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W H Fletcher

Publications and source records attributed to W H Fletcher.

30 records · Page 2Linked to original sources
PubMed1985

Morphologic analysis of long-term bone marrow cultures that support B-lymphopoiesis or myelopoiesis.

A comparative morphological analysis of the Whitlock-Witte long-term B-cell culture and the predominantly myeloid Dexter long-term bone marrow culture demonstrates that similarities and differences exist between the two systems. Cells from the long-term B-cell cultures have a characteristic lymphoid morphology, whereas those from the Dexter cultures are predominantly granulocytes and macrophages along with a few undifferentiated blast cells. A multilayered stromal cell layer is a common feature of both systems. Scanning electron micrographs show the cells in this layer to be large, irregularly shaped and flattened. The data further indicate that there are unique features in the relationship between developing B cells and stromal cells in the long-term B-cell cultures. Large, mononuclear cells are present that have numerous membrane infoldings within which numerous lymphoid cells lie. The relationship of these cells to macrophages and epithelial/reticular cells is discussed.

Animals↗
PubMed1985

Intracellular kinetics of free catalytic units dissociated from adenosine 3',5'-monophosphate-dependent protein kinase in adrenocortical tumor cells (Y-1).

To define the role of cAMP in the actions of ACTH, the dissociation of cAMP-dependent protein kinase and the subsequent intracellular location of its free catalytic units were monitored after exposure of Y-1 cells to ACTH, FSH, or cyclic nucleotide analog. To accomplish this, a fluorescinated cytochemical probe was used that complexes specifically with free catalytic units from cAMP-dependent protein kinase. Also, the effects of hormone or nucleotide on secretion of fluorogenic steroids and DNA synthesis were examined. Y-1 cells dissociated protein kinase in a dose-dependent fashion when exposed to ACTH or cAMP analog, but did not respond to FSH, which was one of the control agents used. After 30 min of treatment with 1.5 X 10(-10) M ACTH, free catalytic units were observed only in the cytoplasm of Y-1 cells, whereas a similar time of exposure to 3 X 10(-10) M ACTH led to the appearance of catalytic units in nucleolus as well as in cytoplasm. ACTH (6 X 10(-10) M) caused a rise in cytoplasmic and nucleolar protein kinase dissociation proportionally greater than that seen in cultures exposed to 3 X 10(-10) M ACTH. Upon treatment with 6 X 10(-10) M ACTH, the amount of free catalytic units in cytoplasm and nucleolus was detectably greater than that in controls within 1 min of stimulation and continued to rise with increasing time of exposure to hormone. The nuclear, mostly nucleolar, content of free catalytic unit appeared to peak after 15 min of stimulation, while cytoplasmic enzyme levels continued to rise up to 60 min. Exposure of Y-1 cells to nucleotide analog caused cAMP-dependent protein kinase dissociation with temporal kinetics and a subcellular distribution similar to that seen after ACTH stimulation. We conclude that actions of ACTH are mediated by cAMP-dependent protein kinases. Further, there appear to be two intracellular pools of protein kinase, one nucleolar, the other cytoplasmic, and these may be independently regulated, with the nucleolar enzyme requiring higher concentrations of ACTH for dissociation than those needed for cytoplasm protein kinase. These observations may be relevant to the fact that more ACTH is required to inhibit DNA synthesis than is necessary to enhance steroid production.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Receptor mediated action without receptor occupancy.

The relationship between occupancy of a specific cell surface receptor and actions initiated in the same cell by that event was tested on ovarian granulosa cells exposed to hCG. Hormone bound to its receptor was identified by immunocytochemistry or by autoradiography and the dissociation of cAMP-dependent protein kinase was followed by direct cytochemistry. Only 25-30% of the granulosa cells bound hCG and in each instance this resulted in protein kinase dissociation. Cells that did not bind hCG nevertheless dissociated protein kinase if they contacted a cell that had bound hormone and dissociated enzyme. Cells that neither bound hormone, nor contacted cells that did so, failed to dissociate protein kinase. These observations establish, in individual cells, a direct relationship of receptor occupancy to receptor-mediated action and indicate that this event can be communicated to receptorless cells, presumably by gap junctions, thereby amplifying the response to hormone. Similar processes may occur in other tissues wherein receptor-bearing cells are capable of intercellular communication.

