Biomedical subjects

L Terracio

Publications and source records attributed to L Terracio.

At least 37 records · Page 2Linked to original sources

PubMed1993

The extracellular matrix components laminin, fibronectin, and collagen IV are present among the epithelial cells forming Rathke's pouch.

Cell-matrix interactions probably play a cooperative role with cell-hormone interactions to ensure normal differentiation of the adenohypophysis. The extracellular matrix (ECM) surrounding adult adenohypophysial cells contains laminin but its embryonic development has not been described. This study was carried out to test the hypothesis that adenohypophysial cells are associated with components in the ECM prior to cellular differentiation in the adenohypophysis. Fetuses were removed from Golden Syrian hamsters every 12 h from embryonic days 8.5-14. Coronal and sagittal 5-microns-thick sections of paraplast-embedded embryos were stained for fibronectin, laminin, or collagen IV using avidin-biotin immunoperoxidase staining. The adenohypophysial anlage was observed initially as a group of epithelial cells (Rathke's pouch) in the roof of the stomatodeum in contact with a basement membrane. The basement membrane stained positively for laminin, collagen IV and fibronectin. Lighter staining for laminin, fibronectin and collagen IV was observed between the developing adenohypophysial cells in Rathke's pouch. Proliferative activity was apparent in the antero-inferior region of Rathke's pouch and resulted in the formation of the bulk of the adenohypophysis. Mesenchyme infiltrates the region between the base of Rathke's pouch and the oral epithelium, thus separating the two. The basement membrane surrounding the pouch appears to become discontinuous in the regions of high proliferative activity. These results show that ECM components appear early during the development of adenohypophysial cells prior to their cellular differentiation into hormone-containing cells. This association between ECM components and developing adenohypophysial cells provides the anatomical basis for cell-ECM interactions to influence adenohypophysial development and differentiation.

Animals↗

PubMed1993

Contractile activity modulates actin synthesis and turnover in cultured neonatal rat heart cells.

In this study, the role that active tension development plays in the formation and maintenance of cardiac myocyte myofibrillar structure and cellular shape was investigated. By use of the calcium channel blocker verapamil, spontaneous contractile activity of neonatal rat heart myocytes was inhibited for 24 to 96 hours. Confocal microscopy of rhodamine phalloidin-stained cells revealed that, within 24 hours of contractile arrest, actin filaments of myofibrils were no longer aligned with one another at their I bands and Z lines. Cellular shape was also affected, with the cells developing a less stellate appearance while remaining attached to the substrate as well as to one another. By 48 hours, actin fibrils were largely absent from these cells. The disappearance of actin was confirmed by measurements of actin synthesis and accumulation rates and by pulse-chase biosynthetic labeling experiments. It was revealed that, although actin synthesis was significantly reduced in arrested myocytes, the rapid disappearance of total cellular actin was largely due to increased rates of actin degradation. Contractile arrest produced by L-type calcium channel blockade with verapamil (or other calcium channel blockers) accelerated actin degradation to a greater extent than K+ depolarization. Chloroquine partially suppressed the accelerated rate of actin degradation, indicating that lysosomal proteolysis may be involved in actin degradative processing. Protein kinase C activation also partially inhibited the accelerated rate of actin degradation but did not restore actin filaments in arrested myocytes. The reformation of actin fibrils and their reassembly into striated myofibrils occurred when contractile activity was restored by removal of verapamil from the culture medium. The period of time required for myocytes to reassemble actin filaments and to regain their elongated morphology was proportional to the period of time that the cells were inhibited from contracting. Data are presented to indicate that active tension development by neonatal cardiac myocytes in culture is critical to the maintenance of filamentous actin structure via mechanisms involving actin assembly, disassembly, and degradation.

Actins↗

PubMed1992

Expression of collagenase and IL-1 alpha in developing rat hearts.

