Biomedical subjects

J F Whitfield

Publications and source records attributed to J F Whitfield.

At least 91 records · Page 5Linked to original sources

PubMed1992

The effect of muscarinic cholinergic agonists on intracellular calcium and progesterone production by chicken granulosa cells.

The effects of the muscarinic cholinergic receptor agonist carbachol on intracellular calcium ion concentration ([Ca2+]i) and progesterone production was determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the calcium-responsive fluorescent dye fura-2. Resting [Ca2+]i was 96 +/- 5 nM (n = 13). There was a 4- to 8-fold increase in [Ca2+]i in 85% (n = 80) of the cells within 10 sec after the addition of a maximally stimulating concentration (2 mM) of carbachol. The initial [Ca2+]i spike was followed by a sustained, but lower, calcium elevation, with superimposed oscillations which returned to resting level after several minutes. Both phases of the calcium transient were inhibited by pretreating the cells with atropine (1 microM), pirenzepine (2 microM) or 4-diphenylacetoxy-N-methylpiperidine methiodide (1 microM). The sustained phase of the response with its superimposed oscillations, but not the initial spike, was inhibited by pretreating the cells with the calcium channel blockers lanthanum (1 mM), cobalt (5 mM), or methoxyverapamil (50 microM), or by incubating the cells in calcium-free medium. Nifedipine (0.5-20 microM) did not affect the carbachol-induced calcium transient. 8-(N,N-Diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (50 microM) blocked the sustained-oscillatory phase of the carbachol-induced [Ca2+]i transient, but did not affect the initial spike when added before carbachol (2 mM). Despite its ability to stimulate [Ca2+]i surges, carbachol (10(-6)-10(-3) M) did not affect basal or LH-stimulated cAMP or progesterone production in 24-h cultures. These studies demonstrate that activation of chicken granulosa cell muscarinic receptors causes a rapid increase in [Ca2+]i through the release of Ca2+ from intracellular stores, followed by a sustained elevation of Ca2+ with superimposed oscillations caused by the influx of extracellular Ca2+. These results also indicate that an increase in [Ca2+]i in chicken granulosa cells does not alone stimulate steroidogenesis, since the carbachol-induced increases in [Ca2+]i were not accompanied by increased progesterone production.

Acetylcholine↗

PubMed1992

Muscarinic cholinergic stimulation elevates intracellular pH in chicken granulosa cells by a Ca(2+)-dependent, Na(+)-independent mechanism.

Various hormones and growth factors act at least in part by raising the cytosolic pH of the target cell. In the present studies we examined the influence of the muscarinic cholinergic agonist carbachol on intracellular pH (pHi) in chicken granulosa cells. The pHi in granulosa cells from the two largest preovulatory follicles of hens was determined spectrofluorometrically using the pH-sensitive dye 2',7'-bis-(carboxyethyl)- 5(6)-carboxyfluorescein (BCECF). Carbachol (0.1-2 mM) induced a concentration-dependent increase in pHi, which reached a maximum of 0.23 +/- 0.02 pH units at a concentration of 1 mM. Cytosolic alkalinization was observed within 10 min of the addition of carbachol and lasted over the 60-min observation period. The effect of carbachol was mimicked by acetylcholine (1 mM) and muscarine (1 mM), but not by nicotine (0.1 mM). The carbachol-induced alkalinization was blocked by pretreating the cells with the muscarinic cholinergic receptor antagonists atropine (1 and 10 microM) and pirenzepine (0.01 and 0.1 mM). The increase in pHi did not appear to be mediated by a conventional mechanism involving the Na+/H+ antiporter, because it was unaffected by replacement of extracellular Na+ with the nonpermeant choline chloride or the presence of antiporter inhibitors, such as amiloride, dimethylamiloride, or ethylisopropylamiloride. However, the mechanism required external Ca2+, but did not involve Ca2+ channels, because it was unaffected by the general Ca2+ channel blocker methoxyverapamil (50 microM). Thus, Ca(2+)-dependent and Na(+)-independent intracellular alkalinization may be a part of the signalling mechanism by which muscarinic receptor activation regulates chicken granulosa cell function.

