonstrated a concentration-dependent activation of the oxidative burst without the use of a second cross-linking antibody. The oxidative burst and stimulated fluid pinocytosis responses induced by monomeric MoAb 197 could be elicited only after the IFN-gamma induction of approximately 8,000 Fc gamma RI receptor equivalents and did not occur in freshly isolated or control-cultured PMN. Competitive blocking of Fc binding of MoAb 197 by human IgG or purified Fc fragments inhibited cellular activation. We believe the ability of MoAb 197 to activate these oxidative burst and fluid pinocytic responses was because of its murine IgG2a subclass, which allowed it to function as a trivalent anti-Fc gamma RI antibody binding through the combination of its two FAB regions and the Fc domain. This study demonstrates that the cross-linking of CD64 can activate PMN oxidative and endocytic responses and supports a role for Fc gamma RI in the human neutrophil inflammatory response.

The effects of geraniin, a tannin, isolated from Geranium thunbergii Sieb. et Zucc. on the morphology and function of macrophages were studied. Geraniin caused a marked retardation of the recovery from fully spread surface membrane and a highly reorganized cytoskeleton of macrophages, whereas endocytotic activity, phagocytosis and pinocytosis in the cells were significantly inhibited.

Diacylglycerols (OAG, diC8) and PMA were found to stimulate fluid pinocytosis (net uptake of FITC-dextran) to a far greater extent than other neutrophil activators, such as the chemotactic agents fNLPNTL and LTB4, the microtubule disassembling agents colchicine and nocodazole, the kinase inhibitor H-7, or D2O. OAG and diC8 produce a dose-dependent increase in the uptake of FITC-dextran, which is up to about 25- to 30-fold the control value of unstimulated neutrophils. The protein kinase inhibitor H-7 alone had a small stimulating effect on the net uptake, and it failed to inhibit stimulation of fluid pinocytosis by PMA, OAG, and diC8. Also, the protein kinase inhibitor staurosporine failed to inhibit fluid pinocytosis stimulated by OAG, diC8, and PMA. Stimulated fluid pinocytosis and vacuolization in response to PMA or diacylglycerols is associated with surface ruffling of neutrophils. Pinocytosis as well as surface ruffling stimulated by PMA, OAG, diC8, or diC10 are suppressed in the presence of cytochalasin D. The results suggest that diacylglycerols may be instrumental in transducing the signal for stimulated pinocytosis and that the surface movements induced by diacylglycerols, and PMA may be instrumental in fluid pinocytosis.

The presence of calcitonin and somatostatin immunoreactive cells has been determined in semithin sections of human bone and bone marrow samples by immunocytochemical techniques. Calcitonin and somatostatin immunoreactive cells were demonstrated at the interface between bone and bone marrow, in close contact with vessels. It has also been shown that exogenous calcitonin and somatostatin affect the ligand-receptor internalisation, depress the incorporation of 3H-thymidine into acid-insoluble material, and exert opposite effects on 35S-methionine incorporation to proteins of bone marrow cells in vitro. The evidence for calcium-dependent action of calcitonin and somatostatin on bone marrow cells has been presented. The regulatory role of calcitonin and somatostatin in bone marrow hemopoiesis is suggested and the implication of these findings for local hormonal regulation of hemopoiesis and bone structure is discussed.

The cytochalasin D-resistant (pinocytic) portion of the entry of two chlamydia strains (Chlamydia trachomatis L2/434/Bu and Chlamydia psittaci GPIC [guinea pig inclusion conjunctivitis]) was examined. By ultrastructural criteria, few organisms of either strain were observed in association with coated host-cell plasma membrane during entry into McCoy cells; this argues against a coated-pit mechanism of entry. When association with a coated membrane was seen, coat material appeared to pinch off ahead of internalizing chlamydiae. However, entry of both strains was substantially reduced by cytosol acidification, a procedure shown to prevent coated-pit vesiculation (K. Sandvig, S. Olsnes, O. W. Petersen, and B. van Deurs, J. Cell Biol. 105:679-689, 1987). No conclusive evidence of displacement of the fluid-phase marker [3H]sucrose from constitutively forming endocytic vesicles was found. Indeed the entry of strain 434 (but not strain GPIC) was accompanied by the influx of a large volume of fluid, suggesting an inducible mechanism. Additionally, entry of strain 434 (but not strain GPIC) was partially inhibitable by amiloride, yet the drug had no effect on the entry of transferrin, a ligand known to enter solely via coated pits. Our findings endorse the view that chlamydial entry can occur via a pathway involving coated pits. However, the unusual morphology of entry and lack of fluid exclusion are consistent with a process whereby although chlamydiae are not fully enclosed by coat material, their entry is dependent on the vesiculation of coated pits. Furthermore, the data support the proposition that a significant proportion of the entry of strain 434 occurs via an inducible pathway independent of coated-pit uptake.