During development, extracellular matrix (ECM) molecules are thought to play a major role in regulating the formation of the heart. The change in the heart from a simple tube to a complex, four-chambered organ requires the modification of both the cellular components as well as the surrounding ECM. Matrix metalloproteinases (MMP), which include collagenases, are enzymes present in the ECM that have the potential to modify the existing ECM during the development of the heart. Using both monoclonal and polyclonal antisera against collagenase, specific temporal and spatial patterns have been documented during critical periods of heart development. The cytokine interleukin 1 alpha (IL-1 alpha), a potent inducer of the MMP expression, was also shown to have a similar staining pattern in the developing heart. The monoclonal anti-rat collagenase (Mab) intensely stained the surfaces of the myocytes in the trabeculae and the ventricular and atrial walls of the 11.5 or 12.5 embryonic day (ED) rat hearts. In contrast, the polyclonal anti-human collagenase (Pab) stained not only the cardiomyocytes but also the hypertrophic endocardial cells. Pab appeared to stain the leading edge of the mesenchymal cells that migrate into the cardiac jelly of the 11.5 or 12.5 ED hearts. Immunohistochemical staining showed IL-1 alpha on the endocardial endothelium and the surface of cardiomyocytes near the cardiac jelly just before or coincident with the appearance of migrating cells. IL-1 alpha was detected on the endocardial endothelium, cardiomyocytes in the trabeculae, and the ventricular and atrial walls, as well as in the myocardial basement membrane of the truncal or atrioventricular region. However, no staining could be detected on the migrating cells in the cardiac cushions. These results indicate the presence of collagenase and IL-1 alpha on the surface of cardiomyocytes and mesenchymal cells at times when the heart is undergoing acute remodeling during septation and trabeculation. These data suggest a role for collagenase/cytokine interaction in tissue remodeling during critical stages of cardiac embryogenesis where modification of the ECM is essential to cardiac morphogenesis.

Animals↗

PubMed1992

The role of beta 1 integrin in spreading and myofibrillogenesis in neonatal rat cardiomyocytes in vitro.

The influence of the extracellular matrix (ECM) on cell behavior, myofibrillogenesis and cytoarchitecture was investigated in neonatal rat cardiac myocytes in vitro. Cell behavior was examined by analyzing cell spreading on different ECM components under a variety of experimental conditions. Area measurements were made on digitized images of cells grown for various time intervals on fibronectin (FN), laminin (LN), collagens I and III (C I+III), plastic, and bovine serum albumin (BSA). The amount of spreading was varied on the different matrices and was maximal on FN greater than LN greater than C I+III greater than plastic greater than BSA. Addition of anti-beta 1 integrin antibodies to myocytes cultured on FN, LN and C I+III blocked spreading outward on the substrates and altered normal myofibrillogenesis, especially on LN. Concomitantly, the integrin antibodies induced the formation of giant pseudopodial processes which protruded upward from the substrates. These pseudopods contained actin polygonal networks which exhibited a regular geometrical configuration. Effects of the ECM on cytoarchitecture was examined by analyzing the temporal and spatial patterns of fluorescence and immunogold labeling of cytoskeletal and integrin proteins as myocytes spread in culture. The first indication of sarcomeric patterns was the appearance at 4 hours of striations formed by lateral alignment of alpha-actinin aggregates into Z bands. At later times, vinculin at 8 hours and beta 1 integrin at 22 hours became co-localized with alpha-actinin at the Z bands and focal adhesions. These data indicate that ECM components influence myocyte spreading and that myofibril assembly and/or stability is associated with ECM-integrin-cytoskeleton associations.

Actinin↗

PubMed1992

Reconstituted basement membrane influences prolactin, LH, and FSH secretion from adult and fetal adenohypophyseal cells in vitro.