Animals↗

PubMed1992

A new, nongenomic estrogen action: the rapid release of intracellular calcium.

We have investigated the effects of steroids on the intracellular calcium ion concentration [Ca2+]i in chicken granulosa cells obtained from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The resting [Ca2+]i in these cells was 100 +/- 5 nM. There was an immediate (i.e. less than 5 sec) 4- to 8-fold increase in [Ca2+]i in all of the 76 cells examined after the addition of 10(-7) M estradiol-17 bdta. Estradiol-17 beta was effective between 10(-10)-10(-6) M. Estradiol-17 alpha, estrone, and estriol (10(-8)-10(-6) M) were as effective as estradiol-17 beta, but the progestins, pregnenolone, and progesterone, and the androgens, testosterone, androstenedione, or 5 alpha-dihydrotestosterone were ineffective at concentrations up to 10(-5) M. The prompt estradiol-17 beta-induced [Ca2+]i spike was not affected by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA or by pretreating them with the Ca2+ channel blockers lanthanum (1 mM), cobalt (5 mM), methoxyverapamil (D600; 50 microM), or nifedipine (20 microM). The estrogen-triggered [Ca2+]i surge was also not affected by pretreating the cells with the conventional estrogen receptor antagonist tamoxifen (10(-5) M), or the RNA and protein synthesis inhibitors actinomycin D (1 microgram/ml) and cycloheximide (1 microgram/ml), but was abolished by pretreating the cells with inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) and U-73,122 (2.5 microM). The closely related, but inactive, compound U-73,343 (1 microM) did not affect the estrogen-triggered [Ca2+]i surge. Estradiol-17 beta (10(-7) M), but not progesterone (10(-5) M), also triggered a large [Ca2+]i surge in pig granulosa cells, which, like the [Ca2+]i surge in chicken granulosa cells, was almost immediate, transient, and unaffected by incubation in Ca(2+)-free medium or pretreatment with methoxyverapamil (D600; 50 microM), lanthanum (1 mM), or tamoxifen (10(-5)M). However, granulosa cells from immature rats primed with diethylstilbestrol or PMSG did not respond to estradiol-17 beta, even at concentrations as high as 10(-5) M, although they promptly generated a [Ca2+]i transient upon exposure to LHRH (10(-5) M). These results suggest that estrogens almost instantaneously trigger the release of Ca2+ from intracellular stores which may be mediated through phosphoinositide breakdown. The striking rapidity of this estrogen-induced internal Ca2+ mobilization is consistent with the activation of a cell surface receptor which is different from the conventional slowly acting, gene-stimulating nuclear estrogen receptor.

Androgens↗

PubMed1992

Calcium signals and cancer.

Calcium, the great signaler, is at the heart of proliferation, differentiation, and cancer. It figures prominently in the five signals that activate proliferatively quiescent normal cells and then trigger chromosome replication and mitosis. Calcium regulates intercellular communication through gap junctions and triggers the terminal differentiation programs of cells such as colon cells and keratinocytes. Calcium is also the killer in the programmed suicide mechanism (apoptosis) of differentiated senescent cells or functionally superfluous cells that is needed to maintain tissue homeostasis. The cancer cells emerging from the multistep carcinogenic process with inactivated or deleted tumor-suppressor genes and/or activated oncogenes are much less dependent than normal cells on external growth factors because they make and secrete their own factors. They also need much less external calcium to proliferate, and they no longer obey calcium signals to differentiate and ultimately die.

Calcium↗

PubMed1991

Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in thymocytes.