The effect of the trimethylxanthine, caffeine, was examined on the growth and endocytosis pathways of the vegetative amoebae of the cellular slime mold Dictyostelium discoideum. Caffeine at concentrations of 1.5-3 mM was found to inhibit axenic growth, fluid-phase pinocytosis, and secretion of lysosomal enzymes. Cell viability was unaffected by incubation for 16 hours with 5 mM caffeine but decreased markedly thereafter. Phagocytosis of the bacterium Escherichia coli by Dictyostelium amoebae was also inhibited by caffeine, although at concentrations twofold to threefold higher. Caffeine rapidly entered into amoebae to reach an equilibrium between extracellular and intracellular concentrations, and it was not appreciably metabolized by Dictyostelium. Inhibition of growth and endocytosis was reversible upon removal of the drug and was partially counteracted by 10 mM adenosine. As caffeine discharged intracellular calcium stores in Dictyostelium (Abe et al., 1988), its inhibitory effect on endocytosis could result from the perturbation of calcium homeostasis. In agreement with this hypothesis, the cation La3+ (10 microM), a Ca2(+)-transport inhibitor, also strongly reduced fluid-phase pinocytosis.

The present study demonstrates new properties of H-7. The protein kinase inhibitor H-7 is a potent activator of several neutrophil functions. Stimulation of initially spherical nonmotile neutrophils elicits vigorous shape changes within a few seconds, increases in cytoskeletal actin, altered F-actin distribution, increased adhesiveness and a relatively small increase in pinocytic activity. H-7 has also chemokinetic activities. Depending on the experimental condition, H-7 may elicit or inhibit neutrophil locomotion. It failed to induce chemotaxis. Thus, the response pattern elicited by H-7 is different from that of other leukocyte activators such as chemotactic peptides, PMA or diacylglycerols. The finding that H-7 can elicit shape changes, actin polymerization and pinocytosis suggests that these events can occur without activation of protein kinase C (PKC). PMA-induced shape changes and stimulation of pinocytosis were not inhibited by H-7.

The early structural changes of F344 rat hepatocytes exposed to the hepatocarcinogen 1,2-dimethylhydrazine (DMH) were characterized in short-term monolayer cultures. Continuous exposure of monolayers to DMH (2-16 mM) caused cytoplasmic vacuoles visible by phase-contrast microscopy in all hepatocytes within 6 hr of exposure. These changes preceded maximal release of lactate dehydrogenase (LDH) which occurred after 48 hr of continuous exposure to cytocidal concentrations of DMH (8-16 mM). Ultrastructurally, hepatocytes exposed to DMH (4 mM, 6 hr) showed a twofold increase in mitochondrial diameter from 340 +/- 70 nm in control hepatocytes to 800 +/- 140 nm in DMH-exposed cells. Hepatocyte monolayers exposed to DMH (4 mM, 6 hr) with subsequent removal of DMH attained normal phase-contrast appearance within 6 hr. Ultrastructural studies showed no significant differences when compared with control hepatocytes and mitochondrial diameters (330 +/- 70 nm) were comparable with control hepatocytes. Pretreatment of hepatocytes with depletors of cellular reduced glutathione concentration, including 1,3-bis(2-chloroethyl)-1-nitrosourea (40 microM) and diethyl maleate (160 microM), did not potentiate hepatocellular vacuolation nor release of LDH from hepatocytes exposed to DMH (0-16 mM, 48 hr). These studies demonstrate a distinctive form of reversible high-amplitude mitochondrial swelling that can be monitored by phase-contrast microscopy of cultured hepatocytes in monolayers. Since DMH-induced mitochondrial swelling and its progression to irreversible injury are not potentiated by depletors of reduced thiols, this response appears distinct from prelethal mitochondrial swelling in hepatocytes subjected to oxyradical-mediated mechanisms of injury.