These investigations tested the hypothesis that secretion of prolactin (PRL), LH, and FSH in vitro is influenced by the substratum on which adult or fetal adenohypophyseal cells are cultured. Adenohypophyses were removed from adult male Golden Syrian hamsters and from fetal hamsters on day 16 of gestation. The glands were dissociated and cultured in a 1:1 mixture of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 medium containing 10% fetal bovine serum (FBS), 25 mM Hepes, and antibiotics. The cells were cultured on three substrata: glass, laminin, and the reconstituted basement membrane of the Engelbreth-Holm-Swarm (EHS) tumor (Matrigel). Medium was collected and replaced every 48 h for 14-22 days. Concentrations of PRL, LH, and FSH in medium were measured by RIA. The substratum influenced hormone secretion. PRL concentrations were elevated in cultures of adult cells on Matrigel in each of four experiments. Adenohypophyseal cells on Matrigel maintained a rounded shape longer than cells on glass or laminin. In studies using fetal adenohypophyseal cells, PRL concentrations were elevated significantly in medium from cultures on Matrigel at and after 2 days as were concentrations of LH and FSH after 6 days. Additional experiments showed that the higher PRL concentrations in medium surrounding adult cells plated on Matrigel were not due to the release of soluble factors from Matrigel, differential cell attachment on Matrigel, the differential presence of adenohypophyseal fibroblasts, nor differential rates of cell proliferation. The results show that Matrigel maintains the secretion of PRL from adult adenohypophyseal cells in vitro more effectively than glass or laminin substrata and support the hypothesis that cell-matrix interactions mediate the observed differences. The results also show that in long-term cultures (14-22 days), fetal adenohypophyseal cells secrete significantly more PRL, LH, and FSH on Matrigel than they secrete when cultured on glass or laminin. Thus, Matrigel influences the function and possibly the maturation of adenohypophyseal cells in vitro. Furthermore, although laminin is the most abundant component in Matrigel, the effects of Matrigel on lactotrophs and gonadotrophs in vitro are probably not attributable solely to its laminin content.

Age Factors↗

PubMed1992

Immunolocalization of ubiquitin conjugates at Z-bands and intercalated discs of rat cardiomyocytes in vitro and in vivo.

Ubiquitin, a highly conserved 76-residue protein found in all eukaryotic cells, can be covalently bound to a wide variety of proteins in the nucleus, cytosol, cytoskeleton, and plasmalemma. This diversity of target proteins reflects a diversity of functions for ubiquitin conjugation. Previous studies have showed enhanced localization of ubiquitin conjugates to Z-bands of normal skeletal muscle and increased ubiquitination in atrophic muscles. These results have implicated a ubiquitin-mediated pathway in protein turnover and degradation in striated muscle. To investigate whether such a pathway might also exist in cardiac striated muscle, we used an affinity-purified polyclonal antibody (conjugate specific) and indirect immunofluorescence to localize ubiquitin conjugates in neonatal and adult rat cardiac myocytes both in vitro and in vivo. In both cultured myocytes and heart tissue, fluorescent ubiquitin conjugates were found in the nucleus as aggregates, in the cytoplasm in a striated pattern indicative of Z-bands, and in intercellular junctions at the intercalated discs between myocytes. Although the acceptor proteins and the physiological significance of ubiquitination at these locations are unknown, the targeting of ubiquitin to specific sites within the nucleus, myofibrils, and sarcolemma could provide a means for selective processing of individual components within these larger macromolecular assemblies, thus implying a regulatory role for ubiquitin conjugation in turnover or stability of proteins in the heart.

Animals↗

PubMed1992

Ultrastructural localization of laminin on in vivo embryonic, neonatal, and adult rat cardiac myocytes and in early rat embryos raised in whole-embryo culture.

The temporal and spatial distribution of the basement membrane component laminin was examined in vivo in developing rat hearts at 11.5 and 15 days of embryonic development (ED), and in neonates and adults, by pre-embedding ultrastructural immunocytochemistry. In addition, the patterns observed at 11.5 days ED were compared to the distribution of laminin in embryos maintained in whole-embryo culture. At 11.5 days ED laminin was localized in punctate patches on the surface of the plasma membrane, with large gaps between areas of staining. The development of myocytes and localization of laminin in the whole embryo-cultured embryos was similar to that found in the in vivo embryos. At 15 days ED, laminin localization was limited to distinct patches of developing extracellular matrix material associated with the sarcolemma. Gaps between areas of localization were shorter than in the 11.5-day hearts. In neonates, distribution of laminin localization was more extensive with fewer gaps and was associated with the developing basement membrane. In adult hearts, laminin was localized along the entire length of the basement membrane and was heaviest in areas of morphological specialization, such as Z-bands, where collagen bundles contacted the sarcolemma.