Animals↗
PubMed1985

Intracellular localization of the catalytic subunit from cyclic AMP-dependent protein kinase in mutant Chinese hamster ovary cells deficient in this enzyme.

A direct cytochemical procedure that specifically locates free catalytic subunits (C) from cAMP-dependent protein kinase has been used to follow the kinetics of kinase dissociation in parental Chinese hamster ovary cells (CHO 10001) and 4 mutant CHO cell lines variously deficient in this enzyme (CHO 10215, 10248, 10260, 10265) (1-5). When cultures of wild-type cells were stimulated with 8BrcAMP a time- and dose-dependent dissociation of kinase was observed. The catalytic unit appeared first in the cytoplasm and nucleolus and with time in the nucleoplasm as well. At peak protein kinase activation (30 min) more than 80% of the cells possessed abundant C in all of these subcellular compartments. The data indicate that both the type I and type II isozymes of the cAMP-dependent protein kinase are localized in similar areas of the cell. Stimulation of the mutant cell lines with 8-BrcAMP revealed that they each contained activatable kinase, determined cytochemically, that paralleled in amount the total assayable protein kinase determined biochemically (1-6). There were differences in the basal (unstimulated) levels of free C in the mutants relative to each other and to wild-type cells. For example, wild-type and mutants 10248, and 10260 had barely detectable cytoplasmic catalytic-subunit in unstimulated cultures whereas mutant 10215 possessed a significant amount of free C. Upon stimulation with 8-BrcAMP, the subcellular distribution of C was in all cases similar; although there were significant quantitative disparities between the wild type and various mutant cell lines. All cell lines examined had roughly equivalent amounts of nucleolar C but differed predominantly in the quantity of cytoplasmic kinase. Specifically, two of the mutant lines (10260 and 10248) contained barely detectable amounts of nucleoplasmic C whereas the other mutants had moderate (10265) to near normal (10215) amounts of nucleoplasmic enzyme. Mutants having only type I isozyme (10248) and those with only type II isozyme (10265) gave similar patterns of free C localization after 8BrcAMP stimulation.

Animals↗
PubMed1984

Hormone-induced intercellular signal transfer dissociates cyclic AMP-dependent protein kinase.

We used co-cultures of porcine ovarian granulosa cells and mouse adrenocortical tumor cells (Y-1) to examine the kinetics of contact-dependent intercellular signal transfer and to assess the molecular mechanisms employed by this process. Exposure to follicle-stimulating hormone (FSH) caused cAMP-dependent protein kinase dissociation in granulosa cells and, with time, in Y-1 cells if, and only if, they contacted a responding granulosa cell. Y-1 cells close to a granulosa cell but not touching it failed to respond similarly. In reciprocal experiments, co-cultures were stimulated with adrenocorticotropic hormone (ACTH). Y-1 cells dissociated protein kinase as did granulosa cells in contact with Y-1 cells; however, granulosa cells that were not in contact with Y-1 cells failed to respond to the hormone. Fluorogenic steroids were secreted by Y-1 cells cultured alone and stimulated with ACTH, but were not secreted by cultures exposed to FSH. Neither hormone caused fluorogenic steroid production by granulosa cells. On the other hand these steroids were secreted in co-cultures stimulated with ACTH and to a lesser degree in co-cultures exposed to FSH. Autoradiography revealed that I125-FSH bound only to granulosa cells, never to Y-1 cells, even if they were in contact with an ovarian cell. The possibility of cell fusion was tested by experiments in which Y-1 cell membranes were labeled with cationized ferritin. These cells were then placed in co-culture with ovarian granulosa cells that had previously been allowed to ingest latex spheres. At regions of gap junctions between Y-1 and granulosa cells ferritin remained attached to the adrenal cell membrane and was never observed to migrate to the granulosa cell membrane. From these data, we conclude that hormone specific stimulation of one cell type leads to protein kinase dissociation in heterotypic partners only if they contact a hormone responsive cell. This signal transfer is bidirectional, exhibits temporal kinetics and occurs in the absence of apparent cell fusion. The only structural feature connecting Y-1 and granulosa cells were gap junctions implying they provided the communication channels; however, alternative mechanisms cannot be excluded. We have not established the identity of the signal being transferred although cAMP is a logical candidate.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Multiparameter morphology as a means of specifically identifying hormone stimulated adrenal cortical tumor cells (Y-1) and granulosa cells in coculture.

