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J T Mahan

Publications and source records attributed to J T Mahan.

At least 19 recordsLinked to original sources
PubMed1995

Integrin and phosphotyrosine expression in normal and migrating newt keratinocytes.

BACKGROUND: Cells interact with the extracellular matrix through a family of cell surface receptors known as integrins. Ligand specificity of a given integrin is determined in part by the type of alpha and the type of beta subunit comprising it. Accumulating evidence suggests that integrin-ligand binding in some systems influences cell behavior through tyrosine phosphorylation of intracellular proteins. METHODS: In this study, we utilized immunohistochemistry to examine the expression of beta 1 and beta 4 integrin subunits as well as tyrosine phosphorylation in normal keratinocytes and in keratinocytes migrating to form a wound epithelium. An adhesion assay was used to determine if freshly isolated keratinocytes could interact with fibronectin and collagen. Polyacrylamide gel electrophoresis followed by immunoblotting was employed to compare beta 1 integrins in migrating and nonmigrating keratinocytes. RESULTS: In normal epidermis, beta 1 and beta 4 localized primarily to basal cells, where both subunits were generally distributed over all parts of the cell periphery. Except for a modest presence in suprabasal cells and a minimal presence adjacent to the epidermal basement membrane, phosphotyrosine (ptyr) had a similar distribution. In migrating keratinocytes, beta 1, beta 4, and ptyr localized most heavily at the interface between the forming wound epithelium and the wound bed. Adhesion assays using keratinocytes from normal epidermis revealed a population of cells that could specifically adhere and spread on fibronectin and type I collagen. Immunoblots of beta 1 subunits from normal and migrating keratinocytes showed no increase in amount of beta 1, nor did the apparent size of beta 1 change in migrating compared to normal cells. CONCLUSIONS: The heavy accumulation of beta 1 and beta 4 at the wound bed interface in migrating cells suggests that these subunits may be involved in attachments of migrating cells to extracellular matrix proteins in the wound. The accumulation of ptyr in the same region further suggests that integrin-ligand interaction in keratinocytes modulates cell behavior through phosphorylated proteins. The fact that freshly isolated newt keratinocytes could adhere and spread on fibronectin or collagen shows that these cells are constitutively activated. This view is supported by the absence of any evidence that the beta 1 in migrating keratinocytes is larger and therefore more mature than beta 1 in normal keratinocytes. By comparison, beta 1 integrins on human keratinocytes are not constitutively activated (Takashima and Grinnell, 1985; Toda et al., 1987; Guo et al., 1990, 1991), a difference that may explain why epidermal wound healing is faster in newts than in humans.

Animals↗
PubMed1994

Migratory interaction of amphibian epidermal cells with components of the basement membrane.

In adult newts, basal epidermal cells adjacent to a fresh wound move toward the damaged area by migrating over the epidermal basement membrane. In an attempt to determine which basement membrane components mediate this migration, small pieces of glass coated with various natural matrices, purified proteins, or fragments of proteins were implanted into skin wounds such that epidermal cells attempting to form a wound epithelium would encounter the implants. Laminin derived from a cell line (M1536-B3) that produces no type IV collagen was inactive as a migration substrate. Migration on recombinant entactin was somewhat better than on laminin but was still only approximately 14% of that on type I collagen. M15 matrix, a laminin and entactin-containing product of M1536-B3 cells, was no better than entactin alone. Type IV collagen was an excellent substrate, producing slightly more migration than corresponding concentrations of type I collagen at nearly all concentrations tested. Migration on type IV lacking the NC1 domain was at least as good as on intact type IV. All the activity in type IV was present in a 95 kD fragment (alpha 1(IV)95) from the carboxy terminal two-thirds of the alpha 1 chain. Approximately 60% of the activity on alpha 1(IV)95 was obtained on implants coated with a 110 amino acid fragment of the alpha 1 chain derived from the carboxy terminal half of alpha 1(IV)95. Adding the synthetic peptide, arg-gly-asp-ser (RGDS) to the medium, blocked migration on fibronectin-coated implants but had no effect on implants coated with type IV, suggesting that migration on type IV involves different cell surface receptors than those mediating migration over fibronectin. Matrigel, a commercial product containing most basement membrane components, was a poor migration substrate. Thus if type IV mediates basal cell migration toward a wound in vivo, there may have to be some alterations in basement membrane structure to allow epidermal receptors to access type IV active site(s).