The effects of topoisomerase II-reactive epipodophyllotoxins etoposide and teniposide as well as amsacrine on the viability of thymocytes in primary culture has been examined. All three drugs were shown to produce DNA cleavage detectable by resolving isolated DNA by pulsed field agarose gel electrophoresis. The DNA cleavage was found to have two components. The first was due to the interaction of the drugs with topoisomerase II, whereas the second component was due to endonuclease cleavage caused by the drug-induced entry of the thymocytes into programmed cell death or apoptosis. This second component of the DNA cleavage was also detected in thymocytes undergoing apoptosis following exposure to the glucocorticoid analogue, dexamethasone. The effect of the drugs on programmed cell death is dependent upon new protein and RNA synthesis, indicating that topoisomerase II has a role in the very first stages of the process. These results are discussed in terms of the use of this class of topoisomerase II-reactive drugs in chemotherapy.

Amsacrine↗

PubMed1991

Changes in cyclic adenosine monophosphate-responsive element binding proteins in rat hepatomas.

We applied Southwestern and Western blotting and gel retardation techniques to investigate the changes that occur in the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) binding (CREB) proteins in rapidly growing, chemically induced 5123tc and 5123D Morris hepatomas. Using the CRE sequences from the c-fos, E2A, and somatostatin gene promoters, we identified in the nuclear proteins from normal unstimulated or proliferating rat liver cells six different protein factors of Mr 34,000, 36,000, 40,000, 47,000, 56,000, and 72,000 capable of binding to the element. The Mr 47,000 protein had the highest specificity for the core CRE, suggesting its importance in cAMP-mediated gene expression. We could not find the Mr 47,000 CREB protein in the 5123tc and 5123D hepatomas. Our efforts to detect this protein in the tumors by (a) using the CRE sequence from different gene promoters, (b) altering the protocol for extracting nuclear proteins, or (c) attempting to restore its DNA-binding property by phosphorylation [with endogenous protein kinase(s), a catalytic subunit of cAMP-dependent protein kinase, and protein kinase C/dephosphorylation (with alkaline phosphatase)] were unsuccessful. The loss of tje Mr 47,000 CREB protein from solid tumors of the Morris hepatoma is likely to be related to the neoplastic properties of the tumor cell rather than to cell growth because the level of this protein remained unchanged during a 6-day period of liver regeneration. The nuclear extract from the Morris hepatoma that did not have the Mr 47,000 CRE-binding factor contained proteins immunologically related to the CREB, c-Jun, and c-Fos proteins. We conclude that the Mr 47,000 factor represents a distinct member of the CRE-binding protein family and that its absence from the hepatomas may lead to aberrant expression of cAMP-inducible genes.

Animals↗

PubMed1991

The direct measurement of protein kinase C (PKC) activity in isolated membranes using a selective peptide substrate.

A protein kinase C (PKC)-selective peptide substrate was used to develop a method for measuring PKC activity directly and quantitatively in isolated cell membranes without prior detergent extraction and reconstitution of the enzyme with phosphatidylserine and TPA in the presence of excess Ca2+. This simple and rapid method can reliably measure changes in membrane-associated PKC activity induced by various bioactive compounds such as hormones and growth factors. Also, this method, which measures PKC activity in its native membrane-associated state, has the advantage of being able to distinguish between active and inactive PKC associated with cell membranes.

Amino Acid Sequence↗

PubMed1991

Role of chloride ions in progesterone production by chicken granulosa cells.