In order to test for biologically valid, direct communication between mouse adrenal tumor and porcine ovarian granulosa cells a method was needed for distinguishing the two cell types in culture. To this end granulosa cells were cultured for 24-48 hours after which they were challenged with rhodamine conjugated latex beads for 30 minutes to 24 hours in monolayer culture. When phagocytosis was allowed to occur for longer than one hour the phagocytic vesicles containing the conjugated beads appeared to fuse with each other and with digestive vacuoles. The resultant vesicles contained many beads and were located within the cell body close to the nucleus. At shorter incubation periods, beads were observed predominantly in the periphery of the cell. Y-1 adrenal cortical tumor cells failed to ingest beads and could be distinguished from the bead labeled granulosa cell in coculture with scanning or thin section electron microscopic as well as fluorescence microscopic techniques. Gap junctions were observed between heterotypic cell pairs; however, no evidence of cell fusion was found. ACTH stimulated granulosa: Y-1 adrenal cocultures initiated cAMP-dependent kinase dissociation in the Y-1 cells and in contacting granulosa cells identified positively by the presence of the beads. We have used the bead labeling technique and a procedure for following the kinetics of cAMP-dependent protein kinase dissociation in order to follow the specific hormonal stimulation of these heterotypic cells in coculture.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Neoplasms↗
PubMed1983

Activation of cyclic adenosine 3':5'-monophosphate-dependent protein kinase in H35 hepatoma and Chinese hamster ovary cells by a phorbol ester tumor promoter.

The ability of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol- 13-acetate (TPA) to lead to the activation of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase was investigated in three cell lines. In the Reuber H35 hepatoma cells, TPA (1.6 microM) led to an increase in the protein kinase activity ratio from a value of 0.13 in control cultures to 0.40 in the stimulated cells within 30 min of addition. A specific fluorescent cytochemical procedure was further utilized to study the intracellular localization of the free catalytic subunit following activation of the holoenzyme by TPA. A marked increase in cytoplasmic and nucleolar fluorescence indicative of the appearance of the free catalytic subunit (activation of cAMP-dependent protein kinase) in these cellular compartments was observed in both Reuber H35 hepatoma and Chinese hamster ovary 10001 cells incubated with TPA (1.6 microM) for 30 min. In the H35 cells monitored with the cytochemical probe for the catalytic subunit, protein kinase was activated within 5 min of the addition of TPA (1.6 microM) with 0.16 microM TPA in the culture medium resulting in the maximal degree of activation. No increase in intracellular fluorescence throughout a 40-min period was observed in a cAMP-dependent protein kinase-deficient mutant strain Chinese hamster ovary 10260 cells incubated with TPA. These studies indicate that, while TPA does lead to the marked activation of cAMP-dependent protein kinase in at least two cell types shown to be responsive to TPA, this activation may be only one of several rapid events which mediate the many specific effects of the phorbol esters.

Animals↗
PubMed1982

Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. I. Development and validation of fluorescinated inhibitor.