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PubMed1994

Further studies on the interaction of migrating keratinocytes with fibrinogen.

If glass implants placed under one edge of a skin wound in the adult newt are coated with fibrinogen (FGN), keratinocytes from the wound periphery migrate onto the implant. To learn more about the site(s) in FGN that permits this migration, we exposed keratinocytes to implants coated with forms of FGN containing modifications or deletions in the 3 most commonly studied cell binding sites; the RGDF sequence at A alpha 95-98, RGDS at A alpha 572-575 and the carboxy terminal 12 amino acids in the gamma A chain. Recombinant FGN with either RGD sequence altered to RGE supported migration as well as unmodified FGN did. Replacement of the carboxy terminal 4 amino acids in the gamma A chain by a 20 amino acid sequence that disrupts the ability of the gamma terminus to mediate platelet aggregation (the gamma' variant) likewise had no effect. Nor did simultaneous antibody blockade of the RGDS, RGDF, and gamma A sites have any effect. At its best, Dhem1, a fragment containing the RGDS and gamma A sites, produced only about half as much migration as the maximum obtained on intact FGN. Dhem2, a fragment differing from Dhem1 only by having a gamma' variant in place of gamma A, was even less active. Two other D fragments, both of which were missing a large part of the A alpha chain, and one of which contained none of the three major binding sites, supported considerable migration, suggesting that loss of the A alpha chain COOH terminus reveals a site that was not exposed in Dhem1 and 2. A alpha chain fragments containing the RGDF or RGDS sequence were active, but a much larger fragment without RGD was inactive. A soluble peptide consisting of the sequence, RGDS, was a potent inhibitor of migration on FGN but RGDF and the gamma A pentapeptide, KQAGD, were minimally effective. Longer versions of these peptides decreased the effectiveness in all cases. These results suggest that under certain circumstances, newt keratinocytes may interact with each of the 3 major binding sites in FGN as well as a site outside these sequences.

Amino Acid Sequence↗
PubMed1992

Divalent cations and extracellular matrix receptor function during newt epidermal cell migration.

Skin explants were placed in plastic dishes coated with fibronectin (FN), fibrinogen (FGN) or collagen. Explants were cultured for 16 h in serum-free medium containing calcium (Ca) and magnesium (Mg), or in medium containing either Ca-only, Mg-only or manganese (Mn)-only. In Ca/Mg, migrating keratinocytes on all test substrata produced a sheet of contiguous cells that formed a robust halo around each explant. When Ca was the only divalent cation added, the halos in FN- and FGN-coated dishes were approximately 70-80% as large as in Ca/Mg. On collagen, however, the halos were significantly smaller than on the other two substrata. This substratum-specific response in Ca-only suggests that migration on collagen is fundamentally different than migration on FN and FGN. Halos as large or larger than those in Ca/Mg formed on all three substrata in Mg-only. In this case, the halos were not in the form of a sheet of contiguous cells, but were composed of dissociated cells that had migrated from the explant. Individual cells likewise migrated from explants cultured in medium containing Mn-only; however, these halos were never as large as in Mg-only. Thus, while exogenous Ca appears to be an absolute requirement for maintenance of cell-cell connections, the cell-substratum interactions that lead to migration can utilize either Ca, Mg or, to a lesser extent, Mn. Additionally we found that migration on the generally nonpermissive protein, BSA, was not improved by the presence of Mn.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1991

Tenascin localization in skin wounds of the adult newt Notophthalmus viridescens.

Earlier studies have shown that the extracellular matrix (ECM) protein tenascin (TN) is present between uninjured epidermal cells of urodele appendages, but is absent from most of the mesenchymally derived ECM. Following appendage amputation, this distribution is reversed. TN is lost from the epidermis and appears in the ECM of the stump and the regeneration blastema. In the present study, monoclonal and polyclonal antibodies to TN were used to localize this protein immunohistochemically in limbs of the adult urodele Notophthalmus viridescens at various stages following skin removal with or without damage to underlying muscle to determine 1) if the loss of TN by the epidermis and its gain by mesenchymal tissues occurs in wounds that do not require regulation by epigenetic mechanisms, and 2) if TN is present in the provisional wound matrix beneath migrating epidermal cells. In addition, skin explants were cultured on TN-coated dishes to learn if TN possesses active sites that can support epidermal cell migration. The results indicate that simple wounding leads to the same TN patterns as occurs following limb amputation. Tenascin loss from the epidermis could be seen as early as 6 hr after wounding, a time during which migrating epidermal cells are moving over the wound bed. During this period, there was no evidence of TN in the provisional wound matrix. In contrast to collagen, which supports considerable epidermal cell migration from skin explants, TN allowed no more migration than did the inactive protein, myoglobin.

