Adhesiveness and proliferation of epithelial cells are differentially modulated by activation and inhibition of protein kinase C in a substratum-dependent manner

In the present study, we have examined the regulation of attachment, onset of proliferation and the subsequent growth, in vitro, of chick retinal pigmented epithelial (RPE) cells as a function of the nature of the substratum and of either the activation or inhibition of protein kinase C (PKC). The RPE cells have an adhesive preference for protein carpets which contain laminin. This preference disappears gradually with time in culture. The adhesion of RPE cells to fibronectin is shown to be a receptor-mediated process which involves the RGD recognition signal. This study also demonstrates that a PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), affects RPE cell adhesion in a substratum-dependent manner. Exposure of RPE cells to TPA lowers the cell attachment efficacy to ECM protein substrata but does not affect cell attachment to plastic. The onset of cell proliferation is accelerated by TPA on all of the substrata tested. The minimal duration of an effective TPA pulse exerting a long-lasting influence on RPE cell proliferation is between 1.5 and 3.5 hr. Stimulation of cell proliferation by TPA in long-term cultures is independent of the nature of the growth substratum. The acceleration of the onset of cell proliferation by TPA is sensitive to 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), an inhibitor of conventional PKC, and thus appears to be dependent on the activation of conventional PKC. H7 also affects cell-cell contacts, causing an alteration in the shape (“squaring”) of RPE cells packed into large colonies. Conversely, the effects of TPA on both the attachment and the long-term proliferation of RPE cells are not dependent a conventional PKC isotype, since H7 cannot abolish the influence of TPA on either process. We conclude that the effect of TPA on long-term proliferation of RPE cells is either dependent on a novel PKC isotype or independent of PKC. © 1993 Wiley-Liss, Inc.     

Effects of a tumour promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on expression of differentiated phenotype in the chick retinal pigmented epithelial cells and on their interactions with the native basement membrane and with artificial substrata

Chick retinal pigmented epithelial (RPE) cells grown in vitro on basement membrane matrices from the Engelbreth-Holm-Swarm tumour (BM-matrigel) do not spread, and they maintain their differentiated phenotype, most notably the heavy pigmentation. Maintenance of the differentiated phenotype by RPE cells on BM-matrigel is promoted not only by the biochemical composition of the gel but also by its mechanical properties, i.e., its low rigidity prevents cell spreading. In this report, RPE cells on BM-matrigel were treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to promote the transformed phenotype and diminish cell traction. In contrast to most cell types TPA treatment induced RPE cells to increase their spread area. TPA promoted RPE cell spreading on BM-matrigel and changed the spatial organization of actin and actin-associated proteins in the cytoskeleton-ECM linkage complexes, uncoupling actin from its extracellular counterpart. TPA did not affect other components of the cytoskeleton in RPE cells. TPA also affected labile adhesions i.e., focal contacts and adherens junctions in statu nascendi, but preformed, stable adherens junctions were resistant to TPA. TPA enhanced proliferation, blocked melanogenesis and thus inhibited differentiation of RPE cells grown on either artificial substrata or their natural basement membrane.     

Substrate ifluences human epidermal melanocyte attachment and sperading in vitro

Summary Previous culture systems for melanocytes have employed serum-supplemented medium and uncoated plastic dishes, prohibiting examination of possible substrate influences on cellular morphology and function. We now report, using a sensitive serum-free system and a quantitative procedure for evaluating cellular morphology, that modification of the plating surface affects human epidermal melanocyte attachment rate and subsequent morphology in vitro. Melanocytes attach and spread more rapidly on surfaces coated with fibronectin or Type I/III collagen or on surfaces previously conditioned by human keratinocytes, dermal fibroblasts, melanocytes, or melanoma cells than do melanocytes on untreated control surfaces. Type IV collagen and laminin, although minimally beneficial for cell attachment, do support a characteristics melanocyte morphology that differs from that seen either on the other coated surfaces or on uncoated plastic controls. Addition of fetal bovine serum at the time of inoculation has no appreciable effect on attachment but markedly improves cell spreading on untreated surfaces, while addition of nerve growth factor with or without serum to this system fails to affect cell attachment or spreading. Our data establish that human epidermal melanocytes are indeed capable of responding morphologically to substrate signals. The ability of several biochemically unrelated surfaces to enhance melanocyte attachment rate and spreading suggests that melanocytes have surface receptors with a variety of specificities. This work is relevant to the development of improved culture systems for melanocytes in vitro and to understanding melanocyte behavior in vivo. This work was supported by the USDA Agricultural Research Service, by a grant from Cheesebrough-Ponds, Inc., and by a Dermatology Foundation Fellowship (Dr. Yaar).     

Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of the differentiated phenotype by epithelial cells in vitro is regulated by both biochemistry and mechanics of the substratum

In this paper I sought to determine how the expression of differentiated traits of chick retinal pigmented epithelial (RPE) cells in vitro can be modulated by varying both the biochemical and the spatial complexity, and the mechanical properties, of the growth substratum. I have used glass derivatized with proteins of a basement membrane extract (nondeformable, two-dimensional substratum) and gels of reconstituted basement membrane extract (viscoelastic, three-dimensional substratum). These two biochemically similar substrata were compared to an inert substratum (untreated glass) and to the native basement membrane of the RPE, i.e., Bruch's Membrane. With immunofluorescence microscopy, I have shown that RPE cells, given space, will spread on their native basement membrane and form stress fibres and focal contacts, analogous to the stress fibres and integrin-, talin-, and vinculin-containing focal contacts of the cells grown on glass. Therefore, the stress fibres and focal contacts present in cultured cells are not artifacts of growth in vitro, but are a natural cellular response to the nondeformability of commonly used tissue culture substrata. The proteins of the basement membrane promote expression of some of the differentiated traits by RPE cells in vitro: however, the fully differentiated phenotype is expressed by RPE cells only when their spreading is prevented by low resilience of a substratum. Basement membrane gels generally are not resilient enough to support RPE cell spreading; however, the cells spread and form stress fibres, and integrin-, talin-, and vinculin-containing focal contacts when they are presented with areas of the gel which locally acquired higher resilience. The extent of cell spreading is determined by the deformability of substratum, hence elastic forces operating within the substratum determine the maximal cell traction allowable and, indirectly, the cytoarchitecture. Therefore, in addition to biochemical composition, the mechanical properties of substrata play important role in regulation of expression of the differentiated phenotype of cells in vitro and, possibly, in vivo.     

Substratum effects on cell dispersal, morphology, and differentiation in cultures of avian neural crest cells

Adhesive extracellular matrix (ECM) molecules appear to play roles in the migration of neural crest cells, and may also provide cues for differentiation of these cells into a variety of phenotypes. We are studying the influences of specific ECM components on crest differentiation at the levels of both individual cells and cell populations. We report here that the glycoproteins fibronectin and laminin differentially affect melanogenesis in cultures of avian neural crest-derived cells. Clusters of neural crest cells were allowed to form on explanted neural tubes for 24 and 48 hr, and then subcultured on uncoated glass coverslips or coverslips coated with fibronectin or laminin. The morphology of cells varied on the three substrata, as did patterns of cell dispersal. Crest cells dispersed most rapidly and extensively on fibronectin. In contrast, cells on laminin dispersed initially, but then assumed a stellate morphology and rapidly formed small aggregates. Cell dispersal was minimal on glass substrata, resulting in a uniformly dense distribution. These patterns of dispersal were similar in subcultures of both 24- and 48-hr clusters, although dispersal of cells from older clusters was less extensive. The rate and extent of melanogenesis correlated with patterns of cell dispersal. Cell from 24-hr clusters underwent melanogenesis significantly more slowly on fibronectin than on the other two substrata. Pigment cells began to differentiate by 2 days of subculture in the cell aggregates on laminin and in the dense centers of cultures on untreated glass. By 5 days, there was significantly more melanogenesis in cultures on laminin and glass than on fibronectin substrata. Melanogenesis in cultures of 48-hr clusters was more rapid and extensive on control (glass) substrata than on fibronectin or laminin, correlating with reduced cell dispersal. We conclude that fibronectin and laminin, which are found along neural crest migratory pathways in vivo, can affect melanogenesis in vitro by regulating patterns of cell dispersal.     

