Cyclic AMP levels in migrating and non‐migrating newt epidermal cells

Dunlap, Mary K.

doi:10.1002/jcp.1041040310

Cited by 18 publications

(13 citation statements)

References 32 publications

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“…The asterisk denotes a band precipitated nonspecifically in all [ 355]methionínelabeled preparations by the Pansorbin treatment . 14C_ molecular weight markers include phosphorylase B (97,400), bovine serum albumin (29,000), ovalbumin (46,000), carbonic anhydrase (30,000), and lactoglobulin A (18,400 7 Anti-bone proteoglycan immunoprecipitate fluorogram (scan) of an SDS gel from media of cultured osteoblasts labeled with 35 504 . Both the 190,000-mol-wt peak (3-cm migration) and the minor peak at 1-cm migration are characteristic of the electrophoretic pattern for purified fetal bovine bone proteoglycans on this SDS gradient gel system (6) .…”

Section: Resultsmentioning

confidence: 99%

Fetal bovine bone cells synthesize bone-specific matrix proteins.

Whitson¹,

Harrison²,

Dunlap³

et al. 1984

The Journal of Cell Biology

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We isolated cells from both calvaria and the outer cortices of long bones from 3-to 5-mo bovine fetuses . The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type I Collagen . In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by imunoprecipitation and fluorography of SIDS polyacrylamide gels . Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (-94%) type I collagen, with small amounts of types III and V collagens . In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and 3-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen . These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro .Several criteria have been used to characterize cells isolated and cultured from bone as osteoblasts, although none have proved specific . These are that (a) freshly isolated bone cells possess high alkaline phosphatase activity (1), (b) bone cells show a strong cyclic AMP response to parathyroid hormone (2), (c) bone cells secrete predominantly type I collagen when grown in the presence of ascorbic acid (3), and (d) bone cells produce and partially mineralize a matrix, given extended time in culture and defined culture conditions (4-9).Recently, the isolation and purification of several noncollagenous matrix proteins from fetal calf bone (10) have provided new tools to identify bone cells with increased certainty . Antibodies against these proteins have been used to establish their tissue specificity in fetal calf bone (11)(12)(13). In this study we confirm the criteria listed above using bone cells obtained from the calvaria and long bones of fetal calves, the first nonrodent system to be explored . We show by indirect im-THE JOURNAL OF CELL BIOLOGY -VOLUME 99 AUGUST 1984 607-614 © The Rockefeller University Press -0021-9525/84/08/0607/08 $1 .00 munofluorescence and in biosynthetic experiments, that these cells produced three bone-specific, noncollagenous proteins ; osteonectin (11), the bone proteoglycan (12), and the bone sialoprotein (13), thus proving conclusively that this methodology can clearly identify bone cells in vitro. MATERIALS AND METHODSCulture Conditions : Fetal calves (3-5 mo in utero [8] and still in the fetal sac) were obtained from Schneider Packing Co ., S...

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Section: Resultsmentioning

confidence: 99%

Fetal bovine bone cells synthesize bone-specific matrix proteins.

Whitson¹,

Harrison²,

Dunlap³

et al. 1984

The Journal of Cell Biology

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“…Interestingly, Dunlap et al [1980] noted that cyclic AMP levels were higher in migrating, wounded newt epithelial cells compared to non-migratory epithelial cells and in human keratinocytes application of dibutyryl cAMP resulted in increased migration rates and up-regulation of metalloproteinase activity [Iwasaki et al, 1994], which may be important for keratinocyte migration [Woodley et al, 1986]. Furthermore, cAMP concentration gradients have been shown in and around cells of the migrating pseudoplasmodia of Dictyostelium discoideum [Brenner, 1977;Saran et al, 1994] and expression of the dominant inhibitory form of the regulatory subunit of PKA, Rm, partially inhibits migration, as well as development [Bonner and Williams, 1994;Harwood, et al, 1992].…”

Section: Discussionmentioning

confidence: 97%

Cyclic AMP‐dependent protein kinase A plays a role in the directed migration of human keratinocytes in a DC electric field

Pullar

Isseroff

Nuccitelli

2001

Cell Motil. Cytoskeleton

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Skin wound healing requires epithelial cell migration for re-epithelialization, wound closure, and re-establishment of normal function. We believe that one of the earliest signals to initiate wound healing is the lateral electric field generated by the wound current. Normal human epidermal keratinocytes migrate towards the negative pole, representing the center of the wound, in direct currents of a physiological strength, 100 mV/mm. Virtually nothing is known about the signal transduction mechanisms used by these cells to sense the endogenous electric field. To elucidate possible protein kinase (PK) involvement in the process, PK inhibitors were utilized. Two important findings have been described. Firstly, addition of 50 nM KT5720, an inhibitor of PKA, resulted in a 53% percent reduction in the directional response of keratinocytes in the electric field, while not significantly affecting general cell motility. The reduction was dose-dependent, there was a gradual decrease in the directional response from 5 to 50 nM. Secondly, addition of 1 microM ML-7, a myosin light chain kinase inhibitor, resulted in an approximate 31% decrease in the distance the cells migrated without affecting directional migration. The PKC inhibitors GF109203X at 4 microM and H-7 at 20 microM and W-7, a CaM kinase inhibitor, did not significantly alter either directed migration or cell migration, although they all resulted in a slight reduction in directional migration. D-erythro-sphingosine at 15 microM, a PKC inhibitor, had virtually no effect on either migration distance or directed migration. These findings demonstrate that divergent kinase signaling pathways regulate general cell motility and sustained directional migration and highlight the complexity of the signal transduction mechanisms involved. The inhibitor studies described in this paper implicate a role for PKA in the regulation of the directional migratory response to applied electric fields, galvanotaxis.

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“…Our data show that optimal migration requires PKA activity. Many chemoattractants elevate cAMP during migration [25], either directly or secondary to changes in cell shape [26], although there is a narrow range for which this increase is stimulatory for migration [12,13]. The regulation of these cAMP levels is very complex.…”

Section: Discussionmentioning

confidence: 99%