Entamoeba histolytica is a primitive eukaryote that lacks mitochondria. Golgi and a well-developed endoplasmic reticulum. Close to half of the cell volume is occupied by pinocytic vesicles, which are in continuous turnover with the plasma membrane and perform functions that in higher eukaryotic cells are taken over by lysosomes. Similar to the latter, the amebal vesicles are acidified. We report here that bafilomycin A1, a specific inhibitor of vacuolar-type (V-) ATPases, suppressed this acidification at submicromolar concentrations; concomitantly, it inhibited pinocytosis. These results strongly suggest the presence of a V-ATPase in pinocytic vesicles of E. histolytica, and thereby support the notion that the V-ATPases in the organelles of higher eukaryotes are derived from an archaic plasma membrane-bound form.

The effect of 5-fluorouracil (5-FU) on murine monocytes and macrophages was the subject of this study. This drug strongly decreased the levels of all types of peripheral blood cell, but had no effect on the composition and cellularity of peritoneal exudate or the expression of Ia antigens or phagocytic activity of peritoneal macrophages. The pinocytic activity of normal macrophages was unchanged, but the pinocytosis of thioglycollate-stimulated macrophages was significantly reduced. The expression of Fc receptors was influenced only after the first day after 5-FU injection. The clearance and organ distribution of radioactive labeled antigen was also not changed. Our findings suggest that tissue macrophages are resistant to 5-FU, but the results obtained with stimulated macrophages suppose differential susceptibility of macrophages. We presume that the immunosuppressive action of 5-FU results from selective depletion and suppression of T and B lymphocytes rather than from changes in immunoregulating functions of macrophages.

Amitriptyline is known to increase the permeability of the blood-brain barrier but the morphological basis of this increase has not been studied. As catecholamines can influence pinocytosis in dog peripheral blood vessels, the effect of amitriptyline on the pinocytotic activity of blood brain microvessels was studied. Amitriptyline, 34 mg.kg-1 i.p., was injected to rats and the parietal cortex of control and treated animals was prepared for ultrastructural study. Pinocytotic vesicles in endothelial cells were quantified. Amitriptyline significantly increased the density of pinocytotic vesicles in capillary endothelial cells. No other morphological changes occurred after amitriptyline treatment. We conclude that the increase in blood-brain barrier permeability due to amitriptyline may be ascribed at least in part to an increase of pinocytotic activity in brain capillary endothelial cells.

Male Wistar rats were divided in free choice conditions into heavy-drinkers consuming greater than 3.5 g/kg of ethanol daily (HD), and light-drinkers consuming less than 2.0 g/kg/day (LD). Subsequent 30 day intragastric administration of 25% ethanol (8-11 g/kg/day) caused an increase in permeability of the BBB to 14C-tyrosine, 14C-tryptophan and 14C-DOPA at all the stages of alcoholization. At late stages of intoxication (20-30 days) the penetration of horseradish peroxidase (HRP) indicating pinocytosis was present. All the changes were more pronounced in LD than in HD rats. Disulfiram, and to a lesser extent phenazepam and diazepam, when repeatedly injected (for 16-30 days) together with ethanol aggravated its effects. Clonidine and haloperidol antagonized ethanol-induced increase of the BBB permeability to labelled compounds and HRP in the two groups of rats. Picamilon, lithium oxybutyrate, chlorpromazine and alpha-tocopherol had little effect on the penetration of radioactive tracers but effectively antagonized the penetration of HRP. The other drugs were without effects on the BBB regulatory and barrier functions altered by chronic alcoholization. In view of the BBB dysfunction in chronic alcoholics the adequacy of the above-mentioned drugs for their treatment is discussed.

A muramyl dipeptide (MDP) has been conjugated directly with various proteins by means of a water-soluble carbodiimide. The enhancement of the antitumour activity of mouse peritoneal macrophages by the MDP-protein conjugates has been investigated to assess the ability of the proteins for targeting MDP to the macrophages. These were activated to inhibit the in-vitro growth of tumour cells much more effectively, when immunoglobulin (IgG), fibronectin (FN), and gelatin conjugates were used than when MDP was used alone. The minimum amount of MDP in both the MDP-gelatin and the MDP-IgG conjugates necessary for macrophage activation was approximately 2000 times lower than the amount of free MDP needed. The macrophages activated by the conjugates exhibited growth inhibitory activity against phenotypically diverse tumour cells. The activity induced by the MDP-gelatin conjugate was higher than that of the MDP-IgG conjugate over the range of MDP concentrations, regardless of the isoelectric point of the gelatin used. When MDP was conjugated with bovine serum albumin (BSA), the antitumour activity of macrophages was reduced as the amount of BSA conjugated increased. With both free MDP and MDP-protein conjugates, the macrophages were more strongly activated, the longer they were pretreated. However, less pretreatment time was needed to potentiate macrophage activation by the MDP-gelatin conjugate than by free MDP. Also, the macrophages pretreated with the MDP-gelatin conjugate could maintain their activated state for longer than those pretreated with free MDP. It is concluded that gelatin is an effective carrier protein for the targeting MDP to macrophages, resulting in their activation.