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PubMed1989

Fibrinogen-mediated epidermal cell migration: structural correlates for fibrinogen function.

Previously we showed that epidermal cells are able to use fibrinogen (FGN) as a migration substratum during wound closure. The goal of the present study was to determine the structural features of FGN that allow this migration. Pieces of glass coated with native, fragmented, or other modified forms of FGN were implanted into full-thickness skin wounds of adult newts such that migrating epidermal cells would encounter the implant. In this system, a coating of FGN allowed considerably more migration than a coating of BSA. At high concentrations, heat-denatured FGN supported as much migration as the same amount of intact FGN. Fraction I-9, a circulating form of FGN missing a 20-30K (K = 10(3) Mr) carboxy-terminal segment of the A alpha chain, was no less effective than intact FGN. Comparison of the isolated D1 and E fragments of FGN showed migration only on D1, but never to the extent seen on intact FGN containing the same amount of D1. Plasmin digestion of D1 in the presence of EDTA, a process which produces D3, a fragment differing from D1 by the loss of the carboxy-terminal 109 amino acids of the gamma chain, caused a significant loss of activity in the D fragment. Migration was good on implants coated with relatively high concentrations of purified A alpha chains but gamma chains were inactive. Migration over intact FGN was almost totally blocked by 230 microM-Arg-Gly-Asp-Ser (RGDS), a peptide known to interact with integrin-type receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

A serum-free primary culture system for studying cell-substrate interactions during newt epidermal cell migration.

To study the interaction of migrating newt epidermal cells with purified extracellular matrix (ECM) molecules we have developed an in vitro migration assay using pieces of newt skin explanted onto culture dishes coated with various ECM molecules and cultured for 18 h in defined serum-free medium. Newt epidermal cells migrate out from explants placed on dishes coated with either collagen, vitronectin, fibronectin, or fibrinogen but not on albumin-coated or uncoated dishes. Explant outgrowth on collagen was best in CEM 2000 medium diluted to 60% of mammalian osmolarity. Other media such as RPMI 1640 or Ex-Cell 300, diluted similarly, may also be used although in our hands CEM 2000 always allowed more migration. We found no migration on collagen when skin explants were incubated in Holtfreter's solution (an amphibian saline solution that we have previously shown allows reepithelialization on amputated newt limbs). Supplementation of Holtfreter's solution with glucose did not improve its ability to support migration. By testing various supplement combinations in conjunction with CEM 2000 and RPMI 1640 we found that neither serum, insulin, selenium, transferrin, nor L-glutamine is required for explant outgrowth. Of the additives tested, outgrowth was stimulated only by insulin. Epidermal cell outgrowth on collagen was inhibited by both puromycin and cycloheximide, indicating the necessity for protein synthesis in this system. Whether the effects of these protein synthesis inhibitors are specifically on migration-related events or on general metabolic requirements is not clear. Inasmuch as there was no correlation (r = -0.227) between DNA synthesis (measured by incorporation of tritiated thymidine) and the amount of outgrowth, we believe that our assay is a measure of cell migration alone rather than a combination of mitosis and migration. This explant outgrowth system represents a new and relatively simple assay that can be used in the study of cell-substrate interactions during newt epidermal cell migration over extracellular matrix molecules in a defined serum-free environment.

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PubMed1988

Newt epidermal cell migration over collagen and fibronectin involves different mechanisms.

Effects of the synthetic peptides, Arg-Gly-Asp-Ser (RGDS), the amino acid sequence representing the fibroblast attachment site in fibronectin, and Arg-Gly-Glu-Ser (RGES), on collagen- and fibronectin-mediated migration in newt epidermal cells were compared. When RGDS at 50 micrograms ml-1 was included in the incubation medium of skin explants, migration in fibronectin-coated dishes was almost totally blocked. In type I collagen-coated dishes, this concentration of RGDS also inhibited migration, but to a lesser degree than on fibronectin. With 250 micrograms ml-1 of RGES in the medium, the reverse was true. Here, migration on collagen was practically non-existent, while migration on fibronectin was affected only moderately. Collagen-mediated migration was sensitive to RGDS even when the peptide was added after migration on the coated substratum was well underway. At a coating concentration of 10 micrograms ml-1 CB3, a cyanogen bromide fragment of the collagen alpha 1(I) chain, which contains no RGD sequences, was as good a migration substratum as intact collagen applied at the same coating concentration. At lower concentrations intact collagen was somewhat better than equivalent concentrations of CB3. The presence of RGDS in the medium throughout an experiment inhibited migration in CB3-coated dishes in a manner similar to its effect in dishes coated with collagen. On both substrata there appeared to be a peptide-sensitive and a peptide-insensitive component to migration. The inhibitory effect of RGES on CB3-mediated migration was also similar to its effect in collagen-coated dishes.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1987