The influence of cold insoluble globulin on platelet morphological response to substrata

Light microscopic studies have been carried out on the attachment and morphological responses of washed human platelets in serum-free medium to fibrinogen-coated, collagen-coated and uncoated tissue culture plastic substrata. Platelets were observed to attach to the substratum, extend filipodia and undergo spreading. Subsequently, lysis of platelets occurred. On uncoated tissue culture plastic substrata, the addition of cold insoluble globulin to the incubations had no effect on the above morphological changes. On the other hand, on the protein coated substrata, there was very little platelet spreading or lysis without the addition of cold insoluble globulin.     

Unique morphology of HeLa cell attachment, spreading and detachment from microcarrier beads covalently coated with a specific and non-specific substratum

A SEM and TEM evaluation of adhesion of HeLa-S3 cells to suspensions of culture microcarriers coated with various substrata revealed two unique cell morphologies. One is similar to that for cells attaching to culture dishes and the other one only appeared with microcarriers stirred under high shear conditions. The usual appearance of a spreading cell is to change from a sphere to the shape of a 'fried egg'. This proceeded in HeLa cells by a radial extension of the filopodia in between which the cytoplasm subsequently filled. Fluorescent antibody staining of actin suggested that more actin was present at the periphery of the spreading edges of the cell than inwards. The above morphology was characteristic of HeLa cell attachment to gelatin-coated microcarriers. However, the morphology of the attachment to microcarriers coated with non-biological substances such as negatively charged sulfonate groups or positively charged polyethyleneimine or even with the attachment protein laminin was quite different. Here the cells attached and began to spread as with gelatin-microcarriers, however, the spreading was not radial but occurred from one or two major regions of the cell periphery. The cell then appeared to constrict with the formation of a substratum attached pedestal upon which the cell body was perched. With time the cell pinched-off from pedestal. Evidence indicated that the pedestal was quite fragile. Furthermore, fluorescent antiactin staining indicated that the initial spreading region contained abundant actin which was depleted upon pedestal formation and detachment. The above in addition to previous kinetic measurements provided the information to classify cell substrate attachment materials into two distinct types. One is specific substrata which promote normal attachment and spreading and appear to interact with specific cell surface proteins. The other is non-specific substrata which in high shear conditions induces pedestal formation followed by pinching-off of the cells. Had previous attachment assays been done under high shear as done with the microcarriers and HeLa cells it is likely that substrata classified as specific might be reclassified into non-specific.     

Herpes simplex virus gE/gI expressed in epithelial cells interferes with cell-to-cell spread

The herpes simplex virus (HSV) glycoprotein heterodimer gE/gI plays an important role in virus cell-to-cell spread in epithelial and neuronal tissues. In an analogous fashion, gE/gI promotes virus spread between certain cell types in culture, e.g., keratinocytes and epithelial cells, cells that are polarized or that form extensive cell junctions. One mechanism by which gE/gI facilitates cell-to-cell spread involves selective sorting of nascent virions to cell junctions, a process that requires the cytoplasmic domain of gE. However, the large extracellular domains of gE/gI also appear to be involved in cell-to-cell spread. Here, we show that coexpression of a truncated form of gE and gI in a human keratinocyte line, HaCaT cells, decreased the spread of HSV between cells. This truncated gE/gI was found extensively at cell junctions. Expression of wild-type gE/gI that accumulates at intracellular sites, in the trans-Golgi network, did not reduce cell-to-cell spread. There was no obvious reduction in production of infectious HSV in cells expressing gE/gI, and virus particles accumulated at cell junctions, not at intracellular sites. Expression of HSV gD, which is known to bind virus receptors, also blocked cell-to-cell spread. Therefore, like gD, gE/gI appears to be able to interact with cellular components of cell junctions, gE/gI receptors which can promote HSV cell-to-cell spread.     