Animals↗

PubMed1991

Application of confocal laser scanning microscopy to the deep pineal gland and other neural tissues.

The study of the deep pineal gland of the Mongolian gerbil and other neuronal tissue from the rat by means of confocal laser scanning microscopy (CLSM) is described. Opical serial sectioning was performed on thick (100-200 microns) sections of the deep pineal gland of the Mongolian gerbil stained immunohistochemically using antisera to S-antigen and tyrosine hydroxylase (TH). Both dual-stained and single-stained material was examined using the fluorochromes fluorescein isothiocyanate (FITC) and Texas Red. High resolution images were obtained showing that pinealocytes have 1-3 processes that extend primarily to other pinealocytes or presumptive pinealocytes. Pinealocytes are located within the deep pineal gland as well as adjacent to the posterior aspect of the medial habenular nuclei. Pinealocyte processes were not seen extending into the habenular nuclei, but rather ended within the deep pineal gland a significant distance from their perikarya. The TH-immunopositive fibers were distributed throughout the deep pineal gland, often forming "baskets" of fibers around pinealocytes rather than being associated primarily with blood vessels. Other uses of the confocal microscope are demonstrated on rat neural tissue reacted with peroxidase/diaminobenzidine (DAB) immunohistochemistry and FITC fluorescence immunohistochemistry (paraventricular nucleus) as well as Golgi-stained neuronal tissue (cerebral cortex). The HRP/DAB and Golgi-stained images were visualized using the reflected image mode of the confocal system.(ABSTRACT TRUNCATED AT 250 WORDS)

3,3'-Diaminobenzidine↗

PubMed1991

Myofibrillar and cytoskeletal assembly in neonatal rat cardiac myocytes cultured on laminin and collagen.

Neonatal rat cardiomyocytes were cultured on extracellular matrix components laminin and collagens I + III to examine effects of extracellular matrix on the assembly of cytoskeletal proteins during myofibrillogenesis. Myofibril assembly was visualized by immunofluorescence of marker proteins for myofibrils (f-actin for I bands and alpha-actinin for Z bands), focal adhesions (vinculin), and transmembrane extracellular matrix receptors (beta 1 integrin) as cells spread for various times in culture. By 4 h in culture, f-actin appeared organized into nonstriated stress-fiber-like structures while alpha-actinin, vinculin and beta 1 integrin were localized in small streaks and beads. Subsequently, striated patterns were observed sequentially in the intracellular cytoskeletal components alpha-actinin, vinculin, f-actin, and then in the transmembrane beta 1 integrin receptor. These data support an earlier model for sarcomerogenesis in which stress-fiber-like structures serve as initial scaffolds upon which alpha-actinin and then vinculin-containing costameres are assembled. This sequential and temporal assembly was the same on both laminin and collagens I + III. A quantitative difference, however, was apparent on the 2 matrices. There was an increased appearance on collagens I + III of rosettes (also called podosomes or cortical actin-containing bodies in other cells) which consisted of an f-actin core surrounded by alpha-actinin, vinculin and beta 1 integrin rims. The increased incidence of rosettes in neonatal myocytes on collagens I + III suggests that these cytoskeletal complexes are involved in recognition and interaction with extracellular matrix components.

Actins↗

PubMed1991

Intracranial self-stimulation motivates weight-lifting exercise in rats.