Role of carbohydrates in cell-substrate interactions during newt epidermal cell migration.

The effect of several solubilized monosaccharides on epidermal cell migration from skin explants of the adult newt was examined. The ability of epidermal cells to migrate on substrates coated with these same sugars or with wheat germ agglutinin (WGA) was also determined. Adding 0.05 M N-acetyl-glucosamine (GlcNAc) to the medium inhibited epidermal cell migration in dishes coated with either type I collagen or fibrinogen. The same concentration of fucose, galactose, or mannose had no effect. In contrast to type I collagen, which supported considerable migration when dried onto the bottom of plastic dishes, epidermal cells were unable to migrate on dishes coated similarly with WGA, mucin (a protein high in sialic acid residues), or bovine serum albumin (BSA) conjugated to galactose, mannose, or GlcNAc. Red blood cell (RBC) binding assays showed that drying WGA onto plastic did not destroy its GlcNAc binding sites--nor did it damage the GlcNAc residues of BSA-GlcNAc. The RBC assay also verified that for both these proteins, substrates with distinctly different cell binding capacities had been tested in the migration experiments. In dishes coated with either WGA or BSA-GlcNAc, red cells bound to dish bottoms in a GlcNAc-specific manner right up to the margins of explants. Other control experiments showed that the failure of migration in WGA- and BSA-GlcNAc-coated dishes could not be explained by competition between adsorbed and desorbing protein for cell surface receptors. This work shows that adhesive bonds between epidermal cell surface GlcNAc and a GlcNAC-specific lectin on the substrate are not by themselves adequate to support cell migration. Nor is GlcNAc, sialic acid, galactose, or mannose alone on the substrate sufficient. In conjunction with our earlier work (Donaldson and Mahan: J. Exp. Zool., 231:211-219, '84; Donaldson, Mahan, Hasty, McCarthy, and Furcht: J. Cell. Biol., 101: 73-78, '85), these observations suggest that factors other than carbohydrate content or capacity to act as a lectin determine whether a given extracellular protein will support migration.

Animals↗
PubMed1987

Newt epidermal cell migration in vitro and in vivo appears to involve Arg-Gly-Asp-Ser receptors.

The effect of a synthetic peptide consisting of Arg-Gly-Asp-Ser (RGDS), the amino acid sequence representing the fibroblast attachment site in fibronectin (FN), was tested on migrating newt epidermal cells. In one approach, skin explants were placed on the bottom of plastic dishes coated with human FN, human fibrinogen (FGN), human serum spreading factor (SF), or bovine type I collagen. The explants were then incubated overnight in serum-free medium with or without RGDS. In these experiments exposure to 50 micrograms ml-1 of RGDS reduced migration over FN, FGN and SF to 2-7% of control levels. Two peptides structurally dissimilar to RGDS (Val-Gly-Ser-Glu and Thr-Pro-Arg-Lys), and two that are structurally similar (Lys-Gly-Asp-Ser and Arg-Gly-Glu-Ser), had no effect on explant migration even when used at concentrations higher than 50 micrograms ml-1. Upon removal of the RGDS peptide, inhibited explants quickly recovered. In collagen-coated dishes 50 micrograms ml-1 of RGDS was much less effective than in dishes coated with the other substrates. Raising the RGDS concentration in collagen-coated dishes tenfold did not greatly increase the RGDS effect. When added to the medium bathing wounded limbs, 50 micrograms ml-1 of RGDS only moderately inhibited wound closure. This concentration of peptide, however, severely inhibited migration from skin explants in newt-plasma-coated-dishes and migration over pieces of newt-plasma-coated plastic placed under one edge of a skin wound. Increasing the RGDS concentration to 500 micrograms ml-1 resulted in almost total suppression of wound closure. Wounds exposed to this same concentration of Lys-Gly-Asp-Ser closed normally. These results indicate that newt epidermal cells possess RGDS receptors and that these receptors are involved in epidermal wound closure in vivo and in migration from skin explants onto plastic coated with FN, FGN, SF and collagen. The relative RGDS-insensitivity of wound closure in vivo and in migration from explants onto collagen may reflect in these instances the presence of a relatively high density of RGDS receptor binding sites on the substrate; the presence of RGDS receptor binding sites of relatively high affinity; or the participation of receptors other than those involved in migration over plastic coated with FN, FGN or SF.