The importance of chloride ions in luteinizing hormone (LH)-stimulated progesterone production by chicken granulosa cells from the two largest preovulatory follicles was investigated in vitro. Reduction of the extracellular chloride concentration from 147.8 mM to 2.8 mM, by substitution with equimolar concentrations of non-permeant glutamate and aspartate, inhibited the ability of LH to stimulate progesterone production and cAMP accumulation during a 4 h incubation. LH-stimulated granulosa cell progesterone production was also suppressed in a concentration-dependent manner by the chloride channel blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS; 10(-8)-5 x 10(-5) M) or 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS; 10(-8)-5 x 10(-5) M). The inhibitory effect was observed within 30 min of the addition of the blockers and was irreversible. DIDS appeared to act at a site(s) proximal to the generation of cAMP, since concentrations of DIDS (10(-8)-10(-6) M) which inhibited LH- and human chorionic gonadotropin-stimulated progesterone production, did not affect progesterone production stimulated by dibutyryl cAMP, 8-bromo cAMP or forskolin. In addition, concentrations of DIDS (10(-8)-10(-6) M) which attenuated LH-stimulated progesterone production also reduced the accumulation of extracellular cAMP. These studies suggest that chloride ions may play an important role in the stimulatory action of LH on chicken granulosa cell progesterone production.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1991

tsLA23-NRK cells need pp60v-src protein-tyrosine kinase activity in G2 phase to initiate mitosis in serum-free medium.

Lowering the temperature from 41 to 36 degrees C stimulates quiescent tsLA23-NRK rat cells (infected with the tsLA23 mutant of the Rous sarcoma virus) in serum-free medium to resume cycling and initiate DNA replication by reactivating the tsLA23-RSV's abnormally thermolabile pp60v-src protein-tyrosine kinase. Inactivating the enzyme in these pp60v-src-stimulated cells by again raising the temperature to 41 degrees C after the cells had initiated DNA replication did not prevent the completion of DNA replication and entry into the G2 phase, but it stopped the initiation of mitosis. Adding serum at the time of the temperature increase replaced the lost pp60v-src activity and the cells were able to continue to mitosis. The G2-arrested cells at 41 degrees C were able to initiate mitosis when pp60v-src was reactivated again by lowering the temperature to 36 degrees C. These observations suggest that protein-tyrosine kinase activity is needed to initiate mitosis and that the tsLA23-NRK cell is a good model for studying the function of this kinase activity in the initiation of mitosis.

Avian Sarcoma Viruses↗

PubMed1991

Characterization of a novel erythropoiesis-inhibiting human protein.

We have isolated an erythropoiesis-inhibiting protein, DIP (differentiation-inhibiting protein), from the blood of a 60-year-old woman suffering from pure red cell aplasia. This protein inhibits the growth and differentiation of normal human and murine BFU-E, but not CFU-E, cells as well as dimethyl sulfoxide-induced hemoglobin synthesis by Friend murine erythroleukemia cells. It appears that DIP primarily affects differentiation rather than proliferation, because it does not inhibit the proliferation of untreated Friend erythroleukemia cells. DIP seems to function like a recently described 45-kDa autocrine differentiation-inhibiting protein factor (ADIF) which is secreted by tsAEV-transformed chicken erythroleukemia cells. Both proteins selectively block the differentiation of normal human and murine BFU-E cells as well as the differentiation (but not the proliferation) of Friend murine erythroleukemia cells. However, the human DIP is not an autocrine product of the patient's bone marrow cells, nor does it affect chicken erythroid cells.

Animals↗

PubMed1991

Polyoma virus middle tumor antigen stimulates membrane-associated protein kinase C at lower levels than required for phosphatidylinositol kinase activation and neoplastic transformation.

Twelve independent rat F111 cell lines expressing the polyoma virus middle tumor antigen (mT) under control of the dexamethasone-regulatable MMTV-LTR promoter were assayed for levels of membrane-associated protein kinase C (PKC) activity. Low background levels of mT antigen expression (approximately 2%), although insufficient for transformation, triggered a dramatic increase in PKC activity. Under the same conditions, levels of the mT-associated phosphatidylinositol kinase activity were low, indicating that this kinase might be a factor limiting transformation in this cell system.

1-Phosphatidylinositol 4-Kinase↗

PubMed1990

Parathyroid hormone fragment [3-34] stimulates protein kinase C (PKC) activity in rat osteosarcoma and murine T-lymphoma cells.