Cultures of rat pleural mesothelial cells (PMC) were exposed to nonlethal doses of UICC chrysotile A. The morphology was studied by optical and electron microscopy. The consequences of chrysotile ingestion on the rate of pinocytosis of horseradish peroxidase (HPR) metabolism and benzo-3-4-pyrene (BP) were studied. Nonlethal doses of chrysotile (5 micrograms/mL) induced a time-dependent vacuolation of PMC; a dose-dependent inhibition of the vacuolation was observed when PMC were pretreated with DMSO. The origin of the vacuoles is not clear, but some features of autophagy and lysosomal storage were observed. Chrysotile fibers did not modify the rate of pinocytosis of HRP. Similarly, the metabolism of BP was unchanged when BP and chrysotile were both added to the culture medium or when PMC were preincubated with the fibers 24 hr prior to the addition of BP.

Treatment of thioglycolate-elicited macrophages with mouse beta-interferon markedly reduces pinocytosis of horseradish peroxidase and fluorescein isothiocyanate (FITC)-dextran but stimulates phagocytosis of IgG-coated sheep erythrocytes. Experiments with FITC-dextran have revealed that the overall decrease in pinocytosis is due to a nearly complete inhibition of pinocytosis in a large fraction of interferon-treated macrophages. In the remaining cells pinocytosis continues at a rate similar to that in untreated control cells. A considerable reduction in the number of cells pinocytosing FITC-dextran was observed within 12 h from the beginning of interferon treatment. Measurement of the overall level of pinocytic activity with horseradish peroxidase showed a progressive decline through 72 h of treatment. In the interferon-sensitive subpopulation, there were marked changes in cytoskeletal organization. Microtubules and 10-nm filaments were aggregated in the perinuclear region while most of the peripheral cytoplasm became devoid of these cytoskeletal structures as observed by fluorescence and electron microscopy. In addition, interferon treatment of macrophages appeared to disrupt the close topological association between bundles of 10-nm filaments and organelles such as mitochondria, lysosomes, and elements of the Golgi apparatus and endoplasmic reticulum. Such alterations in the distribution of microtubules and 10-nm filaments were not seen in the interferon-insensitive subpopulation. We have investigated the mechanism of the interferon-induced enhancement of phagocytic activity by binding IgG-coated sheep erythrocytes to mouse peritoneal macrophages at 4 degrees C and then initiating a synchronous round of ingestion by warming the cells to 37 degrees C. Thioglycolate-elicited macrophages that had been treated with mouse beta-interferon ingested IgG-coated erythrocytes faster and to a higher level than control cells in a single round of phagocytosis. In interferon-treated cultures, phagocytic cups became evident within 30 s of the shift of cultures from 4 degrees to 37 degrees C, whereas in control cultures, they appeared in 2 min. Cytochalasin D, an inhibitor of actin assembly and polymerization, abolished phagocytic activity in both control and beta-interferon-treated macrophages. However, to inhibit phagocytosis completely in thioglycolate-elicited interferon-treated macrophages, twice as much cytochalasin D was required in the treated as in control cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Lysolecithin was found to modify cation-induced pinocytosis in Amoeba proteus. It is shown here that lysolecithin (LPC) in the concentration range of 10(-15) to 10(-10) g/ml has the same effect on Na+ -induced pinocytosis as cAMP and a pinocytosis regulating factor (PRF) which is secreted by the amoeba. Thus, LPC activated Na+-induced pinocytosis in starved amoebae and decreased the sensitivity to the inducer in normal cells. Pinocytosis depressed by treatment with EGTA or dibucaine became normal upon addition of LPC to the inducer. These effects were also obtained with lysolecithin isolated from the amoeba. It is suggested that PRF and amoeba LPC may be closely related and that phospholipase activity of the amoeba may regulate its capacity for pinocytosis.