Amino Acid Sequence↗
PubMed1986

Events in the movement of newt epidermal cells across implanted substrates.

Pieces of coverslip glass, polycarbonate filters, or coverslip plastic, coated with fibrinogen or type I collagen, were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Migratory events were then analyzed by phase contrast and electron microscopy. Phase-contrast microscopy revealed two types of lamellipodia on leading edge cells: one which was attached broadly to the cell body and one attached by a long, thin stalk. Stalkless forms were by far the most common type and we believe they provide the motive force for cell movement. Stalked-forms often moved at distinct angles to the direction of sheet movement, suggesting that they may be sensory appendages. Phase photographs of the leading edge of migrating sheet 4 hours and 8 hours after implantation showed that all cells that were on the leading edge at 4 hours continued to advance for the next 4 hours, demonstrating clearly that under these circumstances the distalmost cells do not become immobile upon contact with the substrate as others have suggested. TEM revealed that migrating sheets were modified monolayers and that regardless of proximodistal location in the sheet, and even in the intact skin adjoining a wound, each epidermal cell adjacent to the substrate puts forth a lamellipodium which underlaps the cell in front. This and the behavior of sheets as they were teased or pulled from the implant suggest strongly that all basal cells contribute to movement of the sheet by interacting with the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Lectin binding to newt epidermis: fluorescent localization and effects on motility.

The ability of seven lectins to bind to newt epidermal cells and influence their motility was examined. Of the seven fluoresceinated lectins applied to frozen sections containing intact newt skin and migrating epidermis (wound epithelium), only Con A (concanavalin A), WGA (wheat germ agglutinin), and PNA (peanut agglutinin) produced detectable epidermal fluorescence. Con A and WGA each heavily labeled all layers of intact epidermis, but PNA bound only to the more superficial layers. In contrast to a single population of labeled cells in migrating epidermal sheets after treatment with Con A, there were both labeled and unlabeled cells after exposure to either WGA or PNA. The wound bed was labeled by both Con A and WGA, but not by PNA. DBA (Dolichos bifloris agglutinin), RCA I (Ricinus communis agglutinin), and UEA (Ulex europaeus agglutinin), did not produce significant fluorescence with either migrating or intact epidermis. In general, inhibitory effects on epidermal motility correlated with the binding studies. Thus, Con A, WGA, and PNA, the lectins which clearly bound to the epidermis, all produced a concentration-dependent depression in the rate of epidermal wound closure. RCA was somewhat paradoxical in that it was moderately inhibitory despite showing essentially no binding. The effects of SBA and UEA were equivocal. DBA had no effect. These results indicate that the inhibition of motility produced by Con A that we have described previously is not peculiar to this mannose-binding lectin, but is shared by at least one lectin with an affinity for D-GlcNAc (WGA), and one with an affinity for B-D-Gal(1-3)-D-GalNAc (PNA).(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1985

Location of a fibronectin domain involved in newt epidermal cell migration.

The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronectin, was studied. Pieces of nitrocellulose coated with intact human plasma fibronectin or proteolytically derived fragments were implanted into wounded limbs so that the coated nitrocellulose served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Epidermal cells migrated very poorly on nitrocellulose pieces coated with (a) a 27-kD amino-terminal heparin-binding fragment, (b) a 46-kD gelatin-binding fragment, (c) a combined 33- and 66-kD carboxy-terminal heparin-binding preparation representing peptide sequences in the A and B chains, respectively, or (d) a 31-kD carboxy-terminal fragment from the A chain, containing a free sulfhydryl group. In contrast, epidermal cells readily migrated onto nitrocellulose coated with a mixture of fragments from the middle of the molecule (80-125kD) that bind neither heparin nor gelatin. Attempts to block migration on fibronectin-coated nitrocellulose using IB10, a monoclonal antibody that blocks Chinese hamster ovary cell attachment to fibronectin, were unsuccessful despite saturation of the epitope against which IB10 is directed. In contrast, a polyclonal anti-fibronectin antibody did inhibit migration. These results show that the ability of fibronectin to support newt epidermal cell migration is not shared equally by all regions of the molecule, but is restricted to a domain in the middle third. They also suggest that the site supporting migration is separate and distinct from the site mediating Chinese hamster ovary cell attachment.