The parathyroid hormone (PTH) fragment [1-34] strongly stimulated both adenylate cyclase and membrane-associated PKC activities in rat 17/2 osteosarcoma cells. By contrast, the PTH [3-34] fragment, which was unable to stimulate adenylate cyclase, remained a potent stimulator of membrane-associated PKC activity in these cells. Both PTH fragments also strongly stimulated membrane-PKC activity in cyc-S49T-lymphoma cells possessing a defective adenylate cyclase system. This ability of PTH [3-34] to stimulate membrane-associated PKC activity could explain the residual bioactivity of this fragment.

Adenosine Triphosphate↗

PubMed1990

The cytotoxicity of Pseudomonas exotoxin A, inactivated by modification of the cell-binding domain I, is restored when conjugated to an erythroid cell-specific targeting agent.

To be capable of selective killing of tumor cells, the non-selective Pseudomonas aeruginosa exotoxin A must have its cell-binding domain inactivated or removed and then be chemically linked to, or genetically fused with, a specific targeting agent. In the present study, epsilon-NH2 groups of lysine residues of the cell-binding domain of exotoxin A were extensively propionylated with N-succinimidyl-3-propionate (NSP). The NSP-treated exotoxin retained its cytocidal ADP-ribosyltransferase activity, but it could no longer bind to, and inhibit the proliferation of, Friend murine erythroleukemia cells. Cytotoxicity (i.e., the ability to inhibit proliferation) for the Friend erythroid cells was restored completely to the NSP-inactivated exotoxin by conjugating it to ADIF, an autocrine factor secreted by chicken erythroleukemia cells which selectively inhibits the differentiation of erythroid cells such as Friend erythroleukemia cells without inhibiting their proliferation.

ADP Ribose Transferases↗

PubMed1990

Transforming signals generated by the polyoma virus tumor antigens.

The secretion of a threshold amount of SAGF can elicit one of the major features of the transformed phenotype, anchorage-independent growth. It appears that, like other cells, NIH 3T3 cells make and secrete SAGF, but not enough to enable them to proliferate in soft agar. The stream of signals from polyoma virus MTAg:pp60c-src:PI-kinase complexes attached to the inner surface of the cell membrane is not enough to raise SAGF secretion to the threshold level for agar growth, but these signals can powerfully enhance the responsiveness to added SAGF. Only when NIH 3T3 cells express all three polyoma T antigens do they secrete enough SAGF and become responsive enough to the factor to form colonies in soft agar. This is in contrast to F111 cells which produce adequate amounts of, and respond to, SAGF after MTAg expression alone. The reasons for this difference remain to be investigated. Obviously, factors expressed during the course of the spontaneous establishment are different for these two lines and they affect the cells' response to MTAg. Clearly, knowing how polyoma virus transforms cells will require a deeper understanding of the deregulation of membrane signaling enzymes by the viral MTAg and the identification and characterization of the autocrine SAGF's that mediate at least part of the neoplastic response to the viral signals.

Animals↗

PubMed1990

Carcinogenic implications of the neighborhood coherence principle (NCP).

A new hypothesis on carcinogenesis is set forth on the basis of the neighborhood coherence principle (NCP). NCP constitutes a general rule of pattern formation and maintenance. According to this principle, a system of interacting cells can produce and maintain a spatial organization by virtue of cell-cell communication. This hypothesis suggests that this homeostasis primarily results from a NCP-like process implying cell-cell communication. Each cell is constrained by its neighbors to maintain the mature phenotype despite its inherent individual variability. If the cell-cell mature communication happens to be impaired, tissue homeostasis is disrupted and a proliferative state can be initiated. A further potential effect may result from the establishment of NCP-like communication specific for proliferative cells allied to paracrine and outocrine factors which can lock the cells into the proliferative mode. Most mechanisms implied in this hypothesis have already been investigated. There is a large body of experimental results supporting the role of junctional communication in cooperative metabolism, growth, differentiation and tumour-related events. This new hypothesis provides a framework within which these known facts may be put in a theoretical perspective; it might well constitute the unifying theory--as yet missing--in carcinogenesis.