The purpose of this study was to determine the feasibility of using a positive reinforcement protocol to motivate weight-lifting exercise in rats. Intracranial self-stimulation was used to induce weight-lifting exercise. Bipolar electrodes were implanted in the ventral tegmental area of rats, and the animals were trained to bar press on a continuous reinforcement schedule for electrical brain stimulation. Animals with response rates of 1,200-1,500 presses/h were then trained with a discriminative light stimulus to alternate between a normally positioned bar and an elevated bar that could be reached only by standing on the hindlimbs. The animals were fitted with a weighted jacket at a starting resistance of 5-10% of their body weight. Weight-training sessions were conducted 5 days/wk for 10 wk. Training consisted of 600 presses/session, alternating every 15 presses between the low and high bars. At the beginning of each subsequent week, the resistance was progressively increased, with some animals eventually training at resistances greater than 50% of their body weight. At the end of the training period, the rats were lifting over 550% of the starting weight. Gastrocnemius size and mean fiber diameter were increased in the weight-lifting animals. This model combines exercise with positive incentive and has the advantages of being relatively easy to implement and not producing any apparent physical or mental trauma in the animal.

Animals↗

PubMed1991

Expression of collagen binding integrins during cardiac development and hypertrophy.

The interaction between components of the extracellular matrix and the cell surface of cardiac myocytes appears to be regulated in part by receptors belonging to the integrin superfamily. The expression of the integrins was investigated at different stages of development of the heart as well as during cardiac hypertrophy. The characterization of the membrane proteins showed that a beta 1-integrin and associated alpha-chains were responsible for the interaction with collagen, laminin, and fibronectin. Immunoprecipitation data indicated that the presence of specific alpha-chains varied with development. These data were correlated with the ability of the isolated myocytes to attach to specific components of the extracellular matrix. The expression of the alpha 1-chain was prominently associated with the recognition of interstitial collagens. The presence of the alpha 1-chain was also associated with stages when collagen synthesis was increased, especially during fetal and neonatal growth and cardiac hypertrophy. Immunohistochemical localization with the antiserum against beta 1-integrin demonstrated its specific localization near the Z lines of cardiac myocytes. The localization both in vitro and in vivo indicated that the beta 1-integrin may play a role in myofibrillogenesis during development. The present immunohistochemical, cell adhesion, and biochemical data clearly indicate that integrins play a major role in the regulation of the interaction between cardiac myocytes and the extracellular matrix during development and disease.

Animals↗

PubMed1991

Collagen expression in mechanically stimulated cardiac fibroblasts.

The cardiac extracellular matrix, composed predominantly of collagenous fibers, forms a stress-tolerant network that facilitates the distribution of forces generated in the heart and provides for proper alignment of cardiac myocytes. Although considerable information exists regarding the morphological organization of the heart extracellular matrix, little is known about the regulation of the synthesis and accumulation of extracellular matrix components. A potentially significant factor in the cardiovascular system is mechanical stimulation including changes in physical tension and pressure. We recently have developed an in vitro model system to elucidate the effects of mechanical stretch on isolated populations of heart cells. In the present study, we have used biochemical and molecular biological techniques to analyze changes in collagen synthesis by cardiac fibroblasts in response to mechanical stretch. These studies show that the ratio of collagen type III to collagen type I increases in mechanically stretched cells. They also show that type III collagen mRNA levels are increased in response to cyclic mechanical stretch for durations as short as 12 hours. Type I collagen mRNA levels were not found to change under the stretch conditions used in this study. Our results emphasize the potential regulatory role of mechanical stimulation in the expression of specific genes in the heart and support previous studies indicating this to be an intriguing in vitro model of cardiac hypertrophy.

Animals↗

PubMed1991

Demonstration by confocal microscopy that unliganded overexpressed glucocorticoid receptors are distributed in a nonrandom manner throughout all planes of the nucleus.