Animals↗
PubMed1984

Inability of newt epidermal cells to migrate over concanavalin A-coated substrates.

Pieces of coverslip glass coated with various proteins were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Despite the fact that concanavalin A (Con A) receptors could be demonstrated on newt epidermal cells with fluorescein isothiocyanate (FITC)-conjugated lectin, Con A-coated implants supported practically no migration, an even poorer response than the modest amount of migration that occurred on uncoated glass. Coomassie blue staining verified that the lectin formed a complete film over the glass, and peroxidase binding assays showed that even after several hours in the wound, the Con A binding sites for mannose were still available. Migration on fibrinogen-coated glass (a good migration substrate) was not affected by placing the implants next to Con A-coated implants. Thus, the failure to migrate on Con A cannot be explained by soluble Con A effects from lectin leaching off the implants. These data suggest that linkages between cell surface mannose and the substrate are not part of the strategy by which newt epidermal cells migrate.

Animals↗
PubMed1984

Epidermal cell migration on laminin-coated substrates. Comparison with other extracellular matrix and non-matrix proteins.

Pieces of coverslip glass coated with various proteins were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for the migrating wound epithelium. Laminin, a protein that has been implicated as an epithelial-specific adhesin, was a moderately good migration substrate. Type-IV collagen, fibrinogen and fibronectin, however, were significantly better. Fetuin, myoglobin, and casein all proved to be very poor substrates, allowing practically no migration. The inability of fetuin, myoglobin, and casein to support migration is further evidence that the considerable migration that occurs on collagen (Donaldson et al. 1982) fibrinogen and fibronectin (Donaldson and Mahan 1983) and the moderate migration on laminin, is a relatively specific response to these proteins and is therefore of special significance. The fact that laminin is a poorer migration substrate than collagen, fibrinogen or fibronectin suggests that the absence of cell surface laminin that has been associated with epithelial movement in several studies (Stanley et al. 1981; Clark et al. 1982; Madri and Stenn 1982; Gulati et al. 1983) may promote motility by allowing epithelial cells to interact directly with other extracellular macromolecules.

Animals↗
PubMed1984

Influence of catecholamines on epidermal cell migration during wound closure in adult newts.

Following removal of a skin patch from each hind limb of a series of adult newts, the limbs were explanted into small dishes of Holtfreter solution containing various combinations of test drugs. Later, the amount of wound epithelium that formed on each limb was determined using a planimeter on wound tracings obtained with the aid of a drawing tube-equipped microscope. All three (-)-catecholamines tested inhibited migration with the following order of effectiveness: isoproterenol greater than epinephrine greater than norepinephrine. The effect was stereospecific with (-)-isoproterenol clearly more effective than the (+)-isomer. Propranolol, a beta antagonist, blocked the effect of (-)-isoproterenol while the alpha antagonist, phentolamine, was less effective. One hour in (-)-isoproterenol inhibited migration to the same degree as continuous exposure for the 6 hr most experiments were allowed to run. Taken together, the data suggest that catecholamines exert their effect on epidermal cell migration via beta 2 adrenergic receptors on the cell surface, a binding site which implicates cAMP in the chain of events.

Animals↗
PubMed1984

Effects of concanavalin A and cholera toxin on epidermal cAMP and migration rate during wound closure in adult newts.

Following removal of a skin patch from each hind limb of a series of adult newts, the limbs were explanted into small dishes of Holtfreter solution containing various combinations of test drugs. Later, the amount of wound epithelium that formed on each limb was determined using a planimeter on wound tracings obtained with the aid of a drawing tube-equipped microscope. Exposure of migrating cells to the plant lectin, concanavalin A (con A), lowered cyclic AMP (cAMP) levels and depressed migration. Exposure to cholera toxin and theophylline (CTX) significantly elevated cAMP levels and significantly depressed migration rate. Exposure of CTX-treated cells to con A tended to lower CTX-elevated cAMP levels while depressing the migration rate well beyond the depression caused by CTX alone. These results provide further evidence that cAMP can regulate the rate of newt epidermal cell migration. They also show that the inhibitory effect of con A on motility in these cells is independent of its effects on cAMP.

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