Interactions of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix-controlled modes of attachment and spreading

We have examined the interaction of adult rat hepatocytes in primary culture, to type IV collagen, fibronectin, and laminin, the major basement membrane proteins of normal rat liver. Culture substrata consisted of glass coverslips, which were covalently derivatized with individual purified basement membrane constituents at varying densities of protein. The attachment of freshly prepared hepatocytes was examined after incubation at 37 degrees C for 30 min as a function of the amount of protein on the coverslips. For each of the three types of substratum under study, distinct modes of cell attachment were observed, with the apparent affinity of hepatocytes for type IV collagen being three-fold greater than for fibronectin and ten-fold greater than for laminin. Cell attachment exhibited saturation on all substrata. Hepatocyte spreading was measured by scanning electron microscopy of cells incubated at 37 degrees for 2 h on similarly prepared coverslips. A five-fold greater surface density of type IV collagen was required for maximal spreading compared with attachment. For cells on fibronectin or laminin the maximal cell spreading reached on type IV collagen did not occur even at coverslip protein densities 10 to 20 times those providing for maximal cell attachment. A very similar qualitative pattern of cell proteins was secreted within a few hours of plating on the various substrata and further studies failed to reveal any evidence that attachment and spreading was mediated by endogenously produced matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnesium-dependent attachment and neurite outgrowth by PC12 cells on collagen and laminin substrata

We report a study of the substratum and medium requirements for attachment and neurite outgrowth by cells of the pheochromocytoma-derived PC12 line. In attachment medium containing both Ca2+ and Mg2+, more than 50% of cells attached within 1 hr to petri dishes coated with native collagen Types I/III or II, native or denatured collagen Type IV, laminin, wheat germ agglutinin (WGA), or poly-L-lysine; attachment to dishes coated with nerve growth factor (NGF) was only about 20% and attachment to uncoated dishes or to dishes coated with fibronectin or gelatin was almost nil. Neither prior culturing in the presence of NGF nor addition of NGF to the attachment medium significantly affected the extent of attachment to collagen or laminin. With Ca2+ (1 mM) as the sole divalent cation, cells attached normally to WGA, polylysine, and NGF, but failed to attach to collagen or laminin. With Mg2+ (1 mM) as the only divalent cation, attachment to all substrata was about the same as in medium with both Ca2+ and Mg2+. Like the ionic requirements, the kinetics of attachment, insensitivity to protease treatment of the cells, and inhibition by low temperature and sodium azide were similar for PC12 attachment to collagen and laminin, suggesting that a common molecular mechanism may underlie attachment to these substrata. The only significant difference observed was that addition of WGA (30 micrograms/ml) to the attachment medium inhibited attachment to collagen but promoted attachment to laminin. Finally, PC12 cells extended neurites on laminin, on native collagens I/III, II, and IV, and on denatured collagen IV; they did not extend neurites on denatured collagens I/III or II, NGF, or WGA. Neurite outgrowth on collagen and laminin occurred with Mg2+ as the sole divalent cation. These results suggest that the same Mg2+-dependent adhesion mechanism operates at the cell body and at the growth cone.     