Animals↗

PubMed1990

The role of signal-transducing events in the proliferative response of cells to a mitogenic viral K-ras protein.

Activated oncogenic ras proteins are powerful mitogenic agents which by themselves can initiate and maintain the proliferation of quiescent cells in the absence of any exogenous growth factors. In an attempt to understand how ras proteins induce proliferation we examined the early events in the G0 to G1 transition caused by the activation of a thermolabile K-ras protein in quiescent, serum-starved tsKSV-transformed NRK cells. We show that ras reactivation, in the absence of exogenous growth factors, triggered a rapid surge in free cytosolic Ca2+ and diacylglycerol production, which led to a transient increase in membrane-associated protein kinase C (PKC) activity which was necessary for G1 transit. Unlike TPA-stimulated PKC activity, the ras-induced increase in PKC was readily extracted from membranes by EGTA. These signal transducing events occurred despite the fact that ras activation did not induce the tyrosine phosphorylation of any known surface receptor. The results indicate that the K-ras protein triggers the G0 to G1 transition by an intracellular mechanism and not indirectly via autocrine stimulation.

Animals↗

PubMed1990

Membrane protein kinase C activity rapidly increases in quiescent tsRSV-infected NRK cells upon reactivation of the mitogenic v-src protein kinase.

The viral src protein kinase, pp60v-src, is a powerful intracellular mitogen which can initiate and maintain the proliferation of quiescent cells in the absence of any exogenous growth factors. In an attempt to understand how pp60v-src induces proliferation, we examined the early events in the G0 to G1 transition caused by the activation of a thermolabile v-src protein in quiescent, serum-starved tsRSV-transformed NRK cells. The reactivation of pp60v-src, in the absence of exogenous growth factors, triggered a rapid biphasic surge of membrane-associated protein kinase C (PKC) activity. Unlike TPA-stimulated PKC activity, the pp60v-src-induced increase in PKC was readily extracted from membranes by EGTA. The down-regulation of PKC activity in these quiescent cells by prolonged exposure to TPA strongly inhibited the ability of the reactivated v-src protein to stimulate DNA replication in serum-deficient medium, suggesting that PKC plays a role in the initial signal by which the viral enzyme induces the G0 to G1 transition in NRK cells.

Animals↗

PubMed1990

Evidence that mammalian ribonucleotide reductase is a nuclear membrane associated glycoprotein.

Epitope-specific antibodies to the M1 and M2 subunits of mammalian ribonucleotide reductase were prepared using peptides predicted to have a high antigenic index. Western blotting demonstrated that the anti-M1 antibody was specific for the 89-kilodalton M1 subunit (and its degradation fragments) and the anti-M2 antibody specifically recognized the 45-kilodalton M2 subunit. Both antibodies inhibited the CDP-reductase activity of the holoenzyme. Using these antibodies, both the M1 and M2 subunits were shown to be localized in the cytoplasm and in the nuclear regions of a number of cell types, including B77 avian sarcoma virus transformed NRK cells, T51B rat liver cells, 5123tc hepatoma cells, and rat liver cells in vivo. In addition, the M1 subunit was found to be localized as a halo around isolated rat liver nuclei. Biochemical analysis of the cytoplasmic fraction of liver cells and a Triton X-100 wash of nuclei from these cells confirmed the location of the enzyme activity in these cellular compartments. The M1 subunit appears to be glycosylated, as indicated by its retention on a Affi-Gel-concanavalin A affinity column. Therefore, in mammalian cells ribonucleotide reductase appears to be not only in the cytoplasm, but is also associated with the nuclear membrane or nuclear lamina. The activity of the enzyme in the membrane fraction changes dynamically during the cell cycle.

Animals↗