Mouse glucocorticoid receptors (GR) that are over-expressed in Chinese hamster ovary (CHO) cells behave like progesterone receptors, in that the unliganded receptor localizes to the nucleus where it resides in a loosely bound docking complex, probably in association with the 90-kDa heat shock protein (hsp90) and hsp70. In this paper we examine the localization of the overexpressed GR within the CHO cell nucleus by confocal microscopy. In hormone-free cells the receptor distributes in a mottled pattern throughout all planes of the nucleus. The receptor is not present in nucleoli and shows no preferential localization in the periphery vs. the center of the nucleus. The mottled distribution in each plane of the nucleus demonstrates clearly that there are regions that do not contain receptor; thus, the distribution of the GR is not random. When triamcinolone acetonide is added to the CHO cells, there is no detectable change in receptor distribution. Overexpressed receptors that have either no hormone-binding activity or no DNA-binding activity because of point mutations localize in the same mottled pattern as the wild-type receptor. These observations are consistent with the proposal that the overexpressed GR can enter the nucleus in its unliganded state and proceed to loci distributed throughout the nucleus, where it is retained in an inactive docking complex until the binding of hormone triggers its progression to high affinity sites where the primary events in transcriptional activation occur. As there is no detectable change in localization with the addition of ligand, we suggest that the docking complex may be located very near or possibly at the site where the primary events in transcriptional activation occur.

Animals↗

PubMed1991

Characterization of platelet-derived growth factor beta-receptor expressing cells in the vasculature of human rheumatoid synovium.

Platelet-derived growth factor (PDGF) beta-receptor expression in normal and rheumatoid synovia was investigated by double immunofluorescence staining of frozen sections and by in situ hybridization. In the inflamed synovia, PDGF beta-receptor mRNA was present in vascular cells, as well as in discrete stromal cells. PDGF beta-receptor expressing cells in rheumatoid synovia were characterized by double immunofluorescence staining using the PDGFR-B2 monoclonal antibody at a concentration at which this antibody merely stained granular accumulations of PDGF beta-receptors. Granular accumulations of PDGF beta-receptors were articulate in blood vessel cells, but also appeared in discrete stromal cells. Thus, the overall distribution of cells having granular accumulations of PDGF beta-receptors was similar to the distribution of cells expressing PDGF beta-receptor mRNA. Double immunofluorescence stainings showed that: (a) a majority (greater than 90%) of resident macrophages did not express granular PDGF beta-receptor staining, but macrophages were often juxtaposed to PDGF beta-receptor-positive cells; (b) T lymphocytes did not express PDGF beta-receptors, but these cells were frequently found in the proximity of cells stained by PDGFR-B2; (c) in some blood vessels both HLA-DR expressing cells and PDGF beta-receptor expressing cells could be visualized, whereas in other blood vessels, cells expressing only one of these activation markers could be detected; (d) smooth muscle cells in blood vessels contained PDGF beta-receptors; and (e) capillary endothelial cells in the inflamed synovia recurrently displayed granular PDGF beta-receptor staining. The granular accumulations of PDGF beta-receptors may reflect internalization of the receptor as a result of paracrine or autocrine ligand stimulation. In support of such a possibility are the findings that elevated levels of PDGF B chain mRNA were detected by in situ hybridization in the inflamed synovia, and that cells expressing PDGF B chain mRNA were distributed similarly to cells expressing PDGF beta-receptor mRNA. Taken together, the results indicate that PDGF has a role in the inflammatory process in rheumatoid synovitis, most likely by stimulating proliferative events in the vasculature.

Arthritis, Rheumatoid↗

PubMed1990

A fluorescent residualizing label for studies on protein uptake and catabolism in vivo and in vitro.

Residualizing labels are tracers which remain in lysosomes after uptake and catabolism of the carrier protein and have been especially useful for studies on the sites of plasma protein degradation. Thus far these labels have contained radioactive reporters such as 3H or 125I. In the present paper we describe a fluorescent residualizing label, NN-dilactitol-N'-fluoresceinylethylenediamine (DLF). Modification of asialofetuin (ASF) or rat serum albumin (RSA) with DLF affected neither their normal kinetics of clearance from the rat circulation nor their normal tissue sites of uptake and degradation. After injection of DLF-ASF, fluorescent degradation products were recovered nearly quantitatively in liver and retained with a half-life of about 2 days. Fluorescent degradation products from DLF-RSA were recovered in skin and muscle, and were localized in fibroblasts by fluorescence microscopy. These results confirm previous studies with radioactive residualizing labels in which fibroblasts in peripheral tissues were identified as primary sites of albumin degradation. Fluorescent catabolites also accumulated in fibroblasts incubated with DLF-RSA in vitro, and residualized with a half-life of about 2 days. Overall, the data establish that DLF functions efficiently as a fluorescent residualizing label both in vivo and in vitro. The advantages of fluorescent, compared with radioactive, residualizing labels should make them valuable tools for studies on protein uptake and catabolism in biological systems.