Adhesion,spreading, and proliferation of cells on protein carpets: Effects of stability of a carpet

Summary In the present report we have investigated the role that the physical properties of substrata play in modulating the effects which components of extracellular matrix (ECM) exert on adhesion, spreading, and growth of retinal pigmented epithelial cells. By simple modifications of conditions for protein adsorption on glass we obtained a set of substrata all coated with proteins of ECM (protein carpets) but with different physical properties. Using these protein carpets we have shown that their stability (desorption rate) in tissue culture conditions varies according to the technique with which they were prepared. Both semiremovable and immobilized carpets are stable, whereas removable protein carpets desorb readily. Therefore, the protein concentration or composition or both may change with time in tissue culture depending on the technique used to prepare the carpet. In addition, efficacy of cell attachment to given protein may vary depending on whether a technique used to prepare the protein carpet involves denaturation of the protein. Adherent cells quickly remove (clear) weakly adsorbed protein carpets and it seems that the carpet removal is a mechanical process. During the carpet removal cells are rounded, which indicates that a spread cell phenotype normally associated with stress fibers and focal contacts occurs when the substratum is rigid enough to sustain cell traction. In addition, substrata lacking the rigidity to support the spread phenotype do not support cell proliferation either.     

Adhesive substrates for fibronectin

In order to promote cell attachment, fibronectin must first undergo activation by a suitable substrate. In this study, 52 materials have been surveyed for their ability (a) to bind fibronectin, (b) to activate the cell-adhesive property of fibronectin, and (c) to support the growth of cells. Many plastics, polysaccharides, metals, and ceramics were found to support cell growth as well as the fibronectin-dependent attachment of cells. Several other substrates have been identified that were inactive in promoting either cell attachment or growth. Hydrophobic substrates were found to be active in fibronectin activation, whereas hydrophilic substrates were found to be inactive. Since fibronectin binds to substrata of extremely varied chemical composition, it is clear that the binding of fibronectin to such substrata is nonspecific in nature. Since protein pretreatment of all substrata, except collagen and poly(L-lysine), abolished the physical binding of fibronectin, the binding of fibronectin to artificial substrata is probably ascribable to a nonspecific hydrophobic protein-substratum interaction. In contrast, several lines of evidence indicate that the interaction between fibronectin and collagen displays biological specificity. Poly(hydroxyethylmethacrylate)(poly(HEMA)), which has previously been shown to be nonadhesive for cells, is demonstrated here to be unique in its inability to bind fibronectin. Addition of one part per million of an adhesive polymer to poly(HEMA) permits fibronectin binding to occur.     

Effect of modified pectin molecules on the growth of bone cells

The aim of this study was to investigate molecular candidates for bone implant nanocoatings, which could improve biocompatibility of implant materials. Primary rat bone cells and murine preosteoblastic MC3T3-E1 cells were cultured on enzymatically modified hairy regions (MHR-A and MHR-B) of apple pectins. MHRs were covalently attached to tissue culture polystyrene (TCPS) or glass. Uncoated substrata or bone slices were used as controls. Cell attachment, proliferation, and differentiation were investigated with fluorescence and confocal microscopy. Bone cells seem to prefer MHR-B coating to MHR-A coating. On MHR-A samples, the overall numbers as well as proportions of active osteoclasts were diminished compared to those on MHR-B, TCPS, or bone. Focal adhesions indicating attachment of the osteoblastic cells were detected on MHR-B and uncoated controls but not on MHR-A. These results demonstrate the possibility to modify surfaces with pectin nanocoatings.     

In vitro studies on the control of nerve fiber growth by the extracellular matrix of the nervous system