A specific and sensitive procedure has been developed that reliably localizes intracellular sites of free catalytic unit (C) dissociated from cAMP-dependent protein kinase. The method is based on a FITC conjugate (F:PKI) of affinity column-purified heat-stable protein inhibitor (PKI) of free C. The fidelity of this cytochemical probe was determined using cultures of Reuber H-35 hepatoma cells that had been stimulated for 2 h with 0.1 mM DBcAMP, or with diluent, then fixed with anhydrous acetone at -30 degrees C. In these preparations the F:PKI probe complexed with free C in cytoplasm, nucleolus, and, to a minor extent, in nucleoplasm. Binding of the F:PKI molecule to free C was competitively diminished by arginine analogues, guanidinium HCI and polyarginine, each used over a 2-log dose range. When the inhibitor's arginine residues were blocked by reaction with cyclohexanedione it no longer inhibited phosphotransferase activity of free C, and when fluorescinated it failed to localize C in stimulated cells. Similarly, when F:PKI was preabsorbed with excess pure C it no longer functioned as a cytochemical stain. Affinity column-purified antibody to free C also reduced significantly the ability of F:PKI to complex with C in cell cultures stimulated with 0.1 mM DBcAMP. 1 microgram of antibody reduced by approximately 10% the binding of F:PKI to all cell compartments while 5 microgram of antibody diminished binding by greater than 50%. Together, these results indicate that the F:PKI binds specifically, perhaps exclusively, to the catalytic units of cAMP-dependent protein kinase. The cytochemical procedure, unlike its biochemical counterparts, is able to locate the dissociation of cAMP-dependent protein kinase in individual cells of functionally or histologically complex cultures. Also, it reveals variations in the time- and dose-dependent activation of the kinase amongst clonal cells stimulated with cyclic nucleotide analogues or hormones.

Animals↗
PubMed1982

Direct cytochemical localization of catalytic subunits dissociated from cAMP-dependent protein kinase in Reuber H-35 hepatoma cells. II. Temporal and spatial kinetics.

The activation of cyclic AMP-dependent protein kinase has been found to be the predominant mode by which cyclic AMP (cAMP) leads to alterations of a large variety of cellular functions. The activation of the kinase results in the release of the catalytic subunit which as the free enzyme possesses phosphotransferase activity for a variety of specific protein substrates. Using a sensitive and specific cytofluorometric technique we monitored the appearance of free catalytic subunit in Reuber H35 hepatoma cells in culture after incubation with N6-1'-O-dibutyryl-cyclic AMP (DBcAMP), 8-bromoadenosine-3':5'-cyclic monophosphate (8-BrcAMP), and glucagon. The cytochemical method employs the heat-stable inhibitor of the free catalytic subunit which has been conjugated to fluorescein isothiocyanate (F:PKI) and was validated as described in the companion paper (Fletcher and Byus. 1982. J. Cell Biol. 93:719-726). Here we studied the temporal and spatial kinetics of the free catalytic subunit following activation of cAMP-dependent protein kinase by increasing concentrations of DBcAMP,8-BrcAMP, and glucagon. Under similar conditions protein kinase activation was also assessed biochemically in H35 cell supernatants by assaying the protein kinase activity ratio. Incubation of the hepatoma cells with DBcAMP (0.1 mM) led to an increase in the activity ratio from 0.2 in control cultures to a value of nearly 1.0 within a 1- to 2-h period. During this same period using the F:PKI probe, a significant increase in cytoplasmic and nucleolar fluorescence indicative of the release of the free catalytic subunit was coincidentally observed. In contrast to the rapid appearance of catalytic subunit in the cytoplasm and nucleolus of the cell within 5-15 min of the addition of DBcAMP, discernible nucleoplasmic fluorescence did not occur until after 1 h. H35 cell cultures incubated with 8-BrcAMP (0.01-1.0 mM) exhibited a more rapid activation of the protein kinase measured cytochemically compared to the cells treated with DBcAMP. Cultures incubated with 8-BrcAMP had significantly increased cytoplasmic and nucleolar fluorescence compared to unstimulated cells within 1 min of the addition of the analogue and reached a maximal level within 15 min. By employing a microspectrophotometer a distinct dose-dependent increase in cellular fluorescence (i.e., free catalytic subunit) was observed as the concentration of 8-BrcAMP was increased from 0.01 to 1.0 mM at 1, 5, 15, and 60 min following stimulation. The addition of glucagon (10(-6) M) to the culture also led to the activation of cAMP-dependent protein kinase as determined by an increase in the activity ratio. This increase was paralleled throughout the incubation period by a marked elevation in cytoplasmic and nucleolar fluorescence. The results reported herein suggest that both cyclic nucleotide analogues and a polypeptide hormone lead to the activation of cAMP-dependent protein kinase in similar intracellular compartments in Reuber H35 hepatoma cells...

Adenosine Monophosphate↗
PubMed1975

Intercellular communication in the rat anterior pituitary gland. An in vivo and in vitro study.

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.

Animals↗