Animals↗

PubMed1990

Distribution of vinculin in the Z-disk of striated muscle: analysis by laser scanning confocal microscopy.

Vinculin is a major cytoskeletal component in striated muscle, where it has been reported to form a rib-like structure between the cell membrane and the Z-disk termed a costamere. This arrangement of vinculin has been purported to be involved in the alignment of the myofibrils. However, the three-dimensional arrangement of vinculin in relation to the Z-disk of the myofibril was not known. In the present study, we examined the distribution of vinculin in striated muscle with monospecific antibodies using immunofluorescence and laser scanning confocal microscopy. Isolated cardiac and skeletal muscle cells from a variety of species, tissue sections, and neonatal myocytes with developing myofibrils were examined. Optical sectioning in the X-Y and X-Z planes demonstrated that vinculin immunoreactivity was heaviest at the periphery of the cell; however, the immunoreactivity was also distributed within the Z-disk although at a relatively reduced level. This distribution is potentially significant in understanding the physiological significance of vinculin in striated muscle function and in myofibrillogenesis.

Actinin↗

PubMed1990

Different beta 1-integrin collagen receptors on rat hepatocytes and cardiac fibroblasts.

Detergent extracts of primary rat hepatocytes and neonatal cardiac fibroblasts were applied to collagen type I-Sepharose in the presence of 1 mM MnCl2. Elution of bound proteins by 10 mM EDTA yielded one beta 1-integrin heterodimer from hepatocytes with an Mr of 180,000/115,000 under nonreducing conditions. Two beta 1-integrins with Mr's (nonreduced) of 180,000/115,000 and 145,000/115,000 could be isolated from surface-iodinated fibroblasts. A monoclonal antibody, 3A3, directed against the rat homolog of the human integrin VLA-1, precipitated the affinity-purified Mr 180,000/115,000 heterodimer, establishing the relatedness of the Mr 180,000 subunit to the alpha 1-chain of the beta 1-integrin subfamily. Both the alpha 1 beta 1-integrin and the 145,000/beta 1-integrin heterodimers bound specifically to Sepharose beads derivatized with the collagen fragment alpha 1(I) CB3, which lacks RGD sequences. Immunofluorescence staining using the 3A3 monoclonal antibody revealed that the rat alpha 1 beta 1-integrin was present at focal adhesion sites of fibroblasts grown on native collagen type I- but not on fibronectin-coated substrates, although both types of substrates supported the formation of beta 1-integrin containing focal adhesions. Similarly, hepatocytes cultured on substrata coated with collagen type I (but not fibronectin) were stained in a patchy pattern localized to the cell periphery by 3A3 IgG. Furthermore, 3A3 IgG completely inhibited the attachment of hepatocytes to collagen type I, whereas under identical conditions the attachment of fibroblasts to these substrates was inhibited only by approximately 40%. The attachment of both hepatocytes and cardiac fibroblasts to fibronectin was unaffected by the presence of the 3A3 antibody. Collectively these data show that a rat homolog of the human VLA-1 heterodimer both biochemically and functionally fulfills the criteria of a single collagen receptor on rat hepatocytes. In contrast, rat cardiac fibroblasts utilize two different collagen-binding integrins to adhere to collagen, one of which is the rat homolog of the human VLA-1 heterodimer. Furthermore alpha 1(I) CB3 contains cell binding sites for beta 1-integrins.

Animals↗

PubMed1990

Beta 1 integrin-mediated collagen gel contraction is stimulated by PDGF.

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent beta 1 integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-beta 1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin beta 1-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-beta 1 integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the beta 1 integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.

Actins↗