1. Cultured neurons from embryonic chick sympathetic ganglia or dorsal root ganglia grow nerve fibers extensively on simple substrata containing fibronectin, collagens (types I, III, IV), and especially laminin. 2. The same neurons cultured on substrata containing glycosaminoglycans grow poorly. Glycosaminoglycans (heparin) inhibit nerve fiber growth on fibronectin substrata. 3. Proteolytic fragments of fibronectin support nerve fiber growth only when the cell attachment region is intact. For example, a 105 kD fragment, encompassing the cell attachment region, supports growth when immobilized in a substratum, but a 93 kD subfragment, lacking the cell attachment region, is unable to support fiber growth. When it is added to the culture medium, the 105 kD fragment inhibits fiber growth on substrata containing native fibronectin. 4. In culture medium lacking NGF, DRG neurons extend nerve fibers only on laminin and not on fibronectin, collagen or polylysine. Studies with radioiodinated laminin indicate that laminin binds with a relatively high affinity (kd approximately equal to 10(-9) M) to DRG neurons, and to a variety of other neural cells (NG108 cells, PC12 cells, rat astrocytes, chick optic lobe cells). We have isolated a membrane protein (67 kD) by affinity chromatography on laminin columns and are characterizing this putative laminin receptor. 5. Dissociated DRG neurons or ganglionic explants cultured on complex substrata consisting of tissue sections of CNS or PNS tissues extend nerve fibers onto the PNS (adult rat sciatic nerve) but not CNS (adult rat optic nerve) substrata. Other tissue substrata which support fiber growth in vivo (embryonic rat spinal cord, goldfish optic nerve) support growth in culture. While substrata from adult CNS, which support meager regeneration in vivo (adult rat spinal cord) support little fiber growth in culture. 6. Ganglionic explants cultured in a narrow space between a section of rat sciatic nerve and optic nerve grow preferentially onto the sciatic nerve suggesting that diffusible growth factors are not responsible for the differential growth on the two types of tissues. 7. Dissociated neurons adhere better to sections of sciatic nerve than optic nerve. Laminin, rather than fibronectin or heparan sulfate proteoglycan, is most consistently identifiable by immunocytochemistry in tissues (sciatic nerve, embryonic spinal cord, goldfish optic nerve) which support nerve fiber growth. Taken together, these data suggest that ECM adhesive proteins are important determinants of nerve regeneration.     

N-acetylglucosamine and adenosine derivatized surfaces for cell culture: 3T3 fibroblast and chicken hepatocyte response

3T3 fibroblasts and primary chicken hepatocytes were cultured on derivatized polystyrene surfaces to examine the effect of cell-specific ligands on cellular morphology and growth. Surfaces were prepared by derivatizing chloromethylated polystyrene with N-acetylglucosamine (GlcNAc; recognized by the chicken asialoglycoprotein receptor) and adenosine (not recognized by adult hepatocytes). These surfaces were compared with tissue culture polystyrene (TCPS), acid-cleaned glass, and the unmodified chloromethylated polystyrene. The spreading, cytoskeletal structure and growth of the fibroblasts following attachment to these surfaces were examined. The extent of attachment, total protein levels, and DNA contents for surfaces-attached chicken hepatocytes were also measured. Fibroblast spreading was greatest on polymer surfaces derivatized with GlcNAc, whereas cytoskeletal structure and growth rate were independent of surface chemistry. Although chicken hepatocytes attached most efficiently to the GlcNAc derivatized polymer, the total protein and DNA levels of the surface-attached cells were not affected. In anticipation of the application of these polymers for cell culture and hybrid artificial organ design, the GlcNAc-derivatized polystryrene was fabricated into porous microcarriers. Fibroblasts grew avidly on the microcarriers, whereas chicken hepactocytes adhered well to the formed large aggregates arounds the microcarriers.     

Increased gap junctional area in the rat liver after administration of dibutyryl cAMP

Summary Since cAMP has recently been reported to be a possible physiological modulator of cell-to-cell communication, we performed a quantitative freeze-fracture investigation on the hepatocyte gap junctions after administration of a membrane-permeant derivative of this cyclic nucleotide. For this purpose, male rats received two intraperitoneal injections of 100 mg dibutyryl cAMP/kg body weight with a time interval of 2.5 h. Litter mates were injected with saline only. Five hours after the start of the treatment, tissue blocks of the left lateral liver lobe were fixed by immersion and processed for freeze-fracture. By point counting on negatives projected on a square double-lattice test system the relative gap junctional area on contiguous hepatocyte membranes was determined. As compared to control animals, the proportion of the membrane area occupied by gap junctions in dibutyryl cAMP-treated liver parenchyma significantly increased from 4.9% to 6.1%. Within the gap junctions no changes in shape, particle density or packing pattern were observed. Possibly, the enlarged gap junctional area provides structural pathways for the integration of the response of hepatocytes to messages mediated by cAMP.This investigation was supported by grant nr. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium).Research assistant of the National Fund for Scientific Research (Belgium).     

Isolation,characterization, and long-term culture of fetal bovine tracheal epithelial cells

Summary Epithelial cells were isolated from fetal bovine trachea by exposing and stripping the mucosal epithelium from the adjacent connective tissue. The tissue was minced and enzymically dissociated in Ca-Mg-free medium containing dispase and dithiothreitol. The stripping procedure and selective trypsinization produced epithelial cell cultures free of fibroblasts. Seeded on plastic, the plating efficiency was 21.5% with a doubling time of 24 h. Dome formation, evidence of occluding junctions and active ion transport characteristic of epithelial cells, was common. Growth of the cells on glass, collagen, and Engelbreth-Holm-Swarm (EHS) substrate demonstrated a striking difference in morphology. Cells grown on EHS presented a more distinctly three-dimensional growth pattern and many more microvilli when compared to cells grown on glass or collagen. The cells retained their epithelioid characteristics through more than 30 passages as shown by the presence of distinct apical and basolateral membranes, tight junctions, and positive keratin staining. This study was supported in part by grants BRSG S07 RR05408-25, Biomedical Research Support Grant Program, Division of Research Resources, by ES 00159, Center Grant, National Institutes of Environmental Health Sciences, by R23-HL37621, New Investigator Award, National Heart, Lung and Blood Institutes, National Institutes of Health, and by the Health Effects Institute, an organization jointly funded by the U.S. Environmental Protection Agency (Assistance Agreement X-8120059) and automotive manufactures. The contents do not necessarily reflect the views of policies of HEI, EPA, or automotive manufacturers.     

Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures

The ultrastructure of gap and tight junctions and the cell-to-cell transfer of small molecules were studied in primary cultures and freshly isolated sheets of endothelial cells from calf aortae and umbilical veins. In thin sections and in freeze-fracture replicas, the gap and tight junctions in the freshly isolated cells from both sources appeared similar to those found in the intimal endothelium. Most of the interfaces in replicas had complex arrays of multiple gap junctions either intercalated within tight junction networks or interconnected by linear particle strands. The particle density in the center of most gap junctions was noticeably reduced. In confluent monolayers, after 3-5 days in culture, gap and tight junctions were present, although reduced in complexity and apparent extent. Despite the relative simplicity of the junctions, the cell-to-cell transfer of potential changes, dye (Lucifer Yellow CH), and nucleotides was readily detectable in cultures of both endothelial cell types. The extent and rapidity of dye transfer in culture was only slightly less than that in sheets of freshly isolated cells, perhaps reflecting a reduced gap junctional area combined with an increase in cell size in vitro.     

Intermitochondrial junctions in the extraocular muscle of the rat

Summary Intermitochondrial junctions with a spacing of 17–21 nm were observed in the superior rectus muscle of a rat. Periodic rounded densities are aligned midway between the apposed outer mitochondrial membranes at some of these junctions. Such densities have a diameter of about 8–10 nm and a center-to-center spacing of about 26–30 nm. These junctions occur in cases where one mitochondrial profile is enclosed within another or where two profiles are interlocked so that their combined overall form has a smoothly contoured profile. Intermitochondrial junctions seem not to have been previously described in muscle, but have been reported in other kinds of tissues. In agreement with those previous reports, the presently observed intermitochondrial junctions usually involve mitochondria that display atypical features indicative of tissue abnormality or stress. Such junctions were never observed in normal extraocular muscle.This study was supported by Grants EY-00309, EY-01297 and EY-01842 from the National Eye Institute (USPHS) and in part by a grant from the Eye Surgery Fund, Inc. and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc.